High pressure liquid chromatographic assay for alpha hydroxylation of N-nitrosopyrrolidine by isolated rat liver microsomes.
A high-pressure liquid chromatographic assay for alpha 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 degrees. The major product of alpha 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 alpha 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 alpha hydroxylation of N-nitrosopyrrolidine was observed.
D000818:Animals; D001140:Aroclors / Q000494:pharmacology; D001711:Biotransformation; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D006900:Hydroxylation; D066298:In Vitro Techniques; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D009242:N-Nitrosopyrrolidine / Q000378:metabolism*; D009602:Nitrosamines / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344
D001140:Aroclors; D009602:Nitrosamines; D009242:N-Nitrosopyrrolidine