The urinary metabolites of 17alpha-ethynylestradiol-9alpha,11xi-3H in women. Chromatographic profiling and indentification of ethynyl and non-ethynyl compounds

Abstract

Metabolites of 17alpha-ethynylestradiol (EE2) were obtained from human urine following ingestion of tritium-labeled EE2. Over 95% of the recovered activity was found as conjugated steroids and these were separated into four groups by chromatography of the urine extract on Sephadex LH-20 with chloroform-methanol (1/1) + 0.01M NaCl. The two major conjugate fractions appeared to be almost exclusively glucosiduronates. Enzymatic hydrolysis liberated at least ten different EE2 metabolites as shown by chromatography on Sephadex LH-20 with benzene-methanol (85/15). After additional separation and purification of these metabolites, positive identification was obtained for nine radioactive compounds by either gas liquid chromatography-mass spectrometry or reverse-isotope recrystallization. Five were ethynyl compounds: EE2, 2-MeO EE2, 16beta-OH EE2, 2-OH EE2 and 6alpha-OH EE2. The other four were de-ethynylated estrogens: estrone, estradiol-17beta, estriol, and 2-Me-O-estradiol-17beta.

PIP: Hysterectomized women were administered tritiated-17alpha-ethinyl estradiol either iv or orally and their urine collected and assayed for labeled-metabolites. Over 95% of the recovered activity was found as conjugated steroids. These were separated into 4 groups by Sephadex LH-20 chromatography with chloroform-methanol and .01M NaCl. 2 of the groups were almost entirely glucosiduronates. 17alpha-ethinyl estradiol itself was the major component in 3 of the fractions while 2-methoxy-17alpha-ethinyl estradiol was the major component of the 4th. Hydrolysis, chromatography with benzene-methanol, and further purification of each of the 4 fractions yielded similar metabolite patterns. Of the major compounds identified, 5 were ethinyl compounds: 17alpha-ethinyl estradiol, 2-methoxy-17alpha-ethinyl estradiol, 16 beta-hydroxy-17alpha-ethinyl estradiol, 2-hydroxy-17alpha-ethinyl estradiol, and 6 alpha-hydroxy-17alpha-eithinylestradiol; 4 were deethinylated estrogens: estrone, estreadiol-17beta, estriol, and 2-methoxy-estradiol-17 beta.