Clin Endocrinol Metab. 2012 Oct; 97(10): 3792–3798.

Published online 2012 Aug 1. doi: 10.1210/jc.2012-2221

I. A Mediterranean Diet Enriched with Olive Oil Is Associated with Higher Serum
Total Osteocalcin Levels in Elderly Men at High Cardiovascular Risk
José Manuel Fernández-Real, Mónica Bulló, José Maria Moreno-Navarrete, Wifredo
Ricart, Emilio Ros, Ramon Estruch, and Jordi Salas-Salvadó

Abstract
Age-related bone mass loss and decreased bone strength is an almost invariable feature of
human biology, affecting women and men alike as an important determinant of osteoporosis
and fracture risk (1). Nutritional factors are known to be involved in age-related bone loss
associated with osteoblast insufficiency during continuous bone remodeling, in interaction
with a combination of genetic, metabolic, and hormonal factors (2).
Epidemiological studies have shown that the incidence of osteoporosis in Europe is lower in
the Mediterranean basin (3, 4). The traditional Mediterranean diet, rich in fruit and
vegetables, with a high intake of olives and olive products, mainly olive oil, could be one of
the environmental factors underlying this difference.
Some reports have suggested that the consumption of olives (5), olive oil (6), and
oleuropein, an olive oil polyphenol (7), can prevent the loss of bone mass in animal models
of aging-related osteoporosis (8, 9). Recent in vitro studies have shown that oleuropein, the
main phenolic compound in olive leaves and fruit and a constituent of virgin olive oil, reduced
the expression of peroxisomal proliferator-activated receptor-γ, inhibiting adipocyte
differentiation and enhancing differentiation of mesenchymal stem cells into osteoblasts. In
addition, the gene expression of osteoblastogenesis markers, runt-related transcription
factor II, osterix, collagen type I, alkaline phosphatase, and osteocalcin, was higher in
osteoblast-induced oleuropein-treated cells (10).
A protective effect of olive oil and oleuropein has also been observed in experimental
models. Femoral failure load and diaphyseal bone mineral density were increased after
consumption of oleuropein and olive oil in ovariectomized mice (6). In addition to its role as
bone marker, osteocalcin has also been related to glucose homeostasis. Mice lacking
osteocalcin displayed decreased β-cell proliferation, glucose intolerance, and insulin
resistance when compared with wild-type mice (12). We are unaware of studies evaluating
the effects of olive oil on circulating osteocalcin and its possible relationship with insulin
secretion/resistance in humans. The objective of this study was to explore circulating bone
formation and resorption markers in association with the intake of olive oil. For comparison,
we also studied the effects of consuming nuts and the effects of a low-fat diet.

Subjects and Methods

Study subjects
The participants in this study were 127 community-dwelling men aged 55–80 yr randomly
selected from one of the Prevención con Dieta Mediterránea (PREDIMED) study centers
(PREDIMED-Reus) who had at least 2 yr of follow-up. The PREDIMED study is a large,
parallel-group, randomized, controlled trial aimed to assess the effect of the Mediterranean
diet on the primary prevention of cardiovascular diseases. Full details of the PREDIMED
protocol have been published elsewhere (13). Subjects were elderly without prior
cardiovascular disease but having a diagnosis of type 2 diabetes or harboring at least three
cardiovascular risk factors, namely hypertension; dyslipidemia [low density lipoprotein (LDL)
cholesterol level of ≥ 4.14 mmol/liter [≥ 160 mg/dl] (or treatment with hypolipidemic drugs)
or high density lipoprotein (HDL) cholesterol level ≤1.04 mmol/liter (≤40 mg/dl)]; overweight
[body mass index (BMI) ≥ 25 kg/m2]; or a family history of premature cardiovascular disease.
Exclusion criteria were any severe chronic illness; alcohol or drug abuse; a BMI greater than
40 kg/m2; and loss of more than 5% body weight in the last year. For the present analysis,
we further excluded current smokers, subjects treated with insulin, those with secondary
hypertension, subjects using medications known to affect bone or calcium metabolism, such
as corticosteroids, bisphosphonates, thiazides, and vitamin or mineral supplements; and
those receiving large amounts of aspirin or anticoagulant medication that would interfere
with vitamin K absorption. Renal function was systematically assessed in all subjects, and
only subjects with normal renal function (as estimated using serum creatinine concentration
or estimated glomerular filtration rate) were included in this study.

Design of the study

Participants were randomly assigned to three intervention groups: advice on the MedDiet
plus supplementation with virgin olive oil (MedDiet+VOO); advice on the MedDiet plus
supplementation with mixed nuts (MedDiet+nuts); and advice on a low-fat diet (control
group). Dietitians gave personalized dietary advice to participants randomized to the
different diets, with instructions directed to upscale the score, including, among others, the
following: 1) abundant use of olive oil for cooking and dressing; 2) increased consumption
of fruit, vegetables, legumes, and fish; 3) reduction in total meat consumption,
recommending white meat instead of red or processed meat; 4) preparation of homemade
sauce with tomato, garlic, onion, and spices with olive oil to dress vegetables, pasta, rice,
and other dishes; 5) avoidance of butter, cream, fast food, sweets, pastries, and sugar-
sweetened beverages; and 6) in alcohol drinkers, moderate consumption of red wine.
Participants allocated the low-fat diet received recommendations to reduce all types of fat
from both animal and vegetable sources. In the MedDiet+VOO intervention group, those
individuals taking refined olive oil were advised to change this type of oil by an extra virgin
olive oil that retains all the phytochemical and antioxidants compounds. Those taking other
sources of vegetable oil also were advised to change the oil to extra virgin olive oil. Refined
oil does not have such an amount of phytochemicals. The advice on virgin olive oil was to
consume at least 50 ml/d, whereas mixed nuts (walnuts, almonds, and hazelnuts) were
provided in daily allotments of 30 g (13). The dietary changes that occurred in the
PREDIMED study have been previously described (14). Energy restriction was not advised
nor physical activity promoted. Changes in physical activity were not observed during the
follow-up (13). The local institutional review board approved the study protocol and all
participants provided written informed consent.

Procedures
At baseline, 1 yr, and 2 yr of dietary intervention, a short-questionnaire about lifestyle
variables, medical conditions, and medication use was obtained. Once a year, usual dietary
intakes were assessed during the study using a previously validated, semiquantitative, 137-
item food frequency questionnaire. Energy and nutrient intakes were calculated from
Spanish food composition tables, as described (15). Physical activity was evaluated using
the validated Spanish version of the Minnesota Leisure Time Physical Activity Questionnaire
(16).

Biochemical analyses
Biochemical measurements were performed at baseline and after 2 yr of follow-up on fasting
blood samples. Serum levels of glucose, total cholesterol, HDL-cholesterol, and triglycerides
were measured by standard enzymatic methods using a biochemistry autoanalyzer. The
LDL cholesterol concentration was calculated by the Friedewald's formula [total cholesterol
− ([HDL-cholesterol] + ([triglycerides]/5) (in milligrams per deciliter)]. Fasting insulin was
measured by a chemiluminiscent immunoassay (Linco Research, St. Charles, MO; intra and
inter-assay coefficients of variation 6 and 10%, respectively). Insulin resistance was
estimated by the homeostasis model assessment (HOMA) method as HOMA-IR. Altered β-
cell function was studied using HOMA-BCF, as previously described (17). Serum total and
uncarboxylated osteocalcin levels were measured by electrochemiluminiscence
immunoassay (N-mid osteocalcin, electrochemiluminescence immunoassay; Roche,
Indianapolis, IN; the intra- and interassay coefficients of variation of 3.6 and 6.6%,
respectively). Human cross-linked C-telopeptide of type I collagen (CTX) and procollagen I
N-terminal propeptide (P1NP) concentrations in serum were measured by commercial
ELISA kits (E90665Hu and E90639Hu, respectively; Uscn, Life Science Inc., Wuhan, China)
according to the manufacturer's protocol. The intra- and interassay coefficients of variation
were less than 10% and less than 12%, respectively.

Statistical analyses
Means (SE) or percentages were used to describe the participants' baseline characteristics.
χ2 tests and one-way ANOVA were used to compare the qualitative traits and means of

Table 1.
Anthropometric and biochemical characteristics of study subjects at baseline and after 2 yr
of follow-up
 Control diet MedDiet+nuts MedDiet+VOO Pa

Before After P Before After P Before After P

n 34 51 42
Age (yr) 68.4 ± 6 67.6 ± 6 67.9 ± 6.9 0.85
2
BMI (kg/m ) 28.8 ± 29.1 0.13 28.6 ± 28.7 0.13 28.6 ± 28.6 ± 0.75 0.83
3.08 ± 3.1 3.3 ± 2.8 2.9
3.009
Waist 102.6 103.1 0.42 101.7 102.7 0.06 102.5 103.02 0.47 0.74
circumference ± 8.7 ± 8.3 ± 8.1 ± 7.8 ± 7.03 ± 7.8
(cm)
SBP (mm Hg) 152 ± 146 ± 0.08 149 ± 147.3 0.4 155.6 149.9 0.02 0.23
18 15 14.9 ± ± 20 ± 16
16.3
DBP (mm Hg) 82.2 ± 79.7 0.11 83.5 ± 80 ± 0.01 82.2 ± 79.4 ± 0.09 0.51
10 ± 9.6 9.6 8.8 10.7 10.9
Cholesterol 197.7 198.1 0.93 205.4 199.9 0.03 204.8 194.3 0.04 0.66
(mg/dl) ± 39.1 ± ± 36.6 ± ± 43.7 ± 36.2
40.1 38.7
LDL-cholesterol 118.8 121.4 0.65 124.2 125.9 0.43 128.4 118.9 0.04 0.55
(mg/dl) ± 37.4 ± ± 33.4 ± ± 37.4 ± 27
34.8 33.1
HDL-cholesterol 48.1 ± 46.4 0.17 54.7 ± 51.3 0.01 54.3 ± 51.4 ± 0.07 0.06
(mg/dl) 9.4 ± 9.8 13.7 ± 15.2 14.7
11.4
Triglycerides 151.5 157.2 0.62 129.2 112.6 0.01 115.6 123.8 0.22 0.12
(mg/dl) ± 91.5 ± ± 72 ± ± ± 62.3
107.5 51.7 68.01
Fasting glucose 126.6 127.8 0.74 109.6 109.9 0.81 121.6 128.8 0.24 0.01
(mg/dl) ± 30.8 ± ± 26.2 ± ± 33.4 ±
36.8 26.6 44.05
Fasting insulin 6.1 ± 5.2 ± 0.13 6.1 ± 5.7 ± 0.52 4.9 ± 5.7 ± 0.11 0.34
(mU/liter) 3.9 2.8 4.6 4.01 2.5 4.4
HOMA-IR 1.92 ± 1.6 ± 0.14 1.64 ± 1.45 0.13 1.5 ± 1.79 ± 0.11 0.35
1.3 0.95 1.23 ± 1.1 0.8 1.5
HOMA-BCF 39.6 ± 45.4 0.34 50.3 ± 52.7 0.62 33.2 ± 44.7 ± 0.01 0.22
28.8 ± 44.06 ± 16.5 31.5
43.7 42.08
Serum calcium 9.71 ± 9.42 0.001 9.67 ± 9.51 0.02 9.59 ± 9.51 ± 0.30 0.45
(mg/dl) 0.42 ± 0.47 ± 0.44 0.41
0.40 0.43
Serum 3.20 ± 3.38 0.14 3.23 ± 3.26 0.62 3.36 ± 3.36 ± 0.95 0.57
phosphate 0.50 ± 0.47 ± 0.59 0.63
(mg/dl) 0.69 0.40
CTX (ng/ml) 1.13 ± 0.37 0.0001 0.99 ± 0.44 0.0001 1.02 ± 0.54 ± 0.0001 0.49
0.83 ± 0.84 ± 0.52 0.36
0.25 0.33
P1NP (ng/ml) 243.2 240.1 0.92 253.2 260.4 0.82 205.4 277 ± 0.01 0.39
± ± ± ± ± 108.3
167.3 105.4 177.3 131.2 143.2
UC osteocalcin 1.9 ± 1.9 ± .93 1.6 ± 1.9 ± 0.06 1.7 ± 1.8 ± 0.36 0.84
(ng/ml) 1.7 1.8 0.7 1.01 1.3 1.6
Osteocalcin 9 ± 9.1 9.3 ± 0.72 8.3 ± 9.12 0.32 6.9 ± 3 8.4 ± 0.007 0.27
(ng/ml) 6.9 4.9 ± 5.4 3.8

quantitative variables, respectively. Fasting glucose and insulin levels and markers of insulin
resistance were logarithmically transformed to reduce skewness and the geometric mean
and 95% confidence intervals were used. Mean differences in glucose and insulin metabolic
markers during follow-up were normally distributed. Interaction tests for age (product terms,
age osteocalcin, age undercarboxylated osteocalcin) showed that there were no statistically
significant differences by age regarding the associations between osteocalcin and glucose
and insulin concentrations. Analyses were performed using the SPSS software version 17.0
(SPSS Inc., Chicago, IL).

Results
The clinical and biochemical characteristics of the study subjects are shown in Table 1.
Baseline characteristics (age, BMI, waist circumference, lipid profile, fasting insulin, and
osteocalcin) were similar in all intervention groups. Serum glucose, however, was
significantly lower in the group allocated to MedDiet+nuts. After intervention for 2 yr, total
cholesterol, HDL-cholesterol, and fasting triglycerides decreased significantly in subjects
allocated to the MedDiet+nuts group.
Open in a separate window
Data are expressed as mean ± SD. DBP, diastolic blood pressure; SBP, systolic blood
pressure; UC, undercarboxylated.
a
P values for comparisons of baseline parameters.
Fasting insulin concentration and HOMA values tended to decrease in the control group and
in participants in the MedDiet+nuts group, whereas a significant increase in HOMA β-cell
was observed in the MedDiet+VOO group (P = 0.01). In subjects not taking oral antidiabetic
drugs, baseline osteocalcin concentrations were positively associated with higher fasting
insulin concentrations and HOMA-BCF at follow-up, even after adjustment for BMI, physical
activity, intervention group, presence of type 2 diabetes mellitus, and baseline values of
each dependent variable. Undercarboxylated osteocalcin tended to increase in subjects in
the MedDiet+nuts group (P = 0.06) in parallel with the tendency toward decreased HOMA-
IR. However, these changes became nonsignificant when subjects taking oral antidiabetic
drugs (n = 17 in the control group, n = 12 in the in the MedDiet+nuts group, and n = 16 in
the in the MedDiet+VOO group) were excluded from the analysis.
Total osteocalcin increased in the MedDiet+VOO group (P = 0.007) but not in the
MedDiet+nuts or control groups (Table 1 and Figures 1 and and2).2). Baseline total
osteocalcin varied widely (Fig. 1). Interestingly, although the bone resorption marker CTX
decreased significantly in all study groups (P = 0.0001), the bone formation marker P1NP
increased significantly only in the MedDiet+VOO group (P = 0.01, Table 1). These findings
were in parallel to nonsignificant changes in serum calcium in this group, whereas serum
calcium decreased significantly in the other two groups.

Fig. 1.
Effects of the MedDiet+VOO on individual HOMA-BCF and circulating osteocalcin
concentrations (upper panels). Error bars show median and 95% confidence intervals.
Fig. 2.
Effects of the MedDiet+nuts (left panels) and control diet (right panels) on HOMA-BCF and
circulating osteocalcin concentrations. Error bars show median and 95% confidence
intervals.
The increase in total osteocalcin after the MedDiet+VOO was still significant when subjects
taking oral antidiabetic drugs were excluded from the analysis (8.8 ± 4.2 vs. 6.8 ± 3.5
ng/ml, P = 0.013). Olive consumption was positively associated with both baseline total
osteocalcin concentrations (r = 0.23, P = 0.02) and follow-up osteocalcin (r = 0.21, P = 0.04)
in the total cohort (n = 127).
In the MedDiet+VOO group, total osteocalcin increased significantly (8.7 ± 4.5 vs. 6.9 ± 2.7
ng/ml, P = 0.02) in subjects not taking statins (n = 24), subjects in whom a statin was added
during follow-up (10.5 ± 4.7 vs. 8.2 ± 3.5 ng/ml, P = 0.018, n = 8), or subjects who
maintained the same treatment, whether taking statins or not (8 ± 3.5 vs. 6.5 ± 2.9 ng/ml, P =
0.02, n = 32). Interestingly, serum P1NP levels also increased in these subjects (280.1 ±
100 vs. 198.1 ± 142 ng/ml, P = 0.012). In contrast, no significant changes were observed in
total osteocalcin or serum P1NP in the MedDiet+nuts group or in the control group in
subjects taking or not taking statins. On the other hand, the changes in serum osteocalcin
were similar in subjects taking or not antihypertensives.
Discussion
To the best of our knowledge, the present randomized clinical trial is the first assessing the
effects of the MedDiet on total osteocalcin concentrations in humans. The main finding is
that consumption of a MedDiet enriched with olive oil, but not a MedDiet enriched with nuts
or a control diet, was associated with a significant increase of total osteocalcin
concentrations that paralleled an increase of HOMA-BCF. These findings were strengthened
by the simultaneous observation of increased serum P1NP in subjects taking olive oil,
whereas circulating CTX levels decreased. The concentration of P1NP in serum is a
sensitive indicator of the synthesis of type I collagen and is mostly affected by changes in
bone metabolism. Also in line with preserved bone metabolism, we observed no significant
changes in serum calcium in subjects taking olive oil, whereas serum calcium decreased
significantly in the other two groups.
Recent findings disclosed that mice lacking osteocalcin displayed decreased β-cell
proliferation, glucose intolerance, and insulin resistance when compared with wild-type mice
(12). There is previous evidence in the literature of increased bone density in type 2
diabetes, characteristically associated with hyperinsulinemia and insulin resistance (18). We
have described that circulating osteocalcin was positively associated with insulin secretion
in humans (19), and these findings have been confirmed by other authors (20).
There is limited information in the literature concerning the effects of olive oil on circulating
osteocalcin concentration in humans. In a recent study, participants were randomly assigned
into two groups, one receiving vitamin K2 supplementation and the other placebo capsules
containing olive oil. After 12 months, there were no between-group differences in bone loss
rates at the total hip or any other measurement site. Serum levels of undercarboxylated
osteocalcin decreased in the treatment vs. the placebo group (P < 0.001) (21), suggesting
that in fact placebo (olive oil) led to a significant increase of undercarboxylated osteocalcin
when compared with vitamin K2. We did not find significant changes in undercarboxylated
osteocalcin after consumption of natural olive oil, in agreement with another study designed
to test the effects of diets enriched with corn oil or an olive oil/sunflower oil mixture on vitamin
K metabolism and vitamin K-dependent proteins in young men (22).
Interestingly, we also found that a higher intake of olives was associated with higher serum
osteocalcin concentrations. Taken together, our findings concur with experimental reports
that associate the consumption of olives (5), olive oil (6), and oleuropein (7) with the
prevention of bone mass loss in animal models of osteoporosis (8, 9). In vitro studies have
also shown that oleuropein intake leads to increased osteocalcin concentrations (10).
We have found decreased circulating CTX with all interventions. On the contary, several
studies have reported elevations in serum and urinary markers of bone resorption after
modest (5–15%) weight reduction over 6–12 months in both pre- and postmenopausal
women and adult men and women induced via moderate energy restriction (reviewed in
Ref. 23).
Serum calcium concentration was found to decrease in subjects under a low-fat diet and
MedDiet+nuts. Individuals vary in their ability to absorb the calcium they consume. In one
study, fractional calcium absorption was positively associated with dietary fat intake (r =
0.29, P = 0.001) (24). Subjects in the lowest tertile of the ratio of dietary fat to fiber had 19%
lower fractional calcium absorption values (24). These differences could be behind
decreased serum calcium concentration in subjects under a low-fat diet and MedDiet+nuts
but not in those subjects consuming MedDiet+VOO.
Statins have been described to increase circulating osteocalcin (11). For this reason, we
controlled for statin intake. We found that statin intake did not influence significantly the
results regarding osteocalcin or P1NP changing levels in subjects of the MedDiet+VOO
group.
The strengths of this study include the randomization of study subjects in a representative
sample of men followed up during 2 yr. We did not measure bone density or β-cell function
by using indexes more reliable and sensitive than HOMA-β-cell, and these are study
limitations. Potentially important confounding factors, such as thyroid function or serum
testosterone concentration were not systematically measured in these subjects.
Furthermore, we performed only single measurements at baseline and at 2 yr of follow-up.
However, the findings of P1NP were in parallel to those of osteocalcin.
In summary, the consumption of a MedDiet enriched with virgin olive oil for 2 yr is associated
with increased serum osteocalcin concentrations that parallel an increase in β-cell function
in elderly men at high cardiovascular risk, suggesting a protective effect on bone. To discern
whether increased osteocalcin is the cause or the consequence of increased β-cell function
awaits further studies, ideally in healthy volunteers.

Acknowledgments
Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y Nutrición is
an initiative of the Instituto de Salud Carlos III (Madrid, Spain). J.M.F.-R. participated in the
experimental design and wrote and edited the manuscript; M.B. recollected the samples and
performed the biochemical analysis; J.M.M.-N. performed the biochemical analysis; W.R.,
E.R., and R.E. revised the manuscript; and J.S.-S. designed the experiments and revised
the manuscript.
This work was partially supported by research grants from the Ministerio de Educación y
Ciencia (Grant SAF2008-02073) and by CIBERobn (CIBER Patofisiología Obesidad y
Nutrición).
Disclosure Summary: J.S.-S. is a nonpaid member of the Scientific Advisory Board of the
International Nut Council. E.R. is a nonpaid member of the Scientific Advisory Board of the
California Walnut Commission. The other authors have no conflict of interest affecting the
conduct or reporting of the work submitted.

Footnotes
Abbreviations:
BMI Body mass index
CTX C-telopeptide of type I collagen
HDL high-density lipoprotein
HOMA homeostasis model assessment
HOMA-BCF HOMA-β-cell function
HOMA-IR
HOMA insulin resistance index
LDL low-density lipoprotein
P1NP procollagen I N-terminal propeptide
PREDIMED Prevención con Dieta Mediterránea.
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Nutrients. 2017 May; 9(5): 472.

Published online 2017 May 9. doi: 10.3390/nu9050472

 II. Transcriptomics and the Mediterranean Diet: A Systematic
Review
Luis V. Herrera-Marcos,1,2 José M. Lou-Bonafonte,2,3,4 Carmen Arnal,2,4,5 María A.
Navarro,1,2,4and Jesús Osada1,2,4,*

Abstract

1. Introduction
The traditional Mediterranean diet (considered by United Nations Educational, Scientific and
Cultural Organization (UNESCO) as a heritage of humanity) used to be the food
consumption pattern in countries around the Mediterranean basin during the decade of the
1960s. Epidemiological and clinical studies have established the health benefits of the
Mediterranean diet, which is associated with a lower incidence of atherosclerosis,
cardiovascular diseases, some types of cancer, and overall mortality [1,2].
The Mediterranean diet is largely vegetarian in nature, and olives and olive oil derived from
the olive tree (Olea europea) are important components. The medicinal properties of its
leaves and fruit have been known since antiquity. In particular, olive oil consumption has
been associated with a decreased risk of cardiovascular disease and certain cancers [3,4].
It is now well-established that, in olives and extra virgin olive oil (EVOO), not only the
monounsaturated fatty acid constituents but also the minor phenolic components and other
phytochemicals have important health benefits [5]. In this regard, research over the past
decade has highlighted the beneficial effects of a range of phenolic compounds [6],
particularly for cardiovascular disease, metabolic syndrome, and inflammatory conditions.
In acute or long-term administration, both in vivo and in vitro studies have shown that EVOO
components have positive effects on metabolic parameters, such as plasma lipoproteins,
oxidative damage, inflammatory markers, pro-thrombotic markers, platelet function, and
antimicrobial activity. These bioactive compounds may influence gene expression at
different levels such as transcription, maturing and stability of RNAs, their translation into
proteins, and post-translational modifications [7]. Accordingly, they may regulate pathways
and proteins related to those pathological entities. However, the underlying mechanisms
remain unknown.
Transcriptional studies have been particularly addressed due to RNA constant chemical
properties of water-solubility and the ability to recognize complementary molecules of RNAs,
in contrast to proteins where such uniform patterns do not exist. In an attempt to better
understand the interactions between nutrients and gene expression, high throughput gene
expression using DNA chips or microarrays has been introduced and applied to analyze
transcriptomes. In contrast to genomic studies, there is not a single transcriptome for an
organism but one for each cell, and it may change in certain environmental circumstances
[8]. The discovery of a large number of non-coding RNAs (approximately 70,000) (Figure 1)
with regulatory functions opens a new field of study for nutrient action and emphasizes the
study of transcriptomics as an end-point of regulatory control. In this review, special attention
will be paid to the changes in a number of tissues considering the different transcriptional
programs that they have undergone [9], and that their responses to nutritional stimuli are
established upon diverse transcription factors and may have distinct physiological purposes
[8].
Figure 1
Current perspective of potential types of RNA in a hypothetical human cell. Prepared using
information from [9,10,11,12].
The present review has adhered to systematic review guidelines [13], and the keywords
used to search PubMed [14] are reflected in Table 1. It covers the up-to-date knowledge of
nutrigenomic studies dealing with the Mediterranean diet, olive oil, or its components. It
identified 165 hits from November 1945 to 7 April 2017. The search was refined by
eliminating duplicate documents. The 53 papers obtained were critically reviewed to verify
whether they analyzed transcriptomics and the Mediterranean diet or its components (Figure
2). Documents that failed to meet this criterion were excluded. Thus, this review covers the
works related to the effects of Mediterranean diet and transcriptomics in 37 papers.

Figure 2
Flow chart displaying the stages used to select the references considered. EndNote X7.7
(Bld 9325 Thomson Reuters: New York, NY, USA, 2016).

Table 1
Combinations of keywords used to search the PubMed database.

Number of Number of
Olive Oil Mediterranean Diet
References References
DNA arrays 1 DNA arrays 1
Microarray 28 Microarray 7
Microarrays 11 Microarrays 3
Gene expression Gene expression
26 11
profile profile
Transcriptional Transcriptional
11 6
profile profile
RNA profile 8 RNA profile 1
Transcriptome 19 Transcriptome 12
Transcriptomics 2 Transcriptomics 12
RNA seq or RNAseq 0 RNA seq or RNAseq 1
mRNA-Seq 1 mRNA-Seq 1
RNA sequencing 1 RNA sequencing 0
Accessed on 7 April 2017.

2. Technical Considerations
Table 2 reflects 37 published studies corresponding to 34 transcriptomic analyses. In most
of them, the tool of choice was the microarray, and Affymetrix platforms were those most
preferred. Agilent, Ilumina, Applied Biosystems, and home-made low-density arrays were
less frequently used. Just four papers used RNA seq [15,16,17,18] and there was one serial
analysis of gene expression [19]. Equally variable was the question employed in the search,
as reflected in the intervention column. This review will be categorized according to the
Mediterranean diet components. Human peripheral blood mononuclear cells (PBMC) and a
wide range of tissues were employed in these studies. Another complication is the
consequence of rapid updating, with increasing numbers of transcripts added in recent
versions. Assuming all these limitations, the information gathered should be considered in
progress, but a glimpse of a powerful tool to delineate nutritional components is clearly
envisioned.

Table 2
Study characteristics.

Number
Dietary
Species Tissue Platform of References
Intervention
Probes
Human studies
GeneChip
Ex vivo Adipose tissue arrays 17,699 MUFA vs. SFA [20]
Affymetrix
 Number
Dietary
Species Tissue Platform of References
Intervention
Probes
PrimeView ™
Med diet vs.
Ex vivo PBMC arrays 36,000 [21]
previous diet
Affymetrix
GeneChip
MUFA vs. SFA
Ex vivo PBMC arrays 17,699 [22]
vs. Med diet
Affymetrix
Oligo
Phenolic
Microarrays
Ex vivo PBMC 45,220 compounds, [23]
(G4112A)
postprandial
Agilent
HT-12 v4 47,000
Phenolic
Illumina V2
Ex vivo PBMC compounds, [24]
MicroRNA 1146 postprandial
Illumina
Gene 1.1 ST MUFA vs. SFA,
Ex vivo PBMC 56,249 [25]
Array Affymetrix postprandial
Genome Survey
Microarray V2.0 Virgin olive oil,
Ex vivo PBMC 32,878 [26]
Applied postprandial
Biosystems
GeneChip
Low fat vs. Med
Ex vivo PBMC U133A 2.0 18,400 [27]
diet
Affymetrix
Genome Survey
Long-term
Microarray V2.0
Ex vivo PBMC 32,878 virgin olive oil [28]
Applied
consumption
Biosystems
HT-12 v4
OO vs. EPA or
Ex vivo PBMC BeadChip 47,000 [29]
DHA
Illumina
Cells of human origin
Custom-made
Colon cancer Phenolic
In vitro Oligo 700 [30]
cells compounds
Microarray chip
Erythroleukemic
cell line K562 20 ×
In vitro RNA seq Hydroxytyrosol [15,16]
and 106reads
keratinocytes
Oligo
JIMT1 breast Microarrays Phenolic
In vitro 45,220 [31,32]
cancer cells (G4112F) compounds
Agilent
 Number
Dietary
Species Tissue Platform of References
Intervention
Probes
Serial Analysis Counts
Mesenchymal
In vitro of Gene not Oleuropein [19]
stem cells
Expression shown
Human genome
Fish oil vs. OO
In vitro Prostatic tumors 133A 2.0 22,000 [33]
or corn oil
Affymetrix
TRL from
Low density
In vitro SMC 4376 MUFA vs. SFA [34]
array
vs. Med diet
Animal studies
C.
elegans Gene-
C. Tyrosol (250
Whole organism Chip ® Genome 22,500 [35]
elegans µM)
Arrays
Affymetrix
Chip Array
Flounder Liver Tokyo 14,461 OO vs. LO [36]
University
MouseRef-8 v2 Hydroxytyrosol
Mouse Adipose tissue 25,600 [37]
Illumina (0.03%)
Agilent Mouse 39,430
Cerebral cortex GE 44K v2 and Phenolic
Mouse [38]
Cerebelum Agilent mice 1247 content of OO
miRNA array
GeneChip
OO vs. palm or
Mouse Intestine MOE430 2 39,000 [39]
safflower
Affymetrix
GeneChip
Unsaponifiable
Mouse Liver MOE430A 22,690 [40]
fraction of OO
Affymetrix
GeneChip
Mouse Liver MOE430A 22,690 Oleanolic acid [41]
Affymetrix
GeneChip
Mouse Liver MOE430A 22,690 Maslinic acid [42]
Affymetrix
OO vs.
23 × Western or
Mouse Liver RNA seq [17]
106reads Long-chain
MUFA
 Number
Dietary
Species Tissue Platform of References
Intervention
Probes
Codelink
Fish oil vs. OO
Rat Colon UniSetRat I GE 9028 [43,44]
or corn oil
Healthcare
Rat Liver Array Affymetrix 12,500 Fish oil vs. OO [45]
Rat U34 MUFA vs. SFA,
Rat Liver 26,334 [46]
Affymetrix long-term
Expression
Virgin olive oil,
Array 230
Rat Liver 31,000 fasting vs. [47]
version 2.0
postprandial
Affymetrix
RGU34A OO vs. corn oil,
Rat Liver GeneChip 8800 long-term [48]
Affymetrix regimen
Counts
Mammary OO vs butter or
Rat RNA seq not [18]
glands safflower
shown
Expression
Mammary
Rat Array 230 2.0 31,000 Hydroxytyrosol [49]
tumors
Affymetrix
OO vs. corn
Mammary Rat Exon 1.0 ST
Rat 1 × 106 and low fat [50]
tumors Array Affymetrix
diets
OO vs. beef
Porcine genome
Swine Muscle 23,937 tallow, coconut [51]
array Affymetrix
or soybean oils
Open in a separate window
DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; LO: linseed oil; Med:
Mediterranean; MUFA: monounsaturated fatty acids; OO: olive oil; PBMC: peripheral blood
mononuclear cells; SFA: saturated fatty acids; SMC: smooth muscle cells; TRL: triglyceride-
rich lipoproteins.

3. Transcriptional Analysis of Mediterranean Diet Consumption or Its Components in Human

Studies
PBMC play a crucial role in the initiation and progression of atherosclerosis [52] and are
easily accessible cells. For these reasons, the majority of human transcriptional studies have
been performed using them as samples with different experimental designs and types of
subjects. Adipose tissue biopsies and other cells of human origin have also been employed
in order to test in vitro actions.

3.1. Influence of a Mediterranean Diet

A six-week intervention of a Mediterranean-inspired diet was able to reduce the established
biomarkers of inflammation and resulted in a trend toward normalizing the microbiota in
Crohn’s disease patients. A transcriptomic approach showed that the cumulative effect of
small changes in many genes combined to have a beneficial effect [21]. In another
experiment, the effects of a Mediterranean-type diet, and the replacement of saturated fatty
acids (SFA) with monounsaturated fatty acids (MUFA) in a Western-type diet, were tested
in abdominally overweight men and women allocated to an eight-week, completely
controlled SFA diet (19% daily energy as SFA), a MUFA diet (20% daily energy MUFA using
refined olive oil), or a Mediterranean diet (21% daily energy MUFA using EVOO).
Consumption of the MUFA and Mediterranean diets, compared with the SFA diet, decreased
the expression of oxidative phosphorylation genes, plasma connective tissue growth factor,
and apolipoprotein B concentrations. Compared with the Mediterranean and SFA diets, the
MUFA diet changed the expression of genes involved in B-cell receptor signaling and
endocytosis signaling. Participants who consumed the Mediterranean diet had lower
concentrations of proinflammatory proteins at the end of the intervention. Thereby, the
replacement of SFA with MUFA may improve health, by reducing metabolic stress and
oxidative phosphorylation activity in PBMC [22]. The Mediterranean diet may have additional
antiatherogenic effects by lowering proinflammatory plasma proteins due to other biological
active compounds [53].
The influence of postprandial ingestion of a single dose of olive oil (50 mL) on gene
expression was analyzed in PBMC cells from six healthy male volunteers. Compared with
the fasting state, upregulated expressions were observed in genes related to metabolism,
cellular processes, and cancer, and downregulation was described for genes related to
atherosclerosis (e.g., USP48 and OGT) and associated processes such as inflammation
(e.g., AKAP13 and IL-10) and DNA damage (e.g., DCLRE1C and POLK) [26].
The long-term (3-month) effect of a traditional Mediterranean dietary pattern on the whole
transcriptomic response was explored in a subsample (n = 34) of the Prevención Con Dieta
Mediterránea (PREDIMED) study, where participants consuming a low-fat diet as a control
group were compared to two intervention groups assigned to a traditional Mediterranean
diet supplemented with either virgin olive oil or nuts. The Mediterranean diet with virgin olive
oil elicited changes in 241 genes (139 upregulated and 102 downregulated genes), while
the supplementation with nuts modified the response of 312 genes (165 upregulated and
147 downregulated genes), and the low-fat diet changed the expression of 145 genes (100
upregulated and 45 downregulated genes) compared to the initial status. Using functional
annotation analysis, 12 pathways were differentially expressed, and 43% of pathways were
modulated by both Mediterranean interventions; the most prevalent pathways were related
to atherosclerosis and hypertension (i.e., ADRB2, IL7R, IFNgamma, MCP1, TNFalpha) and
were often associated with an improvement in systemic markers for oxidation and
inflammation [7,27]. Thereby, these changes in the transcriptomic response of genes related
to cardiovascular risk may be crucial in the mechanisms of Mediterranean diet benefits.

3.2. Influence of Monounsaturated vs. Saturated Fatty Acid Containing Diets

The influence of the nature of administered fat on the adipose tissue transcriptome was
studied in an 8 week parallel controlled-feeding trial using abdominally overweight subjects
at risk of metabolic syndrome. The inclusion of refined olive oil to provide 20% of their intake
as monounsaturated fatty acids compared to the 11% of the saturated diet led to a more
anti-inflammatory gene expression profile [20]. No information on the source of saturated fat
was provided. Considering that adipose tissue plays an interesting role in lipid metabolism
and inflammation, this replacement of dietary saturated fat with a monounsaturated fatty
acid diet could prevent adipose tissue inflammation and may reduce the risk of inflammation-
related diseases where this tissue is particularly relevant e.g., metabolic syndrome.
The different postprandial response comparing SFA and MUFA was tested in a cross-over
study in which 17 lean and 15 obese men received two 95 g fat shakes differing in the type
of fat, and their gene-expression profiles were compared in the fasting and 4 h postprandial
states. During fasting, 294 genes were differently expressed between lean and obese
people. The fat challenge increased differences to 607 genes after SFA (palm oil) and 2516
genes after MUFA coming from high-oleic acid sunflower oil. In both groups, SFA decreased
expression of cholesterol biosynthesis and uptake genes and increased cholesterol efflux
genes and MUFA increased inflammatory genes such as Toll-like receptor signaling and
interleukin 8-chemokine receptors 1/2 IL8-CXCR1/2 pathways and peroxisome proliferator-
activated receptor alpha PPAR-alpha targets involved in beta-oxidation [25]. These results
indicate that a fat challenge magnifies differences in health status of subjects, especially
after MUFA, and these types of fatty acids exert distinct effects on lipid handling pathways
in PBMC cells.
The effect of the type of fat on gene expression in human coronary artery smooth muscle
cells was studied by incubating them with postprandial triglyceride-rich lipoproteins prepared
from the plasma of healthy volunteers who had received meals enriched in refined olive oil,
butter, or a mixture of vegetable and fish oils. Sixty-six genes were regulated by the
incubation of lipoproteins obtained from the butter group, 55 after olive oil consumption, and
47 with vegetable and fish oils. Lipoproteins obtained from subjects consuming butter and
vegetable and fish oils induced the expression of genes involved in inflammation, particularly
macrophage-inhibiting cytokine-1, while those coming from the olive oil group promoted a
less atherogenic gene profile [34].
The effect of the source of dietary fat on prostate cancer progression was tested in male
immunodeficient mice injected with LAPC-4 human prostate cancer cells. Two weeks after
injection, mice were randomized to Western diets (35% kilocalories from fat) containing the
different fat sources—fish oil, olive oil, corn oil, and animal fat—and euthanized when tumor
volumes reached 1000 mm3. The fish oil group had decreased gene expression in pathways
related to mitochondrial physiology and insulin synthesis/secretion, slowed tumor growth,
and improved survival compared with that of the mice consuming the other diets [33].
Therefore, the type of dietary fat consumed may be important for controlling tumor
progression.
Thus, the fatty acid composition of diets modified gene expression patterns of adipocytes,
PBMC, coronary artery smooth muscle, and human prostate cancer cells. The inclusion of
MUFA from olive oil led to a program with lower expression of inflammatory genes and,
consequently, to a less atherogenic profile, and tumor progression was controlled when
compared to other sources of fat.

3.3. Influence of Monounsaturated vs. n-3 Fatty Acid Containing Diets

The effect of six week supplementation with either olive oil, eicosapentaenoic acid (EPA), or
docosahexaenoic acid (DHA) on gene expression was tested in subjects with mild elevation
in plasma lipoprotein-phospholipase A2 (Lp-PLA2) at baseline and six weeks after receiving
olive oil 6.0 g/day (n = 16), EPA 1.8 g/day (n = 16), or DHA 1.8 g/day (n = 18). Neither DHA
nor olive oil produced changes, but EPA significantly affected gene expression in the
following pathways: (1) interferon signaling; (2) receptor recognition of bacteria and viruses;
(3) G protein signaling, glycolysis, and glycolytic shunting; (4) S-adenosyl-l-methionine
biosynthesis; and (5) cyclic adenosine monophosphate (cAMP)-mediated signaling
including cAMP responsive element protein 1 (CREB1), as well as many other individual
genes, such as hypoxia inducible factor 1 alpha subunit(HIF1A) [29]. Thus, EPA
supplementation was associated with significant effects on gene expression involving the
interferon pathway, as well as downregulation of CREB1 and HIF1A, which may relate to its
beneficial effect on cardiovascular risk reduction.

3.4. Influence of the Administration of Olive Oil Enriched in Phenolic Compounds

These compounds have been the focus of many transcriptomic analyses. Using PBMC, the
presence of virgin olive oil phenolic compounds was tested in 20 patients with metabolic
syndrome consuming two virgin olive oil-based breakfasts with high and low (398 and 70
mg/kg, respectively) contents of phenolic compounds in a double-blinded, randomized,
crossover design. Olive oil phenolic compounds differentially expressed 98 genes (19
upregulated and 79 downregulated). Among these, several genes that seemed to be
involved in inflammatory processes mediated by transcription factor NF-kappaB, activator
protein-1 transcription factor complex AP-1, cytokines, mitogen-activated protein kinases
(MAPK), or arachidonic acid pathways were repressed by phenolic compounds, thereby
switching the activity of PBMC to a less deleterious inflammatory profile [23]. Recently,
D’Amore et al. used a similar approach in healthy subjects and in patients with metabolic
syndrome. They reported that phenolic compounds modulate mRNA and miRNA levels
participating in metabolism, inflammation, and cancer. They confirmed the switch of PBMC
to a less deleterious inflammatory phenotype with more information, since they used an
advanced version of microarray and the inclusion of miRNAs. Furthermore, they found a
more pronounced effect in healthy subjects when compared to patients [24]. These results
indicate that changes in gene expression are part of the postprandial protective action of
olive oil consumption and phenolic compounds exert an influence. In addition, these studies
pose the question that the response may change depending on the studied population.
A mid-term (3 weeks) intervention with feasible daily doses of 50 mL explored the genes
that responded to virgin olive oil consumption in order to ascertain the molecular
mechanisms underlying its beneficial action in the prevention of atherosclerosis. To this end,
gene expression profiles from healthy individuals were examined after moderate and regular
consumption of virgin olive oil, as the main fat source in a diet controlled for antioxidant
content. The response to virgin olive oil consumption was confirmed for 10 upregulated
genes (ADAM17, ALDH1A1, BIRC1, ERCC5, LIAS, OGT, PPARBP, TNFSF10, USP48,
and XRCC5) [28].
Overall, these studies indicate that the consumption of virgin olive oil with phenolic
compounds, either in a postprandial regimen or over a relatively short period, influences the
expression of genes related to atherosclerosis development and progression, and that this
effect is observed at the moderate doses consumed in this dietary pattern. Whether this
action is due to a particular phenolic compound or to a combination of them has not been
addressed. The translation into proteins has not been analyzed.

3.5. Influence of the Administration of Isolated Phenolic Compound Extracts

This possibility has been explored only at the cell culture level. Indeed, EVOO extracts
composed of hydroxytyrosol, secoiridoids, and lignans were tested in vitro in a colon cancer
cell line engineered to overexpress estrogen receptor beta (HCT8-beta8). The extracts
showed an antiproliferative effect through the interaction with estrogen-dependent signals
involved in tumor cell growth. Specifically, the ability of the extracts to inhibit cell proliferation
was superimposable to the activation of the estrogen receptor beta, similar to what was
observed after 17beta-estradiol incubation [30]. Using HER2-gene amplified JIMT-, a cell
line used to explore resistance against HER1/2-targeted drugs, the anti-cancer activity of
EVOO phenolic extracts from 14 Spanish single-variety oils positively correlated with the
phenolic index (i.e., total content of phenolic components) and with a higher content of
secoiridoids, rather than lignans. These changes using a transcriptome approach were
found to be associated with changes at the level of the cell cycle and p53 pathways. Indeed,
phenolic extracts differentially modulated the expression of stress-sensing, G2-M check-
point-related GADD45 genes, and of p53-related CDKN1A, CDKN1C, and PMAIP-1 genes.
Secoiridoid-rich extracts inhibited mitosis by promoting gap2-mitosis cell cycle arrest,
induced hyperacetylation of histone H3 at lysine, and activated the mitogen-activated protein
kinases MEK1 and p38. Thus, secoiridoids may be adjuvants in breast cancer therapy,
particularly in refractory to HER-targeting therapies and may facilitate the development of a
series of new agents to combat this cancer [31]. In fibroblasts, phenolic extracts mainly
containing the secoiridoids oleuropein aglycon and decarboxymethyl oleuropein aglycon
were found to activate endoplasmic reticulum stress and the unfolded protein response,
spermidine and polyamine metabolism, and sirtuin-1 and NRF2 signaling. These
secoiridoids also activated AMP-activated protein kinase and suppressed crucial genes
involved in the Warburg effect and the self-renewal capacity of cancer cells triggering anti-
aging transcriptomic signatures [32]. In bone-marrow mesenchymal stem cells, oleuropein
was found to upregulate the expression of 60% of adipogenesis-repressed genes [19].
Hydroxytyrosol, the main phenolic compound in minor constituents of olive oil, has been
shown to be a potent antioxidant and has anti-atherogenic and anti-cancer properties. Using
genome-wide mRNA-seq, the treatment of keratinocytes with 20 µM hydroxytyrosol resulted
in the upregulation of numerous antioxidant proteins and enzymes, including heme
oxygenase-1, glutaredoxin, and glutathione peroxidase. This may account for the reduction
in oxidative stress and suggests a mechanism for the chemoprevention of cancer by
hydroxytyrosol. Alteration in the expression of transcription factors such
as STAT3, STAT6, SMAD7, and ETS-1 may also contribute to the anti-cancer and anti-
inflammatory effects. Furthermore, the downregulation of the telomerase reverse
transcriptase subunit, observed in the erythroleukemic cell line K562 treated with this
compound [15], together with changes in the complement system, the Warburg effect, and
chromatin remodeling, are other candidate genes involved in the prevention of cancer by
hydroxytyrosol [16].
These in vitro studies clearly indicate that extracts enriched in hydroxytyrosol, secoiridoids,
and lignans are able to inhibit cell proliferation by modulating cell cycle expression.
Hydroxytyrosol itself has been shown to exert transcriptional changes related to transcription
factors. However, the action of secoiridoids cannot be neglected and a combined action is
expected. Direct proof of their action in controlled clinical trials is still missing.

4. Transcriptional Analysis of Mediterranean Diet Consumption or of Its Components in Non-

Human Studies
Several animal models have been tested, as well as a wide range of tissues. In these
experimental settings, not only was the influence of olive oil studied but that of its minor
components as well. In these experiments, it was possible to delineate the physiological role
of genes with unknown function as well.

4.1. Influence of Monounsaturated vs. n-3 and n-6 Polyunsaturated Fatty Acid Containing
Diets
Japanese flounders were used to identify hepatic genes involved in adaptation to n-3
unsaturated fatty acid-depleted diets and the possibility of breeding them to be resistant to
this condition. They were fed diets supplemented with fish oil, linseed oil (LO), or olive oil for
6 weeks. The latter two groups showed significantly retarded growth, lower feed intake,
lower protein efficiency ratio, and lower hepatosomatic index. As a result of the
administration of a diet with n-3 highly unsaturated fatty acid deficiency, 169 transcripts
changed their expression, particularly those involved in signal transduction (23.2%), cellular
processes (21.1%), metabolism (including glucose, lipid, and nucleotide; 15.5%), transport
(11.3%), regulation of transcription (10.5%), and immune response (4.2%). Several genes
encoding serine/threonine kinases (such as protein kinase C and calmodulin-dependent
kinase), nuclear hormone receptors (such as vitamin D receptor, retinoic acid receptor) and
receptors for cytokines (bone morphogenic protein and transforming growth factor beta)
were also influenced. Among 169 transcripts, 57 genes were upregulated and 38 genes
were downregulated in the LO group, whereas in the olive oil group, nine genes were
upregulated and 87 genes were downregulated [36]. These results indicate that changes in
gene expression induced by n-3 deficiency are not restored by the supplementation of LO
or olive oils.
The influence of dietary fat on colon tumor formation was explored in Sprague-Dawley rats
assigned to three dietary groups differing only in the type of fat (corn oil/n-6 PUFA, fish oil/n-
3 PUFA, or olive oil/n-9 monounsaturated fatty acid) receiving the carcinogen azoxymethane
for 12 h and 10 weeks. Only the consumption of n-3 PUFA exerted a protective effect at the
initiation and promotional stages, estimated as DNA adduct formation and aberrant crypt
foci, respectively. Colonocyte gene expression profiles were different at both the initiation
and promotional stages of tumor development since the number of differentially expressed
genes in each of the three diets increased with the progression of colon cancer, but each
dietary lipid source exhibited its own unique transcriptional profile. The authors observed
that the chemopreventive effect of fish oil was due to the direct action of n-3 PUFA and not
to a reduction in the content of n-6 PUFA [43], andthey [44] found that the mediators of stem
cell homeostasis, e.g., ephrin B1 and bone morphogenic protein 4, were involved. In other
carcinogenesis models, the effects of a low-fat, high-corn-oil, or high-EVOO diet from
weaning or from induction on the susceptibility of the mammary gland to experimental
malignant transformation were tested. Whereas rats consuming EVOO modified the
expression of metabolism genes, those consuming corn oil showed downregulated
expression of genes related to the immune system and apoptosis, and increased
proliferation and lower apoptosis in the mammary glands, thereby favoring mammary
tumorigenesis [50]. Recently, Govindarajah et al. [18] tested the effects of prenatal high-fat
diet (olive oil, butterfat, or safflower) exposure on cancer susceptibility in prepubertal
mammary glands of rats postnatally treated with dimethylbenz[alpha]anthracene (DMBA).
High-fat diet exposure significantly increased the tumor volume. Transcriptome profiling
identified 43 differentially expressed genes in the mammary glands of the butterfat group as
compared with controls. Differences in the expression patterns
of Lrrn1, NF1, DBF4, Cadm4, Tmem45b, and Btn1a1, among high-fat diet groups,
supported the existence of certain diet-dependent cancer modifier networks underlying
differential susceptibility to mammary cancer risk in adult life. Collectively, these findings
suggest that colon and mammary gland tumorigenesis is modulated by dietary fat and
involves changes in gene expression.
A short-term—two-week—nutritional effect of dietary fat on the liver mRNA expression
profile was tested in rats consuming isocaloric diets containing 22% of calories as butter or
olive and fish oils together with butter (10%, 6%, and 6% of the total energy, respectively).
The latter diet induced 72 and inhibited 180 genes related to lipolysis or lipogenesis and
newly identified responders from other metabolisms. Some of these genes were also
reported to be similarly modulated by the action of fibrates, but without the complete gene
pattern of these agents [46]. Likewise, the increased expression of many PPAR-dependent
enzymes mediating fatty acid oxidation followed the substitution of dietary monounsaturated
or polyunsaturated fatty acids (olive or menhaden oils as 40% of calories) for carbohydrates
in corpulent leptin receptor-deficient rats. This fact, together with the reduced hepatic
expression of sterol regulatory element-binding protein-1c (SREBP-1c) and the enzymes
related to lipid synthesis, could explain the observed decrease in hepatic triglyceride content
and lipogenesis [45]. A three-week regimen was selected to explore the influence of the
amount of fat (5% and 70%), provided by olive, corn, and echium oils on gene expression
in male Sprague-Dawley rats. At the low fat content, compared with the corn oil group,
differences in hepatic gene expression signatures associated with greater fatty acid
synthesis, carbohydrate-responsive element-binding proteins, and SREBP-1c signaling,
and increased fatty acid transport was observed in the olive oil group. With the higher fat
diet (70%), when compared to the corn oil group, rats receiving the olive oil-enriched diet
showed increased antioxidant pathways and lower expression of genes linked to
inflammation and fibrosis, despite the increased macrosteatosis, but with no further hepatic
damage [48]. Thereby, a Mediterranean diet high in olive oil may reduce the risk of non-
alcoholic fatty liver disease progression to nonalcoholic steatohepatitis.
The effects of 3% of dietary fat from different sources, i.e., beef tallow, soybean oil, olive oil,
and coconut oil, on gene expression in Longissimus dorsi muscle was explored in growing-
finishing crossbred pigs (LandracexLarge WhitexDuroc). The type of dietary fat modified the
fatty acid composition, and six genes involved in the insulin signaling pathway were
differentially expressed depending on the dietary group. In particular, the genes encoding
the cAMP-dependent protein kinase regulatory, type II, alpha, and the catalytic subunit of
protein phosphatase 1, beta isoform showed the highest expression levels in the olive oil
group [51]. Thus, a slight change in the source of fat is able to induce changes in gene
expression in pig muscle.
Even more, not all MUFA behave alike. Indeed, using a transcriptomics approach, Yang et
al. found that long-chain MUFA are particularly active at upregulating PPAR signaling
pathways in the liver of low-density lipoprotein receptor-deficient mice [17].

4.2. Influence of the Administration of Olive Oil Enriched in Phenolic Compounds

The challenge for the liver of the postprandial phase was characterized by analyzing the
gene expression profile in response to a bolus of 5 mL of EVOO in male Wistar rats.
Compared with the fasting state, hepatic A2m, Slc13a5, and Nrep mRNA expressions were
significantly modified at four and eight hours after fat ingestion and showed the highest
significant associations with postprandial plasma triglycerides [47]. These results highlight
the rapid changes in hepatic gene expression produced by a postprandial regimen and the
discovery of new proteins involved in the process.
Middle-aged C57Bl/6J mice fed for 6 months with EVOO rich in phenols (6 mg/kg/day)
showed cognitive and motor improvement compared to controls fed the same olive oil
deprived of phenolic compounds. To evaluate whether these behavioral modifications were
associated with changes in gene and miRNA expression, two areas involved in cognitive
and motor processes were chosen: the brain cortex and cerebellum, and their gene and
miRNA profiling were analyzed by microarrays. Most of the gene expression changes were
restricted to the cerebral cortex. Compared to low phenolic content administration, the genes
modulated by aging were mainly downregulated by high phenolic olive oil, and this diet
significantly upregulates genes associated with synaptic plasticity and with motor and
cognitive behavior, such as Notch1, Bmps, Ngfr, Glp1r, and Crtc3. The agrin pathway was
also significantly modulated and miRNAs were mostly upregulated in old animals consuming
low-phenolic olive oil compared to young. However, mice fed the phenolic-enriched olive oil
displayed a miRNA expression profile similar to that observed in the cortex of young mice
and sixty-three miRNAs were significantly downregulated, particularly mir-484, mir-27, mir-
137, mir-30, mir-34, and mir-124 [38]. These results indicate that a food rich in phenols can
modulate, at the molecular level, the expression of genes and miRNAs involved in neuronal
function and synaptic plasticity, along with cognitive, motor, and emotional behavior.

4.3. Influence of the Administration of Isolated Phenolic Compound Extracts

Since tyrosol was found to increase the lifespan of Caenorhabditis elegans, a transcriptomic
approach was designed to search for candidate genes or signaling pathways potentially
involved in its effects on longevity. Analyses of microarrays identified 208 differentially
expressed genes (206 up- and two downregulated) when comparing nematodes treated with
or without the compound. The regulation of growth, transcription, reproduction, lipid
metabolism, and body morphogenesis were target genes [35]. The pattern of expression
found overlapped with that elicited by quercetin and tannic acid, known lifespan enhancers
in this model, and raise the notion of key cellular mechanisms directly related to longevity
and the possibility of manipulating them by using these compounds.
As already mentioned, adipose tissue, where inflammation, oxidative stress, and the
secretion of adipocytokines contribute to cardiovascular risk, is a target organ in
cardiometabolic prevention. Long-term supplementation to mice of hydroxytyrosol at
nutritionally-relevant doses, i.e., 0.03%, modulated the antioxidant network in the adipose
tissue, as mediated by glutathione and associated enzymes, and reduced circulating leptin
[37]. These effects, also confirmed in cultured adipocytes, indicate that low, physiological
concentrations of hydroxytyrosol were able to blunt the alteration of oxidative stress.
In a rat model of experimental mammary carcinoma induced by DMBA, the administration
of 0.5 mg/kg hydroxytyrosol for six weeks was found to inhibit the experimental mammary
tumor growth and proliferation rate, with results comparable to those of doxorubicin. It also
altered the expression of genes related to apoptosis, cell cycle, proliferation, differentiation,
survival, and transformation pathways. It modified the Wnt signaling pathway [49].
Altogether, these studies provide evidence that tyrosol and hydroxytyrosol are able to control
growth, oxidative stress, and differentiation in different experimental models.

4.4. Influence of the Administration of Other Minor Components of Virgin Olive Oil
In apolipoprotein E (Apoe)-deficient mice, an olive oil-enriched diet reduced fatty liver
disease, and the degree of hepatic steatosis was associated with
hepatic Fsp27 and Syt1expressions [54]. The influence of the unsaponifiable fraction in
virgin olive oil on hepatic gene expression was also tested in these mice using two
isoenergetic, isonitrogenous diets containing either 10% (w/w) refined olive oil or olive oil
enriched with the unsaponifiable fraction. Eleven genes with remarkably modified
expression (signal log2 ratio >3 or <−3) were selected and confirmed by quantitative
polymerase chain reaction. Orosomucoid and serum amyloid A2 were upregulated by the
unsaponifiable fraction to a variable extent, depending on the mouse genetic background,
in the absence of hepatic steatosis and inflammation. Fabp5 and Mt2 were also strongly
upregulated. Several proteases were markedly suppressed by the unsaponifiable-fraction-
enriched olive oil diet. The findings indicate that these genes are good targets of the
unsaponifiable fraction of olive oil [40]. Two studies were carried out to investigate the effects
of two triterpene components of the unsaponifiable fraction, maslinic and oleanolic acid, both
present in virgin olive oil at concentrations ranging from 3 to 49 and 3 to 78 mg/kg,
respectively. The effect of maslinic acid on hepatic gene expression was tested in Apoe-
deficient mice with a C57BL/6J genetic background receiving 10% (w/w) fat diets. In mice
receiving the maslinic-enriched diet, Cyp2b9, Cyp2b13, and Dbp expressions appeared to
be significantly increased, and Marco, a gene strongly upregulated by the absence of APOE,
was significantly decreased. Dbpwas upregulated to an extent depending on the genetic
background of the mice and was negatively associated with the expression of Marco. Thus,
maslinic acid is active in controlling hepatic gene expression and some of its effects are
modulated by the presence ofAPOE [42]. The other triterpene, oleanolic acid, administered
to Apoe-deficient mice, increased the hepatic area occupied by lipid droplets with no change
in oxidative stress. Bmal1 and the other core component of the circadian clock, Clock,
together with Elovl3, Tubb2a, and Cldn1 hepatic expressions, were significantly increased,
while Amy2a5, Usp2, Per3, and Thrsp were significantly
decreased. Bmal1and Cldn1 expressions were positively associated with lipid droplets.
Increased Clockand Bmal1 expressions were also observed in F344 rats, but not in Apoa1-
deficient mice. These results indicate that the core liver clock components, Clock-Bmal1,
are targets of oleanolic acid independently of the diets provided, and this compound requires
APOA1-HDL for its hepaticaction [41]. Overall, these results highlight the important
biological effects of the terpenes that are constituents of the unsaponifiable fraction of virgin
olive oil, whereas oleanolic and maslinic acids are particularly active in controlling the
circadian clock and inflammation genes, respectively.

5. Outlook
Future studies are in progress. For instance, “New dietary strategies addressing the specific
needs of elderly population for a healthy aging in Europe” (NU-AGE), an interdisciplinary
European research network including research centers on nutrition and aging and food
industries, will explore the transcriptome of elderly people following the Mediterranean diet
for one year [55]. This and other designs will complete our understanding of the complex
interaction of nutrients and gene expression.

6. Conclusions
Nutrition is undoubtedly the most important environmental factor that living beings face. On
this basis, a complex process of adaptation from a genomic point of view is expected.
Therefore, nutritional regulation of many genes will exist, and its failure may be behind the
development of many diseases. The large number of gene expression changes observed in
nutritional interventions due to the increased sensitivity of using transcriptomics, as
compared to well-established biomarkers, opens up a new era of dietary intervention
studies. A representative overview of the magnitude of this task is represented in Figure 3.
To accomplish this endeavor, an international consortium is required that should be
committed to four goals: a well-defined composition of chemical compounds in food;
bioavailability studies to characterize in vivo levels of minor components; characterization of
all types of RNA expression in all tissues at feasible doses, and a bioinformatics effort to
provide information in a friendly environment. Thus, an integrative combination of programs
executed at different levels (mRNA, lncRNA, circRNA, miRNA) may be envisioned and even
a dynamic adaptation according to different time points. Nowadays, considering the low
number of known biological functions of mRNAs, let alone non-coding RNAs, the process
will be long, and during it, some gene functions will be linked to many dietary components.

Figure 3
Potential transcriptomic changes in different cells upon exposure to the Mediterranean diet.
The influence of Mediterranean diets or their components on miRNA has only been studied
in two reports. No study has addressed the regulation of lncRNA or circRNA expressions.
Since olive oil is an important component of this dietary pattern, much effort has been
devoted to understanding the effect of MUFA, the major components of this oil. The
replacement of SFA with MUFA reduced metabolic stress and prevented the expression of
inflammatory genes in different tissues (PMBC, adipocytes, PBMC, coronary artery smooth
muscle, and human prostate cancer cells), and consequently to a less atherogenic profile
and control of tumor progression when compared to other sources of fat. In animal models,
colon and mammary gland tumorigenesis has been found to be really sensitive to this issue.
This similar response in different models and experimental conditions suggests a conserved
mechanism in different species.
Minor components of virgin olive oil have been particularly studied and different authors
have found that the consumption of virgin olive oil with phenolic compounds, either in a
postprandial regimen or over a relatively short period of time, influences the expression of
genes related to atherosclerosis progression, and that this effect is observed at the
moderate doses consumed in this dietary pattern. Whether this action is due to a single or
a combination of phenolic compounds awaits an answer. Not much effort has been devoted
to the translation of theses transcriptional changes into proteins. However, in vitro studies
clearly indicate that extracts enriched in hydroxytyrosol, secoiridoids, and lignans are able
to inhibit cell proliferation by modulating cell cycle expression. We need to explore whether
these compounds act synergistically, as well as their action in controlled clinical trials.
Despite their being more abundant than phenolic compounds in virgin olive oil, terpenes
have received less attention. In animal models, oleanolic and maslinic acids were
particularly active in controlling the circadian clock and inflammation genes, respectively.
Thus, the term “MUFA-rich oil” no longer appears appropriate for encompassing all the oils
to which it is currently applied, paying no attention to the active minor components (phenolic
and terpenic compounds).
The results reviewed provide at least a partial molecular basis for the reduced risk of
cardiovascular disease and cancer observed in Mediterranean countries. The nutrient
complexity of the Mediterranean diets, differences among the studies and the diversity in
the responses depending on the specific genetic makeup makes this field an open arena
with enormous possibilities. Increasing and consolidating the nutrigenomic knowledge of this
diet warrants further research in order to provide sound, personalized, and optimized
nutritional recommendations.

Acknowledgments
We thank Martha Messman for her assistance in manuscript editing. The work of this group
was supported by grants from the Spanish Ministerio de Economía y
Competitividad, Agencia Estatal de Investigación—European Regional Development Fund
(2013-41651-R and 2016-75441-R) and the European Social Fund—Gobierno de
Aragón (B-69). CIBER de Fisiopatología de la Obesidad y Nutrición (CIBEROBN) is an
initiative of the Instituto de Salud Carlos III, Spain.

Author Contributions
L.V.H.-M., J.M.L.-B., C.A. and M.A.N. carried out the search and prepared the draft, and
J.O. supervised the work and draft and prepared the final version of the manuscript.

Conflicts of Interest
The authors declare no conflict of interest.

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Nutr J. 2015; 14: 94.

III. Effect of consuming a grape seed supplement with

abundant phenolic compounds on the oxidative status of
healthy human volunteers
Felix Grases, 1,2 Rafel M. Prieto,1,2 Rafel A. Fernández-Cabot,1 Antonia Costa-Bauzá,1,2Ana
M. Sánchez,3 and Marin Prodanov3

Abstract

Background
Reactive oxygen species (ROS) and free radicals can react with membrane lipids, nucleic
acids, and proteins, leading to cellular damage. Humans and other organisms have diverse
antioxidant systems that protect against ROS. Enzymatic antioxidants include superoxide
dismutase, glutathione peroxidase, glutathione transferase, and catalase [1]; nonenzymatic
antioxidants include low molecular mass molecules, such as ascorbic acid, vitamin E, uric
acid, N-acetylcysteine, carotenoids, phenols, and phytates [2]. When ROS are generated in
excess or in amounts that overwhelm antioxidant defense mechanisms, oxidative stress and
cell damage may occur. Oxidative stress is associated with the pathogenesis of numerous
human diseases, including neurodegenerative diseases [3], psychiatric conditions [4],
rheumatoid arthritis [5] cancer [6], and renal lithiasis [7]. Thus, there is great emphasis on
antioxidant supplement therapy that targets ROS [8].
Grape (Vitis vinfera L.) seed extracts have high levels of numerous anti-oxidants and are
considered among the most powerful plant-derived antioxidant foods. Their anti-oxidant
activity is mainly attributed to flavonoids and 3 different flavan-3-ols (flavanols): catechin,
epicatechin, and epicatechin gallate and its polymers. Flavan-3-ols can donate electrons or
protons to ROS and act as scavengers [9, 10]. Some authors [11] claimed that the
antioxidant activity of these compounds is due to the presence of hydroxyl groups at
positions 3, 5, 4′, and 5′ (and position 7 to a lesser extent) of the benzopyran structure, and
those of the gallic acid moiety in the case of the galloylated forms (Fig. 1). Plumb et al. [12]
proposed that antioxidant activity increased from monomers to trimers, and then decreased
from trimers to tetramers. Grape seed procyanidins are particularly interesting because of
the vast diversity of their polymeric and galloylated forms, which are based on several basic
elemental units [13].

Fig. 1
Chemical structure of the elemental flavan-3-ol units of grape seed extracts
The main problem in studying individual grape seed procyanidins is their separation and
quantification. Normal-phase HPLC can separate flavan-3-ols by molecular mass, but is
limited to qualitative or semi-quantitative analysis. Reversed-phase HPLC is mostly used for
fine separation of flavan-3-ols, but only allows separation and quantification of monomers,
dimers, some nongalloylated trimers, and occasionally tetramers and monogalloylated
trimers. In most cases, prior purification of the sample is necessary [14, 15]. Increasing the
chromatographic resolution by use of two dimensional chromatography allows separation of
heptamer nongalloylated, hexamer monogalloylated, and pentamer digalloylated
procyanidins [16], but only provides qualitative results. Even with advances in the
development of microbore columns and ultra-high pressure HPLC (UPLC) [17], the main
HPLC technique currently used for measurement of flavan-3-ols is based on a C18 bonded
stationary phase with 5 μm particle size, and this only provides partial quantitative estimates
of some individual flavan-3-ols.
Urine antioxidant capacity is usually assessed by complex procedures, including assays of
total phenolics (Folin–Ciocalteu assay), ferric-reducing antioxidant power (FRAP), oxygen
radical absorbance capacity (ORAC) [18], or by determination of the concentrations of
individual antioxidant compounds such as malondialdehyde, ascorbic acid, or uric acid.
Measurement of urinary redox potential is a simpler method for assessment of urine
antioxidant capacity [19].
In the present study, we determined the major phenolic compounds of exGrape® grape
seed extract by an improved HPLC method and analyzed the effect of consumption of this
product on urine oxidative status by measurement of urinary redox potential in healthy
volunteers.

Methods

Extract characterization, reagents, and apparatuses

The reference substances, (+)-catechin (purity > 99 %), (−)-epicatechin (purity > 99 %), (−)-
epicatechin-3-gallate (purity > 97.5 %), procyanidin dimer B1 (purity > 80 %), and
procyanidin dimer B2 (purity > 90 %), were from Extrasynthèse (Genay, France). Gallic acid
(99 % purity) was from Sigma-Aldrich (St. Louis, Missouri, USA). Procyanidin dimers B3-
B7 were purified from natural extracts by high-speed counter current chromatography
(HSCCC) according to Köhler et al. [20] and Esatbeyoglu et al. [21], and were characterized
by nuclear magnetic resonance (NMR). Purified procyanidin extract from cocoa (Breko
GmbH, Bremen, Germany) was used as a complex reference sample for the identification
of procyanidin trimer C1 [EC-(4α-8)-EC-(4α-8)-EC], tetramer [EC-(4α-8)-EC-(4α-8)-EC-(4α-
8)-EC], pentamer [EC-(4α-8)-EC-(4a-8)-EC-(4α-8)-EC-(4α-8)-EC], and hexamer [EC-(4α-
8)-EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC].
Grape seed extract constituents were assessed by the reversed phase (RP) HPLC method
of Prodanov et al. [15], with adaptation for a 3 μm particle size stationary phase to increase
the separation efficiency [22]. Two HPLC apparatuses were used: (i)HPLC/PAD/FL, a
Varian 920-LC Galaxie (Varian Instruments, Walnut Creek, California, USA) that was
coupled to a photodiode array (PDA) and fluorescence (FL) detectors in series and (ii) an
Agilent series 1100 HPLC/PAD/MS (Palo Alto, California, USA) that was coupled to a
quadrupole mass spectrometer (MS) (Hewlett-Packard series 1100 MSD) with an
electrospray interface (ESI). Separation was performed on an ACE-3-C18-AR
(200 mm × 4.6 mm, 3 μm particle size) column from Advanced Chromatography
Technologies (Aberdeen, UK) and a guard-column (20 × 4.6 mm) of the same material. The
mobile phase was a linear gradient of solvent A (water:acetic acid, 98:2) to solvent B
(acetonitrile:acetic acid, 98:2) as follows: from 0 to 20 % of B in 80 min, from 20 to 28 % of
B in 35 min, from 28 to 100 % of B in 5 min, isocratically 100 % of B for 10 min, from 100 to
0 % of B in 5 min, and equilibration of the column for the next analysis for 10 min. The total
run time was 140 min. The flow rate of the mobile phase was 0.6 mL/min. Chromatograms
were acquired by measurement of absorption at 280 nm (by the PDA) and fluorescence at
316 nm (273 nm excitation). For ESI, the drying gas was N2 (10 L/min at 340 °C), the
nebulizing pressure was 40 psi, and the capillary tension was 4000 V. Mass spectra were
obtained by scanning negative ions from m/z less than 200 at 100 V, m/z 200–1000 at
200 V, and m/z 1000–2500 at 250 V. Mass spectra were recorded from m/z 100 to 2500.
Gallic acid, (+)-catechin, (−)-epicatechin, (−)-epicatechin-3-O-gallate, and procyanidin
dimers B1 and B2 were identified by co-elution and comparison with the masses and UV
spectra and retention times of commercial references. Procyanidin dimers B3, B4, B5, and
B7 were identified by comparison of the UV absorbance spectra, fluorescence following
excitation at 273 nm, molecular ions, and retention times of reference substances isolated
by HSCCC from natural extracts [20, 21] and characterized by NMR. Procyanidin trimer
C1 [EC-(4α-8)-EC-(4α-8)-EC], tetramer [EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC], pentamer
[EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC], and hexamer [EC-(4α-8)-EC-(4α-8)-EC-
(4α-8)-EC-(4α-8)-EC-(4α-8)-EC] were identified by comparison of the UV absorbance
spectra, fluorescence following excitation at 273 nm, molecular ions, and retention times of
the corresponding complex reference sample from a purified procyanidin extract from cocoa
[23]. For the procyanidin pentamer [EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC], both the
single- and the double-charged ions were present, but for the procyanidin hexamer [EC-(4α-
8)-EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC-(4α-8)-EC], only the double-charged ion was present
in the mass spectra. Procyanidin trimers, C2 [C-(4α-8)-C-(4α-8)-C] and [EC-(4α-8)-EC-(4α-
8)-C], were identified based on mass, UV absorbance spectra, fluorescence following
excitation at 273 nm, retention time, and order of elution [15]. The galloylated procyanidin
dimers B1-3-G and B2-3′-G were identified by mass, UV absorbance spectra, retention times,
and order of elution [15].
Gallic acid, (+)-catechin, (−)-epicatechin and (−)-epicatechin-3-gallate were quantified by
calibration curves of standards. All procyanidin nongalloylated dimers to hexamers were
referred to the calibration curve of the procyanidin dimer B1 from the fluorescence
chromatograms. The quantities of galloylated procyanidin dimers B1-3-G and B2-3′-G were
calculated based on a calibration curve for the (−)-epicatechin-3-gallate from the 280 nm
absorbance chromatograms.

Human studies
Forty-six non-smokers healthy volunteers (20 males and 26 females, mean age: 34 years,
age range: 17 to 60 years) were invited to participate. Individuals who reported consuming
antioxidant supplements or omega-3 polyunsaturated fatty acids and those with addiction to
alcohol or drugs were excluded. None of the participants received any pharmacological
treatment during urine collection. Fruits and vitamin supplements were discontinued 24 h
prior to collection of the first urine sample. All subjects were instructed to maintain their usual
diets and to not participate in sports activities during the two day study period. The subjects
consumed the biscuits with the 100 % of compliance. All subjects provided written informed
consent and the study protocol (IB 2030/13 PI) was approved by the Ethics Investigation
Committee of the Balearic Islands (Spain).
The first day, each volunteer ate eight traditional biscuits (Quely from Quely S.A., Mallorca
Islands, Spain) during the day (4 in breakfast and 4 in dinner) with no plant-derived phenol
supplementation. The biscuits, of 9 g of weight, are made with wheat, sunflower oil, yeast,
olive oil and salt. An overnight urine sample, starting prior to sleep and ending with the first
morning urine while still in a fasting state, was obtained the next day.
The second day each volunteer ate eight traditional biscuits supplemented with 0.6 % (wt/wt)
of red grape (Vitis vinfera L.) seed extract (Quely Cor from Quely S.A.,) during the day,
corresponding to about 250 mg of phenols, the phenols content of the biscuits was
measured by Folin’s method and expressed as gallic acid. This represent a total polyphenol
intake about 2 g/d. The red grape seed extract (exGrape®) was purchased from La
Gardonnenque-Groupe Grap’Sud® (Cruviers-Lascours, France). The second urine sample
was collected on the third morning as described above.
At 2 h after collection of each non filtered urine sample, the redox potential was measured
at 25 °C using a Crison potentiometer (Crison Instruments S.A., Barcelona, Spain), with a
platinum electrode as the working electrode and a saturated calomel electrode as the
reference electrode. Results are shown as means ± standard errors and compared using
Student’s paired t-test.

Results
Table 1 shows the chemical composition, the mass spectral data and the content of of the
grape seed extract exGrape® used in this study. Figure 2 illustrates a typical ultraviolet
absorption chromatogram (280 nm) of the studied extract, with indications of the main
identified compounds. The results indicates that this extract mainly consists of flavan-3-ol
monomers and oligomers (17.46/100 mg), and very small amounts of gallic acid and some
other unidentified compounds. Epicatechin, catechin, procyanidin dimers B1 to B4, and the
procyanidin trimer C2 were the major components, and these had concentrations of 0.65–
1.70/100 g. Lower amounts of other nongalloylated procyanidin trimers and the procyanidin
tetramer [EC-EC-EC-EC] were also present. The improved HPLC method detected the
procyanidin pentamer [EC-EC-EC-EC-EC] and hexamer [EC-EC-EC-EC-EC-EC], but their
amounts were lower than the limit needed for quantification by the fluorescence detector.
The improved HPLC method also detected the procyanidin pentamer [EC-EC-EC-EC-EC]
and hexamer [EC-EC-EC-EC-EC-EC], but their amounts were also lower than the limit
needed for quantification. The total amount of the nongalloylated procyanidin monomers and
oligomers was 9.11/100 mg and the amount of the galloylated forms (represented only by
the epicatechin gallate and the common peak of the monogalloylated dimers B1-3-G and B2-
3′-G), was 0.65/100 mg.

Table 1
Chemical composition, mass spectral data (negative ionisation mode) and content of the
main identified constituents of exGrape® seed extract (quantitaive data are reffered to the
extrac as it is)

Rt Compound [M-H]− [M-2H]2− Content

(min) (m/z) (m/z) (mg/100 mg)
8.3 Gallic acid 169.0 0.09
32.2 PC2 (B3) [C-C] + PC3 (C2) [C-C-C] 577.0, 865.1 0.65
34.8 PC2 (B1) [EC-C] 577.0 1.19
36.3 C 289.0 1.47
41.3 PC3 [EC-EC-C] 865.1 0.30
47.1 PC2 (B4) [C-EC] 577.1 0.64
54.4 PC2 (B2) [EC-EC] 577.0 1.69
61.7 EC 289.0 1.70
72.4 PC2-G (B1-3-G + B2-3′-G) 729.1 0.51
74.8 PC2 (B7) [EC-C] 577.1 0.23
76.0 PC3 (C1) [EC-EC-EC] 865.1 0.83
81.6 PC4 [EC-EC-EC-EC] 1153.1 0.28
 Rt Compound [M-H]− [M-2H]2− Content
(min) (m/z) (m/z) (mg/100 mg)
83.2 PC5 [EC-EC-EC-EC-EC] 1441.1 720.2 0.11a
85.0 PC6 [EC-EC-EC-EC-EC-EC] - 864.2 0.02a
92.5 EC-G 441.1 0.14
111.2 PC2 (B5) [EC-EC] 577.2 0.22
∑ nongalloylated flavan-3-ol monomers 3.17
∑ nongalloylated flavan-3-ol dimers 4.53
∑ nongalloylated flavan-3-ol monomers and oligomers 9.11
∑ galloylated flavan-3-ol monomer and oligomers 0.65
∑ flavan-3-ols 17.46
Open in a separate window
Rt retention time, C (+)-catechin, EC (−)-epicatechin, PCx procyanidin oligomers (subscript
x - number of elemental units (x = 2 (dimer), 3 (trimer), etc.), PCxG galloylated
procyanidin, Ggalloyl unit
a
Values below the limit of quantification

Fig. 2
Ultraviolet (280 nm) absorption HPLC chromatogram of the grape seed extract exGrape®
(all abbreviations are the same as those shown in Table 1)
Figure 3 shows the redox potential of the urine from 46 healthy volunteers at 12 h after
consumption of eight traditional biscuits and at 12 h after consumption of eight traditional
biscuits with grape seed extract, the redox potential range observed before consuming the
phenolic compound rich biscuits was 76 mV and after consuming the phenolic compound
rich biscuits of 114 mV. The urinary redox potential was 33 % lower after consumption of
the grape seed extract, indicating that consumption of this polyphenol-rich supplement
significantly increased the antioxidant capacity of urine.
Fig. 3
Urine redox potential (mV) of 46 volunteers who ate eight biscuits without phenols on day-1
and eight biscuits supplemented with a red grape seed extract (250 mg polyphenols) on
day-2. Values indicate means ± standard errors. * p < 0.001 by Student’s paired t-test

Discussion
Table 1 shows that the exGrape® seed extract has a composition typical of grape seed
extracts, in that the monomers catechin and epicatechin are most abundant, followed by the
nongalloylated procyanidin dimers and trimers with C4-C8 interflavan bonds [14, 15].
Epicatechin and epicatechin-based oligomers (procyanidin dimer B2 and trimer C1) are the
most abundant among the other catechin-containing derivatives. The natural fluorescence
of the nongalloylated flavan-3-ols was greater, so the procyanidin tetramer, [EC-EC-EC-EC],
was quantified whereas the other two procyanidin oligomers, [EC-EC-EC-EC-EC] and [EC-
EC-EC-EC-EC-EC], were simply separated and detected. To the best of our knowledge this
is the first time that pentamer and hexamer procyanidins were separated and detected in a
single RP HPLC run with direct injection of an unpurified grape seed extract. The content of
flavan-3-ol monomers and oligomers was within the normal limits for this type of extract,
even though the level was almost 2-times lower than reported by Shafiee et al. [24] for the
same extract 10 years ago. However, the difference with regard to the total content of
nongalloylated flavan-3-ol monomers and dimmers was minimal, suggesting that the
differences are most probably due to use of different analytical methods. In particular,
Shafiee et al. [24] used the vanillin method for assessment of oligomer procyanidins, and
this method is based on vanillin condensation of all procyanidins in the sample, including
high molecular mass procyanidin polymers. In contrast, we only considered the major and
well-resolved procyanidin peaks, and these do not account for all nongalloylated
procyanidins.
Grape seed extracts differ from other flavan-3-ol-rich extracts because of their high content
of galloylated compounds. In some red varieties of grapes, these can account for up to 18 %
of the total procyanidin content [14]. Some authors consider these types of procyanidins as
stronger antioxidants than the corresponding nongalloylated analogues [11, 12], and this
has led to increased interest in the galloylated forms. Nevertheless, aside from the present
study, there is little quantitative data on the levels of galloylated and nongalloylated
compounds in grape seed extracts. Thus, even with our improved chromatographic
resolution achieved by use of the 3 μm particle size stationary phase, we were only able to
separate and unambiguously quantify the epicatechin gallate and a peak containing two
monogalloylated dimers, the B1-3-G and B2-3′-G. Perhaps, the most noteworthy difference
we found is that the epicatechin gallate content was 10-fold lower than reported by Shafiee
et al. [24] for the same extract. Nevertheless, we should note that we detected many mono-
and digalloylated procyanidin oligomers in the extract, but their separation was poor and
their amounts were close to or below to limits of detection.
Plant-derived phenols are important dietary antioxidants, and the main dietary sources are
fruits and fruit juices. Tea, red wine, vegetables, legumes, cereals, and chocolate also
contribute to total polyphenol intake, which may be as high as 1 g/day [25]. Grape seed
extract is a concentrated source of polyphenols [26]. Clinical studies found that consumption
of polyphenols can contribute to the prevention of cardiovascular disease [27], some types
of cancer [28], osteoporosis [29], and calcium oxalate papillary renal stones [30]. The
antioxidant activity of polyphenols prevents oxidative membrane damage through metal
chelation and scavenging of ROS [24]. The polyphenol content of urine is an indicator of the
intake of polyphenol-rich foods, such as cacao [31]. Moreover, total urinary phenols were
significantly increased at 4 h after the ingestion of red wine that contained 125 mg of
polyphenols [gallic equivalent] [32]. The flavonoid concentration in urine, determined by
liquid chromatography–mass spectrometry, may reflect the intake of fruits and vegetables,
and the total urinary excretion of flavonoids over 24 h may be a biomarker for fruit and
vegetable intake [33].
Urine is the product of blood filtration, and its composition (including that of ions and low
molecular mass molecules, such as salts, vitamins, and minerals) mirrors that of blood,
despite every subject will have different metabolic rate and therefore different levels of
phenolic metabolites could be found in the urine. Thus, a high level of antioxidant tissue
destruction may be reflected by decreased urinary excretion of low molecular mass
antioxidants. If antioxidants cannot prevent free radical damage or if radical formation is
excessive, the oxidant/antioxidant ratio increases and leads to oxidative stress [34]. The
urinary total antioxidant capacity determined is dependent on the levels of non-enzymatic
antioxidants, which would be predominantly phenolic metabolites in this case, rather than
host derived free radicals, oxidants or electrophiles. One method used to determine the
antioxidant capacity of a biological fluid, such as urine, is based on the reduction of ferric to
ferrous ions; ferrous ions form complexes with specific dyes and generate a product that
can be measured spectrophotometrically [35, 36]. The redox potential of an aqueous
solution is a measure of its ability to be oxidized or reduced, and a more negative potential
indicates a greater reducing capacity. In contrast, strong oxidizing agents have more positive
redox potentials, so a decrease in the urine antioxidant status may reflect an increase in
urinary redox potential. Individuals with high oxidative stress will therefore excrete urine with
low levels of antioxidants and high redox potential. The correlation of the antioxidant
capacity of urine, evaluated using ferric ion and the dye 1, 10-phenantroline (FRAP method)
[35, 36] with the redox potential using the platinum electrode, was already demonstrated
[19]. The correlation was statistically significant by a linear regression equation between
antioxidant capacity (mM of Fe2+) or FRAP method versus the redox potential (mV)
(y = −0.073x + 16.941; R:0.522; p < 0.001) [19]. It demonstrates the suitability of the latter
procedure to determine the antioxidant capacity of urine.
The results obtained in this study demonstrate that a single dietary intervention with a grape
seed supplement significantly lowered the redox potential of urine, reflecting an overall
increase in antioxidant capacity. Other studies have reported similar results after
consumption of polyphenol-rich beverages and/or foods [32, 37–39].
Conclusions
The ExGrape® extract has a composition typical of grape seed extracts, in that the
monomers catechin and epicatechin are the most abundant constituents, followed by the
nongalloylated procyanidin dimers and trimers with C4-C8 interflavan bonds. Epicatechin and
epicatechin-based oligomers (procyanidin dimer B2 and trimer C1) are the most abundant
among the other catechin-containing derivatives. Among the nongalloylated flavan-3-ols, we
quantified the procyanidin tetramer [EC-EC-EC-EC] and separated and detected two other
procyanidin oligomers, [EC-EC-EC-EC-EC] and [EC-EC-EC-EC-EC-EC]. The phenolic
content of urine is an indicator of the intake of plant-derived phenol-rich foods. A single
dietary intervention -- ingestion of biscuits rich in plant-derived phenols -- significantly
lowered the redox potential of urine, and this reflects an overall increase in antioxidant
capacity. The clinical benefits of more sustained dietary interventions with grape seed
extract remain to be established.

Acknowledgments
This work was supported by grant CTQ2010-18271 from the Ministerio de Ciencia e
Innovación (Gobierno de España), by FEDER funds (European Union), and by grant 9/2011
from the Conselleria d’Educació, Cultura i Universitat (Govern de les Illes Balears).
CIBER Fisiopatología Obesidad y Nutrición (CB06/03), Instituto de Salud Carlos III, Spain,
also provided support.
The authors want to thank Quely S.A. (Inca, Spain) for supply the traditional biscuits
(Quely) and the biscuits supplemented with red grape seed extract (Quely Cor).

