Study on the inactivation mechanism of androgens in rheumatoid arthritis: excretory rate of free and conjugated 17-ketosteroids.

Unconjugated, sulpho- and glucurono-conjugated androgen hormone metabolites have been determined in the urine of patients with rheumatoid arthritis. An increase in the excretory rate of unconjugated 5 beta-reduced 17-ketosteroids and a decrease in that of 17-ketosteroid conjugates, especially in dehydroepiandrosterone sulphate and in the sum of dehydroepiandrosterone, etiocholanolone and androsterone glucuronoside were observed. In contrast to unconjugated metabolites, there was less significant change in the 5 beta-metabolite conjugates in urine. Corticosteroid treatment resulted in an additional decrease of metabolite excretion by patients. Further study is necessary to determine the causative factors in the altered steroid pattern observed in this severe, non-endocrine disease.     

Dehydroepiandrosterone and dehydroepiandrosterone sulfate dynamics in obesity.

Dehydroepiandrosterone (D) and dehydroepiandrosterone sulfate (DS) dynamics were studied in three obese female subjects following a single injection of [4-14-CA1D and [7 alpha-3-H]-DS tracers. Dynamic parameters were calculated simultaneously by both the urinary and blood method of compartmentalization; Estimates for the urinary secretion and production rates of D were found to be high, and those of DS varied within normal range. Calculation of the conversion factors, rho DDS and rho DSD, by the urinary method revealed a noraml extraglandular DS yields D conversion, while that for D yields DS appeared deficient in obese female subjects. Estimates of inner and outer pool distribution volumes were extremely increased for free D; in contrast to this, moderately increased inner and decreased outer pool volumes of DS were observed. The metabolic clearance rates of D were normal or decreased and those for DS were greater than normal. The blood production rates of both B and DS were higher in obese female subjects than those estimated for normal women in our previous study; These observations suggest a considerable uptake of unconjugated D by adipose tissue, an overall poor D yields DS conversion and an accelerated DS metabolism in obese female subjects.     

Determination of urine steroid profile in untrained men to evaluate recovery after a strength training session

Intense physical exercise is an important modifier of hormone metabolism. The aim of this study was to evaluate the variations in the urine profile of glucuroconjugated steroids (androgens, estrogens, and corticosteroids) as a consequence of a session of strength exercises. The subjects were a group (N = 20) of untrained male university students. They performed 3 sets of 10 repetitions, with a 3-minute recovery time between sets, at 70-75% of 1 repetition maximum (1RM). Four urine samples were collected per subject: before the session, immediately after, 3 hours after, and 48 hours after the session. They were assayed using a gas chromatograph coupled with a mass spectrometer. The concentrations of the different hormones were determined according to the urine creatinine level (ng steroid per mg creatinine). The substances assayed were testosterone, epitestosterone (Epit), androstenedione, dehydroepiandrosterone (DHEA), androsterone, etiocholanolone, beta-estradiol, estrone, tetrahydrocortisone (THE), and tetrahydrocortisol (THF). The results showed a significant decline after exercise with respect to the rested state in the urinary excretion of testosterone, Epit, DHEA, androsterone, and etiocholanolone. At 48 hours, there was a significant increase in the urinary excretion of Epit, androstenedione, androsterone, etiocholanolone, estrone, and THE. The androsterone + etiocholanolone/THE + THF ratio decreased after exercise, increased significantly (p < 0.05) at 3 hours, and returned to near resting levels at 48 hours. The data suggest that the performing a strength session at 70-75% of maximum strength provoked a state of fatigue in the subjects, from which they recovered 48 hours after the exercise.     

