Cover page

Table of Contents

We dedicate this edition to our families who supported us during its creation, and to our scientific families (our mentors and colleagues) who make working in the field of nutritional sciences exciting, worthwhile, and a wonderful life’s profession.

Title page

List of Contributors

Peter J. Aggett
School of Health and Medicine
Physics Building
Lancaster University
Lancaster LA1 4YD
UK

Janice Albert
Nutrition and Consumer Protection Division
Food and Agriculture Organization of the United Nations
Viale delle Terme di Caracalla
Rome 00153
Italy

Lindsay H. Allen
USDA-ARS Western Human Nutrition Research Center
University of California, Davis
430 W. Health Sciences Drive
Davis, CA 95616
USA

John J.B. Anderson
Department of Nutrition
Gillings School of Global Public Health
University of North Carolina
Chapel Hill, NC 27599-7461
USA

Arne Astrup
Faculty of Science
University of Copenhagen
Rolighedsvej 30
DK-1958 Frederiksberg
Denmark

Thiane G. Axelsson
Department of Clinical Science, Intervention and Technology
Divisions of Baxter Novum and Renal Medicine
Karolinska Institutet
Karolinska University Hospital
Huddinge
Stockholm 141 86
Sweden

Lynn B. Bailey
Department of Foods and Nutrition
University of Georgia
273 Dawson Hall
Athens, GA 30602
USA

Joseph L. Baumert
Department of Food Science and Technology
University of Nebraska
237 Food Industry Building
Lincoln, NE 68585-0919
USA

Prasad Bellur
Monsanto Research Centre
#44/2A, Vasants Business Park
Bellary Road NH 7, Hebbal
Bangalore 560092
India

Claire E. Berryman
Department of Nutritional Sciences
110 Chandlee Laboratory
The Pennsylvania State University
University Park, PA 16802
USA

Lucien Bettendorff
University of Liège–GIGA-Neurosciences
Av. de l’Hôpital 1 B36
Liège 4000
Belgium

Sekhar Boddupalli
Monsanto Vegetable Seeds
Woodland, CA 95695
USA

Annalies Borrel
UNICEF Office of Emergency Programmes-Humanitarian Policy and Advocacy
New York
USA

Jennie Brand-Miller
Boden Institute of Obesity, Nutrition, Exercise, and Eating Disorders
University of Sydney
Sydney, NSW 2006
Australia

Ronette R. Briefel
Mathematica Policy Research
1100 1st Street NE, 12th Floor
Washington, DC 20002-4221
USA

Alan L. Buchman
Department of Medicine
Feinberg School of Medicine
Northwestern University
Chicago, IL 60611
USA

Louise M. Burke
Australian Institute of Sport – Sports Nutrition
PO Box 176
Leverrier Crescent
Belconnen
ACT 2617
Australia

Leah E. Cahill
Department of Nutrition
Harvard School of Public Health
Building 2
655 Huntington Avenue
Boston, MA 02115
USA

Philip C. Calder
Institute of Human Nutrition
University of Southampton Faculty of Medicine
IDS Building MP887 Southampton General Hospital
Tremona Road
Southampton SO16 6YD
UK

Robert Carter, III
Military Nutrition Division
US Army Research Institute of Environmental Medicine
42 Kansas Street
Natick, MA 01760-5007
USA

Krista Casazza
Department of Nutrition Sciences
The University of Alabama at Birmingham
1675 University Blvd, WEBB 439
Birmingham, AL 35294-3360
USA

Marie A. Caudill
Division of Nutritional Sciences
Cornell University
228 Savage Hall
Ithaca, NY 14853
USA

Samuel N. Cheuvront
Military Nutrition Division
US Army Research Institute of Environmental Medicine
42 Kansas Street
Natick, MA 01760-5007
USA

Michal Chmielewski
Department of Nephrology, Transplantology and Internal Medicine
Medical University of Gdansk
ul. Debinki 7
20-811 Gdansk
Poland

Dallas L. Clouatre
Glykon Technologies Group, LLC
1112 Montana Avenue #541
Santa Monica, CA 90403
USA

Paul M. Coates
Office of Dietary Supplements
National Institutes of Health
6100 Executive Blvd, Room 3B01, MSC 7517
Bethesda, MD 20892-7517
USA

Stephen Colagiuri
Boden Institute of Obesity, Nutrition, Exercise, and Eating Disorders
University of Sydney
Sydney, NSW 2006
Australia

Karen D. Corbin
UNC Nutrition Research Institute
Department of Nutrition
University of North Carolina at Chapel Hill
500 Laureate Way, Rm 2218
Kannapolis, NC 28081
USA

Joseph Cornelius
Monsanto Company
800 North Lindbergh Blvd
St. Louis, MO 63167
USA

Vanessa R. da Silva
Department of Foods and Nutrition
University of Georgia
273 Dawson Hall
Athens, GA 30602
USA

Sai Krupa Das
Jean Mayer USDA Human Nutrition Research Center on Aging
Tufts University
711 Washington Street
Boston, MA 02111-1524
USA

Jeanne H.M. de Vries
Division of Human Nutrition
WU Agrotechnology & Food Sciences
Wageningen University
Bomenweg 2 # 307/214
6703HD Wageningen
The Netherlands

Alan M. Diamond
Department of Pathology
University of Illinois at Chicago
840 S. Wood Street, Suite 130 CSN
Chicago, IL 60612
USA

Kelly A. Dougherty
Department of Pediatrics
Gastroenterology, Hepatology, and Nutrition
The Children’s Hospital of Philadelphia
University of Pennsylvania, Perelman School of Medicine
Philadelphia, PA 19104
USA

