In vivo metabolism of [3H]equilin in the pregnant mare.

[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.

Biosynthesis of estrogens in pregnant and non-pregnant guinea pigs.

Guinea pig ovaries obtained from pregnant and non-pregnant animals, incubated in the presence of a NADPH-generating system, converted [3H]-dehydroepiandrosterone and [14C]testosterone to estradiol. Under the same conditions, no radioactive estrogen could be identified in adrenal and placental incubations. The rate of formation of estradiol from dehydroepiandrosterone was linear during the 30-min incubation. The initial rates of formation for estradiol were 415 X 10(-13) and 10.4 X 10(-13) mol/min/g tissue for the ovaries from pregnant and non-pregnant guinea pigs, respectively. The formation of estrone was shown only in the pregnant animal, and the initial rate of formation was 36.0 X 10(-13) mol/min/g tissue. These in vitro studies suggest that the major site of estrogen formation in both pregnant and non-pregnant guinea pigs is the ovary.

Formation of steroids by the pregnant mare. VI. Metabolism of [14C]farnesyl pyrophosphate and [3H]dehydroepiandrosterone injected into the fetus.

A mixture of [4,8,12-14C]farnesyl pyrophosphate and [3H]dehydroepiandrosterone was injected into a horse fetus im during laparotomy, after which maternal urine was collected for 6 days. Steroid conjugates in the urine were extracted with Amberlite XAD-2 resin, hydrolyzed, and separated into phenolic and neutral fractions. Estrone, 17 alpha-estradiol, equilin [3-hydroxy-1,3,5(10),7-estratetraen-17-one], and 17 alpha-dihydroequilin [1,3,4(10),7-estratetraene-3,17 alpha-diol] were isolated from the phenolic fraction and their radiochemical purities were established. Only estrone and 17 alpha-estradiol contained both 3H and 14C, while the B ring unsaturated estrogens, equilin and 17 alpha-dihydroequilin, contained only 14C. From the neutral fraction, 14C-labeled 3 beta-hydroxy-5 alpha-pregnan-20-one, 5 alpha-pregnane-3 beta-20 beta-diol, and 5 alpha-pregnane-3 beta, 20 alpha-diol were isolated. These results together with our previous findings demonstrate that the route of biosynthesis of both the ring B saturated and unsaturated estrogens is the same up to the stage of farnesyl pyrophosphate. Thus, the bifurcation in the classical pathway of steroid biosynthesis is occurring at a point after the formation of farnesyl pyrophosphate and before the formation of squalene and cholesterol.

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.

Metabolic clearance rate of equilin sulfate and its conversion to plasma equilin, conjugated and unconjugated equilenin, 17 beta-dihydroequilin, and 17 beta-dihydroequilenin in normal postmenopausal women and men under steady state conditions.

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.

Pharmacokinetics of 17 beta-dihydroequilin sulfate and 17 beta-dihydroequilin in normal postmenopausal women.

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.

Pharmacokinetics of equilin and equilin sulfate in normal postmenopausal women and men.

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.

Transport of equine estrogens: binding of conjugated and unconjugated equine estrogens with human serum proteins.

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.

Metabolism of [3H]equilin-[35S]sulfate and [3H]equilin sulfate after oral and intravenous administration in normal postmenopausal women and men.

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.

Radioimmunoassay of plasma equilin and estrone in postmenopausal women after the administration of premarin.

