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.     

In vivo uptake and metabolism of 3-h-5alpha-androstane-3alpha,17beta-diol and of 3-h-5alpha-androstane-3beta,17beta-diol by human prostatic hypertrophy.

Tritiated 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) and 5alpha-androstane-3beta,17beta-diol (3beta-diol) respectively were administered to patients with benign prostatic hypertrophy (bph) undergoing prostatectomy. In prostate and skeletal muscle homogenates and in plasma the total radioactivity content as well as the formation of metabolites were measured. Histological examination of each ectomized prostate was performed to evaluate the cellular composition of the tissue. After 3alpha-diol injection, a higher uptake of radioactivity in the prostate was obtained than after 3beta-diol. Within 30 min the 3alpha-isomer was very efficiently converted to 5alpha-DHT, while most of the 3beta-isomer remained unchanged. There was, however, also after administration of the 3beta-diol a substantial biconversion to 5alpha-DHT as has been confirmed by recrystallization to constant specific radioactivity. Only after 3beta-diol epiandrosterone was detected in small but significant amounts. 3alpha-diol administration resulted in distinct concentrations of 3beta-diol, whereas the conversion of 3beta-diol to the 3alpha-isomer was insignificant. When comparing the histological composition of the prostatic tissue with the accumulation of radioactivity and the formation of metabolites only a weak correlation between glandular structure and radioactivity uptake after 3alpha-diol administration could be revealed.     

Urinary 5 alpha-androstane-3 alpha,17 beta-diol radioimmunoassay: a new clinical evaluation.

A rapid specific and reliable RIA for urinary 5 alpha-androstane-3 alpha,17 beta-diol (Adiol) is described using chromatographical purification and a specific antibody. Values are reported under some physiological and pathological conditions in 179 individuals. In 43 normal adult men, the mean (+/-SD) urinary Adiol excretion was 193 +/- 77 micrograms/24 h, and in 29 normal women it was 44 +/- 23 micrograms/24 h. These values are significantly different (P less than 0.01). In 49 hirsute women, urinary Adiol Excretion was elevated (137 +/- 51 micrograms/24 h) and significantly different from this value in normal women (P less than 0.01). The urinary Adiol excretion in 10 postmenopausal women was very low (less than 5 micrograms/24 h). In normal adult subjects, the theoretical contribution to urinary Adiol of the major secreted androgens was calculated. Whereas dehydroisoandrosterone and dehydroisoandrosterone sulfate yield the same amount of urinary Adiol in both sexes, testosterone is the main precursor of Adiol in men and androstenedione is the main precursor in normal premenopausal and hirsute women. However, the amount of Adiol recovered in the 24-h urine depends not only on the secretion rate of androstenedione and testosterone but is also related to the testosterone 5 alpha-reductase activity present in androgen target cells, especially in sexual skin.     

Simultaneous determination of testosterone, 5alpha-androstan-17beta-ol-3-one, 5alpha-androstane-3alpha,17beta-diol and 5alpha-androstane-3beta,17beta-diol in plasma of adult male rabbits by radioimmunoassay(1).

A simultaneous radioimmunoassay procedure for plasma testosterone (T), 5alpha-androstan-17beta-ol-3-one (DHT), 5alpha-androstane-3alpha,17beta-diol (3alphaol), and 5alpha-androstan-3beta,17beta-diol (3betaol) is described in this report. Peripheral plasma concentrations of T, DHT, 3alphaol, and 3betaol were 1.16 +/- 0.26, 0.49 +/- 0.15, 0.17 +/- 0.03, and 0.24 +/- 0.04 ng/ml, respectively, in adult male rabbits. In contrast, T, DHT, 3alphaol, and 3betaol peripheral plasma concentrations in dexamethasone-treated castrate rabbits were 0.07 +/- 0.01, 0.02 +/- 0.01, 0.07 +/- 0.01, and 0.05 +/- 0.01 ng/ml, respectively. These results represent the first simultaneous measurement of T, DHT, 3alphaol, and 3betaol in peripheral plasma of any male vertebrate. Moreover, we suggest that DHT, 3alphaol, and 3betaol represent the unidentified immunoreactive material found in peripheral plasma of male rabbits by Falvo and Nalbandov (1).     

Formation of 5alpha-androstane-3alpha,17beta-diol by normal and hypertrophic human prostate.

The 3-keto reduction of [1,2-3H]dihydrotestosterone to 3alpha- and 3beta-androstanediols was assessed in homogenates of 40 prostates obtained either at surgery or at medicolegal autopsy from men who had died suddenly. Formation of both androstanediols was demonstrable in cytosol and in microsomes, and both NADH and NADPH were effective cofactors for the two reactions. Formation of the two steroids was not influenced by storage of the gland for up to 8 h prior to processing. When NADPH was cofactor, the formation of 3alpha- and 3beta-androstanediol was significantly higher in microsomes and cytosol from hypertrophic than from normal glands.     