Metabolism of C19-steroids by homogenates of normal rat and mouse adrenal tissue and of the Snell transplantable rat adrenocortical tumour 494

C(19)-steroid metabolism in homogenates of adrenal tissue from rats and mice has been studied. Production of these compounds from [7alpha-(3)H]cholesterol by rat adrenal tissue appeared to follow a route independent of pregnenolone. The major products of [7alpha-(3)H]-dehydroepiandrosterone metabolism by rat adrenal tissue were 5alpha-reduced steroids, principally androsterone, epiandrosterone and 5alpha-androstanedione. No differences in metabolism of [7alpha-(3)H]dehydroepiandrosterone or [4-(14)C]pregnenolone were detected between adrenal tissue from Sprague-Dawley, Wistar and Osborne-Mendel rats, but experiments with the Snell rat adrenocortical tumour 494 showed that this tissue had low 5alpha-reductase activity. In contrast, the major products of [7alpha-(3)H]dehydroepiandrosterone metabolism by mouse adrenal tissue were 5beta-reduced steroids. Differences were observed between LACA and NH strains of mice in that there was a lower metabolism of androstenedione by NH mouse adrenal and a considerable difference in the proportions of aetiocholanolone and epiaetiocholanolone produced.     

The extraction of plasma 3-hydroxy-17-oxo steroid sulphates and the measurement of the constituent dehydroepiandrosterone sulphate and androsterone sulphate

1. A simple method for the extraction of 17-oxo steroid sulphates of plasma is described; glucosiduronates and orthophosphates are extracted, but to a smaller extent. 2. Four methods of analyses of the extracts are given and are relatively simple. Three of these are specific for steroid sulphates and two measure the sulphate conjugates directly. 3. Values for dehydroepiandrosterone sulphate and androsterone sulphate concentrations of normal and pathological plasmas are given.     

The in vitro synthesis of 7-hydroxy dehydroepiandrosterone by human mammary tissues

Normal and tumorous human mammary tissues were incubated in vitro with [7α-³H] dehydroepiandrosterone and [7α-³H] dehydroepiandrosterone sulphate. Tritium-labelled 7α-hydroxy dehydroepiandrosterone was identified as a principal metabolite from four of the five studies with normal tissue and all seven studies with tumorous tissue.     

Studies on the biosynthesis in vivo and excretion of 16-unsaturated C19 steroids in the boar

1. In one experiment [7alpha-(3)H]pregnenolone was infused continuously for 12min into the left spermatic artery of a sexually mature boar and blood was collected during this period by continuous drainage from the spermatic vein. After infusion, the testis was removed and immediately cooled to -196 degrees C. 2. From both the testicular tissue and the spermatic venous plasma, (3)H-labelled 16-unsaturated C(19) steroids were isolated and characterized and their radiochemical purity was established. 5alpha-Androst-16-en-3alpha- and 3beta-ol occurred mainly as sulphate conjugates and to a lesser extent as free steroids. Only traces of these alcohols occurred as glucosiduronate conjugates. 5alpha-Androst-16-en-3-one was found in the free (ether-extractable) fraction. 3. The isotope concentration of each of the (3)H-labelled 16-unsaturated C(19) steroids in testicular tissue was different from that in spermatic venous plasma. 4. The ratios of tritiated 5alpha-androst-16-en-3alpha- and 3beta-ol (free steroids) to their respective sulphate conjugates in the testicular tissue were less than the ratios of the same compounds in the spermatic venous plasma. The possibility that the sulphates are partially hydrolysed by testicular sulphatases before secretion is discussed. 5. In a second experiment, a continuous close-arterial infusion of [7alpha-(3)H]pregnenolone into the left testis was performed over a 200min period and all the urine that accumulated during the infusion was collected for analysis. 6. No (3)H-labelled 16-unsaturated C(19) steroids were detected in the urine as free steroids. Only a trace of 5alpha-androst-16-en-3alpha-ol was detected conjugated as glucosiduronate, whereas the corresponding 3beta-alcohol occurred mainly as glucosiduronate and to a lesser extent as sulphate. 7. The absence of 5alpha-androst-16-en-3beta-ol glucosiduronate in the spermatic venous blood and its presence in considerable amount in the urine may be attributed to hepatic glucuronyl transferase activity.     

Metabolism of [17-2H]pregnenolone into 5-[17 beta-2H, 17 alpha-18O]androstene-3 beta, 17 alpha-diol and other products by incubation with the microsomal fraction of boar testis under 18O2 atmosphere.

