Estradiol isolation

Testosterone and estradiol are present m the body m only minute amounts and their isolation and identification required heroic efforts In order to obtain 0 012 g of estradiol for study for example 4 tons of sow ovaries had to be extracted ... 

WS-7528 [132147-69-4][VI] a nonsteroidal estrogen, is an isoflavone which has been isolated from Streptomjces sp. No. 7528 and is an estrogen agonist. It inhibits [3ff]-estradiol binding to its receptor in rat uterine cytosol at an inhibitor for 50% of the rats tested (IC q) concentration of 5.7 nM. It also induces the growth of estrogen-dependent human breast cancer cell line MCE-7 (7). 

The general reaction procedure and apparatus used are exactly as described in Procedure 2. Ammonia (465 ml) is distilled into a 2-liter reaction flask and to this is added 165mlofisopropylalcoholandasolutionof30g(0.195 mole) of 17/ -estradiol 3-methyl ether (mp 118.5-120°) in 180 ml of tetrahydrofuran. The steroid is only partially soluble in the mixture. A 5 g portion of sodium (26 g, 1.13 g-atoms total) is added to the stirred mixture and the solid dissolves in the light blue solution within several min. As additional metal is added, the mixture becomes dark blue and a solid (matted needles) separates. Stirring is inefficient for a few minutes until the mass of crystals breaks down. All of the sodium is consumed after 1 hr and 120 ml of methanol is then added to the mixture with care. The product is isolated as in Procedure 4h 2. After being air-dried, the solid weighs 32.5 g (ca. 100% for a monohydrate). A sample of the material is dried for analysis and analyzed as described in Procedure 2 enol ether, 91% unreduced aromatics, 0.3%. The crude product may be crystallized from acetone-water or preferably from hexane. 

Although estrone and estradiol (26) have both been isolated from human urine, it has recently been shown that it is the latter that is the active compound that binds to the so-called estrogen receptor protein. Reduction of estrone with any of a large number of reducing agents (for example, any of the complex metal hydrides) leads cleanly to estradiol. This high degree of stereoselectivity to afford the product of attack at the alpha side of the molecule is characteristic of many reactions of steroids. 

To a solution of 2.3 ml of phosphorus oxychloride in 50 ml of dry pyridine is added a solution of 2.2 grams of 3-N-bis((3-chloroethyl)carbamate of estradiol while stirring and at a temperature of about -10°C. The reaction mixture is allowed to stand at about 0°C for I /i hours, whereupon it is hydrolyzed by pouring it into ice-water. The main part of the pyridine is evaporated in vacuo, whereupon the residue is poured into 100 ml of cold 3.5 N hydrochloric acid with stirring. The precipitate thus obtained is isolated and washed with 0.1 N hydrochloric acid and water. 

Fig. 12.2. Comparison of ATP-dependent transport activity between rats and humans determined in isolated bile canalicular membrane vesicles. Key 1, SN-38 glucuronide (carboxylate) 2, SN-38 glucuronide (lactone) 3, E3040 (6-hydroxy-5,7-dimethyl-2-methyl-amino-4-(3-pyridylmethyl) benzothiazole) glucuronide 4, 170 estradiol-170-D-glucuro-nide 5, grepafloxacin glucuronide 6, leuko-...

Important studies were performed by Trush and coworkers [42], who showed the advantages of applying lucigenin-amplified CL for the measurement of superoxide production by mitochondria in unstimulated monocytes and macrophages as well as by isolated mitochondria [43,44]. Later on, these authors have shown that mitochondrial superoxide production measured by lucigenin-amplified CL increased in the liver of rats treated with the promoter of hepatocarcinogenesis ethinyl estradiol [45], in liver from obese mice [46], and in children with Down syndrome [47]. 

Estradiol. The first neuroactive steroid receptor type to be recognized was that for estradiol [3]. In vivo uptake of [3H] estradiol, and binding to cell nuclei isolated from hypothalamus, pituitary and other brain regions, revealed steroid specificity closely resembling that of the uterus, where steroid receptors were first discovered [3]. Cytosolic estrogen receptors isolated from pituitary and brain tissue closely resemble those found in uterus and mammary tissue. A hallmark of the estrogen receptor is its existence... 

Teo S, Vore M. 1990. Mirex exposure inhibits the uptake of 17beta-estradiol (beta-D-glucuronide), taurocholate, and L-alanine into isolated rat hepatocytes. Toxicol Appl Pharmacol 104(3) 411- 420. 

