Acetaminophen Project - Revision history

Bes: /* Abstract */

2021-05-12T13:32:06Z

Abstract

Bes at 13:28, 12 May 2021

2021-05-12T13:28:03Z

Wfox: /* Introduction */

2021-05-10T19:26:11Z

Introduction

Wfox: /* Introduction */

2021-05-10T19:23:29Z

Introduction

Wfox: /* HPLC */

2021-05-10T19:22:54Z

HPLC

Wfox: /* Early Acetaminophen Trial Sample Preparation */

2021-05-10T19:22:23Z

Early Acetaminophen Trial Sample Preparation

Wfox: /* Early Acetaminophen Trial Sample Preparation */

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Early Acetaminophen Trial Sample Preparation

Wfox: /* Pharmacokinetics & Pharmacodynamics */

2021-05-10T19:21:26Z

Pharmacokinetics & Pharmacodynamics

Wfox: /* Discussion */

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Discussion

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Discussion

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 ''<small>The following document was completed by William B Fox in Spring of 2021 in partial fulfillment of his requirements for a Bachelor's Degree of Science in Biochemistry, Monmouth College, Monmouth, Il 61462. The research was done under the supervision of Bradley E. Sturgeon.</small>''
 ==Abstract==
-Acetaminophen(APAP) is an active ingredient in many over-the-counter and prescription painkillers, such as Tylenol and Oxycodone. APAP is also responsible for approximately 50% of the cases of acute liver-failure in the United States and Great Britain(1). Treatments for acetaminophen-induced liver injury(AILI) are limited. The current mechanism for AILI is the production of hepatotoxic NAPQI as a metabolite in an enzymatic, two-electron oxidation(1). However, evidence also supports a one electron oxidation(2). In an effort to reduce the mortality rate of acetaminophen overdose, we are investigating the identity of single-electron oxidation products and their bioactivity as an alternative mechanism for AILI. An oxidation product of APAP was isolated via high performance liquid chromatography (HPLC) and subsequent flash chromatography. The evidence of a radical intermediate was proven via EPR. The dimer was oxidized in order to test bioactivity and a novel product was observed in the resultant chromatography. These oxidation products can be isolated, identified, and investigated further in order to assess bioactivity.
+Acetaminophen(APAP) is an active ingredient in many over-the-counter and prescription painkillers, such as Tylenol and Oxycodone. APAP is also responsible for approximately 50% of the cases of acute liver-failure in the United States and Great Britain(1). Treatments for acetaminophen-induced liver injury(AILI) are limited. The current mechanism for AILI is the production of hepatotoxic NAPQI as a metabolite in an enzymatic, two-electron oxidation(1). However, evidence also supports a one electron oxidation(2). In an effort to reduce the mortality rate of acetaminophen overdose, we are investigating the identity of single-electron oxidation products and their bioactivity as an alternative mechanism for AILI. An oxidation product of APAP was isolated via high performance liquid chromatography (HPLC) and subsequent flash chromatography. The evidence of a radical intermediate was proven via EPR. The dimer was further oxidized and a novel product was observed. These oxidation products can be isolated, identified, and investigated further in order to assess the overall mechanism of AILI.
 ==Introduction==

