Electron spin resonance studies on the degradation of hydroperoxides by rat liver cytosol

Incubation of tBuOOH (in the concentration range 200 microM to 20 mM) with rat liver post-microsomal supernatant in the presence of the spin trap DMPO gives three radical species, which can be observed by electron spin resonance spectroscopy. The first of these is the ascorbyl radical (which decreases in concentration with time), the other two are identified as spin adducts of alkoxyl and carbon-centred radicals; these latter species increase in concentration with time. Addition of NADH, but not NADPH, led to an increase in concentration of the alkoxyl and carbon-centred radical adducts and a decrease in the concentration of the ascorbyl radical. Results obtained in the presence of iron chelators and other ligands suggest that the generating system is an NADH-dependent enzyme that reduces tBuOOH by one-electron to give initially the tBuO. radical. Results from experiments carried out on dialysed cytosol samples lend support to this conclusion.