Clinical analysis of steroids. XXV. Assay of estradiol 17-sulfate 2-hydroxylase activity by high-performance liquid chromatography.
A simple method for the assay of the 2-hydroxylation enzyme of estradiol 17-sulfate in rat liver microsomes has been established by using reversed-phase high-performance liquid chromatography with electrochemical detection. The technique devised involves elution with 50 mM acetate buffer, pH 5.0--methanol (3:2, v/v) using an ODS SIL column and monitoring the potentials at 1.1 V vs. the silver/silver chloride reference electrode. The calibration curve of the relationship between the amounts of 2-hydroxyestradiol 17-sulfate (5-50 ng) injected and peak heights (cm) of the chromatogram was linear. By this method, some kinetic parameters of estradiol 17-sulfate 2-hydroxylase were measured.
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D019363:Cytochrome P-450 CYP1A1; D066298:In Vitro Techniques; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D013250:Steroid Hydroxylases / Q000032:analysis*
D013250:Steroid Hydroxylases; D019363:Cytochrome P-450 CYP1A1; C020120:estrogen 2-hydroxylase