Epitope Characterization of Malondialdehyde−Acetaldehyde Adducts Using an Enzyme-Linked Immunosorbent Assay

Abstract

Malondialdehyde (MDA) and acetaldehyde react together with proteins in a synergistic manner and form hybrid protein adducts, designated as MAA adducts. In a previous study, a polyclonal antibody specific for MAA-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 chemistry of MAA adduct formation was further characterized. When several synthetic analogs were tested for their ability to inhibit antibody binding in a competitive ELISA, the results indicated that the major determinant of antibody binding was a highly fluorescent cyclic adduct composed 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-dicarbaldehyde derivative of an amino group of a protein. Examination of MAA adduct formation with a variety of proteins indicated that in addition 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 binding as assessed in a competitive ELISA, although the efficiency of inhibition of antibody binding by these fluorescent epitopes on MAA-adducted proteins varied depending upon the particular protein. However, when these MAA-adducted proteins were hydrolyzed with Pronase, the concentration 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 accessibility of the antibody to bind the epitope on these various derivatized proteins. In summary, a cyclic fluorescent adduct of defined structure has been identified as the epitope recognized by our MAA adduct antibody. In addition to this specific adduct, MAA adducts are also comprised of other nonfluorescent products.

Detection of circulating antibodies against malondialdehyde-acetaldehyde adducts in patients with alcohol-induced liver disease

“…15,16 The chemical structure of these adducts has been characterized to some extent showing the presence of a linear adduct to the ⑀-amino group of lysine containing acetaldehyde and MDA in 1:1 ratio (2-formyl-3-alkylamino-butanol), and a cyclic adduct (4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde) where the two aldehydes are present in 2:1 ratio (Fig. 1).…”

“…MAA-protein adducts were generated by reacting 1 mg/mL of human serum albumin (HSA) with 1 mmol/L of acetaldehyde and 1 mmol/L MDA, obtained by the acid hydrolysis of MDA-bis-dimethylacetal as previously described. 15,16 The reaction was conducted at 37°C for 72 hours in tightly sealed polypropylene tubes. Acetaldehyde-modified HSA (HSA-AcA) was obtained under reducing conditions by incubating 1 mg/mL HSA solution in phosphate-buffered saline (PBS) pH 7.4 with 10 mmol/L acetaldehyde and 10 mmol/L sodium cyanoborohydride for 1 hour at room temperature.…”

“…Hexyl-MAA (1-hexyl-4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde) was synthesized via the Hantzsch reaction as previously reported. 15 …”

“…14 Recent studies from our laboratory have shown that malonildialdehyde (MDA), a product of ethanol-induced lipid peroxidation, and acetaldehyde react together with proteins to form mixed malonildialdehyde-acetaldehyde (MAA) adducts. 15,16 One of the major MAA protein adducts has been identified as the 4-methyl-1,4-dihydropyridine-3,5-dicarbaldehyde derivative of amino groups (Fig. 1).…”

“…1). 15 MAAprotein conjugates have strong immunogenic properties and induce the production of antibodies against both the MAA epitopes and the carrier protein. 17 MAA adducts have been detected in livers of ethanol-fed rats, 16 and their presence is associated with the development of circulating anti-MAA antibodies in alcohol-treated rats.…”