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Serum ethinyl estradiol (EE2), sex hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG) concentrations were studied in healthy young women randomly allocated to one of two low-dose combination oral contraceptives containing 30 micrograms EE2 and either 75 micrograms gestodene (F) or 150 micrograms desogestrel (M) per unit. There was either no (formerly non-pill users) or one (pill users) wash-out cycle before the study started with a pill-free pretreatment cycle in which the hormone status and basal SHBG and CBG levels were measured. Treatment was for three months. During treatment cycles 1 and 3, there were three test days each. Seven serum samples were obtained up to four hours and one sample 24 hours after intake of the first, tenth and the last (21st) pill. Additional samples were taken prior to morning ingestion of pills 5 and 15. For each individual and each test day, a representative serum pool has been constructed for SHBG and CBG analysis. EE2 concentrations were analyzed in all individual samples by means of a specific and sensitive RIA using anti-EE2-6 beta-CMO-BSA antiserum. Area under the curves (AUC) up to 4 and 24 hours, Cmax and tmax were evaluated and compared between the two treatment groups (n = 40 for F, n = 43 for M). SHBG and CBG concentrations were measured using commercially available immunoassay kits. Groups were large enough to detect a difference in group means of 75% of one standard deviation (alpha = 0.05, 1-beta = 0.9) of target variables, which is equivalent to 28 pg EE2/ml for Cmax, 69 pg.h.ml-1 for AUCEE2 0-4h, 257 pg.h.ml-1 for AUCEE2 0-24h, 39 nmol/l SHBG and 13.4 micrograms CBG/ml. Results clearly demonstrate that there were no differences between the two treatment groups in any of the target variables at any of the six test days distributed over a three-month period. Mean SHBG and CBG pretreatment levels of about 70 nmol/l and 37 micrograms/ml, respectively, increased to about 210 nmol/l and 88 micrograms/ml during the first treatment cycle and to about 230 nmol/l and 93 micrograms/ml during the third treatment cycle. Whereas the time of maximum EE2 serum levels did not differ significantly between test days, Cmax, AUCEE2 0-4h and AUCEE2 0-24h values increased by 30-35% or 40-50%, respectively, when test days 10 and 21 were compared to test day 1. Similar results were found for the third treatment cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

The concept of a weighted pool for estimating the area under the curve (AUC) is presented and set in relationship to the trapezoidal rule. An example from a pharmacokinetic study on ethinyl estradiol is used to demonstrate the use of variance component analysis for relating the intraindividual variance of the AUC, trapezoidal rule and weighted pool to the variance of the determination process. Depending on the sampling times, the theoretical variance of the weighted pool is greater than the theoretical variance of the trapezoidal rule. In the example presented, it was shown that this difference is of no importance in relation to the interindividual variance of the AUC, which dominates the total variance. In the example study, routine quality control samples were also determined in each assay, which allowed independent confirmation of the discussed results on the intraindividual variance of the AUCs.

pS2 is a major estradiol-inducible gene in MCF-7 breast cancer cells. In this study we tested the effects of tamoxifen and ZK 119010, a novel nonsteroidal antiestrogen, on pS2 gene expression with or without a pretreatment of cells with estradiol (10(-11), 10(-8) M). Estradiol increased pS2 expression in MCF-7 cells approximately 12-fold. Tamoxifen (10(-6) M) reduced estradiol-induced pS2 expression to 78% of the stimulated level, while ZK 119010 was 50% effective. Given alone, either of the two antiestrogens in the above concentrations evoked a pS2 gene expression in MCF-7 cells significantly above background levels. From these data we conclude that the antiestrogens tamoxifen and ZK 119010 possess both antagonistic and agonistic potencies in MCF-7 cells. However, the antiestrogenic potency of ZK 119010 seems to be higher than that of tamoxifen.

