Sensitive Immunochemical Assays for Monitoring Acetaminophen Toxicity in Humans

Roberts, Dean W.; Benson, Robert W.; Pumford, Neil R.; Potter, David W.; Poulsen, Henrik Enghusen; Hinson, Jack

doi:10.1021/bk-1990-0451.ch029

Cited by 43 publications

(57 citation statements)

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“…2, with an average of 3.3 paired samples per subject (range [1][2][3][4][5][6][7][8][9][10][11][12][13][14]. Data from acute overdoses are distinguished from subjects with chronic ingestions by legend markers, and no apparent pattern differentiates the two groups.…”

Section: Urinary Apap-cysmentioning

confidence: 99%

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Prolonged Acetaminophen-Protein Adduct Elimination During Renal Failure, Lack of Adduct Removal by Hemodiafiltration, and Urinary Adduct Concentrations After Acetaminophen Overdose

et al. 2014

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Elevated concentrations of serum acetaminophenprotein adducts, measured as protein-derived acetaminophen-cysteine (APAP-CYS), have been used to support a diagnosis of APAP-induced liver injury when histories and APAP levels are unhelpful. Adducts have been reported to undergo first-order elimination, with a terminal half-life of about 1.6 days. We wondered whether renal failure would affect APAP-CYS elimination half-life and whether continuous venovenous hemodiafiltration (CVVHDF), commonly used in liver failure patients, would remove adducts to lower their serum concentrations. Terminal elimination half-lives of serum APAP-CYS were compared between subjects with and without renal failure in a prospective cohort study of 168 adults who had ingested excessive doses of APAP. APAP-CYS concentrations were measured in plasma ultrafiltrate during CVVHDF at times of elevated serum adduct concentrations. Paired samples of urine and serum APAP-CYS concentrations were examined to help understand the potential importance of urinary elimination of serum adducts. APAP-CYS elimination half-life was longer in 15 renal failure subjects than in 28 subjects with normal renal function (41.3±2.2 h versus 26.8±1.1 h [mean ± SEM], respectively, p < 0.001). CVVHDF failed to remove detectable amounts of APAP-CYS in any of the nine subjects studied. Sixty-eight percent of 557 urine samples from 168 subjects contained no detectable APAP-CYS, despite levels in serum up to 16.99 μM. Terminal elimination half-life of serum APAP-CYS was prolonged in patients with renal failure for reasons unrelated to renal urinary adduct elimination, and consideration of prolonged elimination needs to be considered if attempting back-extrapolation of adduct concentrations. CVVHDF did not remove detectable APAP-CYS, suggesting approximate APAP-protein adduct molecular weights≥50,000 Da Toxicol. (2015) 11:169-178 DOI 10.1007 in the minority of instances was most compatible with renal adduct production and protein shedding into urine rather than elimination of serum adducts.

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Section: Urinary Apap-cysmentioning

confidence: 99%

“…Measurement of circulating APAP-protein adducts, in the form of protein-derived 3-cysteinyl-acetaminophen, was described more than 25 years ago [3]. Methodology has improved since then, but general principles remain the same.…”

Section: Introductionmentioning

confidence: 99%

Prolonged Acetaminophen-Protein Adduct Elimination During Renal Failure, Lack of Adduct Removal by Hemodiafiltration, and Urinary Adduct Concentrations After Acetaminophen Overdose

et al. 2014

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“…When generated NAPQI depletes GSH, NAPQI covalently binds to cysteine residues on intracellular proteins and induces the formation of 3-(cysteine-S-yl) APAP adducts in hepatocytes. 37,38 This process is the initial and irreversible step in the development of cell injury. 38,39 Among several adducts, selenium-binding protein with a molecular weight of 56 kDa, a major APAP adduct, reflects the amounts of generated NAPQI and APAP adducts, 40,41 and can determine the magnitude of APAP-induced direct liver injury.…”

Section: Il-1ra and Apap-induced Liver Injury T Ishibe Et Almentioning

confidence: 99%

Reduced acetaminophen-induced liver injury in mice by genetic disruption of IL-1 receptor antagonist