Footnotes
Competing interests
The University of Balearic Islands has an agreement to develop biscuits supplemented with
red grape seed extract with Quely S.A. (Inca, Spain), whose responsible are FG, RMP and
ACB. The other authors declare that they have no competing interests.
Authors’ contributions
FG took part in planning the study design, and drafting and writing of manuscript. RMP took
part in planning the study design and drafting and writing of the manuscript. RAFC and ACB
were responsible for human studies and took part in planning the study design. AMS and
MP contributed to extract characterization, study design and contributed to writing of the
manuscript. All authors read and approved the final manuscript.

Contributor Information
Felix Grases, Phone: +34 971 173257, Email: se.biu@sesargf.
Rafel M. Prieto, Email: se.biu@oteirp.mlefar.
Rafel A. Fernández-Cabot, Email: se.oohay@cflegnalefar.
Antonia Costa-Bauzá, Email: se.biu@atsoc.ainotna.
Ana M. Sánchez, Email: se.mau@zehcnas.airamana.
Marin Prodanov, Email: se.mau@vonadorp.niram.
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Front Endocrinol (Lausanne). 2014; 5: 58.

IV. Peptides and Food Intake

Carmen Sobrino Crespo,1 Aránzazu Perianes Cachero,1 Lilian Puebla Jiménez,1Vicente
Barrios,2,3 and Eduardo Arilla Ferreiro1,*

Abstract

Introduction
Nutrients created by the digestion of food are proposed to activate G-protein-coupled
receptors on the luminal side of enteroendocrine cells, e.g., the L-cells. This stimulates the
release of gut hormones. The hormones released from the gut and the adipose tissue (AT)
play an important role in the regulation of food intake and energy expenditure (1).
Many circulating signals, including gut hormones, can influence the activity of the arcuate
nucleus (ARC) neurons directly, after passing across the median eminence. The ARC is
adjacent to the median eminence, a circumventricular organ with fenestrated capillaries and
hence an incomplete blood–brain barrier (BBB) (2). The ARC plays a crucial role in the
regulation of food intake and energy homeostasis. The ARC contains two populations of
neurons with opposing effects on food intake (3). Medially located orexigenic neurons
express neuropeptide Y (NPY) and agouti-related protein (AgRP) (4, 5). Anorexigenic
neurons (i.e., those inhibiting appetite) in the lateral ARC express alpha-melanocyte-
stimulating hormone (α-MSH) derived from pro-opiomelanocortin (POMC) and cocaine and
amphetamine-regulated transcript (CART) (6). The balance between the activities of these
neuronal circuits is critical to body weight regulation.
In contrast, other peripheral signals influence the hypothalamus indirectly via afferent
neuronal pathways and brainstem circuits. In this context, the gastrointestinal vagal afferents
are activated by mechanoreceptors and chemoreceptors, and converge in the nucleus of
the tractus solitaries (NTS) of the brainstem. Neuronal projections from the NTS, in turn,
carry signals to the hypothalamus (1, 7). Gut hormones also alter the activity of the
ascending vagal pathway from the gut to the brainstem. In the case of ghrelin and peptide
tyrosine tyrosine (PYY), there is some evidence for a direct action of both on the ARC and
an action via the vagus nerve and brainstem.
The axons of these neurons project to “second-order” neurons, located in part in the
paraventricular nucleus (PVN), where the anorexigenic substances thyrotropin-releasing
hormone (TRH), corticotropin-releasing hormone (CRH), and oxytocin are secreted, and in
part, in the lateral hypothalamic (LH) and perifornical area (PFA), where the orexant
molecules melanin-concentrating hormone (MCH) and orexins are produced. When adipose
signals reach the ARC, anorexigenic peptides are released which activate a catabolic circuit.
In contrast, the activation of the anabolic pathway leads to the release of orexigenic peptides
and occurs when adiposity signal concentrations in the brain are low, thus indicating the
urgency to replenish fuel stores (8).
Peripheral Gut Hormones

Orexigenic peptides

Ghrelin
Ghrelin is a peptide consisting of 28 amino acids and is unusual among peptide hormones
in that Ser3 is n-octanoylated.
Ghrelin is present in X/A-like cells, which account for approximately 20% of the endocrine
cell population in adult oxyntic glands (9). Ghrelin-immunoreactive cells are also found in
the duodenum, jejunum, ileum, and colon. In the intestine, the ghrelin concentration
gradually decreases from the duodenum to the colon. This peptide is also secreted from
other organs such as the hypothalamus and pancreas of rats and, in addition, ghrelin mRNA
is expressed in various organs (10, 11). The best known factor for the regulation of ghrelin
secretion is feeding. The plasma ghrelin concentration increases when fasting, and
decreases after food intake. The factors involved in the regulation of ghrelin secretion have
not yet been identified. Blood glucose levels may be a most probable candidate; thus, oral
or intravenous administration of glucose decreasing plasma ghrelin concentrations exhibits
a nocturnal increase and are low in obese people and high in lean people (12–18).
Exogenous growth hormone (GH) decreases stomach ghrelin mRNA expression and
plasma ghrelin concentration, but does not affect stomach ghrelin stores (19).
The localization of ghrelin receptors on vagal afferent neurons in the rat nodose ganglion
suggests that ghrelin signals from the stomach are transmitted to the brain via the vagus
nerve (20, 21). In summary, ghrelin is secreted primarily from the stomach in response to
hunger and starvation, circulates in the blood and serves as a peripheral signal, informing
the central nervous system (via vagus nerve) to stimulate feeding. Ghrelin-containing
neurons are also found in the ARC of the hypothalamus, a region involved in appetite
regulation (1). In fact, intracerebroventricular (ICV) injection of ghrelin increases cumulative
food intake and decreases energy expenditure, resulting in body weight gain (21–24). To
stimulate the release of the orexigenic peptides, ghrelin-containing neurons send efferent
fibers onto NPY and AgRP-expressing neurons. On the other hand, to suppress the release
of the anorexigenic peptide, ghrelin-containing neurons send efferent fibers onto POMC
neurons. The ARC is also a target of leptin, an appetite-suppressing hormone produced in
AT (25). Leptin directly inhibits the appetite-stimulating effects of NPY and AgRP, whereas
hypothalamic ghrelin blocks leptin-induced reduction of feeding. Thus, ghrelin and leptin
have a competitive interaction in the regulation of feeding.
Ghrelin is only a hunger signal from peripheral tissues. Intravenous and sub-cutaneous
injections of ghrelin increase food intake; likewise, peripherally injected ghrelin stimulates
hypothalamic neurons and food intake (22, 23, 26–28). Because the rate at which peripheral
ghrelin passes the BBB has been shown to be very low, peripheral ghrelin must activate the
appropriate hypothalamic regions via an indirect pathway (29).
In addition, it has been reported that the enzyme ghrelin O-acyl transferase, essential for
ghrelin acylation, is modulated by nutrient availability, depends on specific dietary lipids as
acylation substrates. Thus, this mechanism may act as a nutrient sensor by using
absorbable fatty acids to signal to the brain that high caloric food is available, leading to
optimization of nutrient partitioning (30).

Anorexigenic agents
Peptide tyrosine tyrosine
Peptide tyrosine tyrosine has 36 amino acids, contains several tyrosine residues, and
requires C-terminal amidation for biologic activity.
Low levels of PYY are detected in enteroendocrine cells in the stomach, and levels increase
distally along the small and large intestine, reaching their highest levels in cells in the colon
and rectum (31). PYY is released from enteroendocrine L-cells lining the distal
gastrointestinal tract in proportion to caloric intake. Plasma levels of PYY rise within 30 min
of a meal and, in humans, circulating levels plateau at 1–2 h post-prandially, remaining
elevated for up to 6 h (32). Protein-rich meals cause the greatest increase in PYY levels
compared to other macronutrients (33, 34). The anorectic effects of PYY3–36 appear to be
mediated centrally via the ARC. PYY1–36 and PYY3–36 exert their effects through the NPY
family (35). PYY1–36 binds with similar affinity to NPY receptors. However, PYY3–36 is a
selective high affinity at the Y2 receptor subtype (Y2R) (36) thought to be the receptor
responsible for reduction of food intake by PYY. A vagal brainstem-mediated pathway may
also be involved in the actions of circulating PYY3–36. PYY is present in enteric nervous
plexus neurons innervating the gastrointestinal tract and the Y2R receptor has been
identified on the vagus nerve (37), the effects of PYY3–36 on satiety and central control of
appetite are clear. Most are mediated via anorectic neuronal populations in the ARC, but
vagal/brainstem-mediated pathways and peripheral effects of PYY on gastric emptying and
intestinal motility may also play a part.

Pancreatic polypeptide
Pancreatic polypeptide (PP) is an amidated 36-amino acid peptide that belongs to the “PP-
fold” family of peptides. It is released post-prandially under vagal control by pancreatic islet
PP cells (38). PP is comparable to other anorectic intestinal peptides such as PYY, being
secreted in proportion to caloric intake.
Pancreatic polypeptides bind to all the members of the Y receptor family, but have the
highest affinity for the Y4 receptor subtype (39). The effects of PP are likely to be mediated
by the ventromedial hypothalamus (VMH) and paraventricular hypothalamus (PVN) and
brainstem [area postrema (AP) and ARC] (40), in areas central to the control of appetite.
The anorectic effects of PP in humans appear to be independent of changes in gastric
motility (41).

Cholecystokinin
Cholecystokinin (CCK) is released post-prandially from the small intestine (42), and has also
been shown to co-localize with PYY in L-cells (43). It is released in response to saturated
fat, long chain fatty acids, amino acids, and small peptides that would normally result from
protein digestion (44, 45). CCK1 receptors are present in peripheral tissues such as the
pancreas, gallbladder, and on vagal afferent nerve fibers innervating the gut (46).
Furthermore, CCK1 receptors have been identified in areas within the CNS involved in the
regulation of food intake such as the NTS, AP, and dorsomedial hypothalamus (47). The
CCK2 receptor has a different distribution. It is found in the cortex, hypothalamus, vagal
afferents, and gastric mucosa, once again encompassing several areas known to be
involved in appetite regulation. In humans, intravenous administration of physiological doses
of CCK reduces food intake and increases the perception of fullness (48).
Leptin
Leptin is a 167-amino acid protein known to suppress appetite and regulate energy
expenditure. Leptin is secreted mainly by adipocytes (49), but has also been found in the
stomach (50) and the pituitary gland (51). Nevertheless, AT remains its main source
responsible for 95% of leptin production (51). Circulating leptin levels are positively
correlated with body mass index (BMI) and AT mass. Adipocytes possess large numbers of
GH receptors (GHR) and it is known that GH directly regulates leptin gene expression (52).
Furthermore, the production of leptin is influenced by several regulators, being stimulated
by insulin and blood glucose but inhibited by sympathetic activity, lipolytic catecholamines,
and free fatty acids (FFA). Leptin production correlates positively with AT mass (53) and is
independent of the adiposity. In these reason, leptin levels are higher in women than in men
(54). In humans, there is a highly organized pattern of leptin secretion over a 24-h period. In
general, the circadian pattern is characterized by basal levels between 08:00 and
12:00 hours, rising progressively to peak between 24:00 and 04:00 hours, and receding
steadily to a nadir by 12:00 hours (55). The nocturnal rise in leptin secretion is entrained to
mealtime probably due to cumulative hyperinsulinemia of the entire day (54). Leptin is
secreted in a regular pulsatile fashion with an interpeak interval of about 44 min, and the
circadian rhythm is attributable solely to increased pulse height. Circulating leptin is
transported across the BBB via a saturable process. Starvation reduces transport, whereas
refeeding increases the transport of leptin across the BBB (56, 57).
Leptin is transported across the BBB by a saturable transporter system (58) and exerts its
anorectic effect via the ARC, where both NPY/AgRP and POMC/CART neurons express
leptin receptors (59). Leptin inhibits NPY/AgRP neurons and activates POMC/CART
neurons (60, 61), resulting in reduced food intake. It also influences in the secretion of
neuropeptides engaged in energy homeostasis such as CRH, TRH, and BDNF (62–65).
Additionally, leptin stimulates the adrenergic system, thus increasing energy expenditure
(66). In addition to its interaction with central neural processes, there is evidence of a
synergy between leptin and the episodic satiety factor CCK discussed previously (67). Leptin
has been shown to enhance the satiating effect of CCK (68). In addition, leptin-induced
down-regulation of the hippocampal somatostatinergic system may potentiate its
anorexigenic effect (69).

Amylin
Amylin is a 37-amino acid peptide, also known as islet amyloid polypeptide. In mammals,
amylin is co-released with insulin from pancreatic β-cells in response to food intake and has
an anorectic effect (70). Amylin seems to decrease food intake through both central and
peripheral mechanisms and, indirectly, by slowing gastric emptying. The AP plays a
predominant role in peripheral amylin’s satiating effect, involving a direct activation of AP
neurons by blood-borne amylin. Its anorectic effect may, in part, be due to reduced
expression of orexigenic neuropeptides in the LH area (71). There is evidence that amylin
may also exert its effects through serotonergic, histaminergic, and dopaminergic systems.

Insulin
Insulin is produced in β-cells of the pancreatic islets of Langerhans and enters the brain from
the circulation acting on hypothalamic neuron located mainly in the ARC, to reduce energy
intake. There is also a specific insulin transport across the BBB regulated by a saturable
mechanism that involves insulin receptors in brain microvessels. ICV infusion or systemic
injection of insulin results in a dose-dependent suppression of food intake. Central action of
insulin promotes anorexia because it decreases NPY and stimulates POMC expression (72).
Insulin binds to its receptors highly expressed in POMC/CART and NPY/AgRP neurons.
Insulin and leptin both activate POMC neurons, but they seem to differentially regulate
AgRP, with leptin inhibiting and insulin stimulating its synthesis (73). Insulin deficiency is
associated with increased NPY, while insulin administration inhibits hypothalamic NPY
expression.

Glucagon-like peptides
Pre-proglucagon gene expression is limited to α-cells in the pancreas, L-cells in the gut, and
neurons in the brain stem nucleus of the NTS. Whereas post-translational processing of
proglucagon in the pancreas leads to the formation of glucagon and the major proglucagon
fragment, proteolytic cleavage in the L-cells of the gut and in the NTS yields the peptides
glicentin, oxyntomodulin (OXM), glucagon-like peptides 1 and 2 (GLP-1 and -2) (74). After
a meal, GLP-1 and GLP-2 are secreted in parallel in the circulation. GLP-1, is perhaps best
known as a gut-derived incretin hormone. GLP-1 is a 30-amino acid peptide and one of
several cleavage products of the pre-proglucagon gene. It is secreted by the
enteroendocrine L-cells of the distal intestine in response to incoming nutrients (75). GLP-1
is also a neurotransmitter synthesized by a small population of neurons in the NTS in the
caudal brainstem (76). Food ingestion promotes the release of GLP-1 from L-cells in the
intestine, which activates vagal afferents. (77). GLP-2 is a peptide highly conserved across
different mammalian species. Its main biological effects are related to the regulation of
energy absorption and maintenance of mucosal morphology, and function and integrity of
the intestine. In the gastrointestinal tract, GLP-2 increases the uptake of luminal nutrients,
including sugars and lipids, by augmenting the activity and expression of nutrients
transporter. Its influence on appetite regulation is unclear but recent studies have shown
that intraperitoneal injection of GLP-2 reduces food intake in mice (78).

Oxyntomodulin
Oxyntomodulin is a 37-amino acid peptide released post-prandially from L-cells in proportion
to caloric intake. OXM (79) causes a reduction of neuronal activity in the ARC, PVN, and
supraoptic nucleus. This pattern of activation is distinct from that of GLP-1 under the same
conditions (80), implying that these two hormones act via different hypothalamic pathways.
OXM reduces food intake in normal weight human volunteers when administered
intravenously or subcutaneously (81). There is evidence that OXM may increase energy
expenditure in humans (82).

Bombesin
Bombesin is a tetradecapeptide that was isolated from amphibian skin and is similar in
structure to mammalian gastrin-releasing peptide (GRP) and neuromedin B (83, 84).
Bombesin (85, 86) and GRP administration (87) decrease food intake in lean human
subjects but not in obese women (88). Peripheral or central injection of bombesin reduces
food intake that is not blocked by vagotomy (89, 90). Bombesin also activates the
sympathetic nervous system (91). In animals that have been starved or have ventromedial
hypothalamic lesions, bombesin produces a profound drop in temperature because the
sympathetic nervous system cannot be activated (91, 92).
Obestatin
Recently, it has been demonstrated that preproghrelin undergoes additional proteolytic
cleavage, generating a 23-amino acid peptide, which has been named obestatin. In contrast
to ghrelin, obestatin has anorexigenic effects, reduces gastric emptying, inhibits jejunal
contractions, and suppresses body weight gain (93). However, several recent studies
performed in rats and mice under various experimental conditions did not reproduce these
results (94, 95). Pan et al. (96) reported that obestatin is unable to cross the BBB and is
rapidly degraded in the circulation; this was confirmed by Vergote et al. (97). An alternative
hypothesis is that obestatin exerts its effects on eating and drinking through direct
interactions with the gastrointestinal system. Indeed, Zhang et al. (98) observed decreased
contractile activity of jejunum muscle strips in vitro and suppression of gastric emptying in
vivo after obestatin treatment. Thus, the inhibition of jejunal contraction could generate an
afferent vagal signal to induce satiety in the brain. Recently, Fujimiya et al. (99) supposed
that obestatin may act on the obestatin receptor on vagal afferent nerve terminals, and
corticotropin-releasing factor (CRF) and urocortin-2 neurons in the hypothalamus may
mediate the action of obestatin to inhibit the gastroduodenal motility via CRF1-R and CRF2-
R in the brain (100).

Central Hypothalamic Peptides

The hypothalamus contains several important nuclei that are associated with energy
homeostasis and feeding regulation. The LH is a feeding center, the VMH is the satiety
center, and the ARC is an integrated center for feeding regulation.

Hypothalamic orexigenic peptides

Neuropeptide Y
The ARC is the major site of expression for NPY within neurons in the hypothalamus that
project to PVN, dorsomedial hypothalamus (DMH), LH, and other hypothalamic sites.
Although NPY can produce diverse effects on behavior and other functions, its most
noticeable effect is the stimulation of feeding after central administration (101). NPY
synthesis in the ARC and its release into the PVN, the most abundant projection, is regulated
by inhibitory afferent signals such as leptin and insulin and stimulatory signal as
glucocorticoids. The NPY neurons are potential hypothalamic targets for leptin and inhibition
of the synthesis, and probably release of NPY seems to partly explain the ability of leptin to
induce hypophagia and weight loss. Insulin has been shown to inhibit NPY synthesis and
secretion in the PVN. Five G-protein-coupled NPY receptors have been identified – Y1, Y2,
Y4, Y5, and Y6. Y5 receptors have been implicated as important receptors that mediate the
feeding effects of NPY (102, 103). The Y5 receptor is expressed at relatively high levels in
the LHA, close to the site where NPY acts most potently to stimulate feeding (104).

Agouti-gene related protein

Agouti-gene related protein is a 132-amino acid peptide. Within the CNS, AgRP is expressed
exclusively in the ARC and AgRP mRNA co-localizes with NPY mRNA in 95% of NPY
positive cells in this nucleus (105). Rossi et al. have shown that like NPY, AgRP is an
orexigenic peptide when injected ICV (106) or directly into the PVN or DMH (107). Uniquely,
AgRP acts as an endogenous antagonist of the melacortin-3 (MC3R) and melacortin-4
receptor (MC4R) (108). It is likely AgRP plays a modulatory role in feeding. It may be that
AgRP is more important during conditions of high energy requirements, such as pregnancy
and lactation, under which it has been shown to be more highly expressed (109).

Melanin-concentrating hormone
Melanin-concentrating hormone is an orexygenic cyclic 19-amino acid neuropeptide. Within
the hypothalamus (MCH), it is highly expressed in the LH and zona incerta (110) and has
orexigenic effects after ICV infusion (111). Interest regarding the effector mechanisms by
which MCH is orexigenic has largely focused on the MCHR1 receptors in the nucleus
accumbens shell (AcbSh) where MCH injection decreases neuronal firing in medium spiny
neurons (112). The nucleus accumbens is thought to be involved in motivational aspects of
eating.

Hypocretins/orexins
The hypocretins (1 and 2; also known as orexins A and B) are excitatory neuropeptides that
are produced in cell bodies of the LH area, but have extensive projections to many regions.
The hypocretin/orexins bind to orexin receptor 1 and 2 (OXR1 and OXR2), which arise from
two separate genes. The distribution of the two receptors is different. Within the
hypothalamus, OX1R is the most highly expressed in the PVN (113).
Orexins are appetite-stimulating neuropeptides. Orexin neuronal cell bodies are present in
the LH and DMH, and orexin-containing neuronal fibers are distributed in several nuclei, with
abundant projections to the ARC. Orexin-containing neurons project to NPY-containing
neurons in the ARC, and NPY neurons express the OX1R (114). Furthermore, orexins
increase the cytosolic Ca2+ concentration in NPY neurons isolated from the ARC (115).
These results indicate that NPY neurons receive excitatory signals from orexin-containing
neurons in the LH. The distribution of the two receptors is also different; within the
hypothalamus, OX1R is most highly expressed in the VMH and OX2R is most highly
expressed in the PVN (113).

Galanin
Galanin is a 29-amino acid C-terminally amidated (30 amino acid, non-amidated in humans),
found in the brain and the gut. Galanin coexists with GABA, noradrenaline, 5-
hydroxytryptamine (5-HT), and NPY in several regions of the brain. Hypothalamic galanin
neurology is found largely in the PVN, supraoptic nucleus of hypothalamus (SON), and ARC.
Many galanin-positive fibers as well as galanin-positive neurons have been demonstrated
in the dorsal vagal complex, suggesting that galanin produces its effects by involving vagal
neurons. The nucleus of the solitary tract is the major source of the galanin terminals in the
dorsal vagal complex. There are two cloned galanin subtype receptors: GalR1 and GalR2
are majorly distributed in the hypothalamus, PVN, amygdale, hippocampus, brainstem,
spinal cord, peripheral nervous system, and other tissues (116). This peptide participates in
modulating learning, memory, feeding, inflammation, pain threshold control, sexual
behavior, insulin, and pituitary hormone release (117–119). Acute central administration of
galanin has been reported to increase fat consumption.

Galanin-like peptide
Galanin-like peptide (GALP) is a novel 60-amino acid peptide, with residues 9–21 being
identical to the biologically active N-terminal (1–13) portion of galanin (120). In
situ hybridization studies have shown that GALP mRNA is distributed within the
periventricular regions of the ARC (121, 122) in the median eminence, and in the pituitary
gland of the rat (123). NPY-containing axon terminals are closely apposed (opposed) to
GALP-containing neurons in the ARC (124). Moreover, Cunningham et al. (125)
demonstrated, using double-label in situ hybridization, that GALP-containing neurons in the
macaque expressed the NPY Y1 receptor, suggesting that NPY regulates GALP neurons in
the ARC. However, whether NPY activates GALP is yet to be determined.

Cerebellin1
Cerebellin1 (Cbln1) is highly expressed in the hypothalamus. ICV administration of Cbln1
increases food intake and the release of NPY from hypothalamic explants and reduces
plasma thyroid-stimulating hormone (TSH) levels after postinjection in rats without adverse
behavioral effects. Cbln1 mRNA expression levels were increased in the ventromedial
nucleus of the hypothalamus in fasted rats. These data suggest that Cbln1 is a novel
orexigenic peptide, which may mediate its effects via hypothalamic NPY (126).

Hypothalamic anorexigenic peptides

Cocaine and amphetamine-regulated transcript

Cocaine and amphetamine-regulated transcript is a neuropeptide which appears to be a
powerful physiological anorexic signal. CART mRNA was identified on the basis of its
increase following cocaine or amphetamine treatment in rats (127). CART peptide is
localized in specific areas of the hypothalamus including the periventricular nucleus,
dorsomedial nucleus, perifornical regions, lateral nucleus, and the ARC. In the PVN, CART
mRNA is co-localized with vasopressin and CRF-containing neurons (128).

Melanocortins
The melanocortins are bioactive peptides derived from the precursor molecule POMC via
tissue-specific post-translational cleavage (56). The POMC gene is expressed at
physiologically significant levels in a number of mammalian tissues including anterior and
intermediate pituitary, skin, the immune system, and hypothalamic neurons. The repertoire
of products derived from POMC by any tissue is determined by the specificities of the
convertases expressed in the tissue (129, 130). The intermediate lobe of the pituitary yields
α-melanocytes-stimulating hormone (α-MSH), a peptide which activates melanocortin (MC)
3 and MC4 MC receptors and inhibits food intake. The MC3R and MC4R receptors are found
in areas known to be involved in regulation of energy balance, but also in other regions such
as cerebral cortex and hippocampus. Bioactive peptides generated in hypothalamic neurons
act as endogenous ligands for the MC4R, a key molecule underlying appetite control and
energy homeostasis (131).

Glucagon-like peptides
In the brain, release of GLP-1 within the nucleus of the solitary tract NTS and from
projections of GLP-1 neurons to the PVN leads to GLP-1 receptor activation, which
promotes satiety and anorexia. Activated GLP-1 neurons of the NTS also project to the ARC
to modulate vagal motor outflow to the pancreas and other tissues not depicted, increasing
insulin secretion from the β-cells in states of hyperglycemia and suppresses glucagon from
the α-cells, leading to lowering of blood glucose. Systemic GLP-1 may also access the brain
via leaks in the BBB such as the subfornical organ and the AP, as demonstrated to occur in
rats. The intravenous administration of GLP-1 to normal and obese humans decreases food
intake in a dose-dependent manner (132) as well as reducing gastric emptying (133, 134).
These effects are thought to be mediated through vagal and brainstem pathways since
peripheral administration of GLP-1 activates neurons within the brainstem in rats (1, 135).
The distribution of the co-localized peptide GLP-2 displays a perfect overlap with GLP-1 in
the CNS, with the highest concentration in the diffuse ventral part of the dorsomedial nucleus
(76, 136). When injected into the lateral ventricle, GLP-2 has a marked inhibitory effect on
feeding. The effect of GLP-2 on feeding is both behaviorally and pharmacologically specific
(76). The CNS GLP-2R is essential for the control of feed behavior. Glp-2r deletion in POMC
neurons increases food intake with amplified meal frequency and accelerates gastric
emptying, suggesting that CNS GLP-2 is a key satiety signal for the physiological short-term
control of feeding behavior and gastric motility and contributes to the long-term homeostatic
control of energy balance (or body weight). Moreover, activation of GLP-2R signaling
suppresses food intake and gastric emptying through the MC4R signaling pathway. Guan
et al. (137) findings suggest that gastric emptying is a key process for the short-term control
of feeding behavior and POMC neuron-mediated suppression of food intake may be
executed through decelerating gastric emptying (137).

Corticotropin-releasing factor
Corticotropin-releasing factor or CRH is a 41-amino acid mammalian neurohormone that is
best known as the major physiological regulator of pituitary ACTH secretion. CRH is highly
expressed in PVN neurons and, when centrally injected, inhibits food intake and reduces
body weight in rats. Peripheral administration of human CRH increases energy expenditure
and fat oxidation in humans. Leptin infusion stimulates CRH expression, while pretreatment
with a CRH antagonist attenuates the leptin-induced reduction of food intake and body
weight.

Neurotensin
Neurotensin (NT) is a 13-amino acid peptide. NT is produced in the ARC, PVN, and DMH
of the hypothalamus and its microinjection into the PVN decreases food intake. NT neurons
appear to play an anorectic role downstream of leptin as ICV leptin infusion into the PVN
stimulates NT synthesis in association with reduced food intake (138, 139). These results
suggest that leptin action may be mediated, at least in part, by NT.

Nesfatin-1
In the early 1990s, a protein was identified in mouse (140) and human cell lines (141) and
termed nucleobindin or NEFA (DNA binding/EF-hand/acidic amino acid-rich region). Until
now, two nucleobindins have been identified, namely nucleobindin1 (NUCB1) and
nucleobindin2 (NUCB2 or NEFA). NUCB2 contains a 24-amino acid N-terminal signal
peptide and a 396-amino acid sequence that is highly conserved in rodents and humans
(142), pointing toward its physiological relevance. NUCB2 was localized on the plasma
membrane and in the cytoplasm.
In 2006, Oh and colleagues (143) were the first to describe that putative post transcriptional
processing of NUCB2 by the enzyme pro-hormone convertase (PC)-1/3 results in nesfatin-
1 (amino acid 1–82), nesfatin-2 (amino acid 85–163), and nesfatin-3 (amino acid 166–396)
(143). So far, the biological activity has only been demonstrated in nesfatin-1 and the
fragment nesfatin-124–53.
The initial report described the expression of NUCB2 mRNA substantiated by nesfatin-1
inmmunohistochemistry in rat hypothalamic and brainstem nuclei involved in the regulation
of ingestive behavior, such as the PVN, supraoptic nucleus, ARC, LH, zona incerta, and
NTS (143). Nesfatin-1 inmmunopositive neurons co-localize with a number of brain
transmitters (8, 12–17).
Nesfatin-1 directly inhibits ARC neurons containing NPY. A recent study provided
compelling evidence for the involvement of an oxytocin pathway in the inhibitory effect of
nesfatin-1 on food intake (144). Nefastatin-1 is also likely to act in series through the
recruitment of the central MC and corticotrophin-releasing factor 2 (CRF2) signaling systems
to reduce food intake. The anorexic action of peripheral nesfatin-1/NUCB2 may be mediated
by vagal afferents projecting to the nucleus of the solitary tract in addition to a potential
hormonal action via crossing of the BBB (145).
Central nesfatin-1/NUCB2 mediates its anorexigenic effect via activation of
melanocortin3/4 and CRF2 signaling and also by hyperpolarizing neurons containing the
orexigenic peptide, NPY. Nesfatin-1 also activates the hypothalamic magnocellular
oxytocinergic system, which could reduce food intake and delay gastric emptying. Peripheral
nesfatin-1 can reach the brain via the circulation and crossing of the BBB and/or a direct
action on circumventricular organs as well as on the modulation of vagal afferent activity.
Immunostaining in the peripheral tissues confirmed the expression of nesfatin-1/NUCB2
protein in the rat stomach and, additionally, in pancreatic endocrine islets of Langerhans,
testis, and pituitary gland. Similarly, nesfatin-1-immunopositive cells of the endocrine
pancreas exclusively co-localize with insulin in β-cells (146, 147). These findings suggest a
differential release of nesfatin-1 and ghrelin from the stomach and nesfatin-1 and insulin
from the pancreas, which warrants further investigation. The prominent and exclusive
endocrine distribution of nesfatine-1/NUCB2 in cells of the stomach and pancreas support
the fact that nesfatin-1 may act as a gut–brain peptide to influence food intake and glucose
homeostasis. The anorexic action of peripheral nesfatin-1/NUCB2 may be mediated by
vagal afferents projecting to the nucleus of the solitary tract, in addition to a potential
hormonal action through crossing of the BBB (145).

Pituitary Hormones

Vasopressin
Vasopressin significantly reduces food intake over a 4 h period in experimental animals. The
reduction in food intake, particularly in the first 30 min of feeding, is not significantly impaired
by vagotomy, suggesting that its peripheral mechanism of action is different from that of
CCK or enterostatin (148).

Melanocyte-stimulating hormone
In yellow mice, that overexpress AgRP, treated with melanocyte-stimulating hormone (MSH)
there is a substantial increase in food intake and weight gain which is 30–100 times greater
than that of the acylated form (α) of MSH. In contrast, injection of αMSH produces a much
more potent darkening of the melanocyte than does dMSH.

Growth hormone
Following treatment with GH, hypophysectomized animals increase their food intake and
growth. This finding could be a direct effect of GH on feeding centers or a may be due to a
second stimulation by an enhanced flux of amino acids into new proteins that leads to an
increase in feeding.
Growth hormone stimulates lipolysis in the AT and, particularly, in the visceral and sub-
cutaneous depots (149–152). Hormone-sensitive lipase (HSL or LIPE) is a crucial enzyme
implicated in this process. GH may also modulate the expression of the lipid droplet
associating protein, CIDE-A (cell-death-inducing DFF45-like effector). CIDE proteins have
been associated with lipid droplets, where they facilitate lipid accumulation and inhibit
lipolysis. Unlike the AT, GH induces FFA uptake into skeletal muscle by up-regulation of
LPL expression (153, 154). The re-sterification of triacylglycerides (TAG) from FFAs results
in generation of intermediates such as diacylglycerol and ceramides that activate PKC
isoforms. PKC can down-regulate insulin signaling by several mechanisms (155, 156). GH
secretion is diminished in obesity, where increased FFA levels may have a suppressive
effect on GH secretion. The hyperinsulinemia associated with this pathological situation may
also contribute to decreased GH secretion (156). Thus, in this manner, the GH-induced
increase in FFA uptake and TAG synthesis could result in insulin resistance. These data
also suggest that GH induces a shift in substrate utilization from glucose to lipids in the
skeletal muscle.

Implications for Obesity and Metabolic Syndrome

Obesity
The etiology of obesity is believed to be extremely complex and includes a combination of
excess dietary calories and decreased physical activity, coupled with either some
predisposing genetic factors or metabolic disorders (157–159).

Ghrelin
Although the role of ghrelin in the etiology of obesity is not understood, it is considered a
vital target because of its capacity to induce a positive energy balance state (160, 161).
Supporting the relevance of the ghrelin pathway regarding obesity, recent studies by Wortley
et al. and Zigman et al. (162, 163) show that the absence of both ghrelin or its receptor GHS-
1a protects mice against diet-induced obesity. In addition, ghrelin immunization in rats has
been reported to reduce body weight gain (164) and catalytic anti-ghrelin antibodies in
C57BL/6 mice reduce refeeding for 6 h after a 24-h starvation and maintain high levels of
energy expenditure (165). Nevertheless, the immunization against ghrelin failed to cause
long-term body weight reduction (166) and some studies with mice from a pure C57BL/6
background knockout for ghrelin or ghrelin receptor suggest only negligibly small differences
in food intake and body weight under caloric restriction or a high-fat diet compared with wild-
type mice (167). Finally, the absence of ghrelin in ob/ob mice does not seem to decrease
food intake or body weight in this mouse model, although lowering blood glucose
substantially (168, 169).

Leptin
Obese patients with three risk factors for metabolic syndrome have lower leptin levels (170).
Mutations in the leptin receptor (Ob-R) gene are responsible for monozygotic obesity in
rodents and humans. In obesity, there is also a defective transport of leptin across the BBB,
which suggests the existence of central leptin resistance (171). This resistance could be
inherited or secondary to obesity, associated with a less efficient transport of leptin via the
BBB or abnormalities in leptin signaling (171).

Insulin
Insulin is a signal of satiety and obesity (172). Reduced expression or deletion of insulin
receptors in the brain leads to hyperfagia and obesity (173).

Hypothalamic–pituitary–adrenal axis
There is a neuroendocrine integration of the stress centers in the CNS with centers that
control appetite (174). Acute stress exerts an anorexigenic effect though stimulation of
POMC/CART neurons by increased CRH levels and an additional decrease of NPY
secretion (174, 175). CRH activates the hypothalamic–pituitary–adrenal axis with an
increase in cortisol secretion which, in turn, inhibits the activation of the HPA axis. This is
responsible for the anorexigenic effect of glucocorticoids in the case of acute stress. Chronic
stress is associated with chronic activation of the HPA axis and prolonged glucocorticoid
secretion. Chronically elevated levels of glucocorticoids exert orexigenic effects caused by
inhibition of CRH and stimulation of NPY expression (174, 175). Several studies have also
shown increased responsiveness of the HPA axis in obese patients to different stimuli and
in a dynamic test with neuropeptides and small doses of dexamethasone (176). Abdominal
obesity is also associated with attenuated negative feedback in the HPA axis (173).

Growth hormone
Growth hormone secretion is consistently reduced in obesity (177, 178). Consequently, low
GH secretion could further contribute to accumulation of abdominal fat (156). Obesity-
induced hyperinsulinemia, hypoadiponectinemia, leptin resistance, and increased bioactive
insulin-like growth factor-1 (IGF-1) and FFA levels could suppress GH secretion from the
pituitary by various mechanisms mentioned above. Reduced GH secretion further increases
fat accumulation and, thus, exacerbates the obesity condition. Moreover, reduced GHR
expression and increased expression of truncated GHR (ΔGHR) in the AT results in a GH-
resistant state that also contributes to the complications associated with obesity (156).

Thyroid hormones
The hypothalamic–pituitary–thyroid (HPT) axis may play a direct role in appetite regulation.
Hypothyroidism reduces energy expenditure and causes weight gain, while hyperthyroidism
exerts the opposite effect (179). TRH, after peripheral or central administration, exerts a
direct anorexigenic effect (180). Similarly, central administration of TSH causes inhibition of
food intake in rats. Triiodothyronine (T3) which directly stimulates food intake at the
hypothalamic level, can also cross the BBB and reach the CNS directly and then be
transported to the ARC (181). Peripheral administration of T3 increases NPY and reduces
POMC expression (179). Direct injection of T3 into the VMN also exerts an orexigenic effect
in rats (179).

Metabolic syndrome
Metabolic syndrome is due to cluster of cardiovascular risk factors that includes obesity,
hypertension, insulin resistance, and glucose and lipid metabolic abnormalities (182, 183).
Metabolic syndrome is associated with an increased risk of cardiovascular disease (CVD)
and type 2 diabetes mellitus, even before the development of glucose intolerance
(169, 183, 184).

Galanin
The effect of the galanin peptide family on the metabolic syndrome includes increased food
consumption and the preference for a high-fat diet, which elevates the probability of obesity
and dyslipidemia, and decreased insulin resistance and blood pressure to relieve the risk
for type 2 diabetes mellitus and hypertension (185).

Adiponectin and leptin

Adiponectin, an anti-atherogenic and anti-inflammatory adipocytokine involved in glucose
and lipid metabolism, improves insulin sensitivity (186). Lower levels of adiponectin were
observed in patients with high blood pressure, hyperglycemia, low HDL-C, and
hypertriglyceridemia, and in obese patients with MS (187). Brooks et al. showed that a low
level of circulating adiponectin may be used as a possible biomarker for MS (188). Leptin,
an anti-obesity adipocytokine, regulates body weight by modifying energy levels and
increasing the metabolic rate while decreasing food intake. Most overweight and obese
patients show resistance to leptin at the receptor level, and therefore, have higher leptin
levels than non-overweight individuals (189). Serum leptin levels in patients with MS are
higher than those in healthy controls (190). Adiponectin and leptin levels show an inverse
correlation with each other (191).
García-Cardona et al. studied the correlation between obesity and insulin resistance and
methylation frequency of the leptin and adiponectin promoters in obese adolescents, with
the aim of identifying epigenetic markers that might be used as tools to predict and follow-
up the physiological alterations associated with the development of the metabolic syndrome.
Obese adolescents without insulin resistance showed higher and lower circulating levels of
leptin and adiponectin, respectively, along with increased plasmatic concentrations of insulin
and triglycerides. The methylation frequency of CpG sites located at −51 and −31 nt relative
to the transcription start site of the leptin gene dropped dramatically in obese adolescents
with insulin resistance (192).

Neuropeptide Y
Bray et al. studied positive association of an NPY gene variant (880I/D) with obesity in
Mexican-American families (193). Additionally, there have been many studies examining the
functional Leu7Pro polymorphism (rs16139). This single nucleotide polymorphism (SNP)
has been associated with a large number of conditions related to obesity and metabolic
syndrome traits, including increased BMI in adults (194), development of obesity in young
adults (195), risk of hypertension (196), high plasma low-density lipoprotein-cholesterol
(LDL-c) in children and adults (196, 197), and elevated plasma TAG (198). This variant has
been associated with metabolic syndrome in patients with coronary artery disease (199).
This SNP has also been shown to correlate with high birth body weight in preschoolers, the
risk of an accelerated atherosclerotic process or carotid atherosclerosis in adults (196, 200),
and the risk of type 2 diabetes mellitus in adults (201). Additionally, Josune-Olza et al.
validated the association between the SNPs NPY rs16147 genotype and BMI in Spanish
children, observing higher BMI values in TT homozygotes as compared with heterozygous
C allele carriers (202).
Pro-opiomelanocortin
Yoo et al. (203) demonstrated that high POMC methylation in cord blood was associated
with lower birth weight and children with high POMC methylation in cord blood showed
higher TAG and higher insulin concentrations in blood. Thus, POMC methylation status in
cord blood may be an early predictive marker of future metabolic syndrome.

Conclusion
The control of energy balance depends critically on the CNS. The various CNS regions that
control energy homeostasis are accessible to numerous circulating factors discussed above.
Within these central locations are specific neuronal populations that recognize these signals
and act in the network to integrate the multiple inputs, and help to regulate appetite (68). In
particular, the hypothalamus is a center of integration of several nutrient signals. It can sense
and integrate variations in adiposity and gastric hormones, as well as nutrients, and also
receives neuroanatomical projections from other nutrient sensors, mainly within the
brainstem. In addition, it also integrates these signals with cognitive forebrain-descending
information to coordinate neuroendocrine, behavioral, and metabolic effectors of energy
balance.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or
financial relationships that could be construed as a potential conflict of interest.

Acknowledgments
This work was supported by grants from Ministerio de Ciencia y Tecnología (SAF 2010-
22277), CIBERobn (CB03/06), Fundación Endocrinología y Nutrición, and by Fondo de
Investigación Sanitaria PI13/02195.

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Nutrients. 2014 Jan; 6(1): 231–248.

V. The Mediterranean Diet and Nutritional Adequacy: A

Review
Itandehui Castro-Quezada,1 Blanca Román-Viñas,2,3 and Lluís Serra-Majem1,2,*

Abstract

1. Introduction
The Mediterranean diet is known to be one of the healthiest dietary patterns [1]. The
Mediterranean diet is a plant-based pattern, where vegetables, fruits, cereals (preferably as
whole grain), legumes, and nuts should be consumed in high amount and frequency. The
Mediterranean dietary pattern (MDP) also includes moderate consumption of fish and
shellfish, white meat, eggs, and dairy products. On the contrary, consumption of red meat,
processed meats, and foods rich in sugars and in fats should be small in both quantity and
frequency. The principal source of dietary lipids of the MDP is olive oil and an adequate daily
intake of water should be guaranteed, as well as moderate consumption of wine is
recommended. Seasonality, biodiversity, the use of traditional and local food products are
also important elements in this pattern. In addition, the Mediterranean diet has also
qualitative cultural and lifestyle elements, such as conviviality, culinary activities, physical
activity, and adequate rest [2]. It encloses a beneficial fatty acid profile with a high content
of monounsaturated fatty acids (MUFA) and a higher MUFA/saturated fatty acids (SFA) ratio
than non-Mediterranean diets [3,4]. High consumption of dietary fiber [5], low glycemic index
and glycemic load [6], anti-inflammatory effects [7], and antioxidant compounds [8,9], may
act together to produce favorable effects on health status.
The Mediterranean Diet is associated with a lower incidence of mortality from all-causes
[10,11,12], and is also related to lower incidence of cardiovascular diseases [13], type 2
diabetes [14], certain types of cancer [15], and neurodegenerative diseases [10,11].
Finding a dietary pattern that fulfills the nutritional requirements of a population is a priority
in order to establish nutritional recommendations [16]. Nutritional adequacy is defined as the
sufficient intake of essential nutrients, needed to fulfill nutritional requirements for optimal
health. According to the criterion of adequacy defined, the requirement for a given nutrient
may be at a lower or higher intake amount. The criteria that are generally used to define
adequacy of intake are: the prevention of deficiency diseases, the prevention of chronic
diseases or the reduction of risk for diet associated diseases, subclinical nutritional health
conditions identified by specific biochemical or functional measures, or requirements to
maintain physiological balance [17]. Nutritional adequacy emerges from the comparison
between the nutrient requirement and the intake of a certain individual or population. As
neither the real intake nor the real requirement for one individual is known, the assessment
of nutrient intake adequacy of an individual or population is based on the probability of
adequacy [16].
The Mediterranean diet used to be sufficiently caloric and rich in vitamins and minerals,
derived from vegetables and fruits, whole-meal cereals, nuts, virgin olive oil and fish, which
made the risk of deficient micronutrient intakes quite infrequent. This explains why
inadequate intakes of the B group vitamins (B1, B2, niacin, B6, folates, or B12) were rare in
the Mediterranean basin, and intakes of antioxidant vitamins (vitamins E and C) and
carotenes were also high [18,19]. However, people from Mediterranean countries are
changing the traditional Mediterranean diet and include low nutrient dense foods (such as
sugared soft drinks, sweets, bakery products, salted snacks) or vary their food processing
methods (such as refinement of flour) towards a less healthy diet. These changes may have
contributed to an increased risk of deficient intakes for some vitamins, especially folates,
vitamins A and D, as well as inadequate intakes for the rest of the vitamins, in particular
among certain population groups or collectives [18,19].
Nutritional adequacy may be used to determine the risk of deficiency of the nutrient
assessed, in terms of low intakes or high intakes (for instance, the adverse effects of high
levels of sodium intake may be applicable to reducing the risk of certain chronic diseases or
conditions, such as hypertension) [20]. However, the complexity of the relationships
between dietary intake and the pathology cannot be attributed to a single nutrient but, rather,
to multiple nutrients and foods. Thus, the correct exposure has to be measured to
understand such a relationship, and not only nutrients, but also foods, and the interaction
between them, are of concern for this kind of evaluation. Food pattern analysis, such as the
MDP, is then a key issue to investigate the linkages between nutrition and disease [21].
The objective of this study was to review the available evidence on the Nutritional Adequacy
of the Mediterranean diet, its assessment, the general nutritional adequacy in different
European and Mediterranean countries, and compared to a Western dietary pattern, as well
as in children.

2. Methods
A scientific literature search was conducted on MEDLINE (National Library of Medicine,
Bethesda, MD, USA) for relevant articles about the Mediterranean diet and nutritional
adequacy published from January 2000 to June 2013. We used the keywords
“Mediterranean diet”, “pattern”, “adequacy”, “nutritional”, “nutrient”, “intake”, “assessment”,
and combinations, such as “Mediterranean diet and nutritional adequacy” or “Mediterranean
diet and nutrient adequacy”. We narrowed the search to studies published in English, and
limited to those conducted in humans. We focused the search on articles referring to the
Mediterranean diet as a whole and excluded studies regarding specific foods of this diet.
We limited the search to studies published in English and to those conducted in humans.
Additional publications were identified from references provided in original papers. Only 15
articles were finally selected for this review.

Nutritional Adequacy Assessment

The quality of the diet can be estimated in terms of food or food group intakes and diet
patterns, or in terms of nutrient intake and the level of compliance with the nutrient
requirements. When evaluating the diet in terms of nutrient adequacy, diverse types of
analyses are used. The method used depends on the purpose of the analysis (to assess
individuals or a population), on the nutrient under study and the type of distribution of the
nutrient intake [22,23]. Recommendations used for the comparison will be country specific
and evidence based.
Most countries around the world recommend nutrient intake values for their populations but
the amount of nutrients and the terms used to describe the requirement vary between them
[24]. To avoid confusion, a standardized terminology of these recommendations has been
proposed by a group of experts of the United Nations University (UNU), in collaboration with
the Food and Agriculture Organization (FAO), the World Health Organization (WHO), and
the United Nations Children’s Fund (UNICEF). The expert group proposed to use the
Nutrient Intake Values (NIVs) as a common set of terms and definitions in all countries
[24,25,26,27]. The NIVs encompass the following terms: the Average Nutrient Requirement
(ANR), the Individual Nutrient Level (INLx), and the Upper Nutrient Level (UNL) (Figure 1)
[28].

Figure 1
Graph of Nutrient intake values and the risk of nutrient inadequacy or excess a. This image
shows: (1) Average Nutrient Requirement (ANR); (2) Individual Nutrient Level (INLx); and
(3) Upper Nutrient Level (UNL). a Adapted from: Institute of Medicine. Dietary Reference
Intakes: The Essential Guide to Nutrient Requirements; National Academy Press:
Washington, DC, USA, 2006 [20].
The Average Nutrient Requirement (ANR) is defined as the average or median usual intake
value that is estimated to meet the requirement for a specific criterion in a life stage and
gender group. The ANR is equivalent to the Estimated Average Requirement (EAR) used
by the Institute of Medicine (IOM-USA). The INLx is the recommended nutrient level for all
healthy individuals in a specific subpopulation. The committees frequently add 2 SD to ANR,
which will cover the needs of most of the population (i.e., 98%), assuming that the
distribution is symmetrical. The INL98 is equivalent to the Recommended Dietary Allowance
(RDA) used by the IOM. Finally, most nutrients will have an Upper Nutrient Level (UNL),
which is the highest intake that can be daily tolerated without risk of adverse health effects
[28,29]. The term employed by the IOM is Tolerable Upper Intake Level (UL). Finally, an
Adequate Intake (AI) is estimated if there is not enough scientific evidence to establish
values for ANR and INLx. The AI has been included in IOM recommendations, but not in the
standardized terminology proposed by the UNU [29]. These nutritional requirements are
applied both to the nutritional assessment and to the planning of dietary interventions on an
individual- and population-based level [16].
According to the IOM guidance, the prevalence of inadequate intakes for groups can be
estimated by two methods: the probability approach and EAR (ANR) cut-point method.
Regardless of the method actually chosen to estimate the prevalence of inadequacy, the
ANR (or EAR) is the appropriate NIV to use when assessing the adequacy of group intakes
[30,31].
The probability approach method requires the estimation of the probability of inadequate
intakes for each individual in a population subgroup, averaging the probabilities, and then
using this average as an estimate of the prevalence of inadequacy [23]. The EAR cut-point
method measures the prevalence of inadequate intakes as the proportion of the population
with usual intakes below the average nutrient requirement (ANR or EAR). It provides a good
approximation of prevalence; however, it is necessary to fulfill the following conditions:
intakes and requirements for the nutrient under study must be independent, the distribution
of nutrient requirement must be distributed symmetrically, and the variance of the distribution
of requirements should be smaller than the variance of the usual intake distribution [32].
Although certain nutrients are known to have a role in the etiology of several nutrient
deficiencies and chronic diseases, the complexity of the relationship between dietary intake
and disease cannot be reduced to the study of the effect that certain nutrients have on
health. As such, not only nutrients, but also foods, and the interaction between them, are of
concern for such an evaluation. Diet indexes (defined as a composite score of foods,
nutrients, or both) were the first methods used in nutritional epidemiology to assess the
effect that a combination of nutrients or foods may exert on health [22]. The Nutrient
Adequacy Ratio (NAR) is an index of adequacy, which compares the individual’s daily intake
of a nutrient with the INL98 for that nutrient [33]. Mean Adequacy Ratio (MAR) calculates the
average for the Nutrient Adequacy Ratio values for the selected nutrients for a certain
individual [33].
Diet indexes are known as a priori, as they are built based on dietary guidelines or
recommendations. On the other hand, the a posteriori approach consists in defining food
patterns once the dietary data are collected and using specific statistical analyses to identify
the relevant actual food patterns of the study population [21]. Factor analysis or cluster
analysis are the main statistical procedures used to analyze dietary data and identify dietary
patterns [34]. Both a priori hypothesis-oriented diet indexes and a posteriori defined
patterns have been related to the incidence of health outcomes (hard clinical end-points)
and biomarkers in epidemiological or clinical studies. Some of these dietary patterns have
been related to nutrient adequacy. This approach parallels a validation study, based on the
rationale that if the classification of participants according to their adherence to the dietary
pattern is able to determine whether or not they fail to reach the optimal nutrient intake, the
use of the dietary pattern is sufficiently valid [21].
Some diet indices, a priori defined, have been correlated with the adequacy of certain
nutrients for example, the revised Diet Quality Index (DQI) [35], Healthy Eating Index (HEI)
[36], Dietary Diversity Score (DDS) [37], and the Food Variety Score (FVS) [38]. Similarly,
the Mediterranean diet has been quantified in diet indices established a priori that attempt
to make a global evaluation of the quality of the diet based on a traditional Mediterranean
reference pattern [39]. For example, the Mediterranean Adequacy Index (MAI) was
developed to assess how close a diet is to the Healthy Reference National Mediterranean
Diet (HRNMD). Alberti et al. found that MAI values of diets in elderly participants from 10
European countries, followed for 10 years, were inversely associated with total mortality
[40]. For children and youths, Serra Majem et al. developed the Mediterranean Diet Quality
Index (KIDMED index) to assess the adequacy of the MDP in this age group [41,42].
Referring to the a posteriori defined analysis, the studies evaluating nutrient intake
adequacy associated with dietary patterns showed that the Prudent pattern (defined by
factor analysis as a diet rich in vegetables, fruits, legumes, whole grains, and fish) was valid
to assess the intake adequacy of α-carotene, lycopene, and lutein, for men [43], and for
assessing β-carotene, vitamin C, vitamin B6, and folic acid, for women [44].
Apart from the method used to identify dietary patterns, the micronutrients with less
probability of being effectively assessed are vitamin B12 and vitamin E. Nevertheless,
scientific evidence shows that some diet indices a priori or a posteriori defined are tools with
fair to moderate validity to assess micronutrient intake adequacy [21].

3. Results

3.1. Prevalence of Nutritional Adequacy in Europe and Some Mediterranean Countries

Recently, Roman Viñas et al. estimated the prevalence of nutrient intake inadequacy in
Europe using nutrient intake data already published [45]. The analysis of a number of
micronutrients in adult and elderly European populations, showed a mean prevalence of
inadequacy at or below 10% of the population for zinc, iron, and vitamin B12 (only in the
elderly population); a prevalence between 11% and 20% for copper in the adult and elderly
populations, for vitamin B12 in the adult population, and for vitamin C in elderly Europeans.
Finally, micronutrients with a prevalence of inadequacy above 21% of the population were
vitamin D, folic acid, calcium, selenium, and iodine, in the adults and elderly, and vitamin C
in the adults only [45]. Previously Elmadfa et al. found no large differences of vitamin intake
between four regions of Europe (North, South, Central-East, and West). However, the lowest
intake levels of cobalamin and the highest intake levels of vitamin D were reported in the
Northern region. Concerning the intake values of calcium, phosphorus, and iron, no large
differences between the regions were observed. The intake of zinc was lower in the Western
region than in the other regions and selenium intake was lower in the Northern region [46].
Another study conducted in eight European countries, evaluated the adequacy of nutrient
intake in different age groups. Mensink et al. found that proportions below the EAR of
calcium and copper were low. Inadequate intake of iodine was high in several countries, and
for older adults in France and Germany the proportion below de EAR was 40%–60% [47].
Mean intakes of selenium were below the EAR in almost all countries, with high proportions
with intakes below the EAR. Regarding iron, high proportions of inadequate intakes were
found in teenage girls and women aged 11–50 years. Intakes of vitamin A, B1, B2, B6, B12,
E, and C, were generally adequate. However, the proportions of the population with vitamin
D intakes below the recommendations were exceptionally high, although the authors
mention that in Mediterranean countries, vitamin D can be obtained from conversion through
the skin stimulated by UV radiation, and therefore, the proportion that should be obtained
from food is unknown. In Spain, more than 5% of intakes below the LRNI were found only
for potassium, in women, and vitamin A, for elderly women [47].
The EURopean micronutrient RECommendations Aligned (EURRECA) Network of
Excellence explored the process of setting micronutrient recommendations to address the
variance in recommendations across Europe. Data on intake of vitamin C, vitamin D, vitamin
B 12, folic acid, calcium, zinc, and iron (males only), from seven European countries were
used for the assessment of inadequacy by applying the cut-point method. The highest ratios
of inadequate intakes were found in countries such as Finland and Sweden, in males, and
in Ireland and the United Kingdom, among females. In Mediterranean countries (Spain,
Portugal, and Italy), the intake of calcium was in general adequate. In Spain, more than 20%
of the male population presented three of seven vitamins and minerals with inadequate
intakes, and for female population inadequate intake of four from six nutrients assessed was
observed [17].
In the African Mediterranean countries the information is scarce. In Morocco, nutrient intake
was evaluated among pregnant women. The mean daily intakes of energy and some
nutrients were adequate. However, iron, folate, zinc, and calcium intakes were inadequate
for the majority of women and more markedly in rural area [48]. On the other hand, Tunisian
migrants living in the south of France, have also reflected better diet quality, variety, and
adequacy than the local-born French, showing lower prevalence of chronic diseases
compared with local-born French [49]. Information from other countries from the Maghreb
and Middle East Mediterranean shows that, in Turkey, the average diet was inadequate to
meet recommended daily intake of calcium, iron, riboflavin, vitamin A, and animal protein
intakes in children. Deficient intakes of calcium, iron, and vitamin A were also found among
adolescents and pregnant women [50]. In Iran, no national data on nutrient intake by age or
sex groups are available, data is obtained through small surveys in many parts of the
country. Results show that low-income families have low intakes of vitamins A and B2 (in
some areas up to 50% and 70%, respectively). Iodine and iron deficiencies are the most
frequent nutrient deficiencies in Iran [51,52].
As we have seen in this section, the nutrient intake varies according to the geographical
zone. European countries have different nutrient intakes according to the culture, availability,
and accessibility of food. It seems that among Europeans, countries in the Mediterranean
basin have good nutrient intake quality, however, some countries of the Middle East
Mediterranean and North Africa have severe deficiencies of essential nutrients. More
research is needed on the adherence to the Mediterranean dietary pattern (MDP) and
prevalence of nutrient adequacy in European and especially in Mediterranean countries.

3.2. Nutritional Adequacy of the Mediterranean and Western Dietary Patterns

The relationship between nutrient adequacy and the MDP and the Western dietary pattern
(WDP) has been assessed in Spain by Serra-Majem et al. in a cohort study, Seguimiento
de la Universidad de Navarra (SUN). The study assessed nutrient intake adequacy of certain
vitamins (vitamins B12, B6, B3, B2, B1, A, C, D, and E) and minerals (Na, Zn, iodine, Se,
folic acid, P, Mg, K, Fe, and Ca). The probability of intake adequacy for nutrients was
estimated by the probability approach and using the NIVs [4].
The WDP was correlated with the intake of red and processed meat, eggs, sauces,
precooked food, fast food, energy soft drinks, sweets, whole dairy, and potatoes, and
showed a negative correlation with the consumption of low-fat dairy (Table 1). The food
groups identified in the MDP were olive oil, poultry, fish, low-fat dairy, legumes, fruits, and
vegetables (Table 1) [4].

Table 1
Correlation between baseline food consumption and factors representing the Mediterranean
and Western dietary patterns in the Seguimiento de la Universidad de Navarra (SUN) cohort
study (n = 17,197) [4].

Dietary Patterns **
Food groups *
Factor 1 (Western) Factor 2 (Mediterranean)
Olive oil - 0.32
Poultry - 0.38
Red meat 0.54 -
Processed meat 0.5 -
Eggs 0.37 -
Fish - 0.59
Sauces 0.42 -
Pre-cooked food 0.41 -
Fast food 0.57 -
Caloric soft drinks 0.35 -
Commercial sweets 0.4 -
Whole fat dairy 0.43 -
Low fat dairy −0.31 0.37
Legumes - 0.3
Vegetables - 0.68
Fruits - 0.54
Potatoes 0.45 -
Open in a separate window
* Presented in g/day; ** Correlation coefficients < 0.3 were omitted for simplicity.
Subjects who scored high on the WDP were less likely to achieve adequate intakes of iodine,
vitamin E, magnesium, iron, vitamin A, Se, vitamin C, and folic acid than those with a lower
score. Furthermore, it was found that when the score on the WDP was high, the number of
unmet nutrient intakes increased (Figure 2). Subjects in the highest quintile of WDP had a
2.5-fold increased risk for ≥10 NIVs unmet when compared to the lowest score on the WDP
(OR: 2.48, 95% IC: 1.13–5.43, p-trend <0.001) [4].
Figure 2
Average number of nutrients with intakes not meeting recommended levels across quintiles
of Western Diet pattern score; adjusted for age and sex [4].
Conversely, it was observed that higher scores of adherence to the MDP were associated
with lower percentage of energy coming from total fat and SFA intakes. Ratio of MUFA to
SFA increased with increased scores of adherence to the MDP (p for trend <0.001). Protein
intake (as percentage of energy) increased across categories of adherence to the MDP [4].
Carbohydrate intake was low (43%–44%), showing a similar value across all the quintiles,
on the contrary, consumption of dietary fiber increased according to the levels of adherence
to the MDP. All the nutrients studied (except for sodium), showed increasing values with
increasing scores to the MDP. Therefore, subjects with a higher score for the MDP had a
better nutrient profile, with a lower prevalence of individuals showing inadequate intakes of
micronutrients (Figure 3). People who scored high on the MDP were more likely to achieve
adequate nutrient intakes of Zn, iodine, vitamin E, Mg, Fe, vitamin B1, vitamin A, Se, vitamin
C, and folic acid, than those with a lower score. This population included premenopausal
women and iron requirements are highly skewed due to menstruation, in order to address
this issue, iron intake was log transformed for the estimation of nutritional adequacy.
Therefore, according to their results, the MDP could address the potential risk of inadequate
iron intake in women of reproductive age; however, more research is needed to confirm their
results. Furthermore, it was found that subjects in the highest quintile of the MDP had lower
risk for failing to meet ≥10 NIVs (OR: 0.02, 95% CI: 0.00–0.16, p-trend <0.001) when
compared to the lowest category of adherence to the MDP [4].
Figure 3
Average number of nutrients with intakes not meeting recommended levels across quintiles
of the Mediterranean Diet pattern score; adjusted for age and sex [4].
Therefore, the MDP was directly associated with the MUFA/SFA ratio, showing a healthier
profile of the quality of fat intake when comparing to other studies conducted in non-
Mediterranean countries. Even more, as adherence to the Mediterranean diet increases, the
probability of not fulfilling the nutrient recommendations decreases [4].
Another study conducted in 328 subjects (18–75 years) from Catalonia (Northeastern
Spain), analyzed the association between different biomarkers and two Mediterranean diet
(MD) adherence indexes. Bach-Faig et al. found that subjects with higher MD adherence
had significantly higher plasma concentrations of beta-carotene, folates, vitamin C, alpha-
tocopherol, and HDL cholesterol [53].
In France, Maillot et al. conducted a study by applying individual diet modeling in a
representative sample of adults to evaluate the smallest dietary changes needed to fulfill a
whole set of nutrient recommendations by each individual. Authors found that the inclusion
of foods typical of the Mediterranean Diet were strictly necessary to achieve French nutrient
recommendations [54].

3.3. Nutritional Adequacy in Children and the Mediterranean Diet

The Mediterranean diet has been associated with nutritional adequacy in Children. A cross-
sectional study conducted in Spain, the enKid study, assessed individuals aged from six to
24 years (n = 3166) [55]. Information on dietary habits, lifestyle, and socioeconomic status
was collected. To assess the compliance with a Mediterranean Diet model, a short
questionnaire was used (KIDMED index), which allowed to classify subjects according to
the quality of the Mediterranean Diet categorized as: High, Medium, or Poor (Table 2) [42].
The nutrient intake adequacy was assessed as the percentage of population with intakes
below two-thirds of the recommended nutrient intakes.

Table 2
KIDMED test to assess the Mediterranean Diet adherence [41].

KIDMED test Scoring

Takes a fruit or fruit juice every day +1
Has a second fruit every day +1
Has fresh or cooked vegetables regularly once a day +1
Has fresh or cooked vegetables more than once a
+1
day
Consumes fish regularly (at least 2–3/week) +1
Goes >1/week to a fast food restaurant (hamburger) −1
Likes pulses and eats them >1/week +1
Consumes pasta or rice almost every day (5 or more
+1
per week)
Has cereals or grains (bread, etc.) for breakfast +1
Consumes nuts regularly (at least 2–3/week) +1
Uses olive oil at home +1
Skips breakfast −1
Has a dairy product for breakfast (yoghurt, milk, etc.) +1
Has commercially baked goods or pastries for
−1
breakfast
Takes two yoghurts and/or some cheese (40 g) daily +1
Takes sweets and candy several times every day −1
Adherence to the Mediterranean
KIDMED Index
Diet
Score ≤ 3 points Poor
Score 4–7 points Medium
Score ≥ 8 points High
Open in a separate window
In the enKid study, the authors found that total energy intake did not change according to
the KIDMED Index, with the exception of male adolescents aged 15 to 24 years, who
showed a tendency towards increased levels. Consumption of fiber, calcium, iron,
magnesium, potassium, phosphorus, and practically all the vitamins with the exception of
vitamin E, increased according to the KIDMED Index. Results showed that the proportion of
children with inadequate intake of calcium, iron (in females), magnesium, vitamin B6
(excluding males aged 6–14 years), vitamin C and A (in females), decreased when the
scores of the KIDMED Index increased (Table 3) [41].

Table 3
Percentage of inadequate intakes (<2/3 INL) in scholar children according to Mediterranean
Diet adherence [41].

KIDMED Index for 6–14 years

Poor ≤ 3 (%) Medium 4–7 (%) High ≥ 8 (%) p-trend

Men/Women

Energy 4.8/13.3 1.9/6.9 1.3/6.3 0.303/0.467

Protein 0.0/0.0 0.0/0.0 0.0/0.0 -

Calcium 4.8/26.7 2.6/10.4 0.4/4.2 0.027/<0.000

Iron 0.0/33.3 0.8/23.8 0.0/15.4 0.300/0.008

Magnesium 19.0/0.0 9.8/4.2 4.6/2.9 0.004/0.707

Thiamin 0.0/0.0 0.4/0.4 0.0/0.4 0.464/0.871

Riboflavin 0.0/0.0 1.1/1.2 0.4/0.8 0.559/0.881

Niacin 0.0/0.0 0.4/0.4 0.0/0.4 0.464/0.871

Vitamin B6 0.0/33.3 3.0/10.8 2.9/5.0 0.724/<0.000

 KIDMED Index for 6–14 years

Poor ≤ 3 (%) Medium 4–7 (%) High ≥ 8 (%) p-trend

Folate 14.3/46.7 9.8/32.3 5.0/23.8 0.021/0.010

Vitamin B12 0.0/0.0 0.0/0.0 0.0/0.0 -

Vitamin C 47.6/13.3 18/15.4 5.4/4.6 ˂0.000/˂0.000

Vitamin A 57.1/80.0 63.9/61.5 59.6/54.2 0.523/0.024

Vitamin D 100.0/100.0 95.9/99.6 95.8/97.1 0.618/0.024

Vitamin E 28.6/66.7 43.2/60.8 36.3/57.1 0.394/0.310

A study conducted in Spain showed that 20 healthy male adolescents aged 11–14 with a
diet based on the MDP allowed to maintain adequate zinc serum levels despite the content
of dietary phytate, which is present in vegetables, cereals and legumes [56]. In the same
age group, there is evidence that when a MDP was consumed, a drastic increase in iron
absorption was observed among the subjects when compared to their habitual diet [57].
Furthermore, in male adolescents another study has found significant increases in calcium
absorption, calcium retention, and a considerable decrease in urinary calcium excretion with
a Mediterranean type diet intervention when comparing to a basal diet [58].

4. Discussion
The Mediterranean diet has been associated with nutritional adequacy in adult population
and children. Greater adherence to the MDP was related with a higher prevalence of
individuals showing adequate intakes of micronutrients. The MDP had similarities with the
healthiest patterns [43,44,59,60] defined in non-Mediterranean countries: a positive
correlation with intakes of fruits, green leafy vegetables, poultry, and fish, and certain
lifestyle habits, such as non-smoking and being more physically active [61]. However, when
the association of the dietary patterns with their nutrient intake profiles was analyzed,
differences arose, especially in relation to fat intake. Prudent and healthy patterns had lower
intakes of total and saturated fat [44,62,63], and some studies found even lower intakes of
MUFA [62,63]. Healthy patterns showed higher percentages of energy coming from proteins
and carbohydrate and lower percentages of energy coming from fat [44,64] when compared
the highest quintile to the lowest. Japanese traditional diets share a number of features with
the Mediterranean diet in some food groups as cereals, beans, seafood, vegetables, and
fruits. On the other side, both diets also differ in view of fat intake, where in Japan was
extremely low in the past. Alcohol consumption is quite different due to the type of alcohol,
one is wine and the other is sake or alcohol made of rice or corn [65]. Many of the
characteristics of the diet in Okinawa are shared with the MDP, for example, the low intake
of saturated fat, high antioxidant intake, and low glycemic load in these diets are likely
contributing to a decreased risk for cardiovascular disease, some cancers, and other chronic
diseases through multiple mechanisms, including reduced oxidative stress [66,67].
Therefore, the choice of the Mediterranean or the Japanese diet would be according to the
circumstances and local availability, and trying to combine the best ingredients of both diets
according to one’s habits and preferences. A new Japomediterranean diet that includes olive
oil, wine, fish, beans, nuts and seeds, soya, vegetables, fruits, bread, rice, seaweed, dairy
products, and mushrooms, could be the option for a promising future. Otherwise, keeping
our own (Mediterranean or Japanese) traditional diet would be the best choice for our health
and for our culture [67].
There are some limitations to this review, first, the evidence is very limited and almost all
studies have been conducted in Spain. Second, to our knowledge, there are only few studies
that relate nutrient intake of the MDP with biomarkers in adolescent population. Third, in the
studies selected, the MDP was analyzed in different ways. Depending on how the
Mediterranean diet is defined, the type of food and nutrient intake or other indices, such as
glycemic index or glycemic load, could change. A more precise and quantitative definition
of the Mediterranean diet is required if the adherence to this dietary pattern is intended to
be accurately measured [39]. Therefore, more research is encouraged in order to give
evidence-based recommendations on the Mediterranean diet and nutritional adequacy.

5. Conclusions
The Mediterranean Diet is a pattern with high nutritional quality; apart from better dietary fat
quality [4], anti-inflammatory effects [7] and the increased quantity of antioxidants [68,69],
we should also add the factor of enhanced nutritional adequacy. It has been demonstrated
that higher levels of adherence to a Mediterranean dietary pattern are associated to a
reduced risk of inadequate intakes. Therefore, health promotion strategies should be
prioritized to promote the Mediterranean Diet, especially in population groups that are
vulnerable to micronutrient deficiencies [32,70].

Acknowledgments
The authors want to thank the English revision made by Lyndel Aplvor. This report has been
supported by the official funding agency for biomedical research of the Spanish government,
Instituto de Salud Carlos III (ISCIII), through grants provided to research networks (RTIC
G03/140, RTIC RD 06/0045 and through Centro de Investigación Biomédica en Red de
Fisiopatología de la Obesidad y Nutrición [CIBERobn]), and by grants from Centro Nacional
de Investigaciones Cardiovasculares (CNIC 06/2007), Fondo de Investigación Sanitaria–
Fondo Europeo de Desarrollo Regional (PI04-2239, PI 05/2584, CP06/00100, PI07/0240,
PI07/1138, PI07/0954, PI 07/0473, PI10/01407, PI10/02658, PI11/01647, and P11/02505),
Ministerio de Ciencia e Innovación (AGL-2009-13906-C02 and AGL2010-22319-C03),
Fundación Mapfre 2010, Agencia Canaria de Investigación, Innovación y Sociedad de la
Información-EU FEDER (PI 2007/050), Consejería de Salud de la Junta de Andalucía
(PI0105/2007), Public Health Division of the Department of Health of the Autonomous
Government of Catalonia, Generalitat Valenciana (ACOMP06109, GVACOMP2010-181,
GVACOMP2011-151, CS2010-AP-111, and CS2011-AP-042), and Regional Government
of Navarra (P27/2011). I.C.-Q. is supported by a scholarship of the National Council on
Science and Technology of México (CONACYT).

Conflicts of Interest
The authors declare no conflicts of interest.

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Nutr Diabetes. 2017 Mar; 7(3): e247.

VI. Overweight and obesity in Mexican children and

adolescents during the last 25 years
S Hernández-Cordero,1 L Cuevas-Nasu,1 M C Morán-Ruán,1 I Méndez-Gómez
Humarán,2M A Ávila-Arcos,1 and J A Rivera-Dommarco1,*

Abstract

Introduction
Prevalence of overweight (OW) and obesity (OB) in all age groups has increased throughout
most countries in recent decades, with childhood OB representing a public health challenge.
Worldwide, in 2010 an estimated 42 million children were OW, and 35 million were living in
developing countries.1
OB in childhood has immediate consequences on health including hyperlipidemia,
hypertension and abnormal glucose tolerance as well as orthopedic, neurological,
pulmonary, gastroenterological, endocrine and hepatic disorders, especially when OB is
severe.2, 3, 4 Other consequences of OB are psychological effects and social stigmatization
that obese youth face, which can produce serious consequences for emotional and physical
health.4, 5 There is an established link between OB during childhood and its persistence into
adolescence and adulthood.2 OW+OB are well-recognized risk factors for
noncommunicable diseases in adults, such as hypertension, type 2 diabetes and
cardiovascular diseases, among others.3, 4 It is expected that the increase of childhood
OW+OB will be followed by the occurrence of chronic diseases at younger ages, with the
associated disabilities and early death as well as increased expenses for families and
country health systems.4, 5
The burden of childhood OB on the health system is also undeniable and cannot yet be fully
estimated. Health problems will be seen in the next generation of adults as obese children
today become obese adults.5
In Mexico, data from three national surveys conducted in 1988, 1999 and 2006, using the
International Obesity Task Force (IOTF) classification system described the upward trends
on OW and OB in school-age children and adolescents at the national level.6
The present paper provides the most recent prevalence estimates of OW+OB in Mexican
children and adolescents aged 0–19 years using data from the 2012 Mexican National
Health and Nutrition Survey and describes trends in OW and OB in the last 13–24 years,
using the World Health Organization Child Growth Standard to define OW and OB.

Materials and methods

Study population and National Health and Nutrition Survey

Prevalence of OW+OB was calculated using data from the most recent National Health and
Nutrition Survey–2012 (Encuesta Nacional de Salud y Nutrición or ENSANUT-2012
according to its acronym in Spanish). For trend analysis, data from previous National Health
and Nutrition Surveys obtained in 1988, 1999, and 2006, along with the 2012 data, were
used. ENSANUT-2012 was conducted from October 2011 to May 2012 by the National
Institute of Public Health in Mexico. A nationally representative sample of the population was
selected using a stratified, multistage probability sample design. The sample is
representative at state, regional, and urban and rural levels. Information collected during the
ENSANUT-2012 comprises anthropometric data (weight and height, among others). A more
detailed description of ENSANUT methods has been published elsewhere.7 Informed
consent was obtained from all participants and/or parents or primary caregiver (in case of
young children), and the protocol was reviewed and approved by the Institutional Review
Board of the National Institute of Public Health.
The study population for the present analyses includes children <19 years of age classified
according to three different age groups: preschool children (<5 years of age), school-aged
children (5–11 years) and adolescents (12–19 years). ENSANUT-2012 overall nonresponse
rate was 8.7%. A total of 1.25% of examined children and adolescents had missing data for
body mass index (BMI) either because of lack of one or both measurements, implausible
data or in the case of pregnancy in adolescent girls. BMI plausible data were considered at
a range of −5.0 and +5 s.d., eliminating all those with BMI values <10 or >38 for preschool
children and school-age children and BMI <10 or >58 for adolescents. Excluded from the
analyses were all cases where z-score for height and age was<−6 or >+6 s.d. For
ENSANUT-2012, data for analyses were available for 10 658 preschool children, 16 351
school-age children and 13 992 adolescents (Table 1). Weight and height were measured
with the same validated and standardized methods as well as same criteria were used to
define plausible anthropometric data for the samples from previous surveys included in the
analyses. For previous National Health and Nutrition Surveys of 1999 and 2006,
nonresponse rate was 17.6%8 and 0.6%,9 respectively. Unfortunately, there is no
information of 1988 survey's nonresponse rate.

Table 1
Sample size and descriptive characteristics of children and adolescents by age group
(National Health and Nutrition Surveys 1988, 1999, 2006 and 2012)a
Characterist Preschoolers (0–4 years) School-age children (5–11 Adolescents (12–19 years)
ic years)

n (sampl n (thousand % n (sampl n (thousand % n (sampl n (thousand %

e) s) e) s) e) s)
1988
Total 6794 8268.1 100 4917 5776.5 100
Sex
Male 3455 4216.8 51. – – –
0
Female 3339 4051.4 49. 4917 5776.5 100
0
Area
Urban 5702 6518.8 78. 4248 4770.8 82.
8 6
Rural 1092 1749.3 21. 669 1005.7 17.
2 4
HLCI-Q
Q1 1189 1847.0 22. 828 1104.8 20.
3 0
Q2 1228 1452.7 17. 914 1139.8 20.
6 6
Q3 1191 1280.7 15. 1204 1330.7 24.
5 1
Q4 1116 1203.6 14. 832 893.3 16.
6 2
Q5 1134 1270.8 15. 939 1058.1 19.
4 1

1999
Total 7473 10125.9 100 11 274 15405.7 100 5070 7559.0 100
Sex
Male 3786 5083.1 50. 5568 7546.7 49. – – –
2 0
Female 3687 5042.9 49. 5706 7859.0 51. 5070 7559.0 100
8 0
Area
Urban 4406 7138.8 70. 6336 10596.0 68. 3015 5410.9 71.
5 8 6
Characterist Preschoolers (0–4 years) School-age children (5–11 Adolescents (12–19 years)
ic years)

n (sampl n (thousand % n (sampl n (thousand % n (sampl n (thousand %

e) s) e) s) e) s)
Rural 3067 2987.1 29. 4938 4809.6 31. 2055 2148.1 28.
5 2 4
HLCI-Q
Q1 1850 2240.5 22. 2836 3410.0 22. 1088 1404.6 18.
1 1 6
Q2 1529 1896.3 18. 2166 2767.3 18. 961 1284.8 17.
7 0 0
Q3 1633 2103.4 20. 2390 3149.7 20. 1096 1551.8 20.
8 4 5
Q4 1242 1811.5 17. 2050 2842.3 18. 930 1340.9 17.
9 4 7
Q5 1020 1839.5 18. 1552 2904.6 18. 875 1809.8 23.
2 9 9

2006
Total 7697 9400.1 100 15 045 15749.4 100 14 445 18320.1 100
Sex
Male 3945 4765.7 50. 7518 7834.5 49. 7088 9163.3 50.
7 7 0
Female 3752 4634.3 49. 7527 7914.9 50. 7357 9156.7 50.
3 3 0
Area
Urban 5366 6933.8 73. 10 112 11340.6 72. 10 146 13542.0 73.
8 0 9
Rural 2331 2466.2 26. 4933 4408.8 28. 4299 4778.1 26.
2 0 1
HLCI-Q
Q1 1923 2317.3 24. 3953 4068.2 25. 3115 3952.9 21.
7 8 6
Q2 2000 2264.5 24. 3607 3534.9 22. 3308 3797.3 20.
1 4 7
Q3 1616 1841.7 19. 3071 2908.9 18. 3001 3587.1 19.
6 5 6
Q4 1302 1740.5 18. 2586 2913.0 18. 2743 3565.8 19.
5 5 5
Q5 829 1208.0 12. 1773 2276.4 14. 2221 3341.8 18.
9 5 2

2012
Total 10 658 10785.1 100 16 351 16444.1 100 13 992 18102.8 100
Sex
Male 5314 5418.9 50. 8195 8327.4 50. 7041 9232.1 51.
2 6 0
Female 5344 5366.2 49. 8156 8116.7 49. 6951 8870.7 49.
8 4 0
Area
Urban 6569 8036.8 74. 10 126 12262.9 74. 9017 13659 75.
5 6 5
 Characterist Preschoolers (0–4 years) School-age children (5–11 Adolescents (12–19 years)
ic years)

n (sampl n (thousand % n (sampl n (thousand % n (sampl n (thousand %

e) s) e) s) e) s)
Rural 4089 2748.3 25. 6225 4181.3 25. 4975 4443.8 24.
5 4 5
HLCI-Q
Q1 2646 2163.4 20. 3827 3146.2 19. 2827 3018.0 16.
1 1 7
Q2 2413 2115.6 19. 3630 3124.5 19. 2860 3268.3 18.
6 0 1
Q3 2223 2176.0 20. 3347 3252.7 19. 2899 3541.5 19.
2 8 6
Q4 1947 2342.9 21. 3088 3519.3 21. 2763 3923.6 21.
7 4 7
Q5 1429 1987.2 18. 2459 3401.5 20. 2643 4351.3 24.
4 7 0
Open in a separate window
Abbreviations: HLCI, Household Living Condition Index; Q, Quintile.
a
Age groups defined as preschoolers: 0–4 years; school-age children: 5–11 years; and
adolescents 12–19 years.

Definition of OW+OB for all nutritional surveys

All anthropometric information was measured using standardized techniques and
equipment.7 BMI was calculated as weight (kg) divided by height (m) squared. We defined
risk of overweight (RO), OW and OB among all age groups based on the World Health
Organization (WHO) Child Growth Standard for preschool children10 and 2007 WHO growth
reference for school-age children and adolescents.11 For preschool children, the RO
category was defined as z-score of BMI<1 s.d. and 2 or less s.d. OW and OB was defined
as z-score +2 s.d. For school-aged children and adolescents, OW was defined as z-score
of BMI>1 s.d. and 2 or less s.d.; OB as BMI z-score >2 s.d.9, 10

Other variables for analysis of ENSANUT-2012

Area of residence was classified according to the number of inhabitants, considering those
areas with a population of 2500 or more as urban areas and those with <2500 persons as
rural areas.
The Household Living Condition Index (HLCI) was used as a proxy of socioeconomic status
and was constructed using the principal component analysis.8 The HLCI was constructed
considering household characteristics (number of rooms, running water, WC and
construction materials) as well as household amenities (washing machine, microwave,
stove, television and so on). The first factor was used as the HLCI, explaining 40.5% of the
variance. The index was further divided into quintiles of HLCI to be considered as categorical
variables in the statistical analysis.

Statistical analysis
For ENSANUT-2012 OW+OB prevalence analysis, we performed frequencies at a national
level by age group, sex, area of residence (rural and qurban areas) and HLCI. Trends for
OW+OB from the 1988, 1999, 2006 and 2012 surveys were calculated with a standardized
proportions difference using the asymptotic normal distribution.12 School-age children of
both sexes and male adolescents were not measured in 1988; thus, trends were estimated
only from 1999 to 2012 in school-age children and for adolescents during the entire 1988–
2012 period, but only for females.
To study trends in prevalence of OW and OB, descriptive analyses were performed using
frequencies and their respective 95% confidence intervals (CIs) stratified at the national level
for the four geographic regions, rural and urban areas, and HCLI categories. Multinomial
logistic regression models with fixed effects12 were used to estimate differences among
categories. Prevalence was further divided by age group blocks. Trends for OW+OB from
the 1988, 1999 and 2006 surveys were calculated using the standardized difference
between proportions.12 Due to the fact that the duration of the time periods between surveys
differed, in order to compare the prevalence we present the changes as percentage points
(pp) and pp per year.
Data were analyzed using the Statistical Program SPSS v. 15.0 with the complex sampling
software (SPSS Inc., Chicago, IL, USA) and Stata v.12.1 (Stata Corp., College Station, TX,
USA).

Results

Overweight and obesity: National Health and Nutrition Survey 2012

Overall among children 0–19 years of age, 28.8% presented either RO (preschool children)
or OW, or OB in 2012. Table 2 shows detailed prevalence estimates of RO, OW+OB for
preschool children and OW and OB for school-age children and adolescents. Results are
presented by year of survey, sex (for school-age children and adolescents), area of
residence (urban/rural) and HLCI. For comparison purposes we provide the OW+OB
estimates using the IOTF definition13 as Supplementary Material, Supplementary Table 1.

Table 2
Prevalence of risk of overweight, overweight and obesity by age group, sex, area of
residence HLCI (Surveys 1988, 1999, 2006 and 2012)a , b , c , d , e
Va 1988 1999 2006 2012
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n (t % 9 n (t % 9 n (t % 9 n (t % 9 n (t % 9 n (t % 9 n (t % 9 n (t % 9
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us % us % us % us % us % us % us % us %
an C an C an C an C an C an C an C an C
ds) I ds) I ds) I ds) I ds) I ds) I ds) I ds) I
9 3
) )
HL Q 32 1 ( 10 5 ( 24 1 ( 10 6 (
CI 1 7.7 6 1 5.8 . 4 9.0 6 1 6.0 . 4
. 4 4 . . 3 8 .
8 . 0 0 . 8
3 , 3 ,
, 7 , 9
1 . 1 .
9 4 9 5
. ) . )
8 1
) )
Q 35 1 ( 19 1 ( 28 1 ( 17 1 (
2 8.0 9 1 0.9 0 8 8.4 8 1 3.3 1 8
. 6 . . . 5 . .
5 . 4 5 3 . 0 6
8 , 4 ,
, 1 , 1
2 2 2 3
2 . 1 .
. 7 . 8
6 ) 6 )
) )
Q 36 2 ( 25 1 ( 36 1 ( 22 1 (
3 0.5 0 1 5.3 4 1 1.1 9 1 3.3 2 1
. 7 . 1 . 7 . 0
7 . 7 . 9 . 3 .
2 7 0 3
, , , ,
2 1 2 1
4 8 3 4
. . . .
7 2 2 7
) ) ) )
Q 39 2 ( 30 1 ( 43 2 ( 40 1 (
4 4.8 1 1 1.3 6 1 0.0 0 1 8.2 9 1
. 8 . 3 . 7 . 6
6 . 5 . 7 . 6 .
2 8 9 6
, , , ,
2 1 2 2
5 9 3 3
. . . .
5 6 7 1
) ) ) )
Q 39 2 ( 33 1 ( 48 2 ( 42 1 (
5 1.7 1 1 2.8 8 1 0.8 1 1 7.3 9 1
. 8 . 2 . 8 . 6
9 . 6 . 8 . 4 .
1 8 4 4
, , , ,
2 2 2 2
 Va 1988 1999 2006 2012
ria
bl
e
Overweigh Obesity Overweigh Obesity Overweigh Obesity Overweigh Obesity
t t t t

n (t % 9 n (t % 9 n (t % 9 n (t % 9 n (t % 9 n (t % 9 n (t % 9 n (t % 9
ho 5 ho 5 ho 5 ho 5 ho 5 ho 5 ho 5 ho 5
us % us % us % us % us % us % us % us %
an C an C an C an C an C an C an C an C
ds) I ds) I ds) I ds) I ds) I ds) I ds) I ds) I
6 6 5 2
. . . .
2 2 6 7
) ) ) )
M 38 2 ( 21 1 ( 39 2 ( 24 1 (
al 98. 1 2 83. 1 1 12. 1 2 12. 3 1
e 0 . 0 1 . 0 9 . 0 2 . 2
s 3 . 9 . 6 . 3 .
a 2 9 5 5
n , , , ,
d 2 1 2 1
fe 2 3 2 4
m . . . .
al 4 0 8 2
e ) ) ) )
s
Open in a separate window
Abbreviations: BMI, body mass index; CI, confidence interval; HLCI, Household Living
Condition Index; Q, Quintile; WHO, World Health Organization.
a
Percentage and 95% CI.
b
Age groups defined as: preschoolers: 0–4 years; school-age children: 5–11 years; and
adolescents: 12–19 years.
c
For preschoolers, overweight category refers to risk of overweight; obesity category
includes overweight and obesity.
d
For preschoolers at risk of overweight: z-score of BMI/age >1 s.d. (WHO); overweight and
obesity: z-score of BMI/age ⩾2 s.d. (WHO). For school-aged children and adolescents:
overweight z-score of BMI/age >1 s.d. and ⩽2 s.d. (WHO); obesity: z-score of BMI/age
>2 s.d. (WHO).
e
No data available for school-age children in 1988.

