equilin and equilin-sulfate

To determine whether estrogen regulates retinoic acid (RA) production and signaling in the human endometrium as it does in the rodent uterus, we investigated the effects of estrogens on the expression of RA-metabolizing enzymes, retinoid receptors, and biomarker genes in the post- and premenopausal human endometrium. Real-time quantitative PCR revealed that retinaldehyde dehydrogenase (RALDH) 2, a critical enzyme in RA biosynthesis, was induced 4-fold by estrogen replacement therapy with either Premarin or a mixture of estrone and equilin sulfates for 3 months. Estrogen replacement therapy also increased the expression of the RA receptor RAR alpha 1.9-fold. In parallel, there was a marked increase in the expression of two RA-regulated genes, cellular retinoic acid-binding protein II and tissue transglutaminase. In the premenopausal endometrium, the levels of RALDH1, RALDH2, RAR alpha, and cellular retinoic acid-binding protein II were increased in the estrogen-dominated proliferative phase, and the transcripts for the RA catabolic enzyme retinoic acid 4-hydroxylase (CYP26A1) and tissue transglutaminase were significantly increased in the secretory phase. Our results suggest that estrogen coordinately up-regulates RA production and signaling in the human endometrium. This coordinate mechanism may play a role in the antiproliferative effects that counterbalance the estrogen-induced endometrial proliferation.

Topics: Aldehyde Oxidoreductases; Biomarkers; Cytochrome P-450 Enzyme System; Endometrium; Enzyme Induction; Equilin; Estrogen Replacement Therapy; Estrogens; Estrogens, Conjugated (USP); Estrone; Female; Homeostasis; Humans; Isoenzymes; Middle Aged; Placebos; Polymerase Chain Reaction; Postmenopause; Premenopause; Receptors, Retinoic Acid; Retinal Dehydrogenase; Retinoic Acid 4-Hydroxylase; RNA, Messenger; Signal Transduction; Transglutaminases; Tretinoin

To study the impact of low-dose unopposed esterified estrogens on menopausal symptoms and quality of life.. In a long-term, 2-year, randomized, double-blind, placebo-controlled study, 204 postmenopausal women were treated with esterified estrogens 0.3 mg daily or placebo. Menopausal symptoms were assessed with a modified Kupperman index at baseline, 3, 6 and thereafter every 6 months. In a second 12-week, open-label, short-term pilot study, 25 postmenopausal women with moderate to severe vasomotor symptoms were treated with esterified estrogens 0.3 mg daily for 12 weeks. Vasomotor symptoms and quality of life were assessed using the Greene scale and Quality of Life Menopause Scale (QUALMS).. In the long-term study, significant (p < 0.05) reductions in total symptom scores were observed at each time point with esterified estrogens compared with placebo. Somatic symptom scores (hot flushes, night sweats, vaginal dryness) decreased significantly (p < 0.01) in patients treated with esterified estrogens 0.3 mg compared to baseline and placebo. In the short-term, open-label pilot study, the incidence of vasomotor symptoms was significantly (p < 0.01) reduced with esterified estrogens 0.3 mg from week 4 until the study end. Significant (p < 0.05) improvements versus baseline were seen in the somatic and vasomotor/sleep domains and in the total quality-of-life score.. Esterified estrogens 0.3 mg given daily provide adequate menopausal symptom relief and improved quality of life in postmenopausal women.

