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

Abstract

This report examines the physico-chemical properties of mono and triglutamate forms of 5-methyltetrahydrofolaten (5CH3–H4–PteGlun) in human gastric juice and under artificial conditions, typical of the upper gastrointestinal tract. Degradation of 5CH3–H4–PteGlun to 5-methyldihydrofolaten (5CH3–5,6-H2PteGlun) and on to the C9–N10 scission product p-aminobenzoylglutamate (P-ABG), and the salvage of 5CH3–H4–PteGlun from 5CH3–5,6-H2PteGlun 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-H2PteGlun and prevent loss of vitamin activity as indicated by P-ABGn formation. This process is less efficient at pH 7.0, although under such conditions 5CH3–5,6-H2PteGlun 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.