Androgen metabolism in sheep

³H-Testosterone (³H-T) plus ¹⁴C-androst-4-ene-3.17-dione (A-dione) and ³H-epi-testosterone (17α-hydroxy-4-androsten-3-one) (epiT) plus ¹⁴C-T were injected intravenously into two male sheep with bile fistulae, respectively. Urine and bile samples were collected at intervals for 4–8 hours and analyzed by the use of DEAE-Sephadex A-25 and Lipidex 5000 columns, TLC, and paper chromatography; the aglycones were identified by co-crystallization with authentic standards.Five fractions were obtained from urine and bile: unconjugated, glucosiduronates, sulfates, sulfo-glucosiduronates and disulfates. In urine, the major conjugates were glucosiduronates, while sulfates predominated in bile. About 80–90% of recovered radioactivity was found to be either glucosiduronates or sulfates. Among the metabolites identified, epi-T was the principal one, accounting for 10–15% of the administered doses. Conversion to 17α-hydroxysteroids thus appears to be a major route of metabolism of the androgens administered in sheep. Other metabolites in the glucosiduronate and sulfate fractions were androsterone, etiocholanolone (3α-hydroxy-5β-androstan-17-one), 5β-androstane-3α, 17β-diol, two unknown diols and polar metabolites. The results indicated that androgen metabolism is somewhat unusual in sheep, as compared with other animals and the human.     

The metabolic fate of chlormadinone acetate in the baboon.

The metabolic fate of chlormadinone acetate (17alpha-acetoxy-6-chloro-4, 6-pregnadiene-3, 20-dione; CAP) was studied in intact and biliary fistula baboons. The steroid was labeled with 3H at position 1 and with 14C at the carboxyl moiety of the 17alpha-acetate, thus affording the opportunity to ascertain the loss of the 17alpha-acetoxy group and the fate of both labels. The averages of the radioactivity excreted, given as percentages of the amounts injected, and the standard deviations were as follows: In the urine of intact animals after 6 hours, 5.7 +/- 0.2% and 5.5 +/- 0.7% of the 3H and 14C were recovered, respectively. After 6 days, there was 17.5% of the 3H and 16.2% of the 14C in the urine plus 15.3% of the 3H and 16.4% of the 14C in the feces. In baboons with biliary fistulas, the total radioactivity excreted was 7.8 +/- 0.7% of the 3H and 11.6% of the 14C in the urine, and 30.9 +/- 4.4% of the 3H and 30.7% of the 14C in the bile after 6 hours. Glucosiduronates were the predominant conjugates in the urine and bile. The similarity in the urinary excretion of radioactivity in the first 6 hours in intact and biliary fistula animals, the relatively low excretion of radioactivity in the bile and after 6 days in the urine, and the low fecal excretion suggest that the metabolites of CAP are not involved in an extensive enterohepatic circulation in the baboon. Deacetylation of the 17alpha-acetate in CAP was detected in the early collection periods of the urine and bile and constituted a very small percentage of the injected compound. No significant oxygenation of CAP at position 1 was detected. The metabolism of CAP is discussed and compared to our previously reported data on the metabolism of progesterone, ethynodiol diacetate and medroxyprogesterone acetate and the data on other progestogens reported in the literature. It appears that the excretion of CAP is significantly slower in the baboon than that of the other progestogens. The amounts of glucosiduronates of CAP and/or its metabolites formed in vivo are less than those formed with the other progestogens. Also, the extent of deacetylation of the 17alpha-acetate of CAP is much less than that of the 3beta-acetate of ethynodiol diacetate.     

