EPITOPE CHARACTERIZATION OF MALONDIALDEHYDE-ACETALDEHYDE ADDUCTS USING AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY

Malondialdehyde (MDA) and acetaldehyde react together with proteins in a synergistic manner and form hybrid protein adducts, designated as M AA adducts. In a previous study, a polyclonal antibody specific for MA A-protein adducts was used in an immunoassay to detect the presence of MAA adducts in livers of ethanol-fed rats. In the present study, the specific epitope recognized by the antibody was defined and the chemis try of MAA adduct formation was further characterized. When several sy nthetic analogs were tested for their ability to inhibit antibody bind ing in a competitive ELISA, the results indicated that the major deter minant of antibody binding was a highly fluorescent cyclic adduct comp osed of two molecules of MDA and one of acetaldehyde. The structure of this adduct was shown to be a 4-methyl-1,4-dihydropyridine-3,5-dicarb aldehyde derivative of an amino group of a protein. Examination of MAA adduct formation with a variety of proteins indicated that in additio n to this specific fluorescent adduct, MAA adducts were also comprised of other nonfluorescent products. The amount of fluorescent epitopes present on a given protein was the major determinant of antibody bindi ng as assessed in a competitive ELISA, although the efficiency of inhi bition of antibody binding by these fluorescent epitopes on MAA-adduct ed proteins varied depending upon the particular protein. However, whe n these MAA-adducted proteins were hydrolyzed with Pronase, the concen tration of these modified proteins necessary to achieve 50% inhibition of antibody binding in a competitive ELISA fell into a much narrower range of values, indicating that protein hydrolysis equalized the acce ssibility of the antibody to bind the epitope on these various derivat ized proteins. In summary, a cyclic fluorescent adduct of defined stru cture has been identified as the epitope recognized by our MAA adduct antibody. In addition to this specific adduct, MAA adducts are also co mprised of other nonfluorescent products.