Metabolism of radioactive 17alpha-ethynylestradiol by women

Data on the urinary excretion and subsequent fractionation of radioactivity derived from 6,7-tritiated-17alpha-ethinyl estradiol (tritiated EE) are presented. Tritiated EE was administered orally to 9 women. 17alpha-ethinyl estradiol 2-methoxy-17alpha-ethinyl estradiol, 2 -hydroxy-17alpha-ethinyl estradiol 3-methyl ether, and D-homoestradiol-1 7abeta were identified as urinary metabolites by reverse isotope dilution. The extent of D-homoannulation was much less than that reported previously with rabbits.     

Oxidative metabolism and de-ethynylation of 17alpha-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.     

Plasma estradiol 17-sulfate and 2-hydroxyestradiol 17-sulfate levels and their metabolic clearance rates in rats

By using highly specific antisera against estradiol 17-sulfate (E2-17-S) and against 2-hydroxyestradiol 17-sulfate (2-OH-E2-17-S), plasma concentrations of these sulfates in Wistar rats were determined. The plasma levels of E2-17-S and 2-OH-E2-17-S in the male were 23.5 +/- 5.3 and 21.6 +/- 6.2 pg/ml, respectively. During the estrus cycle of the female, the plasma concentration of E2-17-S reached its highest level 69.0 +/- 11.8 pg/ml, during the diestrus stage, and its lowest level 36.9 +/- 6.6 pg/ml, during the proestrus stage. Similar tendencies were observed in the case of 2-OH-E2-17-S. To examine the dynamic behavior of both sulfates, the plasma metabolic clearance rate (MCRp) of E2-17-S and 2-OH-E2-17-S were determined by infusion experiments. MCRp of E2-17-S and 2-OH-E2-17-S in male rats were 102 and 653 ml/h (means), respectively, and in female rats were 115 and 644 ml/h (means), respectively. The low MCRp values of both sulfates imply their slow metabolic turn-over.     

Antiestrogenic activity of vitamin A in in vivo uterotrophic assay

All-trans-retinoic acid (ATRA), the primary active metabolite of vitamin A, was examined for its antiestrogenic activity in rats using an in vivo uterotrophic assay. All rats were ovariectomized 2 weeks prior to receiving 5 mg/kg/day ATRA or 0.3 micro g/kg/day ethynyl estradiol (EE) subcutaneously once a day for 3 consecutive days. Rats were sacrificed 1, 3, 6, 12 or 24 h after the last treatment. EE increased uterine weight and the coinjection of ATRA with EE significantly suppressed this effect 3 and 24 h after treatment. mRNA expression was examined during this 24-h period and the mRNA expression levels of estrogen receptor alpha (ER alpha), retinoic acid receptor beta (RAR beta), retinoid X receptor gamma (RXR gamma) and cellular retinol-binding protein I (CRBP I) were found to have significantly increased in the ATRA+EE group compared with those in the EE group. This is the first report on the antiestrogenic activity of ATRA determined using an in vivo adult rat uterotrophic assay. The up-regulation of RAR or RXR mRNA expression level was probably responsible for the antiestrogenic activity of ATRA.     

Some new aspects of 17alpha-estradiol metabolism in man

17Alpha-estradiol (1,3,5(10)-estratriene-3,17alpha-diol) together with a tracer dose of the tritium-labeled compound was administered orally and sublingually to male volunteers. The serum concentrations of 17alpha-estradiol (free and liberated by enzymatic hydrolysis) were quantified by GC/MS, and the serum total radioactivity and urinary radioactivity excretion were determined. After oral administration, 17alpha-estradiol was rapidly and intensively conjugated; only tiny quantities of the free steroid (<1% of total) appeared in serum. Sublingual administration resulted in temporary (up to 3 h p.a.) higher serum levels of the free compound. The metabolite patterns obtained by TLC of extracts from serum and urine demonstrated that 17alpha-estradiol is the subject of a poor phase I metabolism in man. A great discrepancy was found in the serum concentrations of 17alpha-estradiol (free + conjugated) determined by GC/MS and the serum radioactivity expressed in 17alpha-estradiol equivalents. By TLC analysis of the steroid conjugates extracted from serum, various 17alpha-estradiol conjugate peaks were found. By enzymatic hydrolysis with beta-glucuronidase/aryl sulfatase from Helix pomatia they were only partially cleaved. Thus, the difference between the serum radioactivity and the 17alpha-estradiol levels determined by GC/MS had to be attributed to an incomplete conjugate hydrolysis. It has been shown with the synthesized 17alpha-estradiol sulfate conjugates that only the 3-sulfate is cleaved by enzymatic hydrolysis, whereas the 17-sulfate group resists enzymatic hydrolysis. The methanolysis procedure (acetyl chloride in MeOH) has proved to be an efficient method for cleaving both the 3-sulfate group and the 17-sulfate group. In contrast to the 17alpha-estradiol conjugates in serum, the urinary conjugates were intensively split by the enzyme preparation. From this, it has to be concluded that the serum conjugates were deconjugated and newly reconjugated before urinary excretion.     

