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.     

Steroid estrogen conjugates in hens' urine II. identifications of some minor conversion products of intramuscularly injected [4-14c]estrone

[4-¹⁴C]Estrone was injected intramuscularly into two mature laying Rhode Island Red hens. Radioactive steroids and steroid conjugates recovered from the urine on Amberlite XAD-2 columns were fractionated on columns (100 cm) of DEAE-Sephadex A-25 by NaCl gradients. The presences of the following were confirmed, the figures in brackets indicating average proportions as per cent of total radioactivity recovered after Sephadex column chromatography: -the 3-β-glucuronides of estrone (10. 9) and of estradiol-17α plus estradiol-17β(9.8); the 17-β-glucuronides of estradiol-17α plus estradiol-17β (2.1); the 3-sulfates of estrone (14. 5) and of estradiol-17α plus estradiol-17β (27. 4); and the disulfates of estradiol-17α plus estradiol-17β (2. 3). The following additional conjugates were identified:-a β-glucuronide of 16-epiestriol (0.2) and a β-glucuronide of 16-ketoestradiol-17β (0. 2); the 3-sulfates of 16-epiestriol (1. 4), of 17-epiestriol (0. 9), of 16, 17-epiestriol (0. 7), of 16-keto-estradiol-17β (1. 1), and of 2-methoxyestrone (0. 7). Some evidence was obtained for the presence of 16, 17-epoxy-estratrienol-3-sulfate (1.9).     

Identification of radioactive 17beta-estradiol-17-beta-D-glucuronide and 17alpha-estradiol-17-beta-D-glucuronide in the urine of the domestic fowl after intramuscular injection of [4-14C]estrone.

Two previously uncharacterized radioactive estrogen conjugates, 17beta-estradiol-17-beta-D-glucuronide (3-hydroxyestra-1,3,5(10)-trien-17beta-D-glucopyranosiduronate) and 17alpha-estradiol-17beta-D-glucuronide (3-hydroxyestra-1,3,5(10)-trien-17alpha-yl-beta-D-glucopyranosiduronate), have been identified in small but significant amounts in avian urine and in a ratio of approximately 2:1 after intramuscular injection of [4-14C]estrone.     

Incorporation of 3H and 14C from (6 alpha-3H)penicillin N and (10-14C,6 alpha-3H) penicillin N into deacetoxycephalosporin C.

1. In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium mutant M-0198, 3H and 14C were incorporated from singly- and doubly-labelled penicillin N into deacetoxycephalosporin C. 2. The deacetoxcephalosporin C obtained from the above feeding experiments was converted into two different crystalline derivatives, namely N-phthalimidodeacetoxycephalosporin C bisbenzhydryl ester and N-phthalimidodeacetoxycephalosporin C bisdicyclohexylamine salt and recrystallized to constant specific activity or constant ratio of specific activity. 3. That 3H is incorporated at C-7 in the biosynthesized deacetoxycephalosporin C was shown by the loss of radioactivity (95.2%) after methoxylating the derived N-phthalimidodeacetoxycephalosporin C bisbenzyhydryl ester. 4. Deacetoxycephalosporin C was also the product of the cell-free reaction conducted in the presence of ferrous ions and ascorbic acid, as shown by two-dimensional paper electrophoresis-chromatography; these additives appreciably improved the efficiency of conversion.     

Boron estrogens: Synthesis,biochemical and biological testing of estrone and estradiol-17β 3-carboranylmethyl ethers

Synthesis, biochemical and biological testing of the first carborane derivatives of estrogens are described. Estrone 3-carboranylmethyl ether was synthesized in two steps from estrone. Reduction of estrone 3-carboranylmethyl ether with sodium borohydride provided estradiol-17β 3-carboranylmethyl ether. Enzyme kinetic measurements showed that estrone 3-carboranylmethyl ether is a substrate for human placental 17β-hydroxy-steroid dehydrogenase with Km = 5×10^?6M, and Vmax = 0.016 μmol min^?1 μg^?1. The relative affinity constant of estradiol-17β 3-carboranylmethyl ether for rat uterine estrogen receptor was 0.5 (compared with a value of 100 for estradiol-17β). Consistent with its low affinity for estrogen receptor, the dose-dependent uterotropic response to estradiol-17β 3-carboranylmethyl ether in castrated female rats was one sixtieth that of estradiol-17β. None of the tested rats had a toxic reaction to estradiol-17β 3-carboranylmethyl ether. These results demonstrate that exceptionally stable carborane derivatives of estrogens can be synthesized with preservation of their biochemical and biological properties. Boron-containing estrogens may be useful for thermal neutron capture therapy of cancers with estrogen receptors to concentrate boron in the cell nucleus.     

Dynamic appearance of [4-14C] dehydroepiandrosterone and [7 alpha-3H] dehydroepiandrosterone sulphate metabolites in urine of normal and obese female subjects.

