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Many aging men experience reduced energy and libido related to non-optimal testosterone levels. We conducted a randomized double-blind trial with TrigozimR fenugreek extract to assess impact on plasma and saliva testosterone, and some subjective effects. 95 men (40-80y) completed a 12-week intervention, taking 3 tablets daily with 0 mg (placebo; n = 22), 600 mg (n = 21), 1200 mg (n = 25) and1800 mg (n = 27) fenugreek extract and essential nutrients. Samples were collected at weeks 0, 2, 6, and 12. Participants answered a pre- and post-intervention questionnaire on lifestyle and libido. We measured total testosterone (HPLC-MS/MS) and sex hormone binding globulin (ELISA), calculated free testosterone index (FTI), and measured saliva testosterone. Plasma total testosterone and FTI increased after any dose of TrigozimR vs. baseline (13.0%, p = 1.0x10-4 and 16.3%, p = 6.2x10-6), but not vs. placebo (9.0%, p = 0.122 and 11.3% p = 0.059). 1800 mg TrigozimR resulted in 12.2% increased FTI (p = 0.025). Saliva testosterone concentration increased after any dose of TrigozimR vs. baseline (31.1%, p = 2.3x10-4) and vs. placebo (37.2%, p = 0.042). 1800 mg TrigozimR for 12 weeks resulted in 19.6% (p = 0.006) increased saliva testosterone. Compliance was confirmed by enhanced plasma concentration of 25-hydroxy vitamin D3. We observed no subjective effects or side-effects of TrigozimR. TrigozimR increased saliva and plasma testosterone concentration during intervention but only for saliva vs. placebo. Saliva may be preferred for measuring free testosterone due to no protein-bound testosterone., Competing Interests: TEG is CEO and stockowner in Vitas Ltd. CAD is stockowner, consultant, and board member in the contract laboratory Vitas Ltd where the intervention was performed and where most of the laboratory analyses were carried out. CAD is CEO and stockowner in the consulting company DBG Ltd responsible for design and execution. We received funding from the commercial source Vitas Ltd. This does not alter our adherence to PLOS ONE policies on sharing data and materials. SL was paid for performing the statistical analyses., (Copyright: © 2024 Lee-Ødegård et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The capacity of an individual to respond to changes in food intake so that postprandial metabolic perturbations are resolved, and metabolism returns to its pre-prandial state, is called phenotypic flexibility. This ability may be a more important indicator of current health status than metabolic markers in a fasting state., Aim: In this parallel randomized controlled trial study, an energy-restricted healthy diet and 2 dietary challenges were used to assess the effect of weight loss on phenotypic flexibility., Methods: Seventy-two volunteers with overweight and obesity underwent a 12-wk dietary intervention. The participants were randomized to a weight loss group (WLG) with 20% less energy intake or a weight-maintenance group (WMG). At weeks 1 and 12, participants were assessed for body composition by MRI. Concurrently, markers of metabolism and insulin sensitivity were obtained from the analysis of plasma metabolome during 2 different dietary challenges-an oral glucose tolerance test (OGTT) and a mixed-meal tolerance test., Results: Intended weight loss was achieved in the WLG (-5.6 kg, P < 0.0001) and induced a significant reduction in total and regional adipose tissue as well as ectopic fat in the liver. Amino acid-based markers of insulin action and resistance such as leucine and glutamate were reduced in the postprandial phase of the OGTT in the WLG by 11.5% and 28%, respectively, after body weight reduction. Weight loss correlated with the magnitude of changes in metabolic responses to dietary challenges. Large interindividual variation in metabolic responses to weight loss was observed., Conclusion: Application of dietary challenges increased sensitivity to detect metabolic response to weight loss intervention. Large interindividual variation was observed across a wide range of measurements allowing the identification of distinct responses to the weight loss intervention and mechanistic insight into the metabolic response to weight loss., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Self-reported adherence to the Mediterranean diet has been modestly inversely associated with incidence of type 2 diabetes (T2D) in cohort studies. There is uncertainty about the validity and magnitude of this association due to subjective reporting of diet. The association has not been evaluated using an objectively measured biomarker of the Mediterranean diet., Methods and Findings: We derived a biomarker score based on 5 circulating carotenoids and 24 fatty acids that discriminated between the Mediterranean or habitual diet arms of a parallel design, 6-month partial-feeding randomised controlled trial (RCT) conducted between 2013 and 2014, the MedLey trial (128 participants out of 166 randomised). We applied this biomarker score in an observational study, the European Prospective Investigation into Cancer and Nutrition (EPIC)-InterAct case-cohort study, to assess the association of the score with T2D incidence over an average of 9.7 years of follow-up since the baseline (1991 to 1998). We included 22,202 participants, of whom 9,453 were T2D cases, with relevant biomarkers from an original case-cohort of 27,779 participants sampled from a cohort of 340,234 people. As a secondary measure of the Mediterranean diet, we used a score estimated from dietary-self report. Within the trial, the biomarker score discriminated well between the 2 arms; the cross-validated C-statistic was 0.88 (95% confidence interval (CI) 0.82 to 0.94). The score was inversely associated with incident T2D in EPIC-InterAct: the hazard ratio (HR) per standard deviation of the score was 0.71 (95% CI: 0.65 to 0.77) following adjustment for sociodemographic, lifestyle and medical factors, and adiposity. In comparison, the HR per standard deviation of the self-reported Mediterranean diet was 0.90 (95% CI: 0.86 to 0.95). Assuming the score was causally associated with T2D, higher adherence to the Mediterranean diet in Western European adults by 10 percentiles of the score was estimated to reduce the incidence of T2D by 11% (95% CI: 7% to 14%). The study limitations included potential measurement error in nutritional biomarkers, unclear specificity of the biomarker score to the Mediterranean diet, and possible residual confounding., Conclusions: These findings suggest that objectively assessed adherence to the Mediterranean diet is associated with lower risk of T2D and that even modestly higher adherence may have the potential to reduce the population burden of T2D meaningfully., Trial Registration: Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12613000602729 https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=363860., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Authors declare support from the UK Medical Research Council, British Heart Foundation, Wellcome Trust, European Research Council, Swedish Research Council and National Institute for Health Research Cambridge Biomedical Research Centre for the submitted work. FI is a member of PLOS Medicine’s editorial board. PWF reports support for the submitted work from Novo Nordisk Foundation and consulting for Zoe Limited. JD reports grants, personal fees and non-financial support from Merck Sharp & Dohme (MSD), grants, personal fees and non-financial support from Novartis, grants from Pfizer and grants from AstraZeneca outside the submitted work. JD sits on the International Cardiovascular and Metabolic Advisory Board for Novartis (since 2010); the Steering Committee of UK Biobank (since 2011); the MRC International Advisory Group (ING) member, London (since 2013); the MRC High Throughput Science ‘Omics Panel Member, London (since 2013); the Scientific Advisory Committee for Sanofi (since 2013); the International Cardiovascular and Metabolism Research and Development Portfolio Committee for Novartis; and the Astra Zeneca Genomics Advisory Board (2018). ASB reports institutional grants outside the submitted work from AstraZeneca, Bayer, Biogen, BioMarin, Bioverativ, Novartis and Sanofi. Authors otherwise report no financial relationships with any organisations that might have an interest in the submitted work in the previous three years and no other relationships or activities that could appear to have influenced the submitted work.”, (Copyright: © 2023 Sobiecki et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Insulin secretion following ingestion of a carbohydrate load affects a multitude of metabolic pathways that simultaneously change direction and quantity of interorgan fluxes of sugars, lipids and amino acids. In the present study, we aimed at identifying markers associated with differential responses to an OGTT a population of healthy adults. By use of three metabolite profiling platforms, we assessed these postprandial responses of a total of 202 metabolites in plasma of 72 healthy volunteers undergoing comprehensive phenotyping and of which half enrolled into a weight-loss program over a three-month period. A standard oral glucose tolerance test (OGTT) served as dietary challenge test to identify changes in postprandial metabolite profiles. Despite classified as healthy according to WHO criteria, two discrete clusters (A and B) were identified based on the postprandial glucose profiles with a balanced distribution of volunteers based on gender and other measures. Cluster A individuals displayed 26% higher postprandial glucose levels, delayed glucose clearance and increased fasting plasma concentrations of more than 20 known biomarkers of insulin resistance and diabetes previously identified in large cohort studies. The volunteers identified by canonical postprandial responses that form cluster A may be called pre-pre-diabetics and defined as "at risk" for development of insulin resistance. Moreover, postprandial changes in selected fatty acids and complex lipids, bile acids, amino acids, acylcarnitines and sugars like mannose revealed marked differences in the responses seen in cluster A and cluster B individuals that sustained over the entire challenge test period of 240 min. Almost all metabolites, including glucose and insulin, returned to baseline values at the end of the test (at 240 min), except a variety of amino acids and here those that have been linked to diabetes development. Analysis of the corresponding metabolite profile in a fasting blood sample may therefore allow for early identification of these subjects at risk for insulin resistance without the need to undergo an OGTT., Competing Interests: TG is the CEO and stockowner in Vitas Ltd. CD is a founder, stockowner, board member and consultant in Vitas Ltd. DS was an employee of biocrates life sciences AG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fiamoncini, Donado-Pestana, Duarte, Rundle, Thomas, Kiselova-Kaneva, Gundersen, Bunzel, Trezzi, Kulling, Hiller, Sonntag, Ivanova, Brennan, Wopereis, van Ommen, Frost, Bell, Drevon and Daniel.)