The pharmacokinetics of gestodene (GEST) and ethinylestradiol (EE2) were determined in 14 healthy women (age 18 to 32 years) during a treatment period of three months with a new tri-step combination oral contraceptive (Milvane). Prior to this treatment period, the same women received a single administration of a coated tablet containing 0.1 mg GEST together with 0.03 mg EE2. There was a wash-out phase of one week between both treatments. Following single dose administration, a mean terminal half-life of 18 h was observed for GEST. The total clearance was 0.9 ml x min-1 x kg-1 and the volume of distribution was 84 l. During a treatment cycle, GEST levels in the serum accumulated by a factor of 8 as compared to single dose administration. Steady-state drug levels were reached during the second half of each cycle. As compared to single dose administration, the following changes were observed for GEST at the end of treatment cycles one and three: prolonged terminal half-life (20 to 22 h), reduced total (0.16 ml x min-1 x kg-1) and free clearance (ca. 27 ml x min-1 x kg-1), reduced volume of distribution (ca. 18 l). A concomitant EE2-induced increase in the SHBG concentrations by a factor of three as compared to pretreatment values was observed during a treatment cycle and appeared to be mainly responsible for the changes in the pharmacokinetics of GEST. Marked changes were also seen for the serum protein binding of GEST. After single dose administration, the free fraction of GEST was 1.3% and the fractions bound to SHBG and albumin were 69.4% and 29.3%, respectively. At the end of cycle one, the free fraction was only 0.6% and the fractions bound to SHBG and albumin were 81.4% and 18.0%, respectively. There was no difference in corresponding pharmacokinetic parameters and in the serum protein binding of GEST at the end of cycles one and three. On the last day of treatment cycles one and three, the AUC(0-4h) values of EE2 were 299.2 and 278.1 pg x ml-1 x h, respectively, which corresponds to an about 30% increase as compared to single dose administration, where an AUC(0-4h) value of 216.1 pg x ml-1 x h was found. Total and free testosterone concentrations decreased during treatment cycles one and three by about 36% and 60%, respectively, compared with the corresponding values measured prior to treatment. The fraction of unbound testosterone thus decreased from 0.5% to 0.3% during treatment.In Germany, researchers examined blood samples from 14 healthy young women, who first took a single oral dose of 0.1 mg gestodene and .03 mg ethinyl estradiol, then Milvane, a new tri-step combination oral contraceptive, for 3 months, to learn in detail the pharmacokinetics of gestodene. A 1-week wash-out phase occurred between the 2 treatments. The mean terminal half-life for gestodene after receiving the single dose was 18 hours. It volume of distribution stood at 84. Total clearance was 0.9 ml x min-1 x kg-1. Gestodene serum levels accumulated by a factor of 8 when compared to levels after single-dose administration. Drug levels reached a steady-state by the second part of each treatment cycle. At the end of treatment cycles 1 and 3, in comparison with single-dose administration, gestodene had a prolonged terminal half-life (20-22 h), reduced total and free clearance (0.16 ml x min-1 x kg-1 and ca. 27 ml x min-1 x kg-1, respectively), and reduced volume of distribution (ca. 18 1). When comparing single-dose administration and 3-month treatment levels, ethinyl estradiol increased sex hormone binding globulin (SHBG) levels by a factor of 3, apparently causing the changes in the pharmacokinetics of gestodene. Further, the free fraction of gestodene fell from 1.3 to 6%, and the fractions bound to SHBG increased from 69.4 to 81.4% while the fractions bound to albumin fell from 29.3 to 18%. The ethinyl estradiol values of the area under the curve stood at 299.2 pg x ml-1 x h at 0 hours of the last day of treatment cycle 1 and 3. At 4 hours, it was 278.1 pg x ml-1 s h. Between pretreatment and treatment cycles 1 and 3, total and free testosterone levels fell by almost 36% and 60%, respectively, resulting in a decrease of the fraction of unbound testosterone from 0.5 to .03% during treatment. These findings showed that pharmacokinetic parameters did not differ between cycles 1 and 3, indicating that equilibrium had been already reached after one cycle and that prolonged treatment should not result in any other changes.

