Measurement of enzyme activities of cytochrome P-450 isoenzymes by high-performance liquid chromatographic analysis of products.
A method is described for the qualitative and quantitative determination of the isoenzymes of cytochrome P-450 from rat liver microsomes. Microsomes are incubated with the endogenous steroid 17 beta-testosterone, which results in the formation of a number of stereo-specific hydroxylation products of testosterone. The hydroxylated products were identified using standards or by comparison with data from the literature. The products can be analysed by reversed-phase gradient high-performance liquid chromatography. The assay has been optimised for pH, linearity and time of incubation. An evaluation of the assay was performed for different kinds of microsomes, microsomal dilution and specificity for particular cytochrome P-450 isoenzymes.
Reinerink
E J
EJ
Laboratory for Toxicology, National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands.
Doorn
L
L
Jansen
E H
EH
Van Iersel
A A
AA
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
D007527:Isoenzymes; D013739:Testosterone; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System