The metabolism of estrone and estradiol-17β and their 3-sulfates by female guinea pig liver microsomes

The metabolism, by female guinea pig liver microsomes of estrogen 3-sulfates (estrone-3-sulfate and 17β-estradiol-3-sulfate) was compared to that of the unconjugated estrogens, estrone and estradiol-17β. Metabolites identified indicated that 16β-hydroxylated products (16β hydroxyestrone and 16 epiestriol) arose mainly from the free estrogens while 16α-hydroxy steroid sulfates (16α hydroxyestrone-3-sulfate and estriol-3-sulfate) were predominantly formed from the sulfated estrogens. These results show that the sulfate moiety at position 3 of the steroids directs 16-hydroxylation from the β to the α configuration.     

Metabolites of estradiol-17β in guinea pig uterus late in pregnancy

The metabolism of estradiol-17β by the guinea pig uterus late in pregnancy was studied $\text{in vivo}$ and $\text{in vitro}$.Whole uteri were examined for estrogen metabolites one hour following an intravenous injection of [³H]-estradiol-17β or uterine sections were examined after incubation for one hour at 37°C in medium containing [³H]-estradiol-17β.In both instances uterine tissue metabolized estradiol-17g to five products: estrone, estrone-3-sulfate, 17β-estradiol-3-sulfate, estrone-3-glucuronide and 17β-estradiol-3-glucuronide. Of the total radioactive products 11 – 43% were glucuronides, 17 – 26% were sulfates and 4 – 17% was estrone. These results indicate that the guinea pig uterus actively transforms estradiol-17β into glucuronides and sulfates late in pregnancy.     

Preparation of specific antisera to estrone-3-glucuronide and estriol-3-glucuronide

The preparation and antigenic properties of estrone-3-glucuronide- and estriol-3-glucuronide-bovine serum albumin conjugates in which the hapten is linked to the carrier protein through an (O-carboxymethyl)oxime bridge at the C-6 position on the steroid nucleus, have been described. Antibodies raised against the two immunogens in the rabbit possessed high specificity to estrone-3-glucuronide and estriol-3-glucuronide, respectively, exhibiting little cross-reactivities with other estrogen conjugates and no cross-reactions with related steroids except for free estrogens, their 3-methyl ethers and 3-sulfates. The cross-reactive antibodies were eliminated by partial immunoadsorption on affinity chromatographic media using the estrone-3-methyl ether 17-(O-carboxymethyl)oxime- and estriol-3-methyl ether 16 (or 17)-hemisuccinate-aminohexyl Sepharose conjugates, respectively. The purified antisera exhibited no cross-reactivities with free estrogens and ring A conjugates of estrone and estriol.     

Structure-activity relationships of 9β-estrogens

The 9β isomers of estradiol-17β, estradiol-17α, estrone and 17-ethinylestradiol-17β were synthesized and compared with their 9α-counterparts in the rat uterine cytosol estrogen receptor, utero-tropic, and gonadotropin release inhibition assays. Except for 17-ethinyl-9β-estradiol-17β which was as active as its 9α isomer in the uterotropic assay, none of the 9β estrogens exhibited any biological activity which was equal to or greater than their 9α counterparts. For examples, 9β-estradiol-17β was $\text{1}\text{10}$ as active as estradiol-17β, and 9β-estrone was $\text{1}\text{4}$ as active as estrone in the uterotropic assay.     

Identifications of radioactive steroid estrogen conjugates in blood plasma of laying hens after intramuscular injection of (4--14C)-estrone.

[4--14C] Estrone was injected intramuscularly into six laying hens. Fifty minutes later the hens were exsanguinated. The plasmas were examined for conjugates of radioactive phenolic steroids by recovery on columns of Amberlite XAD-2 or by extraction with tetrahydrofuran followed by chromatography on a column of DEAE-Sephadex A-25 in a gradient of NaCl. The biggest Sephadex chromatographic fraction (50,4% of total) contained about 42% of its radioactivity as estradiol-17alpha-3-sulfate and 18% as estradiol-17beta-3-sulfate and the remaining 40% was identified tentatively as estradiol-17alpha-17-sulfate plus a small proportion of estradiol-17beta-17-sulfate. The second biggest Sephadex chromatographic fraction (12.7% of total) was a mixture of conjugates not further identified. Minor fractions identified comprised estrone-beta-glucuronide (2.8%), estradiol-17alpha-3-beta-glucuronide (2.8%), estradiol-17beta-3-beta-glucuronide (2.3%) and estrone sulfate (6.0%). Evidence was obtained for the presence of small proportions of estradiol-17alpha disulfate and estradiol-17beta disulfate.     

