Characterization of hamster liver nicotine metabolism. I. Relative rates of microsomal C and N oxidation.

A high pressure liquid chromatographic procedure has been developed for the determination of the two principal N- and C-oxidation products of nicotine in hamster liver subcellular fractions. Advantage was taken of the fact that cyanide ion forms a stable adduct with the microsomal metabolite that is the precursor of cotinine. The rate and extent as well as the sensitivity of inhibition were similar for cotinine, the 5'-cyanonicotine adduct, and an as yet unidentified microsomal metabolite which is presumed to be the initial microsomal metabolite on the pathway to cotinine formation. The rates of nicotine-N'-oxidase and nicotine-5'-hydroxylase activities exhibited differential response to inhibitors as well as differential susceptibility to proteolytic digestion. Data are presented which indicate that low levels of nornicotine contamination in stock nicotine resulted in the artifactual formation of methylcyanonornicotine adduct. No evidence consistent with the formation of nornicotine by isolated microsomes was obtained.