Testosterone (T) has traditionally been the most commonly reported doping agent by doping control laboratories. The screening of T misuse is performed by the quantification of six endogenous androgenic steroids and the ratio T/E included in the Athlete Biological Passport (ABP). The inclusion of additional metabolites can improve the screening capabilities of ABP. In this study, the potential of 3α-glucuronide-6β-hydroxyandrosterone (6OH-Andros3G) and 3α-glucuronide-6β-hydroxyetiocholanolone (6OH-Etio3G) as markers of T oral administration was evaluated. These glucuronides have been shown to be resistant to enzymatic hydrolysis and their quantification by means of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was reported as the only way to obtain feasible results. Urine samples were collected from five volunteers before and after the oral administration of 40mg of T undecanoate and were analyzed by a LC-MS/MS method recently developed. Concentration of 6OH-Andros3G and 6OH-Etio3G compounds and those of the glucuronides of T (TG), epitestosterone (EG), androsterone and etiocholanolone were established and different concentration ratios were calculated. The detection windows (DWs) for the T administration obtained by each selected ratio were compared to the one of TG/EG. The results showed that four out of the nine tested markers presented DWs much larger for all volunteers than those obtained by the World Anti-Doping Agency established T/E marker or other alternative markers. The 6OH-Andros3G/EG, 6OH-Etio3G/EG, 6OH-Andros3G/TG and 6OH-Etio3G/TG markers were able to identify the T abuse up to 96h after the administration, extending our detection capability for the misuse up to 84h more than the classic marker. The importance of these markers was also highlighted by their prolonged capacity to detect the T misuse in the case of one volunteer whose TG/EG barely exceeded his individual threshold. As a consequence, the four markers presented in this study seem to have an exceptional potential as biomarkers of T oral administration.

In Europe, chemical castration has been adopted as a treatment for paraphilia since the 1930s. Among the various chemical castration agents, luteinizing hormone-releasing hormone (LHRH) agonists are now used widely because of their effectiveness and safety. In South Korea, a legislation of chemical castration to control the sexual impulses of sexual offenders was enforced in July 2011. Most of these subjects are treated with leuprorelin acetate, an LHRH agonist, for chemical castration. Despite this, there are few studies that address the long-term influence of LHRH agonists on testosterone (T) and epitestosterone (E) levels in chemical castration subjects. In order to analyze the urinary levels of T in chemical castration subjects, whose T levels are extremely low, we developed and validated an analytical method for the detection of both T and E in human urine using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. The urine samples were hydrolyzed, extracted, and analyzed by LC-MS/MS with electrospray ionization in the positive-ion mode. The limits of detection were 0.02 ng/mL and the limits of quantitation were 0.05 ng/mL, which provided great sensitivity. The established method was applied to urine samples from chemical castration subjects and healthy male volunteers. The chemical castration subjects showed significantly lower urinary T levels than the control subjects. In addition, the urinary E levels were also lower in the chemical castration subjects; however, the T/E ratios were constant and did not show a notable decrease because of the simultaneous decrease in both urinary T and E. The urinary T levels and T/E ratio did not exceed the doping control criteria for exogenous T ingestion for any subject. This study shows the trend of urinary T and E levels in long-term treated chemical castration subjects by establishing a highly sensitive LC-MS/MS method, that provides useful information for monitoring chemical castration.

The steroid glucuronide conjugates of 16,16,17-d(3)-testosterone, epitestosterone, nandrolone (19-nortestosterone), 16,16,17-d(3)-nortestosterone, methyltestosterone, metenolone, mesterolone, 5alpha-androstane-3alpha,17beta-diol, 2,2,3,4,4-d(5)-5alpha-androstane-3alpha,17beta-diol, 19-nor-5alpha-androstane-3alpha,17beta-diol, 2,2,4,4-d(4)-19-nor-5alpha-androstane-3alpha,17beta-diol and 1alpha-methyl-5alpha-androstane-3alpha/beta,17beta-diol were synthesized by means of the Koenigs-Knorr reaction. Selective 3- or 17-O-conjugation of bis-hydroxylated steroids was performed either by glucuronidation of the corresponding steroid ketole and subsequent reduction of the keto group or via a four-step synthesis starting from a mono-hydroxylated steroid including (a) protection of the hydroxy group, (b) reduction of the keto group, (c) conjugation reaction and (d) removal of protecting groups. The mass spectra and fragmentation patterns of all glucuronide conjugates were compared with those of the commercially available testosterone glucuronide and their characterization was performed by gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy. For mass spectrometry the substances were derivatized to methyl esters followed by trimethylsilylation of hydroxy groups and to pertrimethylsilylated products using labelled and unlabelled trimethylsilylating agents. The resulting electron ionization mass spectra obtained by GC/MS quadrupole and ion trap instruments, full scan and selected reaction monitoring experiments are discussed, common and individual fragment ions are described and their origins are proposed.

