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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
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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.
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These quartets are much more broadly planned than earlier works
by Mozart in the same form. Not only are the separate movements
generally longer; the middle section of the first movements is
intricate and extended, and the minuets are not less seriously
treated than the other movements. The treatment of the separate
instrumental parts is, of course, distinguished and fine.
It would be difficult to characterize each one distinctly. The first, in G

major (K. 387), is marked by a certain decisive clearness throughout.
The two themes of the first movement are especially clearly
differentiated. The development section is long and rather severe. It
will be noticed that the minuet takes the second place in the cycle,
as in many of Haydn’s quartets. The final movement is in fugal style
and not unrelated in spirit to the final movement of the great Jupiter
symphony.
The second quartet, in D minor (K. 421), takes both from its tonality
and from the nature of its themes a thin veil of melancholy. The
opening theme is poignantly expressive, but the fire of it is often
covered. The characteristic width of its intervals is used throughout
the entire movement, with a strange effect of yearning, now
resigned, now passionately outspoken. The andante, in F major, is
tinged with the same melancholy. The trio of the minuet is one of the
few places where Mozart made use of pizzicato effects. The last
movement is a series of variations on a melancholy little theme cast
in the rhythm of the Siciliana, one of the Italian rhythms already
made use of by Handel and Gluck, among others.
The third quartet, in E-flat major (K. 428), is on the whole reserved
and classical in spirit. The opening theme, given in unison, has a
gentle dignity which marks the whole first movement. The measures
following the second theme are especially smooth and lovely in their
slowly falling harmonies. In the second movement, andante con
moto, there is a constant shifting of harmonies, and a somewhat
restless interchange of parts among the instruments. The trio of the
minuet, in C minor, is subtly woven over a drone bass. The final
movement is a lively rondo.
The fourth, in B-flat major (K. 458), is, in the first movement, very like
Haydn, light-hearted and wholly gay. The following minuet, adagio,
and rondo need hardly be specially mentioned. The A major quartet
(K. 464), the next in the series, is in a similar vein. The slow
movement, again the third in the cycle, is in the form of variations;
and the last is full of imitations and other contrapuntal devices.
The last of these quartets, in C major (K. 465), is the most profound
and the most impassioned. The boldness of Mozart’s imagination in
harmonies is in most of his work likely to fail to impress the modern
ear. One hears but half-consciously the subtlety of his modulations.
But here and there in his work the daring of the innovator still has
power to claim our attention; as in the andante of the last pianoforte
sonata in F major (K. 533), and still more in the introduction of this
quartet. The sharp harmonies of the first few measures roused
hostility; and the discussion as to their grammatical propriety was
continued for more than half a century after Mozart’s death.
The whole quartet is full of an intensity of feeling. The andante has

that quality of heart-melting tenderness which sprang only from
Mozart’s genius. One cannot but place the four movements with the
three great symphonies, as something not only immortal, but
precious and inimitable in the world’s treasure of instrumental music.
This series of six quartets did not make a decidedly favorable

impression upon the general public. The next quartet from his pen
was in a much more conventional manner, as if Mozart had tried to
suppress the genius in him which prompted him ever to new
discoveries in his art. The quartet in D major (K. 499) was composed
on the 19th of August, 1786. It is beautifully worked in detail, light in
character. No special reason is known why he should have written
and published a single quartet like this; and it has been thought that
he hoped by it to rouse the public to enthusiasm for his instrumental
works.
There remain three more quartets to mention. These were written for
Frederick William II of Prussia, at whose court Mozart had been a
frequent attendant during the early spring of 1789. The first quartet
was completed in Vienna, in June, 1789. The other two were written
about a year later. In Köchel’s index the three are Nos. 575, in D
major, 589, in B-flat major, and 590, in F major.
All are very plainly written with a king in mind who played the
violoncello. In most of the movements the 'cello is given a very
prominent part, frequently playing in unusually high registers as in
the announcement of the second theme in the first movement of the
first of these quartets; in the trio and the finale as well. In many
places the viola plays the bass part, leaving the 'cellist free to be
soloist, as in the opening measures of the Larghetto in the second
sonata. Thus these quartets, fine and free in style as they are, are
not the fullest expression of Mozart’s genius, as the series of six
dedicated to Haydn may be taken to be.
There are, as we have said, twenty-three quartets in all. The majority

