Occurrence of hydroxysteroid oxidoreductases in liver of turtles.

1. Hydroxysteroid oxidoreductases have been partially purified from the cytosol fraction (105,000 g supernatant) of liver from a fresh-water turtle (Podocnemis expansa) and a sea-water turtle (Chelonia mydas mydas) by precipitation with ammonium sulphate (AS, 10-80% saturation). 2. The following enzymes were detected (substrates in brackets): 3 alpha-hydroxysteroid oxidoreductase (androsterone), 3 beta-hydroxysteroid oxidoreductase (DHEA) and 17 beta-hydroxysteroid oxidoreductase (testosterone, oestradiol-17 beta). NAD as well as NADP were effective as cofactors. 3. In fresh-water turtle, highest activities of the 3 alpha-enzyme were measured in the 20% AS fraction (cofactor NAD), of the 3 beta-enzyme in the 60% AS fraction (cofactor NAD) and of the 17 beta-enzyme in the 40% AS fraction (cofactor NADP). 4. In sea-water turtle, highest activities were observed for all three enzymes in the 60% AS fraction. 5. Generally, enzyme activities were higher in sea-water turtles than in fresh-water turtles. The most active enzyme in both turtles was found to be the 3 alpha-hydroxysteroid oxidoreductase, followed by the 17 beta- and the 3 beta-hydroxysteroid oxidoreductases.

Steroid metabolism in an androblastoma (Sertoli-Leydig cell tumor): a histopathological and biochemical study.

Steroidogenesis in a histologically and ultrastructurally clearly defined Sertoli-Leydig cell tumor ( androblastoma ) was studied by incubation of tumor slices with tritiated and unlabeled pregnenolone. Metabolites were isolated by thin-layer and gel-column chromatography and identified by gas chromatography associated with a radioactivity detector and/or by gas chromatography-mass spectrometry. Steroids were detected by mass chromatography as the trimethylsilyl- or tert-butyldimethylsilyl ethers. The following steroids were found: progesterone, 20 alpha-dihydroprogesterone, 17 alpha-hydroxypregnenolone, 5-pregnene-3 beta,20 alpha-diol, 4-androstene-3,17-dione, dehydroepiandrosterone, testosterone, androsterone, and etiocholanolone. The two latter C19-steroids were found to be the principal metabolites. Neither estradiol-17 beta nor estrone were detected. There was evidence from ultramicroscopic studies that in addition to typical Leydig cells, neoplastic stromal and sex cord cells are involved in androgen metabolism.

[Biochemical and morphologic studies on prostaglandins E and F in the normal and inflamed gingiva]. / Biochemische und morphologische Untersuchungen über die Prostaglandine E and F in der normalen und entzündlich veränderten Gingiva.

With the help of a radioimmunoassay for prostaglandins E and F, an attempt was made to determine the actual concentration of these substances in normal and inflammatorily altered gingival tissue. The content of PGE and PGF was significantly higher in the inflammatorily altered gingiva than in normal tissue. Morphologic examination using immunologic methods showed prostaglandin E particularly in the monocytes.

Studies on the metabolism of steroid hormones in a virilizing adrenal cortex adenoma.