[17-2H]Pregnenolone was incubated with the microsomal fraction of boar testis under an 18O2 atmosphere. The metabolites were analyzed by gas chromatography-mass spectrometry, and the following six metabolites labeled with 2H or 18O (or both) were identified: 17 alpha-[17-18O]hydroxypregnenolone, [17-18O]dehydroepiandrosterone, 5-[17-18O]androstene-3 beta, 17 beta-diol, 16 alpha-[16-18O]hydroxy[17-2H]pregnenolone, 5-[17 beta-2H, 17-18O]androstene-3 beta,17 alpha-diol, and 5,16-[17-2H]androstadien-3 beta-ol. The time course of the formation of these metabolites from pregnenolone was also studied using 14C-labeled substrate. The results obtained from these experiments suggest that the first three metabolites were synthesized by a well-documented pathway--pregnenolone yields 17 alpha-hydroxypregnenolone yields dehydroepiandrosterone yields 5-androstene-3 beta,17 beta-diol--, and that 16 alpha-hydroxypregnenolone, 5-androstene-3 beta,17 alpha-diol and 5,16-androstadien-3 beta-ol were synthesized from [17-2H]pregnenolone with retention of 17-2H.     

Relevance of the selective oestrogen receptor modulators tamoxifen, toremifene and clomiphene in doping field: endogenous steroids urinary profile after multiple oral doses

The present study was performed to investigate the influence of the intake of selective oestrogen receptor modulators on the urinary endogenous steroids profile. For this purpose the circadian variability of luteinizing hormone, follicle-stimulating hormone, testosterone, 5α-androstan-3α,17β-diol, 5β-androstan-3α,17β-diol, epitestosterone, 4-androstenedione, androsterone and etiocholanolone were measured on eight subjects (four males and four females) by gas chromatography–mass spectrometry and chemiluminescent immunometric assay techniques before and after oral administration of multiple doses of either tamoxifen (80 mg for 2 days) or toremifene (120 mg for 2 days) or clomiphene (100 mg for 2 days). The individual baseline variability of the steroids studied was set up by collecting the urine samples every 3 h, for 3 days prior to the treatment; whereas the evaluation of the effects of the oral administration of multiple doses of selective oestrogen receptor modulators on the steroid urinary profile was assessed by collecting urine samples every three hours for at least five days from the first administration.The results of our measurements showed that, only in male subjects, the relative urinary concentrations of testosterone, epitestosterone and 4-androstenedione were significantly altered generally after the second day of drug administration. While no significant effects were recorded in both sexes on the luteinizing hormone, follicle-stimulating hormone, androsterone, etiocholanolone, 5α-androstan-3α,17β-diol and 5β-androstan-3α,17β-diol urinary levels and on testosterone/epitestosterone, 5α-androstan-3α,17β-diol/5β-androstan-3α,17β-diol and androsterone/etiocholanolone ratios.     

Formation of the A/B cis ring junction of ecdysteroids in the locust, Schistocerca gregaria.

1. The mechanism of formation of the A/B cis ring junction of ecdysteroids in the locust Schistocerca gregaria, was investigated by incorporation of [4-14C,3 alpha-3H], [4-14C,4 alpha-3H] and [4-14C,4 beta-3H]cholesterol into 20-hydroxyecdysone in fifth-instar larvae and into ecdysteroid conjugates in ovaries of maturing adult females. 2. In both systems there was retention of the 4 alpha-3H atom in the ecdysteroid and elimination of the 3 alpha- and 4 beta-3H atoms. 3. The 3H retained in the ecdysone formed from [4 alpha-3H]cholesterol in the ovarian system was probably located at C-4. The results are interpreted by postulating the involvement of a 3-oxo-delta 4 intermediate in ecdysteroid biosynthesis in insects.     