Competitive protein-binding assay of estrone, estradiol-17 or estradiol-17 in human or ruminant plasma

A procedure for the assay of estrone, estradiol-17β or estradiol-17α in plasma is described. The technique also appears applicable to the assay of estriol in plasma. The procedure uses a semi-automatic extraction of plasma, rapid micro-alumina column chromatography and competitive binding of the estrogens to stable proteins of sheep uterine cytosol. The use of alumina column chromatography results in consistently low blanks. The assay has been evaluated for the measurement of estradiol-17β and estrone in human and sheep plasma, and for estradiol-17α and estrone in goat plasma. The change in binding affinity of estradiol-17α relative to estradiol-17β when incubated in sheep uterine cytosol at two different temperatures (25°C and 4°C), makes it possible to differentiate the two epimers of estradiol. Measurement of estradiol-17β down to 10 pg and of estrone and estradiol-17α to 25 pg are maintained in routine analyses. The specificity of the procedure was thoroughly checked by various methods, including comparison with spectrophotofluorimetric analysis.     

Steroidal control of rat uterine 17β-hydroxysteroid dehydrogenase activity

The interconversion of estradiol-17β and estrone in the rat uterus is due to the action of 17β-hydroxysteroid dehydrogenase. Whole uteri or 800 × g supernatant fractions of the uteri were incubated in the presence of [³H] estradiol-17β and NAD at 37°C for 3 h or 1 h, respectively. In the mature rat uterus the oxidation of estradiol-17β and estrone was dependent on the stage of the estrous cycle, suggesting hormonal control. The 17β-hydroxysteroid dehydrogenase activity was highest at estrus (200 fmol estrone) and lowest at diestrus (80 fmol estrone). An enhancement of activity occurred when adult rats at each stage of the estrous cycle were administered estradiol-17β, while progesterone administration at each stage resulted in decreased enzyme activity. The uterine 17β-hydroxysteroid dehydrogenase activity of estradiol-17β treated ovariectomized rats was time and dose dependent but decreased when progesterone was administered with or without estradiol-17β administration. These results suggest that estradiol-17β caused an increase in enzyme activity that was inhibitable by progesterone in the rat uterus. The increased 17β -hydroxysteroid dehydrogenase activity may reflect a specific response of the rat uterus to estradiol-17β.     

Luteinizing hormone concentrations in anestrous ewes administered various estrogens

Twenty four anestrous ewes were evenly assigned to one of six groups and administered either sesame oil, estradiol-17β, estradiol-17α, estrone, estradiol benzoate or estradiol valerate. All estrogen treated ewes received 50 μg of the respective estrogen. Blood plasma was collected for 28 hours post-treatment and quantified for luteinizing hormone (LH) by radioimmunoassay. An estrogen induced LH surge was detected in at least three of the four ewes administered either estradiol-17β, estrone, estradiol benzoate or estradiol valerate whereas only one of the four estradiol-17α treated ewes and none of the ewes administered sesame oil had an LH surge. The interval from treatment to peak LH was similar for estradiol-17β (17.3±2.7 hours), estrone (18.5±1.0 hours) and estradiol benzoate (19.0±0.6 hours) treated ewes but delayed 7 to 9 hours for ewes administered estradiol valerate (26.0±1.2 hours).     

Mass fragmentographic determination of eleven estrogens in the body fluids of pregnant and nonpregnant subjects

Mass fragmentographic determinations of 11 estrogens in urine, bile, or plasma of pregnant and nonpregnant subjects were made. The estrogens (estriol, estrone, 2-methoxyestrone, estradiol-17beta, estradiol-17alpha, 16-epiestriol, 17-epiestriol, 16-alpha-hydroxyestrone, 16beta-hydroxyestrone, 16oxoestradiol-17beta, and 15alpha-hydroxyestrone) were quantitatively determined in bile from 1 male and 3 postmenopausal women, in the urine of a nonpregnant woman, and in a 20 ml pool of late pregnancy plasma obtained from 10 women. The specificity of mass fragmentography as compared with gas chromatography is considered better because a characteristic ion is monitored rather than the total ion current measured by flame ionization detection and reliable measurements can be made in the presence of larger amounts of impurities, resulting in a shortened fractionation procedure.     

