5-Methyl tetrahydrofolate

Metabolism and Mobilization. On entry of vitamin B 2 into the cell, considerable metaboHsm of the vitamin takes place. Co(III)cobalamin is reduced to Co(I)cobalamin, which is either methylated to form methylcobalamin or converted to adenosylcobalamin (coenzyme B>22)- The methylation requires methyl tetrahydrofolate. 

The methylation of deoxyuridine monophosphate (dUMP) to thymidine monophosphate (TMP), catalyzed by thymidylate synthase, is essential for the synthesis of DNA. The one-carbon fragment of methy-lene-tetrahydrofolate is reduced to a methyl group with release of dihydrofolate, which is then reduced back to tetrahydrofolate by dihydrofolate reductase. Thymidylate synthase and dihydrofolate reductase are especially active in tissues with a high rate of cell division. Methotrexate, an analog of 10-methyl-tetrahydrofolate, inhibits dihydrofolate reductase and has been exploited as an anticancer drug. The dihydrofolate reductases of some bacteria and parasites differ from the human enzyme inhibitors of these enzymes can be used as antibacterial drugs, eg, trimethoprim, and anti-malarial drugs, eg, pyrimethamine. 

When acting as a methyl donor, 5-adenosylmethionine forms homocysteine, which may be remethylated by methyltetrahydrofolate catalyzed by methionine synthase, a vitamin Bj2-dependent enzyme (Figure 45-14). The reduction of methylene-tetrahydrofolate to methyltetrahydrofolate is irreversible, and since the major source of tetrahydrofolate for tissues is methyl-tetrahydrofolate, the role of methionine synthase is vital and provides a link between the functions of folate and vitamin B,2. Impairment of methionine synthase in Bj2 deficiency results in the accumulation of methyl-tetrahydrofolate—the folate trap. There is therefore functional deficiency of folate secondary to the deficiency of vitamin B,2. 

Supplements of 400 Ig/d of folate begun before conception result in a significant reduction in the incidence of neural mbe defects as found in spina bifida. Elevated blood homocysteine is an associated risk factor for atherosclerosis, thrombosis, and hypertension. The condition is due to impaired abihty to form methyl-tetrahydrofolate by methylene-tetrahydrofolate reductase, causing functional folate deficiency and resulting in failure to remethylate homocysteine to methionine. People with the causative abnormal variant of methylene-tetrahydrofolate reductase do not develop hyperhomocysteinemia if they have a relatively high intake of folate, but it is not yet known whether this affects the incidence of cardiovascular disease. 

Methyl tert-butylhydroquinone, 20 105 Methyl-tertiary-butyl ether. See Methyl-tert-butyl ether (MTBE) Methyltestosterone, registered for use in aquaculture in Australia, 3 222t Nb-Methyl tetrahydrofolic acid, 25 802 2-Methyltetrahydrofuran (METHF),... 

Fig. 14.1 Cellular pathway of methotrexate. ABCBl, ABCCl-4, ABC transporters ADA, adenosine deaminase ADP, adenosine diphosphate AICAR, aminoimidazole carboxamide ribonucleotide AMP, adenosine monophosphate ATIC, AICAR transformylase ATP, adenosine triphosphate SjlO-CH -THF, 5,10-methylene tetrahydrofolate 5-CHj-THF, 5-methyl tetrahydro-folate DHFR, dihydrofolate reductase dTMP, deoxythymidine monophosphate dUMP, deoxy-uridine monophosphate FAICAR, 10-formyl AICAR FH, dihydrofolate FPGS, folylpolyglutamyl synthase GGH, y-glutamyl hydrolase IMP, inosine monophosphate MTHFR, methylene tetrahydrofolate reductase MTR, methyl tetrahydrofolate reductase MTX-PG, methotrexate polyglutamate RFCl, reduced folate carrier 1 TYMS, thymidylate synthase. Italicized genes have been targets of pharmacogenetic analyses in studies published so far. (Reproduced from ref. 73 by permission of John Wiley and Sons Inc.)...

