Synthesis of novel europium-labeled estradiol derivatives for time-resolved fluoroimmunoassays.

The O-(5-carboxypentyl)-, O-(4-aminobutyl)-, O-(6-aminohexyl)oximes of 2- and 4-formylestradiol as well as the 4-carboxyethylthioether derivative of estradiol were synthesized. These estradiol derivatives were characterized using IR-, 1H-, and 13C NMR spectroscopy. All synthesized estradiol derivatives were labeled with europium chelates. These "tracers" were purified and tested in a competitive time-resolved fluoroimmunoassay with monoclonal antibody (SSI 57-2) raised against the 6-O-(carboxymethyl)oxime-bovine serum albumin derivative of estradiol. All the tested europium-labeled estradiol-4-derivatives were bound by the antibody, whereas tracers linked via position 2 were not recognized by this antibody. It was observed that tracers conjugated via C-4 gave more sensitive standard curves than tracers conjugated via C-6. Especially, the estradiol-4-thioether derivative was found to be highly useful in time-resolved fluoroimmunoassays of estradiol while using this antibody.     

Development of a sensitive, direct luminescent enzyme immunoassay for plasma estradiol-17 beta

A rapid, sensitive, precise, chemiluminescent enzyme immunoassay for estradiol-17 beta has been developed and validated. Antibodies were produced in rabbits using estradiol-17 beta-6-(O-carboxymethyl)oxime coupled to bovine serum albumin, purified and immobilized on polystyrene beads (6.4 mm diameter). The same derivative was used to prepare the enzymatic tracer by coupling with horseradish peroxidase. The assay, direct on the serum sample, featured a 4-h binding step at 4 degrees C followed by the chemiluminescent detection using luminol/H2O2. The detection limit was 0.15 pg/tube and the assay was carried out on 20-100 microliter of sample, allowing measurement of estradiol-17 beta in plasma concentrations from 1.5 to 500 pg/ml. The method fulfills all the standard requisites of precision and accuracy and the results agree well with a radioimmunoassay procedure on extracted serum.     

Sensitive liquid chromatographic method using fluorescence detection for the determination of estradiol 3- and 17-glucuronides in rat and human liver microsomal incubations: formation kinetics

We have developed a sensitive and specific HPLC-fluorescence assay for the determination of estradiol-3-glucuronide and estradiol-17-glucuronide in human and rat liver microsomal incubations. The method utilizes a mobile phase comprised of acetonitrile and 50 mM ammonium phosphate buffer (35:65, v/v) that is pumped though a phenyl column at 1 ml/min; the run time is less than 15 min. Calibration curves for both metabolites were linear over the range 20-4000 pmol. The intra- and inter-day coefficients of variation were <6%. In both rat and human liver microsomes, the formation of estradiol-3-glucuronide displayed atypical kinetics (consistent with activation), while estradiol-17-glucuronide formation was consistent with classical Michaelis-Menten kinetics. Overall, the assay described is a sensitive and reproducible method for the determination of estradiol glucuronides in liver microsomal preparations.     

Synchronization of follicular wave emergence in the seasonally anestrous ewe: the effects of estradiol with or without medroxyprogesterone acetate

Fertility is often lower in anestrous compared to cyclic ewes, after conventional estrus synchronization. We hypothesized that synchronization of ovarian follicular waves and ovulation could improve fertility at controlled breeding in anestrous ewes. Estradiol-17beta synchronizes follicular waves in cattle. The objectives of the present experiments were to study the effect of an estradiol injection, with or without a 12-d medroxyprogesterone acetate (MAP) sponge treatment, on synchronization of follicular waves and ovulation in anestrous ewes. Twenty ewes received sesame oil (n=8) or estradiol-17beta (350 microg; n=12). Eleven ewes received MAP sponges for 12d and were treated with oil (n=5) or estradiol-17beta (n=6) 6d before sponge removal. Saline (n=6) or eCG (n=6) was subsequently given to separate groups of ewes at sponge removal in the MAP/estradiol-17beta protocol. Estradiol treatment alone produced a peak in serum FSH concentrations (4.73+/-0.53 vs. 2.36+/-0.39 ng/mL for treatment vs. control; mean+/-S.E.M.) after a short-lived (6 h) suppression. Six of twelve ewes given estradiol missed a follicular wave around the time of estradiol injection. Medroxyprogesterone acetate-treated ewes given estradiol had more prolonged suppression of serum FSH concentrations (6-18 h) and a delay in the induced FSH peak (32.3+/-3.3 vs. 17.5+/-0.5 h). Wave emergence was delayed (5.7+/-0.3 vs. 1.4+/-0.7d from the time of estradiol injection), synchronized, and occurred at a predictable time (5-7 vs. 0-4d) compared to ewes given MAP alone. All ewes given eCG ovulated 3-4d after injection; this predictable time of ovulation may be efficacious for AI and embryo transfer.     

