Associations of vitamin D status with dietary intakes and physical activity levels among adults from seven European countries: the Food4Me study.

PURPOSE: To report the vitamin D status in adults from seven European countries and to identify behavioural correlates. METHODS: In total, 1075 eligible adult men and women from Ireland, Netherlands, Spain, Greece, UK, Poland and Germany, were included in the study. RESULTS: Vitamin D deficiency and insufficiency, defined as 25-hydroxy vitamin D3 (25-OHD3) concentration of <30 and 30-49.9 nmol/L, respectively, were observed in 3.3 and 30.6% of the participants. The highest prevalence of vitamin D deficiency was found in the UK and the lowest in the Netherlands (8.2 vs. 1.1%, P < 0.05). In addition, the prevalence of vitamin D insufficiency was higher in females compared with males (36.6 vs. 22.6%, P < 0.001), in winter compared with summer months (39.3 vs. 25.0%, P < 0.05) and in younger compared with older participants (36.0 vs. 24.4%, P < 0.05). Positive dose-response associations were also observed between 25-OHD3 concentrations and dietary vitamin D intake from foods and supplements, as well as with physical activity (PA) levels. Vitamin D intakes of ≥5 µg/day from foods and ≥5 µg/day from supplements, as well as engagement in ≥30 min/day of moderate- and vigorous-intensity PA were associated with higher odds (P < 0.05) for maintaining sufficient (≥50 nmol/L) 25-OHD3 concentrations. CONCLUSIONS: The prevalence of vitamin D deficiency varied considerably among European adults. Dietary intakes of ≥10 µg/day of vitamin D from foods and/or supplements and at least 30 min/day of moderate- and vigorous-intensity PA were the minimum thresholds associated with vitamin D sufficiency.

Application of dried blood spots to determine vitamin D status in a large nutritional study with unsupervised sampling: the Food4Me project.

An efficient and robust method to measure vitamin D (25-hydroxy vitamin D3 (25(OH)D3) and 25-hydroxy vitamin D2 in dried blood spots (DBS) has been developed and applied in the pan-European multi-centre, internet-based, personalised nutrition intervention study Food4Me. The method includes calibration with blood containing endogenous 25(OH)D3, spotted as DBS and corrected for haematocrit content. The methodology was validated following international standards. The performance characteristics did not reach those of the current gold standard liquid chromatography-MS/MS in plasma for all parameters, but were found to be very suitable for status-level determination under field conditions. DBS sample quality was very high, and 3778 measurements of 25(OH)D3 were obtained from 1465 participants. The study centre and the season within the study centre were very good predictors of 25(OH)D3 levels (P<0·001 for each case). Seasonal effects were modelled by fitting a sine function with a minimum 25(OH)D3 level on 20 January and a maximum on 21 July. The seasonal amplitude varied from centre to centre. The largest difference between winter and summer levels was found in Germany and the smallest in Poland. The model was cross-validated to determine the consistency of the predictions and the performance of the DBS method. The Pearson's correlation between the measured values and the predicted values was r 0·65, and the sd of their differences was 21·2 nmol/l. This includes the analytical variation and the biological variation within subjects. Overall, DBS obtained by unsupervised sampling of the participants at home was a viable methodology for obtaining vitamin D status information in a large nutritional study.

Hydroxytyrosol is the major anti-inflammatory compound in aqueous olive extracts and impairs cytokine and chemokine production in macrophages.

Substances in olive products contribute to improved health as suggested by epidemiological data. In this study we assessed the effects of hydroxytyrosol (HT) on inflammatory mediators, cytokines and chemokines, and identified anti-inflammatory constituents of aqueous olive extracts, I.E., olive vegetation water (OVW). Murine macrophages (RAW264.7 cells) were stimulated with lipopolysaccharide (LPS) in the absence or presence of substances; inflammatory mediators [nitric oxide (NO), prostaglandin E2 (PGE2), cytokines, interleukins, chemokines] were determined by the Griess reaction, EIA, or multiplex ELISA (Luminex technology). Expression of inflammatory genes was determined by RT-PCR. Aqueous olive extracts were fractionated by preparative HPLC and the fractions investigated for their effects on NO and PGE2 production. Results were further analyzed by principal component analysis. HT inhibited production of NO and PGE2 with an IC50 of 11.4 and 19.5 µM, respectively, reflecting strong anti-inflammatory activity. HT and OVW diminished secretion of cytokines (IL-1 α, IL-1 ß, IL-6, IL-12, TNF- α), and chemokines (CXCL10/IP-10, CCL2/MCP-1). HT and OVW concentration-dependently reduced the expression of genes of inducible nitric oxide synthase (iNOS), IL-1 α, CXCL10/IP-10, MIP-1 ß, matrix metalloproteinase-9, and prostaglandin E2 synthase (PGES). The effects of HT were partly mediated VIA the NF- κB pathway, as shown by RT-PCR analysis. HT was identified as the main bioactive compound of OVW. The data provide a molecular basis for elucidating the effects of HT on inflammatory processes. The effects of HT on NO and chemokine production point to their impact on chronic inflammatory processes in endothelium or arthritis.

