Radioimmunoassays for androsterone, 5alpha-androstane-3alpha, 17beta-diol and 5alpha-androstane-3beta, 17beta-diol

Androsterone (3alpha-hydroxy-5alpha-androstan-17-one), 5alpha-androstane-3alpha, 17beta-diol and 5alpha-androstane-3beta, 17beta-diol were conjugated at C-16 through sulfur to bovine and human serum albumin. Rabbits injected with these conjugates produced antibodies suitable for radioimmunoassays of these hormone metabolites. Samples were purified on Sephadex LH-20 columns. Levels of these steroids were measured in a rat blood serum pool and in ovarian tissue extract pools.     

Adult sheep blood metabolizes dihydrotestosterone to 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta, 17 beta-diol

The metabolism of 5 alpha-dihydrotestosterone by adult sheep blood was investigated. Erythrocytes contain 3 alpha- and 3 beta-hydroxysteroid dehydrogenase activities. The mean rate of reduction of 5 alpha-dihydrotestosterone by erythrocytes established in 15-min incubations was 0.66 +/- 0.36 (s.d.) mumol ml-1 erythrocytes h-1 and at equilibrium after a 60-min incubation, 90.6 +/- 5.1% of the substrate was reduced. The reduction of 5 alpha-dihydrotestosterone was shown to be dependent upon extracellular glucose and the intracellular cofactor NADPH. The proportion of the two reduction products was determined at equilibrium after separation by paper partition, chromatography and favoured 5 alpha-androstane-3 alpha, 17 beta-diol (96.0%) to 5 alpha-androstane-3 beta, 17 beta-diol (4.0%). The identities and proportions of the two products were confirmed by recrystallization procedures. The fact that erythrocytes can significantly metabolize the androgen 5 alpha-dihydrotestosterone is evidence for the recognition of blood as a major component of steroid endocrine homeostasis in sheep.     

Determination of 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol in human plasma by radioimmunoassay

5 alpha-Androstane-3 alpha,17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol) were measured in human peripheral plasma by radioimmunoassay using celite microcolumn purification. The antisera used for the assay were obtained by immunization of rabbits with 3 alpha,17 beta-dihydroxy-5 alpha-androstane-6-(O-carboxymethyl) oxime: BSA for 3 alpha-diol and 3 beta,17 beta-dihydroxy-5 alpha-androstane-15 alpha-carboxymethyl: BSA for 3 beta-diol. The concentrations (pg/ml +/- SD) of the two diols in normal male and female plasma are respectively: 216 +/- 51 and 49 +/- 32 for 3 alpha-diol, 239 +/- 76 and 82 +/- 45 for 3 beta-diol. Comparison of these results with published ones shows that 3 beta diol concentrations were significantly lower. The high specificity of the assay is due to chromatography on celite microcolumns, allowing elimination of 5-androstene-3 beta,17 beta-diol from the plasma sample.     

Serum 5-androstene-3 beta,17 beta-diol sulphate and 5 alpha-androstane-3 alpha,17 beta-diol sulphate in hirsute females with polycystic ovarian disease

Serum sulphates of 5-androstene-3 beta,17 beta-diol (5-ADIOL-S), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-DIOL-S) and dehydroepiandrosterone (DHEA-S), unconjugated androstene-dione (AD) and testosterone (T), sex hormone binding globulin (SHBG), free androgen index (FAI), 17 alpha-hydroxyprogesterone (17OHP), luteinising hormone (LH) and follicle stimulating hormone (FSH) were measured by specific radioimmunoassay in 28 hirsute women with polycystic ovarian disease (PCO) and in normal women (n = 73). Mean levels of steroids measured were significantly elevated, and SHBG significantly depressed, in the women with PCO with values (mean +/- SE) for 5-ADIOL-S (516 +/- 51 vs 267 +/- 10 nmol/l), 3 alpha-DIOL-S (130 +/- 9 vs 52 +/- 2 nmol/l), DHEA-S (7.3 +/- 0.5 vs 4.4 +/- 0.2 mumol/l), AD (11.3 +/- 1.1 vs 3.4 +/- 0.2 nmol/l), T (3.3 +/- 0.2 vs 1.5 +/- 0.1 nmol/l) and 17OHP (5.1 +/- 0.8 vs 2.8 +/- 0.2 nmol/l). SHBG levels were 31 +/- 2.9 vs 65 +/- 2.5 nmol/l, and the free androgen index [100 x T (nmol/l) divided by (SHBG nmol/l)] was 12.5 +/- 1.4 vs 2.4 +/- 0.1. The mean LH to FSH ratio was also elevated at 2.8 +/- 0.3. These studies suggest that the measurement of 5-ADIOL-S and DHEA-S may indicate adrenal gland involvement in PCO while 3 alpha-DIOL-S appears to be a reflection of peripheral androgen metabolism. A comprehensive biochemical profile of PCO should thus include the analysis of these sulphoconjugates as well as unconjugated steroids.     