Studies on flavonoid metabolism. Biliary and urinary excretion of metabolites of (+)-[U-14C]catechin

1. The biliary and urinary excretion of (+)-[U-(14)C]catechin was studied in normal male rats after a single injection of the flavonoid. 2. In rats large amounts of radioactivity (33.6-44.3% of the dose in 24h) were excreted in the bile as two glucuronide conjugates [one of which was a (+)-catechin conjugate] and three other unconjugated metabolites. 3. Excretion of radioactivity in the urine when the bile duct was not cannulated amounted to 44.5% of the dose. 4. In both the urine and bile the new metabolites showed maximum excretion in the (1/2)-1(1/2)h after intravenous injection of [(14)C]catechin. 5. The metabolites m-hydroxyphenylpropionic acid, p-hydroxyphenylpropionic acid, delta-(3-hydroxyphenyl)-gamma-valerolactone and delta-(3,4-dihydroxyphenyl)-gamma-valerolactione originate from the action of the intestinal micro-organisms on the biliary-excreted metabolites of (+)-catechin. These phenolic acid and lactone metabolites are then reabsorped and excreted in the urine. 6. It is proposed that, depending on the route of administration of (+)-catechin, there exists an alternative pathway, involving biliary excretion, for the metabolism of (+)-catechin.     

16Beta-hydroxylation of dehydroepiandrosterone sulphate by homogenates of human foetal liver.

The chemical syntheses of the 3-sulphates of 16 alpha-acetoxy-, 16 alpha-hydroxy- and 16 beta-hydroxy-dehydroepiandrosterone as their triethylammonium salts are described preparatory to studies of the metabolism of [7 alpha-3 H]dehydroepiandrosterone sulphate by homogenates of human foetal liver. Five main radioactive products of the incubation were separated by partition chromatography on a Celite column. Two were identified as 16 alpha-and 16 beta-hydroxydehydroepiandrosterone 3-sulphates by crystallization to constant specific radioactivity before and after solvolysis. The yields of the conversion to the two epimers were 24.4% (16 alpha) and 1.8% (16 beta).     

Use of bioconversion for the preparation of [4-14C]-labeled 7 alpha- and 7 beta-hydroxylated derivatives of dehydroepiandrosterone and epiandrosterone

The 7 alpha- and 7 beta-hydroxylated derivatives of [4-14C]-dehydroepiandrosterone were prepared with use of the yeast-expressed human cytochrome p4507B1. Epiandrosterone (EPIA), 5 alpha-androstane-3beta,17 beta-diol, and 5 alpha-androstane-3,17-dione were obtained after incubation of [4-14C]-5 alpha-dihydrotestosterone with Escherichia coli-expressed (3beta,17 beta)-hydroxysteroid dehydrogenase from Pseudomonas testosteroni. The 7 alpha- and 7 beta-hydroxylated derivatives of [4-14C]-EPIA produced were prepared after incubation with mycelium of Rhizopus nigricans. Each labeled steroid was purified by chromatography and identified by crystallization to constant specific activity after isotopic dilution with each authentic steroid carrier. Production yields and radio-purity measurements allowed the use of such procedures for the preparation of the described radio-steroids for studies of metabolism and mode of action.     

Androgen metabolism by hepatic and renal tissues of the fetal rhesus monkey

Liver and kidney from fetal monkeys (day 125 of gestation) were fractionated into low speed pellets, microsomal and cytosolic fractions. Liver cytosols converted as much testosterone (T) to 5 beta-androstane-3 alpha,17 beta-diol (5 beta-diol) at 0 degrees C as at 4 degrees-45 degrees C without exogenous cofactors. The principal product formed from 5 alpha-dihydrotestosterone (5 alpha-DHT) was 5 alpha-diol. A 1000-fold molar excess of radioinert 5 beta- or 5 alpha-DHT inhibited 5 beta-diol formation from [3H]T by cytosols and increased 5 beta-DHT formation. Similarly, using 5 alpha-DHT as substrate, 5 alpha-diol formation was inhibited. Microsomal and low speed pellets with added cofactors formed products which recrystallized with either etiocholanolone or androsterone from [3H]T or [3H]DHT, respectively. Little product was formed without cofactor. Whole liver homogenates produced 5 beta-reduced products from [3H]T in the presence of an NADPH generating system whereas kidney homogenates produced 5 alpha-reduced products. These data provide new information on the capacity of fetal monkey liver and kidney to metabolize androgens. The 3 alpha-reductases are cytosolic. The 5 alpha- and 5 beta-reductases are mostly in the low speed pellet but are sufficiently represented in cytosols to mediate diol formation. The 17-hydroxysteroid dehydrogenases are in the microsomal fraction. Our results suggest that 5 alpha-DHT is the active androgen in fetal liver since testosterone is metabolized to 5 beta-DHT and 5 beta-diol which are inactive androgens.     