← Older revision		Revision as of 13:28, 12 May 2021
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		+	''<small>The following document was completed by William B Fox in Spring of 2021 in partial fulfillment of his requirements for a Bachelor's Degree of Science in Biochemistry, Monmouth College, Monmouth, Il 61462. The research was done under the supervision of Bradley E. Sturgeon.</small>''
	==Abstract==		==Abstract==
	Acetaminophen(APAP) is an active ingredient in many over-the-counter and prescription painkillers, such as Tylenol and Oxycodone. APAP is also responsible for approximately 50% of the cases of acute liver-failure in the United States and Great Britain(1). Treatments for acetaminophen-induced liver injury(AILI) are limited. The current mechanism for AILI is the production of hepatotoxic NAPQI as a metabolite in an enzymatic, two-electron oxidation(1). However, evidence also supports a one electron oxidation(2). In an effort to reduce the mortality rate of acetaminophen overdose, we are investigating the identity of single-electron oxidation products and their bioactivity as an alternative mechanism for AILI. An oxidation product of APAP was isolated via high performance liquid chromatography (HPLC) and subsequent flash chromatography. The evidence of a radical intermediate was proven via EPR. The dimer was oxidized in order to test bioactivity and a novel product was observed in the resultant chromatography. These oxidation products can be isolated, identified, and investigated further in order to assess bioactivity.		Acetaminophen(APAP) is an active ingredient in many over-the-counter and prescription painkillers, such as Tylenol and Oxycodone. APAP is also responsible for approximately 50% of the cases of acute liver-failure in the United States and Great Britain(1). Treatments for acetaminophen-induced liver injury(AILI) are limited. The current mechanism for AILI is the production of hepatotoxic NAPQI as a metabolite in an enzymatic, two-electron oxidation(1). However, evidence also supports a one electron oxidation(2). In an effort to reduce the mortality rate of acetaminophen overdose, we are investigating the identity of single-electron oxidation products and their bioactivity as an alternative mechanism for AILI. An oxidation product of APAP was isolated via high performance liquid chromatography (HPLC) and subsequent flash chromatography. The evidence of a radical intermediate was proven via EPR. The dimer was oxidized in order to test bioactivity and a novel product was observed in the resultant chromatography. These oxidation products can be isolated, identified, and investigated further in order to assess bioactivity.

