High-performance liquid chromatographic assay of hydroxyl free radical using salicylic acid hydroxylation during in vitro experiments involving thiols

A HPLC method was developed to monitor the production of hydroxyl free radical (degrees OH) produced during in vitro experiments: (i) a chemical reaction involving EDTA chelated ferric ion and various exogenous and endogenous thiols [glutathione (GSH) and its metabolites], and (ii) an enzymatic reaction corresponding to the breakdown of GSH catalyzed by gamma -glutamyltransferase (GGT). The method relies upon the use of a selective trapping reagent of degrees OH: salicylic acid (SA). The three resulting dihydroxylated products, i.e,, 2,3-dihydroxybenzoic acid (DHB), 2,5-DHB and catechol, were measured in an ion-pairing reversed-phase HPLC system coupled with amperometric detection; the sum of the three concentrations was used to quantify the production of degrees OH during in vitro experiments. Resulting data demonstrate that degrees OH is produced during Fenton-like reactions involving thiols and GSH catabolism via GGT. (C) 2001 Elsevier Science B.V. All rights reserved.