Epitestosterone Research Articles

Development of a Derivatization Method for Investigating Testosterone and Dehydroepiandrosterone Using Tandem Mass Spectrometry in Saliva Samples from Young Professional Soccer Players Pre- and Post-Training

In the last decade, high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) combined with electrospray ionization (ESI) has been widely used for determining low concentrations of steroids, and derivatization has often been employed to enhance detection. In the present study, endogenous steroids were extracted using a Strata-XL polymeric reverse phase cartridge. The isolated steroids were reacted with 2-hydrazino-1- methylpyridine (HMP) at 50 °C for 30 min. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used in a positive mode with multiple reaction monitoring (MRM) for the quantification of testosterone (T) and its precursor, dehydroepiandrosterone (DHEA), in saliva samples collected from twenty young Saudi professional soccer players. The analytes were separated on an ACE Ultracore 2.5 Superphenylhexyl column (150 × 3.0 mm id). The extraction recovery during the pre-treatment was >89% and gave <±20% for inter- and intra-assay precision and accuracy. The limits of quantification (LOQ) were found to be 20 pg/mL for (T and DHEA) and 50 pg/mL for Epitestosterone (EPI). The results showed no significant variation in the concentration of T between pre and post training, whereas DHEA was significantly increased after short-term exercise. These results could indicate that there is no correlation between T and its precursor DHEA level following short term physical activity. EPI concentrations could not be detected with a LOQ of 50 pg/mL in the saliva samples.

Simultaneous profiles of sulfonated androgens, sulfonated estrogens and sulfonated progestogens in postpubertal boars (sus scrofa domestica) measured by LC–MS/MS

Sulfonated steroids (s-St) have been usually regarded as inactive metabolites but are progressively considered as precursors for the intra-tissue formation of bioactive steroids. Moreover, independent effects without preceding removal of the sulfate group have been observed. We use the porcine testicular-epididymal compartment as a model to investigate the still largely unknown s-St physiology as the boar exhibits an intriguingly broad s-St spectrum predominantly originating from the testis. The application of LC–MS/MS in steroidomics enables the determination of unconjugated and intact sulfonated steroids with currently highest specificity and good sensitivity, allowing the concurrent measuring of numerous analytes in larger quantities of samples. Profiles (6h, 20min intervals) were generated for sulfonated 5-androstene-3ß,17ß-diol (Adiol-S), androsterone (A-S), dehydroepiandrosterone (DHEA-S), epiandrosterone (EA-S), epitestosterone (ET-S), estrone (E1-S), estradiol-17β (E2-S), pregnenolone (P5-S), 17αOH-pregnenolone (OHP5-S) and unconjugated testosterone (T) in four unstimulated and four hCG-stimulated boars. Moreover, concentrations were measured in individual samples collected from testicular afferent and efferent blood to differentiate between testicular vs. extratesticular origin. Highest concentrations were found for EA-S, followed by ET-S, Adiol-S and DHEA-S, which mostly exceeded the levels of E1-S and A-S. Lowest concentrations were obtained for E2-S, P5-S and OHP5-S. The analytical profile also included sulfonated T, 5α-dihydrotestosterone and cholesterol. However, their concentrations were below the limit of quantification. Profiles of quantifiable s-St were consistent with a wave-like pattern associated with T pulses. In postpartal females (n=5) concentrations of all analytes assessed were undetectable, suggesting that in pigs the adrenals are not a quantitatively significant source of s-St.

Determination of Free and Deconjugated Testosterone and Epitestosterone in Urine Using SPME and LC–MS/MS

A thin sheet of polydimethylsilosane membrane was used as an extraction phase for solid-phase microextraction. Compared with fiber or rod solid-phase microextraction geometries, the thin film exhibited much higher extraction capacity without sacrificing extraction time due to its higher area-to-volume ratio. The analytical method involved direct extraction of unconjugated testosterone (T) and epitestosterone (ET) followed by separation on a C18 column and detection by selected reaction monitoring in positive ionization mode. The limit of detection was 1 ng/l for both T and ET. After method validation, free (unconjugated) T and ET were extracted and quantified in real samples. Since T and ET are extensively metabolized, the proposed method was also applied to extract the steroids after enzymatic deconjugation of urinary-excreted steroid glucuronides. The proposed method allows quantification of both conjugated and unconjugated steroids, and revealed that there was a change in the ratio of T to ET after enzymatic deconjugation, indicating different rates of metabolism.

