Separation of acetanilide and its hydroxylated metabolites and quantitative determination of "acetanilide 4-hydroxylase activity" by high-pressure liquid chromatography. |
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Authors: | T M Guenthner M Negishi D W Nebert |
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Institution: | Developmental Pharmacology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014 USA |
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Abstract: | A simple and very sensitive method for the separation of 4-hydroxyacetanilide, 3-hydroxyacetanilide, 2-hydroxyacetanilide, and acetanilide was developed with the use of high-pressure liquid chromagraphy. Each of these phenolic derivatives can be separated completely from acetanilide and from one another. A simple assay for “acetanilide 4-hydroxylase activity” is thus described. The limit of sensitivity for cytochrome P-450-mediated acetanilide 4-hydroxylase activity is estimated to be 1.0 pmol/min/mg microsomal protein, thereby allowing this assay to be useful in detecting monooxygenase activity in “low level” nonhepatic tissues. Hepatic acetanilide 4-hydroxylase activity is induced about fourfold in mice by 3-methylcholanthrene. Although acetanilide 2-hydroxylase activity is about seven times lower than the 4-hydroxylase activity, the 2-hydroxylase is also induced about three- or fourfold in mice by 3-methylcholanthrene. The “2-hydroxylase activity” cannot, however, be strictly quantitated under the conditions described herein. The Km values of both the 3-methylcholanthrene-induced and control 4-hydroxylase activity are about 0.55 mm; Vmax values for 3-methylcholanthrene-treated and control mice, respectively, are 4.9 ± 1.1 and 1.1 ± 0.31 nmol/min/mg microsomal protein. The 4-hydroxylase in the liver of both 3-methylcholanthrene-treated and control mice appears to represent two or more catalytic activities, i.e., two or more forms of P-450 having widely differing affinities for the substrate acetanilide. |
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