of the early ones were written under the influence of a certain mode
or style, as experiments or as test pieces; and the last four were
written with the purpose of pleasing the public or of suiting the
special abilities of a king of Prussia. Only the six quartets dedicated
to Haydn may be taken as what Mozart felt to be his best effort in the
form, the expression, perfect as far as he could make it, of his
highest ideals. As such they are almost unique in his music.
With the quartets may be mentioned the four great quintets for
strings, written, two in the spring of 1787, one in December, 1790,
and one in April, 1791. Of the combination of five string parts Haydn
made little use. Boccherini, however, had written at least one
hundred and twenty-five quintets. He was himself a 'cellist and, as
might be expected, the added instrument in his quintets was a 'cello.
Mozart added another viola to the group. Though this added no new
strand of color to the whole, it rather complicated the problems
offered by the quartet. As Otto Jahn has carefully explained, with the
volume of sound thus thickened, there came a need for even more
active movement of the separate parts. Since the additional part was
among the middle voices, the outer voices must be spread as far
apart as possible so as to allow sufficient freedom of movement to
the inner. The extra viola might be treated as a bass part to the first
and second violins, or as the upper part above the other viola and
the 'cello. Mozart made use of this possibility of contrast nowhere
more clearly than in the opening pages of the quintet in G minor.
The four quintets are respectively in C major (K. 515), G minor (K.
516), D major (K. 593), and E-flat major (K. 614). Of these that in G
minor is clearly the most remarkable; and it is indeed conspicuous
above almost all his instrumental music, for the passionate intensity
of the moods which it voices. Needless to say it still holds its place
as one of the supreme master-works in chamber music. More than a
similarity of key unites it to the symphony in G minor. The themes in
both works seem much alike, and both are equally broad in form and
full of harmonic color.
FOOTNOTES:
[62] L. Piquot: Notice sur la vie et les ouvrages de Luigi Boccherini; Paris, 1851.
[63] These are Nos. 1-18, inclusive, in Pohl’s index. The opus numbers by which
Haydn’s quartets are usually designated are taken from the thematic index
prefixed to the complete Trautwein Edition of 1844. These first quartets are: Opus
1, Nos. 1-6; opus 2, Nos. 1-6; and opus 3, Nos. 1-6.
[64] C. F. Pohl: ‘Joseph Haydn,’ Vol. I, p. 331.
[65] Eugène Sauzay: Étude sur le quatuor. Paris, 1861.
[66] Cf. C. F. Pohl: op. cit., Vol. II, p. 293.
[67] Étude sur le quatuor.
[68] Adagio, allegro, minuetto. The finale rondo was added some years later. Cf.
‘W. A. Mozart,’ by de Wyzewa and de Saint-Foix: Paris, 1912.
[69] Cf. Wyzewa and Saint-Foix: op cit. Gassmann was born in Bohemia in 1723
and died in Vienna in 1774. A great many of his works in manuscript are in the
libraries at Milan. He had been appointed to a place in Vienna in 1762, and was
hardly likely, therefore, to be in Milan when Mozart was; but he had lived at one
time in Milan and came back there occasionally from Vienna to superintend
performances of his operas.
CHAPTER XVI
THE STRING QUARTET: BEETHOVEN
Beethoven’s approach to the string quartet; incentives; the six
quartets opus 18—The Rasumowsky quartets; opera 74 and 95—
The great development period; the later quartets, op. 127 et seq.:
The E-flat major (op. 127)—The A minor (op. 132); the B-flat
major (op. 130); the C-sharp minor (op. 131); the F major (op.
135).
I
Beethoven’s six quartets, opus 18, were first published in 1800. He
had already experimented in other forms of chamber music, not only
for strings alone. The sextet for two clarinets, two bassoons, and two
horns; the quintet, opus 16, for piano, oboe, clarinet, horn, and
bassoon; the string quintet, opus 4; three trios for violin, viola, and
violoncello; the trio, opus 11, for piano, clarinet, and violoncello; and
sonatas for violin and piano, and violoncello and piano, had already
been written. The pianoforte sonatas up to that in B-flat, opus 22,
and the first symphony had likewise been completed. Beethoven
thus turned to the composition of string quartets after an experience
with almost all other branches of music had made him master of the
art of composition.
Apart from any inner development in the man which waited thus long
before attempting expression in that form in which the very last and
in some ways the most remarkable of his thoughts were to find
utterance, one or two external circumstances probably turned his
attention to the string quartet. One was undoubtedly the morning
musicales at the house of his friend and patron, Prince Lichnowsky,
where such music was especially in demand, and where Beethoven
must constantly have heard the quartets of Haydn and Mozart.[70]
Another was his personal acquaintance with Emanuel Aloys Förster,
[71] a composer of quartets for whom Beethoven had a high regard.
These first quartets appeared in two groups of three. They are not
arranged in the order of their composition. For example, that in D
major, the third in the first set, is probably the oldest of the six. But
the series presents little evidence of development within its limits,
and there is hardly reason to attach serious importance to the order
in which the various quartets were created. Besides, with the
exception of the quartet in C minor, No. 4, the entire series is
expressive of much the same mood and intention. If one quartet is at
all distinguished from the others, it is only by a few minor details,
usually of biographical or otherwise extrinsic significance. The
technique is that of Haydn and Mozart, lacking, perhaps, the assured
grace of the earlier masters; the character, one of cheerfulness, with
only here and there a flash of the emotional imperiousness with
which Beethoven took hold of music.
No. 1, in F major, is known as the ‘Amenda’ quartet. Beethoven had

