The Research Institute on Nutrition and Food Security at the University of Barcelona
(INSA-UB) was founded in 2005 by twenty-two research groups from the Faculties of
Pharmacy and Food Science; Biology; Chemistry; and Geography and History, as well
as other UB-affiliated centers and hospitals. Most of the groups at the Institute
are, or at least are part of, the research groups established by the Government of
Catalonia. INSA-UB was founded to meet the current societal need for research, training
and service provision in the sectors related to the agro-alimentary industry. Researchers
at the Institute are experts in different fields of nutrition; food analysis and control;
food safety and the study of the social and economic impact of food.
The main objectives of the institute are to promote research in the fields in which
it works; to encourage collaboration between researchers and the establishment of
multidisciplinary teams; to promote participation in research programs and institutional
administration, particularly in European research projects; to encourage the development
of joint projects with companies in the sectors related to its scope; to make available
all the social potential of the UB in this area, especially the training of technicians
and specialists and provision of services; to promote the transfer of knowledge and
the dissemination of research results between society and government; and to advise
consumers, businesses and public authorities on nutrition, food safety and quality.
The Second Annual Workshop hosted by INSA-UB titled, “Cocoa and Chocolate: Science
and Gastronomy” provided a forum where the latest findings around three aspects relating
to cocoa (gastronomy, food technology and nutritional health) could be disseminated,
and to understand its complexity.
Cocoa is a product that originates from the beans of Theobroma cacao. It contains
macronutrients, such as proteins, lipids and carbohydrates, micronutrients, and bioactive
compounds, such as flavanols, fiber, and theobromine . However, in addition to
its nutritional characteristics, its consumption is associated with pleasure or with
different emotional changes. The gastronomical interest in cocoa is increasing due
to its sensory properties and use in new cuisine, as well as new tendencies towards
high-priced chocolates and delicacies containing high amounts of cocoa. The chocolate
industry and its market are large and wide due to cocoa products having reached a
critical mass. Finally, in terms of nutrition and health, its bioactives (epicatechin
and procyanidins) infuse cocoa with healthy properties, which can provide beneficial
protection towards cardiovascular risk factors, brain and immune functions, and cancer
prevention, among others [2,3,4,5,6].
The “Cocoa and Chocolate: Science and Gastronomy” workshop took place at the headquarters
of INSA-UB, located at the Food and Nutrition Torribera Campus, University of Barcelona
(Santa Coloma de Gramanet, Barcelona, Spain). Although the main invited scientific
presenters focused on different aspects of cocoa and chocolate, the selected oral
and poster presentations reflected the research related to the Institute on nutrition
and food safety, and provided an opportunity to share research and create new partnerships
with members of the Institute and participants from other institutions and/or industries.
More than 150 delegates from 16 different institutions attended to the II Annual Workshop
on 9 November 2016.
The workshop opened with the results of the research grant awarded in 2014 by INSA-UB.
The different sessions focused on cocoa and chocolate, divided thematically into:
(1) history and gastronomy; (2) science and technology; and (3) nutrition and health.
The session regarding history and gastronomy as they related to cocoa and chocolate
discussed some of the issues surrounding the worldwide importance of cocoa products
in the market; the outlook of different cultures around chocolate; and how industry
uses these concepts to exploit the sector and sell products. This session also discussed
the use of chocolate throughout different historical periods and cuisines around the
world. This last presentation provided a technological point of view and looked at
the manner in which industry had used history to evolve from using a metate, the first
tool used to produce chocolate, towards the development of new manufacturing processes
to arrive at the current movement of “Bean to Bar”. Other technological approaches
addressing the structures and textures of chocolate though sharp melting and the melting
behavior of crystals to obtain new textures of chocolate were also discussed. The
workshop also offered three different practical sessions focused on historical and
new concepts of cocoa and chocolate. The first practical session used tempering chocolate
to view the effects of crystallization in cocoa on the final quality of the chocolate
coating. The second practical focused on the application of chocolate in the mythical
creations of modern cuisine, where participants practiced formulating different textures
of chocolate, including spherifications and foams. The third practical allowed participants
to taste cocoa and cocoa products by its passage through history and different types
of cultivation. The last part of the workshop highlighted the wide range of health
effects attributed to cocoa consumption, including its positive effects on chronic
diseases, cognitive function and cardiovascular disease, and emphasized the role of
the main bioactive cocoa compounds responsible for these effects.
This Conference Report contains the summary of lectures (Section 2) and selected abstracts
(Section 3) detailing in each subsection the title of the presentation followed by
the author’s names. All the author’s affiliations are detailed in the appendix section
at the final of the manuscript (Appendix A).
2. Summary of Lectures
2.1. The History and Consumption of Chocolate in Catalonia, Spain
Did you know that the average person in Catalonia consumes 3.2 kg of chocolate per
year, which is slightly above the Spanish average of between three and 3.1 kg, per
person per year? In contrast, the consumption of chocolate in countries, such as Germany,
Switzerland, Belgium, the USA and Japan is between eight and 13 kg, per person per
year. What are the causes of this imbalance between some countries and others? Furthermore,
did you know that 90% of the population worldwide likes chocolate?
Chocolate, therefore, has massive product acceptance, or as marketing experts’ say,
“the product has a critical mass”. Only 10% of those asked stated that they did not
like chocolate. Few products are as widely popular with consumers, a fact that should
be exploited by the chocolate and cocoa product sector. However, despite being such
a popular and universal product, there are many unknown elements for the immense majority
Another important aspect to consider is the industrial point of view. In Spain, the
entire chocolate and cocoa products industry has to appeal to the public to buy chocolate
as part of their “gift” expenditure; while in France, the focus is based on one’s
own consumption, gastronomic pleasure or the hedonistic factor (“I indulge myself”).
This can be seen by the much higher levels of chocolate consumption in France than
in Spain. As chocolate bars can be elements of socialization and thus become the undisputed
leaders in the market for middle and upper-middle class people, it is the area a company
wants to be managed effectively.
For instance, it is interesting to analyze the strategy used by one of the most important
chocolate industries in Spain. Valor, a family business located in Villajoyosa with
over 130 years of history, focuses on “WHO”, being understood as a variable response:
“who may my consumers be” rather than on “WHAT”, being understood as the product on
offer. This is called market-driven management or company management with market orientation.
2.2. Cooking with Chocolate: Catalan Cuisine
Chocolate was introduced to European gastronomy from the colony of New Spain (specifically,
from the territory that currently corresponds to Mexico and Central America). It expanded
out of Spain thanks to monastic orders, and the fame that it acquired in the Spanish
Court and at the tables of the nobility and the commercial bourgeoisie. Its basic
consumption was as a beverage (taken in water and later in milk) that was sweetened
(with honey or sugar), flavored (with vanilla, cinnamon, or other additives) and with
different degrees of viscosity (watery or thick). Special tableware associated with
its preparation and consumption was even designed. This consisted of the chocolate
grinder; the jicaras in which the hot chocolate was served; and a tray called the
mancerina (in honor of Pedro Álvarez de Toledo y Leiva, VI Marqués de Mancera and
Viceroy of Peru who made it fashionable) where it was accompanied with puff pastry
or pancakes. Its consumption became popular with the appearance of the first chocolatiers.
One of the characteristics of Baroque cuisine was the use of “table chocolate”, obtained
by grinding of cocoa or chocolate with nuts and spices, in making sauces. Undoubtedly,
the Mexican moles, especially the “poblano mole”, are the best-known chocolate-based
sauces. In Europe, it was used in the preparation of chocolate sauces that basically
served to accompany bush meat dishes. Catalan cuisine is one of a few European cuisines
that incorporate chocolate in their sauces, not only for meat but also fish and seafood,
as well as mixed “turf and surf” dishes.
In the nineteenth century, the solidification of chocolate led to the development
of new processes that produced chocolate bars and chocolate sweets, and thus expanded
its use in confectionery.
Today, a study on the use of chocolate in the traditional cuisine of the Catalan coast
is being coordinated by the Catalan Coast Cuisine Museum, located in Can Ganga in
Tossa de Mar (Costa Brava, Spain) and is managed by the town hall and the University
2.3. Technology of Cocoa and Chocolate: From Cocoa to Chocolate
This talk was given from the personal point of view of the managing director of a
chocolate production facility. This lecture explained the process of manufacturing
chocolate and its evolution from the “metate”, the first utensil used for its manufacture,
to the current tendency: “Bean to bar”.
Major milestones in the evolution of the manufacturing process were introduced: from
the great discoveries of the nineteenth century that allowed the popularization of
the product; to its mass production in the second half of the twentieth century due
to the development of mechanical technology and product sophistication; and the introduction
of robotics at the end of the twentieth century. Finally, the incipient “return to
origins” model was explained, which is driven in part by the new consumer values of
the twenty-first century and the rapid development promoted by social networks.
