Eoin P. Quinlivan

MTHFR 677C→T genotype is associated with folate and homocysteine concentrations in a large, population-based, double-blind trial of folic acid supplementation

BACKGROUND The methylenetetrahydrofolate reductase (MTHFR) genotype is associated with modification of disease and risk of neural tube defects. Plasma and red blood cell (RBC) folate and plasma homocysteine concentrations change in response to daily intakes of folic acid supplements, but no large-scale or population-based randomized trials have examined whether the MTHFR genotype modifies the observed response. OBJECTIVE We sought to determine whether the MTHFR 677C→T genotype modifies the response to folic acid supplementation during and 3 mo after discontinuation of supplementation. DESIGN Northern Chinese women of childbearing age were enrolled in a 6-mo supplementation trial of different folic acid doses: 100, 400, and 4000 μg/d and 4000 μg/wk. Plasma and RBC folate and plasma homocysteine concentrations were measured at baseline; after 1, 3, and 6 mo of supplementation; and 3 mo after discontinuation of supplementation. MTHFR genotyping was performed to identify a C→T mutation at position 677 (n = 932). RESULTS Plasma and RBC folate and homocysteine concentrations were associated with MTHFR genotype throughout the supplementation trial, regardless of folic acid dose. MTHFR TT was associated with lower folate concentrations, and the trend of TT < CC was maintained at even the highest doses. Folic acid doses of 100 μg/d or 4000 μg/wk did not reduce high homocysteine concentrations in those with the MTHFR TT genotype. CONCLUSION MTHFR genotype was an independent predictor of plasma and RBC folate and plasma homocysteine concentrations and did not have a significant interaction with folic acid dose during supplementation. This trial was registered at clinicaltrials.gov as NCT00207558.

Folate synthesis in plants: the first step of the pterin branch is mediated by a unique bimodular GTP cyclohydrolase I.

GTP cyclohydrolase I (GCHI) mediates the first and committing step of the pterin branch of the folate-synthesis pathway. In microorganisms and mammals, GCHI is a homodecamer of ≈26-kDa subunits. Genomic approaches identified tomato and Arabidopsis cDNAs specifying ≈50-kDa proteins containing two GCHI-like domains in tandem and indicated that such bimodular proteins occur in other plants. Neither domain of these proteins has a full set of the residues involved in substrate binding and catalysis in other GCHIs. The tomato and Arabidopsis cDNAs nevertheless encode functional enzymes, as shown by complementation of a yeast fol2 mutant and by assaying GCHI activity in extracts of complemented yeast cells. Neither domain expressed separately had GCHI activity. Recombinant tomato GCHI formed dihydroneopterin triphosphate as reaction product, as do other GCHIs, but unlike these enzymes it did not show cooperative behavior and was inhibited by its substrate. Denaturing gel electrophoresis verified that the bimodular GCHI polypeptide is not cleaved in vivo into its component domains, and size-exclusion chromatography indicated that the active enzyme is a dimer. The deduced tomato and Arabidopsis GCHI polypeptides lack overt targeting sequences and thus are presumably cytosolic, in contrast to other plant folate-synthesis enzymes, which are mitochondrial proteins with typical signal peptides. GCHI mRNA and protein are strongly in expressed unripe tomato fruits, implying that fruit folate is made in situ rather than imported. As ripening advances, GCHI expression declines sharply, and folate content drops, suggesting that folate synthesis fails to keep pace with turnover.

Epigenetic regulation of hepatic endoplasmic reticulum stress pathways in the ethanol-fed cystathionine beta synthase-deficient mouse.

