The Dietary Supplement Label Database: Recent Developments and Applications.

Objective: To describe the history, key features, recent enhancements, and common applications of the Dietary Supplement Label Database (DSLD). Background and History: Although many Americans use dietary supplements, databases of dietary supplements sold in the United States have not been widely available. The DSLD, an easily accessible public-use database was created in 2008 to provide information on dietary supplement composition for use by researchers and consumers. Rationale: Accessing current information easily and quickly is crucial for documenting exposures to dietary supplements because they contain nutrients and other bioactive ingredients that may have beneficial or adverse effects on human health. This manuscript details recent developments with the DSLD to achieve this goal and provides examples of how the DSLD has been used. Recent Developments: With periodic updates to track changes in product composition and capture new products entering the market, the DSLD currently contains more than 71,000 dietary supplement labels. Following usability testing with consumer and researcher user groups completed in 2016, improvements to the DSLD interface were made. As of 2017, both a desktop and mobile device version are now available. Since its inception in 2008, the use of the DSLD has included research, exposure monitoring, and other purposes by users in the public and private sectors. Future Directions: Further refinement of the user interface and search features to facilitate ease of use for stakeholders is planned. Conclusions: The DSLD can be used to track changes in product composition and capture new products entering the market. With over 71,000 DS labels it is a unique resource that policymakers, researchers, clinicians, and consumers may find valuable for multiple applications.

Vitamin B6 nutritional status and cellular availability of pyridoxal 5'-phosphate govern the function of the transsulfuration pathway's canonical reactions and hydrogen sulfide production via side reactions.

The transsulfuration pathway (TS) acts in sulfur amino acid metabolism by contributing to the regulation of cellular homocysteine, cysteine production, and the generation of H2S for signaling functions. Regulation of TS pathway kinetics involves stimulation of cystathionine ß-synthase (CBS) by S-adenosylmethionine (SAM) and oxidants such as H2O2, and by Michaelis-Menten principles whereby substrate concentrations affect reaction rates. Although pyridoxal phosphate (PLP) serves as coenzyme for both CBS and cystathionine γ-lyase (CSE), CSE exhibits much greater loss of activity than CBS during PLP insufficiency. Thus, cellular and plasma cystathionine concentrations increase in vitamin B6 deficiency mainly due to the bottleneck caused by reduced CSE activity. Because of the increase in cystathionine, the canonical production of cysteine (homocysteine â†’ cystathionine â†’ cysteine) is largely maintained even during vitamin B6 deficiency. Typical whole body transsulfuration flux in humans is 3-7 µmol/h per kg body weight. The in vivo kinetics of H2S production via side reactions of CBS and CSE in humans are unknown but they have been reported for cultured HepG2 cells. In these studies, cells exhibit a pronounced reduction in H2S production capacity and rates of lanthionine and homolanthionine synthesis in deficiency. In humans, plasma concentrations of lanthionine and homolanthionine exhibit little or no mean change due to 4-wk vitamin B6 restriction, nor do they respond to pyridoxine supplementation of subjects in chronically low-vitamin B6 status. Wide individual variation in responses of the H2S biomarkers to such perturbations of human vitamin B6 status suggests that the resulting modulation of H2S production may have physiological consequences in a subset of people. Supported by NIH grant DK072398. This paper refers to data from studies registered at clinicaltrials.gov as NCT01128244 and NCT00877812.

Pyridoxine supplementation does not alter in vivo kinetics of one-carbon metabolism but modifies patterns of one-carbon and tryptophan metabolites in vitamin B-6-insufficient oral contraceptive users.