Testosterone metabolites in dog bile

Testosterone-1,2-3H was injected intravenously into a male dog with a bile fistula and bile and urine collected. The radioactivity was excreted preponderantly in bile (52% of the injected dose) in 6 hours; only 12% appeared in the urine. Methods to study the biliary metabolites of testosterone in this and other animals were developed. Satisfactory conjugate patterns were obtained by fractionation on DEAE-Sephadex A-25 columns using two different elution systems. In addition to an unchanged fraction, six different monoglucuronide fractions were separated. No other conjugates were isolated. Lipidex 5000 column chromatography, TLC and paper chromatography were used for the isolation and purification of aglycone metabolites, which were further identified by co-crystallization methods. The biliary metabolites of testosterone were epiandrosterone (3beta-hydroxy-5alpha-androstan-17-one), etiocholanlone (3alpha-hydroxy-5beta-androstan-17-one), 5alpha-androstan-3beta, 17beta-diol, 5beta-androstan-3alpha, 17beta-diol and 5beta-androstan-3beta,17beta-diol.     

Metabolism of testosterone sulfate in the rat: analysis of biliary metabolites.

Following intraperitoneal injection of a mixture of testosterone-7-3-H-17-sulfate and testosterone-4-14-C into male and female rats with bile fistulas, biliary metabolites were separated and purified by a combination of column chromatography, enzymic hydrolysis or solvolysis of the conjugate fractions and identification of the liberated aglycones. The injected steroids were extensively metabolized and excreted predominantly in the bile. The major portion of the 3H was excreted in the disulfate fraction in both sexes. Solvolysis of the disulfate revealed the sex-specific aglycone pattern: 5alpha-Androstane-3beta,17beta-diol was the major metabolite in the male rat, whereas 5alpha-androstane-3alpha,17beta-diol and polar steroids were found in the female. In marked contrast, testosterone was metabolized in a different way than testosterone sulfate. 14-C radioactivity was distributed in monoglucosiduronate, monosulfate, and diconjugate fractions. Analysis of the aglycones showed that polar steroids were the main metabolites in the male. In the female, testosterone was metabolized to polar steroids, androsterone, and 5alpha-androstane-3alpha,17beta-diol.     

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.     

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).     

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.     

Disposition and metabolism of 16 beta-ethyl-17 beta-hydroxy-4-estern-3-one (TSAA-291), a new antiandrogen, in rats.

Matabolic fate of a new antiandrogen, 16 beta-ethyl-17 beta-hydroxy-4-estren-3-one (TSAA-291), was studied in rats. 14C-TSAA-291 intramuscularly injected as an aqueous suspension was absorbed gradually to give an increase in the plasma level which attained a plateau at 0.5 h, persisted till 8 h and then declined with an approx. half-life of 3.6 days. The drug was widely distributed in tissues, with the concns. almost equal to or higher than that in the plasma. The 14C-drug was eliminated mostly as metabolites within 10 days after dosing with higher activities found in the feces than in urine. Biliary 14C effectively underwent enterohepatic cycling. Biliary metabolites of TSAA-291 were characterized by the combined use of deuterium labeling and GLC-MS analysis. The metabolites identified were as follows: the parent drug, monohydroxy TSAA-291 having the additional hydroxy function in the steroid skeleton, 17 beta-hydroxy-16 beta-(1 xi-hydroxyethyl)-4-estren-3-one, 16 beta-ethyl-17 beta-hydroxy-5 beta-estran-3-one, 16 beta-ethyl-17 beta-hydroxy-5 alpha-estran-3-one, 16 beta-ethyl-5 beta-estrane-3 alpha, 17 beta-diol, 16 beta-ethyl-5 alpha-estrane-3 alpha, 17 beta-diol, 16 beta-ethyl-3 alpha-hydroxy-5 beta-estran-17-one and 16 beta-ethyl-3 alpha-hydroxy-5 alpha-estran-17-one. Monoketodihydroxy and/or trihydroxy metabolites were also detected in the bile.     

Formation of 5alpha-reduced C19 steroids from progesterone in vivo by 5alpha-reduced pathway in older prepubertal rat testis.