The compounds that are the immediate methyl gronp donors are methyltetra hydrofate (CH3-FH4) and S-adenosyl methionine (SAM) (see Figure 15.2). These are involved in, at least, five key reactions or processes which are summarised in Figure 15.4. Complexity arises in the topic of methyl group transfer in formation and reformation of the methylating compounds 5-adenosylmethione and methyl tetrahydrofolate. There are four important reactions in the formation utilisation and then the reformation of 5-adenosylmethionine as follows ... 

Figure 15.5 Four reactions involved in methylatlon. The reactions are (1) formation of S-adenosylmethlonIne (SAM) (11) transfer of methyl group to an acceptor (111) conversion of S-adenosylmethlonIne to homocysteine (Iv) conversion of homocysteine to methionine using methyl tetrahydrofolate as the methyl donor with the formation of FH4.

Figure 15.6 Formation of methyl tetrahydrofolate and SAM from serine. Reaction (1) is described in Appendix 8.3. Reaction (ii) is Figure 15.5 and the several reactions represent in reaction (iv) are discribed in Figure 15.4.

A Shibukawa, DK Lloyd, IW Wainer. Simultaneous chiral separation of leu-covorin and its major metabolite 5-methyl-tetrahydrofolate by capillary electrophoresis using cyclodextrins as chiral selector. Estimation of the formation constants and mobility of the solute—cyclodextrin complexes. Chromatographia 35 419-429, 1993. 

Incubation of tryptamine derivatives with 5-methyl-tetrahydrofolic acid and an enzyme preparation from brain gives tryptolines. Dopamine and its derivatives form related tetrahydroisoquinolines such as the product that arises from reaction with acetaldehyde (see Eq. 30-5). This product has been found in elevated amounts in alcoholics (who synthesize excess acetaldehyde), in phenylketonurics, and in L-dopa-treated patients with Parkinson disease.1136... 

Vitamin B12 activates methyl groups for methionine biosynthesis by binding them to the Co ion at the sixth position. The methyl group donor to Bi2 is 5-methyl tetrahydrofolate. The methyl-Bi2 donates its methyl group to homocysteine, forming methionine. 

Figure 21-3. The methionine synthase reaction. Methionine synthase catalyzes the remethylation of homocysteine to methionine. In the first half reaction (1), a methyl group is transferred from 5-methyl tetrahydrofolate (5-MTHF) to the reduced form of cobalamin [Cob(I)], generating methyl-cobalamin [Methyl-Cob(III)] and tetrahydrofolate (THF). During the second half reaction (2), the methyl group is transferred from methylcobalamin to homocysteine, generating methionine. During the catalytic reaction, Cob(I) occasionally becomes oxidized, producing an inactive form of cobalamin, cob(II)alamin [Cob(II)]. The enzyme methionine synthase reductase (MTRR) then reactivates Cob(II) through reductive methylation, producing methyl-Cob(III). SAM, 5-adenosylmethionine SAH, 5-adeno-sylhomocysteine.

The most widely used methyl donor for enzymatic methyl transfer is the cofactor S-adenosyl-L-methionine (SAM). The methyl moiety on the L-methionine is supplied by another known methyl donor, N5-methyl tetrahydrofolate.30 To date, numerous enzymes that perform SAM- dependent methylation reactions have been described in plants, and several reports attempting to sort out their evolutionary relationships have been published.31- 3... 

Cyanocobalamine is a component of several coenzymes and has an effect on nucleic acid formation through its action in cycling 5-methyl-tetrahydrofolate back into the folate pool. The most important dietary sources of the vitamin are animal products. Vitamin Bl2 is also produced by many microorganisms. It is not surprising that vitamin B12 deficiency of dietary origin only occurs in vegetarians. 

Verwei, M., Arkbage, K., Havenaar, R., Van den Berg, H., Witthoft, C., and Schaafsma, G. (2003), Folic acid and 5-methyl-tetrahydrofolate in fortified milk are bioaccessible as determined in a dynamic in vitro gastrointestinal model,/. Nutr., 133, 2377-2383. 

