Vitamin B-6 and riboflavin, their metabolic interaction, and relationship with MTHFR genotype in adults aged 18-102 years.

BACKGROUND: The generation of the active form of vitamin B-6, pyridoxal 5'-phosphate (PLP), in tissues is dependent upon riboflavin as flavin mononucleotide, but whether this interaction is important for maintaining vitamin B-6 status is unclear. OBJECTIVE: To investigate vitamin B-6 and riboflavin status, their metabolic interaction, and relationship with methylenetetrahydrofolate reductase (MTHFR) genotype in adulthood. METHODS: Data from 5612 adults aged 18-102 y were drawn from the Irish National Adult Nutrition Survey (NANS; population-based sample) and the Trinity-Ulster Department of Agriculture (TUDA) and Genovit cohorts (volunteer samples). Plasma PLP and erythrocyte glutathione reductase activation coefficient (EGRac), as a functional indicator of riboflavin, were determined. RESULTS: Older (≥65 y) compared with younger (<65 y) adults had significantly lower PLP concentrations (P < 0.001). A stepwise decrease in plasma PLP was observed across riboflavin categories, from optimal (EGRac ≤1.26), to suboptimal (EGRac: 1.27-1.39), to deficient (EGRac ≥1.40) status, an effect most pronounced in older adults (mean ± SEM: 76.4 ± 0.9 vs 65.0 ± 1.1 vs 55.4 ± 1.2 nmol/L; P < 0.001). In individuals with the variant MTHFR 677TT genotype combined with riboflavin deficiency, compared with non-TT (CC/CT) genotype participants with sufficient riboflavin, we observed PLP concentrations of 52.1 ± 2.9 compared with 76.8 ±0.7 nmol/L (P < 0.001). In participants with available dietary data (i.e., NANS cohort, n = 936), PLP was associated with vitamin B-6 intake (nonstandardized regression coefficient ß: 2.49; 95% CI 1.75, 3.24; P < 0.001), supplement use (ß: 81.72; 95% CI: 66.01, 97.43; P < 0.001), fortified food (ß: 12.49; 95% CI: 2.08, 22.91; P = 0.019), and EGRac (ß: -65.81; 95% CI: -99.08, -32.54; P < 0.001), along with BMI (ß: -1.81; 95% CI: -3.31, -0.30; P = 0.019). CONCLUSIONS: These results are consistent with the known metabolic dependency of PLP on flavin mononucleotide (FMN) and suggest that riboflavin may be the limiting nutrient for maintaining vitamin B-6 status, particularly in individuals with the MTHFR 677TT genotype. Randomized trials are necessary to investigate the PLP response to riboflavin intervention within the dietary range. The TUDA study and the NANS are registered at www.ClinicalTrials.gov as NCT02664584 (27 January 2016) and NCT03374748 (15 December 2017), respectively.Clinical Trial Registry details: Trinity-Ulster-Department of Agriculture (TUDA) study, ClinicalTrials.gov no. NCT02664584 (January 27th 2016); National Adult Nutrition Survey (NANS), ClinicalTrials.gov no. NCT03374748 (December 15th 2017).

DNA methylation of hypertension-related genes and effect of riboflavin supplementation in adults stratified by genotype for the MTHFR C677T polymorphism.

BACKGROUND: The interaction between genetic, epigenetic and environmental factors plays an important role in the aetiology of hypertension. GWAS and observational studies link the C677T polymorphism in methylenetetrahydrofolate reductase (MTHFR) with hypertension, while riboflavin, the MTHFR cofactor, has been shown to reduce blood pressure and global DNA methylation in homozygous (TT genotype) individuals. It is currently unclear whether riboflavin modulates DNA methylation of other hypertension-related genes. OBJECTIVES: To compare DNA methylation of hypertension-related genes in adults stratified by MTHFR genotype and effect of riboflavin intervention in adults with the variant MTHFR 677TT genotype. METHOD: Pyrosequencing was carried out for hypertension-related genes (ACE, AGTR1, GCK, GNA12, IGF2, MMP9 and NOS3) in blood samples from participants in previous trials (CC, n = 40; TT, n = 40). The effect of intervention with riboflavin (1.6 mg/d for16 weeks) or placebo on DNA methylation was investigated in adults with the variant MTHFR 677TT genotype (n = 80). RESULTS: Individuals with the MTHFR 677TT v CC genotype had significantly higher average DNA methylation at NOS3 (+1.66%, P = 0.044). In response to riboflavin supplementation in TT individuals, there was an increase in average DNA methylation at IGF2 (+1.09%, P = 0.019) and a decrease at ACE (-0.44%, P = 0.021) in females only. Specific CpG sites were hypomethylated in GNA12 and hypermethylated in AGTR1. CONCLUSION: This study provides the first RCT evidence that riboflavin alters DNA methylation of hypertension-related genes in adults with the MTHFR 677TT genotype, providing some insight into mechanisms linking hypertension with the genotype-specific response of BP to riboflavin.

