equilin and dihydroequilin

To determine the relative bioavailability of the estrogenic components of a generic brand of conjugated estrogens marketed in Chile in comparison to that of Conpremin (Premarin in the United States).. A randomized cross-over study was conducted on 16 healthy postmenopausal women receiving single oral doses of either two Conpremin 0.625-mg tablets or two 0.625-mg tablets of the generic brand, with a 14-day wash-out interval between doses. A gas chromatography tandem mass spectrometry assay was used to determine estrogen components.. The peak plasma concentrations of unconjugated and total estrone and equilin, unconjugated 17 beta-dihydroequilin and 17 beta-estradiol were higher and occurred earlier with the generic conjugated estrogens than with Conpremin. The 90% confidence limits for both variables lay outside the accepted bioequivalence limits of 80-125%. Additionally, no measurable plasma concentration of unconjugated delta 8,9-dehydroestrone or 17 beta-delta 8,9-dehydroestradiol was seen after administration of the generic conjugated estrogens.. These pharmacokinetic results indicate that the generic tablets do not have the modified-release characteristics of Conpremin tablets. In addition, the absence of delta 8,9-dehydroestrone and 17 beta-delta 8,9-dehydroestradiol in the plasma indicates that the generic form is not compositionally equivalent to Conpremin.

Topics: Adult; Biological Availability; Chile; Cross-Over Studies; Drugs, Generic; Equilin; Estradiol; Estrogens, Conjugated (USP); Estrone; Female; Humans; Middle Aged; Postmenopause; Therapeutic Equivalency; United States

To determine the independent biologic effects of 17 alpha-dihydroequilin sulfate.. Prospective randomized study.. University of Southern California Medical Center.. Twenty-one postmenopausal women, mean age 50 +/- 2 (+/-SEM) years, and mean body mass index 27 +/- 2.. Women were randomized to receive daily oral doses of either 1.25 mg of estrone sulfate (E1S), 0.2 mg of 17 alpha-dihydroequilin sulfate, or a combination. Three blood and urine samples were obtained before and after 30 and 90 days of treatment.. After 30 and 90 days of treatment, E1S alone increased sex hormone-binding globulin (SHBG) levels significantly, 19.7% +/- 6.0% and 61.3% +/- 13.0%, whereas 17 alpha-dihydroequilin sulfate reduced SHBG levels, 20.8% +/- 68% and 12.4% +/- 7.5%, respectively. Nevertheless, the combination of E1S and 17 alpha-dihydroequilin sulfate significantly increased SHBG levels, 103% +/- 27.9% and 98.2% +/- 19.1%, compared with baseline at 30 and 90 days. Fewer changes were evident with corticosteroid-binding globulin (CBG). After 90 days of treatment, CBG levels significantly increased 30.9% +/- 5.5% with E1S, decreased by 7.2% +/- 5.0% with 17 alpha-dihydroequilin sulfate, and, with the combination, significantly increased by 10.5% +/- 2.4% compared with baseline. Changes in lipids and lipoproteins were more variable. However, high-density-lipoprotein cholesterol increased significantly with E1S at 30 and 90 days compared with baseline, 96.5% +/- 39% and 91.5% +/- 22.6%, and with the combination increased 66.4% +/- 13.3% and 79.2% +/- 24.4%, respectively. Fewer changes were evident with 17 alpha-dihydroequilin sulfate alone, decreasing 4.4% +/- 22% and 2.6% +/- 21.3%. Urinary ratios of bone collagen equivalents-creatinine and calcium-creatinine decreased in all three groups. However, the combination group resulted in a significantly greater percentage decrease in bone collagen equivalents-creatinine than with E1S alone.. 17 alpha-Dihydroequilin sulfate could modify some of the first-pass effects of conjugated equine estrogens and act synergistically with other conjugated equine estrogens to reduce bone resorption.

Topics: Calcium; Cholesterol, HDL; Collagen; Creatinine; Equilin; Estrone; Female; Follicle Stimulating Hormone; Humans; Middle Aged; Prospective Studies; Sex Hormone-Binding Globulin; Transcortin

Celecoxib has been reported to switch the human SULT2A1-catalyzed sulfonation of 17β-estradiol (17β-E2) from the 3- to the 17-position. The effects of celecoxib on the sulfonation of selected steroids catalyzed by human SULT2A1 were assessed through in vitro and in silico studies. Celecoxib inhibited SULT2A1-catalyzed sulfonation of dehydroepiandrosterone (DHEA), androst-5-ene-3β, 17β-diol (AD), testosterone (T) and epitestosterone (Epi-T) in a concentration-dependent manner. Low μM concentrations of celecoxib strikingly enhanced the formation of the 17-sulfates of 6-dehydroestradiol (6D-E2), 17β-dihydroequilenin (17β-Eqn), 17β-dihydroequilin (17β-Eq), and 9-dehydroestradiol (9D-E2) as well as the overall rate of sulfonation. For 6D-E2, 9D-E2 and 17β-Eqn, celecoxib inhibited 3-sulfonation, however 3-sulfonation of 17β-Eq was stimulated at celecoxib concentrations below 40 μM. Ligand docking studies in silico suggest that celecoxib binds in the substrate-binding site of SULT2A1 in a manner that prohibits the usual binding of substrates but facilitates, for appropriately shaped substrates, a binding mode that favors 17-sulfonation.