We tested the hypothesis that the pathogenesis of alcoholic liver injury is mediated by epigenetic changes in regulatory genes that result from the induction of aberrant methionine metabolism by ethanol feeding. Five‐month‐old cystathionine beta synthase heterozygous and wild‐type C57BL/6J littermate mice were fed liquid control or ethanol diets by intragastric infusion for 4 weeks. Both ethanol‐fed groups showed typical histopathology of alcoholic steatohepatitis, with reduction in liver S‐adenosylmethionine (SAM), elevation in liver S‐adenosylhomocysteine (SAH), and reduction in the SAM/SAH ratio with interactions of ethanol and genotype effects. Hepatic endoplasmic reticulum stress signals including glucose‐regulated protein‐78 (GRP78), activating transcription factor 4, growth arrest and DNA damage‐inducible gene 153 (GADD153), caspase 12, and transcription factor sterol response element binding protein‐1c (SREBP‐1c) were up‐regulated in ethanol‐fed mice with genotype interactions and negative correlations with the SAM/SAH ratio. Immunohistochemical staining showed reduction in trimethylated histone H3 lysine‐9 (3meH3K9) protein levels in centrilobular regions in both ethanol groups, with no changes in trimethylated histone H3 lysine‐4 levels. The chromatin immunoprecipitation assay revealed a decrease in levels of suppressor chromatin marker 3meH3K9 in the promoter regions of GRP78, SREBP‐1c, and GADD153 in ethanol‐treated heterozygous cystathionine beta synthase mice. The messenger RNA expression of the histone H3K9 methyltransferase EHMT2 (G9a) was selectively decreased in ethanol‐fed mice. Conclusion: The pathogenesis of alcoholic steatohepatitis is mediated in part through the effects of altered methionine metabolism on epigenetic regulation of pathways of endoplasmic reticulum stress relating to apoptosis and lipogenesis. (HEPATOLOGY 2009.)

Mechanism of the antimicrobial drug trimethoprim revisited

We tested the hypothesis that the mechanism of action of the antifolate drug trimethoprim is through accumulation of bacterial dihydrofolate resulting in depletion of tetrahydrofolate coenzymes required for purine and pyrimidine biosynthesis. The folate pool of a strain of Escherichia coli (NCIMB 8879) was prelabeled with the folate biosynthetic precursor [3H]‐p‐aminobenzoic acid before treatment with trimethoprim. Folates in untreatedE. coli were present as tetrahydrofolate coenzymes. In trimethoprim‐treated cells, however, a rapid transient accumulation of dihydrofolate occurred, followed by complete conversion of all forms of folate to cleaved catabolites (pteridines and para‐aminobenzoylglutamate) and the stable nonreduced form of the vitamin, folic acid. Both para‐aminobenzoylglutamate and folic acid were present in the cell in the form of polyglutamates. Removal of trimethoprim resulted in the reconversion of the accumulated folic acid to tetrahydrofolate cofactors for subsequent participation in the one‐carbon cycle. Whereas irreversible catabolism is probably bactericidal, conversion to folic acid may constitute a bacteriostatic mechanism since, as we show, folic acid can be used by the bacteria and proliferation is resumed once trimethoprim is removed. Thus, the clinical effectiveness of this important drug may depend on the extent to which the processes of either catabolism or folic acid production occur in different bacteria or during different therapeutic regimes.—Quinlivan, E. P., McPartlin, J., Weir, D. G., Scott, J. Mechanism of the antimicrobial drug trimethoprim revisited. The FASEB J. 14, 2519–2524 (2000)

The relationship between increased folate catabolism and the increased requirement for folate in pregnancy

Objectives To estimate the rate of folate catabolism in pregnant and nonpregnant women and to derive the recommended dietary allowance for folate.

