Estriol-3-sulfate Research Articles

Detection of Vaginal Metabolite Changes in Premature Rupture of Membrane Patients in Third Trimester Pregnancy: a Prospective Cohort Study

Premature rupture of membranes (PROM) is usually associated with pregnant and neonatal complications. Most of the PROM cases are caused by ascending asymptomatic genital infection. In China, PROM (15.3%) is more common than spontaneous preterm labor (7.3%) and leads to more adverse pregnancy outcomes. Here, we designed a prospective cohort study to measure the metabolomics changes in vaginal swab samples and explored their potential contribution to PROM. A total of 260 differentially expressed metabolites were identified and further analyzed. In the PROM group, N-acetyl-d-galactosamine and sucrose were downregulated (P = 0.0025, P = 0.0195, respectively), both of which are the upstream metabolites of the glycolysis pathway. Furthermore, estriol 3-sulfate 16-glucuronide (P = 0.0154) and 2-methoxy-17beta-estradiol 3-glucosiduronic acid (P = 0.004), two final metabolites in steroid hormone biosynthesis, were both downregulated in the PROM group. Finally, we found two catechin metabolites (epigallocatechin-7-glucuronide, P = 0.0009; 4′-methyl-epigallocatechin-7-glucuronide, P = 0.01) as well as DL-citrulline (P = 0.0393) were also significantly downregulated in the PROM group compared with the healthy control (HC) group, which are related to important antioxidant and anti-inflammatory activities in the human body. Altogether, metabolite changes in glycolysis, steroid hormone biosynthesis, and antioxidant/anti-inflammatory pathways may contribute to (or be a consequence of) vaginal dysbiosis and PROM. Metabolite pathway analysis is a new and promising approach to further investigate the mechanism of PROM and help prevent its unfavorable pregnant outcomes at a functional level. Trial registration number: ChiCTR2000034721

Characterizing the steroidal milieu in amniotic fluid of mid-gestation: A LC–MS/MS study

Growth and development of an embryo or fetus during human pregnancy mainly depend on intact hormone biosynthesis and metabolism in maternal amniotic fluid (AF). We investigated the hormonal milieu in AF and developed a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the determination of 14 sulfated and 6 unconjugated steroids in AF. 65 A F samples (male: female = 35: 30) of mid-gestation ranging from 16th week of gestation to 25th week of gestation were analyzed. Reference data of 20 steroid levels in AF of healthy women were provided. 13 sulfated and 3 unconjugated steroids were for the first time quantified in AF by LC–MS/MS. Highest concentrations were found for pregnenolone sulfate (PregS: mean ± SD, 8.6 ± 3.7 ng/mL), 17α-hydroxypregnenolone sulfate (17OHPregS: 4.9 ± 2.0 ng/mL), epitestosterone sulfate (eTS: 7.3 ± 3.6 ng/mL), 16α-hydroxydehydroepiandrosterone sulfate (16OH-DHEAS: 21.5 ± 10.7 ng/mL), androsterone sulfate (AnS: 9.2 ± 7.4 ng/mL), estrone sulfate (E1S: 3.0 ± 3.0 ng/mL), estriol 3-sulfate (E3S: 8.1 ± 4.0 ng/mL) and estriol (E3: 1.2 ± 0.4 ng/mL). Only testosterone (T) showed a significant sex difference (p < 0.0001). Correlations between AF steroids mirrored the steroid metabolism of the feto-placental unit, and not only confirmed the classical steroid pathway, but also pointed to a sulfated steroid pathway.

Development of a sensitive and robust online dual column liquid chromatography-tandem mass spectrometry method for the analysis of natural and synthetic estrogens and their conjugates in river water and wastewater.

