Acetaminophen Manuscript - Revision history

Matt1014: /* Results */

2022-07-07T13:11:42Z

Results

Matt1014 at 13:10, 7 July 2022

2022-07-07T13:10:39Z

Matt1014: /* Discussion */

2022-07-07T13:08:45Z

Discussion

Matt1014: /* Results */

2022-07-07T13:07:25Z

Results

Matt1014: /* Results */

2022-07-07T13:06:56Z

Results

Matt1014: /* Experimental Procedures */

2022-07-07T13:05:53Z

Experimental Procedures

Matt1014: /* Introduction */

2022-07-07T13:04:36Z

Introduction

Matt1014: /* Introduction */

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Introduction

Matt1014: /* Introduction */

2022-07-07T13:03:56Z

Introduction

Matt1014: /* Abstract */

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Abstract

← Older revision		Revision as of 13:11, 7 July 2022
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	Compound A, the APAP dimer (diAPAP) and earliest formed metabolite during APAP oxidation, was further investigated using H-NMR. The zoomed in aromatic regions are shown for both the experimentally purified APAP dimer as well as a purchased ortho-linked APAP dimer with the purpose of being used as a standard (Figure 4). The fact that compound A holds similarity too, but also unique splitting patterns from the ortho-linked APAP dimer standard indicates that the enzymatic oxidation of APAP yields a mixture of diAPAP isomers. This was further visulaized by through normal phase HPLC, in which poor but sufficient separation allowed for the visualization of two chemically unique absorbance peaks (Figure 6). The two geometric isomers were attributed to the prescence of an ortho-linked diAPAP and an ether-linked diAPAP.		Compound A, the APAP dimer (diAPAP) and earliest formed metabolite during APAP oxidation, was further investigated using H-NMR. The zoomed in aromatic regions are shown for both the experimentally purified APAP dimer as well as a purchased ortho-linked APAP dimer with the purpose of being used as a standard (Figure 4). The fact that compound A holds similarity too, but also unique splitting patterns from the ortho-linked APAP dimer standard indicates that the enzymatic oxidation of APAP yields a mixture of diAPAP isomers. This was further visulaized by through normal phase HPLC, in which poor but sufficient separation allowed for the visualization of two chemically unique absorbance peaks (Figure 6). The two geometric isomers were attributed to the prescence of an ortho-linked diAPAP and an ether-linked diAPAP.

−	[[File:stacked_diapap_NMR.png\|~~350px~~\|thumb\|left\|Figure 4:]] [[File:diAPAP_Chromatography.png\|350px\|thumb\|center\| Figure 6: Compound A chromatography was attempted using both reverse phase HPLC (red) as was done prior and normal phase HPLC (black).]]	+	[[File:stacked_diapap_NMR.png\|500px\|thumb\|left\|Figure 4:]] [[File:diAPAP_Chromatography.png\|350px\|thumb\|center\| Figure 6: Compound A chromatography was attempted using both reverse phase HPLC (red) as was done prior and normal phase HPLC (black).]]