Ishibe

Kimura

Ishida

et al. 2009

Laboratory Investigation

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Acetaminophen (APAP) induced increases in intrahepatic expression of interleukin (IL)-1a, IL-1b, and IL-1 receptor antagonist (IL-1ra), when administered intraperitoneally. These observations prompted us to define the pathophysiological roles of IL-1ra in APAP-induced liver injury. Compared with wild-type (WT) mouse-derived hepatocytes, IL-1ra-deficient (IL-1ra KO)-derived hepatocytes exhibited more resistance against APAP but not APAP-derived major toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Moreover, the amounts of a major APAP adduct (selenium-binding protein), an indicator of NAPQI generation from APAP, was significantly lower in IL-1ra KO mice than WT mice with depressed intrahepatic expression of CYP1A2, CYP2E1, and CYP3A11, the enzymes crucially involved in NAPQI generation from APAP. These observations would indicate that IL-1ra deficiency impaired APAP metabolism. IL-1a and IL-1b were expressed to similar extents in livers of untreated IL-1ra KO and WT mice. By contrast, the intranuclear amount of p65 of NF-kB, which can suppress the gene expression of CYP1A2, CYP2E1, and CYP3A11, was higher in untreated IL-1ra KO than WT mice. Moreover, when mice were intraperitoneally administered APAP (200 mg/kg), IL-1ra KO mice exhibited attenuated APAP-induced liver injury as evidenced by reductions in serum alanine transferase levels and histopathological changes such as centrilobular necrosis, hemorrhages, and leukocyte infiltration. Finally, when given 12 h before APAP challenge, IL-1a repressed the intrahepatic expression of CYP1A2, CYP2E1, and CYP3A11, eventually reducing APAP-induced liver injury, along with reduction in APAP adducts. Collectively, NF-kB was activated without any stimuli by the genetic disruption of IL-1ra, and suppressed cytochrome P450 enzyme expression, thereby reducing APAP-induced liver injury.

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“…Previous methods that have been used to investigate covalent binding of APAP to proteins in vivo include several immunoassays (Roberts et al, 1987;Bartolone et al, 1988;Matthews et al, 1996;James et al, 2001) and the use of radiolabeled APAP (Axworthy et al, 1988;Zhou et al, 1996;Qiu et al, 1998). Still, these methods often lack the necessary sensitivity when concerning human serum samples (Zhou, 2003) quantify covalent protein binding of APAP.…”

Section: Adductmentioning

confidence: 99%

Liquid Chromatography/Tandem Mass Spectrometry Detection of Covalent Binding of Acetaminophen to Human Serum Albumin

Damsten¹,

Commandeur²,

Fidder³

et al. 2007

Drug Metab Dispos

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ABSTRACT:Covalent binding of reactive electrophilic intermediates to proteins is considered to play an important role in the processes leading to adverse drug reactions and idiosyncratic drug reactions. Consequently, both for the discovery and the development of new drugs, there is a great interest in sensitive methodologies that enable the detection of covalent binding of drugs and drug candidates in vivo. In this work, we present a strategy for the generation and analysis of drug adducts to human serum albumin. Our methodology is based on the isolation of albumin from blood, its digestion to peptides by pronase E, and the sensitive detection of adducts to the characteristic cysteine-proline-phenylalanine (CPF) tripeptide by liquid chromatography/tandem mass spectrometry. We chose acetaminophen (APAP) as a model compound because this drug is known to induce covalent binding to proteins when bioactivated by cytochromes P450 to its reactive N-acetyl-p-benzoquinoneimine metabolite. First, by microsomal incubations of APAP in presence of CPF and/or intact albumin, in vitro reference adducts were generated to determine the mass spectrometric characteristics of the expected CPF adducts and to confirm their formation on pronase E digestion of the alkylated protein. When applying this methodology to albumin isolated from blood of patients exposed to APAP, we were indeed able to detect the corresponding CPF adducts. Therefore, this strategy could be seen as a potential biomonitoring tool to detect in vivo reactive intermediates of drugs and drug candidates, e.g., in the preclinical and clinical development phase.

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Sensitive Immunochemical Assays for Monitoring Acetaminophen Toxicity in Humans

Cited by 43 publications

References 0 publications

Prolonged Acetaminophen-Protein Adduct Elimination During Renal Failure, Lack of Adduct Removal by Hemodiafiltration, and Urinary Adduct Concentrations After Acetaminophen Overdose

Prolonged Acetaminophen-Protein Adduct Elimination During Renal Failure, Lack of Adduct Removal by Hemodiafiltration, and Urinary Adduct Concentrations After Acetaminophen Overdose

Reduced acetaminophen-induced liver injury in mice by genetic disruption of IL-1 receptor antagonist

Liquid Chromatography/Tandem Mass Spectrometry Detection of Covalent Binding of Acetaminophen to Human Serum Albumin

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