Topics: Androstenediol; Binding Sites; Celecoxib; Cyclooxygenase 2 Inhibitors; Dehydroepiandrosterone; Epitestosterone; Equilin; Estradiol; Humans; Models, Molecular; Molecular Docking Simulation; Pyrazoles; Recombinant Proteins; Sulfonamides; Sulfotransferases; Testosterone

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

The presence of natural estrogen hormones as trace concentrations in the environment has been reported by many researchers and is of growing concern due to its possible adverse effects on the ecosystem. In this study, municipal biosolids, poultry manure (PM) and cow manure (CM), and spent mushroom compost (SMC) were analyzed for the presence of seven estrogen hormones. 17α-estradiol, 17β-estradiol, 17α-dihydroequilin, and estrone were detected in the sampled biosolids and manures at concentrations ranging from 6 to 462 ng/g of dry solids. 17α-estradiol, 17β-estradiol, and estrone were also detected in SMC at concentrations ranging from 4 to 28 ng/g of dry solids. Desorption experiments were simulated in the laboratory using deionized water (milli-Q), and the aqueous phase was examined for the presence of estrogen hormones to determine their desorption potential. Very low desorption of 0.4% and 0.2% estrogen hormones was observed from municipal biosolids and SMC, respectively. An estimate of total estrogen contribution from different solid waste sources is reported. Animal manures (PM and CM) contribute to a significant load of estrogen hormones in the natural environment.

Topics: Agaricales; Animals; Cattle; Environmental Monitoring; Equilin; Estradiol; Estrogens; Estrone; Manure; Poultry; Refuse Disposal; Risk Assessment; Soil; Soil Pollutants

The degradation of the mixture of steroid hormones including seven estrogens (17α-estradiol, 17β-estradiol, 17α-dihydroequilin, 17α-ethinyl estradiol, estriol, estrone and equilin) and five progestins (levonorgestrel, gestodene, trimegestrone, medrogestone and progesterone) by ozonation in aqueous solution is investigated. The ozonation process provides high removal (up to 100%) of hormones and estrogenicity in the treated water. Computational methods such as quantum chemistry calculations (QCCs) are applied to interpret the observed results. Quantum chemistry descriptors computed for steroid hormones explain the nature of the reactions and differences in reactivities between estrogen and progestin hormones within the framework of the Density Functional Theory (DFT). Computed molecular descriptors were combined with physical properties to develop qualitative structure activity relationship (QSAR) models (using multiple linear regression algorithm). The developed models have correlation coefficients (R(2)) of 0.994 for estrogens and 0.997 for progestins, and could be used to predict the removal efficiencies for similar compounds. The frontier molecular orbitals (the HOMO and the LUMO) have a major impact on the reactivity of steroid hormones. The susceptibility of certain functional groups to ozone and possible reactive sites for all steroids was discussed by Frontier Molecular Orbital approach.

Topics: Equilin; Estradiol; Estrogens; Estrone; Ethinyl Estradiol; Models, Chemical; Ozone; Progestins; Water Pollutants, Chemical

Ultrasound assisted degradation of estrogen hormones was examined in a batch reactor using a 2 kW (20 kHz) sonication unit. The degradation of estrogens follow a pseudo first order rate kinetics, and the order of degradation is 17alpha-dihydroequilin > equilin >17alpha-ethinyl estradiol >17alpha-estradiol >17beta-estradiol > estrone > estriol. Effect of solution alkalinity and salinity on the sonochemical degradation of estrogen hormones is examined. At alkalinity concentration of 10 mM, no adverse effect on the degradation rate constants of estradiols (17alpha-estradiol, 17beta-estradiol, and 17alpha-ethinyl estradiol) was observed, whereas equilin compounds showed a decrease in their degradation rate constants. Significant inhibitory effects were observed for all the compounds at high alkalinity concentration of 120 mM and which could be due to the scavenging of OH(*) radicals in the bulk solution. The presence of salinity (0.17 M) enhanced the estrogen degradation except for the equilin compounds. Simultaneous presence of high alkalinity (120 mM) and salinity (0.17 M) also increased the degradation of estrogen hormones than the case when only alkalinity (120 mM) was present, indicating the diffusion of analytes to the cavity interface where most of the degradation occurs under these conditions. A mechanistic approach was used to model the degradation behavior of estrogen hormones under different solution alkalinity and salinity conditions.

