Search

(1) Background: Antifolate methotrexate (MTX) is the most common disease-modifying antirheumatic drug (DMARD) for treating human rheumatoid arthritis (RA). The mitochondrial-produced formate is essential for folate-mediated one carbon (1C) metabolism. The impacts of MTX on formate homeostasis in unknown, and rigorously controlled kinetic studies can greatly help in this regard. (2) Methods: Combining animal model (8-week old female C57BL/6JNarl mice, n = 18), cell models, stable isotopic tracer studies with gas chromatography/mass spectrometry (GC/MS) platforms, we systematically investigated how MTX interferes with the partitioning of mitochondrial and cytosolic formate metabolism. (3) Results: MTX significantly reduced de novo deoxythymidylate (dTMP) and methionine biosyntheses from mitochondrial-derived formate in cells, mouse liver, and bone marrow, supporting our postulation that MTX depletes mitochondrial 1C supply. Furthermore, MTX inhibited formate generation from mitochondria glycine cleavage system (GCS) both in vitro and in vivo. Folinate selectively rescued 1C metabolic pathways in a tissue-, cellular compartment-, and pathway-specific manner: folinate effectively reversed the inhibition of mitochondrial formate-dependent 1C metabolism in mouse bone marrow (dTMP, methionine, and GCS) and cells (dTMP and GCS) but not methionine synthesis in liver/liver-derived cells. Folinate failed to fully recover hepatic mitochondrial-formate utilization for methionine synthesis, suggesting that the efficacy of clinical folinate rescue in MTX therapy on hepatic methionine metabolism is poor. (4) Conclusion: Conducting studies in mouse and cell models, we demonstrate novel findings that MTX specifically depletes mitochondrial 1C supply that can be ameliorated by folinate supplementation except for hepatic transmethylation. These results imply that clinical use of low-dose MTX may particularly impede 1C metabolism via depletion of mitochondrial formate. The MTX induced systematic and tissue-specific formate depletion needs to be addressed more carefully, and the efficacy of folinate with respect to protecting against such depletion deserves to be evaluated in medical practice.

Folate, an essential nutrient found naturally in foods in a reduced form, is present in dietary supplements and fortified foods in an oxidized synthetic form (folic acid). There is widespread agreement that maintaining adequate folate status is critical to prevent diseases due to folate inadequacy (e.g., anemia, birth defects, and cancer). However, there are concerns of potential adverse effects of excess folic acid intake and/or elevated folate status, with the original concern focused on exacerbation of clinical effects of vitamin B-12 deficiency and its role in neurocognitive health. More recently, animal and observational studies have suggested potential adverse effects on cancer risk, birth outcomes, and other diseases. Observations indicating adverse effects from excess folic acid intake, elevated folate status, and unmetabolized folic acid (UMFA) remain inconclusive; the data do not provide the evidence needed to affect public health recommendations. Moreover, strong biological and mechanistic premises connecting elevated folic acid intake, UMFA, and/or high folate status to adverse health outcomes are lacking. However, the body of evidence on potential adverse health outcomes indicates the need for comprehensive research to clarify these issues and bridge knowledge gaps. Three key research questions encompass the additional research needed to establish whether high folic acid or total folate intake contributes to disease risk. 1) Does UMFA affect biological pathways leading to adverse health effects? 2) Does elevated folate status resulting from any form of folate intake affect vitamin B-12 function and its roles in sustaining health? 3) Does elevated folate intake, regardless of form, affect biological pathways leading to adverse health effects other than those linked to vitamin B-12 function? This article summarizes the proceedings of an August 2019 NIH expert workshop focused on addressing these research areas.

BackgroundFormate is an important metabolite that serves as a donor of one-carbon groups to the intracellular tetrahydrofolate pool. However, little is known of its circulating concentrations or of their determinants.ObjectiveThis study aimed to define formate concentrations and their determinants in a healthy young population.DesignSerum formate was measured in 1701 participants from the Trinity Student Study. The participants were men and women, aged 18 to 28 y, enrolled at Trinity College, Dublin. Formate concentrations were compared with other one-carbon metabolites, vitamin status, potential formate precursors, genetic polymorphisms, and lifestyle factors.ResultsSerum formate concentrations ranged from 8.7 to 96.5 µM, with a mean of 25.9 µM. Formate concentrations were significantly higher in women than in men; oral contraceptive use did not further affect them. There was no effect of smoking or of alcohol ingestion, but the TT genotype of the methylenetetrahydrofolate reductase (MTHFR) 677C→T (rs1801133) polymorphism was associated with a significantly decreased formate concentration. Formate was positively associated with potential metabolic precursors (serine, methionine, tryptophan, choline) but not with glycine. Formate concentrations were positively related to serum folate and negatively related to serum vitamin B-12.ConclusionsFormate concentrations were sensitive to the concentrations of metabolic precursors. In view of the increased susceptibility of women with the TT genotype of MTHFR to give birth to infants with neural tube defects as well as the effectiveness of formate supplementation in decreasing the incidence of folate-resistant neural tube defects in susceptible mice, it will be important to understand how this genotype decreases the serum formate concentration. This trial was registered at www.clinicaltrials.gov as NCT03305900.