BACKGROUND: Low chronic vitamin B-6 status can occur in a subset of women who use oral contraceptives (OCs) with uncertain metabolic consequences. An insufficiency of cellular pyridoxal 5'-phosphate (PLP), which is the coenzyme form of vitamin B-6, may impair many metabolic processes including one-carbon and tryptophan metabolism. OBJECTIVE: We investigated the effects of vitamin B-6 supplementation on the in vivo kinetics of one-carbon metabolism and the concentration of one-carbon and tryptophan metabolites in vitamin B-6-deficient OC users. DESIGN: A primed, constant infusion of [(13)C5]methionine, [3-(13)C]serine, and [(2)H3]leucine was performed on 10 OC users (20-40 y old; plasma PLP concentrations <30 nmol/L) before and after 28 d of supplementation with 10 mg pyridoxine hydrochloric acid/d. In vivo fluxes of total homocysteine remethylation, the remethylation of homocysteine from serine, and rates of homocysteine and cystathionine production were assessed. Targeted metabolite profiling was performed, and data were analyzed by using orthogonal partial least-squares-discriminant analysis and paired t tests adjusted for multiple testing. RESULTS: Pyridoxine supplementation increased the mean ± SD plasma PLP concentration from 25.8 ± 3.6 to 143 ± 58 nmol/L (P < 0.001) and decreased the leucine concentration from 103 ± 17 to 90 ± 20 nmol/L (P = 0.007) and glycine concentration from 317 ± 63 to 267 ± 58 nmol/L (P = 0.03). Supplementation did not affect in vivo rates of homocysteine remethylation or the appearance of homocysteine and cystathionine. A multivariate analysis showed a clear overall effect on metabolite profiles resulting from supplementation. Leucine, glycine, choline, cysteine, glutathione, trimethylamine N-oxide, and the ratios glycine:serine, 3-hydroxykynurenine:kynurenine, 3-hydroxykynurenine:3-hydroxyanthranilic acid, and 3-hydroxykynurenine:anthranilic acid were significant discriminating variables. CONCLUSIONS: Consistent with previous vitamin B-6-restriction studies, fluxes of one-carbon metabolic processes exhibited little or no change after supplementation in low-vitamin B-6 subjects. In contrast, changes in the metabolic profiles after supplementation indicated perturbations in metabolism, suggesting functional vitamin B-6 deficiency. This study was registered at clinicaltrials.gov as NCT01128244.

High provitamin A carotenoid serum concentrations, elevated retinyl esters, and saturated retinol-binding protein in Zambian preschool children are consistent with the presence of high liver vitamin A stores.

BACKGROUND: Biomarkers of micronutrient status are needed to best define deficiencies and excesses of essential nutrients. OBJECTIVE: We evaluated several supporting biomarkers of vitamin A status in Zambian children to determine whether any of the biomarkers were consistent with high liver retinol stores determined by using retinol isotope dilution (RID). DESIGN: A randomized, placebo-controlled, biofortified maize efficacy trial was conducted in 140 rural Zambian children from 4 villages. A series of biomarkers were investigated to better define the vitamin A status of these children. In addition to the assessment of total-body retinol stores (TBSs) by using RID, tests included analyses of serum carotenoids, retinyl esters, and pyridoxal-5'-phosphate (PLP) by using high-pressure liquid chromatography, retinol-binding protein by using ELISA, and alanine aminotransferase (ALT) activity by using a colorimetric assay. RESULTS: Children (n = 133) were analyzed quantitatively for TBSs by using RID. TBSs, retinyl esters, some carotenoids, and PLP differed by village site. Serum carotenoids were elevated above most nonintervened reference values for children. α-Carotene, ß-carotene, and lutein values were >95th percentile from children in the US NHANES III, and 13% of children had hypercarotenemia (defined as total carotenoid concentration >3.7 µmol/L). Although only 2% of children had serum retinyl esters >10% of total retinol plus retinyl esters, 16% of children had >5% as esters, which was consistent with high liver retinol stores. Ratios of serum retinol to retinol-binding protein did not deviate from 1.0, which indicated full saturation. ALT activity was low, which was likely due to underlying vitamin B-6 deficiency, which was confirmed by very low serum PLP concentrations. CONCLUSIONS: The finding of hypervitaminosis A in Zambian children was supported by high circulating concentrations of carotenoids and mildly elevated serum retinyl esters. ALT-activity assays may be compromised with co-existing vitamin B-6 deficiency. Nutrition education to improve intakes of whole grains and animal-source foods may enhance vitamin B-6 status in Zambians.