Most of the dietary folate undergoes reduction and methylation within the intestinalmucosa and what enters theportal bloodstream is alargely 5-methyl-tetrahydrofolate. Single doses of more than about 200 /xg of folic acid saturate the intestinal dUiydrofolate reductase, so that free folic acid is absorbed and circulates in the bloodstream. It can be taken up by tissues, reduced to tetrahy-drofolate, and utilized. 

There is considerable enterohepatic circulation of folate, equivalent to about one-third of the dietary intake. Methyl-tetrahydrofolate is secreted in the bUe, then reabsorbed in the jejunum together with food folates. In experimental animals, bUe drainage for 6 hours results in a reduction of serum folate to 30% to 40% of normal (Steinberg et al., 1979). There is very litde loss of folate jejunal absorption is very efficient, and the fecal excretion of 450 nmol (200 /xg) of folates per day largely represents synthesis by intestinal flora and does not reflect intake to any significant extent. 

Methyl-tetrahydrofolate from the intestinal mucosa circulates bound to albumin and is the main vitamer for uptake by extrahepatic tissues. Small amounts of other one-carbon substituted folates also circulate (about 10% to 15% of plasma folate is 10-formyl-tetrahydrofolate) and are also available for tissue uptake. There are two mechanisms for tissue uptake of folate ... 

The reduced folate transporter is a transmembrane protein with a high affinity for methyl-tetrahydrofolate and a low affinity for other vitamers. It is especially active in enterocytes and renal tubule epithelium, but is also found in other cells (Sirotnak and Tolner, 1999). 

Demethylated tetrahydrofolate monoglutamate is released hy extrahepatic tissues and is transported hound to a plasma folate binding protein similar to that in milk. It has a very low affinity for methyl-tetrahydrofolate and other one-carbon substituted derivatives. It functions mainly to return folate to the liver, where it is either conjugated for storage or methylated to 5-methyl-tetrahydrofolate that is secreted in the bile. 

The principal substrate for glutamylation is free tetrahydrofolate one-carbon substituted folates are poor substrates. Because the main circulating folate, and the main form that is taken up into tissues, is methyl-tetrahydrofolate, demethylation by the action of methionine synthetase (Section 10.3.3) is essential for effective metabolic trapping of folate. In vitamin B12 deficiency, when methionine synthetase activity is impaired, there wUl be impairment of the retention of folate in tissues. 

There is very little urinary loss of folate, some 5 to 10 nmol of microbiologically active material per day. Not only is most folate in plasma bound to proteins (either folate binding protein for unsubstituted folate or albumin for methyl-tetrahydrofolate), and thus protected from glomerular filtration, but also the renal brush border has a high concentration of folate binding protein that acts to reabsorb any that is filtered. 

Methylene-, methenyl-, and 10-formyl-tetrahydrofolates are freely interconvertible. The two activities involved - methylene-tetrahydrofolate dehydrogenase and methenyl-tetrahydrofolate cyclohydrolase - form a trifunctional enzyme with 10-formyl-tetrahydrofolate synthetase (Paukert et al., 1976). This means that single-carbon fragments entering the folate pool in any form other than as methyl-tetrahydrofolate can be readily available for any of the biosynthetic reactions shown in Figure 10.4. 

Methylene-Tetrahydrofolate Reductase The reduction of methylene-tetrahydrofolate to methyl-tetrahydrofolate, shown in Figure 10.7, is catalyzed hy methylene-tetrahydrofolate reductase, a flavin adenine dinucleotide-dependent enzyme during the reaction, the pteridine ring of the substrate is oxidized to dihydrofolate, then reduced to tetrahydrofolate by the flavin, which is reduced by nicotinamide adenine dinucleotide phosphate (NADPH Matthews and Daubner, 1982). The reaction is irreversible under physiological conditions, and methyl-tetrahydrofolate - which is the main form of folate taken up into tissues (Section 10.2.2) - can only be utilized after demethylation catalyzed by methionine synthetase (Section 10.3.4). 