An online ultra-high-performance-liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) method for detection and quantification of natural and synthetic estrogens and their conjugates in aqueous matrices was developed. Target compounds include the natural estrogen estradiol (E2) and its main metabolites estrone (E1) and estriol (E3), the synthetic estrogens ethinylestradiol (EE2) and diethylstilbestrol (DES) and their conjugates estrone 3-sulfate (E1-3S), estriol 3-sulfate (E3-3S), estradiol 17-glucuronide (E2-17G), estrone 3-glucuronide (E1-3G), and estriol 16-glucuronide (E3-16G). After pH adjustment, sample filtration and addition of internal standards (IS), water samples (5mL) were preconcentrated on a Hypersil GOLD aQ column after which chromatographic separation was achieved on a Kinetex C18 column using methanol and water as a mobile phase. The experimental parameters, such as sample loading flow rate, elution time, the percentage of organic solvent in the aqueous-organic eluent mixture, pH, and volume of analyzed samples, were optimized in detail. The benefits of the method compared to previously published methods include minimum sample manipulation, lower detection limits, reduced total analysis time, and overall increased method accuracy and precision. Method detection limits (MDLs) are in subnanogram per liter, complying with the requirements of the EC Decision 2015/495 (Watch list) for hormones listed therein. Applicability of the developed method was confirmed by analysis of river and raw wastewater samples taken directly from urban sewerage systems before being discharged into the river. Graphical abstract Sheme of online SPE-UHPLC-MS/MS system.

Sulfonation of Maternal Steroids is a Conserved Metabolic Pathway in Vertebrates

All vertebrate embryos develop in the presence of maternally derived steroids, and maternal steroids have been hypothesized to link phenotype of the offspring to maternal physiology. In placental vertebrates, it is known that maternally derived steroids are metabolized during development via the sulfonation pathway. We used eggs from the red-eared slider turtle (Trachemys scripta) to determine whether the same metabolic pathway is used to metabolize maternally derived steroids in an oviparous vertebrate. To examine the relationship between estradiol and estrogen sulfates during development, levels of maternally derived estradiol were compared with levels of estradiol sulfate, estrone sulfate, and estriol sulfate at oviposition and after 20 days of embryonic development. Estrone sulfate was the only detectable estrogen sulfate. At oviposition, levels of both estradiol and estrone sulfate varied seasonally with clutches from later in the nesting season having significantly higher concentrations of both steroids. Levels of estrone sulfate increased during development, demonstrating that the sulfonation of maternally derived steroids occurs in oviparous vertebrates as well as in placental vertebrates. We also found that exogenous estrone sulfate increases the production of female hatchlings, thereby demonstrating the ability of this metabolite to influence embryonic development. To examine the role of sulfonation in the metabolism of maternal progesterone and testosterone, we characterized the metabolic fate of both steroids by applying tritiated forms of each steroid at oviposition and characterizing metabolites after 20 days of incubation. Similar to what was demonstrated for estradiol, both progesterone and testosterone are converted to sulfonated metabolites during embryonic development. These data suggest that steroid sulfates, both those that are maternally derived and those resulting from the metabolism of maternal steroids, are a key component of the mechanism underlying steroid-mediated maternal effects.

