European Journal of Nutrition (v.56, #2)

Public health relevance of drug–nutrition interactions by Szabolcs Péter; Gerjan Navis; Martin H. de Borst; Clemens von Schacky; Anne Claire B. van Orten-Luiten; Alexandra Zhernakova; Renger F. Witkamp; André Janse; Peter Weber; Stephan J. L. Bakker; Manfred Eggersdorfer (23-36).
The public health relevance of drug–nutrition interactions is currently highly undervalued and overlooked. This is particularly the case for elderly persons where multi-morbidity and consequently polypharmacy is very common. Vitamins and other micronutrients have central functions in metabolism, and their interactions with drugs may result in clinically relevant physiological impairments but possibly also in positive effects. On 12 April 2016, the University Medical Center Groningen (The Netherlands), as part of its Healthy Ageing program, organized a workshop on the public health relevance of drug–nutrient interactions. In this meeting, experts in the field presented results from recent studies on interactions between pharmaceuticals and nutrients, and discussed the role of nutrition for elderly, focusing on those persons receiving pharmaceutical treatment. This paper summarizes the proceedings of the symposium and provides an outlook for future research needs and public health measures. Since food, pharma and health are closely interconnected domains, awareness is needed in the medical community about the potential relevance of drug–nutrition interactions. Experts and stakeholders should advocate for the integration of drug–nutrition evaluations in the drug development process. Strategies for the individual patients should be developed, by installing drug review protocols, screening for malnutrition and integrating this topic into the general medical advice.
Keywords: Public health; Drug–nutrient interactions; Micronutrient deficiency; Microbiota; Health benefits

Celiac disease: understanding the gluten-free diet by Karla A. Bascuñán; María Catalina Vespa; Magdalena Araya (449-459).
The only effective and safe treatment of celiac disease (CD) continues being strict exclusion of gluten for life, the so-called gluten-free diet (GFD). Although this treatment is highly successful, following strict GFD poses difficulties to patients in family, social and working contexts, deteriorating his/her quality of life. We aimed to review main characteristics of GFD with special emphasis on factors that may interfere with adherence to it.We conducted a search of various databases, such as PubMed, Google Scholar, Embase, and Scielo, with focus on key words such as “gluten-free diet”, “celiac disease”, “gluten” and “gluten-free diet adherence”. Available literature has not reached definitive conclusions on the exact amount of gluten that is harmless to celiac patients, although international agreements establish cutoff points for gluten-free products and advise the use of clinical assessment to tailor the diet according to individual needs. Following GFD must include eliminating gluten as ingredient as well as hidden component and potential cross contamination in foods. There are numerous grains to substitute wheat but composition of most gluten-free products tends to include only a small number of them, especially rice. The diet must be not only free of gluten but also healthy to avoid nutrient, vitamins and minerals deficiencies or excess. Overweight/obesity frequency has increased among celiac patients so weight gain deserves attention during follow up. Nutritional education by a trained nutritionist is of great relevance to achieve long-term satisfactory health status and good compliance.A balanced GFD should be based on a combination of naturally gluten-free foods and certified processed gluten-free products. How to measure and improve adherence to GFD is still controversial and deserves further study.
Keywords: Celiac disease; Gluten; Gluten-free diet; Adherence to diet

Hyperactive platelets, in addition to their roles in thrombosis, are also important mediators of atherogenesis. Antiplatelet drugs are not suitable for use where risk of a cardiovascular event is relatively low. It is therefore important to find alternative safe antiplatelet inhibitors for the vulnerable population who has hyperactive platelets in order to reduce the risk of cardiovascular disease. Potent antiplatelet factors were identified in water-soluble tomato extract (Fruitflow®), which significantly inhibited platelet aggregation. Human volunteer studies demonstrated the potency and bioavailability of active compounds in Fruitflow®. Fruitflow® became the first product in Europe to obtain an approved, proprietary health claim under Article 13(5) of the European Health Claims Regulation 1924/2006 on nutrition and health claims made on foods. Fruitflow® is now commercially available in different countries worldwide. In addition to its reduction in platelet reactivity, Fruitflow® contains anti-angiotensin-converting enzyme and anti-inflammatory factors, making it an effective and natural cardio-protective functional food.
Keywords: Tomato; Water-soluble tomato extract; Fruitflow® ; Human platelets; Platelet activation; Polyphenols; ADP; Blood pressure; Angiotensin-converting enzyme; EU regulation 1924/2006; EFSA health claim

Postnatal high-fat diet enhances ectopic fat deposition in pigs with intrauterine growth retardation by Honglin Yan; Ping Zheng; Bing Yu; Jie Yu; Xiangbing Mao; Jun He; Zhiqing Huang; Daiwen Chen (483-490).
Intrauterine growth retardation (IUGR) and postnatal nutrition are risk factors for adult metabolic syndrome. However, the influences of long-term high-fat diet (HFD) intake on ectopic fat deposition in non-adipose tissues in IUGR pigs remain unclear. The present study was to determine whether HFD consumption would enhance ectopic fat deposition in IUGR pigs.At day 28, IUGR and control pigs were fed ad libitum to either a regular diet or a HFD. Lipid store, enzymatic activities and mRNA expression of lipid metabolism-related factors in liver and semitendinosus muscle (SM) were quantified at postnatal day 178.Feeding a HFD to IUGR pigs but not to control pigs significantly increased daily weight gain, carcass fat mass, plasma leptin level and lipid content and lipoprotein lipase (LPL) activity and mRNA abundances of LPL and peroxisome proliferator-activated receptor gamma (PPARγ) in liver and SM, but decreased daily feed intake and mRNA expression of hormone-sensitive lipase (LIPE) and carnitine palmitoyl transferase-1 (CPT-1) in liver and SM (P < 0.05). Compared with control pigs, IUGR pigs had a lower body weight but higher plasma levels of total cholesterol (TC) and insulin (P < 0.05). HFD-fed pigs exhibited greater body weight, plasma concentrations of triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C), regardless of birth weight (P < 0.05).Our results suggested that IUGR increased the vulnerability of HFD-fed pigs to ectopic fat deposition via enhanced fatty acid flux toward ectopic sites and reduced lipolysis and fatty acid oxidation.
Keywords: Intrauterine growth retardation; Ectopic fat deposition; Liver; Skeletal muscle; Pigs

The present placebo-controlled, double-blind, randomized trial aimed to investigate whether a natural mineral water rich in magnesium sulphate and sodium sulphate (Donat Mg) may help to improve bowel function.A total of 106 otherwise healthy subjects with functional constipation were randomly assigned to consume 300 or 500 mL of a natural mineral water as compared to placebo water, over a course of 6 weeks. The 300-mL arms were terminated due to the results of a planned interim analysis. Subjects documented the complete spontaneous bowel movements, spontaneous and overall bowel movements/week, stool consistency, gastrointestinal symptoms and general well-being in a diary. Change in the number of complete spontaneous bowel movements was defined as the primary outcome.For the 75 subjects in the 500-mL arms, the change in the number of complete spontaneous bowel movements per week tended to be higher in the active group when compared to placebo after 6 weeks (T2 = 1.8; p value = 0.036; one-sided). The mean number of spontaneous bowel movements significantly increased over the course of the study, with significant differences between study arms considering the whole study time (F test = 4.743; p time × group = 0.010, 2-sided). Stool consistency of spontaneous bowel movements (p < 0.001) and the subjectively perceived symptoms concerning constipation (p = 0.005) improved significantly with the natural mineral water as compared to placebo.The daily consumption of a natural mineral water rich in magnesium sulphate and sodium sulphate improved bowel movement frequency and stool consistency in subjects with functional constipation. Moreover, the subjects’ health-related quality of life improved.EudraCT No 2012-005130-11.
Keywords: Bowel function; Stool frequency and consistency; Natural mineral water; Clinical trial

Plasma vitamin D biomarkers and leukocyte telomere length in men by Bettina Julin; Irene M. Shui; Jennifer Prescott; Edward L. Giovannucci; Immaculata De Vivo (501-508).
Vitamin D may reduce telomere shortening through anti-inflammatory and anti-cell proliferation mechanisms. In women, higher plasma 25-hydroxyvitamin D (25(OH)D) has been shown to be associated with longer telomere length, but the relationship has not been assessed in men.We conducted a cross-sectional analysis of 25(OH)D, 1,25-dihydroxyvitamin D (1,25(OH)2D) and relative leukocyte telomere length (LTL) among 2483 men [1832 men for 1,25(OH)2D] who were selected as cases and controls in three studies of telomeres and cancer nested within the Health Professionals Follow-up Study. We also genotyped 95 SNPs representing common genetic variation in vitamin D pathway genes. LTL was measured by quantitative PCR, and z-scores within each study were calculated. Associations were assessed by linear as well as logistic regression adjusting for age and other potential confounders.Age (P-trend < 0.0001), pack-years of smoking (P-trend = 0.04) and body mass index (P-trend = 0.05) were inversely associated with LTL. Neither 25(OH)D nor 1,25(OH)2D was associated with LTL (multivariable-adjusted P-trend 0.69 and 0.41, respectively, for the linear regression model). One SNP in the retinoid X receptor alpha gene was associated with long LTL (P = 0.0003).In this cross-sectional study of men, 25(OH)D and 1,25(OH)2D were not associated with relative LTL.
Keywords: Cross-sectional; Men; Telomeres; Vitamin D; Vitamin D pathway SNPs