Topics: Double-Blind Method; Endometrial Hyperplasia; Equilin; Estrogen Replacement Therapy; Estrone; Female; Hot Flashes; Humans; Middle Aged; Placebos; Postmenopause; Quality of Life; Surveys and Questionnaires; Sweating; Uterine Hemorrhage; Vaginal Diseases

The fragmentation pathways of five estrogens (estradiol, estrone, equilin sulfate, 17 a-dihydroequilin sulfate and equilenin sulfate) have been studied with high resolution and high mass accuracy using electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF/MS) in the negative ion mode. Molecular weights were obtained from [M-H](-) ions in the product ion spectra. The results indicate that the five structurally similar estrogens have similar fragmentation pathways. Using their stable isotope forms as internal reference compounds, the accurate mass and composition of the fragment ions were determined. During collision-induced dissociation (CID), cleavage is initiated by loss of oxygen atoms from carbon-17, after which D and C rings cleave sequentially and rearrange to finally form stable conjugate structures with highly abundant characteristic fragment ions at m/z 183 (accompanied by m/z 181), m/z 169 and m/z 145 (accompanied by m/z 143). Understanding these characteristic fragmentation pathways of estrogens will be helpful in identifying the structures of steroid hormones in general.

Topics: Chemical Fractionation; Equilenin; Equilin; Estradiol; Estrogens; Estrone; Ions; Spectrometry, Mass, Electrospray Ionization

Steroid hormones and their metabolites are currently undergoing clinical trials as potential therapeutics for traumatic brain injury (TBI). To support this work, it is necessary to develop improved procedures for differentiating isobaric species in this compound class. Equilin sulfate (E-S), estrone sulfate (E1-S), 17α-dihydroequilin sulfate (ADHE-S), and 17β-dihydroequilin sulfate (BDHE-S) are primary constituents in hormone replacement therapies, such as Premarin, which are among pharmaceuticals being investigated for TBI treatment. The latter three compounds are isomers and can be difficult to differentiate in trace analytical determinations. In this work, a systematic study of the fragmentation of ADHE-S, BDHE-S, E1-S, and E-S under different stages of higher order tandem mass spectrometry (MS(n)) and variation of collision energy, allowed optimization of conditions for distinguishing the isomeric structures. For epimeric variants (e.g., ADHE-S versus BDHE-S; α- versus β-stereoisomerization in the C-17 position), differentiation was achieved at MS(4) and fragmentation was demonstrated through MS(5). Computational analysis was performed to further explore differences in the fragmentation pathways due to changes in stereochemistry.

Topics: Computer Simulation; Equilin; Estrogens, Conjugated (USP); Estrone; Humans; Isomerism; Models, Molecular; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

Identification of biomarkers potentially provides prognostic information that can help guide clinical decision-making. Given the relationship between estrogen exposure and endometrial cancer, especially low grade endometrioid carcinoma, we hypothesized that high expression of genes induced by estrogen would identify low risk endometrioid endometrial cancers. cDNA microarray and qRT-PCR verification were used to identify six genes that are highly induced by estrogen in the endometrium. These estrogen-induced biomarkers were quantified in 72 endometrial carcinomas by qRT-PCR. Unsupervised cluster analysis was performed, with expression data correlated to tumor characteristics. Time to recurrence by cluster was analyzed using the Kaplan-Meier method. A receiver operating characteristic (ROC) curve was generated to determine the potential clinical utility of the biomarker panel to predict prognosis. Expression of all genes was higher in endometrioid carcinomas compared to non-endometrioid carcinomas. Unsupervised cluster analysis revealed two distinct groups based on gene expression. The high expression cluster was characterized by lower age, higher BMI, and low grade endometrioid histology. The low expression cluster had a recurrence rate 4.35 times higher than the high expression cluster. ROC analysis allowed for the prediction of stage and grade with a false negative rate of 4.8% based on level of gene expression in endometrioid tumors. We have therefore identified a panel of estrogen-induced genes that have potential utility in predicting endometrial cancer stage and recurrence risk. This proof-of-concept study demonstrates that biomarker analysis may play a role in clinical decision making for the therapy of women with endometrial cancer.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Biomarkers, Tumor; Body Mass Index; Carcinoma, Endometrioid; Cluster Analysis; Endometrial Neoplasms; Equilin; Estrogen Replacement Therapy; Estrogens, Conjugated (USP); Estrone; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genetic Association Studies; Humans; Middle Aged; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Prognosis; Randomized Controlled Trials as Topic; Reverse Transcriptase Polymerase Chain Reaction; ROC Curve

