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Glycine undergoes decarboxylation in the glycine cleavage system (GCS) to yield C[O.sub.2], N[H.sub.3], and a 1-carbon unit. C[O.sub.2] also can be generated from the 2-carbon of glycine by 10-formyltetrahydrofolate-dehydrogenase and, after glycine-to-serine conversion by serine hydroxymethyltransferase, from the tricarboxylic acid cycle. To evaluate the relative fates of glycine carbons in C[O.sub.2] generation in healthy volunteers (3 male, 3 female, aged 21-26 y), primed, constant infusions were conducted using 9.26 [micro]mol x [h.sup.-1] x [kg.sup.-1] of [1,2-[sup.13]C]glycine and 1.87 [micro]mol x [h.sup.-1] x [kg.sup.-1] of [5,5,5-[sup.2][H.sub.3]]leucine, followed by an infusion protocol using [1-[sup.13]C]glycine as the glycine tracer. The time period between the infusion protocols was >6 mo. In vivo rates of whole-body glycine and leucine flux were nearly identical in protocols with [1,2-[sup.13]C]glycine and [5,5,5-[sup.2][H.sub.3]]leucine and with [1-[sup.13]C]glycine and [5,5,5-[sup.2][H.sub.3]]leucine tracers, which showed high reproducibility between the tracer protocols. Using the [1-[sup.13]C]glycine tracer, breath C[O.sub.2] data showed a total rate of glycine decarboxylation of 96 [+ or -] 8 [micro]mol x [h.sup.-1] x [kg.sup.-1], which was 22 [+ or -] 3% of whole-body glycine flux. In contrast, infusion of [1,2-[sup.13]C]glycine yielded a glycine-to-C[O.sub.2] flux of 146 [+ or -] 37 [micro]mol x [h.sup.-1] x [kg.sup.-1] (P = 0.026). By difference, this implies a rate of C[O.sub.2] formation from the glycine 2-carbon of 51 [+ or -] 40 [micro]mol x. [h.sup.-1] x [kg.sup.-1], which accounts for ~35% of the total C[O.sub.2] generated in glycine catabolism. These findings also indicate that ~65% of the C[O.sub.2] generation from glycine occurs by decarboxylation, primarily from the GCS. Further, these results suggest that the GCS is responsible for the entry of 5,10-methylenetetrahydrofolate into 1-carbon metabolism at a very high rate (~96 [micro]mol x [h.sup.-1 x [kg.sup.-1]), which is ~20 times the demand for methyl groups for homocysteine remethylation.

Glycine is a precursor of purines, protein, glutathione, and 1-carbon units as 5,10-methylenetetrahydrofolate. Glycine decarboxylation through the glycine cleavage system (GCS) and glycine-serine transformation by serine hydroxymethyl- transferase (SHMT) require pyridoxal 5'-phosphate (PLP; active form of vitamin B-6) as a coenzyme. The intake of vitamin B-6 is frequently low in humans. Therefore, we determined the effects of vitamin B-6 restriction on whole-body glycine flux, the rate of glycine decarboxylation, glycine-to-serine conversion, use of glycine carbons in nucleoside synthesis, and other aspects of 1-carbon metabolism We used a primed, constant infusion of [1,2-[sup.13][C.sub.2]glycine and [5,5,5- [sup.2][H.sub.3]leucine to quantify in vivo kinetics in healthy adults (7 males, 6 females; 20-39 y) of normal vitamin B-6 status of marginal vitamin B-6 deficiency. Vitamin B-6 restriction lowered the plasma PLP concentration from 55 [+ or -] 4 nmol/L (mean [+ or -] SEM) to 23 [+ or -] 1 nmol/L (P < 0.0001), which is consistent with marginal deficiency, whereas the plasma glycine concentration increased (P< 0.01 ). SHMT-mediated conversion of glycine to serine increased from 182 [+ or -] 7 to 205 [+ or -] 9 [micro]mol x [kg.sup.-1] x [h.sup.-1] (P < 0.05), but serine production using a GCS-derived 1-carbon unit (93 [+ or -] 9 vs. 91 [+ or -] 6 [micro]mol x [kg.sup.-1] x [h.sup.-1]) and glycine cleavage (163 [+ or -] 11 vs. 151 [+ or -] 8 [micro]mol x [kg.sup.-1] x [h.sup.-1]) were not changed by vitamin B-6 restriction. The GCS produced 1- carbon units at a rate (~140-170 [micro]mol x [kg.sup.-1] x [h.sup.-1]) that greatly exceeds the demand for remethylation and transmethylation processes (~4-7 [micro]mol x [kg.sup.-1] x [h.sup.-1]). We conclude that the in vivo GCS and SHMT reactions are quite resilient to the effects of marginal vitamin B-6 deficiency, presumably through a compensatory effect of increasing substrate concentration.

