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Analysis in Nutrition
Research
This page intentionally left blank
Analysis in Nutrition
Research
Principles of Statistical Methodology
and Interpretation of the Results
Edited by
George Pounis
Nutrition Consultant in the Greek Food Industry, Athens, Greece
Academic Press is an imprint of Elsevier
125 London Wall, London EC2Y 5AS, United Kingdom
525 B Street, Suite 1650, San Diego, CA 92101, United States
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Contents
List of Contributors ............................................................................................................................. xv
Preface ................................................................................................................................................ xix
Acknowledgments............................................................................................................................... xxi
PART 1 ANALYSIS IN NUTRITION RESEARCH

CHAPTER 1 Design of Observational Nutrition Studies........................................... 3
George Pounis
1.1 Introduction to Observational Nutrition Studies ........................................................3
1.2 Ecological Nutrition Studies.......................................................................................5
1.2.1 Description........................................................................................................ 5
1.2.2 Challenges ........................................................................................................ 5
1.2.3 Example of Ecological Study on Diet and Cancer.......................................... 6
1.3 Cross-Sectional Nutrition Studies ..............................................................................6
1.3.1 Description........................................................................................................ 6
1.3.2 Sampling........................................................................................................... 8
1.3.3 Challenges ...................................................................................................... 10
1.3.4 Example of the National Health and Nutrition Examination Survey ........... 11
1.4 CaseeControl Nutrition Studies...............................................................................12
1.4.1 Description...................................................................................................... 12
1.4.2 Sampling......................................................................................................... 12
1.4.3 Challenges ...................................................................................................... 14
1.4.4 An Example in the Study of Hodgkin Lymphoma........................................ 15
1.5 Cohort Nutrition Studies...........................................................................................15
1.5.1 Description...................................................................................................... 15
1.5.2 Sampling......................................................................................................... 16
1.5.3 Challenges ...................................................................................................... 17
1.5.4 Example of the Nurses’ Health Study ........................................................... 18
References........................................................................................................................ 19
CHAPTER 2 Study Design in Experimental Settings .............................................. 23
Monica Dinu, George Pounis and Francesco Sofi
2.1 Introduction...............................................................................................................24
2.2 Experimental Designs ...............................................................................................24
2.2.1 Single-Arm Studies ........................................................................................ 25
2.2.2 Parallel Studies ............................................................................................... 25
2.2.3 Crossover Studies ........................................................................................... 27
2.3 Planning a Dietary Intervention ...............................................................................29
2.3.1 Definition of the Research Question, Hypothesis, and Main Objectives...... 30
v
vi CONTENTS
2.3.2 Primary and Secondary Outcomes................................................................. 30

2.3.3 Study Population ............................................................................................ 30
2.3.4 Selection of the Study Design........................................................................ 35
2.3.5 Measurements................................................................................................. 35
2.3.6 Study Protocol ................................................................................................ 35
2.4 Conducting the Dietary Intervention........................................................................37
2.4.1 Recruitment and Screening of Participants.................................................... 37
2.4.2 Compliance of Participants ............................................................................ 37
2.4.3 Data Management........................................................................................... 38
2.4.4 Statistical Analysis ......................................................................................... 39
2.5 Conclusions ...............................................................................................................39
References.........................................................................................................................39
Further Reading ................................................................................................................41
CHAPTER 3 Collection and Management of Dietary Data ..................................... 43
Pauline M. Emmett, Louise R. Jones, Kate Northstone, George Pounis and
Caroline M. Taylor
3.1 Introduction...............................................................................................................44
3.2 Dietary Information ..................................................................................................45
3.2.1 Methods for Collecting Dietary Information................................................. 45
3.2.2 Choice of Method........................................................................................... 54
3.2.3 Types of Collected Dietary Information ........................................................ 54
3.3 Data Management and Dietary Analysis..................................................................57
3.3.1 Portion Sizes................................................................................................... 57
3.3.2 Estimations of Nutrient Content of Foods..................................................... 58
3.3.3 Configuration and Labeling of Datasets ........................................................ 60
3.3.4 Handling of Food Group Data ....................................................................... 61
3.4 Data Manipulation ....................................................................................................64
3.4.1 Validation, Reproducibility, Calibration, and Biomarkers ............................ 64
3.4.2 Measurement Error, Misreporting, and Outliers............................................ 65
3.4.3 Energy Adjustment......................................................................................... 67
References ........................................................................................................................ 67
CHAPTER 4 Dietary Pattern Analysis ..................................................................... 75
Claudia Agnoli, George Pounis and Vittorio Krogh
4.1 Introduction...............................................................................................................76
4.2 Types of Dietary Pattern Analysis............................................................................77
4.3 A Priori Dietary Pattern Analysis.............................................................................77
4.3.1 Description...................................................................................................... 77
4.3.2 Key Aspects for the Development of a Dietary Score .................................. 79
4.3.3 Dietary Assessment Methodologies for a Priori Dietary Pattern Analysis... 81
CONTENTS vii
4.3.4 Evaluation of the Quality of an a Priori Dietary Pattern .............................. 81

4.3.5 Examples of a Priori Dietary Patterns ........................................................... 82
4.4 A Posteriori Dietary Pattern Analysis ......................................................................86
4.4.1 Description...................................................................................................... 86
4.4.2 Factor Analysis............................................................................................... 86
4.4.3 Examples of Factor Analysis ......................................................................... 89
4.4.4 Cluster Analysis ............................................................................................. 89
4.4.5 Examples of Cluster Analysis ........................................................................ 91
4.4.6 Comparison of Factor and Cluster Analysis.................................................. 91
4.4.7 Reproducibility and Validity of a Posteriori Dietary Pattern Analysis......... 92
4.5 Hybrid Dietary Pattern Analysis ..............................................................................94
4.5.1 Description...................................................................................................... 94
4.5.2 Examples of Hybrid Dietary Pattern Analysis .............................................. 94
4.6 Challenges in Dietary Pattern Analysis....................................................................95
References.........................................................................................................................96
CHAPTER 5 Statistical Analysis of Retrospective Health and Nutrition Data.... 103
George Pounis
5.1 Introduction...........................................................................................................104
5.2 Hypothesis Testing................................................................................................105
5.3 Descriptive Statistics ............................................................................................106
5.3.1 Categorical Data .........................................................................................107
5.3.2 Continuous Data .........................................................................................107
5.4 Assessment of Normality .....................................................................................111
5.5 Confidence Interval...............................................................................................113
5.6 Pearson Chi-Square Test.......................................................................................115
5.7 Statistical Tests for Comparison of Means ..........................................................117
5.7.1 t Test ...........................................................................................................117
5.7.2 One-Way Analysis of Variance ..................................................................118
5.8 Pearson Correlation Coefficient ...........................................................................120
5.9 Nonparametric Tests .............................................................................................121
5.10 Linear Regression Analysis..................................................................................122
5.10.1 Simple Linear Regression ........................................................................122
5.10.2 Multiple Linear Regression Analysis.......................................................130
5.11 Logistic Regression Analysis ...............................................................................135
5.11.1 Odds Ratio ................................................................................................135
5.11.2 Simple Binary Logistic Regression Analysis ..........................................136
5.11.3 Multiple Binary Logistic Regression Analysis........................................137
5.11.4 Examples ..................................................................................................138
References.......................................................................................................................140
viii CONTENTS
CHAPTER 6 Statistical Analysis of Prospective Health and Nutrition Data ....... 145
George Pounis
6.1 Introduction.............................................................................................................145
6.2 Descriptive Statistics...............................................................................................146
6.3 Measures to Calculate the Occurrence of a Health Outcome................................146
6.3.1 Incidence....................................................................................................... 146
6.3.2 Relative Risk ................................................................................................ 148
6.4 Survival Analysis ....................................................................................................149
6.4.1 Basic Concepts ............................................................................................. 149
6.4.2 KaplaneMeier Analysis ............................................................................... 150
6.4.3 Log-Rank Test .............................................................................................. 152
6.4.4 Cox Regression Analysis ............................................................................. 155
References ...................................................................................................................... 159
CHAPTER 7 Meta-Analysis of Nutrition Studies .................................................. 163
Emmanouil Bouras, Konstantinos K. Tsilidis, George Pounis and
Anna-Bettina Haidich
7.1 Introduction.............................................................................................................164
7.2 Methodology of Meta-Analysis in Nutrition Research..........................................165
7.2.1 Defining the Search Strategy........................................................................ 166
7.2.2 Study Selection Procedure ........................................................................... 169
7.2.3 Quality Assessment ...................................................................................... 169
7.2.4 Data Extraction............................................................................................. 170
7.3 Statistical Methodologies Applied in Meta-Analysis of Nutrition Studies ...........170
7.3.1 Statistical Measures of Effect Included in Meta-Analysis .......................... 171
7.3.2 Choice of Meta-Analytical Method ............................................................. 173
7.3.3 Statistical Heterogeneity .............................................................................. 175
7.3.4 Small-Study Effects...................................................................................... 177
7.3.5 Software for Meta-Analysis ......................................................................... 178
7.4 Presentation and Interpretation of Results .............................................................178
7.4.1 Study Selection............................................................................................. 179
7.4.2 Study Characteristics.................................................................................... 179
7.4.3 Forest Plot..................................................................................................... 179
7.4.4 Assessing Heterogeneity .............................................................................. 182
7.4.5 Risk for Bias................................................................................................. 184
7.4.6 Funnel Plot ................................................................................................... 184
7.5 Limitations and Biases............................................................................................187
7.5.1 Challenges .................................................................................................... 189
References ...................................................................................................................... 191
CONTENTS ix
CHAPTER 8 Principles of Research Publication ................................................. 197