ESTRADIOL METABOLISM DURING EMBRYONIC DEVELOPMENT IN TRACHEMYS SCRIPTA

Event Abstract Back to Event ESTRADIOL METABOLISM DURING EMBRYONIC DEVELOPMENT IN TRACHEMYS SCRIPTA Ryan T. Paitz1, Ashley R. Sawa1 and Rachel M. Bowden1* 1 Illinois State University, United States Vertebrate embryos are exposed to a variety of maternal steroids during development that can have persistent effects well beyond development. In oviparous species yolk steroids can be quite abundant in eggs at oviposition, declining as development proceeds. The presence of estradiol during embryonic development influences numerous traits of the developing embryo, but recent work in an oviparous reptile indicates that embryos have the ability to metabolize maternal estradiol present in eggs, associated with the previously observed decline in yolk steroids. We characterized estradiol metabolism by red-eared slider (Trachemys scripta) embryos throughout embryonic development to better understand the fate of maternal steroids during development. Tritiated estradiol was applied to eggs at oviposition and we then measured the amount of estradiol and estradiol metabolites in various egg components across embryonic development. Results indicate that the majority of the estradiol was metabolized to several estrogen sulfates, including estriol sulfate, estradiol sulfate and estrone sulfate. Also, metabolite levels in the yolk and albumen decreased throughout development. The developmental decrease in the yolk and albumen samples is consistent with the transfer of metabolites to the embryo as later stages of egg development occur. Characterization of the metabolites present in the embryo is currently ongoing (supported by NSF grant IOS-0952840 to RMB). Keywords: Estrogens, steroid conjugation, turtle, yolk steroids Conference: ISAREN 2011: 7th International Symposium on Amphibian and Reptilian Endocrinology and Neurobiology, Ann Arbor, United States, 11 Jul - 13 Jul, 2011. Presentation Type: Invited Symposium Topic: Hormones and development Citation: Paitz RT, Sawa AR and Bowden RM (2011). ESTRADIOL METABOLISM DURING EMBRYONIC DEVELOPMENT IN TRACHEMYS SCRIPTA. Front. Endocrinol. Conference Abstract: ISAREN 2011: 7th International Symposium on Amphibian and Reptilian Endocrinology and Neurobiology. doi: 10.3389/conf.fendo.2011.03.00037 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 21 Jul 2011; Published Online: 09 Aug 2011. * Correspondence: Dr. Rachel M Bowden, Illinois State University, Normal, United States, rmbowde@ilstu.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Ryan T Paitz Ashley R Sawa Rachel M Bowden Google Ryan T Paitz Ashley R Sawa Rachel M Bowden Google Scholar Ryan T Paitz Ashley R Sawa Rachel M Bowden PubMed Ryan T Paitz Ashley R Sawa Rachel M Bowden Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Contribution of Glucuronic Acid and Sulfonic Acid Moieties during Photocatalytic Degradation of Estrogen Conjugates

Contribution of glucuronic acid and sulfonic acid moieties during the photocatalytic degradation of estrogen conjugates, one of the endocrine disrupting chemicals, has been investigated. Estrogens were subjected to photocatalytic degradation using titanium dioxide immobilized on glass beads as a catalyst, whose time courses were measured by HPLC or liquid chromatography (LC)/MS/(MS). Estradiol and estrone, which have an unconjugated phenolic hydroxy group at the C-3 position, were gradually degraded by UV irradiation and nearly disappeared within 6 hr. 3-Desoxyestradiol, which does not have a phenolic hydroxy group at C-3 position, was also degraded like estradiol. The corresponding 17- or 3-glucuronide was degraded faster than the respective genin, estradiol or estrone. The double conjugate, estriol 3-sulfate 16-glucuronide, was also easily degraded within 3 hr. On the other hand, the degradation of estrogen 3-sulfate did not start within 2.5 hr but the reaction was completed within 6 hr. These data showed that the glucuronic acid moiety on the estrogen skeleton and sulfonic acid moiety at phenolic hydroxy group play an important role for this degradation reaction.

Analysis and occurrence of estrogen in wastewater in Japan

This paper describes an analytical procedure for free estrogens and their conjugates in domestic wastewater. The procedure demonstrated in this study is innovative in terms of levels of detection and quantification of the following substances: estrone (E1); 17beta-estradiol (E2); 17alpha-ethynylestradiol (EE2); estriol (E3); estrone-3-sulfate (E1-S); beta-estradiol 3-sulfate (E2-S); estriol 3-sulfate (E3-S); estrone beta-D-glucuronide (E1-G); beta-estradiol 17-(beta-D)-glucuronide (E2-G); estriol 3-(beta-D)-glucuronide (E3-G); beta-estradiol 3-sulfate 17-glucuronide (E2-SandG); and estradiol 3,17-disulfate (E2-diS). The detection limits of this method ranged from 0.1 to 1.4 ng/l. The recovery efficiencies of the estrogens in the analysis from influent and effluent of the secondary settling tank in a wastewater treatment plant (WWTP) were higher than 94% for the free estrogens, but were less than 50% for the conjugated estrogens. The field study using this method was conducted at twenty WWTPs in Japan. The median concentrations of the estrogens ranged from ND to as high as >100 ng/l. In the influent and secondary effluent samples, the concentrations of E1, E2 and E3 were the same levels as those previously reported. We found that the conjugated estrogens exist at higher concentrations in the influent and the secondary effluent than in the other studies, and that the concentrations of the conjugated estrogens were higher than those of the free estrogens.