sent an earlier form of it (completed in 1799) to his friend Karl
Amenda. A year later he wrote Amenda, saying that he had greatly
altered it, knowing now for the first time how truly to write a quartet.
The later arrangement differs from the original in details of
workmanship, not in spirit. There are four movements, in
conventional form and sequence: an allegro con brio, 3:4; an adagio
affettuoso ed appassionato, in D minor; a scherzo and a final rondo.
Amenda had a story to tell of the adagio, to the effect that when
Beethoven had completed the quartet he played this movement to a
friend and asked him afterward of what it made him think. It seemed
to the friend to have represented the parting of two lovers.
Beethoven is reported then to have said that in composing it he had
had in mind the scene in the tomb from ‘Romeo and Juliet.’
The second quartet of the series, in G major, is known as the

Komplimentier quartet, because of the graceful character of the
opening theme, and, indeed, of the whole first movement. The third,
in D major, is not less cheerful. The final movement is a virtuoso
piece for all the instruments. The triplet rhythm is akin to the last
movement of the Kreutzer sonata, and to that of the sonata for
pianoforte, opus 31, No. 3; both of which originated not later than in
1801. Similarly the whole of the fifth quartet—in A major—is in
brilliant concert style. There is a minuet instead of a scherzo,
standing as second, not third movement, as was frequently the case
in the works of Haydn and Mozart, and, indeed, in later works of
Beethoven. The third movement is an andante and five variations in
D major. Finally the first movement of the sixth and last of the series
—in B-flat major—cannot but suggest a comparison with the first
movement of the pianoforte sonata, opus 22, in the same key, which
originated about the same time. Both are very frankly virtuoso music.
The last movement of the quartet is preceded by a short adagio, to
which Beethoven gave the title, La malinconia. This whispers once
again for a moment not long before the end of the lively finale in
waltz rhythm.
The fourth quartet of the series is alone in a minor key. It is of more

serious nature than those among which it was placed, and may be
related in spirit, at least, to the many works in the same key (C
minor) which seem like successive steps in a special development.
Paul Bekker suggests in his ‘Beethoven’[72] that we may consider a
C minor problem in Beethoven’s work; and points to sonatas, opus
10 and 13, the pianoforte trio, opus 1, the string trio, opus 9, the
pianoforte concerto, opus 37, the duet for piano and violin, opus 30,
and finally the fifth symphony and the overture to Coriolanus, all of
which are in C minor, and all of which follow closely one after the
other. Whether or not the quartet in question may be thus allied with
other works, there is evidence that it is closely connected with an
early duet for viola and violoncello (with two obbligato Augengläser)
which originated in 1795 or 1796. Riemann[73] is of the opinion that
both the duet and the quartet are rearrangements of some still earlier
work. The first movement is weakened by the similarity of the first
and second themes. The second is a delightful Andante scherzoso,
quasi allegretto, in C major, 3:8. The third movement is a little minuet
and the last a rondo.
II
The year after the publication of these first quartets appeared the
quintet for strings, in C major, opus 29. This is the only original string
quintet of Beethoven’s, except the fugue written for a similar group of
instruments in 1817, probably as a study. The quintet, opus 4, is a
rearrangement of the octet for wind instruments, written in 1792,
before coming to Vienna. The quintet, opus 104, is an arrangement
of the trio in C minor, opus 1, which Beethoven made in 1817,
following an anonymous request, and which he regarded
humorously.
In 1808 were printed the three great quartets opus 59, dedicated to
Count (later Prince) Rasumowsky. Beethoven’s earlier patron, Prince
Lichnowsky, had left Vienna, and the famous quartet under the
leadership of Schuppanzigh, which had played such a part in his
Friday morning musicales, was now engaged by Rasumowsky,
Russian Ambassador to the court at Vienna. Rasumowsky
commissioned Beethoven to write three quartets in which there was
to be some use of Russian melodies.
Between the quartets, opus 18, and these so-called Russian