A RIA for the measurement of plasma equilin (3-hydroxy-1,3,5-(10)7-estratetraen-17-one) and estrone in postmenopausal women and other estrogen-deficient women on exogenous equine estrogen replacement therapy is described. Antiserum against estriol-3,16,17-trihemisuccinate-HSA and high specific activity [2,4-3H]equilin and [6,7-3H]estrone were used in the assay procedure. Specificity of the assay was achieved by separation of equilin from estrone by chromatography on micro-Celite partition columns using silver nitrate as the stationary phase. The sensitivity of the standard curves for both equilin and estrone was 10-20 pg, and the smallest amount of estrone and equilin that could be measured accurately in the plasma was 250 pg/ml for both steroids. The coefficient of variation for both steroids ranged from 1-8%, over a range of 250-5000 pg/ml plasma. The interassay coefficients of variation for equilin and estrone were 4.6% and 2.4%, respectively. After the administration of 10 mg Premarin iv, maximum concentrations of 4 and 11.2 ng/ml for equilin and estrone, respectively, were obtained after 20 min. Thereafter, both steroids disappeared from the plasma gradually. When 10 mg Premarin were administered orally, equilin and estrone appeared in the blood gradually, and maximum levels of 560 and 1400 pg/ml were reached after 3 and 5 h for equilin and estrone, respectively. Equilin gradually disappeared, and by 24 h, only small amounts (125 pg/ml) were detectable. The levels of estrone declined more rapidly, though it was still detectable after 24 h. These preliminary results indicate that equilin sulfate is converted to circulating unconjugated equilin in a manner similar to the conversion of circulating estrone sulfate to estrone.

Loss of IGF-II imprinting in endometrial tumors: overexpression in carcinosarcoma.

The genomic imprinting of the maternal allele defines the monoallelic expression of the IGF-II gene in most human tissues. The loss of imprinting (LOI) leading to biallelic overexpression of IGF-II has been reported in several human malignancies, including uterine leiomyosarcoma. To ascertain if LOI occurs in endometrial malignancies, the allelic expression of the IGF-II gene was examined in samples of normal human endometrium (n=22) and endometrial tumors (n=12) by assessing the ApaI polymorphism in cDNA segments amplified by RT-PCR. The biallelic overexpression of IGF-II mRNA, involving activation of all four (P1-P4) promoters, was detected in one normal endometrium and in one endometrial carcinosarcoma. Low level biallelic expression of IGF-II was also detected in two samples of hormone-unresponsive/Type II endometrial carcinomas. The level of IGF-I mRNA in these four samples was low. The IGF-IR mRNA was overexpressed in all endometrial cancers including the carcinosarcoma sample, but not in normal endometrium. These data suggest that LOI associated with overexpression of IGF-II and concomitant overexpression of IGF-IR may play a role in the rare carcinosarcoma of the endometrium.

Applicability of the product isolation and the radiometric aromatase assays for the measurement of low levels of aromatase: lack of aromatase activity in the human endometrium.

The purpose of this investigation was to assess the applicability of two well established procedures: (i) the product isolation assay and (ii) the radiometric 3H2O assay for the determination of very low levels of aromatase activity. The methods were validated and used to assess the capacity of normal and neoplastic human endometrium to synthesize oestrogens from androgens. Using the product isolation assay, various specimens (n = 27) of normal and neoplastic endometrium were incubated with [1,2,6,7-3H]testosterone either by a standard incubation procedure or by a superfusion technique. Following the incubation, carrier oestrone and oestradiol or [14C]oestrone and [14C]oestradiol were added, and the oestrogens were isolated and purified by paper chromatography and high-performance liquid chromatography. The radiochemical purity of oestrone and oestradiol was checked by the isotope dilution technique. In all samples, the 3H associated with oestrone and oestradiol failed to recrystallize as oestrone and oestradiol. No radioactivity was detectable in the oestrone and oestradiol crystals after acetylation. Similarly, 16 endometrial samples were tested for aromatase activity by the 3H2O release assay using [1 beta-3H]androstenedione as substrate. The results indicate that 3H2O was indeed released during these incubations, but this activity could not be inhibited by the aromatase inhibitor 4-hydroxyandrostenedione, by excess substrate or by heat inactivation of the tissue. Furthermore, the release of 3H2O from [1 beta-3H]androstenedione under the incubation conditions used (Dulbecco's modified Eagle's medium or RPMI-1640 containing fetal bovine serum and NADPH) also occurred in the absence of any tissue. This activity was not inhibited by 4-hydroxyandrostenedione nor by excess substrate. The results demonstrate that the human endometrium does not contain detectable levels of aromatase activity and that the radiometric assay can give rise to false-positive results if used for detection of very low levels of aromatase activity.

