Abstract
A sensitive assay procedure for determining the rate of liver microsomal N-demethylation with chlorcyclizine as substrate is presented. The product, norchlorcyclizine, formed via the N-demethylation of chlorcyclizine, is acetylated by reaction with 3H-acetic anhydride and the reaction product, 3H-acetylnorchlorcyclizine, is quantified. The method is extremely sensitive, utilizing very small amounts of tissue in the incubation, 3H-acetic anhydride of high specific activity for acetylation and an extraction procedure to reduce the blank. The N-demethylation of chlorcyclizine is similar to other microsomal drug and steroid oxidations in that reduced nicotinamide adenine dinucleotide phosphate and Mg++ are required for maximum activity and the rate of demethylation increases with age, is greater in the male than in the female rat and can be stimulated by treatment with phenobarbital. The assay is extremely sensitive since enzyme activity can be determined with as little as 3 mg of liver (0.06 mg of microsomal protein) per ml of incubation mixture and since 0.64 mµmol of norchlorcyclizine formed per ml of incubation mixture, an amount which is 4 times that detectable at zero time, can be measured. This sensitivity will allow the use of this method for studies on the mechanism of microsomal hydroxylations for which only small amounts of enzyme are available.
Footnotes
- Received January 28, 1972.
- Accepted March 27, 1972.
- © 1972 by The Williams & Wilkins Co.
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