Adam Drewnowski
School of Public Health & Community Medicine
University of Washington
Box 353410
Seattle, WA 98195-3410
USA

Johanna T. Dwyer
Jean Meyer Human Nutrition Research Center on Aging
Tufts University
Box 783 Tufts Medical Center
800 Washington Street
Boston, MA 02111-1524
and Office of Dietary Supplements
National Institutes of Health
Bethesda, MD
USA

Ahmed El-Sohemy
Department of Nutritional Sciences
University of Toronto
150 College Street, Room 350
Toronto, Ontario M5S 3E2
Canada

Guylaine Ferland
Université de Montréal
Centre de recherche
Institut universitaire de gériatrie de Montréal
4565 chemin Queen-Mary
Montréal, Québec H3W 1W5
Canada

James C. Fleet
Department of Foods and Nutrition
Purdue University
700 West State Street
West Lafayette, IN 47906-2059
USA

Michael R. Flock
Department of Nutritional Sciences
110 Chandlee Laboratory
The Pennsylvania State University
University Park, PA 16802
USA

Edward A. Frongillo
Department of Health Promotion, Education, and Behavior
University of South Carolina, Columbia
800 Sumter Street
Columbia, SC 29208
USA

Amy Gorin
Department of Psychology
University of Connecticut
406 Babbidge Road, Unit 1020
Storrs, CT 06269-1020
USA

Jesse F. Gregory, III
Food Science and Human Nutrition Department
University of Florida
PO Box 110370
Gainesville, FL 32611-0370
USA

Kristina A. Harris
Department of Nutritional Sciences
110 Chandlee Laboratory
The Pennsylvania State University
University Park, PA 16802
USA

Robert P. Heaney
Creighton University Medical Center
601 North 30th Street – Suite 4841
Omaha, NE 68131
USA

William C. Heird
Department of Pediatrics
USDA-ARS Children’s Nutrition Research Center
Baylor College of Medicine
1100 Bates Street
Houston, TX 77030-2600
USA

Helen L. Henry
Department of Biochemistry
University of California
Riverside, CA 92521
USA

Daniell B. Hill
Department of Medicine
Center for Translational Research
University of Louisville
505 South Hancock Street
Louisville, KY 40292
USA

Simone D. Holligan
Department of Nutritional Sciences
110 Chandlee Laboratory
The Pennsylvania State University
University Park, PA 16802
USA

Roberta R. Holt
Department of Nutrition
University of California, Davis
One Shields Avenue,
Davis, CA 95616
USA

Lindsay M. Jaacks
Department of Nutrition
UNC Gillings School of Global Public Health
2212 McGavran-Greenberg Hall
135 Dauer Drive
Chapel Hill, NC 27599
USA

Wei Jia
Department of Nutrition
University of North Carolina at Greensboro
North Carolina Research Campus
500 Laureate Way
Kannapolis, NC 28081
USA

Elizabeth J. Johnson
Jean Mayer USDA Human Nutrition Research Center on Aging
Tufts University
711 Washington Street
Boston, MA 02111-1524
USA

Ian T. Johnson
Institute of Food Research
Norwich Research Park
Colney, Norwich NR4 7UA
UK

Carol S. Johnston
Nutrition Program
College of Nursing and Health Innovation
Arizona State University
500 North 3rd Street
Phoenix, AZ 85004
USA

Alexandra M. Johnstone
Rowett Institute of Nutrition and Health
Greenburn Road
Bucksburn
Aberdeen AB21 9SB
UK

Peter J.H. Jones
Richardson Centre for Functional Foods
University of Manitoba
Smartpark Research and Technology Park
196 Innovation Drive
Winnipeg, Manitoba R3T 6C5
Canada

Jaya Joshi
Monsanto Research Centre
#44/2A, Vasants Business Park
Bellary Road NH 7, Hebbal
Bangalore 560092
India

Carl L. Keen
Department of Nutrition
University of California, Davis
One Shields Avenue
Davis, CA 95616
USA

Robert W. Kenefick
Military Nutrition Division
US Army Research Institute of Environmental Medicine
42 Kansas Street
Natick, MA 01760-5007
USA

James B. Kirkland
Department of Human Health and Nutritional Sciences
College of Biological Sciences
University of Guelph
Guelph, Ontario N1G 2W1
Canada

Penny M. Kris-Etherton
Department of Nutritional Sciences
110 Chandlee Laboratory
The Pennsylvania State University
University Park, PA 16802
USA

Toshinobu Kuroishi
Department of Nutrition and Health Sciences
University of Nebraska–Lincoln
316 Ruth Leverton Hall
Lincoln, NE 68583-0806
USA

Shantala Lakkanna
Monsanto Research Centre
#44/2A, Vasants Business Park
Bellary Road NH7, Hebbal
Bangalore 560092
India

Alice H. Lichtenstein
Jean Mayer USDA Human Nutrition Research Center on Aging
Tufts University
150 Harrison Avenue
Boston, MA 02111
USA

Bengt Lindholm
Department of Clinical Science, Intervention and Technology
Divisions of Baxter Novum and Renal Medicine
Karolinska Institutet
Karolinska University Hospital
Huddinge
Stockholm 141 86
Sweden

Brian L. Lindshield
Department of Human Nutrition
Kansas State University
208 Justin Hall
Manhattan, KS 66502
USA

Joanne R. Lupton
Department of Nutrition and Food Science
Texas A&M University, College Station
213 Kleberg Center
2253 TAMU
College Station, TX 77843-2253
USA