Either [3H] progesterone (0.5 or 5 nmol/5 muCi), 5alpha-[3H] pregnane-3,20-dione (5 nmol/5 muCi) or [14C] progesterone (6.6 nmol/0.2 muCi) plus 5alpha-[3H]-pregnane-3,20-dione (1 or 6.6 nmol/0.6 muCi), suspended in 0.05 ml of physiological saline solution, was injected into each testis of 32- and 90-day-old rats. Following injection, radioactive metabolites in testis and spermatic vein blood were extracted, isolated, measured and identified by column and paper chromatographies, with derivative formation and recrystallization to constant specific activity. In the blood and testis of older prepubertal rats, major 17-OH-C21 and C19 metabolites of progesterone were 5alpha-reduced steroids such as 3alpha, 17alpha-dihydroxy-5alpha-pregnan-20-one, 5alpha-androstane-3alpha,17beta-diol and androsterone. Following injection of [14C] progesterone plus 5alpha-[3H] pregnane-3,20-dione into 32-day-old rat testis, no significant augmentation of the isotope from progesterone was observed in 5alpha-reduced C19 steroids as compared with 5alpha-reduced 17-OH-C21 steroids, indicating that 5alpha-reduced C19 steroids were mainly formed from 5alpha-reduced 17-OH-C21 steroids in older prepubertal testis. In the blood and testis of adult rats, small amounts of 5alpha-reduced metabolites were shown to be produced from progesterone, while active 17alpha-hydroxylation of 5alpha-pregnane-3,20-dione followed by C17-C20-lyase reaction was demonstrated. These findings seem to indicate that formation of 5alpha-reduced C19 steroids from progesterone by the 5alpha-reduced pathway is a major pathway of androgen biosynthesis in older prepubertal rat testis in vivo.     

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.     

Metabolism of metandienone in man: identification and synthesis of conjugated excreted urinary metabolites, determination of excretion rates and gas chromatographic-mass spectrometric identification of bis-hydroxylated metabolites

After oral administration of metandienone (17 alpha-methyl-androsta-1,4-dien-17 beta-ol-3-one) to male volunteers conjugated metabolites are isolated from urine via XAD-2-adsorption, enzymatic hydrolysis and preparative high-performance liquid chromatography (HPLC). Four conjugated metabolites are identified by gas chromatography-mass spectrometry (GC/MS) with electron impact (EI)-ionization after derivatization with N-methyl-N-trimethyl-silyl-trifluoroacetamide/trimethylsilyl-imidazole (MSTFA/TMS-Imi) and comparison with synthesized reference compounds: 17 alpha-methyl-5 beta-androst-1-en-17 beta-ol-3-one (II), 17 alpha-methyl-5 beta-androst-1-ene-3 alpha,17 beta-diol (III), 17 beta-methyl-5 beta-androst-1-ene-3 alpha,17 alpha-diol (IV) and 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol (V). After administration of 40 mg of metandienone four bis-hydroxy-metabolites--6 beta,12-dihydroxy-metandienone (IX), 6 beta,16 beta-dihydroxy-metandienone (X), 6 beta,16 alpha-dihydroxy-metandienone (XI) and 6 beta,16 beta-dihydroxy-17-epimetandienone (XII)--were detected in the unconjugated fraction. The metabolites III, IV and V are excreted in a comparable amount to the unconjugated excreted metabolites 17-epimetandienone (VI), 6 beta-hydroxy-metandienone (VII) and 6 beta-hydroxy-17-epimetandienone (VIII). Whereas the unconjugated excreted metabolites show maximum excretion rates between 4 and 12 h after administration the conjugated metabolites III, IV and V are excreted with maximum rates between 12 and 34 h.     

Androgen glucuronyl transferase activity in rat liver, evidence for the importance of hepatic tissue in 5 alpha-reduced androgen metabolism

