Physicochemical effect of pH and antioxidants on mono- and triglutamate forms of 5-methyltetrahydrofolate, and evaluation of vitamin stability in human gastric juice: Implications for folate bioavailability

This report examines the physico-chemical properties of mono and triglutamate forms of 5-methyltetrahydrofolate_n (5CH₃-H₄-PteGlu_n) in human gastric juice and under artificial conditions, typical of the upper gastrointestinal tract. Degradation of 5CH₃-H₄-PteGlu_n to 5-methyldihydrofolate_n (5CH₃-5,6-H₂PteGlu_n) and on to the C₉-N₁₀ scission product p-aminobenzoylglutamate (P-ABG), and the salvage of 5CH₃-H₄-PteGlu_n from 5CH₃-5,6-H₂PteGlu_n was examined with emphasis on the influence of pH and ascorbic acid. At pH 3.5, physiological levels of ascorbic acid (50 μMol/l) salvage both 5CH₃-H₄PteGlu₁ and 5CH₃-H₄PteGlu₃ from acid labile 5CH₃-5,6-H₂PteGlu_n and prevent loss of vitamin activity as indicated by P-ABG_n formation. This process is less efficient at pH 7.0, although under such conditions 5CH₃-5,6-H₂PteGlu_n is rendered relatively stable. Ascorbic acid (50 μMol/l) reduces degradation of 5CH₃-H₄PteGlu₁ and 5CH₃-H₄PteGlu₃ at both pH 3.5 and 7.0. However, irrespective of ascorbic acid, 5CH₃-H₄PteGlu₁ and 5CH₃-H₄PteGlu₃ are both more stable at pH 3.5 than pH 7.0. There is a clear differential between mono- and polyglutamate stability, since, irrespective of pH or the presence of ascorbic acid, 5CH₃-H₄PteGlu₁ is inherently more stable than 5CH₃-H₄PteGlu₃. While ascorbic acid clearly stabilises mono- and triglutamate forms of methylfolate in human gastric juice, a factor in gastric juice removes any differential instability as seen in buffer solution. We speculate that this endogenous factor could be either a binding protein or some other antioxidant. These in vitro findings provide information that may be useful in evaluating the in vivo bioavailability of natural polyglutamate forms of the vitamin.