title: A sensitive immunochemical assay for acetaminophen-protein adducts.
abstract: The hepatotoxicity of acetaminophen may be mediated by the reactive metabolite N-acetyl-p-benzoquinone imine which binds covalently to protein primarily as 3-(cystein-S-yl)acetaminophen. We have developed an avidin biotin-amplified competitive enzyme-linked immunosorbent assay to detect protein-bound acetaminophen. This assay utilizes antisera from rabbits immunized with 3-(N-acetyl-L-cystein-S-yl)acetaminophen coupled via the carboxyl group to primary amino groups on the protein keyhole-limpet hemocyanin. The competitive enzyme-linked immunosorbent assay utilizes metallothionein derivatized with N-acetyl-p-benzoquinone imine (acetaminophen-bound metallothionein) and quantitation was obtained by competition of acetaminophen-derivatives for a limited amount of antibody in the presence of excess solid phase acetaminophen-bound metallothionein. Synthetic 3-(N-acetyl-L-cystein-S-yl)acetaminophen, acetaminophen bound to mouse 9,000 X g supernatant, 100,000 X g supernatant, microsomes, as well as acetaminophen-bound metallothionein were inhibitory. The 50% inhibition for 3-(N-acetyl-L-cystein-S-yl)acetaminophen was 110 fmol/well. In contrast, free acetaminophen was 6200 times less efficient as an inhibitor. The mean 50% inhibition for protein-bound acetaminophen was 2.89 pmol/well. The utility of the method to detect acetaminophen-protein adducts in biological samples was confirmed by detection of NADPH-dependent binding of acetaminophen to microsomal proteins.
mesh_terms: D000082:Acetaminophen / Q000032:analysis / Q000378:metabolism* / Q000633:toxicity; D000818:Animals; D003545:Cysteine / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D008099:Liver / Q000187:drug effects; D008433:Mathematics; D008722:Methods; D051379:Mice; D008862:Microsomes, Liver / Q000032:analysis; D009249:NADP / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits
chemical_list: D011506:Proteins; C043967:acetaminophen-cysteine conjugate; D000082:Acetaminophen; D009249:NADP; D003545:Cysteine