Asim Maqbool
Department of Pediatrics
Gastroenterology, Hepatology, and Nutrition
The Children’s Hospital of Philadelphia
University of Pennsylvania, Perelman School of Medicine
Philadelphia, PA 19104
USA

Luis Marsano
Department of Medicine
Center for Translational Research
University of Louisville
505 South Hancock Street
Louisville, KY 40292
USA

Elizabeth J. Mayer-Davis
Department of Nutrition
UNC Gillings School of Global Public Health and School of Medicine
2212 McGavran-Greenberg Hall
135 Dauer Drive
Chapel Hill, NC 27599
USA

Craig J. McClain
Department of Medicine
Center for Translational Research
University of Louisville
505 South Hancock Street
Louisville, KY 40292
USA

Stephen A. McClave
University of Louisville School of Medicine
401 East Chestnut Street, Ste 310
Louisville, KY 40202
USA

Donald B. McCormick
Department of Biochemistry and Program in Nutrition and Health Science
Rollins Research Center
Emory University
Atlanta, GA 30322
USA

Margaret A. McDowell
National Institute of Health
Division of Nutrition Research Coordination
Two Democracy Plaza, Rm 629
6707 Democracy Blvd, MSC 5461
Bethesda, MD 20892-5461
USA

Joshua W. Miller
Department of Pathology and Laboratory Medicine
University of California Davis Medical Center
Research III
4645 2nd Avenue
Suite 3200A
Sacramento, CA 95817
USA

John A. Milner
Nutritional Sciences Research Group
National Cancer Institute
6130 Executive Blvd, Suite 3164 EPN
Rockville, MD 20852
USA

Pablo Monsivais
UKCRC Centre for Diet and Activity Research
Box 296
Cambridge Institute of Public Health
Forvie Site
Cambridge, CB2 0SR
UK

Scott J. Montain
Military Nutrition Division
US Army Research Institute of Environmental Medicine
42 Kansas Street
Natick, MA 01760-5007
USA

Tim R. Nagy
Department of Nutrition Sciences
The University of Alabama at Birmingham
1675 University Blvd, Webb 439
Birmingham, AL 35294-3360
USA

Marguerite A. Neill
The Warren Alpert Medical School
Brown University
Box G-A1
Providence, RI 02912
USA

Holly Nicastro
Nutritional Sciences Research Group
National Cancer Institute
6130 Executive Blvd, Suite 3164 EPN
Rockville, MD 20852
USA

Forrest H. Nielsen
Grand Forks Human Nutrition Research Center
USDA-ARS-NPA
2420 2 Avenue N, Stop 9034
Grand Forks, ND 58202-9034
USA

Anthony W. Norman
Department of Biochemistry and Division of Biomedical Sciences
University of California
Riverside, CA 92521
USA

Marga C. Ocké
The National Institute for Public Health and Environment
PO Box 1
3720 BA Bilthoven
The Netherlands

Thomas M. O’Connell
LipoScience Inc.
2500 Sumner Blvd
Raleigh, NC 27616
USA

Christine M. Olson
Division of Nutritional Sciences
Cornell University
376 Martha Van Rensselaer Hall
Ithaca, NY 14853
USA

Andrea A. Papamandjaris
Nestlé Inc.
Medical and Scientific Unit
North York, Ontario M2N 6S8
Canada

Elizabeth P. Parks
Department of Pediatrics
Gastroenterology, Hepatology, and Nutrition
The Children’s Hospital of Philadelphia
University of Pennsylvania, Perelman School of Medicine
Philadelphia, PA 19104
USA

Sue D. Pedersen
LMC Endocrinology Centre
Suite 102
5940 MacLeod Tr SW
Calgary, Alberta T2H 2G4
Canada

David L. Pelletier
Division of Nutritional Sciences
Cornell University
212 Savage Hall
Ithaca, NY 14853
USA

W. Todd Penberthy
Department of Molecular Biology and Microbiology
University of Central Florida College of Medicine
Orlando, FL 32816
USA

Paul B. Pencharz
Departments of Paediatrics and Nutritional Sciences
Research Institute
The Hospital for Sick Children
University of Toronto
Toronto, Ontario M5G 1X8
Canada

Barry M. Popkin
Department of Nutrition
Carolina Population Center
University of North Carolina at Chapel Hill
University Square, CB# 8120
123 W. Franklin Street
Chapel Hill, NC 27516-3997
USA

Harry G. Preuss
Department of Biochemistry
Georgetown University Medical Center
Washington, DC 20057
USA

Joseph R. Prohaska
Department of Biomedical Sciences
University of Minnesota Medical School Duluth
1035 University Drive
Duluth, MN 55812
USA

Charles J. Rebouche (Retired)
Department of Pediatrics
University of Iowa
Iowa City, IA 52242
USA

Patrick Ritz
Gérontopôle de Toulouse
Unité de Nutrition
CHU Larrey
TS 30030
31059 Toulouse
France

Susan B. Roberts
Jean Mayer USDA Human Nutrition Research Center on Aging
Tufts University
711 Washington Street
Boston, MA 02111-1524
USA

Robert B. Rucker
Department of Nutrition
University of California, Davis
One Shields Avenue
3415 Meyer Hall
Davis, CA 95616-8575
USA

Katelyn A. Russell
Food Science and Human Nutrition Department
University of Florida
PO Box 110370
Gainesville, FL 32611-0370
USA

Kate Sadler
Friedman School of Nutrition Science and Policy
Feinstein International Center
Tufts University
200 Boston Avenue
Medford, MA 02155
USA