The metabolism of estradiol 17-sulfate by pheochromocytoma tissue

The metabolism of estradiol 17-sulfate by subcellular localization enzymes of pheochromocytoma tissue obtained from a 41-year old female was investigated. In any incubations under the presence of NADH and NADPH, metabolites hydroxylated at the C-2, C-4, C-6 beta, C-7 alpha and C-7 beta positions were produced. These hydroxylations are considered to occur without cleavage of the sulfate group. The 2-hydroxylation at the substrate concentration of 100 microM by mitochondria, microsomes and cytosol fractions occurred at rates of 141, 222 and 167 pmol/mg protein/30 min, respectively; the corresponding rates for the 4-hydroxylation were 24, 40 and 38 pmol/mg protein/30 min. Mitochondrial 2- and 4-hydroxylations were enhanced by addition of calcium ion (Ca2+) into the incubation medium.     

Binding of the contraceptive steroids medroxyprogesterone acetate and ethynyloestradiol in blood of various species

This study compares the binding of medroxyprogesterone acetate (MPA) and 17a-ethynyloestradiol to plasma proteins of various species (baboons, monkeys, dogs, rabbits, guinea-pigs, rats) with that in man. Plasma samples were collected from each species and subjected to centrifugation, filtration; equilibrium dialysis and polyacrylamide gel electrophoresis (PAGE). The results confirm the presence in the plasma of the species examined of a protein with a high capacity and low affinity for ethynyl estradiol and MPA. This protein appears to be the albumin. Ethynyl estradiol was bound to a greater extent than MPA in each species. There was a lack of binding of ethynyl estradiol to SHBG (sex hormone binding globulin) in any species, nor was binding reduced by dihydrotestosterone. This was attributed to a general steric effect of the introduction of the 17-alpha ethynyl group. It was also shown that more than 90% of ethynyl estradiol is loosely bound, mainly to serum albumin, with similar apparent association constants; in all species except dog and guinea-pig, binding occurred only to albumin. For MPA, less binding of MPA than ethinyl estradiol occurred in all species except the rabbit, and binding was significantly lower in the guinea-pig than in the other species. PAGE showed that binding occurred only to albumin. Stability of MPA-albumin complexes on PAGE varied and appeared to be less stable than the ethynyl estradiol-albumin complex. These variations in stability may account for the differences in biological activities of the steroids in different species.     

Metabolism of [6,7-3H, 35S] estradiol 17-sulfate in rats

To confirm whether or not the sulfo group of estradiol 17-sulfate (ES) is removed during in vivo metabolism in rats, the doubly labeled conjugate [6,7-3H, 35S] ES was injected into rats, and its biliary and urinary metabolites were determined by reverse isotope dilution method (RIDM). In male rats, the major radioactivity was detected in biliary disulfate fraction, which was composed of mainly ES and its two minor metabolites, 2-hydroxyestradiol 17-sulfate (2-OH-ES) and 2-methoxyestradiol 17-sulfate (2-MeO-ES). In female rats, in contrast, the radioactivity was dispersed into three fractions:biliary monosulfate, biliary disulfate, and urinary monosulfate fractions (Frs.) In both monosulfate Frs., 7beta-hydroxyestradiol 17-sulfate was detected as the major metabolite followed by 6alpha-, 6beta-, and 15beta-hydroxyestradiol 17-sulfates. Like male rats, 2-OH-ES and 2-Meo-ES as the minor products were detected in biliary disulfate fraction. The isotope ratios of ES and its metabolites in both sexes were essentially the same as that of the dose except that of 6alpha-hydroxylated metabolite, which may be derived from the loss of the tritium labeled at C6. These results confirm the occurrence of the direct metabolism of ES in rats.     