Increased dietary levels of α-linoleic acid inhibit mammary tumor growth and metastasis by Marianela Vara-Messler; Maria E. Pasqualini; Andrea Comba; Renata Silva; Carola Buccellati; Annalisa Trenti; Lucia Trevisi; Aldo R. Eynard; Angelo Sala; Chiara Bolego; Mirta A. Valentich (509-519).
The aim of this study was to determine whether α-linolenic acid (ALA ω-3 fatty acid) enriched diet affects growth parameters when applied to a syngeneic model of mammary carcinoma.BALB/c mice were divided and fed with: 1) a chia oil diet, rich in ALA or 2) a corn oil diet, rich in linoleic acid (LA ω-6 fatty acid). Mice were subcutaneously inoculated with a tumor cell line LM3, derived from a murine mammary adenocarcinoma.After 35 days, tumor incidence, weight, volume and metastasis number were lower in the ALA-fed mice, while tumor latency time was higher, and the release of pro-tumor metabolites derived from ω-6 fatty acids decreased in the tumor. Compared to the control group, a lower number of mitosis, a higher number of apoptotic bodies and higher T-lymphocyte infiltration were consistently observed in the ALA group. An ALA-rich diet decreased the estrogen receptor (ER) α expression, a recognized breast cancer promotor while showing an opposite effect on ERβ in tumor lysates.These data support the anticancer effect of an ALA-enriched diet, which might be used as a dietary strategy in breast cancer prevention.
Keywords: α-linolenic acid (ALA); Chia oil; Mammary carcinoma; LOXs metabolites; Estrogen receptor

Intakes of whole grain in an Italian sample of children, adolescents and adults by Stefania Sette; Laura D’Addezio; Raffaela Piccinelli; Sinead Hopkins; Cinzia Le Donne; Marika Ferrari; Lorenza Mistura; Aida Turrini (521-533).
There is wide evidence that regular consumption of whole grain foods may reduce the risk of chronic diseases. The aim of this work was to quantify the intake of whole grains and identify main dietary sources in the Italian population.Whole grain intakes were calculated in a sample of 2830 adults/older adults and of 440 children/adolescents from the last national survey INRAN-SCAI 2005–06. Food consumption was assessed from a 3-day food record. The whole grain content of foods was estimated mainly from quantitative ingredient declarations on labels.Mean whole grain intakes were 3.7 g/day in adults/older adults and 2.1 g/day in children/adolescents. Overall, 23 % of the sample reported consumption of whole grain foods during the survey, among which mean whole grain intakes ranged from 6.0 g/day in female children to 19.1 g/day in female older adults. The main sources of whole grains were breakfast cereals in children/adolescents (32 %) and bread in adults/older adults (46 %). Consumption of whole grain among adults was associated with significantly higher daily intakes and adequacy of dietary fibre, several vitamins (thiamine, riboflavin, vitamin B6) and minerals (iron, calcium, potassium, phosphorus, zinc, magnesium) compared to non-consumption. Among children, whole grain intake was associated with significantly higher intakes of iron and magnesium.The study reveals very low whole grain intakes across all age groups of the Italian population. Considering the positive association in consumers between whole grain intakes and fibre and micro-nutrient intakes, public health strategies to increase whole grain consumption should be considered.
Keywords: Whole grain; Nutrients; Food source; Diet quality

Hepatic DNA hydroxymethylation is site-specifically altered by chronic alcohol consumption and aging by Stephanie A. Tammen; Lara K. Park; Gregory G. Dolnikowski; Lynne M. Ausman; Simonetta Friso; Sang-Woon Choi (535-544).
Global DNA hydroxymethylation is markedly decreased in human cancers, including hepatocellular carcinoma, which is associated with chronic alcohol consumption and aging. Because gene-specific changes in hydroxymethylcytosine may affect gene transcription, giving rise to a carcinogenic environment, we determined genome-wide site-specific changes in hepatic hydroxymethylcytosine that are associated with chronic alcohol consumption and aging.Young (4 months) and old (18 months) male C57Bl/6 mice were fed either an ethanol-containing Lieber–DeCarli liquid diet or an isocaloric control diet for 5 weeks. Genomic and gene-specific hydroxymethylcytosine patterns were determined through hydroxymethyl DNA immunoprecipitation array in hepatic DNA.Hydroxymethylcytosine patterns were more perturbed by alcohol consumption in young mice than in old mice (431 differentially hydroxymethylated regions, DhMRs, in young vs 189 DhMRs in old). A CpG island ~2.5 kb upstream of the glucocorticoid receptor gene, Nr3c1, had increased hydroxymethylation as well as increased mRNA expression (p = 0.015) in young mice fed alcohol relative to the control group. Aging alone also altered hydroxymethylcytosine patterns, with 331 DhMRs, but alcohol attenuated this effect. Aging was associated with a decrease in hydroxymethylcytosine ~1 kb upstream of the leptin receptor gene, Lepr, and decreased transcription of this gene (p = 0.029). Nr3c1 and Lepr are both involved in hepatic lipid homeostasis and hepatosteatosis, which may create a carcinogenic environment.These results suggest that the location of hydroxymethylcytosine in the genome is site specific and not random, and that changes in hydroxymethylation may play a role in the liver’s response to aging and alcohol.
Keywords: Alcohol; Aging; DNA hydroxymethylation; hmeDIP-Chip; Liver

The synergistic effect between the Mediterranean diet and GSTP1 or NAT2 SNPs decreases breast cancer risk in Greek-Cypriot women by Maria G. Kakkoura; Maria A. Loizidou; Christiana A. Demetriou; Giorgos Loucaides; Maria Daniel; Kyriacos Kyriacou; Andreas Hadjisavvas (545-555).
Xenobiotic metabolism is related to the interplay between diet and breast cancer (BC) risk. This involves detoxification enzymes, which are polymorphic and metabolise various dietary metabolites. An important characteristic of this pathway is that chemoprotective micronutrients can act not only as substrates but also as inducers for these enzymes. We investigated whether functional GSTP1 (p.Ile105Val-rs1695), NAT2 (590G>A-rs1799930) SNPs and GSTM1 and GSTT1 deletion polymorphisms could modulate the effect of the Mediterranean diet (MD) on BC risk, in Greek-Cypriot women.Genotyping was performed on women from the MASTOS case–control study of BC in Cyprus. A 32-item food-frequency questionnaire was used to obtain dietary intake information. A dietary pattern, which closely resembles the MD (high loadings of vegetables, fruit, legumes and fish), was previously derived with principal component analysis and was used as our dietary variable. GSTT1 null genotype increased BC risk compared with the homozygous non-null GSTT1 genotype (OR 1.21, 95 % CI 1.01–1.45). Increasing adherence to the MD reduced BC risk in women with at least one GSTP1 Ile allele (OR for Ile/Ile = 0.84, 95 % CI 0.74–0.95, for Ile/Val = 0.73, 95 % CI 0.62–0.85) or one NAT2 590G allele (OR for 590 GG = 0.73, 95 % CI 0.63–0.83, for 590 GA = 0.81, 95 % CI 0.70–0.94). p interaction values were not, however, statistically significant.The homozygous null GSTT1 genotype could be a risk allele for BC among Greek-Cypriot women. The anticarcinogenic effects of the high adherence to MD against BC risk could also be further enhanced when combined with the wild-type alleles of the detoxification GSTP1 or NAT2 SNPs.
Keywords: Mediterranean diet; GSTP1; GSTM1; GSTT1; NAT2; Breast cancer

Zinc enhances the number of regulatory T cells in allergen-stimulated cells from atopic subjects by Eva Rosenkranz; Ralf-Dieter Hilgers; Peter Uciechowski; Arnd Petersen; Birgit Plümäkers; Lothar Rink (557-567).
The trace element zinc is essential for immune function and its regulation. Since zinc deficiency and allergic hyperresponsive reactions are often accompanied, the influence of zinc on allergen-induced cell growth, CD4+ regulatory T (Treg) cell numbers and cytokine expression during allergic immune reactions was investigated.Peripheral blood mononuclear cells (PBMCs) from non-atopic and atopic subjects were treated with timothy grass allergen pre-incubated with or without zinc. Proliferation was determined by analyzing the incorporation of 3H-thymidine. Intracellular zinc and Foxp3 levels and cell surface antigens were measured by FACS, cytokine expression by ELISA and real-time PCR.Incubation with 50 μM zinc sulfate (Zn50) enhances cytosolic zinc concentrations in CD3+ T cells. The data also reveal that the combination of Zn50 plus allergen significantly reduces PBMC proliferation of atopic subjects. Additionally, Zn50 plus allergen enhances Th1 cytokine responses shown by increased interferon (IFN)-γ/interleukin (IL)-10 ratios as well as enhanced tumor necrosis factor-α release. In response to allergen, zinc increases Treg cells and upregulates the mRNA expression of cytotoxic T-lymphocyte antigen-4 in atopic subjects. Interestingly, Zn50 alone leads to an increase of CD4+CD25high(hi)+ cells in atopic and non-atopic subjects.Zinc may regulate unwanted hyperresponsive immune reactions by suppressing proliferation through a significant shift from IL-10 to the Th1 cytokine IFN-γ, and enhanced regulatory T cell numbers. Therefore, zinc supplementation may be a promising tool for the therapy of allergies, without negatively affecting the immune system.
Keywords: Zinc; Nutritional immunology; Allergy; Regulatory T cells; Treg; Foxp3

