BACKGROUND Serine and glycine are major one-carbon donors. In eukaryotic cells one-carbon metabolism is compartmentalized; the glycine cleavage enzymes are mitochondrial, while the cytosolic and mitochondrial forms of serine hydroxymethyltransferase (SHMT) are independently transcribed. DESIGN One-carbon donors ([3-13C]serine, [2, 3, 3-d3]serine, or [2-13C]glycine) were administered in separate infusions to healthy males (n = 5). Monocyte DNA was subsequently isolated for the determination of deoxynucleotide enrichment by LC-MS/MS. RESULTS Total thymidine and deoxyadenosine enrichment was similar for [3-13C]serine and [2, 3, 3-d3]serine [Table]. M+2 thymidine enrichment (i.e., that deriving from cytosolic SHMT) constituted 23% of [2, 3, 3-d3]serine derived enrichment. Thymidine enrichment from [2-13C]glycine was lower than from either labeled serine while deoxyadenosine enrichment from [2-13C]glycine was overestimated, as [2-13C]glycine was also directly incorporated into purines. Percentage molar enrichment (mean ± SD) of thymidine (dT) and deoxyadenosine (dA). Values in brackets are molar enrichments expressed as a percentage of total one-carbon enrichment: {enrichment / (enrichment from [2, 3, 3-d3]serine + enrichment from [2-13C]glycine)} CONCLUSION The majority (~84%) of one-carbon units used for thymidine synthesis are mitochondrial derived. Supported by NIH DK56274 and GCRC grant M01-RR00082

Cysteine synthesis from homocysteine is catalyzed by two pyridoxal 5'-phosphate (PLP)-dependent enzymes. This suggests that vitamin B-6 status might affect cysteine and glutathione homeostasis, but it is unclear whether this occurs in humans. We assessed the effects of vitamin B-6 status on static and kinetic parameters of cysteine and glutathione metabolism in healthy female (n=5) and male (n=4) volunteers (20-30 y) before and after 4 wk of dietary vitamin B-6 restriction (<0.5 mg vitamin B-6/d). Rates of reactions related to cysteine metabolism were measured from blood sampled during primed, constant infusions of [(13)C(5)]methionine, [3-(13)C]serine, and [(2)H(2)]cysteine that were conducted after an overnight fast at baseline and after the dietary protocol. Vitamin B-6 restriction reduced the concentration of PLP (55.1+/- 8.3 vs. 22.6+/-1.3 nmol/L; P=0.004) and increased concentrations of cystathionine (124%; P<0.001) and total glutathione (38%; P<0.008) in plasma. Concentrations of plasma homocysteine, cysteine, cysteinylglycine, and C-reactive protein (an indicator of systemic inflammation) were not affected by dietary vitamin B-6 restriction. The rate of cysteine synthesis via transsulfuration was below detection limits in this protocol. Neither the fractional synthesis rate of cystathionine nor whole-body cysteine flux was affected by vitamin B-6 restriction. These data indicate that glutathione homeostasis is altered by dietary vitamin B-6 deficiency and appears to be unrelated to cysteine flux under conditions of minimal amino acid intake as evaluated in this study.

The transsulfuration pathway, which aids in regulating homocysteine concentration and mediates cysteine synthesis, may be sensitive to vitamin B-6 status because cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CGL) require pyridoxal 5'-phosphate (PLP). To assess relations between vitamin B-6 and transsulfuration, we evaluated the effects of dietary pyridoxine (PN) on the hepatic concentration of relevant metabolites and in vitro activity of CBS and CGL. Growing rats were fed AIN-93G- or AIN-76A-based diets that ranged from adequate to deficient in vitamin B-6 (2, 1, 0.5, 0.1, or 0 mg of PN/kg diet, n = 5). This design allowed assessment of the effects of supplemental methionine (AIN-76A) vs. cysteine (AIN-93G) in common research diets over a range of vitamin B-6 levels. CBS activity, assayed in the presence or absence of added S-adenosylmethionine, was independent of diet type and PN level. CGL activity was independent of diet type but proportional to dietary PN. Rats fed deficient (0 and 0.1 mg PN/kg) diets exhibited only approximately 30% of the CGL activity of those fed the 2 mg PN/kg diets. Hepatic cystathionine increased from 20 to 30 nmol/g for the 1-2 mg PN/kg diets to approximately 85 nmol/g for the 0 mg PN/kg diet; however, cysteine was reduced only in B-6-deficient rats consuming the AIN-93G diet (means of 30-40 nmol/g for adequate to 11.6 nmol/g for 0 mg PN/kg AIN-76A diet). In spite of these effects, hepatic glutathione concentration increased in vitamin B-6 deficiency. These results suggest that vitamin B-6-dependent changes in transsulfuration do not limit hepatic glutathione production.

