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High Pressure Liquid Chromatographic Assay for Hydroxylation of N-Nitrosopyrrolidine by Isolated Rat Liver Microsomes¹

Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, New York 10595

A high-pressure liquid chromatographic assay for hydroxylation of N-nitrosopyrrolidine by isolated hepatic microsomes was developed. Mixtures consisting of N-nitrosopyrrolidine, microsomes, and an NADPH-generating system were incubated at 37°. The major product of 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 hydroxylation of N-nitrosopyrrolidine 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-Nitrosopyrrolidine exhibited type II spectral changes upon interaction with isolated hepatic microsomes. A close correspondence between binding affinity and hydroxylation of N-nitrosopyrrolidine was observed.

¹ Supported by Grants CA-012376 and RR-05775 from the National Cancer Institute. This is paper 10 of the series, "A Study of Chemical Carcinogenesis."

² Recipient of National Cancer Institute, Research Career Development Award 5KO4 CA-00124.

Received 5/30/78. Accepted 8/ 3/78.