The serum concentrations of gestodene (CAS 60282-87-3) as well as the binding of this progestin to serum proteins were studied in 40 women who took a low-dose oral contraceptive (Femovan, Femodene) containing 30 micrograms ethinyl estradiol (CAS 57-63-6) and 75 micrograms gestodene for 3 months. On days 1, 10, and 21 of the first and the third treatment cycle, respectively, 7 blood samples were drawn before and up to 4 h after pill intake; additional samples were taken prior to morning ingestion of pill on days 2, 5, 11, 15 and 22 of these cycles. Gestodene levels were measured by means of a specific radioimmunoassay and were evaluated for Cmax, tmax, and AUC up to 4 and 24 h. Independent of the test day and the treatment cycle studied, mean maximum gestodene serum levels were found about 0.8 to 0.9 h after pill intake. During the first treatment cycle, mean values of Cmax, AUC(0-4h), and AUC(0-24h) amounted to 4.3 ng.ml-1, 9.3 ng.ml-1.h, and 27.3 ng.ml-1.h on test day 1; these values increased by 250-400% and by 300-500%, respectively, when days 10 and 21 were compared to day 1. On day 1 of the third treatment cycle, these pharmacokinetic parameters were higher by almost a factor of two as compared to the corresponding data obtained on the beginning of the first cycle whereas the increase of these values between day 1 and the subsequent test days (200-300%) was slightly lower in cycle 3 as compared to cycle 1.(ABSTRACT TRUNCATED AT 250 WORDS)In Germany, health workers drew serum samples from 40 healthy young women who used a low dose oral contraceptive (OC) (Femovan, Femodene) with 30 mcg ethinyl estradiol and 75 mcg gestodene for 3 treatment cycles to measure gestodene levels, the free fraction of gestodene, and its distribution over the binding proteins in serum pools from all women. Mean maximum gestodene levels occurred .8 to .9 hours after taking the pill. The mean maximum gestodene level on test day 1 was 4.3 ng ml-1 during the first treatment cycle. This figure increased significantly to 7.4 ng ml-1 during the third cycle. Both day 1 levels were significantly lower than the levels obtained on day 10 (10.4 and 13.1 ng ml-1, respectively) and day 21 (12.1 and 13.4 ng ml-1, respectively). The area under the curve zero to 4 hours after pill intake was 9.3 ng ml-1. These figures rose 250-400% and 300-500%, respectively, on days 10 and 21. During the third treatment cycle, figures for days 10 and 21 were somewhat lower than those during the first cycle. 78% of total gestodene was bound to sex hormone binding globulin (SHBG) and 21% was bound to albumin on day 1 of the first treatment cycle. Just 1% of gestodene continued to be free. ON day 21, the fraction of gestodene bound to SHBG rose to 87%, that bound to albumin was 13%, and 0.5% remained free. Gestodene was not redistributed over its binding proteins during the third treatment cycle. These results indicated that pharmacokinetic accumulation and the increase in serum SHBG concentration and binding capacity for gestodene allows researchers to understand changes in gestodene levels during longterm treatment with a low dose OC containing gestodene.

Studies on tenascin expression in hormonally dependent growing tissues of breast and endometrium suggested that its expression parallels the progression of normal or malignant proliferative alteration of the tissue. With the study presented here we addressed the question of whether antiprogestin-induced terminal differentiation down-regulates tenascin expression. By comparative immunolocalization of tenascin in sections of untreated 7,12-dimethylbenz[alpha]anthracene-induced tumors, tumors grown in ovariectomized animals, tamoxifen-treated tumors, and antiprogestin-treated tumors, we obtained the following results. (a) The entire extracellular space of the stromal mesenchyme was filled by tenascin immunoreactivity in cases of untreated control tumors. (b) Both ovariectomy and antiestrogen treatment with tamoxifen did not affect the overall staining pattern and resulted in a slight increase of the arbitrarily judged staining intensity. (c) Within antiprogestin-treated tumors tenascin-like immunoreactivity predominantly was restricted to fiber-like, collagenous connective tissue structures, which appeared in the stromal compartment as a result of the antiprogestin treatment. In large areas of the tumor composed of apparently secretory active tumor cells we failed to immunolocalize tenascin. Our results provide further evidence that expression of tenascin reflects both benign and malignant proliferative alterations of the tissue, whereas its down-regulation is correlated to differentiation of the tissue. Additionally, evidence is provided that the mechanism of tumor growth inhibition by antiprogestins indeed is induction of terminal differentiation of tumor cells.

A 65-year old patient, suspected to be suffering from an androgen producing ovarian tumour, was treated preoperatively with the GnRH agonist triptorelin (500 micrograms/day s.c.) for 7 days. After an initial rise, gonadotrophin levels were suppressed under this treatment. The elevated serum testosterone concentrations were reduced by approx. 50% by the triptorelin injections. After the extirpation of the tumour (histologically a Leydig cell tumour of the ovary without signs of malignancy), primary cell cultures which secreted testosterone and androstenedione were prepared. Coincubation of the tumour cells with the GnRH agonist triptorelin had no effect on their androgen secretion. Treatment of the tumour cells with high concentrations (10(-5) M) of a GnRH antagonist, however, resulted in a 100% increase of their testosterone and androstenedione secretion. GnRH-binding sites of low affinity (Ka = 0.54 x 10(5) M-1) and high capacity (B max = 1364 x 10(-12) M/mg membrane protein) were identified in the tumour. These findings suggest that GnRH analogues might modify androgen secretion of sex-cord stromal tumours of the ovary via the suppression of endogenous gonadotrophin secretion and possibly also via direct effects on the tumour cells.