Muscle-specific deletion of signal transducer and activator of transcription 5 augments lipid accumulation in skeletal muscle and liver of mice in response to high-fat diet by Myunggi Baik; Mi Sun Lee; Hyeok Joong Kang; Seung Ju Park; Min Yu Piao; Trang Hoa Nguyen; Lothar Hennighausen (569-579).
Growth hormone (GH) controls liver metabolism through the transcription factor signal transducer and activator of transcription 5 (STAT5). However, it remains to be fully understood to what extent other GH/STAT5 target tissues contribute to lipid and glucose metabolism. This question was now addressed in muscle-specific STAT5 knockout (STAT5 MKO) mice model.Changes in lipid and glucose metabolism were investigated at physiological and molecular levels in muscle and liver tissues of STAT5 MKO mice under normal diet or high-fat diet (HFD) conditions.STAT5 MKO mice exhibited an increased intramyocellular lipid (IMCL) accumulation in the quadriceps in HFD group. Decreased lipolytic hormone-sensitive lipase transcript levels may contribute to the increased IMCL accumulation in STAT5 MKO mice. STAT5 MKO induced hepatic lipid accumulation without deregulated STAT5 signaling. The upregulation of lipoprotein lipase and Cd36 mRNA levels, an increased trend of very low-density lipoprotein receptor mRNA levels, and elevated circulating concentrations of free fatty acid, triglyceride, and total cholesterol support the increase in hepatic lipid accumulation.STAT5 MKO in conjunction with a HFD deregulated both lipid and glucose metabolism in skeletal muscle, and this deregulation induced hepatic fat accumulation via increased circulating glucose, FFA, and TG concentrations. Our study emphasizes that muscle-specific STAT5 signaling is important for balancing lipid and glucose metabolism in peripheral tissues, including muscle and liver and that the deregulation of local STAT5 signaling augments HFD-induced lipid accumulation in both muscle and liver.
Keywords: STAT5 muscle deletion; Lipid and glucose metabolism; Intramyocellular lipid accumulation; Hepatic lipid accumulation

Vitamin D intake of Dutch infants from the combination of (fortified) foods, infant formula, and dietary supplements by Janneke Verkaik-Kloosterman; Marja H. Beukers; Martine Jansen-van der Vliet; Marga C. Ocké (581-590).
Due to changes in the Dutch fortification policy for vitamin D and the vitamin D supplementation advice for infants (10-μg/d for 0–4 year olds), a partially virtual scenario study was conducted to evaluate the risk of excessive vitamin D intake assigning all infants to a 100 % adherence to the supplementation advice and considering the current fortification practice.Food consumption data from the Nutrition Intake Study (2002; N = 941, 7–19 months) were combined with Dutch food composition data from 2011 to estimate vitamin D intake from (fortified) foods. For infants 0–6 months of age, the consumption volume infant formula was estimated from energy requirement and body weight. All subjects were assigned to take a daily 10 µg vitamin D supplement, according the Dutch supplementation advice for infants. Habitual vitamin D intake was estimated using the Statistical Program to Assess Dietary Exposure and compared with the tolerable upper intake levels (ULs) set by the European Food Safety Authority.The median habitual total vitamin D intake was 16–22 µg/day for infants aged 0–6 months (increasing with age) and 13–21 µg/day for infants aged 7–19 months (decreasing with age). About 4–12 % of infants aged 7–11 months exceeded the UL. At the 99th percentile, the intake was 2–4 µg above the UL, depending on age. Infants aged 0–6 and 12–19 months did not exceed the UL.In case of combined intake from infant formula, (fortified) foods, and supplements, vitamin D intakes above the UL are possible among some infants during a limited time period.
Keywords: Vitamin D; Infant; Excessive intake; Food; Supplements; Infant formula

Ellagic acid in Emblica officinalis exerts anti-diabetic activity through the action on β-cells of pancreas by Noor Fatima; Rahman M. Hafizur; Abdul Hameed; Shakil Ahmed; Maliha Nisar; Nurul Kabir (591-601).
The present study was undertaken to explore the possible anti-diabetic mechanism(s) of Emblica officinalis (EO) and its active constituent, ellagic acid (EA), in vitro and in vivo.Neonatal streptozotocin-induced non-obese type 2 diabetic rats were treated with a methanolic extract of EO (250 or 500 mg/kg) for 28 days, and blood glucose, serum insulin, and plasma antioxidant status were measured. Insulin and glucagon immunostaining and morphometry were performed in pancreatic section, and liver TBARS and GSH levels were measured. Additionally, EA was tested for glucose-stimulated insulin secretion and glucose tolerance test.Treatment with EO extract resulted in a significant decrease in the fasting blood glucose in a dose- and time-dependent manner in the diabetic rats. It significantly increased serum insulin in the diabetic rats in a dose-dependent manner. Insulin-to-glucose ratio was also increased by EO treatment. Immunostaining of pancreas showed that EO250 increased β-cell size, but EO500 increased β-cells number in diabetic rats. EO significantly increased plasma total antioxidants and liver GSH and decreased liver TBARS. EA stimulated glucose-stimulated insulin secretion from isolated islets and decreased glucose intolerance in diabetic rats.Ellagic acid in EO exerts anti-diabetic activity through the action on β-cells of pancreas that stimulates insulin secretion and decreases glucose intolerance.
Keywords: Emblica officinalis ; Non-obese type 2 diabetes; β-Cell function; Antioxidant; Ellagic acid; Insulin secretion

Green tea consumption and glutathione S-transferases genetic polymorphisms on the risk of adult leukemia by Ping Liu; Min Zhang; Xing Xie; Jie Jin; C. D’Arcy J. Holman (603-612).
Green tea may have a beneficial role of inhibiting leukemia. Glutathione S-transferases (GSTs) are known to detoxify certain carcinogens. We investigated the roles of green tea consumption and polymorphisms of GSTM1, GSTT1 and GSTP1 on the risk of adult leukemia, and to determine whether the associations varied within GSTs genotypes.A multicenter case–control study was conducted in China, 2008–2013. It comprised 442 incident, hematologically confirmed adult leukemia cases and 442 outpatient controls, individually matched to cases by gender, birth quinquennium and study site. Data were collected by face-to-face interview using a validated questionnaire. Genetic polymorphisms were assayed by PCR.An inverse association between green tea consumption and adult leukemia risk was observed. Compared with non-tea drinkers, the adjusted odds ratios (95 % confidence intervals) were 0.50 (0.27–0.93), 0.31 (0.17–0.55) and 0.53 (0.29–0.99) for those who, respectively, consumed green tea >20 years, ≥2 cups daily and dried tea leaves >1000 g annually. In assessing the associations by GSTs genotypes, risk reduction associated with green tea consumption was stronger in individuals with the GSTT1-null genotype (OR 0.24; 95 % CI 0.11–0.53) than GSTT1-normal carriers (OR 0.67; 95 % CI 0.42–1.05; P interaction = 0.02). GSTM1 and GSTP1 did not significantly modify the inverse association of leukemia with green tea.The results suggest that regular daily green tea consumption may reduce leukemia risk in Chinese adults regardless of GSTM1 and GSTP1 polymorphic status. The association between green tea and adult leukemia risk varied with GSTT1 genotype and highlights further study.
Keywords: Green tea; Adult leukemia; GST; Genetic polymorphism; Gene–diet interaction

Role of selected amino acids on plasma IGF-I concentration in infants by Manja Fleddermann; Hans Demmelmair; Veit Grote; Martin Bidlingmaier; Philipp Grimminger; Maximilian Bielohuby; Berthold Koletzko (613-620).
Insulin-like growth factor-I (IGF-I) is related to growth and its secretion is modified by protein intake in early infancy. We examined the relationship of dietary protein and circulating amino acids on plasma IGF-I levels and early growth. Healthy formula-fed infants (n = 213) were randomly assigned to receive either a protein-reduced infant formula with alpha-lactalbumin-enriched whey and free tryptophan and phenylalanine (IF) or an isocaloric standard formula without free amino acids (CF) for the first 120 days of life. A group of breastfed (BF) infants was studied as a non-randomized reference cohort. Biochemical variables were measured shortly after birth (subpopulation) and at an age of 120 days. A path analysis was used to explore the relationship between IGF-I, insulin and amino acids. Results are derived from secondary analyses of a randomized controlled trial. Plasma concentrations of IGF-I at 120 days were significantly higher in IF than in CF infants [58.5 (15.0) vs. 53.7 (9.95) ng/mL; p = 0.020]. BF infants showed lower IGF-I concentrations of 41.6 (10.7) ng/mL. All amino acids but Thr and Cit had a more marked effect on insulin than on IGF-I level. Considering weight, sex and feeding group, Trp explained an equal percentage of variance of IGF-I and insulin (total R 2 12.5 % of IGF-I and 12.3 % of insulin), while branched-chain AA explained an up to twofold higher variance of insulin than IGF-I. Compared to CF, IF explained 18.9 % of the IGF-I level (p = 0.03), while for insulin no direct effect was detectable.Higher IGF-I concentrations and growth velocities in infants receiving protein-reduced IF indicate that the protein concentration of an infant formula alone does not control IGF-I levels and growth. Other components (e.g., selected amino acids) of infant formulae might control directly or indirectly via insulin influence IGF-I.
Keywords: Infant formula; Growth regulation; Alpha-lactalbumin; Insulin-like growth factor-I; Human milk