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 Acetaminophen (''Figure 1''), often referred to as "APAP" is an active ingredient in many over-the-counter and prescription painkillers, such as Tylenol and Oxycodone. The mechanism of pain relief has still not been proven thoroughly. The drug works to raise the pain threshold for a person, effectively increasing the body's requirement for more pain signals in order to feel pain. One of the players in pain are prostaglandins (PG). These are lipid molecules that are made at the site of tissue damage that cause inflammation, and inflammation causes pain (3). Prostaglandins do many other physiological jobs such as protecting the lining of the stomach (3). Enzymes that produce prostaglandins are called cyclooxygenases. These enzymes are often the target for analgesics. APAP originally was thought to inhibit prostaglandin formation in the brain, but was later disputed (3). This was due to the finding that APAP does not suppress PG formation in broken cells, but intact cells(3). Thus, APAP is regarded as a weak inhibitor of both COX-1 and COX-2 enzymes. However, APAP does not have a harmful effect on the gastrointestinal tract, does not affect platelet function, and does not cause breathing issues in asthmatics and therefor has been theorized to be be a selective COX-2 inhibitor (3). Further research showed that APAP is 4.4 times more likely to be selective for COX-2 than COX-1(3). Since this is the case, long term usage of APAP should be cautioned due to potential cardiovascular issues (3).
-APAP is also responsible for approximately 50% of the cases of acute liver-failure in the United States and Great Britain(1). Treatments for acetaminophen-induced liver injury(AILI) are limited. The current mechanism for AILI is the production of hepatotoxic NAPQI as a metabolite in an enzymatic, two-electron oxidation(1). At the overdose of acetaminophen, glutathione is depleted by binding to NAPQI. When glutathione is depleted 70% or more, NAPQI can no longer be detoxified by that pathway, and the excess of NAPQI binds covalently to cellular protein macromolecules in the liver, causing cell death and hepatic necrosis [4]. Currently, there is only one approved treatment for acetaminophen overdose and that is a compound called N-acetylcysteine (NAC). Administration of NAC (<8 hours after ingestion) is crucial for decreasing the risk of hepatotoxicity. There are several side effects after administration and caution should be used when dealing with the compound. It is thought to provide cysteine for glutathione synthesis and possibly to form an adduct directly with the toxic metabolite of acetaminophen, N-acetyl-p-benzoquinoneimine [5]. However, this has not been proven in vivo.
+APAP is also responsible for approximately 50% of the cases of acute liver-failure in the United States and Great Britain(1). Treatments for acetaminophen-induced liver injury(AILI) are limited. The current mechanism for AILI is the production of hepatotoxic NAPQI as a metabolite in an enzymatic, two-electron oxidation(1). At the overdose of acetaminophen, glutathione is depleted by binding to NAPQI. When glutathione is depleted 70% or more, NAPQI can no longer be detoxified by that pathway, and the excess of NAPQI binds covalently to cellular protein macromolecules in the liver, causing cell death and hepatic necrosis (4). Currently, there is only one approved treatment for acetaminophen overdose and that is a compound called N-acetylcysteine (NAC). Administration of NAC (<8 hours after ingestion) is crucial for decreasing the risk of hepatotoxicity. There are several side effects after administration and caution should be used when dealing with the compound. It is thought to provide cysteine for glutathione synthesis and possibly to form an adduct directly with the toxic metabolite of acetaminophen, N-acetyl-p-benzoquinoneimine (5). However, this has not been proven in vivo.
 Evidence also supports a one electron oxidation(2). A radical intermediate was detected via ESR in the 1980s(6,7). Specifically, a phenoxyl radical was detected and subsequent polymeric material formed(6,7). Thus, this radical chemistry could be forming biologically active products that contribute to hepatotoxicity. The oxidation products were also identified shortly after via nuclear magnetic resonance(2). However, there lacks evidence in the literature regarding isolation and further experimentation of these oxidation products. Therein lies the reason for investigating the identity of single-electron oxidation products and their bioactivity as an alternative mechanism for AILI. In order to determine the identities and properties of the single electron oxidation products, it is necessary to first synthesize them. The single electron oxidation can be accomplished by using a system of horseradish peroxidase (HRP) and hydrogen peroxide. HRP carries out two, single-electron oxidations of the substrate, APAP in this case. Cytochrome P450s (CYPs) metabolize countless fo endogenous and exogenous chemicals in the liver including APAP. CYPs  do not bind, oxidize, and move on when encountering substances in the liver. These enzymes behave similar to a peroxidase- such as HRP. The HRP reaction ran ''in vitro'' (''Figure 2'') mimics the reactions that occur in the liver.
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 ===Pharmacokinetics & Pharmacodynamics===
-Therapeutic doses in adults range from 1-4 g/day. The minimum lethal dose is 5-15 g, with acute lethal doses ranging from 13-25 g [8]. An acetaminophen dose of 150 mg/kg causes liver injury. Peak plasma concentrations are seen within 2 hours of ingestion. The half-life is usually 1-3 hours. The metabolites of acetaminophen stay in the body from 4-5 hours[9]. Hepatotoxicity usually develops 24-36 hours after ingestion, whereas renal insufficiency may develop in 2 to 4 days [10].
+Therapeutic doses in adults range from 1-4 g/day. The minimum lethal dose is 5-15 g, with acute lethal doses ranging from 13-25 g (8). An acetaminophen dose of 150 mg/kg causes liver injury. Peak plasma concentrations are seen within 2 hours of ingestion. The half-life is usually 1-3 hours. The metabolites of acetaminophen stay in the body from 4-5 hours (9). Hepatotoxicity usually develops 24-36 hours after ingestion, whereas renal insufficiency may develop in 2 to 4 days (10).
-Acetaminophen is absorbed from the gastrointestinal tract and exhibits dose-dependent kinetics (first order). Plasma protein binding for acetaminophen is between 20 and 50%, and passage of the blood-brain barrier is restricted. Elimination of the metabolized drug is though the kidney [10,11].
+Acetaminophen is absorbed from the gastrointestinal tract and exhibits dose-dependent kinetics (first order). Plasma protein binding for acetaminophen is between 20 and 50%, and passage of the blood-brain barrier is restricted. Elimination of the metabolized drug is though the kidney (10,11).
 ====Metabolism and Excretion====
-About 90% of acetaminophen is biotransformed by cytochrome P-450 in the liver. Main metabolites are sulphate (about 52%) and glucuronide (about 42%) conjugates. About 4% of the drug is biotransformed to N-acetyl-p-benzoquinoneimine (NAPQI), which is a highly reactive cytotoxic intermediate. NAPQI is detoxified by conjugation with glutathione and excreted in the urine as cysteine and mercapturic acid [10,11].90-100% of an ingested dose is excreted in urine during about 24 h [12]. Approximately 2 % of acetaminophen is excreted unchanged [13].
+About 90% of acetaminophen is biotransformed by cytochrome P-450 in the liver. Main metabolites are sulphate (about 52%) and glucuronide (about 42%) conjugates. About 4% of the drug is biotransformed to N-acetyl-p-benzoquinoneimine (NAPQI), which is a highly reactive cytotoxic intermediate. NAPQI is detoxified by conjugation with glutathione and excreted in the urine as cysteine and mercapturic acid (10,11).90-100% of an ingested dose is excreted in urine during about 24 h (12). Approximately 2 % of acetaminophen is excreted unchanged (13).
 {|