Impact of the MTHFR C677T polymorphism on one-carbon metabolites: Evidence from a randomised trial of riboflavin supplementation.

Homozygosity for the C677T polymorphism in MTHFR (TT genotype) is associated with a 24-87% increased risk of hypertension. Blood pressure (BP) lowering was previously reported in adults with the TT genotype, in response to supplementation with the MTHFR cofactor, riboflavin. Whether the BP phenotype associated with the polymorphism is related to perturbed one-carbon metabolism is unknown. This study investigated one-carbon metabolites and their responsiveness to riboflavin in adults with the TT genotype. Plasma samples from adults (n 115) screened for the MTHFR genotype, who previously participated in RCTs to lower BP, were analysed for methionine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), betaine, choline and cystathionine by liquid chromatography tandem mass spectrometry (LC-MS/MS). The one-carbon metabolite response to riboflavin (1.6 mg/d; n 24) or placebo (n 23) for 16 weeks in adults with the TT genotype was also investigated. Plasma SAM (74.7 ± 21.0 vs 85.2 ± 22.6 nmol/L, P = 0.013) and SAM:SAH ratio (1.66 ± 0.55 vs 1.85 ± 0.51, P = 0.043) were lower and plasma homocysteine was higher (P = 0.043) in TT, compared to CC individuals. In response to riboflavin, SAM (P = 0.008) and cystathionine (P = 0.045) concentrations increased, with no responses in other one-carbon metabolites observed. These findings confirm perturbed one-carbon metabolism in individuals with the MTHFR 677TT genotype, and for the first time demonstrate that SAM, and cystathionine, increase in response to riboflavin supplementation in this genotype group. The genotype-specific, one-carbon metabolite responses to riboflavin intervention observed could offer some insight into the role of this gene-nutrient interaction in blood pressure.

Riboflavin supplementation alters global and gene-specific DNA methylation in adults with the MTHFR 677 TT genotype.

DNA methylation is important in regulating gene expression and genomic stability while aberrant DNA methylation is associated with disease. Riboflavin (FAD) is a cofactor for methylenetetrahydrofolate reductase (MTHFR), a critical enzyme in folate recycling, which generates methyl groups for homocysteine remethylation to methionine, the pre-cursor to the universal methyl donor S-adenosylmethionine (SAM). A polymorphism (C677T) in MTHFR results in decreased MTHFR activity and increased homocysteine concentration. Previous studies demonstrated that riboflavin modulates this phenotype in homozygous adults (MTHFR 677 TT genotype), however, DNA methylation was not considered. This study examined DNA methylation, globally and at key MTHFR regulatory sites, in adults stratified by MTHFR genotype and the effect of riboflavin supplementation on DNA methylation in individuals with the 677 TT genotype. Samples were accessed from participants, screened for the MTHFR C677T polymorphism, who participated in observational (n = 80) and targeted riboflavin (1.6 mg/day) RCTs (n = 80). DNA methylation at LINE-1 and key regulatory regions of the MTHFR locus were analysed by pyrosequencing in peripheral blood leukocytes. LINE-1 (+1.6%; p = 0.011) and MTHFR south shelf (+4.7%, p < 0.001) were significantly hypermethylated in individuals with the MTHFR 677 TT compared to CC genotype. Riboflavin supplementation resulted in decreased global methylation, albeit only significant at one CpG. A significant reduction in DNA methylation at the MTHFR north shore (-1.2%, p < 0.001) was also observed in TT adults following intervention with riboflavin. This provides the first RCT evidence that DNA methylation may be modulated by riboflavin in adults with the MTHFR 677 TT genotype.

Blood pressure in treated hypertensive individuals with the MTHFR 677TT genotype is responsive to intervention with riboflavin: findings of a targeted randomized trial.

