Introduction to Human

Second Edition

Edited on behalf of The Nutrition Society by Michael J Gibney Susan A Lanham-New Aedin Cassidy Hester H Vorster

A John Wiley & Sons, Ltd., Publication

Introduction to The Nutrition Society Textbook Series

Introduction to Human Nutrition Nutrition and Introduction to Human Nutrition: a global perspective on Core concepts of nutrition and nutrition Molecular nutrition Body composition The regulation of food intake metabolism Integration of metabolism 1: Energy Nutrition and metabolism of and amino acids Integration of metabolism 2: and and metabolism of carbohydrates Integration of metabolism 3: and amino acids Nutrition and metabolism of lipids Dietary reference standards and lactation The Growth and aging and trace elements Gastrointestinal tract Measuring food intake Cardiovascular system Food composition The skeletal system Food and nutrition: policy and regulatory issues The immune and infl ammatory systems Nutrition research methodology The sensory systems : a public issue of growing importance Physical activity Food and nutrition-related : the global challenge Overnutrition Undernutrition The brain

Public Health Nutrition An overview of nutrition General principles of clinical nutrition Nutrition Metabolic and nutritional assessment Overnutrition Assessment of nutritional status at individual and Undernutrition level Metabolic disorders Assessment of physical activity Eating disorders Overnutrition Adverse reactions to Undernutrition Nutritional support Eating disorders, and food fads Ethical and legal issues PHN strategies for nutrition: intervention at the level of Gastrointestinal tract individuals The liver PHN strategies for nutrition: intervention at the The pancreas ecological level The kidney Food and nutrition guidelines and bone marrow Fetal programming The lung Cardiovascular Immune and infl ammatory systems Cancer and blood vessels The skeleton Traumatic diseases A defi ciency Infectious diseases defi ciency Malignant diseases defi ciency Pediatric nutrition Maternal and child health Cystic fi brosis Breast feeding Clinical cases Adverse outcomes in pregnancy and electrolytes Introduction to Human Nutrition

Second Edition

Edited on behalf of The Nutrition Society by Michael J Gibney Susan A Lanham-New Aedin Cassidy Hester H Vorster

A John Wiley & Sons, Ltd., Publication This edition fi rst published 2009 First edition published 2002 © 2009, 2002 by The Nutrition Society

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Library of Congress Cataloging-in-Publication Data

Introduction to human nutrition / edited on behalf of the Nutrition Society by Michael J. Gibney . . . [et al.]. – 2nd ed. p. ; cm. – (The human nutrition textbook series) Includes bibliographical references and index. ISBN 978-1-4051-6807-6 (pbk. : alk. paper) 1. Nutrition. I. Gibney, Michael J. II. Nutrition Society (Great Britain) III. Series. [DNLM: 1. Nutrition 2. Food. QU 145 I623 2009] QP141.I665 2009 612.3–dc22 2008035123

A catalogue record for this book is available from the British Library.

Set in 10 on 12 pt Minion by SNP Best-set Typesetter Ltd., Hong Kong Printed in Singapore by Fabulous Printers Pte Ltd

1 2009 Contents

Contributors vii Series Foreword ix Preface xi Acknowledgments xii 1. Introduction to Human Nutrition: A Global Perspective on Food and Nutrition 1 HH Vorster 2. Body Composition 12 P Deurenberg 3. Energy Metabolism 31 A Astrup and A Tremblay 4. Nutrition and Metabolism of Proteins and Amino Acids 49 NK Fukagawa and Y-M Yu 5. Digestion and Metabolism of Carbohydrates 74 J Mathers and TMS Wolever 6. Nutrition and Metabolism of Lipids 86 BA Griffi n and SC Cunnane 7. Dietary Reference Standards 122 KM Younger 8. The Vitamins 132 DA Bender 9. Minerals and Trace Elements 188 JJ Strain and KD Cashman 10. Measuring Food Intake 238 UE MacIntyre 11. Food Composition 276 HC Schönfeldt and JM Holden 12. Food and Nutrition: Policy and Regulatory Issues 293 MJ Gibney and A McKevitt 13. Nutrition Research Methodology 305 JA Martínez and MA Martínez-González 14. Food Safety: A Public Health Issue of Growing Importance 324 A Reilly, C Tlustos, J O’Connor, and L O’Connor 15. Food and Nutrition-Related Diseases: The Global Challenge 350 HH Vorster and MJ Gibney Index 361

Contributors

Professor Arne Astrup Dr Bruce A Griffi n Head, Department of Human Nutrition, Reader in Nutritional Metabolism, Faculty of , Nutritional Sciences Division, University of Copenhagen, Faculty of Health and Medical Sciences, Copenhagen, Denmark University of Surrey, Guildford, UK Dr David A Bender Sub-Dean (Education), Joanne M Holden University College London Medical School, Data Laboratory, London, UK Beltsville, Maryland, USA Professor Kevin D Cashman Department of Food and Nutritional Sciences, Una E MacIntyre University College Cork, Institute for Human Nutrition, Ireland University of Limpopo, Medunsa, Dr Stephen C Cunnane South Africa Departments of Medicine, Physiology and and Research Center on Aging, Dr Aideen McKevitt Université de Sherbrooke School of Biomedical Sciences, Canada University of Ulster, Northern Ireland Professor Paul Deurenberg Associate Professor in Nutrition, Professor J Alfredo Martínez Department of Human Nutrition, Intitute of Nutrition and Food Sciences, Wageningen University, University of Navarra, The Netherlands Spain Visiting Professor, University Tor Vergata, Rome, Italy Professor Miguel A Martínez-González Nutrition Consultant, Singapore Department of Preventive Medicine and Public Health, Professor Naomi K Fukagawa University of Navarra, Department of Medicine, Spain University of Vermont, Burlington, Vermont, USA Professor John Mathers Human Nutrition Research Centre, Professor Michael J Gibney Institute for and Health Department of Clinical Medicine, University of Newcastle, UK Trinity College, Dublin, Ireland Dr Judith O’Connor Food Safety Authority of Ireland, Dublin, Ireland viii Contributors

Dr Lisa O’Connor Professor Hester H Vorster Food Safety Authority of Ireland, Director of the Centre of Excellence for Nutrition Dublin, Ireland Faculty of Health Sciences, North-West University Alan Reilly Potchefstroom, South Africa Food Safety Authority of Ireland, Dublin, Ireland Dr Thomas MS Wolever Department of Nutritional Sciences, Professor Hettie C Schönfeldt Faculty of Medicine, School of Agricultural and Food University of Toronto, University of Pretoria, Canada South Africa Dr Kate M Younger Professor JJ (Sean) Strain Lecturer in Human Nutrition, Professor of Human Nutrition, School of Biological Sciences, Northern Ireland Centre for Food and Health, Dublin Institute of Technology, University of Ulster, Ireland Coleraine, Northern Ireland Dr Yong-Ming Yu Christina Tlustos Department of Surgery, Food Safety Authority of Ireland, Massachusetts General Hospital and Shriners Burns Dublin, Ireland Hospital, Harvard Medical School, Angelo Tremblay Boston, Massachusetts, USA Preventive and , Laval University, Ste-Foy, Québec, Canada Series Foreword

The early decades of the twentieth century were a medium for the publication of primary research on period of intense research on constituents of food all aspects of human and nutrition by scien- essential for normal growth and development, and tists from around the world. Recognizing the needs of saw the discovery of most of the vitamins, minerals, students and their teachers for authoritative reviews amino acids and essential fatty acids. In 1941, a group on topical issues in nutrition, the Society began pub- of leading physiologists, and medical lishing Nutrition Research Reviews in 1988. In 1997, scientists recognized that the emerging discipline of we launched Public Health Nutrition, the fi rst inter- nutrition needed its own learned society and the national journal dedicated to this important and Nutrition Society was established. Our mission was, growing area. All of these journals are available in and remains, “to advance the scientifi c study of nutri- electronic, as well as in the conventional paper form tion and its application to the maintenance of human and we are exploring new opportunities to exploit the and animal health”. The Nutrition Society is the largest web to make the outcomes of nutritional research learned society for nutrition in Europe and we have more quickly and more readily accessible. over 2000 members worldwide. You can fi nd out To protect the public and to enhance the career more about the Society and how to become a member prospects of nutritionists, the Nutrition Society is by visiting our website at www.nutsoc.org.uk committed to ensuring that those who practice as The ongoing revolution in initiated by nutritionists are properly trained and qualifi ed. This large-scale genome mapping and facilitated by the is recognized by placing the names of suitably quali- development of reliable, simple-to-use molecular fi ed individuals on our professional registers and biological tools makes this a very exciting time to be by the award of the qualifi cations Registered Public working in nutrition. We now have the opportunity Health Nutritionist (RPHNutr) and Registered Nutri- to obtain a much better understanding of how spe- tionist (RNutr). Graduates with appropriate degrees cifi c interact with nutritional intake and other but who do not yet have suffi cient postgraduate expe- lifestyle factors to infl uence expression in indi- rience can join our Associate Nutritionist registers. vidual cells and tissues and, ultimately, affect our We undertake accreditation of university degree pro- health. Knowledge of the polymorphisms in key genes grams in public health nutrition and are developing carried by a patient will allow the prescription of accreditation processes for other nutrition degree more effective, and safe, dietary treatments. At the programs. population level, molecular epidemiology is opening Just as in research, having the best possible tools is up much more incisive approaches to understanding an enormous advantage in teaching and learning. the role of particular dietary patterns in disease cau- This is the reasoning behind the initiative to launch sation. This excitement is refl ected in the several sci- this series of human nutrition textbooks designed for entifi c meetings that the Nutrition Society, often in use worldwide. This was achieved by successfully collaboration with sister learned societies in Europe, launching the fi rst series in multiple languages includ- organizes each year. We provide travel grants and ing Spanish, Portuguese and Greek. The Society is other assistance to encourage students and young deeply indebted to Professor Mike Gibney and his researchers to attend and participate in these team of editors for their tireless work in the last 10 meetings. years to bring the fi rst edition of this series of text- Throughout its history a primary objective of the books to its successful fruition worldwide. We look Society has been to encourage nutrition research and forward to this new edition under the stewardship of to disseminate the results of such research. Our fi rst Dr Susan Lanham-New in equal measure. Read, learn journal, The Proceedings of the Nutrition Society, and enjoy. recorded, as it still does, the scientifi c presentations made to the Society. Shortly afterwards, The British Professor Ian McDonald Journal of Nutrition was established to provide a President of the Nutrition Society

Preface

The Nutrition Society Textbook Series started ten The study of human nutrition needs a solid base in years ago as an ambitious project to provide under- the physiology and of human metabo- graduate and graduate students with a comprehen- lism and that is the basis of the textbook Nutrition and sive suite of textbooks to meet their needs in terms Metabolism. The present textbook is designed to serve of reference material for their studies. By all accounts two needs. Firstly, many will use this book as an intro- the project has been successful and the Nutrition duction to human nutrition and go no further. Stu- Society Textbook Series have been adapted by all of dents in , , agriculture and the the best academic nutrition units across the globe. like may take introductory modules to human nutri- The series has been translated into Spanish and tion and leave the subject there but be well informed in Portuguese. the area. Those who will go on to study human nutri- This second edition of Introduction to Human tion will fi nd within this textbook an introduction to Nutrition is an update of the very basic foundations the many areas of and health that they will go on for the study of human nutrition. Although little has to study in greater depths using the remaining text- changed, all authors have made whatever updates are books in the Nutrition Society series. Besides the basic necessary and we have made some re-arrangements biology, students will be introduced to the concept of of some chapters. The study of human nutrition at and to the dual challenges to the global universities across the globe is rapidly expanding food supply, both over and under nutrition. as the role of diet in health becomes more evident. As I write, I am handing over the leadership of Indeed, the sequencing of the human genome has the Nutrition Society Textbook Series to Dr Susan highlighted the narrower range of genes controlling Lanham-New at the University of Surrey who has , emphasising the critically important agreed to take on this important task for the Society. role of the environment including diet in human I would like to thank all those with whom I have health. Moreover, we now recognize the important worked with on this project and to wish Sue and her role that diet plays in interacting with our genome new team all the very best. both in utero and in the immediate period of post natal development. Michael J Gibney

The Nutrition Society Textbook Series Editors

Outgoing Editor-in-Chief Incoming Editor-in-Chief Professor Michael J Gibney Susan A Lanham-New University College Dublin, Ireland University of Surrey, UK

Assistant Editor Assistant Editor Julie Dowsett Jennifer Norton University College Dublin, Ireland The Nutrition Society, UK Acknowledgments

With grateful appreciation to all those who have time to make this edition possible. Very special thanks served on the International Scientifi c Committee and must go to Mike Gibney and Julie Dowsett, for their the Textbook Editors, without whom this task would effort and dedication in seeing this textbook of the be insurmountable and to all the authors who gave second edition through to publication. 1 Introduction to Human Nutrition: A Global Perspective on Food and Nutrition

Hester H Vorster

Key messages

• Human nutrition is a complex, multifaceted scientifi c domain • The hundreds of millions of food- and nutrition-insecure people indicating how substances in foods provide essential nourish- globally, the coexistence of undernutrition and overnutrition, and ment for the maintenance of life. inappropriate nutritional behaviors are challenges that face the • To understand, study, research, and practice nutrition, a holistic nutritionist of today. integrated approach from molecular to societal level is needed. • Nutrition practice has a fi rm and well-developed research and • Optimal, balanced nutrition is a major determinant of health. It knowledge base. There are, however, many areas where more can be used to promote health and well-being, to prevent ill- information is needed to solve global, regional, communal and health and to treat disease. individual nutrition problems. • The study of the structure, chemical and physical characteristics, • The development of ethical norms, standards, and values in and physiological and biochemical effects of the more than nutrition research and practice is needed. 50 found in foods underpins the understanding of nutrition.

1.1 Orientation to human nutrition renaissance with the observation by scientists that intakes of certain foods, later called nutrients, and The major purpose of this series of four textbooks on eventually other substances not yet classifi ed as nutri- nutrition is to guide the nutrition student through the ents, infl uence the of the body, protect exciting journey of discovery of nutrition as a science. against disease, restore health, and determine people’s As apprentices in nutrition science and practice stu- response to changes in the environment. During dents will learn how to collect, systemize, and classify this period, nutrition was studied from a medical knowledge by reading, experimentation, observation, model or paradigm by defi ning the chemical struc- and reasoning. The road for this journey was mapped tures and characteristics of nutrients found in foods, out millennia ago. The knowledge that nutrition – their physiological functions, biochemical reactions what we choose to eat and – infl uences our and human requirements to prevent, fi rst, defi ciency health, well-being, and quality of life is as old as diseases and, later, also chronic noncommunicable . For millions of years the quest for diseases. food has helped to shape human development, the Since the late 1980s nutrition has experienced a organization of society and history itself. It has infl u- second renaissance with the growing perception that enced wars, population growth, urban expansion, the knowledge gained did not equip mankind to solve economic and political theory, religion, science, med- the global problems of food insecurity and malnutri- icine, and technological development. tion. The emphasis shifted from the medical or path- It was only in the second half of the eighteenth ological paradigm to a more psychosocial, behavioral century that nutrition started to experience its fi rst one in which nutrition is defi ned as a basic human

© 2009 HH Vorster. 2 Introduction to Human Nutrition right, not only essential for human development but tual framework, illustrating the complex interactions also as an outcome of development. between internal or constitutional factors and exter- In this fi rst, introductory text, the focus is on prin- nal environmental factors which determine nutri- ciples and essentials of human nutrition, with the tional status and health, is given in Figure 1.1. main purpose of helping the nutrition student to On a genetic level it is now accepted that nutrients develop a holistic and integrated understanding of dictate phenotypic expression of an individual’s gen- this complex, multifaceted scientifi c domain. otype by infl uencing the processes of transcription, translation, or post-translational reactions. In other 1.2 An integrated approach words, nutrients can directly infl uence genetic (DNA) expression, determining the type of RNA formed Human nutrition describes the processes whereby (transcription) and also the proteins synthesized cellular , cells, tissues, organs, systems, and (translation). For example, , a the body as a whole obtain and use necessary sub- macronutrient, increases transcription for the synthe- stances obtained from foods (nutrients) to maintain sis of glucokinase, the iron increases structural and functional integrity. For an under- translation for the synthesis of ferritin, while vitamin standing of how humans obtain and utilize foods and K increases post-translational carboxylation of glu- nutrients from a molecular to a societal level, and of tamic acid residues for the synthesis of prothrombin. the factors determining and infl uencing these pro- Nutrients, therefore, infl uence the synthesis of struc- cesses, the study and practice of human nutrition tural and functional proteins, by infl uencing gene involve a spectrum of other basic and applied scien- expression within cells. tifi c disciplines. These include , Nutrients also act as substrates and cofactors in all , biochemistry, chemistry, physics, food of the metabolic reactions in cells necessary for the science, , physiology, , growth and maintenance of structure and function. , psychology, sociology, political science, Cells take up nutrients (through complex mecha- , agriculture, , communi- nisms across membranes) from their immediate cations, and economics. Nutrition departments are, environment, also known as the body’s internal envi- therefore, often found in Medical (Health) or Social ronment. The composition of this environment is Science, or Pharmacy, or Agriculture Faculties at carefully regulated to ensure optimal function and tertiary training institutions. The multidisciplinary survival of cells, a process known as , of the science of nutrition, lying in both which gave birth to a systems approach in the study the natural (biological) and social scientifi c fi elds, of nutrition. demands that students of nutrition should have a Nutrients and are provided to the internal basic understanding of many branches of science and environment by the circulating blood, which also that they should be able to integrate different con- removes metabolic end-products and harmful sub- cepts from these different disciplines. It implies that stances from this environment for through students should choose their accompanying subjects the , the kidneys, and the large bowel. (electives) carefully and that they should read widely The concerted function of different organs and in these different areas. systems of the body ensures that nutrients and oxygen are extracted or taken up from the external environ- 1.3 A conceptional framework for ment and transferred to the blood for transport and the study of nutrition delivery to the internal environment and cells. The digestive system, for example, is responsible for the In the journey of discovery into nutrition science it of food and beverages, the breakdown will often be necessary to put new knowledge, or new (digestion and ) of these for extraction applications of old knowledge, into the perspective of nutrients, and the absorption of the nutrients into of the holistic picture. For this, a conceptual frame- the circulation, while the respiratory system extracts work of the multidisciplinary nature of nutrition oxygen from the air. These functions are coordinated science and practice may be of value. Such a concep- and regulated by the endocrine and central nervous Global Perspective on Food and Nutrition 3

Levels of human function Accompanying scientific disciplines (factors) of study

Constitutional

• Cell nucleus DNA RNA • Molecular biology, foods (Genetic level)

• Cells: metabolism • Internal environment • Biochemistry, foods • Circulation • Biochemistry, physiology, foods • All systems, also • Physiology, pathology, foods, pharmacology, etc. • Central • Psychology, pathology, foods, pharmacology, etc.

Nutritional status Health/ill-health

External environment • /insecurity • Foods, agriculture, food systems, sociology, • characteristics; care anthropology, economics, politics, policy, etc. • Social circumstances • Economic circumstances • , , politics Figure 1.1 Conceptual framework • Agriculture, health services for a holistic, integrated understand- (resources; ideologies) ing of human nutrition. systems in response to the chemical and physical tions. A medical, natural science or biological model composition of the blood and internal environment, underpinned the study of the relationships between and to cellular needs. nutrition and health or ill-health. During the second The health or disease state of the different organs renaissance, these aspects are not neglected, but and systems will determine the nutrient requirements expanded to include the study of all other external of the body as a whole. environmental factors that determine what and how The central nervous system is also the site or “head- much food and nutrients are available on a global quarters” of the higher, mental functions related to level. These studies are underpinned by social, behav- conscious or cognitive, spiritual, religious, and cul- ioral, economic, agricultural, and political sciences. tural behaviors, which will determine, in response to The study of human nutrition therefore seeks to the internal and external environments, what and understand the complexities of both social and bio- how much will be eaten. What and how much is eaten logical factors on how individuals and will further depend on what is available, infl uenced maintain optimal function and health, how the by a of factors determining food security. All of quality, quantity and balance of the food supply are these factors, on an individual, household, commu- infl uenced, what happens to food after it is eaten, and nity, national, or international level, shape the exter- the way that diet affects health and well-being. This nal environment. integrated approach has led to a better understanding During the fi rst renaissance of nutrition, emphasis of the causes and consequences of , and was placed on the study of nutrients and their func- of the relationship between nutrition and health. 4 Introduction to Human Nutrition

1.4 Relationship between nutrition nutrients. The study of nutrients, the ABC and and health numeric calculations of nutrition, will form a major part of the student’s nutrition journey, and should Figure 1.2 shows that individuals can be broadly cat- include: egorized into having optimal nutritional status or ● the chemical and physical structure and character- being undernourished, overnourished, or malnour- istics of the nutrient ished. The major causes and consequences of these ● the food sources of the nutrient, including food nutritional states are indicated. It is important to composition, the way in which foods are grown, realize that many other lifestyle and environmental harvested, stored, processed and prepared, and the factors, in addition to nutrition, infl uence health and effects of these on nutrient composition and nutri- well-being, but nutrition is a major, modifi able, and tional value powerful factor in promoting health, preventing and ● the digestion, absorption, circulatory transport, treating disease, and improving quality of life. and cellular uptake of the nutrient, as well as regu- lation of all these processes 1.5 Nutrients: the basics ● the metabolism of the nutrient, its functions, storage, and excretion People eat food, not nutrients; however, it is the com- ● physiological needs (demands or requirements) for bination and amounts of nutrients in consumed the nutrient in health and disease, and during foods that determine health. To read one must special circumstances (pregnancy, lactation, sport know the letters of the alphabet; to do sums one must events), as well as individual variability be able to count, add, subtract, multiply, and divide. ● interactions with other nutrients, nonnutrients To understand nutrition, one must know about (phytochemicals), , and drugs

Nutritional situation Health consequences, outcomes

Optimum nutrition Food-secure individuals with Health, well-being, normal development, adequate, balanced and prudent high quality of life diets

Undernutrition: Food-insecure individuals living • Decreased physical and mental development in poverty, ignorance, politically • Compromised immune systems unstable environments, • Increased infectious diseases disrupted societies, war • Vicious circle of undernutrition, underdevelopment, poverty

Overnutrition Overconsumption of food, , , cardiovascular especially macronutrients, plus: disease, mellitus, certain • low physical activity cancers: chronic NCDs, often characterized • smoking, stress, alcohol abuse by overnutrition of macronutrients and undernutrition of

Malnutrition : Individuals Double burden of infectious diseases plus NCDs, and communities previously often characterized by overnutrition of food insecure → confronted with macronutrients and undernutrition of abundance of palatable micronutrients foods → some undernourished, others too many macronutrients and too few micronutrients

Figure 1.2 Relationship between nutrition and health. NCD, noncommunicable disease. Global Perspective on Food and Nutrition 5

● the consequences of underconsumption and over- not suffi cient to understand and address the global consumption of nutrients problem of malnutrition facing mankind today. This ● the therapeutic uses of the nutrient perception has resulted in the cultivation of social ● factors infl uencing food and nutrition security and science disciplines to support knowledge from the food safety. biological sciences to address global malnutrition. There are more than 50 known nutrients (includ- ing amino acids and fatty acids) and many more 1.6 Global malnutrition chemicals in food thought to infl uence human func- tion and health (Box 1.1). Nutrients do not exist in It is a major tragedy that millions of people currently isolation, except for water and others in some phar- live with hunger, and fear . This is despite maceutical preparations. In foods, in the gut during the fact that food security or “access for all at all times, digestion, fermentation and absorption, in the blood to a sustainable supply of nutritionally adequate and during transport, and in cells during metabolism, safe food for normal physical and mental develop- nutrients interact with each other. Therefore, a par- ment and healthy, productive ” is a basic human ticular nutrient should not be studied in isolation, but right embedded in the constitution of most develop- integrated with other nutrients and seen in the context ing countries. It is also despite the fact that suffi cient of total body function. The study of nutrition also food is produced on a global level (see Box 1.2). Food includes how to determine nutrient requirements to make recommendations for intakes and how nutri- tional status is monitored by measuring intakes, Box 1.2 , body composition, biochemical markers refl ecting nutritional status, and the clinical Food insecurity: when people live with hunger, and fear starvation. signs of malnutrition. Food security: access for all, at all times, to a sustainable, This knowledge of nutrients and their functions affordable supply of nutritionally adequate and safe food for normal physical and mental development and healthy, produc- will enable the nutritionist to advise individuals what tive lives. and how much to eat. However, this knowledge is

Box 1.1 Classes of nutrients for human nutrition

Class/category Subclass/category Nutrient examples

Carbohydrates (macronutrients) Glucose, , galactose , maltose, and dietary fi ber Proteins (macronutrients) and animal source proteins Amino acids (n = 20): aliphatic, aromatic, -containing, acidic, basic and oils (lipids) Saturated fatty acids Palmitic and stearic acid (macronutrients) Monounsaturated fatty acids Oleic (cis) and elaidic (trans) fatty acids Polyunsaturated fatty acids (n-3, n-6, Linoleic, α-linolenic, arachidonic, eicosapentaenoic, n-9) docosahexaenoic acid Minerals (micronutrients) Minerals and electrolytes , , , , iron, , Trace elements , , , , fl uoride, Vitamins (micronutrients) soluble (A), calciferols (D), (E),

Water soluble Ascorbic acid (C), (B1), ribofl avin (B2), (B3),

(B6), , cobalamin (B12) Water Water Water 6 Introduction to Human Nutrition insecurity is an obstacle to human rights, quality of date of 2015; the blueprint of these goals was agreed life, and human dignity. It was estimated that, during to by all the world’s countries and leading develop- the last decade of the twentieth century, 826 million ment institutions. people were undernourished: 792 million in develop- ing countries and 34 million in developed countries. A 2001 report from the FAO indicated that in In developing countries, more than 199 million chil- 1997–1999 there were 815 million undernourished dren under the age of 5 years suffer from acute or people in the world, of whom 777 million were in chronic protein and energy defi ciencies. An estimated developing countries, 27 million in transitional coun- 3.5–5 billion people are iron defi cient, 2.2 billion tries and 11 million in the industrialized countries. iodine defi cient, and 140–250 million defi - The annual decrease in undernourished people from cient. This has led to several global initiatives and the 1990–1992 period was 6 million. To reach the commitments, spearheaded by a number of United World Food Summit’s goal of halving the number of Nations organizations, to reduce global undernutri- undernourished in developing countries by 2015, it is tion, food insecurity, hunger, starvation, and micro- estimated that the annual decrease required is 22 nutrient defi ciencies. Some progress has been made million. in reducing these numbers, but the problems are far Clearly, this is a huge challenge for food and nutri- from solved. Some of the initiatives are: tion scientists and practitioners. It would need a holistic approach and understanding of the complex, interacting factors that contribute to malnutrition on ● the 1990 United Nations Children’s (Emergency) Fund (UNICEF)-supported World Summit for different levels. These include immediate, intermedi- Children, with a call to reduce severe and moderate ate, underlying, and basic causes: malnutrition among children under 5 years of age by ● individual level or immediate causes: food and half the 1990 rate by the year 2000, including goals nutrient intake, physical activity, health status, social for the elimination of micronutrient malnutrition structures, care, taboos, growth, personal choice ● the 1992 World Health Organization/Food and ● household level or intermediate causes: family size Agriculture Organization (WHO/FAO) Interna- and composition, gender equity, rules of distribu- tional Conference on Nutrition that reinforced tion of food within the household, income, avail- earlier goals and extended them to the elimination ability of food, access to food of death from ● national level or underlying causes: health, educa- ● the 1996 FAO-supported World Food Summit tion, sanitation, agriculture and food security, during which 186 heads of state and governments war, political instability, urbanization, population pledged their political will and commitment to a growth, distribution and confl icts, war, natural plan of action to reduce the number of undernour- disasters, decreased resources ished people to half their 1996 number by 2015 ● international level or basic causes: social, economic ● the establishment in 1997 of the Food Insecurity and political structures, trade agreements, popula- and Vulnerability Information and Mapping System tion size, population growth distribution, environ- (FIVIMS) and their Interagency Working Group mental degradation. (IAWG), which consists of 26 international organi- zations and agencies with a shared commitment to To address these causes of undernutrition food- reduce food insecurity and vulnerability and its insecure and hungry communities and individuals multidimensional causes rooted in poverty; infor- must be empowered to be their own agents of food mation about these initiatives can be accessed at: security and livelihood development. Complicating http://www.fao.org/ the task of fi ghting food insecurity and hunger are ● Millennium Development Goals: the United Nations natural disasters such as droughts, fl oods, cyclones articulated eight goals, ranging from halving extreme and extreme temperatures, ongoing wars and regional poverty and hunger, halting the spread of the human confl icts, as well as the devastating impact of HIV and immunodefi ciency virus (HIV)/acquired immuno- AIDS, especially in sub-Saharan Africa. defi ciency syndrome (AIDS) and providing univer- In many developing countries, indigenous people sal primary education, to be reached by the target have changed their diets and physical activity patterns Global Perspective on Food and Nutrition 7 to those followed in industrialized countries. Supple- be aware of the dynamics within particular mentary feeding programs in these countries communities responsible for nutritional problems. have often been associated with increasing trends These would include household food security, towards obesity, insulin resistance, and the emergence socioeconomic background, education levels, of chronic diseases of lifestyle in some segments of childcare practices, sanitation, water, energy these populations, while other segments are still sources, healthcare services, and other quality-of- undernourished. life indicators. The nutritionist will The coexistence of undernutrition and overnutri- design, implement, and monitor appropriate, com- tion, leading to a double burden of infectious and munity-participatory programs to address these chronic, noncommunicable diseases, and the multi- problems. factorial causes of malnutrition, call for innovative ● The public health or public nutritionist covers the approaches to tackle both undernutrition and overnu- health and care practice areas but will also be con- trition in integrated nutrition and health-promoting cerned with food security (agricultural) and envi- programs, focusing on optimal nutrition for all. ronmental issues on a public level. The public health or public nutritionist will, for example, be responsible for nutrition surveillance, and the 1.7 Relationship between nutrition design, implementation, and of dietary science and practice guidelines that address relevant public health prob- lems. A background knowledge in economics, The journey through the scientifi c domain of nutri- agriculture, political science, and policy design is tion will, at a specialized stage, fork into different essential for the formulation and application of roads. These roads will lead to the different scopes or nutrition policy in a country. branches of nutrition science that are covered in the second, third, and fourth texts of this series. These Many developing countries will not have the capac- different branches of nutrition science could lead to ity or the fi nancial resources to train and employ the training of nutrition specialists for specifi c prac- professionals for different specialties. However, future tice areas. specialized training and employment of different pro- The main aim of nutrition professionals is to apply fessionals could result in a capacity to address nutri- nutrition principles to promote health and well- tional problems more effectively. being, to prevent disease, and/or to restore health (treat disease) in individuals, families, communities 1.8 Nutrition milestones: the development and the population. To help individuals or groups of of nutrition as a science people to eat a balanced diet, in which food supply meets nutrient needs, involves application of nutri- Ancient beliefs tion principles from a very broad fi eld to almost every Throughout human existence people have attributed facet of human life. It is therefore not surprising that special powers to certain foods and developed beliefs these different branches or specialties of nutrition and taboos regarding foods. These were often based have evolved and are developing. They include clini- on climatic, economic, political, or religious circum- cal nutrition, community nutrition, public health, stances and principles, but also on observations and public nutrition. It can be expected that there will regarding the relationship between the consumption be overlap in the practice areas of these specialties. of certain foods and health. ● The clinical nutritionist will counsel individuals Recorded examples are ancient Chinese and Indian from a biomedical–disease–behavioral paradigm to philosophers who advised on the use of warming and promote health, prevent disease, or treat disease. cooling foods and spices for certain conditions and The clinical nutritionist will mostly work within the for “uplifting the soul,” the Mosaic laws documented health service (facility-based settings such as hospi- in the Old Testament which distinguished between tals, clinics, private practice). clean and unclean foods, the fasting and halal prac- ● The community nutritionist, with additional skills tices of Islam, and the Benedictine monks from from the psychosocial behavioral sciences, should Salerno who preached the use of hot and moist versus 8 Introduction to Human Nutrition cold and dry foods for various purposes. Hippocrates, that beriberi in Japanese sailors could be prevented the father of modern medicine, who lived from 460 by supplementing their polished diets with to about 377 bc, and later Moses Maimonides, who bread, and, eventually, the isolation of the responsible lived in the twelfth century, urged people to practice factor, thiamine or vitamin B1, by Funk in 1911. abstemiousness and a prudent lifestyle. They, and Others are the Nobel Prize-winning discovery by others, advised that, for a long and healthy life, one Minot and Murphy in 1926 that pernicious is should avoid too much fat in the diet, eat more , a nutritional disorder due to a lack of in get ample , and be physically active – advice that the diet, the description of kwashiorkor as a protein- is still incorporated in the modern, science-based defi ciency state by Cecily Williams in 1935, and dietary guidelines of the twenty-fi rst century! the discovery of resistant starch and importance of colonic fermentation for humans by nutritionists of Cultural beliefs the Dunn Clinical Nutrition Centre in the 1980s. The perception that food represents more than its The history of modern nutrition as practiced today constituent parts is still true. Eating together is an is an exciting one to read, and students are encour- accepted form of social interaction. It is a way in aged to spend some time on it. It is often character- which cultural habits and customs, social status, ized by heartbreaking courage and surprising insights. kinship, love, respect, sharing, and hospitality are An example of the former is the carefully documented expressed. Scientists and nutrition professionals clinical, metabolic, and pathological consequences of realize that, when formulating dietary guidelines for hunger and starvation by a group of Jewish doctors traditional living people, cultural beliefs and taboos in 1940 in the Warsaw ghetto: doctors who them- should be taken into account and incorporated. There selves were dying of hunger. An example of the latter are numerous examples of traditional food habits and is the studies by Price, an American dentist, who tried diets, often based on what was available. Today, with to identify the dietary factors responsible for good the world becoming a global village, cultures have dental and overall health in people living traditional learned from each other, and dietary patterns associ- lifestyles. He unwittingly used a fortigenic paradigm ated with good health, such as the Mediterranean in his research, examining the strengths and factors diet, are becoming popular among many cultures. that keep people healthy, long before the term was defi ned or its value recognized. The fi rst renaissance: development of At present, thousands of nutrition scientists an evidence base examine many aspects of nutrition in laboratories The knowledge of the specifi c health effects of par- and fi eld studies all over the world and publish in ticular diets, foods, and nutrients is now fi rmly based more than 100 international scientifi c nutrition jour- on the results of rigid scientifi c experimentation. nals. This means that nutrition science generates new Nutrition developed gradually as a science, but knowledge based on well-established research meth- advanced with rapid strides during the twentieth odologies. The many types of experiments, varying century. There are numerous meticulously recorded from molecular experimentation in the laboratory, examples of how initial (often ancient and primitive) through placebo-controlled, double-blinded clinical observations about diet and health relationships led interventions, to observational epidemiological sur- to the discovery, elucidation of function, isolation, veys, and experiments based on a health (fortigenic) and synthesis of the different nutrients. Perhaps the or a disease (pathogenic) paradigm, will be most often quoted example is ’s descrip- addressed in this volume (Chapter 13). The peer- tion in 1772 of how citrus fruit could cure and prevent review process of published results has helped in the in seamen on long voyages. The anti-scurvy development of guidelines to judge how possible, factor (ascorbic acid or ) was only isolated probable, convincing, and applicable results from in 1921, characterized in 1932, and chemically syn- these studies are. New knowledge of nutrients, foods, thesized in 1933. Other examples of nutritional mile- and diet relationships with health and disease is, stones are the induction of beriberi in domestic fowl therefore, generated through a process in which many by Eijkman in 1897, the observation of Takaki in 1906 scientists examine different pieces of the puzzle all Global Perspective on Food and Nutrition 9 over the world in controlled scientifi c experiments. different substances in plant foods, not yet classifi ed Therefore, nutrition practice today has a fi rm research as nutrients, will also be examined. These substances base that enables nutritional professionals to practice are produced by for hormonal, attractant, and evidence-based nutrition. chemoprotective purposes, and there is evidence that many of them offer protection against a wide range The second renaissance: of human conditions. It is possible that new functions solving global malnutrition of known nutrients, and even new nutrients, may be There is little doubt that improved nutrition has con- discovered, described, and applied in the future. tributed to the improved health and survival times experienced by modern humans. However, global Clinical and community nutrition fi gures on the prevalence of both undernutrition and Today, the focus has moved from simple experiments overnutrition show that millions of people do not with clear-cut answers to studies in which sophisti- have enough to eat, while the millions who eat too cated statistics have to be used to dissect out the role much suffer from the consequences of obesity. It is of specifi c nutrients, foods, and diets in multifactorial tempting to equate this situation to the gap between diseases. Nutrition epidemiology is now established the poor and the rich or between developing and as the discipline in which these questions can be developed countries, but the situation is much more addressed. A number of pressing problems will have complex. Obesity, a consequence of overnutrition, is to be researched and the results applied, for example: now a public health problem not only in rich, devel- ● the biological and sociological causes of childhood oped, food-secure countries but also in developing, obesity, which is emerging as a global public health food-insecure countries, especially among women. problem Undernutrition, the major impediment to national ● the nutrient requirements of the elderly: in the year development, is the biggest single contributor to 2000, more than 800 million of the ’s inhabit- childhood death rates, and to impaired physical ants were older than 60 years; to ensure a high- growth and mental development of children in both quality life in the growing elderly population, much developing and developed countries. Moreover, a more needs to be known about their nutrient combination of undernutrition and overnutrition requirements in the same communities, in single , and ● the relationships between nutrition and immune even in the same individual is often reported. function and how improved nutrition can help to Examples are obese mothers with undernourished defend against invading ; in the children and obese women with certain micronutri- light of the increasing HIV/AIDS pandemic, more ent defi ciencies. The perception that these global information in this area is urgently needed problems of malnutrition will be solved only in inno- ● dietary recommendations: despite suffi cient, con- vative, multidisciplinary, and multisectorial ways has vincing evidence about the effects of nutrients and led to the second, very recent renaissance in nutrition foods on health, nutritionists have generally not research and practice. been very successful in motivating the public to change their diets to more healthy ones. We need 1.9 Future challenges for nutrition to know more about why people make certain food research and practice choices in order to design culturally sensitive and practical dietary guidelines that will impact posi- Basic, molecular nutrition tively on dietary choices. The food-based dietary The tremendous development in recent years of guidelines that are now being developed in many molecular biology and the availability of sophisticated countries are a fi rst step in this direction. new techniques are opening up a fi eld in which nutri- ent–gene interactions and dietary manipulation of Public health nutrition genetic expression will receive increasing attention The single most important challenge facing mankind (see Chapter 15). The effects of more than 12 000 in the future is probably to provide adequate safe 10 Introduction to Human Nutrition food and clean water for all in an environmentally Functional foods: a new development safe way that will not compromise the ability of future Functional foods are new or novel foods, developed generations to meet their needs. In addition to the to have specifi c health benefi ts, in addition to their hundreds of millions not eating enough food to meet usual functions. Examples are spreads with added their needs for a healthy, active life, an additional 80 phytosterols, to lower serum low-density lipoprotein million people have to be fed each year. The challenge cholesterol and the risk of coronary heart disease, and to feed mankind in the future calls for improved agri- the development of starchy products with resistant culture in drought-stricken areas such as sub-Saharan starch and lower glycemic indices, to help control Africa, the application of in a respon- blood glucose levels. The development and testing of sible way, interdisciplinary and intersectorial coop- functional foods is an exciting new area. These foods eration of all involved, and a better distribution of the may help to improve or restore nutritional status in food supply so that affordable food is accessible by many people. However, much more should be known all. The need for sustained economic growth in poor about suitable biomarkers to test their effi cacy, vari- countries is evident. ability in human response to specifi c food products, Nutritionists have an important part to play in safety, consumer understanding, and how their ensuring food security for all, a basic human right, in health messages must be formulated, labeled, and the future. One of their main functions would be to communicated. educate and inform populations not to rely too heavily on animal products in their diet, the produc- tion of which places a much heavier burden on the Food safety environment than plant foods. A major challenge The continued provision of safe food, free from would be to convince political leaders and govern- microorganisms, toxins, and other hazardous sub- ments that addressing undernutrition (the major stances that cause disease, remains a huge challenge. obstacle in national development) in sustainable pro- Recent experiences with suffering from bovine grams should be the top priority in developing and spongiform encephalopathy (BSE or mad cow disease) poor communities. Another challenge is to develop or from foot-and-mouth disease, or birds infected models based on the dynamics within communities with the infl uenza A virus (bird fl u), have shown how and, using a human rights approach, to alleviate quickly a national problem can become an interna- undernutrition without creating a problem of over- tional one because of global marketing of products. nutrition. There are examples where such models, The list of possible hazardous substances in foods incorporated into community development pro- emphasizes the need for continuous monitoring of grams, have been very successful (e.g., in Thailand). the food supply by health offi cials (Figure 1.3).

Microbial contamination Bacteria and mold (fungi) producing toxins and aflatoxins Toxins cause “food poisoning” and aflatoxins are carcinogenic

Natural toxins Agricultural residues Such as cyanide in , solanine in potatoes; Pesticides such as DDT or hormones used to

can be produced by abnormal circumstances, promote growth such as bovine somatotrophin could be inhibitors or antivitamins

Environmental contamination Intentional additives and minerals Artificial sweeteners Criminal adulteration, industrial Preservatives Substances from packaging materials Phytochemicals Changes during and processing of foods Modified carbohydrates (for functional foods)

Figure 1.3 Potential hazardous substances in food. DDT, dichloro-diphenyl-trichloroethane. Global Perspective on Food and Nutrition 11

1.10 Perspectives on the future Box 1.3 Future challenges that require exceptional leadership

Nutrition research and practice, although it has been • Basic molecular nutrition around for many years, is in its infancy as a basic and • Nutrient–gene interactions • applied scientifi c discipline. The present and future Role of phytochemicals in health • New nutrients? New functions? nutrition student will take part in this very exciting • Community and public health nutrition second renaissance of nutrition and see its matura- • Childhood obesity tion. However, to infl uence effectively the nutrition • Requirements of the elderly and health of individuals and populations, the nutri- • Dietary recommendations • tionist will have to forge links and partnerships with Nutrition of patients with human immunodefi ciency virus/ acquired immunodefi ciency syndrome other health professionals and policy-makers, and • Public nutrition will have to develop lateral thinking processes. The • To feed mankind magnitude and complexity of nutritional problems • Food security facing mankind today demand concerted multidisci- • Functional foods • plinary and multisectorial efforts from all involved to To ensure that novel foods are effective and safe • Food safety solve them. Therefore, the principal message to take • Continuous monitoring on a nutrition science journey is that teamwork is • Partnerships with other disciplines essential: one cannot travel this road on one’s own; • Leadership partners from different disciplines are needed. Another essential need is the continuous develop- ment of leadership in nutrition. Leaders on every level The student in nutrition, at the beginning of this of research and practice are necessary to respond to journey of discovery of nutrition as a science, must the existing challenges of global malnutrition and to make use of the many opportunities to develop lead- face future challenges. ership qualities. May this be a happy, fruitful, and The modern advances in molecular biology and lifelong journey with many lessons that can be applied biotechnology on the one hand, and the persistence in the research and practice of nutrition to make a of global malnutrition on the other, increasingly difference in the life of all. demand a re-evaluation of ethical norms, standards, and values for nutrition science and practice. Direc- tion from responsible leaders is needed (Box 1.3). Further reading There is an urgent need for ethical guidelines and a Websites code of conduct for partnerships between food industries, UN agencies, governments, and academ- http://whq.libdoc.who.int/trs/who_trs_916 http://www.who.int/nutrition/en ics. These partnerships are necessary for addressing http://www.ifpri.org global malnutrition in sustainable programs. http://fao.org/ag/agn/nutrition/profi les_en.stm 2 Body Composition

Paul Deurenberg

Key messages

• Body composition data are used to evaluate nutritional status, • Several direct, indirect, and doubly indirect techniques are avail- growth and development, water homeostasis, and specifi c able to measure body composition, each with its own distinct disease states. advantages and disadvantages. • composition is studied at atomic, molecular, cellu- • The choice of method will be infl uenced by the availability of lar, , and whole body levels. The levels are interrelated. instrumentation, invasiveness, and radiation danger to subjects, • A “normal weight” human body consists of approximately 98% price, accuracy required, and application objectives. oxygen, , , , and calcium; of 60–70% • Interpretation and application of data from body composition water, 10–35% fat (depending on gender), 10–15% protein, and measurements should be carried out with care and should take 3–5% minerals. into account the limitations of the method used, age, gender, • The variation in body composition between individuals is large, and ethnic group. mainly because of variations in fat mass. Variations in fat-free mass are smaller.

2.1 Introduction into the changes occurring during growth and devel- opment. They also form the basis for a number of Mankind has long been fascinated with the composi- methods now widely used to assess body composition tion of the human body. Centuries ago, the Greeks in vivo. dissected human cadavers to obtain an insight into Today, it is known that many diseases and disor- the structure and build of the human body, and draw- ders are related to abnormal body composition or to ings from the Middle Ages of gross muscle structures changes in body composition. The most common of grace the walls of many famous art galleries. They are these conditions is obesity, in which the amount of prized not only for their artistic merit, but also for body fat is excessively high, leading to abnormalities what they reveal of the work of the dissectionists of in and carbohydrate metabolism, high blood that era. With progress in the development of analyti- pressure, and adult-onset diabetes. At the other end cal chemical methods in the twentieth century, these of the nutritional spectrum, energy and protein mal- studies of body composition were applied to body nutrition results in a decrease in the amount of fat tissues, fetuses, and cadavers of newborns. Scientists and protein stores in the body, and many diseases are such as Mitchell, Widdowson, and Forbes performed related to abnormalities in total or to the the most important work of chemical analyses in distribution of body water across the intracellular and adult cadavers during the 1940s and 1950s. Today, extracellular spaces. neutron activation analysis allows the chemical com- Because of the high variability between subjects in position of the human body to be studied in vivo. chemical body composition, mainly due to the high These early chemical analyses of the body gave insights variation in body fat stores, the concept of fat-free

© 2009 P Deurenberg. Body Composition 13 mass (FFM) was introduced at the end of the nine- Table 2.1 Body composition at the atomic level of a 70 kg reference teenth century. If body composition data are expressed man as a proportion of the FFM, data become much more Atomic element Amount (kg) Amount (% body weight) consistent between individuals. For example, the fraction of water in the FFM (0.73 ± 0.02) is very Oxygen 43 61 consistent across individuals, whereas the between- Carbon 16 23 subject variation is two to three times higher if Hydrogen 7 10 Nitrogen 1.8 2.6 expressed per kilogram of body weight. This high Calcium 1.0 1.4 variability in body components led to the defi nition 0.6 0.8 of a “reference man,” an imaginary person with a Total 69.4 98.8 given body composition. In this chapter a (global) description of the com- position of the healthy human body is given and dis- Box 2.1 cussed at the following levels: The water content in the body varies with age. In a fetus, the water content slowly decreases from more than 90% after conception to ● atomic about 80% before delivery at about 7 months of gestation. A ● molecular newborn has about 70% body water, which is about 82% of the ● cellular fat-free mass. This value slowly decreases further to 72% of the ● tissue fat-free mass until the body is chemically mature at age 15–18 years. In general, males have more body water (related to body ● whole body. weight) than females, as their body fat content is lower. Of the many methods available to measure body composition, a few are highlighted and a short description of each is given. For more detailed infor- still forms the basis for many in vivo techniques that mation, the books by Forbes (1987) and Heymsfi eld are used to assess body composition. et al. (2005) on human body composition are recom- mended for further reading. Molecular level The chemical elements in the human body are bound 2.2 Five levels of body composition in and, in very global terms, the main com- partments are water, lipids, proteins, minerals, and Human body composition can be studied at the carbohydrates. The total amount of water in the body atomic, molecular, cellular, tissue, and whole body is high and, depending on the body fat content, can level. These fi ve levels are related to each other. For be as high as 60–70% of total body weight. Total body example, information at the atomic level can be used, water can be divided into intracellular water and subject to certain assumptions, to provide informa- extracellular water, and the ratio of the two is an tion at the whole body level. important health parameter that is disturbed in many diseases (Box 2.1). Atomic level Lipids appear in the human body in different Many chemical elements () are found in the forms. Essential structural lipids such as the phospho- human body, but the six elements oxygen, carbon, lipids (cell membranes) and sphingomyelin (nervous hydrogen, nitrogen, calcium, and phosphorus are the system) form only a minor part of the total lipids in most abundant and together account for more than the body. The nonessential lipids, mostly triglycerides 98% of body weight (Table 2.1). Indeed, the 11 most or triacylglycerol (fat), are the most abundant. They common elements account for 99.5% of the atomic are the energy store of the adult human body, insulate body composition. This information was initially against cold, protect vital organs such as the kidneys based on chemical analysis of carcasses, but today the against mechanical damage, and, to a certain extent, information can also be obtained by in vivo neutron enhance the body’s appearance. In a “normal weight” activation analysis (IVNAA). The classical chemical healthy adult, the amount of body fat varies between cadaver analysis, as carried out mainly in the 1940s, 10% and 25% in men and between 15% and 35% in 14 Introduction to Human Nutrition

Table 2.2 Body composition at the molecular level of a 70 kg refer- Table 2.3 Body composition at the tissue level of a 70 kg reference ence man man

Component Amount (kg) Amount (% body weight) Tissue/organ Amount (kg) Amount (% body weight)

Water Muscle 28 40 Extracellular 18 26 Adipose tissue 15 21.4 Intracellular 24 34 Blood 5.5 7.9 Lipid Bone 5 7.1 Essential 1.5 2.1 Skin 2.6 3.7 Nonessential 12 17 Liver 1.8 2.6 Protein 10.1 14.4 Total 57.9 82.7 3.7 5.3 Carbohydrate 0.5 0.6 Total 69.8 99.4 Tissue level women. In severe obesity body fat can be as high as Cells with equal functions form tissues, including 60–70% of body weight. muscular, connective, epithelial, and nervous tissue. Body protein varies between 10% and 15%. It is Bones are connective tissue and consist mainly of higher in males than in females, as males generally have hydroxyapatite, [Ca3(PO4)2]3Ca(OH)2, bedded in a more muscles. There is no protein storage in the body protein matrix. A rather simple body composition and, generally speaking, loss of protein coincides with model at the tissue level would be: a loss of functionality given the high protein content Body weight = adipose tissue + skeletal muscle and high protein turnover rates in vital organs. + bone + organs + rest The amount of minerals in the body varies between 3% and 5%, again dependent on body fat. Calcium Several of these components can now be measured and phosphorus are the two main minerals. They are with, for example, computed tomography (CT) or found mainly in bones. Carbohydrates are found in magnetic resonance imaging (MRI) for adipose tissue; the body as glucose (blood ) and , a creatinine excretion or N-methyl- excretion in muscle and liver cells that serves as in 24 h for skeletal muscle; dual-energy X-ray a short-term energy store. The amount of carbohy- absorptiometry (DXA) for bones; and MRI or ultra- drates in the body rarely exceeds 500 g. Table 2.2 gives sound for organs. Body composition at the tissue level the body composition of the reference man at a is given in Table 2.3. molecular level. Whole body level Cellular level Body composition measurements at the whole body At the cellular level, body composition can be level use simple body parameters to give an insight described in terms of body cell mass, extracellular into body composition. Formulae, based on statistical fl uids, and extracellular solids. The body cell mass relationships that have been established in earlier includes the cells with all their contents, such as water, studies between body parameters (e.g., skinfold thick- proteins, and minerals. Extracellular fl uid contains ness) and information on body composition (e.g., about 95% water, which is plasma in the intravascular body fat by density), also enable the assessment of space and interstitial fl uid in the extravascular space. body composition. Another example is the assess- Extracellular solids are mainly proteins (e.g., colla- ment of body water based on weight, height, age, and gen) and minerals (bone minerals and soluble miner- gender. als in the extracellular fl uid). Body composition at the cellular level is not easy to measure, owing to its 2.3 Relationships between different complex nature. As will be discussed later, the 40K levels of body composition method can be used to assess body cell mass and some dilution techniques, for example bromide dilution, The fi ve levels of body composition are interrelated. can be used to assess extracellular water. This means that information at one level can be trans- Body Composition 15

Table 2.4 Methods used to determine body composition Box 2.2 Direct Indirect Doubly indirect Adipose tissue is made of adipocytes, which are cells that store triglycerides in the form of small fat droplets. Adipose tissue con- Carcass analyses Densitometry Weight/height indices tains about 80% triglycerides and some 1–2% protein (), IVNAA oxide Skinfolds/ultrasound and the remaining part is water plus electrolytes. During weight dilution loss adipose tissue decreases: the actual fat loss will be about 80% 40K counting Circumferences/diameters of the actual weight loss. More-compartment Impedance models DXA Infrared interactance CT/MRI scans Creatinine excretion lated to another level. This is important as it forms IVNAA, in vivo neutron activation analysis; DXA, dual-energy X-ray the basis of many techniques used to determine body absorptiometry; CT, computed tomography; MRI, magnetic resonance composition. In the context of this chapter, only a few imaging. examples are given. After determining the amount of calcium in the body by, for example, IVNAA (atomic level), the amount of bone can be calculated assuming ● In direct methods, the body component of interest that a certain amount of total body calcium is in the is determined directly without or with only minor skeletal tissue. Determination of total body potassium assumptions. Examples are chemical carcass analy- (by 40K or IVNAA) enables the assessment of the body ses and IVNAA for the atomic components. cell mass, as most of the body potassium is known to ● In indirect techniques, the body component of be intracellular. Skinfold thickness measurements interest is determined indirectly. Examples are the (total body level) enable the assessment of body fat determination of body protein from body nitrogen, (molecular level). Formulae used for these calcula- assuming a constant conversion factor of 6.25 from tions are component based, property based, or some- nitrogen to protein, and the determination of body 40 times a combination. Component-based formulae cell mass using K. In both examples, assumptions are based on fi xed relationships between components. are used. These assumptions may not be valid in An example is the calculation of total body water the given situation or for the subject(s) under study from measured hydrogen: the chemical formula of and hence could lead to biased results. water determines the factor. Property-based formulae ● Doubly indirect methods rely on a statistical rela- are based on established statistical relationships tionship between easily measurable body para- between variables. An example is the prediction of meter(s) and the body component of interest. body fat percentage (body composition parameter) Examples are the assessment of skeletal muscle mass from skinfold thickness (property) (Box 2.2). by creatinine excretion and the assessment of body Property-based formulae tend to be population spe- fat from skin-fold thickness. Table 2.4 gives an over- cifi c, which limits the widespread application. view of the most common methods. Most body composition techniques that are in use today are based on assumptions, often derived 2.5 Direct methods from carcass analyses or experimentally derived from observational studies. Violation of these assumptions Carcass analysis leads to biased results, and some methods are more The (chemical) analysis of carcasses is a time- prone to bias than others. In the following short consuming and requires very precise description of different methodologies, the most approaches to the task. The carcass has to be carefully important assumptions are highlighted. dissected into the different tissues that are then exactly weighed, after which the chemical analyses have to be 2.4 Body composition techniques performed. To avoid errors it is important that no unaccounted water losses occur during the analytical Body composition techniques can be described in work. As early as the nineteenth century, it was rec- terms of direct, indirect, and doubly indirect ognized that the variation in chemical body composi- methods. tion was reduced when results were expressed as a 16 Introduction to Human Nutrition fraction of the fat-free body. The data on the chemical example, from total body nitrogen total body protein composition of only a few human cadavers form the can be calculated as 6.25 times the total nitrogen, basis for the assumptions that are normally used in assuming that body protein consists of 16% nitrogen. indirect methods. These chemical analyses were per- The advantage of the method is that the chemical formed in fi ve men and one woman. It was concluded body composition can be determined in vivo and can that, on the basis of FFM, the mean amounts of water, be compared with other, indirect, techniques. For protein, and minerals in the body are 72.6%, 20.5%, fundamental studies and for validation of existing and 6.9%, respectively. The variability in these fi gures techniques in special groups of subjects, for example is about 13% for protein and minerals and 4% for in different ethnic groups, elderly subjects, obese sub- water. Although one can question the quality of these jects, or in the diseased state, the methodology can be data as a basis for other methods (low number, high of great importance. The disadvantage of IVNAA is variation in age, variation in gender, some carcasses not only the price. The subject is irradiated, with the were not analyzed immediately after death), they radiation dose used depending on the number and form the basis for many indirect and doubly indirect kind of elements to be determined. It is relatively body composition methods. Chemical carcass analy- low for nitrogen (0.26 mSv) but high for calcium sis also revealed that the amount of potassium in the (2.5 mSv). FFM is fairly constant. This fact is used as the basis for the calculation of the amount of FFM or for body cell mass from total body potassium, determined by 2.6 Indirect methods 40K scanning. In the 1980s, cadaver studies were performed again Densitometry in the “Brussels study.” Unfortunately, only informa- The densitometric method assumes that the body tion at a tissue level and not at atomic or molecular consists of two components, a fat mass, in which all level was collected. However, the need for cadaver “chemical” fat is located, and the FFM, which consists studies has greatly diminished given that the same of (fat-free) bones, muscles, water, and organs. information can now be obtained in vivo by Chemically, the FFM consists of water, minerals, IVNAA. protein, and a small amount of carbohydrate, the last often being neglected. The density of the fat mass is In vivo neutron activation analysis 0.900 kg/l and, from carcass analysis data, the density IVNAA is a relatively new body composition tech- of the FFM can be calculated as 1.100 kg/l, depending nique that allows the determination of specifi c chemi- on the relative amount of minerals, protein, and cal elements in the body. The body is bombarded with water in the FFM (Box 2.3). fast neutrons of known energy level. The neutrons The density of the total body depends on the ratio can be captured by chemical elements (as part of mol- of fat mass to FFM. Once the density of the body has ecules) in the body, resulting in a transition state of been determined, the percentage of fat in the body higher energy for that element – energy that is fi nally (BF%) can be calculated by Siri’s formula (Box 2.4): emitted as gamma rays. For example, capture of BF% = (495/body density) − 450 neutrons by nitrogen results in the formation of the isotope 15N, which will emit the excess energy as Body density can be determined by several tech- gamma rays: niques, the oldest and perhaps most accurate being underwater weighing. Behnke fi rst used the tech- 14N + 1n → 15N* + gamma rays nique, showing that excess body weight in American where 14N is nitrogen with atomic mass 14, 15N is football players was not the result of excess fat but of nitrogen with atomic mass 15, and 1n is a neutron. enlarged muscle mass. With IVNAA, many elements in the body can be In underwater weighing, the weight of the subject determined, including calcium, phosphorus, nitro- is fi rst measured in air and then while totally immersed gen, oxygen, potassium, and . in water. The difference between weight in air and The information obtained at the atomic level weight under water is the upwards force, which equals can be converted to more useful information. For the weight of the displaced water (Archimedes’ law), Body Composition 17

Box 2.3

The density of the fat-free mass (FFM) can be calculated if its com- position is known. In the calculation example below it is assumed that the FFM consists of 73.5% water, 19.6% protein, and 6.9% minerals with densities (at 37°C) of 0.993, 1.340, and 3.038 kg/l, respectively. In addition, it is assumed that the volumes of the separate com- partments can be added up to the total volume of the FFM (in fact, the compartments do not “mix”). Thus, the volume of the FFM equals the sum of the other compartments:

FFMvolume = Watervolume + Mineralvolume + Proteinvolume As volume is weight/density, the equation can be written as:

100/DensityFFM = 73.5/0.993 + 6.9/3.038 + 19.6/1.340 From this, the density of the FFM can be calculated as 1.0999 kg/l. It is obvious that differences in composition of the FFM will result in a different density.

Box 2.4

Siri’s formula can be derived assuming that the body consists of fat mass (FM) and fat-free mass (FFM). If body weight is assumed to be 100% and body fat is x%, then FFM is 100 − x%. It is assumed Figure 2.1 Underwater weighing. The subject is submerged com- that the volumes of these two compartments can be added up to pletely and breathes via a respirometer (1) for simultaneous residual total body volume. Then: lung volume measurement. Weight (W) (2) under water (uw) is recorded and density (D) is calculated as Dbody = Wair/(Wair − Wuw). = + Bodyvolume FMvolume FFMvolume Corrections are made for water temperature and lung volume: percent- age of fat in the body = 495/D − 450. As volume is weight/density, the equation can be written as: body 100/body density = x/0.9 + (100 − x)/1.1 From this, body fat percentage (BF%) can be calculated as: fat is assumed to be maximal 3% of BF%. This error is mainly due to violation of the assumption that the BF% = 495/density − 450 density of the FFM equals 1.100 kg/l in the subject The general formula to calculate BF% from body density (Db) is: under study. It can be argued that in certain subjects ⎛ × ⎞ ⎛ ⎞ or groups of subjects this assumption may be vio- =×1 DDFFM FM − DFM BF% ⎝⎜ ⎠⎟ ⎝⎜ ⎠⎟ Db DDFFM− FM DDFFM− FM lated, as for example in young children and in preg- In general, a lower density of the FFM than 1.1 kg/l will result in an nant women. Use of Siri’s formula will then lead to overestimation of BF% if Siri’s formula is used. It is likely that the biased conclusions. Some laboratories have attempted density of the FFM is lower in elderly people, owing to bone to use water displacement instead of underwater mineral loss (osteoporosis). weighing, but the technique failed, mainly because of Densitometry (using Siri’s equation) overestimates body fat the diffi culty in accurately reading the water level in compared with a four-compartment model (see Figure 2.7). the tank. An air-displacement method has been commer- from which, after correction for the water tempera- cially available since 1995. This method measures ture (density), the displaced water volume (and thus body volume after placing the subject in a small, air- the body volume) can be calculated. Corrections tight chamber and increasing the pressure by adding must be made for residual lung volume and air in the a known amount of air into the chamber. Boyle Gay- gut. Figure 2.1 shows an underwater weighing. The Lussac’s law enables the calculation of body volume. technique gives very reproducible results within about Corrections are made for temperature and humidity 1% of BF%. The absolute error in determined body changes, and lung volume is assessed simultaneously. 18 Introduction to Human Nutrition

Research to date has generally shown good agreement Box 2.5 between underwater weighing and air displacement. Air displacement is better accepted by the volunteers, A person with a body weight of 75 kg is given an exactly weighed but some experience diffi culties because of the dose of 15 g deuterium oxide. This deuterium oxide is allowed to breathing pattern to be followed or because of be equally distributed in the body water compartment for about 3–5 hours. Then, blood is taken and the deuterium concentration claustrophobia. in the sample is determined. Assuming the plasma level to be 370 mg/kg, the “deuterium space” can be calculated as Dilution techniques 15 000/370 = 40.5 kg. As deuterium exchanges in the body with Carcass analyses revealed that the amount of water in hydroxyl groups from other molecules, the deuterium space has to the FFM is relatively constant at about 73%. Total be corrected for this nonaqueous dilution (4–5%). Thus, total body water is 0.95 × 15 000/370 = 38.5 kg. Assuming a hydration of body water (TBW) can be determined by dilution the fat-free mass of 73%, the body fat percentage of this 75 kg techniques. Dilution techniques are generally based weight subject would be: 100 × [75 − (38.5/0.73)/75] = 29.7%. on the equation:

C1 × V1 = C2 × V2 = Constant where C is the tracer (deuterium oxide, tritium, or Box 2.6 18O water) concentration and V is the volume. When a subject is given a known amount of a tracer 7 × (C1 V1), which is known to be diluted in a given 6 body compartment, the volume of that body com- 5 partment can be calculated from the dose given and 4 the concentration of the tracer in that compartment 3 after equilibrium has been reached. Suitable tracers 2 for the determination of TBW are deuterium oxide, 1 18 tritium oxide, and O-labeled water. Other tracers 0 can also be used, such as alcohol and , but they -1 are less suitable because they are partly metabolized Bias in body fat percent 70 71 72 73 74 75 (alcohol) or because they are actively excreted from Percent water in FFM the body (urea) during the dilution period. After giving a subject the tracer and allowing around 3–5 For the computation of body composition from dual-energy X-ray absorptiometry, especially body fat and lean tissue, several hours for equal distribution throughout the body, assumptions are made, one of which is a constant hydration of the determination of the concentration of deuterium in fat-free mass (FFM). The fi gure shows that the bias in calculated blood, saliva, or urine allows the calculation of TBW body fat percentage depends on the hydration of the FFM. (Box 2.5). Reference is a four-compartment model. Alternatively, other tracers can be used, such as tritium oxide and 18O-labeled water, and the tracer can be given intravenously, which is advantageous when the subject has gastrointestinal disorders. The jects with a larger than 73% water content in the FFM reproducibility of the method is 1–3%, depending on (pregnant women, morbid obese subjects, and patients the tracer used and the analytical method chosen. with edema), the factor 0.73 will result in an overesti- From TBW, the FFM, and hence fat mass, can be mation of the FFM. A three-compartment model of calculated, assuming that 73% of the FFM is water: the body that contains fat mass, water, and dry FFM has a lower bias than a two-compartment model. BF% = 100 × (Weight − TBW/0.73)/Weight An overestimation of body fat by densitometry, for The precision for estimations of body fat is about example because of a relatively high amount of water 3–4% of body weight. As with the densitometric in the FFM, will be counteracted by an underestima- method, this error is due to violations of the assump- tion using the dilution method (see also Box 2.6). tion used (i.e., that the relative amount of water in the The use of tracers that do not cross the cell mem- FFM is constant and equals 73% of the FFM). In sub- brane enables the determination of extracellular Body Composition 19 water (ECW). Commonly used tracers in this respect 3 are bromide or sodium-24. Intracellular water (ICW) cannot be determined directly and is calcu- lated as the difference between TBW and ECW. Men Total body potassium Chemical carcass analysis has revealed that the amount of potassium in the fat-free body is relatively con- 2 stant, although the amount of potassium in different tissues varies widely. The determination TBK:FFM of total body potassium (TBK) is relatively easy, owing to the natural occurrence of three potassium isotopes Women (39K, 40K, and 41K), in constant relative amounts, of which 40K is radioactive (gamma emission). Counting the emission of the gamma rays from the body reveals the amount of radioactive potassium, from which 1 TBK and hence FFM can be calculated. The chamber 25 45 65 85 in which the subject is scanned has to be carefully Age (years) shielded to avoid any background radiation (cosmic Figure 2.2 Difference in total body potassium (TBK) content of the radiation). The scanning of the body for potassium fat-free mass (FFM) between men and women and the relationship with age. lasts for 20–30 min and the reproducibility is 2–3%. Several authors have shown that the amount of potassium in the FFM is different between males and females, is lower in obese subjects, and is probably instrument’s software generates a two-dimensional also age dependent. Thus, TBK is much more useful picture of the body or the body compartment under as a measure of body cell mass (BCM) than as a study. The software can calculate several body com- measure of FFM. However, this discrepancy can be ponents: bone mineral content and bone mineral used to calculate the “quality” of FFM, defi ned as the density, lean mass, and adipose tissue fat mass. These ratio of cellular to extracellular components of FFM, calculations are possible for each of the body parts, or operationally as BCM/FFM. Thus, when TBK is e.g., for legs, trunk, spine, femur, and arms. However, used to assess BCM, and another method such as the method cannot distinguish between subcutane- hydrodensitometry or DXA is used to assess FFM ous adipose tissue and discrete adipose tissue sites independently, it can be shown that the quality of such as perirenal adipose tissue. The reproducibility FFM declines with age, along with the quantity (Figure of DXA is very high, varying from about 0.5% for 2.2). When potassium values are used to calculate bone mineral density to about 2% for total body com- intracellular water, BCM, or FFM, assuming constant position. The reproducibility for regional body com- amounts of potassium in these body components, the position is less. The method is quick and easy to same errors can occur as with densitometry and dilu- perform and places very few demands on the subject. tion techniques. The radiation dose (0.02 mSv) is only a fraction of the Although the technique is easy to apply in patients, radiation dose of a normal chest radiograph, and the high cost of the scanning instrumentation limits hardly higher than the normal background. Apart its use other than in research settings. from repeated scanning, the radiation dose should not be a limiting factor in terms of volunteers being Dual-energy X-ray absorptiometry exposed to hazardous levels of radiation. A disadvan- During DXA (also known as DEXA), the body or part tage of the method is that the attenuation of the X- of the body is scanned with X-rays of two distinct rays depends on the thickness of the tissue. Therefore, levels of energy. The attenuation of the tissues for the correction for the body size has to be made. Compared two different levels of radiation depends on its chemi- with traditional methods, DXA scanning is easy and cal composition and is detected by photocells. The widely available which, in turn, leads to prediction 20 Introduction to Human Nutrition equations for body composition based on DXA. However, as with other methods, DXA relies on certain assumptions (Box 2.6) and there are many publications showing that the error in body composi- tion measurements using DXA can be considerable (Figure 2.3). Moreover, identical machines, even using the same software versions, can give different results in scanning the same person.

Multicompartment models Two-compartment models, consisting of fat mass and FFM, lack validity in many situations where the composition of the body is “abnormal.” Examples already mentioned are pregnancy, morbid obesity, and the elderly. A combination of techniques often results in more valid estimates, as is the case when, for example, body density and body water are com- bined. In this particular case, the body is divided into three compartments: Body weight = Fat mass + Body water + Dry fat-free mass In this three-compartment model the variation of the water content in the FFM is accounted for. There are fewer assumptions in this model, leading to more valid results. Modern techniques such as DXA enable the valid and precise measurement of bone mineral, from which total body mineral can be estimated. When the mineral content of the body is com- bined with body density and body water, a four- compartment model of the body is generated: Body weight = Fat mass + Water + Minerals + Protein In this model, most of the variation in the amounts of the chemical components is accounted for, result- ing in a very reliable body composition measure (Box 2.7). Four-compartment models can also be obtained using other techniques. For example, the measure- ment of calcium, phosphorus, and nitrogen with IVNAA in combination with TBW provides informa- tion for a model consisting of fat, minerals, protein, Figure 2.3 Dual-energy X-ray absorptiometer (DXA) scan using a and water. In the literature, models based on six HOLOGIC whole-body DXA (QDR-4500). Subcutaneous body fat, bone, compartments are also described. However, they do and muscle are distinguished by different colors. not provide much additional information and the increased technical error negates the methodological advantage. More-compartment models enable the best possi- ble estimate of body composition for populations as Body Composition 21

Box 2.7

FM FM FM M M + P P

FFM TBW TBW

The fi rst bar represents a two-compartment model of body compo- Figure 2.4 Magnetic resonance imaging scan at the L4 level in an sition, in which the body is divided into fat mass and fat-free mass obese subject. The white areas in the image are adipose tissue. Sub- (FFM). In the second bar, the FFM is divided into water and a “dry” cutaneous adipose tissue and intra-abdominal adipose tissue are sepa- FFM, consisting of protein and mineral. The third bar shows a four- rated by the abdominal muscles. compartment model in which the body is divided into water, protein, mineral, and fat. The four-compartment model shown has only minor assumptions and provides body composition data that amounts of tissues with different attenuation, for are very accurate. example adipose tissue against nonadipose tissue. The CT technique was introduced for body composi- tion assessments in the 1980s and is now widely used, well as for individuals. Although some studies com- predominantly for measurements of body fat distri- paring body composition from four-compartment bution. Figure 2.4 shows a scan of the abdomen at the models show that mean values generally agree with level of the umbilicus, made by MRI, a technique that simpler approaches, there are also studies showing gives comparable information. The precision of the directional bias of two-compartment body composi- calculation of a tissue area or tissue volume from the tion models. For this reason, more-compartment same scan(s) is very accurate, with an error of about models should ideally be used as a reference (gold 1%. Partial volume effects (pixels that contain tissue standard). However, only a limited number of labo- with different attenuation) may infl uence the accu- ratories can perform all of the necessary measure- racy and reproducibility of the method. ments for the calculation of maximum compartment A single CT scan provides only relative data, for models. Moreover, the data are expensive to collect, example in a scan of the abdomen the relative amount and measurements are time-consuming and not very of visceral adipose tissue to subcutaneous adipose practical in clinical situations. tissue. Multiple CT scanning allows the calculation of tissue volumes. From adipose tissue volumes (tissue Imaging techniques level) and an assumed density and composition of the CT scanning enables the visualization of tissues in adipose tissue, the amount of fat mass (molecular cross-sectional slices of the body. The thickness of level) can be calculated. Multiplying tissue volumes those slices can vary, but is normally about 1 cm. with specifi c densities of these tissues (determined in During CT scanning a source of X-rays rotates per- vitro) allows a recalculation of the body weight, a pendicularly around the body or a body segment, necessary but not suffi cient exercise for validation of while photodetectors, opposite to the source, register a whole body technique. Research in this area has the attenuation of the X-rays after they have passed shown that the CT technique allows the determina- through the body in the various directions. The infor- tion of total body composition, with an error of mation received by the photodetectors is used to gen- estimate for fat mass of 3–3.5 kg (compared with erate images. Software enables the calculation of the densitometry). 22 Introduction to Human Nutrition

CT scanning is expensive and, because of the rela- Table 2.5 Classifi cation of weight in adults according to body mass tively high level of radiation, the method is limited to index subjects for whom scanning is indicated on clinical Classifi cation Body mass index (kg/m2) Risk of comorbidities grounds. An alternative method to CT scanning is MRI, which has the advantage that no ionizing radia- <18.5 Low tion is involved. Normal range 18.5–24.9 Average > During MRI, the signals emitted when the body is Overweight 25.0 Preobese 25.0–29.9 Increased placed in a strong magnetic fi eld are collected and, as Obese class I 30.0–34.9 Moderate with CT scanning, the data are used to generate a Obese class II 35.0–39.9 Severe visual cross-sectional slice of the body in a certain Obese class III >40 Very severe region. The determination of adipose tissue versus nonadipose tissue is based on the shorter relaxation Reproduced with permission of the World Health Organization. time of adipose tissue than of other tissues that contain more protons or differ in resonance frequency. MRI tion specifi c). The Quetelet index or BMI is the most has the advantage over CT scanning that the subject is widely used index today. Its correlation with body fat not exposed to ionizing radiation. However, the time is high (depending on the age group r = 0.6–0.8) and necessary to make an MRI image is relatively long the correlation with body height is generally low. The (minutes versus seconds using CT), which has impli- World Health Organization (WHO) promotes the cations for the quality of the image. Any movement of BMI as a crude indicator for weight judgment. In the subject, even the movements of the intestinal tract Table 2.5 the cut-off points for underweight, normal when making images in the abdominal region, will weight, overweight, and obesity according to the decrease the quality of the image. WHO are given. These cut-off values are based on the As with CT scanning, images can be combined to relation of BMI with mortality and with risk factors obtain information on total body composition. Infor- for disease as found in Caucasian populations. For mation about organ size can be obtained with a high non-Caucasian populations other cut-off values may accuracy. For example, MRI is used to study the con- apply (WHO, 2004). tribution of various organs to the resting metabolic The cut-off values for BMI as in Table 2.5 cannot rate of the total body. be used in children. In younger children, weight com- Both CT scanning and MRI are expensive, and pared with height is relatively low, and so is the BMI. therefore their use will remain limited to a few labo- During growth, the increase in weight is larger than ratories and for very specifi c situations. the increase in height and, consequently, the BMI increases with age during the pubertal phase of life. There are age-related BMI cut-off values for obesity 2.7 Doubly indirect methods for children. The BMI can also be used as a predictor for the Anthropometry percentage of body fat. Several studies have been pub- Weight/height indices lished in which a good relationship between the BMI A weight/height index aims to correct body weight and the amount of body fat (either as fat mass or as for height. As a measure of body composition, for body fat percentage) was demonstrated. The relation- example body fat, a weight/height index should have ship between BMI and body fat percentage is age and a high correlation with body fat, but also a low cor- gender dependent and is different among certain relation with body height, otherwise in short people ethnic groups (Box 2.8). When using such age- and body fat would be systematically overestimated or gender-specifi c prediction equations, body fat per- underestimated. centage can be predicted with an error of 3–5%. This In the literature, a number of weight/height indices error is similar to the prediction error of other doubly have been proposed. Examples are the Quetelet index indirect methods, for example skinfold thickness or or body mass index (BMI: weight/height2), the Broca total body bioelectrical impedance measurements. index [weight/(height – 100)], and the Benn index The disadvantage of these prediction formulae is that (weight/heightp, in which the exponent p is popula- they obviously cannot be used in certain subjects or Body Composition 23

Box 2.8 Box 2.9

Recent studies have shown that the relationship between body From Table 2.6 it can be seen that for the same amount of subcu- mass index (BMI) and body fat percentage differs among ethnic taneous fat (identical skinfold thickness) women have more body groups. For example, compared with Caucasian populations some fat than men. This is because of the higher internal (organ) fat Asian populations have 3–5% more body fat for the same BMI, content in women. It can also be seen (in both females and males) age, and gender. These differences can be explained by differences that at equal skinfold thickness older people have more body fat: in body build or frame size, subjects with a smaller frame having with age the amount of internal fat increases. more body fat at the same BMI. These differences can have important consequences for the defi nition of obesity (based on BMI cut-off values) and the preva- lence of obesity in a population. In Indonesia, obesity has recently been redefi ned as BMI ≥ 27 kg/m2. At this BMI, Indonesians have a similar body fat to Caucasians with a BMI of 30 kg/m2. The lower- ing of the cut-off point for obesity from 30 to 27 kg/m2 increased the prevalence of obesity from less than 5% to over 10%. Recently an Expert Consultation of the World Health Organiza- tion (WHO) resulted in new guidelines to redefi ne “action points” in non-Caucasian populations. For this not only was the different relationship between BMI and body fat percentage important, but also the high levels of cardiovascular risk factors at low BMI values (WHO, 2004). groups of subjects such as pregnant women or body- builders. However, other predictive methods also have their limitations in these subjects. TBW can also be predicted from weight and height, and numerous prediction formulae have been pub- lished. The standard error of estimate of these equa- tions is, however, considerable. Figure 2.5 Measurement of the biceps skinfold. Skinfold thickness measurements Body fat is located both internally and subcutane- total body fat are skinfolds on the upper arm biceps ously. If one assumes a constant relationship between (Figure 2.5) and triceps, under the scapula (subscapu- subcutaneous fat and total body fat, then total body lar) and above the iliac crest (suprailiac). The sum of fat can be estimated by measuring the amount of the more skinfolds is normally used to reduce the error subcutaneous adipose tissue. The amount of subcu- in measurement and to correct for possible differ- taneous tissue can be estimated by measuring the ences in subcutaneous body fat distribution between thickness of the subcutaneous fat layer at different subjects within the same age and gender group. sites of the body using a skinfold caliper, infrared Various prediction formulae for body fat from skin- interactance, or ultrasound measurements. In a given fold thickness have been published. For children, in age group, the relation between subcutaneous fat and whom the relationship between skinfold thickness total fat is indeed relatively constant. However, the and body fat depends on biological age, separate for- relationship is different between males and females, mulae must be used. females having relatively more internal fat (Box 2.9). Measuring skinfolds adequately requires a trained Thus, it is possible by using age- and gender-specifi c and experienced observer, otherwise excessively large prediction equations to assess the total amount of errors in the assessment of the body fat can occur. A body fat by measuring skinfolds at different sites of disadvantage of the method is that the subject has to the body. be partly undressed. This hampers the use of the Skinfolds can be measured all over the body. The method in epidemiological studies. In bed-ridden or most often measured skinfolds for the assessment of seriously ill patients the measurement of the trunk 24 Introduction to Human Nutrition

Table 2.6 Body fat percentage from the sum of four skinfolds (biceps, triceps, subscapular, suprailiac) in males and females of different agesa

Age (Males) Age (Females)

Skinfolds (mm) 17–29 30–39 40–49 ≥50 60–87 17–29 30–39 40–49 ≥50 60–87

20 8 12 12 13 24 14 17 20 22 34 30 13 16 18 19 27 20 22 25 27 37 40 16 19 21 23 29 23 26 28 30 39 50 19 22 25 27 31 27 28 31 33 41 60 21 24 27 29 32 29 31 33 36 42 70 23 25 29 32 33 31 33 35 38 43 80 25 27 31 34 34 33 34 37 40 44 90 26 28 33 36 35 35 36 38 41 45 100 28 29 34 37 36 36 37 40 43 46 110 29 30 36 39 37 38 39 41 44 47 120 30 31 37 40 37 39 40 42 45 48 130 31 32 38 42 38 40 41 43 46 48 140 32 33 39 43 38 41 42 44 47 49 150 33 34 40 44 39 42 43 45 48 49 a Based on data from Durnin and Womersley (1974) for the age groups 17 to ≥50 and on Visser et al. (1994) for the elderly.

skinfold thicknesses can be diffi cult. This can be over- gender, and level of body fatness. Given the possible come by measuring only the skinfold thickness at the error in densitometry (3%), this means that in extreme upper arm, for example the triceps. However, the cases body fat from skinfolds can be as much as 10– error can be large because the triceps does not neces- 15% off. sarily represent the total amount of subcutaneous fat. With advancing age, the triceps skinfold becomes less Other anthropometric variables representative of total body fat. Measurements of widths of skeletal diameters provide In elderly subjects, the correlation between skin- an indication of the amount of skeletal mass. There fold thickness and total body fat as measured by are formulae that allow the calculation of the skeletal densitometry is generally lower than in young and mass from body height, wrist diameter, and knee middle-aged adults. This is due to an increased diameter. The current reference data for ideal weight amount of internal fat in the elderly. Obese subjects in the USA use the elbow diameter to classify people are diffi cult to measure and the error is large even into three types of body build. when measured by trained observers. This is also the In elderly subjects, the measurement of stature can case in subjects with edema, in whom the thickness be diffi cult owing to kyphosis and/or shrinkage of the of the subcutaneous adipose tissue is easily overesti- spinal vertebrae. Knee height can then be used to mated. In patients with human immunodefi ciency predict the maximal stature during lifetime. Arm virus (HIV) lipodystrophy, peripheral subcutaneous span is also used for that purpose. However, one has fat may be almost absent, while abdominal fat is to realize that the current prediction formulae are increased. In this situation, skinfolds can be very mis- developed in younger populations, in which the rela- leading as indicators of total body fat, and should be tionship between stature and surrogate measure- used only to assess regional fat. ments may be different. In addition, the prediction The calculation of the body fat percentage once the error (3–5 cm) is relatively high. Knee height can also skinfolds have been measured is very simple. For a be used by itself (without predicting total stature), given skinfold thickness, the amount of body fat can when comparing stature-adjusted body composition be read from a table (Table 2.6). between young and old people. The prediction error in body fat percentage is 3– Circumferences of the extremities or the trunk are 5% compared with densitometry, depending on age, used to obtain information on body composition. Body Composition 25

From the mid-arm circumference, in combination skinfold thickness measurements have a better pre- with the triceps skinfold thickness, information on dictive value. muscle mass and fat mass of the upper arm can be Internal abdominal body fat can also be assessed obtained. Circumferences of the trunk at waist, hip, with ultrasound. Studies have shown that ultrasound and upper thigh level are used as indicators of body measurements provide a better method than anthro- fat distribution. The WHO suggests an upper limit pometry to assess internal abdominal fat depots. waist-to-hip circumference ratio above 0.85 for females and 1.00 for males for abdominal fat distribu- Bioelectrical impedance tion. Diameters can also be used to gain insights into In bioelectrical impedance, a small alternating current body fat distribution. A high sagittal diameter com- is applied to the body. It is assumed that the body pared with transverse diameter of the abdomen is consists of different components, of which water and indicative of an enlarged amount of visceral fat. dissolved electrolytes are able to conduct the current. However, it has to be kept in mind that the relation- Hence, body impedance is a measure of body water. ship between these anthropometric parameters of The electrical resistance or impedance of an electro- body fat distribution and the intra-abdominal fat lyte solution depends on several factors, of which the determined by CT or MRI scan is generally low. most important are the amount of electrolytes (pure Changes in internal fat detected by MRI are only water does not conduct the current), the kind of weakly associated with changes in these anthropo- electrolytes, and the temperature of the solution. If metric parameters of fat distribution. currents of low frequency (<5 kHz) are used, body impedance is a measure of ECW, as a low-frequency Infrared interactance current cannot penetrate the , which The principle of infrared interactance is based on acts, with its layers of protein, lipids, and proteins, as differences in absorbance and refl ection of infrared an electrical capacitor. With increasing frequencies light in different tissues. When the absorbance the capacitor features of the cell membrane diminish of near-infrared light (700–1100 nm) is measured and gradually ICW also participates in the conduc- at well-defi ned sites of the body, information on tance of the current, resulting in lower impedance the thickness of the subcutaneous adipose tissue values at higher frequencies. Hence, at higher fre- layer can be obtained. In the prediction formulae quencies, TBW is measured. TBW and ECW can be used, many other parameters are included, such as predicted from impedance at high and low frequency, weight, height, age, and gender, and it has been argued respectively, using empirically derived prediction for- that the prediction of body fat depends more on mulae. Other parameters are often taken into consid- these parameters than on the measured infrared eration, such as body weight, age, and gender. interaction. Most prediction equations are based on statistical relationships between empirically measured im- Ultrasound measurements pedance index values (height2/impedance) and body Ultrasound measurements can also be used to study water values obtained by dilution techniques such as several aspects of body composition. With ultrasound deuterium oxide dilution (for TBW) and bromide measurements, the thickness of the subcutaneous fat dilution (for ECW). As body water in healthy subjects layer can be determined and total body fat can be is an assumed fi xed part (73%) of the FFM, bioelectri- calculated. A good refl ection signal depends heavily cal impedance measurements can also be used for the on the absence of connective tissue between adipose prediction of the FFM and hence body fat percentage. tissue and muscle. The main diffi culty in ultrasound For those prediction equations, the impedance index measurements is the exact application of the ultra- was related to measures of FFM, normally obtained sound transducer perpendicular to the tissue without by densitometry or by DXA. any pressure. In the literature, several studies report Body impedance depends on the frequency of the a good correlation between skinfold thicknesses mea- current used and on body water distribution between sured by calipers and those measured by ultrasound. the extracellular and intracellular space and between The correlation of skinfolds with total body fat was the different geometrical body compartments (legs, higher than when using ultrasound, suggesting that trunk, and arms). This calls for extreme caution in the 26 Introduction to Human Nutrition

Total body electrical conductivity Box 2.10 Total body electrical conductivity (TOBEC) was devel- The relative validity of impedance prediction formulae can be oped in the 1970s. The principle of the method is that demonstrated by a simple calculation example. A man, aged 35 conductive material (body water and dissolved elec- years, of height 170 cm, weight 75 kg, and measured impedance (from foot to hand) 400 Ω, has a predicted fat-free mass (FFM) of trolytes) that is placed in an electromagnetic fi eld will 64.7 kg according to Lukaski et al. (1986) and a predicted FFM of cause an inductive current, which is related to the 60.5 kg according to Segal et al. (1988). Both prediction formulae amount of conductive material. In practice, the subject were developed in US populations and were cross-validated. The lies on a stretcher, which enters the inner space of an instrument used was the same and the method of reference in both electric wire coil, through which a high-frequency studies was underwater weighing. current (2.5–5 MHz) passes. The measurement is very quick (it takes only seconds), painless, and without any risk to the subject. The reproducibility of a mea- surement is within 2% and the error in the predicted interpretation of calculated body composition values FFM was found to be about 3 kg in a group of adult in situations where body water distribution can be subjects, which is similar to, for example, skinfold disturbed, as is the case, for example, in dialysis thickness measurements or impedance measurements. patients and in ascites. In general, prediction formulae The TOBEC method is especially suitable for mea- based on impedance values are strongly population surements in infants and young children, in whom specifi c, and age and gender are important contribu- bioelectrical impedance measurements are diffi cult or tors. Differences between populations and individuals impossible to perform, owing to movement. The main are partly caused by differences in body build (e.g., disadvantage of the method is the high price. relatively long legs), which is not surprising, as the legs contribute most to total body impedance relative to other parts of the body (Box 2.10). Creatinine excretion and Currently available impedance analyzers vary in N-methyl-histidine excretion their electrical features and in their principles. Many In the muscle cell, phosphate, necessary for companies have developed impedance analyzers the energy metabolism of the cell, degenerates to cre- for personal use, anticipating considerable interest atinine at a constant daily rate of about 2%. It is among the public in determining their body fat per- assumed that 1 g of excreted creatinine is equivalent centage. There are instruments that measure imped- to 18–22 kg of muscle mass. As the cell cannot recycle ance from foot to foot while standing on a weighing creatinine, the kidneys excrete it. Since metabolized scale and provide not only body weight but also body creatine phosphate is not the only source of urinary fat percentage. Other instruments measure imped- creatinine (e.g., creatinine in ingested is also ance from hand to hand and allow the reading of excreted immediately), the validity of the method is body fat percentage, using a built-in software program dubious. A day-to-day coeffi cient of variation in the in which weight, height, age, and gender have to be excretion of creatinine of almost 20% is reported, entered. Combinations of foot-to-foot and hand-to- when the subject is “free living” and the urine is hand impedance analyzers are also marketed. sampled over constant periods of 24 hours. The high As for all other impedance analyzers, the incorpo- variation is due to the ingestion of creatinine with rated formulae are population specifi c and have a nonvegetarian , differences in physical activity prediction error of 4–5%. This means that, apart levels, and variation in creatinine excretion within the from a systematic error (prediction formula is not phase of the . After careful standard- valid), the value can be as much as 10% off in extreme ization, which includes a controlled diet, the day-to- cases. This kind of error is similar to the possible error day variability in excretion can be decreased to about in skinfold thickness measurements, and hence 5%. To obtain a reliable assessment of the creatinine impedance is no better than skinfold thickness mea- excretion, sampling of urine over multiple 24 hour surements. The advantage of impedance analyzers is periods is necessary. that there is no need to undress and measurements The excretion of 3-methylhistidine has also been are less prone to observer bias. proposed as a measure for muscle mass. FFM deter- Body Composition 27 mined by densitometry correlates well with excreted the required accuracy. The use in epidemiological 3-methylhistidine. The chemical determination of studies is different than that in clinical situations or 3-methylhistidine is, however, more complicated in physiological research. Table 2.7 provides a “buyer’s than that of creatinine. A unique feature of 3- guide” to the several methods discussed in this methylhistidine is that it gives a measure of muscle chapter. It is diffi cult to generalize as to which method protein breakdown. Given the greater expense of should be used in a given study. Apart from the measuring 3-methylhistidine and the limited benefi t factors mentioned in Table 2.7, availability plays for muscle mass estimates, it is probably best to use an important role. Some situations are discussed it primarily for turnover studies. below. The main disadvantages of creatinine and 3- For the description of body fatness of a large general methylhistidine excretion as measures for body com- population group, the calculation of the body fat per- position are the large variability in excretion, the centage from the BMI may be as good as or even necessity to follow a controlled (meat-free) diet for better than the more expensive information obtained several days before and during the urine collections, from bioelectrical impedance or the laborious mea- and the diffi culties associated with collecting 24 hour surement of skinfold thicknesses. Whichever method urine samples. is to be used for the prediction of body fat percentage in the population, it is important to remember that Use and misuse of body composition data the formulae used should have been validated in the Information on the body composition of groups of population under study. The fact that a formula is subjects or individuals is important, as body compo- cross-validated by the authors who published the sition is an indicator of nutritional status and also formula does not mean that the formula is valid in provides information about acute water homeostasis. another population. The use of the Durnin and Depending on what information is needed, several Womersley (1974) equations for estimating body fat methods are available. However, all have their advan- from the sum of four skinfolds may be correct if the tages and limitations. The price of the method population is adult but younger than about 60 years. (both the instrument and the required personnel), the In older subjects, the amount of body fat is likely to eventual stress and danger (e.g., radiation) for the be underestimated with these formulae. Therefore, subject, and the time necessary to obtain the informa- their use and thoughtless interpretation in the elderly tion determine the choice of the method, as well as would lead to completely wrong conclusions about

Table 2.7 Buyer’s guide to different methods used to determine body composition

Method Accuracy Expenses Radiation Time Convenience for subject

Carcass analysis + + + − − Neutron activation + + + − −− − + ++ + Densitometry + + + + + +/− Dilution method + + +/− (−) ++ 40K method + + − + ++ + DXA + + + +/− − + ++ + More-compartment models + + + −− −+ CT scanning + + − − − + ++ + MRI scanning + + − + + + Anthropometry + + + ++ + + Infrared interactance + + ++ ++ + Bioelectrical impedance ++ + + ++ + + + TOBEC +− + ++ + Creatinine/N-methylhistidine excretion ++ −−

DXA, dual-energy X-ray absorptiometry; CT, computed tomography; MRI, magnetic resonance imaging; TOBEC, total body electrical conductivity. + + +, excellent; + +, very good; +, good; +/–, reasonable; –, bad; – –, very bad. 28 Introduction to Human Nutrition body composition in the elderly and changes in body among other factors, and there are no universally composition with age. For the same reason, the BMI valid prediction equations based on impedance. as an indicator of body fatness is only suitable when Choice of method to assess body composition in a corrections for age and gender are made; for example, population would also take into consideration within- a BMI of 25 kg/m2 at the age of 20 years refl ects a and between-observer variability. Thus, impedance much lower percentage of body fat than at the age of may be better than skinfold thickness measurements 70 years. Body fat in females is always higher than in a study of 1000 people, where variability between body fat in males with the same BMI and age. Recent technicians is important. One may argue that, for studies have shown that the relationship between population studies, a slight overestimation or under- BMI and body fat percentage also differs among estimation of body fat is not important. However, if, ethnic groups. Figure 2.6 gives a good insight on how for example, obesity is defi ned as a certain amount of misleading a low BMI can be. The biodata also show body fat that is achieved at a certain distinct level of the differences in body build, which is at least part of BMI, even minor differences in body fat percentage the reason for the paradox. Prediction equations or in BMI will result in large differences in the preva- based on impedance are dependent on body build, lence of obesity.

Dutch Asian

Age (years) 21 22

Height (cm) 175.6 158.0

Weight (kg) 77.2 43.9

Relative sitting height 0.53 0.55

Wrist (cm) 5.6 4.4

Knee (cm) 9.1 8.4

Arm span (cm) 177.0 157.5

Waist (cm) 82.0 62.2

Hip (cm) 104.0 87.0

BMI 25.0 17.6

BF (%) 31.1 29.6

Figure 2.6 The difference in the relationship between BMI and body fat percentage across populations is best demonstrated in this fi gure and the given biodata. Note that the two young women are the same age and their percentage body fat as determined by a chemical four- compartment model (bias free!) is the same. The Asian woman has relatively shorter legs and a more slender body build (determined as height/(sum of knee and wrist diameter). Relative leg length and “slenderness” are main determining factors in the BMI/percentage body fat relationship in addition to physical activity level. Body Composition 29

20

10

0 Figure 2.7 Individual differences in mea- sured body fat percentage compared with a chemical four-compartment model in 20

Bias in body fat percentage (% body weight) young and 20 elderly females using various techniques. y-axis, BF% from four-compart- ment model minus: ᮀ, densitometry (Siri); –10 ᭺, deuterium dilution; ᭝, DXA; +, three- Young females Elderly females compartment model (Siri); ᭿, skinfold thick- (22 years) (72 years) ness; ᭞, bioelectrical impedance.

In physiological studies where, for example, report changes in impedance values instead of changes energy metabolism has to be corrected for body in predicted body composition. Another example is composition, a systematic bias in body composition the very low change in impedance after water removal results may lead to completely wrong conclusions. in patients with ascites. Despite the fact that a consid- When a body composition formula or method sys- erable amount of water is removed from the body, the tematically overestimates the FFM in obese subjects, change in total body impedance is low as the trunk a “normal” resting metabolic rate in obese subjects hardly contributes to total body impedance. would be underestimated after “correction” for dif- All methods have their limitations, doubly indirect ferences in body composition (expressed as kJ/kg more than indirect methods. Figure 2.7 shows the FFM). This may lead to the conclusion that obese individual errors in body fat percentage from den- subjects have lower metabolic rates, which might sitometry, DXA, deuterium dilution, a chemical have caused their excess adipose tissue stores. This three-compartment model, skinfold thicknesses, and argument does not even imply whether the FFM can bioelectrical impedance in young and elderly women be treated as one entity in different populations compared with body fat percentage obtained from a instead of different components such as the heart, four-compartment model. It is obvious that errors liver, or muscles (Gallagher et al., 1998). These con- can be considerable, both at a group level and espe- siderations are important for comparative studies cially at an individual level in the elderly. Many of between groups (e.g., obese versus nonobese, elderly these errors can be explained by the violation of versus young or middle-aged), but also for longitudi- assumptions. This clearly shows that information on nal studies (e.g., weight-loss studies). body composition must be used with an awareness of The use of bioelectrical impedance to predict the limitations of the techniques. changes in FFM, TBW, or body fat during weight loss is questionable. The difference in body water distri- 2.8 Perspectives on the future bution (anatomically as well as intracellular/extracel- lular) before and after weight loss may be considerable, Given the importance of body composition to evalu- leading to a different and systematic bias of the pre- ate nutritional status and to gain information on diction equation. In such a study it may be better to certain disease processes, it can be expected that more 30 Introduction to Human Nutrition easy-to-use methods and instrumentation will be ments on 481 men and women aged from 17 to 72 years. Br J developed and become available in the future. Nutr 1974; 32: 77–97. Forbes GB. Human Body Composition. Springer, New York, 1987. Application and interpretation of data from these Gallagher D, Belmonte D, Deurenberg P, Wang Z-M, Krasnow N, methods should be used with an awareness of pos- Pi-Sunyer FX, Heymsfi eld SB. Organ-tissue mass measurement sible limitations, as mentioned above. There is a by MRI allows accurate in vivo modeling of REE and metabolic active tissue mass. Am J Physiol 1998: 275: E249–258. growing perception that, because of differences in Heymsfi eld SB, Lohman TG, Wang ZW, Going SB. Human body build, frame size, and possibly also other vari- Body Composition, 2nd edn. Human Kinetics, Champaign, IL, ables, there are differences in the relationship between 2005. Lukaski HC, Bolonchuk WW, Hall CB, Siders WA. Validity of tet- BMI and body fat percentage among ethnic groups. rapolar bioelectrical impedance method to assess human body Some Aboriginal and Asian people have a higher fat composition. J Appl Physiol 1986; 60: 1327–1332. percentage and therefore greater risk for several Segal KR, Van Loan M, Fitzgerald PI, Hodgdon JA, Van Itallie TB. Lean body mass estimation by bio-electrical impedance analysis: chronic diseases at a given BMI than Caucasian popu- a four site cross-validation study. Am J Clin Nutr 1988; 47: lations. Conversely, Africans often have higher bone 7–14. density than Caucasians. Much more research is Visser M, Heuvel van den E, Deurenberg P. Prediction equations for the estimation of body composition in the elderly using needed to defi ne the optimal BMI values that will anthropometric data. Br J Nutr 1994; 71: 823–833. correlate with optimal health and the lowest risk of WHO Expert Consultation. Appropriate body-mass index for disease for different populations. Asian populations and its implications for policy and interven- tion strategies. Lancet 2004; 363: 157–163. Acknowledgment Further reading This chapter has been revised and updated by Paul Siri WE. Body composition from fl uid spaces and density: analysis Deurenberg based on the original chapter by Paul of methods. In: Brozek J, Henschel A, eds. Techniques for Deurenberg and Ronenn Roubenoff. For more infor- Measuring Body Composition. National Academy of Sciences, mation on this topic visit www.nutritiontexts.com Washington, DC, 1961: 223–244. Snijder WS, Cook MJ, Nasset ES, et al. Report of the Task Group on Reference Man. Pergamon Press, Oxford, 1984. Wang Z-M, Pierson RN, Heymsfi eld SB. The fi ve-level model; a References new approach to organise body composition rsearch. Am J Clin Nutr 1992; 56: 19–28. Durnin JVGA, Womersley J. Body fat assessed from total body World Health Organization. Obesity: Preventing and Managing the density and its estimation from skinfold thickness: measure- Global . WHO, Geneva, 1998. 3 Energy Metabolism

Arne Astrup and Angelo Tremblay

Key messages

• Energy balance in the body is the balance between how much composition, and level of physical activity, consistent with long- energy is consumed and how much is expended. Positive balance term good health. This includes the energy needs for optimal is when intake exceeds expenditure and is associated with growth and development in children, and the needs of pregnancy increases in body energy stores (weight gain). During negative and lactation (deposition of tissue and of ). balance, as in periods of starvation, body energy stores are • Body mass index (BMI) classifi es weight relative to height squared depleted. and is the most accepted and widely used crude index of obesity. • Energy intake corresponds to the energy content of macro- A BMI of 18–24.9 kg/m2 is regarded as normal for adults, nutrients in foods. Carbohydrate provides 16.8 kJ/g, protein also between 25 and 29.9 kg/m2 as overweight, and >30 kg/m2 as 16.8 kJ/g, and fat 37.8 kJ/g. In addition, alcohol provides obese. 29.4 kJ/g. • Energy expenditure can be measured by direct methods (calor- • Total energy expenditure constitutes approximately two-thirds of imetry) or indirect methods, in which oxygen consumption and the energy expended by the body to maintain basic physiological production are used to calculate energy expendi- functions plus the thermic effect of a and energy expended ture. However, the modern gold standard is measurement by during physical movement. The basic physiological functions doubly labeled water, which is a noninvasive method used to include heart beat, muscle function, and respiration (resting or measure total energy expenditure over periods of 7–14 days basal metabolic rate). The thermic effect of a meal is about 10% while subjects are living in their usual environments. of the caloric value of the meal needed to digest, metabolize, • Hunger is the physiological need to eat and results in actions to and store ingested macronutrients. The energy expended during attempt to obtain food for consumption. Appetite is a psychologi- physical activity is energy expended when skeletal muscles are cal desire to eat and is related to the pleasant sensations that used for any type of physical movement. In infants and children, are often associated with food. Thus, hunger is more of an the cost of growth is added. intrinsic instinct, whereas appetite is often a learned response. • Energy requirement is the amount of needed to balance energy expenditure in order to maintain body size, body

3.1 Introduction results in maintenance of body weight and body energy stores. This regulation of energy balance is Defi nition and conceptualization of achieved over the long term despite large fl uctuations energy balance in both energy intake and energy expenditure within The average adult human consumes close to 1 000 000 and between days. The accuracy and precision by calories (4000 MJ) per year. Despite this huge energy which the body maintains energy balance is high- intake, most healthy individuals are able to strike a lighted by the fact that even a small error in the system remarkable balance between how much energy is can have detrimental consequences over time. If consumed and how much energy is expended, thus energy intake chronically exceeds energy expenditure resulting in a state of energy balance in the body. This by as little as 105 kJ/day, then, over time, a person accurate balance between energy intake and energy will become substantially obese. The achievement of expenditure is an example of homeostatic control and energy balance is driven by the fi rst law of thermo-

© 2009 A Astrup and A Tremblay. 32 Introduction to Human Nutrition dynamics, which states that energy can be neither energy cost of arousal. Because of the diffi culty in destroyed nor created. This principle necessitates that achieving BMR under most measurement situations, when energy intake equals energy expenditure, body resting metabolic rate (RMR) is frequently measured energy stores must remain constant. This chapter using the same measurement conditions stated for explains how the body is able to achieve this state of BMR. Thus, the major difference between BMR and energy balance through control of energy intake and RMR is the slightly higher energy expended during energy expenditure. In addition, the various ways that RMR (~ 3%) owing to less subject arousal and non- body energy stores can be measured and some exam- fasting conditions. Because of this small difference, ples of conditions in which energy balance may be the terms basal and resting metabolic rate are often disrupted are summarized. Particular emphasis is used interchangeably. RMR occurs in a continual placed on obesity, which is the end-result of a positive process throughout the 24 hours of a day and remains energy balance and is now considered one of the relatively constant within individuals over time. In major nutritional disorders. the average adult human, RMR is approximately 4.2 kJ/min. Thus, basal or resting metabolic rate is the Components of energy balance largest component of energy expenditure and makes Energy intake up about two-thirds of total energy expenditure. Energy intake is defi ned as the caloric or energy In addition to RMR, there is an increase in energy content of food as provided by the major sources of expenditure in response to food intake. This increase dietary energy: carbohydrate (16.8 kJ/g), protein in metabolic rate after food consumption is often (16.8 kJ/g), fat (37.8 kJ/g), and alcohol (29.4 kJ/g). referred to as the thermic effect of a meal (or meal- induced thermogenesis) and is mainly the energy that Energy storage is expended to digest, metabolize, convert, and store The energy that is consumed in the form of food or ingested macronutrients, named obligatory thermo- can either be stored in the body in the form of genesis. The measured thermic effect of a meal is fat (the major energy store), glycogen (short-term usually higher than the theoretical cost owing to a energy/carbohydrate reserves), or protein (rarely facultative component caused by an activation of used by the body for energy except in severe cases of the sympathoadrenal system, which increases energy starvation and other wasting conditions, as discussed expenditure through peripheral β-adrenoceptors. later in the chapter), or be used by the body to fuel The energy cost associated with meal ingestion is pri- energy-requiring events. marily infl uenced by the composition of the food that is consumed, and also is relatively stable within indi- Energy expenditure viduals over time. The thermic effect of a meal usually The energy that is consumed in the form of food is constitutes approximately 10% of the caloric content required by the body for metabolic, cellular, and of the meal that is consumed. The third source of mechanical work such as breathing, heart beat, and energy expenditure in the body is the increase in muscular work, all of which require energy and result metabolic rate that occurs during physical activity, in heat production. The body requires energy for a which includes exercise as well as all forms of physical variety of functions. The largest use of energy is activity. Thus, physical activity energy expenditure needed to fuel the basal metabolic rate (BMR), which (or the thermic effect of exercise) is the term fre- is the energy expended by the body to maintain basic quently used to describe the increase in metabolic rate physiological functions (e.g., heart beat, muscle con- that is caused by use of skeletal muscles for any type traction and function, respiration). BMR is the of physical movement. Physical activity energy expen- minimum level of energy expended by the body to diture is the most variable component of daily energy sustain life in the awake state. It can be measured after expenditure and can vary greatly within and between a 12 hour fast while the subject is resting physically individuals owing to the volitional and variable nature and mentally, and maintained in a thermoneutral, of physical activity patterns. quiet environment. The BMR is slightly elevated In addition to the three major components of above the metabolic rate during sleep, because energy energy expenditure, there may be a requirement for expenditure increases above basal levels owing to the energy for three other minor needs. Energy Metabolism 33

● The energy cost of growth occurs in growing indi- occur in very inactive individuals as well as in highly viduals, but is negligible except within the fi rst few active individuals provided that adequate energy months of life. sources are available. It is also important to think ● Adaptive thermogenesis is heat production during of energy balance in terms of the major sources of exposure to reduced temperatures, and occurs in energy, i.e., carbohydrate, protein, and fat. For humans, e.g., during the initial months of life and example, carbohydrate balance occurs when the body during fever and other pathological conditions, but balances the amount of carbohydrate ingested with also as a contributor to daily energy expenditure. that expended for energy. ● Thermogenesis is increased by a number of agents in the environment, including in foods and bever- 3.2 Energy intake ages. Nicotine in tobacco is the most important one, and heavy smokers may have a 10% higher Sources of dietary energy energy expenditure than nonsmokers of similar body size and composition and physical activity. As mentioned above, the sources of energy in the food Caffeine and derivatives in , , and choco- we eat include the major macronutrients: protein, car- late, capsaicin in hot chilies, and other substances bohydrate, and fat, as well as alcohol. Carbohydrate in foods and drinks may possess minor thermo- and protein provide 16.8 kJ of energy for each gram; genic effects that affect energy expenditure. alcohol provides 29.4 kJ/g, whereas fat is the most energy dense, providing 37.8 kJ/g. Note that 4.2 kJ is Energy balance defi ned as the amount of heat that is required to raise Energy balance occurs when the energy content of the temperature of 1 liter of water by 1°C. The energy food is matched by the total amount of energy that is content of food can be measured by bomb calori- expended by the body. An example of energy balance metry, which involves combusting a known weight of would be the scenario cited at the outset of this food inside a sealed chamber and measuring the chapter in which, over a year, the average adult con- amount of heat that is released during this process. sumes and expends 1 000 000 calories, resulting in no Thus, 1 g of pure fat would release 37.8 kJ during its net change in the energy content of the body. When complete combustion, whereas 1 g of pure carbohy- energy intake exceeds energy expenditure, a state of drate would release 16.8 kJ. Thus, if the gram quanti- positive energy balance occurs. Thus, positive energy ties of any type of food are known, the energy content balance occurs when excessive overfeeding relative to can easily be calculated. For example, if a protein-rich energy needs occurs, and the body increases its overall nutrition snack contains 21 g of carbohydrate, 6 g of fat, and 14 g of protein, then the total energy content energy stores. Examples of positive energy balance × + × + × = include periods around major festivals when overeat- is (21 16.8) (6 37.8) (14 16.8) 814.8 kJ. The ing and inactivity generally prevail, and during preg- macronutrient composition of food is typically nancy and lactation when the body purposefully assessed in the percentage contribution of each mac- increases its stores of energy. When energy intake is ronutrient to the total number of calories. If a food has lower than energy expenditure, a state of negative a carbohydrate content of 21 g, which is 352.8 kJ, and energy balance occurs, for example during periods of the total energy content is 820 kJ the proportion of starvation. In this regard, evidence suggests that, energy derived from carbohydrate is 43%; the fat under conditions of substantial energy imbalance, be content is 6 g, or 226.8 kJ, equivalent to 28% of the it positive or negative, energy expenditure may reach energy; and the protein contributes 14 g, 235.2 kJ and a level that is beyond what could be predicted by body 29% of the energy. weight changes. This so-called “adaptive thermogen- esis” might contribute to the occurrence of resistance Regulation of food intake to lose fat in the context of obesity treatment or the Appetite, hunger, and satiety achievement of a new body weight plateau following The quality and quantity of food that is consumed are overfeeding. It is important to note that energy closely regulated by the body. Food intake is regulated balance can occur regardless of the levels of energy by a number of factors involving complex inter- intake and expenditure; thus, energy balance can actions among various hormones, neuroendocrine 34 Introduction to Human Nutrition factors, the central nervous system, and organ systems (e.g., brain and liver), and environmental and exter- nal factors. Postabsorptive Sensory Cognitive Appetite is usually defi ned as a psychological desire Postingestive to eat and is related to the pleasant sensations that are Early Late often associated with specifi c foods. Scientifi cally, Food appetite is used as a general term of overall sensations related to food intake. Satiation Satiety Hunger is usually defi ned as the subjective feeling Figure 3.1 The satiety cascade by John Blundell (1987). The four that determines when food consumption is initiated categories of mechanisms are sensory, cognitive, postingestive, and and can be described as a nagging, irritating feeling postabsorptive. The sensoric phase includes stimuli mediated via that signifi es food deprivation to a degree that the sensory fi bers in the cranial nerves and relates to the palatability of the ingested meal including smell, taste, temperature, and texture. The next eating episode should take place. cognitive phase of the satiety cascade represents learned, known, and Satiety is considered as the state of inhibition over assumed properties of the ingested food. In the postingestive phase eating that leads to the termination of a meal, and is the gastrointestinal tract is involved in a number of satiety signals both related to the time interval until the next eating episode. via chemo- and mechanoreceptors and appetite-regulating peptides Thus, hunger and satiety are more intrinsic instincts, from the gut and pancreas either entering circulation and acting directly in the CNS or signaling via the vagus nerve. Important satiety whereas appetite is often a learned response. signals in the postabsorptive phase include circulating nutrients, The internal factors that regulate the overall feeling signals derived from differences in energy turnover, substrate oxida- of hunger and satiety include the central nervous tion, and neurohormonal factors. Reprinted from Blundell et al. system (primarily the hypothalamus and the vagus (1987), copyright 1987 with permission of Elsevier. nerve), the major digestive organs such as the and liver, and various hormones. In addition, envi- ronmental factors (e.g., meal pattern and composi- tion, food availability, smell and sight of foods, and drink in the stomach and intestine and the resul- climate), emotional factors (e.g., stress), and some tant pressure that they exert may regulate food intake. diseased states (e.g., anorexia, trauma, infection) may This effect is known as gastrointestinal distension. infl uence the feelings of both hunger and appetite. In addition, the stomach produces a hormone called The factors that infl uence appetite include factors cholecystokinin (CCK) in response to food intake, external to the individual (e.g., climate, weather), which may, in turn, regulate food intake. Furthermore, specifi c appetite cravings, specifi c learned dislikes or when subjects have fat or carbohydrate infused avoidance (e.g., alcohol), intrinsic properties of food directly into the , they report feelings (e.g., taste, palatability, texture), cultural practices or of satiety. This suggests that factors in the intestine preferences, specifi c effects of some drugs and dis- regulate food intake. Indeed, receptors in the intes- eases, and metabolic factors such as hormones and tine have been identifi ed that recognize the presence neurotransmitters. Some of these factors are described of specifi c macronutrients; these receptors are linked in further detail below. to the brain and therefore can communicate directly The classic way to describe the complex appetite- with the central nervous system, resulting in regula- regulating system is the satiety cascade put forth by tion of energy balance. In addition, other gastrointes- John Blundell. The satiety cascade describes four dis- tinal hormones, such as glucagon-like peptide-1 and tinctly different but overlapping categories of mecha- -2 (GLPs), CCK, and glucose-dependent insulinotro- nisms involved in acute within-meal feeling of satiety pic polypeptide (GIP) are likely to play a role in the (referred to as satiation) and the inbetween-meal mediation of gut events and brain perception of satiety (Figure 3.1). hunger and satiety.

Factors infl uencing food intake Central nervous system factors Digestive factors The main contributory factor regulating food intake Several factors in the digestive system exert a short- in the central nervous system is the hypothalamus. term infl uence over food intake. The presence of food The hypothalamus is linked to specifi c parts of the Energy Metabolism 35 brain that are known to modify feeding behavior, gut- and pancreas-derived satiety signals CCK, peptide specifi cally the paraventricular nuclei and the nigro- YY (PYY), GLP-1, oxyntomodulin (OXM), and pan- striatal tract. These areas of the brain respond to creatic polypeptide (PP). Many of the peripheral various neurotransmitters as well as sympathetic satiety signals have receptors in the arcuate nucleus nervous system activity. In general, food intake will (ARC) of the hypothalamus, which plays an impor- decrease as sympathetic nervous system activity tant role in appetite regulation. The ARC contains increases, and vice versa. neuropeptide Y (NPY)- and agouti-related peptide (AgRP)-expressing neurons acting to stimulate food Circulating factors intake along with the adjacent pro-opiomelanocortin After consumption of a meal, food is broken down (POMC) and cocaine- and amphetamine-regulated into its basic components (i.e., carbohydrate is broken transcript (CART)-expressing neurons which inhibit down to glucose, protein to amino acids, and fats or feeding. Besides the ARC, the nucleus of the solitary triglycerides to and fatty acids) and the cir- tract (NTS) and the area postrema (AP) receive appe- culating levels of some of these breakdown products tite-regulating inputs from vagal afferents and circu- increase in the blood. Consequently, glucose, amino lating factors and are connected to the hypothalamic acids, glycerol, and fatty acids are further metabo- nuclei controlling food intake. lized, primarily in the liver, or used for immediate energy (e.g., in muscle or brain). There is evidence to External factors suggest that this resultant metabolism, especially in Various nonphysiological or external factors are also the liver, may in turn regulate food intake. After meal known to modify food intake, and these effects may consumption, the circulating levels of nutrients fall be mediated through the intrinsic factors described (within minutes for glucose, several hours for triglyc- above. Psychological factors such as depression may erides) and the feelings of hunger return. The link lead to either increased or decreased food intake, or from nutrient metabolism to central control of food changes in the consumption of specifi c types of foods. intake occurs through signals from the liver to the Environmental factors are also important, the most brain via the vagus nerve. Thus, circulating factors obvious being food availability. Even when food is provide a link between the digestive system and the available, some of the specifi c properties of foods central nervous system, which provides another make them more or less appealing, thereby modifying system for regulating food intake. food intake. Important physical characteristics of food include taste, texture, color, temperature, and Signals from the periphery presentation. Other cultural infl uences in the envi- is a hormone that is produced by fat cells ronment, such as time of day, social factors, peer and communicates with the central nervous system infl uence, and cultural preferences, can also play a through leptin receptors in the hypothalamus. Reduced role in infl uencing food intake. production of leptin, or lack of sensitivity of the hypo- thalamus to leptin, may regulate food intake and play a key role in the etiology of rare forms of obesity in 3.3 Energy expenditure humans. Leptin and the other peripheral hormones with a central effect on appetite are divided into two Concept of energy expenditure broad categories: (1) the so-called adiposity signals, The process of energy expenditure and the oxidation which are tonically active providing information on or combustion of food for energy in the body is anal- body fat stores to the CNS and (2) the satiety signals ogous to a woodstove that burns to release heat which are released in response to food intake and are in a controlled fashion. In this analogy, large chunks thought to be involved in short-term regulation of of wood are fed to the stove and the wood is gradually energy intake. Currently known adiposity signals are combusted in the presence of oxygen to release carbon insulin, leptin, and adiponectin, which are considered dioxide, water vapor, and heat. Similarly, in the body, as long-acting signals reducing energy intake. Among the food consumed is oxidized or combusted in the the satiety signals are the hunger hormone ghrelin, presence of oxygen to release carbon dioxide, water, which is secreted in the stomach, and the short-acting and heat. When ingested food is used for energy, 36 Introduction to Human Nutrition however, the release and transfer of energy occur amount of heat that had to be produced by the animal through a series of tightly regulated metabolic path- to melt the measured amount of ice. ways in which the potential energy from food is released slowly and gradually over time. This process Measurement of energy expenditure ensures that the body is provided with a gradual and Lavoisier’s device was the fi rst calorimeter that was constant energy store, rather than relying on a sudden used to measure heat production. This approach is release of energy from an immediate combustion of termed direct calorimetry because heat production ingested food. As a simple example of how the body is measured directly. Direct calorimeters have been uses food for energy, consider the combustion of a designed for measuring heat production in humans, simple glucose : but this approach is technically demanding, especially in human studies, and is now infrequently used. C H O + 6O → 6H O + 6CO + Heat 6 12 6 2 2 2 Indirect calorimetry measures energy production via Similar chemical reactions can be described for the respiratory gas analysis. This approach is based on combustion of other sources of energy, such as fat oxygen consumption and carbon dioxide production and other types of carbohydrates. These types of reac- that occurs during the combustion (or oxidation) of tion occur continuously in the body and constitute protein, carbohydrate, fat, and alcohol, as shown in energy expenditure. As discussed previously, the three the example of glucose combustion. Respiratory gas major sources of energy expenditure in the body are analysis can easily be achieved in humans either over to fuel RMR, the thermic effect of meals, and physical short measurement periods at rest or during exercise activity. As discussed in more detail below, energy using a face mask, mouthpiece, or canopy system for expenditure can be measured by assessment of total gas collection, and over longer periods of 24 hours heat production in the body (direct calorimetry) or (and longer) with subjects living in a metabolic by assessment of oxygen consumption and carbon chamber. BMR is typically measured by indirect calo- dioxide production (indirect calorimetry). rimetry under fasted conditions while subjects lie quietly at rest in the early morning for 30–40 min. Historical aspects of energy expenditure The thermic effect of a meal is typically measured by The burning or combustion of food in the body was monitoring the changes in metabolic rate by indirect originally described in the classic experiments of calorimetry for 3–6 hours following consumption of Lavoisier, who worked in France in the late eight- a test meal of known caloric content. The energy eenth century. Lavoisier discovered that a candle expended in physical activity can be measured under would burn only in the presence of oxygen. In addi- laboratory conditions, also using indirect calorimetry tion, he was the fi rst to describe how living during standard activities. In addition, free-living produced heat in a similar way, as they required physical activity-related energy expenditure over oxygen for life and combusted food as they released extended periods of up to 2 weeks can be measured heat. His experiments were the fi rst to document the by the combination of doubly labeled water (DLW) heat production of living organisms. Working before to measure total energy expenditure (see below), and the invention of electricity, he built the fi rst calorim- indirect calorimetry to measure resting energy expen- eter in which a small animal was placed in a sealed diture and the thermic effect of a meal. Indirect calo- chamber. Lavoisier packed ice into a sealed pocket rimetry has an added advantage in that the ratio of around the chamber (he could only perform these carbon dioxide production to oxygen consumption studies in the winter when ice was collected from the (the respiratory quotient, or RQ) is indicative of the ground), and then placed the chamber and ice layer type of substrate (i.e., fat versus carbohydrate) being inside an insulated chamber. Lavoisier then collected oxidized, for example carbohydrate oxidation has a and measured the volume of melting water. Since the RQ of 1.0 and fat oxidation has a RQ close to 0.7. ice layer was insulated from the outside world, the Energy expenditure can be assessed from indirect only way that the ice could melt was by the increase calorimetry in a simple, less accurate way by ignoring in heat produced by the living animal. Lavoisier the contribution of protein oxidation or by collecting therefore measured the volume of melted ice water, urine during the measurement to analyze the excreted and, by so doing, was able to calculate accurately the nitrogen. The latter approach is preferable because it Energy Metabolism 37 gives a more accurate estimate of energy expenditure where 17, 17.5, and 38.9 are the heat produced (kJ) and RQ. by the combustion of 1 g of protein, glycogen, and triglyceride, respectively. Step 1 The equations are produced by the insertion of the First, the contribution. of protein oxidation to oxygen heat equivalent for carbohydrate and fat, and are consumption. (VO2) and carbon dioxide production valid even though there is a quantitative conversion (VCO2) is estimated based on the knowledge that the of carbohydrate to lipid (de novo lipogenesis) or nitrogen content of protein is 1/6.25: glyconeogenesis. . = × × The caloric equivalent for O2 is similar to the three .VO2(prot) n 6.25 0.97 main substrates: 21 kJ/l O2 for carbohydrate, 19 kJ/l VCO2(prot) = n × 6.25 × 0.77 O2 for fat, and 17.8 kJ/l O2 for protein (which con- where V is volume, 0.97 and 0.77 are liters of O2 con- tributes only modestly to energy expenditure). Energy sumed and CO2 produced by the biological oxidation expenditure can therefore be calculated with reason- of 1 g of protein, respectively, and prot is protein. able accuracy by the equation: . Energy expenditure (kJ/min) = 20 kJ/l × VO (l/min) Step 2 . . 2 Next,. nonprotein. VO2 (VO2(nonprot)) and nonprotein With pure fat oxidation the RQ is 0.707, with pure VCO2 (VCO2(nonprot)) are calculated: carbohydrate oxidation it is 1.0, and with pure protein . . . = − oxidation it is approximately 0.8. .VO2(nonprot) V.O2 VO. 2(prot) = − VCO. 2(nonprot) VCO2 VCO2(prot) = × + × Step 5 .VO2(nonprot) C 0.828 F 2.03 Oxidation of protein (P), carbohydrate (C), and fat VCO = C × 0.828 + F × 1.43 2(nonprot) (F) can be calculated by the following equations, where C and F are grams of oxidized carbohydrate where n is the unit g/min: and fat, respectively, and can be found by solving the = × P (g/min) 6.25 n. . two equations with two unknowns; O2 and CO2 pro- = × − × − C (g/min) 4.55 V.CO2 3.21 . VO2 2.87 duced by the combustion of 1 g of carbohydrate is = × − × − × F (g/min) 1.67 VO 1.67 VCO 1.92 n 0.828 liters, whereas the combustion of 1 g triglycer- 2 2 ide consumes 2.03 liters O2 and produces 1.43 liters CO2. The protein oxidation (P) is n × 6.25 g. 3.4 Factors that infl uence energy expenditure Step 3 The RQ is defi ned as: Resting metabolic rate . . Each of the components of energy expenditure is VCO /VO 2 2 determined by various factors. RMR is highly variable Nonprotein RQ (RQ(nonprot)) is calculated by the between individuals (±25%), but is very consistent equation: within individuals (<5%). Since RMR occurs pre- . . dominantly in muscle and the major organs of the RQ = VCO /VO (nonprot) 2(nonprot) 2(nonprot) body, the main source of individual variability in Step 4 RMR is an individual’s amount of organ and muscle Next, energy expenditure can be calculated: mass. Thus, fat-free mass (FFM; the total mass of the body that is not fat, i.e., predominantly organs and Energy expenditure (kJ/min) muscle) explains 60–80% of the variation in RMR = + − [19.63. 4.59 (RQ(nonprot). 0.707] between individuals. This concept can be explained × VO2(nonprot) + 18.78 × VO2(nonprot) using the woodstove analogy; the larger the wood- stove (or FFM), the larger the amount of heat pro- or duction (or the larger the RMR). Since FFM is a het- Energy expenditure (kJ/min) = erogeneous mixture of all nonfat body components, 17 × P + 17.5 × C + 38.9 × F the metabolic rate associated with each kilogram of 38 Introduction to Human Nutrition

FFM is dependent on the quality of the FFM, in terms Table 3.1 Variation in total energy expenditure (TEE) as a function of of hydration and relative contribution of the different resting metabolic rate (RMR) among various populations organs that make up the FFM. For example, skeletal Study group Average TEE/RMR (range) muscle constitutes approximately 43% of total mass in an adult, but contributes only 22–36% of the RMR, 5-year-old children in Arizona, USA 1.37 (1.15–1.70) whereas the brain, which constitutes approximately Obese women in the UK 1.39 (1.20–1.77) Elderly women in Vermont, USA 1.42 (1.25–1.82) only 2% of mass, contributes 20–24% of the RMR. In 5-year-old children in Vermont, USA 1.44 (1.11–1.77) addition, the metabolic cost of each kilogram of FFM Elderly men in Vermont, USA 1.50 (1.30–2.11) decreases with developmental progression, probably Obese Pima Indians 1.56 (1.03–1.99) Adolescents in the UK 1.56 owing to developmental increases in the muscle mass Dutch adults 1.64 to organ mass ratio within FFM. Thus, the relation- Obese women in New York, USA 1.68 ship between RMR and FFM is not linear across all Young men in Boston, USA 1.70 (1.38–2.32) Obese women in New York, USA 1.73 ages and is estimated to be 331.8 kJ/kg between the Elderly men in Boston, USA 1.74 ages of 0 and 2.5 years, 151.2 kJ/kg in children aged Young men in the UK 1.88 (1.44–2.57) 4–7 years, 88.2 kJ/kg during adolescence, and 151.2 kJ/ Young men in Boston, USA 1.98 (1.57–2.60) Mount Everest climbers 2.0 kg in adulthood. Tour de France cyclists 5.3 Although fat mass is generally thought to be meta- Burns patients 1.3 bolically inert, it signifi cantly contributes to varia- tions in RMR. This is likely explained, at least in Range of TEE/RMR is given in parentheses for studies in which the individual data were reported. part, by neurobiological effects (e.g., changes in sympathetic nervous system activity) resulting from variations in fat mass which affect the metabolism of metabolic rate) and sympathetic nervous system other tissues. RMR is also infl uenced by fat mass, activity. even though fat mass is generally thought to be meta- Several prediction equations have been developed bolically inert. Fat mass contributes in the order of to estimate RMR from other simple measures. These 42.0–54.6 kJ/kg to RMR. This difference is indepen- equations are often useful for making estimates in dent of the gender difference in FFM; in other clinical situations when measurement of RMR cannot words, if one studied a group of males and females of be achieved, or for estimating energy needs for other identical FFM and similar age, RMR would be individuals. The classic equations of Harris and higher in males than in females by around 210.0 kJ/ Benedict are frequently used for this purpose. These day. This gender difference is consistent across the equations were developed from limited measures per- lifespan, and the source of the difference is not formed in the early 1900s, and predict RMR from age, well understood (Table 3.1). More active people height, and weight, and may be of limited accuracy. tend to have a higher RMR than inactive individuals. More recent equations have been developed in larger This difference may be explained in part by the resid- groups of subjects and can predict RMR from body ual effects of chronic exercise on metabolic rate. In weight (Table 3.2). These new equations have been other words, RMR appears to be elevated because of shown to be more accurate. the long-lasting effects of the thermic effect of exer- cise. However, other factors are also involved, since Thermic effect of feeding the higher RMR in more active individuals persists The thermic effect of meal ingestion is primarily long after the last bout of exercise has been com- infl uenced by the quantity and macronutrient quality pleted. Collectively, FFM, fat mass, age, gender, and of the ingested calories. The thermic effect of food has physical activity explain 80–90% of the variance in also been termed meal-induced thermogenesis, or the RMR. In addition, a portion of the unique variance specifi c dynamic action of food. The increase in meta- in RMR across individuals has been ascribed to bolic rate that occurs after meal ingestion occurs over genetic factors, although the specifi c source of this an extended period of at least 5 hours; the cumulative genetic variation has not yet been identifi ed. Other energy cost is equivalent to around 10% of the energy factors that have been shown to infl uence metabolic ingested. In other words, if one consumed a mixed rate include thyroid hormones (higher levels increase meal of 2.1 MJ, the body would require 210.0 kJ to Energy Metabolism 39

Table 3.2 Simple equations for estimating resting metabolic rate Table 3.3 Examples of metabolic equivalent (RMR) from body weight according to gender and age (MET) values for various physical activities

RMR (kJ/day) Activity MET

Age (years) Equation for males Equation for females Basketball 8.0 Chopping wood 6.0 0–3 (60.9 × wt) − 54 (61.0 × wt) − 51 Cleaning house 2.0–4.0 3–10 (22.7 × wt) + 495 (22.5 × wt) + 499 Cycling for pleasure 8.0 10–18 (17.5 × wt) + 651 (12.2 × wt) + 746 Gardening 5.0 18–30 (15.3 × wt) + 679 (14.7 × wt) + 496 Kayaking 5.0 30–60 (11.6 × wt) + 879 (8.7 × wt) + 829 Mowing lawn (power mower) 4.5 >60 (13.5 × wt) + 487 (10.5 × wt) + 596 Painting house 4.0–5.0 Playing musical instrument 2.0–4.0 Running slowly (8–11 km/h) 8.0–10.0 wt, body weight (kg). Running quickly (14–16 km/h) 16.0–18.0 Soccer 7.0–10.0 Strength training 6.0 Stretching 4.0 digest, process, and metabolize the contents of the Tennis 6.0–8.0 meal. The thermic effect of feeding is higher for Skiing 7.0–14.0 protein and carbohydrate than for fat. This is because, Swimming laps 6.0–12.0 Walking 3.0–5.0 for fat, the process of energy storage is very effi cient, Water skiing 6.0 whereas, for carbohydrate and protein, additional energy is required for metabolic conversion to the appropriate storage form (i.e., excess glucose con- running, typing), and the intensity at which the par- verted to glycogen for storage, and excess amino acids ticular activity is performed. The metabolic cost of from protein converted to fat for storage). In addition physical activities is frequently expressed as metabolic to the obligatory energetic cost of processing and equivalents (METs), which represent multiples of storage of nutrients, a more variable facultative ther- RMR. Thus, by defi nition, sitting quietly after a 12 mogenic component has been described. This com- hour fast is equivalent to 1 MET. Table 3.3 provides ponent is mainly pertinent to carbohydrates, which MET values for other typical physical activities. through increased insulin secretion produce a dipha- The cumulative total daily energy cost of physical sic activation of the sympathoadrenal system. The activity is highly variable both within and between initial phase is an insulin-mediated increase in sym- individuals. Therefore, physical activity provides the pathetic activity, which produces a β-adrenoceptor- greatest source of plasticity or fl exibility in the energy mediated increase in energy expenditure. The second expenditure system, and is the component through and later phase occurs when a counter-regulatory which large changes in energy expenditure can be increase in plasma epinephrine is elicited by the achieved. falling blood glucose. This increase in epinephrine has a similar slight stimulatory effect on energy Total energy expenditure: measurement expenditure. As a result of the mediation by β- by doubly labeled water adrenoceptors the thermic effect of carbohydrate-rich The integrated sum of all components of energy meals can be slightly reduced by pharmacological β- expenditure is termed total energy expenditure. Until adrenoceptor antagonists. recently, there was no good way to measure total energy expenditure in humans living under their Energy expenditure related to physical habitual conditions. Total energy expenditure can be activity measured over 24 hours or longer in a metabolic Physical activity energy expenditure encompasses all chamber, but this environment is artifi cial and is not types of activity, including sports and leisure, work- representative of the normal daily pattern of physical related activities, general activities of daily living, and activity. The DLW technique can be used to obtain fi dgeting. The metabolic rate of physical activity is an integrated measure of all components of daily determined by the amount or duration of activity energy expenditure over extended periods, typically (i.e., time), the type of physical activity (e.g., walking, 7–14 days, while subjects are living in their usual 40 Introduction to Human Nutrition environment. The technique was fi rst introduced in energy balance, total energy intake must be equiva- the 1950s as an isotopic technique for measuring the lent to total energy expenditure. This aspect of the carbon dioxide production rate in small animals. technique has been used as a tool to validate energy Unfortunately, it was not possible to apply the tech- intakes using other methods such as food records and nique to humans because the dose required was cost dietary recall. For example, it has been known for prohibitive given the relatively poor sensitivity of the some time that obese subjects report a lower than required instrumentation at that time. It was not for expected value for energy intake. At one time it was another 20 years that the inventors of this technique thought that this was due to low energy requirements described the feasibility of applying the technique to in the obese due to low energy expenditure and measure free-living energy expenditure in humans, reduced physical activity. However, using DLW, it and 10 years later this concept became a reality. has now been established that obese subjects system- The DLW method requires a person to ingest small atically underreport their actual energy intake by 30– amounts of “heavy” water that is isotopically labeled 40% and actually have a normal energy expenditure, 2 18 with deuterium and oxygen-18 ( H2O and H2 O). relative to their larger body size. These forms of water are naturally occurring, stable The major disadvantages of the technique are the (nonradioactive) isotopes of water that differ from periodic nonavailability and expense of the 18O the most abundant form of water. In deuterium- isotope (around —500–600 for a 70 kg adult), the need labeled water, the hydrogen is replaced with deute- for and reliance on expensive equipment for analysis rium, which is an identical form of water except that of samples, and that the technique is not well suited deuterium has an extra neutron in its nucleus com- to large-scale epidemiological studies. Furthermore, pared with hydrogen, and is thus a heavier form of although the technique can be used to obtain esti- water; similarly, 18O-labeled water contains oxygen mates of physical activity energy expenditure, it does with an additional two extra neutrons. Thus, these not provide any information on physical activity pat- stable isotopes act as molecular tags so that water can terns (i.e., type, duration, and intensity of physical be tracked in the body. After a loading dose, deute- activity periods during the day). rium-labeled water is washed out of the body as a The DLW technique has been validated in humans function of body water turnover; 18O is also lost as a in several laboratories around the world by compari- function of water turnover, but is lost via carbon son with indirect calorimetry in adults and infants. dioxide production as well. Therefore, using a number These studies generally show the technique to be of assumptions, the rate of carbon dioxide produc- accurate to within 5–10%, relative to data derived by tion and energy expenditure can be assessed based on indirect calorimetry for subjects living in metabolic the different rates of loss of these isotopes from the chambers. The theoretical precision of the DLW tech- body. nique is 3–5%. However, the experimental variability The major advantages of the DLW method are that is ±12% under free-living conditions, owing to fl uc- the methodology is truly noninvasive and nonobtru- tuations in physical activity levels, and ±8% under sive (subjects are entirely unaware that energy expen- more controlled sedentary living conditions. The diture is being measured), and measurement is good accuracy and reasonable precision of the tech- performed under free-living conditions over extended nique therefore allow the DLW method to be used periods (7–14 days). Moreover, when used in combi- as a “gold standard” measure of free-living energy nation with indirect calorimetry for assessment of expenditure in humans against which other methods resting metabolic rate, physical activity-related energy can be compared. expenditure can be assessed by the difference (i.e., total energy expenditure minus resting metabolic 3.5 Energy requirements rate, minus the thermic effect of meals = physical activity energy expenditure). The additional power of How much energy do we need to sustain life and assessing total energy expenditure with the DLW maintain our body energy stores? Why do some method is that this approach can provide a measure people require more energy and others less? In other of total energy intake in subjects who are in energy words, what are the energy requirements of different balance. This is because, by defi nition, in a state of types of people? Based on our earlier defi nition of Energy Metabolism 41 energy balance, the energy needs or energy require- RMR. The PAL factor has been assessed in a variety of ments of the body to maintain energy balance must types of individual. A low PAL indicates a sedentary be equal to total daily energy expenditure. Total daily lifestyle, whereas a high PAL represents a highly active energy expenditure is the sum of the individual com- lifestyle. The highest recorded sustained PAL in ponents of energy expenditure as discussed previ- humans was recorded in cyclists participating in the ously, and represents the total energy requirements of Tour de France road race. These elite athletes could an individual that are required to maintain energy sustain a daily energy expenditure that was up to balance. Until recently, there was no accurate way to fi ve times their RMR over extended periods. Smaller measure total energy expenditure or energy needs of animals, such as migrating birds, have a much higher humans. The DLW technique has provided a truly ceiling for achieving higher rates of total energy expen- noninvasive means to measure accurately total daily diture, which can reach up to 20 times their RMR. energy expenditure, and thus energy needs, in free- Factors such as body weight, FFM, and RMR living humans. Before DLW, energy requirements account for 40–60% of the variation in total energy were usually assessed by measurement or prediction expenditure. Total energy expenditure is similar of RMR, the largest component of energy require- between lean and obese individuals after taking into ments. However, since the relationship between RMR account differences in FFM. Thus, fatness has small, and total energy expenditure is highly variable because but important, additional effects on total energy of differences in physical activity, the estimation of expenditure, partly through RMR, as discussed previ- energy needs from knowledge of RMR is not that ously, but also by increasing the energetic cost of any accurate and requires a crude estimate of physical physical activity. activity level. Nevertheless, reasonable estimates can With regard to age, some studies suggest that only be made to estimate daily energy budgets for indi- a limited change in total energy expenditure (relative viduals (Table 3.4). to RMR) occurs from childhood to adulthood, but Following the validation of DLW in humans, this that a decline occurs in the elderly. Recent data also technique has been applied to many different popula- suggest a gender-related difference in total energy tions. Total energy expenditure is often compared expenditure, in addition to that previously described across groups or individuals using the ratio of one’s for RMR. In a meta-analysis that examined data from total energy expenditure to RMR, or physical activity a variety of published studies, absolute total energy level (PAL). Thus, for example, if the total energy expenditure was signifi cantly higher in males than in expenditure was 12.6 MJ/day and the RMR was females by 3.1 MJ/day (10.2 ± 2.1 MJ/day in females, 6.3 MJ/day, the PAL factor would be 2.0. This value 13.3 ± 3.1 MJ/day in males), and nonresting energy indicates that total energy expenditure is twice the expenditure remained higher in men by 1.1 MJ/day.