The apolipoprotein E gene ε4 allele (APOE ε4) and higher circulating level of C-reactive protein (CRP) have been extensively investigated as risk factors for Alzheimer's disease (AD). Paradoxically, APOE ε4 has been associated with lower levels of blood CRP in middle-aged and older populations. However, few studies have investigated this intriguing relation and its impact on neurological markers for AD in younger ages, nor across the whole lifespan. Here, we examine associations of blood CRP levels, APOE ε4, and biomarkers for AD in a cognitively healthy lifespan cohort (N up to 749; 20-81 years of age) and replicate the findings in UK Biobank (N = 304 322; 37-72 years of age), the developmental ABCD study (N = 10 283; 9-11 years of age), and a middle-aged sample (N = 339; 40-65 years of age). Hippocampal volume, brain amyloid-β (Aβ) plaque levels, cerebrospinal fluid (CSF) levels of Aβ and tau species, and neurofilament protein light protein (NFL) were used as AD biomarkers in subsamples. In addition, we examined the genetic contribution to the variation of CRP levels over different CRP ranges using polygenic scores for CRP (PGS-CRP). Our results show APOE ε4 consistently associates with low blood CRP levels across all age groups (p < 0.05). Strikingly, both ε4 and PGS-CRP associated mainly with blood CRP levels within the low range (<5mg/L). We then show both APOE ε4 and high CRP levels associate with smaller hippocampus volumes across the lifespan (p < 0.025). APOE ε4 was associated with high Aβ plaque levels in the brain (FDR-corrected p = 8.69x10 -4 ), low levels of CSF Aβ42 (FDR-corrected p = 6.9x10 -2 ), and lower ratios of Aβ42 to Aβ40 (FDR-corrected p = 5.08x10 -5 ). Blood CRP levels were weakly correlated with higher ratio of CSF Aβ42 to Aβ40 (p = 0.03, FDR-corrected p = 0.4). APOE ε4 did not correlate with blood concentrations of another 9 inflammatory cytokines, and none of these cytokines correlated with AD biomarkers. CONCLUSION: The inverse correlation between APOEε 4 and blood CRP levels existed before any pathological AD biomarker was observed, and only in the low CRP level range. Thus, we suggest to investigate whether APOEε 4 can confer risk by being associated with a lower inflammatory response to daily exposures, possibly leading to greater accumulation of low-grade inflammatory stress throughout life. A lifespan perspective is needed to understand this relationship concerning risk of developing AD., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The effect of personalised nutrition advice on discretionary foods intake is unknown. To date, two national classifications for discretionary foods have been derived. This study examined changes in intake of discretionary foods and beverages following a personalised nutrition intervention using these two classifications., Methods: Participants were recruited into a 6-month RCT across seven European countries (Food4Me) and were randomised to receive generalised dietary advice (control) or one of three levels of personalised nutrition advice (based on diet [L1], phenotype [L2] and genotype [L3]). Dietary intake was derived from an FFQ. An analysis of covariance was used to determine intervention effects at month 6 between personalised nutrition (overall and by levels) and control on i) percentage energy from discretionary items and ii) percentage contribution of total fat, SFA, total sugars and salt to discretionary intake, defined by Food Standards Scotland (FSS) and Australian Dietary Guidelines (ADG) classifications., Results: Of the 1607 adults at baseline, n = 1270 (57% female) completed the intervention. Percentage sugars from FSS discretionary items was lower in personalised nutrition vs control (19.0 ± 0.37 vs 21.1 ± 0.65; P = 0.005). Percentage energy (31.2 ± 0.59 vs 32.7 ± 0.59; P = 0.031), percentage total fat (31.5 ± 0.37 vs 33.3 ± 0.65; P = 0.021), SFA (36.0 ± 0.43 vs 37.8 ± 0.75; P = 0.034) and sugars (31.7 ± 0.44 vs 34.7 ± 0.78; P < 0.001) from ADG discretionary items were lower in personalised nutrition vs control. There were greater reductions in ADG percentage energy and percentage total fat, SFA and salt for those randomised to L3 vs L2., Conclusions: Compared with generalised dietary advice, personalised nutrition advice achieved greater reductions in discretionary foods intake when the classification included all foods high in fat, added sugars and salt. Future personalised nutrition approaches may be used to target intake of discretionary foods., Trial Registration: Clinicaltrials.gov NCT01530139 . Registered 9 February 2012.

Introduction: Carbohydrate intake and physical activity are related to glucose homeostasis, both being influenced by individual genetic makeup. However, the interactions between these 2 factors, as affected by genetics, on glycaemia have been scarcely reported., Objective: We focused on analysing the interplay between carbohydrate intake and physical activity levels on blood glucose, taking into account a genetic risk score (GRS), based on SNPs related to glucose/energy metabolism., Methods: A total of 1,271 individuals from the Food4Me cohort, who completed the nutritional intervention, were evaluated at baseline. We collected dietary information by using an online-validated food frequency questionnaire, a questionnaire on physical activity, blood biochemistry by analysis of dried blood spots, and by analysis of selected SNPs. Fifteen out of 31 SNPs, with recognized participation in carbohydrate/energy metabolism, were included in the component analyses. The GRS included risk alleles involved in the control of glycaemia or energy-yielding processes., Results: Data concerning anthropometric, clinical, metabolic, dietary intake, physical activity, and genetics related to blood glucose levels showed expected trends in European individuals of comparable sex and age, being categorized by lifestyle, BMI, and energy/carbohydrate intakes, in this Food4Me population. Blood glucose was inversely associated with physical activity level (β = -0.041, p = 0.013) and positively correlated with the GRS values (β = 0.015, p = 0.047). Interestingly, an interaction affecting glycaemia, concerning physical activity level with carbohydrate intake, was found (β = -0.060, p = 0.033), which also significantly depended on the genetic background (GRS)., Conclusions: The relationships of carbohydrate intake and physical activity are important in understanding glucose homeostasis, where a role for the genetic background should be ascribed., (© 2021 The Author(s). Published by S. Karger AG, Basel.)

To elucidate the contributions of specific lipid species to metabolic traits, we integrated global hepatic lipid data with other omics measures and genetic data from a cohort of about 100 diverse inbred strains of mice fed a high-fat/high-sucrose diet for 8 weeks. Association mapping, correlation, structure analyses, and network modeling revealed pathways and genes underlying these interactions. In particular, our studies lead to the identification of Ifi203 and Map2k6 as regulators of hepatic phosphatidylcholine homeostasis and triacylglycerol accumulation, respectively. Our analyses highlight mechanisms for how genetic variation in hepatic lipidome can be linked to physiological and molecular phenotypes, such as microbiota composition., (© 2021 The Authors. Published under the terms of the CC BY 4.0 license.)