Intervention with riboflavin was recently shown to produce genotype-specific lowering of blood pressure (BP) in patients with premature cardiovascular disease homozygous for the 677C→T polymorphism (TT genotype) in the gene encoding the enzyme methylenetetrahydrofolate reductase (MTHFR). Whether this effect is confined to patients with high-risk cardiovascular disease is unknown. The aim of this randomized trial, therefore, was to investigate the responsiveness of BP to riboflavin supplementation in hypertensive individuals with the TT genotype but without overt cardiovascular disease. From an available sample of 1427 patients with hypertension, we identified 157 with the MTHFR 677TT genotype, 91 of whom agreed to participate in the trial. Participants were stratified by systolic BP and randomized to receive placebo or riboflavin (1.6 mg/d) for 16 weeks. At baseline, despite being prescribed multiple classes of antihypertensive drugs, >60% of participants with this genotype had failed to reach goal BP (≤140/90 mm Hg). A significant improvement in the biomarker status of riboflavin was observed in response to intervention (P<0.001). Correspondingly, an overall treatment effect of 5.6±2.6 mm Hg (P=0.033) in systolic BP was observed, with pre- and postintervention values of 141.8±2.9 and 137.1±3.0 mm Hg (treatment group) and 143.5±3.0 and 144.3±3.1 mm Hg (placebo group), whereas the treatment effect in diastolic BP was not significant (P=0.291). In conclusion, these results show that riboflavin supplementation targeted at hypertensive individuals with the MTHFR 677TT genotype can decrease BP more effectively than treatment with current antihypertensive drugs only and indicate the potential for a personalized approach to the management of hypertension in this genetically at-risk group. Clinical Trial Registration- URL: http://www.clinicaltrials.gov. Unique identifier: ISRCTN23620802.

Incremental cholecalciferol supplementation up to 15 µg/d throughout winter at 51-55° N has no effect on biomarkers of cardiovascular risk in healthy young and older adults.

Two separate, identical, double-blind, randomized, placebo-controlled intervention studies were carried out in the south and north of Ireland (51-55°N). Men and women aged 20-40 y (n = 202) and ≥64 y (n = 192) received cholecalciferol at doses of 0 (P), 5 (D3-5), 10 (D3-10), or 15 (D3-15) µg/d (0-600 IU) during wintertime. Serum 25-hydroxyvitamin D [s25(OH)D], intact parathyroid hormone, systolic and diastolic blood pressure, fasting lipids, glucose and insulin, HOMA-IR, high-sensitivity CRP, matrix metalloproteinase-9, and its inhibitor (tissue inhibitor metalloproteinase-1) were measured at baseline (October) and 22 wk later at endpoint (March). Vitamin D receptor Fok I and Taq I genotypes were analyzed and dietary intakes of vitamin D and calcium were assessed. In young adults, s25(OH)D decreased from baseline to endpoint (P < 0.001), except in the D3-15 group, who maintained the baseline concentration of ~70 nmol/L. Older adults had lower s25(OH)D at baseline (median, 54.2 nmol/L) and concentrations increased in the D3-10 and D3-15 groups (P < 0.001). There were no significant effects of supplementation on cardiovascular disease (CVD) risk biomarkers in either age group. Fasting glucose and total and HDL cholesterol were lower (P < 0.05) in older adults with the Fok 1 ff genotype than in those with FF or Ff. Putative effects of vitamin D on cardio-metabolic health will only be evident at higher intakes than the current RDA and possibly in individuals at particular risk of low s25(OH)D and/or CVD risk.

Riboflavin offers a targeted strategy for managing hypertension in patients with the MTHFR 677TT genotype: a 4-y follow-up.

BACKGROUND: We recently reported that the elevated blood pressure (BP) observed in patients with cardiovascular disease who are homozygous for the 677C→T polymorphism (TT genotype) in the gene encoding methylenetetrahydrofolate reductase (MTHFR) was responsive to supplementation with riboflavin-the cofactor for MTHFR. OBJECTIVE: The objective was to investigate the effect of riboflavin on BP targeted at patients with the TT genotype 4 y after initial investigation, during which time major changes in the clinical guidelines for antihypertensive therapy were introduced. DESIGN: A total of 83 patients (representing all 3 genotypes) who participated in a placebo-controlled riboflavin intervention for 16 wk in 2004 agreed to take part. Nested within this follow-up, those with the TT genotype (n = 31) proceeded to intervention with riboflavin (1.6 mg/d for 16 wk) or placebo, conducted in a crossover style whereby the 2004 treatment groups were reversed. RESULTS: At follow-up in 2008, as in 2004, patients with the TT genotype had higher systolic BP (P < 0.01), with a nonsignificant trend noted for higher diastolic BP (P = 0.051). Despite the marked changes in antihypertensive therapy that had occurred, BP remained unchanged in patients with the TT genotype at the time of follow-up. Riboflavin supplementation (administered in 2004 and 2008) produced an overall decrease in systolic (-9.2 ± 12.8 mm Hg; P = 0.001) and diastolic (-6.0 ± 9.9 mm Hg; P = 0.003) BP. CONCLUSIONS: Optimizing riboflavin status offers a low-cost targeted strategy for managing elevated BP in this genetically at-risk group. These findings, if confirmed in the general population, could have important implications for the prevention of hypertension.

