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.     

Steroid levels after intramuscular injection of radioactive estradiol-17beta, estrone, progesterone and testosterone in the bovine

Eight 2 year old Hereford cows from days 8 to 12 of the estrous cycle were injected intramuscularly with 5 ml of corn oil containing 5 mg of estradiol-17beta (two cows), estrone (two cows), progesterone (two cows) or testosterone (two cows). Each cow treated with estradiol received 494 microc of estradiol-17beta-6, 7 H3 and each cow treated with estrone received 492 microc of estrone-6, 7 H3. Each cow treated with progesterone or testosterone received 400 muc of H3 compound labeled in the 7 position. Total urine was collected by urethral catheterization of the cows treated with estrogens. Blood samples for plasma and serum were collected via jugular cannulae. Blood and urine samples from estrogen-treated cows were collected hourly for the first 24 hr, at 2 hr intervals for the next 26 hr, at 4 hr intervals for the next 12 hr and at 12 hr intervals until background was reached. Blood samples were collected hourly from 1 to 8 hr after injection from progesterone or testosterone-treated cows. Plasma and serum levels of radioactive estradiol-17beta, estrone, progesterone and testosterone were similar. Blood levels of radioactivity peaked at 2 hr post-injection in cows receiving estradiol-17beta and at 3 hr in cows receiving estrone. Blood levels of labeled estradiol-17beta and estrone were nondetectable by 54 hr and 83 hr, respectively. Peak urinary excretion of radioactivity was reached at 7 hr for estradiol-17beta and at 14 hr for estrone and nondetectable levels were reached by 95 hr for estradiol-17beta and 14 hr for estrone. At these times, 15.5% of the total dose of radioactive estradiol-17beta and 17.5% of the injected estrone had been excreted in the urine. Peak blood and urinary excretion levels were reached earlier for radioactive estradiol-17beta than for estrone, and excretion of estradiol-17beta was completed more rapidly. No difference was found in plasma and serum levels for any steroid studies; thus, endogenous steroid titers in blood plasma and serum are not different in the cow.     

The effect of estradiol-17beta and estrone administration on GnRH-induced LH release during the early postpartum in dairy cattle

The effect of high plasma concentrations of estradiol-17beta or estrone, similar to those observed in late gestation, on the gonadotropin releasing hormone (GnRH)-induced luteinizing hormone (LH) release was studied in early postpartum dairy cows. Twenty dairy cows in late gestation were assigned to four groups of five cows each. Treatment groups were 1) no exogenous estrogens, 2) 20 mg estradiol-17beta (E(2)beta) daily, 3) 30 mg estrone (E(1)) daily and 4) 20 mg E(2)beta and 30 mg E(1) daily. Steroids were dissolved in ethanol (vehicle). Injections of the vehicle or steroids were given in two daily subcutaneous injections for seven consecutive days starting immediately following parturition. All cows (Groups 1-4) were given 100 mug GnRH intramuscularly on days 2, 10, 18 and 26 postpartum. Blood for plasma determination of E(2)beta, E(1), progesterone (P) and LH was collected daily from parturition to completion of vehicle or steroid injection and on alternate days thereafter. In addition, blood was collected on GnRH treatment days prior to GnRH and at 30-min intervals thereafter for four hours. Concentrations of hormones were determined by validated radioimmunoassays (RIA's). Effects of treatment (T), days postpartum (D) and the interaction between T and D (T x D) on the amount of LH released (area under the curve) in response to GnRH were significant (P < 0.01). More LH was released over all days combined in Group 1 compared to the other groups. LH release to GnRH increased as time postpartum increased in Groups 1 and 3, but at a ratelower for Group 3 than Group 1 (P < 0.05). In contrast, LH release to GnRH was greater (P < 0.05) on day 2 postpartum for Groups 2 and 4 compared to Groups 1 and 3, but less on days 10 and 18 postpartum. Average LH release was less (P < 0.05) on day 10 for Groups 2 and 4 than for day 2 postpartum. By day 26 postpartum, however, LH release in Groups 2 and 4 was greater than in Group 3. In summary, E(2)beta appeared to stimulate LH release early postpartum with a subsequent inhibition of LH release after prolonged E(2)beta administration, and E(1) administration did not stimulate LH release early postpartum.     

