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At least 19 recordsLinked to original sources
PubMed1977

Determination of placental estriol in urine by gas-liquid chromatography, with equilenin as internal standard.

Placental estriol in urine can be determined by rapid enzymatic hydrolysis, extraction with neutral XAD-2 resin, and quantitation by gas-liquid chromatography, with equilenin as the internal standard. Glucose, methenamine mandelate, hydrochlorothiazide, phenolphthalein, or salicylate do not interfere. Analytical recovery of estriol added to urine was 92-108%. Day-to-day precison (CV), established by 15 repilicate analyses at a mean concentration of 21.3 mg/liter, was 6.6%.

17-Ketosteroids↗
PubMed1976

Human placental 3beta-hydroxysteroid dehydrogenase: delta5-isomerase. Demonstration of an intermediate in the conversion of 3beta-hydroxypregn-5-en-20-one to pregn-4-ene-3,20-dione.

3beta-Hydroxypregn-5-en-20-one (pregnenolone) and NAD+ were incubated with a solubilized preparation of the coupled enzyme 3beta-hydroxysteroid:NAD(P) oxidoreductase-3-ketosteroid delta4,delta5-isomerase (3beta-hydroxysteroid dehydrogenase: delta5-isomerase) from the mitochondrial fraction of human placenta. Unconverted pregnenolone, pregn-4-ene-3,20-dione (rogesterone), and a small but detectable amount of pregn-5-ene-3,20-dione were isolated from the medium by Sephadex LH-20 chromomatography. The identification of pregn-5-ene-3,20-dione, confirmed by mass fragmentography, has provided the first direct evidence for the formation of the hypothetical delta5,3-ketone intermediate in the conversion of pregnenolone to progesterone. When tritium-labeled pregnenolone and [4-14C]pregnenolone were incubated simultaneously the 3H:14C ratio in isolated pregn-5-ene-3,20-dione was 4.6 times greater than in isolated progesterone and pregnenolone, indicating a kinetic isotope effect in the enzymatic isomerization of tritium-labeled pregn-5-ene-3,20-dione. Exposure of the enzyme to two steroids which inhibit the overall enzyme reaction, 2alpha-cyano-17beta-hydroxy-4,4,17alpha-trimethylandrost-5-en-3-one (cyanoketone) and 3-hydroxyestra-1,3,5(10),6,8-pentaen-17-one (equilenin), increased the relative yield of labeled pregn-5-ene-3,20-dione as well as the recovery of radioactivity remaining as unconverted pregnenolone, suggesting that both the dehydrogenase and isomerase activities were inhibited. Exposure of the enzyme to equilenin increased the ratio of isolated pregn-5-ene-3,20-dione radioactivity to progesterone radioactivity as progesterone synthesis was inhibited. Equilenin also diminished the tritium isotope effect on the isomerase reaction. Both findings suggest that it is possible to inhibit the isomerase to a greater extent than the dehydrogenase. In order to measure the rate of progesterone produced by the coupled enzymes, we have modified a radiochemical method which involves precipitation of pregnenolone by digitonin. Digitonin precipitation proved to be effective in separating unconverted pregnenolone from the steroid products of both enzyme reactions, progesterone and pregn-5-ene-3,20-dione. Neither the steroidal inhibitors nor the kinetic isotope effect altered the accuracy of the method for routine measurement of the overall rate of conversion of delta5,3beta-hydroxysteroid to delta4,3-ketosteroid.

Female↗
PubMed1992

Nature of the intermediate in the 3-oxo-delta 5-steroid isomerase reaction.

The role of Tyr-14 of 3-oxo-delta 5-steroid isomerase (KSI) was probed by analysis of the spectra of 3-amino-1,3,5(10)-estratrien-17 beta-ol (4) and equilenin (5) bound to the active site of KSI. The ultraviolet spectrum of 4 bound to KSI is identical to that for 4 in neutral solution. This observation indicates that Tyr-14 does not protonate the amine group of 4 at the active site. By analogy, it is argued that the 3-oxo group of steroid substrates for KSI is not protonated during the reaction. In contrast, the fluorescence excitation spectra of 5 bound to KSI show characteristics of an ionized phenol, even at pH values as low as 3.8. It is concluded that the pKa of equilenin is perturbed from its value in solution of 9 to less than or equal to 3.5 at the active site of KSI. Similarly, the pKa of the intermediate dienol in the KSI reaction should be lowered to less than or equal to 4.5 when it is bound to KSI. Thus, the function of Tyr-14 as an electrophilic catalyst is likely the stabilization of the anion of the dienol by hydrogen bonding rather than by proton transfer.

