Individual Estrogen Research Articles

Identification of conjugated estrogen metabolites in dog plasma following administration of estriol-2,4,6,7- 3H.

Following the constant infusion of 2,4,6,7- 3H-estriol in male dogs for a period of 90 minutes, the radioactive metabolites present in arterial plasma were separated by solvent partition, DEAE-Sephadex, Celite partition and thin layer chromatography. The identities of the individual estrogens and estrogen conjugates were confirmed by specific activity determinations after chromatography in several different solvent systems, enzyme hydrolysis and chromatography of the unconjugated steroids and their derivatives. All of the unconjugated radioactivity in the plasma after 90 minutes was identified as free estriol. Five conjugated metabolites of estriol were isolated. Three monoconjugates were identified as estrlol-3-glucosiduronate, estriol-16α-glucosiduronate and estriol-3-sulfate. Two polyconjugates of estriol were tentatively identified as estriol-3-sulfate-16α-glucosiduronate and an estriol diglucosiduronate.

EFFECT OF AMPICILLIN ADMINISTRATION ON THE EXCRETION OF TWELVE OESTROGENS IN PREGNANCY URINE

The excretion of twelve oestrogens in urine, pooled daily from a group of pregnant women, was determined before, during and after ampicillin administration (2 g/day, for 3 days). On the second day of ampicillin administration total oestrogen excretion fell to 67% of the mean control value, oestriol excretion to 69% and that of the other eleven individual oestrogens to an average of 62% of the mean control values. In general, on the third day of treatment and on the two post-treatment days this decrease tended to be corrected. The patterns of change in the urinary levels of the individual metabolites provided no clear lead to the basic mechanism of ampicillin impairment of oestrogen excretion. However, as the drug affected all their excretion in more or less the same way as it did that of oestriol, it is possible that ampicillin interferes primarily with their enterohepatic circulation in the mother as has been established with reasonable certainty in the case of oestriol.

Effect of ampicillin administration on the excretion of twelve oestrogens in pregnancy urine.

The excretion of twelve oestrogens in urine, pooled daily from a group of pregnant women, was determined before, during and after ampicillin administration (2 g/day, for 3 days). On the second day of ampicillin administration total oestrogen excretion fell to 67% of the mean control value, oestriol excretion to 69% and that of the other eleven individual oestrogens to an average of 62% of the mean control values. In general, on the third day of treatment and on the two post-treatment days this decrease tended to be corrected. The patterns of change in the urinary levels of the individual metabolites provided no clear lead to the basic mechanism of ampicillin impairment of oestrogen excretion. However, as the drug affected all their excretion in more or less the same way as it did that of oestriol, it is possible that ampicillin interferes primarily with their enterohepatic circulation in the mother as has been established with reasonable certainty in the case of oestriol.

Identification of some plasma metabolites of 6, 7- 3H-estrone glucosiduronate in male dogs

Following the constant infusion of 6, 7- 3H-estrone glucosiduronate in male dogs for a period of 120 minutes, the radioactive metabolites present in the plasma were separated by solvent partition, DEAE-Sephaphadex, Celite partition and thin layer chromatography. The identities of the individual estrogens and estrogen conjugates were confirmed by specific activity determinations after chromatography in several different solvent systems, enzyme hydrolysis and chromatography of the unconjugated steroids and their derivatives. Most of the radioactivity in the plasma was identified as estrone glucosiduronate. The major metabolite present was estradiol-17β-3-glucosiduronate. Small amounts of estradiol-17α-3-glucosiduronate and free estrone were also identified. Three other minor conjugates were separated, but positive identification could not be made.

Oestrone, oestradiol-17beta and oestriol levels in human foetal plasma during gestation and at term.