Vitamin D3 supplementation does not modify cardiovascular risk profile of adults with inadequate vitamin D status by Eric Seibert; Ulrike Lehmann; Annett Riedel; Christof Ulrich; Frank Hirche; Corinna Brandsch; Jutta Dierkes; Matthias Girndt; Gabriele I. Stangl (621-634).
The Nutrition Societies in Germany, Austria, and Switzerland recommend a daily intake of 20 µg vitamin D3 for adults when endogenous synthesis is absent. The current study aimed to elucidate whether this vitamin D3 dose impacts cardiovascular risk markers of adults during the winter months.The study was conducted in Halle (Saale), Germany (51o northern latitude) as a placebo-controlled, double-blinded, randomised trial (from January to April). A total of 105 apparently healthy subjects (male and female, 20–71 years old) were included. Subjects were randomly allocated to two groups. One group received a daily 20-µg vitamin D3 dose (n = 54), and the other group received a placebo (n = 51) for 12 weeks. Outcome measures included blood pressure, heart rate, concentrations of renin, aldosterone, serum lipids and vascular calcification markers, and haematologic variables such as pro-inflammatory monocytes.Blood pressure and systemic cardiovascular risk markers remained unchanged by vitamin D3 supplementation, although serum 25-hydroxyvitamin D3 increased from 38 ± 14 to 73 ± 16 nmol/L at week 12. The placebo and vitamin D groups did not differ in their final cardiovascular risk profile.Daily supplementation of 20 µg vitamin D3 during winter is unlikely to change cardiovascular risk profile.
Keywords: Vitamin D3 ; Supplementation; Cardiovascular risk; Monocyte subsets; Adults

Nutrition is indispensable for cell survival and proliferation. Thus, loss of nutrition caused by serum starvation in cells could induce formation of reactive oxygen species (ROS), resulting in cell death. Liquiritigenin (LQ) is an active flavonoid in licorice and plays a role in the liver as a hepatic protectant.This study investigated the effect of LQ, metformin [an activator of activated AMP-activated protein kinase (AMPK)] and GW4064 [a ligand of farnesoid X receptor (FXR)] on mitochondrial dysfunction and oxidative stress induced by serum deprivation as well as its molecular mechanism, as assessed by immunoblot and flow cytometer assays.Serum deprivation in HepG2, H4IIE and AML12 cells successfully induced oxidative stress and apoptosis, as indicated by depletion of glutathione, formation of ROS, and altered expression of apoptosis-related proteins such as procaspase-3, poly(ADP-ribose) polymerase, and Bcl-2. However, LQ pretreatment significantly blocked these pathological changes and mitochondrial dysfunction caused by serum deprivation. Moreover, LQ activated AMPK in HepG2 cells and mice liver, as shown by phosphorylation of AMPK and ACC, and this activation was mediated by its upstream kinase (i.e., LKB1). Experiments using a chemical inhibitor of AMPK with LKB1-deficient Hela cells revealed the role of the LKB1–AMPK pathway in cellular protection conferred by LQ. LQ also induced protein and mRNA expression of both FXR as well as small heterodimer partner, which is important since treatment with FXR ligand GW4064 protected hepatocytes against cell death and mitochondrial damage induced by serum deprivation.AMPK activators such as LQ can protect hepatocytes against oxidative hepatic injury and mitochondrial dysfunction induced by serum deprivation, and the beneficial effect might be mediated through the LKB1 pathway as well as FXR induction.
Keywords: AMPK; FXR; Liquiritigenin; Mitochondria; Nutrition deprivation

Consumption of Bifidobacterium animalis subsp. lactis BB-12 in yogurt reduced expression of TLR-2 on peripheral blood-derived monocytes and pro-inflammatory cytokine secretion in young adults by Huicui Meng; Zhaoyong Ba; Yujin Lee; Jiayu Peng; Junli Lin; Jennifer A. Fleming; Emily J. Furumoto; Robert F. Roberts; Penny M. Kris-Etherton; Connie J. Rogers (649-661).
Probiotic bacteria modulate immune parameters and inflammatory outcomes. Emerging evidence demonstrates that the matrix used to deliver probiotics may influence the efficacy of probiotic interventions in vivo. The aims of the current study were to evaluate (1) the effect of one species, Bifidobacterium animalis subsp. lactis BB-12 at a dose of log10 ± 0.5 CFUs/day on immune responses in a randomized, partially blinded, 4-period crossover, free-living study, and (2) whether the immune response to BB-12 differed depending on the delivery matrix.Healthy adults (n = 30) aged 18–40 years were recruited and received four treatments in a random order: (A) yogurt smoothie alone; smoothie with BB-12 added (B) before or (C) after yogurt fermentation, or (D) BB-12 given in capsule form. At baseline and after each 4-week treatment, peripheral blood mononuclear cells (PBMCs) were isolated, and functional and phenotypic marker expression was assessed.BB-12 interacted with peripheral myeloid cells via Toll-like receptor 2 (TLR-2). The percentage of CD14+HLA-DR+ cells in peripheral blood was increased in male participants by all yogurt-containing treatments compared to baseline (p = 0.0356). Participants who consumed yogurt smoothie with BB-12 added post-fermentation had significantly lower expression of TLR-2 on CD14+HLA-DR+ cells (p = 0.0186) and reduction in TNF-α secretion from BB-12- (p = 0.0490) or LPS-stimulated (p = 0.0387) PBMCs compared to baseline.These findings not only demonstrate a potential anti-inflammatory effect of BB-12 in healthy adults, but also indicate that the delivery matrix influences the immunomodulatory properties of BB-12.
Keywords: Probiotics; BB-12; Delivery matrix; Inflammation; TNF-α

Effect of olive oil phenolic compounds on the expression of blood pressure-related genes in healthy individuals by Sandra Martín-Peláez; Olga Castañer; Valentini Konstantinidou; Isaac Subirana; Daniel Muñoz-Aguayo; Gemma Blanchart; Sonia Gaixas; Rafael de la Torre; Magí Farré; Guillermo T Sáez; Kristina Nyyssönen; Hans Joachim Zunft; Maria Isabel Covas; Montse Fitó (663-670).
To investigate whether the ingestion of olive oil having different phenolic contents influences the expression of blood pressure-related genes, involved in the renin–angiotensin–aldosterone system, in healthy humans.A randomized, double-blind, crossover human trial with 18 healthy subjects, who ingested 25 mL/day of olive oils (1) high (366 mg/kg, HPC) and (2) low (2.7 mg/kg, LPC) in phenolic compounds for 3 weeks, preceded by 2-week washout periods. Determination of selected blood pressure-related gene expression in peripheral blood mononuclear cells (PBMNC) by qPCR, blood pressure and systemic biomarkers.HPC decreased systolic blood pressure compared to pre-intervention values and to LPC, and maintained diastolic blood pressure values compared to LPC. HPC decreased ACE and NR1H2 gene expressions compared with pre-intervention values, and IL8RA gene expression compared with LPC.The introduction to the diet of an extra-virgin olive oil rich in phenolic compounds modulates the expression of some of the genes related to the renin–angiotensin–aldosterone system. These changes could underlie the decrease in systolic blood pressure observed.
Keywords: Olive oil; Phenolic compounds; Blood pressure; Nutrigenomics; RAAS

Metabolic changes in serum metabolome in response to a meal by Aahana Shrestha; Elisabeth Müllner; Kaisa Poutanen; Hannu Mykkänen; Ali A. Moazzami (671-681).
The change in serum metabolic response from fasting state to postprandial state provides novel insights into the impact of a single meal on human metabolism. Therefore, this study explored changes in serum metabolite profile after a single meal.Nineteen healthy postmenopausal women with normal glucose tolerance participated in the study. They received a meal consisting of refined wheat bread (50 g carbohydrates, 9 g protein, 4.2 g fat and 2.7 g dietary fibre), 40 g cucumber and 300 mL noncaloric orange drink. Blood samples were collected at fasting and five postprandial time points. Metabolic profile was measured by nuclear magnetic resonance and targeted liquid chromatography–mass spectrometry. Changes over time were assessed with multivariate models and ANOVA, with baseline as control.The metabolomic analyses demonstrated alterations in phospholipids, amino acids and their breakdown products, glycolytic products, acylcarnitines and ketone bodies after a single meal. More specifically, phosphatidylcholines, lysophosphatidylcholines and citrate displayed an overall declining pattern, while leucine, isoleucine, methionine and succinate increased initially but declined thereafter. A sharp decline in acylcarnitines and ketone bodies and increase in glycolytic products postprandially suggest a switch in the body’s energy source from β-oxidation to glycolysis. Moreover, individuals with relatively high postprandial insulin responses generated a higher postprandial leucine responses compared to participants with lower insulin responses.The study demonstrated complex changes from catabolic to anabolic metabolism after a meal and indicated that the extent of postprandial responses is different between individuals with high and low insulin response.
Keywords: Metabolomics; Postprandial changes; Insulin; Amino acid; Acylcarnitine; Glycolytic products; Phosphatidylcholine