2.4. Structures and Textures of Chocolate
Chocolate is made up of cocoa butter crystals formed as a continuous body in which
tiny particles of sugar, cacao mass, and other ingredients are dispersed. Sharp melting
and the quick release of flavor and sweetness/bitterness are determined by the melting
behavior of cocoa butter crystals. Cocoa butter exhibits six different polymorphic
forms, referred to as I–VI. Among them, form V is industrially promoted through specific
dynamic thermal treatments (tempering), as this polymorph provides the desired melting,
textural, and mouth-feel characteristics of chocolate, so that this form may also
be maintained over time. Nevertheless, some undesired external factors, such as storage
at high temperatures, temperature fluctuation or oil migration processes may cause
the blooming phenomenon, which is caused by the formation of form VI needle-shaped
cocoa butter crystals and is of significant concern for the chocolate and confectionery
industries. Dynamic thermal treatments may be also applied for the development of
new chocolate textures, such as the velvet effect. Thermal treatments enable the formation
of thin layers of cocoa butter crystals with much smaller particle sizes and a lower
melting point compared to normally-tempered chocolate, which creates the soft-mouth
2.5. Health Implications of Regular Cacao Consumption
Until recently, chocolate and cocoa were a most restrained pleasure. Their consumption
was often linked to negative effects like increased blood lipids, weight gain, or
skin problems. Scientific research over the last two decades; however, has depicted
a very different scenario. Indeed, consuming cocoa products has shown positive effects
on blood pressure; endothelial function; antioxidant status; platelet activity or
insulin resistance; in addition to improvements in mood and cognitive function. These
beneficial health effects have been attributed to the polyphenolic fraction of cocoa,
which is rich in flavanols and procyanidins, since it is commonly accepted that a
diet rich in flavonoids promotes health and delays the onset of different non-transmittable
pathologies, such as cardiovascular disease, Type 2 diabetes mellitus, obesity, etc.
However, other bioactive compounds in cocoa products such as methylxanthines and dietary
fiber should not be overlooked. The author reviewed the scientific evidence for the
beneficial effects of cocoa and chocolate consumption on some of the most common chronic
diseases, looked at the mechanisms of action involved and considered the contribution
of non-phenolic bioactive compounds.
2.6. Effects of Cocoa on Cardiovascular Health
Evidence based on epidemiological studies suggests that dietary flavonoids may play
a critical role in the prevention of coronary heart disease (CHD). Cocoa (Theobroma
cocoa) and its derived products, such as cocoa powder, represent a very rich source
of dietary flavonoids, containing a higher content per serving than tea, red wine,
legumes or fruit. The health benefits associated with cocoa consumption have been
related to their protective effect mainly on cardiovascular disease, but also in other
related diseases such as diabetes and age-related cognitive decline. Observational
studies have shown that the Kuna India population from the San Blas Islands of Panama
has very low rates of hypertension and cardiovascular disease, effects that have been
related to their high cocoa intake. Epidemiological and clinical trials have also
evaluated the effects of cocoa intake on different cardiovascular risk factors. Several
studies have confirmed that cocoa intake reduces blood pressure in normotensive and
hypertensive subjects. In fact, dark chocolate intake increases nitric oxide (NO)
generation that leads to vasodilatation and reduces systolic and diastolic blood pressure
by 2.77 mmHg and 2.20 mmHg, respectively, whereas white chocolate does not reduce
blood pressure. Another mechanism by which flavanols may lower blood pressure is through
the inhibition of the angiotensin-converting enzyme (ACE). In addition, cocoa intake
improves lipid profile and insulin sensibility; reduces platelet activity and function;
and ameliorates endothelial dysfunction and arterial stiffness. At least part of these
beneficial effects have been attributed the anti-inflammatory and anti-oxidant properties
of the polyphenols contained in cocoa. Other beneficial effects may also be due to
changes in gut microflora. Recent population-based studies have observed an inverse
relationship between all-cause mortality, and cardiovascular morbidity and mortality.
In conclusion, cocoa consumption ameliorates cardiovascular risk factors, including
diabetes, and reduces cardiovascular morbidity and mortality. However, the beneficial
effects may be even higher if cocoa consumption is included into a well-balanced healthy
diet, such as the Mediterranean diet.
2.7. The Food Metabolome Biomarker: A New Strategy for Evaluating the Intake of Dietary
Bioactive Compounds, Foods and Dietary Patterns. Summary of the Project FRI-2013
The study of the relationship between dietary bioactive components (i.e., phytochemical)
intake and health status requires accurate measurements of dietary exposure. Thus,
although biomarkers cannot completely replace traditional methods of dietary assessment,
there is broad consensus that the application of Metabolomics in the detection and
identification of novel and robust biomarkers of exposure to diet and/or consumption
of healthy foods can improve and validate these methods.
The main aim of this project was to define the technical and scientific aspects of
“Food Metabolome” as a new strategy for identifying biomarkers that allow the evaluation
of exposure to healthy diets.
A pilot study was carried out with 45 students who twice collected 24 h urine samples.
In addition, a validated food frequency questionnaire and three-day food recall was
obtained from each participant. Differences within the information provided by the
traditional methods of collecting dietary information were analyzed with respect to
the food metabolome. The composition of the food metabolome was compared according
to the stratification of the population based on the consumption of foods or dietary
patterns. The relevance and originality of this project lies in merging the new powerful
nutrimetabolomic fingerprint approach with well-known dietary epidemiology techniques
to produce new insights into the biomarkers of healthy dietary intake patterns in
a student population under free-living conditions.
3. Selected Abstracts
In this section, the abstracts were presented orally or as a conference poster. A
total of five oral presentations and 26 conference posters were selected for inclusion.
3.1. Oral Presentations
3.1.1. Histamine Intolerance Management in Clinical Practice: Do Putrescine Contents
Justify the Exclusion of Certain Foods?
Background and objectives: Histamine intolerance is a disorder in the homeostasis
of histamine due to reduced enzymatic intestinal activity, which causes an accumulation
of this amine in plasma and the appearance of multi-faced allergy-like clinical symptoms.
Current clinical strategy treatments are based on the limitation of histamine containing
food and/or the supplementation with exogenous diamine oxidase enzyme (main enzyme
of histamine intestinal metabolism). Histamine distribution in food is wide and variable,
even among different batches of the same product. However, some foods (presumably
without or with small amounts of this amine) are usually excluded from these restrictive
diets for being related to the onset of symptoms. The presence of other bioactive
amines such as putrescine, could be co-responsible for triggering adverse effects
by competing for the same metabolic pathway. The aim of this work was to study the
occurrence of putrescine in some foods of plant origin where their exclusion was not
justified by their histamine content. Batch origin, storage conditions (one week at
four degrees Celsius) and peel removal were studied in order to assess their contribution
to putrescine content.
Methodology: Three vegetables, courgette (n = 24), paprika (n = 10) and cucumber (n
= 9), were selected out of the group of totally excluded vegetal products recommended
by the clinical management of histamine intolerance, despite the presumable absence
of histamine. The content of histamine and putrescine in these items were determined
in triplicate by ultra-high-pressure liquid chromatography with fluorescence detection
Results and conclusions: Histamine was not found in any of the analyzed products.
Putrescine was present in all samples of courgette (ranging from three to 13 mg/kg);
paprika (one to 145 mg/kg); and cucumber (one to seven mg/kg). In general, there were
no statistically significant differences in putrescine content among products in the
same batch; however, those differences were significant when different origins were
considered. The elimination of the courgette and cucumber peels did not influence
putrescine content. A significant increase in putrescine levels was observed in practically
all samples subjected to one-week refrigerated storage, with an average increase up
to 53% in the case of courgette. Although the absence of histamine in courgette, paprika
and cucumber has been confirmed, putrescine found in these products could potentially
explain their association with the symptomatology of histamine intolerance as it competes
with histamine detoxifying enzymes. The increase of putrescine during domestic storage
also needs to be taken into account, and product freshness is a key parameter to be
considered in minimizing putrescine intake in the dietary framework of histamine intolerant
3.1.2. Home Cooking and Ingredients Synergism Improve Lycopene Isomers in Sofrito
Background and objectives: Tomato products rich in lycopene Z-isomers are of interest
since these carotenoids present more bioavailability and antioxidant capacity than
the all-E lycopene forms. Intrinsic food properties, processing, and the interaction
between dietary components are all factors that can influence the content, type and
bioavailability of carotenoids. The aim of this study was to evaluate how the content
of carotenoids and their isomerization in tomato-based Mediterranean sofrito, might
be affected by the process of home cooking, as well as by the presence of other ingredients.
Methodology: A full factorial design 24 was applied to clarify the contribution of
extra virgin olive oil (5%–10%), onion (20%–40%), and garlic (2%–4%); and cooking
duration (30–60 min) on the carotenoid composition of sofrito. The identification
of the carotenoids was based on retention time; chromatography with standards; UV/VIS
absorption spectrum: λmax, %III/II and %Ab/II; and mass spectrum. High pressure liquid
chromatography with diode array detection (HPLC-DAD) was used to provide quantitative
results, in conjunction with external calibration curves with standards.
Results and conclusions: The main factors associated with a higher production of 5-Z-lycopene,
9-Z-lycopene and 13-Z-lycopene in sofrito were the cooking duration and onion content.
Onion proved to be the most interesting ingredient in the sofrito formulation due
to its effect on enhancing lycopene isomerization. This vegetable could be explored
as an ingredient to improve the bioavailability of lycopene in tomato products.
Acknowledgments: National Council for Scientific and Technological Development (CNPq,
Brazil); The Ministry of Education, Culture and Sport (MECD), Spanish Ministry of
Economy and Competitiveness (MEC), CIBER Physiopathology of Obesity and Nutrition
(CIBEROBN) and Generalitat de Catalunya (Spain).
3.1.3. Biomarkers of Cocoa Intake: Multimetabolite Biomarker Models as a Novel Strategy
to Improve Dietary Assessment
Background and objectives: There is a growing body of evidence on the beneficial effects
of cocoa consumption on the cardiovascular system. Untargeted metabolomics was used
as a hypothesis-generating tool.
The aim of this work was to contribute to the identification of biomarkers related
to cocoa ingestion (biomarkers of intake).
