1-carbon Units Research Articles

Platelet Metabolic Profiling Reveals Glycolytic and 1-Carbon Metabolites Are Essential for GP VI-Stimulated Human Platelets-Brief Report.

Evolving evidence suggests that besides signaling pathways, platelet activation involves a complex interplay between metabolic pathways to support thrombus growth. Selective targeting of metabolic checkpoints may inhibit platelet activation and provide a novel antiplatelet strategy. We, therefore, examined global metabolic changes that occur during the transition of human platelets from resting to an activated state to identify metabolites and associated pathways that contribute to platelet activation. We performed metabolic profiling of resting and convulxin-stimulated human platelet samples. The differential levels, pathway analysis, and PCA (principal component analysis) were performed using Metaboanalyst. Metascape was used for metabolite network construction. Of the 401 metabolites identified, 202 metabolites were significantly upregulated, and 2 metabolites were downregulated in activated platelets. Of all the metabolites, lipids scored highly and constituted ≈50% of the identification. During activation, aerobic glycolysis supports energy demand and provides glycolytic intermediates required by metabolic pathways. Consistent with this, an important category of metabolites was carbohydrates, particularly the glycolysis intermediates that were significantly upregulated compared with resting platelets. We found that lysophospholipids such as 1-palmitoyl-GPA (glycero-3-phosphatidic acid), 1-stearoyl-GPS (glycero-3-phosphoserine), 1-palmitoyl-GPI (glycerophosphoinositol), 1-stearoyl-GPI, and 1-oleoyl-GPI were upregulated in activated platelets. We speculated that platelet activation could be linked to 1-carbon metabolism, a set of biochemical pathways that involve the transfer and use of 1-carbon units from amino acids, for cellular processes, including nucleotide and lysophospholipid synthesis. In alignment, based on pathway enrichment and network-based prioritization, the metabolites from amino acid metabolism, including serine, glutamate, and branched-chain amino acid pathway were upregulated in activated platelets, which might be supplemented by the high levels of glycolytic intermediates. Metabolic analysis of resting and activated platelets revealed that glycolysis and 1-carbon metabolism are necessary to support platelet activation.

The role of energy, serine, glycine, and 1-carbon units in the cost of nitrogen excretion in mammals and birds

The efficiency with which dietary protein is used affects the nitrogen excretion by the animal and the environmental impact of animal production. Urea and uric acid are the main nitrogen excretion products resulting from amino acid catabolism in mammals and birds, respectively. Nitrogen excretion can be reduced by using low-protein diets supplemented with free amino acids to ensure that essential amino acids are not limiting performance. However, there are questions whether the capacity to synthesize certain nonessential amino acids is sufficient when low-protein diets are used. This includes glycine, which is used for uric acid synthesis. Nitrogen excretion not only implies a nitrogen and energy loss in the urine, but energy is also required to synthesize the excretion products. The objective of this study was to quantify the energy and metabolic requirements for nitrogen excretion products in the urine. The stoichiometry of reactions to synthesize urea, uric acid, allantoin, and creatinine was established using information from a publicly available database. The energy cost was at least 40.3, 60.7, 64.7, and 65.4 kJ/g excreted N for urea, uric acid, allantoin, and creatinine, respectively, of which 56, 56, 47, and 85% were retained in the excretion product. Data from a broiler study were used to carry out a flux balance analysis for nitrogen, serine, glycine, and so-called 1-carbon units. The flux balance indicated that the glycine intake was insufficient to cover the requirements for growth and uric acid excretion. The serine intake was also insufficient to cover the glycine deficiency, underlining the importance of the de novo synthesis of serine and glycine. One-carbon units are also a component of uric acid and can be synthesized from serine and glycine. There are indications that the de novo synthesis of 1-carbon units may be a “weak link” in metabolism, because of the stoichiometric dependency between the synthesized 1-carbon units and glycine. The capacity to catabolize excess 1-carbon units may be limited, especially in birds fed low-protein diets. Therefore, there may be an upper limit to the 1-carbon-to-glycine requirement ratio in relation to nutrients that supply 1-carbon units and glycine. The ratio can be reduced by increasing uric acid excretion (i.e., reducing protein deposition) or by dietary supplementation with glycine. The hypothesis that the 1-carbon-to-glycine requirement ratio should be lower than the supply ratio provides a plausible explanation for the growth reduction in low-protein diets and the positive response to the dietary glycine supply.

