Acetaminophen-protein adduct detection

Muldrew, K. L., James, L. P., Coop, L., McCullough, S. S., Hendrickson, H. P., Hinson, J. A., and Mayeux, P. R. (2002). Determination of acetaminophen-protein adducts in mouse liver and serum and human serum after hepatotoxic doses of acetaminophen using high-performance liquid chromatography with electrochemical detection. Drug Metab. Dispos. 30 446-451. 

Clinical data also support the association of covalent binding and toxicity. Early studies utilized immunoassays to detect adducts in the blood samples of patients with acetaminophen overdose (Hinson et al. 1990). The highest levels of adducts were found in the patients with the most severe toxicity. The recent development of a highly sensitive and specific HPLC-EC assay for detection of acetaminophen protein adducts (3-cysteine-acetaminophen in proteins) has allowed for further... 

We perceived the need for sensitive assays that do not rely on the use of radioisotopes or extensive analytical methodology and that could accurately detect protein-bound acetaminophen in biological fluids in the presence of unbound acetaminophen. To this end, we recently developed sensitive avidin biotin-amplified ELISA (A-B ELISA) and particle concentration fluorescence immunoassays (PCFIA) which use antiserum specific for the major acetaminophen-protein adduct associated with toxicity (13-16). These assays are new tools to study the relation between formation of the 3-Cys-A protein adduct and acetaminophen-induced toxicity. In this report we review how these assays were developed, validated in laboratory animals, and used to quantify 3-Cys-A protein adduct formation in human acetaminophen overdose patients. 

This antiserum has also been used to detect acetaminophen-protein adducts in Western blots of serum and liver fractions from acetaminophen-dosed animals (1 ) and to localize the 3-Cys-A adduct in target tissues (17 and Bucci et al. this volume). 

Immunohistochemical Detection of Antigenic Biomarkers in Microwave-Fixed Target Tissues Acetaminophen—Protein Adducts... 

To evaluate the amount of acetaminophen bound to proteins, utilizing the competitive A-B ELISA or PCFIA, inhibition by unknown samples was compared with an assay standard prepared by derivatizing protein with NAPQI. For some experiments, 3-(N-acetyl-L-cystein-S-yDacetaminophen was used as an assay standard, in which case, the values obtained were corrected for differences in the relative inhibitory potency of 3-(N-acetyl-L-cystein- yl)acetaminophen and 3-Cys-A protein adduct (120 fmol/well and 2300 finol/well, respectively) (14). After dialysis, unknown samples were diluted to a final concentration of approximately 4 / g protein/assay well, assayed in duplicate, and expressed as nmoles of 3-Cys-A per mg of protein. The ELISA and PCFIA were shown to have similar limits of detection (20 pmole/mg protein) and to recognize the same itope as demonstrated by similar relative inhibitory potencies for N-acetylcysteine-acetaminophen, acetaminophen-bound... 

Acetaminophen (paracetamol) is a commonly used analgesic which is hepatotoxic at high doses in humans and in laboratory animals. Toxicity is believed to be mediated by the reactive metabolite N-acetyl-p-benzoquinone imine which binds to protein thiols as 3-(cystein-S-yl)acetaminophen adducts. Ultrasensitive immimoassays for 3-(with parallel elevations in serum adducts and serum levels of the liver-specific transaminase ALT. This suggested that the serum adducts were of hepatic origin and could be monitored as a biomarker of acetaminophen toxicity. Analysis of serum samples from acetaminophen overdose patients demonstrated a positive correlation between immunochemically detectable serum adducts and hepatotoxicity. 

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