5alpha-androstane-3alpha,17beta-diol is formed in tammar wallaby pouch young testes by a pathway involving 5alpha-pregnane-3alpha,17alpha-diol-20-one as a key intermediate

The synthetic pathway by which 5alpha-androstane-3alpha,17beta-diol (5alpha-adiol) is formed in the testes of tammar wallaby pouch young was investigated by incubating testes from d 20-40 males with various radioactive precursors and analyzing the metabolites by thin-layer chromatography and HPLC. [(3)H]Progesterone was converted to 17-hydroxyprogesterone, which was converted to 5alpha-adiol by two pathways: One involves the formation of testosterone and dihydrotestosterone as intermediates, and the other involves formation of 5alpha-pregnane-3alpha,17alpha-diol-20-one (5alpha-pdiol) and androsterone as intermediates. Formation of 5alpha-adiol from both [(3)H]testosterone and [(3)H]progesterone was blocked by the 5alpha-reductase inhibitor 4MA. The addition of nonradioactive 5alpha-pdiol blocked the conversion of [(3)H]progesterone to 5alpha-adiol, and [(3)H]5alpha-pdiol was efficiently converted to androsterone and 5alpha-adiol. We conclude that expression of steroid 5alpha-reductase in the developing wallaby testes allows formation of 5alpha-reduced androgens by a pathway that does not involve testosterone as an intermediate.     

Production of testosterone, 5 alpha-androstane-3 alpha, 17 beta-diol and androsterone by dispersed testicular interstitial cells and whole testes in vitro.

The production of 5 alpha-androstane-3 alpha, 17 beta-diol (androstanediol), androsterone and testosterone by whole rat testes and testicular interstitial cells dispersed with collagenase was studied in vitro. Luteinizing hormone stimulated the production of each of the androgens by cells prepared from 31- to 34-day-old rats. Half maximum stimulation of the production of each androgen occurred with approximately 3.5 ng NIH-LH-B9/ml medium. Androstanediol was the predominant product then androsterone and then testosterone. Luteinizing hormone stimulated the production of testerone, but not androstanediol or androsterone by dispersed interstitial cells from 200-day-old rats. The time-course of production and the effect of the concentration of cells on the production of these androgens suggested that in dispersed testicular interstitial cells from immature animals androstanediol and androsterone are formed, at least partially, by the metabolism of testosterone. In these experiments LH-stimulated testosterone production increased during incubation for 15--60 min and then remained constant up to 180 min. The concentrations of androstanediol and androsterone increased in a linear manner during incubation for 60--180 min. Varying the number of cells incubated yielded a positive correlation between cell concentration and the ratio 5 alpha-reduced androgen : testosterone produced. Luteinizing hormone stimulated production of each androgen by whole tests obtained from rats at 30--175 days of age. The serum concentration of testosterone in these rats increased abruptly at 50 days of age. Significant changes in androgen production in vitro also observed at this age included: (1) increased production of the three steroids when incubated in either the presence or absence of LH and (2) testosterone production, either in the presence or absence of LH, which represented a greater percentage of the total production of the three androgens.     

Virilization of the urogenital sinus of the tammar wallaby is not unique to 5alpha-androstane-3alpha,17beta-diol

The androgen 5alpha-androstane-3alpha,17beta-diol (5alpha-adiol) is synthesized in testes and secreted into plasma of male tammar wallaby pouch young and appears to virilize the urogenital sinus. To provide insight into its mechanism of action, a dose response study showed that administration of 1 microg 5alpha-adiol monoenanthate per g body wt. per week for 3 weeks to 24-day-old female pouch young induced prostate bud formation equivalent to that of males of the same age. Administration of this same dose of the enanthates of testosterone, dihydrotestosterone, and 5alpha-adiol to female pouch young caused equivalent virilization of the urogenital sinus. The fact that 5alpha-adiol does not exert a unique effect, together with our earlier findings in this species that 5alpha-adiol and testosterone are converted to dihydrotestosterone in the urogenital sinus and that virilization of the urogenital sinus is prevented by the androgen receptor antagonist flutamide, suggest that 5alpha-adiol is a circulating precursor for dihydrotestosterone formation in this tissue.     

Plasma levels of 5alpha-androstane-3alpha,17beta-diol in boys during adolescent growth.