Synthesis of 5 alpha-androstane-3 alpha,17 beta-diol 3- and 17-glucuronides

The glucuronidation of 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol) was carried out by three different methods: The Koenigs-Knorr reaction using methyl-2,3,4-tri-O-acetyl-1 alpha-bromo-1-deoxy-beta-D-glucuronate, by employing methyl-2,3,4-tri-O-acetyl-1-O-(trichloroacetimidoyl)-alpha-D-gl ucopyranuronate (imidate procedure), and by the reaction of 2,3,4-tri-O-acetyl-alpha-D-glucopyranuronate catalyzed by trimethylsilyl trifluoromethanesulfonate (triflate method). The Koenigs-Knorr method gave the beta-anomers of both the 3- and 17-glucuronides. The imidate procedure also resulted in the beta-anomers of the 3- and 17-glucuronides, but in lower yield. The triflate method, however, yielded only the alpha-anomers of the 3- and 17-glucuronides. The structural assignments of these compounds were made from NMR spectral data obtained with a 500 mHz instrument.     

Age-related changes in conversion of 5 alpha-androstan-17 beta-ol-3-one to 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol by rat testicular cells in vitro

Conversion of labelled 5 alpha-androstane-17 beta-ol-3-one (DHT) by isolated testicular cells from rats of different ages was examined under saturating substrate conditions in vitro (5--10 micrograms DHT/ml in a 24 h incubation). Two detectable metabolites of DHT were produced by testicular cells in vitro. 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) and 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol). Production of these diols during a 24 h period was linear, and the amounts formed were directly related to the cell number. The amount of 3 alpha- and 3 beta-diols formed by testicular cells of rats of different ages increased from Day 10 to Day 25, then declined. Testicular cells from rats 10 to 20 days of age converted DHT mainly to 3 alpha-diol, but thereafter 3 beta-diol was the predominant testicular metabolite of DHT.     

A direct radioimmunoassay for 5 alpha-androstane-3 alpha,17 beta-diol 17-glucuronide.

Synthesis of the 11 alpha-hemiglutaryl derivative of 5 alpha-androstane-3 alpha,17 beta-diol 17-glucuronide (androstane-diol-17G) starting from androsta-4,9(11)-diene-3,17-dione through a 10-step sequence and the preparation of its bovine serum albumin conjugate is described. By using this conjugate, antiserum was raised in rabbits which proved to be very specific for androstanediol-17G. A direct radioimmunoassay using a double antibody procedure is described for the measurement of androstanediol-17G from plasma without prior chromatography.     

Metabolism of 5 alpha-androstane-3 beta,17 beta-diol to 17 beta-hydroxy-5 alpha-androstan-3-one and 5 alpha-androstan-3 alpha,17 beta-diol in the rat.

Significant metabolism of 5 alpha-androstane-3 beta,17 beta-diol to 17 beta-hydroxy-5 alpha-androstan-3-one was recorded in several tissues and organs from rats and humans. This bioconversion was further investigated in rat testis homogenates. 5 alpha-Androstane-3 beta,17 beta-diol was readily metabolized to 17 beta-hydroxy-5 alpha-androstan-3-one with NAD and/or NADP added as cofactors. When a NADPH generating system was included in the incubation, 5 alpha-androstane-3 beta,17 beta-diol was metabolized to 5 alpha-androstan-3 alpha,17 beta-diol. Only small amounts of 17 beta-hydroxy-5 alpha-androstan-3-one accumulated under the latter condition.     

Serum levels of 5-androstene-3 beta,17 beta-diol sulphate, 5 alpha-androstane-3 alpha, 17beta-diol sulphate and glucuronide, in late onset 21-hydroxylase deficiency

