Nutrition and Chronic Conditions

nutrients
Nutrition and
Chronic Conditions
Edited by
Omorogieva Ojo
Printed Edition of the Special Issue Published in Nutrients
www.mdpi.com/journal/nutrients
Nutrition and Chronic Conditions
Nutrition and Chronic Conditions
Special Issue Editor

Omorogieva Ojo
MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade

Omorogieva Ojo
University of Greenwich
UK
Editorial Office
MDPI
St. Alban-Anlage 66
4052 Basel, Switzerland
This is a reprint of articles from the Special Issue published online in the open access journal Nutrients
(ISSN 2072-6643) in 2018 (available at: https://www.mdpi.com/journal/nutrients/special issues/
nutrition and chronic conditions)
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Contents
About the Special Issue Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Preface to ”Nutrition and Chronic Conditions” . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Omorogieva Ojo, Osarhumwese Osaretin Ojo, Fajemisin Adebowale and Xiao-Hua Wang
The Effect of Dietary Glycaemic Index on Glycaemia in Patients with Type 2 Diabetes:
A Systematic Review and Meta-Analysis of Randomized Controlled Trials
Reprinted from: Nutrients 2018, 10, 373, doi:10.3390/nu10030373 . . . . . . . . . . . . . . . . . . . 1
Hanan A. Alfawaz, Kaiser Wani, Abdullah M. Alnaami, Yousef Al-Saleh, Naji J. Aljohani,
Omar S. Al-Attas, Majed S. Alokail, Sudhesh Kumar and Nasser M. Al-Daghri
Effects of Different Dietary and Lifestyle Modification Therapies on Metabolic Syndrome
in Prediabetic Arab Patients: A 12-Month Longitudinal Study
Edgar Denova-Gutiérrez, Paloma Mu ñoz-Aguirre, Nitin Shivappa, James R. Hébert,

Lizbeth Tolentino-Mayo, Carolina Batis and Simón Barquera
Dietary Inflammatory Index and Type 2 Diabetes Mellitus in Adults: The Diabetes Mellitus
Survey of Mexico City
Reprinted from: Nutrients 2018, 10, , doi:10.3390/nu10040385 . . . . . . . . . . . . . . . . . . . . . 31
Estefania Sanchez-Rodriguez, Elena Lima-Cabello, Sara Biel-Glesson, Jose R.

Fernandez-Navarro, Miguel A. Calleja, Maria Roca, Juan A. Espejo-Calvo, Blas
Gil-Extremera, Maria Soria-Florido, Rafael de la Torre, Montserrat Fito, Maria-Isabel
Covas, Juan de Dios Alche, Emilio Martinez de Victoria, Angel Gil and Maria D. Mesa
Effects of Virgin Olive Oils Differing in Their Bioactive Compound Contents on Metabolic
Syndrome and Endothelial Functional Risk Biomarkers in Healthy Adults: A Randomized
Double-Blind Controlled Trial
Li-Li Wang, Qi Wang, Yong Hong, Omorogieva Ojo, Qing Jiang, Yun-Ying Hou,
Yu-Hua Huang and Xiao-Hua Wang
The Effect of Low-Carbohydrate Diet on Glycemic Control in Patients with Type 2
Diabetes Mellitus
Renée Wilson, Jinny Willis, Richard B. Gearry, Alan Hughes, Blair Lawley, Paula Skidmore,
Chris Frampton, Elizabeth Fleming, Angie Anderson, Lizzie Jones, Gerald W. Tannock and
Anitra C. Carr
SunGold Kiwifruit Supplementation of Individuals with Prediabetes Alters Gut Microbiota and
Improves Vitamin C Status, Anthropometric and Clinical Markers
Blanka Klı́mová and Martin Vališ

Nutritional Interventions as Beneficial Strategies to Delay Cognitive Decline in Healthy
Older Individuals
v
Magali Leyvraz, Carmelle Mizéhoun-Adissoda, Dismand Houinato, Naby Moussa Baldé,
Albertino Damasceno, Bharathi Viswanathan, Mary Amyunzu-Nyamongo, Jared Owuor,
Arnaud Chiolero and Pascal Bovet
Food Consumption, Knowledge, Attitudes, and Practices Related to Salt in Urban Areas in Five
Sub-Saharan African Countries
Reprinted from: Nutrients 2018, 10, 1028, doi:10.3390/nu10081028 . . . . . . . . . . . . . . . . . . 105
Christian R. Juhl, Helle K. M. Bergholdt, Iben M. Miller, Gregor B. E. Jemec,

Jørgen K. Kanters and Christina Ellervik
Lactase Persistence, Milk Intake, and Adult Acne: A Mendelian Randomization Study of 20,416
Danish Adults
Christian R. Juhl, Helle K. M. Bergholdt, Iben M. Miller, Gregor B. E. Jemec,

Jørgen K. Kanters and Christina Ellervik
Dairy Intake and Acne Vulgaris: A Systematic Review and Meta-Analysis of 78,529 Children,
Adolescents, and Young Adults
Lukasz Szternel, Magdalena Krintus, Katarzyna Bergmann, Tadeusz Derezinski and

Grazyna Sypniewska
Association between Fasting Glucose Concentration, Lipid Profile and 25(OH)D Status in
Children Aged 9–11
Tatiana S. Collese, Gabriela Vatavuk-Serrati, Marcus Vinicius Nascimento-Ferreira,

Augusto César Ferreira De Moraes and Heráclito Barbosa Carvalho
What is the Validity of Questionnaires Assessing Fruit and Vegetable Consumption in Children
When Compared with Blood Biomarkers? A Meta-Analysis
Namrata Sanjeevi, Leah M. Lipsky and Tonja R. Nansel

Cardiovascular Biomarkers in Association with Dietary Intake in a Longitudinal Study of Youth
with Type 1 Diabetes
Silvana Perez-Leon, M. Amalia Pesantes, Nathaly Aya Pastrana, Shivani Raman,

Jaime Miranda and L. Suzanne Suggs
Food Perceptions and Dietary Changes for Chronic Condition Management in Rural Peru:
Insights for Health Promotion
Maaike J. Bruins, Peter Van Dael and Manfred Eggersdorfer

The Role of Nutrients in Reducing the Risk for Noncommunicable Diseases during Aging
vi
About the Special Issue Editor
Omorogieva Ojo has a Ph.D. in nutrition from the University of Greenwich, London, a post-graduate
diploma in diabetes from the University of Surrey, Roehampton and a graduate certificate in Higher
Education from the University of Greenwich. Prior to these qualifications, Dr. Ojo was awarded
his BSc and MSc in animal science from the University of Ibadan, Nigeria. He is currently a Senior
Lecturer in Diabetes Care and Management in the Faculty of Education and Health, the University
of Greenwich and he teaches across a range of courses and programmes. He was previously a
nutrition specialist at the Home Enteral Nutrition Team, Lewisham Primary Care Trust, London, a
post-doctoral research fellow in the School of Science, University of Greenwich, London, and taught
at the College of Agriculture, Asaba, Nigeria. He is an internationally acclaimed expert in nutrition
and diabetes and these are the primary focus of his teaching and research activities, including Ph.D.
supervision. Dr. Ojo has been a keynote speaker at international conferences and he is on the Editorial
Board of many international journals including Nutrients.
vii
Preface to ”Nutrition and Chronic Conditions”
This book on Nutrition and Chronic Conditions aims to provide insight into the effect of nutrition
in the development, care, and management of chronic conditions. This is in recognition of the impact
that nutrition has on chronic conditions, such as diabetes, cardiovascular disease, dementia, stroke,
and inflammatory bowel disease, which continues to generate interest among researchers. There is
evidence that diet is a modifiable risk factor for these diseases, which manifest either as single entities
or in co-morbid states in individuals and populations around the world. In particular, the prevalence
of diabetes and cardiovascular disease is on the increase, especially in developed countries, but also
in developing economies, partly due to lifestyle changes, including diet. For example, ischaemic
heart disease is the leading cause of death globally. When combined with stroke, these conditions
accounted for 15 million deaths in 2015 and are the world’s greatest killers. In addition, there were an
estimated 422 million adults who were living with diabetes in 2014 compared to 108 million in 1980.
These chronic conditions and their associated complications have significant implications
for morbidity and mortality, and incur huge costs to the health services around the world.
The composition of the diet, the proportion and types of macronutrients and micronutrients present
in the diet are major contributors to these diseases. In addition, the beneficial effects of nutritional
interventions have been well documented although differences remain among researchers with
respect to their overall impact. The evaluation of the role of nutrition in chronic conditions draws
on its effect on body weight and body composition, glycaemic and insulin excursions, vascular
remodeling, and gastro-intestinal dysfunction.
Internationally acclaimed experts in the field of nutrition and chronic conditions have
contributed chapters to this book that should provide the evidence base for practice and research.
Therefore, this book is aimed at patients, students, and healthcare professionals, including nurses,
doctors, public health practitioners, and dietitians/nutritionists. The book has sixteen chapters
covering original research and reviews, which should guide healthcare professionals in their areas
of practice. These include evaluations of the effects of various nutritional interventions, dietary
and lifestyle modifications on cognitive decline, anthropometric parameters, metabolic syndrome,
diabetes, and glycaemic control. In addition, assessment of food consumption, knowledge, attitudes,
and practices related to salt, dairy intake and acne vulgaris, food perceptions, and dietary changes in
chronic conditions are the key topics of interest in this book.
Omorogieva Ojo
ix
nutrients
Review
The Effect of Dietary Glycaemic Index on Glycaemia
in Patients with Type 2 Diabetes: A Systematic
Review and Meta-Analysis of Randomized
Controlled Trials
Omorogieva Ojo 1, *, Osarhumwese Osaretin Ojo 2 , Fajemisin Adebowale 3 and Xiao-Hua Wang 4
1 Department of Adult Nursing and Paramedic Science, University of Greenwich, London SE9 2UG, UK
2 Healthcare, Care UK, HMP Wormwood Scrubs, London W12 0AE, UK; [email protected]
3 Department of Animal Production and Health, Federal University of Technology, PMB, Akure 704,
Ondo State, Nigeria; [email protected]
4 The School of Nursing, Soochow University, Suzhou 215006, China; [email protected]
* Correspondence: [email protected]; Tel.: +44-020-8331-8626; Fax: +44-020-8331-8060
Received: 31 January 2018; Accepted: 15 March 2018; Published: 19 March 2018
Abstract: Background: The increasing prevalence of diabetes in the United Kingdom and worldwide
calls for new approaches to its management, and diets with low glycaemic index have been proposed
as a useful means for managing glucose response. However, there are conflicting reports and
differences in the results of studies in terms of their effectiveness. Furthermore, the impact of
low-glycaemic index diets and their long-term use in patients with type 2 diabetes remains unclear.
Objectives: The objective of this study was to conduct a systematic review and meta-analysis of
the effect of low-glycaemic index diets in patients with type 2 diabetes. Methods: Search methods:
Randomised controlled studies were selected from a number of databases (EBSCOHost with links
to Health Research databases, PubMed, and grey literature) based on the Population, Intervention,
Comparator, Outcomes and Study designs (PICOS) framework. The search terms included synonyms
and Medical Subject Headings (MeSH) and involved the use of Boolean operators (AND/OR) which
allowed the combination of words and search terms. Selection criteria: As per the selection criteria,
the following types of articles were selected: studies on randomised controlled trials, with year
of publication between 2008 and 2018, including patients with type 2 diabetes. Thus, studies
involving patients with gestational and type 1 diabetes were excluded, as were observational studies.
Nine articles which met the inclusion criteria were selected for the systematic review, whereas
only six articles which met the criteria were included in the meta-analysis. Data collection and
analysis: Studies were evaluated for quality and risk of bias. In addition, heterogeneity, meta-analysis,
and sensitivity tests of the extracted data were carried out using Review Manager 5.3 (Review
Manager, 2014). Results: The findings of the systematic review showed that the low-glycaemic
index (low-GI) diet resulted in a significant improvement (<0.05) in glycated haemoglobin (HbA1c)
in two studies: low-GI diet Δ = −0.5% (95% CI, −0.61% to −0.39%) vs. high-cereal fibre diet
Δ = −0.18% (95% CI, −0.29% to −0.07%); and low-GI legume diet Δ = −0.5% (95%, −0.6%
to −0.4%) vs. high-wheat fibre diet Δ = −0.3% (95% Cl, −0.4 to −0.2%). There was a slight
improvement in one study (low glycaemic response = 6.5% (6.3–7.1) vs. control = 6.6% (6.3–7.0) and no
significant difference (p > 0.05) in four studies compared with the control diet. Four studies showed
improvements in fasting blood glucose in low-GI diets compared to higher-GI diets or control: low-GI
diet = 150.8 ± 8.7 vs. higher-GI diet = 157.8 ± 10.4 mg/dL, mean ± SD p = 0.43; low-GI diet = 127.7
vs. high-cereal fibre diet = 136.8 mg/dL, p = 0.02; low-GI diet = 6.5 (5.6–8.4) vs. standard diabetic
diet = 6.7 (6.1–7.5) mmol/L, median and interquartile range p > 0.05; and low-GI diet = 7.3 ± 0.3 vs.
conventional carbohydrate exchange diet = 7.7 ± 0.4 mmol/L, mean ± SEM (Standard Error of Mean)
p < 0.05. The results of the meta-analysis and sensitivity tests demonstrated significant differences
(p < 0.001 and p < 0.001, respectively) between the low-GI diet and the higher-GI diet or control
Nutrients 2018, 10, 373; doi:10.3390/nu10030373 1 www.mdpi.com/journal/nutrients

Nutrients 2018, 10, 373
diet in relation to glycated haemoglobin. Differences between the low-GI diet and higher-GI diet or
control were significant (p < 0.05) with respect to the fasting blood glucose following meta-analysis.
Conclusion: The low-GI diet is more effective in controlling glycated haemoglobin and fasting blood
glucose compared with a higher-GI diet or control in patients with type 2 diabetes.
Keywords: glycaemic index; glycated haemoglobin; fasting blood glucose; type 2 diabetes;
randomised controlled trials; meta-analysis; systematic review
1. Introduction
The increasing prevalence of diabetes and its impact on morbidity and mortality have become
global problems [1,2]. About 422 million adults worldwide were reported to live with diabetes in
2016, and the global prevalence rose from 4.7% in 1980 to 8.5% in 2014 [1]. In the United Kingdom,
the prevalence of type 2 diabetes more than doubled from 2.39% in the year 2000 to 5.32% in 2013 [2].
The management of type 2 diabetes and its related complications, including retinopathy, kidney
dysfunction, neuropathy, and foot problems accounts for about 10% of the entire National Health
Service (NHS) budget in the UK [2]. Presently, 11% of U.S. adult population has diabetes and the
total estimated costs associated with the condition in 2012 were US $245 billion due to direct medical
costs and reduced worker productivity [3]. Several factors, including genetic predisposition and
environmental factors, have been implicated in the aetiology of diabetes [3,4]. This is particularly true
in the case of type 2 diabetes, which accounts for over 90% of all forms of diabetes and where lifestyle
has a profound effect on its manifestation [5]. Usually, lifestyle factors such as diet and physical
activities can be modified in terms of the choices that individuals make. The composition of diet with
respect to the quality of the nutrients including carbohydrates, protein, fats, minerals and vitamins is
important in determining nutritive value and usefulness in human health [6].
1.1. Description of the Intervention

Foods that are composed of carbohydrates which break down quickly during the process of
digestion (such as white bread) and that are rapidly absorbed into the blood stream are often termed
as foods with high glycaemic index (GI) [7–9]. Foods with high GI not only rapidly increase blood
glucose, but also insulin responses following the consumption of food [10]. In contrast, foods with
a low glycaemic index such as legumes, lentils, and oats usually contain carbohydrates which break
down slowly during digestion and are slowly assimilated [8,9]. Therefore, these foods have a slower
impact on blood glucose levels and insulin response.
The GI is a measure of the percentage of the area under curve (AUC) with respect to 2-h
blood glucose following the ingestion of a test diet compared with a standard diet (usually glucose
or bread) [7]. It can also be viewed as a reflection of the relative rate of digestibility of the
available carbohydrates of the food compared with a reference food, which is often glucose [11,12].
Differences exist in literature as to what constitutes a low-GI diet and a high-GI diet. Values such as
GI ≤ 40 and GI ≤ 55 for the low-GI diet and GI ≥ 70 for the high-GI diet have been reported [7,13].
1.2. How the Intervention Might Work

The GI value of food is not based on the characteristics of the individual that consumed it, instead,
it depends on the food consumed [9,14,15]. Therefore, dietary management approaches which target
weight loss and improved glycaemic control (including glycated haemoglobin and fasting blood
glucose) in patients with type 2 diabetes may rely on the use of diets with low glycaemic index
instead of using standard low-fat diet [16]. The foods with low GI may contribute to glycaemic control
compared to foods with high GI through the promotion of insulin sensitivity, reducing fluctuations
in blood glucose levels and reducing daily insulin requirements [8]. While glycated haemoglobin
2
Nutrients 2018, 10, 373
(HbA1C) provides a measure of the average glycaemia over the preceding 3 months, the fasting blood
glucose is a measure of blood glucose level following at least 8 h of fasting, and is usually taken before
breakfast [17].
1.3. Why It Is Important to Do This Review

Strategies for managing diabetes often rely on lifestyle modifications, including dietary
interventions and pharmacological approaches. There is also evidence that the consumption of
diets with high glycaemic index and glycaemic load over a long period of time may have implications
for metabolism and health, including chronic hyperglycaemia and hyperinsulinaemia, which can lead
to insulin resistance and diabetes [14]. In addition, studies involving populations in China and the
USA have shown that women with a high intake of food with a high glycaemic index were more at
risk of developing type 2 diabetes compared with women on diets with low glycaemic index [14,18,19].
However, there are inconsistencies and controversies with respect to the use of GI of food as a guide
in the selection of foods for patients with diabetes [8,12,20–22]. Evidence from previous studies on
the role of diets with low GI on health and health-related outcomes have produced mixed results [16].
While some studies have found the high-GI diet to be related to poorer short-term metabolic outcomes,
greater hunger, less satiety, and greater food intake [10], the results from other studies have been
different, either not finding the same association or finding an inverse relationship [12,23]. Jung and
Choi [13] demonstrated the beneficial effects of low-GI diet on glucose control in relatively short-term
trials in patients with type 2 diabetes, although the long-term effects of low-GI diets remain unclear.
This view is further reinforced by Thomas and Elliott [8] who noted that the effects of low-GI diets in
managing patients with diabetes have demonstrated mixed results, from small but clinically useful
effects on the medium-term glycaemic control in diabetes, to only modest secondary benefit. The review
by Thomas and Elliott [8], which was published more than 7 years ago and involved patients with
type 1 and type 2 diabetes, found that there was a significant decrease in HbA1c in a low-GI diet
compared with control. Some of the studies included in this review involved children, and the primary
outcome measures were HbA1c and fructosamine. Another review on glycaemic index and type 2
diabetes included only observational studies [22].
However, the current systematic review is based only on randomised controlled trials and involves
only adults with type 2 diabetes, and the outcomes of interest are HbA1c and fasting blood glucose.
In addition, there is currently no globally agreed form of diet for managing patients with diabetes [8].
Therefore, research on how best to understand the quality and composition of carbohydrates and other
nutrients in foods will be essential in developing diets that will one day be useful to patients with
diabetes and acceptable to the global community.
1.4. Objectives
This is a systematic review and meta-analysis which evaluates the effect of the low-glycaemic
index diet in patients with type 2 diabetes
Research question: Is a low-GI diet effective in improving glycaemia in patients with type 2
diabetes compared with a higher-GI diet?
2. Methods
2.1. Types of Studies

Only studies involving randomised controlled trials were selected for this review (Table 1).
2.2. Types of Participants

The participants in the studies selected were adult patients with type 2 diabetes (Table 2).
3
Nutrients 2018, 10, 373
2.3. Types of Interventions

The effect of low-glycaemic index diet was compared with higher-glycaemic index diet or control
(conventional carbohydrate exchange, high-cereal fibre diet, high-wheat fibre diet, standard diabetic
diet, American diabetes association diet) in adult patients with type 2 diabetes. The higher-GI or
control diets were classified as having a higher glycaemic index based on the lower GI values of the
intervention diets (low-GI diet).
2.4. Types of Outcome Measures

The following were the outcome measures of interest:
Blood glucose parameters: Glycated haemoglobin (%), fasting blood glucose (mg/dL).
Search Methods for Identification of Studies

The Population, Intervention, Comparator, Outcomes and Study designs (PICOS) framework
was used to identify articles in the various databases [24,25]. The search terms included synonyms
and Medical Subject Headings (MeSH) and involved the use of Boolean operators (AND/OR) which
allowed the combination of words and search terms (Table 1).
Table 1. Search terms and search strategy.
Combining
Patient/Population Intervention Comparator Study Designs
Search Terms
Low-glycaemic Higher-glycaemic Randomised
Patients with diabetes
index diet index diet or control controlled trial
#1 Randomised
controlled trial OR
Patients with diabetes Glycaemic index OR
controlled clinical trial
OR type 2 diabetes OR glycemic index OR
OR randomized OR
diabetes OR diabetes glycaemic load OR Column 1 and
placebo OR drug
complications OR glycaemic indices or Column 2 and
therapy OR randomly
diabetes mellitus, glycaemic index Column 3
OR trial OR groups
type 2 OR number or glycaemic
#2 “Animals”
diabetes mellitus index numbers
NOT “Humans”
#3 #1 NOT #2
2.5. Electronic Searches

A number of research databases were used to search for relevant articles for this review.
These included EBSCoHost research databases with links to Health Research databases which
incorporate Academic Search Premier, Medline, the Psychology and Behavioural Sciences Collection,
PSYCInfo, and the Cumulative Index to Nursing and Allied Health Literature (CINAHL) Plus.
In addition, Pubmed was searched for useful articles (Figure 1).
2.6. Searching Other Resources

The Web of Science database which encompasses the BIOSIS citation index was searched for
conference papers, and the reference list of articles were also searched.
2.7. Selection of Studies

Only primary research on randomised controlled studies carried out between 2008 and 2018
were included in this review (Table 2). This period was chosen because the search period for the
previous systematic review and meta-analysis by Thomas and Elliot study [8] ended in March 2009.
Earlier search conducted from 2009 to 2018 did not yield enough studies for the current review.
4
Nutrients 2018, 10, 373
In addition, only studies involving adults with type 2 diabetes and the use of the dietary glycaemic
index were included. Studies written in English from across the world have been included as diabetes
is a worldwide problem.
Identification
Records identified Records identified Reference list of articles
through EBSCOHost through PubMed (n = 2) 2008–2018
2008–2018 2008–2018 Web of Science (n = 196)
(n =2347) (n = 3543)
Records after de-duplication (5540)

Screening
Further Screening using inclusion and Exclusion Criteria:
Full-text articles assessed for

Eligibility
eligibility N = 2 (Studies involving
(N =24) ketogenic diets)
N = 7 (Studies on glycaemic
load)
15
N = 6 (Studies involving
carbohydrate diets)
Studies included in systematic
review
(N = 9)
Included
N = 1 (Study results that
could not be extracted due

3 to statistical mode of
presentation)
Studies included in Meta-analysis
N = 2 (Studies not providing

(N = 6)
data before intervention)
Figure 1. PRISMA flow chart showing the selection of articles.
Therefore, other studies involving patients with type 1 diabetes or gestational diabetes and animal
studies were excluded from this review (Table 2). Similarly, studies involving children with diabetes or
healthy adults without diabetes were also excluded. Studies which were not randomised and those
involving dietary supplements have been excluded from this review.
5
Nutrients 2018, 10, 373
Table 2. Criteria for considering studies for the review based on the Population, Intervention,
Comparator, Outcomes and Study designs (PICOS) structure.
Inclusion Criteria Exclusion Criteria

Studies involving patients with type 1 diabetes
Adult patients (≥18 years) with or gestational diabetes and animal studies.
Population
type 2 diabetes Studies involving children with diabetes or
healthy adults.
Intervention Low-glycaemic index diet Studies involving dietary supplements
Higher-glycaemic index diet
Comparator Studies involving additional supplements
and/or control
Blood glucose parameters:
Outcomes Glycated haemoglobin, fasting Qualitative outcomes
blood glucose
Observational studies
Letters
Types of study: quantitative Randomised controlled trials
Comments
Reviews
2.8. Evaluation of Quality

The quality of the peer-reviewed articles was evaluated using the checklists for quantitative
studies [26] and the experience of the authors.
2.9. Data Extraction and Management

Data from the selected articles were extracted separately by all the authors based on an agreed
framework and verified by all the authors following completion.
2.10. Meta-Analysis Methods

The meta-analysis of data was carried out using Review Manager (RevMan) 5.3 software [24].
Changes in means and standard deviations between the baseline values and final results for each
outcome of interest in the low-GI diet and the higher-GI diet or control for the different studies were
determined. In addition, the number of participants in the intervention and control groups in each
study were included in a table and entered into the RevMan software for analysis. A heterogeneity test
was carried out in order to evaluate the evidence of variability of the intervention effects [27]. For the
heterogeneity test, a p value of 0.1 was used to determine statistical significance [27]. The heterogeneity
statistic I2 value was <50 and this indicated low heterogeneity for the studies included in both the
glycated haemoglobin and fasting blood glucose analyses. Therefore, the fixed effects model was used
for both the meta-analysis and the sensitivity tests.
A fail-safe number was also calculated for both outcomes of interest. A sensitivity analysis
involving a repeat of the meta-analysis of studies that were definitely known to be eligible was also
conducted [27]. This process involved removing some of the studies from the primary analysis in order
establish that the findings from the systematic review were not dependent on unclear decisions [27].
In this case, the sensitivity tests were carried out for both glycated haemoglobin and fasting blood
glucose by repeating the meta-analysis after removing the studies with the most weight in order to
confirm whether the results were stable.
2.11. Assessment of Risk of Bias in Included Studies

The assessment tool used for evaluating the risk of bias was a domain-based evaluation tool [27].
The process involved the separate critical assessment of the various domains including the random
sequence generation (selection bias), allocation concealment (selection bias), blinding of participants
and personnel (performance bias), blinding of outcome assessment (detection bias), incomplete
6
Nutrients 2018, 10, 373
outcome data (attrition bias), selective reporting (reporting bias), and other bias [27]. The risk of
bias was assessed by Review Manager 5.3 software [24].
3. Results
With respect to the systematic review, only nine articles [28–36] met the criteria for inclusion
(Table 3). However, six of these studies [28–31,35,36] were used for the meta-analysis to test the effect
of low-GI diet on glycated haemoglobin and fasting blood glucose in patients with type 2 diabetes.
The others were excluded as they did not meet the criteria for meta-analysis such as reporting their
results in the form of median and interquartile or not providing data before intervention.
The length of study ranged from 2 weeks to 22 months. In terms of the interventions,
these involved comparing the low-GI diet with the higher-GI diet or control (conventional carbohydrate
exchange, high-cereal fibre diet, high-wheat fibre diet, standard diabetic diet, American diabetes
association diet) (Table 3).
In most of the studies [28,30,34–36] that reported dietary glycaemic index values, the low-GI
diet had significantly (p < 0.05) lower values than the higher-GI diet or the control. The study by
Jenkins et al. [29] also showed that the low-GI diet resulted in lower GI values than the high-cereal
fibre diet, although the level of statistical difference was not stated (Table 3). However, differences in
dietary GI values were not significant (p > 0.05) in one study [31], while data were not available in two
of the studies [32,33] although the authors stated that the intervention diets involved low glycaemic
index or low glycaemic response.
In studies reporting on the effect of dietary GI on the HbA1c levels, diets with low GI were shown
to result in a significant improvement (p < 0.05) in HbA1c levels in two studies [29,30] compared with
the higher-GI diet or the control. One study [33] reported slight improvement in HbA1c in the low-GI
diet group compared with control, while differences between low-GI diet and higher-GI diet or control
were not significantly different in four studies [31,34–36].
The effect of low-GI diets on fasting blood glucose compared to higher-GI diets or control diets
was evident in seven of the studies selected [28–30,32–34,36]. While four studies [28,29,34,36] showed a
greater improvement in fasting blood glucose in the low-GI diet compared with higher-GI diet, some of
the differences were not statistically significant. Furthermore, there was a lower fasting blood glucose
level in the higher-GI diet or control compared with low-GI diet in two studies [30,33]. The fasting
blood glucose levels were not significantly different in the low-GI diet compared with control in one
other study [32].
7
Table 3. Summary of studies included in the systematic review.
Length Study Sample Diabetes Duration Glycated Haemoglobin

Citation Age (Years) Interventions Blood Glucose Dietary Glycaemic Index
of Study Type Size (Years) (HbA1c) %
*# Baseline ## Baseline
Low-GI diet = 148.9 ± 8.2 vs. Low-GI diet = 63 ± 6 vs.
# Low-GI (Glycaemic higher-GI diet 147.8 ± 10.7 higher-GI diet = 66 ± 4
Parallel Low-GI diet versus
Gomes et al. [28] 1 month 20 # 42.4 ± 5.1 Index) diet (4.8 ± 1.5) No data 30 days 30 days
Nutrients 2018, 10, 373
Design higher-GI diet

higher-GI diet (4.9 ± 1.6) Low-GI diet = 150.8 ± 8.7 vs. Low-GI diet = 54 ± 4 vs.
higher-GI diet = 157.8 ± 10.4 higher-GI diet = 72 ± 3
p = 0.43 p = 0.005
#### Week 0
Low-GI diet =
* (Mean) Week 0
Low-GI diet Δ = −0.5% 80.8 (79.6–82.0) vs.
Low-GI diet = 138.8 vs.
(95% CI, −0.61% to high-cereal fibre diet =
# Low-GI diet = 60 (10) # Low-GI diet = 8.3 (6.5) high-cereal fibre diet = 141.2
Parallel Low-GI diet versus −0.39%) vs. high-cereal 81.5 (80.4–82.7)
Jenkins et al. [29] 6 months 210 High-cereal fibre High-cereal fibre Week 24
Design high-cereal fibre diet fibre diet Δ = −0.18% (95% Week 24
diet = 61 (9) diet = 7.2 (5.9) Low-GI diet = 127.7 vs.
CI, −0.29% to −0.07%) Low-GI diet =
high-cereal fibre diet = 136.8
p < 0.001 69.6 (67.7–71.4) vs.
p = 0.02
high-cereal fibre diet =
83.5 (82.4–84.7)
*#### Baseline #### Baseline
Low-GI legume diet = 141 Low-GI legume diet = 80
(135–147) (95% CI) vs. (79–82) (95% CI) vs.
Low GI legume diet
high-wheat fibre diet = 134 high-wheat fibre diet = 78
## Low-GI legume diet = ## Low-GI legume Δ = −0.5% (95%, −0.6% to
8
(127–141) (95% CI) (77–80) (95% CI)
Parallel 58 (1.3) diet = 9.2 (8.0) Low-GI legume diet vs. −0.4%) vs. high-wheat
Jenkins et al. [30] 3 months 121 End of study End of study
Design High-wheat fibre High-wheat fibre high-wheat fibre diet fibre diet Δ = −0.3% (95%
Low-GI legume diet = Low-GI legume diet = 66
diet = 61 (1.0) diet = 8.6 (0.8) Cl, −0.4 to −0.2%)
132(126–138) (95% CI) vs. (64–67) (95% CI) vs.
p < 0.001
high-wheat fibre diet = 127 high-wheat fibre diet = 82
(121–133) (95% CI) (81–83) (95% CI)
p = 0.001 p < 0.001
## Baseline ## Baseline
Low-GI diet = Low-GI diet =
8.74 ± 0.29% vs. baseline 79.35 ± 1.36 vs. ADA diet
Low-GI diet vs. American ADA diet = 8.1 ± 0.28% = 82.03 ± 1.31
12 Parallel
Ma et al. [31] 40 # 53.53 ± 8.40 # 9.32 ± 9.66 Diabetes Association diet 12 months No data 12 months
months Design
(ADA) Low-GI diet = Low-GI diet =
8.39 ± 0.30% vs. 12-month 76.64 ± 1.46 vs. ADA diet
ADA diet = 7.67 ± 0.28% = 80.36 ± 1.40
p = 0.08 p = 0.07
Table 3. Cont.
Length Study Sample Diabetes Duration Glycated Haemoglobin

Citation Age (Years) Interventions Blood Glucose Dietary Glycaemic Index
of Study Type Size (Years) (HbA1c) %
*# Low-GI diet first day (127
± 30) vs. higher-GI diet (148
Gonçalves Reis Cross-over Low-GI diet vs. higher-GI ± 62) (p < 0.05)
2 weeks 12 # 60 ± 8 # 12 ± 7 No data No data
and Dullius [32] study diet By the second day FBG levels
Nutrients 2018, 10, 373
had the same average value

(132 mg/dL) (p = 0.78)
### Baseline **### Baseline
Low-GR = 6.5% (6.1–6.9) Low-GR = 7.3 (6.4–8.1)
Low-GR (Glycaemic
Stenvers et al. 22 Cross-over Control = 6.5% (6.2–6.9) Control = 6.8 (6.1–7.4)
20 # 60 ± 7 ### 5 (1–9) Response) liquid formula No data
[33] months study 12 weeks 12 weeks
versus free choice (control)
Low-GR = 6.5% (6.3–7.1) Low-GR = 7.2 (6.5–7.7)
Control = 6.6% (6.3–7.0) Control = 7.0 (6.7–7.8)
### Low-GI diet = **### Low-GI diet = ### Low-GI diet =
20 (12 6.63 (6.08–7.0)% 6.5 (5.6–8.4) 49 (48–51)
Cross–over Low-GI diet versus
Visek et al. [34] 3 months men + # 62.7 ± 5.8 # 7 ± 4.1 Standard diabetic diet = Standard diabetic diet = 6.7 Standard diabetic diet = 68
study standard diabetic diet
8 women) 6.45 (6.18–6.91)% (6.1–7.5) (61–72)
(p > 0.05) (p > 0.05) (p < 0.01)
## Baseline
Low-GI diet = 6.2 ± 0.8%
## Baseline Low-GI diet =
Higher-GI diet = 6.2 ± 1%
60.3 ± 0.4 Higher-GI diet
Low-GI diet = 60.6 ± 1.0 Outcomes
9
Wolever et al. 12 Parallel Low-GI diet vs. higher-GI = 61.5 ± 0.4 Study Low-GI
162 Higher-GI diet = No data Low-GI diet = No data
[35] months Design diet diet = 55.1 ± 0.4
60.4 ± 1.1 6.34 ± 0.05%
Higher-GI diet = 63.2 ±
Higher-GI diet =
0.4 p < 0.001
6.34 ± 0.05%
p > 0.05
## Baseline
**## Baseline
Low-GI diet =
Low-GI diet = 7.33 ± 2.23
7.68 ± 1.13% # Week 12 Low-GI diet =
Low-GI diet vs. CCE = 7.01 ± 1.79
Parallel CCE = 7.51 ± 1.24% 57 ± 6
Yusof et al. [36] 12 weeks 100 53.5 No data conventional carbohydrate Week 12
Design Week 12 Week 12 CCE = 64 ± 5
exchange (CCE) Low-GI diet = 7.3 ± 0.3
Low-GI diet = 7.2 ± 0.1% p < 0.001
CCE = 7.7 ± 0.4
CCE = 7.2 ± 0.2%
p < 0.05
p > 0.05
Abbreviations: ADA (American Diabetes Association); CCE (conventional carbohydrate exchange); FGB (fasting blood glucose); glycated haemoglobin (HbA1c); GI (glycaemic index);
GR (glycaemic response); low-GR (low glycaemic response); Δ (change); * (FBG, mg/dL); ** FBG (mmol/L); # (Mean ± SD); ## (mean ± SEM); ### (Median) (25th–75th percentile);
#### (Mean and 95% CI, confidence interval).
Nutrients 2018, 10, 373
3.1. Assessment of Risk of Bias in Included Studies

In terms of the selection bias (random sequence generation and allocation concealment) 100% of
the studies showed low risk of bias (Figures 2 and 3). With respect to the other risks of bias (blinding,
incomplete outcome data, selective reporting and other potential sources of bias), all the studies
demonstrated 100% low risk of bias or unclear risk of bias (Figures 2 and 3) except for the Gomes et al.
study [17] which demonstrated high risk of bias in relation to the blinding of participants and personnel.
Figure 2. A risk of bias summary.
Figure 3. A risk of bias graph.
10
Nutrients 2018, 10, 373
3.2. Effect of Low Glycaemic Index on Glycated Haemoglobin

The test of the overall effect of the low-GI diet compared to higher-GI diet or control on HbA1c
showed that the low-GI diet was more effective both in the meta-analysis (p < 0.001) and in the
sensitivity test (p < 0.001) (removing the study with the most weight) (Figure 4).
There was low heterogeneity (p = 0.33) in the studies used to evaluate the effect of low-GI diet on
glycated haemoglobin. The I2 test showed I2 = 13% for meta-analysis and I2 = 33% for the sensitivity
test, again, confirming low heterogeneity of the studies included.
The result of the sensitivity test (Figure 4) showed that the effect of the low-GI diet on glycated
haemoglobin was reliable. According to Nfs0.05 = (∑Z/1.64)2 − S (Z representing the Z value of
each single study; S representing the number of all enrolled studies) to calculate the fail-safe number
(Nfs0.05 )24 , the Nfs0.05 was 16. That is, another 16 negative studies would be needed to reverse this
result, thus indicating that the result is stable.

Figure 4. A forest plot showing the effect of low-GI diet on glycated haemoglobin (%).
3.3. Effect of Low Glycaemic Index on Fasting Blood Glucose

With respect to evaluating the effect of the low-GI diet on fasting blood glucose compared to
the higher-GI diet or control diet, the meta-analysis favoured the low-GI diet. However, while the
differences were significant in the meta-analysis (p < 0.05), this was not so in the sensitivity test
(p = 0.15) (removing the study with the most weight) (Figure 5). These sensitivity results (Figure 5)
indicate that the effect of the low-GI diet on fasting blood glucose was not very reliable. The fail-safe
number (Nfs0.05 ) with respect to fasting blood glucose was 5.
Figure 5. A forest plot showing the effect of low-GI diet on fasting blood glucose (mg/dL).
11
Nutrients 2018, 10, 373
Regarding the heterogeneity test, the high p value (p = 0.36) and the chi-squared statistic (2.04)
relative to the degree of freedom (2) would suggest that there was low heterogeneity between the
studies. This was confirmed by the results of the I2 test. The heterogeneity test for fasting blood glucose
showed I2 = 2% for meta-analysis and I2 = 45% for the sensitivity test, indicating low heterogeneity of
the studies included.
4. Discussion
Based on the findings of the systematic review, the low-GI diet resulted in greater improvement
in glycated haemoglobin [29,30] and fasting blood glucose [28,29,34,36] compared to higher-GI diet
or control in patients with type 2 diabetes. In the study by Yusof et al. [36], although the effect on
HbA1c was not significantly different between the low-GI diet and the higher-GI diet or control,
the improvement within the low-GI group was more pronounced and of clinical benefit. However,
compared with the American Diabetes Association (ADA) diet, the low-GI diet achieved equivalent
control of HbA1c using less medication [31] and for patients on diet alone with optimal glycaemic
control, long-term HbA1c was not affected by altering the GI [35]. The findings of this systematic
review confirm the results of earlier systematic review by Thomas and Elliot [8] which suggests that
lowering the glycaemic index of food may improve glycated haemoglobin in patients with diabetes.
However, the difference between this review and the Thomas and Elliot [8] study is with respect to
the second outcome measure. While fasting glucose was the outcome of interest in this review, it was
fructosamine in the review by Thomas and Elliot [8]. Regarding the risk of bias, 100% of the studies
showed low risk of selection bias while all the studies demonstrated 100% low risk of bias or unclear
risk of bias in relation to blinding, incomplete outcome data, selective reporting, and other potential
sources of bias except for one study that showed a high risk of bias with respect to the blinding of
participants and personnel.
There was no statistically significant difference between the low-GI and higher-GI diets in relation
to HbA1c and fasting blood glucose in the study by Visek et al. [34]. Acute glycaemic control was
variable over the 3-day period in the study by Gonçalves Reis and Dullius [32] following the adoption
of low-GI diet. While the first day of the study demonstrated a significant difference in acute glycaemic
control, differences were not significant on the second or third day [32]. Stenvers et al. [33] found
no beneficial effect of the low-glycaemic response liquid meal with respect to fasting blood glucose.
The differences observed between these studies in terms of their results may be due to the limitations of
using HbA1c in identifying the daily changes in glycaemia [17]. The use of HbA1c may not detect the
harmful effects of excessive postprandial hyperglycaemic excursions and the risk of hypoglycaemia [7].
According to Chiu and Taylor [7], the contribution of postprandial glucose may be up to 70% in
daily hyperglycaemia, and the contribution of fasting glucose increases as glycaemic control worsens.
This may explain why the association between dietary GI and HbA1c is not consistent across studies [7].
Therefore, the use of a meta-analysis of both fasting blood glucose and HbA1c levels to evaluate the
effect of dietary GI in patients with diabetes is a useful strategy of eliminating the limitation of using
HbA1c to assess GI exposure. Other factors to consider include the nature of the starch, particle size,
pH, the amount of fibre, fat, and protein, and the cooking method and time, which may affect the GI
of food and its effect on blood glucose response, and lead to differences in outcomes of studies [15].
The differences in the classification of low- and high-GI diets may also have effect on outcomes of
studies [37].
The Food and Agricultural Organisation [9] has recommended the use of the glycaemic index
of foods in clinical applications in patients with diabetes, and that the glycaemic index be used as
a useful indicator of the impact of food on the blood glucose response. Recently, the American
Diabetes Association (ADA) [38] recommended that patients with diabetes consume carbohydrates
from vegetables, fruits, legumes, whole grains, and dairy products. The ADA [38] also recommended
that emphasis should be placed on foods which are higher in fibre and lower in glycaemic load as
opposed to other sources, especially those with added sugar. The glycaemic load is the product of
12
Nutrients 2018, 10, 373
the glycaemic index of food or diet and the grams of available carbohydrate in that food or diet
divided by 100 [9,15]. These recommendations are in line with the findings of the current systematic
review and meta-analysis, which showed that low-GI diets were more effective in controlling glycated
haemoglobin than higher-GI diets. The position of the ADA [38] with respect to the mixed results of
glycaemic index or glycaemic load in patients with diabetes is also true in relation to the findings of
the studies included in this review. Foods with low GI have demonstrated beneficial effects on glucose
control in short-term trials in patients with type 2 diabetes [13]. However, the higher intake of sucrose
or fructose and the long-term use of high-GI diets can place higher metabolic demands on the body in
relation to higher insulin requirements [6,39,40].
5. Strengths and Limitation of the Study

This review used a systematic approach and meta-analysis to provide contemporary evidence on
the positive effects of low glycaemic index diet on fasting blood glucose and glycated haemoglobin.
The limitation is in the number of studies included in the meta-analysis. In addition, most of the
studies had relatively small sample sizes. The availability and inclusion of more studies would have
increased its wider application.
6. Conclusions
The findings of this systematic review and meta-analysis have shown that low-GI diets are more
effective in controlling HbA1c and fasting blood glucose compared with higher-GI diets or control
diets in patients with type 2 diabetes. Although the outcomes of the individual studies were sometimes
different with respect to the variables of interest, the results of the meta-analysis and sensitivity tests
have demonstrated significant differences (p < 0.001 and p < 0.001, respectively) between low-GI diets
and higher-GI diets or control diets in relation to glycated haemoglobin. In addition, differences
between the low-GI diet and the higher-GI diet or control were significant (p < 0.05) with respect to the
fasting blood glucose following meta-analysis.
7. Perspectives for Future Research

It will useful to evaluate the long-term effectiveness of low-glycaemic index diet in patients with
type 2 diabetes.
Author Contributions: The concept for this article was agreed upon by O.O., O.O.O., F.A. and X.-H.W. O.O.
O.O.O., F.A., and X.-H.W. participated in the searches and in extracting data from the articles reviewed. O.O.
and X.-H.W. carried out the data analysis. O.O. wrote the initial draft, which was reviewed and revised by O.O.,
O.O.O., F.A., and X.-H.W.
Conflicts of Interest: The authors declare no conflict of interest.
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© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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15
nutrients
Article
Effects of Different Dietary and Lifestyle
Modification Therapies on Metabolic Syndrome
in Prediabetic Arab Patients: A 12-Month
Longitudinal Study
Hanan A. Alfawaz 1,2 , Kaiser Wani 1,3 , Abdullah M. Alnaami 1,3 , Yousef Al-Saleh 4 ,
Naji J. Aljohani 5 , Omar S. Al-Attas 1,3 , Majed S. Alokail 1,3 , Sudhesh Kumar 6
and Nasser M. Al-Daghri 1,3, *
1 Prince Mutaib Chair for Biomarkers of Osteoporosis, Biochemistry Department, College of Science,
King Saud University, Riyadh 11451, Saudi Arabia; [email protected] (H.A.A.);
[email protected] (K.W.); [email protected] (A.M.A.); [email protected] (O.S.A.-A.);
[email protected] (M.S.A.)
2 Department of Food Science and Nutrition, College of Food Science & Agriculture, King Saud University,
Riyadh 11451, Saudi Arabia
3 Biomarkers Research Program, Biochemistry Department, College of Science, King Saud University,
Riyadh 11451, Saudi Arabia
4 College of Medicine, King Saud bin Abdulaziz University for Health Sciences, Riyadh 11461, Saudi Arabia;
[email protected]
5 Specialized Diabetes and Endocrine Center, King Fahad Medical City, Faculty of Medicine, King Saud bin
Abdulaziz University for Health Sciences, Riyadh 11525, Saudi Arabia; [email protected]
6 Division of Metabolic and Vascular Health, Clinical Sciences Research Institute, University Hospitals
Coventry and Warwickshire Trust, Walsgrave, Coventry CV2 2DX, UK; [email protected]
* Correspondence: [email protected]; Tel.: +966-11-467-5939
Received: 12 February 2018; Accepted: 15 March 2018; Published: 20 March 2018
Abstract: This three-arm, randomized, controlled study aimed to determine the differences in the
effects of general advice (GA) on lifestyle change, intensive lifestyle modification programme (ILMP)
and GA + metformin (GA + Met) in reducing the prevalence of full metabolic syndrome (MetS)
in subjects with prediabetes; 294 Saudis with prediabetes (fasting glucose 5.6–6.9 mmol/L) were
initially randomized, 263 completed 6 months and 237 completed 12 months. They were allocated
into three groups: GA group which received a standard lifestyle change education; ILMP which
followed a rigorous lifestyle modification support on diet and physical activity; and a GA + Met
group. Anthropometric and biochemical estimations were measured. Full MetS (primary endpoint)
and its components (secondary endpoint) were screened at baseline, 6 and 12 months. Full MetS
in the ILMP group decreased by 26% (p < 0.001); in GA + Met group by 22.4% (p = 0.01) and in GA
group by 8.2% (p = 0.28). The number of MetS components decreased significantly in the ILMP and
GA + Met groups (mean change 0.81, p < 0.001 and 0.35, p = 0.05, respectively). Between-group
comparison revealed a clinically significant decrease in MetS components in favor of the ILMP group
(−0.58 (−0.88–0.28), p < 0.001). This study highlights the clinical potency of ILMP versus other
diabetes prevention options in reducing MetS in Saudi adults with elevated fasting glucose.
Keywords: impaired glucose regulation; lifestyle modifications; metabolic syndrome; type 2

diabetes; metformin

Nutrients 2018, 10, 383
1. Introduction
Type 2 diabetes mellitus (T2DM) has a huge impact on the health status of patients and the
overall health care cost of the country. One big opportunity to reduce such an impact is to reduce
the incidence of the disease by focusing on high-risk people such as those with impaired glucose
regulation. This condition is characterized either by impaired fasting glucose (IFG) (fasting glucose
levels 5.6–6.9 mmol/L) or impaired glucose tolerance (IGT) (2-h oral glucose tolerance test (OGTT)
7.8–11 mmol/L) [1]. Every year, about 5–10% of people with IGT progress to T2DM [2]. Fortunately,
landmark clinical trials like the Diabetes Prevention Programme (DPP) [3] and others [4,5] have
revealed the effectiveness of lifestyle modifications in reducing the incidence of T2DM. The major focus
of these interventions were weight loss and physical activity. However, a recent meta-analysis [6,7]
concluded that when translated into routine clinical settings, these expensive programmes have more
effect on weight reduction and less effect on diabetes risk reduction. Clearly, such intervention studies
need to be done, not only to add to the literature to better predict the outcomes on a global health-care
perspective but also to devise large-scale effective and low cost intervention programmes on a regional
level such as in Saudi Arabia, where data are limited.
Identification of individual risk factors associated with the increased effectiveness of lifestyle
intervention programmes may help in devising them in a more economical and practical way. Metabolic
Syndrome (MetS), an amalgam of cardiovascular risk factors, is a major health problem globally. It is
defined by the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) as the
presence of at least three out of five risk factors (central obesity, hyperglycemia, low HDL-cholesterol,
hypertriglyceridemia and elevated blood pressure) [8]. The prevalence of MetS is increasing rapidly
worldwide, including Saudi Arabia, as a result of rapid economic growth and Westernization of
diet [9,10].
The risk of developing incident T2DM in patients with MetS is manifold compared to those
without this condition [11]. Also, all components of MetS are independent risk factors for developing
T2DM [12]. Prevention of T2DM in subjects with impaired glucose regulation through lifestyle
modifications in diet, physical activity or by giving drugs like metformin have been studied in the past,
however, the applicability of these treatments in prevention or reversal of MetS is largely unknown,
particularly in a population like Saudi Arabia where the prevalence of both T2DM and MetS is
high [13,14]. We hypothesize that lifestyle modifications in diet and physical activity have a role
in preventing or reversal of Mets in subjects with impaired glucose regulation. Hence, this study
aimed to investigate the differences in the effectiveness of lifestyle modifications (diet and physical
activity) and drug therapy (metformin), in an adult Saudi population with impaired glucose regulation,
in preventing or reversing full MetS and its individual components.
2. Materials and Methods

This is a 12 months, 2-center, 3-arm randomized controlled (1:1:1), lifestyle intervention study
conducted from April 2013 until March of 2017. This lifestyle intervention programme was approved
by the Ethics Committee of the College of Science, King Saud University; Riyadh, Saudi Arabia
(Reference# 8/25/220355) and funded by the National Plan for Science and Technology; Riyadh,
Saudi Arabia (Grant# 12-MED2881-02). All procedures followed were in accordance with the ethical
standards of the responsible committee on human experimentation (institutional and national) and
with the Helsinki Declaration of 1975, as revised in 2008. Written informed consent was obtained from
each participant prior to inclusion in this 12-month interventional study.
2.1. Study Population

A total of 294 Saudi males and females (age range 25–60) attending King Khalid University
Hospital and King Salman Hospital in Riyadh, Saudi Arabia, agreed to take part in this lifestyle
intervention Programme. The criteria for selection was a fasting glucose level of 5.6 to 6.9 mmol/L,
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identified as one of the five components of MetS (NCEP ATP III criteria) [15]. Interested participants
were referred to the concerned physician who used the guidelines stated under section “prevention
or delay of type 2 diabetes” in “standards for medical care in diabetes” [16] to screen the candidates.
Subjects who were already on anti-hyperglycemic treatment; pregnant or lactating women; with known
renal, hepatic, pulmonary, cardiac, etc., complications were excluded.
A computer-generated serial number, randomly assigned to one of the three intervention groups
(GA, ILMP, and GA + Met), was given blindly to each participant. All participants were allocated (1:1:1)
to receive one of the three interventions. True allocation concealment was done since the research
personnel involved cannot adjust randomization. The total duration of this lifestyle intervention
programme was 12 months. Fasting blood samples and anthropometric data was collected at
recruitment, at 6-month and at the end of the programme. Out of the 294 initially recruited, the
data for 217 was used in this study. A flow chart summarizing the programme is provided in Figure 1.
Figure 1. Flow chart detailing the participation of subjects and their allocation to treatment groups.
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2.2. Intervention
All participants had an orientation session with concerned physician and a dietician where they
were educated about risk of developing T2DM and the current scenario of diabetes worldwide and
Saudi Arabia. They were advised to adopt lifestyle modifications in their dietary habits; weight
reduction; exercise; increased physical activity etc. This knowledge sharing included distributing
pamphlets and booklets with information related to lifestyle modifications in earlier programmes,
done elsewhere [17,18]. In addition to this, every four months, seminars were conducted at the
auditorium of the respective hospitals in which the investigators of the current study educated the
participants about lifestyle modifications to prevent T2DM. This intervention process was unified
across the two study centers.
The participants in the general advice (GA) group received the usual instructions to lifestyle
change as described above. In addition to this the participants in intensive lifestyle modification
programme (ILMP) group were followed with a rigorous lifestyle modification support published
earlier [19,20], which included:
(a) Individual consultation with the dietician was done to assess the participant’s food intake.
Special dietary charts were supplied to participants explaining how to reduce the total fat intake
to less than 30% of energy consume and increase the fiber intake to 15 g/1000 Kcal.
(b) Guidelines for physical activity were supplied as pamphlets to each participant. Also, each
participant was given a pedometer (081564483, Patterson Medical) and recommended at least
5000 steps per day to gradually increase as tolerance develops.
(c) Individual consultation with an expert on vitamin D to educate about the benefits of optimal
levels of vitamin D for good health. They were recommended to expose to sunlight for at least
30 min either before 10 a.m. and/or after 3 p.m. twice a week.
The intervention in ILMP group was monitored regularly which were scheduled every three
months during the course of the programme.
The third intervention group (GA + Met) was provided with the same general advice and were
given 500 mg of metformin hydrochloride, twice a day. The participants in ILMP and GA + Met groups
were regularly contacted by research assistants to reinforce instructions through phone calls.
2.3. Anthropometric and Biochemical Measurements

Anthropometrics were collected at recruitment (baseline), 6-month and 12-month. The anthropometrics
included height (cm), weight (kg), waist and hip circumferences (cm), systolic and diastolic blood
pressure by standard methods. Fasting blood samples, taken at each time point, were sent immediately
to Prince Mutaib Chair for biomarkers in Osteoporosis (PMCO), King Saud University (KSU), Riyadh
where they were processed, aliquoted and stored at recommended temperature for further analysis.
Fasting blood glucose and lipid profile was quantified using routine biochemical tests in
an automated biochemistry analyzer (Konelab 20, Thermo-Fischer scientific, Helsinki, Finland).
The reagents were supplied ready to use by Thermo Fischer (catalog# 981379 for glucose; 981812
for total cholesterol; 981823 for HDL-cholesterol and 981301 for triglyceride). The imprecision,
calculated as the total CV, was ≤5%, ≤3.5%, ≤4% and ≤4% for these tests respectively. 25(OH)vitamin
D was quantified using COBAS e-411 autoanalyzer (Roche Diagnostics, Indianapolis, IN, USA).
Glycated haemoglobin (HbA1c) was quantified in DCA vantage analyzer (Siemens, Munich, Germany).
The imprecision of the HbA1c assay was ≤3.6% in the important clinical ranges. The standards and
controls used for these biochemical assays were routinely tested by Quality assurance department of
KSU, for highly reproducible research data.
2.4. Outcome Variables

For the purpose of this study, the status of MetS and its five components were evaluated at follow
up (6-month and 12-month) versus baseline. MetS was defined by the criteria set in “The National
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Cholesterol Education Programme Adult Treatment Panel III” (NCEP ATP III) as having atleast three
of the five components [15]:
(a) Central obesity-waist circumference of >101.6 cm in males and >88.9 cm in females.

(b) Hyperglycemia-fasting glucose > 5.6 mmol/L.
(c) Low HDL-Cholesterol < 1.03 mmol/L in males and <1.30 mmol/L in females.
(d) Hypertriglyceridemia-fasting triglycerides > 1.7 mmol/L.
(e) Hypertension-systolic blood pressure > 130 mmHg and/or diastolic blood pressure > 85 mmHg.
Two other variables were tested in this study for the intervention effects between groups. One was
the total number of MetS components (taken as a continuous variable) and the other was the MetS
risk-score. The MetS risk-score was constructed for the evaluation of continuous MetS status, calculated
using the formula with cut-off values employed to define each component of MetS, with consideration
to age and gender as follows:
MetS risk-score = ((waist/101.6 for males or 88.9 for males) + (fasting glucose/5.6) −
(HDL-Cholesterol/1.03 for males or 1.30 for females) + (triglyceride/1.7) + (systolic BP/130) + (diastolic
BP/85)) × (Age/45 for males or 50 for females).
The Receiver Operating Characteristic (ROC) analysis was employed to test this score for
predicting MetS in our data, with full MetS positive (≥3 MetS components) versus full MetS negative
(<3 MetS components). The ROC revealed an area under the curve (AOC) of 0.890 with 95% confidence
interval of 0.86 and 0.92 and a p-value of <0.001. The cut-off of MetS risk-score for predicting MetS,
obtained in ROC analysis, was 3.85 (Supplementary Figure S1, Table S1–S2).
2.5. Data Analysis

As expected with longitudinal studies, the data in this study had random missing values which
are a limitation for utilizing any test on repeated measure data. Hence, the missing data (<5% of
the total data points in any variable) was dealt with the last observation carried forward (LOCF)
method. However, as much as possible, the LOCF was minimized by removing the data of the subjects
lost to follow up at 6-month or 12-month and also by removing ones with >5% missing data in any
variable (Figure 1). The remaining data (n = 217) was analyzed using SPSS 21. Continuous normally
distributed variables were summarized as mean ± standard deviation while median (25th percentile,
75th percentile) was used for continuous non-normal variables. Simple One-way ANOVA and
Kruskal-Wallis one-way ANOVA were used to test the differences between the three treatment groups
at baseline. The status of MetS and its five components were evaluated as present/absent at all
three time-points, which were presented as frequency (% of the present in the respective group) and
chi-square test (McNemar 2 × 2 contingency table) was used to calculate the p-value of the difference
in percentages. The intervention effect within each group was shown as Odds ratio (95% confidence
interval) and respective p-value representing odds of having MetS and its components independently
at follow-up compared to baseline and this data was generated by Generalized Estimating Equation
(GEE) in SPSS for repeated measures of nominal data. Finally, the intervention effect between the
groups was shown as mean Change (95% confidence interval), p-value for the total number of MetS
components (taking MetS components as scalar quantity) and MetS risk-score by mixed repeated
measures ANCOVA. p-Values were considered significant at <0.05.
3. Results
A total of 294 (98 in each group) Saudi adults with impaired fasting glucose were initially randomized
and 237 (94 in GA, 75 in ILMP and 68 in GA + Met) completed the entire 12 months of this
intervention. The most common reasons for drop out included loss to follow-up and poor compliance.
After excluding persons with missing data >5% in any parameter, the data for 217 subjects (85 in GA,
73 in ILMP and 59 in GA + Met) were used for analysis (Figure 1).
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3.1. Anthropometric and Biochemical Characteristics at Baseline and over Time

Table 1 shows the anthropometric, glycemic, and lipid characteristics of the study participants at
baseline, 6-month, and 12-month, according to treatment groups. The mean change in fasting glucose
from baseline to end of study for ILMP and GA + Met groups decreased significantly (−0.39 mmol/L,
p = 0.003 and −0.81 mmol/L, p < 0.001 respectively). This was not observed in the GA group
(−0.005 mmol/L, p = 0.65). Weight was significantly reduced in the GA + Met group from baseline to
12 months (mean change of −4.15 kg, p < 0.001). ILMP group also showed a significant reduction in
weight (mean change = −1.86 kg, p = 0.015) while the average weight for GA group increased from
baseline to the end of the study by 0.49 kg. Waist, systolic blood pressure and triglycerides significantly
reduced from baseline to 12 months in the ILMP group (−1.61 cm, p = 0.004; −2.59 mmHg, p = 0.049
and −0.23 mmol/L, p = 0.03 respectively). At baseline, the three treatment groups were significantly
different in waist (p < 0.01), systolic blood pressure (p = 0.01), diastolic blood pressure (p = 0.02), fasting
glucose (p < 0.01), HbA1c (p < 0.01) and vitamin D levels (p = 0.03).
Table 1. Anthropometric and biochemical characteristics at baseline and follow-up.
Treatment (n) GA (85) ILMP (73) GA+Met (59)

pB
Female/Male 64/21 51/22 42/17
Anthropometrics
Age (years) 42.3 ± 11.2 43.4 ± 7.8 42.6 ± 6.9 0.74
Weight (Kg)
Baseline 81.7 ± 13.9 79.6 ± 15.9 80.4 ± 14.9
6-month 82.3 ± 13.9 78.7 ± 15.9 77.6 ±13.9
12-month 82.2 ±13.4 77.7 ±16.2 76.3 ±14.1 0.67
Change at 6 months 0.61 −0.93 −2.86 **
Change at 12 months 0.49 −1.86 * −4.15 **
BMI (kg/m2 )
Baseline 32.6 ± 5.8 31.3 ± 6.4 32.1 ± 5.7
6-month 32.8 ± 5.9 31.0 ± 6.7 31.0 ± 5.4
12-month 32.8 ± 5.7 30.6 ± 6.6 30.4 ± 5.3 0.18
Change at 6 months 0.26 −0.32 −1.14 **
Change at 12 months 0.21 −0.71 * −1.68 **
Waist (cm)
Baseline 95.6 ± 6.8 97.9 ± 13 103.6 ± 12.5
6-month 95.7 ± 6.7 97.7 ± 13.5 102.6 ± 12.8
12-month 95.5 ± 6.2 96.3 ± 13 102.6 ± 12.3 <0.01
Change at 6 months 0.12 −0.25 −0.95
Change at 12 months −0.09 −1.61 ** −0.96
Hips (cm)
Baseline 110.2 ± 7.9 111. 9 ± 12 111.0 ± 12
6-month 110.5 ± 7.9 111.0 ± 11.6 109.5 ± 10.9
12-month 110.6 ± 7.6 109.9 ± 12.2 109.6 ± 10.9 0.60
Change at 6 months 0.26 −0.86 * −1.48 **
Change at 12 months 0.44 −1.98 ** −1.39 *
Systolic BP (mmHG)
Baseline 120.0 ± 12.1 122.1 ± 15.8 127.4 ± 11.6
6-month 117.8 ± 14.4 120.0 ± 18.5 129.2 ± 11.1
12-month 119.2 ± 15.8 119.5 ± 16.6 129.3 ± 10.7 0.01
Change at 6 months −2.25 −2.12 1.86
Change at 12 months −0.86 −2.59 * 1.97
Diastolic BP (mmHG)
Baseline 76.4 ± 9.7 76.0 ± 11.9 80.6 ± 9.0
6-month 76.4 ± 12.1 76.0 ± 12.1 81.2 ± 11.1
12-month 77.1 ± 13.7 74.6 ± 12.8 83.3 ± 9.2 0.02
Change at 6 months 0.06 0.14 0.68
Change at 12 months 0.71 −1.38 2.73
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Table 1. Cont.
Treatment (n) GA (85) ILMP (73) GA+Met (59)

pB
Female/Male 64/21 51/22 42/17
Glycemic Profile
Fasting Glucose (mmol/L)
Baseline 6.0 ± 0.4 6.1 ± 0.4 6.6 ± 0.5
6-month 6.1 ± 0.7 5.7 ± 0.8 6.0 ± 1.3
12-month 5.9 ± 0.9 5.7 ± 0.8 5.8 ± 1.7 <0.01
Change at 6 months 0.09 −0.40 ** −0.56 **
Change at 12 months −0.05 −0.39 ** −0.81 **
HbA1c
Baseline 5.6 ± 0.5 5.8 ± 0.4 5.6 ± 0.5
6-month 5.7 ± 1.6 5.6 ± 0.4 5.1 ± 1.5
12-month 5.6 ± 1.5 5.5 ± 1.0 5.0 ± 1.7 <0.01
Change at 6 months 0.06 −0.22 −0.47 **
Change at 12 months −0.06 −0.30 −0.53 **
Lipid Profile
Total Cholesterol (mmol/l)
Baseline 4.8 ± 1.0 5.2 ± 1.3 4.8 ± 1.2
6-month 4.8 ± 1.2 5.0 ± 1.1 4.9 ± 1.0
12-month 4.6 ± 1.1 5.0 ± 1.0 4.9 ± 1.2 0.06
Change at 6 months −0.05 −0.19 0.04
Change at 12 months −0.22 −0.24 0.06
HDL-Cholesterol (mmol/l)
Baseline 1.1 ± 0.3 1.2 ± 0.4 1.2 ± 0.4
6-month 0.93 ± 0.4 1.2 ± 0.4 1.1 ± 0.4
12-month 0.96 ± 0.4 1.2 ± 0.4 1.2 ± 0.3 0.49
Change at 6 months −0.16 0.02 −0.02
Change at 12 months −0.13 0.03 0.01
Triglycerides (mmol/l)
Baseline 1.4 (1.1, 2.1) 1.5 (1.1, 1.8) 1.6 (1.2, 2.0)
6-month 1.5 (1.1, 2.1) 1.3 (1.1, 1.8) 1.6 (1.3, 2.2)
12-month 1.4 (1.1, 2.0) 1.2 (1.0, 1.7) 1.6 (1.3, 2.0) 0.75
Change at 6 months 0.11 −0.12 0.03
Change at 12 months −0.03 −0.23 * 0.00
25(OH) vitamin D (nmol/l)
Baseline 41.7 (24.0, 73.0) 47.3 (30, 67.2) 57.0 (38, 96)
6-month 45.0 (28.5, 72.6) 54.1 (40, 68) 62.4 (41, 95)
12-month 48.3 (28.3, 74.7) 56.1 (40, 72.2) 62.4 (42, 96) 0.03
Note: Data presented as Mean ± SD for continuous normal variables and medians (25th–75th percentile) for
continuous non-normal variables; * and ** represent significant mean change at p < 0.05 and p <0.01 respectively.
p B represents difference between treatment groups at baseline. GA is “general advice group”, ILMP is “intensive
lifestyle monitoring programme group”, GA + Met is “Metformin group”, BMI is “body mass index”, BP is “blood
pressure”, HbA1c is “glycated haemoglobin”, HDL is “high density lipoprotein”. p < 0.05 is considered significant.
3.2. Prevalence of MetS and Its Components at Baseline and Overtime

Table 2 shows the percentage of subjects having different components of MetS and full MetS in
the three treatment groups at baseline, follow-up and percentage of subjects in which there was
a change in status over time. MetS and its components were evaluated as binomial variables.
Component 1 (central obesity) and component 4 (hypertriglyceridemia) showed the lowest changes
at follow-up in all the three groups (−1.2%, −1.4% and −1.7% in central obesity; and +3.5%, −4.1%
and 0% in hypertriglyceridemia for groups GA, ILMP and GA + Met respectively). Component 3
(Low HDL-Cholesterol) increased by 7.1% (n = 6) in GA group at end of the study compared to
baseline while it decreased by 6.8% (n = 5) and 3.4% (n = 2), respectively, in ILMP and GA + Met
groups. Component 5 (Hypertension) increased by 2.4% (n = 2) and by 8.5% (n = 5, p = 0.035)
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in GA and GA + Met groups, respectively, while it decreased by 1.4% (n = 1) in the ILMP group.
The highest change was seen in component 2 (hyperglycemia) where from baseline to end of the study,
hyperglycemia was reduced by 22.4% (n = 19), 38.4% (n = 28) and 39% (n = 23), respectively, in GA,
ILMP, and GA+ILMP groups respectively. Full MetS was also significantly reduced by 8.2% (n = 7),
26% (n = 19, p < 0.001), and 22.4% (n = 13, p = 0.013), respectively, in GA, ILMP, and GA + Met groups.
Table 2. Prevalence of MetS and its 5 components at baseline and follow-up.
Central Hyper Low