Various dietary components have been studied in relation to overall mortality; however, little is known about the relationship between the inflammatory potential of overall diet and mortality. We examined the association between the dietary inflammatory index (DII) and mortality in the National Health and Nutrition Examination Survey III follow-up study. The DII was computed from baseline dietary intake assessed using 24-h dietary recalls (1988–1994). Mortality was determined from the National Death Index records through 2006. Cox proportional hazards regression was used to estimate hazard ratios (HRs) and 95 % confidence interval (95 % CI). During the follow-up, 2795 deaths were identified, including 1233 due to cardiovascular disease (CVD), and 615 due to cancer, 158 of which were due to digestive-tract cancers.Multivariate Cox proportional hazards regression analyses, adjusting for age, race, diabetes status, hypertension, physical activity, body mass index, poverty index, and smoking, revealed positive associations between higher DII scores and mortality. Comparing subjects in DII tertile 3 versus tertile 1, significant associations were noted for all-cause mortality (HRTertile3vs1 1.34; 95 % CI 1.19–1.51, P trend < 0.0001), CVD mortality (HRTertile3vs1 1.46; 95 % CI 1.18–1.81, P trend = 0.0006), cancer mortality (HRTertile3vs1 1.46; 95 % CI 1.10–1.96, P trend = 0.01), and digestive-tract cancer mortality (HRTertile3vs1 2.10; 95 % CI 1.15–3.84, P trend = 0.03).These results indicate that a pro-inflammatory diet, as indicated by higher DII scores, was associated with higher risk of all-cause, CVD, and cancer mortality.
Keywords: Dietary inflammatory index; Mortality; NHANES III

Relation between neonatal malnutrition and gene expression: inflammasome function in infections caused by Candida Albicans by Thacianna Barreto Da Costa; Natália Gomes De Morais; Joana Maria Bezerra De Lira; Thays Miranda De Almeida; Suênia Da Cunha Gonçalves-De-Albuquerque; Valéria Rêgo Alves Pereira; Milena De Paiva Cavalcanti; Célia Maria Machado Barbosa De Castro (693-704).
To investigate the effects of neonatal malnutrition followed by nutritional replacement on the signaling mechanisms developed by the inflammasome complex by analyzing the expression of the targeted TLR2, TLR4, NLRP3, caspase-1 and release of IL-1β and IL-18 by alveolar macrophages infected in vitro with Candida albicans.Male Wistar rats (n = 24), 90–120 days, were suckled by mothers whose diet during lactation contained 17 % protein in the nourish group and 8 % protein in the malnourished group. After weaning, both groups were fed a normal protein diet. Macrophages were obtained after tracheostomy, through the collection of bronchoalveolar lavage fluid. The quantification of the expression levels of targets (TLR2, TLR4, NLRP3 and caspase-1) was performed by real-time RT-PCR. Production of cytokines was performed by ELISA.The malnourished animals during lactation showed reduced body weight from the fifth day of life, remaining until adulthood. Further, the model applied malnutrition induced a lower expression of TLR4 and caspase-1. The quantification of the TLR2 and NLRP3, as well as the release of IL-1β and IL-18, was not different between groups of animals nourished and malnourished. The system challenged with Candida albicans showed high expression levels of all targets in the study.The tests demonstrate nutritional restriction during critical periods of development, although nutritional supplementation may compromise defense patterns in adulthood in a timely manner, preserving distinct signaling mechanism, so that the individual does not become widely vulnerable to infections by opportunistic pathogens.
Keywords: Neonatal malnutrition; Programming; Macrophage; Candida albicans ; Toll-like receptors; NOD-like receptors

Taurine supplementation regulates Iκ-Bα protein expression in adipose tissue and serum IL-4 and TNF-α concentrations in MSG obesity by Luiz Carlos Caetano; Maria Lúcia Bonfleur; Rosane Aparecida Ribeiro; Tarlliza Romanna Nardelli; Camila Lubaczeuski; Juliana do Nascimento da Silva; Everardo Magalhães Carneiro; Sandra Lucinei Balbo (705-713).
Obesity is usually associated with low-grade inflammation, which impairs insulin action. The amino acid, taurine (TAU), regulates glucose homeostasis and lipid metabolism and presents anti-inflammatory actions. Here, we evaluated whether inflammatory markers are altered in the serum and retroperitoneal adipose tissue of monosodium glutamate (MSG) obese rats, supplemented or not with TAU.Male Wistar rats received subcutaneous injections of MSG (4 mg/kg body weight/day, MSG group) or hypertonic saline (CTL) during the first 5 days of life. From 21 to 120 days of age, half of each of the MSG and CTL groups received 2.5 % TAU in their drinking water (CTAU and MTAU).At 120 days of age, MSG rats were obese and hyperinsulinemic. TAU supplementation reduced fat deposition without affecting insulinemia in MTAU rats. MSG rats presented increased pIκ-Bα/Iκ-Bα protein expression in the retroperitoneal adipose tissue. TAU supplementation decreased the ratio of pIκ-Bα/Iκ-Bα protein, possibly contributing to the increased Iκ-Bα content in MTAU adipose tissue. Furthermore, MSG obesity or supplementation did not alter TNF-α, IL-1β or IL-6 content in adipose tissue. In contrast, MSG rats presented lower serum TNF-α, IL-4 and IL-10 concentrations, and these alterations were prevented by TAU treatment. MSG obesity in rats was not associated with alterations in pro-inflammatory markers in retroperitoneal fat stores; however, reductions in the serum concentrations of anti-inflammatory cytokines and of TNF-α were observed. TAU treatment decreased adiposity, and this effect was associated with the normalization of circulating TNF-α and IL-4 concentrations in MTAU rats.
Keywords: Cytokines; Monosodium glutamate; Obesity; Taurine supplementation

Methylation patterns of Vegfb promoter are associated with gene and protein expression levels: the effects of dietary fatty acids by Roberto Monastero; Sara García-Serrano; Ana Lago-Sampedro; Francisca Rodríguez-Pacheco; Natalia Colomo; Sonsoles Morcillo; Gracia M. Martín-Nuñez; Juan M. Gomez-Zumaquero; Eduardo García-Fuentes; Federico Soriguer; Gemma Rojo-Martínez; Eva García-Escobar (715-726).
We have investigated the epigenetic regulation by dietary fatty acids of Vegfb levels in rats’ white adipose tissue and 3T3-L1 cells. A group of rats were assigned to three diets, each one with a different composition of saturated, monounsaturated and polyunsaturated fatty acids. Samples of white adipose tissues were taken for the methylation and expression studies. Additionally, 3T3-L1 cells were treated with palmitic, oleic, and linoleic fatty acids. After treatment, cells were harvested and genetic material was extracted for the analysis of Vegfb levels.We report evidence of changes in the methylation levels of the CpG island at the Vegfb promoter and in the Vegfb expression levels in vivo and in vitro by dietary fatty acid, with the main contribution of the linoleic fatty acid. Vegfb promoter methylation levels were closely related to the Vegfb gene expression.According to our results, the regulation of Vegfb gene expression by dietary fatty acids may be mediated, at least in part, by epigenetic modifications on Vegfb promoter methylation. Considering the deep association between angiogenesis and tissue growth, we suggest the nutriepigenetic regulation of Vegfb as a key target in the control of the adipose tissue expansion.
Keywords: Adipose tissue; Dietary fatty acids; Gene expression; Nutriepigenetic regulation; Vegfb

The effect of three different ad libitum diets for weight loss maintenance: a randomized 18-month trial by Anette Due; Thomas M. Larsen; Huiling Mu; Kjeld Hermansen; Steen Stender; Søren Toubro; David B. Allison; Arne Astrup (727-738).
To test the effect of three diets in their ability to sustain weight loss and improve type 2 diabetes (T2D) and cardiovascular disease (CVD) risk markers after 18-month intervention. Following a ≥8 % weight loss, 131 healthy, overweight/obese (BMI ± SD 31.5 ± 2.6 kg/m2) men (n = 55) and women (n = 76) aged 28.2 ± 4.8 years were randomized to either 1. Moderate fat (40 E%) with 20 E% MUFA and low in glycemic index (GI) (MUFA, n = 54), 2. Low fat (25 E%) and medium in GI (LF, n = 51) or 3. Control (35 E% fat) and high in GI (CTR, n = 26) all with similar protein content, and all provided ad libitum. First 6-month intervention with 100 % food provision (previously reported) following 12 months of moderately intensive intervention with 20 % food provision now reported. Attrition rate was higher in MUFA (63 %) than in LF (37 %, P = 0.019) and CTR (42 %, P = 0.09) group. Weight regain in completers was not different between groups (mean ± SEM), MUFA 7.1 ± 2.1 % versus LF 5.6 ± 1.3 % versus CTR 7.2 ± 1.5 %, nor was body fat regain, MUFA 4.8 ± 1.0 % versus LF 4.7 ± 0.8 % versus CTR 5.7 ± 0.6 %. The MUFA group reduced LDL/HDL ratio by −0.47 ± 0.09 compared with −0.23 ± 0.11 in LF (P < 0.05) and 0.06 ± 0.14 (P < 0.005) in CTR groups.Weight regain or body composition did not differ between diets over 18 months. No effects on risk markers for T2D or CVD were found, with the exception of an improvement in the LDL/HDL ratio by the MUFA diet compared to the CTR diet. The LF diet was generally more satisfactory and the MUFA diet seemed more difficult to follow.
Keywords: Weight loss; Weight maintenance; Mediterranean diet; Cardiovascular disease; Dietary intervention