Gregory S. Patience, George Pounis, Paul A. Patience and Daria C. Boffito
8.1 Introduction.............................................................................................................198
8.2 Citation Impact and Metrics ...................................................................................198
8.3 Article Elements .....................................................................................................200
8.3.1 Title............................................................................................................... 200
8.3.2 Abstract......................................................................................................... 203
8.3.3 Introduction .................................................................................................. 204
8.3.4 Methods and Results .................................................................................... 205
8.3.5 Discussion and Conclusions......................................................................... 207
8.4 Web Tools for Writing............................................................................................207
8.4.1 Word Choices ............................................................................................... 208
8.4.2 Thesauri ........................................................................................................ 208
8.4.3 Grammar ....................................................................................................... 210
8.4.4 Translation .................................................................................................... 211
8.5 Reporting Data and Analysis..................................................................................211
8.5.1 Dietary Evaluation and Analysis.................................................................. 212
8.5.2 Statistical Analysis ....................................................................................... 212
8.5.3 Graphs........................................................................................................... 213
8.5.4 Tables............................................................................................................ 217
8.6 Publishing Process ..................................................................................................218
8.6.1 Selecting the Journal .................................................................................... 218
8.6.2 A Winning Cover Letter ............................................................................... 218
8.7 Authorship Criteria and Acknowledgments ...........................................................220
8.8 Strengthening the Reporting of Observational Studies in Epidemiology and
Consolidated Standards of Reporting Trials Statements........................................223
8.9 Conclusions .............................................................................................................224
References.......................................................................................................................225
PART 2 CHALLENGES IN NUTRITION SCIENCE

CHAPTER 9 Mediterranean Diet: A Health-Protective Dietary Pattern
for Modern Times .............................................................................. 233
Dimitra Mastorakou, Mikael Rabaeus, Patricia Salen, George Pounis and
Michel de Lorgeril
9.1 Introduction.............................................................................................................234
9.2 Scientific Definition of Mediterranean Diet...........................................................234
9.2.1 Historical Overview...................................................................................... 234
9.2.2 The Traditional Mediterranean Diet ............................................................ 235
x CONTENTS
9.3 Evidence on Health Benefits of the Mediterranean Diet .......................................235

9.3.1 Cardiovascular Diseases ............................................................................... 235
9.3.2 Cancer........................................................................................................... 236
9.3.3 Cognitive Function ....................................................................................... 237
9.3.4 Nonalcoholic Fatty Liver Disease................................................................ 237
9.3.5 Rheumatoid Arthritis.................................................................................... 238
9.3.6 Pulmonary Function ..................................................................................... 238
9.4 Food Components of Mediterranean Diet..............................................................239
9.4.1 Fish and Marine Omega-3 Polyunsaturated Fats......................................... 239
9.4.2 Plant Omega-3 Fats ...................................................................................... 240
9.4.3 Olive Oil ....................................................................................................... 241
9.4.4 Fruits and Vegetables ................................................................................... 241
9.4.5 Nuts and Seeds ............................................................................................. 242
9.4.6 Dietary Fiber................................................................................................. 243
9.4.7 Wine.............................................................................................................. 244
9.5 Mediterranean Diet Adherence in Modern Times .................................................244
9.5.1 Level of Adherence in Modern Populations................................................ 244
9.5.2 Challenges of Adhering to the Mediterranean Diet .................................... 246
9.6 Shifting to the Mediterranean Diet in the Modern Context ..................................246
9.6.1 Updated Mediterranean Diet Recommendations ......................................... 246
9.6.2 Focus on Sustainability ................................................................................ 248
9.7 Conclusions .............................................................................................................250
References.......................................................................................................................250
CHAPTER 10 Polyphenol-Rich Diets in Cardiovascular Disease Prevention ..... 259
Junichi Sakaki, Melissa Melough, Sang Gil Lee, George Pounis and
Ock K. Chun
10.1 Introduction...........................................................................................................260
10.2 Polyphenols: Classes, Structures, and Chemical Properties ................................261
10.3 Polyphenols in Human Diet .................................................................................261
10.3.1 Major Dietary Sources .............................................................................261
10.3.2 Analytical Techniques to Determine Phenolic Contents .........................264
10.3.3 Estimation of Polyphenol Intakes in Human Populations.......................265
10.4 Dietary Polyphenols and Cardiovascular Disease ...............................................267
10.4.1 Cardiovascular Disease Incidence and Mortality: Evidence From
Epidemiological Studies...........................................................................267
10.4.2 Cardiovascular Disease Biomarkers: Evidence From
Epidemiological Studies...........................................................................272
10.4.3 Cardiovascular Disease Biomarkers: Evidence From Interventional
Studies ......................................................................................................274
CONTENTS xi
10.4.4 Biological Functions of Polyphenols in Cardiovascular Disease

Prevention .................................................................................................280
10.5 Challenges in Polyphenol Research .....................................................................287
10.6 Conclusion ............................................................................................................288
References.......................................................................................................................289
Chemspider Websites......................................................................................................298
CHAPTER 11 Hydration and Health...................................................................... 299
Adam D. Seal, Hyun-Gyu Suh, Lisa T. Jansen, LynnDee G. Summers and
Stavros A. Kavouras
11.1 Introduction...........................................................................................................300
11.1.1 Terminology and Measurement of Hydration Status...............................300
11.2 Hydration and Kidney Health ..............................................................................301
11.2.1 Introduction ..............................................................................................301
11.2.2 Hydration and Kidney Function...............................................................302
11.2.3 Arginine Vasopressin and Chronic Kidney Disease ................................302
11.2.4 Mesoamerican Nephropathy.....................................................................303
11.2.5 Other Kidney Diseases .............................................................................303
11.2.6 Water Intake Intervention in Chronic Kidney Disease............................304
11.3 Hydration and Glucose Regulation ......................................................................304
11.3.1 Introduction ..............................................................................................304
11.3.2 Implications of Arginine Vasopressin in Glucose Regulation.................304
11.3.3 Other Considerations in Hydration and Glucose Regulation ..................306
11.4 Implications of Fluid Balance and Obesity .........................................................306
11.4.1 Introduction ..............................................................................................306
11.4.2 Sugar-Sweetened Beverages ....................................................................307
11.4.3 Hydration Status and Obesity ..................................................................307
11.5 Hydration and Cardiovascular Health ..................................................................309
11.5.1 Introduction ..............................................................................................309
11.5.2 Cardiovascular Effects..............................................................................310
11.6 Hydration and Oxidative Stress............................................................................310
11.6.1 Introduction ..............................................................................................310
11.6.2 Implications of Poor Fluid Balance .........................................................311
11.7 Hydration and Risk of Bladder Cancer................................................................311
11.7.1 Introduction ..............................................................................................311
11.7.2 Possible Causes ........................................................................................312
11.8 Conclusion ............................................................................................................312
References.......................................................................................................................313
xii CONTENTS
CHAPTER 12 Diet, Healthy Aging, and Cognitive Function................................. 321