Topics: Equilin; Estradiol; Estrogens; Hydrogen-Ion Concentration; Salinity; Sonication; Water; Water Pollutants, Chemical

Three municipal wastewater treatment plants (WWTPs) in southeastern Pennsylvania were sampled to determine the presence and concentrations of 12 natural and synthetic estrogen hormones in the wastewater influent and effluent. The target estrogens were 17alpha-estradiol, estrone, estriol, equilin, 17alpha-dihydroequilin, 17beta-estradiol, 17alpha-ethinyl estradiol, gestodene, norgestrel, levonorgestrel, medrogestone, and trimegestone. One WWTP uses a biofilm reactor (packed-bed trickling filter),and the other two use suspended-growth media (continuously stirred activated sludge reactor and sequential batch reactor). Estrone was detected in all the three plants; estriol and estradiol were detected at two WWTPs; and 17 alpha-dihydroequilin and 17 alpha-ethinyl estradiol were detected at one WWTP. The concentration of estrogens in the influent and effluent of the three treatment plants ranged from 1.2 to 259 ng/L and 0.5 to 49 ng/L, respectively. The percentage removal of estrogens from the aqueous phase ranged from 41 to 99%, except in the case of 17alpha-dihydroequilin; the removal of 17alpha-dihydroequilin was negligible. The suspended-growth media systems showed higher removal efficiencies for estrogens than the biofilm system. The analytical method uses a Varian C-18 solid-phase extraction (Varian Inc., Palo Alto, California), followed by a derivatization with bis(trimethylsilyl)trifluoroacetamide. The detection limits for the estrogen compounds ranged from 0.1 to 10 ng/L using a sample size of 1 L. The method recoveries ranged from 71 to 120%, and the relative standard deviation ranged from 6 to 14% for all the hormones.

Topics: Equilin; Estrogens; Ethinyl Estradiol; Gas Chromatography-Mass Spectrometry; Industrial Waste; Pennsylvania; Sewage; Solid Phase Extraction; Solubility; Waste Disposal, Fluid; Water Pollutants, Chemical

In most studies showing cardio- and vasculoprotective effects of estrogens, 17beta-estradiol was used and little information on possible effects of different estrogen metabolites is yet available. We investigated whether particular estrogen metabolites are effective in counteracting inflammatory activation of human endothelium. Human endothelial cells were incubated with 17alpha-dihydroequilenin, 17beta-dihydroequilenin, delta-8,9-dehydroestrone, estrone and 17beta-estradiol and stimulated with interleukin (IL)-1alpha. The expression of IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1) was determined. 17beta-dihydroequilenin and 17beta-estradiol at a concentration of 1 microM reduced IL-1alpha-induced up regulation of IL-6, IL-8 and MCP-1 close to control levels. When both compounds were used in combination an additive effect was observed. 17alpha-dihydroequilenin and delta-8,9-dehydroestrone showed a similar anti-inflammatory effect only when used at 10 microM whereas estrone had no effect. The effect of 17beta-dihydroequilenin on IL-1alpha-induced production of IL-6, IL-8 and MCP-1 was reversed by the estrogen receptor antagonist ICI 182,780. 17beta-dihydroequilenin also inhibited IL-1alpha-induced translocation of p50 and p65 to the nucleus of the cells. We have identified the estrogen metabolite 17beta-dihydroequilenin, as an inhibitor of inflammatory activation of human endothelial cells. Characterization of specific estrogens--as shown in our study--could provide the basis for tailored therapies, which might be able to achieve vasoprotection without adverse side effects.

Topics: Anti-Inflammatory Agents; Base Sequence; Cells, Cultured; Chemokine CCL2; Dose-Response Relationship, Drug; Endothelial Cells; Equilin; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogens; Fulvestrant; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Molecular Sequence Data; NF-kappa B; RNA, Messenger

Most effluents from wastewater treatment works (WwTWs) contain estrogenic chemicals that include steroidal estrogens and xenoestrogens. We investigated the nature of mixtures of estrogenic contaminants taken up by two species of fish exposed to two WwTWs effluents. Sexually immature rainbow trout, Oncorhynchus mykiss, and sexually mature roach, Rutilus rutilus, were exposed to tap water, river water, or one of two estrogenic WwTWs effluents for up to 10 days, when the fish were sacrificed and tissues removed for chemical analysis. Estrogenic contaminants in the bile and gonads were hydrolyzed, concentrated by solid-phase extraction, and fractionated by RP-HPLC. Active fractions were detected and quantified using a yeast estrogen receptor transcription screen (YES assay) and the identities of estrogenic components in the fractions determined by GC-MS. Bile from rainbow trout exposed to either tap water or river water contained low amounts of 17beta-estradiol (E2) and estrone (E1) with a total estrogenic activity (mean+/-standard error) of 10+/-5 and 31+/-9 ng of E2 equivalents/mL (ng of E2eq/mL) for male and female fish, respectively. In effluent-exposed trout the total estrogen content of bile was considerably higher with the following composition and concentrations (ng of E2eq/mL) of individual estrogens: E2 (male, 591+/-125; female, 710+/-207), E1 (male, 338+/-75; female, 469+/-164), ethinylestradiol, EE2 (male, 32+/-2; female, 40+/-6), nonylphenol (NP) and short-chain NP polyethoxylates (male, 21+/-4; female, 22+/-3). An additional estrogenic compound, 17beta-dihydroequilenin (DHQ), was identified for the first time in effluent-exposed fish, and was present in trout bile at concentrations of (male) 40+/-9 and (female) 30+/-5 ng of E2 eq/mL. DHQ, E2, E1, and EE2, but not NP or NP polyethoxylates, were also detected in bile of effluent-exposed roach, and the concentrations of all these steroidal estrogens in ng of E2eq/mL were lower in male (E2, 62+/-2; E1, 35+/-11; EE2, 10+/-2; DHQ, 1+/-1) compared with female (E2, 740+/-197; E1, 197+/-37; EE2, 40+/-6; DHQ, 8+/-2) roach. The synthetic estrogen EE2 was also detected in the testes and ovaries of effluent-exposed roach. This study shows that a mixture of estrogenic contaminants present in WwTWs effluents bioconcentrate in fish tissues, resulting in the induction of vitellogenin, and are likely to contribute to feminizing effects in wild fish living in U.K. rivers. The composition of the mixture of estrogen

