Folinate Supplementation Ameliorates Methotrexate Induced Mitochondrial Formate Depletion In Vitro and In Vivo.

(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.

Knowledge gaps in understanding the metabolic and clinical effects of excess folates/folic acid: a summary, and perspectives, from an NIH workshop.

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.

Lifestyle, metabolite, and genetic determinants of formate concentrations in a cross-sectional study in young, healthy adults.

Background: Formate 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. Objective: This study aimed to define formate concentrations and their determinants in a healthy young population. Design: Serum 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. Results: Serum 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. Conclusions: Formate 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.

Biomarkers of Nutrition for Development-Folate Review.

The Biomarkers of Nutrition for Development (BOND) project is designed to provide evidence-based advice to anyone with an interest in the role of nutrition in health. Specifically, the BOND program provides state-of-the-art information and service with regard to selection, use, and interpretation of biomarkers of nutrient exposure, status, function, and effect. To accomplish this objective, expert panels are recruited to evaluate the literature and to draft comprehensive reports on the current state of the art with regard to specific nutrient biology and available biomarkers for assessing nutrients in body tissues at the individual and population level. Phase I of the BOND project includes the evaluation of biomarkers for 6 nutrients: iodine, iron, zinc, folate, vitamin A, and vitamin B-12. This review represents the second in the series of reviews and covers all relevant aspects of folate biology and biomarkers. The article is organized to provide the reader with a full appreciation of folate's history as a public health issue, its biology, and an overview of available biomarkers (serum folate, RBC folate, and plasma homocysteine concentrations) and their interpretation across a range of clinical and population-based uses. The article also includes a list of priority research needs for advancing the area of folate biomarkers related to nutritional health status and development.

The Dihydrofolate Reductase 19 bp Polymorphism Is Not Associated with Biomarkers of Folate Status in Healthy Young Adults, Irrespective of Folic Acid Intake.

BACKGROUND: Dihydrofolate reductase (DHFR) is essential for the conversion of folic acid to active folate needed for one-carbon metabolism. Common genetic variation within DHFR is restricted to the noncoding regions, and previous studies have focused on a 19 bp deletion/insertion polymorphism (rs70991108) within intron 1. Reports of an association between this polymorphism and blood folate biomarker concentrations are conflicting. OBJECTIVE: In this study, we evaluated whether the DHFR 19 bp deletion/insertion polymorphism affects circulating folate biomarkers in, to our knowledge, the largest cohort to address this question to date. METHODS: Healthy young Irish individuals (n = 2507) between 19 and 36 y of age were recruited between February 2003 and February 2004. Folic acid intake from supplements and fortified foods was assessed with the use of a customized food intake questionnaire. Concentrations of serum folate and vitamin B-12, red blood cell (RBC) folate, and plasma total homocysteine (tHcy) were measured. Data were analyzed with the use of linear regression models. RESULTS: Folic acid intake was positively associated with serum (P < 0.0001) and RBC (P = 0.0005) folate concentration and was inversely associated with plasma tHcy (P = 0.001) as expected. The DHFR 19 bp polymorphism was not significantly associated with either serum (P = 0.82) or RBC (P = 0.21) folate, or plasma tHcy (P = 0.20), even in those within the highest quintile of folic acid intake (>326 µg folic acid/d; P = 0.96). A nonsignificant trend toward lower RBC folate by genotype (P = 0.09) was observed in the lowest folic acid intake quintile (0-51 µg/d). CONCLUSION: In this cohort of healthy young individuals, the DHFR 19 bp deletion allele did not significantly affect circulating folate status, irrespective of folic acid intake. Our data rule out a strong functional effect from this polymorphism on blood folate concentrations.

Common Variants at Putative Regulatory Sites of the Tissue Nonspecific Alkaline Phosphatase Gene Influence Circulating Pyridoxal 5'-Phosphate Concentration in Healthy Adults.

