Utilization of the nitroxyl radical TEMPOL to assess scavenging activities of lipid-soluble antioxidants against radicals initiated by the thermal decomposition of 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) in ethanol].

Abstract

To develop a simple and sensitive method for assessing the radical-scavenging activity of lipophilic antioxidants, the decay of the electron spin resonance (ESR) signal of 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl radical (TEMPOL) was investigated as an indicator of radical reactions. The ESR signal of TEMPOL was decreased in ethanol, but not in acetonitrile, by the pyrolysis of 2,2'-azobis(2,4-dimethylvaleronitrile). Signal decay in ethanol was suppressed by degassing and did not occur in the presence of the spin trapping agent 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO). Spin trapping with DEPMPO showed the formation of peroxyl, alkoxyl, and alkyl radical adducts during the reaction, with peroxyl radical adducts decreasing in the presence of TEMPOL. These results indicate that TEMPOL signal decay occurs by reactions involving ethanol-derived peroxyl radicals. The TEMPOL signal decay was remarkably inhibited by 0.01 mmol/L 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), which is lower than the Trolox concentration used in the ESR-spin trapping method reported previously. Inhibition by methyl gallate was markedly stronger than Trolox, while the effects of 2,6-di-tert-butyl-p-cresol and resveratrol were minor. The order of inhibition of TEMPOL signal decay by antioxidants correlated to some extent with the order of suppression of peroxyl radical adduct formation by DEPMPO spin trapping. Therefore, the peroxyl radical-scavenging activities of lipid-soluble antioxidants may be evaluated with high sensitivity by examining the inhibitory activity of TEMPOL decay caused by radical reactions in ethanol. The measurement time was less than 5 min.