Table 3.4 Typical daily energy budgets for a sedentary and a physically active individual of identical occupation, body weight, and resting metabolic rate of 6.0 MJ/day (4.2 kJ/min)

Minutes per day MJ per day

Activity Activity index Sedentary Active Sedentary Active

Sleep 1.0 480 480 2.0 2.0 Daily needs 1.06 120 120 5.3 5.3 Occupational 1.5 480 480 3.0 3.0 Passive recreation 2.0 360 300 3.0 2.5 Exercise 12.0 0 60 0 3.0 Total 1440 1440 8.6 11.1 PAL = 1.4 PAL = 1.8

Thus, the sedentary individual would need to perform 60 min of vigorous activity each day at an intensity of 12.0 to increase the physical activity level (PAL) from a sedentary 1.4 to an active and healthy 1.8. 42 Introduction to Human Nutrition

Individuals who have sedentary occupations and ages 5.1 MJ/day, while the currently recommended do not participate frequently in leisure pursuits that intake for these children is 6.2 MJ/day. Thus, newer require physical activity probably have a PAL factor estimates of the energy requirements of infants are in the region of 1.4. Those who have occupations needed based on assessment of total energy expendi- requiring light activity and participate in light physi- ture data. cal activities in leisure time probably have a PAL Several laboratories have reported measurements around 1.6 (this is a typical value for sedentary people of total energy expenditure in young, healthy, free- living in an urban environment). Individuals who living children around the world. Despite marked dif- have physically active occupations and lifestyles prob- ferences in geographical locations, the data are similar, ably have a PAL greater than 1.75. It has been sug- although environmental factors such as season and gested that the optimal PAL that protects against the sociocultural infl uences on physical activity can infl u- development of obesity is around 1.8 or higher. ence total energy expenditure and thus energy require- Increasing one’s physical activity index from 1.6 to ments. In the average 5 year old child weighing 20 kg, 1.8 requires 30 min of daily vigorous activity, or total energy expenditure is approximately 5.5–5.9 MJ/ 60 min of light activity (Table 3.4). day, which is signifi cantly lower than the existing rec- ommended daily allowance for energy in children of this age, by approximately 1.7–2.1 MJ/day. Thus, as 3.6 Energy balance in various conditions with infants, newer estimates of energy needs in chil- dren are needed based on assessment of total energy Infancy and childhood expenditure data. Changes in energy intake during infancy have been well characterized. During the fi rst 12 months of life, Aging energy intake falls from almost 525 kJ/kg per day in In the elderly, two different problems related to the fi rst month of life to a nadir of 399 kJ/kg per day energy balance can be recognized. In one segment of by the eighth month, then rises to 441 kJ/kg per day the elderly population there is a decline in food intake by the 12th month. However, total energy expendi- that is associated with dynamic changes in body com- ture in the fi rst year of life is relatively constant at position where there is a tendency to lose FFM, which around 252–294 kJ/kg per day. In infants, the large leads to loss in functionality. In others there is a difference between total energy expenditure and tendency to gain fat mass, which increases the risk energy intake is explained by a positive energy balance for obesity, , and noninsulin- to account for growth. In the fi rst 3 months of life it dependent diabetes. These two opposing patterns is estimated that the energy accretion due to growth suggest that the ability to self-regulate whole body is 701.4 kJ/day, or approximately 32% of energy energy balance may diminish with aging. Thus, pre- intake, falling to 151.2 kJ/day, or 4% of energy intake, scription of individual energy requirements may serve by 1 year of age. Individual growth rates and early as a useful tool to prevent the age-related deteriora- infancy feeding behavior are at least two known tion of body composition. Other special consider- factors that would cause variation in these fi gures. ations in the elderly relate to meeting energy needs in There is now substantial evidence to suggest that special populations, such as those with Alzheimer’s existing recommendations may overestimate true and Parkinson’s disease, which frequently can lead energy needs, based on measurement of total energy to malnourished states and a diminishing of body expenditure in infants. In the fi rst year of life, tradi- weight. It was thought that these neurological condi- tional values of energy requirements overestimate tions may lead to body weight loss because of an those derived from measurement of total energy associated hypermetabolic condition in which meta- expenditure and adjusted for growth by 11%. Between bolic rate may increase above normal, thus increasing 1 and 3 years of age the discrepancy is more striking, energy needs. However, more recent studies have where the traditional values for requirements are 20% clearly shown that the wasting or loss of body weight higher than those derived from total energy expendi- often associated with these conditions is explained by ture and adjusted for growth. For example, in 3 year a reduction in food intake, probably owing to a loss old children total energy expenditure by DLW aver- in functionality. Energy Metabolism 43

Energy requirements in physically this vigorous training program leading to a reduction active groups in spontaneous physical activity and/or a reduction in voluntary physical activities, similar to that The DLW technique has been used to assess energy observed in several animal studies. Thus, it should requirements in highly physically active groups of not automatically be assumed that energy require- people. The most extreme case is a study that assessed ments are elevated by participation in activity the energy requirements of cyclists performing in the programs, and the ultimate change in energy 3 week long Tour de France bicycle race. The level of requirements may be dictated by the intensity of the total energy expenditure recorded (PAL factor of training program and the net sum of change in 5.3, or approximately 35.7 MJ/day) was the highest the individual components of energy expenditure. recorded sustained level in humans. In another study An important area of research is to identify the involving young male soldiers training for jungle optimal program of exercise intervention in terms of warfare, energy requirements were 19.9 MJ/day exercise mode, type, duration, and intensity that can (PAL factor of 2.6). The total energy expenditure of have optimal effects on all components of energy four mountaineers climbing Mount Everest was balance. 13.6 MJ/day (PAL 2.0–2.7), which was similar to energy expenditure during on-site preparation prior Energy requirements in pregnancy to climbing (14.7 MJ/day). Total energy expenditure and lactation in free-living collegiate swimmers was almost 16.8 MJ/ Pregnancy and lactation are two other examples in day in men and 10.9 MJ/day in women. In elite female which energy metabolism is altered in order to achieve runners previously performed studies of energy positive energy balance. The specifi c changes in intake suggested unusually low energy requirements. energy requirements during pregnancy are unclear However, in a study in nine highly trained young and the various factors affecting this change are women, free-living energy expenditure was 11.9 ± complex. Traditional government guidelines suggest 1.3 MJ/day, compared with the reported energy intake that energy requirements are raised by 1.3 MJ/day of 9.2 ± 1.9 MJ/day. This study suggests that elite during pregnancy. This fi gure is based on theoretical female runners underreport true levels of energy calculations based on the energy accumulation asso- intake and confi rms the absence of energy-saving ciated with pregnancy. However, these fi gures do not metabolic in this population. include potential adaptations in either metabolic effi - Regular participation in exercise is traditionally ciency or PAL during pregnancy. In a study that per- thought to elevate energy requirements through the formed measures in 12 women every 6 weeks during additional direct cost of the activity, as well as through pregnancy the average increase in total energy an increase in RMR. However, in some situations expenditure was 1.1 MJ/day. The average energy cost energy requirements are not necessarily altered by of pregnancy (change in total energy expenditure plus participation in regular physical activity. For example, change in energy storage) was 1.6 MJ/day. However, in a study of an elderly group of healthy volunteers, there was considerable variation among the 12 there was no signifi cant change in total energy ex- subjects for the increase in average total energy penditure in the last 2 weeks of an 8 week vigorous expenditure (264.6 kJ/day to 3.8 MJ/day) and the endurance training program. The failure to detect average energy cost of pregnancy (147 kJ/day to an increase in total energy expenditure occurred 5.2 MJ/day). despite a 10% increase in RMR (6703.2 ± 898.8 to Metabolic adaptations during lactation have been 7404.6 ± 714 kJ/day), as well as an additional 630 kJ/ examined in well-nourished women using the DLW day associated with the exercise program. These technique. The energy cost of lactation was calculated increases in energy expenditure were counteracted by to be 3.7 MJ/day. Just over half of this energy cost was a signifi cant reduction in the energy expenditure of achieved by an increase in energy intake, while the physical activity during nonexercising time (2.4 ± 1.6 remainder was met by a decrease in physical activity versus 1.4 ± 1.9 MJ/day). The lack of increase in total energy expenditure (3.2 MJ + 873.6 kJ/day at 8 weeks energy expenditure in this study is probably explained of lactation compared with 3.9 + 1.1 MJ/day in the by a compensatory energy-conserving to same women prior to pregnancy). 44 Introduction to Human Nutrition

Energy requirements in disease and trauma status at opposite ends of the spectrum. For example, The DLW technique has been used in various studies cerebral palsy is associated with reduced fat mass and to assess the energy requirements of hospitalized FFM, whereas half of patients with myelodysplasia are patients. Information on energy requirements during obese. It is unclear whether the abnormal body com- hospitalization for disease or trauma is important position associated with these conditions is the end- because: result of inherent alterations in energy expenditure and/or food intake, or whether alterations in body ● energy expenditure can be altered by the disease or composition are an inherent part of the etiology of injury the specifi c disability. In addition, it is unclear how ● physical activity is often impaired or reduced early in life total energy expenditure may be altered ● both underfeeding and overfeeding of critically ill and whether reduced energy expenditure is involved patients can lead to metabolic complications; there- with the associated obese state. Nevertheless, pre- fore, correct assessment of energy requirements scription of appropriate energy requirements may be during recovery is an important part of therapy. a useful tool in the improvement of nutritional status The metabolic response during recovery from a in developmental disabilities. burn injury includes an increase in RMR, although Total energy expenditure has been shown to be this is not necessarily a function of the extent of the lower in adolescents with both cerebral palsy and burn. The widely used formulae to predict energy myelodysplasia, partly owing to reduced RMR but needs in burn patients are not based on measurement primarily to reduced physical activity. Based on of energy expenditure and estimate that most patients measurements of total energy expenditure, energy require 2–2.5 times their estimated RMR. However, requirements of adolescents with cerebral palsy and using the DLW technique, total energy expenditure myelodysplasia are not as high as previously specu- was 6.7 + 2.9 MJ/day in 8 year old children recovering lated. In nonambulatory patients with cerebral palsy, from burn injury, which was equivalent to only 1.2 energy requirements are estimated to be 1.2 times times the nonfasting RMR. The lower than expected RMR, and in the normal range of 1.6–2.1 times RMR values for total energy expenditure in children recov- in ambulatory patients with cerebral palsy. ering from burns suggest that RMR is not as elevated in burn patients as previously speculated, and that RMR is not a function of burn size or time after the 3.7 Obesity injury, probably owing to improvements in wound care which reduce heat loss. In addition, energy Basic metabolic principles requirements in patients recovering from burn injury Obesity is the most common form of a disruption in are reduced because of the sedentary nature of their energy balance and now constitutes one of the major hospitalization. and most prevalent disorders of nutrition. Because of In a study of patients with anorexia nervosa, total the strong relationship between obesity and health energy expenditure was not signifi cantly different risks, obesity is now generally considered a disease by than controls (matched for age, gender, and height). health professionals. However, physical activity-related energy expendi- Although the body continuously consumes a mixed ture was 1.3 MJ/day higher in anorexia nervosa diet of carbohydrate, protein, and fat, and sometimes patients, which was compromised by a 1.3 MJ/day alcohol, the preferred store of energy is fat. There is lower RMR. Thus, energy requirements in anorexia a clearly defi ned hierarchy of energy stores that out- nervosa patients are normal, despite alterations in the lines a preferential storage of excess calories as fat. For individual components of total energy expenditure. alcohol, there is no storage capacity in the body. Thus, In infants with cystic fi brosis, total energy expendi- alcohol that is consumed is immediately oxidized for ture was elevated by 25% relative to weight-matched energy. For protein, there is a very limited storage controls, although the underlying mechanism for this capacity and, under most situations, protein metabo- effect is unknown. lism is very well regulated. For carbohydrate there is Developmental disabilities appear to be associated only a very limited storage capacity, in the form of with alterations in energy balance and nutritional glycogen, which can be found in the liver and in Energy Metabolism 45 muscle. Glycogen provides a very small and short- tion in the energy balance system – a failure of the term energy store, which can easily be depleted after regulatory systems to make appropriate adjustments an overnight fast or after a bout of exercise. Most between intake and expenditure. It is now becoming carbohydrate that is consumed is immediately used clear that the increased health risks of obesity may be for energy. Contrary to popular belief, humans cannot conferred by the distribution of body fat. In addition, convert excess carbohydrate intake to fat. Instead, the infl uence of altered body fat and/or body fat dis- when excess carbohydrates are consumed, the body tribution on health risk may vary across individuals. adapts by preferentially increasing its use of carbohy- Thus, obesity is best defi ned by indices of body fat drate as a fuel, thus, in effect, burning off any accumulation, body fat pattern, and alterations in excessive carbohydrate consumption. Large excesses health risk profi le. of carbohydrate may induce de novo lipogenesis, The body mass index (BMI) is now the most but normally this process is quantitatively minor. accepted and most widely used crude index of obesity. However, no such adaptive mechanism for fat exists. This index classifi es weight relative to height squared. In other words, if excess fat is consumed, there is no The BMI is therefore calculated as weight in kilo- mechanism by which the body can increase its use of grams divided by height squared in meters, and fat as a fuel. Instead, when excess fat calories are con- expressed in the units of kg/m2. Obesity in adults is sumed, the only option is to accumulate the excess fat defi ned as a BMI above 30.0 kg/m2, while the normal as an energy store in the body. This process occurs at range for BMI in adults is 18.5–24.9 kg/m2. A BMI in a very low metabolic cost and is therefore an extremely the range of 25–30 kg/m2 is considered overweight. In effi cient process. To store excess carbohydrate as children, it is more diffi cult to classify obesity by BMI glycogen is much more metabolically expensive and because height varies with age during growth; thus, therefore a less effi cient option. There is another age-adjusted BMI percentiles must be used. important reason why the body would prefer to store One of the major disadvantages of using the BMI fat rather than glycogen. Glycogen can only be stored to classify obesity is that this index does not distin- in a hydrated form that requires 3 g of water for each guish between excess muscle weight and excess fat gram of glycogen, whereas fat does not require any weight. Thus, although BMI is strongly related to such process. In other words, for each gram of glyco- body fatness, at any given BMI in a population, there gen that is stored, the body has to store an additional may be large differences in the range of body fatness. 3 g of water. Thus, for each 4 g of storage tissue, the A classic example of misclassifi cation that may arise body stores only 16.8 kJ, equivalent to just 4.2 kJ/g, from the use of the BMI is a heavy football player or compared with the benefi t of fat which can be stored body-builder with a large muscle mass who may have as 37.8 kJ/g. a BMI above 30 kg/m2 but is not obese; rather, this Thus, a typical adult with 15 kg of fat carries man has a high body weight for his height resulting 567.0 MJ of stored energy. If the adult did not eat and from increased FFM. was inactive, he or she might require 8.4 MJ/day for Since the health risks of obesity are related to body survival, and the energy stores would be suffi cient for fat distribution, and in particular to excess abdominal almost 70 days. This length is about the limit of human fat, other anthropometric indices of body shape are survival without food. Given that glycogen stores useful in the defi nition of obesity. Traditionally, the require 4 g to store 4.2 kJ (3 g of water plus 1 g of gly- waist-to-hip ratio has been used as a marker of upper cogen = 16.8 kJ), we can calculate that to carry this versus lower body-fat distribution. More recent much energy in the form of glycogen requires 135 kg studies suggest that waist circumference alone pro- of weight. It is no wonder therefore that the body’s vides the best index of central body-fat pattern and metabolism favors fat as the preferred energy store. increased risk of obesity-related conditions. The rec- ommended location for the measurement of waist Defi nition of obesity circumference is at the midpoint between the lowest Obesity has traditionally been defi ned as an excess point of the rib cage and the iliac crest. The risk of accumulation of body energy, in the form of fat or obesity-related diseases is increased above a waist cir- adipose tissue. Thus, obesity is a disease of positive cumference of 94 cm in men and above 80 cm in energy balance, which arises as a result of dysregula- women. 46 Introduction to Human Nutrition

Etiology of obesity: excess intake or ● increased use of television and computers for enter- decreased physical activity tainment and leisure activities Stated simply, obesity is the end-result of positive ● use of elevators and escalators rather than using energy balance, or an increased energy intake relative stairs to expenditure. It is often stated, or assumed, that ● increased fear of crime, which has reduced the like- obesity is simply the result of overeating or lack of lihood of playing outdoors physical activity. However, the etiology of obesity is ● poor urban planning, which does not provide not as simple as this, and many complex and interre- adequate cycle lanes or even pavements in some lated factors are likely to contribute to the develop- communities. ment of obesity; it is extremely unlikely that any Thus, the increasing prevalence, numerous health single factor causes obesity. Many cultural, behav- risks, and astounding economic costs of obesity clearly ioral, and biological factors drive energy intake and justify widespread efforts towards prevention. energy expenditure, and contribute to the homeo- The relationship between obesity and lifestyle static regulation of body energy stores, as discussed factors refl ects the principle of energy balance. Weight earlier in the chapter. In addition, many of these maintenance is the result of equivalent levels of energy factors are infl uenced by individual susceptibility, intake and energy expenditure. Thus, a discrepancy which may be driven by genetic, cultural, and hor- between energy expenditure and energy intake de- monal factors. Obesity may develop very gradually pends on either food intake or energy expenditure, over time, such that the actual energy imbalance is and it is becoming clear that physical activity provides negligible and undetectable. the main source of plasticity in energy expenditure. Although there are genetic infl uences on the various In addition, lifestyle factors such as dietary and activ- components of body-weight regulation, and a major ity patterns are clearly susceptible to behavioral mod- portion of individual differences in body weight can ifi cation and are likely targets for obesity prevention be explained by genetic differences, it seems unlikely programs. A second, yet related, reason that control that the increased global prevalence of obesity has of the obesity epidemic will depend on preventive been driven by a dramatic change in the gene pool. It action is that both the causes and health consequences is more likely and more reasonable that acute changes of obesity begin early in life and track into adulthood. in behavior and environment have contributed to the For example, both dietary and activity patterns rapid increase in obesity, and genetic factors may be responsible for the increasing prevalence of obesity important in the differing individual susceptibilities are evident in childhood. to these changes. The most striking behavioral changes that have occurred have been an increased reliance on Role of physical activity and energy high-fat and energy-dense fast foods, with larger expenditure in the development of obesity portion sizes, coupled with an ever-increasing seden- tary lifestyle. The more sedentary lifestyle is due to an Although it is a popular belief that reduced levels of increased reliance on technology and labor-saving energy expenditure and physical activity lead to the devices, which has reduced the need for physical activ- development of obesity, this hypothesis remains con- ity for everyday activities. Examples of energy-saving troversial and has been diffi cult to prove. There are devices are: certainly good examples of an inverse relationship between physical activity and obesity (e.g., athletes ● increased use of automated transport rather than are lean and nonobese individuals), as well as good walking or cycling examples of the positive relationship between obesity ● central heating and the use of automated equip- and physical inactivity (obese individuals tend to be ment in the household, e.g., washing machines less physically active). However, not all studies provide ● reduction in physical activity in the workplace supporting evidence. For example, several studies due to computers, automated equipment, and elec- suggest that increased television viewing (as a marker tronic mail, which all reduce the requirement for for inactivity) increases the risk of obesity, whereas physical activity at work others do not. Similar to the results for physical activ- Energy Metabolism 47 ity, some studies suggest that a low level of energy differences in energy expenditure and physical activ- expenditure predicts the development of obesity, and ity and their impact on the development of obesity others do not support this hypothesis. are different at the various stages of maturation. This Physical activity is hypothesized to protect people hypothesis is supported by previous longitudinal from the development of obesity through several studies in children, showing that a reduced energy channels. First, physical activity, by defi nition, results expenditure is shown to be a risk factor for weight in an increase in energy expenditure owing to the cost gain in the fi rst 3 months of life, but not during of the activity itself, and is also hypothesized to the steady period of prepubertal growth. Secondly, increase RMR. These increases in energy expenditure there could be individual differences in the effect of are likely to decrease the likelihood of positive energy altered energy expenditure on the regulation of energy balance. However, the entire picture of energy balance balance. Thus, the effect of energy expenditure on the must be considered, particularly the possibility that etiology of obesity could vary among different sub- increases in one or more components of energy groups of the population (e.g., boys versus girls, dif- expenditure can result in a compensatory reduction ferent ethnic groups) and could also have a differential in other components (i.e., resting energy expenditure effect within individuals at different stages of devel- and activity energy expenditure). Secondly, physical opment. It is conceivable that susceptible individuals activity has benefi cial effects on substrate metabo- fail to compensate for periodic fl uctuations in energy lism, with an increased reliance on fat relative to expenditure. Third, explanations related to the meth- carbohydrate for fuel utilization, and it has been odology can also be offered because of the complexity hypothesized that highly active individuals can main- of the nature of physical activity and its measure- tain energy balance on a high-fat diet. ment. The success of controlled exercise interven- Cross-sectional studies in children and adults have tions in improving body composition indicates an shown that energy expenditure, including physical extremely promising area for the prevention of activity energy expenditure, is similar in lean and obesity. However, further studies are required to elu- obese subjects, especially after controlling for differ- cidate the specifi c effects of different types of exercise ences in body composition. Children of obese and on the key features of body weight regulation. lean parents have also been compared as a model of preobesity. Some studies show that children of obese 3.8 Perspectives on the future parents had a reduced energy expenditure, including physical activity energy expenditure, whereas another Much is known about how the body balances energy study did not. A major limitation of the majority of intake and expenditure. There are, however, areas studies that have examined the role of energy expen- that need further research. The technology to deter- diture in the etiology of obesity is their cross-sectional mine total energy expenditure with doubly labeled design. Because growth of individual components of water has been standardized. Most of the data from body composition is likely to be a continuous process, using this method have been obtained in populations longitudinal studies are necessary to evaluate the rate living in industrialized countries. More studies on of body fat change during the growing process. Again, infants, children, adolescents, adults, pregnant and some longitudinal studies support the idea that lactating women, and the elderly living in developing reduced energy expenditure is a risk factor for the countries are indicated. Doubly labeled water is an development of obesity, whereas others do not. expensive method. There is a need to develop more Finally, intervention studies have been conducted to cost-effective methods that can be used in fi eld studies determine whether the addition of physical activity and to determine the energy cost of specifi c activities can reduce obesity. These studies tend to support of people throughout the life cycle in developing the positive role of physical activity in reducing countries. Obesity has recently been defi ned as a body fat. disease by the World Health Organization. The Several possibilities could account for such dis- growing problem of obesity worldwide, and in chil- crepant fi ndings. First, the ambiguous fi ndings in the dren and in people who were previously food inse- literature may be explained by the possibility that cure and malnourished, needs to be addressed with 48 Introduction to Human Nutrition better information about the behavioral and cultural Reference factors that infl uence energy balance. This demands a more holistic, integrated approach to the study of Blundell JE, Rogers PJ, Hill AJ. Evaluating the satiating power of obesity in the future. foods: implications for acceptance and consumption. In: Solms J, Booth DA, Pangbourne RM, Raunhardt O, eds. Food Acceptance and Nutrition. Academic Press, London, 1987: 205–219. Acknowledgment Further reading This chapter has been revised and updated by Arne Astrup and Angelo Tremblay based on the original Bray G, Bouchard. D, eds. Handbook of Obesity, 3rd edn. Informa chapter by Michael I Goran and Arne Astrup. Healthcare, New York, 2008. DeFronzo RA, Ferrannini E, Keen H, Zimmet P. International Textbook of Diabetes Mellitus, 3rd edn. John Wiley & Sons, Chichester, 2004. 4 Nutrition and Metabolism of Proteins and Amino Acids

Naomi K Fukagawa and Yong-Ming Yu

Key messages

• Protein is the most abundant nitrogen-containing compound in metabolism, including synthesis, breakdown, inter- the diet and the body. Proteins are formed when L-α-amino acids conversions, transformations, oxidation, and synthesis of other polymerize via formation. nitrogen-containing compounds and urea. These processes are • Amino acids have similar central structures with different side- infl uenced by genetics, phase of life cycle, physical activity, chains determining the multiple metabolic and physiological dietary intake levels, how energy needs are met, route of delivery roles of free amino acids. of nutrients, disease, hormones, and products. • Indispensable (essential) amino acids cannot be synthesized • Protein and amino acid requirements can be determined by nitro- by humans from materials ordinarily available to cells at a gen excretion and balance, factorial estimations, and/or tracer speed commensurate with the demands of human growth and techniques. maintenance. • Existing recommendations on requirements differ by various • The requirements for indispensable amino acids can be defi ned authorities because of a lack of data when some were formu- as “the lowest level of intake that achieves nitrogen balance or lated, different interpretations of data, and different criteria for that balances the irreversible oxidative loss of the amino acid, judging adequate intakes. without requiring major changes in normal protein turnover and • The United Nations plans to publish new recommendations for where there is energy balance with a modest level of physical protein and amino acids in the near future. Those made by the activity.” For infants, children, and pregnant and lactating Institute of Medicine, US National Academies of Science, in 2002 women, requirements would include protein deposited and are cited in this chapter. secretion of milk proteins. • Apparent protein digestibility, measured in the past as the differ- • “Conditionally” indispensable amino acids are those for which ence between nitrogen intake and fecal nitrogen output, under- there are measurable limitations to the rate at which they can be estimates “true” digestibility because fecal nitrogen is derived, in synthesized because their synthesis requires another amino acid part, from endogenous nitrogen sources. and because only a number of tissues are able to synthesize • Tracer techniques have shown that “true” digestibility of most them, and probably only in limited amounts. The metabolic dietary proteins is high. The quality of food protein can be demands for these amino acids may rise above the biosynthetic assessed as the protein digestibility-corrected amino acid score. capacity of the . • Animal protein foods generally have higher concentrations of • Protein and amino acid requirements are determined by the pro- indispensable amino acids than plant foods. is often the cesses of protein synthesis, and maintenance of cell and organ most limiting amino acid, followed by sulfur amino acids (methio- protein content, as well as the turnover rates of protein and nine and cystine) and and .

4.1 Introduction acids through synthesis of peptide bonds contributes to the formation and structural framework of pro- Protein is the most abundant nitrogen-containing teins. These may contain two or more polypeptide compound in the diet and in the body. It is one of the chains forming multimeric proteins, with the indi- fi ve classes of complex present in cells vidual chains being termed subunits. Proteins are the and tissues, the others being DNA, RNA, polysaccha- workhorses in cells and organs and their building rides, and lipids. The polymerization of L-α-amino blocks are the amino acids, which are joined together

© 2009 NK Fukagawa and Y-M Yu. 50 Introduction to Human Nutrition according to a sequence directed by the base sequence amino acids, such as (Tables 4.2 and 4.3), of the DNA (the genome), and so they serve as the play multiple roles. It is not surprising, therefore, that currency of protein nutrition and metabolism. The inappropriate intakes of proteins and/or of specifi c Human Genome Project completed in 2000 revealed amino acids can have important consequences for that the human genome consists of only 30 000 genes, tissue and organ function, and the maintenance of whereas there may be hundreds of thousands of pro- health and the well-being of the individual. teins that are responsible for giving a human its par- This chapter begins with a short historical perspec- ticular characteristics and uniqueness. A new fi eld of tive and then moves in Sections 4.3 and 4.4 to discuss nutrition research has now opened up and is referred the structure, chemistry, and classifi cation of amino to as “nutrigenomics,” which is the study of how acids. Section 4.5 is concerned with the biology of nutrition and interact to infl uence health. protein and amino acid requirements, with Sections Proteins and amino acids fulfi ll numerous functions, 4.6 and 4.7 describing how the requirements are many of which are summarized in Table 4.1. Some established and how they may be met, respectively. Finally, Section 4.8 examines how factors other than dietary protein can infl uence the requirements for Table 4.1 Some functions of amino acid and proteins proteins and amino acids.

Function Example 4.2 A historical perspective Amino acids Substrates for protein Those for which there is a codon The early history of protein metabolism and nutrition synthesis is closely tied to the discovery of nitrogen and its Regulators of protein ; ; ; glutamine distribution in nature. The reason for this is that pro- turnover Regulators of enzyme Glutamate and NAG synthase teins, on average, contain about 16% nitrogen by activity (allosteric) weight (to convert nitrogen to protein it is necessary and PAH activation to multiply by 6.25). Daniel Rutherford, in Edinburgh, Precursor of signal Arginine and nitric oxide can be regarded as the discoverer of nitrogen, which transducer Methylation reactions he called “phlogisticated air” in his Doctorate in Neurotransmitter Tryptophan (serotonin); glutamine fl uxes ; glutamate Precursor of “physiologic” Arg (creatinine); Glu-(NH2) purines Table 4.2 Multiple functions of an amino acid; glutamine as an molecules example Histidine/β- (carnosine) Cysteine//glutamate (glutathione) Substrate of protein synthesis (codons: CAA, CAG) Anabolic/trophic substance for muscle; intestine (“competence Transport of nitrogen Alanine; glu-(NH2) Regulator of gene Amino acid depletion and factor”) transcription synthase gene activation Controls acid–base balance (renal ammoniagenesis) Regulator of mRNA Leucine: alters activity of initiation Substrate for hepatic ureagenesis translation factor 4E-BP and P70 (6SK) via Substrate for hepatic/renal gluconeogenesis mTOR signaling pathway Fuel for intestinal enteroctyes Fuel and precursor and important for generation of Proteins cytotoxic products in immunocompetent cells Enzymatic catalysis Branched chain ketoacid scavenger dehydrogenase Substrate for citrulline and arginine synthesis Transport B12 binding proteins; ceruloplasmin; Nitrogen donor (, amino , coenzymes) apolipoproteins; albumin Nitrogen transport (1/3 circulating N) (muscle; lung) Messengers/signals Insulin; growth hormone; IGF-1 Precursor of GABA (via glutamate) Movement Kinesin; actin; myosin Shuttle for glutamate (CNS) Structure Collagens; elastin; actin Preferential substrate for GSH production? Storage/sequestration Ferritin; metallothionein Osmotic signaling mechanism in regulation of protein synthesis? Immunity Antibodies; cytokine, chemokines Stimulates glycogen synthesis Growth; differentiation; Peptide growth factors; transcription L-Arginine NO metabolism gene expression factors Taste factor (umami)

IGF-1, insulin-like growth factor-1; NAG, N-acetyl glutamate; PAH, CNS, central nervous system; GABA, γ-aminobutyric acid; GSH, growth- phenylalanine hydroxylase; glu-(NH2), glutamine. stimulating hormone; NO, nitric oxide. Nutrition and Metabolism of Proteins 51

Table 4.3 Biochemical roles of amino acids not directly related to The nutritional importance of nitrogenous com- protein metabolism ponents in the diet was fi rst recognized in 1816 by Magendie. He described experiments in dogs that Amino acid Biochemical function received only sugar and olive oil until they died within Integration of carbon and nitrogen metabolism a few weeks. It was concluded that a nitrogen source Leucine, , Ubiquitous nitrogen donors and was an essential component of the diet. Magendie’s metabolic fuel Ubiquitous nitrogen donor, insightful views on nitrogen metabolism and nutri- extracellular tion were followed by studies carried out by the Glutamate Transporter of four-carbon units French scientifi c school, including Justus von Leibig, Glutamine See Table 4.2 Alanine Ubiquitous nitrogen donor, who investigated the chemical basis of protein meta- extracellular bolism and discovered that urea was an end-product Transporter of three-carbon units of protein breakdown in the body. Later, Leibig Aspartate Ubiquitous nitrogen donor Transfer form of nitrogen from founded a school of biochemical studies in Gissen cytoplasmic amino acids to and later in Munich, Germany, from which Carl Voit urea emerged as a distinguished scientist and laid the Single carbon metabolism foundations of modern studies of body nitrogen Methionine Donor and acceptor of methyl groups balance. He, in turn, trained many famous scientifi c Important role in single-carbon celebrities, including Max Rubner, from Germany, metabolism who studied the specifi c dynamic action of proteins Glycine Donor of methylene groups and their effects on energy metabolism, and Wilbur Donor of hydroxymethylene Atwater and Graham Lusk, from the USA, who studied groups food composition, protein requirements, and energy Neurotransmitter synthesis metabolism. Through their work, and that of others, Histidine Precursor for histamine synthesis theories of protein metabolism were proposed and Phenylalanine and Precursors for tyramine, dopamine, epinephrine, and challenged, leading to the more or less contemporary norepinephrine synthesis view which was established through the seminal work Tryptophan Precursor for serotonin synthesis of Rudolf Schoenheimer, conducted at Columbia Glutamate Precursor for γ-aminobutyric acid synthesis University, New York, in the mid-1930s and early Miscellaneous 1940s. He applied the new tracer tool of stable isotope- Arginine Immediate precursor for urea enriched compounds, especially amino acids, in the Precursor for nitric oxide synthesis study of dynamic aspects of protein turnover and Cysteine Potential intracellular thiol buffer amino acid metabolism. Stable isotopes (such as 13C, Precursor for glutathione and 18 15 taurine synthesis O, and N) are naturally present in our environ- Glycine Nitrogen donor for heme ment, including the foods we eat, and they are safe to synthesis use in human metabolic studies. Using this approach, Histidine/β-alanine Precursors for carnosine synthesis Schoenheimer established the fundamental biological principle of a continued tissue and organ protein loss and renewal, which forms the basis for the dietary Medicine thesis in 1792. The fi rst amino acid to be need for protein or supply of amino acids and a utiliz- discovered was cystine, which was extracted from a able form of nitrogen. urinary calculus by Wallaston in England in 1810. It was not until 1935 that threonine, the last of the so- called nutritionally indispensable (essential) amino 4.3 Structure and chemistry of acids for mammals, including man, was discovered amino acids by WC Rose at the University of Illinois. Finally, the term “protein” was invented by the Swedish chemist With the exception of , the amino acids Jons Jakob Berzelius (1779–1848) and this was later that make up peptides and proteins have the accepted and promoted by the infl uential Dutch same central structure (Figure 4.1; the A in this fi gure chemist Gerhardus Mulder in 1838. and 52 Introduction to Human Nutrition

(a) (b) Tyrosine Proline

HO CH2 A COOH N H Histidine Tryptophan

CH2 A CH2 A

N N H H N H Phenylalanine

CH2 A

(c)

Figure 4.1 Structures of some of the nutritionally important amino acids. All are components of proteins and they are coded by DNA. (a) Nutritionally indispensable (essential) includes also tryptophan and histidine; (b) nutritionally conditionally indispensable; (c) nutritionally dispensable.

H amino acids within the linear peptide structure, while | the side-chains distinguish the physical and chemical subsequent fi gures represent the – C – COOH moiety). properties of each chemical class of amino acid. In | addition, some features of the amino acid side-chains NH 2 are critical to the metabolic and physiological roles of The carboxylic acid and amino nitrogen groups are free, as opposed to protein-bound, amino acids (Table the components of the peptide bond that links the 4.3; Figures 4.1 and 4.2). These roles are refl ections of Nutrition and Metabolism of Proteins 53

● dopamine is formed from tyrosine and fulfi lls a neurotransmitter function ● ornithine can be formed from glutamate and serves as both an intermediate in the urea cycle and a precursor of the polyamines spermine and spermi- dine, which are used in DNA packaging. Amino Finally, other amino acids (Figure 4.3) appear in pro- teins via a post-translational modifi cation of a specifi c amino acid residue in the polypeptide chain that is being formed during protein synthesis. N In addition to serving the function as precursors for protein synthesis, amino acids also serve as signal- ing molecules modulating the process of protein synthesis. The translation of mRNA into protein in skeletal muscle is initiated from (1) the binding of met-tRNA to the 40S ribosomal subunit to form the 43S preinitiation complex; (2) the subsequent binding of this complex to mRNA and its localization to the AUG start codon; and (3) the release of the initiation factors from the 40S ribosomal complex to allow the formation of the 80S ribosomal complex via the joining of the 60S ribosomal subunit. Then the 80S ribosomal complex proceeds to the elongation stage Figure 4.2 Physiologically important amino acid metabolites. Both of translation. The formation of the 43S preinitiation the metabolic relationship between alanine and and complex is mediated by a heterotrimeric complex of their transamination partners, the keto acids pyruvate and α- eIF–4F proteins. The signaling pathway regulating ketoglutarate, and the similarity between the catabolic oxidation mRNA translation involves the protein kinase termed β pathway of the branched-chain amino acids and the -oxidation the mammalian target of rapamycin (mTOR). mTOR pathway of saturated fatty acids are shown. regulates the formation of the eIF–4F complex via a series of phosphorylation–dephosphorylation pro- either their specifi c chemical properties or specifi c cesses of the downstream targets. The mTOR signal- metabolic interrelationships. Examples of the former ing pathway is traditionally considered to be solely are the facility of methionine to donate a methyl group involved in mediating the action of hormones. Recent in one-carbon metabolism, the propensity for the studies revealed that the branched-chain amino acids, amide group of glutamine to serve as a nitrogen source especially leucine, serve a unique role in regulating for pyrimidine synthesis, or the sulfhydryl group of mRNA translation via the same mTOR-signaling cysteine forming disulfi de bonds for cross-linking. pathway. Increased availability of leucine activates the The former metabolic relationship allows alanine mTOR and its downstream targets. However, inhibi- and glutamate (and glutamine) to provide a link tion of the mTOR pathway by rapamycine partially between carbohydrate and protein metabolism; the inhibits the stimulatory effect of leucine on protein latter enables the branched amino acids to function synthesis, indicating the involvement of an mTOR- when required, as a “universal” fuel throughout the independent signaling pathway by leucine in the reg- body. ulation of protein synthesis. The detailed mechanisms Some of these amino acid and nitrogen compounds involved in these regulations, especially those of the are derivatives of other amino acids: mTOR-independent pathways, remain an active fi eld ● creatine is formed from glycine, arginine, and of research. methionine and serves in intracellular energy Furthermore, individual amino acids play multiple transduction regulatory roles in health and diseased conditions. 54 Introduction to Human Nutrition

L L NG,NG-dimethyl- -arginine NG,NG-dimethyl- -arginine has been well established. The physiology of the argi- (ADMA) (SDMA) nine–nitric oxide pathway has also been an active area of investigation. In general, these nonprotein func- CH3 CH3 tions of amino acids serve important functions in the HN N CH H C HN N 3 3 maintenance of (1) immune and other protective C C functions; (2) digestive function; and (3) cognitive and neuromuscular function. It is also worth noting NH NH that these functions are primarily exerted by nutri- CH CH tionally dispensable amino acids. Hence, the de novo 2 2 synthesis pathways and/or the amount of exogenous

CH2 CH2 supply of these amino acids or their precursors are important in modulating the physiological and patho- CH CH 2 2 physiological conditions.

A A 4.4 Classifi cation of amino acids Ornithine “Indispensability” as a basis of classifi cation For most of the past 65 years amino acids have been divided into two general, nutritional categories: indis- pensable (essential) and dispensable (nonessential). This categorization provided a convenient and gener- ally useful way of viewing amino acid nutrition at the time. The original defi nition of an indispensable amino acid was: One which cannot be synthesized by the animal organism out of materials ordinarily available to the cells at a speed commensurate with the demands for normal growth.

Figure 4.3 Some amino acids that arise via a post-translational modi- There are three important phrases in this defi nition: fi cation of a polypeptide-bound amino acid. These amino acids are not ordinarily available, at a speed and for normal coded by DNA but are important determinants of the structural and growth. functional characteristics of proteins. Shown are (1) the formation of The phrase “ordinarily available” is an important hydroxyproline, from proline, involved in the maturation of the differ- ent types of collagens in cells; (2) the methylation of a specifi c histidine qualifi er within this defi nition because a number in the muscle protein actin (it could be that this modifi cation gives this of nutritionally essential amino acids, for example protein its ability to function effectively in the contractile activities of the branched-chain amino acids, phenylalanine and the skeletal muscles that help us to move about); and (3) the methyla- methionine, can be synthesized by transamination of tion of arginine to form asymmetric and symmetric dimethylarginine, their analogous α-keto acids. However, these keto which serve as an endogenous nitric oxide synthase inhibitor and play important roles in modulating nitric oxide production and organ blood acids are not normally part of the diet and so are not fl ow in health and diseased conditions. “ordinarily available to the cells.” They may be used in special situations such as in nitrogen-accumulating diseases, including renal failure, where they may assist For example, glycine is an important anti-infl amma- in maintaining a better status of body nitrogen tory, immunomodulatory, and cytoprotective agent metabolism. through the glycine receptor on the cell surface. The The phrase “at a speed” is equally important role of cysteine in regulating glutathione synthesis because there are circumstances in which the rate of and its role in protection against oxidative damage synthesis of an amino acid may be constrained, such Nutrition and Metabolism of Proteins 55 as by the availability of appropriate quantities of tory , such as cats, the further loss of some “nonessential” nitrogen. Further, the rate of synthesis critical enzyme(s) renders these animals particularly becomes of particular importance when considering dependent on dietary sources of specifi c amino acids, a group of amino acids, exemplifi ed by arginine, such as arginine. The lack of arginine in a single meal cysteine, proline, and probably glycine. These amino when given to a cat can be fatal. However, even within acids are frequently described as being conditionally this view, the important term is “” indispensable. That is, their indispensability is depen- because some amino acids can be synthesized from dent upon the physiological or pathophysiological precursors that are structurally very similar. For condition of the individual. example, methionine can be synthesized both by Finally, the phrase “normal growth” is critical in transamination of its keto acid analogue and by two respects. First, it serves to emphasize that the defi - remethylation of homocysteine. According to this nitions were originally constructed in the context of metabolic assessment of amino acids, threonine and growth. For example, for the growing rat arginine is lysine are the only amino acids that cannot be formed an indispensable amino acid, but the adult rat does via transamination or via conversion from another not require the presence of arginine in the diet and so carbon precursor. In this narrower metabolic view, it becomes a dispensable amino acid at that later stage they are truly indispensable amino acids. A contem- of the life cycle. Of course, if the capacity to synthesize porary nutritional classifi cation of amino acids in arginine is compromised by removing a signifi cant human nutrition is given in Table 4.5. part of the intestine which produces citrulline, a Strictly speaking, a truly dispensable amino acid is precursor of arginine, then the adult rat once again one that can be synthesized de novo from a nonamino requires arginine as part of an adequate diet. Second, acid source of nitrogen (e.g., ammonium ion) and a by confi ning the defi nition to growth, this fails to carbon source (e.g., glucose). Accordingly, and from consider the importance of amino acids to pathways a knowledge of biochemical pathways, the only true of disposal other than protein deposition. This aspect metabolically indispensable amino acid is glutamic of amino acid utilization will be considered below. acid, and possibly also glycine. This is because they can be synthesized from glucose and ammonium Chemical and metabolic characteristics as , in the case of glutamate, and from carbon bases of classifi cation dioxide and ammonium ions, in the case of glycine. It is also possible to classify amino acids according to However, the in vivo conditions may differ in both their chemical and metabolic characteristics rather qualitative and quantitative terms from studies in than on the basis of their need for growth. Examination test-tubes or in isolated cells in culture; amino acid of the amino acids that are generally considered to be metabolism in vivo is inherently more complex than nutritionally indispensable for humans and most is immediately evident from a simple consideration other mammals indicates that each has a specifi c of biochemical pathways alone. structural feature, the synthesis of which cannot be accomplished owing to the absence of the necessary Table 4.5 The dietary amino acids of nutritional signifi cance in mammalian enzyme(s) (Table 4.4). Indeed, in obliga- humans

Indispensable Conditionally indispensable Dispensable Table 4.4 Structural features that render amino acids indispensable components of the diet of mammals Valine Glycine Glutamic acid (?) Isoleucine Arginine Alanine Amino acid Structural feature Leucine Glutamine Serine Lysine Proline Leucine, isoleucine, valine Branched aliphatic side-chain Methionine Cystine Asparagine Phenylalanine Tyrosine Lysine Primary amine Threonine (Taurine)a Threonine Secondary alcohol Tryptophan (Ornithine)a Methionine Secondary thiol Histidine (Citrulline)a Tryptophan Indole ring

Phenylalanine Aromatic ring a Histidine Imidazole ring Nonproteinogenic amino acids, which have nutritional value in special cases. 56 Introduction to Human Nutrition

Sources of nonspecifi c nitrogen as urea and purine and pyrimidines. Glutamate for humans and glutamine provide a critical entry of the In earlier texts it would have been stated that, given ammonia from the nitrogen cycle into other amino a suffi cient intake of the indispensable amino acids, acids. It is, therefore, important to examine briefl y all that is then additionally needed to support body the way in which the human body may obtain this protein and nitrogen metabolism would be a source NSN so as to maintain the nitrogen economy of the of “nonspecifi c” nitrogen (NSN) and that this could individual. be in the form of a simple nitrogen-containing Ammonia can be introduced into amino acids by mixture, such as urea and diammonium citrate. ubiquitous glutamate ammonia ligase (glutamine However, this is no longer a suffi cient description of synthetase) that catalyzes the following reaction: what is actually required to sustain an adequate state + Glutamate + NH 4 + ATP of protein nutriture in the human. This can be illus- + → Glutamine + ADP + Pi + H (4.1) trated by considering the nitrogen cycle, on which all life ultimately depends (Figure 4.4). From this it can and (2) via the glutamate dehydrogenase reaction: be seen that some organisms are capable of fi xing + α-Ketoglutarate + NH + NADPH atmospheric nitrogen into ammonia, and plants 4 ∫ L-Glutamate + NADP + H O (4.2) are able to use either the ammonia or soluble 2 + (which are reduced to ammonia) produced by However, because Km for NH 4 in this reaction is high nitrifying bacteria. However, vertebrates, including (>1 mM), this reaction is thought to make only a humans, must obtain dietary nitrogen in the form modest contribution to net ammonia in of amino acids or other organic compounds, possibly the mammal.

Reduction by Amino acids some anaerobic and other bacteria reduced nitrogen–carbon compounds Synthesis in plants and microorganisms

Degradation by animals and Ammonia – ϩ microorganisms NO3 NH Denitrification 4 by some bacteria (e.g., Klebsiella, Azotobacter, Rhizobium)

Nitrification Nitrification by bacteria by soil bacteria (e.g., Nitrobacter) (e.g., Nitrosomonas) Nitrite – NO2

Figure 4.4 The nitrogen cycle. The most abundant form of nitrogen is present in air, which is four-fi fths molecular nitrogen (N2). The total amount of nitrogen that is fi xed in the exceeds 1011 kg annually. Reproduced from Lehninger AL, Nelson DL, Cox MM. Principles of Biochemistry, 2nd edn. New York: Worth, 1993. Nutrition and Metabolism of Proteins 57

In bacteria and plant chloroplasts, glutamate is nitrogen available to the mammalian organism; this produced by the action of glutamate synthase, accord- glutamate would be derived ultimately from plant ing to the reaction: protein. In this sense, glutamate or its lower homo- + logue, aspartic acid, which could supply the α-amino α-Ketoglutarate + glutamine + NADPH + H nitrogen for glutamate, or its derivative, glutamine, → 2 Glutamate + NADP (4.3) would be required as a source of α-amino nitrogen. The sum of the glutamate synthase (eqn 4.3) While additional research is necessary to determine and glutamine synthetase (eqn 4.1) reactions is, whether glutamate, or one of these metabolically therefore: related amino acids, would be the most effi cient + source of α-amino nitrogen, these considerations α-Ketoglutarate + NH + NADPH + ATP 4 potentially offer a new perspective on the NSN com- → Glutamate + NADP + ADP + P (4.4) i ponent of the total protein requirement. In 1965, a Hence, the two reactions combined (eqn 4.4) give United Nations expert group stated: a net synthesis of one molecule of glutamate. However, The proportion of nonessential amino acid because glutamate synthetase is not present in animal nitrogen, and hence the E/T [total essential or tissues, a net incorporation of ammonia nitrogen via indispensable amino acids to total nitrogen] this nitrogen cycle arises primarily from glutamate ratio of the diet, has an obvious infl uence on rather than from glutamine. A net accumulation of requirements … . To make glutamine would be achieved via the glutamine syn- the best use of the available food supplies there thetase reaction that uses ammonia, which would be is an obvious need to determine the minimum derived from various sources including glutamate or E/T ratios for different physiological states … . other amino acids or via hydrolysis of urea by the Finally, the question arises whether there is an microfl ora on the intestinal lumen. optimal pattern of nonessential amino acids. A net incorporation of ammonia into glycine might also be achieved via the glycine synthase (glycine This statement can just as well be repeated today, cleavage) reaction, as follows: but clearly recent studies are beginning to provide + + deeper metabolic insights into the nature of the NSN CO + NH H + NAD + N ,N - 2 4 5 10 needs of the human body. Methylenetetrahydrofolate + + + ∫ Glycine NAD Tetrahydrofolate (4.5) “Conditional” indispensability The glycine could then be incorporated into proteins A contemporary nutritional classifi cation of amino and into such compounds as glutathione, creatine, acids in human nutrition is given in Table 4.5 and and the porphyrins, as well as being converted to some points should be made here about the “condi- serine. The nitrogen of serine would then either be tionally” indispensable amino acids, a term that is available for cysteine (and taurine) synthesis or be used to indicate that there are measurable limitations released as ammonia via the serine dehydratase reac- to the rate at which they can be synthesized. There are tion. However, the glycine cleavage reaction appears several important determinants. First, their synthesis to be more important in glycine than for requires the provision of another amino acid, either as its synthesis. Therefore, the glycine–serine pathway of the carbon donor (e.g., citrulline in the case of argi- ammonia incorporation into the amino acid economy nine synthesis or serine in the case of glycine synthe- of the organism would appear to have only a limited sis) or as a donor of an accessory group (e.g., the sulfur effect on a net nitrogen input into the amino acid group of methionine for cysteine synthesis). The economy of the body. Serine can be formed from ability of the organism to synthesize a conditionally glucose via 3-phosphoglycerate, which comes from essential amino acid is, therefore, set by the availabil- carbohydrate metabolism, and its nitrogen obtained ity of its amino acid precursor. Second, some of these from glutamic acid synthesis via transamination with amino acids are synthesized in only a limited number 2-ketoglutarate. of tissues. The best example of this is the crucial This suggests, therefore, the possibility that gluta- dependence of the synthesis of proline and arginine mate is a key amino acid in making net amino on intestinal metabolism. Third, most evidence sug- 58 Introduction to Human Nutrition gests that, even in the presence of abundant quantities partially reverse this decline in the amount of protein of the appropriate precursors, the quantities of condi- in skeletal muscles and improve overall function. tionally essential amino acids that can be synthesized The protein requirement of adults is usually con- may be quite limited. Thus, there are circumstances, sidered to be the continuing dietary intake that is just for example in immaturity and during stress, under suffi cient to achieve a “maintenance” of body nitro- which the metabolic demands for the amino acids rise gen, often measured only over relatively short experi- to values that are beyond the biosynthetic capacity of mental periods. For infants and growing children the organism. This appears to be the case with regard and pregnant women an additional requirement is to the proline and arginine nutrition of severely needed for protein deposition in tissues. However, burned individuals, and cysteine and perhaps glycine this concept is oversimplifi ed since the chemical com- in the nutrition of prematurely delivered infants. position of the body is in a dynamic state and changes occur in the nitrogen content of individual tissues and organs in response to factors such as diet, hor- 4.5 Biology of protein and amino monal balance, activity patterns, and disease. Thus, acid requirements proteins are being continually synthesized and degraded in an overall process referred to as turnover. Body protein mass The rate of turnover and the balance of synthesis and A major and fundamental quantitative function of degradation of proteins, in addition to the mass of the dietary α-amino acid nitrogen and of the indis- protein, are also important determinants of the pensable amino acids is to furnish substrate required requirements for nitrogen and amino acids, and these for the support of organ protein synthesis and the aspects will be discussed in the following section. maintenance of cell and organ protein content. Therefore, in the fi rst instance the body protein mass Turnover of proteins and amino is a factor that will infl uence the total daily require- acid metabolism ment for protein. Adult individuals of differing size Protein synthesis, degradation, and turnover but who are otherwise similar in age, body composi- The principal metabolic systems responsible for the tion, gender, and physiological state would be maintenance of body protein and amino acid homeo- expected to require proportionately differing amounts stasis are shown in Figure 4.5. They are: of nitrogen and indispensable amino acids. Changes ● protein synthesis in the distribution and amount of body protein that ● protein breakdown or degradation occur during growth and development and later on ● amino acid interconversions, transformation, and during aging may be considered, therefore, as an eventually oxidation, with elimination of carbon initial approach for understanding the metabolic dioxide and urea production basis of the dietary protein and amino acid needs. ● amino acid synthesis, in the case of the nutritionally (For more detailed considerations of body composi- dispensable or conditionally indispensable amino tion please refer to Chapter 2.) acids. Direct measures of total body protein cannot yet be made in living subjects, although there are various Dietary and nutritional factors determine, in part, indirect measures from which it is possible to obtain the dynamic status of these systems; such factors a picture of the body nitrogen (protein) content at include the dietary intake levels relative to the host’s various stages of life. From these approaches it is clear protein and amino acid requirements, the form and that body nitrogen increases rapidly from birth during route of delivery of nutrients, i.e., parenteral (venous) childhood and early maturity, reaching a maximum and enteral (oral) nutritional support, and timing of by about the third decade. Thereafter, body nitrogen intake during the day, especially in relation to the decreases gradually during the later years, with the intake of the major energy-yielding substrates, which decline occurring more rapidly in men than in are the carbohydrates and fats in foods. Other factors, women. A major contributor to this age-related including hormones and immune system products, erosion of body nitrogen is the skeletal musculature. also regulate these systems. This will be a topic for Strength training during later life can attenuate or discussion in the following volume. Changes in the Nutrition and Metabolism of Proteins 59

f

b

A

Figure 4.5 The major systems in amino acid uptake, utilization, and catabolism, with an indication of the processes involved and some factors that can affect them. TNF, tumor necro- sis factor, IL, interleukin. rates and effi ciencies of one or more of these systems lead to an adjustment in whole body nitrogen (protein) balance and retention, with the net direc- tion and the extent of the balance depending upon the sum of the interactions occurring among the pre- vailing factor(s). In effect, there are two endogenous nitrogen cycles that determine the status of balance in body Figure 4.6 The two endogenous nitrogen cycles that determine the protein: status of body protein (nitrogen) balance. (Adapted from Waterlow JC. The mysteries of nitrogen balance. Nutr Res Rev 1999; 12: 25–54, ● the balance between intake and excretion with permission of Cambridge University Press.) ● the balance between protein synthesis and break- down (Figure 4.6). In the adult these two cycles operate so that they are 4–5 g protein/kg per day, respectively. Three points effectively in balance (nitrogen intake = nitrogen relevant to nutritional requirements may be drawn excretion and protein synthesis = protein break- from these data. First, the higher rate of protein syn- down), but the intensity of the two cycles differs, the thesis in the very young, compared with that in the fl ow of nitrogen (and amino acids) being about three adult, is related not only to the fact that a net deposi- times greater for the protein synthesis/breakdown tion of protein occurs during growth, which may component than for nitrogen intake/excretion cycle. account for about 30% of the total amount of protein Protein synthesis rates are high in the premature synthesized in the 6 month old infant, but also to a newborn, possibly about 11–14 g protein synthesized high rate of protein turnover (synthesis and break- per kilogram of body weight per day, and these rates down) associated with tissue remodeling and repair, decline with growth and development so that in term as well as to removal of abnormal proteins. In the babies and young adults these rates are about 7 g and adult the protein turnover is associated with cell and 60 Introduction to Human Nutrition organ protein maintenance since there is no net tissue growth under most circumstances. Second, as will be seen later, at all ages in healthy subjects the rates of Diet adequate in protein whole body protein synthesis and breakdown are considerably greater than usual intakes (the latter are about 1–1.5 g protein/kg per day in adults) or those levels of dietary protein thought to be just necessary to meet the body’s needs for nitrogen and amino acids (about 0.8 g protein/kg per day). It follows, Diet low therefore, that there is an extensive reutilization in protein Nitrogen balance within the body of the amino acids liberated during the course of protein breakdown. If this were not the case it might be predicted that we would be obligate carnivores and this, undoubtedly, would have changed the course of human . Third, although not evident from this discussion alone, there is a general AB as well as functional relationship between the basal Energy intake energy metabolism or resting metabolic rate and the Figure 4.7 Relationship between nitrogen balance and energy intake rate of whole body protein turnover. Protein synthe- with diets of different protein levels. Between energy intake A (low) sis and protein degradation are energy-requiring pro- and B (higher) the two lines are parallel. (Reproduced from Munro HN, cesses, as will be described elsewhere in these volumes, Allison JB, eds. Mammalian Protein Metabolism, vol. I. New York: Academic Press, 1964: 381 with permission.) and from various studies, including interspecies com- ponents, it can be estimated that about 15–20 kJ (4– 5 kcal) of basal energy expenditure is expended in energy intakes (from sources such as carbohydrates association with the formation of each gram of new and lipids) are suffi cient to meet or balance the needs protein synthesis and turnover. In other words, for amino acids, nitrogen, and the daily energy expen- protein and amino acid metabolism may be respon- diture or, in the case of growth, the additional energy sible for about 20% of total basal energy metabolism. deposited in new tissues. Because basal metabolic rate accounts for a signifi cant proportion of total daily energy expenditure, it should Amino acids as precursors of be clear from this discussion that there are signifi cant, physiologically important quantitative interrelationships between energy and nitrogen compounds protein metabolism and their nutritional require- As already pointed out, amino acids are also used for ments. For these reasons it would not be diffi cult to the synthesis of important nitrogen-containing com- appreciate that both the level of dietary protein and pounds that, in turn, play critical roles in cell, organ, the level of dietary energy can infl uence the balance and system function. In carrying out these particular between rates of protein synthesis and protein break- roles the amino acid-derived metabolites also turn down and so affect body nitrogen balance. Their over and they need to be replaced ultimately by the effects are interdependent and their interactions can nitrogen and indispensable amino acids supplied by be complex. This can be illustrated by the changes in protein intake. Estimates on the quantitative utiliza- body nitrogen balance that occur for different protein tion of these precursor and nonproteinogenic roles of and energy intakes (Figure 4.7); as seen here, the level amino acids in human subjects are limited but it is of energy intake, whether above or below require- possible to give some examples. ments, determines the degree of change in the nitro- gen balance that occurs in response to a change in ● Arginine is the precursor of nitric oxide (NO); the nitrogen intake. Conversely, the level of nitrogen total amount of NO synthesized (and degraded) intake determines the quantitative effect of energy per day represents less than 1% of whole body intake on nitrogen balance. Therefore, optimum arginine fl ux and less than 1% of the daily arginine body protein nutrition is achieved when protein and intake. Nutrition and Metabolism of Proteins 61

● In contrast, the rate of synthesis and degradation of that particular attention should be paid to such creatinine is relatively high and accounts for 10% amino acids in nutritional therapy in these groups of the whole body fl ux of arginine and for 70% of of patients. the daily intake of arginine. ● Similarly, the synthesis and turnover of glutathione Urea cycle enzymes and urea production (a major intracellular thiol and important antioxi- Finally, with reference to the major processes shown dant, formed from glutamate, glycine, and cyste- in Figure 4.5, the urea cycle enzymes, which are dis- ine) accounts for a high rate of cysteine utilization tributed both within the mitochondrion and in the such that it greatly exceeds the equivalent of the cytosol (Figure 4.8), are of importance. The produc- usual daily intake of cysteine. Since continued glu- tion of urea may be viewed largely, but not entirely, tathione synthesis involves a reutilization of endog- as a pathway involved in the removal of amino nitro- enous cysteine, a low intake of dietary methionine gen and contributing to an adjustment of nitrogen and cyst(e)ine would be expected to have an unfa- loss to nitrogen intake under various conditions. The vorable infl uence on glutathione status and synthe- fi ve enzymes of urea associate as a tightly sis. This has been shown experimentally to be the connected , called a metabalon, for case, especially in trauma patients and those suffer- conversion of potentially toxic ammonia as well as ing from acquired immunodefi ciency syndrome removal of excess amino acids via their oxidation (AIDS). Because glutathione is the most important with transfer of the nitrogen to arginine and ulti- intracellular that protects cells against mately urea. This is especially important when the damage by reactive oxygen species, this would mean supply of protein or amino acids is high owing to

Mitochondrion

+ – CPS Carbamyl NH43+ HCO + 2ATP + 2ADP + Pi Mg2+, K+ phosphate

Ornithine OTC N-Acetyl glutamate Pi

Arginine Citrulline Acetyl CoA + Glutamate

Ornithine Fumarate Aspartate

Arginine Argininosuccinate Citrulline AS ASy Arg

Urea Ornithine Figure 4.8 The urea cycle enzymes and their distribution in the liver. CPS, carbamoyl phosphate synthetase; OTC, ornithine transcarbamylase; Asy, argininosuccinic synthetase; AS, argininosuccinate; Arg, arginase. 62 Introduction to Human Nutrition variations in the exogenous intake or when there is a tion of ammonia generated from urea nitrogen high rate of whole body protein breakdown in cata- include (1) citrulline synthesis, (2) l-glutamate dehy- bolic states, as occurs in severe trauma and following drogenase pathway in the mitochondria, and (3) overwhelming infection. glycine synthase. The net formation of amino nitro- Altered intakes of indispensable amino acids and gen from these pathways is quantitatively minimal of total nitrogen result in changes in rates of amino compared with the metabolic fl uxes of these amino acid oxidation and the output of urea nitrogen in acids through their major pathways, such as protein urine. There is a roughly parallel change in urea pro- turnover, dietary intake, and de novo synthesis (of the duction and excretion throughout a relatively wide nutritionally dispensable amino acids only). range of change in the level of dietary nitrogen intake above and below physiological requirement levels. Summary of the metabolic basis for Part of this urea enters the intestinal lumen, where protein and amino acid requirements there is some salvaging of urea nitrogen, via intestinal It should be evident from this account of the underly- hydrolysis of urea to form ammonia. This ammo- ing aspects of the needs for α-amino nitrogen and nium nitrogen can be made available to the host for indispensable amino acids, that the “metabolic” the net synthesis of dispensable or conditionally requirement can usefully be divided: fi rst, into those indispensable amino acids. However, the quantitative needs directly associated with protein deposition, a extent to which this pathway of nitrogen fl ow serves critical issue in infants, early childhood nutrition, and to maintain whole body N homeostasis and retention during recovery from prior depletion due to disease under normal conditions is a matter of uncertainty. or malnutrition; and, second, into those needs associ- The ammonia from urea could also enter the nitrogen ated with the maintenance of body protein balance, moiety of the indispensable amino acids, but this which accounts for almost all of the amino acid would be essentially by an exchange mechanism and requirement in the healthy adult, except for that due so would not contribute to a net gain of these amino to the turnover and loss of the various physiologi- acids in the body. cally important nitrogen-containing products, some The reutilization of urea nitrogen starts from the of which were mentioned above. Quantifying the hydrolysis of the intact urea molecule. By constantly minimum needs for nitrogen and for indispensable 15 infusing the [ N2]-urea tracer, the appearance of the amino acids to support growth should be relatively singly labeled [15N]-urea should represent the extent easy, in principle, because these needs are simply the of urea hydrolysis. A 24 hour constant infusion of product of the rate of protein nitrogen deposition and 15 15 [ N2]-urea revealed a minimal amount of [ N]-urea the amino acid composition of the proteins that are appearance in the plasma, and a linear relationship deposited. Here, it may be pointed out that the gross over a wide range of protein intake versus total urea amino acid composition of whole body proteins production and urea hydrolysis. Furthermore, the shows essentially no difference among a variety of possible metabolic pathways involved in the assimila- mammals, including humans (Table 4.6). Thus, at the

Table 4.6 Essential amino acid composition of mixed body protein of immature mammals

Amino acid composition (mg/g protein)

Lysine Phenylalanine Methionine Histidine Valine Isoleucine Leucine Threonine

Rat 77 43 20 30 52 39 85 43 Human 72 41 20 26 47 35 75 41 Pig 75 42 20 28 52 38 72 37 Sheep 75 42 17 23 53 33 79 47 Calf 69 39 18 27 42 30 74 43

From Reeds PJ. Dispensable and indispensable amino acids for humans. J Nutr 2000; 130: 1835S–1840S. Reprinted with permission of The American Society for Nutrition. Nutrition and Metabolism of Proteins 63

Table 4.7 The involvement of amino acids in physiological systems and metabolic function

System Function Product Precursor

Intestine Energy generation ATP Glu, Asp, Glutamine Proliferation Nucleic acids Glutamine, Gly, Asp Protection Glutathione Cys, Glu, Gly Nitric oxide Arg Mucins Thr, Cys, Ser, Pro Skeletal muscle Energy generation Creatine Gly, Arg, Met Peroxidative protection Taurine (?) Cys Nervous system Transmitter synthesis Adrenergic Phe Serotinergic Try Glutaminergic Glu Glycinergic Gly Nitric oxide Arg Peroxidative protection Taurine (?) Cys Immune system Lymphocyte proliferation (?) Glutamine, Arg, Asp Peroxidative protection Glutathione Cys, Glu, Gly Cardiovascular Blood pressure regulation Nitric oxide Arg Peroxidative protection (?) Red cell glutathione Cys, Glu, Gly

major biochemical level the qualitative pattern of the daily requirement, it is qualitatively and function- the needs of individual amino acids to support ally of considerable importance; health depends on protein deposition would be expected to be generally the maintenance of this component of the protein similar. need. In humans, in contrast to rapidly growing mammals Finally, four physiological systems appear to be such as the rat and pig, the obligatory amino acid critical for health: the intestine, to maintain absorp- needs for the purposes of net protein deposition are tive and protective function; the immune and repair for most stages in life a relatively minor portion of system and other aspects of defense; the skeletal the total amino acid requirement. Hence, most of the musculature system; and the central nervous system. requirement for nitrogen and amino acids is associ- Within each system it is possible to identify critical ated with the maintenance of body protein stores (or metabolic roles for some specifi c amino acids (Table body nitrogen equilibrium). A major portion of the 4.7). Also of note is that, with certain exceptions (the maintenance nitrogen and amino acids needs is involvement of phenylalanine and tryptophan in the directly associated with protein metabolism and maintenance of the adrenergic and serotinergic neu- refl ects two related factors. rotransmitter systems, and methionine as a methyl group donor for the synthesis of creatine, as well as ● Amino acids released from tissue protein degrada- the branched-chain amino acids as nitrogen precur- tion are not recycled with 100% effi ciency. sors for cerebral glutamate synthesis), the necessary ● Amino acid catabolism is a close function of the precursors shown here are the dispensable and con- free amino acid concentration in tissues, and so the ditionally indispensable amino acids. presence of the fi nite concentrations of free amino acids necessary to promote protein synthesis inevi- tably leads to some degree of amino acid catabolism 4.6 Estimation of protein and amino and irreversible loss. acid requirements The other metabolic component of the require- Having considered the biology of protein and protein ment for nitrogen and amino acids, as mentioned requirements, this section now considers how these above, is due to the turnover of functionally impor- factors may be used to estimate the requirement for tant products of amino acid metabolism, which are protein and for amino acids. The fi rst section dis- also necessary to maintain health. Although this may cusses nitrogen balance and the defi nition of protein not necessarily be a major quantitative component of requirements, before discussing how these vary with 64 Introduction to Human Nutrition

age and for various physiological groups. Subsequent ● an appropriate stabilization period to the experi- sections cover the estimation of the requirements for mental diet and periods long enough to establish the indispensable amino acids. reliably the full response to a dietary change ● timing and completeness of urine collections Nitrogen balance and defi nition ● absence of mild infections and of other sources of of requirement stress. The starting point for estimating total protein needs Reference to detailed reviews of the concepts behind has been, in most studies, the measurement of the and techniques involved in the nitrogen balance amount of dietary nitrogen needed for zero nitrogen approach is given in the reading list at the end of this balance, or equilibrium, in adults. In the growing chapter. infant and child and in women during pregnancy and When direct nitrogen balance determinations of lactation, or when repletion is necessary following the protein requirement data are lacking, as is the trauma and infection, for example, there will be an case for a number of age groups, an interpolation of additional requirement associated with the net depo- requirements between two age groups is usually made sition of protein in new tissue and that due to secre- simply on the basis of body weight considerations. A tion of milk. Thus, a United Nations (UN) Expert factorial approach may also be applied; here, the so- Consultation in 1985 defi ned the dietary need for called obligatory urine and fecal nitrogen losses are protein as follows. determined (after about 4–6 days of adaptation to a protein-free diet in adults), summated together The protein requirement of an individual with other obligatory losses, including those via is defi ned as the lowest level of dietary sweat and the integument. For children, estimates of protein intake that will balance the losses nitrogen deposition or retention are also included. In from the body in persons maintaining energy the case of very young infants the recommendations balance at modest levels of physical activity. In for meeting protein requirements are usually based children and pregnant or lactating women, the on estimated protein intakes by fully breast-fed protein requirement is taken to also include the infants. needs associated with the deposition of tissues or the secretion of milk at rates consistent with good health. Protein requirements for various age and physiological groups Most estimates of human protein requirements The protein requirements for young adult men and have been obtained directly, or indirectly, from women have been based on both short- and long- measurements of nitrogen excretion and balance term nitrogen balance studies. This also applies to (Nitrogen balance = Nitrogen intake – Nitrogen healthy elderly people, whose protein requirements excretion via urine, feces, skin, and other minor routes have been judged not to be different from those of of nitrogen loss). It must be recognized that the nitro- younger adults. In order to make practical recom- gen balance technique has serious technical and inter- mendations to cover the requirements for most indi- pretative limitations and so it cannot serve as an viduals, it is necessary to adjust the average or mean entirely secure or suffi cient basis for establishing the requirement for a group by a factor that accounts for protein and amino acid needs for human subjects. the variation in protein requirements among appar- Thus, there are: ently similar individuals in that group. This factor is ● a number of inherent sources of error in nitrogen usually taken to be the coeffi cient of variation (CV) balance measurements that should be considered around the mean requirement and traditionally a ● a number of experimental requirements that must value of 2 × CV (SD/mean) is added to the mean be met if reliable nitrogen balance data are to be physiological requirement, so that the needs of all but obtained. 2.5% of individuals within the population would be These include covered. This adjusted requirement value is taken to ● the need to match closely energy intake with energy be the safe practical protein intake for the healthy need, for the various reasons discussed earlier adult (Table 4.8). Most individuals would require less Nutrition and Metabolism of Proteins 65

Table 4.8 The United Nations (1985 FAO/WHO/UNU) and Institute of . . . . the lowest level of intake of an indispensable Medicine (2002/2005) recommendations for a safe practical protein amino acid that achieves nitrogen balance or intake for selected age groups and physiological states. Reproduced that balances the irreversible oxidative loss with permission from WHO of the amino acid, without requiring major Safe protein changes in normal protein turnover and where level (g/kg/day) there is energy balance with a modest level of physical activity. For infants, children and preg- Group Age (years) UNU IOM nant and lactating women, the requirements for Infants 0.3–0.5 1.47 1.5 the amino acid will include the additional 0.75–1.0 1.15 1.1 amount of the amino acid needed for net protein Children 3–4 1.09 0.95 9–10 0.99 0.95 deposition by the infant, child or fetus and con- Adolescent 13–14 (girls) 0.94 0.85 ceptus and for the synthesis and secretion of 13–14 (boys) 0.97 0.85 milk proteins. Young adults 19+ 0.75 0.80 Elderly 0.75 0.80 The foregoing is an operational defi nition of Women: pregnant 2nd trimester +6 g daily ~1.1 3rd trimester +11 g daily ~1.1 requirement, as in the case of protein. Ideally, a func- lactating 0.6 months ~+16 g daily ~1.1 tional defi nition and determination of these require- 6–12 months 12 g daily ~1.1 ments inherently would be preferable. However, the Values are for proteins such as those of quality equal to a hen’s egg, choice and nature of the functional index or (indices) cow’s milk, meat, or fi sh. (such as maximum resistance to disease or enhanced physical performance) and its quantitative defi nition remain a challenge for future nutrition and health- related research. than this intake to maintain an adequate protein Determination nutritional status. In general, the approaches and methods that have It is worth emphasizing two points. First, the been most often used to determine specifi c indis- current UN recommendations shown in Table 4.8 pensable amino acid requirements are similar to apply to healthy individuals of all ages. However, it is those used for estimation of total protein needs, highly likely that the needs of sick or less healthy i.e., nitrogen excretion and balance and factorial patients would differ from and usually exceed those estimation. Thus, amino acid requirements have of healthy subjects. In this case, the values given in been assessed by nitrogen balance in adults, and by this table can be regarded only as a basis from which determining the amounts needed for normal growth to begin an evaluation of how disease and stress, and nitrogen balance in infants, preschool children, including surgery, affect the needs for dietary protein. and school-aged children. For infants, they have also Unfortunately, the quantitative needs for protein been approached by assessment of the intakes pro- (total nitrogen) in sick, hospitalized patients can be vided by or those supplied from intakes only very crudely approximated at this time. of good-quality proteins. In addition, factorial pre- Second, the values shown in Table 4.8 apply to dictions of the amino acid requirements of infants high-quality food proteins, such as eggs, milk, meat, and adults have been made. One such factorial and fi sh. The differing nutritional value of food pro- approach for use in adults includes the following teins will be considered below. assumptions. Defi nition and determination of ● The total obligatory nitrogen losses (those losses indispensable amino acid requirements occurring after about 4–6 days of adjustment to a Defi nition protein-free diet) are taken to be approximately It is possible to modify slightly the earlier defi nition 54 mg/kg nitrogen per day in an adult, or equiva- for the requirements for protein (nitrogen) for a spe- lent to 0.36 g protein/kg/day. cifi c, indispensable amino acid, which can be stated, ● The average amino acid composition of body pro- therefore, as: teins can be used to estimate the contribution made 66 Introduction to Human Nutrition

by each amino acid to this obligatory nitrogen output (equivalent, therefore, to the obligatory amino acid losses). ● At requirement intake levels, an absorbed amino [13C]-Phenylalanine (indicator) oxidation acid is used to balance its obligatory oxidative loss with an assumed effi ciency of about 70%. This predictive or factorial approach is analogous to the factorial method for estimating the total nitro- gen (protein) requirement of individuals at various ages (where various routes of nitrogen excretion and

nitrogen gains are summated and an effi ciency factor Indicator amino acid oxidation is used to estimate the intake needed to balance this Requirement intake summation). Test amino acid intake Tracer techniques (leucine or lysine) With advances in the routine measurement of stable Figure 4.9 Outline of the concept of the indicator amino acid oxida- isotope enrichment in biological matrices and the tion technique for estimation of indispensable amino acid require- expanded use of tracers enriched with these isotopes ments. Here the indicator is [13C]-phenylalanine and the dietary in human metabolic research, a series of tracer studies requirement is being estimated for either leucine or lysine. was begun at the Massachusetts Institute of Tech- nology, USA, in the early 1980s to determine amino acid requirements in adults. Since that time several an extensive use in studies of human amino acid research groups have used different paradigms in requirements. tracer-based studies of human amino acid require- None of these methods is without its limitations, but ments. These can be distinguished according to the at present the IAAO and IAAB approaches, involving choice of tracer and protocol design applied: tracer studies lasting for a continuous 24 hour day, ● studies involving the use of a labeled tracer of the would appear to be the “reference method” for esti- dietary amino acid being tested and with its rate amino acid requirements in adults. of oxidation (O) at various test intake levels [the direct amino acid oxidation (DAAO) technique, Indispensable amino acid e.g., [13C]-lysine as a tracer to determine the lysine requirement values requirement]; this technique has been used to assess There is still debate and uncertainty about the precise the requirements in adults for leucine, valine, lysine, requirements for amino acids in humans of all ages. threonine, and phenylalanine Three major sets of proposed amino acid require- ● studies involving use of an “indicator” tracer to ment values for healthy subjects should be noted in assess the status of indicator amino acid oxidation this text. First, there are the requirements proposed (IAAO) or indicator amino acid balance (IAAB) by the UN in 1985 for the various age groups, with varying levels of a test amino acid; examples which are presented in Table 4.9. Second, another of the IAAO and IAAB approaches are where the expert group in 1994 (International Dietary Energy rate of [13C]-phenylalanine oxidation (Figure 4.9) Consultancy Group; IDECG) also assessed the amino is measured or a [13C]-leucine balance determined acid needs of infants by using a factorial method and at varying levels of lysine intake to estimate the these turned out to be much lower than those shown lysine requirement in Table 4.9 for infants. It should be noted, however, ● kinetic studies designed to assess the retention of that the 1994 IDECG values approximate the aver- protein during the postprandial phase of amino age requirements, whereas the requirement intakes acid metabolism, using [13C]-leucine as a tracer: the derived from estimates of breast milk intake (shown postprandial protein utilization (PPU) approach; in Table 4.9) would be expected to be well above the this last and promising approach has not yet found requirement for virtually all infants and certainly well Nutrition and Metabolism of Proteins 67

Table 4.9 1985 FAO/WHO/UNUa estimates of amino acid requirements at different ages (mg/kg/day). Reproduced with permission from WHO

Amino acid Infants (3–4 months) Preschool children (2 years) School boys (10–12 years) Adults

Histidine 28 ? ? [8–12] Isoleucine 70 31 28 10 Leucine 161 73 44 14 Lysine 103 64 44 12 Methionine and cystine 58 28 22 13 Phenylalanine and tyrosine 125 69 22 14 Threonine 87 37 28 7 Tryptophan 17 12.5 3.3 3.5 Valine 93 38 25 10 Total 714 352 216 84 Total per g proteinb 434 320 222 111 a FAO/WHO/UNU. Technical Report Series No. 724. Geneva: World Health Organization, 1985. Data taken from Table 4, p. 65, and Table 38, p. 121, and based on all amino acids minus histidine. b Total mg per g crude protein.

above the mean or average requirement. It is not are shown in Table 4.10. The more recent values are surprising, therefore, that the UN and IDECG require- generally very different from the recommendations ments disagreed. This also shows why recommenda- made in 1985. It is important to remain alert in nutri- tions by different national and international expert tion, as these texts will emphasize. At the time of groups differ; they interpret the same data differently, writing the original text, a new UN expert group was use different data, and also may choose to set differ- meeting to consider all of the new data that had been ent criteria for judging the adequacy of intake. accumulated in the past 20 years. It was expected that Further, as is characteristic of various estimates of new recommendations would be made in 2002 or human nutrient requirements in general, it must be 2003, but, since they have not been published, the appreciated that the values given in Table 4.9 are recommendations by the Institute of Medicine of the based on limited data; the values for the preschool US Academies of Science have been added as a sepa- children are derived from a single set of investigations rate column in Table 4.10. The important point, carried out at the Institute for Central America and however, is that all is not set in stone. Nutritional Panama, while those for the school-aged children knowledge continues to advance, and with it the rec- come from studies conducted by a single group of ommendations must change or at least be responsive investigators in Japan. Those for adults are based pri- to this new information. marily on the nitrogen balance studies in men carried out in the 1950s and 1960s. There are multiple reasons for questioning the precise reliability and nutritional 4.7 Meeting protein and amino signifi cance of the adult values, and they include the acid needs facts that adult amino acid requirement values (Table Knowledge of the requirements for the specifi c indis- 4.9) are greatly infl uenced by: pensable amino acids and for total protein provides ● the inappropriate experimental design used earlier the basis for evaluating the relative capacity (or for estimation of requirements quality) of individual protein foods or mixtures of ● the inadequacy of the nitrogen balance technique food protein sources to meet human amino acid and the criterion of nitrogen balance that has been requirements. used to judge the nutritional adequacy of the levels The two major determinants of the nutritional of amino acid intake tested. quality of food proteins are:

Therefore, some contemporary and newly pro- ● the content of indispensable amino acids in the posed amino acid requirement estimates for adults protein 68 Introduction to Human Nutrition

Table 4.10 The earlier and three contemporary suggested patterns of amino acid requirements in healthy adults

Amino Acid United Nationsa 1985 University of Surreyb 1999 MITc 2000 IOMd 2002

Isoleucine 10e (13)f 18 (30) 23 (35) (25) Leucine 14 (19) 26 (44) 23 (65) (55) Lysine 12 (16) 19 (31) 30 (50) (51) Methionine and cystine 13 (17) 16 (27) 13 (25) (25) Phenylalanine and tyrosine 14 (19) 20 (33) 39 (65) (47) Threonine 7 (9) 16 (26) 15 (25) (27) Tryptophan 3.5 (5) 4 (6) 6 (10) (7) Valine 10 (13) 14 (23) 20 (35) (32) a FAO/WHO/UNU. Technical Report Series No. 724. Geneva: World Health Organization, 1985. b Millward DJ. The nutritional value of plant-based diets in relation to human amino acid and protein requirements. Proc Nutr Soc 1999; 58: 249–260. c Young VR, Borgonha S. Nitrogen and amino acid requirements: the Massachusetts Institute of Technology Amino Acid Requirement Pattern. J Nutr 2000; 130: 1841S–1849S, reproduced with permission of the American Society of Nutrition. d US National Academies of Science Institute of Medicine. e Values expressed as mg/kg/d. f Values expressed as mg amino acid/protein required for effectively meeting total protein and amino acid needs.

● the extent to which the indispensable amino acids amino acids have been infused intravenously for pro- are available to the host metabolism. longed periods. This labels the host proteins so that the 15N labeling of ileal proteins allows the calculation Digestibility and intestinal amino of the endogenous contribution of the luminal protein acid metabolism pool. By and large, the results of all these studies lead Traditionally, the assessment of the availability of to the same conclusion; namely, that the true digest- dietary proteins and amino acids under practical con- ibility of most dietary proteins is very high and that ditions has been based on “apparent digestibility,” at least 50% of the fecal nitrogen is derived from host i.e., the difference between nitrogen intake and fecal metabolism rather than from the diet. nitrogen output. However, for two reasons, this Most of the evidence favors the conclusion that method is unsatisfactory for the precise estimation of there is an almost complete digestion of most dietary the digestibility of individual amino acids. First, fecal proteins in the small bowel. It is also quite clear that nitrogen consists largely of bacterial protein, and a considerable amount of amino acid metabolism because the composition of bacterial protein differs occurs in the tissue of the splanchnic bed, in general, markedly from that of common dietary proteins, it and in the intestinal mucosa, in particular, before the gives very little information on the digestibility of amino acids, liberated from food proteins during the different food-derived amino acids. Second, the bac- digestive process, reach organs such as the liver, terial nitrogen is not only derived from undigested kidneys, and skeletal muscles. Calculations based on protein. This is because proteins secreted into the recent isotopic studies suggest that intestinal amino intestinal lumen, as well as the urea nitrogen that has acid utilization (both from the diet and via the blood diffused from the blood, are important contributors supply to the intestine; the mesenteric arterial circula- to colonic nitrogen fl ow. Studies in both animals and tion) can account for as much as 50% of the body’s humans using 15N-labeled amino acids suggest that at utilization of amino acids. It is also important to note least 50% of the fecal nitrogen is derived from the that the degree to which individual amino acids are body rather than directly from undigested dietary utilized by the gut varies markedly (Table 4.11). protein. Among the indispensable amino acids, threonine uti- Recently, 15N-labeled dietary proteins have been lization is particularly high and virtually all of the given to adults and by measuring the fl ow of 15N from dietary glutamate and aspartate are utilized within the the terminal ileum it is possible to calculate the “true” mucosa. In addition, the magnitude of splanchnic digestibility of the dietary source. There have also amino acid metabolism varies with age, being appar- been a number of studies in pigs in which 15N-labeled ently greater in infants and also perhaps in the elderly. Nutrition and Metabolism of Proteins 69

This can affect the effi ciency with which the amino The nutritional signifi cance of these differences acids derived from the protein ingested are used to can be assessed in a number of ways. One useful support overall body nitrogen and amino acid homeo- approach is an amino acid scoring procedure that stasis and balance. compares the content of amino acids in a protein with a reference human amino acid requirement pattern. Protein nutritional quality In 1991 a UN Expert Consultation reviewed the Not all proteins have the same capacity to meet the appropriate methods for measuring quality of food physiological requirements for total nitrogen and the proteins for the nutrition of human populations. This indispensable amino acids. The concentration and consultation concluded that the most appropriate availability of the individual indispensable amino method available was the protein digestibility- acids are major factors responsible for the differences corrected amino acid score (PDCAAS) method, and in the nutritive values of food proteins. Thus, the it was recommended for international use. This amino content and balance of indispensable amino acids acid scoring procedure, including a correction for differ among plant and animal protein foods. For the digestibility, uses the amino acid requirement pattern present purpose a summary is given in Table 4.12, for a 2–5 year old child (as shown in Table 4.9). This listing the four indispensable amino acids that are is the reference amino acid requirement pattern for most likely to be limiting, or in shortest supply and this purpose, expressing the amino acid requirement especially in food proteins of plant origin. As can be in relation to the total protein requirement. seen, lysine is present in a much lower concentration The PDCAAS is estimated from the following in all the major plant food groups than in animal equation: protein foods and is most frequently the most limit- ing amino acid. Concentration of most limiting, digestibility-correccted amino acid in a test protein PDCAAS = Table 4.11 The uptake of dietary amino acids by the visceral tissues Concentration of that amiino acid in the 1991 FAO WHO amino Percentage of intake acid scoring reference ppattern Utilization by the liver Utilization by the (preschool child: see Table 4.9) Amino acid and gut (human) gut (piglet) In addition to establishing the amino acid reference Leucine 26 37 pattern for use in the PDCAAS method, the UN Lysine 32 45 Consultation considered the procedures for measur- Phenylalanine 39 53 Threonine No data 65 ing and estimating amino acids and digestibility. This Glutamine 53 50 approach offers considerable benefi ts over that of Glutamate 88 95 animal bioassays, which traditionally have been used

Table 4.12 The amino acid content of different food protein sources

mg/g protein (mean ± SD)

Food source Lysine Sulfur amino acids Threonine Tryptophan

Legumes 64 ± 10 25 ± 3 38 ± 3 12 ± 4 31 ± 10 37 ± 4 32 ± 4 12 ± 2 Nuts, 45 ± 14 46 ± 17 36 ± 3 17 ± 3 45 ± 12 27 ± 6 29 ± 7 11 ± 2 Animals foods 85 ± 938 4412

From Young VR, Scrimshaw NS, Pellett PL. Signifi cance of dietary protein source in human nutri- tion: Animal and/or plant proteins? In: Waterlow JC, Armstrong DG, Fowder L, Riley, eds. Feeding a World Population of More Than Eight Billion People. Oxford University Press in association with the Rank Prize Funds, Oxford, 1998: 206. 70 Introduction to Human Nutrition to assess the quality of food protein in human diets. protein and amino acid needs. A listing of some calcu- An important benefi t is that the PDCAAS approach lated PDCAAS values for selected food protein sources uses human amino acid requirements as the basis of is given in Table 4.13 and a worked example for a evaluation, which ensures that appropriate levels of mixture of food proteins is presented in Table 4.14. indispensable amino acids will be provided in the diet. The development of an internationally derived In addition, use of the proposed amino acid scoring procedure for evaluating protein quality using the procedure facilitates an evaluation of blending of amino acid scoring concept is a step that had long foods to optimize nitrogen utilization and meet been required. This PDCAAS procedure can be modi- fi ed as new knowledge about specifi c amino acid requirements emerges, as the determination of avail- ability of dietary amino acids is improved, and as the Table 4.13 Protein digestibility-corrected amino acid score (PDCAAS) of wheat, rice, maize, sorghum, and millet factors affecting digestibility and availability are better understood. For the present, the PDCAAS procedure Protein source PDCAAS would appear to be very useful for evaluating the nutritional quality of human food protein sources. Wheat 40 (L) Rice 56 (L) Major sources of food proteins in the diet Maize 43 (L) Sorghum 33 (L) The relative proportions in the diet of food proteins of Millet 53 (L) >100 (S) animal and plant origin differ according to geographi- cal region and other socioeconomic and cultural From Young VR, Scrimshaw NS, Pellett PL. Signifi cance of dietary factors. Broadly, animal protein foods account for 60– protein source in human nutrition: Animal and/or plant proteins? In: 70% of the total protein intake in the developed regions Waterlow JC, Armstrong DG, Fowder L, Riley, eds. Feeding a World (Table 4.15). In contrast, plant proteins make up about Population of More Than Eight Billion People. Oxford University Press in association with the Rank Prize Funds, Oxford, 1998: 207. 60–80% of the total protein intake in developing L, lysine fi rst limiting amino acid; S, sulfur amino acids (methionine regions, with cereals being the dominant source in this and cystine). case. Given the differences in amino acid content of

Table 4.14 Worked example of a protein digestibility-corrected amino acid score (PDCAAS) for a mixture of wheat, chickpea, and milk powder

Analytical data (mg/g protein) Quantities in mixture (mg)

Weight (g) Protein (g/100 g) Lys SAA Thr Trp Digestibility factor Protein (g) Lys TSAA Thr Trp A × B = P AB CDEFG 100 P × CP × DP × EP × F

Wheat 350 13 25 35 30 11 0.85 45.5 1138 1593 1365 501 Chickpea 150 22 70 25 42 13 0.80 33.0 2310 825 1386 429 Milk powder 50 34 80 30 37 12 0.95 17.0 1360 510 629 204 Totals 95.5 4808 2928 3380 1134 Amino acids mg/g protein 50 31 35 12 (total for each amino acid/total protein) Reference scoring pattern used 58 25 34 11 Amino acids scoring for mixture amino acid/g 0.86 1.24 1.03 1.09 protein divided by reference pattern Weighted average protein digestibility sum of [protein × factor (P × G)] 0.85 divided by protein total Score adjusted for digestibility (PDCAAS) (0.85 × 0.86) 0.73 (or 73%) with lysine limiting

From Food and Agriculture Organization of the United Nations. Protein Quality Evaluation. FAO Food and Nutrition Paper 51. FAO, Rome, 1991: table 10. Lys, lysine; SAA, sulfur amino acids; Thr, threonine; Trp, tryptophan; TSAA, total sulfur amino acids. Nutrition and Metabolism of Proteins 71

Table 4.15 Protein supplies per caput per day for selected regions

Plant protein Animal protein protein

Region Total (g) % Total (g) % Total (g) % Total protein (g)

World 26 36 46 64 33 46 72 Developing regions Africa 11 20 46 80 31 54 58 Asia 16 25 49 75 36 56 65 Latin America 32 45 39 55 25 36 70 Developed regions North America 72 64 41 36 25 22 113 Western Europe 62 60 41 40 25 24 103 Oceania 71 69 32 31 19 19 102

From Young VR, Scrimshaw NS, Pellett PL. Signifi cance of dietary protein source in human nutri- tion: Animal and/or plant proteins? In: Waterlow JC, Armstrong DG, Fowder L, Riley, eds. Feeding a World Population of More Than Eight Billion People. Oxford University Press in association with the Rank Prize Funds, Oxford, 1998: 212.

Table 4.16 Calculated mean values per caput for the availability of specifi c indispensable amino acids in developed and developing regions

Amino acid per day (mg) mg/g protein

Region Lys SAA Try Thr Lys SAA Try Thr

Developinga 2947 2160 693 2204 49 36 11 37 Developed and transitionalb 6149 3619 1177 3799 64 38 12 40

From Young VR, Scrimshaw NS, Pellett PL. Signifi cance of dietary protein source in human nutri- tion: Animal and/or plant proteins? In: Waterlow JC, Armstrong DG, Fowder L, Riley, eds. Feeding a World Population of More Than Eight Billion People. Oxford University Press in association with the Rank Prize Funds, Oxford, 1998: 212. a Data for 61 countries. b Data for 29 countries. SAA, sulfur amino acids; TSAA, total sulfur amino acids. food proteins mentioned above it is not surprising that ground. Various environmental, physiological, psy- there are distinct differences in the intakes of the indis- chological, and pathological infl uences affect the vari- pensable amino acids by different population groups ability in physiological requirements for nutrients worldwide. An example of such differences is given in among individuals (Table 4.17). For example, as Table 4.16. As already noted, the four amino acids of already discussed, the growing infant or child requires greatest importance and those most likely to be most higher nutrient intakes per unit of body weight than limiting in intake, relative to requirements, are lysine, does the adult. Besides energy, for which the daily the sulfur amino acids (methionine and cystine), tryp- requirement declines with age because of reduced tophan, and threonine. physical activity, it appears that the nutrient needs of healthy aged subjects do not differ signifi cantly from 4.8 Factors other than diet affecting those of young adults. Nevertheless, a characteristic protein and amino acid requirements of aging is an increased incidence of disease and mor- bidity, which is likely to be far more important than Not everyone of the same age, body build, and gender age per se in determining practical differences between has the same nutrient requirements. These differences the nutrient requirements of younger adults and may be due, in part, to variations in genetic back- elderly people. 72 Introduction to Human Nutrition

Table 4.17 Agent, host, and environment factors that affect protein increased nitrogen retention seen during this period and amino acid requirements and the nutritional status of the is greater than that measured during the preincuba- individual tion phase, and its duration is much longer than Agent (dietary) factors the catabolic period. This may be due, in part, to Chemical form of nutrition (protein and amino acid source) the effect of protein depletion antedating an acute Energy intake episode, which may be the case in poor communities. and preparation (may increase or decrease dietary needs) However, in spite of the potential for disease states to Effect of other dietary constituents increase protein and amino acid needs there are too Host factors few studies that help to assess precisely their quantita- Age tive infl uence on nutrient utilization and dietary Sex requirements. Genetic makeup Pathologic states Drugs 4.9 Perspectives on the future Infection Physical trauma The purpose of this chapter was to provide a general Chronic disease, cancer overview of human protein and amino acid metabo- Environmental factors Physical (unsuitable housing, inadequate heating) lism and a basis for an improved appreciation of the Biologic (poor sanitary conditions) metabolic determinants of the requirements for Socioeconomic (poverty, dietary habits and food choices, physical protein (nitrogen) and for specifi c amino acids. With activity) the recent beginning of the postgenome era, func- tional genomics, proteomics, and metabolomics will take on an increasingly important basic and applied research focus in biology. Thus, it will be even more Thus, superimposed infection, altered gastro- critical for students to understand the physiology of intestinal function, and metabolic changes that often human protein metabolism at its various levels of accompany chronic disease states would all be biological complexity (cell, organ, and whole body) expected to reduce the effi ciency of dietary nitrogen and its nutritional corollaries. and amino acid utilization. The metabolic response There are certain areas of research in protein nutri- to acute infection in healthy young men has been tion where more knowledge will equip nutritionists characterized in experiments involving different types to make the best use of available food supplies. An of intracellular infection, and involves an increased example is the infl uence of the ratio of total essential loss of body nitrogen, together with increased losses or indispensable amino acids to total nitrogen and of several other nutrients including potassium, mag- amino acid requirements for different physiological nesium, phosphorus, and vitamin C. This increased states. Another is the need for a functional defi nition loss clearly implies increased needs for nitrogen, of protein requirements (e.g., indices) for maximum amino acids, and other nutrients. resistance to disease and enhanced physical perfor- In addition to the catabolic response of body nitro- mance. These are some of the challenges facing nutri- gen metabolism to infection and trauma, there is a tionists in the future. It is hoped that this chapter will corresponding anabolic component that is of major serve as an appropriate catalyst for further learning in importance during recovery from these stressful con- this area of human nutrition. ditions. Anabolic responses occur not only during recovery but also in the early phase of illness, when Acknowledgment anabolism is associated with increased production of immunocompetent cells such as phagocytes and other This chapter has been revised and updated by Naomi leukocytes, and the induction of several tissue enzymes K Fukagawa and Yong-Ming Yu based on the original and immunoglobulins. chapter by Vernon R Young and Peter J Reeds. It is During recovery from infection two characteristics dedicated to their memory. For more information on of the anabolic period that follows are that the this topic visit www.nutritiontexts.com Nutrition and Metabolism of Proteins 73

References Further reading

FAO/WHO/UNU. Energy and protein requirements. Report of a Cohen PP. Regulation of the ornithine-urea cycle enzymes. In: Joint FAO/WHO/UNU Expert Consultation.Technical Report Waterlow JC, Stephen JML, eds. Nitrogen Metabolism in Man. Series No. 724. World Health Organization, Geneva, 1985, Applied Science, London, 1981: 215. 1–206. Food and Agriculture Organization of the United Nations. Protein Institute of Medicine, US National Academies of Science. Dietary Quality Evaluation. Food and Nutrition Paper 51. FAO: Rome, Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, 1991. Cholesterol, Protein, and Amino Acids. Institute of Medicine, US Garrow JS, Halliday D, eds. Substrate and Energy Metabolism in National Academies of Science, Washington, DC, 2002. Man. John Libbey, London, 1985. Millward DJ. The nutritional value of plant-based diets in relation Lehninger AL, Nelson DL, Cox MM. Principles of Biochemistry, 2nd to human amino acid and protein requirements. Proc Nutr Soc edn. Worth, New York, 1993. 1999; 58: 249–260. Munro HN, ed. Mammalian Protein Metabolism, vol. III. Academic Munro HN, Allison JB, eds. Mammalian Protein Metabolism, vols I Press, New York, 1969. and II. Academic Press, New York, 1964. Munro HN, ed. Mammalian Protein Metabolism, vol. IV. Academic Reeds PJ. Dispensable and indispensable amino acids for humans. Press, New York, 1970. J Nutr 2000; 130: 1835S–1840S. Waterlow JC, Garlick PJ, Millward DJ. Protein Turnover in Waterlow JC. The mysteries of nitrogen balance. Nutr Res Rev 1999; Mammalian Tissues and in the Whole Body. North-Holland, 12: 25–54. Amsterdam, 1978. Young VR, Borgonha S. Nitrogen and amino acid requirements: the Wolfe RR. Radioactive and Stable Isotope Tracers in Biomedicine: Massachusetts Institute of Technology Amino Acid Requirement Principles and Practice of Kinetic Analysis. Wiley-Liss, New York, Pattern. J Nutr 2000; 130: 1841S–1849S. 1992. Young VR, Scrimshaw NS, Pellett PL. Signifi cance of dietary protein Young VR, Yu Y-M, Fukagawa NK. Energy and Protein Turnover. source in human nutrition: Animal and/or plant proteins? In: In: Kinney JM, Tucker HN, eds. Energy and Protein Turnover in Waterlow JC, Armstrong DG, Fowder L, Riley, eds. Feeding a Energy Metabolism: Tissue Determinants and Cellular Corollaries. World Population of More Than Eight Billion People. Oxford Raven Press, New York, 1992: 439–466. University Press in association with the Rank Prize Funds, Oxford, 1998, 205–222. 5 Digestion and Metabolism of Carbohydrates

John Mathers and Thomas MS Wolever

Key messages

• Carbohydrates are the single most abundant and economic methane in the large bowel. Absorbed SCFAs are metabolized in sources of food energy in the human diet, constituting 40–80% colonic epithelial, hepatic, and muscle cells. of total energy intake in different populations. • For optimum function of the nervous system and other cells, • Carbohydrates are classifi ed according to their degree of poly- blood glucose concentrations are tightly controlled by a group of merization into sugars, , and polysaccharides – hormones (insulin in the absorptive phase; glucagon, epine- the last consisting of with different degrees of resistance phrine, and cortisol in the postabsorptive phase), utilizing to digestion – and dietary fi bers or nonstarch polysaccharides. several possible metabolic pathways for glucose anabolism and • Glycemic carbohydrates are digested (hydrolyzed by enzymes) to catabolism. sugars (monosaccharides) in the small bowel and absorbed and • Intakes of optimum amounts of different types of carbohydrates metabolized. are associated with good health through effects on energy • Nonglycemic carbohydrates are fermented in varying degrees to balance, digestive functions, blood glucose control, and other risk short-chain fatty acids (SCFAs), carbon dioxide, hydrogen, and factors for several chronic diseases.

5.1 Introduction: carbohydrates in foods composition and the type of linkage between sugar residues. Examples of food carbohy- Carbohydrates are one of the four major classes of drates and an overview of their digestive fates are biomolecules and play several important roles in all given in Table 5.1. life forms, including: From birth, carbohydrate provides a large part of the energy in human diets, with approximately 40% ● sources of metabolic fuels and energy stores of the energy in mature breast milk being supplied as ● structural components of cell walls in plants and of lactose. After weaning, carbohydrates are the largest the of arthropods source (40–80%) of the energy in many human diets, ● parts of RNA and DNA in which ribose and with most of this derived from plant material except deoxyribose, respectively, are linked by N-glycosidic when milk or milk products containing lactose are bonds to purine and pyrimidine bases consumed. The carbohydrate contents of some vege- ● integral features of many proteins and lipids table dishes are summarized in Table 5.2. (glycoproteins and glycolipids), especially in cell membranes where they are essential for cell–cell recognition and molecular targeting. 5.2 Digestive fate of dietary carbohydrates Carbohydrates are very diverse molecules that can be classifi ed by their molecular size (degree of poly- As with other food components, the digestive fate of merization or DP) into sugars (DP 1–2), oligosac- particular carbohydrates depends on their inherent charides (DP 3–9), and polysaccharides (DP > 9). The chemical nature and on the supramolecular struc- physicochemical properties of carbohydrates and tures within foods of which they are a part. To be their fates within the body are also infl uenced by their absorbed from the gut, carbohydrates must be broken

© 2009 J Mathers and TMS Wolever. Digestion and Metabolism of Carbohydrates 75

Table 5.1 Classes of food carbohydrates and their likely fates in the human gut

Class DP Example Site of digestion Absorbed molecules

Monosaccharides 1 Glucose Small bowel Glucose 1 Fructose Small bowela Fructose 2 Sucrose Small bowel Glucose + fructose 2 Lactoseb Small bowel Glucose + galactose Oligosaccharides 3 Raffi nose Large bowel SCFA 3–9 Large bowel SCFA Polysaccharides >9 Starches Predominantly small bowelc Glucose >9 Nonstarch polysaccharides Large bowel SCFA a Except where very large doses are consumed in a single meal. b Except in lactose-intolerant subjects, in whom lactose fl ows to the large bowel. c Some starch escapes small bowel digestion (resistant starch). In all these cases, the carbohydrate entering the large bowel becomes a substrate for bacterial fermentation to short-chain fatty acids (SCFAs). DP, degree of polymerization. down to their constituent monosaccharide units, and protein-1 (SGLT1), a process that is powered by Na+/ a battery of hydrolytic enzymes capable of splitting K+-ATPase on the basolateral membrane (Figure 5.1). the bonds between sugar residues is secreted within In contrast, fructose is absorbed by facilitated trans- the mouth, from the pancreas, and on the apical port via the membrane-spanning GLUT5 protein. A membrane of enterocytes. While these carbohydrases member of the same family of transporter proteins, ensure that about 95% of the carbohydrate in most GLUT2, is the facilitated transporter on the basolat- human diets is digested and absorbed within the eral membrane which shuttles all three monosaccha- small intestine, there is considerable variation in rides from the enterocyte towards the blood vessels bioavailability between different carbohydrate classes linking with the portal vein for delivery to the liver. and between different foods. Carbohydrates that are The capacity of the human intestine for transport digested to sugars and absorbed as such in the small of glucose, galactose, and fructose is enormous – esti- bowel are called “glycemic” carbohydrates. mated to be about 10 kg per day – so that this does not limit absorption in healthy individuals. Carbohy- Hydrolysis in the mouth and small bowel drate is usually caused by an inherited The major carbohydrase secreted by the salivary or acquired defect in the brush border oligosacchari- glands and by the acinar cells of the pancreas is the dases. More than 75% of human adults are lactose endoglycosidase α-amylase, which hydrolyzes (digests) intolerant because of a loss (possibly genetically internal α-1,4-linkages in amylose and amylopectin determined) of lactase activity after weaning (primary molecules to yield maltose, maltotriose, and dextrins. lactose intolerance). In such individuals, ingestion of These oligosaccharides, together with the food disac- more than very small amounts of lactose leads to the charides sucrose and lactose, are hydrolyzed by spe- passage of the sugar to the large bowel, where it is cifi c oligosaccharidases expressed on the apical fermented to produce short-chain fatty acids (SCFAs) membrane of the epithelial cells that populate the and gases as end-products. The appearance of hydro- small intestinal villi. Sucrase–isomaltase is a glycopro- gen in the breath after ingestion of lactose is the basis tein anchored via its amino-terminal domain in the for diagnosis of malabsorption of this carbohydrate. apical membrane that hydrolyzes all of the sucrose Diseases of the intestinal tract, such as protein-energy and most of the maltose and isomaltose. The resulting malnutrition, intestinal infections, and celiac disease, monomeric sugars are then available for transport which reduce expression of lactase on the enterocyte into the enterocytes. apical membrane, can result in secondary lactase insuffi ciency. Sucrase–isomaltase activity, which rises Absorption and malabsorption in rapidly from the pylorus towards the jejunum and the small bowel then declines, is inducible by sucrose feeding. About Glucose and galactose are transported across the 10% of Greenland Eskimos and 0.2% of North Amer- apical membrane by the sodium–glucose transport icans have sucrase–isomaltase defi ciency. A missense 76 Introduction to Human Nutrition

Glucose and Fructose galactose Naϩ Gut lumen NSPs 1.2 0.8 3.6 1.1 1.42.4 0.2 0.8 3.2 1.0 4.50.9 1.1 0.1

Apical membrane

Tight junction 0 0.8 0 0.4

Fructose Glucose Naϩ Basolateral and 0.1 1.7 Tr 0.1 Tr 0 Tr 1.8 Tr membrane galactose

ϩ ADP K ϩ Pi

ATP Galactose Sucrose Maltose Lactose Oligosaccharides

Glucose and Fructose galactose Kϩ ϩ 0.1 0 1.0 0 0 1.4 1.7 0 1.9 0 Tr 1.7 Blood Na

GLUT5 GLUT2

SGLT1 Naϩ/Kϩ-ATPase

Figure 5.1 Sugar transporters on enterocytes, showing the transport 4.3 1.3 1.4 0 1.6 0 2.3 0.2 0.2 0 0.1 1.4 0.5 0.4of 0 glucose and 0.6 galactose 0 across the apical membrane. 1999); Tr, trace. 1999); Tr, 10.4 2.2 0.1 0.1 0 0.1 23.0 2.2 0.6 0.6 0 0.9 in SGLT1 is responsible for the very rare glucose–galactose malabsorption syndrome, but such individuals absorb fructose well. In up to 60% of adults, the capacity for facilitated diffusion of fructose appears to be limited, resulting in symptoms of “intestinal distress” when challenged by consumption 52.7 21.3 18.7 1.2 Tr Water Carbohydrate Starch sugars Total Glucose Fructose 77.7 7.6 0.4 5.5 1.9 of 50 g fructose.

(1992). Reproduced with permission from HMSO. 5.3 Glycemic carbohydrates

et al. The rate of uptake of glucose (and other sugars) from the gut is determined by the rate of hydrolysis of oli- gosaccharides and polysaccharides that are suscepti- ble to pancreatic and brush border enzymes. In addi- Curry, chickpea Curry, Shepherd’s pie, 71.7Data from Holland 15.8 14.0 Chili, beans and 72.6 13.1 7.9 Table 5.2 Table g) of some vegetable dishes Carbohydrate composition (g/100 Dish Bhaji, okra Bhaji, Cannelloni, spinachFlan, and mushroom cheese and tomatoPizza, 73.4 49.1 12.6 18.7 51.0 25.2 16.4 NSPs, nonstarch polysaccharides (Englyst method; Englyst et al. NSPs, tion to the primary structure of the polymers, many Digestion and Metabolism of Carbohydrates 77 factors intrinsic to the ingested foods and to the con- complete oxidation of glucose to carbon dioxide and sumer infl uence these rates, including: water occurs under aerobic conditions through the reactions of the glycolytic pathway (in the cell’s cyto- ● food factors plasm), the Krebs cycle, and oxidative phosphoryla- ● particle size tion (in the mitochondrion). The overall reaction can ● macrostructure and microstructure of food, be summarized stoichiometrically as: especially whether cell walls are intact

● amylose–amylopectin ratio of starches C6H12O6 + 6O2 → 6CO2 + 6H2O ● lipid content of food Approximately 40% of the free energy (ΔG) released ● presence (or otherwise) of enzyme inhibitors by this transformation is captured by the production ● consumer factors of ATP (38 moles of ATP per mole of glucose oxi- ● degree of comminution in the mouth dized), which is used for a wide variety of purposes, ● rate of gastric emptying including powering muscle contraction, transporting ● small bowel transit time. substances across membranes against a concentration All three main sugars absorbed from the gut gradient, and synthesis of cell . The (glucose, galactose, and fructose) are transported via remainder of the free energy is released as heat. the portal vein to the liver (glucose concentrations in When the demand for oxygen exceeds supply, as in the portal vein after a meal can rise to almost 10 mM), muscle during intense exercise, anaerobic glycolysis but only glucose appears in signifi cant concentrations produces lactic acid as a major end-product. The rela- in the peripheral circulation. Most of the galactose tive lack of oxygen means that oxidative phosphoryla- and fructose is removed during fi rst pass through the tion cannot keep up with the supply of reduced liver via specifi c receptors on hepatocytes, so that the dinucleotides and, for glycolysis to proceed, NADH blood concentration of these sugars rarely exceeds must be recycled back to NAD+. This is achieved by 1 mM. Within the hepatocytes, galactose is converted the reaction: to galactose-1-phosphate by the enzyme galactoki- + + Pyruvate + NADH + H → Lactate + NAD nase and then to glucose-1-phosphate in three further steps. Fructose is also phosphorylated in hepatocytes which is catalyzed by the enzyme lactate dehydro- (by fructokinase) to fructose-1-phosphate, which is genase. Anaerobic glycolysis provides some or all of subsequently split by aldolase B to yield one molecule the ATP needs for some cells and tissues; for example, of each of the glycolytic intermediates dihydroxyace- erythrocytes, white blood cells, lymphocytes, the tone phosphate and glyceraldehyde. Although the kidney medulla, and eye tissues. The lactate released liver removes some glucose, using the bidirectional from tissues undergoing anaerobic glycolysis is taken transporter GLUT2, most is transported in the periph- up by other tissues that have a high number of mito- eral circulation for utilization by muscle, adipose, and chondria per cell, such as heart muscle, in which the other tissues. lactate is converted back to pyruvate and then enters the Krebs cycle via acetyl coenzyme A. Metabolic utilization of carbohydrate In hepatic and muscle cells some glucose is con- Peripheral tissues utilize glucose and the above-men- verted to glycogen in the glycogenesis pathway. Gly- tioned intermediates from fructose and galactose via cogen is a readily mobilized storage form of glucose glycolysis and the citric acid or Krebs cycle pathways. residues linked with α-1,4-glycosidic bonds into a Glycolysis, a sequence of reactions in which glucose large, branched polymer. Glycogen is a reservoir of is converted to pyruvate, with concomitant produc- glucose for strenuous muscle activity and its synthesis tion of ATP, is the prelude to the and and degradation are important for the regulation of electron transport chain, which together release the blood glucose concentrations. energy contained in glucose. Under aerobic condi- tions pyruvate enters mitochondria, where it is com- Regulation of blood glucose concentration pletely oxidized to carbon dioxide and water. If the The exocrine pancreas (and other tissues) is primed supply of oxygen is limited, as in actively contracting to expect a rise in blood glucose concentration by muscle, pyruvate is converted to lactate. Therefore, peptide hormones such as gastric inhibitory peptide 78 Introduction to Human Nutrition

(GIP) that are secreted from enteroendocrine cells tions in T1DM requires the exogenous supply of within the mucosa of the small bowel. As the glucose insulin by injection. Implanted insulin minipumps or concentration in blood rises above 5 mM after a meal, pancreatic β-cells may offer alternative forms of treat- these peptide hormones amplify the response of the ment in the future. Symptoms of type 1 diabetes β-cells of the endocrine pancreas, resulting in the dis- include the presence of glucose in urine, passage of charge of the hormone insulin from secretory gran- large volumes of urine, body weight loss, and, in ules which fuse with the cell membrane. Insulin has extreme cases, ketosis (excess production of acetone, several effects on metabolism, including facilitating acetoacetate, and β-hydroxybutyrate). Although there the transport, by GLUT4, of glucose into adipocytes is good evidence of genetic predisposition to T2DM, and muscle cells. expression of the disease is due mainly to lifestyle In healthy people, blood glucose concentration (excess energy intakes and low physical activity), (glycemia) is homeostatically controlled within a resulting in obesity, especially when the extra fat is fairly narrow range. It seldom falls below about 5 mM, accumulated on the trunk. The early stages of T2DM even after a prolonged fast, and returns to this value are characterized by insulin insensitivity/resistance, within a couple of hours of a meal. In the absence of i.e., failure of the tissues to produce a normal response uptake from the gut (the postabsorptive state), about to insulin release that can be seen as relatively wide 8 g glucose per hour is provided for those tissues with swings in blood glucose concentrations following a an obligatory demand for glucose – namely, the brain, carbohydrate-containing meal. Raised blood glucose red blood cells, mammary gland, and testis – by concentration sustained for several years is believed breakdown of stores of glycogen in the liver and to be fundamental to the spectrum of complications, muscle and by gluconeogenesis. The brain of an adult including macrovascular () and micro- has a glucose requirement of about 120 g/day. The vascular diseases and problems with the kidneys amount readily available in glycogen approximates (nephropathy), nerves (neuropathy), and eyes (reti- 190 g. In long periods of fasting and starvation glucose nopathy and cataract) experienced by diabetics. must be formed from noncarbohydrate sources by a process known as gluconeogenesis. Gluconeogenesis Dietary management of blood occurs in the liver (responsible for about 90% of glucose concentration gluconeogenesis) and kidney and is the synthesis of Glycemic index glucose from a range of substrates including pyruvate, As an aid to the dietary management of blood glucose lactate, glycerol, and amino acids. Amino acids are concentrations in diabetics, Jenkins and colleagues derived by catabolism of the body’s proteins. All (1981) introduced the concept of the glycemic index amino acids, with the exceptions of lysine and leucine, (GI), which provides a means of comparing quanti- are glucogenic. Triacylglycerols (from adipose tissue) tatively the blood glucose responses (determined are catabolized to release glycerol. These gluconeo- directly by in vivo experiments) following ingestion genic processes are triggered by a fall in blood glucose of equivalent amounts of digestible carbohydrate concentration below about 5 mM and are signaled to from different foods. When a range of carbohydrate- the tissues by the secretion of glucagon and the glu- containing foods was ranked according to their GI cocorticoid hormones. values, there was a strong linear relationship with the rapidly available glucose (RAG) from similar foods Diabetes and its consequences determined in vitro as the sum of free glucose, glucose Diabetes may be diagnosed as an exaggerated response from sucrose, and glucose released from starches over in blood glucose concentration following ingestion of a 20 minute period of hydrolysis with a battery of a fi xed amount of glucose (glucose tolerance test). enzymes under strictly controlled conditions (Englyst The most common forms of diabetes are type 1 dia- method; Englyst et al. 1999). This offers the possibil- betes (T1DM) and type 2 diabetes (T2DM). T1DM ity of assaying foods in vitro for their RAG content, results from the autoimmune destruction of the β- which will be quicker and cheaper than the current cells of the endocrine pancreas (possibly following approach based on GI measurements in vivo. viral exposure), the consequence of which is insulin Studies with glucose and starches enriched with insuffi ciency. Control of blood glucose concentra- the stable isotope carbon-13 have demonstrated that Digestion and Metabolism of Carbohydrates 79 glucose absorption from the gut following a meal con- 0.08 tinues for several hours after blood concentrations have returned to fasting levels. In this later postpran- 0.06 dial period, insulin secretion is suffi cient to ensure that the rate of glucose absorption is matched by the

13 enrichment rate of glucose removal from the circulation. C- 2 0.04

Labeled substrates are being used increasingly to CO 13 investigate the kinetics of digestion, absorption, and [13C]-fructose-labeled meal 0.02 metabolic disposal of glucose and other sugars from a 13 (atoms percent excess) [ C]-glucose-labeled meal range of foods. When continued over several years, Breath high rates of glucose absorption and the subsequent challenge to the capacity of the pancreatic β-cells to 0 100 200 300 400 secrete insulin may be the primary determinants of Time after consumption of test meals (min) 13 insulin resistance and eventual pancreatic failure that Figure 5.2 Enrichment of breath CO2 following ingestion of high- contribute strongly to the etiology of diabetes and sucrose test meals labeled with [13C]-fructose and [13C]-glucose. cardiovascular disease. Such kinetic studies are likely (Redrawn from Daly et al., 2000, with permission of the American to be helpful in identifying foods with slower rates of Society for Nutrition.) intestinal hydrolysis – information that can be used in public health advice or in counseling of individuals. metabolism” to the body. They called these carbohy- drates “unavailable.” This was a very useful concept Fructose because it drew attention to the fact that some carbo- When glucose and fructose are available simultane- hydrate is not digested and absorbed in the small ously after a meal containing sucrose, how does the intestine, but rather reaches the large bowel where it body select which fuel to use fi rst for oxidative is fermented. However, it is now realized that it is purposes? This question has been resolved by experi- misleading to talk of carbohydrate as unavailable ments in which volunteers consumed, on two sepa- because some indigestible carbohydrate can provide rate occasions, high-sucrose test meals which were the body with energy through fermentation in the identical except that one or other of the constituent colon. Thus, “unavailable carbohydrates” are not monomeric sugars was 13C-labeled in each meal. The really unavailable. For this reason, it has been sug- volunteers blew into tubes at intervals after the meals gested by the Food and Agriculture Organization to provide breath samples for measurement of enrich- (FAO 1998) of the United Nations and World Health ment of expired carbon dioxide with 13C. The results Organization that the term “nonglycemic carbohy- showed that, after the high sucrose meal, fructose was drates” is more appropriate. oxidized much more rapidly and extensively than was glucose (Figure 5.2). Nature of carbohydrates that enter This rapid oxidation of fructose may be explained the colon by the fact that, because it is phosphorylated in hepa- Carbohydrates that enter the colon can be classifi ed tocytes, it bypasses 6-phosphofructokinase, one of the either physiologically or chemically. Neither of these key regulatory enzymes in glycolysis. classifi cations is entirely satisfactory because it is dif- fi cult to measure the physiologically indigestible car- 5.4 Nonglycemic carbohydrates bohydrate and this varies in different people. Further, the chemical structure of carbohydrates does not Carbohydrates that are not absorbed in the small always predict their physiological behavior. intestine enter the large bowel, where they are par- tially or completely broken down by bacteria in the Physiological classifi cation of colon by a process called fermentation. McCance and carbohydrates entering the colon Lawrence in 1929 were the fi rst to classify carbohy- Carbohydrates enter the colon because (1) monosac- drates as “available” and “unavailable.” They realized charide transporters do not exist in the intestinal that not all carbohydrates provide “carbohydrates for mucosa or do not function at a high enough rate; (2) 80 Introduction to Human Nutrition the enzymes needed to digest the carbohydrates are Finally, there are many reasons why carbohydrates not present in the small intestine; (3) the enzymes are may not be digested rapidly enough to be completely present but cannot gain access to the carbohydrates; absorbed. Some forms of retrograded or resistant or (4) the enzymes do not digest the carbohydrates starch, or foods with a large particle size, are digested rapidly enough for them to be completely absorbed. so slowly that the time spent in the small intestine is In addition, a small amount of carbohydrate entering not long enough for their complete digestion. Diges- the colon consists of carbohydrate residues occurring tion of these carbohydrates can be altered by factors on mucopolysaccharides (mucus) secreted by the that affect transit time. The presence of osmotically small and large intestinal mucosal cells. active and unabsorbed molecules (such as unabsorbed Some carbohydrates are always nonglycemic sugars) will draw water into the intestine and speed because the human species lacks the enzymes neces- the rate of transit. Substances that increase bulk, such sary for their digestion. However, a signifi cant pro- as wheat , will have similar effects. Transit rate is portion (perhaps up to half ) of all carbohydrates that slowed in old age and in the presence of viscous fi bers. escape digestion in the small intestine have a chemical Drugs may increase or decrease the rate of transit. structure which is such that they could potentially be Certain disorders can also affect transit time, such as digested or absorbed in the small intestine, but they gastroparesis, a complication of type I diabetes. are variably absorbed for various reasons, examples of which are given below. Chemical classifi cation of carbohydrates First, some monosaccharides and sugar alcohols entering the colon are only partially absorbed because of low affi nity for The chemical classifi cation of carbohydrates entering intestinal transporters. Xylose is taken up by the the colon is as follows: glucose transporter, but is only partly absorbed because of a low affi nity. Fructose is poorly absorbed ● Monosaccharides: all except for glucose, fructose, on its own, but readily absorbed in the presence of and galactose are partly or completely unabsorbed. glucose. The surface area of the small intestine avail- Fructose in the absence of a source of glucose able for absorption is reduced by diseases that cause (mono-, di-, or polysaccharide) is partly atrophy of the intestinal mucosa, such as tropical unabsorbed. sprue or celiac disease, or surgical resection of a ● Sugar alcohols: all are partly or completely portion of the intestine (e.g., for Crohn’s disease). An unabsorbed. increased rate of intestinal transit (e.g., high osmotic ● Disaccharides: all except for maltose, sucrose, and load in the small intestinal lumen from undigested lactose are unabsorbed. Lactose is completely or sugars) reduces the time available for absorption to partly unabsorbed in individuals with low intestinal occur. lactase activity. Second, some individuals have a low or absent ● Oligosaccharides: all are unabsorbed except for intestinal lactase activity; thus, lactose is partly or maltodextins. completely nonabsorbed in these individuals. The ● Polysaccharides: all nonstarch polysaccharides are availability of pancreatic amylase may be reduced in unabsorbed. cystic fi brosis or in individuals whose pancreatic ● Resistant starch. mass has been destroyed by, for example, recurrent pancreatitis. Amount of carbohydrate entering Third, although starch (amylopectin or amylose) is the colon potentially digested in the small intestine, if it is It is diffi cult to measure the amount of carbohydrate trapped inside intact cell walls or other plant cell entering the human colon. However, it has been esti- structures, intestinal enzymes may not be able to gain mated that at least 30 g of carbohydrate is required to access to it, and therefore it remains undigested. The support the growth of the bacterial population in the digestibility of the carbohydrates in banana depends colon of an individual on a typical Western diet pro- on the degree of ripeness. The starch in green banana ducing about 100 g stool per day. About half of that is very indigestible, but, as the banana ripens, the amount will come from nonstarch polysaccharide starch is converted to digestible sugars. (NSP, also known as dietary fi ber), 1–2 g from indi- Digestion and Metabolism of Carbohydrates 81 gestible oligosaccharides, and probably about 1–2 g colon could be sampled. Another method was to from intestinal mucopolysaccharides. These compo- study people who have had their colons removed sur- nents add up to only 18–20 g. Where does the other gically and in whom the end of the ileum was sutured 10–12 g come from? It is believed to come from starch, to a in the body wall. In this way, the material because experiments in humans show that about 5– leaving their small intestine could be collected quan- 15% of the starch in foods enters the colon. Typical titatively in a bag attached to their abdomen. With Western diets contain about 120–150 g starch per day, these methods, the amount of carbohydrate leaving and, if 8% of this enters the colon, this will provide the small intestine can be measured directly. These the additional 10–12 g carbohydrate. The amount of methods confi rmed that a substantial amount of carbohydrate entering the colon, however, can be starch enters the colon. increased several-fold, up to 100 g/day or more, by The main forms of resistant starch (RS) are physi- changes in diet such as increased intake of NSP, cally enclosed starch, for example within intact cell non-digestible or partially digestible carbohydrates structures (known at RS1); raw starch granules (RS2); (ingredients in functional foods), total starch, resis- and retrograded amylose (RS3). These kinds of starch tant starch, or slowly digested, low-GI foods. can be identifi ed chemically using methods developed by Englyst and colleagues (Englyst et al. 1996). Resistant starch Resistant starch is starch that escapes digestion in the Dietary fi ber small intestine and enters the colon. However, there Major interest in dietary fi ber began in the early 1970s is controversy over the amounts of resistant starch in with the proposal by Burkitt and Trowell (1975) that foods because there is no universally accepted method many Western diseases were due to a lack of fi ber in for measuring it (different methods yield different the diet. However, the defi nition of dietary fi ber has results). The amount of resistant starch measured been, and continues to be, a source of scientifi c con- chemically is generally less than that observed to enter troversy. Indeed, two consecutive reports from the the colon (or leave the small intestine) in experiments FAO (1997 and 1998) recommended that the term in human volunteers. “dietary fi ber” be phased out. Nevertheless, the term In the 1970s and early 1980s it fi rst became appar- appears to be here to stay because it is accepted by ent that appreciable amounts of starch are not digested consumers, the , and governments. in the small bowel, from experiments showing that A defi nition and method of measuring fi ber is breath hydrogen increased after eating normal starchy important for scientifi c studies and for food-labeling foods. The only source of hydrogen gas in the human purposes. The student must be aware that the defi ni- body is as a product of the anaerobic fermentation of tions and methods of measuring fi ber have changed carbohydrates by colonic bacteria (see below). If a over time, and differ in different parts of the world. person consumed a load of an absorbable sugar such Knowledge of what is meant by the term “fi ber” and as glucose, breath hydrogen did not go up. In contrast, what is included in the measurement is essential for if lactulose (an unabsorbed of fructose proper interpretation of the scientifi c literature (but and galactose) was consumed, breath hydrogen often is not given in the methods section of papers increased rapidly, and the area under the breath and reports). hydrogen curve over an 8–12 hour period was directly Originally, Burkitt and Trowell (1975) defi ned fi ber proportional to the amount of lactulose consumed. If as the components of plant cell walls that are indigest- subjects ate common starchy foods such as white ible in the human small intestine. Later, the defi nition bread or , breath hydrogen levels increased to was expanded to include storage polysaccharides an extent that suggested that 5–10% of the starch was within plant cells (e.g., the gums in some ). fermented in the colon. Subsequently, other ways of Many different methods were developed to measure measuring carbohydrate entering the colon were dietary fi ber, but they measured different things. All developed. In one technique, subjects swallowed a of the methods start with the drying and grinding of tube that was passed through the stomach and along the food and extraction of the fat using an organic to the end of the small intestine so that the material solvent. If the remaining material is treated with leaving the small intestine and about to enter the strong acid, the chemical bonds in starch and many 82 Introduction to Human Nutrition

(but not all) polysaccharides will be broken down to artifi cial, that could be classifi ed as “fi ber” (e.g., poly- release their component sugars. If these are fi ltered dextrose, sucrose polyester, styrofoam). Should these away, the residue is “crude fi ber.” For many years this be included in dietary fi ber? In favor of this is the was the way in which fi ber was measured for food argument that some of these materials have physio- tables. However, acid hydrolysis breaks down many logical properties associated with fi ber, such as stool carbohydrates that would not be digested in the small bulking, or effects on satiety or blood glucose and intestine. So, in more modern methods, the food cholesterol. Against this is the feeling that dietary fi ber residue is digested with amylase to hydrolyze the should include only plant materials that are normally starch to soluble sugars and oligosaccharides. The present in the diet. These are not easy issues and they latter are removed by fi ltration or by centrifugation have not been resolved. to leave a residue containing mainly dietary fi ber, pro- teins, and inorganic materials. Intakes of dietary fi ber, oligosaccharides, The two main methods used to determine dietary and other indigestible sugars fi ber are chemical and gravimetric. In the chemical Vegetarians tend to have higher fi ber intakes than method (used in the UK), the residue is subjected to . The typical intake of dietary fi ber in North acid hydrolysis and the resultant sugars are measured America and northern and central Europe is about colorimetrically, by gas chromatography or by high- 15 g/day. In Scandinavia and Italy, fi ber consumption performance liquid chromatography. The sum of all is 20–30 g/day, whereas in African countries such as these sugars constitutes the NSP. The chemical method Uganda, Kenya, Malawi, and Nigeria intakes may be includes only carbohydrates in the NSP. In the gravi- as high as 50 g/day or more. Naturally occurring metric method (used in the USA and elsewhere), the oligosaccharides are consumed in legumes, , residue is dried and weighed, and the amounts of fennel, chicory, and similar foods. Intakes in Western protein and mineral materials present are subtracted countries are probably up to 2–4 g/day. Fructo- and (after separate analyses). The gravimetric method are now being added to includes the NSP, plus other noncarbohydrate com- certain “functional foods” in a number of countries, ponents such as lignin and waxes. Recently, all coun- and intakes from such sources may increase substan- tries in Europe have recognized the gravimetric tially (up to 10–20 g/day). Many kinds of indigestible method as an approved method for measuring fi ber or partially digested carbohydrates are entering the in foods. food supply in dietetic, diabetic, or functional foods, The main areas of disagreement now with respect including sugar alcohols (polyols, e.g., sorbitol, man- to fi ber are whether indigestible oligosaccharides and nitol, lactitol), polydextrose, resistant starch, hydroge- sugars and nonplant compounds should be included nated starch, and other chemically modifi ed starches and whether the defi nition of fi ber should include and carbohydrates. Thus, the total amount of carbo- a physiological component. In Japan, fructooligo- hydrate entering the colon could become very sub- saccharides (FOSs) are classifi ed as dietary fi ber for stantial for people using these foods. Individually, food-labeling purposes. However, FOSs and similar these ingredients are generally recognized as safe, and compounds, being soluble in water, are not included evidence from populations consuming 50 g and more in the dietary fi ber methods, because they are fi ltered NSP per day suggests that the colon has the capacity out along with the sugars resulting from the starch to adapt to large increases in the load of carbohydrate. hydrolysis. Specifi c methods exist for FOSs and related However, safe upper limits of intake are unknown and compounds, and they could be included as fi ber. the health implications of an increased supply of a Certain animal-derived compounds, such as chitin wide range of carbohydrates to the colon are currently and chitosan, derived from the shells of shrimp and based on inference rather than scientifi c data. crabs, are indigestible, would be included in the gravi- metric fi ber analysis, and could be classifi ed as fi ber. Fermentation in the colon Chitin has some physiological properties, such as The colon contains a complex consisting of cholesterol lowering, which are associated with dietary over 400 known species of bacteria that exist in a sym- fi ber. There are many other indigestible carbohydrate biotic relationship with the host. The bacteria obtain and noncarbohydrate compounds, both natural and the substrates that they require for growth from the Digestion and Metabolism of Carbohydrates 83 host, and return to the host the by-products of their The fi rst step in fermentation is the breakdown of metabolism. The major substrate that the bacteria polysaccharides, oligosaccharides, and disaccharides receive from the host is carbohydrate, mostly in the to their monosaccharide subunits. This is achieved form of polysaccharides. They obtain nitrogen from either by the secretion of hydrolytic enzymes by bac- urea (which diffuses into the colon from the blood) teria into the colonic lumen or, more commonly, by and undigested amino acids and proteins. Fermentation expression of such enzymes on the bacterial surface is the process by which microorganisms break down so that the products of hydrolysis are taken up directly monosaccharides and amino acids to derive energy for by the organism producing the enzyme. To degrade their own metabolism. Fermentation reactions do not the NSP of dietary fi ber, the bacteria may need to involve respiratory chains that use molecular oxygen attach themselves to the surface of the remnants of or nitrate as terminal electron acceptors. Most of the the plant cell walls or other particulate material. fermentation in the human colon is anaerobic, i.e., it Once the monosaccharide is internalized, the proceeds in the absence of a source of oxygen. Different majority of carbohydrate-fermenting species in the bacteria use different substrates via different types colon use the glycolytic pathway to metabolize carbo- of chemical reaction. However, as a summary of the hydrate to pyruvate. This pathway results in the overall process, fermentation converts carbohydrates reduction of NAD+ to NADH. Fermentation reactions to energy, plus various end-products, which include are controlled by the need to maintain redox balance the gases carbon dioxide, hydrogen, and methane, and between reduced and oxidized forms of pyridine the SCFAs acetic (C2), propionic (C3), and butyric nucleotides. The regeneration of NAD+ may be (C4) acids. Acetate, propionate, and butyrate appear achieved in a number of different ways (Figure 5.4). in colonic contents in approximate molar ratios of Electron sink products such as , lactate, 60:20:20, respectively. Most of the SCFAs produced are hydrogen, and succinate are produced by some absorbed and provide energy for the body (Figure bacteria to regenerate oxidized pyridine nucleotides. 5.3). These fermentation intermediates are subsequently The roles of SCFAs in metabolism are discussed fermented to SCFAs by other gut bacteria, and are later in this chapter. Formic acid (C1) and minor important factors in maintaining species diversity in amounts of longer chain SCFAs and branched-chain the ecosystem. SCFAs may also be produced. In addition, lactic and succinic acids and ethanol or methanol may be Fate of short-chain fatty acids intermediate or end-products depending on the Colonic fermentation can be viewed as a way in which conditions of the fermentation. For example, rapid the human host can recover part of the energy of fermentation in an environment with a low pH results malabsorbed carbohydrates. The amount of energy in the accumulation of lactic and succinic acids. recovered from fermentation depends on the fer- mentability of the carbohydrate (which can range from 0% to 100%) and the nature of the products of Carbohydrate fermentation. On a typical Western diet, about 40– 50% of the energy in carbohydrate that enters the colon is available to the human host as SCFAs. The Pyruvate rest of the energy is unavailable to the host, being lost as heat or unfermented carbohydrate or used to produce gases or for bacterial growth (Figure 5.5). 60% Acetate CO2 20% Propionate H2 SCFAs are almost completely absorbed and, while 20% Butyrate CH4 some butyrate is oxidized by colonocytes (the epithe- lial cells lining the colon), most arrives at the liver via the portal vein. Propionate and butyrate are removed Absorbed Breath in fi rst pass through the liver, but increased concen- Feces and utilized Flatus trations of acetate can be observed in peripheral Figure 5.3 Overview of carbohydrate fermentation in the human blood several hours after consumption of indigestible colon. but fermentable carbohydrates. These absorbed 84 Introduction to Human Nutrition

Hexose + NAD H2 CO2

NADH H+ CH 2 ATP 2 4

ATP

Lactate Pyruvate Succinate

+ + NAD NAD NADH2 NADH2 Biotin CO Ethanol Acetyl CoA 2

Propionate Figure 5.4 Summary of biochemical ATP ATP pathways used by the anaerobic bacteria in the colon. Acetyl CoA, acetyl Acetate Butyrate coenzyme A.

Carbohydrate entering the colon the ability of butyrate to induce differentiation and (17.2kJ/g) apoptosis (programmed ) of colon cancer cells. There is some support for the hypothesis that propionate may help to reduce the risk of cardiovas- cular disease by lowering blood cholesterol concen- H2 and CH4 tration and/or by an effect on hemostasis, but the (0.6kJ/g) SCFA evidence so far is not conclusive. Colon (7.2kJ/g) Heat (0.6kJ/g) 5.5 Carbohydrates and dental caries The resident bacteria in the mouth ferment carbohy- drates to yield acidic end-products (mainly lactic acid Unfermented Bacterial but also some formic, acetic, and propionic acids), carbohydrate matter (5.2kJ/g) (3.6kJ/g) which result in a drop in dental plaque pH. When the pH falls below 5.5, the dental enamel dissolves in the plaque fl uid and repeated exposure to periods of very low pH can lead to caries. Not all carbohydrates are Feces equally cariogenic. The sugars found commonly in (8.8kJ/g) human foods, e.g., sucrose, fructose, glucose, and Figure 5.5 Quantitative fate of carbohydrate in the colon. SCFA, maltose, are all readily fermented by bacteria in the short-chain . mouth. Lactose, galactose, and starches are less cario- genic, while sugar alcohols such as xylitol (used as a SCFAs are readily oxidized and contribute modestly sweetener in some confectionery and chewing gums) (up to 10%) to the body’s energy supply. are noncariogenic. Eating sugars with meals reduces There is considerable interest in the possible effects the risk of caries, as does the consumption of cheese, of individual SCFAs on the health of the colon and which provides to prevent demineraliza- the whole body. The strongest evidence to date is for tion and to encourage demineralization of the enamel. an anticancer effect of butyrate, which may be due to ingestion in foods and drinking water or Digestion and Metabolism of Carbohydrates 85 topical application via toothpastes and mouth rinses References prevents dental caries. Too much fl uoride in drinking water can cause fl uorosis, which damages the skeleton Burkitt DP, Trowell HC. Refi ned Carbohydrate Foods and Disease. and teeth. The optimum concentration of fl uoride Academic Press, London, 1975. Englyst KN, Englyst HN, Hudson GL et al. Rapidly available glucose in temperate areas of the world is 1 mg/l, falling to in foods: an in vitro measurement that refl ects the glycemic 0.6 mg/l in tropical climates where fl uid intake is response. Amer J Clin Nutr 1999; 69: 448–454. likely to be greater. Englyst HN, Kingman SM, Hudson GJ et al. Measurement of resist- ant starch in vitro and in vivo. Br J Nutr 1996; 75: 749–755. Food and Agriculture Organization of the United Nations. FAO 5.6 Perspectives on the future food and nutrition paper 66. Carbohydrates in human nutrition. Report of an FAO/WHO Expert Consultation on Carbohydrates, 14–18 April, 1997, Rome, Italy. FAO, Rome, 1998. The carbohydrate structure and amounts in many Holland B, Unwin ID, Buss DH. Vegetable Dishes. Second supple- foods and ingredients can be manipulated to achieve ment to McCance and Widdowson’s The Composition of Foods, specifi c physicochemical properties of benefi t for 5th edn. Cambridge: Royal Society of Chemistry, 1992. Jenkins DJA, Wolever TMS, Taylor RH et al. Glycemic index of food structure and organoleptic effects and to produce foods: a physiological basis for carbohydrate exchange. Am J a diverse range of physiological effects. It can be Clin Nutr 1981;34:362–366. expected that many functional foods of the future will Mccance RA, Lawrence RD. The carbohydrate content of foods. MRC Special Report Series 1929, No. 135. contain such specially selected or modifi ed carbohy- drates, but the metabolic and health consequences of these carbohydrates should be examined in more Further reading detail before health claims can be justifi ed. Future research on carbohydrate nutrition should Asp N-G. Development of dietary fi bre methodology. In: McCleary also focus on the physiological and biochemical (met- BV, Prosky L, eds. Advanced Dietary Fibre Technology. Blackwell Science, Oxford, 2001: 77–88. abolic) effects of the SCFAs produced from nonglyce- Brody T. Nutritional Biochemistry, 2nd edn. Academic Press, San mic carbohydrates. Diego, CA, 1999. To provide a sound evidence base for recommen- Daly ME, Vale C, Walker M et al. Acute fuel selection in response to high-sucrose and high-starch meals in healthy men. Am J Clin dations for intakes of specifi c carbohydrates, the Nutr 2000; 71: 1516–1524. relationships between intakes of different types and Johnson LR. Gastrointestinal Physiology, 5th edn. Mosby, St Louis, quantities of carbohydrate with health and disease, MO, 1997. Rugg-Gunn AJ. Nutrition and Dental Health. Oxford University for example during transition of traditional people Press, Oxford, 1993. and consequent lowering of intakes, should be a fruit- Wolever TMS. The Glycaemic Index: A Physiological Classifi cation ful area for research. of Dietary Carbohydrate. CABI, Wallingford, 2006. 6 Nutrition and Metabolism of Lipids

Bruce A Griffi n and Stephen C Cunnane

Key messages

• Lipids are organic compounds composed of a carbon skeleton • Dietary lipids (fats) are emulsifi ed, lipolyzed (hydrolyzed), and with hydrogen and oxygen substitutions. The most abundant solubilized in the upper small gut before they are absorbed in lipids are sterols or esters of fatty acids with various alcohols such the ileum, entering enterocytes with the help of fatty acid-binding as glycerol and cholesterol. proteins. • Fatty acids are the densest dietary source of energy, but lipids • Lipids are precursors to hormones such as steroids and also have important structural roles in membranes. The processes eicosanoids, and dietary lipids are carriers for fat-soluble controlling the synthesis, modifi cation, and degradation of fatty vitamins. acids contribute to the fatty acid profi le of membrane and storage • Lipids are transported in the blood circulation as lipoprotein lipids. particles: the chylomicrons, very low-density, low-density, and • By enhancing the taste of cooked foods, some dietary lipids are high-density lipoproteins. potentially signifi cant risk factors for obesity and other chronic, • Some polyunsaturated fatty acids are vitamin like because they degenerative diseases that infl uence human morbidity and cannot be synthesized de novo (linoleate, α-linolenate). mortality.

6.1 Introduction: the history of lipids in termed “amphipathic” by Hartley in 1936 and human nutrition renamed “amphiphilic” by Winsor in 1948. The fi rst understanding of how fat was absorbed The term “lipid” was introduced by Bloor in 1943, by emerged in 1879 when Munk studied fat emulsions which time the existence of cholesterol had been and showed that lymph contained TAG after a fatty known for nearly 200 years and individual fats for 130 meal, and even after a meal not containing TAG. In years. Cholesterol was named “cholesterine” (Greek 1905, Knoop deduced that fatty acid β-oxidation for bile-solid) by Chevreul in 1816, although he did probably occurred by stepwise removal of two not discover it. Cholesterol’s association with aortic from the fatty acid. The probable role of two carbon plaques dates at least to Vogel’s work in 1843. Chevreul units as building blocks in the synthesis of fatty acids isolated a mixture of 16- to 18-carbon saturated fatty was recognized by Raper in 1907, but it took until the acids in 1813 that was called margarine because he 1940s for Schoenheimer, Rittenberg, Bloch, and others thought it was a single 17-carbon fatty acid, marga- to confi rm this, using tracers such as deuterated water rate. The mixed triacylglycerol (TAG) of palmitate and carbon-13. The late 1940s was a seminal period (16:0) and stearate (18:0) was also called margarine, in our understanding of how fatty acid oxidation whereas the triglyceride of oleate, stearate, and palmi- occurs. Green and colleagues discovered that ketones tate became known as oleomargarine. Phospholipids were fatty acid oxidation products, and Lehninger were discovered by Thudicum, who isolated and demonstrated the role of mitochondria as the cellular named sphingosine in 1884 and also (phos- site of fatty acid oxidation. Microsomal desaturases phatidylcholine) and kephalin (phosphatidylethanol- were shown to introduce an unsaturated bond into amine). The difference in polarity across phospholip- long-chain fatty acids by Bloomfi eld and Bloch in ids is a key attribute of these molecules and was 1960.

© 2009 BA Griffi n and SC Cunnane. Nutrition and Metabolism of Lipids 87

In 1929, Mildred and George Burr discovered that Table 6.1 Classifi cation of lipids the absence of fat in a diet otherwise believed to Simple lipids (fatty acids Fats (fatty acids esterifi ed with contain all essential nutrients impaired growth and esterifi ed with alcohols) glycerol) caused hair loss and scaling of the skin of rats. This Waxes (true waxes, sterol esters, led to the isolation of the two primary “essential” vitamin A and D esters) polyunsaturated fatty acids, linoleate (18:2n-6) and Complex lipids (fatty acids Phospholipids (contain α-linolenate (18:3n-3). The are a sub- esterifi ed with alcohols plus phosphoric acid and, usually, other groups) a nitrogenous base) class of eicosanoids that were discovered in the early Glycolipids (lipids containing a 1930s by Von Euler, who mistakenly believed that they carbohydrate and nitrogen but originated from the prostate gland. The link between no phosphate and no glycerol) the eicosanoids and polyunsaturates, principally Sulfolipids (lipids containing a sulfur group) arachidonate, was established in the 1960s. Lipoproteins (lipids attached to plasma or other proteins) Lipopolysaccharides (lipids 6.2 Terminology of dietary fats attached to polysaccharides) Derived lipids (obtained by Fatty acids (saturated, hydrolysis of simple or monounsaturated, or Lipids complex lipids) polyunsaturated) Like other organic compounds, all lipids are com- Monoacylglycerols and posed of a carbon skeleton with hydrogen and oxygen diacylglycerols Alcohols (include sterols, substitutions. Nitrogen, sulfur, and phosphorus are steroids, , vitamin A) also present in some lipids. Water insolubility is a key Miscellaneous lipids Straight-chain but not absolute characteristic distinguishing most Squalene lipids from proteins and carbohydrates. There are Vitamins E and K some exceptions to this general rule, since short- to medium-chain fatty acids, soaps, and some complex lipids are soluble in water. Hence, solubility in a “lipid RCOOH, where R is hydrogen in formic acid, CH3 in solvent” such as ether, chloroform, benzene, or acetone acetic acid, or else a chain of one to over 30 CH2 is a common but circular defi nition of lipids. groups terminated by a CH3 group. The various There are four categories of lipids, as classifi ed by names for individual fatty acids (common, offi cial) Bloor: simple, compound (complex), derived, and and their abbreviations are complicated, and the use miscellaneous (Table 6.1). Simple lipids are esters of of one or other form is somewhat arbitrary. The fatty acids with various alcohols such as glycerol or basic rule for the abbreviations is that there are three cholesterol. They include triacylglycerols (TAG = parts: number of carbons, number of double bonds, neutral fats and oils), waxes, cholesteryl esters, and and position of the fi rst double bond. Thus, vitamin A and D esters. Compound lipids are esters the common dietary saturated fatty acid palmitate is of fatty acids in combination with both alcohols and 16:0 because it has 16 carbons and no double other groups. They include phospholipids, glycolip- bonds. The common dietary polyunsaturated fatty ids, cerebrosides, sulfolipids, lipoproteins, and lipo- acid linoleate is 18:2n-6 because it has 18 carbons, two polysaccharides. Derived lipids are hydrolysis products double bonds, and the fi rst double bond is at the sixth of simple or compound lipids, including fatty acids, carbon from the methyl-terminal (n-6). Beyond six monoacylglycerols and diacylglycerols, straight-chain carbons in length, most fatty acids have an even and ring-containing alcohols, sterols, and steroids. number of carbons (Table 6.2). Older fatty acid ter- Miscellaneous lipids include some wax lipids, carot- minology referring to saturated or unsaturated enoids, squalene, and vitamins E and K. carbons in lipids that still occasionally appears includes: aliphatic (a saturated carbon), olefi nic (an Saturated and unsaturated fatty acids unsaturated carbon), allylic (a saturated carbon adja- The main components of dietary fat or lipids are fatty cent to an unsaturated carbon), and doubly allylic acids varying in length from one to more than 30 carbon (a saturated carbon situated between two carbons. They are carboxylic acids with the structure unsaturated carbons). 88 Introduction to Human Nutrition

Table 6.2 Nomenclature of common fatty acids Saturates

Saturated Monounsaturated Polyunsaturated

Formic (1:0) Lauroleic (12:1n-3) Linoleic (18:2n-6) Acetic (2:0) Myristoleic γ-Linolenic (18:3n-6) cis (14:1n-5) -Monounsaturates Propionic (3:0) Palmitoleic Dihomo-γ-linolenic (16:1n-7) (20:3n-6) Butyric (4:0) Oleic (18:1n-9) Arachidonic (20:4n-6) Valeric (5:0) Elaidic Adrenic (22:4n-6) (trans-18:1n-9) Caproic (6:0) Vaccenic (18:1n-7) n-6 Docosapentaenoic (22:5n1-6) Caprylic (8:0) Petroselinic α-Linolenic (18:3n-3) (18:1n-12) Capric (10:0) Gadoleic Stearidonic (18:4n-3) cis-Polyunsaturates (20:1n-11) Lauric (12:0) Gondoic (20:1n-9) Eicosapentaenoic (20:5n-3) Myristic (14:0) Euricic (22:1n-9) n-3 Docosapentaenoic (22:5n-3) Palmitic (16:0) Nervonic (24:1n-9) Docosahexaenoic (22:6n-3) Margeric (17:0) Stearic (18:0) Arachidic (20:0) Behenic (22:0) Lignoceric (24:0)

Figure 6.1 Stick models illustrating the basic structural differences Lengthening of the chain and the introduction of between saturated, cis-monounsaturated, and cis-polyunsaturated additional double bonds beyond the fi rst one occur fatty acids. As shown in two dimensions, the increasing curvature caused by inserting one or more double bonds increases the area from the carboxyl-terminal. The presence of one or occupied by the fatty acid. The physical area occupied by unsaturated more double bonds in a fatty acid defi nes it as “unsat- fatty acids is further accentuated in three dimensions because esteri- urated,” compared with a saturated fatty acid which fi ed fatty acids rotate around the anchored terminal. contains no double bonds. A saturated fatty acid gen- erally occupies less space than an equivalent chain length unsaturated fatty acid (Figure 6.1). Double bonds allow for isomerization or different orientation ucts containing ruminant milk fat. Hence, although (cis or trans) of the adjoining carbons across the they are produced in relatively large quantities double bond (Figure 6.2). In longer chain fatty acids, from the fermentation of undigested carbohydrate in double bonds can also be at different positions in the the colon, as such, they do not become part of the molecule. Hence, unsaturation introduces a large body lipid pools. Medium-chain fatty acids (8–14 amount of structural variety in fatty acids and carbons) arise as intermediates in the synthesis of the resulting lipids. Further details about the features long-chain fatty acids or by the consumption of of the different families of fatty acids are given in oil or medium-chain TAG derived from it. Sections 6.6 and 6.8. Like short-chain fatty acids, medium-chain fatty acids are present in milk but they are also rarely esterifi ed Short- and medium-chain fatty acids into body lipids, except when consumed in large Short-chain fatty acids (less than eight carbons) amounts in clinical situations requiring alternative are water soluble. Except in milk lipids, they are energy sources. Medium-chain fatty acids (8–14 not commonly esterifi ed into body lipids. Short- carbons) are rare in the diet except for coconut and chain fatty acids are found primarily in dietary prod- milk fat. Nutrition and Metabolism of Lipids 89

cis-Monounsaturates long-chain saturates can probably be sustained without a dietary source of these fatty acids. Com- pared with all other classes of dietary fatty acid, espe- cially monounsaturated or polyunsaturated fatty acids, excess intake or synthesis of long-chain satu- rates is associated with an increased risk of cardiovas- cular disease. The most common long-chain cis-monounsatu- rated fatty acids in diet and in the body are oleate trans-Monounsaturates (18:1n-9) and palmitoleate (16:1n-7), with the former predominating by far in both the body’s storage and membrane lipids. As with stearate, most oleate in the human body appears to be of dietary origin. Hence, although humans have the capacity to desaturate Figure 6.2 Stick models comparing a cis- with a trans-unsaturated stearate to oleate, dietary oleate is probably the domi- fatty acid. A cis-unsaturated double bond creates a U-shaped space nant source of oleate in the body. Only plants can and confers curvature to the molecule because, relative to the longi- α tudinal axis of the fatty acid, the two at the double bond further desaturate oleate to linoleate and again to - are on the same side of the molecule. A trans-unsaturated double bond linolenate. As with saturates of >18 carbons in length, does not confer curvature to the molecule because the hydrogens are 20-, 22-, and 24-carbon monounsaturates derived on opposite sides of the double bond. A trans-double bond therefore from oleate are present in specialized membranes tends to give the fatty acid physicochemical properties more like that such as myelin. of a saturated fatty acid. Polyunsaturated fatty acids (PUFAs) Linoleate and α-linolenate are the primary dietary Long-chain saturated and cis-polyunsaturated fatty acids in most diets. Neither monounsaturated fatty acids can be synthesized de novo (from acetate) in animals Long-chain fatty acids (>14 carbons) are the main so are ‘essential’ fatty acids. They can be made constituents of dietary fat. The most common satu- by chain elongation from the two respective 16- rated fatty acids in the body are palmitate and stea- carbon precursors, hexadecadienoate (16:2n-6) and rate. They originate from three sources: directly from hexadecatrienoate (16:3n-3), which are found in the diet, by complete synthesis from acetyl-coenzyme common edible green plants at up to 13% of total A (CoA), or by lengthening (chain elongation) of a fatty acids. Hence, signifi cant consumption of green pre-existing shorter-chain fatty acid. Hence, dietary will provide 16-carbon polyunsaturates or newly synthesized palmitate can be elongated that contribute to the total available linoleate and within the body to form stearate and on to arachidate α-linolenate. (20:0), behenate (22:0), and lignocerate (24:0). In Linoleate is the predominant polyunsaturated fatty practice, little stearate present in the human body acid in the body, commonly accounting for 12–15% appears to be derived by chain elongation of pre- of adipose tissue fatty acids. In the body’s lean tissues existing palmitate. In humans, saturates longer than there are at least three polyunsaturates present in 24 carbons do exist but usually arise only during amounts >5% of the fatty acid profi le (linoleate, ara- genetic defects in fatty acid oxidation, as will be dis- chidonate, docosahexaenoate). In addition, at least cussed later. two other biologically active polyunsaturates are Palmitate and stearate are important membrane present in body lipids [dihomo-γ-linolenate (20:3n- constituents, being found in most tissue phospholip- 6) and eicosapentaenoate (20:5n-3)], although usually ids at 20–40% of the total fatty acid profi le. Brain in amounts between 1% and 3% of total fatty acids. membranes contain 20- to 24-carbon saturates that, Marine fi sh are the richest source of 20- to 22-carbon like palmitate and stearate, are synthesized within the polyunsaturates. α-Linolenate and its precursor, brain and have little or no access to the brain from hexadecatrienoate (16:3n-3), are the only n-3 polyun- the circulation. The normal membrane content of saturates in common terrestrial plants. 90 Introduction to Human Nutrition

Hydrogenated and conjugated fatty from their lower proportion of saturated (straight- acid isomers chain) and higher proportion of unsaturated (bent- The introduction of unsaturation with one double chain) fatty acids. Unsaturated fatty acids usually bond creates the possibility of both positional and have a lower melting point; this facilitates liquefaction geometric isomers in fatty acids. Among long-chain of the fats of which they are a component. TAGs of unsaturated fatty acids, positional isomers exist animal origin are commonly fats, whereas those of because the double bond can be introduced into fi sh or plant origin are usually oils. Animal fats and several different locations, i.e., 18:1n-7, 18:1n-9, fi sh oils frequently contain cholesterol, whereas plant 18:1n-11, etc. Geometric isomers exist because the oils do not contain cholesterol but usually contain two remaining hydrogens at each double bond can be other “phyto” sterols. opposite each other (trans) or on the same side of the TAGs are primarily used as fuels, so dietary fats molecule (cis; Figure 6.2). Thus, there is cis-18:1n-9 (mostly TAGs) are commonly associated with energy (oleate) and trans-18:1n-9 (elaidate), and so on for all metabolism rather than with structural lipids unsaturated fatty acids, with the combinations found in membranes. However, membrane lipids as mounting exponentially as the number of double well as TAGs are extracted with lipid solvents used to bonds increases. determine the fat content of foods, tissues, or plant Trans isomers of monounsaturated or polyunsatu- material. Hence, because organs such as brain are rated fatty acids arise primarily from partial hydroge- rich in membrane phospholipids, when the total nation during food processing of oils, but some also lipids are extracted to determine the organ’s chemical occur naturally in ruminants. The number of trans composition, these organs are said to have a certain isomers increases with the number of double bonds, fat content. On a chemical basis this is true, but this so there is only one trans isomer of oleate but there description often misconstrues the nature of the lipid are three trans isomers of linoleate and seven of α- because the brain in particular contains virtually no linolenate. Virtually all naturally occurring polyun- TAG. saturated fatty acids have double bonds that are Phospholipids methylene interrupted, i.e., have a CH2 group between the two double bonds. However, methylene interrup- Phospholipids contain two nonpolar, hydrophobic tion between double bonds can be lost, again, through acyl tail groups and a single functional head group food processing, and the bonds moved one carbon that is polar and hydrophilic. Hence, they are rela- closer together, becoming conjugated. Thus, the tively balanced amphiphilic lipids and, in this capac- double bonds in linoleate are at the 9–10 and 11–12 ity, are crucial components of biological membranes. carbons, but in conjugated linoleate they are at the The head groups contain phosphorus and amino 9–10 carbons and the 11–12 carbons. Some degree of acids (, serine, ethanolamine), sugars (inosi- further desaturation and chain elongation can occur tol), or an alcohol (glycerol). in conjugated fatty acids, but much less than with (lecithin) is the most abundant phospholipid in methylene-interrupted polyunsaturates. Thus, conju- animal tissues but phosphatidylglycerols (glycosides) gated linoleate is the main conjugated fatty acid that predominate in plant lipids. Phospholipids contain- has attracted considerable attention with respect to its ing a fatty acid amide are sphingolipids. Various phos- potential role in nutritional health. pholipases can hydrolyze the acyl groups or head group during digestion or metabolism. Fats and oils One of the outstanding characteristics that make Fats are esters of fatty acids with glycerol (Table 6.1). phospholipids suitable as major constituents of bio- They usually occur as triesters or triacylglycerols logical membranes is that, in water, they naturally (TAGs), although monoacylglycerols and diacylglyc- aggregate into spherical or rod-like liposomes or ves- erols occur during fat digestion and are used in food icles, with the hydrophilic portion facing outwards processing. Most common dietary fats contain a and the hydrophobic portion facing inwards (Figure mixture of 16- to 18-carbon saturated and unsatu- 6.3). Changing the constituent acyl groups from satu- rated fatty acids. By convention, fats that are liquid at rated to polyunsaturated changes the fl uidity of these room temperature are called oils, a feature arising aggregates because of the greater amount of space Nutrition and Metabolism of Lipids 91

Outer surface

Inner surface

Trans-membrane Saturated protein/receptor phospholipid Phospholipid head groups Unsaturated (asymmetrically distributed) Ion channel phospholipid

Carbohydrate unit Cholesterol

Figure 6.3 Simplifi ed schematic view of a membrane bilayer. The main components are proteins, free cholesterol, phospholipids, and carbohy- drates. There are many different proteins with a myriad of shapes, membrane distribution, and functions, of which three are illustrated. Membrane phospholipids principally help to create the bilayer. They have four types of “head groups” (choline, ethanolamine, serine, and ) that are located at or near the membrane’s two surfaces. The two fatty acids in phospholipids are mixtures of 16- to 22-carbon saturates, monounsaturates, and polyunsaturates in all combinations, with those rich in unsaturated fatty acids occupying more space; hence, their trapezoid shape compared with the narrower, rectangular shape of the more saturated phospholipids. Free cholesterol represents 30–40% of the lipid in most membranes. The many different carbohydrates are on the membrane’s surfaces and are bound to lipids and/or proteins in the membrane.

occupied by more unsaturated fatty acids. At inter- 6.3 Lipids as components of the diet faces between non-miscible polar and non-polar sol- vents, phospholipids also form a fi lm or monolayer. Food or dietary sources of lipids are listed in Table 6.3. Cholesterol is found only in animal lipids, while Sterols a variety of other phytosterols occur in plants. The main sterol of importance in human nutrition is Soyabeans, leafy plants, and lean animal meat are rich cholesterol. It has multiple roles including being: in dietary phospholipids. Animal fat and plant oils from seeds or nuts are rich in TAG. ● a vital component of biological membranes The leafy and fruit components of plants contain ● a precursor to bile salts used in fat digestion phospholipids and sterols, whereas seeds contain tri- ● a precursor to steroid hormones. glycerides. With rare exceptions such as fl axseed Sterols are secondary alcohols belonging to the poly- (linseed), edible green are proportionally much isoprenoids or terpinoids (terpenes), which have a richer in α-linolenate than are seeds. oils are common precursor, isopentenyl diphosphate. Other usually rich in either linoleate or oleate. Common members of the terpinoids include squalene, carot- plant sterols include β-sitosterol, β-sitostanol, and enoids, and dolichols. Bacteria appear to be the only campesterol. Foods enriched with esters of plant life forms not containing cholesterol. Sterols have sterols are used widely to lower blood cholesterol via a common cyclopentano(a)perhydrophenanthrene the inhibition of cholesterol absorption in the gut. skeleton with different substitutions giving rise to the Phospholipids and cholesterol constitute the multiple sterols and steroids. majority of lipids in tissues (gut, kidney, brain, 92 Introduction to Human Nutrition

Table 6.3 Common food sources of lipids is also found in several types of edible seaweed. Tropi- cal fi sh generally have higher arachidonate than do Cholesterol Eggs, shellfi sh, organ Phytosterols Soya products, olive oil cold-water fi sh. Short-chain fatty acids Milk fat Partial hydrogenation is a common feature of (1–6 carbons) unsaturated fatty acids in processed foods. Complete Medium-chain fatty Milk fat, coconut fat hydrogenation makes fats very hard and is more acids (8–14 carbons) expensive than partial hydrogenation. Depending on Long-chain fatty acids Saturates: animal fat, shortening, butter, the applications and the source of the original oil or (16–20 carbons) palm oil, fat, partial hydrogenation is an economical way to Monounsaturates: olive, canola oils Linoleate: sunfl ower, saffl ower, corn oils, control the properties of fats or oils used in food soyabean production. Dietary diacylglycerols and monoacyl- α-Linolenate: fl axseed oil, canola, are used by the food industry for emulsifi ca- soyabean oil, walnuts tion of water- and oil-based components in foods γ-Linolenate: evening primrose oil, borage oil, seed oil such as ice cream and mayonnaise. Stearidonate: blackcurrant seed oil Arachidonate: lean meat and organ lipids 6.4 Digestion, absorption, and transport Eicosapentaenoate: marine cold-water fi sh, shellfi sh, some seaweeds of dietary fat Docosahexaenoate: marine cold-water fi sh, shellfi sh The average daily intake of fat in a Western diet ranges Trans fatty acids: partially hydrogenated between 50 and 100 g and provides between 35% and fats and oils 40% of total energy. It consists mainly of TAG, which forms the principal component of visible oils and fats, and minor quantities of phospholipids and choles- skeletal muscle, etc.) of lean, undomesticated animals. terol esters (CEs). The physical properties of dietary By contrast, in domesticated animals, TAGs or non- fat, such as their hardness at room temperature membrane lipids present in subcutaneous and intra- (melting point) and subsequent metabolic properties muscular adipose tissue deposits are the dominant once in the body, are determined by the number of form of lipid on a weight basis. This is because domes- double bonds in their constituent fatty acids (degree tication usually involves rearing animals with minimal of saturation or unsaturation) and length of the fatty exercise and on higher energy intakes, leading to more acid carbon chain (see Tables 6.2 and 6.3). As men- subcutaneous and visceral TAG obtained through tioned in Section 6.2, fats that are solid at room tem- both fat synthesis and deposition of dietary fat. perature tend to consist of long-chain saturated fats Animal meat lipids are the main dietary source of (>14 carbons, no double bonds), whereas oils consist arachidonate (20:4n-6), although it can also be of long-chain unsaturated fats with several double obtained from tropical marine fi sh. bonds. It has become conventional to refer to dietary Lipoproteins are the main form of lipid in the fats as “lipids” once they have been absorbed into the blood (see Section 6.5). Like lipoproteins, milk lipids body via the small intestine, although it is not incor- also occur as globules consisting of a combination of rect to refer to dietary fat as “dietary lipid.” a mainly TAG core surrounded by a membrane con- taining proteins, cholesterol, and phospholipids. Reception, emulsifi cation, lipolysis, Phospholipids and cholesterol constitute the main solubilization, and absorption lipids of undomesticated edible fi sh, which usually The digestion of dietary fat takes place in three phases, have low amounts of TAG or stored body fat. As in known as the gastric, duodenal, and ilial phases. These domesticated animals, it is likely that subcutaneous involve crude emulsifi cation in the stomach, lipolytic and intramuscular fat deposits of TAG will increase breakdown by lipases and solubilization with bile salts in commercially farmed fi sh. Cold-water marine fi sh in the duodenum and, fi nally, absorption into the epi- are the main dietary source of the long-chain n-3 thelial cells or enterocytes lining the walls of the small (omega-3) polyunsaturates eicosapentaenoate (20:5n- intestine or ileum. Digestion may actually be initiated 3), and docosahexaenoate (22:6n-3), but the former in the mouth under the infl uence of a lingual lipase Nutrition and Metabolism of Lipids 93 secreted by the palate, although its contribution to fashion with the initial removal of a fatty acid from lipolysis in adults is questionable and thought to be position 1 and then position 3 from the glycerol back- more important in young suckling infants, in which bone, generating a 2,3-diacylglycerol, followed by a its release is stimulated by suckling and the presence 2-monoacylglycerol (2-MAG). of milk. It is possible that this lingual lipase is carried into the stomach, where it acts as a human gastric Solubilization of emulsifi ed fat lipase (HGL) that has been shown to degrade up to With the notable exceptions mentioned previously 10% of ingested fat. Although these early products of (Section 6.2), fats are insoluble in water and must be fat digestion, fatty acids and monoacylglycerols, rep- rendered soluble before they can be absorbed in the resent a relatively minor component of fat digested, gut and transported within cells and in the circula- their entry into the duodenum is believed to supply a tion. In each of these situations, this is achieved by major stimulus for the production of the hormone the hydrophobic fat or lipid associating with mole- cholecystokinin (CCK), which inhibits gut motility. cules that are capable of interfacing with both hydro- The stomach serves mainly as an organ of mechan- phobic and hydrophilic environments. Molecules ical digestion and, by churning its contents, produces with these characteristics are called amphipathic mol- a coarse creamy emulsion known as chyme. The cir- ecules, examples of which are phospholipids, bile cular pyloric sphincter muscle that separates the salts, and specialized proteins known as apoproteins stomach from the duodenum and, with other factors, (Figure 6.5). In the small intestine emulsifi ed fats are controls the rate of gastric emptying opens twice a solubilized by associating with bile salts produced in minute to release approximately 3 ml of chyme. Since the liver and stored and released from the gallbladder, emulsifi ed fat in chyme is less dense than the aqueous and phospholipids to form complex aggregates known material, the two fractions separate with the fat col- as mixed micelles. Lipids within cells and in the cir- lecting above the aqueous layer. As a result, the entry culation are solubilized by combining with specifi c of emulsifi ed fat into the duodenum is delayed, allow- proteins known as fatty acid-binding proteins (FABPs) ing suffi cient time for the minor breakdown products and apolipoproteins (ApoA, B, C, E), respectively. to act on CCK. Further details of the structure and function of these The duodenal phase involves the breakdown of the specialized proteins are given in Section 6.5. emulsifi ed fat by a process known as lipolysis and the The action of pancreatic lipase on TAG yields free solubilization of the products of lipolysis. The entry fatty acids and 2-MAG. Fatty acids of short- and of chyme containing minor lipolytic products into medium-chain length (≤14 carbons) are absorbed the duodenum stimulates the: directly into the portal circulation with free glycerol and transported bound to albumin to the liver, where ● release of CCK, which inhibits gut motility they are rapidly oxidized. In contrast, long-chain fatty ● secretion of bile acids from the gall bladder acids (LCFAs; >14 carbons) associate with bile salts in ● release of pancreatic juice containing a battery of bile juice from the gallbladder and are absorbed into lipases. the enterocyte for further processing and packaging Lipolysis is an enzyme-catalyzed hydrolysis that into transport lipoproteins. The primary bile salts, releases fatty acids from lipids (TAGs, phospholipids, cholic and chenodeoxycholic acids, are produced and CEs). It involves the hydrolytic cleavage of bonds from cholesterol in the liver under the action of the between a fatty acid and the glycerol backbone of rate-limiting enzyme 7-α-hydroxylase. These bile TAGs and phospholipids, and cholesterol in CEs, and salts act effectively as detergents, solubilizing lipids by occurs not only in the digestive tract but also in cir- the formation of mixed micelles. These are spherical culating and intracellular lipids (Figure 6.4). The associations of amphipathic molecules (with hydro- hydrolysis of emulsifi ed dietary fat entering the duo- phobic and hydrophilic regions) with a hydrophilic denum is catalyzed by a battery of pancreatic enzymes surface of bile salts and phospholipids that encapsu- including a pancreatic lipase that acts chiefl y on TAG lates a hydrophobic core of more insoluble LCFAs and phospholipase A2 and a cholesterol ester hydro- and 2-MAG (see Figure 6.4). The micelle core will lase acting on phospholipids and CEs. The hydrolysis also contain some lipid-soluble vitamins including of TAG by pancreatic lipase occurs in a sequential tocopherols and carotenoids. The formation of mixed 94 Introduction to Human Nutrition

Plasma Thoracic duct Chylomicrons lymph

Apoproteins and Liver phospholipids Short- and medium-chain fatty acids Gall bladder Cholesterol TAG esters ACAT OH OH

Bile acids ؉ Mixed micelles Bile acid conjugates

Unabsorbed material OH ؉ Feces P P PL Pancreatic Dietary lipase OH fats ؉ OH Hydrophobic lipid core TAGs MAGs Fatty acids

Duodenum Jejunum Ileum Figure 6.4 Reception, emulsifi cation, lipolysis, solubilization, and absorption of fats. ACAT, acyl-CoA-cholesterol acyltransferase; MAG, mono- acylglycerol; TAG, triacylglycerol; PL phospholipid; P, phosphate.

micelles increases the solubility of fat by 100- to 1000- ity for different types of LCFAs. Thus, the absorption fold. They create an acidic microenvironment for the of LCFAs and 2-MAG derived from dietary TAGs lipid core which, through protonation, facilitates the occurs by facilitated diffusion via FABP, which dissociation of LCFAs and 2-MAG from the micelle increases membrane permeation and promotes cel- and diffusion into the enterocyte. lular uptake of LCFAs and monoglycerides. An addi- tional factor that drives the diffusion gradient is the Absorption of solubilized fat rapid re-esterifi cation of LCFAs into 2-MAG and 2- The ilial or absorptive phase involves the transit of MAG into TAGs within the enterocyte by the enzyme dietary fats from mixed micelles into the enterocyte. acyl-CoA-cholesterol acyltransferase (ACAT). The Although originally believed to be a purely passive absorption of dietary TAGs in the small intestine is process, dependent on factors such as the rate of extremely effi cient, with up to 90% being absorbed. gastric emptying, extent of mixing, and gut motility, Dietary cholesterol also associates within mixed the translocation of LCFAs and 2-MAG from the micelles and is absorbed in a similar manner by spe- micelle into the enterocyte is now known to be assisted cifi c sterol-carrying proteins resident in the entero- by the presence of FABPs within the cell membrane cyte membrane. Thus, cholesterol is also absorbed by and the cell. These maintain a diffusion gradient a protein-facilitated mechanism but, in contrast to down which LCFAs and MAGs can fl ow into the cell. dietary TAGs, only about 40% of dietary cholesterol FABPs have numerous roles within cells and specifi c- will be absorbed directly. Nutrition and Metabolism of Lipids 95

Enterohepatic circulation The absorption of fat in the small intestine is depen- dent on the availability of bile acids from biliary which also contain free cholesterol. Both bile acids (>95%) and biliary cholesterol are salvaged by an energy-dependent process in the terminal ileum. This active process of reabsorption via the enterohepatic circulation is tightly controlled by a feedback mechanism that is sensitive to hepatic levels of cholesterol. Thus, the reabsorption of cholesterol downregulates the activity of 7-α-hydroxylase in the liver, shutting down the further production of bile acids. Substances in the lumen of the gut that are capable of binding or competing with bound bile acids, such as naturally occurring plant sterols or Free cholesterol soluble nonstarch polysaccharides (NSPs), prevent Surface lipids their reabsorption which, in effect, interrupts the Phospholipid enterohepatic circulation. This depletes the supply of cholesterol and accelerates the production of bile Protein (apoproteins) acids, depleting the liver of cholesterol (Figure 6.6). Cholesteryl esters To replenish this loss, liver cells respond by increasing Core lipids their uptake of cholesterol from circulating lipopro- Triacylglycerols teins in the blood, with the result of a decrease in Figure 6.5 General lipoprotein structure. (Reproduced from Dur- blood cholesterol. Interruption of the enterohepatic rington PN. Hyperlipidaemia Diagnosis and Management, 2nd edn. circulation helps to explain the cholesterol-lowering Elsevier Science, Oxford, copyright 1995 with permission of Elsevier.) action of some of the earliest known cholesterol-low- ering drugs, but also such dietary constituents as the phytosterols (sitosterol and stanol esters) and soluble fi ber or NSPs. For further details of the control mech- anism see Section 6.5.

