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

Muldrew, Kenneth L.; James, Laura P.; Coop, Leslie; McCullough, Sandra; Hendrickson, Howard P.; Hinson, Jack; Mayeux, Philip R.

doi:10.1124/dmd.30.4.446

Cited by 178 publications

(181 citation statements)

References 34 publications

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“…The levels of NAPQI-CPF adduct present in the serum of patient 444 were estimated to be about 35 pmol/ml of serum. It has been reported previously that significantly higher levels of NAPQI-protein adducts can be found in serum in patients showing severe hepatotoxicity in comparison with serum of patients without or with low hepatotoxicity (Muldrew et al, 2002;James et al, 2006). However, because a different methodology was used in these studies, measuring total NAPQI-Cys adducts after complete hydrolysis of whole serum, the levels of our NAPQI-CPF …”

Section: Lc/tandem Ms Detection Of Apap-albumin Adductsmentioning

confidence: 91%

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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Section: Lc/tandem Ms Detection Of Apap-albumin Adductsmentioning

confidence: 91%

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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“…Serum samples were assayed for APAP adducts using a modification of the previously reported HPLC with electrochemical detection assay for APAP-cysteine derived by proteolytic cleavage of APAP adducts (Muldrew et al, 2002). Assay modifications included centrifugal gel filtration and higher efficiency proteolytic digestion, resulting in improved sensitivity and efficiency of the assay.…”

Section: Methodsmentioning

confidence: 99%

“…In recent studies, our laboratory developed a very precise and sensitive analytical assay for the APAP adducts. In this assay, the liver or serum sample is initially proteolytically hydrolyzed, and the re- leased 3-(cystein-S-yl)-APAP adducts are quantified by high-performance liquid chromatography with an electrochemical detector (Muldrew et al, 2002). Measurement of APAP adducts in clinical serum samples can accurately distinguish between known, well characterized cases of APAP-related ALF and cases of ALF of other etiologies (Davern, et al, 2006).…”

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confidence: 99%

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Pharmacokinetics of Acetaminophen-Protein Adducts in Adults with Acetaminophen Overdose and Acute Liver Failure

et al. 2009

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ABSTRACT:Acetaminophen (APAP)-induced liver toxicity occurs with formation of APAP-protein adducts. These adducts are formed by hepatic metabolism of APAP to N-acetyl-p-benzoquinone imine, which covalently binds to hepatic proteins as 3-(cystein-S-yl)-APAP adducts. Adducts are released into blood during hepatocyte lysis. We previously showed that adducts could be quantified by high-performance liquid chromatography with electrochemical detection following proteolytic hydrolysis, and that the concentration of adducts in serum of overdose patients correlated with toxicity. The following study examined the pharmacokinetic profile and clinical associations of adducts in 53 adults with acute APAP overdose resulting in acute liver failure. A population pharmacokinetic analysis using nonlinear mixed effects (statistical regression type) models was conducted; individual empiric Bayesian estimates were determined for the elimination rate constant and elimination half-life. Correlations between clinical and laboratory data were examined relative to adduct concentrations using nonparametric statistical approaches. Peak concentrations of APAP-protein adducts correlated with peak aminotransferase concentrations (r ‫؍‬ 0.779) in adults with APAP-related acute liver failure. Adducts did not correlate with bilirubin, creatinine, and APAP concentration at admission, international normalized ratio for prothrombin time, or reported APAP dose. After N-acetylcysteine therapy, adducts exhibited first-order disappearance. The mean elimination rate constant and elimination half-life were 0.42 ؎ 0.09 days ؊1 and 1.72 ؎ 0.34 days, respectively, and estimates from the population model were in strong agreement with these data. Adducts were detected in some patient samples 12 days postingestion. The persistence and specificity of APAP-protein adducts as correlates of toxicity support their use as specific biomarkers of APAP toxicity in patients with acute liver injury. Acetaminophen [APAP;acetamide; C 8 H 9 NO 2 ] overdose has recently been identified as a major cause of acute liver failure (ALF) in the United States (Larson et al., 2005). Currently, the diagnosis of APAP overdose is dependent on the history of a large dose of APAP, defined as 7.5 g of APAP in adults (Rumack et al., 1981), supported by an elevated level of APAP in peripheral blood (Smilkstein et al., 1988;Rumack, 2002). Many patients develop ALF rapidly, characterized by encephalopathy and the presence of coagulopathy [international normalized ratio (INR) Ն 1.5]; in these patients, the history of ingestion and specific dosing information may be difficult to obtain. Furthermore, the interpretation of measured APAP concentrations in peripheral blood requires knowledge of the precise time of ingestion of a single large dose of APAP.Overdoses of APAP result in the generation of APAP-protein adducts, which are produced by the binding of the reactive metabolite, N-acetyl-p-benzoquinone imine (Dahlin et al., 1984), to cysteine groups on protein as 3-(cystein-S-yl)-APAP adducts (Ho...