Objective: Higher plasma vitamin C levels are associated with lower type 2 diabetes risk, but whether this association is causal is uncertain. To investigate this, we studied the association of genetically predicted plasma vitamin C with type 2 diabetes., Research Design and Methods: We conducted genome-wide association studies of plasma vitamin C among 52,018 individuals of European ancestry to discover novel genetic variants. We performed Mendelian randomization analyses to estimate the association of genetically predicted differences in plasma vitamin C with type 2 diabetes in up to 80,983 case participants and 842,909 noncase participants. We compared this estimate with the observational association between plasma vitamin C and incident type 2 diabetes, including 8,133 case participants and 11,073 noncase participants., Results: We identified 11 genomic regions associated with plasma vitamin C ( P < 5 × 10 -8 ), with the strongest signal at SLC23A1 , and 10 novel genetic loci including SLC23A3 , CHPT1 , BCAS3 , SNRPF , RER1 , MAF , GSTA5 , RGS14 , AKT1 , and FADS1 . Plasma vitamin C was inversely associated with type 2 diabetes (hazard ratio per SD 0.88; 95% CI 0.82, 0.94), but there was no association between genetically predicted plasma vitamin C (excluding FADS1 variant due to its apparent pleiotropic effect) and type 2 diabetes (1.03; 95% CI 0.96, 1.10)., Conclusions: These findings indicate discordance between biochemically measured and genetically predicted plasma vitamin C levels in the association with type 2 diabetes among European populations. The null Mendelian randomization findings provide no strong evidence to suggest the use of vitamin C supplementation for type 2 diabetes prevention., (© 2020 by the American Diabetes Association.)

Background: Prior research suggested a differential association of 25-hydroxyvitamin D (25(OH)D) metabolites with type 2 diabetes (T2D), with total 25(OH)D and 25(OH)D3 inversely associated with T2D, but the epimeric form (C3-epi-25(OH)D3) positively associated with T2D. Whether or not these observational associations are causal remains uncertain. We aimed to examine the potential causality of these associations using Mendelian randomisation (MR) analysis., Methods and Findings: We performed a meta-analysis of genome-wide association studies for total 25(OH)D (N = 120,618), 25(OH)D3 (N = 40,562), and C3-epi-25(OH)D3 (N = 40,562) in participants of European descent (European Prospective Investigation into Cancer and Nutrition [EPIC]-InterAct study, EPIC-Norfolk study, EPIC-CVD study, Ely study, and the SUNLIGHT consortium). We identified genetic variants for MR analysis to investigate the causal association of the 25(OH)D metabolites with T2D (including 80,983 T2D cases and 842,909 non-cases). We also estimated the observational association of 25(OH)D metabolites with T2D by performing random effects meta-analysis of results from previous studies and results from the EPIC-InterAct study. We identified 10 genetic loci associated with total 25(OH)D, 7 loci associated with 25(OH)D3 and 3 loci associated with C3-epi-25(OH)D3. Based on the meta-analysis of observational studies, each 1-standard deviation (SD) higher level of 25(OH)D was associated with a 20% lower risk of T2D (relative risk [RR]: 0.80; 95% CI 0.77, 0.84; p < 0.001), but a genetically predicted 1-SD increase in 25(OH)D was not significantly associated with T2D (odds ratio [OR]: 0.96; 95% CI 0.89, 1.03; p = 0.23); this result was consistent across sensitivity analyses. In EPIC-InterAct, 25(OH)D3 (per 1-SD) was associated with a lower risk of T2D (RR: 0.81; 95% CI 0.77, 0.86; p < 0.001), while C3-epi-25(OH)D3 (above versus below lower limit of quantification) was positively associated with T2D (RR: 1.12; 95% CI 1.03, 1.22; p = 0.006), but neither 25(OH)D3 (OR: 0.97; 95% CI 0.93, 1.01; p = 0.14) nor C3-epi-25(OH)D3 (OR: 0.98; 95% CI 0.93, 1.04; p = 0.53) was causally associated with T2D risk in the MR analysis. Main limitations include the lack of a non-linear MR analysis and of the generalisability of the current findings from European populations to other populations of different ethnicities., Conclusions: Our study found discordant associations of biochemically measured and genetically predicted differences in blood 25(OH)D with T2D risk. The findings based on MR analysis in a large sample of European ancestry do not support a causal association of total 25(OH)D or 25(OH)D metabolites with T2D and argue against the use of vitamin D supplementation for the prevention of T2D., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: FI and CL are Academic Editors on PLOS Medicine’s editorial board. LAL is Employee and shareholder of Regeneron Pharmaceuticals Inc. ASB has received grants unrelated to this work from AstraZeneca, Biogen, Bioverativ, Merck, Novartis and Sanofi, and personal fees from Novartis. Where authors are identified as personnel of the International Agency for Research on Cancer / World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer / World Health Organization.

Little is known about who would benefit from Internet-based personalised nutrition (PN) interventions. This study aimed to evaluate the characteristics of participants who achieved greatest improvements (i.e. benefit) in diet, adiposity and biomarkers following an Internet-based PN intervention. Adults (n 1607) from seven European countries were recruited into a 6-month, randomised controlled trial (Food4Me) and randomised to receive conventional dietary advice (control) or PN advice. Information on dietary intake, adiposity, physical activity (PA), blood biomarkers and participant characteristics was collected at baseline and month 6. Benefit from the intervention was defined as ≥5 % change in the primary outcome (Healthy Eating Index) and secondary outcomes (waist circumference and BMI, PA, sedentary time and plasma concentrations of cholesterol, carotenoids and omega-3 index) at month 6. For our primary outcome, benefit from the intervention was greater in older participants, women and participants with lower HEI scores at baseline. Benefit was greater for individuals reporting greater self-efficacy for 'sticking to healthful foods' and who 'felt weird if [they] didn't eat healthily'. Participants benefited more if they reported wanting to improve their health and well-being. The characteristics of individuals benefiting did not differ by other demographic, health-related, anthropometric or genotypic characteristics. Findings were similar for secondary outcomes. These findings have implications for the design of more effective future PN intervention studies and for tailored nutritional advice in public health and clinical settings.

Aim: Elevated total cholesterol (TC) and glycated haemoglobin (HbA1c) are risk factors for cardiovascular disease; however, little is known about their determinants in infants. We aimed to describe TC and HbA1c concentrations in infants aged 8-14 months and explore the relation between infant TC, HbA1c, breastfeeding, infant diet, and maternal TC and HbA1c., Methods: In this cross-sectional pilot study, mothers of infants aged 6 and 12 months were invited to complete a food frequency questionnaire and to take home-based dried blood spot samples from themselves and their infants., Results: Among the 143 included infants, the mean (SD, range) concentration was 4.1 (0.8, 2.3-6.6) mmol/L for TC and 4.9 (0.4, 3.7-6.0)% for HbA1c. There was no significant difference between age groups and sexes. There was a positive relation between TC concentrations of all infants and mothers (B = 0.30 unadjusted, B = 0.32 adjusted, P < .001 for both) and a negative relation between infant TC and intake of unsaturated fatty acids in the oldest age group (B = -0.09, P = .03 unadjusted, B = -0.08, P = .06 adjusted). Infant HbA1c was not significantly related to diet or maternal HbA1c., Conclusion: TC and HbA1c concentrations varied widely among infants aged 8-14 months. Infant TC was associated with macronutrient intake and maternal TC., (©2019 The Authors. Acta Paediatrica Published by John Wiley & Sons Ltd on behalf of Foundation Acta Paediatrica.)

