Steroid sulfatase activity in a Peptococcus niger strain from the human intestinal microflora.

A strictly anaerobic gram-positive coccus, identified as Peptococcus niger, that developed sulfatase activity towards steroid-3-sulfate esters was isolated from human fecal material. This strain desulfated the arylsulfate esters estrone-3-sulfate (100%) and beta-estradiol-3-sulfate (50%); only trace amounts of desulfated estriol-3-sulfate were found. In addition, alkylsulfatase activity was found towards the 3 alpha-sulfates of 5 alpha-androstane-17-one and 5 beta-androstane-17-one and towards the 3 beta-sulfates of 5 alpha-androstane-17-one, delta 5-androstene-17-one, 5 alpha-pregnane-20-one, and delta 5-pregnene-20-one, all of which were 100% desulfated. No sulfatase activity was found towards the 17-sulfate esters of beta-estradiol or delta 4-androstene-3-one-17 alpha-ol. The nonsteroid arylsulfate esters paranitrophenyl sulfate, paranitrocatechol sulfate, and phenolphthalein disulfate were desulfated 70, 40, and 40%, respectively. In addition to its sulfatase activity, this strain also developed C-17 oxidoreductase activity towards the estrogens and androsta(e)nes and C-3 oxidoreductase activity towards androsta(e)nes and pregna(e)nes.     

Specificity of bile salt sulfatase activity from Clostridium sp. strains S1.

Clostridium sp. strain S1, an unnamed bile acid-desulfating strain from rat intestinal microflora (S.M. Huijghebaert, J. A. Mertens, and H. J. Eyssen, Appl. Environ. Microbiol. 43:185-192, 1982), was examined for its ability to desulfate different bile acid sulfates and steroid sulfates in growing cultures. Clostridium sp. strain S1 desulfated the 3 alpha-monosulfates of chenodeoxycholic, deoxycholic, and cholic acid, but not their 7 alpha- or 12 alpha-monosulfates. Among the 3-sulfates of the 5 alpha- and 5 beta-bile acids, only bile acid-3-sulfates with an equatorial sulfate group were desulfated. Hence, Clostridium sp. strain S1 desulfated the 3-sulfates of bile acids with a 3 alpha, 5 beta-, a 3 beta, 5 alpha- or a 3 beta, delta 5-structure. In contrast, the bile acid-3-sulfates with a 3 beta, 5 beta- or a 3 alpha, 5 alpha-structure were not desulfated. In addition, Clostridium sp. strain S1 did not hydrolyze the equatorial 3-sulfate esters of C19 and C21 steroids and cholesterol or the phenolic 3-sulfate esters of estrone and estradiol. 23-Nordeoxycholic acid with a C-23 carboxyl group was also not desulfated, in contrast to the 5 beta-bile acid 3 alpha-sulfates with a C-24 or C-26 carboxyl group. Therefore, the specificity of the sulfatase of Clostridium sp. strain S1 is related to the location of the sulfate group on the bile acid molecule, the equatorial orientation of the sulfate group, and the structure of the C-17 side chain, its carboxyl group, and chain length.     

Metabolism of androgens in the seminal vesicles and the different lobes of the prostate in young mature rats

Oxidation and reduction of 4-androstene-3,17-dione (androstenedione), 17 beta-hydroxy-4-androsten-3-one (testosterone), 17 beta-hydroxy-5 alpha-androstan-3-one (DHT), 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-A'diol) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-A'diol) were measured in homogenates from ventral (VP), dorsal (DP) and lateral prostate (LP), the coagulating gland (CG) and seminal vesicle (SV) of the intact sexually mature rat using NAD(H) or NADP(H) as cofactors. The specific activity of the various enzymes varied significantly between the different organs. 5 alpha-Reductase activity was highest in the DP and the CG, and undetectable in the LP. 17 beta-Hydroxysteroid oxidoreductase (17 beta-HSOR) activity was mainly confined to the LP. 3 alpha-Hydroxysteroid oxidoreductase (3 alpha-HSOR) activity was also highest in the LP. In the VP the highest 3 alpha-HSOR activity was recorded using NAD(H) as cofactor. In the other organs, similar or higher enzymatic activities were measured using NADP(H) as added cofactor. 3 beta-Hydroxysteroid oxidoreductase (3 beta-HSOR) activity was high in the LP and low or undetectable in the other tissues. Our results indicate that isoenzymes of 3 alpha-HSOR, 3 beta-HSOR and 17 beta-HSOR are present in the accessory sex organs of the rat.     