BACKGROUND: Vitamin B-6 interconversion enzymes are important for supplying pyridoxal 5'-phosphate (PLP), the co-enzyme form, to tissues. Variants in the genes for these enzymes [tissue nonspecific alkaline phosphatase (ALPL), pyridoxamine 5'-phosphate oxidase, pyridoxal kinase, and pyridoxal phosphatase] could affect enzyme function and vitamin B-6 status. OBJECTIVES: We tested whether single-nucleotide polymorphisms (SNPs) in these genes influence vitamin B-6 status markers [plasma PLP, pyridoxal (PL), and 4-pyridoxic acid (PA)], and explored potential functional effects of the SNPs. METHODS: Study subjects were young, healthy adults from Ireland (n = 2345). We measured plasma PLP, PL, and PA with liquid chromatography-tandem mass spectrometry and genotyped 66 tag SNPs in the 4 genes. We tested for associations with single SNPs in candidate genes and also performed genome-wide association study (GWAS) and gene-based analyses. RESULTS: Seventeen SNPs in ALPL were associated with altered plasma PLP in candidate gene analyses (P < 1.89 × 10(-4)). In the GWAS, 5 additional ALPL SNPs were associated with altered plasma PLP (P < 5.0 × 10(-8)). Gene-based analyses that used the functional linear model ß-spline (P = 4.04 × 10(-15)) and Fourier spline (P = 5.87 × 10(-15)) methods also showed associations between ALPL and altered plasma PLP. No SNPs in other genes were associated with plasma PLP. The association of the minor CC genotype of 1 ALPL SNP, rs1256341, with reduced ALPL expression in the HapMap Northern European ancestry population is consistent with the positive association between the CC genotype and plasma PLP in our study (P = 0.008). No SNP was associated with altered plasma PL or PA. CONCLUSIONS: In healthy adults, common variants in ALPL influence plasma PLP concentration, the most frequently used biomarker for vitamin B-6 status. Whether these associations are indicative of functional changes in vitamin B-6 status requires more investigation.

Tryptophan catabolism and vitamin B-6 status are affected by gender and lifestyle factors in healthy young adults.

BACKGROUND: Abnormalities of tryptophan (Trp) metabolism through the kynurenine (Kyn) pathway have been reported in various diseases; however, nutritional and lifestyle factors that affect this pathway in healthy individuals are not well documented. OBJECTIVE: Our aim was to examine the effect of vitamin B-6 status and lifestyle factors including the use of vitamin B-6 supplements, alcohol, smoking, and oral contraceptives on Trp and its Kyn metabolites in a cohort of 2436 healthy young adults aged 18-28 y. METHODS: Anthropometric and lifestyle data were collected by questionnaire. Participants provided blood samples for analysis of Trp, Kyn, anthranilic acid, kynurenic acid (KA), 3-hydroxykynurenine (HK), 3-hydroxyanthranilic acid (HAA), and xanthurenic acid (XA). Vitamin B-6 species were also measured. RESULTS: Serum Trp metabolites were 10-15% higher among men (n = 993) compared with women (n = 1443; P < 0.0001), except for HK and XA. In all participants, serum Trp was positively associated with plasma pyridoxal 5'-phosphate (PLP; r = 0.28, P < 0.0001), reaching a plateau at PLP concentrations of ∼83 nmol/L. HK was inversely associated with PLP (r = -0.14, P < 0.01). Users of vitamin B-6 supplements (n = 671) had 6% lower concentrations of HK than nonusers (n = 1765; P = 0.0006). Oral contraceptive users (n = 385) had lower concentrations of KA (20.7%) but higher XA (24.1%) and HAA (9.0%) than did nonusers (n = 1058; P < 0.0001). After adjustment for gender and other lifestyle variables, XA concentrations were 16% higher in heavy drinkers (n = 713) than in never or occasional drinkers (n = 975; P = 0.0007). Concentrations of 2 other essential amino acids, methionine and arginine, also were positively associated with serum Trp (r = 0.65 and 0.33, respectively; P < 0.0001). CONCLUSIONS: In this population of healthy young adults, gender has the largest influence on serum Kyn metabolite concentrations. The significant covariance of Trp with unrelated amino acids suggests that protein intake may be an important consideration in evaluating Kyn metabolism.

Maternal choline concentrations during pregnancy and choline-related genetic variants as risk factors for neural tube defects.

BACKGROUND: Low maternal choline intake and blood concentration may be risk factors for having a child with a neural tube defect (NTD); however, the data are inconsistent. This is an important question to resolve because choline, if taken periconceptionally, might add to the protective effect currently being achieved by folic acid. OBJECTIVE: We examined the relation between NTDs, choline status, and genetic polymorphisms reported to influence de novo choline synthesis to investigate claims that taking choline periconceptionally could reduce NTD rates. DESIGN: Two study groups of pregnant women were investigated: women who had a current NTD-affected pregnancy (AP; n = 71) and unaffected controls (n = 214) and women who had an NTD in another pregnancy but not in the current pregnancy [nonaffected pregnancy (NAP); n = 98] and unaffected controls (n = 386). Blood samples to measure betaine and total choline concentrations and single nucleotide polymorphisms related to choline metabolism were collected at their first prenatal visit. RESULTS: Mean (±SD) plasma total choline concentrations in the AP (2.8 ± 1.0 mmol/L) and control (2.9 ± 0.9 mmol/L) groups did not differ significantly. Betaine concentrations were not significantly different between the 2 groups. Total choline and betaine in the NAP group did not differ from controls. Cases were significantly more likely to have the G allele of phosphatidylethanolamine-N-methyltransferase (PEMT; V175M, +5465 G>A) rs7946 (P = 0.02). CONCLUSIONS: Our results indicate that maternal betaine and choline concentrations are not strongly associated with NTD risk. The association between PEMT rs7946 and NTDs requires confirmation. The addition of choline to folic acid supplements may not further reduce NTD risk.

