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 Toxicity

Acetaminophen (APAP) is one of the most widely used analgesics and antipyretics. Similarly, APAP overdose remains to be a large problem in the United States and is responsible for a majority of the acute liver failures annually (Lee at al; Bernal et al).

Introduce NAPQI, Conjugation, and GSH Depletion

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 commonly accepted mechanism of APAP induced hepatotoxicity revolves around a suggested two electron oxidation of APAP into the electrophilic metabolite, NAPQI, via Cytochrome P450 enzymes (CYPs) (Gillette et al. 1981). NAPQI was determined to be the main deleterious metabolite of APAP oxidation in the liver through indirect measurement of its conjugation and detoxification with glutathione (Dahlin et al. 1984). The oxidation of APAP occurs in a dose dependent manner and leads to the depletion of intracellular glutathione levels (???). The hepatotoxic effects occur only after intracellular glutathione levels have been depleted (???); however, studies concerning diethyl maleate, which depletes hepatic glutathione without causing toxicity, signified that glutathione depletion is not the direct cause of hepatotoxicity (Mitchell et al. 1973a, b). Clinical data of APAP conjugation with protein thiol groups is highly correlated to severity of hepatotoxicity (???). Still, there is significant evidence that suggests that covalent binding is not the mechanism of hepatotoxicity (???). This leads to the ...

Introduce 1e- Oxidation Potential and CYP Ability to do this.

Moreover, CYPs have been suggested to catalyze APAP through both a one electron oxidation and a two electron oxidation mechanisms (Potter and Hinson 1987). Oxidation reactions mediated by CYPs involving phenols have been confirmed to occur by both mechanisms of single electron transfers and hydrogen atom transfers (HATs) (???). This could provide grounds for an alternatively toxic metabolism of APAP other than NAPQI, produced via a SAT mechanism followed by radical polymerization. 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 at the molecular level by synthesizing and then characterizing its metabolites in order to consider an alternative mechanism of APAP induced liver injury.

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".

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.

Electrochemical Oxidation of Acetaminophen

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

Discussion

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