quartets, Beethoven had written, among other things, a number of
his great pianoforte sonatas, including opus 27, opus 31, opus 53,
and most of opus 57, the Kreutzer sonata for violin and piano, the
second and third symphonies, and Fidelio. These are the great
works of the second period of his creative activity; and the qualities
which are essential in them are, as it were, condensed, refined and
assembled in the three quartets, opus 59. They may be taken as the
abstract of his genius at that time.
Nothing gives more striking evidence of the phenomenal power of

self-development within Beethoven than a comparison of opus 18
with opus 59, or, again, of the latter with the five last quartets. Of
course, to compare the early with the late sonatas, or the first two
symphonies with the ninth, will astonish in a like measure. But there
are intermediate sonatas and symphonies by which many steps
between the extremes can be clearly traced. The quartets stand like
isolated tablets of stone upon which, at three distinct epochs in his
life, Beethoven engraved the sum total of his musicianship. The
quartets opus 74 and opus 95 hardly serve to unite the Russian
quartets with opus 127.
The Russian quartets are regular in structure, but they are as broad
as symphonies by comparison with opus 18. The style is bold,
though the details are carefully finished, and the instruments are
treated polyphonically, each being as prominent and as important as
the others. This in particular marks an advance over the earlier
works in the same form. There is in them, moreover, an emotional
vigor, which, expressed in broad sweeps and striking, often strident,
harmonies, worked in the opinion of many contemporaries a
barbarous distortion of the hitherto essentially delicate form. To
interpret them is but to repeat what has been already made familiar
by the sonatas of the period, by the Eroica, and by Fidelio. It is the
same Beethoven who speaks here with no less vigor, though with
necessarily finer point.
The first quartet begins at once with a melody for violoncello,

unusually long and broad even for Beethoven. It has in itself no
Russian quality; but the monotonous accompaniment of chords,
repeated with but the harmonic change from tonic to dominant for
eighteen measures, suggests a primitive sort of art, a strumming
such as may well be practised by Russian peasants in their singing.
The harsh dissonances created by the long F’s in the melody, and a
little later by the whole-note D, against dominant seventh harmony
will not pass unnoticed. Such clashes between melody and harmony
can be found in other works of about the same period; for example,
at the return of the first theme in the third section of the first
movement of the Eroica; and the much-discussed, prolonged D-flat
of the oboe against the entrance of the A-flat melody in the second
movement of the fifth symphony. Elsewhere in this quartet the same
procedure makes a striking effect; namely, in the approach to the
second theme, where, however, the long G’s—sharply accented—
are in the nature of a pedal point. The second theme—in C major—is
cognate with the first. The interweaving of the instruments in its
statement is noteworthy. The first phrase is anticipated by the first
violin, and then sung out broadly by the viola; from which the first
violin immediately takes away the second phrase. The second violin
and 'cello, and even the viola, after its first phrase, interchange with
each other the broad C’s which lie at the foundation of the whole.
The line of the melody itself and the suave flow of polyphony will
suggest certain passages in Richard Wagner’s Die Meistersinger.
The second movement (allegretto vivace e sempre scherzando, B-

flat major, 3:8) has in rhythm at least a strong Russian flavor. Here
again there are repeated chords in the accompaniment even more
barbaric in effect than those in the first movement. The 'cello alone
gives in the opening measures the rhythmical key to the whole; and
in the next measures the solo violin (2d) announces, staccato and
pianissimo, the chief melodic motive. The effect of the whole
movement is at once fantastical and witty. The following Adagio in F
minor, in essence and in adornment one of the full expressions of a
side of Beethoven’s genius, dies away in a long cadenza for the first
violin which, without ending, merges into a long trill. Softly under this
trill the 'cello announces the Russian melody upon which the
following wholly good-humored and almost boisterous finale is built.
The second quartet of the series is less forceful, and far more
sensitive and complicated. The key is E minor. Two incisive, staccato
chords, tonic and dominant, open the movement. One remembers
the opening of the Eroica. There follows a full measure of silence
and then the melodic kernel of the first movement, pianissimo in
unison—a rising figure upon the tonic triad (which will again recall for
an instant the Eroica) and a hushed falling back upon the dominant
seventh. Again the full measure of silence, and again the rising and
falling, questioning, motive, this time in F major. After an agitated
transitional passage, the first violin gives out the second theme, a
singing melody in G major. But the threefold first theme—the incisive
chords, the measure of silence, and the questioning figure—carry
the burden of the work, one of mystery to which the second theme is
evidently stranger. At the beginning of the middle section, and again
at the beginning of the long coda, the chords and the breathless
silences assume a threatening character, now hushed, then
suddenly angry, to which the figure reluctantly responds with its
unanswered question.
The second movement (Adagio, E major) must, in Beethoven’s own