In vitro metabolism of 4-androstene-3,17-dione and testosterone by rat submaxillary gland.

Tritiated 4-androstene-3,17-dione and testosterone were incubated with submaxillary gland homogenates of male and female rats. The metabolism was predominately reductive. In 15 and 180 min incubations submaxillary tissue converted 4-androstene-3,17-dione chiefly to androsterone. Less testosterone, 17 beta-hydroxy-5 alpha-androstan-3-one, 5 alpha-androstane-3,17-dione, 5 alpha-androstane-3 alpha, 17 beta-diol, and 4-androstene-3 alpha, 17 beta-diol were also identified. Testosterone was converted to the same products plus 4-androstene-3,17-dione. 5 alpha-Androstane-3 alpha, 17 beta-diol was the major testosterone metabolite. Qualitatively the metabolism by male and female submaxillary gland was similar.     

Metabolism of dehydroisoandrosterone and androstenedione by human A-549 alveolar type II epithelial-like cells

The A-549 cell line was initiated from an explant of human lung carcinoma tissue. The biochemical characteristics of these cells are similar to those of normal alveolar type II epithelial cells. To gain some insight into the steroid-metabolizing capabilities of A-549 cells, the metabolism of tritium-labeled dehydroisoandrosterone and androstenedione by these cells was studied. The metabolism of dehydroisoandrosterone led to the exclusive formation of 5-androstene-3 beta,17 beta-diol. The major product of androstenedione metabolism was testosterone; and, 5 alpha-reduced steroids also were formed, viz. 5 alpha-androstane-3,17-dione, androsterone, isoandrosterone, 5 alpha-dihydrotestosterone, 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol. Estrogens, viz., estrone and estradiol-17 beta, were not products of androstenedione metabolism by A-549 cells. The rates of metabolite formation from either dehydroisoandrosterone or androstenedione were linear as a function of incubation time up to 3 h, and with cell number up to 1 X 10(6) cells/ml. The apparent Km of 17 beta-hydroxysteroid oxidoreductase for dehydroisoandrosterone was 11 microM, and that for androstenedione was 13 microM. The predominant formation of 5-androstene-3 beta,17 beta-diol from dehydroisoandrosterone, and testosterone from androstenedione is a likely indication that the principal C19-steroid-metabolizing enzyme in A-549 cells is 17 beta-hydroxysteroid oxidoreductase; the other steroid-metabolizing enzymes expressed in these cells are 5 alpha-reductase, 3 beta-hydroxysteroid oxidoreductase and 3 alpha-hydroxysteroid oxidoreductase. The findings of this study demonstrate that A-549 cells express steroid-metabolizing enzymatic activities that are qualitatively similar to those found in other human pneumonocytes and human lung tissue, except for 3 beta-hydroxysteroid oxidoreductase-5----4-isomerase activity, which is not expressed in these cells with dehydroisoandrosterone as the substrate.     

