Effects of catechol estrogen methyl ethers on lipid metabolism in prepubertal rats

Thirty-five day old ovariectomised rats were given daily subcutaneous injections (0.05-5.0 micrograms/100 gm body weight) of estradiol (E2) 2-methoxyestradiol (2-ME2) or 4-methoxyestradiol (4-ME2) for six days. At the end of the last injection, the animals were sacrificed and serum lipoproteins were analysed. It was observed that cholesterol decreased significantly in normal fed animals who received E2 and 4-ME2, while no effect was seen in cholesterol fed animals. In the E2 treated group there was a decrease in esterified and free cholesterol, while in the 4-ME2 group only esterified cholesterol decreased. High density lipoproteins were significantly elevated in the E2 treated group. However, there was an increase in very low density lipoproteins and a decrease in low density lipoproteins in 2-ME2 and 4-ME2 treated groups. These results suggest that catechol estrogens may play an important role in the lipoprotein metabolism and atherosclerotic diseases, and the mechanism of action may differ from that of estradiol.     

Estriol concentrations in plasma of normal, non-pregnant women.

Using a rabbit antisera directed against estriol-3-0-carboxy methyl ether complexed to BSA, an immunoassay for estriol (1) was developed. The mean plus or minus SE concentration of estriol in 18 women in days 5-7 of their cycle was 7.9 plus or minus 0.6 pg/ml which was significantly (P less than 0.01) less than the mean value of 11.1 plus or minus 0.8 pg/ml in 15 women in days 20-22 of the cycle. In 3 of 6 women in whom plasma samples were drawn frequently during their cycle, an estriol peak occurred coincident with the estradiol peak. In 3 women from whom plasma was obtained several times during the course of a day estriol levels did not appear to vary significantly. In 8 women who were on oral contraceptives the mean level of estriol was 7.6 plus or minus 1.5 pg/ml. In 8 post-menopausal women the mean level was 6.0 plus or minus 1.2 pg/ml which is significantly (P less than 0.01) less than the mean luteal phase value but not less (P greater than 0.1) than the follicular phase or oral contraceptive user values. We conclude that some of the circulating estriol is directly secreted by the ovary of normal women.     

Synthesis of estrogen methyl ethers by extractive alkylation.

A rapid method for the quantitative preparation of a number of estrogen methyl ethers is described. Estrogen in aqueous base is extracted as an ion pair with the tetrahexylammonium oin into methylene chloride where irreversible alkylation (extractive alkylation) by methyl iodide occurs. Gas chromatography (GC) - mass spectrometry (MS) provided the basis for identification of the methylated products. Estrone (1) and estradiol were easily 3-0-methylated whereas estriol gave a dimethylated product. Further experiments suggested that dimethylation of 2-hydroxyestrone in reasonable yield was possible.     

Changes in plasma levels of estrone sulfate and estrone in the pregnant ewe around parturition

A method for the extraction, separation and measurement of estrone sulfate and estrone in a single plasma sample is described. The method has been applied to the determination of plasma levels of estrone sulfate and estrone in pregnant ewes over the period 60 hr before to 20 hr after parturition. The study revealed that the plasma levels of estrone sulfate and estrone began to increase about 40 hr before parturition, reached a peak at parturition and then declined rapidly to levels below the sensitivity of the method by 15 hr postpartum. The peak level of estrone sulfate recorded at parturition was 103 pmol (38 ng) per ml of plasma which was approximately 30 times greater than the corresponding peak level of estrone.     

Estrogen effects on NADH oxidase and superoxide dismutase in prepubertal female rats

Thirty-four day old, ovariectomised rats were treated with increasing doses of estradiol, 2-hydroxyestradiol 2,3-dimethyl ether (23E₂), 4-hydroxyestradiol 3,4-dimethyl ether (34E₂) and 4-methoxy-estradiol (4ME₂) for five days by subcutaneous injection. Superoxide dismutase, phenol activated NADH oxidase and uterine dry weights were determined. Only estradiol was found to be uterotrophic and increased NADH oxidase activity in these experiments. Both 23E₂ and 34E₂ treatment reduced the enzyme activity significantly. Though 4ME₂ showed a decrease in NADH oxidase at 0.05μg/100gm body weight there was no further decrease at higher dose (5μg/100gm). The Superoxide dismutase (SOD) in uterus and liver was unaffected by estradiol, while 23E₂, 34E₂, and 4ME₂ significantly reduced SOD in both liver and uterus. These results indicate that 23E₂, 34E₂ and 4ME₂, in spite of their non-uterotrophic property, affect uterine metabolism. Furthermore, in view of the reports indicating the importance of SOD levels in various tumors and since catecholestrogens are observed to reduce SOD levels in liver and uterus, it is suggestive that catecholestrogens may play an important role in the pathophysiology of certain tumors.     