Krasimira Aleksandrova, George Pounis and Romina di Giuseppe
12.1 Introduction...........................................................................................................321
12.2 Definition and Epidemiology of Cognitive Decline ............................................322
12.3 Bioactive Components of a Healthy Diet and Cognitive Decline.......................323
12.3.1 Antioxidant Vitamins ...............................................................................323
12.3.2 Polyphenols ..............................................................................................323
12.3.3 Vitamins of the B Complex .....................................................................324
12.3.4 Unsaturated Fatty Acids ...........................................................................325
12.3.5 Minerals ....................................................................................................326
12.4 Dietary Patterns and Cognitive Decline...............................................................326
12.4.1 The Mediterranean Diet ...........................................................................327
12.4.2 The DASH Diet Plan................................................................................327
12.4.3 The MIND Diet ........................................................................................327
12.5 Modern Approaches in the Study of Diet and Cognitive Health ........................328
12.6 Challenges.............................................................................................................329
12.7 Conclusion ............................................................................................................330
References.......................................................................................................................330
CHAPTER 13 Diet and Bone Health...................................................................... 337
Kate Maslin and Elaine Dennison
13.1 Introduction...........................................................................................................338
13.2 Brief Overview of Bone Physiology ....................................................................338
13.2.1 Overview of Bone Structure and Function ..............................................338
13.2.2 Bone Modeling and Remodeling .............................................................338
13.2.3 Assessment of Bone Outcomes in Research............................................339
13.3 Calcium and Vitamin D........................................................................................339
13.3.1 Calcium and Bone, an Overview .............................................................339
13.3.2 Vitamin D and Bone, an Overview..........................................................340
13.3.3 Phosphorus................................................................................................341
13.3.4 Magnesium ...............................................................................................342
13.3.5 Potassium..................................................................................................343
13.3.6 Vitamin K .................................................................................................343
13.3.7 Other Nutrients .........................................................................................343
13.4 Life Course Perspective on Nutrition and Bone ..................................................344
13.4.1 Osteoporosis: Burden and Epidemiology ................................................344
13.4.2 Etiology of Osteoporosis and Role of Diet .............................................344
13.4.3 Maternal Nutrition and Bone Outcomes..................................................345
13.4.4 Childhood and Adolescence.....................................................................346
13.4.5 Bone Health in the Older Adult: Menopause and Beyond .....................348
13.5 Challenges in Nutrition and Bone Research ........................................................351
References.......................................................................................................................352
CONTENTS xiii
CHAPTER 14 Diet and Lung Health ...................................................................... 355

Foteini Malli, Themis Koutsioukis, George Pounis and
Konstantinos I. Gourgoulianis
14.1 Introduction ........................................................................................................356
14.2 Diet and Pulmonary Function ............................................................................357
14.3 Diet and Asthma.................................................................................................357
14.3.1 Dietary Patterns ......................................................................................358
14.3.2 Vitamins..................................................................................................358
14.3.3 Minerals ..................................................................................................359
14.3.4 Fatty Acids..............................................................................................359
14.3.5 Probiotics ................................................................................................359
14.3.6 Phytochemicals.......................................................................................360
14.4 Diet and Chronic Obstructive Pulmonary Disease ............................................360
14.4.2 Vitamins..................................................................................................360
14.4.3 Minerals ..................................................................................................361
14.4.4 Fatty Acids..............................................................................................361
14.4.5 Probiotics and Dietary Fibers.................................................................361
14.4.6 Phytochemicals.......................................................................................362
14.5 Diet and Lower Respiratory Tract Infections ....................................................362
14.5.1 Vitamins..................................................................................................363
14.5.2 Minerals ..................................................................................................364
14.5.3 Fatty Acids..............................................................................................364
14.5.4 ProbioticsePrebiotics .............................................................................364
14.5.5 Phytochemicals.......................................................................................365
14.6 Diet and Tuberculosis.........................................................................................365
14.6.2 Vitamins..................................................................................................365
14.6.3 Minerals ..................................................................................................366
14.6.4 Fatty Acids..............................................................................................366
14.6.5 Probiotics ................................................................................................366
14.6.6 Phytochemicals.......................................................................................366
14.7 Diet and Lung Cancer ........................................................................................367
14.7.2 Vitamins..................................................................................................367
14.7.3 Minerals ..................................................................................................368
14.7.4 Fatty Acids..............................................................................................368
14.7.5 Phytochemicals.......................................................................................368
14.8 Diet and Cystic Fibrosis.....................................................................................369
14.8.1 Vitamins..................................................................................................369
xiv CONTENTS
14.8.2 Minerals ..................................................................................................369