A radioimmunoassay using as antibody against testosterone-3-carboxymethyloxime-BSA is described for the measurement of androstanediol (5alpha-androstane-3alpha,17beta-diol) in human plasma. Sephadex LH 20-column chromatography was used to separate other steroids croos-reaching with the antibody from androstanediol. The sensitivity of the assay was 7 pg and the recovery of labelled androstanediol was 65.1%. The intra-assay coefficient of variation was 9.9%. The existence of 5alpha-androstane-3alpha,17beta-diol in the androstanediol fraction could be demonstrated by gas chromatography and mass spectrometry (GC-MS). Plasma concentration of this substance was measured in 53 normally developing pre-pubertal and pubertal boys and in 13 adult men. The mean concentrations significantly rose from puberty stage 1 to 2, and stage 2 to 3. Although there was no significant difference between the plasma concentrations at stages 3 and 4, an increment from stage 4 and 5 was highly significant. Levels in adult males were significantly higher than those in the stage 5 of normal puberty.     

Urinary 5 alpha-androstane-3 alpha, 17 beta-diol (5-Ad) determination in healthy children and adults

The authors suggest a method for the evaluation of the urinary 5 alpha-androstane-3 alpha, 17 beta-diol (5 Ad) carried out by gas-liquid chromatography. 5-Ad is separated from its isomers, first by means of an adsorption chromatography on alumina and then by means of a high performance liquid chromatography on porous silica. The reliability criteria of the method have been studied according to the advice of the French Society of Clinical Biology. This method, applied to 175 healthy individuals of both sexes, grouped according to age starting from three years, allowed to determine the usual values of urinary excretion of 5-Ad in relation to age.     

Steroid 5alpha-reductase 1 promotes 5alpha-androstane-3alpha,17beta-diol synthesis in immature mouse testes by two pathways

5alpha-Androstane-3alpha,17beta-diol (androstanediol) is the predominant androgen in immature mouse testes, and studies were designed to investigate its pathway of synthesis, the steroid 5alpha-reductase isoenzyme involved in its formation, and whether testicular androstanediol is formed in embryonic mouse testes at the time of male phenotypic development. In 24-26-day-old immature testes, androstanediol is formed by two pathways; the predominant one involves testosterone --> dihydrotestosterone --> androstanediol, and a second utilizes the pathway progesterone --> 5alpha-dihydroprogesterone --> 5alpha-pregnane-3alpha-ol-20-one --> 5alpha-pregnane-3alpha,17alpha-diol-20-one --> androsterone --> androstanediol. Formation of androstanediol was normal in testes from mice deficient in steroid 5alpha-reductase 2 but absent in testes from mice deficient in steroid 5alpha-reductase 1, indicating that isoenzyme 2 is not expressed in day 24-26 testes. The fact that androstenedione and testosterone were the only androgens identified after incubation of day 16 and 17 embryonic testes with [3H]progesterone implies that androstanediol formation in the testis plays no role in male phenotypic differentiation in the mouse.     

Circulating unconjugated 5 alpha-androstane-3 alpha, 17 beta-diol in elderly men and patients with prostatic tumours

Circulating levels of 5 alpha-androstane-3 alpha,17 beta-diol (diol) were measured in 24 elderly men (50-80 years) without prostatic tumours and compared with 32 patients (54-84 years) with benign prostatic hypertrophy (BPH) and 32 patients (51-85 years) with carcinoma of the prostate (Ca). The mean +/- s.e.m. for serum concentrations of diol in patients with BPH, Ca and normals were 813 +/- 43, 524 +/- 35 and 635 +/- 28 pmol/l, respectively. There were significant differences between the mean values of these groups. The increased level of diol in BPH patients, when compared to the normals, corresponds to our previous report of elevated levels of its precursor, namely 5 alpha-dihydrotestosterone (DHT), in these patients. Although the mean values for diol were significantly different between the BPH and Ca patients, the scattered values within each group could not be utilized as an index to differentiate the two groups of patients.     

Penile development is initiated in the tammar wallaby pouch young during the period when 5alpha-androstane-3alpha,17beta-diol is secreted by the testes

Virilization of the urogenital tract is under the control of testicular androgens in all mammals. In tammar young, prostate differentiation begins between d 20 and d 40 under the control of the testicular androgen 5alpha-androstane-3alpha,17beta-diol (5alpha-adiol), but uncertainties exist about the control of penile development. We performed longitudinal studies up to d 150 of pouch life to define normal penile development and the effects of androgen administration and castration. In control animals the male phallus was longer than the female phallus by d 48. Closure of the urethra in males begins around d 60 and continues to at least d 150. Administration of supraphysiological doses of testosterone to females caused penile development equivalent to that of the male and also induced partial closure of the urethral groove by d 150. Castration of male pouch young at d 25 prevented penile development, whereas the penis in males castrated at d 40, 80, or 120 had partial closure of the urethral groove. Administration of 5alpha-adiol to females from d 20-40 also caused partial closure of the urethral groove and some growth of the phallus at d 150, whereas 5alpha-adiol treatment from d 40-80 or 80-120 caused some penile growth but had little effect on urethral development. These findings, together with the fact that we found no sex differences in plasma levels of testosterone, dihydrotestosterone, 5alpha-adiol, dehydroepiandrosterone, or androstenedione from d 51-227, clearly indicate that the action of 5alpha-adiol between d 20 and 40 imprints later differentiation of the male penis.     