[4-14C]-Dehydroepiandrosterone and [7 alpha-3H]-Dehydroepiandrosterone sulphate were injected simultaneously to normal and obese female subjects. The percentage recovery of 14C and 3H radioactivies in dehydroepiandrosterone sulphate, androsterone sulphate, etiocholanolone sulphate, androsterone glucuronoside and etiocholanolone glucuronoside was determined in the day-to-day urine collections for 72 hr. Results showed a normal total 3H recovery and a poor 14C recovery in urinary conjugates of obese patients. The rate of appearance of 3H activity was not identical in the individual metabolites of normal subjects, and it was not normal in obesity. Overweight subjects exhibited an acceleration in [7 alpha-3H]-Dehydroepiandrosterone sulphate metabolism to androsterone glucuronoside. The observation regarding the rate of appearance of urinary conjugates bearing 14C isotope correlate with our previous finding in which a glandular overproduction of free dehydroepiandrosterone was found and an uptake of this steroid by the adipose tissue was suggested. Our results showed that the poor recovery of 14C radioactivity in urine of obese female subjects was not an aspecific consequence of illness.     

Synthesis of 17 beta-(3'-thiophenyl)-5 beta-androstane-3 beta,14 beta-diol 3-D-glucopyranoside, an anti-inotropic cardiac glucoside

In a continuing effort to synthesize cardiac glucoside analogs with modified 17 beta-functional groups for pharmacologic testing, we used 3 beta-benzyloxy-5 beta-androst-15-en-17-one as an efficient intermediate. This report describes a preparation of 17 beta-(3'-thiophenyl)-5 beta-androstane-3 beta,14 beta-diol 3-D-glucopyranoside.     

Identifications of radioactive steroid estrogen conjugates in blood plasma of laying hens after intramuscular injection of (4--14C)-estrone.

[4--14C] Estrone was injected intramuscularly into six laying hens. Fifty minutes later the hens were exsanguinated. The plasmas were examined for conjugates of radioactive phenolic steroids by recovery on columns of Amberlite XAD-2 or by extraction with tetrahydrofuran followed by chromatography on a column of DEAE-Sephadex A-25 in a gradient of NaCl. The biggest Sephadex chromatographic fraction (50,4% of total) contained about 42% of its radioactivity as estradiol-17alpha-3-sulfate and 18% as estradiol-17beta-3-sulfate and the remaining 40% was identified tentatively as estradiol-17alpha-17-sulfate plus a small proportion of estradiol-17beta-17-sulfate. The second biggest Sephadex chromatographic fraction (12.7% of total) was a mixture of conjugates not further identified. Minor fractions identified comprised estrone-beta-glucuronide (2.8%), estradiol-17alpha-3-beta-glucuronide (2.8%), estradiol-17beta-3-beta-glucuronide (2.3%) and estrone sulfate (6.0%). Evidence was obtained for the presence of small proportions of estradiol-17alpha disulfate and estradiol-17beta disulfate.     

Synthesis of 11beta-(4-dimethylaminophenyl)-17beta-hydroxy-17alpha- (3-methyl-1-butynyl)-4, 9-estradien-3-one and 11beta-(4-acetophenyl)- 17beta-hydroxy-17alpha-(3-methyl-1-butynyl)-4, 9-estradien-3-one: two new analogs of mifepristone (RU-486)

From the structure activity relationship, two new analogs, 2 and 3, of the potent progesterone antagonist mifepristone 1 have been designed. The syntheses of these two analogs have been achieved in eleven steps through modified synthetic sequences and improved procedures starting from (+)-estrone. In comparison with mifepristone 1, the relative binding affinities of compound 2 for the progesterone receptor was found to be more, whereas that of compound 3 was less.     

17 beta-(cyclopropylamino)-androst-5-en-3 beta-ol, a selective mechanism-based inhibitor of cytochrome P450(17 alpha) (steroid 17 alpha-hydroxylase/C17-20 lyase)

A new compound, 17 beta-(cyclopropylamino)-androst-5-en-3 beta-ol, MDL 27,302, has been designed and synthesized as a mechanism-based inhibitor of cytochrome P450(17 alpha). The time-dependent inactivation of human testicular P450(17 alpha) is irreversible by dialysis and requires the cofactor, NADPH; Kiapp. 90 nM (determined on cynomolgous monkey testis enzyme). Inactivation was not affected by the nucleophile DTT, suggesting retention of the inhibitor in the enzyme active site during the inactivation process. Inhibition is specific to the cyclopropylamino compound, since the isopropylamino- and cyclobutylamino-analogs were not inhibitory. Enzymatic specificity of MDL 27,302 for P450(17 alpha) was demonstrated by its failure to inhibit steroid 21-hydroxylase and the cholesterol side chain cleavage enzyme (P450scc). Both the 17 alpha-hydroxylase and C17-20 lyase activities of cytochrome P450(17 alpha) of human testis microsomes were inhibited by MDL 27,302.     

The identification of 14 alpha,17 beta-dihydroxyandrost-4-en-3-one monohydrate and 14 alpha,17 beta-dihydroxyandrosta-1,4-dien-3-one monohydrate, metabolites of androstenedione in Mucor piriformis.