SUMMARY Marked rises in both unconjugated and sulphoconjugated oestrone, oestradiol-17β and oestriol were observed in human foetal plasma between mid-gestation and term. Significant arterio-venous differences for the individual oestrogens in the unconjugated form were found in the umbilical cord plasma. No consistent arterio-venous differences were found for the oestrogen sulphates. This indicates that all three oestrogens are secreted from the placenta into the foetal circulation in the unconjugated form. Mean unconjugated oestrogen (oestrone + oestradiol-17β + oestriol) levels rose from 22·7 ng/ml at 17–20 weeks of gestation to 108·9 ng/ml at term in umbilical venous plasma and from 4·3 ng/ml to 23·3 ng/ml in umbilical arterial plasma. This represents a secretion rate of approximately 30 mg oestrogen/day into the umbilical vein at term. Mean oestrogen sulphate levels rose from 128 ng/ml to 313 ng/ml in the cord plasma during the same period. Of the three oestrogens measured, oestriol was quantitatively the major oestrogen in foetal plasma. It consistently represented about 78% of the unconjugated fraction and 95% of the sulphate fraction at all stages of gestation. The method of delivery did not have a significant effect on the oestrogen levels in uncomplicated pregnancies. Similar oestrogen levels were found in foetal heart blood after either hysterotomy or spontaneous abortion at 16–20 weeks of gestation, and no significant differences were found for oestrogen levels in cord plasma after elective Caesarean section at 38–39 weeks when compared with oestrogen levels after normal delivery at term. A significant rise in foetal unconjugated oestrogens at a time when foetal corticosteroids are increasing may be of physiological importance for foetal maturation in women.

Measurement of steroid hormones using intracellular receptor proteins

The high affinity saturable estrogen receptor of the uterus has been successfully employed by a number of groups to assay individual estrogens in blood. Such measurements have elucidated alterations occurring during the normal menstrual cycle, during normal and abnormal pregnancy, during the estrus cycle of a variety of mammals, in a few abnormal menstrual cycles, and in normal men. A few studies possible associated with estrogen excess in men were also reported.

Simultaneous occurrence of individual estrogen- and androgen receptors in female and male target organs.

Reserachers at the Max-Planck-Institut fur Zellbiologie in Wilhelmshaven Germany studied possible relationships between estradiol receptors and dihydrotestosterone receptors in uteri seminal vesicles and prostates. Tissues were removed from 12-week-old calves and 6-month-old castrated male pigs. The dihydrotestosterone receptor remained in solution while the estradiol receptor was removed from target organ extracts by absorption to the coupling product of (4-diazobenzyl) cellulose andestradiol in 2- or 4- position. The 2 receptors are individual but concomitant in the target organs of both sexes. On the other hand a 1000-fold excess of estradiol completely inhibited the binding of dihydrotestosterone to its receptor. A 1000-fold excess of dihydrotestosterone resulted in a 42% reduction of receptor-bound estradiol. Thus although the receptors are individual a cross-affinity also exists. With physiological concentrations the cross-affinity may be of only negligible importance. The beneficial effects of high estrogen doses to prostate cancer patients may be due to competitive inhibition of androgens.

Automated Fluorometry as Applied to the Assay of Individual Estrogens in Purified Urinary Extracts

Abstract Using the Kober-lttrich fluorescence procedure, automated fluorometry was applied to the assay of individual estrogens and their 3-methyl ether derivatives in the purified urinary extracts of nonpregnant women. The values obtained by using this new automated scheme were compared with those obtained by the manual time-decay modification of the Slaunwhite-Engel fluorescence method; the comparison shows excellent correlation. A ratio of the corrected peak reading at 546 mµ. excitation to the corrected peak reading at 490 mµ. excitation was used not only to follow the effect of introducing additional purification stages in the development of assay procedures, but also to follow impurity levels in sample analyses. The same assembly of modules can be used for samples dissolved in acidified water, 70% methanol, and, with a simple change of manifold, in 1 N sodium hydroxide. This fully automated assay provides precise conditions in all stages.

Inhibiting Activity of Estrogens on Capillary Permeability

Although certain estrogens involving Premarin, a mixed preparation of natural conjugated estrogens, have been widely used for years as hemostatic agents, the mechanism of their activity has never been completely clarified. Control studies recently completed by us revealed that this hemostatic activity was due to the ability of estrogens to inhibit kallikrein-induced enhancement of capillary permeability and thus to restore normal capillary physiology. Our tests indicatedd that most estrogens were able to inhibit enhancement of capillary permeability to a greater or lesser degree, depending upon the chemical structure of individual estrogen. Of tested estrogen preparations, Premarin had the greatest activity.

