Hydroxylation of salicylate and phenylalanine as assays for hydroxyl radicals: a cautionary note visited for the third time.

Hydroxylation of salicylate to 2,3- and 2,5-dihydroxy-benzoates (DHBs) is widely used as an index of hydroxyl radical (OH.) formation in vivo and in vitro. Several recent studies indicate that peroxynitrite can lead to generation of DHBs from salicylate and it is uncertain as to whether or not OH. is involved. A similar problem may occur in the use of phenylalanine as an OH. detector. Hence formation of hydroxylation products from salicylate (or phenylalanine) may not in itself be a definitive index of OH. generation, especially in cases where such generation in physiological systems is decreased by inhibitors of nitric oxide synthase. Determination of salicylate (or phenylalanine) nitration products can allow distinction between peroxynitrite-dependent aromatic hydroxylation and that involving "real" OH..