A sensitive immunochemical assay for acetaminophen-protein adducts

Area of Science:

• Biochemistry
• Toxicology
• Immunology

Background:

• Acetaminophen hepatotoxicity is linked to its reactive metabolite, N-acetyl-p-benzoquinone imine.
• This metabolite covalently binds to proteins, forming adducts like 3-(cystein-S-yl)acetaminophen.

Purpose of the Study:

• To develop a sensitive assay for detecting protein-bound acetaminophen.
• To quantify acetaminophen-protein adducts in biological samples.

Main Methods:

• Developed an avidin biotin-amplified competitive enzyme-linked immunosorbent assay (ELISA).
• Utilized antisera against 3-(N-acetyl-L-cystein-S-yl)acetaminophen conjugated to keyhole-limpet hemocyanin.
• Employed acetaminophen-bound metallothionein as a competitor in the ELISA.

Main Results:

• The assay detected synthetic 3-(N-acetyl-L-cystein-S-yl)acetaminophen with 110 fmol/well 50% inhibition.
• Protein-bound acetaminophen showed 2.89 pmol/well 50% inhibition, significantly more sensitive than free acetaminophen.
• Successfully detected NADPH-dependent acetaminophen binding to microsomal proteins.

Conclusions:

• The developed ELISA is effective for detecting and quantifying acetaminophen-protein adducts.
• This assay is valuable for investigating acetaminophen-induced hepatotoxicity mechanisms.
• The method confirms the formation of toxic acetaminophen adducts in biological systems.