Mary C. Williams

The urinary metabolites of 17α-ethynylestradiol-9α,11-3H in women chromatographic profiling and identification of ethynyl and non-ethynyl compounds

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.

Human urinary and liver conjugates of 17α-ethynylestradiol

Abstract Chromatographic profiles of the conjugates of 17α-ethynylestradiol (EE 2 ) were obtained from the urine of castrate and normal women given tritium labeled EE 2 orally. Five distinct radioactive peaks were observed, with considerable quantitative variation between individuals. Glucosiduronate fractions comprised the dominant excreted radioactivity. In vitro incubation of normal human liver also produced five conjugate types. Simultaneous intravenous/oral ( 14 C/ 3 H) administration of EE 2 demonstrated conversion to identical urinary conjugate and free steroid products, with a greater excretion of the intravenous material. Authentic 3-glucuronide of EE 2 was synthesized and its position in the chromatographic system relative to the urinary conjugate peaks demonstrated.

Relative rates of 2- and 4-hydroxyestrogen synthesis are dependent on both substrate and tissue

1. introduction 2. Materials and methods The variety of physiological effects attributed to estrogens, as well as the pathological characteristics of estrogen-sensitive human breast tumors, have prompted numerous studies on the possible metabolic conver- sion of the parent estrogens to activated metabolites. Most of these efforts have focused on the formation and properties of the catecholestrogens of El and E?. The enzyme system catalyzing their synthesis is a cytochrome WSOdependent monooxygenase localized primarily in liver, but also found in numerous estrogen-sensitive mammalian tissues [ 1,2] including human mammary neoplasma [3]. Little information is available on the substrate specificity of this microsomal enzyme system. It is usually described as an estrogen 2-hydroxylase, since derivatives of 2-OHE, and 2-OHEa are the principal products identified from incubations of El and Ez, respectively, with microsomes from rat tissues [4,7]. The objective of this study was to determine if differ- ences in the structure of estrogens of major therapeutic interest would significantly affect the relative rates of formation of their 2- and 4-hydroxy metabolites. Evi- dence was also sought for the biosynthesis of cate- cholestrogens by microsomes from a cultured line of human mammary tumor cells whose growth was known to be stimulated by these hormones [2]. Moxestrol, the 2- and 4-hydroxy derivatives of moxestrol [8] and ethynylestradiol were generously provided by Drs J. P. Raynaud and G. Deltour (Roussel-UCLAF, Romainville). The method in [9] was used for the synthesis of 4.hydroxyequilenin. Other catecholestrogens and their monomethyl ethers were obtained from Steraloids (Wilton NH) or pre- pared by established procedures [ 11. Hepes, NADPH and Lascorbic acid were purchased from Sigma (St Louis MD). [ 3H] SAM (13.5 Ci/mmol) was obtained from New England Nuclear (Boston MA). Spectro- analyzed grade dimethylsulfoxide and HPLC grade n-heptane were from Fisher Scientific (Fair Lawn NJ) and USPgrade absolute ethanol was from US Industrial Chemicals (New York NY).

Chromatographic patterns of urinary ethynyl estrogen metabolites in various populations

Radioactive mestranol (ME) and/or ethynylestradiol (EE) were administered to women in Nigeria, Sri Lanka, and the USA, and the types and patterns of radioactive urinary conjugates examined by Sephadex LH-20 chromatography. There are no differences in the total excretion of urinary radioactivity over 3 days. Consistent geographic differences appear to be present in the proportion of 3-, 17-, and 3,17-glucuronides. If confirmed on larger population samples, these observations may indicate significant geographic differences in the hepatic metabolism of ethynyl estrogens. High performance liquid chromatographic patterns of the urinary aglycone metabolites of ME and EE were examined in a number of women. The separation was accomplished on a Chromegaprep Diol column with a gradient of isopropanol in heptane. Ethynyl estrogen metabolism shows considerable individual variation. EE is usually the principal compound escreted following ME or EE administration. Unmetabolized ME is present in the ME profiles. The profiles of EE and ME are similar, with EE demonstrating a more complex pattern. Oxidative metabolism occurs chiefly at positions 2, 6 and 16 and is fairly extensive in the USA subjects. The Sri Lankan women generally show less of the oxidative products and the Nigerian group display a notable lack of oxidative metabolism. There is no difference in the metabolic patterns of long-term oral contraceptive users vs. non-users. Using silver sulfoethylcellulose column chromatography, from 14.1 to 34.7% of the excreted radiolabeled aglycones are non-ethynyl (i.e., either D-homo or de-ethynylated estrogens).

Oxidative metabolism and de-ethynylation of 17α-ethynylestradiol by baboon liver microsomes

Incubations of tritiated 17alpha-ethynylestradiol (EE2) with liver explants of baboon and mouse showed the primate species to be more efficient in the removal of the ethynyl group. Liver microsomes from sexually immature male and female baboons were then incubated with tritiated EE2 and estradiol (E2). Each hormone bound irreversibly to the microsomal pellet. Addition of glutathione reduced the irreversible or covalent association. Incubations with E2 demonstrated significant conversion to estrone (E1). The EE2 experiments demonstrated a conversion to estrone only in the presence of an NADPH-generating system, and the addition of SKF-525A reduced the conversion of EE2 to E1. The cleavage reaction appears to be an oxidative event.

Problems in the Measurement of Putative Serum Immune Complexes by the Method of Beaumont

ABSTRACT: A methodological investigation of the procedure used by Beaumont et al for measuring a “serum immune complex” precipitated by 25% ammonium sulfate and alleged to contain an ethynyl‐estradiol binding immunoglobulin has found major problems with reproducibility and with the correlation of total protein as measured by the Lowry method and the IgG content as determined by a specific nephelometric procedure.

Metabolites of estradiol-17β in bovine liver: Identification of the 17-β-d-glucopyranoside of estradiol-17α

Abstract An investigation of the unidentified polar metabolites from the livers of steers treated with [4-14-C]-estradiol-17 β or its 3-benzoate revealed them to consist principally of glycosidic derivates of estradiol-17α. The major polar metabolite was the 17-β- d -glucopyranoside of estradiol-17α. The 3-β- d -glucosiduronate of estradiol-17α, and other 17-glycosides of estradiol-17α and estradiol-17β were also characterized. This study demonstrates for the first time the presence of estrogen glycosides in bovine liver tissue.

Synthesis of 17β-d-glucopyranosiduronic acid of 17α-ethynylestradiol

Abstract The synthesis of the 17β- d -glucuronide of 17α-ethynylestradiol was an unexpected product resulting from the use of CdCO 3 in the improved Koenigs-Knorr reaction between 17-alpha-ethynylestradiol and methyl (1α-bromo-triacetyl-glucuronate). Sephadex LH-20 was found to be effective in the purification of the synthetic product. Identification was obtained through mass spectrometry, infrared spectroscopy, ultraviolet spectroscopy, nuclear magnetic resonance spectroscopy, enzymatic and chemical analysis, and gas-liquid chromatography.