To elucidate the role of the liver in 5 alpha-reduced androgen metabolism, we used a rat liver glucuronyl transferase assay to determine the conversion of 17 beta-hydroxy-5 alpha-androstane-3-one (DHT), 5 alpha-androstane-3 alpha, 17 beta-diol (androstanediol), and androsterone to their glucuronide metabolites. Serum levels of the two isomers of androstanediol glucuronide (androstanediol 3- and 17-glucuronide) were also measured. Using 5 microM unconjugated steroid as substrate, the production rate (pmol/mg/min) for each product from its respective unconjugated steroid was 6.9 +/- 0.4 for DHT glucuronide, 101 +/- 3.3 for androstanediol 3-glucuronide, 71 +/- 2.0 for androstanediol 17-glucuronide, and 181 +/- 11 for androsterone glucuronide. Production rates for androstanediol glucuronide were 800 times greater for rat liver than for rat prostate, when examined under similar conditions. In the presence of either 0 or 5 microM unlabeled androstanediol, about 60% of the androstanediol glucuronide formed by rat liver was androstanediol 3-glucuronide. In normal male rat serum, 69 +/- 8% (mean +/- SEM) of total androstanediol glucuronide was androstanediol 3-glucuronide. We have previously shown that rat prostate forms androstanediol 17-glucuronide, but not androstanediol 3-glucuronide. The results from the present study indicate that rat liver forms both androstanediol glucuronide isomers, and does so in about the same ratio as is found in rat serum. The rate of glucuronidation is also much greater in rat liver than in rat prostate. While other sites of glucuronidation are possible, these results are consistent with the hypothesis that DHT and other unconjugated androgens formed in rat prostate are conjugated to glucuronic acid mainly in the liver.     

The metabolism of steroids in the fatty liver induced by orotic acid feeding.

1. The metabolism of 4-[4-14C]androstene-3,17-dione, 4-[4-14C]pregnene-3,20-dione, 5alpha-[4-14C]androstane-3alpha,17beta-diol, [4-14C]cholesterol, 7alpha-hydroxy-4-[6beta-3H]cholesten-3-one, 5beta-[7beta-3H]cholestane-3alpha,7alpha-diol and [3H]lithocholic acid was studied in the microsomal fraction of livers from control and orotic acid-treated male rats. 2. As a result of the treatment the orotic acid-fed rats had fatty livers and subnormal concentrations of cholesterol and triglycerides in serum. 3. The 6beta- and 7alpha-hydroxylation of 4-androstene3,17-dione, and the 2alpha-, 2beta- and 18-hydroxylation of 5alpha-androstane-3alpha,17beta-diol, and the 5alpha-reduction of 4-androstene-3,17-dione and 4-pregnene-3,20-dione were decreased by 40--50% in orotic acid-fed rats. Other oxidative and reductive reactions of the steroid hormones were not significantly affected. 4. The 12alpha-hydroxylation of 7alpha-hydroxy-4-cholesten-3-one was decreased by about 50%, whereas the 7alpha-hydroxylation of cholesterol and the 26-hydroxylation of 5beta-cholestane-3alpha,7alpha-diol were not significantly decreased. The 6beta-hydroxylation of lithocholic acid was stimulated by 40%. 5. The results are discussed in relation to present knowledge of the heapatic drug-metabolizing enzymes and to the recent findings of an abnormal bile acid metabolism in liver disease.     

Dehydroepiandrosterone sulfate in plasma: hydrolysis, extraction and radioimmunoassay.

At pH 4.5, the hydrolysis of 3beta-hydroxy-5-androsten-17-one 3-sulfate (DHA-SO4) to DHA was complete within 75 min at 120 degrees or 4h at 100 degrees. In the same conditions, the 3alpha-SO4 of androsterone was stable, and only 8.5% of the 3beta-SO4 of epiandrosterone hydrolysed to epiandrosterone. Of the sulfates of 5-androstene-3beta, 17beta-diol, 100% of the 3beta-mono-SO4, 2% OF THE 17BETA-MONO-SO4 and none of the 3beta, 17beta-di-SO4 was converted to 5-androstenediol. Denatured plasma proteins adsorbed DHA. The recovery of DHA from plasma diluted 1:100, 58.7 +/- 6.2% (mean +/- S.D.). In similar conditions the recovery of cholesterol from plasma diluted 1:20, was 0.12 - 1.76% (mean, 0.44%). A radioimmunoassay for DHA in extracts of hydrolysed plasma is described. Results for normal subjects in the age range 17-45y were 192 +/- 73mug/dl (22 men) and 158 +/- 57mug/dl (40 women).     