Androgen metabolism by rat epididymis. Metabolic conversion of 3H 5alpha-androstane-3alpha,17beta-diol, in vitro

The epididymis of adult rats metabolizes 3H 5alpha-androstane-3alpah,17beta-diol (3alpha-diol) by experiments in vitro. After incubation of tissue slices at 37 degrees C for 2 hours, 2% of the radioactivity was found in the water-soluble fraction whereas 98% was found to be ether soluble (free steroids). Further investigation of the free steroids showed the following to be present: 3alpha-diol 39.9%, DHT (17beta-hydroxy-5alpha-androstan-3-one) 33.7%, androsterone (3alpha-hydroxy-5alpha-androstan-17-one) 9.2%, 3beta-diol (5alpha-androstane-3beta,17beta-diol) 2.6%, 5alpha-A-dione (5alpha-androstan-3,17-dione) 1.1%, delta 16-3alpha-ol (5alpha-androst-16-en-3alpha-ol) 1.0%, delta16-3beta-ol (5alpha-androst-16-en-3beta-ol) 2.6%, delta 16-3-one (5alpha-androst-16-en-3-one) 2.9%, and polar compounds 3.3%. When segments of the epididymis (caput and cauda) were incubated in the same way, qualitatively similar metabolites were formed but a greater amount of 3alpha-diol was metabolized by the cauda epididymis. This increase was mainly accounted for by an increased formation of delta 16 compounds (14.3% in cauda, 4.3% in caput). This is most probably due to the presence of larger numbers of mature spermatozoa, which, as we have previously shown, form delta16 steroids from 3alpha-diol and DHT (5).     

Androgen and 19-norsteroid profiles in human preovulatory follicles from stimulated cycles: an isotope dilution-mass spectrometric study

Follicular fluid was aspirated from preovulatory follicles of women under ovarian stimulation for in vitro fertilization and analyzed by a highly specific technique based on gas chromatography-mass spectrometry associated with stable isotope dilution. 19-Nortestosterone and 19-norandrostenedione were identified and quantified for the first time in human follicular fluid. There was a strong positive correlation between 19-nortestosterone and estradiol-17 beta and between 19-norandrostenedione and estrone concentrations, thus indicating a common cellular origin. The accumulation of 19-norsteroids in follicular fluid confirms that they are weakly active intermediates in the multistep enzymatic conversion of androgen to estrogen. Testosterone concentrations were significantly lower than those obtained by radioimmunoassay; cross-reaction with substantially higher levels of 19-nortestosterone seems to be at the origin of this discrepancy. Androstenedione concentrations were similar to those reported in the literature and it was therefore confirmed that an estradiol/androstenedione concentration ratio above 20 is favourable for oocyte cleavage. Other and some newly estimated androgens are: testosterone sulfate, 5-androstene-3 beta, 17 beta-diol 3-sulfate and disulfate, dihydrotestosterone sulfate, epitestosterone, 19-hydroxyandrostenedione, 5 alpha-androstane-3 alpha, 17 beta-diol, 5 alpha-androstane-3 beta, 17 beta-diol, 5 alpha-androstane-3,17-dione and androsterone. Dehydroepiandrosterone sulfate was by far the most abundant androgen in this type of follicles.     

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.     

Synthesis of [75Se]trimethylselenonium iodide from [75Se]selenocystine

N-Acetylglucosamine-6-sulfate sulfatase activity was assayed by incubation of the radiolabeled monosaccharide N-acetylglucosamine [1-14C]6-sulfate (GlcNAc6S) with homogenates of leukocytes and cultured skin fibroblasts and concentrates of urine derived from normal individuals, patients affected with N-acetylglucosamine-6-sulfate sulfatase deficiency (Sanfilippo D syndrome, mucopolysaccharidosis type IIID), and patients affected with other mucopolysaccharidoses. The assay clearly distinguished affected homozygotes from normal controls and other mucopolysaccharidosis types. The level of enzymatic activity toward GlcNAc6S was compared with that toward a sulfated disaccharide and a sulfated trisaccharide prepared from heparin. The disaccharide was desulfated at the same rate as the monosaccharide and the trisaccharide at 30 times that of the monosaccharide. Sulfatase activity toward glucose 6-sulfate and N-acetylmannosamine 6-sulfate was not detected. Sulfatase activity in fibroblast homogenates with GlcNAc6S exhibited a pH optimum at pH 6.5, an apparent Km of 330 mumol/liter, and inhibition by both sulfate and phosphate ions. The use of radiolabeled GlcNAc6S substrate for the assay of N-acetylglucosamine-6-sulfate sulfatase in leukocytes and skin fibroblasts for the routine enzymatic detection of the Sanfilippo D syndrome is recommended.     

