HYDROXYLATION OF AROMATIC-COMPOUNDS AS INDEXES OF HYDROXYL RADICAL PRODUCTION - A CAUTIONARY NOTE REVISITED

While setting up an intracerebral microdialysis system to estimate the extent of oxidative stress induced by the neurotoxin, N-methylphenylp yridinium ion (MPP(+)), we encountered a problem in the use of hydroxy benzoic acids as traps of hydroxyl radicals. Using either 2-hydroxyben zoate (salicylate) or 4-hydroxybenzoate as trapping agents, we observe d a nonspecific, that is, nontissue derived, production of hydroxyl ra dicals as measured by the hydroxylation products, 2,3- and 2,5-dihydro xybenzoate from 2-hydroxybenzoate and 3,4-dihydroxybenzoate from 4-hyd roxybenzoate. This production of dihydroxybenzoates was 10 times that expected due to the administration of MPP(+), thus making it impossibl e to interpret our results. Careful investigation of the various compo nents of the microdialysis system indicated that contact of the microd ialysate with metal surfaces resulted in dihydroxybenzoic acid formati on. These results should serve as a reminder to perform stringent test s of the experimental system prior to experiments with biological tiss ues to evaluate the contribution of hydroxyl radical production from n onbiological sources. Therefore, along with the possibility of enzymat ic production of dihydroxybenzoates, artefactual production by compone nts of the experimental apparatus must be considered before assuming t hat one is measuring hydroxyl radical production by a biological syste m.