14.8.3 Fatty Acids..............................................................................................370
14.8.4 Probiotics ................................................................................................370
14.8.5 Phytochemicals.......................................................................................370
14.9 Diet and Interstitial Lung Diseases....................................................................370
14.10 Maternal Diet in Early Life and Lung Health ...................................................371
14.10.1 Dietary Patterns ....................................................................................371
14.10.2 Vitamins................................................................................................371
14.10.3 Fatty Acids............................................................................................372
14.10.4 Probiotics ..............................................................................................372
14.11 Challenges in Diet and Lung Health Research..................................................372
References.......................................................................................................................373
Further Reading ..............................................................................................................381
Index ................................................................................................................................................. 383
List of Contributors
Claudia Agnoli
Epidemiology and Prevention Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
Krasimira Aleksandrova
Nutrition, Immunity and Metabolism Senior Scientist Group, Department of Nutrition and
gerontology, German Institute of Human Nutrition Potsdam-Rehbruecke (DIfE), Nuthetal,
Germany
Daria C. Boffito
Department of Chemical Engineering, Polytechnique Montréal, Montréal, QC, Canada
Emmanouil Bouras
Department of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece
Ock K. Chun
Department of Nutritional Sciences, University of Connecticut, Storrs, CT, United States
Michel de Lorgeril
Laboratoire Cœur et Nutrition, TIMC-IMAG, School of Medicine, University of Grenoble-Alpes,
Grenoble, France
Elaine Dennison
MRC Lifecourse Epidemiology Unit University of Southampton, United Kingdom
Romina di Giuseppe
Institute of Epidemiology, University Kiel, Kiel, Germany
Monica Dinu
Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy
Pauline M. Emmett
Centre for Academic Child Health, Population Health Sciences, Bristol Medical School,
University of Bristol, Bristol, United Kingdom
Konstantinos I. Gourgoulianis
Respiratory Medicine Department, University of Thessaly, School of Medicine, Larissa Greece
Anna-Bettina Haidich
Department of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece
Lisa T. Jansen
University of Arkansas, Fayetteville, AR, United States
Louise R. Jones
Population Health Sciences, Bristol Medical School, University of Bristol, Bristol, United
Kingdom
Stavros A. Kavouras
Hydration Science Lab, Arizona State University, Phoenix, AZ, United States
xv
xvi LIST OF CONTRIBUTORS
Themis Koutsioukis
Vittorio Krogh
Epidemiology and Prevention Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy
Sang Gil Lee
Department of Food Science and Nutrition, Pukyong National University, Busan, Korea
Foteini Malli
Technological Institute of Thessaly, Nursing Department, Larissa, Greece
Kate Maslin
MRC Lifecourse Epidemiology Unit University of Southampton, United Kingdom
Dimitra Mastorakou
Leicester, United Kingdom
Melissa Melough
Kate Northstone
Population Health Sciences, Bristol Medical School, University of Bristol, Bristol, United
Kingdom
Gregory S. Patience
Department of Chemical Engineering, Polytechnique Montréal, Montréal, QC, Canada
Paul A. Patience
Department of Electrical Engineering, Polytechnique Montréal, Montréal, QC, Canada
George Pounis
Alimos, Athens, Greece
Mikael Rabaeus
Geneva, Switzerland
Junichi Sakaki
Patricia Salen
Laboratoire Cœur et Nutrition, TIMC-IMAG, School of Medicine, University of Grenoble-Alpes,
Grenoble, France
Adam D. Seal
Francesco Sofi
Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy
Unit of Clinical Nutrition, Careggi University Hospital, Florence, Italy
Don Carlo Gnocchi Foundation, Onlus IRCCS, Florence, Italy
LIST OF CONTRIBUTORS xvii
Hyun-Gyu Suh
LynnDee G. Summers
Caroline M. Taylor
Centre for Academic Child Health, Population Health Sciences, Bristol Medical School,
University of Bristol, Bristol, United Kingdom
Konstantinos K. Tsilidis
University of Ioannina School of Medicine, Ioannina, Greece
Preface
Analysis in Nutrition Research: Principles of Statistical Methodology and Interpretation of the Results
describes, in a comprehensive manner, the methodologies of the quantitative analysis of data origi-
nating specifically from nutrition studies. The book summarizes various study designs in nutrition
research, research hypotheses, the proper management of dietary data, and analytical methodologies,
with a specific focus on how to interpret the results of any given study. In addition, it provides a
comprehensive overview of the methodologies used in study design and the management and analysis
of collected data, paying particular attention to all of the available, modern methodologies and
techniques.
Readers will find an overview of the recent challenges and debates in the field of nutrition research
that will define major hypotheses for research in the next 10 years. Nutrition scientists, researchers,
and undergraduate and postgraduate students will benefit from this thorough publication on the topic.
xix
Acknowledgments
It is a pleasure to thank all of the people who made this title possible.
I am grateful to the contributors who kindly participated in preparing the various chapters of this
book and shared with us their knowledge, experience, and expertise.
I would not have achieved this ambitious goal without the support of Elsevier and I wish to thank
Megan Ball, who trusted me with the editorial role.
I want to extend my sincere gratitude to my colleagues from Elsevier, who supported me in the
various steps of this publication.
My final thoughts go to my family and Eleftheria. I would not have gone so far without their love
and encouragement.
Georgios
xxi
PART
ANALYSIS IN
NUTRITION
RESEARCH 1
CHAPTER
DESIGN OF OBSERVATIONAL
NUTRITION STUDIES
1
George Pounis
Alimos, Athens, Greece
CHAPTER OUTLINE
1.1 Introduction to Observational Nutrition Studies...................................................................................3
1.2 Ecological Nutrition Studies..............................................................................................................5
1.2.1 Description ..................................................................................................................5
1.2.2 Challenges...................................................................................................................5
1.2.3 Example of Ecological Study on Diet and Cancer.............................................................6
1.3 Cross-Sectional Nutrition Studies ......................................................................................................6
1.3.1 Description ..................................................................................................................6
1.3.2 Sampling.....................................................................................................................8
1.3.3 Challenges.................................................................................................................10
1.3.4 Example of the National Health and Nutrition Examination Survey .................................11
1.4 CaseeControl Nutrition Studies .......................................................................................................12
1.4.1 Description ................................................................................................................12
1.4.2 Sampling...................................................................................................................12
1.4.3 Challenges.................................................................................................................14
1.4.4 An Example in the Study of Hodgkin Lymphoma ...........................................................15
1.5 Cohort Nutrition Studies..................................................................................................................15
1.5.1 Description ................................................................................................................15
1.5.2 Sampling...................................................................................................................16
1.5.3 Challenges.................................................................................................................17
1.5.4 Example of the Nurses’ Health Study ...........................................................................18
References ............................................................................................................................................19
1.1 INTRODUCTION TO OBSERVATIONAL NUTRITION STUDIES

The science of human nutrition that is frequently described as “nutrition science” or “nutrition” is the
science of food, the nutrients and other substances therein, their action, interaction and balance in
relation to health and disease, and the processes by which the human organism ingests, absorbs,
transports, utilizes and excretes food substances [1,2].
Analysis in Nutrition Research. https://doi.org/10.1016/B978-0-12-814556-2.00001-4

Copyright © 2019 Elsevier Inc. All rights reserved.
3
4 CHAPTER 1 DESIGN OF OBSERVATIONAL NUTRITION STUDIES
Systematic observation along with accurate and systematic measurement has been the basis for the
development of science in various fields from mathematics and physics to sociology and medicine.
Although nutrition science is relatively young compared with others, it has been firmly tied to
observation and the measurement since the mid-1950s, when the first evidence-based conclusions
regarding the association of nutrition and health were determined [1e13].
As part of nutrition science, nutritional epidemiology incorporates valuable information related to
the methodologies and techniques of observation and measurement applied to generate evidence-based
conclusions regarding the interaction between diet and health, among others. A considerable pro-
portion of dietary risk factors for various diseases have been identified and studied through epide-
miological health and nutrition studies.
An observational nutrition study may be defined as the detailed investigation and analysis of in-
formation provided by a retrospective or prospective systematic observation and measurement of a
sample’s dietary factors (exposures) and health characteristics (outcomes), in which the researcher
does not willingly influence the collected information.
Observational nutrition studies have an epidemiological character and aim to generalize conclu-
sions derived from the investigation and analysis of sample data from the reference population [6,14].
They are often named population-based studies. According to the nature of the collected data, they are
divided into retrospective and prospective studies [6,14,15]. Types of retrospective surveys include
ecological, cross-sectional, and caseecontrol studies whereas prospective surveys include follow-up
and longitudinal studies, which are generally labeled cohort studies.
All of these study designs provide valuable descriptive information about adherence to dietary
patterns, the consumption of foods and nutrients, the presence of certain dietary behaviors, and other
dietary characteristics in a population base. In addition, they aim to test hypotheses related to the
association of dietary exposures with health outcomes. Although by themselves these studies are not
enough to prove a cause-and-effect relationship between a dietary factor and a health outcome, as-
sociations observed mainly in cohort surveys assign the potential existence of causality. In these cases,
further investigation through intervention trials and meta-analysis is essential when it is ethically and
technologically feasible.
Evidence-based nutrition science or practice is well-promoted as the preferable and most accurate
methodology to make decisions in all related disciplines; it aims to maintain or improve the health of
individuals, groups, and populations [10]. Observational nutrition studies are a first and important step
in the evidence-based concluding chain of nutrition science.
All of the various types of observational nutrition studies have certain advantages and limitations
that derive from their nature, and which should be considered throughout the stages of the survey (i.e.,
design, implementation, analysis, presentation, and publication of results and conclusions). In addi-
tion, the methodology of the collection, the management and statistical analysis of data, and the
presentation and interpretation of the results vary among the different designs. These issues are
addressed in other chapters of the book (i.e., Chapters 3e8).
The following sections offer an overview of the various types of observational nutrition studies
illustrating their basic concepts and design methodologies, providing survey examples from the
literature.
1.2 ECOLOGICAL NUTRITION STUDIES 5
1.2 ECOLOGICAL NUTRITION STUDIES