A radioimmunoassay for the determination of progesterone in saliva using a 125J-labelled progesterone derivate has been developed. The assay is characterized by the excellent sensitivity of 3.12 pg progesterone/ml. During the menstrual cycle of normal women the biphasic rhythm of progesterone in saliva could not been clearly demonstrated. In the follicle phase high peaks of progesterone have been shown and even in the luteal phase individual low values have been observed. The circadian rhythms of progesterone in saliva are characterized by irregular oscillations. The saliva flow rate is not responsible for the progesterone concentration. The measurement of progesterone in serum seems to be more useful in the diagnosis of sterility.

Jung-Hoffmann und Kuhl (1989) berichteten kurzlich uber den an einem kleinen Probandenkollektiv erhobenen Befund vergleichsweise hoherer Serumkonzentrationen des Athinylostradiols (EE2) nach Einnahme von Femovan ((F) 30 μg EE2,75 μg Gestoden; n = 11) als nach Einnahme von Marvelon ((M) 30 μg EE2, 150 μg Desogestrel; n = 11). Die trotz gleicher EE2-Dosis beobachteten unterschiedlichen EE2-Serumkonzentrationen wurden mit einer von der jeweiligen Gestagenkomponente abhangigen, unterschiedlichen Metabolisierung und Elimination des EE2 erklart. Eine andere Arbeitsgruppe (Humpel et al., 1990; Kuhnz et al., 1990) beschrieb dagegen identische EE2-Serumkonzentrationen unter der Behandlung mit beiden Kontrazeptiva.

To investigate the involvement of tenascin, an extracellular matrix glycoprotein, in epithelial growth and malignancy, its specific distribution pattern in the human uterus was examined immunohistochemically. During the proliferative phase of the menstrual cycle, this antigen was found as a sharp band around the endometrial glands. The immunoreactivity persisted until the early postovulatory phase of the menstrual cycle, but was not detectable in the glandular or stromal compartment during this later secretory stage, instead endometrial arterioles were immunostained. In marked contradistinction, when antibodies directed against tenascin were applied to sections of endometrial adenocarcinoma, almost the entire extracellular space stained, whereas the neoplastic cells themselves were nonreactive, whatever the degree of tumor differentiation. In precancerous proliferative lesions of the endometrium, tenascin's presence was variable. It was detectable around some superficial glands demonstrating cystic hyperplasia and around all deeply situated glands at the endometrial/myometrial interface. In cases of adenomatous hyperplasia, tenascin immunolocalized throughout the extracellular space of the stroma and the staining intensity was increased as the hyperplasia became more atypical. We therefore conclude that tenascin may be a stromal marker for epithelial proliferative states including those associated with malignancies of the endometrium.

The synthesis of [2H8]estradiol, [2H7]estrone, [2H6]2-hydroxyestrone and [2H6]4-hydroxyestrone from estrone (as a source) is described. The high isotopical purity renders the labelled compounds as suitable carriers and internal standards for quantitative gas chromatography — mass spectrometry. The content of protonium-form (i.e. natural) estrogens in the labelled derivatives ranged from 0.12 % to 2.58 %. The perfcrmance of these compounds in quantitative assays using selected ion monitoring has been established; and this allows the determination of estrogens from biological material in the lower picogram range.

[4- 14 C]-Oestradiol was incubated with liver slices of two male subjects. Under the protection of ascorbic acid the metabolites formed were separated into the free steroid, sulphate, glucuronide and thioether fractions. Using different Chromatographic and hydrolytic procedures the metabolites were isolated and identified by various microchemical reactions. Apart from oestradiol (31%) and oestrone (31%) the 2-substituted oestrogens 2-hydroxy-oestradiol (5.3%), 2-hydroxyoestrone (2.6%), 2-hydroxyoestrone 2-methyl ether (2.2%) and 2-hydroxyoestradiol 2-methyl ether (2.2%) were found to be the main metabolites (13%). They were present as free steroids (7.3%), sulphates (4.8%), glucuronides (0.3%) and thioethers (0.1%). Conjugation of the catechol oestrogens with either sulphuric acid or with glucuronic acid was only found at the 2-hydroxy group. The amount of ring B and ring D hydroxylated compounds did not exceed 3.8 and 0.5%, respectively. This means that the metabolism of oestradiol mainly proceeds via 2-hydroxylation and further metabolism of the 2-hydroxylated oestrogens. The intermediary metabolism of catechol oestrogens formed after incubation of oestradiol was essentially the same as that after direct incubation of [4- 14 C]-2-hydroxyoestradiol. When [4- 14 C]-oestradiol was administered orally to the same two male subjects 4 weeks after the liver excision the following excretion pattern of metabolites was found in the 72 h urine: 2-substituted oestrogens were the main catabolic products (11%), followed by oestrone and oestradiol (8.0%) and 16α-hydroxylated compounds (6.4%). All metabolites were mainly present as glucuronides.