Self-efficacy, the system of beliefs that individuals possess which enables them to exercise control and actions when faced with a specific task, is an essential component to language learning, as it has the potential to promote the self-regulatory capacity required for successful language acquisition and performance. One way in which self-efficacy mediates language learning is exhibited in how individuals dedicate resources such as effort and time to overcoming tasks. The aim of this study was to determine the influence of Irish language self-efficacy beliefs on performance and resources allocated by Irish adults (N=450) on an Irish language reading test. Over two testing phases, three groups were formed based on results in phase one: a control group; a group of high performers; and a group of low performers. Manipulated comparative feedback and false results produced highly significant effects on performance and resources allocated, with low performing participants receiving a positive intervention consisting of false inflated results, leading to improved performance and increased time dedicated to task completion. High performers who received a negative intervention consisting of falsely deflated results saw a significant decrease in performance and the time allocated post-manipulation. The control group also saw a decrease in average performance scores, making the low performing group’s performance even more noteworthy. Overall, findings show that self-efficacy beliefs, though initially closely aligned with actual performance, can in fact be manipulated to influence performance outcomes.

BackgroundNeural tube defects (NTDs), which are among the most common congenital malformations, are influenced by environmental and genetic factors. Low maternal folate is the strongest known contributing factor, making variants in genes in the folate metabolic pathway attractive candidates for NTD risk. Multiple studies have identified nominally significant allelic associations with NTDs. We tested whether associations detected in a large Irish cohort could be replicated in an independent population.MethodsReplication tests of 24 nominally significant NTD associations were performed in racially/ethnically matched populations. Family-based tests of fifteen nominally significant single nucleotide polymorphisms (SNPs) were repeated in a cohort of NTD trios (530 cases and their parents) from the United Kingdom, and case-control tests of nine nominally significant SNPs were repeated in a cohort (190 cases, 941 controls) from New York State (NYS). Secondary hypotheses involved evaluating the latter set of nine SNPs for NTD association using alternate case-control models and NTD groupings in white, African American and Hispanic cohorts from NYS.ResultsOf the 24 SNPs tested for replication, ADA rs452159 and MTR rs10925260 were significantly associated with isolated NTDs. Of the secondary tests performed, ARID1A rs11247593 was associated with NTDs in whites, and ALDH1A2 rs7169289 was associated with isolated NTDs in African Americans.ConclusionsWe report a number of associations between SNP genotypes and neural tube defects. These associations were nominally significant before correction for multiple hypothesis testing. These corrections are highly conservative for association studies of untested hypotheses, and may be too conservative for replication studies. We therefore believe the true effect of these four nominally significant SNPs on NTD risk will be more definitively determined by further study in other populations, and eventual meta-analysis.

AbstractBackgroundNeural tube defects (NTDs) are common birth defects (~1 in 1000 pregnancies in the US and Europe) that have complex origins, including environmental and genetic factors. A low level of maternal folate is one well-established risk factor, with maternal periconceptional folic acid supplementation reducing the occurrence of NTD pregnancies by 50-70%. Gene variants in the folate metabolic pathway (e.g., MTHFR rs1801133 (677 C > T) and MTHFD1 rs2236225 (R653Q)) have been found to increase NTD risk. We hypothesized that variants in additional folate/B12 pathway genes contribute to NTD risk.MethodsA tagSNP approach was used to screen common variation in 82 candidate genes selected from the folate/B12 pathway and NTD mouse models. We initially genotyped polymorphisms in 320 Irish triads (NTD cases and their parents), including 301 cases and 341 Irish controls to perform case–control and family based association tests. Significantly associated polymorphisms were genotyped in a secondary set of 250 families that included 229 cases and 658 controls. The combined results for 1441 SNPs were used in a joint analysis to test for case and maternal effects.ResultsNearly 70 SNPs in 30 genes were found to be associated with NTDs at the p

This study assesses the influence of sources of self-efficacy construction on Irish language abilities in civil servants working under the requirements of the Official Languages Act 2003. Through a series of focus groups within a Government department, participants with varying abilities and interests in the Irish language were assessed on the determining factors in self-assessing their Irish language skills. It was found that self-efficacy is a more accurate predictor of language beliefs than previous performances for Irish speakers, and that sources such as social persuasion and vicarious experiences have the potential to raise self-efficacy beliefs in non-Irish speakers. It was also found that the Act has not led to an increased engagement with the Irish language, but has only resulted in an increased deference to perceived expert language models.