Preschool children
Prevalence of RO and OW+OB was 23.8% (95% CI: 22.5, 25.1%) and 9.7% (95% CI: 8.9,
10.6%), respectively (Table 2). Differences in prevalence by sex were already observed at
this early age, with a higher combined prevalence of RO and OW+OB in boys (35.2%) than
in girls (31.8% P<0.001). This difference was mainly due to a higher prevalence of RO in
boys (25.3%) than in girls (22.3% data not shown). There was no difference in RO or
OW+OB either by area of residence or by HLCI.
School-age children
The combined prevalence of OW and OB in boys and girls in this age group was 34.4%
(95% CI: 33.3, 35.6%) with the prevalence of OW being 19.8% (95% CI: 18.8, 20.9% Table
2). The combined prevalence of OW and OB was higher in boys (36.9%) than in girls
(32.0%); P<0.001 (data not shown). The difference in the combined prevalence is due to the
higher prevalence of OB among boys (17.4%, 95% CI: 16.0, 18.8%) than in girls (11.8%,
95% CI: 10.8, 12.8% P<0.001; Table 2).
The prevalence of both OW and OB was higher in boys and girls living in urban areas (Table
2). For girls, there was a trend for both OW and OB, increasing the prevalence as the HLCI
increased. The highest prevalence was found in girls from the highest quintile (OW: HLCI
lowest quintile: 14.9 (95% CI: 12.6, 17.5%)) vs HLCI highest quintile: 24.8% (95% CI: 21.1,
29.0% OB: HLCI lowest quintile: 7.9% (95% CI: 6.2, 9.9%)) vs HLCI highest quintile: 16.2
(95% CI: 13.6, 19.2% Table 2). For boys, the trend of increasing OW and OB was more
pronounced for the latter (OW: HLCI lowest quintile 15.7; 95% CI: 13.4, 18.3%) vs HLCI
highest quintile 20.3% (95% CI: 17.2, 23.9%); OB: HLCI lowest quintile 8.2% (95% CI: 6.3,
10.6%) vs HLCI highest quintile 22.7% (95% CI: 19.2, 26.8% Table 2).

Adolescents
Combined prevalence of OW and OB in this age group was 35.8% (95% CI: 34.0, 37.6%)
and 34.1% (95% CI: 32.4, 35.8%) for female and male adolescents, respectively, being
slightly higher in females (P=0.03; data not shown). The difference in combined prevalence
by sex is mainly due to the higher prevalence of OW in females compared with that in males
(23.7 vs 19.6, P<0.001, for females and males, respectively; Table 2). Prevalence of both
OW and OB was higher in female and male adolescents living in urban areas, whereas the
prevalence of OB was higher for adolescents (both male and female) in the highest quintile
of the HLCI (OB prevalence for females: lowest HLCI quintile 8.2% (95% CI: 6.5, 10.2%),
highest HLCI quintile 13.2 (95% CI: 10.9, 15.9%), males: lowest HLCI quintile 6.8% (4.8,
9.5%), highest HLCI quintile: 19.4% (16.4, 22.7% Table 2).

Trends of OW and OB

Prevalence of OW and OB in female children and adolescents during the last 24 years
Overall trends of OW and OB for girls and female adolescents using the available
information are presented in Figure 1. Complete information for the four surveys (1988,
1999, 2006 and 2012) is only available for preschool girls and female adolescents, whereas
for school-aged girls there is information from only the 1999–2012 surveys. Prevalence of
OW and OB increased in all age groups, with the highest rate of increase among female
adolescents followed by school-aged girls.
Figure 1
Prevalence of overweight and obesity in girls and female adolescents by age group and
survey: 1988–2012.1,2,3,4
For preschool girls, there has been a statistically significant increase in all years except
between 1999 and 2006, years when the prevalence of RO and OW decreased (RO and
OW combined prevalence change from 1999 to 2006: −6.1±1.0 pp-, P<0.001; or −0.87 pp
per year). Overall change in the combined prevalence was 6.3±1.0 pp (P<0.001; 0.26 pp
per year) in the last 24 years, showing the highest increase between 1988 and 1999
(combined prevalence change: 8.4±1.0 pp, P<0.001; 0.76 pp per year) compared with
change from 2006 to 2012 (3.9±1.0 pp, P<0.001; 0.65 pp per year).
For school-aged girls, overall change in the combined prevalence of OW and OB from 1999
to 2012 was 6.2±0.8 pp (P<0.001; 0.5 pp per year), demonstrating the highest increase from
1999 to 2006 (6.5±0.8 pp, P<0.001; 0.9 pp per year), with no change from 2006 to 2012
(combined prevalence change: −0.3±0.7, P=0.65; −0.06 pp per year).
The combined prevalence of OW and OB in adolescent females increased (24.7±0.8
pp, P<0.001; 1.0 pp per year) from 1988 to 2012, showing the highest and marked increase
between 1988 and 1999 (17.2±0.9 pp (P<0.001; 1.6 pp per year). The rate of increase began
to slow down from 1999 onward; however, there has been a steady increase since then with
statistical significance between each survey (combined prevalence change from 1999 to
2006: 5.1±0.9 pp, P<0.001; 0.7 pp per year); change from 2006 to 2012: 2.4±0.8
pp, P=0.002 (0.4 pp per year).

Trends by age group, HLCI and area of residence

Preschool children (boys and girls; change from 1988 to 2012)
Increase in the combined prevalence of RO and OW in preschool children occurred at all
socioeconomic levels, without demonstrating any difference by HLCI level (data not shown).
When changes in the combined prevalence of RO and OW by area of residence were
analyzed, the rate of increase was higher among children living in urban areas (1988
prevalence 25.6%, 95% CI: 24.3, 27.1% 2012 prevalence 34.2, 95% CI: 32.5, 36.0%);
change 1988–2012: 8.6±0.8 pp, P<0.001 (0.36 pp per year) than in children living in rural
areas (1988 prevalence 30.3, 95% CI: 27.1, 33.7% 2012 prevalence: 31.4, 95% CI: 29.5,
33.4%. Change from 1988 to 2012 was 1.1±1.6 pp, P=0.48 (0.05 pp per year; Figure 2).
Figure 2
Combined prevalence of risk of overweight and overweight in preschool-age children (both
sexes) by area of residence and year of survey: 1988–2012.1, 2, 3
School-age children (change from 1999 to 2012)
Combined prevalence of OW and OB increased in both rural and urban areas from 1999 to
2012. However, the rate of increase was more pronounced in girls living in rural areas
(change in prevalence 1999–2012: rural: 7.5±1.0 pp, P<0.001; 0.58 pp per year); urban:
4.9±1.0 pp, P<0.001 (0.38 pp per year; Figure 3). This trend was not seen among boys in
whom the rate of change of combined prevalence of OW and OB was similar between area
of residence (change in prevalence 1999–2012: rural: 7.9±1.0 pp, P<0.001 (0.60 pp per
year); urban: 8.1±1.0 pp, P<0.001 (0.62 pp per year). For both boys and girls, the combined
prevalence of OW and OB increased at a higher rate in the lower quintiles of the HLCI
compared with the highest quintile (change of prevalence 1999–2012: girls—lowest HLCI
quintile: 10.5±1.3 pp, P<0.001; highest HLCI quintile: 4.0±2.3 pp, P=0.07; boys—lowest
HLCI quintile: 8.8±1.4 pp, P<0.001; highest HLCI quintile: 2.0, P=0.37; Figure 4).
Open in a separate window
Figure 3
Combined prevalence of overweight and obesity in school-age girls (a) and boys (b) by area
of residency and year of survey: 1999–2012.1,2,3,4,5 (a) Girls. (b) Boys.
Figure 4
Combined prevalence of overweight and obesity in school-age girls (a) and boys (b) by
household living conditions index (HLCI) and year of survey: 1999–2012.1,2,3,4,5 (a) Girls. (b)
Boys.
Female adolescents (change from 1988 to 2012)
Combined prevalence of OW and OB increased 16±1.4 pp from 1988 to 1999 in rural areas
(P=0.01) and 18.4±1.0 in urban areas. A smaller increase in the prevalence from 1999 to
2006 was seen in both areas (rural: 2.8±1.3, P=0.03; urban: 5.9±1.1, P=0.01). This rate of
increase decreased during the last 6 years but was still on the rise among female
adolescents living in urban areas (change in prevalence from 2006 to 2012: 2.6±1.0
pp, P=0.01; Supplementary Figure 1A).
For HLCI, prevalence of OW and OB increased >11 pp between 1988 and 1999 in all
quintiles except the fourth, which showed an increase of 22.3±1.9 pp, P=0.01. From 1999,
the rate of increase at all HLCI levels decreased and the prevalence for 2006 with respect
to 1999 for Q1 and Q2 increased 3.8±1.6 pp (P=0.01) and 10.8±1.5 pp (P=0.01),
respectively. Between 2006 and 2012, females in Q1 and Q4 showed an increase in the
prevalence of OW and OB of 4.7±1.6 pp, P=0.003 and 3.8±1.8 pp, P=0.04, respectively
(Supplementary Figure 1B).
Male adolescents (change from 2006 to 2012)
In male adolescents, no changes were observed in the combined prevalence of OW and OB
between surveys from 2006 to 2012 (change in prevalence: 1.1±0.8 pp, P=0.16), with no
difference by area of residence or HLCI (data not shown).

Discussion
Prevalence of OW+OB in children and adolescents presented in this article are the most
recent estimates in Mexico. Among children and adolescents <19 years of age the combined
prevalence of OW (and RO for preschool children) and OB was as high as 28.8% by 2012.
Combined prevalence of RO and OW+OB in children <5 years of age was 33.5% (RO:
23.8%, OW+OB: 9.7%) and for school-age children and adolescents was 36.9% (OW:
19.5%, OB: 17.4%) and 35.8% (OW: 23.7%, OB: 12.1%), respectively. By 2012, the highest
prevalence was seen among children and adolescents living in urban areas and those from
the highest socioeconomic level. In the last 13–24 years, the prevalence of OW and OB has
increased at the highest rate among female adolescents followed by school-aged girls.
Increase in OW and OB has been more pronounced among those children from the lowest
socioeconomic level in all age groups except for preschool children and those from urban
areas for preschool children and adolescents, whereas for school-aged girls, the increase
has been higher among those living in rural areas.
When comparing prevalence from other countries using the WHO classification systems
(WHO Child Growth Standard for preschool children10 and WHO growth reference for
school-age children and adolescents WHO 2007,11 childhood prevalence of OW and OB in
Mexican children is among the highest. A longitudinal study carried out in Cuba
demonstrated that the combined prevalence of RO and OW in children <5 years of age was
17.3% in 201114 and for Colombian preschoolers was 25.2% in 2005 according to a recent
systematic review.15 Prevalence of OW and OB in school-age children in Brazilian boys was
34.8% in 2009 and 18.9% in 2010 in Colombian children (both sexes), lower than the
prevalence in Mexico during the same time (2012), but the prevalence of OW and OB in
Chilean children (both sexes) in 1997 was 37.7%, much higher than the prevalence shown
in Mexican boys (28.2%) and girls (25.5%) during the same year (1999). Prevalence of
OW+OB in adolescents was relatively low in Colombian adolescents in 2010 (16.7%)
compared with Chile in 2005 (31.0%), and Mexican males (34.1%) and females (35.8%) in
2012.15
In 2012, the combined prevalence of OW and OB was higher in preschool and school-age
boys than in girls, whereas in adolescents, such prevalence was higher among females than
males. Similar sex differences have been reported by Rivera et al.15 who found that in those
studies reporting data stratified by sex, prevalence of OW and OB differed by sex, but the
differences varied across age groups. As in our study, in school-age children, more boys
were identified as OW or OB than girls in Brazil. On the other hand, in adolescents, unlike
in Mexico, the prevalence of OW and OB was higher among adolescent Brazilian males
than in their female counterparts.
Results of the latest Mexican nutritional survey (2012) show that the combined prevalence
of OW and OB in school-aged children and adolescents was higher among those in the
highest quintile of the HLCI, a wealth indicator, whereas in preschool children there were no
differences according to socioeconomic status indicator. Clustering of OW and OB by
socioeconomic status varies depending upon the study population. There is growing
evidence that the distribution of OW and OB varies by socioeconomic status and area of
residence, which depends on the economic development of the countries.16 In some
countries such as Russia,17 China,17 Croatia,18Estonia,18 Latvia18 and
19
Botswana, prevalence of OW and OB was higher among more affluent families, similar to
our results. In other countries such as the USA16, 20 and Spain,21 the highest rates of OW
and OB occur among the most disadvantaged groups.
An important finding of our study is that although the prevalence of OW and OB is still higher
in the population with higher socioeconomic status, the rate of increase of such prevalence
in the last 24 years has been higher in school-aged children and adolescents from the lowest
quintile of socioeconomic status. Some explanations for this rapid increase among the
poorer strata is supported by the increasing evidence that low-income families tend to
consume inexpensive sources of calories due to the relative cost of nutritious foods both in
money and preparation time. The source of these calories is usually from energy-dense
foods with high-fat content and sugar as well as with poor nutritional quality (low content of
vitamins and minerals).20 A recent analysis in Mexico shows that patterns of intake are
different depending on the level of income. The results indicate that the cost per calorie
(defined as the amount of money allocated to consume 1 calorie) decreased between 1992
and 2010. Households with lower-income levels make consumption decisions that allow
them to obtain a higher level of calories at a lower price, even if this represents a lower
dietary quality.22 The lower the socioeconomic level, the greater the percentage of purchase
and consumption of high-energy-dense foods as well as the lower the purchase and
consumption of low-energy-dense foods (mainly fruit and vegetables and low-fat/-sugar
foods).22 This phenomena has been reported by others, indicating that there is a negative
association between income and dietary quality. Thus, as incomes decrease, nutrient-poor
energy-dense foods become the best way to provide daily calories at an affordable cost, to
the detriment of healthier foods (such as fruits and vegetables), which are more expensive.23
There is little information in terms of changes in physical activity among children and
adolescents in the last 24 years. There are few studies in Mexican preschool24 and school-
aged children,25 and adolescents.26 These studies indicate that low physical activity level
and sedentary behavior are common among these age groups. In addition, there is evidence
that some of the determinants of low physical activity and sedentary behavior such as
urbanization, use of innovation in electronic media (computers, televisions) among others
have increased in the Mexican population during the last 20 years.27
Another relevant result of this study is the trend of the combined prevalence of OW and OB
among preschool girls shown to decrease between 1999 and 2006. As reported previously
by Rivera et al.,28 the observed decrease in prevalence for this group may be the result of
the decline in the prevalence of low height for age (stunting) observed during the same time
period, which was about 0.86 pp per year.
Finally, it is worth mentioning that among school-age girls there was an apparent plateau in
the combined prevalence of OW and OB from 2006 to 2012. This trend has been reported
in developed countries like the USA where the prevalence shown in some age groups such
as school-age children and adolescents appears to be leveling off 29 or Denmark where
comparison of prevalence from 1998 to 2011 showed that the prevalence rates of OW and
OB among Danish infants, children and adolescents were largely still at a plateau with
tendencies for a decline among children and adolescents.30Results in the same direction
were previously reported in a review of prevalence of OW and OB in nine countries
(Australia, China, England, France, The Netherlands, New Zealand, Sweden, Switzerland
and USA), where prevalence of OW and OB was stabilizing.31 It may be too early to conclude
that this is happening with the prevalence of OW and OB in school-age children. Follow-up
in regard to this trend with further National Nutrition Surveys will be useful to answer this
question. In addition, although Mexico is demonstrating a plateau in OW and OB in school-
age children, a public health crisis remains as a result of the high prevalence of childhood
OB.
This paper describes the most recent trends of childhood OB in Mexico. It includes
information from different age groups during childhood from four nationally representative
surveys during the last 25 years. Mexico is one of the few countries in the Latin American
and Caribbean region with repeated National Nutrition Surveys across time, allowing us to
explore trends.
Another strength of this study is that we used BMI as the indicator of body fatness. Although
the use of BMI as a measure of health risk related to body fat has been criticized, especially
in children,32 there is evidence that BMI in children is highly correlated with body fat mass
and widely used as a valid indirect measurement of adiposity in children. There has been
an increased number of growth references expressed as a function of age and sex. 31, 32
An additional strength of the study is the use of the WHO Child Growth Standard for
preschool children10 and the 2007 WHO growth reference for school-age children and
adolescents11 with their respective definitions of OW and OB, thus facilitating comparison
with data from other countries.
One limitation of our study is that not all age groups (lack of information in school-age
children on the 1988 national survey) and sometimes not both sexes, particularly boys and
adolescent males (school-age boys and male adolescents were not measured in 1988 and
2006), have complete information for the analysis of prevalence and trends in the last 25
years, limiting our conclusions for those groups for trends. However, we studied complete
trends during the last 25 years for girls and indicated, when necessary, the time frame of
trends in cases where information on both sexes was included.

Conclusions
Prevalence of childhood OB in Mexico is one of the highest worldwide. Even though the
prevalence shown here indicates a higher prevalence of OW and OB among those in urban
areas and those from more affluent families, changes in prevalence of OW and OB
presented here suggest that, as in other countries, in Mexico the burden of OB is shifting
toward groups with a lower socioeconomic level.

Footnotes
Supplementary Information accompanies this paper on the Nutrition & Diabetes website
(http://www.nature.com/nutd)
The authors declare no conflict of interest.

Supplementary Material

Supplementary Table 1
Click here for additional data file.(399K, pdf)

Supplementary Figure S1
Click here for additional data file.(177K, ppt)

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Nutrients. 2018 Jan; 10(1): 71.

 VII. Food Addiction and Binge Eating: Lessons Learned from
Animal Models
Marta G. Novelle* and Carlos Diéguez*

Abstract

1. Introduction
Eating disorders (ED), defined as disturbances in eating habits characterised by insufficient
or excessive food intake causing energy imbalance, are associated with high comorbidity
and have serious health consequences. Therefore, although the prevalence for ED has
remained stable, the high mortality rate, the association with other psychiatric disorders, and
an increased level of awareness of eating disorders between the general population and
clinicians have encouraged researchers to investigate the genetic, neurochemical, and
physiological substrates implicated in ED [1,2]. In a highly obesogenic environment, much
attention has been given to ED characterised by compulsivity and overeating, including
binge eating disorder (BED), certain forms of obesity, and the newly proposed construct of
“food eating addiction” [3]. Throughout this review we will briefly cover current knowledge of
the neurobiology of feeding behaviour, focusing on non-homeostatic circuits, and we will
look over the controversy about the misconception of “food addiction”. Finally, we will
explore new evidences learned from animal models in order to get a better understanding
of BED, recently integrated as a novel diagnosis into the Diagnostic and Statistical Manual
of Mental Disorders (DSM-5).

2. Understanding the Neurobiology of Eating Behaviour. Eating beyond Metabolic Needs

Food intake is an essential behaviour for survival and highly regulated by homeostatic,
hedonic and learned cues. Consequently, eating behaviour depends on a simultaneous
functioning of homeostatic pathway together with a more flexible non-homeostatic one,
whose functions can vary between individuals according to previous experiences and/or
epigenetic variations [4,5,6,7,8].
New insights argue that the impact of the modern food environment is mainly on cortico-
limbic brain systems dealing with reward, emotion and cognition. Signals from the cognitive
and rewarding brain may override classic homeostatic regulation leading to development of
obesity or eating disorders. These stimuli follow pathways that include, but are not limited
to, corticolimbic regions within the amygdala, hippocampus, and thalamus; mesostriatal
dopamine (DA)-gated circuits within the nucleus accumbens (NAc) and the ventral
tegmental area (VTA); and prefrontal cortex (PFC) regions predominantly within the
orbitofrontal projections [9,10,11,12].
In this context, Berridge and collaborators described three aspects of reward: liking, wanting,
and learning, that despite being tightly linked they can be dissociable in terms of their neural
substrates yet. So, while liking and wanting, respectively, refer to the hedonic impact of and
the motivation for a reward, the learning process comprises the associations with and
predictions about rewards [13,14]. Animal models have mainly associated opioid,
cannabinoid, orexin and γ-aminobutyric acid (GABA) systems as mediators in the “liking”
experience, via coordinated activity in a network of hedonic hotspots in the nucleus
accumbens, ventral pallidum and brainstem. Moreover, these neurotransmitters can be also
implicated in other processes of the reward regulation, as opioids enhancing the “wanting”
[15]. On the other hand, the mesolimbic dopamine system is crucial in the “wanting” and
“learning” components [13,16]. It should be pointed out that “liking” and “wanting” systems
are essentially pure “go” systems. That means, once they are activated cannot be
diminished by satiety influences, they never generate a strong “stop” signal to halt intake,
they only tone down the intensity of the “go” [17]. Interestingly, the incentive sensitization
theory of addiction proposed by Robinson and Berridge, is based on a pathological incentive
motivation (wanting) for drugs even after the discontinuation of drug use, that can be
manifest in behaviour via either implicit (as unconscious wanting) or explicit (as conscious
craving) processes, depending on circumstances. These features are linked with learning
mechanisms that normally direct motivation to specific and appropriate targets [18,19,20].
Likewise, excessive “wanting” and “liking” for food, notably hyper-palatable food, may play
a role in overeating. Moreover, as in in drug dependence, the attractive and rewarding
properties of hyper-palatable foods do not remain confined to the reward itself. Reward-
related cues, in this case food cues, can be attributed with excessive incentive salience and
become signals that draw attention and trigger overconsumption [21]. In other words, the
wanting of addiction is connected less with pleasure (liking foods), and more with the
negative reinforcement created by their withdrawal. Consequently, wanting and craving high
sugar and high sugar/high fat foods are more about trying to prevent the return of negative
feelings [22,23].

2.1. The Role of Opioid System, More Than “Liking” Regulation

‘Liking’ and ‘disliking’ of a food was determined by carefully observing the orofacial
expressions of rats drinking caloric test solutions. The original idea to observe rats’ facial
expressions to measure how much pleasure (or aversion) they are getting from a given food
was inspired by earlier human studies and later adapted for rodents [24,25,26].
As we already have commented above, endogenous opioid system is crucial in “liking”
aspect of the reward process. Despite of opioids are involved in a broadly distributed neural
network, affecting both homeostatic and hedonic mechanisms; the dominant view is that
opioids, especially the mu-opioid system, regulate the “hedonics of feeding” by their
modulation of the palatability of food regardless of the caloric value presented. Opioids
stimulated ingestion of “attractive” diets in sated rats, enhanced the “dessert effect” when a
palatable food was offered at the end of a regular meal [27]. On the other hand, opioid
antagonists attenuate appetite for palatable food. Thus, craving for palatable food could be
considered as a form of opioid-related addiction [28,29].
All these conclusions are largely based on evidence obtained from animal models. Some
studies tried to elucidate if opioids stimulate intake of specific macronutrients or of preferred
foods. Several works showed that when rats could select the macro composition of the diets,
after an injection of morphine, a µ-opioid receptor agonist, animals had higher preference
for fat intake, instead of carbohydrate intake [30], in contrast, the opioid antagonist, naloxone
preferentially decreased fat intake [31]. According to these data, opioids regulate the intake
of specific macronutrients. However, when the baseline dietary preferences of the rats were
considered, after morphine injections, it was observed that opioidergic modulation of feeding
may be driven more by individual preference than by macronutrient, since morphine
primarily stimulated carbohydrate intake in the carbohydrate-preferers, and stimulated fat
intake in the fat-preferers [32]. Glass et al. reported a complementary result with naloxone
injections: the intake of the preferred diet was reduced by naloxone at lower doses than
those required to reduce intake of the less-preferred diet [33]. Lately, it was observed that
the opioid effects of preferred versus non-preferred food were dependent on the site of
injection. Therefore, while the naltrexone (NTX, preferential μ-opioid receptor antagonist)
injections in the central nucleus of the amygdala caused a decrease in intake of the preferred
food, injections in the paraventricular nucleus of hypothalamus caused a decrease in the
intake of both foods [34]. The role of µ-opioid stimulation is not only region-dependent. While
µ-opioid stimulation via microinjection of DAMGO (µ-agonist) within the rostrodorsal
quadrant of NAc medial shell can double the hedonic impact of sweet tastes, stimulation in
other sub regions of medial shell does not increase “liking” reactions to sweet food [35,36].
Finally, it should be noted that hedonic enhancement is also receptor (mu, delta, kappa)
dependent [37]; although μ- and κ-opioid receptor stimulation increased the ‘liking’, only the
μ-opioid receptor stimulation increased the incentive motivation for food [38]. Other
researchers also provided some evidence of the role of opioid system either macronutrient
or preference-specific effects on food intake. These studies showed that nucleus
accumbens opioidergic effects were influenced by the relative preference for specific foods,
foods high in fat or sugar. When both high-fat and high-sugar foods were available
simultaneously, opioid stimulation increased intake more for the high-fat food [39,40,41].
However, Mena and collaborators showed that intra-PFC μ-receptor stimulation augments
the reward valuation of carbohydrate-enriched foods, along with several behavioural
changes such as a high-arousal and stress-like state. The authors suggest that this
carbohydrate hyperphagia could represent an attempt to suppress a stress-like aversive
state, since PFC is significantly activated by stress [42]. One mechanism that could
explained the opioid-mediated overconsumption of palatable foods is through delaying the
satiety systems; either the melanocortin or oxytocin systems [27]. These preferences and
craving for appetizing foods were also observed in several human studies [43,44,45,46].
Beyond “liking” process, opioid stimulation directly causes increased cue-triggered ‘wanting’
as well as dopamine stimulation. Therefore, opioid mechanisms can also regulate incentive
motivational, to wit propensity to seek palatable foods [37,38,47]. In fact, for example, the
injection of non-selective opioid receptor antagonist, nalmefene, blocked the anticipatory
negative contrast in the binge-eating procedure as well as highly palatable food binge eating
[48]. External food-related cues, learned cues, can also precipitate the desire for food,
increasing the food craving independently of homeostatic needs. In this context, using
Pavlovian-instrumental transfer (PIT) paradigm, that can model mechanisms responsible for
producing “cue-triggered wanting” or craving [49], it was shown that opioid stimulation
caused “wanting” as well as dopamine [47]. Additionally, NTX, and GSK1521498 (μ-opioid
receptor antagonist) were tested on food seeking behaviour using chocolate-flavoured pellet
reinforcement. Both compounds reduced food intake, but only GSK1521498 reduced the
seeking responses for chocolate before ingestion, suggesting that μ-opioid system has a
crucial role on incentive motivational mechanisms controlling food seeking [50]. Noteworthy,
the same compound also reduced motivational responding in binge-eating obese people in
a placebo-controlled trial, although subjective liking increased following drug treatment [51].
It has been hypothesized that μ-opioid receptors localized on the GABAergic interneurons
in the VTA may act to decrease dopamine release in the NAc to reduce food seeking and
incentive motivation for food [38,52].
Additional studies have also confirmed the role of opioid systems in other brain structures in
the motivational mechanisms underlying eating behaviour. Therefore, when central
amygdala (CeA) was stimulated by DAMGO infusions (µ-opioid agonist) caused elevated
incentive motivation in subjects naturally attracted both by a predictive cue (sign-trackers)
and by a reward contiguous goal cue (goal-trackers); also, an increase in “wanting”
behaviour under PIT model [53,54]. On the other hand, likewise, μ-opioid receptors within
the medial prefrontal cortex (mPFC) mediate an important function in overeating. In fact,
naltrexone microinfused into the mPFC selectively reduced the consumption and the
motivation to obtain highly palatable food, but not standard chow [55], while intra-PFC
DAMGO engendered “high-drive-like” effects [56]. In this context, Baldo group has
suggested that neuroadaptations of the opioid system in mPFC could explain partly the
development of binge-like eating [42,57,58]. Such as it has been recently demonstrated,
mPFC exerts top-down control over midbrain dopaminergic interactions with the striatum
and an increase in the mPFC can suppress natural reward-related behaviour [59]. Finally, it
has been also reported that δ-opioid receptors in the NAc-Shell are involved in the effects
of predictive learning on choice between actions. In fact, under a PIT paradigm, the
treatment with δ-opioid receptor antagonist naltrindole, blocked this behaviour [60,61].

2.2. Dopamine System, the “Want” Pathway

The association between dopamine and food intake appears to date from ancient times and
it has been postulated to be linked to and play a key role in human evolution. Specifically, it
was proposed that that increased levels of dopamine were part of a general physiological
adaptation of our ancestors due to an increased consumption of meat around two million
years ago and later enhanced by further changes in macronutrient intake about 80,000 years
ago [62]. At present there are clear evidences that dopamine pathways and dopamine
receptors are involved in energy homeostasis. In fact, there are clear evidences linking all
the five subtypes dopamine-receptors to energy balance and metabolic homeostasis. In this
regard the FDA have approved a D2-agonist, bromocriptine, as adjunctive treatment for type
2 diabetes [63]. Moreover, most of all the FDA-approved antiobesity drugs, including
liraglutide, appears to act largely through dopaminergic pathways [64]. On the contrary
chronic consumption of dopamine antagonists as in patients with schizophrenia leads to
enhanced eating and weight gain. In general, the relevance of dopamine in the integrated
control of the homeostatic pathways involved in food intake is beyond any doubt. Detailed
studies on dopamine involvement in leptin- and ghrelin-elicited changes in food intake are
well established [65]. This dopamine effects appear to be mediated by both D1R and D2R,
being the latest one the most relevant. Moreover, heteromers of GHSR1:DRD2 have been
implicated in obsessive eating associated to Prader-Willi Syndrome [66].
Another property inherently relevant to feeding behaviour is the concept regarding to
reinforcement, motivation and incentive salience [67,68,69,70]. While “liking” is closer to
sensory processes, “wanting” is closer to decision making and motor action, by reflecting
the cue-driven inclination to choose one behaviour over another to optimize reward.
Dopaminergic projections from the VTA to the NAc and prefrontal cortex are the most
important component of the implicit or unconscious “wanting” system [71]. Part of dopamine
hypothesis of reward is based on the initial work conducted by Wise and collaborators,
where animals subjected to DA antagonist pimozide (specially D2 receptor) showed a
decrease in self-stimulation in ways that implied a devaluation of reward, a decrease in the
pleasure of the reinforcer [72,73]. Through the years many other groups have arrived at
same conclusions, DA is required for normal motivation and reward, and has a crucial role
in feeding behaviour; in fact, animals lacking dopamine throughout the brain and body do
not eat [74,75], although as it has been confirmed its role is brain-region dependent.
The NAc is a brain region in the ventral striatum that appears to play a crucial role in
behaviours related to natural reinforcers and incentive as well as initiating key intracellular
plasticity mechanisms required for learning about food resources [76,77]. Moreover,
dopamine dynamics differ substantially between the NAc core and NAc shell in relation to
distinct aspects of appetitive and aversive motivational states [78]. Pharmacological
blockade of D1 and D2 dopamine receptors in the NAc affects motor conduct and has small
effects on feeding patterns, but does not reduce the amount of food consumed. These
effects can be interpreted as reflecting a more selective role for dopamine transmission in
the anticipatory/approach phase versus the consummatory phase of feeding [79]. According
to this idea, Salamone and colleagues carried out several interesting studies examining in
deep the behavioural effects of moderate NAc dopamine depletions. They found that
dopamine depletion reduced the motor effort to obtain food reward, but approach or intake
did not decrease when food was clearly available [80,81,82], and still animals can have
hedonic responses for food in the absence of dopamine [83]. Therefore, dopamine system
would be responsive to reward predictors and seemingly unresponsive to the reward “itself”
[73,84]. Nevertheless, restoring dopamine signalling selectively to the dorsal striatum,
composed of the caudate and putamen, is sufficient to allow feeding, locomotion, and
reward-based learning [85]. Besides, an increase of D2 receptors in the striatum are
correlated with an optimal goal-directed behaviours and motivation [86,87].
In this context, the downregulation of striatal dopamine D2 function has been proposed to
explain the reward deficiency or reward hyposensitivity theory [88]. According to this, a
reduced D2R expression in the striatum, observed both in human and animal models, is a
neuroadaptive response in order to compensate the overconsumption of palatable foods
[89,90,91,92]. Furthermore, this reduced sensitivity potentially could predict a cause of
excessive eating and/or obesity [89,90,92,93]. Consistent with the reward deficiency theory,
some studies reported obese versus lean adults show lower striatal DA D2-like receptor
availability [94,95], moreover obese adults have less capacity of nigrostriatal neurons to
synthesize DA [96] and less striatal responsivity to tastes of high-fat/sugar beverages [97].
Also, patients with BED tend to have reduced level of DA in the brain [98]. Contrarily, other
groups have shown higher striatal DA in obese individuals [77,99]. Despite some
discrepancies regarding to striatal dopaminergic levels, the A1 allele of the D2/ANKK1 Taq1
polymorphism has been correlated with reduced D2R availability in the striatum, obesity and
compulsive behaviour [77,100]. Likewise, D2 receptors were reported to provide a target for
ameliorating binge eating behaviour in a rat model after NAc deep brain stimulation
[101,102]. Therefore, while DA receptor 2 antagonism in NAc increases binge-like feeding
[101], activation of serotonin 2C receptors in DA neurons inhibits this binge behaviour in
mice [103].
It is important to note that in addition to its role in motivational processes, cortico-mesolimbic
dopamine pathway also play an important role in mediating learning [14,104,105]. Changes
in learning pathways might change rewarded responses. Learning processes are highly
influenced by emotional and motivational components and required for reward prediction,
for making anticipatory responses, for guidance by cues, and for goal-directed action. These
cues from the environment, such as the sight and smell of food, or even advertisements for
food, are learned and become associated with future reward [11,14]. A range of animal
studies has demonstrated that food-associated cues can promote eating in the absence of
metabolic requirements [106,107]. Thus, food-predictive cues can stimulate eating in adults
and children, even when they are full [6,108]. The basis of cue-potentiated feeding (CPF)
behaviour is Pavlovian conditioning. A feature of CPF is that it tends to be specific for the
cued food and does not increase intake generally, which has similarities with the “cravings”
experienced by binge eaters [109]. Recent studies have shown that dopamine has a
selective role in stimulus–reward learning that is specifically associated with the attribution
of incentive salience to reward cues. This fact could explain as individuals who attribute
reward cues with incentive salience find it more difficult to resist such cues, a feature
associated with reduced impulse control [110].
The mPFC receives information about cues in the environment via the sensory cortices, but
also about the internal motivation to eat via dopamine neurones of the VTA [56,58]. A group
of mPFC neurons, which contain the dopamine D1R receptors, are activated during hunger-
induced food intake, and their stimulation and inhibition, increases and reduces feeding
respectively. The main target of the D1R-containing subset of mPFC neurones is the medial
BLA [111]. On the other hand, it has been hypothesised that the transition from voluntary
drug use to more habitual and compulsive drug use represents a transition at the neural
level from PFC to striatum; and a progression in the striatum from ventral to more dorsal
domains, involving its dopaminergic innervation [112]. Moreover, reduced dopaminergic
modulation has been suggested to impair inhibitory control over food intake and to increase
risk of overeating in humans [95].

2.3. Are There Other Neurotransmitters or Hormones That Can Modify “Liking” and/or
“Wanting” Behaviours?
In addition to the endogenous dopamine and opioid systems, various hormonal and
neuropeptide systems influence performance in one or more of the food motivation
behavioural paradigms described earlier. Signals such as leptin, insulin, ghrelin, glucagon-
like peptide-1 (GLP-1) and melanin concentrating-hormone (MCH), orexins, oxytocin,
serotonin between others, are involved in hunger and satiety signalling as well as reward-
related neurocircuitry. The review of this topic is complex and beyond the aim of this paper,
but detailed reviews of this topic can be found elsewhere
[113,114,115,116,117,118,119,120,121,122,123].

3. Can We Talk about “Food Addiction”?

The concept of food addiction has been suggested for the first time by Randolph in 1956
[124], but only recently it has received due attention, mainly because of its correlation to the
increasing rate of obesity. “Addiction is defined as a chronic, relapsing brain disease that is
characterised by compulsive drug seeking and use, regardless of unhealthy consequences”
[125]. This chronic relapsing disorder is comprised of three steps: preoccupation/anticipation
(craving), binge/intoxication, and withdrawal/negative effect. These three stages interact
with each other, becoming more intense, and eventually leading to the pathological state
known as addiction. Not all drugs produce the same pattern of addiction, but lately the
progression of this behaviour triggers alterations in normal brain function and consequently
induces neuroplasticity in all the structures implicated [22,23,126,127]. Remarkably,
addiction induces neuronal changes in prefrontal cortical and basal ganglia activities,
leading to reductions in control and decision-making skills, and causes a chronic
perturbation in brain reward homeostasis mainly in the mesolimbic dopamine system.
Moreover, the opioid, GABAergic and glutamatergic neurocircuitries play a key role in the
development of addiction [92,128,129].
In the context of environments saturated with food, where clearly, obesity has become a
worldwide problem in a short time, and the binge eating disease is the eating disorder with
more incidence [130,131] several questions have been put forward. Can overeating become
a pathologic attachment to food? If so, can clinicians and researchers assert that food
addiction is a new category of psychiatric disorder or brain disease? Compulsive sexual
behaviour, pathologic gambling, and hedonic overeating are important problems, but are
they addictions?
Although in some cases excessive consumption of food can fit with all DSM-required criteria
according to some, food addiction was not included in newest edition of the of the DSM
manual [132]. Although it can share some symptoms with BED and obesity, and includes
behavioural patterns similarly to substance use disorders [129]. One fundamental distinction
between currently accepted addictive substances and food is the fact that food is necessary
for survival. Notably, there is a still an ongoing debate between the scientific community
about whether food addiction is a misnomer and the phenomenon could be more accurately
categorised by an alternative designation [92,129,133,134,135,136,137,138,139,140,141].
Therefore, while some researchers see overeating as substance use disorders, where
people are addicted to sugar, salt, additives and high fat content [140,142,143]; other
suggest increased food intake related to obesity or eating disorders should be considered
as a behavioural addiction [136,144,145]. Different views on this topic are almost
unavoidable if we accept that it is quite unlikely that any animal model of food addiction can
mimicked to a large extent food/eating addiction in humans. However, some researchers
have tried to study the food addiction in animals by following the three-criteria model
proposed by Deroche-Gamonet et al. [146]. Tolerance, reduction in the effect of a drug
resulting from of repeated exposure to the substance, was observed after extended access
to a palatable diet [147] and also some data suggest that there is a cross-tolerance between
sweet solutions and opioids [148]. In this context, Woods hypothesizes humans must learn
to tolerate the intake of food in order to minimize its impact on the body, as they learn
responses to help them tolerate the administration of dangerous drugs [149]. Regarding to
the withdrawal component of addiction, negative effects after the abrupt discontinuation or
decrease in intake of drugs, conflicting results have been reported depending on the kind of
food. This is discussed below in Section 4.1.3. Many other aspects related to “food addition”
concept have been studied in animal models too. By using a so-called time-out model Ghizta
and colleagues have attempted to elucidate the difficulty to limit intake or food seeking; after
prolonged training, animals exposed to palatable diet increase their food seeking responses
[150]. Moreover, some studies have demonstrated that animals continue to seek the food
despite adverse consequences [89,151].
For the first time, the DSM-5 grouped a disorder not involving substance use (gambling
disorder) together with substance use disorders in a new category entitled: “Substance
Related and Addictive Disorders” [132]. In this context, our group agreed to other
researchers, and based on the current studies, consider that there is no enough evidence
to conclude that a specific food, food ingredient or food additive can be addictive. Although
is too early to draw definitive conclusions regarding the “food addiction” concept and further
work is necessary, we consider that we should talk about “eating addiction” or more precise
“addictive eating behaviour” [136,152]. In agreement with other authors [153], we believe
that some animal models from drug addiction research, as the three-criteria model [146],
should be applied in eating disorder field. These models may provide us new neuronal
mechanisms in order to elucidate differences between “food addiction” and other compulsive
eating behaviours.

4. Binge Eating Disorder, a “Full-Fledged” Pathology

The newest edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5)
has introduced important changes in the diagnostic system for eating disorders; trying to
improve the ability for clinicians to arrive at a precise diagnosis [132]. Perhaps the most
significant improvement with the DSM-5 is that Binge Eating Disorder (BED) is now
considered as proper diagnosis in parallel to other main eating disorders as Anorexia
Nervosa and Bulimia Nervosa [154]. BED is the most prevalent eating disorder (between 2–
5% of the adult population) and more common in women than men. BED is characterised
by compulsive episodes of disproportionate consumption of highly palatable foods together
with a strong sense of loss of control. Binge-eating episodes are often accompanied by
feelings of anxiety, shame, disgust and guilt, high risk of suicide, but they are not followed
by compensatory purging behaviours. Although BED is often associated with obesity many
BED patients have normal body weights [130]. Based on these characteristics this kind of
behaviour could be described as an addiction-like behaviour; that is, an “eating addiction”
[136]. However, despite of existing research suggests that there is an overlap between BED
and the “eating addiction” neither all people with BED meet Yale Food Addiction Scale
(YFAS) criteria for this addiction nor all people with eating addiction meet criteria for BED
[152,155,156]. In this context, Hoebel’s group has modelled in rodents all the aspects of
food addiction; “Bingeing”, “withdrawal”, “craving” and “cross-sensitization” with drugs of
abuse. These researches have reported that sugar has addictive properties similar to
psychostimulants and opioids, and confirmed it is possible to talk about sugar addiction as
a different disorder from BED [157,158,159,160].
Since BED is now considered as proper diagnosis, there is a renew interest on the animal
models used in order to uncover the neurobiological basis of BED. Herein we will review the
most relevant features of some of these models.

4.1. Lessons Learned from Animal Models

Animal models can provide invaluable insight for neurobiological disorder research,
especially when the aetiology is well characterised or when there are potentially genes
associated. Current animal models of BED can only provide some few characteristics of the
human disease. These models cannot reproduce all social context that influence human
eating behaviour; neither some psychological aspects, such as sense of lack of self-control,
blame or guilt. Moreover, there is not really a consensus about the criteria an animal model
should fulfil [161]. This is major drawback considering the existence of strong differences
with some rodent strains, e.g., obesity-prone vs. diet-resistant, that could be at the root of
some discrepancies. In addition, one of the main features of BED is the much higher
incidence in females and at specific stages of life as adolescence. The fact that the precise
correlation between age of laboratory rats and human is still a subject of debate and the
generalized use of male rats are further limiting factors. Despite this, these models have
contributed to the understanding of the eating disorders, mainly trying to elucidate the
neurobiological mechanisms implicated.
Many of the results obtained from animal models of eating disorders have been already
discussed throughout the different epigraphy of this article. Likewise, more in detail reviews
about this topic have been written over the last years
[129,162,163,164,165,166,167,168,169,170]. Therefore, we will look briefly the plausible
role of the genetic and environment background, and the relevance of different dietary
macronutrients used in BED models that have not been discussed yet. Finally, we will
highlight the state of the art of new drugs for the treatment of BED.

4.1.1. Genetic Factors

Although the genetic study of BED is still in early stages and there are no enough genome-
wide association studies (GWAS) focused exclusively on BED, preliminary evidences
suggests that could there be predisposing risk factors [171,172,173,174]. In fact, there are
good evidences that heritable factors make a significant contribution to the risk of developing
eating disorders [175]. Patrono et al. showed that exposure to environmental conditions
induces compulsion-like eating behaviour, depending on genetic background. Therefore,
when they compared two mice inbred strains, C57BL/6J and DBA/2J, they observed that
only DBA mice shift from motivated behaviour to compulsive eating behaviour. They
hypothesised that this result could be explained as a genetic vulnerability (low accumbal D2
receptors availability observed in this study) [176]. This same year, the Bryant group has
reported two genetic factors implicated in the development of BED in mice models. They
observed that C57BL/6NJ but not C57BL/6J mice showed rapid and robust escalation in
palatable food consumption and identified Cyfip2 (cytoplasmic FMR1-interacting protein 2)
as a major genetic factor in preclinical BED; suggesting that could be associated with
maladaptive feeding in humans. Based on the association of Cyfip1 with Prader-Willi
syndrome, one hypothesis is that Cyfip1 polymorphisms also affect BED and hyperphagia
[177]. At the same time, they showed Csnk1e (casein kinase 1 epsilon) deletion increased
binge eating behaviours by enhancing opioid-induced locomotor activity [178]. Moreover,
these authors have also reported a robust strain difference in BED (C57BL/6J versus
DBA/2J strain) in accordance with previous results. Therefore, D2J showed escalation in
consumption, conditioned place preference for the food-paired side and compulsive-like
eating in an anxiety-provoking environment relative to B6J [179]. In this same context, rat
strain differences in binge eating proneness have been examined. Results showed that the
Sprague-Dawley female strain is particularly vulnerable to binge eating behaviours, while
the Wistar female rat strain is particularly resistant to BED. Moreover, Sprague-Dawley
males showed low risk for binge eating too. Taken together these results highlight the key
role of sex and genetic backgrounds in the propensity to binge eating behaviours [180].
Despite of oestradiol reduces meal size and is associated with reduced binge frequency,
women are more likely to suffer from BED. In a fat binge eating animal model it was observed
that ovarian hormones, oestradiol and progesterone, have a tonic inhibitory effect on food
intake, but the normal cyclic inhibitory effect on eating was disrupted during binge-type
eating episodes. This observation suggests that these inhibitory effects of oestradiol could
be compromised by binge-type consumption of large fatty meals [181]. Concurrently, other
study has reported that bingeing also attenuated oestradiol tonic effects. While both tonic
and cyclic inhibition of chow intake was maintained after cyclic treatment of ovariectomised
rats with oestradiol alone or co-administrated with progesterone, neither tonic nor cyclic
inhibition of binge-type consumption was observed once bingeing was fully established
[182]. Both studies indicate that oestrogens are the primary ovarian hormones responsible
for food intake and body weight regulation under binge-type conditions in rats. The
oestrogen metabolite, 2-hydroxyestradiol (2OHE2), has been hypothesised may enhance
bingeing in rodent models due to its possible interference with DA signalling. In fact, 2OHE2
may competitively inhibit degradation of DA and/or mimic or enhance D2 receptor actions
[183]. Corwin’s group showed as chronic administration of 2OHE2 to ovariectomised female
rats can exacerbate the induction of binge-type eating. Although they found that 2OHE2
attenuates the increase of food intake associated with ovariectomy, eating behaviour turns
toward a binge-type pattern, when 2OHE2 is administrated before binge development and
during all binge process. Therefore, this study suggests that chronic exposure to 2-
hydroxyestradiol facilitates “learning to binge” [184]. The binge-eating proneness is also
dependent on age. For both rat strains studied, Otsuka Long Evans Tokushima Fatty
(OLETF) and lean control strain, Long Evans Tokushima Otsuka (LETO), the onset of binge
eating behaviour was observed earlier in adolescents, with bigger binge size too. Higher
impulsivity in adolescents could explain their increased vulnerability to BED and overeating.
Furthermore, OLETF rats at both ages ate more than LETO rats. This is in accordance with
the co-morbidity of BED and obesity reported in humans [185]. However, not always the
binge eating behaviour is dependent on susceptibility to obesity, as animal models have
confirmed [186,187]. The development of aberrant eating behaviour during adolescence has
been also studied in other animal models (Sprague-Dawley strain). After postnatal treatment
with tricyclic antidepressant clomipramine, female rats showed more vulnerability to exhibit
binge-like eating behaviour during adolescence [188]. Sex differences in coping strategies
with anxiety have been described clinically. Female strategy, more consistent with harm
avoidance, has been associated with BED [189]. On the other hand, it has been proposed
that cognitive deficits in executive function, inhibitory control, attention, and mental flexibility
may play a role in development and sustaining of binge eating behaviour. When studying a
limited access rat model of binge-like behaviour, Chawla et al. found aberrant gene
expression of brain derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B
(TRKB) in the hippocampus (HPC)-prefrontal cortex (PFC) pathway. Moreover, they
observed reductions in the expression of insulin receptor in the CA3 region of the
hippocampus, up-regulation of serotonin-2C receptors in the orbitoprefrontal cortex and
decreased dopamine receptor 2 expression in the nucleus accumbens (NAc). Altered
expression of genes in the neural pathway HPC-PFC-NAc could be explained as
consequence of engaging in the binge behaviour. Authors have also speculated that animals
binge prone could have cognitive deficits and these contribute to their vulnerability [190].
Similar results were recently found in BED patients. Based on GWAS it has been possible
to identify genes involved in neuropeptide/neurotrophic pathways including neurotensin,
GLP1 and BDNF-TRKB signalling as potential therapeutic targets [191]. On the other hand,
evidence of epigenetic modification affecting the N/OFQ (Nociceptin/Orphanin FQ) and
corticotropin-releasing factor (CRF) systems in response to food restriction and stress
exposure has been demonstrated. These mechanisms could be designed to maximize the
possibility of survival in the presence of food restrictions, but in a society where nutrients are
easily available, alteration of N/OFQ and CRF mechanisms may contribute to BED
development [192].

4.1.2. Environmental Factors

Among the many environmental factors that can influence eating disorders together with the
high palatable food availability, the high incidence of stress in western societies is probably
one of the most relevant [169,193]. It is well documented with different animal models that
stress can influence feeding behaviour [193,194,195,196,197,198] and increase the
susceptibility to developing eating disorders [167]. Most of the animal models have focused
the investigation on the regulation of the stress neurohormone corticotropin-releasing factor
(CRF) system. Calvez et al. have found that stress differentially regulates brain expression
of CRF in binge-like eating prone (BEP) and resistant (BER) female rats. Therefore, in
response to stress, the BER rats significantly enhanced expression of CRF mRNA in the
paraventricular nucleus of the hypothalamus and strongly increased the plasma
corticosterone levels. Conversely, the BEP rats did not displayed stress-induced activation
of the HPA axis, but they demonstrated high CRF mRNA expression in the oval and
anteroventral bed nucleus of the stria terminalis (BNST), an important region for the
motivated behaviour, in response to stress [199]. Moreover, BEP rats showed different
behavioural and hormonal responses to stress. They showed higher motivation for palatable
food and perceived hedonic value [200]. Function of BNST was also assessed in other
models. Indeed, frustration stress manipulation increased BNST neuronal activity and CRF
receptors of BNST seem to have a critical role in stress-induced binge eating disorder [201].
The role of other extra-hypothalamic CRF1 receptors, as those in central amygdala, has
been also implicated in development of binge eating [202]. The early emotional environment
also impacts on eating behaviour. Early attachment experiences increase the risk of eating
disorders. In fact, the impact of perinatal programming on the mechanisms regulating body
weight homeostasis has been fully studied [203]. This same year, an interesting work has
shown that late gestation prenatal stress rewires neural circuits in female mice, leading to
binge-like behaviour [204]. Authors reported that overexpression of maternal CRF, during
late gestation affects the hypothalamus of female offspring, altering the expression levels of
genes responsible for DNA methylation and predisposes adolescent female offspring to BE-
like phenotype. Offspring with BE-like behaviour presented hypomethylation of
hypothalamic miR-1a and downstream dysregulation of the melanocortin system through
Pax7/Pax3. Finally, authors showed that although stress-related epigenetic predisposition
can increase the vulnerability to BED, it can be prevented from being triggered with a methyl
balanced diet during adolescence.

4.1.3. Food Models: Sugar Model, Fat Model, Sweet-Fat Model

The sugar bingeing model was proposed by Avena and collaborators [158,205]. This model
uses sucrose, glucose and saccharin in different liquid solutions. After some days under this
food animals modify the feeding behaviour and it is possible to induce neurochemical
changes in the brain. Even, this model increases DA in NAc [206] and cessation of sweet
food availability induces withdrawal-like behaviour [93,207]. Following withdrawal from ad
libitum access to sugar diet, animals also showed depressive-like behaviour [208,209].
However, in a Wistar rat model of binge eating based on daily 10-min access to a sweet fat
diet, there was no increased anxiety-like behaviour after diet withdrawal. In this same model,
it was observed that pre-treatment with the cannabinoid type 1 receptor antagonist
SR147778 dose-dependently reduced binge-like intake, while binge-like intake was
unaffected by pre-treatment with the corticotropin-releasing factor type 1 receptor antagonist
R121919 [210]. Sugar model has also been reported to increase the cross-sensitization with
other substances [129,205]. Recently, it was observed as binge-like sucrose consumption
reduces the dendritic length and complexity of principal neurons in basolateral amygdala
(BLA) in an adolescent rat model. These maladaptive changes observed in dendritic
architecture of BLA principal neurons, particularly on apical dendrites, show the importance
of the BLA in encoding emotional salience [211]. Furthermore, sugar models have provided
evidence of the relevance of dietary restraint as an antecedent to sugar binges, in
concordance with human studies [212] and can reproduce one human feature of binge
eating known as “eating when not physically hungry” [213]. The studies suggest that, as with
sugar models, a similar addiction-like state may emerge with fat intake. Insight in this issue
was obtained with the limited access model proposed by Corwin [214,215]. In this
experimental paradigm, the rats are given sporadic (generally 3 times per week, binge
group) or time-limited (daily access control group) access to palatable food, in addition to
the continuously available chow. The palatable food typically is a bowl of pure vegetable
shortening, but other palatable food can also be tested including sucrose solutions, various
concentrations of fat presented as solid emulsions, high-fat diets, and fat/sucrose mixtures.
In terms of neurochemical changes, it has been reported as binge eating of fat affects DA
signalling in the accumbens and γ-aminobutyric acid (GABAB) receptors. In fact, licking of
100% corn oil increases DA and its metabolites in the NAc, similar to that seen in sugar-
bingeing animals [216]. On the other hand, peripheral administration of the D2-like
antagonist raclopride stimulated fat intake in intermittent-access rats and had no effect in
daily-access rats. These results further implicate D2 receptors in the consumption of fatty
food, but also indicate as well as uncover the role of differential pre- and post-synaptic D2
signalling under binge and control conditions [215,217]. In this same study, Corwin and
colleagues have reported the role of GABABreceptors in fat binge eating models was
studied. GABAB agonist baclofen reduced intake of shortening, as well as high-fat solid
emulsions, in rats with both daily and sporadic brief access at doses that stimulated or had
no effect on chow intake [217,218]. However, baclofen did not have effect on intake of sugar
solutions or when sugar concentration was high [186,219,220]. Corwin’s model as well, has
shown an increase in progressive-ratio responding in rats under binge eating fat, suggesting
enhanced motivation [215]. Moreover, a history of fat bingeing may predispose to exhibit
more robust addiction like behaviour to other abuse substances [221]. In contrast to sugar
models, animals under fat bingeing models did not display withdrawal-associated symptoms
[222], and even after several weeks fat-fed animals did not show significant changes in body
weight, supporting the idea that BED and obesity are two different pathologies [129]. It has
been proposed, that lack of opiate-like withdrawal signs in fat-bingeing rats may be caused
by fat-induced endogenous galanin activation, which can inhibit the relevant opioid effects
[163]. Finally, many studies have used a sweet-fat model (“cafeteria-diet”), which is probably
the model most similar to the huge availability and diversity of foods that we have nowadays.
According to sugar model, sweet-fat food also induced a downregulation of DA mesolimbic
pathway [89]. Moreover, after exposure to cafeteria diet, following acute withdrawal animals
showed increase in locomotor activity, stress and anxiety-related behaviour
[207,208,223,224].

4.1.4. Neuropharmacology of Binge-Eating Behaviour

The treatment of binge eating disorder is challenging and require a big-picture treatment
plan to meet the individual needs; combination of psychotherapy and medication is usually
the best treatment strategy [130]. Oral lisdexamfetamine dimesylate (LDX), a prodrug of
dextroamfetamine, is currently the only drug to be approved in the USA for the treatment of
moderate to severe BED in adult patients [225]. When given acutely in a female rat BED
model, LDX attenuated binge-eating of chocolate without influencing the consumption of
normal chow or affecting the bodyweight of the animals [226] and reduced impulsiveness
and perseverative behaviour of binge-eating rats in a novel food reward/punished
responding conflict model [227,228]. Pharmacological characterisation indicated that LDX
attenuates binge-eating in part by indirect activation of α1-adrenergic and possibly also
dopamine D1 receptors in the CNS, but α2-adrenergic and D2 receptors are not involved
[226]. Other novel pharmacological treatment approaches also give us additional information
regarding the underlying neuro-pathways implicated in BED. For instance, the D2 receptor
antagonist raclopride reduced sucrose intake [220]. Methylphenidate (MPH), which inhibits
the monoamine uptake transporters for DA and norepinephrine, also reduces sucrose
bingeing episodes in animal models. Concomitant with this, MPH treatment leads to
increased DA transporter and D2 receptor binding in the NAc shell [229]. GS 455534, an
aldehyde dehydrogenase-2 inhibitor that reduces DA synthesis has also been shown to
selectively reduce binge consumption of sugar. Moreover, GS 455534 is associated with an
attenuation of nucleus accumbens dopamine levels in sugar-bingeing rats model [230]. In
addition, it was observed that monoamine stabilizer (−)-OSU6162, which restores striatal
dopaminergic dysfunction, could be a novel BED treatment in rodent models since reduces
both binge-like eating and cue-controlled food seeking in rats [231]. Selective serotonin
reuptake inhibitors (SSRIs) have been also studied. Fluoxetine non-selectively reduced the
intake of normal food and highly palatable food in a model of binge-eating that included
cyclic caloric restriction and stress [232]. Moreover, it has been reported that fluoxetine
and D-fenfluramine (a serotonin releasing agent) reduced binge-eating in mice on an
intermittent high fat diet by enhancing midbrain dopamine neuronal activity [103].
Sibutramine, a monoamine reuptake inhibitor (MRI), removed from the worldwide market
because of an increased risk of myocardial infarction and stroke, has been also studied in
BED animal models [226,232], where reduced the binge eating behaviours. On the other
hand, selective 5-HT2C receptor agonist lorcaserin, developed and approved as an anti-
obesity agent, was recently shown to attenuate binge-eating in mice fed a high fat diet by
stimulating dopamine neuronal activity [103]. In a recent study, it has been reported for the
first time that trace amine-associated receptor-1(TAAR1) may represent a novel target for
the treatment of BED. RO5256390, a TAAR1 partial agonist, completely blocked
compulsive-like eating and conditioned rewarding properties of palatable food when studied
in a rat model with limited access to a highly palatable sugar diet [233].
As we have already mentioned previously, the opioid system plays a key role in binge
behaviours. In fact, opioid receptor antagonists, including naltrexone, nalmefene and
GSK1521498 decreased both normal food intake and compulsive or excessive eating in
several different binge-eating models in rats [55,226,234]. However, despite the positive
pre-clinical evidences, placebo-controlled clinical studies in binge-eating disorder with opioid
receptor antagonists have been disappointing [130].
The orexin system plays a role in eating disorders characterised by compulsive binge-type
episodes, such as BED. For instance, in two different rat compulsive binge-eating models
orexin receptor 1 (OXR1) antagonist SB-334867 and GSK1059865 specially reduced
palatable versus normal food intake [226,235]. However, JNJ-10397049 a selective orexin
receptor 2 (OXR2) antagonist, failed to affect the intake of palatable food and the dual
OX1/OX2 receptor antagonist SB-649868 selectively reduced binge-eating of highly
palatable food but did not affect the chow food pellet intake [235]. In this same context, it
was observed that SB-334867, a selective OXR1 antagonist, decreased the binge-like
consumption behaviour in an ad-lib feeding animal model [236].
The combination of pharmacological agents acting on different neural pathways may also
provide novel therapy for BED. Modulation of GABAergic neural systems also represents a
possible approach for BED treatment. Baclofen, a GABAB agonist, attenuated the intake of
highly palatable food in several different animal models of binge-eating [219,220,226,237].
Recent studies have suggested Nociceptin/Orphanin FQ (N/OFQ) system, a functional
antagonist of corticotrophin-releasing factor, as therapeutic target either obesity or BED. In
fact, intracerebroventricular injections of N/OFQ at low doses significantly reduced BE in
rats [238]. On the other hand, nociceptin receptor antagonist LY2940094 [239] and SB
612111 [240] inhibits excessive feeding behaviour in rodents. Interestingly, a novel drug for
the treatment of BED, may be BD-1063, a selective sigma1 receptor antagonist. Although
the selectivity of this compound has not been well characterised, BD-1063 blocked
compulsive binge-eating in rats trained to obtain a sugary or highly palatable diet [241].

5. Conclusions and Future Perspectives

This review briefly presents the latest knowledge regarding the neurobiology of non-
homeostatic pathways implicated in food intake regulation. Throughout the review we have
tried to discuss the most relevant aspects that can help us to understand the dysregulation
of brain reward systems in eating disorders, by focusing mainly on BED. However, me must
be aware that in most instances it is far from clear whether the changes are related to the
disease, consequence of altered homeostasis or a premorbid trait. During recent years,
research in animal models of compulsive overeating has provided us essential knowledge
to understand the neurobiology mechanisms underlying eating disorders, but there are still
many features waiting to be elucidated. The main brain structures implicated are shown
schematically in Figure 1. Among the mechanisms involved the role of dopamine appears
clear-cut. Recent neuronal mapping using single-cell RNA-seq. have shown the existence
of a higher diversity of neurones than originally expected with up to 62 neuronal subtypes
producing glutamatergic, dopaminergic or GABAergic markers for synaptic
neurotransmission having been identified. The challenge now is to associate the different
subsets of neurones to specific neurobiological process and their alterations to specific
disease-related process. After BED has been recognised as a proper psychiatric disorder,
clinical and basic research in BED is in an exciting phase among other reasons because of
the availability of new neuroimaging tools that allowed us to correlate basic neurobiological
aspects to functionality in the human brain. Development of well-characterised models and
new tools such as chemogenetics and optogenetics should provide us with a much better
mechanistic insight and possible therapeutic targets for BED. Finally, we should not forget
that any functional aspect of the brain is based on the complex interaction among many
different neuronal pathways. It is expected that as soon as larger knowledge is gather from
the connectome project [242] we will be in a much better position to tackle questions related
to complex diseases such as BED and how nutrient intake, in terms of total amount and
schedules, can influence cognitive process.

Figure 1
Schematic representation of main brain structures implicated in hedonic and homeostatic
food intake regulation. Main dopaminergic pathways, mesolimbic and mesocortical pathway,
are represented with red lines, and other minor dopaminergic connections with broken red
lines. Endogenous opioids peptides modulate the dopaminergic pathways through opioid
receptors (µ, κ, δ). Hedonic pathways: PFC, prefrontal cortex; NAc, nucleus accumbens;
VTA, ventral tegmentum area; LH, lateral hypothalamus; Amy, Amygdala; Hipp,
Hippocampus; SN, substantia nigra. Homeostatic pathways: Arc, arcuate nucleus; MCH,
melanin concentrating-hormone.

Acknowledgments
This work has been supported by grants from Ministerio de Economía y Competitividad (CD
BFU2014-55871), Xunta de Galicia (ED431, 2017/030). Centro de Investigación Biomédica
en Red (CIBER) de Fisiopatología de la Obesidad y Nutrición (CIBERobn). CIBERobn is an
initiative of the Instituto de Salud Carlos III (ISCIII) of Spain which is supported by FEDER
funds.

Author Contributions
Both authors substantially contributed to the conception of the manuscript. M.G. Novelle and
C. Diéguez contributed to the literature search and writing. Both revised and approved the
final version.

Conflicts of Interest
The authors declare no conflict of interest.
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VIII. Subjective craving and event-related brain response to

olfactory and visual chocolate cues in binge-eating and
healthy individuals
I. Wolz,1,2 A. Sauvaget,3,4 R. Granero,2,5 G. Mestre-Bach,1,2 M. Baño,1,2 V. Martín-
Romera,5M. Veciana de las Heras,6 S. Jiménez-Murcia,1,2 A. Jansen,7 A. Roefs,7 andF.
Fernández-Arandaa,1,2

Abstract
In our modern society, food is omnipresent; it can be easily purchased and rapidly
consumed, without the need of further processing steps. The ubiquity of food in the
environment may be highly problematic for some individuals, leading to obesity or even
eating disorders. Binge eating disorder (BED) and bulimia nervosa (BN) are both marked by
recurrent binges and high experienced craving1. Craving is defined as a strong and
irresistible desire to consume a specific substance and often leads to loss of control over
food intake2. External cues such as the sight or smell of food are known to trigger craving
and overeating3. The purpose of this study was to look at differences in the processing of
and reaction to high-palatable food stimuli in patients with binge-eating (BE) when compared
to healthy normal-eating adults. This could help to better understand the susceptibility to BE
and cognitive processes which may protect from this kind of behaviour.
Chocolate is one of the most heavily craved foods and perceived as highly problematic with
regard to the controllability of its intake4. However, it is not yet clear how exactly chocolate
consumption influences physiological, psychological and biological functioning. There is
some evidence suggesting that it is not the pharmacological effect of chocolate alone, nor
its high sugar content, which produce these strong cravings in humans, but rather the
sensory experience, a combination of different factors such as aroma, caloric content, and
texture5. So far, however, little attention has been paid to the role of odour in the generation
of craving.
With regard to olfactory cue-induced craving, research has shown that the smell of food
leads to both intensified craving and increased food intake in restrained as well as in normal
eaters6. Although, in contrary to taste, food odour is not a primary reinforcer, specific odours
might get associated to the taste and reward response of food through conditioning 7.
Furthermore, the combination of odour and taste are known to create flavour and thus
together influence the reward value of food in the orbito-frontal cortex8,9,10. There is evidence
to suggest that a combination of olfactory and visual stimuli lead to a more powerful craving
response than visual stimuli alone11. In line with this, a study on reward processing found
that a combination of viewing and tasting chocolate led to greater activation in the orbito-
frontal cortex than the sum of these two modalities when presented separately, which was
denominated as “supralinearity”12. These findings indicate that olfaction might be an
important player in cue-induced craving and increase the craving response to visual stimuli.
As to the effect of food odours on brain activity, basic studies in humans have reported a
reduction of low frequency electroencephalogram (EEG) activity in response to different
odours13,14; for chocolate odour in specific reduced frontal theta activity (4–7 Hz) was found
in comparison to other odours or to neutral olfactory stimulation15. The authors explained
this by a more relaxed state when participants are exposed to chocolate odour; it is however
unclear how this reduction in theta frequency may relate to chocolate craving. A recent study
found increases in theta power to be related to associative appetitive learning of images to
food stimuli16; investigating differences in theta power in response to chocolate odour
between patients with binge-eating and healthy controls might be helpful to conclude on the
importance of odour in stimulus-induced craving.
With regard to visual stimuli, incentive salience of food cues, which corresponds to the
craving or “wanting” for these stimuli17, has been studied using event-related potentials
(ERP) of EEG. The Late Positive Potential (LPP) is an ERP scaling the motivational value
of pictures in that more arousing stimuli lead to higher amplitudes18. It is enhanced for
substance-related stimuli in people with substance use disorders19 and can be seen as an
indicator of “motivated attention” towards salient stimuli20. In a similar way, food stimuli
compared to neutral stimuli in general lead to higher LPP amplitudes21,22, which illustrates
their high incentive salience in healthy, normal-weight individuals. A much debated question
is therefore, how motivated attention towards food relates to craving and BE. Contradictory
results regarding processing of food stimuli in disordered eating were reported in studies
using behavioural measures of attentional bias23. ERPs have the potential of a more exact
timing and it seems that people with overeating or BE compared to normal-eating individuals
may have enhanced attention to food in early ERPs, but there is inconsistency regarding
later components, such as the LPP (for a review see Wolz et al., 2015b).
Low inhibitory control is another important aspect of overeating, loss of control over food
intake and addictive behaviours24. Therefore, another component which might be helpful for
the understanding of food processing in binge-eating is the N2, an early negative-going
anterior-frontal ERP. It has been related to the dorsal anterior cingulate cortex and its conflict
monitoring function during Go/No-Go tasks25,26. In passive picture viewing paradigms, the
N2 has been associated to stimulus discrimination, the automatic evaluation of affective
valence, and arousal27,28. Another recent study found that N2 amplitudes were associated
with the suppression of the emotional response while viewing neutral and negative pictures,
which the authors interpreted as an indicator of conflict29. However, with regard to the
processing of food stimuli, anterior negative deflections in the N2 time-range in two studies
showed that higher amplitudes were related to an increased reactivity to food stimuli30,31. It
was furthermore suggested by Asmaro and colleagues that this anterior negative deflection
might be related to top-down cognitive control mechanisms over the desire to consume
chocolate31.
The main aim of this study was to compare BEP to HC with regard to their craving and
neurophysiologic response towards visual chocolate stimuli. Thereby it was hypothesized
that chocolate pictures would evoke higher craving (self-report) and LPP and N2 amplitudes
than neutral pictures (hypothesis 1a) and that there would be a positive relation between
self-reported craving and LPP and N2 amplitudes (hypothesis 1b). With regard to group
differences, it was hypothesized that BEP compared to HC would have more craving (self-
report), more motivated attention (LPP) and less cognitive control (N2) resources
(hypothesis 2). Moreover, we expected an increase of state chocolate craving throughout
the experiment (hypothesis 3). A second aim was to explore the influence of chocolate odour
on the processing of visual chocolate stimuli; we hypothesized that olfactory and visual
stimuli would have an additive effect, leading to a potentiated response (hypothesis 4). The
third aim was to test if there are differences between BEP and HC in the response to
chocolate odour alone. We expected a higher subjective craving reaction towards odour
stimuli in BEP than in HC (hypothesis 5); regarding theta frequency, due to the explorative
nature of this aim, no directional hypothesis was put forward.
Materials and Methods

Participants
The HC group (n = 20, age 20–56 years, see Supplementary Table S1 for means (M)
and standard deviations (SD)) was recruited from students of the University of Barcelona
and through a snowball system from hospital staff. The BEP group (n = 19, age 19–56 years)
was recruited from consecutive referrals to the ED unit of Bellvitge University Hospital and
diagnosed by means of semi-structured face-to-face interviews to either BN (n = 12) or BED
(n = 7) according to the criteria of the fifth edition of the Diagnostic and Statistical Manual
(DSM; American Psychiatric Association 2013). See Supplementary Tables S1 and S2 for
a description and comparison of the two groups with regard to socio-demographic and
clinical variables.
Exclusion criteria for all participants were: being male, younger than 18 years, current or life-
time history of chronic illness (which could influence electrophysiology) or neurological
condition (abnormal EEG activity), having used in the last 24 hours psychoactive medication
or drugs that may interfere with smell-taste capacity or cortical activity, current substance
dependence, lifetime diagnosis of psychotic disorder, functional anosmia (value <16.5 in
“Sniffin’ Test”), and pregnancy. Additionally, in the HC group, an exclusion criteria was a
lifetime diagnosis of ED, assessed by means of Structured Clinical Interview for DSM-IV
Axis I Disorders (SCID-I) (First et al. 1997) or being obese (Body Mass Index [BMI] ≥ 30) or
underweight (BMI < 18.5).

Procedure
The study was conducted in compliance with the Declaration of Helsinki (1975) and the
Spanish legislation and norms, after revision and approval by the local ethics committee
(CEIC Ciutat Sanitària i Universitària de Bellvitge). All participants signed informed consent.
The study participation consisted of two parts of approximately one hour each, which were
realized either in one or in two separate sessions (50% of HC and 63% of BEP did two
sessions). The first part was to check inclusion criteria, participants were weighed and
measured (height and head circumference), and were tested regarding their olfactory
capacity. In the second part participants filled in a laboratory questionnaire (momentary
mood and hunger (1-item Likert scale from 1 to 9), food eaten this day, menstrual cycle, and
intake of coffee, alcohol and drugs in the last 24 hours), then the EEG electrodes were
placed on the participant’s scalp and she did the experimental task with a duration of
26 minutes. Participants were instructed to have a normal meal two hours before doing the
second session and then to refrain from eating until completion of the experiment.

Study design and experimental task

Hypotheses were tested in a controlled mixed study design. The intra-factors “odour type”
and “picture type” were presented at random and counter-balanced order, with 4
combinations of chocolate versus neutral valence in the two sensory modalities (neutral
odour - neutral picture, neutral odour - chocolate picture, chocolate odour - neutral picture
and chocolate odour - chocolate picture). Each condition was done twice, in two consecutive
sections with 8 blocks of 56 trials each, leading to a total of 224 neutral and 224 chocolate
picture trials (see Fig. 1).
Figure 1
Experimental task and conditions.
EEG = Electroencephalogram; FCCQ-S = Food Chocolate Craving Questionnaire;
VAS = visual analogue scale.
Visual stimuli were 56 pictures each of chocolate products and neutral office items, used
and kindly placed at our disposal by Frankort and colleagues (2014). Pictures were
presented in random order on a grey computer screen using the stimulus delivery software
Presentation (Neurobehavioral Systems); one block consisted of 56 trials, each starting with
a fixation cross (500 ms), followed by the picture (1200 ms). Each visual block was preceded
by a 1 min olfactory exposure to either chocolate or neutral odour, presented by a laboratory
assistant; the participant had her eyes closed during this exposure. During the olfactory
stimulation, participants were exposed to the smell of either a piece of chocolate or a pencil
(as in Frankort et al.11). For a graphical description of the task see Fig. 1.

Assessment

Self-report measures
Subjective chocolate craving was assessed on three different levels: trait craving was
assessed during the baseline assessment session and state craving was assessed before
and after the experimental manipulation through the Food Chocolate-Craving
Questionnaire (FCCQ) – State and Trait Version32,33 (see Supplementary Material, section
S1.2., for a more detailed description and psychometrical evaluation of these scales). The
momentary craving reaction was assessed through a visual analogue scale (VAS) scaling
from 0 (very little) to 100 (very strong) asking “At this moment, how much desire to eat do
you have?” at 17 time points throughout the experiment, once at baseline and then after
each of the odour and visual presentations in the eight blocks.
Additional psychometric questionnaires were used in order to assess difficulties in emotion
regulation (Difficulties in Emotion Regulation Scale34), food addiction (Yale Food Addiction
Scale35), eating disorder pathology (Eating Disorder Inventory-II36) and general
psychopathology (Symptom Checklist-90 revised37); olfactory capacity was assessed using
the “Sniffin’ Sticks” test38 (for a description and reliability measures of the psychometric
scales and of the “Sniffin’ Sticks” test see Supplementary Material, section S1).
Electrophysiology
EEG was recorded continuously throughout the experimental task using PyCorder
(BrainVision). 60 active Ag/AgCI electrodes were inserted into an EEG recording cap
(EASYCAP GmbH), distributed after the 10–20 system, the Cz electrode was used as online
reference. Four electrodes were placed next to the eyes in order to control for eye
movements. Impedances were reduced to be smaller than 20 KOhm using the SuperVisc
high-viscosity electrolyte gel for active electrodes. A sampling rate of 500 Hz and an online
filter between 0.1 and 100 Hz were used.
The N2 was measured at electrodes AFz (central N2), AF3, F1, F3 (left N2) and AF4, F2,
F4 (right N2) as the amplitude and latency of the maximum negative peak in the time window
180–350 ms after visual stimulus onset. The time window for the LPP was set to 300–
1000 ms after visual stimulus onset and measured as the maximum positive peak at centro-
parietal electrode sites: Pz (central LPP), CP1, CP3, P1, P3, P5 (left LPP) and CP2, CP4,
P2, P4, P6 (right LPP). See Supplementary Material, section S2, for a more detailed
description of ERP analysis steps and for a description of the analysis of theta power.

Statistical Analysis
Statistical analyses were conducted with SPSS20 for Windows. For a description of the
sample, socio-demographic and clinical variables were compared between the two groups
using analysis of variance (ANOVA).
In order to test hypotheses 1a, 2 and 4, for momentary craving in response to picture stimuli,
the mean VAS value of the two blocks for each condition was used in an analysis of
covariance (ANCOVA) adjusted for baseline momentary craving, with the repeated factors
“odour prime” (chocolate/neutral) and “picture type” (chocolate/neutral) and the between
subjects factor “group” (HC/BEP). For each of the ERPs (N2 and LPP) the additional factor
“localization” was added, wherefore a 2(“odour prime” chocolate/neutral) × 2(“picture type”
chocolate/neutral) × 3(“localization” central, right, left) × 2(“group” BEP/HC) ANOVA was
calculated for main effects of picture type (hypothesis 1a) and group (hypothesis 2). For
hypothesis 4, interaction effects were analysed. Pairwise comparisons were used to follow
up main and interaction effects.
Partial correlations including the covariates age and baseline-mood were calculated for each
group to look at the relation between dependent measures, i.e. between subjective craving
ratings and electrophysiological brain response (hypothesis 1b). Correlations were
considered as moderate for r > 0.24 (corresponds to d > 0.5) and large for r > 0.3
(corresponds to d > 0.8)39,40.
For hypothesis 3, state chocolate craving (FCCQ-S) before and after the experimental task
was compared using repeated measures (“time” pre/post) ANOVA with the between
subjects factor “group” (BEP/HC).
For hypothesis 5 regarding momentary craving in response to odour stimuli, an ANCOVA
adjusted for baseline momentary craving, with the repeated factor “odour type”
(chocolate/neutral) and the between subjects factor “group” (HC/BEP) was calculated using
the mean of the four VAS ratings assessed after odour presentation. For the QEEG data,
theta frequency was compared by use of a 2(“odour type” neutral/chocolate odour) ×
2(“group” BEP/HC) ANOVA to test for main and interaction effects.
Comparisons were considered significant with p < 0.05 after Bonferroni-Finner correction to
avoid Type-I errors. Mauchley’s Test was used to test for sphericity and if the assumption
was not met (p < 0.05), Greenhouse-Geisser corrected values were used. Effect sizes were
calculated as partial Eta squared (ηp2) for ANOVA or Cohen’s d for mean differences (MD),
while ηp2 > 0.01 or d > 0.2 are considered as small, ηp2 > 0.06 or d > 0.5 as moderate
and ηp2 > 0.14 or d > 0.8 as large40.

Results

Ethical Standards
The authors assert that all procedures contributing to this work comply with the ethical
standards of the relevant national and institutional committees on human experimentation
and with the Helsinki Declaration of 1975, as revised in 2008.

Baseline measures
Groups did not differ in their olfactory capacity and all of the participants had values within
the normal range (TDI > 30 points, see Supplementary Table S1). As expected, BMI was
significantly different between groups; it was however not included as a covariate since it is
a characteristic of the patient group and therefore is taken into account when comparing the
two groups. BEP had higher values than HC in trait craving, difficulties in emotion regulation,
food addiction, eating disorder symptomatology and general psychopathology
(see Supplementary Table S2). BEP reported lower mood (MD = −1.46, p < 0.01) and more
hunger (MD = 1.83, p < 0.01) than HC at baseline before doing the experimental task.
However, the time since they had had their last meal did not differ between the two groups
(p = 0.13) and correlations between baseline-hunger and dependent variables (VAS, FCCQ-
S total and ERPs) were low (rs ≤ 0.3). Age and baseline-mood were both found to have an
influence on the correlations between dependent variables, but since the estimated means
were very similar (variation of <10%) when adjusting the models by age and baseline-mood
into ANCOVAS, in order to simplify the models and to maintain statistical power, it was
decided to calculate ANOVAS without adjustments referring to the principle of parsimonia41.

Subjective craving
An ANOVA to compare effects of time and group on state craving showed a significant
increase in the FCCQ-S through the experiment, as seen in a significant main effect of time.
A significant main effect of group showed that BEP patients reported higher craving than HC
at both time points. Apart from the desire and positive reinforcement subscales which did
not differ significantly between groups these results were mirrored by all subscales. There
were no significant interactions (see Table 1). This supported hypothesis 3 that there would
be an increase in state chocolate craving throughout the experiment.

Table 1
State craving for chocolate measured by the FCCQ-S directly before and after the
experimental manipulation in the two study groups.
 Mean
HC; n = 20 BEP; n = 19 Group Effect Time Effect Group*Tim
e
Interaction
Pre post Pre Post F1,37 p F1,37 p F1,37 p
Desire 4.60 8.95 6.16 10.1 2.27 0.140 66.0 <0.00 1.99 0.735
6 5 1
Positive 6.15 7.35 6.83 9.06 1.58 0.217 22.0 <0.00 1.12 0.166
reinforceme 4 1
nt
Negative 4.90 6.30 8.47 9.00 10.9 0.002 5.49 0.025 1.13 0.295
reinforceme 0
nt
Lack of 4.40 4.90 8.32 9.42 21.1 <0.00 5.14 0.029 0.73 0.398
control 4 1
Hunger 6.20 8.40 8.63 10.4 5.17 0.029 28.6 <0.00 0.30 0.585
2 3 1
Total score 26.3 35.9 38.5 48.3 12.7 0.001 56.7 <0.00 0.01 0.942
0 0 8 7 0 1 1

BEP = binge-eating patients; BMI = body mass index; HC = healthy controls; MD = mean

difference. Significant comparisons are marked in bold.
For momentary craving towards visual stimuli, results showed a significant main effect for
“picture type” (F1,36 = 8.27, p < 0.01, ηp2 = 0.19). Chocolate pictures induced higher
momentary craving than neutral pictures, which supported hypothesis 1a. A significant main
effect of “odour prime” (F1,36 = 8.34, p < 0.01, ηp2 = 0.19) showed that a preceding chocolate
odour led to higher momentary craving than a preceding neutral odour. Pairwise
comparisons showed that chocolate compared to neutral odour did not have a significant
effect on the rating towards neutral pictures (MD = 2.26; p = 0.21) but it did affect the rating
towards chocolate pictures (MD = 5.26; p < 0.001), which gave support for hypothesis 4.
There was no main effect for “group”, or significant interaction (all F < 1.0 and p > 0.32),
wherefore hypothesis 2 was not supported with regard to higher subjective craving in BEP
as compared to HC. See Supplementary Table S3 for M and SD of momentary craving
ratings towards picture stimuli.
Similar to the results for visual stimuli, for the reaction towards odour stimuli a significant
main effect of “odour type” (F1,36 = 8.34, p < 0.01, ηp2 = 0.19) showed more craving in
response to chocolate odour than neutral odour, but no main effect of “group” nor an
interaction between “group” and “odour type” (F = 2.18 and p = 0.15) was found, wherefore
hypothesis 5 suggesting higher subjective craving in BEP than HC in response to chocolate
odour was not supported.

Electrophysiological data
Two patient data sets had to be excluded from the electrophysiological analyses because
of poor data quality. The mean number of segments per condition for the whole sample was
between 100 and 102 for all conditions. N2 and LPP mean amplitudes and latencies
according to conditions and groups are shown in Table 2 and Fig. 2.

Figure 2
Event-related potentials in response to chocolate and neutral pictures.
The graphs show grand averages of stimulus-locked electrophysiological activity from
200 ms before to 1200 ms after stimulus onset. First row: N2 amplitudes (μV) at left (AF3),
central (AFz) and right (AF4) anterior-frontal electrode sites. Second row: LPP amplitudes
(μV) at left (P3), central (Pz) and right (P4) posterior electrode sites. HC = Healthy control;
BEP = Binge-eating patients; LPP = Late Positive Potential.

Table 2
N2 amplitudes and latencies at central anterior electrodes and LPP amplitudes and
latencies at right posterior electrodes for binge-eating patients (BEP) and healthy
controls (HC).

Odour Picture Peak amplitude (μV) Latency (ms)

Prime Type HC (n = 20) BEP HC (n = 20) BEP (n = 17)
(n = 17)
M SD M SD M SD M SD
Neutral −4.2 2.3 −2.8 2.0 246.0 29.6 246.5 27.51
N2 Neutral
8 0 6 9 9 1 5
 Odour Picture Peak amplitude (μV) Latency (ms)
Prime Type HC (n = 20) BEP HC (n = 20) BEP (n = 17)
(n = 17)
M SD M SD M SD M SD
Chocolat −5.2 2.4 −4.1 2.1 251.3 44.3 248.8 32.62
e 3 2 3 4 7 8 5
Neutral −4.2 2.1 −2.7 2.0 241.2 31.1 241.2 22.08
Chocolat 2 7 6 2 1 7 7
e Chocolat −5.0 2.3 −4.6 2.2 241.4 29.4 250.6 35.67
e 1 6 0 8 1 7 9
Neutral 3.65 2.3 2.46 1.7 509.2 81.3 507.4 84.23
0 1 7 5 9
Neutral
Chocolat 4.88 2.8 3.80 1.7 447.9 58.5 494.4 126.3
LP e 5 9 0 3 4 6
P Neutral 3.40 1.8 2.42 1.5 479.1 73.5 539.1 116.7
Chocolat 7 3 4 6 1 1
e Chocolat 4.96 2.9 3.80 1.5 463.3 64.1 492.1 119.6
e 9 6 3 6 0 3

MD = mean difference; SD = Standard deviation.

