Assay for Microsomal α-Hydroxylation of N′-Nitrosonornicotine and Determination of the Deuterium Isotope Effect for α-Hydroxylation¹

A high-pressure liquid chromatographic assay was developed for microsomal α-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°. After addition of 2,4-dinitrophenylhydrazine reagent, the mixtures were analyzed by reverse-phase high-pressure liquid chromatography. The 2,4-dinitrophenylhydrazones of 4-hydroxy-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. K_m'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 V_max'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 α-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 (V_{max h}/V_{max d}) was 2.4 to 2.7 for 5′-hydroxylation and 1.2 for 2′-hydroxylation.