Topics: Animals; Bile; Dose-Response Relationship, Drug; Equilin; Estradiol; Estradiol Congeners; Estrogens; Estrone; Ethinyl Estradiol; Female; Fishes; Gonads; Industrial Waste; Male; Phenols; Receptors, Estrogen; Rivers; Water Pollutants, Chemical; Water Supply

In the present study, neuronal PC12 cells and hippocampal HT22 cells maintained in culture were used to test the neuroprotective effect of equine estrogens estrone, 17beta-estradiol, 17alpha-estradiol, equilin, 17beta-dihydroequilin, 17alpha-dihydroequilin, equilenin, 17beta-dihydroequilenin, 17alpha-dihydroequilenin, Delta(8)-estrone(,) and Delta(8),17beta-estradiol against glutamate toxicity.. The HT22 and PC12 cells were grown in Dulbecco modified Eagle medium supplemented with 5% horse serum, 10% fetal bovine serum, and 10 mM HEPES. The undifferentiated PC12 cells were plated on collagen-coated, 96-well plastic plates at 10,000 cells per well, and the HT22 cells were plated on uncoated 96-well plates at 2500 cells per well. Twenty-four hours after plating, various concentrations of estrogens (0.1-40 microM) and glutamate (1-10 mM) were added in a total volume of 100 microL. After 24 hours, cell viability was determined using the MTS cell proliferation assay. Results were verified in some experiments by using the lactate dehydrogenase cytotoxicity assay.. The results indicate that cell toxicity in both cell lines was directly proportional to the concentration of glutamate. The lowest dose of glutamate that reduced cell viability by 50% under these conditions was 1.8 mM for HT22 cells and 3 mM for PC12 cells. All estrogens tested were neuroprotective against glutamate-induced cell death in a typical dose-related manner. However, these estrogens differed extensively with respect to their neuroprotective potencies. In both cell lines, the Delta(8)-ring B unsaturated estrogens were the most neuroprotective, whereas the classic estrogens 17beta-estradiol, estrone, and 17alpha-estradiol were the least potent. The order of potency was Delta(8),17beta-estradiol > Delta(8)-estrone > 17beta-dihydroequilenin > 17alpha-dihydroequilenin > equilenin > 17beta-dihydroequilin = equilin > 17alpha-dihydroequilin > 17beta-estradiol > estrone > 17alpha-estradiol in PC12 cells and Delta(8),17beta-estradiol > Delta(8)-estrone > equilenin = 17beta-dihydroequilenin > 17beta-dihydroequilin > equilin > 17alpha-dihydroequilenin > 17alpha-dihydroequilin > 17alpha-estradiol = 17beta-estradiol > estrone in HT22 cells.. Our data indicate that the neurotoxic effects of glutamate can be inhibited differentially by various equine estrogens. The less estrogenic (uterotropic) Delta(8) estrogens were the most effective neuroprotectors, and further chemical modifications of these estrogens may provide compounds that are useful for preventing neurodegenerative diseases in both women and men.

Topics: Animals; Cell Line; Dose-Response Relationship, Drug; Equilenin; Equilin; Estradiol; Estradiol Congeners; Estrone; Glutamic Acid; Hippocampus; Neurons; Neuroprotective Agents; PC12 Cells; Rats; Structure-Activity Relationship

So far, most epidemiological studies investigating breast cancer risk and hormone replacement therapy have been conducted with conjugated equine estrogens (CEE). Recent trials indicate that the addition of progestogens may increase breast cancer risk. In the present study, we compared the effects of the human estrogen 17beta-estradiol (E(2)) with those of the main equine components of CEE, i.e. equilin (Eq) and 17alpha-dihydroequilin (Dheq) on the proliferation of human breast cancer cells. The proliferative effect of progestogen addition was also investigated.. The well-established human breast cancer cell line MCF-7 was used as an in vitro model. The proliferative effect of E(2), Eq and Dheq was tested in the concentration range 0.01-10 nmol/l. The progestogens progesterone, medroxyprogesterone acetate (MPA) and norethisterone (NET) were continuously combined with 0.1 nmol/l estrogen at concentrations of 0.01 nmol/l, 1 nmol/l, 0.1 mumol/l and 10 mumol/l. Proliferation was measured after 7 days by the adenosine triphosphate (ATP) chemosensitivity test.. All three estrogens increased the proliferation of MCF-7 cells by between 40 and 180%. The most proliferatively potent estrogen was E(2), followed by Eq and Dheq, which showed a slightly lower proliferative activity than E(2). The addition of progesterone inhibited E(2)-induced proliferation by about 30%, but only at the high non-physiological concentration of 10 mumol/l. All three progestogens inhibited Eq-induced proliferation, although their effect tended to be low, with values between 5 and 40%. No progestogen reduced Dheq-induced proliferation by more than 20%. In contrast, MPA slightly increased the proliferation rate by about 5% at the high physiological concentration of 0.1 mumol/l when combined with Dheq. The same held true when MPA and NET were added at the high pharmacological concentration of 10 mumol/l, causing increases of about 10%.. Our results indicate that equine estrogens have a proliferative action similar to that of 17beta-estradiol. Continuous addition of progestogens does not result in any major reduction of proliferative potency. Some progestogens may even enhance the estrogen-induced proliferation of pre-existing breast cancer cells, particularly when combined with certain equine estrogens. However, in none of the tested circumstances do progestogens increase the proliferative effect of estradiol, and progesterone has no deleterious effect even at pharmacological levels, in contrast to progestogens.

