Assay of cholesterol 7 alpha-hydroxylase utilizing a silica cartridge column and 5 alpha-cholestane-3 beta, 7 beta-diol as an internal standard.
A simplified and accurate assay of cholesterol 7 alpha-hydroxylase activity using 5 alpha-cholestane-3 beta,7 beta-diol as an internal standard is described. Endogenous microsomal cholesterol was used as the substrate. Following incubation and addition of the internal standard, lipids extracted from the incubation mixture were applied to Bond-Elut silica cartridge columns. 7 alpha-Hydroxycholesterol, 7 beta-hydroxycholesterol, 7-ketocholesterol and the internal standard were quantitatively recovered by eluting the column with 6 ml of benzene-ethyl acetate (2:3, v/v) after removal of cholesterol with 6 ml of benzene-ethyl acetate (9:1, v/v). After trimethylsilylation, the mass of 7 alpha-hydroxycholesterol was determined by capillary gas chromatography with selected-ion monitoring. The method permits a faster, easier and more sensitive determination of the activity of cholesterol 7 alpha-hydroxylase in small samples.
Yamashita
H
H
Department of Surgery I, Kyushu University, Faculty of Medicine, Fukuoka, Japan.
Kuroki
S
S
Nakayama
F
F
D000818:Animals; D002777:Cholestanols; D002790:Cholesterol 7-alpha-Hydroxylase / Q000378:metabolism*; D002849:Chromatography, Gas; D005410:Flame Ionization; D008297:Male; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012015:Reference Standards; D013250:Steroid Hydroxylases / Q000378:metabolism*
D002777:Cholestanols; C007426:dihydroxycoprostane; D009249:NADP; D013250:Steroid Hydroxylases; D002790:Cholesterol 7-alpha-Hydroxylase