Background: Existing evidence for the prospective association of vitamin D status with type 2 diabetes (T2D) is focused almost exclusively on circulating total 25-hydroxyvitamin D [25(OH)D] without distinction between its subtypes: nonepimeric and epimeric 25(OH)D3 stereoisomers, and 25(OH)D2, the minor component of 25(OH)D. We aimed to investigate the prospective associations of circulating levels of the sum and each of these three metabolites with incident T2D., Methods: This analysis in the European Prospective Investigation into Cancer and Nutrition (EPIC)-InterAct case-cohort study for T2D included 9671 incident T2D cases and 13,562 subcohort members. Plasma vitamin D metabolites were quantified by liquid chromatography-mass spectrometry. We used a multivariable Prentice-weighted Cox regression to estimate hazard ratios (HRs) of T2D for each metabolite. Analyses were performed separately within country, and estimates were combined across countries using random-effects meta-analysis., Results: The mean concentrations (SD) of total 25(OH)D, nonepimeric 25(OH)D3, epimeric 25(OH)D3, and 25(OH)D2 were 41.1 (17.2), 40.7 (17.3), 2.13 (1.31), and 8.16 (6.52) nmol/L, respectively. Plasma total 25(OH)D and nonepimeric 25(OH)D3 were inversely associated with incident T2D [multivariable-adjusted HR per 1 SD = 0.81 (95% CI, 0.77, 0.86) for both variables], whereas epimeric 25(OH)D3 was positively associated [per 1 SD HR = 1.16 (1.09, 1.25)]. There was no statistically significant association with T2D for 25(OH)D2 [per 1 SD HR = 0.94 (0.76, 1.18)]., Conclusions: Plasma nonepimeric 25(OH)D3 was inversely associated with incident T2D, consistent with it being the major metabolite contributing to total 25(OH)D. The positive association of the epimeric form of 25(OH)D3 with incident T2D provides novel information to assess the biological relevance of vitamin D epimerization and vitamin D subtypes in diabetes etiology.

Background/objectives: Sufficient vitamin A levels are important for many functions-and both too little and too much may have detrimental health effects. The aim of the study was to describe the distribution of retinol levels in Norwegian adolescents, the relation between lifestyle factors and retinol levels, and the relation between retinol levels and bone mineral density (BMD)., Subjects/methods: Serum retinol was measured in 414 girls and 474 boys aged 15-19 years, participating in the Tromsø Study: Fit Futures. Questionnaires regarding health and lifestyle factors were filled in, and physical examinations, body composition, and bone mineral density measurements (DEXA) performed. Multiple regression analyses were used to discover associations between retinol and exposure variables., Results: Retinol levels ranged from 0.26 to 6.46 μmol/L with a median (2.5-97.5 percentile) of 2.35 (1.01-4.67) μmol/L. There was no gender difference. In the multivariate models, fat mass, albumin level, physical activity, and lunch habits were positively associated with retinol levels in boys. In girls, fat mass and height were negatively associated with retinol levels, and lean mass, vitamin D, calcium, total cholesterol, and the use of contraceptives were positively associated with retinol levels (p < 0.05). The models explained 18.3% and 14.6% of the variation (R 2 ) in girls and boys, respectively. Retinol levels were not independently associated with BMD., Conclusion: Retinol levels in Norwegian adolescents are higher than reported elsewhere, and are to a low degree explained by lifestyle and physical measurements. No independent association with BMD was found.

Elevated hepatic ceramide levels have been implicated in both insulin resistance (IR) and hepatic steatosis. To understand the factors contributing to hepatic ceramide levels in mice of both sexes, we have quantitated ceramides in a reference population of mice, the Hybrid Mouse Diversity Panel that has been previously characterized for a variety of metabolic syndrome traits. We observed significant positive correlations between Cer(d18:1/16:0) and IR/hepatic steatosis, consistent with previous findings, although the relationship broke down between sexes, as females were less insulin resistant, but had higher Cer(d18:1/16:0) levels than males. The sex difference was due in part to testosterone-mediated repression of ceramide synthase 6. One ceramide species, Cer(d18:1/20:0), was present at higher levels in males and was associated with IR only in males. Clear evidence of gene-by-sex and gene-by-diet interactions was observed, including sex-specific genome-wide association study results. Thus, our studies show clear differences in how hepatic ceramides are regulated between the sexes, which again suggests that the physiological roles of certain hepatic ceramides differ between the sexes., (Copyright © 2018 by the American Society for Biochemistry and Molecular Biology, Inc.)

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) composition in skeletal muscle have been linked to insulin sensitivity. We evaluated the relationships between skeletal muscle PC:PE, physical exercise and insulin sensitivity. We performed lipidomics and measured PC and PE in m. vastus lateralis biopsies obtained from 13 normoglycemic normal weight men and 13 dysglycemic overweight men at rest, immediately after 45 min of cycling at 70% maximum oxygen uptake, and 2 h post-exercise, before as well as after 12 weeks of combined endurance- and strength-exercise intervention. Insulin sensitivity was monitored by euglycemic-hyperinsulinemic clamp. RNA-sequencing was performed on biopsies, and mitochondria and lipid droplets were quantified on electron microscopic images. Exercise intervention for 12 w enhanced insulin sensitivity by 33%, skeletal muscle levels of PC by 21%, PE by 42%, and reduced PC:PE by 16%. One bicycle session reduced PC:PE by 5%. PC:PE correlated negatively with insulin sensitivity (β = -1.6, P < 0.001), percent area of mitochondria (ρ = -0.52, P = 0.035), and lipid droplet area (ρ = 0.55, P = 0.017) on EM pictures, and negatively with oxidative phosphorylation and mTOR based on RNA-sequencing. In conclusion, PC and PE contents of skeletal muscle respond to exercise, and PC:PE is inversely related to insulin sensitivity.

Vitamin D is a steroid hormone precursor that is associated with a range of human traits and diseases. Previous GWAS of serum 25-hydroxyvitamin D concentrations have identified four genome-wide significant loci (GC, NADSYN1/DHCR7, CYP2R1, CYP24A1). In this study, we expand the previous SUNLIGHT Consortium GWAS discovery sample size from 16,125 to 79,366 (all European descent). This larger GWAS yields two additional loci harboring genome-wide significant variants (P = 4.7×10 -9 at rs8018720 in SEC23A, and P = 1.9×10 -14 at rs10745742 in AMDHD1). The overall estimate of heritability of 25-hydroxyvitamin D serum concentrations attributable to GWAS common SNPs is 7.5%, with statistically significant loci explaining 38% of this total. Further investigation identifies signal enrichment in immune and hematopoietic tissues, and clustering with autoimmune diseases in cell-type-specific analysis. Larger studies are required to identify additional common SNPs, and to explore the role of rare or structural variants and gene-gene interactions in the heritability of circulating 25-hydroxyvitamin D levels.

Diet-quality scores (DQS), which are developed across the globe, are used to define adherence to specific eating patterns and have been associated with risk of coronary heart disease and type-II diabetes. We explored the association between five diet-quality scores (Healthy Eating Index, HEI; Alternate Healthy Eating Index, AHEI; MedDietScore, MDS; PREDIMED Mediterranean Diet Score, P-MDS; Dutch Healthy Diet-Index, DHDI) and markers of metabolic health (anthropometry, objective physical activity levels (PAL), and dried blood spot total cholesterol (TC), total carotenoids, and omega-3 index) in the Food4Me cohort, using regression analysis. Dietary intake was assessed using a validated Food Frequency Questionnaire. Participants ( n = 1480) were adults recruited from seven European Union (EU) countries. Overall, women had higher HEI and AHEI than men ( p < 0.05), and scores varied significantly between countries. For all DQS, higher scores were associated with lower body mass index, lower waist-to-height ratio and waist circumference, and higher total carotenoids and omega-3-index ( p trends < 0.05). Higher HEI, AHEI, DHDI, and P-MDS scores were associated with increased daily PAL, moderate and vigorous activity, and reduced sedentary behaviour ( p trend < 0.05). We observed no association between DQS and TC. To conclude, higher DQS, which reflect better dietary patterns, were associated with markers of better nutritional status and metabolic health., Competing Interests: T.E.G. is founder, stockowner and C.E.O. of Vitas; C.A.D. is founder, board member, stockowner, and consultant for Vitas Ltd. None of the other authors reports a conflict of interest.