Development of a monoclonal antibody for use in radioimmunoassay of ethynylestradiol

Production of monoclonal antibodies (MABs) to a 17 alpha-ethynyl-1,3,5 (10)-estratriene-3,17 beta-diol (ethynylestradiol, EE2) hapten is described. Culture antibodies generated by hybridized cells tested in enzyme-linked immunosorbent assay (ELISA) bound the 6-oxoethynyl-estradiol-6-(0-carboxymethyl) oxime-bovine serum albumin conjugate (EE2-6-0 CMO-BSA) but not BSA. On radioimmunoassay (RIA) with tritiated ethynylestradiol (3H-EE2), some of the antibodies demonstrated high binding affinity (Ka = 2.8 X 10(10) M-1) to EE2. Estradiol-17 beta, estrone and estriol did not show any displacement of 3H-EE2 from the MABs even at high concentration (1 X 10(4) ng/mL). Among the widely used progestins, norethynodrel and norethisterone exhibited considerable cross-reactivity (25.7-100% and 0.3-54.1%, respectively) but not levonor-gestrel with the MABs. The high affinity demonstrated by the MABs to EE2 3-sulfate but not to EE2 17-sulfate and EE2 3,17-disulfate suggests the dominant role of the 17 beta-hydroxyl group in immunogenicity.     

Embryotoxicity of sex steroidal hormone combinations in nonhuman primates: I. Norethisterone acetate + ethinylestradiol and progesterone + estradiol benzoate (Macaca mulatta, Macaca fascicularis, and Papio cynocephalus)

Two sex steroid hormone combinations which have been used clinically as tests for detection of early pregnancy were examined for embryotoxic effects in macaques and baboons. Norethisterone acetate and ethinyl estradiol (NEA + EE) were orally administered to rhesus and cynomolgus monkeys and baboons at dosages ranging from one to 1,000 times the human dose equivalent (HDE) during days 20-50 of pregnancy. Progesterone and estradiol benzoate (P + EB) were delivered by two to six intramuscular injections to rhesus and cynomolgus monkeys between gestational days 20 and 35 at 0.1-25 X HDE. Fetuses were examined following cesarean section at 100 +/- 2 days (NEA + EE) or at term (P + EB). The results showed increased embryolethality over controls at 100-1,000 X HDE (NEA + EE) and at 10 and 25 X HDE (P + EB). Besides growth retardation, isolated cases of minor nongenital malformations were observed only in cynomolgus monkeys following treatment with both hormone combinations mainly at embryolethal dose levels and were considered spontaneous in nature. Virilization of female cynomolgus fetuses following NEA + EE treatment was manifested as two cases of clitoral enlargement in the 300 X HDE group and two cases of increased anogenital distance with reduced vaginal opening in the 1,000 X HDE group. The highest dose of NEA + EE was also maternally toxic, as two maternal deaths occurred at the end of the treatment period. One dead female cynomolgus fetus exposed to P + EB (10 X HDE) also exhibited masculinized external genitalia.(ABSTRACT TRUNCATED AT 250 WORDS)     

The physiological role of estradiol 17-sulfate during pregnancy

To clarify the physiological role of estradiol 17-sulfate (ES) during pregnancy, experiments were conducted and the following results were obtained: (1) serum or urinary ES levels rose as a function of gestational age; (2) placental microsomes showed fairly high 2- and 4-hydroxylase activity for ES; and (3) the catechol products, 2- and 4-hydroxy-ES, had a strong inhibitory effect upon the in vitro production of lipid peroxides. These results suggest that ES actsas a precursor to the catechol metabolites which maintain normal gestation. This is coincident with the negative correlation of serum levels in ES and lipid peroxides observed in late pregnancy.     