Glycine Enhances Satellite Cell Proliferation, Cell Transplantation, and Oligonucleotide Efficacy in Dystrophic Muscle.

The need to distribute therapy evenly systemically throughout the large muscle volume within the body makes Duchenne muscular dystrophy (DMD) therapy a challenge. Cell and exon-skipping therapies are promising but have limited effects, and thus enhancing their therapeutic potency is of paramount importance to increase the accessibility of these therapies to DMD patients. In this study, we demonstrate that co-administered glycine improves phosphorodiamidate morpholino oligomer (PMO) potency in mdx mice with marked functional improvement and an up to 50-fold increase of dystrophin in abdominal muscles compared to PMO in saline. Glycine boosts satellite cell proliferation and muscle regeneration by increasing activation of mammalian target of rapamycin complex 1 (mTORC1) and replenishing the one-carbon unit pool. The expanded regenerating myofiber population then results in increased PMO uptake. Glycine also augments the transplantation efficiency of exogenous satellite cells and primary myoblasts in mdx mice. Our data provide evidence that glycine enhances satellite cell proliferation, cell transplantation, and oligonucleotide efficacy in mdx mice, and thus it has therapeutic utility for cell therapy and drug delivery in muscle-wasting diseases.

Impaired folate 1-carbon metabolism causes formate-preventable hydrocephalus in glycine decarboxylase-deficient mice.

Ventriculomegaly and hydrocephalus are associated with loss of function of glycine decarboxylase (Gldc) in mice and in humans suffering from non-ketotic hyperglycinemia (NKH), a neurometabolic disorder characterized by accumulation of excess glycine. Here, we showed that ventriculomegaly in Gldc-deficient mice is preceded by stenosis of the Sylvian aqueduct and malformation or absence of the subcommissural organ and pineal gland. Gldc functions in the glycine cleavage system, a mitochondrial component of folate metabolism, whose malfunction results in accumulation of glycine and diminished supply of glycine-derived 1-carbon units to the folate cycle. We showed that inadequate 1-carbon supply, as opposed to excess glycine, is the cause of hydrocephalus associated with loss of function of the glycine cleavage system. Maternal supplementation with formate prevented both ventriculomegaly, as assessed at prenatal stages, and postnatal development of hydrocephalus in Gldc-deficient mice. Furthermore, ventriculomegaly was rescued by genetic ablation of 5,10-methylene tetrahydrofolate reductase (Mthfr), which results in retention of 1-carbon groups in the folate cycle at the expense of transfer to the methylation cycle. In conclusion, a defect in folate metabolism can lead to prenatal aqueduct stenosis and resultant hydrocephalus. These defects are preventable by maternal supplementation with formate, which acts as a 1-carbon donor.

Structural basis of inhibition of the human serine hydroxymethyltransferase SHMT2 by antifolate drugs.

Serine hydroxymethyltransferase (SHMT) is the major source of 1-carbon units required for nucleotide synthesis. Humans have cytosolic (SHMT1) and mitochondrial (SHMT2) isoforms, which are upregulated in numerous cancers, making the enzyme an attractive drug target. Here, we show that the antifolates lometrexol and pemetrexed are inhibitors of SHMT2 and solve the first SHMT2-antifolate structures. The antifolates display large differences in their hydrogen bond networks despite their similarity. Lometrexol was found to be the best hSHMT1/2 inhibitor from a panel antifolates. Comparison of apo hSHMT1 with antifolate bound hSHMT2 indicates a highly conserved active site architecture. This structural information offers insights as to how these compounds could be improved to produce more potent and specific inhibitors of this emerging anti-cancer drug target.

Evaluation of Oxidative Stress Parameters and Energy Metabolism in Cerebral Cortex of Rats Subjected to Sarcosine Administration.