Equilin-3-sulfate and delta8,9-dehydroestrone-3-sulfate are two isomers found in equine conjugated estrogens that differ in structure only by the position of a double bond in the steroid B-ring. These geometric isomers were not resolved on a C18 column during the analysis of conjugated estrogen drug products by LC-MS using acetonitrile-ammonium acetate buffer as the mobile phase. While no separations of these two isomers were observed on C18 or other alkyl-bonded silica based phases using a variety of mobile phase conditions, partial separations were achieved on phenyl bonded silica phases with a resolution of 1.5 on a diphenyl phase, and baseline separations were readily achieved on two carbonaceous phases with resolutions routinely exceeding three on graphitic carbon-coated zirconia (Zr-CARB) and resolutions as high as 19 on porous graphitic carbon (Hypercarb). An examination of a selected few conjugated estrogens in the complex drug substance by LC-MS on Hypercarb is presented.

Topics: Chromatography, Liquid; Equilin; Estrogens, Conjugated (USP); Estrone; Graphite; Spectrometry, Mass, Electrospray Ionization; Spectrophotometry, Ultraviolet; Zirconium

The metabolism of equilin sulfate was determined in female dogs receiving 2.5 mg/kg of [3H]equilin sulfate alone or in a preparation that contained all the components that are present in the conjugated equine estrogen product Premarin. The pharmacokinetic parameters of total radioactivity indicated that the drug is rapidly absorbed and it has a moderate half-life in plasma. The total radioactivity in plasma following administration of [3H]equilin sulfate as part of a mixture of conjugated equine estrogens had significantly lower peak concentration (Cmax), a lower area under the curve (AUC), a longer terminal half-life (t1/2) and a longer mean residence time (MRT) than when [3H]equilin sulfate was given alone, indicating that the other components in the conjugated equine estrogen preparation altered the pharmacokinetics of equilin sulfate. An average of 26.7 +/- 4.4% of the administered radioactive dose was excreted in urine of dogs receiving [3H]equilin sulfate. Again, a significantly lower percentage (21.4 +/- 6.3%, P = 0.023) was eliminated in urine of dogs receiving [3H]equilin sulfate in the conjugated equine estrogen preparation, indicating that the absorption of equilin sulfate was perhaps altered by the other components in the conjugated equine estrogen preparation. Metabolite profiles of plasma and urine were similar. Equilin, equilenin, 17 beta-dihydroequilenin, 17 beta-dihydroequilin, 17 alpha-dihydroequilenin and 17 alpha-dihydroequilin were present in both matrices. 17 beta-Dihydroequilin and equilin were the two major chromatographic peaks in plasma samples. 17 beta-Dihydroequilenin and 17 beta-dihydroequilin were the major metabolites in urine. In conclusion, following oral administration of [3H]equilin sulfate to dogs, the radioactivity is rapidly absorbed. The disposition of equilin sulfate is altered by the other components that are present in the conjugated equine estrogen preparation Premarin. The reduction of the 17-keto group and aromatization of ring-B are the major metabolic pathways of equilin in the dog.

Topics: Animals; Biotransformation; Chromatography, High Pressure Liquid; Dogs; Drug Combinations; Equilin; Estrogens, Conjugated (USP); Female; Gas Chromatography-Mass Spectrometry; Half-Life; Metabolic Clearance Rate; Radioisotope Dilution Technique; Tritium