words, be played with much feeling. The chief melody is like a
chorale. It is played first by the first violin, the other instruments
adding a note-for-note, polyphonic accompaniment. It is then
repeated by second violin and viola, in unison, while the first violin
adds above it a serious, gently melodious counterpoint. Other more
vigorous episodes appear later, but the spirit of the movement is
swayed by the sad and prayerful opening theme.
In the trio of the following scherzo another Russian theme is used as

the subject of a fugue. The last movement is unrestrainedly joyful
and vigorous, beginning oddly in C major, but turning presently to the
tonic key (E minor), from which the rondo unfolds in more and more
brilliant power.
The last of this series of quartets—in C major—is for the most part
wholly outspoken. There is little obscurity in meaning, none in form.
At the basis of the slow introduction lies a series of falling half-tones,
given to the 'cello. The first allegro is almost martial in character. The
second movement—andante con moto quasi allegretto, in A minor—
is in the nature of a Romanza; and the frequent pizzicato of the 'cello
suggests the lutenist of days long gone by. There follows a Minuet
instead of a Scherzo; and at the end there is a vigorous fugue.
Between these three quartets and the final series beginning with
opus 127, stand two isolated quartets: opus 74, in E-flat major, and
opus 95, in F minor. Neither indicates a considerable change in
Beethoven’s method, or in his attitude towards his art. The former,
composed in 1809 and dedicated to Prince Lobkowitz, is in the spirit
of the last of the Rasumowsky quartets; that is, outspoken, vigorous,
and clear. The relatively long, slow introduction alone hints at a tragic
seriousness; but it serves rather to show from what the composer
had freed himself, than to expose the riddle of the piece. The first
theme of the first movement is stalwart and well-built; the second, of
rather conventional character, chiefly made up of whirring scale
groups. In the development section there are many measures of
arpeggio figures, at first pizzicato, later growing into arco; by reason
of which the quartet has been given the name of Harfenquartett
(Harp-quartet). In connection with this passage Dr. Riemann has
remarked that all such experiments in sound effects [such as
pizzicato, harmonics and playing on special strings] serve only to
reveal the actual lack of different tone-colors in the quartet; and,
indeed, distract the attention from the ‘drawing’ [i.e., the pure lines of
the various parts] which is peculiarly the affair of the quartet.
The second movement is an adagio in rondo form; the third a

scherzo, with an astonishing trio in 6/8 time; and the last consists of
a theme, oddly syncopated so that the groundwork of the harmonic
progressions may be traced only on the unaccented beats of the
measures, together with five variations.
The quartet in F minor, opus 95, was completed in October, 1810. In

the autograph copy Beethoven gave the work the title Quartett
serioso, omitted in the engraved editions. Theresa Malfatti is
supposed to have refused Beethoven’s offer of marriage in April of
this year. He confided himself rather freely in his friend Zmeskall von
Domanowecz, during these months. The fact that the quartet, opus
95, was held to be serioso by Beethoven, and furthermore that he
dedicated it to Zmeskall, are at least some sort of evidence that the
work sprang from his recent disappointment in love. However, the
first movement is rather spiteful than mournful. It is remarkable for
conciseness. It is, indeed, only one hundred and fifty measures long,
and there are no repetitions. The dominant motive is announced at
once by all four instruments in unison, and is repeated again and
again throughout the movement, like an irritating thought that will not
be banished. There is a second theme, in D-flat major, which
undergoes little development.
The second movement, an allegretto in D major, 2/4, is highly

