Barry Shane

Folylpolyglutamate Synthesis and Role in the Regulation of One-Carbon Metabolism

The physiological importance of folylpolyglutamates is now well established. These derivatives are the intracellular substrates and regulators of one-carbon metabolism, and their synthesis is required for normal folate retention by tissues. Over the last few years, a considerable amount of information has been obtained on the mechanism by which these compounds are synthesized, on how this synthesis is regulated, and on the effects of the polyglutamate chain on the interaction of folate substrates and inhibitors with folate-dependent enzymes. Many regulatory implications have been suggested by these studies, but the physiological relevance of some of these observations remains to be explored. Folates in mammalian tissues are metabolized to polyglutamates of chain lengths considerably longer than that required for folate retention, but the metabolic advantages of this are not entirely clear. Several in vivo model systems have been developed to explore the functioning of specific folylpolyglutamate chain lengths in metabolic cycles of one-carbon metabolism, and these are likely to shed further light on this point. The role of folate-binding proteins in folate transport, the metabolic role of glutamylhydrolases, and the role of folylpolyglutamates in putative multifunctional protein complexes are also areas that are being actively pursued at present and are likely to produce new insights in the future. Recent studies on the retention of antifolates by cells and on their substrate efficacy for folylpolyglutamate synthetases have also suggested mechanisms for the differential cytotoxicity of these agents for different tissues.

Methionine synthase D919G polymorphism is a significant but modest determinant of circulating homocysteine concentrations

Elevation in plasma homocysteine concentration has been associated with vascular disease and neural tube defects. Methionine synthase is a vitamin B12‐dependent enzyme that catalyses the remethylation of homocysteine to methionine. Therefore, defects in this enzyme may result in elevated homocysteine levels. One relatively common polymorphism in the methionine synthase gene (D919G) is an A to G transition at bp 2,756, which converts an aspartic acid residue believed to be part of a helix involved in co‐factor binding to a glycine. We have investigated the effect of this polymorphism on plasma homocysteine levels in a working male population (n = 607) in which we previously described the relationship of the C677T “thermolabile” methylenetetrahydrofolate reductase (MTHFR) polymorphism with homocysteine levels. We found that the methionine synthase D919G polymorphism is significantly (P = 0.03) associated with homocysteine concentration, and the DD genotype contributes to a moderate increase in homocysteine levels across the homocysteine distribution (OR = 1.58, DD genotype in the upper half of the homocysteine distribution, P = 0.006). Unlike thermolabile MTHFR, the homocysteine‐elevating effects of the methionine synthase polymorphism are independent of folate and B12 levels; however, the DD genotype has a larger homocysteine‐elevating effect in individuals with low B6 levels. This polymorphism may, therefore, make a moderate, but significant, contribution to clinical conditions that are associated with elevated homocysteine. Genet. Epidemiol. 17:298–309, 1999.

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 folates 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.

FOLATE CHEMISTRY AND METABOLISM

The vitamin folic acid was initially investigated as a dietary antianemia factor distinct from the pernicious anemia factor [1] and a recent historical review details early studies on the isolation of the vitamin and on the establishment of its role as a cofactor in one-carbon metabolism [2]. Because of the role of folate coenzymes in the synthesis of DNA precursors, folate antagonists have found widespread clinical use as antiproliferative and antimicrobial agents. It has been known for many years that the pernicious anemia that results from defects in vitamin B12 availability is due to induction of a secondary functional folate deficiency [3], but the metabolic interrelationship between vitamin B12 and folate remains controversial. More recently, the demonstration [4,5]. that periconceptional supplementation with low doses of folic acid reduces the incidence of neural tube defects has generated considerable clinical and public health interest, with proposals that the food supply be supplemented with folic acid, although the metabolic basis for this effect is not understood. Regimens for the treatment of folate-related disorders, the use of folate antagonists, and the advantages or potential disadvantages of folate supplementation require an understanding of normal folate metabolism and of the metabolic consequences of derangements in folate metabolism. This chapter presents an overview of current knowledge of the mechanisms by which folate is handled in the body and how tissue folate levels and homeostasis are regulated. The role of folates as coenzymes in one-carbon

Human Methionine Synthase cDNA CLONING, GENE LOCALIZATION, AND EXPRESSION

Human cDNAs for methionine synthase (5-methyltetrahydrofolate:L-homocysteine S-transmethylase; EC 2.1.1.13) have been isolated from fetal and adult liver and HepG2 libraries. The cDNAs span 7.2 kilobases (kb) and consist of a 394-base pair upstream untranslated region, a 3795-base pair open reading frame encoding a 1265-residue 140.3-kDa protein, and about 3 kb of 3′ region. The deduced protein sequence shares 53 and 63% identity with the Escherichia coli and the presumptive Caenorhabditis elegans proteins, respectively, and contains all residues implicated in B12 binding to the E. coli protein. Several potential polymorphisms and a cryptic splice deletion were detected in the coding region of the cDNAs. A polymorphism that results in a D919G modification in the protein is fairly common in human DNA samples. Northern analyses of poly(A) mRNA indicated two major species of about 8 and 10 kb in human tissues and some minor, partially spliced species. mRNA levels were highest in the pancreas, skeletal muscle, and heart of the adult and in the kidney in the fetus and were low in adult liver. Genomic clones were isolated and the 5′ region was analyzed. Exon 1 is preceded by a number of potential promoter sites, including an E box, CAAT boxes, and a GC box, but this region lacks a TATA element. The human methionine synthase gene was localized to chromosome region 1q42.3-43 by in situ hybridization.