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.

In this manuscript we have characterized the androgen receptor from human benign prostatic hypertrophied (BPH) tissue. BPH tissue was obtained fresh from surgery, ground, and frozen in liquid nitrogen. Tissue was homogenized in 10 mM Tes buffer (pH 7.4 at 25 degrees C) containing 0.25 M sucrose, 20 mM Na2MoO4, 12 mM monothioglycerol, and 1.5 mM EDTA. Cytosol was labeled with (3H)-R1881 (0.5-10 nM) +/- a 100-fold excess of R1881 and a 500-fold excess of triamcinolone acetonide to identify specific androgen receptor sites. An exchange assay was developed, whereby maximum binding was achieved by raising the temperature to 30 degrees C for 15 min. A high-affinity binding protein was detected (Kd = 1.3 +/- 0.8 nM) which had a binding capacity of 15 +/- 7 fmol/mg protein. Binding was specific for androgens with the following Ki (nM) values: R1881 (0.09), dihydrotestosterone (6), testosterone (50), progesterone (92), estradiol (100), epitestosterone (860), and cortisol (greater than 1,000). Under high ionic conditions the sedimentation coefficient of the receptor was 4.1 S. Higher S values were not observed in the presence of the following protein inhibitors: leupeptin (20 or 150 microM), iodoacetamide (10 mM), DFP (5 mM), PMSF (1 mM), bacitracin (0.1 mM), antipain (0.1 mM), aprotinin (1 TIU/ml). Gel filtration analysis revealed a Stokes radius of 6.5 nm. These data indicate a Mr of 120,000 and a f/fo of 2.01. The receptor eluted from a chromatofocusing column at a pH of 6.8. The receptor bound to phosphocellulose and DNA cellulose after being heat-treated for 15 min at 30 degrees C. These results suggest that BPH tissue contains an androgen receptor which is similar to receptors in other androgen-responsive tissues.

Neuroendocrine tumors (NETs) are uncommon tumors which can secrete specific hormone products such as peptides, biogenic amines and hormones. So far, the diagnosis of NETs has been difficult because most NET markers are not specific for a given tumor and none of the NET markers can be used to fulfil the criteria of high specificity and high sensitivity for the screening procedure. However, by combining the measurements of different NET markers, they become highly sensitive and specific diagnostic tests. The aim of the work was to identify whether urinary steroid hormones can be identified as potential new biomarkers of NETs, which could be used as prognostic and clinical course monitoring factors. Thus, a rapid and sensitive reversed-phase high-performance liquid chromatographic method (RP-HPLC) with UV detection has been developed for the determination of cortisol, cortisone, corticosterone, testosterone, epitestosterone and progesterone in human urine. The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The limits of detection and quantification were 0.5 and 1 ng mL-1 for each steroid hormone, respectively. Linearity was confirmed within a range of 1-300 ng mL-1 with a correlation coefficient greater than 0.9995 for all analytes. The described method was successfully applied for the quantification of six endogenous steroid levels in human urine. Studies were performed on 20 healthy volunteers and 19 patients with NETs. Next, for better understanding of tumor biology in NETs and for checking whether steroid hormones can be used as potential biomarkers of NETs, a chemometric analysis of urinary steroid hormone levels in both data sets was performed.