The in vitro metabolism of progesterone, 4-androstene-3,17-dione, testosterone and 17 beta-hydroxy-5 alpha-androstan-3-one by fetal rhesus monkey testes.

Homogenates prepared from fetal rhesus monkey testes were incubated with progesterone, 4-androstene-3,17-dione, testosterone and 17 beta-hydroxy-5 alpha-androstan-3-one. The major progesterone metabolite was 17-hydroxy-4-pregnene-3,20-dione. Testosterone also accumulated in the progesterone incubations. 4-Androstene-3,17-dione was converted chiefly to testosterone. Testosterone was not actively metabolized by the fetal monkey testis. 17 beta-Hydroxy-5 alpha-androstan-3-one was actively converted primarily to 5 alpha-androstane-3 beta,17 beta-diol.     

Efficient preparation of steroidal 5,7-dienes of high purity.

Protected forms of dehydroepiandrosterone, delta 5 cholenic acid, (25R)-26-hydroxycholesterol and diosgenin were converted to the corresponding delta 5,7 dienes by successive treatment with 1,3-dibromo-5,5-dimethylhydantoin (dibromantin), tetrabutylammonium bromide and tetrabutylammonium fluoride. The crude products, which contained the delta 5,7 species contaminated by minor amounts of the delta 5 and delta 4,6 steroids, were purified by silica gel-AgNO3 chromatography to give the following steroids in approximately 99% purity and at least 50% yield: 3 beta-acetoxyandrosta-5,7-dien-17-one, methyl 3 beta-acetoxychola-5,7-dien-24-oate, (25R)-3 beta,26-diacetoxycholesta-5,7-diene and (25R)-3 beta-acetoxyspirosta-5,7-diene. Analogous treatment of acetate derivatives of pregnenolone and stigmasterol gave 3 beta-acetoxypregna-5,7-dien-20-one and 3 beta-acetoxystigmasta-5,7,22-triene in approximately 50% yield but of lower purity. Full 1H and 13C NMR assignments are given for seven delta 5,7 steroid acetates and the corresponding delta 5 starting materials. Coupling constants for rings A, B and C of delta 5,7 steroids are presented and stereochemical assignments have been made for the following 1H NMR signals: the C-11 protons of delta 5,7 steroids, the C-16 protons of sterols and bile acids, the C-22 and C-23 protons of bile acid esters and the C-28 protons of stigmasterol derivatives.     

Guinea pig liver 3-hydroxyhexobarbital dehydrogenase. Purification and properties.

3-Hydroxyhexobarbital dehydrogenase, which catalyzes the reversible oxidation of 3-hydroxyhexobarbital to 3-oxohexobarbital, has been purified 470-fold from the soluble fraction of guinea pig liver with a yield of 47%. The specific activity of the purified enzyme is 9.4 units/mg of protein. Results of polyacrylamide gel disc electrophoresis and isoelectric focusing indicated that the purified enzyme preparation is a single and homogeneous protein. NADP+ served as preferred co-factor, but NAD+ is also utilized in the presence of phosphate ion. The guinea pig liver enzyme possessed a relatively narrow substrate specificity in comparison with the rabbit liver enzyme. It is very distinctive that guinea pig liver 3-hydroxyhexobarbital dehydrogenase catalyzes the dehydrogenation of 17beta-hydroxysteroids such as testosterone, 4-androstene-3beta,17beta-diol, 5alpha-androstane-3alpha,17beta-diol, 5alpha-androstane-3beta,17beta-diol, 5alpha-androstan-17beta-ol-3-one, and 5beta-androstane-3alpha,17beta-diol.     