Methodology: An untargeted high-pressure liquid chromatography-time of flight mass
spectrometry (HPLC-Q-ToF-MS) metabolomics strategy was applied in urine samples collected
in acute and short-term clinical trials, as well as in observational studies. To improve
the prediction of cocoa consumption, a combined urinary metabolite model was constructed
using stepwise logistic regression analysis. Receiver operating characteristic (ROC)
curves were performed to evaluate the predictive capacity of candidate biomarkers.
Results and conclusions: Dietary cocoa fingerprinting was characterized by using a
complex metabolic pattern linked to cocoa phytochemicals (alkaloids and polyphenols)
and processing-derived compounds. A large proportion of metabolites were characteristic
of cocoa exposure independently of the study design. The area under the curve (AUC)
values (95% confidence interval (CI)) for the model were 95.7% (89.8%–100%) and 92.6%
(81.9%–100%) in the training and validation sets, respectively, whereas the AUC for
individual metabolites were <90%.
Discriminating metabolites of cocoa exposure were replicated among three studies with
different design, increasing the level of evidence from observed associations. The
predictive capacity of dietary exposition was improved using multimetabolite combined
models compared individually with the same compounds.
Acknowledgments: This work has been supported by Spanish Ministry of Economy and Competitiveness
(MINECO) and co-founded by the Spanish Federation of Rare Disease FEDER: AGL2009-13906-C02-01;
the CONSOLIDER-INGENIO 2010 Program, FUN-C-FOOD (CSD2007-063); PCIN-2014-133; and
the PI13/01172 Project (Plan Nacional de I+D+i 2013–2016) by the “ISCIII-Subdirección
General de Evaluación y Fomento de la Investigación”. We also thank the award of 2014SGR1566
from the Agency for Management of University and Research Grants (AGAUR). Mar Garcia-Aloy
thanks the AGAUR for the predoctoral FI-DGR 2011 fellowship. Rosa Vazquez-Fresno thanks
the Training of Research Staff FPI fellowship; Rafael Llorach and Mireia Urpi-Sarda
thank the “Ramón y Cajal” program (RYC-201007334 and RYC-2011-09677, respectively),
all from the MINECO and Fondo Social Europeo.
3.1.4. Cocoa Consumption and Its Health Impact in University Students
Background and objectives: Although cocoa is well recognized as an excellent source
of polyphenols with multiple benefits in human health; and has shown immunomodulatory
actions in rats, there are no observational data relating to cocoa consumption and
The aim of the present study was to associate cocoa consumption, obtained through
a validated food frequency questionnaire (FFQ), with several health indicators in
Methodology: A sample of 270 university students from the University of Barcelona
and Egas Moniz Cooperativa de Ensino Superior completed a validated FFQ for cocoa
consumption  and a survey about their health status. The values of these health
variables were then compared against students grouped according to their low (L),
moderate (M) and high (H) cocoa consumption.
Results and conclusions: University students had an average consumption of cocoa of
about 13.2 g/day, mostly derived from chocolate bars, but also from dairy products.
The proportion of people suffering chronic diseases was lower in the M group than
in the L group. Likewise, the proportion of students with flu was lower in group H
than those in the L and M groups. More importantly, the proportion of students with
allergies was much lower in the M and H groups than those in the L group, with moderate
cocoa intake associated with a lower presence of allergic symptoms.
Acknowledgments: The authors would like to thank Joan Vila from the Hospital del Mar
Medical Research Institute (IMIM) for the technical and scientific support in data
3.1.5. The Effects of Sofrito on FGF21 (Fibroblast Growth Factor 21) Expression and
Background and objectives: Obesity is a worldwide health problem due to its associated
comorbidities. Fibroblast growth factor 21 (FGF21) is a hormone considered to be a
promising therapeutic candidate for the treatment of obesity, and in the maintenance
of metabolic homeostasis in healthy people. Previously published data suggests the
exertion of FGF21 pleiotropic function on glucose/lipid metabolism.
Some polyphenols or polyphenol-rich foods modulate FGF21 expression. Our hypothesis
was that FGF21 could link dietary polyphenols with their healthy metabolic effects.
Accordingly, our objective was to study the effects of sofrito on FGF21 function in
Methodology: Lean and obese Zucker rats were fed for eight weeks with a sofrito-supplemented
diet. Serum and different tissues were collected after sacrifice, and FGF21 expression
and signaling were analyzed. The expression of FGF2; the FGF21 receptors (FGFR1 and
FGFR4); the co-receptor KLB; and the mRNA levels of the FGF21 target genes c-fos and
Egr1, were analyzed in liver and white adipose tissue (WAT).
Results and conclusions: Obese individuals demonstrate FGF21 resistance as they have
increased levels of FGF21 due to its overexpression in the liver in response to a
down regulation of receptor expression in target organs. Our results reproduced this
FGF21 resistance in obese rats; this phenotype was partially reverted in obese rats
fed with a sofrito-supplemented diet shown by an induction of FGFR1 and KLB mRNA levels
in visceral WAT. A down regulation of FGF21 expression in liver that correlated with
increased mRNA levels of c-fos and Egr1 was also noted. Thus, the results suggested
that a sofrito-supplemented diet improved FGF21 signaling in obese Zucker rats.
Acknowledgments: Ministerio de Educación, Becas The National Commissions for Scientific
and Technological Research (CONICYT-Chile); SAF2013-41093, AGL2010-22319-C03; AGL2013-49083-C3-1-R
from Spanish Ministry of Economy and Competitiveness; Instituto de Salud Carlos III
(ISCIII), CIBER Physiopathology of Obesity and Nutrition (CIBEROBN).
3.2. Poster Sessions
3.2.1. The Evaluation of Anti-Inflammatory Activity Using Zebrafish Larvae and Its
Application to Compounds in Food
Background and objectives: Inflammation is a normal protective response of the innate
immune system to tissue injury. Based on the principle of generating a mechanical
injury to attract leukocytes to damaged zones, zebrafish embryos and larvae have proven
to be suitable for the investigation of the kinetics of inflammation in vivo. The
principal aim of this work was to develop an experimental zebrafish larvae model to
evaluate inflammatory activity quickly and efficiently, as well as the application
of this model in order to study the anti-inflammatory activity of compounds present
Methodology: Inflammation was induced after four days post-fertilization in zebrafish
larvae by tail transection and incubation with bacterial lipopolysaccharides in order
to obtain leukocyte migration to the zone of injury. Migrating zebrafish leukocytes
were detected in situ by myeloperoxidase staining and image analysis.
Results and conclusions: Optimal parameters (exposure time; temperature; image analysis;
and quantification of migration) were established to evaluate anti-inflammatory activity.
Using our optimized zebrafish larvae assay, we found that compounds with a well-known
anti-inflammatory activity (indomethacin, doxepin, piroxicam, dibenzoidolium DPI)
significantly inhibited migration. This effect was also observed for some natural
substances such as naringenin, beta-carotene, oleuropein, quercetin-3-beta-glucoside,
malvidin-beta-glucoside and kaempferol-3-beta-glucoside.
Finally, our data demonstrated that zebrafish larvae provide a rapid and reliable
model in which to quantify neutrophil migration in vivo. We further proposed that
this model might prove useful for screening the anti-inflammatory activity of natural
Acknowledgments: This research was funded by Spanish Ministry of Economy and Competitiveness
AGL2013-49083-C3-1-R. Scholarship granted by the Ecuadorian government through the
National Secretariat for Higher Education, Science, Technology and Innovation (SENESCYT)
3.2.2. In Vivo Screening of Probiotics and Prebiotics Potential on a Model of Acute
Gastroenteritis by Rotavirus
Background and objectives: Group A rotaviruses are the most common causative agents
of acute gastroenteritis in children under two years old. Previous reports have suggested
breastfeeding and the use of probiotics and prebiotics as protective agents for ameliorating
the clinical course of rotavirus infection. This study was designed to establish the
utility of the suckling rat rotavirus infection model to evaluate the protective role
of probiotics and prebiotics.
Methodology: From the first day of life, Lewis neonatal suckling rats received daily
particular strains of probiotics and prebiotics. At the end of the first week of life,
the heterologous simian rotavirus SA11 was inoculated orally in the supplemented groups
and in the non-supplemented group. Rotavirus infection was evaluated daily by clinical
indexes based on color, texture and the amount of feces obtained. Fecal samples were
used to quantify viral shedding and specific immunity by means of antibody production.
Other variables, such as intestinal architecture impairment, changes in gut permeability,
and gene expression of particular molecules in the intestine, were also evaluated.
Results and conclusions: Two different probiotics and prebiotics were tested to study
their differential impact. The behaviors of all strains and prebiotic molecules assessed
differed substantially, from the protection of diarrhea to scarce effects. Furthermore,
the mechanisms involved in such effects were also shown to be product-specific. In
conclusion, these results demonstrated the suitability of this model for the screening
of supplements like probiotics and prebiotics as anti-infective agents in the case
of rotavirus acute gastroenteritis and for the investigation of their mechanisms of
3.2.3. Impact of Cocoa Diet on the Gut Microbiota in a Rat Oral Sensitization Model
Background and objectives: It is well known that there exists an interrelation among
dietary compounds, microbiota and food allergies. Previous studies have reported that
cocoa intake produces changes on intestinal microbiota and induces immune tolerance
in a rat oral sensitization model. Taking these facts into consideration, our aim
was to investigate the modifications on gut microbiota from a cocoa-enriched diet
in an oral sensitization model.
