Determination of hydroxyl free radical formation in human platelets using high-performance liquid chromatography with electrochemical detection

The formation of the hydroxyl free radical (HFR) can be quantified indirect ly, by measuring two products of the hydroxylation of salicylic acid, 2,3-d ihydroxybenzoate (2,3-DHB) and 2,5-dihydroxybenzoate (2,5-DHB). In this stu dy, we used reversed-phase high-performance liquid chromatography with elec trochemical (coulometric) detection to measure 2,3- and 2,5-DHB levels in h uman platelets. The limits of detection of the method were 10 and 5 fmol on column for 2,3-DHB and 2,5-DHB, respectively. We tested the technique by m easuring increases in dihydroxybenzoate levels after exposure of platelets to experimentally induced oxidative stress. Then, we measured platelet leve ls of 2,3- and 2,5-DHB in patients with Parkinson's disease, under therapy with L-DOPA, and in normal subjects. We also measured platelet concentratio ns of L-DOPA and its major metabolite, 3-O-methyldopa (3-OMD). Parkinsonian patients showed increased levels of both 2,3- and 2,5-DHB. Platelet levels of 2,3-DHB were positively correlated with platelet levels of L-DOPA and 3 -OMD. The technique we describe proved simple and extremely sensitive and m ay represent a useful tool for the study of oxidative stress in humans. (C) 1999 Published by Elsevier Science B.V. All sights reserved.