developed and unusual. It opens with a four-measure phrase of
detached, descending notes, for 'cello alone, which may be taken as
a motto for the movement. This is followed by a strange yet lovely
melody for first violin which is extended by a long-delayed cadence.
After this the viola announces a new theme, suggestive of the
opening motive, which is taken up by the other instruments one after
the other and woven into a complete little fugue, with a stretto. Once
again, then, the 'cello gives out the lovely, and somewhat
mysterious, opening phrase, this time thrice repeated on descending
steps of the scale, and punctuated by mournful harmonies of the
other instruments. The viola announces the fugue theme again, in F
minor; and the fugue is resumed with elaborate counterpoint. And at
the end of this, again the 'cello motive, once more in the tonic key,
and the strange melody sung early by the first violin.
The movement is not completed, but goes without pause into the
next, a strangely built scherzo, allegro assai vivace, ma serioso. The
vivace evidently applies to the main body of the movement, which is
in a constantly active, dotted rhythm. The serioso is explained by the
part of the movement in G-flat major, which one may regard as the
trio. This is merely a chorale melody, first given by the second violin.
The lower instruments follow the melody with note-for-note
harmonies; the first violin adds to each note of the melody an
unvarying formula of ornamentation. All this is done first in the key of
G-flat major, then in D major. The opening section is then repeated,
and after it comes the chorale melody, a little differently scored; and
a coda, piu allegro, brings the movement to an end.
The last movement is preceded by a few introductory measures,
which are in character very like the Lebewohl motive in the sonata,
opus 81. And the progression from the introduction into the allegro
agitato is not unlike the beginning of the last movement of the same
sonata. The allegro itself is most obviously in hunting-song style,
suggesting in the first melody Mendelssohn, in parts of the
accompaniment the horns at the beginning of the second act of
Tristan und Isolda. The second theme is a horn-call. Just before the
end the galloping huntsmen pass far off into the distance, their horns
sound fainter and fainter, finally cease. Then there is a mad coda, in
alla breve time.
III
There follows between this quartet and the quartet, opus 127, a
period of fourteen years, in which time Beethoven composed the
seventh, eighth, and ninth symphonies, the last pianoforte sonatas,
the Liederkreis, and the Mass in D. He turned to the quartet for the
last expression in music of what life had finally come to mean to him,
stone-deaf, miserable in health, weary and unhappy. There is not
one of the last five quartets which does not proclaim the ultimate
victory of his soul over every evil force that had beset his earthly
path.
In November, 1822, Prince Nikolaus Galitzin, a man who held

Beethoven’s genius in highest esteem, asked him if he would
undertake the composition of three quartets. In the spring of the
following year Ignaz Schuppanzigh returned to Vienna after a seven-
years’ absence and resumed his series of quartet concerts. Whether
these two facts account for Beethoven’s concentration upon the
composition of quartets alone during the last two years of his life is
not known. Before the receipt of Prince Galitzin’s invitation
Beethoven had written to Peters in Leipzig that he expected soon to
have a quartet to send him. But no traces of quartet composition are
to be found before 1824. Probably, then, the quartet in E-flat major,
opus 127, was composed in the spring of 1824. In 1825 the quartet
in A minor, opus 132, and later that in B-flat major, opus 130, were
composed. These three quartets were dedicated to Prince Galitzin.
The final rondo of the quartet in B-flat major was written considerably
later (was, indeed, the last of Beethoven’s compositions). Originally
the last movement of this quartet was the fugue, now published
separately as opus 133, which the publishers felt made the work too
long and too obscure. Beethoven therefore wrote the final rondo to
take its place.
There is much internal evidence that while Beethoven was at work

on the last two of the quartets dedicated to Prince Galitzin he was
likewise at work on the quartet in C-sharp minor, opus 131,
dedicated to Baron von Stutterheim. The quartet in F major, opus
135, was written later in 1826. It was dedicated to Johann Wolfmeier.
The first performance of opus 127 was given by the Schuppanzigh

quartet[74] on March 7, 1825. On September 9th of the same year,
Schuppanzigh led the first private performance of opus 132 at the
inn Zum Wilden Mann. It was first publicly performed at a concert
given by Linke on November 6, and was well received. Opus 132
was publicly performed first (in its original form, i.e., with the fugue
finale) on March 21, 1826. The second and fourth movements were
encored.
Of the five last quartets the first and last are formally the most clear;
the intermediate three, especially those in A minor and C-sharp
minor, are perhaps the most intricate and difficult music to follow and
to comprehend that has been written. All but the last are very long,
and thus tax the powers of attention of the average listener often
beyond endurance. Their full significance is discerned only by those
who not only have made themselves intimately familiar with every
note and line of them, but who have penetrated deep into the most
secret mysteries of the whole art of music.
Opus 127 begins with a few measures—maestoso—which, as Dr.