The MCRs of 17 beta-dihydroequilin sulfate and 17 beta-dihydroequilin were determined in normal postmenopausal women by single iv injection of either 17 beta-[3H]dihydroequilin sulfate ([3H]17 beta-EqS) or 17 beta-[3H]dihydroequilin ([3H]17 beta-Eq). After the administration of [3H]17 beta-EqS, blood was drawn at various time intervals, and the plasma obtained was fractionated into the unconjugated, sulfate, and glucuronide fractions. The bulk of radioactivity was present in the sulfate fraction, and from this [3H]17 beta-EqS, [3H]equilin sulfate, [3H]equilenin sulfate, and 17 beta-[3H]dihydroequilenin sulfate were isolated and purified, and their concentrations were measured. The disappearance of [3H]17 beta-EqS from plasma can be described as a function of two exponentials. The half-life of the initial fast component was 5 +/- 0.2 min; this component represents the distribution and transfer from a space, with a mean volume (V1) of 6 +/- 0.5 L. The value for the rate constant (k) of total removal from this space was 300 +/- 20 U/day, of which 35 +/- 2% was irreversible. The mean half-life of the slower component of 17 beta-EqS was 147 +/- 15 min, and the mean MCR was 376 +/- 93 L/day.m2. Similarly, after the administration of [3H]17 beta-Eq, the disappearance of radioactivity as 17 beta-Eq from plasma also had two components. The half-lives of the fast and slow component were 5.5 +/- 0.8 and 45 +/- 2.0 min, respectively. The MCR of 17 beta-Eq was 1252 +/- 103 L/day.m2. From both series of experiments, unconjugated and sulfate-conjugated equilin, equilenin, and 17 beta-dihydroequilenin were isolated and purified, and their concentrations were measured. No 17 alpha-reduced metabolites were detected. These results indicate that 17 beta-EqS is cleared twice as fast as equilin sulfate (MCR, 176 L/day.m2), whereas the more potent estrogen 17 beta-Eq is cleared 2 times slower than equilin. The slower elimination and greater estrogenic activity of 17 beta-Eq support the hypothesis that the major in vivo activity of equilin sulfate present in conjugated equine estrogen preparations is expressed via its metabolites 17 beta-EqS and 17 beta-Eq.

Topics: Equilin; Estrogens, Conjugated (USP); Female; Humans; Metabolic Clearance Rate; Middle Aged; Postmenopause; Reference Values

The constant infusion of [3H]equilin sulfate ([3H]EqS) was used to estimate the MCR of equilin sulfate (EqS) and to measure the conversion of this estrogen to equilin (Eq), equilenin (Eqn), equilenin sulfate (EqnS), 17 beta-dihydroequilin (17 beta-Eq), 17 beta-dihydroequilin sulfate (17 beta-EqS), 17 beta-dihydroequilenin (17 beta-Eqn), and 17 beta-dihydroequilenin sulfate (17 beta-EqnS) in normal postmenopausal women and men. Infusion of [3H]EqS was started in five postmenopausal women and two men 30 min after a priming dose and continued at a constant rate of 12-15 microCi/h for 3 h. Blood samples were taken 15 min before the end of infusion, at the end of the infusion, and 15 min after the end of infusion. Unconjugated and sulfate-conjugated Eq, Eqn, 17 beta-Eq, and 17 beta-Eqn were isolated from plasma. The mean MCR of EqS was calculated to be 280 +/- 24 L/day or 170 +/- 18 L/day.m2. The mean conversion ratios for precursor EqS to product 17 beta-EqS, EqnS, 17 beta-EqnS, 17 beta-Eq, Eq, Eqn, and 17 beta-Eqn were 0.300, 0.190, 0.100, 0.020, 0.016, 0.008, and 0.004 respectively. In both the sulfate-conjugated and unconjugated forms, 17 beta-Eq was the most abundant metabolite formed. 17 beta-Eq estrogen is a potent uterotropic agent and has a much higher affinity for estrogen receptors than Eq. Its formation may be of importance in the overall biological activity of EqS present in conjugated equine estrogen preparations.