The microorganism Mucor piriformis transforms androst-4-ene-3,17-dione into a major and several minor metabolites. X-ray crystallographic analysis of two of these metabolites was undertaken to determine unambiguously their composition and chirality. Crystals belong to the orthorhombic space-group P2(1)2(1)2(1), with a = 7.199(4) A and a = 6.023(3) A, b = 11.719(3) A and b = 13.455(4) A, c = 20.409(3) A and c = 20.702(4) A for the two title compounds, respectively. The structures have been refined to final R values of 0.060 and 0.040, respectively.     

Enzyme-assisted synthesis and structure characterization of glucuronide conjugates of methyltestosterone (17 alpha-methylandrost-4-en-17 beta-ol-3-one) and nandrolone (estr-4-en-17 beta-ol-3-one) metabolites

A new and useful method based on enzyme-assisted synthesis was developed for producing 3 alpha-O-beta-D-glucuronide conjugates from synthetic phase I metabolites of methyltestosterone and nandrolone. The formed glucuronide conjugates of 17 alpha-methyl-5 alpha-androstane-3 alpha,17 beta-diol (I), 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol (II), 5 alpha-estran-3 alpha-ol-17-one (III), and 5 beta-estran-3 alpha-ol-17-one (IV) are urinary metabolites, indicating the human misuse of the above-mentioned anabolic androgenic steroids (AAS). The common lack of reference material precludes the use and validation of these biomarkers in human doping control. Liver microsomes from Aroclor 1254-induced rats were used as a highly active source of mammalian UDP-glucuronosyltransferases (UGT, EC 2.4.1.17). After purification by protein precipitation, liquid-liquid extraction (dichloromethane), C-18 solid-phase extraction, and lyophilization, the steroid glucuronide structures were characterized by (1)H and (13)C NMR spectroscopy and tandem mass spectrometry. The enzymatic method was highly stereoselective, producing a single major conjugate from the parent steroids I-IV. The stereochemically pure steroid glucuronide conjugates were recovered in milligram amounts (1.0-2.8 mg, yield 12-29%), which is sufficient for veterinary and human doping control analyses; for pharmaco-, toxico-, and enzyme kinetic studies in the pharmaceutical industry; for clinical laboratories; and for forensic medicine. A new sensitive LC-MS method was developed for controlling the product purity in syntheses, as well as for enzyme kinetic characterization of AAS-metabolizing UGT activities in rat liver toward the aglycones I-IV. In this study, the UGT enzymes responsible for the formation of 3 alpha-O-linked glucuronides from the substrates I, II, III, and IV exhibited the specific enzyme activity values: 25, 124, 48, and 212 nmol/mg microsomal protein in a 2-h incubation, respectively.     

The uptake of radioactivity by the uterus of the rabbit following the sytemic administration of (3H) estradiol-17beta and (3H) estrone and the identification and subcellular localization of radiometabolites in the uterus

In an effort to determine the relevance of the uterine oxido-reduction of estrogens to their action in the rabbit uterus, the uterine uptake of radioactivity administered subcutaneously as [³h] estradiol-17β or [³H]estrone and the subcellular distribution of radio-metabolites in the uterine tissue were studied. The animals were killed 20 min, 1, 3 and 9 hr after the administration of 0.1 μg tritiated steroid and the relative proportions of radioactive estradiol-17β and estrone in plasma and in ‘cytosol’, ‘mitochondrial/microsomal’ and ‘nuclear’ fractions of the uterine homogenates were studied. Despite the presence of a high proportion of estrone in chloroform extract of plasma, very little was found in the fractions from uterine tissue irrespective of the steroid administered. Highest levels of uterine estrone were found in the ‘mitochondrial/microsomal’ preparation. There was no apparent difference in the pattern of uptake of radioactivity administered as [³H] estradiol-17β or [³H] estrone. The presence of high levels of 17β-hydroxysteroid dehydrogenase activity in the rabbit uterus may be responsible for the apparent difference between these results and those of similar experiments using the rat.     

Conversion,in vitro,of (7n-3H) testosterone to estrone and estradiol-17β and their 3-sulfäte conjugate by the guinea-pig placenta

Different cellular fractions of guinea-pig placenta were incubated in the presence of (7n-³H) testosterone. Microsomal aromatization of ³H-testosterone into estrone and estradiol-17β was demonstrated in the presence of NADPH. The predominance of estrone after incubation with 17β-hydroxylated precursors, (7n-³H) testosterone and (6,7-³H) estradiol-17β, indicate that there is a microsomal 17β-hydroxysterold dehydrogenase activity. In this report, cytosolic sulfurylation of estrogens is demonstrated. This latter activity represents a quite original characteristic of the placental metabolism of estrogens in guinea-pigs. In contrast with the human placenta where there is considerable sulfatase activity, the guinea-pig placenta can sulfurylate estrogens.