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 metabolites through enzymatic methods of in vitro oxidation, which was visualized through HPLC pairing with a diode array detector. Characterization of these APAP oxidation products were carried out using electron spin resonance (ESR), electrospray ionization tandem mass spectrometry (ESI-MS/MS), and 400 MHz proton nuclear magnetic resonance (H-NMR). Our findings provide structural and mechanistic insight into the radical propagation of potentially deleterious APAP metabolites formed in the liver during times of APAP overdose.

Introduction

The introduction should state the purpose of the investigation and its relation to other work in the field. Background material should be brief and relevant to the research described. Detailed or lengthy reviews of the literature should be avoided.

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 (1,2). The quantitative difference between a safe dose and a toxic dose is narrow, and varies by individual based on environmental, genetic, and metabolic factors. Hence, it is deceptively easy to take too much APAP and cause a trip to the emergency room, permanent liver damage, or even death.

While APAP itself is harmless, APAP metabolic pathways have been investigated by numerous studies which allude to the 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 the critical initiating events for APAP induced hepatotoxicity by way of causing mitochondrial permeability transition (MPT), which is an increase in permeability of the inner membrane that may lead to apoptosis or necrosis of liver cells (3). 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.

APAP may undergo a one electron oxidation to yield the APAP phenoxyl radical (N-acetyl-p-benzosemiquinone imine; NAPSQI) or a direct two electron oxidation to produce N-acetyl-p-benzoquinone imine (NAPQI) via Cytochrome P450 enzymes (CYPs) (4). The result leads to hepatotoxicity after intracellular supplies of glutathione have been used up. The commonly accepted mechanism of oxidative metabolite formation during APAP overdose revolves around the direct two electron oxidation of APAP into NAPQI (5, 6). NAPQI is then purported to be reduced and detoxified via glutathione (GSH) conjugation through glutathione transferase, leading to cysteine and mercapturic acid metabolites to be formed (7). The formation of NAPQI has been indirectly identified as a metabolite of APAP oxidation within the liver through its conjugation with glutathione (6). The uncertainty associated with this indirect metabolic identification raises hesitance to NAPQI being the only actor during APAP overdose. Moreover, the oxidation of APAP by one electron to form NAPSQI may be independently reduced by GSH, reforming APAP and forming glutathione disulfide (GSSG) (8). Under scenarios of depleted glutathione reserves, this pathway may yield a different set of metabolites, which could play bioactive roles responsible for the MPT and resulting hepatotoxicity observed following APAP overdose.

Possible metabolites associated with a one electron oxidation of APAP in the liver have been identified using model systems such as enzymatic or electrochemical methods of oxidation in place of liver microsomes. Following a one electron oxidation by a model enzyme, Horseradish peroxidase (HRP), studies have been done to characterize the APAP radical intermediate in solution through electron spin resonance (ESR) and the potential for this radical to from a different set of metabolites that are not NAPQI (9,10). Characterization of the postulated metabolites allowed for the identification of a set of APAP polymers consistent with a mechanism of radical polymerization (11). Deeper investigation into such metabolites and their radical intermediates 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 elucidate a better understanding of the chemistry occurring during this reaction.

Experimental Procedures

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Acetaminophen, H2O2 (30%), dimethyl sulfoxide, and Horseradish peroxidase (Type ??) were purchased from Sigma Aldrich. All other reagents and solvents were of the highest grade available…. Dioxane, buffers, methanol, dichloromethane

Acetaminophen Oxidation

Acetaminophen oxidations were carried out with 50mM acetaminophen, 0-50mM H2O2, 0-9.7nm HRP, in 25% dioxane to aqueous buffer (pH 9.2). Samples were equilibrated at room temperature and then initiated by the addition of H2O2. Reactions were allowed to go until completion. Acetaminophen oxidation products were then analyzed and isolated.

Liquid Chromatography

The analytical HPLC system consisted of reversed-phase column and a normal phase column. A binary solvent system with a flow rate of 1.0 mL/minute containing Solvent A, 0.1% trifluoroacetic acid, and Solvent B, 100% acetonitrile, allowed for the separation and quantification of acetaminophen metabolites following oxidation. For reverse phase HPLC, Solvent A was held at 100% for the first minute of the run, followed by a linear transition to 100% solvent B until minute 15 at which Solvent B was held constant for 3 more minutes. For normal phase HPLC, Solvent B was held at 100% for the first minute of the run, followed by a linear transition to 50% solvent A until minute 15 at which Solvent A was held constant for 3. BOth methods gave a total run time of 18 minutes.

The automated flash chromatography system used to purify Acetaminophen metabolites for NMR and MS analysis consisted of … Purification was carried out via a 5.5mg C18 column from () with a flow rate of 18 mL/minute. Solvent A was held constant at 100% for 1 column volume, followed by a linear transition to 75% Acetonitrile over the course of 29 column volumes. Acetaminophen metabolite fractions were collected and the solvent mixtures were removed using a rotary evaporator.

Quadruple Time-of-Flight Mass Spectrometry

Electrospray ionization was utilized with a quadruple time-of-flight functioning as a mass analyzer for the analysis of acetaminophen metabolites solubilized in methanol. Samples were introduced to the mass spectrometer using a direct inject UPLC system… WESTERN

Nuclear Magnetic Resonance Spectroscopy

H-NMR spectra were obtained using a 400 MHz Bruker WM-500 NMR spectrometer. Spectra were obtained at room temperature from solutions of samples in dimethyl sulfoxide-d6. KNOX

Electron Spin Resonance

ESR spectra were measured at room temperature using an ESR spectrometer fitted

with a flat cell containing an immobilized enzyme system. The immobilized enzyme system was made using ## HRP bound to aquigel beads. PARAMERTERS Computer simulation of the experimental ESR spectra were carried out using WINSIM.

Computational Work

WebMO was utilized to gather electron spin density information of the acetaminophen radical intermediate. Molecular orbital calculations were gathered using B3LYP/EPR-II as a basis set.

Results

The results should be presented concisely. Tables and figures should be designed to maximize the presentation and comprehension of the experimental data. The same data should not be presented in more than one figure or in both a figure and a table. Detailed interpretation of results should be reserved for the discussion section of an Article.

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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Acknowledgment

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Abbreviations

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