Very few studies have examined the association between beverage intake patterns and healthy lifestyle characteristics. Most of the research that has been carried out focuses on the consumption of soft drinks or alcohol and ignores the overall beverage pattern. The aim of this study is to evaluate the association between consumption of different types of beverage and physical exercise practice and MedDiet adherence. Cross-sectional information about fluid intake from different types of beverages was collected in 1262 men and women between 18 and 70 years old, using a 24-h fluid-specific diary over seven consecutive days. Physical exercise was evaluated with a self-reported questionnaire, and MedDiet adherence was assessed using a validated 14-item questionnaire. Both variables were classified into three categories.Individuals with greater adherence to the MedDiet showed a higher intake of water and wine and a lower consumption of sweet regular beverages. Participants who engaged in more physical exercise consumed more water, milk and derivatives, juices and wine and less sweet regular beverages. Compared to the lowest category, the possibility of meeting the EFSA recommendations of total fluid intake was greater in individuals with eight or more points on the MedDiet adherence questionnaire [OR 1.94; 95 % CI 1.25–3.01] and in those who practice physical exercise three times a week or more [OR 1.71; 95 % CI 1.22–2.39]. Participants with a healthier lifestyle had a lower risk of exceeding the WHO’s free-sugar recommendations only from beverages.Participants with greater adherence to the MedDiet and who engaged in more physical exercise exhibit a healthier pattern of fluid intake.
Keywords: Fluid intake; Mediterranean diet; Physical exercise; Adult; Beverage

Lebanese children are iodine deficient and urinary sodium and fluoride excretion are weak positive predictors of urinary iodine by Hala Ghattas; Sirine Francis; Carla El Mallah; Dareen Shatila; Karina Merhi; Sani Hlais; Michael Zimmermann; Omar Obeid (749-755).
To assess iodine and fluoride status among Lebanese children.A nationally representative cross-sectional study of 6- to 10-year-old schoolchildren was conducted using multistage cluster sampling. Spot urine samples were collected from 1403 children, and urinary iodine, fluoride, creatinine and sodium levels were measured. Salt samples from markets (n = 30) were tested for iodine concentration by titration. Median urinary iodine concentration was 66.0 µg/l, indicating mild deficiency, and almost 75 % of Lebanese children had a urinary iodine concentration (UIC) <100 µg/l. UIC was higher among children from private schools and in areas of higher socioeconomic status. Most salt samples were fortified at levels far below the legislated requirement, and 56 % of samples contained less than 15 ppm iodine. Fluoride-to-creatinine ratio (F/Cr) was 0.250 (0.159–0.448) mg/g. There were weak positive correlations between UIC and urinary sodium (r 2 = 0.039, P value <0.001) and UIC and urinary fluoride (r 2 = 0.009, P value <0.001).Lebanese elementary school children are iodine deficient due to inadequately iodized salt. The weak correlation between UIC and urinary sodium suggests most dietary iodine does not come from iodized salt. The poor correlation between UIC and urinary fluoride suggests that fluoride intake is not affecting iodine metabolism. Efforts are needed in Lebanon to improve industry compliance with salt fortification through improved monitoring and enforcement of legislation.
Keywords: Urinary iodine excretion; Urinary fluoride; Iodine/creatinine ratio; Fluoride/creatinine ratio; Sodium/creatinine ratio; Lebanon

Relative validation of 24-h urinary hippuric acid excretion as a biomarker for dietary flavonoid intake from fruit and vegetables in healthy adolescents by Katharina J. Penczynski; Danika Krupp; Anna Bring; Katja Bolzenius; Thomas Remer; Anette E. Buyken (757-766).
A biomarker for dietary flavonoid intake from fruit and vegetables (FlavFV) is needed to elucidate the relevance of flavonoids from these sources for the prevention of chronic diseases. Urinary hippuric acid (HA)—a major metabolite of flavonoids—is promising in this respect as it was shown to satisfyingly indicate fruit and vegetable consumption in different age groups. Therefore, we validated urinary HA as a biomarker for intake of FlavFV.Analyses included data from 287 healthy adolescents of the DONALD Study (aged 9–16 years) for whom a minimum of two pairs of HA measurements from 24-h urine samples (test method) and FlavFV intake estimated from 3-day weighed dietary records (reference method) existed. Agreement between both methods was assessed by Spearman correlation and cross-classification analyses. Possible confounders of the association were identified by linear regression models. Analyses were performed using a split-sample approach allowing for consecutive exploration (n = 192) and confirmation (n = 95) of results.Agreement between urinary HA excretion and FlavFV intake was moderate according to correlation analysis in the exploratory sample (r unadjusted = 0.47, P < 0.0001). Yet, 79 % of the subjects were classified into same/adjacent quartiles, and only 5 % were misclassified into opposite quartiles. These findings were corroborated by analyses in the confirmatory sample (r unadjusted = 0.64; 88 % in same/adjacent vs. 4 % in opposite quartiles). Body surface area (BSA) was the only relevant covariate in the exploratory sample, and its adjustment improved cross-classification estimates in both subsamples.BSA-adjusted 24-h urinary HA excretion represents a suitable biomarker of habitual FlavFV intake in healthy adolescents.
Keywords: 24-h Urine; Adolescents; Biomarker of intake; Flavonoids; Hippuric acid; Relative validation

Dietary magnesium intake and the risk of diabetes in the Japanese community: results from the Takayama study by Kie Konishi; Keiko Wada; Takashi Tamura; Michiko Tsuji; Toshiaki Kawachi; Chisato Nagata (767-774).
Several experimental studies showed that magnesium intake improved insulin resistance and glucose uptake in diabetes patients. However, epidemiological studies on the association between magnesium intake and diabetes risk have yielded inconsistent results. We investigated whether magnesium intake is related to the risk of developing diabetes in a population-based cohort study in Japan.Study subjects were participants in the Takayama study. A total of 13,525 residents in Takayama City, Japan, responded to a self-administered questionnaire in 1992 and to a follow-up questionnaire seeking information about diabetes in 2002. Magnesium and other nutrient intakes were estimated from a validated food frequency questionnaire administered at the baseline.During a follow-up of 10 years, 438 subjects reported diabetes newly diagnosed by physician. Compared with women in the low quartile of magnesium intake, women in the high quartile were at a significantly reduced risk of diabetes (HR 0.50; 95 % CI 0.30–0.84; P-trend 0.005) after adjustments for covariates. In men, there was no association between magnesium intake and the risk of diabetes.These results suggest that diets with a high intake of magnesium may decrease the risk of diabetes in women.
Keywords: Magnesium; Diabetes; Prospective studies; Diet; The Japanese

Acute citrulline malate supplementation improves upper- and lower-body submaximal weightlifting exercise performance in resistance-trained females by Jordan M. Glenn; Michelle Gray; Lauren N. Wethington; Matthew S. Stone; Rodger W. Stewart Jr.; Nicole E. Moyen (775-784).
Citrulline malate (CM) is a nonessential amino acid that increases exercise performance in males. However, based on physiological differences between genders, these results cannot be extrapolated to females. Therefore, the purpose of this investigation was to evaluate effects of acute CM supplementation on upper- and lower-body weightlifting performance in resistance-trained females. Fifteen females (23 ± 3 years) completed two randomized, double-blind trials consuming either CM (8 g dextrose + 8 g CM) or a placebo (8 g dextrose). One hour after supplement consumption, participants performed six sets each of upper- (i.e., bench press) and lower-body (i.e., leg press) exercises to failure at 80 % of previously established one-repetition maximum. Immediately after each set, repetitions completed, heart rate and rating of perceived exertion (RPE) were recorded.Repeated-measures analysis of variance indicated that subjects completed significantly (p = .045) more repetitions throughout upper-body exercise when consuming CM versus placebo (34.1 ± 5.7 vs. 32.9 ± 6.0, respectively). When consuming CM, similar significant (p = .03) improvements in total repetitions completed were observed for lower-body exercise (66.7 ± 30.5 vs. 55.13 ± 20.64, respectively). Overall RPE score was significantly lower (p = .02) in upper-body exercise when subjects consumed CM versus placebo (7.9 ± 0.3 and 8.6 ± 0.2, respectively). The supplement consumed exhibited no significant effects on heart rate at any time point.Acute CM supplementation in females increased upper- and lower-body resistance exercise performance and decreased RPE during upper-body exercise. These data indicate that athletes competing in sports with muscular endurance-based requirements may potentially improve performance by acutely supplementing CM.
Keywords: Ergogenic aid; Sports nutrition; Amino acids; Resistance exercise; Women; Nitric oxide

N- and S-homocysteinylation reduce the binding of human serum albumin to catechins by Angelo Zinellu; Salvatore Sotgia; Bastianina Scanu; Dionigia Arru; Annalisa Cossu; Anna Maria Posadino; Roberta Giordo; Arduino A. Mangoni; Gianfranco Pintus; Ciriaco Carru (785-791).
The dietary flavonoids epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) have been shown to interact with circulating albumin for transport in blood to different body tissues. This interaction may modulate their bioavailability and effectiveness.Using affinity capillary electrophoresis to assess binding constants (K b), we investigated whether posttranslational modification of human serum albumin (HSA) through N- and S-homocysteinylation, commonly observed in hyperhomocysteinemia, may modify its interaction with catechins.S-Hcy HSA had lower Kb values toward EC (14 %), EGC (18 %), ECG (24 %) and EGCG (30 %). Similarly, N-Hcy HSA had lower Kb values toward EC (17 %), EGC (22 %), ECG (23 %) and EGCG (32 %). No differences were observed in the affinity between catechins, albumin and mercaptalbumin.Therefore, HSA posttranslational modifications typical of hyperhomocysteinemia reduce its affinity to catechins, potentially affecting their pharmacokinetics and availability at the active sites.
Keywords: Affinity capillary electrophoresis; Catechins; Albumin; Binding constant

