This article is available online at http://dmd.aspetjournals.org ABSTRACT:Acetaminophen-induced hepatotoxicity has been attributed to covalent binding of the reactive metabolite N-acetyl-p-benzoquinone imine to cysteine groups on proteins as an acetaminophen-cysteine conjugate. We report a high-performance liquid chromatography with electrochemical detection (HPLC-ECD) assay for the conjugate with increased sensitivity compared with previous methods. Previous methods to quantitate the protein-bound conjugate have used a competitive immunoassay or radiolabeled acetaminophen. With HPLC-ECD, the protein samples are dialyzed and then digested with protease. The acetaminophen-cysteine conjugate is then quantified by HPLC-ECD using tyrosine as an internal reference. The lower limit of detection of the assay is approximately 3 pmol/mg of protein. Acetaminophen protein adducts were detected in liver and serum as early as 15 min after hepatotoxic dosing of acetaminophen to mice. Adducts were also detected in the serum of acetaminophen overdose patients. Analysis of human serum samples for the acetaminophen-cysteine conjugate revealed a positive correlation between acetaminophen-cysteine conjugate concentration and serum aspartate aminotransferase (AST) activity or time. Adducts were detected in the serum of patients even with relatively mild liver injury, as measured by AST and alanine aminotransferase. This assay may be useful in the diagnostic evaluation of patients with hepatotoxicity of an indeterminate etiology for which acetaminophen toxicity is suspect.Acetaminophen (N-acetyl-p-aminophenol; APAP 1 ; Paracetamol) is the most commonly used drug for the treatment of pain and fever. Although safe at therapeutic doses, in overdose, acetaminophen produces severe hepatotoxicity. The mechanism of toxicity is by initial metabolism to the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) by cytochrome P450 . At therapeutic doses, the reactive metabolite is detoxified by glutathione, but following overdoses, glutathione is depleted and the metabolite covalently binds to proteins as 3-(cystein-S-yl)-acetaminophen (APAP-CYS) (Streeter et al., 1984). Covalent binding to protein is thought to be a critical step in the development of hepatotoxicity.A number of methods have been used to quantify the amount of acetaminophen covalently bound to proteins. In initial studies showing the correlation between acetaminophen toxicity and covalent binding, Jollow and coworkers (1973) used radiolabeled acetaminophen. Subsequently, polyclonal antisera were raised that recognized the acetaminophen-cysteine adducts (Bartolone et al., 1987;Roberts et al., 1987), and the relationship between covalent binding and toxicity was studied extensively. coworkers (1989, 1990) developed a competitive enzyme-linked immunosorbent assay to quantify acetaminophen covalent binding in the liver and serum of treated mice. In addition, Western blot assays were performed. Although these latter assays were not quantitative, they were useful in comparing t...