← Older revision		Revision as of 13:10, 7 July 2022
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	According to the ESR data collected, APAP appears to be most likely to react at the ortho carbon position to form an ortho-linked dimer (diAPAP). This is supported by previous APAP dimer identification performed by Potter in 1985; however, there was an additional geometric isomer of the APAP dimer that Potter did not characterize (11). The additional APAP dimer was found to be an ether-linked diAPAP molecule. Additionally, the diAPAP ESR indicates the mechanism of APAP oxidation by HRP is APAP mediated or APAP induced. In other words, APAP drives the reaction forward. To clarify, diAPAP does function as a substrate for the peroxidase, but does not yield large product fromation when compared to spiking the solution with low levels of APAP. Introducing APAP to the oxidation of diAPAP yielded far greater product outcomes as well as the depletion of diAPAP. It can be reasoned that APAP may be oxidized into a radical intermediate, which then directly adducts to diAPAP to form immediate APAP trimer or stimulates diAPAP oxidation by stealing an electron from diAPAP to generate the diAPAP radical and become reduced back to APAP.		According to the ESR data collected, APAP appears to be most likely to react at the ortho carbon position to form an ortho-linked dimer (diAPAP). This is supported by previous APAP dimer identification performed by Potter in 1985; however, there was an additional geometric isomer of the APAP dimer that Potter did not characterize (11). The additional APAP dimer was found to be an ether-linked diAPAP molecule. Additionally, the diAPAP ESR indicates the mechanism of APAP oxidation by HRP is APAP mediated or APAP induced. In other words, APAP drives the reaction forward. To clarify, diAPAP does function as a substrate for the peroxidase, but does not yield large product fromation when compared to spiking the solution with low levels of APAP. Introducing APAP to the oxidation of diAPAP yielded far greater product outcomes as well as the depletion of diAPAP. It can be reasoned that APAP may be oxidized into a radical intermediate, which then directly adducts to diAPAP to form immediate APAP trimer or stimulates diAPAP oxidation by stealing an electron from diAPAP to generate the diAPAP radical and become reduced back to APAP.
		+
		+	==References==
		+	1. Lee, W. M. (2013) [[Media:Lee; Drug Induced Liver Failure.pdf\|'''Drug-induced acute liver failure''']]. ''Clin Liver Dis.'' 17, 575-586.
		+
		+	2. Bernal, W., Lee, W. M., Wendon, J., Larsen, F. S., and Williams, R. (2015) [[Media:A curable disease.pdf\|'''Acute liver failure: A curable disease by 2024?''']]. ''J. Hepatol.'' 62, 112-120.
		+
		+	3. Hinson, J. A., Reid, A. B., McCullough, S. S., and James, L. P. (2004) [[Media:MPT.pdf\|Acetaminophen‐Induced Hepatotoxicity:'''Role of Metabolic Activation, Reactive Oxygen/Nitrogen Species, and Mitochondrial Permeability Transition''']]. ''Drug Metabolism Reviews'' 36(3-4), 805–822.
		+
		+	4. Potter, D. W. and Hinson, J. A. (1987) [[Media:CYP versus HRP.pdf\|'''Mechanisms of acetaminophen oxidation to N-acetyl-P-benzoquinone imine by horseradish peroxidase and cytochrome P-450''']]. ''J Biol Chem.'' 262, 966–973.
		+
		+	5. Gillette, J. R., Nelson, S. D., Mulder, G. J., Jollow, D. J., Mitchell, J. R., Pohl, L. R., and Hinson, J. A. (1981) '''Formation of chemically reactive metabolites of phenacetin and acetaminophen.''' ''Adv Exp Med Biol.'' 136, 931–950.
		+
		+	6. Dahlin, D. C., Miwa, G. T., Lu, A. Y., and Nelson, S. D. (1984) [[Media:NAPQI discovery.pdf\|'''N-acetyl-p-benzoquinone imine: a cytochrome P-450-mediated oxidation product of acetaminophen''']]. ''Proc. Natl. Acad. Sci. U. S. A.'' 81, 1327-1331.
		+
		+	7. Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gillette, J. R., and Brodie, B. B. (1973b) '''Acetaminophen-induced hepatic necrosis. IV. Protective role of glutathione'''. ''J Pharmacol Exp Ther.'' 187, 211–217.
		+
		+	8. Ross, D., Norbeck, K., and Moldeus, P. (1985) [[Media:Glutathione radical fate.pdf\|'''The Generation and Subsequent Fate of Glutathionyl Radicals in Biological Systems''']]. ''Journal of Biological Chemistry'' 260(28), 15028-15032.
		+
		+	9. West, P. R., Harman, L. S., Josephy, P. D., and Mason, R. P.(1984) [[Media:West 1984.pdf\|'''Acetaminophen: enzymatic formation of a transient phenoxyl free radical''']]. ''Journal of Biochemical Pharmacology'' 33(18), 2933-2936.
		+
		+	10. Fischer, V., West, P. R., Harman, L. S., and Mason, R. P. (1985) [[Media:second APAP ESR.pdf\|'''Free-Radical Metabolites of Acetaminophen and a Dimethylated Derivative''']]. ''Environmental Health Perspectives''64, 127-137.
		+
		+	11. Potter, D. W., Miller, D. W., and Hinson, J. A. (1985) [[Media:J._Biol._Chem.-1985-Potter-12174-80.pdf\|'''Identification of Acetaminophen Polymerization Products Catalyzed by Horseradish Peroxidase.''']] ''J. Biol. Chem.'' 260(12), 174-180.
		+
		+	12. Aqueta, A. and Collins, A. (2016) [[Media:nutrients.pdf\|'''Polyphenols and DNA Damage: A Mixed Blessing''']]. ''Nutrients'' 8(785), 1-21.