Belgian primary school children’s hydration status at school and its personal determinants by Nathalie Michels; Karen Van den Bussche; Johan Vande Walle; Stefaan De Henauw (793-805).
Dehydration has been related to several health aspects, and children are especially vulnerable. Since children spend a large time at school, we aim to examine children’s hydration status at school-start and its change during the school-day by objective measures. To identify subpopulations at risk, determinants of hydration were tested.In 371 Belgian 7–13-year-old children, hydration was measured by (1) urinary osmolality at school-start and by a pooled school-day sample; (2) body water% by impedance; (3) parental reported beverage consumption; (4) urination frequency. Linear regression analyses were used to test putative predictors of hydration status: age, sex, parental education, region (Dutch-speaking versus French-speaking part of Belgium), diet quality and adiposity.A mean osmolality of 888 mosmol/kg was found in the school-start sample and 767 mosmol/kg in the school-day sample. This resulted in, respectively, 76 and 54 % of the children being dehydrated (>800 mosmol/kg). In 45 % of the children, the hydration level decreased over the school-day. Also the body water% as derived from bio-impedance (57 % ±4), the reported average daily beverage intake (911 ml) and the lower urination frequency during weekdays versus weekend days confirmed the low hydration status in our school population. Boys, Walloon children and those with higher adiposity were at increased risk of low hydration level. Diet quality was not the predictor of hydration status.Hydration status at school appeared problematic in this population. This emphasizes the need for more resources and attention by school management and governmental organizations. Herein, especially Walloon schools and boys should be reached.
Keywords: Hydration; Urinary osmolality; Sociodemographic; Adiposity; Diet quality

A possible role for ghrelin, leptin, brain-derived neurotrophic factor and docosahexaenoic acid in reducing the quality of life of coeliac disease patients following a gluten-free diet by Francesco Russo; Guglielmina Chimienti; Caterina Clemente; Carla Ferreri; Antonella Orlando; Giuseppe Riezzo (807-818).
A gluten-free diet (GFD) has been reported to negatively impact the quality of life (QoL) of coeliac disease (CD) patients. The gut–brain axis hormones ghrelin and leptin, with the brain-derived neurotrophic factor (BDNF), may affect QoL of CD patients undergoing GFD. Our aims were to evaluate whether: (a) the circulating concentrations of leptin, ghrelin and BDNF in CD patients were different from those in healthy subjects; (b) GFD might induce changes in their levels; (c) BDNF Val66Met polymorphism variability might affect BDNF levels; and (d) serum BDNF levels were related to dietary docosahexaenoic acid (DHA) as a neurotrophin modulator.Nineteen adult coeliac patients and 21 healthy controls were included. A QoL questionnaire was administered, and serum concentrations of ghrelin, leptin, BDNF and red blood cell membrane DHA levels were determined at the enrolment and after 1 year of GFD. BDNF Val66Met polymorphism was analysed.Results from the questionnaire indicated a decline in QoL after GFD. Ghrelin and leptin levels were not significantly different between groups. BDNF levels were significantly (p = 0.0213) lower in patients after GFD (22.0 ± 2.4 ng/ml) compared to controls (31.2 ± 2.2 ng/ml) and patients at diagnosis (25.0 ± 2.5 ng/ml). BDNF levels correlated with DHA levels (p = 0.008, r = 0.341) and the questionnaire total score (p = 0.041, r = 0.334).Ghrelin and leptin seem to not be associated with changes in QoL of patients undergoing dietetic treatment. In contrast, a link between BDNF reduction and the vulnerability of CD patients to psychological distress could be proposed, with DHA representing a possible intermediate.
Keywords: BDNF; Coeliac disease; DHA; Gluten-free diet; Ghrelin; Leptin; Psychological distress

Metabolic syndrome and selenium during gestation and lactation by Fátima Nogales; M. Luisa Ojeda; Paulina Muñoz del Valle; Alejandra Serrano; M. Luisa Murillo; Olimpia Carreras Sánchez (819-830).
Selenium (Se) has a dual role in metabolic syndrome (MS) development as it has an antioxidant action against both “good” and “bad” reactive oxygen species. This study evaluates Se body profile in dams which present MS during gestation and lactation, in order to elucidate a normal dietary Se’s implication in this pathology.Rats were randomized into control (C) and fructose (F) groups. The rich fructose diet (65 %) during gestation and lactation periods induced MS in dams. Se body distribution was determined by atomic absorption spectrophotometry, and the hepatic activity of the four antioxidant enzymes and the bimolecular oxidation were determined by spectrophotometry. The cardiac activity was monitored using the indirect tail occlusion method. Lipid and glucidic profile was also analyzed.Despite the fact that the diet supplied has 0.1 ppm of Se, the minimal dietary requirement for rats, F dams ate less amount of food, and therefore, they had lower Se retention. However, they had normal levels of Se in serum and milk. Dams with MS had Se depletion in heart and muscle joint to hypertension and a lower heart rate, and Se repletion in liver and kidney. Despite the increase in hepatic glutathione peroxidase (GPx) and catalase activity found, lipid oxidation occurred—probably because superoxide dismutase activity was diminished. In heart, the activity and expression of the selenoprotein GPx1 were decreased.With these results, it is not possible to elucidate whether a dietary Se supplementation or a Se-restricted diet are good for MS; because despite the fact that GPx activity is increased in liver, it is also found, for the first time, that heart Se deposits are significantly decreased during MS.
Keywords: Metabolic syndrome; Selenium; Gestation; Lactation

Antiproliferative activity of the ellagic acid-derived gut microbiota isourolithin A and comparison with its urolithin A isomer: the role of cell metabolism by Antonio González-Sarrías; María Ángeles Núñez-Sánchez; Rocío García-Villalba; Francisco A. Tomás-Barberán; Juan Carlos Espín (831-841).
Urolithins, metabolites produced by the gut microbiota from ellagic acid, have been acknowledged with cancer chemopreventive activity. Although urolithin A (Uro-A) has been reported to be the most active one, 10–50 % of humans can also produce the isomer isourolithin A (IsoUro-A). However, no biological activity for IsoUro-A has been reported so far. Herein, we describe for the first time the antiproliferative effect of IsoUro-A, compared to Uro-A, against both human colon cancer (Caco-2) and normal (CCD18-Co) cell lines.Cell proliferation was evaluated by MTT and Trypan blue exclusion assays. Cell cycle was analyzed by flow cytometry and apoptosis measured by the Annexin V/PI method. Finally, urolithins metabolism was analyzed by HPLC–DAD-MS/MS.IsoUro-A inhibited the proliferation of Caco-2 cells in a time- and dose-dependent manner, though it was significantly lower than Uro-A (IC50 = 69.7 ± 4.5 and 49.2 ± 3.8 μM at 48 h, respectively). Both urolithins arrested Caco-2 cell cycle at S and G2/M phases and induced apoptosis at concentrations previously found in human colon tissues. Notably, Caco-2 cells glucuronidated more efficiently IsoUro-A than Uro-A (~50 vs. ~20 % of conversion after 48 h, respectively). Both Uro-A and IsoUro-A glucuronides did not exert antiproliferative effects. In addition, cell growth inhibition was higher in Caco-2 than in normal cells.IsoUro-A exerts strong antiproliferative activity, which is reduced by the extensive glucuronidation at 9-position in cancer cells. Further studies are needed to elucidate whether the in vitro structure–activity relationship found for Uro-A and IsoUro-A plays any role in humans.
Keywords: Urolithins; Isourolithin A; Ellagic acid; Cell cycle; Apoptosis; Colon cancer

Fish consumption and frying of fish in relation to type 2 diabetes incidence: a prospective cohort study of Swedish men by Alice Wallin; Daniela Di Giuseppe; Nicola Orsini; Agneta Åkesson; Nita G. Forouhi; Alicja Wolk (843-852).
Epidemiological evidence on the association between fish consumption and risk of type 2 diabetes is heterogeneous across geographical regions. Differences related to fish consumption pattern could possibly help explain the discrepancy between the findings. We therefore aimed to investigate the association between fish consumption (total, fried, specific fish items) and type 2 diabetes incidence, taking exposure to contaminants present in fish (polychlorinated biphenyls and methyl mercury) into consideration.The population-based Cohort of Swedish Men, including 35,583 men aged 45–79 years, was followed from 1998 to 2012. We estimated hazard ratios (HRs) with 95 % confidence intervals (CIs) using Cox proportional hazards models.During 15 years of follow-up, 3624 incident cases were identified. Total fish consumption (≥4 servings/week vs. <1 serving/week) was not associated with type 2 diabetes in multivariable-adjusted analysis (HR 1.00; 95 % CI 0.85–1.18); however, a statistically non-significant inverse association was observed after adjustment for dietary contaminant exposures (HR 0.79; 95 % CI 0.60–1.04). Fried fish (≥6 servings/month vs. ≤1 servings/month) and shellfish consumption (≥1 serving/week vs. never/seldom) were associated with HRs of 1.14 (95 % CI 1.03–1.31) and 1.21 (95 % CI 1.07–1.36), respectively.We observed no overall association between total fish consumption and type 2 diabetes. The results indicated that dietary contaminants in fish may influence the relationship. Fried fish and shellfish consumption were associated with higher type 2 diabetes incidence. These findings suggest that more specific advice on fish species sub-types (varying in contamination) and preparation methods may be warranted.
Keywords: Fish; Fried foods; Polychlorinated biphenyls; Methyl mercury; Type 2 diabetes; Cohort study