Sarcosine is an N-methyl derivative of the amino acid glycine, and its elevation in tissues and physiological fluids of patients with sarcosinemia could reflect a deficient pool size of activated 1-carbon units. Sarcosinemia is a rare inherited metabolic condition associated with mental retardation. In the present study, we investigated the acute effect of sarcosine and/or creatine plus pyruvate on some parameters of oxidative stress and energy metabolism in cerebral cortex homogenates of 21-day-old Wistar rats. Acute administration of sarcosine induced oxidative stress and diminished the activities of adenylate kinase, GAPDH, complex IV, and mitochondrial and cytosolic creatine kinase. On the other hand, succinate dehydrogenase activity was enhanced in cerebral cortex of rats. Moreover, total sulfhydryl content was significantly diminished, while DCFH oxidation, TBARS content, and activities of SOD and GPx were significantly enhanced by acute administration of sarcosine. Co-administration of creatine plus pyruvate was effective in the prevention of alterations provoked by sarcosine administration on the oxidative stress and the enzymes of phosphoryltransfer network. These results indicate that acute administration of sarcosine may stimulate oxidative stress and alter the energy metabolism in cerebral cortex of rats. In case these effects also occur in humans, they may contribute, along with other mechanisms, to the neurological dysfunction of sarcosinemia, and creatine and pyruvate supplementation could be beneficial to the patients.

Moderate Vitamin B-6 Restriction Does Not Alter Postprandial Methionine Cycle Rates of Remethylation, Transmethylation, and Total Transsulfuration but Increases the Fractional Synthesis Rate of Cystathionine in Healthy Young Men and Women1–3

AbstractMethionine is the precursor for S-adenosylmethionine (SAM), the major 1-carbon donor involved in >100 transmethylation reactions. Homocysteine produced from SAM must be metabolized either by remethylation for recycling of methionine or transsulfuration to form cystathionine and then cysteine. Pyridoxal 5′-phosphate (PLP) serves as a coenzyme in enzymes involved in transsulfuration as well as for primary acquisition of 1-carbon units used for remethylation and other phases of 1-carbon metabolism. Because the intake of vitamin B-6 is frequently low in humans and metabolic consequences of inadequacy may be amplified in the postprandial state, we aimed to determine the effects of marginal vitamin B-6 deficiency on the postprandial rates of remethylation, transmethylation, overall transsulfuration, and cystathionine synthesis. Healthy, young adults (4 male, 5 female; 2035 y) received a primed, constant infusion of [1-13C]methionine, [methyl-2H3]methionine, and [5,5,5-2H3]leucine to quantify in vivo kinetics at normal vitamin B-6 status and after a 28-d dietary vitamin B-6 restriction. Vitamin B-6 restriction lowered the plasma PLP concentration from 49 ± 4 nmol/L (mean ± SEM) to 19 ± 2 nmol/L (P < 0.0001). Mean remethylation, transsulfuration, and transmethylation rates did not change in response to vitamin B-6 restriction; however, the responses to vitamin B-6 restriction varied greatly among individuals. The plasma cystathionine concentration increased from 142 ± 8 to 236 ± 9 nmol/L (P < 0.001), whereas the fractional cystathionine synthesis rate increased by a mean of 12% in 8 of 9 participants. Interrelationships among plasma concentrations of glycine and cystathionine and kinetic results suggest that individual variability occurs in normal postprandial 1-carbon metabolism and in the response to vitamin B-6 restriction.