Determination of epitestosterone in human urine by off-line immunoaffinity solid-phase extraction coupled with high performance liquid chromatography

Epitestosterone (ET) has been used as a masking agent and prohibited by the World Anti-Doping Agency (WADA) because its administration will decrease the urinary T/ET ratio, a marker of testosterone (T) administration. In this study, an off-line immunoaffinity extraction coupled with high performance liquid chromatography (HPLC) was developed to quantify the endogenous steroid ET in human urine. The immunoaffinity column (IAC) was prepared by immobilizing the anti-ET monoclonal antibodies on CNBr-activated Sepharose 4B, which can remove the contaminations and non-target compounds from matrix to enrich the target analyte ET. The mobile phase was ammonium acetate (10 mM, pH 4.0)/acetonitrile (45/55, v/v) at an isocratic flow of 1.0 mL/min and the UV absorbance detection wavelength was 244 nm for the detection of ET. The IAC showed good reliability and durability since it had been used for more than 100 runs in a year. The limit of quantification (LOQ) was 1 ng/mL. Satisfied repeatability and precision of the day-to-day and within-day were obtained with the RSD values less than 10%. Results of the recovery of the urine samples were ranged from 98% to 102% with repeatability less than 9%, indicating that the method developed can be used for the real urine sample analysis.

Development of a Validated HPLC Method for the Simultaneous Determination of Anabolic Steroids in Biological Fluids

A reversed phase high performance liquid chromatographic (HPLC) method is developed and validated for the simultaneous determination of anabolic steroids: testosterone (TES), epitestosterone (EPI), and nandrolone (NAN). The analytical column, Inertsil C8, 5 µm, 250 × 4 mm, was operated at ambient temperature. Isocratic elution was performed using a mixture of 50% buffer solution CH3COOH 0.11% –CH3COONa 7.5 mmol/L, pH = 4, 45% CH3CN and 5% CH3OH, at a flow rate of 1.1 mL/min. UV detection was performed at 238 nm. The detection limits of the method were 2.4 ng for NAN, 3.6 ng for TES, and 2.6 ng for EPI in blood plasma, and 2.7 ng for NAN, 1.1 ng for TES, and 3.8 ng for EPI in urine, per 20 µL injection volume. Alprazolam was used as internal standard at a concentration of 2 ng/µL. Validation of the method was performed in terms of accuracy and precision: intra-day assay (n = 6) and inter-day assay (n = 3 × 6) and was found to be satisfactory, with high accuracy and precision results. Sample preparation involved solid phase extraction on Nexus cartridges with high recoveries. The developed method was successfully applied to the analysis of urine samples of one female and 9 male volunteers.

Quantification of testosterone and epitestosterone in human urine samples by stir bar sorptive extraction – thermal desorption – gas chromatography/mass spectrometry: Application to HIV‐positive urine samples

A simple method is described for the measurement of testosterone (T) and epitestosterone (ET) in human urine samples. The deconjugated steroids were extracted directly from the samples by stir bar sorptive extraction (SBSE) and derivatized in situ on the stir bar by headspace acylation prior to thermal desorption and GC/MS. Extraction and derivatization parameters, namely salt addition, temperature, and time, were optimized to improve the recovery of T and ET by SBSE. The limits of quantification (S/N 10) were 0.9 ng/mL for T and 2.8 ng/mL for ET. Quantification of the steroids in urine samples was performed using standard addition to avoid the influence of matrix effects. The method was applied for the measurement of urinary T and ET in a group of healthy volunteers and HIV+ patients. Decreased levels of T were detected in the HIV+ group, whereas the excretion of ET was comparable for the two groups. Further clinical research is required to elucidate the biomarker significance of the T/ET ratio in HIV infection.