Is low iron status a risk factor for neural tube defects?

BACKGROUND: Folic acid supplements can protect against neural tube defects (NTDs). Low folate and low vitamin B12 status may be maternal risk factors for having an NTD affected pregnancy. However, not all NTDs are preventable by having an adequate folate/ B12 status and other potentially modifiable factors may be involved. Folate and vitamin B12 status have important links to iron metabolism. Animal studies support an association between poor iron status and NTDs, but human data are scarce. We examined the relevance of low iron status in a nested NTD case-control study of women within a pregnant population-based cohort. METHODS: Pregnant women were recruited between 1986 and 1990, when vitamin or iron supplementation in early pregnancy was rare. Blood samples, taken at an average of 14 weeks gestation, were used to measure ferritin and hemoglobin in 64 women during an NTD affected pregnancy and 207 women with unaffected pregnancies. RESULTS: No significant differences in maternal ferritin or hemoglobin concentrations were observed between NTD affected and nonaffected pregnancies (case median ferritin 16.9 µg/L and hemoglobin 12.4 g/dl versus 15.4 µg/L and 12.3g/dl in controls). As reported previously, red cell folate and vitamin B12 concentrations were significantly lower in cases. Furthermore, there was no significant association of iron status with type of NTD lesion (anencephaly or spina bifida). CONCLUSION: We conclude that low maternal iron status during early pregnancy is not an independent risk factor for NTDs. Adding iron to folic acid for periconceptional use may improve iron status but is not likely to prevent NTDs.

Evaluation of common genetic variants in 82 candidate genes as risk factors for neural tube defects.

BACKGROUND: Neural 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. METHODS: A 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. RESULTS: Nearly 70 SNPs in 30 genes were found to be associated with NTDs at the p < 0.01 level. The ten strongest association signals (p-value range: 0.0003-0.0023) were found in nine genes (MFTC, CDKN2A, ADA, PEMT, CUBN, GART, DNMT3A, MTHFD1 and T (Brachyury)) and included the known NTD risk factor MTHFD1 R653Q (rs2236225). The single strongest signal was observed in a new candidate, MFTC rs17803441 (OR = 1.61 [1.23-2.08], p = 0.0003 for the minor allele). Though nominally significant, these associations did not remain significant after correction for multiple hypothesis testing. CONCLUSIONS: To our knowledge, with respect to sample size and scope of evaluation of candidate polymorphisms, this is the largest NTD genetic association study reported to date. The scale of the study and the stringency of correction are likely to have contributed to real associations failing to survive correction. We have produced a ranked list of variants with the strongest association signals. Variants in the highest rank of associations are likely to include true associations and should be high priority candidates for further study of NTD risk.

Do high blood folate concentrations exacerbate metabolic abnormalities in people with low vitamin B-12 status?

BACKGROUND: In elderly individuals with low serum vitamin B-12, those who have high serum folate have been reported to have greater abnormalities in the following biomarkers for vitamin B-12 deficiency: low hemoglobin and elevated total homocysteine (tHcy) and methylmalonic acid (MMA). This suggests that folate exacerbates vitamin B-12-related metabolic abnormalities. OBJECTIVE: We determined whether high serum folate in individuals with low serum vitamin B-12 increases the deleterious effects of low vitamin B-12 on biomarkers of vitamin B-12 cellular function. DESIGN: In this cross-sectional study, 2507 university students provided data on medical history and exposure to folic acid and vitamin B-12 supplements. Blood was collected to measure serum and red blood cell folate (RCF), hemoglobin, plasma tHcy, and MMA, holotranscobalamin, and ferritin in serum. RESULTS: In subjects with low vitamin B-12 concentrations (<148 pmol/L), those who had high folate concentrations (>30 nmol/L; group 1) did not show greater abnormalities in vitamin B-12 cellular function in any area than did those with lower folate concentrations (≤30 nmol/L; group 2). Group 1 had significantly higher holotranscobalamin and RCF, significantly lower tHcy, and nonsignificantly lower (P = 0.057) MMA concentrations than did group 2. The groups did not differ significantly in hemoglobin or ferritin. Compared with group 2, group 1 had significantly higher mean intakes of folic acid and vitamin B-12 from supplements and fortified food. CONCLUSIONS: In this young adult population, high folate concentrations did not exacerbate the biochemical abnormalities related to vitamin B-12 deficiency. These results provide reassurance that folic acid in fortified foods and supplements does not interfere with vitamin B-12 metabolism at the cellular level in a healthy population.