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 ==Introduction==
 [[File:APAP Structure.png|400px|thumb|'''Figure 1''': The structure of acetaminophen]]
-Acetaminophen (''Figure 1''), often referred to as "APAP" is an active ingredient in many over-the-counter and prescription painkillers, such as Tylenol and Oxycodone. The mechanism of pain relief has still not been proven thoroughly. The drug works to raise the pain threshold for a person, effectively increasing the body's requirement for more pain signals in order to feel pain. One of the players in pain are prostaglandins. These are lipid molecules that are made at the site of tissue damage that cause inflammation, and inflammation causes pain (3). Prostaglandins do many other physiological jobs such as protecting the lining of the stomach (3). Enzymes that produce prostaglandins are called cyclooxygenases. These enzymes are often the target for analgesics. APAP originally was thought to inhibit prostaglandin formation in the brain, but was later disputed (3). This was due to the finding that APAP does not suppress PG formation in broken cells, but intact cells(3). Thus, APAP is regarded as a weak inhibitor of both COX-1 and COX-2 enzymes. However, APAP does not have a harmful effect on the gastrointestinal tract, does not affect platelet function, and does not cause breathing issues in asthmatics and therefor has been theorized to be be a selective COX-2 inhibitor (3). Further research showed that APAP is 4.4 times more likely to be selective for COX-2 than COX-1(3). Since this is the case, long term usage of APAP should be cautioned due to potential cardiovascular issues (3).
+Acetaminophen (''Figure 1''), often referred to as "APAP" is an active ingredient in many over-the-counter and prescription painkillers, such as Tylenol and Oxycodone. The mechanism of pain relief has still not been proven thoroughly. The drug works to raise the pain threshold for a person, effectively increasing the body's requirement for more pain signals in order to feel pain. One of the players in pain are prostaglandins (PG). These are lipid molecules that are made at the site of tissue damage that cause inflammation, and inflammation causes pain (3). Prostaglandins do many other physiological jobs such as protecting the lining of the stomach (3). Enzymes that produce prostaglandins are called cyclooxygenases. These enzymes are often the target for analgesics. APAP originally was thought to inhibit prostaglandin formation in the brain, but was later disputed (3). This was due to the finding that APAP does not suppress PG formation in broken cells, but intact cells(3). Thus, APAP is regarded as a weak inhibitor of both COX-1 and COX-2 enzymes. However, APAP does not have a harmful effect on the gastrointestinal tract, does not affect platelet function, and does not cause breathing issues in asthmatics and therefor has been theorized to be be a selective COX-2 inhibitor (3). Further research showed that APAP is 4.4 times more likely to be selective for COX-2 than COX-1(3). Since this is the case, long term usage of APAP should be cautioned due to potential cardiovascular issues (3).
 APAP is also responsible for approximately 50% of the cases of acute liver-failure in the United States and Great Britain(1). Treatments for acetaminophen-induced liver injury(AILI) are limited. The current mechanism for AILI is the production of hepatotoxic NAPQI as a metabolite in an enzymatic, two-electron oxidation(1). At the overdose of acetaminophen, glutathione is depleted by binding to NAPQI. When glutathione is depleted 70% or more, NAPQI can no longer be detoxified by that pathway, and the excess of NAPQI binds covalently to cellular protein macromolecules in the liver, causing cell death and hepatic necrosis [4]. Currently, there is only one approved treatment for acetaminophen overdose and that is a compound called N-acetylcysteine (NAC). Administration of NAC (<8 hours after ingestion) is crucial for decreasing the risk of hepatotoxicity. There are several side effects after administration and caution should be used when dealing with the compound. It is thought to provide cysteine for glutathione synthesis and possibly to form an adduct directly with the toxic metabolite of acetaminophen, N-acetyl-p-benzoquinoneimine [5]. However, this has not been proven in vivo.