Methodology: Lewis rats were orally sensitized with ovalbumin plus cholera toxin and
were fed either a 10% cocoa diet or a standard diet. Fecal microbiota was analyzed
through a metagenomic study after four weeks of the dietary intervention.
Results and conclusions: Gut microbiota did not undergo many changes due to the oral
sensitization but showed differences in a cocoa-enriched diet. In this sense, the
cocoa diet reduced the absolute abundance of bacteria from Firmicutes and Proteobacteria
phyla, and increased that of Tenericutes and Cyanobacteria phyla. In particular, there
was an increase in bacteria belonging to the RF39 order (Mollicutes class, Tenericutes
phylum) and those belonging to the Streptophyta order (Chloroplast class, Cyanobacteria
phylum). Even though no changes were observed in the absolute abundance of the Bacteroidetes
phylum, cocoa intake increased the relative abundance of the Prevotella genus and
In conclusion, a cocoa diet inhibited oral sensitization and modified the gut microbiota
in a rat oral sensitization model, suggesting that these changes in microbiota composition
might be partially responsible for the tolerogenic effect of cocoa.
Acknowledgments: This study was financially supported with funding from the Spanish
Ministry of Economy and Competitiveness (AGL2011-24279).
3.2.4. Diet and Sleep in Spanish Children and Adolescents with Attention Deficit Disorder
Background and objectives: Nutrient deficiencies and unhealthy diets have been related
to attention-deficit/hyperactivity disorder (ADHD). Sleep disorders also influence
ADHD symptoms. To date, little is known about the impacts of diet and sleep on ADHD
symptoms. The aim of this study was to assess the relationship between diet and sleep
disturbances in a sample of children and adolescents with ADHD.
Methodology: A total of 120 children and adolescents (60 newly diagnosed with ADHD,
and 60 controls) were studied in a sex- and age-matched case-control study. ADHD diagnosis
was conducted by trained psychiatrists in accordance with the Diagnostic and Statistical
Manual of Metal Disorders (DSM-IV-TR). Food consumption and nutrient intake (measured
by a food frequency questionnaire) and sleep (measured with the sleep disturbance
scale for children, a sleep diary and actigraphy) were objectively measured. Lineal
regression was used to determinate associations between diet and sleep.
Results and conclusions: Sleep disorders were more prevalent in ADHD children compared
to the control group. It was found that a lower consumption of fiber, vegetable protein,
thiamin, vitamin D, iron, folate, potassium, and magnesium was associated with a higher
prevalence of total sleep problems (p < 0.05). In the same way, a lower consumption
of fiber, vegetable protein, thiamin, vitamin B6, vitamin E, iron, folate, magnesium
and zinc intake was associated with a higher prevalence of “Excessive Daytime Sleepiness”
(p < 0.05). Our results are in accordance with other authors who concluded that nutritional
deficiencies such as that of B vitamins, magnesium and zinc may impair sleep by altering
neural responses via circulating intestinal hormones (e.g., insulin, CCK ghrelin)
or by affecting the synthesis of serotonin and melatonin.
Acknowledgments: All phases of this study were supported by grant PI11/2009 from the
Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Spain; the Consejo
Nacional de Ciencia y Tecnología (CONACYT-México); and the Consejo Nacional de Ciencia
Tecnología e Innovación (SENESCYT-Ecuador).
3.2.5. Trans-Lycopene from Tomato Juice Attenuates Immune-Inflammatory Biomarkers:
A Dose-Response Intervention Trial
Background and objectives: The aim was to evaluate the effects of carotenoids from
tomato juice (TJ) on the expression of inflammatory biomarkers, by performing a four-week
dose-response nutritional trial in a population at high cardiovascular risk.
Methodology: The study was an open, prospective, randomized, cross-over, and controlled
clinical trial. Twenty-eight volunteers (mean age 69.7 ± 3.1 years; mean BMI 31.5
± 3.6 kg/m2) at high cardiovascular risk were assigned to consume daily for four weeks
in random order: 200 mL (low-dose, LD) or 400 mL (high-dose, HD) of TJ made with 5%
common olive oil, or water as a control (C). Blood samples were collected at baseline
(B) and after each intervention. Endpoints included changes in plasmatic carotenoids;
pro-inflammatory cytokines C-reactive protein (CRP) and interferon-γ (IFN-γ); chemokines
interleukin-8 (IL-8), eotaxin, and CXCL motif chemokine 10 (CXCL10); inter-cellular
adhesion molecule 1 (ICAM-1); and vascular-cell adhesion molecule 1 (VCAM-1).
Results and conclusions: Compared with the control group, both TJ interventions induced
significant decreases in ICAM-1, VCAM-1, CRP, and IL-8 (p ˂ 0.05), and also showed
a trend to reduce eotaxin, IFN-γand CXCL10, in a dose dependent manner. These decreases
were significantly correlated, mainly with the trans isomeric form of lycopene, while
the other carotenoids present in TJ were not associated with any significant changes
in these molecules.
Acknowledgments: This work was supported by the Interagency Council on Science and
Technology (CICYT) (AGL2010-22319-C03; AGL2010-22319-C02; AGL2010-22319-C01; AGL2013-49083-C3-1-R)
and the Instituto de Salud Carlos III (ISCIII), CIBER Physiopathology of Obesity and
Nutrition (CIBEROBN) from the Spanish Ministry of Economy and Competitiveness (MEC)
and Generalitat de Catalunya (GC) 2014 SGR 773. Mariel Colmán Martínez thanks the
University, Research and Information Society Department of the Generalitat de Catalunya
(FI-DGR 2013); Miriam Martínez Huélamo would like to thank the Spanish Ministry of
Science and Innovation (MICINN) predoctoral program; Palmira Valderas-Martínez thanks
the trainee research staff grant (APIF) predoctoral fellowship from the University
of Barcelona, and Sara Arranz thanks the “Sara Borrell” postdoctoral program (CD10/00151).
3.2.6. Validation of a UHPLC-FL Method for the Determination of Histamine and Methylhistamine
in Urine: A New Approach for the Diagnosis of Histamine Intolerance
Background and objectives: Histamine intolerance is a disorder in the homeostasis
of histamine due to the reduced intestinal activity of the diamine oxidase (DAO) enzyme
that causes an accumulation of this amine in plasma and the appearance of adverse
effects. A new approach for the diagnosis of this intolerance could be through the
determination of histamine and its metabolites in urine. The aim of this work was
to develop and validate a rapid method in which to unequivocally determine histamine
and methylhistamine in human urine by Ultra High Performance Liquid Chromatography
and Fluorimetric detection (UHPLC-FL).
Methodology: Purification and concentration of 24 h urine samples were achieved by
solid-phase extraction using a mixed cation exchange cartridge. Chromatographic separation
was performed using a Waters Acquity™ UHPLC equipment with an Acquity UHPLC™ BEH C18
column coupled to a fluorimetric detector.
Results and conclusions: The method provided a satisfactory linearity and chromatographic
sensitivity with a detection limit lower than 0.035 mg/L and a quantification limit
falling below 0.045 mg/L for both analyses. The precision, in terms of relative standard
deviation, was lower than 5.5% and the accuracy, as mean recovery, was higher than
99% for both analyses. The UHPLC-FL method described has been demonstrated as a reliable
procedure for determining histamine and methylhistamine in less than 11 min of chromatographic
elution. The applicability of the method was studied in urine samples from volunteers,
resulting in a reliable tool for the determination of both compounds and, is therefore
a potential new approach for the routine diagnosis of histamine intolerance.
3.2.7. Dietary Management of Histamine Intolerance: Influence of Putrescine in the
Metabolism of Histamine by Diamine Oxidase Enzyme
Background and objectives: Histamine intolerance is a disorder in the homeostasis
of histamine due to a reduced intestinal activity of diamine oxidase enzyme (DAO),
which causes an accumulation of this amine in plasma. Current clinical strategies
used for the treatment of this disorder are based on the exclusion of histamine containing
foods and/or exogenous DAO enzyme supplementation. However, some foods without histamine
or with low levels of this amine are excluded from these diets because patients relate
them to the development of symptoms. Two hypotheses may be considered in order to
explain this correlation: (a) certain foods can provoke the release of endogenous
histamine; and (b) the presence of other amines, such as putrescine, in foods can
trigger symptoms. In order to study this second hypothesis, the aim of this work was
to evaluate the ability of putrescine to compete with histamine for the DAO enzyme.
If putrescine was also metabolized by DAO, it could potentially increase the intestinal
absorption of histamine and facilitate the appearance of symptomatology.
Methodology: The kinetic in vitro study of the metabolism of histamine and putrescine
was carried out in buffer (0.05 M and pH 7) supplemented with different concentrations
of both amines and DAO enzyme of porcine origin. The solutions were kept at a constant
temperature with regular stirring (37 °C, 200 rpm). Aliquots taken at 10 min intervals
up to three hours were analyzed by ultra-high-pressure liquid chromatography with
fluorescence detection (UHPLC-FL).
Results and conclusions: It was found that putrescine was also a substrate of the
DAO enzyme, although the rate of histamine metabolism was in all cases greater than
the rate of putrescine, independent of the proportion of both amines in the solution.
Individually, the reduction substrate at the 30 min test was 100% for histamine and
approximately 50% for putrescine, which was not completely metabolized until the 120
min test. When the joint metabolism of both amines was considered, a decrease in the
disappearance rate of histamine as the proportion of putrescine increased was observed.
The results confirmed the hypothesis that putrescine from food reduces the metabolism
rate of histamine by the DAO enzyme. These results may explain how high levels of
putrescine in foods promote the intestinal absorption of histamine and its accumulation
in plasma, which results in triggering symptoms of histamine intolerance. Therefore,
in the dietary management of this intolerance, both histamine and putrescine contents
should be considered.