Estrogen metabolism in the Asian elephant (Elephas maximus)

Estradiol-17β metabolism was studied in two female Asian elephants (Elephas maximus). In an initial study, 500 μCi of tritiated estradiol-17β was injected iv into a single animal, and 0, 30 and 60 min serum samples were collected as well as all excreted urine and feces for 24 hr. In a second study, 1.5 mg unlabeled estradiol-17β was injected iv into a second animal and 0, 5, 15, 30, and 60 min serum samples and a 30 min urine sample were collected postinjection. Analyses of samples from both studies demonstrated a rapid conversion of free estradiol to conjugated forms in the serum. The first (5 min) serum sample following the injection of unlabelled estradiol contained unconjugated estradiol: conjugated estradiol: conjugated estrone at a ratio of 60: 29: 10, respectively, and at 30 min a ratio of 33: 43: 24. The urinary estrogen metabolites were in the conjugated form with an estradiol: estrone ratio of 60: 40. No radiolabeled estrogen was found in the fecal samples during the 24 hr following administration of the radiolabeled estradiol. These data indicate a rapid clearance of circulating free estradiol in the elephant, with a major metabolite in the serum and urine being estradiol conjugate. © 1992 Wiley-Liss, Inc.     

Radioimmunoassay for Zearalenone and Zearalanol in Human Serum: Production, Properties, and Use of Porcine Antibodies

To produce antigens susceptible to raise antibodies for resorcylic acid lactones, the 6′-carboxymethyloxime derivatives of zearalenone and zearalanone were bound to bovine serum albumin. Pigs could be immunized by using these antigens, the best titer in antibodies being obtained with the zearalenone antigen. The porcine antibodies were specific for the resorcylic acid lactones of structural resemblance with zearalenone. This specificity made the antibodies usable for a radioimmunoassay of zearalenone and zearalanol, which may be found in human and animal sera. The range of the assay was between 0.25 and 10 ng. The limit of detection was 5 ppb (5 ng/ml) in human serum.     

Competitive protein-binding assay of estrone, estradiol-17 or estradiol-17 in human or ruminant plasma

A procedure for the assay of estrone, estradiol-17β or estradiol-17α in plasma is described. The technique also appears applicable to the assay of estriol in plasma. The procedure uses a semi-automatic extraction of plasma, rapid micro-alumina column chromatography and competitive binding of the estrogens to stable proteins of sheep uterine cytosol. The use of alumina column chromatography results in consistently low blanks. The assay has been evaluated for the measurement of estradiol-17β and estrone in human and sheep plasma, and for estradiol-17α and estrone in goat plasma. The change in binding affinity of estradiol-17α relative to estradiol-17β when incubated in sheep uterine cytosol at two different temperatures (25°C and 4°C), makes it possible to differentiate the two epimers of estradiol. Measurement of estradiol-17β down to 10 pg and of estrone and estradiol-17α to 25 pg are maintained in routine analyses. The specificity of the procedure was thoroughly checked by various methods, including comparison with spectrophotofluorimetric analysis.     

Metabolism of sex hormones in the aortic wall

The metabolism of labeled sex hormones was examined in human, canine, and rat aortas. Isolated arterial tissue converted estrone to estradiol, estradiol to estrone, and estrone sulfate to estrone and estradiol. The arterial wall also appeared to metabolize testosterone to androstenedione and an unidentified, relatively nonpolar derivative. Both estrogens and testosterone appeared to enter the arterial wall rapidly. No competition in arterial uptake between the two hormones was apparent. No specific arterial binding of estradiol could be demonstrated. The concentration of estradiol-(3)H in the canine aorta exceeded that in the plasma 1-6 hr after estradiol-(3)H administration. The uptake and disappearance of estradiol-(3)H in the aorta generally resembled the patterns observed in body tissues other than the adrenal gland and uterus. The uptake of estradiol-(3)H was greatest in the adrenal gland while its retention was maximum in the uterus.     