Neoformation of boldenone and related steroids in faeces of veal calves

Conflicting findings regarding the boldenone content of bovine faeces suggest it may be synthesized de novo in emitted faeces. We tested this hypothesis by analysing uncontaminated urine, fresh and various forms of dried faeces from 10 calves (not given boldenone) by liquid chromatography/tandem mass spectrometry for 17α- and 17β-boldenone (α and β BOL); 1,4-androstadiene-3,17-dione (ADD); 4-androstene-3,17-dione (AED), testosterone (T) and epitestosterone (ET). Urine contained no α BOL, β BOL or ADD. The analysed substances were variably present in the rectal faeces, and at generally higher levels in faeces scraped from skin or stall floor. In pooled rectal faeces naturally dried for 13 days, α BOL, ADD, AED and ET levels were extremely high (much higher than accounted for by increases due to drying), and β BOL and T were absent. It is concluded that de novo synthesis of α BOL and metabolites occurs naturally in bovine faeces and only uncontaminated urine should be analysed for illegal boldenone.

Simultaneous Determination 9 Anabolic Steroids Residues in Animal Muscle Tissues by Gas Chromatography‐Mass Spectrometry

A method was developed for determining 9 anabolic steroids (ASs) residues in animal muscle tissues by gas chromatography‐mass spectrometry (GC/MS). After undergoing enzymolysis, being homogenized, and then being picked‐up by ultrasound the sample was extracted with tert‐Butyl methyl ether, cleaned up through solid‐phase extraction (SPE) based on the reverse phase (RP) principle, and after derivatization of the analyst of interest, analyzed by GC/MS under selective ion monitoring (SIM). The limits of the detection (LOD) of the GC/MS method used for testing epitestosterone (ETS), nandrolon (17β‐NT), 17α‐methyl‐testosterone (MTS), testosterone 17‐propionate (PTS), 17β‐estradiol 3‐benzoate (BES), estrone (ESN), 17β‐estradiol (17β‐ES), 17α‐ethynylestradiol (EES), and estriol (EST) in animal muscle ranged from 0.10 to 0.33 µg/kg and the limits of quantification (LOQ) were from 0.24 to 0.82 µg/kg. The experiments using spiked samples, such as pork, beef, chicken, and fish, made it clear that at addition level of 2.0 µg/kg, the average recovery of the ASs ranged from 62.5% to 80.5%, and the coefficient of variation ranged from 12.5% to 26.8%, while at an addition level of 5.0 µg/kg, the average recovery was from 70.8% to 86.4%, and the coefficient of variation was from 8.8% to 18.4%.

Development of a faster determination of 10 anabolic steroids residues in animal muscle tissues by liquid chromatography tandem mass spectrometry

A method had been developed for determination of residues of 10 anabolic steroids (ASs) in animal muscle tissues by liquid chromatography tandem mass spectrometry (LC/MS/MS). After enzymolysis, the sample was extracted with tert-butyl methyl ether, cleaned up through reverse solid-phase extraction and further determined by LC/MS/MS under multiple reaction monitoring (MRM) mode. The limits of detection (LOD) of LC/MS/MS method used for testing epitestosterone (ETS), nandrolone (17 β-NT), 17 α-methyl-testosterone (MTS), testosterone 17-propionate (PTS), medroxyprogesterone (MED), progesterone (PG), estrone (ESN), 17 β-estradiol (17 β-ES), 17α-ethynylestradiol (EES) and estriol (EST) in animal muscle ranged from 0.06 to 0.22 μg/kg, and the limits of quantification (LOQ) were from 0.12 to 0.54 μg/kg. Experiments on spiked samples of pork, beef, chicken and fish showed that at addition level of 1.0 μg/kg, the average recoveries of the ASs ranged from 64% to 77%, and coefficients of variation from 7.1% to 20.3%, while at addition level of 2.0 μg/kg, the average recoveries ranged from 70% to 89%, and coefficient of variation from 7.1% to 19.1%.

Extraction of testosterone and epitestosterone in human urine using aqueous two-phase systems of ionic liquid and salt

Based on aqueous two-phase systems (ATPS) consisting of 1-butyl-3-methylimidazolium chloride, a hydrophilic ionic liquid (IL), and K 2HPO 4, a new and simple extraction technique, coupled with a reversed-phase high performance liquid chromatography (RP-HPLC), was developed for the simultaneous concentration and analysis of testosterone (T) and epitestosterone (ET) in human urine. Under the optimal conditions, the extraction efficiencies for both analytes were 80–90% in a one-step extraction. The method required only 3.0 mL of urine and a single hydrolysis/deproteinization/extraction step followed by direct injection of the IL-rich upper phase into HPLC system for analysis. The method has been satisfactorily applied to the analysis of T and ET in human urine with detection limits of 1 ng/mL and linear ranges of 10–500 ng/mL for both compounds. Compared with conventional liquid–liquid extraction or solid phase extraction, this new method is much “greener” due to no use of volatile organic solvent and low consumption of IL. The proposed extraction technique opens up new possibilities in the separation of other drugs.