Hyperglycemia Hypertension MetS
Obesity Triglyceridemia HDL-Cholesterol
Baseline 63 (74.1) 85 (100) 64 (75.3) 36 (42.4) 14 (16.5) 62 (72.9)
6-month 61 (71.8) 76 (89.4) 71 (83.5) 36 (42.4) 19 (22.4) 63 (74.1)
GA
12-month 62 (72.9) 66 (77.6) 70 (82.4) 33 (38.8) 16 (18.8) 55 (64.7)
(85)
% change-6 −2.3 −10.6 8.2 0.0 5.9 1.2
% change-12 −1.2 −22.4 7.1 −3.5 2.4 −8.2
Baseline 45 (61.6) 73 (100) 47 (64.4) 23 (31.5) 22 (30.1) 45 (61.6)
6-month 46 (63.0) 56 (76.7) 43 (58.9) 21 (28.8) 22 (30.1) 36 (49.3)
ILMP
12-month 44 (60.3) 45 (61.6) 42 (57.5) 20 (27.4) 21 (28.8) 26 (35.6)
(73)
% change-6 1.4 −23.3 −5.5 −2.7 0.0 −12.3
% change-12 −1.4 −38.4 −6.8 −4.1 −1.4 −26.0 **
Baseline 47 (79.7) 59 (100) 39 (66.1) 25 (42.4) 22 (37.3) 49 (83.1)
6-month 46 (78.0) 43 (72.9) 42 (71.2) 24 (40.7) 29 (49.2) 42 (71.2)
GA + Met
12-month 46 (78.0) 36 (61.0) 37 (62.7) 25 (42.4) 27 (45.8) 38 (64.4)
(59)
% change-6 −1.7 −27.1 5.1 −1.7 11.9 * −11.9
% change-12 −1.7 −39.0 −3.4 0.0 8.5 * −22.4 *
Note: Data presented as n (%). % change (6 for 6-month and 12 for 12-month) represents the overall percentage
change in respective treatment groups; * and ** represents significant change at p < 0.05 and p < 0.01 respectively.
3.3. Odds of Having MetS and Its Components at Follow-Up Compared to Baseline
Table 3 shows the odds of having MetS and its individual components at follow-up compared to
baseline in each group. Supplementary Table S3 shows the odds of having MetS for these covariates,
independent of each other.
Table 3. Odds ratio representing the risk of having MetS and its individual components at follow-up
compared to baseline in respective groups.
Baseline 6-Month 12-Month

Reference O.R. (95% C.I.) p O.R. (95% C.I.) p
Central Obesity
Model a 1.00 0.89 (0.8, 1.0) 0.15 0.94 (0.7, 1.2) 0.65
GA Model b 1.00 0.73 (0.5, 1.1) 0.13 0.85 (0.4, 1.7) 0.65
Model c 1.00 0.71 (0.5, 1.1) 0.12 0.84 (0.4, 1.8) 0.65
Model a 1.00 1.06 (0.8, 1.4) 0.65 0.94 (0.7, 1.2) 0.65
ILMP Model b 1.00 1.08 (0.8, 1.5) 0.65 0.92 (0.7, 1.3) 0.654
Model c 1.00 1.25 (0.5, 3.2) 0.64 0.80 (0.3, 2.2) 0.67
Model a 1.00 0.90 (0.6, 1.3) 0.56 0.90 (0.5, 1.5) 0.71
GA + Met Model b 1.00 0.88 (0.6, 1.4) 0.56 0.88 (0.4, 1.7) 0.70
Model c 1.00 0.74 (0.3, 2.1) 0.56 0.74 (0.2, 3.5) 0.71
Low HDL-Cholesterol
Model a 1.00 1.66 (0.9, 3.2) 0.13 1.53 (0.8, 2.8) 0.18
GA Model b 1.00 1.80 (0.8, 3.8) 0.13 1.64 (0.8, 3.4) 0.18
Model c 1.00 1.83 (0.8, 4.0) 0.13 1.66 (0.8, 3.5) 0.18
Model a 1.00 0.75 (0.4, 1.3) 0.34 0.79 (0.4, 1.4) 0.43
ILMP Model b 1.00 0.75 (0.4, 1.4) 0.34 0.79 (0.4, 1.4) 0.43
Model c 1.00 0.74 (0.4, 1.4) 0.34 0.79 (0.4, 1.4) 0.43
Model a 1.00 1.27 (0.7, 2.4) 0.47 0.86 (0.4, 1.7) 0.67
GA + Met Model b 1.00 1.27 (0.6, 2.4) 0.47 0.86 (0.4, 1.7) 0.67
Model c 1.00 1.27 (0.7, 2.4) 0.47 0.86 (0.4, 1.7) 0.67
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Table 3. Cont.
Baseline 6-Month 12-Month

Reference O.R. (95% C.I.) p O.R. (95% C.I.) p
Hypertriglyceridemia
Model a 1.00 1.00 (0.7, 1.5) 1.00 0.86 (0.6, 1.3) 0.49
GA Model b 1.00 1.00 (0.6, 1.5) 1.00 0.86 (0.6, 1.3) 0.49
Model c 1.00 1.00 (0.6, 1.6) 1.00 0.86 (0.5, 1.3) 0.49
Model a 1.00 0.88 (0.5, 1.5) 0.62 0.82 (0.5, 1.4) 0.44
ILMP Model b 1.00 0.87 (0.5, 1.5) 0.62 0.80 (0.5, 1.4) 0.44
Model c 1.00 0.86 (0.5, 1.5) 0.62 0.80 (0.5, 1.4) 0.44
Model a 1.00 0.93 (0.6, 1.5) 0.78 1.00 (0.6, 1.7) 1.00
GA + Met Model b 1.00 0.93 (0.5, 1.6) 0.78 1.00 (0.6, 1.7) 1.00
Model c 1.00 0.92 (0.5, 1.6) 0.78 1.00 (0.6, 1.8) 1.00
Hypertension
Model a 1.00 1.46 (0.8, 2.6) 0.19 1.18 (0.6, 2.1) 0.59
GA Model b 1.00 1.48 (0.8, 2.7) 0.19 1.18 (0.6, 2.2) 0.59
Model c 1.00 1.80 (0.8, 4.2) 0.18 1.28 (0.5, 3.1) 0.59
Model a 1.00 1.00 (0.7, 1.5) 1.00 0.94 (0.6, 1.4) 0.76
ILMP Model b 1.00 1.00 (0.6, 1.6) 1.00 0.93 (0.6, 1.5) 0.76
Model c 1.00 1.00 (0.5, 2.2) 1.00 0.88 (0.4, 2.1) 0.76
Model a 1.00 2.13 (1.1, 4.0) 0.019 1.86 (1.1, 3.1) 0.016
GA + Met Model b 1.00 2.25 (1.1, 4.5) 0.020 1.95 (1.1, 3.4) 0.017
Model c 1.00 2.85 (1.2, 6.7) 0.017 2.36 (1.2, 4.6) 0.012
MetS
Model a 1.00 1.06 (0.6, 1.8) 0.83 0.68 (0.4, 1.2) 0.21
GA Model b 1.00 1.07 (0.6, 2.0) 0.83 0.64 (0.3, 1.3) 0.21
Model c 1.00 1.08 (0.6, 2.1) 0.83 0.62 (0.3, 1.3) 0.21
Model a 1.00 0.61 (0.4, 0.9) 0.035 0.34 (0.2, 0.5) <0.001
ILMP Model b 1.00 0.56 (0.3, 0.9) 0.033 0.29 (0.2, 0.5) <0.001
Model c 1.00 0.52 (0.3, 0.9) 0.033 0.25 (0.1, 0.4) <0.001
Model a 1.00 0.50 (0.3, 0.9) 0.049 0.37 (0.2, 0.8) 0.006
GA + Met Model b 1.00 0.49 (0.2, 0.9) 0.047 0.36 (0.2, 0.7) 0.006
Model c 1.00 0.46 (0.2, 0.9) 0.043 0.32 (0.1, 0.7) 0.005
Note: Data presented as O.R. (95% C.I.) for Odds ratio (95% confidence interval). Model “a” is univariate. Model “b”
is adjusted at age, sex and BMI at baseline. Model “c” is adjusted with additional covariates like waist, systolic and
diastolic BP, fasting glucose, HbA1c, and vitamin D (baseline). Hyperglycemia as a component of MetS is excluded
in this analysis as this component is present in all subjects at reference (baseline).
The odds of central obesity and hypertriglyceridemia showed marginal changes in all groups,
with odds ratio (95% confidence interval (C.I.)) of 0.84 (0.4, 1.8) and 0.86 (0.5, 1.3), respectively, in GA
group; 0.80 (0.3, 2.2) and 0.80 (0.5, 1.4) in ILMP group and 0.74 (0.2, 3.5) and 1.0 (0.6, 1.8) in GA + Met
group. The odds of low HDL-cholesterol increased (1.66 (0.8, 3.5)) in GA group, but was reduced both
in ILMP (0.79 (0.4, 1.4)) and GA + Met (0.86 (0.4, 1.7)) groups. The odds of hypertension increased
in GA + Met group (2.36 (1.2, 4.6), p-value = 0.012) and GA group (1.28 (0.5, 3.1)) but was modestly
decreased in ILMP group (0.88 (0.4, 2.1)). The odds of full MetS was significantly reduced both in
ILMP (0.25 (0.1, 0.4), p-value < 0.001) and GA + Met (0.32 (0.1, 0.7), p-value = 0.005) groups only.
Figure 2 shows the odds ratio of MetS and its components at the end of the study compared to
baseline for different treatment groups. The odds for MetS were significantly reduced in ILMP group
followed by GA + Met group.
3.4. Intervention Effects in Total Number of MetS Components and MetS Risk Factor
Table 4 shows the intervention effects in the total number of MetS components and the MetS
risk-score. The number of MetS components significantly decreased from baseline to the end of the
study in the ILMP group (mean change (standard error)) of 0.81 (0.13), p-value < 0.001. Similarly,
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it decreased in GA + Met group by 0.35 (0.18), p-value = 0.05. Between-group comparison revealed
that the decrease in the number of MetS components in ILMP was statistically more significant than
GA (p < 0.001). Between (GA + Met) and GA groups, this decrease was not significant. Intervention
effects between groups for MetS risk score showed the same trend between ILMP vs. GA group (0.31
(−0.53, −0.09); p = 0.003).
Figure 2. Odds ratio (OR) of MetS and its components for three treatment groups. The model is adjusted
for age, sex, BMI and baseline covariates: waist, systolic and diastolic BP, fasting glucose, HbA1c, and
vitamin D.
Table 4. Intervention effects in number of MetS components and the MetS risk-score.
Treatment Groups Intervention Effects: Mean Change (95% C.I.), p

GA ILMP GA + Met ILMP-GA (GA + Met)-GA
No. of MetS Components
Baseline 3.15 (0.10) 2.91 (0.11) 3.11 (0.15)
6-month 3.14 (0.11) 2.41 (0.12) 2.93 (0.16)
12-month 2.88 (0.11) 2.10 (0.12) 2.77 (0.16)
−0.58 (−0.88, −0.28), −0.12 (−0.53, 0.27), 1.0
<0.001
6M −0.02 (0.12) −0.49 (0.13) ** −0.18 (0.17)
12 M −0.28 (0.12) −0.81 (0.13) ** −0.35 (0.18) *
MetS Risk-score
Baseline 4.22 (0.08) 4.13 (0.09) 4.28 (0.10)
6-month 4.25 (0.07) 3.82 (0.08) 4.22 (0.09)
12-month 4.12 (0.07) 3.72 (0.08) 4.16 (0.10)
−0.31 (−0.53, −0.09), −0.11 (−0.42, 0.19), 1.0
0.003
6M 0.04 (0.07) −0.31 (0.07) * −0.06 (0.09)
12 M −0.09 (0.07) −0.41(0.08) ** −0.12 (0.10)
Note: Data presented as Mean (Standard error) for baseline, 6 months and 12 months. Changes at time-intervals
are presented as mean change (standard error), where 6 M (6-month minus baseline) and 12 M (12-month minus
baseline). Overall change in ILMP and GA + Met groups versus GA group are reported as Mean Change (95%
confidence interval). Values were adjusted for baseline covariates age, sex and BMI, systolic and diastolic BP, waist,
fasting glucose, HbA1c, and vitamin D (baseline values). * represents p-value < 0.05 and ** represents p-value < 0.01.
p < 0.05 considered significant.
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Nutrients 2018, 10, 383
Figure 3 shows the intervention effects of the three treatment strategies on MetS, represented by
bar graphs showing changes in total number of MetS components and MetS risk-score overtime.
Figure 3. Intervention effects in total number of MetS components (A) and MetS risk-score (B) for
three treatment strategies calculated at baseline and follow-up. Data is adjusted for age, sex and BMI,
systolic and diastolic BP, waist, fasting glucose, HbA1c, and vitamin D (all baseline values). The overall
change in ILMP and GA + Met groups versus GA group reported as Mean Change (95% confidence
interval). Within group intervention effect is shown as * for p < 0.05 or ** for p < 0.01.
4. Discussion
In this study, Saudi adults with impaired glucose regulation were given treatment based on
lifestyle modifications in diet and exercise or low doses of metformin to assess the status of MetS and
its individual components over time. Changing lifestyle and reducing the burden of chronic diseases in
this population is of great public health importance, however, the impact of such programmes on full
MetS and components of MetS in the pre-diabetic Saudi population has not been studied. It is notable
that, for the past two decades since these lifestyle intervention studies started, the study outcome
variables in most of them has been the incidence of diabetes. In this study, we chose a broader set of
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Nutrients 2018, 10, 383
outcome measures and focused on the status of MetS and its components together, as well as those
independently considered as risk factors for the incidence of T2DM. To the best of our knowledge,
this is the first such study in Saudi Arabia which emphasizes the changes in the status of MetS and its
components through lifestyle modifications. A similar study was done by the Diabetes Prevention
Program (DPP) research group in 2005 [21] and focused on lifestyle intervention and metformin-based
therapies for MetS.
Mean fasting glucose was significantly reduced in the ILMP and GA + Met groups while in
the GA group this reduction was modest. Similarly, the prevalence of hyperglycemia was reduced
significantly by 38.4% and 39% in the ILMP and GA + Met groups, respectively. This improvement in
mean fasting glucose is similar to previous studies done in USA [22], Italy [23], and Iran [24] where,
on an average, fasting glucose was reduced by 6 mg/dl from baseline to the end of the study in the
dietary intervention group. Lifestyle modifications, including replacing high-glycemic index diet with
low-glycemic index diet like vegetables, dairies, and whole grains, etc. might have played a role in the
reduction in mean fasting glucose [25]. However, as expected, the mean reduction in fasting glucose in
the ILMP group is lesser than that found in the GA + Met group. Though the molecular mechanism
of the action of metformin remains debated, a consensus is on the direct action of metformin on
hepatic glucose production and improving insulin sensitivity in muscle and fat cells [26]. Furthermore,
metformin’s action is not limited to glucose reduction and insulin sensitivity, it is also an effective
weight loss drug due to its effect on appetite and its regulation of fat oxidation, storage in liver and
adipose tissue [27]. In our study, the mean weight loss from baseline to the end of the study was
4.15 kg in GA + Met group and this was higher than the ILMP group (1.86 kg).
The ILMP group showed modest improvements in all of the other components of MetS, namely,
central obesity, low HDL-cholesterol, hypertriglyceridemia and hypertension. Waist circumference
also decreased from baseline to the end of study in the ILMP group by an average of 1.86 cm.
This change in waist circumference is close to a mean decrease of 2.7 cm found in a meta-analysis
of six interventions [28]. The odds for low HDL-cholesterol was 1.66 (95% confidence interval 0.8 to
3.5) in the GA group, indicating a higher risk in the absence of an intensive lifestyle change program.
Similarly, the odds for hypertriglyceridemia at the end of the study versus baseline in the metformin
group was 1.0 (0.6, 1.8), which together with the odds obtained for low HDL-cholesterol indicated
that GA + Met induce less effects on lipid levels than the ILMP. This was in agreement with studies
like [29] which reported significantly larger improvement in lipid indexes in the ILMP group than in
the GA + Met group.
The prevalence of hypertension was significantly higher at the end of the study compared to
baseline in the GA + Met group (O.R. 2.36 (1.2, 4.6), p-value 0.012). The literature on the effect of
metformin on hypertension has yielded conflicting results. While some [30] suggest metformin has no
intrinsic effect on blood pressure, others, like a recent meta-analysis from 26 studies [31], suggested
that metformin could effectively lower systolic blood pressure especially in those with IGT. There
are also reports on the role of metformin in reversing pulmonary hypertension through inhibition of
aromatase synthesis [32]. In any case, the findings in this study related to hypertension in the GA + Met
group should be looked at keeping in mind that most of the study participants (72.3% of all subjects
and 71.2% of GA + Met group) are females. Women normally tend to have lower blood pressure than
men, related to sex differences in concentrations of angiotensin II and sodium reabsorption in distal
nephron [33].
The decrease in the prevalence of full MetS at the end of the study compared to baseline was
greater in ILMP (−26%, p< 0.001) and GA + Met groups (−22.4%, p = 0.013) than the GA group
(−8.2%, p = 0.281). Furthermore, the analysis of the number of MetS components as well as MetS
risk-score suggests that although ILMP was superior to GA + Met, both are more effective compared
to GA. The biological mechanism whereby ILMP or metformin exerts its protective effect from MetS is
complex and unclear; however, greater intakes of dietary fibers, low-glycemic index foods, vitamins,
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more physical activity, etc. in ILMP all contribute to the reduction in low-grade inflammation, normally
associated with MetS [34,35].
The MetS risk-score, employed in testing the hypothesis of the study regarding MetS reversal/
reduction with three different treatments, had high discriminative power for presence/absence of
MetS (AOC of 0.890, 95% C.I. of 0.86 to 0.92 and a p-value of <0.001, Supplementary Figure S1,
Tables S1–S2). A continuous risk-score for predicting MetS has been developed by approaches such as
taking standardized residuals (z-scores) or scores from principal component analysis of risk factors [36,37].
These scores, although precise, are complex and require specialized statistical software. While the
authors attempted an easy-to-calculate continuous MetS risk-score, it should be used with caution as
the values apply to the population and local laboratory reference values.
The authors acknowledge certain limitations of the study. Firstly, the study was done on subjects
with IGT and, hence, the results may not hold true for those with normal glucose regulation but having
full MetS manifestations. Such intensive lifestyle change programmes should be conducted in these
categories to have a better understanding of the benefits. Secondly, the study does not give details
about the actual changes in lifestyle that each participant in this programme carried out in terms of
variables related to diet or physical activity, measured before intervention and follow up. Despite
the study being based on self-monitoring; it showed a significant reversal in MetS in the ILMP group
which suggests that, if guided properly, people are willing to change from a sedentary to a healthy
lifestyle. The study also offered a unique statistical approach in assessing MetS and its components
and by evaluating the change in the total number of MetS components as an outcome variable. Also,
the MetS risk-score devised in this study, if further researched, may provide a simplistic estimate for
assessing MetS in an individual.
5. Conclusions
Intensive lifestyle modifications or low dose metformin for a period of 12 months significantly
reduces MetS manifestation in individuals with prediabetes, with lifestyle modifications being superior
to metformin, as the latter’s potency is limited to weight loss and reduction of hyperglycemia, while the
former improves all the components of MetS together as well as independently.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/10/3/383/s1,

Figure S1: ROC curve depicting power of MetS_risk factor (RS) for predicting full metabolic syndrome; Table S1:
Data showing area under the curve (AUC); Table S2: Data showing coordinates of the ROC curve; Table S3: Odds
ratio representing the risk of having metabolic syndrome and its individual components in all samples.
Acknowledgments: The authors thank Deanship of Scientific Research, KSU and Prince Mutaib Bin Abdullah
Chair for Biomarkers of Osteoporosis (PMCO) for its continuous support. We also acknowledge Hamza Saber for
his efforts in maintaining/providing good quality samples for analysis during this study. The authors are also
thankful to Malak Nawaz Khan Kattak and Syed Danish for critical comments on data analysis. This work was
supported by the National Plan for Science and Technology (NPST), Riyadh, Saudi Arabia (grant# 12-MED2881-02).
Author Contributions: Study conception and design: N.M.A.-D., H.A.A.; Study execution: H.A.A., N.J.A., Y.A.S.,
O.S.A.-A., M.S.A.; Sample analysis: K.W., A.M.A.; Manuscript writing: K.W.; Statistical analysis: K.W.; Manuscript
review: H.A.A., N.M.A., S.K.
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nutrients
Article
Dietary Inflammatory Index and Type 2 Diabetes
Mellitus in Adults: The Diabetes Mellitus Survey
of Mexico City
Edgar Denova-Gutiérrez 1 , Paloma Muñoz-Aguirre 2 , Nitin Shivappa 3,4,5 , James R. Hébert 3,4,5 ,
Lizbeth Tolentino-Mayo 1 , Carolina Batis 6 and Simón Barquera 1, *
1 Nutrition and Health Research Center, National Institute of Public Health, Cuernavaca 62100, Mexico;
[email protected] (E.D.-G.); [email protected] (L.T.-M.)
2 Center for Research on Population Health , National Institute of Public Health, Cuernavaca 62100, Mexico;
[email protected]
3 Cancer Prevention and Control Program, University of South Carolina, Columbia, SC 29208, USA;
[email protected] (N.S.); [email protected] (J.R.H.)
4 Department of Epidemiology and Biostatistics, Arnold School of Public Health, University of South
Carolina, Columbia, SC 29208, USA
5 Connecting Health Innovations LLC, Columbia, SC 29250, USA
6 CONACYT-Nutrition and Health Research Center, National Institute of Public Health, Cuernavaca 62100,
Mexico; [email protected]
* Correspondence: [email protected]; Tel.: +52-(777)-329-3017 or +52-(777)-311-2219
Received: 23 February 2018; Accepted: 19 March 2018; Published: 21 March 2018
Abstract: Diet and inflammation are both associated with type 2 diabetes mellitus (T2DM). In the
present study, we aimed to assess the relation between the dietary inflammatory index (DII) and
the presence of T2DM in Mexican adults participating in the Diabetes Mellitus Survey administered
in Mexico City (DMS-MC). The study involved 1174 subjects (48.5% men) between 20–69 years of
age. A validated semi-quantitative food frequency questionnaire was employed to evaluate dietary
intake and to compute DII. The DII is based on scientific evidence about the association between
dietary compounds and six established inflammatory biomarkers. Multivariate logistic regression
models were used to estimate the odds ratios (ORs) and 95% confidence intervals (95% CIs) of DII
in relation to T2DM. Our results suggest that subjects in the highest quintile of the DII had higher
odds of T2DM (OR = 3.02; 95% CI: 1.39, 6.58; p = 0.005) compared to subjects in the lowest quintile
of DII scores. Assessing possible effect modification, an association with T2DM was evident when
comparing DII quintile 5 to quintile 1 for participants aged ≥ 55 years (OR = 9.77; 95% CI: 3.78, 25.50;
p = 0.001). These results suggest that a pro-inflammatory diet is associated with significantly higher
odds of T2DM among adult Mexicans.
Keywords: type 2 diabetes mellitus; dietary inflammatory index; obesity; Mexican population; survey
1. Introduction
Low-grade, systemic inflammation, which provides the substrate for many chronic diseases,
is characterized by elevated pro-inflammatory markers, including interleukin-6 (IL-6), and tumor
necrosis factor-alpha (TNF-α) [1]. Chronic systemic inflammation has been linked to non-communicable
diseases such as cancer, cardiovascular disease, and diabetes [2]. Globally, type 2 diabetes mellitus
(T2DM) has become a serious public health problem, affecting 382 million people in 2013 [3] and had
led to a loss of approximately 64 million disability adjusted life-years (DALYs) in 2015 [4]. Currently,
80% of people with T2DM live in low-income and middle-income countries [5]. In Mexico, T2DM
prevalence has reached 14.7% of the population and two million DALYs lost in 2013 [5,6].

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Recent evidence indicates that risk factors can induce chronic inflammation, which are also related
with T2DM such as adiposity [7], a sedentary lifestyle [8], and diet [9]. In the past several decades,
certain individual dietary factors have been widely investigated regarding their association with
diabetes risk and inflammation, including high intake of saturated fatty acids, sugar-sweetened
beverages, and starchy food, combined with low consumption of fruits, vegetables, and whole
grains [10]. More recently, alternative approaches have been used in order to capture the complicated
nutrient interactions and cumulative effects in the food matrix [11]. Based on evidence linking diet with
inflammation and chronic conditions such as T2DM, we used a literature-derived, population-based
dietary inflammatory index (DII) [12] to evaluate the potential inflammatory properties of diet. The DII
has been validated with different inflammatory biomarkers [13,14]. Furthermore, it has been associated
with components of metabolic syndrome [15], as well as a variety of chronic disease outcomes [16,17].
To the best of our knowledge, no study has yet explored the relation between the DII and T2DM
in a Mexican population. Thus, we aimed to explore the association between DII and the prevalence of
T2DM in adults participating in the Diabetes Mellitus Survey in Mexico City 2015 (DMS-MC 2015).
2.1. Study Design

The present analysis was conducted with data from the DMS-MC 2015, a cross-sectional
probabilistic population-based survey, designed and implemented by our group at the National
Institute of Public Health (INSP by its Spanish acronym), representative of adults aged 20–69 years
living in Mexico City. The DMS-MC 2015 sample was randomly stratified into clusters according to city
district. In the first stage, 16 primary sampling units were selected and by means of a probabilistic and
systematic sampling, six secondary sampling units were designated. Then, six houses per secondary
sampling unit were included. Finally, per household, we evaluated up to two adults between
20–69 years of age. Fasting venous blood samples were collected in a subsample of participants
randomly selected based on estimations of the Mexican National Health and Nutrition Survey 2012 [18].
For the present analysis, we excluded participants with >10% blank items on their food frequency
questionnaires, and who did not consume between 600 kcal and 5500 kcal daily (n = 10), determined
with the standard deviation method suggested by Rosner [19]. Additionally, we excluded participants
with incomplete biomarkers data or with missing information on other important covariates (n = 142).
Finally, we excluded those subjects with more than 12 months past their T2DM diagnosis date (n = 90).
A total of 1174 individuals were included in our final sample.
This study was managed according to the Declaration of Helsinki guidelines. The Research,
Ethics and Biosecurity Committee at INSP reviewed and approved the study protocol (No. 1658) and
informed consent forms (No. B04). Written informed consent was obtained from each participant.
2.2. Dietary Assessment

To assess dietary intake, a previously validated semi-quantitative food frequency questionnaire
(SFFQ) [20] was used. The instrument describes the consumption of 140 foods over the past seven
days prior to the interview. For each food, a commonly used portion size was specified on the
SFFQ. Frequency of food consumption was characterized by set categories ranging from never to
six. First, frequency was expressed as times per day, but then was converted into portion size per
day. To compute the energy (kcal/day) and daily nutrient intake, we multiplied the frequency of
consumption of each food by the estimated nutrient content with a comprehensive database of food
contents, compiled by the INSP [21]. The SFFQ was administered by interviewers and were collected
by personnel trained using standardized data collection and entry procedures.
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2.3. Dietary Inflammatory Index (DII) Assessment

SFFQ-derived dietary data was used to calculate DII scores for each participant. A complete
description of the DII is available elsewhere [12]. Briefly, the body of literature on DII consists
of all qualifying publications between 1950 and 2010 reporting one or more associations between
dietary components and the following inflammatory markers: IL-1β, IL-4, IL-6, IL-10, TNF-α
and C-reactive protein [12]. A total of 45 different food parameters were identified as being
related to the six inflammatory biomarkers in the literature review. Each was assigned a “food
parameter-specific inflammatory effect score” through a process of counting the number of studies
reporting a pro-inflammatory, anti-inflammatory, and no inflammatory effect on one or more of the six
inflammatory markers, and weighing the scores by study design and size of the literature for each
food parameter/inflammatory marker relation. In previous analyses, the DII was positively correlated
with circulating level of high-sensitivity C-reactive protein (hs-CRP) [13,22].
To calculate DII scores for the participants of this study, the dietary data was first linked to
the world database that provided estimates of a mean intake and standard deviation for each food
parameter [12]. These then became the multipliers to express an individual’s exposure relative to
the “standard global mean” as a z-score. This was achieved by subtracting the “standard global
mean” from the amount reported and dividing this value by the standard deviation. Since data was
skewed to the right (a common occurrence with dietary data), we converted this value into a centered
percentile score. The centered percentile score from every individual was multiplied by the food
parameter-specific inflammatory effect score in order to obtain a food parameter-specific DII score for
an individual. All of the food parameter-specific DII scores were then summed to create the overall
DII score for every participant in the study [12]. DII scores for individuals in the DMS-MC 2015 were
calculated using the 27 food items and nutrients (out of the 45 possible items) for which we had intake
data available from the SFFQ: carbohydrate, protein, fat, alcohol, fiber, cholesterol, saturated fatty
acids, mono-unsaturated fatty acids, poly-unsaturated fatty acids, omega 3 fat, omega 6 fat, trans fat,
niacin, thiamin, riboflavin, vitamin B12, vitamin B6, iron, magnesium, zinc, vitamin A, vitamin C,
vitamin E, folic acid, beta carotene, garlic, and onion. Since the DII was calculated per 1000 calories
of food consumed we used the energy-standardized version of the world database to control for the
effect of total energy intake.
2.4. Biomarkers Assessment

A fasting venous blood sample (fasting time was ≥8 h) from an antecubital vein was collected from
each participant. Serum aliquots were stored in cryovials and transported to a laboratory, where the
aliquots were stored at −70 ◦ C until they were used for analysis.
Plasma triglycerides were measured with a colorimetric method following enzymatic hydrolysis
performed with the lipase technique. Total cholesterol, high-density lipoprotein-cholesterol (HDL-c),
and low-density lipoprotein-cholesterol (LDL-c) were measured using the colorimetric method
following enzymatic assay. Additionally, plasma glucose was measured with the enzymatic
colorimetric methods by using glucose oxidize. Finally, the proportion of hemoglobin A1c (HbA1c)
was determined using the immunocolorimetric method [23].
2.5. Type 2 Diabetes Mellitus Definition

For the present study, subjects who declared to have a previous T2DM physician-diagnosis
independent of their survey glucose concentration were called “previously diagnosed”. Of these,
respondents who were diagnosed with T2DM less than 11 months prior to administration of the
SFFQ, had fasting glucose concentrations ≥126 mg/dL (at the moment of the survey) and poor
glycemic control (HbA1c (%) ≥ 6.5) [24]. These subjects were considered participants with T2DM.
Furthermore, subjects whose glucose concentration in the fasting blood sample taken during the survey
was ≥ 126 mg/dL and had levels of (HbA1c (%) ≥ 6.5) were defined as displaying fasting glucose
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and/or HbA1c values consistent with T2DM diagnostic criteria and were also defined as participants
with T2DM.
2.6. Anthropometric and Blood Pressure Assessment

Participants’ height and weight were measured by trained personal using standardized
procedures. Height was measured by using a conventional stadiometer (SECA 213, Medical Measuring
Systems and Scales, Hamburg, Germany) to the nearest 0.1 cm and body weight was measured with
a previously calibrated electronic (SECA 874, Medical Measuring Systems and Scales, Hamburg,
Germany) scale with a precision of 0.1 kg. Body mass index (BMI = weight (kg)/height (m2 )) was
calculated based on measured weight and height. We defined overweight/obesity as BMI ≥25 kg/m2 .
Waist circumference was measured to the nearest 0.1 cm at the high point of the iliac crest at the end of
normal expiration, with a measuring tape, which was placed below any clothing, directly touching the
participant’s skin. Abdominal obesity was defined as a waist circumference of ≥90 cm in men and
≥80 cm in women [25].
Subjects’ blood pressure was measured twice by a trained personal using an automatic medical
grade monitor (OMROM HEM-907, OMROM Mexico, Mexico City, Mexico). The first measurement
was taken after five minutes of rest, while participants were sitting with the dominant arm supported
at heart level. The second measurement was taken in the same way, five minutes after the first.
2.7. Physical Activity Assessment

Physical activity was evaluated with a previously used and validated [26] short version of the
international physical activity questionnaire (s-IPAQ). The questionnaire includes 9 items that assesses
time spent performing moderate-intense physical activity for at least 10 min for each activity over seven
days. The s-IPAQ data was analyzed in agreement with IPAQ protocol [27], as follows: first, physical
activity interval duration gathered in hours was converted into minutes; second, data which was
described as a weekly frequency was transformed into an average daily time; and third, subjects whose
responses were “do not know”, or “refused”, or had “missing data” for time duration or frequency
were removed from the present analysis. Based on the reported time spent performing moderate to
intense physical activity, participants were classified as inactive (<150 min/week), physically active
(150–299 min/week), or highly active (≥300 min/week) according to the World Health Organization
(WHO) physical activity guidelines [28].
2.8. Socioeconomic Status and Education Assessment

Socioeconomic status (SES) was constructed by combining eight variables that assessed
household characteristics, goods, and available services including: construction materials of the
floor, ceiling, and walls; household goods (stove, microwave, washing machine, refrigerator and
boiler); and electrical goods (television, computer, radio and telephone). The index was divided into
tertiles and used as a proxy for low, medium, and high SES. Education level was stratified into three
groups according to the highest level of education obtained: primary or less, secondary/high school,
and/or higher education.
2.9. Other Participant Characteristics

Participants completed two self-administered questionnaires (home and individual level) and
delivered detailed information regarding their demographic characteristics (e.g., age, sex, education,
marital status), self-perception of body weight, past medical history, current medication use, lifestyle
information (e.g., diet, physical activity, smoking status, alcohol consumption, etc.), depression
symptoms, sleep quantity, and information on reproductive history (for females).
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2.10. Statistical Analysis

We conducted descriptive analyses of the main characteristics of interest to assess adherence
to model assumptions. One-way analysis of variance (ANOVA) was used to test for differences
for general characteristics across quintiles of DII, while, chi-square tests were used to evaluate the
distribution of qualitative variables across DII quintiles. To evaluate the magnitude of the association
between specific DII and diabetes, we estimated multivariable adjusted odds ratios (OR) 95% CI using
logistic regression models. In all multivariate models, the first quintile of the DII score was considered
the reference. The Mantel–Haenszel extension chi-square test was used to assess the overall trend of
OR across increasing quintile of DII scores.
Additionally, to assess possible effect modification, we conducted a stratified analysis by age
groups (<55 years vs. ≥55 years), sex, BMI (<25.0 kg/m2 vs. ≥25.0 kg/m2 ), and physical activity
(inactive vs. active or highly active). We tested the significance using a likelihood ratio test by
comparing a model with the main effects of each intake and the stratified variable and the reduced
model interaction terms with only the main effects.
All p-values presented are two-tailed, p < 0.05 was considered significant. All analyses were
performed using STATA software (College Station, StataCorp LP, TX, USA), version 13.0.
3. Results
Participants’ baseline characteristics are shown in Table 1. A total of 1174 subjects (48.5% men)
between 20–69 years were included in the present analysis. The overall prevalence of T2DM was 13.6%;
of these, 10.1% were previously diagnosed individuals, whereas 3.5% were individuals with glucose
and/or HbA1c values consistent with T2DM definition at the time of the survey. The mean age among
participants with T2DM was 52.3 years. Subjects with T2DM had a significantly higher prevalence of
obesity, abdominal obesity, had lower levels of physical activity and intake of cereal fiber, and also,
as expected, levels of total cholesterol, triglycerides, and glycated hemoglobin were all significantly
higher in participants with T2DM.
Table 1. Characteristics of the study population: The Diabetes Mellitus Survey of Mexico City, 2015.
Overall Study Non-T2DM Subjects T2DM Subjects

Variables p-Value a
(n = 1174) (n = 973) (n = 201)
Sex, %
Men 48.5 48.4 48.7
0.17
Women 51.5 51.6 51.3
Age (years) b 39.9 (0.48) 38.0 (0.46) 52.3 (0.83) <0.001
Socioeconomic status, %
Low 21.9 20.2 33.5
Medium 36.4 36.1 37.4 <0.001
High 41.7 43.7 29.1
Education, %
Elementary and secondary education 19.3 16.5 37.4
High school 27.2 26.5 32.0 <0.001
Bachelor’s degree or higher 53.5 57.0 30.6
Smoking status, %
Current 45.6 45.2 48.8
Past 11.6 12.1 7.8 0.41
Never 42.8 42.7 43.4
Physical activity, %
Inactive 23.0 22.8 24.4
0.17
Active/highly active 77.0 77.2 75.6
Family history of DM2, % 41.9 38.2 66.5 <0.001
Hypertension, (%) 15.9 11.5 44.1 <0.001
Body mass index (kg/m2 ) b 28.8 (0.22) 28.2 (0.23) 31.2 (0.48) <0.001
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Table 1. Cont.
Overall Study Non-T2DM Subjects T2DM Subjects

Variables p-Value a
(n = 1174) (n = 973) (n = 201)
Body mass index, %
Normal (<25.0 kg/m2 ) 25.5 28.2 9.1
Overweight (≥25.0 to <30.0 kg/m2 ) 40.2 39.9 41.8 <0.001
Obesity (≥30.0 kg/m2 ) 34.3 31.9 49.1
Abdominal obesity, % 43.1 38.9 70.2 <0.001
Glucose (mg/dL) b 107.7 (2.0) 91.3 (0.53) 213.8 (7.7) <0.001
Glycated hemoglobin (HbA1c %) b 5.9 (0.07) 5.3 (0.02) 9.8 (0.23) <0.001
Triglycerides (mg/dL) b 205.6 (6.3) 189.8 (6.3) 307.3 (15.1) <0.001
Total cholesterol (mg/dL) b 189.1 (1.5) 186.2 (1.5) 207.8 (4.0) <0.001
High density lipoprotein (mg/dL) b 42.3 (0.41) 42.8 (0.49) 39.9 (0.68) 0.39
Low density lipoprotein (mg/dL) b 84.0 (2.1) 80.9 (2.0) 87.6 (4.9) <0.001
Dietary variables
Energy intake (kcal/day) c 2224 (2106–2342) 2329 (2196–2462) 1801 (1673–1927) <0.001
Carbohydrates (% energy) c 55.4 (54.7–56.1) 55.1 (54.3–55.9) 56.8 (55.4–58.1) 0.06
Total fats (% energy) c 31.1 (30.5–31.7) 31.3 (30.6–31.9) 30.2 (29.3–31.2) 0.03
Saturated fats (% energy) c 11.6 (11.3–11.9) 11.8 (11.5–12.0) 11.2 (10.7–11.6) 0.001
Monounsaturated fatty acids (% energy) c 10.9 (10.7–11.1) 11.0 (10.7–11.2) 10.4 (10.0–10.8) 0.03
Polyunsaturated fatty acids (% energy) c 7.0 (6.8–7.2) 7.0 (6.8–7.2) 7.0 (6.8–7.3) 0.61
Fiber (g/day) c 27.7 (25.9–29.6) 28.3 (26.3–30.3) 25.6 (23.8–27.4) <0.001
Alcohol intake (g/day) c 9.4 (7.4–11.3) 10.4 (8.2–12.6) 5.0 (2.5–7.5) 0.003
Magnesium (mg/day) c 409.7 (386.5–432.9) 417.7 (392.3–443.1) 377.3 (351.4–403.2) <0.001
a p <0.05, difference in mean and proportion between diabetic and non-diabetic subjects. Values were determined
using a student’s t-test for continuous variables and Chi-square test for categorical variables. b Mean and (standard
error); c Mean and 95% confidence intervals (95% CI). T2DM, type 2 diabetes mellitus; HbA1c, hemoglobin A1c.
According to DII score quintiles, subjects with the most pro-inflammatory diet were significantly
older, less educated, had lower SES, smoked less, had a higher prevalence of obesity, abdominal obesity,
and T2DM (Table 2). The most pro-inflammatory diet was characterized by a higher consumption
of carbohydrates (56.1%; (95% CI: 54.3, 57.8) vs. 53.8% (52.6, 55.1)), red meat and processed meat
(78.3 g/day (66.3, 90.2) vs. 40.1 g/day (33.7, 46.5)), refined cereals (173.9 g/day (147.1, 200.6) vs.
109.2 g/day (96.0, 122.5)), and soft drinks (401.7 mL/day (343.3, 460.1) vs. 69.5 mL/day (51.6, 87.4))
compared to the most anti-inflammatory diet. All of these differences were statistically significant. Also,
significantly decreasing trends were observed for the anti-inflammatory nutrients: fiber, vitamin C,
vitamin D, and magnesium (Table 3).
After adjusting for age and sex, the odds of having T2DM, across all DII score quintiles were
1.00, 1.63, 1.80, 1.84, and 2.29 (95% CI: 1.11, 4.75; p = 0.01). Finally, after additional adjustment for
co-variables, we observed that subjects with the most pro-inflammatory diet had approximately three
times greater odds of having T2DM (OR: 3.02, 95% CI: 1.39, 6.58; p = 0.005), compared to individuals in
the lowest DII quintile (Table 4).
Additionally, a sensitivity analysis included individuals with glucose and/or HbA1c values
consistent with T2DM definition at the time of the survey. In this case, we observed that subjects with
the most pro-inflammatory diet had greater odds of having T2DM (OR = 3.56; 95% CI: 1.13, 9.11) (data
not shown).
In addition, we evaluated the effect-modifying role of age; we observed a greater association with
T2DM contrasting DII extreme quintiles (Q5 vs. Q1 ) for participants aged ≥ 55 years (OR = 9.77; 95%
CI: 3.78, 25.50; p = 0.001) (Figure 1).
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Table 2. Characteristics of participants according to quintiles of the dietary inflammatory index: The Diabetes Mellitus Survey of Mexico City, 2015.
Dietary Inflammatory Index

Quintile 1: Most Quintile 5: Most
Quintile 2 Quintile 3 Quintile 4
Anti-Inflammatory Pro-Inflammatory p-Value a
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(n = 235) (n = 235) (n = 235) (n = 235) (n = 234)

Mean DII-density −3.05 −1.85 −0.81 0.24 1.79 <0.001
Sex, %
Men 24.7 37.5 38.9 40.1 48.9
<0.001
Women 75.3 62.5 61.1 59.9 51.1
Age (years) b 39.9 (0.91) 42.9 (1.2) 45.5 (1.11) 46.2 (1.08) 48.5 (1.05) 0.001
Socioeconomic status, %
Low 21.5 22.8 21.5 22.3 34.8
Medium 32.2 39.0 38.5 41.0 34.3 <0.001
High 46.3 38.2 40.0 36.7 30.9
Education, %
Elementary and secondary education 10.6 26.1 27.3 27.6 34.4
37
High school 24.5 21.3 27.2 29.8 31.7 <0.001
Bachelor’s degree or higher 59.9 52.6 45.5 42.6 33.9
Smoking status, %
Current 52.9 51.8 47.8 39.5 31.7
Past 10.5 6.1 10.0 17.9 15.2 <0.001
Never 36.6 42.1 42.2 42.6 53.1
Physical activity, %
Inactive 25.7 27.8 29.5 26.5 23.8
0.14
Active/highly active 74.3 72.2 70.5 73.5 76.2
Body mass index (kg/m2 ) b 28.1 (0.38) 28.3 (0.40) 28.7 (0.47) 28.9 (0.45) 29.8 (0.48) 0.007
Body mass index, %
Normal (<25.0 kg/m2 ) 29.2 25.3 23.5 25.4 20.8
Overweight (≥25.0 to <30.0 kg/m2 ) 38.5 38.8 41.4 38.6 36.2 0.005
Obesity (≥30.0 kg/m2 ) 32.3 35.8 35.1 36.0 43.0
Table 2. Cont.

Anti-Inflammatory Pro-Inflammatory p-Value a
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(n = 235) (n = 235) (n = 235) (n = 235) (n = 234)

Abdominal obesity, %
Yes 39.3 47.2 48.3 50.3 54.2 <0.001
Glucose (mg/dL) b 100.9 (3.6) 103.8 (2.8) 108.8 (3.6) 112.4 (3.9) 117.2 (4.7) <0.001
Glycated hemoglobin (HbA1c %) b 5.7 (0.10) 5.8 (0.12) 6.2 (0.14) 6.2 (0.13) 6.7 (0.11) 0.006
Triglycerides (mg/dL) b 200.6 (10.8) 188.3 (12.3) 217.9 (13.0) 203.1 (11.6) 216.7 (13.1) 0.96
Total cholesterol (mg/dL) b 187.7 (3.1) 181.6 (3.0) 192.4 (3.5) 190.9 (3.2) 196.4 (3.3) 0.02
High density lipoprotein (mg/dL) b 42.8 (1.03) 41.2 (0.81) 42.1 (0.75) 42.1 (0.76) 41.7 (0.71) 0.17
Low density lipoprotein (mg/dL) b 81.2 (4.4) 85.2 (3.4) 80.2 (4.7) 88.7 (4.4) 88.9 (4.8) 0.79
Type 2 Diabetes Mellitus, %
Yes 6.4 11.1 13.3 16.2 22.8 <0.001
a p-values were determined using analysis of variance (ANOVA) test for continuous variables and Chi-square test for categorical variables. b Data are given as means, with standard
38
error (SE) in parentheses, unless otherwise specified.
Table 3. Nutrient and food consumption according to quintiles of the dietary inflammatory index: in the Diabetes Mellitus Survey of Mexico City, 2015.

Variables Quintile 2 Quintile 3 Quintile 4 p-Value a
Anti-Inflammatory Pro-Inflammatory
Mean (95% CI) Mean (95% CI) Mean (95% CI) Mean (95% CI) Mean (95% CI)
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Carbohydrates intake (% energy) b 53.8 (52.6, 55.1) 55.0 (53.7, 56.4) 56.0 (55.2, 58.4) 56.1 (54.7, 57.5) 56.1 (54.3, 57.8) <0.001
Protein intake (% energy) 14.4 (13.9, 14.8) 13.9 (13.4, 14.6) 13.7 (13.1, 14.0) 13.0 (12.6, 13.5) 12.6 (11.7, 13.7) <0.001
Total fat intake (% energy) 31.8 (30.9, 32.9) 30.8 (29.8, 31.6) 30.3 (29.2, 31.3) 31.2 (30.1, 32.3) 31.3 (29.9, 32.7) 0.08
Saturated fats (% energy) 11.4 (10.9, 11.9) 11.7 (11.2, 12.2) 11.3 (10.9, 11.8) 11.3 (10.8, 11.8) 12.3 (11.5, 13.2) 0.51
Monounsaturated fats (% energy) 11.2 (10.8, 11.6) 10.8 (10.4, 11.2) 10.6 (10.1, 11.1) 10.9 (10.4, 11.4) 10.9 (10.3, 11.4) 0.20
Polyunsaturated fats (% energy) 7.6 (7.3, 7.9) 7.2 (6.9, 7.5) 6.9 (6.6, 7.2) 6.8 (6.5, 7.1) 6.7 (6.5, 7.0) <0.001
Fiber (g/day) 42.7 (40.0, 45.4) 31.5 (27.8, 35.2) 24.7 (23.5, 25.9) 24.2 (22.6, 25.9) 13.3 (12.7, 14.0) <0.001
Alcohol consumption (g/day) 15.2 (9.4, 21.0) 11.8 (7.2, 16.4) 6.6 (2.2, 11.0) 6.6 (1.2, 13.2) 2.5 (1.2, 3.9) <0.001
Magnesium (mg/day) 600.3 (568.8, 631.8) 453.5 (402.1, 504.8) 365.5 (350.3, 380.7) 355.9 (333.7, 388.2) 219.5 (208.6, 230.5) <0.001
Vitamin C (mg/day) 374.4 (344.5, 404.2) 288.0 (258.2, 317.8) 194.5 (181.0, 208.2) 160.6 (141.4, 179.7) 82.0 (72.9, 91.2) <0.001
Vitamin A (μg/day) 1900.2 (1666.1, 2134.2) 1219.9 (1071.6, 1368.2) 830.1 (772.7, 887.6) 701.5 (635.1, 767.9) 396.3 (360.0, 432.6) <0.001
Vitamin E (mg/day) 13.0 (12.3, 13.6) 8.9 (7.6, 10.1) 6.5 (6.2, 6.8) 6.5 (6.2, 7.3) 3.4 (3.4, 3.9) <0.001
Vitamin D (μg/day) 7.7 (7.1, 8.3) 5.1 (4.4, 5.7) 4.4 (3.9, 4.9) 3.8 (3.4, 4.2) 2.5 (2.5, 3.1) <0.001
Vegetables (g/day) 370.0 (321.2, 418.8) 245.5 (212.8, 278.4) 139.4 (121.3, 157.5) 112.2 (95.9, 128.5) 69.3 (60.0, 78.6) <0.001
Fruits (g/day) 327.6 (293.3, 362.0) 259.9 (225.9, 294.0) 178.8 (162.2, 195.5) 134.5 (114.3, 154.6) 74.2 (64.4, 84.0) <0.001
Legumes (g/day) 67.4 (49.5, 85.3) 51.0 (29.2, 72.6) 31.6 (26.0, 37.2) 33.7 (26.6, 40.8) 21.2 (16.2, 26.1) <0.001
39
Fish and seafood (g/day) 24.1 (15.0, 33.1) 13.9 (4.7, 23.0) 10.8 (5.9, 15.7) 10.4 (5.1, 15.7) 3.2 (1.5, 4.9) <0.001
Dairy products (g/day) 242.5 (209.9, 275.1) 225.8 (188.9, 262.6) 177.9 (142.3, 213.5) 172.5 (134.3, 210.8) 139.2 (109.3, 169.1) <0.001
Red meat and processed meat (g/day) 40.1 (33.7, 46.5) 46.9 (41.3, 52.6) 55.0 (44.4, 65.5) 55.3 (41.8, 68.7) 78.3 (66.3, 90.2) <0.001
Eggs (g/day) 28.9 (23.8, 33.9) 30.8 (25.1, 36.5) 32.2 (25.2, 39.2) 35.5 (28.4, 42.6) 34.3 (26.6, 41.9) <0.001
Refined cereals (g/day) 109.2 (96.0, 122.5) 145.0 (129.8, 160.3) 145.4 (125.5, 165.3) 158.2 (122.7, 193.6) 173.9 (147.1, 200.6) <0.001
Potatoes (g/day) 9.7 (5.2, 14.2) 11.8 (7.7, 15.8) 11.5 (7.8, 15.3) 8.3 (4.9, 11.7) 7.1 (5.1, 9.2) 0.87
Soft drinks (mL/day) 69.5 (51.6, 87.4) 146.9 (109.6, 184.1) 219.5 (175.7, 263.3) 262.4 (202.2, 322.6) 401.7 (343.3, 460.1) <0.001
a p-values were determined using ANOVA test. b Data are given as means, with 95% CI in parentheses.
Nutrients 2018, 10, 385
Table 4. Odds ratio (OR) and 95% confidence intervals (CI) for the relation between the dietary
inflammatory index and T2DM in the Diabetes Mellitus Survey of Mexico City, 2015.

Quintile 1: Most Quintile 5: Most p-Value
Anti-Inflammatory Pro-Inflammatory
OR OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI)
Model I 1.0 1.55 (0.86, 2.78) 1.80 (0.90, 3.56) 1.78 (0.93, 3.53) 2.29 (1.11, 4.75) 0.01
Model II 1.0 1.73 (0.94, 3.20) 1.88 (0.91, 3.89) 1.97 (1.06, 3.64) 3.00 (1.38, 6.55) 0.005
Model III 1.0 1.80 (0.95, 3.38) 2.01 (0.97, 4.13) 2.10 (1.07, 3.78) 3.02 (1.39, 6.58) 0.005
Model I: Adjusted by age and sex. Model II: Model I plus physical activity (inactive vs. active/highly active); hours of
television watching; tobacco use (current, past, and never); socioeconomic status (low, medium, and high); education
(elementary and secondary education, high school, and Bachelor’s degree or higher); family history of diabetes
mellitus (yes vs. no); persona history of hypertension (yes vs. no), medication use (yes vs. no), multivitamin use
(yes vs. no), and alcohol intake (gr/day). Model III: Model II plus body mass index (<25.0 vs. ≥25.0 kg/m2 ).
%* OR 95% CI pvalue

%)
%( Sex1
%'
%& Male 3.85 1.10, 13.46
0.01

%%
%$ Female 3.06 0.91, 10.80
-
, Age2
+ < 55 years 2.88 1.05, 8.08
* 0.001
) ≥ 55 years 9.77 3.78, 25.50
(
' BMI3
&
% < 25.0 kg/m2 2.66 0.53-6.42
0.13
$
≥ 25.0 kg/m2 5.10 1.66, 15.60

.))

/))

.&) $!&
/&) $!&

!

Physical activity4
Low 3.16 1.16, 7.02
0.02

2.85 0.90, 11.01
Figure 1. Subgroup analysis. Odds ratios (95% CI) for the association between extreme quintiles of
the dietary inflammatory index (DII) and type 2 diabetes mellitus (T2DM). 1 Adjusted for age (years),
physical activity (inactive vs. active/highly active), hours of television watching; tobacco use (current,
past, and never), socioeconomic status (low, medium, and high), education (elementary and secondary
education, high school, and Bachelor’s degree or higher), family history of diabetes mellitus (yes vs. no),
personal history of hypertension (yes vs. no), medication use (yes vs. no), multivitamin use (yes vs. no),
alcohol intake (gr/day), body mass index (<25.0 vs. ≥25.0 kg/m2 ). 2 Adjusted for sex, physical activity
(inactive vs. active/highly active), hours of television watching; tobacco use (current, past, and never),
socioeconomic status (low, medium, and high); education (elementary and secondary education, high
school, and Bachelor’s degree or higher), family history of diabetes mellitus (yes vs. no), personal
history of hypertension (yes vs. no), medication use (yes vs. no), multivitamin use (yes vs. no), alcohol
intake (gr/day), body mass index (<25.0 vs. ≥25.0 kg/m2 ). 3 Adjusted for age (years), sex, physical
activity (inactive vs. active/highly active), hours of television watching, tobacco use (current, past,
and never), socioeconomic status (low, medium, and high); education (elementary and secondary
education, high school, and Bachelor’s degree or higher), family history of diabetes mellitus (yes vs. no),
personal history of hypertension (yes vs. no), medication use (yes vs. no), multivitamin use (yes vs. no),
alcohol intake (gr/day). 4 Adjusted for age (years), sex, hours of television watching, tobacco use
(current, past, and never); socioeconomic status (low, medium, and high); education (elementary and
secondary education, high school, and Bachelor’s degree or higher), family history of diabetes mellitus
(yes vs. no), personal history of hypertension (yes vs. no), medication use (yes vs. no), multivitamin
use (yes vs. no), alcohol intake (gr/day), body mass index (<25.0 vs. ≥25.0 kg/m2 ).
40
Nutrients 2018, 10, 385
4. Discussion
In the Mexico City Diabetes Mellitus Survey, we observed that participants in the DII
score highest quintile, representing the most pro-inflammatory diet, had higher odds of T2DM
(independent of other diabetes risk factors) compared with participants in the DII score lowest
quintile (maximum anti-inflammatory potential). When we stratified by age (<55 years vs. ≥55 years),
we observed positive associations between DII and T2DM, with larger magnitude of association among
participants ≥55 years of age.
In the present study, we used a previously derived and validated DII score [13] to appraise the
capacity of a pro-inflammatory diet on T2DM. The DII scores computed from a Mexican population
included 27 food parameters, ranged from −5.49 to +4.12 with a mean of −0.68. This result is
consistent with prior publications conducted in Italy, France, and Spain [16,17,29,30]. For example,
Ramallal and colleagues at the University of Navarra cohort study reported a DII score ranging from
−5.14 to +3.97 [30].
We also evaluated associations between food groups and nutrient intake and the DII, and found
that subjects in the highest DII quintile (most pro-inflammatory diet) consumed more red and processed
meat, eggs, refined cereals, and soft drinks, and had a lower intakes of vegetables, fruits, fish,
and seafood. Similar to these results, the “PREvención con DIeta MEDiterránea” (PREDIMED) study
found that consumption of vegetables and fruit was less frequent among men and women in the
highest DII quintile [29].
Regarding nutrient intake, we observed that subjects with the most pro-inflammatory diet
had lower intakes of polyunsaturated fatty acids, fiber, magnesium, and some vitamins. Similar
to our results, the PREDIMED study found that subjects in the highest DII quintile consumed fewer
polyunsaturated fatty acids, vitamins, and fiber. Moreover, other studies using the dietary pattern
or dietary score approach, have observed an inverse association between healthy diets or patterns
(mainly characterized by fruits, vegetables, whole grains and fiber) and inflammation and T2DM,
as well as a positive association with Western patterns or unhealthy diet scores [9,10,30,31].
In the present analysis, we studied the association between DII and T2DM. We observed that
subjects in the highest DII quintile had approximately three times greater odds of T2DM compared
with subjects in the lowest DII quintile. The findings in our analysis agree with prior studies that
evaluate the relation between diet and T2DM. For example, in the Insulin Resistance Atherosclerosis
Cohort study, including subjects with a diet high in red meat, low-fiber cereals, fried potatoes, eggs,
cheese, and low in wine had approximately 4.5 times greater risk of T2DM comparing extreme
quartiles of the dietary pattern [31]. Similar results were observed in the Nurses’ Health Study [32],
where a pattern higher in sugar-sweetened soft drinks, refined grains, and processed meat but low in
vegetables was associated with an increased risk of T2DM (OR = 3.09; 95% CI: 1.99, 4.79; compared to
diet characterized by lower consumption of these dietary components). On the other hand, multiple
prospective studies have reported an inverse association between the adherence to Mediterranean
diet (with anti-inflammatory effects) [33] and the risk of T2DM [34]. Furthermore, a recent study [35]
that evaluated the relation between DII and the risk of prediabetes found that subjects in the highest
DII tertile had higher odds of prediabetes (OR = 18.88; 95% CI: 7.02, 50.82) compared to those who
consumed a more anti-inflammatory diet. Additionally, most of the highly consumed food groups in
the most pro-inflammatory DII quintile (red and processed meat, refined cereals, and soft drinks) have
been associated with T2DM and with inflammatory markers [10]. For example, soft drinks have been
linked to T2DM and inflammatory biomarkers due to their significant contribution to the glycemic
load [36]. Whereas, low intakes of whole grains, fruits, and vegetables in the highest DII quintile
have been related with reduced diabetes risk, probably mediated by a reduction of CRP, and certain
interleukins, and by an improvement in the endothelial function [37].
Other results in the present study indicate that a pro-inflammatory effect of diet on T2DM
could be particularly unfavorable among older (≥55 years) or inactive individuals (<150 min/week).
Although not statistically significant, there was a suggestion of an interaction between DII and
41
Nutrients 2018, 10, 385
overweight/obesity in relation to T2DM. Despite the lack of significance, this result could be
biologically important. Similar results were obtained in the Multiethnic Cohort (MEC) Study. In this
study, the stratified analysis suggests that, among men, the risk for T2DM increased with higher
consumption of fat and meat patterns among overweight (Hazard ratio = 1.49; 95% CI: 1.23, 1.81)
and obese (HR = 1.57; 95% CI: 1.16, 2.12) individuals [38]. In women, similar results were observed,
although these results were not statistically significant. In our case, this finding should be interpreted
with caution; it is difficult to determine, with our study design, the role of BMI in the causal sequence
(i.e., whether it is a confounder or an effect modifier). The relation between DII and T2DM remained
strong after adjustment for BMI, indicating that the DII may be associated with diabetes in all
individuals, but especially among obese subjects.
The connection between DII and T2DM could be explained through the effect of a pro-inflammatory
diet on insulin resistance that has been linked to the inflammatory process [39]. In this sense, prior
studies suggest a positive relationship between markers of chronic inflammation, including CRP, IL-1β,
IL-6, and TNF-α, and insulin resistance [39]. Furthermore, it has been established that insulin resistance
increases levels of these adhesion molecules in diabetic and nondiabetics individuals [40]. On the other
hand, the functional effects of food or food groups and nutrients like meat, processed meat, refined
cereals, and soft drinks, which corresponded with high DII scores in our study, have been also shown
to influence systemic inflammation [32].
Strengths of our study include: (1) the random stratified cluster design of the DMS-MC; (2) the use
of previously validated questionnaires; (3) the inclusion of many potential demographic, behavioral,
and other factors as potential effect modifiers or confounders in the multivariate analysis; and (4)
the use of a validated DII score specially constructed to assess the inflammatory potential of any
diet [12,13]. As reported in other studies [17,29], the DII can be adapted for use in different populations
including the Mexican population providing results that can be compared to those from studies based
in diverse populations in many parts of the world.
Some potential weaknesses of our study need to be highlighted. Because of its cross-sectional
design, this study cannot infer causality. Therefore, these results need to be further investigated in
future longitudinal studies. Although the SFFQ used in this study had been previously validated [20],
other possible limitations are related to information bias. In this regard, it is important to note that
dietary factors used to calculate the DII were evaluated from a single measurement of SFFQ, which is
subject to random error that would tend to underestimate the true association between DII and T2DM
in our study. As observed in previous studies [29], some dietary components such as saffron, thyme,
turmeric, and others were not available to compute the DII in the present analysis; however, as they
are not habitually consumed in large quantities or common in Mexican diet, they may not have
an important impact on the DII score. Another probable weakness could be related to the T2DM
definition; nevertheless, we provided appropriate allowance for the prevalent ascertainment in the
present analysis. In other words, prevalent cases of T2DM (previously diagnosed) were included only
if they were recently diagnosed (less than 11 months), had fasting glucose concentrations ≥126 mg/dL,
and had poor glycemic control (HbA1c ≥ 6.5) at the time of the survey. Additionally, we conducted
a sensitivity analysis including only subjects diagnosed at the time of the survey and we observed
similar results (OR = 3.56; 95% CI: 1.13, 9.11). We adjusted for many potentially confounding factors,
however, residual confounding due to measurement error, mainly in assessing some self-reported
lifestyle variables or due to insufficient control in statistical models, might have produced some degree
of bias in our results. However, it is improbable that such a bias would explain the consistently robust
association observed between DII and T2DM.
5. Conclusions
Our data suggest that a higher DII score (revealing a more pro-inflammatory diet) was associated
with increased odds of T2DM compared with DII scores in the lowest quintile of the DII (indicative of
an anti-inflammatory diet) among participants from the DMS-MC. In addition, we observed that
42
Nutrients 2018, 10, 385
the magnitude of the association appeared to be more pronounced among overweight/obesity

subjects, older individuals, and those with low levels of physical activity. Further longitudinal
investigations evaluating this relationship are needed to determine causality. Finally, the DII may be
an important tool to characterize the diet of the Mexican population and further explore associations
with non-communicable diseases.
Acknowledgments: This study was possible thanks to an unrestricted research grant obtained by SB from
Novo Nordisk (number Project: 1295. Cities Changing Diabetes: Mexico City Representative Diabetes survey)
and a NIH-Fogarty grant (NIH-Fogarty RO3 TW009061) to the National Institute of Public Health. Shivappa and
Hébert were supported by grant number R44DK103377 from the United States National Institute of Diabetes and
Digestive and Kidney Diseases.
Author Contributions: S.B. and E.D.-G. designed the study; E.D.-G., S.B., L.T.-M. conducted the research; E.D.-G.,
P.M.-A. and N.S. performed the statistical analyses; E.D.-G. wrote the first draft of the manuscript; E.D.-G., P.M.-A.,
N.S., J.R.H., L.T.-M., C.B., and S.B. critically reviewed and commented on the manuscript. All authors read and
approved the final version of the manuscript.
Conflicts of Interest: E. Denova-Gutiérrez, P. Muñoz-Aguirre, L. Tolentino-Mayo, C. Batis, S. Barquera, declare
no conflict of interest. James R. Hébert owns controlling interest in Connecting Health Innovations LLC (CHI),
a company planning to license the right to his invention of the dietary inflammatory index (DIITM ) from the
University of South Carolina in order to develop computer and smart phone applications for patient counseling
and dietary intervention in clinical settings. Dr. Nitin Shivappa is an employee of CHI.
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Effects of Virgin Olive Oils Differing in Their
Bioactive Compound Contents on Metabolic
Syndrome and Endothelial Functional Risk
Biomarkers in Healthy Adults: A Randomized
Double-Blind Controlled Trial
Estefania Sanchez-Rodriguez 1 , Elena Lima-Cabello 2 , Sara Biel-Glesson 3 ,
Jose R. Fernandez-Navarro 3 , Miguel A. Calleja 3 , Maria Roca 4 , Juan A. Espejo-Calvo 5 ,
Blas Gil-Extremera 6 , Maria Soria-Florido 7 , Rafael de la Torre 8 , Montserrat Fito 7,9 ,
Maria-Isabel Covas 9,10 , Juan de Dios Alche 2 , Emilio Martinez de Victoria 11 , Angel Gil 1,9, * and
Maria D. Mesa 1
1 Department of Biochemistry and Molecular Biology II, Institute of Nutrition and Food Technology
“José Mataix”, Biomedical Research Center, University of Granada, Parque Tecnológico de la Salud,
Avenida del Conocimiento s/n, 18100 Armilla, Granada, Spain; [email protected] (E.S.-R.);
[email protected] (M.D.M.)
2 Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín (CSIC),
Profesor Albareda 1, 18008 Granada, Spain; [email protected] (E.L.-C.);
[email protected] (J.d.D.A.)
3 Fundación Pública Andaluza para la Investigación Biosanitaria de Andalucía Oriental “Alejandro Otero”
(FIBAO), Avenida de Madrid 15, 18012 Granada, Spain; sbiel@fibao.es (S.B.-G.);
[email protected] (J.R.F.-N.); [email protected] (M.A.C.)
4 Food Phytochemistry Department, Instituto de la Grasa, Consejo Superior de Investigaciones Científicas
(CSIC), University Campus Pablo de Olavide, 41013 Sevilla, Spain; [email protected]
5 Instituto para la Calidad y Seguridad Alimentaria (ICSA), Avenida de la Hispanidad 17, 18320 Santa Fe,
Granada, Spain; [email protected]
6 Department of Medicine, University of Granada, Avenida de la Investigación 11, 18071 Granada, Spain;
[email protected]
7 Cardiovascular Risk and Nutrition Research Group, Hospital del Mar Medical Research Institute (IMIM), Dr.
Aiguader 88, 08003 Barcelona, Spain; [email protected] (M.S.-F.); mfi[email protected] (M.F.)
8 Integrative Pharmacology and Systems Neuroscience Research Group, IMIM (Hospital del Mar Research
Institute), Universitat Pompeu Fabra (CEXS-UPF), Dr. Aiguader 88, 08003 Barcelona, Spain; [email protected]
9 Spanish Biomedical Research Networking Centre, Physiopathology of Obesity and Nutrition (CIBEROBN),
Instituto de Salud Carlos III, Monforte de Lemos 3-5, 28029 Madrid, Spain; [email protected]
10 NUPROAS Handelsbolag, Nackã, Sweden, NUPROAS HB, Apartado de Correos 93, 17242 Girona, Spain
11 Department of Physiology, Institute of Nutrition and Food Technology “José Mataix”, Biomedical Research
Center, University of Granada, Parque Tecnológico de la Salud, Avenida del Conocimiento s/n,
18100 Armilla, Granada, Spain; [email protected]
Received: 10 April 2018; Accepted: 14 May 2018; Published: 16 May 2018
Abstract: The aim of this study was to evaluate the effect of virgin olive oils (VOOs) enriched with
phenolic compounds and triterpenes on metabolic syndrome and endothelial function biomarkers
in healthy adults. The trial was a three-week randomized, crossover, controlled, double-blind,
intervention study involving 58 subjects supplemented with a daily dose (30 mL) of three oils:
(1) a VOO (124 ppm of phenolic compounds and 86 ppm of triterpenes); (2) an optimized VOO
(OVOO) (490 ppm of phenolic compounds and 86 ppm of triterpenes); and (3) a functional olive oil
(FOO) high in phenolic compounds (487 ppm) and enriched with triterpenes (389 ppm). Metabolic
syndrome and endothelial function biomarkers were determined in vivo and ex vivo. Plasma high