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 ===HPLC===
-Reverse phase high pressure liquid chromatography (HPLC) was performed on an Agilent Technologies 1100 Series instrument using a C18 column. The experimental method consisted of a 30-minute long run with mobile phase beginning at 100 percent acidified water to 100 percent 30% volume to volume acetonitrile. UV/Vis spectroscopy was used as a detector for absorbance at 270 nm.
+Reverse phase high pressure liquid chromatography (HPLC) was performed on an Agilent Technologies 1100 Series instrument using a C18 column at 28˚C. The experimental method consisted of a 30-minute long run with mobile phase beginning at 100 percent acidified water to 100 percent 30% volume to volume acetonitrile. UV/Vis spectroscopy was used as a detector for absorbance at 270 nm.
 ===Flash Chromatography===

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 ====Early Acetaminophen Trial Sample Preparation====
-A 2 mM acetaminophen pH 5 stock solution was prepared in 100 mL of nanopure water. Stock solutions of Hydrogen peroxide (0.413 M), and HRP were created. The experimental solutions of acetaminophen (2 mM), H<sub>2</subO<sub>2</sub>  (0.25 M, 0.5 M, 1 M, and 2M), and 10 uL of horseradish peroxidase was to each of a reaction vial. (NOTE: this method was to visualize the reaction on the HPLC. In order to obtain a more efficient reaction, the method was updated.)
+A 2 mM acetaminophen pH 5 stock solution was prepared in 100 mL of nanopure water. Stock solutions of Hydrogen peroxide (0.413 M), and HRP were created. The experimental solutions of acetaminophen (2 mM), H<sub>2</sub>O<sub>2</sub>  (0.25 M, 0.5 M, 1 M, and 2M), and 10 uL of horseradish peroxidase was to each of a reaction vial. (NOTE: this method was to visualize the reaction on the HPLC. In order to obtain a more efficient reaction, the method was updated.)
 ====Recent Acetaminophen Trial Sample Preparation====

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 ==Discussion==
-The current mechanism for AILI is the production of hepatotoxic NAPQI as a metabolite in an enzymatic, two-electron oxidation(1). However, evidence shown above supports a one electron oxidation(2). The polymeric material formed during experimentation could have a hepatotoxic effect due to the nature of its size. Polymeric material was observed even in the early oxidation reactions (''Figure 5''). The oxidation products could also play a role in liver injury, this is plausible because the "dimer" was isolated and oxidized. There was a product that was not seen in the original APAP oxidation, so this product could also be bioactive. The only way to know for sure is to repeat the isolation, identification, and experimentation processes shown in this project. So far the first oxidation product has been isolated and oxidized, however it needs to be identified via mass spectrometry. Once this is completed the second, third, and fourth products can be isolated, identified, and tested for bioactivity. Ascorbate could be used in addition to the original HRP/H<sub>2</sub>O<sub>2</sub> experiment to model GSH-APAP interactions. Theoretically, once the products have been investigated thoroughly a new proposal of how AILI occurs in the liver and further treatments can be investigated.
+The current mechanism for AILI is the production of hepatotoxic NAPQI as a metabolite in an enzymatic, two-electron oxidation(1). However, evidence shown in this project supports a one electron oxidation(2). The polymeric material formed during experimentation could have a hepatotoxic effect due to the nature of its size. Polymeric material was observed even in the early oxidation reactions (''Figure 5''). The oxidation products could also play a role in liver injury, this is plausible because the "dimer" was isolated and oxidized (''Figure 7''). There was a product that was not seen in the original APAP oxidation, so the dimer is biologically active and its resulting product could also be bioactive. The only way to know for sure is to repeat the isolation, identification, and experimentation processes shown in this project. So far the first oxidation product has been isolated and oxidized, however it needs to be identified via mass spectrometry. Once this is completed the second, third, and fourth products can be isolated, identified, and tested for bioactivity. Ascorbate could be used in addition to the original HRP/H<sub>2</sub>O<sub>2</sub> experiment to model GSH-APAP interactions and gain further insight. Theoretically, once the products have been investigated thoroughly a new proposal of how AILI occurs in the liver and further treatments can be investigated.
 ==References==