Plasma estrone sulfate levels in postmenopausal women

Estrone sulfate levels were measured in the plasma of 63 postmenopausal women. The assay method involved prior extraction of the free estrogens, enzyme hydrolysis of the estrone sulfate with sulfatase and radioimmunoassay of the estrone liberated. The plasma levels ranged from 37 to 320 pg/ml (expressed as free estrone) with a mean value of 178 ± 79 pg/ml. As observed in premenopause, estrone sulfate is quantitatively the most important circulating estrogen in postmenopausal women.     

Specific antisera for the radioimmunoassay of estriol 3-sulfate

Antisera were raised in male guinea pigs against 6-oxoestriol 3-sulfate O-carboxymethyloxime-bovine serum albumin (BSA) conjugate. The antisera to this antigen exhibited high affinity (Ka=4.7 X 10(9)M-1) and excellent specificity for estriol 3-sulfate, showing slight cross-reactions (less than 0.43%) with other estrogen sulfates, and no cross-reactivities with free estrogens and other steroids (less than 0.01%) except cholesterol sulfate (0.22%). A standard curve using [6, 7-3H]-estriol 3-sulfate as the radioactive ligand showed high sensitivity in the range of 10-1000 pg estriol 3-sulfate.     

A rapid specific radioimmunoassay for unconjugated estriol in plasma.

A rapid, non-chromatographic radioimmunossaay for unconjugated estriol in pregnancy plasma has been developed which utilizes a commonly available antiestrogen antisera. Estradiol-17beta and estrone demonstrate 135% relative cross-reactivity with our antiserum, as compared with 100% for estriol. Specificity is achieved by purification of estriol with solvent partitioning using benzene: petroleum ether (1:1). The results obtained using this method are similar to a radioimmunoassay utilizing a highly specific, but commercially unavailable, antiestriol antiserum. The method is precise, with coefficients of variation ranging from 3.0 to 8.2%.     

A radioimmunoassay method for urinary catechol estrogens

A radioimmunoassay method for urinary catechol estrogens is described. The specific nature of the antisera allows direct analyses of acid hydrolyzed urine. A LH-20 Sephadex column chromatography can be employed for individual determinations of 2-hydroxyestrone and 2-hydroxyestradiol. The excretion of catechol estrogens during menstrual cycles ranged from 14.48 to 50.15 μg per 24 hours, whereas, during the last trimester of pregnancies, the values ranged from 129.30 to 1758.20 μg per 24 hours.     

A non-chromatographic radioimmunoassay of estrone and estradiol-17beta serum

A sensitive and efficient non-chromatographic procedure employing the Girard reagent and solvent-partitioning has been developed for the accurate radioimmunoassay (RIA) of estrone (E1) and estradiol-17β(E2) in a single 1.0 ml specimen of male or female serum. Using standard curves which permitted the discrimination of zero from 0.75–1.5 pg (p=0.05), the following mean procedural blanks (pg ± S.D.) were determined (1.0 ml water, n= 24): estrone, 2. 1 ± 1.1 (range 0–4.1); estradiol 1.0± 1.1 (range 0–3.6).A comparison of RIA of estrogens (1) in serum after separation by the Girard procedure and by TLC yielded correlation coefficients of 0.99 and 0.98 for E1 and E2 respectively. The following results (pg/ml ± S.D.) were obtained on RIA of E1 and E2 in 12 different 1.0 ml specimens of male and female serum using the Girard procedure: male. E1 (32.0 ± 9.2), E2 (24.1 ± 10.9); female, E1 (108.5 ± 60.8), E2 (126.4 ± 63.2).The intra-assay variability (c.v.) was found to be 12.6% for E1 and 9.4% for E2. The interassay variability was 14.2% for both estrogens.Twenty-four assays of E1 and E2 can be completed by one person in 2 working days.     

Synthesis of new steroid haptens for radioimmunoassay. part I. 15β-Carboxyethylmercaptotestosterone-bovine serum albumin conjugate. measurement of testosterone in male plasma without chromatography

A radioimmunoassay (RIA) procedure has been developed for measurement of testosterone in male plasma after ether:chloroform (4:1) extraction of the plasma sample without resorting to chromatography. The highly specific anti-testosterone serum was generated from both rabbits and sheep immunized with 15β-carboxyethylmercapto-testosterone-BSA conjugate. The synthesis of 15β-carboxyethylmercaptotestosterone and the preparation of its BSA conjugate are described. The high affinity (K_a = 2.38 × 10⁹ liters/mole) antiserum binds 50% of 50 picograms of tritiated testosterone at working dilutions of 1:100,000 to 1:200,000. Both 5α and 5β-dihydrotestosterone compounds exhibited less than 2% cross-reaction. The only other steroids that showed minor cross-reaction were 11β-hydroxytestosterone (3.8%), progesterone (2.1%), corticosterone (1.6%), and deoxycorticosterone (7.7%).     