Nutrients 2018, 10, 626
density lipoprotein cholesterol (HDLc) increased after the OVOO intake. Plasma endothelin-1 levels
decreased after the intake of the three olive oils, and in blood cell cultures challenged. Daily intake
of VOO enriched in phenolic compounds improved plasma HDLc, although no differences were
found at the end of the three interventions, while VOO with at least 124 ppm of phenolic compounds,
regardless of the triterpenes content improved the systemic endothelin-1 levels in vivo and ex vivo.
No effect of triterpenes was observed after three weeks of interventions. Results need to be confirmed
in subjects with metabolic syndrome and impaired endothelial function (Clinical Trials number
NCT02520739).
Keywords: olive oil; virgin olive oil; olive oil polyphenols; maslinic acid; oleanolic acid;
cardiovascular diseases; endothelial function; phenolic compounds; triterpenes; metabolic syndrome
1. Introduction
Metabolic syndrome (MS) is a cluster of associated metabolic and clinical disturbances that
tend to occur together [1]. This syndrome is commonly represented by the combination of obesity
(particularly abdominal), hyperglycemia, dyslipidemia, and hypertension [2]. MS is associated with
an increased risk of cardiovascular disease (CVD), which is the main cause of disability and mortality
in industrialized countries, and is associated with a chronic inflammatory response characterized by
abnormal cytokine production leading to endothelial dysfunction [3].
There is consolidated clinical evidence that the Mediterranean diet (MD) is associated with
a lower risk of CVDs, including myocardial infarction, stroke and cardiovascular death [4].
Mayneris-Perxachs et al. [5] proposed the MD as a successful tool for the prevention and treatment of
MS and related comorbidities. A large study in a high cardiovascular risk population has found that the
MD supplemented with virgin olive oil (VOO) protects people from vascular disease, suggesting a key
role of olive oil [6]. A recent systematic review and meta-analysis provided evidence that olive oil might
exert beneficial effects on endothelial function and biomarkers of inflammation, thus representing a key
ingredient contributing to the MD cardiovascular-protective effects [7]. Olive oil is not only a source of
monounsaturated fatty acids (MUFAs) but also an important source of bioactive compounds, such as
phenols and triterpenes [8,9]. Previous studies suggested a protective effect of olive oil phenolic
compounds on endothelial dysfunction [10], whereas olive oil triterpenes could be useful for the
prevention of multiple diseases related to cell oxidative damage [11]. Maslinic and oleanolic acids
are the principal triterpenes found in VOO. The potential of these olive oil triterpenic acids for use as
a therapeutic strategy to improve vascular function and treating CVD has been recently reviewed [12].
However, to our best knowledge, no clinical trial has been performed to provide evidence of their
benefits in healthy adults, according to EFSA requirement [13]. The present study which acronyms is
NUTRAOLEUM, aimed to evaluate the effect of VOO enriched with bioactive compounds, such as
phenolic compounds and triterpenes, on MS and endothelial function biomarkers in healthy adults.
We reported that daily intake of VOO enriched in phenolic compounds during three weeks improved
plasma high density lipoprotein cholesterol levels (HDLc), one of the features of metabolic syndrome,
although no differences were found at the end of the three interventions. In addition, VOO with at
least 124 ppm of phenolic compounds, regardless of the triterpenes content, improved the systemic
endothelin-1 levels in vivo and ex vivo, while no additional effect of triterpenes was observed.
2.1. Subjects
Information about subjects, sample size and their eligibility and dietary control have been
referenced in detail elsewhere [14]. In brief, fifty-eight intention-to-treat subjects were eligible.
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Nutrients 2018, 10, 626
The inclusion criteria were as follows: people in good health on the basis of a physical examination and
basic biochemical and hematological analyses, and willingness to provide written informed consent.
The exclusion criteria were as follows: smoking, intake of antioxidant supplements, aspirin or any
other drug with established antioxidant properties, hyperlipidemia, obesity (body mass index (BMI)
>30 kg/m2 ), diabetes, hypertension, celiac or other intestinal disease, any condition limiting mobility,
life-threatening diseases, or any other disease or condition that would impair compliance. Five subjects
declined to participate for personal reasons before olive oil type allocation. Fifty-three subjects (27 men
and 26 women) aged from 20 to 50 years from the general population of Granada were enrolled in the
study from February 2014 to July 2014 and were assigned into groups. Two subjects did not complete
the study. After the first intervention, one subject refused to continue for personal reasons, and the
other did not follow the protocol correctly. At the end of the experimental period, 51 subjects remained
in the study (Figure 1) [14]. All subjects provided written informed consent according to the principles
of the Declaration of Helsinki, and the local institutional review board, the Ethics Committee Research
Centre of Granada, approved the protocol (13/11 C38).
Figure 1. CONSORT Based Flow Diagram of the recruitment, enrollment and randomization process.
2.2. Study Design

The NUTRAOLEUM study has been designed to evaluate the effects of VOO high in phenolic
compounds and enriched with triterpenes, maslinic and oleanolic acids, from olive exocarp,
on MS features and endothelial function risk biomarkers in comparison with a standard VOO.
The characteristics of the olive oils used in the study, the design of the study, and the detailed
study procedures of the sustained consumption study have been previously published [14]. In brief,
the trial was a randomized, crossover, controlled and double-blind clinical trial involving three oils:
(1) an optimized VOO high in phenolic compounds (OVOO) (490 ppm of phenolic compounds and
86 ppm of triterpenes) produced from Picual olives (Andalucía, Spain); (2) a functional olive oil (FOO),
that was the same OVOO high in phenolic compounds (487 ppm) and enriched with triterpenes
(389 ppm) from olive exocarp; and (3) a VOO obtained from the OVOO after washing to eliminate
the majority of phenolic compounds (124 ppm of phenolic compounds and 86 ppm of triterpenes).
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Nutrients 2018, 10, 626
San Francisco de Asís Cooperative (Montefrío (Granada), Spain) provided the olive oils used in the
present study. Daily doses of 30 mL of the three types of raw olive oils, as recommended by the
US Food and Drug Administration [15], were distributed over three meals. Olive oils were blindly
prepared in special containers; the three types of olive oil were labeled “A”,”B”, and “C”. Containers
with the corresponding 30 mL olive oil daily dose and enough amount of the same oil for cooking
during each intervention period were delivered to the subjects at the beginning of each intervention
period. The subjects were randomly assigned to three orders of administration of olive oil, paired
by gender and age, using the block-randomization method of a software program for sequence
generation [14]. The randomization lists were concealed in a lightproof sealed envelope. The sealed
envelopes were kept by the independent statistician during the study, avoiding the breaking of the
seal. Thus, the subjects, investigators, and outcome assessors were blinded and could not foresee
the treatment allocation throughout the study. Hence, blinding of outcome assessment was also
ensured. According to previous published studies, olive oils were sequentially administered over three
periods of 3 weeks [16] preceded by two weeks of washout periods [17] during which the subjects
were requested to avoid olives and olive oil consumption. A nutritionist personally advised subjects
on replacing all types of habitually consumed raw fats using only the assigned oil. The advantage
of using a crossover design is that each subject served as his or her own control. Olive oils were
specially prepared for the trial and differed only in phenolic compound and triterpenes contents
(Table 1). The oils were prepared in dark, sealed containers and were similar in appearance and
color, thus ensuring blinding of the subjects and study personnel. The trial has been registered at
ClinicalTrials.gov ID: NCT02520739.
Table 1. Characteristics of the administered olive oils.
VOO OVOO FOO

Fatty Acid Profile (%)
C18:0 2.3 2.2 2.1
C18:1n9 78.9 78.2 78.4
C18:2n6 6.6 6.8 6.9
C18:3n3 0.6 0.7 0.7
C20:0 0.4 0.4 0.4
C20:1 0.3 0.4 0.4
C22:0 0.1 0.1 0.1
C24:0 <0.1 <0.1 <0.1
Total phenolic compounds (ppm) 124 490 487
Hydroxytyrosol and derivates 105 424.0 423.0
Lignanes 18.2 61.3 59.2
Flavonoids 0.7 3.4 3.2
Simple phenols 0.0 0.9 0.9
Total triterpenes (mg/kg) 86.5 86.3 388.8
Maslinic acid 47.3 47.3 217.7
Oleanolic acid 39.2 39.1 171.1
Ursolic acid <10 <10 <10
α-tocopherol (ppm) 174 183 176
Squalene (mg/100 g) 529.2 536.2 545.5
Total pigments (ppm) 15.73 17.59 16.78
Total carotenoid pigments (ppm) 7.08 6.79 6.97
Total sterols (ppm) 1437 1396 1460
FOO, functional olive oil; OVOO, optimized virgin olive oil; VOO, virgin olive oil.
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2.3. Evaluation of Dietary Intake

Subjects completed a 3-day dietary record at baseline and during each intervention period [18].
Energy consumption and dietary intakes of macro- and micronutrients data were processed using CSG
software (General ASDE) and the Spanish Food Composition Database (BEDCA) for the subjects who
completed the intervention [19].
2.4. Blood Sample Collection

Fasting venous blood samples were collected at the beginning of the study (baseline) and before
(pre-intervention, after the washout period) and at the end (post-intervention) of each olive oil
intervention period using EDTA-coated tubes. Three-milliliter aliquots were stored at 4 ◦ C for the
ex vivo experiments. The rest of the blood samples were centrifuged (4 ◦ C, 10 min at 1750× g),
and plasma aliquots were immediately frozen and stored at −80 ◦ C until analysis.
2.5. Ex-Vivo Whole Blood Cultures

An aliquot of blood samples (as indicated above) were collected using lithium–heparin tubes
(BD Vacutainer System, Heidelberg, Germany) from a subsample of 36 subjects. Blood was diluted
1:3 with Dulbecco’s modified Eagle’s medium and agitated gently in 3-mL tubes (Greiner Bio-one,
Solingen, Germany) within 3 h after collection. One-milliliter aliquots were seeded in each well of
24-well plates (Nunc, VWR International GmbH, Langenfeld, Germany) and cultured for 24 h at 37 ◦ C
under an atmosphere of 5% CO2 . From each blood drawing, we performed triplicate incubations in
parallel with positive and negative controls, separate cultures that included phytohaemagglutinin
(PHA, 10 μg/mL), E. coli lipopolysaccharide (LPS, 1 μg/mL) and phorbol 12-myristate 13-acetate plus
ionomycin (PMA, 25 ng/mL + IO, 1 μg). The same lots of PHA, LPS, PMA + IO and phosphate buffered
saline were used in all experiments. Blood cultures were removed from each well and centrifuged at
700× g for 5 min at 20 ◦ C. The resulting supernatants (plasma) were aliquoted and pooled from eight
subjects from each of the three assigned orders of administration of olive oil and stored at −20 ◦ C until
further analysis of endothelin-1 [20,21].
2.6. Measurement of Metabolic Syndrome Biomarkers

In the fasting state, anthropometric measurements (weight, height and waist circumference)
were determined at baseline and before and after each intervention period by the same member of
the professional staff. For all measurements, the subjects did not wear shoes. MS biomarkers were
determined as primary outcomes. BMI was calculated as weight (kg) divided by height squared (m2 ).
Total cholesterol, triacylglycerols and serum glucose were determined by standard enzymatic methods
using a PENTRA-400 autoanalyzer (ABX-Horiba Diagnostics, Montpellier, France). Plasma HDLc was
measured as soluble HDLc as determined using an accelerator selective detergent method (ABX-Horiba
Diagnostics). Plasma low density lipoprotein cholesterol (LDLc) concentrations were calculated using
the Friedewald formula. Systolic (SBP) and diastolic (DBP) blood pressures were measured with
a mercury sphygmomanometer after a minimum of 10 min resting in the seated position; the average
of two measurements was recorded. The pulse pressure was calculated as the difference between SBP
and DBP. Total cholesterol, triacylglycerols, serum glucose, HDLc and LDLc could only be measured
for 46 subjects.
2.7. Measurement of Selected Plasma Hormones and Endothelial Function Biomarkers

A Milliplex Map Kit, human monoclonal antibody kits (EMD Millipore Corporation, Billerica,
MA, USA) were used according to the manufacturer’s instructions in conjunction with a Luminex®
200 system with the XMap technology (Luminex Corporation, Austin, TX, USA) to determine the
concentrations of the following biomarkers as secondary outcomes: adiponectin (coefficient of variation
50
Nutrients 2018, 10, 626
(CV): 10.3%), resistin (CV: 7.7%), soluble intercellular adhesion molecule (sICAM-1) (CV: 6.1%) and
soluble vascular adhesion molecule (sVCAM-1) (CV: 5.4%), (Cat. #HADK1MAG-61K).
Endothelin-1 was determined as a secondary outcome by ELISA (CV): 7.2%) (R&D Systems,
Minneapolis, MN, USA; Cat. DET100) in both plasma and whole blood culture supernatants.
2.8. Measurements of Triterpenes and Phenolic Compounds in Urine

To ensure the subjects’ compliance with the assigned intervention, triterpenes (maslinic and
oleanolic acid) derivatized with 2-picolylamine (see Appendix A) and olive oil phenolic compounds
(hydroxytyrosol and metabolites) were analyzed in 24-h urine from 12 random subjects by liquid
chromatography coupled to a mass spectrometer [22].

Baseline data are presented as the mean values ± standard error of the mean (SEMs) unless
otherwise indicated. The normality of variables was assessed using Q-Q graphs. The χ2 test was used
for categorical variables to determine differences in the baseline. One-factor ANOVA or Kruskal-Wallis
tests were used (depending on whether the normality assumption was met) for continuous variables
to determine differences among the three olive oil interventions, in terms of baseline characteristics,
nutrient intake and for the ex vivo endothelin-1 experiment.
Biochemical parameters are presented as adjusted mean values ± SEMs and were analyzed
using a linear mixed-effects model (LMM). The normality of the residues was evaluated using Q-Q
graphs. Missing data were imputed using appropriate methods. The outliers for each intervention
were removed if kurtosis > 1 and asymmetry > 1 in the distribution of the responses. In all cases, more
than 80% of the data were analyzed.
Variables with a skewed distribution were logarithm-transformed for analysis (nutritional
variables and resistin). A LMM was used to compare variables before and after each intervention
(pre- vs. post-interventions, intra-treatment effect) and to compare the results between the groups
after the 3-week intervention (inter-treatment effect), adjusting for age, gender, pre-intervention
and period as fixed effects and for subjects and hospital as random effects. The same model was
also used to compare changes of the variables (post-intervention minus pre-intervention) without
adjusting for pre-intervention. Carryover effects were assessed as the interaction between period and
intervention [23]. The multiple comparison post hoc is given by the estimated means in the model
(adjusted by Sidak). This statistical model (LMM) takes into account all the possible confounders
(covariates) which are included on it. In addition, the baselines are used as outcomes but without effect,
so they can act as a control and take into account a possible carryover effect [24]. Within the LMMs,
the factor treatment, time and the random effect considered by participant are taken into account in
the structure of the data.
In addition, an interaction term was checked for differences on the effect part intervention by
gender. Model goodness-of-fit was tested using residual plots. The Bayesian Information Criterion
was used to assess model reduction and the selection of variables and interactions. We performed all
analysis on an intention-to-treat basis. A p < 0.05 value was considered significant. Statistical Package
for the Social Sciences version 20 software was used to perform the statistical analysis (SPSS Inc.,
Chicago, IL, USA).
3. Results
3.1. Baseline Characteristics

Tables 2 and 3 show the clinical and biochemical characteristics, and the average daily nutritional
intakes, respectively, of the subjects grouped according to the sequence of olive oil administration at the
beginning of the study. No differences were observed among the three groups of subjects at baseline.
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Nutrients 2018, 10, 626
Table 2. Clinical and biochemical characteristics of subjects at baseline according to olive oil
administration sequence.
Characteristics Sequence 1 Sequence 2 Sequence 3

Age, years 32 ± 2 29 ± 2 28 ± 2
Gender, male n (%) 12 (60) 10 (53) 8 (42)
BMI, kg/m2 24 ± 1 24 ± 1 24 ± 1
Waist circumference, cm 80 ± 2 78 ± 3 77 ± 2
Males 80 ± 3 82 ± 4 82 ± 2
Females 81 ± 5 73 ± 3 73 ± 2
HDLc, mg/dL 58 ± 2 58 ± 3 59 ± 2
Males 55 ± 3 51 ± 3 52 ± 3
Females 64 ± 2 65 ± 5 64 ± 3
LDLc, mg/dL 117 ± 9 107 ± 7 102 ± 5
Total cholesterol, mg/dL 192 ± 10 180 ± 7 175 ± 7
Triacylglycerols, mg/dL 87 ± 7 81 ± 13 67 ± 5
Glucose, mg/dL 90 ± 2 92 ± 2 87 ± 2
Adiponectin, mg/L 11.40 ± 1.38 12.49 ± 1.69 17.20 ± 2.78
Resistin, μg/L 16.38 ± 1.88 16.57 ± 1.82 15.26 ± 1.69
SBP, mmHg 121 ± 2 120 ± 3 118 ± 3
DBP, mmHg 77 ± 2 74 ± 2 71 ± 2
Pulse pressure, mmHg 44 ± 2 46 ± 3 47 ± 2
Endothelin-1, pg/mL 1.35 ± 0.08 1.36 ± 0.10 1.38 ± 0.12
sICAM, ng/mL 74.48 ± 4.50 62.26 ± 3.16 66.10 ± 4.63
sVCAM, ng/mL 459 ± 21 459 ± 30 443 ± 25
Values are expressed as the means ± SEMs. ANOVA and χ2 tests were used to compare results between groups.
Sequence 1: OVOO, VOO and FOO olive oil, n = 20; Sequence 2: VOO, FOO and OVOO olive oil, n = 19; Sequence 3:
FOO, OVOO and VOO olive oil, n = 19. BMI, body mass index; DBP, diastolic blood pressure; FOO, functional
olive oil; HDLc, high density lipoprotein cholesterol; LDLc, low density lipoprotein cholesterol; n, number of
observations; OVOO, optimized virgin olive oil; SBP, systolic blood pressure; SEM, standard error of the mean;
sICAM-1, soluble intercellular adhesion molecule; sVCAM-1, soluble vascular cell adhesion molecule; VOO, virgin
olive oil.
Table 3. Average daily energy and selected nutrient intake of subjects at baseline according to olive oil
administration sequence.
Nutritional Characteristics Sequence 1 Sequence 2 Sequence 3

Energy, kcal 1976 ± 90 2151 ± 138 2074 ± 109
Total carbohydrates, g 200 ± 10 213 ± 16 214 ± 13
Proteins, g 80 (16–222) 96 (37–258) 93 (21–215)
Total fat, g 86 ± 4 102 ± 10 89 ± 6
MUFA, g 31 (12–80) 33 (6–85) 33(6–89)
PUFA, g 13 (3–43) 11 (1–44) 14 (2–49)
SFA, g 21 (6–64) 27 (11–80) 26 (4–66)
Vitamin A, μg retinol equivalents 440 (111–1552) 512 (109–158) 539 (7–1669)
Vitamin C, mg ascorbic acid 70 (4–400) 88 (11–428) 81 (3–335)
Vitamin D, μg 2 (0–41) 2 (0–40) 2 (0–34)
Vitamin E, mg α-tocopherol equivalents 9 (2–29) 9 (1–67) 11 (1–39)
Cholesterol, mg 284 ± 26 354 ± 31 287 ± 24
Alcohol, g 0 (0–52) 0 (0–48) 0 (0–84)
Selenium, μg 31 (5–130) 34 (1–114) 36 (3–117)
Values are expressed as the means ± SEMs or as medians (range). ANOVA was used to compare results between
groups for those variables that followed normality, and the Kruskal Wallis test was used for those that did not. Data
of 51 subjects were obtained from the 3-day dietary record at baseline. Sequence 1: OVOO, VOO and FOO olive oil,
n = 54; Sequence 2: VOO, FOO and OVOO olive oil, n = 42; Sequence 3: FOO, OVOO and VOO olive oil, n = 57.
FOO, functional olive oil; MUFA, monounsaturated fatty acids; n, number of observations; OVOO, optimized virgin
olive oil; PUFA, polyunsaturated fatty acids; SEM, standard error of the mean; SFA, saturated fatty acids; VOO,
virgin olive oil.
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3.2. Nutritional Analysis

Table 4 shows the average daily energy and selected nutrient intakes after the three olive oil
interventions. No differences were observed among the three interventions (Table 4).
Table 4. Average daily energy and selected nutrient intake of subjects after the three olive
oil interventions.
Nutritional Characteristics VOO OVOO FOO

Energy, kcal 1983 (873–4342) 2006 (697–4561) 1914 (780–3457)
Total carbohydrates, g 199 (47–408) 203 (30–531) 175 (38–533)
Proteins, g 77 (24–180) 80 (17–200) 73 (25–207)
Total fat, g 89 (27–244) 94 (24–257) 91 (20–227)
MUFA, g 44 (8–119) 44 (8–113) 45 (9–123)
PUFA, g 11 (4–37) 12 (4–36) 13 (3–41)
SFA, g 25 (7–74) 27 (6–82) 24 (5–69)
Vitamin A, μg retinol equivalents 433 (34–1477) 427 (26–1504) 452 (39–1515)
Vitamin C, mg ascorbic acid 48 (0–280) 60 (0–255) 55 (1–305)
Vitamin D, μg 1.5 (0–32) 1.2 (0–82) 1.2 (0–81)
Vitamin E, mg α-tocopherol equivalents 10 (2–30) 11 (3–32) 11 (2–32)
Cholesterol, mg 285 (11–981) 253 (28–853) 272 (15–1011)
Alcohol, g ethanol 0 (0–97) 0 (0–54) 0 (0–78)
Selenium, μg 30 (0–260) 31 (0–198) 31 (1–160)
Values are expressed as median (range). ANOVA was used to compare intakes between interventions. Data for
51 subjects were obtained from the 3-day dietary record at baseline. Data for 51 subjects were obtained from the
3-day dietary record at baseline. FOO, functional olive oil; MUFA, monounsaturated fatty acids; n, number of
observations; OVOO, optimized virgin olive oil; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids;
VOO, virgin olive oil.
3.3. Plasma Metabolic Syndrome and Endothelial Function Biomarkers

Table 5 shows MS and endothelial function biomarker data before and after the three interventions.
All clinical and biochemical MS biomarkers were within normal values at the beginning and at the end
of the study. BMI, waist circumference, pulse pressure, and fasting plasma glucose, adiponectin and
resistin concentrations were unchanged during the study.
When comparing pre- vs. post-intervention data (intra-treatment effect), HDLc levels significantly
increased only after the OVOO intervention (p = 0.041), and only in females (p = 0.005). However, no
differences were observed between the three interventions. Total cholesterol increased after the FOO
intervention (p = 0.021). LDLc was unaffected. Fasting plasma triacylglycerol concentrations increased
after the VOO and OVOO interventions (p = 0.037 and p = 0.002, respectively) but not after the FOO
intervention. However, plasma triacylglycerols were low at the beginning of the study (78 ± 5 mg/dL).
On the other hand, SBP decreased after the VOO intervention (p = 0.019) and increased after the FOO
intervention (p = 0.004), while DBP and pulse pressure were unchanged after the three interventions.
Plasma endothelin-1 concentrations decreased after the VOO, OVOO, and FOO interventions (p = 0.006,
p = 0.006 and p = 0.014, respectively), and the plasma concentrations of sICAM-1 and sVCAM-1 were
unchanged after the three interventions.
When analyzing the inter-treatment effects, LDLc levels were higher after the FOO intervention
compared with the OVOO intervention (p = 0.033), and SBP was higher after the FOO intervention
compared with the VOO intervention (p = 0.001).
The changes in metabolic clinical variables and endothelial function biomarkers (Supplementary
Table S1) were similar in all the subjects after the three interventions except SBP, which increased up
to 118 mmHg after the FOO but decreased after the VOO and OVOO interventions (up to 115 and
116 mmHg, respectively) (p < 0.001). The results show differences by gender for all interventions.
However, no interactions were observed between gender and intervention.
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Table 5. Metabolic syndrome and endothelial function biomarkers before and after each olive oil intervention in healthy adults.
VOO OVOO FOO

Characteristics
Pre-Intervention Post-Intervention Pre-Intervention Post-Intervention Pre-Intervention Post-Intervention
BMI, kg/m2 23.9 ± 0.1 24 ± 0.1 23.9 ± 0.1 24 ± 0.1 24 ± 0.1 24 ± 0.1
Nutrients 2018, 10, 626
Waist circumference, cm 77.4 ± 0.7 77.2 ± 0.7 77.2 ± 0.7 76.8 ± 0.7 77.4 ± 0.7 76.9 ± 0.7
HDLc, mg/dL 58 ± 2 59 ± 2 57 ± 2 60 ± 2 * 58 ± 2 60 ± 2
Males 52 ± 2 53 ± 2 52 ± 2 52 ± 2 52 ± 2 54 ± 2
Females 64 ± 2 65 ± 2 63 ± 2 67 ± 2 * 65 ± 2 66 ± 2
LDLc, mg/dL 105 ± 4 108 ± 4 a,b 104 ± 4 106 ± 4 a 104 ± 4 111 ± 4 b
Total cholesterol, mg/dL 179 ± 5 182 ± 5 177 ± 5 183 ± 5 178 ± 5 186 ± 5 *
Triacylglycerols, mg/dL 72 ± 7 75 ± 7 * 74 ± 7 81 ± 7 * 75 ± 7 75 ± 7
Glucose, mg/dL 91 ± 2 91 ± 2 91 ± 2 91 ± 2 90 ± 2 91 ± 2
Adiponectin, mg/L 13.74 ± 1.7 12.8 ± 1.7 12.48 ± 1.7 13.6 ± 1.71 12.62 ± 1.71 13.74 ± 1.71
Resistin, μg/L 14.1 ± 1.1 14.1 ± 1.1 14.5 ± 1.1 13.8 ± 1.1 13.8 ± 1.1 14.1 ± 1.1
SBP, mmHg 117 ± 3 115 ± 3 *,a 119 ± 3 116 ± 3 a,b 114 ± 3 118 ± 3 *,b
DBP, mmHg 72 ± 2 72 ± 2 75 ± 2 73 ± 2 72 ± 2 74 ± 2
Pulse pressure, mmHg 45 ± 1 43 ± 1 44 ± 1 42 ± 1 42 ± 1 45 ± 1
Endothelin-1, pg/mL 1.53 ± 0.15 1.38 ± 0.15 * 1.58 ± 0.15 1.41 ± 0.15 * 1.49 ± 0.15 1.35 ± 0.15 *
54
sICAM-1, ng/mL 68.09 ± 3.19 67.11 ± 3.19 65.17 ± 3.20 67.73 ± 3.19 65.1 ± 3.20 66.7 ± 3.21
sVCAM-1, ng/mL 451 ± 22 443 ± 23 435 ± 22 451 ± 22 431 ± 22 442 ± 23
Values are expressed as the adjusted means ± SEMs. LMM was used to compare pre-intervention vs. post-interventions with each oil data, and data after the three interventions
(post-interventions). * Significant differences between pre-intervention vs. post-intervention data within each intervention with the three olive oils. Different superscript letters indicate
significant differences between post-intervention results (a,b ). p < 0.05 was considered significant. DBP, diastolic blood pressure; FOO, functional olive oil; HDLc, high density lipoprotein
cholesterol; LDLc, low density lipoprotein cholesterol; OVOO, optimized virgin olive oil; SBP, systolic blood pressure; SEM, standard error of the mean; sICAM-1, soluble intercellular
adhesion molecule; sVCAM-1, soluble vascular cell adhesion molecule; VOO, virgin olive oil.
Nutrients 2018, 10, 626
3.4. Plasma Endothelin-1 Ex Vivo Experiments

Before the three interventions, pooled blood cultures challenging with PHA, LPS, or PMA + IO
induced a potent increase in the supernatant concentrations of endothelin-1 in whole blood cultures.
Figure 2 shows that the supernatant endothelin-1 concentration changes (post-intervention minus
pre-intervention data) in whole blood cultures from the subjects were similar after the VOO and
OVOO interventions and were significantly lower after the FOO intervention: −26.7 ± 15.3 pg/mL,
−41.0 ± 12.9 pg/mL, and −119.5 ± 28.5 pg/mL for the VOO, OVOO and FOO interventions,
respectively (p = 0.035), when stimulating with PHA; −30.3 ± 6.5 pg/mL, −58.2 ± 10.1 pg/mL
and −109.6 ± 9.7 pg/mL for the VOO, OVOO, and FOO interventions, respectively, when stimulating
with LPS (p = 0.002); and −38.9 ± 3.4 pg/mL, −50.8 ± 8.5 pg/mL and −87.0 ± 11.1 pg/mL for the
VOO, OVOO, and FOO interventions, respectively, when stimulating with PMA+IO (p = 0.015).
Figure 2. Plasma endothelin-1 ex vivo changes (post-intervention minus pre-intervention data) when
stimulated with PHA, LPS or PMA + IO in whole blood cultures from healthy adults. Values are
expressed as the means ± SEMs. ANOVA was used to compare differences between interventions
and induction treatments. The Tukey post-hoc test was used for multiple comparisons among groups.
p < 0.05 was considered significant. FOO, functional olive oil; IO, ionomycin; OVOO, optimized virgin
olive oil; PHA, phytohemagglutinin; PMA, phorbol 12-myristate 13-acetate; VOO, virgin olive oil.
Supplemental Figure S1 shows the supernatant endothelin-1 concentrations that were induced
ex vivo with PHA, LPS, and PMA + IO in whole blood cultures from the subjects before and after the
three interventions. After the VOO, OVOO, and FOO interventions, challenging with LPS or PMA
+ IO induced a significantly lower increase of endothelin-1 secretion in whole blood cultures, while
challenging with PHA induced a significantly lower increase of endothelin-1 secretion only after the
OVOO and FOO interventions.
3.5. Biomarkers of Intervention Compliance

Recoveries of triterpenes in urine were consistent with the olive oils consumed in each intervention.
Figure 3 shows urinary triterpenes changes (post-intervention minus pre-intervention data) for each
olive oil intervention. The amounts of both triterpenic acids in urine after the FOO intervention were
55
Nutrients 2018, 10, 626
about four times higher than those recovered after the VOO and OVOO interventions (p = 0.004 and
p < 0.001, respectively, for maslinic acid, and p = 0.026 and p < 0.001, respectively, for oleanolic acid).
Urine concentrations of total hydroxytyrosol (the sum of hydroxytyrosol and its glucuronide and
sulfate conjugates) were higher after the OVOO (2444 mM, p = 0.003) and FOO (2876 mM, p = 0.002)
interventions than after the VOO (115 mM, p = 0.011) intervention.
Figure 3. In vivo urinary triterpenes changes (post-intervention minus pre-intervention data) for each
olive oil intervention in healthy adults. Values are expressed as the means ± SEMs. ANOVA was
used to compare differences between the three interventions. Different superscript letters indicate
significant differences between the interventions for oleanolic acid (a,b ) and for maslinic acid (c,d ).
p < 0.05 was considered significant. FOO, functional olive oil; OVOO, optimized virgin olive oil; VOO,
virgin olive oil.
4. Discussion
The NUTRAOLEUM study is the first human nutritional clinical trial concerning the effects of
VOO that is high in phenolic compounds and enriched with triterpenes, maslinic and oleanolic acids,
from olive exocarp, on MS features and endothelial function risk biomarkers in comparison with
standard VOO in healthy subjects.
The PREDIMED study reported that a MD supplemented with at least 50 g of dietary VOO
caused a reversion of MS after a median follow-up of 4.8 years [25], reducing the rate of CVD events
by 30% compared with a low-fat diet control group [26]. In addition, this MD enriched with VOO
and without energy restrictions reduced the diabetes risk among individuals at high cardiovascular
risk [27]. Consumption of VOO close to 2.7, 164 or 366 ppm/day of phenolic compounds in humans,
a 0.03% of hydroxytyrosol in a rodent model, and 50 ppm/day of hydroxytyrosol in a murine model
have been reported to improve the blood lipid profile, although results in mice were not conclusive [28],
possibly due to differences in the phenolic content of the olive oil and the physio-pathology of the
studied animals. Polyphenols from olive oils have been shown to provide additional benefits on
HDLc, other than those provided by the MUFA content. However, contradictory data exists on these
benefits. In 2015, a meta-analysis reported no effect on HDLc concentration after the intake of VOO
with at least 150 ppm of phenolic compounds [29], while, in accordance with our results, a recent
systematic review [30] concluded that plasma HDLc was increased in different studies consuming
from 2.28 to 75 g/day of olive oil. In the Eurolive Study, a European multicenter study, three olive oils
(refined, medium and high) differing in their phenolic compound content (2.7, 164 and 366 ppm/day of
phenolic content, respectively) increased HDLc and decreased triacylglycerol concentrations [16]. The
increase in HDLc was linear with the olive oil polyphenol content. This additional benefit has also been
56
Nutrients 2018, 10, 626
described in healthy and hypercholesterolemic subjects treated with polyphenol-enriched VOO [31]
when excluding patients treated with hypolipidemic medication. Additionally, the increase of olive oil
polyphenols in the lipoprotein fraction may increase HDL size, stability, and antioxidant status [32].
It has been reported that the daily intake of olive oils enriched with its own polyphenols (250 ppm
or 500 ppm), as well as the intake of olive oil enriched in polyphenols from thyme (250 ppm/day)
decrease LDLc and improve the lipoprotein subclass distribution and associated ratios [33]. Other
authors have described that a one-year intervention with a MD enriched with VOO improved several
LDL characteristics related to its atherogenicity (resistance against oxidation, size, composition, and
cytotoxicity) but did not modify the plasma LDLc concentrations in a subsample of subjects at high
cardiovascular risk in the PREDIMED study [34]. Our results are consistent with this null effect of
VOO on LDLc. On the other hand, besides the weak increase in plasma triacylglycerols observed
after interventions with both VOO and OVOO, this was without clinical significance given that levels
were low at baseline and remain low at the end of the three interventions, and thus it did not increase
cardiovascular risk in these healthy subjects (triacylglycerols < 150 mg/dL) [35]. Our results are
in contrast with the triacylglycerol improvement reported in the EUROLIVE Study [16]. A recent
meta-analysis and systematic review focused on high polyphenol VOO concluded no effect on plasma
triacylglycerol concentrations [29]; in addition, Saibandith et al. [36] stated that the effect of olive oil
polyphenols on plasma triacylglycerols remained unclear. However, longer studies are needed to
clarify this issue.
To the best of our knowledge, this is the first clinical trial evaluating the effect of olive
oil triterpenes on plasma lipids and endothelial function biomarkers. Previous animal studies
have reported different effects of oleanolic acid on plasma lipids, depending on the experimental
animal model [37]. Based on our results, FOO enriched with triterpenes did not modify plasma
HDLc, LDLc, or triacylglycerols, but increased plasma total cholesterol concentrations, although
no inter-group significance was found. However, although these modifications do not represent
an increase in cardiovascular risk since the values are low enough to be considered safe (total
cholesterol < 190 mg/dL) [35], further human clinical trials are needed to demonstrate the potential
benefit of olive phenolic compounds and triterpenes on plasma lipid concentrations over longer
intervention periods.
Endothelial dysfunction is a critical early event in the development of atherosclerosis [38].
An imbalance between vasodilating and vasoconstricting molecules, such as nitric oxide and
endothelin-1, respectively, contributes to the pathogenesis of hypertension and its complications [39].
Our results indicate that plasma endothelin-1 levels decreased after the VOO, OVOO and FOO
interventions, and these results were also confirmed throughout ex vivo blood culture experiments.
Although endothelin-1 is mainly produced from vascular endothelial cell, several studies have
suggested that blood cells, such as polymorphonuclear neutrophils [40] and T-Cells [41], and also
macrophages [42] are responsible from circulating levels of endothelin-1. In addition, ex vivo
experiment has demonstrated the release of the mature peptide after LPS and LPS + PMA
stimulation [40]; Mencarelli et al. [43] have confirmed these results. As demonstrated here, endothelin-1
production was decreased after the three interventions. This effect may be beneficial for cardiovascular
risk affected people, since the effect of endothelin-1 has been documented on endothelial and
inflammatory cells, which contributes to pathophysiological processes such as vascular hypertrophy,
cell proliferation, fibrosis and inflammation [44–46]. In humans, the consumption of VOO has shown
benefits on blood pressure and endothelial function [47]. In agreement with our results, a meta-analysis
stated that olive oils with at least 150 ppm of phenolic compounds exert a moderate effect on lowering
SBP and no effects on DBP [29]. Regarding triterpenes, besides the weak SBP increase observed after
this intervention, no clinical significance was observed as blood pressure remained under 130 mmHg
and did not increase cardiovascular risk [35]. It is reported that SBP varies to a greater degree than
DBP [48,49]. For this reason, we calculated pulse pressure and found no significant effect of FOO.
A beneficial effect of triterpenes on endothelial function [50] and blood pressure [51] has been described
57
Nutrients 2018, 10, 626
in animal models of hypertension, but further studies are required to explore the mechanisms involved
in the effect of specific components of VOO on endothelin-1 regulation.
In vitro studies showed that minor olive oil components, specifically hydroxytyrosol and its
metabolites, down-regulate the secretion of E-selectin, p-selectin, sICAM-1, and sVCAM-1, affecting
endothelial function [52]. However, and in agreement with our results, no differences in sICAM-1 or
sVCAM-1 plasma concentrations were reported after 50 mL of VOO or refined olive oil consumption
containing (161 or 14.67 ppm/day of phenolic compounds, respectively) in coronary heart disease
patients [53]. Another study found a significant reduction in sICAM-1 but not in sVCAM-1
concentrations after the intake of 50 mL/day of olive oil [54]. Recently, it has been proposed that the
intake of 25 mL/day of VOO containing 366 ppm of phenolic compounds modulates the expression
of several genes related to the renin-angiotensin-aldosterone system [55]. Therefore, further longer
studies are needed to reach final conclusions about the effect of VOO minor compounds on molecules
modulating endothelial function.
One of the limitations of the present study is that young and healthy subjects are recruited as
target population. The present intervention does not appear sufficient to draw definitive conclusions.
Therefore, new studies in older subject affected by metabolic syndrome and endothelial dysfunction
would be interesting in order to evaluate the beneficial effect of VOO components. Although no
differences in dietary intakes were observed during the three interventions, measurements of dietary
intake relied on self-reporting and were therefore subjective. Another limitation of this study is that
dietary records for the washout periods were not recorded, thus we cannot analyzed dietary intakes
during these periods. In addition, our subjects live in the south of Spain, where MD and VOO are
highly consumed. Therefore, a 3-week intervention is not long enough to cause significant changes.
Further studies are required to find conclusions related to the bioactive compounds presents in olive
oils, and to explore the mechanisms involved in these effects of specific components of VOO on MS
and endothelial function.
5. Conclusions
In conclusion, olive oil rich in polyphenols increased HDLc levels in females, although no
differences were found at the end of the three interventions, and improved an endothelial function
biomarker both in vivo and ex vivo. No additional benefits were obtained from triterpenes VOO
enrichment after 3-wk supplementation. However, further longer studies are warranted on olive
oil triterpenes and their health benefits in older subjects particularly in those affected by metabolic
syndrome and endothelial dysfunction.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/10/5/626/s1,

Figure S1: Plasma endothelin-1 ex vivo secretion in whole blood cultures from healthy adults before and after
3 weeks of intervention with the three olive oils and PHA, LPS or PMA + IO stimulation. Table S1: CONSORT
2010 checklist of information to include when reporting a randomized trial.
Author Contributions: J.A.E.-C., B.G.-E., M.S.-F., R.d.l.T., M.F., M.-I.C., J.d.D.A., E.M.d.V., A.G., and M.D.M.
designed the research. E.S.-R., S.B.-G., J.R.F.-N., M.A.C., and M.D.M. conducted the research. E.L.-C. and J.d.D.A.
were responsible for the ex vivo analyses. M.R. carried out the olive oil analyses. R.d.l.T. carried out urine phenolic
and triterpenic acid measurements. E.S.-R. carried out biochemical analyses. E.S.-R., E.L.-C., R.d.l.T., J.d.D.A.,
E.M.d.V. and M.D.M. analyzed the data and performed the statistical analysis. E.S.-R. wrote the paper. E.S.-R.,
E.M.d.V., A.G. and M.D.M. had primary responsibility for the final content. All authors read and approved the
final manuscript. This paper contains results included in the doctoral thesis of Estefanía Sánchez Rodríguez,
which was written within the context of Nutrition and Food Sciences at the University of Granada.
Acknowledgments: The “NUTRAOLEUM Study” has been supported by the grant ITC-20131031 from the I+D
FEDER-INTERCONNECTA (CDTI) and Junta de Andalucía, Spain”. We thank ACER CAMPESTRES S.L., SAN
FRANCISCO DE ASIS Coop and AGROINSUR S.L., for the funding provided. The authors thank Pilar Jiménez,
Alberto Guarnido, María Molina and Elizabeth García for administering the questionnaires to the subjects;
Isabel Mérida, Isabel Hinojosa, Agustín Martín García, and María Luz Abarca for collecting the biological samples;
and Victoria Martín Laguna and Laura Campaña Martín for aliquoting the samples. The authors also thank
María Cruz Rico Prados for sample analysis and Llenalia M. García Fernández for her contributions to the
statistical analysis.
58
Nutrients 2018, 10, 626
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
Appendix A
Analysis of Triterpenes in Urine

For the analysis of maslinic acid (MA) and oleanolic acid (OA), aliquots of 250 μL of urine
were transferred into 15-mL screw-capped glass tubes and spiked with 1 ng/mL of d3 -OA, 20 μL of
β-glucuronidase from Escherichia coli and 200 μL of 0.1 M phosphate buffer pH 6.0. After overnight
incubation in a water bath at 37 ◦ C, 50 mg of NaHCO3 /Na2 CO3 (1:2, w/w) was added to each tube
before extraction. The samples were then subjected to a liquid-liquid extraction with 2 mL of methyl
tert-butyl ether. The mixture was homogenized in a shaker rotator for 20 min and centrifuged at
3500 rpm for 5 min at room temperature. The organic phase was transferred to clean tubes and
evaporated (40 ◦ C) under a stream of nitrogen. The extracts were then derivatized with 50 μL
of 2-picolylamine (1 μg/μL in ACN). The reaction mixture was incubated for 10 min at 60 ◦ C on
a heating block and then dried under a nitrogen stream. Samples were reconstituted in 100 μL of
ACN-H2 O MilliQ grade (1:1). Derivatized OA and MA in urine was quantified using an Acquity
UPLC system, (Waters Associates, Milford, MA, USA) for the chromatographic separation, the column
was coupled to a triple quadrupole (Quattro Premier) mass spectrometer provided with an orthogonal
Z-spray-electrospray interface (ESI) (Waters Associates). Nitrogen was used as the drying and
nebulizing gas. The desolvation gas flow was set to approximately 1200 L/h, and the cone gas
flow was set to 50 L/h.
Capillary voltages of 3 kV and 2.5 kV were used in positive and negative ionization mode,
respectively. The nitrogen desolvation temperature was set to 450 ◦ C, and the source temperature was
set to 120 ◦ C. The collision gas was argon, and the flow rate was 0.21 mL/min.
The liquid chromatography separation was performed at 55 ◦ C using an Acquity CSH
phenyl-hexyl column (100 mm, 2.1 mm i.d., 1.7 μm) (Waters Associates) operating at a flow rate
of 300 μL min−1 . Water and methanol, both containing formic acid (0.01% v/v) and ammonium
formate (1 mM), were selected as mobile phase solvents. For the target detection of derivatized OA
and MA, a gradient program was used to separate the analytes; the percentage of organic solvent
was linearly changed as follows: 0 min, 70%; 0.5 min, 70%; 7 min, 98%; 9 min, 98%; 9.5 min, 70%;
and 11 min, 70%.
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62
nutrients
Article
The Effect of Low-Carbohydrate Diet on Glycemic
Control in Patients with Type 2 Diabetes Mellitus
Li-Li Wang 1 , Qi Wang 1 , Yong Hong 1 , Omorogieva Ojo 2 , Qing Jiang 1 , Yun-Ying Hou 1 ,
Yu-Hua Huang 3 and Xiao-Hua Wang 1, *
1 School of Nursing, Medical College, Soochow University, Suzhou 215006, China;
[email protected] (L.-L.W.); [email protected] (Q.W.); [email protected] (Y.H.);
[email protected] (Q.J.); [email protected] (Y.-Y.H.)
2 Department of Adult Nursing and Paramedic Science, University of Greenwich, London SE9 2UG, UK;
[email protected]
3 Medical College, Soochow University, Suzhou 215006, China; [email protected]
Received: 8 April 2018; Accepted: 17 May 2018; Published: 23 May 2018
Abstract: Objective: In China, a low-fat diet (LFD) is mainly recommended to help improve blood
glucose levels in patients with type 2 diabetes mellitus (T2DM). However, a low-carbohydrate diet
(LCD) has been shown to be effective in improving blood glucose levels in America and England.
A few studies, primarily randomized controlled trials, have been reported in China as well. Method:
Firstly, we designed two ‘six-point formula’ methods, which met the requirements of LCD and LFD,
respectively. Fifty-six T2DM patients were recruited and randomly allocated to the LCD group
(n = 28) and the LFD group (n = 28). The LCD group received education about LCD’s six-point
formula, while the LFD group received education about LFD’s six-point formula. The follow-up
time was three months. The indicators for glycemic control and other metabolic parameters were
collected and compared between the two groups. Results: Forty-nine patients completed the study.
The proportions of calories from three macronutrients the patients consumed met the requirements
of LCD and LFD. Compared to the LFD group, there was a greater decrease in HbA1c level in the
LCD group (−0.63% vs. −0.31%, p < 0.05). The dosages of insulin and fasting blood glucoses (FBG)
in the third month were lower than those at baseline in both groups. Compared with baseline values,
body mass index (BMI) and total cholesterol (TC) in the LCD group were significantly reduced in the
third month (p < 0.05); however, there were no statistically significant differences in the LFD group.
Conclusions: LCD can improve blood glucose more than LFD in Chinese patients with T2DM. It can
also regulate blood lipid, reduce BMI, and decrease insulin dose in patients with T2DM. In addition,
the six-point formula is feasible, easily operable, and a practical educational diet for Chinese patients
with T2DM.
Keywords: diabetes mellitus; diet; carbohydrate; blood glucose; HbA1c; fasting blood glucose;
postprandial blood glucose
1. Introduction
Dietary intervention is a strategy to manage diabetes mellitus (DM) [1], as it can reduce the
burden on islet cells and thus improve blood glucose levels, lipid profiles, and cognitive status [2–4].
However, good adherence to diabetic diets is the premise of diet therapy. In China, a low-fat diet (LFD)
is mainly recommended to help improve blood glucose levels in patients with type 2 diabetes mellitus
(T2DM) [5]. Studies have shown that LFD could reduce glycated hemoglobin (HbA1c) by as much as
0.8–2.8% [6–8].

Nutrients 2018, 10, 661
On the other hand, a low-carbohydrate diet (LCD) is a dietary strategy that refers to carbohydrate
intake of between 30–200 g/day or calories from carbohydrates/total calories of <45%, supplementing
instead with fat or protein [9]. This has been found to be effective in the treatment of obesity,
and apart from significantly reducing weight, it can also effectively improve blood lipid and insulin
resistance [10]. In recent years, the American Diabetes Association and Diabetes UK have both
confirmed the effectiveness of LCD in reducing weight, improving blood glucose, and regulating blood
lipid in patients with DM [11,12]. In Japan, Yamada [13] reported that HbA1c and triglyceride (TG)
levels in patients with T2DM decreased significantly in the LCD group without calorie-restriction,
compared to the LFD group with calorie-restriction. This indicates that LCD made patients with DM
have less desire to eat due to a feeling of satiety. However, only limited studies relating to the use of
LCD in patients with DM, especially randomized controlled trials, have been reported in China.
Based on research evidence, only 29.8% of Chinese patients with T2DM comply with a diabetic
diet advised by their doctors and dietitians [14]. In addition, we found that certain types of foods were
strictly limited and patients with DM were finding it hard to understand the caloric values of foods
consumed, thus making it difficult to adhere to the diet. Thus, it is necessary to develop an easy and
more effective method to support these patients. Firstly, we designed the ‘six-point formula’ to help
patients master LCD and LFD. We then let them record details of their diets and hand over to us the
task of calculating the caloric values of foods. Based on this, we explored the effect of two DM diets
(LCD and LFD) on hyperglycemia.
2.1. Subjects
Participants with T2DM were recruited from the community and the First Affiliated Hospital of
Soochow University. The inclusion criteria were the following: Patients older than 18 years, had been
diagnosed with T2DM, had no change in oral antidiabetic drugs or insulin in half a month before the
intervention, were able to communicate, had volunteered to participate in this study, and are able to
provide informed consent. Those excluded were patients who ate nuts regularly (≥4 day/week ) [15];
were allergic to food, especially nuts; had difficulty in chewing nuts (such as those with few teeth);
received other dietary interventions or had severe conditions including indigestion, heart failure,
renal failure, malignant tumours, severe cerebrovascular disease, ketosis, digestive dysfunction, liver
dysfunction or severe gallbladder and pancreatic diseases; and those whose fasting blood glucose
(FBG) were more than 16.7 mmol/L [16] during the interventions.
2.2. Study Design

This study is a prospective, single-blind randomized controlled trial (RCT) performed between
December 2015 to December 2016. The recruited patients were randomly allocated to receive either
LCD or LFD using a table of random numbers. Before the intervention, all subjects underwent
a one-week [17] washout period to diminish the effect of background diets on the study. The patients
were blinded when assigned to groups. This study followed the Declaration of Helsinki and the
Guidelines for Good Clinical Practice and was approved by the ethics committee of the First Affiliated
Hospital of Soochow University (No. 2015106). All enrolled patients signed a consent form.
2.3. Sample Size Calculation

Evidence from the literature showed that changes in the HbA1c level for six months were 0.6 ± 0.5%
in the LCD group and 0.2 ± 0.5% in the calorie-restricted group [13]. Therefore, we calculated 25 patients
for each group, with α = 0.05 and power = 0.80. In view of the sample loss of 10%, the number for each
group was 28. Finally, we recruited 28 patients for each group in the study.
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Nutrients 2018, 10, 661
2.4. Biochemical Parameters and Analyses

Glycated hemoglobin provides an estimate of glycemic control for the past three months and
is predictive of clinical outcomes [18]. HbA1c was measured at baseline and at the end of the third
month. Blood samples were obtained to measure HbA1c at the nursing School of Soochow University
and measured by high-performance liquid chromatography using Afinion AS100 Analyzer (Alere, Inc.,
Shanghai, China) in the molecular laboratory of the nursing school of Soochow University. Fasting
blood glucose (FBG) and postprandial 2-h blood glucose levels were measured by collecting the
peripheral blood from fingers using rapid glycaemic apparatus by patients once a week at home.
Fasting blood samples were also collected for various biochemical assays, including total
cholesterol (TC), performed as per the experimental protocol in hospitals.
Hypoglycemic episodes in this study were determined by the self-reported hypoglycemic
symptoms of patients with or without a measured plasma glucose concentration <70 mg/dL
(3.9 mmol/L) or only a measured plasma glucose concentration <70 mg/dL (3.9 mmol/L). Therefore,
all episodes of abnormal low plasma glucose concentration that exposed the individual to potential
harm and other clinical incidents, including severe hypoglycemia, documented symptomatic
hypoglycemia, asymptomatic hypoglycemia, probable symptomatic hypoglycemia and relative
hypoglycemia referred to the self-reported hypoglycemic symptoms of patients without a measured
plasma glucose concentration <70 mg/dL (3.9 mmol/L), were considered [19]. In this study,
the modification of hypoglycemic agents referred to change in the quantities of insulin dosages
the participants used at baseline and in the third month. Researchers collected data of modification of
hypoglycemic agents at every follow up.
2.5. Anthropometric Measurements

Body mass index (BMI) was calculated as weight (in kilograms) divided by height (in meters
squared). At baseline and in the third month, the weight and height of patients were measured by
a unified measuring device at the nursing school of Soochow University.
2.6. Diet Record

Patients maintained a diet record, including a detailed diet of any day over the weekend and two
working days. The composition of the diets was calculated using the Chinese CDC nutrition calculator
V2.63 software (Development team of Fei Hua nutrition software, Beijing, China) and the quantities
and distributions of energy from three macronutrients intake was determined. This also enabled an
understanding of the patients’ dietary adherence.
2.7. Intervention
Firstly, our team developed a preliminary dietary education handbook for patients with T2DM
based on evidence from literature and guidelines regarding T2DM dietary management [5,20].
Secondly, two endocrinologists, four diabetic nurse specialists, and one dietician reviewed and
modified the handbook. Finally, five T2DM inpatients of different ages and educational levels reviewed
the handbook to ensure that patients with T2DM understood it and that it could help improve their
dietary adherence. The major content of the handbook was a concise formula that included six points.
Detailed contents of the six-point formula are shown in Figure 1. Other educational contents about
foods included how to distinguish vegetables and staple food (such as potato and broad bean); ways
to cook food; and symptoms, prevention and treatment of hypoglycemia.
In the one-on-one education session, the researcher and the patients reviewed the handbook.
Using the LCD handbook, the researcher focused on instructing patients to restrict intake of staple
food/meal (1 Liang) per day in the LCD group. The reduced staple food/meal was replaced by
consuming 60 g/day nuts for males and 50 g/day for females, respectively. Nuts were uniformly
purchased, weighed, vacuum-packed, and distributed every two weeks.
65
Nutrients 2018, 10, 661
For patients in the LFD group, we provided participants with a handbook about LFD, and instructed
them on a pithy formula of six points.
Follow up was conducted once a week in the first month of the intervention and once every two
weeks in the second and third months. The duration of follow up was about 10 min. The main focus
of the follow-up was to review the patients’ compliance to the diet program and to support them to
adhere to it (in patients with poor compliance). It also involved collecting data of the modification
of hypoglycemic agents and the occurrence of hypoglycemia. If a patient’s diet did not meet the
requirements of the dietary program in the intervention period, they were excluded from the study.
Figure 1. The detailed contents of the six-point formula of two groups. Notes: 1 jin = 10 liang = 500 g,
Chinese conventional units of weight. Staple food/meal refers to foods rich in carbohydrates, mainly
three kinds of steamed bread, noodles and rice in China. LFD: Low-fat diet; LCD: Low-carbohydrate diet.

Statistical analyses were performed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA).
For continuous variables, the results were described as the mean ± standard deviation (SD) and
comparisons were performed using Independent Samples t-test, paired samples t-test or the Wilcoxon
rank-sum test. For categorical variables, the results were presented as frequency (percentages);
comparisons between groups were made using the Chi-squared test or Fisher’s exact test. The trends
in the FBG and postprandial 2 h blood glucose in two groups during the intervention were described
by the fold line diagram. Intention-To-Treat (ITT) of HbA1c was performed to ensure the reliability of
research results. A p value of < 0.05 was considered statistically significant.
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Nutrients 2018, 10, 661
3. Results
3.1. Study Participants

On the basis of inclusion and exclusion criteria, 56 T2DM participants were recruited and
randomly allocated to the LCD group (n = 28) and the LFD group (n = 28). Four participants in
the LCD group and three participants in the LFD group withdrew from the study. In the LCD group,
two participants didn’t like nuts, one showed poor adherence (<4 day/week, and one was lost during
follow-up. In the LFD group, two showed poor adherence to the diet program (<4 day/week) and
one was lost during follow-up. Finally, the data of 24 in the LCD group and 25 in the LFD group were
analyzed (Figure 2). The mean age of patients were (63.94 ± 10.79) years and 26 (53.1%) were men.
The general characteristics of the enrolled participants in each group are shown in Table 1 There were
no statistically significant differences in any of the parameters between the two groups (p > 0.05).
Figure 2. Flow diagram of the patients.
Table 1. Baseline characteristics.
LCD (n = 24) LFD (n = 25)

Variables t/χ2 p
x ± SD/n (%) x ± SD/n (%)
Demographic data
Age, years 66.79 ± 9.12 61.20 ± 11.71 1.860 a NS
Gender, Male 13 (54.2) 13 (52.0) 0.023 b NS
Married 23 (95.8) 22 (88)
Marital Status Unmarried 0 (0) 1 (4.0) 1.728 c NS
Widowhood 1 (4.2) 2 (8.0)
Education level, years 9.63 ± 4.10 8.36 ± 3.12 1.219 a NS
67
Nutrients 2018, 10, 661
Table 1. Cont.
LCD (n = 24) LFD (n = 25)

Variables t/χ2 p
x ± SD/n (%) x ± SD/n (%)
On the job 4 (16.7) 10 (40.0)
Occupation status 3.267 b NS
Retirement 20 (83.3) 15 (60.0)
Living by oneself 1 (4.2) 4 (16.0)
Living with spouse 21 (87.4) 19 (76.0)
Residential status 3.733 c NS
Living with children 1 (4.2) 2 (8.0)
Living with mother 1 (4.2) 0 (0)
Medical insurance, No 1 (4.2) 1 (4.0) 0.001 b NS
Value 19 (79.2) 14 (56.0)
Family Support 2.988 b NS
Ordinary 5 (20.8) 11 (44.0)
Never exercise 1 (4.2) 2 (8.0)
Exercise Never regular exercise 11 (45.8) 12 (48.0) 0.400 c NS
Regular exercise 12 (50.0) 11 (44.0)
Clinical data
Smoking, yes 2 (8.3) 5 (20.0) 1.361 d NS
SBP, mmHg 131.42 ± 10.89 130.84 ± 14.83 0.155 a NS
DBP, mmHg 77.54 ± 10.48 76.40 ± 10.43 0.382 a NS
Family history of diabetes, yes 12 (50.0) 9 (36.0) 0.980 b NS
Diabetes duration, years 12.79 ± 6.49 9.10 ± 6.52 1.985 a NS
Oral antilipemic agents, yes 8 (33.3) 11 (44.0) 0.587 a NS
Oral antidiabetic drugs or/and insulin 22 (91.7) 22 (88.0) -d NS
Complications, yes 9 (37.5) 5 (20.0) 1.838 b NS
Accompanying diseases, yes 17 (70.8) 19 (76.0) 0.168 b NS
p value for comparison between treatments diets by Independent Samples t-test or Chi-square test. a t-test;
b Chi-square test; c Likelihood Ratio; d Fisher’s Exact Test. NS: Differences are not significant; SBP: Systolic blood
pressure; DBP: Diastolic blood pressure.
3.2. Dietary Adherence
3.2.1. Comparison of Dietary Adherence

Dietary adherence was assessed mainly from two aspects: the days of adherence to the dietary
program per week and macro-nutrient allocation and their quantities. The Wilcoxon rank-sum test was
performed to compare dietary compliance in the two groups (LCD versus LFD). The result showed
that there was no difference in self-reported dietary compliance per week (p > 0.05, Table 2).
Table 2. Comparison of dietary adherence between the two groups.
LCD (n = 24) LFD (n = 25) Z p

4 d/W 3 (12.5) 7 (28.0) 4.449 NS
5~6 d/W 7 (29.2) 10 (40.0)
7 d/W 14 (58.3) 8 (32.0)
p value for comparison by Wilcoxon rank-sum test. Z: Wilcoxon rank-sum test; NS: Differences are not significant.
3.2.2. Proportions of Calories from Three Macronutrients the Patients Consumed

Prior to the intervention, the total energy and the proportions of calories from the three major
nutrients were not significantly different between the two groups (LCD versus LFD). After the
intervention, compared to the LFD group, the calories from carbohydrates decreased, while those from
fat significantly increased in the LCD group (p < 0.05). In addition, the percentage of calories from
68
Nutrients 2018, 10, 661
carbohydrates (39%) met the standard of LCD (<45%). The 26% of calories from fat met the standard of
LFD, while the calories from protein were almost similar in the two groups (p > 0.05, Table 3) (Figure 3).
Table 3. Comparison of the calories from three macronutrients consumed by the patients.
Variables LCD (n = 24) LFD (n = 25) T p

Total calorie intake/day 1796.0 ± 186.6 1768.8 ± 138.7 0.421 NS
Carbohydrate-calorie (Kcal) 948.8 ± 130.9 922.5 ± 145.1 0.485 NS
Baseline
Fat-calorie (Kcal) 538.9 ± 92.4 542.0 ± 94.8 −0.084 NS
Protein-calorie (Kcal) 306.6 ± 56.7 303.3 ± 41.8 0.166 NS
Total calorie intake/day 1808.0 ± 190.7 1731.5 ± 109.6 1.257 NS
Carbohydrate-calorie (Kcal) 695.2 ± 106.6 970.2 ± 101.1 −6.747 <0.001 **
3rd month
Fat-calorie (Kcal) 763.1 ± 99.1 442.8 ± 52.0 10.320 <0.001 **
Protein-calorie (Kcal) 350.3 ± 64.4 317.4 ± 52.0 1.433 NS
p value for comparison by Independent Samples t-test. ** p < 0.01 NS: Differences are not significant.

Figure 3. The percentage of the calories from carbohydrates (39%) met the standard of LCD (<45%) in
the LCD group, while the 26% calories from fat met the standard of LFD. LCD: Low-carbohydrate diet;
LFD: Low-fat diet
3.3. Effect of LCD on Glycemic Control
Glycated Hemoglobin
Compared to the baseline, HbA1c levels in both the LCD group and LFD group decreased
significantly (0.63 ± 1.18% and 0.31 ± 0.70%), respectively. At the baseline, HbA1c levels were
not significantly different between the two groups. However, after the intervention, HbA1c levels
in the LCD group decreased significantly (p < 0.05, Table 4), when compared to the LFD group.
The Intention-To-Treat (ITT) in relation to HbA1c levels was performed to ensure the stability of the
above results. The ITT results were found to be in agreement with the earlier findings (Table 5).
Table 4. Comparison of glycated hemoglobin (%) between the two groups.
Study Period LCD (n = 24) LFD (n = 25) t p

Baseline 7.43 ± 1.39 7.79 ± 1.20 −0.971 NS
3rd month 6.80 ± 0.83 7.48 ± 1.15 −2.350 0.023 *
MD 0.63 ± 1.18 0.31 ± 0.70 - -
t 2.601 2.213 - -
p 0.016 * 0.037 * - -
p value for comparison by Independent Samples t-test or paired samples t-test. * p < 0.05. NS: Differences
are not significant.
69
Nutrients 2018, 10, 661
Table 5. Comparison of glycated hemoglobin (%) between the two groups in ITT.
Study Period LCD (n = 28) LFD (n = 28) T p

Baseline 7.39 ± 1.29 8.16 ± 1.59 −1.994 NS
3rd month 6.85 ± 0.79 7.89 ± 1.63 −3.017 0.004 **
MD 0.54 ± 1.12 0.28 ± 0.67 - -
t 2.556 2.194 - -
p 0.017 * 0.037 * - -
p value for comparison by Independent Samples t-test or paired samples t-test. * p < 0.05; ** p < 0.01;
ITT: Intention-To-Treat; NS: Differences are not significant.
3.4. Fasting Blood Glucose
3.4.1. Changing Trends of Fasting Blood Glucose

The changing trends of the FBG in the two groups during the intervention are described by
the fold line diagram (Figure 4). The results showed that the change of FBG in the LCD group
decreased significantly for the first four weeks and then decreased steadily after the fourth week.
In contrast, the FBG in the LFD group demonstrated dynamic fluctuation, although it was lower than
the baseline value.
Figure 4. The changing trends of the FBG in the LCD and LFD Groups. FBG: fasting blood glucoses
3.4.2. Comparison of Fasting Blood Glucose levels

Compared to the baseline, FBG levels of the two groups significantly improved (p < 0.01). But the
differences between the two groups with respect to FBG was not statistically significant (p > 0.05)
(Table 6).
Table 6. Comparison of fasting blood glucose (mmol/L) between the two groups.

Baseline 8.28 ± 1.64 7.55 ± 0.75 1.469 NS
3rd month 6.87 ± 0.65 6.70 ± 0.57 0.793 NS
t 4.873 3.889 - -
p <0.001 ** 0.003 ** - -
p value for comparison by Independent Samples t-test or paired samples t-test. ** p < 0.01. NS: Differences are
not significant.
3.5. Postprandial Two-Hour Blood Glucose
3.5.1. Trends in Postprandial Two-Hour Blood Glucose

The changing trends of the postprandial 2-h blood glucose of the two groups during the
intervention are described by the fold line diagram (Figure 5.). Both groups showed fluctuation
in this indicator.
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Nutrients 2018, 10, 661
Figure 5. The changing trends of the postprandial 2-h blood glucose in the LCD and LFD Groups.
3.5.2. Comparison of Postprandial Two-Hour Blood Glucose

Compared to the baseline, the postprandial 2-h blood glucose in the two groups improved
significantly (p < 0.01). However, there was no significant difference between the two groups (p > 0.05)
(Table 7).
Table 7. Comparison of postprandial 2-h blood glucose (mmol/L) in the groups.

