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

Abstract

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.

Keywords: metabolomics; one-carbon metabolism; oral contraceptives; tryptophan catabolism; vitamin B-6.

FIGURE 1

Consolidated Standards of Reporting Trials diagram of study participants. OC, oral contraceptive; PLP, pyridoxal 5′-phosphate; TSH, thyrotropin (thyroid-stimulating hormone).

FIGURE 2

Score plots from a multilevel orthogonal partial least-squares–discriminant analysis for the overall pooled data of one-carbon metabolites and kinetic variables, tryptophan metabolites, and biomarkers of inflammation analyzed in plasma samples at baseline and after 28 d of vitamin B-6 supplementation in OC users. (A) Preprandial state. (B) Postprandial state. Each data point represents a function of the entire metabolite profile of each participant. The cumulative R² and Q² for the preprandial state were 0.962 and 0.905, respectively. The cumulative R² and Q² for the postprandial state were 0.928 and 0.847, respectively. t[1], principal component 1; to[1], orthogonal score vector 1.

FIGURE 3

S-plots showing the relative contribution of variables for the separation between groups. (A) Preprandial state. (B) Postprandial state. The p(corr)[1] axis represents the correlation of the variables with the model classes, whereas the p[1] axis represents the magnitude of variables of the discriminating component of the orthogonal partial least-squares–discriminant analysis. Variables that showed a high p(corr)[1] (less than −0.6 and >0.6) and high p[1] were considered to be the variables that most strongly contributed to differences between groups, which are potential biomarkers (closed circles with labels). Negative p[1] values indicate variables that declined after pyridoxine supplementation, whereas positive p[1] values designate variables that increased because of supplementation. AA, anthranilic acid; Chol, choline; GSH, total glutathione; HAA, 3-hydroxyanthranilic acid; HK, 3-hydroxykynurenine; KYN, kynurenine; TMAO, trimethylamine N-oxide.