Determination of oestriol, oestrone and oestradiol in pregnancy urine by thin-layer chromatography

A method is described for the determination of individual oestrogens in human pregnancy urine by means of thin-layer chromatography. After acidic hydrolysis, the urine was extracted with ether, washed with Na 2CO 3 and the extract chromatographed on Kieselgel G layer with the solvent system benzene-ethanol (9:1). Quantitation was carried out using Ittrich's colour reaction in the presence of adsorbent. Physiological excretion during the Ist to the Xth month of pregnancy is presented. The findings are in good agreement with those reported in the literature.

Effect of SU4885 on the production of oestrogens in the rat ovary.

19-Hydroxylation of the steroid molecule is an obligatory step in the conversion of androgens to oestrogens. It has been shown (Földes, Koref, Fehér & Steczek, 1964) that the urinary excretion of individual oestrogens decreases after the administration of 2-methyl-1,2-bis-(3-pyridyl)-1-propanone (SU4885). The aim of the present communication is to show that SU 4885 inhibits the transformation of androstenedione to oestrogens in the rat ovary in vitro. The following trivial names are used: androstenedione = androst-4-ene-3,17-dione; oestradiol = oestra-1,3,5(10)-triene-3,17β-diol; oestrone = 3-hydroxy-oestra-1,3,5(10)-trien-17-one. Female rats of the Wistar strain, weighing 180–250 g., received two injections of 50 units chorionic gonadotrophin (HCG, Choriogonin, Richter, Budapest) 24 and 16 hr. before the ovaries were removed. For each assay, 18 animals were used, six of which also received s.c. 10 mg. SU 4885 (Metopirone, Ciba, Basel) 40, 24 and 16 hr. before death. The ovaries were removed, halved and distributed (12 halved ovaries, 250–350 mg./vessel) between six vessels each

Free estrogens in dog plasma during the estrous cycle and pregnancy.

SummarySolvent partitioning, paper and thin-layer chromatography, and fluorometry were employed to isolate, identify and quantitate the free estrone, estradiol-17α, estradiol-17β, estriol and 16-epiestriol in dog plasma during the estrous cycle and pregnancy. There was a continuous decline in both individual and combined estrogen concentrations from early proestrus to late estrus. Throughout pregnancy the levels of estradiol-17α and 16-epiestriol remained fairly constant. Estrone and estradiol-17β attained peak levels at 3–4 weeks and declined thereafter. Estriol was the major metabolite and by the last week of pregnancy showed a 43% increase over the initial level. All of the estrogens except estradiol-17α rose substantially during the first week post-partum.

Abnormal urinary estrogen levels in hydatidiform moles determined by gas-liquid chromatography

Twenty-four hour urine samples from patients with hydatidiform moles were analyzed by gas chromatography for estrone, estradiol, and estriol. The individual estrogen levels were far below the expected value for comparable periods in pregnancy and approximated those of normal menstruating nonpregnant women. However, the E 1/E 2 ratio varied greatly from both the normal nonpregnancy and pregnancy ranges. The low estrogen levels and the abnormal E 1/E 2 ratios appear to be sensitive indices of severe placental dysfunction as early as the twelfth week of gestation.

COLOUR REACTIONS FOR THE IN SITU CHARACTERISATION OF STEROID OESTROGENS ON THIN-LAYER CHROMATOGRAMS.

ABSTRACT Methods are described for the in situ characterisation of 32 steroid oestrogens on thin-layer chromatograms. The methods are suitable for the detection of a) phenols, b) benzene rings, c) o-dihydroxyphenols, d) unsaturated bonds, e) ketones, f) α,β-diketones and g) individual oestrogens. The sensitivity of the different methods varies with various oestrogens and is between 0.2 and 15.0 μg.