The excretion pattern of different 2-hydroxyoestrogen monomethyl ethers (2-methoxyoestrone, 2-hydroxyoestrone 3-methyl ether, 2-methoxyoestradiol, 2-hydroxyoestradiol 3-methyl ether and the sum of the isomeric monomethyl ethers of 2-hydroxyoestriol) was quantitatively determined in urines of pregnant women. The analytical procedure involved several chromatographic purification steps and microchemical reactions, i.e. oxidative demethylation of the monomethyl ethers to the catechol compounds (2-hydroxyoestrone, 2-hydroxyoestradiol and 2-hydroxyoestriol) and conversion of 2-hydroxyoestrone and 2-hydroxyoestradiol to the corresponding phenazine derivatives. The phenazines were quantitated by U.V.-spectrometry and 2-hydroxyoestriol was determined by gas-chromatography. The specificity of the procedure was established by various spectrometric, chromatographic and microchemical methods. The amounts of the different oestrogens varied within the following ranges: 2-methoxyoestrone = 20–360 μg/24 h; 2-hydroxyoestrone 3-methyl ether = 5–50 μg/24 h; 2-methoxyoestradiol =

Urinary 2-hydroxyestrone was quantitatively determined in the course of several normal human pregnancies. The urine was subjected to hot acid hydrolysis and after chromatographic purification, 2-hydroxyestrone was converted into the corresponding phenazine derivative, which was submitted to a final column chromatography and then quantitated by UV-spectrometry. For correction of procedural losses 2-hydroxyestrone-4-14C was used as internal standard. The urinary 2-hydroxyestrone of different subjects varied within a wide range especially at mid-pregnancy between 100 and 2500 μg/24 h. The day-to-day variation of the excretion of 2-hydroxyestrone was mostly less than 30% of the total value of that day, but sometimes could even reach 60%. The investigation of 2-hydroxyestrone and total estrogens at regular intervals throughout several pregnancies, showed that the excretion of 2-hydroxyestrone generally reached a maximum during the second trimester, while the excretion of the total estrogens steadily increased ...

2-Hydroxyoestriol was isolated from human late-pregnancy urine and quantitatively determined by g.l.c. The purification procedure involved two chromatographic steps on silica gel columns and two paper Chromatographic separations using ascorbic acid impregnated stationary phases. The identity of the 2-hydroxyoestriol isolated was proved by gas chromatography-mass spectrometry and by a microchemical reaction. The urinary excretion of 2-hydroxyoestriol at the end of pregnancy, being in the order of 0.1 mg/24 h, was found to be lower than the conc. of 2-hydroxyoestrone in the same urine with only one exception.

Polar metabolites extracted from the effluents of viable porcine uterine strips superfused with either 6,7-3H-estrone or 6,7-3H-estradiol were identified as a 1:1 mixture of 6 alpha-hydroxyestrone and 7 alpha-hydroxyestrone by paper chromatography in various systems, derivatization and crystallizations to a constant specific activity. The hydroxylated compounds are the only derivatives detected after estrone superfusion. The major metabolite of estradiol released in short-time experiments is estrone followed by its 6 alpha- and 7 alpha-hydroxylated derivatives.

An assay for the quantitative determination of 2-hydroxyestrone and 2-methoxyestrone in human urine is described. The analytical procedure involves several purification steps: XAD-2 column chromatography of urine (1 ml), hot acid hydrolysis under reducing conditions, extraction with benzene/ethyl acetate, separation of monophenolic steroids from catecholestrogens by the formation of borate complexes, and partition between different solvents. Quantitation is achieved by radioimmunoassay using highly specific antibodies. For correction of procedural losses [6, 7-3H2] 2-hydroxyestrone and [6, 7-3H2] 2-methoxyestrone are used as internal standards. The method is highly specific as checked by comparison to a double-isotope-derivative method and a newly developed gas chromatography-mass spectrometry procedure. Using this method the urinary excretion of 2-hydroxyestrone and 2-methoxyestrone was studied in children, men, cycling, pregnant and postmenopausal women. Special interest was focussed on the molar ratios of 2-hydroxyestrone to 2-methoxyestrone and the so called "total estrogens" which vary markedly within the different groups investigated. The excretion of 2-hydroxyestrone is especially favoured in women during the menstrual cycle and pregnancy.