Effect of adiposity on vitamin D status and the 25-hydroxycholecalciferol response to supplementation in healthy young and older Irish adults.

There is increasing epidemiological evidence linking sub-optimal vitamin D status with overweight and obesity. Although increasing BMI and adiposity have also been negatively associated with the change in vitamin D status following supplementation, results have been equivocal. The aim of this randomised, placebo-controlled study was to investigate the associations between anthropometric measures of adiposity and the wintertime serum 25-hydroxycholecalciferol (25(OH)D) response to 15 µg cholecalciferol per d in healthy young and older Irish adults. A total of 110 young adults (20-40 years) and 102 older adults ( ≥ 64 years) completed the 22-week intervention with >85 % compliance. The change in 25(OH)D from baseline was calculated. Anthropometric measures of adiposity taken at baseline included height, weight and waist circumference (WC), along with skinfold thickness measurements to estimate fat mass (FM). FM was subsequently expressed as FM (kg), FM (%), FM index (FMI (FM kg/height m2)) and as a percentage ratio to fat-free mass (FFM). In older adults, vitamin D status was inversely associated with BMI (kg/m2), WC (cm), FM (kg and %), FMI (kg/m2) and FM:FFM (%) at baseline (r - 0·33, - 0·36, - 0·33, - 0·30, - 0·33 and - 0·27, respectively, all P values < 0·01). BMI in older adults was also negatively associated with the change in 25(OH)D following supplementation (ß - 1·27, CI - 2·37, - 0·16, P = 0·026); however, no such associations were apparent in younger adults. Results suggest that adiposity may need to be taken into account when determining an adequate wintertime dietary vitamin D intake for healthy older adults residing at higher latitudes.

Maintenance of wintertime vitamin D status with cholecalciferol supplementation is not associated with alterations in serum cytokine concentrations among apparently healthy younger or older adults.

Epidemiological studies have shown that low vitamin D status results in impaired immune function and is associated with the prevalence of autoimmune and inflammatory conditions. Vitamin D supplementation has been shown to reduce circulating concentrations of inflammatory markers in such conditions. However, the possible beneficial effect of vitamin D supplementation in the general population, particularly for those individuals living at high latitudes where hypovitaminosis D is common during wintertime, remains unclear. The aim of this study was to assess the effect of vitamin D supplementation using doses of 5, 10, and 15 µg/d cholecalciferol (D3) compared with placebo on cytokine concentrations throughout winter in apparently healthy younger (aged 20-40 y) and older (aged ≥64 y) adults. A total of 211 younger and 202 older adults completed the 22-wk intervention (from October to March) with >85% compliance. Serum concentrations of 25-hydroxycholecalciferol [25(OH)D3], high sensitivity C-reactive protein, IL-6, IL-10, soluble CD40 ligand, TGFß, TNFα, and fibrinogen were measured using ELISA. 25(OH)D3 concentrations significantly decreased in the placebo and 5 and 10/d µg D3 groups in the younger cohort and in the placebo group in the older cohort. Whereas 15 µg/d D3 supplementation maintained 25(OH)D3 concentrations in the younger cohort (baseline, 75.9 nmol/L; postintervention, 69.0 nmol/L) and significantly increased concentrations in the older cohort (baseline, 55.1 nmol/L; postintervention, 73.9 nmol/L), it had no significant effect on cytokine concentrations (ANCOVA, P > 0.05). The long-term effects of low vitamin D status remain to be elucidated and optimization of vitamin D status in otherwise healthy individuals may potentially have lasting beneficial effects on the immune system.

Cholecalciferol supplementation throughout winter does not affect markers of bone turnover in healthy young and elderly adults.