1.2.1 DESCRIPTION
An ecological nutrition study is the first level of systematic and comparative observation and mea-
surement of the dietary characteristics of large populations (mainly geographically oriented), usually
in parallel with the systematic and comparative observation and measurement of health-related indices
in the same populations [6]. The experience of variations in an index of health status between pop-
ulations with variations in the average value of a dietary factor introduces the concept of the potential
association between the dietary exposure and the health outcome. This remains to be confirmed in
other observational and experimental settings.
Implementation of this type of survey requires at least that a population-based measure of a dietary
factor and an index of health status be available for two or more populations. For instance, a country
with 20% per capita intake of calories from fats has a lower incidence of colon cancer than does a
country with 45% per capita intake of calories from fats. This introduces the concept that the con-
sumption of fat in these populations may have a role in explaining the variation presented in their
incidences of colon cancer.
Although the major aim of ecological studies is to characterize populations over a dietary factor
and an index of health status rather than evaluating the association between a dietary exposure and a
health outcome [14]. More often, this survey type is used to explore geographical differences in the
diet and health of large populations. It is also used to compare changes in diet and the health status of
populations over time. Sometimes an ecological study is the only design that can be planned if the
dietary data of the study population are unavailable at an individual level.
An advantage of this survey type is that the average population-based diets and the per capita
consumption of food tend to be stable over time [14]. Moreover, the indices of diseases are mainly
derived from large samples and are under limited biases. For these reasons, it can be hypothesized that
variations in health indices and dietary factors in the studied populations may have longer time of pre-
occurrence.
However, a serious limitation is that the same variations can also be attributed to determinants of
disease other than dietary, such as genetic, environmental, clinical, and lifestyle. This poses a major
disadvantage of ecological studies along with the limited opportunity to reproduce results, especially
for international surveys.
Despite the inability of this study design to validate a cause-and-effect relationship between a
dietary exposure and a health outcome, it has been proved to be effective in generating scientific
hypotheses for further observational and experimental studies.
1.2.2 CHALLENGES
Among the major concerns for the designers of an ecological nutrition study is the identification of
appropriate population-based measures of the dietary characteristics and health status of the pop-
ulations under investigation.
Estimates of the population dietary intake are often retrieved from preexisting data generated by
systematic measures and evaluations performed for other reasons (i.e., economic studies, census).
Sources of dietary information include national and international data about the per capita
6 CHAPTER 1 DESIGN OF OBSERVATIONAL NUTRITION STUDIES
consumption of foods, data from household surveys, and individual survey data from representative
sample populations.
An important consideration for the survey’s investigators is a study of the methodology used for
data collection and management. The use harmonized national and international population-based
dietary data ensures their comparability. This purpose is assisted by using common data collection
and management methodologies.
Another challenge to conducting an ecological study is evaluating the level of accuracy of
population-based dietary data. Biases related to the methodology of data acquisition should be limited.
For instance, frequently, the data for the per capita consumption of food in a country are retrieved by
economic studies for food sales, imports, and exports. Because of their aims and methodology, these
surveys cannot control for the amount of food that is wasted. It may be true that a significant amount of
food that has been sold to consumers was not actually consumed. The percentage of wasted food
among countries with different socioeconomical standings may vary significantly. This may lead to
biased conclusions regarding variations observed in food consumption and the health status of pop-
ulations under investigation.
1.2.3 EXAMPLE OF ECOLOGICAL STUDY ON DIET AND CANCER

In 1975, Armstrong and Doll published findings of their ecological study on the association of dietary
factors with the incidence of various types of cancer and cancer mortality rates [16]. This was one of
the first observational studies on the topic, and despite the methodological limitations derived from its
design, it has been a reference work for further studies on the topic (i.e., more than 1700 citations in
Scopus Metrics).
The researchers used data on the incidence rate of 27 cancer types in 23 countries for individuals
aged 35e64 years, derived from the Union for International Cancer Control in 1966 and 1970 [17,18].
Data on the cancer mortality rates for 14 cancer types in 32 countries were taken from Segi et al. and
the World Health Organization (1967e69, 1970) [19e21]. This information was studied in association
with data on the international per capita consumption of various foods derived from various reference
sources.
The results of the study were presented grouped by the type of cancer, and graphs showing related
trends were used. Various correlations were noted between dietary variables and cancer incidence or
mortality. The researchers pointed out in their conclusions that the most strong associations were of
meat and total fat with colon, rectum, and breast cancer. Interestingly, they also stressed the limitations
of their study and noted that other population characteristics might act as confounding factors in some
of the observed associations.
1.3 CROSS-SECTIONAL NUTRITION STUDIES

1.3.1 DESCRIPTION
Cross-sectional nutrition studies are often named descriptive surveys; they are a basic type of
observational study. They mainly collect and analyze dietary and health data from a sample population
at the certain time point in the participants’ recruitment [22,23]. For this reason, information provided
by participants is a snapshot of the sample’s dietary or other characteristics at the time of recruitment.
1.3 CROSS-SECTIONAL NUTRITION STUDIES 7
Cross-sectional studies in nutrition science provide descriptive results for the degree of exposure to
dietary factors of a sample population. They evaluate current dietary habits of participants and offer
valuable information about the consumption of foods and other dietary components. Energy, water,
and nutrient intake, adherence to dietary patterns, and other dietary factors can be estimated
[6,24e27]. Moreover, by collecting biological samples and biobanking [28,29], markers of food and
nutrient bioavailability and metabolism can be measured in the sample population base. Frequently,
participants undergo anthropometric screening and the related information is collected and analyzed
[30e32].
Because these studies run in parallel with other health-related studies, they provide important
information for the prevalence of a disease or health condition [33]. It is crucially important that the
diagnostic criteria used in the survey are the most up-to-date and in accordance with the recent
literature. There are several expressions of prevalence; a simple one is:
Number of diagnosed cases of a disease or a health condition
at a certain time point in the sample population
Prevalence ¼ 100
sample size
For example, when we read that the prevalence of obesity was 25% in November 2015 in a sample
population, we may understand that of the 100 participants who were screened at that specific time
point, 25 were obese.
During a cross-sectional nutrition survey, the degree of exposure to various potential risk factors
such as lifestyle, socioeconomic, environmental, genetic, and clinical is usually measured in parallel at
the same time point of the study sample’s recruitment [8,34e36]. The choice of the factor that will be
assessed depends on the aims of the survey. It is highly essential that all screening tools used to assess
the risk factors be valid and accurate.
The time frame of a cross-sectional study is limited to a time point, so it is impossible to identify
cause-and-effect relationships between dietary exposures and health outcomes [33,37,38]. The defi-
nition of causality in an observed association requires at least that the dietary exposure occurred before
the diagnosis of a disease or health condition [6,14,15]. This condition is not confirmed by a cross-
sectional design.
Statistical methodologies are available to evaluate the association of a dietary exposure and a health
outcome and they are also widely used by researchers, such as linear regression modeling and
generalized linear modeling (see Chapter 5). However, the interpretation of significant associations in
cross-sectional settings should be limited by the study’s inability to express causality. When these
associations are supported by literature data (i.e., in vitro or other studies) that offer biological
plausibility, they may guide further hypothesis testing and studies in other survey designs (i.e., cohort
studies, meta-analyses, and randomized control trials).
Weakness points of this type of observational study also relate to dietary assessment methodologies
and tools, which sometimes fail to evaluate the dietary habits of participants accurately. In particular,
bias related to the recall of dietary information from sample members is evident whereas potential
alterations of long-term dietary habits resulting from the previous presence of a disease or a health
condition cannot be thoroughly investigated. Thus, the observed statistical associations between di-
etary exposures and health outcomes may be biased. These issues are addressed in detail in Chapter 3,
where the collection and management of dietary data are discussed.
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LESSON XLIII.
HOW THEY BEHAVE.
ABOVE THE WAVE.