The biosynthesis of some androst-16-enes from C21 and C19 steroids in boar testicular and adrenal tissue

1. The formation of androst-16-enes from [4-(14)C]progesterone has been investigated with long-term incubations and short-term kinetic studies. After 4hr., 1.7 and 10.3% respectively of 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes were formed in boar testis minces, but much smaller yields were obtained in boar adrenal. Both tissues formed small quantities of androsta-4,16-dien-3-one. 2. The amounts of androst-4-ene-3,17-dione and testosterone isolated were small, suggesting that androst-16-ene formation may occur preferentially in the boar testis. 3. In the absence of tissue no radioactive androst-16-enes were formed. 4. Incubation of both [4-(14)C]pregnenolone and [7alpha-(3)H]progesterone resulted in 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes containing (3)H/(14)C ratios of near unity and confirmed that both C(21) steroids were precursors. A similar incubation with 17alpha-hydroxy[4-(14)C]-progesterone and [7alpha-(3)H]progesterone gave the same Delta(16)-alcohols, but they contained only (3)H, indicating that side-chain cleavage of pregnenolone and progesterone occurred before 17alpha-hydroxylation. 5. Dehydroepiandrosterone, testosterone, testosterone acetate and 16-dehydroprogesterone were not found to be precursors of Delta(16)-steroids. 6. A pathway is proposed for the biosynthesis of 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes from pregnenolone and progesterone; this may involve androsta-4,16-dien-3-one as an intermediate, but excludes 17alpha-hydroxyprogesterone, testosterone and dehydroepiandrosterone.     

Androgen metabolism in the rhesus monkey.

A mixture of 3H-testosteron (T) and 14C-4-androstene-3, 17-dione (A) was injected intravenously into 2 (I and II) rhesus monkeys (Macaca mulatta). A third monkey (III) was injected with 3H-T only. Urine and bile samples were collected at intervals for 6 hours following the injection. The excretion, conjugation and aglycone metabolites of the steroids injected were studied using these samples. Of the injected dose, animal I (male) excreted 32% 3H and 23% 14C in the bile and 30% 3H and 21% 14C in the urine in 6 hours. Animal II (female), however, had a comparatively higher biliary excretion (66% 3H, 40% 14 C), but a urinary excretion (18% 3H, 13% 14C) comparable to that of animals I and III. The averages in the bile of the 3 animals were: unconjugated compounds 3%, glucosiduronates 78%, sulfates 9%, sulfoglucosiduronates 5% and disulfates 3%; and in urine, 5% unconjugated, 92% glucosiduronates and 3% sulfates. The aglycones obtained following hydrolysis were separated gy chromatography on Lipidex 5000, further purified by thin layer and paper chromatography and identified by co-crystallization. The major matabolites from 3H-T were androsterone and 5beta-androstane-3alpha,17beta-diol, whereas that from 14C-A was androsterone. Other metabolites identified were: etiocholanolone (3beta-hydroxy-5-beta-androstan-17-one); T, epitestosterone (epi-T), (17alpha-hydroxy-4-androsten-3-one); epiandrosterone (3-beta-hydroxy-5alpha-androstan-17-one) and 5alpha-androstane-3alpha, 17beta-diol. The results indicate that while androgen metabolism in the rhesus monkey is similar to that of the baboon and human in conjugate and metabolite formation, the rate of excretion was significantly different, resembline more closely that of the baboon than the human.     