Catalysis↗
PubMed1975

Mammalian 3-oxosteroid delta4-delta5-isomerase: a membrane-bound enzyme. I. Fluorescence study of the relationship between the enzymatic binding site, phospholipids, water and ions.

The microsomal membranes and the proteolipidic particles obtained by disruption of the microsomes by alkaline-earth ions at molar concentration have been compared by measuring the fluorescence properties of 1-anilino-naphthalene-3-sulfonate and naphthyl-1-phenylamine. The protein lipid arrangement of these two systems appears to be not essentially different. The study of fluorescence polarization of an hydrophobic probe (perylene) in function of Mg2+ concentration suggests a possible mechanism of disruption of the membrane by Mg2+ involving the strong structure-making effect of the ion. The comparison of the fluorescence polarization changes of perylene and equilenine (a competitive inhibitor of the isomerase) with the ionic concentration indicates that there is no direct relation between the bulk lipidic phase and the enzymatic binding site properties. Moreover, the emission of equilenine is completely quenched by I-, in contrast with the napththyl-1-phenylamine and perylene probes, which clearly demonstrates the accessibility of the catalytic site to water molecules and ions.

Anilino Naphthalenesulfonates↗
PubMed1979

Separation and quantitation of esterified estrogens in bulk mixtures and combination drug preparations using high-performance liquid chromatography.

A high-performance liquid chromatographic method for esterified estrogens is described. By using a facile acid hydrolysis extraction procedure for the sample preparation, the compounds are chromatographed as their free phenolic forms. The separation of structurally similar compounds, such as equilenin, equilin, estrone, and estradiol, was achieved with a reversed-phase column and a methanol--water mobile phase. Several samples of bulk mixtures and tablets were assayed; the results compared favorably with those obtained using the USP XIX method. The method was rapid, and the detector response was linear over a wide concentration range. A relative standard deviation of +/- 5% indicates the reliability and accuracy of the proposed method.

Chromatography, High Pressure Liquid↗
PubMed1975

Steroid hormone modulation of 3H-prostaglanding E1 binding to bovine corpus leteum cell membranes.

The specific binding of 3H-prostaglandin E1 (3H-PGE1) to bovine corpus luteum cell membranes was not affected by cholesterol or various progestins at concentrations of up to 9.0x10-minus-6M. At concentrations above 2.5 x 10-minus-6M; estrone, 17beta-estradiol (but not 17alpha-estradiol or 17beta-estradiol glucuronide), estroil, equilin, D-equilenin, 17-ethynyl estradiol, diethylstilbestrol, cortisol, corticosterone, deoxycorticosterone and aldosterone inhibited specific binding of 3H-PGE1. On the other hand, testosterone and dihydrotestosterone (DHT) (but not androstenedione) significantly enhanced 3H-PGE1 binding. These findings permitted the following correlations between steroid structure and modulation of 3H-PGE1 binding: steroids with a free phenolic ring and a 17beta-hydroxyl or 17-keto group or C-21 steroids with a C-20 ketone and a C-21 hydroxy group decrease, whereas C-19 steroids with a C-17 hydroxy group enhance specific binding of 3H-PGE1. PGE receptors are heterogeneous with respect to affinity for 3H-PGE1. The steroids that decreased 3H-PGE1 binding caused a lowering to a complete loss of low affinity PGE receptors. Steroids that increased 3H-PGE1 binding caused appearance of new low affinity PGE receptors. Association rate constants for 3H-PGE1 binding were decreased by 17beta-estradiol (61%) and increased by DHT (59%).

Aldosterone↗
PubMed1975

Oestrogens, LH, PMSG, and prolactin in serum of pregnant mares.