Baseline 10.67 ± 2.33 10.08 ± 1.29 0.818 NS
3rd month 9.00 ± 1.80 8.58 ± 0.80 0.761 NS
t 4.690 3.786 - -
p <0.001 ** 0.003 ** -
p value for comparison between treatments diets by Independent Samples t-test or paired samples t-test. ** p < 0.01.
NS: Differences are not significant.
3.6. Effect of LCD on Other Metabolic and Anthropometric Indicators

Compared to the baseline, body mass index (BMI) and total cholesterol (TC) in the LCD group
improved significantly in the third month (p < 0.05). However, there were no similar results in the LFD
group. After the intervention, the metabolic indicators were not significantly different between the
two groups (Table 8).
Table 8. Comparison of other metabolic indicators between the two groups.
Variables Study Period LCD (n = 24) LFD (n = 25) t p

Baseline 24.29 ± 3.36 24.62 ± 5.17 −0.261 NS
BMI 3rd month 23.52 ± 2.70 23.47 ± 3.11 0.060 NS
(Kg/m2 ) t 2.756 1.235 - -
p 0.011 * NS - -
Baseline 4.85 ± 0.87 4.55 ± 1.04 1.101 NS
TC 3rd month 4.49 ± 0.86 4.63 ± 0.99 −0.521 NS
(mmol/L) t 2.540 −0.363 - -
p 0.018 * NS - -
p value for comparison by Independent Samples t-test or paired samples t-test. * p < 0.05; BMI: Body mass index;
TC: total cholesterol. NS: Differences are not significant.
71
Nutrients 2018, 10, 661
3.7. Hypoglycemia and Medication Changes
3.7.1. Frequency of Hypoglycemia

The frequencies of hypoglycemia during the three-month period in the two groups showed no
significant differences (p > 0.05), before and after the intervention. In addition, there were no significant
differences (p > 0.05) between the two groups before and after the interventions (Table 9)
Table 9. Comparison of the frequencies of hypoglycemia between the two groups.
Time LCD (n = 24) LFD (n = 25) t P

Baseline 0.21 ± 0.59 0.52 ± 0.77 −1.596 NS
3rd month 0.04 ± 0.20 0.36 ± 0.86 −1.798 NS
t 1.282 0.778 - -
p NS NS - -
p value for comparison by Independent Samples t-test or paired samples t-test. NS: Differences are not significant.
3.7.2. The Dosages of Insulin Used

When compared to the baseline, the dosage of insulin used in the two groups decreased
significantly after the intervention (p < 0.05, Table 10), although there was no significant difference
between the two groups (p > 0.05).
Table 10. Comparison of insulin dose (insulin unit, IU) between the two groups.
Times LCD (n = 7) LFD (n = 13) t P

Baseline 31.14 ± 16.38 29.00 ± 12.27 0.332 NS
3rd month 28.29 ± 13.74 26.62 ± 11.20 0.294 NS
t 2.765 3.023 - -
p 0.033 * 0.011 * - -
p value for comparison by paired samples t-test. * p < 0.05. NS: Differences are not significant.
3.7.3. The Changes of Other Antidiabetic Drugs

There was no significant difference between the two groups in the third month (p > 0.05, Table 11).
Table 11. Comparison of other antidiabetic drugs between the two groups.
LCD (n = 23) LFD (n = 11) χ2 p

No change 20 (87.0%) 11 (100%)
Reduction 2 (8.7%) 0 (0) 2.482 NS
Addition 1 (4.3%) 0 (0)
p value for comparison between treatments diets by Chi-square test. NS: Differences are not significant.
4. Discussion
The use of LCD in human nutrition and health is a dietary strategy that ensures that carbohydrate
intake is restricted. However, in a Chinese dietary plan, most staple foods have high glycemic
index [20,21]. Therefore, it would seem that LCD may not be accepted easily among Chinese patients
with diabetes mellitus (DM). In consideration, we initially designed the ‘six-point formula’ to help
patients improve dietary adherence. We found that the participants showed good adherence to the
intervention, and no significant difference with respect to dietary adherence between two groups (LCD
versus LFD) was observed. The proportions of energy provided by the three macronutrients met the
requirements of LCD and LFD. It was indicated that the ‘six-point formula’ of the DM diet was feasible
for Chinese T2DM patients.
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Nutrients 2018, 10, 661
4.1. Effect of LCD on Glycemic Control

High levels of HbA1c, FBG, and postprandial 2h blood glucose levels are some of the most difficult
challenges faced by patients with T2DM and these parameters could be used as the main indicators to
establish glycemic control [5].
HbA1c levels can reflect blood glucose levels in 2~3 months before blood extraction and long-term
glycemic control of patients [5]. The result of this study showed that HbA1c levels in LCD (8.5%)
decreased significantly (p < 0.05) compared to that in LFD (4%). The reason might be due to the
decreased level of high glycemic index foods, the total amount of foods rich in carbohydrates, and the
increased intake of nuts, which could help improve hyperglycemia and insulin sensitivity [22–24].
Yamada et al. [13] showed that HbA1c levels were significantly decreased by as much as 7.9% in the
LCD group and by only 2.6% in the calorie-restricted group. Mayer et al. [25] also found LCD led
to a relative improvement in HbA1c than LFD. However, some studies have shown that LFD could
decrease HbA1c by 0.8–2.08% [7,8]. These values were less than the result of our study, which might
be due to the effect of the ‘six-point formula’ that was simple and easy to remember, helped patients
master the methods of the DM diet better, and improved dietary compliance and hyperglycemia.
Fasting blood glucose and postprandial 2-h blood glucose are important indicators for the
diagnosis and monitoring of DM [5]. The fold line diagram in this study showed that FBG significantly
decreased during the first four weeks in the two groups. While FBG steadily decreased in the LCD
group, there was dynamic fluctuation after the initial first month in the LFD group. A reason for the
same might be that the patients in the two groups showed keen interest in the ‘six-point formula’ at
the beginning of the intervention, which helped improve their dietary adherence and promote FBG
control. In addition, nuts could stabilize blood glucose levels [23,26,27], which may have contributed
to the steady decrease of FBG in the LCD group. Postprandial 2h blood glucose obviously decreased
in the LCD group, which might have resulted from its relationship to limited carbohydrates [20,22].
4.2. Other Metabolic Indicators

Nuts are high-fat diets with high-energy levels, but they do not increase the weight of patients [27]
because they increase a feeling of satiety and lead to a strong dietary compensation effect [28].
In addition, energy absorption efficiency of the nuts is low and the total energy does not increase [28].
This study further confirmed that BMI in the LCD group decreased. The result is in agreement with
the results of Li et al. [23] and Barbour et al. [29].
Diabetes is significantly related to dyslipidemia [5]. While we pay attention to blood glucose
levels, it is also necessary to regulate blood lipids. Lovejoy et al. [30] found that TC level in diets
enriched in almonds was lower by 21%. Our study found that the TC level decreased significant
by 7.4% in the LCD group, which might be related to the effect of some ingredients of the nuts
consumed [27].
4.3. Hypoglycemia and Medication Changes

We found that the insulin dose used by patients in the LCD group during the intervention period
decreased, consistent with a study by Westman et al. [31], which found that patients could reduce or
terminate the use of hypoglycemic agents by controlling the intake of carbohydrates. But there were
no significant differences between group comparisons.
In this study, hypoglycemia is used as a safety indicator. Although there was no statistical change
in the frequency of hypoglycemia in within-group comparison and no difference in between-group
comparison, the frequencies of hypoglycemia were reduced in the two groups.
5. Limitation
There are some limitations to the study. Firstly, the method used to evaluate the energy intake
of food may not have been robust enough. At the baseline, we obtained data of caloric intake from
73
Nutrients 2018, 10, 661
patients’ memories, which meant that it was probably underestimated. Secondly, measurement
differences might exist in FBG and postprandial 2-h blood glucose levels, which were measured at
home by the patients themselves using different blood glucose meters. Thirdly, the prolonged effect of
LCD on the prognosis of DM was not observed due to short follow-up time. Finally, a control group
without a treatment was not considered in the study design.
6. Conclusions
LCD can improve blood glucose more than LFD in Chinese patients with T2DM. It can also
regulate blood lipids, reduce BMI, and decrease insulin doses in patients with T2DM. In addition,
the six-point formula is feasible, easily operable, and is a practical educational diet for Chinese patients
with T2DM.
Author Contributions: L.-L.W. and Q.W. collected and analyzed data and wrote the initial draft, which was
revised by X.-H.W., Y.-H.H., Y.H., O.O., Q.J., Y.-Y.H. All authors participated in research design and quality control.
L.-L.W. and Q.W. contributed equally to this study. X.-H.W. was the corresponding author.
Acknowledgments: We thank the patients with T2DM who participated in the study. Thanks also to Huijuan Zhou,
Xiaoyan Zhang and Li Wang, who provided us with study sites to ensure that the study was conducted. This study
was supported by Suzhou Science and Technology Project, China (Grant number SYS201513).
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nutrients
Article
SunGold Kiwifruit Supplementation of Individuals
with Prediabetes Alters Gut Microbiota and Improves
Vitamin C Status, Anthropometric and
Clinical Markers
Renée Wilson 1 , Jinny Willis 2 , Richard B. Gearry 1,3 , Alan Hughes 4 , Blair Lawley 4 ,
Paula Skidmore 5 , Chris Frampton 1 , Elizabeth Fleming 5 , Angie Anderson 5 , Lizzie Jones 5 ,
Gerald W. Tannock 3,4,6 and Anitra C. Carr 7, *
1 Department of Medicine, University of Otago, Christchurch 8140, New Zealand;
[email protected] (R.W.); [email protected] (R.B.G.);
[email protected] (C.F.)
2 New Zealand Nursing Organisation, Christchurch 8140, New Zealand; [email protected]
3 Microbiome Otago, University of Otago, Dunedin 9054, New Zealand; [email protected]
4 Department of Microbiology and Immunology, University of Otago, Dunedin 9054, New Zealand;
[email protected] (A.H.); [email protected] (B.L.)
5 Department of Human Nutrition, University of Otago, Dunedin 9054, New Zealand;
[email protected] (P.S.); liz.fl[email protected] (E.F.); [email protected] (A.A.);
[email protected] (L.J.)
6 Riddet Centre of Research Excellence, Massey University, Palmerston North 4442, New Zealand
7 Department of Pathology & Biomedical Science, University of Otago, Christchurch 8140, New Zealand
Received: 11 June 2018; Accepted: 2 July 2018; Published: 12 July 2018
Abstract: Kiwifruit are a nutrient dense food and an excellent source of vitamin C. Supplementation
of the diet with kiwifruit enhances plasma vitamin C status and epidemiological studies have shown
an association between vitamin C status and reduced insulin resistance and improved blood glucose
control. In vitro experiments suggest that eating kiwifruit might induce changes to microbiota
composition and function; however, human studies to confirm these findings are lacking. The aim of
this study was to investigate the effect of consuming two SunGold kiwifruit per day over 12 weeks
on vitamin C status, clinical and anthropometric measures and faecal microbiota composition in
people with prediabetes. This pilot intervention trial compared baseline measurements with those
following the intervention. Participants completed a physical activity questionnaire and a three-day
estimated food diary at baseline and on completion of the trial. Venous blood samples were collected
at each study visit (baseline, 6, 12 weeks) for determination of glycaemic indices, plasma vitamin
C concentrations, hormones, lipid profiles and high-sensitivity C-reactive protein. Participants
provided a faecal sample at each study visit. DNA was extracted from the faecal samples and
a region of the 16S ribosomal RNA gene was amplified and sequenced to determine faecal microbiota
composition. When week 12 measures were compared to baseline, results showed a significant
increase in plasma vitamin C (14 μmol/L, p < 0.001). There was a significant reduction in both
diastolic (4 mmHg, p = 0.029) and systolic (6 mmHg, p = 0.003) blood pressure and a significant
reduction in waist circumference (3.1 cm, p = 0.001) and waist-to-hip ratio (0.01, p = 0.032). Results also
showed a decrease in HbA1c (1 mmol/mol, p = 0.005) and an increase in fasting glucose (0.1 mmol/L,
p = 0.046), however, these changes were small and were not clinically significant. Analysis of faecal
microbiota composition showed an increase in the relative abundance of as yet uncultivated and
therefore uncharacterised members of the bacterial family Coriobacteriaceae. Novel bacteriological
investigations of Coriobacteriaceae are required to explain their functional relationship to kiwifruit
polysaccharides and polyphenols.

Nutrients 2018, 10, 895
Keywords: vitamin C; blood pressure; waist circumference; glucose; glycaemic control; HbA1c;
kiwifruit; gut microbiota; Coriobacteriaceae
1. Introduction
Diabetes is one of the largest global health crises, affecting 415 million adults worldwide, with type
2 diabetes mellitus (T2DM) accounting for at least 90% of all cases of diabetes [1]. People with
diabetes are at a higher risk of developing a number of disabling and life-threatening health
conditions [1]. Prediabetes reflects a stage between normal glucose tolerance (NGT) and T2DM.
Those with prediabetes are at high risk of progressing to T2DM, although this is not inevitable [2].
In routine practice, prediabetes is characterized by an increase in HbA1c. Results from the 2008/2009
New Zealand Adult Nutrition Survey (NZANS) provided data on the prevalence of diabetes and
prediabetes using the American Diabetes Association (ADA) criteria and found that the prevalence of
diabetes and prediabetes in New Zealand was 7.0% and 25.5% respectively [3]. The alarmingly high
prevalence of people with prediabetes necessitates further research to explore modifiable causative
factors for prediabetes.
Hyperglycaemia (elevated blood glucose concentrations) and insulin resistance are implicated in
the pathogenesis of micro- and macrovascular complications [4]. Maintaining normal blood glucose
levels through diet is one of the main goals in the management of a glucose metabolism disorder
(diabetes or prediabetes) [5]. Higher plasma vitamin C is associated with reduced insulin resistance and
improved blood glucose control [6–10]. Since hyperglycaemia is associated with increased oxidative
stress, a role for antioxidants such as vitamin C in the prevention of T2DM and/or the reduction
of complications is a reasonable proposition. However, there have been mixed findings reported in
randomised controlled trials (RCTs) of supplementation with vitamin C on glycaemic control and
insulin sensitivity [11–13]. A recent meta-analysis of 15 RCTs investigating vitamin C supplementation
and insulin resistance and biomarkers of glycaemic control (fasting glucose, HbA1c) found that doses
of ≥200 mg/day vitamin C significantly reduced glucose concentrations in patients with T2DM,
particularly if the intervention was for more than 30 days and in older individuals [14]. Furthermore,
a recent 12 month RCT found that treating those with T2DM with both metformin and vitamin C was
more effective at reducing HbA1c and risk factors for diabetes-related long-term complications than
treating with metformin alone [15].
SunGold kiwifruit are one of the best dietary sources of vitamin C (160 mg/100 g) among fruit and
vegetables [16]. Regular consumption of two SunGold kiwifruit has been shown to significantly
increase the plasma vitamin C levels in men with inadequate levels of plasma vitamin C [17].
Virtually all fruits represent a source of sugars and therefore moderate consumption is recommended
in people with T2DM. However, there are differing amounts of sugar and other nutrients in various
fruit and portion sizes vary markedly. Kiwifruit, eaten as a whole fruit, have a low glycaemic impact
and are, therefore, thought to be a suitable choice for those with prediabetes and T2DM [18].
In addition to having a positive effect on glycaemic control, gold kiwifruit contain dietary fibre
(1.4 g/100 g in raw Zespri SunGold kiwifruit) [16] and polyphenols which resist digestion by human
enzymes and are degraded by bacteria in the digestive tract, stimulating the growth or activity of
certain bacteria in the colon. The fibre in kiwifruit comprises both soluble (e.g., pectin) and insoluble
(e.g., hemicelluloses and celluloses) components which make up cell walls [19].
T2DM has been reported to be associated with an alteration in the usual balance of bowel
bacterial species, described as a bacterial dysbiosis [20]. Differences in the gut microbiota have been
demonstrated between individuals with T2DM and healthy individuals in previous research [21,22].
Further, the finding of differences in gut microbiota between healthy individuals, individuals with
prediabetes and those with T2DM suggests a potential association between gut ecology and the
progression from normal glucose tolerance to T2DM [23]. A logical extension is the hypothesis that
77
Nutrients 2018, 10, 895
manipulation, by dietary change, of the relative abundances of particular bacterial taxa within the gut
microbiota could play a role in managing T2DM [24].
On the basis of in vitro experiments, SunGold Kiwifruit consumption has been speculated to
affect the composition and functioning of the bowel microbiota [25]. However, the outcomes of this
study need to be confirmed in human intervention studies. Therefore, the aim of the present study
was to determine whether gold kiwifruit consumption by a prediabetic cohort altered gut microbiota
composition, increased plasma vitamin C concentrations and improved glycaemic control or prevented
further deterioration of glucose intolerance.
2.1. Study Participants

The study was approved by the Southern Health and Disability Ethics Committee (consent
no. 16/STH/87) and was included in the Australian New Zealand Clinical Trials Registry
(ACTRN12616000858493). Written informed consent was obtained from all participants. Individuals
≥18 years meeting the inclusion criteria detailed below were recruited by a range of methods including:
advertisements in local newspapers, flyers in general practice and other primary care health settings,
e-mailing staff at local large businesses including the Canterbury District Health Board (CDHB),
participants from previous studies were contacted, study information was posted to those who
had attended prediabetes education classes and flyers placed at local businesses in Christchurch,
New Zealand.
A total of 41 individuals underwent a screening questionnaire and venous blood test to measure
HbA1c to ascertain eligibility for the study. Twenty-six participants were enrolled and 24 participants
completed the study. Four participants (one of whom did not complete the trial) were excluded from
the gut microbiota analyses due to starting a course of antibiotics during the trial. One patient left the
study due to a family bereavement. Another participant was admitted to hospital for a pre-existing
condition and had to conclude involvement in the study.
2.1.1. Inclusion Criteria

Participants (≥18 years) who met the ADA diagnostic criteria for prediabetes (HbA1c result of
39–46 mmol/mol) at baseline were recruited. HbA1c is routinely used as the diagnostic test for diabetes
in New Zealand [26]. The red blood cell has a 90–120 day lifespan and during this time haemoglobin is
glycated in proportion to the mean exposure to glucose [26]. Accordingly, a three-month intervention
period was chosen for this trial.
2.1.2. Exclusion Criteria

Individuals unable to give informed consent, those with an HbA1c outside the diagnostic range for
prediabetes (HbA1c result of 39–46 mmol/mol), those with a previous diagnosis of diabetes, or those on
diabetes medications such as Metformin. In addition, individuals who had taken antibiotics in the last
month, those with a medical history of significant gastrointestinal disease (for example, inflammatory
bowel disease), previous bowel resection, those with a known kiwifruit allergy, women who were
pregnant, breastfeeding or planning a pregnancy and those planning to travel overseas in the three
months post selection (trial period) were also excluded.
2.2. Study Design

This was a pilot intervention trial where baseline measurements were used as the control measures
for comparison. Participants were screened with a questionnaire and venous blood test to measure
HbA1c. If eligible they started a lead in phase where they were asked to not eat any kiwifruit (green or
gold) for seven days. During this time, they also completed an estimated food diary by writing down
78
Nutrients 2018, 10, 895
everything they ate or drank over three non-consecutive days (two weekdays and one weekend day)
to analyse their usual dietary intake.
After the lead-in phase, Zespri SunGold kiwifruit (Gold3, Actinidia chinensis) were delivered to
participants weekly. Participants were provided with twice as many fruit as were required and were
advised that extra fruit could be shared with family and friends but to ensure that they had enough
to last them until the next delivery. The participants were asked to store the fruit in a refrigerator
or cool place. The kiwifruit were selected to be of the same size and quality. The average weight of
one SunGold kiwifruit was 132 g (whole fruit) and 95 g for the flesh (28% accounts for the skin, [16]).
Participants were asked to consume the flesh of two SunGold kiwifruit every day for twelve weeks.
Strategies to avoid forgetting the fruit were discussed such as eating them at the same time every day
and having two kiwifruit out on the bench every day so the participants knew whether or not they had
eaten them. Participants were asked not to eat any kiwifruit other that the two study fruit during the
12 weeks. As the study was a free-living situation, participants were asked to consume the fruit as part
of their usual diet. Participants were asked not to eat the skin and to eat the flesh raw and not crushed
or blended in a smoothie. Participants were asked to maintain their normal dietary and lifestyle habits
for the duration of the trial.
Following screening and the lead-in week there were three study visits; baseline, week six and
week twelve (Figure 1). Table 1 shows the information that was collected at each study visit.
Lead-In Mid-Point Study

Baseline
Screening Phase Completion
(week 0) (week 6)
(1 week) (week 12)
Figure 1. Study timeline.
Table 1. Information and samples collected at each study visit.
Week 0 Week 6 Week 12

Lead-In Phase
(Baseline) (Study Mid-Point) (Study Completion)
Demography Changes to
Medical history Changes to medications and
Medications medications and supplements
Questionnaires Food diary
Supplements supplements Anthropometry
Anthropometry Anthropometry Physical activity
Physical activity Food diary
Fasting glucose Fasting glucose
vitamin C vitamin C
HbA1c HbA1c
Blood Tests Fasting glucose
Lipids Lipids
Hormones Hormones
hs-CRP hs-CRP
2.3. Anthropometric Measures
2.3.1. Weight (kg)

Participants were asked to remove their footwear and heavy outer clothing such as jackets and
were weighed to the nearest 0.1 kg on calibrated Tanita scales (Model BWB-800A, Tanita Corporation,
Tokyo, Japan).
2.3.2. Height (m)

Measured once at baseline to the nearest mm using calibrated height measures.
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2.3.3. BMI (kg/m2 )

Calculated by weight in kilograms divided by height in metres squared.
2.3.4. Waist Circumference (cm)

The WHO STEPwise Approach to Surveillance protocol for measuring waist circumference was
used. The measurement was made at the approximate midpoint between the lower margin of the last
palpable rib and the top of the iliac crest [27]. The tightness of the tape was controlled by using a Gulick
II Measuring tape (Model 67020, Country Technology Inc., Gays Mills, WI, USA). Three measurements
were taken at each study visit. The measurements for each participant were averaged. The coefficient
of variation associated with the measurement error for waist circumference was 0.65%.
2.3.5. Hip Circumference (cm)

Measured to the nearest mm around the widest portion of the buttocks with the tape parallel to
the floor using a Gulick II Measuring tape, as described above. Three measurements were taken at
each study visit. The measurements for each participant were averaged. The coefficient of variation
associated with the measurement error for hip circumference was 1.86%.
2.3.6. Waist-To-Hip Ratio

Calculated by dividing the waist circumference by the hip measurement.
2.3.7. Fat Mass (%)

Measured using the BIA 450 Bioimpedance Analyser (Biodynamics Corporation, Seattle, WI, USA).
Patient assessments were conducted using a connection between the individual’s wrist and ankle and
the analyser, using standard ECG sensor pad electrodes (CONMED Corporation, Utica, NY, USA).
One participant had a pacemaker so was excluded from this measure.
2.3.8. Blood Pressure (mmHg)

Measured using an automated blood pressure monitor (Bp TRU, BTM-300, Omron Healthcare Co.,
Ltd., Muko, Kyoto, Japan). Three measurements were taken at each study visit. The measurements for
each participant were averaged. The coefficient of variation associated with the measurement error for
systolic and diastolic blood pressure were 2.99% and 5.16% respectively.
2.4. Blood Parameters

Venous blood samples were collected after a 12-hour fast and were analysed for fasting glucose
as an additional measure of glycaemic control. EDTA plasma was collected and immediately stored
on ice and centrifuged for 10 minutes at 1000× g (2500 rpm) at 4 ◦ C. The plasma was removed and
immediately frozen at −80 ◦ C for batch analysis of vitamin C.
2.4.1. Glucose
Fasting glucose was measured in blood collected in fluoride oxalate venoject tubes by standard
methods (Glucose Hexokinase Enzymatic Assay, Abbott c series analyser, Abbott Park, IL, USA) at
an IANZ laboratory. The coefficient of variation associated with the measurement of glucose in plasma
is 1.5% at 6.78 mmol/L [28].
2.4.2. Vitamin C
Frozen plasma samples were rapidly defrosted, acidified and stabilised with perchloric acid and
a metal chelator. The supernatants were then treated with a reducing agent prior to analysis to recover
any vitamin C that had become oxidised during handling or storage [29]. The samples were analysed by
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the gold standard method of high performance liquid chromatography with electrochemical detection
as described previously [17,29].
2.4.3. HbA1c
Determined in EDTA blood by standard methods (Bio-rad Variant HPLC, Bio-Rad, Hercules, CA, USA)
at an IANZ laboratory. The coefficient of variation associated with the measurement of HbA1c at the IANZ
is 0.66% at HbA1c of 38 mmol/mol [30].
2.4.4. Lipid Parameters

Total cholesterol, HDL-cholesterol, LDL-cholesterol and triglycerides were determined in lithium
heparin blood by standard methods (Abbott c series analyser, Abbott Park, IL, USA) at an IANZ laboratory.
2.4.5. hs-CRP
The inflammatory marker hs-CRP was measured using end-point nephelometry at an IANZ laboratory.
2.4.6. Hormones
EDTA plasma was collected and centrifuged for 10 minutes at 1000× g (2500 rpm) at 4 ◦ C. The plasma
was frozen at −80 ◦ C and stored for batched analyses. Ghrelin, leptin and adiponectin were determined by
the Christchurch Heart Institute, Department of Medicine, University of Otago, Christchurch.
Ghrelin was measured by an in-house RIA following extraction from plasma using Sep Pak C18
cartridges, as described previously [31]. The assay recognises the total circulating ghrelin (i.e., both
octanoyl and non-octanoyl forms). The cross reactivities of other peptides in the assay, including
vasointestinal peptide, prolactin, galanin, growth hormone releasing hormone, neuropeptide Y, brain
natriuretic peptide, atrial natriuretic peptide, endothelin-1 and angiotensin II were all less than 0.03%.
The RIA had a mean detection limit of 10.8 ± 0.8 pmol/L and mean ED50 of 136.2 ± 10.0 pmol/L over
23 consecutive assays.
Leptin and adiponectin were measured using commercial ELISA from BioVendor (Brno, Czech Republic),
Research and Diagnostic products (RD191001100 Human Leptin ELISA and RD191023100 Human
Adiponectin ELISA) according to the manufacturer’s instructions.
Insulin was measured using the Roche Cobas e411 method in an IANZ laboratory. After storage
at −80 ◦ C, thawed plasma was pre-treated using 25% polyethylene glycol to precipitate antibodies.
2.5. Faecal Microbiota Analysis

Participants provided a faecal sample at their study appointment, which they had collected at
home in a sterile collection bottle no more than 24 h prior to their study visit. The sample was either
stored in an insulated bag with an ice pack or in a home refrigerator. Samples were transported
in a coolie bag with an ice pack to their study appointment. All samples were processed within
24 h of the patient collecting their sample. The samples were processed in a microbiological sterile
hood into four aliquots of 0.5–1.0 g of faeces and stored in sterile 1.5 mL Eppendorf tubes at −80 ◦ C
for batched analyses. Faecal sample collection methodology was adapted from the International
Human Microbiome standard operating procedures and with the Human Microbiome Project
Methodology (www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/document.cgi?study_id=phs000228.v3.
p1&phd=3190#sec114).
2.5.1. DNA Extraction and Sequence Analysis

DNA was extracted from stool using the MoBio PowerSoil DNA Isolation kit according to the
Human Microbiome Project standard operating procedures (www.ncbi.nlm.nih.gov/projects/gap/cgi-
bin/document.cgi?study_id=phs000228.v3.p1&phd=3190#sec114). Genomic DNA was submitted to
Argonne National Laboratories for barcoded amplification of the V4 region of the bacterial 16S rRNA
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gene and sequencing on a MiSeq (Illumina, San Diego, CA, USA) instrument. DNA sequences were
processed using the QIIME v. 1.9.1 and vsearch v.2.0.2 suite of programs [32,33]. Genus level taxonomy
was obtained by filtering Operational Taxonomic Unit (OTU) tables containing taxonomic data
generated using the RDP classifier, extracting representative sequences and using BLAST to identify
genus level matches, where possible, within the NCBI database. Alpha- and beta-diversity analysis
of phylogenetic data compared the coverage and richness (number of phylotypes) and similarity or
difference in the composition of the stool microbiota between subjects and treatment groups.
2.5.2. Faecal Water Content

Water content of faeces reflects gut transit time. Shorter transit time means more water in faeces.
Shorter transit time means less time for bacterial replication and might favour species with shorter
doubling times. For each faecal sample, approximately 200 mg of faeces was placed in a pre-weighed
microfuge tube, the weight was recorded and the tube with cap open was placed in a 37 ◦ C incubator.
The tubes were dried until a constant dry weight was obtained and percentage water content was
then calculated.
2.6. Questionnaires
2.6.1. Demographic Information

Participants recorded their date of birth, sex, ethnicity and qualification. Information on medical
history, alcohol consumption and smoking status was also collected.
2.6.2. Medication and Supplement Use

At baseline participants were asked to record any medications (prescription or non-prescription)
they were currently taking, the dose, the number per day and the length of time they had been taking
the medication. They were also asked if they were taking any dietary supplements, how frequently
they took the supplement and when their last dose was. At weeks 6 and 12 they were asked if they
had stopped taking any medications/supplements or if they had started taking a new medication or
supplement and if so the details were recorded.
2.6.3. Dietary Intake

Participants completed a three day (two weekdays and one weekend day) estimated food diary
during their lead-in week and during their final week of consuming the SunGold kiwifruit (week
12). Participants’ daily dietary intake was calculated as an average over the three days. Participants
were asked to record everything they ate or drank over the three days; describing each item in detail,
including cooking details and any salt, sugar, spices and sauces they may have added before eating.
They were also asked to record the brand name of each food, drink or cooking ingredient and also if
possible to attach the wrappers of foods to provide the nutrition information. Tips were provided to
assist with estimation of portion sizes such as using household measures, for example, two rounded
teaspoons of sugar. A book with photos of commonly eaten foods of different portion sizes was also
provided. Participants were also encouraged to take photos of their meals in addition to completing
the food diary with estimated portions. Once completed the diary was reviewed during their study
appointment to add any missing information if necessary.
The food diaries were entered into Kaiculator (version 1.08d), a nutrient analysing programme
developed by the Department of Human Nutrition at the University of Otago, New Zealand. Kaiculator
uses the 2014 version of the New Zealand food composition database, NZ FOODfiles. The methodology
for entering the food diaries has been described for our previous observational study [34]. Average total
daily energy and fibre were calculated along with percent energy values for total fat, carbohydrate
and protein. Dietary vitamin C was also analysed. Food groups for fresh fruit and total fruit were
created and calculated for comparison of fruit intake prior to (lead-in phase) and when consuming
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the two SunGold kiwifruit daily (week 12). At week 12 both fresh and total fruit, with and without
the SunGold kiwifruit, were created for comparison with baseline fruit intake. Kiwifruit consumption
recorded in the week 12 food diary was used as an indication of compliance.
Percent energy from macronutrients per day was calculated from the average total daily dietary
intake as follows [35]:
• percent energy from fat = (fat (g/day) × 37.7 kJ/g)/energy (kJ/day)

• percent energy from carbohydrate = (carbohydrate (g/day) × 16.7 kJ/g)/energy (kJ/day)
• percent energy from protein = (protein (g/day) × 16.7 kJ/g)/energy (kJ/day)
2.6.4. Physical Activity

A physical activity questionnaire was collected to monitor that participants maintained their
usual activity levels. Participants completed the self-administered International Physical Activity
Questionnaire (IPAQ) short form. The questionnaire asks about physical activity over the previous
seven days. Participants were scored as MET-minutes per week using the IPAQ scoring protocol [36].

Standard descriptive statistics including means, medians, standard deviations, interquartile
ranges, frequencies and percentages were used to describe the baseline demographic, anthropometric,
laboratory and questionnaire data. The changes in these measures over time were compared by paired
t-tests and Wilcoxon Signed rank tests as appropriate. Changes in taxonomic relative abundance (RA)
and diversity data was statistically analysed using Wilcoxon signed rank tests and are presented as
medians and interquartile ranges. The association between vitamin C concentrations and intakes and
demographic, dietary, laboratory and anthropometric measures were tested by Pearson’s correlation
coefficients. Changes in anthropometric and laboratory measures were compared between the
two vitamin C adequacy groups by one-way ANOVA. A two-tailed p < 0.05 was taken to indicate
statistical significance.
3. Results
The average age of the participants was 66 years and ranged from 44 to 85 years old. There was
an equal number of male and female participants and the majority of participants were European
(81%) (Table 2). There was a mix of educational qualifications, which is expected given the age range
of the participants. There were fewer participants currently smoking (15%) compared to the number of
participants who reported being ex- (39%) or non-smokers (46%). The majority of participants reported
consuming alcohol (73%) (Table 2).
Table 2. General characteristics of study participants.
Characteristics n = 26
Age (years) (mean ± SD) 66 ± 9
Gender
Female % (n) 50 (13)
Male % (n) 50 (13)
Ethnicity
European % (n) 81 (21)
Māori % (n) 8 (2)
Samoan % (n) 4 (1)
Asian/Chinese % (n) 4 (1)
Other % (n) 4 (1)
Qualification
No qualification % (n) 27 (7)
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Table 2. General characteristics of study participants.
Characteristics n = 26
Secondary school % (n) 19 (5)
Post-secondary certificate, diploma or trade diploma % (n) 42 (11)
University % (n) 12 (3)
Smoking Status
Current smoker % (n) 15 (4)
Ex-smoker % (n) 39 (10)
Non-smoker % (n) 46 (12)
Alcohol Status
Current drinker % (n) 73 (19)
Ex-drinker % (n) 12 (3)
Non-drinker % (n) 15 (4)
3.1. Dietary Intakes: Macronutrients, Micronutrients, Fruit Intake

There were no significant differences in daily energy, fibre, protein, total fat and total available
carbohydrate when week 12 was compared to baseline (Table 3). There was a non-significant trend towards
an increase in total available sugars and when investigated further results showed the daily intake of
fructose and glucose to be significantly higher at week 12 compared to baseline (p < 0.001) and the intake
of sucrose to be significantly lower at week 12 compared to baseline (p = 0.018). Starch intake was not
significantly different at week 12 compared to baseline. Significant changes in micronutrient intake are
included in Table 3. There was a significant increase in vitamin C (p < 0.001) and E (p = 0.037) intakes
(Table 3). There was a significantly higher intake of folate at week 12 compared to baseline (p = 0.012) and
when investigated further this was attributed to folate naturally occurring in foods (p = 0.002).
At baseline the amount of fruit eaten (fresh and total) is the same whether or not kiwifruit is
excluded, as participants were instructed not to eat any kiwifruit during the lead-in phase of the trial,
during which time the baseline food diaries were completed (Table 3). Analysis of total and fresh fruit
intake including and excluding kiwifruit consumption showed that participants were substituting
some of their usual fruit intake for kiwifruit during the study (Table 3). At week 12 compliance with
the intervention was calculated from the food diary data to be 91%. This was calculated for every
individual as the percentage of the six kiwifruit consumed over three days.
Table 3. Dietary Intake of participants at week 0 and week 12.
Daily Dietary Intake Week 0 (n = 26) Week 12 (n = 24)

Macronutrients
Energy (KJ) 7407 ± 2759 7176 ± 1683
Fibre (g) 23 ± 8 23 ± 8
Protein (g) 81 ± 24 80 ± 22
Protein (% of energy) 19 ± 4 19 ± 4
Total fat (g) 72 ± 35 68 ± 18
Total fat (% of energy) 35 ± 6 36 ± 6
Total carbohydrate (g) 190 ± 71 184 ± 49
Total carbohydrate (% of energy) 43 ± 5 43 ± 6
Total available sugars (g) 82 ± 33 86 ± 23
Fructose (g) 16 ± 8 22 ± 6 ***
Glucose (g) 14 ± 6 20 ± 5 ***
Sucrose (g) 35 ± 25 29 ± 16 *
Lactose (g) 14 ± 8 20 ± 5
Maltose (g) 3.0 ± 1.8 2.8 ± 1.5
Total starch (g) 108 ± 43 97 ± 34
Micronutrients †
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Table 3. Cont.
Daily Dietary Intake Week 0 (n = 26) Week 12 (n = 24)

vitamin C (mg) 79 ± 35 347 ± 70 ***
vitamin E (mg) 8.5 ± 3.9 10.3 ± 3.3 *
Total folate (μg) 271 ± 126 337 ± 119 *
Folate (naturally occurring) (μg) 236 ± 105 291 ± 98 **
Food Groups
Fresh fruit including kiwifruit (g) 131 ± 91 237 ± 68 ***
Fresh fruit excluding kiwifruit (g) 131 ± 91 72 ± 55 ***
Total fruit including kiwifruit (g) 184 ± 101 274 ± 104 **
Total fruit excluding kiwifruit (g) 184 ± 101 108 ± 94 **
Values presented as mean ± SD. Paired sample t-tests were used to compare dietary data between times. * Significant
at the 0.05 level, ** significant at the 0.01 level, *** significant at the 0.001 level. † Micronutrients that were significantly
different when week 12 concentrations were compared to baseline.
3.2. Dietary Vitamin C Intakes

At baseline the majority of participants (n = 20) met the New Zealand RDI for vitamin C of 45 mg/day
(Figure 2). There was only one participant who had a dietary intake below the New Zealand Estimated
Average Requirement (EAR) (30 mg/day) and there were no participants reaching the New Zealand
Ministry of Health suggested dietary target (SDT) to reduce chronic disease risk, that is, 220 mg/day for
men and 190 mg/day for women (Figure 2). At the end of the study the majority of participants (n = 22) had
dietary intakes reaching the SDT and the other participants had intakes above the Recommended Dietary
Intake (RDI) (n = 2) (Figure 2).
100%
80%
% of Individuals
60% ш SDT
40% RDI-<SDT
EAR-<RDI
20%
<EAR
0%
Week 0 Week 12
Time
Figure 2. Dietary vitamin C intake of individuals at weeks 0 and week 12 meeting the estimated average
requirement (EAR) (30 mg/day), recommended dietary intake (RDI) (45 mg/day) and suggested dietary
target (SDT) to reduce chronic disease risk (220 mg/day for men and 190 mg/day for women) [36].
3.3. Anthropometry and Blood Pressure

From baseline to the end of the study there were no significant changes in weight, BMI or fat mass.
There was a significant decrease in waist circumference at week 6 of 0.9 cm (p = 0.019) and 3.1 cm at
week 12 (p = 0.001) compared to baseline (Table 4). Although waist-to-hip ratio was significantly lower
at week 12 compared to baseline (p = 0.032), the difference was small and not clinically significant
(Table 4). Blood pressure also decreased over the duration of the study (Table 3). At week 6 diastolic and
systolic blood pressure had decreased from baseline by 3 mmHg (p = 0.040) and 5 mmHg (p = 0.026)
respectively (Table 4). At week 12 systolic and diastolic blood pressure were 4 mmHg (p = 0.029) and
6 mmHg (p = 0.003) lower than baseline (Table 4). At baseline 42% (n = 11) of participants recorded
taking medication that lowers blood pressure. There were changes to blood pressure medication for
only two of these participants during the trial. One participant had their blood pressure medication
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dose reduced by half from week 6 to 12 and another participant had it increased between baseline and
week 6 but then reduced back to the baseline dose between week 6 and 12.
Table 4. Anthropometric, blood pressure and physical activity measures.
Characteristics Week 0 (n = 26) Week 6 (n = 26) Week 12 (n = 24)

Anthropometry
Weight (kg) 80.2 ± 19.8 80.1 ± 20.0 77.9 ± 18.6
BMI (kg/m2 ) 29.4 ± 7.3 29.4 ± 7.4 28.6 ± 7.0
Fat mass (%) 34.3 ± 6.6 34.4 ± 6.6 34.0 ± 6.9
Waist circumference (cm) 98.6 ± 15.3 97.7 ± 15.2 * 95.5 ± 14.6 ***
Waist-to-hip ratio 0.90 ± 0.09 0.90 ± 0.09 0.89 ± 0.09 *
Blood Pressure
Diastolic (mmHg) 76 ± 8 73 ± 9 * 72 ± 10 *
Systolic (mmHg) 129 ± 14 124 ± 17 * 123 ± 18 **
Physical Activity
(met-minutes/week) 3598 ± 5273
Values presented as mean ± SD. Wilcoxon Signed Ranks tests were used to compare physical activity between
times. Paired sample t-tests were used for all other variables. * Significant at the 0.05 level, ** significant at the 0.01
level, *** significant at the 0.001 level. In addition to the missing data for the two participants who did not complete
week 12 there were missing data at baseline and week 6 for fat mass (one participant) and for physical activity at
week 12 (one participant).
3.4. Biochemical Indices

There was a small but statistically significant decrease in HbA1c of 1 mmol/mol at week 12 compared
to baseline (p = 0.005) (Table 5). This decrease, however, would not be considered clinically significant.
Although fasting glucose significantly increased by 0.2 mmol/L at week 6 (p = 0.001), it was only 0.1 mmol/L
higher than baseline at week 12 (p = 0.046), which is not clinically significant (Table 5).
Table 5. Laboratory measures of participants at week 0 and week 12.
Biochemical Indices Week 0 (n = 26) Week 12 (n = 24)

HbA1c (mmol/mol) 43 ± 2 42 ± 2 **
Fasting Glucose (mmol/L) 5.4 ± 0.7 5.5 ± 0.8 *
Plasma vitamin C (μmol/L) 50 ± 19 64 ± 13 ***
Total cholesterol (mmol/L) 5.2 ± 1.3 5.1 ± 1.3
HDL cholesterol (mmol/L) 1.35 ± 0.23 1.35 ± 0.23
LDL cholesterol (mmol/L) 3.3 ± 1.0 3.3 ± 1.1
Triglycerides (mmol/L) 1.1 ± 0.5 1.1 ± 0.4
Cholesterol (total/HDL) ratio 3.9 ± 0.9 3.8 ± 0.9
hs-CRP (mg/L) 1.8 (0.6–3.2) 0.9 (0.5–2.2)
Insulin (pmol/L) 51 (31–73) 42 (31–67)
Ghrelin (pmol/L) 162 (110–204) 154 (119–206)
Leptin (ng/mL) 38 (27–71) 35 (25–75)
Adiponectin (μg/mL) 9 (6–11) 10 (7–12)
Values presented as mean ± SD or median and interquartile range (25th to 75th percentiles). Wilcoxon Signed
Ranks tests were used to compare hs-CRP and hormones (insulin, ghrelin, leptin and adiponectin) between times.
Paired sample t-tests were used for all other variables. * Significant at the 0.05 level, ** significant at the 0.01 level,
*** significant at the 0.001 level. In addition to the two participants who did not complete the trial there was missing
data at baseline and week 6 for fasting glucose (one participant), plasma vitamin C (one participant), and insulin at
week 12 (one participant).
3.5. Plasma Vitamin C Levels

At baseline, none of the participants met the criteria for plasma vitamin C deficiency (<11 μmol/L).
There were three participants (13%) with marginal (11–23 μmol/L) and seven (29%) with inadequate
(24–49 μmol/L) plasma vitamin C concentrations (Figure 3). Over half of the cohort (n = 14) had either
adequate (42%, 50–69 μmol/L) or saturating (17%, ≥70 μmol/L) plasma vitamin C concentrations
at baseline (Figure 3). Consistent with a previous observational study [37], baseline fasting glucose
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(r = −0.450, p = 0.024), insulin (r = −0.520, p = 0.008) and waist circumference (r = −0.399, p = 0.048)
were inversely associated with plasma vitamin C concentrations.
100%
90%
80%
70%
% of Individuals
60%
Saturating
50%
Adequate
40%
Inadequate
30%
20% Marginal
10%
0%
Week 0 Week 12
Time
Figure 3. Plasma vitamin C status of individuals at weeks 0 and 12 classified as having saturating
(≥70 μmol/L), adequate (50–69 μmol/L), inadequate (24–49 μmol/L) and marginal (11–23 μmol/L)
plasma vitamin C. There were no participants classified as having deficient (<11 μmol/L) plasma
vitamin C concentrations [38].
After 12 weeks supplementation, plasma vitamin C concentrations were significantly higher

compared to baseline (mean increase of 14 μmol/L, p < 0.001) indicating compliance with kiwifruit
consumption (Table 5). The increase in plasma vitamin C concentrations was greatest in those who had
started the trial with inadequate plasma vitamin C concentration (increase of 22 μmol/L, p = 0.004),
compared to those who already had adequate plasma vitamin C concentrations at baseline (increase of
7 μmol/L, p = 0.029). At the end of the study, there were no participants with marginal plasma vitamin
C and only three participants (13%) had inadequate plasma vitamin C concentrations. Fourteen (58%)
of the participants had adequate and 7 (29%) of the participants had saturating plasma vitamin C
concentrations at week 12 (Figure 3).
3.6. Faecal Microbiota

The faecal samples of four participants (one of whom did not complete the trial) were excluded
from analysis due to starting a course of antibiotics during the trial. For this analysis, there were
22 participants at week 6 and 21 participants at week 12, due to another participant not completing
the trial.
All DNA samples passed quality check and each sample was amplified and barcoded twice to
reduce the possibility of losing a sample to sequence failure. Only a single amplification product
failed to sequence (1/152 = 0.7%; well within accepted rate of 4%), however the duplicate reaction
from this sample sequenced well. Thus, all submitted samples generated good sequence data. A total
of 5,527,067 sequences were available for further analysis following quality checking procedures.
Each sample generated an average of 72,725 sequences (range 39,216–117,799).
Rarefaction curves showed good coverage of microbiota composition (Figure 4A). Comparison
of study time points at a rarefaction level of 35,000 sequences per sample did not show a statistically
significant difference in alpha diversity (the complexity in composition of the microbiota) using a variety of
metrics (Table 6).
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Table 6. Diversity (at 35,000 sequences/sample) of participants at week 0, 6 and 12.
Diversity Measure Week 0 (n = 22) Week 6 (n = 22) Week 12 (n = 21)

Observed species 250 (213–316) 279 (232–306) 241 (217–283)
Whole tree (PD) 19 (17–23) 21 (18–22) 20 (17–22)
Shannon index 5.1 (4.6–5.4) 5.4 (4.7–5.8) 5.0 (4.9–5.3)
Simpson’s diversity 0.95 (0.91–0.96) 0.96 (0.93–0.97) 0.94 (0.93–0.95)
Chao index 318 (257–377) 367 (288–396) 338 (307–389)
Values presented as median and interquartile range (25th to 75th percentiles). Wilcoxon Signed Ranks tests were
used to compare the diversity between times. There were no significant differences.
Beta-diversity (unique fraction metric (Unifrac) weighted and unweighted) was used to compare
similarities in microbiota composition over time. A comparison of distances within and between time
points did not reveal significant changes in community structure with time (Figure 4B,C). Unweighted
unifrac distances (kinds of bacteria and phylogenetic relationships) indicated that the microbiotas of the
participants tended to be more similar to each other after 12 weeks intervention than at baseline or 6 weeks
(Figure 4B). Applying a weighting (based on the relative abundance of OTUs) suggested that the microbiota
of participants are more similar to each other at both 6 and 12 weeks than at baseline (Figure 4C).
Figure 4. Faecal microbiota diversity metrics. (A) Rarefaction curves based on Observed Species metric of
alpha diversity, showing coverage of DNA sequences obtained from faecal DNA. Means and 95% CI are
shown. (B,C) Beta diversity metrics comparing microbiota similarity distances within and between groups.
Means and SEM are shown. (B) Unweighted Unifrac (kinds of bacteria and phylogenetic relatedness).
(C) Weighted Unifrac (kinds of bacteria, phylogenetic relatedness and relative abundances).
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The complete results of taxonomic analysis of the faecal microbiota at the Family level are supplied
in Supplementary Material Table S1. Sequences from members of the bacterial phylum Actinobacteria
and a bacterial family within this taxon, Coriobacteriaceae, were more abundant in faecal samples
collected during the kiwifruit intervention compared to baseline (Figure 5A,B). The original OTU table
contained 10 clusters identified as Coriobacteriacae, including one OTU each in the genera; Atopobium,
Collinsella, Eggerthella, Gordonibacter and Senegalimassilia. The other five OTUs were ‘unidentified’
(i.e., never cultured and characterised). Combining the abundances from these OTUs replicated the
significant increase in relative abundance of the Family Coriobacteriaceae observed with time (Figure 5C).
Thus, it appears that as yet uncultivated member(s) of the Coriobacteriaceae contributed to a significant
increase in relative abundance during the intervention.
Figure 5. Relative abundances of bacterial groups in faeces of participants at baseline and during
supplementation of the diet with kiwifruit. Box and whiskers plots showing individual values and
means as horizontal lines. (A) Phylum Actinobacteria. (B) Family Coriobacteriaceae. (C) OTUs of
uncharacterized bacteria of the Family Coriobacteriaceae. Statistical evaluation by Wilcoxon matched
pairs test using Prism 7.
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Dietary intervention of two SunGold kiwifruit per day resulted in significantly greater faecal
water content at weeks 6 (76%, p < 0.001) and 12 (74%, p = 0.01) compared to baseline (68%).
4. Discussion
In this study, consuming two SunGold kiwifruit per day was associated with a significant increase in
plasma vitamin C and fasting glucose, and a decrease in HbA1c, however, these latter two changes were
small and, although statistically significant, were not clinically significant. There was a significant reduction
in both diastolic and systolic blood pressure. A significant reduction in waist circumference and waist-to-hip
ratio was also seen, despite small and non-significant reductions in average BMI and fat mass.
Consumption of two SunGold kiwifruit per day (total of 190 g flesh) for three months provided
approximately 300 mg of vitamin C daily from the kiwifruit alone. This increase in dietary vitamin
C from the SunGold kiwifruit resulted in most participants meeting the New Zealand Ministry of
Health’s suggested dietary target to reduce chronic disease risk [35]. This increase in dietary vitamin C
resulted in a significant increase in plasma vitamin C concentrations (mean increase of 14 μmol/L).
The increase in plasma vitamin C concentrations was greatest in those who had started the trial with
inadequate plasma vitamin C concentration (i.e., <50 μmol/L), compared to those who already had
adequate plasma vitamin C concentrations at baseline. This result is consistent with findings from an
earlier kiwifruit trial where those with lower baseline plasma vitamin C concentrations showed an
earlier and greater response to supplementation, compared to those with adequate plasma vitamin C
concentrations at baseline, who showed little effect from supplementation [17]. Three participants (13%)
had only adequate plasma vitamin C concentrations at week 12 which may indicate non-compliance
or be related to some lifestyle factor, such as smoking, or chronic disease [39].
While all participants met the ADA criteria for prediabetes using HbA1c (39–46 mmol/mol)
at baseline, there was still a range of anthropometric and laboratory measures for participants.
For example, fasting glucose ranged from 3.9 to 7.5 mmol/L, insulin ranged from 15 to 220 pmol/L
and waist circumference ranged from 70 to 125 cm. Fasting glucose, insulin and waist circumference
were all inversely associated with plasma vitamin C concentrations at baseline. This result is consistent
with previous research [6–10,37,38].
It has been suggested that vitamin C concentrations at baseline may represent a critical factor in
predicting the metabolic responses to nutritional interventions tackling oxidative stress and may in
part explain why there have been inconsistent findings in RCTs investigating supplementation with
vitamin C and glycaemic control [17,39]. Individuals in this study were supplemented with vitamin
C-rich kiwifruit; however, there were only three participants with marginal vitamin C levels and no
participants with deficient levels at baseline. This may explain why no clinically significant differences
in metabolic markers were observed between baseline and week 12. Furthermore, the length of the
intervention may need to be longer than 12 weeks to see biological changes [14].
There was a small statistically significant reduction in both diastolic and systolic blood pressure over the
duration of the study. This result is unlikely to be due to changes in blood pressure medication as only two
participants reported changes to the dose of their blood pressure medication during the study. One of these
participants actually had their blood pressure medication dose reduced by half between week 6 and 12 and
the other participant was taking the same dose at week 12 as they were at baseline despite having it increased
earlier in the study. Those with inadequate vitamin C levels at baseline (i.e., <50 μmol/L) had a greater
reduction in blood pressure (decrease of 7 ± 9 mmHg diastolic and 10 ± 11 mmHg systolic) compared to
those who had adequate levels (decrease of 2 ± 8 mmHg diastolic and 5 ± 10 mmHg systolic) when week
12 was compared to baseline. This may be related to their level of oxidative stress as hypertension has been
related to increased oxidative stress and reduced antioxidant status [40]. Kiwifruit contain a wide range of
natural antioxidants; they are rich in vitamin C and also contain vitamin E, polyphenols and flavonoids
which are all potent antioxidants [41]. This result is consistent with a RCT of smokers, who are also known to
have lower vitamin C concentrations, which showed three kiwifruit per day for eight weeks was associated
with reductions of 10 mmHg in systolic blood pressure and 9 mmHg in diastolic blood pressure [42].
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Nutrients 2018, 10, 895
Despite the small increase in fruit intake at week 12, there were no significant differences in energy,
fibre, protein, total fat and total carbohydrate intake when dietary data at week 12 was compared to baseline.
This result suggests participants did not make major changes to their diet during the trial. The addition
of the SunGold kiwifruit also had no significant impact on their macronutrient intake as it is relatively
low in calories (63 kcal/100 g), protein (1.0 g/100 g), fat (0.3 g/100 g) and carbohydrate (16 g/100 g) [16].
However, SunGold kiwifruit does contain 12 g/100 g of sugar [16], which may have contributed to the
significantly higher intake of fructose (increase of 6 g) and glucose (increase of 6 g) at week 12 compared to
baseline. SunGold kiwifruit also contains vitamin E (1.4 mg/100 g) and folate (31 μg/100 g), so would have
contributed to the higher intake of these vitamins at week 12 compared to baseline.
Although faecal microbiota diversity was stable across the 12-week intervention when viewing
the cohort as a whole, Coriobacteriaceae family members showed a significant increase in relative
abundance during the course of the study. The assignment of these bacteria to uncharacterised
bacterial species was particularly interesting because it suggests that as yet uncultivated bacteria that
use substrates associated with kiwifruit (such as pectins) exist in the human faecal microbiota. Future
work should include efforts to cultivate these bacteria and to determine their functional characteristics.
The Coriobacteriaceae are in general a poorly studied bacterial family, however they can chemically
transform plant polyphenols [43] and thus their activities may be of interest in promoting human
health [44]. Certainly, future work should investigate the potential link between Coriobacteriaceae and
polyphenolic compounds in the faeces of kiwifruit-fed subjects and chemical derivatives of these
substances in the blood circulation. While the increased relative abundances of Coriobacteriaceae were
small, this does not preclude an important role for the bacteria in bowel ecology because low abundance
bacteria in microbial communities can have a disproportionally large effect relative to abundance [45].
Our human intervention trial did not confirm the reported in vitro effect of kiwifruit fermentation [25].
In the vitro study, Bacteroides spp., Parabacteroides spp. and Bifidobacterium spp. were reported to increase
in cultures containing kiwifruit and inoculated with human faeces. Increased Coriobacteriaceae relative
abundances were the only impact of dietary modification seen in the participants in our study.
Dietary intervention in the form of two gold kiwifruit per day led to an increase in stool water
content at both 6 and 12 weeks. This indicates a shortened gut transit time (laxative effect) and has been
linked to decreases in community diversity in other studies. Stool consistency is strongly associated
with gut microbiota richness and composition, enterotypes and bacteria growth rates [46]. In our study,
the observation may be confounded by the fact that kiwifruit contains 1.4 g/100 g of fibre [16], which is
comprised of both soluble and insoluble components at a ratio of approximately 1:3 [19]. The soluble
fibre is made up almost exclusively of pectic polysaccharides that have the ability to retain water
and form gels, which increases the size and softness of the faeces and aids stimulation of peristaltic
movements [19]. The soluble fibre may be the cause of the increase in faecal water seen in this study.
Results from several clinical trials also show that kiwifruit promotes laxation [47–50].
This was the first study to measure changes in the gut microbiota and vitamin C status associated
with a whole fruit (SunGold kiwifruit) in people with prediabetes. The participants were free-living
representing a realistic intervention for the general population. The use of a whole food rather
than an extract/supplement in this study also makes it more affordable and available for people.
Although there was no control group in this study, participants acted as their own control with baseline
measurements for comparison and dietary intake was consistent over time. In this study plasma
vitamin C status corroborated the food diaries as an objective measure of compliance. The study
duration of three months enabled the use of HbA1c as a measure of glycaemic control and allowed us
to investigate temporary and longer-term shifts in the microbiota.
As this was a pilot exploratory study with a limited number of participants it may not have
been sufficient to identify some clinically relevant associations. Additionally, we did not pre-screen
participants for vitamin C intake or status and, as a result, half the study cohort already had adequate
plasma vitamin C status at baseline. Furthermore, because we did not include an arm with vitamin C
supplementation alone, it is difficult to ascertain the contribution of the vitamin C content of the fruit
91
Nutrients 2018, 10, 895
to the observed effects. When assessing dietary intakes there are also limitations around misreporting
and a potential bias in recording “good/bad” foods.
5. Conclusions
Supplementation with two SunGold kiwifruit per day significantly increased plasma vitamin C
concentrations and provided small improvements in several markers of metabolic and cardiovascular
health. The results of faecal microbiota analysis point to the need for novel bacteriological
investigations of Coriobacteriaceae in order to explain their faecal abundances and to investigate
mechanistic relationships with regard to kiwifruit polysaccharides and polyphenols. Due to the
small sample size of this pilot study a larger study is indicated, particularly focusing on recruiting
participants with low vitamin C status at baseline and determinations of polyphenol derivatives in
faeces and blood.
Supplementary Materials: The following is available online at http://www.mdpi.com/2072-6643/10/7/895/s1.

Supplementary Table S1. OTU table collapsed to family level, showing number of curated sequences 1 per family
per sample.
Author Contributions: R.W. conducted participant recruitment and study visits, administration of questionnaires,
collection of clinical data; R.W., P.S., E.F., A.A. and L.J. dietary analysis; A.C.C., vitamin C analysis; B.L., A.H. and
G.W.T. microbiota analysis; C.F. statistical analysis; R.W., G.T., A.C., J.W. and R.B.G. undertook the conception and
writing of the paper.
Funding: This research was funded by Zespri International Ltd.
Acknowledgments: We would like to thank all participants for volunteering their time to take part in the study.
We would also like to thank Sharon Berry for helping take blood samples and Philippa Wadsworth for delivering
the kiwifruit. R.W., J.W., R.B.G., P.S. and G.T. are the recipients of a Zespri International Ltd. grant. A.C. is
supported by a Health Research Council of New Zealand Sir Charles Hercus Health Research Fellowship.
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nutrients
Review
Nutritional Interventions as Beneficial Strategies to
Delay Cognitive Decline in Healthy
Older Individuals
Blanka Klímová 1, * and Martin Vališ 2
1 Department of Applied Linguistics, University of Hradec Kralove, Rokitanskeho 62,
Hradec Kralove 500 03, Czech Republic
2 Department of Neurology, University Hospital Hradec Kralove, Sokolska 581,
Hradec Kralove 500 05, Czech Republic; [email protected]
Received: 22 June 2018; Accepted: 13 July 2018; Published: 15 July 2018
Abstract: Current demographic trends indicate that the population is aging. The aging process
is inevitably connected with cognitive decline, which manifests itself in worsening working
memory, processing speed, and attention. Therefore, apart from pharmacological therapies,
non-pharmacological approaches which can influence cognitive performance (such as physical
activities or healthy diet), are being investigated. The purpose of this study is to explore the types
of nutritional interventions and their benefits in the prevention and delay of cognitive delay in
healthy older individuals. The methods used in this study include a literature review of the available
studies on the research topic found in Web of Science, Scopus, and MEDLINE. The findings show that
nutritional intervention has a positive impact on cognitive function in healthy older people. However,
it seems that the interactions between more than one nutrient are most effective. The results reveal
that specifically the Mediterranean diet appears to be effective in this respect. Moreover, the findings
also indicate that multi-domain interventions including diet, exercise, cognitive training, and vascular
risk monitoring have a far more significant effect on the enhancement of cognitive functions among
healthy older individuals.
Keywords: nutrition; healthy older individuals; cognitive decline; intervention; prevention;

randomized clinical trials
1. Introduction
Currently, the number of older people is rapidly increasing. For instance, in 2017 there
were 962 million people aged 60+ years worldwide, with their number increasing by 3% every
year. The largest proportion of the aging population can be found in Europe (25%). By 2050,
all continents except Africa will have one quarter or more of their population aged over 60 years [1].
This demographic trend of aging population will cause significant social and economic problems [2].
Therefore, there are efforts to develop relevant strategies and guidelines both at national and
international levels in order to delay this process of aging and prolong the active age of older
individuals, as well as to maintain quality of life [3].
The aging process is inevitably connected with cognitive decline, which manifests itself in
worsening working memory, processing speed, and attention [4], i.e., in so-called fluid intelligence,
which deteriorates at different rate in each individual [5]. The rate and severity of cognitive decline
varies from normal age-related change to chronic neurodegenerative diseases such as Alzheimer’s
disease for which at present there is no cure [6]. Therefore, non-pharmacological approaches which
can influence fluid intelligence, such as physical activities or healthy diet, are being investigated [6–8].

Nutrients 2018, 10, 905
Evidence-based studies [9,10] indicate that specifically the Mediterranean diet (MedDiet) rich in
olive oil and nuts, seems to contribute to the prevention of cognitive impairment among healthy
older people. However, other food products, for example, avocados [11], or nutritional supplements
such as consumption of flavanone-rich 100% orange juice [12] or omega-3 fatty acid [13] also play
an important role in the prevention of cognitive decline in healthy older individuals. In addition,
nutritional interventions in comparison with the pharmacological treatment, are usually well-tolerated,
easy-to-implement, cost-effective, and safe for long-term use [13].
The purpose of this study is to explore the types of nutrition intervention and its benefits in the
prevention and delay of cognitive delay in healthy older individuals.
2. Methods
The authors conducted a literature review of the research studies on the basis of the key words in
three acknowledged databases: Web of Science, Scopus, and MEDLINE. This review was performed
over the period from 2014 to June 2018 since several review studies had been already written on
this topic [14,15]. The key words were as follows: diet AND cognitive decline AND healthy older
people, diet AND cognitive decline AND healthy elderly, nutrition intervention AND cognitive
decline AND healthy older people, nutrition intervention AND cognitive decline AND healthy elderly,
nutrition AND cognitive decline AND healthy older people, nutrition AND cognitive decline AND
healthy elderly, diet AND dementia AND healthy older people, diet AND dementia AND healthy
elderly, nutrition intervention AND dementia AND healthy older people, nutrition intervention AND
dementia AND healthy elderly, nutrition AND dementia AND healthy older people, nutrition AND
dementia AND healthy elderly.
The majority of the studies were detected in the Web of Science database (97 studies), followed
by Scopus (66 studies) and MEDLINE (45 studies). However, in MEDLINE it is possible to look only
for clinical trials, and therefore the selection was easier. Altogether, 208 studies were found via the
database search and 11 from other available sources (i.e., web pages, conference proceedings and
books outside the scope of the databases described above). The titles of all studies were then checked
in order to discover whether they focused on the research topic or not and irrelevant studies were
excluded. In addition, the duplicate studies were also excluded. Afterwards, the authors checked the
content of the abstracts whether the study examined the research topic. Thirty-two studies/articles
were selected for the full-text analysis. Only 12 studies were then able to be used for detailed analysis
of the research topic. The selection of these studies is described below (Figure 1).
Figure 1. An overview of the selection procedure.
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Nutrients 2018, 10, 905
The study was included if it was a randomized controlled trial, and matched the corresponding
period, i.e., from 2014 up to June 2018. Furthermore, the study was included if it involved healthy
older people, i.e., those without any cognitive impairment or dementia aged 50+ years, and focused
on the research topic, i.e., nutritional intervention in the delay and prevention of cognitive decline
in healthy older individuals. All studies had to be written in English. Thus, studies such as [5] were
excluded, as well as cross-sectional descriptive and cohort studies, for example [16–18].
3. Results
Altogether 12 randomized controlled trials (RCT) were found. The majority of were from Europe
(i.e., Finland, Germany, Italy, Spain, and the UK) [10,12,13,19–24] and the rest from the USA and
Australia [11,25,26]. The nutrition intervention involved dietary supplements (i.e., cocoa flavanols,
Vitis vinifera, docosahexaenoic acid (DHA)-rich fish oil, or omega-3 fatty acid), vegetable (e.g., avocado),
berry and orange beverages, and the MedDiet. Two of the studies were multidomain lifestyle
intervention studies including, apart from healthy diet, physical exercises, social activities, cognitive
training, and vascular risk management [20,24]. The intervention period in the studies ranged from
five weeks to five years. The subject samples also varied; the smallest sample of subjects consisted
of 37 healthy older individuals and the largest included 775 healthy older people. The efficacy of the
nutrition intervention focused on the prevention and delay of cognitive decline in the studies was
measured with available validated cognitive assessment tools such as a battery of cognitive tests aimed
at assessing working memory, attention, or processing speed. Table 1 below provides an overview of
the main findings of these studies. They are summarized in alphabetical order of their first author.
97
Table 1. Overview of the twelve selected studies focused on cognitive decline and its prevention by nutrition intervention.
Type of The Nutrition

Intervention Number of Main Outcome
Author Objective Intervention And Its Main Findings
Period Subjects Assessments
Frequency
To investigate whether The results indicate that DG
Daily intake of 900 mg
Brickman et al. the enhancement of Functional magnetic dysfunction is a driver of
Nutrients 2018, 10, 905
cocoa flavanols in the 37 healthy older

[25] dentate gyrus (DG) resonance imaging (fMRI), age-related cognitive decline and
intervention vs. 10 mg Three months. individuals, age:
RCT function with dietary a battery of cognitive tests, suggest non-pharmacological
cocoa flavanols in the 50–69 years.
(USA) flavanols improves statistical analysis. means for its amelioration such as
control group.
cognition in older adults. the daily high flavanol intake.
To investigate the The findings reveal that 12 weeks
57 subjects in the
potential beneficial effects Cognigrape® (250 of Cognigrape® supplementation
intervention
Calapai et al. [19] of a Vitis vinifera-based mg/day) in the A battery of cognitive and is safe, can improve physiological
group and 54
RCT dietary supplement on intervention group and 12 weeks. neuropsychological tests, cognitive profiles, and can
subjects in the
(Italy) cognitive function and placebo in the control statistical analysis. concurrently ameliorate negative
control group; age:
neuropsychological status group. neuropsychological status in
55–75 years.
in healthy older adults. healthy older adults.
Three groups: control
(IC)—an interview in
which information
about activities and
98
health was discussed;
goal-setting (GS)—an
interview in which they The Lifetime of Experiences
To evaluate a goal-setting
set behaviour change Questionnaire (LEQ), The results show that at 12-month
intervention aimed at 75 healthy elderly
goals relating to Physical Activities Scale for follow-up, the two goal-setting
Clare et al. [20] promoting increased (IC—27 subjects;
physical, cognitive and the Elderly (PASE), a groups increased their level of
RCT cognitive and physical 12 months. GS—24 subjects;
social activity, health battery of cognitive tests, physical (effect size 0.37) and
(UK) activity and improving GM—24 subjects);
and nutrition; and audio-recordings of the cognitive (effect size 0.15) activity
mental and physical age: 50+ years.
goal-setting with interviews, statistical relative to controls.
fitness, diet and health.
mentoring (GM)—the analysis.
goal-setting interview
followed by bi-monthly
telephone mentoring.
The one-to-one
interviews lasted for 90
min.
Table 1. Cont.

Frequency
1720 mg DHA and 600
To test whether mg eicosapentaenoic
Nutrients 2018, 10, 905
194 subjects in the

docosahexaenoic acid acid or The results show that
Danthiir et al. [26] intervention
(DHA)-rich fish oil slows low-polyphenolic olive A battery of cognitive tests, supplementing older adults with
RCT 18 months. group and 196 in
18-month cognitive oil daily, as capsules in statistical analysis. fish oil does not prevent cognitive
(Australia) the control group;
decline in cognitively the intervention group decline.
age: 65–90 years.
healthy individuals. and placebo in the
control group.
Daily 350 mg
To examine whether eight
consumption of
weeks of daily The results indicate that after the
flavanone-rich 100% A battery of cognitive,
Kean et al. [12] flavanone-rich orange 37 healthy 8-week consumption of flavanone
orange juice and executive function and
RCT juice consumption was Eight weeks subjects, mean rich 100% orange juice, the global
equicaloric low-flavone episodic memory tests,
(UK) beneficial for cognitive age: 67 years. cognitive performance was
(37 mg) orange statistical analysis.
performance in healthy significantly improved (p < 0.05).
flavoured cordial (500
older people.
mL).
Daily intake of 2200 mg
99
long-chain 22 healthy older
To investigate the impact Visuospatial
polyunsaturated subjects in the The findings reveal that the daily
Kulzow et al. [13] of omega-3 fatty acid object-location-memory
omega-3 fatty acids intervention intake of LC-n3-FA has a positive
RCT supplementation on 26 weeks. task (LOCATO), standard
(LC-n3-FA) in the group and 22 in impact on memory functions in
(Germany) memory functions in neuropsychological tests,
intervention group and the control group, healthy older people (p = 0.049).
healthy older adults. statistical analysis.
placebo in the control age: 50–75 years.
group.
The findings show that the intake
631 healthy older
of several vitamins and minerals
Lehtisalo et al. To discuss the success of Dietary intervention subjects in the
remained unchanged or increased
[21] dietary counselling counselling: 3 intervention Food records, statistical
Two years. in the intervention group and that
RCT intervention among individual and 8 group group and 629 in analysis.
the dietary counselling may have a
(Finland) healthy older individuals. sessions. the control group,
positive impact on age-related diet
age: 60–77 years.
quality and cognitive performance.
A drink containing 993 90 cognitively
To evaluate the effect of The results reveal that regular CF
Mastroiacovo et al. mg high flavanol (HF), intact elderly A battery of
flavanol consumption on consumption can reduce some
[22] 520 mg intermediate subjects divided neuropsychological tests,
cognitive performance in Eight weeks. measures of age-related cognitive
RCT flavanol (IF), or 48 mg into three groups blood pressure measures,
cognitively intact elderly dysfunction, possibly through an
(Italy) low flavanol (LF) cocoa (HF, IF, LF); age: statistical analysis.
people. improvement in insulin sensitivity.
flavanols (CFs). 61–85 years.
Table 1. Cont.

