, respectively). However, the rate of overall homocysteine remethylation (ϳ8 mol⅐kg Ϫ1 ⅐h Ϫ1) 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 [3-13 C]serine enrichment based on a conservative correction of plasma [3-13 C]serine enrichment, serine was calculated to contribute ϳ100% of the methyl groups used for total body homocysteine remethylation under the conditions of this protocol. This contribution represented only a small fraction (ϳ2.8%) 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. methionine; methylation cycle; cystathionine ELEVATED PLASMA HOMOCYSTEINE CONCENTRATION is considered an independent risk factor for the development of cardiovascular disease (5,30,32). Accordingly, many investigators have sought to define the genetic and environmental factors that affect plasma homocysteine concentration. Associations exist between plasma homocysteine concentrations and gene polymorphisms, as well as lifestyle and other environmental factors (5). Strong evidence implicates nutritional deficiencies of folate, vitamins B 6 and B 12 , and the methylenetetrahydrofolate reductase (MTHFR) 677C 3 T polymorphism as causes of elevated plasma homocysteine concentration (12, 25). Folate and vitamin B 6 deficiencies and the MTHFR 677C 3 T polymorphism are thought to increase circulating homocysteine concentrations by decreasing the availability of 5-methyltetrahydrofolate (5-CH 3 THF) and thereby inhibiting homocysteine remethylation (24). However, a causal relationship between reduced homocysteine remethylation and hyperhomocysteinemia under these conditions has not been confirmed in humans in vivo.Steady-state plasma homocysteine concentration is not solely a function of the rate of its removal by remethylation but is also affected by the rates of homocysteine production, catabolism through transsulfuration, and loss in renal excretion (24). Specific measurements of homocysteine metabolism through these individual pathways are needed to clarify why homocysteine concentration is elevated by particular genetic variations as well as by nutritional and other environmental conditions. To test the hypothesis that homocysteine remethylation is compromised when 5-CH 3 THF availability is reduced, homocysteine remethylation rates must be measured in individuals affected by these homocysteineelevating factors.Remethylation rates have been measured in humans in vivo by use of methionine tracers labeled with stable isotopes at both the carboxyl and methyl groups or else by measurements of separate methyl-and carboxyl-labeled methionine tracers in primed constant infusion experiments (18,26). By use of these methodologies, the effects of dietary sulfur amino acid intake, sex, age, prandial statu...
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 hydroxymethyltransferase (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-(13)C(2)]glycine and [5,5,5-(2)H(3)]leucine to quantify in vivo kinetics in healthy adults (7 males, 6 females; 20-39 y) of normal vitamin B-6 status or marginal vitamin B-6 deficiency. Vitamin B-6 restriction lowered the plasma PLP concentration from 55 +/- 4 nmol/L (mean +/- SEM) to 23 +/- 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 +/- 7 to 205 +/- 9 micromol x kg(-1) x h(-1) (P < 0.05), but serine production using a GCS-derived 1-carbon unit (93 +/- 9 vs. 91 +/- 6 micromol x kg(-1) x h(-1)) and glycine cleavage (163 +/- 11 vs. 151 +/- 8 micromol x kg(-1) x h(-1)) were not changed by vitamin B-6 restriction. The GCS produced 1-carbon units at a rate (approximately 140-170 micromol x kg(-1) x h(-1)) that greatly exceeds the demand for remethylation and transmethylation processes (approximately 4-7 micromol x kg(-1) x h(-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.
5-Formyltetrahydrofolate (5-CHO-THF) is formed viaa second catalytic activity of serine hydroxymethyltransferase (SHMT) and strongly inhibits SHMT and other folate-dependent enzymes in vitro. The only enzyme known to metabolize 5-CHO-THF is 5-CHO-THF cycloligase (5-FCL), which catalyzes its conversion to 5,10-methenyltetrahydrofolate. Because 5-FCL is mitochondrial in plants and mitochondrial SHMT is central to photorespiration, we examined the impact of an insertional mutation in the Arabidopsis 5-FCL gene (At5g13050) under photorespiratory (30 and 370 mol of CO 2 mol ؊1 ) and non-photorespiratory (3200 mol of CO 2 mol ؊1 ) conditions. The mutation had only mild visible effects at 370 mol of CO 2 mol ؊1 , reducing growth rate by ϳ20% and delaying flowering by 1 week. However, the mutation doubled leaf 5-CHO-THF level under all conditions and, under photorespiratory conditions, quadrupled the pool of 10-formyl-/5,10-methenyltetrahydrofolates (which could not be distinguished analytically). At 370 mol of CO 2 mol ؊1 , the mitochondrial 5-CHO-THF pool was 8-fold larger in the mutant and contained most of the 5-CHO-THF in the leaf. In contrast, the buildup of 10-formyl-/5,10-methenyltetrahydrofolates was extramitochondrial. In photorespiratory conditions, leaf glycine levels were up to 46-fold higher in the mutant than in the wild type. Furthermore, when leaves were supplied with 5-CHO-THF, glycine accumulated in both wild type and mutant. These data establish that 5-CHO-THF can inhibit SHMT in vivo and thereby influence glycine pool size. However, the near-normal growth of the mutant shows that even exceptionally high 5-CHO-THF levels do not much affect fluxes through SHMT or any other folate-dependent reaction, i.e. that 5-CHO-THF is well tolerated in plants. 5-Formyltetrahydrofolate (5-CHO-THF)1 is formed from 5,10-methenyltetrahydrofolate (5,10-CHϭTHF) by a hydrolytic reaction catalyzed by serine hydroxymethyltransferase (SHMT) in the presence of glycine (1, 2). Spontaneous chemical hydrolysis of 5,10-CHϭTHF may be a minor additional source (3). 5-CHO-THF is the most stable natural folate and the most enigmatic, for it is the only one that does not serve as a cofactor in one-carbon metabolism. Instead, 5-CHO-THF is a potent inhibitor of SHMT and most other folate-dependent enzymes in vitro (4, 5). 5-CHO-THF probably acts as a stable storage form of folate in seeds and fungal spores (5-7), but it is not clear what role, if any, it plays in metabolically active tissues (8). This question is particularly pertinent for leaves. Leaf mitochondria have very high levels of SHMT and, during photorespiration, receive a massive influx of glycine (which leads to a matching SHMT-mediated glycine 3 serine flux) (9). Conditions in leaf mitochondria therefore favor 5-CHO-THF formation (Fig. 1). Indeed, 5-CHO-THF can comprise 50% of the folate pool in leaf mitochondria (10, 11), which is far more than in mammalian mitochondria (12)(13)(14). Furthermore, 5-CHO-THF is reported to make up 14 -40% of the folate pool in leaves and o...
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.
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 micromol x kg(-1) x h(-1) [1,2-(13)C2]glycine and 1.87 micromol x kg(-1) x h(-1) [(2)H3]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)CO2 enrichment, were determined for use in kinetic analysis. Serine synthesis by direct conversion from glycine via SHMT occurred at 193 +/- 28 micromol x kg(-1) x h(-1) (mean +/- SEM), which comprised 41% of the 463 +/- 55 micromol x kg(-1) x h(-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)CO2 measurement, glycine decarboxylation (190 +/- 41 micromol x kg(-1) x h(-1)) accounted for 39 +/- 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.
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