Slices of an adreno-cortical adenoma which had been obtained at operation from an 11-year-old girl with clinical signs of virilism were incubated with each of the following steroids: [1,2-3H]progesterone, [4-14C]pregnenolone, [1,2-3H]testosterone, [4-14C]androstenedione and [7-3H]dehydroepiandrosterone, respectively. Isolation and identification of the free radioactive metabolites were achieved by gel column chromatography on Sephadex LH-20, thin-layer chromatography, radio gas chromatography and isotope dilution. After incubation of progesterone, the following metabolites were identified: 11beta-hydroxyprogesterone, 16alpha-hydroxyprogesterone, 17alpha-hydroxyprogesterone, 21-deoxycortisol, corticosterone and cortisol. Pregnenolone was metabolized to 17alpha-hydroxypregnenolone, progesterone, dehydroepiandrosterone, androstenedione and 11beta-hydroxyandrostenedione. When testosterone was used as substrate, 11beta-hydroxytestosterone, androstenedione and 11beta-hydroxyandrostenedione were found as metabolites, whereas androstenedione was metabolized to testosterone and 11beta-hydroxyandrostenedione. After incubation of dehydroepiandrosterone, only androstenedione and 11beta-hydroxyandrostenedione were isolated and identified. From these results, it appears that cortisol was formed in the adenoma tissue via 21-deoxycortisol and corticosterone. Delta4-3oxo steroids of the C19-series arose exclusively from pregnenolone via 17alpha-hydroxypregnenolone and dehydroepiandrosterone, and not from progesterone and 17alpha-hydroxyprogesterone. Calculated on the amounts of metabolites formed, the highest enzyme activities were those of the 11beta-hydroxylase and the 17alpha-hydroxylase. It is interesting to note that only traces of testosterone were detected after incubation of androstenedione, whereas testosterone yielded large amounts of androstenedione.

[The problem of characterization of sterins in the gingiva by means of gas chromatography--mass spectrometry]. / Zur Frage der Charakterisierung von Sterinen in der Gingiva mit Hilfe der Gaschromatografie/Massenspektrometrie.

Methods suitable for demonstrating previously unidentifiable substances in healthy and diseased gingiva via gas chromatography/mass spectrometry were described. The advantage of these methods is that the substances can be directly demonstrated from the tissue available. The steps necessary to isolate and demonstrate the substances were explained on the basis of the phytosterol campesterol.

The determination of 5alpha-androstane-3alpha, 17beta-diol in human plasma by radioimmunoassay.

Antibodies have been raised in rabbits against 3alpha, 17beta-dihydroxy-5alpha-androstane-6-0-carboxymethyloxime coupled with Cohn's fraction IV-4. The antiserum exhibited significant cross reactions with 5beta-androstane-3alph1, 17beta-diol, 5alpha-dihydrotestosterone, and testosterone. No cross reactions were observed with 5alpha-androstane-3beta,17beta-diol and 5-androstene-3beta,17beta-diol. The methodological criteria for the measurement of 5alpha-androstane-3alpha, 17beta-diol in human plasma were as follows: The specificity was ensured by separating the cross reacting steroids by thin layer chromatography. The intraassay and interassay coefficients of variation were found to be 6.2 and 10.2%, respectively. The sensitivity was 30 pg. The recovery of different amounts of 5alpha-androstane-3alpha,17beta-diol added to human plasma (80, 120, and 200 pg) yielded 91.3, 92.5, and 93.5%, respectively. The following concentrations of 5alpha-androstane-3alpha,17beta-diol have been determined in human plasma (mean +/- SD, ng/dl): Normal males: 18.98 +/- 5.9; normal females: 2.65 +/- 0.27; females with idiopathic hirsutism: 11.9 +/- 6.4; prepubertal children: not detectable.

New progesterone metabolites in human myometrium.

Homogenates from human myometrium with an added NADPH regenerating system and ATP were incubated with 4-(14)C progesterone. Six metabolites were identified: 5alpha-pregnane-3, 20-dione, 5beta-pregnane-3, 20-dione, 3alpha-hydroxy-4-pregnen-20-one, 3beta hydroxy-4-pregnen-20-one. 20alpha-hydroxy-4-pregnen-3-one and 20beta-hydroxy-4-pregnen-3-one. The identification of these metabolites was based on their behaviour in paper and thin layer chromatography and especially in radiogaschromatography either as free compounds or after formation of derivatives. The identification of the two allylic metabolites was supplemented by specific microchemical and enzymatic reactions. The formation of 5beta-pregnane-3, 20-dione, 20beta-hydroxy-4-pregnen-3-one and of the two allylic alcohols 3alpha-hydroxy-4-pregnen-20-one and 3beta-hydroxy-4-pregnen-20-one in human myometrium homogenates was demonstrated for the first time.

