Application of A. C.-polarography in a study of p-nitroanisole metabolism and its kinetic properties.

Phase sensitive alternating current polarography was introduced for the simultaneous determination of p-nitroanisole and its metabolites p-nitrophenol and p-nitrocatechol in kinetic studies with rat liver microsomes. The substrate p-nitroanisole disappears rather rapidly while p-nitrophenol is formed. First traces of a second oxidation product, p-nitrocatechol, can be detected only after a few minutes after the initiation of the reaction. This suggest that O-demethylation of p-nitroanisole is the primary reaction which is followed by aromatic ortho hydroxylation of p-nitrophenol. After incubation times longer than 15 minutes, appreciable amounts of p-nitrocatechol are found which shows optical absorption characteristics similar to those of p-nitrophenol (absorption maximum at 440 nm). It is concluded from these kinetic experiments that optical determination of the primary metabolite during the initial reaction phase constitutes a reliable measure of microsomal O-demethylation activity. Phenobarbital induction differentially increases O-demethylation and ring-ortho-hydroxylation activities. From this and respective inhibition studies it is concluded that possibly multiple forms of cytochrome P-450 are involved in the metabolism of either p-nitroanisole or p-nitrophenol.