Topics: Aged; Equilenin; Equilin; Estrogens, Conjugated (USP); Female; Homeostasis; Humans; Male; Middle Aged; Postmenopause; Reference Values

The absorption of equilin sulfate and equilin from the gastrointestinal tract was determined in normal men after the ingestion of [3H]equilin-[35S]sulfate or a mixture of [3H]equilin and equilin-[35S]sulfate, while the metabolism of equilin sulfate was investigated after iv administration of [3H]equilin sulfate to postmenopausal women. After the oral administration of [3H]equilin-[35S]sulfate, equilin sulfate containing both 3H and 35S was isolated from plasma; however, only in the first sample taken at 10 min was the 3H/35S ratio the same as that of the [3H]equilin-[35S]sulfate ingested. The 3H/35S ratio then increased, and by 12 h only traces of equilin-[35S]sulfate were detectable. Similarly, after the ingestion of [3H]equilin and equilin-[35S]sulfate, [3H]equilin-[35S]sulfate was isolated from plasma. The 3H/35S ratio was at all time points greater than the 3H/35S ratio of the ingested mixture. Analysis of urine indicated that over 98% of 35S was not associated with any steroid and was most likely inorganic sulfate. After iv administration of [3H] equilin sulfate to postmenopausal women, equilin, equilenin, 17 beta-dihydroequilin, and 17 beta-dihydroequilenin were isolated from the urine. These results indicate that 1) some of the orally administered equilin sulfate was absorbed from the gut without prior hydrolysis, 2) some equilin sulfate was hydrolyzed in the gut before absorption; 3) equilin was absorbed more efficiently than equilin sulfate; 4) equilin absorbed was readily sulfated and circulated in this form; and 5) equilin sulfate was extensively metabolized, and the metabolites were excreted in the urine mainly conjugated with glucuronic acid.

Topics: 17-Ketosteroids; Administration, Oral; Age Factors; Digestive System; Equilenin; Equilin; Female; Humans; Injections, Intravenous; Intestinal Absorption; Male; Menopause; Middle Aged

In order to determine the relative potency of equilin sulfate (EqS), a major constituent of conjugated equine estrogens, 15 women received oral doses of EqS (0.15, 0.31, and 0.625 mg) for 25 days. Doses of 0.31 and 0.625 mg significantly stimulated hepatic globulins. This stimulatory effect ranged from being 1.5 to 8 times greater than the effects of comparable doses of estrone sulfate and conjugated equine estrogens. A significant stimulation in high-density lipoprotein-cholesterol occurred with as little as 0.15 mg of EqS. Elevations in the high-density lipoprotein/low-density lipoprotein-cholesterol ratio occurred with EqS, which resulted in an approximately 4-fold greater response than that achieved with comparable doses of conjugated equine estrogens. The fasting urinary calcium/creatinine ratio was only significantly lowered with 0.625 mg of EqS and was less potent than conjugated equine estrogens in this regard. It is concluded that EqS is a potent estrogen that contributes significantly to the hepatic stimulatory effects of conjugated equine estrogens. These data also provide support for the suggestion that there may be a dissociation in potency between estrogenic effects on liver and bone.

Topics: 17-Ketosteroids; Adult; Angiotensinogen; Calcium; Cholesterol, HDL; Creatinine; Dose-Response Relationship, Drug; Equilin; Female; Humans; Lipoproteins, LDL; Menopause; Middle Aged; Sex Hormone-Binding Globulin; Transcortin

