ANALYSIS AND BIOCHEMISTRY OF BLOOD FOLATE

Although the analysis of low plasma concentrations of 5-methyltetrahyd rofolate by several specific HPLC methods has been reported, considera bly fewer routine chromatographic techniques exist for the analysis of specific folate coenzymes in the erythrocyte where a nonspecific bioa ssay indicates that the vitamin achieves a level 10 times higher than that in plasma. By using three separate folylpolyglutamate deconjugati on procedures and combining an extraction technique which adequately p reserves all native folate coenzymes with an HPLC technique utilizing fluorescence, diode array, and off-line radioassay detection capable o f resolving all crucial native folates in their monoglutamyl forms, we were unable to demonstrate levels of 5-methyltetrahydrofolate in whol e blood hemolysate beyond what might be expected from the plasma compo nent. While the exact nature of erythrocyte folate could not be ascert ained, we provide evidence that a proportion of it may exist at the fo rmyl level of oxidation. The complex pH and enzymatic interrelationshi p between folate coenzymes at the formyl oxidation level is discussed in terms of our extraction technique and findings, as well as in a bro ader biological context. This paper also describes a simple acid preci pitation technique for measuring plasma 5-methyltetrahydrofolate, as w ell as providing comprehensive data on the chromatographic behavior of all the folylmonoglutamates in reversed-phase and weak anion-exchange modes, including useful spectral data for optimizing detection parame ters and identifying individual coenzymes. 10-Formyltetrahydrofolate a nd 5-methyltetrahydrofolate are the two most important one-carbon-subs tituted folate coenzymes. 10-Formyltetrahydrofolate is un-available co mmercially, probably due to its instability. We chart the chemical syn thesis of this important coenzyme and show that it and what is thought to be 5,10-hydroxymethylenetetrahydrofolate are actually minor produc ts compared to the parent 5,10-methenyltetrahydrofolate and the ultima te reaction product, 5-formyltetrahydrofolate. Since intraerythrocyte folate binds to a specific hemoglobin site, we ascertained the total n umber of binding sites on hemoglobin (B-max) and the equilibrium disso ciation constant (K-d) for 5-methyltetrahydrofolate, 5-formyltetrahydr ofolate, and the antimetabolite methotrexate. Binding affinities were consistent with a low-affinity, low-capacity interaction for all three . It was demonstrated that hemoglobin has a greater affinity for 5-met hyltetrsrhvdrofolate than for the other folate derivatives (K-d = 1.2 x 10(-3) M), while rather surprisingly, methotrexate had a higher affi nity for hemoglobin than did 5-formyltetrahydrofolate (K-d = 2.5 x 10( -3) and 3.7 x 10(-9) M, respectively). (C) 1996 Academic Press, Inc.