The goal of this study was to investigate the effects of oral administration of d-aspartate (D-Asp) to sexually immature male C57BL/6NTacCnrm (B6N) mice on the in vitro fertilisation (IVF) rate with cryopreserved-thawed spermatozoa and on cryopreserved sperm quality as well as on LH, epitestosterone, and testosterone levels. Males were treated at 7 weeks of age with a dose of 20 mM sodium d-aspartate in drinking water for 1.3, 2, 4 or 6 weeks so that they were 8.3, 9, 11 or 13 weeks of age, respectively, at the end of the study. The timepoints for controls were week 0 (start of experiment), 1.3, 2, 4 or 6, whereby mice received no D-Asp. At each timepoint, spermatozoa were cryopreserved for IVF and testes as well as sera were frozen for hormone level measurement. After 2 and 4 weeks of treatment, the IVF rate was significantly higher in the D-Asp group than in the controls (64% vs. 44% and 52% vs. 38%, respectively). Spermatozoa from D-Asp-treated males showed lower morphological abnormalities than their control counterparts. After 2 and especially after 4 weeks of treatment, the hormone levels in sera and testes and the total and progressive motility of the spermatozoa were higher in D-Asp-treated males. Although we did not elucidate the full mechanism leading to an improved IVF rate with spermatozoa from D-Asp-treated males lower morphological abnormalities and increased motility contribute to this observation. Importantly, D-Asp significantly improved the IVF rates as early as 2 weeks after treatment when mice were only 9 weeks old. This implies that sperm can be efficiently cryopreserved from younger males, compared to 13-weeks-old males, which are usually used for sperm cryopreservation. This is of relevance when genetic alterations cause premature death in males as well as high severity levels in older mice and aids in better resource management.

Effects of metabolites of testosterone and of anabolic androgens on bone marrow and thymus of ovariectomized mice were studied. Steroids contained in capsules made of silastic tubing were implanted on the day of castration or were injected at the day of castration (Noralone and T.P.), lymphatic organs were examined two weeks later. It was found that testosterone, 5 alpha-DHT, 3 alpha-diol, Dianabole, T.P., and Noralone caused increases in the relative number of large cells and in activity of 20 alpha SDH in bone marrow. Concomitantly, these steroids caused a marked reduction in thymus cell number and an increase in the responsiveness of the thymus cells to Con A and PHA. The steroid 3 beta-diol increased marrow cell 20 alpha SDH activity but did not affect the thymus cell number. Other steroids tested, Ad-dione 3 alpha or 3 beta-androsterone, 5 beta-DHT, epitestosterone, and progesterone had no effect on thymus or bone marrow.

Uptake and metabolism of [3H]testosterone, mainly to 5 alpha-dihydrotestosterone (5 alpha-DHT and 5 alpha-androstanediol were higher in the caput than in the cauda epididymidis in vitro. The metabolites represented 57, 49 and 47% of the total radioactivity in the caput, corpus and cauda epididymidis, respectively; subcellular distributions of the metabolites in each segment showed 67% of total radioactivity in cytosol and 18% in the nuclei. In both fractions, the amount of 5 alpha-DHT was greater than that of androstanediol while the reverse was true for the mitochondria and microsomes. The distribution of 5 alpha-reductase activity in subcellular fractions was similar to that of the microsomal marker enzyme NADPH: cytochrome C reductase, whilst 3 alpha-hydroxysteroid dehydrogenase was found mainly in the cytosol. Maximal 5 alpha-reductase activity was at pH 5.3, apparent Km values in the microsomal and nuclear fractions were 1.65 +/- 0.7 and 1.75 +/- 0.36 x 10(-6) M respectively, and the Vmax in these preparations was 5.28 +/- 1.19 and 3.1 +/- 0.52 pmol/mg protein/min, respectively. The activity of 5 alpha-reductase was inhibited by Zn2+, Cu2+, Ba2+ and Cd2+ and by epitestosterone, progesterone and 4-androstene-3-one 17 beta-carboxylic acid.

