APPLICATIONS OF LIQUID CHROMATOGRAPHY/ELECTROCHEMISTRY TO THE STUDY OF THE METABOLISM OF HYDROXYLATED ACETANILIDES
Abstract
The goals of this research were to further illustrate the utility of the liquid chromatography/electrochemistry technique in the determination of easily oxidized substances in biological samples. This combination allowed for the selective detection of electroactive species with picogram detection limits. Previous studies in this laboratory with acetaminophen (APAP, N-acetyl-p-aminophenol) and benzidine (4,4'-diaminobiphenyl) have shown that metabolic activation of these compounds is dependent on a two electron oxidation to an electrophilic species. These intermediates react with cellular nucleophiles to form addition products. It was proposed that analogous reactions should occur for similar easily oxidized compounds. Furthermore, if the formation of addition products is dependent on the oxidation reaction and does not involve an enzyme-mediated addition step (as suspected by this laboratory) the extent of reaction should correlate with ease of oxidation. To this end, the metabolism of the hydroxylated metabolites of acetaminophen, 3-hydroxy and 3-methoxyacetaminophen (3-OH and 3-OMe-APAP) was examined in the presence of mouse liver microsomes. Improved analytical methodology was developed for the determination of the urinary metabolites of APAP. Previously reported procedures were either unsuitable for quantitation or did not include the ring-hydroxylated metabolites. A combined TLC/LCEC approach was developed by which unchanged APAP and seven metabolites (including 3-OH and 3-OMe-APAP) could be determined. In experiments with mouse liver microsomes, 3-OH and 3-OMe-APAP were demonstrated to form analogous addition products to those observed with APAP. The extent of reaction was the greatest for the most easily oxidized compound, 3-OH-APAP and the least for the more difficult to oxidize substrate, APAP. Furthermore, addition products were observed to form from 3-OH-APAP in the absence of a microsomal enzyme system, lending further support to the hypothesis that non-enzymatic reactions are responsible for the transfer of the nucleophile to the ring. The expertise acquired in these studies was applied to a study of the microsomal and urinary metabolites of 2 and 3-hydroxyacetanilide (2-HAA and 3-HAA), isomers of acetaminophen. Although structurally similar, these compounds have been reported to exhibit reduced liver toxicity in comparison of APAP, while retaining similar analgesic effects. Little was known about the metabolism of these compounds, direct chemical studies were previously unreported.
Degree
Ph.D.
Subject Area
Analytical chemistry
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