Acetaminophen Induced Toxicity - Revision history

Matt1014: /* Results */

2022-05-19T23:07:54Z

Results

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2022-05-19T22:43:59Z

Results

Matt1014: /* Discussion */

2022-05-13T15:08:58Z

Discussion

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2022-05-13T03:30:39Z

Discussion

Matt1014: /* References */

2022-05-13T03:21:56Z

References

Matt1014: /* Discussion */

2022-05-13T03:18:37Z

Discussion

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2022-05-13T02:59:29Z

Results

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2022-05-13T02:59:05Z

Results

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2022-05-13T02:56:05Z

Results

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2022-05-13T02:54:09Z

Discussion

← Older revision		Revision as of 23:07, 19 May 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: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\|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).]]

← Older revision		Revision as of 22:43, 19 May 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:~~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: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).]]

@@ Line 69: / Line 69: @@
 ==Discussion==
-Clearly, APAP oxidation is not so simple so as to only lead to the formation of one, clear cut, metabolite during oxidation in times of overdose. While NAPQI may be a product of APAP oxidation in the liver, products of a one electron oxidation cascade cannot be written out and remain prevalent. Five major APAP oxidation products were shown to be formed after modeling the CYP mediated reaction with HRP, a classic peroxidase. CYPs have been shown to catalyze one electron oxidations of phenolic compounds in environments of oxidative stress, which is certaintly the case with APAP overdose once glutathione levels have been depleted (). Perhaps the metabolism of APAP after gluthione depletion favors the mechanism of radical polymerization as opposed to the net two electron oxidation of APAP into NAPQI. Considering the former could clear up discrepencies when considering the mystery of how APAP induced liver necrosis disseminates. For instance, while polyphenolic compounds are generally seen as safe supplements, high concentration seem to function antagonistically and were purported to lead to increases in DNA damage (12). If APAP is oxidized via a mechanism of one electron oxidation that yields polymers, then the concentration of polyphenols being formed would be classified as very high. This could elucidate the mechanism of mitochondrial dysfunction often associated with APAP overdose in the liver. If mitochondrial DNA is being damaged, then the kill switches, necrosis or apoptosis, could be signaled for in hepatocytes, thus bringing about APAP induced hepatotoxicity.
+Clearly, APAP oxidation is not so simple so as to only lead to the formation of one, clear cut, metabolite during oxidation in times of overdose. While NAPQI may be a product of APAP oxidation in the liver, products of a one electron oxidation cascade cannot be written out and remain prevalent. Five major APAP oxidation products were shown to be formed after modeling the CYP mediated reaction with HRP, a classic peroxidase. CYPs have been shown to catalyze one electron oxidations of phenolic compounds in environments of oxidative stress, which is certaintly the case with APAP overdose once glutathione levels have been depleted (4). Perhaps the metabolism of APAP after gluthione depletion favors the mechanism of radical polymerization as opposed to the net two electron oxidation of APAP into NAPQI. Considering the former could clear up discrepencies when considering the mystery of how APAP induced liver necrosis disseminates. For instance, while polyphenolic compounds are generally seen as safe supplements, high concentration seem to function antagonistically and were purported to lead to increases in DNA damage (12). If APAP is oxidized via a mechanism of one electron oxidation that yields polymers, then the concentration of polyphenols being formed would be classified as very high. This could elucidate the mechanism of mitochondrial dysfunction often associated with APAP overdose in the liver. If mitochondrial DNA is being damaged, then the kill switches, necrosis or apoptosis, could be signaled for in hepatocytes, thus bringing about APAP induced hepatotoxicity.
 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.

@@ Line 71: / Line 71: @@
 Clearly, APAP oxidation is not so simple so as to only lead to the formation of one, clear cut, metabolite during oxidation in times of overdose. While NAPQI may be a product of APAP oxidation in the liver, products of a one electron oxidation cascade cannot be written out and remain prevalent. Five major APAP oxidation products were shown to be formed after modeling the CYP mediated reaction with HRP, a classic peroxidase. CYPs have been shown to catalyze one electron oxidations of phenolic compounds in environments of oxidative stress, which is certaintly the case with APAP overdose once glutathione levels have been depleted (). Perhaps the metabolism of APAP after gluthione depletion favors the mechanism of radical polymerization as opposed to the net two electron oxidation of APAP into NAPQI. Considering the former could clear up discrepencies when considering the mystery of how APAP induced liver necrosis disseminates. For instance, while polyphenolic compounds are generally seen as safe supplements, high concentration seem to function antagonistically and were purported to lead to increases in DNA damage (12). If APAP is oxidized via a mechanism of one electron oxidation that yields polymers, then the concentration of polyphenols being formed would be classified as very high. This could elucidate the mechanism of mitochondrial dysfunction often associated with APAP overdose in the liver. If mitochondrial DNA is being damaged, then the kill switches, necrosis or apoptosis, could be signaled for in hepatocytes, thus bringing about APAP induced hepatotoxicity.
-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.
+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==

← Older revision		Revision as of 03:21, 13 May 2022
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	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.		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.


	4. :[[Media:J. Biol. Chem.-1990-Ramakrishna Rao-844-7.pdf]]		4. :[[Media:J. Biol. Chem.-1990-Ramakrishna Rao-844-7.pdf]]

← Older revision		Revision as of 02:59, 13 May 2022
Line 57:		Line 57:
	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:diAPAP_chromatography.png\|350px\|thumb\|~~right~~\| Figure 6: Compound A chromatography was attempted using both reverse phase HPLC (red) as was done prior and normal phase HPLC (black).]]	+	[[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 02:56, 13 May 2022
Line 57:		Line 57:
	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:diAPAP_chromatography.png\|350px\|thumb\|right]]

← Older revision		Revision as of 02:54, 13 May 2022
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	==Discussion==		==Discussion==
		+
		+	Clearly, APAP oxidation is not so simple so as to only lead to the formation of one, clear cut, metabolite during oxidation in times of overdose. While NAPQI may be a product of APAP oxidation in the liver, products of a one electron oxidation cascade cannot be written out and remain prevalent. Five major APAP oxidation products were shown to be formed after modeling the CYP mediated reaction with HRP, a classic peroxidase.

	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.		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.