GLUCOSE-DEPENDENT FORMATION OF FREE-RADICAL SPECIES IN LENS HOMOGENATE

Free radicals are thought to be involved in the pathogenesis of many d isease states such as diabetes, cataractogenesis, cancer, and aging. I nvolvement of free radicals in most of these disorders, especially dia betic cataractogenesis, however, is derived from indirect evidence onl y. The purpose of this study was to directly detect the free radicals generated in lens homogenate incubated under hyperglycemic conditions, We investigated the generation of free radicals in lens homogenates i ncubated with 5.5 or 50 mM glucose containing the spin trap 5,5'-dimet hyl 1-pyroline-N-oxide. The spin adducts were identified by electron s pin resonance (ESR) spectroscopy and modulation of the levels of the s pin adducts was achieved by including various antioxidants and metal c helators. Increased ESR signals were observed in the homogenates incub ated with 50 mM glucose compared to those incubated with 5.5 mM glucos e. The results show that the principal spin adduct has hyperfine split ting constants of alpha(N) = 1.41 +/- 0.01 mt and alpha(N) = 2.02 +/- 0.02 mt, indicating that these signals are due to carbon-centered free radicals. Superoxide dismutase, catalase, Trolox, and diethylenetriam ine pentaacetic acid inhibited the signals while EDTA enhanced them. B ased upon the results obtained by the inclusion of various modulators, it is concluded that the generation of the free radicals is mediated by a metal-derived Fenton type reaction. (C) 1996 Academic Press, Inc.