Hepatic Aminopyrine N-Demethylase System: Further Studies of Assay Procedure

Abstract

Abstract-Rat hepatic aminopyrine N-demethylase activity was measured by detecting the amount of formaldehyde produced from aminopyrine. Some optimal conditions for the N-demethylation were determined using both isolated microsomes and whole homogenates, and the standard assay method is described. Formaldehyde production from the substrate by microsomal enzyme system was linear to the amount of micro somes added during 3 min reaction time, whereas long-time incubation caused a decrease in the apparent activity of aminopyrine N-demethylation. The N-demethylase activity observed in normal rat liver homogenate was quite similar to that in microsomes when the activity was expressed on the basis of cytochrome P-450 as molecular activity. Pretreatment of animals with typical inducers, phenobarbital and 3-methylcholanthrene, resulted in alteration of the aminopyrine N-demethylase system, which was detectable in both microsomes and whole homogenates.

Heterogeneous distribution of the cytochrome P-450 monooxygenase system in rat liver lobes.

“…Determination of enzyme activity: Activities of aniline hydroxylation, aminopyrine N demethylation and 7-alkoxycoumarin 0 dealkylation reactions were determined as described previously (17,21,22). GOT (aspartate aminotransferase; EC 2.6.1.1) and GPT (alanine aminotransferase; EC 2.6.1.2) activities were determined essentially as described by La Due et al (23) with slight modification (24).…”

Hepatic Aminopyrine N-Demethylase System: Effect of Cyanide on Microsomal N-Demethylase Activity

“…Thus, the effect of cyanide on the oxidative biotransformation of type I substrates was studied using aminopyrine as a model compound. The time course of formaldehyde production from aminopyrine by microsomal enzymes was linear for about 2-3 min, then the rate decreased with time (17). Addition of cyanide to the incubation mixture resulted in a marked decrease of formaldehyde production ( Fig.…”

“…Micro somal aminopyrine N-demethylase activity showed two distinct phases with respect to aminopyrine concentration (Fig. 3), suggesting the association of two species of cytochrome P-450 with the reactions (17). Individual species of microsomal cytochrome P-450 may have the ability to bind with arninopyrine and/or cyanide but differ in their relative affinity to these compounds.…”

“…for three days. Liver microsomes were prepared as described previously (17), and microsomal protein concentration was determined by the biuret reaction (18) using bovine serum albumin as standard.…”

“…The activities of microsomal N-denethylase and aniline hydroxylase were determined as described previously (15,17). The aliphatic hydroxylase activities of hexobarbital and cyclophosphamide were measured at 20'C by determining the initial rate of NADPH oxi dation (19).…”