ENDOCRINE EVALUATION OF ATHLETE FOR TESTOSTERONE OR ITS ANALOGUES ABUSE

Background. Doping involves the use of substances and methods prohibited by international and national sports institutions. The use of certain substances and methods is harmful to the human organism, so all sports organisations are trying to protect athletes from the harmful side effects of such substances and methods. Methods. The article deals with special protocol, which is used in the case, when the testosterone/epitestosterone ratio in urine is higher than 6 and less then 10. When establishing the use of testosterone or analogues, ketoconazol is used. If after the application the presence of a testosterone (T) to epitestosterone (E) ratio is greater than six (6) to one (1), there is an evidence of doping offence. Conclusions. In the case of T/E greater than 6, it is mandatory that the relevant medical authority conducts an investigation before the sample is declared positive. A full report should be written and should include a review of previous tests, subsequent tests and any results of endocrine investigations.

Development and Method Validation for Testosterone and Epitestosterone in Human Urine Samples by Liquid Chromatography Applications

An isocratic high-performance liquid chromatographic method for the determination of testosterone (T) and epitestosterone (ET) in human urine using liquid-liquid or solid-phase extraction (SPE) is developed and validated. The optimum separation is achieved using a Hypersil C(18) column, water-acetonitrile (57:43, v/v) as the mobile phase and UV-absorbance detection at 245 nm. The recoveries obtained for T and ET in liquid-liquid and SPE demonstrate that these procedures are interchangeable. Quantitation limits for T and ET are 8.6 and 5.4 ng/mL using solvent extraction and 7.3 and 5.7 ng/mL using SPE, respectively. The proposed method is used to evaluate the urinary T, ET, and the T/ET ratio for a healthy male population using liquid-liquid extraction, and the T and ET excretion profile for nine healthy men using SPE.

The effect of epitestosterone on gonadotrophin synthesis and secretion.

The effects of 3-week treatment with increasing doses of epitestosterone (ET) on gonadotrophin gene expression and secretion, on testosterone and 5 alpha-dihydrotestosterone (DHT) levels, and on the weight of testes and prostates, were studied in intact adult male rats. The hormones were delivered by means of silastic capsules of different lengths filled with the steroid. One group of rats received testosterone (T) instead of ET, to compare the results with previous studies concerning the testosterone effect. The controls were given capsules with glucose only. Treatment with ET, as well as with T, significantly reduced the weights of prostates. When the data from ET-treated rats and controls were combined, a significant negative correlation (P < 0.001) was found between the weight of prostates and serum ET. T, in contrast to ET, also decreased significantly the weights of testes, ET treatment caused a significant reduction of serum T levels but only an insignificant decline of DHT levels, independent of the dose. Serum and pituitary (p) luteinizing hormone (LH) levels in the ET-treated rats did not change. Pituitary mRNA contents for the beta LH subunit (beta LH-mRNA) showed a dose-dependent significant increase, up to 170% (P < 0.01), with ET treatment. pFSH decreased with the lowest ET (2 cm) dose (P < 0.05), but no change was observed with the other doses. The mRNA for the common alpha-subunit also increased with the ET load. In conclusion, ET acts at several sites in the regulation of gonadotrophin formation and release. It enhances the steady-state mRNA levels of both gonadotrophins in the pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of simultaneous self-administration of testosterone and epitestosterone in healthy men

Combined self-administration of testosterone (T) and epitestosterone (ET) by athletes counteracts the efficiency of the corresponding urinary glucuronides (G) ratio, (T/ET)G, as an indicator of T abuse. I therefore propose 5-androstene-3 beta, 17 alpha-diol (5A3 beta 17 alpha), the immediate metabolic precursor of ET, as a new reference compound for the expression of relative excretions of T and ET. Thus (T/5A3 beta 17 alpha)G and (ET/5A3 beta 17 alpha)G become potential criteria to indicate joint administration of T and ET, since their respective threshold values (2.5 and 1.5) are exceeded even when (T/ET)G remains below the critical value of 6.