A method is described for the assay of pregnanediol in pregnancy urine as well as in the urine of men, children and non pregnant women. The analytical procedure comprises Amberlite XAD-2 extraction, hot acid hydrolysis, and automated solid injection g.l.c. of the trifluoroacetate derivatives. For low titer urines an additional alumina column Chromatographic step is required. The specificity of the method has been established by g.l.c.-MS; the sensitivity is only limited by the sensitivity of the g.l.c. detector. 0.01 mg pregnanediol/24 h can be determined without difficulty.

For stabilisation 2-hydroxyestrone and 2-hydroxyestradiol-17β are converted to their phenazine derivatives by oxidation with sodium metaperiodate to the 2,3-quinones and subsequent condensation with o-phenylenediamine. The structures of this new class of heterocyclic steroids were established by nuclear magnetic resonance, ultraviolet, infrared and mass spectra. The chemical properties and the chromatographic behaviour of these phenazines are described. The specificity of the oxidation was established by using various oxidants and different estrogens; the condensation was optimised by acetic acid catalysis on a microgram scale.

1. In view of previous experiments in vitro and in vivo, in which 6-hydroxylation of phenolic steroids by various tissues was demonstrated, an attempt was made to isolate 6-hydroxy oestrogens from the urine of pregnant women. 2. During this work it was found that free 6alpha- as well as 6beta-hydroxylated oestrogens were extremely unstable under acidic conditions (pH3); it was therefore necessary to establish experimental conditions under which no rearrangement of the hydroxyl group would take place. 3. By avoiding acid steps during the experimental procedures, a ketonic-phenolic fraction was obtained from enzymically hydrolysed late-pregnancy urine that was subjected to paper chromatography in various systems. 4. By using similar methods on a large scale and partition chromatography on Celite columns, a crystalline material was obtained from 1001. of late-pregnancy urine that was identified as 6alpha-hydroxyoestrone by various chemical reactions and by infrared spectroscopy.

Incubation of androsta-4,16-dien-3-one with 10 000 × g supernatant of human placenta tissue resulted in the formation of a phenolic metabolite. This was isolated and subsequently identified as oestratetraen-3-ol by its melting point, paper chromatography, and the absorption curves of the sulphuric acid water reaction and the Kober reaction. The conversion of androsta-4,16-dien-3-one to oestratetraen-3-ol was approximately 25%, whereas under similar conditions testosterone was aromatised to oestradiol-17β (and oestrone) in a 30% yield. The significance of these results is discussed with regard to the conversion of testosterone to 16,17-epioestriol.

After incubation of 6α-hydroxyandrost-4-ene-3,17-dione with the aromatising enzyme system of human placenta, a 6-hydroxyoestrome was isolated which was identical with the compound formed after reduction of 6-oxo-oestradiol-17β with NaBH4 and subsequent selective oxidation with CrO3. When 6β-hydroxyandrost-4-ene-3,17-dione was incubated under the same conditions, a 6-hydroxyoestrone was isolated which was identical with the compound obtained after catalytic hydration of 6-oxo-oestrone. Since the configuration of the 6-hydroxyl groups in neutral steroids is well established, these experiments permit the definite assignment of configurations to the epimeric 6-hydroxylated phenolic steroids. The incubation of oestradiol-17β with rat-liver microsomes in the presence of NADPH2 and molecular oxygen led to the formation of 6α- and 6β- and hydroxyoestradiol-17β. The amount of 6α-hydroxyoestradiol-17β was about five times greater than that of 6β-hydroxyoestradiol-17β. When 6α- and 6β-hydroxyoestradiol-17β were incubated with rat-liver slices, 6-oxo-oestradiol-17β as well as 6α- and 6β-hydroxyoestrone were formed. Under the same conditions, 6α- and 6β-hydroxyoestrone were metabolised to 6-oxo-oestrone, 6α- and 6β-hydroxyoestradiol-17β. Incubation of 6-oxo-oestradiol-17β led to the formation of 6-oxo-oestrone, 6α- and 6β-hydroxyoestradiol-17β, whereas 6-oxo-oestrone yielded 6-oxo-oestradiol-17β, 6α- and 6β-hydroxyoestrone.