Direct and Functional Biomarkers of Vitamin B6 Status.

Measures of B6 status are categorized as direct biomarkers and as functional biomarkers. Direct biomarkers measure B6 vitamers in plasma/serum, urine and erythrocytes, and among these plasma pyridoxal 5'-phosphate (PLP) is most commonly used. Functional biomarkers include erythrocyte transaminase activities and, more recently, plasma levels of metabolites involved in PLP-dependent reactions, such as the kynurenine pathway, one-carbon metabolism, transsulfuration (cystathionine), and glycine decarboxylation (serine and glycine). Vitamin B6 status is best assessed by using a combination of biomarkers because of the influence of potential confounders, such as inflammation, alkaline phosphatase activity, low serum albumin, renal function, and inorganic phosphate. Ratios between substrate-products pairs have recently been investigated as a strategy to attenuate such influence. These efforts have provided promising new markers such as the PAr index, the 3-hydroxykynurenine:xanthurenic acid ratio, and the oxoglutarate:glutamate ratio. Targeted metabolic profiling or untargeted metabolomics based on mass spectrometry allow the simultaneous quantification of a large number of metabolites, which are currently evaluated as functional biomarkers, using data reduction statistics.

Metabolite profile analysis reveals association of vitamin B-6 with metabolites related to one-carbon metabolism and tryptophan catabolism but not with biomarkers of inflammation in oral contraceptive users and reveals the effects of oral contraceptives on these processes.

BACKGROUND: The use of oral contraceptives (OCs) has been associated with low plasma pyridoxal 5'-phosphate (PLP). The functional consequences are unclear. OBJECTIVES: To determine whether functional vitamin B-6 insufficiency occurs in OC users and is attributable to OCs, we investigated the associations of PLP with metabolites of one-carbon metabolism, tryptophan catabolism, and inflammation in OC users, and evaluated the effects of OCs on these metabolites. METHODS: Plasma metabolite concentrations were measured in 157 OC users (20-40 y of age). Associations between PLP and the metabolites were analyzed through use of generalized additive models and partial least squares-discriminant analysis (PLS-DA). Additionally, data from 111 of the 157 OC users were compared to previously reported data from 11 nonusers, at adequate and low vitamin B-6 status, with use of multivariate ANOVA. RESULTS: PLP showed significant (P < 0.05) negative nonlinear association with homocysteine, glutathione, and ratios of asymmetric dimethylarginine to arginine, 3-hydroxykynurenine to 3-hydroxyanthranilic acid, and 3-hydroxykynurenine to kynurenic acid. PLS-DA supported these conclusions and identified 3-hydroxykynurenine and the 3-hydroxykynurenine-to-kynurenine ratio as discriminating biomarkers in women with PLP ≤30 nmol/L. Among the many differences, OC users had significantly higher plasma pyridoxal (157% at adequate and 195% at low vitamin B-6 status), 4-pyridoxic acid (154% at adequate and 300% at low vitamin B-6 status), xanthurenic acid (218% at low vitamin B-6 status), 3-hydroxyanthranilic acid (176% at adequate and 166% at low vitamin B-6 status), quinolinic acid (127% at low vitamin B-6 status), and nicotinamide (197% at low vitamin B-6 status). Biomarkers of inflammation were not associated with PLP, and no differences were found between the 2 groups. CONCLUSIONS: PLP is associated with biomarkers of one-carbon metabolism and tryptophan catabolism but not with biomarkers of inflammation in OC users. Independent of vitamin B-6 status, OCs have effects on metabolites and ratios of one-carbon metabolism and tryptophan catabolism but not on biomarkers of inflammation. This study was registered at clinicaltrials.gov as NCT01128244. The study from which data for nonusers was derived was registered as NCT00877812.