Targeted Disruption of the Methionine Synthase Gene in Mice

ABSTRACT Alterations in homocysteine, methionine, folate, and/or B12 homeostasis have been associated with neural tube defects, cardiovascular disease, and cancer. Methionine synthase, one of only two mammalian enzymes known to require vitamin B12 as a cofactor, lies at the intersection of these metabolic pathways. This enzyme catalyzes the transfer of a methyl group from 5-methyl-tetrahydrofolate to homocysteine, generating tetrahydrofolate and methionine. Human patients with methionine synthase deficiency exhibit homocysteinemia, homocysteinuria, and hypomethioninemia. They suffer from megaloblastic anemia with or without some degree of neural dysfunction and mental retardation. To better study the pathophysiology of methionine synthase deficiency, we utilized gene-targeting technology to inactivate the methionine synthase gene in mice. On average, heterozygous knockout mice from an outbred background have slightly elevated plasma homocysteine and methionine compared to wild-type mice but seem to be otherwise indistinguishable. Homozygous knockout embryos survive through implantation but die soon thereafter. Nutritional supplementation during pregnancy was unable to rescue embryos that were completely deficient in methionine synthase. Whether any human patients with methionine synthase deficiency have a complete absence of enzyme activity is unclear. These results demonstrate the importance of this enzyme for early development in mice and suggest either that methionine synthase-deficient patients have residual methionine synthase activity or that humans have a compensatory mechanism that is absent in mice.

Biomarkers of vitamin B-12 status in NHANES: a roundtable summary

A roundtable to discuss the measurement of vitamin B-12 (cobalamin) status biomarkers in NHANES took place in July 2010. NHANES stopped measuring vitamin B-12–related biomarkers after 2006. The roundtable reviewed 3 biomarkers of vitamin B-12 status used in past NHANES—serum vitamin B-12, methylmalonic acid (MMA), and total homocysteine (tHcy)—and discussed the potential utility of measuring holotranscobalamin (holoTC) for future NHANES. The roundtable focused on public health considerations and the quality of the measurement procedures and reference methods and materials that past NHANES used or that are available for future NHANES. Roundtable members supported reinstating vitamin B-12 status measures in NHANES. They noted evolving concerns and uncertainties regarding whether subclinical (mild, asymptomatic) vitamin B-12 deficiency is a public health concern. They identified the need for evidence from clinical trials to address causal relations between subclinical vitamin B-12 deficiency and adverse health outcomes as well as appropriate cutoffs for interpreting vitamin B-12–related biomarkers. They agreed that problems with sensitivity and specificity of individual biomarkers underscore the need for including at least one biomarker of circulating vitamin B-12 (serum vitamin B-12 or holoTC) and one functional biomarker (MMA or tHcy) in NHANES. The inclusion of both serum vitamin B-12 and plasma MMA, which have been associated with cognitive dysfunction and anemia in NHANES and in other population-based studies, was preferable to provide continuity with past NHANES. Reliable measurement procedures are available, and National Institute of Standards and Technology reference materials are available or in development for serum vitamin B-12 and MMA.

Molecular cloning, characterization, and regulation of the human mitochondrial serine hydroxymethyltransferase gene.

The human mitochondrial serine hydroxymethyltransferase (mSHMT) gene was isolated, sequenced, and characterized. The 4.5-kilobase gene contains 10 introns and 11 exons, with all splice junctions conforming to the GT/AG rule. The 5′ promoter region contains consensus motifs for several regulatory proteins including PEA-3, Sp-1, AP-2, and a CCCTCCC motif common to many genes expressed in liver. Consensus TATA or CAAT sequence motifs are not present, and primer extension and 5′-rapid amplification of cDNA ends studies suggest that transcription initiation occurs at multiple sites. The mitochondrial leader sequence region of the deduced mRNA contains two potential ATG start sites, which are encoded by separate exons. The intervening 891-base pair intron contains consensus promoter elements suggesting that mSHMT may be transcribed from alternate promoters. 5′-Rapid amplification of cDNA ends analysis demonstrated that the first ATG is transcribed in human MCF-7 cells. However, transfection of Chinese hamster ovary cells deficient in mSHMT activity with the human mSHMT gene lacking exon 1 overcame the cells glycine auxotrophy and restored intracellular glycine concentrations to that observed in wild-type cells, showing that exon 1 is not essential for mSHMT localization or activity and that translation initiation from the second ATG is sufficient for mSHMT import into the mitochondria. Mitochondrial SHMT mRNA levels in MCF-7 cells did not vary during the cell cycle and were not affected by the absence of glycine, serine, folate, thymidylate, or purines from the media.

Folate assay: A comparison of radioassay and microbiological methods

Folate mono- and polyglutamate standards were assayed by a microbiological method and by four commercially available radioassay methods to evaluate the usefulness of radioassay techniques for the quantitation of naturally occurring folates. Folate monoglutamates exhibited different responses in the radioassay procedures, depending on the one-carbon constituent and oxidation state. Folate polyglutamates exhibited an increased response that varied depending on the folate concentration. The varied responses of folate mono- and polyglutamates in the radioassay procedures make this technique unsuitable for the determination of the mixture of folate derivatives that are normally encountered in biological extracts.

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.