Luteinizing hormone concentrations in anestrous ewes administered various estrogens

Twenty four anestrous ewes were evenly assigned to one of six groups and administered either sesame oil, estradiol-17β, estradiol-17α, estrone, estradiol benzoate or estradiol valerate. All estrogen treated ewes received 50 μg of the respective estrogen. Blood plasma was collected for 28 hours post-treatment and quantified for luteinizing hormone (LH) by radioimmunoassay. An estrogen induced LH surge was detected in at least three of the four ewes administered either estradiol-17β, estrone, estradiol benzoate or estradiol valerate whereas only one of the four estradiol-17α treated ewes and none of the ewes administered sesame oil had an LH surge. The interval from treatment to peak LH was similar for estradiol-17β (17.3±2.7 hours), estrone (18.5±1.0 hours) and estradiol benzoate (19.0±0.6 hours) treated ewes but delayed 7 to 9 hours for ewes administered estradiol valerate (26.0±1.2 hours).     

Functional characterization of two anti-estradiol antibodies as deduced from modelling and site-directed mutagenesis experiments

Monoclonal antibodies are now widely used to measure the concentration of steroid hormones in human serum samples. The great development of molecular engineering techniques over the past 10 years has made possible the improvement of specificity and/or sensitivity of selected antibodies. We have obtained two monoclonal antibodies, 17E12E5 and 10G6D6, using estradiol-6-ethyl methoxy carbonyl (EMC)-bovine serum albumin (BSA) as immunogen. To tentatively improve their affinities for natural estradiol, we have initiated their structural and functional studies. For this purpose, we have cloned and sequenced the genes encoding the variable fragments of each antibody. Single chain variable fragments (scFv) were produced into the periplasmic space of E. coli using the pLIP6 expression vector. Mapping of the functional structures of both antibodies was obtained by combination of modelling and mutational analyses together with cross-reaction studies. The two binding pockets are described and models of estradiol complexed to 17E12E5 and 10G6D6 are proposed.     

Active immunization of female rats against 17 beta-estradiol. Preliminary studies on 17 beta-estradiol binding in uterine and pituitary cytosols

Female rats were immunized with 17 beta-estradiol-6-carboxymethyloxime-bovine serum albumin. They developed antibodies to estradiol and, to a very low extent, antibodies to BSA. Anti-estradiol antibodies possessed tight specificity to estradiol-17 beta, without cross-reactivities with other estrogens. It was demonstrated that the specific estradiol binding in uterine and pituitary cytosols gradually decreased when antiserum titres increased. In uterine cytosols, the presence of progesterone receptor was studied using promegestone (R50 20) as ligand. No significant variations in promegestone binding were observed. Competition experiments however, questioned the permanence in immunized rats of the actual progesterone receptor or of a promegestone binding protein.     

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.     

Effect of 3-(3,4-dihydro-6-methoxy-2-naphthyl)2,2-dimethyl pentanoic acid--an antifertility agent--on in vitro uptake of estradiol 17beta-6, 7(3)-H in rat uterus.

The effect of 3-(3,4-dihydro-6-methoxy-2-naphthyl)-2,2-dimethyl-pentanoic acid, a potent nonsteroidal antifertility compound, on the uptake of estradiol-17beta-6, 7-tritiated in vitro in the rat uterus was studied. The estradiol uptake of estrogen-primed and compound-treated groups were the same. When estradiol and the compound were present in medium at the same time, estradiol uptake was significantly (p less than .01) increased. The results indicate that the compound synergizes the effect of estradiol.     

Measurement of excreted steroids in Macaca nemestrina

A practical method for the quantitative measurement of the estrogenic steroid estradiol-17β in the feces of pigtailed macaques (Macaca nemestrina) is described. The method, which includes homogenization, filtration, ether extraction, and sephadex purification, produces an 85.3% recovery of 3H-estradiol. Comparable recovery of 3H-progesterone (64.1%) and 3H-testoster-one (67.9%) is also obtained. Estradiol, measured by radioimmunoassay methodology, has inter- and intra-assay variations of 7.1% and 4.3%, respectively. Linearity of estradiol was checked by varying the amount of feces analyzed. Results show a correlation of 0.9945, Y = 32.526x + 0.205. Accuracy of estradiol, determined by adding radioinert estradiol to the feces before homogenization, was 98.7% with a correlation of 0.9950, Y = 1.027x ? 0.543. The menstrual cycles of several animals were analyzed and corresponded well with serum levels of estradiol-17β. Similar correspondence was obtained in matched fecal and serum samples from pregnant animals. These methods provide a practical and accurate solution to the problem of collecting hormone data in field studies without the potential complications of capture in laboratory research.     