3.2.8. Probiotics with Diamine Oxidase Activity: Histamine Reduction Quantification
and Location of the Responsible Gene
Background and objectives: The use of probiotics with histaminase activity to help
metabolize exogenous histamine in the intestine could be an alternative treatment
for histamine intolerance by reducing DAO, thus broadening the spectrum of beneficial
effects associated with probiotics. The ability to degrade histamine has been described
in different microorganisms, mainly in bacteria used as starters. This activity has
been shown as strain dependent and some authors have proposed that the gene coding
for the histamine oxidase enzyme could be located in a plasmid. To our knowledge,
this activity has not yet been described in any probiotic microorganism. The aim of
this work was to study the histamine oxidase in vitro activity in Lactobacillus gasseri
and L. sakei strains and the possible location of the gene that encodes this enzyme
into a plasmid DNA.
Methodology: Lactic acid bacteria have been identified with histaminase activity in
vitro, specifically, Lactobacillus sakei and L. gasseri strains with reduction rates
of 8%–50% and 70%–85%, respectively. The presence of plasmids was studied in four
L. sakei strains and L. gasseri (1010 LU, LU 1011, 1015 LU, LU 1016) using a commercial
kit (Miniprep plasmid PureYield™ System-A1220, Promega Biotech Ibérica, S.L., Madrid,
Results and conclusions: No plasmid DNA was isolated from any of the tested strains,
indicating that histamine oxidase activity is encoded in the genomic DNA. The fact
that this gene was not located in plasmid DNA in the studied strains does not mean
that it could not be located in a plasmid in other strains or species, so this study
should be extended to other microorganisms. The responsible gene for histamine oxidase
activity of L. gasseri was identified in genomic DNA by sequence alignment using the
Basic Local Alignment Search Tool (BLAST), which allowed comparison between the results
obtained from sequencing and sequences described in the literature for histamine oxidase
activity in lactic bacteria.
3.2.9. Effect of Composition of Tomato-Based Mediterranean Sofrito on Color Changes
during Accelerated Storage
Background and objectives: Color in food is an important quality indicator of the
degradation of product during storage and affects consumer-buying decision. The color
of sofrito is related to the content of carotenoids, which are associated with health
benefits. The objective of this study was to evaluate whether different ingredients
of sofrito formulations might have an effect on color preservation during accelerated
Methodology: The effect of ingredients on the color change kinetics of sofrito was
investigated during storage at 40 °C for 120 days. Eight formulations were made with
the presence and absence of extra virgin olive oil (10%), onion (20%), garlic (2%)
and the possible combinations between them. All samples were cooked for 30 min at
100 °C and packed in glass jars. Color changes during storage were measured by a colorimeter
(model CR-410, Konica Minolta, Ramsey, NJ, USA). The brightness (L*), total color
difference (∆E*), and hue (h°), as well as the kinetic models of zero, first and second
order were evaluated by lineal regression.
Results and conclusions: Results suggested that the presence of onion and extra virgin
olive oil allowed a better preservation of color, as demonstrated by the hue values
and the total color change kinetic model. Formulations containing garlic accelerated
the degradation of color, indicated by good models of L* and suffered the most expressive
total color change during storage. Thus, the presence of onion and extra virgin olive
oil must be encouraged in the sofrito formulation to preserve the color and carotenoid
Acknowledgments: National Council for Scientific and Technological Development (CNPq,
Brazil); The Ministry of Education, Culture and Sport (MECD); Spanish Ministry of
Economy and Competitiveness (MEC); CIBER Physiopathology of Obesity and Nutrition
(CIBEROBN), and Generalitat de Catalunya (Spain).
De la Garza
3.2.10. Maternal Weight and Genetic Variants of the Fatty Acid Desaturase and Elongase
Genes on Children’s Fatty Acids and Cognition
Background and objectives: Maternal polymorphisms (SNPs) in fatty acid desaturase
(FADS) and elongase (ELOVL) genes alter long chain (LC) polyunsaturated fatty acid
(PUFA) availability, compromising fetus supply and therefore cognitive development.
Our aim was to determine how maternal polymorphisms in FADS and ELOVL genes influenced
children’s fatty acids (FAs) and cognition according to maternal weight.
Methodology: Children (n = 72) from the (Early Programming of Obesity) PREOBE cohort
were divided according to maternal pre-pregnancy BMI: Group 1 (normoweight mothers,
n = 31) and Group 2 (overweight/obese mothers, n = 41). Maternal SNPs were genotyped
(seven in FADS1, five in FADS2, three in ELOVL2 and two in ELOVL5). At 18 months in
age, children’s cheek cells were analyzed to measure PUFAs in the phospholipid fraction
and cognition was assessed using the Bayley III Cognitive Scale.
Results and conclusions: Major homozygotes in Group 1 had a higher arachidonic acid/dihomo-γ-linolenic
acid index for rs174537 (FADS1) and higher cognition for rs174545 (FADS1) than minor
allele carriers. Both tendencies persisted in all single-nucleotide polymorphisms
(SNPs) in FADS1 while cognition tendency also persisted in FADS2. Group 2 showed that
major homozygotes had higher cognition for rs2397142 (ELOVL5). Regarding rs2397142,
Group 2 showed higher cognition when mothers carrying minor alleles had high docosahexaenoic
acid intake and high plasma eicosapentaenoic acid/arachidonic acid and docosahexaenoic
acid/arachidonic acid ratios.
Maternal weight, genotype and FAs influence children’s outcome. FADS1 SNPs in normoweight
mothers decreased children’s cognition and enzymatic activity in FA metabolism. Children’s
cognition was also lowered by ELOVL5 SNPs in obese, but not normoweight, mothers with
low n-3 FA levels. A high n-3 FA intake should be promoted, especially in obese pregnancies,
to enhance cognition in children.
3.2.11. Effect of Adipokines Supplementation on Growth and Immunity in Suckling Rats
Background and objectives: Breast milk contains bioactive factors that support the
growth and development of the tissues and organ systems of newborns. Specifically,
they promote the maturation of the digestive and immune systems and their physiological
function. Among these bioactive factors, leptin and adiponectin—metabolic hormones
or adipokines—may regulate physiological and metabolic processes, as well as the inflammatory
response. However, the specific role of these adipokines in early life remains unexplored.
The aim of this study was to establish the effects of leptin and adiponectin supplementation
on rat growth and its development. It was also evaluated for its effect on the intestinal
and systemic antibody response.
Methodology: For this purpose, newborn Wistar rats were supplemented daily by oral
gavage with leptin or adiponectin during the suckling period (21 days). In order to
determine the morphometric variables, the body weight of the animals was assessed
daily. At days 10, 14 and 21, the small intestine, spleen and thymus were obtained
and weighed. Moreover, at day 21, plasma and gut washes were obtained to quantify
systemic immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin G (IgG)
concentrations and intestinal IgA and IgM levels, respectively.
Results and conclusions: Adipokines supplementation did not affect body growth. Leptin
increased intestinal weight, whereas adiponectin increased thymus weight. Regarding
the development of the immune system, leptin and adiponectin differentially modulated
the intestinal and systemic antibody response in early life.
Acknowledgments: This study was supported by the Spanish Ministry of Economy and Competitiveness
3.2.12. Bioavailability of Carotenoids Sofrito in Healthy and Young Men Following
a Diet Rich in Foods with Antioxidant Compounds
Background and objectives: The sofrito is a typical sauce in a Mediterranean diet
that contains tomato as the main component and other plant-based ingredients (olive
oil, onion and garlic) rich in bioactive compounds such as carotenoids. However, bioavailability
is low and various factors such as processing and food matrix may interfere positively
and/or negatively. The aim of this study was to evaluate the bioavailability of carotenoids
in sofrito after a diet rich in antioxidants.
Methodology: A pilot study was performed on twenty-two healthy, non-smoking men, aged
between 18 and 32 years who consumed sofrito (at a dose of 240 g/70 kg). Identification
and quantification of carotenoids in the plasma collected at 0, 5 and 24 h was carried
out by high-pressure liquid chromatography with ultraviolet detector (HPLC-UV), 1100HPLC
HP system (Hewlett-Packard, Waldbronn, Germany).
Results and conclusions: Carotenoids increased significantly (p > 0.05) between zero
and 24 h (3.227 ± 1.738 vs. 7.630 ± 3.933) and between five and 24 h (4.570 ± 2.524
vs. 7.630 ± 3.933). Specifically, lycopene increased 24 h after sauce intake (2.731
± 1.089 vs. 5.816 ± 3.332). However, no significant differences were found among xanthophylls
(0.624 ± 0.083 vs. 0.603 ± 0.221 vs. 1.010 ± 0.838) after intervention. The carotenoids
in plasma increased significantly after sofrito intake, mainly lycopene after 24 h
of sofrito intervention.
Acknowledgments: This study was funded by the Ministry of Economy and Competitiveness
(AGL2013-49083-C3-1-R) and CIBER Physiopathology of Obesity and Nutrition (CIBEROBN).
3.2.13. Tyramine and Histamine Risk Assessment Related to Consumption of Dry Fermented
Sausages by the Spanish Population
Background and objectives: Tyramine and histamine are bioactive amines involved in
the appearance of adverse health effects in at risk population subgroups. A high intake
of tyramine can trigger hypertensive disorders, among others, especially when combined
with the administration of drugs that inhibit the metabolism of this amine (monoamine
oxidase inhibitors (MAOI) or reversible inhibitors of monoamine oxidase A (RIMA)).