Mammalian MTHFD2L Encodes a Mitochondrial Methylenetetrahydrofolate Dehydrogenase Isozyme Expressed in Adult Tissues

Previous studies in our laboratory showed that isolated, intact adult rat liver mitochondria are able to oxidize the 3-carbon of serine and the N-methyl carbon of sarcosine to formate without the addition of any other cofactors or substrates. Conversion of these 1-carbon units to formate requires several folate-interconverting enzymes in mitochondria. The enzyme(s) responsible for conversion of 5,10-methylene-tetrahydrofolate (CH(2)-THF) to 10-formyl-THF in adult mammalian mitochondria are currently unknown. A new mitochondrial CH(2)-THF dehydrogenase isozyme, encoded by the MTHFD2L gene, has now been identified. The recombinant protein exhibits robust NADP(+)-dependent CH(2)-THF dehydrogenase activity when expressed in yeast. The enzyme is localized to mitochondria when expressed in CHO cells and behaves as a peripheral membrane protein, tightly associated with the matrix side of the mitochondrial inner membrane. The MTHFD2L gene is subject to alternative splicing and is expressed in adult tissues in humans and rodents. This CH(2)-THF dehydrogenase isozyme thus fills the remaining gap in the pathway from CH(2)-THF to formate in adult mammalian mitochondria.

Update on Folate and Human Health

Folate is an essential B vitamin found naturally in a variety of foods such as leafy vegetables, citrus fruits, liver, and lentils. There are many forms of folate, and folic acid is the form used in folate supplements and food fortification. Folate is involved in many reactions requiring the transfer of 1-carbon units, being essential for both DNA and RNA synthesis. Although folate was first demonstrated to be important for human health more than 70 years ago, only recently have health professionals begun to understand the full range of its physiological functions. For example, several controlled clinical trials have shown that increased consumption of folic acid may decrease the risk of neural tube defects. Other observational studies suggest inverse relationships between folate and risk for a variety of chronic diseases such as cancer and cardiovascular disease; controlled clinical trials to confirm these associations, however, are either in process or have tended not to support a simple cause-and-effect relationship. Assessment of folate status can involve both biochemical and dietary data, and it is important that clinicians understand the appropriate uses and limitations of these assessment tools. Because of the potential public health benefit of higher folate intakes on decreasing risk of neural tube defects, the Food and Drug Administration launched a national folic acid food fortification program that became fully effective in 1998. Dietary and biochemical analysis suggests that folate status has increased since the initiation of this program, although there is concern that folate intake now may be too high in some individuals. Still others argue that folate intake remains too low to achieve the original intended effect. Clearly, continued monitoring of this program is warranted. Although much has been learned about folate during the past 80 years, there is still much to ascertain about the mechanisms by which it exerts its numerous effects on human health

Structure-Function Relationships in Miscoding by Sulfolobus solfataricus DNA Polymerase Dpo4: GUANINE N2,N2-DIMETHYL SUBSTITUTION PRODUCES INACTIVE AND MISCODING POLYMERASE COMPLEXES

Structure-Function Relationships in Miscoding by Sulfolobus solfataricus DNA Polymerase Dpo4: GUANINE N2,N2-DIMETHYL SUBSTITUTION PRODUCES INACTIVE AND MISCODING POLYMERASE COMPLEXES

Production of 1-Carbon Units from Glycine Is Extensive in Healthy Men and Women

Glycine undergoes decarboxylation in the glycine cleavage system (GCS) to yield CO2, NH3, and a 1-carbon unit. CO2 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 CO2 generation in healthy volunteers (3 male, 3 female, aged 21–26 y), primed, constant infusions were conducted using 9.26 μmol·h−1·kg−1 of [1,2-13C]glycine and 1.87 μmol·h−1·kg−1 of [5,5,5-2H3]leucine, followed by an infusion protocol using [1-13C]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 [1,2-13C]glycine and [5,5,5-2H3]leucine and with [1-13C]glycine and [5,5,5-2H3]leucine tracers, which showed high reproducibility between the tracer protocols. Using the [1-13C]glycine tracer, breath CO2 data showed a total rate of glycine decarboxylation of 96 ± 8 μmol·h−1·kg−1, which was 22 ± 3% of whole-body glycine flux. In contrast, infusion of [1,2-13C]glycine yielded a glycine-to-CO2 flux of 146 ± 37 μmol·h−1·kg−1 (P = 0.026). By difference, this implies a rate of CO2 formation from the glycine 2-carbon of 51 ± 40 μmol·h−1·kg−1, which accounts for ∼35% of the total CO2 generated in glycine catabolism. These findings also indicate that ∼65% of the CO2 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 μmol·h−1·kg−1), which is ∼20 times the demand for methyl groups for homocysteine remethylation.