Liver cell LDL Y Y Y

Y

HMG-CoA reductase Y

LDL Acetate Free cholesterol receptors ؉

7-␣-Hydroxylase ؉>؊

x Excreted Dietary cholesterol ϩ bile acids in feces Figure 6.6 Interruption of the entero- Anion exchange resins/soluble hepatic circulation. LDL, low-density lipoprotein; HMG-CoA, 3-hydroxy-3-methyl- Intestine glutaryl-coenzyme A. 96 Introduction to Human Nutrition

Re-esterifi cation of triacylglycerols in these molecules are rapidly acylated back to 1,2-dia- the enterocyte cylglycerol and fi nally TAGs by the sequential actions Once LCFAs have entered the cell they are activated of three enzymes: CoA ligase, monoglycerol acyl- by acyl-CoA and are re-esterifi ed with glycerol back transferase, and diacylglycerol acyltransferase. In a into TAG and phospholipids by two distinct bio- similar fashion, lysophosphatidylcholine, produced chemical pathways, the 2-MAG and glycerol-3- by the action of pancreatic phospholipase ‘A’ on phosphate (G-3-P) pathways. The difference between dietary phospholipids, is absorbed by the enterocyte these two pathways lies in: and re-esterifi ed back to phosphatidylcholine in the enterocyte by direct acetylation (Figure 6.7). The bulk ● their substrates of activation of free cholesterol absorbed from the intestinal lumen ● the former using 2-MAG and the latter is also re-esterifi ed in the enterocyte by the enzyme α-glycero-3-phosphate ACAT. ● their location within different cellular organelles: the 2-MAGs reside in the smooth endoplasmic Lipoprotein assembly and secretion reticulum and the G-3-P in the rough endoplasmic Plasma lipoproteins are a family of spherical, macro- reticulum molecular complexes of lipid and protein, the princi- ● the periods during which they are most active. pal function of which is to transport endogenous The 2-MAG pathway is quantitatively of greater lipids (synthesized in the liver) and exogenous importance in the enterocyte of the intestine and thus lipids (synthesized in the gut from dietary fats) predominates in the postprandial period, whereas the from these sites of production and absorption to G-3-P pathway is more active in the postabsorptive peripheral sites of utilization (e.g., oxidation in phase in tissues such as liver, muscle, and adipose muscle, incorporation in membranes, or as precur- tissue. Following the absorption of a fatty meal and sors of biologically active metabolites) and storage uptake of 2-MAG into the enterocyte, up to 90% of (e.g., adipose tissue).

Exogenous lipid transport pathway Endogenous lipid transport pathway

Via lymph Liver VLDL Intestine CM

Capillary Energy in muscle Energy in muscle Lipoprotein VLDL lipase FA Lipoprotein CM FA lipase TAG in Hepatic adipose tissue TAG in lipase IDL adipose tissue 50% 50%

Liver CM Liver Other tissues remnant LDL 80% 20%

Figure 6.7 Re-esterifi cation of triacylglycerides in enterocytes. CM, chylomicron; FA, fatty acid; IDL, intermediate-density lipoprotein; LDL, low- density lipoprotein; TAG, triacylycerol; VLDL, very low-density lipoprotein. (Reproduced from Mangiapane EH, Salter AM, eds. Diet, Lipoproteins and Coronary Heart Disease. A Biochemical Perspective. Nottingham University Press, Nottingham, 1999, with permission of Nottingham University Press.) Nutrition and Metabolism of Lipids 97

In the small intestine, the newly re-esterifi ed TAGs chylomicrons containing lipids enriched with poly- and CEs associate with specifi c amphipathic proteins unsaturated fatty acids (PUFAs) are larger than and phospholipids in the enterocyte to form the chylomicrons enriched with , since the largest and most TAG-rich lipoproteins, known as former occupy more space when packaged into a lipo- chylomicrons. The enterocyte is capable of synthesiz- protein. This has implications for the subsequent ing three different apoproteins (apo): apoA-I, apoA- metabolism and fate of these lipoproteins in the cir- IVs and apoB (B-48). The last apoprotein is expressed culation, since TAGs associated with larger chylomi- in two isoforms, the arbitrarily named apoB-100, crons are known to be hydrolyzed more rapidly. It is which is synthesized in the liver, and a shorter relative thought that apoB-48 is produced continuously in the of B-100, which is produced by the enterocyte and is enterocyte-forming pools of apoB-48 in readiness for approximately 48% of the size of B-100 and thus the sudden reception of dietary fat and production of appropriately named apoB-48. While both apopro- chylomicrons. Nevertheless, small chylomicrons can teins are products of the same gene, the mRNA be detected throughout the postabsorptive phase. undergoes post-transcriptional editing in the entero- The onset, duration, and magnitude of postpran- cyte to produce a truncated polypepetide. ApoB-48 is dial lipemia can be monitored in the laboratory after produced in the rough endoplasmic reticulum and a standard fat-containing meal by making serial mea- transferred to the smooth endoplasmic reticulum, surements of serum TAG or more specifi cally TAG where it combines with a lipid droplet, or nascent associated with TAG-rich lipoproteins over a post- chylomicron, and then migrates to the Golgi appara- prandial period of up to 8 or 9 hours (remnants of tus. Here, the apoproteins (A-I, A-IV, and B-48) are chylomicrons can be detected 12 hours after a meal). glycosylated before the chylomicrons eventually leave Alternatively, the levels of apoB-48 or retinyl esters in the enterocyte by exocytosis through the basement serum act as useful markers or tracer molecules membrane, across the intracellular space between the for following the metabolism of chylomicrons in enterocyte and the lacteal, and are fi nally discharged the postprandial period. In normal subjects postpran- into the lymphatic vessels. dial lipemia peaks between 3 and 4 hours and sub- sides to baseline concentration after 5–6 hours. In Postprandial lipemia some cases, postprandial TAG (mainly in chylomi- The turbidity or milkiness of serum or plasma follow- crons) can appear in the blood within 30 min and ing the ingestion of fat marks the arrival of dietary fat peak as early as 1 hour after the ingestion of fat. So now contained in chylomicrons in the blood. The rapid is this rise in TAG that it is believed to represent milky appearance of plasma or serum after the inges- preformed lipid in the enterocyte from the previous tion of fat arises from the chylomicrons, which are of meal that is shunted into the circulation by the incom- a suffi cient size physically to scatter light and create ing fat load. Note that, in addition to the time taken the milky appearance of serum or plasma after a meal. to emulsify, hydrolyze, and absorb dietary fat, re- The size and composition of the chylomicrons pro- esterifi cation of TAG in the enterocyte and lipopro- duced after a fatty meal are determined by the fat tein assembly alone takes about 15 min, although content of the meal. Hence, the nature of fatty acids shunting means that the fi rst TAG can appear within in chylomicron TAG refl ects the nature of fatty acid 30 min, with the fi rst peak after 1 hour. This shunting in the meal. Each chylomicron particle carries a single phenomenon is particularly noticeable during the day molecule of apoB-48 which, unlike its other smaller and gives rise to two or even more peaks, whereas counterparts A-I and A-IV, remains with the chylomi- postprandial peaks following an overnight fast are cron throughout its life in the circulation. There is usually monophasic. little evidence to suggest that the production of apoB- Chylomicrons are not the only TAG-rich lipopro- 48, and thus the number of particles, increases in teins in the postprandial phase. Chylomicrons clearly response to an increased fl ux of dietary fat. Instead, contribute signifi cantly to the extent of postprandial the enterocyte incorporates more TAG into each chy- lipemia, and the rate at which the TAGs in these lipo- lomicron and expands the size of each chylomicron proteins are hydrolyzed is known to be a critical deter- to facilitate the transport of larger amounts of minant of the extent and time-course of postprandial absorbed dietary fat. There is evidence to suggest that lipemia. The TAGs in circulating chylomicrons are 98 Introduction to Human Nutrition

Table 6.4 Plasma lipoproteins: classes, composition, and distribution

Chylomicrons VLDLs LDLs HDLs

Mass (106 Da) 0.4–3.0 10–100 2–3.5 0.2–0.3 Density (g/ml) >0.95 <1.006 1.02–1.063 1.063–1.210 Particle diameter (nm) >90 30–90 22–28 5–12 Apoproteins B-48, A-I, C-I, C-II, C-III, E B-100, E B-100 A-I, A-II Lipids % mass (molecules/particle) Cholesterol 8 (60 000) 22 (10 000) 48 (2000) 20 (100) Triacylglycerols 83 (500 000) 50 (24 000) 10 (300) 8 (20) Ratio of particles Postabsorptive 1 40 1000 10 000 Postprandial 1 25 250 250 000

VLDL, very low-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein. lipolyzed by a rate-limiting lipase known as lipopro- about the same time, evidence emerged that TAG-rich tein lipase (LPL). LPL is tethered to the endothelial lipoproteins, and especially remnants of chylomi- lining of blood vessels in peripheral tissues, most crons, were directly atherogenic, meaning that they notably muscle and adipose tissue, by proteoglycan can damage the endothelial lining of and fi bers, and as such is known as an endothelial lipase. promote the deposition of cholesterol in coronary Several molecules of LPL can interact and lipolyze the arteries. For this reason, there is considerable research TAG from a single chylomicron particle to generate a interest in the metabolic determinants of postprandial chylomicron remnant which is removed by specifi c cell lipemia. This includes the mechanisms that underlie membrane receptors in the liver. The situation is com- the production and removal of TAG-rich lipoproteins, plicated by the fact that TAG-rich lipoproteins from not only in the intestine but also in the liver, since the the liver, known as very low-density lipoprotein production and removal of VLDL can clearly infl u- (VLDL), also contribute to this postprandial lipemia ence postprandial events. The quality and, to a lesser to variable extents in health and disease states. These extent, quantity of dietary fat are extremely important VLDLs containing endogenously produced TAG are in this respect and have a major role to play in modu- similar in lipid composition to chylomicrons but con- lating lipid-mediated atherosclerosis. siderably smaller (Table 6.4). While chylomicrons carry up to 80% of measurable plasma TAG during the 6.5 Circulating lipids: lipoprotein postprandial period, VLDL particles can carry up to structures and metabolism 80% of the measurable protein (mainly as apo-B), and signifi cantly outnumber chylomicrons at all times. Circulating blood lipids are insoluble in water and VLDL-TAG are also metabolized by LPL, which creates must be solubilized for transportation in the extracel- competition for the of endogenously and lular fl uid by combining with bipolar molecules with exogenously derived TAG carried by VLDLs and chy- charged and uncharged regions (apoproteins and lomicrons respectively. phospholipids). This property, known as amphipath- icity, enables these molecules to associate with aqueous Postprandial lipemia: relevance to (hydrophilic) and nonaqueous (hydrophobic) envi- atherosclerosis ronments and thus renders them perfect for It was suggested by Zilversmit in 1979 that atheroscle- enveloping insoluble lipids, chiefl y TAG and CE, in rosis was a postprandial phenomenon. This concept macromolecular lipid–protein complexes called lipo- was based on the fi nding that patients either with or at proteins. It is worth remembering that, in the absence high risk of developing coronary heart disease (CHD) of lipoproteins, TAG would exist in aqueous blood as showed an impaired capacity to remove TAG-rich immiscible oil droplets, while free fatty acids liberated lipoproteins from the circulation after a meal. This from TAG and phospholipids in the absence of the resulted in enhanced postprandial lipemia, which also blood protein albumin would act as detergents and became known as the TAG intolerance hypothesis. At dissolve cell membranes. Nutrition and Metabolism of Lipids 99

Lipoprotein structure: a shopping bag techniques permit the further resolution of VLDL, and groceries low-density lipoproteins (LDLs), and high-density lipoproteins (HDLs) into discrete subclasses, the dis- The general structure of a lipoprotein consists of a tribution of which relates to cardiovascular risk and central core of hydrophobic, neutral lipid (TAG and is determined by genetic and lifestyle factors. CE) surrounded by a hydrophilic coat of phospholip- ids, free cholesterol, and apoproteins. A useful analogy Lipoprotein transport pathways for this arrangement of molecules is that of a “shop- Lipoprotein transport can be described in terms of ping bag and groceries,” with the lipid core represent- the production, transport, and removal of cholesterol ing the groceries and the outer coat the fabric of the or TAG from the circulation. In reality, these two bag. The apoproteins weave in and out of the lipid processes are inseparable because both TAG and cho- core and outer surface layer and form the thread of lesterol are transported together in lipoproteins. the fabric which holds the bag together (see Figure Lipoproteins are in a constant state of change, with 6.5). This clever arrangement of molecules renders lipids and apoproteins constantly shuttling between the hydrophobic lipids soluble for the purpose of different lipoproteins that interrelate through inte- transport in blood. In addition to conferring struc- grated metabolic pathways. A useful analogy here is tural integrity on the lipoprotein particle, apoproteins to think of lipoproteins as railway trains, transporting have a vital role in controlling the metabolism of passengers that represent lipids and apoproteins lipoproteins by acting as ligands for cell membrane within a complex rail network. The trains and receptors and cofactors for key enzymes. passengers are in a constant state of fl ux within Plasma lipoproteins can be subdivided into distinct and between stations. Lipoprotein metabolism is con- classes on the basis of their physical properties and/or trolled by the activity of functional proteins (enzymes, composition, both of which refl ect the physiological cell surface receptors, receptor ligands) that deter- role in the transport of lipids from sites of synthesis mine the rate at which lipoproteins enter and leave (endogenous lipids) and absorption (exogenous the system, and by the physicochemical properties of lipids, absorbed in the gut) to sites of storage (adipose the lipoprotein themselves. This corresponds to all of tissue) and utilization (skeletal muscle). The principal the rate-limiting features of a train journey, the classes of lipoproteins are traditionally defi ned by number of trains, and type of passengers. density, which is determined by the ratio of lipid to All lipoproteins, with the notable exception of protein in the lipoprotein particle. Since lipids tend HDLs, begin life as TAG-rich particles The principal to occupy a greater molecular volume than proteins, transport function of these lipoproteins in the fi rst they are lighter and less dense. Thus, particles with instance is to deliver fatty acids liberated from the high lipid content are larger and less dense (carry TAG to tissues. The enterocytes in the gut are the more lipid groceries) than lipoproteins enriched with producers of lipoproteins which deliver dietary fats protein. This property relates directly to the transport into the blood as chylomicrons (exogenous lipid), function and metabolic interrelationships between whereas the liver is the central terminus for the pro- lipoprotein classes in blood. It can also be used to duction of VLDLs and removal of their cholesterol- separate lipoproteins of different densities because rich end-products, LDLs. VLDLs, although smaller lipoproteins of different density have different fl ota- than chylomicrons, resemble the latter in many ways tion characteristics in the ultracentrifuge (note that and are often referred to as the liver’s chylomicrons. plasma lipoproteins will fl when subjected to cen- While the rate at which the gut produces chylomi- trifugal force, whereas pure proteins sink). Other clas- crons depends largely on the amount of absorbed sifi cation schemes for plasma lipoproteins have dietary fat, the rate of production of VLDL is deter- exploited differences in their net electrical charge mined by the supply of fatty acids in the liver that can (electrophoretic mobility), particle size (exclusion be re-esterifi ed back to TAG for incorporation into chromatography, gradient gel electrophoresis), and VLDL. These fatty acids are derived chiefl y from the immunological characteristics conferred upon systemic circulation in the form of nonesterifi ed fatty the lipoprotein by the types of apoproteins in each acids (NEFAs), and to a lesser extent from the uptake lipoprotein subclass (see Table 6.4). Some of these of circulating lipoprotein remnants. It is noteworthy 100 Introduction to Human Nutrition that, although the liver has the capacity to synthesize activation of LPL, are progressively depleted of their fatty acids, the amount synthesized by de novo lipo- TAG in a stepwise fashion by LPL to become choles- genesis is relatively small in humans on a mixed terol-rich remnants that are removed by specifi c, Western diet. However, the contribution of fatty acids high-affi nity receptors found chiefl y in the liver. from this source may increase in conditions associ- Several molecules of LPL may bind to a single chylo- ated with an overproduction of VLDLs, and has been micron or VLDL particle, although LPL shows greater shown to occur on low-fat, high-carbohydrate diets, affi nity for chylomicrons in preference to VLDL. This and in metabolic disease. situation leads to competition between these TAG- rich lipoproteins and provides a mechanism to explain Metabolic determinants of how VLDL can infl uence the clearance of TAG in the lipoprotein metabolism postprandial period. The metabolism of serum lipoproteins and fate of Lipolyzed chylomicrons form chylomicron rem- their transport lipids is controlled by: nants which, during passage through the liver, bind to specifi c receptors on the surface of hepatocytes that ● the physical and chemical characteristics of the recognize apoE, an apoprotein that is also acquired at lipoprotein, such as its size and lipid and apopro- an early stage from HDLs. Remnant receptors are tein content maintained at a very high level of activity and are not ● the activity of the endothelial LPL and hepatic downregulated through a feedback mechanism (see lipase (HL), so called because they are attached to low-density lipoprotein receptor pathway). This is the surface of endothelial cells lining blood vessels fortunate, since chylomicron remnants have been in peripheral tissues, such as adipose tissue and shown to be capable of depositing their cholesterol in skeletal muscle, and the liver, respectively walls, thus promoting coronary atherosclerosis. ● lipid transfer proteins; cholesteryl ester and phos- The secretion of VLDL from the liver is again fol- pholipid transfer proteins, (CETP and PLTP lowed by the sequential lipolysis of TAG by LPL and respectively). generation of VLDL remnants or, in this case, the ● apoproteins that act as activators of enzymes and further lipolysis of these remnants into LDL. ligands for specifi c lipoprotein receptors on the sur- The remnants and LDLs bind to another receptor in faces of cells (apoB-100 and apoE as ligands for the liver that recognizes both apoE exclusively in the LDLs and remnant receptors in the liver, VLDL remnants and apoB-100 in LDLs, namely the respectively) LDL receptor. Approximately 60% of LDL is removed ● the activity of specifi c lipoprotein receptors on cell by the LDL receptor. The remainder is internalized surfaces. into cells via scavenger receptors. This latter route has Lipoprotein transport is traditionally described in been associated with the development of atheroscle- terms of the forward and reverse transport of choles- rotic disease. terol. Forward transport encompasses the exogenous Whether a VLDL particle is removed as a remnant and endogenous pathways, which describes the arrival or transcends to LDL largely depends on its pedigree, of cholesterol in the blood from either the gut or the i.e., its size and lipid composition. Experiments with liver and carriage back to the liver for processing; the radioactively labeled VLDL have shown that larger, liver has the unique capacity to secrete cholesterol TAG-rich VLDL particles are less likely to be con- either as free cholesterol or as bile acids. Conversely, verted into LDL and are removed as partially delipi- reverse transport describes the HDL pathway and the dated VLDL remnants, whereas smaller VLDLs are effl ux of cholesterol out of peripheral tissues back to precursors of LDL. the liver. This directionality can be misleading because each pathway can direct cholesterol back to the liver. The low-density lipoprotein Both the exogenous and endogenous pathways share receptor pathway a common saturable lipolytic pathway that consists of The incontrovertible link between plasma cholesterol a delipidation cascade in which the TAG-rich lipopro- and CHD is directly responsible for the rapid growth, teins (chylomicrons and VLDLs), after receiving apo- and occasional quantum leaps, in our understanding C (C-II) from HDL, an essential for the of cholesterol homeostasis in relation to diet and Nutrition and Metabolism of Lipids 101

Serum LDL

Liver cell Y Y Y LDL

Y HMG-CoA reductase Y Y LDL Acetate Y Free cholesterol receptors ؉

N-SREBP LDL receptor protein

Transcription Figure 6.8 Low-density lipoprotein (LDL) factor receptor pathway. HMG-CoA, 3-hydroxy-3- Transcription LDL receptor gene ؉ methyl-glutaryl-coenzyme A; SREBP, sterol regulatory element binding protein. disease, the most prolifi c of which was the discovery The metabolic effects of intracellular free cholesterol of the LDL receptor pathway by the Nobel Prize are: winners Goldstein and Brown (1977) (Figure 6.8). All ● it decreases the production of LDL receptors via cells, most notably those in the liver, have a highly SREBP developed and sensitive mechanism for regulating ● it inhibits the synthesis of cholesterol by the intracellular and intravascular levels of cholesterol. enzyme 3-hydroxy-3-methylglutaryl (HMG)-CoA Cells in the liver synthesize approximately 500 mg of reductase cholesterol a day and, although they can import the ● it increases the re-esterifi cation of cholesterol for same quantity from the blood in the form of LDL, in storage as cholesterol esters. the complete absence of LDL, cells could theoretically manufacture suffi cient cholesterol to meet their met- Goldstein and Brown were aided in the discovery abolic needs. However, when stressed, cells will always of the LDL receptor by studying a condition known import cholesterol in preference to synthesizing it as familial hypercholesterolemia, a genetic abnormal- themselves as the former process takes less energy. ity in the LDL receptor gene that produces defects in Cells acquire cholesterol from the blood by the uptake the LDL receptor pathway and considerably elevated and degradation of LDL particles. As the requirement blood cholesterol concentrations in early life (15– for free cholesterol increases within the cell, it increases 20 mmol/l) and premature cardiovascular disease. its production and thus activity of LDL receptors, so They also initiated pioneering studies on the infl u- that more LDL is extracted from the blood, lowering ence of dietary fats on the activity of the LDL pathway, blood cholesterol. Conversely, if the cell becomes which led to a widely accepted explanation for overloaded with cholesterol, it senses that it requires the cholesterol-raising properties of saturated fatty less cholesterol and produces fewer LDL receptors, acids. causing blood cholesterol to increase. Since the pro- duction of LDL receptors is regulated by the intracel- Reverse cholesterol transport (high-density lular level of free cholesterol, anything that increases lipoprotein pathway) free cholesterol within the cell will inadvertently lower The removal of cholesterol from tissues back to the blood LDL cholesterol. Intracellular free cholesterol liver via HDLs represents the only route of elimina- represses the activity of a sterol regulatory element tion for cholesterol from the body. This physiological binding protein (SREBP), a positive nuclear tran- role of HDLs explains, in part, the cardioprotective scription factor that promotes the transcription of the effects of these lipoproteins, as indicated by a strong LDL receptor gene when free cholesterol levels fall. inverse relationship between serum HDL cholesterol 102 Introduction to Human Nutrition

Pre-␤-HDL Free Peripheral tissues LCAT (including lesions) cholesterol

HDL3

LPL Hepatic Liver lipase

CE Figure 6.9 Reverse cholesterol trans- port. CE, cholesterol ester; CETP, choles- terol ester transfer protein; HDL,

HDL2 high-density lipoprotein; LCAT, lecithin– LDL VLDL cholesterol acyltransferase; LDL, low- density lipoprotein; LPL, lipoprotein CE lipase; VLDL, very low-density CETP lipoprotein. and CHD risk in prospective cohort studies. The phospholipid of HDL, where it effectively punches activity of the HDL pathway is infl uenced by genetic a hole in the surface coat to facilitate access to the and dietary factors that can interact to either increase lipid core and delivery of CE to the hepatocyte or reduce the effi ciency of cholesterol removal. This, (Figure 6.9). in turn, may be refl ected in changes in the concentra- tion of serum HDLs and their functional properties. Interrelationships among serum HDLs are synthesized in the gut and liver, and triacylglycerols and low- and increase their particle size in the circulation as a result high-density lipoproteins of the acquisition of cholesterol from two principal Lipids are constantly moving between lipoprotein sources: (1) surface material released from TAG-rich particles. This movement is not totally random but lipoproteins during lipolysis and (2) peripheral infl uenced by the relative lipid composition of the tissues. The particles, which are responsible for lipoproteins and by specifi c lipid transfer proteins removing cholesterol from cells, are very small pre- (LTPs) that act as lipid shuttles. In a normal, healthy HDLs and are disk-shaped particles composed of individual, TAG-rich lipoproteins transfer TAG to phospholipid and apoA-I (ApoA-I is capable of this LDL and HDL in equimolar exchange for CE. This is function on its own). The effl ux of free cholesterol mediated through an LTP called cholesteryl transfer from tissue sites, including deposits of cholesterol in protein (CETP). In this way, CEs are transferred from the coronary arteries, is facilitated by the formation HDL to VLDL for passage back to the liver. Conversely, of a free cholesterol gradient from the cell across the when the concentration of serum TAG and thus TAG- cell membrane to pre-HDLs. The gradient is gener- rich lipoproteins is increased, for example by either ated by the re-esterifi cation of free cholesterol by the the overproduction of TAG in the liver or the impaired enzyme lecithin–cholesterol acyltransferase (LCAT) removal of TAG by LPL, the result is a net transfer of and via the migration of these newly formed choles- TAG into LDL and HDL. As LDL and HDL are over- terol esters into the hydrophobic core of what becomes loaded with TAG they become favored substrates for mature, spherical HDL. The newly acquired choles- the action of HL and are remodeled into smaller and terol is transported back to the liver either directly by denser particles. While small, dense HDL is catabo- HDL or indirectly by transfer to apoB-containing lized rapidly in the liver, lowering serum HDL and lipoproteins VLDL and LDL. Blood vessels in the liver impairing reverse cholesterol transport, small, dense contain a close relative of LPL, i.e., HL. This enzyme LDLs are removed less effectively by LDL receptors acts on smaller lipoproteins and especially the surface and accumulate in serum. Small, dense LDL, by virtue Nutrition and Metabolism of Lipids 103 of its size, has a much greater potential to infi ltrate resistant conditions. Insulin also stimulates the the artery wall and deposit its cholesterol. Even a synthesis of cholesterol by activating HMG-CoA moderately raised concentration of serum TAG reductase and the activity of LDL receptors, although (>1.5 mmol/l) may be inversely associated with the overall effect on cholesterol homeostasis is small reduced HDL cholesterol (<1 mmol/l) and a predomi- in relation to the control of the LDL pathway described nance of small, dense LDL. This collection of fi ndings above. is known as the atherogenic lipoprotein phenotype (ALP) and is a very common but modifi able source of Sex hormones increased CHD risk in free-living populations. The effect of sex hormones on serum lipoproteins is best illustrated by the pronounced differences in lipid Endocrine control of and lipoprotein profi les between adult men and pre- lipoprotein metabolism menopausal women. Men present with higher total All hormones exert an infl uence on lipoprotein serum and LDL cholesterol, higher serum TAG, and metabolism. However, with respect to diet and the lower HDL cholesterol concentrations than premeno- control of postprandial lipid metabolism, insulin has pausal women. This difference in lipid profi les confers by far the greatest impact. Although classically associ- protection against CHD on premenopausal women ated with carbohydrate metabolism and the uptake of so that their CHD risk lags some 10 years behind that glucose into cells, the actions of insulin are critical to of men of the same age. This applies until estrogen the control of postprandial lipid metabolism. Insulin failure at the menopause, when CHD risk in women is secreted in response to the reception of food in the overtakes that of men. Estrogen was the fi rst com- gut and it: pound shown to stimulate LDL receptor activity in cell culture, an effect that not only accounts for lower ● stimulates LPL in adipose tissue LDL levels in women but also the sharp increase in ● suppresses the intracellular lipolysis of stored TAG LDL cholesterol after the menopause, to levels in adipose tissue by inhibiting hormone-sensitive above those of men. Estrogens also stimulate the pro- lipase duction of TAG and VLDL, but any adverse effects ● suppresses the release of VLDL from the liver. must be outweighed by the effi ciency of TAG removal Insulin coordinates the lipolysis of dietary TAG and mechanisms that maintain lower serum TAG levels in uptake of NEFA into adipose tissue. It achieves this women than in men until the menopause. In addition by minimizing the release of NEFA from TAG stores to these effects, estrogen selectively inhibits the activ- in adipose tissue and TAGs produced in the liver by ity of HL, which contributes to the HDLs in women. suppressing the secretion of VLDL. The sensitivity of In direct contrast, the androgenic male hormone tes- the target tissues – liver, adipose tissue, and, perhaps tosterone suppresses LDL receptor activity, raising to a lesser extent, skeletal muscle – to insulin is critical LDL cholesterol. It is also a powerful stimulant of HL to the maintenance of these effects. Failure of insulin activity, and is responsible for lowering HDL choles- action, insulin resistance, in conditions such as obesity terol in men, most notably in male body builders on and diabetes results in dyslipidemia characterized by anabolic steroids, in whom serum HDL can be almost an impaired capacity to lipolyze TAG-rich lipopro- absent. teins (TAG intolerance or enhanced postprandial lipemia). This effect is compounded by the failure of The triacylglycerol hypothesis insulin to suppress the mobilization of NEFA from Dietary effects on serum cholesterol or LDLs alone adipose tissue TAG, which increases the fl ux of NEFA provide an inadequate basis on which to explain the to the liver and stimulates the overproduction of relationship between diet and CHD within popula- VLDL (‘portal hypothesis’). The suppression of VLDL tions. The ability of humans to protect themselves secretion is also abolished so that VLDL is released against an overaccumulation of cholesterol in their into the postprandial circulation and is free to compete vascular system through nutritional changes depends with chylomicrons, augmenting postprandial lipemia to a much greater extent on increasing the effi ciency still further. This series of events gives rise to a dys- of the HDL pathway and utilization of TAG-rich lipidemia or ALP, which is found frequently in insulin- lipoproteins. The latter represent the precursors of 104 Introduction to Human Nutrition potentially harmful cholesterol-rich remnants and of proteins functioning as receptors, transporters, LDLs that contribute to coronary atherosclerosis. The enzymes, ion channels, etc. Some lipids, i.e., PUFAs, effects of diet, and in particular dietary fats, in modu- confer the feature of “fl uidity” to membranes, whereas lating the clearance of TAG-rich lipoproteins in the others, i.e., cholesterol and saturated fatty acids, have postprandial period is of paramount importance in the opposite rigidifying effect. Membranes have preventing the accumulation of atherogenic remnants extraordinarily diverse fatty acid profi les and phos- and development of proatherogenic abnormalities in pholipid composition depending on their tissue and LDL and HDL. The actions of insulin coordinate the subcellular location. They are also the body’s reservoir metabolism of TAG-rich lipoproteins but can become of both fat-soluble vitamins and eicosanoid precur- defective through energy imbalance, weight gain, sors such as arachidonate. and ultimately obesity. As a consequence, the most Most of the body’s cholesterol is present in the common abnormalities in lipoproteins to increase unesterifi ed form in membranes, where it represents risk in populations arise from a primary defect in the 35–45% of total lipids. Skin, plasma, and adrenal metabolism of TAG, induced through insulin resis- cortex contain 55–75% of cholesterol in the esterifi ed tance and not cholesterol per se. Equally important is form. Bile also contains free cholesterol and bile salts the fact that these metabolic defects originate, in part, derived from cholesterol. through nutrient–gene interactions and are thus highly amenable to dietary modifi cation. Storage lipid pool Triacylglycerols are the main energy storage form of 6.6 Body lipid pools lipids and they are the principal component of body fat. TAG-containing fatty acids destined for Lipids in the human body exist in two major pools: oxidation are also present in measurable but much structural lipids in membranes and storage lipids in lower amounts in all tissues that can oxidize long- body fat. The lipid composition and metabolic fate of chain fatty acids, i.e., muscle and heart. TAG is syn- these two pools are quite distinct, although many of thesized by the intestine and liver, where it is subse- the fatty acids occupying both pools are the same. The quently incorporated into lipoproteins (see Section main components of both membrane and storage 6.4) for the transport of lipids to and from other lipids are the long-chain (16–24 carbons) saturated, tissues. monounsaturated, and polyunsaturated fatty acids. The main fatty acids in the TAG of adult human Although several of the major long-chain fatty acids body fat are palmitate (20–30%), stearate (10–20%), in the body are common to both membrane and oleate (45–55%), and linoleate (10–15%). The fatty storage lipids, namely palmitate, stearate, oleate, and acid profi le of adult body fat always refl ects the profi le linoleate, three important distinctions exist between of dietary fat. Only rarely would this result in other membrane and storage lipids. fatty acids being more prevalent in body fat than the four listed here. At birth, the fatty acid profi le of body 1 Membrane lipids are not usually hydrolyzed to fat is unusual in having very low linoleate (<3%) and release free fatty acids for energy metabolism. α-linolenate (<1%) but a higher proportion of long- 2 Membrane lipids contain a much higher propor- chain polyunsaturates than later in life. Body fat occu- tion of long-chain PUFAs. pies several discrete sites that expand and contract as 3 Membrane lipids are more diverse and rarely needed. Body fat is about 82% by weight TAG, making include TAGs, which are the main component of it by far the main body pool of palmitate, stearate, storage lipids. oleate, and linoleate. The main sites of body fat are subcutaneous and Structural lipid pool intravisceral, and they have different rates of response Biological membranes surrounding cells and subcel- to stimuli for accumulation or release of fatty acids. lular organelles exist primarily as lipid bilayers (Figure Within a given site, growing evidence suggests that 6.3). The lipids in both the inner and outer surfaces PUFAs are more easily released from adipose tissue of membranes are composed mainly of phospholip- TAG than are saturated fatty acids, especially during ids and free cholesterol, which interface with a myriad fasting or longer term energy defi cit. Nutrition and Metabolism of Lipids 105

Plasma and milk lipids also rises rapidly in brain lipids, followed a little later In a way, plasma and milk lipids are an exception to by an increasing content of long-chain saturates and the general rule distinguishing membrane and storage monounsaturates as myelin develops. Adipose tissue α lipids. Plasma and milk lipids are present mostly as contains very little linoleate or -linolenate at birth lipoproteins, comprising mostly phospholipids and but their content increases rapidly with milk feeding. cholesterol in the surrounding membrane and TAG Plasma cholesterol is relatively low at birth and in in the core (see Section 6.5). Plasma lipids contain the infancy, but increases by more than twofold by only signifi cant pool of free fatty acids or NEFAs in adulthood. the body. Free fatty acids are not components of lipo- In general, regardless of the profi le of dietary fatty proteins but are transported bound to albumin. They acids, saturated and monounsaturated fatty acids pre- are liberated mostly from adipose tissue when plasma dominate in adipose tissue, whereas there is a closer glucose and insulin are low. Plasma also contains pro- balance between saturates, monounsaturates, and portionally more fatty acids esterifi ed to cholesterol polyunsaturates in structural lipids. Long-chain (cholesteryl esters) than are found in tissues. PUFAs such as docosahexaenoate are present in high concentrations in specialized membranes, including Whole body content and organ profi le of those of the retina photoreceptor, in synapses of the fatty acids brain, and in sperm. An estimate of the whole body content of lipids in a healthy adult human is given in Table 6.5. Additional body fat is deposited during pregnancy, but the fatty 6.7 Long-chain acid composition remains similar to that of nonpreg- Synthesis nant adults and refl ects dietary fat intake. The total lipid content of plasma rises in the third trimester, with a Synthesis of fatty acids occurs in the cytosol. It begins proportionally greater increase in saturated fatty acids with acetyl-CoA being converted to malonyl-CoA by than PUFAs. This downward trend in the percentage of acetyl-CoA carboxylase, an enzyme dependent on PUFA towards term has led to some concern about the biotin. Malonyl-CoA and a second acetyl-CoA then β possible adverse consequences for the fetus of defi - condense via -ketothiolase. This is subsequently ciency of PUFA. However, the actual amount of PUFA reduced, dehydrated, and then hydrogenated to yield in blood lipids rises but less so than for saturated fatty a four-carbon product that recycles through the same acids; resulting in a proportional decrease in PUFA. series of steps until the most common long-chain Soon after birth, body lipid composition starts to fatty acid product, palmitate, is produced (Figure change. Brain cholesterol rises moderately from under 6.10). Acetyl-CoA is primarily an intramitochondrial 40% to nearly 50% of brain lipids. Docosahexaenoate product. Thus, the transfer of acetyl-CoA to the cytosol for fatty acid synthesis appears to require its conversion to citrate to exit the mitochondria before being reconverted to acetyl-CoA in the cytosol. Table 6.5 Body fat content of major fatty acids in humans There are three main features of long-chain fatty Fatty acid Content (g) acid synthesis in mammals:

Palmitic acid 3320 1 inhibition by starvation Stearic acid 550 2 stimulation by feeding carbohydrate after fasting Oleic acid 6640 3 general inhibition by dietary fat. 1560 Arachidonic acid 80 Carbohydrate is an important source of carbon for α-Linolenic acid 130 Eicosapentaenoic acid <10 generating acetyl-CoA and citrate used in fatty acid Docosahexaenoic acid <10 synthesis. Enzymes of carbohydrate metabolism also Total 12 300 help to generate the NADPH needed in fatty acid Data are based on a 70 kg adult human with 20% (14 kg) body fat. synthesis. Acetyl-CoA carboxylase is a key control Fat tissue contains about 88% actual fatty acids by weight, yielding point in the pathway and is both activated and induced about 12.3 kg fatty acids in this example. to polymerize by citrate. Acetyl-CoA carboxylase is 106 Introduction to Human Nutrition

C—C—CO (Malonyl SA) + C—CO (Acetyl SS) R—CH2—CH2—CH2—CO—CoA

FAD Condensation

FADH

CO2 + C—CO—C—CO (Acetoacetyl SA) — R—CH2—CH—CH—CO—CoA

First reduction H2O

C—COH—C—CO (β-Hydroxybutyryl SA) R—CH2—CHOH—CH2—CO—CoA

Dehydration NAD1

H2O NADH C—C— C—CO (Crotonyl SA)

R—CH2—CO—CH2—CO—CoA Second reduction CoA

C—C—C—CO (Butyryl SA) Figure 6.10 Principal steps in fatty acid synthesis. The individual steps occur with the substrate being anchored to the acyl carrier protein. SA, S-acyl carrier protein; SS, S-synthase. R—CH2—CO—CoA CH2—CO—CoA Figure 6.11 Principal steps in β-oxidation of a saturated fatty acid. The steps shown follow fatty acid “activation” (binding to coenzyme inhibited by long-chain fatty acids, especially PUFAs A) and -dependent transport to the inner surface of the mito- such as linoleate. This is probably one important chondria. Unsaturated fatty acids require additional steps to remove the double bonds before continuing with the pathway shown. FAD, negative feedback mechanism by which both starva- fl avin adenine dinucleotide; FADH reduced fl avin adenine dinucleo- tion and dietary fat decrease fatty acid synthesis. High tide; R, 12 carbons. amounts of free long-chain fatty acids would also compete for CoA, leading to their β-oxidation. normal food intake can markedly alter tissue fatty Elongation of palmitate to stearate, etc., can occur in acid profi les. mitochondria using acetyl-CoA, but is more com- monly associated with the endoplasmic reticulum Oxidation where malonyl-CoA is the substrate. β-Oxidation is the process by which fatty acids are Humans consuming >25% dietary fat synthesize utilized for energy. Saturated fatty acids destined for relatively low amounts of fat (<2 g/day). Compared β-oxidation are transported as CoA esters to the outer with other animals, humans also appear to have a leafl et of mitochondria by FABP. They are then relatively low capacity to convert stearate to oleate translocated inside the mitochondria by carnitine and linoleate or α-linolenate to the respective longer acyl-transferases. The β-oxidation process involves chain polyunsaturates. Hence, the fatty acid profi les repeated dehydrogenation at sequential two-carbon of most human tissues generally refl ect the intake of steps and reduction of the associated fl avoproteins dietary fatty acids; when long-chain n-3 PUFAs are (Figure 6.11). Five ATP molecules are produced present in the diet, this is evident in both free-living during production of each acetyl-CoA. A further 12 humans as well as in experimental animals. Neverthe- ATP molecules are produced after the acetyl-CoA less, fatty acid synthesis is stimulated by fasting/ condenses with oxaloacetate to form citrate and goes refeeding or weight cycling, so these perturbations in through the tricarboxylic acid cycle. Nutrition and Metabolism of Lipids 107

The effi ciency of fatty acid oxidation depends on starvation, diabetes, and a very high-fat, low- the availability of oxaloacetate and, hence, concurrent carbohydrate “ketogenic” diet. carbohydrate oxidation. β-Oxidation of saturated fatty acids appears to be simpler than oxidation of Carbon recycling unsaturated fatty acids because, before the acetyl- Carbon recycling is the process by which acetyl-CoA CoA cleavage, it involves the formation of a trans derived from β-oxidation of one fatty acid is incor- double bond two carbons from the CoA. In contrast, porated into another lipid instead of completing the β-oxidation of unsaturated fatty acids yields a double β-oxidation process to carbon dioxide. In principle, bond in a different position that then requires further all fatty acids undergo this process to some extent but isomerization or hydrogenation. From a biochemical it is most clearly evident for two PUFAs, linoleate and perspective, this extra step appears to make the oxida- α-linolenate. Carbon recycling captures the over- tion of unsaturated fatty acids less effi cient than that whelming majority of α-linolenate carbon, i.e., about of saturated fatty acids. However, abundant in vivo 10 times more than is incorporated into docosahexae- and in vitro research in both humans and animals noate, which remains in the body of suckling rats 48 clearly shows that long-chain cis-unsaturated fatty hours after dosing with uniformly 13C-labeled α-lino- acids with one to three double bonds (oleate, linole- lenate. Carbon recycling of linoleate in the rat cap- ate, α-linolenate) are more readily β-oxidized than tures similar amounts of the linoleate skeleton to saturated fatty acids of equivalent chain length, such those of arachidonate, the main desaturation and as palmitate and stearate. The oxidation of PUFA chain-elongation product of linoleate. Hence, carbon and monounsaturates in preference to saturates has recycling appears to be a ubiquitous feature of the potential implications for chronic diseases such as metabolism of PUFA, although its biological signifi - coronary artery disease because their slower oxida- cance is still unclear. tion implies slower clearance from the blood, thereby providing more opportunity for esterifi cation to cho- Peroxidation lesterol and subsequent deposition in the vessel wall. Peroxidation (auto-oxidation) is the nonenzyme-cat- In peroxisomes, fatty acid β-oxidation is a trun- alyzed reaction of molecular oxygen with organic cated process by which long-chain PUFAs are chain compounds to form peroxides and related breakdown shortened. This peroxisomal detour has been identi- products. PUFAs are particularly vulnerable to peroxi- fi ed as an obligatory step in the endogenous synthesis dation at the double bonds. Initiating agents such as of docosahexaenoate from eicosapentaenoate. pre-existing peroxides, transition metals, or ultraviolet Odd-carbon long-chain fatty acids are relatively or ionizing radiation produce singlet oxygen. Singlet uncommon but, when β-oxidized, yield propionyl- oxygen can then abstract hydrogen at the double bonds CoA, the further β-oxidation of which requires biotin of polyunsaturates to produce free (peroxy) radicals, and vitamin B12 as coenzymes. which abstract further hydrogens from the same or different fatty acids and propagate the peroxidation Ketogenesis and ketosis process. Eventually, this leads to termination by the Large amounts of free fatty acids inhibit glycolysis formation of stable degradation products or hydro- and the enzymes of the tricarboxylic acid cycle, peroxides (Figure 6.12). Trans isomers are frequently thereby impairing production of oxaloacetate. When formed during the process. Hydroperoxides can form insuffi cient oxaloacetate is available to support the further hydroperoxy radicals or can be reduced by continued oxidation of acetyl-CoA, two acetyl-CoA , which contain thiol groups, i.e., glutathi- molecules condense to form a ketone, acetoacetate. one and cysteine. Peroxidation of dietary fats gives rise Acetoacetate can be spontaneously decarboxylated to to aldehydes, i.e., 2-undecenal, 2-decenal, nonanal, or form acetone, a volatile ketone, or converted to a third octanal, which have a particular odor commonly ketone, β-hydroxybutyrate. When glucose is limiting, known as rancidity. ketones are an alternative source of energy for certain Since peroxidation is a feature of polyunsaturates, organs, particularly the brain. They are also effi cient it is a potential hazard facing most membranes and substrates for lipid synthesis during early postnatal dietary lipids. Antioxidants such as are development. Conditions favoring ketogenesis include usually present in suffi cient amounts to prevent or 108 Introduction to Human Nutrition

acyl chain into which the double bond is inserted. HH Although myristate (14:0) and palmitate can be X• converted to their monounsaturated derivatives, XH myristoleate (14:1n-5) and palmitoleate (16:1n-7) respectively, commonly it is only the fatty acids of 18 or more carbons that undergo desaturation. The Δ9 H • desaturases in all organisms, except for anaerobic bac- teria, use oxygen and NADPH to introduce a cis double bond at carbons 9 and 10 of stearate. This is • H accomplished by an enzyme complex consisting of a series of two cytochromes and the terminal desatu- rase itself. The acyl-CoA form of fatty acids is the O2 usual substrate for the desaturases, but fatty acids esterifi ed to phospholipids can also be desaturated H OO • in situ. All mammals that have been studied can convert stearate to oleate via Δ9 desaturase. However, in the O O absence of dietary oleate, young rats may have insuf- • fi cient capacity to sustain normal tissue oleate levels. Normal values depend on the reference, which can Endoperoxide H OOH vary widely depending on the source and amount of oleate in the diet. Nevertheless, it is important to dis- tinguish between the existence of a given desaturase O O Hydroperoxide + R• and the capacity of that pathway to make suffi cient of the necessary product fatty acid. Hence, as with the HH long-chain polyunsaturates and, indeed, with other Malondialdehyde nutrients such as amino acids (see Chapter 4), it is Figure 6.12 Principal steps in peroxidation of a polyunsaturated fatty important to keep in mind that the existence of a acid. pathway to make a particular fatty acid or amino acid does not guarantee suffi cient capacity of that pathway to make that product. This is the origin of the concept block peroxidation in living tissues. Humans and of “conditional essentiality” or “indispensability.” animals readily detect peroxidized fats in foods by Both plants and animals are capable of desaturating their disagreeable odor and avoid them. However, at the 9–10 carbon (Δ9 desaturase) of stearate, result- modeling the effects of peroxides produced in vivo ing in oleate. However, only plants are capable of and in vitro is particularly challenging because lipid desaturating oleate to linoleate and then to α-lino- peroxidation undoubtedly is an important part of lenate. Once linoleate and α-linolenate are consumed several necessary biological processes such as activa- by animals, their conversion to the longer chain tion of the immune response. PUFAs of their respective families proceeds primarily by an alternating series of desaturation (Δ6 and Δ5 Desaturation, chain elongation, desaturases) and chain-elongation steps (Figure 6.13). and chain shortening Sequential desaturations or chain elongations are also One important characteristic of long-chain fatty acid a possibility, resulting in a large variety, though low metabolism in both plants and animals is the capacity abundance, of other PUFAs. to convert one to another via the processes of desatu- During dietary defi ciency of linoleate or α-lino- ration, chain elongation, and chain shortening. lenate, oleate can also be desaturated and chain elon- Plants and animals use desaturases to insert a gated to the PUFA eicosatrienoate (20:3n-9). Hence, double bond into long-chain fatty acids. There are most but not all PUFAs are derived from linoleate or several desaturases, depending on the position in the α-linolenate. Nutrition and Metabolism of Lipids 109

w 6 Polyunsaturates w 3 Polyunsaturates

Linoleic α-Linolenic

Δ6 Desaturation

γ-Linolenic Stearidonic

Chain elongation

Dihomo-γ-Linolenic ω3-Eicosatrienoic

Δ5 Desaturation

Arachidonic Eicosapentaenoic Figure 6.13 Conversion of linoleic (18:2n-6) and α Chain elongation -linolenic (18:3n-3) acids to their respective longer chain, more unsaturated polyunsaturates. In mem- branes, linoleic and arachidonic acids are the prin- Adrenic ω3-Docosapentaenoic cipal n-6 polyunsaturates, while docosahexaenoic acid is the principal n-3 polyunsaturate. Hence, these two families of fatty acids have different Chain elongation, affi nities for the desaturation and chain-elongation peroxisomal chain shortening enzymes. This pathway is principally based in the endoplasmic reticulum but appears to depend on peroxisomes for the fi nal chain shortening, which involves 24 carbon intermediates that are not ω6-Docosapentaenoic Docosahexaenoic illustrated.

Chain elongation of saturated and unsaturated bacteria are the only organisms known to have this fatty acids occurs primarily in the endoplasmic retic- capability. As in chemical hydrogenation practiced by ulum, although it has also been demonstrated to the food industry, biohydrogenation in the rumen can occur in mitochondria. Unlike the desaturation steps be incomplete, resulting in the formation of small immediately before and after, the elongation steps do amounts of trans isomers, particularly of oleate, lino- not appear to be rate limiting in the metabolism of leate, and α-linolenate, which are found in milk fat. linoleate or α-linolenate. Despite the capacity to insert at least three double Eicosanoids bonds in both n-3 and n-6 polyunsaturates, there is Eicosanoids are 20-carbon, oxygen-substituted no proof that a Δ4 desaturase exists to insert the fi nal cyclized metabolites of dihomo-γ-linolenate, arachi- double bond in docosapentaenoate (22:5n-6) or doc- donate, or eicosapentaenoate. They are produced via osahexaenoate (Figure 6.13). Rather, it appears that a cascade of steps starting with the cyclooxygenase or the precursors to these two fatty acids undergo a lipoxygenase enzymes present in microsomes. The second elongation, repeated Δ6 desaturation followed main cyclooxygenase products comprise the classical by chain shortening in peroxisomes. This unexpect- prostaglandins, prostacyclin and the thromboxanes. edly convoluted series of steps is corroborated by the The main lipoxygenase products are the leukotrienes docosahexaenoate defi ciency observed in disorders (slow-reacting substances of anaphylaxis) and the of peroxisomal biogenesis such as Zellweger’s noncyclized hydroperoxy derivatives of arachidonate syndrome. that give rise to the hepoxylins and lipoxins (Figure 6.14). Hydrogenation Eicosanoids are considered to be fast-acting local Opposite to the desaturation process is hydrogena- hormones, the presence of which in the plasma and tion or removal of unsaturated bonds in lipids. Rumen urine is largely a spillover from localized production, 110 Introduction to Human Nutrition

Membrane arachidonic acid

Figure 6.14 The arachidonic acid cascade Phospholipase A2 is a fundamental component of cell signal-

ing during injury. Phospholipase A2 is immediately activated and the free arachi- Free arachidonic acid donic acid thus released is accessible to a controlled peroxidation process involving several cyclooxygenases (constitutive or Lipoxygenase Cyclooxygenase inducible) and lipoxygenases. Over 50 metabolically active products are poten- tially produced, depending on the tissue involved, the type of cell that has been stimulated, and the type of injury. Only the HPETEs PGG2 main classes of these metabolites are Peroxidase shown. Before excretion, they are further metabolized to stable products that are not shown. Several of the cyclooxygenase products are competitive with each other, HETEs PGH2 Hepoxylins such as the platelet-aggregating and blood vessel wall-constricting effects of Leukotrienes Lipoxins thromboxane A2 (TXA2) produced in plate- lets, versus the opposite effects of prosta-

PGs PGI2 cyclin (PGI2) derived from the blood vessel wall. HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; TXA2 PG, ; TX, thromboxane. usually in response to an injury or a stimulus that derived from dihomo-γ-linolenate (1 series) and from releases the free precursor, most commonly arachido- eicosapentaenoate (3 series) often have effects that nate. The site of highest eicosanoid concentration oppose those derived from arachidonate (2 series) appears to be the seminal fl uid, although some species (Figures 6.14 and 6.15). Thus, unlike prostaglandin have no detectable eicosanoids in semen. Eicosanoids E2, prostaglandin E1 has anti-infl ammatory actions, are second messengers modulating, among other reduces vascular tone, and inhibits platelet aggrega- pathways, protein phosphorylation. The lung is a tion. Fourth, varying the ratio of the precursor fatty major site of eicosanoid inactivation. acids in the diet is an effective way to modify eico- Four important characteristics of eicosanoid action sanoid production. Thus, eicosapentaenoate and should be noted. First, individual eicosanoids often dihomo-γ-linolenate inhibit the synthesis of 2 series have biphasic actions as one moves from very low eicosanoids derived from arachidonate. This occurs through to higher, often pharmacological, concentra- by inhibiting arachidonate release from membranes tions. Thus, effects can vary dramatically depending by phospholipase A2 and its cascade through the not only on the experimental system but also on the cyclooxygenases and lipoxygenases. The overproduc- eicosanoid concentration used. Second, several of the tion of 2 series eicosanoids is associated with higher more abundant eicosanoids arising from the same blood pressure, increased platelet aggregation, and precursor fatty acid have opposite actions to each infl ammatory processes, and can be effectively inhib- other. For instance, prostacyclin and thromboxane ited by dietary approaches using oils rich in eicosap-

A2 are both derived from arachidonate but the entaenoate and γ-linolenate (18:3n-6), the precursor former originates primarily from the endothelium to dihomo-γ-linolenate. and inhibits platelet aggregation, while the latter orig- Stable analogues of some classical prostaglandins inates primarily from platelets and is a potent plate- have specialized medical applications, including the let-aggregating agent. Third, competing eicosanoids termination of pregnancy and the closing of a patent Nutrition and Metabolism of Lipids 111

1 series 3 series PGs LTs Dihomo-γ- Figure 6.15 Arachidonic acid is not linolenic acid the only 20-carbon polyunsaturated fatty acid that can be metabolized via the cyclooxygenases and lipoxygen- γ Cyclooxygenase Lipoxygenase ases; both dihomo- -linolenic acid 2 series 4 series (20:3n-6) and eicosapentaenoic acid Arachidonic acid LTs PGs (20:5n-3) are well-established precur- sors as well, and produce prostaglan- dins (PGs) and leukotrienes (LTs) that are frequently competitive with those Eicosapentaenoic acid produced from arachidonate, thereby neutralizing the effects of the arachido- 3 series 5 series nate cascade (see Figure 6.14). This PGs LTs provides a critical balance in the overall reaction to cell injury. ductus arteriosus shortly after birth. Many anti-infl am- followed by refeeding a carbohydrate-rich meal is the matory and anti-pyretic drugs are inhibitors of eico- classic way to stimulate fatty acid synthesis. Insulin is sanoid synthesis. One potentially dangerous side-effect implicated in this process. When repeated, fasting/ of inhibiting eicosanoid synthesis is gastric erosion refeeding or weight cycling induces a gradual increase and bleeding. Receptor antagonists of leukotrienes are in the proportion of saturated and monounsaturated effective in reducing the symptoms of asthma. compared with PUFAs in tissues, especially body fat. This shift occurs because of the increase in fatty acid 6.8 Nutritional regulation of long-chain synthesis, easier oxidation of polyunsaturates, and the fatty acid profi les and metabolism inhibition of desaturation and chain elongation by fasting. The implications of such an alteration in tissue Phospholipids of all cellular and subcellular mem- fatty acid profi les have not yet been extensively studied, branes contain a diverse range of long-chain fatty but probably involve changes in insulin sensitivity and acids, the profi le of which is subject to both dietary other hormone effects. Protein defi ciency also inhibits infl uence and endogenous control. A few organs, desaturation and chain elongation of PUFAs. notably the brain, maintain extraordinarily strict Copper supplementation increases Δ9 desaturase control of their membrane composition. However, activity in animals, resulting in higher oleate levels. the fatty acid profi le of most organs is usually respon- This effect was fi rst observed when copper was used sive to the infl uence of changes in dietary fatty acid to reduce gastrointestinal infection in pigs, but also composition and other nutritional variables, yet led to softer back fat. Opposite to the effects of copper maintains the vital “gatekeeper” functions of all mem- supplementation, copper defi ciency inhibits synthesis branes. Hence, when changes in dietary fat alter of both oleate and docosahexaenoate. membrane fatty acid profi les, appropriate membrane fl uidity can be maintained by the addition or removal Polyunsaturated fatty acids of other lipids such as cholesterol. Insuffi cient energy There are four key features of the nutritional regula- intake and the presence of disease have important tion of the profi les and metabolism of PUFAs. These consequences for fatty acid synthesis, desaturation, attributes govern the effects of defi ciency or excess of and chain elongation and, consequently, tissue fatty one or more of these families of fatty acids almost as acid profi les. much as their level in the diet. These key features are: Saturates and monounsaturates Inadequate energy intake increases macronutrient 1 specifi city within families oxidation, including fatty acids. Short-term fasting 2 competition between families 112 Introduction to Human Nutrition

3 substrate and end-product inhibition NADH. This effect is severe enough that inherited 4 cofactor nutrients. forms of zinc defi ciency such as acrodermatitis enteropathica cause a precipitous decline in plasma Specifi city arachidonate, greater than usually observed with An n-6 PUFA cannot be converted to an n-3 or n-9 dietary defi ciency of n-6 polyunsaturates. PUFA. Thus, defi ciency of one family of polyunsatu- rates cannot be corrected by excess of those in a dif- 6.9 Nutritional and metabolic effects of ferent family and, indeed, is exacerbated by excess dietary fatty acids intake of the other families. Two types of issue exist in relation to the nutritional Competition and health implications of individual dietary lipids. The three families of PUFAs appear to use a common series of desaturases and chain elongases. The prefer- 1 Whether synthesized endogenously or only ence of these enzymes is for the more unsaturated obtained from the diet, what are the specifi c mem- fatty acids so, everything else being equal, more α- brane, precursor, or metabolic effects of dietary linolenate will be desaturated than linoleate or oleate. lipids beyond that of providing energy? However, in practice, more linoleate is consumed 2 Whether synthesized endogenously or obtained than α-linolenate and, as a result, more arachidonate from the diet, does an excess amount of a dietary is produced endogenously than eicosapentaenoate. lipid have benefi cial or deleterious implications for Furthermore, this competition for desaturation and health? chain elongation between linoleate and α-linolenate Short- and medium-chain fatty acids can lead to exacerbation of symptoms of defi ciency of one or other fatty acid family. Thus, as has been Short-chain fatty acids (1–6 carbons) are mostly demonstrated both clinically and experimentally, derived from carbohydrate fermentation in the large excess linoleate intake using sunfl ower oil is a common bowel and appear to be mainly used for energy, way to accelerate defi ciency of n-3 PUFA. although they are also substrates in several pathways. Butyrate may have an important role as an energy Inhibition substrate for enterocytes. Medium-chain fatty acids Excess linoleate or α-linolenate intake appears to (8–14 carbons) naturally appear in mammalian milk inhibit production of the respective long-chain prod- and are almost exclusively used as energy substrates. ucts in the same fatty acid family, i.e., high α-lino- They may also be chain elongated to palmitate. lenate intake inhibits synthesis of docosahexaenoate. Likewise, the main end-products of desaturation and Saturated fatty acids chain elongation tend to inhibit further metabolism Palmitate and stearate constitute a major proportion through this pathway, so arachidonate inhibits its of the acyl groups of membrane phospholipids and own synthesis. Similarly, dietary defi ciency of linole- all mammals have the capacity to synthesize them. ate increases activity of the Δ6 and Δ5 desaturases, Hence, empirically, they presumably have an impor- presumably to restore depleted levels of long-chain tant function in energy metabolism, cell structure, n-6 polyunsaturates such as arachidonate. normal development, and growth. The 20- to 24- carbon saturates are also important constituents of Cofactors myelin. However, in any of these functions, it is The cofactor requirements of the desaturation chain- unlikely that a dietary source of saturates is necessary. elongation enzymes are not yet well understood, but In fact, the brain is unable to acquire saturated a few relationships are known. The desaturases are fatty acids from the circulation and relies on its metalloenzymes containing iron, and iron defi ciency own endogenous synthesis for these fatty acids. therefore inhibits desaturase activity. is Furthermore, chronic excess intake and/or synthesis needed for microsomal desaturase activity in vitro. of palmitate and stearate is associated with an Zinc defi ciency inhibits Δ6 and Δ5 desaturation, appar- increased risk of diabetes and coronary artery ently by interrupting the fl ow of electrons from disease. Nutrition and Metabolism of Lipids 113

Monounsaturated fatty acids the diet of all mammals, including humans. Offi cial Little is known about the nutritional or health impli- dietary guidelines generally recommend a dietary cations of palmitoleate (16:1n-7), but there is a bur- source of linoleate at 1–2% of energy intake. It has geoning interest in the main dietary monounsatu- taken much longer to demonstrate that n-3 PUFAs rated fatty acid, oleate, and the health implications of are required by humans. Although this now seems olive oil. In the context of the same total fat intake, widely accepted among nutrition researchers, some the main benefi t of higher oleate intake seems to be countries, including the USA, still do not yet offi cially α that this reduces intake of palmitate and stearate and recognize that, as a minimum, -linolenate is a that this helps to lower serum cholesterol. required nutrient. As with other nutrients, the require- ment for polyunsaturates varies according to the stage Partially hydrogenated fatty acids of the life cycle, with pregnancy, lactation, and infancy Partially hydrogenated fatty acids contain a large pro- being the most vulnerable. Symptoms of linoleate portion of trans fatty acids that are not naturally defi ciency are virtually impossible to induce in healthy occurring but arise directly from food processing. adult humans, so the concept of “conditional indis- Hence, unlike saturates and cis-unsaturated fatty pensability or dispensability” of PUFAs has recently acids, they are not a necessary component of the diet emerged to replace the older but ambiguous term except in the small amounts found in cow’s milk. “.” Linoleate appears to be condi- Their physical characteristics make them economi- tionally dispensable in healthy nonpregnant adults, cally suitable for inclusion in a wide variety of baked, but is not in pregnancy, lactation, or infancy. fried, and oil-based foods, from which they can easily Because of the competition between the two fami- contribute up to 10% of dietary fat depending on lies of PUFAs, defi ciency of n-3 PUFA is commonly food selection. Epidemiological evidence and some induced by an excess of dietary linoleate. Hence, dis- α experimental studies show that common dietary trans cussion of the requirements for linoleate and -lino- fatty acids raise LDL cholesterol and lower HDLs in lenate has focused on their ratio in the diet. The ratio healthy adults, so the main nutritional concern is that of n-6 to n-3 polyunsaturates in human milk (5:1 to they may contribute to an increased risk of cardiovas- 10:1) has been widely viewed as a suitable reference cular disease (see Section 6.11). for this ratio in the general diet. In most affl uent Trans fatty acids have also been experimentally countries, this ratio remains much higher, at about shown to compete with and impair the metabolism 20:1, and has been implicated in subclinical defi ciency of other dietary long-chain fatty acids, but the rele- of n-3 polyunsaturates. There is recent evidence to vance of these observations in humans is unclear. suggest that it is the absolute amounts of long-chain Trans fatty acids can be present in baby foods at rela- n-3 and n-6 fatty acids that are important in predict- tively high concentrations but, so far, there is no evi- ing health outcomes, and not the dietary ratio of these dence of deleterious effects on growth or development. PUFAs. Some information on the metabolism of trans fatty Essential fatty acid defi ciency acids in humans has been gained from tracer studies, but fundamental information, such as the rate at The fi rst experimental model of defi ciency of polyun- which they are oxidized, is still unknown. saturates was total fat defi ciency. The elimination of dietary fat had to be extreme because the traces of fat Polyunsaturated fatty acids found in starch and dietary proteins were suffi cient Unlike saturates and monounsaturates, a dietary to prevent reproducible symptoms of fat defi ciency. source of n-6 and n-3 polyunsaturates is a necessity The defi ciency symptoms are now well known and for normal growth and development. As with other involve dry, scaly skin, growth retardation, and repro- essential nutrients, this has given rise to assessment of ductive failure. Most of these gross symptoms are α the dietary requirements for polyunsaturates and the relieved by linoleate and arachidonate. Although - implications of inadequate dietary intake of them. linolenate cannot be synthesized de novo, it has little It has been accepted for over 50 years that n-6 effect on these gross symptoms. However, careful polyunsaturates, particularly linoleate, are required in studies using a diet that is extremely defi cient in n-3 polyunsaturates and contains an excess of n-6 poly- 114 Introduction to Human Nutrition unsaturates led to defi ciency of n-3 polyunsaturates, an inherited disease is Zellweger’s syndrome. This characterized by delayed and impaired neuronal condition causes severe mental retardation and early development and impaired vision. These symptoms death. It is a disorder of peroxisomal biogenesis and have been traced in many species to the inadequate one outcome is markedly impaired synthesis of doco- accumulation of docosahexaenoate in the brain and sahexaenoate. Dietary supplementation with docosa- eye. Hence, the main function of n-3 polyunsaturates hexaenoate appears to partially restore neurological appears to hinge on synthesis of docosahexaenoate. development. In contrast, the function of n-6 polyunsaturates Epidemiological evidence shows that chronic involves independent roles of at least linoleate and degenerative diseases of affl uence are directly associ- arachidonate. ated with the defi ciency of n-3 PUFAs. Indeed, coun- Human cases of defi ciency of PUFAs, usually tries with relatively high rates of these diseases usually involve a clinical disorder, often involving weight loss, have an adequate to perhaps unnecessarily higher trauma such as surgery, or a disease requiring paren- intake of linoleate. High intakes of linoleate have been teral nutrition. However, reports of these cases are implicated in death from coronary artery disease and uncommon and describe dissimilar characteristics, several types of cancer because these diseases are asso- leading one to question whether the same defi ciency ciated with low intakes of n-3 polyunsaturates. Mental exists. Recent investigations into the amount of PUFA illnesses such as schizophrenia may also be associated in the whole body and the rate at which they can be with low intake of n-3 polyunsaturates and respond to oxidized suggest that traumatic or disease-related supplements of n-3 polyunsaturates. A more balanced processes leading to weight loss affect metabolism of ratio of intake of n-6 and n-3 polyunsaturates might polyunsaturates more severely than simple dietary achieve a reduction in the rate of these degenerative defi ciency in a weight-stable, healthy individual. For diseases but has not yet been widely investigated. example, defi ciency of linoleate has been long sus- Diets in Paleolithic times contained no processed pected but diffi cult to demonstrate in cystic fi brosis. food and probably balanced amounts of n-3 to n-6 Despite poor fat digestion, intake levels of linoleate polyunsaturates and a lower level of saturates. Such may not be inadequate but its β-oxidation could well diets would be predicted to lead to a lower incidence be abnormally high owing to the chronic infectious of degenerative disease. Since the brain has a very challenge. high energy requirement, it has also been speculated that human brain evolution beyond that of other pri- Clinical importance of polyunsaturates mates was dependent on a reliable and rich source of Infant brain and visual development is dependent on dietary energy and a direct source of long-chain poly- adequate accumulation of docosahexaenoate. The unsaturates, particularly docosahexaenoate. 1990s saw intense clinical and experimental assess- ment of the role of docosahexaenoate in early brain development and a widespread concern that many 6.10 Cholesterol synthesis and regulation infant formulae do not yet contain docosahexaenoate. Several clinical studies and extensive use of formulae Cholesterol and the brain containing docosahexaenoate and arachidonate have Mammalian brain function is dependent on special- shown that they are safe. Many but not all such studies ized membranes designed for signal transmission. show an improvement in visual and cognitive scores Greater cognitive sophistication in humans appears compared with matched formulae containing no to depend on a much greater number of connections docosahexaenoate or arachidonate. The infant brain and, consequently, greater potential for signal pro- and body as a whole clearly acquire less docosahexae- cessing. Like the membrane lipids of most other noate when only α-linolenate is given. As a whole, mammalian organs, brain lipids contain a relatively these data suggest that docosahexaenoate is a condi- high proportion of cholesterol, which increases from tionally indispensable fatty acid. about 40% of the lipid content in neonates to nearly Aside from questionable defi ciency of polyunsatu- 50% in adults. rates in cystic fi brosis (see above), one of the most Unlike other organs, the mammalian brain is prob- graphic examples of their defi ciency being caused by ably unique in being unable to acquire appreciable Nutrition and Metabolism of Lipids 115 amounts of cholesterol from the circulation, i.e., from Over 30 years ago Keys and Hegsted made the land- the diet or from synthesis outside the brain. This has mark observation that variation in the concentration been extensively studied in the young rat and sup- of serum cholesterol across seven different countries porting, although inconclusive, evidence is also avail- was positively related to the amount of energy derived able for the pig. The brain has suffi cient capacity to from saturated fat. Conversely, they found that intake synthesize cholesterol from acetyl-CoA derived pri- of dietary PUFA was inversely related to serum cho- marily from either glucose or ketones. Hence, it lesterol. From this fi nding they were able to formulate achieves the required level of cholesterol apparently equations that enabled them to predict the quantita- entirely by endogenous synthesis. In neonates, ketones tive effect of saturated and polyunsaturated fat on appear to play a greater role as substrates for brain serum cholesterol (Figure 6.16). In simpler terms, the cholesterol than in adults, in whom their main func- ratio of PUFAs to saturated fatty acids (SFAs), the tion seems to be as an alternative fuel to glucose. P : S ratio, was used with effect to predict changes in Among the common dietary long-chain fatty acids serum cholesterol. Although still effective today, the that would give rise to ketones during fat oxidation, equations and P:S ratio are being superseded by PUFAs, particularly linoleate and α-linolenate, appear advanced knowledge of the biological effects of indi- to be the best substrates for ketogenesis, since carbon vidual fatty acids. It is now well established that satu- from these fatty acids readily appears in brain choles- rated fats with between 12 and 16 carbon atoms, terol in suckling rats. namely lauric, myristic, and palmitic acids, are par- ticularly hypercholesterolemic, whereas stearic acid, 6.11 Effect of diet on serum lipids an extremely abundant SFA in most diets, is relatively and lipoproteins neutral in its effects on serum cholesterol. [Note that stearic acid is desaturated to monounsaturated fatty Diet and serum cholesterol acids (MUFAs) by Δ9 desaturase]. The cholesterol- Diet exerts a profound infl uence on blood lipids and raising effect of SFAs arises chiefl y from an increase lipoproteins and, as such, should always be a major in LDL cholesterol and is about twice as potent as the component of strategies for the primary prevention hypocholesterolemic effect of dietary PUFAs. Para- of diseases in which lipids play an etiological role, doxically, SFAs actually increase serum HDL choles- such as CHD. Nevertheless, despite convincing epide- terol. The polyunsaturates are divisible into two main miological evidence and the existence of credible series on the basis of the position of the fi rst double biochemical mechanisms to support a relationship bond from the methyl end of the fatty acid chain, the α between dietary fat and serum cholesterol, the parent fatty acids being linoleic (C18:2n-6) and - outcome of prospective intervention trials designed linolenic (C18:3n-3) acids. The cholesterol-lowering to test this relationship within populations has been effects of dietary PUFA is largely attributable to the disappointing. effects of linoleic acid in lowering LDL. Historically,

LDL receptors Blood LDL-C

Intracellular Cholesterol pool of free ACAT esters cholesterol

Saturated fatty acids Figure 6.16 Infl uence of dietary fatty acids on serum cholesterol through Intracellular Cholesterol differential effects on free cholesterol and pool of free low-density lipoprotein (LDL) receptor ACAT esters cholesterol activity. ACAT, acyl-CoA-cholesterol acyl- transferase; LDL-C, LDL cholesterol; MUFA, monounsaturated fatty acid; PUFA, poly- MUFAs and PUFAs unsaturated fatty acid. 116 Introduction to Human Nutrition monounsaturated fat was considered to be neutral of intake these fats are unlikely to exert adverse effects with respect to its effects on lipids and lipoproteins on serum lipoproteins. and was omitted from the predictive formulae of Keys and Hegsted. However, further studies, prompted by Plant sterols and soluble interest in the role of MUFAs in the Mediterranean nonstarch polysaccharides diet, have shown that MUFA-enriched diets may These compounds may be grouped together as they decrease LDL cholesterol, although possibly to a lesser share a similar mode of action on LDL cholesterol, extent than linoleic acid, and increase HDL choles- which is to reduce the availability of dietary and terol. An additional benefi t of MUFAs is thought to biliary cholesterol for absorption in the gut. This be conferred by the presence of a single double bond action interrupts the enterohepatic circulation and in MUFAs, which when incorporated into the mem- upregulates the production and activity of LDL recep- brane phospholipids of LDL, protect this lipoprotein tors (see Section 6.5). Plant sterols and their esters from oxidative modifi cation, an essential prerequisite such as those incorporated into margarines (stanols step in the deposition of cholesterol in the artery wall. and stanol esters), despite being nearly identical in In this regard, there has been concern that increasing structure to cholesterol, are poorly absorbed and dietary PUFA will impose additional oxidative stress interfere with the reabsorption of cholesterol origi- on LDL. While this idea forms part of the rationale nating from bile (~1 g/day) and dietary sources for limiting the amount of dietary PUFA to less than (300 mg/day) by either coprecipitation or competi- 10% of energy intake, there is as yet no convincing tion. Margarines or spreads (30–40 g/day) containing evidence of adverse effects from increasing the level plant sterols or their derivatives have been shown to of PUFA in tissues and circulating lipoproteins. reduce LDL cholesterol by up to 14% in controlled trials. Soluble NSPs such as those found in gums and Trans fatty acids gelling agents from fruit (gum arabic and pectins) act While saturated fats consist of straight chains of in a similar way and have been shown to be equally carbon atoms which pack together tightly in the effi cacious in reducing LDL cholesterol. phospholipids of cell membranes and lipoproteins, in contrast, carbon double bonds in the cis confi gura- Dietary cholesterol tion in MUFAs and PUFAs introduce a bend or kink There is a popular misconception that dietary choles- into the carbon chain. This alters the physical proper- terol correlates directly with serum cholesterol, when ties of the phospholipids containing these fatty acids, in fact dietary cholesterol, within a range of normal by, for example, increasing their packing volume, a dietary consumption (100–400 mg/day), has only a physical property that contributes to an increase in very small impact on blood cholesterol levels. Eggs membrane fl uidity. The partial hydrogenation of represent the principal source of dietary cholesterol MUFAs and PUFAs, most notably during the indus- in most diets (1 egg = 150–250 mg cholesterol); trial processing of foods for the purpose of solidifying in their absence, most Western diets would contain unsaturated oils, results in a loss of this kink as the considerably less than 100 mg cholesterol/day. The fatty acid assumes a straighter, trans confi guration classic but extreme egg-feeding studies showed that that resembles that found in SFAs. This likeness in feeding of up to six eggs per day (900 mg cholesterol) chemical structure is thought to account for the SFA- increased LDL cholesterol acutely. However, the body like effects of trans fatty acids such as elaidic acid effectively counters this effect with sensitive, compen- (trans isomer of oleic acid) on serum lipids (see satory mechanisms to deal with an increasing load of Figures 6.1 and 6.2). The results of prospective cohort dietary cholesterol, one of which is to reduce the studies such as the Nurses’ Health Study showed that amount of cholesterol absorbed in the gut. This com- high levels of trans fats in excess of 7% energy pensation effectively abolishes any dose–response increased serum LDLs and reduced HDLs (Willet et relationship between dietary cholesterol, over a prac- al. 1993). However, despite the continued use of par- tically realistic range of intakes, and serum choles- tially hydrogenated fats in food products, the average terol. Two factors that may infl uence the variability in intake of trans fatty acids in most Western diets does response to dietary cholesterol are dietary saturated not exceed 2% of total energy intake, and at this level fatty acids, which have been shown to augment the Nutrition and Metabolism of Lipids 117 cholesterol-raising effects of dietary cholesterol, and acid in the food industry, together with a widespread a phenomenon of increased susceptibility to dietary resistance to the consumption of fi sh, has increased cholesterol in some individuals for some, as yet, the ratio of n-6 to n-3 PUFAs in northern Europe and unknown reason. the USA since the 1970s. This situation has major To place these dietary infl uences on blood choles- implications for the development of abnormalities in terol in perspective with other cholesterol-lowering circulating lipoproteins, since defi ciency in eicosapen- strategies, a metaanalysis of dietary intervention trials taenoic acid and docosahexaenoic acid could help to undertaken by the World Health Organization promote an increase in plasma TAG. This could occur (WHO) revealed that dietary modifi cation could through an overproduction of endogenous TAG achieve reductions in serum cholesterol of between (VLDL) in the liver and intolerance to dietary (exoge- only 4% and 5%. This fi nding is in sharp contrast to nous) fat, and lead to the development of dyslipidemia the potent effects of cholesterol-lowering drugs, which known as an ALP (see Section 6.5). The frequency of can reduce serum cholesterol by 30–40% and have this dyslipidemia is believed to be very rare in been shown, unequivocally, to reduce the incidence of Mediterranean countries that have a high intake of death from CHD. It also highlights the need to address dietary n-3 PUFA and an n-6:n-3 ratio closer to 1. other risk factors which are more responsive to dietary High-carbohydrate diets have been shown to increase change. plasma TAG. Carbohydrate-induced hypertriacylglyc- erolemia is not, as was originally thought, a short- Fat quantity versus quality: importance term adaptive response in the liver, as it changes its of the ratio of n-6:n-3 polyunsaturated pattern of oxidation from fat to carbohydrate, but a fatty acids real phenomenon associated with the overconsump- The underlying principle for a reduction in total fat tion of the non-milk extrinsic sugars, sucrose and intake is to reduce energy intake from the consump- fructose, most notably in individuals with insulin- tion of the most energy-dense macronutrient in order resistant dyslipidemia. There is evidence to suggest to prevent weight gain and ultimately obesity. The that this effect can be avoided by limiting the intake of current recommendation for the UK is to reduce sucrose and increasing the intake of slowly absorbed energy derived from fat to 35% or less. Since weight carbohydrate with a low impact on blood glucose. gain is associated with raised plasma TAGs and abnor- The results of several metaanalyses of dietary inter- malities in circulating lipoproteins, reducing total fat vention trials support dietary MUFAs as the most intake should, in theory, reduce blood lipids. However, favored substitute for dietary saturated fatty acids, in practice there is little evidence to support such an and even linoleic acid in areas of high intake. effect within populations. Metaanalyses have revealed that little benefi t is to be gained, at least in terms of Effects of n-3 polyunsaturated fatty acids changes in blood lipids, by reducing total fat without from plants and fi sh altering the composition of dietary fatty acids. The current dietary recommendation for the intake Metaanalyses have also helped to resolve the issue of of long-chain n-3 PUFAs (eicosapentaenoic acid/doc- what represents the most appropriate replacement osahexaenoic acid) in the UK is 450 mg/day (SACN, nutrient for SFAs. Since PUFAs, and specifi cally lin- 2004). This was to increase intake by consuming two oleic acid, were shown to counter the actions of SFAs portions of fi sh per week, one of which should be oily and were abundant in natural sources such as sun- (e.g., mackerel, sardines). This recommendation was fl ower and corn oils, they were an obvious fi rst choice. based on evidence from a host of epidemiological and The alternative was to substitute fat with dietary car- intervention studies, which showed that regular fi sh bohydrate. There have been problems associated with consumption could reduce the risk of sudden cardiac both of these approaches. First, increasing dietary lin- death. Since this an acute end-point of CHD, the ben- oleic acid excludes the lesser abundant, but more met- efi ts of fi sh oil have been ascribed to the prevention abolically active, n-3 PUFA, and especially the longer of fatal cardiac arrhythmia, and, to a lesser extent, chain (C20–C22) members of this series derived from coronary thrombosis, but not to any favorable effects marine oils [C20:5 (eicosapentaenoic acid) and C22:6 on blood lipids. However, there is also convincing (docosahexaenoic acid)]. Overemphasis on linoleic evidence to show that fi sh oil supplementation (1 g/ 118 Introduction to Human Nutrition day for 3 years) reduces the incidence of death from PUFA at the same time. However, in practice, this will CHD in healthy, free-living subjects. This longer term be diffi cult to achieve, not least because of a mass benefi t may be linked to the effects of eicosapentae- resistance to the increased consumption of oily fi sh noic acid/docosahexaenoic acid on a host of other and diminishing fi sh stocks. An obvious alternative cardiovascular risk factors, including plasma TAGs would be to increase the intake of the shorter chain and lipoproteins. precursor of eicosapentaenoic acid/docosahexaenoic Long-chain n-3 PUFAs exert multiple effects on acid, α-linolenic acid (C18:3n-3). The latter is derived lipid metabolism, the most notable of which is the from plant seeds such as fl ax and rapeseed, and is capacity to decrease postabsorptive plasma TAG levels desaturated and elongated to its longer chain relatives by 20–30%. -enriched diets have also been in the body. Unfortunately, the rate of conversion to shown to attenuate the magnitude and duration of eicosapentaenoic acid and especially docosahexaenoic postprandial lipemia following the ingestion of a fat- acid is slow, and the effi ciency of conversion is reduced containing meal. These effects are frequently accom- by high levels of linoleic acid, which competes more panied by benefi cial changes in circulating LDLs and effectively than α-linolenic acid for desaturation. HDLs, and the correction of an ALP. There is, as yet, no evidence to suggest that the rate Widespread knowledge of the favorable effects of of conversion of dietary α-linolenic acid to eicosa- eicosapentaenoic acid and docosahexaenoic acid has pentaenoic acid and especially docosahexaenoic acid raised awareness of the need to increase intakes of is suffi cient to achieve fi sh oil-like effects on blood these fatty acids, and to reduce the amount of n-6 lipids (Table 6.6).

Table 6.6 Effects on plasma lipids of substituting dietary saturated fats with polyunsaturated fatty acids, monounsaturated fatty acids, and carbohydrates

LDL-C HDL-C TAG TC:HDL

SFA (C12–C16)

a n-6 PUFA Linoleic acid (sunflower/corn)

n-3 PUFA b LC = EPA/DHA (fish oil) SC = linolenic acid (/soyabean/rape)

n-9 MUFA Oleic acid (olive oil/rape)

Carbohydrate Sucrose/fructose?

a n-6 PUFA in excess of 10% energy. b Increase in response to redistribution of LDL subsclasses. PUFA: polyunsatruated fatty acids; MUFA: monounsaturated fatty acids. SFA: saturated fatty acids; LC: long chain; EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid; short chain; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; TAG: triacylglycerols; TC: total cholesterol. Nutrition and Metabolism of Lipids 119

How do dietary fatty acids infl uence serum TAG by inhibiting the enzymes phosphatidic acid cholesterol and triacylglycerols? phosphatase and diacylglycerol acyltransferase. They In common with other physiological systems, lipo- may also selectively increase the degradation of apoB- protein metabolism is coordinated by interplay 100, further reducing the production of TAG-rich between the activity of specifi c genes and hormones VLDLs. (Note that apoB-100 is produced constitu- that determines the production and activity of func- tively, so that the production of VLDL is driven by the tional proteins (enzymes, receptors, lipid transfer, and supply of substrates for the synthesis of TAG). In apoproteins). Effects on these functional proteins addition, long-chain n-3 PUFAs accelerate the clear- ultimately regulate the quantity and quality of circu- ance of TAG-rich lipoproteins from the circulation in lating lipids and lipoproteins. While there have been the postprandial phase by stimulating the activity of signifi cant advances in knowledge of the modulatory LPL. Together, these effects are thought to underlie effects of dietary fatty acids on hormones and gene the ability of these fatty acids to correct the lipopro- expression, evidence for the effects of dietary fats on tein abnormalities associated with an ALP. It is also functional proteins is by far the most advanced. possible that many of the effects of eicosapentaenoic acid/docosahexaenoic acid on blood lipids and other Saturated fatty acids and low-density cardiovascular risk factors are mediated through an lipoprotein cholesterol increase in the sensitivity of tissues to the action of The most well-elucidated mechanism to explain how insulin. However, there is, as yet, no convincing evi- different dietary fats produce variable effects on LDL- dence to support such an effect in adipose tissue, liver, cholesterol is through the LDL receptor pathway, the or skeletal muscle. control of which has already been described (see Section 6.5). The ability of the cell to regulate its pool Nutrient–gene interactions of free cholesterol depends to a large extent on the It has been estimated that diet could account for up nature of the fatty acids available for esterifi cation by to 50% of the variation in blood lipids and lipopro- the enzyme ACAT, an intracellular relative of LCAT. tein levels between individuals. This would mean that ACAT favors unsaturated fatty acids (MUFAs and genetic differences must explain the remaining 50%. PUFAs) as substrates for esterifi cation, which utilizes In real terms, interactions between diet and genes free cholesterol within the cell. The resulting reduc- represent a sizeable proportion of each of these unre- tion in intracellular free cholesterol stimulates the alistically discrete fractions. transcription of the LDL receptor gene and produc- tion of new LDL receptors through the SREBP mech- Fixed genetic polymorphisms anism, and fall in circulating LDL as already described. Variation in the structure of specifi c genes between Conversely, SFAs are poor substrates for ACAT, and individuals (genetic heterogeneity) has been shown to their presence in the cell exerts the opposite effect on give rise to differences in dietary responsiveness. A free cholesterol levels, thus increasing circulating LDL few common polymorphisms have been identifi ed in cholesterol and total serum cholesterol (Figure 6.8). genes associated with lipoprotein metabolism, the Fatty acids may also exert direct effects on the activity best example of which is apoE. ApoE facilitates the of LDL receptors by altering the composition of uptake of TAG-rich lipoproteins (chylomicron rem- membrane phospholipids and thus membrane fl uid- nants and VLDL) via the remnant and LDL receptors ity. Alternatively, there is evidence to suggest that and, thus, in part, determines the removal of TAG dietary PUFA could upregulate LDL receptors indi- from the circulation. The gene for apoE is polymor- rectly, by increasing the cholesterol content (litho- phic, which means that it exists in multiple forms genicity) of bile and in this way accelerate the excre- between individuals. This polymorphism generates tion of cholesterol. several isoforms of the protein that express variable affi nities for their receptors and thus variable poten- Long-chain n-3 polyunsaturated fatty acids tial to remove TAG-rich lipoproteins from the circu- and serum triacylglycerols lation. In this way, apoE genotype can modulate the Long chain n-3 PUFAs have potent effects in the liver, response of an individual to any dietary fat that exerts where they suppress the production of endogenous an infl uence on TAG-rich lipoproteins, giving rise to 120 Introduction to Human Nutrition

Table 6.7 Effect of apoprotein E phenotype on serum cholesterol

Phenotype (gene frequency) Receptor binding affi nity Hepatic free cholesterol LDL receptor activity LDL-C

↓↑ E4 (ε4 15%) + + +↑ (feedback) →→→ E3 (ε3 77%) + + + ↑↓ E2 (ε2 8%) ↓ (feedback)

Carriage of ε4 allele is associated with increased risk of coronary heart disease and greater changes in low-density lipoprotein cholesterol (LDL-C) in response to increased dietary fat and cholesterol in men.

the concept of genetic susceptibility to diet (Table for the “ideal” fat intake will continue, but this seems 6.7). misguided because humans in good health in differ- ent cultures and geographical locations ultimately Modulation of gene expression consume a wide range of total fat and fatty acid ratios; Dietary fatty acids and/or their intracellular (eico- their overall health is based on much more than their sanoid) derivatives can also infl uence the expression fat intake. Nevertheless, pursuit of an ideal fat intake of genes (rate of gene transcription) by interacting or composition will satisfy the of many research- with specifi c nuclear receptors within the nucleus of ers, consumers, and government agencies but, in all cells. These nuclear receptors control the rate of gene likelihood, will not greatly alter the impact of fats on transcription by binding to specifi c regions of DNA disease processes. These would include: known as responsive elements. Genes associated with ● the role of the quantity and quality of dietary fat on the production of functional proteins can be either postprandial lipemia, specifi c lipoproteins, and the stimulated or repressed according to the nature of the risk of CHD nuclear transcription factor and its binding substrate ● the metabolic roles of the short-chain fatty acids (PUFAs or derivative). Peroxisome proliferator-acti- (1–6 carbons) vated receptors (PPARs) represent examples of nuclear ● the effect of trans fatty acids in baby foods, and the receptors that may utilize long-chain PUFAs as sub- level of intake of trans fatty acids that will increase strates. PPARs can be found in all tissues of the body, the risk of CHD in adults but notably in the liver, where they control the syn- ● the appropriate amounts of n-6 to n-3 polyunsatu- thesis of lipid and apoproteins (PPAR-α), and in rates to prevent, reverse, and/or treat chronic adipose tissue (PPAR-γ), where they control the dif- degenerative diseases ferentiation of adipocytes and insulin-sensitive mobi- ● the requirements of docosahexaenoate and other lization and synthesis of TAG. SREBPs represent long-chain PUFA in infant and enteral feeding another example of nuclear transcription proteins ● the conditional indispensability of particular fatty that control cholesterol and fatty acid metabolism acids throughout the life cycle (especially during within the cell. pregnancy and lactation, and in the aged) ● the effects of particular dietary fatty acids and com- 6.12 Perspectives on the future binations of fatty acids on hormone and gene expression. Future research on fatty acids and their role in health and disease will be largely dictated by progress in More knowledge in all of these areas will lead to a intervention studies using fatty acid supplements in better understanding of the mechanisms through chronic diseases of infl ammation, brain degeneration, which dietary lipids infl uence blood lipids and lipo- cancer, and heart disease. Some of these studies will proteins and therefore the risk of chronic diseases. It be based on effects of dietary fats on gene expression will also lead to improved recommendations regard- that have yet to be discovered; others will be based on ing the quantity and quality of dietary fats that are information that we already have. No doubt the search commensurate with optimum human health. Nutrition and Metabolism of Lipids 121

Acknowledgment Further reading

This chapter has been revised and updated by Bruce Cunnane SC. Alpha-linolenate acid in human nutrition. In: Griffi n based on the original chapter by Bruce Griffi n Cunnane SC, Thompson LU, eds. Flaxseed in Human Nutrition. AOCS Press, Champaign, IL, 1995: 99–127. and Stephen Cunnane. Dolecek TA. Epidemiological evidence of relationships between dietary polyunsaturated fatty acids and mortality in the Multiple Risk Factor Intervention Trial. Proc Soc Exp Biol Med 1992; 200: References 177–182. Durrington PN. Hyperlipidaemia Diagnosis and Management, 2nd Bloomfi eld DK & Bloch K. The formation of Δ9-unsaturated fatty edn. Elsevier Science, Oxford, 1995. acids. J Biol Chem 1960; 235: 337-345. Griffi n BA The effects of n-3 PUFA on LDL subfractions. Lipids Burr MM & Burr GO. A new defi ciency disease produced by the 2001; 36: S91–S97. rigid exclusion of fat from the diet. J Biol Chem 1929; 82: Griffi n BA How relevant is the ratio of dietary n-6 to n-3 345-367. polyunsaturated fatty acids to cardiovascular disease risk? Goldstein JL & Brown MS. Atherosclerosis: the LDL receptor Evidence from the OPTILIP Study. Curr Opin Lipidol 2008; 19: hypothesis. Metabolism 1977; 26: 1257-1275. 57–62. Hegsted DM, McGrandy RB, Myers ML et al. Quantitative effects Lands WEM. Impact of daily food choices on of dietary fat on serum cholesterol in man. Am J Clin Nutr 1965; and disease prevention. In: Hamazaki H, Okuyama H, eds. Fatty 17: 281-295. Acids and Lipids: New Findings. Karger, Basel, 2001: 1–5. Keys A, Anderson JT & Grande F. Prediction of serum cholesterol Lee A, Griffi n BA. Dietary cholesterol, eggs and coronary heart responses of man to changes in fats in the diet. Lancet 1977; 2: disease in perspective. Br Nutr Found Bull 2006; 31: 21–27. 959-966. Mangiapane EH, Salter AM, eds. Diet, Lipoproteins and Coronary Ponticorvo L, Rittenberg D & Bloch K. The utilisation of acetate Heart Disease. A Biochemical Perspective. Nottingham University for the synthesis of fatty acids, cholesterol and protoporphyrin. Press, Nottingham, 1999. J Biol Chem 1949; 179: 839-842. Simopoulos AP. Evolutionary aspects of diet and essential fatty Scientifi c Advisory Committee on Nutrition (SACN) 2004. Report acids. In: Hamazaki H, Okuyama H, eds. Fatty Acids and Lipids: published for the Food Standards Agency and the Department New Findings. Karger, Basel, 2001: 18–27. of Health by TSO. ISBN 0-11-243083-X. Willett WC. Eat, Drink and Be Healthy: The Harvard Medical Willet WC, Stamfer MJ, Manson JE et al. Intake of trans fatty acids School Guide to Healthy Eating. Simon and Schuster, New York, and risk of coronary heart disease among women, Lancet 1993; 2001. 341: 581–585. Zilversmit DB. Atherogenesis is a postprandial phenomenon. Circulation 1979; 60: 473-485. 7 Dietary Reference Standards

Kate M Younger

Key messages

• This chapter discusses the development of terminology and the • The methods used to determine requirements are discussed. change in conceptual approaches to setting nutrient recommen- These include deprivation studies, radioactive tracer studies, dations from adequate to optimum nutrition. balance studies, factorial methods, measurement of nutrient • The interpretation and uses of dietary recommendations are levels in biological tissues, biochemical and biological markers, discussed. and animal experiments. • The chapter describes how reference values can be used to assess the adequacy of the nutrient intakes of population groups.

7.1 Introduction past become obsolete as they are replaced by new fi gures based on new data or new interpretations of The fi rst attempt to set standards for nutrient intakes existing data. was by the Food and Nutrition Board of the National Research Council of the USA in 1941, which pub- 7.2 Terminology and conceptual lished recommended daily allowances (RDAs) in 1943 approaches to setting nutrient to “provide standards to serve as a goal for good recommendations nutrition.” The fi rst UK RDAs followed in 1950, pub- lished by the British Medical Association, and many From the time of their fi rst issue in the 1940s and other countries and international agencies now throughout the next 50 years, the concepts and ter- publish dietary standards that are intended to allow minology of RDAs remained unchanged. The basis on the adequacy of the nutrient intakes of groups or which these RDAs were built was the statistical distri- populations to be assessed by comparison with the bution of individual requirements to prevent defi - standards. ciency criteria for the target nutrient. The peak of the As the amount known about human requirements curve of the Gaussian distributions of such require- and nutrient functions has increased, so too has the ments is the “average requirement,” with half the size of the documents describing the recommenda- population having requirements above this value and tions, from a mere six pages dealing with 10 nutrients the other half having lower requirements. The RDA in 1943 to the series of weighty books, each dealing was taken to be a point on that distribution that was with the dietary reference intakes (DRIs) of only a few equal to the mean or “average requirements” plus 2 of more than 30 nutrients, published by the Institute standard deviations (SDs) (Figure 7.1). By setting the of Medicine of the USA. Furthermore, continuing recommendation close to the upper end of the distri- research and the development of more informed bution of individual requirements, the needs of most interpretations of the expanding body of data avail- of the population would be met. If the standard were able necessitate the regular revision and updating of set to meet the apparent needs of almost everyone, the recommendations; thus, the “standards” of the the resultant value would be so high as to be unat-

© 2009 KM Younger. Dietary Reference Standards 123

2.5% 95% 2.5% However, since the 1980s, changes have occurred in both of these areas.

Changes in terminology Two basic changes occurred with regard to terminol- ogy. The fi rst was that the term “recommended dietary allowance” was altered and the second was that new terms were introduced so that the adequacy of diets could be evaluated from several perspectives. The Number of individuals reason for changing the terminology was in effect to a bcre-emphasize some of the basic concepts underlying Nutrient requirements the term RDA. “Recommended” has a prescriptive air Figure 7.1 Frequency distribution of individual requirements for a about it and there were concerns that consumers nutrient. (a) The mean minus a notional 2 standard deviations (SDs); might see this as something that had to be met daily intakes below this will be inadequate for nearly all of the population. and met precisely. The term “allowance” reinforces (b) The mean; the midpoint of the population’s requirement. (c) The the perception of a prescriptive approach. Thus, the mean plus a notional 2 SDs; the intake that is adequate for nearly all UK adopted the term dietary reference value (DRV), of the population. Note that, in practice, because insuffi cient data exist to establish reliable means and SDs for many nutrient requirements, the EU introduced the term population reference the reference intakes describing the points a and c on the curve are intake (PRI), the USA and Canada introduced the generally set, in the case of a, at the level that is judged to prevent term (DRI), and Australia the appearance of signs of defi ciency (biochemical or clinical), and, in and New Zealand now use the term nutrient intake the case of c, at the level above which all individuals appear to be value (NIV). All are precisely equivalent to the origi- adequately supplied. Thus, it is unlikely that even 2.5% of the popula- tion would not achieve adequacy at intake level c. nal concept of the RDA, a term that many countries prefer to continue to use. Two new terms were introduced: a minimum tainable at population level. If the standard were set requirement and a safe upper level. The minimum at the point of the average of all individual require- requirement represents the average requirement ments, then half the population would have require- minus 2 SDs (point a in Figure 7.1). A defi nition ments in excess of the standard. In a normal distribu- describing this point is given in Figure 7.1 along with tion, some 2.5% of points lie at the upper and lower the various terms used to defi ne this point (Box 7.1). tails outside that range of the mean plus or minus 2 The concept of an upper safe limit of intake has SDs. Thus, by setting the RDA to this point of the gained importance in view of the increased opportu- mean plus 2 SDs, we are setting the standard for nity for people to consume high levels of nutrients 97.5% of the population. The consumption of most from fortifi ed foods or supplements. The recently nutrients at levels somewhat greater than actually revised US DRI set “tolerable upper intake” levels required is generally not harmful; hence, setting rec- (ULs) that are judged to be the highest level of nutri- ommendations at the population average require- ent intake that is likely to pose no risk of adverse ment plus a notional 2 SDs is logical if the aim is to health effects in almost all individuals in a group. The describe an intake that is adequate for almost every- current European and UK recommendations also one. However, this is spectacularly inappropriate in address this concern in the case of those nutrients for the case of recommendations for energy intake, since which toxic levels have been reported. The terms used even relatively small imbalances in energy intake over by different recommending bodies to describe the expenditure will lead, over time, to overweight and various points on the distribution of individual ultimately obesity, an increasing problem in most requirements for a nutrient are given in Box 7.2, while populations. Recommendations for energy intake are precise defi nitions may be found in the relevant pub- therefore given only as the estimated population lications referred to. average requirement. The World Health Organization (WHO) has taken Thus, for almost half a century, these were the a rather different approach, defi ning population safe terms used and the underlying conceptual approaches. ranges of intake. “Normative requirement” is now 124 Introduction to Human Nutrition

Box 7.1 Terms used to describe points a, b, and c on the frequency distribution

abc

European Communities Scientifi c Committee for Food Lowest threshold intake Average requirement Population reference Population reference intakes (1993) (LTI) (AR) intake (PRI) US Food and Nutrition Board, Recommended daily National Academy of Sciences, allowance (RDA) National Research Council Recommended daily allowances (1989) US Food and Nutrition Board, Estimated average Recommended daily Institute of Medicine, requirement (EAR) allowance (RDA) National Academies of Health, Canada Dietary reference intakes (1997–2005) British Committee on Medical Aspects of Food Policy Lower reference Estimated average Reference nutrient intake (COMA) nutrient intake (LRNI) requirement (EAR) (RNI) Dietary reference values (1991) World Health Organization/Food and Agriculture Recommended nutrient Organization (WHO/FAO) intake (RNI) National Health and Medical Research Council (NHMRC), Estimated average Recommended dietary Australia and New Zealand requirement (EAR) intake (RNI) Nutrient reference values (2006) United Nations University (UNU) Average nutrient Individual nutrient level

Nutrient intake values (2007) requirement (ANR) (INLx; in this case INL98)

used to describe the population mean normative Box 7.2 Additional terms used requirement (which would allow the maintenance of, European population Acceptable range of intakes or a desirable, body store or reserve); “maximum” to reference intake refer to the upper limit of safe ranges of population (1993) mean intakes; and “basal” for the lower such limit, US recommended daily Safe intake and adequate intake below which clinically detectable signs of inadequacy allowance (1989) would be expected to appear. These WHO require- US dietary reference Adequate intake (AI) and tolerable intake (1997–2005) upper intake level (UL), acceptable ments are revised in groups of nutrients at different macronutrient distribution range times (see Further reading), and in those that date (AMDR) from 1974 the term “recommended intake” or “rec- British dietary reference Safe and adequate intake ommended nutrient intake” is used to describe the value (1991) average requirement plus an amount that takes into World Health Recommended intake Organization (1974– account interindividual variability and hence is con- 1996) sidered to be suffi cient for the maintenance of health World Health Basal, normative, and maximum in nearly all people. Organization (1996–) population requirement ranges, More recently, the United Nations University mean intake goals (UNU) has published a suggested harmonized National Health and Acceptable macronutrient approach and methodologies for developing nutrient Medical Research distribution range (AMDR) and Council (2006) suggested dietary target (SDT) recommendations, together with proposed terminol- United Nations Upper nutrient level (UNL) ogy (Box 7.1), that could be used worldwide to University (2007) promote objectivity, transparency, and consistency among those setting and using nutrient recommen- Dietary Reference Standards 125 dations. Their preferred term, nutrient intake value In the USA, the reference value for calcium is based (NIV), refers to dietary intake recommendations on optimizing bone calcium levels, which is a move based on research data; the term “nutrient” was chosen away from the traditional approach of focusing on in order to distinguish these from dietary compo- preventing defi ciency symptoms. An example of nents such as cereals, and the term “value” is intended attempts to set the reference standard for optimizing to emphasize the potential usefulness for both assess- a biochemical function is a level of folic acid that ing dietary adequacy (and hence dietary planning) would minimize the plasma levels of homocysteine, a and policy-making. The individual nutrient level potential risk factor for cardiovascular disease. Another

(INLx) is fl exible, in that x refers to the chosen per- might be the level of zinc to optimize cell-mediated centile of the population for whom this intake is suf- immunity. An example of a possible reference stan- fi cient; for example 98% (mean or median requirement dard to optimize a risk factor for a disease is the level

+ 2 SDs), written as INL98, but it could be set lower in of sodium that would minimize hypertension or the the case of certain nutrients. level of n-3 polyunsaturated fatty acids (PUFAs) to lower plasma triacylglycerols (TAGs). The amount of Changes in conceptual approach folic acid to minimize the population burden of neural tube defect would be an example of a reference value When a committee sits to make a recommendation to minimize the incidence of a disease. At present, for a standard in relation to nutrient intakes, it begins there is much debate as to the best approach to choos- with a distribution of requirements. In the past, ing criteria for setting reference standards for minerals although the choice of criteria for requirement might and vitamins, and this is an area that is likely to con- vary between committees, the orientation was always tinue to court controversy. An important point to note the same: requirements were set at a level that should in this respect is that, while minimizing frank defi - prevent defi ciency symptoms. More recently, the ciency symptoms of micronutrients is an acute issue concern for health promotion through diet has led to in many developing countries, any evolution of our the introduction of the concept of optimal nutrition, concepts of desirable or optimal nutrient require- in which the optimal intake of a nutrient could be ments must lead to a revision of the estimate of the defi ned as that intake that maximizes physiological numbers of those with inadequate nutrition. and mental function and minimizes the development of degenerative diseases. It should be borne in mind that, although this may appear simple enough to 7.3 Interpretation and uses of defi ne in the case of single nutrients, things clearly dietary recommendations become more complex when considering all nutrients When using dietary recommendations, several impor- together, in all possible physiological situations. tant points need to be considered. Genetic variability may also, increasingly, be taken The nutrient levels recommended are per person into account; for example, the requirement for folate per day. However, in practice this will usually be of those carrying certain variants of the MTHFR gene achieved as an average over a period of time (days, (around 10% of the population tested thus far) might, weeks, or months) owing to daily fl uctuations in the arguably, need to be set higher than for the rest of the diet. As stated above, the setting of a range of dietary population. recommendations should encourage appropriate It is now recognized that there are several levels for interpretation of dietary intake data, rather than the considering the concept of optimal nutrition, i.e., the inappropriate assumption that the value identifi ed to level that: meet the needs of practically all healthy people is a ● prevents defi ciency symptoms, traditionally used to minimum requirement for individuals. If an individ- establish reference nutrient intakes ual’s nutrient intake can be averaged over a suffi cient ● optimizes body stores of a nutrient period then this improves the validity of the compari- ● optimizes some biochemical or physiological son with dietary recommendations. However, in the function case of energy intakes, such a comparison is still inap- ● minimizes a risk factor for some chronic disease propriate: dietary reference values for energy are ● minimizes the incidence of a disease. intended only for use with groups, and it is more 126 Introduction to Human Nutrition useful to compare an individual’s energy intake with milk compared with formula , but rather the some measure or calculation of their expenditure in reverse. order to assess adequacy. The dietary recommendations for infants post- In the case of a group, the assumption can be made weaning and for children and adolescents are gener- that the quality of the diet can be averaged across the ally based on less robust scientifi c evidence than those group at a given time-point, and therefore that appar- for adults, for whom much more good information is ently healthy individuals within a group may com- available. In the absence of reliable data, values for pensate for a relative defi ciency on one day by a children are usually derived by extrapolation from relative excess on another. It should also be remem- those of young adults. The calculation of nutrient bered that allowances may need to be made for body requirements is generally based on energy expendi- size, activity level, and perhaps other characteristics ture because metabolic requirements for energy prob- of the individual or group under consideration, since ably go hand in hand with those for nutrients in the recommended intakes are designed for “reference” growing children. In the case of infants post-weaning populations. on mixed diets, values are obtained by interpolation Another assumption made when setting recom- between values known for infants younger than 6 mendations for a particular nutrient is that the intake months and those calculated for toddlers aged 1–3 of all other nutrients is adequate, which in an appar- years. Thus, the dietary recommendations for children ently healthy population eating a varied diet is prob- and adolescents need to be approached with some ably reasonable. caution, being more suitable for planning and labeling Recommendations are not intended to address the purposes than as a description of actual needs. needs of people who are not healthy: no allowance is Finally, assessment of the dietary adequacy of made for altered nutrient requirements due to illness people at the other end of the population age range or injury. For example, patients confi ned to bed may is made diffi cult by the lack of data on healthy elderly require less energy owing to inactivity, and may people. One of the normal characteristics of aging is require higher micronutrient intakes because of an that various body functions deteriorate to some illness causing malabsorption by the gut. Certain extent, and disease and illness become more common nutrients may also be used as therapeutic agents, for as people age. Until more data are available, the example n-3 fatty acids can have anti-infl ammatory assumption is made that, except for energy and a few effects. These clinical aspects are considered elsewhere nutrients, the requirements of the elderly (usually in these texts. defi ned as those over 65 years old) are no different One complication arising in the formulation of from those of younger adults. dietary recommendations is caused by the fact that Bearing the above points in mind, dietary recom- various groups of people within a population may mendations can be useful at various levels. have different nutrient requirements. Therefore, the population is divided into subgroups: children and ● Governments and nongovernment organizations adults by age bands, and by gender. For women, allow- (NGOs) use dietary recommendations to identify ances are also made for pregnancy and lactation. the energy and nutrient requirements of popula- Infants are recommended to be fully breast-fed for tions and hence allow informed decisions on food the fi rst few months of life. This poses a problem for policy. This could include the provision of food aid the bodies setting the dietary recommendations, or supplements (or rationing) when the diet is which have to set standards for those infants who are inadequate, fortifi cation of foods, providing appro- not breast-fed. The dietary recommendations for priate nutrition education, introducing legislation formula-fed infants are based on the energy and concerning the food supply, infl uencing the import nutrients supplied in breast milk, but, because the and export of food, subsidies on certain foods or bioavailability of some nutrients is lower in formula for producers of food, and so on. than in breast milk, the amounts stated appear higher ● The food industry requires this information in the than those that might be expected to be achieved by development and marketing of products. The breast-feeding. This should not therefore be inter- industry is aware of consumers’ increasing interest preted as an inadequacy on the part of human (breast) in the nutritional quality of the food that they buy, Dietary Reference Standards 127

and has responded by providing foods to address eat to meet their own perceived needs. At the very particular perceived needs, and more informative least, consumers should be able to compare prod- food labels. ucts to get their money’s worth. ● Researchers and the health professions need to assess the nutritional adequacy of the diets of 7.4 The use of reference values to assess groups (or, cautiously, of individuals) by compar- the adequacy of the nutrient intakes of ing dietary intake survey data with the dietary refer- population groups ence values (see below). Once the limitations of the dietary assessment data have been taken into Ideally, this is accomplished by discovering the distri- account (see Chapter 10), this information can be bution of intakes of a nutrient in the population used to attempt to improve people’s nutrient intakes group (e.g., by carrying out a dietary survey), and by bringing them more into line with the dietary comparing these intakes with the distribution of recommendations. The formulation of dietary requirements for that nutrient within the same popu- advice or guidelines depends on an appreciation of lation. In practice, reliable data with which to plot the the existing situation: the solution can only be second of these distributions have rarely been col- framed once the problem is characterized. lected, and therefore what must be used is an estima- ● Institutions and caterers use dietary recommenda- tion of the average requirement together with an esti- tions to assess the requirements of groups and mation of the variance in that requirement, i.e., the devise nutritionally adequate menus. This is a great standard deviation (based on whatever scientifi c evi- deal more easily said than done, mainly because of dence is available), that is used to plot the population the fi nancial constraints involved and, often, the distribution of requirements as shown in Figure 7.1. food preferences of the population being catered When considering how to assess the adequacy of for. nutrient intakes of populations it is important ● The public needs this information to help in the to compare the intakes with the most appropriate interpretation of nutrition information on food level of requirement as defi ned in dietary labels that may describe nutrient content in both recommendations. absolute terms (g, mg, etc.) and as a percentage of It is not useful to compare usual intakes with the the recommended dietary allowance (RDA) for that RDA (PRI, RNI, i.e., the average requirement plus a nutrient (usually per 100 g or per “serving”). It is notional 2 SDs) at the population level since this thought that the latter is more meaningful to con- approach leads to overestimates of the prevalence of sumers, even though the concepts involved in inadequacy. (It may, however, be justifi ed to compare setting the dietary recommendations are rather an individual’s intake with the RDA.) Furthermore, complex (making it diffi cult to judge which level of this approach might be seen to encourage the con- recommendation should be used as the standard) sumption of higher intakes, which could be toxic in and they can be open to misinterpretation (see the case of certain nutrients. above). Since 1998, some UK manufacturers and Comparison of the population intake with the retailers have provided information about guide- average requirement [AR; estimated average require- line daily amounts (GDAs) for energy, some nutri- ment (EAR)] is now considered to be the best estima- ents, , and fi ber. These were developed by the tion of dietary adequacy; if the average intake is less Institute of Grocery Distribution (IGD, a UK than the average requirement, then it is clear that research and training body for the food and grocery there could be a problem in that population. Accord- chain) and are derived from the DRVs [and the ingly, using the average requirement as a cut-off point, British Committee on Medical Aspects of Food the proportion of individuals in the group whose Policy (COMA) and Scientifi c Advisory Council usual intakes are not meeting their requirements can on Nutrition (SACN) recommendations for salt be calculated, allowing the problem to be quantifi ed. intake], but are much simplifi ed. Unless consumers However, this approach cannot be used in the case of are provided with nutrition information in the energy since energy intakes and requirements are most appropriate form on food labels, they cannot highly correlated (the effects of an imbalance being make informed choices as to what foods to buy and quickly obvious to the individual). 128 Introduction to Human Nutrition

The lowest defi ned intake level [lowest threshold US EAR 60 and 75 mg/day for women and men, intake (LTI), lower reference nutrient intake (LRNI), respectively). Similarly, variations in calcium recom- i.e., the average requirement minus a notional 2 SDs] mendations exist because some committees choose to is not regarded as being useful in the context of assess- use zero calcium balance as the criterion of adequacy, ing the adequacy of population nutrient intakes. This while others use maximum skeletal calcium reserves. is because it would identify only those individuals In some cases, one recommending body will include who were almost certainly not meeting their require- a nutrient among its dietary recommendations while ment, and by the same token would omit to include others will not; for example, vitamin E, the require- many in the population who would be at appreciable ment for which depends directly on the dietary intake risk of nutrient inadequacy (in other words, those and tissue levels of PUFAs, which are highly skewed. whose intake was below the average requirement). The vitamin E requirement corresponding to the Finally, the tolerable upper levels of intake defi ned highest levels of PUFA intake would be much higher for certain nutrients can also be used as cut-off points than that needed by those with much lower (but ade- to identify those individuals at risk of consuming quate) intakes. To set the high value as the recom- toxic levels of a nutrient. mendation might suggest to those with lower polyunsaturate intakes that they should increase their 7.5 Methods used to determine intake of vitamin E (unnecessarily). Thus, in Britain requirements and set dietary and Europe, only “safe and adequate” intakes have recommendations been set, based on actual intakes in healthy popula- tions, which should be at least 3 mg/day for women In order to derive the most accurate and appropriate and 4 mg/day for men. In contrast, the US RDA (DRI) dietary recommendations, committees of experts are has been raised to 15 mg/day as α-, based established that look at the scientifi c evidence and use on induced vitamin E defi ciency studies in humans their judgment to decide which nutrients to consider and measures of lipid peroxidation. and then, for each nutrient, make decisions in respect There are even some examples of dietary compo- of the: nents that have not traditionally been regarded as essential nutrients having recommendations set for ● criterion by which to defi ne adequacy them, as in the case of choline. The US DRI defi nes ● estimation of the average amount required to meet an adequate intake for choline (of 450 and 550 mg/ that criterion of adequacy day for women and men, respectively), on the basis ● estimated standard deviation of requirement in the that endogenous synthesis of this compound is not population under consideration (i.e., the shape of always adequate to meet the demand for it (for the frequency distribution over the range of require- the synthesis of acetylcholine, phospholipids, and ments: broad, narrow, skewed, etc.). betaine). Dietary intake data for choline and the sci- The problem of different committees identifying entifi c evidence for inadequacy are limited; thus, different criteria of adequacy is illustrated by vitamin dose–response studies would need to be done before C (ascorbic acid). Experimental evidence (the Shef- an average requirement could be derived. It is proba- fi eld and Iowa studies) has shown that an intake of ble that further dietary components will be included approximately 10 mg/day is required to prevent the in dietary recommendations as research data accu- defi ciency disease scurvy in adult men. At intakes mulate. Potential candidates include the fl avonoids below 30 mg/day, serum levels are negligible, rising and some other antioxidant compounds. steeply with intakes of between 30 and 70 mg/day, after which they begin to plateau (and urinary excre- tion of the unmetabolized vitamin increases). The 7.6 Methods used to question facing the committees drafting dietary refer- determine requirements ence values is whether to choose a level of intake that allows some storage of the vitamin in the body pool Deprivation studies (e.g., EU AR 30 mg/day for adults), or one that more This is the most direct method and involves removing nearly maximizes plasma and body pool levels (e.g., the nutrient from the diet, observing the symptoms Dietary Reference Standards 129 of defi ciency, and then adding back the nutrient until in the gut (generally in the case of those nutrients of the symptoms are cured or prevented. Diffi culties which the uptake is regulated) or the rate of excretion with this approach are as follows. First, that the exper- in the urine (in the case of very soluble nutrients) or iment may need to continue for several years owing feces, or both. However, there comes a point beyond to the presence of body stores of the nutrient, and which balance cannot be maintained; therefore, it can often requires a very limited and therefore boring be proposed that the minimum intake of a nutrient dietary regimen. Second, unpredicted long-term at which balance can be maintained is the subject’s adverse consequences may result. Third, such experi- minimum required intake of that nutrient. However, ments are not ethical in vulnerable groups such as this approach would need to be extended over time children (often the most relevant for study). In some to investigate possible adaptive responses to reduced cases, epidemiological data may be available; for intakes, e.g., absorption could eventually be increased. example, the defi ciency disease beriberi occurs in In the case of calcium, the European consensus is that populations whose average thiamin intake falls below average daily losses are assumed to be 160 mg/day in 0.2 mg/4.2 MJ (1000 kcal). adults, and absorption is assumed to be 30%; thus, around 530 mg would need to be consumed to balance Radioactive tracer studies the losses. Adding or subtracting 30% to allow for This approach makes use of a known amount of the individual variation (the notional 2 SDs explained radioactively labeled nutrient, which is assumed to above) gives (rounded) dietary reference values of disperse evenly in the body pool, allowing the estima- 400, 550 and 700 mg/day (LTI, AR, and PRI, tion of the total pool size by dilution of the isotope respectively). in samples of, for instance, plasma or urine (i.e., if the body pool is large, then the dilution will be greater Factorial methods than if the body pool is small). Specifi c activity, that These are predictions, rather than measurements, of is radioactivity per unit weight of the nutrient in the the requirements of groups or individuals, taking into samples, can be used to calculate pool size as long as account a number of measured variables (factors, the total dose administered is known. The rate of loss hence “factorial”) and making assumptions where can then be monitored by taking serial samples, allow- measurements cannot be made. For example, the ing calculation of the depletion rate. In the case of increased requirements during growth, pregnancy, or vitamin C, the average body pool size of a healthy lactation are calculated by this method; this approach male was found to be 1500 mg, which, on a vitamin is necessitated by the lack of experimental data in C-free diet, depleted at a rate of approximately 3% (of these physiological situations owing to ethical prob- the body pool) per day. This fractional catabolic rate lems. The idea is that the rate of accumulation of was independent of body pool size, and symptoms of nutrients can be calculated and hence the amount scurvy appeared when the body pool fell below required in the diet to allow that accumulation can be 300 mg. The estimated replacement intake needed to predicted. In the case of pregnancy, the requirement maintain the body pool above 300 mg was therefore is estimated to be the amount of the nutrient needed 3% of 300 mg, i.e., 9 mg (similar to the 10 mg found to achieve balance when not pregnant plus the amount to be needed to prevent scurvy in the earlier Sheffi eld accumulated daily during the pregnancy, all multi- experiment). plied by a factor accounting for the effi ciency of absorption and assimilation (e.g., 30% for calcium). Balance studies For lactation, the calculation for energy is based on These rely on the assumption that, in healthy the amount in the milk secreted daily, which is individuals of stable body weight, the body pool of increased by a factor accounting for the effi ciency of some nutrients (e.g., nitrogen, calcium, and sodium) conversion from dietary energy to milk energy (reck- remains constant. Compensation mechanisms equal- oned to be 95%), from which total is subtracted an ize the intake and output of the nutrient over a wide allowance for the contribution from the extra fat range of intakes, thereby maintaining the body pool. stores laid down during pregnancy, which it is desir- Thus, day-to-day variations of intake are compen- able to reduce in this way. The diffi culty with this sated for by changes in either the rate of absorption approach is that the theoretical predictions do not 130 Introduction to Human Nutrition necessarily take account of physiological adaptations reductase depends on ribofl avin and, when activity is (e.g., increased effi ciency of absorption in the gut) measured in both the presence and absence of excess that may reduce the predicted requirement. This ribofl avin, the ratio of the two activities (the erythro- would apply particularly in the case of pregnancy, as cyte glutathione reductase activation coeffi cient, shown by the ability of women to produce normal EGRAC) refl ects ribofl avin status: if perfectly suffi - babies even in times of food shortage. cient, the ratio would be 1.0, whereas defi ciency gives values greater than 1.0. Measurement of nutrient levels in biological tissues Biological markers Some nutrient requirements can be defi ned according These are measures of some biological function that to the intakes needed to maintain a certain level of is directly dependent on the nutrient of interest; the nutrient in blood or tissue. For many water- again, not always easy to fi nd, hence the recent sug- soluble nutrients, such as vitamin C, blood levels gestion that some functional indices be considered refl ect recent dietary intake, and the vitamin is not that are not necessarily directly dependent on the generally measurable in plasma at intakes less than nutrient. Iron status is assessed according to a battery about 40 mg/day. This level of intake has therefore of biological markers, including plasma ferritin been chosen as the basis for the reference in some (which refl ects body iron stores), serum transferrin countries such as the UK. This approach is not, saturation (the amount of plasma transferrin in rela- however, suitable for those nutrients of which the tion to the amount of iron transported by it is reduced plasma concentration is homeostatically regulated, in defi ciency), plasma-soluble transferrin receptor such as calcium. In the case of the fat-soluble vitamin (an index of tissue iron status), and the more tradi- retinol, the dietary intake required to maintain a liver tional tests such as blood hemoglobin (now consid- concentration of 20 μg/g has been used as the basis of ered to be a rather insensitive and unreliable measure the reference intake. To do this, the body pool size of iron status since it indicates only frank anemia, and needed to be estimated; assumptions were made as to also changes as a normal response to altered physio- the proportion of body weight represented by the logical states such as pregnancy). liver (3%) and the proportion of the body pool of Vitamin K status is assessed by measuring pro- retinol contained in the liver (90%). The fractional thrombin time (the length of time taken by plasma to catabolic rate has been measured as 0.5% of the body clot), which is increased when vitamin K levels fall pool per day, so this would be the amount needing to since the synthesis of prothrombin in the liver depends be replaced daily. The effi ciency of conversion of on vitamin K as a cofactor. This test is clinically useful dietary vitamin A to stored retinol was taken to be in patients requiring anticoagulant therapy (e.g., 50% (measured range 40–90%), giving an EAR of using warfarin, which blocks the effect of vitamin K), around 500 μg/day for a 74 kg man. in whom the drug dosage must be closely monitored. Biochemical markers In many respects, biochemical markers represent the Animal experiments most satisfactory measure of nutrient adequacy since These are of limited use in defi ning human nutrient they are specifi c to the nutrient in question, are sensi- requirements because of species differences (e.g., rats tive enough to identify subclinical defi ciencies, and can synthesize vitamin C, so it is not a “vitamin” for may be measured precisely and accurately. However, them), differences in metabolic body size (i.e., the such markers are available for only a few nutrients, proportions of metabolically active tissue, such as mostly vitamins, at present. One well-established muscle, and less active tissue, such as adipose tissue, example of a biochemical marker is the erythrocyte gut contents), and differences in growth rates (young glutathione reductase activation test for ribofl avin animals generally grow far more rapidly than humans, status. Erythrocytes are a useful cell to use for enzyme e.g., cattle reach adult size in about 1 year). However, assays since they are easily obtainable and have a animals have provided much of the information on known life-span in the circulation (average 120 days), the identifi cation of the essential nutrients, and their aiding the interpretation of results. Glutathione physiological and biochemical functions. Furthermore, Dietary Reference Standards 131 animals can be used in experiments that would not Food and Agriculture Organization/United Nations University/ be possible in humans, such as lifelong modifi cations World Health Organization. Energy and Protein Requirements. Report of a Joint FAO/WHO/UNU Expert Consultation. in nutrient intake; it is merely the setting of human Technical Report Series 724. WHO, Geneva, 1985. requirements for which they are inappropriate. Food and Agriculture Organization/World Health Organization. Human Vitamin and Mineral Requirements. Report of a joint FAO/WHO expert consultation. Bangkok, Thailand. FAO, Rome, 7.7 Perspectives on the future 2002. Institute of Medicine (USA). Dietary Reference Intakes for Calcium, As the amount known about human requirements Phosphorus, Magnesium, Vitamin D and Fluoride. National Academy Press, Washington, DC, 1997. and nutrient functions increases, so too will the com- Institute of Medicine (USA). Dietary Reference Intakes for Thiamin, plexity of dietary recommendations. It is probable Ribofl avin, Niacin, , Folate, Vitamin B12, Pantothenic that further dietary components will be included in Acid, Biotin and Choline. National Academy Press, Washington, DC, 1998. dietary recommendations as research data accumu- Institute of Medicine (USA). Dietary Reference Intakes for Water, late. Potential candidates include the fl avonoids and Potassium, Sodium, and . National Academy some other antioxidant compounds. Furthermore, Press, Washington, DC, 1998. Institute of Medicine (USA). Dietary Reference Intakes for Vitamin continuing research and the development of more A, Vitamin K, Arsenic, , Chromium, Copper, Iodine, Iron, informed interpretations of the expanding body of Manganese, Molybdenum, , , and Zinc. data available necessitate the regular revision and National Academy Press, Washington, DC, 2000. Institute of Medicine (USA). Dietary Reference Intakes for Vitamin updating of the recommendations. C, Vitamin E, Selenium and Carotenoids. National Academy The general conclusion that can be drawn here is Press, Washington, DC, 2000. that no single criterion of nutrient status can be used Institute of Medicine (USA). Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein and to defi ne human requirements for all nutrients. This Amino Acids (Macronutrients). National Academy Press, is not surprising when one considers the range of Washington, DC, 2005. roles that the different essential nutrients play in Institute of Medicine (USA). Dietary Reference Intakes: The Essential Guide to Nutrient Requirements. National Academy Press, humans. Washington, DC, 2006. National Health and Medical Research Council. Nutrient Reference Values for Australia and New Zealand Including Recommended Further reading Dietary Intakes. Wickliffe Ltd, Wellington, 2006. Available online at http://www.nhmrc.gov.au/publications/synopses/_fi les/n35. Department of Health. Dietary Reference Values for Food Energy and pdf. Nutrients for the United Kingdom. Report on Health and Social National Research Council, Food and Nutrition Board, Commission Subjects 41. Committee on Medical Aspects of Food Policy. on Life Sciences. Recommended Dietary Allowances, 10th edn. HMSO, London, 1991. National Academy Press, Washington, DC, 1989. Department of Health. Nutrition and Bone Health: with Particular United Nations University. International harmonisation of Reference to Calcium and Vitamin D. Report on Health and approaches for developing nutrient-based dietary standards. In: Social Subjects 49. Committee on Medical Aspects of Food and King JC, Garza, C, eds. Food and Nutrition Bulletin, vol. 28, no. Nutrition Policy. The Stationery Offi ce, London, 1998. 1 (supplement). International Nutrition Foundation for The Dietary reference intake texts available online at: http://lab. United Nations University, Tokyo, 2007. Available online at nap.edu/nap-cgi/discover.cgi?term=dietary%20reference% http://www.unu.edu/unupress/food/FNBv28n1_Suppl1_fi nal. 20intakes&restric=NAP. pdf. EC Scientifi c Committee for Food Report. Nutrient and Energy World Health Organization. Handbook on Human Nutritional Intakes for the European Community. 31st Series. Director Requirements. Monograph Series No. 61. WHO, Geneva, 1974. General, Industry, Luxembourg, 1993. World Health Organization. Diet, Nutrition and the Prevention of Expert Group on Vitamins and Minerals. Safe Upper Limits Chronic Diseases. Technical Report Series 797. WHO, Geneva, for Vitamins and Minerals. Food Standards Agency, London, 1990. 2003. World Health Organization. Trace Elements in Human Nutrition Food and Agriculture Organization/World Health Organization. and Health. WHO in collaboration with FAO, AEA, Geneva, Requirements for Vitamin A, Iron, Folate and Vitamin B12. Report 1996. of a Joint FAO/WHO Expert Consultation. Food and Nutrition Series. FAO, Rome, 1988. 8 The Vitamins

David A Bender

Key messages

• The vitamins are a chemically disparate group of compounds with • Where relevant, this chapter will deal with each of the vitamins a variety of functions in the body. under the following headings: • What they have in common is that they are organic compounds • vitamers that are required for the maintenance of normal health and meta- • absorption and metabolism bolic integrity. • metabolic functions and other uses • Vitamins are required in very small amounts, of the order of mil- • defi ciency ligrams or micrograms per day, and thus can be distinguished • requirements from the essential fatty acids and the essential amino acids, • assessment of status which are required in larger amounts of grams per day. • toxicity and drug interactions.

8.1 Introduction Two factors were found to be essential: one was found in the cream and the other in the watery part of milk. In order to demonstrate that a compound is a vitamin, Logically, they were called factor A (fat-soluble, in the it is necessary to demonstrate both that deprivation cream) and factor B (water-soluble, in the watery part of experimental subjects will lead to the development of the milk). Factor B was identifi ed chemically as an of a more or less specifi c clinical defi ciency disease amine, and in 1913 the name “vitamin” was coined and abnormal metabolic signs, and that restoration for these “vital amines.” of the missing compound will prevent or cure the Further studies showed that “vitamin B” was a defi ciency disease and normalize metabolic abnor- mixture of a number of compounds, with different malities. It is not enough simply to demonstrate that actions in the body, and so they were given numbers as a compound has a function in the body, since it may well: vitamin B1, vitamin B2, and so on. There are gaps normally be synthesized in adequate amounts to meet in the numerical order of the . When what requirements, or that a compound cures a disease, might have been called vitamin B3 was discovered, it since this may simply refl ect a pharmacological action was found to be a chemical compound that was already and not indicate that the compound is a dietary known, nicotinic acid. It was therefore not given a essential. number. Other gaps are because compounds that were The vitamins, and their principal functions and assumed to be vitamins and were given numbers, such defi ciency signs, are shown in Table 8.1; the curious as B4 and B5, were later shown either not to be vitamins, nomenclature is a consequence of the way in which or to be vitamins that had already been described by they were discovered at the beginning of the twentieth other workers and given other names. century. Early studies showed that there was some- Vitamins C, D and E were named in the order of thing in milk that was essential, in very small amounts, their discovery. The name “vitamin F” was used at one for the growth of animals fed on a diet consisting of time for what we now call the essential fatty acids; purifi ed fat, carbohydrate, protein, and mineral salts. “vitamin G” was later found to be what was already

© 2009 DA Bender. The Vitamins 133

Table 8.1 The vitamins, their principal functions and defi ciency diseases

Vitamin Functions Defi ciency disease

A Retinol Visual pigments in the retina; cell differentiation; Night blindness, xerophthalmia; keratinization of skin β-Carotene β-carotene is an antioxidant D Calciferol Maintenance of calcium balance; enhances Rickets (poor mineralization of bone); osteomalacia + intestinal absorption of Ca2 and mobilizes (demineralization of bone) bone mineral E Tocopherols Antioxidant, especially in cell membranes Extremely rare: serious neurological dysfunction K Phylloquinone Coenzyme in formation of γ-carboxyglutamate in Impaired blood clotting, hemorrhagic disease Menaquinones enzymes of blood clotting and bone matrix B1 Thiamin Coenzyme in pyruvate and 2-keto-glutarate Peripheral nerve damage (beriberi) or central nervous dehydrogenases, and transketolase; poorly system lesions (Wernicke–Korsakoff syndrome) defi ned function in nerve conduction B2 Ribofl avin Coenzyme in oxidation and reduction reactions; Lesions of corner of mouth, lips, and tongue; prosthetic group of fl avoproteins seborrheic dermatitis Niacin Nicotinic acid Coenzyme in oxidation and reduction reactions, Pellagra: photosensitive dermatitis, depressive Nicotinamide functional part of NAD and NADP psychosis B6 Pyridoxine Coenzyme in transamination and decarboxylation Disorders of amino acid metabolism, convulsions Pyridoxal of amino acids and glycogen phosphorylase; Pyridoxamine role in steroid hormone action Folic acid Coenzyme in transfer of one-carbon fragments Megaloblastic anemia B12 Cobalamin Coenzyme in transfer of one-carbon fragments Pernicious anemia (megaloblastic anemia with and metabolism of folic acid degeneration of the spinal cord) Functional part of coenzyme A and acyl carrier Peripheral nerve damage (burning foot syndrome) protein H Biotin Coenzyme in carboxylation reactions in Impaired fat and carbohydrate metabolism, dermatitis gluconeogenesis and fatty acid synthesis C Ascorbic acid Coenzyme in hydroxylation of proline and lysine Scurvy: impaired , loss of dental in collagen synthesis; antioxidant; enhances cement, subcutaneous hemorrhage absorption of iron

known as vitamin B2. Biotin is still sometimes called in adequate amounts to meet requirements. These vitamin H. Vitamin K was discovered by Henrik Dam, include carnitine, choline, inositol, taurine, and in Denmark, as a result of studies of disorders of ubiquinone. blood coagulation, and he named it for its function: Two compounds that are generally considered to koagulation in Danish. be vitamins can be synthesized in the body, normally As the chemistry of the vitamins was elucidated, so in adequate amounts to meet requirements: vitamin they were given names as well, as shown in Table 8.1. D, which is synthesized from 7-dehydrocholesterol in When only one chemical compound has the biologi- the skin on exposure to , and niacin, which is cal activity of the vitamin, this is quite easy. Thus, synthesized from the essential amino acid tryptophan.

vitamin B1 is thiamin, vitamin B2 is ribofl avin, etc. However, both were discovered as a result of studies With several of the vitamins, a number of chemically of defi ciency diseases that were, during the early related compounds found in foods can be intercon- twentieth century, signifi cant public health problems: verted in the body, and all show the same biological rickets (due to vitamin D defi ciency and inadequate activity. Such chemically related compounds are sunlight exposure) and pellagra (due to defi ciency of called vitamers, and a general name (a generic descrip- both tryptophan and preformed niacin). tor) is used to include all compounds that display the same biological activity. 8.2 Vitamin A Some compounds have important metabolic func- tions, but are not considered to be vitamins, since, as Vitamin A was the fi rst vitamin to be discovered, ini- far as is known, they can be synthesized in the body tially as an essential dietary factor for growth. It has 134 Introduction to Human Nutrition a role in vision, as the prosthetic group of the light- acid (preformed vitamin A); and a variety of caro- sensitive proteins in the retina, and a major role in the tenes and related compounds (collectively known as regulation of gene expression and tissue differentia- carotenoids) that can be cleaved oxidatively to yield tion. Defi ciency is a major public health problem in retinaldehyde, and hence retinol and retinoic acid. large areas of the world, and prevention of vitamin A Those carotenoids that can be cleaved to yield retinal- defi ciency is one of the three micronutrient priorities dehyde are known as A carotenoids. of the World Health Organization (WHO) (the other Preformed vitamin A (mainly as retinyl esters) is two are iron and iodine). found only in foods of animal origin. The richest source by far is liver, which may contain suffi cient Vitamers and international units vitamin A to pose a potential problem for pregnant Two groups of compounds, shown in Figure 8.1, have women, since retinol is teratogenic in excess. Caro- vitamin A activity: retinol, retinaldehyde, and retinoic tenes are found in green, yellow, and red fruits and

CH3 CH3 H3C CH3 CH2OH Retinol CH3

CH3 CH3 H3C CH3 H C O Retinaldehyde CH3

CH3 CH3 CH3 H3C CH3 H3C CH3 COO-

all-trans-Retinoic acid CH3 CH3 9-cis-Retinoic acid H3C COO-

H3C CH3 CH3 H3C CH3

H3C CH3 CH3 CH3 CH3 α-Carotene

H3C CH3 CH3 H3C CH3

CH CH H3C CH3 CH 3 3 3 β-Carotene Figure 8.1 The major vitamin A vitamers and vitamin A active carotenoids. The Vitamins 135 vegetables, as well as in liver, margarine, and milk and absorbed, and even at high levels of intake this falls milk products. In addition to their role as precursors only slightly. However, in people with a very low fat of vitamin A, carotenoids have potentially useful anti- intake (less than about 10% of energy from fat), oxidant action, and there is epidemiological evidence absorption of both retinol and carotene is impaired, that diets that are rich in carotenoids (both those that and low-fat diets are associated with vitamin A are vitamin A active and those that are not) are associ- defi ciency. ated with a lower incidence of cancer and cardio- Dietary retinyl esters are hydrolyzed by lipases in vascular disease. However, intervention studies with the intestinal lumen and mucosal brush border mem- β-carotene have been disappointing, and it is not pos- brane, then re-esterifi ed to form retinyl palmitate sible to determine desirable intakes of carotene other before release into the circulation in chylomicrons. than as a precursor of vitamin A. Tissues can take up retinyl esters from chylomi- Retinoic acid is a metabolite of retinol; it has crons, but most retinol is in the chylomicron rem- important biological activities in its own right and nants that are taken up by the liver. Here retinyl esters will support growth in vitamin A-defi cient animals. are hydrolyzed, and the vitamin may either be secreted The oxidation of retinaldehyde to retinoic acid is irre- from the liver bound to retinol binding protein, or be versible. Retinoic acid cannot be converted in vivo to transferred to stellate cells in the liver, where it is retinol, and does not support either vision or stored as retinyl esters in intracellular lipid droplets. in defi cient animals. Some 50–80% of the total body content of retinol is Some 50 or more dietary carotenoids are potential in the stellate cells of the liver, but a signifi cant amount sources of vitamin A: α-, β-, and γ-carotenes and may also be stored in adipose tissue. cryptoxanthin are quantitatively the most important. The main pathway for catabolism of retinol is Although it would appear from its structure that one oxidation to retinoic acid (which, as discussed molecule of β-carotene will yield two of retinol, below, has important biological activities in its this is not so in practice. Nutritionally, 6–12 μg of β- own right, distinct from the activities of retinol). carotene is equivalent to 1 μg of preformed retinol. The main excretory product of both retinol and For other carotenes with vitamin A activity, 12–24 μg retinoic acid is retinoyl glucuronide, which is secreted is equivalent to 1 μg of preformed retinol. in the bile. Conventionally, the total amount of vitamin A in As the intake of retinol increases, and the liver con- foods is expressed as μg retinol equivalents, calculated centration rises above 70 μmol/kg, a different pathway from the sum of μg of preformed vitamin A + 1/6 becomes increasingly important for the catabolism of × μg β-carotene + 1/12 × μg other provitamin A retinol in the liver. This is a microsomal cytochrome carotenoids. Recent studies on the absorption of caro- P450-dependent oxidation, leading to a number of tenes and their bioeffi cacy as vitamin A precursors polar metabolites that are excreted in the urine and have led to the defi nition of retinol activity equiva- bile. At high intakes this pathway becomes saturated, lents. 1 μg retinol activity equivalent = 1 μg preformed and excess retinol is toxic since there is no further retinol, 12 μg β-carotene or 24 μg other provitamin capacity for its catabolism and excretion. A carotenoids. Before pure vitamin A was available for chemical Carotene dioxygenase analysis, the vitamin A content of foods was deter- Like retinol, carotenoids are absorbed dissolved in mined by biological assays and the results were lipid micelles. The biological availability and absorp- expressed in standardized international units (IU): tion of dietary carotene varies between 5% and 60%, 1 IU = 0.3 μg of retinol, or 1 μg of retinol = 3.33 IU. depending on the nature of the food, whether it is Although obsolete, IU are sometimes still used in cooked or raw, and the amount of fat in the meal. food labeling. As shown in Figure 8.2, β-carotene and other pro- vitamin A carotenoids are cleaved in the intestinal Metabolism and storage of vitamin A and mucosa by carotene dioxygenase, yielding retinalde- pro-vitamin A carotenoids hyde, which is reduced to retinol, then esterifi ed and Retinol is absorbed from the small intestine dissolved secreted in chylomicrons together with retinyl esters in lipid. About 70–90% of dietary retinol is normally formed from dietary retinol. 136 Introduction to Human Nutrition

H3C CH3 CH3 H3C CH3

H3C CH3 CH3 CH3 CH3 β-Carotene O2 Carotene dioxygenase

H3C CH3 CH3 H3C CH3

H3C CH3 O O CH3 CH3 CH3

CH3 CH3 H3C CH3 H C O

2 × Retinaldehyde CH3

NAD(P)H H2O + O2 Retinol + Aldehyde NAD(P) dehydrogenase oxidase H2O2

CH3 CH3 CH3 CH3 H3C CH3 H3C CH3 - CH2OH COO

Retinol all-trans-Retinoic acid CH3 CH3 Figure 8.2 The oxidative cleavage of carotene to yield retinol and retinoic acid. Carotene dioxygenase (EC 1.13.11.21), retinol dehydrogenase (EC 1.1.1.105), retinaldehyde oxidase (EC 1.2.3.11).