Levels of oestrone, equilin and equilenin (E1), oestradiol (E2) LH, PMSG and prolactin were measured by radioimmunoassay in serum from pregnant mares. Levels of E1 were always greater than those of E2. Both E1 and E2 remained at low levels until Day 80, increased significantly (P less than 0-05) by Day 120 to reach peak levels at Day 210 or 240 and then declined until parturition. Maximum levels of oestrogens observed in this study were 828 +/- 151 pg/ml for E1 and 71 +/- 18 pg/ml for E2 at Days 210 and 240 respectively. Spikes of LH release were observed in early pregnancy in most mares. Levels of PMSG were first detectable at Days 32 or 36 and reached a peak of 67-1 i.u./ml 64 days after the last mating. Levels of LH were very low (often non-detectable) from Day 210 of gestation until parturition. Levels of prolactin were extremely variable and showed no clear fluctuating patterns during pregnancy.

Animals↗
PubMed1992

Supercritical fluid chromatography of selected oestrogens.

The separation of selected oestrogens (oestrone, equilin, alpha-oestradiol, beta-oestradiol and d-equilenin) using capillary supercritical fluid chromatography (SFC) was studied. Three different stationary phases (SB-methyl-100, SB-biphenyl-30 and SB-cyanopropyl-50) were studied for the separation of the compounds. A baseline separation of the oestrogens was achieved on a SB-cyanopropyl-50 column using a carbon dioxide density gradient at an oven temperature of 73 degrees C. Typical analysis time on a 7 m column was 21 min. Retention times of each oestrogen decreased with an increase in either mobile phase density or oven temperature. Accuracy and precision of the SFC method were in the 1-5.5% range. The SFC method was applied to three different dosage forms containing oestrogens.

Chromatography, Liquid↗
PubMed1976

Novel pyrazolo, isoxazolo, and thiazolo steroidal systems and model analogs containing dimethyoxylaryl (or dihydroxylaryl) groups and derivatives. Synthesis, spectral properties, and biological activity.

The total syntheses of a series of vicinal-substituted dimethoxy and dihydroxy heterosteroids of the equilenin type and model analogs are described. A novel class of pyrazolo steroidal N-glucosides has also been synthesized. Compounds prepared were screened in vitro for growth inhibition of different microorganisms. Of these, 1-alpha-d-glucopyranosyl-4,5-dihydro-7-methyoxy-1H-benz[g]indazole tetraacetate (3) was quite active. For example, N-glucoside 13 inhibited the growth of Bacillus subtilis, Pseudomonas fluorescens, Staphylococcus aureus, and KB cells at moderate concentrations.

Bacillus subtilis↗
PubMed1979

Use of borohydride reduction in the separation of estrogen carbonyls.

The chromatographic behavior of the estrogen carbonyls, equilenin, equilin, and estrone, and their respective sodium borohydride reduction products has been studied. The separation of the 17 alpha-hydroxy reduced compounds is superior to the separations achieved for the parent carbonyls using both reversed-phase and normal-phase systems. The reductions appear quantitative by the chromatographic systems used but other work indicates that a small quantity of 17 alpha-hydroxy isomer is produced. The separations developed here have been used in the identification of 9-dehydroestrone, an impurity in estrone, and in the identification of the estrogens in a commercial aqueous suspension of estrogenic substances.

Borohydrides↗
PubMed1975

Formation of steroids by the pregnant mare. V. Metabolism of 14C-isopentenylpyrophosphate and 3H-dehydroisoandrosterone injected into the fetus.

A mixture of 1-14C-isopentenylpyrophosphate and 3H-dehydroisoandrosterone was injected into a horse fetus intramuscularly during laparotomy, after which maternal urine was collected for 4 days. Steroid conjugates in the urine were extracted with Amberlite XAD-2 resin, hydrolysed and separated into phenolic and neutral fractions. From the phenolic fraction estrone, 17alpha-estradiol, equilin and equilenin were isolated. Only estrone and 17alpha-estradiol contained both 3H and 14C, while the ring B unsaturated estrogens contained only 14C. From the neutral fraction 14C-labeled 3beta-hydroxy-5alpha-pregnan-20-one, 5alpha-pregnane-3beta,20beta-diol and 5alpha-pregnan-3beta, 20alpha-diol were isolated. These results demonstrate that the route of biosynthesis of both the ring B saturated and unsaturated estrogens is the same up to the stage of isopentenylpyrophosphate. Thus, the bifurcation in the classical pathway of steroid biosynthesis reported previously by us is occurring at a point after the formation of isopentenylpyrophosphate and prior to the formation of squalene.