A mathematical model of tryptophan metabolism via the kynurenine pathway provides insights into the effects of vitamin B-6 deficiency, tryptophan loading, and induction of tryptophan 2,3-dioxygenase on tryptophan metabolites.

Vitamin B-6 deficiency is associated with impaired tryptophan metabolism because of the coenzyme role of pyridoxal 5'-phosphate (PLP) for kynureninase and kynurenine aminotransferase. To investigate the underlying mechanism, we developed a mathematical model of tryptophan metabolism via the kynurenine pathway. The model includes mammalian data on enzyme kinetics and tryptophan transport from the intestinal lumen to liver, muscle, and brain. Regulatory mechanisms and inhibition of relevant enzymes were included. We simulated the effects of graded reduction in cellular PLP concentration, tryptophan loads and induction of tryptophan 2,3-dioxygenase (TDO) on metabolite profiles and urinary excretion. The model predictions matched experimental data and provided clarification of the response of metabolites in various extents of vitamin B-6 deficiency. We found that moderate deficiency yielded increased 3-hydroxykynurenine and a decrease in kynurenic acid and anthranilic acid. More severe deficiency also yielded an increase in kynurenine and xanthurenic acid and more pronounced effects on the other metabolites. Tryptophan load simulations with and without vitamin B-6 deficiency showed altered metabolite concentrations consistent with published data. Induction of TDO caused an increase in all metabolites, and TDO induction together with a simulated vitamin B-6 deficiency, as has been reported in oral contraceptive users, yielded increases in kynurenine, 3-hydroxykynurenine, and xanthurenic acid and decreases in kynurenic acid and anthranilic acid. These results show that the model successfully simulated tryptophan metabolism via the kynurenine pathway and can be used to complement experimental investigations.

Metabolite profile analysis reveals functional effects of 28-day vitamin B-6 restriction on one-carbon metabolism and tryptophan catabolic pathways in healthy men and women.

Suboptimal vitamin B-6 status, as reflected by low plasma pyridoxal 5'-phosphate (PLP) concentration, is associated with increased risk of vascular disease. PLP plays many roles, including in one-carbon metabolism for the acquisition and transfer of carbon units and in the transsulfuration pathway. PLP also serves as a coenzyme in the catabolism of tryptophan. We hypothesize that the pattern of these metabolites can provide information reflecting the functional impact of marginal vitamin B-6 deficiency. We report here the concentration of major constituents of one-carbon metabolic processes and the tryptophan catabolic pathway in plasma from 23 healthy men and women before and after a 28-d controlled dietary vitamin B-6 restriction (<0.35 mg/d). liquid chromatography-tandem mass spectrometry analysis of the compounds relevant to one-carbon metabolism showed that vitamin B-6 restriction yielded increased cystathionine (53% pre- and 76% postprandial; P < 0.0001) and serine (12% preprandial; P < 0.05), and lower creatine (40% pre- and postprandial; P < 0.0001), creatinine (9% postprandial; P < 0.05), and dimethylglycine (16% postprandial; P < 0.05) relative to the vitamin B-6-adequate state. In the tryptophan pathway, vitamin B-6 restriction yielded lower kynurenic acid (22% pre- and 20% postprandial; P < 0.01) and higher 3-hydroxykynurenine (39% pre- and 34% postprandial; P < 0.01). Multivariate ANOVA analysis showed a significant global effect of vitamin B-6 restriction and multilevel partial least squares-discriminant analysis supported this conclusion. Thus, plasma concentrations of creatine, cystathionine, kynurenic acid, and 3-hydroxykynurenine jointly reveal effects of vitamin B-6 restriction on the profiles of one-carbon and tryptophan metabolites and serve as biomarkers of functional effects of marginal vitamin B-6 deficiency.

K16-biotinylated histone H4 is overrepresented in repeat regions and participates in the repression of transcriptionally competent genes in human Jurkat lymphoid cells.