Only a proportion of carotene undergoes oxidation a molar basis, is considerably lower than that of in the intestinal mucosa, and a signifi cant amount of retinol, not twofold higher as might be expected. In carotene enters the circulation in chylomicrons. Caro- addition to poor absorption of carotene, three factors tene in the chylomicron remnants is cleared by the may account for this. liver; some is cleaved by hepatic carotene dioxygenase, again giving rise to retinaldehyde and retinyl esters; ● The intestinal activity of carotene dioxygenase is the remainder is secreted in very low-density lipopro- relatively low, so that a relatively large proportion of teins (VLDLs), and may be taken up and cleaved by ingested β-carotene may be absorbed unchanged. carotene dioxygenase in other tissues. ● Other carotenoids in the diet may inhibit carotene Central oxidative cleavage of β-carotene, as shown dioxygenase and reduce the formation of retinol. in Figure 8.2, should yield two molecules of retinal- ● The principal site of carotene dioxygenase attack is dehyde, which can be reduced to retinol. However, as the central bond of β-carotene, but asymmetric noted above, the biological activity of β-carotene, on cleavage also occurs, leading to the formation of 8′-, The Vitamins 137

10′- and 12′-apo-carotenals, which are oxidized to dized to 11-cis-retinaldehyde. This reacts with a lysine yield retinoic acid, but are not precursors of retinol residue in opsin, forming the holoprotein rhodopsin. or retinaldehyde. As shown in Figure 8.3, the absorption of light by rhodopsin causes isomerization of the retinaldehyde Plasma retinol binding protein bound to opsin from 11-cis to all-trans, and a confor- Retinol is released from the liver bound to an α- mational change in opsin. This results in the release of globulin, retinol binding protein (RBP); this serves to retinaldehyde from the protein and the initiation of a maintain the vitamin in aqueous solution, protects it nerve impulse. The overall process is known as bleach- against oxidation and delivers the vitamin to target ing, since it results in the loss of the color of rho- tissues. RBP is secreted from the liver as a 1:1 complex dopsin. The all-trans-retinaldehyde released from with the thyroxine-binding prealbumin, transthy- rhodopsin is reduced to all-trans-retinol, and joins the retin. This is important to prevent urinary loss of pool of retinol in the pigment epithelium for isomeri- retinol bound to the relatively small RBP, which would zation to 11-cis-retinol and regeneration of rhodop- otherwise be fi ltered by the kidney, with a consider- sin. The key to initiation of the visual cycle is the able loss of vitamin A from the body. availability of 11-cis-retinaldehyde, and hence vitamin Cell surface receptors on target tissues take up A. In defi ciency both the time taken to adapt to dark- retinol from the RBP–transthyretin complex, trans- ness and the ability to see in poor light are impaired. ferring it on to an intracellular RBP. The receptors The excited form of rhodopsin (metarhodopsin II) also remove the carboxy-terminal arginine residue initiates a G-protein cascade leading to hyperpolar- from RBP, so inactivating it by reducing its affi nity for ization of the outer section membrane of the rod or both transthyretin and retinol. As a result, apo-RBP cone, caused by the closure of sodium channels is fi ltered at the glomerulus; most is reabsorbed in the through the membrane, and the initiation of a nerve proximal renal tubules and hydrolyzed. The apopro- impulse. tein is not recycled. During the development of vitamin A defi ciency in Retinoic acid and the regulation of experimental animals, the plasma concentration of gene expression RBP falls, whereas the liver content of apo-RBP rises. The main function of vitamin A is in the control of The administration of retinol results in release of cell differentiation and turnover. All-trans-retinoic holo-RBP from the liver. This provides the basis of acid and 9-cis-retinoic acid are active in the regulation the relative dose–response (RDR) test for liver reserves of growth, development, and tissue differentiation; of vitamin A (see below). they have different actions in different tissues. Like the steroid hormones and vitamin D, retinoic acid Metabolic functions of vitamin A interacts with nuclear receptors that bind to response and carotenes elements (control regions) of DNA, and regulate the The fi rst function of vitamin A to be defi ned was in transcription of specifi c genes. vision. More recently, retinoic acid has been shown to There are two families of nuclear retinoid recep- have a major function in regulation of gene expres- tors: the retinoic acid receptors (RARs) bind all-trans- sion and tissue differentiation. retinoic acid or 9-cis-retinoic acid, and the retinoid X receptors (RXRs) bind 9-cis-retinoic acid, and some Vitamin A in vision of the other physiologically active retinoids as well. In the retina, retinaldehyde functions as the prosthetic RXR can form active dimers with RARs, RXRs group of the light-sensitive opsin proteins, forming (homodimers), and the receptors for rhodopsin (in rods) and iodopsin (in cones). Any one (vitamin D), thyroid hormone, long-chain polyun- cone cell contains only one type of opsin, and hence saturated fatty acid (PUFA) derivatives [the peroxi- is sensitive to only one color of light. Color blindness some proliferators-activated receptor (PPAR)], and results from loss or mutation of one or other of the one for which the physiological ligand has not yet cone opsins. been identifi ed (the COUP receptor). In the pigment epithelium of the retina, all-trans- The result of this is that a very large number of retinol is isomerized to 11-cis-retinol and then oxi- genes are sensitive to control by retinoic acid in 138 Introduction to Human Nutrition

CH3 CH3 CH3 H3C CH3 H3C CH3 CH2OH

CH3 CH3 H3C cis all-trans-Retinol 11- -Retinol CH2OH

CH3 H3C CH3

CH3 H3C 11-cis-Retinaldehyde HC O

+ C O H3N

CH3 NH H3C CH3 Lysine residue in opsin

CH H C 3 3 C O Rhodopsin HC N (visual purple) NH

Light 10–15 s

CH3 CH3 H3C CH3 H C O C N

NH CH3 Photorhodopsin 45 ps Bathorhodopsin 30 ns

Lumirhodopsin 75 µs Metarhodopsin I

10 ms GTP Transducin-GDP Pi Metarhodopsin II GTPase H C CH CH3 CH3 3 3 H Transducin-GTP C O min GDP Metarhodopsin III CH3 Inactive Active all-trans-Retinaldehyde phosphodiesterase phosphodiesterase Opsin cGMP 5'GMP Na+ channel open Na+ channel closed Figure 8.3 Role of vitamin A and the cyclic GMP cascade in the visual cycle. Retinol isomerase (EC 5.2.1.3), phosphodiesterase (EC 3.1.4.35). The Vitamins 139 different tissues, and at different stages in develop- to green light; this is followed by impairment of the ment, and retinoic acid is essential for the normal ability to adapt to dim light, then an inability to see responses to vitamin D, thyroid hormone and long- at all in dim light: night blindness. More prolonged chain PUFA derivatives. or severe defi ciency leads to the condition called Unoccupied RXRs can form dimers with calcitriol xerophthalmia: keratinization of the cornea, followed and other receptors; these bind to hormone response by ulceration – irreversible damage to the eye that elements on DNA, but do not lead to activation of causes blindness. At the same time there are changes transcription. This means that vitamin A defi ciency in the skin, with excessive formation of keratinized will impair responses to vitamin D and thyroid tissue. hormone more markedly than might be expected Vitamin A also plays an important role in the dif- simply from lack of 9-cis-retinoic acid to form active ferentiation of immune system cells, and mild defi - heterodimers. ciency, not severe enough to cause any disturbance of Vitamin A in excess may also impair responsiveness vision, leads to increased susceptibility to a variety of to vitamin D and other hormones, since high concen- infectious diseases. At the same time, the synthesis of trations of 9-cis-retinoic acid will lead to the forma- RBP is reduced in response to infection (it is a nega- tion of RXR–RXR homodimers, leaving too few RXRs tive acute-phase protein), so that there is a reduction to form heterodimers with vitamin D and other in the circulating concentration of the vitamin, and receptors. There is epidemiological evidence that hence further impairment of immune responses. habitually high intakes of vitamin A are associated Signs of vitamin A defi ciency also occur in protein– with poor bone health in later life as a result of energy malnutrition, regardless of whether or not the impaired responsiveness to vitamin D. intake of vitamin A is adequate. This is due to impair- ment of the synthesis of plasma RBP; functional The antioxidant function of carotenes vitamin A defi ciency can occur secondary to protein– At least in vitro, and under conditions of low oxygen energy malnutrition; even if liver reserves of the availability, carotenes can act as radical-trapping anti- vitamin are adequate, it cannot be mobilized. oxidants. There is epidemiological evidence that high intakes of carotene are associated with a low incidence Vitamin A requirements and of cardiovascular disease and some forms of cancer, reference intakes although the results of intervention trials with β- There have been relatively few studies of vitamin A carotene have been disappointing, with an increased requirements in which subjects have been depleted of incidence of lung cancer among those taking carotene the vitamin for long enough to permit the develop- supplements. ment of clear defi ciency signs. Current estimates of The problem is that although carotene is an anti- requirements are based on the intakes required to oxidant at a low partial pressure of oxygen, as occurs maintain a concentration in the liver of 70 μmol in most tissues, at a high partial pressure of oxygen, retinol/kg, as determined by measurement of the rate as occurs in the lungs, it is an autocatalytic pro- of metabolism of isotopically labeled vitamin A. This oxidant, acting as a source of oxygen radicals. The UK is adequate to maintain normal plasma concentra- Food Standards Agency specifi cally advises smokers tions of the vitamin, and people with this level of liver not to take carotene supplements. reserves can be maintained on a vitamin A-free diet for many months before they develop any detectable Vitamin A defi ciency: night blindness signs of defi ciency. and xerophthalmia The average requirement to maintain a concentra- Worldwide, vitamin A defi ciency is a major public tion of 70 μmol/kg of liver is 6.7 μg retinol equiva- health problem and the most important preventable lents/kg body weight, and this is the basis for cause of blindness; the WHO estimates that some 256 calculation of reference intakes. million children under 5 years old show subclinical defi ciency and 2.7 million have xerophthalmia. Assessment of vitamin A status The earliest signs of clinical defi ciency are associ- The only direct assessment of vitamin A status is by ated with vision. Initially, there is a loss of sensitivity liver biopsy and measurement of retinyl ester reserves. 140 Introduction to Human Nutrition

This is an invasive procedure that cannot be consid- needs for 4–6 months. About 1% of children so ered for routine investigations and population surveys. treated show transient signs of toxicity, but this is Status can also be assessed by clinical and functional considered an acceptable risk in view of the high tests, the plasma concentrations of retinol and RBP, prevalence and devastating effects of defi ciency. and the response to a test dose of vitamin A, the RDR The chronic toxicity of vitamin A is a more general test. cause for concern; prolonged and regular intake of In fi eld surveys, clinical signs of vitamin A defi - more than about 7.5–9 mg/day by adults (and signifi - ciency, including Bitot’s spots, corneal xerosis, corneal cantly less for children) causes signs and symptoms ulceration, and keratomalacia, can be used to identify of toxicity affecting: those suffering from vitamin A defi ciency. The earliest ● the central nervous system: headache, nausea, ataxia signs of corneal damage are detected by conjunctival and anorexia, all associated with increased cerebro- impression cytology (CIC); however, abnormalities spinal fl uid pressure only develop when liver reserves are seriously ● the liver: hepatomegaly with histological changes depleted. in the liver, increased collagen formation and The ability to adapt to dim light is impaired early hyperlipidemia in defi ciency, and dark adaptation time is sometimes ● bones: joint pains, thickening of the long bones, used to assess vitamin A status. However, the test is hypercalcemia and calcifi cation of soft tissues not suitable for use on children (the group most at ● the skin: excessive dryness, scaling and chapping of risk of defi ciency) and the apparatus is not suited to the skin, desquamation and alopecia. use in the fi eld. The fasting plasma concentration of retinol remains The recommended upper limits of habitual intake of constant over a wide range of intakes and only falls retinol, compared with reference intakes, are shown signifi cantly when liver reserves are nearly depleted. in Table 8.2. As discussed above, habitual high intakes Therefore, although less sensitive to subtle changes of vitamin A, albeit below these prudent upper levels within the normal range than some methods of of intake, may be associated with impaired respon- assessing nutritional status, measurement of plasma siveness to vitamin D, poor mineralization of bone retinol provides a convenient and sensitive means of and the early development of osteoporosis. detecting people whose intake of vitamin A is inade- quate to maintain normal liver reserves. Teratogenicity of vitamin A The RDR test is a test of the ability of a dose of The synthetic retinoids (vitamin A analogues) used in retinol to raise the plasma concentration several hours dermatology are highly teratogenic. After women after chylomicrons have been cleared from the circu- have been treated with them, it is recommended lation. It depends on the fact that apo-RBP accumu- that contraceptive precautions be continued for 12 lates in the liver in vitamin A defi ciency. The RDR is months, because of their retention in the body. By the ratio of the plasma concentration of retinol 5 h after the dose to that immediately before it was given. An RDR greater than 20% indicates depletion of liver Table 8.2 Prudent upper levels of habitual vitamin A intake μ retinol to less than 70 mol/kg. Upper limit of intake Reference intakesa Age group (μg/day) (μg/day) Toxicity of vitamin A There is only a limited capacity to metabolize vitamin Infants 900 350–375 A. Excessively high intakes lead to accumulation in 1–3 years 1800 400 4–6 years 3000 400–500 the liver and other tissues, beyond the capacity of 6–12 years 4500 500–700 normal binding proteins, so that free, unbound, 13–20 years 6000 600–700 vitamin A is present. This leads to liver and bone Adult men 9000 600–1000 damage, hair loss, vomiting, and headaches. Single Adult women 7500 600–800 doses of 60 mg of retinol are given to children in Pregnant women 3000–3300 700 developing countries as a prophylactic against vitamin a Reference intakes show range for various national and international A defi ciency: an amount adequate to meet the child’s authorities. The Vitamins 141 extrapolation, it has been assumed that retinol is also nuclear receptors that regulate gene expression. teratogenic, although there is little evidence. In case– Defi ciency, leading to rickets in children and osteo- control studies, intakes between 2400 μg/day and malacia in adults, continues to be a problem in north- 3300 μg/day during pregnancy have been associated ern latitudes, where sunlight exposure is poor. with birth defects. Other studies have not demon- There are relatively few sources of vitamin D, mainly strated any teratogenic effect at this level of intake, oily fi sh, with eggs, liver, and butter providing modest and it has been suggested that the threshold plasma amounts; fortifi ed milk, containing ergocalciferol, is concentration associated with teratogenic effects is available in some countries. As a result, strict vegetar- unlikely to be reached with intakes below 7500 μg/ ians are especially at risk of defi ciency, especially in day. Nevertheless, pregnant women are advised not to northern latitudes with little sunlight exposure. consume more than 3000 μg/day (American Pediatric Although meat provides apparently negligible Association recommendation) or 3300 μg (UK quantities of vitamin D, it may be an important Department of Health recommendation). source, since what is present is largely the fi nal active metabolite, calcitriol, which is many times more Interactions of vitamin A with drugs and potent on a molar basis than is cholecalciferol. other nutrients Historically, there was considerable confusion between Vitamers and international units vitamins A and D, and for many years it was not clear The normal dietary form of vitamin D is cholecalcif- which acted in which system. By the 1950s it was erol (also known as calciol). This is also the com- believed that the problem had been solved, with pound that is formed in the skin by ultraviolet (UV) clearly defi ned functions of vitamin A in vision, and irradiation of 7-dehydrocholesterol. Some foods are vitamin D in calcium homeostasis and bone develop- enriched or fortifi ed with (synthetic) ergocalciferol, ment. However, both have overlapping effects on a which undergoes the same metabolism as cholecalcif- number of systems, including bone metabolism and erol and has the same biological activity. Early studies immune system function. It is now known that this is assigned the name vitamin D1 to an impure mixture the result of formation of retinoid–vitamin D recep- of products derived from the irradiation of ergosterol; tor heterodimers, so that in some systems both are when ergocalciferol was identifi ed it was called required in appropriate amounts for normal regula- vitamin D2, and when the physiological compound tion of gene expression. was identifi ed as cholecalciferol it was called vita-

Chlorinated hydrocarbons, as contained in agricul- min D3. tural pesticides, deplete liver retinol. Metabolites of Like vitamin A, vitamin D was originally measured polychlorinated biphenyls bind to the thyroxine in international units of biological activity before the binding site of transthyretin, and in doing so impair pure compound was isolated: 1 IU = 25 ng of chole- the binding of RBP. As a result there is free RBP- calciferol; 1 μg of cholecalciferol = 40 IU. bound retinol in plasma, which is fi ltered at the glom- erulus and hence lost in the urine. Habitual use of Absorption and metabolism barbiturates may also lead to defi ciency as a result of Vitamin D is absorbed in lipid micelles and incorpo- induction of cytochrome P450, which catalyzes the rated into chylomicrons; therefore, people on a low- catabolism of retinol. fat diet will absorb little of such dietary vitamin D as is available. Indeed, it is noteworthy that at the time 8.3 Vitamin D that rickets was a major public health problem in Scotland, herrings (a rich source) were a signifi cant Vitamin D is not strictly a vitamin, since it can be part of the diet: it can only be assumed that the diet synthesized in the skin, and indeed under most condi- was so low in fat that the absorption of the vitamin tions endogenous synthesis is the major source of the was impaired. vitamin: it is only when sunlight exposure is inade- quate that a dietary source is required. Its main func- Synthesis of vitamin D in the skin tion is in the regulation of calcium absorption and As shown in Figure 8.4, the steroid 7-dehydrocholes- homeostasis; most of its actions are mediated by terol (an intermediate in the synthesis of cholesterol 142 Introduction to Human Nutrition OH 2 CH OH OH Tachysterol Calcitriol HO C 3 H OH (1,25-dihydroxycholecalciferol) Kidney, 1-hydroxylase Light 2 2 CH CH Previtamin D 3 Calcidiol Vitamin D CH OH HO HO Liver, calciol (cholecalciferol) (25-hydroxycholecalciferol) Slow 25-hydroxylase Kidney, Light OH thermal isomerization 24-hydroxylase OH 2 CH HO 7-Dehydrocholesterol HO Figure 8.4 Figure D synthesis and metabolism. Vitamin 24-Hydroxycalcidiol The Vitamins 143

Table 8.3 Nomenclature of vitamin D metabolites

Trivial name Recommended name Abbreviation

Vitamin D3 Cholecalciferol Calciol –

25-Hydroxycholecalciferol Calcidiol 25(OH)D3

1α-Hydroxycholecalciferol 1(S)-Hydroxycalciol 1α(OH)D3

24,25-Dihydroxycholecalciferol 24(R)-Hydroxycalcidiol 24,25(OH)2D3

1,25-Dihydroxycholecalciferol Calcitriol 1,25(OH)2D3

1,24,25-Trihydroxycholecalciferol Calcitetrol 1,24,25(OH)3D3

Vitamin D2 Ergocalciferol Ercalciol –

25-Hydroxyergocalciferol Ercalcidiol 25(OH)D2

24,25-Dihydroxyergocalciferol 24(R)-Hydroxyercalcidiol 24,25(OH)2D2

1,25-Dihydroxyergocalciferol Ercalcitriol 1,25(OH)2D2

1,24,25-Trihydroxyergocalciferol Ercalcitetrol 1,24,25(OH)3D2

The abbreviations shown in column 3 are not recommended, but are frequently used in the literature.

that accumulates in the skin but not other tissues) yield ercalcitriol. The nomenclature of the vitamin D undergoes a non-enzymic reaction on exposure to metabolites is shown in Table 8.3. UV light, yielding previtamin D, which undergoes a The fi rst stage in vitamin D metabolism occurs in further reaction over a period of hours to form cho- the liver, where it is hydroxylated to form the 25- lecalciferol, which is absorbed into the bloodstream. hydroxy derivative calcidiol. This is released into the In temperate climates there is a marked seasonal circulation bound to a vitamin D binding globulin. variation in the plasma concentration of vitamin D; There is no tissue storage of vitamin D; plasma cal- it is highest at the end of summer and lowest at the cidiol is the main storage form of the vitamin, and it end of winter. Although there may be bright sunlight is plasma calcidiol that shows the most signifi cant in winter, beyond about 40° N or S there is very little seasonal variation in temperate climates. UV radiation of the appropriate wavelength for cho- The second stage of vitamin D metabolism occurs lecalciferol synthesis when the sun is low in the sky. in the kidney, where calcidiol undergoes either 1- By contrast, in summer, when the sun is more or less hydroxylation to yield the active metabolite 1,25- overhead, there is a considerable amount of UV light dihydroxyvitamin D (calcitriol) or 24-hydroxylation even on a moderately cloudy day, and enough can to yield an apparently inactive metabolite, 24,25- penetrate thin clothes to result in signifi cant forma- dihydroxyvitamin D (24-hydroxycalcidiol). Calcidiol tion of vitamin D. 1-hydroxylase is also found in other tissues that are In northerly climates, and especially in polluted capable of forming calcitriol as an autocrine or para- industrial cities with little sunlight, people may well crine agent. not be exposed to enough UV light to meet their vitamin D needs, and they will be reliant on the few Regulation of vitamin D metabolism dietary sources of the vitamin. The main function of vitamin D is in the control of calcium homeostasis and, in turn, vitamin D metabo- Metabolism to calcitriol lism in the kidney is regulated, at the level of 1- or Cholecalciferol, either synthesized in the skin or from 24-hydroxylation, by factors that respond to plasma foods, undergoes two hydroxylations to yield the concentrations of calcium and phosphate. In tissues active metabolite, 1,25-dihydroxyvitamin D or cal- other than the kidney that hydroxylate calcidiol to citriol, as shown in Figure 8.4. Ergocalciferol from calcitriol, the enzyme is not regulated in response to fortifi ed foods undergoes similar hydroxylation to plasma calcium. 144 Introduction to Human Nutrition

● Calcitriol acts to reduce its own synthesis and ● differentiation of monocyte precursor cells; increase formation of 24-hydroxycalcidiol, by regu- ● modulation of cell differentiation, proliferation and lating the expression of the genes for the two apoptosis. hydroxylases. In most of its actions, the role of calcitriol seems to ● is secreted in response to a be in the induction or maintenance of synthesis of fall in plasma calcium. In the kidney it acts to calcium binding proteins, and the physiological effects increase the activity of calcidiol 1-hydroxylase and are secondary to changes in intracellular calcium decrease that of 24-hydroxylase. In turn, both cal- concentrations. citriol and high concentrations of calcium repress Calcitriol acts like a steroid hormone, binding to a the synthesis of parathyroid hormone; calcium also nuclear receptor protein, commonly as a heterodimer inhibits the secretion of the hormone from the with the RXR (vitamin A) receptor, then binding to parathyroid gland. hormone response elements on DNA and modifying ● Calcium exerts its main effect on the synthesis the expression of one or more genes. and secretion of parathyroid hormone. However, The best-studied actions of vitamin D are in the calcium ions also have a direct effect on the kidney, intestinal mucosa, where the intracellular calcium reducing the activity of calcidiol 1-hydroxylase. binding protein induced by vitamin D is essential for ● Phosphate also affects calcidiol metabolism; the absorption of calcium from the diet. Vitamin D throughout the day there is an inverse fl uctuation also acts to increase the transport of calcium across of plasma phosphate and calcitriol, and feeding the mucosal membrane by recruiting calcium trans- people on a low-phosphate diet results in increased port proteins to the cell surface. circulating concentrations of calcitriol. Calcitriol also raises plasma calcium by stimulating the mobilization of calcium from bone. It achieves Metabolic functions of vitamin D this by activating osteoclast cells. However, it acts later The principal function of vitamin D is to maintain to stimulate the laying down of new bone to replace the plasma concentration of calcium; calcitriol the loss, by stimulating the differentiation and recruit- achieves this in three ways: ment of osteoblasts.

● increased intestinal absorption of calcium ● reduced excretion of calcium by stimulating resorp- Vitamin D defi ciency: tion in the distal renal tubules (due to increased rickets and osteomalacia calbindin D synthesis) Historically, rickets is a disease of toddlers, especially ● mobilization of bone mineral. in northern industrial cities. Their bones are under- mineralized as a result of poor absorption of calcium There is a growing body of evidence that low vitamin in the absence of adequate amounts of calcitriol. D status (but not such a degree of defi ciency as to When the child begins to walk, the long bones of the disturb calcium homeostasis) is associated with legs are deformed, leading to bow-legs or knock knees. impaired glucose tolerance, insulin resistance and More seriously, rickets can also lead to collapse of the non-insulin dependent diabetes mellitus, as well as ribcage and deformities of the bones of the pelvis. obesity and the low grade chronic infl ammation asso- Similar problems may also occur in adolescents who ciated with (especially abdominal) obesity. There is are defi cient in vitamin D during the adolescent also evidence poor vitamin D status is a factor in the growth spurt, when there is again a high demand for etiology of some cancers. Calcitriol has a variety of calcium for new bone formation. permissive or modulatory effects; it is a necessary, but Osteomalacia is the adult equivalent of rickets. It not suffi cient, factor, in: results from the demineralization of bone, rather than ● synthesis and secretion of insulin, parathyroid, and the failure to mineralize it in the fi rst place, as is the thyroid hormones; case with rickets. Women who have little exposure to ● inhibition of production of interleukin by activated sunlight are especially at risk from osteomalacia after T-lymphocytes and of immunoglobulin by acti- several , because of the strain that preg- vated B-lymphocytes; nancy places on their marginal reserve of calcium. The Vitamins 145

Osteomalacia also occurs in the older people. Here hypervitaminosis and hypercalcemia. Increased sun- again the problem may be inadequate exposure to light exposure will improve vitamin D status without sunlight, but there is also evidence that the capacity the risks of toxicity, but excessive sunlight exposure is to form 7-dehydrocholesterol in the skin decreases a cause of skin cancer. The main problem in trying to with advancing age, so that older people are more balance improved vitamin D status through increased reliant on the few dietary sources of vitamin D. sunlight exposure, and increased risk of skin cancer, Although vitamin D is essential for prevention and is that there is very little information on the amount treatment of osteomalacia in older people, there is less of sunlight exposure required for the synthesis of a evidence that it is benefi cial in treating the other given amount of vitamin D. common degenerative bone disease of advancing age, osteoporosis, which is due to a loss of bone matrix, Vitamin D toxicity rather than enhanced release of calcium from bone During the 1950s, rickets was more or less totally with no effect on the organic matrix, as is seen in eradicated in Britain and other temperate countries. osteomalacia. The result is negative calcium balance This was due to enrichment of a large number of and loss of bone mineral, but secondary to the loss of infant foods with vitamin D. However, a small number organic matrix, owing to progressive loss of estrogens of infants suffered from vitamin D poisoning, the and androgens, rather than failure of the vitamin D most serious effect of which is an elevated plasma system. concentration of calcium. This can lead to contrac- tion of blood vessels, and hence dangerously high Vitamin D requirements and blood pressure, and calcinosis, that is the calcifi cation reference intakes of soft tissues, including the kidney, heart, lungs, and It is diffi cult to determine requirements for dietary blood vessel walls. vitamin D, since the major source is synthesis in the Some infants are sensitive to intakes of vitamin D skin. Before the development of methods for mea- as low as 50 μg/day. To avoid the serious problem of surement of calcidiol the diagnosis of subclinical vitamin D poisoning in these susceptible infants, the rickets was by detection of elevated alkaline phospha- extent to which infant foods are fortifi ed with vitamin tase in plasma; nowadays, the main criterion of ade- D has been reduced considerably. Unfortunately, this quacy is the plasma concentration of calcidiol. means that a small proportion, who have relatively In older people with little sunlight exposure, a high requirements, are now at risk of developing dietary intake of 10 μg of vitamin D/day results in a rickets. The problem is to identify those who have plasma calcidiol concentration of 20 nmol/l, the lower higher requirements and provide them with end of the reference range for younger adults at the supplements. end of winter. Therefore, the reference intake for older The toxic threshold in adults is not known, but people is 10 μg/day, whereas average intakes of vitamin those patients suffering from vitamin D intoxication D from unfortifi ed foods are less than 4 μg/day. who have been investigated were taking supplements There is little evidence to establish what are appro- providing more than 250 μg/day. priate plasma concentrations of calcidiol; certainly Although excess dietary vitamin D is toxic, exces- the lower end of the reference range for young adults sive exposure to sunlight does not lead to vitamin D at the end of winter in a temperate climate is a mini- poisoning. There is a limited capacity to form the malist goal, and is not much higher than the level at precursor, 7-dehydrocholesterol, in the skin, and which biochemical signs of defi ciency occur. However, a limited capacity to take up cholecalciferol from unfortifi ed foods will not meet even this goal. the skin. Furthermore, prolonged exposure of previ- There is increasing evidence that high vitamin D tamin D to UV light results in further reactions to status is associated with a lower incidence of various yield lumisterol and other biologically inactive cancers, diabetes, and the metabolic syndrome, sug- compounds. gesting that desirable intakes are higher than current reference intakes. Widespread fortifi cation of foods Interactions with drugs and other nutrients would improve vitamin D status, but might also put As discussed above, vitamin D receptors form het- a signifi cant proportion of the population at risk of erodimers with RXR, so that vitamin D-dependent 146 Introduction to Human Nutrition functions require adequate, but not excessive, vitamin Synthetic α-tocopherol does not have the same A status. A number of drugs, including barbiturates biological potency as the naturally occurring com- and other anticonvulsants, induce cytochrome pound. This is because the side-chain of tocopherol

P450, resulting in increased catabolism of calcidiol has three centers of asymmetry and when it is synthe- (and retinol), and cause drug-induced osteomalacia. sized chemically the result is a mixture of the various The antituberculosis drug isoniazid inhibits cholecal- isomers. In the naturally occurring compound all ciferol 25-hydroxylase in the liver, and prolonged three centers of asymmetry have the R-confi guration, administration can lead to the development of and naturally occurring α-tocopherol is called all-R, osteomalacia. or RRR-α-tocopherol. Strontium is a potent inhibitor of the kidney 1- hydroxylase, and strontium intoxication can lead to Absorption and metabolism the development of vitamin D-resistant rickets or Tocopherols and tocotrienols are absorbed unchanged osteomalacia. Although there is normally little expo- from the small intestine, in micelles with other dietary sure to potentially toxic intakes of strontium, its lipids, and incorporated into chylomicrons. The salts are sometimes used to treat chronic lead major route of excretion is in the bile, largely as gluc- intoxication. uronides and other conjugates. There are two mechanisms for tissue uptake of 8.4 Vitamin E vitamin E. Lipoprotein lipase releases the vitamin by hydrolyzing the triacylglycerols in chylomicrons and Although vitamin E was identifi ed as a dietary essen- VLDLs, while separately there is uptake of low-density tial for animals in the 1920s, it was not until 1983 that lipoprotein (LDL)-bound vitamin E by means of LDL it was clearly demonstrated to be a dietary essential receptors. Retention within tissues depends on intra- for human beings. Unlike other vitamins, no unequiv- cellular binding proteins, and it is likely that the dif- ocal physiological function for vitamin E has been ferences in biological activity of the vitamers are due defi ned; it acts as a lipid-soluble antioxidant in cell to differences in the affi nity of these proteins for the membranes, but many of its functions can be replaced different vitamers. by synthetic antioxidants. There is epidemiological evidence that high intakes of vitamin E are associated Metabolic functions of vitamin E with lower incidence of cardiovascular disease, The main function of vitamin E is as a radical- although in many intervention trials vitamin E trapping antioxidant in cell membranes and plasma supplements have been associated with increased lipoproteins. It is especially important in limiting all-cause mortality. radical damage resulting from oxidation of PUFAs, by Vegetable oils are rich sources of vitamin E, but reacting with the lipid peroxide radicals before they signifi cant amounts are also found in nuts and seeds, can establish a chain reaction. The tocopheroxyl most green leafy vegetables, and a variety of fi sh. radical formed from vitamin E is relatively unreactive and persists long enough to undergo reaction to yield Vitamers and units of activity non-radical products. Commonly, the vitamin E Vitamin E is the generic descriptor for two families of radical in a membrane or lipoprotein is reduced back compounds, the tocopherols and the tocotrienols to tocopherol by reaction with vitamin C in plasma. (Figure 8.5). The different vitamers have different The resultant monodehydroascorbate radical then biological potency. The most active is α-tocopherol, undergoes enzymic or non-enzymic reaction to yield and it is usual to express vitamin E intake in terms of ascorbate and dehydroascorbate, neither of which is mg α-tocopherol equivalents. This is the sum of mg a radical. α-tocopherol + 0.5 × mg β-tocopherol + 0.1 × mg The stability of the tocopheroxyl radical means that γ-tocopherol + 0.3 × mg α-. The other it can penetrate further into cells, or deeper into vitamers have negligible vitamin activity. plasma lipoproteins, and potentially propagate a The obsolete international unit of vitamin E activ- chain reaction. Therefore, although it is regarded ity is still sometimes used: 1 IU = 0.67 mg α- as an antioxidant, vitamin E may, like other tocopherol equivalent; 1 mg α-tocopherol = 1.49 IU. antioxidants, also have pro-oxidant actions at high The Vitamins 147 δ-Tocotrienol β-Tocotrienol γ-Tocotrienol α-Tocotrienol 3 3 3 3 CH CH CH CH O O O O 3 3 3 3 3 CH CH CH CH CH C C C 3 3 3 HO HO HO H H H γ-Tocopherol δ-Tocopherol α-Tocopherol β-Tocopherol 3 3 3 3 CH CH CH CH O O O O 3 3 3 3 3 3 CH CH CH CH CH CH C C 3 3 HO HO HO HO Figure 8.5 Figure The vitamin E vitamers, tocopherols and tocotrienols. H H 148 Introduction to Human Nutrition concentrations. This may explain why, although epi- They suffer from severe damage to nerve and muscle demiological studies have shown a clear association membranes. between high blood concentrations of vitamin E and Premature infants are at risk of vitamin E defi - lower incidence of atherosclerosis, the results of inter- ciency, since they are often born with inadequate vention trials have generally been disappointing. In reserves of the vitamin. The red blood cell membranes many trials there has been increased all-cause mortal- of defi cient infants are abnormally fragile, as a result ity among those taking vitamin E and other antioxi- of unchecked oxidative radical attack. This may lead dant supplements. to hemolytic anemia if they are not given supple- The tocotrienols have lower vitamin activity than ments of the vitamin. tocopherols, and indeed it is conventional to consider Experimental animals that are depleted of vitamin only γ-tocotrienol as a signifi cant part of vitamin E E become sterile. However, there is no evidence that intake. However, because of their unsaturated side- vitamin E nutritional status is in any way associated chain, the tocotrienols also have a hypocholesterol- with human fertility, and there is certainly no evi- emic action not shared by the tocopherols. They act dence that vitamin E supplements increase sexual to reduce the activity of 3-hydroxy-3-methylglutaryl- potency, prowess, or vigor. coenzyme A (HMG CoA) reductase, the rate-limiting enzyme in the pathway for synthesis of cholesterol, by Vitamin E requirements repressing synthesis of the enzyme. It is diffi cult to establish vitamin E requirements, partly because defi ciency is more or less unknown, Vitamin E defi ciency but also because the requirement depends on the In experimental animals vitamin E defi ciency results intake of PUFAs. It is generally accepted, albeit with in a number of different conditions. little experimental evidence, that an acceptable intake of vitamin E is 0.4 mg α-tocopherol equivalent/g ● Defi cient female animals suffer the death and reab- dietary PUFA. sorption of the fetuses. This provided the basis of the original biological assay of vitamin E. Indices of vitamin E status ● In male animals defi ciency results in testicular The plasma concentration of α-tocopherol is used to atrophy and degeneration of the germinal epithe- assess vitamin E status; since most vitamin E is trans- lium of the seminiferous tubules. ported in plasma lipoproteins, it is the concentration ● Both skeletal and cardiac muscle are affected in per gram total plasma lipid, or better per mole defi cient animals. This necrotizing myopathy is cholesterol, that is useful, rather than the simple sometimes called nutritional muscular dystrophy – concentration. an unfortunate term, since there is no evidence that Erythrocytes are incapable of de novo lipid synthe- human muscular dystrophy is related to vitamin E sis, so peroxidative damage resulting from oxygen defi ciency. stress has a serious effect, shortening red cell life and ● The integrity of blood vessel walls is affected, with possibly precipitating hemolytic anemia in vitamin E leakage of blood plasma into subcutaneous tissues defi ciency. This has been exploited as a method of and accumulation under the skin of a green fl uid: assessing status by measuring the hemolysis of red exudative diathesis. cells induced by dilute hydrogen peroxide relative to ● The nervous system is affected, with the develop- that observed on incubation in water. This gives a ment of central nervous system necrosis and axonal means of assessing the functional adequacy of vitamin dystrophy. This is exacerbated by feeding diets rich E intake, albeit one that will be affected by other, unre- in PUFAs. lated, factors. Plasma concentrations of α-tocopherol Dietary defi ciency of vitamin E in human beings is below 2.2 mmol/mol cholesterol or 1.1 μmol/g total unknown, although patients with severe fat malab- plasma lipid are associated with increased susceptibil- sorption, cystic fi brosis, some forms of chronic liver ity of erythrocytes to induced hemolysis in vitro. disease or (very rare) congenital lack of plasma β- An alternative method of assessing functional anti- lipoprotein suffer defi ciency because they are unable oxidant status, again one that is affected by both to absorb the vitamin or transport it around the body. vitamin E and other antioxidants, is by measuring the The Vitamins 149 exhalation of pentane arising from the catabolism of selenium acts both to remove the cause of lipid per- the products of peroxidation of n-6 PUFAs or ethane oxidation and to recycle vitamin E. arising from n-3 PUFAs. 8.5 Vitamin K Higher levels of intake There is good epidemiological evidence that intakes Vitamin K was discovered as a result of investigations of vitamin E are associated with a lower risk of ath- into the cause of a bleeding disorder (hemorrhagic erosclerosis and ischemic heart disease. High concen- disease) of cattle fed on silage made from sweet clover trations of vitamin E will inhibit the oxidation of and of chickens fed on a fat-free diet. The missing PUFAs in plasma lipoproteins, and it is this oxidation factor in the diet of the chickens was identifi ed as that is responsible for the development of atheroscle- vitamin K, whereas the problem in the cattle was that rosis. The plasma concentrations of α-tocopherol the feed contained dicumarol, an antagonist of the that appear to be benefi cial would require an intake vitamin. of 17–40 mg/day, which is above what could be Since the effect of an excessive intake of dicumarol achieved by eating normal diets. Individual interven- was severely impaired blood clotting, it was isolated tion trials of vitamin E supplements have generally and tested in low doses as an anticoagulant, for use in been disappointing, and metaanalysis shows a signifi - patients at risk of thrombosis. Although it was effec- cant increase in all-cause mortality among people tive, it had unwanted side-effects, and synthetic taking vitamin E (and other antioxidant) supple- vitamin K antagonists were developed for clinical use ments. This presumably refl ects the fact that the stable as anticoagulants. The most commonly used of these tocopheroxyl radical can penetrate deeper into tissues is warfarin, which is also used, in larger amounts, to and plasma lipoproteins, and increase radical damage. kill rodents. However, it is also possible that the plasma concentra- tion of α-tocopherol is a surrogate marker for some Vitamers other protective factor in the diet. Three compounds have the biological activity of Interactions with other nutrients vitamin K (Figure 8.6): Vitamin C in plasma and extracellular fl uid is impor- ● phylloquinone, the normal dietary source, found in tant in reducing the tocopheroxyl radical in cell green leafy vegetables; membranes and plasma lipoproteins back to tocoph- ● menaquinones, a family of related compounds syn- erol. There is also evidence that a variety of lipid- thesized by intestinal bacteria, with differing lengths soluble antioxidants may be important in the of the side-chain; antioxidant action of vitamin E in membranes and ● menadiol and menadiol diacetate, synthetic com- lipoproteins, including ubiquinone and synthetic pounds that can be metabolized to phylloquinone. antioxidants used in food processing, such as butyl- ated hydroxytoluene and butylated hydroxyanisole. Dietary sources, bacterial synthesis Synthetic antioxidants will prevent or cure a number and metabolism of the signs of vitamin E defi ciency in experimental Phylloquinone has a role in , and animals. therefore it is found in all green leafy vegetables; the There is a considerable overlap between the func- richest sources are spring (collard) greens, spinach, tions of vitamin E and selenium. Vitamin E reduces and Brussels sprouts. In addition, , rapeseed, lipid peroxide radicals to unreactive fatty acids; the cottonseed, and olive oils are relatively rich in vitamin selenium-dependent enzyme glutathione peroxidase K, although other oils are not. reduces hydrogen peroxide to water, thus lowering the About 80% of dietary phylloquinone is normally intracellular concentration of potentially lipid-dam- absorbed into the lymphatic system in chylomicrons, aging peroxide. A membrane-specifi c isoenzyme of and is then taken up by the liver from chylomicron glutathione peroxidase will also reduce the tocopher- remnants and released into the circulation in VLDLs. oxyl radical back to tocopherol. Thus, vitamin E acts Intestinal bacteria synthesize a variety of menaqui- to remove the products of lipid peroxidation, whereas nones, which are absorbed to a limited extent from 150 Introduction to Human Nutrition

O

CH3

O 3

Phylloquinone (vitamin K1)

O

CH3

O n CH3 Menaquinone (vitamin K2) C O OH O

CH3 CH3

OH O Menadiol diacetate Figure 8.6 The vitamin K vitamers, phylloqui- Menadiol (vitamin K3) C O (acetomenaphthone) none (vitamin K ), menaquinone (vitamin K ), and CH 1 2 3 menadiol (a synthetic compound, vitamin K3). the , again into the lymphatic system, lished. It is the cofactor for the carboxylation of cleared by the liver, and released in VLDLs. It is often glutamate residues in the postsynthetic modifi cation suggested that about half of the requirement for of proteins to form the unusual amino acid γ- vitamin K is met by intestinal bacterial synthesis, but carboxyglutamate, abbreviated to Gla (Figure 8.7). there is little evidence for this, other than the fact that In the presence of warfarin, vitamin K epoxide about half of the vitamin K in liver is phylloquinone cannot be reduced back to the active hydroquinone, and the remainder a variety of menaquinones. It is but accumulates and is excreted as a variety of conju- not clear to what extent the menaquinones are bio- gates. However, if enough vitamin K is provided in logically active. It is possible to induce signs of vitamin the diet, the quinone can be reduced to the active K defi ciency simply by feeding a phylloquinone-defi - hydroquinone by the warfarin-insensitive enzyme, cient diet, without inhibiting intestinal bacterial and carboxylation can continue, with stoichiometric action. utilization of vitamin K and excretion of the epoxide. The synthetic compound menadiol is absorbed High doses of vitamin K are used to treat patients largely into the hepatic portal system, and undergoes who have received an overdose of warfarin, and at alkylation in the liver to yield menaquinone-4, which least part of the resistance of some populations of rats is released together with phylloquinone and other to the action of warfarin is due to a high consumption menaquinones in VLDLs. of vitamin K from maram grass, although there are also genetically resistant populations of rodents. Metabolic functions of vitamin K Prothrombin and several other proteins of the Although it has been known since the 1920s that blood clotting system (factors VII, IX and X, and vitamin K was required for blood clotting, it was not proteins C and S) each contain between four and until the 1970s that its precise function was estab- six γ-carboxyglutamate residues per mole. γ-Carboxy- The Vitamins 151

– – – – H2C COO HC COO OOC CHCOO CH O2 CH CO CH 2 Vitamin K 2 2 2 NH HC C O epoxidase NH HC C O Nonenzymic HN CHC O

Glutamate residue Glutamate carbanion γ-Carboxyglutamate residue

OH O

CH3 CH3 O R R OH O Vitamin K hydroquinone Vitamin K epoxide

NADP+ Disulphide Sulphydryl quinone Vitamin K quinone Vitamin K epoxide reductase reductase reductase NADPH Sulphydryl Disulphide O

CH3

R O Vitamin K quinone Figure 8.7 Role of vitamin K in the carboxylation of glutamate. Vitamin K epoxidase (EC 1.14.99.20), warfarin-sensitive epoxide/quinone reduc- tase (EC 1.1.4.1), warfarin-insensitive quinone reductase (EC 1.1.4.2).

glutamate chelates calcium ions, and so permits the acterized protein simply known as bone matrix Gla binding of the blood clotting proteins to lipid mem- protein. Osteocalcin is interesting in that as well as γ- branes. In vitamin K defi ciency, or in the presence of carboxyglutamate, it also contains hydroxyproline, so an antagonist such as warfarin, an abnormal precur- its synthesis is dependent on both vitamins K and C; sor of prothrombin (preprothrombin) containing in addition, its synthesis is induced by vitamin D, and little or no γ-carboxyglutamate is released into the the release into the circulation of osteocalcin provides circulation. Preprothrombin cannot chelate calcium a sensitive index of vitamin D action. It constitutes or bind to phospholipid membranes, and so is unable some 1–2% of total bone protein, and modifi es the to initiate blood clotting. Preprothrombin is some- crystallization of bone mineral. The matrix Gla times known as PIVKA: the protein induced by protein is found in a variety of tissues, and acts to vitamin K absence. prevent mineralization of soft connective tissue. The fetal warfarin syndrome involves neurological Other vitamin K-dependent proteins as well as bone abnormalities. The vitamin K- It has long been known that treatment of pregnant dependent carboxylase is expressed in different brain women with warfarin or other anticoagulants can regions at different times during embryological devel- lead to bone abnormalities in the child: the fetal war- opment, and the product of the growth arrest-specifi c farin syndrome. Two proteins in bone matrix contain gene 6 (Gas6) is a Gla-containing growth factor γ-carboxyglutamate: osteocalcin and a less well char- that is important in the regulation of growth and 152 Introduction to Human Nutrition

development, and the regulation of apoptosis and cell 8.6 Vitamin B1 (thiamin) survival. Historically, thiamin defi ciency affecting the periph- Vitamin K defi ciency and requirements eral nervous system (beriberi) was a major public Apart from deliberate experimental manipulation, health problem in south-east Asia following the intro- vitamin K defi ciency is unknown, and determination duction of the steam-powered mill that made highly of requirements is complicated by a lack of informa- polished (and therefore thiamin-depleted) rice widely tion on the importance of menaquinones synthesized available. There are still sporadic outbreaks of defi - by intestinal bacteria. ciency among people whose diet is rich in carbohy- The classical way of determining vitamin K status, drate and poor in thiamin. More commonly, thiamin and monitoring the effi cacy of anticoagulant therapy, defi ciency affecting the heart and central nervous is by measuring the time required for the formation system is a problem in people with an excessive con- of a fi brin clot in citrated blood plasma after the addi- sumption of alcohol, to the extent that there was a tion of calcium ions and thromboplastin: the pro- serious suggestion in Australia at one time that thrombin time. A more sensitive index is provided by thiamin should be added to beer. direct measurement of preprothrombin in plasma, The structures of thiamin and the coenzyme most commonly by immunoassay using antisera thiamin diphosphate are shown in Figure 8.8. against preprothrombin that do not react with Thiamin is widely distributed in foods, with pork prothrombin. being an especially rich source; potatoes, whole-grain Based on determination of clotting time, and direct cereals, meat, and fi sh are the major sources in most measurement of prothrombin and preprothrombin, diets. Like other water-soluble vitamins, thiamin is an intake of 1 μg/kg body weight per day is consid- readily lost by into cooking water; further- ered adequate; this forms the basis of reference intakes more, it is unstable to light, and although bread and of between 65 and 80 μg/day for adults. fl our contain signifi cant amounts of thiamin, much A small number of newborn infants have very low of this can be lost when baked goods are exposed to reserves of vitamin K and are at risk of potentially sunlight in a shop window. fatal hemorrhagic disease. It is therefore generally rec- Thiamin is also destroyed by sulfi tes, and in potato ommended that all neonates should be given a single products that have been blanched by immersion in prophylactic dose of vitamin K. sulfi te solution there is little or no thiamin remaining. , including tannic acid in tea and betel Toxicity and drug interactions nuts, also destroy thiamin, and have been associated There is no evidence that phylloquinone has any sig- with thiamin defi ciency. nifi cant toxicity. However, high intakes can overcome the effects of warfarin and other anticoagulants. This means that patients who are being treated with war- farin could overcome the benefi cial effects of their Thiamin medication if they took supplements of vitamin K. H3C N NH2 CH The danger is that if their dose of warfarin is increased 3 CH2 CH2OH to counteract the effects of the vitamin supplements N and they then stop taking the supplements, they C N H2 S would be receiving considerably too much warfarin and would be at risk of hemorrhage. Thiamin diphosphate It is unlikely that a normal diet could provide a H C N NH O O suffi cient excess of vitamin K to lead to problems, but 3 2 CH3 H2 - habitual consumption of especially rich sources could CH2 C O P O P O N result in intakes close to those that antagonize thera- C N O- O- peutic warfarin. A diet containing relatively large H2 S amounts of foods prepared with vitamin K-rich oils Figure 8.8 Thiamin (vitamin B1) and the coenzyme thiamin may pose a risk. diphosphate. The Vitamins 153

Thiaminases that catalyze base exchange or Thiamin triphosphate has a role in nerve conduc- hydrolysis of thiamin are found in microorganisms tion, as the phosphate donor for phosphorylation of (including some that colonize the gut), a variety of a nerve membrane sodium transport protein. plants, and raw fi sh. The presence of thiaminase in fermented fi sh is believed to be a signifi cant factor in Thiamin defi ciency the etiology of thiamin defi ciency in parts of south- Thiamin defi ciency can result in three distinct east Asia. syndromes:

Absorption and metabolism of thiamin ● a chronic peripheral neuritis, beriberi, which may or may not be associated with heart failure and Thiamin is absorbed in the duodenum and proximal edema jejunum, and then transferred to the portal circula- ● acute pernicious (fulminating) beriberi (shoshin tion by an active transport process that is inhibited by beriberi), in which heart failure and metabolic alcohol. This may explain why alcoholics are espe- abnormalities predominate, with little evidence of cially susceptible to thiamin defi ciency. peripheral neuritis Tissues take up both free thiamin and thiamin ● Wernicke’s encephalopathy with Korsakoff’s psy- monophosphate, then phosphorylate them further to chosis, a thiamin-responsive condition associated yield thiamin diphosphate (the active coenzyme) and, especially with alcohol and narcotic abuse. in the nervous system, thiamin triphosphate. Some free thiamin is excreted in the urine, increas- In general, a relatively acute defi ciency is involved ing with diuresis, and a signifi cant amount may also in the central nervous system lesions of the Wernicke– be lost in sweat. Most urinary excretion is as thio- Korsakoff syndrome, and a high energy intake, as in chrome, the result of non-enzymic cyclization, as well alcoholics, is also a predisposing factor. Dry beriberi as a variety of products of side-chain oxidation and is associated with a more prolonged, and presumably ring cleavage. less severe, defi ciency, and a generally low food intake, There is little storage of thiamin in the body, and whereas higher carbohydrate intake and physical biochemical signs of defi ciency can be observed activity predispose to wet beriberi. within a few days of initiating a thiamin-free diet. The role of thiamin diphosphate in pyruvate dehy- drogenase means that in defi ciency there is impaired Metabolic functions of thiamin conversion of pyruvate to acetyl-CoA, and hence Thiamin has a central role in energy-yielding metabo- impaired entry of pyruvate into the citric acid cycle. lism, and especially the metabolism of carbohydrates. Especially in subjects on a relatively high carbohy- Thiamin diphosphate (also known as thiamin pyro- drate diet, this results in increased plasma concentra- phosphate, see Figure 8.8) is the coenzyme for three tions of lactate and pyruvate, which may lead to oxidative decarboxylation reactions: pyruvate dehy- life-threatening lactic acidosis. The increase in plasma drogenase in carbohydrate metabolism, α-keto- lactate and pyruvate after a test dose of glucose has glutarate dehydrogenase in the citric acid cycle, and been used as a means of assessing thiamin nutritional the branched-chain keto-acid dehydrogenase involved status. in the metabolism of leucine, isoleucine, and valine. These three enzymes are multienzyme complexes that Dry beriberi catalyze oxidative decarboxylation of the substrate Chronic defi ciency of thiamin, especially associated linked to reduction of enzyme-bound lipoamide, and with a high carbohydrate diet, results in beriberi, eventually reduction of NAD+ to NADH. which is a symmetrical ascending peripheral neuritis. Thiamin diphosphate is also the coenzyme for Initially, the patient complains of weakness, stiffness transketolase, in the pentose phosphate pathway of and cramps in the legs, and is unable to walk more carbohydrate metabolism. This is the major pathway than a short distance. There may be numbness of the of carbohydrate metabolism in some tissues, and an dorsum of the feet and ankles, and vibration sense important alternative to glycolysis in all tissues, being may be diminished. As the disease progresses, the the source of half of the NADPH required for fatty ankle jerk refl ex is lost, and the muscular weakness acid synthesis. spreads upwards, involving fi rst the extensor muscles 154 Introduction to Human Nutrition of the foot, then the muscles of the calf, and fi nally ated with alcohol misuse, the more usual presentation the extensors and fl exors of the thigh. At this stage is as the Wernicke–Korsakoff syndrome, due to central there is pronounced toe and foot drop: the patient is nervous system lesions. unable to keep either the toe or the whole foot Initially, there is a confused state, Korsakoff’s psy- extended off the ground. When the arms are affected chosis, which is characterized by confabulation and there is a similar inability to keep the hand extended: loss of recent memory, although memory for past wrist drop. events may be unimpaired. Later, clear neurological The affected muscles become tender, numb, and signs develop: Wernicke’s encephalopathy. This is hyperesthetic. The hyperesthesia extends in the form characterized by nystagmus and extraocular palsy. of a band around the limb, the so-called stocking and Post-mortem examination shows characteristic brain glove distribution, and is followed by anesthesia. lesions. There is deep muscle pain, and in the terminal stages, Like shoshin beriberi, Wernicke’s encephalopathy when the patient is bed-ridden, even slight pressure, can develop acutely, without the more gradual as from bedclothes, causes considerable pain. development of Korsakoff’s psychosis, among previously starved patients given intravenous glu- Wet beriberi cose and seriously ill patients given parenteral The heart may also be affected in beriberi, with dilata- hyperalimentation. tion of arterioles, rapid blood fl ow, and increased pulse rate leading to right-sided heart failure and Thiamin requirements edema, so-called wet beriberi. The signs of chronic Because thiamin has a central role in energy-yielding, heart failure may be seen without peripheral neuritis. and especially carbohydrate, metabolism, require- The arteriolar dilatation probably results from high ments depend mainly on carbohydrate intake, and circulating concentrations of lactate and pyruvate have been related to “non-fat calories.” In practice, as a result of impaired activity of pyruvate requirements and reference intakes are calculated on dehydrogenase. the basis of total energy intake, assuming that the average diet provides 40% of energy from fat. For Acute pernicious (fulminating) beriberi: diets that are lower in fat, and hence higher in carbo- shoshin beriberi hydrate, thiamin requirements may be somewhat Heart failure without increased cardiac output, and higher. no peripheral edema, may also occur acutely, associ- From depletion/repletion studies, an intake of at ated with severe lactic acidosis. This was a common least 0.2 mg of thiamin/1000 kcal is required to presentation of defi ciency in Japan, where it was prevent the development of defi ciency signs and called shoshin (meaning acute) beriberi; in the 1920s maintain normal urinary excretion, but an intake of some 26 000 deaths a year were recorded. 0.23 mg/1000 kcal is required for a normal transketo- With improved knowledge of the cause and lase activation coeffi cient (see below). improved nutritional status, the disease has become Reference intakes are calculated on the basis of more or less unknown, although in the 1980s it 100 μg/MJ (0.5 mg/1000 kcal) for adults consuming reappeared among Japanese adolescents consuming a more than 2000 kcal/day, with a minimum require- diet based largely on such high-carbohydrate, low- ment for people with a low energy intake of 0.8– nutrient, foods as sweet carbonated drinks, “instant” 1.0 mg/day to allow for metabolism of endogenous noodles, and polished rice. It also occurs among substrates. alcoholics, when the lactic acidosis may be life- threatening, without clear signs of heart failure. Acute Assessment of thiamin status beriberi has also been reported when previously The impairment of pyruvate dehydrogenase in starved subjects are given intravenous glucose. thiamin defi ciency results in a considerable increase in the plasma concentrations of lactate and pyruvate. Wernicke–Korsakoff syndrome This has been exploited as a means of assessing Whereas peripheral neuritis, acute cardiac beriberi thiamin status, by measuring changes in the plasma and lactic acidosis occur in thiamin defi ciency associ- concentrations of lactate and pyruvate after an oral The Vitamins 155 dose of glucose and mild exercise. The test is not intense yellow color, ribofl avin is widely used as a specifi c for thiamin defi ciency since a variety of other food color. conditions can also result in metabolic acidosis. Although it may be useful in depletion/repletion Photolytic destruction studies, it is little used nowadays in assessment of Photolysis of ribofl avin leads to the formation of nutritional status. lumifl avin (in alkaline solution) and lumichrome (in Whole blood total thiamin below 150 nmol/l is acidic or neutral solution), both of which are biologi- considered to indicate defi ciency. However, the cally inactive. Exposure of milk in clear glass bottles changes observed in depletion studies are small. Even to sunlight or fl uorescent light can result in the loss in patients with frank beriberi the total thiamin con- of signifi cant amounts of ribofl avin. This is poten- centration in erythrocytes is only 20% lower than tially nutritionally important. Lumifl avin and lumi- normal, so whole blood thiamin is not a sensitive chrome catalyze oxidation of lipids (to lipid perox- index of status. ides) and methionine (to methional), resulting in the Although there are several urinary metabolites of development of an unpleasant fl avor, known as the thiamin, a signifi cant proportion is excreted either “sunlight” fl avor. unchanged or as thiochrome, and therefore the urinary excretion of the vitamin (measured as thio- Absorption and metabolism chrome) can provide information on nutritional Apart from milk and eggs, which contain relatively status. Excretion decreases proportionally with intake large amounts of free ribofl avin bound to specifi c in adequately nourished subjects, but at low intakes binding proteins, most of the vitamin in foods is as there is a threshold below which further reduction in fl avin coenzymes bound to enzymes, which are intake has little effect on excretion. released when the protein is hydrolyzed. Intestinal The activation of apo-transketolase in erythrocyte phosphatases then hydrolyze the coenzymes to liber- lysate by thiamin diphosphate added in vitro has ate ribofl avin, which is absorbed in the upper small become the most widely used and accepted index of intestine. The absorption of ribofl avin is limited and thiamin nutritional status. Apo-transketolase is after moderately high doses only a small proportion unstable both in vivo and in vitro, so problems may is absorbed. arise in the interpretation of results, especially if Much of the absorbed ribofl avin is phosphorylated samples have been stored for any appreciable time. An in the intestinal mucosa and enters the bloodstream activation coeffi cient >1.25 is indicative of defi ciency, as ribofl avin phosphate, although this does not seem and <1.15 is considered to refl ect adequate thiamin to be essential for absorption of the vitamin. status. About 50% of plasma ribofl avin is free ribofl avin, which is the main transport form, with 44% as fl avin

8.7 Vitamin B2 (ribofl avin) adenine dinucleotide (FAD) and the remainder as ribofl avin phosphate. The vitamin is largely protein- Ribofl avin defi ciency is a signifi cant public health bound in plasma; free ribofl avin binds to both problem in many areas of the world. The vitamin has albumin and α- and β-globulins; both ribofl avin and a central role as a coenzyme in energy-yielding the coenzymes also bind to immunoglobulins. metabolism, yet defi ciency is rarely, if ever, fatal, since Uptake into tissues is by passive carrier-mediated there is very effi cient conservation and recycling of transport of free ribofl avin, followed by metabolic ribofl avin in defi ciency. trapping by phosphorylation to ribofl avin phosphate, The structures of ribofl avin and the ribofl avin- and onward metabolism to FAD. derived coenzymes are shown in Figure 8.9. Ribofl avin phosphate and FAD that are not bound Milk and dairy products are important sources, to proteins are rapidly hydrolyzed to ribofl avin, which providing 25% or more of total ribofl avin intake in diffuses out of tissues into the bloodstream. Ribofl a- most diets, and it is noteworthy that average ribofl a- vin and ribofl avin phosphate that are not bound to vin status in different countries refl ects milk con- plasma proteins are fi ltered at the glomerulus; renal sumption to a considerable extent. Other rich sources tubular resorption is saturated at normal plasma con- are eggs, meat, and fi sh. In addition, because of its centrations. There is also active tubular secretion of 156 Introduction to Human Nutrition

OH OH OH

CH2 C C C CH2OH H H H

H3C N N O NH H3C N O

OH OH OH OH

CH2 C C C CH2 O P O H H H OH H3C N N O

NH Riboflavin monophosphate H3C N (flavin mononucleotide) O NH2 N OH OH OH OH OH N CH2 C C C CH2 O P O POCH2 O N N H H H OH OH H3C N N O

NH OH OH H3C N Flavin adenine dinucleotide O

Figure 8.9 Ribofl avin (vitamin B2) and the fl avin coenzymes, ribofl avin monophosphate and fl avin adenine dinucleotide. the vitamin; urinary excretion of ribofl avin after at which there is quantitative excretion of the moderately high doses can be two- to threefold greater vitamin. than the glomerular fi ltration rate. There is very effi cient conservation of ribofl avin in Under normal conditions about 25% of the urinary defi ciency, and almost the only loss from tissues will excretion of ribofl avin is as the unchanged vitamin, be the small amount that is covalently bound to with a small amount as glycosides of ribofl avin and enzymes and cannot be salvaged for reuse. There is its metabolites. only a fourfold difference between the minimum con- centration of fl avins in the liver in defi ciency and the Ribofl avin balance level at which saturation occurs. In the central nervous There is no signifi cant storage of ribofl avin; apart system there is only a 35% difference between defi - from the limitation on absorption, any surplus ciency and saturation. intake is excreted rapidly, so that once metabolic requirements have been met urinary excretion of Metabolic functions of the fl avin ribofl avin and its metabolites refl ects intake until coenzymes intestinal absorption is saturated. In depleted animals, The metabolic function of the fl avin coenzymes is as the maximum growth response is achieved with electron carriers in a wide variety of oxidation and intakes that give about 75% saturation of tissues, reduction reactions central to all metabolic processes, and the intake to achieve tissue saturation is that including the mitochondrial electron transport chain, The Vitamins 157 and key enzymes in fatty acid and amino acid oxida- mouth (angular stomatitis), a painful desquamation tion, and the citric acid cycle. The fl avin coenzymes of the tongue, so that it is red, dry, and atrophic remain bound to the enzyme throughout the catalytic (magenta tongue), and a seborrheic dermatitis, with cycle. The majority of fl avoproteins have FAD as the fi liform excrescences, affecting especially the nasola- prosthetic group rather than ribofl avin phosphate; bial folds, eyelids, and ears. some have both fl avin coenzymes and some have There may also be conjunctivitis with vasculariza- other prosthetic groups as well. tion of the cornea and opacity of the lens. This last is Flavins can undergo a one-electron reduction to the only lesion of aribofl avinosis for which the bio- the semiquinone radical or a two-electron reduction chemical basis is known: glutathione is important in to dihydrofl avin. In some enzymes formation of dihy- maintaining the normal clarity of crystallin in the drofl avin occurs by two single-electron steps, with lens, and glutathione reductase is a fl avoprotein that intermediate formation of the semiquinone radical. is particularly sensitive to ribofl avin depletion. Dihydrofl avin can be oxidized by reaction with a The main metabolic effect of ribofl avin defi ciency substrate, NAD(P)+, or cytochromes in a variety of is on lipid metabolism. Ribofl avin-defi cient animals dehydrogenases, or can react with molecular oxygen have a lower metabolic rate than controls and require in oxygenases and mixed function oxidases a 15–20% higher food intake to maintain body weight. (hydroxylases). Feeding a high-fat diet leads to more marked impair- ment of growth and a higher requirement for ribofl a- Flavins and oxidative stress vin to restore growth. Reoxidation of the reduced fl avin in oxygenases and mixed function oxidases proceeds by way of formation Resistance to malaria in ribofl avin defi ciency of the fl avin radical and fl avin hydroperoxide, with the Several studies have noted that in areas where malaria intermediate generation of superoxide and perhy- is endemic, ribofl avin-defi cient subjects are relatively droxyl radicals and hydrogen peroxide. Because of this, resistant and have a lower parasite burden than ade- fl avin oxidases make a signifi cant contribution to the quately nourished subjects. The biochemical basis of total oxidant stress of the body. Overall, some 3–5% of this resistance to malaria in ribofl avin defi ciency is the daily consumption of about 30 mol of oxygen by not known, but two possible mechanisms have been an adult is converted to singlet oxygen, hydrogen proposed. peroxide, and superoxide, perhydroxyl, and hydroxyl radicals, rather than undergoing complete reduction ● The malarial parasites may have a particularly high to water in the electron transport chain. There is thus requirement for ribofl avin. Some fl avin analogues a total production of some 1.5 mol of reactive oxygen have antimalarial action. species daily, potentially capable of causing damage to ● As a result of impaired antioxidant activity in membrane lipids, proteins, and nucleic acids. erythrocytes, there may be increased fragility of erythrocyte membranes or reduced membrane fl u- Ribofl avin defi ciency idity. As in sickle cell trait, which also protects Although ribofl avin is involved in all areas of metabo- against malaria, this may result in exposure of the lism, and defi ciency is widespread on a global scale, parasites to the host’s immune system at a vulner- defi ciency is not fatal. There seem to be two reasons able stage in their development, resulting in the for this. One is that, although defi ciency is common, production of protective antibodies. the vitamin is widespread in foods and most diets will provide minimally adequate amounts to permit main- Ribofl avin requirements tenance of central metabolic pathways. The second, Estimates of ribofl avin requirements are based on more important, reason is that in defi ciency there is depletion/repletion studies to determine the minimum extremely effi cient reutilization of the ribofl avin that intake at which there is signifi cant excretion of the is released by the turnover of fl avoproteins, so that vitamin. In defi ciency there is virtually no excretion only a very small amount is metabolized or excreted. of the vitamin; as requirements are met, so any excess Ribofl avin defi ciency is characterized by lesions of is excreted in the urine. On this basis the minimum the margin of the lips (cheilosis) and corners of the adult requirement for ribofl avin is 0.5–0.8 mg/day. 158 Introduction to Human Nutrition

At intakes of 1.1–1.6 mg/day urinary excretion rises an increase in the EGR activation coeffi cient and sharply, suggesting that tissue reserves are saturated. increased urinary excretion of ribofl avin, with reduced A more generous estimate of requirements, and the tissue concentrations of ribofl avin phosphate and basis of reference intakes, is the level of intake at FAD, despite feeding diets providing more ribofl avin which there is normalization of the activity of the red than is needed to meet requirements. Although there cell enzyme glutathione reductase; the activity of this is no evidence that patients treated with these drugs fl avoprotein is especially sensitive to ribofl avin nutri- for a prolonged period develop clinical signs of ribo- tional status. Normal values of the activation coeffi - fl avin defi ciency, long-term use of chlorpromazine is cient are seen in subjects whose habitual intake of associated with a reduction in metabolic rate. ribofl avin is between 1.2 mg/day and 1.5 mg/day. Ribofl avin defi ciency is sometimes associated Because of the central role of fl avin coenzymes in with hypochromic anemia as a result of impaired energy-yielding metabolism, reference intakes are iron absorption. A greater proportion of a test dose sometimes calculated on the basis of energy intake: of iron is retained in the intestinal mucosal cells 0.14–0.19 mg/MJ (0.6–0.8 mg/1000 kcal). However, bound to ferritin, and hence lost in the feces, rather in view of the wide range of ribofl avin-dependent than being absorbed, because the mobilization of iron reactions, other than those of energy-yielding metab- bound to ferritin in mucosal cells for transfer to olism, it is diffi cult to justify this basis for the calcula- transferrin requires oxidation by a fl avin-dependent tion of requirements. enzyme. Ribofl avin depletion decreases the oxidation of Assessment of ribofl avin nutritional status dietary vitamin B6 to pyridoxal; pyridoxine oxidase is The urinary excretion of ribofl avin and its metabo- a fl avoprotein and is very sensitive to ribofl avin deple- lites (either basal excretion or after a test dose) can be tion. It is not clear to what extent there is functional used as an index of status. However, ribofl avin excre- vitamin B6 defi ciency in ribofl avin defi ciency. This is tion is only correlated with intake in subjects who are partly because vitamin B6 nutritional status is gener- in nitrogen balance. In subjects in negative nitrogen ally assessed by the metabolism of a test dose of tryp- balance there may be more urinary excretion than tophan, and kynurenine hydroxylase in the tryptophan would be expected, as a result of the catabolism of oxidative pathway is a fl avoprotein; ribofl avin defi - tissue fl avoproteins, and loss of their prosthetic ciency can therefore disturb tryptophan metabolism groups. Higher intakes of protein than are required quite separately from its effects on vitamin B6 nutri- to maintain nitrogen balance do not affect the require- tional status. ment for ribofl avin or indices of ribofl avin nutri- The disturbance of tryptophan metabolism in tional status. ribofl avin defi ciency, due to impairment of kynuren- Glutathione reductase is especially sensitive to ine hydroxylase, can also result in reduced synthesis ribofl avin depletion. The activity of the enzyme in of NAD from tryptophan, and may therefore be a erythrocytes can therefore be used as an index of factor in the etiology of pellagra. ribofl avin status. Interpretation of the results can be complicated by anemia, and it is more usual to use 8.8 Niacin the activation of erythrocyte glutathione reductase (EGR) by FAD added in vitro. An activation coeffi - Niacin is not strictly a vitamin, since it can be synthe- cient of 1.0–1.4 refl ects adequate nutritional status, sized in the body from the essential amino acid tryp- whereas >1.7 indicates defi ciency. tophan. Indeed, it is only when tryptophan metabo- lism is deranged that dietary preformed niacin Interactions with drugs and other nutrients becomes important. Nevertheless, niacin was discov- The phenothiazines such as chlorpromazine, used in ered as a nutrient during studies of the defi ciency the treatment of schizophrenia, and the tricyclic anti- disease pellagra, which was a major public health depressant drugs such as imipramine, are structural problem in the southern USA throughout the fi rst analogues of ribofl avin, and inhibit fl avokinase. In half of the twentieth century, and continued to be a experimental animals, administration of these drugs problem in parts of India and sub-Saharan Africa at doses equivalent to those used clinically results in until the 1990s. The Vitamins 159

Several studies have investigated the equivalence of Vitamers and niacin equivalents dietary tryptophan and preformed niacin as precur- Two compounds, nicotinic acid and nicotinamide, sors of the nicotinamide nucleotides, generally by have the biological activity of niacin. When nicotinic determining the excretion of niacin metabolites in acid was discovered as the curative and preventive response to test doses of the precursors, in subjects factor for pellagra, it was already known as a chemical maintained on defi cient diets. The most extensive compound, and was therefore never assigned a such study was that of Horwitt et al. in 1956. They number among the B vitamins. The name niacin was found that there was a considerable variation between coined in the USA when it was decided to enrich subjects in the response to tryptophan and niacin, maize meal with the vitamin to prevent pellagra; it and in order to allow for this individual variation they was considered that the name nicotinic acid was not proposed the ratio of 60 mg of tryptophan equivalent desirable because of its similarity to nicotine. In the to 1 mg of preformed niacin. Changes in hormonal USA the term niacin is commonly used to mean spe- status may result in considerable changes in this ratio, cifi cally nicotinic acid, and nicotinamide is known as with between 7 and 30 mg of dietary tryptophan niacinamide; elsewhere “niacin” is used as a generic being equivalent to 1 mg of preformed niacin in late descriptor for both vitamers. Figure 8.10 shows the pregnancy. structures of nicotinic acid and niacin, as well as the The niacin content of foods is generally expressed nicotinamide coenzymes, NAD and as mg niacin equivalents; 1 mg niacin equivalent = mg NADP. preformed niacin + 1/60 × mg tryptophan. Because The nicotinamide ring of NAD can be synthesized most of the niacin in cereals is biologically unavail- in the body from the essential amino acid tryptophan. able (see below), it is conventional to ignore pre- In adults almost all of the dietary intake of trypto- formed niacin in cereal products. phan, apart from the small amount that is used for Because endogenous synthesis from tryptophan is net new protein synthesis, and synthesis of the neu- more important than preformed dietary niacin, the rotransmitter serotonin, is metabolized by this main dietary sources of niacin are generally those that pathway, and hence is potentially available for NAD are also rich sources of protein. It is only when the synthesis. dietary staple is a cereal such as maize, which is

COOH CONH2

N N Nicotinic acid Nicotinamide

NH2 CONH2 N O O N N CH2 O P O POCH2 O N N OH OH OH OH

OH OH O Nicotinamide adenine dinucleotide Figure 8.10 The niacin vitamers, nicotinic acid and Phosphorylated nicotinamide, and the coenzyme nicotinamide in NADP adenine dinucleotide. 160 Introduction to Human Nutrition remarkably lacking in tryptophan, that problems of Catabolism of NAD(P) defi ciency occur. Trigonelline in coffee beans is The catabolism of NAD+ is catalyzed by four demethylated to nicotinic acid during roasting, and enzymes: moderate coffee consumption may meet a signifi cant ● NAD glycohydrolase, which releases nicotinamide proportion of niacin requirements. and ADP-ribose; ● NAD pyrophosphatase, which releases nicotin- Unavailable niacin in cereals amide mononucleotide; this can be either hydro- Chemical analysis reveals niacin in cereals (largely in lyzed by NAD glycohydrolase to release nicotin- the bran), but this is biologically unavailable, since it amide, or reutilized to form NAD; is bound as niacytin – nicotinoyl esters to a variety of ● ADP-ribosyltransferases; macromolecules. In wheat bran some 60% is esteri- ● poly(ADP-ribose) polymerase. fi ed to polysaccharides, and the remainder to poly- peptides and glycopeptides. The activation of ADP-ribosyltransferase and Treatment of cereals with alkali (e.g., by soaking poly(ADP-ribose) polymerase by toxins, oxidative overnight in calcium hydroxide solution, as is the tra- stress or DNA damage may result in considerable ditional method for the preparation of tortillas in depletion of intracellular NAD(P), and may indeed Mexico) and baking with alkaline baking powder provide a protective mechanism to ensure that cells releases much of the nicotinic acid. This may explain that have suffered very severe DNA damage die as a why pellagra has always been rare in Mexico, despite result of NAD(P) depletion. The administration of the fact that maize is the dietary staple. DNA-breaking carcinogens to experimental animals Up to 10% of the niacin in niacytin may be biologi- results in the excretion of large amounts of nicotin- cally available as a result of hydrolysis by gastric amide metabolites and depletion of tissue NAD(P); acid. addition of the compounds to cells in culture has a similar effect. Chronic exposure to such carcinogens Absorption and metabolism and mycotoxins may be a contributory factor in the Niacin is present in tissues, and therefore in foods, etiology of pellagra when dietary intakes of trypto- largely as the nicotinamide nucleotides. The post- phan and niacin are marginal. mortem hydrolysis of NAD(P) is extremely rapid in animal tissues, so it is likely that much of the niacin Urinary excretion of niacin and metabolites of meat (a major dietary source of the preformed Under normal conditions there is little or no urinary vitamin) is free nicotinamide. excretion of either nicotinamide or nicotinic acid. Nicotinamide nucleotides present in the intestinal This is because both vitamers are actively reabsorbed lumen are not absorbed as such, but are hydrolyzed from the glomerular fi ltrate. It is only when the con- to free nicotinamide. Many intestinal bacteria centration is so high that the reabsorption mecha- have high nicotinamide deamidase activity, and nism is saturated that there is any signifi cant excre- a signifi cant proportion of dietary nicotinamide tion of niacin. may be deamidated in the intestinal lumen. Both Nicotinamide in excess of requirements for nicotinic acid and nicotinamide are absorbed from NAD synthesis is methylated by nicotinamide N- the small intestine by a sodium-dependent saturable methyltransferase. N1-Methylnicotinamide is actively process. secreted into the urine by the proximal renal The nicotinamide nucleotide coenzymes can be tubules. N1-Methylnicotinamide can also be meta- synthesized from either of the niacin vitamers and bolized further, to yield methylpyridone-2- and from quinolinic acid, an intermediate in the metabo- 4-carboxamides. lism of tryptophan. In the liver, synthesis of the coen- Nicotinamide can also undergo oxidation to nico- zymes increases with increasing intake of tryptophan, tinamide N-oxide when large amounts are ingested. but not preformed niacin. The liver exports nicotin- Nicotinic acid can be conjugated with glycine to form amide, derived from turnover of coenzymes, for nicotinuric acid (nicotinoyl-glycine) or may be meth- uptake by other tissues. ylated to trigonelline (N1-methylnicotinic acid). It is The Vitamins 161 not clear to what extent urinary excretion of trigonel- calcium concentrations by releasing calcium from line refl ects endogenous methylation of nicotinic intracellular stores, acting as second messengers in acid, since there is a signifi cant amount of trigonelline response to nitric oxide, acetylcholine, and other in foods, which may be absorbed, but cannot be neurotransmitters. utilized as a source of niacin, and is excreted unchanged. Pellagra: a disease of tryptophan and niacin defi ciency Metabolic functions of niacin Pellagra became common in Europe when maize The best-defi ned role of niacin is in the metabolism of was introduced from the New World as a convenient metabolic fuels, as the functional nicotinamide part of high-yielding dietary staple, and by the late the coenzymes NAD and NADP, which play a major nineteenth century it was widespread throughout role in oxidation and reduction reactions. The oxi- southern Europe, north and south Africa, and the dized coenzymes have a positive charge on the nico- southern USA. The proteins of maize are particularly tinamide ring nitrogen and undergo a two-electron lacking in tryptophan, and as with other cereals little reduction. The oxidized forms are conventionally or none of the preformed niacin is biologically shown as NAD(P)+ and the reduced forms either as available. + NAD(P)H2 or, more correctly, as NAD(P)H + H , Pellagra is characterized by a photosensitive der- since although it is a two-electron reduction, only one matitis, like severe sunburn, typically with a butterfl y- proton is incorporated into the ring, the other remain- like pattern of distribution over the face, affecting all ing associated with the coenzyme. parts of the skin that are exposed to sunlight. Similar In general, NAD+ acts as an electron acceptor in skin lesions may also occur in areas not exposed to energy-yielding metabolism, being oxidized by the sunlight, but subject to pressure, such as the knees, mitochondrial electron transport chain, while the elbows, wrists, and ankles. Advanced pellagra is also major coenzyme for reductive synthetic reactions is accompanied by dementia (more correctly a depres- NADPH. An exception to this general rule is the sive psychosis), and there may be diarrhea. Untreated pentose phosphate pathway of glucose metabolism, pellagra is fatal. which results in the reduction of NADP+ to NADPH, The depressive psychosis is superfi cially similar to and is the source of half the reductant for fatty acid schizophrenia and the organic psychoses, but clini- synthesis. cally distinguishable by sudden lucid phases that In addition to its coenzyme role, NAD is the source alternate with the most fl orid psychiatric signs. It is of ADP-ribose for the ADP-ribosylation of a variety probable that these mental symptoms can be explained of proteins and poly(ADP-ribosylation) and hence by a relative defi cit of the essential amino acid tryp- activation of nucleoproteins involved in the DNA tophan, and hence reduced synthesis of the neuro- repair mechanism. transmitter 5-hydroxytryptamine (serotonin), and In the nucleus, poly(ADP-ribose)polymerase is not to a defi ciency of niacin per se. activated by binding to breakage points in DNA. The enzyme is involved in activation of the DNA repair Additional factors in the etiology of pellagra mechanism in response to strand breakage caused by Pellagra also occurs in India among people whose radical attack or UV radiation. In cells that have suf- dietary staple is jowar (Sorghum vulgare), even though fered considerable DNA damage, the activation of the protein in this cereal contains enough tryptophan poly (ADP-ribose) polymerase may deplete intracel- to permit adequate synthesis of NAD. Here the lular NAD to such an extent that ATP formation is problem seems to be the relative excess of leucine in impaired, leading to cell death. the protein, which can inhibit the synthesis of NAD ADP-ribose cyclase catalyzes the formation of from tryptophan. It is likely that leucine is a factor in cyclic ADP-ribose from NAD, and of nicotinic acid the etiology of pellagra only when the dietary intakes adenine dinucleotide phosphate from NADP (by cat- of both tryptophan and niacin are low, a condition alyzing the exchange of nicotinamide for nicotinic that may occur when sorghum is the dietary staple, acid). Both of these compounds act to raise cytosolic especially at times of food shortage. 162 Introduction to Human Nutrition

Although the nutritional etiology of pellagra is well niacin metabolites, neither of which is wholly established, and tryptophan or niacin will prevent or satisfactory. cure the disease, additional factors, including defi - Niacin toxicity ciency of ribofl avin or vitamin B6, both of which are required for synthesis of NAD from tryptophan, may Nicotinic acid has been used to lower blood triacyl- be important when intakes of tryptophan and niacin glycerol and cholesterol in patients with hyperlipid- are only marginally adequate. emia. However, relatively large amounts are required During the fi rst half of the twentieth century, of the (of the order of 1–6 g/day, compared with reference 87 000 people who died from pellagra in the USA intakes of 18–20 mg/day). At this level of intake, there were twice as many women as men. Reports of nicotinic acid causes dilatation of blood vessels and individual outbreaks of pellagra, both in the USA and fl ushing, with skin irritation, itching, and a burning more recently elsewhere, show a similar gender ratio. sensation. This effect wears off after a few days. This may well be the result of inhibition of trypto- High intakes of both nicotinic acid and nicotin- phan metabolism by estrogen metabolites, and hence amide, in excess of 500 mg/day, also cause liver reduced synthesis of NAD from tryptophan. damage, and prolonged use can result in liver failure. Several bacterial, fungal and environmental toxins This is especially a problem with sustained-release activate ADP-ribosyltransferase or poly(ADP-ribose) preparations of niacin, which permit a high blood polymerase, and it is possible that chronic exposure level to be maintained for a relatively long time. to such toxins will deplete tissue NAD(P) and hence be a contributory factor in the development of pella- 8.9 Vitamin B6 gra when intakes of tryptophan and niacin are marginal. Apart from a single outbreak in the 1950s, due to

overheated infant milk formula, vitamin B6 defi ciency Niacin requirements is unknown except under experimental conditions. On the basis of depletion/repletion studies in which Nevertheless, there is a considerable body of evidence the urinary excretion of niacin metabolites was mea- that marginal status and biochemical defi ciency may sured after feeding tryptophan or preformed niacin, be relatively widespread in developed countries. the average requirement for niacin is 1.3 mg of niacin Vitamin B6 is widely distributed in a variety of equivalents/MJ energy expenditure, and reference foods. However, a considerable proportion of the intakes are based on 1.6 mg/MJ. vitamin in plant foods may be present as glucosides, Average intakes of tryptophan in Western diets will which are probably not biologically available, although more than meet requirements without the need for a a proportion may be hydrolyzed by intestinal dietary source of preformed niacin. bacteria. When foods are heated, pyridoxal and pyridoxal Assessment of niacin status phosphate can react with the ε-amino groups of lysine Although the nicotinamide nucleotide coenzymes to form a Schiff base (aldimine). This renders both function in a large number of oxidation and reduc- the vitamin B6 and the lysine biologically unavailable; tion reactions, this cannot be exploited as a means of more importantly, the pyridoxyl-lysine released assessing the state of the body’s niacin reserves, during digestion is absorbed and has antivitamin B6 because the coenzymes are not fi rmly attached to antimetabolic activity. Overall, it is estimated that their apoenzymes, as are thiamin pyrophosphate, some 70–80% of dietary vitamin B6 is available. ribofl avin, and pyridoxal phosphate, but act as cosub- strates of the reactions, binding to and leaving the Vitamers enzyme as the reaction proceeds. No specifi c meta- The generic descriptor vitamin B6 includes six vita- bolic lesions associated with NAD(P) depletion have mers: the alcohol pyridoxine, the aldehyde pyridoxal, been identifi ed. the amine pyridoxamine, and their 5′-phosphates. The two methods of assessing niacin nutritional There is some confusion in the older literature, status are measurement of the ratio of NAD/ because at one time “pyridoxine,” which is now used NADP in red blood cells and the urinary excretion of specifi cally for the alcohol, was used as a generic The Vitamins 163 descriptor, with “pyridoxol” as the specifi c name for metabolic trapping. Much of the ingested pyridoxine the alcohol. The vitamers are metabolically intercon- is released into the portal circulation as pyridoxal, vertible and, as far as is known, they have equal bio- after dephosphorylation at the serosal surface. Unlike logical activity; they are all converted in the body to other B vitamins, there seems to be no limit on the the metabolically active form, pyridoxal phosphate. amount of vitamin B6 that is absorbed. 4-Pyridoxic acid is a biologically inactive end-product Most of the absorbed vitamin is taken up by the of vitamin B6 metabolism. liver by passive diffusion, followed by metabolic trap- ping as phosphate esters, which do not cross cell Absorption and metabolism membranes, then oxidation to pyridoxal phosphate. The phosphorylated vitamers are dephosphorylated The liver exports both pyridoxal phosphate (bound by membrane-bound alkaline phosphatase in the to albumin) and pyridoxal (which binds to both intestinal mucosa; pyridoxal, pyridoxamine, and pyri- albumin and hemoglobin). Free pyridoxal remaining doxine are all absorbed rapidly by passive diffusion. in the liver is rapidly oxidized to 4-pyridoxic acid, Intestinal mucosal cells have pyridoxine kinase and which is the main excretory product. pyridoxine phosphate oxidase (Figure 8.11), so that Extrahepatic tissues take up pyridoxal and pyri- there is net accumulation of pyridoxal phosphate by doxal phosphate from the plasma. The phosphate is

- CH2OH O CH2OH H2 H2 HO C OH Kinase O P O C OH O- Phosphatase N CH3 N CH3 Pyridoxine Pyridoxine phosphate

Oxidase

- COO– HC O O HC O H2 H2 H2 HO C OH HO C OH Kinase O P O C OH Oxidase O- Phosphatase N CH3 N CH3 N CH3 4-Pyridoxic acid Pyridoxal Pyridoxal phosphate

Transaminases Oxidase

- H2C NH2 O CH2NH2 H2 H2 HO C OH Kinase O P O C OH O-

N CH3 Phosphatase N CH3 Pyridoxamine Pyridoxamine phosphate

Figure 8.11 Interconversion of the vitamin B6 vitamers. Pyridoxal kinase (EC 2.7.1.38), pyridoxine phosphate oxidase (EC 1.1.1.65), pyridoxamine phosphate oxidase (EC 1.4.3.5). 164 Introduction to Human Nutrition hydrolyzed to pyridoxal, which can cross cell mem- The cause of the convulsions was severe impair- branes, by extracellular alkaline phosphatase, then ment of the activity of the pyridoxal phosphate- trapped intracellularly by phosphorylation. Tissue dependent enzyme glutamate decarboxylase, which concentrations of pyridoxal phosphate are controlled catalyzes the synthesis of the inhibitory neurotrans- by the balance between phosphorylation and mitter γ-aminobutyric acid (GABA), together with dephosphorylation. accumulation of hydroxykynurenine as a result of

Some 80% of the body’s total vitamin B6 is pyri- impaired activity of kynureninase, which is also pyri- doxal phosphate in muscle, mostly associated with doxal phosphate dependent. glycogen phosphorylase. This does not function as a Moderate vitamin B6 defi ciency results in a number reserve of the vitamin and is not released from muscle of abnormalities of amino acid metabolism, espe- in times of defi ciency; it is released into the circula- cially of tryptophan and methionine. In experimental tion (as pyridoxal) in starvation, when glycogen animals, a moderate degree of defi ciency leads to reserves are exhausted and there is less requirement increased sensitivity of target tissues to steroid for phosphorylase activity. Under these conditions it hormone action. This may be important in the devel- is available for redistribution to other tissues, and opment of hormone-dependent cancer of the breast, especially the liver and kidneys, to meet the increased uterus, and prostate, and may therefore affect the need for transamination of amino acids to provide prognosis. Vitamin B6 supplementation may be a substrates for gluconeogenesis. useful adjunct to other therapy in these common cancers; certainly, there is evidence that poor vitamin

Metabolic functions of vitamin B6 B6 nutritional status is associated with a poor prog- Pyridoxal phosphate is a coenzyme in three main nosis in women with breast cancer. areas of metabolism: Vitamin B6 requirements ● in a wide variety of reactions of amino acids, espe- Most studies of vitamin B6 requirements have fol- cially transamination, in which it functions as the lowed the development of abnormalities of trypto- intermediate carrier of the amino group, and decar- phan and methionine metabolism during depletion boxylation to form amines and normalization during repletion with graded ● as the cofactor of glycogen phosphorylase in muscle intakes of the vitamin. Although the tryptophan load and liver, where it is the phosphate group that is test is unreliable as an index of vitamin B6 nutritional catalytically important status in fi eld studies, under the controlled conditions ● in the regulation of the action of steroid hormones. of depletion/repletion studies it gives a useful indica- Pyridoxal phosphate acts to remove the hormone– tion of the state of vitamin B6 nutrition. receptor complex from DNA binding, and so ter- Since the major role of vitamin B6 is in amino acid minate the action of the hormones. In vitamin B6 metabolism it is likely that protein intake will affect defi ciency there is increased sensitivity and respon- vitamin B6 requirements. Adults maintained on siveness of target tissues to low concentrations of vitamin B6-defi cient diets develop abnormalities of steroid hormones, including estrogens, androgens, tryptophan and methionine metabolism more quickly, cortisol, and vitamin D. and their blood vitamin B6 falls more rapidly, when their protein intake is relatively high (80–160 g/day in Vitamin B6 defi ciency various studies) than on low protein intakes (30–50 g/

Defi ciency of vitamin B6 severe enough to lead to day). Similarly, during repletion of defi cient subjects, clinical signs is extremely rare, and unequivocal defi - tryptophan and methionine metabolism and blood ciency has only been reported in one outbreak, during vitamin B6 are normalized more rapidly at low than the 1950s, when babies were fed on a milk preparation at high levels of protein intake. that had been severely overheated during manufac- From such studies the average requirement for ture. Many of the affected infants suffered convul- vitamin B6 is estimated to be 13 μg/g dietary protein, sions, which ceased rapidly following the administra- and reference intakes are based on 15–16 μg/g dietary tion of vitamin B6. protein. The Vitamins 165

Requirements of infants The tryptophan load test

Estimation of the vitamin B6 requirements of infants The tryptophan load test for vitamin B6 nutritional presents a problem, and there is a clear need for status (the ability to metabolize a test dose of trypto- further research. Human milk, which must be assumed phan) is one of the oldest metabolic tests for func- to be adequate for infant nutrition, provides only tional vitamin nutritional status. It was developed as some 2.5–3 μg of vitamin B6/g protein. This is very a result of observation of the excretion of an abnor- much lower than the requirement for adults, although mal colored compound, later identifi ed as the trypto- there is no reason why infants should have a lower phan metabolite xanthurenic acid, in the urine of requirement. defi cient animals. Based on the body content of 3.7 μg (15 nmol) Kynureninase (see Figure 8.12) is a pyridoxal phos- of vitamin B6/g body weight, and the rate of weight phate-dependent enzyme, and its activity falls mark- gain, a minimum requirement for infants over the edly in vitamin B6 defi ciency, at least partly because it fi rst 6 months of life is 100 μg/day to establish undergoes a slow mechanism-dependent inactivation tissue reserves, and an additional 20% to allow for that leaves catalytically inactive pyridoxamine phos- metabolic turnover. Even if the mother receives phate at the active site of the enzyme. The enzyme can daily supplements of 2.5 mg of vitamin B6 through- only be reactivated if there is an adequate supply of out lactation, thus more than doubling her normal pyridoxal phosphate. This means that in vitamin B6 intake, the infant’s intake ranges from 100 μg/day to defi ciency there is a considerable accumulation of 300 μg/day over the fi rst 6 months of life. At 1 month both hydroxykynurenine and kynurenine, suffi cient this is only 8.5 μg/g protein, rising to 15 μg/g by to permit greater metabolic fl ux than usual through 2 months. kynurenine transaminase, resulting in increased for- mation of kynurenic and xanthurenic acids. Xanthurenic and kynurenic acids, and kynurenine Assessment of vitamin B6 status and hydroxykynurenine, are easy to measure in urine, Fasting plasma total vitamin B (measured microbio- 6 so the tryptophan load test [the ability to metabolize logically), or more specifi cally pyridoxal phosphate, is a test dose of 2–5 g (150–380 μmol/kg body weight) widely used as an index of vitamin B nutritional 6 of tryptophan] has been widely adopted as a con- status. Despite the fall in plasma pyridoxal phosphate venient and very sensitive index of vitamin B in pregnancy, which has been widely interpreted as 6 nutritional status. However, because glucocorticoid indicating vitamin B depletion or an increased 6 hormones increase tryptophan dioxygenase activity, requirement, the plasma concentration of pyridoxal abnormal results of the tryptophan load test must be phosphate plus pyridoxal is unchanged. This suggests regarded with caution, and cannot necessarily be that determination of plasma pyridoxal phosphate interpreted as indicating vitamin B defi ciency. alone may not be a reliable index of vitamin B nutri- 6 6 Increased entry of tryptophan into the pathway will tional status. overwhelm the capacity of kynureninase, leading to About half of the normal dietary intake of vitamin increased formation of xanthurenic and kynurenic B is excreted as 4-pyridoxic acid. Urinary excretion 6 acids. Similarly, estrogen metabolites inhibit kyn- of 4-pyridoxic acid will largely refl ect the recent intake ureninase, leading to results that have been misinter- of the vitamin rather than the underlying nutritional preted as vitamin B defi ciency. status. 6 The methionine load test Coenzyme saturation of transaminases The metabolism of methionine includes two pyri-

The most widely used method of assessing vitamin B6 doxal phosphate-dependent steps: cystathionine status is by the activation of erythrocyte transami- synthetase and cystathionase (see Figure 8.16). nases by pyridoxal phosphate added in vitro. An acti- Cystathionase activity falls markedly in vitamin B6 vation coeffi cient for alanine transaminase >1.25, or defi ciency, and as a result there is an increase in the for aspartate transaminase >1.8, is considered to indi- urinary excretion of homocysteine and cystathionine, cate defi ciency. both after a loading dose of methionine and under 166 Introduction to Human Nutrition

COO– + O2 H2C C NH3 H Kynurenine aminotransferase Kynurenine Kynurenic acid N H Tryptophan dioxygenase and O formylkynurenine formamidase 2 Tryptophan NADPH Kynurenine hydroxylase NADP+

Hydroxykynurenine Xanthurenic acid Kynurenine aminotransferase H2O Kynureninase Alanine

3-Hydroxyanthranilic acid 3-Hydroxyanthranilic acid oxidase

Aminocarboxymuconic semialdehyde

Total oxidation Quinolinic acid via acetyl CoA for NAD synthesis Figure 8.12 Oxidative pathway of tryptophan: the basis of the tryptophan load test. Tryptophan dioxygenase (EC 1.13.11.11), formylkynurenine formamidase (EC 3.5.1.9), kynurenine hydroxylase (EC 1.14.13.9), kynureninase (EC 3.7.1.3).

basal conditions. However, as discussed below, homo- All of the studies that suggested that oral contra-

cysteine metabolism is more affected by folate status ceptives cause vitamin B6 defi ciency used the metabo- than by vitamin B6 status and, like the tryptophan load lism of tryptophan as a means of assessing vitamin B6 test, the methionine load test is probably not reliable nutritional status. When other biochemical markers

as an index of vitamin B6 status in fi eld studies. of status were also assessed, they were not affected by oral contraceptive use. Furthermore, most of these Non-nutritional uses of vitamin B6 studies were performed using the now obsolete high- Several studies have suggested that oral contraceptives dose contraceptive pills.

cause vitamin B6 defi ciency. As a result of this, supple- Oral contraceptives do not cause vitamin B6 defi - ments of vitamin B6 of 50–100 mg/day, and some- ciency. The problem is that estrogen metabolites times higher, have been used to overcome the side- inhibit kynureninase and reduce the activity of kyn- effects of oral contraceptives. Similar supplements urenine hydroxylase. This results in the excretion of have also been recommended for the treatment of the abnormal amounts of tryptophan metabolites, similar

premenstrual syndrome, although there is little evi- to what is seen in vitamin B6 defi ciency, but for a dif- dence of effi cacy from placebo-controlled trials. ferent reason. The Vitamins 167

Doses of 50–200 mg of vitamin B6/day have an antiemetic effect, and the vitamin is widely used, Structure and vitamers alone or in conjunction with other antiemetics, to The structure of vitamin B12 is shown in Figure 8.13. minimize the nausea associated with radiotherapy The term corrinoid is used as a generic descriptor for and to treat pregnancy sickness. There is no evidence -containing compounds of this general struc- that vitamin B6 has any benefi cial effect in pregnancy ture that, depending on the substituents in the pyrrole sickness, or that women who suffer from morning rings, may or may not have vitamin activity. The term sickness have lower vitamin B6 nutritional status than “vitamin B12” is used as a generic descriptor for the other pregnant women. cobalamins, that is, those corrinoids having the bio-

Doses of vitamin B6 of 100 mg/day have been logical activity of the vitamin. Some of the corrinoids reported to be benefi cial in the treatment of the carpal that are growth factors for microorganisms not only tunnel syndrome or tenosynovitis. However, most of have no vitamin B12 activity, but may be antimetabo- the reports originate from one centre and there lites of the vitamin. appears to be little independent confi rmation of the Although was the fi rst form in usefulness of the vitamin in this condition. which vitamin B12 was isolated, it is not an important naturally occurring vitamer, but rather an artifact due Vitamin B6 toxicity to the presence of cyanide in the charcoal used in the

In experimental animals, doses of vitamin B6 of extraction procedure. It is more stable to light than 50 mg/kg body weight cause histological damage to the other vitamers, and hence is used in pharmaceuti- dorsal nerve , and doses of 200 mg/kg body cal preparations. Photolysis of cyanocobalamin in weight lead to the development of signs of peripheral solution leads to the formation of aquocobalamin or neuropathy, with ataxia, muscle weakness, and loss of hydroxocobalamin, depending on pH. Hydroxoco- balance. The clinical signs of vitamin B6 toxicity in balamin is also used in pharmaceutical preparations, animals regress within 3 months after withdrawal of and is better retained after parenteral administration these massive doses, but sensory nerve conduction than is cyanocobalamin. velocity, which decreases during the development of Vitamin B12 is found only in foods of animal origin, the neuropathy, does not recover fully. although it is also formed by bacteria. There are Sensory neuropathy has been reported in seven no plant sources of this vitamin. This means that patients taking 2–7 g of pyridoxine/day. Although strict vegetarians (vegans), who eat no foods of there was some residual damage, withdrawal of these animal origin, are at risk of developing dietary vitamin extremely high doses resulted in a considerable B12 defi ciency, although the small amounts of recovery of sensory nerve function. Other reports vitamin B12 formed by bacteria on the surface of have suggested that intakes as low as 50 mg/day fruits may be adequate to meet requirements. Prepa- are associated with neurological damage, although rations of vitamin B12 made by bacterial fermentation these studies were based on patients reporting symp- that are ethically acceptable to vegans are readily toms rather than objective neurological examination. available. There have been no reports of nerve damage in chil- There are claims that and some plants (espe- dren with vitamin B6-dependent homocystinuria, cially some algae) contain vitamin B12. This seems to or other inborn errors of metabolism, who take be incorrect. The problem is that the offi cially recog- 200–300 mg/day. nized, and legally required, method of determining

vitamin B12 in food analysis is a microbiological assay

8.10 Vitamin B12 using organisms for which vitamin B12 is an essential growth factor. However, these organisms can also use

Dietary defi ciency of vitamin B12 occurs only in strict some corrinoids that have no vitamin activity. There- vegans, since the vitamin is found almost exclusively fore, analysis reveals the presence of something that in animal foods. However, functional defi ciency (per- appears to be vitamin B12, but in fact is not the active nicious anemia, with spinal cord degeneration) as a vitamin and is useless in human nutrition. Biologi- result of impaired absorption is relatively common, cally active vitamin B12 has been identifi ed in some especially in older people with atrophic gastritis. preparations of algae, but this seems to be the result 168 Introduction to Human Nutrition

O

H2C C NH2 O O H3C H2 H2 H2N C CH2 H3C C C C NH2 H2C O N H2 H2N C C N CH3 Co H C N 3 CH3 H3C O N H2 H2C C C NH2 CH3 H2N C C CH3 H2 O CH2 CH2 C O NH

CH2 O N CH3 H3C C O P O Figure 8.13 Vitamin B12. Four coordination sites on H the central cobalt are occupied by nitrogen O OH N atoms of the ring, and one by the nitrogen of CH3 the dimethylbenzimidazole side-chain. The sixth coordination site may be occupied by cyanide (cyanocobalamin), a hydroxyl ion (hydroxocobala- O min), water (aquocobalamin), or a methyl group HO CH2 (methylcobalamin).

of bacterial contamination of the lakes where the Gastric acid and pepsin play a role in vitamin B12 algae were harvested. nutrition, serving to release the vitamin from protein binding, so making it available. Atrophic gastritis is a relatively common problem of advancing age; in the Absorption and metabolism of vitamin B12 early stages there is failure of acid secretion but more Absorption or less normal secretion of intrinsic factor. This can

Very small amounts of vitamin B12 can be absorbed result in vitamin B12 depletion due to failure to release by passive diffusion across the intestinal mucosa, but the vitamin from dietary proteins, although the

under normal conditions this is insignifi cant; the absorption of free vitamin B12 (as in supplements or major route of vitamin B12 absorption is by attach- fortifi ed foods) is unaffected. In the stomach, vitamin ment to a specifi c binding protein in the intestinal B12 binds to cobalophilin, a binding protein secreted lumen. in the saliva. This binding protein is intrinsic factor, so called In the duodenum cobalophilin is hydrolyzed,

because in the early studies of pernicious anemia it releasing vitamin B12 to bind to intrinsic factor. Pan- was found that two curative factors were involved: an creatic insuffi ciency can therefore be a factor in the

extrinsic or dietary factor, which is now known to be development of vitamin B12 defi ciency, since failure to vitamin B12, and an intrinsic or endogenously pro- hydrolyze cobalophilin will result in the excretion of duced factor. Intrinsic factor is a small glycoprotein cobalophilin-bound vitamin B12 rather than transfer secreted by the parietal cells of the gastric mucosa, to intrinsic factor. Intrinsic factor binds the various

which also secrete hydrochloric acid. vitamin B12 vitamers, but not other corrinoids. The Vitamins 169

Vitamin B12 is absorbed from the distal third of the The other clinical feature of vitamin B12 defi ciency, ileum. There are intrinsic factor–vitamin B12 binding which is rarely seen in folic acid defi ciency, is degen- sites on the brush border of the mucosal cells in this eration of the spinal cord; hence the name “perni- region; neither free intrinsic factor nor free vitamin cious” for the anemia of vitamin B12 defi ciency. The B12 interacts with these receptors. spinal cord degeneration is due to a failure of the In plasma, vitamin B12 circulates bound to transco- methylation of one arginine residue in myelin basic balamin I, which is required for tissue uptake of the protein. About one-third of patients who present with vitamin, and transcobalamin II, which seems to be a megaloblastic anemia due to vitamin B12 defi ciency storage form of the vitamin. also have spinal cord degeneration, and about one- third of defi cient subjects present with neurological

Enterohepatic circulation of vitamin B12 signs but no anemia. There is a considerable enterohepatic circulation of The most common cause of pernicious anemia is vitamin B12. A third plasma vitamin B12 binding failure of the absorption of vitamin B12, rather than protein, transcobalamin III, is rapidly cleared by the dietary defi ciency. Classical pernicious anemia is due liver, with a plasma half-life of the order of 5 min. to failure of intrinsic factor secretion, commonly the

This provides a mechanism for returning vitamin B12 result of autoimmune disease, with production of anti- and its metabolites from peripheral tissues to the liver, bodies against either the gastric parietal cells or intrin- as well as for clearance of other corrinoids without sic factor. Atrophic gastritis with increasing age also vitamin activity, which may arise from either foods or leads to progressive failure of vitamin B12 absorption. the products of intestinal bacterial action, and be Dietary defi ciency of vitamin B12 does occur, rarely, absorbed passively across the lower gut. in strict vegetarians (vegans). The rarity of vitamin

These corrinoids are then secreted into the bile, B12 defi ciency among people who have no apparent bound to cobalophilins; 3–8 μg (2.25–6 nmol) of dietary source of the vitamin suggests that bacterial vitamin B12 may be secreted in the bile each day, about contamination of water and foods with vitamin B12- the same as the dietary intake. Like dietary vitamin producing organisms will provide minimally adequate

B12 bound to salivary cobalophilin, the biliary coba- amounts of the vitamin. The fruit bat develops lophilins are hydrolyzed in the duodenum, and the vitamin B12 defi ciency when fed on washed fruit vitamin binds to intrinsic factor, so permitting reab- under laboratory conditions, but in the wild micro- sorption in the ileum. Although cobalophilins and bial contamination of the outside of the fruit provides transcorrin III have low specifi city, and will bind a an adequate intake of the vitamin. variety of corrinoids, intrinsic factor binds only cobalamins, and so only the biologically active vitamin Vitamin B12 requirements is reabsorbed. Most estimates of vitamin B12 requirements are based on the amounts given parenterally to maintain normal Metabolic functions of vitamin B12 health in patients with pernicious anemia due to a

There are three vitamin B12-dependent enzymes in failure of vitamin B12 absorption. This overestimates human tissues: methylmalonyl-CoA mutase (dis- normal requirements, because of the enterohepatic cussed below under methylmalonic aciduria), leucine circulation of vitamin B12; in people lacking intrinsic amino-mutase, and methionine synthetase (discussed factor, or secreting anti-intrinsic factor antibodies, in Section 8.11). the vitamin that is excreted in the bile will be lost in the feces, whereas normally it is almost completely Vitamin B12 defi ciency: pernicious anemia reabsorbed.

Vitamin B12 defi ciency causes pernicious anemia; the The total body pool of vitamin B12 is of the order release into the bloodstream of immature precursors of 2.5 mg (1.8 μmol), with a minimum desirable body of red blood cells (megaloblastic anemia). As dis- pool of about 1 mg (0.3 μmol). The daily loss is about cussed below, vitamin B12 defi ciency causes functional 0.1% of the body pool in subjects with normal entero- folate defi ciency; this is what disturbs the rapid mul- hepatic circulation of the vitamin; on this basis tiplication of red blood cells, causing immature pre- requirements are about 1–2.5 μg/day and reference cursors to be released into the circulation. intakes for adults range between 1.4 μg and 2.0 μg. 170 Introduction to Human Nutrition

Assessment of vitamin B12 status bolic reactions; it is therefore metabolically closely Measurement of plasma concentrations of vitamin related to vitamin B12. Defi ciency of either causes megaloblastic anemia, and the hematological effects B12 is the method of choice, and several simple and reliable radioligand binding assays have been devel- of vitamin B12 defi ciency are due to disturbance of folate metabolism. oped. A serum concentration of vitamin B12 below 110 pmol/l is associated with megaloblastic bone Apart from liver, the main dietary sources of folate marrow, incipient anemia, and myelin damage. Below are fruits and vegetables. Although folate is widely 150 pmol/l there are early bone marrow changes, distributed in foods, dietary defi ciency is not uncom- abnormalities of the deoxyuridine monophosphate mon, and a number of commonly used drugs can (dUMP) suppression test (see Section 8.11) and cause folate depletion. More importantly, there is methylmalonic aciduria after a valine load. good evidence that intakes of folate considerably higher than normal dietary levels reduce the risk of

The Schilling test for vitamin B12 absorption neural tube defects, and, where cereal products are

The absorption of vitamin B12 can be determined by not fortifi ed with folate by law, pregnant women are the Schilling test. An oral dose of [57Co] or [58Co]- recommended to take supplements. There is also evi- vitamin B12 is given with a parenteral fl ushing dose of dence that high intakes of folate may be effective in 1 mg of non-radioactive vitamin to saturate body reducing plasma homocysteine in subjects genetically reserves, and the urinary excretion of radioactivity is at risk of hyperhomocystinemia (some 10–20% of the followed as an index of absorption of the oral mate- population), which may reduce the risk of ischemic rial. Normal subjects excrete 16–45% of the radioac- heart disease and stroke. tivity over 24 h, whereas patients lacking the intrinsic factor excrete less than 5%. Vitamers and dietary equivalence The test can be repeated, giving the intrinsic factor As shown in Figure 8.14, folic acid consists of a orally together with the radioactive vitamin B12; if the reduced pterin linked to p-aminobenzoic acid, impaired absorption was due to a simple lack of forming pteroic acid. The carboxyl group of the p- intrinsic factor, and not to anti-intrinsic factor anti- aminobenzoic acid moiety is linked by a peptide bond bodies in the saliva or gastric juice, then a normal to the α-amino group of glutamate, forming pteroyl- amount of the radioactive material should be absorbed glutamate (PteGlu). The coenzymes may have up to and excreted. seven additional glutamate residues linked by γ- peptide bonds, forming pteroyldiglutamate (PteGlu2), Methylmalonic aciduria pteroyltriglutamate (PteGlu3), etc., collectively Methylmalonyl-CoA is formed as an intermediate in known as folate or pteroyl polyglutamate conjugates the catabolism of valine and by the carboxylation of (PteGlun). propionyl-CoA arising in the catabolism of isoleu- “Folate” is the preferred trivial name for pteroyl- cine, cholesterol, and (rare) fatty acids with an odd glutamate, although both “folate” and “folic acid” may number of carbon atoms. Normally, it undergoes be used as a generic descriptor to include various vitamin B12-dependent rearrangement to succinyl- polyglutamates. PteGlu2 is sometimes referred to as CoA, catalyzed by methylmalonyl-CoA mutase. folic acid diglutamate, PteGlu3 as folic acid trigluta- Vitamin B12 defi ciency leads to an accumulation of mate, and so on. methylmalonyl-CoA, which is hydrolyzed to methyl- malonic acid, which is excreted in the urine. Urinary O COO– excretion of methylmalonic acid, especially after a H H2 H H2 H loading dose of valine, provides a means of assessing N C N C N CH vitamin B nutritional status. HN 12 CH2 H2N N N Tetrahydrofolate CH2 8.11 Folic acid H C O Folic acid functions in the transfer of one-carbon (Glu)n fragments in a wide variety of biosynthetic and cata- Figure 8.14 Tetrahydrofolate (folic acid). The Vitamins 171

Tetrahydrofolate can carry one-carbon fragments Despite this, there is very little fecal loss of folate; attached to N-5 (formyl, formimino, or methyl jejunal absorption of methyl-tetrahydrofolate is a groups), N-10 (formyl), or bridging N-5–N-10 (methy- very effi cient process, and the fecal excretion of some lene or methenyl groups). 5-Formyl-tetrahydrofolate 450 nmol (200 μg) of per day represents syn- is more stable to atmospheric oxidation than is folate, thesis by intestinal fl ora and does not refl ect intake to and is therefore commonly used in pharmaceutical any signifi cant extent. preparations; it is also known as folinic acid, and the synthetic (racemic) compound as leucovorin. Tissue uptake of folate The extent to which the different forms of folate Methyl-tetrahydrofolate circulates bound to albumin, can be absorbed varies; on average only about half of and is available for uptake by extrahepatic tissues, the folate in the diet is available, compared with more where it is trapped by formation of polyglutamates, or less complete availability of the monoglutamate. To which do not cross cell membranes. permit calculation of folate intakes, the dietary folate The main circulating folate is methyl-tetrahydro- equivalent has been defi ned as 1 μg mixed food folates folate, which is a poor substrate for polyglutamylation; or 0.6 μg free folic acid. On this basis, total dietary demethylation by the action of methionine synthetase folate equivalents = μg food folate + 1.7 × synthetic (see below) is required for effective metabolic trap-

(free) folic acid. ping of folate. In vitamin B12 defi ciency, when methio- nine synthetase activity is impaired, there will Absorption and metabolism of folate therefore be impairment of the uptake of folate into About 80% of dietary folate is as polyglutamates; a tissues. variable amount may be substituted with various one-carbon fragments or be present as dihydrofolate Folate excretion derivatives. Folate conjugates are hydrolyzed in the There is very little urinary loss of folate, only some small intestine by conjugase (pteroylpolyglutamate 5–10 nmol/day. Not only is most folate in plasma hydrolase), a zinc-dependent enzyme of the pancre- bound to proteins (either folate binding protein atic juice, bile, and mucosal brush border; zinc defi - for unsubstituted folate or albumin for methyl- ciency can impair folate absorption. tetrahydrofolate), and thus protected from glomeru- Free folate, released by conjugase action, is absorbed lar fi ltration, but the renal brush border has a high by active transport in the jejunum. The folate in milk concentration of folate binding protein, which acts to is mainly bound to a specifi c binding protein; the reabsorb any fi ltered in the urine. protein–tetrahydrofolate complex is absorbed intact, The catabolism of folate is largely by cleavage of the mainly in the ileum, by a mechanism that is distinct C-9–N-10 bond, catalyzed by carboxypeptidase G. from the active transport system for the absorption The p-aminobenzoic acid moiety is amidated and of free folate. The biological availability of folate from excreted in the urine as p-acetamidobenzoate and p- milk, or of folate from diets to which milk has been acetamidobenzoyl-glutamate; pterin is excreted either added, is considerably greater than that of unbound unchanged or as a variety of biologically inactive folate. compounds. Much of the dietary folate undergoes methylation and reduction within the intestinal mucosa, so that Metabolic functions of folate what enters the portal bloodstream is largely 5- The metabolic role of folate is as a carrier of one- methyl-tetrahydrofolate. Other substituted and carbon fragments, both in catabolism and in biosyn- unsubstituted folate monoglutamates, and dihydrofo- thetic reactions. These may be carried as formyl, late, are also absorbed; they are reduced and methyl- formimino, methyl or methylene residues. The major ated in the liver, then secreted in the bile. The liver sources of these one-carbon fragments and their also takes up various folates released by tissues; again, major uses, as well as the interconversions of the sub- these are reduced, methylated and secreted in the stituted folates, are shown in Figure 8.15. bile. The major point of entry for one-carbon fragments The total daily enterohepatic circulation of folate is into substituted folates is methylene-tetrahydrofolate, equivalent to about one-third of the dietary intake. which is formed by the catabolism of glycine, serine, 172 Introduction to Human Nutrition

Sources of one-carbon units Synthesis using one-carbon units Serine Serine

(a) (b) Glycine Methylene-THF Methyl-THF Methionine

Choline TMP + Dihydrofolate

Histidine Formimino-THF Methenyl-THF

Formate Formyl-THF Purines

CO2 Figure 8.15 Interconversion of the principal one-carbon substituted folates; sources of one-carbon fragments are shown on the left, and pathways in which one-carbon units are used and free tetrahydrofolate is regenerated on the right. (a) Methylene-tetrahydrofolate reductase (EC 1.5.1.20); (b) methionine synthetase (EC 2.1.1.13).

and choline. Serine is the most important source of Thymidylate synthetase and substituted folates for biosynthetic reactions, and dihydrofolate reductase the activity of serine hydroxymethyltransferase is The methylation of dUMP to thymidine monophos- regulated by the state of folate substitution and the phate (TMP), catalyzed by thymidylate synthetase, is availability of folate. The reaction is freely reversible, essential for the synthesis of DNA, although pre- and under appropriate conditions in liver it functions formed TMP arising from the catabolism of DNA can to form serine from glycine as a substrate for be reutilized. gluconeogenesis. The methyl donor for thymidylate synthetase is Methylene-, methenyl-, and 10-formyl-tetrahydro- methylene-tetrahydrofolate; the reaction involves folates are freely interconvertible. This means that reduction of the one-carbon fragment to a methyl when one-carbon folates are not required for syn- group at the expense of the folate, which is oxidized thetic reactions, the oxidation of formyl-tetrahydro- to dihydrofolate. Dihydrofolate is then reduced to folate to carbon dioxide and folate provides a means tetrahydrofolate by dihydrofolate reductase. of maintaining an adequate tissue pool of free Thymidylate synthase and dihydrofolate reductase folate. are especially active in tissues with a high rate of cell By contrast, the reduction of methylene-tetra- division, and hence a high rate of DNA replication hydrofolate to methyl-tetrahydrofolate is irreversible, and a high requirement for thymidylate. Because of and the only way in which free folate can be formed this, inhibitors of dihydrofolate reductase have been from methyl-tetrahydrofolate is by the reaction of exploited as anticancer drugs (e.g. methotrexate). methionine synthetase (see below). Chemotherapy consists of alternating periods of The Vitamins 173

administration of methotrexate to inhibit tumor ● Methionine synthetase is a vitamin B12-dependent growth, and folate (normally as 5-formyl-tetrahydro- enzyme, for which the methyl donor is folate, leucovorin) to replete tissues and avoid folate methyl-tetrahydrofolate. defi ciency; this is known as leucovorin rescue. ● Homocysteine methyltransferase utilizes betaine (an intermediate in the catabolism of choline) as the methyl donor, and is not vitamin B Methionine synthetase and 12 dependent. the methyl-folate trap In addition to its role in the synthesis of proteins, Both enzymes are found in most tissues, but only the methionine, as the S-adenosyl derivative, acts as a vitamin B12-dependent methionine synthetase is methyl donor in a wide variety of biosynthetic reac- found in the central nervous system. tions. As shown in Figure 8.16, the resultant homo- The reduction of methylene-tetrahydrofolate to cysteine may be either metabolized to yield cysteine methyl-tetrahydrofolate is irreversible, and the major or remethylated to yield methionine. source of folate for tissues is methyl-tetrahydrofolate. Two enzymes catalyze the methylation of homo- The only metabolic role of methyl-tetrahydrofolate is cysteine to methionine: the methylation of homocysteine to methionine, and

Methylated product Acceptor

S-Adenosylmethionine S-Adenosylhomocysteine Methyltransferases PPi + Pi Methionine adenosyltransferase Adenosine ATP CH3 S SH

CH2 CH2 CH2 Methionine synthetase CH2 + + HC NH3 HC NH3 Homocysteine COO– COO– Methionine Methyl THF Homocysteine Tetrahydrofolate Serine

Cystathionine b -synthetase

H2O

Cystathionine H2O

g -Cystathionase

+ α-Ketobutyrate + NH4

Cysteine Figure 8.16 Methionine metabolism. Methionine synthetase (EC 2.1.1.13), methionine adenosyltransferase (EC 2.5.1.6), cystathionine synthetase (EC 4.2.1.22), cystathionase (EC 4.4.1.1). 174 Introduction to Human Nutrition this is the only way in which methyl-tetrahydrofolate disease. However, a number of intervention trials with can be demethylated to yield free folate in tissues. folate supplements have shown no reduction in death Methionine synthetase thus provides the link between from myocardial infarction, nor any decrease in all- the physiological functions of folate and vitamin B12. cause mortality, despite a signifi cant decrease in Impairment of methionine synthetase activity in plasma homocysteine. Similarly, in countries where vitamin B12 defi ciency will result in the accumulation there has been mandatory enrichment of fl our with of methyl-tetrahydrofolate, which can neither be uti- folate for some years, there is no evidence of reduced lized for any other one-carbon transfer reactions nor mortality from cardiovascular disease. It is possible be demethylated to provide free folate. that elevated plasma homocysteine is not so much a This functional defi ciency of folate is exacerbated cause of atherosclerosis (although there are good by low tissue concentrations of methionine and an mechanisms to explain why it might be atherogenic) accumulation of homocysteine, since the transulfura- as the result of impaired kidney function due to early tion pathway to form cysteine from homocysteine is atherosclerosis. If this is so, the lowering of plasma regulated by the availability of cysteine: it is a biosyn- homocysteine by increasing folate intake would thetic pathway rather than a pathway for disposal of not be expected to affect the development of methionine and homocysteine. atherosclerosis.

Methylene-tetrahydrofolate reductase Folate in pregnancy and During the 1980s a considerable body of evidence Elevated blood homocysteine is a signifi cant risk accumulated that spina bifi da and other neural tube factor for atherosclerosis, thrombosis, and hyperten- defects (which occur in about 0.75–1% of pregnan- sion, independent of factors such as dietary lipids and cies) were associated with low intakes of folate, and plasma lipoproteins. About 10–15% of the popula- that increased intake during pregnancy might be pro- tion, and almost 30% of people with ischemic heart tective. It is now established that supplements of disease, have an abnormal variant of methylene- folate begun periconceptually result in a signifi cant tetrahydrofolate reductase, which is unstable, and reduction in the incidence of neural tube defects, and loses activity more quickly than normal. As a it is recommended that intakes be increased by result, people with the abnormal form of the enzyme 400 μg/day before conception. (Closure of the neural have an impaired ability to form methyl-tetrahydro- tube occurs by day 28 of pregnancy, which is before folate (the main form in which folate is taken up by the woman knows she is pregnant.) The studies were tissues) and suffer from functional folate defi ciency. conducted using folate monoglutamate and it is Therefore, they are unable to remethylate homocys- unlikely that an equivalent increase in intake could be teine to methionine adequately and develop achieved from unfortifi ed foods. In many countries hyperhomocysteinemia. there is mandatory enrichment of fl our with folate, People with the abnormal variant of methylene- and there has been a 25–50% decrease in the number tetrahydrofolate reductase do not develop hyperho- of infants born with neural tube defects since the mocysteinemia if they have a relatively high intake of introduction of fortifi cation. The true benefi t is folate. This seems to be due to the methylation greater than this, since some affected fetuses abort of folate in the intestinal mucosa during absorption; spontaneously and there are few data on the number intestinal mucosal cells have a rapid turnover of therapeutic terminations of pregnancy for neural (some 48 h between proliferation in the crypts and tube defects detected by antenatal screening; there- shedding at the tip of the villus), and therefore it is fore, supplements are recommended. Where folate not important that methylene-tetrahydrofolate enrichment is not mandatory, the advice is that all reductase is less stable than normal, as there is still an women who are, or may be about to become, preg- adequate activity of the enzyme in the intestinal nant, should take supplements of 400 μg/day. mucosa to maintain a normal circulating level of methyl-tetrahydrofolate. Folate and cancer This has led to the suggestion that supplements of Much of the regulation and silencing of gene expres- folate will reduce the incidence of cardiovascular sion that underlies tissue differentiation involves The Vitamins 175 methylation of CpG islands in DNA, and there is Assessment of folate status evidence that some cancers (and especially colorectal Measurement of the serum or red blood cell concen- cancer) are associated with under-methylation of tration of folate is the method of choice, and several CpG islands as a result of low folate status. A number simple and reliable radioligand binding assays have of small studies have suggested that folate supple- been developed. There are problems involved in ments may be protective against colorectal cancer, but radioligand binding assays for folate, and in some no results from large-scale randomized controlled centers microbiological determination of plasma or trials have yet been reported, and to date there is no whole blood folates is the preferred technique. Serum evidence of a decrease in colorectal cancer in coun- folate below 7 nmol/l or erythrocyte folate below tries where folate enrichment of fl our is mandatory. 320 nmol/l indicates negative folate balance and early depletion of body reserves. At this stage the fi rst bone Folate defi ciency: megaloblastic anemia marrow changes are detectable. Dietary defi ciency of folic acid is not uncommon and, as noted above, defi ciency of vitamin B12 also leads to functional folic acid defi ciency. In either case, it is cells Histidine metabolism: that are dividing rapidly, and therefore have a large the formiminoglutamate test requirement for thymidine for DNA synthesis, that The ability to metabolize a test dose of histidine pro- are most severely affected. These are the cells of the vides a sensitive functional test of folate nutritional bone marrow that form red blood cells, the cells of status; formiminoglutamate (FIGLU) is an interme- the intestinal mucosa and the hair follicles. Clinically, diate in histidine catabolism, and is metabolized by folate defi ciency leads to megaloblastic anemia, the the folate-dependent enzyme formiminoglutamate release into the circulation of immature precursors of formiminotransferase. In folate defi ciency the activity red blood cells. of this enzyme is impaired, and FIGLU accumulates and is excreted in the urine, especially after a test dose Megaloblastic anemia is also seen in vitamin B12 defi ciency, where it is due to functional folate defi - of histidine: the so-called FIGLU test. ciency as a result of trapping folate as methyl-tetra- Although the FIGLU test depends on folate nutri- hydrofolate. However, the neurological degeneration tional status, the metabolism of histidine will also be of pernicious anemia is rarely seen in folate defi ciency, impaired, and hence a positive result obtained, in and indeed a high intake of folate can mask the devel- vitamin B12 defi ciency, because of the secondary defi ciency of free folate. About 60% of vitamin B12- opment of megaloblastic anemia in vitamin B12 defi - ciency, so that the presenting sign is irreversible nerve defi cient subjects show increased FIGLU excretion damage. after a histidine load.

Folate requirements The dUMP suppression test Depletion/repletion studies to determine folate Rapidly dividing cells can either use preformed TMP requirements using folate monoglutamate suggest for DNA synthesis, or synthesize it de novo from a requirement of the order of 80–100 μg (170– dUMP. Stimulated lymphocytes incubated with [3H]- 220 nmol)/day. The total body pool of folate in adults TMP will incorporate the label into DNA. In the is some 17 μmol (7.5 mg), with a biological half-life presence of adequate amounts of methylene-tetrahy- of 101 days. This suggests a minimum requirement drofolate, the addition of dUMP as a substrate for for replacement of 37 μg (85 nmol)/day. Studies of thymidylate synthetase reduces the incorporation of the urinary excretion of folate metabolites in subjects [3H]-TMP as a result of dilution of the pool of labeled maintained on folate-free diets suggest that there is material by newly synthesized TMP and inhibition of catabolism of some 80 μg (170 nmol) of folate/day. thymidylate kinase by thymidine triphosphate. Because of the problems in determining the bio- In normal cells the incorporation of [3H]-thymi- logical availability of the various folate polyglutamate dine into DNA after preincubation with dUMP is conjugates found in foods, reference intakes allow a 1.4–1.8% of that without preincubation. By contrast, wide margin of safety, and are based on an allowance cells that are defi cient in folate form little or no thy- of 3 μg (6.8 nmol)/kg body weight. midine from dUMP, and hence incorporate nearly as 176 Introduction to Human Nutrition

3 much of the [ H]-thymidine after incubation with come by adding vitamin B12 to foods as well as dUMP as they do without preincubation. folate. Whereas gastric acid is essential for the

Either a primary defi ciency of folic acid or func- release of vitamin B12 bound to dietary proteins, tional defi ciency secondary to vitamin B12 defi ciency crystalline vitamin B12 used in food enrichment is will have the same effect. In folate defi ciency, addition free to bind to cobalophilin without the need for of any biologically active form of folate, but not gastric acid. vitamin B12, will normalize the dUMP suppression of ● Antagonism between folic acid and the anticonvul- 3 [ H]-thymidine incorporation. In vitamin B12 defi - sants used in the treatment of epilepsy is part of ciency, addition of vitamin B12 or methylene- their mechanism of action; about 2% of the popu- tetrahydrofolate, but not methyl-tetrahydrofolate, lation have (drug-controlled) epilepsy. Relatively will normalize dUMP suppression. large supplements of folic acid (in excess of 1000 μg/ day) may antagonize the benefi cial effects of some Drug–nutrient interactions of folate anticonvulsants and may lead to an increase in the Several folate antimetabolites are used clinically, as frequency of epileptic attacks. If enrichment of a μ cancer chemotherapy (e.g., methotrexate), and as food such as bread with folate is to provide 400 g/ antibacterial (trimethoprim) and antimalarial (pyri- day to those who eat little bread, those who eat a methamine) agents. Drugs such as trimethoprim and relatively large amount may well have an intake in μ pyrimethamine act by inhibiting dihydrofolate reduc- excess of 1000 g/day. There is, however, no evi- tase, and they owe their clinical usefulness to a con- dence of a signifi cant problem in countries where siderably higher affi nity for the dihydrofolate reduc- enrichment of fl our has been mandatory for some tase of the target organism than the human enzyme; years. nevertheless, prolonged use can result in folate defi ciency. 8.12 Biotin A number of anticonvulsants used in the treatment of epilepsy, including diphenylhydantoin (phenytoin), Biotin was originally discovered as part of the complex and sometimes phenobarbital and primidone, can called bios, which promoted the growth of yeast and, also cause folate defi ciency. Although overt megalo- separately, as vitamin H, the protective or curative blastic anemia affects only some 0.75% of treated factor in “egg white injury,” the disease caused in epileptics, there is some degree of macrocytosis in humans and experimental animals being fed diets 40%. The megaloblastosis responds to folic acid sup- containing large amounts of uncooked egg white. The plements, but in about 50% of such patients treated structures of biotin, biocytin, and carboxy-biocytin with relatively high supplements for 1–3 years there (the active metabolic intermediate) are shown in is an increase in the frequency of epileptic attacks. Figure 8.17. Biotin is widely distributed in many foods. It is Folate toxicity synthesized by intestinal fl ora, and in balance studies the total output of biotin in urine plus feces is three There is some evidence that folate supplements in to six times greater than the intake, refl ecting bacterial excess of 400 μg/day may impair zinc absorption. In synthesis. It is not known to what extent this is avail- addition, there are two potential problems that have able to the host. to be considered when advocating either widespread use of folate supplements or enrichment of foods Absorption and metabolism of biotin with folate for protection against neural tube defect Most biotin in foods is present as biocytin (ε-amino- and possibly cardiovascular disease and cancer. biotinyllysine), which is released on proteolysis, then ● Folate supplements will mask the megaloblastic hydrolyzed by biotinidase in the pancreatic juice and

anemia of vitamin B12 defi ciency, so that the pre- intestinal mucosal secretions, to yield free biotin. The senting sign is irreversible nerve damage. This is extent to which bound biotin in foods is biologically especially a problem for older people, who may available is not known.

suffer impaired absorption of vitamin B12 as a result Free biotin is absorbed from the small intestine by of atrophic gastritis. This problem might be over- active transport. Biotin circulates in the bloodstream The Vitamins 177

O onyl-CoA carboxylase, and methylcrotonyl-CoA carboxylase to form the active holoenzymes from HN NH (inactive) apoenzymes and free biotin. Biotin also has a role in the control of the cell cycle, and acts via cell surface receptors to regulate the - S C O expression of key enzymes involved in glucose metab- Biotin O olism. In response to mitogenic stimuli there is a con- O siderable increase in the tissue uptake of biotin, much of which is used to biotinylate histones and other HN NH nuclear proteins.

Biotin defi ciency and requirements H C O S C N CH Biotin is widely distributed in foods and defi ciency is O unknown, except among people maintained for many NH Biotinyl lysine (biocytin) months on total , and a very small number of people who eat large amounts of uncooked O egg. Avidin, a protein in egg white, binds biotin -OOC NNH extremely tightly and renders it unavailable for absorption. Avidin is denatured by cooking and then loses its ability to bind biotin. The amount of avidin H C O S C N in uncooked egg white is relatively small, and prob- CH lems of biotin defi ciency have only occurred in people O Carboxybiocytin NH eating a dozen or more raw eggs a day, for some years. Figure 8.17 Biotin, biotinyl-lysine (biocytin) and the role of biocytin The few early reports of human biotin defi ciency as a carbon dioxide carrier. are all of people who consumed large amounts of uncooked eggs. They developed a fi ne scaly dermatitis and hair loss (alopecia). of the skin both free and bound to a serum glycoprotein that showed an absence of sebaceous glands and atrophy has biotinidase activity, catalyzing the hydrolysis of of the hair follicles. Provision of biotin supplements biocytin. of 200–1000 μg/day resulted in cure of the skin Biotin enters tissues by a saturable transport system lesions and regrowth of hair, despite continuing the and is then incorporated into biotin-dependent abnormal diet providing large amounts of avidin. enzymes as the ε-amino-lysine peptide, biocytin. There have been no studies of providing modest Unlike other B vitamins, where concentrative uptake doses of biotin to such patients, and none in into tissues can be achieved by facilitated diffusion which their high intake of uncooked eggs was followed by metabolic trapping, the incorporation of not either replaced by an equivalent intake of cooked biotin into enzymes is relatively slow, and cannot be eggs (in which avidin has been denatured by heat, and considered part of the uptake process. On catabolism the of which are a good source of biotin) or of the enzymes, biocytin is hydrolyzed by biotinidase, continued unchanged, so there is no information permitting reutilization. from these case reports of the amounts of biotin required for normal health. More recently, similar Metabolic functions of biotin signs of biotin defi ciency have been observed in Biotin functions to transfer carbon dioxide in a small patients receiving total parenteral nutrition for pro- number of carboxylation reactions. The reactive longed periods after major resection of the gut. The intermediate is 1-N-carboxy-biocytin (Figure 8.17), signs resolve following the provision of biotin, but formed from bicarbonate in an ATP-dependent reac- again there have been no studies of the amounts of tion. A single enzyme acts on the apoenzymes of biotin required; intakes have ranged between 60 μg/ acetyl-CoA carboxylase, pyruvate carboxylase, propi- day and 200 μg/day. 178 Introduction to Human Nutrition

Glucose metabolism in biotin defi ciency day and 200 μg/day. Since dietary defi ciency does not Biotin is the coenzyme for one of the key enzymes of occur, such intakes are obviously more than adequate gluconeogenesis, pyruvate carboxylase, and defi ciency to meet requirements. can lead to fasting hypoglycemia. In addition, biotin acts via cell surface receptors to induce the synthesis 8.13 Pantothenic acid of phosphofructokinase and pyruvate kinase (key enzymes of glycolysis), phospho-enolpyruvate Pantothenic acid (sometimes known as vitamin B5, carboxykinase (a key enzyme of gluconeogenesis) and and at one time called vitamin B3) has a central role glucokinase. in energy-yielding metabolism as the functional Rather than the expected hypoglycemia, biotin moiety of coenzyme A (CoA) and in the biosynthesis defi ciency may sometimes be associated with hyper- of fatty acids as the prosthetic group of acyl carrier glycemia as a result of the reduced synthesis of gluco- protein. The structures of pantothenic acid and CoA kinase. Glucokinase is the high Km isoenzyme of are shown in Figure 8.18. hexokinase that is responsible for uptake of glucose Pantothenic acid is widely distributed in all food- into the liver for glycogen synthesis when blood con- stuffs; the name derives from the Greek for “from centrations are high. It also acts as the sensor for everywhere,” as opposed to other vitamins that were hyperglycemia in the β-islet cells of the pancreas; originally isolated from individual especially rich metabolism of the increased glucose 6-phosphate sources. As a result, defi ciency has not been unequivo- formed by glucokinase leads to the secretion of cally reported in human beings except in specifi c insulin. There is some evidence that biotin supple- depletion studies, most of which have used the antag- ments can improve glucose tolerance in diabetes. onist ω-methyl-pantothenic acid.

