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

Laurent Diez, Marie Hélène Livertoux, Avishay Abraham Stark, Maria Wellman-Rousseau, Pierre Leroy

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106 Scopus citations

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 γ-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.

Original languageEnglish
Pages (from-to)185-193
Number of pages9
JournalJournal of Chromatography B: Biomedical Sciences and Applications
Volume763
Issue number1-2
DOIs
StatePublished - 5 Nov 2001

Keywords

  • Hydroxyl free radical
  • Salicylic acid
  • Thiols

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