Effect of tamoxifen treatment on the endometrial expression of human insulin-like growth factors and their receptor mRNAs.

The effect of tamoxifen (Tam) treatment on the endometrial expression of IGF-I and II/IGF-IR and IIR mRNAs was assessed by measuring the levels of expression of these mRNAs in the endometrial samples of Tam-treated postmenopausal breast cancer patients by RT-PCR assays. The levels of these mRNAs were compared with those in normal endometrium and in Type I and Type II endometrial carcinomas (EC). The promoter-specific transcript profiles of IGF-I and II were also elucidated. The levels of IGF/IGF-R mRNAs in the endometrium from Tam-treated patients were found to be comparable with the high levels of these mRNAs observed in the proliferative and early secretory phase endometrium and in the samples of Type I EC. These results indicate that Tam acts as an estrogen agonist in inducing the endometrial expression of these genes. Tam stimulated transcription from the multiple promoters of IGF-I and II, with the exception of IGF-II P2 promoter, in which case the transcription across exon 4 appeared to be inhibited. The profiles of IGF-I and II transcripts of endometrium from Tam-treated patients were similar to Type I EC but differed considerably from those of Type II EC. These results suggest that the Tam-associated ECs are likely to be similar to type I EC and will therefore have a more favorable prognosis.

Discordant expression of insulin-like growth factors and their receptor messenger ribonucleic acids in endometrial carcinomas relative to normal endometrium.

The inappropriate expressions of insulin-like growth factors (IGF-I and II) and IGF-I receptor (IGF-IR) are implicated in the malignant growth of many cancers. To determine changes, if any, in the levels of expression of IGFs and IGF receptor genes in neoplastic endometrium, relative to normal endometrium, the mRNA levels of IGF-I and II and of IGF-IR and IIR were measured in samples of endometrial carcinomas (EC) and normal endometrium, through all phases of the menstrual cycle, by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assays. In normal endometrium, the mRNA levels of IGF-I were elevated in the proliferative and early secretory phases. The IGF-II mRNAs were relatively high in the proliferative phase, but unaltered through early and late secretory phases. Significantly elevated levels of IGF-II transcripts were observed during the menstrual phase, suggesting a possible role of IGF-II in endometrial regeneration. A positive correlation between the levels of IGF-I and IGF-IR mRNAs, apparent in the samples of normal endometrium, was not observed in endometrial carcinomas. The IGF-IR and IIR mRNA levels were elevated in endometrial carcinoma samples. On the other hand, the IGF-I and II mRNA levels were conspicuously low in many carcinoma samples, which were not associated with hyperplasia (type II EC), but relatively elevated in two other carcinoma samples, associated with adenomatous hyperplasia (type I EC). These results albeit with few samples suggest the possibility that the overexpressed receptor, IGF-IR, could be activated differently in two types of endometrial carcinomas, namely ligand-dependently in type I ECs and ligand-independently in type II ECs.

Endometrial transcripts of human insulin-like growth factors arise by differential promoter usage.

By the application of RT-PCR, we have demonstrated that in the human endometrium mRNAs for insulin-like growth factors, IGF-I and II, and their receptors are expressed not only in the intact endometrium, but also in the freshly isolated stromal and epithelial cells. The expression of multiple transcript forms of the IGF-I and II at various phases of the menstrual cycle, occurs by differential use of all four IGF-I transcriptional start sites, and two of the four known promoter sites of the IGF-II gene. The complete spectrum of transcripts is displayed by the proliferative phase and the menstrual phase endometrium. During the secretory phase, the exon 1 upstream start site of the IGF-I gene and the P2 promoter of the IGF-II gene are not used. Irrespective of the phase of the menstrual cycle, the stromal cells always display the same transcriptional patterns of both growth factor genes as those of the intact endometrium. In contrast, the epithelial cells do not express IGF-I transcript originating from the exon 2 upstream initiation site. These results indicate that the expressions of the IGF-I and II genes in the intact endometrium and stromal and epithelial cells are modulated at the transcriptional level during the menstrual cycle by differential usage of promoters and start sites.