Lipid metabolism in biotin defi ciency Absorption, metabolism, and metabolic The skin lesions of biotin defi ciency are similar to functions of pantothenic acid those seen in defi ciency of essential fatty acids, and About 85% of dietary pantothenic acid is as CoA and serum linoleic acid is lower than normal in biotin- phosphopantetheine. In the intestinal lumen these are defi cient patients owing to impairment of the hydrolyzed to pantetheine; intestinal mucosal cells elongation of PUFAs as a result of reduced activity of have a high pantetheinase activity and rapidly hydro- acetyl-CoA carboxylase. lyze pantetheine to pantothenic acid. The intestinal The impairment of lipogenesis also affects the absorption of pantothenic acid seems to be by simple tissue fatty acid composition, with an increase in the diffusion and occurs at a constant rate throughout the proportion of palmitoleic acid, mainly at the expense length of the small intestine; bacterial synthesis may of stearic acid, apparently as a result of increased fatty contribute to pantothenic acid nutrition. acid desaturase activity in biotin defi ciency. Although The fi rst step in pantothenic acid utilization is phos- dietary protein and fat intake also affect tissue fatty phorylation. Pantothenate kinase is rate limiting, so acid composition, the ratio of palmitoleic to stearic that, unlike vitamins that are accumulated by meta- acid may provide a useful index of biotin nutritional bolic trapping, there can be signifi cant accumulation status in some circumstances. of free pantothenic acid in tissues. It is then used for Biotin defi ciency also results in an increase in the synthesis of CoA and the prosthetic group of acyl normally small amounts of odd-chain fatty acids carrier protein. Pantothenic acid arising from the turn- (mainly C15:0 and C17:0) in triacylglycerols, phos- over of CoA and acyl carrier protein may be either pholipids, and cholesterol esters. This is a result of reused or excreted unchanged in the urine. impaired activity of propionyl-CoA carboxylase, leading to an accumulation of propionyl-CoA, which Coenzyme A and acyl carrier protein can be incorporated into lipids in competition with All tissues are capable of forming CoA from panto- acetyl-CoA. thenic acid. CoA functions as the carrier of fatty acids, as thioesters, in mitochondrial β-oxidation. The Safe and adequate levels of intake resultant two-carbon fragments, as acetyl-CoA, then There is no evidence on which to estimate require- undergo oxidation in the citric acid cycle. CoA also ments for biotin. Average intakes are between 10 μg/ functions as a carrier in the transfer of acetyl (and The Vitamins 179

Pantothenic acid Coenzyme A (CoASH)

H2 H2 O C OH O CNHC C SH CH2 CH2 -SH group forms thioesters with fatty acids CH2 CH2 NH NH C O C O CHOH CHOH NH H3C C CH3 H3C C CH3 2 CH 2 CH2 N N OH O O- - N N O POP O CH2 O O O

O OH -O P O Figure 8.18 Pantothenic acid - O and coenzyme A. other fatty acyl) moieties in a variety of biosynthetic rather the subjects were given and other and catabolic reactions, including: rich sources of all vitamins as part of an urgent program of nutritional rehabilitation. ● cholesterol and steroid hormone synthesis Experimental pantothenic acid depletion, together ● long-chain fatty acid synthesis from palmitate and with the administration of ω-methyl-pantothenic elongation of PUFAs in mitochondria acid, results in the following signs and symptoms after ● acylation of serine, threonine and cysteine residues 2–3 weeks: on proteolipids, and acetylation of neuraminic acid. ● neuromotor disorders, including paresthesia of the Fatty acid synthesis is catalyzed by a cytosolic multi- hands and feet, hyperactive deep tendon refl exes, enzyme complex in which the growing fatty acyl and muscle weakness. These can be explained chain is bound by thioester linkage to an enzyme- by the role of acetyl-CoA in the synthesis of the bound 4′-phosphopantetheine residue, rather than to neurotransmitter acetylcholine, and impaired free CoA, as in β-oxidation. This component of the formation of threonine acyl esters in myelin. fatty acid synthetase complex is the acyl carrier Dysmyelination may explain the persistence and protein. recurrence of neurological problems many years after nutritional rehabilitation in people who had Pantothenic acid defi ciency and safe and suffered from burning foot syndrome adequate levels of intake ● mental depression, which again may be related to Prisoners of war in the Far East in the 1940s, who either acetylcholine defi cit or impaired myelin were severely malnourished, showed, among other synthesis signs and symptoms of vitamin defi ciency diseases, a ● gastrointestinal complaints, including severe vom- new condition of paresthesia and severe pain in the iting and pain, with depressed gastric acid secretion feet and toes, which was called the “burning foot syn- in response to gastrin drome” or nutritional melalgia. Although it was ten- ● increased insulin sensitivity and a fl attened glucose tatively attributed to pantothenic acid defi ciency, no tolerance curve, which may refl ect decreased antag- specifi c trials of pantothenic acid were conducted, onism by glucocorticoids 180 Introduction to Human Nutrition

● decreased serum cholesterol and decreased urinary , bats, the passeriform birds, and most fi shes. excretion of 17-ketosteroids, refl ecting the impair- Ascorbate is synthesized as an intermediate in the gulo- ment of steroidogenesis nolactone pathway of glucose metabolism; in those ● decreased acetylation of p-aminobenzoic acid, sul- vertebrate species for which it is a vitamin, one enzyme fonamides and other drugs, refl ecting reduced of the pathway, gulonolactone oxidase, is absent. availability of acetyl-CoA for these reactions The vitamin C defi ciency disease, scurvy, has been ● increased susceptibility to upper respiratory tract known for many centuries and was described in the infections. Ebers papyrus of 1500 bc and by Hippocrates. The Crusaders are said to have lost more men through There is no evidence on which to estimate panto- scurvy than were killed in battle, while in some of the thenic acid requirements. Average intakes are between long voyages of exploration of the fourteenth and 3 mg/day and 7 mg/day, and since defi ciency does not fi fteenth centuries up to 90% of the crew died from occur, such intakes are obviously more than adequate scurvy. Cartier’s expedition to Quebec in 1535 was to meet requirements. struck by scurvy; the local native Americans taught him to use an infusion of swamp spruce leaves to Non-nutritional uses of pantothenic acid prevent or cure the condition. Blood levels of pantothenic acid have been reported Recognition that scurvy was due to a dietary defi - to be low in patients with rheumatoid arthritis; some ciency came relatively early. James Lind demonstrated workers have reported apparently benefi cial effects of in 1757 that juice and juice were pro- supplementation, but these reports remain uncon- tective, and Cook kept his crew in good health during fi rmed and there are no established pharmacological his circumnavigation of the globe (1772–1775) by uses of the vitamin. stopping frequently to take on fresh fruit and vegeta- Pantothenic acid defi ciency in rats leads to a loss of bles. In 1804 the British Navy decreed a daily ration fur color and at one time pantothenic acid was known of lemon or juice for all ratings, a requirement as the “anti-grey hair factor.” There is no evidence that that was extended to the merchant navy in 1865. the normal graying of hair with age is related to pan- The structure of vitamin C is shown in Figure 8.19; tothenic acid nutrition, or that pantothenic acid sup- both ascorbic acid and dehydroascorbic acid have plements have any effect on hair color. Its use in vitamin activity. Monodehydroascorbate is a stable shampoo is not based on any evidence of effi cacy. radical formed by reaction of ascorbate with reactive Pantothenic acid has very low toxicity; intakes of up oxygen species, and can be reduced back to ascorbate to 10 g/day of calcium pantothenate (compared with a by monodehydroascorbate reductase. Alternatively, 2 normal dietary intake of 2–7 mg/day) have been given mol of monodehydroascorbate can react together to for up to 6 weeks with no apparent ill-effects. yield 1 mol each of ascorbate and dehydroascorbate. Dehydroascorbate may either be reduced to ascorbate 8.14 Vitamin C (ascorbic acid) or undergo hydration to diketogulonate and onward metabolism. Vitamin C is a vitamin for only a limited number of Vitamin C is found in fruits and vegetables. Very vertebrate species: humans and the other primates, the signifi cant losses occur as vegetables wilt, or when

CH2OH CH2OH CH2OH HO CH O HO CH O HO CH O O O O

OH OH .O OH O O Figure 8.19 Vitamin C (ascor- Ascorbate Monodehydroascorbate Dehydroascorbate bic acid, monodehydroascor- (semidehydroascorbate) bate and dehydroascorbate). The Vitamins 181 they are cut, as a result of the release of ascorbate is recovered from subjects receiving high intakes of oxidase from the plant tissue. Signifi cant losses of the the vitamin, this is the result of bacterial metabolism vitamin also occur in cooking, both through leaching of unabsorbed vitamin in the intestinal lumen. into the cooking water and also atmospheric oxida- The fate of the greater part of ascorbic acid is excre- tion, which continues when foods are left to stand tion in the urine, either unchanged or as dehydro- before serving. ascorbate and diketogulonate. Both ascorbate and dehydroascorbate are fi ltered at the glomerulus then Absorption and metabolism of vitamin C reabsorbed. When glomerular fi ltration of ascorbate There is active transport of the vitamin at the intesti- and dehydroascorbate exceeds the capacity of the nal mucosal brush border membrane. Both ascorbate transport systems, at a plasma concentration of and dehydroascorbate are absorbed across the buccal ascorbate between 70 and 85 μmol/l, the vitamin is mucosa by carrier-mediated passive processes. excreted in the urine in amounts proportional to Intestinal absorption of dehydroascorbate is carrier intake. mediated, followed by reduction to ascorbate before transport across the basolateral membrane. Metabolic functions of vitamin C Some 80–95% of dietary ascorbate is absorbed at Ascorbic acid has specifi c roles in two groups of usual intakes (up to about 100 mg/day). The frac- enzymes: the copper-containing hydroxylases and the tional absorption of larger amounts of the vitamin is 2-oxoglutarate-linked iron-containing hydroxylases. lower, and unabsorbed ascorbate from very high It also increases the activity of a number of other doses is a substrate for intestinal bacterial meta- enzymes in vitro, although this is a non-specifi c bolism, causing gastrointestinal discomfort and reducing action rather than refl ecting any metabolic diarrhea. function of the vitamin. In addition, it has a number About 70% of blood ascorbate is in plasma and of non-enzymic effects due to its action as a reducing erythrocytes, which do not concentrate the vitamin agent and oxygen radical quencher. from plasma. The remainder is in white cells, which have a marked ability to concentrate it. Copper-containing hydroxylases Both ascorbate and dehydroascorbate circulate in Dopamine β-hydroxylase is a copper-containing free solution, and also bound to albumin. About 5% enzyme involved in the synthesis of the catechol- of plasma vitamin C is normally dehydroascorbate. amines norepinephrine (noradrenaline) and epi- Both vitamers are transported into cells by glucose nephrine (adrenaline) from tyrosine in the adrenal transporters, and concentrations of glucose of the medulla and central nervous system. The enzyme order of those seen in diabetic hyperglycemia inhibit contains Cu+, which is oxidized to Cu2+ during the tissue uptake of ascorbate. hydroxylation of the substrate; reduction back to Cu+ There is no specifi c storage organ for ascorbate; specifi cally requires ascorbate, which is oxidized to apart from leukocytes (which account for only 10% monodehydroascorbate. of total blood ascorbate), the only tissues showing a Some peptide hormones have a carboxy-terminal signifi cant concentration of the vitamin are the amide that is hydroxylated on the α-carbon by a adrenal and pituitary glands. Although the concentra- copper-containing enzyme, peptidylglycine hydroxy- tion of ascorbate in muscle is relatively low, skeletal lase. The α-hydroxyglycine residue then decomposes muscle contains much of the body’s pool of 900– non-enzymically to yield the amidated peptide and 1500 mg (5–8.5 mmol). glyoxylate. The copper prosthetic group is oxidized in Diketogulonate arising from dehydroascorbate can the reaction, and, as in dopamine β-hydroxylase, undergo metabolism to xylose, thus providing a route ascorbate is specifi cally required for reduction back for entry into central carbohydrate metabolic path- to Cu+. ways via the pentose phosphate pathway. However, oxidation to carbon dioxide is only a minor fate of Oxoglutarate-linked iron-containing ascorbate in humans. At usual intakes of the vitamin, hydroxylases less than 1% of the radioactivity from [14C]-ascorbate Several iron-containing hydroxylases share a common 14 is recovered as carbon dioxide. Although more CO2 reaction mechanism, in which hydroxylation of the 182 Introduction to Human Nutrition substrate is linked to decarboxylation of 2- monodehydroascorbate. At physiological concentra- oxoglutarate. Many of these enzymes are involved in tions of ascorbate, both Fe3+ and Cu2+ ions are reduced the modifi cation of precursor proteins to yield the by ascorbate, yielding monodehydroascorbate. Fe2+ fi nal, mature, protein. This is a process of postsyn- and Cu+ are readily reoxidized by reaction with hydro- thetic modifi cation of an amino acid residue after it gen peroxide to yield hydroxide ions and hydroxyl has been incorporated into the protein during synthe- radicals. Cu+ also reacts with molecular oxygen to sis on the ribosome. yield superoxide. Thus, as well as its antioxidant role, ascorbate has potential pro-oxidant activity. However, ● Proline and lysine hydroxylases are required for the because at high levels of intake the vitamin is excreted postsynthetic modifi cation of procollagen in the quantitatively, is it unlikely that tissue concentrations formation of mature, insoluble, collagen, and will rise high enough for there to be signifi cant for- proline hydroxylase is also required for the post- mation of oxygen radicals. synthetic modifi cation of the precursor proteins of osteocalcin and the C1q component of Vitamin C defi ciency: scurvy complement. The vitamin C defi ciency disease scurvy was formerly ● Aspartate β-hydroxylase is required for the postsyn- a common problem at the end of winter, when there thetic modifi cation of the precursor of protein C, had been no fresh fruit and vegetables for many the vitamin K-dependent that hydrolyzes months. activated factor V in the blood-clotting cascade. Although there is no specifi c organ for storage of ● Trimethyl-lysine and γ-butyrobetaine hydroxylases vitamin C in the body, signs of defi ciency do not are required for the synthesis of carnitine. develop in previously adequately nourished subjects Ascorbate is oxidized during the reaction of these until they have been deprived of the vitamin for 4–6 enzymes, but not stoichiometrically with the decar- months, by which time plasma and tissue concentra- boxylation of 2-oxoglutarate and hydroxylation of the tions have fallen considerably. The earliest signs of substrate. The purifi ed enzyme is active in the absence scurvy in volunteers maintained on a vitamin C-free of ascorbate, but after some 5–10 s (about 15–30 cycles diet are skin changes, beginning with plugging of hair of enzyme action) the rate of reaction begins to fall. At follicles by horny material, followed by enlargement this stage the iron in the catalytic site has been oxi- of the hyperkeratotic follicles, and petechial hemor- dized to Fe3+, which is catalytically inactive; activity is rhage with signifi cant extravasation of red cells, pre- restored only by ascorbate, which reduces it back to sumably as a result of the increased fragility of blood Fe2+. The oxidation of Fe2+ is the consequence of a capillaries. side-reaction rather than the main reaction of the At a later stage there is also hemorrhage of the enzyme, which explains how 15–30 cycles of enzyme gums, beginning in the interdental papillae and pro- activity can occur before there is signifi cant loss of gressing to generalized sponginess and bleeding. This activity in the absence of ascorbate, and why the con- is frequently accompanied by secondary bacterial sumption of ascorbate is not stoichiometric. infection and considerable withdrawal of the gum from the necks of the teeth. As the condition pro- Pro-oxidant and antioxidant roles of ascorbate gresses, there is loss of dental cement, and the teeth Ascorbate can act as a radical-trapping antioxidant, become loose in the alveolar bone and may be lost. reacting with superoxide and a proton to yield hydro- Wounds show only superfi cial healing in scurvy, gen peroxide, or with the hydroxy radical to yield with little or no formation of (collagen-rich) scar water. In each instance the product is the monodehy- tissue, so that healing is delayed and wounds can droascorbate radical. Thus, as well as reducing the readily be reopened. The scorbutic scar tissue has only tocopheroxyl radical formed by interaction of about half the tensile strength of that normally α-tocopherol in membranes with lipid peroxides, formed. ascorbate acts to trap the oxygen radicals that would Advanced scurvy is accompanied by intense pain in otherwise react to form lipid peroxides. the bones, which can be attributed to changes in bone At high concentrations, ascorbate can reduce mineralization as a result of abnormal collagen syn- molecular oxygen to superoxide, being oxidized to thesis. Bone formation ceases and the existing bone The Vitamins 183 becomes rarefi ed, so that the bones fracture with The requirement for vitamin C to prevent clinical minimal trauma. scurvy is less than 10 mg/day. However, at this level The name scurvy is derived from the Italian scor- of intake wounds do not heal properly because of the butico, meaning an irritable, neurotic, discontented, requirement for vitamin C for the synthesis of colla- whining, and cranky person. The disease is associated gen in connective tissue. An intake of 20 mg/day is with listlessness and general malaise, and sometimes required for optimum wound healing. Allowing for changes in personality and psychomotor performance individual variation in requirements, this gives a ref- and a lowering of the general level of arousal. These erence intake for adults of 30 mg/day, which was the behavioral effects can be attributed to impaired syn- British recommended daily allowance (RDA) until thesis of catecholamine neurotransmitters, as a result 1991. of low activity of dopamine β-hydroxylase. The 1991 British reference nutrient intake (RNI) Most of the other clinical signs of scurvy can be for vitamin C is based on the level of intake at which accounted for by the effects of ascorbate defi ciency on the plasma concentration rises sharply, showing that collagen synthesis, as a result of impaired proline and requirements have now been met, tissues are satu- lysine hydroxylase activity. Depletion of muscle car- rated and there is spare vitamin C being transported nitine, due to impaired activity of trimethyllysine and between tissues, available for excretion. This criterion γ-butyrobetaine hydroxylases, may account for the of adequacy gives an RNI of 40 mg/day for adults. lassitude and fatigue that precede clinical signs of The alternative approach to determining require- scurvy. ments is to estimate the total body content of vitamin C, then measure the rate at which it is metabolized, Anemia in scurvy by giving a test dose of radioactive vitamin. This is Anemia is frequently associated with scurvy, and may the basis of both the former US RDA of 60 mg/day be either macrocytic, indicative of folate defi ciency, or for adults and the Netherlands RDA of 80 mg/day. hypochromic, indicative of iron defi ciency. Indeed, it also provides an alternative basis for the Folate defi ciency may be epiphenomenal, since the RNI of 40 mg/day. major dietary sources of folate are the same as those The problem lies in deciding what is an appropriate of ascorbate. However, some patients with clear meg- body content of vitamin C. The studies were per- aloblastic anemia respond to the administration of formed on subjects whose total body vitamin C was vitamin C alone, suggesting that there may be a role estimated to be 1500 mg at the beginning of a deple- of ascorbate in the maintenance of normal pools of tion study. However, there is no evidence that this is reduced folates, although there is no evidence that any a necessary, or even a desirable, body content of the of the reactions of folate is ascorbate dependent. vitamin. It is simply the body content of the vitamin Iron defi ciency in scurvy may well be secondary to of a small group of people eating a self-selected diet reduced absorption of inorganic iron and impaired rich in fruit. There is good evidence that a total body mobilization of tissue iron reserves (see below). At the content of 900 mg is more than adequate. It is three same time, the hemorrhages of advanced scurvy will times larger than the body content at which the fi rst cause a signifi cant loss of blood. signs of defi ciency are observed, and will protect There is also evidence that erythrocytes have a against the development of any signs of defi ciency for shorter half-life than normal in scurvy, possibly as a several months on a completely vitamin C-free diet. result of peroxidative damage to membrane lipids There is a further problem in interpreting the results. owing to impairment of the reduction of tocopher- The rate at which vitamin C is metabolized varies with oxyl radical by ascorbate. the amount consumed. This means that as the experi- mental subjects become depleted, so the rate at which Vitamin C requirements they metabolize the vitamin decreases. Thus, calcula- Vitamin C illustrates extremely well how different tion of the amount that is required to maintain the criteria of adequacy, and different interpretations of body content depends on both the way in which the experimental data, can lead to different estimates results obtained during depletion studies are extrapo- of requirements, and to reference intakes ranging lated to the rate in subjects consuming a normal diet between 30 and 90 mg/day for adults. and the amount of vitamin C in that diet. 184 Introduction to Human Nutrition

An intake of 40 mg/day is more than adequate to Without a differential white cell count, leukocyte maintain a total body content of 900 mg of vitamin ascorbate concentration cannot be considered to give C (the British RNI). At a higher level of habitual a meaningful refl ection of vitamin C status. The dif- intake, 60 mg/day is adequate to maintain a total body ferent types of leukocyte have different capacities to content of 1500 mg (the former US RDA). Making accumulate ascorbate. This means that a change in the allowances for changes in the rate of metabolism with proportion of granulocytes, platelets, and mononu- different levels of intake, and allowing for incomplete clear leukocytes will result in a change in the total absorption of the vitamin gives the Netherlands RDA concentration of ascorbate/106 cells, although there of 80 mg/day. may well be no change in vitamin nutritional status. The current US reference intake (75 mg for women Stress, myocardial infarction, infection, burns, and and 90 mg for men) is based on intakes required to surgical trauma all result in changes in leukocyte dis- saturate leukocytes with vitamin C. tribution, with an increase in the proportion of gran- ulocytes, and hence an apparent change in leukocyte ascorbate. This has been widely misinterpreted to Assessment of vitamin C status indicate an increased requirement for vitamin C in Urinary excretion and saturation testing these conditions. Urinary excretion of ascorbate falls to undetectably low levels in defi ciency, and therefore very low excre- Possible benefi ts of high intakes of tion will indicate defi ciency. However, no guidelines vitamin C for the interpretation of urinary ascorbate have been There is evidence from a variety of studies that high established. vitamin C status and a high plasma concentration of It is relatively easy to assess the state of body reserves the vitamin is associated with reduced all-cause of vitamin C by measuring the excretion after a test mortality. dose. A subject who is saturated will excrete more or At intakes above about 100–120 mg/day the body’s less the whole of a test dose of 500 mg of ascorbate capacity to metabolize vitamin C is saturated, and over 6 h. A more precise method involves repeating any further intake is excreted in the urine unchanged. the loading test daily until more or less complete Therefore, it would not seem justifi able to recom- recovery is achieved, thus giving an indication of how mend higher levels of intake. However, in addition depleted the body stores were. to its antioxidant role and its role in reducing the tocopheroxyl radical, and thus sparing vitamin Blood concentrations of ascorbate E, vitamin C is important in the absorption of The plasma concentration of vitamin C falls relatively iron, and in preventing the formation of nitrosa- rapidly during experimental depletion studies to mines. Both of these actions depend on the presence undetectably low levels within 4 weeks of initiating a of the vitamin in the gut together with food, and vitamin C-free diet, although clinical signs of scurvy intakes totaling more than 100 mg/day may be may not develop for a further 3–4 months, and tissue benefi cial. concentrations of the vitamin may be as high as 50% of saturation. In fi eld studies and surveys, subjects Iron absorption with plasma ascorbate below 11 μmol/l are consid- Inorganic dietary iron is absorbed as Fe2+ and not as ered to be at risk of developing scurvy, and anyone Fe3+; ascorbic acid in the intestinal lumen will both with a plasma concentration below 6 μmol/l would maintain iron in the reduced state and chelate it, thus be expected to show clinical signs. increasing the amount absorbed. A dose of 25 mg of The concentration of ascorbate in leukocytes is vitamin C taken together with a meal increases the correlated with the concentrations in other tissues, absorption of iron by around 65%, while a 1 g dose and falls more slowly than plasma concentration in gives a ninefold increase. This occurs only when depletion studies. The reference range of leukocyte ascorbic acid is present together with the test meal; ascorbate is 1.1–2.8 mol/106 cells; a signifi cant loss of neither intravenous administration of vitamin C nor leukocyte ascorbate coincides with the development intake several hours before the test meal has any effect of clear clinical signs of scurvy. on iron absorption. Optimum iron absorption may The Vitamins 185 therefore require signifi cantly more than 100 mg of There is little evidence of signifi cant toxicity from vitamin C/day. these high intakes. Once the plasma concentration of ascorbate reaches the renal threshold, it is excreted Inhibition of nitrosamine formation more or less quantitatively with increasing intake, and The safety of nitrates and nitrites used in curing meat, there is no evidence that higher intakes increase the a traditional method of preservation, has been ques- body pool above about 110 μmol/kg body weight. tioned because of the formation of nitrosamines by Unabsorbed ascorbate in the intestinal lumen is a reaction between nitrite and amines naturally present substrate for bacterial fermentation, and may cause in foods under the acid conditions in the stomach. In diarrhea and intestinal discomfort. experimental animals nitrosamines are potent car- Ascorbate can react non-enzymically with amino cinogens, and some authorities have limited the groups in proteins to glycate the proteins, in the same amounts of these salts that are permitted, although way as occurs in poorly controlled diabetes mellitus, there is no evidence of any hazard to humans from and there is some evidence of increased cardiovascu- endogenous nitrosamine formation. Ascorbate can lar mortality associated with vitamin C supplements prevent the formation of nitrosamines by reacting in diabetics. non-enzymically with nitrite and other nitrosating Up to 5% of the population are at risk from the reagents, forming NO, NO2, and N2. Again, this is an development of renal oxalate stones. The risk is from effect of ascorbate present in the stomach at the same both ingested oxalate and that formed endogenously, time as the dietary nitrites and amines, rather than an mainly from the metabolism of glycine. Some reports effect of vitamin C nutritional status. have suggested that people consuming high intakes of vitamin C excrete more oxalate in the urine. However, Pharmacological uses of vitamin C no pathway for the formation of oxalate from ascor- Several studies have reported low ascorbate status in bate is known, and it seems that the oxalate is formed patients with advanced cancer, which is perhaps an non-enzymically under alkaline conditions either in unsurprising fi nding in seriously ill patients. With the bladder or after collection, and hence high vitamin very little experimental evidence, it has been sug- C intake is not a risk factor for renal stone gested that very high intakes of vitamin C (of the formation. order of 10 g/day or more) may be benefi cial in enhancing host resistance to cancer and preventing 8.15 Perspectives on the future the development of the acquired immunodefi ciency syndrome (AIDS) in people who are human immu- Current estimates of requirements and reference nodefi ciency virus (HIV) positive. In controlled intakes of vitamins are based on the amounts required studies with patients matched for age, gender, site and to prevent or reverse subtle indices of defi ciency, stage of primary tumors and metastases, and previous and can thus be considered to be amounts required chemotherapy, there was no benefi cial effect of high- to prevent defi ciency, but possibly not to promote dose ascorbic acid in the treatment of advanced optimum nutritional status and health. There is cur- cancer. rently very little evidence on which to base reference High doses of vitamin C have been recommended intakes above those required to prevent (subtle bio- for the prevention and treatment of the common chemical) defi ciency, but indices of enhanced immune cold, with some evidence that the vitamin reduces the system function and whole-body oxidative stress and duration of symptoms. However, the evidence from other biomarkers may do so in due course. controlled trials is unconvincing. There are several compounds that have clearly defi ned functions in the body but can be synthesized Toxicity of vitamin C in apparently adequate amounts, so that they are Regardless of whether or not high intakes of ascorbate not considered to be dietary essentials. These sub- have any benefi cial effects, large numbers of people stances have been receiving increasing attention, habitually take between 1 and 5 g/day of vitamin C and these, in addition to other compounds, are likely supplements (compared with reference intakes of to continue to stimulate interest and discussion in the 40–90 mg/day) and some take considerably more. future. 186 Introduction to Human Nutrition

Biofl avonoids longed periods on total parenteral nutrition. There is The most studied fl avonoids are hesperitin and quer- some evidence that supplements of carnitine may citin. Because they are biologically active, they are increase the ability of muscle to oxidize fatty acids, commonly called biofl avonoids. Most fruits and green and so increase physical work capacity, although other leafy vegetables contain relatively large amounts of studies have shown no effect. fl avonoids; altogether some 2000 have been identi- Choline fi ed, and average intakes of fl avonoids from a mixed diet are of the order of 1 g/day. Choline is important as a base in phospholipids: both There is no evidence that biofl avonoids are dietary phosphatidylcholine (lecithin) in all cell membranes essentials, but they have potentially useful antioxidant and sphingomyelin in the nervous system. In addi- actions. Oxidation of fl avonoids may serve to protect tion, acetylcholine is a transmitter in the central and susceptible nutrients from damage in foods and the parasympathetic nervous systems and at neuromus- intestinal lumen, and they may also act as antioxi- cular junctions. There is some evidence that the avail- dants in plasma and tissues. Epidemiological evidence ability of choline may be limiting for the synthesis of suggests that the intake of fl avonoids is inversely cor- acetylcholine in the central nervous system under related with mortality from coronary heart disease. some conditions. In animals, defi ciency of choline results in fatty infi ltration of the liver, apparently as a Carnitine result of impairment of the export of lipoproteins Carnitine has a central role in the transport of fatty from hepatocytes; prolonged defi ciency may result in acids across the mitochondrial membrane. It is syn- cirrhosis. The kidney can also be affected, with tubular thesized in both liver and skeletal muscle by methyla- necrosis and interstitial hemorrhage, probably as a tion of lysine, followed by two vitamin C-dependent result of lysosomal membrane disruption. hydroxylations. In experimental animals, defi ciency There is no evidence that choline is a dietary essen- of lysine has little effect on plasma and tissue concen- tial for humans, and no condition similar to the trations, but methionine defi ciency can lead to carni- effects of choline defi ciency in experimental animals tine depletion, and carnitine has a methionine-sparing has been reported. Since phosphatidylcholine is found effect in methionine-defi cient animals. in all biological membranes, dietary defi ciency is Defi ciency of vitamin C may result in impaired unlikely to occur except when people are maintained synthesis of carnitine in species for which ascorbate on defi ned diets free from phospholipids. Plasma is a vitamin. concentrations fall during long-term total parenteral The administration of the anticonvulsant valproic nutrition, and it is possible that the impaired liver acid can lead to carnitine depletion. This results in function seen in such patients is partly the result of impaired β-oxidation of fatty acids and ketogenesis, choline depletion. and hence a nonketotic hypoglycemia, with elevated plasma nonesterifi ed fatty acids and triacylglycerols. Inositol There may also be signs of liver dysfunction, with The main function of inositol is in phospholipids; hyperammonemia and encephalopathy. The admin- phosphatidylinositol constitutes some 5–10% of the istration of carnitine supplements in these conditions total membrane phospholipids. In addition to its has a benefi cial effect. structural role in membranes, phosphatidylinositol Although carnitine is not generally nutritionally has a major function in the intracellular responses to important, it may be required for premature infants, hormones and neurotransmitters, yielding two intra- since they have a limited capacity to synthesize it. cellular second messengers, inositol trisphosphate, There is some evidence that full-term infants may also and diacylglycerol. have a greater requirement for carnitine than can There is no evidence that inositol is a dietary essen- be met by endogenous synthesis; infants fed on tial. Infants may have a higher requirement than can carnitine-free soya-milk formula have higher plasma be met by endogenous synthesis. Untreated diabetics concentrations of nonesterifi ed fatty acids and triacyl- have high plasma concentrations of free inositol and glycerols than those receiving carnitine supplements. high urinary excretion of inositol, associated with Carnitine depletion, with disturbed lipid metabolism, relatively low intracellular concentrations of inositol, has also been reported in adults maintained for pro- suggesting that elevated plasma glucose may inhibit The Vitamins 187 tissue uptake of inositol. There is some evidence that foods of plant origin, have potentially benefi cial impaired nerve conduction velocity in diabetic neu- effects, although they are not nutrients. Collectively, ropathy in both patients and experimental animals is they are known as phytoceuticals (substances of plant associated with low intracellular concentrations of origin with potential pharmaceutical action) or inositol, and inositol supplements may improve nerve . The following compounds are exam- conduction velocity. However, high intracellular con- ples of phytoceuticals: centrations of inositol also impair nerve conduction ● Many glucosinolates and glycosides either inhibit velocity, and supplements may have a deleterious the enzymes of phase I metabolism of foreign com- effect. pounds (the reactions that activate many potential Taurine carcinogens) or induce the reactions leading to conjugation and excretion of foreign compounds. Until about 1976 it was assumed that taurine was a ● Terpenes that are found in the volatile (essential) oils metabolic end-product, the only function of which of herbs and spices are potentially active as lipid- was the conjugation of bile acids. The occurrence of soluble antioxidants, as are many of the carotenoids changes in the electrical activity of the retina in chil- that are not active as precursors of vitamin A. dren maintained on long-term total parenteral nutri- ● Compounds such as squalene, which are precursors tion without added taurine has shown that it has of cholesterol synthesis, may have a hypocholester- physiological functions, and has raised the question olemic action, by reducing the activity of the rate- of whether or not it should be regarded as a dietary limiting enzyme of cholesterol synthesis, hydroxy- essential. methylglutaryl-CoA reductase. Ubiquinone (coenzyme Q, “vitamin Q”) ● Various water-soluble compounds, including poly- phenols, anthocyanins, and fl avonoids, have anti- Ubiquinone is one of the electron carriers in mito- oxidant action. chondria. Therefore, it has an essential function in all ● Several plants (especially soyabeans) contain com- energy-yielding metabolism and may also have a pounds with estrogenic action () general antioxidant role in membranes. Like vitamin that also have antiestrogenic action and appear to E, it can be anchored in membranes by the hydropho- be protective against the development of hormone- bic tail, with the reactive quinone group at the mem- dependent cancer of the breast and uterus. brane surface. Ubiquinone is readily synthesized in the body, and there is no evidence that it is a dietary essential, or that supplements serve any useful Reference purpose, although they may have non-specifi c anti- oxidant actions and so spare vitamin E. Horwitt MK. J Nutr 1956; 60 (Suppl 1): 1–43.

“Phytoceuticals” Further reading In addition to the compounds with clearly defi ned metabolic functions discussed above, various com- Bender DA. Nutritional Biochemistry of the Vitamins, 2nd edn. pounds naturally present in foods, and especially in Cambridge University Press, Cambridge, 2003. 9 Minerals and Trace Elements

JJ (Sean) Strain and Kevin D Cashman

Key messages

• This chapter defi nes the essential minerals and trace elements. • Health effects and symptoms of both inadequate and toxic • It describes the functions and routes of metabolism within the intakes are described. body of each of the minerals and trace elements in turn. • Methods of assessing the body status of each mineral and trace • Dietary requirements and dietary sources are discussed for each element are reviewed. mineral.

9.1 Introduction can be controlled in the body. In addition, storage in inactive sites or in an unreactive form can prevent an Essential minerals, including the trace elements, are element from causing adverse effects in the body, and inorganic elements (see Figure 9.1) that have a physi- release from storage can be important in times of ological function within the body. These must be dietary insuffi ciency. supplied in the diet (food and fl uids) and vary from All elements have the potential to cause toxic symp- grams per day for the major (macro) minerals through toms, whereas some, the known essential elements milligrams to micrograms per day for the trace in Figure 9.1, have the potential to cause defi ciency elements. symptoms. Even so, defi ciencies of only four of these It has been proposed that the environment (most inorganic elements are known to be prevalent in probably in the primordial sea around hydrothermal human populations. Two of these defi ciencies, iodine vents) in which living organisms evolved was a and iron, are widespread in human populations primary determinant of which elements became whereas the other two, zinc and selenium, only occur essential for life by providing structural integrity and in some population groups under specially defi ned catalytic ability to the fi rst complex organic mole- conditions. Overt clinical signs of defi ciency of any cules. As life evolved from the oceans on to land, a of the other inorganic elements are exceptional in process may have resulted in some humans and mainly occur secondary to other clinical elements becoming relatively more important because conditions. Such observations do not preclude the of superior catalytic abilities over other elements. In possibility that suboptimum status of the great major- any event, the uneven distribution of elements in a ity of the elements indicated in Figure 9.1 is impor- land-based environment meant that effi cient homeo- tant in human nutrition. Indeed, there is an increasing static mechanisms had to be in place to conserve awareness of the potential role of suboptimal as well essential elements and to eliminate excesses of essen- as supraoptimal nutritional status of minerals and tial and nonessential elements. The processes of trace elements in the development of degenerative absorption from the gastrointestinal tract and excre- age-related diseases, such as coronary heart disease, tion with body fl uids, therefore, are major ways in cancer, and osteoporosis. Moreover, other elements, which the concentration and amount of an element which currently have no published dietary recom-

© 2009 JJ Strain and KD Cashman. Minerals and Trace Elements 189

1a 2a 3a 4a 5a 6a 7a 8 1b 2b 3b 4b 5b 6b 7b 8b 1 2 H He 3 4 5 6 7 8 9 10 Li Be B C N O F Ne 11 12 13 14 15 16 17 18 Na Mg Al Si P S Cl Ar 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 K Ca Sc Ti V Cr Mn Fe Co Ni Cu Zn Ga Ge As Se Br Kr 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 Rb Sr Y Zr Nb Mo Tc Ru Rh Pd Ag Cd In Sn Sb Te I Xe 55 56 57 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 Cs Ba La Hf Ta W Re Os Ir Pt Au Hg Tl Pb Bi Po At Rn 87 88 89 104 105 106 Fr Ra Ac

58 59 60 61 62 63 64 65 66 67 68 69 70 71 Lanthanides Ce Pr Nd Pm Sm Eu Gd Tb Dy Ho Er Tm Yb Lu 90 91 92 93 94 95 96 97 98 99 100 101 102 103 Actinides Th Pa U Np Pu Am Cm Bk Cf Es Fm Md No Lr

Figure 9.1 The periodic table of the elements. The widely accepted or putative essential elements are encircled. mendations but are highlighted in Figure 9.1, might various ferrous and nonferrous alloys. It is also used prove to be essential for the optimum health and well- as an alloying agent for aluminum, beryllium, copper, being of humans. lead, and magnesium alloys. Calcium was among the Major constraints to the elucidation of the poten- fi rst materials known to be essential in the diet. All tial roles of minerals and trace elements in the onset foods of vegetable origin contain small but useful of degenerative diseases include diffi culties in assess- amounts of calcium. Animals concentrate calcium ing status, and thereby defi ning requirements, and in milk, and milk and dairy products are the most myriad interactions among minerals and other nutri- important food sources of calcium for many human ent and nonnutrients in the diet. Sometimes, natural populations. experiments of genetic disorders can throw light on the potential roles of minerals in disease processes Absorption, transport, and and these will also be discussed as appropriate in the tissue distribution following sections. The adult human body contains about 1200 g of calcium, which amounts to about 1–2% of body 9.2 Calcium weight. Of this, 99% is found in mineralized tissues, such as bones and teeth, where it is present as calcium Calcium is a metallic element, fi fth in abundance in phosphate (together with a small component of the Earth’s crust, of which it forms more than 3%. calcium carbonate), providing rigidity and structure. Calcium is never found in nature uncombined; it The remaining 1% is found in blood, extracellular occurs abundantly as chalk, granite, eggshell, sea- fl uid (ECF), muscle, and other tissues. shells, “hard” water, bone, and limestone. The metal Calcium is under close homeostatic control, with is used as a reducing agent in preparing other metals processes such as absorption, excretion and secretion, such as thorium, uranium, and zirconium, and is and storage in bone being involved in maintaining the used as a deoxidizer, disulfurizer, or decarburizer for concentration of ionized calcium in the plasma within 190 Introduction to Human Nutrition a tightly regulated range. This tight regulation of Calcium is absorbed in the intestine by two routes, plasma calcium concentration is achieved through a transcellular and paracellular (Figure 9.3). The trans- complex physiological system comprising the interac- cellular route involves active transport of calcium by tion of the calcitropic hormones, such as parathy- the mucosal calcium transport protein, calbindin, and roid hormone (PTH), 1,25-dihydroxycholecalciferol is saturable and subject to physiological and nutri-

[1,25(OH)2D3] and calcitonin, with specifi c target tional regulation via vitamin D. The paracellular route tissues (kidney, bone, and intestine) that serve to involves passive calcium transport through the tight increase or to decrease the entry of calcium into junctions between mucosal cells; it is nonsaturable, the extracellular space (plasma) (Figure 9.2). Only in essentially independent of nutritional and physiologi- extreme circumstances, such as severe malnutrition cal regulation, and concentration dependent. Most or hyperparathyroidism, is the serum ionized calcium calcium absorption in humans occurs in the small concentration below or above the normal range. The intestine, but there is some evidence for a small secretion of these hormones is governed wholly, or in colonic component. Transcellular calcium absorption part, by the plasma concentration of ionized calcium, responds to calcium needs, as refl ected by changes thus forming a negative feedback system. PTH and in plasma calcium concentration, by hormone-

1,25(OH)2D3 are secreted when plasma calcium is mediated up- or down-regulation of calbindin in low, whereas calcitonin is secreted when plasma mucosal cells; for example, reduced plasma calcium calcium is high. evokes a PTH-mediated increase in plasma

Calcium in food occurs as salts or associated with 1,25(OH)2D3, which stimulates increased calbindin other dietary constituents in the form of complexes synthesis in intestinal mucosal cells. of calcium ions. Calcium must be released in a soluble, On average, between 10% and 30% of the calcium and probably ionized, form before it can be absorbed. is absorbed from a mixed diet by healthy adults.

1000 mg Diet GIT

PTH Feces + 100 mg 800 mg 300 mg

Ca2+ Parathyroids

Plasma calcium (9–10.4 mg/100 ml) Kidney 10 000 mg 400 mg Ca2+ +

Thyroid 9 800 mg + 400 mg Ϫ Ϫ +

+ 25(OH)D CT +

Bone 1,25(OH)2D3 200 mg

Figure 9.2 Homeostatic regulation of serum calcium, showing the integration of hormone action at the tissue level. CT, calcitonin; PTH, para- thyroid hormone; 1,25(OH)2D3, 1,25-dihydroxycholecalciferol (to convert from mg/day to mmol/day multiply by 40). Minerals and Trace Elements 191

Mucosal cell Lumen Ca2+ TRPV6/CaT1 CaT1

Calbindin D N 9k N Protein synthesis Nuclear N N receptor Transcellular Ca transport mRNA Tight junction protein complex Paracellular Ca transport ATP ATP Nucleus

ADP ADP + + Na K 1,25 (OH)2D3 Ca-ATPase Na-K-ATPase Blood Figure 9.3 Calcium transport across the intestinal mucosal lining: paracellular calcium transport (between mucosal cells) and transcellular calcium transport (across the mucosal cell).

However, the effi ciency of intestinal calcium absorp- Table 9.1 Factors affecting calcium absorption tion is infl uenced by a variety of physiological factors (Table 9.1). Calcium absorption may also be infl u- Increased absorption Decreased absorption enced by a number of dietary factors (Table 9.1). Physiological factors Vitamin D adequacy Vitamin D defi ciency Metabolic function and essentiality Increased mucosal mass Decreased mucosal mass Calcium defi ciency Menopause Calcium is required for normal growth and develop- Phosphorus defi ciency Old age ment of the skeleton. During skeletal growth and Pregnancy Decreased gastric acid (without maturation, i.e., until the early twenties in humans, a meal) Lactation Rapid intestinal transit time calcium accumulates in the skeleton at an average rate Disease states (e.g., Disease states (e.g., of 150 mg/day. During maturity, the body, and there- hyperparathyroidism, malabsorption syndrome, fore the skeleton, is more or less in calcium equilib- sarcoidosis, idiopathic celiac disease, Crohn’s rium. From the age of about 50 years in men and from hypercalciuria) disease, chronic renal failure, the menopause in women, bone balance becomes diabetes, hypoparathyroidism, primary biliary cirrhosis) negative and bone is lost from all skeletal sites. This Dietary factors bone loss is associated with a marked rise in frac- Lactose (in infants) Phytate ture rates in both sexes, but particularly in women. Casein phosphopeptides (?)a Oxalate Adequate calcium intake is critical to achieving Nondigestible oligosaccharides Large calcium load Small calcium load High habitual calcium intake optimal peak bone mass (PBM) and modifi es the rate Low habitual calcium intake Ingestion without a meal of bone loss associated with aging. Extraskeletal Ingestion with a meal calcium (representing around 1% of total body calcium) plays a role in mediating vascular contrac- a Confl icting data in the literature. tion and vasodilatation, muscle contraction, nerve transmission, glandular secretion, and as an impor- bolic calcium defi ciency probably never exists, at least tant second messenger molecule. not as a nutritional disorder. An inadequate intake or poor intestinal absorption of calcium causes the Defi ciency symptoms circulating ionized calcium concentration to decline Because of the small metabolic pool of calcium (less acutely, which triggers an increase in PTH synthesis than 0.1% in the ECF compartment) relative to the and release. PTH acts on three target organs (either large skeletal reserve, for all practical purposes meta- directly or indirectly) to restore the circulating calcium 192 Introduction to Human Nutrition concentration to normal (Figure 9.2). At the kidney, insuffi ciency in adults, the US Food and Nutrition PTH promotes the reabsorption of calcium in the Board established a tolerable upper intake level (UL) distal tubule. PTH affects the intestine indirectly by of calcium of 2500 mg/day for children, adolescents, stimulating the production of 1,25(OH)2D3 (in the and adults, as well as pregnant and lactating women. kidney), which, in turn, leads to increased calcium absorption. PTH also induces bone resorption (by Genetic diseases signaling osteoclasts), thereby releasing calcium into Two rare inborn errors of vitamin D metabolism, blood. Owing to the action of PTH and 1,25(OH)2D3 vitamin D-dependent rickets types I and II, have an on the target tissues, plasma calcium concentrations associated hypocalcemia that can impair the bone are restored within minutes to hours. calcifi cation process. Type I vitamin D-dependent If, however, there is a continual inadequate intake rickets appears to be caused by in the or poor intestinal absorption of calcium (e.g., because enzyme 25(OH)D3-1-α-hydroxylase [responsible of vitamin D defi ciency), circulating calcium concen- for the synthesis of 1,25(OH)2D3 from 25(OH)D3], tration is maintained largely at the expense of skeletal leading to defective activity of this enzyme, whereas mass, that is, from an increased rate of bone resorp- type II vitamin D-dependent rickets, which is associ- tion. This PTH-mediated increase in bone resorp- ated with normal or elevated levels of 1,25(OH)2D3, tion is one of several important causes of reduced is thought to result from target tissue resistance to the bone mass and osteoporosis. The cumulative effect of action of 1,25(OH)2D3. This resistance arises owing calcium depletion (by whatever mechanism) on the to changes in the vitamin D receptor molecule. Daily skeleton over many years contributes to the increas- vitamin D3 administration seems to be an effective ing frequency of osteoporotic fractures with age. therapy for both disorders. Prolonged inadequate calcium intake in younger A hypercalcemia has been noted in familial benign people reduces the rate of accretion of the skeleton hypercalcemia (types I and III). Type I familial benign and may prevent the attainment of the genetically hypercalcemia, a renal tubular defect in calcium determined maximal PBM. This may increase the risk reabsorption, is caused by a mutation in the gene of osteoporosis as the PBM in adulthood is predictive encoding the calcium-sensing receptor. Type III of bone mass in later life. Chronic inadequate intake familial benign hypercalcemia represents a distinct or poor intestinal absorption of calcium may also play genetic entity. However, the gene(s) responsible for some role in the etiologies of hypertension, including this type of hypocalciuric hypercalcemia is still being pre-eclampsia and colon cancer. Calcium intake may mapped. also play a role in body weight regulation; however, this requires further investigation. Assessing status There is, as yet, no biochemical indicator that refl ects Toxicity calcium nutritional status. Blood calcium concentra- The available data on the adverse effects of high tion, for example, is not a good indicator because it calcium intakes in humans are primarily from the is tightly regulated. There are, however, some poten- intake of calcium from nutrient supplements. The tial indicators of nutritional calcium adequacy, which three most widely studied and biologically important are related to content or metabolism of the skeletal are: calcium reserve. Measures of bone mass may be used as indicators of body calcium status. These include ● kidney stone formation (nephrolithiasis); bone mineral content (BMC, which is the amount of ● the syndrome of hypercalcemia and renal insuffi - mineral at a particular skeletal site such as the femoral ciency, with or without alkalosis (referred to his- neck, lumbar spine, or total body) and bone mineral torically as milk alkali syndrome associated with density (BMD, which is BMC divided by the area of peptic ulcer treatments); the scanned region). Besides their relationship to ● the effect on absorption of other essential minerals, bone mass and strength, BMD and BMC are strong e.g., iron, zinc, magnesium and phosphorus. predictors of fracture risk and are thus functional Based largely on the data concerning the association indicators of calcium status. The US Food and of high calcium intakes with hypercalcemia and renal Nutrition Board used data relating dietary calcium Minerals and Trace Elements 193 intake to BMD and BMC to establish the estimates of Table 9.2 Calcium and phosphorus contents of some common foods calcium intake requirements for pregnancy and lacta- tion. Desirable calcium retention, which is based on Range (mg/100 g) balance data, may be considered a functional indica- Food source Description Ca P tor of nutritional adequacy of calcium in population groups and was used by the US Food and Nutrition Cheese Hard, from milk 400–1200 400–810 Cheese Soft, from milk 60–700 100–790 Board in 1997 to establish recommendations for daily Sardines Tinned, in oil 550 520 calcium intakes. This is based on the concept that Milk Cow’s (3.9, 1.6 and 115–120 92–94 to maximize skeletal strength, optimum bone mass 0.1% fat) Yoghurt Whole milk 160–200 130–170 must be attained through a maximum skeletal calcium Ice cream Dairy 110–130 99–110 reserve. Finally, recent research suggests that bio- Eggs Chicken, raw, whole 57 200 chemical markers of bone turnover that predict bone Chicken, Raw 9–12 190–200 mass changes and fracture risk may be functional duck, turkey indicators of the adequacy of calcium intake. This Beef, mutton, Raw 7–10 60–200 requires more investigation. pork Cod, plaice, Raw 16–51 170–180 Requirements and dietary sources whiting Wheat fl our Whole fl our 38 320 Milk and milk products are the most important Wheat fl our White fl our 15–140 110–120 dietary sources of calcium for most people in Western Bread White 100–180 79–120 Bread Brown 100–140 150–180 countries, with cereal products and fruits and vegeta- Spinach Raw 170 45 bles each making a much smaller contribution (Table Watercress Raw 170 52 9.2). For example, the contribution of dairy products Broccoli Green, raw 56 87 Peas Processed, canned 33 89 to total calcium intake has been estimated as 73% in Rice Raw, white, polished 18–25 54–67 the Netherlands, 51–52% in Germany, 51–52% in the Potatoes Raw 5–6 34–37 USA, 57% in the UK and 44% in Ireland. Tinned fi sh, Soyabean, steamed, or 510–1480 95–270 such as sardines, are rich sources of calcium but do fried not make a signifi cant contribution to intake for most Data from Holland et al. (1995). Reproduced with permission from people. In general, foods of plant origin are not very HMSO. rich sources of calcium. However, owing to the level of consumption, foods of plant origin make a signifi - Calcium requirements, therefore, vary throughout an cant contribution to total calcium intake. For example, individual’s life, with greater needs during the periods in the USA, cereals contribute about 25–27% of total of rapid growth in childhood and adolescence, during calcium intake, whereas in the UK cereals contribute pregnancy and lactation, and in later life. There are about 25% of total calcium intake with about 14% important genetic and environmental infl uences of from bread because of calcium fortifi cation of white calcium requirements. Genetic infl uences include fl our. Increased availability of calcium-fortifi ed foods such factors as bone architecture and geometry, and and dietary supplements containing calcium salts is responsiveness of bone to hormones that mediate the leading to a wider range of rich dietary sources of function of bone as the body’s calcium reserve. calcium. Contributions from nutritional supplements Environmental infl uences include factors such as or medicines may be signifi cant for some people. dietary constituents and the degree of mechanical Given the high proportion of body calcium which is loading imposed on the skeleton in everyday life. present in bone, and the importance of bone as the Because of their effects on urinary calcium losses, major reservoir for calcium, development and main- high intakes of both sodium and protein increase tenance of bone is the major determinant of calcium dietary calcium requirements. needs. Thus, unlike other nutrients, the requirement There is considerable disagreement over human for calcium is considered to relate not to the mainte- calcium requirements, and this is refl ected in the wide nance of the metabolic function of the nutrient but variation in estimates of daily calcium requirements to the maintenance of an optimal reserve and the made by different expert authorities. For example, support of the reserve’s function (i.e., bone integrity). expert committees in the USA and the EU have 194 Introduction to Human Nutrition

Table 9.3 Recommended calcium intakes in the USA and UK balance in humans and may increase the zinc require-

a b ment. Overall, the available data on the interaction UK RNI (1998) US AI (1997) of calcium with these nutrients do not show any Age group (years) mg/day Age group (years) mg/day clinically or functionally signifi cant depletion of the affected nutrient in humans and, in the context of risk 0–1 525 0–0.5 210 assessment, these interactions should probably not be 1–3 350 0.5–1 270 4–6 450 1–3 500 considered adverse effects of calcium. However, such 7–10 550 4–8 800 interactions deserve further investigation. It is well 11–14 M 1000 9–13 1300 established that a defi ciency of vitamin D (arising 15–18 M 1000 14–18 1300 11–14 F 800 19–30 1000 from a lack of exposure to sunlight, inadequate dietary 15–18 F 800 31–50 1000 intake, or both) can result in a reduced effi ciency of 19–50 700 51–70 1200 intestinal calcium absorption that, in turn, can lead >50 700 >70 1200 Pregnancy to a decrease in serum ionized calcium. Pregnancy NI ≤18 1300 19–50 1000 Lactation +550 Lactation 9.3 Magnesium ≤18 1300 19–50 1000 Like calcium, magnesium is an alkaline earth metal. Magnesium is the eighth most abundant element in a Reference nutrient intake (RNI); UK Department of Health (1991). b Adequate intake (AI); US Institute of Medicine (1997). the Earth’s crust. It does not occur uncombined, but Estimates of Ca requirements refer to both males and females unless is found in large deposits in the form of magnesite, stated otherwise. dolomite, and other minerals. The metal is used in M, requirements for males; F, requirements for females; NI, no fl ashlight photography, fl ares, and pyrotechnics. It increment. is one-third lighter than aluminum, and in alloys is essential for airplane and missile construction. established very different recommendations for Magnesium is used in producing nodular graphite in calcium intake (Table 9.3). Much of this divergence cast iron and as an additive to conventional propel- arises because of different interpretations of available lants. The hydroxide (milk of magnesia), chloride, human calcium balance data. The higher recommen- sulfate (Epsom salts), and citrate are used in dations in the USA derive from defi ning calcium medicine. requirements based on desirable calcium retention Magnesium was fi rst shown to be an essential estimated from human calcium balance studies, i.e., dietary component for rats in 1932 and later for that which results in the maximum skeletal calcium humans. This essentiality is a refl ection of the role reserve. that magnesium plays in the stabilization of ATP and other molecules. Since then, nutritionists have come Micronutrient interactions to realize that frank magnesium defi ciency is rare and There is considerable evidence from studies on exper- that it only occurs in clinical settings as a secondary imental animals that excessive calcium intake can consequence of another disease. More recently, mod- impair the nutritional status of other nutrients, erate or marginal defi ciency has been proposed as a particularly iron, zinc, and magnesium, but data on risk factor for chronic diseases such as osteoporosis, humans are not clear. While calcium interacts with cardiovascular disease, and diabetes. These associa- magnesium and phosphorus, and reduces their tions are controversial. absorption, there is no evidence that high calcium intakes are associated with depletion of the affected Absorption, transport and nutrient. Calcium inhibits the absorption of iron in a tissue distribution dose-dependent and dose-saturable fashion. However, Magnesium is the second most common cation found the available human data fail to show cases of iron in the body (about 25 g). It is evenly distributed defi ciency or even decreased iron stores as a result of between the skeleton (50–60% of total) and the soft high calcium intake. There is some evidence that high tissues (40–50% of total). In the skeleton, about one- dietary calcium intakes reduce zinc absorption and third of the magnesium is on the surface of bone. This Minerals and Trace Elements 195 magnesium pool is thought to be exchangeable and Metabolic function and essentiality thus may serve to maintain serum or soft-tissue Magnesium is essential for a wide range of funda- magnesium concentrations in times of need. Body mental cellular reactions, and is involved in at least magnesium is most closely associated with cells; 300 enzymic steps in intermediary metabolism, for only 1% of total body magnesium is extracellular. example in the glycolytic cycle converting glucose to Within the cell, magnesium is found in all of the pyruvate, in β-oxidation of fatty acids, and in protein compartments. synthesis. Magnesium plays an important role in Magnesium homeostasis is maintained by control- the development and maintenance of bone; about ling the effi ciency of intestinal absorption and mag- 60% of total body magnesium is present in bone. nesium losses through the urine. The latter process is Magnesium has also been demonstrated to enhance a stronger regulatory control mechanism for magne- the condensation of chromatin, and given the role of sium. Magnesium absorption is presumed to occur chromosomal condensation in the regulation of gene throughout the small intestine of humans. In normal, activity, magnesium depletion could indirectly affect healthy individuals, magnesium absorption is between gene transcription. 20% and 70% of magnesium in a meal. Magnesium crosses the by three different Defi ciency symptoms mechanisms: passive diffusion, solvent drag (i.e., fol- Magnesium homeostasis can be maintained over a lowing water movement) and active transport. Regu- wide range of intakes in normal, healthy individuals. lation of intestinal nutrient absorption is generally Thus, magnesium defi ciency does not appear to be a thought to occur only for the active component of problem in healthy people. Frank magnesium defi - absorption. The mechanisms controlling intestinal ciency is only seen in humans under two conditions: magnesium absorption are unclear at this time. as a secondary complication of a primary disease state Because of the chemical similarity of magnesium to (diseases of cardiovascular and neuromuscular func- calcium, scientists have examined whether vitamin D tion, endocrine disorders, malabsorption syndromes, status regulates magnesium absorption. It appears muscle wasting) and resulting from rare genetic that only large changes in vitamin D status lead to abnormalities of magnesium homeostasis. Symptoms alterations in magnesium absorption. Only limited of frank magnesium defi ciency include: information is available on the infl uence of dietary components on magnesium in humans. Phosphate ● progressive reduction in plasma magnesium (10– may be an inhibitor of magnesium absorption. Free 30% below controls) and red blood cell magnesium phosphate may form insoluble salt complexes with (slower and less extreme than the fall in plasma magnesium; phosphate groups in phytate may also magnesium) inhibit magnesium absorption. Fiber-rich foods ● hypocalcemia and hypocalciuria have been shown to lower magnesium bioavailability. ● hypokalemia resulting from excess potassium excre- However, it is not clear whether this was an indepen- tion and leading to negative potassium balance dent effect of fi ber or a refl ection of the phytate ● abnormal neuromuscular function. content of these foods. Protein and fructose may enhance magnesium absorption. All of these symptoms are reversible with dietary mag- As mentioned above, the kidney is the principal nesium repletion. Disrupted calcium metabolism is organ involved in magnesium homeostasis. The renal also evident from the effect of magnesium depletion handling of magnesium in humans is a fi ltration– on serum PTH and 1,25(OH)2D3 concentrations. reabsorption process. Approximately 70% of serum Scientists have attempted to demonstrate that magnesium is ultrafi ltrable, and the normal healthy suboptimal intake of magnesium [e.g., below the rec- kidney reabsorbs about 95% of fi ltered magnesium. ommended dietary allowance (RDA) but not frank When an individual is fed a low-magnesium diet, defi ciency] is a contributor to the development of renal output of magnesium is reduced. Excessive chronic maladies such as cardiovascular disease, magnesium loss via urine is a clinical condition con- diabetes mellitus, hypertension, eclampsia and pre- tributing to magnesium depletion in patients with eclampsia, and osteoporosis. However, the results of renal dysfunction. studies in this area are ambiguous. The lack of 196 Introduction to Human Nutrition positive fi ndings may refl ect the lack of sensitive and It has been proposed that Bartter’s syndrome is reliable tools for assessing magnesium status, the a heterogeneous entity with at least two subsets, failure to account for magnesium intake from water Gitelman’s syndrome and “true” Bartter’s syndrome. (in dietary studies), or the diffi culty in attributing True Bartter’s syndrome, a hypokalemic alkalosis with causality to a single nutrient owing to the apparent hypercalciuria, is caused by mutation in the NaK2Cl heterogeneity of causes arising from epidemiological cotransporter gene SLC12A1 on chromosome 15. data relating to most chronic diseases. The fact that Bartter’s syndrome is also caused by mutations in the in 1997 the US RDA for magnesium was raised for K(+) channel gene ROMK on chromosome 11. Gitel- most groups is a refl ection that nutrition scientists man’s syndrome, a hypokalemic alkalosis with hypo- believe that there is a negative consequence to sub- calciuria and hypomagnesemia, is caused by mutations optimal magnesium intake. Additional research is in the thiazibesensitive NaCl cotransporter gene on needed to justify this concern. 16q13.

Toxicity Assessing status Magnesium, when ingested as a naturally occurring Estimating magnesium requirements and establish- substance in foods, has not been demonstrated to ing magnesium–disease relationships depend on exert any adverse effects in people with normal renal accurate and specifi c indicators of magnesium status. function. However, adverse effects of excess magne- Several such indicators have been described. All of sium intake (e.g., diarrhea, nausea, abdominal cramp- these are based on measurement of the magnesium ing) have been observed with intakes from nonfood content in various body pools. Analysis of total mag- sources such as various magnesium salts used for nesium in serum is often used as an indicator of mag- pharmacological purposes. For this reason the US nesium status, although only about 1% of total body Food and Nutrition Board established the tolerable magnesium is present in ECF. It has been suggested UL for adolescents and adults as 350 mg of nonfood that the concentration of ionized magnesium in magnesium. serum may be a more reliable and relevant determi- nant of magnesium defi ciency. In addition, intracel- Genetic diseases lular magnesium concentration (usually measured in Several disease states are associated with magnesium accessible tissues such as erythrocytes and lympho- defi ciency, some of which have genetic roots, for cytes) provides a more accurate assessment of body example hypomagnesemia with secondary hypocal- magnesium status than does the concentration of cemia, primary hypomagnesemia with hypercalciuria, magnesium in serum. The dietary balance approach renal hypomagnesemia 2, Bartter’s syndrome, and is considered to be the best available method for Gitelman’s syndrome. estimating magnesium requirements. Although this Primary hypomagnesemia with hypercalciuria is method is a powerful research tool for the study of caused by a mutation in the paracellin-1 (PCLN1) magnesium homeostasis, it is time, , and gene on chromosome 3. PCLN1 is a component of the labor intensive, and these limit its application to large tight junction complex in and, therefore, populations. None of the currently available proce- has a role in renal magnesium ion reabsorption. dures is perfect for all circumstances. Hypomagnesemia with secondary hypocalcemia is an autosomal recessive disorder and is determined by a Requirements and dietary sources mutation in a gene located on 9q12–q22.2. In 1997, the US RDA [the nutrient intake value that Renal hypomagnesemia 2 is believed to be due to is suffi cient to meet the requirement of nearly all an autosomal dominant isolated renal magnesium (97–98%) individuals in a life-stage and sex group] loss, which is caused by misrouting of the Na+/K(+)- for magnesium was revised upwards for most groups. ATPase gamma-subunit. This small, type I membrane The current RDA for adult women is now 320 mg/day protein is localized on the basolateral membranes of and for adult men is 420 mg/day. An additional value epithelial cells and is expressed in the distal is now part of the US Food and Nutrition Board’s convoluted tubule, the main site of active renal mag- dietary reference intakes, the estimated average nesium reabsorption. requirement (EAR; the nutrient intake value that is Minerals and Trace Elements 197 estimated to meet the requirement of 50% of the appear to be a signifi cant barrier to achieving ade- individuals in a life-stage and sex group). This esti- quate magnesium status. Thus, the current recom- mate was set to 265 and 350 mg/day for adult women mendations for a based on the food and men, respectively. This value is similar to the pyramid are consistent with the goals of reaching the mean magnesium intake reported for women and US RDA for magnesium. men in the USA (228 and 323 mg/day). Collectively, these data suggest that most Americans are not con- Micronutrient interactions suming enough magnesium in their diet and this also As mentioned above, phosphorus as phosphate, espe- appears to be the case for several European popula- cially in phytate, may decrease intestinal magnesium tions. However, while the public health relevance of absorption. In general, calcium intake in the usual this observation is currently being debated, the fact dietary range does not affect magnesium absorption, that there is not a universally accepted reliable mag- but calcium intakes in excess of 2.6 g have been nesium status assessment tool makes it diffi cult to reported to reduce magnesium balance. Magnesium determine the actual consequence of this apparent intake in the usual dietary range does not appear to low intake. alter calcium balance. For those who want to increase their magnesium intake, a number of high magnesium foods and 9.4 Phosphorus dietary practices will lead to adequate intake. Foods with a high magnesium content include whole grains, Phosphorus is never found free in nature, but is legumes, green leafy vegetables, and tofu; meat, fruits, widely distributed in combination with minerals. and dairy products have an intermediate magnesium Phosphate rock, which contains the mineral apatite, content (Table 9.4). The poorest sources of magne- an impure tricalcium phosphate, is an important sium are refi ned foods. Although high levels of source of the element. Phosphorus is most commonly calcium, phosphate, or fi ber may lead to reduced bio- found in nature in its pentavalent form in combina- 3− availability of magnesium, differences in bioavailabil- tion with oxygen as phosphate (PO4 ). Phosphorus ity of magnesium from various food sources does not (as phosphate) is an essential constituent of all known protoplasm and is uniform across most plant and animal tissues. A practical consequence is that, as Table 9.4 Magnesium content of some common foods organisms consume other organisms lower in the Mg content food chain (whether animal or plant), they automati- Food source Description (mg/100 g) cally obtain their phosphorus.

Beef Lean (from six different cuts) 20 Absorption, transport, and Lamb Lean (from six different cuts) 24 tissue distribution Pork Lean (from three different cuts) 22 Chicken Raw, meat only 25 Phosphorus makes up about 0.65–1.1% of the adult Cod, plaice, Raw 22–28 body (~600 g). In the adult body 85% of phosphorus whiting is in bone and the remaining 15% is distributed in Eggs Chicken, whole, raw 12 Cheese Soft and hard varieties 8–45 soft tissues. Total phosphorus concentration in whole Pulses Raw 17–250 blood is 13 mmol/l, most of which is in the phospho- Wheat fl our Whole fl our 120 lipids of erythrocytes and plasma lipoproteins, with Wheat fl our White fl our 20–31 approximately 1 mmol/l present as inorganic phos- Milk Cow’s (3.9, 1.6 and 0.1% fat) 11–12 phate. This inorganic component, while constituting Yoghurt Whole milk 19 Tofu Soyabean, steamed 23–59 only a minute percentage of body phosphorus Green leafy Raw 8–34 (<0.1%), is of critical importance. In adults, this com- vegetables ponent makes up about 15 mmol in total and is Rice Raw, white, polished 32 located mainly in the blood and ECF. It is into the Potatoes Raw 14–17 inorganic compartment that phosphate is inserted on Data from Holland et al. (1995). Reproduced with permission from absorption from the diet and resorption from bone, HMSO. and from this compartment that most urinary 198 Introduction to Human Nutrition phosphorus and hydroxyapatite mineral phosphorus absorption is reduced by ingestion of aluminum- are derived (Figure 9.4). This compartment is also the containing antacids and by pharmacological doses of primary source from which the cells of all tissues calcium carbonate. There is, however, no signifi cant derive both structural and high-energy phosphate. interference with phosphorus absorption by calcium Food phosphorus is a mixture of inorganic and at intakes within the typical adult range. Excretion of organic forms. Intestinal phosphatases hydrolyze the endogenous phosphorus is mainly through the organic forms contained in ingested protoplasm and, kidneys. Inorganic serum phosphate is fi ltered at the thus, most phosphorus absorption occurs as inor- glomerulus and reabsorbed in the proximal tubule. In ganic phosphate. On a mixed diet, absorption of total the healthy adult, urine phosphorus is essentially phosphorus ranges from 55% to 70% in adults. There equal to absorbed dietary phosphorus, minus small is no evidence that this absorption varies with dietary amounts of phosphorus lost in shed cells of skin and intake. Furthermore, there appears to be no apparent intestinal mucosa. adaptive mechanism that improves phosphorus absorption at low intakes. This situation is in sharp Metabolic function and essentiality contrast to calcium, for which absorption effi ciency Structurally, phosphorus occurs as hydroxyapatite in increases as dietary intake decreases and for which calcifi ed tissues and as phospholipids, which are a adaptive mechanisms exist that improve absorption major component of most biological membranes, still further at habitual low intakes. While a portion and as nucleotides and nucleic acid. Other functional of phosphorus absorption is by way of a saturable, roles of phosphorus include: active transport facilitated by 1,25(OH)2D3 the fact that fractional phosphorus absorption is virtually ● buffering of acid or alkali excesses, hence helping to constant across a broad range of intakes suggests that maintain normal pH the bulk of phosphorus absorption occurs by passive, ● the temporary storage and transfer of the energy concentration-dependent processes. Phosphorus derived from metabolic fuels

1400 mg GIT Diet

PTH Feces 200 mg 500 mg 1100 mg

Ca2+ Parathyroids

Plasma phosphorus (3.5 mg/100 ml) Kidney 7000 mg 5000 mg

+

5000 mg 6100 mg

Bone

900 mg Figure 9.4 Homeostatic regulation of serum phosphorus. PTH, parathyroid hormone. Minerals and Trace Elements 199

● by phosphorylation, and hence activation of many ● a suggestion that high phosphorus intakes could catalytic proteins. decrease calcium absorption by complexing calcium in the chyme. As phosphorus is not irreversibly consumed in these processes and can be recycled indefi nitely, the actual Concern about high phosphorus intake has been functions of dietary phosphorus are fi rst to support raised in recent years because of a probable popula- tissue growth (either during individual development tion level increase in phosphorus intake through or through pregnancy and lactation), and second to such sources as cola beverages and food phosphate replace excretory and dermal levels. In both processes, additives. it is necessary to maintain a normal level of inorganic phosphate in the ECF, which would otherwise be Genetic diseases depleted of phosphorus by growth and excretion. Several disease states are associated with phosphorus defi ciency, some of which have genetic roots, for Defi ciency symptoms example X-linked hypophosphatemia, hypophospha- Inadequate phosphorus intake is expressed as hypo- temic bone disease, and Fanconi’s syndrome. phosphatemia. Only limited quantities of phosphate X-linked hypophosphatemia is, as the name implies, are stored within cells, and most tissues depend on inherited as an X-linked dominant trait with the ECF inorganic phosphate for their metabolic phos- mutant gene located in Xp22.2–p.22.1. The classical phate. When ECF inorganic phosphate levels are triad, fully expressed in hemizygous male patients, low, cellular dysfunction follows. At a whole organ- consists of: ism level, the effects of hypophosphatemia include anorexia, anemia, muscle weakness, bone pain, rickets ● hypophosphatemia and osteomalacia, general debility, increased suscep- ● lower limb deformities tibility to infection, paresthesia, ataxia, confusion, and ● rate. even death. The skeleton will exhibit either rickets in Although low serum phosphate is evident early after children or osteomalacia in adults. In both groups, birth, it is only at the time of weight bearing that the disorder consists of a failure to mineralize forming leg deformities and progressive departure from the growth plate cartilage or bone matrix, together with normal growth rate become suffi ciently striking to impairment of chrondroblast and osteoblast func- attract attention and make parents seek medical tion. These severe manifestations are usually confi ned opinion. It is generally accepted that hypophosphate- to situations in which ECF phosphate falls below mia is the consequence of a primary inborn error of approximately 0.3 mmol/l. Phosphorus is so ubiqui- phosphate transport, probably located in the proxi- tous in various foods that near total starvation is mal nephron. required to produce dietary phosphorus defi ciency. Hypophosphatemic bone disease is characterized Toxicity clinically by modest shortening of stature, bowing of the lower limbs, and nonrachitic bone changes (some- Serum inorganic phosphate rises as total phosphorus what resembling metaphyseal chondrodysplasia) intake increases. Excess phosphorus intake from any and biochemically by hypophosphatemia. Although a source is expressed as hyperphosphatemia and, essen- defect in renal transport of phosphate was demon- tially, all the adverse effects of phosphorus excess are strated, the defect appeared to be different from that owing to the elevated inorganic phosphate in the ECF. of X-linked hypophosphatemia. The principal effects that have been attributed to Fanconi’s syndrome is an autosomal dominant dis- hyperphosphatemia are: order. It is characterized by lactic aciduria and tubular ● adjustments in the hormonal control system regu- proteinuria in childhood, with glycosuria and amino- lating the calcium economy aciduria developing in the second decade and osteo- ● ectopic (metastatic) calcifi cation, particularly of the malacia from the start of the fourth decade. Glomerular kidney function deteriorates slowly but is compatible with a ● in some animal models, increased porosity of the normal lifespan. There has been reported linkage of skeleton the disorder to chromosome 15q15.3. 200 Introduction to Human Nutrition

Assessing status, requirements and about 80% of the dissolved matter in seawater. dietary sources Although there is a wide variety of sodium salts, many Previously, dietary phosphorus recommendations of which are used as additives in food processing (e.g., have been tied to those for calcium, usually on an sodium nitrate and monosodium glutamate), sodium equimass or equimolar basis, and this approach was chloride is the major source of sodium in foods. As used in the USA, EU, and UK in establishing recom- sodium and chloride intakes in humans are so closely mended dietary allowances, population reference matched, both will be considered together in this intakes, and reference nutrient intakes, respectively, text. for phosphorus. However, in 1997 the US Food and Salt was of major importance in early civilizations Nutrition Board suggested that a calcium–phospho- and in prehistory. Humans have special taste and salt rus concept of defi ning phosphorus requirements is appetite systems, which led to special culinary uses for of severely limited value, in that there is little merit in salt and made it a much sought-after commodity. having the ratio “correct” if the absolute quantities Nowadays, salt is still used widely to modify fl avor, to of both nutrients are insuffi cient to support optimal alter the texture and consistency of food, and to growth. Therefore, because the phosphorus intake control microbial growth (Table 9.5). directly affects serum inorganic phosphate, and Absorption, transport and because both hypophosphatemia and hyperphospha- tissue distribution temia directly result in dysfunction or disease, the US Food and Nutrition Board considered that the most Sodium is the major extracellular electrolyte and logical indicator of nutritional adequacy of phospho- exists as the fully water-soluble cation. Chloride is rus intake in adults is inorganic phosphate. If serum also mainly found in ECF and is fully water soluble inorganic phosphate is above the lower limits of as the chloride anion. Both ions are readily absorbed normal for age, the phosphorus intake may be con- from the digestive tract. Glucose and anions such as sidered adequate to meet cellular and bone formation citrate, propionates, and bicarbonate enhance the needs of healthy individuals. Current US RDAs for uptake of sodium. The “average” 70 kg male has about phosphorus are infants 100 mg (fi rst 6 months), 90 g of sodium with up to 75% contained in the 275 mg (7–12 months), children 460 mg (1–3 years), mineral apatite of bone. Plasma sodium is tightly 500 mg (4–8 years), 1250 mg (9–18 years), adults regulated through a hormone system, which also 700 mg, pregnant women 1250 mg (<18 years), regulates water balance, pH, and osmotic pressure. 700 mg (19–50 years), and lactating women 1250 mg (<18 years), 700 mg (19–50 years). Phosphates are found in foods as naturally occur- Table 9.5 Sodium-containing additives used in food processing ring components of biological molecules and as food additives in the form of various phosphate salts. The Additive Use phosphorus density of cow’s milk and other dairy Sodium citrate Flavoring, preservative produce is higher than that of most other foods in a Sodium chloride Flavoring, texture, typical diet (Table 9.2). The same is true for diets high preservative in colas and a few other soft drinks that use phos- Sodium nitrate Preservative, color fi xative Sodium nitrite Preservative, color fi xative phoric acid as an acidulant. Sodium tripoliphosphate Binder Sodium benzoate Preservative Micronutrient interactions Sodium eritrobate Antioxidant Sodium propionate Preservative It has been reported that intakes of polyphosphates, Monosodium glutamate Flavor enhancer such as are found in food additives, can interfere with Sodium aluminosilicate Anticaking agent the absorption of iron, copper, and zinc. Sodium aluminum phosphate acidic Acidity regulatory, emulsifi er Sodium cyclamate Artifi cial sweetener 9.5 Sodium and chloride Sodium alginate Thickener and vegetable gum Sodium is the sixth most abundant element in the Sodium caseinate Emulsifi er Sodium bicarbonate Yeast substitute Earth’s crust and salt (sodium chloride) makes up Minerals and Trace Elements 201

Angiotensin and both act to conserve Defi ciency symptoms sodium by increasing sodium reabsorption by the Obligatory losses of sodium are very low, and low kidney. Sodium depletion stimulates the renal pro- plasma sodium or chloride depletion is diffi cult to duction of , which generates active angiotensin induce. Low plasma sodium or chloride is not diet in the circulation. The latter stimulates vasoconstric- related but rather caused by a variety of clinical tion, which increases blood pressure, decreases water conditions, including major trauma and cachexia loss, and stimulates aldosterone release from the and overuse of diuretics. Loss of sodium can also adrenal cortex. Atrial natriuretic hormone counter- ensue as a result of excessive water intake, anorexia acts the sodium retention mechanisms by suppressing nervosa, ulcerative colitis, liver disease, congestive renin and aldosterone release and by inducing water heart failure with edema, and severe infection and and sodium excretion. It also decreases blood pres- diarrhea. Acute diarrhea is the most common cause sure and antagonizes angiotensin. A raised plasma of sodium defi ciency, and oral rehydration depends sodium concentration stimulates the renal reabsorp- on the effi cient enteric uptake of sodium from iso- tion of water and decreases urinary output via tonic glucose/saline solutions and saves many lives antidiuretic hormone from the posterior pituitary. In worldwide. Vomiting, chronic renal disease, renal contrast to sodium, chloride is passively distributed failure, and chronic respiratory acidosis can result in throughout the body and moves to replace anions lost chloride depletion. to cells via other processes. The main excretory route for both sodium and chloride is the urine. Sweat loss of these ions tends Toxicity to be very low except with severe exertion in hot Excessive salt intakes are usually excreted effi ciently in climates. Fecal losses are also low in healthy healthy individuals, whereas high plasma sodium and individuals. chloride are commonly caused by diabetes insipidus, brainstem injury, and dehydration through either Metabolic function and essentiality excessive sweating or defi cient water intake. Excessive The sodium cation is an active participant in the reg- salt intake may have roles in the degenerative diseases ulation of osmotic and electrolyte balances, whereas of coronary heart disease, stroke, gastric cancer, osteo- the chloride anion is a passive participant in this regu- porosis, and bronchial hyperactivity. There are latory system. Each ion, however, has other functions accumulating data from epidemiological studies and within the body. controlled clinical trials to indicate an adverse effect Sodium is involved in nerve conduction, active cel- of sodium intake on blood pressure, and that most lular transport and the formation of mineral apatite people are sodium sensitive. It now appears that low- of bone. Central to its role in water balance, nerve ering this intermediate or surrogate measure (blood conduction, and active transport is the plasma mem- pressure) of disease can be translated into reduced brane enzyme sodium–potassium-ATPase (Na+/K+- morbidity and mortality of cardiovascular disease ATPase). This enzyme pumps sodium out of the cell from long-term follow-up assessed 10–15 years after and at the same time returns potassium to the intra- the original dietary sodium reduction trials. The cellular environment while ATP is hydrolyzed. Signal mechanism linking salt intake with blood pressure is transmission along nerve cells, active transport of unclear but probably relates to sodium homeostasis. nutrients into the enterocyte and muscle contraction/ It has been suggested that extracellular sodium relaxation all depend on the Na+/K+-ATPase pump. In concentrations may adversely affect vascular reac- the muscle there is an additional pump, the sodium– tivity and growth and stimulate myocordial fi brosis. calcium system. The ATP utilized by the sodium Low-sodium diets differ in nutrient composition pump makes up a substantial part of the total meta- from the prevailing diet, and animal experimentation bolic activity and thermogenesis. indicates that low potassium or calcium intake Among the main functions of the chloride anion encourages a salt-induced increase in blood pres- are as dissociated hydrochloric acid in the stomach sure, as does feeding simple carbohydrates (sucrose, and in the chloride shift in the erythrocyte plasma glucose, or fructose). Copper defi ciency in rats has membrane, where it exchanges with the bicarbonate been demonstrated to increase blood pressure ion. independently of sodium intake. Epidemiological and 202 Introduction to Human Nutrition other studies indicate that heavy metals, such as lead Salt intakes are notoriously diffi cult to measure, and mercury, may also contribute to increased blood and urinary sodium excretion is considered to be a pressure. valid measure of sodium intake under circumstances Effi cient sodium conservation mechanisms mean where little sodium is lost in sweat. Sodium in urine is that current sodium intakes in many populations are easily measured, but the collection of complete 24 h unnecessarily high and are probably much higher urinary samples is diffi cult because of subject com- than the generally lower sodium diets eaten during pliance, and the completeness of these collections the long period of human evolution. Clinical studies should be validated using a marker such as para-amino indicate that a high-sodium diet increases calcium benzoic acid. Lithium (as carbonate) fused with excretion and measures of bone resorption, thereby sodium chloride can act as a reliable tracer to estimate suggesting a possible role for high salt intakes in discretionary salt (cooking and table) intakes. osteoporosis. Cross-cultural epidemiology suggests that high salt Requirements and dietary sources intakes are associated with gastric cancer, whereas a Average requirements for sodium and chloride are low-salt diet is regarded as having a potentially favor- estimated to be about 500 and 750 mg/day, respec- able effect in asthma patients. tively. Normal sodium (mostly from salt) intake varies from about 2 g/day to 14 g/day, with chloride Genetic diseases (mostly from salt) intakes generally slightly in excess A number of rare genetic disorders have thrown some of sodium (Table 9.6). Snack and processed foods light on the pathological mechanisms linking sodium have more added salt than unprocessed foods. The balance and hypertension (pathologically elevated amount of discretionary salt added in cooking or at blood pressure). A number of candidate genes have the table appears to vary greatly among individuals been identifi ed in monogenic forms of low renin salt- and among countries. Discretionary salt intakes can sensitive hypertension. These encode for enzymes vary from less than 10% to 20–30% of total salt intake involved in aldosterone biosynthesis or cortisol and these fi gures emphasize the major effect of pro- metabolism and for the epithelial sodium channel. cessed foods on total salt intakes in most populations These genetic defects decrease the ability of the renal (Table 9.7). tubules to excrete sodium. It is possible that similar genetic mechanisms operate in more common forms Micronutrient interactions of hypertension such as essential hypertension and The major interactions between sodium (and chlo- especially salt-sensitive hypertension. Moreover, ride) and other micronutrients are with respect to molecular mechanisms associated with renin–angio- potassium and calcium. Data from animals (and some tensin–aldosterone are central to the pathophysiology clinical studies) indicate that dietary potassium and of this condition. Common essential hypertension, calcium potentiate increases in blood pressure in salt- however, is complex and heterogeneous and has a sensitive experimental models. There is evidence to genetic heritability of about 30%. suggest that the sodium to potassium ratio correlates more strongly with blood pressure than does either Assessing status nutrient alone. As indicated previously, the metabo- The tight regulation of plasma sodium and, in turn, lism of sodium, chloride, and potassium is closely chloride ensures that fl uctuations in the plasma related, and sodium and calcium ions have a close concentration of these ions are minimized and metabolic relationship within cells. changes only occur in certain pathological circumstances. Measurements of plasma sodium, 9.6 Potassium therefore, are of little consequence as far as nutri- tional status is concerned. Total body (excluding Potassium, sodium, and chloride make up the princi- bone) sodium, however, is increased in malnutrition pal electrolytes within the body. In contrast to sodium and trauma and this total exchangeable sodium and chloride, nutritional concerns with potassium can be measured, with some technical diffi culty, using are mainly concerned with the possibility of radioisotopes. underconsumption. Minerals and Trace Elements 203

Table 9.6 Salt intake as NaCl (g/day)

Before 1982 Year Intake From 1988 Year Intake

Communities not using added salt Brazil (Yanomamo Indian) 1975 0.06 New Guinea (Chimbus) 1967 0.40 Solomon Islands (Kwaio) 1.20 Botswana (Kung bushmen) 1.80 Polynesia (Pukapuka) 3.60 Alaska (Eskimos) 1961 4.00 Marshall Islands in the Pacifi c 7.00 Salt-using communities Kenya (Sambura nomads) 5–8 Mexico (Tarahumsa Indian) 3–10 Mexico (Tarahumsa Indian) 1978 5–8 Mexico, rural (Nalinalco) 1992 5.7 Mexico, urban (Tlaplan) 1991 7.18 Denmark 9.8 Denmark 1988 8.00 Canada (Newfoundland) 9.9 Canada 8–10 New Zealand 10.1 Sweden (Göteborg) 10.2 USA (Evans Country, Georgia) 10.6 USA (Chicago) 7.7 Iran 10.9 Belgium 1966 11.4 Belgium 1988 8.4 UK (Scotland) 11.5 Australia 12.0 India (north) 12–15 India 9–11.4 Federal Republic of Germany 13.1 Finland (east) 14.3 Finland 10.6 Bahamas 15–30 Kenya (Samburus, army) 1969 18.6 Korea 19.9 Japan Japan (farmers) 1955 60.3 Japan 1988 8–15 Japan (Akita) 27–30 Japan 1964 20.9

Absorption, transport, and and other factors regulate potassium homeostasis, tissue distribution both within cells and with the external environment. Potassium is the major intracellular electrolyte and Hyperkalemia (too much potassium in the ECF) exists as the fully water-soluble cation. More than stimulates insulin, aldosterone, and epinephrine 90% of dietary potassium is absorbed from the diges- (adrenaline) secretions, which promote the uptake of tive tract. potassium by body cells. The aldosterone hormone Few dietary components affect absorption of potas- also stimulates potassium excretion by the kidney sium, although olive oil can increase and dietary fi ber and, at the same time, conserves sodium. Hypokale- decrease absorption to some extent. The “average” mia has opposite effects, such that more potassium is 70 kg man contains about 120 g of potassium, depend- released from cells. As with sodium, the kidney regu- ing on muscle mass, with men having proportionally lates potassium balance. Urine is the major excretory greater muscle mass, and hence potassium, than route in healthy people, with only small amounts lost women. Almost all of the body potassium is exchange- in the feces and minimal amounts in sweat. able, intracellular concentration being more than 30 times the concentration of the ECF. Potassium is Metabolic function and essentiality distributed within the body in response to energy- Potassium, sodium, and chloride are the major deter- dependent sodium redistribution. Various hormonal minants of osmotic pressure and electrolyte balance. 204 Introduction to Human Nutrition

Table 9.7 Salting (mg/100 g fresh weight) of foods in Western metabolic alterations. Potassium supplementation societies may have a role to play in treating chronic heart failure, and increased potassium intakes can decrease blood Na K Ca Mg pressure via antagonistic metabolic interactions with Maize-based products sodium, resulting in increased sodium excretion, and Corn 4 284 55 41 also via a direct vasodilatory effect. Oral administra- Tortilla, rural 11 192 177 65 tion of potassium salts has been shown to improve Breakfast cereals 866 101 3 11 Processed snacks 838 197 102 56 calcium and phosphorus balance, reduce bone resorp- tion and increase the rate of bone formation. Wheat-based products Natural cereals 39 1166 94 343 Toxicity Tortillas, wheat 622 73 11 17 Breakfast cereals 855 869 81 236 Hyperkalemia, as a result of either a shift of potas- Processed bread (urban) 573 126 47 31 sium from cells to the ECF or excessive potassium Salted bread, made locally (rural) 410 92 10 74 retention, can be caused by major trauma and infec- Sweet bread, made locally (rural) 97 93 87 18 Processed bread (rural) 344 79 213 18 tion, metabolic acidosis, Addison’s disease (aldoste- Processed biscuits 582 80 16 17 rone insuffi ciency) and chronic renal failure. Overuse Pulses of potassium supplements can also result in potas- Unprocessed, cooked 53 373 50 41 sium excess. As with potassium depletion, the most Processed, canned 354 371 27 79 important clinical consequence of potassium excess is cardiac arrest. Reproduced from Sánchez-Castillo and James in Sadler et al. Encyclo- pedia of Human Nutrition, copyright 1999 with permission of Assessing status Elsevier. The plasma concentration of potassium is not a reliable index of whole-body potassium status. Total The concentration difference of potassium and body potassium can be measured by 42K dilution or sodium across cell membranes is maintained by the by whole body counting of the naturally abundant 40K Na+/K+-ATPase pump and is critical for nerve trans- to determine the amount of lean body tissue. More mission and muscle function. The physiological direct measures of tissue potassium can be obtained importance of potassium in the body covers many by muscle biopsies. systems including cardiovascular, respiratory, diges- tive, renal, and endocrine. In addition, potassium is a Requirement and dietary sources cofactor for enzymes involved in inter alia energy Adult requirements for potassium are estimated to be metabolism, glycogenesis, and cellular growth and about 2 g/day. Because of potential benefi cial antago- division. nistic effects against high salt intakes, higher intakes (around 3.5 g/day) of potassium are considered to be Defi ciency symptoms optimal, although chronic intakes above 5.9 g/day The low concentration of potassium in plasma is may be dangerous for individuals with impaired renal tightly regulated. Hypokalemia, however, can result function. Potassium, like sodium and chloride, is from either excessive uptake of potassium by cells or naturally widely distributed in foods (Table 9.8). potassium depletion from the body. Insulin excess, Food processing (through leaching) may decrease catecholamine increases, Cushing’s disease (excess potassium content as well as increasing salt content. steroids), diuretics that enhance potassium loss, Legumes, nuts, , and fresh fruit, especially chronic renal disease, diarrhea, vomiting, and laxative bananas, melons, avocados, and kiwi fruit, are rich abuse can result in hypokalemia. Low potassium sources of potassium. Major vegetable sources of intakes are unlikely to lead to clinical potassium potassium are potatoes and spinach, although cereal depletion and hypokalemia except during starvation and dairy products, which have a lower potassium and anorexia nervosa. content but are consumed in large quantities, are also The activity of nerves and muscles is affected in important dietary sources. In addition, meat and potassium depletion, and other clinical sequelae fi sh contain appreciable quantities of potassium. involve cardiac (including cardiac arrest), renal, and People who eat large quantities of fruit and vegetables Minerals and Trace Elements 205

Table 9.8 Sodium and potassium content of various foods (mg/100 g siderable quantity. The core of the Earth is thought to edible portion) be largely composed of iron and it makes up 4.7% of the Earth’s crust. The most common ore is hematite, Food Na K which is frequently seen as black sands along beaches Legumes and streams. Taconite is becoming increasingly Red kidney beans 18 1370 important as a commercial ore. Because iron is easy Soyabeans 5 1730 to obtain, its discovery is lost in the history of man, Lentils 12 940 many thousands of years ago. The early Greeks were Dried fruit aware of the health-giving properties of iron. Iron has Raisins 60 1020 Figs 62 970 been used for centuries as a health tonic. It is therefore paradoxical that although the need for iron was dis- Nuts covered long ago and although it is the most common Walnuts 7 450 Almonds 14 780 and cheapest of all metals, iron defi ciency is probably the most frequent defi ciency disorder in the world Fruit and vegetables Banana 1 400 and the main remaining nutritional defi ciency in Melon 5–32 100–210 Europe. Iron can exist in oxidation states ranging Potato 11 320 from −2 to +6. In biological systems, these oxidation Spinach 140 500 states occur primarily as the ferrous (Fe2+) and ferric 3+ Meat and fi sh (Fe ) forms and these are interchangeable. Beef, veal, lamb 52–110 230–260 Chicken 81 320 Absorption, transport, and Herring 120 320 tissue distribution Halibut 60 410 Tuna 47 400 The iron content of a typical 70 kg adult man is Mussels 290 320 approximately 4–5 g. Of this content, approximately Miscellaneous two-thirds is utilized as functional iron such as Cow’s milk 55 140 hemoglobin (60%), myoglobin (5%), and various Chocolate 11 300 heme (cytochromes and catalase) and nonheme (NADH hydrogenase, succinic dehydrogenase, aconi- Reproduced from Sánchez-Castillo and James in Sadler et al. Encyclo- tase) enzymes (5%). The remaining iron is found in pedia of Human Nutrition, copyright 1999 with permission of Elsevier. body storage as ferritin (20%) and hemosiderin (10%), the two major iron storage proteins. Only very minor quantities of iron (<0.1%) are found as a transit may have dietary intakes of potassium exceeding chelate with transferrin, the main iron transport 6 g/day. protein in the body. Micronutrient interactions The metabolism of iron differs from that of other minerals in one important respect: there is no phy- As might be expected from the close metabolic inter- siological mechanism for iron excretion. The body has actions among the major electrolytes, potassium and three unique mechanisms for maintaining iron balance sodium dietary interactions may be important in and preventing iron defi ciency and iron overload: determining the risk of coronary heart disease and stroke. Another potentially important interaction ● storage of iron (with ferritin being an important concerns calcium. Potassium appears to have positive reversible storage protein for iron) effects on calcium balance by regulating the acid–base ● reutilization of iron (especially of iron in balance and ameliorating any effects of sodium on erythrocytes) calcium depletion. ● regulation of iron absorption. In theory, therefore, when the body needs more iron, 9.7 Iron absorption is increased, and when the body is iron suffi cient, absorption is restricted. This control is not Iron is a relatively abundant element in the universe. perfect but is still of great importance for the preven- It is found in the sun and many types of stars in con- tion of iron defi ciency and excess. Iron from food is 206 Introduction to Human Nutrition absorbed mainly in the duodenum by an active process Heme iron is absorbed by a different mechanism that transports iron from the gut lumen into the from nonheme iron. The heme molecule is absorbed mucosal cell. When required by the body for meta- intact into the mucosal cell, where iron is released by bolic processes, iron passes directly through the the enzyme heme oxygenase. Its absorption is little mucosal cell into the bloodstream, where it is trans- infl uenced by the composition of the meal, and varies ported by transferrin, together with the iron released from 15% to 35% depending on the iron status of the from old blood cells (i.e., the effi cient iron recycling consumer. Although heme iron represents only 10– system, Figure 9.5), to the bone marrow (80%) and 15% of dietary iron intake in populations with a high other tissues (20%). If iron is not required by the body, meat intake, it could contribute 40% or more of the iron in the mucosal cell is stored as ferritin and is total absorbed iron (Figure 9.6). Many poorer regions excreted in feces when the mucosal cell is exfoliated. of the world consume little animal tissue and rely Any absorbed iron in excess of needs is stored as ferri- entirely on nonheme iron. The absorption of nonheme tin or hemosiderin in the liver, spleen, or bone marrow. iron is strongly infl uenced by dietary components, Iron can be released from these iron stores for utiliza- which bind iron in the intestinal lumen. The com- tion in times of high need, such as during pregnancy. plexes formed can be either insoluble or so tightly bound that the iron is prevented from being absorbed. Alternatively, the complexes can be soluble and iron Absorption of iron absorption is facilitated. Under experimental condi- tions, nonheme iron absorption can vary widely from Plasma: less than 1% to more than 90%, but under more Transferrin iron typical dietary conditions it is usually in the region of 1–20%. The main inhibitory substances and enhanc- Spleen: Bone marrow: ers of iron absorption are shown in Table 9.9. Reticulo-endothelial Iron stores Red blood cell macrophages precursors Metabolic function and essentiality Iron acts as a catalytic center for a broad spectrum of Circulating RBCs metabolic functions. As present in hemoglobin, iron is required for the transport of oxygen, critical for cell Tissue iron respiration. As myoglobin, iron is required for oxygen storage in muscle. Iron is also a component of various Iron losses from body tissue enzymes, such as the cytochromes, that are critical for energy production, and enzymes necessary Figure 9.5 Metabolism of iron. There is a main internal loop with a continuous reutilization of iron and an external loop represented by for immune system functioning. Therefore, these iron losses from the body and absorption from the diet. Adapted from iron-containing molecules ensure that body fuels, Hallberg et al. (1993) with permission of Elsevier. such as carbohydrate, fat, and protein are oxidized to

Heme 10%

Heme 40% Nonheme Nonheme 100% 60% Nonheme 90%

(a) (b) (c) Figure 9.6 Heme and nonheme iron in foods: (a) foods of animal origin; (b) foods of plant origin; (c) dietary iron intake from all foods, daily average. Minerals and Trace Elements 207

Table 9.9 Factors affecting (a) heme and (b) nonheme iron iron is insuffi cient to provide for enough hemoglobin absorption for new erythrocytes and insuffi cient to fulfi ll other physiological functions. During the last stage, free Increased absorption Decreased absorption protoporphyrin, destined for hemoglobin, increases (a) Heme in plasma two- to fi vefold, indicating a lack of tissue Physiological factors iron. The harmful consequences of iron defi ciency Low iron status High iron status occur mainly in conjunction with anemia. Iron defi - Dietary factors ciency anemia is most common in infants, preschool Low heme iron intake High heme iron intake children, adolescents, and women of child-bearing Meat Calcium age, particularly in developing countries. (b) Nonheme The functional effects of iron defi ciency anemia Physiological factors result from both a reduction in circulating hemoglo- Depleted iron status Replete iron status bin and a reduction in iron-containing enzymes and Pregnancy Achlorhydria (low gastric myoglobin. Both factors presumably play a role in the acid) Disease states (aplastic anemia, hemolytic fatigue, restlessness, and impaired work performance anemia, hemochromatosis) associated with iron defi ciency anemia. Other func- Dietary factors tional defects include disturbances in normal ther- Ascorbic acid Phytate moregulation and impairment of certain key steps in Meat, fi sh, seafood Iron-binding phenolic the immune response. For example, there is evidence compounds that iron defi ciency anemia is associated with lower Certain organic acids Calcium T- and B-lymphocyte, macrophage, and neutrophil function. Although the phagocytic uptake of neutro- phils is usually normal, the intracellular killing mech- anism is usually defective. This abnormality is thought to be owing to a defect in the generation of reactive provide the energy necessary for all physiological pro- oxygen intermediates resulting from a decrease in the cesses and movement. The importance of iron as an iron-containing enzyme myeloperoxidase. Iron defi - element necessary for life derives from its redox reac- ciency anemia can also have an adverse effect on psy- tivity as it exists in two stable, interchangeable forms, chomotor and mental development in children, and ferrous (Fe2+) and ferric (Fe3+) iron. This reaction is the mortality and morbidity of mother and infant an essential part of the electron transport chain, during pregnancy. responsible for the generation of ATP during the oxi- dation of substances in intermediary metabolism and for the reductions necessary in the synthesis of larger Toxicity molecules from their components. The very effective regulation of iron absorption pre- vents overload of the tissues by iron from a normal diet, except in individuals with genetic defects, as in Defi ciency symptoms idiopathic hemochromatosis (see below). Excess iron The progression from adequate iron status to iron- via overuse of iron supplements could pose a possible defi ciency anemia develops in three overlapping health risk. The mechanism of cellular and tissue stages. The fi rst stage consists of depletion of storage injury resulting from excess iron is not fully under- iron, which is characterized by a decrease in serum stood. Liabilities may include increased risks for bac- ferritin, which, in turn, refl ects the size of the iron terial infection, neoplasia, arthropathy, cardiomyopa- stores in the liver, bone marrow, and spleen. The thy, and endocrine dysfunctions. However, there is second stage is a decrease in transported iron and is still much debate as to the strength of evidence to characterized by a decline in serum iron and an support a relationship between dietary iron intake increase in the total iron-binding capacity, as transfer- and cancer or cardiovascular disease. rin has more free binding sites than in normal iron Gastrointestinal distress does not occur from status. The third stage develops when the supply of consuming a diet containing naturally occurring or 208 Introduction to Human Nutrition fortifi ed iron. Individuals taking iron at high and, in relation to body weight, they are highest for levels (>45 mg/day) may encounter gastrointes- the young infant. An adult man has obligatory iron tinal side-effects (constipation, nausea, vomiting, losses of around 1 mg of iron/day, largely from the and diarrhea), especially when taken on an empty gastrointestinal tract (exfoliation of epithelial cells stomach. Based largely on the data on gastrointestinal and secretions), skin, and urinary tract. Thus, to effects following supplemental elemental iron intake remain replete with regard to iron, an average adult in apparently healthy adults, the US Food and Nutri- man needs to absorb only 1 mg of iron from the diet tion Board established a tolerable UL of iron of on a daily basis. Similar obligatory iron losses for 45 mg/day. women amount to around 0.8 mg/day. However, adult women experience additional iron loss owing Genetic diseases to , which raises the median daily iron Primary idiopathic hemochromatosis is a hereditary requirement for absorption to 1.4 mg (this covers disorder of iron metabolism characterized by an 90% of menstruating women; 10% will require daily abnormally high iron absorption owing to a failure of absorption of at least 2.4 mg iron to compensate for the iron absorption control mechanism at the intes- their very high menstrual losses). Pregnancy creates tinal level. High deposits of iron in the liver and the an additional demand for iron, especially during the heart can lead to cirrhosis, hepatocellular cancer, con- second and third trimesters, leading to daily require- gestive heart failure, and eventual death. Sufferers of ments of 4–6 mg. Growing children and adolescents this disorder can develop iron overload through con- require 0.5 mg iron/day in excess of body losses to sumption of a normal diet, but would be at much support growth. Physiological iron needs can be higher risk if consuming iron-fortifi ed foods. Thus, translated into dietary requirements by taking into early detection of the disease via genetic screening account the effi ciency at which iron is absorbed from followed by regular blood removal has proven to be a the diet (typically around 10%). Current RDAs for successful treatment. iron (recommended by the US Food and Nutrition Board in 2001) are infants 0.27 mg (fi rst 6 months; Assessing status this is an adequate intake value), 11 mg (7–12 Several different laboratory methods must be used in months), children 7 and 10 mg (1–3 and 4–8 years, combination to diagnose iron defi ciency anemia cor- respectively), teenage boys 8 and 11 mg (9–13 and rectly. The most commonly used methods to assess 14–18 years, respectively), adult men 8 mg (19 years iron status include: and older), teenage girls 8 and 15 mg (9–13 and 14–18 years, respectively), adult women 18 and 8 mg (19–50 ● serum ferritin years and 51 years and older, respectively), pregnant ● transferrin saturation women 27 mg and lactating women 10 and 9 mg ● erythrocyte protoporphyrin (younger than 18 years and 19–50 years, ● mean corpuscular volume respectively). ● serum transferrin receptor Iron is widely distributed in meat, eggs, vegetables, ● hemoglobin or packed cell volume. and cereals, but the concentrations in milk, fruit, and Iron defi ciency anemia is usually defi ned as a hemo- vegetables are low (Table 9.10). The iron content per globin level below the cut-off value for age and sex se of individual foods has little meaning as iron plus at least two other abnormal iron status measure- absorption varies considerably. There are two types of ments. The most commonly used are probably food iron: nonheme iron, which is present in both low serum ferritin, high protoporphyrin, and, more plant foods and animal tissues, and heme iron, coming recently, high serum transferrin receptor. from the hemoglobin and myoglobin in animal prod- ucts. Heme iron represents 30–70% of the total iron Requirements and dietary sources in lean meat and is always well absorbed. Nonheme Daily (absorbed or physiological) iron requirements iron from meat and vegetable foods enters a common are calculated from the amount of dietary iron neces- nonheme iron pool in the gastric juice, from which sary to cover basal iron losses, menstrual losses, and the amount of iron absorbed depends to a large extent growth needs. They vary according to age and sex, on the presence of enhancing and inhibiting sub- Minerals and Trace Elements 209

Table 9.10 Iron content of some common foods Table 9.11 Approximate zinc content of major organs and tissues in the adult man Food source Description Fe content (mg/100 g) Percentage of body Liver Raw, calf 8.0 Tissue Total Zn content (g) Zn (%) Beef Lean (from six 2.1 different cuts) Skeletal muscle 1.53 ~57 Black (blood) Fried 20.0 Bone 0.77 29 sausage Skin 0.16 6 Chicken Raw, meat only 0.7 Liver 0.13 5 Cod, plaice, whiting Raw 0.3–1.0 Brain 0.04 1.5 Eggs Chicken, whole, 1.9 Kidneys 0.02 0.7 raw Heart 0.01 0.4 Pulses Raw 0.6–11.1 Hair <0.01 ~0.1 Wheat fl our Whole fl our 3.9 Blood (plasma) <0.01 ~0.1 Wheat fl our White fl our 1.5–2.0 Milk Cow’s (3.9, 1.6 0.05–0.06 Modifi ed from Mills CF, ed, Zinc in Human Biology, copyright 1998 and 0.1% fat) with kind permission of Springer Science + Business Media. Green leafy Raw 0.7–2.2 vegetables Rice Raw, white, 0.5 polished Brass, nickel, silver, typewriter metal, commercial Potatoes Raw 0.3–0.4 bronze, spring brass, German silver, soft solder, and aluminum solder are some of the more important Data from Holland et al. (1995). Reproduced with permission from alloys. Large quantities of zinc are used to produce die HMSO. castings, used extensively by the automotive, electri- cal, and hardware industries. Zinc is also extensively stances in the meal and on the iron status of the used to galvanize other metals, such as iron to prevent individual. corrosion. is widely used in the manufac- ture of paints, rubber products, cosmetics, pharma- Micronutrient interactions ceuticals, fl oor coverings, plastics, printing inks, soap, The fact that serum copper has been found to be low storage batteries, textiles, electrical equipment, and in some cases of iron defi ciency anemia suggests other products. Zinc sulfi de is used in making lumi- that iron status has an effect on copper metabolism. nous dials, X-ray and television screens, and fl uores- Copper defi ciency impinges on iron metabolism, cent lights. The chloride and chromate are also causing an anemia that does not respond to iron important compounds. In biological systems zinc is supplementation. Interactions between iron and virtually always in the divalent (+2) state. Unlike iron, copper seem to be owing to impaired utilization of zinc does not exhibit any direct redox chemistry. one in the absence of the other. As mentioned above, calcium can inhibit iron absorption under certain cir- Absorption, transport, and cumstances. In aqueous solutions iron impairs zinc tissue distribution absorption, but this interaction does not take place Zinc is ubiquitous in the body. It is the most abundant when iron is added to an animal protein meal, indi- intracellular trace element, with >95% of the body cating different uptake mechanisms for solutions and zinc intracellular. An adult human contains about 2 g solid foods. of zinc, of which about 60% and 30% are in skeletal muscle and bone, respectively, and 4–6% is present 9.8 Zinc in skin (Table 9.11). Zinc turnover in these tissues is slow and, therefore, the zinc in these tissues is not The natural abundance of zinc in the Earth’s crust is accessible at times of deprivation. Because zinc is 0.02%. The principal ores of zinc are sphalerite or essential for the synthesis of lean tissue, it is while this blende (sulfi de), smithsonite (carbonate), calamine is occurring that it may become a limiting nutrient. (silicate), and franklinite (zinc iron oxide). Zinc is Although some zinc may be available in short-term used to form numerous alloys with other metals. zinc deprivation from a mobile hepatic pool, it is 210 Introduction to Human Nutrition generally assumed that the body has no specifi c zinc having a low, medium, or high bioavailability, accord- reserve and is dependent on a regular supply of the ing to the content of zinc, phytate, and animal protein. element. From a mixed animal and plant product diet, 20–30% With essential roles in many fundamental cellular zinc absorption can be expected. The lowest absorp- processes (see below), it is not surprising that whole- tion, 10–15%, is seen from diets prevalent in develop- body zinc content is tightly controlled. Zinc in foods ing countries that are based on cereals and legumes is absorbed via a carrier-mediated transport process, with a high phytate content and with negligible which under normal physiological conditions appears amounts of animal protein. not to be saturated. Zinc is absorbed throughout the small intestine. Proximal intestinal absorption is effi - Metabolic function and essentiality cient, but it has a large enteropancreatic circulation; Zinc has three major groups of functions in the the net intestinal absorption of zinc is achieved by the human body: catalytic, structural, and regulatory. distal small intestine. Zinc is transported in the plasma Most biochemical roles of zinc refl ect its involvement by albumin and α2-macroglobulin, but only 0.1% of in the folding and activity of a large number (up to body zinc is found in plasma. Body zinc content is 10%) of proteins and over 100 different zinc metal- regulated by homeostatic mechanisms over a wide loenzymes have been identifi ed, including RNA range of intakes by changes in fractional absorp- nucleotide polymerase I and II, alkaline phosphatase tion (normally 20–40%) and urinary (0.5 mg/day) and carbonic anhydrases. Important structural roles and intestinal (1–3 mg/day) excretion. For example, for zinc are in the zinc fi nger motif in proteins, but during periods of low zinc intake, absorption is also in metalloenzymes [e.g., copper/zinc superoxide enhanced and secretion of endogenous zinc into the dismutase (Cu/Zn-SOD)]. Zinc is also required by gastrointestinal lumen is suppressed. In contrast, high protein kinases that participate in signal transduction zinc intake is associated with decreased absorption processes and as a stimulator of transacting factors and enhanced secretion of endogenous zinc. Within responsible for regulating gene expression. Zinc plays cells, fl uctuations in zinc content are modulated an important role in the immune system and, though by changes in the amount of the metal associated not a redox-active transition metal, is an antioxidant with the storage protein metallothionein but there in vivo. is a large number and variety of zinc homeostatic proteins found throughout cells. Although zinc Defi ciency symptoms transporters are very important for generating and The clinical manifestations of severe zinc defi ciency maintaining zinc gradients across membranes and in humans are growth retardation, sexual and skeletal within cellular compartments, little is known about immaturity, neuropsychiatric disturbances, derma- many aspects of their functions and regulatory modes titis, alopecia, diarrhea, increased susceptibility to of action. infections, and loss of appetite. Many of these fea- The bioavailability of dietary zinc depends on tures, by and large, represent the dependence on zinc dietary enhancers and inhibitors and host-related of tissues with a high rate of turnover. However, severe factors (Table 9.12). Diets can be roughly classifi ed as zinc defi ciency in humans is rare, and more interest has been focused on marginal zinc defi ciency. This is Table 9.12 Factors affecting zinc absorption more diffi cult to diagnose and often occurs with other micronutrient defi ciencies including iron. The current Increased absorption Decreased absorption understanding of zinc defi ciency is largely based on Physiological factors responses to zinc supplementation. Zinc supplemen- Depleted zinc status Replete zinc status tation has been reported to stimulate growth and Disease state (acrodermatitis enteropathica) development in infants and young children, and Dietary factors reduce morbidity (diarrhea and respiratory infec- Low zinc intake High zinc intake tions) in children, particularly in developing coun- Certain organic acids Phytate tries and can increase both innate and adaptive Certain amino acids Certain metals immunity. In women, low serum zinc concentration Human milk during pregnancy was found to be a signifi cant Minerals and Trace Elements 211 predictor of , and low maternal zinc however, as they are relatively resistant to changes in intake has been associated with an approximately dietary zinc and, moreover, metabolic conditions twofold increased risk of low birth weight and unrelated to zinc status cause them to decline. The increased risk of preterm delivery in poor urban development of zinc defi ciency is different from that women. of many other nutrients because a functional reserve or store of zinc does not seem to be available when Toxicity zinc intake is inadequate. Tissue zinc is conserved by Gross acute zinc toxicity has been described following reduction or cessation of growth in growing organ- the drinking of water that has been stored in galva- isms or by decreased excretion in nongrowing organ- nized containers or the use of such water for renal isms. Depending on the degree of defi ciency, zinc dialysis. Symptoms include nausea, vomiting, and homeostasis can be re-established by adjusting growth fever, and are apparent after acute ingestion of 2 g or and excretion or, with a more severe defi ciency, further more. The more subtle effects of moderately elevated metabolic changes occur, resulting in a negative zinc intakes, not uncommon in some populations, are of balance and loss of tissue zinc. greater concern, because they are not easily detected. Prolonged intakes of supraphysiological intakes of Requirements and dietary sources zinc (75–300 mg/day) have been associated with The US RDA for zinc was based primarily on data impaired copper utilization (producing features such derived from metabolic balance studies. Such studies as microcytic anemia and neutropenia), impaired are technically diffi cult to perform and it is uncertain immune responses and a decline of high-density lipo- whether information from these studies refl ects proteins, but some have argued that even short-term true requirements. A different approach, using the intakes of about 25–50 mg zinc/day may interfere factorial method, was proposed for estimates of zinc with the metabolism of both iron and copper. The US requirements and future RDAs. Factorial calculations Food and Nutrition Board reported that there was no to estimate zinc requirements require knowledge of evidence of adverse effects from intake of naturally obligatory losses, tissue composition, and needs for occurring zinc in food; however, they derived a toler- growth and tissue repair. Current RDAs for zinc (rec- able UL of 40 mg/day for adults older than 19 years, ommended by the US Food and Nutrition Board in which applies to total zinc intake from food, water, 2001) are infants 2 mg [fi rst 6 months; this is an ade- and supplements (including fortifi ed foods). Data on quate intake (AI) value], 3 mg (7–12 months), chil- reduced copper status in humans were used to derive dren 3 and 5 mg (1–3 and 4–8 years, respectively), this UL for zinc. Using similar data but different teenage boys 8 and 11 mg (9–13 and 14–18 years, uncertainty factors, the UL for total zinc intake was respectively), adult men 11 mg (19 years and more), set at 25 mg/day in the EU. teenage girls 8 and 9 mg (9–13 and 14–18 years, respectively), adult women 8 mg (19 years and older), Genetic diseases pregnant women 13 and 11 mg (younger than 18 Acrodermatitis enteropathica, a rare, inborn, auto- years and 19–50 years, respectively) and lactating somal recessive disease, is a disorder of primary zinc women 14 and 12 mg (younger than 18 years and malabsorption. It is characterized by alopecia; vesicu- 19–50 years, respectively). lar, pustular and/or eczematoid skin lesions, specifi - The zinc content of some common foods is given cally of the mouth, face, hands, feet and groin; growth in Table 9.13, whereas Table 9.14 classifi es foods based retardation; mental apathy; diarrhea and secondary on zinc energy density. The bioavailability of zinc in malabsorption, defects in cellular and phagocytic different foods varies widely, from 5% to 50%. Meat, immune function; and intercurrent infections. The seafood (in particular oysters) and liver are good disorder responds very well to zinc therapy. sources of bioavailable zinc. It has been estimated that approximately 70% of dietary zinc in the US diet is Assessing status provided by animal products. In meat products, the Measurement of zinc in plasma or activities of zinc zinc content to some extent follows the color of the metalloenzymes or peptides in blood are frequently meat, so that the highest content, approximately used to measure zinc status. They are not ideal indices, 50 mg/kg, is found in lean red meat, at least twice that 212 Introduction to Human Nutrition

Table 9.13 Zinc content of some common foods Table 9.14 Classifi cation of foods based on zinc energy density

Food source Description Zn content (mg/100 g) Zinc energy mg Zn/1000 kcal Foods

Liver Raw, calf 7.8 Very poor 0–2 Fats, oils, butter, cream cheese, Beef Lean (from six different 4.3 confectionery, soft/alcoholic cuts) drinks, sugar, preserves Lamb Lean (from six different 4.0 Poor 1–5 Fish, fruit, refi ned cereal cuts) products, biscuits, cakes, Pork Lean (from three 2.4 tubers, sausage different cuts) Rich 4–12 Whole grains, pork, poultry, Chicken Raw, meat only 1.1 milk, low-fat cheese, yoghurt, Cod, plaice, Raw 0.3–0.5 eggs, nuts whiting Very rich 12–882 Lamb, leafy and Muscles Boiled 2.1 vegetables, crustaceans, beef Oysters Raw 90–200 kidney, liver, heart, molluscs Crab Boiled 5.5 Eggs Chicken, whole, raw 1.3 Adapted from Solomons, N.W. (2001) Dietary sources of zinc and Cheese Soft and hard varieties 0.5–5.3 factors affecting its bioavailability. Food and Nutrition Bulletin, 22, Pulses Raw 0.2–5.0 138–54 Wheat fl our Whole fl our 2.9 Wheat fl our White fl our 0.6–0.9 Milk Cow’s (3.9, 1.6 and 0.4 phytate-rich diets, but this has not been confi rmed in 0.1% fat) human studies. Yoghurt Whole milk 0.5–0.7 Green leafy Raw 0.2–0.6 vegetables 9.9 Copper Rice Raw, white, polished 1.8 Potatoes Raw 0.2–0.3 Copper occurs in the environment in three oxidation states. Copper (0) metal is used widely in the building Data from Holland et al. (1995). Reproduced with permission from industry (e.g., water pipes, electrical wires) because of HMSO. its properties of malleability, ductibility, and high thermal and electrical conductivity. Brass, an alloy of copper and zinc, is used for cooking utensils and in chicken. However, in many parts of the world, most musical instruments, and bronze, an alloy of copper zinc is provided by cereals. In cereals, most of the zinc and tin, has been used in castings since early times. is found in the outer fi ber-rich part of the kernel. The Copper-based alloys and amalgams are used in dental degree of refi nement, therefore, determines the total bridges and crowns, and copper is a constituent of zinc content. Wholegrain products provide 30–50 mg/ intrauterine contraceptive devices. Copper com- kg, but a low extraction rate wheat fl our contains pounds are widely used in the environment as fertil- 8–10 mg/kg. The bioavailability of zinc can be low izers and nutritional supplements and, because of from plant-based diets, in particular from wholegrain their microbicidal properties, as fungicides, algicides, cereals and legumes, owing to the high content of insecticides, and wood preservatives. Other industrial , a potent inhibitor of zinc absorption. uses include dye manufacturing, petroleum refi ning, , and metal fi nishing. Copper com- Micronutrient interactions pounds in the cuprous (1) state are easily oxidized to A decrease in copper absorption has been reported in the more stable cupric (2) state, which is found most the presence of excessive zinc. Data indicate that the often in biological systems. level necessary to impair bioavailability is >40–50 mg/ The most important copper ores are chalco- day; therapeutic levels (150 mg/day) over extended cite (Cu2S), chalcopyrite (CuFeS2), and malachite periods produce symptoms of copper defi ciency. As [CuCO3 ⋅ Cu(OH)2]. Copper concentrations in soil mentioned above, iron under certain circumstances vary from 5 to 50 mg Cu/kg and in natural water from impairs zinc absorption. Animal studies have sug- 4 to 10 μg Cu/l. Concentrations of copper in water, gested an interaction between calcium and zinc in however, depend on acidity, softness, and the extent Minerals and Trace Elements 213 of copper pipes, and municipal water supplies can stream, with some going directly to other tissues, contain appreciably higher concentrations. The taste especially the kidney. Hepatic copper is mostly incor- threshold of copper ranges from 1 to 5 mg Cu/l, pro- porated into ceruloplasmin, which is then released ducing a slight blue–green color at concentrations into the blood and delivered to other tissues. Uptake >5 mg/l copper. Acute copper toxicity symptoms, of copper by tissues can occur from various sources, mainly nausea and gastrointestinal irritation, can including ceruloplasmin, albumin, transcuprein, and occur at concentrations of >4 mg/l copper. low molecular weight copper compounds. Chaperone proteins are then thought to bind the copper and Absorption, transport, and transfer bound copper across the cell membrane to tissue distribution the intracellular target proteins, for example cyto- About 50–75% of dietary copper is absorbed, mostly chrome c oxidase. The ATPase proteins may form part via the intestinal mucosa, from a typical diet. The of the transfer process. amount of dietary copper appears to be the primary The body of a healthy 70 kg adult contains a little factor infl uencing absorption, with decreases in the over 0.1 g of copper, with the highest concentrations percentage absorption as the amount of copper found in the liver, brain, heart, bone, hair, and nails. ingested increases. High intakes of several nutrients Over 25% of body copper resides in the muscle, which can also infl uence copper bioavailability. These forms a large part of the total body tissue. Much of include antagonistic effects of zinc, iron, molybde- the copper in the body is functional. Storage of copper, num, ascorbic acid, sucrose, and fructose, although however, is very important to the neonate. At birth, evidence for some of these is mainly from animal infant liver concentrations are some fi ve to 10 times studies. Drugs and medication, such as penicillamine the adult concentration and these stores are used and thiomolybdates, restrict copper accumulation during early life when copper intakes from milk in the body and excessive use of antacids can inhibit are low. copper absorption. Although high intakes of sulfur amino acids can limit copper absorption, absorption Metabolic functions and essentiality of copper is promoted from high-protein diets. Copper is a component of several enzymes, cofactors, Ionic copper can be released from partially digested and proteins in the body. These enzymes and proteins food particles in the stomach, but immediately forms have important functions in processes fundamental complexes with amino acids, organic acids, or other to human health (Table 9.15). These include a require- chelators. Soluble complexes of these and other highly ment for copper in the proper functioning of the soluble species of the metal, such as the sulfate or immune, nervous and cardiovascular systems, for nitrate, are readily absorbed. Regulation of absorp- bone health, for iron metabolism and formation of tion at low levels of copper intake is probably by a red blood cells, and in the regulation of mitochon- saturable active transport mechanism, while passive drial and other gene expression. In particular, copper diffusion plays a role at high levels of copper intake. functions as an electron transfer intermediate in Regulation of copper absorption is also effected via redox reactions and as a cofactor in several copper- metallothionein, a metal-binding protein found in containing metalloenzymes. As well as a direct role in the intestine and other tissues. Metallothionein- maintaining cuproenzyme activity, changes in copper bound copper in mucosal cells will be lost when these status may have indirect effects on other enzyme cells are removed by intestinal fl ow. The major regula- systems that do not contain copper. tor of copper elimination from the body, however, is biliary excretion. Most biliary copper is not reab- Defi ciency symptoms sorbed and is eliminated in the feces. The overall Owing to remarkable homeostatic mechanisms, clini- effect of these regulatory mechanisms is a tight cal symptoms of copper defi ciency occur in humans homeostasis of body copper status. Little copper is only under exceptional circumstances. Infants are lost from the urine, skin, nails, and hair. more susceptible to overt symptoms of copper defi - After absorption from the intestinal tract, ionic ciency than are any other population group. Among copper (2) is transported tightly bound to albumin the predisposing factors of copper defi ciency are and transcuprein to the liver via the portal blood- prematurity, low birth weight, and malnutrition, 214 Introduction to Human Nutrition

Table 9.15 Human enzymes and proteins that contain copper

Enzyme or protein Function

Cytochrome c oxidase Mitochondrial enzyme involved in the electron transport chain; reduces oxygen to water and allows formation of ATP; activity is highest in the heart and also high in the brain, liver, and kidney Ceruloplasmin (ferroxidase I) Glycoprotein with six or seven copper atoms; four copper atoms involved in oxidation/reduction reactions; role of other copper atoms not fully known; scavenges free radicals; quencher of superoxide radicals generated in the circulation; oxidizes some aromatic amines and phenols; catalyzes oxidation of ferrous iron to ferric iron; assists with iron transport from storage to sites of hemoglobin synthesis; about 60% of plasma copper bound to ceruloplasmin; primarily extracellular; activity will be low during severe copper restriction Ferroxidase II Catalyzes oxidation of iron; no other functions known; in human plasma is only about 5% of ferroxidase activity Hephaestin Membrane-bound ceruloplasmin homologue; probably a multicopper oxidase required for iron export from the intestine Monoamine oxidase Inactivates catecholamines; reacts with serotonin, norepinephrine (noradrenaline), tyramine, and dopamine; activity inhibited by some antidepressant medications Diamine oxidase Inactivates histamine and polyamines; highest activity in small intestine; also high activity in kidney and placenta Lysyl oxidase Acts on lysine and hydroxylysine found in immature collagen and elastin; important for integrity of skeletal and vascular tissue; use of estrogen increases activity Dopamine β-hydroxylase Catalyzes conversion of dopamine to norepinephrine (noradrenaline), a neurotransmitter; contains two to eight copper atoms; important in brain and adrenal glands Copper, zinc superoxide dismutase Contains two copper atoms; primarily in cytosol, protects against oxidative damage by converting superoxide ion to hydrogen peroxide; erythrocyte concentrations are somewhat responsive to changes in copper intake Extracellular superoxide dismutase Protects against oxidative damage by scavenging superoxide ion radicals and converting them to hydrogen peroxide; small amounts in plasma; larger amounts in lungs, thyroid, and uterus Tyrosinase Involved in melanin synthesis; defi ciency of this enzyme in skin leads to albinism; catalyzes conversion of tyrosine to dopamine and oxidation of dopamine to dopaquinone; present in eye and skin and forms color in hair, skin, and eyes Metallothionein Cysteine-rich protein that binds zinc, cadmium, and copper; important for sequestering metal ions and preventing toxicity Albumin Binds and transports copper in plasma and interstitial fl uids; about 10–15% of copper in plasma is bound to albumin Transcuprein Binds copper in human plasma; may transport copper Blood clotting factors V and VIII Role in clotting and thrombogenesis; part of structure homologous with ceruloplasmin

especially when combined with feeding practices such frank copper defi ciency in human populations, some as cow’s milk or total parenteral nutrition. The most have speculated that suboptimal copper intakes over frequent symptoms of copper defi ciency are anemia, long periods may be involved in the precipitation of neutropenia, and bone fractures, while less frequent chronic diseases, such as cardiovascular disease and symptoms are hypopigmentation, impaired growth, osteoporosis. The pathological signifi cance of subtle increased incidence of infections, and abnormalities changes, in the longer term, in those systems that of glucose and cholesterol metabolism and of electro- respond to copper defi ciency have yet to be defi ned cardiograms. Various attempts have been made to for humans. relate these symptoms to alterations in copper metal- loenzymes (see Table 9.15) and noncopper enzymes Toxicity that may be copper responsive, and to identify the role Acute copper toxicity in humans is rare and usually of copper as an antioxidant, in carbohydrate metabo- occurs from contamination of drinking water, lism, immune function, bone health, and cardiovas- beverages, and foodstuffs from copper pipes or con- cular mechanisms. Notwithstanding the rarity of tainers, or from accidental or deliberate ingestion of Minerals and Trace Elements 215 large amounts of copper salts. Symptoms include Wilson’s disease, which affects 1 in 30 000 in vomiting, diarrhea, hemolytic anemia, renal and liver most populations, is caused by numerous (over 100 damage, sometimes (at about 100 g or more) followed recognized) mutations in the gene for a copper- by coma and death. Clinical symptoms of chronic transporting ATPase. The defect results in impaired copper toxicity appear when the capacity for protec- biliary excretion of copper and accumulation of tive copper binding in the liver is exceeded. These copper in the liver and brain of homozygous indi- symptoms include hepatitis, liver cirrhosis, and viduals or compound heterozygotes. Abnormalities in jaundice. copper homeostatis, however, may also occur in het- Consumption of formula milks, heavily contami- erozygous carriers, who may make up 1–2% of the nated with copper after boiling or storage in brass population. The age of onset is from childhood vessels, is usually a feature of Indian childhood cir- onwards and patients may present in three different rhosis, which occurs in early-weaned infants between ways: with hepatic symptoms (liver cirrhosis and fatty the ages of 6 months and 5 years. Symptoms include infi ltration in the latter stages), with neurological abdominal distension, irregular fever, excessive crying, symptoms (degeneration of the basal ganglia result- and altered appetite, followed by jaundice and often ing in defective movement, slurred speech, diffi culty death. Some believe that a genetic disorder enhances swallowing, face and muscle spasms, dystonia, and susceptibility to this toxicity syndrome, associated poor motor control), or with psychiatric and behav- with excessive dietary exposure to copper and massive ioral problems (including depression and schizophre- accumulation of liver copper. nia, loss of emotional control, temper tantrums, and insomnia). Kayser–Fleischer rings (corneal copper Genetic diseases deposits) in the eyes are generally present in neuro- There are several disorders that result in defi ciency or logical or psychiatric presentations. The phenotypic toxicity from exposure to copper intakes that are differences between Wilson’s disease and Menkes’ adequate or tolerated by the general population. The syndrome are probably owing to the tissue-specifi c most important of these are Menkes’ syndrome, an expression of the ATPase genes. X-linked copper defi ciency that is usually fatal in early If Wilson’s disease is diagnosed early, copper chela- childhood; Wilson’s disease, an autosomal recessive tion therapy, usually with d-penicillamine, can be disorder resulting in copper overload; and acerulo- benefi cial, although neurological symptoms are often plasminemia, an autosomal recessive disorder of iron irreversible and liver disease may be advanced at the metabolism. All three disorders are characterized by time of diagnosis. Zinc supplements limit copper low serum copper and ceruloplasmin. absorption and subsequent accumulation, and this is Menkes’ syndrome, which affects 1 in 300 000 in the treatment of choice for maintenance therapy. most populations, is caused by mutations in the gene Aceruloplasminemia, which affects about 1 in 2 that encodes a novel member of the family of cation- million individuals, is caused by mutations in the transporting p-type ATPases. The gene is expressed ceruloplasmin gene. Ceruloplasmin is involved in in extrahepatic tissues, and symptoms result from iron metabolism and, in the disease, there is an accu- an inability to export copper from cells, particularly mulation of ferrous iron within the recticuloendothe- from intestinal cells and across the placenta. The syn- lial system with pathogenesis mainly linked to the drome has three forms, classic, mild, and occipital slow accumulation of iron in the brain, rather than horn. Among the symptoms of the classic (most other tissues. Symptoms include dementia, speech severe) Menkes’ syndrome are abnormal myelination problems, retinal degeneration, poor muscle tone, with cerebellar neurodegeneration (giving progres- and diabetes. Early therapy with the high-affi nity iron sive mental retardation), abnormal (steely, kinky) chelator desferoxamine can relieve some of the hair, hypothermia, hypopigmentation, seizures, con- symptoms. vulsions, failure to thrive, and connective tissue Disruption of copper metabolism may be involved abnormalities resulting in deformities in the skull, in other neurodegenerative diseases such as the accu- long bones and ribs, and twisted, tortuous arteries. mulation of amyloid β-protein in Alzheimer’s disease Death usually occurs in the severe forms before 3 and the accumulation of modifi ed prion protein in years of age. human prion disease. 216 Introduction to Human Nutrition

About 10% of motor neuron disease cases are Table 9.16 Putative functional indices of copper status familial and 20% of these are owing to autosomal Molecular indices dominant inheritance of mutations in the Cu/Zn- Changes in activity/concentration of Cu-metalloproteins SOD (SODI) gene. It is unclear how changes in Ceruloplasmin oxidase activity of this copper enzyme might be involved Ceruloplasmin protein in the progressive muscle weakness and atrophy Superoxide dismutase of motor neuron disease or in Down’s syndrome, Cytochrome c oxidase Lysyl oxidase where additional Cu/Zn-SOD activity results Diamine oxidase from the SODI gene being present in the extra chro- Dopamine β-monooxgenase mosome 21. Peptidylgycine α-amidating monooxgenase Tyrosinase Assessing status Factor V Factor VIII It is possible to diagnose severe copper defi ciency in Transcuprein infants from plasma or serum copper, ceruloplasmin Biochemical indices protein, and neutrophils. These measures, however, Pyridinium cross-links of collagen cannot be used to detect suboptimal copper status in Various measures of oxidative stress (TBARS) individuals, as such measures are insensitive to small Catecholamines changes in copper status and there are intractable Encephalins problems in interpretation. Ceruloplasmin, the major Polyamines copper protein in plasma or serum, is an acute-phase Physiological indices reactant and is raised by cigarette smoking, oral Immune function contraceptives, estrogens, pregnancy, infections, Hemostasis Cholesterol metabolism infl ammation, hematological diseases, hypertension, Glucose tolerance diabetes, cardiovascular diseases, cancer, and cirrho- Blood pressure sis, and after surgery and exercise. Arterial compliance Currently, there is no adequate measure of subop- Arterial plaque DNA damage and repair timal (or supraoptimal) copper status and this is a Bone density major barrier to determining precise dietary require- ments for copper and the possible role of suboptimal or supraoptimal copper status in the etiology of Requirements and dietary sources chronic disease. Table 9.16 gives some of the func- Although copper is the third most abundant trace tional indices (classifi ed as molecular, biochemical, element, after iron and zinc, in the body, precise and physiological) that might be used to defi ne sub- dietary requirements for copper are still subject to optimal or supraoptimal status in humans. A valid conjecture because of the diffi culty in assessing copper functional index of copper status in humans must status. Current estimates suggest that the require- respond sensitively, specifi cally, and predictably to ments for copper for the great majority of adults are changes in the concentration and supply of dietary below about 1.5 mg copper/day, while most people copper or copper stores, be accessible for measure- can tolerate 3 mg copper/day or more over the long ment and measurable, and impact directly on health. term and 8–10 mg copper/day or more in the shorter As such, indices in Table 9.16 have not been validated term (over several months). Using similar data from and many lack sensitivity and specifi city. Perhaps, the copper supplementation trials where there was an best way forward is to use a combination of measures. absence of any adverse effects on liver function, UL Among the more promising are erythrocyte for copper was derived to be 10 mg/day in the US and super-oxide dismutase activity, platelet cytochrome c 5 mg/day in the EU; the difference owing to the use oxidase, plasma diamine oxidase, plasma peptidyl of different uncertainty factors in the derivation. glycine α-amidating mono-oxygenase, urinary pyri- Estimates of average intakes of copper are about 1.5 dinium cross-links of collagen (may indicate lowered and 1.2 mg copper/day for men and women, respec- activity of the cuproenzyme, lysyl oxidase), and tively, on mixed diets, with higher intakes for those various immunological measures. on vegetarian diets or those consuming water with Minerals and Trace Elements 217 appreciable concentrations of copper. Particularly Elemental selenium is stable and has three allotropic rich food sources of copper include offal, seafood, forms, deep red crystals, red amorphous powder, and nuts, seeds, legumes, wholegrain cereals, and choco- the black vitreous form. late. Milk and dairy products are very low in copper Selenium has many industrial uses, e.g., in elec- and infants are at risk of copper defi ciency if they are tronics, glass, ceramics, pigments, as alloys in steel, as fed exclusively on cow’s milk. catalysts in pharmaceutical production, in rubber vulcanization and in agriculture, as feed supplements Micronutrient interactions and . Because of its increasing use, selenium The major micronutrient interactions with copper has become a potential health and environmental are those involving zinc and iron, high intakes of hazard. The primary pathway of exposure to selenium which can restrict copper utilization in infants and for the general population is food, followed by water adults. The mechanism by which zinc appears to exert (predominantly inorganic selenate and selenite), and on antagonistic effect on copper status is through air (mainly as elemental particulate selenium from the induction of metallothionein synthesis by zinc in combustion of fossil fuels and from volcanic gas). mucosal cells in the intestine. Metallothionein has a particularly strong affi nity for copper. Metallothionein- Absorption, transport and bound copper is not available for transport into the tissue distribution circulation and is eventually lost in the feces when the Absorption of dietary selenium takes place mainly in mucosal cells are sloughed off. Molybdenum also has the small intestine, where some 50–80% is absorbed. a strong interaction with copper and thiomolybdates Organic forms of selenium are more readily absorbed are potent systemic copper antagonists. Although than inorganic forms and selenium compounds from both cadmium and lead can inhibit copper utiliza- plants are generally more bioavailable than those tion, this inhibition only occurs at dietary intakes of from animals, and particularly from fi sh. Some natu- these heavy metals above those normally consumed rally occurring inorganic and organic compounds of by humans. Vitamin E, selenium, and manganese selenium are given in Table 9.17. have metabolic interactions with copper as antioxi- The bioavailability of selenium from water (mainly dants, but data on benefi cial interactions of these on inorganic selenates) and supplements is lower than symptoms of copper defi ciency are largely confi ned from food. The overall bioavailability of selenium to animal studies. Copper defi ciency exerts an effect from the diet depends on a number of factors, includ- on iodine metabolism resulting in hypothyroidism, at ing selenium status, lipid composition, and metals. least in animal models. Inorganic forms of selenium are passively trans- ported across the intestinal brush border, whereas 9.10 Selenium organic forms (selenomethionine and probably sele- nocysteine) are actively transported. On reaching the Selenium is a nonmetallic element that has similar bloodstream, selenium is transported largely bound chemical properties to sulfur and has four natural to protein (mainly very low-density β-lipoprotein oxidation states (0, −2, +4, +6). It combines with other with a small amount bound to albumin) for elements to form inorganic selenides [sodium selenide (−2) Na2Se], selenites [sodium selenite + + ( 4) Na2SeO3] and selenates [sodium selenate ( 6) Table 9.17 Some naturally occurring inorganic and organic com- Na2SeO4], and with oxygen to form oxides [selenium pounds of selenium (+4) dioxide SeO2] and oxyacids [selenic (+6) acid 2− H SeO ]. Selenium replaces sulfur to form a large Selenite {SeO3 ] 2 4 Selenate {SeO 2−] number of organic selenium compounds, parti- 4 Methylselenol (CH3SH) cularly as , the twenty-fi rst amino acid. Dimethylselenide (CH3-Se-CH3) + Selenium is a component of selenoproteins, where it Trimethyselenonium ion [(CH3)3-Se ] also occurs as selenides on the side-chains of seleno- Selenocysteine cysteine at physiological pH. Selenium also displaces Selenomethionine Se-Methyl-selenocysteine sulfur to form the amino acid selenomethionine. 218 Introduction to Human Nutrition deposition in various organs. Liver and kidney are the Selenate major target organs when selenium intake is high Na2SeO4 but, at lower intakes, the selenium content of the liver is decreased. Heart and muscle tissue are other target organs, with the latter, because of its total General body proteins Selenite bulk, accounting for the greatest proportion of body selenium. The total body content of selenium can Selenoproteins Selenomethionine (as selenocysteine) GS-Se-SG vary from about 3 mg to 15 mg depending on dietary intakes. In the body, dietary selenium can be bound to sele- Selenocysteine Selenophosphate GS-SeH nium binding proteins but can also be directly incor- porated into selenoproteins during translation at the ribosome complex using a transfer RNA specifi c for H Se the amino acid selenocysteine; thus, selenocysteine 2 can be considered as the twenty-fi rst amino acid in terms of ribosome-mediated protein synthesis. CH SeH Se-Methyl Sec The major excretion routes of selenium are in urine 3 (mainly as trimethylselenonium ion), in feces (via biliary pancreatic and intestinal secretions, together (CH3)2Se breath with unabsorbed dietary selenium), and in breath (as volatile dimethylselenide). Unlike copper, and partic- ularly iron, which have ineffi cient excretion mecha- + (CH3)3Se urine nisms, selenium is rapidly excreted in the urine. Figure Figure 9.7 Selenium metabolism and excretion. 9.7 gives an overall view of selenium metabolism and excretion. poor selenium status together with the prevention of Metabolic function and essentiality the disease in an at-risk population by supplementa- Selenocysteine is a component of at least 30 seleno- tion with selenium), there are certain epidemiological proteins, some of which have important enzymic features of the disease that are not readily explained functions (Table 9.18). Selenocysteine is generally at solely on the basis of selenium defi ciency. A similar the active site of those selenoproteins with catalytic situation occurs with Kashin–Beck disease, a chronic activity, and functions as a redox center for the osteoarthropathy that most commonly affects growing selenium-dependent glutathione peroxidases (cys- children and occurs in parts of Siberian Russia and tolic, phospholipid hydroperoxide, extracellular, and in China, where it overlaps with Keshan’s disease. gastrointestinal), iodothyronine deiodinases (types I, Although oral supplementation with selenium is II, and III), and thioredoxin reductases. The glutathi- effective in preventing the disease, it is likely that one peroxidase isozymes, which account for about other factors, apart from selenium defi ciency, are 36% of total body selenium, differ in their tissue involved in the etiology of Kashin–Beck disease. There expression and map to different chromosomes. are also some selenium-responsive conditions with symptoms similar to Keshan’s disease that occur in Defi ciency symptoms patients receiving total parenteral nutrition. Keshan’s disease is a cardiomyopathy that affects chil- One explanation for the complex etiology of sele- dren and women of child-bearing age and occurs in nium-responsive diseases in humans is that low areas of China where the soil is defi cient in selenium. selenium status may predispose to other deleterious Despite the strong evidence for an etiological role for conditions, most notably the increased incidence, selenium in Keshan’s disease (i.e., the occurrence of virulence, or disease progression of a number of viral the disease only in those regions of China with low infections. For example, in a selenium-defi cient selenium and, hence, low amounts of selenium animal model, harmless coxsackie virus can become in the food chain, and only in those individuals with virulent and cause myocarditis, not only in the Minerals and Trace Elements 219

Table 9.18 Selenoproteins

Selenoprotein Function

Glutathione peroxidases (GPx1, GPx2, GPx3, GPx4; Antioxidant enzymes: remove hydrogen peroxide, and lipid and phospholipid cystolic, gastrointestinal, extracellular and hydroperoxides (thereby maintaining membrane integrity, modulating phospholipid hydroperoxide, respectively) eiconsanoid synthesis, modifying infl ammation, and likelihood of propagation of further oxidative damage to biomolecules, such as lipids, lipoproteins and DNA) (Sperm) mitochondrial capsule selenoprotein Form of glutathione peroxidase (GPx4): shields developing sperm cells from oxidative damage and later polymerizes into structural protein required for stability/motility of mature sperm

Iodothyronine deiodinases (three isoforms) Production and regulation of level of active thyroid hormone, T3, from thryoxine T4 Thioredoxin reductases (three isoforms) Reduction of nucleotides in DNA synthesis; regeneration of antioxidant systems; maintenance of intracellular redox state, critical for cell viability and proliferation; regulation of gene expression by redox control of binding of transcription factors to DNA Selenophosphate synthetase, SPS2 Required for biosynthesis of selenophosphate, the precursor of selenocysteine, and therefore for selenoprotein synthesis Selenoprotein P Found in plasma and associated with endothelial cells; appears to protect endothelial cells against damage from peroxynitrite Selenoprotein W Needed for muscle function Prostate epithelial selenoprotein (15 kDa) Found in epithelial cells of ventral prostate; seems to have redox function (resembles GPx4), perhaps protecting secretory cells against development of carcinoma DNA-bound spermatid selenoprotein (34 kDa) Glutathione peroxidase-like activity; found in stomach and in nuclei of spermatoza; may protect developing sperm 18 kDa selenoprotein Important selenoprotein, found in kidney and large number of other tissues; preserved in selenium defi ciency

Reprinted with permission from Elsevier (Rayman, MP Lancet, 2000, 356, pp. 233–241). selenium-defi cient host, but also when isolated and the production of antitumorigenic metabolites may injected into selenium-replete animals. A coxsackie explain the lowering of cancer incidence, particularly virus has been isolated from the blood and tissues of prostate cancer, after selenium supplementation in patients with Keshan’s disease and the infection may selenium-replete subjects (those who already had be responsible for the cardiomyopathy of that disease. maximized selenoenzyme activity). Other proposed It has been speculated that similar events linked with mechanisms for a cancer chemoprotective effect of other RNA viruses may explain the emergence of new selenium include antioxidant protection and reduc- strains of infl uenza virus in China and the postulated tion of infl ammation; inactivation of protein kinase crossing-over of the human immunodefi ciency virus C; altered carcinogen metabolism; reduction in DNA (HIV) to humans in the selenium-defi cient popula- damage, stimulation of DNA repair (p53), and altera- tion of Zaire. Many human viral pathogens (e.g., HIV, tion in DNA methylation; cell cycle effects; enhanced coxsackie, hepatitis, and measles viruses) can synthe- apoptosis and inhibition of angiogenesis. Further size viral selenoproteins and, thereby, lower the sele- evidence for any chemoprotective effect of selenium nium available to the host. In any event, selenium against cancer should arise from the Selenium and defi ciency is accompanied by loss of immunocompe- Vitamin E cancer Prevention Trial (SELECT), which tence, with the impairment of both cell-mediated is a large randomized controlled trial investigating the immunity and B-cell function. Covert suboptimal effi cacy of selenium (200 μg of l-selenomethionine) selenium status may be widespread in human popula- and vitamin E (400 IU, dl-α-tocopherol acetate) alone tions, as selenium supplementation in subjects and in combination for the prevention of prostate considered to be selenium replete had marked immu- cancer in over 35 000 healthy men in 435 sites in the nostimulant effects, including increased proliferation USA, Puerto Rico, and Canada and which should of activated T-cells. Such immunostimulant effects or report sometime after 2008. 220 Introduction to Human Nutrition

The evidence for suboptimal selenium status Assessing status increasing the risk of cardiovascular disease is more Plasma or whole blood, hair, and toenail selenium fragmentary, but it has been proposed that optimiz- concentrations can indicate changes in selenium ing the activity of the seleno-dependent glutathione status in humans. Plasma and serum selenium con- peroxidases and, thereby, increasing antioxidant activ- centrations respond rapidly to changes in selenium ity may be a factor. As selenium has well-recognized intakes, whereas erythrocyte selenium is an index of antioxidant and anti-infl ammatory roles, other oxi- longer term or chronic intake. Dietary intake data, dative stress or infl ammatory conditions (e.g., rheu- however, are insuffi cient to determine selenium status matoid arthritis, ulcerative colitis, pancreatitis, and in individuals because of uncertainties about bio- asthma) may benefi t from selenium supplementation. availability and variations in the content and form of In addition, some, but certainly not all, studies have selenium in foodstuffs. Although plasma (or prefera- suggested benefi cial (possibly antioxidant) effects of bly platelet) glutathione peroxidase activities have selenium on mood and in humans. The been used as functional indices to estimate selenium evidence, however, supporting a role for optimum requirements, it has not been established how these selenium status preventing or ameliorating most measurements relate to other biochemical functions infl ammatory conditions is not strong and may be of selenium, such as thyroid metabolism, or immune confounded by other dietary antioxidants, particu- function and their health sequelae. For example, at larly vitamin E, compensating for low selenium higher levels of selenium intake, glutathione peroxi- status. dase activities plateau but immunoenhancement may be evident at supplementation levels higher than Toxicity those needed to optimize the selenoenzyme activity. There is a narrow margin, perhaps not much more Perhaps the best way forward is to select from a than three- or fourfold, between benefi cial and battery of functional indices, such as selenoenzyme harmful intakes of selenium. The dose necessary to activity, plasma thyroid hormone concentrations, cause chronic selenosis in humans is not well defi ned, and immune measures, according to the function or but the threshold for toxicity appears to lie some- disease under investigation. where in the range of 850–900 mg/day. Symptoms of chronic selenium toxicity include brittle hair Requirements and dietary sources and nails, skin lesions with secondary infections, and Dietary intakes of selenium vary widely with geo- odor on the breath, resulting from the expira- graphical spread (Table 9.19). Requirements for tion of dimethyl selenide. Toxicity depends on the selenium have been estimated at intakes required to chemical form of selenium, with most forms having saturate plasma glutathione peroxidase activity low toxicity. Data from animal studies indicate that selenite and selenocysteine are a little more toxic than selenomethionine and much more toxic than other Table 9.19 Dietary selenium intakes worldwide organic selenium compounds (dimethyl selenide, tri- methyselenonium ion, selenoethers, selenobetaine). Country (region) Range (μg/day) Methylation in the body is important for detoxifi ca- Australia 57–87 tion of the element. Canada 98–224 China (Keshan county) 3–22 Genetic diseases China (Enshi county) 3200–6690 Although no important genetic diseases affecting Greece 110–220 Mexico 10–223 selenium status are apparent, polymorphisms in gene New Zealand (Dunedin) 6–70 sequences of some selenoenzymes may determine Portugal 10–100 selenium utilization and metabolic needs, and hence Russia 60–80 dietary requirements. These polymorphisms may UK 30–60 USA 62–216 explain the signifi -cant variation among individuals in the extent of the response to supplementation of After Reilly, Selenium in Food and Health, copyright 1996 with kind selenoenzyme activities. permission of Springer Science + Business Media. Minerals and Trace Elements 221

(which corresponds to lower status and intake than concentrations as a result of adding selenium to fertil- that needed to saturate platelet glutathione peroxi- izers used for grain production and horticulture dase activity) in the vast majority (97.5%) of all indi- and fodder crop and hay production. The resulting viduals in a population. The RDAs for both men and increase in the selenium status of the population is women is 55 μg/day in the USA. In the UK, the refer- largely owing to wheat (bread) consumption but the ence nutrient intake (RNI) has thus been set at 75 and biofortifi cation of vegetables may also have an impact 60 μg/day selenium for men and women, respectively. on public health as, in contrast to wheat, where the Blood selenium concentrations in the UK population major selenocompound is selenomethionine, seleno- have declined by approximately 50% over the past 30 methylselenocysteine is the predominant form in years and current UK intakes are only about 50% of vegetables; the last compound may have important the RNI. As explained previously, however, there is cancer chemoprotective effects (see also Figure 9.8) uncertainty as to what constitutes optimum selenium Fish, shellfi sh, and offal (liver, kidney) are rich sources status and the intakes of selenium in various dietary of selenium, followed by meat and eggs. Animal regimens needed to achieve optimum status. Optimum sources, however, have lower bioavailability of sele- status may not necessarily be refl ected in saturated nium than do plant sources. glutathione peroxidase activity. The UL for adults is set at 400 μg/day in the USA and at 300 μg/day in Micronutrient interactions the EU. Selenium is an antioxidant nutrient and has impor- Selenium enters the food chain through plants that, tant interactions with other antioxidant micronutri- in general, largely refl ect concentrations of the element ents, especially vitamin E (Figure 9.8). Vitamin E, as in the soil on which the plants were grown. The an antioxidant, can ameloriate some of the symptoms absorption of selenium by plants, however, is depen- of selenium defi ciencies in animals. Copper defi ciency dent not only on soil selenium content but also on also increases oxidative stress, and the expression of pH, microbial activity, rainfall, and the chemical form glutathione peroxidase genes is decreased in the of selenium. Higher plants absorb selenium preferen- copper-defi cient animal. tially as selenate and can synthesize organic selenium The metabolic interactions between selenium and compounds, e.g., selenomethionine, and to a lesser other micronutrients, however, extend beyond those extent selenocysteine. Brazil nuts contain high con- between selenium, vitamin E, and other antioxidants. centrations of selenium because of the seleniferous Peripheral deiodination of thyroxine (T4), the pre- soils in the Andes mountains but also the effi ciency dominant hormone secreted by the thyroid, to the of accumulation of selenium by the plants species. more biologically active triiodothyronine (T3) in Selenium concentrations of cereals and staples are extrathyroidal tissues is accomplished through the much lower, but the content and bioavailability of selenium-dependent deiodinase enzymes. Selenium selenium in wheat usually make this a major con- defi ciency, therefore, can contribute to iodine defi - tributor to overall selenium intakes because of the ciency disorders, and goiter complications have been high quantities of wheat consumed as bread and noted in up to 80% of Keshan’s disease casualties after other baked products. Wheat is the most effi cient autopsy. Moreover, higher serum T4 concentrations accumulator of selenium within the common cereal were found in patients with subacute Keshan’s disease crops (wheat > rice > maize > > ). There and in children with latent Keshan’s disease compared are major varietal differences in selenium uptake and with the respective controls. All thyroid hormone for wheat, tomatoes, soybean, and onions, there are concentrations in these studies were within normal up to fourfold differences in uptake of selenium from ranges, suggesting that selenium defi ciency, or even soils amongst cultivars. The ability of plants to accu- suboptimal selenium status, was blocking optimum mulate selenium has been useful for agronomic bio- thyroid and iodine metabolism. fortifi cation, which differs from food fortifi cation Excess selenium intake interferes with zinc bio- where the nutrient is added during food processing. availability, decreases tissue iron stores, and increases The Finnish Policy (1984) has led to a 10-fold increase copper concentrations in the heart, liver, and kidney. in cereal grain selenium concentration as well as Vitamins C and E, sulfur amino acids and sulfate, marked increases in fruit and vegetables and meat arsenic, and heavy metals can decrease the toxicity of 222 Introduction to Human Nutrition bilirubin) Ubiquinol Estrogens (Also urate, SH-proteins, α- Chain-breaking s; S-AA, GSSG (Mg) shunt Hexose monophosphate zed oxidant damage 12 Vitamin A Vitamin E Se Zn Cu Mn Fe Mg Riboflavin Nicotinic acid Folate Vitamin B GSSG, oxidized glutathione; Vitamin C* NADP NADPH GR (riboflavin) Fe Cu Diet GSH S-AA S-AA GSSG Vitamin E Vitamin E* Vitamin C (Se) GSH-Px Ϫ Lutein Phytic acid Astaxanthin Isoflavonoids Other Phenols Other Carotenoids ONOO Alcohols Organo-sulfur compounds GST β-carotene and radicals Constituents of herbs and spices NO PUFA alcohols PUFA peroxides 2 O acids ? 1 Mercapturic Dienes Epoxides Carbonyls Energy 2 O O 2 ? 2 Fe Cu H H oxygen “Activated” “Activated” carotenoids Ferritin (Fe) sugar products Amino acid and ) Mitochondrial dysfunction 12 (Zn) (Fe) (Se) (Cu) SOD Repair DNA and GSH-Px (Cu, Mn) Catalase Transferrin Hemopexin protein damage Haptoglobin Metallothionein Caeruloplasmin DNA repair enzymes riboflavin, niacin and MSR (Mg, Zn, folate, vitamin B and GR, glutathione reductase (EC1.6.4.2); GSH, reduced glutathione; GSH-Px glutathione peroxidase (EC1.11.1.9); GST, glutathione-S-transferase (EC 2.5.1.18); MSR, methionine sulfoxide reductase (EC1.8.4.5); PUFA, polyunsaturated fatty acid sulfur amino acids; SH-proteins, sulfydryl proteins; SOD, superoxide dismutase (EC1.15.1.1); , transition metal-cataly to biomolecules. ? Biological relevance. Preventive scavenging Figure 9.8 Figure Antioxidant defense system (from Strain and Benzie, 1999). (also vitamin A) Minerals and Trace Elements 223 selenium. Conversely, selenium modifi es the toxicity and circulates in the blood to all tissues in the of many heavy metals. In seafoods, selenium is com- body. The thyroid gland traps most (about 80%) of bined with mercury or methyl mercury and this inter- the ingested iodine, but salivary glands, the gastric action may be one of the factors that decreases the mucosa, choroid plexus, and the lactating mammary bioavailability of selenium in these foods. Indeed, gland also concentrate the element by a similar active well-known antagonistic interactions of selenium transport mechanism. Several sulfur-containing with both mercury and arsenic suggest that selenium compounds, thiocyanate, isothiocyanate, and goitrin can promote detoxifi cation effects with respect to inhibit this active transport by competing for uptake these toxins. with iodide, and their goitrogenic activity can be overcome by iodine supplementation. These active 9.11 Iodine goitrogens are released by plant enzymes from thio- glucosides or cyanogenic glucosides found in cassava, Iodine is a nonmetallic element of the halogen kale, cabbage, sprouts, broccoli, kohlrabi, turnips, group with common oxidation states of −1 (iodides), swedes, rapeseed, and mustard. The most important +5 (iodates), and +7 (periodates), and less common of these goitrogen-containing foods is cassava, which states of +1 (iodine monochloride) and +3 (iodine can be detoxifi ed by soaking in water. Tobacco smoke trichloride). Elemental iodine (0) is a soft blue–black also contributes thiocyanate and other antithyroid solid, which sublimes readily to form a violet gas. compounds to the circulation. The principal industrial uses of iodine are in the pharmaceutical industry, medical and sanitary uses Metabolic functions and essentiality (e.g., iodized salt, water treatment, protection from Iodine is an essential constituent of the thyroid radioactive iodine, and disinfectants), as catalysts hormones, thyroxine (T4) and triiodothyronine (synthetic rubber, acetic acid synthesis), and in animal (T3), which have key modifying or permissive feeds, herbicides, dyes, inks, colorants, photographic roles in development and growth. Although T4 is equipment, lasers, metallurgy, conductive polymers, quantitatively predominant, T3 is the more active. and stabilizers (nylon). Naturally occurring iodine The mechanism of action of thyroid hormones minerals are rare and occur usually in the form of appears to involve binding to nuclear receptors that, calcium iodates. Commercial production of iodine is in turn, alter gene expression in the pituitary, liver, largely restricted to extraction from Chilean deposits heart, kidney, and, most crucially, brain cells. Overall, of nitrates (saltpeter) and iodine in caliche (soluble thyroid hormones stimulate enzyme synthesis, oxygen salts precipitated by evaporation), and from concen- consumption and basal metabolic rate and, thereby, trated salt brine in Japan. Iodine is the least abundant affect the heart rate, respiratory rate, mobilization, halogen in the Earth’s crust, at concentrations of and metabolism of carbohydrates, lipogenesis and a 0.005%. The content of iodine in soils varies and much wide variety of other physiological activities. It is of the original content has been leached out in areas probable that iodine has additional roles to those of of high rainfall, previous glaciation, and soil erosion. thyroid hormone activity, for example in antibiotic The concentration of iodine (as iodide and iodate) and anticancer activity, but these are poorly in the oceans is higher, at about 0.06 mg/l. Iodine understood. volatilizes from the surface of the oceans and sea Once iodide (−1) is trapped from the circulation spray as salt particles, iodine vapor or methyl iodide and actively transported to the lumen of the thyroid vapor. Some iodine can then return to land in rain- gland, it is oxidized to I2 (0) and reacts with tyrosine water (0.0018–0.0085 mg iodine/l). There is a large in thyroglobulin protein to form monoiodotyrosine variation of iodine content in drinking water (0.0001– or diiodotyrosine. These reactions are catalyzed by 0.1 mg iodine/l). thyroid peroxidase. The iodinated compounds, in

turn, couple to form T3 and T4, which are secreted Absorption, transport, and from the thyroid into the circulation. tissue distribution Flavonoids, found in many plants, including pearl Iodine, usually as an iodide or iodate compound in millet, and phenol derivatives, released into water food and water, is rapidly absorbed in the intestine from soil humus, inhibit thyroid peroxidase and the 224 Introduction to Human Nutrition organifi cation of iodide. The concentration of iodine occurs in the fetus, infancy or childhood and adoles- in the thyroid gland also affects the uptake of iodide cence. In adulthood, the consequences of iodine defi - into the follicle, the ratio of T3 to T4, and the rate ciency are more serious in women, especially during of release of these hormones into the circulation. pregnancy, than in men. This process is also under hormonal control by the The mildest form of IDD, goiters, range from those hypothalamus of the brain, which produces thyroid- only detectable by touch (palpation) to very large releasing hormone, which then stimulates the pitu- goiters that can cause breathing problems. The enlarge- itary gland to secrete thyroid-stimulating hormone ment of the thyroid gland to produce goiter arises from (TSH), which, in turn, acts on the thyroid gland to stimulation of the thyroid cells by TSH and, without produce more thyroid hormones. the ability to increase hormone production owing to Almost all of the thyroid hormones released from iodine defi ciency, the gland becomes hyperplastic. the thyroid are bound to transport proteins, mainly Apart from congenital hypothyroidism, hypothy- thyroxine-binding globulin. The longer half-life of T4 roidism, and goiter, other features linked to IDDs are ensures that there is a reservoir for conversion to the decreased fertility rates, increased and spon- more active T3 with a much shorter half-life of 1 day. taneous abortion rates, and increased perinatal and The deiodination of T4 to T3 takes place in extra- . The public health signifi cance of thyroidal tissues (mainly the liver). Excretion of iodine defi ciency cannot be underestimated, with iodine is predominantly in the urine. over 1 billion people (worldwide, but mostly in Asia and Africa) estimated to be living in iodine-defi cient Defi ciency symptoms areas and, therefore, at risk of IDDs. Estimates of A defi ciency of iodine causes a wide spectrum of those with IDDs demonstrate the scale of the problem, disorders from mild goiter (a larger thyroid gland with 200–300 million goitrous people, over 40 million than normal) to the most severe forms of endemic affected by some degree of mental impairment and congenital hypothyroidism (cretinism) (severe, irre- some 7 million people with congenital hypothyroid- versible mental, and growth retardation). Collectively, ism. Fortunately, these fi gures should decrease as these manifestations of iodine defi ciency are termed public health programs using preventive interven- iodine defi ciency disorders (IDDs) and symptoms tions with iodized oil (oral or intramuscular injec- differ depending on the life stage at which iodine tion) salt, bread, water, or even sugar have an impact. defi ciency occurs. The most severe disorders (con- Treatment with iodine supplementation in older chil- genital hypothyroidism) arise if the fetus suffers from dren and adults can reverse many of the clinical mani- iodine defi ciency. The clinical features of endemic festations of IDDs, including mental defi ciency, congenital hypothyroidism are either a predominant hypothyroidism and goiter. Although iodine defi - neurological syndrome with severe to profound ciency is the primary cause of IDDs, goitrogenic mental retardation, including defects of hearing and factors limiting bioavailability appear to be superim- speech (often deaf–mutism), squint, and disorders of posed on the primary cause. In addition, genetic stance and gait of varying degrees (neurological con- variation, immunological factors, sex, age, and growth genital hypothyroidism), or predominant features of factors seem to modify expression of the conditions, hypothyroidism and stunted growth with less severe producing a wide range of symptoms and severity of mental retardation (myxedematous congenital hypo- IDDs with similar iodine intakes. thyroidism). Profound hypothyroidism is biochemi- Toxicity cally defi ned as high serum TSH and very low T4 and T3, and is accompanied by a low basal metabolic rate, A wide range of iodine intakes is tolerated by most apathy, slow refl ex relaxation time with slow move- individuals, owing to the ability of the thyroid to ments, cold intolerance, and myxedema (skin and regulate total body iodine. Over 2 mg iodine/day for subcutaneous tissue are thickened because of an accu- long periods should be regarded as excessive or poten- mulation of mucin, and become dry and swollen). tially harmful to most people. Such high intakes are Although congenital hypothyroidism is the severest unlikely to arise from natural foods, except for diets form of IDD, varying degrees of intellectual or growth that are very high in seafood and/or seaweed or com- retardation are apparent when iodine defi ciency prising foods contaminated with iodine. In contrast Minerals and Trace Elements 225 to iodine-replete individuals, those with IDDs or pre- Dietary intakes and requirements viously exposed to iodine-defi cient diets may react to Requirements in infancy and childhood range from sudden moderate increases in iodine intake, such 40 to 150 μg iodine/day. Adult requirements are esti- as from iodized salt. Iodine-induced thyrotoxicosis mated at 150 μg iodine/day, increasing to 175 and (hyperthyroidism) and toxic nodular goiter may 200 μg/day for pregnancy and lactation. The UL for result from excess iodine exposure in these indivi- adults is set at 600 μg/day (EU) and at 1.1 mg/day duals. Hyperthyroidism is largely confi ned to those (USA). over 40 years of age and symptoms are rapid heart Under normal circumstances, about 90% of iodine rate, trembling, excessive sweating, lack of sleep, and intake is from food, with about 10% from water. The loss of weight and strength. concentration of iodine in most foods is low and, in Individuals who are sensitive to iodine, usually general, refl ects the iodine content of the soil, water, have mild skin symptoms, but very rarely fever, sali- and fertilizers used in plant and animal production. vary gland enlargement, visual problems, and skin In most countries other sources, such as iodized salts problems, and, in severe cases, cardiovascular col- or foods, are required. Seafoods and seaweed concen- lapse, convulsions, and death may occur. The occur- trate iodine from seawater and are particularly rich rence of allergic symptoms, for example to iodine sources. In some populations, milk has become a medications or antiseptics, however, is rare. major source of iodine, owing to the use of iodized salt licks and iodine-enriched cattle feed for dairy Genetic diseases herds. Minor amounts may come from adventitious Pendred’s syndrome is an autosomal recessive inher- contamination from iodophor disinfectants (teat- ited disorder with a frequency of 100 or less per dip). Iodine-enriched cattle feed will also increase the 100 000. It is characterized by goiter and profound iodine content of meat for beef herds raised on con- deafness in childhood and is caused by mutations in centrated feedstuffs. Processed foods contribute some the Pendrin gene located on chromosome 7. The gene additional iodine from food additives, such as calcium codes for pendrin, a transporter protein for chloride/ iodate used in the baking industry. iodine transport across the thyroid apical membrane. This results in defective iodination of thyroglobulin. Mutations in another gene, the sodium/iodide sym- Micronutrient interactions porter (NIS) gene, occasionally cause defective iodide From a public health viewpoint, the most important transport and goiter, whereas single nucleotide poly- metabolic interaction of iodine with other micronu- morphisms in the TSH receptor gene may predispose trients is with selenium. Adequate selenium status is individuals to the hyperthyroidism of toxic multi- essential for thyroid hormone metabolism and, there- nodular goiter and Graves’ disease. fore, normal growth development, by ensuring suffi -

cient T3 supply to extrathyroidal tissues. Most T3 is Assessing status formed from T4 by the selenium-dependent de- The critical importance of iodine for the thyroid iodinases. Iodine and selenium defi ciencies overlap in indicates that iodine status is assessed by thyroid various parts of the world and concurrent defi ciencies function. A standard set of indicators (goiter by of both may contribute to the etiologies of Kashin– palpation, thyroid volume by ultrasound, median Beck disease in Russia, China, and Tibet, and myxede- urinary iodine, and whole blood TSH) is used to matous congenital hypothyroidism in Zaire. In addi- determine prevalence in countries with endemic tion, both nutrients are required for normal repro- defi ciency. Measurement of plasma thyroid hormones duction, normal gene expression, synthesis of zeno-

