ESR and HPLC-EC analysis of ethanol oxidation to 1-hydroxyethyl radical: rapid reduction and quantification of POBN and PBN nitroxides

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Extensive ESR spin-trapping studies have shown that ethanol is oxidized to 1-hydroxyethyl radical (HER) by rat and deer mice liver microsomal systems. The ESR detection of POBN/HER nitroxide in bile, and formation of antibodies, which recognize HER adducts in alcoholics, suggest that HER is produced in vivo. In liver, where ethanol is primarily metabolized, only traces of PBN/HER nitroxide are documented. One limitation of the ESR spin-trapping technique, however, is that the nitroxides formed in the presence of cellular reductants can be metabolized to the corresponding ESR “silent” hydroxylamines. Ascorbate and NADPH plus liver microsomes were found to reduce rapidly both POBN/HER and PBN/HER nitroxides to their ESR “silent” hydroxylamine derivatives. An HPLC method with electrochemical detection was developed for the detection and quantification of both POBN/HER and PBN/HER nitroxides, as well as their hydroxylamines. Both the diastereomers of the POBN/HER nitroxide and hydroxylamine can be detected, as can both isomers of the PBN/HER nitroxide, and it is estimated that the sensitivity of the HPLC procedure is in the nM range when using EC detection. The hydroxylamines are stable in ethanol, while pH-dependent auto-oxidation occurs in aqueous buffers. Some of the characteristics associated with HER formation by microsomes as detected with ESR (e.g., sensitivity to SOD and catalase, increase after induction of CYP2E1) are reproduced with the HPLC method. By quantification of the POBN/HER hydroxylamine, the NADPH-dependent rates of HER formation by microsomes from pyrazole-treated rats are estimated to be about 1–1.5 nmol HER per min per mg microsomal protein. This rate is less, as compared to the two electron-dependent rate of acetaldehyde formation by these microsomes, about 10–15 nmol per min per mg protein. Thus, at first approximation, the one electron-dependent rate of ethanol oxidation is about 10% the two electron-dependent rate by isolated microsomes. The HPLC procedure can readily detect the POBN/HER and PBN/HER nitroxides and their hydroxylamine derivatives in the same sample and may be of value in detecting HER spin-trapped adducts under biological reducing conditions.