The goal of this study was to assess various analytical approaches for the simultaneous and efficient extraction of steroid hormones (cortisone, cortisol, prednisolone, corticosterone, testosterone, 17α-methyltestosterone, epitestosterone, progesterone) from urine samples prior to separation based on field-amplified sample stacking MEKC (FASS-MEKC). FASS-MEKC successfully allowed the compounds to be separated within 12 min using a BGE composed of 5 mM sodium tetraborate, 150 mM boric acid, 50 mM SDS, and 15% methanol. Therefore, many procedures such as solid-phase microextraction, SPE, and dispersive liquid-liquid microextraction (DLLME) were tested and compared using a multivariate tool, namely, cluster analysis. Finally, DLLME-FASS-MEKC was validated and proved a good linearity of calibration curves (R2 above 0.9948) in a concentration range from 50 to 1000 ng/mL for all analytes. The LOD was established at 15 ng/mL, whereas the LOQ was 50 ng/mL. The intra- and interday precision, expressed as RSD%, did not exceed 9.97%. The DLLME-FASS-MEKC method was successfully applied to the analysis of urine samples from healthy volunteers and sportsmen. This methodology could prove to be useful in clinical studies and/or doping control depending on the steroid concentrations required in biomedical applications.

Progesterone, epitestosterone (4-androstene-17 alpha-ol-3-one, EpiT) and 4- androstene-3-one-17 beta-carboxylic acid (COOH), three known in vitro inhibitors of 5 alpha reductase, were injected daily for 30 days to male rats to study their effect on some parameters of epididymal function. Progesterone at the dose of 750 and 2000 micrograms/day decreased fertility by 59% and 50% respectively. EpiT at a dose of 1500 micrograms/day decreased fertility by 74%. These treatments did not change the sperm counts in the cauda epididymis. Treatment with COOH did not decrease fertility. Progesterone at 750 and 2000 micrograms/day and EpiT at 750 micrograms/day decreased the weight of the epididymis, prostate and seminal vesicles. None of the compounds tested produced variations in body weight or in the weight of liver and testis. The 5 alpha reductase activity of epididymis, testis and liver was diminished by progesterone treatment, while EpiT decreased only that of testis and liver.

1. Seasonal change in the biosynthesis of androgens by testicular tissue from a scincid lizard Tiliqua rugosa has been studied in vitro using [7-3H]pregnenolone and [4-14C]progesterone as substrates. 2. Evidence is presented for the synthesis of epitestosterone, testosterone, androstenedione, 17 alpha-hydroxyprogesterone, 20 beta-hydroxypregn-4-en-3-one, and 17 alpha, 20 beta-dihydroxypregn-4-en-3-one. 3. Epitestosterone was the major conversion product. Its yield was highest (68%) during summer and lowest (14%) during spring. The yield of testosterone was maximal (10%) during spring (mating season). The production of 20 beta-hydroxypregn-4-en-3-one was highest during spring and undetectable during summer. 4. Histological evidence and changes in testicular weight indicated that spermatogenic activity was greatest during spring. Pronounced regression of the testis occurred immediately after the breeding season and lasted until autumn.

Three different muscles (Longissimus dorsi, Semitendinosus, Extensor carpi ulnaris) of bulls and steers, which represent different parts of the carcass and which have differing properties (function, proportions of fat and connective tissue), were analysed with GC-MS for their contents of testosterone, cortisol, cortisone, pregnenolone, dehydroepiandrosterone, progesterone, hydroxyprogesterone, androstenedione, dihydrotestosterone, epitestosterone and androsterone. No difference in the hormone patterns could be detected between the three muscles. However, the enrichment of beef samples with inter- and intramuscular fat decreased the levels of the polar corticosteroids, whereas the levels of lipophilic steroids were increased. The patterns of the lipophilic sex steroids, their precursors and metabolites, which can be used to determine the sexual origin of beef and which might prove useful in evaluating residues of administered steroid hormones, seem to be less affected by the beef sample's fat content, however.