In in vitro studies, weak estrogenic effects of bisphenol A were found in cell line MCF7, which was established from human breast cancer cells, and in studies with cytosol preparations from isolated uteri. InMCF7 cells, the estrogenic effects were seen at 1-5 ng/ml and were manifested as in increase in cell proliferation and the induction of progesterone receptors. Bisphenol A was 1000-5000 times less potent than estradiol-17. 

In some instances, combinations of Cig and silica columns are also used for better purification of the crude extracts (431, 445). A combination of Cg, silica, and amino solid-phase extraction columns has been successfully employed to fractionate anabolic and catabolic steroid hormone residues from meat in polar and nonpolar neutral and phenolic compounds, and to purify further each fraction effectively (452). Another combination of two solid-phase extraction columns, one using a graphitized carbon black sorbent and the other Amberlite resin in the hydroxyl form, allowed neutral anabolics to be isolated and separated from acidic anabolics and their metabolites (453). A combination of basic alumina column placed in tandem with an ion-exchange column has also been applied for the purification of the crude extracts in the determination of diethylstilbestrol and zeranol (427), and estradiol and zeranol in tissues (450). 

Female Hormones. Closely related 10 the male androgenic hormones, and probably synthesized from them in the female organism, are the estrogenic hormones which are produced principally in the ovary. Although / -estradiol is die normally secreted ovarian hormone, a number or ulher estrogenic substances have been isolated from urine and from animal studies. They include o-cstradiol. esiriol, and estrone. The structures of these hormones are given in Fig. 2. 

Before Edward Doisy earned the Nobel Prize for his research on vitamin K, he studied female hormones. It was at this time in 1936 that he isolated estradiol. In rodents without ovaries,... 

On the academic side, W. H. Perkin made mention of unsuccessful experiments directed towards the preparation of benzocyclobutene in 1888. This compound was eventually prepared by H. Finkelstein, a student of J. Thiele, in 1909, as reported in his long-unknown Ph.D. thesis. Benzocyclobutene and biphenylene were also reported by Lothrop in 1941. Tropylium bromide was synthesized in 1891 by Merling, but its structure was only established in 1954 by Doering and Knox. The isolation and structure determination of the aromatic steroids estrone, estriol, and estradiol arrived only in 1929 (E. A. Doissy, A. Bute-nandt), 1931 (G. F. Marian), and 1935 (E. A. Doissy), respectively, with the advent of X-ray crystallography. 

When steroid hormones were first isolated, people believed that no synthetic hormone could rival the astonishing potency of natural steroids. In the past 50 years, however, many synthetic steroids have been developed. Some of these synthetic hormones are hundreds or thousands of times more potent than natural steroids. One example is ethynyl estradiol, a synthetic female hormone that is more potent than estradiol. Ethynyl estradiol is a common ingredient in oral contraceptives. 

CRITICAL ASSESSMENT OF THE METHOD Using primary cultures of hepatocytes the retention of transporter expression and activity needs to be guaranteed strictly speaking after each preparation. Cryopreserved hepatocytes from one preparation could be a powerful alternative for industrial applications in the future, because transporter expression and activity could only be characterized once. Houle et al. (2003) compared the transporter activities in cropreserved and freshly isolated hepatocytes and found no significant difference in the transport rates of 14C-taurocholate, 3H-estrone sulfate and 3H-estradiol-17 3-D-glucuronide. 

P-Estradiol (=17p-Oestradiol 17 p-Estradiol) (sterol) isolated by Edward Doisy (USA) (Nobel Prize, Medicine, 1943, with Henrik Dam, Vitamin K)... 

Mucuna pruriens cell cultures are known to hydroxylate a variety of phenolic compounds (Pras, 1990). The solubility of the phenolic steroid, 1713-estradiol (Fig. 6.1), is only 12 pM in culture medium and no biotransformation products could be detected after administration to freely suspended cells, immobilized cells or partially purified Mucuna phenoloxidase. Complexation with (3-cyclodextrin dramatically enhanced the solubility of 17(3-estradiol. Alginate-entrapped cells, cell homogenates and the phenoloxidase were able to o-hydroxylate 17(3-estradiol when supplied as the cyclodextrin complex, the most efficient biotransformation being achieved with the isolated enz)mrie (Woerdenbag et at, 1990). 

Beck et al. [21] reported the LC-MS analysis of estrogenie eompounds in eoastal surface water of the Baltic Sea. SPE on Oasis HLB eartridges was applied to isolate the analytes from 5 1 of water. Deteetion limits were from 0.02-1 ng/1. Estrone and ethinyl estradiol were found at 0.1 and 17 ng/1, respeetively. 

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