Frequency
Daily intake of 795 mg 20 healthy subjects
To evaluate effects on
berry beverage with in the intervention The results indicate that the
Nutrients 2018, 10, 905
cognitive functions and

Nilsson et al. [23] polyphenols or dietary group and 20 A battery of cognitive tests, subjects performed better in the
cardiometabolic risk
RCT fibre or 11 mg berry Five weeks. healthy subjects in cardiometabolic tests and working memory test after the
markers with a mixture of
(Spain) control beverage with the control group, statistical analysis. berry beverage compared to after
berries intervention in
no poly phenols or mean age: 50–70 the control beverage (p < 0.05).
healthy older individuals.
dietary fibre. years.
20 healthy subjects The results show that including the
1 avocado daily in the in the intervention daily intake of one avocado may
Scott et al. [11] To explore the effect of the intervention group and group and 20 have a positive impact on cognitive
A battery of cognitive tests,
RCT daily consumption of one 1 potato or 1 cup of Six months. healthy subjects in performance in healthy older
statistical analysis.
(USA) avocado on cognition. chickpeas in the control the control group, individuals, specifically on their
group. mean age: 63 working memory (p = 0.036) or
years. sustained attention (p = 0.033).
Participants were The findings of the intervention
To assess whether randomly assigned to reveal that cognitive benefits were
775 healthy
baseline leukocyte the lifestyle more pronounced with shorter
100
subjects (392 A battery of
Sindi et al. [24] telomere length (LTL) intervention (diet, baseline LTL, particularly for
control, 383 neuropsychological tests,
RCT modified the cognitive exercise, cognitive Two years. executive functioning, indicating
intervention), at blood samples, statistical
(Finland) benefits of a 2-year training, and vascular that the multi-domain lifestyle
the age of 30–77 analysis.
multidomain lifestyle risk management) and intervention was especially
years.
intervention. control (general health beneficial among higher-risk
advice) groups. individuals.
Participants were
randomly assigned to a 447 cognitively
To investigate whether a Mediterranean diet healthy volunteers
Mediterranean diet supplemented with (233 women
Valls-Pedret et al. In an older population, a
supplemented with extra virgin olive oil (1 (52.1%); mean age,
[10] A neuropsychological test Mediterranean diet supplemented
antioxidant-rich foods L/week), a Five years. 66.9 years); three
RCT battery, statistical analysis. with olive oil or nuts is associated
influences cognitive Mediterranean diet groups: two
(Spain) with improved cognitive function.
function compared with a supplemented with intervention
control diet. mixed nuts (30 g/day), groups and one
or a control diet (advice control group.
to reduce dietary fat).
RCT: randomized controlled trial.
Nutrients 2018, 10, 905
Apart from one study [26], results of all RCTs indicate that the nutritional interventions had a
positive impact on the cognitive performance of healthy older individuals, specifically on their working
memory and attention [10,11,23]. Moreover, the findings of the multidomain lifestyle intervention
studies reveal that such interventions exhibit significant impacts on cognitive functioning in later
life [20,24]. The results also show that the change in the cognitive performance can be already detected
after five weeks of intervention [23]. However, this is questionable because according to Kurz and van
Baelen [27], the relevant period for medication assessment by regulatory authorities is 24 weeks at
a minimum.
4. Discussion
The findings described in Table 1 show that nutrition intervention has a positive impact on
cognitive functioning of healthy older people. The trials with the exception of four RCTs [10,19,23,26],
concentrated only on one type of nutritional supplement (i.e., Vitis vinifera, docosahexaenoic acid
(DHA)-rich fish oil, omega-3 fatty acid, avocado, and berry). Three RCTs [12,22,25] focused on
high flavanol-rich drinks, which appeared to have quite a positive effect on cognitive performance
among healthy older individuals. In fact, all nutrients described in Table 1 are part of the traditional
multi-nutrient MedDiet pattern, and the research [28] indicates that the interaction of specific foods
and nutrients, especially in the MedDiet, is more powerful on the aging brain than individual nutrients
or a low-fat diet. In fact, the MedDiet seems to be a nutritional model for healthy dietary habits
since it contains all the needed nutrients: monounsaturated fatty acids, polyunsaturated fatty acids,
antioxidants (e.g., allium sulphur compounds, anthocyanins, beta-carotene-flavonoids, catechins,
carotenoids, indoles, or lutein), vitamins (A, B1, 6, 9, 12, D, E), and minerals (magnesium, potassium,
calcium, iodine, zinc, selenium) [28]. In addition, the combination of these nutrients positively
affects pathological neurodegenerative processes such as oxidative stress, neuroinflammation, insulin
resistance, and reduced cerebral blood flow [29]. Nevertheless, the MedDiet appears to improve
cognitive performance in case there is a high adherence to this diet [30–32]. Furthermore, the MedDiet
has a positive impact on neuropsychological [19] and physical state of older people [16] and on vascular
diseases and diabetes [22–24,33]. Although the results mainly concerned western developed countries,
similar research (however, non-RCT) was conducted among healthy older adults in Asian countries
such as Taiwan [34] and Japan [35]. The findings of these studies also show a positive correlation
between the healthy diet rich in vegetables, soy products, fruit, and fish and the prevention and delay
of cognitive decline in older age. Wright et al. [36] in their study carried out among 2090 African
Americans and whites emphasized that higher diet quality was associated with higher performance
on tests of attention and cognitive flexibility, visuospatial ability, and perceptual speed. This was also
evidenced by Smyth et al. [18] who in their cohort study claimed that higher diet quality is associated
with a reduced risk of cognitive decline. Hosking et al. [37] report that adequate nutrition is essential
for cognitive development in childhood and cognitive ability in childhood is strongly associated with
cognitive performance across the lifetime.
Moreover, it seems that multi-nutrient dietary intervention should be implemented in the delay
of cognitive decline among healthy older individuals. The multi-nutrient intervention is also one of
the priorities discussed at the First WHO Ministerial Conference on Global Action against Dementia in
March 2015 [38]. Furthermore, Shilsky et al. [39] suggest in their study that healthcare systems should
play a more significant role in integrating nutrition care for healthy older individuals and that the
nutrition assessment should be incorporated in the medical records. This was also confirmed by the
discussed study [21], for which findings revealed that the dietary counselling had a positive impact on
age-related diet quality and cognitive performance.
The findings of the reviewed RCTs [20,24] indicate that the multi-domain interventions including
healthy diet, physical exercises, cognitive training, and vascular risk monitoring have a far more
significant effect on the enhancement of cognitive functions among healthy older individuals [40].
In fact, these non-pharmacological strategies (healthy diet, physical exercises and cognitive training)
101
Nutrients 2018, 10, 905
have been confirmed by other research studies [7,8] and they should be all integrated at an optimal
level into daily regime of healthy older individuals in order to prolong their active and quality life.
This was also proposed at the International Conference on Nutrition and the Brain in Washington in
2013 [41].
On the contrary, research implies that dietary patterns rich in fat and sugar, with high intake of
meat/poultry or eggs, have negative, harmful effects on cognitive functioning in older age [34,35,37].
The limitations of the reviewed studies consist in the small sample sizes, different types of
nutrition interventions and outcome measures, and a lack of follow-up assessments, as well as
the fact that not all studies were specifically designed to examine cognitive performance. All these
insufficiencies might generate overestimated conclusions in this review study [42,43]. Therefore, more
RCTs should be conducted to prove the efficacy of nutritional interventions on the prevention and
delay of cognitive decline.
Author Contributions: Conceptualization, B.K. and M.V.; Methodology, M.V.; Software, B.K.; Validation, B.K.
and M.V.; Formal Analysis, B.K.; Investigation, B.K.; Resources, B.K.; Data Curation, B.K.; Writing—Original
Draft Preparation, B.K.; Writing—Review and Editing, M.V.; Visualization, M.V.; Supervision, M.V.; Project
Administration, M.V.; Funding Acquisition, M.V.
Funding: This research received no external funding.
Acknowledgments: This paper was supported by the research project Excellence 2018, Faculty of Informatics and
Management, University of Hradec Kralove, Czech Republic, as well as by MH CZ – DRO (UHHK 00179906) and
PROGRES Q40 run at the Medical Faculty Charles University, Czech Republic.
Conflicts of Interest: The authors declare no conflicts of interest.
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nutrients
Article
Food Consumption, Knowledge, Attitudes, and
Practices Related to Salt in Urban Areas in Five
Sub-Saharan African Countries
Magali Leyvraz 1 , Carmelle Mizéhoun-Adissoda 2 , Dismand Houinato 3 , Naby Moussa Baldé 4 ,
Albertino Damasceno 5 , Bharathi Viswanathan 6 , Mary Amyunzu-Nyamongo 7 , Jared Owuor 7 ,
Arnaud Chiolero 1,8 and Pascal Bovet 1,6, *
1 Institute of Social and Preventive Medicine (IUMSP), Canton University Hospital (CHUV), 1010 Lausanne,
Switzerland; [email protected] (M.L.); [email protected] (A.C.)
2 School of Nutrition and Dietetics, Faculty of Health Science, University of Abomey-Calavi,
Cotonou 01 BP 526, Benin; [email protected]
3 Laboratory of Noncommunicable and Neurologic Diseases Epidemiology, Faculty of Health Science,
University of Abomey-Calavi, Cotonou 01 BP 526, Benin; [email protected]
4 Department of Endocrinology and Diabetes, Donka University Hospital, Conakry, Guinea;
[email protected] or [email protected]
5 Department of Medicine, Eduardo Mondlane University, Maputo, Mozambique; [email protected]
6 Ministry of Health, Victoria, Republic of Seychelles; [email protected]
7 African Institute for Health and Development (AIHD), Nairobi 00100, Kenya;
[email protected] (M.A.-N.); [email protected] (J.O.)
8 Institute of Primary Health Care (BIHAM), University of Bern, 3012 Bern, Switzerland
* Correspondence: [email protected]; Tel.: +41-21-314-72-72
Received: 28 June 2018; Accepted: 3 August 2018; Published: 7 August 2018
Abstract: High salt intake is a major risk factor of hypertension and cardiovascular disease. Improving
knowledge, attitudes, and practices (KAP) related to salt intake in the general population is a key
component of salt reduction strategies. The objective of this study was to describe and compare
the KAP of adults related to salt in urban areas of five countries in sub-Saharan Africa. The survey
included 588 participants aged 25 to 65 years who were selected using convenience samples in the
urban areas of Benin, Guinea, Kenya, Mozambique, and Seychelles. Socio-demographic and food
consumption were assessed using a structured closed-ended questionnaire administered by survey
officers. Height, weight, and blood pressure were measured. Food consumption varied largely
between countries. Processed foods high in salt, such as processed meat, cheese, pizzas, and savory
snacks were consumed rather infrequently in all the countries, but salt-rich foods, such as soups
or bread and salty condiments, were consumed frequently in all countries. The majority of the
participants knew that high salt intake can cause health problems (85%) and thought that it is
important to limit salt intake (91%). However, slightly over half (56%) of the respondents regularly
tried to limit their salt intake while only 8% of the respondents thought that they consumed too
much salt. Salt and salty condiments were added most of the time during cooking (92% and 64%,
respectively) but rarely at the table (11%). These findings support the need for education campaigns
to reduce salt added during cooking and for strategies to reduce salt content in selected manufactured
foods in the region.
Keywords: salt; sodium; hypertension; knowledge; attitudes; practices; diet; Africa; Benin; Guinea;
Mozambique; Kenya; Seychelles

Nutrients 2018, 10, 1028
1. Introduction
Cardiovascular disease (CVD) is the leading cause of deaths worldwide including in low-income
and middle-income countries (LMICs) [1]. It is estimated that high salt intake accounts for 9.5%
of all CVD deaths globally due to its effect on blood pressure [2]. In sub-Saharan Africa (SSA),
approximately 800,000 deaths per year are due to CVD and 6% of these deaths are attributable to high
salt intake [2]. The disease burden related to elevated salt intake is expected to further increase over the
next decade in LMICs [3] due to the growing and aging populations and trends towards urbanization
and westernization of the diet in these countries.
Reducing dietary salt intake in the population is a key strategy to reduce the CVD burden [4].
Strategies to reduce salt intake at the population level include awareness campaigns advising
individuals to reduce their salt consumption, food labeling, and reformulation of selected industrially
produced foods [5]. Improving knowledge, attitudes, and practices (KAP) related to salt intake in the
population is an important part of any salt reduction strategy [6]. In particular, dietary advice can
reduce salt intake, blood pressure, and other CVD risk factors [7,8]. In 2015, 40 countries worldwide
were implementing some organized policy or program to reduce salt consumption in the population
but only one program was implemented in SSA (i.e., in South Africa) [9].
Only a few studies have examined KAP related to salt intake in SSA countries [10–14].
These studies have generally found low levels of KAP related to salt intake. However, most of
these studies were conducted in specific population groups (e.g., hypertensive patients) and in selected
settings. Given the scarcity of data on KAP related to salt intake and the high burden of CVD in
LMICs [15], it was recently recommended that more studies should be conducted to assess dietary
patterns and key sources of sodium in these countries [16].
The objective of this study was to describe and compare the food consumption, KAP related to
salt intake, and associated factors in adults from the general population in urban areas of five SSA
countries. This information is expected to be useful for guiding the development of salt reduction
programs and policies in SSA.

Cross-sectional surveys were conducted in a main city in each of the five countries (Bohicon in
Benin, Conakry in Guinea, Mombasa in Kenya, Maputo in Mozambique, and Victoria in Seychelles)
between January 2012 and April 2013. The countries were selected based on the geographical diversity
and the presence of investigators who expressed interest in examining these issues in the countries.
For four countries, participant selection was based on the convenience of a three-stage sampling
strategy. The first stage was the selection of two areas in the selected cities. The second stage was the
selection of households within each area and the third stage was the selection of one person within a
household. The person was chosen to ensure similar numbers of participants from 25 to 44 years of
age and from 45 to 65 years of age and similar numbers of men and women. Pregnant women and
adults unable to understand the questionnaire were excluded. In Seychelles, participants were selected
from an electronic register of all inhabitants living around Victoria while ensuring similar numbers of
participants 25–44 years of age and 45–65 years of age and similar numbers of men and women.
Survey officers administered a structured closed-ended questionnaire and performed
anthropometric measurements. In view of the lack of a standardized dietary questionnaire in SSA and
the large variety of diets across countries in the region, a questionnaire was developed during a two-day
meeting with the main investigators of each country with several questions being adapted from
World Health Organization (WHO) instruments [17]. Questions assessed household characteristics,
socio-demographic characteristics, health-related behaviors, and frequency of selected common food
items including food items rich in salt (e.g., processed meats, cheeses, pizzas, savory snacks, bread,
soups, and relevant local dishes). The questionnaire also included questions on KAP related to salt
intake. Height, weight, and three blood pressure readings were measured. Informed consent was
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obtained from each participant. In each country, the study was approved by the locally relevant
institutional ethical review boards.
Weight was measured with an electronic weighing scale to the nearest 0.1 kg. Height was
measured with a fixed height rod to the nearest 0.1 cm in all countries. Overweight and obesity were
defined for body mass index (BMI) between 25 and 29 or ≥30 kg/m2 , respectively. Blood pressure
was measured with an electronic blood pressure device and high blood pressure defined as
systolic/diastolic blood pressure ≥140/90 mmHg or taking treatment for hypertension.
We assessed associations between KAP variables and selected predictors using Spearman
correlation coefficients and using stratified analysis. We used the chi-square test to test for differences
between categories. Means for all countries were weighted so that each country had the same weight.
The level of significance was set at 0.05. Analyses were conducted using Stata 14.1 (StataCorp,
College Station, TX, USA).
Publication of data on KAP from this study was delayed because the assessment of salt excretion,
which was originally another goal of this study in addition to KAP, had to be cancelled due to funding
and other issues. Moreover, sample size was limited when compared to the initially four-time larger
anticipated sample size due to discontinued funding by the donor.
3. Results
3.1. Sample Characteristics

A total of 588 adults between 25 and 65 years old participated in the survey. The characteristics of
the participants and their households are described in Table 1. The country samples included similar
proportions of men versus women and younger persons (25–44 years of age) versus older person
(45–65 years of age). This was consistent with the selection strategy of the participants. Slightly more
than half of the participants (54%) were overweight or obese and approximately one-fourth (26%) had
high blood pressure. Nearly all households had electricity (90%), a television set (87%), or a radio
(89%). Women cooked food more often than men with 60% of the women cooking food every day
versus 36% of the men. In addition, 5.4% of the women never cooked versus 20% of the men.
Table 1. Socio-demographic characteristics of the participants 1 .
Characteristics All 2 Benin Guinea Kenya Mozambique Seychelles

Participant characteristics
Total sample size (n) 588 140 119 102 77 150
Female (%) 54 (50−58) 51 (43–60) 52 (43–61) 49 (39–59) 61 (50–71) 58 (50–66)
Age (mean) 42 (41–43) 42 (41–44) 43 (41–45) 43 (41–45) 41 (39–43) 42 (40–44)
25−44 years (%) 57 (53−61) 54 (45–62) 59 (50–67) 54 (44–63) 61 (50–71) 56 (48–64)
45−65 years (%) 43 (39–47) 46 (38–55) 41 (33–50) 46 (37–56) 39 (29–50) 44 (36–52)
Completed primary school (%) 80 (76–83) 56 (47–64) 78 (70–85) 88 (80–93) 77 (66–85) 99 (95–100)
Overweight (%) 30 (26–34) 31 (24–39) 24 (17–32) 24 (16–33) 32 (22–43) 40 (32–48)
Obese (%) 24 (21–28) 24 (17–31) 16 (10–24) 23 (16–32) 29 (20–41) 29 (23–37)
High blood pressure or treatment
26 (22–30) 26 (20–34) 18 (12–26) 25 (18–35) 26 (17–37) 37 (29–45)
for hypertension (%)
Treatment for hypertension (%) 15 (12–18) 9 (5–15) 13 (8–21) 17 (11–25) 17 (10–27) 19 (13–26)
Household assets
Running water (%) 74 (70–78) 68 (60–75) 83 (75–89) 30 (22–40) 90 (80–95) 99 (95–100)
Electricity (%) 90 (87–92) 86 (80–91) 99 (94–100) 70 (60–78) 96 (89–99) 100 (100–100)
Fridge (%) 59 (55–63) 19 (13–26) 71 (62–78) 30 (22–40) 77 (66–85) 100 (100–100)
Radio (%) 89 (86–92) 87 (80–92) 80 (72–87) 86 (78–92) 94 (85–97) 99 (95–100)
Television (%) 87 (84–89) 77 (69–83) 92 (86–96) 66 (56–74) 99 (91–100) 100 (100–100)
Cable television (%) 47 (43–51) 48 (40–56) 79 (71–85) 29 (21–39) 16 (9–26) 65 (57–72)
Bicycle or motorcycle (%) 28 (25–32) 64 (55–71) 29 (21–37) 30 (22–40) 5 (2–13) 13 (8–19)
Car or truck (%) 24 (21–28) 6 (3–12) 40 (32–49) 8 (4–15) 22 (14–33) 43 (36–51)
1 Values are means (95% confidence intervals). 2 Estimates for all were weighted so that data from each country had
the same weight.
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3.2. Food Consumption

The usual consumption of foods in each country is shown in Table 2. The most frequently
consumed staple foods were maize in Benin, rice and bread in Guinea, Mozambique, and Seychelles,
and maize and bread in Kenya. Fish was the most frequent source of animal protein in all countries.
The most frequently consumed beverages were tea in Kenya, Mozambique, and Seychelles, coffee
in Guinea, and fruit juices in Benin. Participants consumed on average 2.7 meals (95% confidence
interval (CI) 2.6–2.7) and 1.2 snacks (95% CI 1.2–1.3) per day and ate 3.0 meals (95% CI 2.8–3.3) outside
the home on a weekly basis.
Table 2. Consumption of selected food items according to country 1 .
Food item Benin Guinea Kenya Mozambique Seychelles

Staple foods
Rice
Maize
Potato
Yam
Bread
Fruits and vegetables
Salad
Vegetables
Fruits
Animal products
Chicken
Red meat
Processed meat
Fish
Eggs
Cheese
Other foods
Soup
Pizza
Breakfast cereals
Savory snacks
Sweets and pastries
Supplements or vitamins
Non-alcoholic beverages
Tea
Coffee
Commercial soft drink
Locally made lemonade
Fresh fruit juice
Non-fresh fruit juice
Milk
1 : <1 times/week, : 1–3 times/week, : 4–6 times/week, : every day.
A number of processed food items with known high salt content including processed meat, cheese,
pizzas, breakfast cereals, and savory snacks were consumed rather infrequently in all the countries.
However, soup and bread, which often contain high amounts of salt, were consumed frequently in
all countries. As mentioned in the next paragraph, salt-rich condiments (e.g., Maggi cubes and food
spreads such as Marmite/Vegemite), which are often added in soups and other dishes, were also used
frequently in all countries.
3.3. Knowledge, Attitudes, and Practices (KAP) Related to Salt

Levels of KAP related to salt intake are shown in Table 3. The majority of the participants knew
that high salt intake can cause health problems (85%) and could name at least one adequate health
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problem that can arise from high salt intake (66%). Most of the participants thought it was important
to limit salt intake (91% ‘very important’ and ‘somehow important’). However, only 56% of the
respondents often tried to limit their salt intake. Moreover, only a small proportion of respondents
thought that they consumed too much salt (8%), while a substantial proportion of the respondents
thought they consumed too little salt (26%).
Table 3. Knowledge, attitudes, and practices related to salt intake according to country (in %).
Knowledge, Attitudes, and Practices All 1 Benin Guinea Kenya Mozambique Seychelles
Knowledge
High salt intake can cause serious health problems
Yes 85 (82–88) 93 (88–97) 66 (57–74) 87 (79–92) 88 (78–93) 92 (86–95)
No 5 (3–7) 2 (1–7) 5 (2–11) 6 (3–13) 4 (1–12) 6 (3–11)
Don’t know 10 (8–13) 4 (2–9) 28 (21–37) 7 (3–14) 8 (4–17) 2 (1–6)
Health problems are associated with high salt intake
≥1 problem known 66 (61–70) 87 (78–92) 64 (52–74) 48 (38–59) 93 (81–98) 50 (41–59)
None known 34 (30–39) 13 (8–22) 36 (26–48) 52 (41–62) 7 (2–19) 50 (41–59)
It is important to limit salt intake
Very important 9 (7–12) 7 (4–13) 20 (13–28) 7 (3–14) 7 (3–15) 5 (2–10)
Somehow important 12 (9–14) 4 (1–8) 23 (16–32) 14 (9–23) 0 (0–0) 17 (12–24)
Not really important 79 (76–82) 89 (83–93) 57 (48–66) 79 (70–86) 93 (85–97) 78 (70–84)
Attitudes
Try to limit salt
Often 55 (51–59) 76 (68–82) 47 (38–57) 54 (44–63) 66 (54–76) 32 (25–40)
Sometimes 16 (13–19) 11 (7–18) 18 (12–26) 26 (18–36) 3 (1–11) 22 (16–29)
Not really 29 (25–33) 13 (8–20) 35 (26–44) 20 (13–29) 31 (21–43) 46 (38–54)
Perceived amount of salt consumed
Too little 26 (22–30) 2 (1–6) 55 (46–64) 36 (27–45) 30 (21–41) 7 (4–12)
About right 59 (55–63) 96 (92–99) 20 (14–28) 52 (43–62) 60 (48–70) 67 (59–74)
Too much 8 (6–10) 1 (0–5) 5 (2–11) 12 (7–20) 10 (5–20) 11 (7–17)
Don’t know 7 (5–9) 1 (0–5) 19 (13–27) 0 (0–0) 0 (0–0) 16 (11–23)
Practices
Salt is added during cooking
Never 2 (1–3) 2 (1–6) 3 (1–8) 2 (0–8) 0 (0–0) 1 (0–5)
Sometimes (1–2 times/week) 7 (5–9) 1 (0–5) 27 (20–36) 3 (1–9) 3 (1–10) 0 (0–0)
Often (most meals) 19 (16–22) 5 (2–10) 18 (12–26) 30 (22–39) 27 (18–38) 15 (10–21)
Always (all meals) 73 (69–76) 92 (86–96) 52 (43–61) 65 (56–74) 70 (59–79) 84 (77–89)
Salty condiments are used during cooking 2
Never 9 (6–11) 6 (3–12) 8 (4–14) 21 (14–31) 4 (1–11) 4 (2–9)
Often (most meals) 22 (18–25) 9 (5–15) 11 (6–18) 24 (17–34) 25 (16–36) 40 (32–48)
Always (all meals) 42 (38–47) 78 (70–84) 50 (41–59) 17 (11–26) 47 (36–58) 18 (13–25)
Salt is added to food at the table
Never 66 (62–70) 71 (63–78) 46 (37–55) 46 (36–56) 82 (72–89) 87 (80–91)
Often (most meals) 6 (4–8) 2 (1–6) 14 (8–21) 8 (4–15) 4 (1–11) 2 (1–6)
Always (all meals) 5 (3–7) 3 (1–7) 8 (4–14) 9 (5–16) 3 (1–10) 2 (1–6)
Consumption of foods high in salt 3
Never 34 (30–38) 1 (0–6) 41 (32–50) 16 (10–24) 83 (73–90) 28 (21–36)
1–2 times/week 15 (13–18) 7 (4–13) 38 (30–48) 18 (11–27) 1 (0–9) 12 (8–18)
3–4 times/week 29 (25–32) 5 (2–10) 9 (5–16) 54 (45–64) 16 (9–26) 58 (50–66)
Every day/almost every day 21 (18–25) 86 (79–91) 6 (3–12) 12 (7–20) 0 (0–0) 2 (1–6)
1 Estimates for all were weighted so that data from each country had the same weight. 2 These condiments included
bouillon cubes, Aromat powder, soy sauce, food spreads (e.g., Vegemite, Marmite), and similar items. 3 These foods
included salted fish, salted meat, salami, salted peanuts, food spreads, pizza, and other typical local meals rich
in salt.
Most participants reported that salt was added to the foods most of the time during cooking
(92% ‘often’ and ‘always’). Salty condiments such as bouillon cubes, aroma enhancing powders, and
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sauces were used frequently in all countries especially in Benin, Guinea, and Mozambique (87%, 61%,
and 72% added ‘often’ and ‘always’). In contrast, few participants reported adding salt to meals at the
table (11% ‘often’ and ‘always’).
3.4. Associations between KAP Related to Salt and Socio-Demographic Characteristics

The distribution of several KAP variables was significantly different between countries (p < 0.001),
but not consistent with sex, age, education, and hypertension treatment (see Supplemental Table S1).
Women added salt and other salty condiments during cooking more often than men (p = 0.045 and
p = 0.006, respectively) and added salt less often at the table (p = 0.043). KAP levels did not differ
significantly between participants who completed primary school vs. those who did not, except for
the use of salty condiments (such as bouillon cubes), which were used more frequently among persons
with lower education levels (p < 0.001). KAP levels did not differ according to anti-hypertensive
treatment, except that salty condiments were used less frequently by treated persons (p < 0.05) and
treated persons thought more often that they consumed too little salt (p < 0.05).
Most of the KAP variables were not associated with each other (data not shown). The small sample
sizes in several categories precluded meaningful statistical analyses. However, lower discretionary use
of salt was associated with higher levels of knowledge related to salt intake (“thinks high salt intake
can cause serious health problems”: ρ = −0.23, p < 0.001, “knows at least one correct salt-related health
problem”: ρ = −0.09, p < 0.05, “thinks it is important to limit salt intake”: ρ = −0.20, p < 0.001).
4. Discussion
Overall, the study shows that the distribution of intake of food items varied widely between
countries. Several processed food items with known or presumably high salt content (such as pizzas
and savory snacks) were consumed rather infrequently (<3 times/week), but soups and bread were
consumed frequently in all countries (>3 times/week), which may suggest substantial salt intake.
The study also shows a fairly good level of KAP in relation to salt intake in urban settings among five
countries in Africa. However, there were some gaps. A fairly modest proportion of persons added
salt at the table. The large majority of participants were aware of health risks related to salt intake
and recognized the importance of limiting dietary salt intake. Yet, less than one in ten participants
believed they consumed too much salt. We did not find substantial associations within KAP variables
or between KAP variables and socio-demographic characteristics, except for an inverse association
between the knowledge of the need to restrict salt intake and the discretionary use of salt at the table.
Food consumption differed largely between countries, which underlies the difficulty of developing
dietary questionnaires and nutritional guidelines that could apply to all countries. However, certain
processed foods such as soups and bread were consumed frequently in all countries. These findings
suggest the need for voluntary or mandatory reformulation strategies to reduce the salt content of
selected manufactured foods that are both commonly consumed and have high salt content. A study
conducted in the early 2000s in South Africa reported that bread was a main source of dietary salt in
this country [18]. As a result, the South African government regulated the maximum levels of salt
permitted in a wide range of industrially processed food categories, including breads, in order to reduce
salt intake in the population [19,20]. Reformulation policies aimed at reducing salt in manufactured
foods can be highly cost-effective [21] and are recommended by the WHO’s Global Action Plan for
the Prevention and Control of Non-Communicable Diseases [4]. Several countries have implemented
policies to reformulate selected manufactured foods with subsequent reductions of the salt intake
at the population level [22–24]. However, none of the five countries included in this survey have
implemented such reformulation strategies [9]. South Africa is the only country in the African region
that has taken regulatory steps to mandatorily reduce the salt content of selected foods [9]. Findings of
our study also emphasize the need for continued education campaigns in order to encourage people
to limit dietary salt intake. Such campaigns are also useful when advocating for studies assessing
sources of salt intake in a particular population and when advocating for corresponding reformulation
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strategies. Policy aimed at reformulating foods frequently consumed and high in salt is a cornerstone
strategy for effective reduction of salt intake in the population.
Knowledge on the detrimental effect of high salt intake was fairly high and higher than could
have been anticipated from previous research [10–14]. However, fairly good knowledge about health
effects of salt did not seem to have translated into strong attitudes and practices with regards to salt
intake reduction. For example, knowledge that salt could be detrimental for health was associated
with only one salt-related practice, i.e., low discretionary use of salt. A study conducted in 2014–2015
in Mozambique also found participants high in knowledge but low in attitudes and practices, in
which is similar to our study [25]. This suggests that campaigns aimed at raising awareness about
the detrimental impact of salt intake on health might better translate in actual salt reduction if
structural measures are also implemented, e.g., programs to reduce salt in the food served in work
or school canteens and measures to limit salt intake in selected manufactured foods (e.g., bread).
This is consistent with a review showing that the implementation of education and awareness-raising
interventions alone is unlikely to be adequate in reducing population salt intake to the recommended
levels, which suggests that behavior change might better occur when combining health education
and public awareness campaigns [26]. The quasi-ubiquitous presence of television and radio in the
surveyed households suggests that these media could be the main instruments to relay such public
awareness campaigns.
The main strengths of this study were the inclusion of population-based samples in five countries
and the use of the same methodology, which allows direct comparison between countries. The study
also has limitations. First, salt intake was not measured in all countries and we cannot report on
the relation between salt-related behaviors and an objective measurement of salt intake. Moreover,
our study did not allow for the quantification of salt added at the table or the contribution of salt in
the form of processed versus non-processed foods. Second, a number of selection biases may have
occurred in the sampling of participants. For example, persons present in a household at the time of
the survey may have been different than persons absent. This may, however, have limited impact,
since food consumption tends to be fairly homogenous at a household level. Individuals from a
low socioeconomic status (e.g., the illiterate persons unable to understand questions) may also have
been under-represented in this study. However, this proportion is likely small and has little overall
impact on the results. Our survey was limited to urban areas and the generalization of the findings
is, therefore, limited to such urban areas. Admittedly, food consumption may differ largely in rural
areas and further studies need to be conducted in these different areas. Yet, urbanization is rapidly
increasing in SSA and findings in this study may reflect dietary habits among large segments of the
population on the continent. Third, the study relied on reported information, which is prone to recall
and other biases that can lead to under-reporting or over-reporting of certain foods and practices.
Fourth, the fairly low numbers of participants precluded meaningful statistical analyses of associations
between KAP variables and socio-demographic characteristics.
Very little data is available on actual salt intake, the sources of salt intake, and KAP related to salt
in SSA. One systematic review aimed to identify all published studies reporting salt intake in countries
of SSA until 2015 [27]. This review found that 81% of the adult populations consumed salt intake above
the recommended maximum 5 g per day [27], which suggests overall high intake in the region. Intake
was higher in urban than in rural populations [27]. With regard to countries included in this study,
the review identified one study in children in Benin in 1996 [28] and one study in Kenya in 1986 in
rural areas [29]. When looking for more recent studies, urinary excretion of salt was assessed in Benin
based on the same study on KAP and in Mozambique using another study. Both studies found high
salt intake (10.2 g and 10.5 g of salt per day in Benin and Mozambique, respectively) [30,31]. A global
modelling study [32] estimated that mean salt intake in all of the five countries in our study, apart
from Kenya, was above the maximum 5 g of salt recommend by the WHO [33].
Future research on KAP related to salt is recommended in these countries among others in the
region. An objective measurement of the actual amount of salt consumed in these populations is
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needed to assess whether salt intake is above recommendations and in which population groups.
In addition, further qualitative studies should examine specific practices that may favor salt intake.
Lastly, analyses of salt intake of foods especially in bread, instant soups and selected local foods as
well as market studies are needed to identify the main sources of dietary salt in these populations in
order to guide reformulation guidelines.
5. Conclusions
In conclusion, our study among adults in urban settings found largely different food consumption
patterns between countries but consumption of salt-rich bread, soup, and salty condiments was
frequent in all countries, which suggests that the salt intake could be substantial in all countries.
We found fairly good knowledge related to the detrimental effects of high salt intake, but mixed
findings related to the attitudes and practices related to the reduction of salt intake. These findings
support the need for both education campaigns to promote knowledge, attitudes, and behaviors for the
control of dietary salt intake and reformulation strategies to reduce salt content of selected frequently
eaten foods high in salt.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/10/8/1028/

s1, Table S1: Knowledge, attitudes, and practices related to salt intake according to sex, age, education, and
hypertension treatment.
Author Contributions: Conceptualization, P.B. and M.A.-N. Study design and Methodology, P.B., D.H.,
A.D., N.M.B., and M.A.-N. Data Analysis, M.L. and P.B. Writing-Original Draft Preparation, M.L. and P.B.
Writing-Review & Editing, C.M.-A., D.H., N.M.B., A.D., B.V., M.A.-N., J.O., and A.C. Supervision, P.B. and A.C.
Project Administration, M.A.-N. and P.B. Funding Acquisition, M.A.-N. and P.B.
Funding: This research benefited partly from an unconditional seed grant by PepsiCo. M.L. led the analysis
and writing of the manuscript during her PhD studies, which was funded by the Federal Food Safety and
Veterinary Office.
Acknowledgments: The authors thank all the survey officers and collaborators involved in data collection as well
as the participants.
Conflicts of Interest: The authors declare no conflict of interest. The funder had no role in the design of the study,
in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish
the results.
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nutrients
Article
Lactase Persistence, Milk Intake, and Adult Acne:
A Mendelian Randomization Study of 20,416
Danish Adults
Christian R. Juhl 1 , Helle K. M. Bergholdt 2 , Iben M. Miller 3 , Gregor B. E. Jemec 3 ,
Jørgen K. Kanters 1, *and Christina Ellervik 2,4,5,6, *
1 Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen,
2100 Copenhagen, Denmark; [email protected]
2 Department of Production, Research, and Innovation, Region Zealand, 4180 Sorø, Denmark;
[email protected]
3 Department of Dermatology, Zealand University Hospital, 4000 Roskilde, Denmark;
[email protected] (I.M.M.); [email protected] (G.B.E.J.)
4 Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen,
2100 Copenhagen, Denmark
5 Department of Laboratory Medicine, Boston Children’s Hospital, 300 Longwood Avenue,
Boston, MA 02115, USA
6 Department of Pathology, Harvard Medical School, Boston, MA 02115, USA
* Correspondence: [email protected] (J.K.K.); [email protected] or
[email protected] (C.E.)
Received: 15 July 2018; Accepted: 3 August 2018; Published: 8 August 2018
Abstract: Whether there is a causal relationship between milk intake and acne is unknown. We tested
the hypothesis that genetically determined milk intake is associated with acne in adults using
a Mendelian randomization design. LCT-13910 C/T (rs4988235) is associated with lactase persistence
(TT/TC) in Northern Europeans. We investigated the association between milk intake, LCT-13910
C/T (rs4988235), and acne in 20,416 adults (age-range: 20–96) from The Danish General Suburban
Population Study (GESUS). The adjusted observational odds ratio for acne in any milk intake vs.
no milk intake was 0.93(95% confidence interval: 0.48–1.78) in females and 0.49(0.22–1.08) in males
aged 20–39 years, and 1.15(95% confidence interval: 0.66–1.99) in females and 1.02(0.61–1.72) in males
above 40 years. The unadjusted odds ratio for acne in TT+TC vs. CC was 0.84(0.43–1.62) in the age
group 20–39 years, and 0.99(0.52–1.88) above 40 years. We did not find any observational or genetic
association between milk intake and acne in our population of adults.
Keywords: acne; acne vulgaris; milk; dairy; diet; Mendelian randomization; adults
1. Introduction
Acne is a common chronic inflammatory skin disease, which is almost universal in adolescence,
with rates up to 85% [1–4]. After adolescence the prevalence decreases but a significant number
of patients are affected by persistent acne or develop new-onset adult acne [5]. Acne is overall
characterized by open comedones, papules, pustules, and nodules [6], but the clinical appearance
varies by age and lifestyle [5,7,8].
The genetic architecture of acne vulgaris is complex and multiple susceptible loci have been
identified reflecting the multifactorial pathogenesis of acne involving the innate immune system,
inflammation, modified lipogenesis, and androgens [9,10]. But there is likely also an environmental
component in the development of acne vulgaris. Several observational studies have investigated the

Nutrients 2018, 10, 1041
association of milk intake with acne in children, adolescents, and young adults [11–16]. However, no
previous observational study has been performed in all adult ages.
In individuals of Northern European descent, the genetic variant LCT-13910 C/T (rs4988235)
located 13,910 base pairs upstream of the lactase (LCT) gene on chromosome 2q21-22, within intron 13
of the adjacent MCM6 gene, shows complete correlation with lactase persistence/non-persistence [17].
The T-allele is the lactase-persistent allele, whereas the C allele is the lactase non-persistent allele.
The inheritance is autosomal recessive manner such that individuals homozygous for CC are unable to
digest lactose, whereas individuals with TC or TT are able to digest lactose.
To investigate whether there is a causal relationship between milk intake and acne, large long-term
randomized trials would be needed, but these are costly and it is difficult to uphold the randomization
over time. Instead the epidemiological Mendelian randomization (MR) design offers a feasible
alternative [18]. The underlying principle in the MR design is that genetic variants are randomly
assorted during gamete formation, which is similar to the random assignment of patients to placebo or
active treatment in a clinical intervention trial. In the MR design, confounders are, therefore, balanced
across the genotypes, and the genotypes will serve as a proxy for lifelong exposure.
In this study, we used the Mendelian randomization design to investigate the long-term effect
of milk intake on acne using the lactase persistent (TT + TC)/non-persistent(CC) LCT-13910 C/T
genotype in 20,416 adult individuals from The Danish General Population study (GESUS).
2.1. Participants
The cross-sectional population study The Danish General Population Study (GESUS) was
conducted from January 2010 to October 2013 in Naestved Municipality, Denmark [19]. Criteria
for invitation were age 20+, Danish citizenship and Danish Civil Registration number (CPR number).
All persons aged 30+ were invited and in the age group 20–30 years only a random 25% selection
were invited. 21,205 adults were enrolled, with an overall participation rate of 43%.
In this present study, 20,850 persons was included of whom 98.9% were of Danish descent and
the rest other Scandinavian or European descent. Individuals with missing values for acne diagnosis
(n = 53) or LCT-13910 C/T genotyping (n = 381) were excluded, resulting in inclusion of 20,416 people.
A prerequisite for attending the health examination was a completed self-reported paper-
questionnaire about demographic information, medical history, smoking, skin-condition, and food
intake, among others. The health examination was performed by trained health professionals and took
place at the department of clinical biochemistry at Naestved University Hospital, Denmark. Body mass
index (BMI) was calculated as kg/m2 . Details about the study design of GESUS have been described
elsewhere [19].
Written informed consent was obtained from all participants. The study conforms to the principles
of the Declaration of Helsinki and is approved by the institutional review board, the ethical committee
of Region Zealand (SJ-113, SJ-114, SJ-191) and the Danish Data Protection Agency.
2.2. Milk Intake

Intake of milk was reported in the questionnaire as: “How much milk do you averagely consume
per week?” and the possible answers were glasses of whole milk (3.5% fat), semi-skimmed milk
(0.5–1.5% fat), skimmed milk (0.1–0.3% fat), butter milk, and lactose free milk. A blank response
in one variable was set as no intake, when any of the other variables were filled. Extreme values
were confirmed/deferred by contacting the participants by phone [19]. Milk intake was divided into
categorical variables, based on glasses of milk per day. Dichotomized variables for types of milk
intake were performed to compare no milk intake with intake of low-fat (0.1–0.3%) and high-fat milk
(0.5–3.5%).
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Nutrients 2018, 10, 1041
2.3. Acne Diagnosis

The acne diagnosis was based on the self-reported questionnaire validated by Dalgard et al. [20]
The questions in the questionnaire were as follows: “During the last week, have you had any of the
following complaints?” one of the complaints was pimples and the possible answers were: No (0);
Yes, a little (1); Yes, quite a lot (2) or Yes, very much (3). The criteria used for the diagnosis of acne was
answering “Yes, quite a lot (2)” or “Yes, very much (3)”. The validation study by Dalgard et al. showed
a high specificity (96%) for a non-healthcare seeking population, but a low sensitivity (<50%) [20].
2.4. Genotyping
Every participant in the GESUS study was genotyped for LCT-13910 C/T variant (rs4988235).
The genotyping was done by KASPar allelic discrimination (LGC Genomics) with a call rate of
99% [21]. The genotype LCT-13910 TC and TT are lactase persistent and CC lactase non-persistent.
The LCT-13910 C/T genotype distribution, in the GESUS population, was in Hardy-Weinberg
equilibrium (Supplementary Table S1).
2.5. Statistical Analyses

Statistical analyses were performed in SAS Enterprise Guide 7.1 (SAS institute Inc., Cary, NS, USA).
The descriptive statistics of all the continuous variables showed normal distributions, except for milk
intake. Transformation of the variable was performed, but this did not improve normal distribution
substantially. Milk intake was instead categorized into quantiles (0, 1–3, 4–7, 8–14, >14 glasses/week)
and dichotomized variables were performed for total milk, low-fat milk and high-fat milk. (any vs.
none). Chi-square test and analysis of variance (ANOVA) was used to test relationships for categorical
variables and differences in means. A p-value < 0.05 was considered significant. The Mendelian
randomization design consisted of three analyses: First, logistic regressions were performed to test
the observational associations of milk intake and acne. The logistic regressions were stratified by
age, based on an empirical data description of milk intake and acne diagnosis (Supplementary Table
S2). This resulted in the two age groups; 20–39 years and 40+ years. Furthermore, stratification by
gender was performed, because of interaction with milk intake (p = 0.003) The logistic regressions
were performed both unadjusted and adjusted for age, body mass index (BMI) and smoking, as BMI
and smoking are related to acne in the literature [22,23]. Second, the median milk intake was studied
for the LCT-13910 C/T genotypes and the difference was tested with the Kruskal-Wallis test. Finally,
logistic regressions were performed for the LCT-13910 C/T genotypes and acne. An interaction test
was performed for milk intake (no/yes) and lactase genotype in an additive (TT; TC; CC) and dominant
(TT/TC; CC) model in both age groups.
2.6. Meta-Analysis of Acne in Adults

The aim was to meta-analyze the association between milk intake and acne in adults.
The search was performed on 11 December 2017 and included all studies up until that date.
Studies were identified in the PubMed database using the search terms: (“Dairy products”[Mesh]
OR “dairy”[All Fields] OR “milk”[Mesh] OR “milk”[All Fields] OR yogurt[All Fields] OR cheese[All
Fields] OR lifestyle[All Fields]) AND (“Acne Vulgaris”[Mesh] OR “Acne”[All Fields]). We identified
241 records. Inclusion criteria was mean age ≥ 30 years, case (acne) and control (non-acne) groups,
and information on odds ratio (95%CI) or raw numbers to calculate the odds ratio. Milk intake was
defined as binary (yes, no), low-fat (yes/no), or high-fat (whole) milk (yes/no). We identified two
studies of milk intake and adult acne [24,25], but only study met the inclusion criteria for case-control
group design [25] which we meta-analyzed with our own results. We calculated pooled fixed and
random effects odds ratios.
117
Nutrients 2018, 10, 1041
3. Results
3.1. Baseline Characteristics

Baseline characteristics of the 20,416 participants by acne, age group and LCT-13910 genotype
are presented in Table 1 and Supplementary Table S2. The acne group consisted of 303 participants,
which were significantly younger, had a higher percentage of smokers, as well as a higher mean milk
intake. The group aged 20–39 years had significantly fewer men, lower BMI, higher prevalence of
acne, and a higher milk intake, compared to the group aged 40+. The lactase genotype groups were
significantly different, with a higher BMI and milk intake in the lactase persistent TC/TT genotypes,
compared to the lactase non-persistent genotype (CC).
3.2. Milk Intake and Acne

The adjusted observational odds ratio for acne in individuals with any milk intake vs. no milk
intake was 0.93(95% confidence interval: 0.48–1.78) in females and 0.49(0.22–1.08) in males aged
20–39 years, and 1.15(95% confidence interval: 0.66–1.99) in females and 1.02(0.61–1.72) in males above
40 years (Tables 2 and 3). Results were similar for unadjusted analyses.
3.3. Lactase Genotype and Milk Intake

In the age group 20–39 years, the median milk intake (Table 4) for the lactase persistent genotypes
was 10 glasses/week (inter-quantile range (IQR) [4:16]) for TT and 10 glasses/week [3:16] for TC
compared to 7 glasses/week [2:14] for the non-persistent lactase genotype CC (p = 6.32 × 10−4 ).
In the age group 40+ (Table 4), results were similar but attenuated with a median milk intake of 5
glasses/week [IQR: 0:10] for TT and 6 glasses/week [0:14] for TC compared to 3 glasses/week [0:14]
for CC (p = 3.78 × 10−12 ).
3.4. Lactase Genotype and Acne

The unadjusted odds ratio for acne in individuals with the lactase persistent genotypes TC/TT
vs. the lactase non-persistent genotype CC was 0.84 (0.43:1.62) in the age group 20–39 years, and 0.99
(0.52–1.88) above 40 years. (Table 5). No interactions were found for the lactase genotype and milk
(no/yes) for both the additive and dominant model, in both age groups.
3.5. Meta-Analysis of Milk Intake and Adult Acne

Combining all age groups in our own study with the study by Landro [25], the pooled fixed
effects odds ratios for acne was 1.04(0.79–1.37) for any milk intake (yes/no), 1.05(0.70–1.58) for whole
milk, and 1.02(0.78–1.34) for low-fat milk intake (Table 6).
118
Table 1. Baseline characteristics for The Danish General Suburban Population study (GESUS) by acne, age groups, and LCT-13910 genotype.
LCT-13910 Genotype
All Acne Control Age Age > 40
n = 20,416 n = 303 n = 20,113 20–39 n = 17,674 CC TC TT
p-Value * p-Value * p-Value
100% 1.5% 98.5% n = 2742 86.6% n = 1246 n = 7377 n = 11,793
**
13.4% 6.1% 36.1% 57.8%
Nutrients 2018, 10, 1041
Acne, 303 141 162 20 119 164

- - - <0.0001 0.4523
n (%) (1.5) (5.1) (0.9) (1.6) (1.6) (1.4)
Age, 56.3 44.2 56.5 35.0 59.7 55.7 56.4 56.4
<0.0001 <0.0001 0.1892
mean (SD), years (13.6) (13.2) (13.5) (4.0) (11.3) (13.2) (13.6) (13.6)
Age: 20–39, 2742 141 2601 166 955 1621
<0.0001 - - -
n (%) (13.4) (46.5) (12.9) (6.1) (34.8) (59.1) 0.2851
Age: > 40, 17674 162 17512 1080 6422 10172
<0.0001 - - -
n (%) (86.6) (53.5) (87.1) (6.1) (36.3) (57.6)
Men, 9294 125 9169 1193 8101 540 3332 5422
0.1327 0.0228 0.1522
n (%) (45.5) (41.3) (44.9) (43.5) (45.8) (43.3) (45.2) (46.0)
Body Mass Index, 26.7 26.9 26.7 25.9 26.9 26.5 26.6 26.8
0.4549 <0.0001 0.0290
mean (SD), kg/m2 (4.7) (5.6) (4.7) (4.9) (4.6) (4.7) (4.6) (4.7)
119
Current Smoker, 3632 75 3557 490 3142 233 1318 2081
0.0014 0.9060 0.6371
n (%) (17.8) (24.8) (17.7) (17.9) (17.8) (18.7) (17.9) (17.7)
Milk Intake, 8.1 10.2 8.1 11.0 7.7 6.1 8.2 8.3
<0.0001 <0.0001 <0.0001
mean (SD), glasses/week (9.1) (14.3) (9.0) (9.9) (8.9) (7.1) (9.1) (9.4)
* The chi-square test or the analysis of variance (ANOVA) test is used to calculate the p-value. ** Rao-Scott modified chi-square test is used for categorized variables and ANOVA for
continuous variables. LCT-13910 genotype (rs4988235): CC = the lactase non-persistent genotype, TC/TT = the lactase persistent genotype.
Table 2. Odds ratio for acne by milk intake in the age group 20–39 years.
Female Male
Milk Intake
Unadjusted Adjusted * Unadjusted Adjusted *
(glasses/week) n Total n Acne n Total n Acne
OR [95% CI] p OR [95% CI] p OR [95% CI] p OR [95% CI] p
0 172 11 1.00 - 1.00 - 122 8 1.00 - 1.00 -
Nutrients 2018, 10, 1041
1–3 274 19 1.09 [0.51:2.35] 0.82 0.98 [0.45:2.13] 0.96 147 4 0.40 [0.12:1.36] 0.14 0.43 [0.13:1.47] 0.18
4–7 375 25 1.05 [0.50:2.18] 0.91 0.98 [0.47:2.06] 0.96 186 11 0.90 [0.35:2.29] 0.82 0.93 [0.36:2.41] 0.89
8–14 397 25 0.98 [0.47:2.05] 0.96 0.90 [0.43:1.90] 0.79 322 7 0.32 [0.11:0.89] 0.03 0.36 [0.13:1.02] 0.06
>14 331 20 0.94 [0.44:2.01] 0.88 0.85 [0.39:1.83] 0.67 416 11 0.39 [0.15:0.98] 0.05 0.40 [0.16:1.02] 0.05
Any 1377 89 1.01 [0.53:1.93] 0.97 0.93 [0.48:1.78] 0.82 1071 33 0.45 [0.20:1.00] 0.05 0.49 [0.22:1.08] 0.08
Low-fat ** 1348 85 0.83 [0.47:1.48] 0.53 0.84 [0.47:1.52] 0.57 1031 31 0.47 [0.23:0.98] 0.04 0.49 [0.23:1.03] 0.06
High-fat *** 58 7 2.06 [0.91:4.67] 0.08 1.42 [0.55:3.69] 0.47 82 5 1.94 [0.74:5.08] 0.18 2.01 [0.75:5.38] 0.17
* Adjusted for age, smoking and body mass index, ** Low-fat (0.1–0.3%), *** High-fat (0.5–3.5%). p-values are calculated as analysis of maximum likelihood estimates.
Table 3. Odds ratio for acne by milk intake for in the age group ≥40 years.
Female Male
120
Milk Intake
Unadjusted Adjusted * Unadjusted Adjusted *
(glasses/week) n Total n Acne n Total n Acne
OR [95% CI] p OR [95% CI] p OR [95% CI] p OR [95% CI] p
0 2802 17 1.00 - 1.00 - 2099 20 1.00 - 1.00 -
1–3 1564 16 1.69 [0.85:3.36] 0.13 1.30 [0.65:2.59] 0.46 1099 10 0.95 [0.45:2.05] 0.90 0.91 [0.42:1.96] 0.80
4–7 2129 22 1.71 [0.91:3.23] 0.10 1.40 [0.74:2.65] 0.31 1592 13 0.86 [0.42:1.73] 0.66 0.82 [0.40:1.68] 0.59
8–14 1816 11 1.00 [0.47:2.14] 1.00 0.76 [0.35:1.63] 0.48 1682 23 1.44 [0.79:2.63] 0.23 1.31 [0.71:2.43] 0.39
>14 1262 12 1.57 [0.75:3.30] 0.23 1.13 [0.53:2.40] 0.75 1629 18 1.16 [0.61:2.20] 0.65 0.99 [0.51:1.91] 0.97
Any 6771 61 1.49 [0.87:2.55] 0.15 1.15 [0.66:1.99] 0.62 6002 64 1.12 [0.68:1.86] 0.66 1.02 [0.61:1.72] 0.94
Low-fat ** 6324 58 1.49 [0.90:2.49] 0.12 1.14 [0.68:1.91] 0.63 5268 57 1.14 [0.72:1.80] 0.59 0.98 [0.61:1.58] 0.94
High-fat *** 590 4 0.82 [0.30:2.26] 0.70 0.97 [0.35:2.68] 0.95 901 10 1.08 [0.56:2.10] 0.82 1.25 [0.64:2.46] 0.51
* Adjusted for age, smoking and body mass index, ** Low-fat (0.1–0.3%), *** High-fat (0.5–3.5%). p-values are calculated as analysis of maximum likelihood estimates.
Table 4. Differences in milk intake, glasses per week, by the LCT-13910 C/T genotype.
LCT-13910 Genotype Lactase n % Median IQR Kruskal-Wallis Test

Age Group 20–39 Years
CC Non-persistent 166 6 7 [2:14] Chi-Square 14.73
TC Persistent 955 35 10 [3:16] DF 2
Nutrients 2018, 10, 1041
TT Persistent 1621 59 10 [4:16] P 6.32 × 10−4

Age Group ≥ 40 Years
CC Non-persistent 1080 6 3 [0:14] Chi-Square 52.83
TC Persistent 6422 36 6 [0:14] DF 2
TT Persistent 10,172 58 5 [0:10] P 3.38 × 10−12
IQR: Inter-quartile range.
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Nutrients 2018, 10, 1041
Table 5. Odds ratio for acne by LCT-13910 C/T genotypes.
Median Milk Intake [IQR]

LCT-13910 C/T Genotype Glasses/Week n Total n Acne OR [95% CI] p-Value
Age Group 20–39 Years
CC 7 [2:14] 166 10 1 -
TC 10 [3:16] 955 50 0.86 [0.43:1.74] 0.68
TT 10 [4:16] 1621 81 0.82 [0.42:1.62] 0.57
TC/TT 10 [3:16] 2576 131 0.84 [0.43:1.62] 0.60
LCT gene (additive model) × milk (no/yes) interaction test 0.36
LCT gene (dominant model) × milk (no/yes) interaction test 0.15
Age Group ≥ 40 Years
CC 3 [0:10] 1080 10 1 -
TC 6 [0:14] 6422 69 1.16 [0.60:2.26] 0.67
TT 5 [0:14] 10,172 83 0.88 [0.46:1.70] 0.70
TC/TT 5 [0:14] 16,594 152 0.99 [0.52:1.88] 0.97
LCT gene (additive model) × milk (no/yes) interaction test 0.32
LCT gene (dominant model) × milk (no/yes) interaction test 0.37
Table 6. Meta-analysis of milk intake and acne in adults.
Low Upper
Dairy Author Year OR p-Value
95% CI 95% CI
Any intake Di Landro [25] 2016 0.88 0.61 1.28 0.52
Any intake Juhl Current 1.27 0.84 1.92 0.26
Fixed effects pooled odds ratio 1.04 0.79 1.37 0.78
Random effects pooled odds ratio 1.05 0.74 1.49 0.80
Whole milk Di Landro [25] 2016 0.84 0.49 1.44 0.53
Whole milk Juhl Current 1.43 0.76 2.70 0.27
Low-fat milk Di Landro [25] 2016 0.90 0.61 1.32 0.59
Low-fat milk Juhl Current 1.15 0.79 1.68 0.47
4. Discussion
Among 20,419 adults from the Danish general population we found no association between milk
intake and acne, observationally or genetically using the lactase persistent/non-persistent LCT-13910
C/T genotype in a Mendelian randomization design.
In the Mendelian randomization design the genetic variant is used as a proxy for the long-term
differences in milk intake, thereby largely avoiding confounding and reverse causation, which can blur
or distort the underlying true association in observational studies [18]. The LCT-13910 fulfilled the
requirements for using the Mendelian randomization design, as the variant is linked to the intermediate
phenotype (milk intake) in a biologically explainable way [17], there is no known pleiotropic effects
of the variant, and the variant was not associated with confounders. The Mendelian randomization
design mimics a randomized clinical trial and takes advantage of the random assortment of alleles at
conception which ensures random distribution of confounding factors thereby circumventing reverse
causation and most confounding. Thus, the Mendelian randomization design provides an estimate of
the long-term effect of milk intake on acne.
We showed that both the milk consumption and the acne diagnosis declined with age, and that
fewer people drank milk as age increased. Nevertheless, we found a crude prevalence of self-reported
acne of 6%. We also investigated milk intake and acne among adults by combining our current
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Nutrients 2018, 10, 1041
study with findings from Landro , but the associations were still null for any milk intake, whole
milk, and low-fat milk. In contrast, observational studies of mostly childhood and adolescent acne
have debated, but largely favored, the association between milk intake and acne [11–13,16,26–29].
These studies were heterogeneous with respect to geographical location, cultural dairy influence,
gender, sample size, reporting of dairy frequency and type, and the ascertainment of participants
(dermatology clinics, general population study, online questionnaire). Some of the studies did not run
adjusted analyses [16,26,27], thus confounding may account for the associations. However, reasons for
the discrepant findings between our study and the study by Landro [25] in adults versus the studies in
adolescent acne [11–13,16,26–29] could also be related to different pathogenesis and the appearance of
acne in different age groups. Milk intake increases levels of insulin-like growth factor-1 (IGF1), which is
hypothesized to be a central link between milk intake and stimulation of the sebaceous gland [30].
Thus, as people age and drink less milk, they may be less exposed to IGF1 and, therefore, also less prone
to the development of acne. The appearance of acne varies by age, such that acne precox and acne
tarda are mostly variants with predominant inflammatory papules, pustules and nodules at the lower
face half [5,31], while adolescent acne is mostly characterized by comedoes and papules/pustules at
the entire face (chest and back) [7]. In contrast, acne in adult smokers is characterized by comedoes
and scars [8] and acne fulminans exhibits a severe clinical picture [32]. Even though the acne diagnosis
was questionnaire based as in many previous observational studies [16,28], it has been validated in
a comparable setting with a high specificity but with a sensitivity of only 50% [20]. However, we did
not have information on anatomical acne location or longer period of acne appearance. Additionally,
our study spanned four years including all seasons, thereby compensating for seasonal variation in
acne appearance [33].
The strength of our study is the homogenous population of largely Danish descent or other
Scandinavian descent making confounding from population substructure less likely. Similarly, the
rs4988235 variant is the most common lactase persistent variant in populations of Northern European
descent, despite the many other lactase persistence haplotypes [34]. A limitation of our study included
the low prevalence of lactase non-persistence (6%) and the fact that some of these individuals drank
milk, which could offset and conceal a true association. Milk consumption was based on self-reported
questionnaire data, with possible recall bias.
5. Conclusions
In conclusion, in the Danish General Suburban Population Study (GESUS) of adults we did
not find any observational or genetic association between milk intake and acne using the lactase
persistent/non-persistent LCT-13910 C/T genotype in a Mendelian randomization design.

s1, Table S1: Hardy-Weinberg equilibrium test; Table S2: Characteristics by age groups.
Author Contributions: Conceptualization, C.E., G.B.J., H.K.M.B., I.M.M., C.R.J., J.K.K.; Methodology, C.E.,
G.B.J., H.K.M.B., I.M.M., C.R.J., J.K.K.; Formal Analysis, C.R.J. , J.K.K., C.E.; Data Curation, C.E. and H.K.M.B.;
Writing-Original Draft Preparation, C.R.J.; Writing-Review & Editing, C.E., G.B.J., H.K.M.B., I.M.M., C.R.J., J.K.K.;
Visualization, C.R.J.; Supervision, C.E., G.B.J., H.K.M.B., I.M.M., C.R.J., J.K.K.; Project Administration, C.E., C.R.J.,
J.K.K.; Funding Acquisition, G.B.J., C.E., J.K.K.
Funding: C.R.J. was funded by Region Zealand. The Danish General Suburban Population Study was funded
by the Region Zealand Foundation, Naestved Hospital Foundation, Edith and Henrik Henriksens Memorial
Scholarship, Johan and Lise Boserup Foundation, TrygFonden, Johannes Fog’s Foundation, Region Zealand,
Naestved Hospital, The National Board of Health, and the Local Government Denmark Foundation.
Conflicts of Interest: The authors declare no conflict of interest. HKMB was partly funded by the Danish Dairy
Research Foundation from 2012–2015; the foundation was not involved in this study.
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nutrients
Article
Dairy Intake and Acne Vulgaris: A Systematic Review
and Meta-Analysis of 78,529 Children, Adolescents,
and Young Adults
Christian R. Juhl 1 , Helle K. M. Bergholdt 2 , Iben M. Miller 3 , Gregor B. E. Jemec 3,4 ,
Jørgen K. Kanters 1, * and Christina Ellervik 2,4,5,6, *
1 Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen,
2100 Copenhagen, Denmark; [email protected]
2 Department of Production, Research, and Innovation, Region Zealand, 4180 Sorø, Denmark;
[email protected]
3 Department of Dermatology, Zealand University Hospital, 4000 Roskilde, Denmark;
[email protected] (I.M.M.); [email protected] (G.B.E.J.)
4 Department of Clinical Medicine, Faculty of Health and Medical Sciences, University of Copenhagen,
2100 Copenhagen, Denmark
5 Department of Laboratory Medicine, Boston Children’s Hospital, 300 Longwood Avenue,
Boston, MA 02115, USA
6 Department of Pathology, Harvard Medical School, Boston, MA 02115, USA
* Corresponding Authors: [email protected] (J.K.K.);
[email protected] or [email protected] (C.E.)
Received: 15 July 2018; Accepted: 7 August 2018; Published: 9 August 2018
Abstract: A meta-analysis can help inform the debate about the epidemiological evidence on dairy
intake and development of acne. A systematic literature search of PubMed from inception to
11 December 2017 was performed to estimate the association of dairy intake and acne in children,
adolescents, and young adults in observational studies. We estimated the pooled random effects
odds ratio (OR) (95% CI), heterogeneity (I2 -statistics, Q-statistics), and publication bias. We included
14 studies (n = 78,529; 23,046 acne-cases/55,483 controls) aged 7–30 years. ORs for acne were
1.25 (95% CI: 1.15–1.36; p = 6.13 × 10−8 ) for any dairy, 1.22 (1.08–1.38; p = 1.62 × 10−3 ) for full-fat
dairy, 1.28 (1.13–1.44; p = 8.23 × 10−5 ) for any milk, 1.22 (1.06–1.41; p = 6.66 × 10−3 ) for whole
milk, 1.32 (1.16–1.52; p = 4.33 × 10−5 ) for low-fat/skim milk, 1.22 (1.00–1.50; p = 5.21 × 10−2 ) for
cheese, and 1.36 (1.05–1.77; p = 2.21 × 10−2 ) for yogurt compared to no intake. ORs per frequency
of any milk intake were 1.24 (0.95–1.62) by 2–6 glasses per week, 1.41 (1.05–1.90) by 1 glass per day,
and 1.43 (1.09–1.88) by ≥2 glasses per day compared to intake less than weekly. Adjusted results were
attenuated and compared unadjusted. There was publication bias (p = 4.71 × 10−3 ), and heterogeneity
in the meta-analyses were explained by dairy and study characteristics. In conclusion, any dairy,
such as milk, yogurt, and cheese, was associated with an increased OR for acne in individuals aged
7–30 years. However, results should be interpreted with caution due to heterogeneity and bias
across studies.
Keywords: meta-analysis; dairy; milk; acne; yogurt
1. Introduction
Acne is a common chronic inflammatory skin disease of sebaceous follicles [1,2]. Clinically,
acne is characterized by the presence of open and closed comedones, papules, pustules, and dermal
tissue damage with eventually heavy scar formation. Follicular hyperkeratosis, modifications of the
sebofollicular microbiome, increase production of sebum with increased amounts of pro-inflammatory

Nutrients 2018, 10, 1049
monounsaturated fatty acids, and Th17-cell-mediated inflammatory responses are all involved in acne
pathogenesis. Sebum production can be induced by insulin-like growth factor-1 (IGF-1) and androgens,
whose adrenal and gonadal synthesis is stimulated by IGF-1 [3]. Although prevalence varies across
studies, acne is common in children and adolescents aged 12–24 years and is moderate to severe in
15–20% of cases [1,4–6].
Heritability of acne alone does not explain high acne prevalence rates of over 80% in western
countries [5,7]. It has long been debated if a Western diet per se or specific dietary components
contribute to the prevalence and severity of acne [4,8]. This has predominantly been investigated
in observational studies and only a few trials exist [9]. In particular, dairy products have been
incriminated. Milk-derived amino acids promote insulin secretion and induce hepatic insulin-like
growth factor-1 (IGF-1) synthesis [10]. IGF-1 has been suggested as the pivotal driver of acne and
stimulates follicular epithelial growth and keratinization [11–13]. IGF-1 gene polymorphism has been
shown to increase susceptibility to acne [14] and IGF-1 plasma levels correlate with acne severity [12].
Several worldwide observational studies have been published on dairy intake and acne in children,
adolescents, and young adults (7–30 years) in various countries [15–27]. Some narrative and systematic
reviews about dairy intake and acne have been published [4,9,28]. Recently, a meta-analysis of dairy
and acne was published [29] but with several methodological flaws, including lack of bias assessment
and inadvertent double-counting of studies due to duplicate publications [19,23,30,31] that caused
inappropriate weighting of results and skewed pooled estimates. So far, no previous meta-analysis has
statistically combined the observational studies in an attempt to estimate the effect of the association
of dairy intake and acne with the heterogeneity across studies, a bias assessment, a stratified analysis
by study characteristics, and publication bias.
The primary objective of this study was therefore to perform a meta-analysis to estimate
the association of acne in children, adolescents, and young adults consuming any dairy products.
Furthermore, our aim was to explore the association between acne and intake of varies types of dairy
(milk, yogurt, cheese), dairy subgroups (full fat, low fat, skim), and various amounts and frequencies of
dairy intake (times per week or day).
2. Methods
This systematic review and meta-analysis was undertaken according to Meta-analysis of
Observational Studies in Epidemiology (MOOSE) guidelines and according to a specified protocol
(Supplementary Materials). The search, selection of studies, full-text reading, and data extraction were
performed by CRJ and verified by CE.
2.1. Search Strategy

The search was performed on 11 December 2017 and included all studies up until that date.
Studies were identified in the PubMed database using the search terms: (“Dairy products”[Mesh] OR
dairy[All Fields] OR milk[Mesh] OR milk[All Fields] OR yogurt[All Fields] OR cheese[All Fields] OR
lifestyle[All Fields]) AND (“Acne Vulgaris”[Mesh] OR Acne[All Fields]). We identified 241 records.
2.2. Eligibility Criteria

All observational studies (case-control, cross-sectional, population-based, retrospective) on
childhood, adolescent, or young adult acne (max age of 30 years) were eligible if they reported
a risk estimate and a 95% confidence interval for acne in a dairy group vs. a non-dairy group, or the
raw numbers from 2 by 2 tables of dairy intake and acne.
2.3. Procedure for Selection of Studies

We screened the title and abstracts of 241 articles (Figure 1). If relevant, we retrieved the full-text
articles. We identified 25 full-text articles, but excluded the following 11 studies: duplicate [19]
(there was a statement in the article by Grossi that it was the same cohort and results as [23]),
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Nutrients 2018, 10, 1049
beliefs/opinions about acne aggravating food items [32,33], semi-fat/whole milk vs. skim milk/no
milk drinkers [34], Chinese ying-yang medicine [35], no control group [36], adult acne (mean age
≥ 30 years) [37,38], milk as part of a Mediterranean diet [39], milk only as a continuous variable
in acne and non-acne groups [40], and poorly defined intake [41]. In total, we included 14 studies.
Two other studies were identified outside the search, but these studies were duplicates and published
simultaneously without a clear statement of which one was the original; therefore, we did not include
these papers [30,31]. The study selection process is shown in a flow diagram (Figure 1).
Figure 1. Flow diagram for meta-analysis.
2.4. Data Extraction and Management

We extracted the following data for each study and entered the information in an excel spreadsheet:
author, year, population, country, age, gender, study design, how outcome was estimated, dairy type
(dairy, milk, yogurt, cheese), dairy subtype (whole (full-fat), low-fat, skim), dairy amount, frequency
of intake (times per day or week), numbers of acne patients and controls subjects in each category of
dairy intake, crude and/or adjusted odds ratio (OR) or prevalence ratio with 95% confidence interval
(CI), raw numbers to calculate crude OR (95% CI).
2.5. Overall and Subgroup Analyses

The primary objective was to perform a meta-analysis to estimate the odds ratio of acne in children,
adolescents, and young adults consuming any dairy compared to those who do not. The secondary
objective was to estimate the odds ratio of acne associated with intake of varies types of dairy (milk,
yogurt, cheese), dairy subgroups (full-fat, low-fat, skim), and various amounts and frequencies of
dairy intake (times per week or day) compared to those who did not consume any dairy/milk.
2.6. Risk of Bias and Study Quality Assessment

The quality of each study was evaluated and scored using the nine-star Newcastle-Ottawa Scale
(NOS), a tool used for quality assessment of nonrandomized studies [42]. Studies were evaluated
based on selection, comparability, exposure, and outcome, and scored by a maximum of nine points.
Scores above five indicate moderate to high study quality. The NOS for cohort and case-control studies
was retrieved from [43].
2.7. Statistical Analyses

The meta-analyses were performed with STATA SE 14.0 (Stata Corp., College Station, TX, USA).
Using raw numbers, we calculated the crude odds ratios OR (95% CI). Analyses were performed for
any dairy intake, any milk intake, full-fat dairy, whole milk, and low-fat/skim milk compared to those
who did not consume any dairy/milk (study specific definitions). For any milk intake, whole milk and
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low-fat/skim milk, analyses of frequencies (times per week or day) were performed using studies by
Adebamowo et al. [15–17], as these studies had identical ascertainment of the frequency of milk intake.
DerSimonian and Laird (D + L) pooled random effects estimates were used. We also present inverse
variance (I-V) fixed effects in supplementary Figures. Heterogeneity was assessed by Cochrane Q
statistic test and I2 -statistical analysis. The I2 -statistical analysis assess what proportion of the observed
variance reflects variance in true effect sizes rather than sampling error [44]. Publication bias was
examined visually by funnel plots and statistically using Egger’s test (one-sided) [45] and by using the
Duval and Tweedie's Trim and Fill to simulate where potential unpublished studies would belong in
the funnel plot and to calculate a hypothetical new pooled odds ratio based on the added simulated
studies. Robustness of the meta-analysis was examined by “leaving-one-out” analysis. Publication
bias and robustness were carried out by use of the statistical program Comprehensive Meta-Analysis
(CMA) version 3 (Biostat, Englewood, NJ, USA) for any dairy intake vs. no dairy intake and any milk
intake vs. no milk intake. Four studies provided adjusted estimates for milk intake, with one study
providing them as odds ratios [46], and three studies as prevalence ratios [15–17]. In a sensitivity
analysis, we used only adjusted prevalence ratios from the studies by Adebamowo et al. [15–17].
Stratification on acne severity was not possible because of too few studies.
3. Results
3.1. Description of the Studies