Synthesis of new steroid haptens for radioimmunoassay. Part IV. 3-O-carboxymethyl ether derivatives of estrogens. Specific antisera for radioimmunoassay of estrone,estradiol-17β, and estriol

The syntheses of 3-O-carboxymethyl ether derivatives of estrone, estradiol-17β, and estriol and the preparation of their bovine serum albumin (BSA) conjugates are described. These conjugates were employed for the generation of specific antisera suitable for radioimmunoassay (RIA) of estrone, estradiol-17β, and estriol. The previous concept that specific antisera for estrogens cannot be obtained by employing estrogens derivatized at the 3-position is unfounded.     

The effect of oral contraceptive steroid therapy on the urinary metabolites of radioactive estrone and estradiol-17beta

Eight urinary metabolites of radioactive estrone and estradiol-17β (estrone, estradiol-17β, 2-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestrone 3-methyl ether, 16α-hydroxyestrone, 2-hydroxyestradiol and estriol) have been measured by reverse isotope dilution from young women on oral contraceptive therapy. There was a decrease in the sum of the 16α-hydroxy1ated metabolites in the Ketodase liberated fraction from the subjects taking ethynylestradiol containing preparations as compared to those taking preparations containing mestranol and those subjects who were taking no oral contraceptives. This report is also the first to document and measure 2-hydroxyestradiol as a urinary metabolite of radioactive estrone and estradiol.     

Boron estrogens: Synthesis,biochemical and biological testing of estrone and estradiol-17β 3-carboranylmethyl ethers

Synthesis, biochemical and biological testing of the first carborane derivatives of estrogens are described. Estrone 3-carboranylmethyl ether was synthesized in two steps from estrone. Reduction of estrone 3-carboranylmethyl ether with sodium borohydride provided estradiol-17β 3-carboranylmethyl ether. Enzyme kinetic measurements showed that estrone 3-carboranylmethyl ether is a substrate for human placental 17β-hydroxy-steroid dehydrogenase with Km = 5×10^?6M, and Vmax = 0.016 μmol min^?1 μg^?1. The relative affinity constant of estradiol-17β 3-carboranylmethyl ether for rat uterine estrogen receptor was 0.5 (compared with a value of 100 for estradiol-17β). Consistent with its low affinity for estrogen receptor, the dose-dependent uterotropic response to estradiol-17β 3-carboranylmethyl ether in castrated female rats was one sixtieth that of estradiol-17β. None of the tested rats had a toxic reaction to estradiol-17β 3-carboranylmethyl ether. These results demonstrate that exceptionally stable carborane derivatives of estrogens can be synthesized with preservation of their biochemical and biological properties. Boron-containing estrogens may be useful for thermal neutron capture therapy of cancers with estrogen receptors to concentrate boron in the cell nucleus.     

Formation of glucuronides of estradiol-17 beta by human mammary cancer cells

The formation of glucuronides of estradiol-17 beta by human mammary cancer cell lines is reported for the first time. When incubated with [3H]estradiol-17 beta (1 nM) for 16 h, ZR-75-1 and T47-D cells formed estradiol-3-glucuronide and estradiol-17 beta-glucuronide in approximately equal proportions, whereas MCF-7 cells formed E2-3-glucuronide only. Yields of monoglucuronides from MCF-7 and ZR-75-1 cells were 0.35 pmol/mg DNA, which represented 20-26% of the yield of estradiol-monosulphates. A HPLC system capable of separating most estradiol monosulphates, monoglucuronides and mixed conjugates, is described.     