The metabolic fate of the anti-androgenic agent, oxendolone, in man

After intramuscular administration of 16 beta-ethyl-17 beta-hydroxy-4-4-[4-14C]estren-3-one (14C-oxendolone; 300 mg) to 3 human subjects, excretion of 14C was very slow and incomplete despite a 20-day sample collection period. During this time, means of 37% and 21% of the administered 14C were recovered in urine and faeces, respectively, and if excretion continued at the same rate, approximately 90% of the administered 14C would have been excreted during 5-12 weeks. Peak plasma 14C concentrations were reached at 3-6 days after dosing, when they represented 0.2-1.1 micrograms equiv./ml, and declined very slowly thereafter with a half-life of 5.0-6.6 days. Concentrations of unconjugated drug-related steroids circulating in plasma never exceeded about 0.1 microgram/ml. Mass spectroscopic analysis of isolated urinary and faecal metabolites indicated that the principal routes of biotransformation of oxendolone in man are similar to those of the endogenous androgens-namely, reduction of the 4,5-double bond, further reduction of the saturated 3-ketone to the 3 alpha-hydroxysteroid, and oxidation of the 17 beta-alcohol to the corresponding ketone, followed by conjugation, mainly with glucuronic acid, and excretion in the urine and bile.     

Bile acids and bile alcohols in a child with hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency: effects of chenodeoxycholic acid treatment

Duodenal bile, urine, plasma, and feces from a child with hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency were analyzed by fast atom bombardment mass spectrometry and gas chromatography-mass spectrometry to investigate the formation and excretion of abnormal bile acids and bile alcohols. The biliary bile salts consisted of glycocholic acid (25%) and of sulfated and glycine conjugated di- and trihydroxycholenoic acids (55%), two C27 bile acids, and eleven sulfated bile alcohols (mainly tetrols, 20%), all having 3 beta,7 alpha-dihydroxy-delta 5 or 3 beta,7 alpha,12 alpha-trihydroxy-delta 5 ring structures. In plasma, sulfated cholenoic acids constituted 65% and unconjugated 3 beta,7 alpha-dihydroxy-5-cholestenoic acid 25% of the total level, 71 micrograms/ml. The urinary excretion of the former was 30.4 mg/day and that of unsaturated bile alcohol sulfates, mainly pentols, 7 mg/day. The predominant bile acid in feces was an unconjugated epimer of 3 beta,7 alpha,12 alpha-trihydroxy-5-cholenoic acid, and small amounts of cholic acid were present. The minimum total excretion was 11.3 mg/day. Treatment with chenodeoxycholic acid resulted in marked clinical improvement and normalized liver function tests. Further studies are needed to define the mechanism of action. Plasma bile acids decreased to 1.6 micrograms/ml and urinary excretion to 3.4 mg/day. Chenodeoxycholic and ursodeoxycholic acids became predominant in all samples. The fecal excretion of unsaturated cholenoic acid sulfates increased to 40 mg/day compared to 89 mg/day of saturated bile acids. The results provide further support for a defective hepatic 3 beta-hydroxy-delta 5-C27-steroid dehydrogenase deficiency, and indicate that the 3 beta-hydroxy-delta 5 bile acids are formed via 7 alpha-hydroxycholesterol. The formation of glycocholic acid may be due to an incomplete enzyme defect or to transformation of the 3 beta-hydroxy-delta 5 structure by bacterial and hepatic enzymes during an enterohepatic circulation.     

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.     