Metabolism of progesterone in uterine tissue of non-pregnant rats in vitro.

The metabolism of labelled progesterone was studied in vitro in uterine tissue of non-pregnant rats with particular emphasis on the influence of substrate concentration. Neither a qualitative nor quantitative difference was found for a steroid tissue ratio between 15 X 10(-6) and 4.2 X 10(-9) to 1 g (substrate amounts between 57.73 and 0.02 nmol); with both concentrations 42 to 44 per cent of progesterone was metabolized to about 35 per cent monohydroxymonoketonic steroids and 4-6 per cent dihydroxylated C21O2-compounds. In both sets of incubations we have isolated and identified the following steroids: 3alpha-hydroxy-5alpha-pregnan-20-one, 3beta-hydroxy-4-pregnen-20-one, 3alpha-hydroxy-4-pregnen-20-one, 20alpha-hydroxy-4-pregnen-3-one, 5alpha-pregnane-3alpha,20alpha-diol and 4-pregnene-3alpha,20alpha-diol. The most abundant metabolite formed in these incubations was 3alpha-hydroxy-4-pregnen-20-one which corresponds to about 30 per cent of the total activity recovered. It is the first time that the presence of 20alpha-hydroxysteroid-oxidoreductase activity is definitely established in this type of tissue. The identification of three allylic alcohols as progesterone metabolites in the rat uterus confirms that delta4-3-hydroxysteroids are important intermediates in the in vitro uterine metabolism of steroids.

Metabolism of 20beta-hydroxy-4-pregnen-3-one in uterine tissue of non-pregnant rats in vitro.

In vitro experiments carried out with uterus preparations of ovariectomized adult rats indicate the presence in this tissue of a 20beta-hydroxy-steroid-oxidoreductase which catalyzes the conversion of 20beta-hydroxy-4-pregnen-3-one to progesterone. Since a hepatic 20beta-hydroxysteroid-oxidoreductase is absent in adult female rats, the myometrial enzyme can be responsible for the biological activity of 20beta-hydroxy-4-pregnen-3-one in these animals. Besides progesterone five metabolites were isolated and identified after incubation of [4-14C]20beta-hydroxy-4-pregnen-3-one with uterine tissue: 20beta-hydroxy-5alpha-pregnan-3-one, 20beta-hydroxy-5beta-pregnan-3-one, 5alpha-pregnane-3alpha,20beta-diol, 4-pregnene-3alpha,20beta-diol and 4-pregnene-3beta,20beta-diol. The conversion of 20beta-hydroxy-4-pregnen-3-one to progesterone permits us to regard all five steroids isolated as progesterone metabolites in the rat uterus. 20beta-hydroxy-5beta-pregnan-3-one is the first C21-metabolite with a 5beta(H)-configuration isolated in the rat uterus, which indicates the presence of 5beta-reductase in this tissue.

A simple radioimmunoassay for the measurement of testosterone glucosiduronate in unextracted urine.

A simple, reliable and rapid radioimmunoassay (RIA) for the determination of testosterone glucosiduronate (TG) in crude urine is described. Two protein-TG complexes were investigated in raising antibodies: a) Bovine serum albumin (BSA)-TG and b) human plasma Cohn's fraction IV-4 (CF)-TG. In rabbits, high titers of antibodies were obtained after the injection of CF-TG. The specificity of the antiserum was sufficiently high (cross reaction with free testosterone 27%, with 5alpha-dihydrotestosterone-glucosiduronate 20%). TG was estimated in small aliquots of male and female urine after evaporation overnight at 50 degrees C in order to eliminate interfering material. The intraassay coefficient of variation (CV) was found to be 6% and the interassay CV 11%. TB has been determined in 40 samples of urine simultaneously by "direct" RIA and by a "classical" RIA following hydrolysis with beta-glucuronidase. The coefficient of correlation was found to be 0.89. The mean excretion of TG in the urine of 26 healthy men amounted to 164+/-51 mug/24 hours with a range from 97 to 346 mug/24 hours. In a group of 16 women a mean urinary excretion of TG of 24+/-10 mug/24 hours was determined. The method allows a technician to assay 40 samples per day.