Glycine plays several roles in human metabolism, e.g. as a 1-carbon donor, in purine synthesis, and as a component of glutathione. Glycine is decarboxylated via the glycine cleavage system (GCS) that yields concurrent generation of a 1 -carbon unit as 5,10-methylenetetrahydrofolate (methyleneTHF). Serine hydroxymethyltransferase (SHMT) catalyzes the interconversion of glycine and serine, another 1-carbon donor. The quantitative role of glycine in human 1-carbon metabolism has received little attention. The aim of this protocol was to quantify whole body glycine flux, glycine to serine flux, and rate of glycine cleavage in humans. A primed, constant infusion with 9.26 [micro] mol.[kg.sup.-1] [h.sup.-1] [[1,2-.sup.13] [C.sub.2] ]glycine and 1.87 [micro] mol.[kg.sup.- 1] [h.sup.-1] [[2.sup.H.sub.3]]leucine was used to quantify the kinetic behavior of glycine in young, healthy volunteers (n = 5) in a fed state. The isotopic enrichment of infused tracers and metabolic products in plasma, as well as breath [13.sup.C][O.sub.2] enrichment, were determined for use in kinetic analysis. Serine synthesis by direct conversion from glycine via SHMT occurred at 193 [+ or -] 28 [micro] mol.[kg.sup.-1]. [h.sup.-1] (mean [+ or -] SEM), which comprised 41% of the 463 [+ or -] 55 [micro] mol.[kg.-1] [h.sup.-1] total glycine flux. Nearly one-half (46%) of the glycine-to-serine conversion occurred using GCS-derived methyleneTHF 1-carbon units. Based on breath [[13.sup.C][O.sub.2] measurement, glycine decarboxylation (190 [+ or -] 41 [micro] mol.[kg.sub.-1] [h.sup.-1]) accounted for 39 [+ or -] 6% of whole body glycine flux. This study is the first to our knowledge to quantify human glycine cleavage and glycine-to-serine SHMT kinetics. GCS is responsible for a substantial proportion of whole body glycine flux and constitutes a major route for the generation of 1-carbon units. J. Nutr. 137: 2647-2652, 2007.

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 ~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 ~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.

In a 10-wk study with nonpregnant women (21-27 y, n = 5-6 per group), subjects were fed a diet containing [approximately]68 nmol/d (30 [[micro]gram]/d) folate from food that was supplemented with folic acid in apple juice to yield a constant intake of 454, 680 or 907 nmol/d (200, 300 or 400 [[micro]gram]/d) to evaluate folate status and long-term in vivo kinetics. Reported here is an additional phase of this protocol conducted to determine the relationship between short-term urinary excretion after a single isotopically labeled dose and various measures of folate nutritional status. It was hypothesized that urinary excretion from a single [glutamate-2H4]folic acid ([2H4]folic acid) dose would increase in proportion to folate nutritional status due to saturable cellular uptake and retention processes along with saturation of renal reabsorption. Each subject was given 1.13 [micro]mol (500 [[micro]gram]) of [2H4]folic acid orally on the morning of d 70 of the study, followed by a complete 24-h urine collection. Urine was analyzed to determine the isotopic enrichment of urinary folate by gas chromatography-mass spectrometry and the concentration of urinary folate by HPLC. Urinary excretion of [2H4]folate was greatest at the 907 nmol/d intake and was positively correlated with serum folate concentration but was not correlated with erythrocyte folate. Excretion of [2H4]folate tended to be greatest when plasma homocysteine concentrations were low ( KEY WORDS: * folate * urinary excretion * homocysteine * stable isotopes * humans