Lisa M. Sanders
Kellogg Company
Battle Creek, MI
USA

Thomas A.B. Sanders
Diabetes & Nutritional Sciences Division
School of Medicine
King’s College London
4.43 Franklin-Wilkins Building
150 Stamford Street
London SE1 9NH
UK

Michael N. Sawka
Military Nutrition Division
US Army Research Institute of Environmental Medicine
42 Kansas Street
Natick, MA 01760-5007
USA

Marion Secher
Gérontopôle de Toulouse
Unité de Nutrition
CHU Larrey
TS 30030
31059 Toulouse
France

Anders Sjödin
Faculty of Science
University of Copenhagen
Rolighedsvej 30
DK-1958 Frederiksberg
Denmark

Noel W. Solomons
Center for Studies of Sensory Impairment
Sensory Impairment, Aging and Metabolism (CeSSIAM)
17a Avenida #16-89, Zona 11
Guatemala City 01011
Guatemala

Sally P. Stabler
Division of Hematology
University of Colorado School of Medicine
12700 E. 19th Avenue
Denver, CO 80045
USA

Virginia A. Stallings
Department of Pediatrics
Gastroenterology, Hepatology, and Nutrition
The Children’s Hospital of Philadelphia
University of Pennsylvania, Perelman School of Medicine
Philadelphia, PA 19104
USA

Susan E. Steck
Department of Epidemiology and Biostatistics
University of South Carolina–Columbia
915 Greene Street, Rm 236
Columbia, SC 29208
USA

Paolo M. Suter
Clinic and Policlinic of Internal Medicine
University Hospital
Rämistrasse 100
8091 Zurich
Switzerland

Deborah F. Tate
Department of Health Behavior and Nutrition
Gillings School of Global Public Health
University of North Carolina at Chapel Hill
Chapel Hill, NC 27599
USA

Robert V. Tauxe
Division of Foodborne, Waterborne and Environmental Diseases
National Center for Emerging and Zoonotic Infectious Diseases
Centers for Disease Control and Prevention
Mailstop C-09
Atlanta, GA 30333
USA

Steve L. Taylor
Department of Food Science & Technology
University of Nebraska
255 Food Industry Bldg
Lincoln, NE 68585-0919
USA

Emily N. Terry
Department of Pathology
University of Illinois at Chicago
840 S. Wood St, Suite 130 CSN
Chicago, IL 60612
USA

Maret G. Traber
School of Biological and Population Health Sciences
Linus Pauling Institute
Oregon State University
307 Linus Pauling Science Center
Corvallis, OR 97331-6512
USA

Federico Tripodi
Monsanto Company
800 North Lindbergh Blvd
St. Louis, MO 63167
USA

Janet Y. Uriu-Adams
Department of Nutrition
University of California, Davis
One Shields Avenue
Davis, CA 95616
USA

Wija A. van Staveren
Division of Human Nutrition
WU Agrotechnology & Food Sciences
Wageningen University
Bomenweg 4 #309/2004
6703HD Wageningen
The Netherlands

Bruno Vellas
Gérontopôle de Toulouse
Unité de Nutrition
CHU Larrey
TS 30030
31059 Toulouse
France

Rohini Vishwanathan
Jean Mayer USDA Human Nutrition Research Center on Aging
Tufts University
711 Washington Street
Boston, MA 02111-1524
USA

Stella Lucia Volpe
Department of Nutrition Sciences
Drexel University College of Nursing and Health Professions
245 N. 15th Street
Bellet Building–Room 521
Mail Stop 1030
Philadelphia, PA 19102
USA

Li Wang
Department of Nutritional Sciences
110 Chandlee Laboratory
The Pennsylvania State University
University Park, PA 16802
USA

Robert A. Waterland
Departments of Pediatrics and Molecular & Human Genetics
USDA/ARS Children’s Nutrition Research Center
Baylor College of Medicine
Houston, TX 77030-2600
USA

Connie M. Weaver
Department of Nutrition Science
Purdue University
1264 Stone Hall
700 W State Street
West Lafayette, IN 47907-2059
USA

Robert Weisell (Retired)
Food and Agriculture Organization of the United Nations
Viale delle Ginestre 8
Ariccia (RM) 00040
Italy

Subhashinee S.K. Wijeratne
Department of Nutrition and Health Sciences
University of Nebraska–Lincoln
316 Ruth Leverton Hall
Lincoln, NE 68586-0806
USA

Gary Williamson
School of Food Science and Nutrition
University of Leeds
Woodhouse Lane
Leeds, LS2 9JT
UK

Rena R. Wing
The Miriam Hospital
Alpert Medical School
Brown University
Providence, RI 02903
USA

Judith Wylie-Rosett
Department of Epidemiology and Population Health
Albert Einstein College of Medicine
Jack and Pearl Resnick Campus
1300 Morris Park Avenue
Belfer Building, Room 1307
Bronx, NY 10461
USA

Parveen Yaqoob
Food and Nutritional Sciences
University of Reading
2-55 Food Biosciences
Reading, RG6 6AH
UK

Helen Young
Friedman School of Nutrition Science and Policy
Feinstein International Center
Tufts University
200 Boston Avenue
Medford, MA 02155
USA

Steven H. Zeisel
UNC Nutrition Research Institute
Department of Nutrition
University of North Carolina at Chapel Hill
500 Laureate Way, Rm 2218
Kannapolis, NC 28081
USA

Janos Zempleni
Department of Nutrition and Health Sciences
University of Nebraska–Lincoln
316 Ruth Leverton Hall
Lincoln, NE 68583-0806
USA