Background: National guidelines emphasize healthy eating to promote wellbeing and prevention of non-communicable diseases. The perceived healthiness of food is determined by many factors affecting food intake. A positive perception of healthy eating has been shown to be associated with greater diet quality. Internet-based methodologies allow contact with large populations. Our present study aims to design and evaluate a short nutritional perception questionnaire, to be used as a screening tool for assessing nutritional status, and to predict an optimal level of personalisation in nutritional advice delivered via the Internet., Methods: Data from all participants who were screened and then enrolled into the Food4Me proof-of-principle study (n = 2369) were used to determine the optimal items for inclusion in a novel screening tool, the Nutritional Perception Screening Questionnaire-9 (NPSQ9). Exploratory and confirmatory factor analyses were performed on anthropometric and biochemical data and on dietary indices acquired from participants who had completed the Food4Me dietary intervention (n = 1153). Baseline and intervention data were analysed using linear regression and linear mixed regression, respectively., Results: A final model with 9 NPSQ items was validated against the dietary intervention data. NPSQ9 scores were inversely associated with BMI (β = -0.181, p < 0.001) and waist circumference (Β = -0.155, p < 0.001), and positively associated with total carotenoids (β = 0.198, p < 0.001), omega-3 fatty acid index (β = 0.155, p < 0.001), Healthy Eating Index (HEI) (β = 0.299, p < 0.001) and Mediterranean Diet Score (MDS) (β = 0. 279, p < 0.001). Findings from the longitudinal intervention study showed a greater reduction in BMI and improved dietary indices among participants with lower NPSQ9 scores., Conclusions: Healthy eating perceptions and dietary habits captured by the NPSQ9 score, based on nine questionnaire items, were associated with reduced body weight and improved diet quality. Likewise, participants with a lower score achieved greater health improvements than those with higher scores, in response to personalised advice, suggesting that NPSQ9 may be used for early evaluation of nutritional status and to tailor nutritional advice., Trial Registration: NCT01530139 .

FFQ, food diaries and 24 h recall methods represent the most commonly used dietary assessment tools in human studies on nutrition and health, but food intake biomarkers are assumed to provide a more objective reflection of intake. Unfortunately, very few of these biomarkers are sufficiently validated. This review provides an overview of food intake biomarker research and highlights present research efforts of the Joint Programming Initiative 'A Healthy Diet for a Healthy Life' (JPI-HDHL) Food Biomarkers Alliance (FoodBAll). In order to identify novel food intake biomarkers, the focus is on new food metabolomics techniques that allow the quantification of up to thousands of metabolites simultaneously, which may be applied in intervention and observational studies. As biomarkers are often influenced by various other factors than the food under investigation, FoodBAll developed a food intake biomarker quality and validity score aiming to assist the systematic evaluation of novel biomarkers. Moreover, to evaluate the applicability of nutritional biomarkers, studies are presently also focusing on associations between food intake biomarkers and diet-related disease risk. In order to be successful in these metabolomics studies, knowledge about available electronic metabolomics resources is necessary and further developments of these resources are essential. Ultimately, present efforts in this research area aim to advance quality control of traditional dietary assessment methods, advance compliance evaluation in nutritional intervention studies, and increase the significance of observational studies by investigating associations between nutrition and health.

Objective: Sufficient vitamin D status during infancy is important for child health and development. Several initiatives for improving vitamin D status among immigrant children have been implemented in Norway. The present study aimed to evaluate the vitamin D status and its determinants in children of immigrant background in Oslo., Design: Cross-sectional study., Setting: Child health clinics in Oslo., Subjects: Healthy children with immigrant background (n 102) aged 9-16 months were recruited at the routine one-year check-up from two child health clinics with high proportions of immigrant clients. Blood samples were collected using the dried blood spot technique and analysed for serum 25-hydroxyvitamin D (s-25(OH)D) concentration using LC-MS/MS., Results: Mean s-25(OH)D was 52·3 (sd 16·7) nmol/l, with only three children below 25 nmol/l and none below 12·5 nmol/l. There was no significant gender, ethnic or seasonal variation in s-25(OH)D. However, compared with breast-fed children, s-25(OH)D concentration was significantly higher among children who were about 1 year of age and not breast-fed. About 38 % of the children were anaemic, but there was no significant correlation between s-25(OH)D and Hb (Pearson correlation, r=0·1, P=0·33)., Conclusions: Few children in the study had vitamin D deficiency, but about 47 % of the children in the study population were under the recommended s-25(OH)D sufficiency level of ≥50 nmol/l.

Mediterranean Diet (MedDiet) adherence has been proven to produce numerous health benefits. In addition, nutrigenetic studies have explained some individual variations in the response to specific dietary patterns. The present research aimed to explore associations and potential interactions between MedDiet adherence and genetic background throughout the Food4Me web-based nutritional intervention. Dietary, anthropometrical and biochemical data from volunteers of the Food4Me study were collected at baseline and after 6 months. Several genetic variants related to metabolic risk features were also analysed. A Genetic Risk Score (GRS) was derived from risk alleles and a Mediterranean Diet Score (MDS), based on validated food intake data, was estimated. At baseline, there were no interactions between GRS and MDS categories for metabolic traits. Linear mixed model repeated measures analyses showed a significantly greater decrease in total cholesterol in participants with a low GRS after a 6-month period, compared to those with a high GRS. Meanwhile, a high baseline MDS was associated with greater decreases in Body Mass Index (BMI), waist circumference and glucose. There also was a significant interaction between GRS and the MedDiet after the follow-up period. Among subjects with a high GRS, those with a high MDS evidenced a highly significant reduction in total carotenoids, while among those with a low GRS, there was no difference associated with MDS levels. These results suggest that a higher MedDiet adherence induces beneficial effects on metabolic outcomes, which can be affected by the genetic background in some specific markers., Competing Interests: K.G. reports he was employed by Sciona Inc. (a provider of genetic testing services) from 2002 to 2008 and is founder/director of the personal genetics services company Eurogenetica Ltd. C.A.D. is cofounder, board member, stock-owner and consultant for Vitas Ltd. () performing the dried blood spot (DBS) analyses. T.E.G. is cofounder, stock-owner and CEO of Vitas. None of the other authors reported a conflict of interest related to the study.

Scope: Previous work highlighted the potential of odd-chain length saturated fatty acids as potential markers of dairy intake. The aim of this study was to assess the reproducibility of these biomarkers and their sensitivity to changes in dairy intake., Methods and Results: Fatty acid profiles and dietary intakes from food frequency questionnaires (FFQs) were measured three times over six months in the Food4Me Study. Reproducibility was explored through intra-class correlation coefficients (ICCs) and within-subject coefficients of variation (WCV). Sensitivity to changes in diet was examined using regression analysis. C15:0 blood levels showed high correlation over time (ICC: 0.62, 95% CI: 0.57, 0.68), however, the ICC for C17:0 was much lower (ICC: 0.32, 95% CI: 0.28, 0.46). The WCV for C15:0 was 16.6% and that for C17:0 was 14.6%. There were significant associations between changes in intakes of total dairy, high-fat dairy, cheese and butter and C15:0; and change in intakes of high-fat dairy and cream and C17:0., Conclusion: Results provide evidence of reproducibility of C15:0 levels over time and sensitivity to change in intake of high-fat dairy products with results comparable to the well-established biomarker of fish intake (EPA+DHA)., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Biomarkers of nutrient intake or nutrient status are important objective measures of foods/nutrients as one of the most important environmental factors people are exposed to. It is very difficult to obtain accurate data on individual food intake, and there is a large variation of nutrient composition of foods consumed in a population. Thus, it is difficult to obtain precise measures of exposure to different nutrients and thereby be able to understand the relationship between diet, health, and disease. This is the background for investing considerable resources in studying biomarkers of nutrients believed to be important in our foods. Modern technology with high sensitivity and specificity concerning many nutrient biomarkers has allowed an interesting development with analyses of very small amounts of blood or tissue material. In combination with non-professional collection of blood by finger-pricking and collection on filters or sticks, this may make collection of samples and analyses of biomarkers much more available for scientists as well as health professionals and even lay people in particular in relation to the marked trend of self-monitoring of body functions linked to mobile phone technology. Assuming standard operating procedures are used for collection, drying, transport, extraction, and analysis of samples, it turns out that many analytes of nutritional interest can be measured like metabolites, drugs, lipids, vitamins, minerals, and many types of peptides and proteins. The advantage of this alternative sampling technology is that non-professionals can collect, dry, and mail the samples; the samples can often be stored under room temperature in a dry atmosphere, requiring small amounts of blood. Another promising area is the potential relation between the microbiome and biomarkers that may be measured in feces as well as in blood.