Topics: Breast Neoplasms; Cell Division; Dose-Response Relationship, Drug; Equilin; Estradiol; Estrogens, Conjugated (USP); Female; Humans; Medroxyprogesterone Acetate; Norethindrone; Progesterone; Progestins; Tumor Cells, Cultured

The pharmacology and SAR of representative equine estogens is described. 17alpha-Dihydroequilenin was found to prevent bone loss after 5 weeks of oral administration to ovariectomized rats. The stereochemical significance of the D-ring and the C/D ring juncture was investigated with a series of benzothiophene-based equilenin analogues.

Topics: Animals; Equilin; Estradiol Congeners; Female; Models, Molecular; Molecular Conformation; Organ Size; Ovariectomy; Rats; Stereoisomerism; Structure-Activity Relationship; Uterus

In on-going studies of 'classical' and ring B-unsaturated oestrogens in equine pregnancy, the products of metabolism of [2,2,4,6,6-2H(5)]-testosterone and [16,16,17-2H(3)]-5,7-androstadiene-3 beta,17 beta-diol with equine placental subcellular preparations and allantochorionic villi have been identified. Using mixtures of unlabelled and [2H]-labelled steroid substrates has allowed the unequivocal identification of metabolites by twin-ion monitoring in gas chromatography-mass spectrometry (GC-MS). Two types of incubation were used: (i) static in vitro and (ii) dynamic in vitro. The latter involved the use of the Oxycell cartridge (Integra Bioscience Systems, St Albans, UK) whereby the tissue preparation was continuously supplied with supporting medium plus appropriate cofactors in the presence of uniform oxygenation. [2H(5)]-Testosterone was converted into [2H(4)]-oestradiol-17 beta, [2H(4)]-oestrone and [2H(3)]-6-dehydro-oestradiol-17 alpha in both placental and chorionic villi preparations, but to a greater extent in the latter, confirming the importance of the chorionic villi in oestrogen production in the horse. On the basis of GC-MS characteristics (M(+) m/z 477/482 (as O-methyl oxime-trimethyl silyl ether), evidence for 19-hydroxylation of testosterone was found in static incubations, while the presence of a 6-hydroxy-oestradiol-17 alpha was recorded in dynamic incubations (twin peaks in the mass spectrum at m/z 504/507, the molecular ion M(+)). It was not possible to determine the configuration at C-6. The formation of small, but significant, quantities of [2H(4)]-17 beta-dihydroequilin was also shown, and a biosynthetic pathway is proposed. In static incubations of placental microsomal fractions, the 17 beta-dihydro forms of both equilin and equilenin were shown to be major metabolites of [2H(3)]-5,7-androstadiene-3,17-diol. Using static incubations of chorionic villi, the deuterated substrate was converted into the 17 beta-dihydro forms of both equilin and equilenin, together with an unidentified metabolite (base peak, m/z 504/506). The isomeric 17-dihydroequilins were also obtained using the dynamic in vitro incubation of equine chorionic villi, together with the 17 beta-isomer of dihydroequilenin. Confirmation of the identity of 17 beta-dihydroequilin and 17 beta-dihydroequilenin was obtained by co-injection of the authentic unlabelled steroids with the phenolic fraction obtained from various incubations. Increases in the peak areas for the non-

Topics: Androstenediol; Animals; Chorionic Villi; Equilenin; Equilin; Estradiol; Estrogens; Female; Gas Chromatography-Mass Spectrometry; Horses; In Vitro Techniques; Placenta; Pregnancy; Testosterone

Premarin, which contains several equine estrogens, as well as estradiol (E2) as a minor component, is widely used for replacement therapy of estrogen deficits, but little is known of its direct actions on brain cells. In mixed glial cultures, apolipoprotein E (apoE) and glial fibrillary acidic protein (GFAP) are induced by estrogens. GFAP induction showed an inverted-U shape E2 dose response, with a maximum induction at 1 pM, whereas apoE mRNA induction was greatest at 100 pM. GFAP and ApoE mRNAs were induced by equine estrogens in the following order: E2=equilin>estrone>17 alpha-dihydroequilenin. However, the induction of apoE secretion by 17 alpha-dihydroequilenin was as effective as by the other estrogens. The greater response of apoE secretion than GFAP mRNA induction to 17 alpha-dihydroequilenin might be therapeutically important because of the glial scarring during brain lesions, in which GFAP induction has a major role in inhibiting neurite outgrowth, whereas apoE secretion supports neurite outgrowth.