Vivian M. Zhao
Nutrition and Metabolic Support Service and Department of Pharmaceutical Services
Emory University Hospital
1364 Clifton Road NE
Atlanta, GA 30322
USA

Thomas R. Ziegler
Emory University Hospital
Nutrition and Metabolic Support Service and
Emory University School of Medicine
1648 Pierce Drive NE
Atlanta, GA 30307
USA

Michael B. Zimmermann
Laboratory for Human Nutrition
Swiss Federal Institute of Technology Zürich
Schmelzbergstrasse 7
LFV E19
Zürich CH-8092
Switzerland

Preface

We are honored to have been asked to edit the tenth edition of Present Knowledge in Nutrition. The first edition was published in 1953, and throughout the book’s history its authors have been a “Who’s Who” of nutritional science. The current volume is no exception. With this edition, we aimed to find productive, knowledgeable, and well-known authors to help us provide integrated information on nutrition, physiology, health and disease, and public-health applications – all in one text. This ambitious goal was set for one purpose: to provide readers with the most comprehensive and current information covering the broad fields within the nutrition discipline. Reflecting the global relevance of nutrition, our authors come from a number of countries. It is hoped that this edition captures the current state of this vital and dynamic science from an international perspective.

New to this edition are chapters on topics such as epigenetics, metabolomics, and sports nutrition – areas that have developed significantly in recent years. The remaining chapters have all been thoroughly updated to reflect developments since the last edition. Suggested reading lists are now provided for readers wishing to delve further into specific subject areas.

To make this edition as accessible and continuously relevant as possible, it is available in both print and electronic formats. An accompanying website (visit ) provides book owners with access to an Image Bank of tables and figures as well as to any updates the authors may post to their chapters in the future.

We hope this volume will be a valuable reference for researchers, health professionals, and policy experts, and a useful resource for educators and advanced nutrition students.

John W. Erdman Jr.
Urbana, Illinois
Ian A. Macdonald
Nottingham, England
Steven H. Zeisel
Chapel Hill, North Carolina

Acknowledgments

A great deal of work and dedication was involved in producing this extensive volume. First and foremost, we thank the authors of the 73 chapters who reviewed and condensed a vast amount of knowledge and literature. Their undertaking was significant, and our gratitude for their dedication cannot be overstated. The editors of the ninth edition, Barbara Bowman and Rob Russell, are thanked for the critical help they provided in the conceptualization of this edition as well as in the author selection. All of the chapters in this edition were externally reviewed by leaders in each chapter’s field; their generous, voluntary assistance was invaluable. We thank the International Life Sciences Institute for continuing to foster the production of Present Knowledge in Nutrition, and we especially thank Allison Worden for her guidance and hundreds of hours of work, and for keeping everything on track.

1

SYSTEMS BIOLOGY APPROACHES TO NUTRITION

JAMES C. FLEET, PhD

Purdue University, West Lafayette, Indiana, USA


Summary
Systems biology is an integrative approach to the study of biology. It integrates information gathered from reductionist experiments and various high-density profiling tools to understand how the parts of the system interact with each other and with other external factors such as diet. The science of nutrition is well suited to a systems biology approach. The tools of systems biology can be applied to settings relevant to nutrition with the goal of better understanding the breadth and depth of the impact that changing nutrient status has on physiology and chronic disease risk. However, there are many challenges to appropriately applying the systems biology approach to nutritional science. Among the challenges are those related to cost, study design, statistical analysis, data visualization, data integration, and model building.

Introduction

Reductionism versus Systems Biology: A Changing Paradigm

Nutrition requires an understanding of disciplines such as physiology, cell biology, chemistry, biochemistry, and molecular biology among others. In contrast to this broad view, we apply reductionist experimental approaches to advance our understanding of specific nutrient functions. However, while these approaches have been useful, significant issues limit their utility. For example, it can be difficult to translate mechanism-focused research in cells into the complex physiology of a whole organism. As a result, biological models developed from reductionist experiments often fail to explain why gene knockout mice studies do not have the expected phenotype (e.g. the facilitated diffusion model used to describe intestinal calcium absorption is being challenged by the results from calbindin D9k and TRPV6 knockout mice (Benn et al., 2008; Kutuzova et al., 2008)). Even after extensive examination of a problem with reductionist approaches, we often find that gaps exist in our understanding. It is clear that re-applying the approaches we have traditionally used to investigate nutritional questions is unlikely to yield a different outcome. Because of this we need new approaches that complement traditional reductionist approaches but which give us a new, broader perspective of how nutrients are influencing human biology. Systems biology is such an approach.

Systems biology has been described as an approach to biological research that combines reductionist techniques with an “integrationist” approach to identify and characterize the components of a system, and then to evaluate how each of the components interacts with one another and with their environment. The goal of the systems biology approach is to integrate many types of information so that you get a more complete view of a system (Kohl et al., 2010). This definition has a flexibility that is very attractive to nutrition. The notion of a “system” can be applied narrowly to a cell, where the parts are individual biochemical and signaling pathways and the “environment” is the growth factors and hormones that regulate these pathways. However, it can be applied more broadly to a person, where the integration relates to the physiologic systems and the “environment” is lifestyle variables such as diet. For example, we know that calcium influences bone metabolism but we know that this relies upon the efficiency of intestinal calcium absorption and renal calcium excretion as well as on hormones produced at various sites (e.g. PTH in the parathyroid gland, 1,25-dihydroxyvitamin D in the kidney). Thus, our understanding of how dietary calcium intake influences bone is enhanced by looking at the interactions between multiple tissues rather than just focusing only on bone.