Background: The effects of vitamin B-6 status on steady-state kinetics of homocysteine metabolism in humans are unclear.Objective: The objective was to determine the effects of dietary vitamin B-6 restriction on the rates of homocysteine remethylation and synthesis in healthy humans.Design: Primed, constant infusions of [C-13(5)]methionine, [3-C-13] serine, and [H-2(3)]leucine were conducted in healthy female (n = 5) and male (n = 4) volunteers (20-30 y) before and after 4 wk of dietary vitamin B-6 restriction (< 0.5 mg vitamin B-6/d) to establish whether vitamin B-6 status affects steady-state kinetics of homocysteine metabolism in the absence of concurrent methionine intake. Effects of dietary vitamin B-6 restriction on vitamin B-6 status, plasma amino acid concentrations, and the rates of reactions of homocysteine metabolism were assessed.Results: Dietary vitamin B-6 restriction significantly reduced plasma pyridoxal 5 '-phosphate (PLP) concentrations (55.1 +/- 8.3 compared with 22.6 +/- 1.3 nmol/L; P = 0.004), significantly increased plasma glycine concentrations (230 +/- 14 compared with 296 +/- 15; P = 0.008), and significantly reduced basal (43%; P < 0.001) and PLP-stimulated (35%; P = 0.004) lymphocyte serine hydroxymethyltransferase activities in vitro. However, the in vivo fluxes of leucine, methionine, and serine; the rates of homocysteine synthesis and remethylation (total and vitamin B-6-dependent); and the concentrations of homocysteine, methionine, and serine in plasma were not significantly affected by dietary vitamin B-6 restriction.Conclusions: Moderate vitamin B-6 deficiency does not significantly alter the rates of homocysteine remethylation or synthesis in healthy young adults in the absence of dietary methionine intake.

The methylenetetrahydrofolate reductase (MTHFR) 677C-->T polymorphism is thought to influence the partitioning of 1-carbon units between methylation and other components of 1-carbon metabolism and to influence the risk and etiology of several major cancers and cardiovascular disease. Our objective was to determine the effect of the MTHFR 677C-->T polymorphism and folate status on the relative rate and extent of in vivo synthesis of DNA precursors. Adequately nourished, healthy women (9 CC, 9 TT) were infused with [3-(13)C]serine and [(13)C(5)]methionine for 9 h before and after 7 wk of consumption of a low-folate diet. Blood was drawn over 5 d for monocyte DNA isolation. Isotopic enrichment of the nucleosides in DNA digests was determined by LC-MS/MS. Maximum thymidine enrichment tended to be higher (P = 0.07) in TT than in CC subjects, suggestive of marginally higher mean thymidylate synthesis. However, the subset of TT subjects who exhibited formyltetrahydrofolate in erythrocytes (an indicator of 1-carbon partitioning) had greater (P = 0.036) thymidine enrichment than CC subjects, who had no erythrocyte formyltetrahydrofolate. Purine enrichment was not affected by genotype or folate depletion. However, the deoxyadenosine to deoxyguanosine enrichment ratio was significantly higher in TT subjects, suggesting a greater relative rate of adenine synthesis. The approximately 40% greater (P = 0.012) labeling of the methyl group of methyldeoxycytidine during folate depletion suggests a change in the origin of this 1-carbon unit. This is the first time that 1-carbon incorporation into human DNA has been measured in vivo after infusion of (13)C-labeled 1-carbon precursors. These findings support the feasibility of further assessment of factors affecting deoxynucleotide synthesis and DNA methylation in human 1-carbon metabolism.

The effects of folate status and the methylenetetrahydrofolate reductase (MTHFR) 677C→T polymorphism on the kinetics of homocysteine metabolism are unclear. We measured the effects of dietary folate restriction on the kinetics of homocysteine remethylation and synthesis in healthy women (20–30 y old) with the MTHFR 677 C/C or T/T genotypes (n = 9/genotype) using i.v. primed, constant infusions of [13C5]methionine, [3-13C]serine, and [2H3]leucine before and after 7 wk of dietary folate restriction (115 μg dietary folate equivalents/d). Dietary folate restriction significantly reduced folate status (∼65% reduction in serum folate) in both genotypes. Total remethylation flux was not affected by dietary folate restriction, the MTHFR 677C→T polymorphism, or their combination. However, the percentage of remethylation from serine was reduced ∼15% (P = 0.031) by folate restriction in C/C subjects. Further, homocysteine synthesis rates of T/T subjects and folate-restricted C/C subjects were twice that of C/C subjects at baseline. In conclusion, elevated homocysteine synthesis is a cause of mild hyperhomocysteinemia in women with marginal folate status, particularly those with the MTHFR 677 T/T genotype.