Riemann has suggested, play something of the same rôle in the first
movement as the Grave of the Sonata Pathétique plays there. The
passing over from the introduction to the allegro is only a trill,
growing softer over subdominant harmony. The allegro is in 3/4 time,
and the first theme, played by the first violin, is obvious and simple,
almost in the manner of a folk-song. Yet there is something
sensuous in its full curves and in the close, rich scoring. The
transitional passage is regularly built, and the second theme—in G
minor—pure melody that cannot pass unnoticed. Everything is
simple and clear. The first section ends in G major, and the
development section begins with the maestoso motive in the same
key, followed, just as at the opening of the movement, with the trill
and the melting into the first theme. This theme is developed, leading
to the maestoso in C major. It is then taken up in that key. The
maestoso does not reappear as the beginning of the restatement
section, the first theme coming back in the original key without
introduction. Instead of the simple note-for-note scoring with which it
was first presented, it is now accompanied by a steadily moving
counterpoint. The second theme is brought back in E-flat major. The
coda is short and simple, dying away pianissimo.
The following movement is an adagio, to be played not too slowly

and in a wholly singing manner. The time is 12/8, the key, A-flat
major. The opening notes, which build up slowly a chord of the
dominant seventh, are all syncopated. The first violin gives only a
measure or two of the melody, which, thus prepared, is then taken
up by the violoncello. The second strophe is sung by the violin.
There is a full cadence.
The first variation opens with the melody for violoncello, only slightly
altered from its original form. The violins add a counterpoint in
dialogue. This variation comes to a full stop. The second brings a
change in time signature (C, andante con moto). The theme, now
highly animated, is divided between the first and second violins. In
the fourth variation (E major, 2/2, adagio molto espressivo) only the
general outline of the theme is recognizable, cut down and much
compressed. The fifth variation brings back the original tempo and
the original key. The violoncello has the theme, only slightly varied in
rhythm, and the first violin a well-defined counter-melody. The sixth
and last variation (in this movement) grows strangely out of the fifth,
in D-flat major, sotto voce, leads to C-sharp minor, and thence to A-
flat major. There is a short epilogue.
The main themes of the Scherzo and Trio which follow are so closely
akin to the theme of the adagio, that the movements may be taken
as further variations. The main body of the Scherzo is in that dotted
rhythm of which Beethoven made frequent use in most of his last
works; and is fairly regular in structure, except for the intrusion, at
the end of the second part, of measures in 2/4 time, in unison, which
may be taken as suggestions of still another fragmentary variation of
the adagio theme. The Trio is a presto in E-flat minor.
The Finale is entirely in a vigorous, jovial and even homely vein. The
themes are all clear-cut and regular; the spirit almost boisterous,
suggesting parts of the ‘Academic Festival Overture’ or the
Passacaglia from the fourth symphony of Brahms.
III
This E-flat major quartet was completed at the latest in January,
1825. Work on the following three quartets—in A minor, B-flat major,
and C-sharp minor—began at once, but was interrupted by serious
illness. About the sixth of May Beethoven moved to Gutenbrunn,
near Baden; and here took up the work again. The A minor was
completed not later than August, the B-flat in September or October,
the C-sharp minor some months later, after his return to Vienna.
The three quartets are closely related. In the first place all show a
tendency on the part of Beethoven to depart from the regular four-
movement type. There are five movements in the A minor, six in the
B-flat major, seven in the C-sharp minor; though in the last, two of
the movements are hardly more than introductory in character. The
Danza Tedesca in G major in opus 130, was written originally in A
major and intended for the A minor quartet. Finally the chromatic
motive, clearly stated in the introduction to the A minor quartet, and
lying at the basis of the whole first movement, may be traced in the
fugue theme in opus 130, and in the opening fugal movement of
opus 131.
The A minor quartet is fundamentally regular in structure. The

opening allegro is clearly in sonata-form; there follows a Scherzo
and Trio. The Adagio consists of a chorale melody, thrice repeated in
higher registers, with regular interludes. A short march and a final
Allegro in A minor conclude the work. But the movements are all
strangely sustained and at the same time intense; and there is a
constant whisper of inner and hidden meanings, which cannot be
grasped without deep study and which leave but a vague and
mysterious impression. The chromatic motive of the introduction has
a more or less cryptic significance; the chorale melody is in an
unfamiliar mode; and there are reminiscences of earlier and even
youthful works. So that the whole proves intricate and even in the
last analysis baffling.
There are eight introductory measures (Assai sostenuto) which are