Systems Biology as Discovery Tool

Systems biology is an approach but within this approach are also three classes of novel tools necessary for a successful systems biology analysis. First, there are the high-density phenotyping platforms that allow simultaneous measurement of whole classes of biological compounds, i.e. omics methods such as genomics, transcriptomics, proteomics, metabolomics, and ionomics (). Next, the information from these platforms must be analyzed to identify the important changes resulting from a treatment. This requires the application of sophisticated statistics. Third, the information must be annotated and integrated with prior knowledge: this is the field of bioinformatics.

 Definitions related to systems biology

Term Description
Genomics The study of the genomes of organisms including influences of DNA sequence variation on biology and the impact of modifying DNA and histones on DNA function (i.e. epigenomics)
Transcriptomics The study of transcripts from the genome including messenger RNA and non-coding RNA such as micro RNA
Proteomics The study of proteins in a biological system including their level, location, physical properties, post-translational modifications, structures, and functions
Metabolomics The study of the unique chemicals (metabolites) that are produced as a result of cellular processes, e.g. small molecules such as lipids, metabolites of intermediary metabolites
Ionomics The study of the mineral nutrient and trace element composition of an organism
Next-generation sequencing High-throughput DNA sequencing technologies that parallelize the sequencing process thereby producing millions of sequences at once
Cluster A graphical representation of relationships between data based on similarities in their concentrations or changes in concentrations
Pathway A graphical representation of biological data organized on the basis of accepted relationships (e.g. glycolysis; signaling through the insulin receptor; lipoprotein transport)
Network A complex graphical representation of biological data that is developed from the experimental data. This will include known relationships (pathways) and new relationships linking pathways

Systems Biology and Omics Tools for Biomarker Discovery

Omics analyses are often used to profile a biological state and then essential elements of the profile are used as a biomarker. Theoretically, the more independent traits one incorporates into a biomarker, the less likely it will be that the biomarker will be influenced by extraneous/confounding factors. To illustrate this point we can look to the field of iron metabolism. Nutritional iron status can be evaluated by measuring serum ferritin (high ferritin = high iron status) but this parameter is confounded by chronic inflammation (high inflammation = high ferritin) that can mask iron deficiency (Wang et al., 2010). The serum levels of other proteins are also affected by the changes in iron status, e.g. hepcidin (high levels = high iron status) and soluble transferrin receptor (low levels = high iron status). Whereas hepcidin is affected negatively by inflammation (Nemeth and Ganz, 2009), transferrin receptor is not (Beguin, 2003). Thus, by simultaneously assessing the serum levels of ferritin, transferrin receptor, and a serum marker of chronic inflammation (e.g. C-reactive protein), one can assess iron status and remove the confounding caused by the inflammation associated with acute or chronic disease. The approach of using omics to identify measurements that can be combined to make an effective biomarker has been applied to the assessment of certain cancers (Sikaroodi et al., 2010) and some argue that this approach may be useful for the assessment of nutrient status or of nutrition-related conditions that have proved resistant to the single marker approach (e.g. micronutrients such as zinc) (Lowe et al., 2009).

Use of Systems Biology to Define New Modes of Regulation by a Nutrient or Metabolic State

A second way to use systems biology is to identify the groups of genes/transcripts/proteins/metabolites coordinately regulated under specific conditions. These groups could be organized within known biological pathways or as random groupings driven by statistical correlation, i.e. networks that expose new relationships not previously recognized from traditional reductionist research.

Understanding the Systems Biology Approach

It is an over-simplification to imply that there is just one way to do systems biology research but this section will attempt to provide a framework for approaching a nutritional research problem from the systems biology perspective (see for a summary of the steps in the framework).

 An overview of the steps in a systems biology analysis.

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Experimental Design

This is the single most important step of a systems biology research project for several reasons (Allison et al., 2006). First, adequate experimental planning is necessary to focus the research and to use resources efficiently. One will likely need multiple time points to collect data on multiple phenotypes. For example, early time points may be more informative for measurement of direct transcriptional regulation (e.g. using transcriptomics or chromatin immunoprecipitation coupled to high-density DNA sequencing [ChIP-seq]). However, later time points will be more informative for evaluating protein production or changes in metabolism. Second, the use of multiple conditions should be examined so that a broader, more representative view of the regulation can be determined. Work from model systems such as yeast, where a knockout line exists for each of the 6700 yeast genes, shows us that combin­ing transcriptomic analysis for all of these lines permits computer modeling that can reveal new biological relationships and coordination of regulatory processes (Beer and Tavazoie, 2004). Third, sample replication is necessary so that the study has sufficient statistical power to detect biologically important differences between treatments. Finally, the experimental plan should control all extraneous variables so that any changes can be unambiguously attributed to the treatment of interest.

High-density Phenotyping Platforms

Genomics

Gene Promoter Analysis. 

Genetic regulation involves coordinated molecular regulation through transcription factor binding sites within groups of promoters (e.g. molecular regulation of cholesterol and lipid metabolism (Desvergne et al., 2006)). A large number of computational methods are available to locate transcription factor binding sites in mammalian gene promoters (Elnitski et al., 2006). Unfortunately, because transcription factor binding sites tolerate sequence heterogeneity, these computational methods have a high false-positive detection rate (Tompa et al., 2005).