Chemical synthesis of two novel diaryl ether dimers of estradiol-17beta

We recently detected the formation of estradiol-17beta (estradiol) dimers, linked together through a diaryl ether bond between the C-3 phenolic oxygen of one estradiol molecule and the 2- or 4-position aromatic carbon of another estradiol, following incubations of [3H]estradiol with human liver microsomes or cytochrome p450 enzymes in the presence of NADPH. Using estradiol as the starting material, we designed a four-step method for the chemical synthesis of these two estrogen dimers with the Ullmann condensation reaction as a key step. Step 1: Synthesis of 2- or 4-bromoestradiol from estradiol. Step 2: Protection of the C-3 phenolic hydroxyl group of the 2- or 4-bromoestradiol. Step 3: The Ullmann condensation reaction between the phenol-protected bromoestradiol and the estradiol potassium salt under our modified reaction conditions (with a 41% product yield). Step 4: Removal of the C-3 benzyl group by catalytic hydrogenation. The chromatographic and various spectrometric properties of the two synthesized compounds were identical to those metabolically formed by human cytochrome p450 3A4.     

Functional characterization of an anti-estradiol antibody by site-directed mutagenesis and molecular modelling: modulation of binding properties and prominent role of the V(L) domain in estradiol recognition

The high-affinity monoclonal anti-estradiol antibody 9D3 presents a specificity defect towards estradiol-3-sulphate and 3-glucuronide conjugates incompatible with use in direct immunoassays. The corresponding single-chain variable fragment (scFv), cloned and produced in E. coli, exhibited a 10-fold lower affinity for estradiol (K(a)=1.2 x 10(9) M (-1)) and a slightly increased specificity defect for the 3-position. Site-directed mutagenesis revealed critical residues involved in estradiol recognition and produced mutants exhibiting up to a 3-fold increase of the binding affinity for estradiol and up to a 2-fold decrease of the cross-reactivity with estradiol-3-sulphate. A comparative model of the antibody 9D3-estradiol complex was built in which the estradiol D-ring is buried into the binding pocket while the 3-, 6- and 7-positions are solvent exposed, agreeing with the lack of specificity for these three positions. Two potential alternative orientations of the A-ring, one close to CDR H3 and L2 loops, and the other one close to CDR H2 and L3 loops, have been considered for the docking of estradiol, none of which could be unambiguously privileged taking into account data from cross-reactivity measurements, photolabelling and mutagenesis studies. For both orientations, estradiol is stabilized by hydrogen bonding of the 17beta-OH group with TyrL36, His89 and GlnH35 in the first case, or TyrL36, only, in the second case and by van der Waals contacts from TyrL91 with alpha- or beta-face of estradiol, respectively, and from ValH95 and GlyH97 with the opposite face. To elucidate the molecular basis of antibody 9D3 specificity, as compared with that of another anti-estradiol antibody 15H11, single variable domains (V(H) and V(L)) and scFv hybrids have been constructed. The binding activity of V(L)9D3 as well as the specificity of the V(L)9D3/V(H)15H11 hybrid, both similar to antibody 9D3, revealed a prominent role of V(L) in estradiol recognition. These findings establish premises for antibody engineering to reduce cross-reactivity, especially with estradiol-3-conjugates.     

Biotinyl-estradiol derivatives in enzyme immunoassays: structural requirements for optimal antibody binding

The use of the avidin/biotin complex in immunoassays is well documented. No comprehensive studies, however, are available on the structural requirements of the linkage between biotin and small molecules to get an optimal antigen-antibody interaction. We have synthesized seven different biotinylated estradiol derivatives. They were evaluated in an antibody- and in an antigen-immobilized enzyme immunoassay system. All three derivatives lacking a spacer group were useless for use in immunoassays, demonstrating the importance of a long distance between the biotin- and estradiol-moiety. In addition, the chemical structure of the linkage at the site of attachment to the steroid skeleton is very important for the antibody recognition: it may either be rigid but identical to that one used in the immunogen (6-carboxymethyloxime), or must be structurally flexible as exemplified by a 6-amido-linkage. A rigid structure (hydrazone) different from that of the immunogen absolutely prevents antibody binding.     