N2 peak amplitudes and latencies

For N2 peak amplitudes, there was a significant main effect for “picture type” (hypothesis
1a) and “localization” and a significant interaction effect between “odour prime”, “picture
type” and “group” (hypothesis 4); an interaction between “picture type” and “group” was
marginally significant (See Supplementary Table S4 for F- and p-values). No further main or
interaction effects were found (all F < 2.86 and p > 0.10).
The main effect of “picture type” was due to higher negative activity in response to chocolate
pictures than to neutral pictures in the whole sample, which was expected by hypothesis 1a.
Highest N2 amplitudes were found at the central localization compared to left and right
lateralized electrodes; left and right hemispheres did not differ.
The interaction effect between “odour prime”, “picture type” and “group” was explained by
BEP having significantly higher amplitudes for chocolate pictures primed by chocolate
compared to neutral odour, which was not found for neutral pictures. No such effect was
found in HC. Another single effect pointed out by this interaction was seen in higher N2
amplitudes in HC versus BEP for neutral but not for chocolate pictures (see Fig. 3).
Therefore, hypothesis 4 of an additive effect of olfactory and visual chocolate stimuli was
supported only for the BEP group, but not for HC.
Figure 3
N2 amplitudes (in μV) in response to the presentation of neutral and chocolate
pictures for individuals with binge-eating and healthy controls.
The graph shows the significant interaction between odour type, picture type and group,
which was explained by enhanced amplitudes in response to chocolate pictures preceded
by chocolate odour as compared to neutral odour for binge-eating patients, which was not
found for healthy controls. Furthermore, there were higher amplitudes during neutral picture
processing in healthy controls as compared to patients with binge-eating, but N2 amplitudes
in response to chocolate pictures did not differ between groups. Error bars represent
standard errors.
The marginally significant interaction between “picture type” and “group” was explained
through lower amplitudes towards neutral stimuli in BEP than in HC. This led to the
conclusion, that BEP have a higher increase in activation when comparing chocolate to
neutral stimuli than HC. Therefore, a post-hoc analysis was conducted to calculate a
difference score by subtracting for each individual the N2 peak amplitude related to neutral
stimuli from the N2 peak amplitude related to chocolate stimuli. An ANOVA of this difference
score including “group” as between subjects factor showed a significant effect of “group”
(F1,35 = 4.95, p < 0.05, ηp2 = 0.12), characterized by a higher difference score, i.e. higher
relative N2 amplitudes in BEP (M = −1.56, SD = 0.88) than in HC (M = −0.87, SD = 0.98).
See Fig. 4 for difference waves.

Figure 4
Difference waves for N2 (left panel) and LPP (right panel) amplitudes.
Electrophysiological activity during processing of chocolate pictures after subtracting activity
during processing of neutral pictures. HC = Healthy control; BEP = Binge-eating patients;
LPP = Late Positive Potential.
For N2 latency the only significant effect was a main effect of “localization”
(see Supplementary Table S4, all other F < 2.5 and p > 0.09), explained through shorter
latencies for the central N2 compared to the left and right lateralized N2 (see Supplementary
Table S4).

LPP peak amplitudes and latencies

For LPP peak values, there was a significant main effect of “picture type” (hypothesis 1a)
and “localization” and a significant interaction between “picture type” and “localization”
(see Supplementary Table S4 for F- and p-values). There were no further main or
interaction effects (all F < 2.1 and p > 0.16). As expected by hypothesis 1a, chocolate
pictures led to significantly higher amplitudes than neutral pictures; however, contrary to
hypothesis 2, there were no differences between HC and BEP in LPP amplitudes. Highest
LPP amplitudes were found at right parietal electrode sites compared to left and central
sites, activation at central sites was higher than at left parietal sites (this difference was not
significant for neutral pictures) (see Supplementary Table S4 for MD and p-values).
Regarding LPP latencies, results showed significant main effects for “picture type” and
“localization” (see Supplementary Table S4). No further main or interaction effects were
found for LPP latencies (all F < 2.1 and p > 0.16). The LPP peaks for chocolate pictures were
earlier in latency than those for neutral pictures. An earlier latency was found for the right
LPP compared to central and left LPPs. Latencies did not differ between left and central
localization (see Supplementary Table S4).

Differences in theta frequency

Contrary to expectations, there were no significant effects for “group” or “odour type” in theta
frequency (all F < 2.1 and p > 0.12).

Correlation analyses
With regard to hypothesis 1b of a positive relation between subjective and
electrophysiological dependent variables, results differed between groups. For HC,
momentary craving was positively correlated with N2 and LPP amplitudes, high correlations
were found for chocolate pictures primed by chocolate odour. Higher state chocolate craving
at baseline went along with higher N2 (rs = 0.41 to 0.76) and higher LPP amplitudes
(rs = 0.12 to 0.48). Surprisingly, for BEP this pattern was different: higher momentary craving
(VAS) and higher state craving (FCCQ-S) predicted smaller N2 (rs −0.02 to −0.41) and LPP
amplitudes (rs = 0.06 to −0.41) (see Supplementary Table S5 for r-values).

Post-hoc tests for P1 peaks

Since from visual inspection it appeared that there are big differences between the two
groups in P1 amplitudes, a post-hoc analysis with regard to P1 peaks was conducted. Peak
amplitudes for the P1 were extracted for each individual at occipital electrodes (Oz and POz)
as the maximum positive peak in the time interval 60–150 ms after stimulus onset. An
ANOVA including the factors “odour prime” (2), “picture type” (2) and “group” (2) showed a
significant main effect of “picture type” (F = 42.87 and p < 0.001) and of “group” (F = 5.57
and p < 0.05). There were higher P1 amplitudes for chocolate (M = 5.47, SD = 0.58) than for
neutral (M = 2.90, SD = 0.51) pictures and HC (M = 5.38, SD = 0.68) had higher amplitudes
than BEP (M = 2.99, SD = 0.74). Because of these group differences, the analyses for N2
and LPP amplitudes were repeated, including the P1 amplitudes of each condition as
covariates. Although F- and p-values changed slightly by controlling for this factor, in the
whole the evidence did not change. The only comparison which lost statistical significance
after controlling for P1 amplitudes was the main effect of “localization” for LPP amplitudes.
Apart from this, the results found before were supported after taking into account the group
differences in P1 amplitudes.
Altogether, the EEG findings support the hypotheses stated in the introduction only partly.
Hypothesis 1a of higher LPP and N2 amplitudes for chocolate than for neutral pictures was
supported for both the HC and BEP group. With regard to the relation between subjective
craving and electrophysiological measures, there were positive correlation coefficients for
HC but for BEP hypothesis 1b was not supported. Furthermore, hypothesis 2 was partially
supported, since BEP had higher relative N2 amplitudes (difference score) in response to
chocolate than HC. However, study results did not show significant differences between
groups with regard to LPP amplitudes, wherefore the hypothesis of more motivated attention
in BEP compared to HC in response to chocolate stimuli was not supported. Hypothesis 4
of an additive effect of olfactory and visual stimuli was supported only for BEP in that
chocolate pictures preceded by chocolate odour led to higher N2 amplitudes than chocolate
pictures preceded by neutral odour. The influence of chocolate odour compared to neutral
odour stimulation was not visible in theta frequency changes for either of the groups
(hypothesis 5).

Discussion
The current study aimed to compare the subjective craving and event-related brain response
of individuals with BE and healthy individuals towards visual and olfactory chocolate stimuli.
Additionally, the study aimed to explore the additive effect of olfactory and visual stimuli on
craving induction.
The first set of analyses referred to subjective data, showing that both groups had an
increase in craving through the experimental manipulation, BEP reported more craving at
baseline and after the experiment than HC. When controlling for baseline craving, chocolate
pictures evoked a higher momentary craving response than neutral pictures in participants
as a whole, but there were no differences between the two groups. These results were
mirrored by ERP amplitudes, with higher amplitudes for chocolate than for neutral pictures
in both groups.
As expected, there was a significantly higher central N2 for chocolate than for neutral stimuli.
There were no group differences in the absolute N2 amplitudes towards chocolate pictures,
but the difference in activation between chocolate minus neutral stimuli was higher for BEP
than for HC, i.e. the relative increase in response to chocolate pictures after accounting for
activation during neutral pictures was higher for patients with binge-eating than for HC. The
N2 has been related to response conflict and cognitive inhibitory control in basic research
during Go/No-Go tasks25,26. In studies looking at food processing in mere picture viewing
tasks, a comparable increased frontal negativity was found in restrained eaters in response
to available food, which was not found in unrestrained eaters30. Another study31 found an
enhanced N2 (labelled as “Anterior Negativity”) for chocolate versus neutral pictures in non-
chocolate cravers, which was significantly reduced after chocolate intake. The authors
interpreted this effect as top-down cognitive control in non-cravers; they did however not
find this effect in chocolate cravers, which seems somehow counter-intuitive. In our study,
although in the absolute amplitudes there were no differences between groups for N2
amplitudes towards chocolate stimuli, BEP had lower amplitudes towards neutral stimuli. In
accord with the N2 interpretation of Asmaro and colleagues31, this interaction effect might
indicate that in the patient group there was a higher relative increase in cognitive control in
response to chocolate images than in HC. In addition, looking at correlations between
craving and N2 amplitudes, in HC self-reported state craving for chocolate at baseline was
a strong predictor of higher N2 amplitudes, ratings of momentary craving were also positively
related to N2 amplitudes of HC. This could indicate that HC individuals with higher craving
use more top-down control in response to chocolate pictures. However, in the patient group
there was a tendency for negative relations between self-reported measures of cravings and
N2 amplitudes. A possible explanation for the N2 results as a whole might be that there is a
non-linear relation between N2 amplitudes and craving. There might be two subgroups of
patients: on the one hand those who, similar to HC, have little increase in N2 amplitudes in
response to chocolate but experience stronger craving for chocolate (higher ratings in VAS),
and on the other hand those who have a high increase in N2 amplitudes related to chocolate
pictures and experience lower craving (seen in VAS ratings). However, these results have
to be regarded with care and replication is needed in order to confirm this hypothesis.
Regarding motivated attention, a late, right lateralized, posterior component consistent with
the LPP was higher in amplitude for chocolate than for neutral pictures. The hypothesis of
BEP having more motivated attention towards chocolate stimuli than HC was however not
supported by our data. In contrast to earlier findings42, no group effects for differences in
LPP peak amplitudes towards chocolate pictures were found. However, this former study
used a mix of high-caloric food pictures in contrast to chocolate pictures only in our study.
Furthermore, the sample was a mere BED sample, while our sample was a mixed sample
of patients with BE symptomatology, including BED and BN. Until now, there is no published
data looking at motivated attention towards food in BN by use of EEG, and it is possible that
they regulate their attention towards food stimuli in a similar way as it was proposed for
obese adults43. This is supported by the correlations between self-reported momentary
craving and LPP amplitudes, which pointed towards a positive association between craving
and motivated attention in HC, but a negative association in BEP.
The second aim referred to a potential “supralinearity” or additive effect of olfactory and
visual stimuli on craving induction. Results of subjective data partly support this hypothesis,
by showing an increased craving response towards chocolate pictures primed by chocolate
odour, while chocolate versus neutral odour did not modulate the self-reported craving
towards neutral stimuli. The influence of a preceding odour stimulus was not visible in the
amplitudes of the visually evoked ERPs (N2 and LPP) in the whole sample. There was
however a higher N2 amplitude for BEP towards chocolate pictures preceded by chocolate
odour than chocolate pictures preceded by neutral odour, which was not found in HC. This
might indicate a higher susceptibility of BEP to the odour of food stimuli. A recent study has
shown that after appetitive conditioning, formerly neutral images lead to increased
amplitudes of the N2-P3 component16. Therefore, a possible explanation of these N2 results
might be an association of chocolate odour to its taste and rewarding effect through classical
conditioning in BEP. Appetitive conditioning of taste to odour has been shown to take place
in the orbito-frontal cortex and thus chocolate odour might potentiate the craving response
to visual cues7 through supralinearity12.
The third study aim was to look at the effect of olfactory stimulation on electrophysiological
activity. Similar to the results for visual stimuli, participants reported more craving after
smelling chocolate than neutral odour. Contrary to our hypotheses, no differences between
groups were found. Furthermore, theta power density was analysed during neutral and
chocolate odour presentation, but results did not show any differences between odour types
or groups regarding this measure.
Although this study has many strengths, such as a sample of individuals with BE
psychopathology, an experimental design and the comparison of subjective and
electrophysiological measures, there are some limitations which have to be considered. First
of all, the total sample was too small to have enough power to discover complex interactions
with small effect sizes, wherefore some group differences may not have been detected.
Second, the patient sample was a mixed sample of BN and BED patients; although both
patient groups struggle with BE and there is a high cross-over between these diagnostic
categories44, there may be other processes underlying each one of these disorders which
differ between the two diagnostic categories. Future studies should compare these two
groups with larger samples in order to see if there are differences in craving induction
depending on diagnosis. Regarding the experimental manipulation, two limitations have to
be mentioned: first, the lack of differences in theta activity may be due to the participants
having their eyes closed during the presentation of the olfactory stimuli, wherefore the
increase in alpha activity may disguise the underlying theta activity. Furthermore, in order to
look at each sensory modality separately, olfactory and visual stimuli were not presented
simultaneously in this experiment, wherefore the real “supralinearity” effect may be
underestimated by the results of this study.
This is an important issue for future research. An olfactometer could be used for a
simultaneous and precise, event-related presentation of odour stimuli, which may allow a
better understanding of the interaction between olfaction and vision in stimulus induced
craving. To better understand the meaning of enhanced ERPs in food processing, future
studies should also look at neural generators of these potentials. This could also be helpful
to inform about possible targets to reduce craving through neuromodulation, as proposed in
recent research45,46,47.
The main conclusions of the current study are that chocolate pictures are related to higher
amplitudes in electrophysiological measures of cognitive control (N2) and motivated
attention (LPP) than neutral pictures; while BEP might have lower baseline N2 activity, they
showed a higher relative increase in response to chocolate cues than HC. When considering
self-report, although BEP reported more craving than HC at baseline and after the
experiment, when controlling for that variable, there were no differences between the two
groups in the craving reaction towards chocolate stimuli (visual and olfactory). Furthermore,
an additive effect of olfactory and visual stimuli on cue-induced craving was partially
supported. Chocolate odour to some extent increased the incentive value and craving
reaction towards visual chocolate images in both healthy and BEP, but BEP seem to be
more susceptible to this effect.

Additional Information
How to cite this article: Wolz, I. et al. Subjective craving and event-related brain response
to olfactory and visual chocolate cues in binge-eating and healthy individuals. Sci. Rep. 7,
41736; doi: 10.1038/srep41736 (2017).
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.

Supplementary Material
Supplementary Material:
Click here to view.(587K, pdf)

Acknowledgments
Financial support was received from Fondo de Investigación Sanitaria -FIS (PI14/290) and
co-funded by FEDER funds - a way to build Europe. IW was supported by a predoctoral
grant of AGAUR (2016FI_B2 00001). G.M.-B. was supported by a predoctoral grant of
AGAUR (2016FI_B 00568). CIBER Fisiopatología de la Obesidad y Nutrición (CIBERobn)
is an initiative of Instituto de Salud Carlos III. The funders had no role in the study design,
data collection and analysis, decision to publish, or preparation of the manuscript.

Footnotes
The authors declare no competing financial interests.
Author Contributions I.W., A.S., S.J.M. and F.F.A. conceptualized and designed the study.
I.W., A.R., A.J. and F.F.A. wrote the main manuscript text. G.M.B. and M.B. contributed
during the data collection process and to the design of the study. R.G., V.M.R. and I.W. did
the statistical analysis of the data. M.V.H. screened participants for neurological disorders.
All authors reviewed the final version of the manuscript.

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Nutrients. 2015 Sep; 7(9): 7914–7924.

IX. Standardization of the Food Composition Database Used

in the Latin American Nutrition and Health Study (ELANS)
Irina Kovalskys,1,2,* Mauro Fisberg,3,4 Georgina Gómez,5 Attilio Rigotti,6 Lilia Yadira
Cortés,7Martha Cecilia Yépez,8 Rossina G. Pareja,9 Marianella Herrera-Cuenca,10 Ioná Z.
Zimberg,11Katherine L. Tucker,12 Berthold Koletzko,13 and Michael Pratt14, on behalf of the
ELANS Study Group‡

Abstract

1. Introduction
The global epidemic of obesity in all age groups and in both developed and developing
countries has raised the need for large-scale multicenter studies on dietary/lifestyle factors.
These studies are essential to substantially improve our knowledge on the complex
relationship existing between energy imbalance, obesity and associated chronic diseases
at large scale and wider geographical heterogeneity [1].
Evaluating dietary consumption is an arduous task. Food intake is a complex phenomenon
and its assessment using available tools is subject to errors inherent to the instruments
themselves, as well as the interviewee and the interviewer. These errors, defined as
systematic and random, should be known and controlled in order to generate more precise
and powerful data to reveal risks associated to unhealthy dietary patterns [2].
Increasingly, literature has been published on the methodological and statistical approaches
to improve the standardization of dietary measurements collected in large multicenter
studies [1,3,4,5]. A major operational issue relates to the comparability of dietary
measurements collected from populations of different countries. Between-country
comparisons are particularly prone to error when different food composition databases and
software are used to estimate nutrient intake [4,5]. Food composition tables are rarely
consistent across countries and many foods are defined or presented in different ways,
making comparisons of the dietary intake difficult.
Although there have been ongoing efforts since 1984 to standardize food composition
databases over the world [6], the lack of a homogeneous and complete Latin American
database represents a handicap for nutritional epidemiology studies on the relationship
between nutrition and health, and the comparison and evaluation of dietary intake within this
world region, similar to the situation for international studies in other parts of the world.
The process of standardization at the food and nutrient assessments was performed to
minimize systematic and random errors in nutrient intake estimations and to allow
comparisons between Latin American countries involved in ELANS (Latin American Nutrition
and Health Study). Therefore, the aim of this paper is to describe the procedures and
rationale for the selection and adaptation of food composition from a single database during
the ELANS study to make cross-country nutritional intake comparisons.

2. Experimental Section

2.1. Study Sample

The Latin American Study of Nutrition and Health/Estudio Latinoamericano de Nutrición y
Salud (ELANS) is a household-based multi-national cross-sectional survey aimed at
investigating food and nutrient intake as well as nutritional and physical activity statuses of
nationally representative samples from urban populations. The project involves eight Latin
American countries (i.e., Argentina, Brazil, Chile, Colombia, Costa Rica, Ecuador, Perú, and
Venezuela) representing a total cohort of 9000 individuals, aged 15.0–65.0 years, stratified
by geographical location (only urban areas), gender, age, and socioeconomic status. The
rationale and design of the study are reported in more detail elsewhere [7]. The overarching
ELANS protocol was approved by the Western Institutional Review Board (#20140605) and
is registered at Clinical Trials (#NCT02226627). Each site-specific protocol was also
approved by the ethical review boards of the participating institutions.

2.2. Dietary Assessment

Dietary intake data were obtained using two 24-h food recalls (24-HR) performed on two
non-consecutive days within one week, totaling a target of 18,000 24-HR recalls. 24-HR was
selected because of its nearly universal applicability across populations with varying literacy
skills and its relatively low burden for participants. As a single 24-HR is limited and generally
inadequate for assessing the diet of individuals, two recalls were chosen to estimate routine
food consumption and to evaluate intra-individual variability in nutrient intake [8].
24-HRs provided detailed information on all food and beverages (including water and
alcoholic beverages), preparations and supplements consumed over previous 24 h, or
usually, the previous day [9]. Information collected included the time of consumption, the
name of the eating occasion, detailed food descriptions allowing accurate food coding, and
the amount eaten. Separate forms were included to report on homemade recipes so name
of dish, total quantities of all ingredients and fraction of dish consumed could be stated.
Each recall was administered in person by trained interviewers, provided with standardized
neutral probing questions according to the Multiple Pass Method [10] to improve the
precision of the information obtained. To assist the participant in specifying and quantifying
foods in household measures, a photographic album containing the most commonly
household utensils and size portions were used. These were specific to each country,
including local food item pictures and common utensils, and standardized within the country.
The household measures obtained in the 24-HR were converted to grams (g) and milliliters
(mL) by trained nutritionists, according to the literature and/or previously standardized
references in each country. Then, this information was transformed into energy,
macronutrient, and micronutrient quantities using the Nutrition Data System for Research
software, version 2013 (NDS-R, Minnesota University, MN, USA). NDS-R is an accurate
nutrient and food group serving calculation software available for research purposes, which
is based on the United States Department of Agriculture (USDA) Nutrient Data Laboratory
as the primary source of nutrient values and nutrient composition, including over 18,000
foods. Among the 150 nutrients available in NDS-R, 19 were initially prioritized in the ELANS
based on their importance in the diet and on fact that they had the most complete information
in the database (Table 1).

Table 1
Energy, micro and macronutrient final outcomes in Latin American Study of Nutrition and
Health (ELANS).

Nutrients (Unit)
Energy (kcal)
Total protein (g)
Macronutrients Total carbohydrate (g)
Total fat (g)
Vitamin A (RAE)
Vitamin D (μg)
Vitamin C (mg)
Micronutrients
Calcium (mg)
Iron (mg)
Sodium (mg)
Fiber (g)
Added sugar (g)
Animal and vegetable proteins (g)
Saturated fatty acids (g)
Monounsaturated fatty acids (g)
Polyunsaturated fatty acids (g)
Trans-fatty acids (g)
Cholesterol (mg)
Open in a separate window

2.3. Quality Control in Coding of Reported Foods/Beverages

A quality control system to minimize error and increase reliability of interviewing and coding
24-HR was performed in all study-sites, according to a theoretical framework presented
elsewhere [3]. All the interviewers were trained using a standard form for the application of
the 24-HR and an explanatory manual for completing this form. All recalls were checked by
a dietitian within three to eight days after the interview. If there was any problem with data
quality (e.g., handwriting, out-of-range quantities, recipe not described), the interviewee was
contacted in order to provide the information and/or to verify the information in the second
recall, and when not resolved, the recall was excluded.
Researchers of each country analyzed the consistency of the data according to the review
of the quantities of key nutrients (kilocalories, carbohydrates, protein, and fat). High or low
intakes were rechecked and verified, or the recall was designated inaccurate. If no
reasonable explanation could be ascertained, the recall was designated as unsatisfactory
and the participant was excluded.

2.4. Food Matching

NDS-R was chosen for ELANS to increase comparability of nutrient composition of foods
consumed by study participants. As NDS-R was generated in the USA, a food matching
standardized procedure was strictly conducted by professional nutritionists in each country
in both 24-HR (Figure 1). This harmonization process included a check of nutritional
equivalency of food items reported by the study subjects of each country against the USA
version of foods available in NDS-R database; if the food item was not available in the NDS-
R database, a food with similar definition, description, and nutrient content was considered.
To ensure maximum similarity with the Latin American foods, USA foods were identified in
the NDS-R database through its general description (i.e., by name, type and mode of
preparation). The values described in the software were compared to the values available
in local food composition tables (Table 2) or nutrition labels and food industry composition
tables. A concordance rate between 80 and 120% for energy and macronutrient content was
required to accept a food selection from this database [11]. Some examples of food matching
performed in ELANS are presented in Table 3.

Figure 1
Standardized food matching procedure applied across Latin American Study of Nutrition and
Health (ELANS) countries.

Table 2
Local food composition tables used in the food matching process.

Country Local Food Composition Tables *

Latin American Network of Food Composition Data System (LATINFOODS)
Argentina
[12]
 Country Local Food Composition Tables *
Brazil Tabela Brasileira de Composição de Alimentos [13]
Chile Tabla de Composición Química de los Alimentos Chilenos [14]
Colombia Tabla de Composición de Alimentos Colombianos [15,16]
Costa
Tabla de Composicin de Alimentos de Centroamerica [17], Infonut [18]
Rica
Tabla de Composición de Alimentos Colombianos [15], Tablas de Uso
Ecuador Práctico de los Alimentos de Mayor Consumo en México [19], Tablas
Peruanas de Composición de Alimentos [20], Llanos et al. [21]
Tabla de composición de alimentos del Instituto de Investigación Nutricional
Peru
[22] (includes the Tablas Peruanas de Composición de Alimentos [20]),
Venezuela Tabla de Composición de Alimentos para Uso Práctico [23]

* All countries also utilized food labels from manufacturers when foods were not available in
any database.

Table 3
Examples of food matching and recipes created in NDS-R for ELANS.

Food/Recipe Ingredients
Food
Aji colorado molido
Peppers, hot chili, red, raw
(peper)
Anón (fruit) Sugar apple (sweetsop or anon)
Arroz doce (sweet
Desserts, miscellaneous, pudding, rice (arroz con leche), plain
rice)
Avena a granel (oat
Ingredient, cooked cereal—dry, oat bran, before cooking
bulk)
Caldo de frijol
Soup, bean, black beans, prepared from ready-to serve can, regular
(beans soup)
Feijão-carioca Beans, brown, canned—drained, regular, boiled, salt—regular, fat
(beans) used as seasoning—oil, soybean-unhydrogenated
Candy, other confections, dulce de guayaba (sweetened guava
Goiabada (sweet)
paste)
Meatball, without sauce, beef, hamburguer or ground beef—10%
Kafta (meat)
fat (90% lean meat)
Mote cocido (corn) Corn, white, cooked from fresh, whole kernel
Ponqué con sabor a
Hispanic, ponque (rum flavored pound no frosting)
ron (cake)
Queque de tienda Cake, yellow or flavored, purchased ready-to-eat, not frosted or
(cake) glazed, after cooking
Food/Recipe Ingredients
Semillas de chan
Seeds, chia
(seeds)
Tacaco (Sechium
Potato, raw, with refuse
tacaco)
Recipe
Açaí (fruit) Fresh figs; soybean oil
Egg, raw, whole; sugar, white granulated; juice or flavored drink,
Bolo de laranja
orange, juice, fresh; oil, soybean—unhydrogenated; baking
(cake)
powder, regular; and flour, white all-purpose, enriched
Buriti (fruit) Egg, raw, yolk only; oil, soybean—unhydrogenated; orange, fresh
Potato, boiled, without skin, before cooking; peppers, hot chili, red,
Chanfainita (mixed raw; vegetables, garlic, fresh; onion, white, yellow or red, raw; beef,
dish) organ meats, kidney; oil, soybean 90%/cottonseed 10%; mint,
spearmint, fresh
Flour, corn, masa, yellow—enriched; brown sugar; cloves (ground);
Chicha (beverage)
cinnamon (ground)
Banana, fresh or ripe; apple, fresh, with skin; papaya, fresh; passion
fruit (maracuya)—fresh; pineapple, fresh; nuts and seeds coconut,
Cholao (beverage) fresh; strawberries, fresh; kiwi green; condensed (sweetened),
regular; jams or preserves, regular; juice or flavored drink,
lemonade and lemon drinks, homemade
Coucus (mixed
Water, tap; cornstarch; cornmeal, dry, yellow (degermed, enriched)
dish)
Flour, corn, masa, white—enriched; oil, soybean—unknown type,
Empanadas de tomato, cooked from fresh; onion, white, yellow or red, cooked,
pipian (pastries) eggs boiled; cornstarch; potato, boiled, without skin; nuts and seeds
peanuts, roasted, dry roasted, unsalted; peppers, sweet red, raw
Ensalada de
Carrots, raw; vegetables, corn, white, cooked from fresh, cob;
verduras cocida sin
broccoli, raw, string or green beans; raw, lemon, juice, fresh
mayonesa (salad)
Rice white, regular cooking, cooked in salted water; onion, white,
Gallo pinto (rice and
yellow or red, cooked, after cooking edible portion; peppers sweet
beans dish)
red cooked, beans black, cooked form dried, after cooking salt
Picadillo de papa Potato boiled with out skin; peppers sweet red cooked; onion with,
(potato dish) yellow or red edible portion after cooking; cilantro fresh, salt
Plátano maduro con Plantains, ripe yellow, boiled or baked after cooking, no salt added;
queso (plantain with cheese queso fresco (Mexican white cheese, margarine regular
cheese) stick salted, soybean palm oils)
Eggs, boiled; coconut, dried (shredded or flaked), unsweetened;
Pudim de coco com
coconut, milk, fresh (liquid from grated meat—water added); milk,
calda de caramelo
condensed (sweetened), regular; milk, whole (3.5%—4% fat);
(pudding)
sugar, white granulated
 Food/Recipe Ingredients
Spaguetti noodles, white, cooked in unsalted water; potato, boiled,
Sopa de fideos con
without skin, before cooking; carrots, raw; celery, raw; leeks, raw;
verduras (soup)
squash, hubbard, before cooking
Open in a separate window
No new food was added to the NDS-R database and no chemical analyses were performed.
Regional foods, recipes, and commercial foods not available in the NDS-R database were
broken down into ingredients and entered into the software as user recipes. These user
recipes were created from the available NDS-R database and documented in a food-
matching control sheet. They were provided by national publications, recipe books, and
culinary websites of each country, and checked against actual data from 24-HR.
When regional foods did not have an exact equivalent or similar food available in the NDS-
R database, one or more foods combined were inserted as a recipe, as the software does
not accept “new foods”. Local teams were responsible for creating a recipe that represents
the same nutritional value as the original version. Some examples of recipes are described
in Table 3. The list of recipes is documented as part of the ELANS procedure of food
matching.
Before beginning the field study, each country study group standardized approximately 600
food/beverages and recipes commonly consumed by its population, according to ELANS
pilot study and other available sources of national food consumption data.

2.5. Data Consistency

After the end of the fieldwork and prior conducting the analysis of food intake, data
consistency was held for each 24-HR to ensure that there was no error of typing or food
entered and to ensure more reliable results. Initially it was performed the consistency of
energy intake. Total daily intake values below 800 kcal/day or above 4000 kcal/day
suggested errors in collection or data entry and were revised. Then, the total intake of
micronutrients was analyzed. The micronutrient composition of the detailed participant´s
food intake with extreme values was compared with the same national food composition
tables used for the verification of energy and macronutrient content. If the concordance rate
was not between 80% and 120%, a routine was performed in Stata version 13 (StataCorp,
College Station, TX, USA) for correction of the values.

3. Conclusions
Until now, no study has evaluated the food and nutrient intake, as well as nutritional status
and physical activity patterns of representative populations in Latin America using a
standardized methodology across a consortium of several participating countries. The Latin
American Study of Nutrition and Health/Estudio Latinoamericano de Nutrición y
Salud (ELANS) is a randomized, cross-sectional, multicenter investigation on nutrition and
physical activity of adolescents and adults in eight Latin American countries.
The standardization of nutritional assessment in ELANS should prevent or minimize bias
(systematic errors) which could affect the pooling of data collected in different centers and
improve between-country comparisons.
Full information on the general and specific criteria applied in this harmonization of food
composition database in ELANS will be freely available and will provide comparability with
similar studies to be performed in other countries.

Acknowledgments
The following are members of ELANS Study Group: Chairs: Mauro Fisberg and Irina
Kovalskys; Co-chair: Georgina Gómez Salas; Core Group members: Attilio Rigotti, Lilia
Yadira Cortés Sanabria, Georgina Gómez Salas, Martha Cecilia Yépez García, Rossina
Gabriella Pareja Torres, and Marianella Herrera-Cuenca; Steering Committee:Berthold
Koletzko, Luis A. Moreno, Michael Pratt, and Katherine L. Tucker; Project
Managers: Viviana Guajardo and Ioná Zalcman Zimberg; International Life Sciences
Institute (ILSI)—Argentina: Irina Kovalskys, Viviana Guajardo, María Paz Amigo, Ximena
Janezic, and Fernando Cardini; Instituto Pensi—Hospital Infantil Sabara—Brazil: Mauro
Fisberg, Ioná Zalcman Zimberg, and Natasha Aparecida Grande de França; Pontificia
Universidad Católica de Chile: Attilio Rigotti, Guadalupe Echeverría, Leslie Landaeta, and
Óscar Castillo; Pontificia Universidad Javeriana—Colombia: Lilia Yadira Cortés Sanabria,
Luz Nayibe Vargas, Luisa Fernanda Tobar, and Yuri Milena Castillo; Universidad de Costa
Rica: Georgina Gómez, Rafael Monge Rojas, and Anne Chinnock; Universidad San
Francisco de Quito—Ecuador: Martha Cecilia Yépez García, María Elisa Herrera Fontana,
Mónica Villar Cáceres, and María Belén Ocampo; Instituto de Investigación Nutricional—
Perú: Rossina Pareja Torres, María Reyna Liria, Krysty Meza, Mellisa Abad, and Mary
Penny; Universidad Central de Venezuela:Marianella Herrera-Cuenca, Maritza Landaeta,
Betty Méndez, Maura Vasquez, Omaira Rivas, Carmen Meza, Servando Ruiz, Guillermo
Ramirez, and Pablo Hernández; Statistical advisor: Alexandre DP Chiavegatto
Filho; Accelerometry analysis: Priscila Bezerra Gonçalves and Claudia Alberico; Physical
activity advisor: Gerson Luis de Moraes Ferrari; We would like to thank the ELANS External
Advisory Board and following individuals who made substantial contributions to ELANS:
Beate Lloyd, Ilton Azevedo, Regina Fisberg and Luis Moreno. The ELANS and researchers
(PIs and advisory board) are supported by a scientific grant from the Coca Cola Company
and by different grants and support from the Instituto Pensi/Hospital Infantil Sabara,
International Life Science Institute of Argentina, Universidad de Costa Rica, Pontificia
Universidad Católica de Chile, Pontificia Universidad Javeriana, Universidad Central de
Venezuela (CENDES-UCV)/Fundación Bengoa, Universidad San Francisco de Quito, and
Instituto de Investigación Nutricional de Peru. The funders had no role in study design, data
collection and analysis, the decision to publish, or the preparation of this manuscript.

Author Contributions
All authors were involved in the conception and design of the study. IZZ, IK and MF
researched the literature and drafted the manuscript. All authors critically reviewed the
manuscript and approved the final version.

Conflicts of Interest
The authors declare no conflict of interest.

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Diabetes Metab Syndr Obes. 2013; 6: 263–273.

X. Effect of tomato consumption on high-density lipoprotein

cholesterol level: a randomized, single-blinded, controlled
clinical trial
Daniel Cuevas-Ramos,1 Paloma Almeda-Valdés,1 Emma Chávez-Manzanera,1Clara Elena
Meza-Arana,2 Griselda Brito-Córdova,1 Roopa Mehta,1 Oscar Pérez-
3 1
Méndez, andFrancisco J Gómez-Pérez

Abstract

Introduction
Cardiovascular diseases (CVDs) are the main causes of death worldwide, with well
recognized risk factors associated with their development.1 Low levels of high-density
lipoprotein cholesterol (HDL-C) rank among the most common lipid abnormalities associated
with CVD.2 Low HDL-C is currently defined as an HDL-C value <40 mg/dL for men and <50
mg/dL for women.3 Factors associated with low HDL-C include cigarette smoking,4 high
triglyceride concentrations,5 a sedentary lifestyle,6 and insulin
resistance.7 Nonpharmacological strategies to increase HDL-C concentration include
increasing alcohol and fish consumption,8,9 weight reduction,3 physical activity,10 and
smoking cessation.8 Some of these strategies are difficult to implement in practice.
Moreover, in low-income countries, these interventions could be costly for the general
population. Vegetable consumption may be an alternative for managing low HDL-C.
Epidemiologic evidence indicates that a high consumption of vegetables reduces the risk of
CVD,11 and particular attention has been paid to tomato-based products. Growing evidence
from several epidemiological studies indicates that lycopene, the major carotenoid in
tomatoes,12 might be more important than other carotenoids in preventing atherosclerosis
and CVD.13,14 The consumption of more than seven servings per week of tomato-based
products has been associated with a 30% reduction in the relative risk of CVD.15 Such
potential benefits to vascular health from a tomato-rich diet could be related to a lowering of
arterial intimal wall thickness,13,16 a reduction in levels of low-density lipoprotein
cholesterol (LDL-C),17 and an inverse correlation with markers of inflammation and vascular
endothelial dysfunction.18 However, HDL-C levels may also be positively influenced by
tomato consumption. In a pilot study, we found that tomato juice consumption did not
increase HDL-C after 1 month (unpublished data); this finding has also been reported
previously.19 In contrast, another study has shown that the daily consumption of 300 g of
uncooked tomatoes during 1 month significantly increased HDL-C levels by
15.2%.20 However, that study was not controlled, blinded, or randomized. Roma tomato
consumption could be an accessible intervention to improve HDL-C levels; however, a
longitudinal clinical trial is necessary to evaluate this association. Therefore, we performed
a randomized, single-blinded, controlled clinical trial to specifically evaluate whether the
consumption of two uncooked tomatoes per day (14 servings a week) during 1 month could
produce a favorable effect on HDL-C. Our data suggest that raw tomato consumption can
increase HDL-C levels in overweight women.

Subjects and methods

Ethics statement
This study was conducted according to the guidelines in the Declaration of Helsinki, and all
procedures involving human patients were approved by our Institutional Human Research
Ethics Committee (REF2039). Written informed consent was obtained from all patients after
a full explanation of the purpose and nature of all procedures was provided.

Study subjects
Between March 1, 2009 and April 30, 2011, workers and patients from the Instituto Nacional
de Ciencias Médicas y Nutrición Salvador Zubirán were invited to participate in the study.
After participants had signed the informed consent, a complete fasting lipid profile was
measured in all participants. Of 432 potentially eligible subjects, 66 (15.2%) fulfilled the
inclusion criteria, defined as age between 18 years and 65 years, low HDL-C level (men <40
mg/dL and women <50 mg/dL), and a normal triglyceride concentration (<150 mg/dL).
Exclusion criteria included a previous diagnosis of diabetes; arterial hypertension; renal,
hepatic, or cardiac insufficiency; hyperuricemia; hyperandrogenic anovulation; thyroid
dysfunction (hypothyroidism or hyperthyroidism); any difficulty in swallowing; or
hospitalization in the previous 6 months. Additionally, subjects taking fibrates, statins,
nicotinic acid, steroids, allopurinol, hormone replacement therapy (testosterone, estrogens,
or progesterone), metformin or other oral hypoglycemic agents, insulin, sibutramine, orlistat,
and nonsteroidal antiinflammatory drugs were also excluded (N = 366). Furthermore, 14
individuals who fulfilled the inclusion criteria declined to participate or were unable to
participate because of acute illness or difficulty in attending the study visits (Figure 1). A final
sample of 52 patients was randomized using a block-designed randomization system with
sealed opaque envelopes for assignment.

Open in a separate window

Figure 1
Flow chart of the study protocol.
Abbreviation: n, number of subjects.

Study design
This was a longitudinal, comparative, randomized, single-blinded, controlled clinical trial.
The protocol included a 2-week run-in period with prescription of an isocaloric diet (50%
carbohydrates, 20% proteins, and 30% fats). After completion of the run-in period,
participants were randomized to consume 300 g of raw cucumber (control group) or the
same amount of uncooked tomatoes (approximately two Roma tomatoes) a day. Patients
were instructed to weigh and prepare the cucumber or the tomato at home. Participants
were instructed to minimize changes in diet and daily habits, specifically physical activity
and smoking. We used cucumber because (1) it was not possible to have a tomato placebo;
(2) cucumber does not have any lycopene; (3) both can be consumed in a similar manner;
and (4) the required quantity can be measured in the same way. After treatment assignment,
we requested participants to avoid mentioning during clinical evaluations whether they were
in the tomato or cucumber arm of the study. One blinded nutritionist performed the
evaluations and the other evaluated adherence. No member of the research team knew the
participant’s study group.

Clinical evaluation
Clinical evaluation consisted of a complete medical history and physical examination
performed by one nurse and one physician unrelated to the study. Resting blood pressure
was measured in the morning by a trained nurse using a mercury sphygmomanometer and
after instructing participants to remain seated at rest for at least 10 minutes. We took the
average of two measurements at every visit. Basal daily physical activity was evaluated with
a questionnaire already validated in the Mexican population.21 The questionnaire quantifies
the level of physical activity (kilocalories per day or in kilojoules if kilocalories are multiplied
by 4.1855) over a 24-hour period as previously described.22 Every subject completed three
questionnaires, recording the physical activity level over 2 workdays and 1 day of the
weekend. These results were analyzed, and the average kilocalories per day and
kilocalories per month were obtained. Smoking was classified as (1) current in those who
smoked more than one cigarette per day (low: 1–14; moderate: 15–24; high: ≥25); (2)
previous smoker (one or more cigarettes per day in the past); or (3) never smoked.

Nutritional evaluation and adherence

The anthropometric measurements were performed by a nutritionist blinded to the
participant’s intervention. After participants removed their shoes and upper garments, body
weight was quantified with a UM-026 Tanita body composition analyzer (Tanita Corporation,
Tokyo, Japan). All subjects were instructed to stand on the central part of the scale during
weight assessment. Height was obtained to the nearest 0.5 cm using a floor scale’s
stadiometer with the patient standing on the central part of the scale. Body mass index (BMI)
was calculated as weight (in kilograms) divided by height (in meters squared). Waist and hip
circumferences were measured with patients standing with their feet together, placing their
arms on their sides with the palms of their hands facing inward, and breathing out gently.
Abdominal circumference was measured to the nearest 0.1 cm at the level of the greatest
frontal extension of the abdomen between the bottom of the rib cage and the top of the iliac
crest. Hip circumference was measured around the maximum circumference of the buttocks.
In addition, nutritional evaluation consisted of three 24 hour food records for each patient at
every visit. Consumption of carbohydrates, proteins, lipids, fiber, simple sugars, fish, omega-
3 acids, and alcohol was calculated with standardized tables.23–26 Another nutritionist
evaluated adherence, asking for the number of days per week that a given patient fully
complied with tomato or cucumber consumption. Adherence was reinforced in every visit.
To detect small changes in weight, physical activity, and diet throughout the study duration,
the clinical and nutritional evaluations were performed every week during the 6-week study
period (Table S1). These measurements were averaged and used for statistical adjustment
as needed.

Biochemical evaluation
Glucose and lipid profiles were measured at the screening visit and again at the end of
follow-up. Laboratory measurements were performed in the Department of Endocrinology
and Metabolism at the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán
using standardized procedures. The measurements were performed with commercially
available standardized methods. Glucose was measured by the glucose oxidase method
(Roche Diagnostics, Indianapolis, IN, USA); serum total cholesterol, triglycerides, HDL-C,
and LDL-C levels were measured by an enzymatic method (Beckman Coulter, Inc, Brea,
CA, USA). The coefficients of variation for total cholesterol and HDL-C were 3.3% and 2.5%,
respectively.

Statistical analysis
The sample size was calculated with the formula for means for two-tailed comparisons.
According to a previous report,20 we expected an increase of at least 6 mg/dL in HDL-C
after 1 month of tomato consumption. With a standard deviation of 5 mg/dL, an alpha level
of 0.05, and a study power of 80%, and adding 20% for potential losses, we calculated that
a total of 48 subjects (24 per group) was required. Normally distributed data, determined
with a Kolmogorov–Smirnov test, were expressed as means and standard deviation,
whereas variables with a skewed distribution were reported as median and interquartile
range. A χ2 test, Student’s unpaired t-test, Wilcoxon signed rank test, or Mann–
Whitney U test was used as appropriate for comparison between groups. Homogeneity of
variance was evaluated with Levene’s test. Correlation coefficients between HDL-C and
dimensional variables were evaluated in all participants and were calculated with the
Spearman’s rho or Pearson’s r tests. To evaluate the effect of tomato consumption on HDL-
C, we used the difference between final and basal levels (indicated as “delta”). A stepwise
linear regression model was used to examine the impact of variables on delta HDL-C levels.
The variables selected for the regression analyses were those that correlated significantly
or those that are known to be associated with plasma HDL-C levels. All reported P-values
were based on two-sided tests, with P ≤ 0.05 considered significant. Analyses were
performed with the Statistical Package for the Social Sciences version 17.0 (SPSS, Inc,
Chicago, IL, USA).

Results
A total of 52 subjects were included in the study. They were randomized to receive tomato
(N = 26) or cucumber (N = 26). Two patients were eliminated after 1 week of follow-up (for
gastric intolerance and poor study compliance). Both requested to be excluded from the
study. The remaining subjects completed 1 month of follow-up (Figure 1). The mean
adherence per month was 27.6 ± 1.9 days and 27.5 ± 2.0 days in the tomato and cucumber
groups, respectively (P = 0.90). A total of 47 (94%) of the subjects declared that they had
followed the assigned intervention for ≥25 days during the month of follow-up (Table 1).

Table 1
Characteristics of the population studied

Parameter Group P-
value
Control (N = 24) Tomato (N = 26)
Gender (female) 19 (38) 22 0.72
(44)
Age (years) 40.3 ± 16.0 43.4 ± 15.5 0.49
Weight (kg) 69.2 ± 11.9 69.5 ± 14.6 0.93
2
BMI (kg/m ) 27.1 ± 4.0 27.1 ± 5.0 0.64
Waist circumference 90.1 ± 9.8 88.6 ± 9.7 0.96
(cm)
Hip circumference (cm) 102.6 ± 8.0 101.8 ± 8.9 0.94
Systolic pressure 112.3 ± 19.3 107.5 ± 17.2 0.28
(mmhg)
Diastolic pressure 74.9 ± 8.4 71.1 ± 10.0 0.16
(mmhg)
Smoking, n (%) 0.34
Never 9 (19.1) 14 (29.8)
1 to 14 per day 6 (12.8) 3 (6.4)
More than 14 per day 0 (0) 0 (0)
In the past 8 (17.0) 7 (14.9)
Diet
Carbohydrates (%) 49.6 ± 6.5 50.9 ± 6.5 0.50
Simple sugars (%) 13.8 ± 5.8 15.1 ± 6.6 0.49
Fiber (%) 25.2 ± 7.4 23.7 ± 4.4 0.38
Fat (%) 33.8 ± 5.5 33.1 ± 5.1 0.64
Proteins (%) 16.4 ± 2.4 15.8 ± 2.5 0.44
Alcohol (g/month) 3.5 ± 1.4 4.9 ± 1.5 0.83
Fish (g/month) 0.3 ± 0.06 0.4 ± 0.05 0.70
Omega-3 (g/month) 0.17 (0.0–0.96) 0.61 (0.25–1.30) 0.02
Daily activity 658 (576.5– 712 (643.1– 0.31
(kcal/month) 729.4) 826.1)
Adherence (days) 0.85
21 1 (4.2) 0 (0.0)
22 0 (0.0) 1 (3.8)
24 1 (4.2) 0 (0.0)
25 1 (4.2) 2 (7.7)
26 2 (8.3) 2 (7.7)
27 4 (16.7) 4 (15.4)
Parameter Group P-
value
Control (N = 24) Tomato (N = 26)
Gender (female) 19 (38) 22 0.72
(44)
28 8 (33.3) 6 (23.1)
29 3 (12.5) 5 (19.2)
30 4 (16.7) 6 (23.1)
Open in a separate window
Notes: Data are presented as mean ± SD or median (interquartile range). Frequencies are
expressed as N (%). Values represent the average from six weekly evaluations throughout
the study (see supplementary table).
Abbreviations: N, number of subjects; BMI, body mass index.

Clinical and anthropometric characteristics

Table 1 shows the characteristics of the subjects studied. A total of 41 individuals (82%)
were women. There were no significant differences between groups with respect to age (P =
0.49) or BMI (P = 0.64). We only identified a significantly higher consumption of omega-3
fatty acids in the tomato group (P = 0.02). During the study, the subjects’ anthropometric
characteristics did not change significantly (Table 1). Table 2 shows the effect of tomato
consumption on lipid profile and anthropometric measurements of the subjects categorized
by group and gender.

Table 2
Effect of tomato consumption on lipid profile and anthropometric measurements

Parameter Group

Tomato (N = 26) Control (N = 24)

Baseline Final P-value Baseline Final P-

value
HDL-C 36.5 ± 7.5 41.6 ± 6.9 <0.0001 36.8 ± 7.2 35.8 ± 7.3 0.08
Men 32.0 ± 4.3 37.3 ± 2.0 0.06 32.6 ± 5.4 32.2 ± 6.7 0.62
Women 37.5 ± 6.2 42.3 ± 7.2 <0.0001 38.06 ± 7.5 36.8 ± 7.3 0.10
Triglycerides 113.4 ± 46.4 122.7 ± 0.18 107.5 ± 36.3 106.9 ± 0.89
21.8 41.5
Men 110 (51.0– 133 (42.0– 0.88 103.0 (77.5– 104.0 0.68
140.7) 136.0) 113.5) (66.5–
145.5)
 Parameter Group

Tomato (N = 26) Control (N = 24)

Baseline Final P-value Baseline Final P-

value
Women 107.5 (77.7– 110.0 0.12 119.0 (73.0– 101.0 0.28
120.0) (82.5– 131.0) (66.0–
165.7) 127.7)
Cholesterol 165.9 ± 44.7 169.7 ± 0.62 162.5 ± 31.0 159.9 ± 0.41
39.7 33.2
Men 132.0 ± 70.1 139.6 ± 0.44 157.0 ± 39.1 160.2 ± 0.61
76.2 36.2
Women 172.0 ± 41.2 174.2 ± 0.74 164.11 ± 159.8 ± 0.26
32.5 29.5 33.4
LDL-C 108.1 ± 38.1 104.5 ± 0.63 103.2 ± 28.0 104.3 ± 0.71
31.0 30.0
Men 63 (44.9– 67 (34.0– 0.59 101.2 ± 37.0 103.2 ± 0.79
111.5) 154.0) 28.9
Women 112.6 ± 37.6 107.6 ± 0.53 103.8 ± 26.4 104.7 ± 0.80
24.9 31.1
BMI (kg/m2) 27.1 ± 5.0 27.0 ± 4.9 0.21 27.1 ± 4.0 26.9 ± 4.2 0.32
Men 26.4 ± 4.5 26.4 ± 4.1 0.87 25.8 ± 3.5 25.7 ± 2.9 0.69
Women 27.2 ± 5.1 27.1 ± 5.2 0.15 27.5 ± 4.2 27.2 ± 4.6 0.37
WC (cm) 88.6 ± 9.7 88.0 ± 10.6 0.38 90.1 ± 9.8 90.8 ± 10.4 0.11
Men 90.1 ± 6.0 90.1 ± 2.2 0.98 92.1 ± 9.9 92.5 ± 9.3 0.57
Women 88.3 ± 10.2 87.7 ± 11.3 0.30 89.6 ± 10.0 90.3 ± 10.8 0.15
HC (cm) 101.8 ± 8.9 101.6 ± 9.4 0.66 102.6 ± 8.0 102.7 ± 7.9 0.66
Men 99.0 ± 7.1 98.5 ± 5.8 0.67 97.9 ± 6.7 98.4 ± 5.8 0.43
Women 102.2 ± 9.2 102.1 ± 9.9 0.77 104.0 ± 8.0 104.0 ± 8.1 0.93
WHR 0.86 ± 0.05 0.86 ± 0.05 0.47 0.88 ± 0.07 0.88 ± 0.07 0.18
Men 0.92 ± 0.0 0.91 ± 0.0 0.65 0.94 ± 0.0 0.93 ± 0.0 0.21
Women 0.86 ± 0.0 0.85 ± 0.0 0.54 0.86 ± 0.0 0.87 ± 0.0 0.01
Open in a separate window
Abbreviations: N, number of subjects; HDL-C, high-density lipoprotein cholesterol; LDL-C,
low-density lipoprotein cholesterol; BMI, body mass index; WC, waist circumference; HC,
hip circumference; WHR, waist-to-hip ratio.

Changes in lipid profile

Baseline values of HDL-C (36.5 ± 7.5 mg/dL versus 36.8 ± 7.2 mg/dL, P = 0.83) and
triglyceride levels (113.4 ± 46.4 mg/dL versus 108.5 ± 36.9 mg/dL, P = 0.54) were similar
between groups. Additionally, at baseline, serum triglycerides (P = 0.77), total cholesterol
(P = 0.82), and LDL-C (P = 0.37) were not different between groups (Table 2). After 1 month
of intervention, a significant increment of HDL-C levels from 36.5 ± 7.5 mg/dL to 41.6 ± 6.96
mg/dL (P < 0.0001) was observed in the group assigned to tomato consumption (Table 2).
The mean increment of HDL-C was 5.0 ± 2.8 mg/dL (range 1–12 mg/dL). Levels of
triglycerides, LDL-C, and total cholesterol did not change significantly. Adherence correlated
positively with the HDL-C increment in the tomato group (r = 0.34, P = 0.01). This
association was not identified with cucumber consumption (r = 0.08, P = 0.71; Figure
2). Figure 3 shows the change in HDL-C levels according to days of adherence (Figure 3A)
and in every case studied (Figure 3B).

Figure 2
Correlation between the change in HDL-C levels and the number of days reported with
complete adherence to cucumber (control group) or tomato consumption.
Note: Dotted lines represent a 95% confidence interval.
Abbreviation: HDL-C, high-density lipoprotein cholesterol.
Open in a separate window
Figure 3
Change in HDL-C after tomato or cucumber consumption.
Notes: (A) Change in HDL-C levels according to days of adherence. The number of subjects
is described in Table 1. (B) Change in HDL-C levels for every case studied (Student’s t-
test, P < 0.0001).
Abbreviations: HDL-C, high-density lipoprotein cholesterol; n, number of subjects.

Independent predictors of HDL-C increment

To identify independent factors related to the change in HDL-C, we performed a linear
regression model using the delta (final-basal) HDL-C level as the dependent variable,
adjusted for those variables that could change HDL-C (Table 3). Results showed that tomato
consumption (β = 5.79, 95% confidence interval [CI] 3.99–7.59; P < 0.0001) and days of
adherence (β = 0.61, 95% CI 0.12-1.11; P = 0.01) were independently and significantly
associated with the increment in HDL-C levels (F = 5.20; r = 0.83; r2 = 0.69; P < 0.0001).

Table 3
Linear regression model to evaluate the independent parameters associated with the
increment of HDL-C

Parameter β ± SE 95% CI P-value

Tomato 5.79 ± 0.88 3.99–7.59 <0.0001
Adherence 0.61 ± 0.24 0.12–1.11 0.01
Smoking −0.29 ± 0.33 −0.97 to 0.38 0.38
Age 0.014 ± 0.04 −0.08 to 0.11 0.77
Gender −1.07 ± 1.80 −4.74 to 2.60 0.55
WHR 7.66 ± 9.02 −10.73 to 26.05 0.40
Triglycerides −0.01 ± 0.01 −0.03 to 0.01 0.46
BMI 0.05 ± 0.12 −0.20 to 0.31 0.66
Physical activity −0.001 ± 0.002 −0.004 to 0.002 0.45
Omega-3 0.13 ± 0.19 −0.26 to 0.52 0.49
Alcohol 0.01 ± 0.07 −0.13 to 0.17 0.81
Simple sugars −0.06 ± 0.07 −0.22 to 0.08 0.39
Fish 0.38 ± 2.00 −3.69 to 4.46 0.84

Notes: Parameters of the model: F = 5.20; r = 0.83; r2 = 0.69; P < 0.0001.

Abbreviations: HDL-C, high-density lipoprotein cholesterol; SE, standard error; CI,
confidence interval; WHR, waist-to-hip ratio; BMI, body mass index.

Discussion
The occidental diet is usually composed of high-glycemic-index and high-fat foods and has
been associated with the development of chronic diseases, including CVDs, cancer, and
diabetes.27 In contrast, the consumption of tomato-based food sources along with fresh
fruit, vegetables, and olive oil is common in a Mediterranean dietary pattern and provides a
variety of nutrients with potential cardiovascular benefits.28 However, investigation
regarding the association between tomato-based food intake and CVD risk has
demonstrated contradictory results. Previous studies have focused on carotenoids,
including lycopene, and their association with either atherosclerosis, different CVD
subtypes, or multiple cardiovascular risk factors.28–32 Ascherio et al33 reported no
association between dietary lycopene and stroke in a large cohort of healthy male
professionals. In contrast, Karppi et al34 recently reported a 59% lower risk of ischemic
stroke associated with tomato consumption. These inconsistent results may be explained
by the following: (1) the considerable variation in the estimation of lycopene intake
depending on the assessment tools used;12 (2) differing absorption, probably because
carotenoids are tightly bound to macromolecules in foods, and therefore, their absorption
may vary;35 (3) differing availability of lycopene, because this depends on the processing
and treatment of the food containing the carotenoid and on the fat content of the meal in
which lycopene is consumed;12 or (4) because some studies analyze the effect of different
sources of dietary tomato in combination, including both healthy and unhealthy foods (for
example, pizza, tomato juice, and fresh tomatoes).19,32Furthermore, the relationship
between the estimated intake and serum lycopene levels is very poor, with Pearson’s
correlation coefficients between 0.1 and 0.3.35,36 For example, one study reported a low
correlation between dietary lycopene levels and plasma lycopene levels. Despite this fact,
the authors confirmed a 30% reduction in the relative risk of CVD.15 With this information in
mind, we aimed to evaluate the change in HDL-C after 1 month of adding two Roma
tomatoes daily to the participants’ regular diet. This intervention was planned to reduce the
variability of a tomato-based diet using only fresh uncooked tomatoes. We used uncooked
tomato because in a pilot study, we did not identify any significant change in HDL-C using
additional methods of preparation (cooked, juiced, or in sauce), a finding that has been
reported previously.19In contrast, an uncontrolled, nonrandomized prospective study
reported that the daily consumption of 300 g of uncooked tomatoes for 1 month significantly
increased HDL-C levels by 15.2%.20 After taking into consideration other variables that
could increase HDL-C, the beta value in the linear regression model analysis indicated that
we could expect a mean increment of 5.79 mg/dL in HDL-C after the consumption of two
daily Roma tomatoes over a 1-month period. The increment in HDL-C levels was
independent of these and other parameters that are known to modify the circulating HDL-C
concentration (Table 3). Furthermore, the increment in HDL-C levels in the group allocated
to tomato consumption showed a direct relationship with compliance. Although mean
alcohol, fish, and omega-3 fatty acid consumption was higher in the tomato group at follow-
up, these differences were not significant (Table 1). The randomized and blinded design of
our clinical study suggests that this variation was by chance, and the regression analysis
results strongly suggest that the increment in HDL-C was mainly attributable to fresh tomato
consumption. Although the increase in HDL-C was not significant in men, a statistical trend
was seen (P = 0.06). We therefore conclude that overweight women can benefit from daily
fresh tomato consumption. To the best of our knowledge, this is the first clinical trial that
specifically evaluates the impact of fresh tomato consumption on HDL-C levels.
According to our results, an intake of 14 servings of fresh tomato per week may have a
similar positive impact (increment between 3.9 mg/dL and 7.5 mg/dL; Table 3) on HDL-C as
physical activity (3.0–3.5 mg/dL), but a smaller effect than alcohol consumption (9.0–13.1
mg/dL) or smoking cessation (9.9 mg/dL).8 Nevertheless, consumption of uncooked tomato
could be recommended as an additional strategy to increase HDL-C levels. The advantage
of fresh tomato consumption is the fact that tomato is available worldwide, and in low-income
countries, it may be an additional affordable strategy for populations with low HDL-C levels.
The underlying mechanism of the increase in HDL-C with raw tomato may or may not be
related to lycopene. Fuhrman et al37 showed that 60 mg of lycopene per day for 3 months
in six men (approximately equivalent to the amount of lycopene in 1 kg of tomatoes) caused
a 14% reduction in plasma LDL-C with no significant change in HDL-C. However, only a
small sample of patients was analyzed, not necessarily with enough statistical power to
show a difference in HDL-C after the intervention. Recently, lycopene has been shown to
yield improvement in HDL-C functionality, with increases in HDL-C subtypes 2 and 3 after a
lycopene-rich diet and supplements. The activity of cholesteryl ester transfer protein
decreased and the activity of lecithin cholesterol acyltransferase increased in the serum of
overweight, middle-aged individuals.38Although the bioavailability of lycopene is higher
after tomatoes are processed, for example, as a paste, and less bioavailability is seen with
raw tomato,39,40 the results of a study by McEneny et al38 suggest that the benefit of raw
tomato consumption in serum HDL-C levels reported here could be explained by regulation
of the activity of key enzymes in HDL-C metabolism and could also be associated with the
improvement in HDL-C functionality after lycopene consumption. Nevertheless, we cannot
confirm this hypothesis in the present study, and we cannot rule out the possible role of
other unidentified nutrients or beta-carotenes.
Although we showed a significant elevation of HDL-C levels after 1 month of tomato
consumption, only two women normalized their level to 50 mg/dL or more, and no men
achieved normal levels (≥40 mg/dL). However, 20 patients (40%) finished the study with
levels >40 mg/dL. Studies have shown that increasing the concentration of HDL-C can slow
and even reverse the progression of coronary atherosclerosis and can reduce
cardiovascular risk in the majority of people with dyslipidemia even if normalization has not
been achieved.41 However, it is necessary to assess whether the consumption of tomatoes
for longer periods of time or at higher daily amounts can normalize HDL-C levels in a greater
proportion of patients. Future prospective studies should evaluate the impact of fresh tomato
consumption on different cardiovascular risk factors and outcomes. These studies may also
confirm the benefit in men.
The main limitation of the present study is that we cannot describe the mechanism of how
fresh tomato consumption increases HDL-C. Second, we evaluated compliance
subjectively; however, participants in both groups reported similar adherence to blinded
researchers. Also, the number of male patients studied was small, which may explain the
lack of significant associations. Another limitation is the fact that we cannot completely rule
out the influence of other nutrients, foods, or cointerventions by participants, and this may
provide alternative explanations for our findings. However, the randomized and longitudinal
design of our study, the absence of loss to follow-up, and the fact that we adjusted the
analyses for the main confounding factors that influence HDL-C levels suggest that the
increment in HDL-C was caused by the increase in tomato consumption. An additional
strength of the study design is that we evaluated patients without hypertriglyceridemia, and
patients without treatments that may influence HDL levels.

Conclusion
In conclusion, raw tomato consumption (14 servings a week for 1 month) showed a favorable
effect on HDL-C levels in overweight women.

Supplementary table

Table S1
Clinical and nutritional characteristics of the population studied throughout the study

Weekly Groups P-
visits val
Control (N = 24) Tomato (N = 26) ue*

1 2 3 4 5 6 1 2 3 4 5 6
Clinical evaluation
Weekly Groups P-
visits val
Control (N = 24) Tomato (N = 26) ue*

1 2 3 4 5 6 1 2 3 4 5 6
Weight 69. 69. 69. 69. 69. 69. 67. 69. 69. 69. 67. 67. 0.76
(kg) 7 ± 9 ± 7 ± 9 ± 7 ± 1 ± 8 ± 0 ± 0 ± 0 ± 7 ± 6 ±
11. 11. 11. 12. 11. 12. 12. 12. 12. 12. 12. 12.
8 9 7 0 9 5 6 4 3 4 4 6
BMI 26. 27. 27. 27. 26. 26. 27. 27. 27. 27. 27. 27. 0.78
(kg/m2) 9 ± 1 ± 0 ± 0 ± 9 ± 9 ± 6 ± 6 ± 7 ± 7 ± 6 ± 6 ±
4.1 4.1 4.0 4.2 4.1 4.2 5.0 4.9 4.8 4.9 4.9 4.9
WC (cm) 89. 89. 90. 90. 89. 89. 89. 89. 89. 89. 88. 88. 0.30
4 ± 6 ± 2 ± 1 ± 9 ± 9 ± 8 ± 8 ± 9 ± 9 ± 9 ± 5 ±
10. 9.9 10. 10. 9.9 9.9 9.7 9.8 10. 10. 10. 10.
0 5 7 5 0 4 6
HC (cm) 102 102 102 102 102 102 102 102 102 102 102 102 0.19
.4 ± .3 ± .1 ± .3 ± .3 ± .4 ± .5 ± .8 ± .7 ± .9 ± .2 ± .3 ±
8.1 8.0 8.1 8.1 7.9 7.9 9.0 8.4 9.2 9.6 9.2 9.5
SBP 95. 98. 100 98. 97. 97. 105 103 101 100 100 101 0.32
(mmhg) 7 ± 5 ± .0 ± 5 ± 1 ± 1 ± .3 ± .0 ± .0 ± .0 ± .7 ± .6 ±
5.3 10. 10. 12. 9.5 13. 12. 13. 11. 11. 16. 12.
6 0 1 8 8 0 1 2 0 1
DBP 67. 72. 70. 71. 68. 70. 67. 68. 68. 70. 70. 71. 0.76
(mmhg) 1 ± 8 ± 0 ± 4 ± 5 ± 0 ± 2 ± 2 ± 4 ± 0 ± 0 ± 1 ±
7.5 7.5 8.1 10. 8.9 10. 8.3 7.6 8.0 10 10 10.
6 0 7
Nutritional evaluation
Carbohyd – 51. 50. 50. 48. 49. – 52. 49. 50. 50. 50. 0.81
rates (%) 1 ± 1 ± 1 ± 6 ± 5 ± 3 ± 8 ± 8 ± 9 ± 9 ±
8.5 8.7 6.4 7.3 9.3 7.6 7.6 7.9 8.3 7.5
Sugars – 15. 13. 13. 12. 13. – 15. 14. 15. 15. 14. 0.43
(%) 7 ± 5 ± 2 ± 4 ± 4 ± 7 ± 3 ± 3 ± 5 ± 6 ±
8.0 7.4 7.0 6.1 6.5 8.0 7.5 7.8 7.0 7.2
Fiber (g) – 23. 26. 25. 25. 25. – 22. 25. 25. 24. 24. 0.70
3 ± 6 ± 6 ± 2 ± 8 ± 7 ± 2 ± 4 ± 4 ± 6 ±
10. 8.6 7.2 7.4 7.7 8.0 7.6 6.7 7.4 7.0
1
Fat (%) – 32. 33. 33. 34. 34. – 32. 33. 33. 33. 32. 0.78
9 ± 3 ± 2 ± 1 ± 7 ± 5 ± 8 ± 0 ± 3 ± 8 ±
7.6 6.9 6.0 7.3 7.8 6.1 6.1 5.7 7.5 6.5
Proteins – 15. 16. 16. 17. 15. – 15. 16. 16. 15. 16. 0.24
(%) 8 ± 3 ± 5 ± 1 ± 7 ± 1 ± 2 ± 0 ± 7 ± 1 ±
2.6 3.1 3.4 2.8 3.1 2.8 2.9 3.3 2.9 3.1
 Weekly Groups P-
visits val
Control (N = 24) Tomato (N = 26) ue*

1 2 3 4 5 6 1 2 3 4 5 6
Alcohol – 4.7 3.0 3.6 2.3 3.8 – 6.4 2.0 7.6 6.4 2.3 0.57
(g/week) ± ± ± ± ± ± ± ± ± ±
1.3 0.9 2.8 1.3 1.1 1.4 1.6 1.9 1.2 1.6
Fish – 0.3 0.2 0.3 0.3 0.3 – 0.3 0.4 0.5 0.4 0.4 0.98
(g/week) 0 ± 6 ± 4 ± 0 ± 0 ± 6 ± 0 ± 0 ± 5 ± 0 ±
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
7 4 8 7 7 4 5 5 5 5
Omega-3 – 0.3 0.2 0.2 0.1 0.8 – 0.4 0.3 1.1 0.5 0.4 0.25
(g/week) 8 ± 2 ± 5 ± 9 ± 1 ± 0 ± 0 ± ± 6 ± 8 ±
0.2 0.0 0.0 0.0 0.2 0.0 0.0 0.5 0.7 0.0
4 6 4 5 6 7
Open in a separate window
Notes: Data represent the mean ± SD.
*
P-values using repeated-measures analysis of variance. Visit 1 was screening. Treatment
started on visit 2 and ended on visit 6.
Abbreviations: N, number of subjects; BMI, body mass index; WC, waist circumference;
HC, hip circumference; SBP, systolic blood pressure; DBP, diastolic blood pressure; SD,
standard deviation.

Acknowledgment
This trial is registered with ClinicalTrials.gov, clinical trial registration number NCT01342666.

Footnotes
Disclosure
The authors report no conflicts of interest in this work.

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Anemia. 2011; 2011: 284050.

XI. A Program of Nutritional Education in Schools Reduced

the Prevalence of Iron Deficiency in Students
María Nieves García-Casal, 1 ,* Maritza Landaeta-Jiménez, 2 Rafael Puche, 1 Irene
Leets, 1Zoila Carvajal, 1 Elijú Patiño, 2 and Carlos Ibarra 1

Abstract

1. Introduction
Programs on nutritional education have been widely used for teaching or reinforcing
knowledge on food habits or healthy life styles in children and are considered a useful
strategy to prevent the appearance of nontransmissible chronic diseases at early ages. The
implementation of nutritional education programs in schools may help to inculcate in children
the ability of identifying a healthy food choice for themselves [1].
It has been established that the triangulation of information amongst the teacher, the
children, and the family is a useful strategy for modifying negative feeding behaviors that
are contributing to the recent increase in the prevalence of overweight, obesity,
hypertension, diabetes, and metabolic syndrome in children, while in the opposite extreme
of the spectrum, nutritional deficits persist as important nutritional problems, especially
regarding micronutrient, and vitamin deficiencies such as iron, calcium, folic acid, and
vitamin A, among others [2, 3].
The inclusion of nutritional education into formal education programs is one of the most used
and recommended strategies, mainly because the children obtain and fix the information in
a easy, fun, and permanent way, but also because they act as multipliers of the information,
bringing the new information to their homes to achieve, in the best case scenario, the
transmission of the information to the whole family group. Some studies indicate that to
obtain a better impact on changing habits on the long term, nutritional education programs
must include the whole community, to assure the permanence of changes [3].
In general, the application of nutritional education strategies obtains a limited success when
implemented as an isolated strategy. In a first stage, the simultaneous application of
supplementation or fortification programs with nutritional education is the ideal approach.
This is in the understanding that the 2 initially mentioned interventions should be temporary
measures, while the more permanent changes in nutritional habits, are achieved with the
aid of nutritional education [4].
Anemia constitutes the most prevalent nutritional deficiency worldwide, especially in children
and women in childbearing age. The main cause of anemia in these age groups is iron
deficiency, although other nutritional deficiencies, such as folic acid, are also becoming
important etiological agents [5]. Another important nutrient during growing and development
periods is vitamin A, essential for vision, immunological function, development and
maintenance of mucosal barriers, and so forth.
The worldwide prevalence of anemia in preschoolers is 47.4%, and 23.1 millions of those
children live in the Americas [6]. In Venezuela, the prevalence of anemia for this age group
is around 30% [5, 7, 8], although a study performed in groups from the marginal
socioeconomic strata reported a 75% prevalence of anemia [9]. Folic acid deficiency is also
high for preschool children reaching 31% in a National survey [10, 11]. Vitamin A deficiency
has a prevalence of 33.3%, affecting 190 millions of children, half of which live in the
Americas [6]. In Venezuela, there are few documented reports on vitamin A deficiency,
which indicate a prevalence of 25–30% in children from low socioeconomic strata of the
population [12, 13].
Due to the importance of these 3 nutrients for growing and development and to the higher
susceptibility to suffer from these deficiencies during infancy and childhood, the objective of
this study was to determine the prevalence of iron, folates, and vitamin A deficiencies in
school children from 6 to 14 years and to evaluate the changes in these parameters after an
intervention of nutritional education.

2. Materials and Methods

The project “Iron deficiency and anemia” was approved by Fundacion Bengoa Review Board
and developed in 17 schools from 3 Venezuelan States (Aragua, Lara, and Táchira) located
in the northwest region of the country. The sample was constituted by scholars from 6 to 14
years of age, selected from the inscription lists of children and adolescents to the schools of
the 3 zones, during September 2007 to November 2008.
The sampling was probabilistic, in which all children from all the schools selected had a
known and not null probability of being part of the sample. The sample size was 1,301
children (678 males and 623 females), and a subsample of 480 individual, were randomly
selected for blood sampling and for biochemical and anthropometric determinations before
and after the intervention of nutritional education. The first blood drawing had 2 objectives:
1 to determine the prevalence of iron, folic acid, and vitamin A deficiencies in the zone and
2 to establish a baseline or time 0, before the intervention. The second blood draw, 6 months
later, allowed to verify any changes in the biochemical parameters after the intervention.
Parents, children, and teachers received detailed information about the procedure and the
protocol, and an informed consent was signed by all participants. After inclusion in the
protocol, a detailed record with personal data, medical, and nutritional records was filled for
each participant.

2.1. Nutritional Education Intervention

The nutritional education program was initiated with a written test to explore the previous
nutritional knowledge of teachers, and from those results the intervention was designed. The
objective was to educate teachers about the importance of a proper nutrition with emphasis
on anemia and iron deficiency prevention, so they can act as multipliers transferring the
information to their students.
The program contents included basic concepts in food and nutrition, how to obtain a
balanced meal, anemia and iron deficiency as public health problems, growth and
development according to what we eat, balanced and varied meals, food iron sources, food
iron absorption enhancers and inhibitors, and menu preparation. The program in each
school consisted in 6 workshops, 2 participative talks, 5 game activities, 1 cooking course,
and 1 recipe contest designed and directed to teachers and/or pupils. In the workshops,
different techniques were applied to obtain a harmonious and participative ambient in which
interventions were encouraged and the knowledge was obtained through “learning by
doing.” The teachers developed original educative strategies (role-playing, songs, poetries,
puppets, etc.), expositions, and participative talks. Each teacher received a set of technical
notes, with the contents to teach and the transfer of knowledge was evaluated by direct
supervision of the teacher, by the activities performed, the techniques used during the
activities, and also by another written test at the end of the intervention.

2.2. Anthropometric and Biochemical Analysis

The subsample of children and adolescents was measured in light clothes at the beginning
and end of the nutritional education intervention, and weight and height were recorded by
trained personnel. The measuring error was less than 1%, within expected limits. The
classification according to nutritional status was performed by the combination of indicators:
weight-age, height-age, and body mass index (BMI-) age. The data was grouped according
to presumptive diagnosis as deficit (lower than percentile 3), at risk of deficit (≥ percentile 3
and < percentile 10), normal (≥ percentile 10 and ≤ percentile 90), at risk of excess (>
percentile 90 and ≤ percentile 97), and excess (> percentile 97). A 24 h food recall interview
was also performed in children and adolescents from 10 to 14 years of age.
To the same group, 2 blood samples were taken at the times mentioned, to perform
hemoglobin and hematocrit in blood and to determine serum concentrations of ferritin,
folates, and retinol. Each sample consisted in 5 mL of blood taken from the antecubital vein
of the arm after cleaning of the zone with isopropyl alcohol. From this, 4 mL were used to
obtain serum and 1 mL was treated with EDTA for hemoglobin and hematocrit
determinations.
Due to logistic problems and improper blood conservation and transport, it was not possible
to determine hemoglobin and perform anemia prevalence analysis. Therefore, the results
reported in this study include hematocrit (determined in a microcentrifuge at the place of
sampling), serum ferritin, folates, and retinol concentrations and the prevalence of iron,
folates, and retinol deficiencies.
Serum samples were obtained by centrifugation of blood samples, within 4 hours of
extraction. Serum was kept at −30°C and protected from light until analysis.
Ferritin determinations were performed by an immunoenzymatic assay [14] developed and
validated in our laboratory. Polystyrene microtiter plates were coated with monoclonal
antibodies to human ferritin. In each assay, ferritin standards and unknown sera were added
and incubated with the same monoclonal antibody conjugated with horseradish peroxidase.
The amount of enzyme retained in each well was measured by adding o-phenylenediamine
dihydrochloride (OPD) and H202, and the reaction stopped by adding sulfuric acid. The
absorbance in each well was measured spectrophotometrically, and ferritin concentration
was calculated from a standard curve.
Serum retinol was assayed by high-performance liquid chromatography (HPLC) according
to the method of Chow and Omaye [15]. Briefly, 100 μL of serum were extracted with
heptane containing BHT, dried under a nitrogen stream, and suspended in methanol for
separation in an HPLC system (Waters corporation, Mildford MA) with a Bondapack C18
column (3.9 × 300 mm), using 100% methanol as mobile phase at 0.8 mL/min. Detection
was carried at 296 nm and retinol concentrations were calculated from a standard curve
using Empower software from Waters.
The microbiological method for the determination of serum folates was an adaptation by the
Centers for Diseases Control and Prevention (Atlanta, USA) of the method described by
O'Broin and Kelleher [16] and Molloy and Scott [17]. It was based in the folate-dependent
growth of a strain of Lactobacillus. Serum samples and bacteria were diluted in a complete
nutritive medium devoid of folates. The growing of the bacteria was dependent of the
presence of folates in the sample and measured by the turbidity developed in the wells at
590 nm, comparing against a standard curve.
The cutoff points used were for iron deficiency, a serum ferritin level <10 μg/L [6], for vitamin
A, deficiency was classified as severe when serum concentration was <10 μg/dL, and
moderate if <20 μg/dL [18]. For folic acid deficiency, it was severe for serum concentrations
below 6.7 nmol/L, and moderate between 6.7 and 13.2 nmol/L [10, 11].

2.3. Statistical Analysis

Samples were analyzed for hematocrit and serum concentrations of ferritin, folates, and
retinol and expressed as mean ± SD and % of prevalence of the respective deficiency. The
analysis of data by sex and geographic location was performed by ANOVA with Bonferroni
as a posttest. For comparisons of groups before and after nutritional education intervention,
paired t-test was used. Differences were considered as significantly different, when P < .05.

3. Results
Table 1 shows the anthropometric and hematological characteristics of the children studied.
At the moment of inclusion in the protocol, the group presented normal ranges, although
close to lower limits, of hematocrit, ferritin, and retinol concentrations. The mean
concentration of folates was below the cutoff point of 13.2 nmol/L, indicating a moderate
folate deficiency in the population studied. The table also shows that after the nutritional
education intervention, all parameters analyzed, except for retinol and folates, increased
significantly (P < .05).

Table 1
Changes in anthropometric and hematological parameters in children before and after the
intervention of nutritional education in schools from Aragua, Táchira, and Lara States,
Venezuela. (n = 427).

Before intervention After intervention

Age (years) 8.68 ± 2.31 9.44 ± 2.44*

Weight (Kg) 28.47 ± 9.69 31.12 ± 12.11*

Height (cm) 127.49 ± 13.80 131.40 ± 15.88*

Hematocrit (%) 37.42 ± 3.32 38.57 ± 2.90*

Ferritin (μg/L) 18.04 ± 12.00 22.56 ± 15.82*

Serum retinol (μg/dL) 22.09 ± 6.61 20.73 ± 5.80

Serum folate (nmol/L) 11.74 ± 7.19 10.58 ± 8.80

*Statistically significant difference between before and after intervention (P < .05).
When classifying children according to their nutritional status before and after intervention,
the percentages diminished for deficit (from 17.1 to 16.3%), at risk of deficit (from 14.2 to
12.9%), and at risk of excess categories (from 5.0 to 2.5%), while the excess category
increased from 13.7% to 15.0% (Table 2). Some of the children classified as at risk of excess
and excess after the intervention were in reality children with chronic undernutrition in
process of weight recovery, but with a chronically diminished height.

Table 2
Classification of children according to nutritional status, by combination of indexes before
and after nutritional education intervention.

Before intervention After intervention

Categories* Total n % n %

Deficit 80 41 17.1 39 16.3

At risk of deficit 65 34 14,2 31 12.9

Normal 248 120 50.0 128 53.3

At risk of excess 18 12 5.0 6 2.5

Excess 69 33 13.7 36 15.0

Total 480 240 100 240 100

*The data was grouped according to presumptive diagnosis as deficit (lower than percentile
3), at risk of deficit (≥ percentile 3 and < percentile 10), normal (≥ percentile 10 and ≤
percentile 90), at risk of excess (> percentile 90 and ≤ percentile 97), and excess (>
percentile 97).
There were no differences in the percentages of excess and deficit between the 3 states
studied, and according to the 24 h food recall questionnaires, the diets were hypocaloric,
with low consumption of fruit and vegetables, high consumption of soft drinks and snacks
and low or no physical activity. Red meat was the main source of heme iron consumed by
57% of the children (54 g/day). Consumption of soft drinks and natural fruit juices was similar
(47%), and coffee and tea was consumed by 22% of the sample. After intervention, there
was a slight improvement in fruit consumption, based on the 24 h food recall questionnaires.
The intervention of nutritional education produced a significant increase in hematocrit and
ferritin concentration, while retinol and folate concentrations showed no significant changes.
There were also no significant differences by gender, and as shown in Table 3, when
distributed by state, children from Táchira presented a better nutritional condition in terms
of hematocrit (data not shown), ferritin, retinol, and folates status than children from Lara.
Children from Aragua State showed no difference compared to Táchira or Lara, probably as
a consequence of the smaller sample size.

Table 3
Changes in ferritin, retinol, and folate concentrations in children before and after the
intervention of nutritional education in schools from Aragua, Táchira, and Lara States,
Venezuela. Classification by State of precedence.

Ferritin (μg/L) Serum retinol μg/dL Serum folate nmol/L

n Before After Before After Before After

interventi interventi interventi interventi interventi interventi
on on on on on on

Aragu 69 12.00 ± 24.33 ± 20.66 ± 17.17 ± 7.43 ± 10.34 ±

a 10.17a 18.34* 6.72a,b 3.69a 4.99 8.90a,b

Táchir 17 21.06 ± 23.60 ± 23.72 ± 22.45 ± 12.99 ± 12.16 ±

a 9 12.81b 16.48 5.95b 5.88b 7.24 9.29b

Lara 17 16.52 ± 20.88 ± 20.50 ± 20.48 ± 11.21 ± 9.19 ±

9 10.62a,b 13.90 6.91a 5.74b 9.35 8.07a

Total42 16.53 ± 22.94 ± 21.66 ± 20.09 ± 10.54 ± 10.56 ±

7 13.17 16.24* 6.64 5.10 7.19 8.75
Open in a separate window
*Statistically significant difference between before and after intervention (P < .05).
a
Different superscript letters, statistically different between states (P < .05).
The prevalence of iron deficiency was 25% at the beginning of the study and was
significantly reduced to 14.3% after the intervention (Figure 1). The prevalence of severe
and moderate folate deficiencies affected 39% and 36% of the sample, respectively, to reach
approximately 75% of the sample being affected by some degree of folate deficiency. This
degree of prevalence was not affected by the nutritional education intervention. The
prevalence of serum retinol deficiency was severe in less than 3% of the cases, but
moderate deficiency affected more than 40% of children. As with folates, retinol levels were
not significantly affected by the intervention.
Figure 1
Prevalence of iron, folate, and vitamin A deficiencies in children, before and after the
intervention of nutritional education in schools from Aragua, Táchira, and Lara States,
Venezuela. (n = 427).

4. Discussion
The results presented in this work show a high incidence of micronutrient deficiencies in
scholars from 17 schools located in 3 noncontiguous states of Venezuela, which could be
indicating a problem of hidden hunger, since anthropometrical parameters were within
normal ranges.
There was high prevalence of iron, folates, and vitamin A deficiencies in children and
adolescents. The prevalence of severe folic acid deficiency affected 39% of the sample and
requires immediate action, which can be achieved by a supplementation program in schools.
The situation of this vitamin in Venezuela has been previously reported [10, 11], and at this
point it probably could be better combated by a combined strategy, consisting in
supplementation, food fortification, and nutritional education in a first stage.
The prevalence of moderate vitamin A deficiency affected 40% of the sample. If this data
were extrapolated to general population, this would constitute an important Public Health
problem. Regarding iron deficiency, the prevalence was 25% at the beginning of the study,
a percentage similar to previously reported data for these age groups [5]. Unfortunately, due
to the problems with blood and the impossibility to obtain reliable results on hemoglobin
concentrations, we were unable to determine if the intervention had effects on anemia
prevalence. However, a very reliable indicator of red cell volume, hematocrit, was
significantly increased after the nutritional education program.
The most efficacious strategies to combat nutritional deficiencies are food fortification and
supplementation programs, although it is generally recognized that nutritional education
should always accompany those initiatives and also that education is the most fundamental
and permanent strategy to achieve changes in food habits and to obtain a balanced nutrition
that includes all the required nutrients during the different life stages [19]. However, it has
been pointed out that the success of educative programs is dependent on the enthusiasm
and commitment of teachers to include the suggested strategies and also of their interest in
master the knowledge required and the time to implement those new contents [20].
Limitations of educational campaigns include a non-immediate, long-term effect or impact
as well as a limited penetration that does not guarantee complete access or coverage [21].
Also, the impact of educational interventions is usually measured by tests that evaluate if
the children received and understood the message [19, 22]. The reports in the literature
about measuring biochemical parameters to evaluate the impact of a nutritional education
intervention are scarce, but these evaluations are a direct measure that “the message was
received, understood and put into practice,” a conduct that probably would benefit not only
the child or the person receiving the information, but also the whole family group.
The analysis of the impact of the nutritional intervention performed was measured not only
by direct observation and supervision of activities and games, where teachers and pupils
could demonstrate the acquisition of the knowledge, but also by the significant reduction in
iron deficiency prevalence, measured as serum ferritin, after the intervention. There were,
however, no changes in serum retinol and folate concentrations, both nutrients with high
prevalence of deficiency. The lack of effect on those 2 nutrients could be due, in part, to the
fact that the intervention was focused on iron and iron rich foods sources. Also, the limited
access to food of animal origin and the low vegetable and fruits consumption could account
for the lack of effect observed. In this group, precooked corn flour (fortified with iron) was
the most consumed food (2 corn breads called “arepas” per day, 100 g/day) and constituted
96% of the iron in the diet, followed by grains. Furthermore, consumption of performed
vitamin A, which should come from animal sources, was limited in this group, as well as a
provitamin A carotenoids, also a good source of folates.
This study shows, through biochemical determinations, that nutritional education initiatives
have an impact improving nutritional health in children. However, the permanence in time of
such habit modifications that assure the maintenance of the biochemical changes achieved
requires more ample interventions that involve the whole community, as well as the
treatment of other external factors that affect the appearance of nutritional deficiencies
[3, 20].
The combination of strategies (education, supplementation, and/or food fortification) is
probably the most effective approach to combat micronutrient deficiencies, especially severe
deficiencies as in the case of folic acid in this study. It would be desirable to perform
strategies in nutritional education at school level, with measuring the impact through
biochemical variables at the family group level. Also, existing educational campaigns should
be reinforced to encourage consumption of vegetables, fruits, and other sources of folates
and vitamin A.