Recently a direct method has been developed to directly determine transcription factor binding sites throughout the genome. This approach starts with a chromatin immunoprecipitation (ChIP) assay where transcription factors are cross-linked to DNA at the site of their binding and the complex is isolated using antibodies to the transcription factor (Collas, 2010). The DNA from the ChIP assay is then used either to probe a genome-wide DNA tiling array (ChIP on chip) or sequenced directly using next-generation sequencing methods (ChIP-seq) (Park, 2009). This approach was recently used to identify 2776 geno­mic positions occupied by the vitamin D receptor (VDR) after treating lymphoblastoid cell lines with 1,25-dihydroxyvitamin D. These VDR binding sites were significantly enriched near autoimmune and cancer-associated genes identified from genome-wide association (GWA) studies (Ramagopalan et al., 2010), suggesting this information will help us understand the relationship between transcriptional regulation and various disease states.

Genetic Mapping and Forward Genetics. 

Forward genetics, the measurement of phenotype and then determining the associations with variations in genotype, is an important approach that has been virtually untapped for the study of nutrient metabolism and function. The basic concept for this approach starts with the fact that natural sequence variations exist within the genome (e.g. single nucleotide polymorphisms or SNP, copy number variations or CNV), and that this variation is heritable. To be useful in forward genetics, these genetic variations must also influence phenotypes, e.g. tissue mineral levels or fatty acid oxidation rate. Finally, unlike the rare mutations that underlie various genetic diseases and cause extreme phenotypes (such as the mutations in copper transporting ATPases responsible for Wilson’s and Menkes disease), the phenotypic changes resulting from the natural variation identified by forward genetics are not fatal but could result in extreme differences between individuals. The goal is to use variations in phenotypes that result from controlled breeding strategies or within pedigrees to map the location of the natural genetic variation that controls the phenotype. The forward genetics approach makes no assumptions about the genes that influence the trait. Rather, it lets variations in phenotype direct us to the regions of the genome containing genetic variants that have a significant biological impact. Forward genetics is particularly useful in instances where we don’t know enough about the metabolism of a nutrient to justify making gene knockout or transgenic mice (i.e. use of reverse genetics) or when mice continue to have normal biology when a candidate gene is deleted (e.g. suggesting redundancies in the system that need to be revealed).

Relating nutritionally important phenotypes to natural variation can be accomplished in two ways: gene mapping and gene association. Linkage analysis within large families and quantitative trait loci (QTL) mapping in controlled crosses between genetically well-characterized inbred mouse lines have been traditionally used to correlate the variation in a phenotype to sequence variations in the genome (Flint et al., 2005). More recently researchers have begun using the genome-wide association (GWA) study approach whereby a multitude of individual variants or haplotypes of variants are examined for their association with a nutritionally relevant trait within large populations of free-living individuals (Manolio, 2010). However, some are concerned that the GWAS approach is subject to false positives and that GWAS findings are difficult to replicate. Regardless of which approach one takes, once the genetic region or candidate polymorphism is identified, additional studies must be conducted to identify the genes that contain the variation controlling the trait, and traditional reductionist research must be done to learn how the genes identified are involved in the regulation of the trait. Because forward genetics approaches are unbiased and hypothesis free, they can lead to the identification of new biological roles for genes and their protein products (Flint et al., 2005).

The promise of forward genetics for nutrition was recently demonstrated for iron metabolism (Wang et al., 2007) where the genes controlling 30% of the variation in spleen iron levels between inbred mouse lines were mapped to chromosome 9. Within this locus, variation was identified in the Mon1a gene and this information was used to determine that Mon1a is a critical component of spleen iron uptake and recycling of red blood cell iron within macrophages. Thus, even though we have learned a tremendous amount about iron metabolism over the last 15 years from traditional approaches (Andrews, 2008), forward genetics permitted researchers to add another piece to this already complex picture.

Epigenomics. 

In addition to the regulation mediated through DNA sequences, DNA and histones can be modified and this will influence gene transcription (Mathers, 2008) (also, see Chapter 2). In humans, DNA is organized into a nucleosome complex with four histone proteins (H2A, H2B, H3, and H4). The amino terminal tails of the histones can be post-translationally modified in a variety of ways. Histone acetylation reduces histone association with DNA and is permissive for gene transcription whereas histone methylation is associated with both transcription repression and activation. DNA can also be methylated at the C5 position of cytosine in regions of DNA called CpG islands. Cytosine- and guanine-rich sequences are located near coding sequences in about 50% of mammalian genes. When the CpG islands are methylated, these regions become more compact and this prevents gene transcription (Attwood et al., 2002). DNA methylation is responsible for X chromosome inactivation, genomic imprinting, and tissue-specific gene transcription that occur during cellular differentiation.

Because of its importance in gene repression, researchers have developed DNA microarrays and next-generation DNA sequencing approaches for epigenetic profiling of CpG islands in the human genome (Fouse et al., 2010). Folate and other micronutrients are involved in the production of the universal methyl donor S-adenosyl methionine, so it has been proposed that dietary inadequacy may have a global influence on DNA methylation (Oommen et al., 2005). However, the evidence for this diet-induced regulatory paradigm is not yet secure.

Transcriptomics

It is now possible to simultaneously measure the primary transcripts and all of the alternatively spliced forms of transcripts produced from each gene in the genome of humans and several model organisms (i.e. the transcriptome). Transcript levels are a reflection of both primary regulation by a treatment and secondary regulation that follows from the initial regulatory events (). There are many high quality options from assessing the transcriptome including spotted cDNA arrays, tiling oligonucleotide arrays, and even direct sequencing of RNA (Kirby et al., 2007; Forrest and Carninci, 2009). Many factors can influence the choice of a transcript profiling platform including cost, reproducibility of results, breadth of transcript coverage, and availability. Readers should consult other reviews for additional discussion of the strengths and weaknesses of various array platforms (Hoheisel, 2006; Kawasaki, 2006).