Structure-activity relationships of estrogens: effects of esterification of the 11 beta-hydroxyl group

Fourteen esters (formate, acetate, propionate, butyrate, hexanoate, heptanoate, and benzoate) located at C-11 of 11 beta-hydroxyesterone and 11 beta-hydroxyestradiol-17 beta were synthesized and evaluated for uterotropic and gonadotropin release inhibition in rats, as well as their ability to displace (3H) estradiol-17 beta from the rat uterine cytosolic estrogen receptor. The most potent uterotropic agent was 11 beta-formoxyestrone which was 1,625 or 2,500 times as active as 11 beta-hydroxyesterone in the uterotropic or gonadotropin release inhibition assay, respectively. 11 beta-Formoxyestrone was 7.5 times as uterotropic as estradiol-17 beta and equal to estradiol-17 beta in inhibiting gonadotropin release. However, the most potent inhibitor of gonadotropin release was 11 beta-acetoxy-estradiol-17 beta which had 133% of the activity of estradiol-17 beta, although it had only 38% of the activity of estradiol-17 beta in the uterotropic assay. Esters larger than the acetoxy group showed sharply decreased activities in either assay. Despite the high estrogenic potency of the 11-formates or 11-acetates, they were rather weak (6% to 35% as active as estradiol-17 beta) in displacing (3H) estradiol-17 beta from the rat uterine cytosolic estrogen receptor.     

A double antibody radioimmunoassay for free and conjugated estradiol-17 beta in cow's milk

A radioimmunoassay for free estradiol-17 beta, conjugated estradiol-17 beta or total (free + conjugated) estradiol-17 beta in defatted milk of cows is described. Conjugated estradiol-17 beta was hydrolyzed by enzymes of Helix pomatia juice. Estrogens were extracted with dichloromethane; no other purification step was required before radioimmunoassay because of the high specificity of the antiserum. Immunoprecipitation was used to separate bound and free estradiol-17 beta. Concentrations measured were corrected for procedural losses on a per sample basis. The assays were shown to be accurate and specific. The sensitivity was 1.3pg/ml for the assay of free estradiol-17 beta (5ml of milk extracted) and 2.9pg/ml for conjugated or total estradiol-17 beta (2 ml of milk hydrolyzed and extracted). Estrogens were measured in the milk of cyclic cows and in cows stimulated with pregnant mare serum gonadotropin (PMSG). A preovulatory increase was clearly observed. Wether or not the ovary was stimulated by PMSG, concentrations of estrogens were higher and the relative increase during the preovulatory peak was greater for conjugated estradiol-17 beta than for the free form. The assay of conjugated or total estradiol-17 beta in defatted milk should be a practical method for assessing preovulatory growth of follicles in cows.     

Superovulatory and endocrine responses in heifers treated with FSH-P at different stages of the estrous cycle

Forty-two Holstein heifers were superovulated with FSH-P (total dose, 30 mg) and cloprostenol. Treatment was initiated on Day 3 (Group D3, n = 11), Day 6 (Group D6, n = 11), Day 9 (Group D9, n = 10) or Day 12 (Group D12, n = 10) of the estrous cycle. Heifers were bled daily for serum progesterone and estradiol-17beta determinations and every 6 h for a 48-h duration at the expected time of estrus for luteinizing hormone (LH) assay. Ova and embryos were flushed from the reproductive tracts and the number of corpora lutea (CL) were recorded after slaughter on Day 7 post-estrus. Mean (+/- SEM) numbers of observed CL were higher (P < 0.05) in Group D9 (33.3 +/- 4.8) than in Group D3 (15.3 +/- 3.8), with Group D6 (17.0 +/- 2.9) and Group D12 (23.9 +/- 7.3) being intermediate. Similarly, mean (+/- SEM) numbers of fertilized embryos were highest (P < 0.05) in Group D9 (13.3 +/- 2.2). There was also a nonsignificant trend for the number of transferable embryos to be greatest in Group D9. Neither serum progesterone concentrations 3 d after the LH peak nor peak serum estradiol 17beta concentrations differed among groups, but both were significantly correlated with numbers of observed CL and total ova and embryos.