Conflict of Interests
They also declare that they have no conflicto of interests.

Acknowledgments
Authors are thankful to children, teachers, and parents that agreed to participate in this
study. The study was partially funded by NUTRIR Project (Nestlé of Venezuela SA).

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ront Psychol. 2016; 7: 61.

XII. “Food Addiction” in Patients with Eating Disorders is

Associated with Negative Urgency and Difficulties to
Focus on Long-Term Goals
Ines Wolz,1,2,† Ines Hilker,1,† Roser Granero,2,3 Susana Jiménez-Murcia,1,2,3Ashley N.
Gearhardt,5 Carlos Dieguez,2,6 Felipe F. Casanueva,2,7 Ana B. Crujeiras,2,7José M.
Menchón,1,3,8 and Fernando Fernández-Aranda1,2,4,*

Abstract

Introduction
Until now there is no clear agreement about the question if FA is a valid and necessary
concept, specifically in the domain of EDs. On the one hand, different components of food
have been studied using animal models, providing evidence that sugar consumption – and
to some extend also high fat food – can lead to addictive behaviors, similar to other
substances of abuse (Teegarden and Bale, 2007; Avena et al., 2008, 2012; Gold and
Avena, 2013). Hyperpalatable foods, characterized by high levels of sugar, fat and salt are
potentially addictive for humans (Gearhardt et al., 2011a; Davis, 2014; Schulte et al., 2015).
Apart from this, neuroimaging techniques have shed light over neural correlates of FA, as
well as on the similarities between substance dependence and addictive-like eating behavior
in humans in terms of reward value and incentive value of respective stimuli (Gearhardt et
al., 2011b; Volkow et al., 2012; Davis et al., 2013; Smith and Robbins, 2013; Imperatori et
al., 2014). On the other hand, the FA construct seems to overlap with common eating
psychopathology, namely binging, and seems to have collinearity with severity of disordered
eating. Furthermore, a much debated question is whether addictive properties intrinsic to
specific foods (physical dependence) or rather the eating behavior per se (psychological
dependence) play a major role in the explanation of addictive-like eating, wherefore the term
“eating addiction” has been proposed in order to underline the behavioral component of
these symptoms (see Hebebrand et al., 2014 for a review). This shows the need for more
research on psychological processes underlying FA.
The Yale Food Addiction Scale (YFAS) was developed in 2009 with the aim to apply the
diagnostic criteria for substance dependence of the fourth revision of the Diagnostic and
Statistical Manual of Mental Disorders (DSM; American Psychiatric Association, 2013) to
eating behavior (Gearhardt et al., 2009a). Since the development of this first validated tool
for the measurement of addictive behaviors toward food, the number of publications about
FA has experienced a constant growth (Gearhardt et al., 2011a). In DSM-5, the chapter on
addictions has undergone reorganization, including now not only substance related
disorders, but also behavioral addictions. FA could be included in this new category in future
revisions of the DSM.
A meta-analysis including 23 studies using the YFAS reports a mean prevalence of FA of
19.9% in adult samples ranging from healthy normal weight, over obesity, to BED, and BN,
in which the highest prevalence of up to 100% was found (Pursey et al., 2014). In a recent
study using the YFAS in ED patients, 72.8% of the sample fulfilled the criteria for FA
compared to 2.4% of healthy controls, those ED patients who report FA showing higher ED
severity and more general psychopathology (Granero et al., 2014). If ED patients with and
without FA differ on basic psychological measures, such as personality and impulsivity traits,
focused approaches to treatment may be helpful. However, there is a lack of literature
analyzing personality vulnerabilities underlying FA.
The idea, that personality characteristics implicated in addictive processes could also
contribute to ED, is not a new concept and has been confirmed by empiric data (Davis and
Claridge, 1998; Lent and Swencionis, 2012). ED patients are more likely than healthy
controls to use addictive substances such as tobacco, but also illicit drugs (Krug et al., 2008),
which supports the notion of an “addictive personality.” Yet, it is possible that this association
is explained by those patients fulfilling the criteria of FA, rather than being typical to all ED
patients. Assuming that FA is comparable to other (substance and/or behavioral) addictions,
it is expectable that, after controlling for ED subtypes, patients having a positive FA
screening will have more addictive-like personality traits than those who do not fulfill the
YFAS criteria for FA.
A recent meta-analysis on temperament in ED (Atiye et al., 2015) shows high harm
avoidance in all ED-types compared to controls, high novelty seeking in BN patients, high
persistence in AN, BN and Other Not Specified Eating or Feeding Disorders (OSFED), and
no differences in reward dependence between patient and control groups. Furthermore, all
types of ED-patients were found to have lower scores in self-directedness than healthy
controls (Fassino et al., 2004). By comparison, the personality profile found in individuals
with substance related and non-substance related addictive disorders, namely gambling
disorder, shows similarities but also differences: high novelty seeking and low self-
directedness was reported transdiagnostically for different drugs (Le Bon et al.,
2004; Pedrero Pérez and Rojo Mota, 2008) and non-substance related addictions (Alvarez-
Moya et al., 2007), harm avoidance in contrast may vary depending on the substance
consumed (Schneider et al., 2015) and on sex (Clinton et al., 2004; Claes et al.,
2012a; Granero et al., 2014). When comparing behavioral addictions (gambling disorder,
compulsive buying) to BN, high novelty seeking is more specifically related to the former
group, whereas low self-directedness is associated to both groups and reward dependence
is not clearly related to either of the groups (Alvarez-Moya et al., 2007; Jiménez-Murcia et
al., 2015). Harm avoidance in general is high in both clinical groups, but may be a more
gender specific trait, with lower values in males than in females (Alvarez-Moya et al.,
2007; Claes et al., 2012a).
Since impulsivity is an important characteristic common to behavioral and substance
addictions (Lawrence et al., 2009; Alvarez-Moya et al., 2011; Kaiser et al., 2012; Jiménez-
Murcia et al., 2013; Ochoa et al., 2013; Torres et al., 2013; Di Nicola et al., 2015),
heightened levels could also be associated with FA. However, high impulsivity has also been
found in ED patients (Davies et al., 2009; Claes et al., 2012b, 2015), wherefore a clarification
is needed of whether this correlate is related to ED in general, or if it relates specifically to
addictive-like eating. In studies using different self-report measures (UPPS, Barratt
Impulsivity Scale) in student populations, high impulsivity was related to higher scores on
the YFAS (Davis et al., 2011); more specifically, negative urgency, lack of perseverance
(Murphy et al., 2014; Pivarunas and Conner, 2015) and attentional impulsivity (Meule et al.,
2012; Raymond and Lovell, 2015), while motor and non-planning impulsivity were related to
FA only in one (Raymond and Lovell, 2015) of these studies. Regarding behavioral response
inhibition tasks, FA was not consistently related to task performance (Meule et al.,
2012, 2014a). These results show that the term “impulsivity” has been referred to in different
ways and with varying meanings, which may explain the discrepant results of self-report
measures of impulsivity when compared to behavioral impulsivity tasks (Cyders and
Coskunpinar, 2011; Meule et al., 2014a) and shows that a clear definition of this construct
is needed. In the following, impulsivity will be defined according to a five factor-model
(Cyders et al., 2007) incorporating the facets lack of premeditation, lack of perseverance,
sensation seeking, positive urgency and negative urgency.
The objectives of the present study were (1) to investigate if ED patients differ in specific
personality traits depending on a positive FA screening according to the YFAS; and (2) to
find a model to predict FA in ED patients using measures of personality and impulsivity.
More specifically, starting from the literature on addictive personality traits, it was
hypothesized that ED patients with FA would have more novelty seeking, similar self-
directedness, reward dependence and harm-avoidance (1a), and higher negative urgency
and lower perseverance than ED patients without FA (1b). The second objective was more
explorative; therefore, we did not make specific hypotheses on which variables would best
predict FA.

Materials and Methods

Participants
Participants (n = 278, 20 males) were recruited from consecutive referrals to the ED Unit of
the Department for Psychiatry of Bellvitge University Hospital during a period comprised
from September 2013 until March 2015. AN (n = 68), BN (n = 110), BED (n= 39), and
OSFED (n = 61) patients were originally diagnosed according to DSM-IV-TR (American
Psychiatric Association, 2000) criteria by means of the Structured Clinical Interview for DSM
Disorders-I (First et al., 1996), conducted by experienced psychologists and psychiatrists.
DSM-IV diagnoses were reanalyzed post hoc using the recent DSM-5 criteria to ensure
diagnoses reflected the current diagnostic criteria (American Psychiatric Association, 2013).
See Table Table11 for sociodemographic variables, for further information on the sample
characteristics see Supplementary Tables S1 and S2.

Table 1
Demographic and selected clinical data for the sample.

Total n = AN n = 68 BN n = OSFED n BED n = χ2 df p

278 110 = 61 39

Gender 92.8% 89.7% 96.4% 95.1% 84.6% 7.46 3 0.059

females

Employ 69.4% 58.8% 70.0% 78.7% 71.8% 6.19 3 0.103

ed

Tobacc 30.6% 29.4% 31.8% 39.3% 15.4% 6.57 3 0.087

o use

Alcohol 10.1% 7.4% 17.3% 6.6% 0% 12.1 3 0.007

abuse

Other 12.9% 10.3% 19.1% 9.8% 5.1% 6.75 3 0.080

drugs
use

Food 74.8% 55.9% 89.1% 62.3% 87.2% 33.0 3 <0.0

addictio 8 01
n:
screeni
ng
positive

Mea SD Mea SD Mea SD Mea SD Mea SD F df p

n n n n n
 Total n = AN n = 68 BN n = OSFED n BED n = χ2 df p
278 110 = 61 39

Age 29.1 10. 26.7 9.2 28.7 9.4 26.6 10. 38.3 10. 14.4 3; <0.0
(years- 4 2 9 27 01
old) 4

Age of 19.7 8.4 17.7 5.1 19.0 7.0 19.8 9.9 24.6 11. 5.8 3; 0.001
onset 8 27
(years- 4
old)

Duratio 9.3 9.0 8.3 9.1 9.8 8.7 6.9 6.9 13.6 11. 4.6 3; 0.004
n of 1 27
illness 4
(years)

BMI 24.5 8.7 16.8 1.5 25.2 6.4 22.7 4.2 38.9 9.1 125. 3; <0.0
(kg/m2) 0 27 01
4

Food 4.76 1.8 3.51 1.7 5.69 1.4 3.74 1.7 5.92 1.3 42.9 3; <0.0
addictio 9 1 6 2 6 3 27 01
n: total 4
criteria
Open in a separate window
AN, anorexia nervosa; BED, binge eating disorder; BMI, Body Mass Index (kg/m2); BN,
bulimia nervosa; OSFED, Other Not Specified Eating or Feeding Disorders; SD, standard
deviation.

Assessment

Yale Food Addiction Scale-Spanish Version -YFAS-S (Gearhardt et al.,

2009b; Granero et al., 2014)
The YFAS measures FA using 25 items which are assigned to seven scales, referring to the
seven criteria for substance dependence defined by the DSM-IV: (1) tolerance, (2)
withdrawal, (3) substance taken in larger amount/period of time than intended, (4) persistent
desire/unsuccessful efforts to cut down, (5) great deal of time spent to obtain substance, (6)
important activities given up to obtain substance, (7) use continued despite
psychological/physical problems (American Psychiatric Association, 2000). The YFAS was
translated into Spanish and validated in the Spanish adult and ED population, with good
validity and reliability scores (Granero et al., 2014).
For the following analyses, we either used the “FA total criteria,” which indicates the number
of fulfilled subscales, or the positive versus negative screening result. If at least three of the
seven criteria are met for a period of the last 12 months and the person feels significantly
impaired and/or suffers due to the described behavior, this is referred to as “positive YFAS
screening score.” Internal consistency for the YFAS in our sample was excellent, Cronbach’s
α = 0.92.

UPPS-P Impulsive Behavior Scale-UPPS (Whiteside and Lynam, 2001; Cyders et al.,
2007)
The UPPS-P measures five facets of impulsive behavior through self-report on 59 items:
positive and negative urgency (tendency to act rashly in response to positive mood or to
distress), lack of perseverance (inability to remain focused on a task), lack of premeditation
(tendency to act without thinking of the consequences of an act) and sensation seeking
(tendency to seek out novel and thrilling experiences). The Spanish translation shows good
reliability (Cronbach’s α between 0.79 and 0.93) and external validity (Verdejo-García et al.,
2010). Reliability as measured by Cronbach’s α for the UPPS-P in the study sample ranged
from very good (negative urgency α = 0.83) to excellent (positive urgency α = 0.91).

Temperament and Character Inventory-Revised-TCI-R (Cloninger, 1994)

The TCI-R is a 240-item self-report questionnaire measuring personality on four
temperament and three character dimensions. The temperament dimensions are harm
avoidance (inhibited, passive vs. energetic, outgoing); novelty seeking (approach to signals
of reward, impulsivity vs. uninquiring, reflective); reward dependence (sociable, socially
dependent vs. tough-minded, socially insensitive) and persistence (perseverant, ambitious
vs. inactive, erratic). Character covers self-directedness (responsible, goal-directed vs.
insecure, inept); cooperativeness (helpful, empathic vs. hostile, aggressive) and self-
transcendence (imaginative, unconventional vs. controlling, materialistic). The original
questionnaire and the Spanish version of the revised questionnaire were validated and show
good psychometric properties (Cloninger, 1994; Gutiérrez-Zotes et al., 2004). Internal
consistency for the TCI-R in the study sample ranged from very good (novelty seeking α =
0.80) to excellent (harm avoidance α = 0.91).

Eating Disorders Inventory-2-EDI-2 (Garner et al., 1983)

The EDI-2 is a 91-item self-report questionnaire that assesses characteristics of AN and BN
on the dimensions drive for thinness, bulimia, body dissatisfaction, ineffectiveness,
perfectionism, interpersonal distrust, interoceptive awareness, maturity fears, asceticism,
impulse regulation and social insecurity. This scale has been validated in a Spanish
population (Garner, 1998), obtaining a mean internal consistency of α = 0.63.

Symptom Check-List 90-Revised-SCL-90-R (Derogatis, 1994)

The SCL-90-R is a self-report questionnaire measuring psychological distress and
psychopathology through 90 items. The items load on nine symptom dimensions:
somatization, obsessive–compulsive, interpersonal sensitivity, depression, anxiety, hostility,
phobic anxiety, paranoid ideation and psychoticism. The global score (Global Severity Index,
GSI), is a widely used index of psychopathological distress. The SCL has been validated in
a Spanish sample obtaining a mean internal consistency of α = 0.75 (Derogatis, 2002).

Behavioral and Substance Addictions

Gambling, kleptomania, stealing and buying behavior and the abuse of alcohol, the use of
tobacco (smoking on an at least a daily basis) and drugs (lifetime use of any drug other than
alcohol and tobacco) were assessed in a clinical interview conducted by psychologists and
psychiatrists experienced in the field of addictive behaviors.

Procedure
This study was approved by the local ethics committee and was conducted according to the
Declaration of Helsinki. After participants signed informed consent, they were evaluated and
diagnosed at the ED Unit of the University Hospital of Bellvitge by experienced psychologists
and psychiatrists, who conducted two semi-structured face-to-face interviews. The first
interview provided information about current ED symptoms, antecedents and other
psychopathological data of interest. The second interview comprised psychometrical
assessment, and weight (assessment of body mass index and body composition) and eating
monitoring (through daily reports completed at home on food intake, purges, and binges).

Statistical Data Analyses

Statistical analyses were conducted with SPSS20 for windows. Since age significantly
differed between groups and ED subtype is known to influence the probability of FA (Granero
et al., 2014), these two variables were entered as covariates. ANOVA, adjusted by
participants’ age and ED subtype, was used to compare the means of the seven TCI-R and
the five UPPS-P subscales between participants classified into the two FA groups (positive
and negative screening score).
Regarding missing data, statistical analyses were performed for subjects with complete
information on each instrument (pair-wise procedure). The number of missing data was very
low in this study: only data from one SCL-90R questionnaire were missing (for one patient
in the YFAS-negative group), one TCI-R (also for one patient in the YFAS-negative group)
and eight UPPS (two patients of YFAS-negative and six patients of YFAS-positive group).
Stepwise binary logistic regression was used to obtain a predictive model for the outcome
presence of a “positive YFAS screening score” (more than three criteria fulfilled and clinical
significance), considering three blocks: the first block included and fixed the participants’
sex, age and diagnostic subtype, the second block automatically selected the TCI-R scales
with a significant prediction on the dependent variable, and the third block selected the
UPPS-P scales with significant contribution. The predictive capacity of each block was
measured through the increase in the Nagelkerke’s pseudo-R2 coefficient and the
goodness-of-fit of the final model through the Hosmer and Lemeshow test (Hosmer et al.,
2013). Due to the multiple statistical comparisons, Bonferroni-Finner correction was included
to avoid the increase in Type-I errors. The measure of the effect size for mean and proportion
comparisons was done through the 95% confidence interval of the parameters and the
Cohen’s-d coefficient (moderate effect size was considered for |d| > 0.50 and high effect
size for |d| > 0.80).
Results

Temperament, Character and Impulsivity Traits in ED Patients with and without Food
Addiction
Table Table22 shows the results of the ANOVA comparing the temperament and character
(TCI-R) and impulsivity (UPPS-P) traits mean scores between patients with positive versus
negative YFAS screening score, adjusted by age and ED subtype. The analysis was carried
out in two steps. In the first step the interaction parameter “positive YFAS screening score”
by ED-subtype was included into the ANOVA to assess whether differences between
individuals with positive and negative YFAS screening score were related to the different ED
subtypes. Since this interaction term was not statistically significant, it was excluded from
the model and the main effects of a “positive YFAS screening score” were estimated and
interpreted. Results show that ED patients with positive FA screening compared to patients
without FA have lower self-directedness (p < 0.01), while novelty seeking (p = 0.915), harm-
avoidance (p = 0.08) and reward dependence (p = 0.56) do not differ significantly between
groups. For a graphical representation and norm comparisons, see Supplementary
Figure S1.

Table 2
Differences on mean scores of personality traits and impulsivity for patients with or without
food addiction: ANOVA adjusted by age and ED subtype.

Adjusted means; SD ANOVA

FA = FA = FA × ED (adjusted by age and ED

negative positive subtype)

1 1
n = 70 n = 208 Fdf=3;2 p Fdf=1;2 p eta2 M |d|
75 75 D
TCI-R: 100. 15.0 100. 15.8 0.36 0.78 0.02 .915 0.00 0.3 0.0
Novelty 39 7 71 3 1 0 2 2
seeking
TCI-R: Harm 112. 19.5 119. 21.0 1.33 0.26 5.24 .080 0.01 7.0 0.3
avoidance 89 4 91 8 6 9 2 5
TCI-R: 99.4 16.8 101. 15.6 0.23 0.87 0.99 0.56 0.00 2.3 0.1
Reward 0 9 78 2 6 2 4 8 5
dependence
TCI-R: 105. 18.3 106. 22.6 0.42 0.73 0.01 0.91 0.00 0.3 0.0
Persistence 90 7 24 8 9 5 0 4 2
TCI-R: Self- 125. 21.6 115. 20.4 0.59 0.62 11.17 0.00 0.04 - 0.4
directedness 32 3 37 6 2 7 0 9.9 7
5
TCI-R: 136. 17.3 134. 16.2 0.29 0.83 1.02 0.56 0.00 - 0.1
Cooperativen 49 3 07 4 5 2 4 2.4 4
ess 3
 Adjusted means; SD ANOVA

FA = FA = FA × ED (adjusted by age and ED

negative positive subtype)

1 1
n = 70 n = 208 Fdf=3;2 p Fdf=1;2 p eta2 M |d|
75 75 D
TCI-R: Self- 63.3 13.2 63.8 14.2 2.00 0.11 0.07 0.91 0.00 0.5 0.0
Transcenden 2 8 8 7 4 5 0 7 4
ce

UPPS: Lack 23.4 6.08 23.3 6.24 0.07 0.97 0.01 0.91 0.00 - 0.0
premeditatio 8 8 4 2 0 0.1 2
n 0
UPPS: Lack 21.4 5.45 23.5 5.96 0.79 0.50 6.22 0.03 0.02 2.1 0.3
perseveranc 4 4 0 3 3 0 7
e
UPPS: 27.5 8.01 25.2 8.80 0.71 0.54 3.41 0.11 0.01 - 0.2
Sensation 0 7 6 0 3 2.2 6
seeking 3
UPPS: 27.0 8.79 29.1 8.99 0.21 0.89 2.34 0.15 0.00 2.0 0.2
Positive UR 7 3 2 9 9 5 3
UPPS: 29.6 6.70 34.3 6.56 0.22 0.88 24.50 <0.0 0.08 4.7 0.7
Negative UR 8 9 1 01 5 0 1∗
Open in a separate window
FA, food addiction; ED, eating disorder; FA × ED, interaction parameter; MD, mean
difference. eta2, partial eta2. 1p: includes Bonferroni-Finner correction for multiple statistical
comparisons. Bold: significant comparison (0.05 level). ∗Bold: moderate (|d| > 0.50) to high
((|d| > 0.80) effect size.
There were significant differences on the UPPS-P subscales lack of perseverance (p < 0.05)
and negative urgency (p < 0.001), with higher values in FA patients compared to patients
without “positive YFAS screening score” (see Table Table22). Lack of premeditation,
sensation seeking and positive urgency did not differ as a function of FA.

Predictive Capacity of Personality in the Explanation of Food Addiction

Table Table33 includes the final predictive model of the presence of a positive YFAS
screening score. The first block, including the covariates sex, age, and diagnostic subtype,
obtained an initial predictive capacity equal to R2 = 0.22. In the second block, the TCI-R
reward-dependence and self-directedness scale scores were selected and fixed, with an
increase in the predictive capacity equal to R2 = 0.08, while the other TCI-R traits didn’t
explain further variance. In the third block, the UPPS-P lack of premeditation and negative
urgency scores were included, and the new increase in the predictive ability was R2 = 0.08,
while the other UPPS-P subscales did not add additional explanative power. The final
predictive model contained in the third block of the logistic regression indicates that after
adjusting for sex, age, and ED subtype, the odds of a “positive YFAS screening score” is
increased by high scores in the reward-dependence and negative urgency scales and low
scores in the lack of premeditation scale, while negative urgency can be seen as the
strongest predictor of FA. This model achieved goodness-of-fit (Hosmer–Lemeshow
test: p = 0.408).

Table 3
Predictive model for the dependent variable: positive screening of food addiction.

Criterion: FA “positive YFAS B SE Wald p OR 95%CI

screening score” (OR)
First block (ΔR2 = 0.221)
Sex (0 = female, - 0.596 0.383 0.536 0.69 0.22 2.22
1 = male) 0.369
Age (years-old) 0.001 0.018 0.006 0.938 1.00 0.97 1.04
Diagnostic AN vs. OSFED - 0.398 0.541 0.462 0.75 0.34 1.63
subtype 0.293
BN vs. OSFED 1.674 0.450 13.829 <0.001 5.33 2.21 12.88
BED vs. OSFED 2.258 0.813 7.707 0.006 9.57 1.94 47.11
Second block (ΔR = 0.078)
2

Sex (0 = female, - 0.658 0.004 0.953 0.96 0.26 3.49

1 = male) 0.039
Age (years-old) 0.001 0.019 0.002 0.964 1.00 0.97 1.04
Diagnostic AN vs. OSFED - 0.420 0.001 0.978 0.99 0.43 2.25
subtype 0.011
BN vs. OSFED 1.644 0.470 12.239 <0.001 5.18 2.06 13.00
BED vs. OSFED 2.064 0.828 6.208 0.013 7.88 1.55 39.95
TCI-R: Reward- 0.023 0.011 3.897 0.048 1.02 1.00 1.05
dependence
TCI-R: Self- - 0.009 12.154 <0.001 0.97 0.95 0.99
directedness 0.030
Third block (ΔR2 = 0.079)
Sex (0 = female, 0.095 0.709 0.018 0.894 1.10 0.27 4.41
1 = male)
Age (years-old) 0.000 0.020 0.000 0.999 1.00 0.96 1.04
Diagnostic AN vs. OSFED - 0.452 0.011 0.916 0.95 0.39 2.31
subtype 0.048
BN vs. OSFED 1.517 0.486 9.757 0.002 4.56 1.76 11.81
BED vs. OSFED 2.004 0.843 5.658 0.017 7.42 1.42 38.70
TCI-R: Reward- 0.026 0.012 4.531 0.033 1.03 1.00 1.05
dependence
 Criterion: FA “positive YFAS B SE Wald p OR 95%CI
screening score” (OR)
TCI-R: Self- - 0.011 2.209 0.137 0.98 0.96 1.01
directedness 0.016
UPPS: Lack - 0.033 4.235 0.040 0.93 0.87 1.00
premeditation 0.069
UPPS: Negative 0.124 0.034 12.992 <0.001 1.13 1.06 1.21
UR
Constant - 2.358 1.070 0.301 0.09
2.439
Open in a separate window
OSFED, Other Not Specified Eating or Feeding Disorders; AN, anorexia nervosa; BN,
bulimia nervosa; BED, binge eating disorder; SE, Standard Error; OR, Odds Ratio; CI,
Confidence Interval.

Discussion
Our first goal was to determine if ED patients with FA differ in personality traits when
compared with ED Patients without FA, after controlling for ED subtypes and age.
Prevalence of FA is high in ED (Gearhardt et al., 2013; Granero et al., 2014; Meule et al.,
2014b), in our sample 74.8% of participants met criteria for FA. Those with comorbid FA
indeed showed a distinct personality profile, although it was different than expected from the
literature regarding “addictive personality traits.” FA was not related to higher values in
novelty seeking, but exclusively to lower self-directedness (1a). With regard to impulsivity,
the hypothesis that ED patients with FA would have higher lack of perseverance and lower
negative urgency was supported by our data (1b).
Lower self-directedness has been found to be a characteristic trait both in individuals with
substance related and non-substance related addictive disorders, and seems to identify
individuals more vulnerable to develop addictive behavior patterns (Alvarez-Moya et al.,
2007; Schneider et al., 2015). In ED patients, low self-directedness is also a characteristic
trait (Fassino et al., 2002; Cassin and Von Ranson, 2005; Alvarez-Moya et al., 2007), but
those with FA seem to be even more marked in this regard. Further support for our results
is provided by another study (Bégin et al., 2012), that examined personality differences
between overweight/obese women with and without FA and found that women with FA were
more similar to women with substance use disorder than women without FA, particularly in
regard to impulsivity and self-directedness.
Research has shown that harm avoidance is common to all ED subtypes and significantly
higher in patients compared to controls (Cassin and Von Ranson, 2005; Lilenfeld et al.,
2006; Atiye et al., 2015). In our study, both ED groups had values beyond the norms of
general population (see Supplementary Figure S1), but no significant association was found
between this temperament factor and a higher rate of FA. According to this data, we can
thus infer that patients high in FA seem to have more problems with goal-orientation and
accountability (as measured by self-directedness) compared to ED patients without FA, but
both groups are comparable in behavioral and social inhibition and fear of uncertainty (as
measured by harm-avoidance). Low self-directedness in patients high in FA implicates that
this group has poor resourcefulness; this may present itself in problems to realistically adapt
behavior to environmental requirements and to remain in accord with individual goals at the
same time. Patients low in self-directedness may also be blaming and unreliable, which
could lead to interpersonal problems in this patient group.
The results of this study further indicate that patients reporting addictive eating patterns have
more difficulties to pursue tasks to the end and to focus on long-term goals, especially when
they are in a negative mood. This is reflected by their high lack of perseverance and high
values of negative urgency and is consistent with the results reported for non-clinical
populations (Murphy et al., 2014; Pivarunas and Conner, 2015). It is interesting to note that
FA patients show high impulsivity related to the regulation of negative emotions (as
measured by negative urgency), but do not show elevated values in impulsivity related
to positive emotions (as measured by positive urgency). Negative emotions may signal a
discrepancy between personal needs and present conditions, which for individuals with high
negative urgency is hard to bear (Cyders and Smith, 2008). This suggests that patients with
FA feel a strong pressure to act immediately when having negative emotions instead of
enduring until a moment more suitable to change. Since the need by itself all too often
cannot be fulfilled immediately, ingestion of rewarding food can be seen as an attempt to
escape these unbearable emotions by other means, which – depending on subjective
expectancies – could also be a drug or another behavior (Fischer et al., 2012; Torres et al.,
2013). Previous research shows that FA is also related to difficulties in emotion regulation
(Gearhardt et al., 2012; Pivarunas and Conner, 2015), which corroborates the results on
impulsive acts related to negative mood states.
Unexpectedly, ED patients with FA did not show elevated levels of novelty seeking when
compared to ED patients without FA. In general, therefore, it seems that the approach to
appetitive stimuli (reward seeking), which is implied by novelty/sensation seeking, does not
differ between ED patients with and without addictive eating behavior. This points out that
FA as assessed by the YFAS is more related to negative rather than to positive
reinforcement, which is in line with results of a former study in normal weight participants
(Meule and Kübler, 2012). It has been proposed that sensation seeking may be related
rather to non-clinical drug use, than to an actual addiction (Torres et al., 2013), which would
explain why patients with FA do not necessarily show elevated levels of sensation/novelty
seeking.
In regard to the study’s second objective, higher values in reward dependence, negative
urgency and lack of premeditation and lower values in self-directedness together explained
about 15% of the variance on having or not a positive FA screening, over and above sex,
age, and diagnostic subtype, while negative urgency was the most important predictor and
reduced the predictive power of the other variables to very small effects. Until now, risk
factors for suffering FA have been established in different samples, e.g., students (Murphy
et al., 2014; Pivarunas and Conner, 2015), obese women with overeating problems (Bégin
et al., 2012) or in ED patients (Gearhardt et al., 2013; Granero et al., 2014; Meule et al.,
2014b), but no study has explored which would be the highest risk population for presenting
FA. Our prediction model suggests that individuals with a high disposition to act rashly to
negative emotions are highly vulnerable for FA and would benefit from a specific approach
for treating FA symptoms.
It is important to bear in mind the cross-sectional nature of our study; we cannot definitely
conclude if the personality traits found to be related to FA precede or succeed FA symptoms,
or if both have one common cause. Further work is required to confirm the interrelations
between different predictors of FA in ED patients. Another limitation of this study is the small
sample size, especially for male patients, wherefore results on effects of gender in FA should
be investigated in future studies with higher sample power. Furthermore, our study only
included one self-report measure of FA, which could be completed by measures of craving,
daily assessments and behavioral food ingestion tests in future studies.
Regarding the YFAS, a key issue is the high prevalence rates of FA in AN patients, which
seems counterintuitive. Nevertheless, looking at the “total criteria fulfilled” (see Table
Table11), it appears that AN patients have a smaller number of total criteria fulfilled
compared to BN and BED; this may indicate to some part a problem of the cut-off criteria of
the YFAS. In addition to this, our results show that the criteria most frequently fulfilled in AN
patients are “important activities given up” (60.3%) and “unable to cut down/stop” (89.7%)
(see Supplementary Table S3). Some of the items of the YFAS, such as those loading on
“important activities given up” and “impairment or distress” may apply to AN in a similar way
as to patients on the bulimic spectrum, wherefore this patient group also scores high on
these criteria. On the other hand, the subscale “unable to cut down or stop” seems to be
systematically misunderstood by AN patients, possibly due to their subjective feeling of
eating too much. This could be addressed in future revisions of the scale and should be born
in mind when employing the YFAS in this patient group.
It has been formerly suggested that FA may merely be an index of ED severity (Davis,
2013; Gearhardt et al., 2014). The data at hand suggests that ED patients with FA apart
from showing a more severe symptomatology may differ from those without FA in the reward
value they expect from food intake. Rather than enjoying the hedonic value of food in good
mood, ED patients scoring high on FA mainly use food to regulate their negative emotions.
It can be hypothesized that the relation between negative emotional states and food intake
is mediated by impulsive personality traits and problems to focus on basic values or personal
goals.
To improve the described emotional dysregulation and inhibition of responses, a training of
emotion regulation strategies such as acceptance of emotional states could be helpful
(Murakami et al., 2015). The importance to integrate work on emotions and emotion
regulation skills into cognitive behavioral psychotherapy has reached increasing recognition
in the last years (Kahl et al., 2012; Moyal et al., 2015), and new therapy approaches for ED
patients have been developed. One example is the Cognitive Remediation and Emotion
Skills Training (CREST), a manualized brief psychotherapy addressing emotion regulation
and recognition (Money et al., 2011; Tchanturia et al., 2015), where patients learn to
differentiate between different emotions and are taught about the communicative function of
negative emotions. Patients with addictive-like eating patterns might benefit from this kind
of training; the findings of our study further suggest that work on value-oriented behavior is
important for patients with FA. Furthermore, this patient group might benefit to a great extend
from learning to endure negative emotions by the use of strategies other than food intake
and by this means they may be able to gradually reduce their dependence on food/eating in
order to regulate negative mood states.
The psychological basis of addictive-like eating compared to mere ED, e.g., the importance
ascribed to body shape, food-related cognitions, emotion regulation, should be further
investigated in future studies. Which situations and emotional states lead to uncontrolled
food intake in each group and the cognitions going along with this behavior could be
investigated in experimental studies or ecological momentary assessment studies.

Author Contributions
IW and IH contributed to the design of the work, acquisition and interpretation of the data.
RG was responsible for statistical analysis and for writing statistical sections of the
manuscript. SJ-M, AG contributed to administering and interpreting the psychological tests
of this study. CD, FC, AC, JM, FF-A participated in the design of the study. All authors (IW,
IH, RG, SJ-M, AG, CD, FC, AC, JM, FF-A) contributed to critically revising the work,
approved the final version of the article to be published and agreed to be accountable for all
aspects of the work in ensuring that questions related to the accuracy or integrity of any part
of the work are appropriately investigated and resolved.

Conflict of Interest Statement

The authors declare that the researchwas conducted in the absence of any commercial or
financial relationships that could be construed as a potential conflict of interest. The reviewer
Özgür Albayrak and handling Editor Astrid Müller declared their shared affiliation, and the
handling Editor states that the process nevertheless met the standards of a fair and objective
review.

Abbreviations
AN anorexia nervosa

ANOVA analysis of variance

BED binge eating disorder

BN bulimia nervosa

DSM Diagnostic and Statistical Manual of Mental Disorders

ED eating disorder

FA food addiction

OSFED other specified feeding or eating disorders

TCI temperament and character inventory

YFAS Yale Food Addiction Scale

Footnotes
Funding. Financial support was received from Fondo de Investigación Sanitaria -FIS
(PI14/290) and co-funded by FEDER funds – a way to build Europe. IW was supported by
a predoctoral grant of AGAUR (2014FI_B 00372). CIBER Fisiopatología de la Obesidad y
Nutrición (CIBERobn) and CIBER Salud Mental (CIBERsam), are both initiatives of
INSTITUTO DE SALUD CARLOS III. The funders had no role in the study design, data
collection and analysis, decision to publish, or preparation of the manuscript.

Supplementary Material
The Supplementary Material for this article can be found online
at: http://journal.frontiersin.org/article/10.3389/fpsyg.2016.00061
Click here for additional data file.(281K, DOCX)

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J Food Sci Technol. 2015 Jul; 52(7): 4196–4205.

XIII. Effect of six different cooking techniques in the nutritional

composition of two fish species previously selected as
optimal for renal patient’s diet
Isabel Castro-González, Ana Gabriela Maafs-Rodríguez, and Fernando Pérez-Gil Romo

Abstract

Introduction
It has been well established that fish consumption has beneficial effects on human health,
particularly associated with the prevention of cardiovascular disease (CVD) (Mozaffarian et
al. 2003); moreover, some studies support the hypothesis that a diet rich in n-3
polyunsaturated fatty acids (n-3 PUFA) may protect against renal deterioration (Friedman et
al. 2008; Friedman 2010; Lauretani et al. 2008; Lauretani et al. 2009; Fassett et al. 2010),
and a regular consumption of fish may reduce Chronic Kidney Disease (CKD) prevalence
(Gopinath et al. 2011). All those advantages have been related to eicosapentaenoic acid
(EPA) and docosahexaenoic acid (DHA), of which fish is the major source in human nutrition
(Agren and Hänninnen 1993; Al-Saghir 2004).
Current dietetic recommendations promote fish consumption (Ansorena et al. 2012): the
American Heart Association (AHA) advices that oily fish should be eaten at least twice per
week, preferably grilled, baked or broiled; and that the methods used to prepare fish should
minimize the addition of saturated and trans fatty acids, as occurs with the use of cream
sauces or hydrogenated fat during frying (Lichtenstain et al. 2006). However, recent studies
have stated that n-3 PUFA content may vary significantly comparing different fish species
according to the preparation method used (Mozaffarian 2003; Al-Saghir 2004; Izquierdo et
al. 2001; Gokoglu 2004; Bakar et al. 2008; Kaya 2008), and therefore it is not enough to
promote fish consumption in a general way, but rather to enrich the current
recommendations with practical information that allows consumers to make an informed
decision about the best way to eat fish.
Consumers have minimal knowledge about nutritive values of raw and cooked fish
(Gokoglu 2004; Mnari-Bhouri et al. 2010), and most information about nutrition content is
available for raw fish (Mnari-Bhouri 2010). For populations with a higher CVD risk
(Svensson 2004), nutritional therapy must include prescriptions of cooking techniques that
enhance the beneficial components (n-3 PUFA), and in the case of renal patients, diminish
harmful nutrients (phosphorus) of consumed fish species.
Many authors have previously examined the effect that different cooking methods have on
nutritional composition of fish; nonetheless they have mainly studied fatty components (total
lipids, fatty acids and cholesterol) and only in a few species, being the most common:
salmon, trout, tuna, cod and mackerel, among others (Agren 1993; Al-Saghir 2004;
Ansorena 2012; Izquierdo et al. 2001; Gokoglu 2004; Bakar 2008; Mnari-Bhouri 2010;
Echarte et al. 2001; Candela 1998; Elmadfa 2006; Moradi 2009). There are only a few
studies that have analyzed other nutrients as well (Izquierdo et al. 2001; Gokoglu 2004;
Kaya 2008; Elmadfa 2006; Kocatepe et al. 2011). None of the above evaluated nutritional
changes with the purpose of improving renal patients’ diets.
Previous studies have identified fish species that could be administered to renal patients
because of their low phosphorus and high n-3 PUFA content (Castro-González 2009;
Castro-González and Miranda-Becerra 2010). However, the analyses of those studies were
conducted in raw samples; therefore it is necessary to evaluate the impact that cooking
techniques have on the nutritional components of those species in order to provide more
accurate information to renal patients.
The aim was to study the impact that six cooking techniques have on the nutritional
composition of two fish species with low content of adverse nutrients in renal diet (Castro-
González et al. 2010; Castro-González 2012).

Materials and methods

Sampling
The fish, crevalle jack (Caranx hippos) and red drum (Sciaenops ocellatus), were obtained
from the largest fish distribution center in Latin America: the La Nueva Viga Market in Mexico
City. Several fillets of fresh fish were obtained from different vendors in order to get six
samples per specie.

Cooking techniques
Fillets from one fish of both species were prepared by six different cooking methods:
Steamed (ST): Fillets were placed on a steamer with boiled water until they were cooked.
This technique took from 5–9 min, and the fish reached a temperature of 76–80 °C.
Foiled (with aluminum) (FO): The fish fillet was completely wrapped in aluminum foil,
allowing the fish to be steamed in its own juices. The wrapped fish was placed in a “comal”
(a Mexican flat sauce pan) and cooked for 6–10 min. The fillet reached a temperature of 82–
93 °C.
Foiled with banana leaf (BL): This procedure was similar to the foiled method, except that
the fish was wrapped in banana leaf, a common Mexican cooking technique. This method
took from 5–8 min and the fillet reached a temperature of 78–92 °C.
Gas oven-baked (GO): The fish was placed in a gas oven and baked for 7–10 min, until it
reached 73–78 °C.
Microwave oven-cooked (MO): The fish fillet was placed in the microwave and cooked for
2–3 min at regular power. The fillet reached a temperature of 75–82 °C.
Fried Lightly (FL): The fish was placed on a frying pan with only 5 ml of Oleic Oil™. After
3 min, the fillet was turned to cook both sides of the fish. The food was cooked for 4–7 min
and it reached a temperature of 82–93 °C.

Chemical analyses
After the cooking processes, the raw (RA) and cooked samples were milled and
homogenized for chemical analyses. The chemical analyses were carried out in the Food
Sciences and Technology Department laboratories in the Instituto Nacional de Ciencias
Médicas y Nutrición Salvador Zubirán in Mexico City. This laboratory is certified by the
Mexican Accreditation Entity (EMA). All analyses were performed in triplicate.
Protein content was determined according to Mexican standard test methods (Norma Oficial
Mexicana 2002), using automatic protein equipment (Kjeltec 1035, Tecator, Höganaäs,
Sweden); this standard method is in accordance with ISO 8968, based on the Association
of Official Analytical Chemists method 991.20 (2010). Phosphorus (P) content was
determined using AOAC methods (Association of Official Analytical Chemists 1990), with a
Beckman spectrophotometer (DU70, Fullerton, San Diego, CA, USA).
Total fat content was calculated gravimetrically (Castro-González 2007). The fatty acids
were analyzed using solvent extraction and gas chromatography (Varian 3400 CX) with a
flame ionization detector.
The index of peroxidisability was calculated for each specie.
The mean of three repetitions are presented here. The results were grouped in descriptive
tables.

Statistical analyses
A One Way Analysis of Variance test (Kruskal-Wallis One Way Analysis of Variance on
Ranks) was performed to determine the difference among nutrient content of raw and
cooked samples of both species separately. To find means that were significantly different
from each other a Tukey test or Holm-Sidak method was performed. For all statistical tests
the probability level was 0.05. SigmaPlot (2008) for Windows statistical software was used.

Results and discussion

The nutritional composition of raw and fish cooked by six different methods is presented in
Table 1, as well as significant differences in the nutritional composition of different cooking
methods. Considering their lipid content, both species are low-fat fish, with a total fat
percentage 2–4 % (Nurnadia et al. 2011).

Table 1
Nutritional composition of raw and cooked crevalle jack and red drum

Raw Steame Foiled Foiled Gas Microwav Fried

(RA) d (ST) (FO) with oven- e oven- lightly
banana baked cooked (FL)
leaf (GO) (MO)
(BL)
Crevalle jack
Total 3.76a 4.22b 3.51ab 4.10b 3.96ab 5.06b 3.98ab
lipids
(g/100 g)
SFA 434.89a 479.96ab 420.33a 497.62a 393.33b 403.01ab 263.98b
b b
(mg/100 g)
MUFA 275.10a 287.83a 250.81a 278.24ab 223.62b 239.30ab 275.72ab
b b
(mg/100 g)
PUFA 295.10b 374.24ab 379.49a 500.25a 341.60a 325.59ab 260.38b
b b
(mg/100 g)
PUFA n- 232.35b 310.55ab 310.78a 417.87a 280.75a 271.48ab 195.23b
b b
3
(mg/100 g)
PUFA n- 49.77ab 49.57ab 55.15ab 64.12a 47.85ab 42.39b 33.60ab
6
(mg/100 g)
226.60b 305.23ab 304.06a 410.27a 276.39a 265.89ab 192.06b
EPA + DHA b b

(mg/100 g)
n-6/n-3 0.21 0.15 0.17 0.15 0.17 0.15 0.17
Protein 19.02a 23.01b 22.63b 24.50b 22.37b 23.81b 23.03b
(g/100 g)
177.43a 151.14b 204.23b 184.89b 232.44b 172.22b 240.31b
Phosphoru
s
(mg/100 g)
Red drum
Total 2.39a 1.67b 3.00b 2.70b 1.38b 2.33a 3.57b
lipids
(g/100 g)
SFA 328.17a 262.78a 482.69a 1150.03 348.19a 399.27ab 1076.22a
b b b b b
(mg/100 g)
MUFA 202.95a 179.79a 342.10a 732.27ab 207.19a 377.66ab 1204.73b
b b b
(mg/100 g)
 Raw Steame Foiled Foiled Gas Microwav Fried
(RA) d (ST) (FO) with oven- e oven- lightly
banana baked cooked (FL)
leaf (GO) (MO)
(BL)
PUFA 208.69a 348.92ab 460.11a 712.25b 273.78a 461.46ab 950.11b
b b
(mg/100 g)
PUFA n- 189.41a 256.15ab 340.66a 519.66b 196.91a 309.53ab 638.70ab
b b
3
(mg/100 g)
PUFA n- 19.28b 84.38ab 101.58a 167.98a 69.77b 151.93ab 206.64a
b
6
(mg/100 g)
189.41a 255.70ab 333.24a 483.19b 194.01a 309.53ab 614.97ab
EPA + DHA b b

(mg/100 g)
n-6/n-3 0.10 0.32 0.29 0.32 0.35 0.49 0.32
a b b b b b
Protein 23.89 26.36 25.91 27.59 24.98 27.29 27.99b
(g/100 g)
173.69a 171.40a 180.86a 175.25a 171.01a 202.22b 226.46b
Phosphoru
s
(mg/100 g)
Open in a separate window
SFA: Saturated fatty acids; MUFA: Monounsaturated fatty acids; PUFA: Polyunsaturated
fatty acids; EPA: Eicosapentaenoic acid; DHA: Docosahexaenoic acid
Values are shown as mean. Different letters indicate significant differences in the nutritional
composition of different cooking methods when compared to each other (*p < 0.05)
For crevalle jack the total lipid content increased in all cooking techniques except in the FO
sample compared to the raw fish, and the highest value was found in MO, with a 134.5 %
increase. In the case of red drum, lipid content increased in FO, BL and FL samples (125.5,
112.9 and 149.3 %, respectively), the lowest lipid content was found in the GO sample with
a 42.3 % reduction. These alterations in lipid level could be explained by the reduction in
water content after cooking and to the lipid content of each specie (Ferreira de Castro 2007,
Hosseini 2014,).
During the cooking process fatty acids undergo reactions as hydrolysis and oxidation, which
not only affect the FA concentration but also the fish flavor, scent, color and texture (Ferreira
de Castro 2007). The index of peroxidiability of crevalle jack is 194.32 and 155.44 for red
drum, which indicates high oxidative instability of its fatty acids when heated and therefore
could also explain the changes in the FA profile of both species (Testi et al. 2006,
Veselý 2009).
Saturated fatty acids (SFA) in crevalle jack decreased in all cooking techniques except in
ST and BL, which increased in 110.3 and 114.4 %, respectively. The lowest value was found
in the FL sample, with a 39.3 % reduction. The concentration of SFA in red drum only
decreased in the ST sample (with a 20 % reduction) when compared to the raw sample. BL
and FL presented more than 300 % increase in SFA content. Frying results can mainly be
attributed to the fatty acid composition of the frying oil and oxidation of the fish fatty acids
(Hosseini 2014, Domínguez et al. 2014).
Crevalle jacks’ monounsaturated fatty acids (MUFA) decreased in the FO, GO and MO
samples, while the samples cooked by the other three techniques presented an increase in
their MUFA content. The lowest value was found in GO sample with a 15.1 % decrease in
its content, while the highest concentration was presented in the ST sample, with a 104.6 %
increase. For red drum all cooking techniques except ST, produced an important increase
in MUFA content, ranging from 207.19 mg/100 g (only 102.0 % above the raw
concentration) to 1,204.73 mg/100 g in the fried lightly sample (with an increase of more
than 590.0 %).
For crevalle jack polyunsaturated fatty acids (PUFA) and n-3 PUFA increased in all cooking
methods except FL. The most important factor reducing the total n-3 PUFA content during
frying is oil absorption by the fish (Hossesini 2014). PUFA increased from 110.3 to 169.5 %
in MO and BL, respectively; while the n-3 PUFA content increased from 116.8 to 179.8 %.
n-6 PUFA content decreased in all cooking methods except in the foiled and foiled with
banana leaf samples. The lowest concentration was found in the fried lightly sample, with a
32.5 % reduction.
As for red drum, all cooked samples presented a higher PUFA content compared to the raw
sample, due to moisture losses that occur during cooking (Ersoy 2006). The highest
increase was found in BL and FL samples, with an increase of 341.2 and 455.2 %,
respectively. Both n-3 and n-6 PUFA increased in all cooking techniques compared to raw
red drum. The lowest increase was found in the GO samples, while the highest concentration
of both n-3 and n-6 PUFA was found in FL, with a 337.2 and 1071.7 % increase,
respectively.
When compared to raw crevalle jack, eicosanoic and docosahexaenoic acids (EPA + DHA)
content increased in all cooking techniques, except in FL. The highest content was found in
the BL sample, with an increase of 181.0 %. For red drum the sum of those fatty acids also
increased in all cooking methods, from 102.4 % in GO to 324.6 % in the FL sample.
Protein content increased in all cooking techniques in both species; BL presented the
highest protein concentration (128.8 %) in crevalle jack, while FL increased the protein
content of red drum up to 117.1 %. These increases occur because cooking produces
important losses of water, which concentrates the protein content (Ayala et al. 2005).
Phosphorus varied greatly among the different cooking methods, crevalle jack presented
slight decreases in ST and MO, and a maximum value of 240.31 mg/100 g in FL, which
represents an increase of 135.4 %. For red drum, ST and GO samples presented a slight
decrease (1.4 and 1.6 %, respectively), while the rest of the cooking methods had a higher
content. The maximum value was observed in the FL sample, with a 130.3 % increase.
Results could be related to water loss during different cooking methods (Ersoy 2009).
Table 1 presents the n-6/n-3 relation of both fish species in raw and cooked samples.
Crevalle jack’s n-6/n-3 relation decreased in all cooking techniques when compared to the
raw sample. The lowest relation was found in ST, BL and MO samples (0.15). The n-6/n-3
relation of red drum increased in all cooking methods. The lowest relation was found in the
FO sample (0.29) and the highest in the MO sample (0.49).
The fatty acid composition of raw and cooked fish is presented for crevalle jack in
Table 2 and for red drum in Table 3. Table 2 also contains the fatty acid composition of the
Oleic Oil™ used for the FL technique. C16:0 (palmitic acid) was the main fatty acid in raw,
ST, MO and GO crevalle jack samples; as well as in raw, FO and BL samples of red drum.
In crevalle jack, C22:6 n-3 (DHA) was the highest fatty acid in FO and BL; and in the ST and
MO samples of red drum. C18:1 n-9 (oleic acid) was the main fatty acid in the FL samples
of both species.

Table 2
Fatty acid content (mg/100 g) in raw crevalle jack and after different methods of cooking,
and fatty acids of Oleic Oil™

Raw Steame Foiled Foiled Gas Microwav Fried Oleic

d w/banan oven- e oven- lightly Oil™
a leaf baked cooked
C6:0 1.53 1.80 0.08 0.01 1.54 1.81 0.19 1.01
C8:0 0.16 0.87 ND 0.07 0.30 0.25 ND 0.02
C10:0 0.11 0.32 ND 0.01 0.20 0.04 ND 0.02
C11:0 0.04 0.33 ND 0.05 0.05 0.21 ND 0.23
C12:0 0.21 1.25 0.16 0.01 0.13 0.33 0.09 0.16
C13:0 0.22 0.10 ND 0.06 0.03 0.07 ND 0.12
C14:0 39.27 47.79 29.68 37.02 35.86 39.07 18.79 0.48
C14:1 ND ND ND ND ND ND ND ND
C15:0 6.91 7.24 5.58 6.85 5.87 6.10 3.54 0.10
C15:1 2.63 3.97 1.98 2.29 3.69 0.87 2.26 ND
C16:0 260.4 283.03 247.8 282.06 233.2 235.10 154.2 25.97
7 1 6 6
C16:1 78.61 82.43 64.24 71.64 65.11 70.78 37.52 0.76
C17:0 12.57 12.45 10.82 14.09 10.74 12.10 6.58 0.15
C17:1 5.45 5.36 4.64 5.52 4.42 4.70 2.67 0.18
C18:0 104.6 116.18 116.7 145.34 98.68 98.75 74.13 12.19
6 6
C18:1n 1.58 0.26 1.06 1.37 0.25 1.19 0.74 ND
-9 trans
C18:1n 181.2 189.56 172.9 189.96 144.3 156.15 228.7 419.2
-9 cis 7 7 6 6 9
C18:2n 1.27 0.52 0.05 0.45 0.48 0.48 0.22 0.84
-6 trans
C18:2n 9.20 11.88 10.60 14.24 9.66 8.96 29.91 80.58
-6cis
C18:3n 2.67 1.01 1.77 2.08 2.16 1.78 1.03 0.16
-6
C18:3n 4.39 4.37 3.76 5.62 3.72 4.31 2.41 0.22
-3
 Raw Steame Foiled Foiled Gas Microwav Fried Oleic
d w/banan oven- e oven- lightly Oil™
a leaf baked cooked
C20:0 5.01 5.44 4.92 5.88 4.25 5.05 3.41 2.27
C20:1 4.38 4.89 4.65 5.81 4.70 4.36 3.09 1.33
C20:2 2.15 1.53 2.45 3.54 2.74 2.12 1.42 0.01
C20:3n 1.35 0.94 1.42 1.98 0.62 1.26 0.76 0.03
-3
C20:3n 2.80 1.96 3.21 4.19 1.99 2.78 1.85 0.01
-6
C20:4n 44.29 46.58 50.17 57.84 43.67 37.81 30.71 ND
-6
C21:0 5.34 0.43 0.72 1.02 0.33 0.76 0.44 0.05
C22:0 0.06 0.20 ND 0.05 0.37 ND ND 0.06
C20:5n 44.49 59.19 56.49 67.61 48.35 50.48 34.00 2.38
-3
C22:1n 1.11 0.65 1.26 1.59 1.06 1.24 0.66 0.07
-9
C22:2 0.34 0.19 ND 0.02 0.12 0.14 ND 0.15
C23:0 0.94 0.37 0.81 0.60 0.39 1.18 0.47 0.45
C24:0 2.12 2.10 2.95 4.34 1.32 2.14 2.06 0.88
C24:1 0.04 0.68 ND 0.05 ND ND ND ND
C22:6n 182.1 246.03 249.1 342.66 228.0 215.40 158.0 0.41
-3 0 0 4 6
Open in a separate window
ND: not detected. Values are shown as mean of three repetitions

Table 3
Fatty acid content (mg/100 g) in raw red drum and after different methods of cooking

Raw Steamed Foiled Foiled Gas Microwave Fried

w/banana oven- oven- lightly
leaf baked cooked
C6:0 10.75 6.31 9.01 134.99 3.63 112.08 59.83
C8:0 ND ND 3.51 0.57 1.68 ND ND
C10:0 ND ND ND ND 0.74 ND ND
C11:0 ND ND 1.26 26.31 0.24 ND ND
C12:0 ND ND 0.05 ND 0.59 ND 0.92
C13:0 ND ND 0.15 ND 0.62 ND ND
C14:0 9.17 3.80 25.27 31.18 18.52 ND 50.85
C14:1 ND ND ND ND ND ND ND
 Raw Steamed Foiled Foiled Gas Microwave Fried
w/banana oven- oven- lightly
leaf baked cooked
C15:0 ND 2.88 7.04 14.87 6.52 ND 18.6
C15:1 ND ND 2.40 5.99 1.14 ND 0.18
C16:0 202.48 157.37 312.96 604.45 192.09 208.88 677.32
C16:1 80.69 55.25 125.52 240.99 51.38 63.36 262.54
C17:0 7.75 7.74 12.09 28.19 10.74 ND 35.09
C17:1 5.16 7.61 7.95 15.52 13.88 ND 10.58
C18:0 74.71 80.38 100.71 192.97 102.72 78.31 206.82
C18:1n- ND 1.65 2.55 ND 12.94 157.20 5.43
9 trans
C18:1n- 87.28 110.44 187.95 439.30 117.82 157.11 879.24
9 cis
C18:2n- ND ND 1.48 ND 0.31 ND 1.16
6 trans
C18:2n- ND 4.93 12.88 24.62 4.55 ND 98.75
6cis
C18:3n- ND ND 1.45 ND 2.04 ND 6.64
6
C18:3n- ND 0.35 6.19 36.46 2.71 ND 18.93
3
C20:0 ND 4.30 5.37 19.32 7.45 ND 17.85
C20:1 7.96 4.85 14.20 30.46 9.38 ND 39.00
C20:2 ND 3.46 3.52 ND 0.79 ND 4.86
C20:3n- ND ND 1.23 ND 0.19 ND 4.80
3
C20:3n- ND 2.16 3.76 ND 0.51 ND 3.40
6
C20:4n- 19.28 82.22 96.37 167.98 67.21 151.93 196.60
6
C21:0 ND ND 3.80 ND 0.55 ND 2.48
C22:0 ND ND ND ND 0.19 ND ND
C20:5n- 21.49 40.47 86.45 130.99 37.73 81.79 190.17
3
C22:1n- 21.87 ND 1.53 ND 0.65 ND 7.76
9
C22:2 ND ND ND ND 1.46 ND ND
C23:0 23.31 ND ND ND 0.60 ND 1.16
C24:0 ND ND 1.47 97.16 1.31 ND 5.29
 Raw Steamed Foiled Foiled Gas Microwave Fried
w/banana oven- oven- lightly
leaf baked cooked
C24:1 ND ND ND ND ND ND ND
C22:6n- 167.91 215.32 246.80 352.20 156.28 227.74 424.80
3
Open in a separate window
ND: not detected. Values are shown as mean of three repetitions

Cooking techniques
Cooking techniques that require heat can affect the nutritional composition of fish species
depending on the temperature reached, the amount of time the food is exposed to the heat
and the methods used to cook it (Agren 1993; Ansorena 2012; Puwastein 1999; Tur
Marí 2004). In the present study all cooking techniques produced important changes in the
nutritional composition of crevalle jack and red drum. However, the species behaved very
differently when submitted to the same cooking techniques. These differences could be
explained by raw composition, temperature, size, exposed surface and degree of
postmortem ageing prior to cooking (Ayala 2005, Ferreira 2007, Gladyshev 2007).
Figure 1a and andbb presents the perceptual content of beneficial (EPA + DHA) and
adverse (protein, phosphorus) nutrients in crevalle jack and red drum. Crevalle jack (Fig. 1a)
presents a slight increase in its perceptual content of EPA + DHA in ST, FO, BL and MO
samples. The protein perceptual content remains stable in all samples; while the phosphorus
also presented variations: a higher perceptual content is found in the FL sample, while the
rest of the cooking techniques present a slight decrease in their perceptual phosphorus
content. In red drum’s case (Fig. 1b), BL presents the highest perceptual concentration of
EPA + DHA, although all cooking methods present an increase in these nutrients. Protein
perceptual content presents a slight decrease in the BL and FL samples; and phosphorus
perceptual content diminishes among the different techniques, being BL the one with the
lowest content. Red drum (Fig. 1b), in general, presents more variations in its perceptual
content of beneficial and adverse nutrients than crevalle jack (Fig. 1a).
Fig. 1
Perceptual content of beneficial (Eicosanoic and docosahexaenoic acids (EPA + DHA)) and
adverse (protein, phosphorus) nutrients in a) crevalle jack fillets under different cooking
techniques and b) red drum fillets under different cooking techniques. RA: raw, ST:
steamed, FO: foiled, BL: foiled with banana leaf, GO: gas oven-baked; MO: microwave
oven-cooked and FL: fried lightly

Steam
Steaming had opposite effects in crevalle jack and red drum regarding total lipids, SFA,
MUFA and n-6 PUFA. It was the only cooking technique that decreased phosphorus content
in both species and increased EPA + DHA concentration, nutritional qualities appropriate for
renal patients. Mnari-Bhouri (2010) found that steaming lightly increased the total lipid
content of both wild and farmed sea bream, and decreased the MUFA concentration in the
wild fish. In the present study similar results were only observed in crevalle jack’s lipid
content and red drum’s MUFA concentration. The same study by Mnari-Bhouri found that
oleic acid (C18:1 n-9c) decreased in sea bream after steaming; n-3 PUFA decreased, and
n-6 PUFA content remained stable. In the present study oleic acid and n-3 PUFA increased
in both species after steaming, and n-6 PUFA decreased in crevalle jack but increased in
red drum. Phosphorus decrease is consistent with the study of Hosseini (2014), who found
that boiling decreased phosphorus content in fish, while Ersoy (2009) reported that minerals
usually increase after cooking. The factors responsible for these results are not known.

Foil and foil with banana leaf

There are few studies that present the effect of foiled with aluminum or banana leaf
techniques on fish. In the present study, when foiled, the nutritional components of crevalle
jack decreased, except for PUFA, protein and phosphorus. Opposite effects were observed
in red drum, were all elements increased. As for the foiled with banana leaf technique; all
components of both species increased. Banana leaf has an elevated content of PUFA, which
could explain the results found in the present study (Rosas and Díaz 1983).

Gas oven
Nutritional components of GO samples presented variations among the two analyzed
species. While total lipids and phosphorus content decreased in red drum, their
concentration increased in crevalle jack. No other component increased in red drum,
although PUFA, n-3 PUFA, EPA + DHA and protein content increased in crevalle jack.
Kocatepe (2011) found that black sea anchovy fat content significantly increased after
baking, and protein concentration slightly decreased. Agren and Hänninen (1993) found that
baking in the oven decreased the fat content of rainbow trout, but increased it in vendace
and pike. The results of the present study revealed that though gas oven increased the lipid
content of crevalle jack, it decreased it in red drum. The nutritional increases found in these
cooking technique can be explained by the exposure to heat and loss of moisture, because
baking is one of the cooking methods with the greatest water loss (Ersoy 2009,
Hosseini 2014).

Microwave oven
The MO technique increased total lipid content, PUFA, n-3 PUFA, EPA + DHA and protein
in crevalle jack but decreased phosphorus content. In red drum’s case, all components
except total lipids increased with this cooking method. Gokoglu and others (2004) found that
protein and fat content of rainbow trout significantly increased when cooked in the
microwave; and Izquierdo et al. (2001) reported similar results in the case of tuna (Thunnus
thynnus), where protein and lipid content also increased in the cooked samples. Results of
the present study are similar, except for lipid content of red drum, which presented a small
decrease after microwave cooking. The study by Gokoglu (2004) revealed a slight increase
in the phosphorus content of rainbow trout, effect that was also found in red drum during the
present study, however, it is noteworthy that crevalle jack’s phosphorus content decreased
when subjected to this cooking technique. The previously mentioned study by Izquierdo et
al. (2001) reported a significant decrease in the content of C20:5 n-3 (EPA) and C22:6 n-3
(DHA) after microwave cooking. In the present study, both species increased their
concentration of EPA and DHA. Higher cooking losses after microwaving are a combination
of liquid and soluble matter lost during cooking, especially water since heat induced protein
denaturation causes less water to be trapped within protein structures (Domínguez 2014).

Fried lightly
When fish is fried there is a fat exchange between the oil and the fish and oil absorption by
fish resulting in modification of the fatty acid profile (Ansorena 2012, Hosseini 2014). Frying
could also cause oxidation of PUFA in the oil and reaction of the lipid oxidation compounds
generated, with molecules that could appear due to proteolytic reactions (Domínguez 2014).
In the present study, the FL technique included the use of Oleic Oil™, a commercial oil brand
made from safflower seed and available in most supermarkets in Mexico. It has a low content
of SFA and PUFA, but considerable amounts of MUFA. Previous studies have found that
frying increases the fat content of fish (Agren 1993; Ansorena 2012; Izquierdo et al. 2001;
Gokoglu 2004; Bakar 2008; Mnari-Bhouri 2010; Kocatepe 2011; Puwastein et al. 1999). In
our study, the frying technique decreased the fatty acid content of crevalle jack, but not the
total lipid content. Similarly, protein and phosphorus concentration increased in this
technique, because of water loss by heating. For the case of red drum, the results are similar
to those previously reported and all nutritional components increased with the frying
technique.

n-6/n-3 relation
The n-6/n-3 relation indicates the proportion and balance between n-6 and n-3 PUFA. Some
studies suggest that a combination of n-3 and n-6 PUFA is associated with lower levels of
inflammation (Deckelbaum 2010); however, a high n-6/n-3 ratio promotes the pathogenesis
of many diseases, including cardiovascular disease, cancer, and inflammatory diseases,
whereas increased levels of n-3 PUFA (a lower omega-6/omega-3 ratio) exert suppressive
effects (Simopoulos 2006). Modern Western diet has a typical n-6/n-3 ratio of 15.0 (Kiecolt-
Glaser 2007; Simopoulos 2008), and the current recommendation for the prevention of
cardiovascular disease and other chronic diseases is around 2.0-5.0 (Simopoulos 2008). In
the present study, the n-6/n-3 ratios ranged from 0.15 to 0.21 in crevalle jack, and from 0.10
to 0.49 in red drum, which indicate that in all cooking techniques of both species the n-3
PUFA content was considerably higher than the n-6 PUFA. The regular consumption of fish
with these characteristics would contribute to the adequate intake of a low n-6/n-3 ratio,
recommended for the risk reduction of some chronic diseases (Simopoulos 2008). The best
way to improve the n-6/n-3 ratio is by increasing the n-3 PUFA intake and not by decreases
in n-6 PUFA (Deckelbaum 2010); therefore the consumption of fish with high n-3 PUFA,
particularly EPA and DHA, is recommended for the prevention of the previously mentioned
pathologies.
Figure 2a and b presents the fatty acid composition (SFA, MUFA, PUFA, n-3 PUFA, n-6
PUFA and EPA + DHA) of both fish species. In crevalle jack’s case (Fig. 2a) fatty acid
components present a similar trend in each cooking technique. In red drum’s case (Fig. 2b),
SFA present a notably high concentration in BL and FL samples; however, the rest of the
fatty acid components behave similarly in each cooking method. When comparing Fig. 2a
and b, different behavior is observed in both species when submitted to the same cooking
techniques.
Fig. 2
Saturated fatty acids (SFA), Monounsaturated fatty acids (MUFA), polyunsaturated fatty
acids (PUFA), n-3 PUFA, n-6 PUFA and eicosanoic + docosahexanoic acid (EPA + DHA) in
raw and cooked a) crevalle jack and b) red drum. RA: raw, ST: steamed, FO: foiled, BL:
foiled with banana leaf, GO: gas oven-baked; MO: microwave oven-cooked and FL: fried
lightly
The most recommended cooking techniques for renal patients are those that diminish or
maintain stable potentially adverse components (phosphorus and protein), and that increase
the concentration of beneficial nutrients (EPA + DHA). Considering this, crevalle jack should
be preferably consumed ST or MO; and red drum should be ST or GO. Nonetheless, all
cooking techniques in both fish species produced a protein increase lower than 30 g/100 g
of fillet, and a phosphorus concentration lower than 241 mg/100 g, which gives room to
include them all in individualized recommendations.

Conclusion
Crevalle jack and red drum behaved different, and sometimes even in an opposite way,
when cooking methods were applied to them. Therefore, it is important to further evaluate
the impact that cooking techniques have on different fish species in order to give specific
recommendations that provide more benefits to renal patients.
This article contributes with relevant results regarding nutritional composition of different fish
species appropriate to renal patients’ diets, after being submitted to cooking techniques.
This information is necessary to provide more variability to their diets and to improve intake
monitoring of several key nutrients in their nutritional management.
Acknowledgments
The authors would like to thank Mr. Jorge Toral from the Cámara Nacional de Comercio de
la Ciudad de México (National Commerce Chamber of Mexico city) for providing the fish
species used in the present study.

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Biomed Res Int. 2018; 2018: 4810394.

XIV. Monoamine Oxidase-A Inhibition and Associated

Antioxidant Activity in Plant Extracts with Potential
Antidepressant Actions
Tomás Herraiz and Hugo Guillén

Abstract

1. Introduction
The enzyme monoamine oxidase (MAO) metabolizes xenobiotic and endogenous amines
and neurotransmitters including serotonin, dopamine, norepinephrine, tyramine, tryptamine,
and the neurotoxin MPTP [1, 2]. It occurs as two isoenzymes, MAO-A and MAO-B, which
play an important role in the central nervous system (CNS) and peripheral organs. MAO-B
is involved in neurodegenerative diseases and MAO-A in psychiatric conditions and
depression. Inhibitors of MAO-B are useful as neuroprotectants, whereas inhibitors of MAO-
A are effective antidepressants although their use may trigger adverse reactions (e.g.,
hypertensive crisis with foods containing tyramine) [1]. On the other hand, the oxidation of
biogenic amines and neurotransmitters by MAO enzymes generates hydrogen peroxide
(H2O2), oxygen radicals, and aldehydes, which are risk factors for cell oxidative injury.
Therefore, the inhibition of MAO may result in protection against oxidative stress and
neurotoxins [1, 3, 4]. Recent investigations have pointed out that plant and food extracts
may inhibit MAO enzymes resulting in the above-mentioned biological effects [3, 5–14]. On
the other hand, as a result of MAO inhibition, those products might be involved in undesirable
interactions with other herbal preparations, foods, or drugs [1].
Hypericum perforatum L. (family Hypericaceae) (St. John's wort) is widely used for health
purposes and their products are commercially available as herbs, nutraceuticals, teas,
tinctures, juices, oily macerates, phytopharmaceuticals, and food additives and supplements
[15, 16]. H. perforatum is popular for treatment of mild and moderate depression [17–19]. It
may trigger adverse pharmacological interactions with others herbs, drugs, or foods [20–
22]. Its ability to alleviate and improve mood disorders and depression is attributed to active
compounds that exhibit antidepressant properties [23, 24]. The most accepted mechanism
of action is monoamine reuptake inhibition but additional mechanisms including monoamine
oxidase inhibition and synergistic effects can be involved [17]. Peganum harmala (family
Zygophyllaceae) and Lepidium meyenii (family Brassicaceae) (maca) are plants with CNS
effects and potential antidepressant actions [14, 25, 26]. P. harmala, native from the
Mediterranean region and Asia and extended to North America areas, is used as a
multipurpose health remedy including CNS disorders. Preparations of this plant may trigger
adverse pharmacological interactions [27]. L. meyenii is an edible plant from the central
Andes whose roots are used as a food energizer and nutraceutical to improve physical and
mental conditions and fertility [28]. The purpose of this work was to study the inhibition of
human MAO-A by extracts of H. perforatum, P. harmala, and L. meyenii (maca) as well as
by their active components that were identified and analyzed by HPLC-DAD-MS and
subsequently evaluate the antioxidant activity which is specifically associated with the
inhibition of MAO. This specific antioxidant activity is determined for the first time in plant
extracts.

2. Materials and Methods

Hypericum perforatum L. plants collected in Ciudad Real (Spain) were dried and separated
in parts: flowers; top aerial portions of the plant including branched stems and leaves but no
flowers; and main stems (central and lower) and roots. They were ground and the powder
used for sample preparation. Commercial herbs and herbal supplements (capsules and
tablets) of H. perforatum were also purchased in local herbal shops.Peganum harmala L.
plant and seeds were collected in Toledo (Spain). Lepidium meyenii (maca) both as powder
and commercial tablets were obtained from Peru and local shops. Hypericin standard (>95%
purity by HPLC) from HWI Analytik GMBH pharma solutions, hyperforin
dicyclohexylammonium salt, quercetin, harmaline, harmine, catalase, clorgyline, 3,3′,5,5′-
tetramethylbenzidine (TMB), and horseradish peroxidase (HRP) type II were purchased
from Sigma-Aldrich.

2.1. Sample Preparation of Plant Extracts

Samples containing H. perforatum (i.e., plant parts, herbal preparation, capsules, or tablets)
(500 mg) were homogenized in 10 mL of water/methanol (1 : 1) by using an Ultra Turrax
homogenizer, centrifuged at 10000 rpm for 10 min, and the supernatant was collected. The
process was repeated twice with the residue and the three supernatant fractions collected,
mixed and analyzed by HPLC as mentioned below. After three consecutive extractions, the
recoveries of hypericin and pseudohypericin were higher than 97%. Samples of L.
meyenii (maca) (500 mg) and P. harmala seeds (500 mg) were homogenized, respectively,
in 10 mL of water/methanol (1 : 1) or 10 mL of 0.6 M perchloric acid : methanol (1 : 1) by using
an Ultra Turrax homogenizer, centrifuged at 10000 rpm for 10 min, and the supernatant was
collected. This process was repeated twice with the residue and the collected supernatants
were mixed and analyzed by HPLC as mentioned below.

2.2. RP-HPLC Analysis of Plant Extracts

The analysis of H. perforatum extracts was performed by RP-HPLC with UV diode array and
fluorescence detection using a HPLC 1050 (Agilent) coupled with a 1100 diode array
detector (DAD) (Agilent) and a 1046A-fluorescence detector. A 150 × 3.9 mm i.d., 4 μm,
Nova-pak C18 column (Waters) was used for separation. Chromatographic conditions were
50 mM ammonium phosphate buffer (pH 3) (buffer A) and 20% of A in acetonitrile (buffer B).
The gradient was programmed from 0% (100% A) to 32% B in 8 min and 100% B at 10 min.
The flow rate was 1 mL/min, the column temperature was 40°C, and the injection volume
was 20 μL. Detection of hypericins was carried out by absorbance at 590 nm and
fluorescence at 236 nm for excitation and 592 nm for emission. The concentration of
hypericin was determined from a calibration curve of response (absorbance at 590 nm)
versus concentration with solutions made in the laboratory from hypericin standard. The
same response factor was applied to pseudohypericin, protohypericin, and
protopseudohypericin. Flavonoids and flavonoid glycosides were analyzed at 265 nm and
355 nm and the concentration of quercetin was determined at 355 nm from a calibration
curve of response versus concentration. The HPLC fraction corresponding to flavonoids and
flavonoid glycosides (7 to 11 min) was collected by successive injections of H.
perforatum extract (herbs) and, after evaporation in vacuum, dissolved in 30% methanol and
used for MAO-A inhibition. The phloroglucinols (hyperforin, adhyperforin, hyperfirin, and
adhyperfirin) were analyzed at 280 nm by using the same column (Nova-pak C18) and
conditions but under isocratic elution with 20% of 50 mM ammonium phosphate buffer, pH
3, and 80% of acetonitrile. The concentration of these compounds was determined from a
calibration curve of hyperforin standard. The analysis of β-carboline alkaloids in P.
harmala and L. meyenii was carried out as previously described [14, 29].

2.3. Identification by HPLC-ESI-Mass Spectrometry

Identification of compounds in H. perforatum extracts was done by HPLC-MS (electrospray-
negative ion mode) by using a 1200 series HPLC-DAD coupled to a 6110 quadrupole-MS
(Agilent). Chromatographic separation was performed on a 150 × 2.1 mm i.d. Zorbax SB-
C18 (5 μm) column (Agilent Technologies). The chromatographic conditions were eluent A:
formic acid (0.1%); B: formic acid (0.1%) in acetonitrile; gradient: 0% to 70% B in 8 min and
100% B at 10 min, flow rate: 0.3 mL/min; T: 40°C; mass range: 50–700 u, and cone voltage:
150 V. For identification of phloroglucinols (e.g., hyperforin), separation was done using a
Nova-pak C18 (4 μm) column with the same eluents and isocratic elution (eluent A, 20% and
eluent B, 80%) at a flow rate of 0.7 mL/min and mass spectra recorded in negative and
positive ionization. Identification of compounds was done on the basis of mass spectra, UV-
vis spectra (DAD) of chromatographic peaks, and coelution with standards. β-Carbolines
in P. harmala and L. meyenii were identified as previously described [14, 29].

2.4. Monoamine Oxidase (MAO-A) Inhibition Assays

MAO assays were performed as elsewhere [8, 11, 14]. Briefly, membrane protein fractions
containing MAO-A (BD-Gentest) were diluted to the desired concentrations in 100 mM
potassium phosphate buffer (pH 7.4). A 0.2 mL reaction mixture containing 0.01 mg/mL
protein and 0.25 mM kynuramine in 100 mM potassium phosphate (pH 7.4) was incubated
at 37°C for 40 min. After incubation, the reaction was stopped by the addition of 2 N NaOH
(75 μL), followed by the addition of 70% HClO4 (25 μL), and the sample was centrifuged
(10000g) for 10 min. The supernatant (20 μL) was injected into the HPLC and the
deamination product of kynuramine (i.e., 4-hydroxyquinoline) formed during enzymatic
reaction determined by RP-HPLC-diode array detection at 320 nm. A response curve of area
versus concentration was constructed to calculate the concentration of 4-hydroxyquinoline.
In order to perform assays of MAO inhibition, aliquots of extracts from plants or commercial
preparations or instead pure compounds were conveniently diluted and added to reaction
mixtures containing kynuramine (0.25 mM) and MAO-A (0.01 mg/mL protein) in 100 mM
potassium phosphate buffer (pH 7.4), with enzymatic reaction and analysis carried out as
above, and compared with the corresponding controls containing solvent. The standard
inhibitor clorgyline was used as a positive control for inhibition (>90% inhibition at 2.5 μM).
Incubations were carried out at least in duplicate from different experiments and the
IC50 values were calculated using GraphPad Prism 4.0.

2.5. Determination of Antioxidant Activity Associated with Monoamine Oxidase (MAO)

Inhibition
Assays (0.2 mL) of reaction mixtures in 70 mM potassium phosphate buffer (pH 7.4),
containing 0.025 mg/mL MAO-A protein and 0.25 mM kynuramine, were incubated at 37°C
for 40 min in the absence (control assays) or in the presence of plant extracts. MAO assays
were also performed in presence of clorgyline (25 μM), a classical inhibitor of MAO-A
(positive control of inhibition), or catalase enzyme (100 μg/mL). After the incubation period,
the reaction mixture was added with activated charcoal (3.5 mg), mixed, and filtered
(0.45 μm). The solution was added with 20 μL of 10 mM tetramethylbenzidine (TMB) in 40%
DMSO and 20 μL of horseradish peroxidase (HRP) type II (1 mg/mL), kept 5 min, and added
with 0.3 mL of 0.5 M H2SO4 solution. The absorbance at 450 nm was measured to determine
TMB diimine, a yellow product resulting from the oxidation of TMB by HRP and the
H2O2 generated in the oxidative deamination catalyzed by MAO. The oxidation of TMB in
the presence of inhibitors of MAO was compared with the corresponding controls without
inhibitors and appropriate blanks showed absence of interferences.

3. Results and Discussion

Commercial preparations of H. perforatum inhibited human MAO-A with similar potency:
IC50 values of 142.3 ± 30.6 μg/mL (herbal preparation), 193 ± 61 μg/mL (capsules), and 173
± 29 μg/mL (tablets) (Figure 1(a)). Regarding plants, H. perforatumextracts from flowers
afforded the highest inhibition (IC50 of 63.6 ± 9.4 μg/mL) followed by aerial stems and leaves
(IC50 143.6 ± 16.5 μg/mL), and the lowest in root extracts (Figure 1(b)). Extracts from the
aerial parts of H. perforatum were analyzed by HPLC-DAD-ESI (electrospray-negative
ionization). They showed the presence of two major naphthodianthrones identified as
pseudohypericin and hypericin (Figure 2(a) and Table 1). Flower extracts had two additional
compounds identified as protopseudohypericin and protohypericin. Phenolics and
flavonoids abounded in H. perforatum extracts (Figure 2(b)). Chlorogenic acid and the
quercetin glycosides rutin, hyperoside, isoquercitrin, miquelianin, acetyl hyperoside, and
quercitrin, as well as free quercetin and biapigenin, were identified by HPLC-DAD (ESI
negative ionization) and DAD (Table 1). On the other hand, flower extracts contained four
phloroglucinols (Figure 2(c)) that were identified by HPLC-DAD-MS (ESI negative and
positive ionization) and DAD as hyperforin, adhyperforin, hyperfirin, and adhyperfirin (Table
1). The presence of these compounds (Figure 3) in the plant agrees with other results
[15, 30, 31]. The content of the main components was determined by HPLC (Table 2).
Concentration of pseudohypericin was higher than hypericin, whereas protopseudohypericin
and protohypericin were minor compounds (0.4 μg/mg of protopseudohypericin and
0.17 µg/mg of protohypericin were detected in flowers). In the plant, the highest content of
hypericins was found in flowers with significantly low levels detected in stems and absence
in roots. Hyperforin was highly abundant in flowers (27.2 μg/mg), whereas the concentration
in commercial preparations ranged from 0.36 to 2.4 μg/mg. In flowers, adhyperforin (1.4 ±
0.07 μg/mg), hyperfirin (4.2 ± 0.02 μg/mg), and adhyperfirin (0.46 ± 0.02 μg/mg) also
appeared. Flavonoids abounded in H. perforatum and most of them were quercetin
glycosides (Figure 2(b)) whose presence was significantly higher in flowers than in other
parts of the plant. The content of free quercetin in flowers was 2.0 μg/mg, whereas a content
of 6.7 μg/mg was determined in commercial preparations.
Figure 1
Inhibition of human monoamine oxidase-A (MAO-A) by extracts of commercial
preparations of H. perforatum (a) (capsules, ■; tablets, ▲; herbs, ▼), and extracts from
different parts of the plant (b) (flowers, ∆; top stems, ◊; main stems (central), ×; roots, ○).
Significant differences (p < 0.05) among extracts at a selected concentration are indicated
with different letters.
Open in a separate window
Figure 2
HPLC chromatograms of extracts from H. perforatum flowers. (a) Detection of hypericins at
590 nm. 1: protopseudohypericin; 2: pseudohypericin; 3: protohypericin; and 4: hypericin.
(b) Detection of phenols and flavonoids at 265 nm. 1: chlorogenic acid; 2: rutin;3:
hyperoside; 4: isoquercitrin; 5: miquelianin; 6: acetyl hyperoside; 7: quercitrin; 8: quercetin;
and 9: biapigenin. (c) Detection of phloroglucinols at 280 nm. 1: hyperfirin, 2: adhyperfirin; 3:
hyperforin; and 4: adhyperforin.
Figure 3
Structures of compounds identified in H. perforatum: hypericins, quercetin, and quercetin
flavonoids and phloroglucinols (hyperforin, adhyperforin, hyperfirin, and adhyperfirin).

Table 1
Compounds identified in H. perforatum.

Compounds ESI-neg. ion (M − H)− UV max (DAD)

Naphthodianthrones    
Pseudohypericin 519 547, 590
Hypericin 503 547, 590
Protopseudohypericin 521 370, 539
Protohypericin 505 370, 539
Phenolic comp.    
Chlorogenic acid 353 324
Rutin 609 256, 355
Hyperoside 463 256, 355
Isoquercitrin 463 256, 355
Miquelianin 477 256, 355
Acetyl hyperoside 505 263, 352
Quercitrin 447 255, 348
Quercetin 301 255, 369
Biapigenin 537 268, 331
Phloroglucinols    
 Compounds ESI-neg. ion (M − H)− UV max (DAD)
Hyperfirina 467 274
a
Adhyperfirin 481 274
a
Hyperforin 535 274
Adhyperforina 549 274
Open in a separate window
a
These compounds gave also their corresponding (M + H)+ and (M + K)+ ions under ESI-
positive ionization.

Table 2
Content (μg/mg)1 of the main active components in H. perforatum samples.