In total, 14 studies were eligible. Figure 1 shows the flow diagram of the selection of articles for the
meta-analysis. The studies were published in 2005–2017 and included a total of 78,529 individuals of
which 23,046 had acne and 55,483 were controls (Table 1). The prevalence of acne ranged from 7–89%
in population studies and 36–83% in case-control studies. Two studies used non-acne dermatological
controls [23,26] and the rest used healthy controls. Five studies were cross-sectional [20,21,27,46,47],
five studies were case-control [18,22–24,26], one study was retrospective [15], and three studies were
longitudinal [16,17,25]. The age-group ranged from 7–30 years. Two studies were only in females [15,17],
three studies only in males [16,26,46], and the rest included both males and females. The studies covered
five continents: Africa [21], Asia [18,24,47], Europe [20,22,23,25–27], North America [15–17], and South
America [46]. Four studies included less than 1000 individuals in total [18,21–23,47], whereas the
rest ranged from 1285 to 46,879 individuals (Table 1). Four studies used the Willet food frequency
questionnaire [15–17,21]. In six studies, acne was self-reported in a questionnaire [15–17,20,25,27],
and in eight studies, acne was a physician verified diagnosis [18,21–24,26,46,47]. Five studies provided
adjusted estimates, including four on milk intake and one on dairy, two of the studies reported odds
ratios, and three studies reported prevalence ratios [15–17,25,46]. The reference group varied among the
articles and included not weekly [15–18,25], not daily [20,21,46], never [23,27], and unclear [24,26,47].
3.2. Findings
Random effects pooled unadjusted odds ratios for acne were 1.25 (95% CI: 1.15–1.36;
p = 6.13 × 10−8 ) for any dairy (Figure 2), 1.22 (1.08–1.38; p = 1.62 × 10−3 ) for full-fat dairy,
1.28 (1.13–1.44; p = 8.23 × 10−5 ) for any milk, 1.22 (1.06–1.41; p = 6.66 × 10−3 ) for whole milk,
1.32 (1.16–1.52; p = 4.33 × 10−5 ) for low-fat/skim milk, 1.22 (1.00–1.50; p = 5.21 × 10−2 ) for cheese,
and 1.36 (1.05–1.77; p = 2.21 × 10−2 ) for yogurt compared to those who did not consume these food
items (Figure 3 and Supplementary Figures S1–S6).
Random effects meta-analyses for acne by frequency of any milk intake compared to an intake
of ≤1 glass of milk per week showed an odds ratio of 1.24 (0.95–1.62) by 2–6 glasses per week,
1.41 (1.05–1.90) by 1 glass per day, and 1.43 (1.09–1.88) by ≥2 glasses per day for any milk; results for
whole milk and low-fat/skim milk were close (Supplementary Figures S7–S10).
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Table 1. Characteristics of included studies for the association of dairy intake with acne in children, adolescents, and young adults.
Age, Acne
First Author Year Study Population Design Gender Country Total n Acne n No Acne n Milk Variables Acne Diagnosis
Year (%)
Adebamowo Any milk, whole milk, low-fat
2005 Population cohort (Nurses’ Health Study II) Retrospective 13–18 F USA 46,879 3412 7.28 43,467 Q
[15] milk, skim milk
Adebamowo Population cohort (GUTS)—offspring of women in Any milk, whole milk, low-fat
2006 Follow-up 9–15 F USA 3756 2588 68.9 1168 Q
[17] the Nurses’ Health Study II milk, skim milk
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Adebamowo Population cohort (GUTS)—offspring of women in Any milk, whole milk, low-fat
2008 Follow-up 9–15 M USA 2759 1856 67.3 903 Q
[16] the Nurses’ Health Study II milk, skim milk
Cerman [22] 2016 Acne patients and healthy controls Case-control 19 F/M Turkey 86 50 58.1 36 Any milk D
Whole milk, low-fat milk,
Duquia [46] 2017 Acne patients vs. healthy controls in the army Cross-sectional 18 M Brasil 2201 1960 89.1 241 P
cheese, yogurt
Acne patients and non-acne dermatology Any milk, whole milk, skim
Grossi [23] 2016 Case-control 10–24 F/M Italy 563 205 36.4 358 D
patient controls milk, cheese/yogurt combined
Ismail [18] 2012 Acne patients and healthy controls Case-control 18–30 F/M Malaysia 88 44 50 44 Any milk, yogurt, cheese D
Acne patients and non-acne dermatology
Karadag [26] 2017 Case-control 21 M Turkey 4595 3836 83.5 759 Milk/cheese combined D
patient controls
South
Jung [24] 2010 Acne patients and age-matched healthy controls Case-control 24 F/M 1285 783 60.9 502 Cheese D
Korea
Okoro [21] 2016 Population cohort Cross-sectional 11–30 F/M Nigeria 450 292 64.9 158 Any milk D
South
Park [47] 2015 Population cohort Cross-sectional 7–12 F/M 693 251 36.2 442 Any milk, cheese, yogurt D
Korea
Ulvestad [25] 2016 Population cohort Follow-up 15–19 F/M Norway 2387 331 13.9 2056 Any milk, full-fat milk Q
Wolkenstein [20] 2015 Population cohort Cross-sectional 15–24 F/M France 2266 1375 60.7 891 Any milk Q
Whole milk, semi-skimmed
Wolkenstein [27] 2017 Population cohort Cross-sectional 15–24 F/M Europe * 10521 6063 57.6 4458 Q
milk, low-fat milk, dairy
130
Age: mean or range. Q: Questionnaire. D: Dermatologist verified. GUTS: Growing Up Today Study. P: Physician verified. USA: United States of America. * 7 countries: Belgium, Czech
and Slovak Republics, France, Italy, Poland, and Spain.
Nutrients 2018, 10, 1049
Figure 2. Meta-analysis of dairy intake and acne vulgaris: individual studies. The figure shows the
individual studies and the unadjusted pooled random effect estimate from the meta-analysis of dairy
intake and acne vulgaris. I2 (%): I-square heterogeneity expressed as percentage. p-value(het): p-value
from Cochran’s Q-statistic assessing heterogeneity. D + L: DerSimonian and Laird pooled random
effects estimates. See Table 1 for references.
Dairy Adjustment Gender Studies(N) Acne(N) Controls(N) Total(N) pvalue(het) I2(%) OR (95% CI) pvalue
Any dairy No All 14 23046 55483 78529 0.05 42 1.25 (1.15, 1.36) 6.13e-08
Full-fat dairy No All 7 7792 35344 43136 0.05 52 1.22 (1.08, 1.38) 1.62e-03
Any milk No All 11 18095 52164 70259 0.01 58 1.28 (1.13, 1.44) 8.23e-05
Whole milk No All 6 7452 33248 40700 0.03 59 1.22 (1.06, 1.41) 6.66e-03
Low-fat/skim milk No All 6 10672 21931 32603 0.06 53 1.32 (1.16, 1.52) 4.33e-05
Cheese No All 4 3037 1229 4266 0.54 0 1.22 (1.00, 1.50) 5.21e-02
Yogurt No All 3 2255 727 2982 0.77 0 1.36 (1.05, 1.77) 2.21e-02
Any milk Yes All 3 7856 45538 53394 0.21 36 1.15 (1.10, 1.20) 3.95e-09
Whole milk Yes All 3 4265 31893 36158 0.04 70 1.14 (1.05, 1.23) 1.97e-03
Low-fat/skim milk Yes All 3 4174 18394 22568 0.48 0 1.18 (1.13, 1.22) 2.71e-17
.8 1 2
Odds ratio (95%CI)
Figure 3. Meta-analyses of dairy intake and acne vulgaris: summary estimates. The figure shows the
unadjusted pooled random effects estimates from each of the meta-analyses, which can be found in the
supplementary material. I2 (%): I-square heterogeneity expressed as percentage. p-value(het): p-value
from Cochran’s Q-statistic assessing heterogeneity.
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Nutrients 2018, 10, 1049
3.3. Sensitivity Analyses, Heterogeneity, Publication Bias, and Qualitative Bias Assessment
The I2 heterogeneity ranged from 0–70% (Figure 2). To explore heterogeneity, we stratified the
analysis for any dairy intake and acne by age, gender, number of cases, continent, design, acne
diagnosis, and reference group (Supplementary Table S1, Supplementary Figures S11–S17). Stratifying
by age did not show any differences. Stratifying by gender showed similar odds ratios in males and
females, but meta-analyses of females had higher heterogeneity. Stratifying by the number of acne
cases showed that larger studies had smaller odds ratios with more narrow confidence intervals,
but higher heterogeneity compared to those of the smaller studies, but the confidence intervals were
overlapping. Stratifying analyses by continent showed that studies from Europe had the smallest
odds ratios, followed by North and South American studies, and with Asian and African studies with
the largest odds ratios. Stratifying by design removed heterogeneity and showed that prospective
studies had the largest odds ratios. Stratifying by ascertainment of acne diagnosis showed that studies
using self-reported acne as an outcome had higher heterogeneity compared to studies with physician
verified diagnoses of acne. Stratifying by reference group showed overall similar summary estimates,
but with the highest heterogeneity in studies with “less than weekly” being the reference group.
The Newcastle-Ottawa qualitative assessment scale of bias with similar items as in the statistical
heterogeneity assessments revealed scores of 2–5 in case-control studies [18,22–24,26] and 2–6 in cohort
studies [15–17,20,21,25,27,46,47] out of a potential max of 9 points (Supplementary Table S2).
Random effects pooled adjusted estimates for any milk, whole milk, and low-fat/skim milk were
similar but attenuated compared to their unadjusted estimates (Supplementary Figures S18–S20).
Leave-one-out analyses for any dairy or any milk intake did not show any gross deviations, but the
retrospective study by Adebamowo [15] influenced the summary estimates the most (Supplementary
Figures S21–S22). Funnel plot and p-value for Egger’s test revealed publication bias for any dairy
(p-Egger = 4.71 × 10−3 ) (Supplementary Figure S23); Duval and Tweedie’s Trim and Fill method
estimated that five studies were missing for “any dairy”, and the imputed point estimate would be
1.16 (1.06–1.28) had these five studies been added. Funnel plot and p-value for Egger’s test revealed
publication bias for any milk (p-Egger = 2.73 × 10−2 ) (Supplementary Figure S24); Duval and Tweedie's
Trim and Fill method estimated that one study was missing for “any milk”, and the imputed point
estimate would be 1.26 (1.11–1.44) had this study been added.
The New-Castle Ottawa qualitative assessment scale of bias revealed a scores of 2–5 in case-control
studies [18,22–24,26] and 2–6 in cohort studies [15–17,20,21,25,27,46,47].
4. Discussion
Intake of any dairy, any milk, full-fat dairy, whole milk, low-fat/skim milk, and yogurt regardless
of amount or frequency were associated with a higher odds ratio for acne compared to no intake in
individuals aged 7–30 years. Intake of cheese was associated with a borderline higher odds ratio for
acne compared to no intake. Stratifying the association of any milk by frequency of intake revealed
that intake of 1 glass of milk or more per day was associated with a higher odds ratio for acne,
whereas 2–6 glasses per week was not, compared to intake less than weekly. Stratified analyses for
any dairy intake and acne fat content demonstrated that full-fat dairy and whole milk had lower
odds ratios, whereas low-fat/skim milk had higher odds ratios than the overall summary estimates;
a likely explanation for this observation could be that the amount of milk consumed for low-fat/skim
milk is higher than that for whole milk. However, results should be interpreted with caution due to
heterogeneity and bias across studies.
The meta-analyses showed considerable heterogeneity reflecting the heterogeneous age and
gender of the participants, various study characteristics, ascertainment of information about milk
intake and acne, reporting of milk intake, and acne severity across the studies. In general, stratifying
on subgroups in sensitivity analyses revealed that heterogeneity diminished for most subgroups,
but also revealed that especially meta-analyses conducted on females, whole milk, North America,
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and questionnaire ascertained acne diagnosis demonstrated high heterogeneity. Prospective studies
and studies with physician-verified diagnosis of acne had low heterogeneity.
Despite the stratifications, confidence intervals were overlapping.
Stratifying on age and gender demonstrated similar odds ratios; however, the gender stratified
analyses had higher odds ratios than in the gender combined analyses. Smaller studies had higher
odds ratios than large studies, African and Asian studies had higher odds ratios than other studies,
and prospective designs had higher odds ratios than other designs. A recent multinational European
online questionnaire study in adolescents showed that acne prevalence did not differ by gender but
differed by country, and acne was more prevalent in younger people and obese people [27]. Intake of
milk varies globally and is largely dependent on genetically determined lactase persistency, which is
high in people of Northern European descent, but lower in people of Southern European descent,
patchy in Africa, and low in the Middle East and Asia [48]. The weaning of the lactase enzyme activity
usually happens in childhood and early adolescent years. How the age of weaning of the lactase
enzyme activity impacts acne development is not known.
We used random effects method in all meta-analyses, which includes between-study variance and
has a higher degree of statistical uncertainty built into the model. Thus, 95% confidence intervals are
wider compared to fixed effects models. Even in these models, the results of the meta-analyses were
significant. There was evidence of publication bias with Egger’s test with an overweight of smaller
studies overestimating the odds ratio compared to the pooled summary estimate. If the meta-analyses
had captured all the relevant studies, we would expect the funnel plots to be symmetric. The selective
reporting may be explained by studies with null-findings or negative results being deliberately not
published because of authors not submitting or editors rejecting them or authors not finding enough
merit in a potential publishable study [49]. Furthermore, some studies reported only the pooled
exposures for different dairy groups rather than showing the stratified results for each of the dairy
groups and/or for each reported frequency of intake [23,25,26], and some studies had only collected
an overall dairy or milk variable with no possibility for stratification [20,22]. However, the trim and fill
method did not change the overall estimates for “any dairy” or “any milk” remarkably.
There are many limitations of the included studies [4]. Self-reported acne with lack of
a physician verified diagnosis of acne [15–17,20,25] may lead to misclassification bias as validity
of self-reported acne is at best only moderate, with sensitivity of 55%, specificity of 72%, positive
predictive value of 70%, and negative predictive value of 57% [50]. Including other dermatology
patients as controls [26] may attenuate associations, as seborrhea may play a role in several
diseases. The observational studies were cross-sectional [20,21,46,47], case-control [18,22–24,26],
retrospective [15], or longitudinal [16,17,25]; thus, in most studies we cannot rule out reverse causation.
Questionnaire ascertainment of dairy intake varied between the articles and only a few studies used
validated food frequency questionnaires [15–17,21]. Despite the food questionnaire used, participants
may deliberately over- or underestimate (information bias) or not accurately remember (recall bias)
when filling out questionnaires about dairy intake and acne. Furthermore, it was not possible to
differentiate acne development, acne triggers, and severity of acne in the meta-analyses. Only a few
studies provided adjusted results [15–17,25] so we based most of the analyses on raw numbers,
which makes it difficult to rule out confounding from other dietary factors (e.g., glycemic index or
calorie intake) or other lifestyle factors previously associated with acne [4,9,28].
Acne prevalence varied remarkably across the included studies, between 7–89%. The retrospective
study by Adebamowo in 2005 with 7.3% acne cases focused on recall data provided by subjects in
the Nurses’ Health Study II (NHS), which were aged 25–42 years old in 1989 when information on
teenage acne was collected [15]; thus, the acne prevalence is likely underestimated and the results from
this study may not be representative. Furthermore, the studies from 2006 and 2008 were offspring
studies from the NHS in girls and boys [16,17]; however, leave-one-out analyses revealed that only the
Adebamowo 2005 study was an outlier [15].
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The observational studies may suffer from bias from confounding and reverse causation [9],
are unable to indicate causality of the relationship between dairy and acne, and unable to prove
preventive effects of abstaining from dairy. Only one study exists on milk intake and acne. The study
is uncontrolled and unblinded and is based on medical students who drank milk or consumed other
potential acne provoking foods. In addition, the total number of people with and without acne lesions
were counted for all foods combined, but with no formal statistical testing [51]. Thus, there is still
a knowledge gap with respect to whether dairy intake is causally associated with acne, acne flare, or
acne severity and to what extent. To answer this question, we would ideally need results from large
clinical randomized double-blind placebo-controlled trials (RCT); however, the question is whether
this is realistically possible ethically, clinically, and/or operationally. Another approach (which no
previous studies have yet undertaken) would be to perform a Mendelian Randomization study of
lactase persistence, dairy intake, and acne using genetic lactase persistence as a proxy for lifetime dairy
intake under the assumption that alleles are randomly distributed at conception [52,53]. Such a study
design mimics an RCT and allows for the causal estimate of dairy intake and acne.
The observational studies all assessed dairy intake as an isolated factor. However, dairy is
part of various individual and cultural specific diets and not a single factor with a single factor
prediction (“reductionist approach” [54]). Instead, other factors which can affect the bioactive
properties of nutrients in dairy and milk intake should be taken into consideration, such as macro-
and micronutrients (fat, protein, carbohydrates, vitamins, sodium, and minerals), the dairy structure
(liquid or solid), fermentation, and processing (holistic approach [55]). Only two studies in the
meta-analysis also reported the glycemic load and glycemic indices of food consumed in conjunction
with milk/dairy products [18,22], but did not report the glycemic load from the dairy consumption
specifically. Hyperglycemic carbohydrates enhance insulin signaling, which promotes insulin and
IGF-1 signaling, which in a synergistic fashion with milk stimulate mTORC1(mammalian target of
rapamycin complex 1) signal transduction [56]. There is accumulating evidence that acne belongs
to the spectrum of mTORC1-driven diseases of civilization including metabolic syndrome, obesity,
insulin resistance, and cancer [57]. A randomized trial has shown that a low-glycemic-load diet
improves symptoms in acne vulgaris patients [58]. Interestingly, no acne was observed in the Kitavan
Islanders (Pacific Ocean) and in the Ache Hunter-Gatherers from Paraguay, who live under Paleolithic
conditions without milk/dairy and hyperglycemic food, although it should be acknowledged that
many other differences exist to Western societies [59]. To present the pathological effects of milk in the
Western diet it is therefore important to provide controlled studies that consider milk consumption in
association with glycemic load and index as part of a mixed diet [60].
Recently, a meta-analysis of dairy and acne was published [29] but with several
methodological flaws, including the inadvertent double-counting of studies (Landro [19]/Grossi [23],
and Tsoy [30]/Tsoy [31]) due to duplicate publications, which caused inappropriate weighting of
results and skewed pooled estimates. Using the double-counted studies by Tsoy, the authors also only
used the most severe category of acne, which caused extremely high odds ratios of 10 and 12 to be
included in the meta-analysis, further skewing the pooled estimates. Furthermore, the meta-analysis
included a study by Agamia [41], which we decided to exclude as the intake of “milk and dairy
produce” was poorly defined as “low” and “high” intake but not defined with any frequency, type,
or amount of milk. The previous meta-analysis also did not provide evidence for the exact search
strategy to be replicated, for the bias assessment using the Newcastle Ottawa scale, for leave-one-out
analyses, or funnel plots of publication bias. As a comparison, in our meta-analysis, we included
the exact search string so it can be replicated, the heterogeneity across studies, a bias assessment
using the Newcastle-Ottawa scale presented with a table, a stratified analysis by study characteristics
presented in figures, the details of the “leave-one-out” analysis presented in figures, and the publication
bias presented in figures. Furthermore, we excluded duplicate studies, and we included four more
papers [24,26,27,47] that were not included in the previous meta-analysis but should have been as the
studies were published before the search for the previous meta-analysis was done in August 2017 [29].
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It is of crucial importance that authors of meta-analyses have a critical judgement of the reliability
and validity of the papers they consider including in a meta-analysis, otherwise the conduct and
assessment of systematic reviews may be hampered.
5. Conclusions
In conclusion, this meta-analysis of observational studies has provided new insight into the
direction and magnitude of the association between dairy intake and acne overall and by dairy type,
amount, and frequency. It has shed light on the knowledge gaps and the limitations of the studies
included compared to previous systematic and narrative reviews with no meta-analysis, heterogeneity
assessment, or bias assessment included [4,9,28].

s1, Figure S1: Meta-analysis of full-fat dairy intake vs. no dairy intake, Figure S2: Meta-analysis of any milk intake
vs. no milk intake, Figure S3: Meta-analysis of whole milk intake vs. no milk intake, Figure S4: Meta-analysis
of low-fat/skim milk intake vs. no milk intake, Figure S5: Meta-analysis of Cheese intake vs. no cheese intake,
Figure S6: Meta-analysis of yogurt intake vs. no yogurt intake, Figure S7: Meta-analyses of frequency of milk
intake and acne, Figure S8: Meta-analyses of amount of total milk intake, Figure S9: Meta-analyses of amount of
whole milk intake, Figure S10: Meta-analyses of amount of low-fat/skim milk intake, Figure S11: Meta-analyses
of any dairy intake vs. no dairy intake—by age group, Figure S12: Meta-analyses of any dairy intake vs. no dairy
intake—by gender group, Figure S13: Meta-analyses of any dairy intake vs. no dairy intake—by number of cases,
Figure S14: Meta-analyses of any dairy intake vs. no dairy intake—by continent, Figure S15: Meta-analyses of any
dairy intake vs. no dairy intake—by design, Figure S16: Meta-analyses of any dairy intake vs. no dairy intake—by
acne diagnosis, Figure S17: Meta-analyses of any dairy intake vs. no dairy intake—by reference group, Figure
S18: Meta-analysis of any milk intake vs. no milk intake—adjusted analysis, Figure S19: Meta-analysis of whole
milk intake vs. no milk intake—adjusted analysis, Figure S20: Meta-analysis of low-fat/skim milk intake vs. no
milk intake—adjusted analysis, Figure S21: Meta-analysis of any dairy intake vs. no dairy intake—leave one out
analysis, Figure S22: Meta-analysis of any milk intake vs. no milk intake—leave one out analysis, Figure S23:
Funnel plot of standard error by log odds ratio—any dairy intake vs. no dairy intake, Figure S24: Funnel plot
of standard error by log odds ratio—any milk intake vs. no milk intake, Table S1: Sensitivity analyses for the
association of dairy intake and acne, Table S2: Study-specific Newcastle-Ottawa quality assessment.
Author Contributions: C.R.J. and C.E. had full access to all the data in the study and take responsibility for the
integrity of the data and the accuracy of the data analysis. Study concept and design: all authors. Acquisition,
analysis of data: C.R.J. and C.E. Interpretation of data: all authors. Drafting of the manuscript: C.R.J. and C.E.
Critical revision of the manuscript for important intellectual content: all authors. Statistical analysis: C.R.J. and
C.E. Administrative, technical, or material support: N.A. Study supervision: G.B.E.J., J.K.K., and C.E.
Funding: C.R.J. was funded by Region Zealand. The Danish General Suburban Population Study was funded
by the Region Zealand Foundation, Naestved Hospital Foundation, Edith and Henrik Henriksens Memorial
Scholarship, Johan and Lise Boserup Foundation, TrygFonden, Johannes Fog’s Foundation, Region Zealand,
Naestved Hospital, The National Board of Health, and the Local Government Denmark Foundation.
Conflicts of Interest: G.B.E.J. has received honoraria from AbbVie, MSD, Pfizer, Pierre-Fabre, and UCB for
participation on advisory boards, and grants from Abbvie, Actelion, Janssen-Cilag, Leo Pharma, Novartis,
and Regeneron for participation as an investigator, and received speaker honoraria from AbbVie, Galderma, Leo
Pharma, and MSD. He has furthermore received unrestricted research grants from AbbVie and Leo Pharma. He
has received travel grants from AbbVie, Celgene, Desitin, and Novartis. GBEJ’s sponsors were not involved in
any parts of this article (design, conduct, collection, management, analysis, interpretation, preparation, approval,
review, decision to submit). H.K.M.B. has received a grant from the Danish Dairy Research Foundation. None of
the funding agencies had any role in the design, analysis, or writing of this article.
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138
nutrients
Article
Association between Fasting Glucose Concentration,
Lipid Profile and 25(OH)D Status in Children
Aged 9–11
Lukasz Szternel 1, *, Magdalena Krintus 1 , Katarzyna Bergmann 1 , Tadeusz Derezinski 2
and Grazyna Sypniewska 1
1 Department of Laboratory Medicine, Nicolaus Copernicus University, Collegium Medicum,
85094 Bydgoszcz, Poland; [email protected] (M.K.); [email protected] (K.B.); [email protected] (G.S.)
2 Outpatient Clinic, “Esculap”, 88140 Gniewkowo, Poland; [email protected]
* Correspondence: [email protected]; Tel.: +48-525-854-023; Fax: +48-525-854-024
Received: 18 August 2018; Accepted: 19 September 2018; Published: 22 September 2018
Abstract: Background: The aim of this study was to assess the relationship between vitamin D status
and the prevalence of dyslipidemia and impaired fasting glucose (IFG) in children. Methods and
Summary: 284 children (150 boys and 134 girls) aged 9–11 were included in the study. Children with
deficient 25(OH)D (25-hydroxycholecalciferol) levels ≤20 ng/mL (50 nmol/L) were characterized by
a more frequent occurrence of impaired fasting glucose (IFG) (Odd ratios (OR) = 1.966, 95% confidence
interval (CI): 1.055–3.663; p = 0.033) when compared to children with 25(OH)D >20 ng/mL. Serum
25(OH)D with concentration lower by 1 ng/mL (2.5 nmol/L) was linked to higher fasting glucose
(by 0.25 mg/dL, 0.013 mmol/L; p = 0.017), higher total cholesterol (TC) by almost 1 mg/dL
(0.96 mg/dL, 0.25 mmol/L; p = 0.006) and higher high-density lipoprotein cholesterol (HDL-C)
(by 0.57 mg/dL, 0.015 mmol/L; p < 0.001). Conclusion: 25(OH)D deficiency may negatively affect
fasting glucose and total cholesterol concentration in children aged 9–11. Vitamin D-deficient children
are twice as likely to develop prediabetes as reflected by impaired fasting glucose when compared to
those with a 25(OH)D level above 20 ng/mL (50 nmol/L).
Keywords: 25(OH)D deficiency; children; impaired fasting glucose; hypercholesterolemia
1. Introduction
The prevalence of obesity and vitamin D deficiency among children makes this population
demographic especially vulnerable to the development of these two pervasive epidemics [1]. Research
suggests an unhealthy diet coupled with a sedentary lifestyle has become the main causal factor
affecting the development of obesity, which in turn is frequently accompanied by dyslipidemia.
A significant amount of data supports the hypothesis that optimal vitamin D concentration is linked to
a favorable lipid profile and has a positive impact on glucose homeostasis [2]. Numerous observational,
epidemiological, and cross-sectional studies indicate an inverse correlation between the concentration
of 25(OH)D (25-hydroxycholecalciferol) and the rate of conversion from a prediabetes state to fully
symptomatic diabetes mellitus [3]. In the USA, the prevalence of prediabetes (or according to the World
Health Organization (WHO) definition, “intermediate hyperglycemia”) among adolescents aged 12–19
reached 13.1% between 2005 and 2006 [4]. It is estimated that annually 5%–10% of adults convert from
a prediabetes state to overt diabetes, despite the fact that reversion to normoglycemia is much more
common in children and adolescents. There are hypotheses indicating potentially beneficial effects
of vitamin D in preventing transformation to fully symptomatic diabetes mellitus [3]. There is no
consistent agreement as to whether vitamin D deficiency causes lipid abnormalities, or if these are just a
consequence of excess adipose tissue mass storing 25(OH)D molecules. The inflammatory process links

Nutrients 2018, 10, 1359
obesity and insulin resistance to the consequences of improper glucose homeostasis [5]. Vitamin D,
being a negative marker of inflammation, seems to be a trigger molecule in the development of fully
symptomatic metabolic syndrome [6]. The effects of vitamin D on lipid and carbohydrate metabolism
may only be fully exploited with strong evidence from clinical trials of vitamin D supplementation.
The aim of this study is to assess the cross-sectional relationship between the status of vitamin D
and the indices of metabolic pathways of lipids and glucose in a pediatric population.
2. Methods
2.1. Characteristics of the Study Participants and the Panel of Laboratory Tests
This cross-sectional study involved 284 presumably healthy children aged 9–11. The recruitment
and blood collection process took place between October and November 2015. The children were
selected on the basis of age (9–11 years old) from four primary schools in the Kujawsko-Pomorskie
region of Poland. The second inclusion criterion was a fasting state (a minimum of 8 h since last meal)
before blood drawing. Whilst school nurses and specialists in internal medicine participated in the
recruitment process, the general health of the child on the day of study was subjectively evaluated
by their parents. Children with any underlying liver, kidney, or endocrine diseases, or who were
receiving drugs that affected vitamin D levels were excluded from the study. Immediately following
blood collection, the blood samples were transported to the laboratory and centrifuged. Serum was
used for further laboratory analysis. Whole blood samples were collected for HbA1c evaluation.
Vitamin D status (total 25(OH)D concentration), lipid panel (total cholesterol (TC), triglycerides (TG),
high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C)), C-reactive
protein (CRP), and glucose status (fasting glucose concentration and glycated hemoglobin) were
evaluated for all participants.
2.2. Laboratory and Anthropometric Measurements

Concentrations of total 25(OH)D were analyzed on an IDS-iSYS automated analyzer
(Immunodiagnostic Systems Holdings PLC (Didcot Way, Boldon, UK)) using IDS-iSYS 25(OH)DS
chemiluminescence assay for the quantitative determination of 25-hydroxyvitamin D and other
hydroxylated metabolites (24,25(OH)2 D3 ) [7]. The percentage of 25(OH)D3 and25(OH)D2
cross-reactivity was 97% and 120%, respectively. Cross-reactivity with epimers (3-epi-25(OH)D3 ,
3-epi-25(OH)D2 ) did not exceed 1% [8]. The reportable range for IDS-iSYS 25(OH)DS assay ranged
between 7 and 125 ng/mL (18–313 nmol/L). The assay used for the determination of 25(OH)D was
traceable to isotope dilution-liquid chromatography/tandem mass spectrometry. Within-run precision
of the IDS-iSYS 25(OH)DS assay was evaluated by modified protocol CLSI EP-5A2 (Clinical and
Laboratory Standard Institute, Evaluation of Precision Performance of Quantitative Measurement
Methods) and ranged between 4.3% and 6.4% [8].
Lipid parameters including TC, TG, LDL-C, and HDL-C, high-sensitivity C-reactive protein
(hs-CRP) and glucose concentration were measured with the use of an ABX Pentra 400 analyzer
(Horiba Medical, Montpellier, France). Glycated hemoglobin was analyzed on a D-10™ Hemoglobin
analyzer (BIO-RAD Diagnostics, Dublin, Ireland). All measurements were performed on fasting blood
samples. The children’s height and weight was measured before blood collection and body mass index
(BMI) percentiles determined using an online BMI calculator based on the “OLAF” project [9].
2.3. Definitions of Decision Criteria for Study Participants

The participants were divided according to the ADA (American Diabetes Association)
recommendations, where a prediabetes condition is recognized when fasting glucose concentration is
between 100 mg/dL and 125 mg/dL (5.6–6.9 mmol/L). On this basis, 234 (82.4%) children had fasting
glucose concentrations <100 mg/dL (<5.6 mmol/L) and 50 (17.6%) children were recognized as having
impaired fasting glucose (IFG), with fasting glucose concentrations ≥100 mg/dL (≥5.6 mmol/L) [10].
140
Nutrients 2018, 10, 1359
Risk of dyslipidemia was assessed in accord with the currently accepted cut-off values for fasting
lipids in children [11]. Vitamin D status represented by 25(OH)D concentration was also evaluated
according to currently accepted recommendations [12].

Statistical analyses were carried out using SPSS (Statistical Package for the Social Sciences
version 20, Armonk, NY, USA) software. The significance level was established at p < 0.05.
The significance of the differences were determined using the χ2 (chi squared) test, or the Fisher
exact probability test for small groups. All quantitative variables, except for TG and CRP, showed
near-normal distributions. TG and CRP variables were logarithmically normalized to allow for further
regression analysis.
The Fisher exact probability test was used to present significant differences in the percentage
of normal glucose concentration and hyperglycemia in three subgroups of vitamin D status.
Considering that only a small group had optimal 25(OH)D concentration (n = 10), the analysis of
variables was undertaken incorporating a dichotomous division into those with deficiency states of
25(OH)D ≤ 20 ng/dL (<50 nmol/L) and those with 25(OH)D > 20 ng/dL (>50 nmol/L), respectively.
The linear regression analysis was tested to estimate the impact of 25(OH)D and selected variables on
glucose and lipid parameters. The incorporation of hs-CRP in logistic and linear regression analysis
was essential to exclude any potential bias arising from possible inflammation status. In the linear
regression analysis, we presented only statistically important models. Moreover, the variables included
in regression analysis were chosen based on results from Pearson correlation coefficients. Multivariate
logistic regression was performed for two-category variables. Because all statistical analyses were
carried out in traditional units, SI units are placed in brackets.
2.5. Compliance with Ethical Standards

The study was approved by the local ethics committee (Nicolaus Copernicus University Collegium
Medicum in Bydgoszcz, Poland) in accord with the Helsinki declaration and proper ethical standards
(REB 338/2015). Informed consent was obtained from the parents.
3. Results
Our study consisted of 284 children: 150 girls (52.8%) and 134 boys (47.2%) aged 9–11.
Characteristics of the study population are shown in Table 1.
Table 1. Characteristics of the study population (n = 284).
Mean (±SD) or Median *p

Parameters n (%)
(27–75th Percentile) (<0.05)
9 years 112 (39.4) -
Age 10 years 97 (34.2) -
11 years 75 (26.4) -
Boys 150 (52.8) -
Sex
Girls 134 (47.2) -
<5 (underweight) 16 (5.6) 26.8 (± 6.9)
BMI ≥5 and <85 (optimal weight) 200 (70.4) 34.4 (± 6.0)
percentiles ≥85 and <95 (overweight) 33 (11.6) 46 (± 6.8) <0.001 **
≥95 (obese) 35 (12.3) 56.1 (± 12.1)
Glucose (<100 mg/dL; 5.6 mmol/L) 234 (82.4) 90 (± 6.5); 5.0 (± 0.36)
Glycemic Glucose (≥100 mg/dL; 5.6 mmol/L) 50 (17.6) 105 (± 6.5); 5.8 (± 0.36)
<0.001
status HbA1c (<5.7 %; 38 mmol/mol) 249 (88.9) 5.3 (± 0.2); 34.5 (±2.2)
HbA1c (≥5.7 %; 38 mmol/mol) 31 (11.1) 5.8 (± 0.16); 40.0 (±1.7)
141
Nutrients 2018, 10, 1359
Table 1. Cont.
Mean (±SD) or Median *p

Parameters n (%)
(27–75th Percentile) (<0.05)
TC (<170 mg/dL; 4.4 mmol/L) 143 (50.3) 148 (± 14.1); 3.85 (± 0.37)
0.919
TC (≥170 mg/dL; 4.4 mmol/L) 141 (49.7) 194 (± 22.0); 5.02 (± 0.57)
TG: 0–9 years (<75 mg/dL; 0.85 mmol/L) 67 (23.6) 53 (± 12.8); 0.60 (± 0.14)
TG: 10–19 years (<90 mg/dL; 1.02 mmol/L) 123 (43.3) 62 (± 15.8); 0.70 (± 0.18)
<0.001
TG: 0–9 years (≥75 mg/dL; 0.85 mmol/L) 46 (16.2) 112 (± 39.0); 1.27 (± 0.44)
TG: 10–19 years (≥90 mg/dL; 1.02 mmol/L) 48 (16.9) 136 (± 42.6); 1.54 (± 0.48)
Lipids
LDL-C (<110 mg/dL; 2.85 mmol/L) 183 (64.4) 86 (± 14.6); 2.23 (± 0.38)
<0.001
LDL-C (≥110 mg/dL; 2.85 mmol/L) 101 (35.6) 129 (± 20.8); 3.34 (± 0.54)
HDL-C (>45 mg/dL; 1.17 mmol/L) 252 (88.7) 62 (± 11.2); 1.60 (± 0.29)
<0.001
HDL-C (≤45 mg/dL; 1.17 mmol/L) 32 (11.3) 40 (± 4.4); 1.04 (± 0.11)
non-HDL-C (<120 mg/dL; mmol/L) 178 (62.7) 96 (± 15.3); 2.47 (± 0.40)
<0.001
non-HDL-C (≥120 mg/dL; mmol/L) 106 (37.3) 139 (± 21.6); 3.59 (± 0.56)
(<1 mg/L) 196 (69.0) 0.2 (0.12–0.40)
hs-CRP <0.001
(≥1 mg/L) 88 (31.0) 2.1 (1.5–3.8)
Optimal (≥30 ng/mL; 75 nmol/L) 10 (3.5) 31.8 (± 1.5); 79.37 (± 3.74)
25(OH)D
Insufficiency (21–29 ng/mL; 52–72 nmol/L) 167 (58.8) 23.7 (± 2.6); 59.16 (± 6.49)
status 0.007
Deficiency (≤20 ng/mL; 50 nmol/L) 107 (37.7) 17.0 (± 2.3); 42.43 (± 5.74)
* Difference between two structure indicators determined by Chi Square test. ** Optimal vs. overweight
and obese. NA: not applicable. BMI: body mass index; HbA1c : glycated hemoglobin; TC: total cholesterol;
TG: triglycerides; LDL-C: low-density lipoprotein cholesterol (direct method); HDL-C: high-density lipoprotein
cholesterol; non-HDL-C: non-high-density lipoprotein cholesterol (TC-(HDL-C)); hs-CRP: high-sensitivity C-reactive
protein; 25(OH)D: 25-hydroxycholecalciferol (calcifediol); p: statistical significance (<0.05). Unit conversion factors:
glucose ((mg/dL) × 0.05551 = mmol/L); HbA1c (10.93 × (HbA1c %) − 23.5 = mmol/mol); TC, HDL-C, LDL-C
((mg/dL) × 0.0259 = mmol/L); TG ((mg/dL) × 0.0113 = mmol/L); 25(OH)D ((ng/mL) × 2.496 = nmol/L).
Overweight and obesity levels were based on BMI percentiles according to age and sex, being
11.6% and 12.3% respectively. Vitamin D deficiency, estimated by total 25(OH)D concentration,
was identified in 37.7% of the children, whereas its insufficiency was identified in 58.8%. Children
with impaired fasting glucose (≥100 mg/dL; 5.6 mmol/L) constituted 17.6%, and an elevated
HbA1c value was seen in 11.1% (Table 1). The most commonly identified lipid abnormality was
hypercholesterolemia, which was 49.7% for TC and 35.6% for LDL-C, respectively.
Our analysis revealed that 25(OH)D concentration affected glucose metabolism as reflected by
changes in fasting glucose concentrations. As can be seen in Table 2, the risk of hyperglycemia (IFG)
was significantly higher (p = 0.033) in children with 25(OH)D deficiency (≤20 ng/mL; 50 nmol/L)
(OR = 1.966, 95% CI: 1.055–3.663).
Table 2. Association of 25(OH)D deficiency with other variables.
95% CI
Variables * p Value OR
Lower Upper
Age 0.791 0.958 0.699 1.314
Sex 0.636 0.885 0.534 1.467
BMI percentiles 0.053 0.665 0.440 1.005
TC 0.169 1.688 0.800 3.562
TG 0.973 1.010 0.565 1.804
HDL-C 0.245 0.611 0.267 1.402
Non-HDL-C 0.624 1.267 0.493 3.257
142
Nutrients 2018, 10, 1359
Table 2. Cont.
95% CI
Variables * p Value OR
Lower Upper
LDL-C 0.257 0.599 0.247 1.454
hs-CRP 0.935 0.975 0.531 1.791
HBA1C 0.290 1.529 0.696 3.357
Glucose 0.069 1.828 0.955 3.499
Glucose ** 0.033 1.966 1.055 3.663
Logistic regression (multivariate): χ2 = 13.5; df = 11 (degrees of freedom); p = 0.258; r2 Cox and Snell = 0.047 (Cox and
Snell’s R squares complex samples logistic regression algorithms); r2 Nagelkerke = 0.064 (Nagelkerke R squares
complex samples logistic regression algorithms) * Statistical significance was determined in logistic regression
analysis. ** Logistic regression (univariate): χ2 = 4.513; df = 1; p = 0.034; r2 Cox and Snell=0.016; r2 Nagelkerke = 0.022.
25(OH)D: 25-hydroxycholecalciferol (calcifediol); BMI: body mass index; TC: total cholesterol; TG: triglycerides;
HDL-C: high-density lipoprotein cholesterol; non-HDL-C: non-high-density lipoprotein cholesterol (TC-(HDL-C));
LDL-C: low-density lipoprotein cholesterol (direct method); hs-CRP: high-sensitivity C-reactive protein; HbA1c :
glycated hemoglobin.
As can be seen in Figure 1, the prevalence of hyperglycemia and hypercholesterolemia was greater
in the vitamin D deficiency subgroup (24.0% and 57.3%, respectively), when compared to children
with concentrations of 25(OH)D over 50 nmol/L (20 ng/mL) (16.4% and 46.2%, respectively).
≤ 50nmol/L
24 %
25(OH)
57.3 %
ytuł osi
16.4 %
> 50nmol/L
25(OH)
46.2 %
0 10 20 30 40 50 60 70
Prevalence 漑%漒
Figure 1. Occurrence of hyperglycemia/hypercholesterolemia in vitamin D-deficient children.
TC: total cholesterol.
Pearson’s correlation coefficients across the whole study group revealed a statistically significant
weak negative correlation between 25(OH)D level and lipid variables (TG: r = −0.160; p = 0.037; HDL-C:
r = −0.209; p < 0.001), and also with glucose concentrations (r = −0.140; p = 0.018). A statistically
significant correlation between 25(OH)D and glucose was identified in the hypercholesterolemia
subgroup (r = −0.253; p = 0.002). A weak negative correlation (r = −0.215; p = 0.010) between 25(OH)D
and TC was found in the normocholesterolemic subgroup (Table 3). A significant negative correlation
of 25(OH)D with HDL-C was observed in children with hyperglycemia (r = −0.359; p = 0.010).
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Nutrients 2018, 10, 1359
Table 3. Pearson’s correlation coefficients in normo-/hyperglycemic and normo-/hypercholesterolemic state.
Vitamin
D/R-Pearson TC TG HDL-C Non-HDL-C LDL-C Glucose HbA1C hs-CRP
Correlation
Glucose: <100 mg/dL (<5.6 mmol/L) ((n = 234)—normoglycemia)
(R) 1 −0.136 0.001 −0.163 −0.066 −0.064 −0.122 0.131 −0.001
25(OH)D
(p) 2 0.037 0.990 0.012 0.314 0.330 0.063 0.046 0.989
Glucose: ≥100 mg/dL (≥5.6 mmol/L) ((n = 50)—hyperglycemia)
(R) 1 −0.251 −0.025 −0.359 −0.066 −0.135 −0.087 −0.008 −0.041
25(OH)D
(p) 2 0.079 0.862 0.010 0.647 0.351 0.547 0.956 0.779
Total cholesterol ≤170 mg/dL (≤4.4 mmol/L) ((n = 143)—normocholesterolemia)
(R) 1 −0.215 −0.019 −0.187 −0.066 −0.035 −0.019 0.050 −0.053
25(OH)D
(p) 2 0.010 0.820 0.026 0.436 0.677 0.821 0.554 0.531
Total cholesterol ≥170 mg/dL (≥ 4.4 mmol/L) ((n =141)—hypercholesterolemia)
(R) 1 −0.030 0.039 −0.181 0.091 0.030 −0.253 0.154 0.051
25(OH)D
(p) 2 0.724 0.644 0.032 0.282 0.722 0.002 0.070 0.546
All (n = 284)
(R) 1 −0.005 −0.160 −0.209 −0.067 −0.078 −0.140 0.091 0.001
25(OH)D
(p) 2 0.932 0.007 <0.001 0.262 0.193 0.018 0.129 0.991
1 R:Pearson correlation. 2 Statistical significance (p < 0.05), established using Pearson’s correlation analysis.
25(OH)D: 25-hydroxycholecalciferol (calcifediol); TC: total cholesterol; HDL-C: high-density lipoprotein cholesterol;
non-HDL-C: non-high-density lipoprotein cholesterol (TC-(HDL-C)); LDL-C: low-density lipoprotein cholesterol
(direct method); HbA1c : glycated hemoglobin; TG: triglycerides (logarithmic transformation); hs-CRP:
high-sensitivity C-reactive protein (logarithmic transformation).
The linear regression model (Table 4) emphasized a significant correlation between 25(OH)D and
glucose, and remained unchanged following adjustment for sex, age, and BMI percentiles.
Table 4. Impact of 25(OH)D concentration on glucose and lipids.
Independent Adjusted β (beta)(Standardized B (Unstandardized

Model * p Value
Variables r2 coefficients) Coefficients)
Model 1 0.021
25(OH)D 0.020 −0.238 0.021
Glucose
CRP log −0.087 −1.058 0.139
Model 1 adj. 0.027
25(OH)D 0.027 −0.142 −0.247 0.017
Glucose
CRP log −0.107 −1.303 0.106
Model 2 <0.001
25(OH)D 0.052 −0.159 −0.955 0.006
TC
TG log 0.183 0.124 0.002
Model 2 adj. 0.002
25(OH)D 0.048 −0.160 −0.958 0.006
TC
TG log 0.192 0.130 0.003
Model 3 <0.001
25(OH)D 0.097 −0.202 −0.565 <0.001
HDL-C
CRP log −0.244 −4.769 <0.001
Model 3 adj. <0.001
0.093
25(OH)D −0.203 −0.568 <0.001
HDL-C
CRP log −0.249 −4.860 <0.001
* Statistical significance established using multiple linear regression analysis unadjusted and adjusted by
sex, age, and BMI percentiles. Model adj.: model adjusted by sex, age, and BMI percentiles. 25(OH)D:
25-hydroxycholecalciferol (calcifediol); hs-CRP: high-sensitivity C-reactive protein (logarithmic transformation); TG:
triglycerides (logarithmic transformation); TC: total cholesterol; HDL-C: high-density lipoprotein cholesterol.
Based on the first model presented, one can estimate that 25(OH)D reduced by 1 ng/mL
(2.5 nmol/L) was related to slight, although significant, elevation of blood glucose concentration by
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Nutrients 2018, 10, 1359
0.238 mg/dL (0.013 mmol/L). After adjustment for sex, age, and BMI, glucose concentration elevation
was equal to 0.247 mg/dL (0.014 nmol/L) as a result of a unit decrease of 25 (OH)D concentration.
Our second model (Table 4) revealed a significant negative correlation between 25 (OH)D
(β = −0.159; p = 0.006) and TC. The model presented showed a significant elevation in TC by
0.958 mg/dL (0.025 mmol/L) linked to a 25(OH)D reduction by 1 ng/mL (2.5 nmol/L). In the adjusted
model, the TC elevation was identical.
The third linear regression model revealed an increase of HDL-C by 0.565 mg/dL (0.015 mmol/L),
as a result of the 1 ng/mL (2.5 nmol/L) decrease in 25(OH)D concentration (β = −0.203; p < 0.001).
Following adjustment, elevation of HDL-C reached 0.568 mg/dL (0.015 mmol/L) (Table 4).
4. Discussion
In the present study, the occurrence of IFG among children with vitamin D deficiency was
almost two-fold higher (OR = 1.966) when compared to children with 25(OH)D higher than
20 ng/mL (50 nmol/L). We also observed a weak negative correlation between 25(OH)D and glucose
concentration both in the whole group and in children with hypercholesterolemia.
We observed weak negative correlations between 25(OH)D and lipid components: TG and HDL-C
in the whole group, HDL-C in the subgroup with hyperglycemia or TC and HDL-C in the subgroups
with normo/hypercholesterolemia. Despite this weak correlation with lipid exponents, our study
revealed a significant association between serum 25(OH)D and TC concentration. We found that
serum 25(OH)D lower by 1 ng/mL (2.5 nmol/L) was linked to elevation in TC concentration by
0.96 mg /dL (0.025 mmol/L) and elevation of HDL-C by 0.57 mg/dL (0.015 mmol/L). The frequent
occurrence of hypercholesterolemia in our study (57.3%), being a notable trend particularly in
vitamin D-deficient children, is also worth noting. Evidence for the occurrence of dyslipidemia
in relation to vitamin D status was inconsistent. It was observed that the concentration of TC was
significantly higher in relation to vitamin D deficiency [13]. Our earlier study showed that HDL-C
concentration was higher among 25(OH)D-deficient children with newly diagnosed asthma [14].
Other cross-sectional studies conducted independently by Reis et al. and Ashraf et al. did not
find any significant association between low concentrations of vitamin D and abnormalities in lipid
parameters [15,16]. A study by Jorde et al. revealed a cross-sectional relationship between 25(OH)D
and serum lipids [17]. Meta-analysis conducted by Hao Wang et al. showed increased concentration
of TC and LDL-C with simultaneously decreased concentrations of HDL-C and TG as influenced by
vitamin D supplementation [18].
In this study we evaluated fasting glucose concentration, where abnormalities in this exponent of
carbohydrate metabolism is considered as a first stage in prediabetes development. This condition
may last several years without any additional symptoms, and depending on genetic or environmental
background, may either revert to a normoglycemic state or develop into overt diabetes [19].
The prevalence of IFG among overweight and obese Hispanic children reported by Goran et al.
ranged between 13% and 47% [20]. Our data indicated an inverse correlation between glucose and
vitamin D concentrations, consistent with an earlier NHANES III (National Health and Nutrition
Examination Survey) study where a negative relation between 25(OH)D and glycemia or diabetes
occurrence was demonstrated [21]. In another study conducted on children and teenagers aged 12–19,
it was seen that adjusted OR of fasting hyperglycemia among children with the lowest 25(OH)D
(<15 ng/mL; 37 nmol/L) was two-and-a-half fold higher (2.54, 95% CI: 1.01–6.40) when compared
to children in the highest 25(OH)D quartile (>26 ng/mL; 65 nmol/L) [15]. Our study showed that
along with the decrease of 1 ng/mL (2.5 nmol/L) of 25(OH)D, serum concentration of fasting glucose
increased by 0.25 mg/dL (0.014 mmol/L). The study conducted by Liu et al. indicated an association
between 25(OH)D and diabetes, in contrast to that presented by Pittas et al., where it was suggested
that there was insufficient data supporting hypotheses on the relation between vitamin D deficiency
and its contribution to the risk of diabetes [22,23].
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The most significant limitation of this study was sample size, which comprised over
100 dyslipidemic and only 50 hyperglycemic subjects. A further limitation of this study was its
single-center and cross-sectional design which made it difficult to assess the influence of vitamin D
on carbohydrate and lipid metabolism, with only the relationship between variables being tested.
Therefore, our findings warrant confirmation in further studies. On the other hand, the strength of the
present study was the simultaneous evaluation of two closely related metabolic pathways of glucose
and lipids, in terms of the prevalence of vitamin D deficiency among children aged 9–11.
5. Conclusions
25(OH)D deficiency may negatively affect fasting glucose and total cholesterol concentration in
children aged 9–11. Vitamin D-deficient children are twice as likely to develop prediabetes represented
by IFG, when compared to those with 25(OH)D concentration above 20 ng/mL (50 nmol/L).
Author Contributions: Conceptualization, L.S. and G.S. Methodology, L.S., G.S. and M.K. Software, L.S. and K.B.
Validation, G.S. Visualization, L.S. Funding Acquisition, G.S. Data Curation, L.S., M.K. and K.B. Formal Analysis,
L.S. and K.B. Investigation, L.S. Project Administration, G.S. and T.D. Resources, L.S. and M.K. Supervision,
T.D. and G.S. Writing—Original Draft Preparation, L.S. Writing—Review & Editing, M.K. and G.S.
Acknowledgments: We would like to thank all of the children and their parents for participating in our study.
Abbreviations
25(OH)D 25-Hydroxycholecalciferol
ADA American Diabetes Association
BMI Body Mass Index
CLSI Clinical and Laboratory Standards Institute
FG Fasting Glucose
HbA1c Glycated Hemoglobin
HDL-C High Density Lipoprotein Cholesterol by direct method
HS-CRP High Sensitivity C-Reactive Protein
IFG Impaired Fasting Glucose
LDL-C Low Density Lipoprotein Cholesterol by direct method
NHANES National Health and Nutrition Examination Survey
TC Total Cholesterol
TG Triglycerides
WHO World Health Organization
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nutrients
Review
What is the Validity of Questionnaires Assessing
Fruit and Vegetable Consumption in Children When
Compared with Blood Biomarkers? A Meta-Analysis
Tatiana S. Collese *, Gabriela Vatavuk-Serrati, Marcus Vinicius Nascimento-Ferreira,
Augusto César Ferreira De Moraes and Heráclito Barbosa Carvalho
YCARE (Youth/Child cArdiovascular Risk and Environmental) Research Group, Department of Preventive
Medicine, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP 01246-903, Brazil;
[email protected] (G.V.-S.); [email protected] (M.V.N.-F.);
[email protected] (A.C.F.D.M.); [email protected] (H.B.C.)
* Correspondence:[email protected]; Tel.: +55-11-3061-7091; Fax: +55-11-3061-8466
Received: 27 July 2018; Accepted: 20 September 2018; Published: 1 October 2018
Abstract: Fruit and vegetable consumption has been associated with improved health outcomes in
children. As an extensive number of questionnaires are currently used to assess fruit and vegetable
consumption, we performed a systematic review of the criterion validity of questionnaires used to
estimate fruit and vegetable consumption in children, considering blood biomarkers as the reference
method. Five electronic databases (MEDLINE, CINAHL, Scopus, PsycINFO, Web of Science) were
searched from database inception to 23 July 2018. The search strategy used the following sets of
descriptors: children; fruits and vegetables; dietary questionnaires; blood biomarkers; and validation
coefficient. The search terms were adapted for use with other databases in combination with
database-specific filters. Potentially eligible articles were selected independently by two reviewers,
separately, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses
(PRISMA) guidelines. Two articles meeting the inclusion criteria were included. The main reason
for study exclusion was the sample age range, which included adolescents. The pooled correlation
coefficient was 0.32 (95% confidence interval: 0.24–0.40).This review provided insights into assessment
methods of fruit and vegetable consumption in children. Although further studies are required,
questionnaires for assessing fruit and vegetable consumption have fair criterion validity in children.
Keywords: systematic review; meta-analysis; children; fruits and vegetables; biomarkers;

vitamins; validity
1. Introduction
International health associations recommend that children have a diet rich in vegetables, fruits,
whole grains, low-fat dairy products, legumes, fish, and lean meat, and low in saturated fat, trans fat,
and cholesterol, to help maintain a healthy weight and promote cardiovascular health [1]. Given the
role that the diet plays in children’s health, it is crucial to accurately determine their dietary intake and
to estimate the relationship between the foods consumed and nutrients.
Regular fruit and vegetable consumption is routinely suggested as a key component of promoting
health because they are an important source of nutrients, such as water, fiber, potassium, folic
acid, vitamins, and phytochemicals [1,2]. Several systematic reviews and meta-analyses have found
consistent associations between fruit and vegetable consumption and lower risk of cardiovascular
disease, diabetes, cancer, and other chronic diseases [3–5]. Despite the well-known benefits of
consuming fruits and vegetables, children are not meeting the recommended level of five servings
per day [6]. Consequently, increasing fruit and vegetable consumption in children is one of the major

Nutrients 2018, 10, 1396
goals of dietary interventions, worldwide [6]. Thus, valid questionnaires to assess fruit and vegetable
consumption are essential to identify eating habit changes in response to interventions, and to analyze
the impact of fruit consumption on health [7].
In nutritional epidemiology, the most used methods to estimate fruit and vegetable intake are
subjective instruments, such as food frequency questionnaires (FFQs) and dietary recalls, because they
are simple, cost-effective methods to assess long-term eating habits for large samples [8,9]. However,
these subjective instruments are susceptible to both random and systematic errors [10,11], especially
when applied to children, due to their limited skills to report their own dietary intake, depending
on parental (or whoever is responsible for their food) literacy and motivation to complete these
questionnaires [12].
As all dietary assessment methods (e.g., 24 h recalls, FFQs, smart-phone apps) have their
own strengths and limitations, dietary questionnaires are usually validated against an objective
measurement, such as blood biomarkers or doubly-labeled water [8,13]. Previous studies proposed
that plasma vitamins, such as vitamin A, E, and C, are a reliable concentration biomarker of usual fruit
and vegetable intake. Hence they are commonly used in validity studies of dietary assessment methods
as independent proxy measures of fruit and vegetable consumption [14], and to evaluate whether
sources of random error are independent of errors associated with measurement by questionnaire
and/or inaccuracies within nutrient databases [15].
However, these plasma vitamins are still relatively recent, and a significant amount of studies
are warranted in nutritional epidemiology [16]. It concerns deciphering whether these biomarkers are
markers of dietary consumption or markers of altered metabolism, as a result of the food intake [16],
or regarding the individual variability in absorption or availability [17]. Validation studies with blood
biomarkers have been carried out in adults [10,11], but relatively few studies were conducted in
children, lacking literature on key issues such as the impact of the period between fruit and vegetable
consumption and the blood biomarker analysis, or the stability of blood biomarkers in children aged
3 years to less than 10 years.
Therefore, the research question of this review was “What is the criteria validity of questionnaires
used to estimate fruit and vegetable consumption in children when compared with blood biomarkers?”
2. Methods
This systematic review was designed and conducted according to the guidelines of the
Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist
(Supplementary Material 1), as described in Reference [18]. At study initiation, no similar systematic
reviews were available in PROSPERO (International Prospective Register of Systematic Reviews) or
the following databases: Biomed Central, Medical Literature Analysis and Retrieval System Online
(MEDLINE), Web of Science, Cumulative Index to Nursing and Allied Health Literature (CINAHL),
Scopus, and PsycINFO. Thus, this protocol was registered in PROSPERO (CRD42017072531).
2.1. Study Selection

Five electronic databases—MEDLINE, CINAHL, Scopus, PsycINFO, and Web of Science—were
searched from inception to 20 December 2017. The searches were registered in the National Center for
Biotechnology Information (US National Library of Medicine, Bethesda, MD, USA), so that continual
updates on new publications would be received until 23 July 2018, and the searches were updated
until this same date. References of selected articles were analyzed, and the corresponding authors
were contacted to identify other relevant studies. The descriptors and medical terms used for the
database search were as follows: (Child OR Children OR school OR boy OR son OR girl OR daughter) AND
(Fruit OR Vegetable) AND (Questionnaire OR 24h Dietary Recall OR 24 h-Dietary-Recall OR 24 h Dietary
Recall OR Recall OR Food Frequency Questionnaire OR FFQ) AND (Biomarker OR Blood OR concentration
biomarker OR serum OR serum marker OR carotenoids OR vitamins) AND (Validation OR Validity OR
coefficient OR reliability).
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Two investigators examined the articles and performed the data screening, the data extraction,
and the quality assessment independently. Inter-reviewer discrepancies were resolved by consensus,
and a third reviewer was consulted for unresolved discrepancies. Relevant articles were obtained in
full and were assessed for eligibility and exclusion criteria.
2.2. Eligibility Criteria

As the goal of this systematic review was to assess evidence, specifically for validation studies of
questionnaires used to estimate fruit and vegetable consumption in children when compared with
blood biomarkers, this type of review must clearly delineate the population in question. Consequently,
the results may apply only to school-aged children.
The literature suggests that children are already able to help their parents to report their food
intake with relative accuracy [19]. However, after 10 years of age, the World Health Organization [20]
classifies the population as adolescent, with more independence and skills, which strongly influence
decision making, peer affiliation, and behavior [21], allowing them to decide on and self-report their
own dietary intake. In this sense, we considered only the children between three years of age until
nine years and 11 months, as presented in Table 1.
Table 1. Eligibility criteria included in the systematic review.
Eligibility Criteria
Children (3 years to less than 10 years of age) in a free-living environment, without any
Population
specific condition (e.g., eating disorder, obesity, diabetes) or children under medical care.
Exposure Fruit and/or vegetable consumption estimated by questionnaire.
Comparator Blood biomarkers for fruit and/or vegetable consumption.
Outcome Validation coefficients of fruit and/or vegetable consumption.
Study Original articles.
Exclusion Criteria
Studies where the participants had different diseases (or disturbances) that could interfere with
fruit and vegetable consumption were excluded. These criteria were set to increase inter-study
comparability. In addition, review articles, conference papers, or books were excluded.
2.3. Data Screening and Extraction

Potentially eligible articles were selected for inclusion according to the following sequence:
1. Articles published in English, Spanish, and Portuguese were identified;

2. Titles of the articles were screened;
3. Abstracts were screened; and
4. The full texts of the articles were reviewed to determine whether they met the inclusion criteria.
Articles were saved in the EndNote Web reference manager software version 3.1 (Thomson
Research Software, Carlsbad, CA, USA), and duplicates were removed. The data extracted included
the author, title of the study, citation and contact details, and study eligibility (Table 1). When studies
did not meet the eligibility criteria, the main reason for exclusion was documented in ink. Thereafter,
a table (Supplementary Material 2) was created by each reviewer with additional details about the
study, such as the sample size, settings, exposure/outcome definition and unit of measurement,
statistical analysis, bias (funding sources, adjustments), key conclusions, authors, miscellaneous
comments from the study authors, references to other relevant studies, limitations, correspondence
required, and miscellaneous comments by the authors reviewing the included studies.
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2.4. Quality Assessment

All retrieved articles were independently assessed and critically appraised using the standardized
checklist of the STROBE-nut (adapted to our review) to identify sources of bias, performance, attrition,
and detection. Data relevant to this review included the study design, characteristics of the research
subjects, dietary assessment methods used, results, discussion, bias assessment, and ethics information.
2.5. Strategy for Data Synthesis

The reporting status of fruit and vegetable consumption in each of the included studies were
determined from that listed within the results section of the included articles. The reporting status
of each study was determined using three predefined categories. The categories were dependent
on the level of accuracy of reported fruit and vegetable consumption, compared to measured blood
biomarkers. When available from included studies, agreement estimates were extracted if the reporting
status of research subjects correlated to various characteristics of the group. These characteristics
included demographic statistics (age and sex), anthropometric characteristics (height, weight, and
body mass index), and the study design. Limitations of each study and the evidence level were
recorded as well. The agreement estimates from study questionnaires were synthesized.
2.6. Statistics
We used the Stata 14 (Stata Corp., College Station, TX, USA) program for all statistical analyses.
Our outcomes were the correlation coefficients between the questionnaire and blood biomarkers,
which we calculated with their corresponding 95% confidence intervals (considering p < 0.05 as
significance level). In addition, we calculated the sample-weighted pooled correlation coefficient
for the meta-analysis with the random-effects model, due to the moderate heterogeneity found in
each study. We estimated the strength of the agreement of the Spearman rank correlation coefficient
cutoff points using the classification for dietary assessment methods: weak < 0.20, fair = 0.21 to 0.40,
moderate = 0.41 to 0.60, good = 0.61 to 0.80, and >0.80, very good [22]. Additionally, we verified
the heterogeneity of the studies using the I-squared test (percentages and p values under 0.05 were
considered significant). Heterogeneity levels of 25%, 50%, and 75% were considered low, moderate, and
high, respectively [23]. We performed the Egger’s regression test to verify potential publication bias
(positive or negative results) and small-study effects. We also generated the funnel plot (Supplementary
Material 3) to examine the potential bias graphically.
3. Results
The literature search provided 680 potentially eligible records: 4 from PsycINFO, 42 from Embase,
71 from Web of Science, 430 from Scopus, and 133 from Medline (see Appendix 1 in the Supporting
Information online). After duplicates (n = 51) were excluded, 629 studies remained and were screened
by title and, posteriorly, by abstract, according to the established inclusion criteria, resulting in
19 records for which the full-text was assessed for eligibility. Of those, the main reasons for exclusion
were the population (studies included participants over 10 years of age) and biomarker (such as
skin carotenoids), as shown in Figure 1. More details about these 19 study articles are stated
in Supplementary Material 4. Consequently, two studies were eligible and were included in this
systematic review.
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Identification
Records identified
(n = 680)
duplicates
excluded (n = 51)
records screened by
title (n = 629)
Preliminary Selection
records excluded
(n = 561)
records screened by
abstract (n = 68)
records excluded
(n = 49)
full-text articles assessed

for eligibility (n = 19)
Eligibility
Records excluded due

to: Sample (n = 12)
Biomarker (n = 3)
Exposure (n = 1)
Idiom (n = 1)
Inclusion
2 articles included in
the systematic review
Figure 1. Flow chart of article identification, retrieval, and inclusion, for the systematic review.
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The two final eligible articles included in this systematic review were published in English,
but they are from different countries, one from Europe and the other from North America (Table 2).
We found one study published in 1993 [24], but Medin et al. [25] was more recent, published in 2016.
Table 2. Description of the included studies.
Sample Sample Size

Survey Study/Article Title Study
Reference Country Age Total Girls
Year Type
(Years) (n) (%)
The accuracy of parental reports of their
children’s intake of fruits and vegetables:
Byers et al.
1990 USA Validation of a food frequency 06 to 10 97 55.7 Validity
(1993) [24]
questionnaire with serum levels of
carotenoids and vitamins C, A, and E.
Associations between reported intakes
of carotenoid-rich foods and
Medin et al. 08 to 09
2013 Norway concentrations of carotenoids in plasma: 121 55.6 Validity
(2016) [25] 12 to 14
A validation study of a web-based food
recall for children and adolescents.
Table 3 shows that both studies asked for the parental report; however, Byers et al. [24] used the
FFQ regarding the previous three months and considered fruit juice together with fruit and vegetable
consumption. Medin et al. [25] used a web-based food recall, for four consecutive nights, but they
assessed only vegetable intake for 4th grade children (8–9 years of age).
Nine different types of biomarkers were used to validate fruit and vegetable consumption.
Both studies analyzed carotenoids (α-carotene, β-carotene, and cryptoxanthin), but Medin et al. [25]
also included lycopene, lutein, and zeaxanthin in the sum of total carotenoids. On the other hand,
Byers et al. [24] analyzed vitamin A (retinol), vitamin E (α-tocopherol), and vitamin C (ascorbic acid).
High-performance liquid chromatography (HPLC), which is considered the gold standard analytical
technique for characterization and analysis of carotenoids in biological and food samples [26], was used
to assess plasma carotenoids in all studies (and for vitamin E in Byers et al. [24]), and spectrophotometry
was used to assess plasma vitamin C (Table 3).
The correlation coefficients between fruit and vegetable consumption and blood biomarkers are
presented in Table 4. The validity coefficients were fair to moderate, varying from 0.24 (for vitamin E)
to 0.47 (carotenoids). Both studies calculated the Spearman rank correlation coefficients and adjusted
the analyses for age, sex, ethnicity, and body mass index.
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Table 3. Validity studies on questionnaires to assess fruit and vegetable consumption with blood biomarkers in children.
Exposure
Period of Supplements Nutritional Biochemical Fasting
Reference Assessment Report Consumption Blood Biomarkers
Report Assessed Database Method Time
Method
Median fruits,
α-Carotene
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fruit juice, and HLPC (carotenoids,

β-Carotene
Byers et al. FFQ (111 food Previous 3 vegetables Not vitamin A and E), Not
Parental Willet Cryptoxanthin
(1993) [24] items) months Girls: 3.7 declared Spectrophotometry required
α-tocopherol
Boys: 2.9 (vitamin C)
Ascorbic Acid
(serving/day)
α-Carotene
Median fruits, Yes + USDA β-Carotene
Medin et al. Web-based 4 consecutive fruit juice, and supplements National β-Cryptoxanthin
Parental HPLC Non-fasting
(2016) [25] food recall nights vegetables: 225.1 containing Nutrient Lycopene
(g/day) carotenoids Database Lutein
Zeaxanthin
Abbreviations: FFQ: food frequency questionnaire; HPLC: high-performance liquid chromatography.
154
Table 4. Outcome of the included studies.
Correlations between diet and Blood