In the case of histamine, intoxication symptoms may occur due to an excessive intake
of this amine or as a result of the intolerance caused by blockage or deficit diamino
oxidase (the enzyme responsible of the intestinal metabolism of histamine). Dry fermented
sausages, which are extensively consumed in Spain, can easily accumulate high levels
of these hazards. The aim of this study was to evaluate the extent that the consumption
of dry, fermented sausages could contribute to the appearance of these adverse health
effects on an at-risk Spanish population.
Methodology: Probabilistic estimation of tyramine and histamine intake was performed
using the Monte Carlo simulation technique (@Risk 7.0), by combining the distributions
of the content of these amines in dry fermented sausages (n = 474) with the consumption
data of the Spanish population (ENIDE, “Encuesta Nacional de Ingesta Dietética Española”).
For the risk assessment, the maximum tolerable safety levels adopted by European Food
Safety Authority (EFSA); the percentage of potential meat product consumers in Spain;
data on MAOI drug usage (Spanish Agency of Medicines and Sanitary Products); and the
prevalence of histamine intolerance were considered.
Results and conclusions: The risk of suffering hypertensive crisis or histamine intoxication
through the consumption of dry, fermented sausages in a healthy population may be
considered negligible. Conversely, the results confirmed that consumption of fermented
sausages carried risk in patients treated with MAOI drugs, as well as in people diagnosed
with histamine intolerance. In particular, individuals undergoing treatment with MAOI
drugs have a high risk of hypertensive crisis, with a probability of 34% exceeding
the safety threshold of tyramine (six milligrams) in a meal. However, given the reduced
actual usage of these drugs, only three out of a million people could suffer these
adverse effects. With regard to histamine intolerance, EFSA has not established a
safe limit for histamine since the risk of the onset of symptoms varies depending
on the degree of DAO deficit. 66% of sausages contained variable amounts of histamine
(<0.01 to 475 mg/kg), therefore their consumption is liable to trigger symptoms. Considering
that approximately 1% of the population is histamine intolerant, the population at
risk would reach 7000 cases per million individuals.
3.2.14. Serum Diamine Oxidase (DAO) Activity Levels in Patients with Migraine
Background and objectives: Histamine intolerance is a disorder in the homeostasis
of histamine due to a reduced enzymatic intestinal DAO activity, which causes an accumulation
of this amine in plasma. Some authors have associated DAO deficiency with several
pathologies of high prevalence in the population, such as migraine, inflammatory and
degenerative intestinal disorders or atopic dermatitis. A better knowledge of serum
DAO levels in the migranous population is important in order to establish the relationship
between histamine intolerance by DAO deficit and migraine. The objective of this study
was to determine the prevalence of DAO deficiency in healthy subjects and in patients
with a confirmed migraine diagnosis.
: The prevalence of DAO deficiency was assessed in a total of 198 volunteers recruited
at the Headache Unit of the Hospital General de Catalunya; 137 had a confirmed migraine
diagnosis (according to current International Headache Society criteria) and 61 healthy
volunteers without clinical criteria for migraine considered as a control group. Blood
samples were collected from all subjects by venipuncture with an ethylenediaminetetraacetic
acid tube after an eight-hour fasting period and the samples were analyzed with enzyme-linked
immunosorbent assay to determine DAO enzyme activity. Values below 80 HDU/mL (Histamine
Degradation Units/mL) were considered as DAO deficient.
Results and conclusions: The mean value of DAO activity of the migraine population
(64.5 ± 33.5 HDU/mL) was significantly lower (p < 0.0001) than that obtained from
healthy volunteers (91.9 ± 44.3 HDU/mL). A high incidence rate of DAO deficiency (88%)
was observed in the group of patients with migraine. In addition, 44% of non migranous
subjects had levels of DAO activity lower than 80 HDU/mL, and no information about
other pathologies associated with DAO deficiency was registered. DAO deficiency was
more prevalent in migraine patients than in the healthy population. Further studies
are needed in order to test whether DAO deficiency is the cause of triggering migraine.
In that case, a free histamine diet and/or DAO supplementation could play an important
role in the treatment of this disorder.
3.2.15. Nutrimetabolomic Approach to Identify Biomarkers for Cocoa-Food Products Consumption
Monitoring in Healthy Volunteers
Background and objectives: Cocoa consumption has been linked to health-promoting activities
mainly associated with cardiovascular disease. Nutrimetabolomics explores the complex
relationship between the consumption of dietary compounds and the maintenance of health
or disease development, with the aim to discover new biomarkers of intake and effect.
In this regard, the aim of this work was to study the urinary cocoa product fingerprint
for future definition of a specific biomarker imprint of cocoa in healthy and young
Methodology: The cocoa-food products intake was defined according to a food frequency
questionnaire, which was previously completed by the free-living healthy volunteers.
Subjects were classified as high (≥5 g/day), medium (between 1.16 and 4.28 g/day),
and low (≤1 g/day) consumers. Urine samples from the subjects were analyzed by high-pressure
liquid chromatography—time of flight mass spectrometry (HPLC-Q-TOF-MS), followed by
multivariate data analysis (orthogonal signal correction for partial least squares
discriminant analysis (OSC-PLSDA) and hierarchical cluster analysis (HCA)). The metabolomics
analysis was carried out using a R package “Metabolite Automatic Identification Toolkit”
(MAIT) that included an in-house food metabolome database.
Results and conclusions: Urinary metabolome showed significant differences between
the three consumer groups. Several metabolites were associated with cocoa-foods consumption,
the most important derived from theobromine metabolism. In addition, microbial polyphenol
metabolites and vanillin-derived metabolites were putatively identified. These metabolites
have previously been associated with cocoa intake in other populations, however, as
far we know, this is the first time that these metabolites have been identified in
a free-living, healthy and young population using a metabolomics approach. This study
reinforced interest in the replication of results, as well as the capacity of metabolomics
to identify the cocoa-food product footprint by combining epidemiological nutritional
data and metabolomics.
Acknowledgments: This work was supported by a FRI-13 award from INSA-UB (2014–2015).
We also thank the EU Joint Programming Initiative A Healthy Diet for a Healthy Life
on Biomarkers BioNHFOODBALL (PCIN-2014-133—Spanish Ministry of Economy and Competitiveness
(MINECO)) and the award of 2014SGR1566 from the Generalitat de Catalunya’s Agency
AGAUR. Mireia Urpi-Sarda would like to thank the “Ramón y Cajal” program from MINECO
and the Fondo Social Europeo.
3.2.16. Food Metabolome Biomarkers Associated with Dietary Patterns in Healthy Volunteers
Background and objectives: There is a growing interest in the field of nutrition epidemiology
to identify biological markers related to the intake of nutrients, foods and dietary
patterns associated with the prevention of diseases. The aim of this study was to
identify urinary biomarkers to describe the dietary patterns in healthy young individuals.
Methodology: This was an observational study undertaken at the Campus de l’Alimentació-Torribera
(University of Barcelona) during the academic year of 2014–2015. Forty-four healthy
volunteers were included in the study, based on the following criteria: non-smoking,
healthy and with an age between 18 and 25 years old. Participants recorded their dietary
habits with a validated food frequency questionnaire (FFQ). Participants collected
24 h urine samples in two different visits (separated by three months). FFQ data were
grouped into 28 categories and a k-means cluster analysis (Metaboanalyst 3.0) was
used to identify the dietary patterns. Urine was analyzed by high-pressure liquid
chromatography—time of flight mass spectrometry (HPLC-q-Tof) (Applied Biosystems).
Differences in the urinary biomarkers between patterns were analyzed using the metabolomics
R package MAIT.
Results and conclusions: K-means analysis identified two dietary patterns; one characterized
by a significant higher intake of whole grain cereals, fruit, vegetables, pulses,
coffee, moderate wine, nuts and dried fruit (p < 0.05) (High Dietary Vegetables Pattern,
HDP); while the other cluster was characterized by a significant higher intake of
refined grain cereals, processed meats, snacks and high energy beverages (p < 0.05)
(Low Dietary Vegetable Pattern, LDP). Urinary food metabolome showed differences between
both dietary patterns. The HDP was characterized by a higher urinary excretion of
urolithin A glucuronide, a characteristic microbial metabolite of nut consumption;
of 4-hydroxyhippuric acid, a microbial metabolite related to higher intake of vegetables
and derived foods; proline betaine, which is related to orange consumption; and trigonelline,
a metabolite linked to coffee consumption. The LDP was characterized by a higher urinary
excretion of tyrosine sulfate, an endogenous metabolite associated to cardiovascular
risk factors. In conclusion, these results indicated that HDP and LDP are reflected
in the urinary metabolome. Among the HDP biomarkers, it is important to note that
microbial derived metabolites could be very useful in evaluating food intake in epidemiological
studies. Furthermore, LDP biomarkers could be linked to future diseases in epidemiological
Acknowledgments: This work was supported by a FRI-13 award from INSA-UB (2014–2015).
We also thank the EU Joint Programming Initiative A Healthy Diet for a Healthy Life
on Biomarkers BioNHFOODBALL (PCIN-2014-133-MINECO-Spain) and the award of 2014SGR1566
from the Generalitat de Catalunya’s Agency AGAUR. Mireia Urpi-Sarda would like to
thank the “Ramón y Cajal” program from MINECO and the Fondo Social Europeo.