	==Funding Information==		==Funding Information==

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 ==Discussion==
-:The purpose of the discussion is to interpret the results and to relate them to existing knowledge in the field in as clear and brief a fashion as possible. Information given elsewhere in the manuscript should not be repeated in the discussion. Extensive reviews of the literature should be avoided.
+:''The purpose of the discussion is to interpret the results and to relate them to existing knowledge in the field in as clear and brief a fashion as possible. Information given elsewhere in the manuscript should not be repeated in the discussion. Extensive reviews of the literature should be avoided.''
 ==Funding Information==

@@ Line 49: / Line 49: @@
 ==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.
+'':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.''
 [[File:APAP_ESR_spectra(2).png|thumb|left|350px|Figure 1: The data was ccollected according to ESR methods described above while perating at 9.8 GHz, 20 mW microwave power, 60 Gauss sweep width, and 0.5 Gpp modulation amplitude (100KHz). A 2mM APAP, 1mM H2O2, phosphate-citrate buffer (pH 5) solution was flowed through the IE system in order to generate the experimental spectra.]]Oxidation of APAP through the IE-ESR system allowed for the detection of a free radical (Figure 1). Low resolution modulation (0.5 G) allowed for a high resolution ESR spectrum with high signal to noise as illustrated in spectrum A. The assignment of hyperfine coupling constants was done using computer software to give a simulation of the full experimental spectrum in spectrum B. Mathematical removal of the signal associated with species 2 yielded the simulated spectrum C while the removal of the species 1 signal resulted in spectrum D. The assignment of the hyperfine coupling values of spectrum B (APAP radical) is summarized in Table # due to the removal of the signal associated with further polymerized APAP radical background noise being mathematically removed. The formation of the radical was dependent upon the presence of APAP, HRP, and H2O2.

@@ Line 51: / Line 51: @@
 :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'''
+[[File:APAP_ESR_spectra(2).png|thumb|left|350px|Figure 1: The data was ccollected according to ESR methods described above while perating at 9.8 GHz, 20 mW microwave power, 60 Gauss sweep width, and 0.5 Gpp modulation amplitude (100KHz). A 2mM APAP, 1mM H2O2, phosphate-citrate buffer (pH 5) solution was flowed through the IE system in order to generate the experimental spectra.]]Oxidation of APAP through the IE-ESR system allowed for the detection of a free radical (Figure 1). Low resolution modulation (0.5 G) allowed for a high resolution ESR spectrum with high signal to noise as illustrated in spectrum A. The assignment of hyperfine coupling constants was done using computer software to give a simulation of the full experimental spectrum in spectrum B. Mathematical removal of the signal associated with species 2 yielded the simulated spectrum C while the removal of the species 1 signal resulted in spectrum D. The assignment of the hyperfine coupling values of spectrum B (APAP radical) is summarized in Table # due to the removal of the signal associated with further polymerized APAP radical background noise being mathematically removed. The formation of the radical was dependent upon the presence of APAP, HRP, and H2O2.
-:[[File:APAPrad_WebMO.png|300px]] using B3YLP/EPR-II basis set
+In order to better understand the oxidation of APAP, both in vitro “beaker” oxidations by HRP at varying pHs (pH 5.0, 7.4, and 9.2) and electrochemical oxidations were carried out. The result was the same product formation whether considering electrochemical or enzymatic methods of oxidation. Figure 2 depicts the HRP mediated oxidation of APAP to completion in 25% dioxane - pH 9.2 buffer since clearly visualizable product formation was maximized under those conditions. Dioxane was utilized as a solvent since it could both solubilize the water insoluble APAP oxidation products and retain the functionality of HRP.
-[[File:pH7.png|600px]]
+Given that, compounds A and B are strongly suggested to be a dimer and trimer of APAP, respectively. Following the same reasoning, compounds C, D, and E are consistent with the formation of two APAP tetramers and a pentamer. Compounds A and B were further oxidized under the same conditions as APAP in order to visualize product outcome by way of their individual oxidation by HRP (Figure 3). The oxidation of compound A yielded a product that eluted off of the reverse phase column at the same time as compound C while the oxidation of compound B yielded a product that elutes at approximately the same time as compound E.
-'''Flash Purification of APAP Metabolites'''
+Compound A, the APAP dimer (diAPAP) and earliest formed metabolite during APAP oxidation, was further investigated using H-NMR. The zoomed in aromatic regions are shown for both the experimentally purified APAP dimer as well as a purchased ortho-linked APAP dimer with the purpose of being used as a standard (Figure 4). The fact that compound A holds similarity too, but also unique splitting patterns from the ortho-linked APAP dimer standard indicates that the enzymatic oxidation of APAP yields a mixture of diAPAP isomers. This was further visulaized by through normal phase HPLC, in which poor but sufficient separation allowed for the visualization of two chemically unique absorbance peaks (Figure 6). The two geometric isomers were attributed to the prescence of an ortho-linked diAPAP and an ether-linked diAPAP.
-'''Nuclear Magnetic Resonance of Early Acetaminophen Metabolism'''
+Oxidation of diAPAP through the IE-ESR system allowed for the detection of a radical intermediate as illustrated at the top of Figure 7. The signal associated with the oxidation of diAPAP gave a broadband polymeric signal. Spiking the solution with a fractional amount of APAP allowed for a larger signal and for unique character to develop, which is shown just below the top spectra. Switching from a modulation amplitude of 1.0 G to 0.5 G gave high resolution as seen in black. Hyperfine coupling constants were then assigned to this highly resolved signal via the use of computer software. The simulation can be visualized in red just below the experimentally collected data. The determined hyperfine coupling constants of diAPAP were exactly half that of the APAP. The ortho protons, aH, and amide nitrogens, aN, had simulated constants of 2.53 G and 0.88 G compared to APAP’s simulated constants of 5.19 G and 1.68 G, respectively.
-'''Reverse Phase versus Normal Phase Separation of diAPAP'''
+Given that, spiking the diAPAP sample with fractional APAP levels resulted in an increase in the radical concentration of diAPAP. The oxidation products associated with the solo diAPAP oxidation and the APAP spiked diAPAP oxidation through the IE-ESR system is portrayed in Figure 8. diAPAP oxidation resulting in the broadband ESR spectra of Figure 8 results in product formation of compound C, an acetaminophen tetramer. However, when spiked with fractional APAP, the main product outcome of diAPAP oxidation was compound B, an acetaminophen trimer, followed by compound C and further conjugated polymers.
 ==Discussion==

