Chemistry and Biochemical Functions

Folic acid (Figure 1) consists of a pterin moiety linked via a methylene group to a para-aminobenzoylgluta-mate moiety. Folic acid is the synthetic form of the vitamin; its metabolic activity requires reduction to the tetrahydrofolic acid (THF) derivative, addition of a chain of glutamate residues in 7-peptide linkage, and acquisition of one-carbon units.

COOH

COOH

para-aminobenzoylglutamate

-pteroic acid-

para-aminobenzoylglutamate

-pteroic acid-

—substituted pterin —>-Figure 1 Structural formula of tetrahydrofolate (THF) compounds. In tetrahydrofolic acid R = H; other substituents are listed in Table 1. The asterisk indicates the site of attachment of extra glutamate residues; the hatched line and double asterisk indicates the N5 and/or N10 site of attachment of one-carbon units.

Table 1 Structure and nomenclature of folate compounds (see Figure 1)

Compound

R

Oxidation state

5-formy ITHF

—CHO

Formate

10-formylTHF

—CHO

Formate

5-formiminoTHF

—CH=NH

Formate

5,10-methenylTHF

—CH=

Formate

5,10-methyleneTHF

—CH2—

Formaldehyde

5-methyITHF

—CH3

Methanol

One-carbon units at various levels of oxidation are generated metabolically and are reactive only as moieties attached to the N5 and/or N10 positions of the folate molecule (Table 1).

The range of oxidation states for folate one-carbon units extends from methanol to formate as methyl, methylene, methenyl, formyl, or formimino moieties. When one-carbon units are incorporated into folate derivatives, they may be converted from one oxidation state to another by the gain or loss of electrons.

The source of one-carbon units for folate One-carbon units at the oxidation level of formate can enter directly into the folate pool as formic acid in a reaction catalyzed by 10-formylTHF synthase (Figure 2). Entry at the formate level of oxidation can also take place via a catabolic product of histidine, formaminoglutamic acid. The third mode of entry at the formate level of oxidation involves the formation of 5-formylTHF from 5,10-methenylTHF by the enzyme serine hydroxy-methyl transferase (SHMT). The 5-formylTHF may be rapidly converted to other forms of folate.

The enzyme SHMT is involved in the entry of one-carbon units at the formaldehyde level of oxidation by catalyzing the transfer of the ^-carbon of serine to form glycine and 5,10-methyleneTHF. Other sources of one-carbon entry at this level of oxidation include the glycine cleavage system and the choline-dependent pathway; both enzyme systems generate 5,10-methy-lene in the mitochondria of the cell.

The removal and use of one-carbon units from folate Single-carbon units are removed from folate by a number of reactions. The enzyme 10-formylTHF dehydrogenase provides a mechanism for disposing of excess one-carbon units as carbon dioxide. (Folate administration to animals enhances the conversion of ingested methanol and formate to carbon dioxide, diminishing methanol toxicity.) Additionally, single-carbon units from 10-formylTHF are used for the biosynthesis of purines (Figure 2).

The one-carbon unit of 5,10-methyleneTHF is transferred in two ways. Reversal of the SHMT reaction produces serine from glycine, but since serine is also produced from glycolysis via phos-phoglycerate this reaction is unlikely to be important. However, one-carbon transfer from 5,10-methyleneTHF to deoxyuridylate to form thymidylic acid, a precursor of DNA, is of crucial importance to the cell. While the source of the one-carbon unit, namely 5,10-methyleneTHF, is at the formaldehyde level of oxidation, the one-carbon unit transferred to form thymidylic acid appears at the methanol level of oxidation. Electrons for this reduction come from THF itself to generate dihydrofolate as a product. The dihydro-folate must in turn be reduced back to THF in order to accept further one-carbon units.

A solitary transfer of one-carbon units takes place at the methanol level of oxidation. It involves the transfer of the methyl group from 5-methylTHF to homocysteine to form methionine and THF. This reaction is catalyzed by the enzyme methionine synthase and requires vitamin B12 as a cofactor. The substance 5-methylTHF is the dominant folate in the body, and it remains metabolically inactive until it is demethylated to THF, whereupon polyglu-tamylation takes place to allow subsequent folate-dependent reactions to proceed efficiently.