Birds, beasts, and insects live where we can easily watch them.
Wherever we go, they are our near neighbors. We can easily find out
many facts about their way of living. For this reason, many people,
from the earliest times, have observed these creatures, and have
written down what they learned about them.
But of fish, people have been able to see and know much less. It has
taken much longer to learn about creatures that hide in the waters,
and are, for the most part, out of our sight. Seeing fish so much less
often, people took less interest in them.
Since great aquariums were built with glass tanks, in which fish can
live at ease and behave in a natural way, we have learned more of
their habits.
These tanks are filled with salt or fresh water, as the fish may
require. The floor of the tank is covered with sand, rock, shells. Then
crabs and small “shell-fish” of many kinds, and larvæ and worms are
put in, such as the fish would find in their native homes.
Caves and grottos, lined with moss and weeds, are built; and the
tank looks like a true part of a river, pond, or sea. Then the water is
kept at the right warmth, and fresh air is driven in as I have told you.
The light is made just right for the fish in the tank, and proper kinds
of fish are put together.
When you go and stand outside of one of these great tanks, this
water world is before you from top to bottom. Now I shall tell you
some of the things which I learned about how fish behave at home.
By such a tank I first learned that nothing can be more easy and full
of grace and majesty than the motion of a fish in the water.
I watched a great fish swimming. I could not see the motion of a
muscle, or the least quiver of fin or tail. His great round eyes shone;
his splendid red fins were still; his spotted back glistened through the
waves; his broad sides gleamed in silver mail.
He never seemed to stir from his majestic calm. He stole across the
line of vision, but without visible effort. He crossed the path of other
fish. He sailed above them or below them; but he seemed to feel
himself all alone, and to notice nothing.
A fisher who had watched pike told me that, when he watched one
lying in a pool, he was sure that the fish saw him. It did not seem to
make any motion. But the fish changed its place. Soon it was gone.
In the next tank to the one I spoke of, were flounders. They swam
with a wave-like motion of the whole body. They did not swim
straight forward. They went up and down in curves. Their motion was
much like the flight of short-winged, heavy birds. In fact, a sole which
had its eyes and mouth set upon a kind of skull-shaped head, on the
upper side of the flat body, looked much like a dim, first idea of a
bird.
The fish in the tank were not fish that fed on each other, and they
never seemed to notice each other at all.
I saw some kinds of fish asleep. A dog-fish lay asleep on the sand,
with his nose in a corner, for half a day. He looked as if he were
dead. Three hermit crabs[32] were crawling about over him. He never
stirred. He did not seem to notice their sharp claws and their jostling
shells.
This sleeping of the dog-fish reminded me of what I had seen in
pools where fish lie basking in the sunshine. Some fish are very fond
of lying in a sunny spot. Others will get into a shadow, and lie there
with great content.
I found in my study of fish life in tanks, that fish are given to fighting.
Some of them fight with their nearest relatives. In a small tank I saw
two fish that fought and bit each other so cruelly that they had to be
parted.
There had been several fish in this tank, and all but two had been
killed in their battles. Then a glass partition had been put in the
middle of the tank, and one fish lived on each side. They swam up to
this clear wall, laid their noses against it, and glared at each other.
Another fact I remarked in fish is the clearness and keenness of their
vision, and the quick way in which they will dart at food. When they
see something that they wish to eat dropping down through the
water, they spring at it with open mouths, and seem never to miss
their mark.
When they need more air than they can get from the water, they
swim to the surface and take a gulp of air and water. When they are
sick, they do not swim, but lie on their sides, as if they feel too weak
to make any effort. When they are dead, the bodies turn over and
float on their backs.
It is said that fish can reproduce parts that they have lost, as crabs
and star-fish can. At least, they can easily live when an eye or part of
their mouth or a fin is gone. They seem to suffer little from such a
loss. A fish that has a wound, or has just lost an eye, will swim about
and eat as if it felt no pain.
Some fishes that live on the bottom have a queer way of lying close
on the sand, with their mouths wide open. They pant as if in great
pain. But they are not in pain, they are sifting their food out of the
water.
Look into their throats, and you will see a network, like fine bones.
That is their sieve to catch small food.
Some fish are timid, and hide at the least sound. Others are fearless,
and lie in plain view, or boldly follow their prey. A fish darting after his
food is a fine sight. His jaws are open; his spines stand in a ridge on
his back; his eyes gleam. His whole body seems alert and full of fire.
You should watch fish for yourselves. You have seen gold and silver
fish in globes. Other fish are often kept in ponds and fountains as
pets. They become very tame. Sometimes they learn to know those
who feed them, and will come when called.
FOOTNOTES:
[32] See Nature Reader, No. 1, Lesson 8.
LESSON XLIV.
FRY AND SCHOOL.
A fish comes from an egg, as a bird does. There are one or two
kinds that are born as little live fishes. But the rule is that the fishes
hatch out of tiny eggs. The mother fish drops these eggs in the
water, or carefully lays them in some place which she prepares for
them.
A CURIOUS CRADLE.
After they are laid, these eggs are called spawn. Before they are
laid, they lie in a solid mass together. There are many thousands of
them in each mass, and they are then called roe. I have told you of
the wonderful number of eggs laid by insects. But fish surpass even
the insects in the number of eggs.
These eggs are subject to very many dangers. Fish, crabs, and
water-fowls eat them. The waves may dash them ashore, and so
they will be dried up. Therefore it is needful that there should be a
great many, so that after all disasters a large number may hatch.
The eggs of fish are of a pearly white, or a cream color. They are
covered with a kind of glue, so that they stick together. They also
stick to weeds, or rock, or sand, where they are laid.
There are very few fish that carry their eggs about with them, after
they are laid. The fish presses its body against the eggs, which stick
to it, and are carried about, until they hatch. You have read of the
crab which took care of its eggs in this way.[33]
As the little fish grows within the egg, the soft skin-like case
becomes very thin. When the time comes for hatching, the little fish
breaks the case, and comes out. As a general rule, the little fish, just
out of the egg, looks and acts like the parent fish. But there are some
that change much between their first and their full-grown states.
Let us now look at some curious fish eggs and fish nests. Of all the
fin family, the dog-fish has the most curious cradle for its young. The
egg-case of the dog-fish is a horny bag, or purse. It is of a gray or
blackish color, about an inch wide, and two inches long. At each of
the four corners, it has a long, stiff, curly stem.
The mother dog-fish swims near the shore to lay her eggs. She
selects a weed, branch, or piece of tree, lying in the water. To this
she ties her egg-cases by means of their long stems, or strings. She
does this by swimming round and round the twig to which she means
to fasten the case. As she drops the case in the water, she ties it, or
binds it, by drawing with her the curly ends, as she swims, about the
branch.
She lays a number of these purses in a place. Then she swims off
and leaves them. It is lucky that she does, for if she stayed near
them, she would eat the little fish as soon as they came out!
I have seen a dog-fish tying its eggs to a branch lying under water,
and it was a queer sight. When the baby fish has grown large
enough to leave the case, it makes a little opening on one end, and
creeps out.
The skate, that is a cousin of the dog-fish, lays a case much like this.
But the case of the skate is of a shape more nearly square. It has
four sharp, curved horns, not long, curly ends; and the little skate
comes out of a hole in the middle, never at the end.
Another sea-fish, the mackerel, has no such protection for its eggs. It
drops thousands of them on the water, and they look like tiny pearl
beads. They sink to the bottom. As they are sticky, they cling
together, and to the sand, until they hatch.
That prettiest of fish, the trout, which lives in so many clear, shady
streams, where there are deep, quiet pools to bask in, is very careful
of her eggs. The mother trout sinks to the bottom of some clean
stream, and selects a nice sandy place. Then, with her tail, she fans
out all the coarse sand and gravel. If there are larger bits of pebble,
she carries them off in her mouth.
When she has made a nice smooth little nest, like a cup, she drops
her eggs into it. Then she covers them lightly with gravel, so that
they will not be floated away. When she has finished one nest, she
swims off to make another.
The black bass of our lakes and ponds makes a smooth bed for its
eggs. It prepares this bed in the shadow of a stone or sunken log.
Several bass will go together, and select and clear out such a bed.
Then they will lay their eggs there, and for days, until the eggs hatch,
will swim about near, to keep watch over them. Eels, cat-fish, perch,
and suckers, come to eat these eggs, and the big bass drive them
away.
The pretty perch does not take such care as this of her eggs. She
drops them in long chains, among grasses, and leaves of water-
plants, at the edge of the pond. When the yellow cowslips are in
bloom, you can find these eggs among the water-plants, like strings
of fairy beads.
When the baby perch come from the eggs, they are very nimble, and
begin at once to eat. When they are no longer than the nail of your
little finger, it is funny to see them in the water, darting after the living
atoms that serve them for food.
They see and know these tiny things in the water, and pounce upon
them with wide-open mouths.
The little fish, from the time they are out of the egg, until they are
about half grown, are called fry. Some fish, as the salmon, get
different names at different periods of their growth. A great many fish
together are called a school. Thousands of fish will come leaping,
rolling, and tumbling along in the water, and we say it is a school of
fish.
FOOTNOTES:
[33] Nature Reader, No. 1, Lesson 3.
LESSON XLV.
SCALES AND TEETH.
I told you that most fish had scales, and that these scales clothed
them in a gleaming, flexible suit of armor. Most of the beautiful color
of fish is found upon their scales. Many of the scales are iris or
rainbow hued. That is, they have the gleam of many colors,
according as the light strikes them. Have you not seen such colored
light in a glass prism?
The scales of different fish vary in shape, size, color, and hardness.
In general, they are horny like your finger nail, but thinner. Their
shape is nearly round, much like a rose petal. They are fastened by
the smaller edge to the skin of the fish. Then each scale laps over
the next one, and so on.
Scales are always so set that they turn or lap, from the head toward
the tail. In the middle of each scale, on the lateral or side line of the
fish, is a little groove or canal. It runs in the direction of a line from
head to tail. Let us see what it is for.
Have you noticed how slippery fishes are? Is it not hard to hold
them? If you rub your finger hard down their bodies, you rub off a
quantity of slime, or stuff like glue, or thin paste. This glue-like stuff is
made near the mouth of the fish. It is supplied to the scales by little
tubes near the mouth. It runs through that little canal in each scale in
a line upon the side of the fish.
This slimy stuff helps the fish to slide easily through the water. It
keeps the scales limber and healthy. It keeps the body supple. It
helps the fish to slip away from creatures that seize it. Also, I am
sure, it helps the fish to slide easily down the throats of birds,
animals, or fish that capture it!
The fact that the scales all lie turned from the head to the tail of the
fish, also makes it easy for birds or other fish to swallow it. But if a
fish is partly swallowed, it cannot be readily cast out, for its scales
rising, make a rough surface, and hinder it. I suppose that was why
the alwife duck I told you of could not get the partly swallowed fish
out of her throat.
Each kind of fish has its own especial shape and color of scales.
Some are pointed, some are rounded, some are flat, some are
curved, some are three-cornered. Some fish have no scales. Some
have such tiny ones that you will not notice them unless you look
sharp. Some fish, as the sturgeon, have great bony plates, like large
limpet shells, laid in lines up and down the body.
Fish not only have their own especial shape of scales, but their
especial color, and such colors have their especial place on the fish.
One flat, brown fish has all the under side of its body white, but spots
of red, like sealing wax, are laid all over the brown side, as if you had
dropped red wax upon it.
When you see a smoked and salted herring hung up for sale, you
are not likely to guess what a beautiful thing it was, when living in its
water home. It had a coat of blue, green, and silver, and gem-like
eyes. The eye of a dead fish is sunken and dull. The eye of a living
fish is full, and gleaming with light.
There is a little fish called a wrasse which looks as if made out of a
rainbow. It is dressed in bright blue, gold, bronze, and white.
The red bream is of a fine rose-red color, with silver sides. Its
luminous eyes are set in golden rings. The perch has dark, shining
bands on its silver coat, and it has gay, red fins. The gray mullet is a
quaker fish, trim, grave, quiet in its style, but lovely in its shape and
in the rainbow lights along parts of its body.
Once I saw in the water a fish called a gurnard. He seemed to have
borrowed a sunset to dress himself in. His scales were deep red and
bronze. There was a vivid blue on the edges of his fins. The fins
were in shape and coloring like a butterfly’s wings.
I never saw a boy who did not think that a trout was one of the
prettiest things ever made. Very much of the beauty of fishes lies in
the wonderful scales.
But you must examine scales for yourselves. If you have a
microscope to look at them with, you will be full of delight and
surprise at what you see. Once I looked through a very powerful
microscope, and thought I was looking at a lump of half-melted gold
set full of fine jewels. But it was the scale of a fish.
I will now leave the scales, and tell you a little about the teeth of fish.
Fish have a great many teeth. Their mouths are for the most part
hard and horny, and so covered with teeth that it is not likely that
they have very much sense of taste. Still there are some kinds of fish
that are almost, if not quite, toothless.
If you open the mouth of a sea-trout you will find that it is set entirely
round, with sharp, strong teeth. Then if you stretch the mouth open
and look into it, you will see that there is another row of teeth set all
round a bone inside the upper jaw. Look still farther in, and you will
see a row of teeth on the middle bone in the roof of the mouth. Look
still beyond, and you will see a row of teeth on each side of the
tongue.
Some fish, besides all these teeth, have the tongue quite covered
with teeth. Others, that eat vegetables, have little fine teeth all down
the sides of the throat. The French call such teeth as these, “teeth in
the velvet.” Some fish have the entire mouth and throat lined with
teeth.
These teeth may all be alike, not some “double” and some “single”
as you have. But sometimes fish have teeth of different patterns. The
most common form of teeth in fish that live on fish or other animal
food, is that of a slim cone, bent a little inward to hold firmly the fish
caught, so that it cannot slip away.
The fish that feed on weeds have short, roundish teeth with a flat
top, to make a good mill with which to grind or crush their food. Fish
that eat insects do not need such large teeth. They have a great
number of little teeth almost as fine as hairs. Rows of these teeth
look like a little brush.
Some fish are said to have “mill” teeth, as the carp, because their
large flat teeth roll upon each other like the stones of a mill grinding
grain.
The ray fish, that feed on crabs, shell-fish, and flat fish, need very
strong teeth for crushing such food. If you should look into their
mouths, you would see that they are made like a mill, and the upper
and under teeth roll against each other and crush fine all that is
between them. By looking at the mouth of fish and examining its
teeth, you can find out what kind of food it used, just as by looking at
the beak and claws of a bird, you can tell what were its habits and
food.
Some fish have the inside of the stomach thick and furrowed, very
like the gizzard of a fowl. This is to aid in cutting or grinding up the
food. The teeth of a carp have much the appearance of the teeth of
sheep and cows, only they are much smaller. They work against a
plate of gristle in the roof of the mouth and reduce the food to pulp.
The strong, sharp teeth of fish are used not only for eating their food,
but for biting and fighting other fish. Two great salmon have been
known to fight until one was killed. The dead one was found to be
badly wounded and torn by the teeth of its enemy.
Pike have very sharp, strong teeth, and a big pike has been known
to seize hold of the foot or nose of a dog, fox, or other animal, that
came to the water’s edge to drink.
The mouth of a dog-fish is set back on the under side of the head.
This mouth has a row of strong, sharp teeth all round it. When the
fish seizes its prey or its enemy with its teeth, it whirls itself over, and
this action enables it to tear out a large piece of the flesh held by its
teeth.
LESSON XLVI.
BIG AND LITTLE BROTHERS.
Yesterday, as I was going along the street, I saw a large globe full of
tiny fish. They were from one to two inches long. I stopped to
examine them. They were sticklebacks. You must know that little Mr.
Stickleback is a very pretty and curious fellow, and well worth buying.
THE LITTLE HOUSE BUILDER.

