Tetrahydrofolic acid assay

B. E. Wright, J. Biol. Chem. 219, 873 (1956). It is probable that the natural cofactors in animal systems also are conjugates of tetrahydrofolic acid. Assays of fresh extracts for folio acid vitamin activity reveal the presence of very little active material in most tissues, but the amounts increase upon autolysis. Both oxidized and reduced pteridines occur as folic acid derivatives containing one or more additional glutamic acid residues. Enzymes that hydrolyze peptide bonds to liberate folic acid activity have been found in many organisms and have been named conjugases [E. L. R. Stokstad, Vitamins 3, 163 (1954)]. 

Patients sustain convulsions and neurological deterioration. The urine contains low levels of the metabolites of serotonin, norepinephrine and dopamine. The reductase also plays a role in the maintenance of tetrahydrofolate levels in brain, and some patients have had low folate levels in the serum and CNS. Treatment has been attempted with tryptophan and carbidopa to improve serotonin homeostasis and with folinic acid to replete diminished stores of reduced folic acid. This therapy is sometimes effective. Diagnosis involves assay of DHPR in skin fibroblasts or amniotic cells. Phenylalanine hydroxylase activity is normal. 

When the purified material was treated with alkali at 100°C. (conditions which destroy the cofactor activity), a fluorescent compound was formed which had an activation peak at 380-400 m/t and a fluorescent peak at 470-480 m/A. Since these fluorescent characteristics resembled those reported for folic acid and folinic acid, various folic acid derivatives were tested for cofactor activity. Tetrahydrofolate was found to be highly active, while folic acid, dihydrofolic acid, and 5-formyltetrahydrofolate were completely inactive. Anhydroleucovorin (5, 10-methenyltetra-hydrofolate), which would be expected to hydrolyze to 10-formyltetra-hydrofolate under the conditions of the assay, showed some activity (Kaufman, 1958b). 

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