Following the incubation of oestradiol-17β with liver slices of male rats two metabolites more “polar” than estradiol-17β were isolated and conclusively identified as 6-hydroxyo estradiol-17β and oestriol.

Isolation and quantitative estimation of the four epimeric oestriols following the injection of 16α- and 16β-hydroxyoestrone in man. 7.4 mg 16α-hydroxyoestrone were administered by intramuscular injection to a normal man. Three phenolic metabolites were isolated from urine and identified as oestriol, 17-epioestriol and 16-epioestriol. The total excretion in urine amounted to 1.762 mg oestriol, 0.031 mg 17-epioestriol and 0.163 mg 16-epioestriol. When 10.0 mg 16β-hydroxyoestrone diacetate were administered to a normal man, the following metabolites were isolated and identified: 16-epioestriol, 16.17-epioestriol and oestriol. The total excretion in urine was 1.377 mg 16-epioestriol, 0.086 mg 16,17-epioestriol and 1.332 mg oestriol. These findings confirm previous experiments with human liver slices and show that in addition to a 17β-hydroxysteroid dehydrogenase a 17α-hydroxysteroid dehydrogenase is present in man. It should be noted, however, that the amount of 17α-hydroxy-compounds excreted in urine was much smaller than that of 17β-hydroxy-compounds.

When 6-chloro-9β,10α-pregna-1,4,6-triene-3,20-dione (trengestone) was incubated with liver slices of male rats, the substrate was rapidly transformed to a polar metabolite; in contrast, liver slices of female rats metabolised trengestone much more slowly, and only traces of a polar metabolite could be detected. After paper and thin layer chromatography in various systems, the polar metabolite was obtained in crystalline form and identified as 6-chloro-16α-hydroxy-9β,10α-pregna-1,4,6-treiene-3,20-dione (16α-hydroxytreengestone) by its infrared spectrum and by gas chromatography-mass spectrometry. After administration of trengestone to male or female rats, the main metabolite in the urine was again a6α-hydroxytrengestone. When trengestone was incubated with liver slices of male or female human subjects, two metabolites were detected, one of which was formed in large amounts. By comparison with the authentic compound, the major metabolite proved to be identical with (20S)-6-chloro-20-hydroxy-9β,10α-pregna-1,4,6-triene-3-one (20α-hydroxytrengestone). After oral administration of radioactive trengestone to man, the amount of total radioactivity in the urine was considerably greater in a human female (50 %) than in two human males (6–8 %). The main metabolite of trengestone was identified as 20α-hydroxytrengestone. When trengestone was given orally to a female rabbit, and to a female Rhesus monkey, both the 20α-hydroxy and the 2β-hydroxy derivatives of trengestone were detected in the urine. These experiments demonstrate considerable differences in the metabolism of the rat, rabbit, monkey, and man.

25 phenolische Steroide, von denen die meisten bereits aus dem Urin schwangerer Frauen isoliert worden sind, konnen getrennt und quantitativ bestimmt werden durch Kombination von Papier-, Dunnschichtund Gas-Chromatographie. Die Methode besteht aus folgenden Schritten: 1. Enzymatische Hydrolyse des Urins. 2. Extraktion des hydrolysierten Urins mit Ather. 3. Reinigung der Atherextrakte mit Natriumhydrogencarbonat und Wasser bei 0° C. 4. Mehrfache Chromatographie des Ruckstandes auf formamidimpragniertem Papier mit Monochlorbenzol, Chloroform, Cyclohexan, Monochlorbenzol/Athylacetat (3∶1) oder Chloroform/Athylacetat (5∶1) bzw. in zwei Bush-Systemen. 5. Acetylierung der aufgetrennten Oestrogene. 6. Dunnschicht-Chromatographie der Steroidacetate. 7. Quantitative Bestimmung der Acetate durch Gas-Chromatographie mit Hilfe eines Flammen-Ionisations-Detektors. Die Zuverlassigkeit der Methode (Richtigkeit, Genauigkeit, Spezifitat und Empfindlichkeit) wurde in Zusatzversuchen gepruft; zu diesem Zweck wurden jeweils 10 μg Steroid mannlichem Urin zugefugt. Die durchschnittliche Wiederfindung der zugesetzten 25 phenolischen Steroide schwankte zwischen 50 und 60%, wahrend der Variations-Koeffizient sich zwischen 5–15% bewegte. Durch mehrere mikrochemische Reaktionen konnte gezeigt werden, das selbst in der Anwesenheit von Urinpigmenten die Methode eine hohe Spezifitat besitzt. Mit Hilfe des angegebenen Verfahrens konnen etwa 1–2 μg Steroid pro 24 h-Urin quantitativ bestimmt werden.