Serum sulphates of 5-androstene-3 beta,17 beta-diol (5-ADIOL-S), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-DIOL-S) and dehydroepiandrosterone (DHEA-S), as well as 5 alpha-androstane-3 alpha,17 beta-diol glucuronide (3 alpha-DIOL-G) and unconjugated androstenedione (AD) and testosterone (T), sex hormone binding globulin (SHBG), free androgen index (FAI) and 17 alpha-hydroxyprogester-one (17OHP) were measured by specific radioimmunoassays (RIA) in 14 women with late-onset 21-hydroxylase deficiency (LOCAH), and in normal women (n = 73). The diagnosis of LOCAH was made on the finding of a (17OHP) response level greater than 30 nmol/l following ACTH stimulation, and/or an elevation of urinary metabolites of 17OHP. Mean values for serum concentrations of all steroids measured and the free androgen index (100 X T nmol/l divided by SHBG nmol/l) were significantly elevated, and SHBG levels depressed in patients with LOCAH. These studies show that in LOCAH, in addition to the unconjugated steroids AD and T, the sulphoconjugated steroids DHEA-S, 5-ADIOL-S and 3 alpha-DIOL-S are increased, as is the glucuronide conjugate 3 alpha-DIOL-G and the index of bioavailable testosterone (FAI), and that mean SHBG levels are depressed. These data suggest that as well as AD, 5-ADIOL-S and DHEA-S may act as pro-hormones for more potent steroids (T and 5 alpha-dihydrotestosterone) in peripheral tissues, while 3 alpha-DIOL-S and 3 alpha-DIOL-G may both reflect peripheral androgen metabolism in patients with LOCAH.     

Synthesis of deuterium-labeled 5 alpha-androstane-3 alpha, 17 beta-diol and its 17 beta-glucuronide.

Using unlabeled androsterone as starting material, 5 alpha-[16,16-2H2]androstan-3 alpha-ol-17-one was synthesized by exchange using deuterated potassium methoxide. This labeled androsterone product was reduced by sodium borodeuteride, which gave predominantly trideuterated 5 alpha-androstane-3 alpha, 17 beta-diol. The labeled androstanediol was conjugated with glucuronide by using the Koenig-Knorr reaction with methyl-1-bromo-1-deoxy-2,3,4-tri-O-acetyl-alpha-D-glucopyranosuronate . The dominant product was identified by thermospray high-performance liquid chromatography/mass spectrometry (MS) and electrospray MS as 5 alpha-[16,16,17-2H3]androstane-3 alpha, 17 beta-diol, 17 beta-glucuronide.     

Androgenic actions of 5 alpha-androstane-3 alpha,17 beta-diol in rat submandibular gland

To evaluate the action of 5 alpha-androstane-3 alpha,17 beta-diol(3 alpha-diol) in rat submandibular gland, 5 alpha-reductase, 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) and oxidative 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDO) activities, and trypsin-like protease activities, were assayed in control, castrated and 3 alpha-diol injected rats. 3 alpha-Diol (1 mg/kg) was injected subcutaneously in castrated male rats daily for 7 days. A 47% decrease of 5 alpha-reductase activity in the nuclei and a 30% decrease of 3 alpha-HSD(O) activities in the cytosol were shown after castration. 3 alpha-Diol restored the 5 alpha-reductase and 3 alpha-HSD(O) activities to 82 and 140% of the control submandibular gland, respectively. 3 alpha-Diol raised the trypsin-like protease activity to near control values in the submandibular gland of castrated rats. Morphological observations also revealed a distinct effect of 3 alpha-diol on the number of granules of granular duct cells. It is concluded that 3 alpha-diol has an androgenic action in the rat submandibular gland. It stimulates the 3 alpha-HSD(O). The 3 alpha-HSD(O) in its turn may be responsible for DHT accumulation in the cells.     

Pituitary metabolism of 5alpha-androstane-3beta-17beta-diol: intense and rapid conversion into 5alpha-androstane-3beta,6alpha,17beta-triol and 5alpha-androstane-3beta,7alpha, 17beta-triol.

In the male rat pituitary, 5alpha-androstane-3beta, 17beta-diol (3beta-diol) is extensively metabolized into polar steroids. They were identified as 5alpha-androstane-3beta, 6alpha-17beta-triol (6alpha-triol) and 5alpha-androstane-3beta, 7alpha, 17beta-triol (7alpha-triol). 6-alpha-Triol represents 53% and 7alpha-Triol 28% of the total 3beta-diol metabolites. The remaining percentage is related to 6beta and 7beta isomers. The biological role of triols is still unknown.     