Clinical implications of methionine synthase inhibition The inhibition of methionine synthase due to vitamin B12 deficiency induces megaloblastic anemia that is clinically indistinguishable from that caused by folate deficiency. The hematological effect in both cases results in levels of 5,10-methyleneTHF that are inadequate to sustain thymidylate biosynthesis. Clinically, it is essential to ascertain whether the anemia is the result of folate deficiency or vitamin B12 deficiency by differential diagnostic techniques. Vitamin B12 is essential for the synthesis of myelin in nerve tissue, a function probably related to methionine production from the methionine synthase reaction and the subsequent formation of S-adenosyl-methionine. Hence, vitamin B12 deficiency probably leads to nervous disorders in addition to the hematological effects. While the latter respond to treatment with folic acid, the neurological effects do not. Thus, inappropriate administration of folic acid in patients with vitamin B12 deficiency may treat the anemia but mask the progression of the neurological defects. Where possible, vitamin B12 and folate statuses should be checked before giving folate supplements to treat megaloblastic anemia. The main objection to fortifying food with folate is the potential to mask

Ingestion food folates (monoglutamates, polyglutamates, and folic acid) Excretion intact folate intact folate

(enterohepatic circulation):

Deconjugation

(conjugases)

folate catabolites + intact folate monoglutamates

IAbsorption (via small bowel)

I Transport (mainly 5-CH3-THF)

5,10-methylene tetrahydrofolate reductase folate catabolites + intact folate

I Transport (mainly 5-CH3-THF)

5,10-methylene tetrahydrofolate reductase

FADH,

5-formyltetrahydrofolate cyclodehydrase

5-CHO-THF

Figure 2 Physiology and metabolism of folate. GAR, glycinamide ribonucleotide; FGAR, formylglycinamide ribonucleotide; AICAR: aminoimidazolecarboxamide ribonucleotide; figlu, formiminoglutamic acid; IMP, inosine monophosphate.

jtFAD

FADH,

5,10-methylene THF dehydrogenase NADPH

5-formyltetrahydrofolate cyclodehydrase

5-CHO-THF

Figure 2 Physiology and metabolism of folate. GAR, glycinamide ribonucleotide; FGAR, formylglycinamide ribonucleotide; AICAR: aminoimidazolecarboxamide ribonucleotide; figlu, formiminoglutamic acid; IMP, inosine monophosphate.

vitamin B12 deficiency in the elderly, who are most prone to it.

In summary, the biochemical function of folate coenzymes is to transfer and use these one-carbon units in a variety of essential reactions (Figure 2), including de novo purine biosynthesis (formylation of glycinamide ribonucleotide and 5-amino-4-imidazole carboxamide ribonucleotide), pyrimidine nucleotide biosynthesis (methylation of deoxyuridylic acid to thy-midylic acid), amino-acid interconversions (the interconversion of serine to glycine, catabolism of histidine to glutamic acid, and conversion of homocysteine to methionine (which also requires vitamin B12)), and the generation and use of formate.

Many of the enzymes involved in these reactions are multifunctional and are capable of channelling substrates and one-carbon units from reaction to reaction within a protein matrix. Another feature of intracellular folate metabolism is the compart-mentation of folate coenzymes between the cytosol and the mitochondria. For instance, 5-methylTHF is associated with the cytosolic fraction of the cell, whereas most of 10-formylTHF is located in the mitochondria. Similarly, some folate-dependent enzymes are associated with one or other compartment, though some are found in both. Metabolic products of folate-dependent reactions, such as serine and glycine, are readily transported between the two locations, but the folate coenzymes are not.

Folate Deficiency and Hyperhomocysteinemia An important consequence of folate deficiency is the inability to remethylate homocysteine (Figure 2). Indeed, there is an inverse correlation between the levels of folate and those of homocysteine in the blood of humans. Many clinical studies, beginning with the observations of children with homocystei-nuria presenting with vascular abnormalities and thromboembolism, have demonstrated an association between hyperhomocysteinemia and an increased risk of premature atherosclerosis in the coronary, carotid, and peripheral vasculatures. Even mild hyperhomocysteinemia is recognized to be an independent risk factor for cardiovascular disease. The risk of heart disease was found to increase proportionately in most, but not all, studies, throughout the full of range of blood homo-cysteine concentrations. An increase in plasma homocysteine of 5 mmoll-1 is associated with a combined odds ratio of 1.3 for cardiovascular disease. Plasma homocysteine is usually shown to be a greater risk factor for cardiovascular disease in prospective studies than in retrospective studies, probably because the populations in the former studies are older. Metabolically, homocysteine may be disposed of by the methionine synthase reaction (dependent on folate and vitamin B12), the transsulfuration pathway (dependent on vitamin B6), and the choline degradation pathway. Marginal deficiencies of these three vitamins are associated with hyperhomocysteinemia. Of the three vitamins, however, folic acid has been shown to be the most effective in lowering levels of homocysteine in the blood. Convincing evidence of the potential role of folate intake in the prevention of vascular disease has come from a significant inverse relationship between serum folate levels and fatal coronary heart disease. While most studies have focused on the homocysteine-lowering effects of folate, other benefits have also been reported. Potential mechanisms include anti-oxidant actions and interactions with the enzyme endothelial nitric oxide synthase.

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