Whether folate status and the methylenetetrahydrofolate reductase (MTHFR) 677C→T polymorphism interact to affect methionine-cycle metabolite concentrations is uncertain. We evaluated the effects of dietary folate restriction on relations among folate status indices and plasma concentrations of methionine cycle metabolites in women with the MTHFR 677 C/C and T/T genotypes. Healthy, normohomocysteinemic women (n = 18; 20–30 y old) of adequate B vitamin status, and equally divided according to MTHFR 677C→T genotype (9 C/C and 9 T/T) were recruited. Folate status indices and methionine cycle metabolites were measured in blood samples collected at baseline and after 7 wk of dietary folate restriction (115 μg dietary folate equivalents/d). Significant negative correlations between plasma total homocysteine concentrations and total or 5-methyl folate concentrations (P = 0.041 and 0.023, respectively) in RBCs were found only in T/T subjects. Formylated folates were detected in RBCs of T/T subjects only, and their abundance was predictive of plasma total homocysteine concentration despite no significant alteration by folate restriction. Plasma concentrations of S-adenosylmethionine and S-adenosylhomocysteine were not significantly affected by dietary folate restriction and the MTHFR 677 T/T genotype. In conclusion, plasma total homocysteine concentrations in subjects with the MTHFR 677 T/T genotype were inversely related to 5-methyl folate concentrations and directly related to formylated folate concentrations in RBCs, even though the latter were not significantly affected by moderate folate restriction.

The addition of folic acid to foods in the United States, Canada and several other countries has yielded improved folate nutritional status and reduced the incidence of neural tube defects. In spite of this success, a number of questions remain regarding the bioavailability of natural and added folates and the level of folate intake from dietary sources needed to meet nutritional requirements. Many aspects of current folate requirements are being expressed in terms of dietary folate equivalents, in which an adjustment is employed to account for differences in mean bioavailability of natural and added forms of folate. In this review, we discuss the scientific rationale behind such assumptions and research needs in the broad area of folate bioavailability. We also consider the merits of fortification strategies, including the biofortification of foods.

Cysteine synthesis from homocysteine is catalyzed by two pyridoxal 5 9 -phosphate (PLP)-dependent enzymes. This suggests that vitamin B-6 status might affect cysteine and glutathione homeostasis, but it is unclear whether this occurs in humans. We assessed the effects of vitamin B-6 status on static and kinetic parameters of cysteine and glutathione metabolism in healthy female ( n ¼ 5) and male ( n ¼ 4) volunteers (20–30 y) before and after 4 wk of dietary vitamin B-6 restriction ( , 0.5 mg vitamin B-6/d). Rates of reactions related to cysteine metabolism were measured from blood sampled during primed, constant infusions of [ 13 C 5 ]methionine, [3- 13 C]serine, and [ 2 H 2 ]cysteine that were conducted after an overnight fast at baseline and after the dietary protocol. Vitamin B-6 restriction reduced the concentration of PLP (55.1 6 8.3 vs. 22.6 6 1.3 nmol/L; P ¼ 0.004) and increased concentrations of cystathionine (124%; P , 0.001) and total glutathione (38%; P , 0.008)in plasma. Concentrations of plasma homocysteine, cysteine, cysteinylglycine, and C-reactive protein (an indicator of systemic inflammation) were not affected by dietary vitamin B-6 restriction. The rate of cysteine synthesis via transsulfuration was below detection limits in this protocol. Neither the fractional synthesis rate of cystathionine nor whole-body cysteine flux was affected by vitamin B-6 restriction. These data indicate that glutathione homeostasis is altered by dietary vitamin B-6 deficiency and appears to be unrelated to cysteine flux under conditions of minimal amino acid intake as evaluated in this study. J. Nutr. 136: 373–378, 2006.