Formation of 5 alpha-products as major C19-steroids in estrogen-responsive mouse Leydig cell tumor lines

Homogenates of estrogen-responsive mouse Leydig cell tumors (T 124958-R and T 22137) or 28- and 120-day-old mouse testes were incubated with [3H]progesterone or [14C]4-androstene-3,17-dione in the presence of NADPH, and progesterone metabolism and enzyme activities were estimated. The growth of T 124958-R tumor transplanted in BALB/c mice was markedly stimulated by estrogenization of host mice, but the growth of T 22137 tumor was evidently suppressed by the estrogenization. The major C21-17-OH-steroids and C19-steroids formed from progesterone by both tumors and the testes of immature mice were 5 alpha-steroids, such as 3 alpha,17-dihydroxy-5 alpha-pregnan-20-one, 5 alpha-androstane-3,17-dione, androsterone, 3 beta-hydroxy-5 alpha-androstan-17-one and 5 alpha-androstane-3 alpha,17 beta-diol. In contrast, the major steroids formed by the testes of adult mice were testosterone and 4-androstene-3,17-dione, and no or little 5 alpha-steroids were produced. 5 alpha-Reductase activities in both tumor cells (40-50 nmol/l X 10(8) cells per h) were also found to be approx. 5-6 times higher than that in Leydig cells of adult mouse testes (8 nmol/l X 10(8) Leydig cells per h), though 17-hydroxylase activity was much higher in the Leydig cells of adult testes (730 nmol/l X 10(8) Leydig cells per h) than in both tumor cells (1-7 nmol/l X 10(8) cells per h). Furthermore, the presence of significant amounts of endogenous androsterone and/or 5 alpha-androstane-3 alpha,17 beta-diol was demonstrated in both tumors by radioimmunoassay. The present results demonstrate for the first time that C19-5 alpha-steroids are major C19-steroid products (immature type of testicular androgen production) in Leydig cell tumor lines.     

Microbial transformations of testosterone to 5alpha-dihydrotestosterone by two species of Penicillium: P. chrysogenum and P. crustosum.

Two species of Penicillium--P. chrysogenum and P. crustosum--were cultured in presence of [3H]testosterone as a substrate. Both species were shown to reduce the 4,5-double bond in testosterone to give dihydrotestosterone (DHT). The steroids produced were 5alpha-dihydrotestosterone, DHT, 3alpha-hydroxy-5beta-androstan-17-one, 3alpha-hydroy-5alpha-androstan-17-one, 4-androstene-3,17-dione, and 5alpha-androstane-3,17-dione. These products implicate the presence of the 5alpha-reductase, with maximal activity at pH 6 and 8, in both species of Penicillium. The presence of DHT in the growth medium and not in the mycelium suggests that DHT is excreted into the medium.     

Configurational analysis and relative binding affinities of 16-methyl-5alpha-androstane derivatives

The four possible isomers 16beta-hydroxymethyl-5alpha-androstane-3beta,17beta-diol 1, 16alpha-hydroxymethyl-5alpha-androstane-3beta,17beta-diol 2, 16beta-hydroxymethyl-5alpha-androstane-3beta,17alpha-diol 3 and 16alpha-hydroxymethyl-5alpha-androstane-3beta,17alpha-diol 4 with proven configuration were converted into the corresponding 16beta-methyl-5alpha-androstane-3beta,17beta-diol 5, 16alpha-methyl-5alpha-androstane-3beta,17beta-diol 6, 16beta-methyl-5alpha-androstane-3beta,17alpha-diol 7, 16alpha-methyl-5alpha-androstane-3beta,17alpha-diol 8, furthermore into the 16beta-methyl-17beta-hydroxy-5alpha-androstane-3-one 13, 16alpha-methyl-17beta-hydroxy-5alpha-androstan-3-one 14, 16beta-methyl-17alpha-hydroxy-5alpha-androstan-3-one 15 and 16alpha-methyl-17alpha-hydroxy-5alpha-androstan-3-one 16. The steric structures of the resulting epimers were determined by means of 1H-, and 13C-NMR spectroscopy. In this way, comparison was possible with the C-16 epimers 5, 6 and 13, 14 prepared earlier by a different route, and the series of isomers could be completed with the steric structures of 16beta-methyl-17alpha-hydroxy-5alpha-androstan-3beta-ol 7 and 16alpha-methyl-17alpha-hydroxy-5alpha 8 and with their 3-keto derivatives 15 and 16. The relative binding affinities of the 16-methyl-5alpha-androstane-3beta,17-diols 5, 6, 7, 8 and 17-hydroxy-16-methyl-5alpha-androstan-3-ones 13, 14, 15, 16 were studied. The introduction of a 16-methyl substituent into 5alpha-androstane molecules substantially decreases the binding affinity to the androgen receptor and 16alpha-methyl derivatives were always bound more weakly than the 16beta-methyl isomers.     

