An application of electron paramagnetic resonance to evaluate nitric oxide and its quenchers

Electron paramagnetic resonance (EPR) spectrometry has been used for detection of free radicals, including nitric oxide (NO). The method was applied to evaluate if lazaroids, nonspecific scavengers of oxygen-derived free radicals, can reduce another radical species, namely, NO. When solutions containing 0 to 10 microM lazaroids (U78517F, U83836E, and U74500A) were mixed with NO (approximately 8.0 microM), and with a spin-trap agent specific to NO (10 microM carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3 oxide, c-PTIO), the spectrum of NO-c-PTIO determined by the EPR decreased with increasing concentrations of lazaroids. An identical study with 1 to 1000 microM methylprednisolone or dexamethasone, which are prototypes of lazaroids, failed to reduce NO. In a separate study, rat red blood cells preloaded with U78517F were exposed to NO. EPR spectrometry demonstrated that hemoglobin in these cells were less nitrosylated compared with the cells not preloaded with U78517F. An ability of lazaroids to quench NO in vivo was evaluated by an intravenous injection of sodium nitrite (4 mumol/kg body weight) into rats pretreated with 10 mg/kg lazaroids (U78517F and U74500A). The nitrosylation of RBC hemoglobin was markedly attenuated in rats pretreated with lazaroids compared with those with vehicle. Thus, these lipophilic compounds protected cells from NO-induced nitrosylation both in vitro and in vivo. Lazaroids may be applicable as anti-NO agents.