Comparison of the antioxidant effects of equine estrogens, red wine components, vitamin E, and probucol on low-density lipoprotein oxidation in postmenopausal women.

OBJECTIVE: Oxidized low-density lipoprotein (LDL) seems to play an important role in the etiology of atherosclerosis. To further study this, we performed two studies: (1) we determined the ability of 10 estrogen components of the drug, conjugated equine estrogen (CEE), trans-resveratrol (t-resveratrol) and quercetin (red wine components), trolox (vitamin E analog), and probucol (a serum cholesterol-lowering drug) to delay or prevent the oxidation of plasma LDL isolated from untreated postmenopausal women, and (2) we assessed the effect of long-term (>1 year) estrogen replacement therapy and hormone replacement therapy on LDL oxidation by ex vivo methods. DESIGN: For the in vivo study, three groups of postmenopausal women were selected based on whether they were on long-term CEE therapy (group A: 0.625 mg CEE; n = 21), on combination CEE plus progestogen therapy (group B: 0.625 mg CEE + 5.0 mg medroxyprogesterone acetate, 10 days; n = 20), or not on any hormone therapy (group C; n = 37). For the in vitro study, only LDL samples obtained from group C were used. The kinetics of LDL oxidation were measured by continuously monitoring the formation of conjugated dienes followed by determination of the lag time. RESULTS: All compounds tested protected the LDL from oxidative damage. The relative antioxidant potency of estrogen components was generally greater than that of the other compounds. The minimum dose (nmoles) required to double the lag time from the control lag time of 57 +/- 2 min was 0.47 for 17beta-dihydroequilenin, 17alpha-dihydroequilenin, Delta 8 -estrone; 0.6 to 0.7 for Delta 8 -17beta-estradiol, equilenin, and quercetin; 0.9 for 17beta-dihydroequilin and 17alpha-dihydroequilin; 1.3 for equilin, estrone, 17beta-estradiol, 17alpha-estradiol; 1.4 for trolox; 1.9 for probucol; and 3.0 for t-resveratrol. The data from the in vivo study indicate that after long-term estrogen replacement therapy (group A) and hormone replacement therapy (group B), the LDL was significantly ( p < 0.01) protected (higher lag time) against oxidation compared with the control (group C). There was no difference between groups A and B. CONCLUSIONS: The oxidation of LDL isolated from postmenopausal women is inhibited differentially by various estrogens and other antioxidants. The unique ring B unsaturated estrogen components of CEE were the most potent, and t-resveratrol, the red wine component, was the least potent. Long-term CEE or CEE + medroxyprogesterone acetate administration to postmenopausal women protects the LDL against oxidation to the same extent. These combined data support the hypothesis that some of the cardioprotective benefits associated with CEE therapy and perhaps red wine consumption may be due to the ability of their components to protect LDL against oxidative modifications.

Metabolism of [3H]equilin in normal and malignant human endometrium and in endometrial adenocarcinoma transplanted into nude mice.

One of the main components of conjugated equine estrogens is equilin sulfate and this estrogen in postmenopausal women is metabolized to 17 beta-dihydroequilin, 17 beta-dihydroequilenin and equilenin. To investigate the possibility that some of these estrogens may be formed directly in the target tissues, we studied the in vitro metabolism of [3H]equilin in various types of normal and malignant human endometrium, including adenocarcinoma grown in athymic nude mice. The results indicate that normal and neoplastic human endometrium can form the above three metabolites. The highest level of 17 beta-reduced products were isolated from the normal secretory endometrium. Equilenin was the most abundant metabolite isolated from both the normal and malignant endometrium. The formation of [3H]equilenin indicates the presence of a 6,8(9) steroid dehydrogenase-isomerase in the human endometrium. The formation of 17 beta-dihydroequilin in the endometrium may be of importance as this estrogen is 8 times more potent as a uterotrophic agent than equilin and estrone.

Pharmacokinetics and pharmacodynamics of a novel estrogen delta8-estrone in postmenopausal women and men.