Scope: The use of biomarkers in the objective assessment of dietary intake is a high priority in nutrition research. The aim of this study was to examine pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0) as biomarkers of dairy foods intake., Methods and Results: The data used in the present study were obtained as part of the Food4me Study. Estimates of C15:0 and C17:0 from dried blood spots and intakes of dairy from a Food Frequency Questionnaire were obtained from participants (n = 1180) across seven countries. Regression analyses were used to explore associations of biomarkers with dairy intake levels and receiver operating characteristic analyses were used to evaluate the fatty acids. Significant positive associations were found between C15:0 and total intakes of high-fat dairy products. C15:0 showed good ability to distinguish between low and high consumers of high-fat dairy products., Conclusion: C15:0 can be used as a biomarker of high-fat dairy intake and of specific high-fat dairy products. Both C15:0 and C17:0 performed poorly for total dairy intake highlighting the need for caution when using these in epidemiological studies., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: Vitamin D plays a major role in Ca and bone metabolism, and its extraskeletal functions are being appraised. Although inadequate vitamin D concentrations have been reported in populations worldwide, too little is known about vitamin D status and its determinants among children in developing countries. We aimed to determine vitamin D status and its determinants in Nepalese children of pre-school age., Design: A community-based, cross-sectional study., Setting: Rural Nepal at latitude 27.39° N., Subjects: Healthy children (n 280) aged 12-60 months, selected randomly from the records of a vitamin A supplementation programme. Blood samples were collected using the dried blood spot technique and analysed for serum 25-hydroxyvitamin D (s-25(OH)D) concentration using liquid chromatography-tandem mass spectrometry. Ca intake and background variables were assessed with a structured questionnaire., Results: Hypovitaminosis D, defined as s-25(OH)D concentration less than 50 nmol/l, was found in 91.1% of the children. S-25(OH)D concentration was not related to gender, socio-economic indicators, sun exposure or nutritional status. Currently breast-fed children had higher s-25(OH)D concentrations (36.4 (sd 13.2) nmol/l) than those who were not (28.6 (sd 9.8) nmol/l, P<0.001). Adjustment for sociodemographic factors did not alter the results., Conclusion: There is widespread vitamin D deficiency among pre-school children in a rural area of Nepal. In our sample, sociodemographic factors did not affect the vitamin D status of children, but prolonged breast-feeding was associated with higher s-25(OH)D concentrations. Further research is required to investigate the health consequences of poor vitamin D status for this population.

Background: In large epidemiological studies it is often challenging to obtain biological samples. Self-sampling by study participants using dried blood spots (DBS) technique has been suggested to overcome this challenge. DBS is a type of biosampling where blood samples are obtained by a finger-prick lancet, blotted and dried on filter paper. However, the feasibility and efficacy of collecting DBS samples from study participants in large-scale epidemiological studies is not known. The aim of the present study was to test the feasibility and response rate of collecting self-sampled DBS and saliva samples in a population-based study of women above 50 years of age., Methods: We determined response proportions, number of phone calls to the study center with questions about sampling, and quality of the DBS. We recruited women through a study conducted within the Norwegian Breast Cancer Screening Program. Invitations, instructions and materials were sent to 4,597 women. The data collection took place over a 3 month period in the spring of 2009., Results: Response proportions for the collection of DBS and saliva samples were 71.0% (3,263) and 70.9% (3,258), respectively. We received 312 phone calls (7% of the 4,597 women) with questions regarding sampling. Of the 3,263 individuals that returned DBS cards, 3,038 (93.1%) had been packaged and shipped according to instructions. A total of 3,032 DBS samples were sufficient for at least one biomarker analysis (i.e. 92.9% of DBS samples received by the laboratory). 2,418 (74.1%) of the DBS cards received by the laboratory were filled with blood according to the instructions (i.e. 10 completely filled spots with up to 7 punches per spot for up to 70 separate analyses). To assess the quality of the samples, we selected and measured two biomarkers (carotenoids and vitamin D). The biomarker levels were consistent with previous reports., Conclusion: Collecting self-sampled DBS and saliva samples through the postal services provides a low cost, effective and feasible alternative in epidemiological studies.

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.

There are no previous studies of the association between prediagnostic serum vitamin D concentration and glioma. Vitamin D has immunosuppressive properties; as does glioma. It was, therefore, our hypothesis that elevated vitamin D concentration would increase glioma risk. We conducted a nested case-control study using specimens from the Janus Serum Bank cohort in Norway. Blood donors who were subsequently diagnosed with glioma (n = 592), between 1974 and 2007, were matched to donors without glioma (n = 1112) on date and age at blood collection and sex. We measured 25-hydroxyvitamin D [25(OH)D], an indicator of vitamin D availability, using liquid chromatography coupled with mass spectrometry. Seasonally adjusted odds ratios (ORs) and 95% confidence intervals (95% CIs) were estimated for each control quintile of 25(OH)D using conditional logistic regression. Among men diagnosed with high grade glioma >56, we found a negative trend (P = .04). Men diagnosed ≤ 56 showed a borderline positive trend (P = .08). High levels (>66 nmol/L) of 25(OH)D in men >56 were inversely related to high grade glioma from ≥2 yr before diagnosis (OR = 0.59; 95% CI = 0.38, 0.91) to ≥15 yr before diagnosis (OR = 0.61; 95% CI = 0.38,0.96). Our findings are consistent long before glioma diagnosis and are therefore unlikely to reflect preclinical disease.

Background: Intrathecal synthesis of IgG is a hallmark of multiple sclerosis (MS). Vitamin D may modulate B-cell function and dampen the synthesis of IgG., Objective: To investigate the relation between vitamin D levels in cerebrospinal fluid and serum and intrathecal synthesis of IgG., Methods: 25-hydroxyvitamin D (25(OH)D) and IgG were assessed in cerebrospinal fluid and serum in 40 patients with MS., Results: There was no significant correlation between the IgG index and 25(OH)D levels in cerebrospinal fluid or serum. The levels of 25(OH)D in cerebrospinal fluid and serum did not differ between patients with and without intrathecal synthesis of IgG. There was a non-significant trend towards a positive correlation between the concentrations of 25(OH)D and IgG in the cerebrospinal fluid, but not in serum., Conclusion: Physiological variation in vitamin D does not exert a major impact on intrathecal synthesis of IgG in MS., (© 2011 John Wiley & Sons A/S.)

Rationale: F2-isoprostanes are a series of prostaglandin F2-like compounds that are formed by free-radical-catalyzed peroxidation of arachidonic acid (ARA). Several F2-isoprostanes, but in particular 8-epi-PGF(2α), are widely used as oxidative stress biomarkers. In this study we have developed an analytical tool for finger-tip blood sampling and analysis of 8-epi-PGF(2α) from dried blood spots (DBS)., Methods: We have applied solid-phase extraction (SPE) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the extraction, separation and detection of 8-epi-PGF(2α) in DBS and have studied the stability of this marker using the DBS collection platform., Results: The mass limit of detection (mLOD) for 8-epi-PGF(2α) extracted from DBS samples was 1.5 pg while the concentration limit of detection (cLOD) and concentration limit of quantitation (cLOQ) were 6 pg/mL and 18 pg/mL, respectively. All values based on triplicate analysis. Sufficient stability of 8-epi-PGF(2α) in DBS was achieved by preconditioning DBS paper with vitamin E and BHT., Conclusions: The developed method is sensitive, specific, robust, efficient, and can accurately measure endogenous concentrations of 8-epi-PGF(2α) in DBS. Thus, it offers an analytical approach to measure 8-epi-PGF(2α) by a novel sample collection technique that is less invasive and costly than conventional techniques., (Copyright © 2012 John Wiley & Sons, Ltd.)

