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 (*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.
Diez
L
L
Thiols and Cellular Functions, Faculté de Pharmacie, Université Henri Poincaré Nancy 1, France.
Livertoux
M H
MH
Stark
A A
AA
Wellman-Rousseau
M
M
Leroy
P
P
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
D017665:Hydroxyl Radical; D005978:Glutathione; D020156:Salicylic Acid