Quantification of total oxidant scavenging capacity of antioxidants for peroxynitrite, peroxyl radicals, and hydroxyl radicals

We have extended the application of our previously reported total oxidant s cavenging capacity (TOSC) assay (Winston et al., Free Radical Biol. Med. 24 , 480-493, 1998) to permit facile quantification of the absorbance capacity of antioxidants toward three potent oxidants, i.e., hydroxyl radicals, per oxyl radicals, and peroxynitrite. Respectively, these oxidants were generat ed by the iron plus ascorbate-driven Fenton reaction, thermal homolysis of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (ABAP), and 3-morpholin osydnonimine N-ethylcarbamide (SIN-1). Each of these oxidants reacts with a lpha-keto-gamma-methiolbutyric acid (KMBA), which is oxidized and yields et hylene. The antioxidant capacity of the compounds tested is quantified from their ability to inhibit ethylene formation relative to a control reaction . Assay conditions were established in which control reactions give compara ble yields of ethylene with each of the oxidants studied. Thus, the relativ e efficiency of various antioxidants could be compared under conditions of quantitatively similar KMBA oxidizing capability by the three oxidants. Red uced glutathione was an efficient scavenger of peroxyl radicals, but scaven ged peroxynitrite and hydroxyl radicals relatively poorly. Uric acid, Trolo x, and ascorbic acid were comparable scavengers of peroxynitrite and peroxy l radicals. Uric acid and Trolox were approximately an order of magnitude l ess efficient as scavengers of hydroxyl radicals. The classical hydroxyl ra dical scavenging agents mannitol, dimethyl sulfoxide, and benzoic acid had much higher TOSC values with hydroxyl than with peroxyl radicals or peroxyn itrite. The very different chemical reactivity toward KMBA by the SLN-1- an d iron-ascorbate-generated oxidants indicates that hydroxyl radical is not a major oxidant produced by the SIN-1 system. The data show that the TOSC a ssay is useful and robust in distinguishing the reactivity of various oxida nts and the relative capacities of antioxidants to scavenge these oxidants. (C) 1999 Academic Press.