17-Ketosteroids↗
PubMed1979

Improved liquid-chromatographic method for determination of serum cortisol.

We describe a specific and precise method for measuring concentrations of cortisol in serum or plasma by liquid chromatography. Cortisol, together with an internal standard, equilenin, is extracted from 1 mL of serum or plasma and analyzed isocratically on a reversed-phase column with a mobile phase of acetonitrile/phosphate buffer (30/70, by vol.), at a flow rate of 2.0 mL/min. The eluted cortisol is detected by its absorption at 254 nm and quantitated by peak height measurements. Each analysis requires no longer than 15 min at the optimum column temperature of 50 degrees C. The lower limit of detection for cortisol is about 2 ng/sample for a standard solution; sensitivity is routinely 5 micrograms/L of serum. Analytical recoveries exceeded 95%, with good day-to-day precision (coefficients of variation between 4 and 7%). Of more than 50 drugs and steroids tested for possible interference, only the steroids cortisone, prednisone, and prednisolone may interfere with the analysis of cortisol.

Chromatography, Liquid↗
PubMed1977

Determination of aqueous solubility and pKa values of estrogens.

Reported estrone pKa and solubility data show wide variation. Improved experimental procedures were designed and used to obtain reproducible results. The pKa values for several estrogens and related compounds also were determined to assess the effects of structural differences on ionization. No evidence was obtained for long-range D to A ring electronic transmission affecting pKa. Significant differences in pKa values resulted only when conjugated unsaturation was added into the B ring of estrone or estradiol. The aqueous solubilities of estrone and 17alpha-estradiol were 0.8 and 3.9 microgram/ml, respectively, at 25degrees.

Chemical Phenomena↗
PubMed1978

Fluorescent probes in model membranes. II. Monolayer studies of 2,2'-(vinylenedi-p-phenylene)bisbenzoxazole, d-3-aminodesoxyequilenin and N-octadecylnaphthyl-2-amino-6-sulfonic acid with host-lipid tetradecanoic acid.

Film studies at the air-water interface have been carried out for pure films of 2,2'-(vinylenedi-p-phenylene)bisbenzoxazole (VPBO), d-3-aminodesoxyequilenin (EQ) and N-octadecylnapthyl-2-amino-6-sulfonic acid (ONS), and for mixed films with tetradecanoic acid for the first two fluorescent probes. Pure film isotherms indicate highly rigid non-monomolecular films for both VPBO and EQ, revealing the presence of strong intermolecular forces. In mixed films with tetradecanoic acid VPBO rapidly segregates with resultant film loss over a wide concentration range. EQ, however, can be stabilized by the host-lipid at low concentrations. This, coupled with an ability to only slightly affect the host-lipid liquid-condensed/liquid-expanded phase change, suggests that EQ can be regarded as "non-perturbing" and should be retained in condensed lipid phases. ONS, because of its unusual polar headgroup, resembled hexadecanoic acid more than octadecanoic acid. While difficulties in spreading ONS precluded the study of mixed films, the indications are that it would be a satisfactory expanded lipid state probe if mixing can be brought about.

17-Ketosteroids↗
PubMed1975

The nuclear dehydrogenation of steroids by intestinal bacteria.

We have postulated that bacteria able to dehydrogenate the bile-acid nucleus are important in the aetiology of cancer of the colon. In this paper we report on screening for the ability to carry out two such reactions. The relevant enzymes are produced by a high proportion of strains of Clostridium paraputrificum, C. tertium and C. indolis, and by small numbers of strains in other clostridial species, but not by organisms of the other genera tested. Strains able to dehydrogenate the bile-acid nucleus represent a high proportion of the lecithinase-negative clostridia isolated from faeces of people living in Britain but a low proportion of those from people living in Uganda or Hong Kong.

Androstenedione↗