in close polyphonic style out of a single motive. This motive is
announced by the violoncello; immediately taken up, transposed, by
the first violin; given again, inverted, by the violoncello; and in this
form answered by the violin. The Allegro begins upon a diminished
seventh chord in which all the instruments take part, and from which
the first violin breaks with a descending and ascending run of
sixteenth notes, founded upon the chord. The first theme is at once
announced by the first violin, a theme which, distinct and full of
character in itself, really rests upon the opening motive, or upon the
harmonies implied in it. A single measure of adagio prepares for
another start with the same material. The violin has another run,
founded upon the diminished seventh chord, rising thereby to F.
Under this the violoncello takes up the first theme, which is
completed by the viola; while, it will be observed, the first violin,
followed by the second, give out the opening motive, inverted, in
augmentation. Later a transitional theme is announced in D minor by
the first violin, closely imitated by the violoncello and the second
violin. The true second theme follows shortly after, in F major, a
peaceful melody, sung by the second violin over an accompaniment
in triplets shared by viola and violoncello.
The movement is fairly regular in structure. The development is short

and is based chiefly upon the opening chromatic motive, with which
indeed the 'cello begins it. The restatement begins in E minor, with
the familiar diminished seventh run for the first violin. The second
theme appears in C major, and is given to the 'cello. There is a long
coda, which, toward the end, swells over a mysterious low trill to a
brilliant climax.
The next movement is really a Scherzo in A major. The instruments

have four measures in unison, each measure beginning with a half-
step which cannot but suggest some relationship to the chromatic
motive of the first movement. But the short phrase of the first violin,
begun in the fifth measure, is the real kernel of the main body of the
movement. The Trio, in E major, is of magical beauty. The first
section is over a droning A, shared by both violins, at first, to which
the viola and 'cello soon join themselves. The melody is decidedly in
folk-song manner, and is played by the first violin in high registers,
and faithfully followed by the second a tenth below, both instruments
maintaining at the same time their droning A.
This melody is supplanted by a lilting dance movement. The short

phrases begin always on the third beat of the measure, and their
accompanying harmonies are likewise syncopated, in the manner
which is frequent with Brahms. The short phrases are arranged at
first in dialogue fashion between first violin and viola. Later the viola
converses, as it were, with itself. Only the 'cello is limited throughout
the section to accompaniment. A few measures in unison between
the 'cello and viola appear twice before the end of the section, the
notes of which may be intended dimly to recall the chromatic motive
of the first movement. A more positive phrase in alla breve time,
played by second violin, viola, and 'cello in unison, brings back an
epilogue echoing the opening phrases of the Trio; after which the
main body of the movement is repeated.

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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

It would be difficult to characterize each one distinctly. The first, in G

The whole quartet is full of an intensity of feeling. The andante has

This series of six quartets did not make a decidedly favorable

There are, as we have said, twenty-three quartets in all. The majority

[64] C. F. Pohl: ‘Joseph Haydn,’ Vol. I, p. 331.

[65] Eugène Sauzay: Étude sur le quatuor. Paris, 1861.

[66] Cf. C. F. Pohl: op. cit., Vol. II, p. 293.

[67] Étude sur le quatuor.

No. 1, in F major, is known as the ‘Amenda’ quartet. Beethoven had

The second quartet of the series, in G major, is known as the

The fourth quartet of the series is alone in a minor key. It is of more

Between the quartets, opus 18, and these so-called Russian

Nothing gives more striking evidence of the phenomenal power of

The first quartet begins at once with a melody for violoncello,

The second movement (allegretto vivace e sempre scherzando, B-

The second movement (Adagio, E major) must, in Beethoven’s own

In the trio of the following scherzo another Russian theme is used as

The second movement is an adagio in rondo form; the third a

The quartet in F minor, opus 95, was completed in October, 1810. In

The second movement, an allegretto in D major, 2/4, is highly

In November, 1822, Prince Nikolaus Galitzin, a man who held

There is much internal evidence that while Beethoven was at work

The first performance of opus 127 was given by the Schuppanzigh

Opus 127 begins with a few measures—maestoso—which, as Dr.

The following movement is an adagio, to be played not too slowly

The A minor quartet is fundamentally regular in structure. The

There are eight introductory measures (Assai sostenuto) which are

The movement is fairly regular in structure. The development is short

The next movement is really a Scherzo in A major. The instruments

This melody is supplanted by a lilting dance movement. The short

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