Genomic DNA Methylation Changes in Response to Folic Acid Supplementation in a Population-Based Intervention Study among Women of Reproductive Age

Folate is a source of one-carbons necessary for DNA methylation, a critical epigenetic modification necessary for genomic structure and function. The use of supplemental folic acid is widespread however; the potential influence on DNA methylation is unclear. We measured global DNA methylation using DNA extracted from samples from a population-based, double-blind randomized trial of folic acid supplementation (100, 400, 4000 µg per day) taken for 6 months; including a 3 month post-supplementation sample. We observed no changes in global DNA methylation in response to up to 4,000 µg/day for 6 months supplementation in DNA extracted from uncoagulated blood (approximates circulating blood). However, when DNA methylation was determined in coagulated samples from the same individuals at the same time, significant time, dose, and MTHFR genotype-dependent changes were observed. The baseline level of DNA methylation was the same for uncoagulated and coagulated samples; marked differences between sample types were observed only after intervention. In DNA from coagulated blood, DNA methylation decreased (−14%; P<0.001) after 1 month of supplementation and 3 months after supplement withdrawal, methylation decreased an additional 23% (P<0.001) with significant variation among individuals (max+17%; min-94%). Decreases in methylation of ≥25% (vs. <25%) after discontinuation of supplementation were strongly associated with genotype: MTHFR CC vs. TT (adjusted odds ratio [aOR] 12.9, 95%CI 6.4, 26.0). The unexpected difference in DNA methylation between DNA extracted from coagulated and uncoagulated samples in response to folic acid supplementation is an important finding for evaluating use of folic acid and investigating the potential effects of folic acid supplementation on coagulation.

Mice Heterozygous for Germ-line Mutations in Methylthioadenosine Phosphorylase (MTAP) Die Prematurely of T-Cell Lymphoma

Large homozygous deletions of 9p21 that inactivate CDKN2A, ARF, and MTAP are common in a wide variety of human cancers. The role for CDKN2A and ARF in tumorigenesis is well established, but whether MTAP loss directly affects tumorigenesis is unclear. MTAP encodes the enzyme methylthioadenosine phosphorylase, a key enzyme in the methionine salvage pathway. To determine if loss of MTAP plays a functional role in tumorigenesis, we have created an MTAP-knockout mouse. Mice homozygous for a MTAP null allele (Mtap(lacZ)) have an embryonic lethal phenotype dying around day 8 postconception. Mtap/Mtap(lacZ) heterozygotes are born at Mendelian frequencies and appear indistinguishable from wild-type mice during the first year of life, but they tend to die prematurely with a median survival of 585 days. Autopsies on these animals reveal that they have greatly enlarged spleens, altered thymic histology, and lymphocytic infiltration of their livers, consistent with lymphoma. Immunohistochemical staining and fluorescence-activated cell sorting analysis indicate that these lymphomas are primarily T-cell in origin. Lymphoma-infiltrated tissues tend to have reduced levels of Mtap mRNA and MTAP protein in addition to unaltered levels of methyldeoxycytidine. These studies show that Mtap is a tumor suppressor gene independent of CDKN2A and ARF.

Dysregulated Hepatic Methionine Metabolism Drives Homocysteine Elevation in Diet-Induced Nonalcoholic Fatty Liver Disease