1. In contrast to drug-metabolizing cytochromes P-450, the corresponding steroidogenic enzymes appear to be highly specific with respect to substrates, reactions catalysed, and the sites in the substrate molecule that are attacked. 2. Recent studies have shown that important exceptions to this generalization are encountered. 3. The conversion of 11-deoxycorticosterone to aldosterone requires three enzymatic reactions, the last of which (aldehyde synthetase), like the first two, requires a P-450 mono-oxygenation. The involvement of P-450 in this third step (from alcohol to aldehyde) was demonstrated by photochemical action spectra and determination of stoichiometry. The three reactions were shown to be catalysed by a single enzyme as demonstrated by immunoprecipitation and the use of a homogenous enzyme. 4. The conversion of pregnenolone and progesterone to the corresponding delta 16-C19 steroids, which are pheromones, is catalysed by a cytochrome P-450 that also catalyses the conversion of progesterone to androstenedione (that is, C21 side-chain cleavage; hydroxylase/lyase). The synthesis of the delta 16 compounds requires cytochrome b5. 5. The conversion of testosterone and epitestosterone to androstenedione is catalysed by P-450b. Studies of kinetic isotope effects using deuterium and 18O2 show that P-450b catalyses this reaction with epitestosterone via the formation of a gem diol, whereas, with testosterone as substrate, one-third of the product is formed via the same mechanism while the remainder results from the mechanism described as dual hydrogen abstraction.

The NADP+-linked oestradiol-17 alpha dehydrogenase (EC 1.1.1.148) present in cell-free extracts of chicken liver was investigated with the aim of separating it from a closely related oestradiol-17 beta dehydrogenase (EC 1.1.1.62) found in the same subcellular fraction. However, its chromatographic behaviour on CM-cellulose and DEAE-cellulose was almost identical with that previously reported for the latter enzyme, including resolution into two peaks on the anion-exchanger. Both peaks contained oestradiol-17 alpha dehydrogenase and oestradiol-17 beta dehydrogenase activity. Further attempts to separate the putative enzymes by dye-ligand chromatography with the use of the dyes Procion Yellow, Reactive Red and Cibachron Blue linked to Sepharose were unsuccessful, and they behaved identically on affinity columns of adenosine 2',5'-bisphosphate-agarose and 17 beta-oestradiol 3-hemisuccinate bound to Sepharose. A previous report of partial separation on Sephadex G-200 was not confirmed. Slab gel electrophoresis of enzyme preparations after affinity chromatography on adenosine 2',5'-bisphosphate-agarose revealed multiple bands in systems containing sodium dodecyl sulphate, whereas analysis by rod gel electrophoresis gave two major and one minor bands that stained coincidently for oestradiol-17 alpha dehydrogenase, oestradiol-17 beta dehydrogenase, epitestosterone dehydrogenase and testosterone dehydrogenase activities. Isoelectric focusing gave four enzymically active peaks that each oxidized oestradiol-17 alpha and -17 beta. Apparent Km values for the two forms of oestradiol-17 alpha dehydrogenase obtained by DEAE-cellulose chromatography were 17 and 23 microM for oestradiol-17 alpha, and 8.7 and 11.0 microM for NADP+. Limited kinetic studies with oestradiol-17 alpha and -17 beta with the use of the mixed-substrate method showed that the total velocity was equal to the sum of the separate velocities. The active-site inhibitor-alkylating agent 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one did not cause time- or temperature-dependent inhibition, in contrast with the reported case of the oestradiol-17 beta dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase activities of the human placental oestradiol dehydrogenase. NADP+ appeared to afford some protection against inhibition. Investigation of substrate specificity with a limited range of steroids suggests that the enzyme(s) from chicken liver differs substantially from the oestradiol-17 beta dehydrogenase from human placenta, and although the evidence is not conclusive it suggests the existence of one enzyme.

Levels of several natural urinary steroids have been determined in the urine of a large number of animals of different cattle categories in the context of steroid abuse in beef production. Bovine animals of different breeds, sex and age included in the Slovene national residue detection plan for steroid abuse were studied. Urine from 120 males and 174 females was analysed. Urinary boldenone, boldione, androstenedione, equiline, medroxyprogesterone, medroxyprogesterone acetate, melengestrol acetate, progesterone, stanozolol, trenbolone, trenbolone acetate, 17α-ethinylestradiol, 17α-methyltestosterone, epitestosterone, 17β-estradiol, testosterone, and nandrolone were determined by LC-MS/MS. Epitestosterone was found in all bulls; while the proportion of animals containing testosterone and androstenedione increased with age. Testosterone was not detected in bulls less than 5 months of age. Epitestosterone levels, however, were not age dependent. The ratio of testosterone to epitestosterone thus increased with age, from 0.13 ± 0.09 at 1-7 months to 0.42 ± 0.10 at 25-38 months. It was significantly (p < 0.01) higher in bulls above 13 months than in younger animals. In contrast to males, no urinary testosterone was found in females, whereas epitestosterone, androstenedione, progesterone and estradiol were present. The proportion of animals of various age groups in which epitestosterone was detected ranged from 68% to 100%, but the differences were not significant. The presence of both estradiol and progesterone in the same sample was not observed in any animal. The results of this study could be helpful in determining physiological urinary steroid levels in order to provide a baseline for the control of steroid abuse in beef production.