In vivo studies on the metabolism of estrone and estradiol-17beta by the brain

³H-Estrone and H-estradiol-17β were infused in separate experiments into the jugular veins of each of 4 ewes. During the infusions blood samples were obtained from the ipsilateral jugular vein and common carotid artery. The blood samples were analyzed for radioactivity as free estrone and estradiol-17β and the conversion of infused precursor to product steroid by brain tissue, the transtissue conversion (ρ^PRE-PRO_AV) and the extraction by brain tissue of infused precursor, the transtissue extraction (1-ρ^PRE-PRE_AV) and the metabolic clearance rates were calculated.The mean ± SE for ρ_AV^1,2 (precursor, estrone = 1; product, estradiol = 2) was 0.09 ±0.03 and the mean ± SE for ρ_AV^2,1 (precursor, estradiol = 2; product, estrone = 1) was 0.08 ±0.02. The mean trans-tissue extraction of estrone was 0.13 ± 0.02 and of estradiol-17β was 0.14- ± 0.02. The transtissue extractions of estrone and estradiol-17β were greater than ρ_AV^1,2 ρ_AV^2,1 respectively in 2 of the 4 ewes.Brain metabolism of estrogens can account for only 2–4% of the total metabolism of these free estrogens from the blood pool.     

Contrasting effects of estradiol-17 beta and 17 alpha-ethinyl estradiol-17 beta on cultured whole embryos

Estradiol-17 beta (E2) and 17 alpha-ethinyl estradiol-17 beta (EE) were compared in terms of their relative capacities to alter growth and developmental patterns of cultured whole embryos during the early stages of organogenesis. Embryos exhibited a notable differential susceptibility to the embryotoxic effects of parents E2 vs EE when these estrogens were added directly to the media at the onset of the culture period. At initial concentrations of 0.1 mM, E2 failed to produce statistically significant effects whereas EE elicited marked embryotoxicity. Inclusion of a P-450-dependent biotransformation system in the culture media resulted in a significant attenuation of the embryotoxic effects of parent E2 vs EE when these estrogens were added directly to the media at the onset of the culture period. At initial concentrations of 0.1 mM, E2 failed to produce statistically embryotoxicity by hepatic S9. The divergent results produced by the two steroids could not be attributed to differences in rates of catecholestrogen generation in the culture medium or by the conceptuses. The results demonstrate definitive dissimilarities between the effects of two steroidal estrogens on developmental parameters and document marked differences in the effects of biotransformation on their embryotoxic potential. The data strongly suggest that the embryotoxicity of these steroids is not mediated via interactions with estrogen receptors. Additionally, the data show that the differential capacity of these two steroids to produce embryotoxic effects is diametrically opposite to earlier reported patterns of their carcinogenic potential in the Syrian hamster kidney.     

The rate of estrone production by mouse blastocysts increases with estradiol-17 beta concentration in medium

Following the finding of 17 beta-hydroxysteroid dehydrogenase activity in mouse blastocysts, the present study examined the relation of estrone (E1) production rate to estradiol-17 beta (E2) concentration in the medium. When Day 5 blastocysts were cultured in 107-6,860 ng E2/ml medium, the amount of E1 formed during the first 5 hours (y) was found to increase linearly with the logarithm of E2 concentration (x), as represented by the equation y = 2,161x - 3,947. However, there was a sharp decline during the next 5 hours of culture except for the 107 ng E2 culture. The E1 production then remained steady for up to 46-58 hours. There was a tendency for further decline during the 46-70-hour period. The results indicate that (1) E2 may be metabolized to E1 and, probably, another unknown steroid; (2) E1 production rate is E2-dose dependent; and (3) the blastocyst possesses the enzymatic capability to change the steroid milieu to suit its own needs and/or to cause local effects in the uterus for its proper implantation.     

In vitro biosynthesis of radioactive estradiol-17 beta, 17-glucosiduronate by rhesus monkey liver.

A method for the preparation of radioactive estradiol-17 beta, 17-glucosiduronate by incubating 3H-estradiol with rhesus monkey liver microsomal preparation in the presence of uridine diphosphoglucuronic acid is described. Small but significant amounts of the conjugate were also obtained from the 150,00 pellet and cytosol fractions. The addition of NADPH to the incubation media increased the yield of radioactive-estradiol-17 beta, 17-glucosiduronate perhaps by preserving the integrity of the C-17-hydroxyl group. As expected, the effect of the reduced nucleotide was more pronounced in the fractions other than the microsome. The biosynthesized conjugate was isolated and purified by multiple column chromatography and the structure was confirmed by derivative formation, enzyme hydrolysis and crystallization of the aglycone.     

Simultaneous quantification of progesterone, estrone, estradiol-17 beta and corticosterone in female American kestrel plasma

Progesterone, estrone, estradiol-17 beta and corticosterone were quantified simultaneously for the first time in female American kestrel (Falco sparverius) plasma. A mean level for each hormone was determined for the laying and non-laying periods of the summer (April-September), and for February. Means were comparable to those of other wild avian species and were significantly higher for the laying period than for the other 2 periods. The mean corticosterone level for February was higher than that for the non-laying summer period. Plasma from laying kestrels, unlike that from other avian species, required lipid removal before column chromatography. Of 2 lipid removal techniques compared, i.e. the cold methanol and hexane:methanol techniques, the latter proved superior.