Holocarboxylase synthetase (HCS) catalyzes the binding of biotin to lysine (K) residues in histones H3 and H4. Histone biotinylation marks are enriched in repressed loci, including retrotransposons. Preliminary studies suggested that K16 in histone H4 is a target for biotinylation by HCS. Here we tested the hypotheses that H4K16bio is a real histone mark in human chromatin and that H4K16bio is overrepresented in repressed gene loci and repeat regions. Polyclonal rabbit anti-human H4K16bio was generated and affinity purified. An extensive series of testing with synthetic and natural targets confirmed that this new antibody is specific for H4K16bio. Using anti-H4K16bio and chromatin immunoprecipitation assays, we demonstrated that H4K16bio is overrepresented in repeat regions [pericentromeric alpha satellite repeats and long terminal repeats (LTR)] compared with euchromatin promoters. H4K16bio was also enriched in the repressed interleukin-2 gene promoter in human lymphoid cells; transcriptional activation of the interleukin-2 gene by mitogens and phorbol esters coincided with a depletion of the H4K16bio mark at the gene promoter. The enrichment of H4K16bio depended on biotin supply; the enrichment at LTR22 and promoter 1 of the sodium-dependent multivitamin transporter (SMVT) was greater in biotin-supplemented cells compared with biotin-normal and biotin-deficient cells. The enrichment of H4K16bio at LTR15 and SMVT promoter 1 was significantly lower in fibroblasts from an HCS-deficient patient compared with an HCS wild-type control. We conclude that H4K16bio is a real phenomenon and that this mark, like other biotinylation marks, is overrepresented in repressed loci where it marks HCS docking sites.

Biotinylation is a natural, albeit rare, modification of human histones.

Previous studies suggest that histones H3 and H4 are posttranslationally modified by binding of the vitamin biotin, catalyzed by holocarboxylase synthetase (HCS). Albeit a rare epigenetic mark, biotinylated histones were repeatedly shown to be enriched in repeat regions and repressed loci, participating in the maintenance of genome stability and gene regulation. Recently, a team of investigators failed to detect biotinylated histones and proposed that biotinylation is not a natural modification of histones, but rather an assay artifact. Here, we describe the results of experiments, including the comparison of various analytical protocols, antibodies, cell lines, classes of histones, and radiotracers. These studies provide unambiguous evidence that biotinylation is a natural, albeit rare, histone modification. Less than 0.001% of human histones H3 and H4 are biotinylated, raising concerns that the abundance might too low to elicit biological effects in vivo. We integrated information from this study, previous studies, and ongoing research efforts to present a new working model in which biological effects are caused by a role of HCS in multiprotein complexes in chromatin. In this model, docking of HCS in chromatin causes the occasional binding of biotin to histones as a tracer for HCS binding sites.

A 96-well plate assay for high-throughput analysis of holocarboxylase synthetase activity.

BACKGROUND: Holocarboxylase synthetase (HCS) catalyzes the covalent binding of biotin to both carboxylases and histones. Biotinylated carboxylases and biotinylated histones play crucial roles in the metabolism of fatty acids, amino acids, and glucose, and in gene regulation and genome stability, respectively. HCS null mammals are not viable whereas HCS deficiency is linked to developmental delays in humans and phenotypes such as short life span and low stress resistance in Drosophila. METHODS: HCS-dependent biotinylation of the polypeptide p67 was detected and quantified in a 96-well plate format using IRDye-streptavidin and infrared spectroscopy. RESULTS: Biotinylation of p67 by recombinant HCS (rHCS) and HCS from human cell extracts depended on time, temperature, and substrate concentration, all consistent with enzyme catalysis rather than non-enzymatic biotinylation. The Michaelis-Menten constant of rHCS for p67 was 4.1±1.5 µmol/l. The minimal concentration of rHCS that can be detected by this assay is less than 1.08 nmol/l. Jurkat cells contained 0.14±0.02 U of HCS activity [µmol of biotinylated p67 formed/(nmol/l HCSh)] in 400 µg of total protein. CONCLUSIONS: We developed a 96-well plate assay for high-throughput analysis of HCS activity in biological samples and studies of synthetic and naturally occurring HCS inhibitors.