3.2.17. Development of an Advanced High Pressure Liquid Chromatography Tandem Mass
Spectrometry (HPLC-MS/MS) Method for the Determination of Carotenoids and Fat-Soluble
Vitamins in Human Plasma
Background and objectives: Although carotenoids and fat-soluble vitamins are of utmost
interest in human health, sensitive and specific methods for their simultaneous determination
are scarce. The aim of this research was to develop and validate a new high-pressure
liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the quantification
of selected carotenoids and fat-soluble vitamins in human plasma.
Methodology: The samples were extracted using a double liquid-liquid extraction method
with n-hexane/butylated hydroxytoluene (100 mg/L). HPLC-MS/MS was carried out in an
Agilent 1100 HPLC system coupled to a QTRAP4000 triple quadrupole mass spectrometer.
Results and conclusions: In 50 min, 16 samples were separated with an excellent resolution
and suitable mass signal intensity. The proposed HPLC-MS/MS method led to improvements
in the limits of detection (0.001 to 0.422 μg/mL) and quantification (0.003 to 1.406
μg/mL) for all 16 analyzed compounds when compared with the most often used HPLC-DAD
methods, in some cases being more than 100-fold lower. The recovery (86.1%–104.8%),
accuracy (85.9%–114.0%), and precision (<14%) met with the acceptance criteria of
the Association of Official Analytical Chemists International.
According to these results, the described HPLC-MS/MS method was adequately sensitive,
repeatable and suitable for the large-scale analysis of compounds in biological fluids.
Acknowledgments: This work was supported by the Interagency Council on Science and
Technology (CICYT) (AGL2013-49083-C3-1-R); the Instituto de Salud Carlos III (ISCIII),
CIBER Physiopathology of Obesity and Nutrition (CIBEROBN) from the Spanish Ministry
of Economy and Competivity (MEC); Generalitat de Catalunya (GC) 2014 SGR 773; and
by the Project LO1208 (TEWEP) of the National Feasibility Programme I of the Czech
Republic. Hrvolová, B. also thanks the student grant n. SGS04/PřF/2016 from the University
of Ostrava, Czech Republic.
3.2.18. Organoleptic Characteristics of Quality Spanish Sparkling Wine “Cava”
Introduction and objectives: During ageing, the enologists describe the evolution
of the sensorial characteristics of cava from fruity (due to acetate and ethyl esters)
to more complex sensations as nuts, warm, and mature fruits due to the particular
ageing from contact with lees (non-viable cells of Saccharomyces cerevisiae).
The characterization of the aroma of Spanish sparkling wine is useful in order to
find strategies to improve cava volatile composition. Furthermore, 200 tons of lees
were produced every year and are considered as a by-product of the enology industry.
With the aim to exploit this by-product, cava was enriched with the volatiles encapsulated
in lees in order to study their behavior in the sparkling wine.
Methodology: Volatile composition was obtained by Headspace–Solid Phase Microextraction
(HS-SPME) coupled to gas chromatography mass spectrometry (GC/MS). The encapsulation
was produced by diffusion.
Results and conclusions: As expected, the aroma of cava was composed of numerous chemical
families such as esters, alcohols, norisoprenoids, terpenes, and acids. In order to
improve the organoleptic quality of cava, the wine was enriched with two volatiles
encapsulated with yeast lees of second fermentation (S. cerevisiae). The release of
the volatiles occurred in only 24 h. Thus, the reutilization of this enology industry
by-product was proposed in order to increase the organoleptic characteristics of wine.
Acknowledgments: Freixenet S.A. for providing samples. The Interagency Council on
Science and Technology (CICYT) projects AGL2011-23872 and AGL2016-78324-R.
3.2.19. Combined Determination of Polyphenols and Pentacyclic Triterpenes from Table
Olives by High Pressure Liquid Chromatography Mass Spectrometry (HPLC-MS)
Background and objectives: Table olives are typical components of the Mediterranean
Diet that could contribute to its beneficial health effects given its high content
of oleic acid, as well as other bioactive compounds. With the aim of increasing knowledge
on the composition of pentacyclic triterpenes and polyphenols in olives, a method
that enables the simultaneous extraction of both groups of compounds has been developed.
Methodology: Polyphenols (hydroxytirosol, tyrosol, and oleuropein) and pentacyclic
triterpenes (maslinic acid, oleanolic acid, and erythrodiol) were simultaneously extracted
from arbequina table olives using ethanol–methanol (1:1 v/v) as a solvent, which includes
a different internal standard for triterpenes and polyphenols. After three consecutive
extractions that included vigorous vortexmixing and centrifugations, the supernatants
were analyzed by high-pressure liquid chromatography atmospheric pressure chemical
ionization mass spectrometry (HPLC-APCI-MS) to determine pentacyclic triterpenes and
by HPLC-electrospray ionization mass spectrometry for polyphenols.
Results and conclusions: The validation results showed that the method was linear
(r > 0.99), sensitive, precise (CV < 15%) and accurate for all analyses, with a recovery
>88% for triterpenes and >94% for polyphenols. The analysis of Arbequina olives indicated
that maslinic acid was the main bioactive compound (2585.6 ± 90.8 mg/kg) followed
by oleanolic acid (741.6 ± 13.5 mg/kg), hydroxytirosol (458.7 ± 88.1 mg/kg) and tirosol
(41.7 ± 9.04 mg/kg), with minor amounts of erythrodiol (9.48 ± 0.34 mg/kg) and oleuropein
(1.38 ± 0.49 mg/kg). The development of this method provides a reliable and accurate
combined extraction of chemically different compound, thus allowing a rapid analysis
of polyphenols and pentacyclic triterpenes in olives.
Acknowledgments: Supported by AGL2013-41188 (Spanish Ministry of Economy and Competitiveness)
and 2014SGR1221 (Generalitat de Catalunya).
3.2.20. Dietary Spray-Dried Plasma (SDP) Supplementation Promotes Anti-Inflammatory
Mediators in Mice Challenged with S. aureus Enterotoxin B
Background and objectives: In rodents, dietary supplementation with spray-dried plasma
(SDP) attenuates lymphocyte activation in mesenteric lymph nodes (MLN) during intestinal
inflammation induced by S. aureus enterotoxin B (SEB). The objective of this study
was to discern the molecular mechanisms involved in the intestinal protective effects
Methodology: Male C57BL/6 mice were fed with 8% SDP or milk proteins for two weeks,
from weaning until day 33. On day 32, mice were given a single SEB dose (i.p., 25
µg/mice) or phosphate buffered saline (PBS). Twenty-four hours later, mice were sacrificed
and MLN lymphocytes were stained and analyzed by flow cytometry. The expression of
cytokines, adhesion molecules and transcription factors were determined in the intestinal
Results and conclusions: SEB increased the MLN cell recruitment and the percentage
of activated Th lymphocytes (p < 0.05), which were attenuated by SDP (p < 0.05). The
enterotoxin did not change the mucosal expression of IL-10 and TGFβ1, but SDP increased
the expression of both cytokines when compared to the SEB group (p < 0.05). The enterotoxin
increased the expression of Madcam-1 and ICAM-1 (p < 0.001) and these effects were
attenuated by SDP (p < 0.05). SEB also decreased Smad2/3 phosphorylation and augmented
NFκB phosphorylation (p < 0.05), which were prevented by SDP (p < 0.05). Our results
indicated that SDP modulates the immune response in challenged animals through the
regulation of transcription factors and adhesion molecules that, in turn, will reduce
intestinal cell infiltration and the magnitude of the inflammatory response.
Acknowledgments: Supported by 2014SGR1221 (Generalitat de Catalunya, Spain) and Fundació
Bosch i Gimpera (UB) FBG 306994 contract.
3.2.21. Urine Metabolome Changes after Beer and Non-Alcoholic Beer Intake
Background and objectives: Moderate alcohol consumption is associated with a decrease
in cardiovascular risk, but fermented beverages seem to confer greater cardiovascular
protection due to their polyphenolic content. Beer contains a wide range of polyphenols
derived from the malt or hops used during brewing, including simple phenolic acids,
hydroxycinnamoylquinics, flavanols, flavonols, flavones, alkylmethoxyphenols, alpha-
and iso-alpha-acids, hydroxyphenylacetic acids, and prenylflavanoids.
The objective of this work was to compare the effects of moderate consumption of beer,
non-alcoholic beer and gin on the overall urine metabolome.
Methodology: A crossover trial with 33 men at high cardiovascular risk was randomized
to receive beer (30 g alcohol/day), the equivalent amount of polyphenols in the form
of non-alcoholic beer, or gin (30 g alcohol/day) for four weeks. Urine samples were
analyzed by liquid chromatography–high-resolution mass spectrometry (HPLC-MS/MS).
A combination of univariate statistical analysis, multivariate analysis (partial least
squares discriminant analysis), data-dependent MS/MS scan, and accurate mass database
matching was used to measure the effect of beer, non-alcoholic beer and gin intake
in the urinary metabolome.
Results and conclusions: A total of 10 metabolites were identified. Eight were exogenous
metabolites related to beer, non-alcoholic beer or gin consumption, but two of them
were related to metabolic endogenic changes: hydroxyadipic acid linked to fatty acid
oxidation, and 4-guanidinobutanoicacid involved in arginine metabolism. Humulinone,
a hop bitter acid normally found in beer, could be a novel biomarker of beer and nonalcoholic
Acknowledgments: This study was supported by The European Foundation for Alcohol Research
(ERAB) EA 1117 and EA 1324 and CIBER Physiopathology of Obesity and Nutrition (CIBEROBN).