DETERMINATION OF ESTRIOL-3-SULFATE-16-GLUCURONIDE IN PREGNANCY SERUM USING LC/TANDEM MS

A determination method for estriol 3-sulfate 16-glucuronide (E3-3S-16G) in pregnancy serum by liquid chromatography/electrospray ionization-tandem mass spectrometry has been developed. E3-3S-16G gave an intense deprotonated ion in the negative-ion mode, which provided a product ion formed from the elimination of the glucuronic acid moiety by the collision induced dissociation. The serum specimen was diluted, heated to remove protein followed by a solid phase extraction using an OASIS HLB cartridge, then subjected to liquid chromatography/tandem mass spectrometry. The recovery of E3-3S-16G from serum was satisfactory and there was no interfering peak derived from the serum components at the elution position of E3-3S-16G, but the ionization efficiency of the analyte was influenced by some substances from the serum and varied among each specimen. To overcome this problem, the standard addition method was employed in the present study and the inter-assay coefficient of variation was acceptable. The developed method was applied to serum samples obtained from pregnant women in their third trimester and gave satisfactory results.

Quantitative and qualitative determination of estrogen sulfates in human urine by liquid chromatography/tandem mass spectrometry using 96-well technology.

A sensitive and robust method to determine five estrogen sulfates in human urine has been developed employing high-throughput solid-phase extraction with 96-well technology, and HPLC coupled with negative turbo ion spray tandem mass spectrometry in the selected reaction monitoring mode. The five estrogen sulfates determined include three major endogenous estrogen sulfates in the human, estrone 3-sulfate (E1-3S), estriol 3-sulfate (E3-3S), and 17 beta-estradiol 3-sulfate (E2-3S), and two biochemical synthetic estrogen sulfates, 17 beta-estradiol 17-sulfate (E2-17S) and 17 beta-estradiol 3,17-disulfate (E2-3,17S). For E2-3,17S, E3-3S, and E2-17S, external standard calibration was used for quantitation, and for the remaining two compounds, internal standard calibration using a stable isotopic labeled internal standard was employed. A total of 96 samples may be prepared with 96-well C18 extraction disk plate techniques performed by a robot within 25 min including the time for evaporation of solvent. The lower level of quantitation (LOQ) for these estrogen sulfates in human urine was determined at 0.2 ng/mL based on 100-microL aliquots of human urine using the optimum tuning parameters for each individual selected precursor ion/product ion transition. The assay was validated with a linear concentration range of 0.2-200 ng/mL, and the interassay accuracy, intraassay precision, and interassay precision do not exceed 8.6%, 12%, and 12%, respectively, by analysis of quality control samples at five concentration levels including the LOQ of 0.2 ng/mL, from four 96-well plates. The target endogenous test articles were qualitatively determined by comparing the full-scan LC/MS/MS mass spectra and retention time in test samples and reference standards. The LOQ is significantly improved compared to previous reports for the targeted compounds using LC/MS/MS. The described simple and automated sample preparation procedure recovered 91% of the target compounds. A total of 192 samples can be analyzed within 1 day (22 h). The method can measure the endogenous estrogen sulfates in urine from both gravid and nongravid subjects.

Simultaneous determination of estriol and estriol 3-sulfate in serum by column-switching semi-micro high-performance liquid chromatography with ultraviolet and electrochemical detection

A column-switching HPLC with semi-microcolumn enabled us a direct and simultaneous analysis of estriol (E3) and estriol 3-sulfate (E3 S) in human serum in combination with ultraviolet (for E3 S) and electrochemical (for E3) detectors. The mobile phases (phosphate buffer pH 7.0) contained 5 m M tetra- n-butylammonium ion (TBA) as a counter ion for E3 S. Serum samples were diluted with 200 m M phosphate buffer (pH 7.0) containing 100 m M TBA, then injected to the pre-column. After serum proteins had flowed out from the pre-column, E3 and E3 S were transferred to the enrichment column. Subsequently the analytes were eluted to the analytical column. Detection limits of E3 and E3 S in human serum were 2.5 ng/ml and 295 ng/ml. Serum E3 and E3 S levels (mean±SD) of umbilical artery from 18 full-term healthy neonates were 33±23 ng/ml and 1.26±0.69 μg/ml, respectively.

