The Role of Folate in Methionine Metabolism

In addition to its role in the synthesis of proteins and the polyamines sper-midine and spermine, the main metabolic role of methionine is as a methyl donor in a wide variety of biosynthetic reactions.

As shown in Figure 10.9, the methyl donor is S-adenosyl methionine, which is demethylated to S-adenosyl homocysteine. After removal of the adenosyl group, homocysteine may undergo one of two metabolic fates: remethylation to methionine or condensation with serine to form cystathionine, followed by cleavage to yield cysteine - the transulfuration pathway (Section 9.5.5). Cystathionine synthetase has a relatively low Km compared with normal intra-cellular concentrations of homocysteine. It functions at a relatively constant rate, and under normal conditions, most homocysteine will be remethylated to methionine.

Figure 10.9. Metabolism of methionine. Methionine adenosyltransferase, EC 2.5.1.6; methionine synthetase, EC 2.1.1.13 (vitamin B12-dependent) and EC 2.1.1.5 (betaine as a methyl donor); cystathionine p-synthetase, EC 4.2.1.22; and y-cystathionase, EC 4.4.1.1.

There are two separate homocysteine methyltransferases in most tissues. One uses methyl-tetrahydrofolate as the methyl donor and has vitamin B12 (cobalamin; 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 betaine (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 essential for the polyglutamylation and intracellular accumulation of folate.

10.3.4.1 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 nitrous 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 methionine, 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 methionine 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.

Both in vivo and with isolated tissue preparations from vitamin B12-deficient animals, additional methionine can alleviate some of the effects on folate metabolism, elevating the liver concentration of folate and restoring a more normal proportion of unsubstituted tetrahydrofolate; increasing the metabolism of histidine, thus reducing the excretion of formiminoglutamate; reducing the excretion of formate and intermediates of purine synthesis; and restoring normal suppression of the incorporation of [3H]TMP into DNA by dUMP. The additional methionine increases the availability of S-adenosyl me-thionine, thus increasing reactivation of the residual methionine synthetase and permitting increased demethylation of methyl-tetrahydrofolate. The increased concentration of S-adenosyl methionine will also restore normal inhibition of methylene-tetrahydrofolate reductase.

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 of vitamin B12 (Krebs etal., 1976).

This has been called the methyl folate trap and appears to explain many of the similarities between the symptoms and metabolic effects of folate and vitamin B12 deficiency, although it does not provide a completely satisfactory explanation (Chanarin et al., 1985).

10.3.4.2 Hyperhomocysteinemia and Cardiovascular Disease Children with homocystinuria caused by a genetic defect of cystathionine syn-thetase suffer multiple thromboses, and if untreated commonly die in their teens. A number of epidemiological studies during the 1990s identified moderate elevation of plasma homocysteine (significantly lower than seen in ho-mocystinuric children) as an independent risk factor for cardiovascular disease (D'Angelo and Selhub, 1997; Selhub et al., 2000; Herrmann, 2001). As shown in Table 10.1, homocysteine has a variety of potential atherogenic, hypertensive,

Table 10.1 Adverse Effects of Hyperhomocysteinemia Atherogenic

• Especially in the presence of transition metal ions, homocysteine can undergo redox cycling ^ O2-, thus a possibility of oxidative damage to LDL

• May react with cysteine-SH groups and modify apolipoproteins, thus impaired uptake by LDL receptors

Hypertensive

Reacts with NO ^ S-nitrosohomocysteine - this reacts with O2 ^ homocysteine + NO3- + O2-

• Generation of O2- and loss of vasodilation action of NO Procoagulant

Inhibits or downregulates anticoagulants

• Decreases prostacyclin synthesis

• Decreases activation of protein C

• Decreases thrombomodulin expression

• Suppresses the expression of heparan sulfate

• Decreases fibrinolysis Activates procoagulants

• Increased Factor V activity

• Increased tissue clotting factor activity

Causes desquamation of vascular endothelium and impairs regeneration

• Endothelium has anticoagulant activity Causes proliferation of vascular smooth muscle

• Vascular smooth muscle, when exposed, has potent procoagulant activity because of tissue clotting factor

Increases platelet coagulability and so activates platelet aggregation Connective tissue abnormalities Inhibition of lysyl oxidase ^ disturbance of collagen and elastin cross-linking (see Section 13.3.3)

In the presence of homocysteine, fibroblasts produce excessively sulfated proteoglycans

• The product is granular rather than fibrillar

• Excessively sulfated proteoglycans will attract and bind e-amino groups of lysine in lipoproteins

LDL, low-density lipoprotein; NO, nitric oxide; SH, sulfhydryl.

and procoagulant actions (Stamler and Slivka, 1996; Welch and Loscalzo, 1998; Herrmann, 2001).

Deficiency of vitamins B6, B12, or folate are all associated with elevated plasma homocysteine, with vitamin B6 deficiency as a result of impaired activity of cystathionine synthetase (Section 9.5.5) and folate and vitamin B12 as a result of impaired activity of methionine synthetase (Section 10.3.4). In subjects with apparently adequate intakes of vitamins B6 and B12, supplements of these two vitamins have little or no effect on fasting plasma homocysteine, although additional vitamin B6 reduces the plasma concentration of homocysteine after a test dose of methionine. By contrast, supplements of folic acid do reduce plasma homocysteine in hyperhomocysteinemic subjects, despite apparently adequate folate status (Ubbink et al., 1994; Ubbink, 1997; Homocysteine Lowering Trialists' Collaboration, 1998; Refsum et al., 1998). This is presumably as a result of overcoming the effect of being homozygous for the thermolabile variant of methylene-tetrahydrofolate reductase (Section 10.3.2.1). As discussed in Section 10.12, this has led to mandatory enrichment of cereal products with folic acid in the United States and some other countries, although it remains to be demonstrated whether or not there is a causative relationship between hyperhomocysteinemia and cardiovascular disease, and therefore whether lowering homocysteine will have any beneficial effect. There is a stronger relationship between homocysteine and cardiovascular disease in cross-sectional and retrospective case-control studies than in prospective studies (Meleady and Graham, 1999).

Although, as shown in Table 10.1, there are plausible mechanisms to suggest that homocysteine is a causative factor in cardiovascular disease, it is possible that hyperhomocysteinemia is a result of the renal damage that is an early event in cardiovascular disease, thus a proxy marker of disease rather than a causative factor (Jacobsen, 1998; Langman and Cole, 1999; Kircher and Sinzinger, 2000). In chronic renal failure, hyperhomocysteinemia is associated with cardiovascular disease, probably because of both impaired excretion of homocysteine and impaired activity of betaine homocysteine methyl-transferase; elevated plasma dimethylglycine predicts plasma homocysteine (McGregor etal., 2001).

10.4 TETRAHYDROBIOPTERIN

Tetrahydrobiopterin is not a vitamin, because it can be synthesized from GTP, as shown in Figure 10.2 (Thony et al., 2000). It is the coenzyme for mixed-function oxidases: phenylalanine, tyrosine, and tryptophan hydroxylases; alkyl glycerol monoxygenase, which catalyzes the cleavage of alkyl glycerol ethers; and nitric oxide synthase in the formation of nitric oxide. In addition to its coenzyme role, tetrahydrobiopterin has a direct effect on neurons, acting to stimulate dopamine release via a cAMP-dependent protein kinase and a calcium channel (Koshimura et al., 2000).

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