Rose hip and its constituent galactolipids confer cartilage protection by modulating cytokine, and chemokine expression.

BACKGROUND: Clinical studies have shown that rose hip powder (RHP) alleviates osteoarthritis (OA). This might be due to anti-inflammatory and cartilage-protective properties of the complete RHP or specific constituents of RHP. Cellular systems (macrophages, peripheral blood leukocytes and chondrocytes), which respond to inflammatory and OA-inducing stimuli, are used as in vitro surrogates to evaluate the possible pain-relief and disease-modifying effects of RHP. METHODS: (1) Inflammatory processes were induced in RAW264.7 cells or human peripheral blood leukocytes (PBL) with LPS. Inflammatory mediators (nitric oxide (NO), prostaglandin E(2) (PGE(2)) and cytokines/chemokines) were determined by the Griess reaction, EIA and multiplex ELISA, respectively. Gene expression was quantified by RT-PCR. RHP or its constituent galactolipid, GLGPG (galactolipid (2S)-1, 2-di-O-[(9Z, 12Z, 15Z)-octadeca-9, 12, 15-trienoyl]-3-O-ß-D-galactopyranosyl glycerol), were added at various concentrations and the effects on biochemical and molecular parameters were evaluated. (2) SW1353 chondrosarcoma cells and primary human knee articular chondrocytes (NHAC-kn) were treated with interleukin (IL)-1ß to induce in vitro processes similar to those occurring during in vivo degradation of cartilage. Biomarkers related to OA (NO, PGE(2), cytokines, chemokines, metalloproteinases) were measured by multiplex ELISA and gene expression analysis in chondrocytes. We investigated the modulation of these events by RHP and GLGPG. RESULTS: In macrophages and PBL, RHP and GLGPG inhibited NO and PGE(2) production and reduced the secretion of cytokines (TNF-α, IFN-γ, IL-1ß, IL-6, IL-12) and chemokines (CCL5/RANTES, CXCL10/IP-10). In SW1353 cells and primary chondrocytes, RHP and GLGPG diminished catabolic gene expression and inflammatory protein secretion as shown by lower mRNA levels of matrix metalloproteinases (MMP-1, MMP-3, MMP-13), aggrecanase (ADAMTS-4), macrophage inflammatory protein (MIP-2, MIP-3α), CCL5/RANTES, CXCL10/IP-10, IL-8, IL-1α and IL-6. The effects of GLGPG were weaker than those of RHP, which presumably contains other chondro-protective substances besides GLGPG. CONCLUSIONS: RHP and GLGPG attenuate inflammatory responses in different cellular systems (macrophages, PBLs and chondrocytes). The effects on cytokine production and MMP expression indicate that RHP and its constituent GLGPG down-regulate catabolic processes associated with osteoarthritis (OA) or rheumatoid arthritis (RA). These data provide a molecular and biochemical basis for cartilage protection provided by RHP.

Effects of 6-month daily supplementation with oral beta-carotene in combination or not with benzo[a]pyrene on cell-cycle markers in the lung of ferrets.

Epidemiological studies have demonstrated that people who eat more fruits and vegetables (rich in carotenoids) and people who have higher serum beta-carotene (BC) levels have a lower risk of cancer, particularly lung cancer. However, the two main human intervention studies of BC supplementation (the ATBC and the CARET trials) revealed an increased risk of lung cancer among smokers and asbestos workers. Previous studies carried out in the ferret have reported that BC effects are related to dose. Here, we treated ferrets with two concentrations of oral BC (0.8 and 3.2 mg/kg body weight per day) for 6 months, using BC in a formulation also containing dl-alpha-tocopherol and ascorbyl palmitate. The effect of the smoke-derived carcinogenic agent benzo[a]pyrene (BP), with or without low-dose BC, was also analysed. We determined the protein levels and mRNA expression levels of activator protein 1 (c-Jun and c-Fos), c-Myc, cyclin D1, proliferating cellular nuclear antigen and retinoic acid receptor beta. We did not find higher levels of cell proliferation markers in the lung of ferrets treated with BC or signals of squamous metaplasia lesions either. On the other hand, although no evident signals of pulmonary carcinogenesis were observed in animals exposed to BP, BC supplementation in these animals may prevent against excess cell proliferation, since this reestablishes Jun protein and cyclin D1 mRNA levels in the lung of BP-exposed animals. In summary, these results show that the combination of BC with alpha-tocopherol and ascorbyl palmitate does not induce pro-oxidant effects in the lung of ferrets.