Bridge-heterologous chemiluminescence enzyme-linked immunosorbent assay of estriol 3-sulfate in pregnancy plasma

A facile and sensitive chemiluminescence enzyme-linked immunosorbent assay (ELISA) of estriol 3-sulfate using a bridge-heterologous system was established. 6α-Hydroxyestriol 3-sulfate 6-hemisuccinate was synthesized as a novel hapten. Antisera were raised in male guinea-pigs against 6α-hydroxyestriol 3-sulfate 6-hemisuccinate-bovine serum albumin (BSA) and 6-oxoestriol 3-sulfate O-carboxymethyloxime-BSA conjugates. Both haptens were coupled to horseradish peroxidase as an enzyme-label reagent. For separation of free and estriol 3-sulfate bound to the antibody, the crude globulin fractions of these antisera were immobilized to CNBr-activated Sepharose-4B. The enzyme activity was measured by chemiluminescent reaction using amino-butylethyl-isoluminol and hydrogen peroxide as a substrate. The immobilized antibody raised against 6α-hydroxyestriol 3-sulfate 6-hemisuccinate-BSA exhibited a high affinity and an excellent specificity for estriol 3-sulfate. The two bridge-heterologous ELISAs were more sensitive than the homologous systems. The specificity and sensitivity (10 pg) of the bridge-heterologous chemiluminescence ELISAs was comparable to those of the radioimmunoassays (RIAs). Results obtained by the ELISA and the RIA in pregnancy plasmas, showed excellent correlation between ELISA and RIA (r = 0.96).

Prevention of co-elution of steroid sulfates with serum proteins from pre-column in column-switching HPLC system.

A method to prevent co-elution of steroid sulfates with proteins in serum from the pre-column in column-switching HPLC was developed. The pre-column, a polymer-coated mixed function column, was used for ion-pair chromatography with 5 mM tetra-n-butylammonium (TBA) ion. As steroid sulfates, estriol 3-sulfate, dehydroepiandrosterone 3-sulfate and pregnenolone 3-sulfate were used. Human serum (25 microl) was diluted with mobile phases including 5, 100 and 500 mM TBA ion, and then injected directly into the pre-column. The peak areas of the steroid sulfates in serum samples were compared with those of the steroid standards without serum. When 25/microl of serum was diluted with mobile phase including 100 or 500 mM TBA ion, the steroid sulfates in serum were retained in the pre-column; however, the steroid sulfates from the same sample diluted with mobile phase containing 5 mM TBA ion were not retained in the pre-column. Addition of an excess amount of counter ion (TBA ion) into the serum sample made it possible to retain the steroid sulfates in the pre-column. This method was applied to column-switching HPLC for measurement of steroid sulfates in serum using a semi-microcolumn as the analytical column.

Specific imaging of hormone-dependent mammary carcinoma in nude mice with [ 131I]-anti-estriol 3-sulfate antibody

We tried to put the estrogen metabolite to use in tumor imaging. The antibody against estriol 3-sulfate (E 3 3-S), which was one of the major metabolites of estrogen in hormone-dependent mammary carcinoma, was prepared and the tissue distribution and imaging of human breast carcinoma with anti-E 3 3-S antibody (Ab) were studied in nude mice. In hormone-dependent breast carcinoma, MCF-7,-bearing nude mice, [ 125I] anti-E 3 3-S Ab localized in tumor with the percentage injected dose/g of 9.29 ± 3.01 (mean ± SD). This value was significantly high compared with that in hormone-independent breast carcinoma, MDA-MB-231,-bearing nude mice. At 72 h after the administration of [ 125I]anti-E 3 3-S Ab to MCF-7 bearing mice, tumor/blood, tumor/liver and tumor/muscle ratios were 0.49, 5.02 and 6.83, respectively. These ratios were supposed to be enough for imaging. In radioimmunoscintigraphy, a MCF-7 tumor was clearly visualized at 120 or 168 h post-injection of [ 131I]anti-E 3 3-S Ab.