Uptake and metabolism of androgens by bovine spermatozoa

Spermatozoa from bovine ejaculates and cauda epiditymidis were incubated with either tritiated 17 beta-hydroxy-5 alpha-androstane-3-one (DHT) or 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol). Examination of the medium incubations demonstrated metabolic conversion of both DHT and 3 alpha-diol when these steriods were incubated with ejaculated sperm. In addition to this interconversion, the following metabolities were identified: 5 alpha-androstane-3 beta, 17 beta-diol, (3 beta-diol), androsterone and 5 alpha-androstane-3, 17-dione (5 alpha-A-dione). Incubations with cauda spermatozoa showed similar metabolic patterns. Androgen binding was exhibited by both sperm types. Examination of the washed cauda sperm pellet, following incubations with 3 alpha-diol showed that the incubated steroid was the most abundantly bound. DHT and 5 alpha-androst-16-en-3 alpha-ol (delta 16-3 alpha-ol1 were also detected. The major part of the radioactivity bound in the sperm pellet was identified as DHT when this steroid was used as the substrate; the remaining radioactivity consisted of 3 alpha-diol and delta 16-3 alpha-ol. Investigations of ejaculated sperm pellets gave similar results apart from the additional identification of 5 alpha-androst-16-en-3 one (delta 16-3-one) and 5 alpha-androst-16-en-3 beta-ol (delta 16-3 beta-ol (delta 16-3 beta-ol).     

Analysis of profiles of unconjugated steroids in rat testicular tissue by gas chromatography-mass spectrometry

A gas chromatographic-mass spectrometric (GC-MS) method for analysis of unconjugated steroids in a rat testis is described. A combined solvent-solid extraction procedure, utilizing Lipidex 1000 and Sep-Pak C18, gives a 25-fold purified extract. Steroids in this extract are fractionated by straight phase high-performance liquid chromatography (HPLC) on a LiChrosorb DIOL column in n-hexane-2-propanol, 92:8 (v/v). Four fractions are collected and the steroids are converted to tert-butyldimethylsilyl (TBDMS), 3-enol-TBDMS, and mixed TBDMS-trimethylsilyl (TMS) derivatives using TBDMS- and TMS-imidazole with sodium formate as catalyst under conditions suitable for the steroids present in the respective fractions. The derivatives are purified by reversed phase HPLC in 100% methanol and are analyzed by GC-MS, using selected ion monitoring of the major ions of high mass. For quantification, a mixture of known amounts of ten 14C-labelled steroids, [3H]estradiol and [2H3]estradiol are added to the testis homogenate. The mean concentrations (ng/g wet wt) of the twelve steroids determined were: 4-androstene-3, 17-dione, 4.0; testosterone, 127; 17 beta-hydroxy-5 alpha-androstan-3-one, 4.5; 5 alpha-androstane-3 alpha, 17 beta-diol, 5.7; 5 alpha-androstane-3 beta, 17 beta-diol, 1.5; progesterone, 5.5; 17 alpha-hydroxyprogesterone, 14.4; 3 beta-hydroxy-5-androsten-17-one, 0.07; 5-androstene-3 beta, 17 beta-diol, 0.25; 3 beta-hydroxy-5-pregnen-20-one, 10.3; 3 beta, 17 beta-dihydroxy-5-pregnen-20-one, 0.95; and estradiol, 0.025. Variations between animals were large whereas testes from the same animal in most cases had similar steroid concentrations.     