The binding of ring B unsaturated equine estrogens, equilin sulfate (EqS), equilin (Eq), and 17 beta-dihydroequilin (17 beta-Eq) with human serum proteins was determined and compared with the binding of estrone sulfate (E1S), estrone (E1), estradiol (E2), testosterone (T), and 5 alpha-dihydrotestosterone (5 alpha-DHT). Undiluted serum or 5% human serum albumin (HSA) was incubated with 3H-labeled steroids at 37 C, then subjected to gel filtration at 4 C. Gel filtration of serum from Premarin-treated postmenopausal women or normal women incubated with Eq, E1, E2, or 5 alpha-DHT showed two peaks of radioactivity associated with proteins with average apparent mol wt of 128,000 and 68,000 and average Stokes radii of 48.6 and 34.9 A. These values correspond to those reported for sex hormone-binding globulin (SHBG) and albumin, respectively. Binding to SHBG and albumin was confirmed by removing SHBG or albumin from the serum with Concanavalin-A Sepharose 4B gel or CM-Affi Gel Blue, respectively. In the case of [3H]EqS and [3H]E1S, binding to SHBG was not detectable, and only a peak of radioactivity associated with albumin was found. However, under these conditions, the binding of estrogens to SHBG in serum from normal men was not detectable. Incubation of the above steroids with 5% HSA followed by gel filtration resulted in a single peak of radioactivity associated with the protein peak. Using ultrafiltration dialysis followed by Scatchard analysis, at least two sets of binding sites were found for the interaction of HSA with EqS or E1S. The high and low affinity binding sites had association constants k1 and k2 of approximately 0.9-1.1 (X 10(5) M-1) and 0.5-0.8 (X 10(4) M-1). In contrast with Eq and E1, only the low affinity binding sites were found (apparent Ka congruent to 1 X 10(4) M-1). The binding constants of some estrogens and androgens to SHBG at 37 C determined by competitive Scatchard analysis using DEAE filter assay and [3H]5 alpha-DHT were 0.15, 0.07, 0.22, 0.29, 2.70, and 4.53 (X 10(9) M-1) for Eq, E1, 17 beta-Eq, E2, T, and 5 alpha-DHT, respectively. These results indicate that the equine estrogens bind to SHBG and albumin in a manner similar to that of E1 and E2, and that the low MCR of EqS reported previously may be due to its binding to albumin.

Topics: Animals; Binding, Competitive; Biological Transport; Blood Proteins; Chromatography, Gel; Dihydrotestosterone; Equilin; Estradiol; Estrogens; Estrone; Female; Horses; Hot Temperature; Humans; Male; Protein Binding; Serum Albumin; Sex Hormone-Binding Globulin; Testosterone

The MCRs of equilin sulfate and equilin were determined in normal postmenopausal women and a normal man by single iv injections of either [3H]equilin sulfate or [3H] equilin. After the administration of [3H]equilin sulfate, blood was drawn at various time intervals, and the plasma obtained was fractionated into the unconjugated, sulfate, and glucuronide fractions. The bulk of radioactivity was present in the sulfate fraction, and from this, [3H]equilin sulfate, [3H]17 beta-dihydro-equilin sulfate, [3H]equilenin sulfate, and [3H]17 beta-dihydroequilenin sulfate were isolated and purified, and their concentrations were measured. The disappearance of radioactivity from plasma as equilin sulfate can be described as a function that is the sum of two exponentials. The initial fast component (half-life, 5.2 +/- 1.2 min) represents distribution and transfer from a space, with a mean volume of 12.4 +/- 1.6 liters. The mean value for the rate constant of total removal from the initial volume is 163 +/- 19 U/day, of which 15.8 +/- 2% is irreversible. The mean half-life of the slower component of equilin sulfate is 190 +/- 23 min, and the mean MCR is 176 +/- 44 liters/day . m2. Similarly, after the administration of [3H]equilin to a normal postmenopausal woman and a man, the disappearance of radio-activity from plasma as equilin could be fitted by a single straight line, consistent with a one-compartment system. The half-life of equilin was approximately 19-27 min, and the MCR of equilin was calculated to be 1982 liters/day/m2 in the normal man and 3300 liters/day/m2 in the normal postmenopausal woman. The bulk of [3H]equilin was very rapidly metabolized to mainly equilin sulfate. Small amounts of 17 beta-dihydroequilin sulfate and 17 beta-dihydroequilin were also isolated from the plasma. The in vivo formation of 17 beta-dihydroequilin and its sulfate may be of importance, as this estrogen is approximately 8 times more potent as a uterotropic agent than equilin sulfate.

Topics: 17-Ketosteroids; Adult; Equilenin; Equilin; Estrogens, Conjugated (USP); Female; Humans; Kinetics; Male; Menopause; Metabolic Clearance Rate; Middle Aged