H. perforatum samples Pseudohypericin Hypericin Hyperforin Quercetin

Plant        
a a a
Stems (top) 0.25 ± 0.03 0.11 ± 0.01 1.48 ± 0.3 0.28 ± 0.12a
Stems (central) 0.1 ± 0.04a 0.04 ± 0.01a 0.59 ± 0.16a 0.19 ± 0.01a
Roots - - 0.77 ± 0.1a -
b b b
Flowers 2.78 ± 0.7 1.58 ± 0.31 27.2 ± 0.6 2.04 ± 0.08b
Commercial prep.        
a a a
Herbs 0.51 ± 0.05 0.11 ± 0.01 1.18 ± 0.03 0.71 ± 0.4a
Capsules 2.41 ± 0.2b 0.83 ± 0.1b 2.42 ± 0.01b 2.4 ± 0.9a
Tablets 2.39 ± 0.2b 2.11 ± 0.2c 0.36 ± 0.1c 6.7 ± 1.7b
Significant differences (p < 0.05) for a compound within a group are indicated with different
letters. 1μg of compound/mg of plant tissue for plants parts and herbs or mg of powder in
capsules and tablets.
The inhibition of MAO-A by H. perforatum extracts indicates occurrence of inhibitors.
Hypericins, hyperforin, and flavonoids are possible contributors to this inhibition and were
evaluated as inhibitors (Figure 4). Hypericin inhibited MAO-A (IC50 of 35.5 ± 2.1 μM or
17.9 μg/mL) (Figure 4(a)). From the concentration in Table 2, hypericin is a weak contributor
to MAO inhibition in H. perforatum extracts. Indeed, the calculated content of hypericin at
IC50 value in assays of flower extract (i.e., 63.6 μg/mL) was 0.1 μg/mL which is low compared
with IC50 of hypericin (17.9 μg/mL). Hyperforin did not inhibit MAO-A (Figure 4(b)). Quercetin
inhibited human MAO-A (Figure 4(b)) with an IC50value of 11.1 ± 0.8 μM (i.e., 3.36 μg/mL).
Then, quercetin was a better inhibitor than hypericin although its potency was still low to
explain entire inhibition of extracts. Thus, the calculated content of quercetin at IC 50 in
assays of flower extract was 0.13 μg/mL which is lower than the IC50 of quercetin
(3.4 μg/mL). When the fraction corresponding to quercetin glycosides and flavonoids (7–
11 min, Figure 2(b)) was collected by RP-HPLC, it inhibited MAO-A (90% inhibition at
700 μg/mL extract) indicating a contribution of these compounds to MAO inhibition in H.
perforatum, probably by additive effects. Then, inhibition of MAO-A could arise from
components such as quercetin and related flavonoids (i.e., quercetin glycosides) which are
abundant in the plant. In addition, minor compounds not identified here could also contribute
to MAO inhibition as major compounds in Table 2 do not explain whole inhibition.
Figure 4
Inhibition of human monoamine oxidase-A (MAO-A) by active components of H. perforatum:
hypericin (a) and quercetin (■) and hyperforin (▲) (b).
Extracts from P. harmala seeds highly inhibited human MAO-A (Figure 5(a)) affording an
IC50 value of 49.9 ± 5.6 μg/L. Chromatographic analysis indicated that inhibition was due to
the presence of the β-carboline alkaloids, harmaline and harmine, that were identified by
HPLC-DAD-MS (Figure 5(c)). The content of these alkaloids determined in seeds was
48.5 mg/g for harmaline and 40.0 mg/g for harmine (this means 2.4 ng/mL and 2.0 ng/mL,
resp., into assays at the IC50). Therefore, the inhibition potency of MAO-A by P.
harmala seeds was 1274 times more potent than that of H. perforatumflowers. As shown
in Figure 5(b), Lepidium meyenii root extracts did not inhibit human MAO-A. L.
meyenii (maca) is a popular plant from the Andes highlands whose roots are increasingly
used for its nutritional and medicinal properties as energizing and to improve mood and
sexual performance [28, 32]. Previous reports have indicated that they contain alkaloids
including β-carbolines [25, 26] that might inhibit MAO. Analysis of extracts for β-carboline
alkaloids gave 25 μg/g (maca powder) and 11.7 μg/g (capsules) of 1-methyl-1,2,3,4-
tetrahydro-β-carboline-3-carboxylic acid as a major compound. This specific β-carboline is
not an inhibitor of MAO-A [8, 11].
Figure 5
Inhibition of human monoamine oxidase-A (MAO-A) by P. harmala seed (a) and L.
meyenii root (maca) extracts (b) (capsules, ▼ and powder, ■). (c) HPLC chromatogram ofP.
harmala seed extract which potently inhibited human MAO-A. Absorbance detection at
254 nm. Compounds identified are harmaline (m/z at 215 (M + H)+, UVmax at 375 nm) and
harmine (m/z at 213 (M + H)+, UVmax at 245 and 322 nm).
MAO generates hydrogen peroxide (H2O2) that is involved in oxidative cell damage and
pathological conditions [1, 3, 4, 33–36]. Then, the inhibition of MAO may result in specific
antioxidant actions [37]. In order to study the antioxidant activity associated with MAO
inhibition, experiments were designed in this research which linked the activity of MAO-A
with the oxidation of tetramethylbenzidine (TMB) by horseradish peroxidase (HRP) and the
H2O2 produced during oxidative deamination catalyzed by MAO (Figure 6). H.
perforatum and P. harmala extracts which inhibited MAO-A as shown above highly
decreased oxidation of TMB. In contrast, L. meyenii root (maca) extracts that did not inhibit
MAO had a low antioxidant activity in this assay. Clorgyline which is a potent inhibitor of
MAO-A highly decreased the oxidation of TMB when used as a control. The same happened
with the presence of catalase in the media that removes H2O2 generated by MAO-A.
Therefore, these results indicate that H. perforatum and P. harmala extracts afforded
specific antioxidant actions associated with a lower production of H2O2 by inhibition of MAO.

Open in a separate window

Figure 6
Antioxidant activity associated with MAO inhibition in assays coupling activity of MAO-A with
the subsequent oxidation of tetramethylbenzidine (TMB) in the presence H2O2generated in
the reaction of MAO, and horseradish peroxidase (HRP). The graph shows the oxidation of
TMB to diimine (absorbance at 450 nm) in control assays (100%), and in the presence of H.
perforatum (herbs and flower extracts, 800 μg/mL), P. harmala seed extracts (0.8 μg/mL), L.
meyenii root (maca) extracts (800 μg/mL), clorgyline (a standard inhibitor of MAO-A)
(25 μM), and catalase (100 μg/mL). ∗Significant differences (p < 0.01) compared to controls.
H. perforatum improves mood disorders and depression [17, 18, 38]. As shown here, it
contains compounds such as hyperforin, hypericins, and flavonoids responsible for
antidepressant effects (Figure 2 and Table 2). However, the specific mechanism for
antidepressant action is not completely understood. The most accepted mechanism is
inhibition of monoamine reuptake [23, 24, 39, 40]. However, some studies suggest a
combination of mechanisms and synergistic effects [17, 41]. P. harmala exerts numerous
biological and pharmacological actions. Their seeds are increasingly used for recreational
purposes owing to their psychoactive and neuroactive effects [14]. The inhibition of human
MAO-A is an established mechanism for antidepressant action [1]. Both irreversible and
reversible inhibitors of MAO-A (e.g., phenelzine and moclobemide) are successfully used as
antidepressants. In this study, H. perforatumextracts inhibited human MAO-A. However, this
inhibition was moderate. It was more than one thousand times lower than that of P.
harmala seed extracts. Sacher et al. have reported that the occupancy of MAO-A sites into
the human brain determined by PET imaging with 11C-harmine binding (i.e., the same β-
carboline responsible for MAO inhibition in P. harmala) was high for a reversible inhibitor of
MAO such as moclobemide but low for H. perforatum extract (St. John's wort) [42]. This
means that the inhibitors of MAO-A in H. perforatum do not bind efficiently to active sites of
MAO-A in the brain in contrast to the β-carboline harmine. The inhibitors of MAO-A inH.
perforatum are flavonoids such as quercetin and their glycosides and the levels of these
compounds that reach the brain might not be enough to occupy the sites of MAO-A in the
brain and inhibit the enzyme [43]. In contrast, the inhibitors of P. harmala are β-carboline
alkaloids including harmine and harmaline which have a very good brain penetration, bind
with high affinity to MAO sites, and exhibit antidepressant effects [44–46]. Therefore, P.
harmala could afford antidepressant effects by MAO inhibition. In this regard, it could be of
interest to investigate the antidepressant effects of H. perforatum and P. harmala alone and
in combination as they have different mechanisms of action.
The inhibition of MAO-A by H. perforatum and P. harmala extracts may contribute to other
biological effects of these plants such as antioxidant actions and adverse pharmacological
reactions. Extracts of these plants exert neuroprotective and anti-inflammatory effects which
have been related to antioxidant activity [6, 9, 47–50]. In this regard by using a new
procedure, results in this work have evidenced that H. perforatum and P. harmala extracts
show antioxidant activity associated with the inhibition of MAO (lower production of H2O2).
On the other hand, one of the major limitations to the use of these plants is their potential
for producing adverse interactions with other herbs, foods, and drugs [17, 20, 21, 27]. The
inhibition of MAO-A may trigger adverse effects under certain circumstances [1, 14].

4. Conclusions
Extracts from H. perforatum inhibited human MAO-A, and extracts from flowers were the
most potent inhibitors. They were studied by HPLC-DAD-MS and contained
pseudohypericin, hypericin, hyperforin, adhyperforin, hyperfirin, and flavonoids. The highest
content of these compounds appeared in flowers. Hypericin was a weak inhibitor of MAO-
A; hyperforin did not inhibit the enzyme and quercetin was a moderate inhibitor. The fraction
of quercetin glycosides and flavonoids contributed to MAO inhibition. P. harmala seed
extracts highly inhibited MAO-A and its potency of inhibition was more than a thousand times
higher than H. perforatum extracts owing to its content in harmaline and harmine
alkaloids. L. meyenii root (maca) extracts did not inhibit MAO-A. The inhibition of MAO-A
may not explain the entire CNS effects attributed to H. perforatum but it is expected to
contribute to these actions in P. harmala.These plants exert antioxidant effects. By using a
new method this work have evidenced that P. harmala and H. perforatum extracts exhibit
antioxidant activity associated with the inhibition of MAO.

Acknowledgments
The authors are grateful to MINECO-FEDER (SAF2015-66690-R and SAF2015-68580-C2-
R) and CSIC (Spain) (Project 200470E658) for supporting this work. The authors are grateful
also to Marta Aguilar Preiss for technical assistance and to Dr. V. Arán for helping with plant
identification and selection.

Conflicts of Interest
The authors declare no competing financial interest.

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J Food Sci Technol. 2017 Apr; 54(5): 1333–1339.

XV. Determination of soluble dietary fibre content of Okara

treated with high hydrostatic pressure and enzymes: a
comparative evaluation of two methods (AOAC and HPLC-
ELSD)
E. Pérez-López,1 I. Mateos-Aparicio, 2
and P. Rupérez1

Abstract

Introduction
Nowadays, by-products are promising sources of bioactive compounds and prebiotics
(Gullon et al. 2009; Mateos-Aparicio 2011; Mateos-Aparicio et al. 2013; Rastall and
Gibson 2015). Among them, Okara from soybean, a by-product of soymilk or tofu industries
(Mateos-Aparicio et al. 2010c; O’Toole 1999) is cheap, abundant and a valuable source of
dietary fibre (50% insoluble dietary fibre, IDF and 4.5% soluble dietary fibre, SDF) (Mateos-
Aparicio et al. 2010c), which makes it a potential prebiotic supplement (Jiménez-Escrig et
al. 2008; Préstamo et al. 2007; Redondo-Cuenca et al. 2008). However, as Okara is mostly
insoluble, different approaches are used to increase its SDF content, which is responsible
for the prebiotic and anti-carcinogenic effects (Charalampopoulos and Rastall 2012), These
include, chemical (Mateos-Aparicio et al. 2010a), enzymatic treatments with food grade
hydrolytic enzymes at atmospheric pressure, such as Ultraflo ® L or Viscozyme ® L (Kasai
et al. 2004; Rovaris et al. 2012; Rupérez et al. 2011; Villanueva et al. 2013), as well as high
hydrostatic pressure (HHP) treatment (Li et al. 2012; Mateos-Aparicio et al. 2010b) are
employed to release soluble carbohydrates from the complex cell wall found in Okara.
Moreover, a combination of HHP and enzymatic treatment has been used, since HHP can
enhance the activity of some enzymes on Okara’s cell wall, achieving up to 3.5-times higher
soluble content after Ultraflo ® L hydrolysis under HHP (Pérez-López et al. 2016a),
measured by a direct HPLC-ELSD method. However, SDF and IDF have not been measured
by the official AOAC method, and a comparison between both methods is needed.
Therefore, the aim of this work is to compare the direct HPLC-ELSD method with the official
AOAC method in samples of Okara treated with HHP and enzymes and to develop the
application of the optimized HPLC-ELSD method for SDF analysis after AOAC procedure.

Materials and methods

Materials, reagents, samples and equipment

A local food processing industry (Toofu-Ya S.L., Arganda del Rey, Madrid, Spain) provided
fresh Okara from soybean [Glycine max (L.) Merr]. Once at the laboratory it was freeze-dried
(Virtis Bench Top 3 L, Hucoa-Erlöss S.A., Madrid, Spain) and defatted by extraction with
ethylic ether in a Soxtec System (Tecator, Höganäs, Sweden). Freeze-dried Okara has a fat
content of 8% before defatting. Before any further treatment, Okara was re-hydrated in water
(1% w/v) (Pérez-López et al. 2016a). Enzymatic treatments were performed with two
commercial food-grade endo-β-1,3(4)-glucanases: Ultraflo ® L, with both xylanase and
cellulase activities, and Viscozyme ® L, with xylanase, cellulase and hemicellulase activities
(Novozymes Spain, S.A., Pozuelo de Alarcón, Madrid, Spain).
All other reagents and carbohydrate standards used were of chromatography grade. All
solutions, including, dilutions and mobile phases were prepared with ultrapure water
(Resistivity 18.2 MΩ cm at 25 °C; Milli-Q Integral 5 Water Purification System from Millipore,
Merck KGaA, Darmstadt, Germany).

HHP treatment of Okara assisted by enzyme

Re-hydrated Okara solution (1%, w/v) was treated with high hydrostatic pressure (HHP) at
400 and 600 MPa, aided by Ultraflo ® L or Viscozyme ® L (concentration 0.025% v/w), at
40 °C for 15 or 30 min (Pérez-López et al. 2016a). HHP treatment was performed in a
Stansted SFP 7100:9/2C equipment, using water as pressure transmitting medium. After
treatment, samples were freeze-dried (Virtis Bench Top 3 L, Hucoa-Erlöss S.A., Madrid,
Spain) for the analysis of dietary fibre by official AOAC method.

Dietary fibre analysis of Okara treated with HHP assisted by enzyme

Soluble and insoluble dietary fibre fractions were determined in HHP + Ultraflo ® L or
HHP + Viscozyme ® L treated samples according to the modified AOAC 991.43 (Association
of Official Analytical Chemists, 1995) enzymatic–gravimetric method with dialysis
(12 kDa MW cut off) (Mañas and Saura-Calixto 1993; Mateos-Aparicio et al. 2010b) to avoid
ethanolic precipitation (in AOAC 991.43 and 2011.25 methods) that may cause an
underestimation of SDF and overestimation of IDF. An aliquot of SDF fraction was taken for
direct HPLC-ELSD analysis.
Dietary fibre analysis by spectrophotometric methods
All the spectrophotometric methods used were previously miniaturized and adapted to
microplate by volume adjusting. According to AOAC method, uronic acids (UA) in SDF
fraction were spectrophotometrically quantified by the Scott method (Scott 1979) (200 µL
total volume and 50–200 mg L−1 galacturonic acid detection range, DR), and neutral sugars
(NS) were determined by the anthrone method (Loewus 1952) (200 µL total volume and,
25–150 mg L−1 glucose DR). Moreover, SDF and IDF fractions after AOAC method were
hydrolysed with H2SO4 (1 M) at 105 °C for 1.5 h and reducing sugars were
spectrophotometrically measured by dinitrosalicylic acid method (DNS) (Miller 1959)
(270 µL total volume and, 250–1500 mg L−1 glucose DR). Absorbance readings were
measured on a Biotek PowerWawe XS spectrophotometer. Thus, SDF was calculated either
as total neutral sugars plus uronic acids (NS + UA) or as reducing sugars (DNS method).
IDF was calculated as reducing sugars (DNS method) and total dietary fibre (TDF) was
calculated as SDF plus IDF.

Soluble carbohydrate analysis by HPLC-ELSD

The soluble fraction from the modified AOAC method for dietary fibre was filtered through
0.45 μm (cellulose acetate, 25 mm diameter, Análisis Vínicos, Tomelloso, Toledo, Spain)
and directly analysed by High Performance Liquid Chromatography with Evaporative Light
Scattering Detector (HPLC-ELSD) on a size-exclusion column, as previously reported for
carbohydrate analysis (Condezo-Hoyos et al. 2015; Pérez-López et al. 2016a). SDF was
quantified from area data and MW estimated from retention times, with log-calibration curves
(Condezo-Hoyos et al. 2015).

Statistical analysis
Results were expressed as mean values ± standard deviation. At least, three different
measurements were accomplished for each mean. Comparison of means was performed
by one-way analysis of variance with a significance level of P < 0.05 according to
Statgraphic version 5.1. (Statpoint Technologies, Inc. Warrenton, Virginia, USA).
Go to:

Results and discussion

Results from the modified AOAC procedure are shown in Tables 1 and and22 and
Fig. 1 (1.1, 1.2). This method can detect differences between samples, as can be done by
direct HPLC-ELSD analysis (Pérez-López et al. 2016a, b). Thus, for each treatment, an
increase in SDF was observed and 600 MPa, at 40 °C for 30 min treatment was the most
effective one with both enzymes, as in a previous work (Pérez-López et al. 2016a). Also, a
synergy between the HHP and the enzymatic treatment has been observed, especially
in Ultraflo ® L (Table 1), which was comparable with our previous experiments (Pérez-López
et al. 2016a, b). 2.9, 1.5 and 1.5-times higher SDF content using 0.025% Ultraflo® L and 1.2,
1.3 and 1.2- times higher using 0.025% Viscozyme ® L were achieved in these conditions
compared to the control, by NS + UA, DNS and HPLC-ELSD methods, respectively.
However, the direct HPLC-ELSD analysis was more sensitive than the AOAC method
followed by spectrophotometric methods. Values were correspondingly 3.6- and 3.4-times
lower than those reported for soluble polysaccharides measured by direct HPLC-ELSD
method in the supernatant after treatment with 0.025% Ultraflo ® L (Pérez-López et
al. 2016a) or Viscozyme ® L at 600 MPa and 40 °C for 30 min (unpublished results).
Furthermore, SDF values were reported to be lower than in previous works on native Okara
using an AOAC method (991.42) for dietary fibre without dialysis (Redondo-Cuenca et
al. 2008) and in Okara treated with HHP + autoclaving using the same AOAC method as in
this study but followed by gas liquid chromatography of monosaccharides (Mateos-Aparicio
et al. 2010b). Changes in the IDF profile of Okara caused by the treatment with HHP and
hydrolytic enzymes have not been assessed before. Results suggested that SDF increase
was due to a hydrolysis of IDF polysaccharides, as this was reported to be 1.4 and 1.6-times
lower in Ultraflo ® L and Viscozyme ® L treatment at 600 MPa for 30 min, respectively
(Tables 1and and2).2). This was attributable to release of CHO from Okara’s intricate cell
wall (Kasai et al. 2004; Mateos-Aparicio et al. 2010c; Villanueva et al. 2013). Considerable
changes in SDF/TDF ratio were detected (from 0.049 to 0.100) in combined treatments, as
in other previous reports (Mateos-Aparicio et al. 2010b; Pérez-López et al. 2016a).

Table 1
Analysis of dietary fibre in Okara treated with HHP and assisted by Ultraflo ® L

Treatment NS + DNS SDF by HPLC-ELSD

conditions UA
H m Ultra SDF SDF IDF TDF SD TD Peak 1 Peak 2 Tota
H i flo®L F/T F/I l
P n % DF DF CH
( O
M % % % % % MW % MW %
P d.w. d.w. d.w. d.w. d.w. (kDa d.w. (kD d.w.
a) ) a)
0. 0 0 1.20 1.57 30.27 31.84 0.0 1.0 1.31 94.3 1.42 22.6 2.73
1 ± 0.0 ± 0.1 ± 2.7 ± 2.7 49 52 ± 0.0 ± 4.3 ± 0. 4 ± ± 0.
8a 9c 8b 9b 9a 6 04a 1.81 09a
4 1 0 2.33 1.05 36.04 37.09 0.0 1.0 2.84 104. 0.79 20.5 3.63
0 5 ± <0. ± <0. ± <0. ± 0.0 28 29 ± 0.0 93 ± ± 0. 1 ± ± 0.
0 01c 01b 01cd 3cd 5b 2.04 07b 2.81 12bc
0.02 2.33 2.12 33.35 35.47 0.0 1.0 3.19 99.5 0.73 23.2 3.92
5% ± <0. ± <0. ± 0.0 ± 0.0 60 64 ± 0.0 9 ± 0 ± 0. 9 ± ± 0.
01c 01d 6c 6c 4cde .91 06b 0.43 09cd
3 0 2.21 0.84 37.10 37.94 0.0 1.0 2.85 105. 0.62 20.0 3.47
0 ± <0. ± <0. ± 0.0 ± 0.0 22 23 ± 0.1 08 ± ± 0. 5 ± ± 0.
01b 01a 2d 2cde 8b 0.84 04b 0.86 14b
0.02 2.22 2.04 33.36 35.40 0.0 1.0 3.14 95.1 0.78 22.5 3.92
5% ± <0. ± <0. ± 0.2 ± 0.2 58 61 ± 0.1 4 ± 1 ± 0. 3 ± ± 0.
01b 01d 2c 2c 0bcd .31 13b 0.74 03cd
6 1 0 2.17 1.57 38.53 40.10 0.0 1.0 3.14 98.4 0.81 22.2 3.95
0 5 ± <0. ± <0. ± 0.0 ± 0.0 39 41 ± 0.0 0 ± 1 ± 0. 1 ± ± 0.
0 01b 01c 3d 3e 4bcd .67 11b 2.19 15cd
 Treatment NS + DNS SDF by HPLC-ELSD
conditions UA
H m Ultra SDF SDF IDF TDF SD TD Peak 1 Peak 2 Tota
H i flo®L F/T F/I l
P n % DF DF CH
( O
M % % % % % MW % MW %
P d.w. d.w. d.w. d.w. d.w. (kDa d.w. (kD d.w.
a) ) a)
0.02 2.32 2.24 35.89 38.13 0.0 1.0 3.41 96.0 0.75 22.4 4.16
5% ± <0. ± <0. ± 0.0 ± 0.0 59 62 ± 0.0 3 ± 1 ± 0. 7 ± ± 0.
01c 01de 5 cd 5cde 2de .12 02b 0.16 04d
3 0 3.28 1.48 37.09 38.56 0.0 1.0 3.09 99.5 0.89 23.8 3.98
0 ± <0. ± <0. ± 0.0 ± 0.0 38 40 ± 0.1 9 ± 1 ± 0. 1 ± ± 0.
01d 01c 6d 6de 5bc .18 14b 1.87 29cd
0.02 3.48 2.34 22.16 24.50 0.0 1.1 3.47 100. 0.73 19.9 4.19
5% ± 0.0 ± <0. ± 0.0 ± 0.0 95 05 ± 0.0 64 ± ± 0. 0 ± ± 0.
1e 01e 7a 7a 8e 3.17 06b 0.17 02d
Open in a separate window
Different letters in each column differ significantly (P < 0.05)
NS + UA Neutral sugars + Uronic acid, DNS 3,5-dinitrosalicylic acid, SDF Soluble Dietary
Fibre, IDF Insoluble Dietary Fibre, TDF Total Dietary Fibre, d.w. dry weight, MW Molecular
Weight, CHO Carbohydrates

Table 2
Analysis of dietary fibre in Okara treated with HHP and assisted by Viscozyme®L

Treatment NS DNS SDF by HPLC-ELSD

conditions +U
A
H m Visco SD SDF IDF TDF SD TD Peak 1 Peak 2 Tot
H i zyme® F F/T F/I al
P n L % DF DF CH
( O
M % % % % % MW % MW %
P d.w. d.w. d.w. d.w. d.w. (kDa d.w. (kDa) d.w.
a) )
0. 0 0 1.20 1.57 30.2 31.8 0.0 1.0 1.31 94.3 1.42 22.64 2.73
1 ± 0. ± 0. 7±2 4± 49 52 ± 0.0 ± 4. ± 0.0 ± 1.8 ± 0.
08 19a .78cd 2.79 9a 36 4cde 1b 09
4 1 0 1.23 1.75 33.5 35.3 0.0 1.0 1.4 ± 84.1 1.34 13.82 2.74
0 5 ± 0. ± 0. 8±6 2± 50 52 0.01 2 ± ± 0.0 ± 1a ± 0.
0 09 1a .46d 6.46 ab
6.3 5bcd 06
 Treatment NS DNS SDF by HPLC-ELSD
conditions +U
A
H m Visco SD SDF IDF TDF SD TD Peak 1 Peak 2 Tot
H i zyme® F F/T F/I al
P n L % DF DF CH
( O
M % % % % % MW % MW %
P d.w. d.w. d.w. d.w. d.w. (kDa d.w. (kDa) d.w.
a) )
0.025 1.32 1.77 23.9 25.7 0.0 1.0 1.43 93.1 1.46 15.89 2.89
% ± 0. ± 0. 4±2 1± 69 74 ± 0.0 8 ± ± 0.0 ± 2.1 ± 0.
18 04a .24b 2.24 1ab 3.95 1de 8a 01
3 0 1.27 1.75 23.2 24.9 0.0 1.0 1.65 91.5 1.19 23.47 2.84
0 ± 0. ± 0. 3±1 8± 70 75 ± 0c 9± ± 0.2 ± 4.5 ± 0.
07 17a .82b 1.82 4.04 abc bc
2
0.025 1.36 1.73 25.5 27.2 0.0 1.0 1.67 89.6 1.16 29.51 2.83
% ± 0. ± 0. 2±4 5± 63 68 ± 0.0 4 ± ± 0.0 ± 6.9 ± 0.
17 07a .22b 4.22 7c 6.35 6ab 3cd 09
6 1 0 1.07 1.74 26.1 27.8 0.0 1.0 1.63 92.1 1.09 31.31 2.72
0 5 ± 0. ± 0. 4±2 7± 62 66 ± 0.2 9 ± ± 0.1 ± 4.3 ± 0.
0 05 04a .1bc 2.1 6bc 6.97 8a 8d 32
0.025 1.3 1.67 22.6 24.3 0.0 1.0 1.61 91.2 1.2 ± 25.36 2.81
% ± 0. ± 0. 5±1 2± 69 74 ± 0.0 6 ± 0.08 ± 2.5 ± 0.
13 06a .74ab 1.75 4bc 5.1 abc
1bcd 09
3 0 1.18 1.71 23.1 24.8 0.0 1.0 1.4 ± 90.6 1.19 19.15 2.59
0 ± 0. ± 0. 2±4 3± 69 74 0.01 3 ± ± 0.1 ± 1.6 ± 0.
11 1a .19b 4.19 ab
0.14 3abc 4ab 13
0.025 1.45 2.11 18.8 20.9 0.1 1.1 1.56 91.2 1.66 15.54 3.22
% ± 0. ± 0. 3±1 3± 00 11 ± 0.2 4 ± ± 0.1 ± 2.6 ± 0.
08 14b .31a 1.32 2bc 0.72 4e a
26
Open in a separate window
Different letters in each column differ significantly (P < 0.05)
NS + UA Neutral sugars + Uronic acid, DNS 3,5-dinitrosalicylic acid, SDF Soluble Dietary
Fibre, IDF Insoluble Dietary Fibre, TDF Total Dietary Fibre, d.w. dry weight, MW Molecular
Weight, CHO Carbohydrates
Open in a separate window
Fig. 1
HPLC-ELSD chromatograms of soluble fibre after modified AOAC method in Okara treated
with HHP, assisted by Ultraflo ® L (1.1) or Viscozyme ® L (1.2). Treatment conditions Okara
1%, 600 MPa for 30 min, a: without enzyme; b: with 0.025% (v/w) enzyme. Retention time
and MW of standards are represented on the chromatogram. ELSD response is expressed
in millivolts (mV)
AOAC official method is frequently used followed by spectrophotometric analysis (Mateos-
Aparicio et al. 2010a,b; Redondo-Cuenca et al. 2008). Here, the use of the HPLC-ELSD
method for analysing the SDF fraction has been suggested instead. HPLC-ELSD
chromatogram of the SDF fraction after AOAC method revealed the existence of one peak
of ≈95 kDa and a smaller one of ≈22 kDa, which presented a gradual decrease in its MW
upon treatment and it was more noticeable in Viscozyme ® L experiment (Fig. 1.2). These
low MW non-digestible carbohydrates could have beneficial prebiotic effects as they are
more easily fermentable by specific bacteria than those with high MW (Charalampopoulos
and Rastall 2012; Jiménez-Escrig et al. 2008; Mateos-Aparicio et al. 2010a, b; Préstamo et
al. 2007; Villanueva et al. 2011). Direct HPLC-ELSD analysis of supernatant in Okara after
treatment with HHP + Ultraflo ® L (without further AOAC method) showed three peaks of
approximately 24, 10 and 0.55 kDa (Pérez-López et al. 2016a). The smallest MW peak
corresponds to an oligosaccharide with a degree of polymerisation of three. Non-
physiological conditions for dietary fibre by AOAC method could release high MW
polysaccharides from Okara’s cell wall, and small MW molecules (<12 kDa) were lost during
dialysis of SDF (Villanueva et al. 2013). Thus, after modified AOAC method with dialysis
only two peaks were detected, with the loss of the smallest peak. Therefore, direct HPLC-
ELSD analysis as reported in Pérez-López et al. (2016a) was found to be cheaper, less time
consuming, more precise and sensitive for monitoring SDF after HHP + enzymes treatment
of samples and could directly assess the molecular weight of the carbohydrates released
without the inherent interferences of the AOAC methods for dietary fibre analysis.

Conclusion
The official AOAC enzymatic–gravimetric method for dietary fibre modified with dialysis,
followed by spectrophotometric analyses, was able to detect and quantify an increase in
SDF (≈ 1.5-times) and a concomitant decrease in IDF of Okara (≈1.6-times) in samples of
Okara treated with HHP and assisted by food-grade enzymes (Ultraflo ® L
or Viscozyme ® L). Analysis of the SDF fraction by HPLC-ELSD method was twice more
sensitive than the spectrophotometric approaches, and revealed the presence of two
carbohydrate peaks (≈95 kDa and ≈22 kDa MW) which could have improved beneficial
health effects. Compared to the AOAC method for dietary fibre, direct HPLC-ELSD analysis
of the supernatant after HHP + enzymatic treatment of samples was reported to be faster,
cheaper and more precise as small MW carbohydrates can also be detected.

Acknowledgements
Project AGL2016-77056-R from the Spanish MINECO supported this research. Elena
Pérez-López acknowledges the predoctoral training programme of the Education, Language
Policy and Culture of the Basque Government (Spain) (Grant No. PRE_2013_1_682) for her
work experience contract at the Department of Metabolism and Nutrition of ICTAN-CSIC in
Madrid. Thanks are given to Mr. Takazumi from Toofu-Ya S.L. for providing the Okara by-
product and to Mr. Martínez-Gutiérrez from Novozymes Spain, S.A. for providing the
enzymes.

Compliance with ethical standards

Conflict of interest
All authors declare there are no conflicts of interest.

Contributor Information
I. Mateos-Aparicio, Phone: 0034 913941807, Email: se.mcu@soetamni.
P. Rupérez, Phone: 0034 915 49 23 00, Email: se.cisc.natci@zerepurp.

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Oxid Med Cell Longev. 2017; 2017: 8361493.

XVI. A Chilean Berry Concentrate Protects against Postprandial

Oxidative Stress and Increases Plasma Antioxidant
Activity in Healthy Humans
Ines Urquiaga, 1 , * Felipe Ávila, 1 ,2
Guadalupe Echeverria, 1 Druso Perez, 1 Sebastian
Trejo, 1 andFederico Leighton 1

Abstract

1. Introduction
Polyphenols widely present in foods such as fruits, vegetables, and red wine have been
proposed as key molecules associated with the beneficial effects of a Mediterranean diet
[1–3]. Considerable evidence has demonstrated that foodstuffs or extracts with a high
polyphenol content can induce protective effects against oxidative stress and inflammation
in cells and animals [4–6]. Dietary intervention and postprandial studies in humans have
also showed protective effects against oxidative stress and inflammation [7–11]. Oxidative
stress has been closely associated with the pathogenesis of numerous chronic diseases as
well as metabolic syndrome [12]. Plasma concentrations of oxidative stress markers such
as malondialdehyde (MDA), 8-isoprostane, and isofurans are significantly increased in
diabetic subjects as well as patients with cardiovascular diseases [13–15]. Additionally,
plasma postprandial concentrations of MDA increase after high-fat meals. MDA can react
through a Michael addition with glutathione, proteins, and nucleic acids, inducing cytotoxicity
in cells and acting as a genotoxic agent [16, 17].
The postprandial oxidative response to eating depends on several factors including the
chemical nature of the macronutrient intake [18, 19], the unsaturation degree of dietary fatty
acids [20], the lipid intake dose [18], phytonutrient quantity [21], gender [22, 23], smoking
habits [24], and race [25], among others. This oxidative response generates an increase in
the plasma concentrations of MDA [26], lipid peroxides [27], protein carbonylation [20, 28],
and hydrogen peroxide [20]. Postprandial oxidative stress induced by intake of high-
saturated fatty acids can also alter the proteomic profile of peripheral blood mononuclear
cells in patients with metabolic syndrome, which has been related to an increase in THBS-
1 expression [29], a glycoprotein that promotes platelet aggregation [30]. This information
underscores the importance of developing functional foods aimed at effectively reducing
levels to the postprandial state [31].
Polyphenols present in red wine and roasted ground coffee effectively protect against
postprandial oxidative stress, decreasing plasma concentrations of MDA after red meat
cutlet intake [10, 32]. This effect has been explained by the ability of polyphenols to prevent
oxidative reactions occurring during digestion primarily at the stomach level, where an acidic
environment can enhance lipid peroxidation reactions [33].
Native Chilean berries possess high antioxidant activity and, among 120 species and
varieties studied, native berries such as maqui (Aristotelia chilensis), murta (Ugni molinae),
and calafate (Berberis microphylla) displayed the highest Oxygen Radical Absorbance
Capacity (ORAC) antioxidant activities [34]. In human endothelial cell cultures, the addition
of maqui, blackberry, or strawberry juice significantly protects cells from hydrogen peroxide-
induced intracellular oxidative stress, with maqui and blackberry inhibiting more effectively
than strawberry [35]. In addition to the in vitro antioxidant capacities, intracellular antioxidant
responses are activated by berry polyphenols, including Chilean wild raspberries (Rubus
geoides Sm.), which induced an increase in the intracellular glutathione content [4].
This work develops and characterizes a Chilean berry concentrate that consists of four
berries produced in Chile, two of which are native species, with a high antioxidant capacity.
In a randomized, crossover study, the effects of ingesting a dilute beverage prepared from
the berry concentrate with a turkey meat burger prepared with or without 6% of the same
concentrate were assessed on postprandial response, in terms of oxidative stress markers
and antioxidant capacity.

2. Methods

2.1. Berry Concentrate Composition

The berry concentrate liquid mixture (BPC-350) was designed by the Center for Molecular
Nutrition and Chronic Diseases, Pontificia Universidad Católica de Chile and provided by
the Bayas del Sur S.A. Company (Purranque, Chile). The BPC-350 had a pH of 3.91 and
65°Bx and was prepared from the concentrate of four different berries, among them
cranberry, blackberry, blueberry, raspberry, murta, and maqui, in a specific formulation to
obtain a high antioxidant capacity mixture with good taste. Murta (Ugni molinae) and
maqui (Aristotelia chilensis) are two native Chilean berries.
Good quality fruit was washed, sorted, and crushed with equipment specific to each
operation. The juice obtained after pressing the crushed fruit mass was filtrated. For
temporary preservation (inactivate enzymes and microorganisms) an operation with heat
exchangers at ~80°C during 60 s was conducted. Concentrated juice production was
performed by evaporation under a vacuum (0.5 bar residual pressure) at 40°C up to a
concentration of 65°Bx, which assured preservation without further pasteurization.

2.2. Antioxidant Capacity and Polyphenol Determination in BPC-350

Antioxidant capacity was measured by ORAC and Ferric Reducing Antioxidant Power
(FRAP) and determined according to Cao et al. [39] and Benzie and Strain [40], respectively.
The ORAC was expressed as μmol Trolox equivalents per g and FRAP was expressed
as μmol Fe+2 equivalents per g. Total polyphenol concentration was determined with Folin-
Ciocalteu method [41] and was expressed as mg of gallic acid equivalents per g. Total
monomeric anthocyanins were determined through the AOAC Official Method 2005.02 [42]
and were expressed as mg of cyanidin 3-glucoside equivalents per g. Identification of the
berry concentrate polyphenols was conducted by HPLC with UVA-visible detection at
280 nm, 360 nm, and 518 nm, comparing the retention times and UV-visible spectra of the
compounds with pure standards. The polyphenol content was quantified by interpolation in
a calibration curve built with the pure standards. This analysis was performed in three
different polyphenol fractions: neutral, acid, and aqueous, according to the method originally
reported by Salagoïty-Auguste and Bertrand [43] and modified for berry juice analysis by
Miranda-Rottmann et al. [35]. In brief, neutral fraction was prepared by adjusting BPC-350
to a pH of 7.0 by adding 1 M NaOH and extracted in triplicate by agitation for 30 min in the
dark with ethyl acetate. The organic phase was collected and evaporated to dryness with
nitrogen and dissolved in HPLC-grade methanol (Merck, Darmstadt, Germany). The acid
phase was collected from the previously obtained aqueous phase and the pH adjusted to
2.0 with 1 M HCl and extracted three times with ethyl acetate, as previously described. The
organic phase obtained constituted the acid fraction and the remaining aqueous phase
contained the anthocyanins. Neutral, acid, and aqueous fractions were analyzed by HPLC
detecting at 360, 280, and 518 nm, respectively.

2.3. Test Meals and Beverage

Ground turkey leg meat was provided by Sopraval S.A (La Calera, Chile). The ground meat
was mixed with vegetable oil (90% soya, 10% sunflower) at 6% w/w and salt at 0.25% w/w
and then cooked as a burger in an electric oven for 20 min at 250°C. Once cold, the burgers
were vacuum packed and stored at −20°C until use. Burgers with BPC-350 were prepared
using the same recipe and included 6% w/w BPC-350 concentrate in the mix of ground meat,
vegetable oil, and salt. The BPC-350 beverage was made with BPC-350 concentrate at 5%
w/v, purified water, and sucralose as sweetener and was then pasteurized.
MDA levels were quantified in representative samples of turkey burgers according to the
Csallany et al. protocol with modifications [44]. Briefly, 2 g of meat were homogenized in
10 mL of Milli-Q water using a tissue homogenizer for 30 s. Then 10 mL of trichloroacetic
acid (TCA, 10%) was added, the solution was homogenized and centrifuged at 10000 rpm
for 10 min at 4°C, and the supernatant was filtered. Of this solution, 700 μL was treated with
500 μL of 0.6% 2-thiobarbituric acid (TBA) solution and incubated at 90°C for 45 min, and
the complex MDA-TBA2 was detected by HPLC using the methodology described below.
2.4. Subjects
Eleven presumably healthy male subjects between 18 and 35 years old were recruited
through an advertisement placed at the Biological Sciences Faculty at the Pontificia
Universidad Católica de Chile, Santiago, Chile. The inclusion criteria were males between
18 and 35 years old that agree to participate in the study and have read and signed the
informed consent form. The exclusion criteria were (1) pharmacological treatment that would
affect lipid profile, arterial pressure, carbohydrate metabolism, or plasma antioxidant profile;
(2) diabetes mellitus, arterial hypertension, or dyslipidemias; or (3) chronic inflammatory
diseases. Two subjects did not complete the study and were excluded from the analysis.
Subjects were evaluated through a medical interview to determine family antecedents and
cardiovascular disease risk factors with special emphasis on evaluation of metabolic
syndrome components, including arterial pressure and anthropometric parameters, among
others. Additionally, each volunteer was evaluated through validated self-reported
questionnaires, including Chilean Mediterranean Diet Index (Chilean-MDI), instrument
developed and validated for the Center of Molecular Nutrition and Chronic Diseases of the
Pontificia Universidad Católica de Chile, and physical activity, through the IPAQ
(International Physical Activity Questionnaire) [45]. The subjects consisted of nine men with
normal blood lipid and glucose levels with a mean age of 20 years and body mass index of
24.9 kg/m2 (further information in Table 1).
Table 1
Anthropometric, clinical, and biochemical characterization of subjects participating in this
study (N = 11).

Parameter Median Range Reference values

Age (year) 20.2 18.7–27.3  
Waist circumference (cm) 87.4 74.0–100.5 <102∗
BMI (kg/cm2) 24.6 20.7–29.4 18.5–24.9∗∗
Systolic BP (mmHg) 117.5 112.5–130.0 ≤130∗
Diastolic BP (mmHg) 70.0 57.5–90.0 ≤85∗
Fasting glucose (mg/dL) 84 75–95 ≤100∗
Triglycerides (mg/dL) 71 37–193 ≤150∗
Total cholesterol (mg/dL) 150 113–177 <200∗
HDL (mg/dL) 49 32–59 >40∗
LDL (mg/dL) 93 51–105 <130∗
GGT (U/L) 18 8–27 9–31°
SGOT (U/L) 21 15–26 5–40°
Leukocytes 5.9 5.0–8.5 4–11°
∗Cutoff
points for metabolic syndrome components according to NCEP-ATPIII definition [36].
∗∗BMI range for normal weight subjects [37].
°Normal range for males aged 18 to 35 years [38].

2.5. Bioethical Statement

This study was approved by the bioethics committee of the Biological Sciences Faculty of
the Pontificia Universidad Católica de Chile and accomplished with the World Medical
Helsinki Declaration.

2.6. Study Design, Treatments, and Procedures

A randomized type I clinical trial with a crossover design was conducted. The basic design
involved administration of a test meal followed by blood sampling. Each subject consumed,
in a random order, three different test meals on three different occasions separated by ≥1
week.
Subjects reported to the laboratory in the morning following a 12 h overnight fast. They were
asked to exclude intake of creams and fried foods and to have no more than 1 cup of alcohol
and not to smoke the day before each intervention. Test meals consisted of (a) Meal 1: 250 g
of ground turkey leg meat burger and 500 mL of purified water (without gas); (b) Meal 2:
250 g of ground turkey leg meat burger and 500 mL of 5% BPC-350 beverage; (c) Meal 3:
250 g of ground turkey leg meat burger prepared with 6% BPC-350 and 500 mL of 5% BPC-
350 beverage. A cannula was inserted into the vein of the forearm and a baseline (time 0)
fasting blood sample was collected in corresponding tubes. The subjects then ate a test
meal within 20 min. Blood samples (15 mL) were drawn every hour for 6 hours after
consumption. Analyses including biochemical profile, lipid profile, and glycaemia were
performed at the Clinic Hospital of the Pontificia Universidad Católica de Chile. Analyses of
oxidative stress markers (MDA and protein carbonyls), antioxidant capacity (FRAP, and
DPPH), and Vitamin C were performed at the Molecular Nutrition and Chronic Diseases
Center of the Pontificia Universidad Católica de Chile. Vitamin C was determined by
spectrophotometry according to Day et al. [46]. During the analysis the researchers were
blinded to exclude possible bias.

2.7. Malondialdehyde Determination

MDA was quantified according to the Templar et al. [47] protocol with some modifications.
Briefly, human plasma was deproteinized using 5% TCA and supernatant was treated with
fresh 0.6% TBA and incubated for 45 min at 90°C. The mixture was cooled at room
temperature (25°C) and 120 μL was injected in the HPLC. The MDA concentration was
determined by interpolating the area of the peak corresponding to the adduct MDA-TBA2 of
the sample into a MDA calibration curve prepared through acid hydrolysis of 1,1,3,3-
tetraethoxypropane. The HPLC measurements were performed using a reverse-phase
HPLC Merck-Hitachi 7000 series (Merck-Hitachi, Darmstadt, Germany) equipped with an
autosampler device. The separation was reached using an Inertsil ODS-3 column (GL
Sciences, Tokyo, Japan) and a mobile phase composed of a 50 mM sodium phosphate
buffer, pH 7 (65%) and methanol (35%). The detection was conducted using a UV-visible
photodiode array detector and a fluorescence detector (λEXC = 515 nm; λEM = 550 nm). The
fluorescence chromatograms were only used for MDA analysis.

2.8. Protein Carbonyl Content

Determination of carbonyl groups in oxidized plasma proteins was assessed by
derivatization with 2,4-dinitrophenylhydrazine, according to the method of Levine et al. [48].
Spectrophotometric measurement of plasma reactive carbonyl derivatives was performed
and calculated using the extinction coefficient of 2,4-dinitrophenylhydrazine-reactive
carbonyl derivatives at 370 nm = 22 × 103 L mol−1 cm−1 and expressed as μmol/mg of
proteins [48].
2.9. Antioxidant Capacity Determination
Antioxidant activity in human plasma was measured using the DPPH method adapted from
Chrzczanowicz et al. [49] with some modifications. Briefly, plasma samples (100 μL) were
deproteinized by adding 300 μL of acetonitrile and centrifuging for 10 min (4°C, 9500 ×g).
Supernatant was immediately collected and 50 μL was transferred to a microplate, then
100 μL of 0.15 mM DPPH was added, and the absorbance was read at 517 nm during a
30 min incubation period at room temperature (25°C).
A negative control was conducted with acetonitrile (at the same concentration) and the
experiments were performed in triplicate. A calibration curve was built with Trolox where the
absorbance values were interpolated and the results were expressed as Trolox equivalents.
To distinguish and compare the differences between each meal, DPPH-DPPH0 was
calculated, where DPPH means the Trolox equivalents at any time and DPPH0 means the
Trolox equivalents at time zero. The FRAP was determined in plasma obtained from heparin
tubes, according to Benzie and Strain [40].

2.10. Statistical Analysis

Subject descriptive characteristics are presented as median and range. Oxidative stress
(MDA and protein carbonyls) and antioxidant status biomarkers (DPPH, FRAP, and Vitamin
C), were analyzed using a 3 (meal) × 7 (time) repeated measures analysis of variance
(ANOVA). Significant interactions and main effects were further analyzed using Tukey's post
hoc tests. For each biomarker, the area under the curve (AUC) was calculated using the
trapezoidal method. One-Way ANOVA and paired Student's t-tests were used to analyze
statistical differences between AUCs. Student's t-test for independent samples was used to
analyze statistical differences between MDA in uncooked and cooked meats. Data
processing and statistical analyses were performed using the SPSS statistical software
package, version 17.0 (SPSS Inc., Chicago, IL) and R (Team RC. R: A Language and
Environment for Statistical Computing. 2014.). p ≤ 0.05 was considered statistically
significant.

3. Results

3.1. BPC-350 Characterization

The BPC-350 antioxidant capacity determined by ORAC and FRAP assays was 453.74 ±
19.95 μmol Trolox equivalents per g and 313.4 ± 16.9 μmol equivalents Fe+2 per g,
respectively (Table 2). Total phenolics determined by Folin-Ciocalteu were 25.56 ± 1.08 mg
gallic acid equivalents per g (GAE/g) and total anthocyanins determined by the pH
differential method were 5.62 ± 0.19 mg cyanidine 3-glucoside equivalents per g (Table 2).
The total quantification of the main compounds present in BPC-350 in terms of the three
polyphenol families analyzed was anthocyanins (5,170 μg/g) > phenolic acids (411.7 μg/g)
> flavonoids (263.4 μg/g).
Table 2
Concentration of total polyphenols, total anthocyanins, and antioxidant capacity of BPC-350
and 5% BPC-350 beverage.
 Assay BPC-350 5% BPC-350 beverage
(mean ± SD) (mean ± SD)
ORAC (µmol Trolox eq/g) 453.74 ± 19.95 14.23 ± 0.19
Total polyphenols (mg GAE/g) 25.56 ± 1.08 1.39 ± 0.05
Total anthocyanins (mg CE/g) 5.62 ± 0.19 0.18 ± 0.03
GAE: gallic acid equivalents; CE: cyanidine 3-glucoside equivalents.
The pasteurized BPC-350 beverage made with BPC-350 concentrate at 5% w/v, purified
water, and sucralose as sweetener had an ORAC of 14.23 ± 0.19 μmol Trolox equivalents
per g, total phenolics of 1.39 ± 0.05 mg GAE/g, and total anthocyanins of 0.18 ± 0.03 mg
cyanidine 3-glucoside equivalents per g (Table 2).
The HPLC analysis of polyphenols from neutral, acid, and aqueous fractions of BPC-350
are displayed in Figure 1. Through HPLC analysis performed by comparing the retention
times and UV-visible spectra with pure standards, it was possible to identify 7 flavonoids, 4
phenolic acids, and 10 anthocyanins present in neutral, acid, and aqueous fractions,
respectively (Figure 1). The principal components of the anthocyanin family were identified
as Cyanidine 3-glucoside and Cyanidin3-O-(6-O-E-p-coumaroyl-2-O-β-D-xylopyranosyl)-β-
D-glucopyranoside-5-O-β-D-glucopyranoside. Alternatively, isoquercetin and ellagic acid
were the principal components of the neutral and acid fractions, respectively (Figure 1).
Open in a separate window
Figure 1
Identification and quantification of polyphenolic compounds present in BPC350 by HPLC-
UV-Vis chromatography. Panel (a) shows the chromatographic profile, detecting at 360 nm
(neutral fraction). Panel (b) shows chromatographic profile detecting at 360 nm (acid
fraction). Panel (c) shows chromatographic profile detecting at 518 nm (aqueous fraction).
Tables show identification and quantification of chromatographic peaks.

3.2. Test Meal Analysis

To determine whether BPC-350 could affect MDA generation during thermal meat
processing, the MDA concentration was quantified in turkey burger samples. Control turkey
burgers processed thermally (cooked) presented MDA concentration 8.7 times higher than
uncooked burgers (Figure 2). However, turkey burgers prepared with 6% BPC-350
concentrate did not increase MDA concentration after cooking (Figure 2).

Figure 2
Malondialdehyde content in experimental foods used to perform the crossover study.
Malondialdehyde concentrations were quantified in uncooked and cooked turkey burgers
prepared with or without BPC-350 at 6% (N = 3). Data were expressed as the mean ±
SD. ∗ indicates significant differences when compared to uncooked turkey burger, uncooked
turkey burger + BPC-350, and cooked turkey burger + BPC-350 (p < 0.05).

3.3. Postprandial Study

To determine the effect of the Chilean berry concentrate BPC-350, a type I clinical trial with
a crossover design was performed with 11 healthy male subjects. On different days, after
fasting overnight, the volunteers consumed the following meals: 250 g of oven-cooked
turkey burger (11.25 μmol of MDA) and 500 mL of water (Meal 1); similar burger (11.25 μmol
of MDA) and 500 mL of 5% BPC-350 beverage (639 mg GAE of polyphenols) (Meal 2); or
250 g of oven-cooked turkey burger with 6% BPC-350 concentrate (383 mg GAE of
polyphenols, 0.5 μmol MDA) and 500 mL of 5% BPC-350 beverage (639 mg GAE of
polyphenols) (Meal 3). Postprandial changes in glucose, triacylglycerol, MDA, protein
carbonyls, DPPH, FRAP, and Vitamin C were measured over the 360 min (6 h) experimental
period at 60 min intervals after meal consumption. No differences were found in glucose and
triacylglycerol patterns, nor in the values of the areas under the curve between the meals
(data not shown). The time profile of MDA per mg of triacylglycerol can be observed in Figure
3(a). The intake of Meals 1 and 2 significantly increased the mean values (N = 11) of MDA
per mg of triacylglycerol during study time, reaching values ~5 and ~3 times higher than
basal levels for Meals 1 and 2, respectively, which remained high after 6 hours (Figure 3(a)).
However, comparing postprandial changes in MDA per mg of triacylglycerol at 5 h and 6 h,
Meal 2 showed significantly lower concentrations of MDA per mg of triacylglycerol than Meal
1 (p < 0.05). Meal 3 did not significantly increase (p > 0.05) the MDA content of plasma in
comparison to the baseline (time 0). Significant differences over the entire time range
analyzed (6 hours after intake) were found when compared with Meals 1 or 2; this treatment
completely prevented MDA accumulation in plasma.

Figure 3
Plasma postprandial malondialdehyde and protein carbonyls in human healthy subjects after
the intake of three different meals: ■ Meal 1 (turkey burger + water); ● Meal 2 (turkey burger
+ 5% BPC 350 beverage); ▲ Meal 3 (turkey burger prepared with 6% BPC-350 + 5% BPC-
350 beverage). Panels (a) and (c) show the time profile of malondialdehyde corrected by
triglycerides and protein carbonyls concentrations after the intake of the three different
meals, respectively. Panels (b) and (d) show the area under the curve of the different time
profiles corresponding to three different meals for malondialdehyde and protein carbonyls,
respectively. Data were expressed as the mean ± SD. In Panels (a) and (c), ∗ shows
significant differences for each time compared to Meal 1 (p < 0.05). In Panels (b) and
(d), ∗ shows significant differences between meals (p < 0.05).
The mean value of the area under the curve of the time profile of volunteer MDA/TG for the
three meals is displayed in Figure 3(b). Compared to Meal 1, the mean value of the area
under the curve of MDA/TG for Meals 2 and 3 presented a statistically significant reduction
(p < 0.05). Meal 3 also presented a statistically significant decreased area under the curve
compared with Meal 2 (p < 0.05).
The time profile of protein carbonyls after intake of the three experimental meals is shown
in Figure 3(c). No significant changes in protein carbonyls were observed after intake of
Meal 1 in the time ranges analyzed. However, decreases in the protein carbonyl
concentrations were observed for Meals 2 and 3. When compared with Meal 1, statistically
significant differences were found at 4 h and 6 h with Meal 2 and at 2 h to 6 h with Meal 3.
The area under the curve of the time profiles for proteins carbonyls (Figure 3(d)) displayed
significant decrease after Meals 2 and 3 compared to Meal 1.
To determine the effect of BPC-350 on antioxidant activity, human plasma was evaluated
with the FRAP and DPPH radical scavenging capacity. Only the DPPH antioxidant assay
presented statistically significant differences between meals. The changes in the scavenging
capacity of DPPH radical activity during the study are exhibited in Figure 4(a). Meal 1
consumption decreased the antioxidant activity of volunteers' plasma along the time range
(p < 0.05) (Figure 4(a)). After intake of Meal 2, plasma antioxidant activity remained constant
until 5 h. Alternatively, Meal 3 consumption increased plasma antioxidant activity 1 hour after
intake, which remained high and only decreased after 6 hours (Figure 4(a)). When compared
with Meal 1, statistically significant differences were found at 3 h with Meal 2 and at 2 h, 5 h
and 6 h with Meal 3.

Figure 4
Changes in the plasma antioxidant activity determined by means of DPPH method before
and after the intake of three different meals. ■ Meal 1 (turkey burger + water); ● Meal 2
(turkey burger + 5% BPC 350 beverage); ▲ Meal 3 (turkey burger prepared with 6% BPC-
350 + 5% BPC-350 beverage). Panel (a) shows the time profile of changes in DPPH
antioxidant activity. Panel (b) shows the area under the curve of the time profiles,
corresponding to the three different meals. Data were expressed as the mean ± SD. In Panel
(a), ∗ shows significant differences for each time compared to Meal 1 (p < 0.05). In Panel
(b), ∗ shows significant differences between meals (p < 0.05).
The area under the curve for changes in volunteer DPPH scavenging activity after meals is
presented in Figure 4(b). The area under the curve of DPPH time profiles for Meals 2 and 3
displayed a significant increase compared with Meal 1 (p < 0.05).
The time profile of Vitamin C after intake of the three experimental meals is shown in Figure
5. Vitamin C did not present statistically significant differences between meals.

Figure 5
Plasma postprandial Vitamin C in human healthy subjects after the intake of three different
meals: ■ Meal 1 (turkey burger + water); ● Meal 2 (turkey burger + 5% BPC 350 beverage);
▲ Meal 3 (turkey burger prepared with 6% BPC-350 + 5% BPC-350 beverage). Data were
expressed as the mean ± SD.

4. Discussion
Numerous epidemiologic and experimental studies have provided significant evidence
regarding the beneficial effects of regular consumption of fruits and vegetables, which is
associated with a high polyphenol content [50–52]. Red wine is one of the most studied and
characterized beverages, generally known for having a high polyphenol content, which is
concomitant with a high antioxidant capacity [3, 9, 53]. Gorelik et al. demonstrated that one
of the functional properties of red wine is the ability to reduce postprandial oxidative
responses (in terms of MDA quantities) in humans, produced by the intake of red meat [9].
This fact has been explained by the inhibition of oxidative reactions that occur at the stomach
level that could be catalyzed by Fe+2 through a Fenton reaction, among others [32, 33].
However, medical recommendation of wine consumption possesses the limitations inherent
to alcoholic beverages and could not be implemented as a public health strategy to reduce
chronic diseases. In this sense, this work aimed to elaborate a nonalcoholic drink with a high
antioxidant capacity that could be similar to the one found in red wine and to determine
whether it could exert protective effects in the postprandial response in terms of oxidative
stress, and antioxidant capacity in healthy humans. Considering the high antioxidant
capacity [34] and evidence supporting the health effects of berry consumption [35, 54], the
Chilean berries were selected for this study. The effects of 5% w/v BPC-350 beverage were
assessed through a crossover study performed with 11 healthy male volunteers, determining
the responses in terms of oxidative stress markers (MDA and protein carbonyls) and
antioxidant status biomarkers (DPPH, FRAP, and Vitamin C) induced by the intake of three
different meals.
The analysis of BPC-350 antioxidant capacity was evaluated using two different antioxidant
assays: ORAC and FRAP (Table 2). These results indicate a high antioxidant capacity in
comparison to other berries [34, 55]. Anthocyanins are the main phenolic compounds
reported in native Chilean berries such as maqui (Aristotelia chilensis) or calafate (Berberis
microphylla) [35, 55, 56]. The analysis of BPC-350 phenolic content by HPLC (Figure 1)
indicated that anthocyanins are the primary polyphenols present in BPC-350 (88.5%), and
flavonoids and phenolic acids account for 4.5% and 7%, respectively. The primary
anthocyanins present in BPC-350 were cyanidin3-O-(6-O-E-p-coumaroyl-2-O-β-D-
xylopyranosyl)-β-D-glucopyranoside-5-O-β-D-glucopyranoside and cyanidin 3-glucoside
(Figure 1), which are present in high amounts in berries such as Sambucus nigra [57]
and Ribes (magellanicum and cucullatum) [56], respectively. In red wine, anthocyanins
represent ~70%, and are primarily malvidin glucosides but also contain a quantity of cyanidin
3-glucoside [58]. The reactivity of the primary anthocyanins against numerous reactive
oxygen species is similar in the 5% w/v BPC-350 beverage and red wine [59], despite this
beverage presenting lower total polyphenols content and antioxidant activity than an
average red wine [60]. This formulation was created to obtain a high quality polyphenols
beverage with good taste.
Volunteers participating in this study were considered normal according to international
recommendations and no subjects with diabetes or metabolic syndrome were detected in
this population (Table 1) [38]. This allows the comparability of data obtained from the
postprandial responses analysis since alterations in glycemia, triacylglycerides, oxidative
stress markers, and serum antioxidant enzyme activity have been reported in cases with
diabetes and metabolic syndrome [61–64].
When volunteers ate the turkey burger with water, plasma MDA/TG levels rose, but when
volunteers drank 5% w/v BPC-350 beverage instead of water they exhibited a 35% reduction
in the area under the curve of plasma MDA/TG concentration during the 6 hour after meal
period. This beverage diminished MDA accumulation in plasma. Interestingly, intake of the
turkey burger with water did not change carbonyls in plasma proteins. However, the 5% w/v
BPC-350 beverage produced a significant decrease in plasma protein carbonyl
concentration. These results indicate that the 5% w/v BPC-350 beverage, under the scheme
of this study, prevented lipid peroxidation and the consequent formation of MDA and MDA-
protein adduct that occurs during digestion at the stomach level [65].
These results agree with the work of Gorelik et al., which analyzed the effect of red wine on
postprandial oxidative stress modulation [9]. They also found decreased plasma MDA levels
after intake of 250 g of red meat turkey cutlets soaked in red wine concentrate after heating
plus 200 mL of wine, compared to eating 250 g of red meat cutlets plus water [9]. However,
the magnitude of the red wine effect was double that of the 5% w/v BPC-350 beverage even
with a similar quantity of polyphenols. The difference could be explained by the diversity in
polyphenol quality, especially on the capacity to quench lipid oxidation. Alternatively, turkey
burgers prepared with vegetable oil (60% polyunsaturated fatty acids) at 6% w/w are
susceptible to oxidation, which could result in a polyphenol quantity insufficient for stopping
the oxidation reactions. Additionally, due to the suspension of polyphenols in 500 mL of 5%
w/v BPC-350 beverage, it is possible that the beverage passed from the stomach to the
intestine too quickly and lacked sufficient contact with the meat to act as an antioxidant.
Reports indicate that MDA derived from meat consumption modified low-density lipoprotein
(LDL) in vivo, and this modification was directly dependent on the increased plasma MDA
level following a meal [33]. Aldehydes such as MDA may react with lysine residues in the
LDL apo B-100 moiety, resulting in a decreased apo B-100 affinity for the LDL receptor [66].
Also, albumin and plasma proteins react with MDA or other electrophiles produced during
lipid oxidation [67]. This reaction has been studied in vitro, demonstrating than the rate of
generation of protein carbonyl is low at physiological pH [68]. This could explain in part the
absence in protein carbonyl increase after the intake of Meal 1, even when this meal
increased significantly the plasma levels of MDA.
Rising evidence supports that compounds like MDA can have specific signaling roles
inducing adaptive responses driven to decrease oxidative damage and improve antioxidant
defenses [69]. In fact, it has been reported an increase in the plasma levels of thiols after
the intake of high-fat meals [70]. This endogenous antioxidant response to postprandial
oxidative stress, proposed as part of a protein oxidation defense [70], involves the activation
of the transcription factor Nrf2, being the master regulator factor [69]. Also, in vitro studies
have demonstrated that carbonylated proteins can be reduced by a nonenzymatic reaction
with sulfhydryl groups present in cysteine and glutathione [71]. So an increase in plasma
thiols after intake of Meal 1 could prevent protein oxidation according to a physiological
mechanism of protection. However, we did not measure plasma thiols so this is a hypothesis
that should be demonstrated.
Similar reasons as Meal 1 could explain in part the decrease in protein carbonyl after eating
the turkey burger with the 5% w/v BPC-350 beverage (Meal 2). But in this case plasma MDA
level was significantly lower than after intake Meal 1. Lower plasma MDA quantity means
less reaction with protein and, therefore, less carbonyl in plasma protein considering the
relatively low reaction rate between proteins and MDA. The proteins synthetized de novo
could contain less carbonyl than older proteins in the basal state.
On the other hand, polyphenolic enriched extracts of the Chilean native berry Rubus
Geoides have shown to increase glutathione levels in AGS cells [4]. Also, in Wistar rats an
increase in plasma glutathione after intake of a mixture of grape seed proanthocyanidin and
docosahexaenoic acid has been reported [72]. In this sense, the intake of BPC-350 rich in
polyphenols could increase plasma glutathione and promote decarbonylation of protein.
Evidence suggests that polyphenols may induce cellular defense genes by a mechanism
that includes Nrf2 activation [4, 73, 74]. As mentioned before, plasma thiols and glutathione
were not measured in this study, so additional experiments are necessary to confirm our
hypothesis.
MDA, a red meat-derived aldehyde, can interact and modify LDL in plasma, possibly
enhancing atherosclerotic plaque production [33]. Ahotupa et al. found that food lipid
peroxides are incorporated into serum triglyceride-rich lipoproteins and LDL, directing the
lipid peroxide flow towards peripheral tissues [75]. They propose that the specific
atherosclerosis-related effects of serum lipoproteins are not explained only by cholesterol
transport but also from the transport of atherogenic lipid peroxides [75].
Meat consumption with plant derived polyphenols (e.g., red wine or coffee polyphenols) can
prevent the appearance of MDA in plasma and LDL modification [9, 10]. Therefore, we
suggest that the harmful consequences of red meat product consumption might be partially
diminished by simultaneous polyphenol addition to meals with red meat. Polyphenol
treatment of red meat during preparation (e.g., cooking and processing) may also
significantly contribute to the prevention of hazardous and deleterious effects of red meat
products.
Other studies have demonstrated the effect of polyphenols, primarily wine, on postprandial
oxidative stress [76]. Natella et al., who used a test meal consisting of “Milanese” meat and
fried potatoes, observed that intake of the meal with 400 mL of red wine provoked a
significant increase in total plasma antioxidant capacity and a reduction in the postprandial
increase of LDL susceptibility to oxidation [77]. In a similar study, Di Renzo et al. used a
McDonald's Meal (N.1 Big Tasty Bacon Sandwich and N.1 small French Fries) with 250 mL
of red wine, which resulted in lower (p < 0.05) values of postprandial ox-LDL than meal
consumption without red wine [78].
When MDA plasma concentrations were analyzed after intake of Meal 3, no significant
differences were observed when comparing MDA basal levels (time 0) with those produced
6 hours after intake, indicating the inhibitory effect of plasma MDA produced by this meal.
The mean values of the area under the curve indicate a complete reduction of MDA
absorption compared with the response after intake of Meals 1 and 2. In agreement with this
information, we determined that the MDA quantity of Meal 3 (turkey burger prepared with
BPC-350 concentrate and cooked) was 22.5 times less than the MDA quantity of Meal 1
and 2 (turkey burger prepared without BPC-350 and cooked).
These data suggest that the strong effect observed for Meal 3 was due to the inhibition
capacity of lipid peroxidation reactions by BPC-350 concentrate when the turkey burger was
thermally processed. There was a dual protective effect produced by the inhibition of lipid
peroxidation reactions, occurring at the stomach level and during thermal food processing.
This fact is also consistent with in vitro studies that have demonstrated anthocyanins ability
to inhibit lipoperoxidation [79, 80].
The effect of BPC-350 intake on the antioxidant capacity was determined in plasma by FRAP
and DPPH measurements. Significant differences for the plasma antioxidant capacity were
found when the different meals were compared using DPPH values. Considering that human
plasma was deproteinized before DPPH antioxidant activity determination, to ensure
reproducibility [49], any effect associated with scavenging of DPPH radical due to changes
in protein expression can be discarded. This implies that the increase in the antioxidant
capacity determined by DPPH after intake of Meals 2 and 3 could be due to low molecular
weight molecules, including polyphenols, Vitamin C, and urate. We did not observe
significant differences between meals comparing Vitamin C plasma concentrations curves
either point to point or calculating the area under the curve. Therefore, considering that BPC-
350 do not contain Vitamin C, the increase in the antioxidant activity induced by BPC-350
intake (especially in Meal 3) could be attributed to other compounds present in this
concentrate or synthesized in the organism after consumption. Human plasma analysis after
intake of blueberries indicated the presence of 19 out of 25 anthocyanins originally present
in the fruit [81], which suggests the possibility of anthocyanins contribution to the antioxidant
activity found in this study. However, the contribution of phenolic acids cannot be discarded,
including those produced from anthocyanin metabolism occurring in the liver and microbiota.
In this sense, a study of urate levels after intake of BPC-350 could clarify the mechanisms
associated with the increased antioxidant activity observed in this study.
The intake of Meal 1 diminished the antioxidant capacity of plasma measured as DPPH,
according to the increase in MDA plasma concentrations. This could be explained by the
utilization of endogenous antioxidants (able to react with DPPH radical) such as urate and
glutathione [82, 83], due to hydroperoxide absorption, which promotes lipid oxidation. The
postprandial state induces immediate oxidative stress that triggers atherogenic changes
including inflammation, endothelial dysfunction, hypercoagulability, and sympathetic
hyperactivity [84].
Postprandial oxidative stress, which occurs after eating meat fat, is associated with a higher
risk for atherosclerosis, diabetes, and obesity. In Western societies, a significant portion of
the day is spent in a postprandial state. Lipid hydroperoxides present in the diet are
absorbed, producing endothelium-dependent vasodilation. Postprandial oxidative stress is
attenuated when dietary antioxidants are supplied with a meal rich in oxidized or oxidizable
lipids. Ingestion of dietary polyphenols, for example, from wine, cocoa, or tea, improves
endothelial dysfunction and lowers LDL lipid susceptibility to oxidation. Polyphenols affect
endothelial function not only as antioxidants but also as modulatory signaling molecules.
The consumption of high-fat and high-iron potentially prooxidant foods such as red meat
produced postprandial oxidative stress, as detected by the increment in plasma MDA and
the reduction in plasma antioxidant capacity. The intake of food- or beverage-derived
polyphenols with the meal prevented plasma oxidative stress, as evidenced by this work and
those of other researchers [9, 76, 85].
The Mediterranean diet is currently considered a healthy dietary pattern. It includes a great
variety of foods, which are eaten in moderation and within a positive social environment.
The way of cooking food in Mediterranean cuisine has been associated with lower
cardiovascular risk. The basis of Mediterranean dishes is the sauté of onion, garlic, and
tomato in olive oil; this source of antioxidants is used to flavor vegetables such as zucchini,
eggplant, potatoes, and haricot verts; cereals such as rice or pasta; legumes such as beans
or chickpeas; and even meat, poultry, or fish. A wide variety of spices and condiments like
lemon, vinegar, parsley, mint, oregano, herbs, cinnamon, and many others, is used for
seasoning salads and different preparations. Polyphenols widely present in characteristic
Mediterranean foods such as fruits, vegetables, and wine red and the way to use them in
the Mediterranean cuisine could explain the beneficial effects of Mediterranean diet.
The results obtained in this study indicate the usefulness of a berry-based drink to decrease
postprandial oxidative stress. Our results emphasize the effectiveness of a berry
concentrate for inhibiting lipoperoxidation reactions occurring at the stomach level but
primarily during thermal treating of foods. The way in which food is prepared is critical to our
health.

Acknowledgments
This work was supported by Fundación Copec-UC (Grants #SC007 and #6C029) and by
Fundación Alimenta.

Competing Interests
The authors declare that there are no competing interests regarding the publication of this
paper.

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Adv Nutr. 2017 Jan; 8(1): 165S–172S.

 XVII. Setting the Lipid Component of the Diet: A Work in
Process1,2,3
Fabiola M Del Razo Olvera, Marco A Melgarejo Hernández, Roopa Mehta, and Carlos A
Aguilar Salinas*

Abstract

Introduction
Dietary fat, which contributes to food palatability and preservation, is an essential
component of a diet, and the presence of differing types and quantities of fat is strongly
associated with different culture and culinary traditions (1). Dietary fats are commonly
classified according to their acyl chains and the quantity of links, and are divided into either
SFAs or unsaturated FAs. Unsaturated FAs are divided further into MUFAs, PUFAs
(including ω-3, ω-6, and certain ω-9 FAs), and trans FAs, which are initially unsaturated, but
become saturated when they are hydrogenated to give them a solid form (2). Foods contain
a mixture of various classes of FAs (saturated and unsaturated fats), limiting the ability of
experts to classify any particular FA as a healthy or unhealthy dietary choice with respect to
coronary disease risk. Thus, the effects of the consumption of a food product on human
health should be measured instead of being predicted from its nutrient composition (3).
Consequently, a recommendation based on the proportions of nutrients in a food item could
be inaccurate (4).
For many decades, the etiology of atherosclerotic cardiovascular disease (CVD) 4 was
associated with the consumption of dietary fats, in particular saturated fat, which was found
to increase LDL cholesterol (5). Saturated fat represents a highly heterogeneous category
of FAs, with chain lengths ranging from 6 to 24 carbons. These include stearic (18:0),
palmitic (16:0), myristic (14:0), and lauric (12:0) acids. The main foods that contain SFAs
are palm and coconut oil, although others foods, such as dairy products (cheese, milk,
yogurt, and butter), meats (pork, poultry, fish, and red and processed meats), vegetable oils,
and nuts, also may contain saturated fat. Nevertheless, these foods also may contain
MUFAs or PUFAs (6). Certain products that contain saturated fat, such as dairy products,
nuts, and vegetable oils, actually may promote better cardiovascular health; in the past, the
presence of metabolic diseases was thought to be related to increased SFA intake (7).
Evidence regarding the association of health outcomes with the long-term intake of various
dietary fats has generated controversy. As a result, there have been differing postures
regarding the recommendations on fat consumption. Consequently, current dietary
guidelines emphasize the quality, rather than the quantity, of dietary fats (8) (Table 1).
TABLE 1
Key messages

• Evidence regarding health outcomes and the long-term intake of various dietary fats has
generated controversy. As a consequence, recommendations regarding fat consumption
differ. In the past, there has been a trend toward a restrictive position for fat intake,
particularly for saturated fats.
 • Current dietary guidelines emphasize the quality, rather than the quantity, of dietary fats
in order prevent nutrition-related chronic diseases.

• Dietary recommendations should focus on dietary patterns instead of particular nutrient

groups. The cultural traditions of users should be incorporated into such
recommendations. Dietary priorities include increasing intake of fruits, vegetables, nuts,
legumes, fish, vegetable oils, yogurt, and minimally processed whole grains; and
decreasing intake of red and processed meats and foods rich in refined grains, added
sugars, salt, or trans fat. The benefits of this approach require outcome-based evidence.

• A low-fat diet is a heterogeneous food pattern and should not be considered synonymous
with a healthy diet.

• The principal contribution of this document is to review the evidence behind current
dietary guidelines and to highlight areas of opportunity to improve the health impact of the
recommendations.

History of the Dietary Guidelines and Current Status: the Fat Controversy
The theory that an increased dietary intake of saturated fats and cholesterol results in an
increase in serum total cholesterol and LDL cholesterol—and therefore an increase in the
risk of heart disease—has been accepted since the Framingham Heart Study (9). The study
reported that high serum cholesterol was a major risk factor for coronary artery disease
(CAD) (10). Several other studies along the same vein were published in the early 1950s.
Kritchevsky (11) showed that concentrations of certain lipoprotein classes were related to
atherosclerotic heart disease, and implicated dietary fat as a factor in this relation. After this,
Keys (12) examined the association between diet and CVD in different countries in the
Seven Countries Study. This study revealed that countries in which fat consumption was the
highest had the most heart disease, supporting the hypothesis that dietary fat was an
important factor in causing heart disease. This observational study gained massive media
attention and had a major influence on the dietary guidelines over the following decades.
Nevertheless, this theory is still in question years later, largely because of the methodology
used and the conclusions that were drawn as a result.
The first formal recommendations regarding the consumption of dietary fat for the overall
population were published in 1957 by the AHA. This guideline recognized that diet plays an
important role in the pathogenesis of atherosclerosis and that the ratio of saturated and
unsaturated fat, in addition to lifestyle variables such as physical activity and smoking, also
could be determinant factors of the disease. However, although the guideline placed limits
on fat intake, it did not distinguish between the different types of fats (13, 14). The guideline
published in 1961 has a form still recognizable today: it recommends the practice of
moderate-intensity exercise; the maintenance of a healthy body weight; an increase in the
intake of PUFAs; and a reduction in the intake of total fat, SFAs, and cholesterol. Additional
specific guidance was included in 1968, and included advice to reduce animal-fat intake and
the incorporation of these dietary recommendations in the diet of the whole family, even
young children (11).
Subsequently, in 1977, the US Senate Select Committee on Nutrition and Human Needs
recommended the following: a reduction in overall fat intake from 40% to 30% of caloric
intake; a reduction in calories from SFA to <10% of total caloric intake and maintenance of
the ratio of SFA, MUFA, and PUFA consumption, with a contribution of 10% for each; a
reduction in total cholesterol intake to <300 mg/d; lower consumption of simple sugars
(≤15% of carbohydrate intake); and a reduction in salt consumption to ≤3 g/d. In essence,
the dietary goals were to eat less fat, cholesterol, and refined and processed sugars, and to
eat more complex carbohydrates, vegetables, fruits, and whole grains. These guidelines
effectively proposed an increase in the consumption of carbohydrates to between 40% and
60% of total energy intake (15).
All of the above recommendations appeared to be consistent with observational studies, in
which the dietary intake of specific types of fat, particularly SFAs, tended to be positively
correlated with CAD (10, 16, 17). In addition, the Medical Research Council Soybean Oil
Study (18) and LA Veterans Study (19), relevant randomized controlled studies, showed that
reducing the intake of SFAs and increasing the intake of PUFAs resulted in decreased serum
total cholesterol concentrations and a lower prevalence of CAD (18, 19). However, it is
important to note that in the Medical Research Council Soybean Oil Study, despite the
numerically fewer deaths and relapses encountered in the intervention group (diet low in fat
and cholesterol) than in the control group, this result was not statistically significant (18).
Furthermore, in the LA Veterans Study, there were important demographic differences
between study groups; this finding led to debate regarding the validity of the results (19).
The findings of the Oslo Diet-Heart Study (20) were in line with the results reported in the
LA Veterans Study (19). This study showed that a diet low in animal fats and cholesterol but
rich in vegetable oil reduced serum cholesterol values on average by 29% over the 5 y study
period. However, despite this change, no differences were found between groups in the
incidence of death from CVD (20). In contrast, The Finnish Mental Hospital Study reported
a beneficial effect on CAD morbidity (45% in men and 35% in women) associated with the
use of a diet low in SFAs and cholesterol and high in PUFAs compared with a control diet
referred to as a normal hospital diet (21, 22). Other studies, such as the Sydney Diet Heart
Study, that were carried out around the same time did not arrive at similar conclusions when
examining similar fat-restrictive diets or diets incorporating corn oil supplementation (23–
25). After this, attempts were made to reduce major cardiac events or reduce recurrence
through the prescription of a diet low in fat and restrictive in cholesterol intake. To date,
these efforts have been unclear because of mixed results.
The 2000 US diet report recommended restricting dietary fat to ≤30% of total caloric intake,
with SFA constituting <10% of these calories and the daily cholesterol intake limited to <300
mg/d. This statement was widely accepted, to the point at which many international
organizations adopted similar recommendations (26, 27). Subsequent guidelines in 2005
and 2010 recommended restricting total fat to 20–35% of total calorie intake. These
guidelines recommended adjusting SFA consumption for adults on the basis of their LDL
concentrations. For those with LDL cholesterol concentrations <130 mg/dL, SFA
consumption should be <10% of calories. However, for individuals with elevated LDL
cholesterol (>130 mg/dL), ≤7% of calorie intake should be derived from SFAs (28). Calories
from SFAs should be replaced with calories from unsaturated fat (29). The USDA
recommendations in 2010 advised consumption of <10% of calories from SFAs, with daily
cholesterol intake limited to ≤300 mg/d. These recommendations also advised people to
keep trans FA consumption as low as possible by limiting foods that contain synthetic
sources of trans fats, such as partially hydrogenated oil, limiting other solid fats, and
reducing the intake of calories from added sugars, as well as reducing sodium intake (30).
Changes in SFA intake were identified as a critical component of the actions to decrease
the incidence of CAD. However, the available data provided by randomized controlled trials
is scant (31). Over 40 y, it was recommended that the consumption of dietary lipids be limited
to <30–35% of total calories with the difference in energy they provide being compensated
for by carbohydrates. But an interesting fact is that, since the late 1970s, with the introduction
of and adjustments in these dietary recommendations, rates of diabetes, obesity, and other
chronic diseases have significantly increased (32).
In order to aid in the prevention of nutrition-related chronic diseases, the recommendations
of the most recent Dietary Guidelines for Americans (2015) (8) emphasize the quality of food
and the health outcomes associated with their regular intake. One change in the report that
has received media attention is that the Dietary Guidelines for Americans did not make a
recommendation on daily dietary cholesterol intake, as it had done in previous reports. The
authors did not find enough scientific evidence to recommend a specific cholesterol
threshold or evidence that foods containing cholesterol should be limited. The principal
contribution of this document is that dietary guidelines are now based on dietary patterns
and their impact on health and disease, instead of the theoretical distribution of
macronutrients (5).
Although the first guidelines attempted to provide recommendations based on the evidence
of their times, more recent observational and controlled dietary intervention studies have
shown the need to move away from the reductionist perspective that focused on single
nutrients or specific foods. Rather, dietary recommendations should take into account
overall diet, food groups, and nutrients, as well as food combinations, frequency, quality,
and quantity (33).

Dietary FAs and Their Impact on Health

The discrepancy in the results examining the effects of dietary fat on health can be explained
by the quality of FAs contained in food (34). For decades, SFAs generally were thought to
have detrimental effects on health (35); therefore, high intake of saturated fat and trans FAs
from industrial sources and dairy products, especially the saturated fats 12:0, 14:0, and 16:0,
was linked to an increased risk of CVD (36). Consequently, only low-fat and fat-free milk
products were recommended by the dietary guidelines as part of a healthy diet in order to
reduce the risk of CVD (29). However, the situation is not as clear cut now. The findings
indicate that the majority of observational studies fail to show an association between the
intake of dairy products and increased risk of CVD, CAD, and stroke, regardless of milk fat
concentrations (37–39). In addition, a recent meta-analysis of randomized studies also
concluded that neither low-fat nor whole-fat dairy foods have significant effects on traditional
CVD risk factors (40). These results agree with pooled analyses of observational studies
that found beneficial or neutral associations between CVDs and total dairy, low-fat dairy,
whole-fat dairy, milk, and cheese (41, 42). Yakoob et al. (43) studied dairy food FAs and
found that the intake of the SFAs pentadecanoic acid (15:0) and heptadecanoic acid (17:0)
and trans palmitoleate (t-16:1n–7) were associated with a lower incidence of diabetes.
These results generally were consistent with those of Micha et al. (34), who found that SFA
consumption was not linked to the incidence of diabetes. Results from short-term
intervention studies on CVD biomarkers indicated that a diet higher in saturated fat from
whole milk and butter increases LDL cholesterol when these are substituted for
carbohydrates or unsaturated FAs; however, they may also increase HDL, and therefore
might not affect or even lower the ratio of total to HDL cholesterol (44, 45). Another meta-
analysis concluded that a modest increase in daily intake of dairy products may contribute
to the prevention of type 2 diabetes mellitus (T2DM), even if these are whole dairy products
(46). The authors found an inverse association between total dairy intake and T2DM risk in
all strata except European studies and studies not adjusting for family history of T2DM. They
also found a nonlinear association between total dairy product intake and T2DM, with most
of the risk reduction occurring with intake up to ∼200 g/d; a higher intake was associated
with a further but more modest decrease in risk. The relation between obesity and
overweight and intake of dairy also has been evaluated. A review showed that a majority of
clinical trials in adults found a protective association between dairy consumption and
overweight and obesity (47). In this review, only one study showed no protective association,
and another study found a protective effect from low-fat dairy but not total dairy. This review
concluded that the consumption of dairy products had no harmful effect on weight status,
contrary to popular belief. The authors did not find low-fat dairy products to be more
beneficial to weight status than regular-fat dairy products. With respect to results from
numerous prospective observational studies and meta-analyses, not all, but most, showed
no association between the intake of milk fat–containing dairy products and the risk of CVD,
CAD, and stroke, although some showed an inverse relation, even with the intake of whole-
dairy products (48). In addition, the Women’s Health Initiative Diet Modification Trial
suggested no benefit from a reduction in SFA intake on the incidence of T2DM (49), Similar
results were described in another meta-analysis by Tong et al., who found that daily
consumption of dairy products had a beneficial effect in the prevention of T2DM
development (50). The differences in the results between studies could be explained by
dissimilarities in their methodology and population.
Studies on the effects of trans FAs suggest that increased consumption of trans FAs is
strongly associated with CVD risk, systemic inflammation, and endothelial dysfunction (51–
53). The principal food sources for trans FAs are margarine, butter, desserts, fried food,
processed meat, and some fast food. It is important to keep in mind that multiple trans FA
isomers exist, and the effect of each isomer is still unclear (54).
Dietary guidelines have consistently recommended replacing calories from SFAs with
calories from PUFAs and MUFAs, which, for the majority of the population, come from
vegetables oils. However, vegetable oils do not only contain PUFAs and MUFAs, and may
actually be an important source of SFAs, as is the case of palm oil and coconut oil (55). The
cardiovascular effect of these vegetable oils may vary depending on the precise combination
of PUFAs, MUFAs, and SFAs (56). Also, it is important to consider how cooking can change
the composition of FAs; for example, vegetable oils are typically heated to high
temperatures, which induces lipid peroxidation in the PUFAs, transforming them into SFAs
(57, 58).
Different types of foods contain different types of fats that are not homogeneous in their FA
or cholesterol content; this heterogeneity may confer different effects on human health and
disease. This may well muddy the evidence regarding the replacement of SFAs with PUFAs
and MUFAs, with such replacement possibly not having a significant impact on health. In a
recent systematic review and meta-analysis, Chowdhury et al. (59) did not find clear
supporting evidence for the high consumption of PUFAs and low consumption of SFAs. This
contradicts numerous previous reports, including the Prevención con Dieta Mediterránea
trial (60), which showed a significant inverse association between a Mediterranean diet
(characterized by fats derived from PUFAs) and risk of CAD (59–62). Another study
conducted in Costa Rica suggested that the type of oil used for cooking and frying is more
important than amount of oil used, so that a decrease in intake of saturated fat may not have
a significant effect on public health (63). Indeed, the European Prospective Investigation into
Cancer and Nutrition study showed that the consumption of extra-virgin olive oil was
associated with a reduced risk of CVD (35). This meta-analysis, while groundbreaking, was
criticized for being biased (64). There are arguments against the replacement of SFAs with
carbohydrates. First, although decreased intake of SFAs lowers LDL cholesterol, it also
lowers HDL cholesterol (65). A recent review by Lawrence (66) highlighted the necessity for
a rational reevaluation of dietary recommendations that focus on minimizing dietary SFAs,
for which the mechanisms of adverse health effects are lacking (65, 66). Second, excess
intake of carbohydrates, such as starches, refined grains, sugar-sweetened beverages,
sweets, and fruit juice are associated with weight gain. Only vegetables, nuts, fruits, and
whole grains have been associated with healthy body weight (67). In addition, a low-fat,
high-carbohydrate diet causes an increase in serum TGs and small, dense LDL particles;
this is an atherogenic lipid profile (66).

