title: High-performance liquid chromatographic assay of hydroxyl free radical using salicylic acid hydroxylation during in vitro experiments involving thiols.
abstract: A HPLC method was developed to monitor the production of hydroxyl free radical (*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 *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 *OH during in vitro experiments. Resulting data demonstrate that *OH is produced during Fenton-like reactions involving thiols and GSH catabolism via GGT.
authors.affiliation: Thiols and Cellular Functions, Faculté de Pharmacie, Université Henri Poincaré Nancy 1, France.
authors.forename: L
authors.initials: L
authors.lastname: Diez
authors.forename: M H
authors.initials: MH
authors.lastname: Livertoux
authors.forename: A A
authors.initials: AA
authors.lastname: Stark
authors.forename: M
authors.initials: M
authors.lastname: Wellman-Rousseau
authors.forename: P
authors.initials: P
authors.lastname: Leroy
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005978:Glutathione / Q000737:chemistry*; D017665:Hydroxyl Radical / Q000032:analysis*; D006900:Hydroxylation; D051379:Mice; D020156:Salicylic Acid / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
chemical_list: D017665:Hydroxyl Radical; D005978:Glutathione; D020156:Salicylic Acid