@@ Line 26: / Line 26: @@
 :Procedures for experimental methods should be described in sufficient detail to enable other investigators to repeat the experiments. Names of product manufacturers (with city, state address, catalog number) should be included if alternate sources are deemed unsatisfactory or if the product is of limited availability. Novel experimental procedures should be described in detail, but previously published procedures should be referred to by literature citation of the original detailed explanation, and should include description of any modifications.
-: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 Oxidation''
+:The automated flash chromatography system used to purify Acetaminophen metabolites for characterization was a Teledyne Isco CombiFlash Rf 200i system. Purification was carried out via a 5.5mg C18 gold column 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 additional column volumes. Acetaminophen metabolite fractions were collected and the solvent mixtures were removed using a rotary evaporator.
-''Liquid Chromatography''
+:''Nuclear Magnetic Resonance Spectroscopy''
-: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.
+:H-NMR spectra were obtained using a 400 MHz Bruker NMR spectrometer. Spectra were obtained at room temperature with samples solubilized in deuterated dimethyl sulfoxide.
-: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.
+:''Computational Work''
+:WebMO and Gaussian were 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==

@@ Line 16: / Line 16: @@
 :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.

@@ Line 15: / Line 15: @@
 :''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.''
-IUPAC NAMES for Acetaminophen (APAP), paracetamol, and intermediates
+: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 non-toxic Metabolism''
+: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.
-: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.
+: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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 :''An abstract should be included with all Articles, Communications, Chemical Profiles, Reviews, and Perspectives. For Articles, Communications, and Chemical Profiles, the abstract should briefly (300 word maximum) present, in one paragraph, the problem and experimental approach and state the findings and conclusions. For Reviews and Perspectives, the abstract should introduce the topic, summarize key points, and state the major conclusions. In all cases, the abstract should be self- explanatory and suitable for reproduction without rewriting. Footnotes or undefined abbreviations may not be used. Avoid the use of jargon, but include keywords relevant to the field to improve indexing and discoverability to potential readers. If a reference must be cited, complete publication data must be given.''
+: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==