Decreased urinary 5 alpha-androstane-3 alpha,17 beta-diol glucuronide excretion in patients with benign prostatic hyperplasia

Urinary testosterone and 3 alpha-androstanediol (3 alpha diol G) glucuronides together with plasma testosterone, 5 alpha-dihydrotestosterone (DHT), and delta 4-androstenedione (delta 4) were measured in 43 normal young men (18-36 yr old), 23 elderly men without clinically evident prostatic pathology (54-89 yr old), 68 elderly men with benign prostatic hyperplasia (BPH group; 54-91 yr old), and 26 elderly men with well differentiated cancer of the prostate (K group; 63-97 yr old). Plasma testosterone decreased slightly with age in all 3 elderly groups (from 591 to 438, 479, and 444 ng/100 ml, respectively). Plasma DHT, on the contrary, was significantly (P less than 0.01) higher in the BPH group than in the other three groups (68 vs. 30, 37, and 32 ng/100 ml, respectively). Plasma delta 4 was significantly lower (P less than 0.01) in the elderly K group than in all other groups (59 vs. 109, 83, and 78 ng/100 ml, respectively). Urinary testosterone glucuronide decreased with age in all 3 elderly groups (from 109 to 55, 38, and 44 micrograms/24 h, respectively) as a result of decreased androgen production rates with age. All 3 elderly groups also had decreased urinary 3 alpha diol G, from 194 to 123, 55, and 118 micrograms/24 h, respectively. The group of elderly patients with BPH had the lowest mean urinary 3 alpha diol G excretion together with the highest mean plasma DHT. This low urinary 3 alpha diol G excretion, which reflects a decrease in both androgen production and DHT metabolism, suggests a decrease in 3 alpha-hydroxysteroid dehydrogenase activity, which, in turn, could explain the increased DHT availability and tissue retention in most target organs. Moreover, the extent of these modifications in androgen metabolism specific to the BPH condition raises the question of an overall alteration of androgen metabolism in patients with BPH which could be the cause of the disease.     

Determination of 5 alpha-androstane-3 alpha, 17 beta-diol in human serum by GC-SIM and influence of age-associated change

5 alpha-Androstane-3 alpha, 17 beta-diol (A3 alpha diol) is a potent androgen, and is an end product of testosterone. Many authors measured A3 alpha diol levels in human plasma by various methods, but the levels of this steroid were very dissimilar. In order to validate such values, it was measured by gas chromatography-selected ion monitoring (GC-SIM) in this study. A3 alpha diol, 5 alpha-Androstane-3 beta, 17 beta-diol (A3 beta diol) and Testosterone (T) in human peripheral serum were measured by GC-SIM at the same time. The TFA-derivatives of these compounds were analyzed after purification of the serum extract by Sephadex LH-20 microcolumn chromatography. The sensitivity was good: (16.7 pg/ml: A3 alpha diol, 26.7 pg/ml: A3 beta diol). The precision (CV = 2.75%: A3 alpha diol, 3.11%: A3 beta diol) and the accuracy were better than ever reported. Serum A3 alpha diol was measured in 131 healthy men aged 15-81 years and 5 healthy women aged 25-60 years. There were remarkable differences between individuals in the serum levels of A3 alpha diol, but the levels in male serum (greater than 20y) showed a significant negative correlation with age (r = -0.560, p less than 0.01). When these healthy men were classified into three age groups of 20-39, 40-59 and 60-79 years, the values (mean +/- SD) for serum A3 alpha diol were 189.3 +/- 77.7 (n = 20), 127.9 +/- 59.5 (n = 28), and 94.9 +/- 52.9 (n = 73) pg/ml, respectively. There were significant differences between the levels of this steroid in all age groups (p less than 0.01). There was a weak but significant correlation between serum A3 alpha diol and T levels (r = 0.3235, p less than 0.01) in healthy men (25-77 years, n = 77). Determination of serum A3 alpha diol was influenced by age. The number of samples strongly influenced the decision of mean value of A3 alpha diol levels. These results suggested that these factors had to be made obvious when this steroid was studied.