Criterion Conflict of
Reference Vitamin A Covariates in Fully Adjusted Model
Vitamin E Vitamin C Validity Interest
Carotenoids Retinol
Age, sex, ethnicity, family history of
Byers et al. (1993) [24] 0.30 0.25 0.24 0.34 Spearman coronary artery disease, BMI, TG, serum Not declared
total cholesterol, total caloric intake
Vegetable Age, sex, ethnicity, family structure,
Medin et al. (2016) [25] - - - Spearman None
intake: 0.47 BMI, parental education level
Abbreviations: BMI: body mass index; TG: serum triglycerides.
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The heterogeneity between studies was moderate (I-squared = 62.9%, p = 0.029), and
the sample-weighted pooled correlation coefficient for biomarkers was fair (r = 0.32, 95% CI:
0.24–0.40), as shown in the Figure 2. We were not able to stratify the analysis into subgroup categories
due to the small number of eligible studies included in the systematic review.
Figure 2. Forest-plot for correlation coefficients of fruit and vegetable consumption assessed by
questionnaires and blood biomarkers.
4. Discussion
At present, there is no strong evidence regarding the validity of questionnaires for assessing fruit
and vegetable consumption in children with blood biomarkers. Thus, we conducted a comprehensive
systematic review of validity studies of questionnaires to assess fruit and vegetable consumption in
children in comparison with blood biomarkers. Our findings indicated that this criteria validity was
fair. This validity stems from the challenges inherent to dietary assessment through questionnaires,
in addition to the fact that in children, we should consider the parent report, which is the subjective
perception of the parents about their children’s feeding habits [13,27].
The limited number studies (n = 2) meeting the inclusion criteria was likely due to the lack of
validated dietary questionnaires, maybe because conducting blood collection in school children is
less feasible, for practical and financial reasons [28], and due to the sample age range, since different
age group definitions were used for children (e.g., together with infants or adolescents). For example,
the study of Biltoft-Jensen et al. [29], which fulfilled our eligibility criteria, except for the population
age range, which was between 8 and 11 years of age. It is correct that 11-year-old individuals are still
children; however, the timing of biological maturation clearly signals entry into adolescence [21], and
with more cognitive abilities that enable the researchers to apply the self-report of fruit and vegetable
consumption. Therefore, it was excluded from our review.
It is important to emphasize that the credibility of our findings does not rely on the number of
studies included in the meta-analysis (n = 2), as we conducted an exhaustive and reproducible literature
search to produce results with the highest level of quality of evidence available. Murad et al. [30] state
that a systematic review or meta-analysis should evaluate the credibility of the methods applied on the
systematic review, and that this credibility depends on whether the review addressed a specific and
objective question; included an exhaustive literature search; clearly indicated the reproducibility of the
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selection and assessment of studies; and declared the results in a convenient way that enables future
research development. Furthermore, even though only two studies were eligible in our systematic
review, each biomarker was considered separately in the analysis; in other words, we evaluated five
different items (n = 5).
The sample-weighted pooled correlation coefficient found in our study (r = 0.32, 95% CI:
0.24–0.40) was fair, and comparisons with other validity studies carried out in children and adolescents
(9–12 years) showed similar Pearson correlation coefficients for vegetable (0.26, p = 0.13) and fruit
intake (0.49; p = 0.003) with total plasma carotenoids [31]. Studies carried out in adults showed a slight
increase in the Spearman correlation coefficients for alpha-carotene (0.42; CI: 0.09–0.96), beta-carotene
(0.40; CI: 0.09–0.99), and total plasma carotenoids (0.51; CI: 0.12–1.00) [32]. However, these correlations
are not so distant from our results, and it is relevant to note that their confidence intervals were broad.
4.1. Dietary Assessment Methods

Measuring fruit and vegetable consumption is complex, with interactions of different food
compounds, cultural, and socioeconomic conditions, in variable proportions [10,33]. Until this moment,
an ideal questionnaire to assess fruit and vegetable consumption is unknown [28]. There is an extensive
number of methods to perform this evaluation; however, all of them have both random and systematic
limitations and errors [10].
It is difficult to determine from the studies included in this review who is the most accurate
reporter of a child’s fruit and vegetable intake, and which method is most accurate and reliable. It is
important to note that mere participation in a research study may have biased the data reported for
each child, because parents may have selectively reported higher consumption of fruit and vegetable
of their child due to their involvement in the study. Reporting methods also depend on the difficulties
associated with the method of reporting, and with the parental educational level.
4.2. Food Frequency Questionnaires

Byers et al. [24] used an FFQ to estimate fruit and vegetable consumption in children. Some
advantages of the use of the FFQ are as follows: it can be given to the children’s parents with limited
explanation; it is a good method to obtain data regarding food eaten less than twice a week; questions
can be included to allow different portion sizes (adjusted); and the food/nutrient database only needs
to cover commonly eaten foods [34].
Disadvantages: it is time consuming; parents may find it difficult to decide how often their child
eats fruit or vegetables; the FFQ often underestimates foods eaten regularly or in large quantities if the
portion sizes used in the questionnaire are based on average intake; if a portion size is specified in the
FFQ, parents may find it difficult to interpret and answer the question meaningfully; and furthermore,
it depends on the level of literacy of the parents [34].
4.3. Web-Based Food Recall

Medin et al. [25] showed a higher correlation coefficient (0.47) for vegetable consumption, using
a web-based food recall for four consecutive nights. The development of technology-based research
tools for the assessment of intake, such as the web-based food recalls, has the potential to reach large
populations and reduce language barriers through the use of images rather than verbal descriptions [19].
Technology allows for greater scale and efficiencies for researchers to rely on regular dietary intake
data, for surveillance and monitoring [19]. The conversion of paper-based methods into web-based
methods may have benefits, including faster completion, greater reach, and the ability to maximize the
collection of complete data [19], which might explain part of the higher correlation coefficient found by
Medin et al. [25], when compared to other studies that used the FFQ. Furthermore, two small details
might have made a great difference in the Medin et al. [25] results: participants were given a personal
gift card as an incentive for participating in the study, which may have changed their engagement in it;
a voice-assisted cartoon character helped the children and parents to complete the questionnaires, with
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pop-up reminders to reduce the problem of food omissions, and in a more friendly way for kids—this
was shown to be a promising tool for application in pediatric populations.
In addition, Medin et al. [25] included four nightly 24-h recalls. This period is more than the
literature recommendation (three recalls per person) [34], and since parents answered the questionnaire
at night (the end of the day), it was easier for them to remember their child’s fruit and vegetable
consumption on the same day, minimizing the memory bias as well [34].
However, web-based food recalls reduce the limitations in terms of misreporting. Thus, there is a
need for continued development of methods, as well as to continue to evolve statistical methods to
mitigate error. Training researchers, clinical staff, and research subjects on the use of the technology
and the method is still highly warranted and might improve results and compliance with the dietary
method [19].
To be confident about the fruit and vegetable data collected, one should combine the FFQ with food
recall methods, trying to ascertain both frequent and infrequent fruit and vegetable consumption [35].
Additionally, if possible, combining technology with both dietary assessment methods is a good
alternative to reduce the burden of data handling and to make this process more friendly and joyful.
4.4. Creativity
The integration of technology, pictures, drawings, cartoons, and puppets into traditional dietary
questionnaires, considering the main factors inherent (Figure 3) to this approach, might provide
additional insights to mitigate errors in the fruit and vegetable assessment.
Figure 3. Main factors regarding the validity of dietary assessment methods in children.
When financial resources are scarce, like in studies conducted in low and middle-income countries,
using a bit of creativity might be a good solution. For example, if it is not possible to combine the
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technology in the process of dietary assessment, a simple art-based method like a “mascot” (drawn on
paper, as a cartoon, as a toy or a hand puppet) can make a great difference in the motivation and
involvement to answer the questionnaire, turning this process into a fun moment for kids [36,37].
Additionally, pictorial assessment, such as the assessment with the help of food photography albums,
food drawings or food replicas (three-dimensional models), might also be a cheaper and smart
alternative to enhance motivation and give visual support to estimate fruit and vegetable portion
sizes [38,39].
4.5. Administration Mode

A recent systematic review of the validity of dietary assessment methods in children when
compared with doubly labeled water suggests that using a parental reporter is the most accurate
method for reporting dietary intake in children aged 4–11 years [13]. However, it should be noted
that parents were the proxy reporters for the children; thus, some bias can be involved in this report.
For example, the parental weight status may influence the accuracy of their report, as observed in a
study with obese parents, who may underestimate the food intake of their obese children [40].
4.6. Blood Biomarkers

It is important to bear in mind that there are issues pertaining to bio accessibility and
bioavailability of vitamin A, E, and C in fruits and vegetables, depending on several factors, such as
genotype, ripening time, cultivation method, and climatic conditions, processing, food source, food
particle size and location, cooking method, and the content of fiber of the fruit or vegetable [41,42].
Moreover, different parts of the same plant may also contain different types and amounts of
vitamins [41].
In addition, knowing that most vitamins have a half-life of one or 2 months [43], it is difficult to
assess their blood availability and to compare it over long previous periods, such as Byers et al. [24]
did with their FFQ. Blood vitamins cannot be translated into absolute levels of fruit and vegetable
consumption, but these biomarkers do correlate with fruit and vegetable consumption in certain
strengths, and they are commonly applied in dietary assessment studies [28].
There are other factors relating to the children that may also affect the measurement and utility
of these biomarkers to properly reflect their fruit and vegetable consumption. For example, genetic
variability, lifestyle or physiologic factors (e.g., colonic microbiota, body mass index, and metabolic
or inflammation disorders) [44], dietary factors (e.g., frequency of intake and nutrient–nutrient
interactions), blood sample (e.g., whole blood, plasma, serum, conditions of sample collection,
transport, treatment, process, and storage), fasting status before examination (which neither of the
studies required), and laboratory analytical methodology (e.g., accuracy, detection of limits, variation
from laboratory to laboratory) [28].
Even with all these factors influencing vitamins blood levels, this method is still considered by the
scientific community as a reference method for fruit and vegetable consumption in epidemiological
and clinical studies.
4.7. Adjustment for Confounders

In addition, appropriate adjustments for confounding variables are critical for understanding
how factors of interest are interrelated within a population, as there may be a range of plausible
measurement errors that have a slight influence on these associations [45]. The two eligible studies
reported adjusting for confounders; however, the variables in the adjusted model differed according
to the study. Hence, we suggest that future studies should consider, at least, the sex, age, body
mass index, parental education, blood cholesterol, and energy and fiber intake to be included in the
fully-adjusted model.
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4.8. Methodological Quality

To the best of our knowledge, there is no evidence regarding the validity of questionnaires
for assessing fruit and vegetable consumption in children with blood biomarkers. However, other
systematic reviews addressing the validity of dietary assessment questionnaires and objective methods,
such as double label water [13] or skin biomarkers [19], have consistently reported low methodological
quality of the studies as well. Other studies in the field of physical activity in children, validating
questionnaires with objective methods (e.g., accelerometers), also reported low methodological quality,
especially regarding the absence of information about missing subjects and how the missing data were
handled [46].
In this review, only two studies were included. Based on the STROBE checklist, the major bias
identified was related to the information bias, particularly due to the heterogeneity between studies
in the applied methodologies. The absence of information about how the study size was calculated,
or how missing data were handled was a flaw in both studies. Byers et al. [24] did not declare any
adjustment for parental education level, nor information on whether fruit and vegetable intake was
reported with or without the inclusion of dietary supplement intake, nor about funding sources that
could influence their results.
4.9. Bias
The findings of our review indicated that more validity studies of fruit and vegetable consumption
need to be carried out in school-aged children. The two eligible studies were published in English and in
high-income countries. Therefore, it indicates a potential risk of publication bias, which can practically
be explained by the expensive cost of conducting a validity study with blood biomarkers [28].
Moreover, most of the 19 remaining full-text articles assessed for eligibility used the plasma
carotenes as a reference method, but only two articles assessed vitamin E [24,47], and one article
assessed vitamin C [24]. This bias is also probably related to the costs of vitamin C, to its very sensitive
biochemical methodology, and to the many difficulties and bureaucratic barriers for achieving the
reactants necessary for this methodology.
As dietary assessment cannot be estimated without error, it is paramount to attempt to understand
their effect on the data collected to minimize the confounding effect [17].
Byers et al. studied a sample of parents from a selective group of volunteers who reported their
children’s food intake once a year. Thus, the validity of their reports was probably higher than other
parents, because their motivation, involvement, and knowledge in the next step of the study could be
different to other parents who have never been involved in a nutritional research [24].
Another relevant issue of concern is the social approval of fruits and vegetables. As they are
widely known as healthy foods [48], parents may have reported their children’s fruit and vegetable
consumption, either consciously or subconsciously, in ways that make them appear favorable to the
researchers [17].
4.10. Heterogeneity between Studies in the Meta-Analysis

A moderate degree of heterogeneity was found in our meta-analyses (I2 = 62.9%, p < 0.029). At first,
we found a higher degree of heterogeneity in our meta-analyses (I2 = 94.1%, p < 0.001), and it was more
consistent with other systematic reviews regarding dietary assessment [9,19]. However, after critical
point-by-point reanalysisin each article, we decided that some characteristics were not very specific to
our eligibility criteria. For example, the study of Biltoft-Jensen et al. [29] that included the 11-year-old
adolescents in the analysis and applied a self-report administration of the questionnaire; or the study of
Royo-Bordonada et al. [47] that analyzed fruit and vegetable consumption as part of the dietary variety
index (together with sausage and other foods that contributed to the dietary variety). Consequently,
we had to exclude these two studies from the meta-analysis, since they had important sources of
heterogeneity and did not fulfill our eligibility criteria [49]. After this exclusion, the remaining
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heterogeneity could be partially explained by the characteristics inherent to the different types of
questionnaires used (e.g., recall period, number of items, recording bias, web-based system) and the
biomarker (e.g., type of biomarker analyzed, duration of measure, different cutoff points).
4.11. Strengths and Limitations

The strengths of the current review included the use of a comprehensive search strategy and data
collected from children aged 3 years to less than 10 years. To the best of our knowledge, this is the
first attempt to provide estimates of the true effect sizes for assessing fruit and vegetable consumption,
and to conduct a meta-analysis. Additionally, screening was performed by two independent authors;
searches were updated and included papers written not only in English, but also in Spanish and
Portuguese. The PRISMA statement was adopted, and a methodological quality rating was performed
separately, to assist in interpreting the findings. The strength of this review was that two independent
authors conducted the data extraction and the methodological quality assessment.
Despite these strengths, limitations should be noted. Although a comprehensive search was
conducted, the agreement analyses were restricted to information from electronically published data,
to ensure the reproducibility of the results.
Even though we considered Spanish and Portuguese for the literature search and screening,
the two eligible articles were published in English. Both dietary habits and parental awareness can
change considerably as children age; therefore, these findings may not be generalized to children
of other ages. Moreover, although the dietary methods applied in each study were different, both
methods are largely used in nutritional epidemiology [50] and in studies regarding fruit and vegetable
consumption by children [51], enabling the meta-analysis, once we defined the correlation coefficients
of these methods as our outcome [30].
5. Conclusions
Our findings suggest that the criterion validity of questionnaires and blood biomarkers
for assessing fruit and vegetable consumption in children is fair. The assessment of food
consumption—especially fruit and vegetable—in children still faces many challenges. However,
having a thorough understanding of the main factors that may influence this assessment can be a
promising path to enhance the validity of dietary assessment methods. Since measurement error is a
key remaining difficulty, validity studies of questionnaires to assess fruit and vegetable consumption
should be conducted with caution and interpreted within the broad context of the field. For example,
careful study design and meticulous development of the dietary assessment tools can be validated
with blood biomarkers, as long as the whole process of blood collection and analysis is adequate
and rigorously controlled. Moreover, informed statistical analysis, with appropriate adjustments, can
reduce the impact of these errors.
Finally, the incorporation of creativity and the development of technology, despite the challenges,
can lead to advancements in the field of dietary assessment methods, and therefore, have an important
role in the promising future of nutritional epidemiology.

s1, Supplementary Material 1: PRISMA checklist; Supplementary Material 2: Extraction form of full-text articles
assessed for eligibility; Supplementary Material 3: Funnel-plot for correlation coefficients of fruit and vegetable
consumption assessed by questionnaires and blood biomarkers; Supplementary Material 4: Main reasons for
exclusion of the full-text articles assessed for eligibility.
Author Contributions: T.S.C., G.V.-S., M.V.N.-F., A.C.F.D.M. and H.B.C. were responsible for the study concept
and study design. T.S.C. and G.V.-S. examined the articles and performed the data screening, the data extraction,
and the quality assessment. T.S.C., M.V.N.-F., and A.C.F.D.M. performed statistical analyses and interpreted the
data. T.S.C. and G.V.-S. drafted the manuscript. T.S.C., G.V.-S., M.V.N.-F., A.C.F.D.M. and H.B.C. supervised the
study. All authors participated in the writing of the paper, provided comments on the drafts, and approved the
final version of the manuscript. All authors agree to be personally accountable for the author’s own contributions
and ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the
author was not personally involved, are appropriately investigated, resolved, and documented in the literature.
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Funding: This research was funded by the State of São Paulo Research Foundation (FAPESP), which granted
Tatiana Sadalla Collese a doctoral scholarship (proc. 2016/13922-1), Gabriela Vatavuk-Serrati a Scientific Initiation
scholarship (proc. 2018/02452-0), Marcus Vinícius Nascimento-Ferreira a doctoral scholarship (proc. 2016/18436-8
and 2017/11732-3). Augusto César Ferreira De Moraes was awarded a post-doctoral scholarship from FAPESP
(proc. 2014/13367-2). Heráclito Barbosa Carvalho received an advanced scientist scholarship from the National
Council of Technological and Scientific Development (CNPq; proc. 300951/2015-9).
Acknowledgments: We gratefully acknowledge all of the researchers from the YCARE Research Group for their
essential contributions and suggestions for improving this work.
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nutrients
Article
Cardiovascular Biomarkers in Association with
Dietary Intake in a Longitudinal Study of Youth with
Type 1 Diabetes
Namrata Sanjeevi *, Leah M. Lipsky and Tonja R. Nansel
Social and Behavioral Sciences Branch, Division of Intramural Population Health Research, Eunice Kennedy
Shriver National Institute of Child Health and Human Development, Bethesda, MD 20817, USA;
[email protected] (L.M.L.); [email protected] (T.R.N.)
Received: 14 September 2018; Accepted: 17 October 2018; Published: 19 October 2018
Abstract: Despite cardioprotective effects of a healthy diet in the general population, few studies
have investigated this relationship in individuals with type 1 diabetes, who are at elevated risks of
cardiovascular disease (CVD) due to hyperglycemia. The objective of this study was to examine
the association of CVD biomarkers with overall diet quality, as measured by the Healthy Eating
Index-2015 (HEI-2015), and its dietary components in youth with type 1 diabetes. Youth with type 1
diabetes (n = 136, 8–16.9 years) were enrolled in an 18-month behavioral nutrition intervention trial.
Dietary intake from three-day diet records, CVD biomarkers (total cholesterol (TC), high-density
lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C); triglycerides (TG),
C-reactive protein (CRP), 8-iso-prostaglandin-F2alpha (8-iso-PGF2α ), systolic and diastolic blood
pressure (SBP and DBP, respectively), and glycated hemoglobin (HbA1c) were assessed at baseline,
6, 12 and 18 months. Linear mixed-effects models estimated associations of dietary intake with
CVD biomarkers, adjusting for HbA1c and other covariates. Separate models estimated associations
of time-varying change in dietary intake with time-varying change in CVD biomarkers. HEI-2015
was not associated with CVD biomarkers, but whole grain intake was inversely associated with TC,
HDL-C and DBP, and a greater increase in whole fruit intake was associated with lower DBP. Added
sugar, saturated fat and polyunsaturated fat were positively related to serum TG, HDL-C, and DBP,
respectively. Findings suggest that the intake of specific dietary components, including whole grains,
whole fruits, added sugar and PUFA, may influence cardiometabolic health in youth with type 1
diabetes, independent of glycemic control.
Keywords: type 1 diabetes; cardiovascular disease; whole grains; glycemic control; serum lipids
1. Introduction
Cardiovascular disease (CVD) is the major cause of mortality and morbidity in patients with type
1 diabetes, whose risk is several-fold higher than the general population [1,2]. This increased risk starts
early in life; 15–45% of youth with type 1 diabetes have at least two CVD risk factors, and subclinical
CVD abnormalities are observed within the first decade of diagnosis [3]. While glycemic control is
central to CVD prevention in type 1 diabetes [4], identifying additional risk modifiers would inform
timely interventions.
Several studies in the general population have indicated associations of dietary intake with
CVD indicators; however, few studies have examined these relationships in individuals with type
1 diabetes. Better cardiometabolic health in the general population is consistently associated with
better overall diet quality [5,6], greater intake of whole plant foods [7–17], and lower intakes of added
sugar [18,19], saturated fat [20,21] and sodium [22,23]. Associations of dietary intake with CVD risk in

Nutrients 2018, 10, 1552
individuals with type 1 diabetes may differ from that of the general population due to the characteristic
glucose fluctuations and hyperglycemia that trigger inflammatory responses, thereby increasing CVD
risk [24,25]. However, only two studies have examined associations of overall diet quality with CVD
risk factors in individuals with type 1 diabetes, finding an inverse association of overall diet quality
with blood pressure in adults [26] and dyslipidemia in youth [27,28]. Furthermore, associations of
CVD biomarkers with the intake of individual food groups and nutrients other than fiber [29–31],
saturated and total fat [31] in adults, and sugar-sweetened beverages [32] in youth have not been
examined. Although diet quality indices reflect overall dietary patterns, a composite score based
on several dietary components may mask associations of individual food groups or nutrients with
cardiovascular health. Understanding associations with individual dietary components also is critical
for developing behavioral targets for reducing CVD risk in patients with type 1 diabetes. Additionally,
although oxidative stress and inflammation are known CVD risk factors [24], only one study has
investigated dietary associations with inflammation in patients with type 1 diabetes, suggesting an
inverse relationship of fiber intake with inflammation. No study has examined associations of diet
with oxidative stress. Thus, the objective of this research was to investigate relationships of CVD
biomarkers (serum lipids, blood pressure, inflammation and oxidative stress) with overall diet quality,
as measured by Healthy Eating Index-2015 (HEI-2015), and its dietary components in youth with type
1 diabetes followed prospectively for 18 months.

This is a secondary analysis of a randomized controlled trial of a family-based behavioral nutrition
intervention conducted from 2010–2013 at a tertiary diabetes center in Boston, Massachusetts. Youth
eligibility criteria included: Age 8.0 to 16.9 years, diagnosed diabetes duration of ≥1 year, daily
insulin dose of ≥0.5 units per kilogram, HbA1c of 6.5% (48 mmol/mol)–10.0% (86 mmol/mol) at the
most recent clinic visit, insulin pump use or ≥3 injections per day, ≥1 clinic visit in the past year,
and able to communicate in English. Youth were excluded due to the daily use of premixed insulin,
the transition to insulin pump therapy three months prior to study, the use of real-time continuous
glucose monitoring three months prior to study, participation in another intervention study six months
prior to study, the use of medications that significantly interfere with glucose metabolism, the presence
of gastrointestinal disease, multiple food allergies, or significant mental illness. Of 622 eligible youth,
24% (n = 148) provided consent and 22% (n = 139) completed baseline measures. Data from one sibling
each of 3 sibling pairs was excluded resulting in 136 youth, and 125 participants were retained through
the study duration of 18 months.
Intervention details have been described previously [33]. The intervention aimed to increase
the intake of whole plant foods (whole fruits, vegetables, whole grains, legumes, nuts, and seeds).
The control group had an equal frequency of contact with the research staff, but did not receive any
nutrition advice besides that included as part of regular type 1 diabetes care.
Youth and parents provided assent and written informed consent, respectively, at the time of
enrollment. Written informed consent was obtained from youth turning 18 during the study. Study
procedures were approved by institutional review boards of the participating institutions. Sample size
was ascertained to detect meaningful differences in dietary intake and HbA1c between the treatment
and control groups [33].
2.1. Measures
2.1.1. CVD Biomarkers

Youth blood and urine samples obtained at baseline, 6, 12 and 18 months were stored at
room temperature for 20–30 min after withdrawal, centrifuged for 15 min at ~3000 RPM at 4 ◦ C,
then aliquoted and frozen at −80 ◦ C for later analysis. Serum concentrations of triglycerides (TG),
total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein
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Nutrients 2018, 10, 1552
cholesterol (LDL-C) were assessed using enzyme linked immunosorbent assay (ELISA). Systolic and
diastolic blood pressure (SBP and DBP) were abstracted from medical records at each visit. A high
sensitivity enzyme-linked immunosorbent assay (Architect c8000, Abbott) with a lower detection
limit of 0.2 mg/L was used to quantify CRP in the serum samples, and 8-iso-prostaglandin F2α
(8-iso-PGF2α ), an indicator of oxidative stress, was determined from urine frozen at −80 ◦ C using
enzyme linked immunosorbent assay (ELISA).
2.1.2. Diet Assessment

Families completed 3-day youth diet records at baseline and 3, 6, 9, 12, and 18 months based on
instructions from research assistants on accurate measurement and reporting of food and beverage
intake. Potion size estimation was facilitated by scales and measuring utensils. Families were asked to
include detailed description of each food item, such as brand names and item labeling (e.g., low fat
or 1% milk). The completed records were reviewed by research staff, and clarified for any missing
information, as needed. A registered dietitian obtained two nonconsecutive 24-h dietary recalls for
visits in which a family did not complete the diet record (1.7% of assessments).
Three-day diet records were analyzed using Nutrition Data System for Research 2012 (NDSR
2012; Nutrition Coordinating Center, University of Minnesota) software to obtain intake estimates of
key dietary components of the Dietary Guidelines of Americans 2015 (DGA 2015), including total and
whole fruits, total vegetables, greens and beans, whole and refined grains, dairy, total protein foods,
seafood, nuts and seeds, sodium, percentage of energy from added sugars and saturated fat, and fatty
acids. Intake of food groups and sodium per 1000 kcal were determined. The dietary subgroup, fatty
acids, is represented as the ratio of the sum of poly- and mono-unsaturated fat (PUFA and MUFA,
respectively) to saturated fat ((PUFA + MUFA): SFA). Overall diet quality was measured using the
Healthy Eating Index-2015 (HEI-2015), an a priori index that measures in adherence to DGA 2015
recommendations. The score is comprised of twelve component scores, which are summed to obtain
the total score that ranges from 0 to 100; a greater score indicates greater conformance to the DGA 2015.
2.1.3. Clinical and Demographic Data

HbA1c was assessed at baseline, 3, 6, 9, 12 and 18 months using a laboratory assay standardized
to the Diabetes Control and Complications Trial (reference range, 4–6%, (20–42 mmol/mol)). Tosoh
(Tosoh Medics, South San Francisco, CA, USA) was used for initial HbA1c assays ensued by Roche
Cobas Integra (Indianapolis, IN, USA). No clinically significant bias was detected between the Tosoh
and Roche methods based on results from the two instruments on identical samples.
Date of diabetes diagnosis, insulin regimen, age, sex, height, weight, Tanner stage and use of
cardiac medications (anticoagulants, ace inhibitors and statins) were abstracted from the medical
records at each study visit. Tanner stage from the previous visit was used for missing visit data. Body
mass index (BMI, kg/m2 ) was computed from height and weight measurements, and baseline diabetes
duration was calculated based on date of diagnosis. Parents reported youth race/ethnicity at baseline.
Items from the Behavioral Risk Factor Surveillance System assessed total moderate and vigorous
physical activity at baseline, 6, 12 and 18 months [34].

Differences in characteristics between treatment groups were analyzed using independent
samples t-tests for continuous variables and Pearson’s chi-square tests for categorical variables. Serum
TG and CRP were log-transformed to improve normality. Linear mixed-effects models estimated
associations of time-varying continuous independent variables (HEI-2015, and intake per 1000 kcal
of total fruits, whole fruits, total vegetables, dark green vegetables, beans, whole grains, dairy,
total protein foods, seafood, nuts and seeds, refined grains and sodium, percentage of energy from
added sugars and saturated fat, and fatty acids), with time-varying continuous dependent variables
(TG, TC, LDL-C, HDL-C, CRP, 8-iso-PGF2α , SBP and DBP). Models included a random intercept
167
Nutrients 2018, 10, 1552
representing the subject-specific baseline variation in the outcome. Covariates included baseline sex,
treatment assignment, and diabetes duration, and time-varying age, body mass index, Tanner stage,
insulin regimen, physical activity, use of cardiac medications, and HbA1c. Adjustments for multiple
comparisons were not performed due to the exploratory nature of the secondary data analysis [35,36].
Since 42.6%, 85.5%, 76.7% and 54.7% of participants reported zero intake of dark green vegetables,
beans, seafood, and nuts and seeds, respectively, these variables were dichotomized as having either
zero or greater than zero intake. Additional models estimated associations of each of the dichotomized
food group variable with CVD risk factors. Further, to enable within-subject analysis of the association
of change in diet with change in CVD biomarkers, the change from baseline in each dietary intake
variable and CVD biomarkers was calculated for each assessment period. Separate models estimated
associations of time-varying change in dietary intake with time-varying change in CVD biomarkers.
Models were controlled for baseline age, sex, treatment assignment, diabetes duration, Tanner stage,
insulin regimen, HbA1c, physical activity, body mass index and use of cardiac medication for the
respective assessment period. SPSS version 21 were used for all analyses.
3. Results
Baseline demographic and diet characteristics for study participants are shown in Tables 1 and 2,
respectively. At baseline, 19.4%, 11.2%, 28.4%, and 3.7% of participants were not within the
recommended concentrations [37,38] for TG (<150 mg/dL), TC (<200 mg/dL), LDL-C (<100 mg/dL)
and HDL-C (≥35 mg/dL), respectively.
Table 1. Baseline demographic characteristics of youth with type 1 diabetes overall and by
treatment assignment.
Overall (N = 136) Treatment (N = 66) Control (N = 70) p-Value

Age, years 12.7 ± 2.6 12.5 ± 2.7 13.0 ± 2.5 0.27
Body mass index, kg/m2 21.3 ± 4.2 21.0 ± 4.1 21.6 ± 4.3 0.37
Weight status 1
Underweight 1 (0.7) 1 (1.5) 0 (0) 0.55
Normal weight 91 (66.9) 42 (63.6) 49 (70.0)
Overweight 28 (20.6) 17 (25.8) 11 (15.7)
Obese 16 (11.8) 6 (9.1) 10 (14.3)
8.1 ± 1.0 8.1 ± 1.1 8.1 ± 1.0
HbA1c, (%, (mmol/mol)) 0.95
(65 ± 11) (65 ± 12) (65 ± 12)
Duration of diabetes, years 6.0 ± 3.1 5.6 ± 2.5 6.4 ± 3.6 0.15
Insulin dose, Units/day 49.8 ± 26.8 46.9 ± 23.9 52.7 ± 29.3 0.24
Tanner stage 2.5 ± 1.4 2.4 ± 1.4 2.6 ± 1.5 0.38
Youth race/ethnicity
Non-Hispanic white 123 (90.4) 58 (87.9) 65 (92.9) 0.17
Non-Hispanic black 5 (3.7) 2 (3.0) 3 (4.3)
Hispanic 7 (5.2) 6 (9.1) 1 (1.4)
American Indian/Alaska Native 1 (0.7) 0 (0) 1 (1.4)
TG (mg/dL) 111.1 ± 56.9 101.3 ± 42.4 120.2 ± 66.8 0.05
TC (mg/dL) 165.2 ± 27.9 162.7 ± 24.3 167.6 ± 30.8 0.32
HDL-C (mg/dL) 56.6 ± 13.6 56.5 ± 14.0 56.6 ± 13.3 0.98
LDL-C (mg/dL) 86.4 ± 24.0 85.7 ± 19.7 87.0 ± 27.5 0.75
CRP (mg/L) 1.1 ± 1.9 0.9 ± 1.4 1.4 ± 2.3 0.09
8-iso-PGF2α (ng/mL) 1.6 ± 1.3 1.7 ± 1.5 1.4 ± 1.1 0.33
SBP (mm Hg) 108.7 ± 7.1 108.6 ± 7.8 108.8 ± 6.5 0.90
DBP (mm Hg) 66.4 ± 5.5 66.7 ± 5.9 66.2 ± 5.2 0.60
TG, triglycerides; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein
cholesterol; CRP, C-reactive protein; 8-iso-PGF2, 8-iso-prostaglandin-F2-alpha; SBP, systolic blood pressure; DBP,
diastolic blood pressure. Data are presented as mean ± SD or n (%). 1 Underweight = BMI%ile < 5; Normal weight
= 5 ≤ BMI%ile < 85; Overweight = 85 ≤ BMI%ile < 95; Obese = 95 ≤ BMI%ile.
168
Nutrients 2018, 10, 1552
Table 2. Baseline dietary characteristics of youth with type 1 diabetes overall and by treatment assignment.
Dietary Components Overall (N = 136) Treatment (N = 66) Control (N = 70) p-Value

HEI-2015 46.05 ± 11.70 45.33 ± 12.44 46.73 ± 11.01 0.49
Total fruits 1 0.44 ± 0.40 0.45 ± 0.38 0.42 ± 0.43 0.70
Whole fruits 1 0.32 ± 0.37 0.32 ± 0.32 0.33 ± 0.41 0.90
Total vegetables 1 0.49 ± 0.35 0.49 ± 0.38 0.49 ± 0.31 0.99
Dark green vegetables 1 0.09 ± 0.12 0.07 ± 0.11 0.10 ± 0.12 0.09
Beans 1 0.03 ± 0.09 0.04 ± 0.12 0.02 ± 0.05 0.19
Whole grains 1 0.52 ± 0.53 0.47 ± 0.38 0.57 ± 0.64 0.26
Dairy 1 0.68 ± 0.33 0.68 ± 0.34 0.67 ± 0.33 0.85
Protein foods 1 2.54 ± 1.24 2.53 ± 1.27 2.55 ± 1.21 0.94
Seafood 1 0.16 ± 0.40 0.11 ± 0.29 0.20 ± 0.48 0.07
Nuts and seeds 1 0.34 ± 0.73 0.33 ± 0.89 0.34 ± 0.54 0.93
Refined grains 1 3.44 ± 1.09 3.58 ± 1.04 3.30 ± 1.13 0.13
Sodium 2 1.69 ± 0.33 1.69 ± 0.31 1.68 ± 0.34 0.85
Added sugars, % kcal 11.97 ± 5.16 11.39 ± 4.47 12.53 ± 5.71 0.20
Saturated fat, % kcal 12.39 ± 2.75 12.37 ± 2.68 12.41 ± 2.83 0.93
Fatty acid 3 1.69 ± 0.49 1.72 ± 0.52 1.65 ± 0.46 0.42
1 Expressed as total number of cup- or ounce-equivalents per 1000 kcal; 2 expressed as grams per 1000 kcal; 3
calculated as (polyunsaturated fat + monounsaturated fat)/saturated fat.
Associations between dietary intake and serum lipids after adjustments of HbA1c and other
covariates are indicated in Table 3. In the adjusted model, serum lipids were not associated with
overall diet quality. However, whole grain intake was inversely associated with TC and HDL-C. Added
sugar and saturated fat intakes were positively associated with TG and HDL-C, respectively. Other
dietary components (including dark green vegetables, beans, seafood, and nuts and seeds) were not
associated with serum lipids. Findings were unchanged when the food groups, dark green vegetables,
beans, seafood, and nuts and seeds, were treated as categorical variables (Table S1).
Associations of dietary intake with inflammation, oxidative stress and blood pressure are shown
in Table 4. These CVD biomarkers were not associated with overall diet quality; however, 8-iso-PGF2α
was inversely associated with refined grain intake, and lower DBP was related to greater whole grain
intake and greater (PUFA + MUFA): SFA. To determine the specific fatty acids associated with DBP,
we further examined the association of the intake of individual fatty acid with DBP. Although MUFA
intake (% kcal) was not associated with DBP (β = 0.08, SE = 0.10, p = 0.38), PUFA intake (% kcal) was
positively related to DBP (β = 0.23, SE = 0.09, p = 0.008). Dark green vegetables, beans, seafood and
nuts and seeds were not associated with inflammation, oxidative stress and blood pressure (Table 4),
and findings were unchanged when these food groups were treated as categorical variables (Table S2).
Associations between time-varying changes in dietary intake with time-varying changes in CVD
biomarkers are indicated in Tables S3 and S4 Greater increase in whole grain intake was associated
with a decrease in LDL-C and HDL-C. Increase in whole fruit intake was inversely associated with
DBP, whereas increase in fatty acid ratio was positively associated with DBP. Increase in time-varying
total vegetable, whole grain and added sugar intake was positively associated with TG, but increase in
time-varying refined grain intake was inversely associated with TG and 8-iso-PGF2α . Further, increase
in time-varying dairy intake was positively associated with SBP.
169
Table 3. Association of diet quality and food group and nutrient intake with serum lipids in youth with type 1 diabetes 1 .
TG 2 (mg/dL) TC (mg/dL) HDL-C (mg/dL) LDL-C (mg/dL)

Dietary Components
β ± SE p β ± SE p β ± SE p β ± SE p
HEI-2015 0.0003 ± 0.001 0.77 −0.16 ± 0.10 0.11 −0.07 ± 0.05 0.16 −0.12 ± 0.09 0.19
Total fruits 3 0.01 ± 0.03 0.78 1.31 ± 3.21 0.68 0.06 ± 1.49 0.97 1.07 ± 3.00 0.72
Nutrients 2018, 10, 1552
Whole fruits 3 0.01 ± 0.04 0.70 3.22 ± 3.96 0.42 0.67 ± 1.86 0.72 1.59 ± 3.68 0.67
Total vegetables 3 0.03 ± 0.03 0.28 2.44 ± 2.83 0.39 0.05 ± 1.36 0.97 −2.74 ± 2.66 0.30
Dark green vegetables 3 −0.02 ± 0.06 0.75 −9.18 ± 6.31 0.15 −2.63 ± 2.85 0.36 −8.50 ± 5.89 0.15
Beans 3 −0.14 ± 0.10 0.18 −17.17 ± 10.77 0.11 −2.81 ± 5.06 0.58 −5.16 ± 10.22 0.61
Whole grains 3 0.01 ± 0.02 0.61 −4.60 ± 2.05 0.03 −1.98 ± 0.99 0.046 −3.42 ± 1.92 0.08
Dairy 3 0.01 ± 0.03 0.79 −1.97 ± 3.78 0.60 −0.64 ± 1.81 0.72 −1.61 ± 3.44 0.64
Protein foods 3 −0.01 ± 0.01 0.30 0.05 ± 0.88 0.95 0.25 ± 0.41 0.55 0.39 ± 0.83 0.63
Seafood 3 0.01 ± 0.03 0.61 0.09 ± 2.66 0.97 −0.90 ± 1.27 0.48 1.77 ± 2.47 0.47
Nuts and seeds 3 0.004 ± 0.02 0.84 0.15 ± 1.74 0.93 0.12 ± 0.85 0.89 0.06 ± 1.64 0.97
Refined grains 3 −0.02 ± 0.01 0.08 0.54 ± 1.05 0.61 0.31 ± 0.51 0.54 1.72 ± 0.98 0.08
Sodium 4 −0.05 ± 0.03 0.10 2.13 ± 3.33 0.52 0.21 ± 1.52 0.89 5.38 ± 3.10 0.08
Added sugars, % kcal 0.004 ± 0.002 0.04 0.22 ± 0.21 0.30 −0.10 ± 0.10 0.32 0.09 ± 0.20 0.64
Saturated fat, % kcal 0.61 0.35 0.04 0.58
170
−0.002 ± 0.003 0.35 ± 0.37 0.34 ± 0.17 0.19 ± 0.35
Fatty acid 5 −0.002 ± 0.02 0.90 −0.50 ± 2.10 0.81 −0.74 ± 0.96 0.45 −1.12 ± 1.93 0.56
TG, triglycerides; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C low-density lipoprotein cholesterol; HEI-2015, healthy eating index-2015. 1 Models were
controlled for baseline treatment assignment, sex, diabetes duration, and time-varying age, body mass index, Tanner stage, insulin regimen, physical activity, use of cardiac medication,
and glycemic control (HbA1c); 2 log-transformed to meet the normality assumption; 3 expressed as total number of cup- or ounce-equivalents per 1000 kcal; 4 expressed as grams per
1000 kcal; 5 calculated as (polyunsaturated fat + monounsaturated fat)/saturated fat.
Table 4. Association of diet quality and food group and nutrient intake with inflammation, oxidative stress and blood pressure in youth with type 1 diabetes 1 .
CRP 2 (mg/L) 8-iso-PGF2α (ng/mL) SBP (mmHg) DBP (mmHg)

Dietary Components
β ± SE p β ± SE p β ± SE p β ± SE p
HEI-2015 0.0002 ± 0.002 0.90 0.002 ± 0.007 0.77 0.01 ± 0.02 0.59 0.01 ± 0.02 0.53
Total fruits 3 0.04 ± 0.06 0.46 −0.23 ± 0.22 0.31 −0.25 ± 0.71 0.73 0.10 ± 0.66 0.88
Nutrients 2018, 10, 1552
Whole fruits 3 0.07 ± 0.08 0.39 −0.17 ± 0.28 0.54 −0.73 ± 0.86 0.40 −0.11 ± 0.88 0.90
Total vegetables 3 −0.02 ± 0.05 0.67 0.06 ± 0.21 0.77 −0.02 ± 0.69 0.97 0.69 ± 0.63 0.28
Dark green vegetables 3 −0.04 ± 0.12 0.72 −0.42 ± 0.50 0.40 −0.52 ± 1.46 0.72 −1.10 ± 1.38 0.42
Beans 3 −0.30 ± 0.21 0.15 0.85 ± 1.05 0.42 −0.64 ± 2.67 0.81 −0.93 ± 2.43 0.70
Whole grains 3 0.04 ± 0.04 0.26 −0.09 ± 0.16 0.60 0.03 ± 0.47 0.96 −0.98 ± 0.46 0.04
Dairy 3 −0.004 ± 0.07 0.95 −0.47 ± 0.27 0.08 0.89 ± 0.81 0.27 −0.99 ± 0.73 0.18
Protein foods 3 −0.01 ± 0.02 0.55 0.09 ± 0.07 0.22 −0.11 ± 0.20 0.59 0.20 ± 0.17 0.23
Seafood 3 0.02 ± 0.05 0.65 −0.09 ± 0.21 0.69 −0.18 ± 0.63 0.78 0.78 ± 0.61 0.20
Nuts and seeds 3 0.02 ± 0.04 0.61 0.25 ± 0.14 0.08 0.29 ± 0.45 0.53 0.21 ± 0.43 0.62
Refined grains 3 −0.02 ± 0.02 0.42 −0.18 ± 0.08 0.04 0.05 ± 0.25 0.85 0.04 ± 0.23 0.86
Sodium 4 0.02 ± 0.06 0.79 −0.42 ± 0.27 0.12 −0.53 ± 0.78 0.50 0.19 ± 0.78 0.80
Added sugars, % kcal −0.00003 ± 0.004 0.99 0.01 ± 0.02 0.58 −0.04 ± 0.05 0.34 −0.01 ± 0.04 0.89
Saturated fat, % kcal 0.43 0.57 0.87 0.13
171
0.01 ± 0.007 0.02 ± 0.03 0.01 ± 0.08 −0.12 ± 0.08
Fatty acid 5 0.02 ± 0.04 0.59 0.19 ± 0.15 0.20 0.19 ± 0.45 0.67 0.98 ± 0.42 0.02
CRP, C-reactive protein; 8-iso-PGF2α , 8-iso-prostaglandin F2α ; SBP, systolic blood pressure; DBP, diastolic blood pressure; HEI-2015, healthy eating index-2015. 1 Models were controlled
for baseline treatment assignment, sex, diabetes duration, time-varying age, body mass index, Tanner stage, insulin regimen, physical activity, use of cardiac medication and glycemic
control (HbA1c); 2 log-transformed to meet the normality assumption; 3 expressed as total number of cup- or ounce-equivalents per 1000 kcal; 4 expressed as grams per 1000 kcal;
5 calculated as (polyunsaturated fat + monounsaturated fat)/saturated fat.
Nutrients 2018, 10, 1552
4. Discussion
In contrast to the general population [5,39], CVD biomarkers were not associated with greater
adherence to DGA 2015, as measured by HEI, in this sample of youth with type 1 diabetes. However,
greater intake of whole grains and whole fruits, and lower added sugar and PUFA were associated
with more favorable CVD biomarkers. Much of the current study findings regarding the association of
intake of specific dietary components with CVD biomarkers are comparable to the general population.
However, CVD etiology in type 1 diabetes differs from that of the general population, as hyperglycemia
is known to increase CVD risk. Findings suggest that these dietary components may be associated
with CVD risk in persons with type 1 diabetes independent of glycemic control.
Several studies in the general population have indicated the protective role of whole grains in
reducing CVD risk factors, including blood pressure [40] and TC [7,8,10]. In contrast, no study has
examined this relationship in individuals with type 1 diabetes. Improved endothelial function due
to whole grain consumption [41] could explain the association of greater intake with lower blood
pressure in this sample. The inverse association of whole grain intake with TC, independent of HbA1c
could be attributed to increased clearance or decreased biosynthesis of cholesterol [42]. This pathway
also is consistent with the inverse relationship between whole grain consumption and HDL-C found
in this study. Although this result is in contrast to previous research in the general population [8,10],
it is comparable to another study finding that a whole grain diet significantly lowered HDL-C in
overweight and obese adults [43]. The inverse association of whole grain intake with HDL-C is not
considered atherogenic in this sample, given the negligible number of participants (3.7%) with low
HDL-C (<35 mg/dL) at baseline. However, it is important to consider the impact of whole grain
consumption on HDL-C in future CVD-related research in patients with type 1 diabetes. Associations
of whole grain intake with cholesterol are further supported by within-subject analyses, where increase
in whole grain intake was inversely related to LDL-C and HDL-C. Further, improved endothelial
function could explain the inverse association of whole fruits with DBP, which is consistent to previous
research in the general population [44]. However, other relationships of time-varying changes in
whole grain, refined grain and dairy intake with that of CVD biomarkers are not supported by known
biological mechanisms. Findings for within-subject analyses should be interpreted with caution, given
the modest degree of individual change over time, resulting in increased chances of spurious findings.
The positive association of added sugar intake with serum TG is consistent with previous literature
in the general population [18,19]. This result could be attributed to hepatic de novo lipogenesis
induction by common sugars [45], resulting in increased serum TG, independent of glycemic control.
This finding is also somewhat comparable to another study in youth with type 1 diabetes, whereby
increased sugar-sweetened beverage intake was related to greater serum lipids [46]. The positive
association of saturated fat intake with HDL-C is comparable to previous research [47], and may be
explained by increased transport rates of HDL-C ester by dietary fat [47]. While additional studies are
needed, these findings indicate some consistency in the relationship of food group and nutrient intake
with serum lipids in individuals with type 1 diabetes as compared with the general population.
The inverse relationship of refined grain intake with oxidative stress contrasts previous research
indicating the detrimental effects of refined grain intake on CVD biomarkers [9]. This may be attributed
to the use of urinary 8-iso-PGF2α as an oxidative stress marker, which is a less sensitive measure than
its metabolite, 2,3-dinor-5,6-dihydro-15-F2t-isoprostane [48]. Future research using more sensitive
markers of oxidative stress could better elucidate the association of refined grain intake with oxidative
stress. Finally, greater DBP was associated with increased PUFA intake. Given PUFA includes omega-6
and omega-3 PUFA, a high intake could be obtained for an individual consuming excess omega-6 and
minimal omega-3 PUFA. Although total omega-6 PUFA intake could not be obtained for the current
study, stimulation of vasoconstriction by omega-6 PUFA-derived eicosanoids [49] could explain the
relationship of PUFA intake with DBP reported in this study.
The study findings are subject to certain limitations. These findings are observational, precluding
inferences on causality. The eligibility criteria for the primary study, recruitment from a single clinic,
172
Nutrients 2018, 10, 1552
22% recruitment rate and predominantly white sample limit generalizability of the results to all youth
with type 1 diabetes. Since youth with poor glycemic control (most recent HbA1c prior to recruitment
>10.0%) were not recruited, the study may have excluded patients most affected by adverse CVD
biomarkers. The lack of adjustment for multiple comparisons could have increased the family-wise
error rate. Nevertheless, the detected associations of diet with CVD biomarkers are supported by
biological mechanisms. Although food records are among the most valid method of assessing dietary
intake, they are susceptible to reporting and response biases [50]. However, this study is strengthened
by the prospective design utilizing repeated measures of exposures and outcomes, assessment of
several CVD risk factors, and adjustment of potentially important covariates in the analyses.
5. Conclusions
Nutrition recommendations for youth with type 1 diabetes include general healthful eating for
reducing risk of complications, and there are limited disease-specific nutrition guidelines. However,
characteristic hyperglycemia in individuals with type 1 diabetes may attenuate dietary influences on
CVD biomarkers. The current study suggests that overall diet quality was not associated with CVD
biomarkers in youth with type 1 diabetes. However, specific dietary components were associated with
CVD biomarkers, independent of glycemic control. Additional studies are needed to corroborate if
intake of greater whole grains, lower added sugar and PUFA may favorably influence CVD biomarkers
in this population. Examining whether these dietary associations differ by glycemic control could
inform efforts that promote cardiovascular health in patients with type 1 diabetes.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/10/10/1552/s1.

Author Contributions: T.R.N. and L.M.L. designed the research and obtained funding. N.S. conceptualized the
research question and wrote the manuscript, and all authors contributed to the manuscript editing, and read and
approved the final manuscript.
Funding: This research was supported by the intramural research program of the National Institutes of
Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development, contract #’s
HHSN267200703434C and HHSN2752008000031/HHSN275002.
Conflicts of Interest: The authors declare no conflicts of interest.
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Article
Food Perceptions and Dietary Changes for Chronic
Condition Management in Rural Peru: Insights for
Health Promotion
Silvana Perez-Leon 1, *, M. Amalia Pesantes 1 , Nathaly Aya Pastrana 2 , Shivani Raman 3 ,
Jaime Miranda 1 and L. Suzanne Suggs 2
1 CRONICAS Center of Excellence in Chronic Diseases, Universidad Peruana Cayetano Heredia, Lima 15074,
Peru; [email protected] (M.A.P.); [email protected] (J.M.)
2 BeCHANGE Research Group, Institute of Public Communication, Università della Svizzera italiana,
6900 Lugano, Switzerland; [email protected] (N.A.P.); [email protected] (L.S.S.)
3 Department of Sociology, Rice University, Houston, TX 77005, USA; [email protected]
Received: 8 September 2018; Accepted: 19 October 2018; Published: 23 October 2018
Abstract: Peru is undergoing a nutrition transition and, at the country level, it faces a double
burden of disease where several different conditions require dietary changes to maintain a healthy
life and prevent complications. Through semistructured interviews in rural Peru with people
affected by three infectious and noninfectious chronic conditions (type 2 diabetes, hypertension,
and neurocysticercosis), their relatives, and focus group discussions with community members,
we analyzed their perspectives on the value of food and the challenges of dietary changes due to
medical diagnosis. The findings show the various ways in which people from rural northern Peru
conceptualize good (buena alimentación) and bad (mala alimentación) food, and that food choices are
based on life-long learning, experience, exposure, and availability. In the context of poverty, required
changes are not only related to what people recognize as healthy food, such as fruits and vegetables,
but also of work, family, trust, taste, as well as affordability and accessibility of foods. In this paper we
discuss the complexity of introducing dietary changes in poor rural communities whose perspectives
on food are poorly understood and rarely taken into consideration by health professionals when
promoting behavior change.
Keywords: dietary changes; health promotion; health behavior; Peru; chronic conditions
1. Introduction
Over the past several decades, low- and middle-income countries (LMICs) have experienced a
nutrition transition. Countries that have faced hunger and malnutrition as their main concerns are
now confronting an additional problem of overweight and obesity and are experiencing increasing
rates of noncommunicable diseases (NCD) such as diabetes and hypertension [1]. Additionally, LMICs
are still struggling to reduce the incidence of infectious diseases that are often caused by contaminated
food or poor hygiene practices. One example is neurocysticercosis (NCC), a neglected tropical disease
(NTD) characterized by parasitic infection of the brain that primarily affects the poor [2].
Peru is undergoing a nutrition transition. The Ministry of Health of Peru has estimated that 20%
of the burden of disease in 2011 was associated with overweight and obesity [3]. NCDs are the main
cause of mortality in Peru [3], while NTDs continue to affect vulnerable groups of the population such
as those living in rural areas with limited access to water and sanitation facilities. In the north of Peru,
the prevalence of type 2 diabetes (T2D) and hypertension (HT) is high (10% and 26%, respectively) [4,5],
and NCC is considered to be endemic in this part of the country [2].

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Type 2 diabetes (T2D) and hypertension (HT) require dietary changes to manage the conditions
and though patients with NCC do not require specific changes in their diet, its prevention has a
close relation with the consumption of uncooked pork and poor sanitary practices. The control of
NCC requires breaking the life cycle of the Taenia solium [6], where the main intervention to stop
transmission in LMICs is improving sanitary practices [7,8]. The prevention of both NCDs and NCC
share the need to promote changes in dietary habits in the population.
Most studies in LMICs have addressed the problem of nutrition transition from the perspective
of accessibility and affordability [9–12] but nutrition is a complex issue since what people eat is not
only about nutritional value, as food choices and preferences are influenced by many factors [13].
The cultural and social contexts in which individuals are brought up, live, and work have a strong
influence on the food choices made, as they affect their views of foods and eating behaviors [13].
Diet is also an important aspect of social life and is related with sharing, belonging to a group, and
celebration [14]. Thus, promoting and supporting dietary changes requires a thorough understanding
of local cultural norms, values, beliefs, and practices that underlie certain unhealthy habits [15].
In this study, we examine the perspectives on the value of food and the challenges of dietary
changes among people living with T2D, HT, and/or NCC from four rural communities in Northern
Peru. We contrast and compare local perspectives of what and why a particular food item is perceived
as “good” or “bad”. Our findings are particularly relevant for culturally appropriate design of
health promotion aiming to achieve the U.N. Sustainable Development Goals for ending all forms of
malnutrition and ensuring healthy lives.
2.1. Study Design

The study is part of a multicountry research for development project that aims to address the
health challenges faced in low- and middle-income countries as a result of the double burden that
NTDs and NCDs place on local health systems [16].
The data were collected as part of a qualitative study aimed at understanding local health
perceptions and use of health facilities of people with chronic conditions and other community
dwellers. Data were collected through semistructured interviews and focus group discussions
using a predetermined set of questions (see Tables S1–S3, interview and focus group guidelines)
and complemented with field notes. This analysis utilized the data related to local dietary habits and
changes in the patient’s life due to the disease (see Table S1 questions 10, 18, 19; Table S2 questions 13,
20; Table S3 questions A4, A5, D1).
2.2. Study Site

All data were collected in the province of Ayabaca, located in the highlands of the Piura region in
northern Peru (Figure S1. Map of Ayabaca). Ayabaca is divided into 10 districts and has an approximate
population of 140,000 (Information is gathered in 2015) [17], with 73% of the population living in
poverty (Information is gathered in 2009) [17]. Two rural communities and their district’s capital towns
were selected for this study: Pingola (Ayabaca district) and Sicacate (Montero district). The main
economic activity in both communities is agriculture. Men predominately work as agricultural farmers,
while women work as housewives (cooking, cleaning, caring for children, etc.) and in some cases,
manage small shops (bodegas).
2.3. Study Participants and Selection Rationale

Study participants were caregivers, head of households, people living with T2D, HT, and/or
NCC, and community dwellers. The latter participated in the focus group discussions. All participants
were 18 years old or older and provided verbal consent.
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Interviews allowed the discovery of opinions and personal experiences on the selected chronic
conditions of the caregivers, heads of households, and patients with T2D, HT, and NCC. The rationale
for interviewing caregivers, heads of household, and patients was to have complementary views of the
condition from each family. All caregivers and head of households were relatives of those affected by
T2D, HT, or NCC. Due to the difficulties to find patients diagnosed with NCC in these communities,
we included persons with epilepsy. Epilepsy is one of the main symptoms for those with NCC, which
is endemic in Ayabaca [2].
Focus groups examined the common perceptions of community members on T2D, HT, and NCC
as well as other health problems, and focus group participants were homogeneous in age and sex.
In order to have the perspective of a vulnerable group within the selected communities, additional
focus groups were conducted with women beneficiaries of JUNTOS, the national cash transfer program
targeting poor women with children under five years old. Some focus groups did not take place
because no participants showed up. Each “failed” focus group was replaced with four individual
interviews. Additionally, when the focus groups had less than six participants, the research team
conducted a “small” focus group and complimented it with additional interviews.
2.4. Recruitment
For semistructured interviews, fieldworkers used word of mouth to find people with the
conditions of T2D, HT, and NCC, their caregiver and head of households, followed by a snowball
sampling methodology.
For the recruitment of focus groups, fieldworkers invited people gathered in different parts of
the communities to participate, e.g., church, marketplace, main square, etc., and asked them to invite
friends of the same age and sex.
2.5. Data Collection

At the beginning of the focus group discussions and interviews, sociodemographic data were
collected from all participants. All interviews and focus groups were audio-recorded.
2.5.1. Data Collection Teams

Data were collected by four people with degrees in social sciences, who had previous experience
in qualitative research in rural areas, and who underwent a four-day training. They were divided
into two teams, one per district, ensuring sex parity [18]. Evidence suggests that the gender of the
interviewer influences participant’s involvement and interaction [19,20] and that matching the gender
of the interviewer and interviewee can facilitate discussion on sensitive topics [20]. For this reason,
teams were made up of one male and one female fieldworker, which allowed the research team
to conduct focus group discussions and interviews by the interviewer who was the same sex of
the interviewee(s).
Data from semistructured interviews and focus groups were complemented with field notes taken
by a doctoral student that accompanied the fieldwork team.
2.5.2. Collection Period

Most data were collected between 2 and 21 February 2017. However, one research team returned
to the field site from 10 to 12 March 2017, to conduct a focus group discussion with young men who
were unavailable during the initial data collection period. Because the data collection period coincided
with the rainy season in these communities, participation in focus groups discussions was low.
2.6. Data Analysis

All interviews and focus group discussions were transcribed and the fieldworkers entered the
data into matrices. The matrices were reviewed by the research assistant and the lead researcher,
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and were then uploaded into ATLAS.ti 8 (Scientific Software Development GmbH, Berlin, Germany),
including the field notes.
A codebook was created (see Table S4: Codebook) and entered into ATLAS.ti 8. To ensure quality
of coding, weekly discussions were held between the lead researcher, two research assistants, and a
doctoral student, in which the codes were analyzed and concerns were addressed.
2.7. Structure of the Analysis

In the interviews and focus groups, participants were asked about the food consumed on a
regular basis, to have an overview of the diet in the communities of the study. To assess community
perceptions of “good” food, we asked informants “¿Para usted cómo es una buena alimentación?”, which
can be translated as “What is a good meal/food/nutrition for you?”. The question translated into
English does not completely preserve its meaning in Spanish. The words “buena alimentación” can have
different uses, for example, in some cases it can be interpreted as having good nutrition and in other
cases it may mean consuming large amounts of food. The intention of this question was to examine the
different meanings the informants give to “buena alimentación”, thus understanding which elements
they consider most relevant when they talk about nutrition, food, and diets.
We also asked informants “¿Por qué es importante tener una buena alimentación?” (Why is it important
to have a good meal/food/nutrition?)” in order to understand community perceptions about the
importance of buena alimentación. In some cases based on the interview, fieldworkers explored if the
food was related to physical strength.
To understand what changes to diet were made by patients after their diagnoses and how family
life was affected because of the diagnosis, both patients and their caregiver/head of households were
interviewed. Patients were asked what changes they had made in their life since they were diagnosed
with their disease (¿Qué cambios ha hecho en su vida desde que fue diagnosticado con diabetes/ hypertension/
neurocisticercosis ?) and what were the most difficult aspects of living with the disease (¿Cuáles son
los aspectos más difíciles de vivir con su enfermedad?). Caregivers and heads of households were asked
about how family life had been affected as a result of living with a person with T2D/HT/NCC (¿Cómo
afecta la vida familiar el vivir con una persona con diabetes / hipertensión / cisticercosis?) and what were the
main changes that had occurred since this relative was diagnosed with T2D/HT/NCC (¿Cuáles son los
principales cambios que han ocurrido desde que él / ella se le diagnosticó diabetes / hipertensión / cisticercosis?).
2.8. Ethics
The study was approved by the Comité Institucional de Ética (Institutional Review Board) at
Universidad Peruana Cayetano Heredia in Lima, Peru (code 393-22-16), and by the Institutional
Review Board at the University of Geneva in Geneva, Switzerland (IRB No. 2016-01242).
3. Results
A total of 138 individuals participated in the study. In total, 16 semistructured interviews
were conducted with individuals living with T2D (n = 4), HT (n = 7), and NCC or epilepsy (n = 5),
the caregivers (n = 8), and head of household (n = 5). Additionally, 12 focus group discussions were
arranged where 82 community dwellers participated as well as 27 individual interviews that replaced
or complimented some focus groups.
3.1. Overview of the Diet in the Communities

As stated previously, the main economic activity in both communities is agriculture. Half of
the production in these communities is for sale and half for selfconsumption or to feed their animals.
The main products grown are maize, bananas, sugar cane, cassava, potatoes, and cultivated pasture
grasses [21].
When asked about the foods consumed on a regular basis, participants sometimes gave a list of
ingredients or food preparations, while other times they described the characteristics of each meal.
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Based on their answers, it appeared that most people have three meals, breakfast, lunch, and dinner,
and that the quantity of food consumed at each meal was usually big in comparison to the amounts
people from urban areas eat:
People are used to eat a lot, not like in the city where for example in the morning they have coffee
and bread. (Female, 70 years old)
We eat a lot. Here is not like the city where they eat bread, here if there is no rice or cassava it is not
a meal. ( . . . ) Here people eat a lot, the big dish and double portion. (Male, 18–34 years group)
For breakfast, participants said they could have rice and fried chicken or rice and fish or egg.
At noon, some people have a snack and later they will have lunch which often consists of rice
and another starchy local food such as potato, yucca, and plantains with legumes (menestras) (see
Figures S2–S4). At dinner, participants stated they would have a soup with noodles, some vegetables,
chicken or with egg. Red meat and pork were not commonly consumed, while chicken and fish (salted
fish) were mentioned as part of the regular diet. Though pork is not consumed in their day-to-day
diets, people often mentioned it as a traditional meal, something easily found in local restaurants.
Vegetables do not appear to be a common ingredient. According to participants, the consumption of
vegetables is related to their availability according to seasonal changes, as one participant explained:
“during the rainy seasons (January, February and March) we eat more vegetables like lettuce and carrot. In April
and May we eat more tamalitos, more corn.”(Man, 56 years old). It is hard to find vegetables in the local
bodegas and if they are available, they are expensive. We observed that noodles were a staple food
in the communities, as they are affordable. Though the interview and focus group participants did
not frequently report this consumption, the observational notes that complemented this information
showed that noodles and cookies are regularly sold products in the small shops of the communities
and noodles are a common ingredient seen in the meals of local people.
Overall, participants described a high consumption of carbohydrates and legumes that they
usually grow. Meat consumption (especially chicken and salted fish) was frequent but in smaller
quantities. Few participants mentioned fruits except for bananas which are part of the local diet.
3.2. What Do People Consider to Be a “Good” and “Bad” Food in These Communities?
As expected, informants responded to the question “¿Para usted cómo es una buena alimentación?
(What is good meal/food/nutrition for you?)”, in different ways. In most cases, they mentioned
specific types of foods they consider to be a buena alimentación (good meal/food/nutrition) or, on
the contrary, foods they view as a “mala alimentacion” (bad meal/food/nutrition). In other cases,
informants mentioned characteristics of their meals (whether meals are “balanced”), certain methods
of food preparation, or aspects of the food production process.
Informants most often responded by mentioning specific foods that they consider to be a buena
alimentación or mala alimentación. In the following tables (Tables 1 and 2) we summarize the most
frequently mentioned food categories and the number of transcripts where each type of food was
mentioned. These tables include responses from both interviews and focus groups, not all the
informants answered this question. Out of 67 transcripts, 62 had a response.
Table 1. “Buena alimentación”.
Type of Food No. of Transcripts n = 62 n (%)

Vegetables (lettuce, cabbage, chard, cauliflower, cucumber, celery, carrot, beet, onion, etc.) 37 (59.7%)
Meat (chicken, beef, pork, lamb, fish, etc.) 28 (45.2%)
Legumes (beans, lentils, peas, etc.) 27 (43.5%)
Grains (rice, wheat/sango, quinoa, cereals, etc.) 24 (38.7%)
Fruits (banana, apple, orange, etc.) 16 (25.8%)
Potatoes, cassava, sweet potatoes 9 (14.5%)
Eggs and dairy (eggs, milk, cheese, yogurt, etc.) 8 (12.9%)
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Table 2. “Mala alimentación”.
Type of Food No. of Transcripts n = 62 n (%)

Meat (pork, chicken, etc.) 18 (29.0%)
Rice 12 (19.4%)
Fried foods 5 (8.1%)
Processed foods (bread, noodles, etc.) 5 (8.1%)
Junk food 3 (4.8%)
Other (potato, sweets, flours, fats, soda, alcohol, unboiled water) 7 (11.3%)
Of those informants who identified meat as a mala alimentación (n = 18), most identified pork
(n = 9) and/or chicken (n = 6) as the most harmful meats. Informants characterized pork as mala
alimentación due to its high fat content and its link to NCC. For those diagnosed with NCC or epilepsy,
almost all (4/5) mentioned pork as a mala alimentación.
Informants also characterized a buena alimentación by mentioning specific methods of food
preparation. Several mentioned that “comidas sancochadas”, or foods that have been cooked in boiling
water, are preferable to fried foods: “(I think a good meal is) usually boiled food, because most of the people
eat fried, or making steamed fish or ceviche. Because if you eat fried food it has fat. For me healthier food is
steamed.” (Woman, 38 years old).
One participant mentioned that buena alimentación consists of maintaining good hygiene in the
process of preparing food: “We need to have a proper hygiene to be able to feed us. If we don’t have the cookware
well cleaned, the hands washed and the nails cut, then we are not going to have a good nutrition”. (Woman,
18–34 years group). The importance of hygienical preparation was also mentioned in a conversation
with a local restaurant owner.
In addition to mentioning aspects of food preparation, informants also characterized buena
alimentación by describing the food production process. Informants considered the foods grown
in their communities or in the “olden days” to be buena alimentación because they are free of
hormones, pesticides, herbicides, fertilizers, and other chemicals. On the other hand, they viewed
foods from the city or “modern” time as mala alimentación because they are produced using the
abovementioned chemicals:
The food here is healthy because the food is natural. Here you can still find natural food, mainly
what comes from the farms because the products do not have fertilizers, so the food is natural. (Man,
70 years old)
They inject them too much, those chicken don’t have nutrients. What is good is milk, lentil, beef,
mutton, pork, which is also nutritious as they eat corn. (Woman, 35 years or older group)
The truth is that we do not know from where they bring the vegetables that are sold in the market, we
do not have trust. ( . . . ) When you grow vegetables in your garden you have trust. But those plants
that come from other places are not recommended to eat because they irrigate them with wastewater.
(Women, 35 years or older group)
The food now is not like before. (Before) it was natural ( . . . ) now it has a lot of chemical, the plant
absorbs the chemical. (Women, 35 years old)
Several informants’ characterized rice (see Table 2) as mala alimentación because they consider that
it requires a greater use of chemicals to grow: “The rice is more chemical, the rice is to fill the stomach.”
(Man, 35 or older years group).
Several also described buena alimentación as eating “balanced” food/meals. While most did not specify
what they meant by “balanced” food/meals, two informants provided the following descriptions:
You have to eat meat, salad and when there is no meat it can be legumes and to drink it can be chicha
morada (typical beverage made of purple corn). (Woman with HT, 48 years old)
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I think the meals have to be balanced. Eating cereals, fruits, vegetables in large amounts, fish and
eating more white than red meat. Red meat is a little more harmful. From time to time you can eat a
desert, like yogurt. (Man, 24 years old)
The reasons provided to the question “¿Por qué es importante tener una buena alimentación? (Why
is it important to have a good meal/food/nutrition?)” are summarized in Table 3. In many cases,
informants mentioned more than one of the listed reasons. Therefore, the categories below are not
independent of one another, rather, they are often interrelated (e.g., “to maintain good health” co-occurs
with “to prevent diseases”). This table includes responses from both interviews and focus groups, not
all the informants answered this question. Out of the 67 transcripts, 36 had a response.
Table 3. Reasons for “buena alimentación”.
Reasons No. of Transcripts n = 36 n (%)

To maintain good health (in general) 18 (50.0%)
To carry out day-to-day activities/tasks 1 16 (44.4%)
To prevent diseases 12 (33.3%)
To have physical strength and endurance 11 (30.6%)
To maintain a strong immune system 2 (5.6%)
To maintain good mental health 2 (5.6%)
Other 2 5 (13.9%)
1 Working in agriculture, domestic work, caring for children, attending school/studying, etc. 2 To maintain
good sexual health, to maintain strong bones, to maintain healthy skin, to control weight, to have good physical
appearance/“estar regias”.
Most often, informants stated that it is important to have buena alimentación in order to maintain
good health in general. Many also expressed that it is important to have buena alimentación in order
to prevent diseases. They often mentioned maintaining good health in conjunction with preventing
diseases: “To not get sick also, have a good health.” (Women with HT, 48 years old). One female informant
specifically mentioned that buena alimentación is important for preventing anemia; however, no one
mentioned T2D, HT, or NCC.
The second most commonly mentioned reason for maintaining buena alimentación was important
for having sufficient strength and endurance to carry out day-to-day labor and activities. Responses
often corresponded to their roles within their family and in the community. Men commonly mentioned
working in agriculture, while women most often mentioned performing domestic tasks and caring
for children.
Because if we are weak we cannot do anything, what are we supposed to do? You cannot work, not
even with your wife you can work. (Man with NCC, 36 years old)
To have strength to work, for example we are weaving and it’s time to have lunch, we make some
food and then again we weave with strength. (Woman with T2D, 68 years old)
In response to a question about what foods give strength to perform agricultural work, responses
commonly mentioned included mote (Peruvian corn), sango (food prepared with corn), and sweet
potato as foods that provide strength and endurance. “The sweet potato is also good, it lasts all day, from
8:00 am when you eat, until 19:00.” (Man with NCC, 36 years) Participants described mala alimentación as
foods that do not provide sufficient strength and endurance to perform daily labors:
The bread does not make your stomach full, for example here you cannot eat bread because the work
is hard. If you eat bread at breakfast it will only last until 10:00 am and you have no strength.”
(Man, 41 years old)
Some participants also mentioned that buena alimentación is important for attending school
and studying.
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3.3. Changes in Diet Due to a Chronic Disease