Behaviour of cardiac glycosides and cardenolides related to digitoxigenin on sephadex LH-20.

The behaviour of six cardenolides and eight cardiac glycosides related to digitoxigenin during column chromatography on Sephadex LH-20 gel has been investigated. Complete resolution was obtained for mixtures of digitoxigenin, gitoxigenin and digoxigenin, but not for those of the 3-epimeric cardenolides. It was possible to achieve a group separation of cardenolides and their glycosides of the digitoxigenin series from those of the digoxigenin or gitoxigenin series.

Application of over-run thin-layer and gas-liquid chromatography in the separation of closely related C19O2 steroids.

An improved method for the separation of epimeric C19O2 steroids and their related allylic alcohols is described. In this method, the steroids are first separated by over-run thin-layer chromatography, and the unresolved groups are further analysed as free or as trimethylsilyl ether derivatives by gas-liquid chromatography. The behaviour of twenty-one C19O2 steroids was investigated by thin-layer chromatography in four systems and by gas-liquid chromatography in four liquid phases. All steroid pairs of similar polarity were resolved by the combination of these two fractionation procedures.

Metabolism of [4-14C] testosterone in the rat uterus in vitro.

In view of the uterine action of androgens we have investigated in vitro the metabolism of [4-14C]-testosterone in uterine tissue of ovariectomized rats. After purification of the extracts on Amberlite XAD-2 the metabolites have been isolated by gel. Five metabolites were isolated and identified during these incubation studies: 4-androstene 3,17-dione, 17beta-hydroxy-5alpha-androstan-3-one, 5 alpha-androstane-3alpha17beta-diol, 4-androstene-3 beta, 17beta-diol and 4-androstene-3alpha, 17beta-diol. Furthermore, two polar C19O3-metabolites and one isopolar to 5 alpha-androstane-3, 17-dione have also been detected. The metabolites were characterized by radioactive gas chromatogrphy, and determination of the relative specific activity in the eluates of Sephadex column chromatography. The identification of allylic alcohols was complemented by their oxidation to 4-androstene-3,17-dione. The present data show that activity of 17beta,3alpha- and 3beta-hydroxysteroid-oxidoreductase and 5alpha-ring-reductase are involved in the metabolism of testosterone in vitro in the rat uterus. The very low 5 alpha-reductase activity under the experimental conditions used in this work explains the formation of allylalcohols as the principal metabolites of testosterone in the rat uterus.

PIP: The metabolism of 4-carbon-14-testosterone was investigated in the rat uterus in vitro. Purification and isolation of 5alpha-dihydrotestosterone, 4-androstene-3,17-dione, 5alpha-androstane-3alpha, 17beta diol were carried out with Amberlite XAD-2, gel column chromatography Sephadex LH 20, and thin-layer chromatography on silica gel. Radioactive gas chromatography was used for characterization and relative specific activity was determined by Sephadex column chromatography. Identification of allylic alcohols was complemented by their oxidation to 4-androstene-3,17-dione. These data reveal that 17beta,3alpha and 3beta-hydroxysteroid-oxidoreductase and 5alpha-ring-reductase activities are involved in testosterone metabolism in vitro in the rat uterus. The low 5alpha-reductase activity under these conditions explains the formation of allylacohols as the principal metabolites of testosterone.

Gel chromatography of steroid oestrogens on sephadex LH-20.

The behavior of 23 steroid oestrogens Sephadex LH-20 using six solvent systems was investigated. The fractions eluted by gel column chromatography were analysed by thin-layer chromatography and spectrometry. The method was used for the radiochemical purity of labelled compounds by isotopic dilution.