Clinical Practice: Proportions of Total Calories or Dietary Patterns

Dietary patterns are defined as nonrandom combinations of different foods and beverages
in diets determined by social or cultural factors (68). The main goal of the dietary guidelines
is to provide advice and strategies to the public to optimize their food selection while taking
into account the traditions and cultural resources of the users (69). Among the dietary
patterns that have been studied are the Mediterranean, Dietary Approaches to Stop
Hypertension (DASH), Western, and vegetarian diets (56). The conventional DASH diet is
rich in fruits and vegetables; high in potassium, magnesium, and fiber; and low in sodium
and SFAs (<7% of energy). The modified DASH diet is similar to a Mediterranean diet, high
in vegetable oils and low in carbohydrates. Al-Solaiman et al. (70) examined the effect of
the DASH diet in obese hypertensive individuals. That study reported that the DASH diet
was more effective in controlling blood pressure than was potassium, magnesium, and fiber
supplementation. Evidence from observational studies and randomized controlled trials
suggests that high fruit and vegetable consumption is associated with a lower incidence of
CAD (3, 71).
Vegetarian diets may result in substantial benefits in weight reduction and arterial blood
pressure compared with nonvegetarian diets (3, 72). Several types of vegetarian diets exist,
including pesco-vegetarian (includes fish), lacto-vegetarian (includes eggs and dairy
products) and vegan (includes no animal products). To date, few studies have focused on
these types of dietary patterns; as a consequence, possible cardiometabolic benefits have
not been confirmed.
A recent review by Mozaffarian (56) highlights the importance of dietary recommendations
that are focused on dietary patterns instead of particular nutrient groups (e.g., dietary fat
and cholesterol intake). Previously, dietary recommendations often focused on total calories,
body weight, and obesity, rather than cardiovascular and metabolic health. This review
emphasizes the importance of targeting specific foods and overall dietary patterns rather
than single isolated nutrients in order to maintain cardiometabolic health. Dietary priorities
include increasing the intake of fruits, vegetables, nuts, legumes, fish, vegetable oils, yogurt,
and minimally processed whole grains; and decreasing the intake of red and processed
meats and foods rich in refined grains, added sugars, salt, or trans fat.
As a result, recommendations should be adjusted to the characteristics of each target
population. Knowledge should be translated into food combinations that provide enough
energy and nutrients to maintain physiologic functions and normal body composition (5).
Food combinations are not randomly selected: individuals set their dietary preference early
in life (73). Food selections become a repetitive, predictable process in which the main
sources of calories are usually limited to 20–30 products. Clusters of food can be identified
with the use of factorial analyses, a useful approach to integrate the combined effects of
mixed foods, instead of considering individual nutrients (74) This approach, known as
“dietary patterns,” has become popular in epidemiologic studies (75). Indicators have even
been developed to qualify the composition of a dietary pattern (e.g., healthy diet indicator,
healthy eating index, or the Program National Nutrition Santé guideline score). The search
for associations between various dietary patterns and multiple health outcomes (i.e.,
cognitive decline, diabetes, obesity, and neoplasia) has been a matter for several meta-
analyses and systematic reviews (76).
Current guidelines have not been effective in the communication of the main messages on
healthy eating patterns. Rather, the majority of these documents suggest “eating plenty of
fruits, vegetables, and complex carbohydrates, and choosing foods that are lower in
saturated fat, salt, and added sugar” (77). These documents are focused on achieving a
caloric content and a prespecified nutrient distribution, as well as limiting the consumption
of fat- or carbohydrate-enriched food products. Instead, they should consider that the intake
of these products is integrated into a network that should be considered as a whole (78).
Certain European governments have designed regionally adapted strategies to improve
nutritional status and promote physical activity by delivering clear-cut messages (79). This
approach is not focused on single components of a diet but, rather, propose a more broad
line of attack to achieve a healthy dietary pattern (77).
Additional evidence to support the use of dietary patterns in guidelines and research has
been provided by the Nutrition and Chronic Diseases Expert Group. This group presented
data on the consumption of key dietary items over a period of 20 y in men and women with
the use of 325 population-based surveys applied in 187 countries. The group assessed 2
types of dietary patterns: one reflecting the consumption of 10 healthy items (fruit,
vegetables, beans and legumes, nuts and seeds, whole grains, skim milk, total PUFAs, fish,
plant n–3 FAs, and dietary fiber) and the other based on unhealthy items (unprocessed red
meats, processed meats, sugar-sweetened beverages, saturated fat, trans fat, dietary
cholesterol, and sodium). Each dietary pattern was assessed for every country stratified by
age, sex, and national income groups. This large amount of information allowed for the
identification of trends in dietary patterns from 1990 to 2010, independent of diet quantity
(caloric intake). Among countries, the largest variation was noted in the mean intake of whole
grain, fruit juice, nuts and seeds, plant n–3 FAs, sugar-sweetened beverages, and
processed meats. Older adults had better dietary patterns than did younger adults; the same
was true for women compared with men. The report confirmed the growing trend in the
consumption of unhealthy options, especially in low-income countries (80). As a result,
policies are needed not only to limit the consumption of unhealthy options, but also to
stimulate the intake of healthy food (56). The aim of dietary guidelines should be to provide
recommendations for disease prevention, not treatment, and to propose healthy options that
can feasibly be adopted by the public.
Guidelines should be translated to messages and strategies easy to implement by the
population, regardless of income and education. Figures and icons (i.e., plates or pyramids)
are a widely used tool to deliver the messages in an integrative manner. Aspects considered
in the educational diagrams depend on the region, culture, and epidemiologic characteristics
of each country. Graphic representations have been used as a visual aid in the process of
dietary guidance, and images are focused on types of foods, instead of nutrients (81). In the
United States, the healthy eating pyramid was published in 2008 in order to give the best
possible advice to the population on healthy eating on the basis of current evidence. In 2010,
the instrument was changed to have a visual representation of a plate (called MyPlate) (82).
As an alternative to the USDA’s nutrition advice, the Harvard School of Public Health
developed the Healthy Eating Plate in 2011. The main difference between these 2 is that
the Harvard Eating Plate emphasizes the intake of whole-grain cereals, low-fat meat,
poultry, fish, healthy oils, and water, and includes advice for increasing physical activity (83).
Other pyramids or plates also have been developed on the basis of the Mediterranean diet
and other dietary patterns. The plate icon recently has been adopted by certain Latin
American countries, including Costa Rica, Uruguay, Cuba, Argentina, and Mexico (81).

Conclusion
Over the past few decades, dietary guidelines have moved from being consensus
documents to becoming evidence-based recommendations. The total amount and the type
of fat has been a focus of these documents for many years. Despite a lack of evidence from
methodologically sound randomized controlled studies, dietary goals for fat intake have
been based on the proportions of the 3 main classes of fat. In addition, the recommendations
were difficult to communicate to the general public.
It is important to remember that dietary data collection is not without its flaws, in particular
memory bias. This is especially important in long-term studies in which changes in diet over
time, coupled with possible memory bias, may even lead to misinformation.
Long-term observational studies have provided evidence regarding the benefits and risks
associated with the regular consumption of the most common food products. Moreover,
these studies have found that food choices follow a common pattern that can be summarized
as a single profile. It is evident that a low-fat diet is a heterogeneous food pattern and should
not be considered synonymous with a healthy diet. Hence, public nutritional
recommendations should leave behind the reductionist information, specifying the intake of
specific amounts of macronutrients. New guidelines typically do not set a threshold for total
fat and cholesterol intake. Instead, they identify the food type and frequency with which
individuals may consume the main sources of fat on the basis of the best available evidence.
Furthermore, recommendations should be integrated into food networks, designed to be
compatible with the most common food patterns of the target population. Randomized
controlled trials are needed to assess the health consequences of the currently
recommended dietary patterns. The interpretation of epidemiologic associations should be
carried out with care, because even strong associations must be confirmed by well-
controlled intervention studies.

Acknowledgments
We thank Armando Tovar for providing us with the opportunity to participate in this
symposium. All authors were involved in the redaction of the paper, and all authors read and
approved the final manuscript.

Footnotes
4
Abbreviations used: CVD, cardiovascular disease; CAD, coronary artery disease; DASH,
Dietary Approaches to Stop Hypertension; T2DM, type 2 diabetes mellitus.

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BMC Obes. 2016; 3: 52.

XVIII. Nutritional quality and marketing strategies of fast food

children’s combo meals in Guatemala
Sofia Mazariegos,1,2 Violeta Chacón,1,2 Adam Cole,3 and Joaquin Barnoya 1,4

Abstract

Background
Globally, more than 41 million children under the age of five are overweight [1]. In 2009, the
prevalence of overweight and obesity among Guatemalan school-age children was 27.1%
and 7.5%, respectively (self-reported heights and weights) [2]. Excessive intake of energy-
dense foods and reduced physical activity are major contributors of childhood obesity.
Children’s fast food consumption is associated with high energy, sodium, and saturated fat
intake [3, 4], and may be a contributing factor to the growing obesity epidemic [5]. To
promote consumption and influence food choice, fast food restaurants may use potentially
misleading health claims [6] and offer toy giveaways [7, 8]. Caregivers perceive them as the
marketing strategy that most influences the decision to purchase less healthy foods [7].
Additionally, fast food restaurants also use convenient combo meals, price incentives, and
prompt delivery as marketing strategies [9]. However, most research on these marketing
strategies is from high income countries, and is lacking from low/middle-income countries
(LMICs) where the obesity epidemic is rapidly spreading.
Despite the overwhelming fast food marketing strategies targeting children, few jurisdictions
have implemented marketing restrictions. Two cities in the United States (San Francisco
and Seattle) have recently made efforts to restrict toy giveaways and implement menu
labeling policies to help consumers make healthier choices [10–13]. Similar to California,
New York City has proposed a policy that requires children’s combo meals with toys or
promotional items to meet certain nutritional criteria [14]. Although preliminary evidence on
the San Francisco toy ordinance does not show that fewer children receive toy giveaways
with their combo meals, restaurants offer healthier default side dishes and drinks. This has
led to a decrease in calories per order purchased by children [15].
In Guatemala, the availability of nutrition information and quality of children’s fast food
combo meals has not yet been documented. Furthermore, there is no evidence of the
prevalence of health claims on children’s combo meals, time to delivery and price incentives
as marketing strategies. Therefore, we sought to assess the use of toy giveaways, time to
delivery and price incentives on combo meals in fast food chains in Guatemala City. In
addition, we sought to compare nutritional quality of children’s combo meals with and without
health claims.

Methods
All (8) major fast food restaurant chains located in Guatemala City, the largest and capital
city of Guatemala, were surveyed. Two fast food restaurant chains did not offer children’s
combo meals and were not included. Therefore the six fast food restaurant chains included
were McDonald’s, Burger King, Wendy’s, Pollo Campero (local fried chicken), Kentucky
Fried Chicken, and Pizza Hut.
We visited one restaurant (conveniently selected) from each chain between 12:00 PM and
3:00 PM over a 2 week-period. We counted the total number of lunch combo meals and
those that were child-oriented (including toy giveaways). We considered children’s combo
meals those that were marketed specifically to children. Children’s lunch combo meal
packages could have the word “kids”, a picture of children, or a licensed character (e.g.,
Spiderman). Each children’s combo meal contained an entrée, side dish, beverage, and
dessert. All children’s lunch combo meals that were listed on the menu board were
purchased. We did not purchase any additional meal items or super-sized portions. The first
brand and type of beverage included in the combo meal and offered by the cashier was
purchased. We then assessed time to delivery and price between the combo meal and the
meal items purchased individually. Hamburger, chicken drumstick, and ham and cheese
pizza combo meals were used for the time to delivery and price comparisons.
Nutrition information was requested at the point of sale, from the restaurant manager, by
checking the restaurant website, or by calling the customer service phone number. We then
classified combo meals as “healthy” or “less healthy” using the UK Nutrient Profile Model
(NPM) [16]. This model measures the nutritional quality of each food or drink, considering
the inclusion of both positive (e.g., protein and fiber) and negative (e.g., sugars and sodium)
nutrients [16]. Less healthy foods have a score of 4 or higher and beverages 1 or higher
[16].
We assessed if combo meals that included toys met international nutritional quality criteria.
Two U.S. standards are available to determine if a fast food combo meal can include a toy
giveaway, the Institute of Medicine (IOM) standard for the National School Lunch and
Breakfast Program [17] and the Model Ordinance for Toy Giveaways at Restaurants
developed by the National Policy & Legal Analysis Network (NPLAN) to Prevent Childhood
Obesity [18]. Both are based on energy (calories), sodium (mg), trans fat (g) content, and
the percentage of total energy from total and saturated fat. Neither standard includes total
sugar; however, we compared the total sugar of combo meals in Guatemala with those that
have been found in children’s combo meals in the U.S [19].
Health claims found on any package (i.e., paper wrapper or cups, children’s combo meal
boxes) of the combo meal items were also counted. We defined a health claim as any text
or figure stating that a food has a particular nutritional property including, but not limited to
energy, protein, fat, carbohydrates, and vitamins or minerals (e.g., “A perfect day to taste
the flavour of vitamins in fruits”) [20]. We then compared the nutrient content of combo meals
with and without claims.
REDCap™ web-based application was used for data entry and STATA (version 13.0, 2013)
for statistical analysis. Median price (interquartile range, IQR), delivery time, and NPM (for
those with nutrition information) were calculated.

Results
We found six different fast food restaurant chains that offered children’s combo meals with
toy giveaways. The fast food restaurant chain that had the most restaurants was Pollo
Campero (Table 1).

Table 1
Fast food chain restaurants in Guatemala1
 n Guatemala City, n (%)

McDonald’s 77 41 (53.2)

Burger King 42 35 (83.3)

Wendy’s 9 8 (88.9)

Pollo Campero 126 75 (59.5)

KFC 3 3 (100.0)

Pizza Hut 39 37 (94.9)

Data from restaurant’s websites, accessed on November, 2013

1

A total of 114 combo meals across all 6 chains were identified and children’s combo meals
per restaurant ranged (Fig. 1) from 9.5% to 25% (p = 0.85) of all combo meals available
(Table 2). Median price was US$3.60 (IQR 3.31 to 3.90, p = 0.15). On average, combo
meals were less expensive (US$1.93, p = 0.01) and took less time (1.44 min, p = 0.19) to be
delivered compared to purchasing meal items separately. All restaurants offered a soft drink
as the first drink option and all combo meals included a toy giveaway.

Fig. 1
Children’s fast food lunch combo meals in Guatemala
Table 2
Delivery time, price, and nutrition information availability of children’s combo meals in fast
food chain restaurants. Guatemala City, Guatemala

Children’s combo meals1

Restaur Total Combo

ant com meals
bo with
meal Childre Delivery time nutrition Source
s, (n) n’s informat of
combo ion (n)% informat
meals, ion
n (%)

Price (Minutes) 3,4 Price (US$) 5

(US$)

(Medi Com Individ Com Individ

an, bo ual bo ual
range) meal items6 meal items6
2 6 6

McDonal 20 5 (25.0) 3.63 2.46 2.75 3.63 3.89 3 (60) Restaur

d’s (3.51 ant
– Manage
3.64) r

Burger 18 4 (22.2) 3.53 2.03 2.63 3.53 4.41 2 (50) Restaur

King (3.31 ant
– Manage
3.79) r

Wendy’s 23 4 (17.4) 3.64 1.33 2.78 3.63 6.88 0 (0) None

(3.64
–
3.64)

Pollo 14 3 (21.4) 3.50 2.86 4.33 3.5 5.22 0 (0) None

Camper (3.50
o –
3.50)
 Children’s combo meals1

KFC 18 3 (16.7) 3.64 2.48 1.8 3.63 6.62 0 (0) None

(3.64
–
3.64)

Pizza 21 2 (9.5) 3.77 21.8 29.08 3.63 6.1 0 (0) None

Hut (3.64 1
–
3.90)
Open in a separate window
1
Children’s combo meals were defined as those that were promoted for children and
contained an entrée, side dish, beverage, and included dessert or toy giveaway
2
U.S. Dollars at an exchange rate of 7.70 Quetzales per 1 US$
3
Time between placing the order and delivery
p = 0.19
4

p = 0.01
5

6
McDonald’s, Burger King, Wendy’s; Kentucky Fried Chicken, Pollo Campero; and Pizza
Hut
We found that nutrition information was not easily accessible and only available for two out
of six fast food restaurants. Five out of 21 children’s combo meals (Table 2) had nutrition
information and all were classified as “less healthy” according to the NPM. Regarding the
National School Lunch and Breakfast Program and the Model Ordinance for Toy Giveaways
at Restaurants (Table 3), combo meals had more sodium, calories from fat, and saturated
fat than either of the nutrition standards (not statistically significant). Moreover our results
were similar to those of a study from the United States with a similar design (Table 3). Three
of the children’s lunch combo meals included a health claim (Table 4, no statistically
significant difference).

Table 3
Nutrition information for selected Guatemalan children’s combo meals with nutrition
standards and previous research. Guatemala City, Guatemala (n = 5)

Nutrient Guatemalan Institute of National Policy & U.S.

children’s Medicine Legal Analysis children’s
combo meals, Nutrition Network to Prevent combo meals,
Median (Range) Standard1 Childhood Obesity Median
Nutrition Standard2 (Range)3
Energy 514 (404 – 725) <650 <550 530 (180 –
(kcal) 880)
 Nutrient Guatemalan Institute of National Policy & U.S.
children’s Medicine Legal Analysis children’s
combo meals, Nutrition Network to Prevent combo meals,
Median (Range) Standard1 Childhood Obesity Median
Nutrition Standard2 (Range)3
Sodium 885 (495 – 1173) ≤640 <640 810 (340 –
(mg) 1960)
Total 46 (36 – 52) - - 37 (0 – 78)
Sugar (g)
Saturated 11 (8 – 13) <10 <10 9 (0 – 18)
fat (%)
Calories 39 (23 – 52) <35 <35 34
from fat
(%)
Trans fat 0.0 (0 – 0) <0.0 <0.5 -
(g)
1
IOM Nutrition Standards for the National School Lunch and Breakfast Program for children
5 to 11 years of age
2
Model Ordinance for Toy Giveaways at Restaurants
3
from O’donnell et al. Am Am J Clin Nutr 2008 Nov; 88(5): 1388-95Developed by the National
Policy & Legal Analysis Network to Prevent Childhood Obesity (NPLAN)

Table 4
Nutrition information for children’s combo meals with or without a health claim. Guatemala
City, Guatemala

Health claim

Yes No

Nutrient (n = 3) (n = 2)

Energy (kcal)1 464 (404 – 514) 705 (685 – 725)

Sodium (mg) 655 (495 – 885) 1058 (943 – 1173)

Total Sugar (g) 52 (46 – 52) 36 (36 – 36)

Saturated Fat (%) 11 (8 – 13) 9 (8 – 11)

 Health claim

Yes No

Calories from fat (%) 41 (39 – 52) 25 (23 – 26)

Trans fat (g) 0.0 (0 – 0) 0 (0 – 0)

1
Median (range) unless otherwise noted

Discussion
According to our findings, nutrition information was not available for most combo meals
offered targeting children. The few that did have information, the nutrition quality was not
optimal according to U.S. nutrition standards [19]. Furthermore, fast food chains are using
toy giveaways that may promote less healthy combo meals to children. Comprehensive
approaches are needed to improve access to healthy options within fast food restaurants
and make these more desirable to children.
Marketing strategies found in our study (i.e., toy giveaways, time to delivery, price incentives,
and health claims) are widely used by fast food chains [8, 21]. Basch et al. [22] found that
these promoted reduced cost combo meals with high sugar, sodium, and fat content. Given
that fast food combo meals with a poor nutritional content contribute to the increase of
childhood obesity [3], restrictions are needed to ensure that nutrient quality of children’s fast
food combo meals meet healthy guidelines. For instance, Guatemala could implement a
policy that requires additional fruit and vegetable options for combo meal side dishes and
low-fat milk as the first beverage alternative. To increase uptake, these could be offered as
the default option rather than french fries and a soft drink. This would likely improve combo
meal nutritional quality.
Fast food restaurants offer combo meals as an efficient and convenient way to purchase a
meal. According to our findings, chains in Guatemala (and most likely elsewhere) offer meal
items that are less expensive and served faster when they are purchased in a combo meal
rather than separately. This suggests restaurants are using combo meals to offer more food
for lower prices, promoting consumption and therefore higher energy intake.
Menu labeling is now being explored as a strategy to reduce calorie consumption. In 2008,
the Board of Health of the New York City Department of Health and Mental Hygiene
implemented regulations mandating chain restaurants to include calorie information on
menus [23]. Other U.S. cities and states have since tried to implement similar policies
[24, 25], and the Patient Protection and Affordable Care Act (ACA) requires menu labeling
at all restaurant chains with 20 or more locations nationally [26, 27]. Guatemalan fast food
chains did not include calorie information on their menus and we found personnel were
evasive when asked for nutrition information. Therefore, we were unable to obtain data for
most of the combo meals we found. Mandatory menu labeling is a promising strategy
providing consumers with knowledge [28] and improving the nutrient content of fast food
combo meals since it could encourage restaurants to reformulate their products to offer
healthier options. Rationale in favor of menu labeling is grounded in considerable evidence
and unintended consequences are unlikely [29]. The Guatemalan Ministry of Health should
support policies requiring fast food chains to provide nutrition information at the point of sale
and on menus in order to support the selection of healthier options. Furthermore, this
information needs to be presented in a way that is easy to understand for consumers
regardless of literacy level.
Consumption of nutrient poor foods, such as fast food combo meals, promoted by toy
giveaways is likely one of the contributing factors to the observed increase in childhood
obesity [30]. Children’s combo meals found in our study included a toy and those that had
nutrition information failed to meet nutrition standards proposed by the IOM and in California.
Guatemala lacks regulation to improve the nutritional quality of children’s combo meals.
However, restricting toy giveaways to children’s combo meals that meet established nutrition
standards is likely to encourage healthier combo meal selections [8, 31].
Health claims create a “halo” effect over food, preventing consumers from seeking further
nutrition information [32]. Likewise, consumers also draw inferences about the nutritional
quality of food with health claims on the package or combo meal [33]. The food industry,
however, is not the only industry using claims as a marketing strategy. The tobacco industry
uses terms like “light” and “smooth” to give the impression that cigarettes are less harmful
[34]. Our results yield that most combo meals included in our study had health claims, even
though they were all classified by our analysis as ‘less healthy’. Therefore, nutrient content
or nutritional quality should be required in order to include health claims in children’s fast
food combo meals to guarantee accuracy and avoid misleading marketing.
Our study has strengths and limitations. To the best of our knowledge this is the first study
to document the prevalence, marketing strategies, and nutritional quality of children fast food
combo meals in a LMIC. In addition, we surveyed local and international fast food chains.
However, we only included children combo meals and therefore our findings are not
generalizable to all combo meals available at fast food chains. In addition, we did not
evaluate how these strategies influence purchasing decisions in Guatemala.

Conclusions
In conclusion, given our findings, Guatemalan public health authorities (and elsewhere)
should consider a comprehensive approach to encouraging healthier choices within fast
food restaurants. Policies are required to include fruit and vegetable options for meal side
dishes and healthier beverage alternatives. Furthermore, policies are required to mandate
fast food chains to provide easily accessible and understandable nutrition information for
combo meals and restrict the use of toy giveaways. Once implemented, research is
warranted to evaluate the implementation and impact of these policies on childhood obesity
rates in Guatemala.

Acknowledgements
We wish to thank Eduardo Villamor for his contribution to this project.

Funding
This work was carried out with the aid of a grant from the International Development
Research Centre, Ottawa, Canada [Project number 107213-001]. Joaquin Barnoya receives
additional support from an unrestricted grant from the American Cancer Society and from
the Foundation for Barnes-Jewish Hospital. Additional support was received from the NIH
Research Grant [# D43 TW009315] funded by the Fogarty International Center and National
Institute of Aging.

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article (and its
Additional files 1 and 2).

Authors’ contributions
SM was responsible for study design, data collection, analysis, and interpretation, and led
the manuscript writing. VC made substantial contributions to study design, data analysis,
and manuscript writing. AC made substantial contributions to data analysis, interpretation,
and manuscript writing. JB made substantial contributions to the study design, data
collection, analysis, and interpretation, and critically revised the manuscript for important
intellectual content. All authors approved the final version of the manuscript and agreed to
be accountable for all aspects of the work in ensuring that questions related to the accuracy
or integrity of any part of the work are appropriately investigated and resolved.

Competing interests
Authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Abbreviations

LMIC Low/middle-income country

NPM Nutrient profile model

Additional files
Additional file 1:(36K, xlsx)
Child-oriented fast food meals in Guatemala. Data on prevalence of child-oriented fast food
meals in Guatemala (XLSX 36 kb)
Additional file 2:(54K, xlsx)
Price and nutrition information of child-oriented fast food meals in Guatemala. Data on price
and nutrition information of child-oriented fast food meals in Guatemala. (XLSX 53 kb)

Contributor Information
Sofia Mazariegos, Email: moc.liamg@sogeirazam.aifosa.
Violeta Chacón, Email: moc.liamg@nocahcateloiv.
Adam Cole, Email: ac.oolretawu@elocga.
Joaquin Barnoya, Phone: (502) 2475
1908, Email: ude.ltsuw.sisoduw@jayonrab, Email: ude.dravrah.tsop@ayonrabj.

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10.1016/j.jada.2010.07.017. [PubMed] [Cross Ref]
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Items in Restaurants and Similar Retail Food Establishments. vol. 2014-27833;
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Sci Rep. 2016; 6: 30917.

 XIX. Polyphenol-rich strawberry extract (PRSE) shows in
vitro and in vivo biological activity against invasive breast
cancer cells
Stefano Amatori,1,2,* Luca Mazzoni,3,4,* Josè Miguel Alvarez-Suarez,3,5 Francesca
Giampieri,2,3Massimiliano Gasparrini,3 Tamara Yuliett Forbes-Hernandez,3,6 Sadia
Afrin, Alfredo Errico Provenzano, Giuseppe Persico, Bruno Mezzetti,4 Augusto
3 1 1

Amici,7 Mirco Fanelli,a,1and Maurizio Battinob,3,8

Abstract
In the last decades, the ability of phytochemicals to modulate apoptosis signaling pathways
has attracted increasing attention as an anti-cancer agent1. The richest dietary sources of
these bioactive compounds are fruits and vegetables, and their intake has been correlated
with a decreased risk of developing several chronic pathologies, including cardiovascular
and neurodegenerative diseases2,3, obesity4, diabetes5, infections6, skin diseases7 and
cancer8, including breast cancer9.
Among fruits, there is growing interest in berries, and in particular strawberries
(Fragaria x ananassa Duch.), due to their nutritional quality and to the numerous bioactive
compounds they contain10. Compared with other non-berry fruits, the strawberry is a rich
source of folate11, vitamin C and several phytochemicals that can influence the nutritional
and organoleptic qualities of this fruit12,13. Its healthy effects are attributed to high levels of
antioxidant compounds, most of which are phenolic compounds such as anthocyanins,
flavonols, flavanols, condensed tannins (proanthocyanidins, ellagitannins, and
gallotannins), hydroxybenzoic and hydroxycinnamic acid derivatives, and hydrolyzable
tannins13,14. The role of strawberry bioactive compounds on cancer prevention seems to
involve different mechanisms, which have not yet been fully elucidated; therefore, further
investigations are needed to clarify the roles of the different strawberries phytochemicals
against cancer cells. Several studies on extracts of strawberries, raspberries, and other fruits
and berries, did not found any correlations between the content of some phytochemicals
and inhibition of cancer cell proliferation15,16. Different studies indeed have shown that the
complex mixtures of phytochemicals present in fruits and vegetables are more effective than
their individual constituents in preventing cancer, through both additive and synergistic
effects17,18. For this reason, it is important to study potential anticancer activity of fruits and
vegetables using whole extracts containing all phytochemicals, not only using purified
molecules or fractions enriched in certain classes of molecules.
The antioxidant capacity has been considered for years as a first line defense against the
earlier stages of the mutagenesis process, through the capacity of these compounds to
scavenge ROS species decreasing DNA oxidative damage, to stimulate antioxidant
enzymes and to enhance DNA repairing. Several studies have recently underlined the ability
of these compounds to modulate the cellular processes linked to cancer progression, such
as cell proliferation, differentiation, apoptosis, cell cycle arrest, intracellular communication,
inflammation and angiogenesis. Nevertheless, only few studies exist on the anticancer effect
of strawberries and, in particular, against breast cancer19. Breast cancer represents the most
common neoplastic disease among women worldwide, with about 1.67 million new cancer
cases in 2012 (25% of all cancers), and is the second leading cause of cancer death among
women in developed regions (198,000 dead, 15.4%)20.
Evidence supports the idea that tumor growth, recurrence and metastasis formation are
dependent on cells with self-renewal properties, termed cancer initiating cells (CICs) or
cancer stem cells (CSCs)21. A17 cells are a highly tumorigenic and invasive cell line,
established from an FVB/neuT transgenic mammary tumor, displaying properties of CICs
and basal-like breast cancer22,23,24,25,26. It has been demonstrated that A17 cells exhibit a
stemness-related gene signature virtually identical to that of mesenchymal stem cells and
are able to induce secondary tumor lesions due to metastasis formation25. In this study we
investigated the biological activity of “Alba” strawberry extract on breast cancer, with a
particular focus on the A17 cellular model.

Results

Phytochemical analysis of PRSE

The nutritional and phytochemical composition of PRSE was characterized by analyzing
vitamin C content, phytochemical levels and the total antioxidant capacity (TAC) of the
extract. Results are reported in Table 1 and show, according to our previous studies27, that
“Alba” extract presents high levels of Vitamin C (0.57 mg/g) and phytochemical compounds
measured as Total Phenolic Content (TPH) (2.26 mg GAEq/g), Total Flavonoids Content
(TFC) (0.61 mg CEq/g) and Total Anthocyanin Content (ACY) (0.46 mg Pg-glcEq/g).

Table 1
Micronutrient composition, phytochemical content and antioxidant capacity of
strawberry extract.

Parameter Quantification
Vitamin C (mg/g FW) 0.57 ± 0.03
TPH (mg GAEq/g FW) 2.26 ± 0.03
TFC (mg CEq/g FW) 0.61 ± 0.02
ACY (mg Pg-glcEq/g FW) 0.46 ± 0.01
Total antioxidant capacity:
TEAC (μmol TE/g FW) 17.97 ± 1.13
FRAP (μmol TE/g FW) 12.62 ± 0.15

However, TFC and ACY values are similar to those of other strawberry cultivars, while the
level of total phenolic compounds results very high if compared to commercial strawberry
varieties previously analyzed27,28.
Moreover, the total antioxidant capacity of PRSE was quantified showing relevant values by
both Trolox Equivalent Antioxidant Capacity (TEAC) (17.97 μmol TE/g) and Ferric Reducing
Antioxidant Power (FRAP) (12.62 μmol TE/g) assays (Table 1).

Biological effects of PRSE on A17 cells

The effects induced by the extract on the survival of A17 cells were analyzed in both dose-
response and time-course experiments. Cells were treated for 24 h, 48 h or 72 h with
concentrations of extract ranging from 0.5 to 5 mg/ml. Interestingly, we found that the extract
reduces the survival of A17 cells in a time and dose-dependent manner (Fig. 1a).
Open in a separate window
Figure 1
Effects induced by PRSE on cellular viability.
(a) Dose-response and time-course experiments on A17 cells. Cells were treated with the
reported concentrations of PRSE for 24 h, 48 h or 72 h. After treatment cell viability was
evaluated by Trypan blue dye exclusion assay and calculated as percentage compared to
the untreated control. (b) Dose-response experiments on normal (WI38 and NIH-3T3) and
breast cancer (MCF-7 and A17) cell lines. Cells were treated with the reported
concentrations of PRSE for 48 h. After treatment cell viability was evaluated by Trypan blue
dye exclusion assay and calculated as percentage compared to the untreated control. Data
are reported as the mean ± standard deviation of three independent experiments. *P < 0.05
respect to normal cell lines by Student’s T test.
Once characterized in A17 cells, the activity of PRSE was investigated also in normal
fibroblast cell lines of both murine (NIH-3T3) and human (WI38) origin and in the human
breast cancer cell line MCF-7 by dose-response studies at 48 h of treatment. Interestingly,
we found that: i) A17 cells show the higher response to PRSE exposure (IC50 of
1.14 ± 0.29 mg/ml) and, most importantly, that ii) normal cells are significantly less sensitive
to PRSE respect to cancer cell lines (mean IC50 of 3.24 ± 0.14 mg/ml and 1.68 ± 0.77 mg/ml,
respectively – Fig. 1b).
Subsequently, possible alterations of cell cycle phase distribution were evaluated by
cytofluorimetric analysis of propidium iodide stained cells. A17 cells treated for 48 h with
different concentrations of PRSE showed, at the concentrations of 0.5 and 1 mg/ml of PRSE
treatments, a reduction of cells in S phase and a concomitant increase of cells in G1 phase
of the cell cycle (Fig. 2). Moreover, treatments with higher doses of PRSE (2.5 mg/ml)
drastically change the biological response of A17 cells, hindering the cell cycle analysis but
showing the appearance of a hypodiploid cellular subpopulation (Fig. 3).

Figure 2
Cell cycle alterations induced by PRSE on A17 cells.
A17 cells were treated for 48 h with the concentrations of PRSE reported in the figure or left
untreated. Cells were stained with propidium iodide and the DNA content of cells was
analyzed by a cytofluorimeter. (a–c) Representative examples of cell cycle distribution. (d)
Cell cycle percentages calculated by FlowJo 8.6.3 software. Data are reported as the
mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.01 respect
to not treated cells by Student’s T test.
Open in a separate window
Figure 3
Percentage of hypodiploid cells in PRSE-treated A17 cells.
A17 cells were treated for 48 h with the concentrations of PRSE reported in the figure or left
untreated. Cells were stained with propidium iodide and the DNA content of cells was
cytofluorimetrically analyzed. Experiments were performed in triplicate. (a–d)
Representative examples of untreated and treated cells. (e) Percentages of hypodiploid cells
calculated by FlowJo 8.6.3 software. Data are reported as the mean ± standard deviation of
three independent experiments. **P < 0.01 respect to not treated cells by Student’s T test.
A17 cell line is an intriguing model of study because of its highly aggressive and invasive
phenotype25. In consideration of the critical role of cellular migration on the multistep process
of tissue invasion29 we decided to investigate the possible interference of PRSE on the
mobility of A17 cells by wounding assay. To avoid cell turnover that could mimic the cell
migration, cells were starved during this assay. After 48 h of culture, A17 cells not subjected
to treatments were able to migrate and partially fill the wound empty area while exposure to
PRSE inhibited the cell migration in a dose-dependent manner (Fig. 4a). Notably, the
inhibition of wound closure was monitored starting from the lowest concentration of PRSE
tested (0.5 mg/ml), and appears almost completely inhibited starting from a concentration of
2.5 mg/ml (Fig. 4a). A quantitative analysis of wound closure was carried out using the NIH
Image J software (Fig. 4b).
Open in a separate window
Figure 4
PRSE effect on the migration ability of A17 cells.
(a) Wound-healing assay of A17 cells untreated or treated with different concentrations of
PRSE. All samples were analyzed after 48 h of treatments. Representative images from
three independent experiments are shown. (b) Quantification of the percentage of wound
closure by Image J software. Data are reported as the mean ± standard deviation of three
independent experiments.

PRSE modulation of gene expression

The biological effects mediated by the extract on A17 cells were also investigated at the
molecular level by analyzing the expression of a panel of genes known to be involved, with
different roles, in the cellular migration, adhesion and invasion processes. For this purpose,
the level of 84 different mouse gene transcripts was analyzed by applying a quantitative RT-
PCR array on A17 cells untreated or treated for 48 h with sub-lethal doses of PRSE. Among
the 84 gene transcripts screened, 12 genes showed a regulation exceeding the 2-fold criteria
compared to the untreated control (Fig. 5a). Genes down-regulated include the colony
stimulating factor 1 (Csf1, −2.42 fold), the melanoma cell adhesion molecule (Mcam, −2.78
fold), the nuclear receptor subfamily 4, group A, member 3 (Nr4a3, −4.42 fold) and SET
nuclear proto-oncogene (SET, −3.38 fold). The transcripts showing the strongest up-
regulation were glycoprotein nmb (Gpnmb, 4.30 fold), Integrin beta 3 (Itgb3, 2.97 fold) and
the chemokine (C-C motif) ligand 7 (CCl7, 2.89 fold). In addition, also cathepsin L (Ctsl, 2.37
fold), chemokine (C-X-C motif) Receptor 4 (Cxcr4, 2.14 fold), HIV-1 tat interactive protein 2
(Htatip2, 2.14 fold), and matrix metalloproteinases 10 and 3 (Mmp10 and Mmp3, 2.62 and
2.61 fold, respectively) showed an up-regulation exceeding the 2-fold criteria. Notably, 18
out of 84 transcripts present in the PCR array were not amplified (see legend of Fig. 5a).
Open in a separate window
Figure 5
Effects of PRSE on the expression of genes involved in migration, adhesion and
invasion processes in A17 cells.
(a) Screening by PCR array of 84 mouse genes involved in the migration, adhesion and
invasion processes (only the 66 genes amplified are displayed). A17 cells were treated for
48 h with sublethal doses of PRSE or left untreated. After RNA extraction and
retrotranscription, the resulting cDNA was applied in the array and amplified. Data are
reported as fold compared to control untreated A17 cells. Genes showing a regulation
exceeding the 2-fold criteria were considered as differentially expressed. Data are reported
as the mean ± standard deviation of three independent experiments. The transcripts of the
following genes were not amplified: Cdh1, Cdh6, Cdh8, Cxcr2, Elane, Etva, Ewsr1, Fgfr4,
Hgf, Il1b, Kiss1, Mmp13, Mmp7, Mycl, Rorb, Timp4, Tnfsf10 and Tshr. (b) Western blot
analysis of total cell lysates obtained from PRSE-treated and untreated (n.t.) A17 cells.
Subsequently, two genes showing significant modulation, Mcam and Nr4a3, were further
investigated at protein level by western blotting (Fig. 5b). Densitometric analysis of the
bands of the two proteins was performed and normalized to alpha-tubulin expression,
confirming the downregulation of Mcam (−1.89 fold at 0.5 mg/ml and −4.25 fold at 1 mg/ml
of extract) and, at a less extent, of Nr4a3 (−2.37 fold at 0.5 mg/ml and −1.54 fold at 1 mg/ml
of extract). In addition, caspase-1 levels were also analyzed by western blotting showing the
upregulation of the protein at both 0.5 mg/ml (+1.89 fold) and 1 mg/ml (+4.16 fold) of PRSE
(Fig. 5b).

In vivo evaluation of PRSE activity

A17 cells were used to generate an orthotopic model of breast cancer in syngeneic female
FVB/N mice. Four weeks mice were fed with 15% strawberry extract-enriched food or regular
food as control (ten mice for each group). Upon the 8th week of age, all the mice underwent
tumor challenge while animals of each group continued to receive their diets. After 5 weeks,
tumors were withdrawn, measured using a caliper and weighted. Results show a significant
reduction in both tumor weight (Fig. 6a) and tumor volume (Fig. 6b) in mice fed with PRSE.
Open in a separate window
Figure 6
Effect of PRSE on A17 tumors in mice.
Ten mice (fed with 15% strawberry extract-enriched food) were housed one mouse/cage,
while ten mice (fed with regular food) were housed in two cages with free access to water
and food. Upon the 8th week of age, all the mice underwent tumor challenge with 2 × 105A17
cells. After cells injection mice were orally treated for further 5 weeks, at the end of which
tumors were withdrawn and both tumor weight (a) and tumor volume (b) were measured.
***P < 0.001 by Student’s T test.

Discussion
The strawberry (Fragaria x ananassa) was chosen as the model in this study for its high
content of antioxidants and bioactive compounds, as well as for its high availability for food
industry and fresh consumption13. In the last decades, growing interest has been focused
on the antioxidant capacity of strawberries so that TAC is considered a quality parameter
and an indicator of bioactive compounds present in the fruit. The antioxidant capacity of the
strawberry indicates that its consumption could contribute to prevent and reduce oxidative
reactions that cause negative effects on human health, playing different roles in the
development of chronic diseases and cancers.
Moreover, even if the high TAC of strawberries has been proved, it has also been
demonstrated that this parameter is strongly influenced by the strawberry genetic
background and is strictly related to the presence of oxygen radical scavengers such as
Vitamin C and polyphenols11,27,28,30,31. Among polyphenols, anthocyanins are quantitatively
the most important phenolic compounds in strawberries13, so that more than 25 different
anthocyanin pigments have been described from different varieties and selections32.
Analyses of TPH, ACY and TFC in the “Alba” cultivar in the present study confirm the high
content of phytochemicals of this strawberry.
Strawberries are important also for their high content of Vitamin C, which is even higher than
that of citrus fruit. Vitamin C content is an essential parameter due to the high number of
biological roles that it plays in humans, lowering the incidence of cardio-and cerebro-
vascular diseases33, of several cancers34, and other health disorders such as lead toxicity35.
When evaluating the Vitamin C content in strawberries, it is important to consider that the
molecule is very labile and in adverse conditions undergoes oxidation, depending on several
factors such as temperature, water and pH36.
The choice of using “Alba” strawberries in this study was also based on the very interesting
results obtained in recent years using this cultivar, both in vitro37,38 and in vivo39,40.
As shown above, the “Alba” strawberry extract is able to strongly decrease cellular viability
of the highly tumorigenic stromal cell line A17, which is characterized by mesenchymal
features and metastasis formation ability26,41.
In particular, it is possible to distinguish two different biological behaviors: i) a cytostatic-like
effect at low doses of PRSE and ii) an acute toxic effect at higher doses. The capacity of the
strawberry extract to activate the apoptotic process, as well as its anti-cancer potential in
other cancer models, was already characterized by other authors19. However, although
these observations are in accordance with the increased hypodiploidy observed in this study
at high PRSE doses, the analysis of DNA fragmentation by laddering assay did not show
the induction of the apoptotic internucleosomic cleavage of DNA (data not shown).
Interestingly, we observed an increased expression level of the precursor form of caspase-
1 that, together with the monitored hypodiploidy, could suggest an activation of the apoptotic
pathway at a very early stage, and thus below the detection limit of the experimental
approaches used. In addition, we found no evidence of caspase-3 modulation and/or
activation (data not shown).
We hypothesized that sub-lethal doses of strawberry extract could be able to inhibit the
known invasive potential of A17 breast cancer cells. Thus, the effect of PRSE on the cellular
migration, which is known to play a pivotal role in invasion, was first analyzed at a biological
level by wounding healing assay. The observation that low doses of “Alba” PRSE were
sufficient to block cell migration gave us the rationale to extend the study at the molecular
level. The subsequent characterization of the expression levels of genes involved in the
cellular migration, adhesion and invasion processes allowed us to obtain an overall view of
changes that take place in several pathways. In particular, the down-regulation of a
subgroup of genes (Csf1, Mcam, Nr4a3 and Set) supports the anti-invasion effect of the
PRSE extract because high expression levels of these genes are often associated to the
invasive phenotype of cells. In the context of human breast cancer it has been already
reported that: i) high expression of Csf1 is correlated to metastasis formation and thus has
been proposed as negative prognostic factor; ii) up-regulation of human Mcam (named
Muc18 in humans) promotes motility, invasiveness and tumorigenesis of human breast
cancer cells; iii) high expression levels of Nr4a3 were found correlated with increased risk
of developing distant metastasis in triple-negative breast cancer patients; iv) Set nuclear
proto-oncogene (named also I2pp2a) is one of the genes down-regulated by the
mushroom Ganoderma lucidum extract and is involved in the suppression of breast-to-lung
cancer metastasis42,43,44,45. Interestingly, the modulation of Mcam and Nr4a3 was confirmed
also at the protein level, further suggesting a role of these proteins in the effect induced by
PRSE on A17 cells.
Likewise, we found two genes whose monitored up-regulation, as a consequence of PRSE
treatment, can be consistent with an anti-invasion effect: i) Htatip2 (named also Tip30), a
putative metastasis suppressor gene which is inversely correlated with lymph node
metastasis in breast cancer patients, and ii) Gpnmb (also named Osteoactivin/HGFIN), a
gene with a controversial role in the metastatic process46,47.
Otherwise, it was not possible to find any correlation between the known activity of the
remaining six genes found up-regulated by PRSE (Ccl7, Ctsl, Cxcr4, Itgb3, Mmp19, Mmp3)
since their functions are generally associated to the acquisition of invasive
features44,48,49,50,51.
As already stated, the role of strawberry bioactive compounds on cancer prevention seems
to involve different mechanisms of action that are still unclear. Previous studies indeed
indicate that the biological activity exerted by berries against cancer cells is probably
mediated by the synergy between different compounds suggesting the involvement of
several molecular pathways. To date, only few studies have tried to investigate the
molecular basis of strawberry activity against breast cancer cells. For example, the role of
p73 in triggering apoptosis of p53-null cells has been recently suggested19. Our study
indicates the involvement of other genes known to play key roles in cellular invasion,
adhesion and migration.
Notably, cell survival studies show that the effect of the extract is significantly higher in breast
cancer cell lines, and in particular A17 cells, with respect to normal cells, suggesting a
therapeutic window for PRSE in vivo. This observation is in accordance with previous
studies on mice that showed efficacy of strawberry methanolic extract against Ehrlich ascites
carcinoma (EAC)19. We thus explored the efficacy of PRSE in inhibiting tumor formation in
vivo exploiting the intriguing orthotopic breast cancer model generated by injection of A17
cells directly into the mammary gland of mice, finding a strong reduction of both tumor size
and tumor volume in mice fed with PRSE from 4 weeks before tumor challenge.
Although the mechanism by which the strawberry extract exerts its biological effect is not
completely understood at the molecular level, as well as which component plays the major
antineoplastic role, our results suggest an interesting anti-invasive potential of “Alba” PRSE
against breast cancer cells both in vitro and in vivo. Further studies will be necessary to
unravel the pathways involved in the biological effects of PRSE and to elucidate the
molecular basis of strawberry extract action, as well as to shed light on the possible
introduction in the diet of “Alba” and, more in general, strawberries as a useful nutrient to
limit (or prevent) tumor formation.

Methods

Preparation of PRSE
Strawberry fruits of the “Alba” variety were collected in the experimental fields of the
Agricultural Faculty of “Università Politecnica delle Marche” located in Agugliano (AN), in
central east Italy (43°31′60″ N-13°22′60″ E). Fruit samples from the selected variety were
hand-picked at the same day-time on different days, corresponding to the ripening times of
the selected clone, from the second to the fourth picking. Fruit samples were selected for
homogenous fruit, avoiding unripe, wounded or shriveled fruits. Within 2 h after harvest,
whole fruits were stored at −20 °C before analyses. For the evaluation of total antioxidant
capacity (TAC), total phenolic content (TPH), total anthocyanin content (ACY) and total
flavonoid content (TFC), a methanolic extract was prepared via homogenization. Frozen
strawberries were thawed for 60 min at 4 °C. Ten gram aliquots of the fruits were added to
100 mL of the extraction solution, consisting of methanol/milliQ water/concentrated formic
acid (80:20:0.1 v/v), and fruits were homogenized using an Ultraturrax T25 homogeniser
(Janke & Kunkel, IKA Labortechnik, Staufen, Germany) for 2 min. Extraction was maximized
by stirring the suspension for 2 h in the dark at room temperature (RT), then the tubes were
centrifuged at 3500 rpm for 15 min, in two sequential times, to sediment solids. Supernatants
were filtered through a 0.45 μm Minisart filter (PBI International, Milan, Italy), transferred to
5.0 ml amber glass vials and stored at –20 °C until analysis. Methanolic extract was
concentrated through a rotary evaporator and stored in aliquots at −80 °C for subsequent
experimental procedures. Vitamin C analysis was conducted as described28.

PRSE analysis
Two methods were used for the determination of the antioxidant capacity (TAC) of
strawberry extracts: the Trolox Equivalent Antioxidant Capacity (TEAC) and the Ferric
Reducing Antioxidant Power (FRAP). TEAC assay consists in the quantification of
strawberry extract free radical scavenging activity against 2,2-azinobis-(3-ethylbenz-
thiazoline-6-sulfonate) radical cation (ABTS+), using Trolox (6-Hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid) as reference standard52,53. Briefly, 1 ml of the
ABTS+ radical solution was mixed with 10 μl of reagent (strawberry extract or standard). The
analysis solution was vortexed for 20 seconds and, after 1–3 min, spectrophotometrically
analyzed at 734 nm measuring the color inhibition of the ABTS+radical. FRAP assay was
carried out according to the protocol proposed by Deighton and coworkers54, with slight
modifications55.
Total phenolic content (TPH) of the strawberry extracts was determined using the Folin-
Ciocalteu colorimetric method, as modified by Slinkard and Singleton56. Briefly, 100 μL of
sample (milliQ water, water diluted strawberry extracts or gallic acid standard solutions)
were added to 500 μL of Folin-Ciocalteau reagent previously diluted in water (dil. 1/10) and
kept at 4 °C, in the dark. The mixture was incubated for 1 to 8 min at RT, then 400 μL of
0.7 M sodium carbonate were added and the mixture was vortexed. The solution was
incubated for 2 h at RT, in the dark and spectrophotometrically analyzed at 760 nm.
The total anthocyanin (ACY) content of the strawberry extracts was determined using a
modified pH differential method57, while total flavonoid content (TFC) was determined by
using a colorimetric method58,59.
Vitamin C content was measured through HPLC analysis immediately after the extraction
procedure. The HPLC system comprised a Jasco (Jasco Inc., Easton, USA) PU-2089 Plus
controller and a Jasco UV-2070 Plus ultraviolet (UV) detector set at absorbance of 260 nm.
An isocratic elution with 50 mM potassium phosphate at pH 3.2 was performed by means of
a Supelcosil LC8 150 × 4.6 mm HPLC column (Sigma-Aldrich S.r.l. Milan, Italy)60.
Quantification of the vitamin C was carried out through a comparison with pure vitamin C
calibration curve. All the analyses were conducted in triplicate.

Cell culture
The stromal cell line A17 was isolated from a murine model of mammary carcinoma induced
by the overexpression of HER-2/neu transgene in the epithelial compartment of mammary
glands in FVB mice, line 23325. A17 cells were cultured in high-glucose Dulbecco’s Modified
Eagle Medium (DMEM - Euroclone, Pero, Italy), supplemented with 20% fetal bovine serum
(Gibco - Hyclone, South Logan, UT, USA), 1% penicillin-streptomycin and 1% glutamine.
WI38, NIH-3T3 and MCF-7 cell lines were kindly provided by Prof. Pier Giuseppe Pelicci
(European Institute of Oncology - Milan, Italy, 2016) and cultured in high-glucose Dulbecco’s
Modified Eagle Medium (DMEM - Euroclone, Pero, Italy), supplemented with 10% fetal
bovine serum (Gibco - Hyclone, South Logan, UT, USA), 1% penicillin-streptomycin and 1%
glutamine.
All the cell lines were grown in a humidified atmosphere at 37 °C and 5% CO2 as previously
described61,62.

Cell viability
Dried PRSE, stored in aliquots at −80 °C, was diluted in culture medium immediately before
use. Cells were seeded in triplicate in 6-well plates 16 hours before PRSE addition. After
treatment with different concentrations of extract, cell viability was measured by a TC10
automatic cell counter (BioRad, Hercules, CA, USA) and compared with an untreated control
to estimate the cell viability. The 50% inhibitory concentration (IC50) value was calculated
with CompuSyn software (ComboSyn, Inc., Paramus, NJ, USA)63. Data are reported as
mean (±SD) resulting from three independent experiments.

Cell cycle analysis

Cell cycle was analyzed using the propidium iodide staining procedure as previously
reported64. Cells were fixed in ice-cold 70% ethanol and stained using a propidium iodide
staining solution (50 mg/ml). Cytofluorimetric acquisitions were carried out with a PAS flow
cytometer (Partec, Münster, Germany) and sample analysis conducted using FlowJo 8.6.3
software (Tree Star, Inc., Ashland, OR, USA). Cell cycle percentage values were calculated
using a Watson pragmatic model.

Wounding assay
Wounding assay was performed as previously described on the same cellular model (A17)65.
Briefly, a linear wound was produced in A17 confluent cellular population by scratching the
bottom of each dish with a sterile pipette tip. After wounding, cells were washed with
phosphate buffer saline (PBS) and incubated in high glucose DMEM containing 0.5% FBS
with different strawberry extract concentrations (0.5, 1, 2.5 and 5 mg/ml). A17 cells were
allowed to migrate for additional 48 h after which images acquisition was carried out by a
LeitzFluovert FU (Leica Microsystems) microscope. Remaining wound areas were
determined using NIH Image J software for calculation of the percentage of wound closure.
Analyses were performed in triplicate.

Gene expression analysis and western blotting

The Tumor Metastasis RT2 Profiler PCR Array (Qiagen, Hilden, Germany), consisting of 84
mouse genes known to be involved in the metastasis formation process, was used to profile
the biological response of A17 cells induced by the PRSE. Briefly, total RNA was extracted
from treated or untreated A17 cells using RNeasy extraction kit and reverse transcribed into
cDNA using an RT2 First Strand Kit (Qiagen). The cDNA was combined with RT2 SYBR
Green qPCR Master Mix (Qiagen), and equally distributed (25 μl) to each well of the PCR
array plate. Real-time PCR assay and subsequent data collection were performed on a
Rotor-Gene 6000 robocycler (Corbett Life Science, Sydney, Australia)66. Transcript relative
enrichments were calculated following manufacturer’s instructions (Qiagen). Experiments
were performed in triplicate.
Western blotting analyses were performed as previously described66 using anti-CD146
(Mcam - #ab75769, lot #GR208953-3 from Abcam), anti-NOR-1 (Nr4a3 - #sc-133840, lot
#G1911 from Santa Cruz Biotechnology), anti-Caspase-1 (#66441A from Pharmingen
International) and anti-αTubulin (#T9026, lot #057K4842 from Sigma) antibodies. Goat anti-
rabbit IgG, horseradish peroxidase conjugate (#G21234, lot #35837A) and goat anti-mouse
IgG, horseradish peroxidase conjugate (#G21040, lot #83E1-1) were purchased from
Molecular Probes. Elaboration of pictures and densitometric analysis were performed using
ImageJ software (ImageJ 1.43u; National Institutes of Health, Bethesda, MD, USA). The
data obtained by the densitometric analysis were normalized to alpha-tubulin protein levels
and expressed as fold changes.

In vivo evaluation of PRSE efficacy

Four weeks old female FVB/N mice were obtained from Animal Care Facilities of the
University of Camerino. Ten mice (fed with 15% strawberry extract-enriched food) were
housed one mouse/cage, while ten mice (fed with regular food) were housed in two cages
with free access to water and food, and kept at temperature of 19–22 °C and relative
humidity of 45–65% under 12 h/12 h light/dark cycle. Upon the 8th week of age, all the mice
were orthotopically injected with 2 × 105 A17 cells. Tumor monitoring was performed twice a
week by palpation. After 5 weeks, the tumors were analyzed, after resection, evaluating both
weight and volume (two perpendicular diameters - a and b - on each tumor were measured
using a caliper and the volumes were calculated by the V = π/6[(a + b)/2]3 formula). All the
experimental procedures carried out in this study were in compliance with the UK Animals
(Scientific Procedures) Act 1986 and associated guidelines, EU Directive 2010/63/EU, and
were approved by the Ethic Committee on Animal Use of the University of Camerino
(protocol number 14/2012).

Additional Information
How to cite this article: Amatori, S. et al. Polyphenol-rich strawberry extract (PRSE)
shows in vitro and in vivo biological activity against invasive breast cancer cells. Sci. Rep. 6,
30917; doi: 10.1038/srep30917 (2016).
Go to:

Acknowledgments
This work was supported by: Associazione a Sostegno degli Studi Oncologici (ASSO), Lega
Italiana per la Lotta contro i Tumori – LILT (to Mirco Fanelli). Stefano Amatori and Francesca
Giampieri were supported by a Fondazione Umberto Veronesi fellowship.

Footnotes
Author Contributions M.F. and M.B.: experimental design, discussion of the data,
manuscript writing and editing. S.A. and L.M.: execution of the experimental plan, statistical
elaboration of the data, manuscript writing. B.M.: strawberry growth, discussion of the data,
manuscript editing. F.G., J.M.A.-S., M.G., T.Y.F.-H. and S.Af.: strawberry analyses,
wounding healing assay, manuscript editing. A.E.P.: execution of cell cycle analysis,
discussion of the data, manuscript editing. G.P.: execution of western blotting, manuscript
editing. A.A.: cell culture, in vivo experiments, manuscript editing.

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Front Plant Sci. 2016; 7: 1020.

XX. Low Temperature and Short-Term High-CO2 Treatment in

Postharvest Storage of Table Grapes at Two Maturity
Stages: Effects on Transcriptome Profiling
Raquel Rosales, Irene Romero, Carlos Fernandez-Caballero, M. Isabel Escribano, Carmen
Merodio, and M. Teresa Sanchez-Ballesta*

Abstract

Introduction
The maintenance and improvement of fruit quality during postharvest life is becoming
increasingly important in response to a market where consumers demand products of high
quality throughout the year. Low temperature storage is one of the most used methods to
prolong postharvest quality and extend the shelf life of a broad range of horticultural
commodities. Nevertheless, their use is sometimes limited depending on susceptibility to
chilling injury and/or fungal decay. In addition to this, it is important to mention that fruit
quality during cold storage is affected by the maturity stage of the fruit at harvest (Shin et
al., 2008). Grapes (Vitis vinifera L.), which are a non-climacteric fruit with a relatively low
rate of physiological activity, are not susceptible to injury at low (not freezing) temperature.
For this reason, storage around 0°C is recommended for mature table grapes during
postharvest. However, the storage life of grapes at low temperature is limited by their high
susceptibility to fungal decay and sensitivity to serious water loss after harvest, which may
result in stem drying and browning, and even shriveling of berries. The use of atmospheres
modified in O2 and CO2 concentrations may extend the storage life of different fruit and
vegetables at low temperature by reducing respiration, maintaining firmness and controlling
decay. In this sense, we have shown that a 3-day treatment with high CO2 levels at 0°C is
efficient in maintaining the quality of table grapes and controlling total decay (Romero et
al., 2006; Sanchez-Ballesta et al., 2006). At transcriptional level, this gaseous treatment
minimized or modified, the activation of cold-response mechanisms observed in non-treated
grapes as a response to temperature shifts at 0°C (Sanchez-Ballesta et al., 2006;
Fernandez-Caballero et al., 2012; Rosales et al., 2013).
The effect of high CO2 levels on the postharvest quality of small fruit and berries, such as
grapes, depends on cultivar, maturity and storage length (Terry et al., 2009). Thus, Nunes
et al. (2002) reported that three-quarters of colored strawberries responded better to
controlled atmosphere storage at low temperature, maintaining greater firmness, better
color, and reduced decay development than fully red fruit. In the case of table grapes, a
delay in the harvest date did not affect to the effectiveness of the 3-day gaseous treatment.
However, it was decisive in the increase of antioxidant activity by inducing anthocyanin
accumulation in non-treated fruit stored at 0°C but not in CO2-treated ones (Romero et
al., 2009). However, storage of early-harvested table grapes at 0°C in air causes a
significant decrease in bound water levels and greater soluble-water K+accumulation in
comparison to CO2-treated, irrespective of the harvest year. Thus, some of the beneficial
effects of the high CO2 treatment could be explained by restricting water mobility, and its
influence on ion and volume homeostasis (Blanch et al., 2014).
With the development of microarray and next generation sequencing technology, global
transcriptome analyses have been used to investigate the molecular regulatory networks
underlying the responses of fruit during postharvest storage. Nevertheless, at the molecular
level, very little is known about fruit response to short-term high-CO2treatment applied during
low temperature postharvest storage. Most previous works have been confined to the level
of individual genes or small groups of genes. Thus, high CO2effects were analyzed using
microarrays in strawberries (Ponce-Valadez et al., 2009) and grape berries (Becatti et
al., 2010). In the case of strawberries, the heterologous cDNA microarray TOM1 was used
to compare gene expression differences between two cultivars treated with 20 kPa CO 2 for
48 h. The cultivar Jewel accumulates acetaldehyde and ethanol in response to elevated
CO2, whereas Cavendish does not accumulate these compounds under the same storage
conditions. Despite the differences between both cultivars in terms of the number of genes
differentially expressed, the distribution of their putative functions was very similar except
for cDNAs with homology to genes encoding transcription factors and to genes involved in
protein synthesis. Likewise, by using microarray, Becatti et al. (2010) reported that in
detached wine grapes (cv. Trebbian, white skinned) a treatment with 30 kPa of CO2 for 3
days at 20°C was effective in altering the general metabolism, being functional categories
related to protein and hormone metabolism, transport and stress highly represented in both
skin and pulp tissues. Moreover, these authors also observed that the skin cells appeared
to undergo more pronounced changes in transcriptome profiling in response to high CO2than
the pulp. Thus, fermentation, CHO metabolism, and redox regulation functional categories
were represented only in the skin. To improve our understanding of the molecular
mechanisms involved in the beneficial effect of short-term high-CO2treatment on the quality
of table grapes stored at low temperature, further transcriptomic studies are needed.
In this work, changes in the transcriptome of Cardinal table grape skin at different stages of
maturity arising from exposure to high levels of CO 2 and 0°C during 3 days were examined
by using a custom made Affymetrix GrapeGen GeneChip™ (Lijavetzky et al., 2012). The
results obtained improve our understanding of how table grapes respond to changes in
atmospheric composition during the postharvest storage period which maintains their
quality.

Materials and methods

Plant material
Table grapes (Vitis vinifera L. cv. Cardinal) were harvested from a commercial orchard in
Sevilla (Spain) twice over 3 weeks. The first harvest began on the 13th June 2005 (early
harvested, maturity index (MI) of 12.45 ± 0.01) and the second on the 5th July 2005 at
commercial maturity (late harvested, MI of 41.08 ± 0.30). The field-packaged bunches were
transported to the laboratory (time zero, t0) and those free from physical and pathological
defects were randomly divided into two lots and stored up to 27 days at 0 ± 0.5°C and 95%
relative humidity (RH) in two sealed neoprene containers of 1 m3capacity. One lot of 6
replicate bunches was kept under normal atmosphere (non-treated fruit) and the other one,
with same size, was kept under a gas mixture of 20 kPa CO2 + 20 kPa O2 + 60 kPa N2 (CO2-
treated fruit). After 3 days, CO2-treated grapes were transferred to air under the same
conditions as the non-treated fruit until the end of the storage period. At time 0 and after 3
days of storage under air or CO2 conditions, the skin of three biological replicates (each
replicate consisting of 2 bunches) were collected, frozen in liquid nitrogen, ground to a fine
powder and stored at −80°C until analysis. A scheme summarizing the experimental system
is depicted in Supplementary Figure S1.

Quality assessments
Berry quality assessment comprised soluble solids contents (SSC), titratable acidity (TA),
pH and total decay. SSC was determined using a digital refractometer Atago PR-101 (Atago
Co. Ltd., Japan) at 20°C and expressed in °Brix. TA was determined by titration with 0.1N
NaOH up to pH 8.1 and results were expressed in % tartaric acid. The pH of the juice was
measured in a pH meter with glass electrode.
Total decay was assessed on the basis of the total decay after removing and weighing the
healthy berries. The weight of the decayed berries was calculated by subtracting healthy
berries from the total cluster weight. Thus, total decay was expressed as the percentage of
decayed berries with respect to the original cluster weight.

Measurement of ion leakage

Membrane permeability was expressed by the relative ion leakage rate according to
Lafuente et al. (1991) with slight modifications. Ten skin dishes (8 mm) were incubated in
10 mL of 0.3 M mannitol in Falcon 50-mL Conical Centrifuge Tubes. The tubes were shaken
at 120 cycles per min and the conductivity of the solutions was measured after 1.5 h with a
Crison 522 model conductivity meter. Tubes containing the mannitol solution and the tissue
were heated to boiling for 15 min to permit complete leakage from the membranes. After
cooling to room temperature with shaking, the total conductivity was measured. Ratios of
ion leakage are expressed as percentage of the total conductivity per hour.

RNA isolation and genechip® hybridization

In 2009, total RNA was extracted from skin samples according to the procedures described
by Zeng and Yang (2002). RNA samples were treated with DNase I recombinant-RNase
free (Roche) for removing possible genomic DNA contamination. Thereafter, final RNA
purification was performed using RNeasy Mini Kit (Qiagen) following the manufacturer's
instructions. Once RNA was purified, samples were analyzed at the Genomics Unit of the
Spanish National Centre for Biotechnology (CNB-CSIC, Madrid). RNA integrity analyses
were performed with an Agilent's Bioanalyzer 2100. A custom Affymetrix GrapeGen
GeneChip® (Lijavetzky et al., 2012) containing 23,096 probe sets corresponding to 18,711
non-redundant transcripts was used. Probe synthesis, microarrays hybridization, washing,
staining and scanning with the GeneChip™ Scanner 3000 were performed according to the
Affymetrix GeneChip® Expression.

Microarray data processing

Microarray data analyzed in this study have been submitted to the Gene Expression
Omnibus database (www.ncbi.nlm.nih.gov/geo/) under the number GSE83275. Three
biological replicates per experiment were processed to evaluate intra-specific variability.
Signal values from all the microarray hybridizations were normalized together by applying
the RMA (Robust Multiarray Average) algorithm using RMAExpress (Bolstad et al., 2003).
Average of expression values for redundant probe sets was performed using GEPAS (Gene
Expression Pattern Analysis Suite) software v4.0 (Herrero et al., 2003). Multivariate analysis
as Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) (ANOVA
test, P < 0.01 after Bonferroni adjusted correction) over the normalized dataset was
performed by using the MeV (MultiExperiment Viewer) TM4 Microarray Software Suite
(Saeed et al., 2003).

Microarray data analysis

SAM (Significance Analysis Microarray) algorithm implemented in TIGR MEV 4.5.1 (Saeed
et al., 2003) was used to identify significantly altered gene expression in response to low
temperature storage in air or high CO2 levels, and to generate false discovery rate (FDR)
values for the analysis. SAM analyses were computed using two-class unpaired comparison
on 1000 permutations. A FDR lower than 0.001 was applied in the analysis and only probe
sets showing at least 1.5-fold change between conditions were considered as significant.
Area-proportional Venn diagrams were generated in order to compare and visualize
selected lists of genes by using BioVenn software (Hulsen et al., 2008). Probe set annotation
was updated according to the 12X grape reference genome, V2 gene prediction
(http://genomes.cribi.unipd.it/grape/; Vitulo et al., 2014).
The Database for Annotation, Visualization and Integrated Discovery (David 6.7; Huang et
al. (2009), http://david.abcc.ncifcrf.gov) was used to classify specifically the genes with an
altered expression either in air or high CO2 levels at 0°C, not taking into account those
common to both cases, into functional groups based on Gene Ontology. Medium stringency
was applied for the analyses. DAVID analysis identifies significantly enriched biological
themes by examining for enrichment in over 40 different publicly available annotation
categories, analyzing up- and down-regulated sets separately. A functional annotation chart
(FACH) was generated by selecting the GOTERM_BP_FAT annotation category in DAVID,
which allows enrichment analysis to highlight the most relevant Gene Ontology (GO) terms
associated with a given gene list. The significance of gene enrichment in annotation terms
was measured using the Expression Analysis Systematic Explorer (EASE) score, which is
a modified Fisher Exact P-value, with application of the Benjamini-Hochberg method to
correct for multiple comparisons, as applied in DAVID, with the threshold set at 0.05 (Huang
et al., 2009). Functional Annotation Clustering (FAC) was then performed using the DAVID
system. This higher level analysis combines single categories with a significant overlap in
gene content and then assigns an enrichment score (ES; defined as the −log 10 of the
geometric mean of the unadjusted p-values for each single term in the cluster) to each
cluster. In this instance, ES greater than 1.3 were considered significant. Fold-enrichment
scores (FE) were also used to indicated the magnitude of enrichment for individual terms,
and FE greater than 1.4 are suggestive of an informative change (Huang et al., 2009).

Quantitative real-time RT-PCR (RT-qPCR)

Total RNA extraction was extracted as described above and treated with DNAse I
recombinant-RNase free (Roche) to avoid DNA contamination. Then, 1 μg of RNA was used
to synthesize cDNA by using the iScriptTM Reverse Transcription Supermix (Bio-Rad).
Transcript levels were determined by RT-qPCR using a iCycler iQ thermal cycler (Bio-Rad)
and SYBR Green dye (Bio-Rad). Gene-specific primers pairs for RT-qPCR (Table 2) were
designed with Primer 3 software (Koressaar and Remm, 2007) using as templates the gene
sequence from the V. vinifera 12X genomic sequence assembly Adam-Blondon et al.,
(2011; available at http://bioinfogp.cnb.csic.es/tools/GrapeGendb/). Each gene was
evaluated at least in two independent runs. In order to calculate the efficiency of the reaction
(optimal range 90–110%) and to establish the most suitable template concentration, cDNAs
synthesized from serial dilutions (from 40 to 2.5 ng) of total RNA were amplified. Standard
curves and linear equations were determined by plotting cycle threshold (Ct) values (y-axis)
against logs of total RNA (x-axis). The efficiency of each individual run was calculated based
on the raw fluorescence data (ΔRn) exported as output file and subsequently imported into
the LinReg PCR program. The transcription levels were calculated relative to the calibrator
sample (time 0) after normalization to the reference gene actin1 from V. vinifera. Correlation
coefficients (r2) between the log2-transformed expression values as measured by microarray
and RT-qPCR were calculated. The specificity of products was validated by dissociation
curve analysis and by agarose gel; and its sequences confirmed at the Genomic Department
of the CIB-CSIC.

Statistical analyses
Data were subjected to ANOVA (one-way analysis of variance) using the Fisher's Least
Significant Difference (LSD) test to determinate the level of significance at P ≤ 0.05
(Statgraphics Centurion XVII, STSC, Rockville, MD).

Results

Effect of CO2 treatment and storage at 0°C on the quality of table grapes harvested at
different stages of maturity
Irrespective of the maturity stage, treatment with high CO2 levels was effective in controlling
total decay after 13 days at 0°C, the point at which the first symptoms of fungus infection
appears (Table (Table1).1). However, as seen in Table Table1,1, the effect of 3-day high
CO2 levels on the quality parameters analyzed depended on the maturity stage. Thus, in the
case of early-harvested grapes, the SSC content decreased in CO2-treated grapes, whereas
the values of TA and pH did not change. However, in the case of non-treated grapes, values
of SSC and TA decreased after 3 days at 0°C, with the pH values increasing. In late-
harvested grapes, no changes were observed in SSC and TA values in any of the samples
analyzed. By contrast, the pH values significantly decreased in both treated and non-treated
samples, with the treated samples maintaining higher values.

Table 1
Soluble solids content (SSC), titratable acidity (TA), pH, ion leakage total decay of
early- and late-harvested table grapes cv. Cardinal treated with 20 kPa CO2 and stored
during 13 days at 0°C.
 Early-harvested Late-harvested

At 13 days 3 days CO2 + At 13 days 3 days CO2 +

harvest Air 0°C 10 days 0°C harvest Air 0°C 10 days 0°C

Total 0a 10.16 ± 4.34 ± 1.55b 0a 8.33 ± 4.41 ± 1.82b

decay 2.54c 0.9c

At 3 days 3 days At 3 days 3 days

harvest Air 0°C CO2 0°C harvest Air 0°C CO2 0°C

Brix 11.90 ± 11.03 ± 11.15 ± 0.18a 15.73 ± 16.28 ± 14.28 ± 0.74b

0b 0.18a 0.34b 0.25b

TA (% 0.96 ± 0b 0.86 ± 0.92 ± 0.03ab 0.38 ± 0.37 ± 0.38 ± 0.01a

tartaric 0.05a 0.01a 0.02a
acid)

pH 3.23 ± 0a 3.32 ± 3.26 ± 0.04a 4.15 ± 3.78 ± 3.90 ± 0.02b

0.03b 0.02c 0.08a

Ion 52.77 ± 62.31 ± 54.09 ± 54.72 ± 47.88 ± 48.00 ± 4.86a

leakage 6.28a 2.03b 1.06ab 0.17b 1.05a
(%)

Values are the mean of three replicate samples ±SE. Different letters within each row and
harvest stage indicate that means are statistically different according to Fisher's protected
LSD test (P ≤ 0.05).
Another difference between both maturity stages is the fact that late-harvested grapes
showed a decrease in ion leakage in treated and non-treated samples, whereas a sharp
increase was observed in the skin of non-treated early-harvested grapes in response to low
temperature storage, which was avoided by the application of CO2 (Table (Table11).

Main variation components in the grape skin transcriptome at two stages of maturity in
response to 3-day high CO2 treatment and low temperature
In order to obtain an overview of how low temperature and high CO2 levels affect the
transcriptome of table grape skin in the first stage of storage and to analyze whether the
maturity stage affects these changes we have used the GrapeGen GeneChip®. As a first
approach to analyze the complexity of the gene expression dataset and to cluster samples
according to their global gene expression profile, we performed a PCA and HCA (Figure
(Figure1)1) over the expression data of the eighteen analyzed samples, corresponding to
three biological replicates at time zero, plus non-treated and CO2-treated samples at two
maturity stages. Given that in all conditions, the transcriptional profiles of the three separate
RNA replicate samples were tightly clustered, the experiment was considered highly reliable
for further analysis. Principal component 1(PC1) explained 53% of the expression variation
and separated samples in relation to the maturity stage (early vs. late harvested), with each
CO2-treated sample close to its respective t0. However, non-treated fruit at both maturity
stages clustered far from their respective samples at t0. PC2 accounted for 16% of the
variance in the data set. Interestingly, samples from non-treated fruit harvested at early
stage grouped near to samples from t0 and CO2-treated late harvested grapes, far from their
respective t0 samples (variation on component 1), while late-harvested non-treated fruit
clustered in the lower part of component 2 far from t0 and CO2-treated late harvested
samples (Figure (Figure1A).1A). HCA confirmed results obtained by PCA, where t0 and 3-
day CO2 treated samples at each maturity stage were clustered together but in independent
branches, with samples from non-treated fruit at an early stage close to t0 and also to treated
samples from late harvested fruit. Curiously, although 3-day non-treated samples at the late
stage were in the same branch as their respective time zero, they were clustered into an
independent group. These results indicated that 3 days of low temperature exposure led to
an intense change in the grape skin transcriptome regardless of the fruit maturity stage but
with different changes in each one, whereas slight differences were observed in CO2 treated
samples in comparison with fruit at time zero (Figure (Figure1B1B).

Figure 1
(A) Principal Component Analysis (PCA) and (B) Hierarchical Cluster Analysis (HCA) of
transcriptome data from the skin of 3-day CO2-treated and non-treated grapes at early and
late harvested stages. Colors in PCA for each condition are consistent with those in HCA.
Three independent biological replicates of each condition were used and the analysis was
carried out based on expression data previously RMA normalized between samples and
after the average of expression values among redundant probe sets.

Transcriptional bases for the response of grapes to low temperature and high levels of
CO2 at two maturity stages
Venn diagrams summarize the number of overlapping differentially expressed genes (SAM
analysis, FDR < 0.001, 1.5-fold change) in the skin of CO2-treated and non-treated early- or
late-harvested table grapes with respect to each sample at t0 (Figures 2A,B). Major changes
in the number of differentially expressed genes occurred in non-treated samples at early
maturity stage where a total of 4990 non-redundant accumulated transcripts were identified,
representing 26.6% of the total analyzed. Among the transcripts, 61% were down-regulated
and 39% up-regulated by 3 days of storage at low temperature. Exposure of late-harvested
table grapes at 0°C for 3 days also prompted major changes in gene expression (3257), with
2131 non-redundant transcripts (66%) up-regulated and 1126 (34%) down-regulated. The
number of genes with a modified expression in response to the 3-day high CO2 treatment in
comparison to t0 fruit was less remarkable than non-treated grapes at both maturity stages,
although major changes also occurred in the early maturity stage (830) compared to the
late-harvested one (632). In both cases, the number of up-regulated genes was higher than
down-regulated ones.

Figure 2
Venn diagrams showing differentially expressed genes (SAM analysis, FDR < 0.001)
in the skin of CO2-treated and non-treated grapes at the early and late maturity stage.
Expression levels for genes up-regulated (bold) and down-regulated (italics) in table grapes
were compared to those of fruit at time zero at early (A) and late (B) maturity stages.
Numbers in brackets are the sum of all induced or repressed genes in the early or late stage.
The sizes of the circles are consistent with the total number of differentially expressed genes
for each condition.
In order to validate the microarray data, expression levels of 12 transcripts were analyzed
by RT-qPCR using three biological replicates of t0, non-treated and CO2-treated samples at
the two maturity stages (Table (Table2).2). Linear regression analyses displayed
reliable r2 correlation coefficients between 0.73 and 0.99, confirming the validity of the
microarray results.

Table 2
Selected genes and primers used for quantitative RT-PCR and comparison between
GrapeGen GeneChip® microarray and RT-qPCR gene expression data.

GrapegenDB Gene Probe Set Fw/R Primer sequence 5′–3′ r2

12Xv2 unique ID Annotation v
VIT_208s0040g0 CAM7 Calmodulin 7 F CCAAGGAGCTAGGGACA 0.
0470 GTG 9
GrapegenDB Gene Probe Set Fw/R Primer sequence 5′–3′ r2
12Xv2 unique ID Annotation v
R CTCGGAATGCTTCTTTCA
GC
VIT_204s0079g0 GSTF1 Glutathione S- F TCCTACCTCGAATGGGT 0.
0690 1 transferase F12 GAG 9
R TTCGACAGCCTCTGCTC
ATA
VIT_203s0038g0 DNAJ Chaperon F GCAGCCTACTCCACCTT 1
2110 protein DNAJ 11 GTC
R ACCAGCACTGGTCAGTC
TCC
VIT_206s0004g0 CRF4 ap2 erf domain- F CCTCCTCCATTCCAACAA 0.
8190 containing GA 9
transcription
factor
R TCCCTCCACTCACCATTA
GG
VIT_209s0018g0 WRKY Putative WRKY F GAAGACGGGGAAGAAAA 1
0240 40 Transcription AGG
Factor 40
R CTTGGGTGGGTCAGTCA
GAT
VIT_202s0012g0 NAC07 NAC domain- F CCATGGCTTATTGCAGG 1
1040 1 containing ACT
protein 71
R CAAATTCAACTTCCCCAG
GA
VIT_203s0088g0 PRP1 Pathogenesis- F GTGGTTCGCACATGCAA 0.
0710 related protein 1 CT 8
R CCTTTGTCAACTAAACGC
ACA
VIT_200s0173g0 RPS7 Ribosomal F CGATCCGTGAAAAAGAT 1
0170 protein S7 TCAA
R ATGAGTCGATCCGCCTA
CAC
VIT_216s0013g0 Kinase family F GTCACTTGATTTCTGTCC 0.
1920 protein CAAT 8
R CCATTCATTATCCACATC
CTCA
VIT_211s0016g0 MBF1c Multiprotein- F CTTGCGAAGATGGAGAA 0.
4080 bridging factor GGT 8
1c-like
 GrapegenDB Gene Probe Set Fw/R Primer sequence 5′–3′ r2
12Xv2 unique ID Annotation v
R CGAGCGACGGACAAGAC
AC
VIT_202s0025g0 ACS6 1- F GTTCCCATGGGTTTGCT 0.
0360 aminocycloprop TTA 9
ane-1-
carboxylate
synthase-like
R GTTGCATCATCCTCCAT
GTG
VIT_213s0067g0 ERD15 Dehydration- F GGAGGAGGAGAAGGAG 0.
2130 induced protein CATC 7
(ERD15)
R GAGCCTTCTCGAAGTGC
CTA
Open in a separate window
Multiple linear regression analysis (r2) was performed for each reported gene including
samples from all comparisons.

Functional analysis of the differential gene expression in non-treated grapes stored for 3
days at 0°C at both maturity stages
So as to understand the biological significance of the molecular changes underlying the
specific effect of low temperature storage in non-treated grapes depending on the maturity
stage, we performed a functional analysis on up- and down-regulated genes using DAVID.
FACH provides data on the over-representation of GO category terms. At the early maturity
stage, up-regulated transcriptional responses to low temperature storage overlapped
broadly with response to abiotic (cold, heat and water deprivation) and biotic (defense
response to bacterium) stimulus (P < 0.05 and fold-enrichment score (FE) between 2.17 and
1.50) (Figure (Figure3A,3A, Supplementary Table S1). Among the genes belonging to
“response to heat” GO term, those coding for HSPs (heat shock proteins) were strongly
represented. The genes implicated in cold tolerance such as HOS15, LOS1, LOS4, and
temperature-induced lipocalin (TIL) related to the “response to cold” process were induced
during the storage of non-treated early-harvested grapes at 0°C. In the case of up-regulated
genes belonging to “response to water deprivation” process, these included genes coding
for aquaporins, cysteine proteinases and dehydration-responsive protein. However, the
most enriched up-regulated genes affected by low temperature were involved in the
“gluconeogenesis” and “chitin catabolic” process (P < 0.05 and FE of 6.62 and 4.54,
respectively). Thus, a phosphoenolpyruvate carboxykinase, a glucose-6-phosphate
isomerase, two glyceraldehyde-3-phosphate dehydrogenases and four chitinases were
induced (Figure (Figure3A,3A, Supplementary Table S1). Genes involved in
“photosynthesis, light harvesting,” “regulation of GTPase activity,” and “NADP metabolic
process” were also differentially expressed in response to low temperature storage (Figure
(Figure3A,3A, Supplementary Table S1). These included genes encoding chlorophyll
binding proteins, GTPases (ARF, RHO, and RAB) and fructose-bisphosphate aldolases.
Another important over-represented GO terms were “lipid transport” (P < 0.05; FE = 2.65),
including eight genes encoding lipid transfer proteins (LTPs), and “translational initiation”
(P < 0.05; FE = 2.30), with ten genes coding for eukaryotic translation initiation factors (eIFs)
(Figure (Figure3A,3A, Supplementary Table S1). Finally, within the “lignin metabolic
process,” transcripts coding for laccases were over-represented (Figure (Figure3A,3A,
Supplementary Table S1).

Open in a separate window

Figure 3
DAVID Functional Annotation Chart (FACH) of normalized and annotated genes up-
and down-regulated (fold change >1.5) in non-treated early- and late-harvested
grapes stored for 3 days at 0°C. (A) FACH of genes up-regulated in early-harvested
grapes. (B) FACH of genes up-regulated in late-harvested grapes. (C) FACH of genes
down-regulated in early-harvested grapes. (D) FACH of genes down-regulated in late-
harvested grapes. Significance is determined by corresponding enrichment scores.
In late-harvested grapes, the transcriptional responses to low temperature were related to
terms associated with stress such as “response to high light intensity” and “cellular response
to phosphate starvation” (P < 0.05; FE = 2.79 and 2.62, respectively) (Figure (Figure3B,3B,
Supplementary Table S2). Among the genes over-represented in these biological
processes, we found genes coding for HSPs, which are different from those activated in
early-harvested grapes, and also SPX (SYG1/Pho81/XPR1) domain proteins. Other
transcripts up-regulated in late-harvested grapes stored at 0°C encoded calreticulin,
chaperone DNAJ and peptidyl-prolyl isomerases, were associated to “protein folding” (P<
0.05; FE = 1.70) (Figure (Figure3B,3B, Supplementary Table S2). Cold storage also
affected the expression of eight genes coding for Rab GTPases related to the “small
GTPases meditated signal transduction” process (FE = 2.08).
The terms over-represented in down-regulated genes in response to low temperature
depend on the maturity stage (Figures 3C,D, Supplementary Tables S1, S2). The most
enriched down-regulated genes affected by low temperature in early-harvested grapes were
involved in “tubulin complex assembly,” “histone deacetylation,” and “GPI anchor
biosynthetic process” (P < 0.05; FE = 4.50, 3.00, and 2.62, respectively). Concerning energy
metabolism, four aldose-1-epimerase related to “galactose metabolic process” (P<
0.05; FE = 2.42) were also down-regulated. Finally, the term “ATP synthesis coupled proton
transport” (P < 0.05; FE = 2.15), including seven vacuolar (V-type) proton ATPases was
over-represented in early-harvested grapes stored at 0°C (Figure (Figure3C,3C,
Supplementary Table S1). By contrast, the analysis of the genes down-regulated by low
temperature storage in late-harvested grapes mainly revealed terms which were related to
response to abiotic and biotic stimulus (Figure (Figure3D,3D, Supplementary Table S2).
Low temperature storage seems to affect protein biosynthesis due to the fact that genes
associated with the most enriched GO term “translational elongation” (P < 0.05; FE = 3.55)
were down-regulated in the skin of late-harvested grapes, including genes coding for two
ribosomal proteins and two elongation factors (Figure (Figure3D,3D, Supplementary
Table S2).
The FAC tool, FAC, clusters functionally related annotations into groups and ranks them
according to importance by giving them enrichment score (ES). This tool showed four
clusters of up-regulated genes and two clusters of down-regulated genes in non-treated
early-harvested grapes, with significant ES ≥ 1.5 and P < 0.05 (Supplementary Table S3).
Clusters of up-regulated genes were related to “photosynthesis,” “gluconeogenesis,”
“defense response to bacterium,” and “NADP metabolic process” GO terms. Down-
regulated genes were involved in clusters related to “ATP synthesis coupled proton
transport” and “GPI anchor biosynthetic process.” In late-harvested grapes, the FAC of up-
regulated genes were clustered into one group related to “cellular response to phosphate
starvation.” There were two FAC down-regulated clusters in non-treated late-harvested
grapes stored at 0°C. Genes were involved in “response to red light” (cluster 1) and
“response to abscisic acid stimulus” (cluster 2) (Supplementary Table S3).

Functional analysis of the differential gene expression in CO2-treated grapes stored for 3
days at 0°C at both maturity stages
The application of high CO2 levels for 3 days at 0°C in early-harvested grapes significantly
induced the expression of genes encoding mainly transcription factors associated with
different GO terms such as “response to chitin,” “ethylene mediated signaling pathway,”
“response to bacterium,” and “regulation of transcription, DNA-dependent” as well as
response to salicylic, acid, jasmonic and abscisic acid stimulus (Figure (Figure4A,4A,
Supplementary Table S4). Thus, we found twenty eight transcription factor genes (Table
(Table3),3), including seven genes coding for ERF (Ethylene Responsive Factors) and five
WRKYs. The gaseous treatment at 0°C also induced the expression of genes associated
with “protein amino acid phosphorylation” GO term (P < 0.05; FE = 1.64), including fourteen
genes encoding kinases mainly belonging to the receptor-like kinases (RLKs) family and
cyclin-dependent kinase (Supplementary Table S4). However, in late-harvested grapes,
only ten genes up-regulated by high CO2 levels were significantly associated with “defense
response to bacterium,” “photosynthesis,” and “generation of precursor metabolites and
energy” GO terms (Figure (Figure4B,4B, Supplementary Table S5).