Secretion by the human testis of epitestosterone, with its sulfoconjugate and precursor androgen 5-androstene-3β,17α-diol

Epitestosterone (ET) and testosterone (T), free and sulfoconjugated, as well as 5-androstene-3β,17α-diol (5AD3β17α) and its 17β-epimer have been analyzed, by gas chromatography-mass spectrometry with stable isotope dillution, in peripheral and spermatic venous plasma of patients with left varicocele. All these androgens are secreted by the testis as evidenced by the significant concentration gradients between peripheral and spermatic venous plasma. Half of the daily ET production is ascribed to the testis, while 95% of T sulfate and roughly 70% of ET sulfate are also of testicular origin. Significant correlations between ET and 5AD3β17α are an indication that the 5-ene pathway is also operative for ET biosynthesis. High ratios of spermatic to peripheral venous plasma levels of ET and 5AD3β17α are also related to the high clearance rates of 17α-hydroxy-androgens.

Long-term administration of testosterone enanthate to normal men: Alterations of the urinary profile of androgen metabolites potentially useful for detection of testosterone misuse in sport

Doses equivalent to 18, 72 and 216 mg testosterone (T)/week were administered for 6 months to eugonadal men. Urinary excretions of androgen glucuronides (G) were quantitatively analyzed by gas chromatography-mass spectrometry with stable isotope dilution during periods of control (without hormone treatment), T administration and recovery. The lowest dosage T administration did not affect the androgen profile, while higher dosages generally increased urinary excretions of T metabolites (TG, T sulfate, glucuronides of androsterone, etiocholanolone, 5α- and 5β-androstane-3α,17β-diol) and decreased excretions of conjugates of epitestosterone (ET) and its precursor androgen 5-androstene-3β,17α-diol. A dose-dependent decrease of urinary LH in response to T was also observed. The ratio (T/ET)G, which is the sole official criterium for assessment of T self-administration by athletes, increased above the threshold value of 6 in most of the subjects, but not all, after the two highest dosage T regimens, and returned to normal during the recovery period. False positive or negative testing emphasizes the need for improvement of testing procedures. In this regard, valuable complementary information may be gained from ratios such as TG/ET(Total), TG/LH, (T/5-androstene-3β,17α-diol)G, ( 5α 5β ) androstane-3α,17α- diol and ( 5α 5β ) androstane-3α,17β- diol .

Dehydroepiandrosterone and Epitestosterone in the Blood of Cows at Term

From Day 268 of gestation till two days after parturition blood samples were collected from the jugular vein of 5 cows with normal and 4 cows with flumethasone induced parturition and centrifuged immediately after sampling. In another experiment blood samples were taken from the jugular and uterine vein of 3 cows during the last week before normal parturition. The dehydroepiandrosterone (DHA) and epitestosterone (ET) concentrations in plasma were measured using radioimmunoassay. In the control group the DHA concentration was in the range of 2 to 5 nmol/l, ET levels varied from 4 to 8 nmol/l before term. In both groups, there was an increase in DHA concentrations during the last periparturient period whereas ET levels increased only after flumethasone induced parturition. The concentrations of both androgens declined after the expulsion of the placenta and they were higher in the uterine vein than in the jugular vein. It is concluded, that both androgens are secreted by the bovine placenta and that the delta 5-pathway of steroidogenesis is active in vivo.

Cytochemical and Cytophysiological Studies of Gonadotropin-Releasing Hormone (GnRH) Target Cells in the Male Rat Pituitary: Differential Effects of Androgens and Corticosterone on GnRH Binding and Gonadotropin Release*