Human placental microsomes converted epitestosterone to estradiol-17 alpha at rates of 23-48 pmol/min X mg protein with a Km of 113 microM. Activity was inhibited 70-90% by concentrations of CO, metyrapone, n-octylamine, 7,8-benzoflavone and 7-ethoxycoumarin which had no effect on the aromatization of 4-androstene-3, 17-dione. Conversely, cyanide and azide were more effective inhibitors of the conversion of the latter androgen. A variety of neutral steroids inhibited the aromatization of epitestosterone with 19-norsteroids being particularly effective, but competitive effects could not be demonstrated. Both 17 beta-hydroxy-4-estren-3-one and 16 alpha-hydroxy-4-androstene-3,17-dione caused a mixed inhibition. A number of phenolic steroids were also inhibitory with 16-oxo compounds being particularly effective. Inhibition by estrone was non-competitive (Ki = 16 microM). The aromatization of epitestosterone resembles placental microsomal oxidase activities against estrone and benzo [a]pyrene in its inhibitor specificity and epitestosterone may be the native substrate for an oxidase also active in the metabolism of aromatic xenobiotic chemicals.

Herein we demonstrate the first application of ozone-induced cleavage of endocyclic C═C double bonds for improved steroid isomer separation using ion mobility-mass spectrometry. Steroids represent a challenging biomolecular class for ion mobility (IM) separations due to their structural rigidity and subtle stereochemical differences. In this work, we compare the effects of ozonolysis on the relative mobilities of a model stereoisomer pair, testosterone and epitestosterone. A solution-phase ozonolysis approach is used due to its simplicity, relatively low cost, and potential for rapid, online analysis. Despite the presence of solvent-based addition products, we observe that these steroids undergo an ozone-based cleavage resulting in unique, stable gas-phase conformations. The resulting resolution between testosterone and epitestosterone, with collision cross section values of 176.6 and 193.3 Ų, respectively, demonstrates a significant improvement in comparison with previous IM-based approaches. The significantly smaller conformation observed for epitestosterone is stabilized by a three-point interaction between the oxygen-containing functional groups and a sodium ion; this same conformation cannot be sterically achieved by testosterone. Identification of this specific structural difference is strengthened by experimental results showing the disappearance of this conformation following in-source water loss, which eliminates the potential for that three-point interaction. Computational modeling of the lowest energy gas-phase structures for these ozone products corroborates the experimental results. In conclusion, this approach provides tremendous potential as a rapid IM separation method for steroid isomers and other endocyclic C═C double bond containing molecules.

Androgen production during the annual reproductive cycle was investigated in the male scincid lizard Tiliqua (Trachydosaurus) rugosa. Concentrations of testosterone and epitestosterone were measured in plasma and testis (incubated and nonincubated) using radioimmunoassay. Morphological and histological techniques were used to determine the anatomical changes in the testis. Mating behavior was observed during spring, and sperm were most numerous in the testis at this time. Histological evidence and changes in testicular weight indicated that spermatogenesis begins in autumn and culminates in spring. Testicular regression occurred soon after mating. Plasma concentrations of both androgens were maximal during spring and minimal during summer. The maximal concentrations were approximately 500 and 150 nmol/liter (epitestosterone and testosterone, respectively). The minimal plasma concentrations were 250 and 15 nmol/liter, respectively. Plasma and testicular androgen cycles followed a pattern similar to that of the spermatogenetic cycle, suggesting the possible involvement of one or both of these androgens in the control of spermatogenesis and mating behavior in these lizards.

Proper storage conditions of biological samples are fundamental to avoid microbiological contamination that can cause chemical modifications of the target analytes. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method through direct injection of diluted samples, without prior extraction, was used to evaluate the stability of phase II metabolites of boldenone and testosterone (glucuronides and sulphates) in intentionally poorly stored equine urine samples. We also considered the stability of some deuterated conjugated steroids generally used as internal standards, such as deuterated testosterone and epitestosterone glucuronides, and deuterated boldenone and testosterone sulphates. The urines were kept for 1 day at room temperature, to mimic poor storage conditions, then spiked with the above steroids and kept at different temperatures (-18 degrees C, 4 degrees C, room temperature). It has been possible to confirm the instability of glucuronide compounds when added to poorly stored equine urine samples. In particular, both 17beta- and 17alpha-glucuronide steroids were exposed to hydrolysis leading to non-conjugated steroids. Only 17beta-hydroxy steroids were exposed to oxidation to their keto derivatives whereas the 17alpha-hydroxy steroids were highly stable. The sulphate compounds were completely stable. The deuterated compounds underwent the same behaviour as the unlabelled compounds. The transformations were observed in urine samples kept at room temperature and at a temperature of 4 degrees C (at a slower rate). No modifications were observed in frozen urine samples. In the light of the latter results, the immediate freezing at -18 degrees C of the collected samples and their instant analysis after thawing is the proposed procedure for preventing the transformations that occur in urine, usually due to microbiological contamination.