Moderate Dietary Vitamin B-6 Restriction Raises Plasma Glycine and Cystathionine Concentrations While Minimally Affecting the Rates of Glycine Turnover and Glycine Cleavage in Healthy Men and Women

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 [1,2-13C2]glycine and [5,5,5-2H3]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 μmol·kg−1·h−1 (P < 0.05), but serine production using a GCS-derived 1-carbon unit (93 ± 9 vs. 91 ± 6 μmol·kg−1·h−1) and glycine cleavage (163 ± 11 vs. 151 ± 8 μmol·kg−1·h−1) were not changed by vitamin B-6 restriction. The GCS produced 1-carbon units at a rate (∼140–170 μmol·kg−1·h−1) that greatly exceeds the demand for remethylation and transmethylation processes (∼4–7 μmol·kg−1·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.

How Does Folic Acid Cure Heart Attacks?

Folic acid (from Latin folium, meaning leaf; pteroylglutamic acid) is a B vitamin that facilitates the transfer of 1-carbon units in numerous biosynthetic reactions for 2 classes of important cellular functions: biological methylation and the contribution of formyl units to the synthesis of nucleotides (Figure). The interest in folic acid for the treatment of cardiovascular disease stems from its critical role in converting homocysteine to methionine.1 Hyperhomocysteinemia was found to be associated with a higher risk of cardiovascular disease in epidemiological studies, and dietary folate fortification lowers plasma homocysteine levels.1–3 Although recent trials have failed to demonstrate a benefit of lowering homocysteine for cardiovascular disease,4,5 it is not time to close the book on folic acid and cardiovascular health. Folic acid has been found to improve endothelial function independent of its homocysteine-lowering effect in several clinical studies.6–8 A dramatic cardioprotective effect of folic acid reported by Moens and colleagues9 in this issue of Circulation could potentially bring folic acid to the center stage in the management of ischemic heart disease. Figure. The role of folic acid in 1-carbon metabolism and its potential mechanisms for cardiac protection. The active metabolites of folic acid, 5,10-methylene tetrahydrofolate (THF) and 5-methyl THF serve as formyl donor and methyl donors for purine synthesis and methylation reactions, respectively. Besides converting homocysteine to methionine, 5-methyl THF improves vascular endothelial function by enhancing nitric oxide bioavailability and protects against oxidative injury. MTHFR indicates methyl tetrahydrofolate reductase; BH4, tetrahydrobiopterin; NOS, nitric oxide synthase; B6, vitamin B6; and B12, vitamin B12. Article p 1810 In this study, Moens et al9 treated rats with a very high dose of folic acid for 1 week (10 mg/d, roughly 400 times over the high recommended clinical dose …

Glycine Turnover and Decarboxylation Rate Quantified in Healthy Men and Women Using Primed, Constant Infusions of [1,2-13C2]Glycine and [2H3]Leucine ,

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 μmol·kg−1·h−1 [1,2-13C2]glycine and 1.87 μmol·kg−1·h−1 [2H3]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 13CO2 enrichment, were determined for use in kinetic analysis. Serine synthesis by direct conversion from glycine via SHMT occurred at 193 ± 28 μmol·kg−1·h−1 (mean ± SEM), which comprised 41% of the 463 ± 55 μmol·kg−1·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 13CO2 measurement, glycine decarboxylation (190 ± 41 μmol·kg−1·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.

Folate Deficiency Induces Cell-Specific Changes in the Steady-State Transcript Levels of Genes Involved in Folate Metabolism and 1-Carbon Transfer Reactions in Human Colonic Epithelial Cells1