Dehydroepiandrosterone sulphate in rat brain: incorporation from blood and metabolism in vivo

The incorporation of [7-³H]dehydroepiandrosterone[³⁵S]sulphate into brain tissue elements from the circulatory system and its metabolic fate in the brain were studied in developing rats. Approximately 0.037 % of [³H] and 0.023% of [³⁵S] were incorporated into the brain within 15 min after the intracardiac injection of the labelled steroid. More than one-half of the incorporated [³H] was recovered as free steroid, whereas the rest was recovered as sulphate. The ³H/³⁵S ratio in the sulphate fraction suggested that the sulphate entered the brain with the sulphate linkage intact. Upon intracerebral injection of the double-labelled steroid, approximately 6 per cent of the radioactivity was recovered in the brain at 30 min after the injection and 1 per cent was recovered at 1 h after the injection. Of the remaining radioactivity recovered from the brain, 5 per cent was found in the free steroid fraction, probably formed by hydrolysis of the sulphate; 90 per cent was in the sulphate ester fraction; and the rest was in the fraction of more polar compounds. To identify the metabolites, [4-¹⁴C]dehydroepiandrosterone sulphate was injected into the rat brain. Significant amounts of radioactivity were found in androstenediol sulphate, which was isolated from the brain. This compound was apparently derived from dehydroepiandrosterone sulphate by reduction of the 17-keto group to a 17β-hydroxyl group without prior hydrolysis. There was suggestive evidence that free androstenediol was also formed in the brain in this experiment.     

Studies on nuclear binding of dehydroepiandrosterone in hepatocytes.

Experiments were conducted to determine if dehydroepiandrosterone (DHEA) specifically binds to liver nuclei or interferes in the binding of other steroid hormones. Hepatocytes were isolated from obese, female Zucker rats that were treated with and without 0.6% DHEA in their diets for 2 weeks. The hepatocytes were incubated with either [3H]DHEA, [3H]estrone, or [3H]corticosterone. The nuclei isolated from the incubated cells from either control or DHEA-treated rats had no binding with [3H]DHEA, but had the expected binding with [3H]estrone and [3H]corticosterone. Furthermore, increasing concentrations of cold, unlabeled DHEA in the incubation media with [3H]estrone or [3H]corticosterone failed to have any effect on the binding of either of these steroid hormones to the nuclei. These results suggest that DHEA treatment does not exert its effects on cellular metabolism through a receptor-mediated mechanism like other steroid hormones or by interfering in the expression of steroid hormones such as estrone and corticosterone.     

Steroid delta 4-5 beta-reductase in human mammary tumors.

Steroid delta 4-5 alpha- and delta 4-5 beta-reductase activity was determined in 16 human mammary tumors and 8 DMBA-induced rat mammary tumors using a spectrophotometric assay. Steroid delta 4-5 alpha-reductase was present in all tumors investigated while delta 4-5 beta-reductase was detected in only 6 estrogen receptor negative human breast tumors and absent in all estrogen receptor positive human breast tumors as well as in all rat mammary tumors. Further support for the presence of delta 4-5 beta-reductase was established by using a dual-labelling technique consisting of incubating tumor slices with [14C] testosterone and adding [3H] etiocholanolone, [3H] testosterone and [3H]-5 alpha-dihydrotestosterone at the end of the reaction. Following extraction and chromic acid oxidation, 4-androstenedione, 5 beta-androstanedione and 5 alpha-androstanedione were isolated and purified, and the constancy of the 14C/3H ratio was used as proof of 5 alpha-reductase and 5 beta-reductase. These results were shown to be consistent with the data obtained using the spectrophotometric assay.     

Synthesis of bile acid monosulphates by the isolated perfused rat kidney.

Perfusion of an isolated rat kidney with labelled bile acids, in a protein-free medium, resulted in the urinary excretion of the labelled bile acid, 3% being converted into polar metabolities in 1h. These metabolities were neither glycine nor taurine conjugates, nor bile acid glucuronides, and on solovolysis yielded the free bile acid. On t.l.c. the metabolite of [24-14C]lithocholic acid had the mobility of lithocholate 3-sulphate. The principal metabolite of [24-14C]chenodeoxycholic acid had the mobility of chenodeoxycholate 7-sulphate; trace amounts appeared as chenodeoxycholate 3-sulphate. [35S]sulphate was incorporated in chenodeoxycholic acid by the kidney, resulting in a similar pattern of sulphation. No disulphate salt of chenodeoxycholic acid was detected. These findings lend support to the hypothesis that renal synthesis may account for some of the bile acid sulphates present in urine in the cholestatic syndrome in man.