Acetaminophen Manuscript

This page is being prepared to submit a manuscript for peer review and publication in the Journal of Chemical Research in Toxicology by Matthew Simonson. The goal of this work is to provide foundational evidence for a mechanism of hepatotoxicity caused by radical polymerization of Acetaminophen within the liver as opposed to soley a mechanism of two electron oxidation into NAPQI.

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Abstract

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Acetaminophen (APAP) is a common analgesic and an active ingredient in many painkillers such as Tylenol and Percocet. Upon overdose, APAP can lead to toxicity in the liver, which accounts for a striking proportion of acute liver failures in the United States annually. Considering the potential for APAP induced hepatotoxicity, our research group has analyzed APAP and its tendency to oxidize into reactive products through both enzymatic and electrochemical methods of in vitro oxidation, which was visualized through HPLC. Characterization of these APAP oxidation products were carried out using electron spin resonance (ESR), quadruple time-of-flight mass spectrometry (qTOF-MS), 400 MHz proton nuclear magnetic resonance (H-NMR), and cyclic voltammetry (CV). Our findings provide structural insight into potentially deleterious APAP oxidation metabolites formed in the liver during times of APAP overdose formed via a mechanism of radical polymerization.

Introduction

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IUPAC NAMES for Acetaminophen (APAP), paracetamol, and intermediates

Introduce APAP and overarching problem

Acetaminophen (Paracetamol, 4'-Hydroxyacetanilide, 4-Acetamidophenol, or APAP) is one of the most widely used analgesics and antipyretics and can be purchased over the counter. Moreover, APAP overdose remains to be a large problem in the United States and is responsible for a majority of acute liver failures annually (Lee at al; Bernal et al). The difference between a safe dose and a toxic dose is narrow. So it is deceptively easy to take too much and cause a trip to the emergency room, permanent liver damage, or even death.

Introduce NAPQI and Toxicity

While APAP itself is harmless, APAP metabolic pathways have been investigated by numerous studies which allude to reactive metabolites of APAP being responsible for liver necrosis. The formation of reactive metabolites coupled with glutathione depletion and alkylation of mitochondrial proteins are suggested to be critical initiating events for APAP induced hepatotoxicity (???). Reactive metabolites of APAP form via oxidation of APAP in the liver, which occurs after other nontoxic metabolic pathways such as glucuronidation and sulfation have been saturated (???). The commonly accepted mechanism of oxidative metabolite formation during APAP overdose revolves around the two electron oxidation of APAP into an electrophilic metabolite, N-acetyl-p-benzoquinone imine (NAPQI), via Cytochrome P450 enzymes (CYPs) (Gillette et al. 1981 Dahlin et al. 1984 Nelson et al 1990). NAPQI is then purported to be reduced and detoxified via glutahione (GSH), leading to cysteine and mercapturic acid metabolites to be formed (???). However, once intracellular glutathione is used up, toxicity is suggested to disseminate through NAPQI conjugation with thiol groups of proteins and down stream signaling (???). The formation of NAPQI was determined to be the main deleterious metabolite of APAP oxidation in the liver through indirect measurement of its conjugation with glutathione (Dahlin et al.). The uncertainty associated with indirect metabolic identification raises hesitance to NAPQI being the only actor during APAP overdose.

lternative Metabolism

Moreover, oxidation reactions mediated by CYPs involving phenols like APAP have been confirmed to occur by both mechanisms of one electron oxidations or two electron oxidations (Potter and Hinson 1987). Oxidizing APAP by one electron instead of two yields a different set of metabolites, which could play bioactive roles responsible for the hepatotoxicity observed following glutathione conjugation and protein alkylation. Both enzymatic and electrochemical methods have been utilized to model CYP metabolism of phenols as opposed to working with liver microsomes. In one such case, the one electron oxidation of APAP was catalyzed using Horseradich peroxidase, which allowed for the detection of a radical intermediate (1984 and Fischer 1985). This radical intermediate following APAP oxidation could also conjugate with protein thiol groups or GSH and propagate down stream damage just as NAPQI is suggested to do (???). Characterization of the APAP oxidation products following one electron oxidation yielded the identification of a set of APAP polymers consistent with a mechanism of radical polymerization, independent of enzymatic mediation (Potter et al. 1984). Further investigation into such metabolites could shed light on the controversial topic of how APAP induced liver necrosis disseminates. Considering that, the purpose of this work is to revisit the oxidation of APAP and its downstream oxidation products in order to illucidate the radical nature of this reaction.

Experimental Procedures

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Results

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Electron Spin Resonance of Radical Acetaminophen Intermediate

Should be cleaned up for better resolution and more descriptive than "melanin".

using B3YLP/EPR-II basis set

Enzymatic Oxidation of Acetaminophen

The enzymatic oxidation of APAP was observed by reverse phase HPLC with a diode array detector set at 270 nm in a 25% dioxane - pH 7 buffer on the left and a 25% dioxane - pH 9 buffer on the right. The chromatogram on top (blue) serves as a 2mM APAP standard prior to reaction. Increasing concentrations of H2O2 were reacted within the APAP/H2O2/HRP system with each red chromatogram down. These concentrations were 0.5mM, 1mM, and 2mM H2O2 respectively. Results indicate the formation of chemically unique products present at both pH 7 and 9, A, B, C, D. However, the formation of these products were maximized in the pH 9 buffer. As H2O2 concentration rose within the pH 7 buffer, increasingly nonpolar products grew in concentration as signified by the hump in the 6-7 minute range.

Flash Purification of APAP Metabolites

Mass Spectrometry of Acetaminophen Metabolites

RERUN MS using HPLC in order to get more peaks

The data represents a partial set of MS molecular ions as a result of the

flash chromatography purified oxidation products isolated from the APAP/H2O2/HRP

oxidation reaction. ESI-MS data are m/z + 1 and contain relative abundance of

each molecular ion.

Nuclear Magnetic Resonance of Early Acetaminophen Metabolism

Reverse Phase versus Normal Phase Separation of diAPAP

Early Metabolite Oxidation

Electrochemical Oxidation of Acetaminophen

Cyclic Voltammetry of diAPAP

Discussion

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