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“…Acetaminophen Protein and Glutathione Adducts-We modified the method of Muldrew et al (26) to measure the levels of APAP protein and APAP glutathione adducts using HPLC with electrochemical detection. For glutathione APAP adducts, liver tissue was homogenized (1:5, w/v) in 10 mM sodium acetate, pH 6.5, and then centrifuged at 16,000 ϫ g for 20 min at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Acetaminophen-induced Liver Injury Is Attenuated in Male Glutamate-cysteine Ligase Transgenic Mice

Botta

Shi

White

et al. 2006

Journal of Biological Chemistry

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Acetaminophen overdose is a leading cause of drug-related acute liver failure in the United States. Glutathione, a tripeptide antioxidant protects cells against oxidative damage from reactive oxygen species and plays a crucial role in the detoxification of xenobiotics, including acetaminophen. Glutathione is synthesized in a two-step enzymatic reaction. Glutamate-cysteine ligase carries out the rate-limiting and first step in glutathione synthesis. We have generated C57Bl/6 mice that conditionally overexpress glutamate-cysteine ligase, and report here their resistance to acetaminophen-induced liver injury. Indices of liver injury included histopathology and serum alanine aminotransferase activity. Male transgenic mice induced to overexpress glutamate-cysteine ligase exhibited resistance to acetaminophen-induced liver injury when compared with acetaminophen-treated male mice carrying, but not expressing glutamate-cysteine ligase transgenes, or to female glutamatecysteine ligase transgenic mice. We conclude that glutamatecysteine ligase activity is an important factor in determining acetaminophen-induced liver injury in C57Bl/6 male mice. Because people are known to vary in their glutamate-cysteine ligase activity, this enzyme may also be an important determinant of sensitivity to acetaminophen-induced liver injury in humans.The tripeptide antioxidant glutathione (GSH; ␥-glutamylcysteinylglycine) is one of the most abundant cellular thiols. It protects cells against oxidative damage from reactive oxygen species, maintains cellular redox status, promotes cell growth, and plays a crucial role in the detoxification of xenobiotics. GSH can directly scavenge free radicals, act as an antioxidant in GSH-mediated reduction of peroxides and act as a co-substrate for glutathione S-transferase-mediated detoxification of reactive intermediates formed during phase I metabolism (1).GSH plays a major role in detoxifying many hepatotoxicants including acetaminophen (APAP), 3 an over-the-counter analgesic and antipyretic (2-5). APAP overdose is responsible for nearly 50% of the acute liver failure cases in the United States (6) and is thus of high public health concern. APAP metabolism has been well defined, making it a good model for drug-induced liver toxicity. APAP is primarily metabolized through sulfation and glucuronidation pathways (7-9). However, a fraction of APAP is bioactivated by cytochrome P-450s to n-acetyl-p-benzoquinoneimine (NAPQI), which can bind to cellular proteins (3, 7, 10 -12). NAPQI also covalently binds to GSH and is either converted back to APAP, or forms the non-toxic APAP-GSH conjugate. APAP overdose results in depletion of hepatic GSH (by as much as 90%) (2). As GSH stores are depleted, increased levels of NAPQI-protein adducts form, and such adducts are thought to be an important contributor to APAP hepatotoxicity (3,7,8,10,11).Pretreatment with N-acetylcysteine (7, 9, 10), a source of cysteine for GSH biosynthesis, attenuates APAP-induced hepatotoxicity. N-Acetylcysteine given soon after APAP (within...

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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

Cited by 178 publications

References 34 publications

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

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

Pharmacokinetics of Acetaminophen-Protein Adducts in Adults with Acetaminophen Overdose and Acute Liver Failure

Acetaminophen-induced Liver Injury Is Attenuated in Male Glutamate-cysteine Ligase Transgenic Mice

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