Steroid metabolism in human breast cancer cell lines

The metabolism of 1,2-³H-androstenedione was studied in 2 cell lines, MCF-7 (estrogen responsive) and BT-20 (estrogen nonresponsive) over 48 hrs. Water soluble and unconjugated metabolites were separated by solvent partition and the former was submitted to chromatography on Sephadex LH-20 and enzyme hydrolysis. The resulting unconjugated steroids were separated by paper chromatography and identities were established by reverse isotope dilution. The unconjugated steroids initially obtained were separated by chromatography and identified by reverse isotope dilution. About 70% of the androstenedione was metabolized by both cell lines. However, the respective conversions to conjugates by MCF-7 and BT-20 were 31% and 0.32%. In the former, glucosiduronates predominated (94%) and consisted of androsterone (55%), etiocholanolone (9.4%) and androstanediol (5α-androstane-3α,17β-diol) (9.3%). Androsterone comprised most of the unconjugated metabolites in both cell lines. Androstanediol was found in both cell lines, 2% in MCF-7 and 12% in BT-20. Testosterone, 5α-androstane-3,17-dione and 3β-hydroxy-5α-androstan-17-one were isolated only from MCF-7. The metabolism of ³H-estriol was studied in a similar way. Both cell lines produced about equal amounts of estriol-3-sulfate (9%) and a compound with properties of estriol-3-glucosiduronate (0.15 – 0.5%). The results worthy of emphasis are: 1. The far greater conjugation of androgens exhibited by the MCF-7 cell lines as compared to the BT-20 cell lines; 2. In MCF-7, the high conversion of androstenedione to etiocholanolone (glucosiduronate form), a metabolite reported to form only in liver and sebaceous cysts; 3. The possible formation in both cell lines of estriol-3-glucosiduronate, normally a metabolite of the intestine.     

Urinary 5 alpha-androstane-3 alpha,17 beta-diol levels in normal and hirsute women: discriminating power and relation to other urinary steroids

The urinary levels of seven steroids, 5 alpha-androstane-3 alpha,17 beta-diol, 5 beta-androstane-3 alpha,17 beta-diol, androsterone, etiocholanolone, tetrahydrocortisone, tetrahydrocortisol and allotetrahydrocortisol were measured in both normal (n = 18) and hirsute (n = 24) women. The results confirmed 5 alpha-androstane-3 alpha,17 beta-diol as the most significant steroid with respect to discrimination between hirsute and normal subjects. Investigation of the inter-steroid relationships, using multivariate techniques established that the mode of steroid metabolism was different between the two groups. Whereas in normal women the strong correlation amongst all the androgen metabolites inferred a predominant hepatic route to 5 alpha-androstane-3 alpha,17 beta-diol formation, the same analogy was not applicable to the hirsute subjects. Excellent agreement was found for the predicted vs actual excretion of 5 alpha-androstane-3 alpha,17 beta-diol in normal women, based on a regression model involving the six other steroids as independent variables. When the same model was used for estimation of 5 alpha-androstane-3 alpha,17 beta-diol levels in thirteen hirsute subjects, misclassified as "normal", 50% gave values which were considerably less than actually measured. It is suggested that this discrepancy, with respect to these hirsute subjects is a reflection of extrahepatic production of 5 alpha-androstane-3 alpha,17 beta-diol due to increased 5 alpha-reductase activity.     

Conversion of testosterone and androstenedione to 5 beta-androstanes by adult male hamster liver cytosol

The incubation of [4-14C]testosterone with adult male hamster liver cytosol at pH 6.7 yielded 5 beta-androstane-3 alpha, 17 beta-diol and small quantities of 5 beta-androstane-3 beta, 17 beta-diol, 17 beta-hydroxy-5 beta-androstan-3-one, 3 alpha-hydroxy-5 beta-androstan-17-one and androstenedione. The use of [4-14C]androstenedione as substrate yielded the same 5 beta-metabolites and also testosterone and a trace of epitestosterone. 5 beta-Androstane-3 alpha, 17 beta-diol was the major metabolite at "low" concentrations of substrate but testosterone and 3 alpha-hydroxy-5 beta-androstan-17-one became the major metabolites as the concentration of the substrate was increased. Small quantities of 5 beta-androstane-3,17-dione and 3 beta-hydroxy-5 beta-androstan-17-one were detected at "high" while 5 beta-androstane-3 alpha, 17 alpha-diol was detected at "low" concentrations of androstenedione. NADPH was more effective than NADH except in the formation of the 3 beta-steroids. Furthermore, the 3 beta-steroids were formed in maximum quantities at a lower pH than the other metabolites. The relative production of the metabolites was consistent with their respective spectrophotometrically determined degree of hydroxyl dehydrogenation.