On the structures of hydroxylated metabolites of estradiol 17-sulfate by rat liver microsomes

The metabolism of estradiol 17-sulfate (ES) by hepatic microsomes of female rats produced four new metabolites in addition to 2- and 4-hydroxyestradiol 17-sulfates (2- and 4-OH-ES), which were detected on an HPLC chromatogram. By comparison with synthetic specimens, three of these compounds were identified as 6alpha-, 6beta-, and 7beta-hydroxyestradiol 17-sulfates. To elucidate the structure of the remaining metabolite, a large-scale incubation of ES was carried out, followed by isolation using preparative HPLC to give the single material, which was assigned as 15beta-hydroxyestradiol 17-sulfate by instrumental analyses. On the other hand, when ES was incubated with the microsomes of male rats, 2-OH-ES was produced accompanied by two minor products: 4-OH-ES and a metabolite of unknown structure. The results show clearly that the metabolism of ES by rat hepatic microsomes is remarkably different between the sexes.     

Regional adipose tissue metabolism in postmenopausal women after treatment with exogenous sex steroids

In order to evaluate the effect of exogenous sex steroids on adipose tissue metabolism, two groups of postmenopausal women were studied. In one of the groups, the effect of 50 micrograms ethinyl estradiol (EE) was investigated given orally alone and in combination with 10 mg norethisterone acetate (NET). This combination is reminiscent of an old high dose oral contraceptive. In the other group, the effect of 3 mg 17 beta-estradiol was evaluated when administered percutaneously alone and in combination with 300 mg micronized progesterone given orally. These substances and doses were chosen to provide a "physiological" hormonal influence. In the femoral region 50 micrograms EE induced an increase in LPL activity. This elevated LPL value was reversed with the addition of 10 mg NET. Moreover, during treatment with 50 micrograms EE, a decrease in norepinephrine stimulated lipolysis was seen in the abdominal region. The percutaneous administration of 17 beta-estradiol with or without micronized progesterone, however, was inert as regards subcutaneous adipose tissue metabolism. Our findings indicate, therefore, that EE in doses used in oral contraception might promote lipid accumulation in the femoral adipose tissue depot.     

In vitro comparative metabolism of 6,7-3H estrone and 6,7-3H estrone-3-sulfate in maternal and fetal guinea-pig liver]

The metabolism of [6,7-3H] estrone and of [6,7(3)H] estrone-3-sulfate have been comparatively studied in the maternal and fetal guinea-pig livers. The appearance of estradiol-17 beta resulting from the activity of the 17 beta-hydroxysteroid-dehydrogenase is more important in the fetal than in the maternal hepatic tissue. This suggests the direct transformation of estrone-3-sulfate into estradio-3-sulfate in the fetus. After incubation of the [3H] estrone, there is an abundant hepatic conjugation. The glycuroconjugated components are predominant, as well in the maternal as in the fetal hepatic tissue. For the latter-one the sulfoconjugation is inexistant. The sulfatasic activity shown after the incubation of [3H] estrone-3-sulfate is very low in the fetal hepatic tissue; in contrast, this activity is higher in the maternal tissue.     

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

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.     

Specific antisera for the radioimmunoassay of estriol 3-sulfate

Antisera were raised in male guinea pigs against 6-oxoestriol 3-sulfate O-carboxymethyloxime-bovine serum albumin (BSA) conjugate. The antisera to this antigen exhibited high affinity (Ka=4.7 X 10(9)M-1) and excellent specificity for estriol 3-sulfate, showing slight cross-reactions (less than 0.43%) with other estrogen sulfates, and no cross-reactivities with free estrogens and other steroids (less than 0.01%) except cholesterol sulfate (0.22%). A standard curve using [6, 7-3H]-estriol 3-sulfate as the radioactive ligand showed high sensitivity in the range of 10-1000 pg estriol 3-sulfate.     

Synthesis of 6- and 7-hydroxyestradiol 17-sulfates: the potential metabolites of estradiol 17-sulfate by female rat liver microsomes.