Looking into this globe made me think of some of the queer things I
have heard and seen of this little fish. Shall I tell you about him?
The stickleback gets his name from the fact that some of his fins,
one on the back and one on the lower part of the body, have
changed to spines or thorns. Different kinds of stickleback have
three, four, ten, fifteen, of these thorns.
This little fellow is one of the smallest and prettiest of fishes. He is
slender, and from one to three inches long. The general length is
about an inch and a half. His body is silver below, green and purple
in bands on the back, and red and gold on the head and sides. His
fins are thin as finest silk. I never saw a prettier little creature.
The stickleback is very greedy, very fearless, full of courage, very
affectionate, and of all fishes is the most devoted parent. Finally, the
stickleback can build a house.
The colors of the stickleback brighten or fade according as he feels
glad or sad. If he has lost his mate, his home, or his young ones, or if
he has been beaten in a fight, he swims off to hide, and his colors
grow dull. If he has beaten his enemy, if his children and mate are
quite safe in the little house where he has put them, he gleams and
glows like a rainbow.
Sticklebacks are so fearless that when they are in a pond no noise or
shadow will alarm them. You can even catch them without a hook
just by dropping among them a string with a worm on it. You may pull
up the string with two sticklebacks fast, one on each end of the
worm. Little English boys take this way of catching these tiny fish for
their globes.
When put into a globe or box, they fight sharply at first. They strike
each other with their spines, whirl, dart, pretend to be beaten, and
then leap back to the fight. They are real little soldiers. Finally, when
they have found out which of them all is the strongest, each one
chooses his own corner, or shelter by a stone for a home, and
defends that against all strangers.
The different varieties of sticklebacks build different kinds of houses.
One makes a nest like a muff among water-weeds. I will tell you of
one kind of nest. The little fish carries straws and bits of grass and
moss, and tucks them down into the gravel and sand. He glues them
with the glue from his skin.
While he is at work he holds and carries his material with his mouth,
and presses it into place with his body. Having laid a floor, he builds
a little hut of woven fibres and moss. This hut is about as large round
as a twenty-five-cent piece. A little door is at the top. He tries the
strength of his house by stirring up the water near it with his tail.
When all is done Mr. Stickleback swims off to find his mate. He
seems to tell her that the house is ready. She is a lazy little creature,
and does nothing but frolic in the water. She goes along to the new
home, and goes in to lay some eggs there. Mr. Stickleback proudly
swims up and down before the home to keep foes away.
The little mate being fond of play does not like to stay in the house
long. She lays a few eggs about the size of poppy seeds. Then she
bites a hole in the house and runs away! Next day little Mr.
Stickleback goes to find her, and coaxes her back. This goes on for
several days, until a great number of bright yellow eggs, like seeds,
are laid in the nest.
After this, it is a whole month before the little fish will hatch.
Meantime other fish and other sticklebacks will eat them, if they get
the chance. All that month the kind, brave little stickleback swims up
and down near his nest, and drives off enemies. He will let no fish,
not even his mate, come near his treasure.
Finally, out come the wee, wee fish. Now the poor little father has a
harder time than ever. The other fish want to eat up the young fry.
The food of sticklebacks is grubs, tiny insects, and very small fry. As
they are very hungry and greedy, they are on the watch to pick up
the new little fish. But the stickleback, however hungry, never eats
his own little ones. He leaves such vile conduct to the dog-fish.
As the little ones grow, they are very active, and want to stray away
from their home. Their father knows they would get into danger, so
he watches, and chases each runaway back into its home bounds,
where he can take care of them all.
Finally, the little ones are so nearly grown that they can fight for
themselves. They can pick up their own food, and make their own
houses. Then they are allowed to go off and swim where they
choose.
As I have told you about a very little fish, I will now tell you
something of a very big fish. The sturgeon is a great fish which lives
in the waters of many countries. He is found in some of our rivers.
The body and head of the sturgeon are partly covered with five long
lines of great bony plates. These plates are grooved, and have a
sharp point in the centre. They make good armor.
Sturgeon are fond of playing and leaping from the water. They are
often speared by people standing on rocks or in boats. I have seen
very big ones taken from the Niagara River. In the Ohio and
Mississippi the curious spoon-bill or paddle-fish sturgeon are found.
In some of our other rivers the shovel-headed sturgeon lives.
The flesh of the sturgeon is good for food. The Russians make
isinglass from the air bladder of the sturgeon, and one or two kinds
of food are prepared from its roe and flesh. Of the isinglass prepared
from the sturgeon, jelly is made. Glue is very plentiful in the skin and
bones of the sturgeon, and a kind of food is made from the spinal
marrow.
I need hardly tell you much about the uses of fish. You all know that
they form a large part of the food of men, and many birds and
beasts. Glue is made from fish-bones and skins.
From many fish are made those substances that I told you farmers
put upon their land to restore the minerals eaten out by the plants.
Oil is also made from fish. From parts of the cod an oil very useful for
medicine is obtained. From other fish, lamp oil and machine oil are
pressed. A little fish in the Caspian Sea is so oily that when it is dried
its body burns with a clear light. These dried fish are used as
candles. When the Cossacks get hungry, and have nothing else for
supper, they eat their fish candles.
That terrible fish, the shark, is very useful after he is dead. From his
fins, fine gelatine is made. From his skin, a leather-like substance
called shagreen is prepared. Shagreen is used in polishing wood,
and for covers of portfolios, instrument-cases, and the like.
From the bones of the shark, buttons and ornaments are made, and
very costly canes from his backbone. His teeth are very hard and
white, and are often mounted in gold and sold for ornaments.
There are many large and interesting books about fisheries,
manufactures of articles from fish, and about the various kinds of
fish, and their habits and uses. I hope you will try to get some of
these books to read. In fact, I hope you will pursue for yourselves all
these studies in nature which we have begun in the Nature Readers.
REVIEW LESSONS.
I.
1. What are some of the things which we get from the earth?
2. Why do we say “Mother Earth”?
3. What do we say are the eldest children of Mother Earth?
4. What is a plant?
5. What is the chief food of plants?
6. What are some of the things that plants can do?
7. How do animals get most of their mineral food?
8. Do plants take their food in liquid or solid form?
9. What partners have plants taken?
10. What is the business of plants?
11. What good does that business do us?
12. How large are some of the largest plants?
13. How small are some of the smallest plants?
14. What can you say of the length of a plant’s life?
15. What two great divisions of plants are there?
16. What are the six parts of a perfect plant?
17. What is a tendril?
18. Are any of the parts of a plant ever wanting?
19. Of what use to a plant is the root?
20. What are the true roots of plants?
21. What are these thicker parts which are usually called roots?
22. What is a plant stem? Of what use is it?
23. Tell me something about leaves.
24. With what does a plant begin and end?
25. Have plants more than one crop of seeds?
26. What is an annual plant?
27. Will you describe the sprouting of the seed?
28. What is a stamen?
29. What is a pistil?
30. Describe the seed-case.
31. What are the only really necessary parts of a flower?
32. Where does the growth of a plant begin?
33. What is a hardy seed?
34. How do plants grow?
35. What can you say about the plant’s need of moisture?
36. What is the blood of the plant?
37. What two kinds of sap are there in a plant?
38. Tell me about the tubes and cells.
39. Through what does the plant breathe?
40. How does the sap get changed in the leaf?
41. What can you say about starch in plants?
42. What about sugar?
43. How does the plant prepare mineral food for you?
44. Will plants thrive in the same soil year after year?
45. How does the farmer rest his fields?
46. How does he give more food in the fields to the plants?
47. What is a seed?
48. Describe an egg.
49. Describe a seed.
50. What can you say of the shape and color of seeds?
51. How are seeds carried about?
52. Tell me something about leaves.
53. Tell me how plants breathe.
54. What is the work of leaf-green?
55. What about carbonic acid gas in the air?
56. What is the plant’s work in purifying the air?
57. What can you tell me of color in plants?
58. What are some of the dyes we get from plants?
59. What motions have plants?
60. What is the cause of motion in plants?
61. What can you say of climbing plants?
62. Describe the strawberry plant.
63. Describe the walking fern.
64. What can you tell me of coal?
65. What is the use of pollen in the plant?
66. Are stamens and pistils always in the same flower?
67. What is necessary to the production of good seed?
68. How do insects convey pollen from flower to flower?
69. What has the flower to tempt insects?
70. What has the plant to keep away certain insects?
71. Tell me about the methods of insects in getting honey.
72. What plants have the pollen carried by the wind?