Metabolism of lutein and zeaxanthin in rhesus monkeys: identification of (3R,6'R)- and (3R,6'S)-3'-dehydro-lutein as common metabolites and comparison to humans.

Lutein and zeaxanthin are xanthophylls that can be found highly concentrated in the macula of the retina. They are thought to protect the macula through their role as blue-light filters and because of their antioxidant and singlet oxygen quenching properties. Examination of metabolites unique to lutein and zeaxanthin such as 3'-dehydro-lutein, and of their stereochemistry may provide insight to the mechanism by which they are formed and by which they exert protection. To evaluate the formation of such metabolites, eleven monkeys were raised on a xanthophyll-free diet, and supplemented with pure lutein or pure zeaxanthin (2.2 mg/kg body weight/d). The period of supplementation ranged between 12 and 92 weeks. At study start and throughout the study, serum samples were taken and analyzed for xanthophylls using different HPLC systems. Xanthophyll metabolites were identified using UV/VIS and HR-MS detection. Lutein and zeaxanthin metabolites were found in detectable amounts with 3'-dehydro-lutein being a common metabolite of both. Using chiral-phase HPLC, two diastereomers, (3R,6'R)-3'-dehydro-lutein and (3R,6'S)-3'-dehydro-lutein, were identified and shown to be present in nearly equimolar amounts. A pathway for their formation from either lutein or zeaxanthin is proposed. These findings were comparable to results obtained with human plasma.

The impact of micronutrient status on health: correlation network analysis to understand the role of micronutrients in metabolic-inflammatory processes regulating homeostasis and phenotypic flexibility.

BACKGROUND: Vitamins and carotenoids are key micronutrients facilitating the maintenance of health, as evidenced by the increased risk of disease with low intake. Optimal phenotypic flexibility, i.e., the ability to respond to a physiological challenge, is an essential indicator of health status. Therefore, health can be measured by applying a challenge test and monitoring the response of relevant phenotypic processes. In this study, we assessed the correlation of three fat-soluble vitamins, (i.e., vitamin A or retinol, vitamin D3, two homologues of vitamin E) and four carotenoids (i.e., α-carotene, ß-carotene, ß-cryptoxanthin, and lycopene), with characteristics of metabolic and inflammatory parameters at baseline and in response to a nutritional challenge test (NCT) in a group of 36 overweight and obese male subjects, using proteomics and metabolomics platforms. The phenotypic flexibility concept implies that health can be measured by the ability to adapt to a NCT, which may offer a more sensitive way to assess changes in health status of healthy subjects. RESULTS: Correlation analyses of results after overnight fasting revealed a rather evenly distributed network in a number of relatively strong correlations per micronutrient, with minor overlap between correlation profiles of each compound. Correlation analyses of challenge response profiles for metabolite and protein parameters with micronutrient status revealed a network that is more skewed towards α-carotene and γ-tocopherol suggesting a more prominent role for these micronutrients in the maintenance of phenotypic flexibility. Comparison of the networks revealed that there is merely overlap of two parameters (inositol and oleic acid (C18:1)) affirming that there is a specific biomarker response profile upon NCT. CONCLUSIONS: Our study shows that applying the challenge test concept is able to reveal previously unidentified correlations between specific micronutrients and health-related processes, with potential relevance for maintenance of health that were not observed by correlating homeostatic measurements. This approach will contribute to insights on the influence of micronutrients on health and help to create efficient micronutrient intervention programs.

Weekday sunlight exposure, but not vitamin D intake, influences the association between vitamin D receptor genotype and circulating concentration 25-hydroxyvitamin D in a pan-European population: the Food4Me study.

SCOPE: Little is known about diet- and environment-gene interactions on 25-hydroxyvitamin D (25(OH)D concentration. This cross-sectional study aimed to investigate (i) predictors of 25(OH)D concentration and relationships with vitamin D genotypes and (ii) whether dietary vitamin D intake and sunlight exposure modified these relationships. METHODS AND RESULTS: Participants from the Food4Me study (n = 1312; age 18-79) were genotyped for vitamin D receptor (VDR) and vitamin D binding protein at baseline and a genetic risk score was calculated. Dried blood spot samples were assayed for 25(OH)D concentration and dietary and lifestyle information collected. Circulating 25(OH)D concentration was lower with increasing genetic risk score, lower in females than males, higher in supplement users than non-users and higher in summer than winter. Carriage of the minor VDR allele was associated with lower 25(OH)D concentration in participants with the least sunlight exposure. Vitamin D genotype did not influence the relationship between vitamin D intake and 25(OH)D concentration. CONCLUSION: Age, sex, dietary vitamin D intake, country, sunlight exposure, season, and vitamin D genetic risk score were associated with circulating 25(OH)D concentration in a pan-European population. The relationship between VDR genotype and 25(OH)D concentration may be influenced by weekday sunlight exposure but not dietary vitamin D intake.