Determination of estrogen 3-sulfates in biological fluids of mammary tumor-bearing rats by radioimmunoassay

Determination of estradiol 3-sulfate (E 2 3-S) and estriol 3-sulfate (E 3 3-S) in plasma, urine or tumor tissues of mammary tumor-bearing rats were performed using the superior radioimmunoassay ( RIA) system. The plasma level of E 2 3-S after tumorigenesis was found to be about one-third of the normal level. As to E 3 3-S, the levels in urine were significantly high both before and after tumorigenesis. Before that, the average level was about 1.5 times, and after that, about 2.5 times as high as the normal level. In tumor tissues, an extremely high level of E 3 3-S (399.6 ± 113.9 pg/g tissue) was determined.

Relationship between the concentrations of estriol sulfate and estrone sulfate in human breast cyst fluid.

Estriol-3-sulfate (E3S) is present in human breast cyst fluid (BCF) in median levels of 8.7-10.4 nmol/L, yet is barely detectable in the serum (less than 0.034 nmol/L). The source of this huge concentration of E3S is unknown. It may accumulate from blood by active transport or be synthesized and concentrated within the cyst. Since estrone sulfate (E1S) and its possible precursor, dehydroepiandrosterone sulfate (DHEAS) are elevated in BCF, E3S may originate via 16 alpha-hydroxylation of E1S. The present study examined the correlations between the levels of DHEAS and E1S with those of E3S in BCF. The sodium and potassium ions were also quantified and related to the steroid concentrations. By linear regression analysis of log-normalized data there was a highly significant correlation between the concentrations of E1S and E3S (n = 355, r = 0.690, P less than 0.001) and between DHEAS and E3S (n = 361, r = 0.577, P less than 0.001). The BCF were classified according to their K/Na ion ratios: type 1, greater than 1.0, type II, less than 0.25, and type III, 0.25-1.0. By Student's t test, the concentrations of E3S differed between each BCF Type (P less than 0.002). This was also true for E1S and DHEAS. Type 1 cysts were associated with the highest estrogen sulfate levels and type II with the lowest levels. The possible physiological importance of this observation resides in reports that the BCF type expressing the highest steroid concentrations has been related to an aporcine-like epithelial lining of the cyst wall and a somewhat higher risk for developing breast cancer. The results suggest that E3S in BCF may originate from E1S, but alternate mechanisms are not precluded.

Relationship between the concentrations of estriol sulfate and estrone sulfate in human breast cyst fluid

Relationship between the concentrations of estriol sulfate and estrone sulfate in human breast cyst fluid

Pharmacokinetics and biotransformation of orally administered oestrone sulphate and oestradiol valerate in post-menopausal women

The pharmacokinetic properties and biotransformation of two orally active oestrogens, piperazine oestrone sulphate (PE 1S, 2.5 mg/day) and oestradiol valerate (E 2V, 2.0 mg/day), given alone or in combination with levonorgestrel (LNG, 250 μg/day) were compared in 8 post-menopausal women, using a randomized cross-over design. The end points measured in peripheral plasma included oestrone (E 1), oestradiol (E 2), oestriol (E 3), oestrone sulphate (E 1S), oestradiol sulphate (E 2S) and oestriol sulphate (E 3S). In addition, LNG and sex-hormone-binding globulin SHBG concentrations were also assessed. The plasma levels of E 3 were invariably below the detection limit (220 pmol/1). The levels of all the other oestrogens analyzed were consistently higher and the area under the curve significantly greater (except in the case of E 3S following PE 1S administration than those recorded after E 2V ingestion. The terminal half-lives of the circulating oestrogens measured after PE 1S administration did not differ from those found after E 2V administration. After 21 days of PE 1S administration (in combination with LNG for the the last 10 days), the maximum levels of all the oestrogens (except those of E 2) were significantly higher than those seen after the first dose. No such difference was observed after E 2V administration. There was no difference between the effects of the two treatment regimens with regard to the E 1/ E 2 ratios, but the E 1/E 1S ratios were significantly lower after PE 1S treatment than after E 2V administration. It is concluded that, compared with an equivalent dose of PE 1S, daily repeated oral administration of E 2V yields consistently lower peripheral plasma levels of E 2 and its principal metabolites. However, in contrast to PE 1S therapy, prolonged administration of E 2V does not result in an accumulation of the circulating oestrogens measured.