Small intestinal lamina propria (SI-LP) CD103(+) dendritic cells (DCs) are imprinted with an ability to metabolize vitamin A (retinol), a property underlying their enhanced capacity to induce the gut-homing receptors CC chemokine receptor-9 and α4β7 on responding T cells. In this study, we demonstrate that imprinting of CD103(+) DCs is itself critically dependent on vitamin A and occurs locally within the small intestine (SI). The major vitamin A metabolite retinoic acid (RA) induced retinol-metabolizing activity in DCs both in vitro and in vivo, suggesting a direct role for RA in this process. Consistent with this, SI-LP CD103(+) DCs constitutively received RA signals in vivo at significantly higher levels than did colonic CD103(+) DCs. Remarkably, SI CD103(+) DCs remained imprinted in mice depleted of dietary but not of systemic retinol. We found that bile contained high levels of retinol, induced RA receptor-dependent retinol-metabolizing activity in bone marrow-derived DCs, and imprinted these cells with the ability to generate gut-tropic T cells. Taken together, these results suggest a novel and unexpected role for bile in SI-LP CD103(+) DC imprinting.

F2-isoprostanes are a family of prostaglandin F2-like compounds that are formed by free-radical-catalyzed peroxidation of arachidonic acid. Several F2-isoprostanes, but in particular 8-epi PGF(2alpha), are widely used as oxidative stress biomarkers. An analytical method based on liquid chromatography with negative electrospray ionization (ESI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of 8-epi PGF(2alpha) concentrations in human plasma, whole blood, erythrocytes and urine. 8-epi PGF(2alpha)-d4, a stable isotope derivative of 8-epi PGF(2alpha), was used as an internal standard (IS). A 50 microL sample was focused on-column and separated on two 3 microm particle size SUPELCOSIL ABZ+Plus HPLC columns (15 cm x 4.6 mm and 7.5 cm x 4.6 mm) connected in series. An Applied Biosystems 4000 Q TRAP LC/MS/MS system with ESI was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 353.4 --> 193.1 (8-epi PGF(2alpha)), 357.4 --> 197.1 (8-epi PGF(2alpha)-d4), used for quantification. The assay was fully validated and found to have adequate accuracy, precision, linearity, sensitivity and selectivity. The mass limit of detection (mLOD) was 1 pg of analyte eluting from the column. The assay has been successfully applied to the analysis of human plasma, whole blood, erythrocytes and urine samples., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

Here, we investigated the importance of hormone-sensitive lipase (HSL) as a retinyl ester hydrolase (REH). REH activity was measured in vitro using recombinant HSL and retinyl palmitate. The expression of retinoic acid (RA)-regulated genes and retinoid metabolites were measured in high-fat diet fed HSL-null mice using real-time quantitative PCR and triple-stage liquid chromatography/tandem mass spectrometry, respectively. Age- and gender-matched wild-type littermates were used as controls. The REH activity of rat HSL was found to be higher than that against the hitherto best known HSL substrate, i.e., diacylglycerols. REH activity in white adipose tissue (WAT) of HSL-null mice was completely blunted and accompanied by increased levels of retinyl esters and decreased levels of retinol, retinaldehyde and all-trans RA. Accordingly, genes known to be positively regulated by RA were down-regulated in HSL-null mice, including pRb and RIP140, key factors promoting differentiation into the white over the brown adipocyte lineage. Dietary RA supplementation partly restored WAT mass and the expression of RA-regulated genes in WAT of HSL-null mice. These findings demonstrate the importance of HSL as an REH of adipose tissue and suggest that HSL via this action provides RA and other retinoids for signaling events that are crucial for adipocyte differentiation and lineage commitment.

Cytochrome P450 oxidoreductase (POR) is the obligate electron donor for all microsomal cytochrome P450 enzymes, which catalyze the metabolism of a wide spectrum of xenobiotic and endobiotic compounds. Point mutations in POR have been found recently in patients with Antley-Bixler-like syndrome, which includes limb skeletal defects. In order to study P450 function during limb and skeletal development, we deleted POR specifically in mouse limb bud mesenchyme. Forelimbs and hind limbs in conditional knockout (CKO) mice were short with thin skeletal elements and fused joints. POR deletion occurred earlier in forelimbs than in hind limbs, leading additionally to soft tissue syndactyly and loss of wrist elements and phalanges due to changes in growth, cell death, and skeletal segmentation. Transcriptional analysis of E12.5 mouse forelimb buds demonstrated the expression of P450s involved in retinoic acid, cholesterol, and arachidonic acid metabolism. Biochemical analysis of CKO limbs confirmed retinoic acid excess. In CKO limbs, expression of genes throughout the whole cholesterol biosynthetic pathway was upregulated, and cholesterol deficiency can explain most aspects of the phenotype. Thus, cellular POR-dependent cholesterol synthesis is essential during limb and skeletal development. Modulation of P450 activity could contribute to susceptibility of the embryo and developing organs to teratogenesis.

The objective of this study was to evaluate the impact of pre-analytical factors on the short and long term stability of ascorbic acid (AA), the main form of vitamin C in whole blood and plasma. The effects of various anticoagulants, acidification, storage temperature and time were tested. A recently developed fast and sensitive HPLC method was used to measure AA levels. AA baseline values observed in heparin plasma were significantly higher than values observed in EDTA, citrate and Stabilyte plasma, as well as in serum. pH and temperature were identified as additional critical pre-analytical factors during the short, medium and long term handling and storage. Thus, assessment of reliable and accurate AA status in biological samples demonstrates to be highly dependent on whether the initial conditions during sample handling are controlled. In conclusion, heparin tubes should be used for blood sample collection. As AA is rapidly degraded, sample collection should be followed by immediate centrifugation and plasma acidification. To avoid further degradation during sample handling, samples should be stored at -70 degrees C without delay and analyzed within 80 days.

Congenital diaphragmatic hernia (CDH) is a developmental anomaly that results in significant mortality and morbidity. The underlying etiology is poorly understood. Insights will arise from an understanding of the mechanisms by which the teratogen nitrofen induces CDH in rodent models. In this study, we use in vitro cell assays in conjunction with whole animal rodent studies to test hypotheses regarding nitrofen's mechanism of action. The first component examined the interaction of nitrofen with various aspects of the retinoid signaling pathway including uptake proteins, binding proteins, receptors, conversion, and degradation enzymes. The second component examined the interactions of nitrofen and vitamins A, C, and E to test the hypothesis that nitrofen was functioning as an antioxidant to interfere with retinoid signaling. Third, we performed a series of experiments examining the interaction of nitrofen and thyroid signaling. Collectively, the data suggest that the primary aspect of retinoid signaling affected by nitrofen is via inhibition of the rate-limiting enzymes controlling retinoic acid synthesis. Retinoid signaling perturbations do not appear to involve oxidative effects of nitrofen. Any substantial roles of nitrofen-induced perturbations of thyroid hormone signaling or receptor function are not supported.

To study the relationship between micronutrient malnutrition and schistosomiasis mansoni, a cross-sectional study was undertaken involving 421 schoolchildren (mean age 12.6 years; 333 from schistosomiasis mansoni-endemic villages (Workemado and Sille) and 88 non-endemic controls from Sheno). Prevalence of schistosomiasis mansoni infection in Workemado and Sille was comparable (90.6% versus 95%, respectively), and prevalence of PPF in Workemado was significantly higher than in Sille (7.0% versus 0.6%, P < 0.001). Compared with non-endemic controls, serum retinol concentrations were significantly lower and hydroperoxides were significantly higher in subjects from schistosomiasis mansoni-endemic areas. Furthermore, serum alpha-tocopherol concentrations in subjects from an area with high prevalence of PPF were significantly reduced while the concentrations in subjects from an area with low prevalence of PPF were comparable to the levels found in non-endemic healthy controls. In conclusion, micronutrient malnutrition and oxidative stress are associated with Schistosoma mansoni infection and levels of schistosomal PPF.

