High Pressure Liquid Chromatographic Assay for a Hydroxylation of N-Nitrosopyrrolidine by Isolated Rat Liver Microsomes

A high-pressure liquid chromatographic assay for a hydroxylation of N-nitrosopyrrolidine by isolated hepatic microsomes was developed. Mixtures consisting of N-nltrosopyrrolidine, microsomes, and an NADPH-generating system were incubated at 37°. The major product of a hydroxylation of N-nitrosopyrrolidine, 2-hydroxytetrahydrofuran, was trapped by the addition of 2, 4-dinitrophenylhydrazine reagent to form 4-hydroxybutyraldehyde-2, 4-dinitrophenylhydrazone. The latter was quantified by reverse-phase high-pressure liquid chromatography. Under optimal conditions, as determined by varying protein and substrate concentrations, the a hydroxylation of N-nitro-sopyrrolidine was linear for at least 90 min and showed characteristics typical of the microsomal mixed-function oxidase system, such as inhibition by CO and induction by pretreatment of male F-344 rats with Aroclor. N-Nitro-sopyrrolidine exhibited type II spectral changes upon interaction with isolated hepatic microsomes. A close correspondence between binding affinity and a hydroxylation of N-nitrosopyrrolidine was observed.