Design and baseline characteristics of the Food4Me study: a web-based randomised controlled trial of personalised nutrition in seven European countries.

Improving lifestyle behaviours has considerable potential for reducing the global burden of non-communicable diseases, promoting better health across the life-course and increasing well-being. However, realising this potential will require the development, testing and implementation of much more effective behaviour change interventions than are used conventionally. Therefore, the aim of this study was to conduct a multi-centre, web-based, proof-of-principle study of personalised nutrition (PN) to determine whether providing more personalised dietary advice leads to greater improvements in eating patterns and health outcomes compared to conventional population-based advice. A total of 5,562 volunteers were screened across seven European countries; the first 1,607 participants who fulfilled the inclusion criteria were recruited into the trial. Participants were randomly assigned to one of the following intervention groups for a 6-month period: Level 0-control group-receiving conventional, non-PN advice; Level 1-receiving PN advice based on dietary intake data alone; Level 2-receiving PN advice based on dietary intake and phenotypic data; and Level 3-receiving PN advice based on dietary intake, phenotypic and genotypic data. A total of 1,607 participants had a mean age of 39.8 years (ranging from 18 to 79 years). Of these participants, 60.9 % were women and 96.7 % were from white-European background. The mean BMI for all randomised participants was 25.5 kg m(-2), and 44.8 % of the participants had a BMI ≥ 25.0 kg m(-2). Food4Me is the first large multi-centre RCT of web-based PN. The main outcomes from the Food4Me study will be submitted for publication during 2015.

CMO1 deficiency abolishes vitamin A production from beta-carotene and alters lipid metabolism in mice.

Carotenoids are currently investigated regarding their potential to lower the risk of chronic disease and to combat vitamin A deficiency in humans. These plant-derived compounds must be cleaved and metabolically converted by intrinsic carotenoid oxygenases to support the panoply of vitamin A-dependent physiological processes. Two different carotenoid-cleaving enzymes were identified in mammals, the classical carotenoid-15,15'-oxygenase (CMO1) and a putative carotenoid-9',10'-oxygenase (CMO2). To analyze the role of CMO1 in mammalian physiology, here we disrupted the corresponding gene by targeted homologous recombination in mice. On a diet providing beta-carotene as major vitamin A precursor, vitamin A levels fell dramatically in several tissues examined. Instead, this mouse mutant accumulated the provitamin in large quantities (e.g. as seen by an orange coloring of adipose tissues). Besides impairments in beta-carotene metabolism, CMO1 deficiency more generally interfered with lipid homeostasis. Even on a vitamin A-sufficient chow, CMO1(-/-) mice developed a fatty liver and displayed altered serum lipid levels with elevated serum unesterified fatty acids. Additionally, this mouse mutant was more susceptible to high fat diet-induced impairments in fatty acid metabolism. Quantitative reverse transcription-PCR analysis revealed that the expression of peroxisome proliferator-activated receptor gamma-regulated marker genes related to adipogenesis was elevated in visceral adipose tissues. Thus, our study identifies CMO1 as the key enzyme for vitamin A production and provides evidence for a role of carotenoids as more general regulators of lipid metabolism.

Beta-carotene and apocarotenals promote retinoid signaling in BEAS-2B human bronchioepithelial cells.

High dose beta-carotene supplementation of smokers was associated with increased lung cancer risk in two intervention trials. It was proposed that generation of apocarotenals in smoke-exposed lungs impaired retinoic acid (RA) signaling, leading to squamous metaplasia and cell proliferation. To test this, we compared RA target gene regulation by retinoids, apocarotenals or beta-carotene by transcriptomics in BEAS-2B cells cultured to promote squamous differentiation. Retinoids, beta-carotene as well as apocarotenals induced known RA target genes. Retinoids upregulated involucrin, indicating that retinoids did not rescue BEAS-2B cells from squamous differentiation. Muc5AC, a marker for mucous differentiation, was transiently induced. beta-Carotene and apocarotenals less strongly induced involucrin and did not induce muc5AC. In summary, apocarotenals or beta-carotene upregulated RA target genes suggesting promotion, not inhibition, of RA signaling in BEAS-2B cells. Furthermore, apocarotenals and beta-carotene regulated gene expression independently of RA signaling. Squamous differentiation is not unequivocally linked to RA deficiency in BEAS-2B cells.