The potential ring-B hydroxylated metabolites of estradiol 17-sulfate (1) by female rat liver microsomes were chemically prepared as authentic compounds. They are 6alpha- and 6beta-hydroxyestradiol 17-sulfates (7 and 9), and 7alpha- and 7beta-hydroxyestradiol 17-sulfates (12 and 16), whose synthetic procedures are described.     

Influence of a cecal volume-reducing intestinal microflora on the excretion and entero-hepatic circulation of steroids and bile acids

From mouse fecal material we have isolated four strictly anaerobic bacteria which, when associated with germfree mice or rats, reduced the cecal volume by 80 and 60%, respectively. This cecal volume-reducing flora did not metabolize estrone-3-sulfate, taurolithocholate-3-sulfate or taurolithocholate but gnotobiotic rats associated with this particular flora (CRF-rats) excreted these compounds faster in feces plus urine than did germfree rats. The time needed for 50% excretion (t1/2) of orally administered estrone-3-sulfate was 32 h in germfree rats versus 13 h in CRF rats; for intraperitoneally injected taurolithocholate-3-sulfate the t1/2 was 63 h in germfree versus 17 h in CRF rats and for taurolithocholate the t1/2 was 199 h in germfree and 96 h in CRF rats. Association of germfree rats with the cecal volume-reducing flora did not change the cecal absorption rate of estrone-3-sulfate, but shortened the 50% small intestinal transit time of [14C]PEG from 10 to 3 h; a value also found in conventional rats. These results stress the important influence of the intestinal microflora on the absorption and excretion of steroids via its effect on the physiology of the whole intestinal tract and point to the deficiencies inherent to the use of germfree animals in excretion studies.     

Estrogen feedback in normal and sulpiride-induced hyperprolactinemic postmenopausal women

Feedback effect of estrogen on gonadotropin secretion was studied in normal and sulpiride-induced hyperprolactinemic postmenopausal women. Twelve normoprolactinemic postmenopausal women were administered 40 micrograms/day of ethinyl estradiol (EE2) orally throughout the study. On the 4th week of the study, daily doses of 200 micrograms EE2 were also given to each subject for 4 days. Twelve postmenopausal women were given sulpiride orally in a daily dose of 150 mg throughout the study. Serum levels of prolactin were raised in all 12 subjects given sulpiride. In the 12 sulpiride-induced hyperprolactinemic postmenopausal women, EE2 was given in the same manner as in normal postmenopausal women. The negative feedback effect of estrogen with low doses of EE2 (40 micrograms/day for 4 weeks) and the positive feedback effect of estrogen after the subsequent administration of EE2 (200 micrograms/day for 4 days) were demonstrated in both normoprolactinemic and hyperprolactinemic groups. The result of the present study suggests that sulpiride-induced hyperprolactinemia does not affect the negative and positive feedback effect of estrogen in postmenopausal women.     

Effects of orchidectomy and different modes of high dose estrogen treatment on circulating "free" and total 1,25-dihydroxyvitamin D in patients with prostatic cancer

Serum levels of total 1,25-dihydroxyvitamin D (1,25(OH)2D), vitamin D binding protein (DBP), sex hormone binding globulin (SHBG), testosterone, estradiol 17 beta (E2) and the "free" 1,25(OH)2D index were measured before and during treatment in prostatic cancer patients treated by orchidectomy (n = 15), with combined i.m. polyestradiol phosphate (PEP) + oral ethinyl estradiol (EE) (n = 10) and with i.m. PEP only for 3 months, followed by addition of oral EE (n = 9). Total concentrations of 1,25(OH)2D and DBP were unaffected by orchidectomy and treatment with i.m. PEP only, but were significantly elevated during treatment including oral EE. SHBG levels were unaffected by orchidectomy, slightly increased by i.m. PEP only and greatly increased by oral EE. The free 1,25(OH)2D index was slightly elevated by treatment including oral EE. Evidence was obtained that the increase in 1,25(OH)2D levels observed during oral estrogen treatment was secondary to the estrogen-augmented increase in DBP and not a result of an estrogen-stimulated synthesis of 1,25(OH)2D. Furthermore, the stimulatory effect of estrogen on DBP concentrations seemed to be dependent on the route of administration of the hormone.