Comparative specificity of antisera raised against estrone, estradiol-17 and estriol using 6-0-carboxy-methyloxime bovine serum albumin derivatives

Antisera for the radioimmunoassay of estrone and estradiol-17β in plasma are usually raised against estradiol-17β coupled to a protein through a derivative at carbon 17. Such antisera cross react with other naturally occurring estrogens, necessitating preliminary chromatographic separation. This difficulty could be overcome by the use of more specific antisera. We have raised antisera against the 6-0-carboxymethyloxime-bovine serum albumin (BSA) derivatives of estrone, estradiol-17β and estriol respectively. We have determined cross reactions with a number of estrogens and other naturally occurring steroids, and have compared the cross reactivity with that of an antiserum raised against estradiol-17β-17-succinyl-BSA. The former antisera show greatly reduced cross reaction with naturally occurring estrogens known or thought to be in relatively high concentration in plasma, as compared with the latter antiserum, but at the expense of greatly increased cross reaction with estrogens substituted at carbon 6. However, since these latter estrogens are thought to be in low concentration in plasma, the use of antisera raised against the 6-0-carboxymethyloxime-BSA derivatives should result in a net gain in specificity. The antisera raised against the estrone and estriol 6-0-carboxymethyloxime-BSA derivatives should be particularly useful.     

Dynamics of estrone glucosiduronate metabolism in dogs.

Interest in the metabolism of estrogen conjugates has been increased by the recent demonstration that some of these conjugates can be hydrolysed in vivo, and are thus a source of physiologically-active circulating estrogens. In this report the metabolism of the quantitatively important conjugate estrone glucosiduronate has been studied in dogs, following the intravenous infusion of 3H-estrone glucosiduronate. Arterial plasma levels of radioactive estrone glucosiduronate and of the radioactive metabolites estrone, estradiol-17beta-3-glucosiduronate and estradiol-17alpha-3-glucosiduronate were measured. The metabolic clearance rate of estrone glucosiduronate (MCREG) was determined, as well as conversion ratios for estrone glucosiduronate to estrone (CREG.E), estradiol-17beta-3-glucosiduronate (CREG.E2betaG) and estradiol-17alpha-3-glucosiduronate (CREG.E2alphaG). Sequential values for the above mentioned conversion ratios were obtained and the relationship to time was analysed. Transfer constants for estrone glucosiduronate to estrone (rhoEG.E) were measured. The mean MCREG was 329 L/day/m2, SE 35. The values for CREG.E2betaG and CREG.E2alphaG did not become constant until 80 minutes following the start of infusion. The mean values at a steady state were: CREG.E.021, SE .002, CREG.E2betaG .068, SE .005, CREG.E2alphaG .022, SE .002. The mean value for rhoEG.E was .057, SE .006.     

The metabolism of estradiol-17β by the pregnant guinea pig uterus in vitro

The in vitro metabolism of [³H estradiol-17β-by the uterus was studied in non-pregnant, prenant (day 30-term) and post-parturant guinea pigs. Following incubation of tissue sections for one hour is Krebs-Ringer phosphate buffer, five major metabolites could be extracted from the medium or tissue depending upon age of gestation: estrone-3-glucuronide, estrone-3-sulfate, estradiol-3-glucuronide and estradiol-3-sulfate. Both sulfated estrogens were detected at each age of gestation studied, whereas the glucuronides, mainly of estrone, were not detected until approximately day 50. Thereafter, as term (day 65–70) was approached, their percentage contribution to total radioactivity increased at the expense of estradiol and the sulfates. Following parturition, total metabolites of estradiol rapidly decreased, particularly the glucuronides. No conjugates were detected in uteri from nonpregnant guinea pigs. In addition, no conjugates were found in the pre-partum mouse, rat and hamster or in human endometrium obtained immediately after birth. The data suggest that, in the guinea pig, a biochemical factor in the termination of normal pregnancy is the control of tissue levels of active estrogen (estradiol) by conjugation with glucuronic acid.     

Production,clearance rates and metabolic fate of estradiol-17β in the plasma of the laying hen