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)

OBJECTIVE

The concentrations of epitestosterone in human serum correlates negatively with that of estradiol. The possible explanation of this relation was addressed, and the influence of epitestosterone on kinetics of estradiol formation in vitro was evaluated.

METHODS

The concentration of epitestosterone was measured in serum of 54 men participating in a screening program for prostate disease. Epitestosterone inhibition of aromatase and 17beta-hydroxysteroid dehydrogenase activities was tested in vitro in the system consisting of human placental microsomes, NADPH or NAD and NADP respectively, and epitestosterone in increasing concentrations. Testosterone, androstenedione, estrone and 17beta-estradiol were utilized as substrates.

RESULTS

A significant negative correlation between epitestosterone and estradiol levels in human male serum was found. No inhibition of aromatase activity was observed; however, inhibition of 17beta-hydroxysteroid dehydrogenase was found preferentially in the direction leading to oxidation of the C-17 hydroxy group. The inhibitory effect of epitestosterone was more pronounced with androgens as substrates.

CONCLUSIONS

Epitestosterone could influence the formation of estradiol in vitro rather by inhibition of 17beta-hydroxysteroid dehydrogenase than by blocking aromatase activity.

The aim of this paper is to present the development and validation of a high-resolution full scan (HR-FS) electrospray ionization (ESI) liquid chromatography coupled to quadrupole Orbitrap mass spectrometer (LC/Q/Orbitrap MS) platform for the screening of prohibited substances in human urine according to World Antidoping Agency (WADA) requirements. The method was also validated for quantitative analysis of six endogenous steroids (epitestosterone, testosterone, 5α-dihydrotestosterone, dehydroepiandrosterone, androsterone and etiocholanolone) in their intact sulfates form. The sample preparation comprised a combination of a hydrolyzed urine liquid-liquid extraction and the dilute & shoot addition of original urine in the extracted aliquot. The HR-FS MS acquisition mode with Polarity Switching was applied in combination of the Quadrupole-Orbitrap mass filter. The HR-FS acquisition of analytical signal, for known and unknown small molecules, allows the inclusion of all analytes detectable with LC-MS for antidoping investigations to identify the use of known or novel prohibited substances and metabolites after electronic data files' reprocessing. The method has been validated to be fit-for-purpose for the antidoping analysis.

1. In the stallion, estrogens were synthesized and sulfated in vivo by the testis. 2. The equine testicular enzyme aromatized androgens and 19-norandrogens with similar velocity, but not 16 alpha-hydroxytestosterone or epitestosterone in contrast to the human placental aromatase. 3. One single enzyme was implicated in the aromatization of androstenedione, testosterone, 19-norandrostenedione and 19-nortestosterone by ETMES. 4. During the process of androstenedione aromatization by ETMES, 19-hydroxyandrostenedione and 19-oxoandrostenedione were released and 4-hydroxyandrostenedione was a competitive inhibitor causing an additional irreversible enzyme inactivation which is what occurs with HPMES. 5. Dihydrotestosterone was a potent competitive inhibitor of aromatase activity.

Twentyfour hour-urinary 17-oxo-steroids (17 KS), epitestosterone (ET) and testosterone (T) were measured before and after a 5-day-HCG-stimulation test performed in 65 healthy male volunteers aged 30 to 76 years. Subjects whose basal urinary T was higher than average physiological values showed a lower increase (p less than 0,001) in ET excretion rate after HCG stimulation than the rest of the casuistry. Neither T nor 17 KS increase after HCG stimulation showed any significant differences in the two subgroups. As ET behaves differently as compared both to the typical testicular androgen, and to the testicular-adrenal metabolites, the adrenal glands may not be the only nor even the main source of ET in the urine of healthy men.