BIOLOGICAL REACTIONS OF PEROXYNITRITE - EVIDENCE FOR AN ALTERNATIVE PATHWAY OF SALICYLATE HYDROXYLATION

Salicylate hydroxylation has often been used as an assay of hydroxyl r adical production in vivo. We have examined here if hydroxylation of s alicylate might also occur by its reaction with peroxynitrite. To test this hypothesis, we exposed salicylate to various concentrations of p eroxynitrite, in vitro. We observed the hydroxylation of salicylate at 37 degrees C by peroxynitrite at pH 6, 7 and 7.5, where the primary p roducts had similar retention times on HPLC to 2,3- and 2,5-dihydroxyb enzoic acid. The product yields were pH dependent with maximal amounts formed at pH 6. Furthermore, the relative concentration of 2,3- to 2, 5-dihydroxybenzoic acid increased with decreasing pH. Nitration of sal icylate was also observed and both nitration, and hydroxylation reacti on products were confirmed independently by mass spectrometry. The spi n trap N-t-butyl-alpha-phenylnitrone (PEN), with or without dimethyl s ulfoxide (DMSO), was incapable of trapping the peroxynitrite decomposi tion intermediates. Moreover, free radical adducts of the type PBN/(CH 3)-C-. and PBN/(OH)-O-. were susceptible to destruction by peroxynitri te (pH 7, 0.1 M phosphate buffer). These results suggest direct peroxy nitrite hydroxylation of salicylate and that the presence of hydroxyl radicals is not a prerequisite for hydroxylation reactions.