Application of a commercially available hair lotion containing high concentrations of oestradiol led to uterine bleeding in a 76-year old patient. Histological examination of the endometrium showed typical proliferation. Oestradiol blood levels were in the normal range of the follicular or luteal phase, LH- and FSH-serum concentrations were suppressed. After discontinuing the application of the assay used (20 pg/ml) and gonadotrophins rose to normal postmenopausal levels. These findings prove that after local application of oestradiol on the scalp, percutaneous resorption of the steroid is significant and systemic oestrogen effects have to be taken into account.

Der Kalium-, Natrium-, Chlorid- und Wassergehalt der Milz erwachsener männlicher Ratten wurde nach Ganzkörperbestrahlung untersucht; die applizierten Dosen lagen im Bereich des 3,5-Tage-Effektes. Es wurde eine Abnahme des Kaliumgehaltes und eine Zunahme des Natriumgehaltes beobachtet; die Veränderungen waren im Bereich von 500 — 12 000 r unabhängig von der Dosis. Der Wassergehalt der Milz nahm nach der Bestrahlung ab, während der Chloridgehalt sich nicht signifikant änderte. Die Ergebnisse werden im Zusammenhang mit dem Mechanismus der Strahlenschädigung nach Ganzkörperbestrahlung besprochen.

16α, 17α-Epoxyoestratrienol wurde bei Ratten in einer Gesamtdosis von 90 mg/Tier subcutan injiziert. In keinem Falle entwickelten sich bei den Versuchstieren Tumoren.

A thin layer chromatography (TLC) screening test is described, which allows the semiquantitative determination of HMMA in urine samples. From the collected 24 hour urine a measured sample is applied without extraction to a special TLC plate, which is developed in one dimension. In comparison to a series of graded standard solutions the daily HMMA output can be estimated semiquantitatively. In 102 patients over 18 years of age, suffering from high blood pressure, the daily HMMA output was estimated by both the Pisano method (Pisano, J.J., Crout, J.R. & Abraham, D. (1962), Clin. Chim. Acta 7,285--291) and the TLC method. Coincident results with both methods were obtained in 99 of these cases. The residual 3 cases show pathological results with the Pisano method and results in the normal range or the warning range, respectively, with the TLC method. These cases can probably be classified as false positive results of the Pisano method. The TLC method is recommended as a screening test for estimating the daily HMMA excretion.

Urinary 2-hydroxyesterone was quantitatively determined in the course of several normal human pregnancies. The urine was subjected to hot acid hydrolysis and after chromatographic purification, 2-hydroxyesterone was converted into the corresponding phenazine derivative, which was submitted to a final column chromatogrphy and then quantitated by UV-spectrometry. For correction of procedural losses 2-hydroxyestrone-4-14C was used as internal standard. The urinary 2-hydroxyestrone of different subjects varied within a wide range especially at mid-pregnancy between 100 and 2500 mug/24 h. The day-to-day variation of the excretion of 2-hydroxyestrone was mostly less than 30% of the total value of that day, but sometimes could even reach 60%. The investigation of 2-hydroxyestrone and total estrogens at regular intervals throughout several pregnancies, showed that the excretion of 2-hydroxyesterone generally reached a maximum during the second trimester, while the excretion of the total estrogens steadily increased up to parturition. When analyzing the urines of different subjects during the last 4 months of pregnancy, no correlation appeared to exist for the excretions of the total estrogens and of 2-hydroxyestrone.

A thin layer chromatography (TLC) screening test is described, which allows the semiquantitative determination of HMMA in urine samples. From the collected 24 hour urine a measured sample is applied without extraction to a special TLC plate, which is developed in one dimension. In comparison to a series of graded standard solutions the daily HMMA output can be estimated semiquantitatively. In 102 patients over 18 years of age, suffering from high blood pressure, the daily HMMA output was estimated by both the Pisano method (Pisano, J.J., Crout, J.R.Abraham, D. (1962), Clin. Chim. Acta 7,285--291) and the TLC method. Coincident results with both methods were obtained in 99 of these cases. The residual 3 cases show pathological results with the Pisano method and results in the normal range or the warning range, respectively, with the TLC method. These cases can probably be classified as false positive results of the Pisano method. The TLC method is recommended as a screening test for estimating the daily HMMA excretion.