Cytochrome P4502E1 hydroxyethyl radical adducts as the major antigen in autoantibody formation among alcoholics

Background & Aims: We have previously reported that alcoholics have increased titers of immunoglobulins reacting with protein adducts of hydroxyethyl free radicals. Because hydroxyethyl radicals are produced during ethanol metabolism by liver microsomes, the aim of this study was to determine whether such antibodies recognize microsomal proteins complexed with hydroxyethyl radicals. Methods: Liver microsomal proteins reacting with the anti-hydroxyethyl radical antibodies were characterized by an enzyme-linked immunosorbent assay and Western blotting. Results: Alcoholic cirrhotics, but not patients with nonalcoholic cirrhosis or healthy subjects, had increased serum levels of immunoglobulin G and A directed against antigens produced in microsomes incubated with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and ethanol. Such immunoreactivity was completely blocked when microsomes were incubated with ethanol in the presence of the spin-trapping agent 4-pyridyl-1-oxide-t-butyl nitrone or by preincubating the sera with hydroxyethyl radical-bound human albumin. Immunoblotting of proteins from human liver microsomes incubated with NADPH and ethanol showed that 86% of the sera from alcoholic cirrhotics reacted with a 52-kilodalton protein, whereas variable reactivity was observed with proteins of 78, 60, and 40 kilodaltons, respectively. The 52-kilodalton protein was identified by immunoblotting and immunoprecipitation as ethanol-inducible cytochrome P4502E1. Conclusions: Antibodies from alcoholic cirrhotics specifically recognized hydroxyethyl radical-cytochrome P4502E1 adducts, suggesting the possible implication of these antigens in the development of autoimmune reactions in alcoholic liver disease.