Isolation of estrone and 11β-hydroxy estrone from a feminizing adrenal carcinoma

This paper reports the isolation and characterization of estrone and 11β-hydroxy estrone from an adrenal carcinoma in a 25 year old male with gynecomastia. The tumor was homogenized and the extract divided into unconjugated neutral, phenolic, and conjugated fractions. The phenolic fraction was further divided into individual estrogens using paper chromatography. Characterization of estrone and 11β-hydroxy estrone was done by comparing the chromatographic behavior and functional groups before and after sodium borohydride reduction as well as acetylation with authentic reference standards. Final confirmation of the identity was obtained by infra-red analysis.

Oestrogen-bestimmungen II. Bestimmung von oestron, oestradiol und oestriol in schwangerenplasma

A method for the quantitative determination of oestrone, oestradiol, and oestriol in pregnancy plasma is described. Separation of the oestrogens is achieved by paper chromatography on acetylated paper. Extracts of the paper are then purified by chromatography on aluminium oxide. The ittrich modification of the Kober reaction is used for the quantitative determination of the individual oestrogens.

Zur bestimmung der östrogene im urin nach der methode von Ittrich

The application of the method described by Ittrich for the estimation of oestrogens in urine has been studied under various conditions. Satisfactory results were obtained when the Ittrich reaction was applied directly to pregnancy urine. The Ittrich method is not superior to the method of Brown in estimating individual oestrogens present in low concentrations in the urine, when the extracted colour complex is measured colorimetrically. In such cases fluorimetric measurement is to be preferred.

ABNORMALITY OF ESTROGEN METABOLISM IN HUMAN SUBJECTS WITH MYOCARDIAL INFARCTION

Estrogen metabolism has been investigated in male subjects with and without previous myocardial infarction. The urinary excretion of estriol, estrone, and estradiol-17β has been measured 1 day before and 4 days after intramuscular injection of estradiol-17β. The excretion of the individual estrogens resulting from the administration of estradiol was determined by subtracting preinjection values from the daily excretion following injection. The resultant values of estriol (T), estrone (O), and estradiol-l7β (D) were expressed as the following ratios:[Formula: see text]These urinary estrogen ratios were found to be significantly higher in subjects with previous myocardial infarction than in control subjects. The ratios in the infarction group were not influenced by bed rest nor by the duration of time following infarction which varied from 1 week to 2 years.

A method for the separation of four phenolic estrogens in human urine by paper chromatography and an evaluation of biological assay of these compounds.

THIS paper presents a method for the estimation of individual urinary estrogens involving enzymatic hydrolysis of conjugates, ether extraction, paper chromatographic separation and purification of estrone, estradiol-3,17β, estriol and l6-epiestriol and finally quantitative estimation of these compounds by bioassay. The method is suitable for the measurement of very small amounts of urinary estrogens such as are excreted in the urine of patients with mammary cancer. The specificity and sensitivity of estrogen bioassay will be examined in this communication. Estrogens are known to be significantly involved in the genesis of mammary neoplasms in animals (1) and these ovarian hormones are sustaining factors of human breast cancer (2). A study of estrogen excretion and metabolism in women with breast cancer may further advance our understanding of the endocrine factors in the growth of these neoplasms and the mechanism of regression of some mammary cancers following endocrine ablative surgery.

ABNORMALITY OF ESTROGEN METABOLISM IN HUMAN SUBJECTS WITH MYOCARDIAL INFARCTION

Estrogen metabolism has been investigated in male subjects with and without previous myocardial infarction. The urinary excretion of estriol, estrone, and estradiol-17β has been measured 1 day before and 4 days after intramuscular injection of estradiol-17β. The excretion of the individual estrogens resulting from the administration of estradiol was determined by subtracting preinjection values from the daily excretion following injection. The resultant values of estriol (T), estrone (O), and estradiol-l7β (D) were expressed as the following ratios:[Formula: see text]These urinary estrogen ratios were found to be significantly higher in subjects with previous myocardial infarction than in control subjects. The ratios in the infarction group were not influenced by bed rest nor by the duration of time following infarction which varied from 1 week to 2 years.