Inhibition of buttermilk xanthine oxidase by folate analogues and derivatives

Folic acid has been reported recently to be an effective agent for the treatment of hyperuricernia, although conflicting data exist. The relative inhibitory activities of this compound and its breakdown products, pterin aldehyde and 6-hydroxymethylpterin, for the enzyme xanthine oxidase have not been clear. In this study, folic acid purified from these two compounds competitively inhibited buttermilk xanthine oxidase under aerobic conditions by a mechanism kinetically distinct from that of pterin aldehyde, with an inhibition constant (K_i) of 0.12 μM. Methotrexate, leucovorin and N⁵-methylH₄folate were competitive inhibitors of the enzyme with K_i values ranging from 12 to 53 μM. MethyleneH⁴folate, H₂folate and H₄folate did not inhibit xanthine oxidase. N⁵-MethylH₄folate could not be evaluated by the reduction of cytochrome c because of the nonenzymatic oxidation of this folate derivative by cytochrome c to a compound, shown to be N⁵-methylH₂folate. Unless high intracellular concentrations of unchanged folic acid, pterin aldehyde or hydroxymethylpterin can be achieved or folic acid proves to be a more effective inhibitor of reduced than of oxidized enzyme, it is unlikely that this compound will be an effective clinical agent for the inhibition of xanthine oxidase.