Determination of estradiol 2- and 4-hydroxylase activities by gas chromatography with electron-capture detection.
A highly sensitive assay has been developed for measuring the rate of formation of 2-hydroxyestradiol and 4-hydroxyestradiol from estradiol by microsomal preparations. Catechol estrogens were converted to heptafluorobutyryl esters, which were separated by capillary column gas chromatography and quantified using electron-capture detection. 2-Hydroxyestradiol 17-acetate was used as an internal standard. The identity of catechol estrogen derivatives was verified by gas chromatography-mass spectrometry using negative-ion chemical ionization. Estrogens were identified by negative molecular ions and/or by characteristic fragments. This procedure permits quantification of catechol estrogens at the subpicogram level. The assay was validated by comparing estrogen 2- and 4-hydroxylase activities in microsomes from hamster and rat liver with values reported previously.
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston 77550-2782.
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Hachey
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Liehr
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002849:Chromatography, Gas; D006224:Cricetinae; D019363:Cytochrome P-450 CYP1A1; D065633:Cytochrome P-450 CYP1B1; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D004563:Electrochemistry; D004958:Estradiol / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D002393:Estrogens, Catechol; D008401:Gas Chromatography-Mass Spectrometry; D066298:In Vitro Techniques; D008297:Male; D008647:Mesocricetus; D008862:Microsomes, Liver / Q000201:enzymology; D051381:Rats; D011919:Rats, Inbred Strains; D012015:Reference Standards; D012044:Regression Analysis; D013250:Steroid Hydroxylases / Q000032:analysis*
D002393:Estrogens, Catechol; D004958:Estradiol; D003577:Cytochrome P-450 Enzyme System; C001390:2-hydroxyestradiol; C014036:4-hydroxyestradiol; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C585499:Cyp1b1 protein, rat; D019363:Cytochrome P-450 CYP1A1; D065633:Cytochrome P-450 CYP1B1; C020120:estrogen 2-hydroxylase