Topics: Animals; Apolipoproteins E; Astrocytes; Brain; Brain Injuries; Cells, Cultured; Coculture Techniques; Dose-Response Relationship, Drug; Equilin; Estradiol; Estrogens; Estrogens, Conjugated (USP); Estrone; Glial Fibrillary Acidic Protein; Gliosis; Horses; Nerve Regeneration; Neuronal Plasticity; Neuroprotective Agents; Rats; RNA, Messenger; Up-Regulation

Equine unsaturated estrogens are the main components of brand formulations indicated for hormonal replacement therapy in both hypogonadic and postmenopausal women. These hormones are produced by the fetoplacental unit during equine gestation. A method is described for the quantitative determination of equilenin (EL), equilin (EQ), 17alpha-dihydroequilin (17dEQ), and estrone (El) in the plasma of a pregnant mare. Blood samples are obtained weekly during pregnancy by jugular venipuncture using sodium ethylenediaminetetracetic as the anticoagulant. For the quantitation of these estrogens, plasma is submitted to enzymatic hydrolysis followed by liquid-liquid extraction. A high-performance liquid chromatographic system equipped with a UV detector set at 220 nm and an ODS Hypersil column is used. The method met precision, specificity, and accuracy requirements. The hormonal levels determined in one target mare throughout pregnancy were 97.91 to 449.13, 116.47 to 266.02, 74.92 to 235.54, and 84.26 to 300.03 ng/mL, reaching a maximum towards the 25th, 20th, 33rd, and 27th weeks, respectively, for E1, EL, EQ, and 17dEQ. The method was successfully tested by quantitating these estrogens in the plasma from a pregnant mare. Its applicability to the study of estrogen bioavailability and bioequivalence is suggested.

Topics: Animals; Chromatography, High Pressure Liquid; Edetic Acid; Equilenin; Equilin; Estrogens; Estrone; Female; Horses; Hydrolysis; Pregnancy; Sensitivity and Specificity

The effects of estradiol and 17alpha-dihydroequilenin on the apical dendrite spine density of pyramidal cells of the CA1 region of rat hippocampus were compared. 17alpha-Dihydroequilenin was as effective as estradiol in increasing spine densities relative to controls. 17alpha-Dihydroequilenin is not uterotrophic like estradiol but does have beneficial effects on the cardiovascular system, suggesting that it may be an effective single-agent hormone replacement therapy to treat menopausal symptoms and reduce chronic disease risk in menopausal women.

Topics: Animals; Cell Count; Dendrites; Equilin; Female; Hippocampus; Ovariectomy; Rats; Rats, Sprague-Dawley

The effect of the estrogen metabolites, 4-hydroxyestrone and 17alpha-dihydroequilin (metabolites of estradiol-17beta and equilin, respectively), were examined for antioxidant effects on plasma and lipoprotein lipid peroxidation . Lipid peroxidation was evaluated by products of both fatty acid (thiobarbituric acid-reactive substances [TBARS]) and cholesterol (oxysterols) oxidation from lipoproteins or whole plasma. Although all estrogens significantly reduced lipid peroxidation, 4-hydroxyestrone was far more potent than either equilin or 17alpha-dihydroequilin in inhibiting TBARS formation in lipoproteins induced by Cu2+. Similar effects were also noted on TBARS formation in THP-l macrophages in culture. However, 17alpha-dihydroequilin (along with equilin) strongly inhibited oxysterol formation, whereas 4-hydroxyestrone was ineffective. These studies suggest that different estrogens might act preferentially on distinct lipid substrates in exhibiting antioxidant effects.

Topics: Antioxidants; Cell Line; Cholesterol; Equilin; Fatty Acids; Humans; Hydroxyestrones; Lipid Peroxidation; Lipoproteins, LDL; Lipoproteins, VLDL; Macrophages; Sterols; Thiobarbituric Acid Reactive Substances

Our purpose was to determine the effects of 17 alpha-dihydroequilenin on plasma lipid and lipoprotein, glucose, and insulin concentrations; coronary artery vasomotor function; and reproductive organ and mammary gland proliferation in atherosclerotic male and female rhesus macaques.. Fifty adult female and 33 adult male rhesus macaques were randomized to treatment by lifetime dietary cholesterol exposure and ratio of total plasma cholesterol to high-density lipoprotein cholesterol. The female treatment groups were intact female controls (n = 9), ovariectomized controls (n = 16), ovariectomized plus 0.3 mg/kg/day 17 alpha-dihydroequilenin (n = 17) and ovariectomized plus subcutaneous estradiol (n = 7). The male treatment groups were control (n = 16) and 1.25 mg/kg/day 17 alpha-dihydroequilenin (n = 17). Treatment lasted 5 weeks. Longitudinal assessments of plasma lipid and lipoprotein and glucose and insulin concentrations were performed. Coronary artery vasomotor function was assessed by quantitative coronary angiography 1 week after initiation of treatment. Morphologic and immunohistochemical assessments of proliferation index values of reproductive organs and mammary glands were done at necropsy.. 17 alpha-Dihydroequilenin prevented endothelium-dependent vasoconstriction in males (p < 0.05) and ovariectomized females (p < 0.08). 17 alpha-Dihydroequilenin treatment increased plasma apolipoprotein A-1 concentrations (p < 0.05) and lowered fasting insulin concentrations (p < 0.05) without changing fasting plasma glucose concentrations in males. 17 alpha-Dihydroequilenin had no other effects on plasma lipid and lipoprotein concentrations in either males or females. It had no trophic effects on uterus, endometrium, or breast. There was no effect on either prostatic or testicular weight.. 17 alpha-Dihydroequilenin may represent a single-agent hormone therapy for reduction of ischemic hear disease risk for both menopausal women and men. It has no apparent trophic effects on reproductive organs or mammary glands of female and male rhesus macaques.