This article presents a historical perspective on the different methods used to measure folate status in populations and clinical settings. I discuss some of the advantages and limitations of these procedures. For >50 y researchers have used microbiological assay methods to assess folate status in clinical settings and in population-based studies, such as NHANES. Serum and red blood cell folate values obtained with the Lactobacillus casei assay have formed the basis for current ranges and cutoffs for the establishment of folate sufficiency and for the current dietary reference intakes for folate. Over the past 30 y competitive folate protein binding assays, which are available in kit form, have supplanted microbiological assays in many clinical laboratories because of their ease of use. Several NHANES cycles have used these assays. Folate concentrations obtained with these kits are lower than those from microbiological assays and show a wide variation between different protein binding assay kits. This variation has complicated the setting of values for normal ranges of folate status and the comparison of status changes between different NHANES cycles. The recent development of mass spectrometry methods for folate opens up the possibility of measurement of individual folate vitamers such as folic acid. Past experience with microbiological and competitive protein binding assays indicates some of the technical problems that research will need to address before this promise becomes reality.

The development of a battery using different cement-based electrolytes to provide a low but potentially sustainable source of electricity is described. The current, voltage, and lifespan of batteries produced using different electrolyte additives, copper plate cathodes, and (usually) aluminium plate anodes were compared to identify the optimum design, components, and proportions to increase power output and longevity. Parameters examined include water/cement ratio, anode to cathode surface area ratio, electrode material, electrode spacing, and the effect of sand, aggregate, salts, carbon black, silica fume, and sodium silicate on the electrolyte. The results indicate that the greatest and longest lasting power can be achieved using high proportions of water, carbon black, plasticiser, salts, and silica fume in the electrolyte and using a magnesium anode and copper cathode. This cell produced an open-circuit voltage of 1.55 V, a resistor-loaded peak current over 4 mA, maintaining over 1 mA for 4 days, and a quasi steady current of 0.59 mA with a lifespan of over 21 days.

Impaired folate-mediated 1-carbon metabolism has been linked to multiple disease outcomes. A better understanding of the nutritional and genetic influences on this complex biochemical pathway is needed to comprehend their impact on human health. To this end, we created a mathematical model of folate-mediated 1-carbon metabolism. The model uses published data on folate enzyme kinetics and regulatory mechanisms to simulate the impact of genetic and nutritional variation on critical aspects of the pathway. We found that the model predictions match experimental data, while providing novel insights into pathway kinetics. Our primary observations were as follows: 1) the inverse association between folate and homocysteine is strongest at very low folate concentrations, but there is no association at high folate concentrations; 2) the DNA methylation reaction rate is relatively insensitive to changes in folate pool size; and 3) as folate concentrations become very high, enzyme velocities decrease. With regard to polymorphisms in 5,10-methylenetetrahydrofolate reductase (MTHFR), the modeling predicts that decrease MTHFR activity reduces concentrations of S-adenosylmethionine and 5-methyltetrahydrofolate, as well as DNA methylation, while modestly increasing S-adenosylhomocysteine and homocysteine concentrations and thymidine or purine synthesis. Decreased folate together with a simulated vitamin B-12 deficiency results in decreases in DNA methylation and purine and thymidine synthesis. Decreased MTHFR activity superimposed on the B-12 deficiency appears to reverse the declines in purine and thymidine synthesis. These mathematical simulations of folate-mediated 1-carbon metabolism provide a cost-efficient approach to in silico experimentation that can complement and help guide laboratory studies.