Most dietary folate is reduced and methylated to methyl-tetrahydro-folate in the intestinal mucosa (Section 10.2.1). Intestinal mucosal ceUs have a rapid turnover, typicaUy 48 hours from proliferation in the crypt to shedding at the tip of the vUlus. This means that an unstable variant of the enzyme, which loses activity over a shorter time than the normal enzyme, is probably irrelevant in ceUs that have such a rapid turnover. A high intake of folate would therefore result in a relatively high rate of supply of methyl-tetrahydrofolate to cells, arising from newly absorbed folate, so that impaired turnover of folate within cells would be less important. 

There are two separate homocysteine methyltransferases in most tissues. One uses methyl-tetrahydrofolate as the methyl donor and has vitamin B12 (cohalamin Section 10.8.1) as its prosthetic group. This enzyme is also known as methionine synthetase it is the only homocysteine methyltransferase in the central nervous system. The other enzyme utilizes hetaine (an intermediate in the catabolism of choline Section 14.2.1) as the methyl donor and does not require vitamin B12. 

Unlike most enzymes utilizing or metabolizing tetrahydrofolate, methionine synthetase has equal activity toward methyl-tetrahydrofolate mono- and polyglutamates. As discussed in Section 10.2.2, demethylation of methyl-tetrahydrofolate is essendal for the polyglutamylation and intracellular accumulation of folate. 

The Methyl Folate Trap Hypothesis The reduction of meth-ylene-tetrahydrofolate to methyl-tetrahydrofolate is irreversible (Section 10.3.2.1), and the major source of folate for tissues is methyl-tetrahydrofolate. The only metabolic role of methyl-tetrahydrofolate is the methylation of homocysteine to methionine, and this is the only way in which methyl-tetrahydrofolate can be demethylated to yield free tetrahydrofolate in tissues. Methionine synthetase thus provides the link between the physiological functions of folate and vitamin B12. 

Impairment of methionine synthetase activity, for example, in vitamin B12 deficiency or after prolonged exposure to nitrous oxide (Section 10.9.7), will result in the accumulation of methyl-tetrahydrofolate. This can neither be utilized for any other one-carbon transfer reactions nor demethylated to provide free tetrahydrofolate. 

Experimental animals that have been exposed to ititrous oxide to deplete vitamin B12 show an increase in the proportion of liver folate present as methyl-tetrahydrofolate (85% rather than the normal 45%), largely at the expense of unsubstituted tetrahydrofolate and increased urinary loss of methyl-tetrahydrofolate (Horne et al., 1989). Tissue retention of folate is impaired because methyl-tetrahydrofolate is a poor substrate for polyglutamyl-folate synthetase, compared with unsubstituted tetrahydrofolate (Section 10.2.2.1). As a result of this, vitamin B12 deficiency is frequently accompanied by biochemical evidence of functional folate deficiency, including impaired metabolism of histidine (excretion of formiminoglutamate Section 10.3.1.2) and impaired thymidylate synthetase activity (as shown by abnormally low dUMP suppression Section 10.3.3.3), although plasma concentrations of methyl-tetrahydrofolate are normal or elevated. 

This functional deficiency of folate is exacerbated by the associated low concentrations of methionine and S-adenosyl methioitine, although most tissues (apart from the central nervous system) also have betaine-homocysteine methyltransferase that may be adequate to maintain tissue pools of methionine. Under normal conditions S-adenosyl methioitine inhibits methylene-tetrahydrofolate reductase and prevents the formation of further methyl-tetrahydrofolate. Relief of this inhibition results in increased reduction of one-carbon substituted tetrahydrofolates to methyl-tetrahydrofolate. 

The activity of 10-formyl-tetrahydrofolate dehydrogenase, which catalyzes the oxidation of 10-formyl tetrahydrofolate to CO2 and tetrahydrofolate, is reduced at times of low methionine availability as a means of conserving valuable one-carbon fragments. Therefore, there is no sink for one-carbon substituted tetrahydrofolate, and increasing amounts of folate are trapped as methyl-tetrahydrofolate that cannot be used because of the lack ofvitantin B12 (Krebs etal., 1976). 

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