Immunization against 5alpha-androstane-3alpha,17beta-diol affects ovarian folliculogenesis in Merino ewes

The 5alpha-reduced androgens have been implicated as antagonists of follicular development. In this experiment, we examined the effect of active immunization against 5alpha-reduced androgen on follicular development in ewes. During the breeding season, cyclic Merino ewes were either actively immunized three times against 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) or served as controls. Six to nine weeks after the last immunization, they were treated with PGF(2alpha) analog (PG, 125mg cloprostenol i.m.) and luteolysis was induced. Fourteen days after the PG treatment, the ewes were either killed (mid-luteal phase) or treated a second time with PG and killed 24h later (early follicular phase). At slaughter, blood samples were collected and ovaries recovered. All CL and follicles larger than 2mm were dissected and their size and appearance were recorded. Follicular fluid was collected and concentrations of estradiol-17beta (E(2)), progesterone (P), androstenedione (A(4)), testosterone (T), dihydrotestosterone (DHT), 5alpha-androstane-3alpha-ol,17beta-one (androsterone: 3alpha-ol) and 3alpha-diol were determined by RIA. Immunization induced antibodies primarily to DHT and its 5alpha-reduced substrates 3alpha-diol and 3alpha-ol but not to E(2), P, A(4) or T. Immunization increased ovulation rate, size of ovulatory follicles and weight of CL. Immunization appeared to increase ovulation rate by decreasing the incidence of atresia in large preovulatory follicles. Regardless of their physiological status follicles contained only low levels of DHT; 3alpha-ol and 3alpha-diol were not detected in most follicles. Immunization did not appear to affect levels of DHT or other steroids in the follicular fluid. In conclusion, the induction of antibodies to 5alpha-reduced androgens increases ovulation rate by enhancing follicular viability during the preovulatory period in ewes. However, this effect is not brought about by the direct immune-neutralization of DHT or its 5alpha-reduced substrates 3alpha-ol and 3alpha-diol at the ovarian level.     

Metabolism and binding in vitro of 5 alpha-androstane-3 beta, 17 beta-diol and of 5 alpha-androstane-3 alpha, 17 beta-diol in cell fractions of rat ventral prostate and liver.

Rat ventral prostate and liver were investigated for the binding in vitro to particulate fractions and for the metabolism of 5 alpha-androstane-3 beta, 17 beta-diol. Comparative investigations were carried out on the metabolism of 5 alpha-androstane-3 alpha, 17 beta-diol. Preparations of the liver were investigated in order to establish the organ specificity of the method. In the prostate, the bulk of the metabolites of 5 alpha-androstane-3 beta, 17 beta-diol was present as steroids of high polarity. Of the less polar metabolites, 17 beta-hydroxy-5 alpha-androstan-3-one, 3 beta-hydroxy-5 alpha-androstan, 17-one and 5 alpha-androstane-3 alpha, 17 beta-diol were detectable. The binding of a 5 alpha-androstane-3 beta, 17 beta-diol to mitochondria and microsomes was unspecific. In the liver, among the less polar metabolites, 3 beta-hydroxy-5 alpha-androstan-17-one was the main metabolite, and the binding was unspecific. The main metabolite in the prostate homogenate of 5 alpha-androstane-3 alpha, 17 beta-diol was 17 beta-hydroxy-5 alpha-androstan-3-one. The portion of highly polar steroids was very low. The portion of unmetabolized hormone was distributed almost equally among the different cell preparations except the nuclei, in which 17 beta-hydroxy-5 alpha-androstan-3-one was higher and 5 alpha-androstane-3 alpha, 17 beta-diol was lower than in the remaining cell fractions.     

A neuroactive steroid 5alpha-androstane-3alpha,17beta-diol regulates androgen receptor level in astrocytes

Optic nerve head (ONH) astrocytes from patients with glaucomatous optic neuropathy exhibit increased production of 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), a neuroactive metabolite of 5alpha-dihydrotestosterone (5alpha-DHT). To determine whether ONH astrocytes are androgen target cells, and whether 3alpha-diol is capable of regulating astrocyte functions, we studied the response of human ONH astrocytes to 3alpha-diol compared with 17beta-hydroxy-17alpha-methyl-estra-4,9,11-trien-3-one (R1881), a synthetic 5alpha-DHT agonist. In ONH astrocytes, both 3alpha-diol and R1881 increased protein levels of androgen receptor (AR) and glial fibrillary acidic protein (GFAP), however, only R1881 also increased the AR mRNA level and astrocyte proliferation. Both R1881 and 3alpha-diol rapidly activate the mitogen-activated protein kinase (MAPK) signaling pathway in ONH astrocytes, as confirmed by phosphorylation of extracellular signal-regulated kinase (ERK). 3Alpha-diol also activates the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. 3Alpha-diol regulates the increase of AR protein level and the phosphorylation through the PI3K/Akt pathway, whereas R1881 regulates them through the MAPK/ERK pathway. Our findings demonstrate that human ONH astrocytes are androgen target cells and respond to androgens by the rapid activation of cell signaling. The activation of the PI3K/Akt pathway by 3alpha-diol may regulate various properties of astrocytes, including cell motility and survival, and may play a role in the formation and maintenance of the reactive phenotype of ONH astrocytes in glaucoma.