ESR AND HPLE-EC ANALYSIS OF ETHANOL OXIDATION TO 1-HYDROXYETHYL RADICAL - RAPID REDUCTION AND QUANTIFICATION OF POBN AND PBN NITROXIDES

Extensive ESR spin-trapping studies have shown that ethanol is oxidize d to l-hydroxyethyl radical (HER) by rat and deer mice liver microsoma l systems. The ESR detection of POBN/HER nitroxide in bile, and format ion of antibodies, which recognize HER adducts in alcoholics, suggest that HER is produced in vivo. In liver, where ethanol is primarily met abolized, only traces of PBN/HER nitroxide are documented. One limitat ion of the ESR spin-trapping technique, however, is that the nitroxide s formed in the presence of cellular reductants can be metabolized to the corresponding ESR ''silent'' hydroxylamines. Ascorbate and NADPH p lus liver microsomes were found to reduce rapidly both POBN/HER and PB N/HER nitroxides to their ESR ''silent'' hydroxylamine derivatives. An HPLC method with electrochemical detection was developed for the dete ction and quantification of both POBN/HER and PBN/HER nitroxides, as w ell as their hydroxylamines. Both the diastereomers of the POBN/HER ni troxide and hydroxylamine can be detected, as can both isomers of the PBN/HER nitroxide, and it is estimated that the sensitivity of the HPL C procedure is in the nM range when using EC detection. The hydroxylam ines are stable in ethanol, while pH-dependent auto-oxidation occurs i n aqueous buffers. Some of the characteristics associated with HER for mation by microsomes as detected with ESR (e.g., sensitivity to SOD an d catalase, increase after induction of CYP2E1) are reproduced with th e HPLC method. By quantification of the POBN/HER hydroxylamine, the NA DPH-dependent rates of HER formation by microsomes from pyrazole-treat ed rats are estimated to be about 1-1.5 nmol HER per min per mg micros omal protein. This rate is less, as compared to the two electron-depen dent rate of acetaldehyde formation by these microsomes, about 10-15 n mol per min per mg protein. Thus, at first approximation, the one elec tron-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 de rivatives in the same sample and may be of value in detecting HER spin -trapped adducts under biological reducing conditions. (C) 1998 Elsevi er Science Inc.