3.2.22. Are the Olive Oil Sesquiterpenes Influenced by Hydric Stress?
Background and objectives: The virgin olive oil (VOO) market has evolved in the last
few years with the appearance of quality labels as denominations and protected indications
of origin. For this reason, it is necessary to find analytical traceability markers
to avoid fraudulent claims made to consumers. Volatile compounds could be used for
this purpose, especially the products of the lipoxygenase pathway, but most of them
are affected by several technological parameters during olive processing which reduces
their usefulness. Nevertheless, semi-volatile compounds from the secondary metabolism
of olive fruit are also found in the oil: the sesquiterpene hydrocarbons, which are
not significantly modified by the oil extraction process, could be a suitable tool
for the geographical and varietal authentication of VOO. To corroborate the usefulness
of sesquiterpenes as traceability markers, it was necessary to evaluate their dependence
on agronomic factors that could affect VOO sesquiterpene composition. The evaluation
of irrigation, as one of the essential agronomic factors was the aim of the present
Methodology: Sesquiterpene composition was evaluated by solid phase microextraction–gas
chromatography (SPME-GC/MS) in VOO samples from three consecutive crop years (2009–2011)
that were obtained from the same olive cultivar (Arbequina) and region and subjected
to four different irrigation treatments.
Results and conclusions: Sesquiterpene distribution in VOO was influenced by markedly
different water availability and depended on their structure. Cyclic sesquiterpenes
were proportionally related to hydric stress while linear sesquiterpenes and some
terpene ketones are favored by water availability.
3.2.23. Polyphenol Exposure Is Inversely Associated with Geriatric Conditions, Frailty
and Mortality within the Invecchiare in Chianti (InCHIANTI) Cohort: Relevance to Polyphenol
Background and objectives: There is growing evidence on the health-protective role
of dietary polyphenols intake on aging, but the assessment of dietary polyphenols
intake from self-reported questionnaires tends to be inaccurate and unreliable. A
promising alternative is the use of urinary polyphenols concentration as a more accurate
measure of intake. The aim was to investigate the association between dietary polyphenols
and urinary polyphenol concentrations; and cognitive and physical decline, frailty
phenotype, and all-cause mortality among older adults aged ≥65 years or more within
the Invecchiare in Chianti (InCHIANTI) study, an Italian cohort.
Methodology: The dietary intake of total polyphenols and resveratrol was estimated
using a validated food frequency questionnaire and an ad hoc database of polyphenols
in food composition. The urinary concentration of total polyphenols and resveratrol
was determined using the Folin–Ciocalteu colorimetric and mass spectrometry methods,
as biomarkers of the dietary total of polyphenols and resveratrol, respectively.
Results and conclusions: The highest tertile of total urinary polyphenols was inversely
associated with the risk of cognitive and physical decline, frailty and total mortality,
in comparison with the lowest tertile. However, no association with total dietary
polyphenols was observed. Additionally, habitual dietary exposure of resveratrol was
associated with a lower risk of developing frailty in a combination of both measures
(diet and biomarker), as well as individually. In conclusion, these results suggested
the protective effect of a polyphenol-rich diet against cognitive and physical decline,
frailty and all-cause mortality in older persons. Furthermore, it demonstrates the
importance of assessing dietary polyphenol exposure, whenever possible, using nutritional
biomarkers and not only self-reported questionnaires.
Acknowledgments: This research was supported by the Consolider Ingenio 2010 Program
Acronym: FUN-C-FOOD (CSD2007-063); the International Nut and Dried Fruit Council Foundation
(INC) (FBG307906); and the Joint Programming Initiative—A Healthy Diet for a Healthy
Life (JPI HDHL) Acronym: FOODBALL (Ministry of Economy and Competitiveness (PCIN-2014-133).
3.2.24. Norovirus Shedding among Food and Healthcare Workers Exposed to the Virus
in Outbreak Settings
Background and objectives
: Noroviruses (NoVs) are the leading cause of nonbacterial outbreaks of gastroenteritis
worldwide. Individuals who are asymptomatically infected may facilitate the transmission
of NoVs. Our aim was to evaluate the occurrence of NoVs infections among workers exposed
to the virus in different outbreak settings.
Methodology: We screened feces from food handlers and healthcare workers related to
gastroenteritis outbreaks, and shedding concentrations over time were calculated from
serial samples of infected individuals. Sequence analyses of the capsid P2 domain
and region C were used to evaluate the link between asymptomatic employees and outbreak
Results and conclusions
: Of all employees, 59.1% were positive for NoVs, and more than 70% of them were asymptomatic.
Asymptomatic infections were significantly more frequent among food handlers compared
to healthcare workers. Mean viral loads were similar between symptomatic and asymptomatic
individuals, starting at 7.51 ± 1.80 and 6.49 ± 1.93 log10 genome copies/g, respectively,
and decreasing to 5.28 ± 0.76 and 4.52 ± 1.45 log10 genome copies/g after 19 days.
In the setting of a NoVs outbreak, workers show a high risk of becoming infected.
Since shed amounts of viruses without symptoms are also high, reinforcement of hygiene
practices among workers is relevant to reduce the risk of virus secondary transmissions.
Acknowledgments: Thanks to the Working Group for the Study of Outbreaks of Acute Gastroenteritis
in Catalonia; the physicians who reported outbreaks; and the Epidemiological Surveillance
Units of the Department of Health of the Government of Catalonia and the Public Health
Agency of Barcelona.
3.2.25. Effects of Different Doses of Polyphenols from Dealcoholized Red Wine on Endothelial
Function in Subjects with Metabolic Syndrome and High Cardiovascular Risk
Background and objectives: Several studies have pointed out that mortality and risk
from cardiovascular disease (CVD) are higher in subjects with Metabolic Syndrome (MetS),
which is considered as a cluster of risk factors. Epidemiological studies and intervention
clinical trials have shown that dealcoholized red wine (DRW) and moderate consumption
of red wine (RW) are inversely associated with cardiovascular risk factors.
Methodology: A randomized, open prospective trial, running in parallel with a controlled
clinical trial, was performed with 54 subjects with MetS to evaluate the effects of
polyphenols. Doses of 375 mL/day of DRW, dealcoholized red wine with grape extract
(DRWEx) or water were administered during three months. Anthropometric measurements
and blood biochemical analysis were carried out at the baseline and after each intervention.
Levels of endothelial progenitor cells (EPC), circulating endothelial cells (CEC)
and leukocyte cell membrane receptors were analyzed by flow cytometry.
Results and conclusions: After DRW and DRWEx interventions, we observed a decrease
in body weight, body mass index, waist circumference, total cholesterol concentration,
and an improvement on blood pressure. Moreover, there was an increase in the number
of EPCs and a significant decrease in levels of CECs and T-lymphocyte expression.
We concluded that the non-alcoholic fraction of wine, rich in polyphenols, may reduce
cell adhesion molecules and CEC, known markers of CVD severity and increase EPC, a
marker of endothelial regeneration, in a population at high risk of CVD due to MetS.
These features may explain why DRW and moderate RW consumption suggest an improvement
in the condition of the vascular endothelium and possibly contributes in delaying
the development of atherosclerotic plaques.
Acknowledgments: Authors would like to express their gratitude for financial support
from the INCOMES project supported by the Spanish Ministry of Economy and Competitiveness
through the INNPRONTA program, as well as the Interagency Council on Science and Technology
(CICYT) (AGL2013-49083-C3-1-R); Generalitat de Catalunya FI-DGR 2013/FI-DGR 2014;
the Instituto de Salud Carlos III (ISCIII) and CIBER Physiopathology of Obesity and
Nutrition (CIBEROBN) from the Spanish Ministry of Science and Innovation (MICINN).
on behalf of the PREDIMED Study Investigators
3.2.26. Urine Polyphenol Excretion Is Inversely Correlated with Body Weight in the
“Prevención con Dieta Mediterránea” (PREDIMED) Population
Background and objectives: The high prevalence of overweight and obesity is one of
the most common causes of morbidity and mortality. Although polyphenol intake has
been associated with their protection against several chronic diseases, only a few
human studies have investigated the role of polyphenols in body weight. Our aim was
to investigate whether urinary polyphenol levels are associated with obesity parameters.
Methodology: A longitudinal study was performed with 573 participants from the PREDIMED
(Prevención con Dieta Mediterránea) trial (ISRCTN35739639). Total polyphenol excretion
(TPE), as a biomarker of polyphenol intake, was determined by the Folin–Ciocalteu
method in urine samples. Participants were categorized into five groups according
to TPE at five years. Multiple linear regression models were used to assess the relationships
between TPE and obesity parameters.
Results and conclusions: After a five-year follow-up, significant inverse correlations
were observed between five-year TPE and body weight; body mass index; waist circumference;
and waist-to-height ratio, after adjustments for potential confounders. Compared with
those in the lowest quintile, participants in the top TPE quintile showed a lower
prevalence of obesity.
A greater polyphenol intake may therefore contribute to the reduced risk of obesity
in elderly people who are at high cardiovascular risk.
Acknowledgments: This study was supported by the Interagency Council on Science and
Technology (CICYT) (AGL2016-75329-R) from the Spanish Ministry of Economy and Competitiveness
(MEC); the Generalitat de Catalunya (GC) 2014 SGR 773; and the Instituto de Salud
Carlos III (ISCIII) and CIBER Physiopathology of Obesity and Nutrition (CIBEROBN).
CIBEROBN is an initiative of ISCIII, Spain. X. Guo received support from China Scholarship
Council (CSC). A. Medina-Remón thanks the “Juan de la Cierva” postdoctoral program
(JCI-2012-13463) from MEC.