The detection and measurement of D-norgestrel in human milk using Sephadex LH 20 chromatography and radioimmunoassay

An accurate sensitive method for the assay of D-norgestrel in human milk is described. The steroid is isolated from an ether extract of milk by Sephadex LH 20 chromatography in the system iso-octane-benzene-methanol (70:20:10 v/v). The radioimmunoassay utilises a specific antibody produced in rabbits against D-'norgestrel 3-(O-carboxymethyl) - oxime coupled to bovine serum albumin with D-norgestrel 3-(0-carboxymethyl) -oxime/ [125I]-iodohistamine conjugate as radioligand. Accuracy, sensitivity and blank value are satisfactory. Milk samples were obtained from three subjects treated with 30 microgram/day D-norgestrel, treatment commencing two weeks following parturition. Significant amounts of D-norgestrel were found, ranging between 92-135 pg/ml milk at the end of the first two-week treatment regimen. In two of three subjects, lower, but significant concentrations (53 pg and 35 pg/ml respectively) of steroid were found at the end of four weeks treatment. In the third subject, D-norgestrel could not be detected at this time. As a check on the specificity of the assay, three samples were submitted to additional chromatographic purification on alumina thin layer in the system benzene-cyclohexane-ethanol (70:27:3 v/v). Although this additional chromatographic step yielded somewhat lower values, agreement between the respective sets of results was good. The significance and implications of these findings are discussed.     

An assay for urinary estriol-16α-glucuronide based on antibody-enhanced chemiluminescence

An immunoassay for estriol-16α-glucuronide in pregnancy urine is described that utilizes antibody-enhanced chemiluminescence. The steroid glucuronide was covalently conjugated with the chemiluminescent marker aminobutyl-ethyl-isoluminol. The light yield of this conjugate upon oxidation was augmented by specific antibody, and this effect was inhibited by addition of the homologous steroid glucuronide (10–100 pg) in a dose-dependent manner. The assay does not require separation of bound and free ligand and proved satisfactory with respect to sensitivity, precision and accuracy. Assay results obtained by radioinununoassay and chemiluminescence immunoassay were in good agreement (r = 0.98; n = 25).     

4-hydroxyestrone,isolation and identification in human urine

Portions of pregnancy and midcycle urines were submitted to hot acid hydrolysis, extracted with benzene/ethyl acetate and the extracts washed with ascorbic acid buffer. From the remaining organic phase the catecholestrogens were removed with borate buffer and further purified on Sephadex LH-20 columns. After derivatisation 4-hydroxyestrone was separated from the isomeric 2-hydroxy compound by gas chromatography. The mass spectra of the 4-hydroxyestrone peak were identical with that of authentic 4-hydroxyestrone. After treatment of the extracts with sodium borohydride 4-hydroxyestradiol-17β was identified by GC-MS. By the addition of trace amounts of tritiated 4-hydroxyestrone a recovery of 40% was calculated. On the basis of this recovery and the peak heights of the gas chromatograms an excretion of 4 μg (midcycle) and 40 μg (pregnancy) of 4-hydroxyestrone/24 h was estimated.     

Simultaneous radioimmunoassay of testosterone,dihydrotestosterone, 5α-androstane-3α, 17β-diol and 5α-androstane-3β, 17β-diol in the plasma of adult male rats

A single thin layer chromatography and three antibodies were used for the specific radioimmunoassay of four androgens in pooled rat plasma (Sprague-Dawley adult males). The following values were found (pg/ml ± SD). Testosterone : 3, 138 ± 173; dihydrotestosterone : 374 ± 20; 5α-androstane-3α 17β-diol : 284 ± 24; 5α-androstane-3β, 17β-diol : 223 ± 11.     

In vitro effects of estrogens on rat prostatic 5 alpha-reduction of testosterone

The inhibitory effects of a variety of estrogens on rat prostate testosterone Δ4–5α-reductase activity were measured by a specific $\text{in vitro}$ assay. The conversion of ³H-testosterone (initial concentration 2.8 × 10^?9 M) to labelled 5α-dihydrotestosterone and 5α-androstane-3α, 17β-diol was used as a measure of Δ4?5a-reductase activity. At a concentration of 1.8 × 10^?6 M, estradiol was the most potent inhibitor (83.4%) of the estrogens tested. Various ester derivatives, e.g. 3-acetate, 3-phosphate, were effective inhibitors. The 17-glucuronide and 3-sulfate conjugates were less effective inhibitors. The estriol isomers exerted similar degrees of inhibition (40–60%). The 3-methoxy derivatives of estradiol and estriol were poor inhibitors. The introduction of certain groups into the steroid structure, e.g. 15α-hydroxy and 6-ketone, greatly decreased the inhibitory effect of estradiol. The nature of the oxygen function at carbon 17 did not greatly influence the inhibitory effects.     