 A schematic demonstrating the interactions between the various levels of regulation within a cell. Regulatory events occur at the level of transcription (gene space), RNA translation (protein space), protein stability or interactions (protein space), and protein function (gene space, metabolic space). Inorganic elements are involved in all of these processes (ionomic space), and events occurring in one regulatory space can influence events occurring within another regulatory space (e.g. lipid metabolites bind to protein transcription factors with zinc finger DNA binding domains such as PPAR gamma and this interaction regulates gene transcription). Systems biology attempts to model these complex interactions. Reduced omic phenotyping (e.g. in just the gene space or the metabolic space) – combined with previously published research findings – can be used to infer regulatory interactions between these levels.

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For some nutrients that are known to have a direct impact on gene transcription through the activation of a nuclear receptor (e.g. vitamin D and VDR, vitamin A and the retinoic acid receptor bioactive lipids and the peroxisome proliferator-activated receptor, [PPAR]), transcriptomics is a primary endpoint for understanding the impact of the nutrient on biology. A simple example of the value of transcriptomics comes from the area of lipid metabolism. The synthesis of fatty acids and cholesterol is regulated by the sterol regulatory element-binding proteins (SREBP)-1a, -1c, and -2. Horton et al. (2003) used transcriptomics to study transgenic mice overexpressing each SREBP isoform and mice lacking all three nuclear SREBPs (i.e. in SREBP cleavage activating protein (SCAP)-deficient mice). They found several hundred transcripts that were changed in the liver and from this data they defined a subset of 33 genes as SREBP targets that included 20 new SREBP target genes. Thus, in one short, directed set of experiments they dramatically expanded our understanding of how lipid metabolism is regulated.

Proteomics

The proteome refers to all of the proteins expressed and functional in a system. Unfortunately, the methods to assess the proteome cannot measure the entire proteosome simultaneously. As a result, experiments usually measure one or more subproteomes, e.g. the phosphoproteome reflecting the proteins that are targets for protein kinases, proteins within subcellular compartments (e.g. mitochondrial proteome) or specific tissues (serum proteome), or proteins with specific physical properties (e.g. the membrane proteome).

There are two approaches to proteomics. The first is a “top-down” approach whereby whole proteins are studied using multidimensional separation techniques, e.g. separation by isoelectric point followed by size separation (2D polyacrylamide gel electrophoresis [PAGE]) or tandem mass spectrometry (Reid and McLuckey, 2002). In the top-down approach the proteins are isolated, then fragmented (e.g. through trypsin digestion), and the peptide fragments are compared with a database to determine the identity of the protein. In contrast, the “bottom-up” approach digests proteins at the outset, isolates and identifies the peptide fragments using mass spectrometry methods, and then relates the peptide fragments to databases of known proteins to determine the identity of the proteins in a complex mixture.

The major challenge to proteomics is that the spectrometry methods are not standardized. This leads to problems of reproducibility across and within labs. In addition, there are some challenges to separating signal from noise that limit peak detection and quantification. Finally, some proteomic methods are not very sensitive. Specifically, 2D PAGE approaches are often used for serum proteomics and biomarker discovery. However, the ability of radioimmunoassay to detect proteins in serum is 100- to 1000-fold greater than 2D PAGE methods. Even with this weakness, 2D PAGE has been useful for identifying serum biomarkers of nutritional status. For example, Fuchs et al. (2007) used this approach to identify biomarkers of a cardioprotective response to isoflavone supplementation in the peripheral blood mononuclear cells of postmenopausal women.

Metabolomics

Evaluating the metabolome gives a snapshot of the physiology of a cell or organism by simultaneously measuring the levels of metabolites within a biological space (also, see Chapter 4). Like proteomics, the metabolome is assessed by coupling separation techniques (e.g. electrophoresis, chromatography) with sophisticated detection methods (e.g. mass spectro­metry, nuclear magnetic resonance imaging). As such, it suffers from the same problems as proteomics, namely the lack of method standardization and reproducibility. Also, like proteomics, the entire metabolome is too complex for one method to measure all possible metabolites simultaneously, and so submetabolomes based on location or chemical characteristics are commonly analyzed. While many studies use metabolomics for biomarker discovery, this approach can also be used to better understand the impact of physiologic conditions on the flow of information through specific metabolic pathways. For example, in a study of diet-induced insulin resistance, Li et al. (2010) identified many serum and liver metabolites as different between safflower-oil-fed wild-type and glycerol-3-phosphate acyltransferase deficient mice. Many of these were not previously known to be associated with insulin resistance and they point to the utility of metabolomics analysis for identifying biochemical pathways important in understanding the pathophysiology of diabetes. In addition to the standard metabolomic approach that measured changes in steady-state levels of metabolites, others have used radio- or stable isotopes to label compounds and trace their metabolic fate. This is a more dynamic approach that can give a picture of how physiologic states or treatments affect the flow of compounds through specific metabolic pathways (Hellerstein, 2004).

Ionomics

Mineral elements are involved at all levels of biological regulation, e.g. in transcription factors (zinc), in enzymes (zinc, iron, copper, calcium), and in establishing electrochemical gradients in cells (calcium, sodium, potassium). It is also well established that direct and indirect interactions exist between mineral elements that can affect biology (Hill and Matrone, 1970). Because the mineral elements are integrated into the overall biology of a cell (i.e. with links to the metabolome, proteome, transcriptome, and ultimately the genome: et al.