Micronutrients and women of reproductive potential: required dietary intake and consequences of dietary deficiency or excess. Part II--vitamin D, vitamin A, iron, zinc, iodine, essential fatty acids.

Part II of this review considers additional micronutrients. Vitamin D is a fat soluble vitamin found in foods of animal origins (fatty fish, liver oil) or fortified products (milk, cheese). Vitamin D deficiency is common in African-American women living in northern latitudes. Vitamin D supplementation may be needed to reach desired 25-(OH)D3 concentrations of >50 nmol/L. In foods of animal origin, preformed Vitamin A is present; in plants (fruits and vegetables) vitamin A precursors (ß-carotenoids) are present. Vitamin A supplementation is usually not warranted, and in developing countries should not exceed 3000 µg (10,000 IU)/day. Iron in the form of haem-iron is found in meat, fish and poultry; non-haem (inorganic) iron is found in vegetables, fruits and grains. Iron supplementation may be necessary in the third trimester, earlier in pregnancy or in non-pregnant states if serum ferritin is <20 µg/L or haemoglobin <10.9 g/dL. Zinc is available in red meat, seafood including oysters and unpolished grains; supplementation is not necessary. To assure adequate iodine, food is fortified worldwide with iodated salt. If urinary iodine levels are low, supplementation is needed. Essential fatty acids requirements can be met by one to two portions of fish per week.

Folic acid and L-5-methyltetrahydrofolate: comparison of clinical pharmacokinetics and pharmacodynamics.

There is a large body of evidence to suggest that improving periconceptional folate status reduces the risk of neonatal neural tube defects. Thus increased folate intake is now recommended before and during the early stages of pregnancy, through folic acid supplements or fortified foods. Furthermore, there is growing evidence that folic acid may have a role in the prevention of other diseases, including dementia and certain types of cancer. Folic acid is a synthetic form of the vitamin, which is only found in fortified foods, supplements and pharmaceuticals. It lacks coenzyme activity and must be reduced to the metabolically active tetrahydrofolate form within the cell. L-5-methyl-tetrahydrofolate (L-5-methyl-THF) is the predominant form of dietary folate and the only species normally found in the circulation, and hence it is the folate that is normally transported into peripheral tissues to be used for cellular metabolism. L-5-methyl-THF is also available commercially as a crystalline form of the calcium salt (Metafolin(R)), which has the stability required for use as a supplement. Studies comparing L-5-methyl-THF and folic acid have found that the two compounds have comparable physiological activity, bioavailability and absorption at equimolar doses. Bioavailability studies have provided strong evidence that L-5-methyl-THF is at least as effective as folic acid in improving folate status, as measured by blood concentrations of folate and by functional indicators of folate status, such as plasma homocysteine. Intake of L-5-methyl-THF may have advantages over intake of folic acid. First, the potential for masking the haematological symptoms of vitamin B(12) deficiency may be reduced with L-5-methyl-THF. Second, L-5-methyl-THF may be associated with a reduced interaction with drugs that inhibit dihydrofolate reductase.

Micronutrients and women of reproductive potential: required dietary intake and consequences of dietary deficiency or excess. Part I--Folate, Vitamin B12, Vitamin B6.

This two-part review highlights micronutrients for which either public health policy has been established or for which new evidence provides guidance as to recommended intakes during pregnancy. One pivotal micronutrient is folate, the generic name for different forms of a water-soluble vitamin essential for the synthesis of thymidylate and purines and, hence, DNA. For non-pregnant adult women the recommended intake is 400 µg/day dietary folate equivalent. For women capable of becoming pregnant an additional 400 µg/day of synthetic folic acid from supplements or fortified foods is recommended to reduce the risk of neural tube defects (NTD). The average amount of folic acid received through food fortification (grains) in the US is only 128 µg/day, emphasising the need for the supplemental vitamin for women of reproductive age. Vitamin B12 (cobalamin) is a cofactor required for enzyme reactions, including generation of methionine and tetrahydrofolate. B12 is found almost exclusively in foods of animal origin (meats, dairy products); therefore, vegetarians are at greatest risk for dietary vitamin B12 deficiency and should be supplemented. Vitamin B6 is required for many reactions, primarily in amino acid metabolism. Meat, fish and poultry are good dietary sources. Supplementation beyond routine prenatal vitamins is not recommended.