"An In-Vitro Approach to Identifying Potential Drug Candidates to Prevent Acetaminophen Toxicity"
Youngblood, Vaughn.
2017
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Abstract: Abstract Hepatic toxicity due to acetaminophen (APAP) is the leading
cause of acute liver failure in the United States. Acetaminophen is largely metabolized by
sulfotransferases and glucuronyl transferases. Once these enzymes are saturated, APAP is
metabolized by CYP450 enzymes, particularly CYP2E1, into a hepatotoxic metabolite called
N-acetyl- ρ-benzoquinone imine (NAPQI). Quantita... read moretively measuring the accumulation of this
toxic metabolite has been an elusive topic in previous literature. The analysis of the
complex that NAPQI forms with hepatic glutathione (NAPQI-GSH) is the most reliable approach
to quantitate metabolite formation. Using HPLC, incubations of increasing concentrations of
APAP using human liver microsomes were performed in order to quantify toxic metabolite. To
determine if the NADPH-regenerating system within the reaction was functioning as expected,
incubations with midazolam, a benzodiazepine which undergoes CYP450 metabolism to
hydroxylated metabolites, were performed Concentrations of 2 μM, 5 μM, 20 μM, 50 μM, 200
μM, 500 μM, and 1mM APAP were used for incubations. Incubations consisted of
NADPH-regenerating system including NADP+, MgCl2, sodium citrate, glucose-6-phosphate
(G6P), and G6P dehydrogenase. Phosphate buffer and 1mM APAP (stock) were used as standards.
Mobile phase consisted of 80% potassium phosphate (pH 2.2) and 20% methanol. A
reverse-phase C18 column was used to process the reactions (Waters Nova-Pak® C18 (4μm;
3.9x150mm)). UV absorbance was measured at 254nm. Midazolam incubations were performed
using a Waters Nova-Pak® C18 (4μm; 3.9x150mm) HPLC column. The NAPQI-GSH metabolite peak
was not able to be clearly defined with the specified method. HPLC analysis of APAP
incubations yielded no significant metabolite peak formation. Further investigation using a
similar HPLC method is needed.
Thesis (M.S.)--Tufts University, 2017.
Submitted to the Dept. of Pharmacology & Experimental Therapeutics.
Advisors: David Greenblatt, and Margery Beinfeld.
Keyword: Pharmacology.read less - ID:
- 5999nf93x
- Component ID:
- tufts:22771
- To Cite:
- DCA Citation Guide EndNote