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Analysis in Nutrition Research:

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Publisher: Charlotte Cockle

PART 1 ANALYSIS IN NUTRITION RESEARCH

2.3.2 Primary and Secondary Outcomes................................................................. 30

4.3.4 Evaluation of the Quality of an a Priori Dietary Pattern .............................. 81

CHAPTER 8 Principles of Research Publication ................................................. 197

PART 2 CHALLENGES IN NUTRITION SCIENCE

9.3 Evidence on Health Benefits of the Mediterranean Diet .......................................235

10.4.4 Biological Functions of Polyphenols in Cardiovascular Disease

CHAPTER 12 Diet, Healthy Aging, and Cognitive Function................................. 321

CHAPTER 14 Diet and Lung Health ...................................................................... 355

14.8.2 Minerals ..................................................................................................369

1.1 INTRODUCTION TO OBSERVATIONAL NUTRITION STUDIES

Analysis in Nutrition Research. https://doi.org/10.1016/B978-0-12-814556-2.00001-4

1.2 ECOLOGICAL NUTRITION STUDIES

1.2.3 EXAMPLE OF ECOLOGICAL STUDY ON DIET AND CANCER

1.3 CROSS-SECTIONAL NUTRITION STUDIES

ABOVE THE WAVE.

THE LITTLE HOUSE BUILDER.

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