Effects of 4-hydroxy-4-androstene-3,17-dione and 10-propargylestr-4-ene-3,17-dione on the metabolism of androstenedione in human breast carcinoma and breast adipose tissues

The effects of 4-hydroxy-4-androstene-3,17-dione (4-OH-A) and 10-propargylestr-4-ene-3,17-dione (PED) on the aromatization of androstenedione (A) and the conversion of A to testosterone (T) were studied in incubations with breast carcinoma and breast adipose tissues. Parallel studies were carried out to determine the effects of 4-OH-A and PED on A metabolism in tissue from 5 patients with breast carcinoma. At 11 μM, both compounds fully inhibited aromatization, whereas the conversion of A to T was decreased in only 2 incubations.Studies with varying concentrations of 4-OH-A and PED demonstrated that both compounds inhibited estrone (E₁) formation by 80% at a concentration of 0.085 μM, with maximum effect at 0.34 μM. 90% inhibition of estradiol (E₂) formation was observed at inhibitor concentrations of 0.17 μM or greater. T formation was slightly affected at 0.67 μM, but was progressively inhibited with increasing 4-OH-A or PED concentrations, reaching 70% at 11 μM.Similar experiments with 4-OH-A in breast adipose tissue homogenates showed that a concentration of 0.1 μM was sufficient to inhibit aromatization while T inhibition required 11 μM.4-OH-A and PED are selective inhibitors of aromatization in human breast tissues and may provide a mechanism for controlling estrogen responsive processes.     

A radioimmunoassay for the measurement of 5-androstene-3β,17β-diol in plasma

A reliable radioimmunoassay for the determination of 5-androstene-3β, 17β-diol in plasma is described. Antisera were obtained by immunization of rabbits with 3β,17β-dihydroxy-5-androsten-16-one coupled to bovine serum albumin in position 16. The antiserum was characterized by titer, affinity, and specificity. Only dehydroepi-androsterone (24.3 %) and pregnenolone (2.7 %) showed a small crossreactivity. The assay method consisted of extraction with ether, thin-layer chromatography and endpoint determination.The reliability of the method was studied. The interassay variability was 7.5 % at a concentration of 1.22 μg/l. The limit of detection was 0.068 μg/l. Specificity was achieved by Chromatographic separation of the crossreacting steroids. Mass recovery experiments with 250 and 500 pg were performed, in which 99.0 ± 4.6 % of the smaller and 97.6 ± 11.3 % of the greater mass were recovered. In 45 healthy adult males plasma concentrations between 0.44 and 1.80 μg/l were found. The median was 1.06 μg/l. Stimulation of the Leydig cells with human chorionic gonadotropin (HCG) increased plasma concentrations by 93 % (average in 12 males). Therapeutic castration in 8 men caused an average decrease of 55.4 % in plasma values.     

Radioimmunoassay of serum medroxyprogesterone acetate (Provera) in women following oral and intravaginal administration.

A radioimmunoassay (RIA) method for measuring medroxyprogesterone acetate (MPA, Provera) in serum has been developed utilizing benzene:iso-octane extraction, 3H-MPA to assess procedural losses, goat anti-MPA-3-(0-carboxymethyl) oxime-bovine serum albumin serum and dextran-coated charcoal separation. Control serum blanks were undetectable, 200 pg/ml of MPA was measurable with a high reliability, and intra- and interassay coefficients of variation were 6 and 13 percent, respectively. MPA added to control serum was quantitatively recovered. Serum MPA levels measured in 2 women after ingestion of 10 mg MPA rose to 3.4 to 4.4 ng/ml within 1 to 4 hours after oral intake and fell rapidly thereafter to 0.3 to 0.6 ng/ml within 24 hours. Insertion of Silastic intra-vaginal rings (IVRs), containing 100 or 200 mg of MPA, into 4 women for periods of 3 weeks resulted in a rapid rise of serum MPA after insertion, rather stable MPA levels of 0.9 to 1.6 ng/ml while the IVRs were in place, and a rapid decline of serum MPA following IVR removal. Serum estradiol-17beta and progesterone concentrations, measured about 3 times a week in these patients, indicated that ovulation was consistently inhibited. The serum MPA levels observed in this study were approximately 5 times lower than those reported by other investigators using a double-antibody RIA of MPA in unextracted serum.