Figure 4
DAVID Functional Annotation Chart (FACH) analysis of normalized and annotated
genes up- and down-regulated (fold change >1.5) in 3-day CO2-treated early- and late-
harvested grapes. (A) FACH of genes up-regulated and down-regulated (>1.5-fold) in
early-harvested grapes. (B) FACH of genes up-regulated in late-harvested grapes.
Significance is determined by corresponding enrichment scores.

Table 3
Transcription factors up- and down-regulated in the skin of 3-day CO2-treated grapes
at early stage.

GrapegenDB 12Xv2 Probe set annotation Fold

unique ID change
UP-REGULATED
VIT_207s0141g00270 Auxin-induced protein 22D 1.81
VIT_207s0005g01450 bZIP transcription factor 53 3.91
VIT_212s0055g00420 bZIP transcription factor 1.48
VIT_206s0004g08190 Ethylene-responsive transcription factor 2.06
CRF1
VIT_201s0011g03070 AP2/ERF and B3 domain-containing 2.35
transcription factor RAV1-like
VIT_202s0234g00130 Ethylene responsive element binding factor 2.93
1A
GrapegenDB 12Xv2 Probe set annotation Fold
unique ID change
VIT_207s0005g03230 Ethylene responsive element binding factor 2.19
1B
VIT_216s0013g01110 Ethylene-responsive transcription factor 4 4.36
VIT_216s0013g01120 Ethylene responsive element binding factor 5 1.60
VIT_200s0662g00040 Ethylene-responsive transcription factor 2.90
ERF060
VIT_216s0013g00900 Ethylene-responsive transcription factor 1.90
ERF105
VIT_207s0031g00220 Floral homeotic protein APETALA 2 1.51
VIT_208s0007g07550 GATA zinc finger 2.01
VIT_210s0003g01770 Heat stress transcription factor A-4a-like 1.63
VIT_213s0156g00260 Homeobox-leucine zipper protein HAT14-like 2.87
VIT_211s0016g04080 Multiprotein-bridging factor 1c 2.18
VIT_205s0049g01020 Myb transcription factor 2.25
VIT_203s0180g00210 Myb transcription factor 44 2.15
VIT_218s0001g11170 Myb transcription factor 73 2.85
VIT_200s0299g00060 Myb transcription factor 93 1.91
VIT_204s0008g05760 WRKY transcription factor 18 1.74
VIT_215s0046g02190 WRKY transcription factor 22 2.08
VIT_209s0018g00240 WRKY transcription factor 40 1.53
VIT_208s0040g03070 WRKY transcription factor 44 1.81
VIT_208s0058g01390 WRKY transcription factor 70 1.94
VIT_217s0000g01920 NF-X1-type zinc finger protein NFXL1-like 1.64
VIT_206s0004g04180 zinc finger protein ZAT11 2.67
VIT_218s0001g09230 Zinc-finger protein 1 ZAT10-like 1.86
DOWN-REGULATED
VIT_213s0019g03550 Floral homeotic protein APETALA 2 −1.63
VIT_204s0008g06000 Ethylene-responsive transcription factor −1.95
ERF003
VIT_215s0048g02870 Homeobox-leucine zipper protein HB-7 −2.57
VIT_206s0004g02800 Homeodomain-leucine zipper protein −1.72
Revoluta (REV)
VIT_212s0142g00360 MAD-box transcripion factor −1.77
Open in a separate window
The term “regulation of transcription, DNA-dependent” was also over-represented in genes
down-regulated by high CO2 levels in early-harvested grapes (Figure (Figure4A,4A,
Supplementary Table S4). This term included five transcription factors, belonging to the
homeobox-leucine zipper, MAD-box, ERF, and AP2/EREBP families (Table (Table3).3).
However, in the case of late-harvested grapes, no significant GO terms were over-
represented in down-regulated genes by the gaseous treatment.
The FAC of genes up-regulated by high CO2 levels in early- and late-harvested grapes,
revealed three clusters including GO categories of “response to chitin” and “regulation of
transcription” (Cluster 1, ES = 5.79), “ethylene mediated signaling pathway” (Cluster 2; ES =
3.98) and “protein phosphorylation” (Cluster 3; ES = 1.81) (Supplementary Table S6). In
late-harvested grapes, up-regulated ones were clustered into a group related to “defense
response to bacterium” (ES = 2.58) (Supplementary Table S6).

Discussion
To date, no studies have directly compared how low temperature and a short-term high-
CO2 treatment affect the gene expression of table grapes harvested at two different maturity
stages. In table grapes, minimum maturity requirements vary depending on cultivar, growing
area and market. According to Codex Standards (codexstan255-2007) and the Economic
Commission for Europe Standards (UNECE, 2010) for table grape maturity, the berries must
be sufficiently developed and display satisfactory ripeness. In order to satisfy this
requirement, the fruit must reach at least 16°Brix. Likewise, fruits with lower refractometric
indices are accepted provided the sugar/acid ratio is at least equal to: (a) 20:1, if the Brix
level is greater than or equal to 12.5° and less than 14°Brix, (b) 18:1, if the Brix level is
greater than or equal to 14° and less than 16°Brix. In this work, maturity stages were
selected to cover the effect of high CO2 levels on immature table grapes as well as on those
meeting the level of maturity required.
It has been reported that in general, SSC, TA, and pH values remained quite constant in
different varieties of grapes stored at 0°C under several conditions of controlled atmosphere
(Artés-Hernández et al., 2006). However, our results indicated that the 3-day treatment with
high CO2 levels affected the quality parameters analyzed depending on the maturity stage,
although in both cases was effective controlling total decay. One of the common features
accompanying chilling and senescence is increased membrane permeability, expressed as
increasing leakage of ions which is used as an indicator of membrane damage. In Flame
Seedless grapes, postharvest dip treatment with spermine (Champa et al., 2014) and
preharvest salicylic acid application (Champa et al., 2015) extended cold storage
postharvest and reduced the rate of membrane electrolyte leakage induced at low
temperature. The significant increase in ion leakage observed in non-treated early-
harvested grapes, in contrast with the values in CO2-treated fruit, seems to support our
previous studies in which we reported that the 3-day gaseous treatment minimized or
modified the activation of defense mechanisms that took place in non-treated grapes with a
maturity stage near to the early-harvested grapes used in this study, as a response to
temperature shifts at 0°C (Sanchez-Ballesta et al., 2006; Fernandez-Caballero et al., 2012;
Rosales et al., 2013). Likewise, the differences observed between early- and late-harvested
grapes could be related to the fact that the maturity stage affects the sensibility of fruit to
develop chilling injury. Thus, chilling injury index in pre-yellow and yellow mango fruits was
significantly lower than that of green fruits (Zhao et al., 2009b). Similarly, persimmon fruit
(Salvador et al., 2005) and cucumber (Qian et al., 2013) were more susceptible to chilling
injury at the earlier developmental stage.
As it is known, researchers have traditionally focused on transcriptional regulation as the
main determinant of protein levels and, thus, cellular function. However, although the
correlation between mRNA and protein abundance in the cell in a wide range of organisms
is generally considered to be positive, it has been found to vary greatly between studies, cell
types and organisms (reviewed by Plotkin, 2010). In our study, microarray analysis of the
effect of low temperature and high CO2 levels on the skin of table grapes stored at two
maturity stages, revealed substantial changes in the transcriptome as a response to storage
at 0°C. By contrast, the proportion of genes which changed expression because of the 3-
day treatment with high CO2 levels at 0°C was much lower at both maturity stages. These
findings are in concordance with our previous works where the analysis of individual genes
showed that the activation of cold-stress responses in the first stage of grape storage at 0°C
in non-treated fruit, which was less noticeable in CO2-treated grapes (Sanchez-Ballesta et
al., 2006; Rosales et al., 2013).

Tolerance to low temperature of table grapes depends on maturity stage

The majority of transcripts up-regulated by low temperature in the skin of early-harvested
grapes were related to GO terms associated with response to abiotic and biotic stress.
Among them, the induction of HOS15, LOS1, LOS4, TIL, and HSP gene expression in
response to low temperature storage could be related to the mechanisms activated in non-
treated grapes to overcome low temperature storage. The expression of HOS15, a WD40-
repeat protein, was induced by abiotic stresses including cold stress. Zhu et al. (2008)
suggested that HOS15 functions as a repressor to control gene expression important to cold
tolerance through chromatin modification. Likewise, the los1 mutant of Arabidopsis, which
is dysfunctional in the translational elongation factor 2, has reduced freeze tolerance and
has an impaired ability to translate proteins at 0°C (Guo et al., 2002). Furthermore, LOS4, a
DEAD-Box RNA helicase, also was found to be critically involved in chilling sensitivity of
Arabidopsis (Gong et al., 2002). On the other hand, the up-regulation of HSPs was a
common effect of low temperature storage on both early- and late-harvested grapes. The
higher tolerance of different crops to chilling injury has been attributed to the production and
accumulation of HSPs (Sanchez-Bel et al., 2012; reviewed by Aghdam et al., 2013). Yun et
al. (2012) reported the induction of HSPs, cold regulated (COR) and temperature-induced
lipocalins (TILs) gene expression in citrus fruit stored at low temperature, an observation
which hints at the participation of these genes in promoting tolerance toward cold stress.
In V. vinifera, a transcriptome analysis of berry ripening under high temperatures revealed
the establishment of a thermotolerance response marked by the induction of twenty-one
HSPs (Carbonell-Bejerano et al., 2013).
It is important to emphasize that “gluconeogenesis” and the “chitin catabolic process”
showed the highest enrichment score in the FACH analysis of genes up-regulated by low
temperature in the skin of early-harvested grapes. Gluconeogenesis is a fundamental
metabolic process, allowing organisms to make sugars from non-carbohydrate stores such
as lipids and protein. Phosphoenolpyruvate carboxykinase, which plays a pivotal role in
gluconeogenesis by catalyzing the conversion of the C4 dicarboxylic acid oxaloacetic acid
to phosphoenolpyruvate, has previously been associated with cold stress response (Xuan
et al., 2013). Furthermore, although it is known that glyceraldehyde-3-phosphate
dehydrogenases (GAPCs) act in glycolysis and gluconeogenesis, previous studies have
demonstrated that they have a role to play in plant response to various stresses, such as
cold, drought and hypoxia (reviewed by Kosová et al., 2011; Sanchez-Bel et al., 2012). In
plants, GAPCs have been localized in the nucleus during cold stress (Bae et al., 2003), and
their capacity to bind to DNA has been observed, in particular, to the coding sequence of
the NADP-dependent malate dehydrogenase gene (Hameister et al., 2007). This fact seems
to indicate that, in addition to its role in the gluconeogenesis and glycolysis, GAPCs may be
involved in mediating stress signaling and signal transduction to the nucleus. On the other
hand, it is important to note that the up-regulation of the four chitinases preceded the visual
appearance of fungal attack in early-harvested grapes which took place after 13 days at
0°C. Furthermore, in a previous work we observed that expression levels of chit1b increased
in the skin of table grapes after 3-day storage at 0°C and the overexpression of CHIT1b
in Escherichia coli showed in vitro cryoprotective activity and retained catalytic activity at
subzero temperature (Fernandez-Caballero et al., 2009), indicating a putative protective role
during storage at low temperature.
In this study, the “lipid transport” process seems to play an important role in the response of
early-harvested grapes at 0°C, inducing eight genes encoding LTPs. Transcript levels of
LTPs increased in response to drought, salt and cold stresses (Jung et al., 2003).
Stabilization of membranes, cuticle deposition and/or changes in cell wall organization, are
believed to be responses to these stress factors. In peach fruit, Pavez et al. (2013) reported
that expression of genes encoding antioxidant enzymes, HSPs and LTPs increased during
cold storage and remained high after fruits were subjected to room temperature
reconditioning. Likewise, the functions of this protein family as an enhancer of the
phospholipids transfer between membranes and possibly as a molecule binding acyl chains
are crucial in promoting tolerance to cold stress (Zhang et al., 2010).
On the other hand, environmental stress leads to the global reduction of protein synthesis,
while the translation of selected stress responsive proteins can be maintained (Bavli-
Kertselli et al., 2015). In these sense, our results showed the induction of ten genes coding
eIFs in the skin of early-harvested grapes stored at 0°C. Translation initiation in eukaryotes
depends on eIFs, with IF3 playing a central role in the polypeptide chain elongation
interacting with many other translation initiation factors and being its expression induced by
environmental stress (Kawaguchi and Bailey-Serres, 2002). Among the ten eIFs induced by
low temperature, three eIF3 were identified, with IF3 considered the most important in terms
of the selective translation of cold-shock mRNAs in E. coli (Giuliodori et al., 2004). In
addition, a proteomic study indicated that banana fruit with down-regulated eIF5A showed
the appearance of severe chilling injury, while the protein was significantly up-regulated in
the ethylene-pretreated fruit without any chilling injury symptom (Li et al., 2015).
In reference to down-regulated genes by low temperature in the skin of early-harvested
grapes, our results showed repression in the expression of five genes encoding tubulin
folding cofactors. Microtubules constitute ubiquitous cytoskeletal elements required for a
wide variety of cellular processes. They assemble from heterodimers consisting of α- and β-
tubulins, and cofactors are required for the production of correctly folded and assembly-
competent tubulin molecules (Gao et al., 1993). On winter wheat, Abdrakhamanova et al.
(2003) showed that efficient cold acclimation was accompanied by an initial, partial
disassembly of microtubules which was sufficient to trigger efficient cold acclimation. These
authors suggest that microtubules, in addition to their role as effectors of the cold response,
must have a function related to the efficient sensing of low temperature, culminating in the
induction of the acclimation machinery. On the other hand, five genes coding for histone
deacetylases were down-regulated in early-harvested grapes stored at 0°C. In maize, the
expression of histone deacetylases was up-regulated during cold acclimation (Hu et
al., 2011). HDA6 regulates the expression of several long-term cold stress-responsive
genes and plays a role in the acquisition of freezing tolerance in plants (To et al., 2011).
Our results indicated the down-regulation of seven V-ATPase genes in early-harvested
grapes stored at 0°C. One of the primary events of chilling injury in plants appears to be an
inhibition of V-ATPase activity, which leads to an acidification of cytoplasm (Yoshida et
al., 1999). Dietz et al. (2001) indicated that low-temperature induced a decline in V-ATPase
activity and that the concomitant decrease in proton motive force could affect the solute
compartmentation and possibly the hardiness of plants to low temperature. In this sense,
we have observed that low temperature storage significantly increased the water-soluble
K+ pool in the skin of table grapes, while high CO2 levels maintained it (Blanch et al., 2014).
Likewise, in a proteomic study of cold acclimation of Arabidopsis, Minami et al. (2009) found
many V-ATPases located in the microdomains which were down-regulated after chilling
exposure, suggesting that these V-ATPases might contribute to cold or freezing tolerance
by means of membrane trafficking regulation. Our data indicate that non-treated table
grapes respond to low temperature by modulating, the expression of several genes to
overtake cold stress together with other genes associated with the cellular stress which
occurs during storage at a non-optimal temperature.
In the case of table grapes harvested at the optimal maturity standards, low temperature
storage induced the expression of genes related to “response to phosphate starvation,”
including SPX2 and SPX4. At the molecular level, recent studies have shown that several
proteins carrying the SPX domain are essential for maintaining phosphorus homeostasis in
plants (reviewed by Secco et al., 2012). Likewise, constitutive overexpression of OsSPX1 in
tobacco plants resulted in decreased total leaf phosphorus concentration as well as the
accumulation of free proline and sucrose, providing improved cold tolerance compared with
the wild-type (Zhao et al., 2009a). In relation to stressful storage conditions, genes up-
regulated in late-harvested grapes were also associated with the “protein folding” GO term.
Protein folding stability is undoubtedly one of the most challenging problems in organisms
undergoing stressful conditions. Thus, efficient protein repair systems and general protein
stability facilitate survival under sudden changes in the environment. Among the genes
involved in this term, it is interesting to note that in addition to chaperones DNAJ, genes
encoding peptidyl-prolyl cis-trans isomerases (PPIases) were up-regulated. PPIases
represent an important type of protein foldase, catalyzing the reversible conversion of
peptidyl prolyl bond from cis to transwhich is a rate-limiting step in folding proteins (Fischer
and Schmid, 1999). Four structurally distinct subfamilies of PPIases (cyclophilins, FKBP,
parvulins, and PP2A phosphatase activators) were characterized (Lu et al., 2007). Our
results showed that genes coding for different cyclophilins and FKBP subfamilies were up-
regulated by low temperature in late-harvested grapes, suggesting that a larger number of
PPIases are required in order to facilitate the efficient folding of proteins during storage.
Different members of cyclophilin subfamily were directly linked to multiple stresses. In this
sense, Budiman et al. (2011) proposed that foldase and chaperone activities of PPIases are
the main requirements with which to overcome the cold-stress problem in microorganisms
caused by protein folding.
It is important to note that the number of genes repressed in late-harvested grapes stored
at 0°C was almost three times lower than those of early-harvested grapes. Among them,
genes related to the response to biotic and abiotic stress together with the GO term
“translation elongation” (60 s ribosomal protein and elongation factors) were affected. Earlier
studies have shown that the translational machinery can be modified by thermal stress.
Heat-stressed seedlings of Brassica napus have been shown to have low levels of
translation elongation factors (Dhaubhadel et al., 2002). Likewise, whereas the EF-1α
mRNA levels increased at low temperature in maize leaves, time-course experiments over
24 h at 5°C showed that in the case of roots, the overall mRNA level of EF-lα was transiently
decreased (Berberich et al., 1995).

Role of 3-day high CO2 levels in table grape response to low temperature
The transcriptomic analysis showed that both early- and late-harvested grapes presented
more up-regulated genes than down-regulated ones as a response to high CO2 levels at
0°C. However, Becatti et al. (2010) observed that in the case of detached wine grapes, the
application of high CO2 levels (30%) for 3 days at 20°C induced the repression of more
genes in the skin and pulp compared to those activated. Similar results were obtained in a
RNA-Seq analysis performed with nectarines stored under controlled atmospheres (15%
CO2) for 21 days at 4°C (Sanhueza et al., 2015).
The application of a 3-day treatment with high CO2 levels at 0°C in early-harvested grapes
significantly induced the expression of twenty eight transcription factors related to the GO
terms “response to chitin,” “ethylene mediated signaling pathway,” and “regulation of
transcription, DNA-dependent.” Because stress gene induction occurs primarily at the level
of transcription, transcription factors interact with cis-elements in the promoter regions of
several stress-related genes and thus up-regulate the expression of many downstream
genes resulting in imparting abiotic stress tolerance (Agarwal and Jha, 2010). Plants devote
a large portion of their genome capacity to transcription, with the V. vinifera genome coding
in excess of 1200 transcription factors classified into 58 families (Grimplet et al., 2012; Jin
et al., 2014). In this study, we found the induction of transcription factor genes belonging to
different families such as ERF (Ethylene Responsive Factors), a subfamily of the APETALA2
(AP2)/ERF transcription factor family, as well to the WRKY, MYB, basic-domain leucine-
zipper (bZIP), heat stress transcription factor and zinc finger. Expression data from different
plants species, including V. vinifera, have indicated that different members of the
transcription factors participate in plant responses to cold stress. After 4 h of cold treatment,
a total of 70 up-regulated and 18 down-regulated transcription factors were identified in
leaves of V. amurensis, a wild grapevine species with remarkable cold-tolerance, while 68
up-regulated and 43 down-regulated transcription factors were identified in V. vinifera (Xin
et al., 2013). In shoot apices of V. vinifera cv. Muscat Hamburg, Wang et al. (2014) observed
the induction of fifteen VvWRKYs in response to cold stress. To our knowledge, however,
there is no information available about the participation of transcription factors on the
response of fruit to high CO2 levels and low temperature. In this sense, we observed that
the application of 3-day high CO2 levels at 0°C induced the expression of CBF1 and CBF4 in
the pulp of table grapes and CBF4 in the rachis (Fernandez-Caballero et al., 2012). The fact
that CBFs also belong to the AP2/ERF transcription factor family seems to indicate that this
family plays a prominent role in the beneficial effect of the gaseous treatment in table grapes.
Likewise, the thermotolerance response of Muscat Hamburg berries ripened at high
temperature was coincident with the up-regulation of ERF transcription factors, suggesting
their participation in the maintenance of the acclimation response (Carbonell-Bejerano et
al., 2013).
In early-harvested grapes, the gaseous treatment also induced genes that encoded kinases
belonging mainly to the RLKs family. Phosphorylation mediated by kinases is one of the
most ordinary mechanisms by which environmental cues are transduced and protein
function is regulated in eukaryotic cells. It is known that RLKs mediate cold acclimation.
Thus, a null mutant for a gene encoding a plasma-membrane RLK, showed a reduction in
cold-induced gene expression as well as in the capacity to cold acclimate (Yang et al., 2010).
Dehydrins are also proteins involved in cold acclimation and are post-translationally modified
by phosphorylation. They accumulate in response to diverse abiotic stresses, including low
temperature, and act as molecular chaperones (Hara, 2010). In a recent work, we observed
that high CO2 levels induced the accumulation of DHN44 in the skin of table grapes and in
vitro assays indicated that this dehydrin can be phosphorylated (Navarro et al., 2015).
In the early stage, the most enriched down-regulated genes in CO2-treated grapes codified
for five transcription factors belonging to the homeobox-leucine zipper, MAD-box and ERF
families. This finding, together with the fact that transcription factors are mainly up-regulated
in treated grapes, seems to indicate that the gaseous treatment could be an active process
requiring the regulation of transcription factors.
In late-harvested grapes, only ten genes up-regulated by high CO2 levels at 0°C were related
to the GO terms “defense response,” “photosynthesis,” and “generation of precursor
metabolites and energy.” Proteomic analyses indicated that tolerant plants can induce
several protective mechanisms more efficiently than sensitive ones because they are able
to maintain sufficient rates of several metabolic processes, especially those associated with
energy metabolism under adverse stress conditions (Ingle et al., 2005; Dumont et al., 2011).
Maintaining sufficient rates of processes associated with energy metabolism is extremely
important for an efficient stress acclimation since it is an active process associated with de
novo biosynthesis of several stress-protective compounds (Bartels and Sunkar, 2005).
Finally, as we have already mentioned, the gaseous treatment was effective controlling total
decay in table grapes at both maturity stages. Curiously, in early- and late-harvested grapes
the GO term “defense to bacterium” was over-represented although different genes up-
regulated were included in each case.

Conclusions
The transcriptional responses of table grapes to low temperature and high CO 2 levels
depend on the stage of maturity. While it is true that cold storage had similar effect on fruit
quality regardless of the date of harvest and that high CO 2 treatment was effective in
controlling total decay and maintaining quality at the end of the storage period in both
maturity stages (data not shown), our results indicate that they do so through different
mechanisms and the modifications in the transcriptome profile seem to be different. Major
modifications in the transcriptome profile of early- and late-harvested grapes storage at 0°C
are linked to biotic and abiotic stress-responsive terms. However, specific transcriptional
modifications were observed depending on the date of harvest. The results obtained in this
study indicate that in both cases there is a reprogramming of the transcriptome during the
first stage of storage at a non-optimal temperature. Thus, in early-harvested grapes changes
associated with gluconeogenesis, photosynthesis, mRNA translation and lipid transport
occurs, whereas the maintenance of protein folding stability and intracellular membrane
trafficking seems to play an important role in late-harvested grapes. Similarly, the cellular
response to low temperature stress seems to be related to maintaining the ion homeostasis,
as reflected in the inhibition of key transport proteins such as V-ATPases in early-harvested
grapes, which are thought to participate in the very early response to cold stress by
regulating membrane trafficking.
In order to maintain early-harvested table grape quality, the high CO2 treatment seems to
be an active process requiring the activation of transcription factors, as well as protein
kinases implicated in the regulation of protein function. Likewise, although the number of
genes significantly associated with GO-terms in late-harvested grapes under high CO2levels
was low, there seems to be an active process associated with maintaining the energy of the
fruit.

Author contributions
RR, RNA extraction, RT-qPCR analysis and improved the manuscript. IR, table grape quality
assessments, edited and improved the first draft of the manuscript. CF, RNA extraction and
table grape quality assessments. ME and CM, improved the manuscript. MS, designed the
research, data analysis and prepared the first draft of the manuscript. All authors have read
and approved this manuscript.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or
financial relationships that could be construed as a potential conflict of interest.

Acknowledgments
This work was supported by the European Union under the 7th Framework Programme FP7-
PEOPLE-2012-CIG no. 321694 and by CICYT projects AGL2011-26742 and AGL2014-
53081-R. RR, IR, and CF were supported by a postdoctoral JAE contract from the CSIC, a
postdoctoral Juan de la Cierva contract from the MICINN and a predoctoral contract from
the MEC, respectively. The work benefited from the networking activities within the
European COST Action FA1106-QualityFruit. Authors thank Dr. José Miguel Martínez-
Zapater (ICVV, CSIC, Spain) and “Genoma España” for providing the custom-made Vitis
vinifera GeneChip.

Supplementary material
The Supplementary Material for this article can be found online
at: http://journal.frontiersin.org/article/10.3389/fpls.2016.01020
Click here for additional data file.(35K, XLSX)
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Click here for additional data file.(216K, PDF)
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Int J Mol Sci. 2016 Jul; 17(7): 1103.

XXI. Strawberry Achenes Are an Important Source of Bioactive

Compounds for Human Health
María Teresa Ariza,1,* Patricia Reboredo-Rodríguez,2 Luca Mazzoni,3Tamara Yuliett
Forbes-Hernández,4 Francesca Giampieri,4 Sadia Afrin,4 Massimiliano Gasparrini,4Carmen
Soria,1 Elsa Martínez-Ferri,1 Maurizio Battino,4 and Bruno Mezzetti3,*
David Arráez-Román, Academic Editor

Abstract

1. Introduction
Strawberries (Fragaria × ananassa, Duch.) are among the most widely consumed fruits in
the world. They are a very rich source of bioactive compounds including vitamins (such as
vitamin C), β-carotene and phenolic compounds (phenolic acids, flavonoids and
anthocyanins) [1]. These bioactive compounds, such as antioxidants, reduce oxidative
stress by neutralizing the overproduction of reactive oxygen species (ROS), which are
related with the occurrence of several diseases [2,3].
In epidemiological studies, strawberry consumption has been associated with health
benefits [4,5,6], such as the prevention of inflammation [7], oxidative stress [8,9],
cardiovascular diseases [10,11], diabetes [12], cancer [13,14] and obesity [15]. These
healthy effects have been related with the antioxidant activity of phenolic compounds, mainly
ellagitannins [13,14] and anthocyanins [16,17,18,19,20,21,22,23,24,25,26].
Healthy properties of strawberries are not only associated with the amount of bioactive
compounds, but also with the degree of transformation during digestion. In fact, in strawberry
fruits, the chemical nature and/or the integrity of bioactive compounds can be altered by the
specific conditions of the gastrointestinal tract, the activity of gastrointestinal enzymes [27]
and by the action of the local microbiota [28], affecting their uptake throughout the digestive
tract [29]. For example, a major part of the polyphenols ingested is not absorbed through
the gut barrier [30], while anthocyanins are directly and quickly absorbed from the stomach
[31,32,33] and from the small intestine [28,34,35].
The so-called strawberry is actually an aggregate fruit consisting of a swollen and fleshy
floral receptacle that supports a cluster of real dry fruits or achenes containing the seeds
[36]. It has been reported that phenolic composition in achenes and flesh differs [17,37],
achenes having up to 10-fold higher antioxidant activity and amount of phenolic compounds,
especially ellagic acid derivatives [17,38]. In this sense, achenes could themselves
represent a significant source of bioactive compounds for human health. However, after
intake, achene contribution to the antioxidant capacity of the whole fruit is unknown and
could be negligible due to the barrier interposed by their hard and relatively thick pericarp
with ligno-cellulosic structure [39].
In the present study, we have compared strawberry phenolic composition and antioxidant
capacity between achenes and raw fruit, before and after simulated in vitro digestion, in
order to characterize and quantify the release and bioaccessibility of phenolic compounds
in both types of tissues.

2. Results

2.1. Achene Contribution to Total Fruit Weight

Fresh weight of “Camarosa” strawberry fruits was 21.9 ± 0.8 g and achenes represented
0.75% of total fresh weight (i.e., 0.16 ± 0.02 g). Considering that 90% of total fresh fruit
weight is water [17], achenes would then represent the 7.5% of total fruit dry weight. These
figures were used to calculate the relative contribution of achenes and flesh to antioxidant
content and capacity in a whole fruit (see Table 1).

Table 1
Estimation of relative contribution (%) of total phenolic compounds (TPCs), flavonoids
(TFCs) and anthocyanins (ACs) and antioxidant capacity by FRAP, DPPH and TEAC in non-
digested flesh and achenes on a whole fruit.

TPC TFC AC FRAP DPPH TEAC

Flesh 59 77 92 47 55 19

Achene 41 23 8 53 45 81

2.2. Antioxidant Composition in Non-Digested Raw Fruits and Achenes

There were differences in the levels of antioxidant compounds between non-digested flesh
and achenes. Achenes displayed significantly higher total phenolic compounds (TPCs) and
total flavonoid compounds (TFCs) than flesh (more than 10 and four times higher,
respectively; see Table 2). No significant differences were found between flesh and achenes
in anthocyanin content (AC). The differences in antioxidant composition in achenes and
flesh translated into a different contribution of flavonoids and anthocyanins respect to TPC
(Figure 1). Thus, TFCs were 44.15% and AC 14.33% of TPC in flesh, whereas in achenes,
these percentages were 22.71% and 1.82%, respectively. In addition, antioxidant capacity
analyzed by FRAP, DPPH and TEAC was, respectively, 17-, 12- and 64-times higher in
achenes than in flesh (Table 2).

Figure 1
Relative contribution of total flavonoids (TFCs) and anthocyanins (ACs) to total content of
phenolic compounds (TPCs) in six different extracts (non-digested flesh, non-digested
achenes, flesh and achenes after gastric digestion and flesh and achenes after total
digestion).

Table 2
Content of total phenolics (TPCs; mg GAE/g DW), total flavonoids (TFCs; mg CE/g DW),
and total anthocyanins (ACs; mg Pel-glc/g DW), and antioxidant capacity (in mg TE/g DW),
determined by FRAP, DPPH and TEAC in flesh (F) and achenes (A) extracts before (non-
digested) and after gastric (gastric Fraction) and total digestion (intestinal fraction). All
analyses were performed in triplicate. Data represent the mean ± Standard error (SE). Post
hoc comparisons were done by Tukey’s test. (*) and (ns) indicate significant (p < 0.05) and
non-significant differences, respectively.
 Gastric Intestinal
Non-Digested Ee 1 R (%) 2
Fraction Fraction
Fles Ache Achen Fles Ache Fles Ache Fles Ache
Flesh
h ne e h ne h ne h ne
37.68 382.8 255.0 600.34 41.17 16.55
TPC ± 7 ± 2 ± ± 33.51 ± ± 0.39 6.8 1.6 16.1 2.8
0.95 4.69 * 4.40 * 0.61 *
22.04 93.92 135.3 133.66 14.40
7.68 ±
TFC ± ± 4.70 2 ± ± ± 6.1 1.4 10.6 5.7
1.60 ns
0.49 * 4.08 9.13 ns 0.13
0.00
5.4 ± 6.97 ± 41.58 17.59 ± 0.00 ±
AC ± 7.7 2.5 0.0 0.0
0.78 0.79 ns ± 5.37 2.42* 0.00 ns
0.00
2474. 4522.9
149.4 1254. 101.3 75.25
FRA 22 ± 1 ±
0 ± 45 ± 1 ± ± 1.95 8.4 1.8 8.1 1.7
P 38.92 223.18
18.69 46.66 1.87 *
* *
1192. 3931.6
100.5 119.2 90.04 32.85
DPP 21 ± 0 ±
4 ± 5 ± ± ± 2.15 1.2 3.3 75.5 0.8
H 62.09 771.70
4.49 14.30 4.58 *
* *
1286. 2100.6
20.13 2174. 286.8 86.57
TEA 39 ± 8 ± 108.
± 79 ± 2 ± ± 4.69 1.6 13.2 4.1
C 110.3 298.81 0
7.64 46.74 ns 2.79 *
9*
Open in a separate window
1
Ee: Extraction efficiency of gastric conditions (gastric fraction vs. non-digested
extracts); 2 R (%): % recovery of compounds from gastric to intestinal fraction.
Although achenes showed a higher amount of TPCs and TFCs (Table 2), there were
differences in the composition of individual compounds between flesh and achenes.
Phenolic acids (caffeic, chlorogenic and ellagic acids) and anthocyanins (Cya 3-glc, Pel 3-
glc and Pel 3-rut) analyzed by HPLC represented 22% and 4% of TPC in flesh and achenes,
respectively (Table 3). The amount of caffeic and chlorogenic acid was significantly higher
in flesh than in achenes, whereas the reverse was true for ellagic acid. However, although
in the case of Pel 3-rut no significant differences were found, Cya 3-glc and Pel 3-glc
displayed higher values in achenes (three-fold and 2.1-fold, respectively; see Table 3).

Table 3
Content (mg/g DW) of phenolic acids (caffeic acid, chlorogenic acid and ellagic acid) and
anthocyanins (Cyanidin 3-glucoside, Pelargonidin 3-glucoside and Pelargonidin 3-
rutinoside) in flesh (F) and achene (A) extracts before (non-digested) and after gastric
(gastric fraction) and total digestion (intestinal fraction). All analyses were performed in
triplicate. Data represent the mean ± Standard Error (SE). Post hoc comparisons were done
by Tukey’s test. (*) and (ns) indicate significant (p < 0.05) and non-significant differences,
respectively.
 Non- Gastric Intestinal
Ee 1 R (%) 2
Digested Fraction Fraction
Fles Ache Fles Ache Fles Ache Fles Ache Fles Ache
h ne h ne h ne h ne h ne
Phenolic acids
0.45 0.28 0.16
Caffeic 0.40 ± 0.21 ± 0.49 ±
± ± ± 0.6 0.5 58.3 236.6
acid 0.03 * 0.02 ns 0.04 *
0.04 0.02 0.01
1.42 1.25 0.57
Chlorogen 1.22 ± 1.06 ± 0.75 ±
± ± ns ± 0.9 0.9 45.5 70.6
ic acid 0.11 * 0.09 0.07 ns
0.12 0.11 0.05
1.26 0.77 0.19
Ellagic 2.09 ± 0.71 ± 0.35 ±
± ± ± 0.6 0.3 24.4 48.6
acid 0.18 * 0.06 ns 0.03 *
0.11 0.07 0.02
Anthocyanins
0.65 1.98 0.00
Cyanidin 1.98 ± 2.57 ± 0.76 ±
± ± ± 3.0 1.3 0.0 29.5
3-glc 0.11 * 0.01 * 0.11 *
0.07 0.11 0.00
4.10 6.28 13.55 0.90
Pelargoni 8.55 ± 1.43 ±
± ± ± 0.88 ± 1.5 1.6 14.3 10.5
din 3-glc 0.07 * 0.22 ns
0.36 0.11 * 0.07
0.51 1.85 0.44
Pelargoni 0.32 ± 4.26 ± 0.00 ±
± ns ± ± 3.6 13.2 24.0 0.0
din 3-rut 0.05 0.18 * 0.00 *
0.04 0.33 0.02
Open in a separate window
1
Ee: Extraction efficiency of gastric conditions (Gastric fraction vs. non-digested
extracts); 2 R (%): % recovery of compounds from gastric to intestinal fraction.
It is remarkable that, despite the achenes representing only 7.5% of the total fruit dry weight,
their relative contribution to the total antioxidant content and capacity of the whole fruit is
quite high (Table 1), accounting for 41% of TPC and 23% of TFC and even up to 81% of the
fruit antioxidant capacity.

2.3. Bioaccessibility of Antioxidant Compounds after in Vitro Digestion

Flesh and achenes differed in TPC, AC, FRAP, DPPH values in the gastric fraction (Table
2). Hence, TPC, FRAP and DPPH data were more than two-, three- and 32-fold,
respectively, higher in achenes compared with flesh, but the reverse was true for AC (two-
fold higher in flesh). However, in the intestinal fraction, the opposite tendency was observed
(Table 2). Antioxidant compounds and antioxidant capacity in both flesh and achenes were
higher in gastric than in intestinal fractions (Table 2). Thus, the recovery after total digestion
of TPC, TFC and AC was 16.1%, 10.6% and 0%, in flesh and 2.8%, 5.7% and 0% in
achenes, respectively.
In addition, in gastric fraction, antioxidant capacity by FRAP and DPPH assays was higher
in achenes than in flesh (Table 2), whereas, for TEAC, no differences were found. In
intestinal fraction, flesh showed significantly higher values of antioxidant capacity than
achenes (Table 2). When gastric and intestinal fractions were compared, the antioxidant
capacity in the intestinal fractions from both flesh and achenes was decreased (Table 2).
Thereby, FRAP, DPPH and TEAC was reduced by 92%, 24% and 87% in flesh and by 98%,
99% and 96% in achenes, respectively.
When flesh and achenes were subjected to gastric digestion, no significant differences were
observed in the caffeic, chlorogenic and ellagic acid content (Table 3). However, the amount
of anthocyanins (Cya 3-glc, Pel 3-glc and Pel 3-rut) was significantly higher in achenes than
in flesh. In both flesh and achenes, there was a major trend to lower values of phenolic acids
and anthocyanins in the intestinal fraction except for caffeic acid in achenes (Table 3). The
decrease of phenolic acids was more marked in flesh than in achenes, resulting in lower
percentages of recovery. It is remarkable that Cya 3-glc in flesh and Pel 3-rut in achenes
were not detected after intestinal digestion.

2.4. Relationship between Antioxidant Compounds and Antioxidant Capacity

To assess the relationship between variables, data from non-digested and digested extracts
(i.e., gastric and intestinal fraction) from flesh and achenes were analyzed jointly. Most of
the parameters evaluated were highly and significantly correlated (Table 4). However, no
correlation was found between AC and FRAP and DPPH. Regression analysis showed that
most of the variation observed in FRAP was absorbed by TPCs (R2 = 94.4%; Figure 2a), of
which TFCs accounted for 64% and ACs did not contribute. Variation in TEAC was mainly
explained by TFCs (R2 = 90%; Figure 2b), and ACs accounted for 54%. Variation in DPPH
was partially related with TPCs and TFCs (R2 = 51% and 15%, respectively).

Figure 2
Linear relationship between total phenolic content and FRAP (a) and total flavonoid content
and TEAC (b).

Table 4
Regression coefficients and p-values of TPCs, TFCs, and total ACs and antioxidant capacity
by three different assays (DPPH, FRAP and TEAC).

DPPH FRAP TEAC

0.507 0.944 0.657
TPC
0.010 0.005 0.010
0.146 0.641 0.900
TFC
0.020 0.005 0.010
AC 0.002 0.080 0.545
 DPPH FRAP TEAC
0.246 0.060 0.010

2.5. Efficiency of Extraction Conditions

Taking into consideration that extraction conditions could affect antioxidant release, the
comparison between non-digested and gastric extracts is interesting.
The majority of compounds analyzed in flesh and achenes were released to a greater extent
after gastric digestion in comparison with non-digested extracts. This translated into values
of extraction efficiency (Ee) far above one (Table 2), especially in flesh.
In relation to specific compounds, the release of anthocyanins (Cya 3-glc, Pel 3-glc and Pel
3-rut; Table 3) was higher in the gastric extract, whereas the reverse was true for specific
phenolic acids (caffeic, chlorogenic and ellagic acids), in both flesh and achenes.

3. Discussion
The present study gives new insights into the relevance of strawberry achenes as a source
of antioxidant and bioactive compounds providing a new profitable product for human health.
As reported in previous studies [17,37], our results showed that strawberry antioxidant
properties are linked either to flesh or to achenes. Despite achenes representing a small
fraction of total fruit weight, their contribution to total antioxidant content and capacity of the
fruit is remarkable. Although in the whole fruit, phenolic compounds, flavonoids and
anthocyanins are mainly in the flesh, we found that strawberry antioxidant capacity (i.e.,
FRAP and TEAC assays) was mainly attributable to achenes, suggesting that not only the
amount of antioxidants, but mostly the type, accounts for strawberry ROS detoxification
capacity. In fact, although achenes were enriched in phenolic compounds (including
flavonoids) compared to flesh, in consonance with a higher antioxidant capacity (FRAP;
DPPH and TEAC), only 20% of total phenolic compounds were flavonoids, whereas, in the
flesh, flavonoids represented 60%, indicating that non-flavonoid phenolic compounds are
preferably in the achenes. Similarly, anthocyanins were mostly in the flesh. Even more,
specific phenolics and anthocyanins are not equally present in flesh and achenes,
suggesting some degree of compartmentation of antioxidant compounds in the strawberry
fruit. These results are in agreement with the higher antioxidant content of seeds in
comparison to the edible part of fruits [40,41].
The differences between flesh and achenes in antioxidant composition and capacity might
be due to several factors, such as the nature of the matrix where the antioxidants are
embedded. Intrinsically achenes have more antioxidant compounds than flesh, but different
matrices can determine the effectiveness of digestive enzymes, and, therefore, affect the
release of bioactive compounds under the physiological conditions during the digestion [42].
In this sense, the amount of antioxidants in gastric digested flesh, and achenes were quite
higher than in non-digested samples, suggesting that in vitro digestion provide better
conditions (i.e., low pH and pepsin; [43]) for the release of phenolic compounds from the
matrix and/or for their stability [44]. These results are pointing out that phenolic quantification
in non-digested extracts of strawberries is underestimated by the extraction methods
commonly used. Nevertheless, gastric conditions favored a greater release of antioxidants
in the flesh than in achenes (i.e., higher Ee), indicating that the matrix exerts a differential
resistance to the release of antioxidant compounds. However, gastric conditions do not
always translate into a higher release of specific compounds, such as caffeic, chlorogenic
and ellagic acids, pointing to chemical transformation and/or degradation. In contrast, the
specific anthocyanins analyzed were increased in gastric fraction probably due to higher
stability under low pH [45,46].
A drastic decrease of phenolic compounds and flavonoids was observed in the intestinal
fraction, but, in agreement with Clifford [47], anthocyanins were completely absent. The low
amount of phenolic compounds indicates that the intestine imposes a limitation to their
availability that can result either from chemical modifications or physical barriers [48].
Chemical effects could arise from antioxidant degradation or from alteration of the
antioxidant properties caused by intestinal conditions (i.e., in our study, pH 7.7, bile salts,
and pancreatin) [49] since the action of the local microbiota is not considered in this study,
although it contributes significantly to the digestive process.
Physical barriers, represented by the dialysis membrane, can limit the uptake of high
molecular weight compounds [50]. Although chemical and physical aspects might be
interacting at the intestine level determining the recovery in the bloodstream, our results in
specific compounds (phenolic acids and anthocyanins) are pointing out that this effect is
complex and depends on each molecule. Thus, not all the phenolics showed the same
percentage of recovery, reaching up to 236% for caffeic acid in achenes, indicating that
molecule reassembly could be occurring. This result might be relevant for human health,
since it suggests that beneficial compounds could derive from the distinct precursors
provided by the food matrices.
Nevertheless, the low rate of antioxidant recovery in the intestinal fraction does not
necessarily involve a low absorption, since some phenolic compounds, such as
anthocyanins, can be absorbed through the gastric wall [28,32,33]. Even more, in vivo
models have revealed that antioxidants can pass through the gut membrane either by
passive diffusion or by specific membrane transporters after the interaction with certain
glucosides (active glucose transporter: SGLT1) [47]. Therefore, antioxidant absorption could
be underestimated after in vitro digestion.
In both achenes and flesh, the amount of antioxidants was closely related with the
antioxidant capacity: the higher the antioxidants, the higher the antioxidant capacity at all
levels during the digestive process. However, not all antioxidant compounds contribute
equally to the total antioxidant capacity and are not similarly involved in the different radical
scavenging mechanisms. Thus, the FRAP method detects exclusively electron transfer
mechanisms (SET), whereas TEAC and DPPH combine SET and Hydrogen Atom Transfer
(HAT) mechanisms [51]. In contrast to other reports attributing antioxidant capacity mainly
to anthocyanins [52], our results showed that anthocyanins only contribute 54% to TEAC
but neither to DPPH (in disagreement with Espin et al. [53]) nor to FRAP, suggesting that
anthocyanins antioxidant activity should be associated to HAT mechanisms. On the other
hand, the high correlation between FRAP and total phenolic compounds (that include
flavonoids) indicate that SET mechanisms are mainly related either to non-flavonoids
(~30%) and non-anthocyanins flavonoid (~60%) phenolic compounds. Moreover, the
minimal relationship between DPPH and total phenolic compounds (50%) and total
flavonoids (15%) is indicative of its dependence on both non-flavonoids phenolic compounds
(~35%) and other non-phenolic antioxidants (~50%).
In summary, this work highlights not only the relevance of achenes in the strawberry
antioxidant capacity, but their potential healthy properties under physiological-like
conditions. These results give new insights for strawberry profitability and open a wide range
of applications for improving human health (i.e., pharmaceutical uses). Thus, achenes might
become a valuable product representing a new concept to be introduced in strawberry
cultivation and breeding programs. In this sense, some strawberry varieties, which, at
present, have been discarded because of misshapen fruits (i.e., “Camarosa”), could become
interesting for achene production [54]. Moreover, achene antioxidant composition can be
introduced as a new feature in breeding programs for characterization of strawberry
cultivars, as for raw fruits [55], and would confer a differential healthy value to each variety.
Apart from the healthy benefits, the use of achenes as a commercial/profitable product
would allow the utilization of industrial processing waste [17] and would also entail great
advantages to producers, since it is a non-perishable fruit, with easy conservation and
transportation, and suitable to be added to different types of food.

4. Materials and Methods

4.1. Strawberry Material and Sample Preparation

Strawberries (Fragaria × ananassa Duch cv. Camarosa) were planted in mid-October 2014
at the IFAPA-Churriana experimental greenhouse (Málaga, Spain). Three replications of 50
fresh and fully-ripened strawberry fruits were harvested. Fruit weight was calculated and all
the achenes per fruit were manually separated from the flesh and weighed. Afterwards, both
fractions were lyophilized by Labconco Freezone 12 L mod. 78670 (Kansas City, MO, USA).
Flesh was ground into a fine powder with Mixer B-400 (Büchi Labortechnik, Flawil,
Switzerland), and samples were stored at −20 °C until analysis.
To obtain the hydroalcoholic extracts for the analysis of phytochemicals content and total
antioxidant capacity, 1 g of lyophilized sample (flesh or achenes) were added to 100 mL of
80% methanol aqueous solution acidified with 0.1% formic acid and stirred for 2 h in the
dark at room temperature. Extracts were centrifuged at 3000 rpm for 15 min, and the
supernatant was filtered and stored at −80 °C until analysis.
The in vitro digestion of lyophilized flesh and achenes was performed according to the
method developed by Gil-Izquierdo et al. [42]. Briefly, the method consisted of an initial
pepsin-HCl digestion for 2 h for simulating gastric conditions, (pH~1.8) followed by an
intestinal digestion with pancreatin and bile salts for 2 h, both in a 37 °C shaking water bath.
During the intestinal phase, a dialysis membrane (molecular weight cut-off of 12,000 Da,
containing the amount of NaHCO3 necessary to titrate the mixture of post-gastric digestion
fraction + pancreatin-bile to pH 7.8) was introduced in a beaker containing an aliquot of 20
mL of the post-gastric fraction (Figure 3). Gastric and intestinal fractions, from both raw fruit
and achenes, were collected, concentrated under vacuum, purified (C-18 SepPaks Vac 6cc
cartridge; Waters Assoc., Milford, MA, USA) and stored at −80 °C. Before analysis, gastric
and intestinal fractions were opportunely re-suspended in a methanol: water solution and
results were referred to the dried weight before re-suspension.
Open in a separate window
Figure 3
Scheme of the in vitro digestion general procedure used with strawberry and achene
samples.

4.2. Measurement of Antioxidant Compounds

4.2.1. Total Phenolic, Flavonoid and Anthocyanin Content

Total phenolic content was determined by the Folin–Ciocalteu method [56,57]. Briefly, 100
µL of samples or gallic acid standard solution were mixed with 500 µL of Folin–Ciocalteu
reagent (10%), incubated for 3 min, and then 400 µL of sodium acetate solution (7 M) was
added. After 2 h of incubation at room temperature in the dark, the absorbance at 760 nm
was measured in a UV-Vis spectrophotometer (model DU® 6400 Spectrophotometer,
Beckman, Fullerton, CA, USA). Results were expressed as gallic acid equivalents per gram
of dry weight (mg GAE/g DW).
Total flavonoid content was measured by a colorimetric method described by Dewanto et al.
[58]. Briefly, 250 µL of samples or catechin standard solution were mixed to 1.25 mL of MilliQ
water (Millipore, Bedford, MA, USA) and 75 µL of NaNO2 5% solution. After 6 min, 150 µL
of AlCl3·6H2O, 10% solution, was added, allowed to stand for 5 min, and then 500 µL NaOH
1 M were added. The volume was brought to 2.5 mL with water, and the absorbance was
measured at 510 nm. Results were expressed as catechin equivalents per gram of dry
weight (mg CAE/g DW).
Total anthocyanin content was measured by the pH differential method [59]. Briefly, 0.025
M potassium chloride (KCl) buffer, pH 1.0 (buffer 1) and 0.4 M sodium acetate buffer
(CH3CO2Na), pH 4.5 (buffer 2) were prepared. After that, two dilutions (1/10 v/v) of the
samples or pelargonidin-3-glucoside (Pel-3-glu) standard solutions were prepared, one with
buffer 1 and the other with buffer 2. These dilutions were let to rest for 15 min, before
measuring the absorbance of each extract at 500 and at 700 nm to correct for haze, against
a blank represented by MilliQ water. The final absorbance (A) of the diluted samples was
calculated as follows:
A = [(A510 − A700) pH1.0 − (A510 − A700) pH4.5] (1)
Total anthocyanin content (AC) was calculated using the formulation reported by Cerezo et
al. [60]:
Total Anthocyanins = (A × MW × df × 1000)/(ε × 1) (2)
where MW: Pel-3-glc molecular weight; df: dilution factor; ε: molar extinction coefficient of
Pel-3-glc. Results were expressed as Pelargonidin-3-glucoside equivalents per gram of dry
weight (mg Pel-glc/g DW).

4.2.2. Total Antioxidant Capacity

Several methods for determination of antioxidant capacity were used: FRAP, DPPH, and
TEAC assays.
The FRAP (ferric reducing/antioxidant power) assay was carried out according to the
protocol proposed by Deighton et al. [61]. The FRAP reagent solution was prepared as
follows: 10:1:1 of sodium acetate (300 mM, pH 3.6), TPTZ (10 mM in HCl 40 mM) and ferric
chloride (20 mM). Briefly, 100 μL of sample or Trolox standard solution were added to 900
μL FRAP reagent. The mixture was vortexed, and, after 4 min, the absorbance was read at
593 nm against blank.
DPPH radicals (radical-scavenging activity) were determined based on the method
described by Kumaran and Karunakaran [62]. Briefly, 50 µL of Trolox standard solution or
samples were added to 1.450 mL of 3 mM DPPH solution. Subsequently, the mixture was
vortexed and after 1 h of incubation into the dark, and its absorbance was read at 515 nm.
TEAC (Trolox Equivalent Antioxidant Capacity) assay was performed according to Re et al.
[63]. The ABTS radical solution was produced by reacting 7 mM ABTS aqueous stock
solution with 2.45 mM K2S2O8, and maintained in the dark at 25 °C for 12 h before use.
Immediately before analysis, the working solution was obtained by diluting the stock solution
1:50 with PBS buffer, pH 7.4. Briefly, 10 µL of alternatively blank, Trolox standard or MilliQ
water diluted strawberry extract were added to 1 mL of the ABTS working solution into 1.5
mL eppendorfs. The mixture was vortexed for 20 s and after 1–3 min, the absorbance (A)
was read at 734 nm, measuring the colour inhibition of the ABTS radical. The percentages
of inhibition were calculated according the following equation:
% inhibition = (A control − A sample/A control) × 100 (3)
FRAP, DPPH and TEAC results were expressed as Trolox equivalents per gram of dry
weight (µmol TE/g DW).
4.3. Determination of Phenolic Acids and Anthocyanins by HPLC
Phenolics and anthocyanins were extracted by incubation of 7.5 mg of dry frozen powder in
300 µL of 96% methanol aqueous solution acidified with 0.001% formic acid, followed by
centrifugation at 3000 rpm for 15 min, filtering and immediate analysis on a JASCO HPLC
system (Jasco Corp., Tokyo, Japan) equipped with a quaternary gradient pump (JASCO
PU-2089 Plus) and a multi-wavelength UV detector (JASCO UV-2070 Plus). System control
and peak integration was done by the ChromNAV software (Jasco, Tokyo, Japan).
Samples were diluted appropriately and filtered using a 0.45 µm GHP Acrodisc Minispike
filter (Pall Life Sciences, Ann. Arbor, MI, USA) and separated on an Aqua Luna C18 reverse
phase column (250 mm × 4.6 mm; Phenomenex, Torrance, CA, USA) with a particle size of
5 μm protected by a C18 ODS guard column (4.0 mm × 3.0 mm; Phenomenex). The injection
volume was set to 20 µL and the flow rate was 1.0 mL/min−1.
Phenolic acids were analysed following the method of Schieber et al. [64] with minor
modifications. The mobile phase consisted of 2% (v/v) acetic acid in Milli-Q water (eluent A)
and of acetic acid in water and acetonitrile (1:49:50, v/v/v; eluent B). The gradient program
was as follows: 10% B to 55% B (50 min), 55% B to 100% B (10 min), 100% B to 10% B (1
min), and 10% B for 5 min before injecting the next sample. The phenolic acids were
quantified using calibration curves from standards of chlorogenic, caffeic and ellagic acids.
Values were expressed as milligrams of the corresponding phenolic acid per gram of dry
weight (mg/g DW).
Analysis of anthocyanins was carried out following the method described in Fredericks et al.
[65]. The mobile phase consisted of water/formic acid/acetonitrile (87:10:3, v/v/v; eluent A)
and water/formic acid/acetonitrile (40:10:50, v/v/v; eluent B). The gradient programme was
as follows: from 10% to 25% B (10 min), from 25% to 31% B (5 min), from 31% to 40% B (5
min), from 40% to 50% B (10 min), from 50% to 100% B (10 min), 100% B isocratic (5 min),
and from 100% to 10% B (1 min).
Anthocyanins were quantified using calibration curves generated from Cyanidin-3-glucoside
(Cya 3-glc) and Pelargonidin-3-glucoside (Pel 3-glc) standards. Pelargonidin-3-rutinoside
(Pel 3-rut) was quantified using the Pel 3-glc calibration. All anthocyanins were calculated
as milligrams per gram of dry weight (mg/g DW).

4.4. Statistical Analysis

Data were subjected to analysis of variance (randomized complete design; ANOVA).
Differences between mean values were assessed by Tukey honest significant difference
(HSD) with the analytical software STATISTIX 9.0 (Analytical Software, Tallahassee, FL,
USA). Prior to ANOVA, normality and homogeneity were tested by the Kolmogorov–Smirnoff
test and Cochran’s C test, respectively. Pearson’s correlation and regression analysis were
performed to analyse the relationship between variables.

5. Conclusions
It has long been known that strawberries provide benefits for human health, but the present
study highlights that, despite strawberry achenes representing a small fraction of the fruit,
their contribution to total fruit antioxidant content and capacity is very important, providing a
new profitable product for improving human health. Moreover, this study revealed that
strawberry antioxidant compounds are compartmented in the fruit, achenes having different
quantity and quality, and enriched in non-flavonoids, pointing to differences in the ability for
radical scavenging between flesh and achenes. Our results showed that commonly used
extraction methods underestimate antioxidant quantity, and that in vitro digestion is a good
approximation to test how antioxidants are released from different food matrices and are
able to be incorporated into the bloodstream under physiological conditions. This study
reveals the importance of considering achenes in breeding programs and in further studies
on strawberry varieties at different environmental ranges, since strawberry fruit antioxidant
composition varies depending on the cultivars and environmental conditions [55].

Acknowledgments
This research was funded by the RF2011-00016, TRA201300.6 and
PP.AVA.AVA201601.10 projects, co-financed by INIA and/or the Fondo Europeo de
Desarrollo Regional (FEDER 2007–2013, FEDER 2014-2020) and Fondo Social Europeo
(FSE 2007-2013). Dr. Ariza is supported by IFAPA Junta de Andalucía (20%), and by the
FSE 2007–2013 (80%) under the topic “Andalucía se mueve con Europa”. Francesca
Giampieri was supported by a Fondazione Umberto Veronesi Fellowship. Patricia
Reboredo-Rodríguez acknowledges the pre-doctoral and short-stay fellowships from the
University of Vigo.

Author Contributions
This article is the work of two cooperating groups. This work was done in Italy under the
guidance of Maurizio Battino. María Teresa Ariza, Patricia Reboredo--Rodríguez, Luca
Mazzoni, Tamara Yuliett Forbes-Hernandez, Sadia Afrin, Francesca Giampieri and
Massimiliano Gasparrini performed the experiments and analyzed the data together with
Carmen Soria, Elsa Martínez-Ferri, Bruno Mezzetti and Maurizio Battino. Bruno Mezzetti
and Maurizio Battino contributed reagents/materials/analysis tools. María Teresa Ariza and
Elsa Martínez-Ferri wrote the paper.

Conflicts of Interest
The authors declare no conflict of interest.

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Sensors (Basel). 2015 Nov; 15(11): 27854–27868.

XXII. Determination of the Mineral Composition and Toxic Element

Contents of Propolis by Near Infrared Spectroscopy
M. Inmaculada González-Martín,1,* Olga Escuredo,2 Isabel Revilla,3 Ana M. Vivar-
Quintana,3M. Carmen Coello,2 Carlos Palacios Riocerezo,4 and Guillermo Wells Moncada5
W. Rudolf Seitz, Academic Editor

Abstract

1. Introduction
Propolis are resinous substances collected from the buds and wounds of plants and
transformed by honeybees. They use exuded resins as well actively secreted substances
by plants that include lipophilic materials on leaves and leaf buds, gums,
lattices, etc.Propolis is used in hives to reinforce the structural integrity of the hive, to seal
entrances in wintertime, to reduce vibrations inside the hive, and also as an antiseptic agent.
Propolis has different sensorial and physico-chemical properties but most propolis share
considerable similarity in their general chemical composition: 50% resin, 30% wax, 10%
volatile oils, 5% pollen and 5% other organic compounds [1,2]. The composition of propolis
is very complex and varied depending on the phytogeographic diversity of the collection area
and the season [2,3]. Propolis is a natural source of antioxidants, which protect oils and
serum lipoprotein oxidation, highlighting its effects on antibody production and strengthening
the immune system [4]. The presence of phenolic compounds, terpenes, steroids and amino
acids in propolis has been studied extensively [5,6,7,8,9]. However, there is less information
on the content of trace elements in propolis, especially the possible presence of toxic
minerals, which can significantly affect its nutritional properties. Trace elements justify many
virtues of propolis, as participating in metabolism, vitamin and fermentative processes,
contributing to the healing of anemia, preventing arteriosclerosis and increasing the immune
capacity of the body [4]. The mineral contents in propolis is used as a distinguishing feature
of the geographical areas where it is produced [10,11], as an indicator of environmental
pollution [12] and to develop reliable traceability methods [13]. The presence of toxic
elements in propolis is associated with environmental pollution of anthropic origin around
the apiaries through various sources, such as air, water, plants and soil. Some probable
sources for cadmium and lead emissions are industrial sources [14,15,16,17]. Actually,
some plant species are known and well characterized regarding their capacity to accumulate
high levels of heavy metals in their biomass. They are classified as hyperaccumulators
[18,19,20]. Thus, in regions where beekeeping is practiced for commercial purposes, the
identification of bee plants with this characteristic is an important item to be evaluated, in
order to assure that the product fully meets technical and sanitary specifications imposed by
the regulatory agencies and the demanding consumer market in modern times [21].
Moreover, it is known that flavonoids tend to form stable complexes with metals such as
iron, chromium, nickel, copper or lead [22]. This property makes these elements become
one of the main pollutants of propolis.
The determination of the mineral composition of propolis is usually performed by ICP-mass
analysis and also by Electrothermal Atomic Absorption Spectrometry (ET AAS) and UV-Vis
spectrophotometry (UV-Vis) [23], neutron activation [10], electroanalytical methods [13], or
in the case of lead content, using the Graphite Furnace Atomic Adsorption Spectrometry
(GF AAS) method [24]. Thus, using flame atomic absorption spectrometry Formicki et
al. [12] determined the levels of cadmium, iron, magnesium, nickel, lead and zinc in various
bee products collected in Poland; Pierini et al. [13] used a electroanalytical method for
quantification of lead in Argentine propolis; Serra-Bonvehí and Orantes-Bermejo [25]
determined, among others, the levels of arsenic, cadmium, mercury and lead in samples of
propolis collected in southern Spain by ICP-atomic emission spectrometry and Finger et al.
[26], studied the content of cadmium, chromium and lead in Brazilian propolis from Paraná
by electrothermal atomization and flame atomic absorption spectrometry after calcination in
a muffle furnace. Moreover, Gong et al. [11] studied the relationships between the
geographical origin and the content of calcium, aluminum, magnesium, potassium, iron,
sodium, zinc, manganese, strontium, copper, chromium, nickel and toxic elements like
arsenic, cadmium and lead determined by inductively coupled plasma atomic emission
spectrometry after microwave digestion of Chinese propolis. However, there are few studies
evaluating the potential of near infrared spectroscopy (NIR) for quantitative analysis of
propolis. Visible/near infrared spectroscopy (Vis/NIRS) has been applied for the analysis of
chrysin and galangin in Chinese propolis [27] and to explore its applicability for the
determination of antioxidant properties [28].The detection of propolis falsification by the
addition of flavonoid glycosides and tree latex has been carried out by Fourier Transform-
NIR [29]. NIR has been also used to confirm the identity of isolated beeswax propolis [30].
Regarding the mineral characterization and to ensure the safety and quality of this product
for marketing in local and international markets, this paper proposes a fast method for
determining the mineral composition of propolis by using near infrared spectroscopy (NIR),
because this analytical technique is rapid, non-destructive, and requires little or no sample
preparation [31]. Therefore, in this paper, propolis samples from Chile and Galicia and
Castilla-León (Spain) were analyzed with the aim of develop a rapid method of analysis to
quantify some mineral and trace elements, using near-infrared spectroscopy with a remote
reflectance fibre-optic probe applied directly to the sample.

2. Experimental

2.1. Propolis Samples

Propolis samples (N = 91) have been directly collected by beekeepers in Chile (52 samples:
Bio-Bio region) and Spain (39 samples: Galicia and Castilla-León regions), from both
organic and conventional farms (the samples are all from different farms, and are not
duplicated). Samples were collected mostly with mesh and employing the scraping
technique, keeping them frozen until used in the laboratory. For the NIR analysis, of all the
91 samples, 71 were employed for the denominated calibration set, and the other 20 were
used for the external validation.

2.2. Chemical Analysis of the Mineral Composition

The mineral composition of propolis was determined using Inductively Coupled Plasma
Optical Emission Spectrometry (ICP-OES) for aluminum, calcium, iron, potassium,
magnesium, sodium and potassium, and Inductively Coupled Plasma Mass Spectrometry
spectrometry (ICP-MS) for zinc, chromium, nickel, copper and lead. Propolis samples were
crushed in the laboratory before their analysis with a grinder (Knifetec 1095 Sample Mill,
Foss Tecator, Hohnaa, Sweden). Prior to analysis of mineral elements, the mineralization
of the samples (0.2 g) in a microwave system (Ethos Sel Milestone, Ontario, ON, Canada)
was performed and subsequently introduced into a high pressure capsule. In a first phase 5
mL of HNO3 was added and a power of 1000 watts was applied for 5 min. Once the sample
was cool, another 5 mL of HNO3 and 1 mL of 30% H2O2 were added, applying a power of
1000 W for 10 min. The sample was cooled to room temperature, made up to 100 mL with
distilled water and stored at 4 °C until analysis.
The ICP-OES determinations were carried out using Ultima 2 equipment (JobinYvon, Paris,
NJ, USA), performing the calibration with certified standard solutions, ranging from 0.5 to 10
mg/kg. Detection limits were 0.1 mg/kg in solution. The ICP-MS determinations were
performed on an Elan 6000 instrument (Perkin-Elmer, Wellesley, Massachusett, MA, USA).
Do do this an internal standard (Sc, Y, Ho Ge 20 and 100 µg/kg) was added to an aliquot of
previously prepared sample. The calibration was performed with certified standard solutions,
also adding to the same internal standard for the calibration samples and adapted to a range
of 10 to 200 µg/kg. Detection limits were 0.1 µg/kg in solution.

2.3. NIR Spectroscopy

A Foss NIRSystem 5000 (Hillerod, Denmark) with a standard 1.5 m 210/210 bundle fibre-
optic probe (Ref. n° R6539-A) was used. Figure 1 shows a diagram of the NIRS system,
where the main components (optical system, sample module, fiber optic probe) can be
observed. The reflectance detectors receive radiation from diffuse scattering of the sample.
These detectors (four PbS elements) are positioned at 45° from the surface of the sample
(to minimize the specular reflectance). The used spectral range is 1100–2000 nm, since
above 2000 nm significant signal attenuation occurs because of the strong absorption of the
-OH groups that may be present in the optical fiber. The probe employs a remote reflectance
system and uses a ceramic plate as reference. The window is of quartz, with a 5 cm × 5 cm
surface area. The measurement of the spectra was carried out using NIRS technology and
a remote reflectance fibre-optic probe that was applied directly to samples of crushed
propolis. The spectra were recorded at intervals of 2 nm, performing 32 scans for both the
reference and samples. To minimise sampling errors, all the samples were analysed in
triplicate.

Figure 1
Schematic of the used NIRS equipment with fiber optic probe.

2.4. Statistical Data Analysis

2.4.1. Principal Component Analysis

Principal component analysis (PCA) was carried with the NIR spectral data from samples of
the calibration set using WinISI II version 1.50 (Intrasoft International, LLC, Silver Spring,
Maryland, MD, USA). This analysis transforms the original variables (wavelengths) into new
axes called principal components, which are orthogonal, so that the data set presented on
these axes are uncorrelated with each other. Spectra were pretreated by different
techniques, including multiplicative scatter correction (MSC), standard normal variate (SNV),
DT (Detrend) or SNV-DT [32]. These treatments allow minimization of the scattering effect,
since mainly the shift of the maximum and the width changes of the spectra were considered.

2.4.2. Modified Partial Least Squares Regression

The modified partial least squares (MPLS) regression method was used to obtain the NIR
equations for the minerals quantified in propolis. The principal aim was generate models that
allow the prediction of these components in the propolis matrix. Partial least squares (PLS)
regression is similar to principal component regression (PCR), but uses both reference data
and spectral information to form the factors useful for fitting purposes. To optimize the
multivariate regression equations, the spectral scattering effects were taken into account
with several mathematical treatments (MSC; SNV; DT or SNV-DT) [32]. MPLS is often more
stable and accurate than the standard partial least squares algorithm (PLS). Calibrations
were performed by modified partial least squares regression (MPLS) for each component,
after removing the samples for spectral (criterion H) or chemical reasons (criterion T). The
criterion H (Mahalanobis distance) explains the difference of the spectrum of an unknown
sample from the mean spectrum of the set of samples. Samples with an H-value higher than
3 are considered as a different population and are eliminated. Furthermore, the risk of there
being mistakes in the equations under practical conditions is very low or almost null when
the standardised H statistic (Mahalanobis distance) is used during routine analysis of
unknown samples. Using the T > 2.5 criterion, samples that were different from the
population owing to the chemical criterion were removed from the set. In MPLS, the NIR
residuals at each wavelength, obtained after each factor has been calculated, are
standardized (dividing by the standard deviations of the residuals at each wavelength)
before calculating the next factor [33]. In order to select the optimal number of factors and
to avoid over fitting, cross-validation is recommended [34]. The calibration set is divided into
several groups for the cross-validation. Each group is then validated using a calibration
developed on the other samples. Validation errors generated are combined into a root mean
square error of cross-validation RMSECV [35]. It has been reported that the RMSECV is the
best single estimate for the prediction capability of the equation and that this statistic is
similar to the average root mean square error of prediction (RMSEP) from 10 randomly-
chosen prediction sets. In all cases, cross-validation was performed by splitting the
population into six groups. Squared correlation coefficient was considered for the realization
of models. The squared correlation coefficient for predicted versus measured compositions
in cross-validation and the ratio of standard deviation (SD) to RMSECV of data set have
been used. This ratio (of the SD to the RMSECV) is called the ratio of performance to
deviation (RPD). This ratio is desired to be larger than 2 for a good calibration [36]. An RPD
ratio less than 1.5 indicates poor predictions and the model cannot be used for further
prediction. The statistics used to select the best equations were R2, determination
coefficient; RMSEC, root mean square error of calibration; RMSECV, root mean square
error of cross-validation [34].

3. Results and Discussion

3.1. Chemical Composition

The propolis samples had wide ranges in the mineral composition and high standard
deviation values for the studied elements. Table 1 shows the mean concentration and the
range of values of the mineral composition (aluminum, calcium, iron, potassium,
magnesium, sodium, phosphorus, zinc, lead, chromium, nickel and copper) of all the
propolis samples according to the geographical origin obtained by the chemical reference
methods (ICP-OES and ICP-MS).

Table 1
Mineral composition of propolis studied according to geographic origin (mg/kg).
 Castilla-León
Total (N = 91) Chile (N = 52) Galicia (N = 16)
Constituent (N = 23)
Mean Range Mean Range Mean Range Mean Range
43.0– 156.0– 43.0– 78.6–
Al 275.2 354.9a 105.2b 213.2c
833.9 833.9 193.7 518.4
219.1– 274.0– 219.1– 416.7–
Ca 833.4 910.4 563.2 847.3
5173.0 5173.0 1176.1 2169.2
46.1– 181.8– 46.1– 104.5–
Fe 424.6 536.6a 245.8b 295.8b
1538.0 1538.0 656.7 874.0
267.0– 267.0– 359.0– 685.9–
K 978.6 550.0a 1522.1b 1569.4b
4428.3 1841.2 3182.1 4428.3
63.5– 75.1– 63.5– 88.2–
Mg 234.1 261.8 206.4 190.6
1398.0 1398.0 427.0 460.3
116.0– 118.1– 152.3– 116.0–
P 235.0 228.8a 307.5b 198.5a
729.0 402.0 729.0 327.7
0.8– 2.3–
Cr 3.7 3.1 1.4–5.5 2.7 0.8–7.5 5.7
48.9 48.9
Nd– Nd–
Cu 1.8 1.6a Nd–6.2 5.4b 2.8 Nd–7.2
33.4 33.4
Nd– 0.6–
Ni 1.5 1.2 Nd–9.7 1.4 0.5–4.0 2.4
29.9 29.9
Nd– Nd–
Pb 5.8 2.6a Nd–8.0 2.2a Nd–6.0 15.5b
73.9 74.0
5.5– 5.5– 17.4– 11.1–
Zn 62.6 57.8 89.8 54.4
460.7 105.0 460.7 145.3
Open in a separate window
Statistical differences evaluated with Bonferroni test are marked at p < 0.05. Different letters
indicate significant differences between groups. Nd: Not detected, below quantification level
(0.01 mg/kg).
Also, a comparative study of the mineral composition of propolis from other geographical
origins is shown in Table 2.

Table 2
Mineral composition of propolis samples of different geographic origin (mg/kg).

South Spain Argentina Argentina Brazil

China [11]
Constituent [25] [5] [10] [26]
(N = 32)
(N = 25) (N = 10) (N = 96) (N = 42)
Al 308–582 – – 426–1959 Nd–1840
Ca 1773–6683 39–4138 – 404–2637 Nd–4800
Fe 312–1270 101–1697 400–1945 310–2125 –
K 735–4790 101–1697 – 314v1894 410–5490
 South Spain Argentina Argentina Brazil
China [11]
Constituent [25] [5] [10] [26]
(N = 32)
(N = 25) (N = 10) (N = 96) (N = 42)
Mg 301–1405 1115–1031 – 135–1129 500–4650
P 171–611 – – – –
Cr 0.3–3 Nd 0.6–3.7 Nd–12 Nd–19
Cu 2.1–4 Nd – Nd–15 Nd
Ni 0.6–3 Nd – Nd–3 –
Pb 0.07–4 – – 4–55 Nd–160
Zn 163–1236 33-147 11–105 35–386 Nd–500
Poland [37,38] Poland [12] Turkey
Constituent Croatia [39]
(N = 20) (N = 80) [40]
Al – – – –
Ca – 40–317 – 79–118
Fe 28–101 14–251 101 –
K – 51–117 8.2 121–364
Mg 137–823 10–46 – –
P – – – –
Cr – 0–1 – –
Cu – 0.3–6 – 45–96
Ni 2–10 0–0.3 9.8 –
Pb 0.9–3 0.3–64 2.7 –
Zn 18–71 8–933 71.5 176–676
Open in a separate window
Mean value; [5] Lima et al.; [10] Cantarelli et al.; [11] Gong et al.; [12] Formicki et al.; [25]
Serra-Bonvehí, and Orantes-Bermejo; [26] Finger et al.; [37] Roman et al.; [38] Roman et
al.; [39] Cvek et al.; [40] Dogan et al.
The possibility of correlation between all elements was investigated. Potassium, the most
abundant mineral compound, was significantly (p < 0.01) and negatively correlated with
aluminum (r = −0.29) and iron (r = −0.32), and positively correlated with phosphorus (r =
0.28), lead (r = 0.33) and copper (p < 0.05, r = 0.26). Iron was positively correlated (p < 0.01)
with aluminum (r = 0.57), as observed for potassium, and also with calcium (r = 0.36),
magnesium (r = 0.35) and zinc (r = 0.31). Magnesium showed a strong correlation with
calcium (r = 0.97) and a significant correlation (p < 0.01) with phosphorus (r = 0.31). Finally,
regarding the potentially toxic elements beside the significant correlation between lead and
potassium, a strong correlation was found between chromium and nickel (r = 0.90).This
result differs from previously reported by Finger et al. [26] who found a significant correlation
between calcium and potassium and from those reported by Formicki et al. [12] who found
a significant but negative correlation between iron and magnesium. These two works also
found a positive correlation between cadmium and lead that they attributed to a common
polluting source. However in this work the cadmium was not analyzed but a strong
correlation between chromium and nickel was found, metals that were not analyzed in those
works.

3.2. NIR Calibration Equations

For the calibration equations, a set of 71 samples of propolis from different sources (Galicia,
Castilla-León, Chile) was used. Initially, the principal component analysis (PCA) was
performed. In all cases, the spectral variability explained above was 99%. Using both criteria
(criterion H and T) six samples were deleted for the calibration of aluminum, 11 for the
calcium, six for iron, 13 for potassium, seven for magnesium, five for phosphorus, 10 for
chromium, 13 for copper, four for nickel, 12 for lead and seven for zinc. Calibrations were
performed by modified partial least squares regression (MPLS), using the spectral data and
chemical data matrix (obtained with ICP-OES, ICP-MS). The best of the different
mathematical treatments, the concentration range, standard deviation and the calibration
parameters for all elements are shown in Table 3.

Table 3
Statistical descriptors of calibration by NIR of the minerals.

Est
Constitue Math Mea . Est. RMSE RMEC RP
N SD R2
nt treatment n Mi Max C V D
n
Standard
6 257. 123. 0.7
Al MSC 0.0 629.2 56.5 78.8 1.6
5 4 9 9
1,4,4,1
None 6 509. 245. 1247. 0.8
Ca 0.0 102.7 162.1 3.1
1,4,4,1 0 4 9 0 3
None 6 425. 231. 1118. 0.6
Fe 0.0 129.3 147.3 1.6
1,4,4,1 4 6 0 6 9
Detrend
5 772. 559. 2451. 0.9
K only 0.0 126.7 244.3 2.3
8 8 6 7 5
2,4,4,1
None 6 198. 193. 0.7
Mg 0.0 779.4 105.6 160.6 1.2
1,4,4,1 4 4 7 0
Standard
6 236. 103. 0.9
P MSC 0.0 548.4 26.0 40.4 2.6
6 7 9 4
1,4,4,1
SNV
6 0.4
Cr only2,4,4, 2.9 0.8 0.5 5.3 0.6 0.8 1.0
1 8
1
Detrend
5 0.6
Cu only 1.2 1.6 0.0 5.8 0.9 1.2 1.3
8 4
2,10,10,1
None 6 0.5
Ni 1.2 1.0 0.0 4.2 0.7 1.0 1.0
0,0,1,1 7 2
 Est
Constitue Math Mea . Est. RMSE RMEC RP
N SD R2
nt treatment n Mi Max C V D
n
None 5 0.7
Pb 3.5 3.7 0.0 14.6 2.0 3.3 1.1
1,4,4,1 9 0
SNV only 6 0.8
Zn 57.4 28.9 0.0 144.2 10.6 18.7 1.6
2,4,4,1 4 7
Open in a separate window
N, number of samples; SNV, standard normal variate; MSC, multiplicative scatter correction;
SD, standard deviation; Est. Min: minimum value estimated by the model developed; Est
Max: Maximum value estimated by the model developed; RMSEC, root mean square error
of calibration; R2, determination coefficient; RMSECV, root mean square error of cross-
validation; RPD, ratio of performance to deviation.
The quantification of the chemical elements using NIR technology is possible thanks to the
distinct associations of these elements with the organic and inorganic material and with the
water molecules, because of relative strong absorption of the overtones and combination
modes of OH, sulphates and carbonate groups. Since 1981, there have been several reports
on mineral elements in plants determined using NIR [41,42,43,44]; if NIRS can be used for
determining mineral concentrations this is due to the association between minerals and
organic functional groups or the organic matrix [45]. When NIR radiation is absorbed by
molecules the energy is converted to molecular vibration energy. Molecules which are
infrared (IR)-active are those which undergo a change in the dipole moment during
transition, this means that bonds commonly found in biological systems such as C-H, O-H
and N-H bonds are IR active. The prediction of trace elements by NIRS in agricultural
products has been reported even less frequently and has always been used in the context
of plants, only a few reports were found related to the use of NIRS for macro and trace
minerals in both grasses and hay samples [46], in botanical fractions of semi-arid grassland
[47] or legumes [48]. In the case of the propolis, the most likely association of minerals with
organic matter is through flavonoids. Propolis presents a general composition of 40%–50%
resins and balsams (that contain, in turn, 50% flavonoids and phenolic acids), 30%–40%
wax, 10% volatile oils, 5% pollen and 5% minerals and other organic compounds. The
chemical structures of flavonoids favour the formation of very stable complexes with heavy
metals, making propolis a hyperaccumulator of these kinds of elements [18,19,20]. The
correlation between the concentration and that measured at different wavelengths is given
by the expression: y = β0 + β1 Xλ1 + β2 Xλ2 + β3 Xλ3 +…+ βn Xλv, where β are the coefficients
and Xλ1, Xλ2, Xλ3,… Xλn, are the wavelengths at which the correlation of the concentration of
the components is maximum (in + or − value). The higher values of those β coefficients for
each of the parameters studied (Al, Ca, Fe, K, Mg, P, Cr, Cu, Ni, Pb, Zn) together with the
wavelength where these coefficients showed their maxima and minima absorption are: Al
(λ, 1330 and 1556 nm; β, 1986.9 and −2060.8); Ca (λ, 1500 and 1542 nm; β, 21479.9 and
21880.6); Fe (λ, 1228 and 1112 nm; β, 1481.3 and 1224.0); K (λ, 1481.3 and 1224 nm; β,
−106,030.1 and 85,857.3); Mg (λ, 1520 and 1532 nm; β, 40,352.5 and −66,976.4); P (λ,
1554 and 1968 nm; β, 16,557.2 and 19,820.5); Cr (λ, 1366 and 1590 nm; β, 7.8 and −10.6);
Cu (λ, 1244 and 1356 nm; β, 46.3 and −27.5); Ni (λ, 1520 and 1558 nm; β, 25.1 and 20.15);
Pb (λ, 1820 and 1968 nm; β, 436.5 and −266.6); Zn (λ, 1816 and 1976 nm; β, −9692.4 and
6045.0). It is noteworthy that an important number of the mineral elements determined in
this work, showed a correlation between the concentration and the absorbance at
wavelengths in the 1510–1550 nm interval. According to Shi et al., [49], this region of the
spectrum corresponds to the absorbance of two aromatic rings of the basic structure of
flavonoids and to the vibration of the 2nd overtone of the carbonyl group of flavonoids. Other
mineral elements showed correlations with wavelength values near 1400 and 1900 nm
related to the water O-H overtones.
The results showed that it is possible to determine the composition of aluminum, calcium,
iron, potassium, magnesium, phosphorus, chromium, copper, nickel, lead and zinc in the
ranges indicated in samples of propolis of different origins (from Chile and two Spanish
regions) by direct application of a NIR fiber-optic probe on crushed propolis samples without
prior treatment or manipulation.

3.3. Validation

3.3.1. Internal Validation (Prediction)

Models’ evaluations were performed by cross-validation. In this method, the set of calibration
samples is divided into a series of subsets, in our case six. Of these, five were taken for the
calibration set and one for the prediction set. The process is repeated as many times as
there are sets, so that all pass through the calibration set and the prediction set. Using this
process, we validated the models used and checked their prediction capacities. Figure
2 shows the correlation of the values obtained in the laboratory with respect to those
predicted by NIR with remote reflectance fibre-optic probe for aluminum, calcium, iron,
potassium, magnesium, phosphorus, zinc, chromium, nickel, copper and lead in propolis.
The prediction capacity of the model obtained was evaluated with the ratio performance
deviation (RPD) [36].This parameter is defined as the relationship between the standard
deviation of the chemical method (SD ref) and RMSECV, root mean square error of cross-
validation encountered in the NIRS model. Table 3 shows the values obtained for RPD
parameter that were comprehended between 3.1 for calcium and 1.0 for nickel. Therefore,
NIRS technology presents a capacity for prediction that is interesting for the determination
of mineral composition in samples of unknown propolis.
Open in a separate window
Figure 2
Comparison of reference values (mg/kg) with values predicted by the calibration equations
obtained by NIR. R2, determination coefficient; RMSEP, root mean square error of
prediction. (a) Al; (b) Ca; (c) Cr; (d) Cu; (e) Fe; (f) K; (g) Mg; (h) Ni; (i) Pb; (j) P; (k) Zn.

3.3.2. External Validation

The proposed NIR method was verified by applying the developed chemometric model to
20 new samples of different compositions (called external validation set samples). The
recording of the spectra in triplicate and the average spectra was considered. Then,
calibration equations obtained in this work were applied and predicted values were
compared with reference data for aluminum, calcium, chromium, copper, iron, potassium,
magnesium, nickel, phosphorus, lead and zinc determined by OES-ICP and ICP-MS
spectrometry. Table 4 shows the results of external validation corresponding with the
prediction of mineral composition of 20 independent samples.

Table 4
External validation of minerals in propolis by NIR (number of samples: 20).

Constituent Mean SD Est. Min Est. Max RMSEP RMSEP(C) RPD

Al 239.5 91.8 24.6 370.2 114 113.4 0.8
Ca 946.3 290.6 70.8 1283.0 106.5 116.2 2.5
Fe 392.9 164.7 97,3 745.5 164.2 168.5 1.0
K 1052.1 572.3 453.4 2503.5 250.3 258.2 2.2
Mg 198.4 157.6 63.7 441.8 157.1 165.3 1.0
P 237.0 83.3 77.4 364.7 48.1 46.1 1.8
Cr 3.1 0.64 1.7 3.9 0.92 0.90 0.7
Cu 1.37 1.4 0.2 4.5 1.5 1.6 0.9
Ni 1.4 0.6 0.1 2.4 1.3 1.3 0.5
Pb 4.4 3.1 1.2 14.0 1.2 1.4 2.2
Zn 55.4 28.6 7.1 128.2 18.3 24.1 1.2

SD, standard deviation; Est. Min: minimum value estimated by the model developed; Est
Max: Maximum value estimated by the model developed; RMSEP, root mean square error
of prediction; RMSEP(C), root mean square error of prediction corrected with bias; RPD,
ratio of performance to deviation.

4. Conclusions
NIR methodology with a remote reflectance fibre-optic probe is presented as an effective
method of analysis for determining some mineral and toxic trace elements in crushed
propolis. This method is applicable to samples with a wide range of contents of aluminum,
calcium, iron, potassium, magnesium, phosphorus, zinc, chromium, nickel, copper and lead.
This is the first work on the quantification of mineral elements in a propolis matrix with NIR
technology. The method is particularly interesting for the prediction of potentially toxic
elements such as zinc, copper and lead, since it allows a previous detection in a short time
(3 or 4 min). This ensures the safety and quality of propolis for commercialization in national
and international markets.

Acknowledgments
This study was made possible by funds from Project 18KBCN/463AC01 of the University of
Salamanca. The authors with to express their special gratitude to all the beekeepers in this
study for their cooperation.

Author Contributions
M. Inmaculada González-Martín conceived and designed the experiments and wrote the
paper. Olga Escuredo performed the experiments. Isabel Revilla and Ana M. Vivar-Quintana
analyzed the data and wrote the paper. M. Carmen Seijo, Carlos Palacios Riocerezo and
Guillermo Wells Moncada contributed reagents/materials/analysis tools.

Conflicts of Interest
The authors declare no conflict of interest.

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