Recent studies show that the alpha-hydroxyethyl free radical, a highly reactive metabolite of ethanol, is generated in vitro and in vivo in a number of biological systems and organs where active oxygen species are produced. This ubiquitous metabolite of ethanol binds most effectively to proteins, forming stable covalent adducts. Furthermore, these adducts are antigenic, inducing the production of antibodies which recognize the compounds formed. Ethanol expresses its toxic effects in virtually every tissue, including liver, pancreas, heart and brain. However the existence of a single mechanism to account for this broad range of toxic actions has eluded investigators. The studies proposed in this application test the general hypothesis that alpha-hydroxyethyl radical-derived adducts (a) accumulate in tissues and in biological fluids of animals fed alcohol chronically; and (b) have characteristics that allow their use as specific markers of chronic alcohol abuse. The proposed research is designed to (i) characterize the nature of the alpha-hydroxyethyl adducts formed in the reaction between alpha- hydroxyethyl radicals and chemically defined polyaminoacids; (ii) develop antibodies against defined alpha-hydroxyethyl-aminoacid haptens and methodologies for alpha-hydroxyethyl-adduct quantification, (iii) determine the biological half life of alpha-hydroxyethyl-protein adducts and characterize their degradation products in urine; (iv) identify alpha- hydroxyethyl-derived adducts in biological fluids and tissues of animals fed alcohol chronically, (v) investigate the production of alpha- hydroxyethyl free radicals and of alpha-hydroxyethyl-protein adducts by hepatic macrophages; and (vi) quantify alpha- hydroxyethyl derived adducts in the plasma and urine of patients admitted with the diagnosis of alcohol abuse and alcohol dependence. A number of specific hypotheses in relation to these will be tested. Overall, the proposed research investigates the biological and diagnostic significance of a reactive free radical metabolite of ethanol which is generated by active oxygen species acting upon ethanol.