Topics: Animals; Arteriosclerosis; Blood Glucose; Coronary Vessels; Equilin; Female; Genitalia, Female; Gonadal Steroid Hormones; Immunohistochemistry; Insulin; Lipids; Macaca mulatta; Male; Myocardial Ischemia; Organ Size; Sex Characteristics; Vasomotor System

A high-performance liquid chromatographic (HPLC) method with fluorescence detection for the determination of total (unconjugated and conjugated) 17 alpha-dihydroequilenin in male and female rat, female rabbit and male and female rhesus monkey plasma is described here. Plasma sample preparation involved hydrolysis with enzyme (Glusulase), addition of internal standard (14 beta-equilenin) and solvent extraction. The extracts were chromatographed on a C6, 5-microns reversed-phase HPLC column and detection was accomplished with a fluorescence detector operated at an excitation wavelength of 210 nm and an emission wavelength of 370 nm. The assay was linear over a range of 2.5 to 100 ng/ml in male and female rat plasma, and 5 to 500 ng/ml in female rabbit and male and female monkey plasma. The method was specific, accurate and reproducible (percent differences < 14.5; coefficients of variation < 9.5%) in all matrices examined. The applicability of this method was successfully tested by quantifying total plasma concentrations of 17 alpha-dihydroequilenin in ovariectomized female rats, ovariectomized female rabbits and a normal female rhesus monkey receiving 2.0, 8.3 and 0.1 mg/kg, respectively, of 17 alpha-dihydroequilenin sulfate intragastrically.

Topics: Animals; Chromatography, High Pressure Liquid; Equilin; Female; Macaca mulatta; Male; Ovariectomy; Rabbits; Rats; Reference Standards; Solutions; Spectrometry, Fluorescence

The metabolism of 17 beta-dihydroequilin and 17 beta-dihydroequilin sulfate was investigated after intravenous administration of [3H] 17 beta-dihydroequilin and [3H] 17 beta-dihydroequilin sulfate to postmenopausal women. Urine was collected for 3 days and 46.2 +/- 10.5% and 54.5 +/- 8.7% of the injected dose of [3H] 17 beta-dihydroequilin and [17 beta-3H]dihydroequilin sulfate was excreted in the urine respectively. The estrogens present in urine were extracted and fractionated into unconjugated, sulfate, and glucuronide conjugated forms. With both precursors, the major amount (63-64%) of metabolites were excreted in the urine conjugated with glucuronic acid. From the unconjugated, sulfate, and glucuronide fraction, 17 beta-dihydroequilenin, 17 beta-dihydroequilin, equilenin, and equilin were isolated. The conversions with both precursors were similar and 17 beta-dihydroequilenin was the major metabolite isolated from all three fractions; however, the highest levels of all four metabolites were present in the glucuronide fraction. Along with these identifiable metabolites, a large amount (51-81%) of radioactivity was present in the form of metabolites which are more polar than any of the known ring-B unsaturated estrogens. These appear to be polyhydroxy 17 beta-reduced ring-B unsaturated estrogens which remain to be identified. The in-vivo formation of equilenin and 17 beta-dihydroequilenin indicates the presence of the enzyme 6.8(9) steroid dehydrogenase in humans.

Topics: Equilin; Estrogens, Conjugated (USP); Female; Glucuronates; Humans; Middle Aged; Oxygen Radioisotopes; Postmenopause; Sulfates

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

Our purpose was to determine the effect of Premarin (conjugated estrogens) and three of its component estrogens on uterine weight and plasma cholesterol concentrations in surgically menopausal female rats.. A randomized trial of Premarin and three component estrogens--estrone sulfate, 17 alpha-estradiol sulfate, and 17 alpha-dihydroequilenin sulfate--in female rats after oophorectomy.. High-dose Premarin and high- and middle-dose estrone sulfate significantly increased uterine weight relative to untreated controls (high-dose Premarin, 243.34 +/- 0.15 mg; high-dose estrone sulfate, 376.1 +/- 9.36 mg; middle-dose estrone sulfate, 249.0 +/- 6.34 mg; untreated controls, 124.63 +/- 3.17 mg; for all, p < 0.05). 17 alpha-Dihydroequilenin sulfate had no effect on uterine weight relative to controls. All 17 alpha-dihydroequilenin sulfate doses markedly reduced total plasma cholesterol concentrations versus controls (34.02 +/- 3.44 mg/dl, 32.49 +/- 1.08 mg/dl, and 71.55 +/- 5.16 mg/dl vs 90.44 +/- 1.06 mg/dl; for all, p < 0.02). 17 alpha-Dihydroequilenin sulfate had a more pronounced effect on low- or very-low-density lipoprotein cholesterol than total plasma cholesterol or high-density lipoprotein cholesterol concentrations.. 17 alpha-Dihydroequilenin sulfate reduced total plasma cholesterol concentrations without inducing uterine growth in rats after oophorectomy.

