Free radicals are thought to be involved in the pathogenesis of many disease states such as diabetes, cataractogenesis, cancer, and aging. Involvement of free radicals in most of these disorders, especially diabetic cataractogenesis, however, is derived from indirect evidence only. 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 incubated with 5.5 or 50 mM glucose containing the spin trap 5,5'-dimethyl 1-pyroline-N-oxide. The spin adducts were identified by electron spin resonance (ESR) spectroscopy and modulation of the levels of the spin adducts was achieved by including various antioxidants and metal chelators. Increased ESR signals were observed in the homogenates incubated with 50 mM glucose compared to those incubated with 5.5 mM glucose. The results show that the principal spin adduct has hyperfine splitting constants of a(N) = 1.41 ± 0.01 mt and a(N) = 2.02 ± 0.02 mt, indicating that these signals are due to carbon-centered free radicals. Superoxide dismutase, catalase, Trolox, and diethylenetriamine pentaacetic acid inhibited the signals while EDTA enhanced them. Based 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.
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