A single dose of tritiated estradiol-17β (³H-E₂β) was injected i.v. into 5 high egg producing White Leghorn hens, 31 weeks of age, at 19.2 ± 2.1 (mean ± S.D.) hr before oviposition. Blood (2 ml) was sampled at approximately 5 min intervals over 40 min. Whenever possible, metabolites were monitored and identified by the double isotope technique with the addition of the corresponding ¹⁴C-labelled standards to plasma prior to analysis. The metabolic half-life and clearance rate of ³H-E₂β in plasma were 10.9 ± 1.9 min and 118 ± 18 ml/min/kg body weight, respectively. The calculated production rate of E₂β at 19.2 hr before oviposition was 19.5 ± 5.7 ng/min based on the plasma level (93±22 pg/ml) measured at that time. The relative concentrations (% of plasma radioactivity) of the major metabolites isolated at 5.7 ± 0.6 min post injection were, in descending order: estradiol-17β-3-sulfate (E₂β-3S : 14.9 ± 2.7), estradiol-17α-3-sulfate (E₂α-3S; 5.7 ± 0.3), estrone (E₁; 4.6 ± 0.5), estrone sulfate (E₁S; 2.2 ± 0.5), and estradiol-17 α (E₂α; 1.2 ± 0.4). As time proceeded, the relative concentration of E₂α-3S gradually increased so that by 43.2 ± 1.0 min it became the most abundant identifiable metabolite (12.3 ± 1.1) followed by E₂β-3S (9.1 ± 1.7), E₂S (1.2 ± 0.6), E₁ (0.7 ± 0.4) and E₂α (0.3 ± 0.2). These findings are consistent with the view that one of the major pathways of E₂β metabolism in the circulation of the hen is via E₂β

E₂β?3S ?E₁S

E₂α-3S.     

Enzymatic detection of urinary conjugated steroids after gel chromatography

An enzymatic detection method is described for urinary conjugated steroids after chromatographic fractionation with Sephadex G-25. The principle of the method is as follows. Part of a 24-h urine sample, (1–2 ml of urine) is applied directly, to a short column of Sephadex G-25 and eluted with acetate buffer solution. Steroid conjugates in each fraction are hydrolyzed with steroid sulfatase—β-glucuronidase. After enzymatic hydrolysis, an enzymatic color development reagent for steroids, either 3α-hydroxysteroid dehydrogenase or 3β-hydroxysteroid oxidase, are added and the dye formed is measured spectrophotometrically. Excretion patterns of steroid-3β-sulfates, and steroid-3α-glucoronides and steroid-3α-sulfates are shown with some patients' samples. A precision of the assay values for steroid-3α-glucuronide, steroid-3α-sulfate and steroid-3β-sulfates in urine samples and assay values for normal subjects are also studied.This simple enzymatic method for detecting the excreption patterns of urinary conjugated steroids may have a diagnostic value for clinical tests.     

Conversion,in vitro,of (7n-3H) testosterone to estrone and estradiol-17β and their 3-sulfäte conjugate by the guinea-pig placenta

Different cellular fractions of guinea-pig placenta were incubated in the presence of (7n-³H) testosterone. Microsomal aromatization of ³H-testosterone into estrone and estradiol-17β was demonstrated in the presence of NADPH. The predominance of estrone after incubation with 17β-hydroxylated precursors, (7n-³H) testosterone and (6,7-³H) estradiol-17β, indicate that there is a microsomal 17β-hydroxysterold dehydrogenase activity. In this report, cytosolic sulfurylation of estrogens is demonstrated. This latter activity represents a quite original characteristic of the placental metabolism of estrogens in guinea-pigs. In contrast with the human placenta where there is considerable sulfatase activity, the guinea-pig placenta can sulfurylate estrogens.     

The uptake of radioactivity by the uterus of the rabbit following the sytemic administration of (3H) estradiol-17beta and (3H) estrone and the identification and subcellular localization of radiometabolites in the uterus

In an effort to determine the relevance of the uterine oxido-reduction of estrogens to their action in the rabbit uterus, the uterine uptake of radioactivity administered subcutaneously as [³h] estradiol-17β or [³H]estrone and the subcellular distribution of radio-metabolites in the uterine tissue were studied. The animals were killed 20 min, 1, 3 and 9 hr after the administration of 0.1 μg tritiated steroid and the relative proportions of radioactive estradiol-17β and estrone in plasma and in ‘cytosol’, ‘mitochondrial/microsomal’ and ‘nuclear’ fractions of the uterine homogenates were studied. Despite the presence of a high proportion of estrone in chloroform extract of plasma, very little was found in the fractions from uterine tissue irrespective of the steroid administered. Highest levels of uterine estrone were found in the ‘mitochondrial/microsomal’ preparation. There was no apparent difference in the pattern of uptake of radioactivity administered as [³H] estradiol-17β or [³H] estrone. The presence of high levels of 17β-hydroxysteroid dehydrogenase activity in the rabbit uterus may be responsible for the apparent difference between these results and those of similar experiments using the rat.