Topics: Animals; Cholesterol; Dose-Response Relationship, Drug; Equilin; Estradiol; Estrogens, Conjugated (USP); Estrone; Female; Organ Size; Rats; Rats, Inbred F344; Uterus

The present investigation was undertaken to compare the binding affinities (Ka) of the ring B unsaturated equine estrogens (equilin [Eq], equilenin [Eqn], 17 beta-dihydroequilin [17 beta-Eq], 17 beta-dihydroequilenin [17 beta-Eqn], 17 alpha-dihydroequilin [17 alpha-Eq], and17 alpha-dihydroequilenin [17 alpha-Eqn]) and the classic estrogens (estrone [E1], 17 beta-estradiol [17 beta-E2], and 17 alpha-estradiol [17 alpha-E2]) for estrogen receptors in human endometrium and rat uterus. In both species, the ring B unsaturated estrogens bind with cytosol and nuclear receptors with high affinity (Ka x 10(9) M-1). The relative binding affinities of these estrogens were measured by determining the amount of unlabeled estrogen required to reduce by 50% the specific binding of [3H]17 beta-Eq to endometrial cytosol receptors. The order of activity found was 17 beta-Eq greater than 17 beta-E2 greater than 17 beta-Eqn greater than E1 greater than Eq greater than 17 alpha-Eq greater than 17 alpha-E2 greater than 17 alpha-Eqn greater than Eqn. Essentially the same order of activity was observed when the apparent affinity constants of these estrogens for human and rat cytosol and nuclear receptors were determined by a competitive (inhibition) binding assay. Sucrose density gradient analysis indicated that these estrogens form protein complexes with cytosol and nuclear preparation that sediment at approximately 8S and 4S, respectively. The affinity constants for 17 beta-Eq were approximately two- to six-fold higher than E2 in both species. In a rat uterotropic assay, all nine estrogens were uterotropic. These data indicate that all ring B unsaturated estrogens present in conjugated equine estrogen preparations are biologically active and they express their biologic effects in the human endometrium by mechanisms similar to those described for the classic estrogens.

Topics: Animals; Binding, Competitive; Cell Nucleus; Centrifugation, Density Gradient; Cytosol; Endometrium; Equilenin; Equilin; Estrogens; Female; Humans; Rats; Rats, Inbred Strains; Receptors, Estrogen; Uterus

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

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

[3H]Equilin [3H-labeled 3-hydroxy-1,3,5(10), 7-estratetraen-17-one] was administered iv to a pregnant mare in the 10th month of gestation. Maternal urine was collected for 3 days, and blood samples were taken 35 min and 3, 6, 12, and 24 h after the injection. The half-life of the disappearance of radioactivity from the blood was approximately 2.5 h. Over 90% of the administered dose was excreted in the first 24 h. The urine was extracted, hydrolyzed, and fractionated. The bulk of the radioactive material (75%) was present in the phenolic sulfate fraction from which radiochemically pure equilin, equilenin [3-hydroxy-1,3,5(10),6,8-estrapentaen-17-one], 17 alpha-dihydroequilin [1,3,5(10), 7-estratetraen-3,17 alpha-diol], 17 beta-dihydroequilin [1,3,5-(10,7-estratetraen-3,17 beta-diol], 17 alpha-dihydroequilenin [1,3,5(10),6,8-estrapentaen-3,17 alpha-diol], and 17 beta-dihydroequilenin [1,3,5(10),6,8-estrapentaen-3,17 beta-diol] were isolated and identified. Except for equilenin, the above-named steroids were also isolated and identified from the glucuronide fraction. Along with these estrogens, the two classical estrogens, estrone and 17 alpha-estradiol, were also isolated, but both of these estrogens were devoid of any radioactivity. These results indicate that 1) the B ring unsaturated estrogens are not metabolized to the B ring saturated estrogens (classical estrogens), 2) all of the B ring unsaturated estrogens isolated and identified from the pregnant mare's urine are metabolites of equilin, 3) the major metabolite of equilin excreted in the urine was equilin sulfate, 4) from the specific activity of the isolated equilin sulfate and the amount of [3H]equilin injected, the secretion rate of equilin was calculated to be 96 mg/24 h, and 5) the major reduced metabolites of equilin are the biologically less active 17 alpha-reduced products.

Topics: 17-Ketosteroids; Animals; Equilin; Estradiol; Estrone; Female; Glucuronates; Horses; Pregnancy; Pregnancy, Animal

Topics: 17-Ketosteroids; Animals; Cross Reactions; Equilin; Estrogens, Conjugated (USP); Female; Haptens; Horses; Humans; Immune Sera; Methods; Radioimmunoassay; Steroids

Topics: Animals; Body Fluids; Contraceptive Agents, Female; Equilin; Estrogens; Female; Horses; Humans; Pregnancy

Topics: Equilin; Humans; Steroids