Recently a tenth equine estrogen, identified as the sulfate ester of delta8-estrone has been reported to be present in Premarin (a conjugated equine estrogen preparation), and because of its unique ring B unsaturated structure (conjugated double bond in the B ring), we have, in the present study, determined its pharmacokinetics in postmenopausal women and men, its interaction with uterine estrogen receptors and its uterotropic activity. After the administration of [14C]delta8-estrone, blood was drawn at various time intervals, and the plasma fractionated into the unconjugated sulfate and glucuronide fractions. The disappearance of radioactivity as delta8-estrone from plasma can be described as a function of two exponentials. The half-lives of the first and second components were 5+/-0.2 and 40.4 min, respectively. The mean metabolic clearance rate calculated (MCR), was 1711+/-252 l/d m2. From the unconjugated fraction, delta8-17beta-estradiol was also isolated and identified. From the sulfate conjugated fraction, delta8-estrone sulfate and delta8-17beta-estradiol sulfate were isolated in almost equal amounts. No other metabolites of delta8-estrone was detectable in the plasma. Both delta8-estrone and delta8-17beta-estradiol bind with human endometrial and rat uterine estrogen receptors with high affinity. The binding affinities of delta8-17beta-estradiol for human endometrial and rat uterine cytoplasmic receptors were 4 and 25 times higher than those of the parent estrogen delta8-estrone, respectively. Administration of delta8-estrone and delta8-17beta-estradiol (2 microg/100 g body weight) to immature rats significantly (P< 0.05) increased the uterine weight compared to the controls. These data demonstrate that delta8-estrone has estrogenic activity, and that it is further metabolized in man to a single more potent estrogen, delta8-17beta-estradiol. The extent of this activation by 17beta-reduction appears to be greater than that observed with other estrogens. Both estrogens circulate as sulfate conjugates and are very slowly eliminated from the circulation. These data further suggest that delta8-estrone and its major metabolite delta8-17beta-estradiol can contribute to the overall in vivo biological effects of Premarin.

Role of serum androgens and sex hormone binding globulin capacity in the evaluation of hirsutism in women.

Serum samples from 23 hirsute women and 23 non-hirsute women matched for age and deviation from ideal weight were analyzed for total testosterone (T), percent free testosterone (% FT) and sex hormone binding globulin capacity (SHBG). The % FT was assayed by 2 methods, using diluted serum in equilibrium dialysis (EQD) and using undiluted serum in centrifugal ultra-filtration (UF). SHBG was measured by a DEAE cellulose filter assay and T by radioimmunoassay. The two methods for determining % FT correlated well. There was considerable overlap between the hirsute and control groups for all of the measured parameters. The discrimination between the 2 groups provided by the indirect estimate of free testosterone obtained from the ratio of T to SHBG was at least as good as that provided by the free testosterone derived from ultrafiltration or dialysis.

Hyaluronidase activity in human semen: correlation with fertilization in vitro.

OBJECTIVES: To characterize the relationship between hyaluronidase activity and the currently used methods of assessing sperm function and to determine whether the measurement of hyaluronidase activity can provide a reliable index of sperm fertilizing capacity in man. DESIGN: Nonrandomized prospective study. SETTING: Tertiary referral IVF and andrology clinics affiliated with the University of Toronto. SUBJECTS: Four hundred eight samples were collected, 248 from men undergoing investigation in andrology and fertility clinics and 160 from men participating in an IVF program. INTERVENTIONS: Semen samples were treated with NP-40 in buffer to extract hyaluronidase and applied to a circular well cut into a petri dish containing a mixture of hyaluronic acid and agar. Enzyme activity was assessed by measuring the area of substrate hydrolysis. RESULTS: Significant positive correlations were found between hyaluronidase activity and sperm concentration, motility, and the percentage of sperm with normal morphology in the studied samples. In the IVF samples, hyaluronidase activity was found to be related significantly to the fertilization rate. Moreover, we were able to establish values of hyaluronidase below which no fertilization occurred and above which fertilization of at least one oocyte was achieved. CONCLUSION: These results suggest that measurement of hyaluronidase activity may provide a useful method for assessing the integrity of the acrosomal enzyme system, providing a simple and reliable predictor of the fertilizing potential of human sperm.