Abstract

Intravenous administration of the spin-trapping agent alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN) to anesthetized but otherwise untreated rats was used to test for formation of 1-hydroxyethyl radicals in vivo. The only EPR signals observed in bile samples from rats that had received ethanol but no POBN could be attributed to low concentrations of ascorbyl radical. However, when POBN (700 mg/kg, intravenously) was also administered, a nitroxide with a six-line EPR spectrum was readily detected in bile. This spin adduct was proven to be the 1-hydroxyethyl radical adduct of POBN through injection of [1-13C]ethanol to rats, which resulted in the presence of an adduct with a 12-line EPR spectrum. Comparable results were obtained in experiments with isolated perfused rat livers. 1-Hydroxyethyl radical spin adducts of POBN were readily detectable in bile in the presence of only moderate (10-15 mM) concentrations of alcohol. In these experiments, bile samples were collected into a mixture of dipyridyl and bathocuproine disulfonic acid, and the effectiveness of these chelators to prevent ex vivo signal formation was confirmed experimentally. No EPR signals for nitroxide spin adducts were observed in plasma or perfusate, even though high concentrations of POBN and alcohol were present. Taken together, these data indicate that 1-hydroxyethyl radicals are formed in vivo and can be readily detected in bile when high concentrations of POBN are achieved through intravenous injection.