Assay for microsomal alpha-hydroxylation of N'-nitrosonornicotine and determination of the deuterium isotope effect for alpha-hydroxylation.

A high-pressure liquid chromatographic assay was developed for microsomal alpha-hydroxylation (2'-hydroxylation and 5'-hydroxylation) of N'-nitrosonornicotine. N'-Nitrosonornicotine was incubated with rat liver microsomes and a reduced nicotinamide adenine dinucleotide phosphate-generating system at 37 degrees. After addition of 2,4-dinitrophenylhydrazine reagent, the mixtures were analyzed by reverse-phase high-pressure liquid chromatography. The 2,4-dinitrophenylhydrazones of 4-hydroxyl-1-(3-pyridyl)-1-butanone and 4-hydroxy-1-(3-pyridyl)-butanal, which are the products of 2'-hydroxylation and 5'-hydroxylation, were quantified by ultraviolet light detection at 254 nm. Km's for 2'-hydroxylation and 5'-hydroxylation of N'-nitrosonornicotine by liver microsomes from Aroclor-treated male F-344 rats were 1.81 and 1.96 mM, while Vmax's were 0.53 and 1.05 nmol/min/mg protein, respectively. Aroclor pretreatment of rats resulted in a 20-fold induction of 2'-hydroxylation, but only a 1.9-fold induction of 5'-hydroxylation. The deuterium isotope effect for alpha-hydroxylation of N'-nitrosonornicotine was determined by comparing the rates of 2'-hydroxylation and 5'-hydroxylation of N'-nitrosonornicotine, N'-[2',5',5'-D]nitrosonornicotine, N'-[2'-D]nitrosonornicotine, and N'-[5',5'-D]nitrosonornicotine. The deuterium isotope effect (Vmax H/Vmax D) was 2.4 to 2.7 for 5'-hydroxylation and 1.2 for 2'-hydroxylation.