Analysis and biochemistry of blood folate

Although the analysis of low plasma concentrations of 5-methyltetrahydrofolate by several specific HPLC methods has been reported, considerably fewer routine chromatographic techniques exist for the analysis of specific folate coenzymes in the erythrocyte where a nonspecific bioassay indicates that the vitamin achieves a level 10 times higher than that in plasma. By using three separate folypolyglutamate deconjugation procedures and combining an extraction technique which adequately preserves all native folate coenzymes with an HPLC technique utilizing fluorescence, diode array, and off-line radioassay detection capable of resolving all crucial native folates in their monoglutamyl forms, we were unable to demonstrate levels of 5-methyltetrahydrofolate in whole blood hemolysate beyond what might be expected from the plasma component. While the exact nature of erythrocyte folate could not be ascertained, we provide evidence that a proportion of it may exist at the formyl level of oxidation. The complex pH and enzymatic interrelationship between folate coenzymes at the formyl oxidation level is discussed in terms of our extraction technique and findings, as well as in a broader biological context. This paper also describes a simple acid precipitation technique for measuring plasma 5-methyltetrahydrofolate, as well 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 parameters and identifying individual coenzymes. 10-Formyltetrahydrofolate and 5-methyltetrahydrofolate are the two most important one-carbon-substituted folate coenzymes. 10-Formyltetrahydrofolate is unavailable commercially, probably due to its instability. We chart the chemical synthesis of this important coenzyme and show that it and what is thought to be 5,10-hydroxymethylenetetrahydrofolate are actually minor products compared to the parent 5,10-methenyltetrahydrofolate and the ultimate reaction product, 5-formyltetrahydrofolate. Since intraerythrocyte folate binds to a specific hemoglobin site, we ascertained the total number of binding sites on hemoglobin (Bmax) and the equilibrium dissociation constant (Kd) for 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, 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-methyltetrahydrofolate than for the other folate derivatives (Kd = 1.2 x 10(-3) M), while rather surprisingly, methotrexate had a higher affinity for hemoglobin than did 5-formyltetrahydrofolate (Kd = 2.5 x 10(-3) and 3.7 x 10(-2) M, respectively.