Edward D. Helton

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.

The pharmacokinetics of ethynyl estrogens. A review.

Abstract Ethynylestradiol and mestranol play a unique role, as far as estrogens are concerned, among the steroidal contraceptive agents. Knowledge of the pharmacokinetics of these compounds is just beginning to accumulate, more than a decade after their clinical utilization became worldwide. The insights necessary for achieving optimum effectiveness, for perception of potential hazards, for selection of appropriate animal models, and for evaluation of their multiple metabolic actions require a detailed understanding of the circulation, storage, metabolism, and cellular and subcellular effects. This paper represents a summary of the rapidly increasing literature on the pharmacokinetics of the ethynyl estrogens.

Toxic agents resulting from the oxidative metabolism of steroid hormones and drugs.

The oxidative metabolism of some exogenous compounds, and possibly some endogenous compounds as well, can lead to the formation of reactive metabolites. These intermediates react as electrophiles, and they lead in some instances to cell death or cell transformation. Three routes (other routes are also known) of toxicity are discussed. These are the epoxide/dihydrodiol pathway, the catechol/o-quinone pathway, and the alkylation pathway. The possible formation of electrophiles from diethylstilbestrol, from natural estrogens, and from ethynylestradiol is discussed in terms of protein binding. Protein binding is presumptive evidence of electrophile formation, but it does not necessarily indicate that the parent compound is highly cytotoxic, mutagenic, or carcinogenic. Mutagenic and carcinogenic activity is presumed to require reaction of an electrophile with nuclear material. There is evidence for protein binding for these estrogens (diethylstilbestrol, natural estrogens, ethnylestradiol) as a consequence of oxidative metabolism.

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.

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.

Metabolism of ethynyl estrogens

The pharmacokinetics and metabolic conversion of the ethynylated estrogens are reviewed. Special emphasis is given to the comparative pharmacokinetics of ethynyl-estradiol in different populations of women. Similarly, the variability of ethynyl-estradiol and mestranol metabolism in humans resulting from presentation of radio-labeled steroid and purification of the metabolic products is presented and discussed. The concepts of estrogen hepatotoxicity are reviewed with respect to the known phenomenon of estrogen oxidative metabolism and covalent binding. Recent evidence for the metabolic removal of the 17alpha-ethynyl group is discussed, and its relationship to estrogen hepatoxicity is considered and related to the covalent binding phenomenon.

Variability in conjugates of ethynylestradiol produced by in vitro incubation of human liver tissue

Conjugated metabolites of tritiated 911-ethynylestradiol(EE) were examined in human liver tissue obtained at diagnostic needle biopsy from subjects with and without clinical liver disease. Liver tissue was incubated for 24 hours with EE and the extracts were run on Sephadex LH-20. The extract separated into 5 conjugates with different mobilities. The presence and relative proportions of these groups varied greatly between individuals both in normal and diseased subjects. Consistent sex differences were absent. It is suggeested that this variability may be the result of local differences in the metabolic activity of liver tissue. It may be representative of whole liver function as well. A similar variation has been observed in the urinary; pattern of conjugated metabolites.

Synthesis and characterization of the anomeric pair of 17β-glucuronides of ethynylestradiol

Abstract The α- and β-anomers of the 17β- d -glucuronide conjugate of ethynylestradiol were synthesized by the SnCl4-promoted reaction between β-acetoxy GAM and the t-17β-hydroxyl group of EE2-3-acetate. The conjugates were resolved by crystallization and HPLC. Positive identification was established by u.v. spectrophotometry, i.r. and mass spectrometry and 1H- and 134C-n.m.r. The structure of the β-anomer was confirmed by X-ray crystallographic analysis. In addition, the α-anomer was refractory to hydrolysis by bovine β-glucuronidase, establishing a biochemical difference between the conjugate pair.

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.

Metabolism of 17α‐ethynylestradiol by intact liver parenchymal cells isolated from mouse and rat

Liver parenchymal cells isolated by perfusion from female C3H/HeN‐MTV+Nctr mice and Sprague‐Dawley rats were incubated with [6,7‐3H] 17α‐ethynylestradiol (EE2). The incubates were individually fractionated into free steroid (organic phase), steroid conjugates (aqueous), and bound steroids (macromolecular pellet). The rat had significantly less total free radioactive steroid but significantly more total conjugated and irreversibly bound radioactivity than the mouse. However, when the metabolic conversion of EE2 was compared in the rat and the mouse on a cellular basis (metabolic clearance per 106 cells), the rat was found to be less efficient than the mouse. The two species were essentially equivalent in their covalent binding when expressed on a per 106 cell basis. Purification of the free radiolabeled steroids on LH‐20 demonstrated the mouse to have the parent compound and an identifiable 2‐OH‐EE2 fraction. The rat had EE2 and an identifiable 2‐methoxy‐EE2 fraction. A major metabolite fraction for both s...