Hydroxyl radical production by ascorbate and hydrogen peroxide

Ascorbate (AH-) and certain other biological reductants have long been known to produce the cytotoxic hydroxyl radical (OH) when oxidized by hydrogen peroxide (H2O2) in the presence of copper or iron catalysts. The present study documents the in vitro production of the OH solely from the oxidation of AH- by H2O2, independent of mediation by transition metals. Hydroxyl radical generation resulting from the AH-/H2O2 system was quantitatively documented by the specific radical-mediated hydroxylation of salicylic acid, a reaction that was readily assayed with HPLC coupled with electrochemical detection. Two ascorbate-copper complexes (e.g., AH-/Cu2+-EDTA/H2O2 and AH-/Cu2+-EDTA) and a copper/H2O2 system also generated OH, but less effectively than the AH-/H2O2 system. The ability of AH- and H2O2 to generate cytotoxic OH documents a reaction mechanism that may account for cytotoxic activity in some cellular environments where metal catalysts are lacking.