Methionine metabolism plays a central role in methylation reactions, production of glutathione and methylarginines, and modulating homocysteine levels. The mechanisms by which these are affected in NAFLD are not fully understood. The aim is to perform a metabolomic, molecular and epigenetic analyses of hepatic methionine metabolism in diet-induced NAFLD. Female 129S1/SvlmJ;C57Bl/6J mice were fed a chow (n = 6) or high-fat high-cholesterol (HFHC) diet (n = 8) for 52 weeks. Metabolomic study, enzymatic expression and DNA methylation analyses were performed. HFHC diet led to weight gain, marked steatosis and extensive fibrosis. In the methionine cycle, hepatic methionine was depleted (30%, p< 0.01) while s-adenosylmethionine (SAM)/methionine ratio (p< 0.05), s-adenosylhomocysteine (SAH) (35%, p< 0.01) and homocysteine (25%, p< 0.01) were increased significantly. SAH hydrolase protein levels decreased significantly (p <0.01). Serine, a substrate for both homocysteine remethylation and transsulfuration, was depleted (45%, p< 0.01). In the transsulfuration pathway, cystathionine and cysteine trended upward while glutathione decreased significantly (p< 0.05). In the transmethylation pathway, levels of glycine N-methyltransferase (GNMT), the most abundant methyltransferase in the liver, decreased. The phosphatidylcholine (PC)/ phosphatidylethanolamine (PE) ratio increased significantly (p< 0.01), indicative of increased phosphatidylethanolamine methyltransferase (PEMT) activity. The protein levels of protein arginine methytransferase 1 (PRMT1) increased significantly, but its products, monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), decreased significantly. Circulating ADMA increased and approached significance (p< 0.06). Protein expression of methionine adenosyltransferase 1A, cystathionine β-synthase, γ-glutamylcysteine synthetase, betaine-homocysteine methyltransferase, and methionine synthase remained unchanged. Although gene expression of the DNA methyltransferase Dnmt3a decreased, the global DNA methylation was unaltered. Among individual genes, only HMG-CoA reductase (Hmgcr) was hypermethylated, and no methylation changes were observed in fatty acid synthase (Fasn), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (Nfκb1), c-Jun, B-cell lymphoma 2 (Bcl-2) and Caspase 3. NAFLD was associated with hepatic methionine deficiency and homocysteine elevation, resulting mainly from impaired homocysteine remethylation, and aberrancy in methyltransferase reactions. Despite increased PRMT1 expression, hepatic ADMA was depleted while circulating ADMA was increased, suggesting increased export to circulation.

Protein arginine hypomethylation in a mouse model of cystathionine β-synthase deficiency

Accumulation of the homocysteine (Hcy) precursor S‐adenosylhomocysteine (AdoHcy) may cause cellular hypomethylation in the setting of hyperhomocysteinemia because of cystathionine β‐synthase (CBS) deficiency, an inborn error of metabolism. To test this hypothesis, DNA and protein arginine methylation status were assessed in liver, brain, heart, and kidney obtained from a previously described mouse model of CBS deficiency. Metabolite levels in tissues and serum were determined by high‐performance liquid chromatography or liquid chromatography‐electrospray ionization‐tandem mass spectrometry. Global DNA and protein arginine methylation status were evaluated as the contents of 5‐methyldeoxycytidine in DNA and of methylarginines in proteins, respectively. In addition, histone arginine methylation was assessed by Western blotting. CBS‐deficient mice exhibited increased (>6‐fold) Hcy and AdoHcy levels in all tissues examined compared with control levels. In addition, global DNA methylation status was not affected, but global protein arginine methylation status was decreased (10–35%) in liver and brain. Moreover, asymmetric dimethylation of arginine 3 on histone H4 (H4R3me2a) content was markedly decreased in liver, and no differences were observed for the other histone arginine methylation marks examined. Our results show that CBS‐deficient mice present severe accumulation of tissue Hcy and AdoHcy, protein arginine hypomethylation in liver and brain, and decreased H4R3me2a content in liver. Therefore, protein arginine hypomethylation arises as a potential player in the pathophysiology of CBS deficiency.—Esse, R., Imbard, A., Florindo, C., Gupta, S., Quinlivan, E. P., Davids, M., Teerlink, T., Tavares de Almeida, I., Kruger, W. D., Blom, H. J., Castro, R. Protein arginine hypomethylation in a mouse model of cystathionine β‐synthase deficiency. FASEB J. 28, 2686–2695 (2014). www.fasebj.org

In vitamin B12 deficiency, higher serum folate is associated with increased homocysteine and methylmalonic acid concentrations

In the December 11, 2007, issue of PNAS, Selhub et al. (1) suggest that the association between elevated serum folate and methylmalonic acid (MMA) concentrations in vitamin B12 deficiency resulted from disruption of MMA metabolism by folate. An alternative explanation is that the increase in both serum folate and MMA concentrations resulted from … *E-mail: epq{at}ufl.edu