Steroid hormones can differentially modify gonadotropin release stimulated by GnRH. Decreased GnRH-mediated gonadotropin release has been observed in vitro after pretreatment with androgens or glucocorticoids. In this study, we tested this phenomenon further with the use of a new cytochemical stain for a potent biotinylated analog of GnRH ([Biotinyl D-Lys6]GnRH) combined with stains for LH and FSH and gonadotropin RIAs. The first phase of the study involved characterization of the GnRH target cells in monolayer cultures from male rats. Dose-response curves (measured as the ability to release both LH and FSH) showed that biotinylated GnRH (Bio-GnRH) was equipotent with or more potent than unlabeled [D-Lys6]GnRH in parallel cultures. The avidin-biotin complex stain demonstrated that 16% of the 2- to 3-day pituitary monolayer cells were labeled for Bio-GnRH within 10 min of exposure. Double stains for gonadotropins showed that 37% of the LH gonadotropes and 42% of the FSH gonadotropes did not stain for Bio-GnRH. During the second phase of these studies, the cultures were pretreated for 48 h with 1-100 nM 5 alpha-dihydrotestosterone (DHT), 100 nM corticosterone (CT), or 100 nM epitestosterone (ET) to test the effects of these steroids on the number of cells to which Bio-GnRH bound and the gonadotrope secretory response. Compared with ET- or vehicle-pretreated control cultures, DHT and CT both reduced the amount of LH and FSH release stimulated by GnRH. The magnitude of the reduction in LH release was much greater than that in FSH release, especially after pretreatment with CT. DHT and CT had remarkably different effects on the percentages of cells stained for GnRH. Pretreatment with DHT caused a reduction in the percentages of cells staining for bound Bio-GnRH to 9% compared with 14.3% after CT treatment and 16% after vehicle or ET treatment. The counts of the stained gonadotropes suggested that most of the reduction occurred in the LH gonadotrope population. Because both DHT and CT reduced GnRH-mediated gonadotropin release, but only DHT reduced the percentage of cells that bound Bio-GnRH, it is suggested that the two steroids affect gonadotropin release by separate mechanisms.

Antigenic complexes of steroid hormones formed by coupling to protein through position 7: Preparation from Δ 4 -3-oxosteroids and characterization of antibodies to testosterone and androstenedione

A general method for rendering Δ 3-3-oxosteroids antigenic by coupling to a macromolecule through position 7 is described. It involves nucleophilic attack on the 6, 7-dehydroderivatives of the steroids by ambidentate reagents to form 7α-thioether alkanoic acids. These were covalently attached to bovine serum albumin (BSA) by use of the carbodiimide reagent. Addition products with mercapoacetic acid and β-mercaptopropionic acid and their BSA-conjugates, were thus obtained from testosterone androst-4-ene-3, 17-dione, progesterone and 17α-hydroxyprogesterone through the respective 4, 6-dienes. Immunization of rabbits with testosterone-7α-carboxymethyl-thioether-BSA and the homologous testosterone-7α-carboxyethyl-thioether-BSA gave rise to antisera of high affinity for testosterone (Ka=9. 4×10 9 1/mol) that showed little cross-reaction with androstenedione (< 1%) and with a variety of 17-oxoandrostane compounds (⩽ 0.5%). Conversely, immunization with androstenedione-7α -carboxyethyl-thioether-BSA yielded an antiserum with high affinity for androstenedione (Ka = 1. 04 × 10 10 I/mol) but minimal cross-reaction with testosterone (< 0.5%) and 17β-hydroxy-androstane compounds (⩽ 1%). The reaction of anti-testosterone and anti-androstenedione sera with their homologous haptens was not significantly inhibited by the closely related steroids 17α- epit estosterone and dehydroepiandrosterone, or by 11-deoxycorticosterone, progesterone, 17α-hydroxyprogesterone, estrone and estradiol-17β. However, anti-testosterone sera cross-reacted with 5α-dihydrotestosterone (40–50%) and to a lesser extent with 5β-dihydrotestosterone (5%). Analogously, the anti-androstenedione sera cross-reacted with 5α-dihydroandrostenedione (71%) and to a minor extent with 5β-dihydroandrostenedione (8%). A radioimmunoassay procedure for the determination of testosterone in plasma is described, which makes use of the new anti-testosterone serum. Preliminary results suggest that it can be applied to ether extracts from human sera without Chromatographic purification.

The urinary excretion of testosterone and epitestosterone in females following intravenous infusions of testosterone before and during treatment with an antiandrogen (cyproterone).

ABSTRACT Intravenous infusions of 10 mg testosterone (T) in two females (one of them adrenalectomized for Cushing's syndrome) resulted in an increased urinary excretion of T-glucuronide, whereas the excretion of epitestosterone (ET)-glucuronide was not altered significantly. The excretion of T-sulphate increased only slightly. During treatment with 100 mg Cyproterone p. d. a marked elevation of urinary T and ET excretion occurred. A slight increase in unconjugated T and ET was also observed. A second T infusion during Cyproterone treatment did not lead to a further increase of urinary T-glucuronide, but there was a slight increase in urinary excretion of ET-glucuronide and of T-sulphate.