The endogenous anabolic agents, estradiol-17 beta, progesterone and testosterone are steroids that are quickly metabolized by the liver and are not very active when administered orally. Estradiol-17 beta is excreted by the bovine in bile as free estradiol-17 alpha, and by swine, in urine, as glucuronides and sulfates. In ruminants, the primary metabolite of progesterone is pregnandiol and that of testosterone is epitestosterone. In horses, the metabolites of these compounds are primarily 17 ketosteroids. Esters of the endogenous anabolic agents are rapidly hydrolyzed and the nonesterified forms follow the same biotransformation pathways as the natural compounds biosynthesized by the animal. The exogenous anabolic agents, such as trenbolone acetate, zeranol and diethylstilbestrol, may be active by the oral route and are less readily metabolized in the liver than the endogenous anabolics. Their metabolic pathways may be complex and lead to excreted forms after glucuronide conjugation. With respect to biochemical mechanism of action, it can be assumed that the anabolics act like all steroids by way of intracellular receptors. Biotransformations could lead to more reactive molecules that may bind themselves to normal constituents of the organism. Bound metabolites are generally formed later than free metabolites, and are considered less toxic with a lower level of bioavailability.

This presentation is limited to the three groups of steroid sex hormones which alone or in combination have been shown to be anabolic when used in farm animals. It seems essential for realistic evaluation of public health aspects of use of these hormones that the discussions include naturally occurring levels of the hormones. The following topics will be dealt with for each group of hormones: 1. Types and sources; 2. Production rates; 3. Plasma levels; 4. Tissue concentrations; 5. Metabolism and excretion. Gestagens. Progesterone and 20-dihydroprogesterones are mainly produced in ovaries and placenta. Production rates are estimated to 10 and 14 mg/24 hrs in pregnant goats and sheep, respectively. Plasma levels during the luteal phase are of the order of 2--10 ng/ml, during pregnancy somewhat higher. Muscular tissue from calves contain 0.25 mg/g. In dairy cows progesterone is excreted with the milk which contains up to 30 ng/ml; butterfat up to 300 mg/g. In ruminants progesterone is metabolized mainly to androgens excreted with faeces. In pigs large parts are metabolized to pregnanediols excreted with urine. Androgens. Testosterone is mainly secreted by testes. Boar testes also produce large amounts of dehydroepiandrosterone and its sulphate. Production rates have been estimated to be 10 mg and 40--50 mg/24 hrs. in boars and bulls respectively. Plasma levels in bulls and rams are generally 2--10 ng/ml, in boars 2--25 ng/ml. Adipose tissue levels up to 22 ng/g are reported for bulls. In ruminants epitestosterone seems to be a major metabolite excreted mainly with faeces. In boars, urinary 11-deoxy-17-ketosteroids are major metabolites of testicular dehydroepiandrosterone. Castration shows elimination to be rapid. Estrogens. 17beta-Estradiol and estrone are produced in ovaries and placenta and, in large amounts, in boar and stallion testes. Production rates in late pregnancy are estimated to 10 mg oestrone/24 hrs. in goats, 2 mg estrone and up to 28 mg 17beta-estradiol/24 hrs. in sheep. In cows much higher values are found. Boars and stallions produce huge amounts daily. Plasma levels in non-pregnant animals are at the pg/ml level. In late pregnancy levels of 2--4 thousand pg/ml are encountered in sows and cows, in sheep and goats lower levels. Calf muscular tissue contains up to 410 and 610 pg/g of estrone and 17beta-estradiol respectively. In muscle from pregnant heifers corresponding values were 120 and 860 pg/g in the 4th month and 2100 and 370 pg/g in the 9th month of pregnancy. Ruminants in large measure metabolize 17 beta-estradiol and estrone to 17alpha-estradiol which possesses low estrogenic activity. In pigs estrone dominates in blood and urine. Major routes of elimination arre with faeces in ruminants, with urine in pigs and horses. Elimination rates are high. Results obtained during the last few years clearly show that all three groups of steroid sex hormones occur in considerable concentrations in plasma and tissue...

