Difference between revisions of "Acetaminophen Project"

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===HPLC===
 
===HPLC===
After preparing our solutions, we tested them on the HPLC to see where the peaks were at different concentrations of hydrogen peroxide.
 
 
The following link provides insight into how the HPLC works and how a user can correctly use the instrument.
 
 
[[HPLC|HPLC]]
 
  
 
===EPR===
 
===EPR===

Revision as of 21:55, 4 May 2021


Introduction

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. Currently, there is no effective treatment for AILI. The first step towards achieving this would be to definitively determine the mechanism of acetaminophen metabolite toxicity.

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 [3]. 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 stayed in the body from 4-5 hours[4]. Hepatotoxicity usually develops 24-36 hours after ingestion, whereas renal insufficiency may develop 2 to 4 days after toxic ingestion [5].

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 [6,7].

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 [6,7].

Excretion

90-100% of an ingested dose is excreted in urine during about 24 h [7]. At therapeutic dosage, acetaminophen is excreted as glucuronide (45-55%), sulfate (20- 30%), and cysteine (15-55%). Approximately 2 % of acetaminophen is excreted unchanged [8].

Toxicological Mechanism

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 [9].

Treatment for Acetaminophen Overdose

Administration of N-acetylcysteine (NAC) (<8 hours after ingestion) is crucial for decreasing the risk of hepatotoxicity. 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 [10]. However, this has not been proven in vivo.

Materials and Methods

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. In regard, to acetaminophen the oxidation most likely will take place at the hydroxyl group of the phenol. The single electron oxidations result in the formation of a reactive radical intermediate which can move exist in several different resonance structures.

Acetaminophen Standards

In order for our samples to be tested, a 2 mM Acetaminophen pH 5 solution, Hydrogen peroxide stock solution, and horseradish peroxidase solution were made.

2 mM Acetaminophen Buffer Stock Solution

Put the 2 mM Acetaminophen standard into a jar, and add the pH 5 buffer tablet. Stir until dissolved.

Hydrogen Peroxide Stock Solution

Add 5 mL of water into a scintillation vial. Add 283 uL of 30% Hydrogen Peroxide to the water. The solution is now at about 0.413 M.

Horseradish Peroxidase Solution

Preparing The Final samples

Measure out 5 mL of Acetaminophen into four different scintillation vials. Add differing amounts of Hydrogen peroxide solution into each of the vials (3uL, 6uL, 12uL, & 24uL). Add 10uL horseradish peroxidase solution to each of the vials.


HPLC

EPR

An immobilization of peroxidase from horseradish (HRP) was completed using Affi-Gel® 10. This procedure is centered around the isolectric point of the enzyme. The immobilization was completed using a MOPS buffer (pH 7.0) and about 1 mL of Affi-Gel® 10 beads.

Results

The Acetaminophen samples were ran at a wavelength of 263 nm because that is the lambda max for acetaminophen. Running the samples at this wavelength shows us how much absorption occurs at that wavelength.

The figure displays 2mM Acetaminophen Standard, and 3uL, 6uL, 12uL, and 24uL H2O2 added to the Acetaminophen with Horse Radish Peroxidase.

EPR

Discussion

The data from the HPLC was exported as an arw file in order to be analyzed. This file was then loaded into Igor, which we used to make chromatograms. Chromatograms allowed us to gain a better understanding of the how each concentration of Acetaminophen absorbs at 263 nm for our standards, and how each sample absorbs at 263 nm. We used the information from the chromatograms to make standard curves in excel. The slopes of the standard curves gave us an equation we could use to find the concentration of a sample using the absorbance.


Gepasi

We used the software "Gepasi" to analyze the kinetics of the reaction between horseradish peroxidase (HRP) and hydrogen peroxide. This program simulates how the compounds will react with each other. The following link describes how to use "Gepasi":

Gepasi


Copasi

A newer, updated version of Gepasi is Copasi. This software is newer and will be more efficient in getting data to the user. A link on how to navigate and use Copasi is listed below.

Copasi

References

1.) Hinson, Jack A., Dean W. Roberts, and Laura P. James. "Mechanisms of Acetaminophen-Induced Liver Necrosis." Handbook of Experimental Pharmacology Adverse Drug Reactions (2009): 369-405. Web.

2.) Potter, David W., Dwight W. Miller, and Jack A. Hinson. "Identification of Acetaminophen Polymerization Products Catalyzed by Horseradish Peroxidase." Journal of Biological Chemistry 260.22 (1985): 12174-2180. Print

3.) Winek, C.L. (1994) Drug and chemical blood-level data. Winek’s Toxicological Annual, Pittsburgh. Allegheny County Department Laboratories.

4.) Slattery, JT, Wilson, JM, Kalhorn, TF, Nelson, SD. Dose-Dependent Pharmacokinetics of Acetaminophen: Evidence of Glutathione Depletion in Humans. Clin Pharmacol Ther 1987. Apr. 41(4): 413-8

5.) Poisindex, Thomson Micromedex (2005).

6.) Dollery, C. (1993) Therapeutic Drugs, Vol 1 & 2, , ed., London: Churchill Livingstone.

7.) Dart, R.C. ed. (2004) Medical Toxicology, 3rd ed., , Lippincott, Williams & Wilkins.

8.)Haddad, L.M. & Winchester, J.F. (1990) Clinical Management of Poisoningand Drug Overdose, 2nd edn., Philadelphia, PA, USA: W.B. Saunders.

9.) Casarett and Doull’s Toxicology (The Basic Science of Poisons), (1986) Klaassen, C.D., Amdur, M.O., Doull, J., eds., Macmillan Publishing Company.

10.) Lauterburg, B. H., Corcoran, G. B., & Mitchell, J. R. (1983). Mechanism of action of N-acetylcysteine in the protection against the hepatotoxicity of acetaminophen in rats in vivo. The Journal of clinical investigation, 71(4), 980–991. doi:10.1172/jci110853