Vitamin B-6 deficiency causes mild elevation in plasma homocysteine, but the mechanism has not been clearly established. Serine is a substrate in one-carbon metabolism and in the transsulfuration pathway of homocysteine catabolism, and pyridoxal phosphate (PLP) plays a key role as coenzyme for serine hydroxymethyltransferase (SHMT) and enzymes of transsulfuration. In this study we used [[sup.2][H.sub.3]]serine as a primary tracer to examine the remethylation pathway in adequately nourished and vitamin B-6-deficient rats [7 and 0.1 mg pyridoxine (PN)/kg diet]. [[sup.2][H.sub.3]]Leucine and [1-[sup.13]C]methionine were also used to examine turnover of protein and methionine pools, respectively. All tracers were injected intraperitoneally as a bolus dose, and then rats were killed (n = 4/time point) after 30, 60 and 120 min. Rats fed the low-PN diet had significantly lower growth and plasma and liver PLP concentrations, reduced liver SHMT activity, greater plasma and liver total homocysteine concentration, and reduced liver S-adenosylmethionine concentration. Hepatic and whole body protein turnover were reduced in vitamin B-6-deficient rats as evidenced by greater isotopic enrichment of [[sup.2][H.sub.3]]leucine. Hepatic [[sup.2][H.sub.2]]methionine production from [[sup.2][H.sub.3]]serine via cytosolic SHMT and the remethylation pathway was reduced by 80.6% in vitamin B-6 deficiency. The deficiency did not significantly reduce hepatic cystathionine-[Beta]-synthase activity, and in vivo hepatic transsulfuration flux shown by production of [[sup.2][H.sub.3]]cysteine from the [[sup.2][H.sub.3]serine increased over twofold. In contrast, plasma appearance of [[sup.2][H.sub.3]]cysteine was decreased by 89% in vitamin B-6 deficiency. The rate of hepatic homocysteine production shown by the ratio of [1-[sup.13]C]homocysteine/[1-[sup.13]C]methionine areas under enrichment vs. time curves was not affected by vitamin B-6 deficiency. Overall, these results indicate that vitamin B-6 deficiency substantially affects one-carbon metabolism by impairing both methyl group production for homocysteine remethylation and flux through whole-body transsulfuration. J. Nutr. 130: 1115-1123, 2000. KEY WORDS: * one-carbon metabolism * homocysteine * transsulfuration * vitamin B-6 * rats

In a 10-wk study of folate metabolism in nonpregnant women (21-27 y, n =5-6 per group), subjects were fed a diet containing [approximately]68 nmol/d (30 [[micro]gram]/d) folate from food. The remainder of the ingested folate was provided as folic acid in apple juice (as nonlabeled during wk 1-2, as [2H2]folic acid during wk 3-10) to yield a constant intake of 454, 680 or 907 nmol/d (200, 300 or 400 [[micro]gram]/d). Isotopic enrichment of total urinary folate and the primary catabolite para-acetamidobenzoylglutamate (ApABG) was determined. Isotopic enrichment of ApABG served as an indicator of labeling of tissue folates. A kinetic model consisting of fast- and slow-turnover nonsaturable pools and a saturable slow-turnover pool, with provisions for urinary and fecal excretion, catabolism and enterohepatic circulation, yielded a close fit to the data. Mean residence times for total body folate were 212, 169 and 124 d for folate intakes of 454, 680, and 907 nmol/d, respectively. The model predicted that variation in folate intake over this range had little effect on the mass of the large saturable folate pool; however, the fast-turnover nonsaturable pools increased in proportion to folate intake, whereas the slow nonsaturable pool also tended to increase. This model will aid in evaluation of folate turnover and in predicting kinetic consequences of physiologic conditions associated with altered folate requirements. KEY WORDS: * folate * kinetics * modeling * requirements * stable isotopes * humans