ONE-ELECTRON REDUCTION OF VANADATE BY ASCORBATE AND RELATED FREE-RADICAL GENERATION AT PHYSIOLOGICAL PH

The one-electron reduction of vanadate (vanadium(V)) by ascorbate and related free radical generation at physiological pH was investigated by ESR and ESR spin trapping. The spin trap used was 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Incubation of vanadium(V) with ascorbate generated significant amounts of vanadium(IV) in phosphate buffer (pH 7.4) but not in sodium cacodylate buffer (pH 7.4) nor in water. The vanadium(IV) yield increased with increasing ascorbate concentration, reaching a maximum at a vanadium(V): ascorbate ratio of 2:1. Addition of formate to the incubation mixture containing vanadium(V), ascorbate, and phosphate generated carboxylate radical (.COO-), indicating the formation of reactive species in the vanadium(V) reduction mechanism. In the presence of H2O2, a mixture of vanadium(V), ascorbate, and phosphate buffer generated hydroxyl radical (.OH) via a Fenton-like reaction (vanadium(IV)+ H2O2 --> vanadium(V) + .OH + OH-). The .OH yield was favored at relatively low ascorbate concentrations. Omission of phosphate sharply reduced the OH yield. The vanadium(IV) generated by ascorbate reduction of vanadium(V) in the presence of phosphate was also capable of generating lipid hydroperoxide-derived free radicals from cumene hydroperoxide, a model lipid hydroperoxide. Because of the ubiquitous presence of ascorbate in cellular system at relatively high concentrations, one-electron reduction of vanadium(V) by ascorbate together with phosphate may represent an important vanadium(V) reduction pathway in vivo. The resulting reactive species generated by vanadium(IV) from H2O2 and lipid hydroperoxide via a Fenton-like reaction may play a significant role in the mechanism of vanadium(V)-induced cellular injury.