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

Chi hong B. Chen, G. David McCoy, Stephen S. Hecht, Dietrich Hoffmann, Ernst L. Wynder

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)3812-3816
Number of pages5
JournalCancer Research
Volume38
StatePublished - Nov 1 1978

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