Identification of 5 alpha-androstane-3 beta,17 beta-diol and 3 beta-hydroxy-5 alpha-androstan-17-one sulfates as quantitatively significant secretory products of porcine Leydig cells and their presence in testicular venous blood.

By means of high performance liquid chromatography and gas chromatography-mass spectrometry it has been found that 5 alpha-androstane-3 beta,17 beta-diol sulfate and 3 beta-hydroxy-5 alpha-androstan-17-one sulfate (epiandrosterone) are major secretory steroids of the mature boar testes. These same compounds were similarly identified in culture media when porcine Leydig cells were incubated with androstenedione as substrate. In addition, they were seen as the principal secretory products when [3H]androstenedione and [3H]testosterone were used as substrates; and their presence was greatly reduced by an inhibitor of 5 alpha-reductase (N,N-diethyl,4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide). Greater quantities of 5 alpha-androstanediol than epiandrosterone were noted in all instances. These findings provide further evidence of the versatile activity of the boar testes in steroidogenesis.     

Steroid sulfatase and 17 beta-hydroxysteroid oxidoreductase activities in mouse tissues

The metabolism of estrone sulfate and dehydroisoandrosterone sulfate to the free, unconjugated steroids, estrone and dehydroisoandrosterone, was demonstrated in more than thirty different tissues from male and female BALB/c mice. The activity of steroid sulfatase, when expressed per mg tissue, was greatest in both the pituitary gland and the adrenal glands. The pituitary gland, however, had the lowest capacity for hydrolysis of steroid sulfates while the liver had the greatest capacity. 17 beta-Hydroxysteroid oxidoreductase activity also was demonstrated in all mouse tissues by the formation of estradiol-17 beta when using estrone sulfate as the substrate. The highest apparent activity for 17 beta-hydroxysteroid oxidoreductase was found in lung tissue, and the greatest capacity to form estradiol-17 beta from estrone sulfate was found in liver, lungs, kidneys and testes. This study demonstrates that the majority of mouse tissues have steroid sulfatase and 17 beta-hydroxysteroid oxidoreductase activities.     

The structure of the agaran sulfate from Acanthophora spicifera (Rhodomelaceae, Ceramiales) and its antiviral activity. Relation between structure and antiviral activity in agarans

The sulfated agaran isolated by water extraction from the red seaweed, Acanthophora spicifera (Rhodomelaceae, Ceramiales), is made up of A-units highly substituted with sulfate groups on C-2 (28-30%), sulfates on C-2 and 4,6-O-(1'-carboxyethylidene) groups (9-15%), and only the C-2 sulfate groups (5-8%) with small amounts of C-6 sulfate, 6-O-methyl, and nonsubstituted residues. B-units are formed mainly by 3,6-anhydro-alpha-L-galactose (15-16%) and its precursor, alpha-L-galactose 6-sulfate (10-17%), together with lesser amounts of 3,6-anhydro-alpha-L-galactose 2-sulfate, alpha-L-galactose 2,6-disulfate, alpha-L-galactose 2,3,6-tri-sulfate, alpha-L-galactose 2,6-disulfate 3-xylose, 2-O-methyl-alpha-L-galactose, and unsubstituted alpha-L-galactose. Small, but significant quantities of beta-D-xylose were found in all the fractions, together with small amounts to traces of D-glucose. Some of the fractions have high antiviral activity. Attempts to correlate structure and antiviral activity in agarans are presented.