The answers provided by patients and their family members regarding dietary changes and its
implications were complementary and thus the family is the unit of analysis (n = 17). Three themes
emerged in the data regarding the types of changes patients and their caregivers/heads of household
made in their diets and the difficulties they faced in accomplishing these changes.
One of the main responses had to do with dietary changes in the patient’s life. Patients with T2D
or HT and their relatives (n = 12) reported undergoing dietary changes. They spoke about reducing
or eliminating certain types of foods or ingredients such as sugar (5) (sweets, certain fruits, potatoes,
yucca, rice, noodles, and harinas, colloquial way of saying carbohydrates); salt (7), pork (5), and red
meats (3). They also spoke about decreasing their intake of fats/oils (3) or avoiding preparing fried
meals (1). The reduction/elimination of other foods like alcoholic beverages (1) and food coloring (1)
was scarcely mentioned.
Several patients with T2D and HT and their families said the patient had increased their
consumption of vegetables (6) and fruits (1), or they mentioned the types of food they consumed,
such as chicken (1) and beans (1). Some mentioned they had reduced the amount of food they ate at
each meal (3). Two families of patients with HT reported not making any changes in their diet due to
their disease.
The patients with T2D or HT and their families expressed several challenges associated with
making dietary changes. They expressed struggles in adhering to dietary changes (5) due to the
difficulty of eliminating certain foods from their diet and not being able to eat the way they would like
to: “The most difficult (thing) is not being able to eat what I like, what I have been taught to eat”(Woman with
T2D, 68 years old), eating food they did not enjoy like vegetables, not being able to cook their own food
or not being able to buy vegetables: “It is going to be two months or three months that I have not been able to
buy vegetables, I am afraid that my blood pressure will go up, they say because of high blood pressure you can
die.”(Woman with HT, 64 years old). Another challenge was having to decline the food being served in
social occasions (2), like parties or restaurants, which was uncomfortable.
Though most patients with T2D and HT reported making dietary changes, most of the interviewed
families said they cooked the same meal for everyone (7). No case of separate meal preparation
was reported; however, they did mention various ways of adapting the patients´ diets to the
recommendations they received from the health professionals. For example, in some cases the family
separated the patient´s portion before adding spices, and in other cases the entire family adopted a
new diet as a result of the patient’s chronic condition: “Now we cannot prepare some foods, tamal (food
made of corn), tortillas (food made of corn), sweets, those things we cannot prepare. We used to drink sodas, but
now none of us drinks sodas because its poison (...) now (we drink) only water.” (Woman caregiver of TD2,
46 years old). Relatives said that some members of the family would later add spices to the food or
would secretly eat some “unhealthy” types of food: “The food I prepare is tasteless; if they (family) want
they can add more salt.” (Woman with T2D, 52 years) In some cases, patients would try to adapt their diet
with the food already prepared by their family “Everything is already prepared from early in the morning,
so I take out a chicken breast and with a napkin I take out the fat.” (Woman with T2D, 64 years).
Some patients with NCC and their families also mentioned some dietary changes, including the
reduction/elimination of pork (3), duck (2), fish (2), turkey (1), and “bocadillo” (1) (typical sweets from
the district). “They gave me my treatment for epilepsy and told me to not eat pork, which I should not eat until I
die.” (Woman with NCC, 50 years). They also mentioned having difficulties to stop eating some of these
foods: “I do not eat pork now, though I really liked eating pork.” (Man with NCC, 36 years). Two families of
patients with NCC reported not making any changes in their diet.
4. Discussion
Our study shows the various ways in which people from rural northern Peru value food and
the multiplicity of factors that play a role in identifying food as good/healthy or not. Understanding
food- and diet-related contexts like the one in Ayabaca, and similar rural settings facing a nutritional
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transition, and the complexity attached to it, is fundamental to end all forms of malnutrition
(encompassing both undernutrition and overnutrition) and promoting healthy lifestyles and well-being
for all and throughout the life course, as proposed by the sustainable development goals 2 and 3 [22].
Our study highlights the challenges of dietary changes in patients with chronic conditions in
a rural area and the relevance of understanding the local context. In the communities where we
conducted this study, perceptions of buena and mala alimentación are strongly linked to the extent
to which foods enable individuals to perform day-to-day tasks and activities, and this finding is
consistent with other studies in Peru [23]. For this reason, individuals are accustomed to consuming
high-carbohydrate foods such as rice, potato, corn, and grains, often in large portion sizes. On the
other hand, there is conflict between the people recognizing vegetables and fruits as “good” food,
and the insufficient consumption of these fruits and vegetables because they are not available or
are not affordable. As other studies have reported [24,25], we also found that when foods are not
available within the community, participants do not buy them outside due to negative perceptions
about consuming food from an unknown source. Yet, there is an increasing consumption of cheap
processed foods, such as noodles.
Our findings show the complexity of introducing dietary changes in rural communities where
the perspectives on food are poorly understood and rarely taken into consideration when promoting
food- or diet-related behavior change. T2D and HT are diseases for which much of their management
can occur in the community, at the primary care level, and where the prevention of NCC needs
adequate control at the community as well. So the food people intake will have consequences in the
management of their disease or in the prevention of new diseases.
For patients with T2D, HT, and NCC, following dietary recommendations received by health
professionals is challenging. On the one hand, people have difficulties not being able to eat food they
like, the food they grew up with, the food that they perceive as nutritious or tasteful, nor eat certain
foods in the quantities they are used to. On the other hand, the food recommended by the health
professionals are things they do not like to eat. Furthermore, diet modifications involve negotiation or
arrangements with the family, which is key to successfully adapt to the recommended diet [26]. In this
sense, involving the family is a key element for dietary modifications. As Vanstone and colleagues
show in their review, “support at home is universally described as an essential component of successful
dietary modification” [27,28]. Patients in our study know and say they make dietary modifications, but
the compliance of healthy dietary practices seems more complex to accomplish. As what is reported in
other studies [27,28], making dietary changes recommended by health professionals is challenging.
This study helps understand how dietary recommendations for chronic conditions in rural agrarian
communities need to be reconciled with working conditions, access to food, perceptions of food,
and family.
Our results align with some crosscutting themes such as food availability, introducing dietary
changes, and health promotion. What people eat, especially what poor people eat, is not only about
what people identify as healthy, but the availability of such foods. Food availability is one of the four
pillars of food security [29]. Thus, a poor person’s socioeconomic status can exert a strong influence on
food choice behaviors [30–32]. It is necessary to transform food systems into sustainable and sensitive
nutrition systems that will provide a variety of healthy foods, devoting special attention to the most
vulnerable [9].
The availability of cheap unhealthy choices is a growing problem in many LMICs [33–35] and it is
also happening in rural Peru [36]. Understanding local eating habits must take into account the role of
structural factors on food choices [37]. Thus, our study supports the finding that individual choices,
especially those of the poor, are influenced by broader social and environmental factors [32,38].
Our findings are consistent with studies across the globe that show that knowledge is not enough
to influence behavior [39–41]. Theories of human behaviors, such as eating behavior, suggest that a
combination of environmental and personal factors influence behavior [42]. Environmental factors
include cultural determinants (e.g., nationality, ethnicity, identity), physical (e.g., policies, availability,
185
Nutrients 2018, 10, 1563
accessibility), and social (e.g., parental behavior, peer influence, advertisement exposure) [43]. Personal
determinants, such as liking and preference, are the strongest determinants of diet habits [44–46].
Introducing, achieving, and sustaining healthier dietary changes will require a combination of all those
factors, beyond availability and affordability.
This study highlights that patients, caregivers, and community members living in rural Peru have
reasonable knowledge of what a healthy diet consists of, yet making food choices is a complex issue
influenced by work, family, trust, taste, as well as affordability and accessibility. It also highlights
that perceptions of the value of certain foods are not necessarily consistent with the advice received
about healthy diet from health educators and health workers. Health promotion efforts therefore
must include not only factual information about nutrients, but also provide opportunities to taste
new foods, cook in new ways that are sustainable, and learn about what foods and vendors from
outside the community are prepared in ways that respect local preferences. For example, it would be
beneficial for health professionals to have a good idea of what foods are produced locally in order
to recommend these items in the diet, but also to provide a clear understanding of the relationship
between what a person needs versus how much is needed, in accordance with their daily activities
(i.e., caloric expenditure).
Some limitations are worth noting. During the recruitment process, it was not always possible to
achieve the desired sample size of patient, caregiver, and head of household. The participation in focus
groups was low due to the rainy season and there were difficulties recruiting young men in the focus
groups. Additional interviews with community dwellers were conducted so as to reach data saturation.
Additionally, our study did not include the perspective of health workers’ views, which would be
helpful in better understanding their challenges in promoting healthy eating. Furthermore, we did not
collect information about the availability of food at local stores and their prices in a systematic way
that would enable us to fully understand accessibility and affordability of food choices in our sample.
Finally, objective measures of the reality in the homes of participants were not collected and so it was
not possible to know with certainty if perceptions matched the reality in the home. Nonetheless, the
methodology and sampling procedure utilized allow for an overview of the situation regarding food
perceptions in these communities.
5. Conclusions
This study shows the important influence of culture and social conditions on food perceptions
and dietary changes in rural Peru. The findings highlight that poor people in rural northern Peru,
just like others across the globe, make decisions and have knowledge about food that are based on
life-long learning, experience, exposure, and availability. The study also stresses that knowledge
alone does not influence eating behavior and that making dietary change is difficult. Thus, promoting
healthy diets without contextualizing information and recommendations is sure to produce suboptimal
results. Health promotion focused on nutrition for rural, poor communities must take into account the
activities of daily life, access and affordability of foods, but also food taste preferences and traditions.
Supplementary Materials: The following are available online at www.mdpi.com/2072-6643/10/11/1563/s1, Figure

S1: Map of Ayabaca, reproduced from reference [47], Figure S2: Meal made of soup with potatoes, pasta, and
beans, Figure S3: Traditional meal named “Sango” made with maize flour and water with a fires egg, Figure S4:
Meal in a community meeting, containing potatoes, rice, carrots, and onions with one serving of meat, Table S1:
Interview Guide for Patients, Table S2: Interview guide for caregivers and head of household, Table S3: Guide for
focus group discussions, Table S4: Codebook.
Author Contributions: Conceptualization, N.A.P. and L.S.S.; Methodology, M.A.P., N.A.P., and L.S.S.; Formal
Analysis, S.R., S.P.-L., N.A.P., and M.A.P.; Investigation, S.P.-L. and N.A.P.; Resources, J.M.; Data Curation, S.R.
and S.P.-L.; Writing-Original Draft Preparation, S.P.-L.; Writing-Review & Editing, M.A.P., S.R., N.A.P., J.M., and
L.S.S.; Supervision, M.A.P.; Project Administration, M.A.P.; Funding Acquisition, J.M., M.A.P., and L.S.S.
Funding: This research was funded under the r4d Public Health call of the Swiss Programme for Research on
Global Issues for Development by the Swiss National Science Foundation and the Swiss Development Cooperation
grant number 40P740_160366/1.
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Nutrients 2018, 10, 1563
Acknowledgments: We want to thank David Beran, who provided critical feedback on earlier versions of the
manuscript and whose encouragement was key to complete the manuscript.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to
publish the results.
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nutrients
Review
The Role of Nutrients in Reducing the Risk for
Noncommunicable Diseases during Aging
Maaike J. Bruins 1, *, Peter Van Dael 1 and Manfred Eggersdorfer 2
1 Nutrition Science & Advocacy, DSM Nutritional Products, CH-4303 Kaiseraugst, Switzerland;
[email protected]
2 University Medical Center Groningen, 9713 GZ Groningen, The Netherlands; [email protected]
Received: 20 September 2018; Accepted: 27 December 2018; Published: 4 January 2019
Abstract: An increasing aging population worldwide accounts for a growing share of

noncommunicable diseases (NCDs) of the overall social and economic burden. Dietary and nutritional
approaches are of paramount importance in the management of NCDs. As a result, nutrition programs
are increasingly integrated into public health policies. At present, programs aimed at reducing the
burden of NCDs have focused mostly on the excess of unhealthy nutrient intakes whereas the
importance of optimizing adequate essential and semi-essential nutrient intakes and nutrient-rich
diets has received less attention. Surveys indicate that nutrient intakes of the aging population are
insufficient to optimally support healthy aging. Vitamin and mineral deficiencies in older adults
are related to increased risk of NCDs including fatigue, cardiovascular disease, and cognitive and
neuromuscular function impairments. Reviewed literature demonstrates that improving intake for
certain nutrients may be important in reducing progress of NCDs such as musculoskeletal disorders,
dementia, loss of vision, and cardiometabolic diseases during aging. Current knowledge concerning
improving individual nutrient intakes to reduce progression of chronic disease is still emerging with
varying effect sizes and levels of evidence. Most pronounced benefits of nutrients were found in
participants who had low nutrient intake or status at baseline or who had increased genetic and
metabolic needs for that nutrient. Authorities should implement ways to optimize essential nutrient
intake as an integral part of their strategies to address NCDs.
Keywords: chronic disease; noncommunicable disease; nutrient inadequacies and deficiencies; nutrient
interventions; public health; musculoskeletal disorders; dementia; eye disorders; cardiovascular disease
1. Introduction
Globally, significant gains in human longevity have been made in the last couple of decades as
evidenced by an average 5.5-year increase in life expectancy between 2000 and 2016 [1]. In many
countries average life expectancy currently exceeds 80 years [1]. These longevity gains have come at a
cost, however, with the most obvious being an increase in age-related diseases [2]. Noncommunicable
diseases (NCDs) such as diabetes, musculoskeletal disorders, cardiovascular diseases, neurological
disorders, and cancers increase with age, and place a burden on individuals and healthcare systems [3].
Supporting healthy aging by preventing NCDs is a major priority for agencies such as the World
Health Organization (WHO) and United Nations [4,5].
The WHO estimates that NCDs contribute 1.6 billion disability-adjusted life-years (DALYs)
to the global burden of disease and identified unhealthy diets and physical inactivity are among
the main modifiable risk factors, together with excess alcohol and tobacco use [6]. Nutrition is
an important determinant of human health by providing the essential building blocks for growth,
development, and maintenance of a healthy status throughout life [7,8]. In this context, the co-existing
burdens of undernutrition and overnutrition represent a paradigm shift for health authorities

Nutrients 2019, 11, 85
requiring appropriate dietary management recommendations [9]. Modern lifestyles and easy access to
high-energy, low-nutrient rich foods are considered part of the problem [3,10–12]. For example, the
economic costs of unhealthy diets and low physical activity in the EU were calculated to be €1.3 billion
per year [13].
Currently, health authorities mainly target problems associated with obesity and cardiovascular
diseases by focusing on reducing excess intake of calories, sugar, salt, and saturated fats. However,
the importance of a positive message associated with promoting adequate nutrient intake as part of a
balanced diet should not be overlooked [4]. There is considerable variation in the consumption of food
items that need to be encouraged and food items which should be limited, both between and within
different countries. This was reflected in a recent study in European countries showing suboptimal
nutrient-density of diets and significant proportions of the population consuming excess amounts
of salt, sugar and saturated fat, as well as significant proportions of the population not meeting the
required or adequate intakes for various essential nutrients (Table 1) [12].
Table 1. Percentage of adults with nutrient intakes meeting the estimated average requirement (EAR)
or adequate intake (AI) or exceeding the maximum reference value (MRV) [12].
Denmark Czech Republic Italy France

% Meeting EAR or AI EAR or AI
n = 2025 people n = 1869 people n = 2831 people n = 2624 people
Protein, g/d 0.66 g/kg BW 84% 88% 99% 98%
MUFA, E% 10–20 E% 69% 92% 75% 77%

Dietary fiber, g/d 25 19% 4% 12% 9%
Calcium, mg/d 750 70% 31% 43% 62%
Iron, mg/d M: 6; F: 7 92% 96% 98% 98%

Potassium, mg/d 3500 31% 4% 19% 18%
Magnesium, mg/d M: 350; F: 300 46% 25% 20% 23%
Zinc, mg/d M: 7.5; F: 6.2 90% 48% 97% 91%
Vitamin A, μg RE/d M: 570; F490 77% 38% 66% 77%

Vitamin C, mg/d M: 90; F: 80 50% 35% 62% 44%
Vitamin E, mg/d M: 13; F: 11 5% 44% 47% 34%
Vitamin D, μg/d 15 3% 1% 1% 1%
Vitamin B1 , mg/d 0.6 97% 98% 47% 100%
Vitamin B2 , mg/d M: 1.1; F: 0.9 80% 35% 84% 92%

Vitamin B12 , μg/d 4 55% 36% 52% 50%
Folate, μg DFE/d 250 59% 24% 77% 51%

% exceeding MRV MRV
SFA, E% <10 E% 86% 80% 62% 91%
Added sugar, E% <10 E% 32% 21% 24%
Sodium, mg/d <2400 mg/d 80% 98% 13% 85%
RE: retinol equivalents, DFE: dietary folate equivalents, E%: energy percentage, MUFA: mono-unsaturated fatty
acids, SFA: saturated fatty acids. The red, orange, yellow, light green and dark green signals, respectively, represent
≤5%, 6–35%, 36–65%, 66–95%, and ≥96% of people meeting the EAR.
The health consequences of poor nutrition almost certainly accumulate over the lifespan of the
individual. Table 2 presents information regarding some of the more frequently reported chronic
clinical signs associated with certain vitamin and mineral deficiencies in older adults. Clinical signs
and symptoms are mostly nonspecific and difficult to diagnose. During the aging process, a number
of changes occur, such as increased medication use, reduced food intake due to lower food appeal,
191
and compromised nutrient absorption. These complex changes prevent elderly persons from meeting
their nutritional requirements. This consequently leads to increased risk of malnutrition, frailty, and
reduced quality of life (QoL) [14–17].
Table 2. Critical nutrients in older adults [18].
Micronutrient Challenges, Clinical Signs, and Symptoms in Older Adults

Deficiencies common in older adults, often underdiagnosed. Role in reducing elevated
homocysteine, a cardiovascular risk factor. Absorption decreases mainly due to high
Vitamin B12
prevalence of age-related atrophic gastritis. Among the common causes of anaemia in
(cobalamin)
older adults, leading to weakness and fatigue. Low status increases the risk for
cardiovascular disease and cognitive impairment.
Deficiencies common in older adults. Role in reducing elevated homocysteine, a
cardiovascular risk factor. Closely related to vitamin B12 and B6. Among the common
Folate
causes of anaemia in older adults, leading to weakness and fatigue. Deficiencies linked to
depression and dementia.
Deficiencies common in older adults. Role in reducing elevated homocysteine, a
Vitamin B6
cardiovascular risk factor. Closely related to vitamin B12 and folate.
Thiamine Deficiencies common in older adults, often underdiagnosed. Risk factor for heart failure,
(vitamin B1) peripheral neuropathy, and encephalopathy.
Deficiencies common in senior women. Mean intake decreases with age, probably related
Calcium to general change in diet. Associated with low bone mass, rapid bone loss, and high
fracture rates.
Older adults are less exposed to sun and have diminished ability of the skin to synthesize
Vitamin D vitamin and the liver and kidney to hydrolyze vitamin D with age. Deficiency is a risk
factor low bone mass, rapid bone loss, high fracture rates, and muscle weakness.
Prevalence of inadequate intake is very high among adults. May help elderly maintain
Vitamin C
immune cells and function. Smoking increases need.
Women’s iron requirements decrease after the menopause. Deficiencies are mainly seen
Iron among hospitalized, institutionalized, or chronically ill older adults. Among the common
causes of anaemia in older adults, leading to weakness and fatigue.
Deficiency is common in the elderly. Risk factor for immune deficiency and susceptibility
Zinc
to infection in the elderly.
Deficiency deficiency may increase risk of diseases of aging such as cardiovascular disease,
Selenium
reduced immune response, and cognitive decline.
Magnesium Often deficient in older adults. Maintains muscle integrity and function.
Health policies and interventions to improve dietary intake at the population level are essential
to reverse the global trend towards unhealthy dietary patterns and physical inactivity. However,
more individualized approaches may be needed to address persistent nutritional gaps and prevent
future morbidity in high-risk groups such as the older population [19,20]. An estimated 5% to 10%
of community-dwelling adults >70 years of age are undernourished; this proportion rises to 30% to
65% among institutionalized elderly patients. In the older adult population, nutrients of concern
include, among others, calcium, vitamin D, and vitamin B6 and B12 [15,20,21]. Vitamin D deficiency
was found not only to be a problem in the elderly, but to be a global problem common across all
age ranges [22]. Genetic variations play a role in dietary response and genetic variations also play a
role in determining nutrient status and requirements [23]. By understanding the genome that affects
the individual requirements for and response to nutrition, diseases of aging that have a nutritional
component can be addressed in a targeted way.
In general, activities endorsing lifestyles that include healthy diets have usually focused on
limiting the consumption of salt, sugar, and saturated fat. However, focus on the need to meet
adequate dietary intake of essential nutrients through a healthy diet is considered equally important.
This review focuses on the role of nutrients in the risk reduction of NCDs in disorders prevalent
in the aging population and for which the societal costs are substantial [24]. The evidence for a
connection between NCDs and inadequate intake or status of specific nutrients such as vitamins,
192
carotenoids, omega-3 fatty acids, and other bioactive substances is reviewed. Furthermore, the impact
of interventions aimed at correcting these inadequacies will be discussed.
2. Musculoskeletal Health in the Older Adult

The gradual loss of bone mass and disruption of bone architecture associated with osteoporosis
results in an increased risk of bone fractures, particularly of the hip, spine, and wrist. It is an age-related
chronic, complex, multifactorial skeletal disorder which affects both men and women, particularly
postmenopausal women [25]. Osteoporosis places a huge personal and economic burden on society.
In Europe, for example, the disability caused by the disease is greater than that caused by cancers (with
the exception of lung cancer) and is comparable or greater than that caused by a variety of chronic
NCDs, such as rheumatoid arthritis, asthma and hypertension-related heart disease [26].
In a WHO report it was noted that the remaining lifetime risk of an osteoporotic fracture in women
aged 50 years in developed countries was >40% (>20% for hip fracture) [27]. At the time of this report,
osteoporotic fractures had the sixth highest disease burden in the Americas and Europe combined,
as estimated by disability-adjusted life years [27,28]. In 27 countries in the European Union, based
upon the overall epidemiology of 22 million women and 5.5 million men with osteoporosis, it was
calculated that this would result in 3.5 million new bone fractures (hip, 610,000; vertebral, 520,000;
forearm, 560,000; and others 1.8 million) [28]. The economic burden to manage these incident and
prior bone fractures was calculated to be €37 billion.
In the elderly, both micronutrient and macronutrient deficiencies appear to contribute to the
pathogenesis of skeletal fractures as a consequence of age-related bone loss and frailty [16]. Nutrients
that play a role in bone metabolism include vitamin D and vitamin K, calcium, magnesium, phosphorus,
proteins, and fatty acids.
2.1. Vitamin D in Musculoskeletal Health

Vitamin D is involved in bone homeostasis by enhancing calcium and phosphorus absorption
from the intestine and maintaining adequate levels in blood. Low vitamin D levels have been mainly
implicated in musculoskeletal disorders including bone and muscle health [29]. Serum levels of
25(OH)D have been associated with bone turnover markers levels [30].
Vitamin D comprises a group of secosteroids (calciferols), and in humans the two most important
compounds in this group are vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol) [22]. A major
part of vitamin D comes from UV-B induced production in the skin and only about 20% from dietary
intake. Dietary sources are limited to mainly oily fish and foods fortified with the vitamin [31]. Lack
of vitamin D from the diet and increased awareness of the harmful skin effects of excessive sunlight
exposure have contributed to low vitamin D status and even deficiency globally.
Serum 25-hydroxyvitamin D is the most widely used indicator for vitamin D status in clinical
practice and, while 25–50 nmol/L is generally defined as insufficiency with regards to bone health, for
optimal calcium absorption and control of secondary hyperparathyroidism a level closer to 75 nmol/L
has been proposed [16,22,32,33]. Most researchers agree that 25-hydroxyvitamin D levels below
50 nmol/L are associated with lower bone mineral density [22]. Likewise, the effect of vitamin D
deficiency on fracture risk is difficult to quantify, but large population studies found that hip fracture
risk was higher in those with a 25-hydroxyvitamin D level below 50–62.5 nmol/L [34,35]. Based on
a serum 25-hydroxyvitamin D level of <30 nmol/L it was reported that on average 13% of 55,844
European individuals had moderate or severe vitamin D deficiency, and this increased to 40% of
individuals with mild to severe deficiency if a level of <50 nmol/L was included [36]. The authors
noted that vitamin D deficiency was present across Europe and was both a clinical and public health
concern requiring urgent action. Similar levels of vitamin D deficiency and concern have been reported
by many research groups worldwide [22,36–38]. Figure 1 highlights the variable levels of vitamin D
deficiency across Europe [39].
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Figure 1. Europe map of vitamin D deficiency in older adults (mean 25(OH)D status (nmol/L) in adults
aged ≥50 years) (based on: [39]).
The role of vitamin D and related analogues, with or without calcium, for preventing bone
fractures in post-menopausal women and older men was the subject of a Cochrane review [25].
This systematic review included 53 trials and 91,791 older women or men aged over 65 years from
community, hospital, and nursing-home settings, and assessed the impact of vitamin D for the
prevention of hip or other types of fracture. In this analysis vitamin D alone did not appear to have a
significant effect on fracture prevention, whereas vitamin D in combination with calcium significantly
reduced the likelihood of hip fractures (P = 0.01), non-vertebral fractures and any type of fracture.
Hip fracture incidence was particularly reduced in institutionalized residents with a risk reduction of
25%. In a separate systematic review (30 randomized controlled trials (RCTs) involving 5615 subjects
of mean age 61 years), vitamin D supplementation was shown to produce a small but statistically
significant improvement in global muscle strength. The most benefit was observed in individuals
who presented with a 25-hydroxyvitamin D level below 30 nmol/L (compared with those with a level
≥30 nmol/L), and in subjects aged 65 years or older [29].
The economic value of vitamin D supplementation has been the subject of several health economic
evaluations showing that increasing vitamin D status through supplementation or fortification can
prevent fractures and improve QoL in older adults and associated health care costs [40–44].
2.2. Vitamin K in Musculoskeletal Health

Two forms of vitamin K exist: vitamin K1 (phylloquinone, mainly found in green leafy vegetables)
and vitamin K2 (menaquinone, mainly found in fermented dairy and produced by lactic acid bacteria in
the intestine). Vitamin K is required for promoting osteoblast differentiation, upregulating transcription
of specific genes in osteoblasts, and activating bone-associated vitamin K dependent proteins, which
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play critical roles in extracellular bone matrix mineralization. Less is known about vitamin K and
health, but there is growing evidence suggesting a synergistic effect between vitamins K and D in
bone [40]. A number of studies reported that vitamin K is essential for optimization of bone health
with benefits in preventing bone loss [41]. Vitamin K2 supplementation combined with vitamin D
and calcium for 2 years in a randomized placebo-controlled trial resulted in a significant increase
in bone-mineral density and content in older women [42]. In another recent RCT it was found that
combined vitamin K2 , vitamin D and calcium supplementation for 6 months increased the bone mineral
density of lumbar 3 spine vertebra compared to vitamin D and calcium alone in postmenopausal
Korean women [43].
Current research investigating the effect of vitamin D alone or in combination with other nutrients
on fractures, cardiovascular disease, diabetes, cognitive function, immunity, and other benefits
is ongoing in two large scale studies in older adults (DO-HEALTH in Europe, FIND in Finland).
In addition, many research groups engage in basic science to study the combined action of vitamin
K2, vitamin D, and calcium, and their function on the molecular level. More studies are required that
target vitamin D supplementation in combination with other nutrients such as calcium and vitamin K
where it is needed, in people with vitamin D deficiency or older people, who are more likely to be frail
in institutionalized residents.
3. Cognitive Disorders
Dementia is a term that describes a decline in cognitive abilities including memory, and reduction
in a person’s ability to perform everyday activities [44]. Dementia prevalence is forecast to increase
dramatically in future years [45]. At present about 50 million people have dementia worldwide, and
this is projected to reach 80 million by 2030 and 150 million by 2050 [46]. Alzheimer’s disease (AD)
is the most common form of dementia in people aged >60 years, accounting for 60–70% of the total
number of cases and is the major focus of this section [46]. Vascular dementia is the second most
common cause of dementia with at least 20% of dementia cases.
Alzheimer’s disease is a complex, progressive, multifactorial, neurodegenerative disease [24,45].
The presentation generally involves progressive memory loss, impaired thinking, disorientation, and
changes in personality and mood. As the disease advances there is a marked reduction in cognitive
and physical functioning [47,48]. Genetic factors account for about 70% of the risk contributing to AD,
while modifiable factors related to general health and lifestyle may also be involved [48]. Risk factors
for vascular dementia are predominantly modifiable and of vascular origin (including hypertension,
diabetes mellitus, dyslipidemia, and the metabolic syndrome). Managing non-genetic risk factors
effectively may provide opportunity to prevent and treat the progressive cognitive decline associated
with AD [47]. The focus of this section of the review is on nutritional status and its potential role in AD.
The Role of Nutrition in Dementia

In terms of a link between nutrient status in older adults and cognition, evidence exists for
B-vitamins, and vitamin C, D, and E, as well as the omega-3 long chain polyunsaturated fatty
acids (LCPUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), as reviewed by
Antal et al. [48] and summarized here (unless otherwise noted).
Folic acid and vitamin B6 and B12 are important in the nervous system at all ages, but particularly
in elderly people, deficiency contributes to aging brain processes [49]. Low status of folic acid and
vitamins B6 and B12 are among the risk factors for elevated homocysteine. With respect to dementia,
there is reasonable evidence linking lower levels of folic acid, vitamin B6 , vitamin B12 , and higher
concentrations of homocysteine with age-related cognitive decline [50]. One of the mechanisms
involved may the impaired methylation processes due to folic acid and vitamin B12 deficiency that
lead to accumulation of homocysteine affecting mood and some cognitive functions [50]. In several
RCTs supplementation with folic acid, vitamin B12 , and vitamin B6 for at least 2 years has been
investigated [44]. However, the findings of a recent meta-analysis reported that B vitamins had
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little to no effect with respect to preventing cognitive decline [51]. Notably, individuals with high
homocysteine levels had significant cognitive decline and B-vitamins were found to improve memory
only in this subgroup [52]. Also, evidence exists that in elderly subjects with an increased risk of
dementia, B-vitamins can slow brain shrinkage over two years by up to 30% [53]. At present, the
evidence is insufficiently compelling to support B-vitamin supplementation to prevent cognitive
decline and dementia.
Dehydroascorbic acid, a metabolite of vitamin C, is a potent antioxidant, an essential cofactor
in many enzymatic reactions, and has a role in metabolizing cholesterol. Large dietary surveys
undertaken in Germany, the Netherlands, the UK, and the US indicated inadequate vitamin C intake in
up to half of respective populations [54]. As with vitamin E, however, studies of vitamin C in patients
with AD have been equivocal. The overall conclusion of the Team of Alzheimer Drug Discovery
Foundation is that maintaining adequate levels of vitamin C through diet may offer more benefit
than supplementation.
The metabolically active form of vitamin D, 1,25-dihydroxy vitamin D, binds to vitamin D
receptors that are present in brain regions involved in cognition. Proposed mechanisms for the
protective effects of vitamin D against cognitive decline include clearing Aβ peptide, regulating
intraneuronal calcium, anti-inflammatory activity, antioxidative activity, preventing and reducing
ischemia, and regulating choline acetyltransferase neurotrophic agents. There is strong evidence that
patients with AD have lower vitamin D status than healthy controls and that lower vitamin D status is
associated with increased risk of developing dementia. Although vitamin D supplementation alone
was insufficient to improve cognition in a study of patients with newly diagnosed AD, the Vitamin D
Council recommends that middle-aged and older adults maintain vitamin D blood levels in the higher
range of normal (175–200 nmol/L; 70–80 ng/mL).
Vitamin E possesses antioxidant properties which may prevent hyperphosphorylated tau protein
dysfunction and has been shown to reduce the rate of Aβ protein-induced death in cultures of
hippocampal and cortical cells [55]. The γ-tocopherol isomer is particularly effective in scavenging
free radicals that cause inflammation [55]. By scavenging Aβ protein-associated free radicals, vitamin
E may have a neuroprotective effect during oxidative stress. However, while promising in principle,
studies of α-tocopherol supplementation in patients with AD have not been convincing and dietary
vitamin E may provide greater protection against age-related neurodegenerative conditions.
Brain membranes are composed mainly of phospholipids, predominantly the LCPUFAs DHA and
arachidonic acid (ARA). DHA has multiple actions in maintaining neurological function. Lower plasma
DHA has been associated with cognitive decline in both healthy elderly people and AD patients [56].
Investigations to date of the therapeutic potential of supplementation or higher dietary intake of DHA
in patients with AD have produced conflicting results, although it is possible that cognitive impairment
in the study populations was already resistant to intervention. Given the essential role of DHA in the
human brain, a general recommendation to maintain an adequate dietary intake of DHA throughout
adulthood appears to be a reasonable approach to prevent cognitive decline.
Due to a high concentration of oxygen free radicals relative to antioxidative defenses in the brain,
it may be especially vulnerable to oxidative stress and consequent damage to lipids and proteins [57].
AD is also associated with lower levels of acetylcholine in the hippocampal and cortical regions,
resulting in memory impairment. Fruits, vegetables, coffee, and cereal grains contain high levels of
polyphenols. In vitro and animal studies of specific dietary flavonoids and plant extracts have shown
reduction of oxidative stress and inhibition of acetylcholinesterase, suggesting a dual protective role
for polyphenols against cognitive decline and dementia [57]. Although no conclusions can be drawn
about the relative benefits of any particular plant polyphenol over another, the findings emphasize the
importance of life-long consumption of foods with high content of these antioxidants.
Trials that have reported no effect of nutrients generally included older adults who were unlikely
to have a marked decline in cognitive function [52]. Trial design should consider including older
individuals with deficiencies that increases their risk of cognitive decline, and who may benefit from
196
nutrition intervention. Sensitive assessment tools and surrogate markers are needed that examine
specific aspects of brain structure and function such as neuroimaging techniques to advance the
understanding of nutrition interventions that could reduce the risk of dementia.
4. Eye Disorders
Impairments of the essential senses of vision and hearing are the second-leading cause of years of
lived with disability [58]. The most common causes of vision loss among the elderly are age-related
macular degeneration, glaucoma, cataracts, and diabetic retinopathy [59]. Aging is the greatest risk
factor associated with the development of age-related macular degeneration, but also environmental
and lifestyle factors such as smoking, oxidative stress, and diet may significantly affect the risk [60].
Recent studies suggest that increasing exposure to blue light emitted by electronics and energy-efficient
lightbulbs over time could lead to damaged retinal cells which on the long-term can cause vision
problems like age-related macular degeneration [61]. Eye health problems in the ever-increasing aging
generation, and “exposure to blue light” may result in a new NCD.
Carotenoids have a range of functions in human health and, in particular, there is evidence that
they have beneficial effects on eye health [62]. Two dietary carotenoids, lutein and zeaxanthin are
macular pigments found in the human retina [63]. Macular pigment has local antioxidant properties
and absorbs high energy, short wavelength blue light protecting the retina from photochemical
damage [64]. Macular pigment can neutralize ROS, protect against UV-induced peroxidation, and
reduce the formation of lipofuscin and associated oxidative-stress induced damage [63]. Thus, the
carotenoids provide potential benefits for ocular function and health.
Individuals who have low macular pigment optical density levels (0.2 or lower) may benefit from
supplementation with lutein/zeaxanthin which can help increase macular pigment optical density
levels [65–72]. For retinal protection, macular pigment optical density values of 0.4 to 0.6 are desirable,
especially in older adults [73]. Dietary intake of lutein and zeaxanthin may differ with age, sex,
and ethnicity. Across all age groups the intake of lutein is higher than for zeaxanthin and this is
independent of sex and ethnicity. In addition, lower zeaxanthin to lutein ratios are reported for groups
at risk of age-related macular degeneration (e.g., the elderly and females) [74]. A number of studies,
including some in healthy subjects, have demonstrated that lutein/zeaxanthin supplementation
can improve visual performance, including contrast sensitivity, glare tolerance and photo stress
recovery [65–72,75,76].
Age-related macular degeneration is an increasing problem among the elderly and studies of the
effects of lutein/zeaxanthin supplementation have produced mixed results. However, important data
were provided by secondary analyses of the large Age-Related Eye Disease Study 2 (AREDS2) [77,78].
This randomized trial investigated the effect of adding lutein/zeaxanthin 10/2 mg, DHA (350 mg)
+ EPA (650 mg), or both to the original AREDS2 formulation (vitamin C, vitamin E, β-carotene,
zinc, and copper) or to variations of this formulation (excluding β-carotene and/or with reduced
zinc). Participants (n = 4203) were followed for a median 5 years. The primary analysis found
no additional beneficial or harmful effect for lutein/zeaxanthin and/or omega-3 fatty acids on
progression to late age-related macular degeneration compared with the original AREDS1 formula
using β-carotene instead of lutein/zeaxanthin. However, a prespecified secondary analysis found a
significant 26% risk reduction for progression to advanced age-related macular degeneration when
comparing lutein/zeaxanthin supplementation with no lutein/zeaxanthin supplementation in the
quintile with the lowest dietary intake of these two carotenoids (median 0.7 mg/day), as indicated
by a hazard ratio of 0.74 (95% confidence interval 0.59–0.94, p = 0.01). In addition, a post hoc analysis
showed that lutein/zeaxanthin (excluding β-carotene) was more effective than the original AREDS
formulation containing β-carotene but no lutein/zeaxanthin for reducing progression to advanced
age-related macular degeneration (hazard ratio 0.82, 95% CI 0.69–0.96, p = 0.02) [77].
There is also some evidence suggesting there is a relationship between lutein/zeaxanthin
status and the risk of developing nuclear cataracts [79], and in the AREDS2 trial the addition of
197
lutein/zeaxanthin supplementation reduced the risk of cataract surgery in the quintile with the lowest
dietary intake of these carotenoids (hazard ratio 0.68, 95% CI 0.48–0.96, p = 0.03) [80].
If the AREDS2 complex (i.e., vitamin C and E, zinc, copper, lutein/zeaxanthin and omega-3 fatty
acids) was used by all adults aged >55 years, it has been estimated this would result in an average of
about 1 million avoided age-related macular degeneration and cataract events per year in the USA
(based on a risk reduction of 23.6% for age-related macular degeneration and 16.2% for cataracts).
This would result in a net annual cost saving of US$1.2 billion, mostly as a consequence of reduced
healthcare expenditure [81]. Establishing intake recommendations for lutein is an important step
forward to support optimal visual performance and reduce the risk of age-related macular eye disease
in the general population. This would be a relevant contribution to public health in the face of a
globally aging population.
Future studies may include additional assessments of the relationship between macular pigment
and different genotypic and phenotypic forms of age-related macular degeneration, the optimum
dosages of lutein, zeaxanthin, and the possible effects when combined with other nutrients.
5. Cardiovascular Disease
Despite the global decline in cardiovascular mortality, cardiovascular diseases remain the leading
cause of morbidity and mortality, contributing to escalating health care cost [82]. Cardiovascular aging
progresses over decades, influenced by risk factors such as tobacco use, poor physical activity and diet,
resulting in hypertension, dyslipidemia (high triglycerides and lower HDL), elevated fasting blood
glucose, and central obesity [83]. Cardiovascular disease is the major clinical problem in the older
population, with 68% of adults 60–79 years having cardiovascular disease and this increases to 85%
after the age of 80 years [84].
Good nutrition plays an important role in delaying the progression of cardiovascular
disease [85,86]. The adverse effects of excess intakes of saturated and trans fats, cholesterol, added
sugars, and salt in relation to cardiovascular disease progression has been relatively well-established
whereas the effect of addressing inadequate essential nutrients is less well-known. Older adults are
highly susceptible to undernutrition due to the various physiological and socioeconomic factors [87].
In contrast to overnutrition, the potential of addressing undernutrition to optimize cardiovascular
health in older adults has received inadequate attention [88]. Evidence for nutrition in reducing the risk
for cardiovascular aging mostly derives from epidemiological studies, whereas fewer interventions
studies have been performed. The RCTs addressing cardiovascular disease generally have included, but
not exclusively, older adults, not allowing generalizability of results to typical older adults. The authors
have therefore focused on nutrition interventions addressing cardiovascular aging progress, not
restricted to elderly.
5.1. Cardiovascular Events
5.1.1. Diets
Lifestyle changes, including dietary modifications, are recommended as part of the management
strategy to improve lipid profiles and reduce the risk of cardiovascular disease [89–91]. The primary
emphasis of dietary interventions has been on changing dietary macronutrient and salt composition.
The effect of improving micronutrient-richness of the diet in cardiovascular disease control has been
less-well studied. A diet rich in fruits, vegetables, wholegrains, legumes, nuts, fish, poultry, and low-fat
dairy products, and limited consumption of red meat, saturated fat, and added sugar is advocated,
mostly based on positive associations with cardiovascular health [89–91]. Dietary patterns that follow
these principles include the Dietary Approaches to Stop Hypertension (DASH) diet, a diet rich in fiber,
protein, magnesium, calcium, and potassium, and low in total and saturated fats, which has been
shown to reduce low-density lipoprotein (LDL)-cholesterol levels [91], and the Mediterranean diet,
which has been shown to reduce the risk for cardiovascular disease in both primary and secondary
198
settings [92,93]. Regression of coronary artery atherosclerosis has been demonstrated with a program
of intensive lifestyle changes that included a vegetarian diet, exercise, and smoking cessation [94].
In addition to dietary interventions, there has been research into the effects of individual nutrients.
While the evidence for some of these is limited, several interesting findings have been published.
5.1.2. Vitamin D
Low vitamin D has been associated with cardiovascular disease in a number of studies [95].
Few studies have been targeting low vitamin D specifically in the older population. In one study with
post-menopausal women randomized to Vitamin D3 2500 IU or placebo, daily for 4 months, vitamin
D supplementation had no effect on endothelial function, arterial stiffness, or inflammation [96].
Results of a meta-analysis of RCT with older adult participants (≥60 years) suggested that vitamin D
supplementation might protect against cardiac failure but not against MI or stroke [97]. The recent
results of the VITAL trial indicate that daily supplementation of 2000 IU vitamin D did not reduce the
occurrence of cardiovascular events in adults aged ≥50 years [98].
5.1.3. B-Vitamins
B-vitamins have been the subject of substantial research because of their established effects on
normalizing homocysteine levels, an important risk factor for cardiovascular disease. Figure 2 shows
the risk factors including B-vitamin shortages and pathogenetic mechanisms for the effect of high
homocysteine on cardiovascular disease.
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199
Particularly the B-vitamins have been investigated for their potential cardiovascular benefits due
to their established lowering effect on homocysteine levels, a marker for cardiovascular disease risk,
including ischemic stroke. A meta-analysis of 19 RCTs of B vitamins (including folic acid, vitamin B6,
vitamin B12, and B-complex vitamins) found significant reductions in homocysteine levels, however,
no significant effect of vitamin B supplementation on rates of cardiovascular disease, coronary heart
disease, myocardial infarction, cardiovascular death, or all-cause mortality whereas vitamin B reduced
the risk of stroke by 12% [99]. Another meta-analysis of 26 RCTs found that folic acid supplementation
significantly reduced the risk of stroke 7% [100].
There are various reasons for elevated blood homocysteine levels; most people have mild to
moderately elevated serum homocysteine levels due to inadequate intake of folate, vitamin B6 , or
vitamin B12 from the diet, which is reversible when intake of these vitamins is increased. Another
cause are genetic variants of methylenetetrahydrofolate reductase (MTHFR) and methionine synthase
reductase (MTRR) that are associated with elevated homocysteine levels. Elevated homocysteine
levels are a risk factor for developing blood clots in the vasculature and have been implicated
in the pathogenesis of atherosclerosis and deep vein thrombosis [101]. Given that vitamin B
supplementation is associated with normalization of elevated plasma homocysteine levels, many
studies have investigated whether these vitamins may decrease the risk of cardiovascular diseases.
Huang and colleagues undertook a meta-analysis (19 RCTs and 47,921 participants) evaluating the
effects of B vitamin supplementation (search terms: folic acid, folate, vitamin B6 , vitamin B12 , and B
vitamins) on plasma homocysteine levels and cardiovascular and all-cause mortality [99]. The overall
relative risk of a clinical outcome, versus placebo, was 0.98 for cardiovascular disease, 0.98 for CHD,
0.97 for MI, 0.97 for cardiovascular death and 0.88 for stroke; and homocysteine levels were decreased
in all RCTs. Thus, B vitamin supplementation had a significant protective effect for stroke, but not for
any other cardiovascular risk. A more recent meta-analysis of folic acid supplementation (30 RCTs,
82,334 participants) estimated a 10% lower risk of stroke and a 4% lower risk of overall cardiovascular
disease compared with controls [102]. The greatest benefit for cardiovascular disease was observed in
individuals with lower plasma folate levels at baseline and without pre-existing cardiovascular disease
(p = 0.006 for both). While patients with a cardiovascular disease history responded to B-vitamins with
normalization of homocysteine levels, those with the MTHFR 677C > T genotype were less responsive
and may have greater folate requirements than do their counterparts [103].
5.1.4. Vitamin K
Vitamin K plays an important role in anticoagulation and may overcome the detrimental side
effects associated with vitamin K antagonists such as warfarin. Vitamin K may also help to prevent
vascular calcifications, especially in patients on warfarin [104].
5.1.5. Omega-3 LCPUFA

Supplementation of omega-3 LCPUFA increased high-density lipoprotein (HDL) cholesterol
concentration, improved vascular function, and lowered heart rate and blood pressure with DHA
having a greater effect than EPA while both EPA and DHA inhibited platelet activity [105]. Dietary
supplementation with omega-3 LCPUFAs can reduce plasma triglyceride levels by up to 45% [106,107],
with the greatest effect seen in those with the highest baseline levels [106]. Omega-3 LCPUFAs also
cause a modest increase in HDL-C levels, and although they also increase LDL-C levels, this is primarily
an increase in large, less atherogenic, particles [106]. In addition to improving lipid profiles, omega-3
LCPUFAs reduce inflammation, lower blood pressure (blood pressure), and have beneficial effects
on endothelial function and platelet aggregation, all of which could contribute to cardioprotective
effects [106]. However, despite positive effects on intermediate markers, RCTs with omega-3 LCPUFAs
have produced mixed results on cardiovascular morbidity and mortality [108,109]. It must be noted
that these meta-analyses included both primary and secondary prevention studies, before and after
occurrence of events, respectively. One recent meta-analysis of RCTs performed reported a significant
200
reduction in cardiovascular risk only among higher risk populations, such as those with elevated
triglyceride levels (relative risk: 0.84, 95% CI 0.72–0.98) or elevated LDL-cholesterol levels (relative
risk 0.86, 95% CI 0.76–0.98) [108]. Another recent Cochrane meta-analysis of RCTs found that omega3
LCPUFAs reduced cardiovascular events in the main analysis (relative risk: 0.93, 95% CI 0.88–0.97), but
the result was not maintained in sensitivity analyses [109]. The failure of some trials to show effects of
omega-3 LCPUFA on cardiovascular disease was explained by an insufficiently high omega-3 LCPUFA
dose and/or too high omega-3 LCPUFA baseline status to demonstrate effects [110]. RCTs evaluating
the effects of omega-3 LCPUFAs on cardiovascular morbidity and mortality generally enrolled a broad
range of ages while only few RCTs have focused specifically on older adults. The Alpha Omega Trial
that included 60–80-year-olds with previous MI and at least 50% on medication found no significant
effect of approximately 400 mg of omega-3 LCPUFA on cardiovascular events [111]. In the AREDS2
study, 1 g omega-3 LCPUFA given in addition to a standard Vitamin C, Vitamin E, beta-carotene,
zinc oxide, and cupric oxide supplement for 6 months to participants between 50 and 85 years had
no effect on cardiovascular outcomes [112]. The recent results of the VITAL trial showed that in
adults aged ≥50 y daily consuming 840 mg of omega-3 LCPUFA lowered the risk of heart attack by
28%, of fatal heart attack by 50% without significant effect on stroke or cardiovascular deaths [98].
The most pronounced benefits on major cardiovascular event reduction were found in participants
who reported low fish intake at baseline. A recent meta-analysis of RCTs found that omega-3 LCPUFA
supplementation caused a small, but significant, reduction in heart rate (−2.23 bpm, 95% CI −3.07 to
−1.40) [113], which is considered a risk factor for cardiovascular morbidity and mortality [114].
5.1.6. Antioxidants
Inflammation and oxidative stress appear to be key drivers for a number of cardiovascular
diseases and the metabolic syndrome [115,116]. Whereas observations studies suggest that antioxidant
nutrient such as β-carotene and vitamin E are associated with lower cardiovascular disease, the
data of RCTs on antioxidant supplements failed to confirm a significant benefit of antioxidants on
atherosclerotic cardiovascular disease. For instance, supplementation with the antioxidant nutrients
vitamin E, β-carotene, and vitamin C, had no significant effects on cardiovascular outcomes [117].
5.1.7. Vitamin E
A key attribute of vitamin E (a combination of 8 distinct tocopherol/tocotrienol isoforms) is its
antioxidant activity and, as a consequence, its ability to protect poly-unsaturated fatty acids (PUFAs),
lipoproteins, and cell membranes from oxidative damage [118]. Vitamin E has been extensively
investigated for its potential to prevent cardiovascular disease events. Nevertheless, RCTs with
vitamin E had mixed results on various cardiovascular disease endpoints. In the Women’s Health
Study, intake of 600 IU of vitamin E on alternate days in apparently healthy women non-significantly
reduced the risk for cardiovascular events by 7% and significantly reduced the risk for cardiovascular
death by 24% [119]. And among women ages 65 and older, vitamin E supplementation reduced the
risk of major cardiac events by 26% [119]. Data from the same Women’s Health Study suggested
that supplementation with vitamin E may reduce the risk of venous thromboembolism in women,
particularly in those with a prior history or genetic predisposition [120]. RCTs that retrospectively
analyzed the data for the effect of vitamin in E in subgroups of patients with this these genotypes
sometimes showed that these patients are more responsive to vitamin E supplementation [121,122].
5.1.8. Phenolics
Phenolic compounds are bioactive compounds found in plants, and there is evidence that some
may be helpful for reducing cardiovascular risk factors [116]. Flavonoids are polyphenolic compounds
found in fruits, vegetables, tea, and red wine [116]. Amongst the flavonoids, there is some evidence
that flavonols (specifically quercetin) may be effective at reducing blood pressure in hypertensive
patients; however, no effects on other cardiovascular disease risk markers such as endothelin, oxidative
201
stress, or lipid profiles were found [116]. Although an early meta-analysis found that consumption of
flavonols was associated with a lower rate of cardiovascular disease [123], a more recent meta-analysis
and a systematic review do not support such an effect [116,124]. Amongst other phenolic compounds
which might have beneficial cardiovascular effects, resveratrol is a stilbene found in grape skin, red
wine, and peanuts [116]. A systematic review found that resveratrol was associated with reductions
in total cholesterol, LDL-C, triglycerides and apolipoprotein B in a range of patients, including those
with ischemic heart disease [116]. Resveratrol also reduced inflammatory and fibrinolytic biomarkers
in patients with ischemic heart disease [116].
Nutrients have been investigated for their effect on cardiovascular disease progress and as such,
outcomes. B-Vitamins reduced homocysteine levels, a risk factor of cardiovascular disease, without
significant effects on cardiovascular disease events except for the reduced risk for stroke, which was also
reduced by folic acid supplementation. Flavonoids and omega-3 LCPUFA also reduce cardiovascular
disease risk factors although evidence on cardiovascular outcomes is mixed. Possible explanations
include that patients enrolled in the RCTs were already at high risk of cardiovascular disease and
on concomitant medications, with little opportunity for nutrition to reverse the progress. Individual
nutrients like vitamin D, vitamin E and omega-3 LCPUFA on cardiovascular disease prevention
have shown mixed effects. Nutrition interventions have focused mostly on primary prevention of
cardiovascular aging in broad age groups and less on older adults. Recruited participants in the
RCTs were often at high risk of cardiovascular risk factors or preexisting disease, a modest effect of in
patients that already have heart disease or are at high risk of heart disease may be masked by effects
of medication.
5.2. Hypertension
Hypertension is a major public health concern given its link to serious cardiovascular events
such as stroke and ischemic heart disease, the leading causes of worldwide mortality [6]. It has been
estimated that hypertension is responsible for approximately 40% of cardiovascular deaths. By the
year 2025 almost 30% of the global population will be diagnosed with high blood pressure, with 25%
of these cases occurring in developing countries [125]. Hypertension rises dramatically with aging due
to longer exposure to age-associated alterations in vascular function and structure and cardiovascular
risk factors [126].
Hypertension is a multifactorial disease with lifestyle factors such as physical activity, smoking
and drinking habits, diet, bodyweight, and anxiety playing a predominant role. Management of
these is the first step to achieving adequate blood pressure control. Indeed, it has been reported
that two lifestyle modifications can help improve blood pressure control and decrease the number of
cardiovascular outcomes [127].
5.2.1. Diets
In the current healthcare environment, lifestyle changes involving a healthy diet and increased
physical activity are considered pivotal in the management of hypertension. Diets with a high
nutritional value, such as the traditional Mediterranean diet, DASH and the OmniHeart (a variation
of DASH with increased levels of protein) diets, can be important steps on the path to weight loss,
lowering blood pressure, and prevention of hypertension [125]. The benefits of the DASH diet on
blood pressure were reported in a RCT with all participants receiving graded amounts of sodium
(high, intermediate, low). There were dose-response decreases in systolic and diastolic blood pressures,
and age-related increases in blood pressure were blunted [128]. Both the DASH diet and low sodium
markedly decreased blood pressure, and the combined effect was even greater. Findings of the DASH
study also provided additional support that the sodium-to-potassium ratio is stronger associated
with blood pressure outcomes than either nutrient alone among prehypertensive and hypertensive
adults combined. These findings were later confirmed by a systematic review showing that the
202
sodium-to-potassium ratio appears to be more strongly associated with blood pressure outcomes than
either nutrient alone in hypertensive adults [129].
In addition to dietary control there has been research into the effects of other nutrients, including
vitamins, on blood pressure and hypertension. While the evidence for some of these is limited a
number of interesting findings have been published.
5.2.2. Milk peptides

A meta-analysis of 14 RCTs involving 1306 European subjects found that the milk-derived
lactotripeptides isoleucine-proline-proline and valine-proline-proline produced small and statistically
significant reductions in mean systolic blood pressure and diastolic blood pressure [130]. The authors
noted that a similar effect had been seen in Asian populations.
5.2.3. Omega-3 LCPUFAs

The omega-3 LCPUFAs EPA and DHA found in oily fish and fish oils (including capsule
preparations) have been associated with lower blood pressure levels. In a meta-analysis of 70 RCTs,
EPA, and DHA reduced mean systolic blood pressure and mean diastolic blood pressure compared
with placebo. The largest effect was in untreated hypertensive patients [131]. Likewise, in an earlier
meta-analysis (36 trials), intake of fish oil (median dose 3.7 g/d) reduced both mean systolic and
diastolic blood pressure. The antihypertensive effects of doses <0.5 g/d remains to be established [132].
5.2.4. Vitamin C
In short-term studies, vitamin C supplementation reduced systolic and diastolic blood pressure.
Long-term trials on the effects of vitamin C supplementation on BP and clinical events are needed
longer-term trials assessing the effects of vitamin C supplementation on blood pressure and clinical
events in patients with hypertension would seem to be worthwhile [133]
5.2.5. Vitamin D
In a study involving 283 hypertensive patients, vitamin D3 (cholecalciferol) produced a modest but
statistically significant reduction in systolic blood pressure compared with placebo after 3 months [134].
There was no significant effect on diastolic blood pressure.
5.2.6. Flavonols
Flavanols have also been found to lower blood pressure, and there is some evidence suggesting
that they improve endothelial function in patients with ischemic heart disease, but additional studies
are needed [116].
The evidence for nutrients and blood pressure is convincing for lowering sodium and
sodium-to-potassium ratio. Flavanols vitamin C and D may have modest significant effects on blood
pressure lowering.
5.3. Diabetes
Type 2 diabetes has become a global health-related pandemic which is forecast to rise from 425 to
almost 630 million by 2045 [135]. In developing countries, the forecasted increase is more alarming,
particularly in regions which are more rapidly adopting a Western lifestyle. The direct financial burden
on healthcare systems and society is huge, as are the indirect costs from loss of work attendance.
Intensive lifestyle modification, e.g., personalized nutrition and physical activity programs, with
the goal of improving glycaemia and losing excess body weight should be the mainstay of initial
management in individuals with prediabetes [136].
203
5.3.1. Vitamin D
Observational studies have highlighted a link between vitamin D deficiency and type 2 diabetes,
as well as possible future cardiovascular events, whereas results from interventional studies have not
been so conclusive [137]. A recent meta-analysis [137] including a total of 20 RCTs and 2703 participants,
found that vitamin D supplementation was associated with elevated serum vitamin D levels and
significantly decreased insulin resistance. Changes in other parameters such as fasting blood glucose
and hemoglobin A1c (HbA1c) were relatively small and did not achieve statistical significance [137].
In a pilot study in 60 patients with co-existing type 2 diabetes and hypovitaminosis D, vitamin D
improved vitamin D status and several parameters associated with glycemic control such as HbA1c,
mean fasting plasma glucose, and mean post-prandial plasma glucose [138]. In addition, vitamin D in
the study lowered LDL cholesterol levels, systolic blood pressure and diastolic blood pressure.
5.3.2. Vitamin E
Diabetes patients with the haptoglobin 2-2 genotype have elevated risk of cardiovascular disease
events. The haptoglobin 2-2 genotype has inferior antioxidant properties as compared with other
haptoglobin types resulting in elevated levels of oxidative stress, an atherogenic profile and an
increased risk of cardiovascular disease events compared with other Hp genotypes [139]. The RCTs
in diabetes patients that retrospectively analyzed the data for the effect of vitamin in E found
that administration of vitamin E lowered the risk of cardiovascular disease events by 34% and
cardiovascular-related mortality by 53% among patients with the haptoglobin 2-2 genotype [140].
5.3.3. Omega-3 LCPUFA

Cohort studies have shown that in countries where fish consumption is high the prevalence of type
2 diabetes tends to be lower and this has been attributed to the presence of omega-3 LCPUFAs [141].
However, the findings have not been conclusive with respect to providing dietary guidance and a
recent systematic meta-analysis sought to provide more definitive evidence by analyzing different
dosage/compositions of omega-3 LCPUFA supplementation [141]. In total, 20 RCTs recruited
1209 patients with type 2 diabetes. Overall, omega-3 LCPUFA supplementation resulted in a reduction
in triglycerides with the best response with high doses for a longer duration; however, no significant
changes in total cholesterol, fasting plasma glucose, post-prandial plasma glucose, HbA1c, insulin, or
body mass was noted with this regimen. Interestingly, products with a relatively high ratio of EPA to
DHA exhibited an increasing tendency to decrease HbA1c, insulin, total cholesterol, total triglycerides,
and body mass. These findings will be helpful for clinicians and nutritionists who manage patients
with diabetes to provide dietary guidance [141].
5.3.4. Vitamin K
To assess whether vitamin K is a risk factor for the development of type 2 diabetes mellitus,
Beulens and colleagues analyzed a cohort of 38,094 Dutch men and women over a 10-year period [142].
The study showed that both vitamin K1 and vitamin K2 intake were associated with a reduced risk
of type 2 diabetes mellitus. For vitamin K1 the risk reduction occurred at the higher levels of intake,
whereas for vitamin K2 a linear inverse association was established. In older men with diabetes
receiving vitamin K1 supplementation for 36 months, vitamin K1 significantly improved insulin
sensitivity [143].
5.3.5. Chromium
Chromium plays a role in insulin metabolism by activating oligopeptide low-molecular-weight
chromium (LMWCr)-binding substance and activating insulin-dependent kinase activity. A meta-analysis
of the efficacy of chromium supplementation suggest that there is available evidence for chromium on
glycemic control in patients with diabetes [144].
204
Studies in diabetes patients showed that vitamin D supplementation can improve serum vitamin
D levels and significantly decrease insulin resistance. Currently, a large multicenter RCT is ongoing in
the US (Vitamin D and Type 2 Diabetes Study; D2d), hypothesizing that vitamin D will enhance insulin
production, glucose processing and glycemic profiles. Subgroup analyses show that vitamin E may be
promising in reducing the rate of cardiovascular events among diabetes patients with haptoglobin
2-2 genotype who are at increased risk of cardiovascular events. The evidence for omega3 LCPUFA
supplementation on fasting plasma glucose or HbA1C is less conclusive but omega-3 LCPUFA have
promising effects for reduction of triglycerides. The evidence for chromium in glycemic control
is emerging.
6. Conclusions
Inadequate or even deficient nutrient intake and status is still widely prevalent at global level
and, although generally underacknowledged, is a main risk factor for NCDs [20]. Nutrient surveys
indicate that the aging population is at particular risk for poor nutrient intake and status, which may
result in increased risk for chronic fatigue, and cardiovascular, cognitive, and neuromuscular disorders
in older adults. The present paper reviews the evidence for the role of various nutrients in modifying
the risk of development of NCDs throughout aging.
Inadequate vitamin D, calcium and vitamin K intake and status are generally reported in the
aging population and have been associated with musculoskeletal disorders, such as increased bone
fracture risks. Increased vitamin D in combination with increased calcium and possibly also vitamin K
may reduce the risk for hip fractures, thus beneficially impacting musculoskeletal health.
Inadequate B vitamins intake and status, in particular folic acid, vitamins B6 and B12 , have been
associated with age-related cognitive decline, while supplementation has been reported to improve
cognitive performance. Similarly, evidence has been reported for vitamin C, D, and E, as well as
omega-3 LCPUFAs (e.g., DHA) to slow down dementia progression.
Increased intake of lutein and zeaxanthin has been demonstrated to improve macular pigment
optical density measures, a marker of age-related macular degeneration.
Various nutrients have been reported to play a role in reducing the risk for ischemic heart disease,
stroke, myocardial infarction, heart failure, hypertension, and diabetes with varying levels of effect size
and evidence. B-Vitamins reduced homocysteine levels and reduced the risk for stroke. Some but not
all studies reported that higher omega-3 LCPUFAs intakes resulted in reduced risk of cardiovascular
events; most pronounced effects being shown in subjects with low intake or status. Vitamin C and D
may reduce hypertension, omega-3 LCPUFAs may have positive effect on blood lipid profiles, and
omega-3 LCPUFAs, vitamin D, and chromium may reduce diabetes risk factors.
Most pronounced benefits of nutrient interventions were sometimes found in subgroups which
had low baseline intake or status of the nutrient. Genetic factors can affect the status of certain nutrients,
as well as contribute to increased risk for NCDs and raise the needs for certain nutrients [139,145].
Targeted supplementation with nutrients of concern to genetically predisposed subgroups has been
shown to confer benefits as shown by some examples in this review. More research is needed to unravel
the benefits of optimizing nutrition where it is needed, for instance by targeting those at increased risk
for NCDs linked to low nutrition status or genetic profile.
Due to a growing aging global population, related NCDs including musculoskeletal disorders,
dementia, loss of vision, and cardiovascular diseases will place an increasing burden on health systems
and costs. Adequate nutrient status may help to improve health and wellbeing in older populations
and slow the progression of NCDs. Implementing a long-term preventative strategy to promote healthy
aging and break down the barriers to adequate nutrition for older adults could result in significant
healthcare cost savings. Nutrition is increasingly acknowledged and integrated into public health
policies and programs to manage healthy aging. Promoting nutrient-rich diets and adequate nutrient
intakes for healthy aging should be considered part of an integral approach to address NCDs in health
policies. There is a need for public and/or private partnerships where governments, health authorities,
205
academics, and the food sector jointly promote the benefits of healthy nutrient-rich diets and lifestyle
to manage NCDs.
In conclusion, data indicate that inadequate nutrient intake and status is common in older
aged adults and represents a risk for the development of NCDs during aging. Studies for the aging
population have demonstrated that optimizing nutrition can reduce the risk and progress of NCDs.
Although the scientific evidence is not conclusive for all health benefits, it should not prevent health
authorities from promoting balanced and adequate nutrient intakes as integral part of nutrition
strategies to reduce the burden of NCDs associated with inadequate nutrition.
Author Contributions: M.J.B., M.E., and P.V.D. wrote the manuscript.

Acknowledgments: Editorial assistance was provided by Content Ed Net (Switzerland).
Conflicts of Interest: M.J.B. and P.V.D. are employed by DSM Nutritional Products, a manufacturer of vitamins
and supplier to the food, dietary supplement, and pharmaceutical industries. M.E. is a former employee of DSM
Nutritional Products. There were no other conflicts of interest.
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nutrients

Special Issue Editor

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade

ISBN 978-3-03897-602-8 (Pbk)

About the Special Issue Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Preface to ”Nutrition and Chronic Conditions” . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Edgar Denova-Gutiérrez, Paloma Mu ñoz-Aguirre, Nitin Shivappa, James R. Hébert,

Estefania Sanchez-Rodriguez, Elena Lima-Cabello, Sara Biel-Glesson, Jose R.

Blanka Klı́mová and Martin Vališ

Christian R. Juhl, Helle K. M. Bergholdt, Iben M. Miller, Gregor B. E. Jemec,

Christian R. Juhl, Helle K. M. Bergholdt, Iben M. Miller, Gregor B. E. Jemec,

Lukasz Szternel, Magdalena Krintus, Katarzyna Bergmann, Tadeusz Derezinski and

Tatiana S. Collese, Gabriela Vatavuk-Serrati, Marcus Vinicius Nascimento-Ferreira,

Namrata Sanjeevi, Leah M. Lipsky and Tonja R. Nansel

Silvana Perez-Leon, M. Amalia Pesantes, Nathaly Aya Pastrana, Shivani Raman,

Maaike J. Bruins, Peter Van Dael and Manfred Eggersdorfer

Received: 31 January 2018; Accepted: 15 March 2018; Published: 19 March 2018

Nutrients 2018, 10, 373; doi:10.3390/nu10030373 1 www.mdpi.com/journal/nutrients

1.1. Description of the Intervention

1.2. How the Intervention Might Work

1.3. Why It Is Important to Do This Review

2.1. Types of Studies

2.2. Types of Participants

2.3. Types of Interventions

2.4. Types of Outcome Measures

Search Methods for Identiﬁcation of Studies

Table 1. Search terms and search strategy.

2.5. Electronic Searches

2.6. Searching Other Resources

2.7. Selection of Studies

Records identified Records identified Reference list of articles

through EBSCOHost through PubMed (n = 2) 2008–2018

2008–2018 2008–2018 Web of Science (n = 196)

Records after de-duplication (5540)

Further Screening using inclusion and Exclusion Criteria:

Full-text articles assessed for

eligibility N = 2 (Studies involving

(N =24) ketogenic diets)

N = 1 (Study results that

could not be extracted due

Figure 1. PRISMA ﬂow chart showing the selection of articles.

Inclusion Criteria Exclusion Criteria

2.8. Evaluation of Quality

2.9. Data Extraction and Management

2.10. Meta-Analysis Methods

2.11. Assessment of Risk of Bias in Included Studies

Length Study Sample Diabetes Duration Glycated Haemoglobin

Design higher-GI diet

Length Study Sample Diabetes Duration Glycated Haemoglobin

had the same average value

3.1. Assessment of Risk of Bias in Included Studies

Figure 2. A risk of bias summary.

Figure 3. A risk of bias graph.

3.2. Effect of Low Glycaemic Index on Glycated Haemoglobin

3.3. Effect of Low Glycaemic Index on Fasting Blood Glucose

5. Strengths and Limitation of the Study

7. Perspectives for Future Research

Received: 12 February 2018; Accepted: 15 March 2018; Published: 20 March 2018

Keywords: impaired glucose regulation; lifestyle modiﬁcations; metabolic syndrome; type 2