Metabolomic Analysis Reveals Extended Metabolic Consequences of Marginal Vitamin B-6 Deficiency in Healthy Human Subjects

<div><p>Marginal deficiency of vitamin B-6 is common among segments of the population worldwide. Because pyridoxal 5′-phosphate (PLP) serves as a coenzyme in the metabolism of amino acids, carbohydrates, organic acids, and neurotransmitters, as well as in aspects of one-carbon metabolism, vitamin B-6 deficiency could have many effects. Healthy men and women (age: 20-40 y; n = 23) were fed a 2-day controlled, nutritionally adequate diet followed by a 28-day low-vitamin B-6 diet (<0.5 mg/d) to induce marginal deficiency, as reflected by a decline of plasma PLP from 52.6±14.1 (mean ± SD) to 21.5±4.6 nmol/L (P<0.0001) and increased cystathionine from 131±65 to 199±56 nmol/L (P<0.001). Fasting plasma samples obtained before and after vitamin B6 restriction were analyzed by ¹H-NMR with and without filtration and by targeted quantitative analysis by mass spectrometry (MS). Multilevel partial least squares-discriminant analysis and S-plots of NMR spectra showed that NMR is effective in classifying samples according to vitamin B-6 status and identified discriminating features. NMR spectral features of selected metabolites indicated that vitamin B-6 restriction significantly increased the ratios of glutamine/glutamate and 2-oxoglutarate/glutamate (P<0.001) and tended to increase concentrations of acetate, pyruvate, and trimethylamine-N-oxide (adjusted P<0.05). Tandem MS showed significantly greater plasma proline after vitamin B-6 restriction (adjusted P<0.05), but there were no effects on the profile of 14 other amino acids and 45 acylcarnitines. These findings demonstrate that marginal vitamin B-6 deficiency has widespread metabolic perturbations and illustrate the utility of metabolomics in evaluating complex effects of altered vitamin B-6 intake.</p></div

Ratios of selected plasma constituents determined in filtered plasma by ¹H-NMR, before and after vitamin B-6 restriction.

1<p>Mean ± SD, n = 18.</p>2<p><i>P</i> value determined by paired t-test after log₂ transformation. * designates a significant difference at P<0.05.</p

Metabolic patterns separated adequate vitamin B6 at baseline from marginally deficient status after 4 weeks on a restricted diet.

<p>Plasma samples from 23 healthy, young adults collected at baseline and after 4 weeks consuming a vitamin B6-restricted diet were examined by ¹H-NMR spectroscopy followed by multilevel partial least square-discriminant analysis (multilevel PLS-DA). A) Score plot for unfiltered plasma, which contains relatively large signal from lipoproteins. B) Score plot for plasma filtered through a 3 μm pore size filter to remove most of the lipoprotein before ¹H-NMR spectroscopy analysis.</p

S-plots from multilevel PLS-DA show that ¹H-NMR signals from many metabolites contribute to separation of vitamin B6 adequate and vitamin B6 restricted conditions.

<p>Panels A and B: S-plots that respectively correspond with the score plots in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063544#pone-0063544-g001" target="_blank">Figure 1A</a> (Not Filtered) and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063544#pone-0063544-g001" target="_blank">Figure 1B</a> (Filtered) plasma. In these panels, the top 5% of metabolites that contribute to 95% of the separation of baseline and restricted samples are highlighted in red squares. Red-framed points within the ovals represent the most highly discriminating signals. Panels C and D: loading plots illustrating discriminating spectral features from S-plots. Covariance of each discriminating feature is superimposed on the corresponding NMR chemical shift on mean spectra.</p

Estimated concentration of selected plasma constituents before and after vitamin B-6 restriction determined in filtered plasma by Chenomx spectral fitting analysis of ¹H-NMR.

1<p>Mean ± SD, n = 18. Concentrations approximated from the respective singlet resonances using Chenomx spectral fitting analysis.</p>2<p>Adjusted P-value through controlling positive FDR after paired t-tests on changes on log2 transformed concentrations.* designates significant difference at P<0.05. The effect of vitamin B-6 restriction on the overall pattern of constituents in Chenomx analysis was significant in multivariate testing, P = 5.3×10⁻⁷.</p