Specific binding of dehydroepiandrosterone sulfate to a cytoplasmic macromolecule in human fetal membrane.

The cytoplasmic dehydroepiandrosterone 3-sulfate (DHAS)-binding macromolecule in the cytosol fraction of human fetal membrane has been investigated. It was distinguished from serum DHAS-binding component on the basis of binding specificity and molecular weight. Scatchard analysis indicated a single class of binding sites (57 fmol/mg protein) with an apparent dissociation constant of 7.2×10-8M. The cytoplasmic DHAS-binding macromolecule was separated into two moieties by Sephacyl S-300 chromatography. One was eluted at void volume, and the other hand a molecular weight of about 1.7×105. These two moieties had the same specificity for DHAS, and had no affinity for dehydroepiandrosterone or 17β-estradiol. Progesterone, 5α-dihydrotestosterone and sulfated estrogens such as 17β-estradiol 3-sulfate, estriol 3-sulfate and estrone 3-sulfate showed much lower affinity for the DHAS-binding macromolecule. Themperature-dependent nuclear uptake of [3H]DHAS in a cell-free system required the presence of cytosol, suggesting that binding of DHAS to the cytoplasmic macromolecule is a prerequisite for the transfer of DHAS to the nucleus. These data demonstrate the presence in human fetal membrane of a tissue-specific DHAS-binding macromolecule which has properties similar to those of the DHAS-binding protein previously found in rabbit uterine cervix (A. Ito, K. Sakyo, H. Sano, S. Hirakawa and Y. Mori, Chem. Pharm. Bull., 34, 2118 (1986).

Cytoplasmic dehydroepiandrosterone sulfate-binding protein in rabbit uterine cervix.

Dehydroepiandrosterone 3-sulfate (DHAS) binding to a cytoplasmic protein of rabbit uterine cervix was studied in vitro. When [3H]DHAS was incubated with the minced uterine cervix obtained at term pregnancy, it was bound specifically to the cytosol fraction. From Scatchard analysis, the binding capacity and dissociation constant were found to be 33 fmol/mg protein and 5.05×10-8M, respectively. Incubation of the prelabeled cytosol with isolated nuclei resulted in temperature-dependent transfer of DHAS to the nuclei. The cytoplasmic DHAS-binding protein after concentration with (NH4)2SO4 showed a sedimentation constant of 3.6-4.0S, while the DHAS-binding protein in the rabbit serum gave a value of 4.6S. The cytoplasmic DHAS-binding protein was eluted from Sephacryl S-300 mainly in the fractions corresponding to the molecular weight of 1.7×105, and in the void volume to a small extent in the presence of molybdate and glycerol. Dehydroepiandrosterone (DHA) inhibited approx. 50% of [3H]DHAS-specific binding, whereas 17β-estradoil 3-sulfate, estriol 3-sulfate, estrone 3-sulfate, 17β-estradiol, estriol, and 5α-dihydrotestosterone showed lower or no affinity for the DHAS-specific binding site. The content of the DHAS-binding protein in the cervix was found to increase with the progress of pregnancy.These observations suggest that rabbit uterine cervix contains a receptor-like DHAS-specific binding protein int he cytosol fraction which seems to be closely associated with the progress of pregnancy.

A simple radioimmunoassay for estriol 3-sulfate in pregnancy plasma without deconjugation

A highly specific anti-estriol 3-sulfate antiserum was treated with 50% ammonium sulfate, and the crude globulin fraction was coupled to CNBr-activated Sepharose-4B. Addition of 0.1M Tris-HCl buffer (pH 8.3) containing 0.1M glutamine to the solution of antigen-antibody enabled assaying without solvent-extraction or chromatography to remove endogenous interference. Subsequently, a direct radioimmunoassay using [6,7- 3H]-estriol 3-sulfate as a radioactive ligand without deconjugation has been established and applied to the determination of estriol 3-sulfate levels in pregnancy plasma. The increasing plasma levels of estriol 3-sulfate are correlated with estriol levels over the period of gestation. The mean values of sulfated estriol concentration (A) late pregnancy plasma were approximately 7 times as high as unconjugated estriol (B), but individual ratios of A to B showed considerable variability.