(TSH, T4, and T3) provides useful indicators of func- biotic and metabolizing enzymes in the liver, and tional iodine status in the individual. Of these, TSH normal tolerance against cold stress. It is possible that is the most sensitive functional indicator of subopti- hypothyroidism associated with suboptimal selenium mal iodine status. Concentrations of T4 decline in status may explain some of the etiology of cardiovas- more severe iodine defi ciency whereas T3 concentra- cular disease and certain cancers. tions decline only in the most severe of iodine Hypothyroidism is associated with defi ciencies of defi ciencies. other trace elements, including zinc, iron, and copper, 226 Introduction to Human Nutrition while there are close metabolic relationships at the case of human manganese defi ciency has been molecular and transport levels between iodine and reported. vitamin A. Conversely, the widespread disruption of Systemic homeostatic regulation of manganese is metabolism in IDDs can affect the proper utilization brought about primarily through hepatobiliary of a host of other nutrients. excretion rather than through regulation of absorp- tion (e.g., the effi ciency of manganese retention does 9.12 Manganese not appear to be dose dependent within normal dietary levels). Manganese is taken up from blood by the liver Manganese is widely distributed in the biosphere: it and transported to extrahepatic tissues by transferrin constitutes approximately 0.085% of the Earth’s crust, and possibly α2-macroglobulin and albumin. Manga- making it the twelfth most abundant element. nese is excreted primarily in feces. Urinary excretion of Manganese is a component of numerous complex manganese is low and has not been found to be sensi- minerals, including pyroluosite, rhodochrosite, tive to dietary manganese intake. rhodanite, braunite, pyrochite, and manganite. Chemical forms of manganese in their natural depos- Metabolic function and essentiality its include oxides, sulfi des, carbonates, and silicates. Manganese is required as a catalytic cofactor for Anthropogenic sources of manganese are predomi- mitochondrial superoxide dismutase, arginase, and nantly from the manufacturing of steel, alloys, and pyruvate carboxylase. It is also an activator of glycos- iron products. Manganese is also widely used as an yltransferases, phosphoenolpyruvate carboxylase, and oxidizing agent, as a component of fertilizers and fun- glutamine synthetase. gicides, and in dry cell batteries. The permanganate is a powerful oxidizing agent and is used in quantitative Defi ciency symptoms analysis and medicine. Signs of manganese defi ciency have been demon- Manganese is a transition element. It can exist in strated in several animal species. Symptoms include 11 oxidation states from −3 to +7, with the most impaired growth, skeletal abnormalities, depressed common valences being +2, +4, and +7. The +2 reproductive function, and defects in lipid and carbo- valence is the predominant form in biological systems, hydrate metabolism. Evidence of manganese defi - the +4 valence occurs in MnO2, and the +7 valence is ciency in humans is poor. It has been suggested that found in permanganate. manganese defi ciency has never been observed in noninstitutionalized human populations because of Absorption, transport, and the abundant supply of manganese in edible plant tissue distribution materials compared with the relatively low require- The total amount of manganese in the adult human ments of mammals. There is only one report of is approximately 15 mg. Up to 25% of the total body apparent human manganese defi ciency. A male subject stores of manganese may be located in the skeleton was fed a purifi ed diet defi cient in vitamin K, which and may not be readily accessible for use in metabolic was accidentally also defi cient in manganese. Feeding pathways. Relatively high concentrations have been this diet caused weight loss, dermatitis, growth retar- reported in the liver, pancreas, intestine, and bone. dation of hair and nails, reddening of black hair, and Intestinal absorption of manganese occurs through- a decline in concentrations of blood lipids. Manganese out the length of the small intestine. Mucosal defi ciency may be more frequent in infants owing to uptake appears to be mediated by two types of mucosal the low concentration of manganese in human breast binding, one that is saturable with a fi nite capacity milk and varying levels in infant formulae. and one that is nonsaturable. Manganese absorption, probably as Mn2+, is relatively ineffi cient, generally Toxicity less than 5%, but there is some evidence of improve- Manganese toxicity of dietary origin has not been well ment at low intakes. High levels of dietary calcium, documented. Toxicity has been observed only in phosphorus, and phytate impair the intestinal workers exposed to high concentrations of manga- uptake of the element but are probably of limited nese dust or fumes in air. For example, mine-workers signifi cance because, as yet, no well-documented in Chile exposed to manganese ore dust developed, Minerals and Trace Elements 227 possibly as a result of inhalation rather than inges- There is currently no RDA set for dietary manga- tion, “manganic madness,” manifested by psychosis, nese; instead, there is an AI value [these values were hallucinations, and extrapyramidal damage with established by the US Food and Nutrition Board in features of parkinsonism. 2001]: infants 0.003 mg (fi rst 6 months), 0.6 mg (7– In 2001, the US Food and Nutrition Board set the 12 months), children 1.2 and 1.5 mg (1–3 and 4–8 tolerable UL for manganese at 11 mg/day for adults years, respectively), teenage boys 1.9 and 2.2 mg (9–13 (19 years and older). Elevated blood manganese con- and 14–18 years, respectively), adult men 2.3 mg (19 centrations and neurotoxicity were selected as the years and older), teenage girls 1.6 mg (9–18 years), critical adverse effects on which to base their UL for adult women 1.8 mg (19 years and older), pregnant manganese. women 2.0 mg, and lactating women 2.6 mg. The AI was set based on median intakes reported from the Assessing status US Food and Drug Administration Total Diet Study. Progress in the fi eld of manganese nutrition has been hampered because of the lack of a practical method Micronutrient interactions for assessing manganese status. Blood manganese Iron–manganese interactions have been demon- concentrations appear to refl ect the body manganese strated whereby iron defi ciency increased manganese status of rats fed defi cient or adequate amounts of absorption, and high amounts of dietary iron inhibit manganese, but consistent changes in blood or plasma manganese absorption, possibly by competition for manganese have not been observed in depleted or similar binding and absorption sites between nonheme repleted human subjects. Researchers are actively iron and manganese. investigating whether the activities of manganese- dependent enzymes, such as manganese-SOD in 9.13 Molybdenum blood lymphocytes and blood arginase, may be of use in detecting low manganese intake; however, there is Molybdenum does not exist naturally in the pure evidence that these enzymes can be infl uenced by metallic state but rather in association with other ele- certain disease states. ments, or predominantly in solution as the molybdate anion. Insoluble molybdenum compounds include Requirements and dietary sources molybdenum dioxide and molybdenum disulfi de. Relatively high concentrations of manganese have The metal has fi ve oxidation states (2–6), of which been reported in cereals (20–30 mg/kg), brown +4 and +6 are the predominant species. Major bread (100–150 mg/kg), nuts (10–20 mg/kg), ginger molybdenum-containing ores are molybdenum sul- (280 mg/kg), and tea (350–900 mg/kg dry tea). Con- fi tes and ferric molybdenum ores, usually produced centrations of manganese in crops are dependent on as by-products of copper mining operations, while soil factors such as pH, whereby increasing soil pH other molybdenum salts are by-products of uranium decreases plant uptake of manganese. Products of mining. Molybdenum is used mostly in metallurgical animal origin such as eggs, milk, fi sh, poultry, and red applications such as stainless steel and cast iron meat contain low amounts of manganese (Table 9.20). alloys, and in metal–ceramic composites. Molyb- Many and mineral supplements for denum compounds have anticorrosive and lubricant adults provide 2.5–5.0 mg of manganese. properties and can act as chemical catalysts. Molybdenum uptake into plants and hence into the Table 9.20 Dietary sources of manganese food chain occurs mostly from alkaline or neutral soils. Water usually contains little molybdenum except Rich sources Intermediate sources Poor sources near major mining operations. (>20 mg/kg) (1–5 mg/kg) (<1 mg/kg) Absorption, transport, and Nuts Green leafy Animal tissue vegetables tissue distribution Wholegrain cereals Dried fruits Poultry Molybdenum is readily absorbed (40–100%) from Dried legumes Fresh fruits Dairy products foods and is widely distributed in cells and in the ECF. Tea Nonleafy vegetables Seafood Some accumulation can occur in liver, kidneys, bones, 228 Introduction to Human Nutrition and skin. The major excretory route of molybdenum urinary concentrations of sulfi te, hypoxanthine, after ingestion is the urine, with signifi cant amounts zorithine, and other sulfur metabolites, however, are also excreted in bile. generally indicative of impaired activities of the molybdoenzymes. Adult requirements for molybde- Metabolic functions and essentiality num have been estimated at about 45 μg/day (Institute Molybdenum functions as a cofactor for the iron- and of Medicine, USA, 2001). Average intakes tend to be fl avin-containing enzymes that catalyze the hydroxyl- considerably above this value. Milk, beans, bread, and ation of various substrates. The molybdenum cofac- cereals (especially the germ) are good sources tor in the enzymes aldehyde oxidase (oxidizes and of molybdenum, and water also contributes small detoxifi es purines and pyrimidines), xanthine oxidase/ amounts to the total dietary intakes. hydrogenase (production of uric acid from hypoxan- thine and xanthine), and sulfi te oxidase (conversion Micronutrient interactions of sulfi te to sulfate) has molybdenum incorporated as The major micronutrient interactions with molybde- part of the molecule. num are those involving tungsten and copper. Molybdenum supplementation depletes body levels Defi ciency symptoms of the essential trace element, copper, and has been Although there is a clear biochemical basis for the used as a chelating agent for conditions such as essentiality of molybdenum, defi ciency signs in Wilson’s disease, which cause elevated concentrations humans and animals are diffi cult to induce. Naturally of copper in the body. occurring defi ciency, uncomplicated by molybdenum antagonists, is not known with certainty. In animal 9.14 Fluoride experiments, where large amounts of the molybde- num antagonist tungsten have been fed, defi ciency occurs chiefl y in fl uorspar and cryolite, but signs are depressed food consumption and growth, is widely distributed in other minerals. Fluoride is the impaired reproduction, and elevated copper concen- ionic form of fl uorine, a halogen, and the most elec- trations in the liver and brain. tronegative of the elements in the periodic table; the two terms are often used interchangeably. Fluorine Toxicity and its compounds are used in producing uranium In 2001, the US Food and Nutrition Board set the tol- and more than 100 commercial fl uorochemicals, erable UL for molybdenum at 2 mg/day for adults including many well-known high-temperature plas- (aged 19 years and older). Impaired reproduction and tics. Hydrofl uoric acid is extensively used for etching growth in animals were selected as the critical adverse the glass of light bulbs, etc. Fluorochlorohydrocarbons effects on which to base their UL for molybdenum. are extensively used in air conditioning and refrigera- tion. Fluorine is present in small but widely varying Genetic diseases concentrations in practically all soils, water supplies, A rare unborn error of metabolism, resulting in the plants and animals, and is a constituent of all diets. absence of the molybdenum pterin cofactor, may give some clue to the essentiality of molybdenum. These Absorption, transport and patients have severe neurological dysfunction, tissue distribution dislocated ocular lenses, mental retardation, and Fluoride appears to be soluble and rapidly absorbed, biochemical abnormalities, including increased and is distributed throughout the ECF in a manner urinary excretion of xanthine and sulfi te and decreased similar to chloride. The concentrations of fl uorine in urinary excretion of uric acid and sulfate. blood, where it is bound to albumin, and tissues are small. The elimination of absorbed fl uoride occurs Assessing status almost exclusively via the kidneys. Fluoride is freely Determining the body status of molybdenum is dif- fi ltered through the glomerular capillaries and under- fi cult. Homeostatic control of molybdenum ensures goes tubular reabsorption in varying degrees. that plasma concentrations are not elevated, except Fifty percent of orally ingested fl uoride is absorbed after extremely high dietary intakes. Decreased from the gastrointestinal tract after approximately 30 Minerals and Trace Elements 229 minutes. In the absence of high dietary concentra- effect in the metabolism of bacteria in dental plaque tions of calcium and certain other cations with which (i.e., reduced acid production) and on the dynamics fl uoride may form insoluble and poorly absorbed of enamel demineralization and remineralization compounds, 80% or more is typically absorbed. Body during an acidogenic challenge. The ingestion of fl uo- fl uid and tissue fl uoride concentrations are propor- ride during the pre-eruptive development of the teeth tional to the long-term level of intake; they are not also has a cariostatic effect because of the uptake of homeostatically regulated. About 99% of the body’s fl uoride by enamel crystallite and formation of fl uoride is found in calcifi ed tissues (bone and teeth), fl uorhydroxyapatite, which is less acid soluble than to which it is strongly but not irreversibly bound. hydroxyapatite. When drinking water contains 1 mg/l In general, the bioavailability of fl uoride is high, there is a coincidental 50% reduction in tooth decay but it can be infl uenced to some extent by the vehicle in children. Fluoride (at relatively high intakes) also with which it is ingested. When a soluble compound has the unique ability to stimulate new bone forma- such as sodium fl uoride is ingested with water, absorp- tion and, as such, it has been used as an experimental tion is nearly complete. If it is ingested with milk, drug for the treatment of osteoporosis. Recent evi- baby formula, or foods, especially those with high dence has shown an especially positive clinical effect concentrations of calcium and certain other divalent on bone when fl uoride (23 mg/day) is administered or trivalent ions that form insoluble compounds, in a sustained-release form rather than in forms that absorption may be reduced by 10–25%. Fluoride is are quickly absorbed from the gastrointestinal tract. absorbed passively from the stomach, but protein- bound organic fl uoride is less readily absorbed. Defi ciency symptoms The fractional retention (or balance) of fl uoride at The lack of exposure to fl uoride, or the ingestion of any age depends on the amount absorbed and the inadequate amounts of fl uoride at any age, places the amount excreted. In healthy, young, or middle-aged individual at increased risk for dental caries. Many adults, approximately 50% of absorbed fl uoride is studies conducted before the availability of fl uoride- retained by uptake in calcifi ed tissues and 50% is excreted containing dental products demonstrated that dietary in urine. In young children, as much as 80% can be fl uoride exposure is benefi cial, owing to its ability to retained owing to the increased uptake by the develop- inhibit the development of dental caries in both chil- ing skeleton and teeth. In later life, it is likely that the dren and adults. This was particularly evident in the fraction excreted is greater than the fraction retained. past when the prevalence of dental caries in commu- However, this possibility needs to be confi rmed. nities without water fl uoridation was shown to be much higher than that in communities who had their Metabolic function and essentiality water fl uoridated. Both the intercommunity trans- Although there is no known metabolic role in the port of foods and beverages and the use of fl uoridated body for fl uorine, it is known to activate certain dental products have blurred the historical difference enzymes and to inhibit others. While the status of in the prevalence of dental caries between communi- fl uorine (fl uoride) as an essential nutrient has been ties with and without water fl uoridation. This is debated, the US Food and Nutrition Board in 1997 referred to as a halo or diffusion effect. The overall established a dietary reference intake for the ion that difference in caries prevalence between fl uoridated might suggest their willingness to consider fl uorine to and nonfl uoridated area regions in the USA was be a benefi cial element for humans, if not an “essen- reported to be 18% (data from a 1986–1987 national tial nutrient.” survey), whereas the majority of earlier studies The function of fl uoride appears to be in the crys- reported differences of approximately 50%. Therefore, talline structure of bones; fl uoride forms calcium ingestion of adequate amounts of fl uoride is of fl uorapatite in teeth and bone. The incorporation of importance in the control of dental caries. fl uoride in these tissues is proportional to its total intake. There is an overall acceptance of a role for Toxicity fl uoride in the care of teeth. The cariostatic action Fluorine, like other trace elements, is toxic when (reduction in the risk of dental caries) of fl uoride on consumed in excessive amounts. The primary adverse erupted teeth of children and adults is owing to its effects associated with chronic, excessive fl uoride 230 Introduction to Human Nutrition intake are enamel and skeletal fl uorosis. Enamel fl uoridated water, beverages, and some infant formu- fl uorosis is a dose-related effect caused by fl uoride lae that are made or reconstituted with fl uoridated ingestion during the pre-eruptive development of the water, , and some marine fi sh. Because of the teeth. After the enamel has completed its pre-eruptive ability of tea leaves to accumulate fl uoride to concen- maturation, it is no longer susceptible. Inasmuch as trations exceeding 10 mg/100 g dry weight, brewed enamel fl uorosis is regarded as a cosmetic effect, it is tea contains fl uoride concentrations ranging from 1 the anterior teeth that are of most concern. The pre- to 6 mg/l depending on the amount of dry tea used, eruptive maturation of the crowns of the anterior the water fl uoride concentration and brewing time. permanent teeth is fi nished and the risk of fl uorosis Intake from fl uoridated dental products adds is over by 8 years of age. Therefore, fl uoride intake up considerable fl uoride, often approaching or exceeding to the age of 8 years is of most interest. Mild fl uorosis intake from the diet, particularly in young children (which is not readily apparent) has no effect on tooth who have poor control of the swallowing refl ex. The function and may render the enamel more resistant major contributors to nondietary fl uoride intake to caries. In contrast, the moderate and severe forms are toothpastes, mouth rinses, and dietary fl uoride of enamel fl uorosis are generally characterized by supplements. esthetically objectionable changes in tooth color and In 1997 the US Food and Nutrition Board estab- surface irregularities. lished AI values for fl uoride: infants 0.01 mg (fi rst 6 Skeletal fl uorosis has been regarded as having three months), 0.5 mg (7–12 months), children and adoles- stages. Stage 1 is characterized by occasional stiffness cents 0.7, 1.0, and 2.0 mg (1–3, 4–8, and 9–13 years, or pain in joints and some osteosclerosis of the pelvis respectively), male adolescents and adults 3 and 4 mg and vertebrae, whereas the clinical signs in stages 2 (14–18 and 19 years and older, respectively), female and 3, which may be crippling, include dose-related adolescents and adults 3 mg (over 14 years, including calcifi cation of ligaments, osteosclerosis, exostoses, pregnancy and lactation). The AI is the intake value and possibly osteoporosis of long bones, muscle of fl uoride (from all sources) that reduces the occur- wasting, and neurological defects owing to hypercal- rence of dental caries maximally in a group of indi- cifi cation of vertebrae. The development of skeletal viduals without causing unwanted side-effects. With fl uorosis and its severity are directly related to the fl uoride, the data are strong on caries risk reduction level and duration of exposure. Most epidemiological but the evidence upon which to base an actual require- research has indicated that an intake of at least 10 mg/ ment is scant, thus driving the decision to adopt an day for 10 or more years is needed to produce the AI as the reference value. clinical signs of the milder form of the condition. Crippling skeletal fl uorosis is extremely rare. For Micronutrient interactions example, only fi ve cases have been confi rmed in the The rate and extent of fl uoride absorption from the USA since the mid-1960s. gastrointestinal tract are reduced by the ingestion of Based largely on the data on the association of high foods particularly rich in calcium (such as milk or fl uoride intakes with risk of skeletal fl uorosis in chil- infant formulae). dren (>8 years) and adults, the US Food and Nutri- tion Board has established a tolerable UL of fl uoride 9.15 Chromium of 10 mg/day for children (>8 years), adolescents, and adults, as well as pregnant and lactating women. Chromium has an abundance of 0.033% in the Earth’s crust. It is a transition element that can occur in a Assessing status number of valence states, with 0, +2, +3, and +6 being A high proportion of the dietary intake of fl uoride the most common. Trivalent chromium is the most appears in urine. Urinary output in general refl ects stable form in biological systems. The principal ore is the dietary intake. chromite. Chromium is used to harden steel, to manufacture stainless steel, and to form many useful Requirements and dietary sources alloys. It fi nds wide use as a catalyst. Hexavalent chro- Most foods have fl uoride concentrations well below mium is a strong oxidizing agent that comes primarily 0.05 mg/100 g. Exceptions to this observation include from industrial sources. Minerals and Trace Elements 231

Absorption, transport, and symptoms of a patient on total parenteral nutrition tissue distribution were reversed by supplemental chromium. Chromium The human body contains only a small amount of functions primarily through its role in the regulation chromium, less than 6 mg. The kidney, followed of insulin. Adequate dietary chromium leads to a nor- by the spleen, liver, lungs, heart, and skeletal muscle malization of insulin, with reductions in blood glucose are the tissues with the greatest chromium concentration in subjects with elevated blood glucose concentrations. levels, increases in subjects with low blood glucose Absorbed chromium is excreted primarily in urine levels, and no effect on subjects with near-optimal and only small amounts of chromium are lost in the glucose tolerance. Improved insulin function is also hair, sweat, and bile. Therefore, urinary chromium associated with an improved lipid profi le. Supplemental excretion can be used as an accurate estimation of chromium also leads to increased insulin binding and absorbed chromium. At normal dietary chromium increased insulin receptor numbers, and recent evi- intakes (10–40 μg/day), chromium absorption is dence suggests that chromium may be involved in inversely related to dietary intake. Chromium intake the phosphorylation and dephosphorylation of the is approximately 0.5% at a daily intake of 40 μg/day insulin receptor proteins. and increases to 2% when the intake drops to 10 μg/ Defi ciency symptoms day. The inverse relationship between chromium intake and absorption appears to be a basal control The hallmark of marginal chromium defi ciency is mechanism to maintain a minimal level of absorbed impaired glucose tolerance. In studies of patients chromium. It is absorbed in the small intestine, pri- whose total parenteral nutrition solutions contained marily in the jejunum in humans. The mechanism is no chromium or were supplemented with inadequate not well understood, but a nonsaturable passive dif- amounts of chromium, insulin requirements were fusion process seems likely. Ascorbic acid promotes reduced and glucose intolerance was reversed with chromium absorption. chromium chloride supplementation. Two of these Chromium absorption in young and old subjects patients had weight loss that was restored with chro- is similar, but insulin-dependent diabetic patients mium supplementation. Peripheral neuropathy was absorb two to four times more chromium than other seen in one of the patients and it too was reversed apparently healthy subjects. Diabetic subjects appear with chromium supplementation. to have an impaired ability to convert inorganic chro- Toxicity mium to a usable form. Therefore, diabetic subjects require additional chromium and the body responds Trivalent chromium, the form of chromium found in with increased absorption, but the absorbed chro- foods and nutrient supplements, is one of the least mium cannot be utilized effectively and is excreted in toxic nutrients. The chromium often found in paints, the urine. The chromium content of tissues of these welding fumes, and other industrial settings is hexava- patients is also lower. lent and is several times more toxic than the trivalent Chromium is transported to the tissues primarily form. Because trivalent chromium is poorly absorbed, bound to transferrin, the same protein that transports high oral intakes would be necessary to attain toxic iron. It has been hypothesized that iron interferes levels. In 2001, the US Food and Nutrition Board with the transport of chromium in hemochromatosis concluded that there are insuffi cient data to establish and that this may explain the high incidence of dia- a tolerable UL for trivalent chromium. However, betes in hemochromatosis patients, and which may be because of the current widespread use of chromium induced by chromium defi ciency. supplements, more research is needed to assess the safety of high-dose chromium intake from supplements. Metabolic function and essentiality Chromium in the trivalent form is an essential nutri- Assessing status ent that functions in carbohydrate, lipid, and nucleic There is no accurate method for reliable detection of acid metabolism. The essentiality of chromium was marginal chromium defi ciency. Chromium concen- documented in 1977 when the diabetic signs and trations in hair, urine, blood, and tissues can be used 232 Introduction to Human Nutrition to assess recent chromium exposure, but are not long- a biological role or benefi cial action in humans. term measures of chromium status. The only reliable Two elements, fl uorine and lithium, have benefi cial indicator of chromium status is to monitor blood actions when ingested in high (pharmacological) levels of glucose, insulin, lipid, and/or related vari- amounts. Lithium is used to treat bipolar disorder, ables before and after chromium supplementation. A and fl uorine (as fl uoride) is discussed in Section response in blood glucose can often be seen in 2 weeks 9.14 because of its important benefi cial actions in or less, whereas effects on blood lipids may take preventing dental caries in susceptible population longer. groups. Some consider that the circumstantial evidence for chromium is suffi ciently substantial to Requirements and dietary sources warrant special attention in dietary requirement rec- The dietary chromium content of foods varies widely. ommendations, and this element is discussed in The richest dietary sources of chromium are spices Section 9.15. The estimated or suspected requirement such as , brewer’s yeast, mushrooms, of all of these elements (including the essential prunes, raisins, nuts, asparagus, beer, and wine. trace elements, iodine, selenium, and molybdenum) Refi ning of cereals and sugar removes most of the is usually less than 1 mg/day and they are defi ned native chromium, but stainless-steel vessels in as ultratrace elements. Cobalt is not included in the contact with acidic foods may contribute additional list of ultratrace elements because the only require- chromium. ment for cobalt is as a constituent of preformed

There is currently no RDA set for dietary chro- vitamin B12. mium, instead there are AI values [which were estab- These elements are not discussed at length in this lished by the US Food and Nutrition Board in 2001]: chapter and the reader is referred to other reading infants 0.2 μg (fi rst 6 months), 5.5 μg (7–12 months), material. For completeness, three tables, on absorp- children 11 and 15 μg (1–3 and 4–8 years, respec- tion, transport, and storage characteristics (Table tively), teenage boys 25 and 35 μg (9–13 and 14–18 9.21), excretion, retention, and possible biological years, respectively), adult men 35 and 30 μg (19–50 roles of the ultratrace elements (Table 9.22), and years and 50 years and older, respectively), teenage human body content and food sources (Table 9.23) girls 21 and 24 μg (9–13 and 14–18 years, respec- are included here. tively), adult women 25 and 20 μg (19–50 years and 51 years and older, respectively), pregnant women 29 and 30 μg (less than 18 years and 19–50 years, respec- 9.17 Perspectives on the future tively), and lactating women 44 and 45 μg (less than The preceding parts of this chapter have highlighted 18 and 19–50 years, respectively). An AI was set based some issues in the area of minerals and trace elements on representative dietary intake data from healthy for which we have an incomplete understanding. In individuals from the Third Nutrition and Health the future, nutritional scientists, dieticians, and other Examination Survey (NHANES III). health care professionals will have to:

9.16 Other elements ● obtain a greater understanding of the molecular and cellular processes involved in the intestinal In addition to the essential elements discussed in absorption and tissue uptake of certain minerals this chapter, other elements in the periodic table and trace elements may emerge as being essential for human nutrition. ● identify functional markers of mineral and trace For 15 elements, aluminum, arsenic, boron, bromine, element status. These markers could be defi ned as cadmium, chromium, fl uorine, germanium, lead, a physiological/biochemical factor that (1) is related lithium, nickel, rubidium, silicon, tin, and vanadium, to function or effect of the nutrient in target specifi c biochemical reactions have not been defi ned tissue(s) and (2) is affected by dietary intake or and their suspected essentiality is based on circum- stores of the nutrient (which may include markers stantial evidence from data emanating from animal of disease risk). Examples of such indicators models, from essential functions in lower forms or markers are those related to risk of chronic dis- of life, or from biochemical actions consistent with eases, such as osteoporosis, coronary heart disease, Minerals and Trace Elements 233 ; bone a possible -macroglobulin; no 3 2 -histidine and α L probably is both a storage and transport vehicle storage site serum albumin with small amounts bound to undissociated B(OH) organ accumulates physiological amounts of nickel a possible storage site converted into monomethylarsonic acid and dimethylarsinic acid; arsenobetaine not biotransformed; arsenocholine transformed to arsenobetaine undisassociated monomeric silicic acid vanadyl-ferritin; whether transferrin is the transport vehicle and ferritin is the storage vehicle for vanadium remains to be determined; bone is a repository for excess vanadium Bone can serve as a store for lithium Incorporated into metallothionein which ed None identifi Transported in blood principally bound to Transported Boron transported through the body as Transferrin carries aluminum in plasma; bone Transferrin None identifi ed None identifi Before excretion inorganic arsenic is Silicon in plasma believed to exist as None identifi ed; bone might be a repository None identifi Converted into vanadyltransferrin and >90% slightly soluble inorganic forms, 20–30%; inorganic forms with foods, 60–75%; methylated forms, 45–90% or poorly soluble silicates ~1% very low amounts are ingested Lithium chloride highly absorbed: 5% >90% Adults 5–15%, children 40–50% Bone is a repository for lead <10% with food >90% <1% Percentage of ingested absorbedPercentage and storage vehicles Transport Highly absorbed Soluble inorganic forms, >90%; Food silicon near 50%; insoluble ~3%; percentage increases when <10% and absorbed in 3 copyright 1999 with permission of Elsevier. , junctions and pericellular spaces passive diffusion intestine but evidence has been presented for an active transport perhaps involving the system for calcium metals (e.g. zinc) but mechanism is less effi cient for metals (e.g. zinc) but mechanism is less effi cadmium as an amino acid or other low molecular weight transport; occurs in the complex) and energy-driven small intestine this form, probably by passive diffusion the paracellular pathway; also, evidence for active absorption through processes shared with active processes of calcium; probably occurs in proximal duodenum; citrate combined with aluminum enhances absorption rubidium and potassium thought to share a transport system mucosal tissue, then absorption involves a simple movement down a concentration gradient; organic arsenic absorbed mainly by simple diffusion through lipid regions of the intestinal boundary been described phosphate or other anion transport systems; vanadyl has been suggested to use iron transport systems; absorption occurs in the duodenum been described Passive diffusion by paracellular transport via the tight Passive Has not been conclusively determined but probably is by Uncertain; thought to be by passive diffusion in small May share a common absorption mechanism with other Uncertain, evidence for both passive diffusion (perhaps Uncertain; some evidence for passive diffusion through Means of absorption Resembles potassium in its pattern of absorption; Ingested boron is converted into B(OH) Inorganic arsenate becomes sequestered in or on Mechanisms involved in intestinal absorption have not Vanadate has been suggested to be absorbed through Vanadate Mechanisms involved in intestinal absorption have not

et al. Encyclopaedia of Human Nutrition excretion and intestine arsenic as mostly dimethylarsinic acid and organic arsenic as mostly arsenobetaine excretion Major mechanism(s) for homeostasis Absorption LithiumNickel Urinary excretion Both absorption and urinary Table 9.21 Table Absorption, transport and storage characteristics of the ultratrace elements Element Germanium Urinary excretion Lead Absorption Cadmium Absorption Rubidium Excretion through kidney Arsenic Urinary excretion; inorganic Aluminum Absorption Silicon Both absorption and urinary Boron Urinary excretion Tin Vanadium Absorption Reproduced from Nielsen (1999) in Sadler 234 Introduction to Human Nutrition utilization infl uences the response to hormone action, infl transmembrane signaling or transmembrane movement of regulatory cations or anions labile methyl metabolism; regulation of gene expression Regulation of some endocrine function Facilitates iron absorption and/or utilization Facilitates Role in immune function Involved in metallathionein metabolism and Electrolyte balance Possible biological role Possible Cell membrane function or stability such that it Metabolism of methionine, or involved in Enzyme activator , in organic

cis , binds to proteins and amino

in vivo ion − mostly to hemoglobin but some binds a low molecular weight protein in erythrocytes involved in regulating cadmium distribution acids compounds; fi ve naturally occurring boron esters (all compounds; fi antibiotics) synthesized by various bacteria have been characterized forms ester complexes with hydroxyl groups, preferably those adjacent and organisms ranging from microbial to mammalian; methylated end-products include arsenocholine, arsenobetaine, dimethylarsinic acid and methylarsonic acid; arsenite methyltransferase and monomethylarsonic acid methyltransferase use S- adenosylmethionine for the methyl donor phospholipids; aluminum-bound transferrin apparently is a transport molecule None identifi ed None identifi Plasma lead mostly bound to albumin; blood lead binds None identifi ed None identifi Metallothionein, a high sulfhydryl-containing protein Molecules of biological importance Exists as Br Boron biochemistry essentially that of boric acid, which Methylation of inorganic oxyarsenic anions occurs in Aluminum binds to proteins, nucleotides and amounts in bile tract amounts in bile Urine Urine; also signifi cant Urine; also signifi Urine Urine and gastrointestinal Major excretory route after ingestion Urine Urine Urine Urine; also signifi cant Urine; also signifi Bone (100 ng/g) nodes (200 ng/g) Lymph Pituitary gland (135 ng/g) Bone (25 μg/g) Kidney (1–2 μg/g) Liver (1–2 μg/g) Liver (0.3 μg/g) (0.2 μg/g) Pancreas (0.5 μg/g) Testis Liver (4 μg/g) Organs of high content (typical concentration) Liver (40 μg/g) Lung (6.0 μg/g) (5.0 μg/g) Testis Fingernails (15 μg/g) Hair (1 μg/g) (5 μg/g) Teeth Nails (0.35 μg/g) Skin (0.10 μg/g) Lung (35 μg/g) Lithium Adrenal gland (60 ng/g) Lead Aorta (1–2 μg/g) Germanium Bone (9 μg/g) Cadmium Kidney (14 μg/g) Table 9.22 Table Excretion, retention, and possible biological roles of the ultratrace elements Element Bromine Hair (30 μg/g) Boron Bone (1.6 μg/g) Arsenic Hair (0.65 μg/g) Aluminum Bone (1–12 μg/g) Minerals and Trace Elements 235 and folic 12 collagen; role in the initiation of calcifi cation collagen; role in the initiation of calcifi and in collagen formation function acid; role similar to potassium; neurophysiological function metalloenzymes; role in a metabolic pathway involving vitamin B require vanadium for activity; a similar role may exist in higher forms of life Structural role in some mucopolysaccharides or Role in some redox reactions Role similar to potassium; neurophysiological Cofactor or structural component in specifi c Cofactor or structural component in specifi Lower forms of life have haloperoxiclases that 4+ entifi ed in higher forms of life. entifi component 2+ ) and 3 − or VO 4 − complexes with proteins, VO 2+ 2 ) is the form believed to exist in essential for enzymic hydrogenation, 4 3+ by various ligands including amino acids ), vanaclate (H 2− 2+ is absorbed and excreted more readily than Sn copyright 1999 with permission of Elsevier. plasma; magnesium orthosilicate is probably the form of silicon in urine. The bound form of silicon has ed never been rigorously identifi of urease; Ni desulfurization and carboxylation reactions in mostly anaerobic microorganisms (especially histidine and cysteine), proteins (especially albumin) and a macroglobulin called nickeloplasmin important in transport and excretion; Ni especially those associated with iron (e.g. transferrin, hemoglobin) peroxovanadyl [V-OO]; VO peroxovanadyl [V-OO]; 2+ , Sn Vanadyl (VO Vanadyl Silicic acid (SiOH None identifi ed None identifi Binding of Ni amounts in bile amounts in bile amounts excreted through intestinal tract weight complexes Urine; also signifi cant Urine; also signifi Urine; also signifi cant Urine; also signifi Urine Urine; also signifi cant Urine; also signifi Urine as low molecular

et al. Encyclopaedia of Human Nutrition Bone (0.8 μg/g) Kidney (0.2 μg/g) Liver (0.4 μg/g) Kidney (120 ng/g) Liver (120 ng/g) Spleen (120 ng/g) (200 ng/g) Testis Bone (18 μg/g) Skin (4 μg/g) (12 μg/g) Tendon Kidney (5 μg/g) Liver (6.5 μg/g) (20 μg/g) Testis Bone (33 ng/g) Kidney (10 ng/g) Thyroid (30 ng/g) Tin Vanadium Bone (120 ng/g) Silicon Aorta (16 μg/g) Rubidium Brain (4 μg/g) Nickel Adrenal glands (25 ng/g) Reproduced from Nielsen (1999) in Sadler None of the suggested biological functions or roles of any of the ultratrace elements has been conclusively or unequivocally id 236 Introduction to Human Nutrition

Table 9.23 Human body content and defi cient, typical, and rich sources of intakes of ultratrace elements

Apparent defi cient intake Typical human daily Element (species) Human body content dietary intake Rich sources

Aluminum 160 μg/kg (goat) 30–50 mg 2–10 mg Baked goods prepared with chemical leavening agents (e.g. baking powder), processed cheese, grains, vegetables, herbs, tea, antacids, buffered analgesics Arsenic <25 μg/kg (chicks) 1–2 mg 12–60 μg Shellfi sh, fi sh, grain, cereal products <35 μg/kg (goat) <15 μg/kg (hamster) <30 μg/kg (rat) Boron <0.3 mg/kg (chick) 10–20 mg 0.5–3.5 mg Food and drink of plant origin, especially 0.25–0.35 mg/day (human) noncitrus fruits, leafy vegetables, nuts, <0.3 mg/kg (rat) pulses, legumes, wine, cider, beer Bromine 0.8 mg/kg (goat) 200–350 mg 2–8 mg Grain, nuts, fi sh Cadmium <5 μg/kg (goat) 5–20 mg 10–20 μg Shellfi sh, grains, especially those grown on <4 μg/kg (rat) high-cadmium soils, leafy vegetables Germanium 0.7 mg/kg (rat) 3 mg 0.4–3.4 mg Wheat bran, vegetables, leguminous seeds Lead <32 μg/kg (pig) Children less than 10 15–100 μg Seafood, plant foodstuffs grown under high- <45 μg/kg (rat) years old 2 mg, lead conditions Adults 120 mg Lithium <1.5 mg/kg (goat) 350 μg 200–600 μg Eggs, meat, processed meat, fi sh, milk, milk <15 μg/kg (rat) products, potatoes, vegetables (content varies with geological origin) Nickel <100 μg/kg (goat) 1–2 mg 70–260 μg Chocolate, nuts, dried beans and peas, grains <20 μg/kg (rat) Rubidium 180 μg/kg (goat) 360 mg 1–5 mg Coffee, black tea, fruits and vegetables (especially asparagus), poultry, fi sh Silicon <20 mg kg (chick) 2–3 g 20–50 mg Unrefi ned grains of high fi ber content, cereal <4.5 mg/kg (rat) products Tin <20 μg/kg (rat) 7–14 mg 1–40 mg Canned foods Vanadium <10 μg/kg (goat) 100 μg 10–30 μg Shellfi sh, mushrooms, parsley, dill, seed, black pepper, some prepared foods

Reproduced from Nielsen (1999) in Sadler et al. Encyclopaedia of Human Nutrition, copyright 1999 with permission of Elsevier..

hypertension or diabetes. However, for many nutri- status that merit further evaluation. The judicious ents there are as yet no functional indicators that application of methods in molecular biology respond to dietary intake and, in such cases, nutri- (including genomics and proteomics) and nonin- ent requirements are established using more tradi- vasive imaging techniques is likely to provide new tional approaches, such as balance data. The lack of breakthroughs and rapid advances in the nutrition functional markers of mineral and trace element and biology of trace elements status is a signifi cant disadvantage for studies relat- ● evaluate further the specifi c health risks associated ing their intake or status to health outcomes such with marginal defi ciencies of various minerals and as hypertension, cardiovascular disease, osteoporo- trace elements. There is a need to determine reliable sis, diabetes, and other disorders. For example, relationships between mineral status and disease widely used biochemical indicators of essential and then to demonstrate that the incidence or trace element status generally lack both the sensi- severity of specifi c diseases is reversible by repletion tivity and the specifi city that are required to defi ne of mineral status. The development and validation optimal intake at various stages of the life cycle. of reliable assessment tools and functional markers Recent efforts have provided a number of potential of mineral status are the utmost priority for this “sensors” of cellular copper, zinc, and manganese fi eld Minerals and Trace Elements 237

● defi ne the adverse effects of acute and chronic high and Nutrients in the UK. HMSO, Department of Health, London, intakes of some minerals and trace elements. 1991. Hallberg L, Sandstrom B, Aggett PJ. In: JS Garrow, WPT James, A Interest in mineral fortifi cation of foods is higher Ralph, eds. Human Nutrition and Dietetics, 9th edn. Churchill than ever before. Governments worldwide are Livingstone, London, 1993. increasingly tackling the common defi ciencies of Holland B, Welch AA, Unwin ID, Buss DH, Paul AA, Southgate DAT, eds. McCance & Widdowson’s The iron and iodine by adding these minerals to widely Composition of Foods, 5th edn. Royal Society of Chemistry and consumed staple foods such as cereal fl ours, sugar, Ministry of Agriculture, Fisheries and Food. London: HMSO, or soy sauce. The food industry in industrialized 1995. Institute of Medicine (USA). Dietary Reference Intakes for Calcium, countries is manufacturing an increasing number Phosphorus, Magnesium, Vitamin D, and Fluoride. National of functional foods designed to provide the con- Academy Press, Washington DC, 1997. sumer with protection against diseases of major Institute of Medicine (USA). Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, public health signifi cance, such as osteoporosis, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. cancer, and heart disease, and fortifi ed with miner- National Academy Press, Washington DC, 2001. als such as calcium, selenium, zinc, magnesium, and Mills CF, ed. Zinc in Human Biology. Springer-Verlag, London, 1989. copper. The same minerals are added to dietetic Nielsen F. In: MJ Sadler, JJ Strain, B Caballero, eds. Encyclopedia of products, including infant foods, foods for preg- Human Nutrition. Academic Press, London, 1999. nant and lactating women, and enteral feeds for Rayman MP. The importance of selenium to human health. 2000; Lancet 356: 233–241. hospital patients, all designed to cover the nutri- Reilly C. Selenium in Food and Health. Blackie, London, 1996. tional requirements of specifi c consumers. This Reilly C. The Nutritional Trace Metals. Blackwell Publishing, raises the issues not only of the possible health ben- Oxford, 2006 Sánchez-Castillo CP, James WPT. In: MJ Sadler, JJ Strain & B efi ts of fortifi cation but also of possible toxicity. Caballero, eds. Encyclopedia of Human Nutrition. Academic Therefore, there is an ever-increasing emphasis Press, London, 1999. placed on upper safe levels of mineral intake and Strain JJ, Benzie IFF. In: MJ Sadler, JJ Strain & B Caballero, eds. Encyclopedia of Human Nutrition. London: Academic Press, on fortifi cation legislation 1999. ● elucidate the impact of single nucleotide polymor- phisms in the human genome on mineral and trace Further reading element dietary requirements. The key to future applications of the DNA polymorphisms will be to Bowman B, Russel R, eds. Present Knowledge in Nutrition, 8th edn. mine the human genome for DNA sequence infor- ILSI Press, Washington DC, 2001. Passmore R, Eastwood MA, eds. Davidsons and Passmore Human mation that can be used to defi ne biovariation in Nutrition and Dietetics, 8th edn. Churchill Livingstone, London, nutrient absorption and use. However, before this 1986. can be accomplished, a vast amount of nutritional Optimal Nutrition Symposium. A series of papers. Proceedings of the Nutrition Society 1999; 58: 395–512. biology research is needed to correlate gene poly- Sadler MJ, Strain JJ, Caballero B, eds. Encyclopedia of morphism with nutritional outcomes. Human Nutrition, Vol. 3, Parts 1–3. Academic Press, London, 1999. References Website Online Mendelian Genetics in Man (OMIM) website at the Committee on Medical Aspects of Food Policy. Report on Health National Institute for Biotechnology Information: http://www. and Social Subjects 41. Dietary Reference Values for Food Energy ncbi.nlm.nih.gov/Omim 10 Measuring Food Intake

Una E MacIntyre

Key messages

• Measuring the food intake in free-living individuals is a complex • To increase our understanding of the error associated with mea- task. surements of food intake it is also necessary to develop and use • All measurements of food intake are subject to sources of error. physiological and biochemical markers of food intake. • The dietary assessment method used depends on the purpose of • To evaluate food intake data effectively it is important to collect the study. suffi cient additional data to allow individuals to be identifi ed not • The existence of error means that it is always important to be only by age and gender, but also by body mass index, physical aware of and, whenever possible, to assess the nature and activity, and supplement use. magnitude of the error.

10.1 Introduction Assessment of nutritional status Nutritional health is maintained by a state of equilib- The purpose of this chapter is to describe the various rium in which nutrient intake is balanced by nutri- ways in which one can determine what people eat. The tional requirements. Malnutrition occurs when net task may be to fi nd out about the national food supply, nutrient intake is less than requirements (undernutri- the usual intake of a group or a household, or the tion) or exceeds requirements (overnutrition). Both intake of a given individual over a specifi ed period. under- and overnutrition lead to metabolic changes The many reasons for fi nding out about the food which have acute and chronic consequences for that people eat fall into three broad categories: health. 1 Public Health: to evaluate the adequacy and safety There is no ideal tool to measure a person’s nutri- of the food that people eat at national or commu- tional status accurately. Attempts to predict the infl u- nity level and to identify the need for or to evaluate ence of malnutrition based on single measurements nutrition-based intervention programs. fail to consider the many interacting factors between 2 Clinical: to assist with the prevention, diagnosis, nutrition and disease state. For this reason, it is and treatment of diet-related conditions. necessary to look at several different measurements 3 Research: to study the interrelationships between in order to assess a person’s nutritional status. This food intake and physiological function or disease process is known as the A, B, C, D of nutritional conditions under controlled conditions or in fi eld assessment: conditions. ● Anthropometry (discussed in detail in Chapter 2) The kind and amount of food intake data required ● Biochemical and hematological variables differ in each situation and may require data at the ● Clinical and physical assessment national, community, household, or individual level. ● Dietary intake.

© 2009 UE MacIntyre. Measuring Food Intake 239

The rest of this chapter will concentrate on the advantages, and disadvantages. It is, therefore, measurement of food (dietary) intake, but more essential that the purpose of collecting dietary data detailed descriptions of the assessment of nutritional is clearly defi ned, so that the most appropriate status, at the individual and population level, can be dietary assessment method is used. found in the Public Health Nutrition and Clinical It is also essential to recognize that fi nding out what Nutrition textbooks in this series. people eat requires adequate resources. Appropriately Obtaining data on food intake is probably the most trained personnel must be employed not only for the diffi cult aspect of nutritional assessment and is asso- period of data collection but also for the time it takes ciated with several problems: to review, enter, and analyze the data. It may not always be necessary to obtain detailed data on food ● “Food intake” is not a simple measure of one vari- intake in order to answer a particular question. When able, such as weight or height, but requires data on resources are limited it is probably more useful to the intake of many different food items. collect limited data of high quality than to attempt to ● Food intake data are subject to many sources of collect comprehensive dietary data with inadequate variability, since even the same individuals eat dif- resources. Being able to recognize this situation is ferent foods, at different times, in different places, important for maximizing available resources. Table in many different combinations, and with many 10.1 lists the different approaches to measurement of different preparation methods. The net effect of all food intake that are described in this chapter. these sources of variability is that more data are Finally, it is important that the interpretation and needed to generate reliable results than would be application of data derived from food intake studies the case with a less variable measure. take into account the limitations of the data. This ● We are rarely in a position to know the truth about clearly does not improve the quality of the data per food intake. With many biological measurements it se, but maximizes their usefulness for the purpose for is possible to check the results obtained against a which they were originally collected. Recognition of reference method that is known to give accurate the limitations of dietary data involves more than results or by means of an independent measure. For simply stating the limitations. External comparisons example, we can check an infant’s birth weight by to check whether the data are consistent with inde- checking the accuracy of the weighing equipment pendent sources of information on food intake and used to measure it by means of a standard weight to determine the likely direction and magnitude of or, if the information was obtained by means of a any bias are an integral part of the interpretation of questionnaire, we may be able to check the data dietary data. Relevant sources of comparative infor- from offi cial records. With food intake data we have mation may include food supply and expenditure to rely on the individuals who eat the food to data and physiological or biochemical measures provide us with the answers to our questions. We related to nutrient intake. ask individuals to remember what and how much they ate, to estimate how often they eat particular foods, or even, in some situations, to weigh or 10.2 Indirect measurement of measure their food intake for a number of days. For food intake this reason one of the most important consider- Indirect measurements of food intake make use of ations, when obtaining information on food intake information on the availability of food at national, from individuals, is to take all possible steps to regional, or household levels to estimate food intakes, obtain their full cooperation. It is also extremely rather than using information obtained directly from important that individuals understand the purpose individuals who consume the food. Indirect methods of the process and what is expected of them. This are most useful at the population and household may well involve much time and effort on the part levels for determining the amount and types of of the investigator(s), but is essential for high- foods: quality data. ● There are a number of different methods to obtain ● available for consumption at national level dietary intake data. Each method has its purposes, (commodity-level food supply data) 240 Introduction to Human Nutrition

Table 10.1 Approaches to the measurement of food intake in population groups, households, and individuals

Type and nature of data Name of method Used for assessing differences between

Commodity-level food supply data, e.g., production, Food balance sheets Countries and regions of the world imports, and exports (indirect) Product-level food supply data, e.g., retail and Food disappearance data Country, locality, and season wholesale sales data (indirect) Household food expenditure, e.g., money spent on food Household expenditure surveys Country, locality, season, and type of (indirect) household Household food acquisition, e.g., amount of food Household budget surveys Country, locality, season, and type of entering the household (indirect) Household food account household Household food procurement Household food inventory Household food consumption (direct) Household food records Country, locality, season, and type of household Qualitative record of foods (but not amounts) eaten Menu records Geographical, seasonal, and demographic over the course of 1–7 days by individuals (direct) subgroups and individuals Quantitative record of food intake, e.g., record of foods Weighed records and records Geographical, seasonal, and demographic eaten over the course of 1–7 days by individuals estimated in household measures subgroups and individuals (direct) Qualitative or semiquantitative recall, usually of a Food frequency questionnaires Geographical, seasonal, and demographic specifi ed list of foods, eaten in the previous month subgroups and individuals or year by individuals (direct) Quantitative recall of foods eaten on the previous day, Single or multiple 24 hour recalls Geographical, seasonal, and demographic usually obtained from individuals by interview subgroups and individuals (if multiple (direct) recalls obtained) Quantitative recall of habitual intake in the immediate Diet history Temporal and demographic subgroups and past obtained from individuals by interview (direct) individuals

● traded at wholesale or retail levels (product-level human consumption by relevant population statis- food supply data) tics. An analysis of the energy, protein, and fat pro- ● purchased at household level (household-based vided by the food item may also be given. budget/expenditure data). The Food and Agriculture Organization (FAO) has compiled and published food balance sheet data for Commodity level food supply data most countries in the world since 1949. Regularly Food supply data are usually produced at national updated food balance sheet data are available online level from compilations of data from multiple sources. at www.fao.org for most countries for about 100 The primary sources of data are records of agricul- primary crop, livestock, and fi shery commodities and tural production and food exports and imports some products such as sugar, oils, and fats derived adjusted for changes in stocks and for agricultural from them. Table 10.2 shows an extract from the food and industrial use of food crops and food products. balance sheet for the Southern African Development National food supply data are usually referred to as Community for 2003. “food balance sheets” or as “apparent consumption The accuracy of food balance sheets and apparent data.” Food balance sheets give the total production consumption data depends on the reliability of the and utilization of reported food items and show the basic statistics used to derive them, i.e., population, sources (production, stocks, and imports) and utiliza- supply, utilization, and . These tion (exports, industrial use, wastage, and human can vary markedly between countries not only in consumption) of food items available for human con- terms of coverage but also in terms of accuracy. sumption in a country for a given reference period. Several internal and external consistency checks are The amount of each food item is usually expressed built into the preparation of the FAO food balance per caput (per person) in grams or kilograms per year sheets, but users still need to evaluate the data for by dividing the total amount of food available for themselves in the context of the purpose for which Fat/capita/ day (g) 5.79 0.12 0.01 Proteins/ capita/ day (g) Calories/ capita/ day Food/ capita/year (kg) 118.4 960.06 24.82 7.11 10.49 103.23 1.98 0.15 Food quantity (1000 tonnes) 909.45 4.31 37.21 1.1 0.33 50.17 0.24 1.49 0.05 0.03 7.12 0.03 0.26 0.01 0 2.47 5882.29 27.88 171.31 5.18 0.68 26.33 10.22 0.05 0.34 0.01 0 Other uses quantity (1000 tonnes) 322.87 15438.9 73.16 627.56 16.05 0 410.35 1.95 16.46 0.38 0 62.76 0.3 2.2 0.06 Waste Waste quantity (1000 tonnes) Food manufacture (1000 tonnes) , 2003 Seed quantity (1000 tonnes) 42.84 2.57 58.87 4.67 2213.09 Feed quantity (1000 tonnes) 4823.37 473.57 1634 1317.2 356.34 24984.3 5.93 0.08 0 0.01 0 Domestic supply (1000 tonnes) 6099.89 22.68 68.04 19.05 105.43 1779.27 216.22 27.69 495.46 130.45 0 98.52 44.7 648.09 16.2 2.04 16.67 0 156.42 1.67 48.45 0 631.12 18.17 6.54 559.87 10 76.05 12.92 0.07 0 0.39 Export quantity (1000 tonnes) 1164.19 21919.27 4485.34 309.62 400.67 961.91 Stock variation (1000 tonnes) Import quantity (1000 tonnes) 24862.65 8500.95 1847.63 1622.89 33588.34 Production Production quantity (1000 tonnes) 857.34 1432.74 70.97 38.06 2322.99 1.2 0.23 13.39 0 0.48 13.14 excluding beer equivalent) Cereals, Wheat 2041.13 4256.29 183.54 381.07 Reproduced with permission of FAO (http://fao.org). Reproduced with permission of FAO Rice (milled Maize 19463.18 2271.36 1348.92 OatsMillet 33.91 629.77 34.63 11.22 31.07 9 1.08 1.91 Sorghum 1521.78 81.1 180.5 4.11 Table 10.2 Table Extract from the food balance sheet for the Southern African Development Community Items Cereals, otherCereals, 37.9 44.75 5.01 11.61 Barley 277.4 355.47 18.64 20.39 242 Introduction to Human Nutrition they are being used. One of the crucial factors in using Over a period of years, food balance sheets show the data appropriately is to understand the terminol- trends in national food supply and food consumption ogy used. Box 10.1 provides “in principle” defi nitions patterns. They may be used for population compari- of the key terms. sons such as comparing population estimates of fat Food balance sheets provide important data on intake with cardiovascular disease rates. food supply and availability in a country and show In practice, the data needed to compile food balance whether the food supply of the country as a whole is sheets are not always available and imputations or adequate for the nutritional needs of its population. estimates may have to be used at each stage in the calculation of per caput food and nutrient availability. In most industrialized countries reliable data are usually available on primary commodities, but this Box 10.1 is not necessarily the case for the major processed Commodity coverage: all potentially edible commodities whether products. For example, data may be available on fl our used for human consumption or used for nonfood purposes. but not on products such as bread and other cereal Exports: all movements out of the country during the reference products made from fl our that may have quite differ- period. ent nutrient characteristics. The overall impact of Feed: the quantity of the commodity available for feeding livestock incomplete data will vary from country to country, and poultry. Food quantity: the amounts of the commodity and any commod- but it has been suggested that in general underestima- ity derived therefrom available for human consumption during the tion of per caput availability of nutrients is more reference period, e.g., for maize includes maize, maize meal, and likely in less developed countries and overestimation any other products derived from maize that are available for in countries where most of the food supply is con- human consumption. sumed in the form of processed products. Imports: all movements of the commodity into the country, e.g., commercial trade, food aid, donated quantities, and estimates of It is also very important to keep in mind that food unrecorded trade. balance sheets show only data on foods available for Industrial uses: commodities used for manufacture for nonfood consumption, not the actual consumption of foods; purposes, e.g., oils for soap. nor do they show the distribution of foods within the Per caput supply: adjustments are made when possible to the population, for example among different regions resident population for temporary migrants, refugees, and tourists. The fi gures represent only the average supply available for the or among different socioeconomic, age, and gender population as a whole and not what is actually consumed by indi- groups within the population. Food balance sheets viduals. Many commodities are not consumed in the primary form also do not provide information on seasonal varia- in which they are reported in food balance sheets. To take this into tions in food supply. account the protein and fat content shown against primary com- modities are derived by applying appropriate food composition Product-level food supply data factors to the relevant amounts of processed foods, and not by multiplying the quantities shown in the food balance sheet by food In some countries (e.g., Canada and the USA) data composition factors relating to the primary commodity. on per caput food availability are prepared from Production: total domestic production whether produced inside or information on raw and processed foods available at outside the agricultural sector, i.e., includes noncommercial pro- the retail or wholesale level. Such data are derived duction and production from home gardens. Seed: quantity of the commodity set aside for sowing or plant- mainly from food industry organizations and fi rms ing or any other form of reproduction for animal or human engaged in food production and marketing such consumption. supermarkets. Errors arise mainly from inappropriate Stock variation: changes in stocks occurring during the reference conversion factors for processing, the absence of data period at all levels between the production and retail levels, i.e., for some processed products, and the lack of data on stocks held by the government, manufacturers, importers, export- ers, wholesale and retail merchants, and distributors. In practice, food obtained from noncommercial sources such as the available information often only relates to government stocks. home gardens, fi shing, and hunting. Waste: commodities lost through all stages between postharvest Commercial databases such as those produced by production and the household, i.e., waste in processing, storage, the AC Neilsen Company and the electronic stock- and transportation, but not domestic waste. Losses occurring control records from individual supermarkets, from during the manufacture of processed products are taken into account by means of extraction/conversion rates. which they are compiled, have the potential for moni- toring national-, regional-, and local-level trends in Measuring Food Intake 243 the food supply, at a product-specifi c level. Their groups, to identify high-risk groups for nutrition- principal disadvantage at present lies in the costs related conditions, to monitor trends in food patterns associated with processing or otherwise accessing, on over time, and for developing nutrition policy. a regular basis, the very large amounts of data that are A modifi cation of the HBS, known as the list recall involved. method, includes quantities of items purchased, FAO food balance sheets and similar sources of which strengthens the information. The information information are primarily useful for formulating available from HBSs, however, also needs to be con- agricultural and , for monitoring changes sidered in the context of their limitations. in national food supplies over time, and as a basis for ● Information provided by HBSs differs from country forecasting food consumption patterns. They can also to country both in the number of food items be used to make intercountry comparisons of food recorded and in the type of information collected. and nutrient supplies, provided that potential differ- ● Most surveys do not include expenditure informa- ences in data coverage and accuracy are taken into tion on food consumed outside the home. account. ● Most surveys do not collect information on foods Household-based surveys acquired by means other than purchase. For example, food obtained as gifts, produced by the Household-based surveys determine the foods and household itself, or harvested from the wild. beverages available for consumption at family, house- ● Most surveys do not collect information on domes- hold, or institutional levels. Some surveys such as tic wastage, i.e., food given to pets, spoiled food and household expenditure or household budget surveys plate waste, or food provided for guests. determine the amount of money spent on food for a ● It is often diffi cult to estimate the nutrient content given period, while others, such as the food account, of the food available to the household because data food inventory, and food record methods, attempt to are reported only at food group and not individual describe the food available and/or consumed by a food level. household or institution. ● Differences in food coding systems between coun- tries make it diffi cult to compare data between Household food expenditure surveys countries. Household food expenditure surveys determine the amount of money spent on food by a household over Three conclusions emerge from this list. First, the a given period. Household food expenditure data can data obtained from HBSs are not necessarily compa- provide useful information for nutritionists on food rable between countries. Second, most HBSs do not expenditure patterns of different types of households, collect all of the information needed to provide an but without quantitative information this cannot be accurate assessment of the total food supply available translated into nutrient patterns. at household level. Third, provided that the HBS Household budget surveys (HBSs) are conducted methodology remains consistent, HBSs can provide a at regular intervals in many European countries and great deal of valuable information about food pat- many also collect data on food quantities as well as terns over time, in different sociodemographic groups, cost. HBSs have several advantages: and in different parts of the country, and how these relate to social, economic, and technological changes ● they are usually conducted at regular intervals of in the food supply. between 1 and 5 years ● they are conducted on representative samples of Household food account method households In the food account method, the household member ● the food supply information collected can be responsible for the household’s food keeps a record classifi ed by sociodemographic characteristics, of the types and amounts of all food entering the geographical location, and season. household including purchases, gifts, foods produced The large amount of nutrition-related information by the household itself such as from vegetable and collected by these surveys offers the potential to assess fruit gardens, foods obtained from the wild, or from the nutritional patterns of different population other sources. Amounts are usually recorded in retail 244 Introduction to Human Nutrition units (if applicable) or in household measures. be introduced by inaccurate memory or expression of Information may also be collected on brand names answers. and costs. The recording period is usually 1 week but may be as long as 4 weeks. Household food inventory method This method is used to obtain food selection pat- The food inventory method uses direct observation terns from populations or subgroups within a popu- to describe all foods in the household on the day of lation. It has the advantage of being fairly cost-effective the survey. The investigator records the types and and is particularly useful for collecting data from amounts of foods present in a household, whether large samples. It may also be repeated at different raw, processed, or cooked, at the time of the study. times of the year to identify seasonal variations in Information may also be collected on how and where food procurement. food is stored. The food account method does not measure food A food inventory may be combined with the food consumption, wastage, or other uses, nor does it account to determine the changes of food stocks account for foods consumed outside the home. It during the survey period. It may also be used together assumes that household food stocks stay constant with a food procurement questionnaire to describe throughout the recording period, which may not nec- the acquisition of foods in the household. This essarily be the case. For example, food purchases may method is time-consuming for the investigator and be done once a month and therefore stocks may be very intrusive for the respondent, but is useful when depleted in the days preceding the purchase. It also foods are procured by means other than purchase and does not refl ect the distribution of food within the when levels of food security in vulnerable households household and therefore cannot be used to determine need to be assessed. food consumption by individuals within the house- hold. Since the method relies on the respondents Household food record being literate and cooperative, bias may be introduced All foods available for consumption by the household in populations with high levels of illiteracy. The are weighed or estimated by household measures fact of having to record the acquisition may lead to prior to serving. Detailed information such as brand respondents changing their procurement patterns names, ingredients, and preparation methods are either to simplify recording or to impress the also recorded over a specifi c period, usually 1 week. investigator. This method provides detailed information on the food consumption patterns of the household, but it Household food procurement is very time-consuming and intrusive and relies questionnaire/interview heavily on the cooperation of the household. As for A food procurement questionnaire or interview may the other household methods, it does not provide be used as an alternative method to the food account information on distribution of food within the house- method. In this method, the respondent indicates, hold or on individual consumption. When details of from a list of foods, which are used, where these are the household composition are given, estimates of obtained, the frequency of purchase, and the quanti- individual intakes may be calculated. The method ties acquired for a given period. The uses of the food also does not determine foods eaten away from the procurement method are similar to those of the food home nor does it take into account food eaten by account: to describe food acquisition patterns of guests to the home. populations or subpopulations. In contrast to the food account method, it does not require the respon- 10.3 Direct measures of food intake dent to be literate as it may be administered as an interview and it does not infl uence purchasing or Information on food intake can be obtained directly other procurement patterns. from consumers in a number of different ways. Direct The food procurement questionnaire/interview measures are usually used to obtain food intake data does not provide information on actual food con- from individuals but may also be used to obtain data sumption or distribution within the household. As from households. For example, in societies where it is the method relies on recalled information, errors may usual for members of the household to eat out of the Measuring Food Intake 245 same pot this may be the only practical approach Portion size (g) × Frequency × because it does not disrupt the normal pattern of food Nutrient content per g intake. Unlike the indirect measures of food intake, direct measures provide suffi cient information on and summed for all foods eaten by each individual food consumption to convert the food intake into during the study period. energy and nutrient intakes. Direct measurement of food intake can be divided Irrespective of the method used, the process of into two basic approaches: obtaining food intake information and converting 1 reports of foods consumed on specifi ed days: menu this to energy and nutrient data is the same. The records, weighed records, estimated records, and 24 procedure for measuring food and nutrient intake hour recalls involves fi ve steps (Figure 10.1). 2 reports of food intake over a period in the past, 1 obtaining a report of all the foods consumed by which are used to construct typical food patterns: each individual food frequency questionnaires and diet histories. 2 identifying these foods in suffi cient detail to choose Records are usually limited to fairly short periods, an appropriate item in the food tables usually not longer than 7 consecutive days, while 3 quantifying the portion sizes recalls may relate to a single period of 24 hours or 4 measuring or estimating the frequency with which occasionally 48 hours. Diet histories and food fre- each food is eaten quency questionnaires relate to longer periods and 5 calculation of nutrient intake from food tables. their purpose is to obtain an assessment of habitual To convert the information on food intake into intake over the period in question and not a detailed nutrient intake, the nutrient content of each food day-to-day recall of what was eaten during that time. eaten is calculated from food tables as: When records are kept portions may be either weighed

Basic procedure Type of dietary assessment

Step 1 Report RECORD RECALL HISTORY FFQ

Step 2 Identify food Researcher’s choice Subject’s choice from food tables from questionnaire

Weighed/ Average portion Step 3 Quantify Estimated estimated or small/medium/large

Step 4 Frequency As reported Subject's Subject’s choice estimate from questionnaire

Step 5 Calculate intake Food code Food code x Portion weight x Portion weight x Nutrient content/g x Frequency x Nutrient content/g Figure 10.1 The fi ve basic steps in a dietary assessment and the variations according to different methods. FFQ, food frequency questionnaire. 246 Introduction to Human Nutrition

or estimated in terms of standard household mea- Box 10.2 sures. In recalls and questionnaires quantities always have to be estimated. Dimensions, photographs of Accuracy: the extent to which an estimate approximates the true foods, food models, and, sometimes, actual foods may value. be used to assist in this process. Each of these Bias: the extent by which an estimate differs from the true value. Coeffi cient of variation: the standard deviation of a set of obser- approaches has specifi c advantages and disadvantages vations expressed as a percentage of their mean. and no single method of measuring food intake can Habitual intake: an estimate of the long-term average intake of be regarded as the ideal method for all situations. an individual. Until recently, weighed intake recorded over a 7 day Precision: the extent to which a method is repeatable. Usually expressed in terms of the coeffi cient of variation, i.e., the standard period was taken as the reference method against deviation of the results of repeated analyses of the same para- which less detailed methods were compared. It has, meter expressed as a percentage of their mean. however, been realized that this method has its limita- Random errors: errors that are randomly distributed about the tions and that it is not only desirable but also neces- true value. Random errors increase the variability of a set of obser- sary to use physiological and biochemical measures vations but do not affect their mean. Repeatability (reproducibility): a method is repeatable or repro- to determine whether any method of measuring ducible when it gives the same result on repeated measurement. food intake is actually measuring what it sets out to Systematic errors: errors that are not randomly distributed about measure. This will be discussed in more detail in the true value. Systematic errors can increase or decrease the vari- Section 10.6. ability of a set of observations and also affect the estimate of their mean. The effect on the mean is referred to as bias. Validity: a method is valid if it measures what it is intended to Basic concepts measure, i.e., the true value: Before describing the most commonly used direct • Absolute validity or accuracy: terms used to describe the dietary assessment methods it is appropriate to extent to which a method measures the true value. It is not pos- introduce four fundamental concepts relevant to the sible to determine the absolute accuracy of a dietary method by process of dietary assessment and evaluation. A brief comparison with another dietary method. • Face or content validity: a method which gives results that are defi nition of terms related to these concepts is given consistent with other data related to the parameter they are in Box 10.2. Terms are listed in the box in alphabetical intended to measure. order for ease of reference. • Relative (comparative) validity: the extent to which a test method performs in relation to a criterion or reference method, Habitual intake i.e., the dietary method judged to provide the “best” available measure of the true value. The objective of virtually all dietary assessments is to • Validity at group level: a method is valid at the group level if obtain an estimate of the habitual or average long- it provides an unbiased estimate of mean intake for the group. term intake for the group or the individual of interest. • Validity at individual level: a method is valid at the individual Habitual intake represents what is “usual” in the long level if it is able to rank respondents correctly (usually evaluated in terms of the same proportion in the same quintile or tertile term and not simply at a specifi c moment in time. It of the distribution compared with the “true” or more usually a is this level of intake that is relevant for maintenance “reference” value). of energy balance and nutrient status, and for the Variance: statistical term to describe the variation that occurs in a assessment of relationships between nutrient intake set of observations. It is equal to the square of the standard devia- and health in the long term. Habitual intake, however, tion of the individual observations: is diffi cult to measure because food intake varies • Between-person variance: the variance arising from differ- widely from day to day and, to a lesser extent, from ences between individuals. week to week and month to month. Figure 10.2 illus- • Within-person variance: the variance arising from differences trates the energy intake of one individual who main- within individuals. tained a weighed record every sixth day for 1 year. The horizontal line indicates the overall average intake intake over 7 days can differ by as much as 20% from over the year in MJ per day. The open circles show the the overall mean. intake on individual days and the solid circles the average intake over 7 day periods. It is obvious that The nature of error intake on a single day does not provide a reliable There is not, and probably never will be, a method estimate of habitual intake and that even average that can estimate dietary intake without error. This Measuring Food Intake 247

15

MJ/day 10

Figure 10.2 Energy intake of one indi- vidual from weighed records obtained 5 for 1 day every sixth day over 1 year. —, overall mean; , weekly mean; , 10 20 30 40 50 60 intake on individual days. Days does not mean that we should stop collecting dietary Precision/repeatability data but rather that dietary data need independent In the laboratory the precision of a method is given by validation. Methods need to be developed to assess the coeffi cient of variation (CV) of repeated determi- the error structure of dietary datasets so that it can be nations on the same sample made under the same taken into account in analyzing and evaluating the conditions (see Box 10.2). In the context of dietary data. Basically, there are two types of error: random studies we determine whether the same method gives error and systematic error. the same answer when repeated in the same individu- Random error increases the variance of the esti- als, and the terms repeatability and reproducibility are mates obtained and consequently reduces their preci- commonly used to describe the precision of a method. sion (see below). The effects of random error can be It is important to note that it is possible for a method reduced by increasing the number of observations. to have high precision (good repeatability) yet not Day-to-day variation in food intake in individuals is provide an accurate (valid) estimate of intake. one example of a random error that can be reduced by increasing the number of days of observation Accuracy/validity (Figure 10.2). An accurate method is one that measures what the In contrast, the effects of systematic error cannot method intends to measure, i.e., the “truth.” In the be reduced by increasing the number of observations. context of dietary studies the truth represents Systematic error arises from errors that are not ran- the actual intake over the period of the study. For domly distributed in the group or in the data from a example, a valid diet record is a complete and accurate given individual. Inappropriate nutrient data for record of all the food and drink consumed over the some food items will not affect the food intake data period that the record is kept. To be a valid record of for all individuals in the same way. For example, inap- habitual intake it also needs to refl ect what would propriate nutrient data will have a greater effect on have been consumed had the individual not been nutrient intake data of individuals who consume the keeping a record. If the process of recording infl u- food in large amounts than on the data of those who enced what was eaten then the record is not a valid consume only small amounts of the food. Systematic record of habitual intake, although it may be a true error leads to bias in the estimates of intake record of actual intake over the period. Similarly, a obtained. valid 24 hour recall is a complete and accurate account 248 Introduction to Human Nutrition of all food and drink consumed during the specifi ed to provide clear instructions to the respondents not period if it refl ects all foods and drinks consumed in only on how to weigh foods but also on how to the amounts that they were actually consumed. It may describe and record foods and recipes. When respon- not, however, be a valid refl ection of habitual intake dents are responsible for weighing, the investigator if the items consumed were not typical of the indivi- needs to make regular visits to the respondent during dual’s usual intake. the recording period to ensure that the equipment is Determining the validity of a dietary technique has being used correctly and that information is recorded been impossible in the absence of external markers of accurately and in suffi cient detail. intake, except for the 24 hour recall or for individual Weighing can be carried out in two different ways: meals. 1 The ingredients used in the preparation of each Methods for measuring intake on meal or snack, as well as the individual portions of specifi ed days prepared food, must be weighed. Any food waste occurring during preparation and serving or food Menu records not consumed is also weighed. Menu records are the simplest way of recording infor- 2 All food and beverage items are weighed, in the mation on food intake. They only require the respon- form in which they are consumed, immediately dent to write down descriptions of the food and drink before they are eaten, and any previously weighed consumed at each meal and snack throughout the day food that is not consumed is also weighed. for the specifi ed days without quantifying the por- tions. A menu record is useful when information on The fi rst approach is sometimes referred to as the food patterns rather than intake is required over a precise weighing technique and is usually carried out longer period or when respondents have diffi culty in by trained fi eldworkers rather than the respondents providing quantitative information. For example, themselves. It is thus very labor intensive, time- elderly people may have diffi culty in reading the divi- consuming, and expensive to carry out. It is most sions on household scales or in measuring out food appropriate when the food composition tables avail- portions. To derive information on nutrient intake able contain few data on cooked and mixed dishes or from menu records investigators also need to obtain if exposure to contaminants is being assessed. It information on portion sizes of commonly eaten should be noted, however, that the precise weighing foods. Information on portion sizes may be derived technique does not allow for nutrient losses in from existing data or collected in a preliminary study. cooking. To take these into account information on Menu records work well when the diet is relatively cooking losses for the most commonly used cooking consistent and does not contain a great variety of methods must also be available. foods. The method can be used to distinguish differ- The second procedure, which is more widely used, ences in the frequency of use of specifi c foods over involves weighing all food eaten in the form in which time, to determine whether quantitative short-term it is consumed. It is sometimes referred to as the intake records are likely to be representative of habit- weighed inventory method. Using this method the ual intake and as a way of assessing compliance with nutrient content of the diet can be determined either special diets. by chemical analysis of duplicate portions of indi- vidual foods or aliquots of the total food consumed Weighed records or, most often, from tables of food composition. Weighed records require the respondent, or a fi eld- Scales used for weighing food need to be robust and worker, to weigh each item of food or drink at the able to weigh up to 2 kg, accurate to at most 5 g and time it is consumed and to write down a description preferably to 1–2 g. Digital scales are preferred as of this item and its weight in a booklet specially these are more accurate and easier to read than spring- designed for this purpose (sometimes referred to as a balance scales. Record books must have clear instruc- food diary). Weighed records are usually kept for 3, 4, tions, be easy to use, and of a convenient size. They 5, or 7 consecutive days. To obtain accurate informa- should contain guidelines for weighing and examples tion it is necessary to use trained fi eldworkers to illustrating the level of detail required. Figure 10.3 collect the data or to demonstrate the procedures and shows an extract from the instructions and record Measuring Food Intake 249

Seven day food diary instructions

1. Please use this booklet to write down everything you eat or drink for the following seven days. As you will see, each day is marked into sections, beginning with first thing in the morning and ending with bedtime. For each part of the day write down everything that you eat or drink, how much you eat or drink, and a description if necessary. If you do not eat or drink anything during that part of the day, draw a line through the section. 2. You have been provided with: a scale to weigh food, a measuring jug to measure liquids, and a set of measuring spoons to measure small amounts of foods and liquids. 3. Write down everything at the time you eat or drink it. Do not try to remember what you have eaten at the end of the day. 4. Before eating or drinking, the prepared food or drink must be weighed or measured and written in the record book. If you do not consume all the food or drink, what is left must also be weighed or measured and recorded in the book. 5. Please prepare foods and drinks as you always do. Also eat and drink in the same way as normal: eat the foods and drink in the amounts and at the times that you always eat and drink. Try not to change the way you eat and drink at all.

6. We need to know ALL the food and drink you take during these 7 days. So if you eat away from home (e.g., at work, with friends, at a cafe or restaurant) please take your measuring equipment with you so you can still measure your food. Also do not forget to measure food bought at take-aways. 7. Please write down the recipes of homemade dishes such as stews, soups, cakes, biscuits, or puddings. Also say how many people can eat from them or how many biscuits or cakes you get from the recipe. 8. On the next page is a list of popular foods and drinks. Next to each item is the sort of thing we need to know so that we can tell how it is made and how much you had. This list does not contain all foods, so if a food that you have eaten is missing, try to find a food that is similar to it. Please tell us as much about the food as you can. 9. Please tell us the amount and type of oil or fat that you use for cooking, frying, or baking.

Figure 10.3 Extract of instructions and record sheets from a 7 day weighed food record (MacIntyre, 1998) 250 Introduction to Human Nutrition

10. Most packet and tinned foods, like Simba chips, Niknaks, corned meat, tinned pilchards, have weights printed on them. Tins, bottles, and boxes of cold drinks and alcoholic drinks also have weights printed on them. Please use these to show us how much you ate or drank. When possible, please keep the empty packets, bottles, or tins. Please note: we need to know the amount you eat or drink. So, if you do not eat the whole packet or tin of food, or drink the whole bottle of cold drink, please measure the amount you eat or drink. 11. At the end of each day there is a list of snacks and drinks that can easily be forgotten. Please write any extra items in here if you have not already written them down in some part of the day. 12. The research assistant will visit you during the record days to help you if you have any questions or problems. She will collect the equipment and record book after the 7 days. All the information you give us is strictly confidential. It will only be used for research purposes. Only your subject number appears on the record book. Nobody will be able to identify you from the record book.

EXAMPLE Breakfast

Food/drink Description and Amount Amount Amount Code preparation served left eaten

Mealie meal Iwiza. Soft, 1 cup meal 300g porridge and 3 cups water

Milk Fresh, full cream Clover 300ml

Bread Brown 1 ¥ 60g

Margarine Rama, soft 10 ml

Tea Glenn tea bags 1 cup

Milk Fresh full cream 25 ml

Sugar White 2 heaped teaspoons

Figure 10.3 Continued Measuring Food Intake 251

Day no. 1 Date __/___/ ____ Day of week______

Early morning — Before breakfast

Food/drink Description and preparation Amount Amount Amount Code served left eaten

Breakfast

Food/drink Description and preparation Amount Amount Amount Code served left eaten

Mid-morning — Between breakfast and lunch time

Food/drink Description and preparation Amount Amount Amount Code served left eaten

Lunch time

Food/drink Description and preparation Amount Amount Amount Code served left eaten

Mid-afternoon — Between lunch and dinner

Food/drink Description and preparation Amount Amount Amount Code served left eaten

Figure 10.3 Continued 252 Introduction to Human Nutrition

Dinner time

Food/drink Description and preparation Amount Amount Amount Code served left eaten

Late night — up to last thing at night

Food/drink Description and preparation Amount Amount Amount Code served left eaten

Between meals, snacks and drinks if not already written in before Food/drink Description and preparation Amount Amount Amount Code served left eaten

Sweets and chocolates

Biscuits or cakes

Simba chips

Peanuts

Other snacks

Cold drinks

Beer

Tea Coffee

Milk

Figure 10.3 Continued Measuring Food Intake 253 sheets of a 7 day weighed record used in a dietary using appropriate conversion data when available, or intake study in South Africa. by obtaining the necessary information when not. For The strengths of the weighed record are that it pro- example, the investigator can determine the volume of vides the most accurate measurement of portion the measures commonly used in a given household sizes, as food is recorded as it is consumed, it does not and then convert these to weights by weighing food rely on memory, and it gives an indication of food portions of appropriate size or using information habits such as the number and times of meals and about the density (g/ml) of different kinds of foods. A snacks. Weighed records kept for 3 or more days and record book for this kind of study is similar to that for including a weekend day are usually considered to a weighed record study. In some situations a precoded represent habitual intake. record form that lists the commonly eaten foods in Limitations are that weighed records are time-con- terms of typical portion sizes may be appropriate, but suming and require a high level of motivation and an open record form is generally preferred. commitment from both the investigator/fi eldworkers Since there is no need for weighing scales to be and respondents. Respondents may change their food provided the record forms can be distributed by mail habits to simplify measuring and recording or may rather than by interviewers. This is convenient if a not measure and record food items accurately. large number of respondents located over a large geo- Samples of respondents who keep weighed records graphical area is involved. In this situation the follow- may not be representative of the population for three up interview, after completion of the record, could reasons: be conducted by telephone. In situations in which respondents may not be familiar with measuring 1 because of the high respondent burden, respon- foods, the investigator needs to train and provide dents must be volunteers and thus random clear instructions to the respondents and to check sampling cannot be used that respondents are performing measurements and 2 respondents are limited to those who are literate recording correctly during the record period. and who are willing to participate The strengths and limitations of estimated records 3 those who volunteer may have a specifi c interest in are similar to those of the weighed record, but the food intake, e.g., being very health conscious, and method has a lower respondent burden and thus a thus may not be representative of the population. higher degree of cooperation. Loss of accuracy may Metabolic studies carried out to determine absorp- occur during the conversion of household measures tion and retention of specifi c nutrients from mea- to weights, especially if the investigator is not familiar surements of intake and excretion are a specialized with the utensils used in the household. application of the weighed food intake record. In Weighed records are used in countries where metabolic studies all foods consumed by the respon- kitchen scales are a common household item and dents are usually either preweighed or weighed by the quantities in recipes are given by weight, e.g., the UK. investigators at the time of consumption. The foods Estimated records are favored in countries where it is consumed are usually also analyzed for the nutrient customary for recipe books to give quantities by stan- constituents of interest or prepared from previously dard spoons and cups, e.g., the USA and Canada. analyzed ingredients. The dietary literature frequently uses the phrase “diet record” without specifying how portions were quanti- Estimated records fi ed. In these instances, estimated records are most This method of recording food intake is essentially likely to have been used. similar to weighed records except that the amounts of food and beverages consumed are assessed by volume Recalled intake rather than by weight, i.e., they are described in terms Information on dietary intake over a specifi ed period of cups, teaspoons, or other commonly used house- can also be obtained by asking individuals to recollect hold measures, dimensions, or units. Food photo- the types and amounts of food they have eaten. This graphs, models, or household utensils may be used to approach therefore does not infl uence the type of assist quantifi cation. These descriptive terms have food actually consumed in the way that a food record then to be converted to weights by the investigator, may do. However, it is open to misrepresentation of 254 Introduction to Human Nutrition the dietary pattern, with respondents either reporting period. In order to obtain suffi cient information to a “good” dietary pattern in order to project a good quantitatively analyze food intakes from a 24 hour self-image or reporting a “poor” dietary pattern in the recall, a skilled interviewer will use several “passes” or hope of receiving hand-outs or other assistance. stages in questioning the respondent. This procedure Response rates in short-term recall studies tend to has become known as the multiple-pass 24 hour range from 65% to 95% and depend largely on how, recall. This is an interviewing technique consisting of under what conditions, and from whom the infor- three to fi ve steps which take the respondent through mation is obtained. A recall may consist of a face-to- the previous day’s food consumption at different face or telephone interview or of a self-completed levels of detail. All multiple-pass 24 hour recalls com- questionnaire. mence with the respondent simply listing all foods The 24 hour recall is probably the most widely used and beverages consumed during the previous 24 method of obtaining information on food intake hours. The content and number of further steps differ from individuals. It is often used in national surveys from study to study. The US Department of Agricul- because it has a relatively high response rate and can ture (USDA) has developed a fi ve-step multiple pass provide the detailed information required by regula- method comprising the following passes (steps) tory authorities for representative samples of differ- (Conway et al., 2003). ent population subgroups. The 24 hour recall is an attempt to reconstruct Pass 1 Quick list: the respondent lists all food and quantitatively the amount of food consumed either beverages consumed during the preceding 24 hours in the previous 24 hours or on the previous day. This in any order without any prompting or interrup- period is considered to provide the most reliable tions from the interviewer. recall of information. With longer periods memory Pass 2 Forgotten foods list: the interviewer asks about becomes an increasing limitation. Incomplete recalls categories of foods, such as snacks and sweets, are more likely with self-completed records unless which are frequently forgotten. these records are subsequently checked with the Pass 3 Time and occasion: the interviewer asks for respondent by the investigator. An example of a 24 details of the times and names of the eating occa- hour recall sheet is shown in Figure 10.4. sions at which foods were consumed. Traditionally, the food intake has been reviewed Pass 4 Detail: the interviewer asks for details, such as chronologically, i.e., starting from the time the respon- descriptions and preparation methods, and dent wakes up and going through the day until the amounts of foods consumed. following morning. Recalling daily activities often Pass 5 Review: the interviewer goes through the assists the respondent to remember food intakes. information probing for any foods which may have Problems encountered in estimating the amounts of been omitted. foods consumed are similar to those encountered with estimated records. Recalls conducted by means A simplifi ed version of the multiple-pass 24 hour of a face-to-face interview often use aids such as pho- recall consists of three steps: tographs, food models, and household utensils to help the respondent to describe how much food was Pass 1 the respondents provide a list of all foods eaten eaten. In telephone recalls respondents may be pro- on the previous day using any recall strategy they vided with pictures or other two-dimensional aids desire, not necessarily chronological. prior to the interview to help them to describe the Pass 2 the interviewer obtains more detailed infor- amounts consumed. There is, however, very little mation by probing for amounts consumed, descrip- information on how effective these aids are. For this tions of mixed dishes and preparation methods, type of study a standardized interview protocol, which additions to foods such as cream in coffee, and is based on a thorough knowledge of local food habits giving respondents an opportunity to recall food and commonly used foods, is essential when more items that were initially forgotten. than one interviewer is involved. Pass 3 in a third pass the interviewer reviews the list In its simplest form, the 24 hour recall consists of of foods to stimulate reports of more foods and foods and the amounts consumed over a 24 hour eating occasions. Measuring Food Intake 255

Place Food/drink and preparation method Amount Amount Code (home, (HHM) (g) (office use work, (office use only) friends, only) etc.) Did you eat or drink anything when you got up yesterday? What did you have?

Did you eat or drink anything during the morning (before about midday/lunch time)? What did you have?

Did you eat or drink anything in the middle of the day (lunch time)? What did you have?

Did you eat or drink anything during the afternoon (between lunch and dinner time)? What did you have?

Did you eat or drink anything at dinner time? What did you have?

Did you eat or drink anything during the night (after dinner and before you went to sleep)? What did you have?

Is there anything else that you ate or drank yesterday that you haven’t told me already?

Figure 10.4 Example of a 24-hour recall record sheet (Reproduced from Kruger, 2003, with permission of the author). HHM, household measure. 256 Introduction to Human Nutrition

The multiple-pass approach is thought to assist recall habitual dietary intake if only a single 24 hour recall more effectively than chronological cues and thus is conducted. This may be overcome to some extent provide more accurate and complete information. by conducting repeated 24 hour recalls. Another dif- This approach, however, is more time-consuming fi culty is that the 24 hour recall relies on the respon- than the traditional 24 hour recall and may irritate dent to accurately recall and report the types and respondents by seemingly asking about the food amounts of foods consumed. There is a tendency for intake over and over again. Irrespective of the respondents to overestimate low intakes and under- approach used, it is essential that all interviewers are estimate high intakes. This is known as the fl at slope thoroughly trained, that the approach is tested in the syndrome. Respondents may omit certain foods that target population prior to the study, and that the same are considered “bad” or include foods not consumed procedure is used by all interviewers with all respon- but considered “good” (phantom foods) in order to dents throughout the study. impress the interviewer. A major drawback of the 24 hour recall is that it Of the methods so far described weighed records provides information for only a single day and there- should contain the least error as they report all food fore does not take account of day-to-day variation in consumed on specifi ed days with weighed portions. the diet. In large cross-sectional studies in which the Estimating the size of portions increases error and, if aim is to determine average intakes of a group of menu records are quantifi ed with average portions, individuals, a single 24 hour recall may be suffi cient. then the error at the individual level is further When the diets of individuals are assessed or when increased. If food that has already been eaten has to sample sizes are small, repeated 24 hour recalls are be recalled then poor memory can introduce an addi- required. This method is known as multiple 24 hour tional source of error. All methods that report intake recalls. The number of recalls depends on the aim of on specifi ed days are also subject, in individuals, to the study, the nutrients of interest, and the degree the error associated with day-to-day variation in of precision needed. For example, when diets intake, but this error can be reduced by increasing the consist of a limited variety of foods two 24 hour number of days studied. recalls may be suffi cient whereas four or more recalls may be required when diets are complex. Recalls Methods for measuring intake over may also be repeated during different seasons to the longer term take account of seasonal variations. (Note that mul- Food frequency questionnaires tiple 24 hour recalls must not be confused with the Food frequency questionnaires consist of a list of multiple-pass 24 hour recall technique. The multiple- foods and options to indicate how frequently each pass 24 hour recall refers to an interviewing tech- food is consumed. Respondents indicate the frequency nique, whereas the multiple 24 hour recall method of consumption during a specifi ed period by marking refers to repeated 24 hour recalls conducted per the appropriate option column. The food lists may respondent.) contain only a few food items or may contain up to The strengths of the 24 hour recall method are that 200 foods. The type and number of foods included is it has a low respondent burden in comparison with determined by the purpose of the study and the target food records and thus compliance is high, it does not population. For example, a food frequency question- require respondents to be literate, it does not alter naire designed to determine calcium intake would usual food intake, and it is relatively quick and inex- contain only foods which provide calcium, while a pensive to administer and therefore is cost-effective questionnaire to measure overall dietary adequacy when large numbers of respondents are involved. It is would need to contain all foods known to be con- most successful in populations with limited dietary sumed by the target population. Likewise, a food fre- variety and when respondents are able to accurately quency questionnaire designed to assess dietary recall and express the types and amounts of foods intakes of a homogeneous target population with a consumed and when interviewers are skilled in the diet of limited variety will be shorter than one interview technique. designed to assess food intakes of a heterogeneous As already stated a major drawback of the 24 hour population with a variety of food intake patterns. recall is that it does not give an accurate refl ection of There are several types of food frequency question- Measuring Food Intake 257

Box 10.3 ● 1–2 times per week ● 1–2 times per month Food frequency questionnaire (FFQ) (simple/nonquantitative): ● occasionally respondents report usual consumption of foods and beverages from a set list of items for a specifi c period. Portion sizes are not ● never. determined. Semiquantitative food frequency questionnaire: a FFQ which This type of response format requires only that the includes estimation of portion sizes as small, medium, or large. A appropriate columns be marked and is most suitable reference portion (usually medium) may be provided as a guide. for self-administered questionnaires. When appropri- Quantitative food frequency questionnaire: more precise esti- mates of portion sizes are given by reference to portion size mea- ately designed, such questionnaires can be optically surement aids (PSMAs) such as food models and photographs of scanned, which saves time on data entry and checking known weight, household measures, or by direct weighing. procedures (Figure 10.5). List-based food frequency questionnaire: food items are listed Closed response options, however, treat the fre- according to groups or categories of similar foods or foods usually quency of consumption as a categorical variable and eaten together. Meal-based food frequency questionnaire: foods are asked assume that frequency of consumption is constant about according to meals or the time of day at which they are throughout the recall period. The choice of categories consumed. may bias the results: too few categories may underes- Culture-sensitive food frequency questionnaire: a FFQ that timate frequencies whereas too many may overesti- takes account of the food values, beliefs, and behaviors of a mate frequencies. Respondents may have diffi culty in specifi c population or cultural group. matching their food intake to the available categories. For example, when food is purchased on a monthly basis food items such as fresh fruit and vegetables may be consumed every day while the stocks last, but, once naires, which are defi ned in Box 10.3. The type of used up, will not be consumed until purchased in the food frequency questionnaire used depends on the following month. purpose of the study, the target population, and the An alternative method for recording responses is to required level of accuracy of food portion provide columns headed as the number of times per estimation. day, per week and per month, seldom, and never. The period of recall depends on the study objec- From this, the average frequency of consumption and tives. In the past, most food frequency questionnaires the amount of food consumed per day can be calcu- used the preceding year or 6 months as the reference lated. Table 10.3 shows an extract from such a food period. Theoretically, this should eliminate the effects frequency questionnaire. The advantage of this of season. In practice, however, respondents tend to response format is that it allows the respondent to answer according to what is in season or available at describe the frequency of consumption in detail. the time of the study. For example, intake of oranges Responses such as consumption of a food twice a was found to be higher when interviews were carried day for 6 days of the week can be recorded. The out during the citrus season than at other times of the disadvantages are that clear instructions must be year. Information may be more reliable when recall given, making this method more appropriate for period is shorter. Recent recommendations are that interviewer-administered questionnaires than self- recall periods should not be longer than 1 month. If administered questionnaires, and the interview annual intakes are required, the food frequency ques- takes longer and requires more writing and calcula- tionnaire must be repeated in different seasons. It is tions than the closed format, making more room for very important that the respondent understands what errors. the recall period is and that only this period should Most food frequency questionnaires obtain infor- be considered when giving frequencies of intake. mation only on the frequency of consumption of a The frequency of consumption is usually indicated food over a given period and not on the context in by options such as: which the foods were eaten, i.e., on meal patterns. ● more than once a day Meal-based food frequency questionnaires have been ● daily used on the basis that it may be easier for respondents ● 3–4 times per week to provide the information in the context of meals. 258 Introduction to Human Nutrition

N 1 2 3 or 1 2 3 or 4 5 or 6 Less 1–3 Please completely fill one oval in every line. E time times more time times times times than times V times once Please MARK LIKE THIS: E R per month per week per day Flavoured milk drink (cocoa, ™ etc.) Nuts butter or peanut paste Corn chips, potato crisps, Twisties™ etc. Jam, marmalade, or syrups Vegemite™, Marmite™ or Promite™ Dairy products, meats and fish Cheese Ice cream Yoghurt Beef Veal Chicken Lamb Pork Bacon Figure 10.5 Typical layout of a food frequency questionnaire suitable for optical scanning (reproduced with permission of Anti Cancer Council of Victoria, Melbourne, Australia).

The information on meal patterns obtained from factor. Since the cost of administration and respon- such questionnaires is, however, more limited than dent burden are relatively low, they are suitable for that which can be obtained from a dietary history. use when sample sizes are large, particularly if a postal Additional questions, if included, can provide some method is used. information on cooking methods. The success of a food frequency questionnaire Some food frequency questionnaires also attempt depends on how closely the food list and portion size to quantify the frequency information by obtaining descriptions refl ect the food patterns of the target data on portion size. Information on the quantity population. This is sometimes referred to as being of each food consumed may be obtained by asking culture sensitive. Much time and care must be put respondents to indicate on the questionnaire whether into the development of a food frequency question- their usual portions are small, medium, or large naire in order to ensure that it provides an accurate relative to those eaten by others, by asking subjects to refl ection of the dietary intakes of a population. Pre- describe their usual portion size in terms of a stan- liminary studies, using 24 hour recalls, food records, dard portion size described on the questionnaire or indirect methods may be needed to obtain infor- (semiquantitative food frequency questionnaire), or mation on food items, frequency of consumption, by reference to a picture atlas of food portions, food and portion sizes in the target population. Since food models, or actual food portions (quantitative food frequency questionnaires are usually developed for frequency questionnaire). When portion sizes are use in specifi c target populations, a food frequency used, it is important that these refl ect the consump- questionnaire developed for use in one population tion patterns of the population. may not be appropriate for use with another popula- Food frequency questionnaires are mainly used in tion with different food intake patterns. It is also studies designed to look for associations between extremely important that the questionnaire be tested food intake and disease or risk of disease, particularly for repeatability and validity in the target population when specifi c foods rather than the level of consump- before being used, even if it has been tested in a tion of a nutrient are thought to be the important different population. Measuring Food Intake 259

Table 10.3 Extract from a food frequency questionnaire using open ended response options (MacIntyre, 1998)

Offi ce use only

Food Description Amount Times eaten Code Amount/day

Per day Per week Per month Seldom

Maize meal porridge Stiff porridge 4256 Soft porridge 4255 Sour porridge 9999 Do you pour milk on your porridge? YES NO If NO, go to next question on sugar. If YES, what type of milk: fresh, sour, milk powder (Nespray), skim milk powder (name)? If yes, how much milk? Do you pour sugar on your porridge/cereal? YES NO If no, go to ‘samp’ If yes, how much sugar? 9012 Samp (only) 4043 Samp and beans A014 Tshidzimba (samp, beans, and 9816 peanuts) Rice 4040 Macaroni/spaghetti A018 Bread White bread 4001 Brown bread 4002 Fat cakes 4057 Other types of bread Do you spread anything on the bread? YES NO If no, go to next section (chicken, meat, fi sh) IF YES, What do you spread and how much? Margarine Hard 6508 Soft 6521 6509 Other types of spread

Detailed reviews of the design and evaluation of ● an interview to obtain usual frequency methods for use in epidemiological ● a cross-check of this information by food group studies are given by Willett (1998) and Cade et al. ● a 3 day record of food consumed in household (2002). measures. The 3 day record component is now seldom used as Diet history a regular component of a diet history. Its purpose The principal objective of the diet history is to obtain originally was as a way of checking the data obtained detailed information on the habitual intake of an from the diet history interview. A diet history is usually individual. As fi rst proposed by Burke in the 1940s, obtained by an experienced nutritionist by means of the method had several components (Burke 1947): an open interview followed by some kind of cross- 260 Introduction to Human Nutrition check using a standard list of commonly consumed In contrast, the errors that are associated with spe- foods. The interview usually begins with a review of cifi c methods are generally much more dependent on the food that was eaten in a specifi c time-frame (e.g., the nature of the method and the abilities of the yesterday) or on a typical day, and then moves on to respondents, and therefore less easy to control. Errors explore the variations in food intake that occur for of this type include: each meal over a given period. Information on the ● estimation of portion size usual size of food portions is obtained with the aid of ● recall or memory error food models or photographs in the same way as for a ● day-to-day variation in intake 24 hour recall. The time-frame for a diet history can ● effect of survey method on food intake. range from the previous month to the previous year. In practical terms it is easier for respondents to Figure 10.6 illustrates the points in the dietary assess- reconstruct the immediate past, but the past year is ment process at which different kinds of error often used to capture seasonal variation. Whatever the operate. time-frame used, it is important for it to be clearly specifi ed. In the literature the term “diet history” Sampling bias is sometimes used loosely to describe any form of Sampling bias arises when the sample studied is not diet recall, including the 24 hour recall and self- truly representative of the population of interest. The administered food frequency questionnaires, as well importance of minimizing sampling bias depends on as interviewer-administered recalls of habitual or the purpose of the dietary study. Except in method- longer term intake. This broader use can be confusing ological studies, volunteers are not generally appro- and is best avoided. The dependence of the diet priate respondents because frequently the objective is history on both respondent and interviewer skills to study a representative sample of a particular group may make the results obtained less comparable in order to extrapolate the results to the population between individuals than those obtained from other group from which the sample was drawn. For this methods, and for this reason it is often considered purpose it is clearly important that as many as possi- more appropriate to categorize diet history data (e.g., ble of the sample group originally selected participate as high, medium, low) rather than to treat them as in the study. intakes expressed in terms of absolute units per day. The proportion of the sample that agrees to par- The diet history is favored in Scandinavia and the ticipate in the study can vary considerably even with Netherlands, where a structured interview may be the same method. It depends not only on the group used. The structured interview is more standardized being studied but also on the circumstances of the but may miss elements specifi c to the individual or study. In general, response rates tend to be greater in bore the respondents with irrelevant questions. The studies that use methods such as the 24 hour recall open-ended interview allows for tailoring to the indi- and food frequency questionnaires (which make vidual, but risks missing important items. fewer demands on respondents) and lower in studies such as 7 day weighed records (which require much more cooperation, effort, and time from the respon- 10.4 Sources of error in dietary studies dents). It is always important to try to maximize response rates, for example by increasing respondent The major sources of error in dietary studies have motivation, providing specifi c assistance if required, been reviewed in detail by Bingham (1987). Four pos- and by allowing respondents as much fl exibility as sible sources of error occur to some degree with all possible in participation within the context of the dietary methods, but can be minimized by careful study objectives. study design and execution:

● sampling bias Response bias ● response bias Response bias arises when respondents provide ● inappropriate coding of foods incomplete or inappropriate responses. The extent of ● use of food composition tables in place of chemical this problem is not easy to assess, but can be investi- analysis. gated by making measurements that are independent Measuring Food Intake 261

Behavior and interactions Altered food Poor memory Inability to “summarize” dietary patterns Messages from researcher choice False perception of own diet Attitudes to food Socioeconomic status Image management

Report RECORD RECALL HISTORY FFQ

Inadequate Identify food Researcher’s choice Subject’s choice communication/ from food tables from questionnaire descriptions

Weighing errors Weighed/ Average portion Quantify Estimated Poor quantification: Poor estimations estimated or small/medium/large – Questionnaire choices – Subject’s perceptions

Underreporting Frequency As reported Subject’s Subject’s choice Overreporting estimate from questionnaire

Calculate Food code Food code intake x Portion weight x Portion weight Food table values x Nutrient content/g x Frequency differ from actual x Nutrient content/g composition Figure 10.6 The process of dietary assessment showing the different sources of error (shown in italics) and the stages at which they operate in different dietary methods. FFQ, food frequency questionnaire. of dietary intake, both during and after dietary study procedures do not necessarily “standardize” respon- periods. Measurements suitable for this purpose dent response. To date, relatively little work has will be discussed further in the context of validity. focused on the respondent response aspect of dietary Response bias can probably best be minimized by assessment. providing the respondents with clear and well- Since all dietary methods engage the cognitive presented instructions, adequate support, and processes of respondents, an appreciation of the appropriate incentives. Such incentives may include properties of human cognition and its limitations is relevant dietary feedback where this is appropriate or fundamental to improving the accuracy of dietary monetary or other rewards provided that these are assessments. Recently, research into the cognitive within ethical principles. aspects of dietary assessment has been undertaken in In dietary studies that involve more than one inter- an attempt to increase the understanding of how viewer, the training of interviewers and the use of respondents process dietary intake data (Domel, standard procedures for interviewing is one way 1997; Thompson et al., 2002; Vuckovic et al., 2002; of reducing unnecessary random variation (error) Matt et al., 2006). that might otherwise arise because different inter- Some of the important issues in this area that are viewers conduct interviews in different ways. The use relevant to improving the quality of dietary data of standard procedures, however, can also introduce include identifi cation of: systematic error; for example, if one interviewer is assigned to interview all respondents in areas of low ● factors that improve communication between socioeconomic status and another to interview all respondent and investigator respondents in areas of high socioeconomic status. It ● the most effective cues for recall over different is important to recognize also that standard interview periods 262 Introduction to Human Nutrition

● factors that infl uence retention of dietary informa- of this section is simply to review briefl y the kinds of tion over time error that can arise as a consequence of using food ● the ways in which individuals conceptualize foods composition tables to calculate nutrient intake, com- and food quantities. pared with chemical analysis of the diet, and which can lead to both random and systematic errors. Coding Systematic error can result from: Coding refers to the allocation of a specifi c code to each food item. Since the nutritional content of a ● the way in which results are calculated or food varies with different processing and preparation expressed methods, it is vital that the correct codes be assigned ● the analytical method used to each food item. Coding errors arise when the ● the processing and preparation methods in common food that has been consumed is not described in suf- use. fi cient detail to enable unambiguous allocation, by Food composition tables for different countries often the investigator, to a food category in a food composi- use different ways of expressing results and different tion table or database. Food frequency questionnaires analytical methods. The ways in which food items are are often precoded to reduce the time needed for processed or prepared are also likely to differ and for coding and the possibility of coding errors (see Table these reasons different sources will not necessarily 10.3). Making it easy for respondents to describe provide comparable data for the same foods. foods with the level of detail required is therefore Systematic differences, which may not necessarily be an important consideration in study design. This is errors (e.g., when foods are prepared differently in increasingly diffi cult, particularly in industrialized different countries), often only become evident when countries where the food supply now consists of different food composition tables are used to evaluate thousands of different manufactured foods, the names the same diets. of which are often no longer a good guide to their Random error arises from the fact that most foods nutrient content. vary in their composition as a result of changes in Coding errors are also likely to arise when more composition associated with the conditions of pro- than one person is involved in coding and there is duction, processing, storage preparation, and con- no agreed procedure and/or comprehensive coding sumption. The random error associated with the use manual. Coding errors arising exclusively from of food composition databases generally decreases as inadequate description of foods have resulted in the size of the sample group increases. This may not coeffi cients of variation ranging from 3% to 17% for be true, however, in institutional settings where every- different nutrients. Note that a standard procedure one is likely to be consuming food from the same for coding foods, while minimizing differences source. between coders (random error), can also introduce To compare calculated and analyzed data without bias if the coding decisions that are made are not the complication of other sources of error it is neces- based on up-to-date knowledge of the local food sary that the diets are analyzed by collecting a dupli- supply and food preparation methods. Gross errors cate of what has been eaten at the same time as the associated with weights of foods can be checked, diet record. At group level it has been observed that before analysis, by means of computer routines that mean intakes calculated from the food tables are gen- identify values outside a prescribed range and by erally within approximately 10% of the mean ana- using data-checking techniques such as duplicate data lyzed value for energy and macronutrients, but not entry. for micronutrients. However, a large proportion of Use of food composition tables individuals have values that fall outside this range. In general, calculated and analyzed values for nutri- Most dietary studies use food composition tables or ents agree more closely: databases rather than chemical analysis to derive the nutrient content of the foods consumed. Chapter 2 ● for groups than for individuals describes in detail the way in which data on food ● for macronutrients than for micronutrients composition are derived and compiled. The purpose ● when data for locally analyzed foods are used. Measuring Food Intake 263

Estimation of portion size ● commercial or home-made food models: Estimation of portion size has long been recognized ● food pictures or drawings of different portion as an important source of error in dietary studies sizes (Young et al., 1953) with coeffi cients of variation of ● photographs of foods in different portion sizes the differences between estimates and weights of food ● abstract shapes of cardboard, wooden or plastic portions regularly reported to be around 50% for blocks, wedges, circles, balls, and mounds in foods and 20% for nutrients (Nelson and Bingham, various sizes 1997). However, despite the fact that individuals ● household utensils and containers such as cups, are known to vary widely in their ability to estimate spoons, jugs, glasses, bowls, and plates in various portion size, relatively few studies have attempted to sizes quantify the size of this error or to “calibrate” their ● containers and packets of bought foods, e.g., respondents in this respect. The infl uence of some sweet wrappers, potato crisp packets, cold drink factors on the determination of portion size is cans and bottles, yoghurt and ice cream cups, summarized in Box 10.4. milk cartons. In attempts to assist respondents to describe Each PSMA has advantages and disadvantages. The portion sizes, a number of visual aids, known as type of PSMA chosen will depend, among others, portion size measurement aids (PSMAs), have been on the type of study, the target population, whether developed. These include: interviewers go from house to house or respondents

● weighed portions of actual foods go to a research centre, available resources, and the ● allowing respondents to serve out portions of food availability of appropriate PSMAs. Probably the most and direct weighing or measuring of the serving effective method is a combination of PSMAs such as food photographs and household utensils. Irrespec- tive of the type of PSMA used, it is essential that Box 10.4 respondents are able to identify and relate to the Food characteristics PSMA, that PSMAs be tested in the target population • No consistent associations observed with type of food, although prior to their use, and that PSMAs are used con- foods of indeterminate shape are more often associated with sistently throughout the study. larger and liquid foods with smaller errors. • Large portion sizes appear to be more diffi cult to estimate accu- Recall errors rately than small portion sizes. Factors that have been studied in relation to the accu- Visual aids • Household measures may be associated with considerable racy of dietary recall include food consumption errors. patterns, weight status, gender, and age. Many other • Food models produce more reliable results than household mea- characteristics, such as intelligence, mood, attention, sures, but because only one size is usually available they may and salience of the information, however, have also “bias” respondents to report portion sizes similar to those been demonstrated to infl uence performance tests of shown. • Graduated food models and two-dimensional pictures may be general recall, but have not been studied in the context as helpful as three-dimensional models for estimating portion of dietary recall. size. • The range of reference portion sizes available may infl uence the Short-term memory estimates. • The use of multiple photographs results in more accurate esti- Like the ability to estimate portion size, the ability to mates. remember what was eaten varies with the individual. Respondents Studies that have compared the abilities of different • Respondents of all ages have been reported to have diffi culty groups to remember what they have eaten conclude with portion size estimates. that women are generally better than men and that • Women have sometimes, but not invariably, been reported to be younger adults are better than older adults. In short- better able to estimate portion size than men, but this may term recalls of intake (e.g., 24 hour recalls) indivi- simply refl ect the fact that they tend to handle food more often than men. duals more often tend to omit an item or items that they have consumed than to include ones that they 264 Introduction to Human Nutrition have not consumed. For this reason, 24 hour recall the recall and the reference period. The accuracy of studies often provide estimates of food intake that are frequency estimates also deteriorates with time. While lower than food records obtained over the same individuals appear to report more frequently eaten period. The size of the error incurred by the omission foods with greater frequency than less frequently eaten of one or more food items clearly depends on what is foods, there are differences between individuals in the omitted and not only on the proportion of food way that they report the same frequency of consump- omitted. For example, the effect, on 24 hour energy tion. Ranking of individuals on the basis of the usual intake, of omitting a cup of black coffee, a glass of frequency of intake is thus likely to lead to misclassifi - milk, or a bar of chocolate is quite different. cation unless the extent of the differences between The omission of food items in 24 hour recall studies individuals is known and can be taken into account. It can be reduced by appropriate probing by the inter- is diffi cult to see how such misclassifi cation can be viewer in relation to meals, between-meal snacks, and reduced unless it is possible to classify individuals, in other activities on the previous day, but even when some way, in terms of their ability to provide reliable respondents have previously weighed their food the information on habitual long-term intake. average energy intake may still be underestimated by as much as 20%. Day-to-day variation in intake We have already seen that individuals vary consider- Long-term memory ably in their intake of nutrients from day to day (see The diet history and most food frequency ques- Figure 10.2). In addition, the extent of day-to-day tionnaires set out to measure the habitual intake of variation differs between nutrients. The implication an individual over a period of weeks or months. of the fi rst observation is that short-term intake data Individuals are not asked to recall their food intake on (e.g., 24 hour recall data) are unlikely to provide a specifi c days, but to construct a picture of their “usual” reliable estimate of habitual intake for most individu- food consumption pattern over a specifi ed reference als. The implication of the second observation is that period. To provide reliable information individuals the length of time for which dietary data need to be thus need to be able to remember the range of foods collected, in order to estimate habitual intake with that they usually consume, to judge the frequency of any given level of confi dence, varies with the nutrient consumption on a long-term basis, and to be able to of interest. estimate correctly the average amount that is usually Table 10.4 expresses the impact of this variation in consumed. These are complex cognitive tasks. terms of the number of days of dietary information As in the case of 24 hour recalls, no attempt is needed to classify 80% of individuals into the correct usually made to assess how well individuals are able third of the distribution. It is clear from this table that to perform these various tasks. From the limited not only 24 hour recalls but also 7 day records are amount of data available from comparative studies likely to be inadequate to classify 80% of individuals between diet histories and long-term diet records, it correctly into the appropriate third of the distribution appears that the two methods do not give concordant for most micronutrients. This is an important reason, results in individuals. Food frequency questionnaires although not the only reason, why short-term records are subject to the same diffi culties, and have the added are only rarely used for epidemiological studies, in problem that estimates of portion size are based on preference to food frequency questionnaires, despite standard measures or, in the case of mailed question- the loss of detail and precision inevitably associated naires, are made in the absence of visual aids such as with the use of the latter. food models or photographs. When respondents are asked to report their intake Effect on usual diet over a period of weeks they rely largely on generic Recall methods clearly cannot change what has already knowledge of their diet and tend to report items that been eaten, but what has been eaten can be misre- they are likely to have eaten or items that they rou- ported either consciously or unconsciously. When tinely eat, rather than items that they specifi cally individuals are asked to keep records, however, they remember having eaten during the reference period. may also alter their normal habits as a consequence This tendency increases with the time interval between of the recording process. One obvious reason for Measuring Food Intake 265

Table 10.4 Number of days of records required to enable 80% of men defi ned, since this will determine the kind of informa- to be assigned into their correct third of the intake distribution tion and the length of time for which it needs to be collected from each individual. Often, the purpose of British civil Random sample Random sample Nutrient servants of British men of Swedish men the study also determines the level of precision that is required to meet the study objectives and therefore Energy 7 5 7 the sample size. These two considerations are the Protein 6 5 7 most important ones in determining the method to Fat 9 9 7 Carbohydrate 4 3 3 be used, because both the method and the size of the Sugar 2 2 – sample have implications for the human and fi nancial Dietary fi ber 6 10 – resources needed for the study. P:S ratio 11 – – Cholesterol 18 – – Purpose of the study Alcohol 4 – 14 Vitamin C – 6 14 When dietary data are collected to describe the diet Thiamin – 6 15 of a group for comparison with that of another group Ribofl avin – 10 – or groups, it is possible to use either a short-term Calcium – 4 5 method such as a 24 hour recall or record, or a longer- Iron – 12 9 term method such as food records obtained over P:S ratio, ratio of polyunsaturated to saturated fatty acids in the several days, a diet history, or a questionnaire about diet. (Reproduced from Margetts BM, Nelson M. Design Concepts in habitual intake. The fi nal choice will depend on . Oxford: Oxford University Press, 1991, with factors such as the importance of a representative permission from Oxford University Press.) study sample, the resources available, and the level of precision required. Usually, the most effi cient doing so would be to simplify the process of record- approach is to measure the diet of as many individu- ing. Other reasons may include a desire to eat less in als as possible for 1 day. order to lose weight or to be seen to conform with However, if the purpose of the dietary study is to dietary recommendations. If this is what happens in determine the proportion of individuals in the group practice, then what is measured in short-term dietary who are at risk of dietary inadequacy or excess, rela- records may be actual intake or desired intake, but not tive to some standard of reference, then a single day usual intake. of information on each individual is no longer ade- Many studies have now demonstrated that there is quate because it is necessary to have a reliable esti- a tendency, in most population subgroups, for short- mate of the distribution of habitual intake in the term dietary records to provide estimates of energy group. As Figure 10.2 shows, a single day of intake is intake that are on average around 16% lower than generally not a reliable measure of an individual’s would be expected on the basis of measured and/or habitual intake. estimated levels of energy expenditure. To determine the distribution of habitual food These studies will be discussed further in the intake in a group, at least 2 days (preferably not section on precision and validity. The fact that for consecutive) of information from each individual or some groups measurements of energy intake and a representative subsample of individuals from the energy expenditure agree quite closely indicates that group of interest are needed. If several days of intake it is possible to achieve recording without a concomi- are available they can be used to derive a mean intake tant change in diet when there is full cooperation for each individual and from this the distribution of from respondents, and highlights the importance of average intakes for the group. Alternatively, statistical efforts to achieve such cooperation. techniques can be used to adjust 1 day intake data, for the day-to-day variation that occurs in individuals, to 10.5 Choosing a dietary provide a better estimate of the underlying distribu- assessment method tion of habitual intake for the group than is given by the 1 day data (Dodd et al., 2006). While the use of It is not possible to decide which dietary method to appropriate statistical techniques can improve esti- use until the purpose of the study has been clearly mates of the proportion of individuals at risk of 266 Introduction to Human Nutrition defi ciency by adjusting for within-person variation, resources available it is better either to abandon the they do not enable at-risk individuals to be study or to redefi ne the question than to collect inad- identifi ed. equate data. When the purpose of the study is to assess the diet of specifi c individuals it is necessary to obtain dietary 10.6 Repeatability and validity information over at least a week and preferably longer. This is best done by obtaining either multiple 24 hour This section looks at ways in which it is possible to recalls or 24 hour food records over an extended assess the repeatability and validity of dietary period. The minimum number of days needed to methods. obtain an estimate of nutrient intake with a specifi ed level of confi dence differs for different nutrients. Repeatability Information on energy intake, which tends to show Assessing the repeatability (also referred to as the less day-to-day variation than other nutrients, can be reproducibility) of a laboratory method is relatively obtained over a shorter period (days) than informa- straightforward because, with care, it is possible to tion on a nutrient for which day-to-day intake is reproduce both what is measured and the conditions much more variable, such as vitamin A (weeks). of measurement. This is almost always impossible in the case of a dietary intake measurement. Individuals Precision do not eat exactly the same quantities or the same In studies of groups, precision is primarily a function foods on different days or weeks. of sample size, while in studies of individuals it is a All measures of repeatability obtained by applying function of the number of days of information avail- the same method to the same individuals on more able. Precision increases with sample size and with the than one occasion include not only measurement number of days for which information is collected, error but also real day-to-day or week-to-week vari- but so does the cost of the study. Precision therefore ability in intake. needs to be defi ned in relation to the purpose of the While at fi rst sight it might appear easier to measure study. the repeatability of recall methods such as the 24 hour Usually, what is required of the nutritionist is to be recall and diet histories, this process also introduces able to provide the statistician with an estimate of the additional sources of variation since the interviews level of difference that it is important to be able to have to be conducted at different times and possibly detect (in nutritional, not statistical terms) and an by different interviewers. Measures of repeatability estimate of the variance or standard deviation for the for all dietary methods will thus tend to give an over- measurement(s) in question. For example, when estimate of the extent of measurement error because looking for differences in energy intake between two they will always include an element of variation due groups, would a difference of 500 kJ or 1500 kJ be to real differences in what is being measured and in regarded as biologically signifi cant? the conditions under which it is being measured. Since the variance of a dietary measurement Usually, the repeatability of a dietary method is depends not only on the real variation within or determined by repeating the same method on the between respondents but also on the error of the mea- same individuals on two separate occasions, that is, surement, the precision of a dietary estimate can be by a test–retest study. The interval between tests improved not only by increasing sample size but also depends on the time-frame of the dietary method by reducing measurement error. being assessed, but should generally be short enough to avoid the effects of seasonal or other changes in Resources food habits and long enough to avoid the possibility It is inevitable that the resources available, both fi nan- of the fi rst interview or recording period infl uencing cial and human, also infl uence the choice of method. the second one. They should not, however, be the primary consider- The difference between the results obtained on ation. The method used should be determined by the the two occasions can be expressed in a number of question to be answered. If the method or methods different ways. Table 10.5, which was compiled from needed to answer the question are beyond the data reported in the literature, shows various mea- Measuring Food Intake 267

Table 10.5 Measures of repeatability for energy intake obtained for a 3 day food record, a dietary history, and a food frequency question- Validity naire (FFQ) Demonstrating that a dietary method measures what Measure of 3 day food Dietary it is intended to measure is even more diffi cult than repeatability record history FFQ demonstrating that a method is repeatable, because in effect it “requires that the truth be known.” Mean 156 105 954 This is almost always impossible unless it is possi- difference (kJ/day) Mean difference (% of 1.6 1.1 12.5 ble to observe, surreptitiously, what is consumed over overall mean intake) short periods such as 24 hours or at most a few days. Coeffi cient of variation 16.5 18.6 28.5 Observation is usually only feasible in institutional of the differences settings or in situations specially set up to allow unob- within individuals trusive observation of what people eat. (%) Coeffi cient of ±3266 ±1819 ±4294 For methods that are designed to obtain informa- repeatability (kJ) tion on habitual longer-term intake, such as the diet Correlation coeffi cient – 0.86a 0.70a history or food frequency questionnaires, unobtru- Individuals classifi ed in 56 – 60* sive observation is impossible. This is a problem that the same quartile or has been faced by all investigators of dietary assess- tertile* on both occasions (%) ment methods and until relatively recently was usually “solved” by assessing one dietary method in relation a Intraclass correlation. to another dietary method, usually a 7 day weighed dietary record, which was considered to be the best available or criterion measure. Comparison with sures of repeatability for energy intake obtained with another dietary method provides at best only a rela- different dietary methods repeated after an interval tive form of validity and at worst information that is of time. unrelated to validity but refl ects either real differences The different measures of repeatability provide or similar errors between the methods. For example, different information. The correlation coeffi cient is comparison of data from a single 24 hour recall or a widely quoted but is not a good measure of repeat- diet history with data from a 7 day weighed record ability since a good correlation may be obtained even for the same individuals does not compare the same if one set of measurements has been systematically information because the time periods are not con- biased and has a different mean from the other set. current. However, because of the lack of a suitable The mean difference is not a good measure of repeat- external standard against which true validity could be ability in individuals since it depends primarily on judged before the 1980s it was usually assumed that whether the differences are random or systematic. most dietary intake data, and weighed records in par- Measures that refl ect the differences between repeated ticular, provided valid data. Usually, a method was measurements within individuals are to be preferred. judged acceptable if the mean intake, as measured by The coeffi cient of variation of the differences within both methods, did not differ signifi cantly and if cor- individuals and the coeffi cient of repeatability (which relations for nutrient intake in individuals exceeded is simply twice the standard deviation of the differ- 0.5. The magnitude of the coeffi cient of variation ences and represents the 95% confi dence limits of of the differences within individuals was generally agreement) give much better measures of their mag- ignored. nitude. They are also more readily interpreted in Table 10.6 shows data from three studies that practical terms than either a correlation coeffi cient or provide additional information on agreement. All the percentage of individuals classifi ed in the same three studies compared data from a food frequency quintile, quartile, or tertile. If the standard deviation questionnaire with multiple days of food intake of the difference within individuals is of the order of records. When different methods are compared the 20–30% of mean intake, one is unlikely to describe mean differences tend to be higher (there is greater the method as precise or repeatable even if the mean bias) than those found in repeatability studies. difference at group level is only 1%. However, the range of values obtained for other 268 Introduction to Human Nutrition

Table 10.6 Measures of relative validity for energy intake obtained from a food frequency questionnaire (FFQ) compared with multiple days of records in three different studies

7 days of weighed 12 days of weighed 15 days of records in Measure of validity records records household measures

Mean difference (kJ/day) (FFQ – record) 800 926 351 Mean difference (% of overall mean intake) 6 11 4 Coeffi cient of variation of the differences within individuals (%) 32.7 26.6 16.9 Coeffi cient of repeatability (kJ) ±8248 ±4542 ±2950 Correlation coeffi cient – 0.69 0.71 Individuals classifi ed in the same quintile or tertile* on 43* 42 44 both occasions (%) measures of agreement is generally similar to that measures are also subject to errors of measurement obtained in repeatability studies. Agreement at the and classifi cation, but these errors are not related individual level is also not high, with coeffi cients of to the errors inherent in dietary intake assessment variation for differences in individuals ranging from methodologies. 17% to 33% in these studies and less than 50% of The three most widely used measures to assess the respondents classifi ed in the same quintile of intake. validity of dietary intake data are urinary nitrogen to Note that even good agreement between two dietary validate protein intake, energy expenditure as mea- methods does not necessarily indicate validity, but sured by the doubly labeled water (DLW) method to may merely indicate similar errors. validate energy intake in weight-stable individuals, and the ratio of energy intake to basal metabolic rate Biological measures to validate energy and to identify “plausible” records of food intake. nutrient intake It is now clearly recognized that to assess the validity Urinary nitrogen of any dietary method, including weighed records, it One of the fi rst to suggest an external measure as a is necessary to compare the dietary data with one or means of validating dietary intake data was Isaakson more objective measures that refl ect but are indepen- (1980), who proposed urinary nitrogen as an inde- dent of food intake. At the group level such measures pendent measure of protein intake according to the include food supply or food expenditure data, and at equation: the individual level biochemical or physiological mea- Reported protein intake (g) = sures that refl ect energy and nutrient intake. The (24 hour urinary N + 2) × 6.25 (g) latter are often referred to as biological or biochemical markers and include energy expenditure, urinary Like the 24 hour recall, a single 24 hour urine collec- breakdown products of protein, sodium, and potas- tion does not necessarily refl ect what is “usual.” sium, plasma levels of vitamins, tissue levels of miner- However, it appears that urinary nitrogen excretion is als, and the fatty acid composition of subcutaneous less variable from day to day than dietary protein adipose tissue. intake, and that while 16 days of food intake are needed to assess habitual protein intake only eight 24 Biological marker – any biochemical index in an hour urine collections are needed to assess nitrogen easily accessible biological sample that in health excretion with the same level of confi dence. gives a predictive response to a given dietary Although fewer 24 hour urine collections may component. be needed they are, in general, no more acceptable (Bingham, 1987). to respondents than 24 hour food records and also Biological markers are assumed to be objective, require access to laboratory facilities. Nevertheless, i.e., they do not rely on memory, or the respondents’ they can provide a practical independent assessment, ability to express themselves, and are free of biases not only of protein but also of potassium and sodium introduced by the presence of the interviewers. These intake. Measuring Food Intake 269

Very few validation studies have attempted to eval- The main advantage of the DLW method is that it uate how well the different methods rank individuals. makes minimal demands on the respondents and It has been observed that the correlation between does not in any way interfere with their normal daily urinary nitrogen and dietary nitrogen measured by activities and therefore their habitual level of energy diet records was better (0.65 and 0.79) than between expenditure. Its main disadvantage is that the cost of urinary nitrogen and dietary nitrogen measured by the DLW required for each estimate is exceedingly food frequency questionnaires (0.15 and 0.24). high and the method also requires access to sophisti- cated laboratory equipment for mass spectrometric Doubly labeled water method analysis. It is, therefore, not available for use on a The DLW technique allows the measurement of routine basis for the validation of dietary intake energy expenditure in free-living respondents over data. several days with minimal inconvenience to the respondent and with a high level of accuracy and Ratio of energy intake to basal metabolic rate precision. Under controlled conditions the DLW Because of the limitations of the DLW method, method gives a small overestimate of 2–3% compared another approach that is used compares the energy with whole body calorimetry, and under fi eld condi- intake (EI) reported from published studies with the tions bias is not expected to exceed 5%. presumed requirements for energy expenditure, both The DLW method requires that the respondent intake and expenditure being expressed as multiples drinks a small measured dose of water enriched with of the basal metabolic rate (BMR). The relevant naturally occurring stable isotopes of deuterium and equation is: 18O. The two isotopes disperse throughout the body, EI:BMR = EE:BMR (PAL) and are metabolized and then gradually lost from the body. Since the deuterium labels the body water pool, where PAL is the physical activity level. To determine and the 18O labels both the water and the bicarbonate whether reported energy intake is a “plausible” pools, the difference between the disappearance rates measure of actual diet during the measurement period of deuterium and 18O can be used to calculate carbon (i.e., represents either the habitual diet or is a low/ dioxide production. The level of both isotopes is high energy intake obtained simply by chance) an determined, using mass spectrometry, in a small equation was developed by Goldberg and colleagues sample of urine collected each day for between 5 and (1991) to calculate the 95% confi dence limits of 28 days. Energy expenditure is calculated from carbon agreement between EI:BMR and PAL. This equation dioxide production using calorimetric equations. allowed for variation in EI, BMR, and PAL and also Further details of the DLW technique and the main for the length of the dietary assessment period and factors infl uencing its accuracy and precision are pro- study sample size. vided in Chapter 3. For a group, if mean reported EI:BMR is below the Using the DLW technique several investigators lower 95% confi dence limit (cut-off) for the given have compared self-reported dietary energy intake study period and sample size, then there is defi nitely with energy expenditure based on the equation: bias to the underestimation of energy intake. However, the identifi cation of individual under- Energy expenditure (EE) = Energy intake (EI) reporters is much more diffi cult, since reported EI can ± Change in the body energy store deviate quite markedly from energy expenditure (EE) Differences between measured energy intake and before it falls outside the limitations of the methods. expenditure varied from –44% to +28% depending Figures 10.7 and 10.8 illustrate the limitations of both on the population subgroup studied. This fi nding techniques. Figure 10.7 shows the energy intake and confi rms the need to include one or more indepen- DLW EE of 264 women. The solid lines indicate EI: dent measures of validity in all dietary studies EE of 0.76 and 1.24. These are the 95% confi dence to ascertain the level applicable to the particular limits of agreement between EI and EE, allowing for group under study, since it is not readily predicted day-to-day variation in food intake and within-subject on the basis of gender, age, or body mass index variation on repeat DLW measurements. Only women (BMI). with an EI:EE ratio above 1.24 can be confi dently 270 Introduction to Human Nutrition

20 Women 3.0 UR UR AR AR OR 2.5 OR 15 2.0 est 10 1.5 EI (MJ) EI:BMR 1.0 5 0.5

0 0 1.0 1.5 2.0 2.5 3.0 015 01520 PAL (EE:BMR ) DLW EE (MJ) meas Figure 10.7 Reported energy intake (EI) against energy expenditure Figure 10.8 Energy intake–estimated basal metabolic rate (EI:BMRest) (EE) measured by doubly labeled water (DLW) in 264 women aged against physical activity level [PAL; energy expenditure–measured 18–90 years. The solid lines represent the 95% confi dence limits of BMR (EE:BMRmeas)]. Respondents are designated as acceptable report- the expected agreement between EI and EE (±24%). UR, underreport- ers (AR), overreporters (OR), or underreporters (UR) by the direct com- ers; AR, acceptable reporters; OR, overreporters (after Black, 2000, parison of EI:EE. The horizontal lines indicate the lower Goldberg = = reproduced with permission). cut-off for PAL 1.55 and 1.95, 7 day records, and n 1 (after Black, 2000, reproduced with permission).

identifi ed as overreporters, and those below 0.76 as application to other populations must be done with underreporters. In Figure 10.8, the same data are caution. expressed as EI:BMR and EE:BMR, and each respon- ● The ratio does not take differences in physical activ- dent is represented by the same symbol as in Figure ity into account (Black, 2000). 10.7. The line at EI:BMR = 1.05 indicates the Gold- ● Using a single cut-off point to identify under- berg cut-off for n = 1 and PAL = 1.55, which has been reporters has been found to have poor sensitivity widely used to identify low energy reporters (LERs). for underreporting (fails to identify underreport- These data demonstrate that only about 50% of ers), especially at high levels of energy intake (Black, underreporters (as defi ned by EI:EE = 0.76) are iden- 2000). tifi ed as LERs. The second line at EI:BMR of 1.35 is ● Cut-off values differ among studies. Thus it is dif- the Goldberg cut-off for n = 1 and PAL = 1.95. This fi cult to select an appropriate value and to compare cut-off identifi es more of the underreporters, but also studies. includes some of the acceptable reporters. To improve ● Cut-off values apply only to individuals in energy on identifi cation of underreporters it is necessary balance. They cannot be applied to growing chil- to have information on physical activity to enable dren or to adults trying to lose weight (Gibson, respondents to be classifi ed into different levels of 2005). activity and to calculate cut-offs appropriate for each activity level. Characteristics of low energy reporters While the ability to separate the food and nutrient A number of studies have examined the characteris- intake data of those with and without plausible energy tics of low energy reporters (LERs). Associations intakes is a very important step in the evaluation of between low energy reporting and a large number of dietary intake data, it does have limitations. factors including high body weight, high BMI, obesity, ● The equations for the estimation of BMR have dieting, and awareness of body image were found in been derived for Western populations and their every study that looked at these measures. Associations Measuring Food Intake 271 with other sociodemographic factors of the type nor- Allowing for the effects of underreporting mally included in nutrition studies, such as gender, Although techniques for handling biased dietary data age, education, socioeconomic status, and smoking, have been developed, most are complex However, were inconsistent. the following suggestions serve to promote critical examination of data and wariness in drawing 10.7 Evaluation of food intake data conclusions. If the proportion of individuals who report implau- Recognizing the impact of underreporting sibly low intakes of energy differs between population As indicated in Section 10.5, dietary studies are often subgroups of interest, then any comparisons between conducted in order to compare food and nutrient them that do not take this into account will be biased. intake between different groups in the population, to One way to draw attention to the possibility of bias determine the proportion of individuals at risk of between groups is to report not only the mean or dietary inadequacy or excess, or to determine the median energy intake of the groups being compared habitual intake of individuals. but also the EI:BMR ratio. If differences are evident In each case it is important fi rst to assess the valid- then the groups should be compared both with and ity of the data. For most investigators the use of the without LERs included. One problem that arises is Goldberg cut-offs is currently the most practical that by subdividing the groups the sample size is option to indicate whether, and to what extent, the reduced and imprecision increased, so that a differ- results are likely to be biased. However, to use the ence of biological signifi cance may be missed, not Goldberg cutoffs effectively dietary studies need to because it does not exist but because the sample size include: is too small to detect it statistically. When dietary inadequacy or excess is the question ● measurements of weight and height, to be able to of interest, it is again important to consider estimate BMR from equations LERs separately. Energy intake is highly correlated ● questions on activity level to provide guidance on with the intake of many nutrients and, consequently, suitable PALs for evaluation of both mean and indi- intake of nutrients is also likely to be underestimated vidual data. in underreporters and more likely to indicate While the characteristics of “true” underreporters inadequacy relative to recommendations for nutrient (as opposed to LERs identifi ed by a single EI:BMR intake. An alternative approach is to compare cut-off) remain to be confi rmed, the associations nutrient intake per unit energy for both groups. If consistently observed between high BMI, weight con- this differs between LERs and the rest of the popula- sciousness, and low energy reporting suggest that, in tion it provides evidence that the reporting of food addition, questions on self-perception of body shape, intake is also likely to be selective. The nutrients for dieting, and dietary restraint may also help in identi- which signifi cant differences are observed can also fying true underreporters. provide clues as to the types of food likely to be It cannot be overemphasized that it is always involved. important to examine all dietary intake data critically Studies that have examined macronutrient intake because false conclusions generate false hypotheses between respondents above and below a given value that may take years to be disproved. of EI:BMR have generally found that the percentage A classic case was the luxus konsumption hypoth- of energy derived from protein was higher and that esis, namely that lean individuals are energy prodigal from fat lower in LERs than in non-LERs. Results for and obese individuals energy effi cient. This hypothe- carbohydrate have been more variable, but, when sis was generated by studies apparently showing that separated into starch and sugars, energy from starch obese persons did not consume more energy than tended to be higher and energy from sugars lower in their lean controls. Subsequently, DLW studies dem- LERs. also tends to be higher for onstrated beyond doubt that obese persons recruited most nutrients in LERs than in non-LERs, providing for studies of obesity grossly underreported their further indication of differences in food patterns food intake. between the two groups. 272 Introduction to Human Nutrition

Table 10.7 Nutrient intakes in low energy reporting (LER) and non-LER The second limitation is that all estimates of adults in the 1995 Australian National Nutrition Survey dietary adequacy/inadequacy obtained by compari- Nutrient LER Non-LER Total son with reference values for nutrient requirements (n = 1291) (n = 9451) (n = 10 851) depend on how the estimate is derived (see Energy (MJ) 4.62 10.19 9.24 Chapter 7). Protein (g/MJ) 11.0 9.8 9.9 However, irrespective of the approach that is used Fat (g/MJ) 8.1 9.0 9.0 to assess dietary adequacy, unless the extent of under- Starch (g/MJ) 15.9 14.9 15.0 reporting is known and taken into account, the pro- Sugars (g/MJ) 12.9 12.4 12.4 portion of individuals at risk of inadequacy will be Vitamin (μg/MJ) 159 122 127 Ribofl avin (mg/MJ) 0.26 0.22 0.23 overestimated. While it may become possible to dis- Folate (μg/MJ) 37 28 29 tinguish more reliably in population-based studies Vitamin C (mg/MJ) 19 13 13 valid from invalid reports of dietary intake, this still Calcium (mg/MJ) 106 90 92 does not enable population-based estimates of inad- Iron (mg/MJ) 1.8 1.5 1.5 equacy to be made unless those who provide valid Data used from the Australian Bureau of Statistics (abs.gov.au) (ABS, intakes are also representative of the population as a 1998). whole. All the evidence available to date suggests that this is highly unlikely. When the principal objective of a dietary survey is Table 10.7 illustrates these general trends with data to identify the proportion of the population who may from the 1995 Australian National Nutrition Survey. have inadequate intakes of energy and nutrients, it is Twelve per cent of men and 21% of women in this essential that the dietary intake information is inter- survey were identifi ed as LERs. The median energy preted in the light of appropriate biological measures intake in non-LERs was approximately 6% higher in of nutritional status. men and 10% higher in women than for all men and women, and vitamin and mineral intake approxi- 10.9 Assessing food intake mately 5–10% higher in non-LER men and 6–15% higher in non-LER women. Differences of this order Nutritionists usually analyze dietary intake data by of magnitude are important in the context of the converting the information on food intake into nutri- assessment of dietary adequacy. ent intake using relevant food composition databases. Relatively few studies have reported on differences This approach simplifi es the process of analysis and in foods eaten, but there appears to be a general ten- enables the resulting data to be compared with energy dency for LERs to report more foods such as meat, and nutrient requirements (see Chapter 7). Describing fi sh, vegetables, salads, and fruit, and fewer cakes, food intake in terms of foods rather than nutrients biscuits, sugar, confectionery, and fats. presents two practical diffi culties that do not exist when food intake is analyzed in terms of nutrients. 10.8 Assessment of dietary adequacy First, the variety of foods consumed is much greater than the range of nutrients for which food composi- Methods for evaluating dietary adequacy are described tion data are available. Second, while essentially all in Chapter 7. This section simply draws attention to individuals in a group contribute to nutrient intake the limitations of these methods. data, not all individuals contribute food data for all The fi rst limitation is that the evaluation of nutri- foods, i.e., not all individuals are “consumers” of the ent intake can provide only an estimate of the risk of same foods. nutrient inadequacy for a population or an indivi- There are, however, several uses for which informa- dual. None of the methods can identify the specifi c tion on food intake is more relevant or for which individuals who have a nutrient defi ciency. Individu- information on food intake is needed in conjunction als with a nutrient defi ciency or excess can be identi- with data on nutrient intake. For example, food regu- fi ed only on the basis of biochemical or clinical latory authorities and agencies concerned with food measures of nutritional status. safety and nutritional surveillance require data on the Measuring Food Intake 273 availability and intake of foods in addition to infor- but may also be converted to nutrients to provide mation on nutrient intake. Similarly, nutritional epi- information on the nutrient contribution of individ- demiologists are also interested in the relationship ual foods or groups of foods. of different foods and dietary patterns with specifi c Tracking changes in the food sources of nutrients health outcomes. The use of dietary data in the context and nonnutrients is particularly important in the of epidemiological studies is covered in the textbook context of technological developments in food pro- Public Health Nutrition (Gibney et al. 2004). duction and manufacture that result in the addition The analysis and presentation of food intake data of nutrients to foods, in the development of foods for depends on the objectives of the study. When the specifi c functional purposes, and in the genetic modi- purpose is to examine intakes of specifi c foods, intakes fi cation of foods. A specifi c example of the need for of foods may be expressed as means, medians, or individual food, rather than nutrient, intake data is frequency distributions of intakes, as the number or provided by exposure assessments to dietary non- percentage of respondents consuming specifi c foods, nutrients such as food additives, pesticide residues, or as the percentage contribution of food items to the and other possible food contaminants. total food intake, energy intake, or intake of nutrients of interest. Since not all members of a sample consume 10.10 Food safety assessments a given food, it is always important to indicate whether the total sample size or only the number of respon- Safety assessments for food additives are expressed in dents consuming the food has been used in statistical terms of the acceptable daily intake (ADI) estimated calculations. on the basis of lifetime exposure. While it is clearly Although intakes of individual food items may be not possible to collect food consumption data over reported, food intake data are usually reduced to the lifetimes of individuals, it is important that the more manageable proportions by grouping foods into dietary data used for the purpose of estimating accept- appropriate categories. While this can be done in able levels of intake over a lifetime refl ect, as far as is different ways, for example in terms of composition, possible, the habitual level of intake of the foods being biological origin, or cultural use, the process is assessed. relatively straightforward within a given culture or For the purpose of food safety assessments only the country. It is more diffi cult, however, to develop a intake of “consumers” is of interest. It follows, there- classifi cation that can be used consistently across dif- fore, that the dietary data need to be adequate to ferent countries or food cultures. National food clas- obtain both an accurate estimate of the proportion of sifi cation systems tend to differ not only because the the population who are consumers and of the average type and range of foods differs but also because the habitual intake of consumers. Because the frequency same foods are used in different ways. For the purpose of consumption varies between foods (some foods are of comparing food intake patterns between countries eaten by most people on most days, but many other or regions, it is, therefore, necessary to develop a food foods are eaten less frequently), the duration of the classifi cation or coding system that allows food data dietary recording period infl uences both the esti- from individual regions or countries to be assigned in mate of the proportion of consumers and the average a consistent way. intake of consumers. Intake data for 1 day will inevi- The United Nations University Food and Nutrition tably underestimate the true number of consumers Program for an International Network of Food Data for most foods and overestimate the average habitual Systems (INFOODS) was developed for the purpose intake of those consumers because not all foods are of supporting work on the classifi cation and naming eaten every day. However, it appears that 75% or more conventions for individual foods and food groups of household menu items are normally consumed (see Chapter 2). within a 14 day period and that a 14 day diary pro- Indirect information on food consumption, such vides a good estimate of the habitual intake of most as that provided by FAO food balance sheets and by foods by consumers. data from household budget and similar surveys, is Most studies of the food intake of individuals, usually presented in terms of foods or food groups, however, do not last for 14 days because of the 274 Introduction to Human Nutrition increased cost and nonresponse associated with such supplements (both nutrient and nonnutrient) and a long study period. For the purpose of food safety drugs (both social and medicinal). assessment an approach that combines a 3 day food intake record with a food frequency questionnaire has Acknowledgment the potential to give estimates for the intake of con- sumers that are similar to those obtained from 14 day This chapter has been revised and updated by Una E records. MacIntyre based on the original chapter by Ingrid HE Rutishauser and Alison E Black. 10.11 Perspectives on the future

It is unlikely that either the measurement or the eval- References uation of food intake will become less complex in future. If anything, the reverse is likely to be true given Australian Bureau of Statistics. Australian National Nutrition the increasing diversity in the food supply and the Survey. ABS, Canberra, 1998. Bingham SA. The dietary assessment of individuals; methods, increasing recognition of the need to be able to assess accuracy, new techniques and recommendations. Nutr Abstr Rev accurately not only the intake of foods and nutrients (Series A) 1987; 57: 705–742. but also the intake of nonnutrient constituents of Black AE. The sensitivity and specifi city of the Goldberg cut-off for EI:BMR for identifying dietary reports of poor validity. Eur J foods and dietary supplements. While the existence of Clin Nutr 2000; 54: 395–404. errors in association with measurements of food Burke BS. The dietary history as a tool in research. J Am Diet Assoc intake is now widely appreciated, much work still 1947; 23: 1041–1046. Cade J, Thompson R, Burley V, Warm D. Development, validation remains to be done in this area. and utilisation of food-frequency questionnaires: a review. Publ Other aspects of food intake measurement that also Health Nutr 2002; 5: 567–587. require further development in the immediate future Conway JM, Ingwersen LA, Vinyard BT, Moshfegh AJ. Effectiveness of the US Department of Agriculture 5-step multiple-pass are likely to include the following. method in assessing food intake in obese and nonobese women. As all direct methods of food intake measurement Am J Clin Nutr 2003; 77: 1171–1178. involve interaction between investigators and indi- Dodd KW, Guenther PM, Freedman LS, et al. Statistical methods for estimating usual intake of nutrients and foods: a review of viduals and our understanding of the cognitive aspects the theory. J Am Diet Assoc 2006; 106: 1640–1650. of these interactions is still limited, more work is Domel SB. Self-reports of diet: how children remember what needed to improve the communications aspect of they have eaten. Am J Clin Nutr 1997: 65 (suppl): 1148S– 1152S. dietary assessment. Gibney MJ, Margetts BM, Kearney JM, Arab L. Public Health As the food supply becomes more complex indi- Nutrition. Blackwell Publishing, Oxford, 2004; 67–75. viduals will no longer be able to describe the foods Gibson RS. Principles of Nutritional Assessment, 2nd edn. Oxford University Press, Oxford, 2005. they have eaten in adequate detail unless technologi- Goldberg GR, Black AE, Jebb SA et al. Critical evaluation of energy cal developments such as the use of barcodes and intake data using fundamental principles of energy physiology. similar systems of food identifi cation become an inte- I. Derivation of cur-off limits to identify under-recording. Eur J Clin Nutr 1991; 41: 569–581. gral part of dietary assessment. Isaksson B. Urinary nitrogen output as a validity test in dietary As the number of food constituents of interest, in surveys. Am J Clin Nutr 1980; 33: 4–5. relation to health, increases it is important that appro- Kruger R. The determinants of overweight among 10–15 year old school children in the North West Province. University of priate physiological and biochemical markers are also Potchefstroom for CHE. Unpublished PhD Thesis, 2003. developed for these constituents, as well as for the MacIntyre UE. Dietary intakes of Africans in transition in the nutrient constituents of foods. North West Province. University of Potchefstroom for CHE. Unpublished PhD thesis, 1998. Finally, since food intake data serve no useful Matt GE, Rock CL, Johnson-Kozlov M. Using recall cues to improve purpose unless they can be appropriately evaluated measurement of dietary intakes with a food frequency question- it is essential that dietary studies include suffi cient naire in an ethnically diverse population: an exploratory study. J Am Diet Assoc 2006; 106: 1209–1217. ancillary information to allow this to occur. This Nelson M, Bingham SA. Assessment of food consumption and means routinely collecting information not only on nutrient intake. In: Margetts BM, Nelson M, eds. Design Concepts age, gender, body size, and physiological status, but in Nutritional Epidemiology, 2nd edn Oxford University Press, Oxford, 1997. also on key aspects of lifestyle such as physical activity Thompson FE, Subar AF, Brown CC, et al. Cognitive research and the consumption of nonfood items such as enhances accuracy of food frequency questionnaire reports: Measuring Food Intake 275

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Black AE. Critical evaluation of energy intake using the Goldberg cut-off for EI:BMR. A practical guide to its calculation, use and limitations. Int J Obes 2000; 24: 1119–1130. 11 Food Composition

Hettie C Schönfeldt and Joanne M Holden

Key messages

• Reliable good-quality composition data of foods for human con- • Factors such as sampling, variability and analytical methods sumption are critical resources for a variety of applications. involved must be considered when developing such tables. • These data are required for a spectrum of users ranging from • Inadequacies of food composition tables can be minimized by international to national, regional, household, and individual calculating nutrient losses and gains during food processing and levels. preparation. • In general, data obtained on food intake by individuals, or groups • New activities in food composition include: of individuals, are used to estimate the consumption of nutrients • future composition tables could include bioavailability and the and to establish nutritional requirements and health guidelines. glycemic index • The determination of the consumption of nutrients can be • harmonizing food composition tables regionally achieved either by analyzing the foods consumed directly (by far • focusing on within species the most accurate, but also the most costly method) or by using • investigation of the composition of specifi c traditional and food composition tables/databases. ethnic foods • The food described in the food composition table should be rec- • bioactives in foods and their effect on health and well-being ognizably similar to that being consumed by the individual or • food composition data and their role in nutrition and health group. claims.