Genetic variation in folate-regulating enzymes contributes to the risk of cardiovascular disease (CVD). The cytoplasmic serine hydroxymethyltransferase (cSHMT) enzyme is proposed to regulate a key metabolic intersection in folate metabolism. We hypothesized that a variant in cSHMT (cSHMT 1420C [right arrow] T) affects CVD risk, and that the effect depends on a linked step in the metabolic pathway catalyzed by methylenetetrahydrofolate reductase (MTHFR). A nested case-control study of incident CVD was conducted within the all-male Normative Aging Study cohort. Of the incident CVD cases, 507 had DNA samples; 2 controls/case were selected by risk set sampling (matched on age and birth year). A significant gene-gene interaction (P-values 0.0013, 0.0064) was found between MTHFR and cSHMT, and there was little or no change in the coefficients in covariate-adjusted models. The effect of MTHFR 677C [right arrow] T genotype on CVD risk varied by cSHMT 1420C [right arrow] T genotype. Among men with cSHMT 1420C [right arrow] T TT genotype, the odds ratios (OR) for CVD risk for MTHFR 677C [right arrow] T CT and TT genotypes compared with the MTHFR 677C [right arrow] T CC genotype were 3.6 (95% CI, 1.7-7.8) and 10.6 (95% CI, 2.5-46.0), respectively. Among men with the cSHMT 1420C [right arrow] T CC/CT genotype, the corresponding ORs were 1.0 (95% CI, 0.8-1.2) and 1.3 (95% CI, 0.9-1.8). Plasma total homocysteine concentrations were highest in the subgroup of men with both polymorphisms, MTHFR 677C [right arrow] T TT and cSHMT 1420C [right arrow] T TT, consistent with a higher risk of CVD in this subgroup. A more complete understanding of the molecular mechanism awaits identification of the functional effect of the polymorphism. J. Nutr. 135: 1989-1994, 2005. KEY WORDS: * homocysteine * folate * men * cardiovascular disease

The effects of folate status and the methylenetetrahydrofolate reductase (MTHFR) 677C[right arrow]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 [[sup.13][C.sub.5]]methionine, [3-[sup.13]C]serine, and [[sup.2][H.sub.3]]leucine before and after 7 wk of dietary folate restriction (115 [micro]g 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[right arrow]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. KEY WORDS: * methionine * one-carbon metabolism * S-adenosylmethionine * S-adenosylhomocysteine * women

The methylenetetrahydrofolate reductase (MTHFR) 677C [right arrow] 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 [right arrow] 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-[sup.13C]]serine and [[sup.13][C.sub.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 ~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 [sup.13C]-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. KEY WORDS: * thymidine * methyldeoxycytidine * deoxypurine * LC-MS/MS * isotope

Hyperhomocysteinemia in humans is associated with genetic variants of several enzymes of folate and one-carbon metabolism and deficiencies of folate and vitamins [B.sub.12] and [B.sub.6]. 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-[sup.13][C.sub.5]]-methionine tracer is used in conjunction with a [3-[sup.13]C]serine tracer to simultaneously measure rates of total and folate-dependent homocysteine remethylation. In young female subjects, plasma [U-[sup.13][C.sub.4]]homocysteine enrichment, a surrogate measure of intracellular [U-[sup.13][C.sub.5]]methionine enrichment, reached ~90% of the plasma [U-[sup.13][C.sub.5]]methionine enrichment. Methionine-methyl and -carboxyl group fluxes were in the range of previous reports (~25 and ~17 [micro]mol*[kg.sup.-1]*[h.sup.-1], respectively). However, the rate of overall homocysteine remethylation (~8 [micro]mol*[kg.sup.-1]*[h.sup.-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-[sup.13]C]serine enrichment based on a conservative correction of plasma [3-[sup.13]C]serine enrichment, serine was calculated to contribute ~100% of the methyl groups used for total body homocysteine remethylation under the conditions of this protocol. This contribution represented only a small fraction (~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. methionine; methylation cycle; cystathionine

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the methyl donor for the synthesis of methionine from homocysteine. A common C677T mutation in the MTHFR gene renders the enzyme ~50% less active than the wild-type enzyme as shown in in vitro studies using cell extracts. We developed an immortalized cell culture model to determine whether the lower in vitro activity imparted by the homozygous (T/T) genotype is demonstrated in situ when exposed to adequate and marginal physiologic concentrations of folate and riboflavin. T/T MTHFR activity was compared with that of C/C genotype cell extracts by an in vitro assay and in intact cells by measuring the distribution of folate forms, the accumulation of homocysteine in the medium and the synthesis of methionine from formate and homocysteine. Under adequate nutrient conditions, the in vitro activity of the T/T MTHFR enzyme was approximately half that of the C/C genotype. Similarly, the proportion of 5-methyltetrahydrofolate in cells with the T/T genotype was approximately half that of the cells with wild-type MTHFR. In contrast, homocysteine accumulation in the culture medium was low and not different between genotypes, nor was there a difference in methionine synthetic capacity. Significant differences were observed between genotypes only when the supply of both folate and riboflavin was limited in the medium, which resulted in increased homocysteine accumulation and decreased methionine production in the T/T genotype. These data are consistent with the current understanding of the molecular interaction of the MTHFR mutant with folate substrates and the FAD prosthetic group. KEY WORDS: * folate * methylenetetrahydrofolate reductase * homocysteine * immortalized cells