Although there have been several studies of the effect of vitamin D status on bone turnover in the elderly, the findings are unclear, and, furthermore, to date very few have investigated this in young adults. The objective of these randomized, placebo-controlled, double-blind, 2-center intervention studies was to investigate the effect of cholecalciferol supplementation (0, 5, 10, and 15 microg cholecalciferol/d) throughout winter time on indices of vitamin D status and bone turnover in young (aged 20-40 y; n = 215) and elderly (aged > or = 64 y; n = 204) adults, with relatively high mean calcium intakes of 976 and 874 mg/d, respectively. Fasting serum concentrations of 25-hydroxyvitamin D [25(OH)D], parathyroid hormone (PTH), osteocalcin, bone-specific alkaline phosphatase, and carboxyterminal collagen crosslinks were measured by enzyme immunoassays at baseline and endpoint. Fok I and Taq I vitamin D receptor (VDR) genotypes were determined by real-time PCR. Endpoint serum 25(OH)D increased (P < 0.0001) in a dose-related manner with increasing supplemental cholecalciferol (up to 15 microg/d) in 20-40-y olds and up to 10 microg/d in > or = 64-y olds. Endpoint serum PTH was lower (P < 0.05) in the 3 cholecalciferol-supplemented groups compared with that in the placebo group in > or = 64-y olds, but cholecalciferol supplementation did not affect other markers in either cohort and there was no significant interaction with VDR genotype. In conclusion, cholecalciferol supplementation alone throughout winter did not affect bone turnover markers in apparently healthy young and elderly adults, even when stratified by VDR genotype.

Estimation of the dietary requirement for vitamin D in free-living adults >=64 y of age.

BACKGROUND: Older adults may be more prone to developing vitamin D deficiency than younger adults. Dietary requirements for vitamin D in older adults are based on limited evidence. OBJECTIVE: The objective was to establish the dietary intake of vitamin D required to maintain serum 25-hydroxyvitamin D [25(OH)D] concentrations above various cutoffs between 25 and 80 nmol/L during wintertime, which accounted for the effect of summer sunshine exposure and diet. DESIGN: A randomized, placebo-controlled, double-blind, 22-wk intervention was conducted in men and women aged >/=64 y (n = 225) at supplemental levels of 0, 5, 10, and 15 microg vitamin D(3)/d from October 2007 to March 2008. RESULTS: Clear dose-related increments (P < 0.0001) in serum 25(OH)D were observed with increasing supplemental vitamin D(3) intakes. The slope of the relation between total vitamin D intake and serum 25(OH)D was 1.97 nmol . L(-1) . microg intake(-1). The vitamin D intake that maintained serum 25(OH)D concentrations >25 nmol/L in 97.5% of the sample was 8.6 microg/d. Intakes were 7.9 and 11.4 microg/d in those who reported a minimum of 15 min daily summer sunshine exposure or less, respectively. The intakes required to maintain serum 25(OH)D concentrations of >37.5, >50, and >80 nmol/L in 97.5% of the sample were 17.2, 24.7, and 38.7 microg/d, respectively. CONCLUSION: To ensure that the vitamin D requirement is met by the vast majority (>97.5%) of adults aged >/=64 y during winter, between 7.9 and 42.8 microg vitamin D/d is required, depending on summer sun exposure and the threshold of adequacy of 25(OH)D. This trial was registered at http://www.controlled-trials.com/ISRCTN20236112 as ISRCTN registration no. ISRCTN20236112.

Estimation of the dietary requirement for vitamin D in healthy adults.

BACKGROUND: Knowledge gaps have contributed to considerable variation among international dietary recommendations for vitamin D. OBJECTIVE: We aimed to establish the distribution of dietary vitamin D required to maintain serum 25-hydroxyvitamin D [25(OH)D] concentrations above several proposed cutoffs (ie, 25, 37.5, 50, and 80 nmol/L) during wintertime after adjustment for the effect of summer sunshine exposure and diet. DESIGN: A randomized, placebo-controlled, double-blind 22-wk intervention study was conducted in men and women aged 20-40 y (n = 238) by using different supplemental doses (0, 5, 10, and 15 microg/d) of vitamin D(3) throughout the winter. Serum 25(OH)D concentrations were measured by using enzyme-linked immunoassay at baseline (October 2006) and endpoint (March 2007). RESULTS: There were clear dose-related increments (P < 0.0001) in serum 25(OH)D with increasing supplemental vitamin D(3). The slope of the relation between vitamin D intake and serum 25(OH)D was 1.96 nmol x L(-1) x microg(-1) intake. The vitamin D intake that maintained serum 25(OH)D concentrations of >25 nmol/L in 97.5% of the sample was 8.7 microg/d. This intake ranged from 7.2 microg/d in those who enjoyed sunshine exposure, 8.8 microg/d in those who sometimes had sun exposure, and 12.3 microg/d in those who avoided sunshine. Vitamin D intakes required to maintain serum 25(OH)D concentrations of >37.5, >50, and >80 nmol/L in 97.5% of the sample were 19.9, 28.0, and 41.1 microg/d, respectively. CONCLUSION: The range of vitamin D intakes required to ensure maintenance of wintertime vitamin D status [as defined by incremental cutoffs of serum 25(OH)D] in the vast majority (>97.5%) of 20-40-y-old adults, considering a variety of sun exposure preferences, is between 7.2 and 41.1 microg/d.