Intracellular folate homeostasis is essential for the 1-carbon transfer reactions necessary for DNA synthesis and biological methylation reactions in colonic epithelial cells. Perturbed 1-carbon transfer reactions resulting from folate depletion predispose normal colonic epithelial cells to neoplastic transformation while inhibiting the growth of colon cancer cells. Using an in vitro model of folate deficiency, we determined the effects of folate deficiency on the steady-state transcript levels of genes involved in intracellular folate metabolism and 1-carbon transfer reactions in HCT116 and Caco2 human colon adenocarcinoma cells. In HCT116 cells, folate depletion was associated with changes in transcript levels of genes favoring increased folate uptake and intracellular folate retention, the provision of metabolically more effective substrates for folate-dependent enzymes, and reduced folate hydrolysis and efflux. In HCT116 cells, folate depletion was associated with changes in transcript levels of genes favoring the preferential shuttling of the flux of 1-carbon units to the methionine cycle over the nucleotide synthesis pathway. In Caco2 cells, some adaptive responses in response to folate depletion were not as apparent as in HCT116 cells, and in some cases, the direction of change was counterintuitive. In Caco2 cells, the metabolic priority in response to folate depletion was to shuttle the available folate pools to the nucleotide biosynthesis pathway at the expense of biological methylation reactions. In both HCT116 and Caco2 cells, folate depletion was associated with the conservation of the existing pattern and extent of DNA methylation.

Rare Neurodevelopmental Abnormalities of Sarcosinemia May Involve Glycinergic Stimulation of a Primed N-Methyl-d-Aspartate Receptor

Sarcosinemia is a relatively rare autosomal recessive disorder that has a varied phenotypic presentation; rarely, it is associated with neurodevelopmental and neurological abnormalities. Sarcosine is a key intermediate in 1-carbon metabolism, and its elevation in blood and urine could reflect a deficient pool size of activated 1-carbon units. Sarcosine is also an inhibitor of an important glycine transporter in brain and is under clinical investigation as a glycinergic intervention for conditions with presumed N-methyl-d-aspartate (NMDA) receptor hypofunction, such as schizophrenia. Preclinical research with a mouse model that is used to study pharmacological modulation of endogenous NMDA receptor-mediated tone may clarify, at least in some instances, varied phenotypic presentations of sarcosinemia that are often clinically benign. Sarcosine's effectiveness as a glycinergic agonist intervention for NMDA receptor hypofunction depends on an interaction between genetic background and a stressful environmental insult. Thus, neurodevelopmental and neurological abnormalities may manifest rarely in sarcosinemia in the context of relatively unique genetic factors and fetal insult or stress.

Methylenetetrahydrofolate Reductase 677C→T Polymorphism and Folate Status Affect One-Carbon Incorporation into Human DNA Deoxynucleosides

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.

Abnormal serine hydroxymethyl transferase activity in the temporal lobes of schizophrenics

We studied the kinetics of the enzyme serine hydroxymethyl transferase (SHMT) and the concentration of its metabolic substrates serine and glycine, in the postmortem brains of controls and schizophrenics. The K m of SHMT, and the concentration of serine and glycine were all significantly higher in the temporal lobes of brain tissues from schizophrenics than in those from controls. These differences were not observed in the frontal is discussed in relation to the production of glycine and 1-carbon units from which purines and thereby adenosine is produced. Both glycine and adenosine are potent neuromodulatory substances for the release of dopamine and glutamate, neurotransmitters which have been implicated in the pathophysiology of schizophrenia.

Glycine Metabolism: III. A FLAVIN-LINKED DEHYDROGENASE ASSOCIATED WITH THE GLYCINE CLEAVAGE SYSTEM IN PEPTOCOCCUS GLYCINOPHILUS

Abstract One of the four proteins involved in the cleavage of glycine to 1-carbon units and the transfer of electrons from glycine to diphosphopyridine nucleotide is a flavoprotein. Precipitation of the protein with acid ammonium sulfate in the presence of 1 m potassium bromide separated the coenzyme from the apoenzyme. The latter was fully reactivated by incubation with flavin adenine dinucleotide, but showed no activity with flavin mononucleotide. The flavoprotein could be reduced either by glycine or by reduced diphosphopyridine nucleotide. When reduced by the latter, no protein other than the flavoprotein was required for the electron transfer, but to achieve a measurable rate of reduction by glycine it was necessary to add the three other proteins plus tetrahydrofolate. Dihydrofolate was entirely inactive in the system.