Hyperhomocysteinemia in humans is associated with genetic variants of several enzymes of folate and one-carbon metabolism and deficiencies of folate and vitamins B12 and B6. In each case, hyperhomocysteinemia might be caused by diminished folate-dependent homocysteine remethylation, but this has not been confirmed in vivo. Because published stable isotopic tracer approaches cannot distinguish folate-dependent from folate-independent remethylation, we developed a dual-tracer procedure in which a [U-13C5]-methionine tracer is used in conjunction with a [3-13C]serine tracer to simultaneously measure rates of total and folate-dependent homocysteine remethylation. In young female subjects, plasma [U-13C4]homocysteine enrichment, a surrogate measure of intracellular [U-13C5]methionine enrichment, reached approximately 90% of the plasma [U-13C5]methionine enrichment. Methionine-methyl and -carboxyl group fluxes were in the range of previous reports (approximately 25 and approximately 17 micromol.kg(-1).h(-1), respectively). However, the rate of overall homocysteine remethylation (approximately 8 micromol.kg(-1).h(-1)) was twice that of previous reports, which suggests a larger role for homocysteine remethylation in methionine metabolism than previously thought. By use of estimates of intracellular [3-13C]serine enrichment based on a conservative correction of plasma [3-13C]serine enrichment, serine was calculated to contribute approximately 100% of the methyl groups used for total body homocysteine remethylation under the conditions of this protocol. This contribution represented only a small fraction (approximately 2.8%) of total serine flux. Our dual-tracer procedure is well suited to measure the effects of nutrient deficiencies, genetic polymorphisms, and other metabolic perturbations on homocysteine synthesis and total and folate-dependent homocysteine remethylation.

Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) are important for homocysteine remethylation. This study was designed to determine the influence of genetic variants (MTHFR 677C-->T, MTHFR 1298A-->C, and MTRR 66A-->G), folate, and vitamin B-12 status on plasma homocysteine in women (20-30 y; n = 362). Plasma homocysteine was inversely (P < 0.0001) associated with serum folate and plasma vitamin B-12 regardless of genotype. Plasma homocysteine was higher (P < 0.05) for women with the MTHFR 677 TT/1298 AA genotype combination compared with the CC/AA, CC/AC, and CT/AA genotypes. Women with the MTHFR 677 TT/MTRR 66 AG genotype had higher (P < 0.05) plasma homocysteine than all other genotype combinations except the TT/AA and TT/GG genotypes. There were 5.4-, 4.3-, and 3.8-fold increases (P < 0.001) in risk for plasma homocysteine in the top 5, 10, and 20%, respectively, of the homocysteine distribution for subjects with the MTHFR 677 TT compared with the CC and CT genotypes. Predicted plasma homocysteine was inversely associated with serum folate (P = 0.003) and plasma vitamin B-12 (P = 0.002), with the degree of correlation dependent on MTHFR 677C-->T genotype. These data suggest that coexistence of the MTHFR 677 TT genotype with the MTRR 66A-->G polymorphism may exacerbate the effect of the MTHFR variant alone. The potential negative effect of combined polymorphisms of the MTHFR and MTRR genes on plasma homocysteine in at-risk population groups with low folate and/or vitamin B-12 status, such as women of reproductive potential, deserves further investigation.

Little is known about homocysteine metabolism in intestine. To address this question, we investigated homocysteine metabolism under conditions of folate adequacy and folate deprivation in the Caco-2 cell line, a model of human intestinal mucosal cells. Caco-2 cells were cultured in media enriched with [3-(13)C]serine and [U-(13)C(5)]methionine tracers, and enrichments of intracellular free amino acid pools of these amino acids as well as homocysteine, cystathionine, and cysteine were measured by using gas chromatography/mass spectrometry. Homocysteine transsulfuration plus folate-dependent and total remethylation were quantified from these amino acid enrichments. Homocysteine remethylation accounted for 19% of the intracellular free methionine pool in cells cultured with supplemental folate, and nearly all one-carbon units used for remethylation originated from the three carbon of serine via folate-dependent remethylation. Labeling of cystathionine and cysteine indicated the presence of a complete transsulfuration pathway in Caco-2 cells, and this pathway produced 13% of the intracellular free cysteine pool. Appearance of labeled homocysteine and cystathionine in culture medium suggests export of these metabolites from intestinal cells. Remethylation was reduced by one-third in folate-restricted cell cultures (P < 0.001), and only approximately 50% of the one-carbon units used for remethylation originated from the three carbon of serine under these conditions. In conclusion, the three carbon of serine is the primary source of one-carbon units used for homocysteine remethylation in folate-supplemented Caco-2 cell cultures. Remethylation is reduced as a result of folate restriction in this mucosal cell model, and one-carbon sources other than the three carbon of serine contribute to remethylation under this condition.