11.1 Introduction used today as there are limited up-to-date tables avail- able. Worldwide there are currently over 150 food Although the amount, quality and availability of food composition tables or nutrient databases, or their composition data vary among countries and regions, electronic/magnetic equivalents, in use. Many tables in general most developing countries still do not have are based on the data from the United States adequate and reliable data. This is despite the fact that Department of Agriculture’s (USDA) National the components of specifi c foods have been published Nutrient Database for Standard Reference, SR, for over 150 years. Over time food composition data available on the Nutrient Data Laboratory’s web site: have assumed more scientifi c, academic, and political www.ars.usda.gov/nutrientdata. A comprehensive list importance owing to their utility. Refer to Table 11.1 of the food composition tables available can be for practical examples of the uses of food composi- obtained from the Food and Agriculture Organization tion data. It was only in 1961 that a regional food of the United Nations (FAO) homepage on the World composition table was developed and published for Wide Web (http://www.fao.org/infoods/directory). Latin America, followed by a food composition table EuroFIR, the European Food Information Resource for Africa (1968), the Near East (1970), and Asia Consortium, is a partnership between 40 universities, (1972). The data in these tables were based on a very research institutes, and small to-medium-sized enter- limited number of samples, a limited number of prises from 25 countries in Europe. EuroFIR aims to nutrients and, in today’s terms, outdated analytical develop and integrate a comprehensive cohort and methodologies. However, these tables are still being validated network of databanks of food composition

© 2009 HC Schönfeldt and JM Holden. Food Composition 277

Table 11.1 Examples of the uses of food composition data

Level Examples

International Role of food in the provision of nutrients and/or the estimation of adequacy of the dietary intake of population groups Investigation of relationships between diet, health, and nutritional status, e.g., epidemiologists correlate patterns of disease with dietary components Evaluation of nutrition education programs Nutrition intervention and food fortifi cation programs such as in food assistance programs; foods are distributed or enriched to address the specifi c nutritional needs of populations, e.g., iodine or vitamin A In food trade nutritional labeling National Monitoring at governmental level, the availability of foods produced and estimating the individual intake for specifi c dietary requirements, e.g., protein and energy Food balance sheet data are used to provide data on food available nationally for the whole population and are useful in monitoring trends in food consumption over time Researchers work to improve the food supply by selecting or developing new strains or cultivars, improving cultivation, harvesting, preservation, and preparation Estimation of adequacy of the dietary intake of groups within populations Investigation of relationships between diet, health, and nutritional status Evaluation of nutrition education programs Food and nutrition training Nutrition education and health promotion Nutrition intervention and food fortifi cation programs Food and nutrition regulation and food safety Nutrition labeling of foods Regional (infl uenced by meal Institutions such as hospitals, schools, dormitories, and troops/armies (ration scale) formulate nutritionally patterns and food balanced diets to the individuals in their care preferences) Food industries regulate the quality of their foods by routinely analyzing the components in their products Food industries change and improve their products to appeal to new customers by improving nutrient content or sensory appeal through the change in ingredients Product development Household Household food surveys provide data on household food consumption Household budget surveys Household food economics Individual Dietary intake of the individual is assessed to understand present health and to monitor changes in dietary intake Impact of interpretation of choice and preference via data composition Individual energy expenditure is the only true measurement of energy need, e.g., in the management of a sportsman’s diet or in obesity Personal dietary needs and goals with associated likes and dislikes can be assessed on an individual basis Individual nutritional balance studies Therapeutic or restricting diets with specifi c nutrient contents, e.g., management of diabetes and hypertension, can only be described on an individual basis Individual shoppers scan the ingredient list and nutrient content on the labels of packaged foods data for Europe. This network, although comprehen- Biodiversity for Food and Nutrition led by the FAO sive, has at present limited access to the broader nutri- and Biodiversity International focused on genetic tion society. diversity within species and of underutilized, unculti- There is still a continued need to carry out food vated, and indigenous foods. The investigation has analyses as the number of foods consumed all over the highlighted the need for composition data of foods, world, especially unique foods, is still several times not only at species level, but also at subspecies level. greater than the number for which analytical data The limited amount of composition data for under- exist. The recent (2008) Cross Cutting Initiative on utilized, uncultivated, and indigenous foods playing 278 Introduction to Human Nutrition important roles in the consumption patterns in under- foods that form a major part of the food supply and developed and developing countries increases this that are major contributors to the diet in the forms need for food composition analysis. Food analyses are most commonly obtained or consumed, and as many also needed under the following circumstances: as possible of the less frequently consumed foods. For instance, in the USA the number of foods contribut- ● when the data in existing tables are based on a ing to quartiles of critical nutrient intakes was identi- single or very limited number of samples fi ed as the following: 9 foods contribute to 25% of ● when the content of a nutrient or other food com- food intake, 34 foods to 50%, 104 to 75%, and 454 to ponent is not available in an existing food table approximately 100%. ● when there is no information available on which Databases can be compiled directly, where the foods are important sources of a nutrient or another compiler initiates sampling and analyses to obtain the food component of interest data, or indirectly by drawing on the following sources ● when there is no information on the loss or gain of of data, in order of preference: nutrients in foods during preparation by the methods being used by the population under ● original analytical values investigation ● imputed values derived from analytical values from ● when it is necessary to check the comparability of a similar food, e.g., values for “boiled” used for the various food composition tables being used in “steamed” a multicenter study ● calculated values derived from recipes, calculated ● when the method available to determine a particu- from the ingredients and corrected for preparation lar nutrient is considerably improved factors ● when scientifi c evidence is found correlating newly ● borrowed values (refers to using data originally recognized food components to health generated or gathered by someone else) from other ● when new foods are produced or existing foods are tables and databases. reformulated. Today, database compilers normally draw on a com- bination of the direct and indirect methods. 11.2 Foods Description of foods Food composition tables normally consist of a list of The food described in the food composition table selected foods with data on the content of selected should be recognizably similar to that being con- nutrients in each food. For a food composition table to sumed by the individual or group. The precise descrip- be of value in estimating nutrient content, a signifi cant tion of foods is a diffi cult task and much is required portion of the foods consumed by the group or indi- to ensure that foods are described adequately. The vidual being studied, as well as the nutrients of interest, introductory material (description and explanation) should be present in the table. To a large extent this in a printed table may be almost as important as the relationship is critical in determining the quality of the data values. By using several words to describe a food, information obtained by using the tables, assuming called an extended or multifaceted description, the that the data in the tables are of a desirable quality. chance of misinterpreting the data is reduced. As internationalization of food composition data con- Criteria for inclusion in tables tinues, linguistic aspects of defi ning foods, with one The identifi cation of potential contributions of foods defi nition meaning different things in different cul- to the diet of the population group being studied is tures and even from place to place within countries, unquestionably the fi rst step in identifying and select- are highlighted. For instance, sorbet or sherbet is ing which foods should and should not be included made by beating whisked egg whites into the partly in the production of a database. However, common frozen mixture such as in apple sorbet and lemon sense dictates that it is unreasonable to expect that sorbet. However, the term sorbet is preferred to all foods consumed by all individuals at all times be sherbet, since the latter can also refer to a fl avored, included in a specifi c food composition table at any sweet, sparkling powder or drink, or a drink of sweet one time. Therefore, most tables aim to include all diluted fruit juice. The name tortilla is also applied to Food Composition 279 a variety of foods in Latin America. In Africa morogo databanks, and is therefore language independent. In is a collective term used for a variety of indigenous Table 11.2 an example of the application of LanguaL green leafy vegetables harvested from the veld. Using is presented. More information can be obtained from scientifi c names for food items is not necessarily a the LanguaL (Langua aLimentaria or “language of solution, since the relationship between common food”) homepage (www.langual.org). LanguaL is an name and scientifi c name is neither consistent nor international framework for food description, which universally unique, for example the German tables the European LanguaL Technical Committee has group and apples in the same genus, while the administered since 1996. The thesaurus is organized British and US tables separate them. into 14 facets of the nutritional and/or hygienic Many structured food description systems have quality of foods. These include the biological origin, been proposed. These systems should be adapted to the methods of preparation or conservation. The the specifi c purpose (e.g., nutrient content, pesticide European LanguaL Technical Committee has linked regulation) for which they are intended. For example, LanguaL to other international food categorizing and the FAO Committee report, INFOODS Guidelines for coding systems including the CIAA Food Catgorizing Describing Foods: A Systematic Approach to Des- System, Codex Classifi cations, and E-numbers used cribing Foods to Facilitate International Exchange of for additive identifi cations. Food Composition Data, published in 1991, was designed to facilitate interchange of food composi- Classifi cation of foods tion data between nations and cultures. The system is Most food composition tables are organized accord- a broad, multifaceted, and open-ended description ing to the classifi cation of foods into food groups, mechanism using a string of descriptors for foods. with food items listed alphabetically within each The International Food Data System Project food group. For example, the fruit group could start (INFOODS) Nomenclature and Terminology Com- with apples and end with tangerines. A simple coding mittee has developed guidelines for describing foods system could supplement the alphabetically listed to facilitate international exchange of food composi- foods (used in the British tables), but it presents a tion data. INFOODS is a comprehensive effort, begun problem when a new food is introduced and all within the United Nations University Food and Nutri- the codes have to change. Although food groups of tion Program to improve data on the nutrient com- different countries and organizations are never position of food from all parts of the world. In line completely identical, they are usually recognizably with the FAO’s lead role in classifi cation of agricul- similar. However, problems normally arise with the tural activities and products, and to facilitate interna- description of cooked mixed dishes where a dish tional data comparability and exchange, FAOSTAT can be equally well described by one or more food has developed and standardized the Harmonized group. In some tables, particularly those for educa- Commodity Description and Coding System in 1996. tional purposes, there are subgroups based on the The coding system has developed multipurpose content of specifi c nutrients such as high-fat and low- goods’ nomenclature used as the basis for trade sta- fat dairy products. Table 11.3 provides an example of tistical nomenclatures all over the world. major food groups that are used by the FAO for their In 1975 the Food and Drug Administration (FDA) food balance sheets and regional food composition of the USA developed a controlled vocabulary for tables. food description, based on the principle of a faceted thesaurus, where each food indexed is described by a Sampling of foods for inclusion in tables set of standard terms grouped in facets, characteristic Food sampling concerns the selection of the individ- of the product type of a food source and process ual units of foods, food products, or bulk foodstuffs applied to food ingredients. Examples are the biologi- from the food supply or source, whether it be the cal origin, the methods of cooking and conservation, marketplace, manufacturing outlet, fi eld or the homes and technological treatments. It is an automated of the members of the study population. (Sampling method for describing, capturing, and retrieving data also concerns the selection of the representative about food, adapted to computerized national and aliquot from the individual unit or homogenized international food composition and consumption mixture in the laboratory just before analysis.) In- 280 Introduction to Human Nutrition

Table 11.2 Example of the international use of LanguaL

Facet Code English term French term Danish term Hungarian term

Product type A0178 Bread Pain Brød Kenyér Food source B1418 Hard wheat Blé de force (Triticum Hård hvede (Triticum Kemény búza (Triticum aestivum) aestivum) aestivum) Part of plant or C0208 Seed or kernel, skin Graine ou grain sans Frø eller kerne, Szénhidrát vagy hasonló animal removed, germ removed enveloppe et sans skaldele (pericarp/ vegyület (endosperm) germe caryopse) fjernet, kim fjernet (endosperm) Physical state, E0105 Whole, shape achieved Entier façonné épais Hel, facon dannet Egész, formázott, 1.5–7 cm shape, or by forming, thickness de 1,5 à 7 cm ved formning, közötti vastagság form 1.5–7 cm tykkelse 1.5–7 cm Extent of heat F0014 Fully heat treated Transformation Fuldt varmebehandlet Teljesen hókezelt treatment thermique complète Cooking method G0005 Baked or roasted Cuit au four Bagt eller ovnstegt Sütött vagy piritott Treatment H0256 Carbohydrate fermented Fermenté au niveau Kulhydratfermenteret Szénhidrátos fermentált applied des glucides Preservation J0003 No preservation method Sans traitement de Igen konservering Tartósitási eljárást nem method used conservation alkalmaztak Packing medium K0003 No packing medium used Sans milieu de Intet Csomagoló eszközt nem conditionnement pakningsmedium alkalmaztak anvendt Consumer group/ P0024 Human food, no age Alimentation Levnedamiddel uden Emberi fogyasztásra szánt dietary use/ specifi cation humaine courante aldersspecifi kation élelmiszer, label claim kormeghatározás nélkül

Table 11.3 Major food groups that are utilized by the Food and One of the major objectives of food sampling is to Agriculture Organization provide representative mean values for individual Cereals and grain products components in foods. The sampling process is Starchy roots, tubers and fruits described in detail in Table 11.4. Grain legumes and products Food sampling is a critical step in any food com- Nuts and seeds position program. For any research project, personnel Vegetables and vegetable products and fi nancial resources are always limited. The selec- Fruits Sugars and syrups tion, procurement, shipping, and storage of sample Meat, poultry, and units require a signifi cant portion of available Eggs resources. Therefore, sampling must follow a specifi c Fish and shellfi sh and detailed statement of the objectives and proce- Milk and milk products dures to ensure that the selection of units is suffi cient Oils and fats Beverages in number and weight and representative of the foods Miscellaneous of interest. If sampling or sample preparation is done incorrectly then all subsequent analyses are a waste of time and money, as a mistake in sampling can only context sampling can be defi ned as the selection and be corrected by repurchasing and repreparing of a collection of items of food defi ned in number, size, new sample. Pilot studies, conducted by the and nature to represent the food under consideration. investigator(s) or published in the scientifi c literature, The objectives for sampling will, in the large part, can be used as the basis of sampling decisions for the determine the type and nature of the sampling plan. current study. Food Composition 281

Table 11.4 Sampling process of foods for food composition data

1 Prioritizing foods for inclusion 4 Defi nition of sampling size May be based on: Amount of material required: ● type ● objective of analyses ● frequency and ● analyses of individual samples or composite samples of the food ● amount of specifi c foods or products consumed ● number of components to be measured; determine the number and ● quality and quantity of existing data weight of aliquots needed as required by the chemical methods ● appropriateness of prior analytical methods or ● policy for saving reserve or archive aliquots ● perceived benefi t/risk of particular foods as sources of components of interest 5 Protocol for sample collection Foods should be typical of the usual preparation and consumption May be affected by: practices ● changes in the forms of foods or Correct units of foods should be selected ● levels of components, including reformulation or fortifi cation Protocols should be tested for the adequacy of and Levels of available resources will impact on the process of setting transportation facilities, sample unit documentation and labeling, priorities and packaging and short-term preservation requirements 2 Defi ning prioritized foods Policies for the substitution of units should be in place in the event of Within the context of the objective, defi ne the specifi c unavailable sample units characteristics of the food that may contribute to the variability of Sampling among ethnic or native populations may impose additional the estimate: restrictions owing to cultural or religious customs ● uncooked or raw foodstuffs versus cooked forms of the food Samples should be clearly coded for identifi cation. Documentation ● composition of prepared or multicomponent foods (i.e. mixed should start from the planning stage, throughout purchasing, dishes) transporting, preparation and the combining of samples, to analyses ● individual brands or cultivars or generic value including storage condition, use of reference samples, recording of data obtained (duplicate or triplicate values), as well as manipulation 3 Defi nition of sampling unit of the data, e.g. expression of the data on a wet (as eaten) basis, as Collection of units (packages, bunches, or items) representative of opposed to the content of a freeze-dried sample. Correction factors the total population of food units: applied or calculations (e.g. N × Jones factor = protein) should be ● sample units must be taken from the available types and forms recorded for each foodstuff analyzed of the food for which the composition estimates are being Documentation and handling of sample units should be under the determined. Production, consumption or sales statistics may be careful control of the principal co-coordinator and all laboratory used. The population of items may be supplied to or distributed personnel should be informed before the start of the project of the through an entire nation or region or be only typical of a reasons for handling the samples in a specifi c manner. The samples particular subpopulation (e.g. ethnic group or tribe) should preferably be marked with three-digit random codes for ● select sample units from all the various types of food and analysts to ensure that analyses are unbiased. Values should only be geographical or manufacturing locations of food consumed by decoded as results become available, by the principal investigator. the population of interest. The units may be selected according This will improve the reliability of the results if performed on a to the relative importance (e.g., frequency of consumption) for double-blind basis given types ● sample units that are collected can be analyzed as individual units or may be combined together or composited and analyzed. The analysis of composite samples reduces the costs associated with the analysis of individual samples, but information about the variability of the component in that food will be lost

Variability in foods: regional and the following factors: brand, cultivars or species, other differences season, climate, geographical location (e.g., soil type), Foods are biological materials and, as such, have a treatment, method of husbandry, harvesting, naturally variable composition. Even processed foods preservation state, stage of maturity, enrichment/for- produced under highly controlled circumstances tifi cation standards, preparation methods, food color, show some variability. Therefore, a database must be variation in recipes and formulations, distribution able to predict the composition of a single sample of and marketing practices, and other factors. For food within the limits defi ned by its natural variabil- critical components variability may affect the suffi - ity. Variability may be contributed by one or more of ciency, defi ciency, or excess of the intake of a given 282 Introduction to Human Nutrition component. Estimates of variability must be based on source of that nutrient should be analyzed or re- sampling and analyses specifi cally planned to yield analyzed. Food regulation sometimes limits the choice such data. The intended use of the data should deter- of methods. mine the specifi city and level of precision for the The choice of method selected should be that which estimates. most closely refl ects the nutritive content of the food- For instance, it was found that the nutrient com- stuff analyzed. A basic understanding of the chemis- position of whole milk in South Africa differed among try of the nutrients, the nature of the food substrate the fi ve localities investigated between winter and (the way in which the nutrient is distributed and summer, with the fat-soluble vitamins showing the held in the food matrix) to be analyzed, the effect of greatest variation of all the nutrients. Vitamin A is processing and preparation on both the food matrix commonly regarded as one of the micronutrients that and the nutrient, and the expected range of concen- are defi cient in most developing countries and spe- tration of the nutrient determine the choice of cifi cally in disadvantaged schoolchildren in South method. An understanding of the role of the nutrient Africa. Considering the results of the nutrient com- in the diet of individuals or populations is also a position of whole milk, a recommendation was made prerequisite. to the health authorities to fortify summer milk with The basic principle is that the method used should retinol in the South African school-feeding interven- provide information that is nutritionally appropriate. tion program, where milk is served as a mid-morning For instance, traditionally, carbohydrate was esti- snack to 5 million primary school children. mated by difference, that is, by directly measuring the There is a growing recognition that the composi- percentage of protein (from the nitrogen content), tion of commodities such as meat and cereals tends fat, ash, and water, and deducting these from 100 to to change over time. This necessitates updating food provide the percentage of carbohydrate. This method composition data every 5–10 years. In most countries is inadequate for all nutritional purposes as it com- this has not been possible. Changes in nutrient com- bines in one value all of the different carbohydrate position of red meat consumed are due to consumer species: sugars, oligosaccharides, and polysaccharides demand for leaner cuts, changes in breeding for faster (starch and non-starch), together with all of the errors growth, and higher proportions of marketable meat in the other determinations, as the physiological as well as changes in feed to meet scientifi c standards effects of all of the components are quite different. or due to economic reasons. Therefore, the sum of the individually analyzed car- bohydrates is widely recommended today. In studying the relationship between particular 11.3 Nutrients, nonnutrients and energy foods and health or disease, the biological action of related nutrients may be crucial information for par- Analytical methods ticular uses of food composition data. For example, a Judgment should be made on the availability of suit- study on the role of vitamin A and carotenoids in lung able methods of analyses for nutrients and whether cancer requires more information than the vitamin A the resources, laboratory equipment, and experience activity expressed in retinol equivalents. At the very are adequate before deciding which nutrients should least, vitamin A and provitamin A activity are required be included in a nutrient database. If the methods separately. Information on provitamin A could be available are not well developed, one should recon- divided into the various provitamin A carotenoids, sider the importance of the nutrient and whether it and it may also be desirable to have information on justifi es using limited resources, in most instances other carotenoids present. This is also true for the and countries, to develop the method and train the vitamers of other vitamins, including vitamin B6 staff accordingly. It will not be cost-effective to analyze (pyridoxal, pyridoxal phosphate, and pyridoxamine), food for a particular nutrient, however high in prior- folic acid (with a side-chain with one, three, or seven ity, if methods yield confl icting values. This implies glutamic acid residues), vitamin D (D2 or D3), vitamin that, as new or improved methods for measuring a E (various tocopherols and tocotrienols) and vitamin nutrient emerge, foods that are important in the food K (with various numbers of saturated and unsatu- supply and are known or suspected to be a good rated isoprene units in the side-chain). Food Composition 283

The EuroFIR BASIS bioactives database includes its National Nutrient Databank website (http://www. critically assessed composition data on the bioactives ars.usda.gov/nutrientdata). The database contains present in edible plants and plant-based foods as well values for 385 food items for fi ve subclasses of fl avo- as compilation of critically assessed data on their noids namely fl avonols, fl avones, fl avanones, fl avan- biological effect (http://www.eurofi r.net). 3-ols, and anthocyanidins. A European network established to compile and Criteria for inclusion in tables evaluate data on natural food plant toxicants, the EU As the number of nutrients is reasonably infi nite, it is AIR Concerted Action NETTOX, has previously iden- to some extent easier to choose and prioritize food tifi ed 31 major compound classes called the NETTOX: items. Core nutrients for a nutritional database include a list of toxicant classes with 307 major food plants the major proximate constituents, those that are essen- listed in Europe. This list, now known as the EuroFIR tial, and those for which there are recommended NETTOX plant list (http://www.eurofi r.net) has intakes. The inclusion of micronutrients, especially recently been published after being updated to include trace elements, fatty acid profi les, amino acid compo- additional plant parts. The list now includes 550 sition and the various forms of vitamins is normally benefi cial biological effect outputs of the bioactive limited by the resources available. Many databases give compounds of 328 edible plants. This list facilitates limited coverage of the carbohydrates and carotenoids calculations of exposure to bioactive compounds in foods, but methods are available and this limitation such as fl avanols, phenolic compounds, phytosterols, will probably disappear in the future. carotenoids, isofl avones, and . Nutrients to be included in the food composition For a food composition database to include all table will depend on the proposed use of the table. these substances will imply that there may be an over- For instance, when assessing nutrient intake, two emphasis on “nonnutrients.” In general, levels of pes- types of nutrients can often be distinguished: those ticides, residues, toxicants, and additives in food, with nutrients that are found in small quantities in a large the exception of those that contribute to energy and number of foods, such as iron and most of the B nutrients, are often not reported in food composition vitamins, and those that are found in large quantities tables. in a small number of foods, such as cholesterol and vitamin A. The FAO limits the inclusion of nutrients Modes of expression in the table for group feeding schemes to 11 nutrients An increasing amount of attention is being paid to per 100 g of edible portion. The nutrients that have how data are presented in food composition tables. been selected as the most important for developing Interchange and compatibility of food composition countries are energy, protein, fat, calcium, iron, databases are only possible if the data are uniformly vitamin A, thiamine, ribofl avin, niacin, folate, and expressed. To overcome ambiguities in the naming of vitamin C. nutrients and also to allow for the transfer of data Complete coverage of all nutrients in a single food among food composition tables, INFOODS has database is unlikely, as priorities are set according to developed a system for identifying food components, the importance of a food in the provision of a nutri- referred to as tags. The term “tag” refers to the signifi - ent, resulting normally in analyses of proximates and cant part of a generic identifi er. Generic identifi ers are major nutrients. However, with the growing interest predefi ned word-like strings of characters used to dis- in the role of biologically active compounds, residues, tinguish one element type from another. An example and toxicants in food there is increased pressure to of a tagname and its defi nition is presented in Table include these in special-purpose food composition 11.5. The latest information on this system is available tables. Phytochemicals or phytoprotectants, often on the World Wide Web via the INFOODS home page used in functional foods, are bioactive compounds (http://www.fao.org/infoods). As already mentioned, found in food that may have benefi ts to human LanguaL is a multilingual system that provides a stan- health. dardized language for describing food products using A provisional database for food fl avonoid compo- faceted classifi cation. Each food is described by a set sition has been developed and is maintained by the of standard, controlled terms chosen from facets United States Department of Agriculture (USDA) on characteristic of the nutritional and/or hygienic 284 Introduction to Human Nutrition

Table 11.5 Example of an INFOODS tagname and its defi nition for a quality. The quality of the analytical data is affected food component by various factors, including how the food samples were selected (sampling plan) and handled before Energy, total metabolizable; calculated from the energy-producing analysis, use of appropriate analytical method and food components analytical quality control, and adequacy of number of Unit: kJ. The value for may be expressed in kilocalories samples to address variability. In addition, complete instead of the default unit of kiloJoules. However, if expressed in food description and identifi cation of the compo- kilocalories, kcal must be explicitly stated with the secondary nents analyzed are also important. < > tagname Unit The data quality evaluation system developed by Note: It would be confusing and would imply additional the USDA is based on the evaluation of fi ve catego- information that does not exist if two values, i.e. one ries: sampling plan, sample handling, analytical expressed in kilocalories and the other expressed in kiloJoules, were included for a single food item when one value has simply method, analytical quality control, and the number of been calculated from the other using the conversion equation: individual samples analyzed. Detailed documentation 1 kcal = 4184 kJ. Consequently, one or the other should be used, of all the steps within each category is important but not both for evaluating that category. Each category gets a Synonyms: kiloJoules; kilocalories; calories; food energy maximum rating of 20 rating points. A quality index Comments: In addition to a value for the quantity of total (QI) is generated by combining points of all the fi ve metabolizable energy, includes a description or listing categories and confi dence codes (CCs) ranging from of the conversion factors used to calculate this energy value from A to D indicating relative confi dence in the data the proximate quantities. The conversion factors may be described quality are assigned. These confi dence codes could be by a keyword, or the conversion factors may be listed using released with the data and thus provide an indication secondary tagnames within . (More than one tagname may exist for a single food item if the values were of the data quality to the user of the data. Confi dence calculated from the proximate components using different code “A” indicates data of highest quality, while con- conversion factors.) fi dence code “D” suggests data of questionable quality. These procedures can be used to guide the planning and conducting of food analysis projects. quality of a food, for example the biological origin, method of cooking and conservation, and techno- logical treatments. 11.4 Information required on sources of Other problems related to the method of expres- data in tables sion of nutrients may arise from the long-standing It is important to have information on the source of convention of using protein values derived by apply- the data in a food composition table to be able to ing a factor to measured total nitrogen values and check its appropriateness for the study and to confi rm from the calculation of energy values using energy its authenticity. The four major categories of sources conversion factors. Calculation of total carbohydrate of data are: content by difference as opposed to the sum of the individual carbohydrates is no longer the norm. The ● primary publications, e.g., peer-reviewed articles in bases of expression in databases are the most com- scientifi c literature monly used units (such as g) per 100 g of edible ● secondary publications, e.g., reviews or published portion. In some instances unit per 100 g of dry mass compilations with compositional data is presented, or unit per 100 ml. However, some tables ● unpublished reports ranging from analytical records list nutrient content per or household to documents prepared with limited circulation, measure, either as purchased or as prepared. e.g., confi dential reports for clients or internal use within a company Quality of data ● unpublished analytical data that can be either spe- The quality of food composition data is critical for cifi cally commissioned analyses for the generation the accuracy of the estimates of compounds in food. of nutrient data or analytical data that were not In particular, analytical data obtained from scientifi c particularly generated for the purpose of generat- literature and laboratory reports can be evaluated for ing food composition data. Food Composition 285

Data in food composition tables may be original org/infoods). In other tables, such as those in the analytical values, imputed, calculated, or borrowed. Netherlands, where sources of the data are given in Original analytical values are those taken from pub- the references, information on how the data have lished literature or unpublished laboratory reports. been obtained can also be found. However, this is not Unpublished reports may include original calculated the case for all tables of food composition. values, such as protein values derived by multiplying the nitrogen content by the required factor, energy values using energy conversion factors for some con- 11.5 Overcoming the inadequacies of stituents of food, and “logical” values, such as the food composition tables content of cholesterol in vegetable products, which can be assumed to be zero. Imputed values are esti- Nutrient losses and gains during food mates derived from analytical values for a similar processing and preparation food or another form of the same food. This category In the absence of analytical data for all forms of foods includes those data derived by difference, such as nutrient values can be estimated by calculation using moisture and, in some cases, carbohydrate and values standard algorithms that have been experimentally for chloride calculated from the sodium content. Cal- derived. Since the content of nutrients per unit mass culated values are those derived from recipes by cal- of food changes when foods are prepared, such losses culation from the nutrient content of the ingredients and gains can be classifi ed in two ways. The fi rst can corrected by the application of preparation factors. be described by a food yield factor, when the weight Such factors take into account losses or gain in weight of the primary ingredients at the precooking stage is of the food or of specifi c nutrients during preparation compared with the weight of the prepared food at the of the food. Borrowed values are those derived from cooking stage and also with the fi nal weight of other tables or databases without referring to the the food as consumed at the post-cooking stage. The original source. When a value for the content of a weight of the food can be increased due to the hydra- specifi c nutrient in a food is not included, there is a tion of the dry form of a food (e.g., rice, macaroni) “–” or “0” value and, when a table has no values for a with cooking liquid, (e.g., water or broth) or increased particular nutrient, the value is regarded as being “not due to the absorption of fat during frying of the food included.” In some tables, e.g., the National Nutrient (e.g., potato). Alternatively, the weight of the food can Database for Standard Reference, SR, of the USDA, decrease due to dehydration during cooking as a “0” value is a true zero, meaning the particular nutri- result of evaporative and drip losses. ent was not detected by the analytical method used; The second, the nutrient retention factor, is related “–” indicates a missing value. to changes in the amount of specifi c nutrients when The proportion of the various types of data differs foods are prepared. Changes in the nutrient levels can between tables and for different nutrients (Table occur due to partial destruction of the nutrient as a 11.6). Details on food tables can be obtained from the result of the application of heat, alkalization, etc. Also, Food and Agriculture Organization (http://www.fao. for some dietary components (e.g. β-carotene) the amount of available component may increase due to the breakdown of cell walls in the plant-based sample. Although original analytical data would be the most Table 11.6 Proportion of various types of data in food composition tables desirable type of data for foods at all stages of prepa- ration, they are seldom available. Efforts are in prog- McCance and South African food ress in several regions to revise the nutrient losses and Widdowson tables, UK composition table gain factors, including nutrient retention and yield Types of data (developed country) () factors, in order to compare and harmonize them and Analyses 70% 41% in 1999 (improved thereby improve the quality of food composition data from 18% in 1991) calculated. 10% 49% As food composition data are frequently lacking Calculated 15% 10% for cooked foods, estimates based on the use of Estimated 5% – these factors for calculating the nutrient content of 286 Introduction to Human Nutrition prepared foods from raw ingredients are made. Thus, composition table or nutrient database. However, it is the nutrient composition of a prepared or cooked seldom possible to construct a food composition table food is calculated from the analytical data of uncooked with only such data. A plan of action should be devel- food by applying suitable nutrient retention and yield oped by the compilers of the database to deal with factors. To obtain the nutrient content per 100 g of missing food items and values for particular nutrients. cooked food, the nutrient content per 100 g of raw Very often, values of a biologically similar food are food is multiplied by the percentage retained after used. For composite or mixed dishes the composition cooking, and this is divided by the percentage retained of the dish is estimated by calculation from a standard after cooking, divided by the percentage yield* of the recipe and applying appropriate nutrient retention cooked product: factors and, in some cases, adjusting for changes in moisture content due to cooking loss or gain in the Nutrient content of cooked food per 100 g = different cooking procedures. If a food item forms an [(nutrient content of raw food × retention important part of the population’s diet and analysis is factor)/yield of cooked food] × 100 not possible, existing food composition databases The retention factor accounts for the loss of solids should be searched to see whether data on the same or from foods that occurs during preparation and a similar food item could be borrowed. If a value for a cooking. The resulting values quantify the nutrient nutrient is missing a similar approach can be followed, content retained in a food after nutrient losses due to as it is more desirable to have a slightly incorrect esti- heating or other food preparations. This is called the mated value of lower quality than no value at all. A true retention method and is calculated as follows: value of “–” or “0” assigned to missing nutrient values may lead to underestimation of nutrient intakes, % True retention = [(nutrient content per g of especially if those nutrients make a signifi cant cooked food × g cooked food)/(nutrient content contribution to the diet. per g of raw food/g of food before cooking)] × 100 The following example uses only the yield factor to Bioavailability and glycemic index predict the nutrient content of the cooked food. The Nutrient composition information in food composi- yield factors for different foods are reported in the tion tables indicates the amount of nutrients as ana- USDA Agriculture Handbook 102 and for cooked lyzed in that specifi c food sample and does not give an it is 92%. Selected nutrient values in SR 21 for indication of the absorption or bioavailability of the 100 g of raw carrots are 0.93 g of protein, 33 mg nutrient from that food item. However, when dietary of calcium and 5.9 mg of ascorbic acid. Using the reference intakes such as recommended dietary allow- yield factor the composition of 100 g of cooked ances (RDAs) are drawn up, the recommendation carrots is calculated as 0.93 g/0.92 = 1.01 g protein, makes provision for the amount of ingested nutrient 33 mg/0.92= 36 mg calcium and 5.9 mg/0.92 = 6.4 mg that may not be absorbed. The concept of bioavail- of ascorbic acid. This compares favorably to the deter- ability has developed from observations that measure- mined values for carrots of 0.76 g of protein and 30 mg ments of the amount of a nutrient consumed do not of calcium, but less so for ascorbic acid at a value of necessarily provide a good index of the amount of a 3.6 mg, probably because it is heat sensitive; therefore, nutrient that can be utilized by the body. The bioavail- applying the nutrient retention factor for ascorbic ability of a nutrient can be defi ned as the proportion acid (70%) would have resulted in a more accurate of that nutrient ingested from a particular food that prediction (5.9 × 0.7/0.92 = 4.9) of 4.9 mg/100 g) can be absorbed and is available for utilization by the (http://www.ars.usda.gov/nutrientdata). body for normal metabolic functions. This is not simply the proportion of a nutrient absorbed, and Missing values in food composition tables cannot be equated with solubility or diffusibility in In general, original analytical data provide informa- in vitro-simulated physiological systems. Bioavailability tion of the highest quality for inclusion in a food is not a property of a food or of a diet per se, but is the result of the interaction between the nutrient in ques-

*Yield of cooked food (%) = (weight of edible portion cooked food/ tion, other components of the diet and the individual weight of raw food) × 100 consuming the diet. Owing to the many factors Food Composition 287 infl uencing bioavailability, tables of food composition specifi c glycemic response. A more recently proposed cannot give a single value for a nutrient’s bioavailabil- identifi cation of a food’s GI value lies in indicating ity. Most research until now has centered upon inor- the specifi c food’s category of GI as high, medium, or ganic constituents, particularly iron, but the concept low. Accurate numerical values of a food’s GI are dif- is applicable to virtually all nutrients. Iron incorpo- fi cult to obtain as various factors, including human rated into heme is more readily absorbed than iron in subject variability both between and within subjects the nonheme form, and these two forms of iron are during analysis as well as response during ingestion sometimes listed separately in food composition of the food, can differ signifi cantly. tables. Yet, such information does not take into Both bioavailability and GI are food indices that account, for example, the effect of ascorbic acid are infl uenced not only by the characteristics of the (vitamin C) and organic acids (citric, malic, tartaric, food, but also by the response of the individual to the and lactic acid) on nonheme iron absorption. Iron food (i.e., absorption, metabolism, and excretion of absorption is also increased in a state of iron defi - the metabolites). For example, quantitative analysis of ciency and research has shown that vitamin A and iron carotenoids alone could lead to a misinterpretation of intake has to be increased simultaneously to alleviate vitamin A value. Therefore, the bioavailability of test anemia. In the coming years, it can be expected that foods in a single mixture may be investigated using much more work will be carried out on bioavailability the digestive system of nutrient-depleted rats (i.e., than in the past, because of its key role in relating measuring retinol accumulation factor as a measure functional nutritional status to nutrient intake. of total bioavailability), or in humans Future research will probably also focus more on using the relative dose–response test. Advances in the measurement of the bioavailability of food con- analytical chemistry such as improvements in analyti- stituents. Several vitamins and minerals, such as cal methods, information science, computer hardware calcium, iron, zinc, and a number of B vitamins, are and software will assist in fi lling these gaps in special- already being studied, with limited attention to carot- purpose databases in the future. enoid bioavailability. Inhibitors of absorption and the effects of processing and storage on the foodstuffs How to calculate a recipe not included in must be determined. As bioavailability is also infl u- the database enced to a large extent by the meal in which a food If the composition of a composite or mixed dish is constituent is consumed, this means that more infor- not known, it can be estimated by calculation from a mation will be needed not only on daily food con- standard recipe and applying appropriate nutrient sumption but also on intake of other constituents at retention factors and, in some cases, adjusting for individual meals. changes in moisture content due to cooking loss or There is an increasing demand from users of food gain during cooking. The following guidelines are composition tables for information on the glycemic suggested. index (GI) of food, which is used as a tool in the selection of food in the management of diabetes, as ● Identify the ingredients of the recipe from the most opposed to the previous system of carbohydrate appropriate foods available in the food composi- exchange. The GI is a food-specifi c measure of the tion database table. relative tendency of carbohydrate in food to induce ● Quantify the ingredients in mass (g). postprandial glycemia. The body’s response to a 50 g ● Calculate the nutrient values for the specifi c amount carbohydrate dose induced by either glucose or white of each ingredient. bread is taken as the reference and assigned a value of ● Add up the nutrient values of the individual a 100. Responses to all other foods are rated in com- ingredients. parison and listed in tabular format. New datasets ● Calculate the nutrient composition for 100 g of the with complementary values, based on the GI and recipe. available carbohydrate content of food, have been ● Apply suitable retention factors to the mineral and proposed, of which one is a measure of the relative vitamin nutrient values if the recipe food is cooked. glycemic response to a given mass of whole food and Note that if individual ingredients are in a cooked the other is the mass of a food responsible for a given form this step is not necessary. 288 Introduction to Human Nutrition

● Compare the moisture content of the calculated Table 11.7 Calculation of the composition of a dish from a recipe recipe with a similar cooked composite dish. If the Recipe for scrambled eggs with onions moisture content differs by more than 1%, adjust 2 large eggs 1 the moisture content of the recipe food. All of /6 cup whole milk 1 the nutrients of the recipe food must be adjusted /8 teaspoon salt 1 (concentrated or diluted) according to either the /4 cup chopped raw onions decrease or increase in moisture content. 2 teaspoons oil Add milk and salt to eggs and beat with a fork. Fry onions in the ● Assign to a suitable food group and list. oil. Pour egg mixture into frying pan with the onions, and stir This is only an estimation of the make-up of a com- mixture with a fork while cooking until it solidifi es. Makes one serving. posite or mixed dish of unknown composition. Refer to Table 11.7 for an example of the calculation of the Calculation of nutrient content of scrambled eggs from nutrient composition of a dish from a recipe. However, if this values for raw ingredients Step 1: Add nutrient levels for the specifi ed quantities of ingredients dish is a very important part of the diet of an indi- The nutrients in the raw eggs, whole milk, salt, raw onions vidual or group and the information is crucial in and oil are added together assessing the adequacy of the diet, analysis should be Step 2: Readjust quantities of those nutrients in the raw ingredients considered. that are lost during cooking due to evaporation or heat Nutrient loss on cooking Eggs Milk Onions Accurate estimation of portion size Food composition tables and databases are mainly Thiamin (%) 15 10 15 Ribofl avin (%) 5 used in nutritional epidemiology to estimate the Niacin (%) 5 composition of foods consumed by individuals. All Ascorbic acid (%) 25 20 subjects have diffi culties in estimating the exact Folacin (%) 30 portion sizes of food consumed. This issue is further complicated by the difference between the weight of Step 3: Determine weight of the recipe before cooking 1 large egg = 57 g; 57 g × 2 eggs = 114 g; refuse factor to a product as purchased and that of the actual item calculate weight without shell of 11%; consumed (e.g., in meat after cooking there is at least 1 14 g × 0.89 = 101.46 g = × 1 = a 25% cooking loss, without bone and with or without 1 cup whole milk 244 g /6 40.66 g = × 1 = visible fat). Standardized portion sizes for individual 1 teaspoon salt 5.5 g /8 0.69 g = × = foods within countries may help, but a set of standard 1 cup chopped raw onions 170 g 0.25 42.5 g 1 teaspoon oil = 4.53 g × 2 = 9.06 g food models (small, medium, and large) for use in Total weight = 194.37 g dietary assessment may be of more value. Step 4: Determine weight of recipe after cooking Weight loss during cooking due to evaporation is estimated to be 8% 11.6 Description of food composition Weight of recipe after cooking = 194.37 g × 0.92 = 179 g tables and databases and how to Step 5: Determine the nutrient levels of the recipe per 100 g and retrieve data per serving Divide the nutrient levels by 1.79 to determine the nutrient A food composition table or database is easier to use content of 100 g scrambled eggs The calculated nutrient levels represent the nutrient content if the format allows easy access to the data available. of one serving Advances in information technology have led to more and more food composition tables being available in electronic form, progressively replacing the printed Electronic data and access to them are more limited format. Printed food composition tables, although in remote areas in these countries, and a higher level limited by physical proportions such as the size of of computer literacy and equipment is necessary, both the written text and the printed table, continue which is generally seen as a luxury and not a to be popular in developing and underdeveloped necessity. countries. The printed word is seen as authoritative However, electronic databases have many advan- and only a limited level of literacy or knowledge on tages over printed tables, including virtually unlim- nutrition is necessary to be able to access the data. ited capacity to store information, rapid access to Food Composition 289 individual data items, and easy sorting and manipula- checking routines should be incorporated in the data tion of data for use in a wide range of calculations. entry process and subsets of data entered into the However, the ease of accessing data in an electronic computer should be compared with the original or a computerized database is dependent on the data- written records. Where mistakes are found, the extent base access software and not only on the way in which of the error should be determined, because it could data are stored. The development of relational data- involve data for the previous (or next) subject or day, bases has led to the opening up of possibilities to link or those previously (or subsequently) entered by the different databases in regions and countries with each operator involved. In addition to such checks, fre- other. This has led to the identifi cation of new chal- quency distributions of all amounts of food and food lenges such as food identifi cation, compatibility of codes should be carried out. The Food Surveys data, data interchange and data quality. Research Group of the Agricultural Research Service of the USDA has developed an automated method for collecting and processing dietary intake data. The 11.7 Converting foods to nutrients three computer systems, Automated Multiple Pass Method (AMPM), Post-Interview Processing System Entering data (PIPS) and Survey Net collect, process, code, review, Before the computer age, the conversion of food con- and analyze data for nutrient intakes. The system has sumption into nutrient intake had to be done manu- been used for the National Health and Nutrition ally, which was a laborious and time-consuming task. Examination survey since 2002. Later, much of the work, especially for larger surveys, was done on mainframe computers, and has since Converting data in food intake to passed on to microcomputers, because of their ready nutrient intake accessibility and ease of use. Data on food and nutri- A crucial aspect of food composition research is the ent intakes were often subsequently transferred to a transmission of information from those working in mainframe computer, where they were combined food composition and analysis to those working with other survey data for further analysis. Today, in food monitoring, to scientists trying to improve the there is little that cannot be done on a microcom- food supply, to workers in epidemiological, training puter, including data manipulation such as sorting and nutrition programs, and to regulators. Yet there and calculations. is little discussion in the scientifi c literature of the Before proceeding to calculate nutrient intake from issues relating directly to the compilation of food data on food consumption, it is necessary to ensure composition databases, which are the primary means that mistakes that have crept into the data set during of transmission of food composition data to most acquiring, coding, merging, transcription, and storage professionals in the fi eld. If good food statistics are are reduced to an acceptable level. Regardless of the available in a country, as well as access to food intake method used for the collection of data on food con- and food composition databases, estimates of a higher sumption, consideration should be given to how the quality can be made regarding the nutrient intake of data will be entered into the computer. Suitable forms the individual or population as a whole. However, few should be designed for the collection of data. These data on food composition exist for the 790 million can be on paper or in a personal computer-based people in developing countries who are chronically program that can save time and eliminate errors asso- undernourished and where malnutrition in the form ciated with the transcription of data from paper to of defi ciencies of iron, iodine, and vitamin A is rife. the computer. The use of carefully prepared forms, with information to guide those collecting the data, 11.8 Perspectives on the future can reduce the chance of error during the collection of data and, if a separate process, during entry into No universal food database system has been devel- the computer. The collection and entry of data are oped that fulfi lls all of the needs of compilers and subject to human and computer error; therefore, pro- users of food databases, despite the fact that it would cedures must be developed to ensure that the quality represent the primary scientifi c resource from which of data is as high as possible. Editing and error- all other nutritional studies fl ow. However, recent 290 Introduction to Human Nutrition international collaboration has considerably improved Europe has moved much closer to obtaining this goal, the development and compatibility of food composi- preceded by the ASEAN Food Composition Tables tion data. It is essential for the development of nutri- (2000) (www.fao.org/infoods/tables_asia_en.stm). tional sciences that this resource be maintained and improved to serve at both national and international Focusing on biodiversity within species levels. The quest for continued improvement in The FAO has begun a study on the development of quality of representative food composition data are at baseline data for the Nutritional Indicator for the core of most food composition programs. Biodiversity – 1. food composition. The aim is to collect food composition data at the inter- and intra- Recent advances in food composition species level for regions and countries. The process Harmonizing of regional food includes obtaining information on food composition composition tables data at the interspecies level (variety, cultivar, breed) High-quality, comprehensive food composition data and on underutilized and wild foods at the species for foods commonly consumed is important across level, as well as reviewing all available food composi- an ever increasing list of applications, e.g., in epide- tion data at national, regional, and international miological research studying the effect of specifi c levels. The data collected are reported in a template, foods on health and well-being. Integrated, compre- naming the country and the INFOODS regional data hensive, and validated food composition databanks centre. Table 11.8 gives an example of the format of from individual countries within a region will con- reporting at the national level. For the baseline report- tribute immeasurably towards shifting the barriers of ing, at the beginning of 2008 data from 254 publica- current scientifi c understanding. Towards this end tions from 49 countries were included.

Table 11.8 Template for reporting on the nutritional indicator of biodiversity in the food composition literature

Number of foods on subspecies level with following number of components

Publication Material examined References 1 2–9 10–30 >30 1. Food composition databases (FCDB) Reference database of national FCDB User database of national FCDB Other national FCDB 2. Literature National peer-reviewed journals Indicate journals and years National laboratory reports Indicate laboratories and years Reports from national research institutes Indicate research institutes and years National conference presentations (incl. posters) Indicate conferences and years Theses Indicate universities and years Other (specify) Indicate publication and years Material examined Letter Material examined A B References Number Full reference DOI, CiteXplore ID1, other international publication code 1 2 Food Composition 291

Investigating specifi c traditional and oxidative damage) and cardiovascular disease (inhib- ethnic foods iting oxidative damage to low-density lipoprotein Traditional and ethnic foods refl ect cultural inheri- cholesterol). Apart from containing antioxidants, tance and in many cases form key components in the plant foods contain other compounds, not classifi ed dietary patterns in many countries. In many instances as traditional essential nutrients, but as bioactives. traditional foods include underutilized vegetable These bioactives, backed up with substantial evidence, species and in the current evolved world there are may play a role in health promotion. still many of these species and subspecies of which Thousands of plant bioactives have been identifi ed the nutrient information is lacking. Traditional and and the major classes of plant bioactives are fl avo- ethnic foods contribute considerably to the diet of noids and other phenolic compounds, carotenoids, many populations, and may have signifi cant health plant sterols, glucosinolates, and other sulfur- contributions. Research and analysis on these foods containing compounds (http://www.eurofi r.net). The has slowly emerged as a matter of great interest, but USDA has prepared several Special Interest databases with fi nancial constraint on such type of research, on fl avonoids, proanthocyanidins, isofl avones, and much is still to be done. ORAC (antioxidant powers assayed by oxygen radical Ethnic minority populations have become signifi - absorption capacity assay). The ORAC database con- cant parts of the population in many countries, and tains values for total phenols also. similarly in many developing countries traditional foods form a major component of these populations’ Nutrition and health claims diet. Inequalities in health status are observed in these Focusing on the relationship between diet and health, subpopulations compared with the general popula- consumers are demanding more information on tion. These inequalities, which could be due to socio- the food they purchase and consume. Not only has economic status, have highlighted the need for the there been an increase in demand for nutrition expansion of nutrient data on ethnic and traditional information, but the increased prevalence of non- foods. A limited budget is mostly all that is available communicable diseases such as cardiovascular disease for the analysis of these foods, which is one of the and diabetes mellitus, as a consequence of obesity, has main reasons why there are limited data available. led to increase in the need for nutrition communica- Often composition data for ethnic foods are derived tion and guidance in making healthy food choices. or borrowed from other food composition tables or Food labeling has become an important communica- derived from recipes. Variation and modifi cation in tor to the consumer, with the provision that it is based recipes and cooking practices between individuals are on the truth and not misleading. The Codex Alimen- also some of the complications to consider when tarius Commission (http://www.codexalimentarius. the composition of ethnic and traditional foods is net) aims to strengthen local and regional efforts investigated. towards harmonizing and simplifying the process of making a nutritional or health claim. Bioactives in foods and their effect on health Towards this end they proposed the following areas and well-being for further development: Dietary constituents commonly found in foods with ● labeling to allow consumers to be better informed health-promoting or benefi cial effects when ingested about the benefi ts and content of foods are part of the emerging evidence that drives consum- ● measures to minimize the impact of marketing on ers, researchers, and the food industry in their quest unhealthy dietary patterns for validated information. It is generally recognized ● more detailed information about healthy consump- that a diet high in plant foods is associated with tion patterns including steps to increase the con- decreased incidence of certain diseases such as cancers sumption of fruits and vegetables and cardiovascular disease. One of the several plausi- ● production and processing standards regarding the ble reasons for this decrease in incidence of disease is nutritional quality and safety of products. the antioxidant properties of plant-derived foods, which may prevent some of the processes involved in Nutrition and health claims are used to present the development of cancer (protecting DNA from products as having an additional nutritional or health 292 Introduction to Human Nutrition benefi t. In most cases, consumers perceive products Ireland J, Van Erp-Baart AMJ, Charrondière UR, Møller A, Smithers carrying certain claims to be better for their health and G, Trichopoulou A. Selection of food classifi cation system and food composition database for future food consumption surveys. well-being. Nutrient profi ling is the fi rst step towards European Journal of Clinical Nutrition 2002; 56 (Suppl. 2): a possible health claim. At present, different systems S33–S45. for the setting of nutrient profi ling range from a simple Rand WM, Pennington JAT, Murphy SP, Klensin JC. Compiling Data for Food Composition Data Bases. United Nations University algorithm to a scientifi cally complicated approach. It Press, Tokyo, 1991. is diffi cult to develop a single system that refl ects both Southgate DAT. Food composition tables and nutritional databases. the nutrition contribution of a food or food group to In: Garrow JS, James WPT, Ralph A, eds. Human Nutrition and Dietetics, 10th edn, Churchill Livingstone, Edinburgh, 2000: the diet and the effect of the matrix on nutrient bio- 303–310. availability. This discussion is continuing. Truswell AS, Bateson DJ, Madafi glio KC, Pennington JA, Rand WM, Klensin IC. (1991) INFOODS guidelines for describing foods: a systematic approach to describing foods to facilitate interna- tional exchange of food composition data. Journal of Food Further reading Composition and Analysis 1991; 4: 18–38. Verger Ph, Ireland J, Møller A, Abravicius JA, Den Henauw S, Naska A. Improvement of the comparability of dietary intake assess- Brussard JH, Löwik MRH, Steingrímsdóttir L, Møller A, Kearney J, ments using currently available individual food consumption De Henauw S, Becker W. A European food consumption survey surveys. European Journal of Clinical Nutrition 56 (Suppl. 2): method – conclusions and recommendations. European Journal S18–S24, 2002. of Clinical Nutrition 2002; 56 (Suppl. 2): S89–S94. Food and Agriculture Organization of the United Nations. Expert Websites Consultation on Nutrition Indicators for Biodiversity 1. Food Composition. Rome, 2008. Commission: http://www.codexalimentarius. Greenfi eld H, Southgate DAT. Food Composition Data. Production. net Management and Use. Food and Agriculture Organization of the Eurocode2: http://www.eurofi r.org/eurocode United Nations, Rome, 2003 EuroFIR: http://www.eurofi r.net Greenfi eld H. 1990. Uses and abuses of food composition data. Food and Agriculture Organization of the United Nations: Food Australia 42 (8) (Suppl.). http://www.fao.org Gry S, Holden J. Sampling strategies to assure representative values INFOODS: United Nations University of International Food Data in food composition data. Food, Nutrition and Agriculture 1994; Systems Project: http://www.fao.org/infoods/directory 12: 12–20. LanguaL: http://www.langual.org Klensin JC. INFOODS Food Composition Data Interchange United States Department of Agriculture National Nutrient Handbook. United Nations University Press, Tokyo, 1992. Databank: http://www.ars.usda.gov/nutrientdata 12 Food and Nutrition: Policy and Regulatory Issues

Michael J Gibney and Aideen McKevitt

Key messages

• The human food supply is highly regulated and while in the past demand-driven approaches, efforts are made to create a demand there was an emphasis on food safety, there is now a rapidly for a new food-purchasing pattern through a nutrition communi- expanding regulatory base covering nutrition. cation process. • Any policy decision in the nutrition regulatory framework needs • A nutrition communication process should always be built on to be informed by up-to-date and relevant data on prevailing actual studies of consumers attitudes and beliefs, and a number food and nutrient intake patterns. These metrics are compared of tools are commonly used to communicate nutrition and health with agreed standards for optimal food and nutrient intake and messages including nutritional labeling and nutrition claims. on the basis of any discrepancy, public health nutrition programs • Globalization of the food supply has been accompanied by evolv- encompassing regulatory issues are initiated. ing governance issues that have produced a regulatory environ- • Public health nutrition programs can be supply driven or demand ment at national and global level led by large national, driven. In the supply-driven option, the government takes the international agencies in order to facilitate trade and to establish decision centrally to alter some properties of foods the most and retain the confi dence of consumers in the food supply common approach being mandatory food fortifi cation. In chain.

12.1 Introduction 12.2 Reference points in human nutrition

Few areas of our lives are more regulated than that of Chapter 10 in this textbook outlines the many options the food supply and within that regulatory frame- that are available for measuring food intake and con- work, three distinct divisions are evident: food chemi- verting those data into nutrient intakes. Such data are cals, food microbial hazards, and nutrition. In the fundamental to the development of nutrition-related past, the chemistry and microbiology aspects of food regulatory policy. The more detailed the level at which regulation tended to dominate but in recent times, data are collected, the more useful they are for advis- the regulatory environment for nutrition has begun ing and informing policy. Prevailing dietary habits, as to receive increasing attention given that (a) the role measured through dietary surveys, represent the fi rst of diet in noncommunicable chronic disease has been reference point for nutrition policy. The second set of so extensively accepted and woven into policy and (b) reference points are those targets set out by expert food producers have made efforts to develop innova- committees that will move populations toward ever- tive products to help reduce the burden of disease healthier diets. Chapter 7 of this textbook describes risk. The present chapter is intended to provide new the basic principles involved in setting out target students of nutrition with a brief insight into the values for the assessment of dietary intakes, primarily present direction of food regulation as it relates to for micronutrients. These are defi ned using variable dietary choices. terms across the globe but, generally, all defi nitions

© 2009 MJ Gibney and A McKevitt. 294 Introduction to Human Nutrition employ the term “reference” and hence they can be interim attainable targets in realistic public health classifi ed as reference nutritional data. Such data were nutrition programs that can be implemented over a historically developed to ensure the adequacy of the defi ned and reasonable period of time. In summary, human diet from the point of view of micronutrients. it is not possible to develop a meaningful nutrition However, as our knowledge of diet and chronic disease regulatory framework without access to both nutrient has evolved, a second set of reference nutrition values intake data and dietary reference data. had to be developed, this time to minimize the risk of chronic disease. Table 12.1 shows a non-exhaustive 12.3 Exploration of dietary patterns list of these nutrients and the risk factor or chronic disease they are associated with. With a given set of population nutrient intake data In addition to these data, recommendations are and a given set of nutritional reference values, it is made for ideal body weight and there are specifi c sets possible to divide the population into those closest to of dietary guidelines for such life stages as lactation, some nutritional ideal and those furthest from such pregnancy, aging, etc. However, the regulatory envi- an achievement. These two contrasting groups can ronment in nutrition is dominated by the above now be laid against one another and a wide range of nutrients and also the micronutrients. data, listed in Box 12.1, can be examined. The whole purpose of devising these two sets of Based on these comparisons and using appropriate metrics – nutrient intakes and nutrient reference statistical techniques, it is possible to begin to discern values – is to fi rst measure where we are in relation to the reasons why one group are near achieving some our nutritional well-being and second to set targets nutritional ideal and why another are set far off the to move the population toward a healthier diet. There mark. These reasons now feed into policy advice and is however, a very slight antagonism between the begin to form the nucleus of a nutrition regulatory establishment of an ideal pattern of nutrient intake structure that may help the population improve their and developing public health nutrition programs to diet. Given that the focus of this text is nutrition, it achieve that goal. The reason is that the former does would be worthwhile to single out food patterns for so in isolation from the real world of everyday eating. a more critical analysis. The following is a hypotheti- Its focus is on experimental studies that, for example, cal fi nding in relation to three foods that appear to be help delineate the optimal balance of dietary fatty important in determining the nutritional adequacy of acids to minimize plasma cholesterol. That optimal “achievers” and “non-achievers” of some nutrient may be very signifi cantly different from prevailing goal. dietary habits and to attempt to bridge the gap too Look at Table 12.2. At fi rst glance C seems unim- fast might produce a public health nutrition program portant and A and B seem to be important and going that is unrealistic. Thus nutritionists can look at pre- in opposite directions. These are very typical data that vailing intakes against ideal intakes and then set out emerge from such analyses and they hide two very important statistics that should always be sought in studies of this nature. The fi rst missing statistic is Table 12.1 Nutrients and associated risks “% consumers” and the second is the “intake among

Nutrient Effects

Saturated fatty acids Blood cholesterol Monounsaturated fatty acids Blood cholesterol Box 12.1 n-6 polyunsaturated fatty acids Blood cholesterol Trans unsaturated fats Blood cholesterol Nutrients n-3 polyunsaturated fatty acids Blood cholesterol Foods Total fat Obesity Eating habits Sodium Blood pressure Anthropometry Sugar Dental caries Socioeconomic data Fiber Digestive disorders Physical activity Folic acid Neural tube defects Education Fruits and vegetables Certain cancers Others Policy and Regulatory Issues 295 consumers only.” Now reconsider the above data with Supply-driven nutrition policy takes the food these additional statistics and look at Table 12.3. supply and in some way modifi es it so that individuals Now everything has changed with the fi ve consum- consuming a habitual diet will have their nutrient ers converting population average intakes into con- intake altered without having to make any major sumer-only intakes. For any program in public health changes in food choice. Mandatory fortifi cation of nutrition, three important strategies which are often foods with micronutrients is by far the best example lost are (a) strategies to increase or decrease the fi ve of supply-driven food nutrition policy. There are people eating a target food, (b) strategies to alter the certain essential prerequisites to the development of frequency with which a target food is consumed, and a successful supply-driven fortifi cation program. (c) the portion size when the food is eaten. Thus were These are shown in Box 12.2. we to look solely at population averages, food C was Let us now consider these factors for a typical for- of no interest. Now it is of interest if not intriguing: tifi cation process, the mandatory addition of folic “achievers” universally eat this food while only 30% acid to fl our in the USA to reduce the incidence of of “non-achievers” partake of it, and, among the small the neural tube birth defect, spina bifi da (Box 12.3). group of “non-achievers” who do eat the food, they Let us contrast the data in Box 12.3 with the evi- eat it at a much higher level (which might be the same dence linking saturated fatty acids (SFAs) to plasma amount more frequently or a higher amount less cholesterol shown in Box 12.4. frequently).

Box 12.2 12.4 Options to change food and nutrient intakes a There is unequivocal evidence that the lack of a particular nutrient very strongly predisposes to some serious condition Once the above analysis is complete and peer reviewed, b The evidence is based on properly conducted human nutrition defi nite directions in the consumption of nutrients intervention trials c The effect of the nutrient in question on the problem to be and foods become apparent. In this section we focus solved is not dependent on other conditions being met on some of the options but the reader should always d There are no adverse effects from the fortifi cation strategy bear in mind that all options are possible and none is e The scale of the problem is a true public health issue exclusive. Broadly speaking we can think of two con- trasting options: “supply-driven” nutrition policy and “demand-driven” nutrition policy. Box 12.3

a+b There is certainly unambiguous evidence from randomized controlled trials involving high-risk women who had a previ- Table 12.2 Achievers and non-achievers of nutrient goals ous spina bifi da baby that folic acid signifi cantly reduces the risk of a second event Achievers Non-achievers c The effect is independent of any other factor from age, (g/day population average) smoking, weight, and ethnicity, and so on d There is some concern that fortifying with folic acid might Food A 100 40 cause some B12 defi ciency to go undiagnosed but the scale of Food B 20 60 that problem is not enough to halt the fortifi cation program Food C 50 50 e This problem is a truly important public health issue

Table 12.3 Achievers and non-achievers of nutrient goals with consumer-only intakes

Achievers Non-achievers Achievers Non-achievers Achievers Non-achievers

g/day population average % consumers Consumer-only intake

Food A 100 40 20 80 500 50 Food B 20 60 50 50 40 120 Food C 50 50 100 30 50 150 296 Introduction to Human Nutrition

Box 12.4 beliefs. The only way that this can be understood is to study what consumers feel and believe before we can a+b There is certainly strong evidence that elevated levels of expect them (a) to listen to our communication, (b) SFAs can raise plasma LDL cholesterol. However, within the believe it, (c) understand it, or (d) care about it. The category SFAs, some individual fatty acids are more potent present section assumes that is a given. In terms of than others and these are not uniquely found in one single dietary source of fat nutrition communication, there are three very impor- c The effect of SFAs is to some extent also dependent on the tant areas to consider: nutrition labeling, nutrition simultaneous intakes of trans unsaturated fatty acids and claims, and nutrition profi ling. different forms of unsaturated fatty acids d+e There are no adverse effects known and the problem is not Nutrition labeling truly important In most countries, packaged foods bear a label listing particular nutrients in particular ways. The number of nutrients listed can vary either because of the prevailing food policy or because it suits a manufac- turer to have more or less nutritional information From the data in Boxes 12.3 and 12.4 it is easy to imparted to the consumer. The standard format is to defend the folic acid option but less easy to defend express the target nutrients per 100 g of the food or the SFA option for supply-driven policy. It should be per some specifi ed portion of the food. Generally, borne in mind that a supply-driven policy effectively nutrition labeling was a “back of pack” issue, generally takes away from the individual the right to choose in considered less important. Today, it is becoming this regard and thus there are always social and some- increasingly a “front of the pack” issue, with visuals times ethical dimensions to this approach. to immediately let the consumer see what a typical Demand-driven nutrition policy is based on edu- serving supplies in terms of target nutrients and then cating the consumer to demand newer and healthier to express these as a percentage of some reference types of foods from the food supply. This is a chicken- intake. Often colors are used where a serving greatly and-egg situation. Consumers may want something exceeds some nutritional standard (red) or green if it that is not within the scope of industry to produce is well below. either for economic or technical reasons. Equally Nutrition labels fulfi ll a very important role in many companies have developed food products with nutrition communication – helping the consumer see very obvious health benefi ts which were market fail- the nutrient content of the food. However, there are ures because the consumer saw no benefi t. The success aspects that are not so obvious which we need to of this area is thus very market driven. Industry made bear in mind. Comparisons of the nutritional com- spreadable fats low in SFAs, which consumers liked. position of different foods are often diffi cult to They developed immune-boosting , choles- interpret. For example, in choosing a packaged sand- terol-lowering phytosterols, high-fi ber ready-to-eat wich, the consumer can unite their gastronome cereals and cereal bars, juices with various antioxi- preferences with nutritional data to make a choice. dants, low-fat milks, n-3 PUFA-enriched eggs, and so On the other hand, if the choice was a carton of on. For demand-driven food supply to work, we need ready-to-eat soup versus a sandwich, the comparison to invoke a major new area of public health nutrition is much more diffi cult; when it comes to making a – communication. decision on any two foods versus another set of two foods, then the decision process is exceeded for almost 12.5 Nutrition communication everybody. Another limitation of nutritional labeling is that fresh foods are often not packaged and thus are One of the great attractions of the science of human not labeled for nutritional content. The same is true nutrition is the breadth of topics to be covered from for meals, snacks outside the home, in bars, restau- molecular biology, through population science to rants, canteens, delicatessens, and the like. Notwith- communication. The greatest mistake a nutrition standing the shortcomings, nutrition labeling is a very regulatory policy initiative can make is for scientists positive step in helping consumers make informed to think they know the consumer and his or her choices. Policy and Regulatory Issues 297

Nutrition claims in lowering the risk of a disease or condition, they In general, claims in the fi eld of food and health can need to be able to make that claim and to prevent be divided in several ways in matrix form. The fi rst others who have not done this research from simply division is into claims which are “generic” (any manu- adopting that claim. In that way, they stand a chance facturer can use it if they meet the criteria) and claims of developing a market leader and of recouping their which are “unique,” that is specifi c to a brand which research investment. This approach is perfectly under- has some unique attribute on which a claim can be standable but it does cause problems for smaller com- made. In the USA, the Food and Drug Administration panies and for industrial sectors in less developed (FDA) has favored generic claims such as “saturated countries for which such high stakes are unthinkable. fats raise cholesterol” or “calcium helps bone health.” The terms and conditions for the use of such claims The FDA accepts petitions in this area where industry has led to a third area in nutrition communication – groupings put forward a scientifi c case as to why such nutrition profi ling. a generic claim might be used. If accepted, the regula- Nutrition profi ling tor can now decide what the conditions for making a claim are. For example, a typical serving of the food This is by far the newest area and without doubt the would have to achieve a minimum percentage of most controversial. In the EU, the law now requires some reference value before a claim could be made. A that for a food to make a claim, it must meet certain product where a serving size gave 1% of the require- nutritional standards. This is often referred to as the ment for calcium would surely not be allowed to Jelly Bean Rule – that is, if you added zinc to jelly make any claim on bone health. beans, would one support the promotion of jelly bean The other type of claim can be classifi ed into three consumption on the grounds that increased zinc levels (Table 12.4) intake may assist in minimizing poor immune func- At the time of writing, there are developments in tion. The idea is that if the food supply needs zinc to different parts of the world as to how such claims can be added, then a more suitable vehicle needs to be be handled. As one goes up from level 1 to level 3, the found. In principle this is not complex. In the real scientifi c rigor must increase exponentially. Quite world it is an intellectual minefi eld. In terms of devel- probably, level 2 and level 3 will need to be accompa- oping nutrient profi les for whatever reason, there are nied by signifi cant supportive evidence from human two approaches in operation. One seeks to take a dietary intervention studies. Again, such claims will single set of criteria and apply that universally to all require that certain specifi ed attributes of the food be foods. This has been the approach of what is called met before a claim can be made, and different parts the UK Traffi c Light System. All foods are classifi ed of the globe are taking various approaches to these into three types, which can be described as good issues. As with many aspects of labeling communica- (green), bad (red), or neither (orange). Inevitably, the tion, some refl ection will help reveal the complexity application of such a simple system to something as of the task. If companies are to innovate and develop complex as the human food chain leads to exceptions. new foods with enhanced nutritional properties or Walnuts might get a red color because of their high functions, they need to invest in research and devel- fat content, and yet walnuts have been shown along opment. If their research, industry supporting human with other nuts to be protective against heart disease. intervention studies, shows clear evidence of an effect Maybe walnuts are now exceptionally excluded from a red sign. But the process goes on to exceptionally include or exclude and the objectivity of the simple Table 12.4 Nutrition claims approach becomes gradually replaced with the sub- jectivity of exceptions. Level 1. Nutrition This product is a rich source of some content omega-3 fats The second nutrient-profi ling approach is to take Level 2. Function This product is rich in omega-3 fats, which each food category separately and devise nutritional claim promote heart health standards for each category. An example of this is the Level 3. Disease This product is rich in omega-3 fats, which Swedish Keyhole Method. For breads, certain stan- reduction claim reduce the risk of cognitive impairment dards are set and breads that meet these standards in older people carry the keyhole symbol, which consumers recognize 298 Introduction to Human Nutrition as a mark of nutritional quality. The huge advantage be reviewed continuously to take account of issues of this system is that the standards are not universal. such as food sources from new areas with differing One is judging packet soups against packet soups as climates, growing and harvesting techniques, and an example but not against mayonnaise or chocolate public health infrastructure. In addition, there are or breakfast cereals. At present, the area of nutrition very many national approaches to food regulation profi ling is very much at the development stage and refl ecting different perceptions about the value of it remains to be seen how exactly this progresses. new technology, different degrees of protection given Besides the use of nutrient profi ling for permitting by governments to food producers, and even different claims, there is interest in its use in deciding signifi - interpretations of the science involved in the regula- cant nutrition policy issues about individual foods. tory process. The implication of globalization for Advertising of foods is one critically important area food regulation therefore requires both international where this approach may be applied. cooperation among national food regulators and the effective balancing of gains from trade with regula- 12.6 Global players in food and tory differences. nutrition regulation UN and UN agencies Food and nutrition regulation spans the entire food Globally, a range of agencies plays a role in food and chain – from processing of seeds, to planting seeds, to nutrition regulation. The UN was established in 1945 tilling crops, storage and harvest of crops, and sale of as was the Food and Agriculture Organization (FAO), crops. Animal and fi sh farming are equally complex. which was established as a specialized UN agency. The Where these primary products enter the realm of the principal role of the FAO is the provision of food food processor, another range of regulations apply, security for all. Coupled with this is its mandate to for example what is permitted to be added to the food, raise nutrition levels and agricultural productivity in what must be in a food (nutrition), what must not be order to raise the standard of living for rural com- in a food (pesticides), the physical and biological munities and thereby contribute to the growth of the environment in which the food is processed, in addi- global economy. tion to packaging, labeling, transport, storage, sales, The World Health Organization (WHO) is another and advertising. agency of the UN and is a sister organization of the Globalization is one of the driving forces shaping FAO. Established in 1948, its objective is the attain- the world economy and the pace of globalization of ment by all peoples of the highest possible levels of the food trade has accelerated in the past decades. health. The WHO is the directing and coordinating New methods and technologies in food production authority for health within the UN system. The WHO and processing have contributed to this acceleration. is governed by its member states through the World Productivity of animals and crops has risen to unprec- Health Assembly (WHA), which is composed of rep- edented levels. Globalization of the food trade bene- resentatives from each member state. The WHO con- fi ts consumers in terms of quality, affordability, and siders that freedom from hunger and malnutrition is guaranteed supply. It also offers diversity of products, a basic human right and alleviation of these global which can contribute to improved nutrition and problems is fundamental for human and national health. Globalization has been accompanied by evolv- development. While the WHO has traditionally ing governance issues that have produced regulation focused on nutritional defi ciency and associated mor- at a national and global level in an attempt not only bidity and mortality, the issue of malnutrition char- to facilitate trade but also to establish and retain the acterized by obesity and the long-term implications confi dence of the consumer in the food supply chain. of unbalanced dietary and lifestyle practices that The distances that food and feed are now transported result in chronic diseases such as cardiovascular potentially create conditions more conducive to con- disease, cancer, and diabetes has assumed increasing tamination of the supply chain, where even a single importance in recent years. Countries, particularly source can have serious consequences. developing countries where both under- and over- Modern food and nutrition regulation must deal nutrition coexist, are of particular concern. In light of with this range of activity on a global scale and must these challenges and trends the WHO aims to build Policy and Regulatory Issues 299 and implement a science-based, comprehensive, inte- The FAO also recognized the need for international grated, and action/policy-oriented “Nutrition Agenda” agreement on food standards, labeling requirements, at the global, regional and country levels that addresses methods of analysis, etc. In 1963, the Sixteenth the whole spectrum of nutrition problems towards World Health Assembly approved the establishment attaining the Millennium Development Goals of the Joint FAO/WHO Food Standards Program (MDGs) and other nutrition-related international and adopted the statutes of the Codex Alimentarius commitments, including the prevention of the diet- Commission (CAC). related chronic diseases. The Millennium Declaration The CAC is the pre-eminent global food standards (later restated as MDGs with specifi c measurable organization and has had an important impact on targets that should be met by 2015) was signed by 147 food producers, processors, and consumers. The heads of state in 2000 and passed unanimously by the principal aims of Codex are to protect consumers’ members of the UN General Assembly. The MDGs health, ensure fair practices in the food trade by the seek to eliminate hunger, poverty, maternal and of science-based and safety malnutrition with particular emphasis on maternal standards, guidelines, and recommendations, and and fetal undernutrition and malnutrition, and promote coordination of all food standards work micronutrient malnutrition. The UN Children’s Fund undertaken by governmental and international (UNICEF) was established by the UN General organizations. The harmonization of food standards Assembly in 1946. UNICEF provides long-term facilitates trade between countries and underpins it humanitarian and developmental assistance to with a guarantee that food that is traded will be children and mothers in developing countries with safe and of the same quality as the same product special emphasis on pregnancy, and made elsewhere. Membership of CAC is open to all the fi rst 3 years of life. member nations and associate members of the FAO and/or WHO. By 2007, some 174 countries and one FAO/WHO and Codex Alimentarius Member Organization (European Community) were In the 1950s food regulators, traders, consumers, members. CAC meetings are held yearly and alter- and experts were looking increasingly to the FAO and nately at the FAO headquarters in Rome and the WHO for leadership about the plethora of food regu- WHO headquarters in Geneva. At these meetings lations that were impeding trade and that for the draft and fi nal standards, guidelines, and codes of most part were not providing adequate protection for practice are adopted. Each member of the Commis- consumers. As a result, the Joint FAO/WHO Expert sion has one vote. Decisions of the Commission are Committee on Food Additives (JECFA) was estab- taken by a majority of votes cast. Representation is on lished in 1956. Its remit now covers the evaluation of a country basis. National delegations are led by senior contaminants, naturally occurring toxicants and resi- offi cials appointed by their governments. Delegations dues of veterinary drugs in food. In the early 1960s a may include representatives of industry, consumers’ Joint FAO/WHO Meeting on Pesticide Residues organizations, and academic institutions. Countries (JMPR) was set up to provide independent scientifi c not members of the Commission sometimes attend advice to the FAO and WHO with recommendations in an observer capacity. A number of international from panels of independent experts on the use of governmental organizations and international NGOs pesticides in agriculture and safe levels of residues also attend in an observer capacity. These organiza- in foods. The Joint FAO/WHO Meeting on tions may put forward their points of view at every Microbiological Risk Assessment (JEMRA) began stage except in the fi nal decision, which is taken by in 2000. The aim of JEMRA is to optimize the use of member governments. The Commission and member microbiological risk assessment as the scientifi c basis governments have established country Codex for risk management decisions that address microbio- Contact Points and many member countries have logical hazards in foods. Other examples of ad hoc National Codex Committees to coordinate activities joint expert consultations on new or emerging food nationally. and nutrition problems are the Joint FAO/WHO Codex Alimentarius is the Latin name for food law Expert Consultation on Acrylamide or the ad hoc or food code. The main aim of Codex is to defi ne Committee on Foods derived from Biotechnology. international standards, codes of practice, and other 300 Introduction to Human Nutrition guidelines and recommendations. The main work on standard setting is carried out in more than 20 Codex WTO: Sanitary and phytosanitary measures Committees and Task Forces. These include commit- and technical barriers to trade tees dealing with “vertical” and “horizontal” stan- The General Agreement on Tariffs and Trade (GATT) dards, task forces dedicated to a particular task of began in 1948. Countries subsequently agreed to limited duration and regional coordinating commit- lengthy “rounds” of negotiations to develop rules for tees. In addition, the experts’ meetings organized and “non-tariff barriers” to trade. The completion of the supported by the FAO and the WHO, JMPR, JEMRA, Uruguay Round of Multilateral Trade Negotiations and JECFA provide the scientifi c basis (risk assess- 1986–1994 led to the formation of the World Trade ment) for the work of the CAC. At the beginning the Organization (WTO) on 1 January 1995. The Uruguay CAC concentrated on commodity standards called Round Agreements (which began at Punta del Este, “vertical standards,” for example standards for cereals; Uruguay) for the fi rst time incorporated agriculture fats and oils; fi sh and fi sh products; fresh fruits and and food under its rules. Two of the Uruguay Round vegetables; processed and quick frozen fruits and veg- Agreements relevant to international food regulation etables; fruit juices; meat and meat products; milk are the Agreement on the Application of Sanitary and and milk products; sugars, cocoa products, and choc- Phytosanitary Measures (SPS) and the Agreement on olate. In the 1980s it was generally agreed that diver- Technical Barriers to Trade (TBT). The SPS Agreement sifi cation of food products was occurring so rapidly allows governments to take scientifi cally justifi ed that the setting of detailed standards was in fact sanitary and phytosanitary measures to protect hindering trade. Thus a move toward “horizontal” human health. The agreement commits members to standards began. “Horizontal standards” are general base these measures on internationally established standards that have application across a wide range of guidelines and risk assessment procedures. The SPS foods, for example general principles: food additives Agreement has chosen the standards guidelines and and contaminants; food labeling; food , recommendations established by the CAC for food methods of analysis and sampling; pesticide residues, additives, veterinary drug and pesticide residues, con- residues of veterinary drugs in foods; food import taminants, methods of analysis and sampling, and and export inspection and certifi cation systems; codes and guidelines of hygienic practice. A national nutrition and foods for special dietary uses. These standard that provides a greater level of protection standards are then published in one of the Codex’s 13 than Codex is considered to be a trade barrier unless volumes. Codex standards pass through various stages the WTO decides that the stricter national standard of ratifi cation by members – the eight-step process – is based on a risk assessment that demonstrates that the fi nal one being that of acceptance. When members the Codex standard, guideline, or recommendation accept a Codex standard they are committed to provides insuffi cient protection or that the country allowing products conforming to that standard on to maintaining the stricter standard has other scientifi c their market. justifi cation. The TBT agreement seeks to ensure that A major concern of national governments is that technical regulations and product standards includ- food imported from other countries should be safe ing packaging, marking and labeling requirements, and not jeopardize the health of consumers or and analytical procedures for assessing conformity pose a threat to health and safety of their animal and with technical regulations and standards do not create plant populations. So governments of importing unnecessary obstacles to trade. The importance of countries introduce laws and regulations to reduce or Codex standards is also stated in the Technical eliminate such threats. In the food area these Regulations and Standards provisions contained in measures could become disguised barriers to trade as Article 2 of the TBT Agreement. So, although CAC well as being discriminatory. One of the main prin- standards are not enshrined in international law, ciples of the Codex Alimentarius is that harmoniza- WTO endorsement of these standards through the tion of food laws and adoption of internationally SPS and TBT agreements has effectively made them agreed standards would result in fewer barriers to mandatory, and Codex standards are the benchmarks trade and freer movement of food products among standards against which national measures and countries. regulations are evaluated. Both the SPS and TBT Policy and Regulatory Issues 301

Agreements call on the WTO member countries to countries are still faced with resource constraints to seek harmonization of regulations based on the work effective participation in Codex activities. The FAO of the CAC, and foods which meet CAC Codex stan- and WHO technical assistance programs support the dards, recommendations and guidelines should be efforts of developing countries to strengthen their traded freely in international trade. national food safety systems to protect local consum- Adherence to Codex standards has become critical ers and to take advantage of international food trade as they are used as guidelines for the resolution of opportunities. In addition, the FAO/WHO Codex disputes under the enhanced WTO dispute settlement Trust Fund supports the participation of countries in procedure. Annex 2 of the WTO covers all disputes Codex technical committee meetings, and countries arising from the GATT and WTO agreements. A have been funded to attend sessions of the CAC. dispute is triggered when a WTO member complains that another member(s) has failed to live up to the Europe obligations of the GATT or WTO agreements, i.e., a Having considered the global agencies and institu- benefi t guaranteed under one or other of these agree- tions that impact on food and nutrition regulation, ments has been “nullifi ed or impaired” by another the EU will be considered as an example of evolution member(s). The dispute settlement procedure encour- toward a modern system of food and nutrition ages the governments involved to discuss their prob- regulation. lems and settle the dispute by themselves. The fi rst The EU is an association of 27 Member States that stage is therefore consultations between the govern- have agreed to integrate and coordinate much of their ments concerned. If the governments cannot resolve economic policy and some other policy areas. The their dispute they can ask the WTO director-general original European Economic Community (EEC) was to mediate or try to help. If consultations fail, the formed following the signing of the Treaty of Rome complaining country can ask for a panel to be in 1957 and consisted of six Member States, increas- appointed. If the panel decides that the disputed trade ing over time to 9, 12, 15, 25, and 27 Member States measure does break a WTO agreement or an obliga- in 2007 following the accession of Romania and Bul- tion, it recommends that the measure be made to garia. The emphasis in the early years was to concen- conform with WTO rules. The panel may suggest how trate on the free movement of foodstuffs through the this could be done. Either side can appeal a panel’s common market. EU food regulation developed in an ruling. Appeals are limited to points of law and legal uncoordinated fashion over a period of more than 40 interpretation — they cannot re-examine existing years and resulted in a fragmented framework char- evidence or examine new issues. The appeal can acterized by different national traditions of member uphold, modify, or reverse the panel’s legal fi ndings states as well as different policy areas such as trade and conclusions. If a member does not comply with and agriculture to which they were linked. WTO recommendations on bringing its practice in From a consumer health point of view, the domi- line with WTO rules, then trade compensation or nant areas were related to food safety, in particular sanctions, for example in the form of duty increases and microbiology. Nutrition issues in EU or suspension of WTO obligations, may follow. An policy were dominated by compositional standards interesting case that illustrates the working of the for infant foods and clinical foods. The Community Dispute Settlement Understanding is the long- may not act in a policy area unless given the power to running dispute between the EU and the USA do so by Treaty and the Treaty of Rome did not explic- and Canada concerning the EU ban on the use of itly mention consumer protection or public health. growth-promoting hormones in beef and the import These goals were added to the Single European Act of meat treated with hormones (http://www.wto. and the Maastricht Treaty. org/english/tratop_e/dispu_e/cases_e/ds320_e.htm). Three institutions, the European Commission, the Setting international food standards requires the Council of the European Union, and the European participation of all countries. In recent years there has Parliament – the interinstitutional triangle – take been a signifi cant increase in the membership of the decisions in the legislative fi eld. The main differences Codex. Developing countries now constitute a signifi - in the decision-making process are related to cant proportion of total membership. However, many whether the Council decides by qualifi ed majority or 302 Introduction to Human Nutrition unanimity and the degree to which the European Par- the eight regrouped scientifi c committees was created. liament is involved in the process. In legislative initia- The Green Paper on the general principles of food law tives, the Commission has sole right of initiating policy. was published in 1997 to launch a debate on the future The legislative process usually starts with the expecta- development of EU food law. Its aim was to provide tion that the Community should act in a particular the Commission with a solid background for a major policy area. The prompt for action often comes from program of new or amending legislation it would later external pressure perhaps in response to pressure from propose in the 2000 White Paper on food safety. In a particular Member State, the Council of Ministers, January 2002 Regulation (EC) No. 178/2002 laying the European Parliament, trade associations, research down general principles and requirements of food on risks and hazards, technical developments, etc. law, establishing the European Food Safety Authority These infl uences build up pressure for action. and laying down procedures in matters of food safety In 1974, a Scientifi c Committee for Food (SCF) was was adopted. The Regulation sets out the general prin- established by the European Commission “to advise ciples of EU food law, establishes a European Food the Commission on any problem relating to the pro- Safety Authority, and establishes a rapid alert system tection of the health and safety of persons arising or for the notifi cation of direct or indirect risks to human likely to arise from the consumption of food, in par- health deriving from feed or food, and sets down clear ticular on nutritional, hygienic and toxicological procedures for the handling by the Commission and issues.” The SCF was located in the Directorate General the Member States of food safety emergencies and Industry (DG111). At various times, scientifi c com- crises. The main principles of EU food law contained mittees were criticized on a number of grounds by the in the Regulation includes all food and feed at all European Parliament and by industry and consumer stages of production, processing, and distribution; NGOs. Following the bovine spongiform encepha- food law must be based on a system of risk analysis lopathy (BSE) crisis in the UK, there was a further founded on risk assessment, risk management, and decrease in confi dence in the scientifi c committees risk communication; the precautionary principle will and, with the new powers in public health granted by be applied in the case of a potential risk to human the Maastricht Treaty, the European Parliament forced health where there is scientifi c uncertainty as to what the Commission to totally revise the structures of the measures to take; public authorities at all levels will scientifi c committees. Indeed, the Santer Commission apply the principle of transparency in consulting with in its very fi rst year almost collapsed under pressure and informing the public on actual or potential risks from the European Parliament to speed up the reform and the actions that are taken or proposed to deal with and to restore consumer confi dence in the issuing of them. The Regulation provides for a system to allow scientifi c advice to the Commission. A major reorga- the traceability of all food and feed at all stages of the nization of the Commission’s services ensued. The food and feed chain; food operators are responsible at responsibility for monitoring the implementation of all stages of the food chain for ensuring that the food food safety legislation and for providing scientifi c they produce complies with the requirements of advice, hitherto jointly share by the Commissioners food law and must verify that the requirements are for Agriculture and Industry, was transferred to the met; food business operators have an obligation, when Commissioner for Consumer Affairs. The rationale at they have reason to believe that food that has been the time was that it was necessary to separate monitor- imported, produced, processed, manufactured, or dis- ing, compliance with and enforcement of the law from tributed is not in compliance with requirements, to the law-making function itself. This latter function withdraw the product from the marketplace, to remained for a time with the Agriculture and Industry inform the responsible public authorities, and to Commissioners. Two years later, however, the legisla- inform consumers of the reasons for withdrawal; food tion function on food safety was transferred to the imports and exports must meet the requirements of Health and Consumer Protection Commissioner. The EU food law. Commission also announced that the way in which scientifi c advice on food safety was provided at Euro- European Food Safety Authority pean level would be reorganized and strengthened. A The primary responsibility of the European Food Scientifi c Steering Committee to oversee the work of Safety Authority (EFSA) is to provide independent Policy and Regulatory Issues 303 scientifi c advice on all matters with a direct or indi- egy encompasses work not only in the health sector rect impact on food safety. The Authority has been but across all policy areas. In the nutrition arena, the given a wide brief, so that it can cover all stages of scientifi c community has estimated that an unhealthy food production and supply, from primary produc- diet and a sedentary lifestyle might be responsible for tion to the safety of animal feed, right through to the up to one-third of the cases of cancers, and for supply of food to consumers. It gathers information approximately one-third of premature deaths due to from all parts of the globe, keeping an eye on new cardiovascular disease in Europe. Nutrition and phys- developments in science. It shares its fi ndings and ical activity are key determinants for the prevalence listens to the views of others through a network (advi- of obesity, which continues to rise in the EU among sory forum) that will be developed over time, as well children and adults. as interacting with experts and decision-makers on In terms of nutrition the two main objectives are to many levels. Although the Authority’s main “cus- collect quality information and make it accessible to tomer” is the Commission, it is open to respond to people, professionals, and policy-makers, and to estab- scientifi c questions from the European Parliament lish a network of Member State expert institutes to and the Member States and it can also initiate risk improve dietary habits and physical activity habits in assessments on its own behalf. The Authority carries Europe. The long-term objective is to work toward the out assessments of risks to the food chain and indeed establishment of a coherent and comprehensive com- can carry out scientifi c assessment on any matter that munity strategy on diet, physical activity, and health, may have a direct or indirect effect on the safety of which will be built progressively. It will include the the food supply, including matters relating to animal mainstreaming of nutrition and physical activity into health, animal welfare, and plant health. The Authority all relevant policies at local, regional, national, and also gives scientifi c advice on non-food and feed, European levels and the creation of the necessary sup- genetically modifi ed organisms (GMOs), and on porting environments. At Community level, such a nutrition in relation to Community legislation. strategy would cut across a number of Community policies and needs to be actively supported by them. It EU nutrition and public health would also need to actively engage all relevant stake- With regard to public health, the Community’s role holders, including the food industry, civil society, and is to complement national policies, to encourage the media. Finally, it would need to be based on sound cooperation between the Member States and to lend scientifi c evidence showing relations between certain support to their action when it comes to improving dietary patterns and risk factors for certain chronic public health, preventing human disease, and reduc- diseases. The European Network on Nutrition and ing risks to human health. In keeping with the prin- Physical Activity, which the Commission established, ciple of subsidiarity, Community action in the fi eld will give advice during the process. The Community of public health is designed to fully respect the respon- approach is inspired by the WHO’s Global Strategy on sibilities of the Member States for the organization Diet Physical Activity and Health, which was adopted and delivery of health services and medical care. unanimously by the World Health Assembly. In 2000 the European Commission adopted a In 2005, the Commission launched a new forum, Communication on the Health Strategy of the Euro- called “Diet, Physical Activity and Health – a Euro- pean Community. This described how the Commis- pean Platform for Action.” The platform brought sion was working to achieve a coherent and effective together all relevant players active at European level approach to health issues across all the different policy that were willing to enter into binding and verifi able areas and emphasized that health services must meet commitments that could help to halt and reverse the population’s needs and concerns, in a context current obesity trends. This included retailers, food characterized by the challenge of aging and the growth processors, the catering industry, the advertising busi- of new medical techniques, as well as the more inter- ness, consumer and health NGOs, the medical profes- national dimension of health care (contagious dis- sions, and the EU presidency. It enables all individual eases, , increased mobility of obesity-related initiatives to be more promptly shared persons, services and goods). A new Health Strategy among potential partners and across the EU as a for the EU 2008–2013 was adopted in 2007. The Strat- whole. In December 2005 the Commission published 304 Introduction to Human Nutrition a Green Paper called “Promoting healthy diets and European Parliament: http://www.europarl.europa.eu physical activity: a European dimension for the pre- International Life Sciences Institute: http://www.ilsi.org Institute of Food Science and Technology (IFST): http://www.ifst. vention of overweight, obesity and chronic diseases.” org This was followed in May 2007 by the Commission’s Food Standards Agency (FSA): http://www.foodstandards.gov.uk white paper outlining strategies/initiatives in the area European Food Information Council: http://www.eufi c.org European Union: http://.europa.eu of diet, physical activity, and health aimed at promot- ing good health and quality of life and reducing risks EU food agencies of disease. Nutrition is clearly recognized as having a France – L’Agence Française de Sécurité Sanitaire des Aliments: key role in public health and, together with lifestyle, http://www.afssa.fr has a central position within the strategy and actions Ireland – Food Safety Authority of Ireland: http://www.fsai.ie of the Community in public health. Sweden – National Food Administration: http://www.slv.se UK – Food Standards Agency: http://www.foodstandards.gov.uk

12.7 Perspectives on the future US sites Arbor Nutrition Guide: http://www.arborcom.com/ The food we eat is an area of everyday life that is very Centre for Disease Control and Prevention, Atlanta (CDC): http:// heavily regulated from the food safety point of view, www.cdc.gov including chemical and microbial hazards, but Centre for Food Safety and Applied Nutrition: http://www.cfsan. fda.gov/list.html increasingly from the nutritional point of view. The Centre for Nutrition Policy and Promotion: http://www.usda. bedrock of sensible nutrition regulation planning is gov/cnpp the availability of good data and the intelligent use of Environmental Protection Agency (EPA): http://www.epa.gov Food and Drug Administration (FDA): http://www.fda.gov/default. that data to both inform and challenge the policy- htm makers. Globalization of the food supply has been Food and Nutrition Information Center, USDA: http://www.nal. accompanied by evolving governance issues that have usda.gov/fnic Food and Safety Inspection Service (FSIS): http://www.fsis.usda. produced a regulatory environment at the national gov and global level led by large national, and interna- Iowa State University Extension, including a Food Safety Project: tional agencies in order to facilitate trade and to http://www.extension.iastate.edu/foodsafety Institute of Food Technologists – a non-profi t scientifi c society: establish and retain the confi dence of consumers in http://www.ift.org the food supply chain. United States Department of Health and Human Services: http:// www.os.dhhs.gov United States Department of Agriculture (USDA): http://www. Further reading usda.gov

Websites Worldwide BEUC European Consumers Organisation: http://www.beuc. Codex Alimentarius: http://www.codexalimentarius.net org/Content Consumers International: http://www.consumersinternational.org CIAA: http://www.ciaa.be/asp/index.asp/ Dept. of Plant Agriculture, University of Guelph, Ontario, Canada: DG-SANCO: www.europa.eu.int/comm/dg24 http://www.plant.uoguelph.ca/safefood EUROPA: http://europa.eu Food and Agriculture Organization of the United Nations (FAO): European Commission: http://ec.europa.eu/index_en.htm http://www.fao.org European Court of Justice: http://curia.europa.eu/en/index.htm Food Standards Australia New Zealand: http://www.foodstandards. European Food Safety Authority: http://www.efsa.europa.eu gov.au EUROPA Food and Feed Safety: http://ec.europa.eu/food/ International Food Information Council (IFIC): http://www.ifi c. index_en.htm org European Food Information Council: http://www.eufi c.org International Standards Organization (ISO): http://www.iso.ch Eur-Lex The portal to European Union Law: http://eur-lex.europa. World Health Organization (WHO): http://www.who.int eu/en/index.htm World Trade Organization: http://www.wto.org 13 Nutrition Research Methodology

J Alfredo Martínez and Miguel A Martínez-González

Key messages

• This chapter identifi es critical aspects and factors involved in • It defi nes indicators and markers of dietary intake and metabo- nutritionally orientated investigations as well as the measure- lism in human studies. ment qualities concerning research procedures. • It helps to choose methods to investigate the causal relationships • It describes how to select methods and techniques as well as between diet and disease. animal models to assess nutrient utilization and functions.

13.1 Introduction The primary purpose is to provide a primer in nutri- tion research methods early in a student’s career to Research is a meticulous process to discover new, or allow a more critical review of the many studies that, collate old, facts by the scientifi c study of a subject or from time to time, a student will need to consider in through a critical investigation. In this context, nutri- the course of their study. tion research involves advances in knowledge con- cerning not only nutrient functions and the short- or 13.2 Statistical analysis and long-term infl uences of food and nutrient consump- experimental design tion on health, but also studies on food composition, dietary intake, and food and nutrient utilization by In all areas of research, statistical analysis of results the organism. and data plays a pivotal role. This section is intended The design of any investigation involves the selec- to give students some of the very basic concepts of tion of the research topic accompanied by the formu- statistics as it relates to research methodology. lation of both the hypotheses and the aims, the preparation of a research protocol with appropriate Validity and detailed methods and, eventually, the execution Validity describes the degree to which the inference of the study under controlled conditions and the drawn from a study is warranted when account is analysis of the fi ndings leading to a further hypothe- taken of the study methods, the representativeness of sis. These stages of a typical research program are the study sample and the nature of its source popula- commonly followed by the interpretation of the tion. Validity can be divided into internal validity and results and subsequent theory formulation. Other external validity. Internal validity refers to the subjects important aspects concerning the study design are the actually sampled. External validity refers to the exten- selection of statistical analyses as well as the defi nition sion of the fi ndings from the sample to a target of the ethical commitments. population. This chapter begins with a review of some of the important issues in statistical analysis and experi- Accuracy mental design. The ensuing sections look at in vitro Accuracy is a term used to describe the extent to techniques, animal models, and fi nally human studies. which a measurement is close to the true value, and

© 2009 JA Martínez and MA Martínez-González. 306 Introduction to Human Nutrition

Figure 13.1 Accuracy and precision. it is commonly estimated as the difference between Table 13.1 Estimation of sensitivity and specifi city the reported result and the actual value (Figure 13.1). True condition or outcome True condition or outcome present absent

Reliability Test + A B Reliability or reproducibility refers to the consistency Test − C D A D or repeatability of a measure. Reliability does not Sensitivity= Specificity = AC+ BD+ imply validity. A reliable measure is measuring some- thing consistently, but not necessarily estimating its true value. If a measurement error occurs in two sepa- rate measurements with exactly the same magnitude subjects with the condition who are correctly and direction, this measurement may be fully reliable classifi ed as having the condition. Specifi city is the but invalid. The kappa inter-rate agreement statistic proportion of persons without the condition who are (for categorical variables) and the intraclass cor- correctly classifi ed as being free of the condition by relation coeffi cient are frequently used to assess the test or criteria. Sensitivity refl ects the proportion reliability. of affected individuals who test positive, while speci- fi city refers to the proportion of nonaffected individ- Precision uals who test negative (Table 13.1). Precision is described as the quality of being sharply defi ned or stated; thus, sometimes precision is indi- Data description cated by the number of signifi cant digits in the Statistics may have either a descriptive or an inferen- measurement. tial role in nutrition research. Descriptive statistical In a more restricted statistical sense, precision methods are a powerful tool to summarize large refers to the reduction in random error. It can be amounts of data. These descriptive purposes are improved either by increasing the size of a study or served either by calculating statistical indices, such as by using a design with higher effi ciency. For example, the mean, median, and standard deviation, or by a better balance in the allocation of exposed and using graphical procedures, such as histograms, box unexposed subjects, or a closer matching in a case– plots, and scatter plots. Some errors in the data col- control study usually obtains a higher precision lection are most easily detected graphically with the without increasing the size of the study. histogram plot or with the box-plot chart (box-and- whisker plot). These two graphs are useful for describ- Sensitivity and specifi city ing the distribution of a quantitative variable. Nominal Measures of sensitivity and specifi city relate to the variables, such as gender, and ordinal variables, validity of a value. Sensitivity is the proportion of such as educational level, can be presented simply Nutrition Research Methodology 307

tabulated as proportions within categories or ranks. Box 13.1 Example of hypothesis testing Continuous variables, such as age and weight, are customarily presented by summary statistics describ- Among a representative sample of 7097 European men, the ing the frequency distribution. These summary statis- authors found that each 10 unit increase in the leisure-time physi- 2 tics include measures of central tendency (mean, cal activity was associated with −0.074 kg/m in BMI. Physical activity was measured in units of MET-hours/week (1 MET-hour is median) and measures of spread (variance, standard the energy expenditure during 1 resting hour). deviation, coeffi cient of variation). The standard What is the probability of fi nding, in such a sample, a BMI deviation describes the “spread” or variation around 0.074 kg/m2 lower (or still lower) for those whose energy expendi- the sample mean. ture is 10 MET-hours higher, if the actual difference in the whole European population were 0? This probability is the p-value; Hypothesis testing the smaller it is, the stronger is the evidence to reject the null hypothesis. The fi rst step in hypothesis testing is formulating a In this example, the p-value was 0.001, i.e., chance would hypothesis called the null hypothesis. This null explain a fi nding like this, or even more extreme, in only 1 out of hypothesis can often be stated as the negation of the 1000 replications of the study. The conclusion is that we reject the research hypothesis that the investigator is looking null hypothesis (population difference in BMI = 0) and (provision- ally) accept the hypothesis that states that lower physical activity for. For example, if we are interested in showing that, during leisure time is associated with a higher BMI. We call this the in the European adult population, a lower amount alternative hypothesis. and intensity of physical activity during leisure time has contributed to a higher prevalence of overweight and obesity, the research hypothesis might be that Table 13.2 Right and wrong decisions in hypothesis testing there is a difference between sedentary and active adults with respect to their body mass index (BMI). Truth (population) The negation of this research hypothesis is called the Decision Null hypothesis Alternative hypothesis null hypothesis. This null hypothesis simply main- Null hypothesis Right decision Type II error tains that the difference in BMI between sedentary (probability = 1 – α) (probability = β) and active individuals is zero. In a second step, we Alternative Type I error Right decision calculate the probability that the result could have hypothesis (probability = α) (power = 1 – β) been obtained if the null hypothesis were true in the population from which the sample has been extracted. This probability is usually called the p-value. Its patible with the null hypothesis. A large p-value maximum value is 1 and the minimum is 0. The indicates that the data are compatible with the null p-value is a conditional probability: hypothesis. Many authors accept that a p-value lower than 0.05 provides enough evidence to reject the null p-Value = prob(differences ≥ differences found | hypothesis. The use of such a cut-off for p leads to null hypothesis (H ) were true) 0 treating the analysis as a decision-making process. where the vertical bar (|) means “conditional to.” In a Two possible errors can be made when making such more concise mathematical expression: a decision (Table 13.2). A type I error consists of rejecting the null hypoth- p-Value = prob(difference ≥ data | H ) 0 esis, when the null hypothesis is in fact true. Con- The above condition is that the null hypothesis was versely, a type II error occurs if the null hypothesis is true in the population that gave origin to the sample. accepted when the null hypothesis is in fact not true. The p-value by no means expresses the probability The probabilities of type I and type II errors are called that the null hypothesis is true. This is a frequent alpha (α) and beta (β), respectively. and unfortunate mistake in the interpretation of p-values. Power calculations An example of hypothesis testing is shown in The power of a study is the probability of obtaining Box 13.1. Hypothesis testing helps in deciding whether a statistically signifi cant result when a true effect of a or not the null hypothesis can be rejected. A low p- specifi ed size exists. The power of a study is not a value indicates that the data are not likely to be com- single value, but a range of values, depending on the 308 Introduction to Human Nutrition

assumption about the size of the effect. The plot of ● the expected proportion in each group and, conse- power against size of effect is called a power curve. quently, the expected magnitude of the true effect The calculations of sample size are based in the prin- ● the beta error (or alternatively, the power) that is ciples of hypothesis testing. Thus, the power of a required study to detect an effect of a specifi ed size is the com- ● the alpha error. plementary of beta (1 − β). The smaller a study is, the The p-value has been the subject of much criticism lower is its power. Calculation of the optimum sample because a p-value of 0.05 has been frequently and size is often viewed as a rather diffi cult task, but it is arbitrarily misused to distinguish a true effect from an important issue because a reasonable certainty that lack of effect. Until the 1970s most applications of the study will be large enough to provide a precise statistics in nutrition and nutritional epidemiology answer is needed before starting the process of data focused on classical signifi cance testing, involving a collection (Box 13.2). decision on whether or not chance could explain the The necessary sample size for a study can be observed association. But more important than the estimated taking into account at least three inputs: simple decision is the estimation of the magnitude of the association. This estimation includes an assess- ment about the range of credible values for the asso- Box 13.2 Example of sample size calculation ciation. This is more meaningfully presented as a confi dence interval, which expresses, with a certain Let us suppose that we want to compare the proportion of subjects degree of confi dence, usually 95%, the range from the who develop a given outcome depending on whether they have smallest to the largest value that is plausible for the been assigned to diet A or diet B. We expect that 5% of subjects in the group assigned to diet A and 25% of those assigned to diet B true population value, assuming that only random will develop the outcome of interest. We are willing to accept a variation has created discrepancies between the true type I error with a 5% probability and a type II error with a 10% value in the population and the value observed in the probability. A simplifi ed equation* for sample size (n) calculation sample of analyzed data. would be: 2 ()zzpqαβ+ 2 n = 2 Options for statistical approaches to ()− 2 ppAB data analysis ()+ 2 ×× = 1.. 96 1 28 2 0 .. 15 0 85 Different statistical procedures are used for describing n 22 ()005..− 025 or analyzing data in nutritional epidemiology (Table n = 65 13.3). The criteria for selecting the appropriate

where zα/2 and zβ are the values of the normal distribution corre- procedure are based on the nature of the variable = = sponding to alpha 0.05 (zα/2 1.96) and beta 0.10 (zβ 1.28), considered as the outcome or dependent variable. PA and PB are the expected proportions, p is the average of both Three main types of dependent variable can be proportions (0.05+0.25/2 = 0.15) and q = 1 − p. Therefore, in this example: considered: quantitative (normal), qualitative (very often dichotomous), and survival or time-to-event = = zα/2 0.05 (two tailed) 1.96 variables. zβ = = 1.28 0.10 (one tailed) Within bivariate comparisons, some modalities p = 0.05 (q = 0.95) A 1 deserve further insights (Table 13.4). pB = 0.25 (q2 = 0.75) The validity of most standard tests depends on the These values are substituted in the equation and thus the required sample size for each group is obtained (65). Therefore, we shall assumptions that: need 130 participants, 65 in each group. ● the data are from a normal distribution *When the outcome is a quantitative variable, sample means (xA ● the variability within groups (if these are com- and xB) replace proportions in the denominator, while the product 2 pared) is similar. terms pAqA and pBqB are replaced by the respective variances (s ) of the two groups in the numerator: Tests of this type are termed parametric and are to 2 22 ()zzSSαβ+ +⎡ ⎤ some degree sensitive to violations of these assump- = 2 ⎣ AB⎦ n 2 ()xxAB− tions. Alternatively, nonparametric or distribution- free tests, which do not depend on the normal Nutrition Research Methodology 309

Table 13.3 Common statistical methods used in nutritional epidemiology

Dependent variable (“outcome”) Univariate description Bivariate comparisons Multivariable analysis (Katz, 2006)

Mean, standard deviation t-Tests (two groups) Multiple of variance (more than two groups) Regression and correlation (two quantitative Quantitative (normal) variables)

Proportion, odds Chi-squared Multiple logistic regression McNemar paired test Conditional logistic regression Fisher’s exact test (matched data) Cross-tables Odds ratio Qualitative (dichotomous)

100 Kaplan–Meier (product- Log-rank test Proportional hazards model (Cox 90 limit) estimates and (Mantel–Haenszel) regression) 80 plots 70

60

50

40

Cumulative survival (%) 30 20 0 18016014012010080604020 Length of follow-up Survival

Table 13.4 Common statistical methods for comparison of means

Two samples More than two samples Parametric Nonparametric Parametric Nonparametric

Independent Student’s t-test Mann–Whitney U-test Kruskal–Wallis test samples Paired Welch test (unequal variances) Wilcoxon test Bonferroni, Scheffé, Tamhane, Dunnet, Friedman’s test or related Satterthwaite test (unequal Sidak, or Tukey post-hoc tests samples variances) Analysis of variance for repeated Paired t-test measurements General linear models ANCOVA (analysis of covariance)

distribution, can be used. Nonparametric tests are A common problem in nutrition literature is mul- also useful for data collected as ordinal variables tiple signifi cance testing. Some methods to consider because they are based on ranking the values. Relative in these instances are analysis of variance together to their parametric counterparts, nonparametric tests with multiple-comparison methods specially designed have the advantage of ease, but the disadvantage of to make several pairwise comparisons, such as the less statistical power if a normal distribution should least signifi cant difference method, the Bonferroni be assumed. Another additional disadvantage is that and Scheffé procedures, and the Duncan test. Analysis they do not permit confi dence intervals for the differ- of variance can also be used for replicate measure- ence between means to be estimated. ments of a continuous variable. 310 Introduction to Human Nutrition

Correlation is the statistical method to use when earlier cases than to later cases. The multivariate studying the association between two continuous method most appropriate in this setting is the pro- variables. The degree of association is ordinarily mea- portional hazards model (Cox regression) using a sured by Pearson’s correlation coeffi cient. This calcu- time-to-event variable as the outcome (Table 13.3). lation leads to a quantity that can take any value from −1 to +1. The correlation coeffi cient is positive if 13.3 In vitro studies higher values of one variable are related to higher values of the other and it is negative when one vari- Scientifi c research involves studies across a reduction- able tends to be lower while the other tends to be ist spectrum. As studies become more reductionist, higher. The correlation coeffi cient is a measure of the more and more confounding factors are stripped scatter of the points when the two variables are away. In vitro studies represent part of the reduction- plotted. The greater the spread of the points, the lower ist approach in nutrition research. The range of tech- the correlation coeffi cient. Correlation involves an niques used is large. estimate of the symmetry between the two quantita- ● Chemical analysis studies provide data on nutrient tive variables and does not attempt to describe their and nonnutrient content of foods. relationship. The nonparametric counterpart of ● Digestibility techniques, in which a substrate is Pearson’s correlation coeffi cient is the Spearman rank exposed to enzymes capable of digesting the sub- correlation. It is the only nonparametric method that strate, help to refi ne the gross chemical analytical allows confi dence intervals to be estimated. data to predict nutritional potential. To describe the relationship between two continu- ● Intact organs such as the liver of experimental ous variables, the mathematical model most often animals can be used in studies such as perfused used is the straight line. This simplest model is known organ studies. In such studies, the investigator can as simple linear regression analysis. Regression control the composition of material entering an analysis is commonly used not only to quantify the isolated organ and examine the output. Sections of association between two variables, but also to make organs can also be used, such as the everted gut sac predictions based on the linear relationship. Nowa- technique. A small section of the intestine is turned days, nutritional epidemiologists frequently use the inside out and placed in a solution containing some statistical methods of multivariable analysis (Table test material. Uptake of the test material into the 13.3). These methods usually provide a more accurate gut can be readily measured. view of the relationship between dietary and non- ● Another approach is the construction of mechani- dietary exposures and the occurrence of a disease or cal models that mimic an organ, usually the gut (in other outcome, while adjusting simultaneously for nutrition research). Many of these models success- many variables and smoothing out the irregularities fully predict what is observed in vivo and have that very small subgroups can introduce into alterna- advantages such as cost and fl exibility in altering tive adjustment procedures such as stratifi ed analysis the experimental conditions with great precision. (Katz, 2006). System biology is a recently launched platform to Most multivariate methods are based on the integrate metabolic pathways using computational concept of simple linear regression. An explanation biology. of the variation in a quantitative dependent variable (outcome) by several independent variables (expo- The application of molecular biology techniques to sures or predictors) is the basis of a multiple- tissue and cell culture systems has provided research- regression model. However, in many studies the ers with powerful strategies to evaluate and establish dependent variable or outcome is quite often dichoto- metabolic pathways and regulatory roles of nutrient mous (diseased/nondiseased) instead of quantitative and nonnutrient components of food. Thus, North- and can also be explained by several independent ern, Southern and Western blotting techniques to factors (dietary and nondietary exposures). In this quantitate specifi c RNA, DNA, and proteins in tissues case, the statistical multivariate method that must be in response to nutrients are common tools in the applied is multiple logistic regression. In follow-up nutrition laboratory. The infl uence of some nutrients studies, the time to the occurrence of disease is also or nutritional conditions on ribosomal dynamics as taken into account. More weight can be given to well as on cell hyperplasia or hypertrophy processes Nutrition Research Methodology 311 has been estimated through RNA, DNA, or protein/ a part of our knowledge regarding nutrition concepts DNA values, respectively. stems from animal experiments, which are often Furthermore, molecular biological approaches extrapolated to humans and referred to as animal have allowed numerous in vitro discoveries that have models. There are many reasons for choosing an aided our understanding of the genetic basis of animal study over a human study. We can and do nutrient functions and metabolic states in vivo. The subject animals to experimental conditions that we polymerase chain reaction (PCR) can be used for would ethically not be allowed to apply to humans. DNA and/or messenger RNA (mRNA) amplifi cation For example, to study the manner in which a nutrient to determine the genetic background and/or gene infl uences the scale and histopathology of atheroscle- expression in very small cellular samples. Transfec- rosis, animal studies are needed. Just as studies with tion studies allow the insertion of DNA into cells to humans are governed by the rules of ethics commit- examine nutrient function. Thus, cell lines that usually tees, so too are studies with animals. These rules lack the expression of a particular gene can be trans- involve the regulation of facilities, accommodation fected with DNA containing the gene promoter, as and animal care, competence, alternatives to animal well as all or part of the transfected gene of interest, experimentation, anesthesia and euthanasia proce- to study the interactions of various nutrients with the dures, registration, supply of animals, and the involve- expression of a particular gene. Conversely, knockout ment of an ethical committee. cell lines allow us to investigate the consequences of In general, the use of animals as models for human losing a specifi c gene. In either case, nutrient function nutrition research can be examined from three at the cell level and the cell–gene level may be studied aspects: and provide defi nitive results. Gene regulation by ● the animal model nutrients has been assessed in different isolated cells ● the experimental diet and its delivery and tissues using appropriate indicators and markers ● the experimental techniques available. of gene expression RNA levels. The integration of biochemical and molecular The animal model technologies into nutrition research allows the poten- Many species have been used in the study of nutrition. tial for an integrated perspective Many are pure-bred strains such as the Wistar rat, examining the interactions among DNA, RNA protein, the Charles River mouse, or the New Zealand white and metabolites. Following the completion of the rabbit. Some animal models have been specially human genome sequence, new fi ndings about selected to exhibit particular traits, making them very individual genes functions and their involvement in useful models for research. The Wattanable rabbit has body homeostasis is emerging. Thus, technologies to defective low-density lipoprotein (LDL) receptor achieve a simultaneous assessment of thousands of function, making this animal model very useful for gene polymorphisms, the quantitation of mRNA studying the role of diet in infl uencing LDL receptor- levels of a large number of genes (transcriptomics) as mediated arterial disease. The ob/ob mouse develops well as proteins (proteomics), or metabolites (metab- gross obesity because of an alteration in a genetic olomics) is rapidly progressing. Advances in DNA and profi le (leptin synthesis). In recent times there has RNA microarray-based tools as well in the application been a rise in the use of transgenic animal models that of classic two-dimensional gel electrophoresis, various have been produced through advanced molecular Liquid chromatography-mass spectrometry (LC-MS) genetic techniques. In such models, specifi c genes can techniques, image scanning, or antibody arrays is be inserted or deleted to fulfi ll specifi c functions. For contributing to unraveling the intimate mechanisms example, the peroxisome proliferator-activated recep- involved in nutritional processes. studies tor-alpha (PPAR-α) is not expressed in one knockout constitute a rising methodology to be applied in mouse model, giving rise to fat accumulation. Another nutritional research. example of a transgenic mouse presents an overex- pression of the Cu/Zn-superoxide dismutase enzyme. 13.4 Animal models in nutrition research The experimental diet and its delivery Whole animal systems have been used in measuring The nature of the diet and its mode of delivery are the utilization, function, and fate of nutrients. Thus, centrally important in understanding the role of 312 Introduction to Human Nutrition animal models in human nutrition issues. There are create problems when there is a need to examine the several types of diets offered to laboratory animals. effects of specifi c nutrients, such as fatty acids. The Commercially available diets made to internation- fatty acids naturally present in barley cannot be ally accepted nutritional norms are often referred to as ignored. In the case of the rapeseed oil diet in Table chow diets or laboratory chow. For the vast majority 13.5, 40 g of the 56 g of fat per kilogram of diet comes of laboratory animals in studies where nutrient intake from the rapeseed oil, but 16 g (or 28.6%) comes is not the central area of interest, such chow diets are from barley lipid. used. However, when nutrition is the area of research, To deal with this, more refi ned diets are used. An special diets will almost always have to be formulated. example of such a diet is given in Table 13.6. In this The type of diet that needs to be formulated will instance, the authors were examining how different depend on the nature of the research question. dietary fats infl uence blood cholesterol in normal Terms such as semipurifi ed, purifi ed, and chemi- mice and in transgenic mice not expressing the gene cally defi ned diets are often used but frequently it is for the cholesteryl ester transfer protein (CETP), diffi cult to know exactly which type of term fi ts dif- which is a key protein in lipid metabolism. In this ferent formulations. The least refi ned experimental instance, the ingredients are almost all pure. Thus, diet uses ingredients such as barley, soybean, and casein is pure protein and nothing else. Similarly, wheat. An example is given in Table 13.5, taken from sucrose is pure carbohydrate and cellulose is pure a study of rapeseed glucosinolates on the iodine status fi ber. The diets differ only in the source of fat. The of piglets. high-fat diet obviously has more fat and thus more The purpose of the study was to assess the effects energy per kilogram of diet. It is thus critically impor- of glucosinolate derived from ground rapeseed. A tant to note that as the energy density goes up, most direct comparison between the ground rapeseed and other things must also go up to ensure a common the control is not possible because the ground rape- concentration, not on a weight-for-weight basis but seed contains twice as much fat as the controls. Thus, on a weight-for-energy basis. A simple illustration is the rapeseed oil diet is included because it contains the level of the mineral mix used: 2.5 g/100 g in the no glucosinolate, but the same amount of fat as the control diet, 3.2 g/100 g in the low-fat diet and control diet. The ingredients used in these diets, in 4.2 g/100 g in the high-fat diet. But when considered general, contain several nutrients. Thus, the main on a weight-for-energy basis, all fi ve diets contain ingredient, barley, contains protein, carbohydrate, 2.0 g/MJ. The only changes are in fat and in maize and fat as well as fi ber and micronutrients. That can starch, which always vary in opposite directions. Variations in diet composition are often the key for the design of nutrition experiments. In this context, Table 13.5 An example of less refi ned experimental diets to test the different feeding regimens can be applied to labora- effects of rapeseed-derived glucosinolate tory animals depending on scientifi c criteria. In ad Control Rapeseed oil Ground rapeseed libitum feeding the animals have free access to food; (g/kg) (g/kg) (g/kg) in controlled feeding animals are offered a limited amount of food (restricted feeding) or receive as Soybean meala 220 240 195 Rapeseed oil 5 40 – much food as can be fed to them (forced feeding). A Ground rapeseed – – 100 specifi c form of restricted feeding is pair feeding, Barley 755 700 685 which involves the measurement of food consumed Mineral/vitamin 20 20 20 by some animals to match or equalize the intake of a Total 1000 1000 1000 test group on the following day. There are many Energy (MJ/kg)b 12.6 13.3 13.0 Protein (g/kg) 183 177 182 reasons why pair feeding is critically important. An Fat (g/kg) 29 56 58 experiment may seek to examine how a new protein Glucosinolate 0 0 1.9 source, rich in some nutrient of interest, infl uences (mmol/kg) some aspect of metabolism. Let us consider a com- aSolvent-extracted soybean meal. pound in the protein source that may reduce blood bMetabolizable energy. LDL cholesterol. A control diet is constructed based From Schone et al. (2001). Reproduced with permission. on casein. In the experimental diets, this casein is Nutrition Research Methodology 313

Table 13.6 An example of more refi ned experimental diets to examine the cholesterolemic effects of fats in transgenic CETP mice

Control diet Low-fat diet High-fat diet

(g/100 g) (g/MJ) (g/100 g) (g/MJ) (g/100 g) (g/MJ)

Casein 20 12 19 12 24 12 L-Cystine 0.03 0.18 0.28 0.18 0.36 0.18 Maize starch 40 24 48 31 13 6 Dextrinized starch 13 8 12 8 16 8 Sucrose 10 6 9 6 12 6 Cellulose 5 3 5 3 6 3 Soybean oil 7 4 0 0 0 0 Saffl ower oil 0 0 2 1.2 2.4 1.2 Experimental oila 0 0 0 02211 Mineral mix 3.5 2.1 3.3 2.1 4.2 2.1 Vitamin mix 1 0.6 0.9 0.6 1.2 0.6 Energy (%) Total fat 16.9 5.7 48.6 Sugar 10.1 10.9 10.5 Starch 54.1 22.3 22.1 Protein 20.2 20.9 20.7 MJ/kg 16.7 15.9 20.1

a Three different experimental oils were used (butter, saffl ower, high oleic saffl ower) for three different high-fat diets varying in types of fatty acids. From Chang and Snook (2001). Reproduced with permission. replaced on an isonitrogenous basis with the test which may include growth curves, nutrient and protein source. Otherwise the diets are identical. After energy balance, nutrient utilization and signalling, several weeks of ad libitum feeding a blood sample is etc., using cellular, molecular or other strategies. taken and the results show that blood cholesterol rose Another approach to investigate nutritional pro- with the experimental diet. Then the researcher begins cesses is to overexpress, inactivate, or manipulate to look at other data and observes that growth rates specifi c genes playing a role in body metabolism in the control rats were far higher than in the experi- (Campión et al. 2004). These new technologies allow mental group because the latter had a much lower the study of the regulation and function of different food intake. Quite simply, the new protein source was genes. The current standard methods for manipulat- unpalatable. The experiment will now have to be ing genes in nutrition research depend on the method carried out as a pair-fed study. The food intake of rats of introducing/blocking genes. Thus, genetic manip- given the experimental diet will be measured each ulation can be sustained for generations by creating day. On the following day the control rats will be germline transmission. In this way, there are examples rationed to that amount. Food intakes and probably of transgenic animals, overexpressing or knocking out growth rates are identical. Only the protein source genes, but still controlling this gene manipulation in differs. Now the researcher can truly reach conclu- a spatial or temporal manner. However, when the aim sions as to the effect of the new protein source on is not to transfer genetic information to subsequent LDL cholesterol metabolism. The intake or supply of generations, the most usual method is gene transfer nutrients may be administered orally, intravenously, to somatic cells. Different viral and nonviral vectors intraperitoneally, or by means of some specifi c tools are used for the in vivo gene transfer, allowing a tran- (gavage, stereotaxis, etc.). sient or permanent overexpression of the gene of interest. The RNAi (interference) approach allows the The experimental techniques available creation of new in vivo models by transient ablation The outcome or variables of interest to be assessed of gene expression by degrading target mRNA. More- condition the experimental techniques to be applied, over, by inserting RNAi encoding sequences in the 314 Introduction to Human Nutrition genome, permanent silencing of the target gene can diffi cult to manipulate human diets since we do not be obtained. Undoubtedly, new models of investiga- eat purifi ed or semi-purifi ed diets. tion will be developed, combining the different genetic manipulation techniques to achieve the creation of Experimental diets in human nutrition new models to understand the function and the regu- intervention studies lation of metabolism, nutritional, and disease-related In the 1950s, an epidemiological study across seven genes. Indeed, research concerning inhibiting/activat- countries presented data to suggest that the main ing the expression of different genes (transgenic/ determinant of plasma cholesterol was the balance of knockout animals), gene transfer, and RNAi applica- saturated, monounsaturated, and polyunsaturated tion is allowing us to specifi cally investigate functions fatty acids (MUFAs and PUFAs). To test this hypoth- and metabolism of regulatory processes. esis, a series of studies was carried out on human volunteers using “formula diets.” Dried skimmed milk 13.5 Human studies powder, the test oil, and water were blended to form a test milk with specifi c fatty acid compositions. The In human nutrition, man is the ultimate court in volunteers lived almost exclusively on these formulae. which hypotheses are both generated and tested. Although this type of study is simple to conduct, it Nutritional epidemiology, through its observational does not represent the true conditions under which studies, demonstrates possible links between diet, normal humans live. At the other end of the spectrum physical activity, and disease (Willett, 1998). It is not of options for manipulating human diets is that of the only way in which such possible links are gener- issuing advice that the subjects verify by way of a food ated but it is a critically important one in modern record. It is diffi cult to prove that subjects actually ate nutrition. Experimental human nutrition takes the what they say they have eaten. Sometimes, adherence hypothesis and through several experiments tries to to dietary advice can be ascertained using tissue understand the nature of the link between nutrients samples (blood, saliva, hair, fat) and biomarkers. For and the metabolic basis of the disease. Once there is example, adherence to advice to increase oily fi sh a reasonable body of evidence that particular nutri- intake can be monitored using platelet phospholipid tional conditions are related to the risk of disease, fatty acids. experimental nutritional epidemiology examines In between these two extremes of formula feeds how population level intervention actually infl uences and dietary advice lies an array of options in which the incidence of disease (see Section 13.6). In effect, convenience is generally negatively correlated with experimental human nutrition and experimental scientifi c exactitude. In the case of minerals and vita- nutrition epidemiology both involve hypothesis mins, it is possible simply to give out pills for the testing. However, the former is more often intended volunteers to take and measure compliance by count- to understand mechanisms and generally involves ing unconsumed pills and perhaps using biomarkers. small numbers. The latter, in contrast, uses very large When it comes to macronutrients this is not generally numbers to examine the public health impact of a possible. Whereas asking someone to take a mineral nutrition intervention that, under the controlled con- supplement should not alter their eating habits, asking ditions of the laboratory, showed promise. someone to consume a liter of milk a day or a bowl of rice bran per day will alter other aspects of the diets Human nutrition experimentation of the volunteers. It will not then be possible to attri- The use of experimental animals for human nutrition bute defi nitively an event to the intervention (1 l/day research offers many possible solutions to experimen- of milk or 1 bowl/day of rice bran). The event could tal problems. However, the defi nitive experiments, have been caused by possible displacement of some where possible, should be carried out in humans. other foods by the intervention. The only option in Studies involving humans are more diffi cult to human intervention experiments is to prepare foods conduct for two major reasons. First, humans vary for volunteers to eat, which differ only in the test enormously compared with laboratory animals. They nutrient. If the objective is to examine the effect of vary genetically and they also vary greatly in their MUFAs relative to saturated fatty acids (SFAs) on lifestyle, background diet, health, physical activity, blood lipids, then fat-containing foods can be pre- literacy, and in many other ways. Second, it is far more pared that are identical except for the source of fat. Nutrition Research Methodology 315

The more foods and dishes that can be prepared in imbalances that could confound conclusions. For this way, the more successful the experiment will be. example, if one has 45 volunteers for three treatments, The fi nal dilemma is where the test foods will be it could be that the 15 assigned to treatment A include consumed. A volunteer may share the test foods, the fi ve heaviest subjects and the fi ve lightest subjects. which are almost always supplied free of charge, with Another treatment may be predominantly one gender. friends or family. To be sure of consumption, volun- In such instances, a minimization scheme can be used. teers may be asked to consume the test meal in some Minimization is a technique in which individuals are supervised space, usually a metabolic suite. This, allocated to treatment groups, ensuring a balance by however, is a very costly option. Nutritional interven- minimizing the differences between groups in the dis- tion studies with different macronutrient distribution tribution of important characteristics (age, weight, of food content within energy-restricted diets are physical activity). To apply minimization, during the typical in nutrition research (Abete et al. 2006). recruitment process the investigators must keep an ongoing analysis of differences between groups in the Study designs in human nutrition major variables that may affect the result and allocate The randomized clinical trial is the most powerful new individuals to the group that leads to a more bal- design to demonstrate cause–effect relationships. It is anced distribution of these characteristics. Another unique in representing a completely experimental option is stratifi ed randomization in which strata are approach in humans. The major strength of random- identifi ed and subjects are randomly allocated within ized trials is that they are able to control most biases each stratum. While stratifi cation and minimization and confounding even when confounding factors are potentially very useful, it is impractical to stratify cannot be measured. The CONSORT statement has individuals for many variables at the same time or to established the CONsolidated Standards Of Reporting try to minimize every conceivable variable that may Trials (http://www.consort-statement.org/). The affect the result. To a considerable extent, the need to CONSORT guidelines comprise a checklist and a fl ow balance groups becomes less important when all sub- diagram offering a standard way for reporting the jects are rotated through all treatments (crossover research and assessing its quality. The major method- designs). For this to happen, the number of experi- ological issues to be considered and reported in a mental periods must equal the number of treatments. randomized trial include the following aspects: enrol- For any given period, all treatments must be repre- ment, allocation, follow-up, and inclusion in analysis sented. An important factor to consider in this type of of participants, sample size, proceedings for the design is whether or not a washout period is needed randomization, blinding of the allocation, blinded between treatments, and its duration. assessment of the outcome, comparability of groups Consider the situation above if the study was to regarding major prognostic variables, ascertainment examine the effect of fi sh oil (treatment A) versus and measurement of end-points, statistical analyses, olive oil (treatment B) on lymphocyte function. If it subgroup analyses, results description, ancillary anal- is deemed necessary that 20 days are needed to alter yses, adverse events, interpretations, generalizability, the membrane phospholipids of lymphocytes, then and overall quality of the reported evidence. it is likely that 30 days will be needed to return to As the researcher designs the options for altering baseline. If it is necessary that each treatment should the intake of nutrient under investigation, so too the commence at baseline, then a washout period, where design of the study requires careful thought. The volunteers resume their normal routine, is needed. metabolic effect of the nutrient in question may be A fi nal consideration is the occasion when it is not infl uenced by age, gender, and other variables, such as possible to balance all confounding factors. Take as an high levels of alcohol intake or physical activity, example a study to examine the effect of supplemental smoking, health status, prescribed drug use, and calcium on bone mineral density in premenopausal family history. On an experiment-by-experiment women. The treatment group will receive a supple- basis, the researcher must decide which attributes will ment of 1000 mg of calcium as a tablet and the control exclude a volunteer (exclusion criteria). will receive a placebo tablet. What factors might one The volunteers recruited can now be assigned to the wish to balance in such a study? Among the possibili- various treatments. When the numbers are small, ran- ties are age, parity, use of oral contraceptives, intake of domly assigning subjects to the treatments may lead to coffee, smoking, and physical activity. To balance these 316 Introduction to Human Nutrition factors adequately is impossible. However, if they are circumstances, in which the scientist manipulates the recorded, then, when the data are being evaluated on conditions to ascertain the effect of such manipula- a statistical basis, they can be included to ascertain tion on the observations. their effect on the measured outcome, bone mineral density. To accomplish this aim, multivariate methods Experimental studies in such as multiple regression or logistic regression nutritional epidemiology should be used (see Section 13.2). It is necessary to consider that in biological experi- mentation, it is not possible for the scientist to control 13.6 Epidemiological designs completely all of the relevant circumstances, and the manipulation will consist of increasing at most the Epidemiology is a health-related science dealing with degree of variation in the factor that the scientist is the distribution and determinants of health and investigating. The ideal will be to obtain two almost illness in populations. Nutritional epidemiology inte- identical sets of circumstances where all factors are grates the knowledge derived from nutrition research, the same. If a strong variation is then introduced in to examine diet–disease relationships at the level of only one of these factors, all of the observed differ- free-living populations. Nutritional epidemiology ences between the two sets that occurred thereafter provides scientifi c evidence to understand the role of would be causally attributed to the single factor that nutrition in the cause and prevention of disease. the investigator had manipulated. The comparison and choice of different epidemio- Experimental epidemiological designs are those in logical study designs depends on exposure measures, which the investigator assigns the exposure to each outcome measures, costs, and expected length of subject. In these studies, the treatment (or exposure) follow-up. The selection of a study method is often is assigned with the aim of attaining maximum com- infl uenced by pragmatic issues such as feasibility, parability between treated and untreated groups as well as by ethical questions. regarding all other characteristics of the subjects apart Epidemiological studies can be divided into two from the treatment or exposure of interest. In epide- broad categories (Figure 13.2): experimental and miological research, the best way to achieve identical nonexperimental (observational) studies. Observa- sets of circumstances is to assign subjects randomly tional studies can be further divided into descriptive to exposure (treatment) or control groups. This and analytical studies. In a wide sense, an experiment process is called randomization. All randomized is a set of observations, conducted under controlled studies are experimental designs. Exposure, from an epidemiological point of view, describes lifestyle or environmental factors that may Experimental Clinical trial be relevant to health. Outcome is another generic term used to describe the health-related events or Yes Field trial variables that are being studied in relation to the effect of an exposure. In nutritional epidemiology, the Yes Randomization Community trial primary exposure of interest is dietary intake, whereas Intervention trial No outcome measures usually involve disease occurrence (quasi-experiment) Assignment of or nutritional status indicators (anthropometry, exposure by clinical signs of disease/health status, biological or investigator Cohort physiological measures or dietary habits). Analytical It is also possible to design experimental studies Case–control No assigning whole population groups to different expo- Cross-sectional sures. These studies are called community trials. Descriptive (prevalence) For example, if a whole town is assigned to receive Nonexperimental an educational program about healthy eating and Ecological (observational) another neighboring town is assigned to control (no (correlation) educational program), this would be a community Figure 13.2 Classifi cation of epidemiological designs. trial; when randomization is used, it is termed “cluster- Nutrition Research Methodology 317 randomization.” However, when the number of ran- needs of the protocol of the study and not the indi- domized units is scarce, even though each unit may vidual needs of the participant. Therefore, random- be large, there would be no guarantee that the groups ized experiments with humans can only be conducted to be compared would be identical. Conversely, if the under strict ethical conditions (see Boxes 13.3 and randomization has been done on an individual basis 13.4). It is not permissible to carry out experimental and the whole sample is large enough, a random studies where the exposure is potentially harmful. scheme will usually accomplish its objective of dis- Therefore, under these conditions, nonexperimental tributing the participants in groups that are essen- (observational) study designs must be applied. The tially homogeneous in all measured and unmeasured design options in nutritional epidemiology must take factors. This balance makes groups directly compa- into account the setting, uses, advantages, and limita- rable and ensures the validity of causal inferences tions (Table 13.7). extracted from a randomized design (individual randomization). Experimental designs in epidemiology In general, experimental studies with individual Experimental epidemiologists try to conduct con- randomization provide the strongest evidence for the trolled studies, and in these studies it is the investiga- effect of an exposure on an outcome. Experimental tor who assigns the exposure. Human studies, however, studies are the inferentially strongest designs to unlike animal studies, involve aspects that the inves- demonstrate causality, but they may raise substantial tigator cannot control. This is particularly so when ethical problems because the scheme of random they are carried out on a free-living population. Two assignment is used to help not the subject, but the study designs dominate this area of epidemiology: experiment. Subjects are exposed only to meet the randomized controlled trials and crossover studies. In these studies, subjects are randomly assigned to

Box 13.3 Sample ethics form for completion prior to research

The proposal respects the fundamental ethical principles including Box 13.4 Sample of an informed consent form human rights and will deal only with individuals adequately informed and willing to participate. Also, all research data partners This form will cover the following aspects: will obtain national authorization from an ethical committee or I ...... (name) equivalent body before any intervention with subjects. The study does not involve any genetic manipulation. I have read the volunteer’s information ● Requested specifi cations: I have felt free to make questions concerning the study ● Human embryos or fetus No Yes I have received enough information ● Use of human embryonic or fetal tissue No Yes I have talked to the following personnel responsible (names . . . ) ● Use of other human tissue No Yes I understand that my participation is on a voluntary basis ● Research on persons No Yes ● If yes, further specify if it involves: I understand that I can withdraw from the study: ● children No Yes 1. If I wish ● persons unable to consent No Yes 2. Without further explanations ● pregnant women No Yes Therefore, I freely confi rm my availability to be involved in the ● healthy volunteers No Yes trial ● Use of nonhuman primates No Yes ● Use of transgenic animals No Yes Date ● Use of other animals No Yes Signature ● Genetic modifi cation of animals No Yes In addition, all the partners agree with the following statement ● Genetic modifi cation of plants No Yes ● Other specifi cations: In implementing the proposed research I shall adhere most ● The regulations, concerning human and medical research, strictly to all national and international ethical and safety will be respected with precise reference to the recommenda- provisions applicable in the countries where the research is tions of the Helsinki (1964), Tokyo (1975), Venice (1983) and carried out. Hong Kong (1989) committees, as well as other EU regula- I shall conform in particular to the relevant safety regulations tions RD 561/1993, 65/65 CEE, 75/318 CEE, Directive 91/507 concerning the deliberate release into the environment of and 89/843 EN-C (ISBN 92-825-9612-2). genetically modifi ed organisms. 318 Introduction to Human Nutrition

Table 13.7 Design options in nutritional epidemiology

Design Setting Uses Advantages Limitations

Clinical trial Secondary prevention Treatment–outcome Strongest evidence for Low external validity (diseased participants) association causality Ethical problems Highest internal validity High cost Very low potential for bias Field trial Primary prevention (healthy Exposure–onset of Strong evidence for causality Very large sample and participants) disease association High internal validity long follow-up Low potential for bias Low external validity Can assess only single- nutrient effects Highest costs Community Group randomization (towns, Evaluation of If multiple, and small groups Low internal validity trial work-sites, schools) community are randomized, it has if the number of interventions or the advantages of an randomized units educational activities experimental design is low Quasi-experiment Intervention study (not Evaluation of High feasibility Diffi culties in fi nding randomized) community More applicable comparable groups interventions or Investigator controls exposure High potential for bias educational activities Underlying trends may alter results Cohort Participants are initially The most powerful Very low potential for bias Large sample and very classifi ed as exposed or observational tool Ability to study rare exposures, long follow-up nonexposed and followed in nutritional complex dietary patterns No ability to study rare up in time to monitor the epidemiology to and multiple outcomes of a outcomes incidence of the outcome. study diet–health single exposure Bias by low follow-up Retrospective or historical associations Allows direct estimation of (attrition) cohort studies are risks and rates Requires collaborative conducted using previously Minimal ethical problems participants collected information (fi les) High costs Case–control Exposure is compared between Practical analytical Ability to study rare outcomes Potential for biased recall subjects with and without tool in nutritional Ability to study multiple of exposure and biased the outcome. Nested epidemiology to potential causes of a single participation of case–control studies are study diet–health outcome controls conducted within an associations No problems with losses to Inability to study rare ongoing cohort using the follow-up exposures and multiple data of cohort members Minimal ethical problems outcomes of a single who develop the disease Low cost exposure (cases) and a sample of Inability to estimate risks nondiseased members and rates (controls) Cross-sectional Past exposure and outcome Estimation of the Highest external validity Diffi cult to assess the are simultaneously assessed prevalence of a Relatively low costs temporal sequence: in a representative sample disease or an Minimal ethical problems very low ability for of the population exposure A wide spectrum of causal inference Population assessment information about diet and Potential for biased in health planning health can be collected participation and Monitoring trends if it response bias is periodically repeated Ecological The unit of analysis is not the Generation of new Ability for assessing exposures Very low internal validity individual but a community. hypothesis and at the community level (“ecological fallacy”) Exposure and/or disease are contextual or Relatively low costs not measured at the multilevel analysis Minimal ethical problems individual level Nutrition Research Methodology 319 either an exposed or a nonexposed group, commonly Moreover, the induction time needed to appraise the referred to as the treatment and the placebo group. effect of a postulated cause may last longer than the The placebo is a substance that is indistinguishable observation period of a randomized trial, thus pre- from the treatment and enables both subjects and cluding the ability of the trial to ascertain the causal investigators to be blinded to the treatment. Changes relationship. in indicators of health or disease status are compared Quasi-experimental studies are those in which between the two groups at the end of the experiment the assignment of exposure is controlled by the to identify the effect of the exposure. investigator, but subjects are not randomly allocated. Crossover designs in epidemiology operate on the They are sometimes called intervention trials (Figure same principles as the repeated-measures designs 13.2). common to basic science research. All study subjects Some randomized trials are referred to as primary receive the treatment and the placebo for equal prevention trials and others as secondary prevention periods, with a washout period in between, and the trials. Primary prevention trials are those conducted order of treatment or placebo administration is among healthy individuals with the aim of preventing selected at random for each study subject. Crossover the onset of disease. For example, in the Women’s designs are appropriate only for studies of treatments Health Initiative (Howard et al., 2006) more than that have no lasting effects, a feature that limits their 48 000 healthy postmenopausal women were ran- utility in nutritional epidemiology. domly assigned to receive either a low-fat diet or In general, experimental epidemiological study placebo to prevent the onset of cardiovascular disease designs are well suited to the identifi cation of causal (CVD). All participants were free of this disease at relationships between specifi c exposures and indica- the start of the study and they were followed up for tors of health or disease status. Application of these several years to assess the incidence of fatal and non- methods is limited, however, by the diffi culty in con- fatal coronary heart disease, fatal and nonfatal stroke, trolling exposures and by the enormous expense and CVD (composite of both). This is an example of associated with population-based intervention trials a primary prevention trial. Primary prevention trials aimed at modifying risk or chronic diseases. It is are also called fi eld trials. perhaps more feasible to apply experimental study Secondary prevention trials are conducted among designs to contrast the effects of pharmacological patients who already suffer from a particular disease doses of specifi c nutrients or food components the and they are randomly assigned to treatment or exposures of which can be more easily controlled. placebo groups to prevent adverse outcomes. For This approach has been increasingly selected from the example, to study the benefi ts of a Mediterranean- 1990s to assess the effects of specifi c micronutrients style diet, in the Lyon Diet Heart Study, patients were (β-carotene, α-tocopherol, folic acid, and other randomized to two different dietary patterns after minerals and vitamins) using large-scale randomized suffering a myocardial infarction (de Lorgeril et al., trials. 1999). The outcome was not the onset of disease but When only one micronutrient is compared with a the incidence of reinfarction or cardiac death during placebo, the study is called a single trial, whereas mul- the follow-up period. tiple or factorial trials involve designs where several micronutrients are compared with a placebo. In a Nonexperimental (observational) 2 × 2 factorial design, two treatments are evaluated epidemiological studies simultaneously by forming four groups (treatment A, When experiments are not feasible or are unethical, treatment B, both treatments, and placebo). other nonexperimental designs are used. In nonex- Experimental studies keep the highest internal perimental (observational) studies the investigator validity among epidemiological designs. However, has no control over the exposure, because the they may lack generalization (i.e., they may have low participants freely assign themselves or not to the external validity) and their applicability to free-living exposure. In nonexperimental studies the investigator populations may be poor insofar as the dietary intake may take advantage of “natural experiments,” where patterns do not correspond to isolated nutrients but exposure only appears in some defi ned groups. An to the combination of more complex food items. example of this would be an “experiment” where 320 Introduction to Human Nutrition dietary intake is culturally determined, such as in dietary exposures or other factors of etiological Indonesia, where the rice consumed is white rather interest in subjects is generally established using than brown, and beriberi is common as a result of interviews, questionnaires, or medical record reviews. vitamin B1 defi ciency. Within the general framework for case–control Nonexperimental (observational) designs can be studies, there are several options for study design and further classifi ed into four main subtypes: control selection. For example, controls may be matched with cases ● cross-sectional studies at an individual level on the basis of age, gender, or ● case–control studies other variables believed to affect disease risk. Match- ● cohort studies ing eliminates variability between cases and controls ● ecological studies. with respect to the matching variables and thus leads Among observational studies, the main differences to a higher effi ciency in the analysis. Nevertheless, between study designs relate to the time when expo- matching does not control for the confounding effects sure and outcome are measured. The initiative of these risk factors on the observed relationship. “STrengthening the Reporting of OBservational Case–control studies are by far the most logistically studies in Epidemiology (STROBE), http://www. feasible of the analytical study designs in epidemiol- strobe-statement.org” provides a check-list to assess ogy, but their application to questions of interest to the methodological quality of the three major epide- nutritionists is limited by the particular nature of miological designs: cohort studies, case–control diet–disease relationships. studies, and cross-sectional studies (Von Elm and The insight to be gained from a comparison of Egger 2004). dietary exposures between cases and controls is limited by the possibility that the dietary patterns of Cross-sectional (prevalence) studies subjects have changed since the time when diet was Cross-sectional or prevalence studies measure both most important to the disease initiation process. Ret- exposure and outcome in the present and at the same rospective case–control studies attempt to overcome point in time. Cross-sectional surveys provide a snap- this limitation by measuring past diet using food fre- shot of descriptive epidemiological data on nutrition, quency or diet history methods. One concern is that identifying nutritional needs in the population and recall of past diet by cases may be infl uenced by their forming a basis for health promotion and disease pre- present disease status. For example, patients who have vention programs at a single point in time. Several had a heart attack may attach an unfair level of impor- countries conduct regular cross-sectional surveys on tance to their intake of specifi c foods, based on representative samples of their populations focusing misinformation. on dietary habits and frequencies of illness. Dietary A primary factor in choosing between a case– factors can then be correlated with prevalence of control design and a cohort design is whether the illness, which may be helpful for national nutrition exposure or the outcome is rare. If the outcome is policy. rare, case–control studies are preferable, because a cohort would need a very large sample to observe a Case–control studies suffi cient number of events. If the exposure is rare, In case–control studies, outcome is measured in the cohort studies are preferable. present, and past exposure is ascertained. Usually the A nested case–control design consists of selecting dietary and lifestyle patterns of patients with a disease as cases only those members of a previously defi ned (cases) are compared with those of age- and gender- cohort who develop the disease during their follow- matched people without disease (controls). up period. A random sample or a matched sample of Subjects are identifi ed and recruited on the basis of non-cases is also selected from the cohort to make up the presence or absence of the disease or the health the control series as the comparison group. outcome variable of interest. Ideally, the controls are randomly selected from the same study base as the Cohort studies cases, and identical inclusion and exclusion criteria In cohort studies exposure is evaluated in the present are applied to each group. The presence of specifi c and outcome ascertained in the future. Nutrition Research Methodology 321

Cohort studies are most commonly longitudinal or Studies considering the individual (instead of the prospective, with subjects being followed forward in population) as the unit of observation are always time over some predefi ned period to assess disease preferable because in an individually based study it is onset. They may also be retrospective (historical possible to relate exposure and outcome measures cohorts), with groups identifi ed on the basis of expo- more directly, preventing many fl aws that are likely to sure sometime in the past and then followed from that invalidate the fi ndings of ecological studies. One of time to the present to establish presence or absence of these fl aws is known as the “ecological fallacy” and it the outcome. The feasibility of retrospective cohorts is the bias resulting because an association observed depends on the availability of good-quality data from between variables on an aggregated level does not pre-existing fi les. The research costs associated with necessarily represent the association that exists at an cohort study designs mean that such studies are less individual level. A major advantage of individually common than other approaches. Nevertheless, a sub- based studies over aggregated studies is that they stantial effort to develop large cohort studies in nutri- allow the direct estimation of the risk of disease in tional epidemiology has been made since the early relation to exposure. 1980s. Cohort studies can assess multiple outcomes, Ecological studies measure diet less accurately whereas case–control studies are restricted to assess- because they use the average population intake as the ing one outcome, but may be able to assess many dif- exposure value for all individuals in the groups ferent exposures. If an absolute measure of the effect that are compared, leading to a high potential for of the exposure on the outcome is required, the only biased ascertainment of diet–disease associations. design that is appropriate is a cohort study, as case– Ecological studies, also termed correlation studies, control studies cannot be used to estimate incidence. may compare indicators of diet and health or disease For example, to ascertain the relationship between within a single population over time to look for olive oil consumption and coronary heart disease, a secular trends, or to compare the disease incidence case–control study would compare the previous con- rates and dietary intake patterns of migrant groups sumption of olive oil between cases of myocardial with those of comparable populations in the original infarction and healthy controls. A cohort study would and new country. Ecological comparisons have been start with a roster of healthy individuals whose base- important in hypothesizing diet and disease associa- line diet would be recorded. They would then be fol- tions. Nevertheless, they are not able to establish lowed up over several years to compare the occurrence causal relationships. of new cases of myocardial infarction between those consuming different levels of olive oil as recorded Defi nition of outcomes and end-points when they were healthy at baseline. Epidemiological outcomes must be clearly defi ned at the outset of a study. For example, a study of diet and Ecological studies CVD may specify that the outcome (CVD) is verifi ed Epidemiological studies can be classifi ed according to by specifi c clinical tests such as cardiac enzyme level whether measurements of exposure and outcome are or electrocardiographic changes. Taking the word of made on populations or individuals. Observational the patient or the doctor is not suffi cient. Two main investigations in which the unit of observation and measures of the frequency for an outcome are used in analysis is not the individual but a whole community epidemiology: prevalence and incidence. or population are called ecological studies. In ecologi- cal studies, measures of exposure routinely collected Prevalence and aggregated at the household, local, district, The prevalence of an outcome is the proportion of regional, national, or international level are compared subjects in a population who have that outcome at a with outcome measures aggregated at the same level. given point in time. The numerator of prevalence is An example of an ecological study would be plotting the number of existing cases and the denominator is the mortality rates for colon cancer in several coun- the whole population: tries against the average intakes of saturated fat in Existing cases these same countries and calculating the correlation Prevalence = between the two variables. Total population 322 Introduction to Human Nutrition

Incidence by the combination of data may be misleading because The incidence of an outcome is the proportion of new the fi ndings may still be invalid. An alternative to cases that occur in a population during a period of combining published epidemiological data is to pool observation. The numerator of incidence is the and analyze the primary data from all available studies number of new cases developing during the follow-up on a topic that meets specifi ed criteria. Ideally, this period, while the denominator is the total population should involve the active collaboration of the original at risk at the beginning of the follow-up time: investigators, who are fully familiar with the data and its limitations. This kind of study conducted with a New cases Incidence = combination of the original data from several studies, Population inititally at risk is the basis of pooled analysis or pooling projects. In When calculated in this fashion, incidence is a pro- a pooled analysis, the range of dietary factors that can portion. However, incidence can also be expressed as be addressed may be considerably greater than in the a rate (velocity or density), when the time during separate analyses because any one study will have few which each person is observed (i.e., person-time of subjects in the extremes of intake and, sometimes, the observation) is included in the denominator. Then it studies will vary in distribution of dietary factors. The is called incidence rate or incidence density and it is advantages of pooled analyses in nutritional epidemi- expressed as the number of new cases per person- ology are so substantial that they are becoming time of observation. common practice for important issues, such as alco- holic intake and breast cancer, body size and breast Other epidemiological methods cancer, or alcohol beverages and coronary heart disease. Epidemiological studies have also been conducted to Analysis of epidemiological data requires careful assess: consumer attitudes to and beliefs about food, consideration of the criteria for acceptable data nutrition, physical activity patterns, and health to quality, but also of the presentation of categorized or provide policy-makers, researchers and the food continuous independent variables and the applica- industry with data to promote health messages con- tion of empirical scores. The study of subgroup analy- cerning the relation between food or nutrient intake sis and interactions and error correction are other and chronic diseases. These surveys seek information issues of interest. Other limitations are the require- about infl uences on food choice, health determinants, ment of sample to be considered as representative, criteria about perceptions of healthy eating, regular compliance, inaccuracies of information in retrospec- sources of nutritional information, expected benefi ts tive studies, and confounding effects by factors that and barriers to healthy diet implementation, in order are simultaneously associated with both the exposure to identify consumers’ knowledge, attitudes, and beliefs and the outcome. concerning food and health interactions and to promote more focused nutrition education messages. 13.7 Perspectives on the future

Meta-analysis and pooled analysis Future nutrition research will develop new methods The role of meta-analysis for systematically combin- for studying those processes whereby cells, tissues and ing the results of published randomized trials has the whole body obtain and utilize substances con- become routine, but its place in observational epide- tained in the diet to maintain their structure and miology has been controversial despite widespread function in a healthy manner. Particular emphasis use in social sciences. Some have argued that the com- will be paid to molecular and cellular based strategies bining of data from randomized trials is appropriate devised to understand better the genetic basis of because statistical power is increased without concern nutritional outcomes. for validity since the comparison groups have been It can also be anticipated that many ongoing large randomized, but that in observational epidemiology cohort studies with tens of thousands of participants the issue of validity is determined largely by con- will provide valuable information on the role of founding and bias rather than limitations of statistical nutrition in disease prevention, and also on nutri- power. Thus, the greater statistical precision obtained tional management of a large number of diseases by Nutrition Research Methodology 323 dietary means, and gene–nutrient and gene– Lorgeril de M, Salen P, Martin JL, Monjaud I, Delaye J, Mamelle N. environment interactions. Moreover, pooling of data , traditional risk factors, and the rate of car- diovascular complications after myocardial infarction: fi nal from several cohort studies may provide a very pow- report of the Lyon Diet Heart Study. Circulation 1999; 99: erful tool to assess the benefi ts of a healthy diet. An 779–785. increasing interest in a dietary pattern approach Martínez-González MA, Estruch R. Mediterranean diet, antioxi- dants, and cancer: the need for randomised trials. European instead of a single nutrient approach will be seen in Journal of Cancer Prevention 2004; 13: 327–335. nutritional epidemiology in the forthcoming decades. Schone F, Leiterer M, Hartung H, Jahreis G, Tischendorf F. Rapeseed In addition, large primary prevention trials using the glucosinolates and iodine in sows affect the milk iodine concen- tration and the iodine status of piglets. British Journal of approach of assessing the effect of an overall dietary Nutrition 2001; 85: 659–670. pattern (Estruch et al., 2006; Howard et al., 2006) are Von Elm M, Egger M. The scandal of poor epidemiological research. growing nowadays and their results will be on the rise BMJ 2004; 329: 868–869. during the next decade (Martinez-Gonzalez, 2004). Nutritional epidemiology will also adopt a wider, Further reading multidisciplinary approach, with more studies con- cerning the impact of factors affecting social determi- Altman DG. Practical Statistics for Medical Research. Chapman & Hall, London, 1991. nants of eating patterns, food supplies, and nutrient Armitage P, Colton T. Encyclopaedia of Biostatistics, 2nd ed. John utilization on health to facilitate the decisions of Wiley and Sons, New York: 2007. policy-makers, food industry managers, investigators, Breslow NE. Statistics. Epidemiologic Reviews 2000; 22: 126– 130. and consumers. Corthésy-Theulaz I, den Dunnen, JT, Ferre P, Geurts JMW, Müller M, van Belzen, N, van Ommen B. Nutrigenomics: the impact of biomics technology on nutrition research. Annals of Nutrition & Metabolism 2005; 49: 355–365. References Fernandez-Jarne J, Martínez E, Prado M, et al. Risk of non-fatal myocardial infarction negatively associated with olive oil con- Abete I, Parra MD, Zulet MD, Martinez JA. Different dietary strate- sumption: a case-control study in Spain. International Journal of gies of weight loss in obesity: role of energy and macronutrient Epidemiology 2002; 31: 474–480. content. Nutr Res Rev 2006; 19: 5–12. Kumanyika SK. Epidemiology of what to eat in the 21st century. Campión J, Milagro FI, Martinez JA. Genetic manipulation in Epidemiology Reviews 2000; 22: 87–94. nutrition, metabolism, and obesity research. Nutr Rev 2004; Kussmann M, Raymond F, Affolter M. OMICS – driven biomarker 62(8): 321–330. Review. discovery in nutrition and health. Journal of Biotechnology 2006; Chang CK, Snook JT. The cholesterolaemic effects of dietary fats 124: 758–787. in cholesteryl ester transfer protein transgenic mice. British Leedy PD. Practical Research: Planning and Designs, Vol. 2. Journal of Nutrition 2001; 85: 643–648. Macmillan, New York, 1980. Estruch R, Martínez-González MA, Corella D, Salas-Salvadó J, Moreno-Aliaga MJ, Marti A, García-Foncillas J, Martínez JA. DNA Ruiz-Gutiérrez V, Covas MI, Fiol M, et al. Effects of a hybridization arrays: a powerful technology for nutritional mediterranean-style diet on cardiovascular risk factors: a and obesity research. British Journal of Nutrition 2001; 86: randomized trial. Annals of Internal Medicine 2006; 145: 1–11. 119–122. Howard BV, Van Horn L, Hsia J, Manson JE, Stefanick ML, Scheweigert FJ. Nutritional proteomics: methods and concepts for Wassertheil-Smoller S, Kuller LH, et al. Low-fat dietary pattern research in . Annals of Nutrition & Metabolism and risk of cardiovascular disease. JAMA 2006; 295: 655–666. 2007; 51: 99–107. Katz MH. Multivariable Analysis: a Practical Guide for Clinicians, Willett W. Nutritional Epidemiology. Oxford University Press, 2nd edn. Cambridge University Press, Cambridge: 2006. London, 1998. 14 Food Safety: A Public Health Issue of Growing Importance

Alan Reilly, Christina Tlustos, Judith O’Connor, and Lisa O’Connor

Key messages

After reading this chapter the student should have an understanding • types and sources of bacterial contamination in foods of: • bacteria • the reasons for increased concern about the safety of food • food-borne viruses • chronic effects of food-borne illness • parasites • vulnerable groups • transmissible spongiform encephalopathies and food • economic consequences of food-borne illness • chemical contamination and food • emerging food-borne pathogens • food safety control programs.