A robust method for the simultaneous quantification of endogenous reduced glutathione (GSH) and oxidized glutathione (GSSG) in as little as 5 microl human plasma employing two-dimensional chromatographic system with parallel Hypercarb columns coupled with dual fluorescence detectors (FLD) has been developed. After sample preparation, 10 microl of supernatant was injected into the chromatographic system. The limits of detection (LOD) of GSH and GSSG were 0.5 and 0.040 pmol on column, respectively. Derivatization of GSH and GSSG with monobromobimane (MBB) and ortho-phthalaldehyde (OPA) provides a sensitivity and specificity that allows analysis after fingertip sampling, blood sampling from infants or multiple blood sampling from mice or other small experimental animals without sacrificing the animal.

A high-throughput ultrasensitive analytical method based on liquid chromatography with positive ion atmospheric pressure chemical ionization (APCI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of all-trans-4-oxo-retinoic acid (at4oxoRA), 13-cis-4-oxo-retinoic acid (13c4oxoRA), 13-cis-retinoic acid (13cRA), all-trans-retinoic acid (atRA) and all-trans-retinol (atROH) in human plasma. A stable isotope of atRA was used as internal standard (IS). The analytes and IS were isolated from 100 microL plasma by acetonitrile mono-phase extraction (MPE) performed in black 96-well microtiterplates. A 100 microL injection was focused on-column and chromatographed on an Agilent ZORBAX SB-C18 rapid-resolution high-throughput (RRHT) column with 1.8-microm particles (4.6 mmx50 mm) maintained at 60 degrees C. The initial mobile phase composition was acetonitrile/water/formic acid (10:90:0.1, v/v/v) delivered at 1.8 mL/min. Elution was accomplished by a fast gradient to acetonitrile/methanol/formic acid (90:10:0.1, v/v/v). The method had a chromatographic total run time of 7 min. An Applied Biosystems 4000 Q TRAP linear tandem mass spectrometer equipped with a heated nebulizer (APCI) ionization source was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 315.4-->297 (4-oxo-retinoic acids), 301.2-->205 (retinoic acids), 305.0-->209 (IS) and 269.2-->93 (retinol) used for quantification. The assay was fully validated and found to have acceptable accuracy, precision, linearity, sensitivity and selectivity. The mean extraction recoveries from spiked plasma samples were 80-105% for the various retinoids at three different levels. The intra-day accuracy of the assay was within 8% of nominal and intra-day precision was better than 8% coefficient of variance (CV) for retinoic acids. Inter-day precision results for quality control samples run over a 12-day period alongside clinical samples showed mean precision better than 12.5% CV. The limit of quantification was in the range of 0.1-0.2 ng/mL and the mass limit of detection (mLOD) was in the range 1-4 pg on column for the retinoic acids. The assay has been successfully applied to the analysis of 1700 plasma samples., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

Methods for retinoid analysis in tissue include direct spectrophotometry or fluorometry and retinoid responsive reporter constructs in the form of cell reporter assays or transgenic reporter animals, but chromatographic methods dominate and posses several superior features in quantitative analysis. The multitude of extraction protocols used can coarsely be divided into manual liquid-liquid extraction protocols and semi- or fully automated solid phase extraction-based protocols. Liquid chromatographic separation in reversed phase dominates although normal phase is also used. Detection is mainly performed with UV detectors although electrochemical and fluorescence detection is also used. Mass spectrometry in combination with LC is more often used in retinoid analysis and is likely to dominate in the future., ((c) 2006 Wiley Periodicals, Inc.)

A method for the simultaneous quantification of reduced and oxidized glutathione in human plasma employing a two-dimensional chromatographic system with parallel porous graphitized carbon (PGC) columns coupled with fluorescence (FLD) and coulometric electrochemical detection (ED) has been developed. Post-sampling oxidation of reduced glutathione (GSH) was prevented by derivatizing the -SH group with monobromobimane (MBB) and the glutathione-bimane adduct (GSMB) was detected by FLD. Oxidized glutathione (GSSG) was detected by ED optimized to give lowest possible limits of detection (LOD). The method is fully validated and is currently used for determination of GSH, GSSG and its redox potential in different clinical studies.

Vitamin C plays a central role in the body. One of its important functions is its role as an antioxidant, and accurate measurements are important for interpretations of this role. However, its reactive nature and instability complicates the assessment, especially in biological samples. A high-throughput chromatographic method using monolithic column and UV-detection was developed for the assessment of plasma ascorbic acid and total ascorbic acid. The method showed excellent analytical sensitivity, specificity, precision, recovery and linearity during the validation study. The method was used for the assessment of ascorbic acid and total ascorbic acid during several clinical studies.

Background: Vitamin K has several biological effects and dietary intake seems to be more important than previously believed because of low bioavailability of the vitamins from the colon., Materials and Methods: Data from the literature were identified on PubMed, and data from NORKOST II (a dietary study from 1997 based on a nation-wide sample of respondents) were used to calculate dietary intake of vitamin K., Results: The dietary intake of vitamin K in Norway seems to be < 50% of what is recommended. The stores of vitamin K are small and T/2 in the body is approximately 1-1.5 day. Vitamin K executes its effects by carboxylation of proteins and as ligand (vitamin K2) for a nuclear transcription factor. Biological effects beyond coagulation include bone formation, neural functioning and blood vessel calcification. Anticoagulation with warfarin inhibits vitamin K-dependent reactions and may have detrimental effects on bone formation., Interpretation: It is possible that the high incidence of osteoporosis in Norway may be due to the low dietary intake of vitamin K, hence it is suggested that the intake of vitamin K should be increased and vitamin K antagonists be replaced with specific thrombin inhibitors. New technology allows measurements of plasma concentration of vitamin K in relation to malabsorption, insufficient diet, and osteoporosis.

To protect the eye from ultraviolet phototoxicity caused by free radicals, ocular components such as the aqueous humor accumulate antioxidants, such as the carotenoids. Lutein and zeaxanthin are the only carotenoids known to be present in the aqueous humor. Due to the small sample volume, pooling of samples from an undesirable large number of animals is often required for sufficient sensitivity and statistically significant differences to be achieved. In this paper we present a rapid, sensitive and robust packed capillary high-performance liquid chromatographic visible detection method for the quantification of lutein and zeaxanthin in the aqueous humor of single mouse eyes.

The cytochrome P450-dependent monooxygenase system catalyzes the metabolism of xenobiotics and endogenous compounds, including hormones and retinoic acid. In order to establish the role of these enzymes in embryogenesis, we have inactivated the system through the deletion of the gene for the electron donor to all microsomal P450 proteins, cytochrome P450 reductase (Cpr). Mouse embryos homozygous for this deletion died in early to middle gestation (approximately 9.5 days postcoitum [dpc]) and exhibited a number of novel phenotypes, including the severe inhibition of vasculogenesis and hematopoiesis. In addition, defects in the brain, limbs, and cell types where CPR was shown to be expressed were observed. Some of the observed abnormalities have been associated with perturbations in retinoic acid homeostasis in later embryogenesis. Consistent with this possibility, embryos at 9.5 dpc had significantly elevated levels of retinoic acid and reduced levels of retinol. Further, some of the observed phenotypes could be either reversed or exacerbated by decreasing or increasing maternal retinoic acid exposure, respectively. Detailed analysis demonstrated a close relationship between the observed phenotype and the expression of genes controlling vasculogenesis. These data demonstrate that the cytochrome P450 system plays a key role in early embryonic development; this process appears to be, at least in part, controlled by regional concentrations of retinoic acid and has profound effects on blood vessel formation.