Metabolism of strawberry mono- and dimeric ellagitannins in rats fed a diet containing fructo-oligosaccharides by Adam Jurgoński; Jerzy Juśkiewicz; Bartosz Fotschki; Krzysztof Kołodziejczyk; Joanna Milala; Monika Kosmala; Katarzyna Grzelak-Błaszczyk; Lidia Markiewicz (853-864).
We investigated the effects of dietary supplementation with strawberry extracts rich in ETs and fructo-oligosaccharides (FOS) on the intestinal microbiota and the formation of bacterial metabolites in the distal intestine, as well as the absorption of ET metabolites and antioxidant status in rats.Rats were allocated into six groups of eight animals each and fed for 4 weeks with a control diet (group C), a control diet supplemented with FOS (group C + FOS) or modifications of these diets, in which a monomeric or dimeric ET-rich extract was added (groups ME and ME + FOS or DE and DE + FOS, respectively).The extract addition, the FOS addition and their interaction significantly affected the total and selected bacterial counts in the caecal digesta (all P < 0.005). The total bacterial count was the highest in group C + FOS, lower in group DE and the lowest in group ME + FOS (10.6, 10.3 and 8.52 log cells/g, respectively; P ≤ 0.05). The total caecal content of ET metabolites was higher in the ME and ME + FOS group than in the DE and DE + FOS group, respectively (67.8 and 89.5 vs. 13.0 and 18.0 µg/g, respectively; P < 0.001). The total plasma concentration of ET metabolites was higher in the ME + FOS and DE + FOS group than in the ME group (248 and 281 vs. 8.13 ng/mL, respectively; P < 0.001).ETs of the monomeric ET-rich extract are more prone to intestinal breakdown than those of the dimeric ET-rich extract, and absorption of their metabolites can be increased by dietary FOS; however, together, they evoke strong antibacterial activity.
Keywords: Bifidobacteria; Fructans; Hydrolysable tannins; Lactic acid bacteria; Nasutins; Urolithins

Middle-aged C57Bl/6J mice fed for 6 months with extra-virgin olive oil rich in phenols (H-EVOO, phenol dose/day: 6 mg/kg) showed cognitive and motor improvement compared to controls fed the same olive oil deprived of phenolics (L-EVOO). The aim of the present study was to evaluate whether these behavioral modifications were associated with changes in gene and miRNA expression in the brain.Two brain areas involved in cognitive and motor processes were chosen: cortex and cerebellum. Gene and miRNA profiling were analyzed by microarray and correlated with performance in behavioral tests.After 6 months, most of the gene expression changes were restricted to the cerebral cortex. The genes modulated by aging were mainly down-regulated, and the treatment with H-EVOO was associated with a significant up-regulation of genes compared to L-EVOO. Among those, we found genes previously 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. miRNAs were mostly up-regulated in old L-EVOO animals compared to young. However, H-EVOO-fed mice cortex displayed miRNA expression profiles similar to those observed in young mice. Sixty-three miRNAs, out of 1203 analyzed, were significantly down-regulated compared to the L-EVOO group; among them, we found miRNAs whose predicted target genes were up-regulated by the treatment, such as mir-484, mir-27, mir-137, mir-30, mir-34 and mir-124.We are among the first to report that a dietary intervention starting from middle age with food rich in phenols can modulate at the central level the expression of genes and miRNAs involved in neuronal function and synaptic plasticity, along with cognitive, motor and emotional behavior.
Keywords: Aging brain; miRNomics; Genomics; Phenolic compounds; Nutraceuticals

DNA methylation is one of the most extensively studied mechanisms within epigenetics, and it is suggested that diet-induced changes in methylation status could be involved in energy metabolism regulation. Conjugated linoleic acid (CLA) and calcium supplementation counteract body weight gain, particularly under a high-fat (HF) diet, in adult mice. The aim was to determine whether the modulation of DNA methylation pattern in target genes and tissues could be an underlying mechanism of action.Mice (C57BL/6J) were divided into five groups according to diet and treatment: normal fat as the control group (12 % kJ content as fat), HF group (43 % kJ content as fat), HF + CLA (6 mg CLA/day), HF + calcium (12 g/kg of calcium) and HF with both compounds. Gene expression and methylation degree of CpG sites in promoter sequences of genes involved in fatty acid metabolism, including adiponectin (Adipoq), stearoyl-CoA desaturase (Scd1) and fatty acid synthase (Fasn), were determined by bisulphite sequencing in liver and epididymal white adipose tissue. Results showed that the methylation profile of promoters was significantly altered by dietary supplementation in a gene- and tissue-specific manner, whereas only slight changes were observed in the HF group. Furthermore, changes in specific CpG sites were also associated with an overall healthier metabolic profile, in particular for calcium-receiving groups.Both CLA and calcium were able to modify the methylation pattern of genes involved in energy balance in adulthood, which opens a novel area for increasing efficiency in body weight management strategies.
Keywords: Calcium supplementation; Conjugated linoleic acid; DNA methylation; Fatty acid synthase; Stearoyl-CoA desaturase; Adiponectin

Effects of proanthocyanidin on oxidative stress biomarkers and adipokines in army cadets: a placebo-controlled, double-blind study by Mariana C. Gonçalves; Magna C. F. Passos; Cyntia F. de Oliveira; Julio B. Daleprane; Josely C. Koury (893-900).
The relatively recent advent of polyphenol supplement for exercise studies has been tested in a variety of forms and doses. However, the dose–response on adipokines and oxidative stress biomarker effect remains unknown. The aim of the present study was to assess the effect of intense, long-duration (48-h) exercise, and a single dose of proanthocyanidin, on plasma leptin, adiponectin, and electronegative low-density lipoprotein (LDL(−)) concentrations.Fifty-four healthy male army cadets (22 ± 2 years) participated in a double-blind, randomized, placebo-controlled study and were distributed between control (CG; n = 27) and supplemented groups (SG; n = 27). Immediately before the start of the exercise, both CG and SG groups received a capsule containing starch (200 mg) or proanthocyanidin (dry Vitis vinifera extract, 200 mg), respectively. Following a 12-h fasting period, the plasma adiponectin, leptin, and LDL(−) concentrations were measured prior to the start of the exercise after 24 and 48 h of military training, and after 24 h of rest. The effects of the proanthocyanidin (supplement), exercise (time), and their interaction were investigated using factorial two-way ANOVA.Plasma leptin concentration was only influenced by exercise (p = 0.001). Plasma adiponectin concentration was influenced by exercise (p = 0.037), and by the exercise x supplement interaction (p = 0.033). LDL(−) was influenced by the supplement (p = 0.001), exercise (p = 0.001), and their interaction (p = 0.001).A single dose of proanthocyanidin (200 mg) was able to reduce LDL(−) concentration and increase plasma adiponectin concentration after 24 h of rest in SG group, indicating its potential protective action.
Keywords: Low-density lipoprotein; Adiponectin; Leptin; Exercise; Polyphenols; Army cadets

The efficacy of early iron supplementation on postpartum depression, a randomized double-blind placebo-controlled trial by Mahdi Sheikh; Sedigheh Hantoushzadeh; Mamak Shariat; Zahra Farahani; Ozra Ebrahiminasab (901-908).
Evaluating early iron supplementation in non-anemic mothers with postpartum depression (PPD).This randomized, double-blind, placebo-controlled trial evaluated 70 mothers with PPD. One week after delivery, the mothers were randomly allocated in the iron-treated (50 mg elemental iron/daily) and placebo-treated groups. After 6 weeks, the improvement of PPD symptoms was compared between the groups.Ferritin significantly increased in the iron-treated group (p < 0.001), but not in the placebo group (p = 0.09). After intervention, ferritin was higher in the iron-treated group (medians: 78.2 vs. 37 mg/dl, p = 0.01). The rate of iron deficiency significantly decreased in the iron-treated group (p = 0.009), but not in the placebo group (p = 0.4). After intervention, the rate of iron deficiency was higher in the placebo group (31.4 vs. 8.5 %, p = 0.01). The Edinburgh Postnatal Depression Scale (EPDS) score significantly decreased in the iron-treated group (p < 0.001), but not in the placebo group (p = 0.13). After intervention, the EPDS score was lower in the iron-treated group (medians 9 vs. 12, p = 0.01). The improvement rate for PPD was significantly higher in the iron-treated group (42.8 vs. 20 %, p = 0.03). After intervention, mothers with continued PPD had lower ferritin than the improved mothers (41.8 vs. 67 mg/dl, p = 0.03). Mothers with continued depression had higher rate of iron deficiency compared to the improved mothers (27.1 vs. 4.5 %, p = 0.02).Early iron supplementation in mothers with PPD significantly improves the iron stores and causes a significant improvement in PPD with a 42.8 % improvement rate during 6 weeks. Continued PPD might be related to the lower postpartum ferritin levels in untreated mothers.
Keywords: Anemia; Deficiency; Depressed; Ferritin; Postnatal