14.1 Introduction 14.2 Factors contributing to food safety concerns In recent years the reported incidence of food-borne diseases has continued to increase worldwide, with a Although it is diffi cult to determine the global inci- number of extremely serious outbreaks occurring on dence of food-borne disease, the World Health virtually every continent (Kaferstein, 2003). In addi- Organization (WHO) estimates that in 2005 alone, tion, various high-profi le food safety issues, including 1.8 million people died from diarrheal diseases, and bovine spongiform encephalopathy (BSE), dioxins, in industrialized countries around 30% of the popu- acrylamide, Escherichia coli O157 and Sudan Red 1 lation is estimated to suffer from food-borne diseases have presented themselves to consumers, industry each year (WHO, 2007). In the USA, for example, an and regulators alike. estimated 76 million cases of food-borne diseases, In a nutritional context, food-borne illness is often resulting in 325 000 hospitalizations and 5000 deaths associated with malnutrition. In recent times food occur each year (Mead et al., 1999). safety issues have been perceived by the public and governments as posing a greater potential risk to con- Changing food supply system sumer health than nutritional aspects of the diet. To The increasing incidence of food-borne diseases is convey positive public health nutritional messages, due to a number of factors, including changes in food nutritionists must understand the scientifi c basis of production on the farm, new systems of food process- “food scares” that affect attitudes to food, nutrition, ing, longer distribution chains, and new food prepa- and health. This chapter aims to highlight the reasons ration and storage methods. Changing lifestyles have for concern about the safety of food, the types and led to a far greater reliance on convenience foods that sources of biological and chemical contaminants are prepared outside the home, and which may have in foods, and possible control and prevention a longer preparation to consumption time. In addi- strategies. tion, the food chain has become longer and more

© 2009 A Reilly, C Tlustos, J O’Connor and L O’Connor. Food Safety 325 complex, giving increased opportunities for food drome (HUS), a life-threatening complication contamination. International trade in foods has of Escherichia coli O157:H7 infection characterized expanded dramatically, and today the Food and by acute renal failure, hemolytic anemia, and Agriculture Organization of the United Nations thrombocytopenia. (FAO) estimates over 500 million tonnes of food, valued around US$400–500 billion, move in interna- Vulnerable groups tional trade annually. Globalization of the food trade Vulnerable groups tend to be more susceptible to presents a major challenge to food safety control food-borne infections and generally suffer more authorities, in that food can become contaminated in severe illness because their immune systems are in one country and cause outbreaks of food-borne some way impaired. The immune system of infants illness in another. It is not unusual for an average and young children is immature. In pregnant women, meal to contain ingredients from many countries that increased levels of progesterone lead to the downreg- have been produced and processed under different ulation of cell-mediated immunity, increasing the standards of food safety. susceptibility of both mother and fetus to infection by intracellular pathogens (Smith, 1999). In older Chronic effects of food-borne illness people, a general decline in the body’s immune Food-borne diseases are classifi ed as either infections response occurs with age, as does a decrease in or intoxications. Food-borne infections are caused stomach acid production. Immune responses in older when viable microorganisms are ingested and people are also adversely affected if that person is these can then multiply in the human body. malnourished through poor diet. Furthermore, age- Intoxications are caused when microbial or naturally related loss of sensory abilities, such as sight and taste, occurring toxins are consumed in contaminated can lead to diffi culties in choosing and preparing foods. Illnesses that relate to the consumption of food. An aging population is one factor infl uencing foods that are contaminated with chemical toxins or the increase in the prevalence of food-borne disease. microorganisms are collectively referred to as food In 1999, 20% of Europe’s population was older than poisoning. 60 years of age, but this is predicted to rise to 35% by The health consequences of food-borne illness are 2050 (Kaferstein, 2003). Other groups in which the varied and depend on such factors as the individual’s immune system may be suppressed, making them susceptibility, the virulence of the pathogen, and the more susceptible to food-borne infection, include type of disease. Symptoms are often mild and self- cancer patients, transplant patients receiving immu- limiting in healthy individuals and people recover nosuppressant drugs, and patients with acquired within a few days from acute health effects. Acute immunodefi ciency syndrome (AIDS). In nonindus- symptoms include diarrhea, stomach pain and trialized countries, political unrest, war, and famine cramps, vomiting, fever, and jaundice. However, in lead to increased malnutrition and can expose poorer some cases microorganisms or their products are populations to increased risk of food-borne disease. directly or indirectly associated with long-term health effects such as reactive arthritis and rheuma- Improved surveillance toid syndromes, endocarditis, Reiter syndrome, Improved surveillance systems lead to an increase in Guillain–Barré syndrome, renal disease, cardiac and the reported incidence of food-borne disease. Using neurological disorders, and nutritional and other information technology, many countries have devel- malabsorptive disorders. It is generally accepted that oped enhanced surveillance systems to gain a better chronic, secondary after-effect illnesses may occur in picture of the true incidence of food-borne disease. 2–3% of cases of food-borne infections and that the International outbreaks are more readily detectable long-term consequences to human health may be with the use of electronic databases for sharing molec- greater than the acute disease. In one salmonellosis ular typing data (such as PulseNet in the USA and outbreak, associated with drinking contaminated EnterNet in Europe) and rapid alert systems, websites, milk, about 2% of patients developed reactive arthri- or list servers. However, even with this enhanced sur- tis. It is estimated that up to 10% of patients with veillance, it is unlikely that statistics refl ect the true hemorrhagic colitis develop hemolytic uremic syn- incidence of food-borne disease worldwide. 326 Introduction to Human Nutrition

Economic consequences of control of food-borne and water-borne diseases such food-borne illness as typhoid, tuberculosis, and brucellosis. However, As well as morbidity and mortality associated with new food-borne pathogens have emerged. Food- food-borne diseases, there are direct economic costs borne organisms such as E. coli O157, Campylobacter incurred, including the cost of medical treatment jejuni, and Salmonella Enteritidis phage type 4 were and industry losses. WHO estimates that in the USA virtually unknown in the 1970s, but have come to diseases caused by the major food-borne pathogens prominence as virulent pathogens associated with have an annual cost of up to US$35 billion. The foods of animal origin. Cyclospora cayetanensis annual cost for illness due to E. coli O157 infections emerged as a food-borne pathogen in 1995, when it in the USA has been estimated at US$405 million, was associated with outbreaks of illness traced to which includes costs for medical care (US$30 million), raspberries imported into the USA from Guatemala. lost productivity (US$5 million) and premature Cryptosporidium parvum emerged as a pathogen of deaths (US$370 million); however, this estimate does worldwide signifi cance during the 1990s and has been not include costs due to pain and suffering, and linked to contaminated drinking water and to a range expenditure on outbreak investigations (Frenzen et of foods including salads, unpasteurized milk, and al., 2005). The cost of salmonellosis in England and apple juice. Some known pathogens such as Listeria. Wales in 1992 was estimated at between US$560 monocytogenes have only recently been shown to be million and US$800 million. Over 70% of costs were predominantly food-borne and, since they can grow directly associated with treatment and investigation at refrigeration temperatures, have increased in of cases, and costs to the economy of sickness related importance with the expansion of the cold chain for to absence from work. In 2006, Cadbury Schweppes, food distribution. Enterobacter sakazakii has recently the world’s largest confectionary company, were been implicated in outbreaks of infection associated forced to recall seven Cadbury-branded products in with powdered infant formula. Many of these emerg- the UK and two in Ireland due to Salmonella con- ing pathogens are of animal origin and do not usually tamination. The estimated cost of the product recall cause serious illness in the animal host. was £30 million, including a £5 million marketing Another concern is that a proportion of food- campaign to rebuild consumer confi dence. In addi- borne illness is caused by pathogens that have not yet tion, Cadbury Ltd in the UK was fi ned £1 million been identifi ed, and therefore cannot be diagnosed. and ordered to pay costs totaling £152 000 for In the USA, it is estimated that unknown food-borne distributing the contaminated chocolate products agents caused 65% of the estimated 5200 annual which led to illness in 42 people being reported, three deaths from food-borne disease (Mead, 1999; Frenzen, of whom were hospitalized (Cadbury press release, 2004). This is of concern since many of today’s 2007). commonly recognized food-borne pathogens were Bearing these fi gures in mind, the true estimates of not recognized as causes of food-borne illness 30 food-borne disease and the likely economic costs are years ago. In this regard, Mycobacterium avium sub- unknown. In industrialized countries only a small species paratuberculosis (Map) is an organism of proportion of cases of food-borne diseases is reported, potential concern. Map is the causative agent of and even fewer are investigated. Very few non- Johne’s disease in cattle, but it has been proposed that industrialized countries have established food-borne Map is also the causative agent of Crohn’s disease in disease reporting systems, and in those that have, only humans, and that it may be transmitted via milk a small fraction of cases is reported. (including pasteurized milk) and possibly other foods. During the 1980s and 1990s, antibiotic-resistant Emerging food safety issues food-borne pathogens emerged that are associated The emergence of new food-borne pathogens is one with the inappropriate use of antibiotics in animal factor leading to increased concern about food safety. husbandry. For example, Salmonella typhimurium During the twentieth century improvements in sewage DT 104 routinely shows resistance to fi ve different treatment, milk pasteurization, and water treatments, antibiotics. Strains of Salmonella and Campylobacter and better controls on animal disease have led to the are showing resistance to fl uoroquinolones since Food Safety 327 these compounds were introduced for use in that viruses accounted for 6% of all food-borne out- animals. breaks and 8% of cases. Hepatitis A accounted for the In recent years a new range of foods has been majority of these, followed by norovirus. Data pub- implicated with food-borne disease. For instance, the lished by the European Food Safety Authority (EFSA) internal contents of an egg were always presumed to revealed that viruses accounted for 10.2% of all food- be safe to eat raw, and uncooked eggs have been tra- borne outbreaks reported during 2006. Caliciviruses ditionally used in many different food products. This (including norovirus) accounted for the majority situation has changed with the emergence of S. Enter- (61.7%) of these food-borne viral outbreaks. itidis infection in egg-laying fl ocks, resulting in Food-borne viruses are generally enteric, being contamination in shell eggs and a major increase transmitted by the fecal–oral route. However, trans- in food-borne illness worldwide associated with mission by person-to-person contact and via con- uncooked eggs. Animal products are no longer the taminated water is common. Hepatitis A and norovirus only focus for food safety controls, as fresh produce are more commonly transmitted via foods than other is emerging as an important vehicle for food-borne food-borne viruses. The most important food-borne disease (McCabe-Sellers and Beattie, 2004). Between viruses are hepatitis A, norovirus, astrovirus, and 1990 and 2003, 12% of food-borne outbreaks in the rotavirus. These are discussed in detail below. USA were linked to produce and produce dishes; the most common produce foods being salads and alfalfa Hepatitis A virus sprouts. Of the produce-associated outbreaks, 40% Hepatitis A is one of the more severe food-borne were due to norovirus or hepatitis A, and 30% were diseases. The illness results from immune destruction caused by bacteria commonly associated with an of infected liver cells, and a few weeks of debility animal reservoir, such as Campylobacter, E. coli O157 are common (Table 14.3). It is a member of the and Salmonella (Dewaal et al., 2006). picornaviruses. Finally, chemical risks to food, such as pesticide Infections are more likely to be asymptomatic or residues, acrylamide, and the use of food additives, mild in young children than in adolescents or adults. continue to concern consumers. The virus can be shed in feces for up to 14 days before the onset of illness. It is therefore possible for an 14.3 Food-borne bacteria infected food handler with poor personal hygiene (hand-washing, in particular) to contaminate food The major cause of food-borne diseases is the con- during this period. The virus may be shed in the feces sumption of microbiologically contaminated foods. for 1–2 weeks after onset of symptoms. There are many types of food-borne pathogens, Food becomes contaminated with this virus via including bacteria, viruses, and parasites. The charac- infected persons or via fecally contaminated water, as teristics of food-borne bacterial intoxications and is usual with shellfi sh. Examples of other foods impli- infections are summarized in Tables 14.1 and 14.2, cated in hepatitis A outbreaks are oysters, raw mussels, respectively. Food-borne pathogens are covered in drinking water, bakery products and caviar. Hepatitis more detail by Doyle et al. (2001). A has been shown to be more heat resistant than most enteric viruses and is also quite resistant to drying. 14.4 Food-borne viruses The virus is susceptible to chlorination treatment, however, and water-borne hepatitis A outbreaks have It is only in recent years that the role of viruses as been linked to untreated water. etiological agents of food-borne illness have emerged. Diffi culties in attributing viral illness to food have Noroviruses mainly been due to the diagnostic diffi culties in Norovirus was the fi rst enteric virus reported to be detecting viruses in an implicated food and under- food-borne. It was formerly known as Norwalk-like reporting owing to the mild nature of illness in many virus (NLV) or small round structured virus (SRSV) cases. A report from the US Centers for Disease and has recently been classifi ed as a member of the Control (CDC) in 2000, on surveillance of food- calicivirus family. Noroviruses are diffi cult to detect, borne disease outbreaks from 1993 to 1997, revealed especially from foods. 328 Introduction to Human Nutrition b intestinal tract of fi sh intestinal tract of fi and mammals smoked and salted sh, honey fi vegetation and cereal-based products, herbs and spices (a) Source (b) Associated foods (a) Soil, sediment, (b) Canned foods, (a) Soil, dust and (b) Cooked rice, cereals anaerobic positive anaerobe positive anaerobe (a) Gram stain Aerobic/ (b) (a) Gram (b) Obligate (a) Gram (b) Facultative = 121 <0.001 min <0.001 min 0.1 min < = = = 25 min

but can form heat-resistant spores (D 5 min at 85°C 5 0.03–2.35 min) extremely heat resistant (can withstand 121°C for 90 min) 100 121 100 121 Group I Spores: D D 0.1–0.2 min Group II Spores: D Emetic toxin: D Yes Heat-sensitive, Yes destroyed by Toxin: Forms spores Heat resistance b a w (a) 10°C (b) 30–35°C (c) 4.3 (d) 0.95 Group I (a) 10°C (b) 30–40°C (c) 4.6 (d) 0.94 Group II (a) 3.3°C (b) 25–37°C (c) 5.0 (d) 0.97 (d) Min. A (a) Min. temp (b) Opt. temp (c) Min. pH μg cells 5 >10 (12–32 μg toxin/kg) vomiting double vision, dryness of the mouth followed culties by diffi swallowing and nally breathing. fi and Vomiting mild diarrhea may occur in the early stages toxin (a) Nausea and (b) (a) Blurred and/or (b) 0.005–0.5 (a) Symptoms (b) Infectious dose but may be as long as 8 days (from days to months) (a) 1–5 h (a) 1–5 h (b) 6–24 h, (a) 12–36 (b) Variable (a) Onset (b) Duration Food-borne illness inactivated by normal cooking temperatures; spores are quite however, heat resistant. Emetic illness is caused by consumption of heat-stable emetic toxin produced by cells growing to high numbers in food. This is most likely to happen when cooked foods are not served while hot or not cooled rapidly caused when food becomes contaminated with spores from the environment, which are not destroyed by initial cooking or processing. If the food is packaged anaerobically and provides a suitable environment for growth, spores will germinate, leading to toxin production. The toxin is heat sensitive, so further heat treatment of the food would prevent illness. The so called “botulinum cook” (heat treatment to 121°C min or equivalent) is for 3 used for low acid canned food products to destroy these spores Vegetative cells are Vegetative Food-borne botulism is

(Emetic) botulinum (proteolytic)

Table 14.1 Table Characteristics of food-borne bacterial intoxications BacteriaBacillus cereus Comment Clostridium Group I Group II (nonproteolytic) Food Safety 329 nose, throat and hands animal origin that have been physically handled and have not received a subsequent bactericidal treatment (a) Exposed skin lesions, (b) Generally foods of positive anaerobe (a) Gram (b) Facultative ), and other factors. w resistant (able to withstand boiling for up min) to 30 e (in the case of A No heat Toxin: (a) 7°C (b) 35–37°C (c) 4.5 (d) 0.83 cells/g 5 <1.0 μg toxin vomiting, abdominal pain and diarrhea (>10 needed to produce cient toxin) suffi (a) Nausea, (b) days (a) 1–6 h (b) 1–2 , water activity. w to grow to levels cells/g) that will 5 (>10 role in transmission. S. aureus is carried in nose/ throat of ~40% of healthy individuals and can be easily transferred to food via the hands. Most implicated foods have been foods that ready-to-eat have been contaminated by poor handling practices and stored at incorrect temperatures, allowing S. aureus cient heat- produce suffi stable staphylococcal toxin Food handlers play a major

aureus Not an exhaustive list. Under otherwise optimal conditions; limits will vary according to strain, temperature, type of acid (in the case of pH), solut Min., minimum; Opt., optimal; A Staphylococcus a b 330 Introduction to Human Nutrition b feces dried and gravy, precooked foods vegetables, fi sh and vegetables, fi soups (a) Soil and animal (b) Meat, poultry, (a) Source (b) Associated foods (a) Soil and dust (b) Meat, milk, anaerobic anaerobe anaerobe (a) Gram positive (b) Obligate (a) Gram stain Aerobic/ (b) (a) Gram positive (b) Facultative = = 1.3–2.8 min) 121 95 forms heat- resistant spores (D forms heat- resistant spores (D 0.03–2.35 min) Heat resistance Heat-sensitive, but Heat-sensitive, but b a w (a) Min. temp (b) Opt. temp (c) Min. pH (a) 10°C (b) 30–35°C (c) 4.3 (d) 0.95 (a) 15°C (b) 43–45°C (c) 5.0 (d) 0.95 (d) Min. A cells cells/g 5 6 >10 >10 abdominal pain diarrhea (a) Diarrhea and severe (b) (a) Symptoms (b) Infectious dose (a) Abdominal pain and (b) (can be 8–22 h) Food-borne illness (a) 12–18 h (a) 12–18 h (b) 24 (a) Onset (b) Duration h (a) 8–16 h (b) 12–14 /g) followed by 6 enterotoxin production in the large intestine. When contaminated food is cooked, sporulation is induced. As the food cools, the spores germinate and vegetative cells continue to unless the food is multiply, cooled quickly and stored under refrigerated conditions consumption of food containing high numbers of cells (>10 by normal cooking temperatures; however, spores are quite heat resistant. The diarrheal enterotoxin is produced when spores germinate in the small intestine after consumption of contaminated food Comment Illness results from Vegetative cells are inactivated Vegetative

(diarrheal) perfringens

Table 14.2 Table Characteristics of food-borne bacterial infections Bacteria Bacillus cereus Clostridium Food Safety 331 made silage raw milk, deli meats, pâté, hot dogs, raw vegetables, ice cream, and seafood deer burgers, raw milk, salad vegetables, and unpasteurized apple juice cattle, fl ies, and cattle, fl water chicken, raw milk, pork, lamb, beef, sh, and water shellfi (a) Soil, improperly soft(b) Fresh , (a) Cattle, sheep, pigs, (b) Undercooked beef (a) Chickens, birds, (b) Undercooked anaerobe negative anaerobe negative microaerophile (b) Facultative (b) Facultative (b) Facultative (b) Facultative (b) Fastidious (b) Fastidious Heat-sensitive (a) Gram positive Heat-sensitive (a) Gram Heat-sensitive (a) Gram (a) 0°C (b) 30–37°C (c) 4.3 (d) 0.90 (a) 7°C (b) 37°C (c) 4.5 (d) 0.97 (a) 32°C (b) 42–43°C (c) 4.9 (d) 0.98 meningitis and/or septicemia. While pregnant women may u- experience a mild fl like illness, infection may result in miscarriage, stillbirth or birth of a severely ill infant. (FG: fever, watery diarrhea, nausea, headache and pains in joints and muscles) abdominal cramps, bloody diarrhea (hemorrhagic colitis), approx. 5% (mostly children) develop hemolytic uremic syndrome (HUS) diarrhea, sometimes bloody diarrhea. Severe abdominal is rare. pain. Vomiting Complications are uncommon, but include bacteremia, reactive arthritis and Guillain–Barré syndrome (a) Flu-like symptoms, (b) Unknown (a) Diarrhea and severe (b) 10–100 cells (a) Moderate to severe (b) 500 cells weeks (FG: 20–27 h) weeks (FG: self- limiting, usually 1–3 days) (a) Up to 10 (b) Days to (a) 1–6 days (a) 1–6 days (b) 4–6 (a) 2–5 days (a) 2–5 days (b) 1–7 causes is one of the most common causes of bacterial food poisoning in many industrialized countries. Although campylobacters are fragile organisms, and do not survive or multiply very well on foods, the low infectious dose means that a small level of contamination may result in illness. Compared with other food-borne bacteria with low infectious doses, relatively few outbreaks ed have been identifi serious illness in individuals with impaired cell-mediated Highly susceptible immunity. individuals include pregnant women, neonates, older people and immunocompromised individuals. The organism is also reported to cause febrile gastroenteritis (FG) in healthy persons. Foods associated with transmission tend to be processed, ready-to-eat foods, with long shelf-lives (>5 days) stored at refrigeration temperatures concern due to the severity of illness. The organism is easily killed by cooking, but the low infective dose means that foods must be cooked thoroughly and protected from cross-contamination

VTEC is of considerable L. monocytogenes

Escherichia coli (VTEC) monocytogenes

Campylobacter C. jejuni Verotoxigenic Listeria 332 Introduction to Human Nutrition b feces, raw poultry, feces, raw poultry, raw meats, and raw seafood eggs, raw milk and other dairy products, raw fruits and vegetables (e.g., alfalfa sprouts and melons) oysters estuarine , sh raw shellfi sh shellfi (a) Source (b) Associated foods (a) Water, soil, animal (a) Water, (b) Raw meats, poultry, (a) Costal waters, raw sh (b) Shellfi (a) Costal and (b) Fish and raw negative anaerobe negative anaerobe negative anaerobe (a) Gram stain (b) Aerobic/ anaerobic (b) Facultative (b) Facultative (b) Facultative (b) Facultative (b) Facultative Heat resistance Heat-sensitive (a) Gram Heat-sensitive (a) Gram Heat-sensitive (a) Gram b a w (a) Min. temp (b) Opt. temp (c) Min. pH (a) 7°C (b) 35–37°C (c) 4.0 (d) 0.93 (a) 10°C (b) 37°C (c) 5.0 (d) 0.97 (a) 5°C (b) 37°C (c) 4.8 (d) 0.94 (d) Min. A cells cells cells 6 6 6 >10 >10 cramps, nausea, vomiting, headache, chills fever, cramps, fever, some cramps, fever, vomiting, and nausea pain, diarrhea, nausea and sometimes vomiting. Can be fatal in older people or those with weakened immune system (a) Diarrhea, abdominal (b) (a) Symptoms (b) Infectious dose (a) Diarrhea, abdominal (b) (a) Fever, abdominal (a) Fever, (b) ~10 <7 days lasts 6–7 days 12–36 h, but may be 6–72 h Food-borne illness (a) 12–24 h (a) 12–24 (b) (a) Onset (b) Duration (a) 1–3 days (b) Diarrhea (a) Usually (b) 2–5 days

O1 S. can be

Salmonella non-O1 is

V. cholerae V. Typhimurium and Typhimurium related to serotypes only a small number account for most human infections, with considered to be the leading cause of seafood- borne bacterial gastroenteritis. It is sh frequently isolated from fi from both marine and brackish-water environments 2400 different Enteritidis predominating. Undercooked food from infected food animals is most commonly implicated. Egg-associated salmonellosis is an important public health problem S. (the organism that causes Asiatic or epidemic ), but causes a disease reported to be less severe than cholera. It has been generally believed that water was the main vehicle for transmission, but an increasing number of cases have been associated with food Comment Although there are approx. cholerae Vibrio parahaemolyticus V.

Continued

serogroup non-O1 parahaemolyticus

Table 14.2 Table Bacteria Salmonella cholerae Vibrio Vibrio Food Safety 333 animals (e.g., pigs, dogs, cats) and water raw milk and water sediment, , sh shellfi crabs (a) Wide range of (a) Wide (b) Undercooked pork, (a) Coastal waters, (b) Oysters, clams, negative anaerobe negative anaerobe (b) Facultative (b) Facultative (b) Facultative (b) Facultative ) and other factors. w e (in the case of A Heat-sensitive (a) Gram Heat-sensitive (a) Gram (a) –1.3°C (b) 25–37°C (c) 4.1 (d) 0.96 (a) 8°C (b) 37°C (c) 5 (d) 0.96 : water activity. w <100 pain and fever. pain and fever. Intestinal pain, especially in young adults, may be confused with appendicitis gastroenteritis, primarily septicemia individuals but in predisposed cells (a) Diarrhea, abdominal (b) Unknown (a) Wound infections, (a) Wound (b) Unknown in healthy weeks (a) 3–7 days (b) 1–3 weeks (a) 16 h (a) 16 (b) Days to is is considered to psychrotrophic and known to be quite resistant to freezing, surviving in frozen food for extended periods. The organism is present in a wide range of animals, especially pigs. Milk and pork have been implicated in outbreaks, especially in countries where pork is eaten raw or undercooked be one of the most invasive and rapidly lethal of human pathogens. Infection starts with a gastrointestinal illness, and rapidly progresses to a septicemic condition. It is mostly associated with the consumption of raw oysters. Human infections are rare, but those at most risk either have underlying illnesses or are immunocompromised

Y. enterocolitica Y.

enterocolitica Not an exhaustive list. Under otherwise optimal conditions; limits will vary according to strain, temperature, type of acid (in the case of pH), solut Heat-sensitive: cells destroyed by typical cooking temperatures; Min.: minimum; Opt.: optimal; A Vibrio vulnifi cus Vibrio vulnifi cus vulnifi V. Yersinia a b 334 Introduction to Human Nutrition

Table 14.3 Characteristics of the illnesses caused by hepatitis A and norovirus

Hepatitis A (picornavirus) Norovirus (calicivirus)

Properties Particles are featureless spheres 28 nm in diameter, single- Particles are spheres 25–35 nm in diameter, single-stranded RNA stranded RNA coated with protein coated with protein that has characteristic cupped surface depressions Infection Infection via intestine to liver, incubation period 15–20 Infection of intestinal lining, incubation period 24–48 h days (mean 28 days) Illness Illness from immune destruction of infected liver cells: Nausea, vomiting, diarrhea, etc., lasting for 24–48 h fever, malaise, anorexia, nausea, abdominal discomfort, often followed by jaundice; severity tends to increase with age: ranges from unapparent infection to weeks of debility, occasionally with permanent sequelae Shedding Shedding of virus peaks during the second half of the During illness (in vomitus and feces), possibly 7 days after onset incubation period (10–14 days), usually ends by 7 days after onset of jaundice Diagnosis Based on detection of IgM class antibody. Hepatitis A Detection of virus in stool ELISA or PCR or of antibody against the virus in the patient’s blood serum (kits available) virus in patient’s blood serum; no standard methods, reagents not readily available for most agents Immunity Immunity is durable (possibly lifelong) after infection Apparently transient

ELISA, enzyme-linked immunosorbent assay; IgM, immunoglobulin M; PCR, polymerase chain reaction.

Exposure is by contact with infected individuals or food from a common supplier occurred in Osaka, fecally contaminated water or other materials. Shell- Japan, in 1991, affecting 4700 teachers and pupils fi sh (bivalve molluscs) have been the predominant from 14 schools in the city. food vehicle. Shellfi sh beds may frequently become contaminated with human feces from sewage dis- Rotaviruses charges. Aerosolization of vomitus-containing virus Serogroup A rotaviruses are the single most impor- particles has been proposed as another mode of trans- tant cause of infantile gastroenteritis worldwide, mission of the virus and may also be a source of food affecting an estimated 130 million infants and causing contamination. Sensitive detection assays have now 873 000 deaths every year. The rotavirus genome revealed that shedding of the virus in feces may con- consists of 11 segments of double-stranded RNA tinue for up to a week after the illness subsides. surrounded by a double-shelled viral capsid. When examined by electron microscopy, the double-shelled Astroviruses particles resemble a wheel-like structure morphologi- Under the electron microscope, astroviruses appear as cally (Latin rota, wheel). The incubation period of the small, round viruses that have surface projections illness is 1–3 days, and the illness is characterized by resembling a fi ve- or six-pointed star (Greek astron, fever, vomiting, and diarrhea. Although the majority star). The illness differs from the norovirus in that the of rotavirus infections involve infants, outbreaks of incubation period is longer (3–4 days), the duration food-borne, and water-borne disease affecting all age of illness is longer (often lasting for 7–14 days), and groups have been reported, albeit infrequently. vomiting is less common, with diarrhea being the predominant symptom. In addition, the very young Other viruses (<1 year) appear to be the most susceptible group, Picornaviruses other than hepatitis A can also be whereas norovirus affects all age groups. Astrovirus transmitted by the food-borne route. Polioviruses are outbreaks have been reported to occur in crèches, transmitted by food but virulent strains of this agent schools, hospital wards, and homes, but in are now extremely rare. Coxsackie virus and echovi- many cases there was no well-defi ned mode of trans- rus have been associated with food-borne outbreaks, mission. One large outbreak linked to contaminated but data are limited. Hepatitis E has been linked to a Food Safety 335 number of water-borne outbreaks but there has been no association with food. One food-borne outbreak of parvovirus linked to consumption of cockles has been reported.

14.5 Food-borne parasites

Food-borne parasitic diseases are a major public health problem affecting millions of people, predomi- nantly in nonindustrialized countries. The incidence of parasitic disease associated with the consumption of foods of animal origin has declined in industrial- ized countries in recent years, where improvements in animal husbandry and meat inspection have led to considerable safety and quality gains. The situation in Figure 14.1 Life cycle of Diphyllobothrium latum. nonindustrialized countries is very different, in that these diseases are associated with poor standards of sanitation and hygiene, low educational standards, the consumption of raw, undercooked, or otherwise and extreme poverty. underprocessed freshwater fi sh or crustaceans con- Parasites are organisms that live off other living taining the infective stages (metacercariae) of these organisms, known as hosts. They may be transmitted parasites. Table 14.4 summarizes the distribution, from animals to humans, from humans to humans, the principal reservoirs, and freshwater fi sh or crus- or from humans to animals. Food-borne parasitic taceans involved in the transmission of these parasites disease occurs when the infective stages of parasites in the food chain. The most important parasites with are eaten in raw or partially cooked protein foods, or respect to the numbers of people affected are species in raw vegetables and fruits that are inadequately of the genera Clonorchis, Opisthorchis, and Paragoni- washed before consumption. These organisms then mus. The diseases caused by food-borne trematodes live and reproduce within the tissues and organs of include cholangiocarcinoma, gallstones, severe liver infected human and animal hosts, and are often disease, and gastrointestinal problems. excreted in feces. The parasites involved in food-borne disease usually have complex life cycles involving one Nematodes or two intermediate hosts (Figure 14.1). The food- The food-borne roundworms of primary importance borne parasites known to cause disease in humans are in humans belong to the phylum Nematoda and are broadly classifi ed as helminths (multicellular worms) known as nematodes. Undercooked or raw fi shery and protozoa (single-celled microscopic organisms). products and pork meat are the usual foods These include the major helminthic groups of trema- involved. todes, nematodes, and cestodes, and some of the Where fi shery products are the food vector, the emerging protozoan pathogens, such as cryptospo- defi nitive hosts of roundworms causing disease in ridia and cyclospora. The illnesses they can cause humans are piscivorous marine mammals such as range from mild discomfort to debilitating illness and seals. Marine invertebrates and fi sh are the two inter- possibly death. mediate hosts and humans are infected when they These infections occur endemically in some 20 consume raw or minimally processed products. Fish countries, where it is estimated that over 40 million are the secondary hosts and are infected when they people worldwide, mainly in eastern and southern consume the invertebrate primary host or fi sh that are Asia, are affected. Of major concern are the fi sh-borne already infected. There are many species of nematodes trematode infections. The trematode species con- and a very large number of species of fi sh, worldwide, cerned all have similar life cycles involving two inter- that are known to act as intermediate hosts. The most mediate hosts. The defi nitive host is man and other common species of nematode causing disease in mammals. Food-borne infection takes place through humans is Anisakis simplex, sometimes referred to as 336 Introduction to Human Nutrition s , are biologically

Opisthorchis viverrini, O. felineu Opisthorchis viverrini, O.

Heterophyes heterophyes

Clonorchis sinensis intestine, resulting in diarrhea and abdominal pain. In some instances the lining of the small intestine breaks down, and the eggs produced by the parasite enter the bloodstream. Once in the bloodstream the eggs can be carried to cant other organs where they can cause signifi heart, and especially in the liver, pathology, brain similar, food-borne trematodes that chronically similar, the infect the bile ducts and, more rarely, pancreatic duct and gallbladder of humans and other mammals and leads to fi brosis leads to fi The liver fl ukes, The liver fl Similar to The parasite can irritate the lining of the small Disease Infl ammation of the bile ducts which eventually Infl and . spp.), ), trout, and ), dace

Glossogobius

Mugil and

Puntius orphoides Puntius

Pseudorasbora parvaPseudorasbora , and others. In Japan,

Plecoglossus altivelis hakonensis Tribolodon including Metacercariae in fi sh muscles Metacercariae in fi fi sh, mainly Cyprinidae, e.g., carp, fi roach and dace, most important being Metacercariae in muscle and subcutaneous tissue especially mullet ( ( species of fi sh genera species of fi whitebait. Metacercariae in gills, n, or tail fi also involved. Metacercariae in muscle and skin Hampala dispar Tilapia Acanthogobius ( Some 10 species of freshwater fi sh Some 10 species of freshwater fi Many species (c. 110) of freshwater sh of family Cyprinidae. fi Freshwater sh, Brackish water and freshwater fi Metacercariae in fi sh muscles Metacercariae in fi Food involved in transmission to humans Freshwater fi sh, e.g., sweetfi sh sh, e.g., sweetfi fi Freshwater ), and

Felis viverrina other mammals that feed sh waste sh and fi on fi species of fi sh-eating species of fi mammals sh or mammals that eat fi sh waste fi ( pelicans, hawks, and black kite eating birds Dogs, cats, and many other Cats, dogs, and other shing cats Dogs, cats, fi Sheep, cattle Dogs, cats, jackals, foxes, Principal reservoirsPrincipal (other than humans) Dogs, cats, pigs, and fi sh- Dogs, cats, pigs, and fi Macao, Japan, Korea and Vietnam. Macao, Japan, Korea and Vietnam. Migrants to other countries found to be infected; cases in Hawaii sh attributed to consumption of fi imported from China States (CIS), eastern and central Europe (Mekong River basin) Africa, Australia, USA and eastern Asia Widespread in China, Taiwan, in China, Taiwan, Widespread Commonwealth of Independent Laos and north-eastern Thailand East, Europe, the Middle East, the Far Mediterranean basin, especially Egypt Distribution Eastern and southern Asia and ukes ukes

viverrini heterophyes yokogawai related species Table 14.4 Table Food-borne trematode infections Parasite Liver fl Clonorchis sinensis Opisthorchis felineus Opisthorchis hepatica Faciola Intestinal fl Heterophyes Metagonimus Food Safety 337 . Heavy infections show symptoms of diarrhea, ascites, anasarca, and abdominal pain, fever, intestinal obstruction numbers of circulating eosinophils, abdominal discomfort and nausea. Sometimes asymptomatic Most infections are light and asymptomatic. Paragonimiasis Nanophyetiasis Diarrhea, usually accompanied by increased ,

Potamon , ), crayfi sh and ), crayfi

Eriocheir such as water cress shrimps. Metacercariae in muscles, gills, liver (hepatopancreas), and boar meat cardia region. Wild suspected as a source of infection Metacercariae in muscles, fi ns, Metacercariae in muscles, fi and kidneys crabs ( and brachyures Parathelphusa Fish, frogs, and toads. Metacercariae in muscle Uncooked contaminated water plants Salmonid and other fi sh. Salmonid and other fi Freshwater and brackish water Freshwater Crustaceans, amphipods, isopods, birds badgers carnivora that feed on crustaceans Cats, dogs, and fi sh-eating Cats, dogs, and fi Sea birds Pigs Dogs, cats, rats (?), and Domestic and wild , Food safety measures for the control of food-borne trematode infections, page 9, copyright 1995 with permission from Elsevier

et al. , 6, Abdussalam of Amur River) and parts of Sakhalin peninsula, north-western USA Cameroon), the Americas (Ecuador to USA), Japan, Korea, Thailand, Laos, China Philippines Oriental countries Siberia, west Africa (Nigeria,

Food Control and spp. Eastern Siberia (mountain tributaries spp. Eastern and southern Asia uke

brevicaeca westermani related species in Asia, Africa and the Americas Lung fl Nanophyetus Spelotrema Haplorchis buski Fasciolopsis Paragonimus Based on data from 338 Introduction to Human Nutrition the herringworm. The other species involved in anisa- nourishment. Infections can be prevented by sanitary kiasis in North America, Europe, and Japan is Pseudo- disposal and treatment of human waste and by terranova decipiens (the codworm or sealworm). thorough cooking and freezing of contaminated pork Nematodes are commonly present in fi sh caught in and beef. the wild, most frequently in the liver and belly cavity, but can also occur in the fl esh. Anisakiasis is an Protozoa uncommon disease because the parasite is killed by The protozoal human parasites are unicellular organ- heating (55°C for 1 min), and by freezing (−20°C for isms that colonize the intestinal epithelium and form 24 h). There is a risk of illness from fi shery products cysts. These are excreted and may survive for long consumed raw, for example sushi, or after only mild periods in the environment. There are fi ve genera of processing, such as salting at low concentrations or concern in foods: Giardia, Entamoeba, Toxoplasma, smoking. Many countries now require that fi sh used Cyclospora, and Cryptosporidium. for these mildly processed products be frozen before Table 14.5 summarizes the distribution, principal processing or before sale. reservoirs and route of transmission of these parasites Trichinella spiralis is the cause of trichinosis in in the food chain. humans. This most commonly results from the con- sumption of contaminated raw or undercooked pork 14.6 Transmissible spongiform or pork products. Since the mid-1980s outbreaks have encephalopathies and food been associated with raw and undercooked horse- meat. Isolated cases have been reported from the con- Transmissible spongiform encephalopathies (TSEs) sumption of bear meat and ground beef in the USA. are fatal degenerative brain diseases which include The incidence of trichinosis can be controlled by BSE in cattle; scrapie in sheep; kuru, Creutzfeldt– avoiding feeding infected waste foods to pigs or by Jakob disease (CJD), and new variant CJD (vCJD) in fully cooking pig swill. Freezing pork products (−15°C humans. They are characterized by the appearance in for 20 days) or thorough cooking (78°C at the thermal the brain of vacuoles – clear holes that give the brain center) before human consumption will destroy tri- a sponge-like appearance – from which the condi- china larvae. tions derive their name. Several theories have been proposed to explain the Cestodes nature of the agents that cause TSE. Prusiner was Cestodes are tapeworms and the species of major awarded the Nobel Prize in 1997 for the prion theory, concern associated with consumption of fi sh is the which postulates that the agent is a proteinaceous fi sh tapeworm, Diphyllobothrium latum. Humans are infectious particle (PrP) that is capable of replication one of the defi nitive hosts, along with other fi sh- without the need of an agent-specifi c nucleic acid. eating mammals. Freshwater copepods and fi sh are The disease-associated prion (PrPSc) has been shown the intermediate hosts. The plerocercoid is present in to have a different helical shape to normal cellular the fl esh of the fi sh and infects humans following the prion protein (PrPC) found on neuronal cells and consumption of raw or minimally processed fi sh. The some other cells, for example lymphoid cells. However, recorded epidemiology of D. latum shows it to be while it is widely acknowledged that PrPSc is very prevalent in many countries worldwide. The inci- closely associated with the causative agent, there is a dence is relatively high in Scandinavia and the Baltic reluctance by some to accept PrPSc as the sole agent region of Europe. Diphyllobothriasis in humans can responsible for transmission. Another hypothesis be prevented by cooking or freezing fi sh before con- suggests the agent is an unconventional virus, while a sumption. Infections with tapeworms are also associ- third suggests that it is a virion that has similar prop- ated with eating undercooked or raw pork and beef. erties to a virus but uses host proteins to coat its Taenia saginata (the beef tapeworm) and Taenia nucleic acid. solium (the pork tapeworm) are unique among para- sites in that they have no vascular, respiratory, or Bovine spongiform encephalopathy digestive systems. Humans are their defi nitive hosts BSE, sometimes referred to as “mad cow disease,” was and they rely solely on the human body for all of their fi rst identifi ed in the UK in 1986. The disease is fatal Food Safety 339

Table 14.5 Food-borne protozoa

Occurrence Transmission Defi nitive host Incubation Infective dose Pathogenesis

Giardia intestinalis Food-borne, water- Humans, 3–25 days Low (~10 cysts) Chronic diarrhea, malabsorption, worldwide borne, person– domestic, and weight loss person wild animals Entamoeba Food-borne, water- Humans 2–4 weeks Very low Amebiasis, abdominal pain, histolytica borne, person– (~1 cyst) fever, diarrhea, ulceration of worldwide person (food the colon (severe cases) handlers) Toxoplasma Food-borne (raw or Humans, cats, 5–23 days ~1–30 cysts Mostly asymptomatic. In severe gondii inadequately several cases: hepatitis, pneumonia, worldwide cooked infected mammals blindness, severe neurological meat), water- disorders. borne, fecal–oral Can also be transmitted (infected cats) transplacentally resulting in a spontaneous abortion, a stillborn, or mental/physical retardation Cyclospora Food-borne, Humans Several days Not known, Often asymptomatic. cayetanensis water-borne to weeks probably very Abdominal cramps, vomiting, worldwide low weight loss, diarrhea Cryptosporidium Food-borne, water- Humans, Diffi cult to Very low (~1 Often asymptomatic. parvum borne, animal– domestic, and defi ne, in cyst) Abdominal cramps, vomiting, worldwide person, fecal–oral wild animals most cases weight loss, diarrhea 3–7 days, occasionally longer to cattle within weeks to months of its onset. The (inherited) and associated with genetic mutations. incubation period is between 2 and 10 years. Affected Less than 1% are iatrogenic (i.e., accidentally trans- animals may display changes in temperament, such as mitted from person to person as a result of medical nervousness or aggression, abnormal posture, lack of or surgical procedures). Classically, sporadic CJD coordination, and diffi culty in standing, decreased occurs in those over 65 years of age and presents as a milk production, or loss of body weight despite con- rapidly progressive dementia with myoclonus (shock- tinued appetite. Most cattle with BSE show a gradual like contractions of isolated muscles), usually fatal development of symptoms over a period of several within 6 months. Surveillance of CJD, a human neu- weeks or even months, although some can deteriorate rological disease, was reinstituted in the UK in 1990 very rapidly. While the original source of the agent to evaluate any changes in the pattern of the disease responsible for BSE remains unknown, currently that might be attributable to BSE. The overall inci- the most plausible explanation is that a novel TSE dence of CJD rose in the UK in the 1990s, although a appeared in the UK cattle population in the 1970s and portion of this increase was due to improved ascer- subsequently spread through contaminated meat and tainment of CJD in older people as a result of the bone meal fed to cattle. The International Offi ce for reinstitution of surveillance. Epizootic Diseases (OIE) reports cases on its website New variant CJD, also referred to as variant CJD (www.oie.int). (vCJD), is a newly recognized disease in humans, which was fi rst diagnosed in the UK in the mid-1990s. Creutzfeldt–Jakob disease and new In contrast to the traditional forms of CJD, vCJD has variant CJD affected younger patients (average age 29 years) and CJD is a fatal disease of humans, fi rst described in the has a longer duration of illness (approximately 14 1920s and found worldwide. CJD is predominantly a months). Early in the illness, patients usually experi- sporadic disease, but about 14% of cases are familial ence behavioral changes, which most commonly take 340 Introduction to Human Nutrition the form of depression or, less often, a schizophrenia- regarding the incubation period between exposure to like disorder. Neurological signs such as unsteadiness, the infective agent and the emergence of symptoms. diffi culty walking, and involuntary movements develop as the illness progresses and, by the time of 14.7 Chemicals affecting food safety death, patients become completely immobile and mute. Chemicals may be present in food owing to their natural occurrence in soil (e.g., cadmium, lead) or The link between BSE and vCJD from fungal contamination (e.g., afl atoxins, ochra- A geographical association exists whereby the major- toxin), from algal contamination [e.g., amnesic shell- ity of BSE cases occurred in the UK and the majority fi sh poisoning (ASP), diarrhetic shellfi sh poisoning of vCJD cases were also reported there. The emer- (DSP), azaspiracid shellfi sh poisoning (AZP), para- gence of BSE preceded vCJD, indicating a temporal lytic shellfi sh poisoning (PSP)], from industrial or association. Studies of stored human brain tissue other pollution [e.g., lead, mercury, polychlorinated internationally have not identifi ed the histopatho- biphenyls (PCBs), dioxins], from agricultural and vet- logical changes characteristic of vCJD before the erinary practices (e.g., pesticides, fertilizers, veteri- current BSE epidemic. Incubation period and patho- nary drugs) or from food processing and packaging logical lesion studies in mice and molecular typing techniques [e.g. acrylamide, polycyclic aromatic studies demonstrate that vCJD is similar to BSE but hydrocarbons (PAHs), 3-monochloropropane-1,2- different from other TSEs. It is now widely accepted diol (3-MCPD), bisphenol A diglycidyl ether that vCJD was transmitted to humans through the (BADGE)] (Box 14.1). consumption of contaminated food. Toxicological assessment of these substances is Estimates of future prevalence of vCJD vary widely largely carried out on an international basis by expert as too little is known about the disease, especially groups such as the Joint Expert Committee on

Box 14.1 Principal groups of chemicals affecting food safety

Principal chemicals affecting food safety

Additives Contaminants Residues

Food additives Microbial toxins Pesticide residues

Food flavorings Plant toxins Veterinary drug residues

Process contaminants Processing aids

Environmental contaminants

Food contact materials

Naturally occurring contaminants Food Safety 341

Food Additives and Contaminants (JECFA) or Joint MTDI represents permissible human exposure as a Meeting on Pesticide Residues (JMPR), both jointly result of the natural occurrence of the substance in organized by the WHO and FAO. These expert groups food and drinking water. advise on acceptable or tolerable levels of intake of One of the most diffi cult issues in food safety is these substances. to advise on the potential risks to human health for substances found in food which are both genotoxic Acceptable and tolerable levels of intake (damaging DNA, the genetic material of cells) and The acceptable daily intake (ADI) level of a chemical carcinogenic (leading to cancer). For these substances, is the daily intake that, during a lifetime, would pose it is generally assumed that even a small dose can have no appreciable risk to the consumer, on the basis of an effect. JECFA addressed this issue in 1978 and all facts known at the time. It is expressed in mg/kg introduced the concept of an “irreducible level,” which of body weight (Box 14.2). it defi ned as “that concentration of a substance which The tolerable weekly intake (TWI) represents per- cannot be eliminated from a food without involving missible human weekly exposure to those contami- the discarding of that food altogether, severely com- nants unavoidably associated with the consumption promising the ultimate availability of major food sup- of otherwise wholesome and nutritious foods. The plies” (FAO/WHO, 1978). term tolerable signifi es permissibility rather than Until now the risk assessors have advised to keep acceptability for the intake of contaminants that have the exposure to such substances at the lowest possible no necessary function in food, in contrast to those of level. This approach is known as the ALARA principle permitted pesticides or food additives. For cumulative (“as low as reasonably achievable”). A disadvantage of toxicants, such as lead, cadmium, and mercury, the this approach is that it cannot be used to compare tolerable intakes are expressed on a weekly basis risks posed by different substances. Furthermore, the to allow for daily variations in intake levels, the application of the ALARA principle does not take into real concern being long-term exposure to the account the effectiveness of a substance and the actual contaminant. (sometimes extremely low) level of occurrence in The maximum tolerable daily intake (MTDI) has food. been established for food contaminants that are not A different approach, “the margin of exposure” known to accumulate in the body, such as tin, arsenic, (MoE) approach, which can be used to assess the risks and styrene. The value assigned to the provisional to human health of exposure to a substance in the absence of a tolerable daily intake or similar guidance value, has recently been endorsed by the EFSA Scien- tifi c Committee (EFSA, 2005) and the WHO/FAO Joint Expert Committee on Food Additives (WHO/ Box 14.2 Levels of intake of a chemical FAO, 2005). The margin of exposure is defi ned as the reference point on the dose–response curve (usually No observed effect Greatest concentration or amount of an level (NOEL) agent, found by study or observation, based on animal experiments in the absence of human ↓ that causes no detectable, usually data) divided by the estimated intake by humans. It adverse, alteration of morphology, enables the comparison of the risks posed by different functional capacity, growth, genotoxic and carcinogenic substances. Differences in development, or lifespan of the target potency of the substances concerned and consump- /Safety factor Uncertainty factor for extrapolating tion patterns in the population are taken into account ↓ animal data to humans when applying the MoE approach. /Safety factor Human interspecies variation ↓ Setting the acceptable daily intake Acceptable daily Daily intake that, during a lifetime, JECFA generally sets the ADI of a substance on the intake (ADI) would pose no appreciable risk to the basis of the highest no-observed-effect level in animal consumer, on the basis of all facts studies. In calculating the ADI, a “safety factor” is known at the time. It is expressed in mg/kg of body weight applied to the no-observed-effect level to provide a conservative margin of safety on account of the inher- 342 Introduction to Human Nutrition ent uncertainties in extrapolating animal toxicity data or to regulate the growth of plants as crop protection to potential effects in humans and for variation within agents. They are classifi ed into the groups shown in the human species. JECFA traditionally uses a safety Box 14.3. factor of 100 (10 × 10) in setting ADI values based on Most pesticides are toxic substances that are highly long-term animal studies. It is intended to provide an selective, especially those developed since the early adequate margin of safety for the consumer by assum- 1980s, and only have an effect on those pests or plants ing that the human being is 10 times more sensitive to which they are applied. Unlike other environ- than the test animal and that the difference in sensi- mental contaminants, pesticides are applied under tivity within the human population is in a 10-fold controlled conditions that should conform to “good range. However, different safety factors apply depend- agricultural practice” (GAP). This defi nes the effec- ing on the substance and test species in question. tive use of pesticides, up to the maximum allowable dose, applied in a manner that ensures the smallest Maximum levels for food commodities amount of residue in the foodstuff. These levels are calculated taking the above- Pesticides can also be toxic to humans since certain mentioned levels into consideration. Depending on biochemical pathways are relatively conserved across the substance, different principles apply. Residues species, as are some enzymes and hormones. In the such as pesticides and residues of veterinary drugs in context of food safety, exposure to pesticides is clas- foodstuffs are limited by setting a maximum residue sifi ed as acute or chronic. An acute intoxication limit (MRL). Additives are regulated by setting usually has an immediate effect on the body, whereas maximum limits or by applying the “quantum satis” a chronic effect may reveal itself over the lifespan. The principle (the least amount required to exert the severity depends on the dose and the toxicity of the desired technological function). pesticide compound or breakdown product. Toxic For contaminants, maximum levels/limits are effects that have been identifi ed include enzyme inhi- established for those foods that provide a signifi cant bition, endocrine disruption, and carcinogenic action, contribution to the total dietary exposure. However, depending on the compound in question. as a general principle the levels in all foods should In Europe the control of pesticides is based on always be kept as low as reasonably achievable (the Council Directive 91/414/EEC. Under this legislation, ALARA principle). pesticides must be evaluated for safety based on dos- In Europe, additives, pesticides, veterinary residues, siers prepared by their manufacturers. If a pesticide is and a wide range of contaminants are regulated by accepted it is placed on a positive list with an MRL EU legislation in the form of directives or regulations assigned to it. that are transposed into national legislation by each In the case of a limited number of highly toxic member state. pesticides, for which the ADI is necessarily based on acute toxicity rather than chronic toxicity, the level of Pesticide residues exposure is considered in relation to the acute refer- Pesticides are chemicals or biological products used ence dose (ARfD). ARfD values are measures of the to control harmful or undesired organisms and plants, maximum level of intake at one meal, or consump-

Box 14.3 Classifi cation of pesticides

Pesticides

Plant growth Insecticides Herbicides Fungicides Rodenticides Molluscides regulators Food Safety 343 tion over a day. This is the maximum intake level, hormone residues in foods of animal origin on human which is judged to result in no adverse toxicological metabolism. effect following such exposure. The ARfD value includes a safety factor to ensure that older people, Environmental and industrial contaminants infants and children, and those under stress due to These contaminants are of environmental origin or illness are protected. are by-products of industrial processes. Polyhalogenated hydrocarbons (PHHs) are a cate- Veterinary drug residues gory of environmental contaminants that includes Veterinary drugs include antibacterial compounds, toxaphene, dioxins, and polychlorinated biphenyls hormones, and nonsteroidal anti-infl ammatory prep- (PCBs). Certain polyhalogenated hydrocarbons are arations. As animal husbandry practices have intensi- manufactured for use in plastics, paints, transformers, fi ed over the past few decades, antibacterial substances and herbicides; although their use is now either have been increasingly used as growth promoters banned or severely restricted. In most industrialized to increase feed conversion effi ciency, and for pro- nations the compounds have become ubiquitous in phylaxis and therapy to prevent outbreaks and treat the environment. Hence, contamination of the food disease. Similarly, hormones are administered to chain is inevitable and it has been estimated that in increase growth rate and meat yield. Table 14.6 shows Western industrialized countries 90% of human the main types of antibacterial and hormonal exposure is through ingestion of contaminated foods compounds. such as fi sh and milk. Veterinary drugs are metabolized in the animal Foods that are rich sources of fats and oils tend to and are excreted in the urine and feces over time as accumulate PHHs because the compounds are lipo- the detoxifi cation process continues. Hence, residue philic and bioaccumulate in lipid-rich tissues and traces of drugs or their metabolites can be found fl uids. Oily fi sh from areas such as the Baltic Sea, in major organs, muscles, and body fl uids. In where levels of PHHs in the water are high, may general, antibacterial drugs are found in greatest contain elevated levels of these contaminants. Simi- concentration in the kidney, lesser concentrations in larly, cows that graze on polluted pasture can accu- the liver and lowest concentrations in the muscle mulate unacceptable concentrations of PHHs in their tissue, whereas hormones tend to concentrate in the milk. A recent incident in Belgium introduced PCBs liver. and dioxins into the food chain via contaminated The excessive use of antibacterial compounds in animal feed resulting from the accidental incorpora- animal husbandry has raised concerns about the tion of industrial oil into the feed ration. The biologi- development of resistant bacteria and the effect that cal half-life of PHHs can range from a matter of this may have on the usefulness of antibiotics in months to 20 years in human adipose tissue. Hence, human medicine. There have also been concerns they are persistent and accumulate in the body. Expo- about the risk of allergic reactions in humans to anti- sure to PHHs can result in a variety of toxic effects bacterial residues in food of animal origin. The use of that can be carcinogenic, including dermal toxicity, hormones has raised issues surrounding the effects of immunotoxicity, reproductive effects, and endocrine disruption. Metals, metalloids, and their compounds have long Table 14.6 Main types of veterinary drugs been associated with food poisoning, with lead and mercury probably the best documented hazards. Antibacterial compounds Hormones Metals are released into the environment as a result of natural geological action and also as a result of Aminoglycosides β-Agonists β-Lactams Resorcylic lactones man-made pollution from industrial processes. Fluoroquinolones Steroids Metals have an affi nity for biological tissue and Macrolides Stilbenes organic compounds, and hence they are often easily Sulfonamides Thyrostat absorbed into the body and can often accumulate in Tetracyclines organs and fat deposits. Table 14.7 shows some of the Quinolones main metals linked with food-borne toxicity. 344 Introduction to Human Nutrition

Table 14.7 Metals in the food chain Acrylamide has been shown to be neurotoxic in humans. It has been shown to induce tumors in labo- Metal Main food sources ratory rats and has been classifi ed as a probable Lead Shellfi sh, fi nfi sh, kidney, liver human carcinogen, and as such several international Cadmium Shellfi sh, kidney, cereals, vegetables bodies have concluded that dietary exposure should Mercury Finfi sh be as low as reasonably achievable. The most signifi - Arsenic Meat, vegetables, seafood cant pathway of formation of acrylamide in foods has been shown to arise from the reaction of reducing sugars with via the Maillard reaction at Lead toxicity has many symptoms, but the main temperatures above c. 120°C. Acrylamide has been issue relates to its effects on the nervous system of found in a wide range of heat-treated foods; it is children. Here, lead interferes with the transmission found in both foods processed by manufacturers and of nervous signals around the body. This can manifest foods that are cooked in the home. Acrylamide has itself in a reduced intelligence quotient (IQ) and been found to be most prevalent in fried potato prod- coordination problems. In adults, exposure to lead ucts (such as French fries and potato chips), cereals, can result in hypertension and other blood effects bakery wares, and coffee. such as anemia. Cadmium is most often accumulated PAHs are a group of over 100 different chemicals from occupational exposure or smoking and is known that are formed during certain technological pro- to affect the respiratory system. However, food expo- cesses and are common environmental contaminants. sure tends to be at a low level over longer periods. In They are formed during incomplete combustion of this regard, cadmium bioaccumulates in the kidney coal and oil. They are also formed during barbecuing and can cause renal damage. Mercury and its com- or grilling meat. Human exposure usually results pounds also bioaccumulate in the body, where they from and from cigarette smoke. Foods are most frequently associated with neural effects and most likely to be contaminated by PAHs are grilled or renal damage. In particular, methylmercury is highly charred meats. PAHs are toxins that have been docu- toxic particularly to the nervous system, and the mented by the WHO as genotoxic, immunotoxic, and developing brain is thought to be the most sensitive carcinogenic. Long-term exposure to foods contain- target organ for methylmercury toxicity. ing PAHs can lead to serious health risks. In a recent Arsenic is most often an occupational hazard, but Europe-wide incident, PAHs were found in pomace it can also be ingested with food and is responsible olive oil, which resulted in a major product recall. for acute and chronic poisoning. The toxicity of 3-Monochloro-propane-1,2-diol (3-MCPD) is a arsenic depends on its oxidation state and the type of member of a group of contaminants known as complex that it forms with organic molecules in the chloropropanols, which includes known genotoxic body. Chronic effects include gastroenteritis, nephri- animal carcinogens such as 1,3-dichloropropan-2-ol. tis, and liver damage. Arsenic is also considered to be 3-MCPD is a by-product in soy sauce and in hydro- a carcinogen. Other metals are also known contami- lyzed vegetable protein produced through acid hydro- nants and their toxic effects are diverse. Although this lysis. It can also be present as a contaminant in some is not an exhaustive list, these metals include alumi- food additives, and in epichlorhydrin/amine copoly- num, copper, tin, zinc, and chromium. mers, used as fl occulants or coagulant aids in water treatment, and may be present in drinking water Process contaminants (opinion of the European Scientifi c Committee on These types of contaminant occur during the process- Food, adopted on 30 May 2001). ing and production of foods, and include acrylamide, PAHs, chloropropanols, and nitrosamines. Microbial toxins Acrylamide is a reactive unsaturated amide that has Food poisoning can occur as a result of the ingestion found several industrial uses. In 2002, it was discov- of food containing preformed toxins that originate ered to occur in a variety of fried and baked foods, from bacterial growth, fungal growth, or algal growth. in particular carbohydrate-rich foods that had been In the case of bacteria the toxin is absorbed into the subjected to high-temperature cooking/processing. bloodstream via the intestine and therefore illness Food Safety 345 results from intoxication rather than infection. In the to suppression of their immune systems and greater case of fungi, several species are involved in the pro- susceptibility to disease. duction of toxic substances during growth on food- The principal fungi that are associated with myco- stuffs. These toxins are known as mycotoxins. Algal toxin production are the genera Aspergillus, Penicil- toxins are usually associated with seafood, most lium, and Fusarium. Aspergillus and Penicillium are notably molluscan shellfi sh. sometimes referred to as storage fungi as they can grow at low water activity levels and are associated with the Bacterial toxins post-harvest spoilage of stored food commodities such Three bacteria are most commonly associated as cereals, nuts, and spices. Fusarium species are plant with preformed toxin production: Clostridium botu- pathogens and can infect plants in the fi eld and produce linum, Staphylococcus aureus and Bacillus cereus (see mycotoxins preharvest. Table 14.8 provides an over- Table 14.1). view of the most important mycotoxins.

Fungal toxins (mycotoxins) Seafood toxins Mycotoxins are secondary metabolites of molds that Fish and fi shery products are nutritious foods and are can induce acute and chronic symptoms, such as desirable components of a healthy diet. Food-borne carcinogenic, mutagenic, and estrogenic effects in illnesses resulting from the consumption of seafood humans and animals. Acute toxicity due to mycotox- are associated with both fi nfi sh and molluscan shell- ins is associated with liver and kidney damage. fi sh. The major risk of acute illness is associated with Chronic toxicity resulting from the exposure of low the consumption of raw shellfi sh, particularly bivalve levels of mycotoxins in the human diet is a major food molluscs. The consumption of these toxic shellfi sh by safety concern. In nonindustrialized countries myco- humans can cause illness, with symptoms ranging toxins have been reported to be responsible for from mild diarrhea and vomiting to memory loss, increased morbidity and mortality in children owing paralysis, and death. Toxins associated with phyto-

Table 14.8 Mycotoxins in the food supply

Mycotoxin Producing fungi Main foods affected Toxicity

Afl atoxins Aspergillus fl avus and A. parasiticus Nuts, cereals, dried fruit, herbs Carcinogenic, hepatotoxic and spices, milk (afl atoxin M1) Ochratoxin A Aspergillus ochraceus, Penicillium Coffee, dried fruit, cereals, beans, Nephrotoxic, immunotoxic verrucosum, and other Aspergillus pulses, wine, beer, grape juice; and Penicillium spp. kidney, liver and blood from animals fed with contaminated feed Patulin Aspergillus clavatus, also several Fruits and grains, predominantly Cytotoxic species of Penicillium, Aspergillus, apples and apple products and Byssochlamys Trichothecenes Fusarium spp. Wheat, maize, barley, oats, rye, Dermotoxic, enterotoxic, hemotoxic, (nivalenol, malt, beer, bread immunotoxic deoxynivalenol, T2-toxin, etc.) Fumonisins Fusarium spp. Cereals, mainly corn Carcinogenic, cytotoxic, hepatotoxic Sterigmatocystin Aspergillus versicolor, A. nidulans, Cereals, green coffee, herbs and Hepatotoxic and nephrotoxic, and other Aspergillus spp. spices, raw meat products carcinogenic Citrinin Penicillium spp., Aspergillus spp. Cereals Nephrotoxic Zearalenone Fusarium graminearum Maize, barley, oats, wheat, rice, Estrogenic effects, feed refusal, sorghum, bread vomiting Moniliformin Fusarium spp. Cereals, maize Nephrotoxic, causes necrosis of the heart muscle 346 Introduction to Human Nutrition plankton are known as phycotoxins. These toxins Adequate cooking of legume seeds such as kidney have been responsible for incidents of wide-scale beans and disposal of the cooking water will remove death of sea-life and are increasingly responsible for the natural toxins present in these food products. human intoxication. Antinutritional factors are those components of Various seafood poisoning syndromes are associ- plants that interfere with metabolic processes and can ated with toxic marine algae and these include para- lead to defi ciencies of key nutrients in the diet. These lytic shellfi sh poisoning (PSP), amnesic shellfi sh are generally classifi ed as enzyme inhibitors and poisoning (ASP), diarrhetic shellfi sh poisoning (DSP), mineral binding agents. Enzyme inhibitors are poly- neurotoxic shellfi sh poisoning, and azaspiracid shell- peptides and proteins that inhibit the activities of fi sh poisoning (AZP). There are also different types digestive enzymes, and most are thermolabile and are of food poisoning associated with fi nfi sh and these reduced by cooking. For example, trypsin inhibitors include ciguatera poisoning, scombroid or histamine may cause poor protein digestion and a shortage of poisoning, and puffer fi sh poisoning. Consumption sulfur-containing amino acids in the diet. Lectins are of raw molluscan shellfi sh poses well-known risks of proteins that occur in beans that alter the absorption food poisoning, but intoxication from fi nfi sh is not of nutrients in the intestinal wall. Cooking beans so well known. Most of the algal toxins associated will inactivate lectins. Tannins (polyphenols) occur with seafood poisoning are heat stable and are not in cereals, specifi cally in the seed coat. These form inactivated by cooking. It is also not possible visually complexes with proteins and inhibition of digestive to distinguish toxic from nontoxic fi sh. Many coun- enzymes. Phytate is a natural component in plants tries rely on biotoxin monitoring programs to protect and on digestion forms insoluble complexes with public health and close harvesting areas when toxin metal ions in the body. The result is reduced bioavail- algal blooms or toxic shellfi sh are detected. In nonin- ability of essential minerals such as iron. In addition, dustrialized countries, particularly in rural areas, a range of natural plant toxins can cause allergic reac- monitoring for harmful algal blooms does not rou- tions in humans, but there is a general lack of knowl- tinely occur and deaths due to red-tide toxins com- edge about their properties and modes of action. monly occur. Table 14.9 provides an overview of the most important types of fi sh poisoning. Food additives Food additives are added to foods for a specifi c tech- Naturally occurring plant toxins nological purpose during manufacture or storage and Certain plants contain naturally occurring com- become an integral part of that foodstuff. Additives pounds that are toxic to humans or that reduce the can be natural or synthetic and are usually catego- bioavailability of nutrients in foods. Examples of rized by their function (Table 14.11). For example, naturally occurring toxins are listed in Table 14.10. preservatives prevent the growth of bacteria, gelling Some species of mushroom also contain toxic com- agents maintain the structure of some foods during pounds, for instance agaritine. Some cereal-based storage, and emulsifi ers maintain the stability of fat diets have restricted bioavailability of nutrients as a structures. Without additives it would not be possible result of the presence of antinutritional factors such to manufacture many of the foods available today, as phytate and tannins or polyphenols. especially convenience foods and low-fat foods. Food processing methods have evolved that reduce Safety considerations involving additives have cen- human exposure to both natural toxins and antinu- tered on allergic reactions and food intolerances. Addi- tritional compounds. For instance, cassava is a staple tives have also been blamed for inducing hyperactivity food of over 500 million people worldwide. Certain in children. Studies have been conducted into allergies bitter varieties of cassava contain high levels of lin- and they often fi nd that the actual prevalence rate is amarin, a cyanogenic glycoside. The consumption of much lower than the perceived prevalence rate. these varieties has been associated with health defects At the international level, additives are controlled such as goiter and paralysis of the legs. Traditional by means of safety evaluation and the development processing of cassava in Africa that involves grating, of a positive list. To ensure transparency and choice, soaking roots in water, and lactic acid fermentation all additives that are used in prepackaged food should completely removes the cyanide. be labeled with their function and their name or Food Safety 347 puffer fi sh (called fugu in puffer fi Japan) are eaten as a c Ocean Indo-Pacifi delicacy, region in the island states of the c South Pacifi Norway, the Netherlands, Norway, Scotland, and Japan Zealand, and Canada Ireland, suspected cases in Most frequent in Japan, where USA, Canada, and Europe Worldwide Tropical reef waters, particularly Tropical Occurrence Worldwide Florida coast and Gulf of Mexico Europe, Japan, Chile, New culty in shellfi sh poisoning as the actions of both toxins are shellfi Mild poisoning results in tingling and numbness similar. ngers, and in severe cases of the lips, tongue, and fi death by asphyxiation due to respiratory paralysis breathing facial fl ushing and sweating, burning–peppery taste facial fl sensations around the mouth and throat, dizziness, nausea, and headache. A facial rash can develop as well as mild diarrhea and abdominal cramps. Severe cases may blur vision and cause respiratory stress and swelling of the tongue. Symptoms usually last for approximately 4–6 h and rarely exceed 1–2 days nausea); neurological (paresthesia of the extremities, circumoral paresthesia, temperature reversal, ataxia, arthralgia, malign headache, severe pruritus, vertigo, and stiffness, convulsions, delirium, hallucinations, photophobia, transient blindness, salivation, perspiration, watery eyes, metallic taste in mouth, blurred vision, hiccups, exacerbation of acne, dysuria); cardiovascular (dyspnea, bradycardia, hypotension, tachycardia) burning of the lips, staggering, drowsiness, and in severe cases respiratory paralysis and throat Initial symptoms are that of an allergic response with Symptoms Abdominal pain, nausea, vomiting, and severe diarrhea histamine. and associated toxins Tetrodotoxinsh poisoning are similar to paralytic Symptoms of puffer fi Scombroid or Azaspiracid Nausea, vomiting, severe diarrhea, and stomach cramps Domoic acid cramps, diarrhea, disorientation, and diffi Vomiting, Ciguatera Gastrointestinal (diarrhea, vomiting, abdominal pain, Associated toxin Saxitoxins Neurotoxic; symptoms include numbness, tingling and Okadaic acid Brevitoxins Nausea, diarrhea, tingling and burning of the lips, tongue,

Gonyaulax , viscera of

Dinophysis

Gambierdiscus

Nitzchia pungens ) and their toxins. Common species fed on marine dinofl agellates ( fed on marine dinofl Tetraodontidea family, particularly those family, Tetraodontidea c species caught in waters of the Indo-Pacifi Ocean regions like marine fi sh species that have not been like marine fi chilled immediately after capture. Commonly involved are members of the e.g., tuna and mackerel, Scombridae family, and a few nonscombrid relatives, e.g., sh, and amberjack sh, dolphin fi bluefi fed on toxigenic dinofl agellates ( fed on toxigenic dinofl marine diatom feeding on dinofl agellates ( feeding on dinofl fi s h crabs and anchovies are amberjack, barracuda, moray eel, Spanish mackerel, and groupers, trevally, snapper spp.) spp.) Gymnodinidum breve toxicus Toxic mussels, clams, and scallops that have Toxic Consumption of fi sh species belonging to the Consumption of fi Consumption of scombroid and scombroid- Mussels, oysters, clams or scallops that have Mussels and clams that have recently fed on a Flesh of toxic reef fi sh from tropical areas Flesh of toxic reef fi Mussels, oysters, clams, scallops, and razor Shellfi sh that have fed on the dinofl agellate sh that have fed on the dinofl Shellfi Implicated foods poisoning poisoning histamine poisoning poisoning poisoning poisoning poisoning poisoning Diarrhetic shellfi sh Diarrhetic shellfi Puffer fi sh fi Puffer Scombroid or Paralytic shellfi sh shellfi Paralytic Amnesic shellfi sh Amnesic shellfi Ciguatera fi sh Ciguatera fi Azaspiracid shellfi sh Azaspiracid shellfi Neurotoxic shellfi sh Neurotoxic shellfi Types of fi sh poisoning 14.9 Table of fi Types Poisoning 348 Introduction to Human Nutrition

Table 14.10 Naturally occurring plant toxins be controlled through the development and imple- mentation of targeted food safety control programs. Compound Food species Common name This can be achieved either through legislation, or the Glycosides: use of standards or codes of practice. At the interna- Linamarin Manihot escaleatum Cassava tional level, the WHO and the FAO have worked since Dhurrin Sorghum spp. Sorghum the 1960s on developing food standards that aim to Prunasin Prunus spp. Cherries protect the health of consumers and facilitate inter- Glycoalkaloids: national trade of foods and animal feeding stuffs. Solanin Solanium tuberosiem Potatoes This work is carried out by the Codex Alimentarius Pyrrolizidine alkaloids: Commission (CAC), an intergovernmental body Acetyllycopsaimine Symphytum spp. Comfrey managed by the FAO and WHO. Food safety stan- Senecionine Senccio jacobata Ragwort dards developed by the CAC serve as the baseline Glucosinolates: for harmonization of global food standards, codes Sinigrin Brassica spp. Cabbage of practice, guidelines, and recommendations. Broccoli Brussels Harmonization of standards and recognition that dif- sprouts ferent national food safety controls are equivalent are enshrined in the international agreements of the World Trade Organization (WTO). Table 14.11 Categories of food additives according to function The purpose of food safety legislation is to protect Acid Flour treatment agent consumers’ health and interests by providing controls Acidity regulatora Gelling agent throughout the food chain. A recent overhaul of EU c Anticaking agent Glazing agent food safety legislation now places the primary respon- Antifoaming agent Humectant sibility for food safety with the food business opera- Antioxidant Modifi ed starch Bulking agent Preservative tor. It also recognizes that food safety must start at Color Propellant gas and (i.e., the farmer) and places packaging gas increased importance on the safety of animal feed. Emulsifi er This concept of food safety control from “farm-to- Emulsifying salt Raising agent Enzymeb Sequestrantd fork” or “gate-to-plate” has been endorsed interna- Firming agent Stabilizere tionally, but implemented differently in different Flavoring Sweetener countries. Flavor enhancer Thickener The traditional “inspection and detection” aspects of food safety control are now being replaced with a These can act as two-way acidity regulators. b Only those used as additives. strategies for prevention of hazards occurring in the c These substances include lubricants intended for the fi nal consumer. fi rst place. In many countries, food businesses are d Inclusion of these terms in this list is without prejudice to any future now legally obliged to adopt the principles of HACCP decision or mention thereof in the labeling of foodstuffs. (hazard analysis and critical control point) in order e This category also comprises foam stabilizers. to predict what biological, chemical or physical Source: European Union Directive 89/107/EEC, http://eur-lex.europa. eu/. © European Communities. hazards are likely to occur in their process, so that they can implement control measures to prevent approval number (E number). However, there are them happening. some exemptions specifi cally applying to additives that are in a foodstuff as a result of carry-over from 14.9 Perspectives on the future an ingredient. As our society changes, so do the bacteria involved in 14.8 Food safety control programs food-borne disease. Changes in food production systems and the globalization of the food supply, as Each nation has a responsibility to ensure that its citi- well as changes in the food we are eating, and where zens enjoy safe and wholesome food. Governments this food is prepared, expose us to an ever-changing aim to identify major food safety issues that can then spectrum of contamination. The global nature of our Food Safety 349 food supply poses greater risks to consumer health References from the mass production and distribution of foods and increased risk for food contamination. New food Abdussalam M, Käferstein FK, Mott K. Food safety measures for development has led to changing vectors for the the control of food borne trematode infections. Food Control 1995; 6: 71–79. spread of disease. Inappropriate use of antibiotics Cadbury Schweppes. Press Release 2007. Available at: http://www. in animal husbandry can lead to the emergence of cadbury.com/media/press/Pages/2006fullyearresults.aspx antibiotic-resistant food-borne pathogens such as CDC. Surveillance for Foodborne Disease Outbreaks – United States, 1993–1997. Morbidity and Mortality Weekly Report 2000; S. typhimurium DT 104. 49: (SS01)1–51. Food safety and nutrition are inextricably linked Dewaal CS, Hicks G, Barlow K, Aldterton L, Vegosen L. Foods because food-borne infections are one of the most associated with food-borne illness outbreaks from 1990 through 2003. Food Protection Trends 2006; 26: 466–473. important underlying factors of malnutrition, espe- Doyle MP, Beuchat LR, Montville TJ, eds. : cially in poorer countries. Repeated episodes of food- fundamentals and frontiers, 2nd ed. American Society for borne infections can, over a period of time, lead to Microbiology, Washington DC, 2001. EFSA. Opinion of the Scientifi c Committee on a request from EFSA malnutrition, with serious health consequences. A related to a harmonised approach for risk assessment of sub- safe food supply is essential for proper nutrition, basic stances which are both genotoxic and carcinogenic. EFSA Journal health and well-being. 2005; 282: 1–31. EFSA. The community summary report on trends and sources of Maintaining a safe food supply is not diffi cult; zoonoses, zoonotic agents, antimicrobial resistance and food- however, it requires attention to detail at all stages of borne outbreaks in the European Union in 2006. Available at: the food chain from agricultural inputs through http://www.efsa.europa.eu/cs/BlobServer/DocumentSet/Zoon_ report_2006_en,0.pdf?ssbinary=true farms, processing, the distribution network to retail- European Commission. Opinion of the Scientifi c Committee on ers and catering outlets to consumers. There can be Food on 3-monochloro-propane-1,2-diol (3-MCPD) updating no gaps in the continuum from farm to fork if con- the SCF opinion of 1994 adopted on 30 May 2001. Available at: http://ec.europa.eu/food/fs/sc/scf/out91_en.pdf sumer protection is to be optimum. To ensure con- FAO/WHO. Evaluation of certain food additives and contaminants. sumer protection, food standards have to be based on 22nd Report of the Joint FAO/WHO Expert Committee on Food sound science and the principles of risk analysis. At Additives. WHO Technical Report Series No. 631. WHO, Geneva, 1978: 14–15. the national level, food safety controls must be well Frenzen PD. Deaths due to unknown foodborne agents. Emerging coordinated and based on proportionate food legisla- Infectious Diseases 2004; 10: 1536–1543. tion. The food industry must also recognize its Frenzen PD, Drake A, Angulo FJ, the Emerging Infections Program Foodnet Working Group. Economic cost of illness due to primary responsibility for producing safe food and Escherichia coli O157 infections in the United States. Journal of for ensuring that foods placed on the market meet the Food Protection 2005; 68: 2623–2630. highest standards of food safety and hygiene. A mul- Käferstein, FK. Actions to reverse the upward curve of foodborne illness. Food Control 2003; 14: 101–109. tisectoral effort on the part of regulatory authorities, McCabe-Sellers BJ, Beattie SE. Food safety: emerging trends in food industries. and consumers alike is required to foodborne illness surveillance and prevention. Journal of the prevent food-borne diseases. American Dietetic Association 2004; 104: 1708–1717. Mead PS, Slutsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C, Griffi n PM, Tauxe RV. Food-related illness and death in the United Sates. Emerging Infectious Diseases 1999; 5: 607– Acknowledgment 625. Smith JL. Foodborne infections during pregnancy. Journal of Food Protection 1999; 62: 818–829. This chapter has been revised and updated by Alan WHO. Food safety and foodborne illness. Fact sheet No. Reilly, Christina Tlustos, Judith O’Connor, and Lisa 237. Available at: http://www.who.int/mediacentre/factsheets/ O’Connor based on the original chapter by Alan fs237/en/ WHO/FAO. Joint FAO/WHO expert committee on food additives Reilly, Christina Tlustos, Wayne Anderson, Lisa sixty-fourth meeting, Rome, 8-–17 February 2005. Available at: O’Connor, Barbara Foley, and Patrick Wall. ftp://ftp.fao.org/es/esn/jecfa/jecfa64_call.pdf 15 Food and Nutrition-Related Diseases: The Global Challenge

Hester H Vorster and Michael J Gibney

Key messages

• This chapter deals with the current situation, trends and types undernutrition and related defi ciency and infectious diseases of nutrition-related diseases in developed and developing (including human immunodefi ciency virus/acquired immunodefi - countries. ciency syndrome HIV/AIDS), and the emergence of NCDs as • It shows that in developed countries excessive intakes of macro- a result of the nutrition transition. It explains the vicious cycle nutrients (overnutrition) and suboptimal intakes of micronutrients of poverty and undernutrition and how this is related to under- (hidden hunger), mainly because of low fruit and vegetable con- development and increased risk of NCDs in the developing sumption, lead to obesity and related noncommunicable diseases world. (NCDs). • Current global challenges for food and nutrition interventions on • The chapter also shows that developing countries are suffering different levels are highlighted. from a double burden of disease because of the persistence of

15.1 Introduction and promotion have responded to this preference by making high energy-dense foods available at increas- The relationship between nutrition and health was ingly affordable prices. This has led to changes in food summarized in Figure 1.2, illustrating that the nutri- consumption patterns which unfortunately coincided tional quality and quantity of foods eaten, and there- with more sedentary, less active lifestyles. The resul- fore nutritional status, are major modifi able factors tant overnutrition of especially macronutrients is the in promoting health and well-being, in preventing major cause of obesity and also, together with obesity, disease, and in treating some diseases. It is now a risk factor for many of the noncommunicable accepted that our nutritional status infl uences disease (NCDs) such as type 2 diabetes, coronary our health and risk of both infectious and non- heart disease, stroke, hypertension, dental disease, communicable diseases. osteoporosis, and some forms of cancer. But it is also accepted that billions of people in both These changes from traditional low-energy dense, developed and developing countries suffer from one high-fi ber diets to the dietary pattern described above or more forms of malnutrition, contributing to the are collectively known as the nutrition transition (NT). global burden of disease. Mankind has an inherent The NT has proceeded gradually over centuries in the preference for palatable, sugary, salty, fatty and smooth developed world and accelerated during the industrial (fi nely textured, refi ned) foods. These foods are revolution with a resultant gradual and then acceler- mostly energy-dense and low in micronutrients. Food ated emergence of the NCDs. However, globalization production, processing, manufacturing, marketing characterized by urbanization, acculturation, global

© 2009 HH Vorster and MJ Gibney. Food and Nutrition-Related Diseases 351 trade, and information exchange has led to a very rapid disease, stroke, diabetes mellitus (type 2), osteoporo- NT in developing countries. The consequence is that, sis, liver cirrhosis, dental caries, and nutrition-induced different from developed countries, obesity and the cancers of the breast, colon, and stomach. They NCDs emerged before the problems of undernutrition develop over time in genetically susceptible individu- and specifi c nutritional defi ciencies have been solved. als because of exposure to interrelated societal, Developing countries now suffer from a double burden behavioral, and biological risk factors. Together with of nutrition-related diseases because of the coexis- tobacco use, alcohol abuse, and physical inactivity, tence of under- and overnutrition. This dual burden is an unhealthy or inappropriate diet is an important further exacerbated by the HIV/AIDS and TB pan- modifi able risk factor for NCDs. Diet, therefore, plays demics in these countries. a major role in prevention and treatment of NCDs. The purpose of this chapter is to describe the major NCDs are sometimes called “chronic diseases,” but nutrition-related diseases in the developed and devel- some infectious diseases such as HIV/AIDS and oping world, to show the interrelationships between tuberculosis are also chronic. They have also been the causes and consequences of under- and over- called “diseases of affl uence,” which is a misnomer nutrition, and to identify the global challenges in because in developed, affl uent countries, they are addressing the heavy burden of malnutrition that more common in lower socioeconomic groups. Some contribute to underdevelopment, disability, and pre- scientists have a problem with the term “noncom- mature death. municable” because lifestyles, including diets, are transferable between populations. The term “non- 15.2 Nutrition-related diseases in communicable” should therefore be seen as no developed countries transfer of an infectious agent from one organism to another. Because of its fi rst emergence in “Westernized” The current situation societies and associations with Western lifestyles, it is often called “Western” diseases, also a misnomer. It is Economic development, education, food security, and becoming more prevalent in developing countries in access to health care and immunization programs other parts of the world. Another misconception is in developed countries have resulted in dramatic that it is a group of diseases affecting only older decreases in undernutrition-related diseases. Un- people. The risk factors for NCDs accumulate fortunately, many of these factors have also led to throughout the life course – from infancy to adult- unhealthy behaviors, inappropriate diets, and lack of hood, and manifest after decades of exposure. The physical activity, which has exacerbated the develop- increase in childhood obesity is especially of concern ment of chronic diseases, also known as noncommu- because it has long-term implications for NCDs in the nicable diseases (NCDs). These NCDs are now the developed world. main contributors to the health burden in developed countries (these are countries with established market Risk factors for NCDs economies). Table 15.1 lists the risk factors for NCDs. The factors In 2002, 28.2 million global deaths (58.6%) were are interrelated and form a chain of events starting from NCDs. In the same year the predicted mortality with societal factors such as socioeconomic status and for 2020 was 49.6 million (72.6% of all deaths). This environments that infl uence behavior, leading to the is an increase from 448 to 548 deaths per 100,000, development of biological risk factors that cause the despite an overall downward trend in mortality rates. NCDs. The biological risk factors often cluster Although the burden will fall increasingly on develop- together. For example, obesity (abnormal body com- ing countries (see 15.3) NCDs remain the major cause position) is associated with insulin resistance, hyper- of death in developed countries. lipidemia, and hypertension, which all contribute to the development of both cardiovascular disease and Defi nition, terminology and characteristics diabetes. Cardiovascular disease is furthermore one of The NCDs that are related to diet and nutrient intakes the complications of untreated diabetes. The mecha- are obesity, hypertension, atherosclerosis, ischemic nisms through which these risk factors contribute to heart disease, myocardial infarction, cerebrovascular the development of NCDs are discussed in detail in 352 Introduction to Human Nutrition

Table 15.1 Risk factors for nutition-related noncommunicable diseases (NCDS)

Societal Behavioral Biological NCDs

Socioeconomic status Smoking Tobacco addiction Lung disease Cultural habits Alcohol abuse Alcohol addiction Cardiovascular disease Environmental factors Lack of physical activity Dyslipidemia Atherosclerosis Inappropriate diets: Hyperlipidemia Cerebrovascular disease inadequate Insulin resistance Stroke fi ber Hypertension Ischemic heart disease micronutrients Obesity (body composition) Myocardial infarction excess Diabetes total fat Osteoporosis saturated fat Dental caries trans fat Cirrhosis cholesterol Diet-induced cancers salt (NaCl) energy appropriate chapters and sections of this series of Box 15.1 The WHO population nutrient intake goals for textbooks. prevention of death and disability from NCDsa

The role of nutrition Dietary factor Recommended goal The evidence that diets and specifi c nutrient defi cien- (food or nutrient) (range) cies and excesses infl uence the development of NCDs Total fat 15 –30% of total energy and may therefore be used in prevention and treatment Saturated fatty acids <10% of total energy is solid. It comes from extensive research which collec- Polyunsaturated fatty acids (PUFAs) 6–10% of total energy n-6 PUFAs 5–8% of total energy tively gave convincing evidence of the relationships n-3 PUFAs 1–2% of total energy between nutrition and NCDs: fi rst, from ecological Trans fatty acids <1% of total energy studies which compared different populations, the Monounsaturated fatty acids (MUFAs) By differenceb effects of migration of populations, food availability Total carbohydrate 55–75% of total energyc d during economic development, and differences in Free sugars <10% of total energy Protein 10–15% of total energy dietary and nutrient intakes. Second, numerous epide- Cholesterol <300 mg per day miological studies have established the associations Sodium chloride (sodium) <5 g per day between diet and biological risk factors of NCDs. Third, (<2 g per day) interventions with specifi c nutrients and foods in Fruits and vegetables ≥400 g per day placebo-controlled trials using both healthy and dis- Total dietary fi ber >25 g per day Non-starch polysaccharides >20 g per day eased subjects confi rmed the relationships seen in epi- demiological studies. And last, molecular and genetic a WHO Technical Support Series No. 916. research has elucidated many mechanisms through b MUFAs are calculated as total fat minus saturated plus polyun- which diet and nutrients affect genetic mutation and saturated plus trans fatty acids). c expression, adding to our knowledge of how nutrition Energy from carbohydrate is the percentage energy available after taking into account that consumed as fat and protein. infl uences NCD development. This body of knowledge d Free sugars refers to all monosaccharides and disaccharides added has led to several sets of international dietary recom- to foods by the manufacturer, cook, or consumer, plus sugars natu- mendations and guidelines to reduce the burden of rally present in honey, syrups, and fruit juices. It does not include nutrition-related NCDs. An example of one such set of sugars present in milk, fruit and vegetables. guidelines from the World Health Organization (WHO) is shown in Box 15.1. These generic recommendations could be used as the basis for the development of country-specifi c strategies and food-based guidelines for dietary prevention of NCDs. Food and Nutrition-Related Diseases 353

Prevention of NCDs in developed countries and have diffi culties with body image and mobility. The complex chain of events where behavioral and Overweight and obese children often become over- lifestyle factors infl uence the development of the bio- weight or obese adults and carry the long-term risk logical risk factors for NCDs, emphasizes the need for of premature morbidity and mortality from NCDs. a multisectorial approach in which all factors in the Children in the developed world are exposed to a food chain are targeted throughout the life course. In addi- environment in which high energy-dense and micro- tion to the medical treatment of some biological risk nutrient-poor foods, beverages, and snacks are avail- factors (such as pharmacological treatment of hyper- able, affordable, and aggressively marketed. This cholesterolemia) and of the NCD itself (such as blood illustrates that to address the problem of childhood glucose control in diabetes) there is convincing obesity, active and responsible partnerships and evidence that primary prevention is possible, cost- common agendas should be formed between all effective, affordable, and sustainable. In the developed stakeholders (for example between governments, world, early screening and diagnosis, and access to NGOs and the food industry). There are indications health care make primary prevention more feasible that dialogue with the food industry is not suffi cient, than in many developing countries. However, over- and that many countries are now considering or coming the barriers to increase physical activity and already implementing legislation to create a more changing dietary behavior towards more prudent, healthy food environment for children. The problems low-fat, high-fi ber diets may be more diffi cult. The of childhood overweight and obesity and consequent strategies and programs to prevent NCDs would be increases in NCDs are not only seen in developed similar in developed and developing countries, countries. They are emerging in developing countries although the context and specifi c focus of different and in some the total number of children affected interventions may vary. Because the future burden of exceeds those in developed countries. Timely inter- NCDs will be determined by the accumulation of ventions are needed to prevent the escalation experi- risks over a lifetime, the life course approach is rec- enced in developed countries. ommended. This will include optimizing the nutri- tional status of pregnant women (see Box 15.3), 15.3 Nutrition-related diseases in breastfeeding of infants, ensuring optimal nutrition developing countries status and growth of children, preventing childhood obesity and promoting “prudent” diets for adoles- The poverty–malnutrition cycle cents, adults, and older people. Addressing childhood Malnutrition in developing countries affects individ- obesity in developed countries is one of the biggest uals throughout the life course: from birth to infancy nutritional challenges these countries is facing today. and childhood, through adolescence into adulthood, Increases in the prevalence of childhood obesity have and into old age. Malnutrition affects, therefore, criti- been documented for most developed countries. cal periods of growth and mental development, In the USA, the National Health and Nutrition maturation, active reproductive as well as economical Examination Surveys (NHANES) showed substantial productive phases. increases over the last two decades in overweight and The health of populations in developing countries obese children aged 2–19 years. More than 15% of is largely determined by their environment. “Environ- American children are currently considered obese. mental” factors include social and economic con- The International Obesity Task Force estimates that ditions depending on and infl uencing availability at least 22 million of the world’s children under 5 and distribution of resources, agricultural and food years of age are overweight or obese. Overweight and systems, availability and access to nutritious food and obesity have dire consequences in children. These safe drinking water, implementation of immuniza- children already display many of the other biological tion programs, exposure to unhygienic surroundings risk factors of NCDs. There are also immediate health and toxins, women’s status and education, as well as consequences such as risks to develop gallstones, hep- the “political” milieu including dictatorships, confl ict, atitis, sleep apnea, and others. Moreover, these chil- and war, which often determine the availability dren have a lack of self-esteem, are often stigmatized of health services. There is a close, interrelated 354 Introduction to Human Nutrition association between undernutrition and poverty in disease. In 2000, more than 150 million preschool developing countries. children in the developing world were underweight, Figure 15.1 illustrates this relationship, also showing while approximately 200 million were stunted. some of the mechanisms responsible for perpetuating Figure 15.1 also shows that these physically and the relationship over generations. mentally underdeveloped children eventually develop Approximately 243 million adults in developing into adults with “decreased human capital” and countries are severely undernourished, with a body decreased competence. These adults are often not able mass index less than 17 kg/m2. This means that high to create enabling environments for themselves or proportions of especially Asian and African preg- their children to escape poverty and undernutrition nant women are undernourished. Intrauterine (fetal) in the next generation. But moreover, these “under- growth retardation is common in these women, developed” adults are at increased risk of obesity and leading to low birthweight babies (weight at full term other NCDs because of early programming (possibly less than 2500 g). Almost a quarter of newborns in the through epigenetic or DNA methylation changes) in developing world (30 million of the 126 million babies the undernourished fetus. It is especially when these born each year) have low birth weights compared with adults are exposed to low micronutrient quality and only 2% in the developed world. These babies, espe- high energy-dense diets that they rapidly become cially when exposed to inappropriate breastfeeding overweight and obese. This phenomenon explains to and weaning practices, leading to further nutritional a certain extent the coexistence of under- and over- insults, have growth impairment and mental under- nutrition in the same household with undernour- development. In addition, because of undernutrition, ished, wasted, and stunted children being cared for by their immune systems are compromised. The result is an overweight or obese mother or care-giver. stunted children that cannot benefi t optimally from education and with an increased risk of infectious Obesity and noncommunicable diseases in developing countries Obesity and other NCDs are increasingly becoming major public health problems in the developing UNDERNUTRITION world. The WHO estimates that almost 80% of all (undernourished pregnant mothers; Fetal growth restriction) deaths worldwide that are attributable to NCDs are already occurring in developing countries. A disturb- POVERTY Low birthweight babies Undernourished infants ing observation is that they often occur at younger Lack of care Unhygienic environments ages than in the developed world. Obesity and other Food insecurity NCDs have similar biological risk factors in devel- oped and developing countries (and will not be dis- •Growth impairment (underweight; stunting) cussed in detail here). However, the context in which Adults with decreased •Mental underdevelopment human capital and competence they develop may differ, being linked with fetal and •Compromised immune system infant undernutrition. Also, underdevelopment and a lack of resources in developing countries limit the availability of diagnostic and therapeutic care of people suffering from NCDs, leading to increased Increased risk of obesity and other NCDs morbidity and mortality. Inappropriate breastfeeding The other two groups of nutrition-related diseases and weaning in developing countries are nutrient defi ciency dis- Increased risk of infectious Micronutrient eases and infectious diseases, which will now be briefl y disease deficiencies (Fe, Zn, Vit A) discussed.

Major nutrient defi ciency diseases in Increased morbidity and mortality developing countries

Figure 15.1 The intergenerational vicious cycle between undernutri- It is estimated that nearly 30% of humanity suffer tion and poverty. from one or more forms of malnutrition. About 60% Food and Nutrition-Related Diseases 355 of the approximately 11 million deaths each year of childhood deaths. In some developing countries, children aged under 5 years in the developing world children die of AIDS-related diseases. HIV/AIDS is an are associated with malnutrition. In addition to the infectious disease that has pandemic proportions in undernutrition related to poverty, hunger, and food developing countries. It will be discussed in more insecurity, leading to stunted physical and mental detail here to illustrate the complex role of nutrition development, specifi c nutrient defi ciencies are causes in this tragic situation. of specifi c diseases (as discussed in Chapters 4, 5, 8, and 9 of this textbook). The major nutrient defi ciency diseases prevalent in developing countries are briefl y 15.4 HIV/AIDS summarized in Box 15.2, to illustrate the scope of the problem and to identify the nutrition challenges in Introduction the developing world for the twenty-fi rst century. Infection with HIV and the consequent development of AIDS is a global pandemic already responsible for Nutrition-related infectious disease in more than half of total deaths in some developing developing countries countries. It is estimated that since the early 1980s Nutrition is a major determinant of the human body’s when the syndrome was described and the virus iden- defense against infectious diseases. Optimal nutrition tifi ed, this infectious disease has already killed more is necessary for the integrity of the physical barriers than 25 million people, including at least half a million (skin, epithelium) against pathogens. Specifi c nutri- children. A third of these deaths occurred in sub- ents furthermore play important roles in defi ning Saharan Africa, where more than half of the 33.4–46 acquired immune function (both humoral and cell- million people currently infected with HIV live. The mediated responses) and to infl uence, modulate, or pandemic has a devastating and tragic social, eco- mediate infl ammatory processes, the virulence of the nomic and demographic impact on previous devel- infectious agent, and the response of cells and tissues opment and health gains in developing countries. It to hypoxic and toxic damage. affects mostly young, sexually active adults in their The immune system and the infl uence of malnutri- reproductive years as well as babies born from infected tion on its functions are discussed in detail in the mothers. To understand the nutritional challenges of clinical nutrition textbook of the series. Given the HIV/AIDS it is necessary to understand how the virus high prevalence of malnutrition (undernutrition) in is transmitted and to follow the clinical course of the developing countries, it is not surprising that infec- infection. The virus characteristics, its binding to cell tious diseases are still dominating mortality statistics surface receptors, its entry into cells of the immune in these countries. In children under 5 years of age system, its replication and transcription, as well as its these are diarrhea and common childhood illnesses genetic variability, and different classes of the virus in which malnutrition could lead to premature have been intensively researched and described,

Box 15.2 Nutrient defi ciency diseases in developing countries: prevalence (scope)

Nutrient Consequence: disease Estimated: 1995–2006

Iron Anemia; poor brain development in infancy Maternal anemia pandemic: more than 80% in some countries; globally, more than 2000 million people Protein, energy Kwashiorkor, marasmus marasmic kwashiorkor, wasting Millions of children are at risk Vitamin A Blindness; increased mortality from infectious diseases 140–250 million children suffer from subclinical defi ciency (children under 5 years especially vulnerable) Iodine Goiter, cretinism (infants) with severe brain damage and In 1999, 700 million people in developing countries; mental retardation remarkable progress made with universal salt iodization Zinc Its role in stunting and life-threatening childhood illnesses Thought to be common in children and during pregnancy is only now becoming clear 356 Introduction to Human Nutrition forming the basis for the development of antiretrovi- in developing countries may increase vulnerability to ral drugs to treat HIV/AIDS. More about this can be infection: the hopelessness and despair of poverty found in the clinical nutrition textbook of this series could lead to alcohol abuse, violence, rape, and irre- or at http://en.wikipedia.org/wiki/HIV. sponsible sexual behaviors, increasing exposure to the virus. In addition, malnutrition could compromise Transmission of HIV the integrity of the immune system, increasing vul- Because there is still no vaccine against HIV and no nerability to infection. Breaking this cycle by appro- cure available, the emphasis is on prevention of trans- priate public health nutrition interventions in poverty mission of the virus. It is transmitted from person to alleviation programs may indirectly also impact on person via certain body fl uids: blood (and blood HIV transmission. products), semen, pre-seminal fl uid, vaginal secre- tions, and breast milk. ● Stage 1: Incubation period The majority of HIV infections are acquired There are no symptoms during this stage and its through unprotected sexual contact when sexual duration is usually 2–4 weeks. secretions of one partner come into contact with ● Stage 2: Acute infection (seroconversion) genital, oral, or rectal mucous membranes of another. There is rapid viral replication during this stage. It The estimated infection risk per 10 000 exposures may last from a week to several months with a mean (without a condom) to an infected source varies from duration of 28 days. The symptoms in this stage 0.5 to 50, depending on the type of exposure. include fever, lymphadenopathy, pharyngitis, rash, The blood transmission route is responsible for myalgia, malaise, headache, and mouth and esoph- infections in intravenous drug users when they share ageal sores. needles with contaminated persons. Although blood ● Stage 3: Asymptomatic or latency stage and blood products are these days mostly checked for This stage may last from a few weeks up to 10 or 20 HIV, unhygienic practices in some developing coun- years, depending on the nutritional status and drug tries, needle prick injuries of nurses and doctors, as treatment of the individual. It is characterized by well as procedures such as tattoos, piercings, and none or only a few symptoms, which may include scarifi cation rituals pose some risk for infection. subclinical weight loss, vitamin B12 defi ciency, Transmission of the virus from an infected mother changes in blood lipids and liver enzymes, and an to her child can occur in utero during pregnancy, increased susceptibility to pathogens in food and during childbirth (intrapartum), or during breast- water. feeding. The transmission rate between untreated ● Stage 4: Symptomatic HIV infection infected mothers and children is approximately 25%. CD4+ cell counts (the immune cells containing the

This risk can be reduced to 1% with combination CD4 receptor, which binds the virus and which is antiretroviral treatment of the mother and cesarean destroyed during viral replication) have decreased section. The overall risk of a breastfeeding mother to from normal values of 1200, to between 200 child is between 20% and 45%. Recent studies have and 500 cells/µl. Wasting is a characteristic symptom shown that this risk can be reduced three- to fourfold and is defi ned as an involuntary loss of more by exclusive breastfeeding for up to 6 months. Exclu- than 10% of baseline body weight. Other symptoms sive breastfeeding for 6 months is therefore the present include loss of appetite, white plaques in the recommendation from the WHO for infected mothers mouth, skin lesions, fever, night sweats, TB, in developing countries “unless replacement feeding shingles, and other infections. Nutrition interven- is acceptable, feasible, affordable, sustainable and safe tions may help to preserve lean body mass, for them and their infants before that time.” “strengthen” the immune system and slow progres- sion to stage 5. The clinical course of HIV infection: ● Stage 5: AIDS progression to AIDS The CD4+ counts are now below 200 cells/µL. The The different stages of HIV infection dictate different immunosuppression is severe and leads to many types of nutritional intervention. Even before infec- possible opportunistic or secondary infections with tion, the vicious cycle of undernutrition and poverty fungi, protozoa, bacteria and/or other viruses. Food and Nutrition-Related Diseases 357

Malignant diseases and dementia may develop. This Box 15.3 Nutritional recommendations for HIV/AIDS is the fi nal stage, and if not treated by antiretroviral drugs and specifi c drugs for the secondary infec- 1 Nutrition recommendations should do no harm tions it invariably leads to death. 2 Optimum nutrition at population level is necessary as part of a set of general measures to reduce the spread of HIV and TB 3 The focus should be on diversifi ed diets including available, Nutrition and HIV/AIDS affordable and traditional foods. However, fortifi ed foods as The role of nutrition in HIV/AIDS is complex. As well as macro- and micronutrient supplements at safe levels (not more than twice daily recommended level) may be helpful mentioned above, malnutrition could contribute to 4 Ready-to-use supplements are effective in increased vulnerability to infection in developing reversing poor nutritional status found in severely affected indi- countries. The virus probably increases nutritional viduals needs, while its effects on the nervous and digestive 5 Because micronutrient defi ciencies may hasten disease progres- system lead to decreased appetite and intakes, sion and facilitate mother-to-child transmission of HIV, multi- vitamin, zinc and selenium supplementations are indicated, but impaired digestion, and malabsorption. The conse- vitamin A supplementation may increase mother-to-child trans- quent loss of lean body mass gave the infection its mission and zinc supplementation may be harmful in pregnant original African name of “thin disease.” There are women indications that improved nutrition may slow the 6 HIV-infected pregnant women, lactating mothers and their progression of HIV infection to AIDS. There is evi- babies need special advice and care to ensure best possible outcomes dence that nutritional support can help in the toler- 7 Established, well-described steps and protocols should be fol- ance of antiretroviral drugs and their side-effects and lowed in public health nutrition interventions and in the thera- assist in the management of some of the secondary peutical (medical) nutritional support of patients infections of AIDS. General Principles from ASSAf (2007). The optimal diet for people living with HIV/AIDS is not known. At least one study (the THUSA study in South Africa) indicated that asymptomatic infected subjects who regularly included animal-derived foods in their diets had better health outcomes than those Box 15.4 Types of nutrition interventions needed on plant-based diets and with high omega-6 polyun- saturated fat intakes. The nutritional recommenda- Stage Nutrition intervention needed tions for people living with HIV/AIDS are therefore Poverty/malnutrition Public Health Nutrition (PHN) pro- evidence informed and not totally evidence based at cycle grams as part of other programs to this stage. Global recommendations have recently alleviate poverty and improve been evaluated by the Academy of Science of South socioeconomic development Africa, and some of their conclusions are summarized Increased vulnerability Targeted nutrition (TN) programs in Box 15.3. for vulnerable groups including advice to pregnant mothers The transmission of the virus and the different stages in the progression of infection to AIDS indicate Decreased TN programs for PLWH, including that different levels of nutrition intervention and immunocompetence food assistance; food-based dietary guidelines plus advice on safe support are needed, as illustrated in Box 15.4. Specifi c foods and drinking water nutrient requirements during HIV infection are Transmission Individual therapeutic or medical discussed in the clinical nutrition textbook of this High risk behavior nutrition (IMN) programs: series. Asymptomatic HIV i Facility based IMN programs People living with HIV with defi ned algorithms (PLWH) ii Home-based care IMN programs 15.5 The global challenge to address AIDS with practical advice malnutrition Food and supplement assistance Background The nutritional problems and diseases facing mankind at the beginning of the twenty-fi rst century have been 358 Introduction to Human Nutrition identifi ed and briefl y discussed in this chapter. In to regularly access, at affordable prices, adequate developed countries these are mainly childhood and (enough, suffi cient), safe (uncontaminated), and adult obesity and the NCDs related to a combination nutritious food to prevent undernutrition and to of overnutrition, lack of activity, smoking, alcohol ensure optimal nutritional status for health, well- abuse, and stressful lifestyles. In developing countries being, a quality life, ability to actively and produc- the magnitude of undernutrition is staggering. tively work and play, and moreover to reach their Moreover, obesity and NCDs have emerged in these mental and physical development potential. This is countries and are increasingly becoming major causes often defi ned as being food and nutrient “secure.” of mortality. This double burden is further exacer- The above situation would be possible if all stake- bated by the HIV/AIDS pandemic. holders in the global community (UN agencies, gov- ernments, NGOs, food industries, academics, civil Dietary patterns responsible for society, and others) worked together in partnerships the problems to create a food and nutrition environment in which The dietary patterns and nutrient intakes responsible healthy food choices were available, acceptable, and and contributing to these problems have been inten- affordable and where consumers were educated, sively researched in epidemiological, clinical, and informed, and motivated to make the right choices. basic molecular studies. There is a huge body of sci- But we do not live in an ideal world, as the high entifi c evidence available to identify the immediate prevalences of nutrition problems indicate. So the defi ciencies and excesses in intakes, as well as all the questions that need to be answered are what should environmental factors associated with suboptimal be done and by whom to rectify the situation? dietary patterns that lead to the nutrition-related dis- Clearly, the time for individual, separate programs eases highlighted in this chapter. Broadly, these dietary to address undernutrition in one way and overnutri- problems can be summarized as: tion and NCDs in another is past. What is needed is a holistic, integrated approach that will promote and ● hunger and food insecurity in developing coun- make optimum nutrition possible. Several UN agen- tries, with infants, pregnant women, and older cies, separately or in combination have developed people being the most vulnerable “strategic directions” and described policy principles, ● “hidden hunger” or micronutrient defi ciencies in strategies to introduce this on different levels in dif- both developed and developing countries, espe- ferent settings, as well as actions to promote healthy cially of iron, vitamin A, zinc, iodine, and all dietary diets. The challenge is huge, for there are many bar- antioxidants riers to overcome: from war, to uncommitted political ● overconsumption of unfortifi ed and refi ned staple agendas, to “unhealthy” food preferences of individu- foods in “low-quality diets” als. The lessons learned from the failure of many ● availability and intake of too many high-fat, sugary, developing countries to be on-track in reaching the and refi ned convenience and fast foods, increasing Millennium Development Goals by 2015 plead for a total fat, saturated fat, trans fat, omega-6 fatty acid, new approach and global leadership. This could be sugars, and salt intake possible in partnerships in which there is recognition ● not enough fi sh and other sources of omega-3 fatty and respect for different agendas, but where partners acids in the diet are willing to develop a common nutrition agenda ● not enough vegetables and fruit and their products and agree on steps to reach common goals. But there in the diet are also many success stories. For example great ● not enough dietary fi ber-rich foods in the diet; strides have been made in the past few decades to ● too little dietary variety reduce child undernutrition in some developing ● over-reliance on dietary supplements in the devel- regions. A global database on child growth and mal- oped world. nutrition covers 95% of the world’s under-5 popula- tion (http://www.who.int/nutgrowthdb). Much is Suggestions to meet the challenge being done by several UN agencies and international In an ideal world, every human being would be able donors and NGOs to improve the global nutrition to exercise their right (often constitutionally defi ned) situation, assisting countries in nutrition surveillance Food and Nutrition-Related Diseases 359

Box 15.5 UN Agencies: Intersectorial nutrition policies and assistance of displaced people to educating consum- strategies to address the double burden of nutrition problems ers how to choose healthy diets. The development of throughout the life course food-based dietary guidelines in both the developed and developing world to assist people to choose an 1 Nutrition surveillance adequate but prudent diet for optimal nutrition is an 2 Combating micronutrient defi ciencies • Fortifi cation example of the latter. There is total agreement in the • Salt iodization body of literature on the nutrition challenges of the 3 Nutrition advocacy twenty-fi rst century that the focus should be on pre- 4 Developing national food-based dietary guidelines vention of nutrition-related diseases to minimize 5 Addressing nutrition and HIV/AIDS their serious economic and social consequences. 6 Addressing obesity and other diet-related diseases 7 Improving maternal, infant and young child nutrition cycle Improving nutrition of school-age children and adolescents Further reading (integrated school-based program, nutrition friendly schools, health promoting schools) ASSAf. HIV/AIDS, TB and Nutrition. Scientifi c inquiry into nutri- • Ensuring appropriate fetal development tional infl uences on human immunity with special reference to • Implementation of new growth reference HIV infection and active TB in South Africa. Pretoria, Academy • Improving infant and young child nutrition (breastfeeding, of Science of South Africa, 2007: 1–283 (www.assaf.org.za). complementary feeding, baby-friendly hospitals, global Murray CJ, Lopez AD, eds. The Global Burden of Disease. A strategy on infant and young child feeding). Comprehensive Assessment of Mortality and Disability from Diseases, Injuries, and Risk Factors in 1999 and Projected to 2020. Geneva, Harvard University Press and WHO, 1996: 1–989. Standing Committee on Nutrition (SCN). Working together to end child hunger and undernutrition. SCN News 2007; 34: 1–80. United Nations Administrative Committee on Coordination, Sub- and implementation of targeted programs. In Box Committee on Nutrition (ACC/SCN), in collaboration with the International Food Policy Research Institute (IFPRI). 4th Report 15.5 one such an example is given, namely the topics of the World Nutrition Situation: Nutrition Throughout the Life of the WHO’s integrated and intersectorial food poli- Cycle. Geneva, IFPR, 2000. cies and strategies to address the double burden of World Cancer Research Fund and American Institute for Cancer Research. Food, Nutrition, Physical Activity and the Prevention nutrition problems throughout the life course. of Cancer: A Global Perspective. Washington DC, AICR, 2007. There is agreement that policies and programs World Health Organization. Global Strategy on Diet, Physical should be implemented at all “levels” – from global Activity and Health. Diet, Nutrition and the Prevention of Chronic Diseases. WHO. Technical Report Series, no 916. Geneva, macrolevels to individual microlevels. These pro- WHO 2007: 1–149 (http://www.who.int/dietphysicalactivity/ grams will include a variety of actions – from food publications/trs916/en/).

Index

Note: page references in italics refer to information in impact on cholesterol 119–20 fi gures or tables. properties 98 appetite 33–4 abdominal girth 25 hormonal regulation 35 acetyl-CoA 105–7, 178–9 metabolic factors 35 acidity regulators 348 neurophysiological factors 34–5 acrylamide 344 arachidonic acid cascade 109–11, 110 additives 10, 346–7, 348 arginine 51, 52, 60 regulation 299–300 arginine—nitric oxide pathways 54 sodium-containing 200 arsenic 233–4, 236, 344 adipose tissue 15 ascorbic acid see vitamin C component lipids 104 asparagine 52 and dietary fat intake 106 aspartate 51, 52 endocrine control 103 aspartic acid 57 afl atoxins 345 assessment of nutritional status see nutritional status assessments agricultural residues 10 astroviruses 334 AIDS/HIV 355–7 atherogenic lipoprotein phenotype (ALP) 103 nutrition needs 61, 357 atherosclerosis alanine 51, 52 and homocysteine levels 174 albumin 214 lipid-mediated 100, 102–3 aluminium 233–4, 236 cholesterol-lowering drugs 91, 95 Alzheimer’s disease 215 nutritional modifi cations 103–4 amino acids postprandial lipemia 98 biochemical roles and functions 50, 51, 63 TAG regulation hypothesis 103–4 discovery and history 50–1 athletes, energy requirements 43 homeostasis and turnover 58–61, 78 ATP production 77 metabolic needs 58–64 avidin 177 metabolites and derivatives 53–4 biochemical structures 51–4 Bacillus cereus 328, 330 body requirements 66–7 bacterial contamination 10, 327 by species 62 emerging pathogens 326–7 estimation and determination 65–7 pathogen characteristics 328–33 in catabolic states 62, 72 toxins 345 infl uencing factors 58, 71–2 Bartter’s syndrome 196 meeting needs 67–9 basal metabolic rate 37–8 classifi cation 54–8 and energy intake ratios 269–70 sources 56–7 beriberi 153–4 digestibility 68–9 bias 260–3 ammonia 56–7, 61–2 bile acids 95 amylopectin 80 bioactives 291 amylose 80 bioavailability of foods, defi ned 286–7 anemia biochemical markers 130 iron-defi ciency 207 bioelectric impedance 25–6 megoblastic 175 biofl avonoids 186 pernicious 168, 169 biological markers 130 and folate 175 biotin 176–8 and scurvy 183 defi ciencies 177 and vitamin B12 168, 169 functions 177 animal meat blood clotting, and vitamin K 150–2 dietary fats 91–2 blood glucose, regulation mechanisms 77–9 drug residues 343 body composition 12–30 animal studies 311–14 levels anthropometry 22–3 atomic 13 antioxidants 291 cellular 14 intracellular 61 molecular 13 mode of action 222 relative relationships 14–15 apoproteins 97, 99 whole body 14 362 Index

body composition (cont.) Campylobacter 331 measurement techniques 15–27 cancer advantages and disadvantages 27 and folate 174–5 anthropometry 22–3 and selenium 219 bioelectric impedance 25–6 and vitamin B6 164 carcass analysis 15–16 and vitamin C 185, 219 creatinine excretion 26–7 carbohydrates 74–85 CT imaging 21–2 classes 74–5 densitometry 16–18 digestive breakdown and absorption 74–6 dilution methods 18–19 fermentation in the colon 82–3 dual-energy X-ray absorptiometry (DEXA) 18, 19– of short-chain fatty acids 83–4 21 food sources 76 infrared interactance 25 malabsorption problems 75–6 MRI imaging 22 metabolic utilization 77 multicompartment models 20–1 oral pH and dental caries 84–5 N-methyl-histidine excretion 26–7 types skinfold thickness 23–4 glycemic 76–9 total body electrical conductivity (TOBEC) 26 nonglycemic 79–84 ultrasound 25 resistant starch and dietary fi bre 81–2 in vivo neutron activation analysis 16 carbon recycling 107 weight/height indices 22–3 carcass analyis 15–16 prospects for future 29–30 cardiovascular disease 351–3 use and misuse of data 27–9 and homocysteine levels 174 body fat and lipids biochemical components 104 ALP fi ndings 102–3 deposition sites 104 cholesterol-lowering drugs 91, 95 measures 27–9 homeostasis and transport 95, 100–3, 102–3 comparative analysis 29 hormonal control 103 densitometry 16–18 nutritional modifi cations 103–4 skinfold thickness 23–4 postprandial regulation 98 see also adipose tissue triacylglycerol (TAG) hypothesis 103–4 body mass index (BMI) 22–3 carnitine 186 obesity measures 45 carotenoids 135–7, 291 body protein mass 58 antioxidant function 139 bone mass, and calcium 191–2 see also vitamin A boron 233–4, 236 case-control studies 318, 320 bowel cancer cassava 346 prevention cell membranes 90–1, 104 folate supplements 174–5 mechanisms, arachidonic acid cascade 109–11, 110 role of butyrate 84 ceruloplasmin 214 Boyle Gay-Lussac’s law 17–18 cestodes 338 brain chemicals affecting foods 340–6 and lipids 105, 114–15 acceptable intake levels 341–2 role of docosahexaenoate 114 classes 340 bread/fl our, fortifi ed 174, 193 chemotherapy, and folate supplements 172–3 BSE (bovine spongiform encephalopathy) 338–9 children bulking agents 348 energy requirements 42 burning foot syndrome 179–80 malnutrition 299 butyrate 83–4 chloropropanols 344 cholecystokinin (CCK) 34, 93 cadmium 233–4, 236, 344 cholera 332 calcitriol 143–4 cholesterol 86 calcium 189–94 biliary 95 absorption and transport 189–91 biosynthesis 114–15 infl uencing factors 191 dietary intake 116–17 daily requirements 193–4, 194 impact on blood lipids 115–16 food sources 193–4, 193 role of MUFAs 116 functions 191 sources 90, 91–2, 114–15 defi ciency conditions 191–2 dietary regulation cf. drug lowering regimes 117 homeostatic regulation 190 functions, brain lipid membranes 105, 114–15 interactions 194 homeostasis 95 nutritional status 192–3 genetic factors 119–20 tissue distribution 189–91 hormonal control 103 toxicity 192 LDL receptor pathways 100–1, 102–3 and vitamin D 144, 192 HDL reverse transport pathways 101–2, 102–3 Index 363

re-esterifi cation processes 96 cystic fi brosis 114 role of plant sterols 91, 116 cystine 52 triacylglycerol (TAG) hypothesis 103–4 cytochrome C oxidase 214 storage, structural pools 104 cholesterol-lowering drugs 117 daily nutrient recommendations see dietary reference effectiveness 117 standards mode of action 95 data accuracy and validity 247–8, 266–8, 305–6 and plant sterols 91, 116 data analysis 305–10 choline 186 densitometry 16–18 chromium 230–2 dental caries chylomicrons 97–8, 100 and fl uoride 229–30 properties 98 role of carbohydrates 84–5 chyme 93 depression, and food intake 35 clinical trials 318 desaturation processes 108–9 Clostridium botulinum 328 developing countries 353–5 Clostridium perfringens 330 impact of HIV/AIDS 355–7 cobalamins 167–70 infectious disease 355 Codex Alimentarius Commission (CAC) 299–300 nutrient defi ciencies 354–5 coding systems 262 obesity and NCDs 354 coenzyme A (CoA) 178–9 poverty and malnutrition 353–4 coenzyme Q 187 DHA see docosahexaenoate cohort studies 318, 320–1 diabetes 78, 351–3 colon and dyslipidemia 103 bacteria 82–3 and vitamin D status 144 carbohydrate breakdown 79–84 diet history information 259–60 colorectal cancer see also nutritional status assessments prevention diet quality assessments 272 butyrate 84 diet-induced disease see nutrition-related diseases folate supplements 174–5 dietary assessment methods see nutritional status assessments community nutrition, clinical roles 7 dietary data, accuracy and validity 247–8, 266–8 community trials 318 dietary fats 90, 92 composite dishes, food composition calculations 287–8 digestion and absorption 92–8 congenital hyperthyroidism 224 nutritional and metabolic effects 112–14 conjugated fatty acid isomers 90 see also lipids CONSORT guidelines 315 dietary fi ber 81–3, 116 contaminants see bacterial contamination; chemicals affecting intake levels 82 foods; viruses and foods dietary patterns 294–5, 358–9 copper 212–17 dietary reference standards 122–31 daily requirements and sources 216–17 concepts and approaches 122–3 functions 213 changes to 125 defi ciency conditions 213–14 defi nitions and terminology 123–5 enzyme components 181, 213, 214 future studies 131 genetic diseases 215–16 identifi cation methods 128–31 interactions 217 animal experiments 130–4 metabolism and absorption 213 balance studies 129 nutritional status measures 216 biochemical markers 130 supplements 111 biological markers 130 toxicity 214–15 deprivation studies 128–9 transport and tissue distribution 213 factorial methods 129–30 corneal conditions 140 radioactive tracer studies 129 coronary heart disease, fi sh oil supplementation 117–18 tissue nutrient level measures 130 correlation 310 interpretation and uses 125–7 coxsackie viruses 334 for population studies 127–8 creatine 53 digestion creatinine 61 carbohydrates 74–6 excretion 26–7 fats 92–8 cretinism 224 food intake regulation 34 Creutzfeldt-Jakob disease (CJD) 339–40 proteins 68–9 cross-sectional studies 318, 320 dihydrofolate reductase 172–3 Cryptosporidium parvum 339 dioxins 343 CT imaging, body composition analysis 21–2 disaccharides 80 cultural beliefs 8 disease cyanide 10 body energy needs 44 Cyclospora cayetanesis 339 evolutionary perspectives on diet 114 cysteine 51, 52, 54, 61 food-borne illnesses 325 364 Index disease (cont.) ethnicity global challenges 350–9 BMI and body fat analysis 23 in developed countries 351–3 food patterns 291 in developing countries 353–5 Europe nutrient intake goals 352–3 food safety regulation 302–3 docosahexaenoate 118 public health policies 303–4 early brain development 114 European Food Safety Authority (EFSA) 302–3 dopamine 53 exercise, and energy balance 43 double labeled water (DLW) measures 39–40, 269 experimental diets 314–15 Down’s syndrome 216 dual-energy X-ray absorptiometry (DEXA) 18, 19–20 Fanconi’s syndrome 199 multicompartment models 20–1 fat-free mass (FFM), density calculations 17 dUMP suppression test 175–6 fats see dietary fats; fatty acids; lipids Duncan test 309 fatty acids dyslipidemia 103 classifi cation and terminology 87–90 dietary intake echovirus 334 effects on serum cholesterol 115–16, 119 ecological studies 318, 321 imbalances of n-3 to n-6 117–18 eicosanoids 109–11 trans fatty acids 113, 116 eicosapentaenoic acid 117–18 digestion and absorption 92–3 elderly and colonic fermentation 83–4 energy requirements 42 metabolism height/weight measures 24 biosynthesis 105–6 emulsifi cation processes 92–3 desaturation 108–9 emulsifi ers 348 hydrogenation 109 energy availability, carbohydrate metabolism 77 ketogenesis and ketosis 107 energy balance oxidation 106–7 concepts and defi nitions 31–2 peroxidation 107–8 components 32–3 role of eicosanoids 109–11 future perspectives 47–8 nutritional and metabolic effects 112–14 in disease and trauma states 44 defi ciencies 113–14 in infancy and childhood 42 impact of trans fats 113 in old age 42 storage 104–5 in physically active individuals 43 as body fat 104 in pregnancy and lactation 43 whole body profi les 105 measures 268–71 transport and circulation 95–8, 100–4 energy expenditure 35–40 see also lipids concepts and defi nitions 32–3, 35–6 fermentation processes, colonic 82–3 historical aspects 36 ferroxidase 214 infl uencing factors fi ber physical activity 39 dietary 81–3 resting metabolic rate 37–8 intake levels 82 thermic effects of feeding 38–9 fi eld trials 318 measurement 36–7, 268–71 fi sh double labeled water (DLW) 39–40, 269 dietary fats 92, 117–18 urinary nitrogen measures 268–9 parasites 338 energy intake 32, 33–5 poisoning 347 dietary sources 33 toxins 345–6 infl uencing factors 34–5 FIVIMS (Food Insecurity and Vulnerability Information and regulation mechanisms 33–4 Mapping System) 6 energy requirements 40–2 fl avins 155–8 Entamoeba histolytica 339 fl avonoids 291 enterohepatic circulation 95 fl avor enhancers 348 environmental contaminants 10, 343–4 fl ukes 336–7 epidemiological designs 316–22 fl uoride 228–30 cross-sectional studies 320 daily requirements and sources 230 experimental studies 316–19 functions 229 non-experimental studies 319–20 defi ciency symptoms 229 error 246–7 metabolism and absorption 228–9 recall problems 263–4 toxicity 229–30 sources 260–2 folic acid 170–6 essential fatty acid defi ciencies 113–14 daily requirements 175 estrogen 103 equivalents and viamers 170–1 ethics and nutrition studies 317 functions 171–5 Index 365

metabolism and absorption 171 concerns and contributing factors 324–7 tissue uptake 171 bacterial pathogens 10, 327, 328–33 nutritional status assessment 175–6 changing supply systems 324–5 food additives 10, 200, 346–7 chemical contaminants 340–6 regulation 299–300 emerging pathogens 326–7 Food and Agriculture Organization (FAO) 6 food additives 346–7 food classifi cation systems 280 food-borne illnesses 325–6 food composition data 262, 276–92 parasites 335–8 benefi ts and uses 277 prion diseases (BSE/vCJD) 338–40 criteria for inclusion 278 setting safe intake levels 341–2 descriptions and classifi cation of foods 278–9 toxins 344–6 sampling methods 279–81 virus contaminants 327, 334–5, 334 data analysis methods 282–4 control programmes 348 data presentation 283–4 European regulation 302–3 data quality 284 future studies 304, 348–9 data sources 284–5 surveillance systems 325 future studies 289–92 UN/UN agencies regulation 298–300 problems food sampling, for food composition tables 279–81 bioavailability 286–7 food supply composite recipes 287–8 system changes 324–5 food preparation effects 285–6 trade agreements and tariffs 300–1 missing values 286 food-borne illnesses 325 portion estimates 288 bacterial contamination 327, 328–33 retrieval of data 288–9 economic consequences 326 food—nutrient data conversion 289 emerging pathogens 326–7 see also food labeling and profi ling parasite infections 335–8 food diaries 249–53 surveillance 325 Food and Drug Administration (FDA) 279 virus contamination 327, 334–5, 334 food frequency questionnaires 256–9 vulnerable groups 325 food intake formiminoglutamate test (FIGLU) 175 assessments 272–3 fructooligosaccharides (FOSs) 82 of dietary adequacy 272 fructose 75, 76–7, 79 measures functional foods 10, 82, 291, 296 choice of method 265–6 fungal toxins 345 direct 244–60 fungicides 342–3 evaluating data 271–2 indirect 239–44 galactose 75–6 sources of error 260–5 gene expression regulation underreporting 271–2 role of fatty acids 120 validity and repeatability 266–71 role of retinol 137 regulation 33–5 genetics, blood lipid metabolism 119–20 appetite and satiety 33–4 germanium 233–4, 236 central nervous system factors 34–5 ghrelin 35 circulatory factors 35 Giardia intestinalis 339 digestive factors 34 glucose 36 external signals 35 absorption 75–6 peripheral signals 35 metabolism 77 thermic effects 38–9 role of biotin 178 food labeling and profi ling 296, 297–8 rate of uptake 76–7, 78 food policy and regulation 293–304 regulation 77–9 base-line dietary patterns 294–5 and diabetes 78 making changes 295–6 glucose-galactose malabsorption syndrome 76 communication and policy dissemination 296–8 glucosinolates 291 nutrition claims 297 GLUT proteins 75–6 nutrition labeling 296–8 glutamate 51, 52, 56–7 European agencies 301–4 glutamine 50, 51, 52, 56–7 UN and UN agencies 298–9 glutathione 61 WHO/FAO and Codex Alimentarius 299–300 glycemic index (GI) 78–9, 186–7 WTO sanitary measures and trade barriers 300–1 glycine 51, 52, 54, 56–7 food preparation, nutrient losses 285–6 glycoalkoids 348 food processing, contaminants 344 glycogen 77 food profi ling 297–8 glycolysis 77 food safety 10, 324–49 glycosides 348 assessments 273–4 goitre 224 366 Index

Graves’ disease 225 genetic diseases 225 growth, amino acid and protein needs 55, 62–3 interactions 225–6 gum arabic 116 metabolism and absorption 223 nutritional status measures 225 hazardous substances 10 toxicity 224–5 health status, and nutrition 4 transport and tissue distribution 223 heavy metals 10, 343–4 iron 205–9, 355 hemochromatosis 208 daily requirements 208–9 hepatitis E 334–5 food sources 208–9 hepatitis-A virus 327, 334 functions 206–7 herbicides 342–3 defi ciency symptoms 207 high-density lipoproteins (HDL) 98–9 genetic diseases 208 properties 98 interactions 209 reverse cholesterol transport 101–2, 102–3 metabolism and absorption 205–6, 207 histidine 51, 175–6 role of vitamin C 184–5 history of nutrition science 7–9 nutritional status measures 208 HIV/AIDS 355–7 toxicity 207–8 nutrition needs 61, 357 transport and tissue distribution 205–6 homocysteine 53, 173–4 isofl avones 291 elevated levels 174 isoleucine 51, 52 household surveys 243–4 expenditure data 243 JECFA (Joint FAO/WHO Expert Committee on Food Additives) food account methods 243–4 299–300 food inventory methods 244 JEMRA (Joint FAO/WHO Meeting on Microbiological Risk food procurement data 244 Assessment) 299–300 Human Genome Project 50 human nutrition science see nutrition studies Kaplan-Meier estimates 309 hunger 33–4 Kashan-Beck disease 218–19 hydrogenation processes 90, 92, 109 Kayser-Fleischer rings 215 partial 92, 109, 113, 116 Keshan’s disease 218–19 hydroxylases, and vitamin C 181–2 ketogenesis 107 hypercalcemia 192 ketosis 107 hypertension, and homocysteine levels 174 Kruskal-Wallis test 309 hyperthyroidism 225 kynureninase 165–6 hypocalcemia 195 hypokalemia 195 lactation, energy requirements 43 hypophosphatemia 199 lactose 75 hypothalamus 34–5 intolerance 75 hypothesis testing 307 LanguaL 279, 280, 283–4 hypothyroidism 224, 225–6 lead 233–4, 236 toxicity 344 lecithin 90 in vitro studies 310–11 lectins 346 in vivo neutron activation analysis (IVNAA) 16 leptin 35 incidence, defi ned 322 leucine 51, 52, 53, 161 industrial contaminants 343–5 leucovorin rescue 173 industrial pollution 10 leukotrienes 109–11 infants linoleate 104, 106, 108, 109, 113, 117, 118 energy requirements 42 defi ciencies 114 and vitamin B6 165 lipemia, postprandial 97–8 and vitamin D 144–5 lipids 86–120 INFOODS (International Food Data System Project) (UN) 273, background history 86–7 279, 283–4, 290 classifi cation and types 87–91 informed consent 317 circulating 98–104 infrared interactance 25 fats and oils 90 inositol 186–7 hydrogenated and conjugated fatty acid isomers 90 insecticides 342–3 long-chain saturated and monounsaturated fatty acids 89 insulin 35 medium- and short-chain fatty acids 88 functions, lipoprotein metabolism 103, 104 milk and plasma lipids 105 intestinal fl ukes 336–7 phospholipids 90–1 iodine 223–6 polyunsaturated fatty acids (PUFAs) 89 daily requirements 225 saturated fatty acids 87–8 food sources 225 simple lipids 87 functions 223–4 sterols 91 defi ciency symptoms 224, 355 unsaturated fatty acids 87–8 Index 367

dietary intake 92 targets and initiatives 6, 298–300 dietary sources 91–2 UN/UN agency responses 298–9 digestion and absorption 92–4 manganese 226–7 metabolism (long-chain fatty acids) 105–11 Mann-Whitney U-test 309 role of eicosanoids 109–11 meat solubilization 93–4 dietary fats 91–2 storage and deposition 96–8 drug residues 343 body lipid pools 104–5 overcooked 344 whole body fatty acid profi les 105 Mediterranean diet 116, 117 structural and cell membrane functions 90–1, 104 medium-chain fatty acids 88–9, 112 transport 95–8, 96 dietary sources 92 HDL reverse cholesterol pathways 101–2, 102–3 megoblastic anemia 175 LDL receptor pathways 100–1, 102–3 memory, recall errors 263–4 lipolysis 93 menadiol 149–50 lipoprotein lipase (LPL) 98 menaquinone 149–50 lipoproteins 92 Menkes’ syndrome 215 assembly and secretion 96–7 mercury 344 classifi cation and distribution 98, 99 meta-analysis 322 VLDLs 98–103 metabolic rates, at rest 37–8 homeostasis 95, 100–3 methionine 51, 52 structures and metabolism 95, 98–9 load test 165–6 metabolic determinants 100 metabolism 172, 173–4 transport pathways 99–100, 102–3 methyl-folate trap 173–4 Listeria monocytogenes 330 methylmalonic aciduria 170 lithium 233–4, 236 microbial toxins 344–5 liver, cholesterol homeostasis 95, 100–3, 101, 102 milk 193 liver fl ukes 336 sunlight exposure 155 long-chain fatty acids 89 milk lipids 105 dietary sources 92 Millenium Development Goals 6, 299, metabolism 358 biosynthesis 105–6 minerals and trace elements 188–237 carbon recycling 107 defi nitions 188 desaturation 108–9 future study areas 232, 236–7 hydrogenation 109 periodic table 189 ketogenesis 107 ultratrace elements 232–6 oxidation 106–7 molds and toxins 345 perioxidation 107–8 molybdenum 227–8 role of eicosanoids 109–11 monosaccharides 75, 80 nutritional regulation 111–12 monounsaturated fatty acids (MUFAs) 89, 113, 116, low energy reporters (LERs) 270–1 117 low-density lipoproteins (LPLs) motor neurone disease 216 hormonal control 103 MRI techniques, body composition analysis 22 infl uence of dietary fats 115–16 mRNA, protein synthesis 53 properties 98 MUFAs see monounsaturated fatty acids (MUFAs) receptor pathways 100–1, 102–3 mycotoxins 345 lung fl ukes 337 lysine 52 NCDs (non-communicable diseases) see nutrition-related diseases mad cow disease 338–9 nematodes 335–8 magnesium 194–7 neurotransmitters, synthesis 51 daily requirements 196–7 niacin 158–62 food sources 197 classifi cation and equivalents 159–60 functions 195 daily requirements 162 defi ciency conditions 195–6 availability 160 genetic diseases 196 functions 161 homeostasis and absorption 194–5 defi ciency conditions 161–2 interactions 197 metabolism and absorption 160–1 nutritional status assessments 196 catabolism 160 tissue distribution 194–5 urinary excretion 160–1 toxicity 196 nutritional status assessment 162 malabsorption syndromes 75–6 toxicity 162 malaria, and ribofl avin 157 nickel 233, 235, 236 malnutrition nicotinamide 159–60 global challenges and perspectives 5–7, 298–9, 357–9 nicotinic acid 159–60 and poverty 353–4 night blindness 139 368 Index

nitrogen in developing countries 354 biochemical precursors 60–1 etiology 46 body requirements 60, 64 health implications 103, 351–3 protein synthesis 56–7 role of physical activity 46–7 sources 56–7 observational studies 319–20 nitrogen cycles 56, 59, 59 oils 90 nitrosamines 344 oleate 104, 108, 113, 118 and vitamin C 185 oligosaccharides 75, 80, 82 nonglycemic carbohydrates 79–84 omega fatty acids see polyunsaturated fatty acids (PUFAs) nonstarch polysaccharide (NSP) see dietary fi ber ORAC database 291 noroviruses 327, 334, 334 oral contraceptives, and vitamin B6 166–7 novel foods see functional foods ornithine 53–4 nutrient recommendations see dietary reference standards osteomalacia 144–5 nutrients oxidation processes, fatty acids 106–7 dietary reference standards 122–31 oysters, pathogens 333 food composition data 262, 276–92 labeling and profi ling 296–8 PAHs 344 see also carbohydrates; lipids; minerals and trace elements; Paleolithic diets 114 proteins; vitamins palmitate 104, 108 nutrition claims 297 pancreas, blood glucose regulation 77–8 nutrition labeling 296 pantothenic acid 178–80 nutrition studies 1–10 non-nutritional uses 180 approaches 2 parasites 335–8 conceptual frameworks 2–3 parathyroid hormone 144 development history 7–9 partially hydrogenated fatty acids 92, 109, 113, 116 disease risk associations 294 patulin 345 global perspectives 5–7 PCBs (polychlorinated biphenyls) 343 and health 4 PDCAAS (protein digestability-corrected amino acid score) key study components 4–5 69–70 reference points 293–4 pectin 116 research areas and challenges 9–10 pellagra 161–2 research methodology 305–23 Pendred’s syndrome 225 experimental design and statistical analysis 305– periodic table 189 10 pernicious anemia 168, 169 future studies 322–3 peroxidation (auto-oxidation) 107–8 use of animal models 311–14 pesticide residues 342–3 use in epidemiological studies 316–22 pesticides 10 use in human studies 314–16 phenylalanine 51, 52 use of in vitro studies 310–11 phospholipids 90–1, 104 scientifi c theory vs clinical practice 7 phosphorus 197–200 see also nutritional status assessments food sources 193, 200 nutrition-related diseases functions 198–9 in developed countries 351–3 defi ciency conditions 199 in developing countries 353–5 genetic diseases 199 nutritional status assessments homeostasis and absorption 197–8 considerations 238–9 interactions 200 direct measures of intake 244–60 nutritional status assessments 200 basic concepts and defi nitions 246–8 tissue distribution 197–8 for longer periods 256–60 toxicity 199 for specifi ed days 248–56 phylloquinone 149–50 future study areas 274 phytate 346 indirect measures of intake 239–44 phytestrogens 187 approaches 240 phytoceuticals 187 commodity-level food supply data 240–2 phytosterols 95 household surveys 243–4 food sources 92 product-level food supply data 242–3 plant proteins 70–1 method choice 265–71 plant sterols 91, 116, 291 repeatability and validity 266–71 plant toxins 346 safety assessments 273–4 plasma lipids nutritionists, roles 7 overview 105, 118 see also cholesterol; triacylglycerols (TAGs) obesity 44–7 polioviruses 334 basic metabolic principles 44–5 pollutants see chemicals affecting foods defi nitions 45 polyhalogenated hydrocarbons (PHHs) 343–4 in developed countries 351–3 polysaccharides 75, 80 Index 369

polyunsaturated fatty acids (PUFAs) 89 pyridoxines 162–4 basic functions 104 pyruvate 77 defi ciencies 113–14 desaturation 108–9 quality of diets 272 dietary intake 113 quasi-experiments 318 impact on serum cholesterol 115–16, 119 questionnaires 256–9 imbalances of n-3 to n-6 114, 117–18 n-3 defi ciencies 113–14 recipes, food composition calculations 287–8 nutrient—gene interactions 119–20 recommendations and standards see dietary reference standards hydrogenation 109 records of food intake 248–53 nutritional and metabolic effects 113–14 reference values for nutrients see dietary reference standards clinical importance 114 regional food differences 281–2 nutritional regulation 111–12 reliability of data 306 oxidation and peroxidation 107–8 research methodology storage 104, 105 animal models 311–14 pooled analysis 322 in vitro studies 310–11 pork, pathogens and parasites 333, 338 statistical analysis 305–10 portion size estimates 263, 288 residues 342–4 potassium 202–5 resistant starch 81 body composition measures 19 response bias 260–2 daily requirements 204–5 resting metabolic rate (RMR) 37–8 food sources 204–5 retinol 134, 135, 137 functions 203–4 see also vitamin A defi ciency symptoms 204 retinol binding protein (RBP) 137 homeostasis and absorption 203 ribofl avin see vitamin B2 (ribofl avin) interactions 205 rickets 144–5, 192 toxicity 204 rotaviruses 334 transport and tissue distribution 203 rubidium 233, 235, 236 poverty and malnutrition 353–4 power calculations 307–8 salivary glands 75 pregnancy Salmonella 332 and anticoagulants 151–2 salt intake trends 203 energy requirements 43 see also sodium and chloride and folate 174 sample size calculations 308 prion diseases 338–40 sampling bias 260 processed foods, partial hydrogenation processes 92 satiety 34 propionate 83–4 saturated fatty acids 89 prostaglandins 109–11 dietary sources 92 proteins 49–72 nutritional and metabolic effects 112, 118 background and discovery 50–1 and LDL cholesterol 119 biochemical roles 51 public health policies 296 amino acid functions 50 see also long-chain fatty acids biochemical structures 49 SCFAs see short-chain fatty acids biochemical synthesis and degradation 58–62 Schilling test 170 body requirements 58–63, 64–5 scurvy 182–3 by age and physiological groups 58, 64–5 seafood pathogens 332, 347 dietary recommendations 63–5 toxins 345–6 during illness and trauma 72 seed oils 91 estimation 63–5 selenium 217–23 infl uencing factors 58, 71–2 daily requirements 220–1 growth needs 55, 62–3 food sources 220–1 metabolic needs 58–64 functions 218 over supply 61–2 defi ciency conditions 218–20 classifi cation of amino acids 54–8 genetic diseases 220 defi ciencies 355 interactions 221–3 food sources 70–1 metabolism and absorption 217–18 and digestibility 68–9 nutritional status measures 220 and nutritional quality 69–70 toxicity 220 worldwide availability 71, 355 transport and tissue distribution 217–18 future considerations 72 selenoproteins 218, 219 protozoa 338, 339 serine 51, 52 public health nutrition 9–10 sex hormones, functions, lipoprotein metabolism 103 role of nutritionists 7 short-chain fatty acids (SCFAs) 83–4, 88 PUFAs see polyunsaturated fatty acids (PUFAs) absorption 83–4 puffer fi sh 347 roles 83 370 Index

silicon 233, 235, 236 traffi c light system food profi ling 297–8 Siri’s formula 16–17, 17 trans fatty acids 107–8, 113, 116 skeletal mass, measures 24 transmissible spongiform encephalopathy (TSE) 338– skinfold thickness measures 23–4 40 sodium and chloride 200–2 triacylglycerols (TAGs) 90 daily requirements 202 dietary sources 91–2 functions 201 functions 104 defi ciency symptoms 201 lipoprotein assembly 96–7 genetic diseases 202 impact of n-3 PUFAs 119 homeostasis and absorption 200–1 postprandial lipemia 97–8 intake trends 205 re-esterifi cation 96 interactions 202 regulation of cholesterol deposition 103–4 nutritional status assessments 202 impact of n-3 PUFAs 119 in processed foods 200 synthesis 104 toxicity 201–2 trichinosis 338 transport and tissue distribution 200–1 triglycerides, dietary sources 91 solanine 10 tryptophan 51, 52, 159–62 soy beans 187 load test 165 spina bifi da 174 tyrosinase 214 squalene 187 tyrosine 51, 52 standards see Codex Alimentarius Commission (CAC); dietary reference standards ubiquinone 187 Staphylococcus aureus 329 ultrasound measurements 25 starch UNICEF (UN Children’s Emergency Fund) 6, 299 digestion 75, 80 United Nations (UN) resistant 81 food and nutrition regulation 298–9 starches, modifi ed 82 global challenges 358–9 statistical analysis 305–10 nutrient recommendations 124–5 stearate 104, 108 University Food and Nutrition Program INFOODS 273, stone-age diets 114 279 strontium 146 unsaturated fatty acids 87–8 studies on food intake see nutritional status assessments partial hydrogenation 92 sucrase-isomaltase defi ciency 75 types 88–9 sugar alcohols 80 urea cycle enzymes 61 surveillance systems 325 urea production 61–2 urinary nitrogen measures 268–9 t-tests 309 tag names 283–4 validity TAGs see triacylglycerols defi ned 305 tannins 346 of diet measures 267–71 tapeworms 338 valine 51, 52 taurine 53, 187 vanadium 233, 235, 236 teeth verotoxigenic Escherichia coli (VTEC) 331 dental caries 85–6 very low-density lipoproteins (VLDL) 98–9 and fl uoride 229–30 hormonal control 103 terpines 187 veterinary drug residues 343 testosterone 103 Vibrio cholerae 332 thiamin see vitamin B1 (thiamin) Vibrio parahaemolyticus 332 threonine 52 Vibrio vulnifi cus 333 thymidylate synthetase 172–3 viruses and foods 327, 334–5, 334 thyroid hormones, and vitamin D 144 astroviruses 334 thyrotoxicosis 225 emerging pathogens 325–6 tin 233, 235, 236 hepatitis-A 327 total body electrical conductivity (TOBEC) 26 noroviruses 327, 334 total body potassium (TBK) 19 rotaviruses 334 total body water (TBW) 18–19 vision toxins 10, 344–6 role of vitamin A 137, 138 bacterial 345 and night blindness 139 fungal 345 vitamins 132–87 microbial 344–5 functions 132–3 naturally occurring 346–7 defi ciency conditions 132–3 seafood 345–6 future study areas 185–7 Toxoplasma gondii 339 vitamin A 133–41 trace elements see minerals and trace elements classifi cation and units 134–5 trade agreements and tariffs 300–1 daily reference levels 139 Index 371

drug/nutrient interactions 141 defi ciency conditions 144–5 functions 137–9 metabolism and absorption 141 defi ciency conditions 139 to calcitriol 143 metabolism and storage 135 metabolites 143 status assessments 139–40 regulation 143–4 teratogenicity 140–1 synthesis (skin) 141–3 toxicity 140 toxicity 145 vitamin B1 (thiamin) 152–5 vitamin E 146–9 daily requirements 154 classifi cation and units 146 functions 153 daily requirements 148 defi ciency conditions 153–4 high intake levels 149 metabolism and absorption 153 functions 146–8 status assessment 154–5 defi ciency conditions 148 vitamin B2 (ribofl avin) 155–8 metabolism and absorption 146 daily requirements 157–8 nutrient interactions 149 functions 156–7 nutritional status assessment 148–9 defi ciency conditions 157 vitamin K 149–52 metabolism and absorption 155–6 dietary sources 149–50 homeostasis 156 functions 150–1 and oxidative stress 157 defi ciency conditions 152 nutrient/drug interactions 158 metabolism and synthesis 149–50 nutritional status assessments 158 toxicity and interactions 152 photolytic destruction 155 vitamin Q 187 vitamin B6 162–7 classifi cation and vitamers 162–3 waist-to-hip ratios 25 daily requirements 164–5 warfarin, contraindications, pregnancy 151–2 functions 164 weight/height indices 22–3 defi ciency conditions 164 Wernicke-Korsakoff syndrome 154 non-nutritional uses 166–7 WHO (World Health Organization) 123–4 metabolism and absorption 163–4 food and nutrition regulation 298–300 nutritional status assessments 165–6 nutrient intake goals (disease prevention) 352 toxicity 167 WHO/FAO (World Health Organization/Food and Agriculture vitamin B12 167–70 Organization) 6, 124, 299–300 daily requirements 169 Codex Alimentarius Commission (CAC) 299– functions 169 300 defi ciency conditions 169 Wilson’s disease 215 metabolism and absorption 168–9 WTO (World Trade Organization), trade agreements and tariffs and folate 174 301–2 nutritional status assessment 170 structure and vitamers 167–8 X-linked hypophosphatemia 199 vitamin C 180–5 xerophthalmia 139 classifi cation and structures 180–1 daily requirements 183–4 Yersinia enterocolitica 333 benefi ts of high doses 184–5 functions 181–2 Zellweger’s syndrome 114 defi ciency conditions 182–3 zinc 209–12 pro-and anti-oxidant roles 182 daily requirements 211–12 metabolism and absorption 181 food sources 211–12 nutritional status assessments 184 functions 210 pharmacological uses 185 defi ciency symptoms 210–11, 355 toxicity 185 genetic diseases 211 vitamin D 141–6 interactions 212 classifi cation and units 141 metabolism and absorption 209–10 daily recommendations 145 nutritional status measures 211 drug/nutrient interactions 145–6 toxicity 211 functions 144 transport and tissue distribution 209–10