Steroid profiling is the most versatile and informative technique adapted by doping control laboratories for detection of steroid abuse. The absolute concentrations and ratios of endogenous steroids including testosterone, epitestosterone, androsterone, etiocholanolone, 5α-androstane-3α,17β-diol and 5β-androstane-3α,17β-diol constitute the significant characteristics of a steroid profile. In the present study we report the influence of various oxidizing adulterants on the steroid profile of human urine. Gas chromatography-mass spectrometry analysis was carried out to develop the steroid profile of human male and female urine. Oxidants potassium nitrite, sodium hypochlorite, potassium permanganate, cerium ammonium nitrate, sodium metaperiodate, pyridinium chlorochromate, potassium dichromate and potassium perchlorate were reacted with urine at various concentrations and conditions and the effect of these oxidants on the steroid profile were analyzed. Most of the oxidizing chemicals led to significant changes in endogenous steroid profile parameters which were considered stable under normal conditions. These oxidizing chemicals can cause serious problems regarding the interpretation of steroid profiles and have the potential to act as masking agents that can complicate or prevent the detection of the steroid abuse.

The evaluation of the relationships between the hormones involved in the urogenital tract cancer, including bladder, kidney, prostate, and testis, could prove important from diagnostic point of view. The determination of the steroid hormone profiles may likely provide a biomarker for discrimination of hormone-related diseases, as well as for differentiation of healthy volunteers from patients with cancer. The aim of the study was to demonstrate the changes in the steroid hormone profile (comprising corticosteroids, androgens and progesterone) in the urine of patients with the urogenital tract cancer versus urine from healthy subjects. A reliable analytical method based on liquid chromatography coupled with mass spectrometry was successfully applied to determine the urinary profiles of 6 endogenous steroids: cortisol, cortisone, corticosterone, testosterone, epitestosterone and progesterone for 92 urogenital tract cancer patients and 100 healthy controls. The obtained data was further evaluated by in-depth chemometric analysis, including the applied standardized Kennard-Stone's algorithm to pre-process the data. Mann-Whitney U test revealed statistically significant (p <0.05) differences in concentration of androgens and progesterone in the case of bladder cancer for male and female population, for male also cortisol and cortisone levels were significantly increased. PCA analysis proved a reasonable trend for differentiating healthy and cancer patients, and finally, applying PLS-DA model we were able to correctly classify 80.56%of cancer patients. Our results indicate that steroid hormone profile determination could be a promising approach for early diagnosis of urogenital tract cancer. However our preliminary results require an extension both in patient number and steroid profile.

In women, hormonal fluctuations related to the menstrual cycle may impose a great source of variability for some biomarkers of testosterone (T) administration, which can ultimately disrupt the sensitivity of their longitudinal monitoring. In this study, the sensitivity of the current urinary and haematological markers of the Athlete Biological Passport (ABP), as well as serum steroid biomarkers was investigated for the monitoring of a 28-days T gel treatment combined with endogenous fluctuation of the menstrual cycle in 14 healthy female subjects. Additionally, the analysis of urinary target compounds was performed on a subset of samples for endogenous/exogenous origin via isotope ratio mass spectrometry (IRMS). In serum, concentrations of T and dihydrotestosterone (DHT) increased significantly during the treatment, whereas in urine matrix the most affected biomarkers were found to be the ratios of testosterone/epitestosterone (T/E) and 5α-androstane-3α,17β-diol/epitestosterone (5αAdiol/E). The detection capability of both urinary biomarkers was heavily influenced by [E], which fluctuated depending on the menstrual cycle, and resulted in low sensitivity of the urinary steroidal ABP module. On the contrary, an alternative approach by the longitudinal monitoring of serum T and DHT concentrations with the newly proposed T/androstenedione ratio showed higher sensitivity. The confirmatory IRMS results demonstrated that less than one third of the tested urine samples fulfilled the criteria for positivity. Results from this study demonstrated that the 'blood steroid profile' represents a powerful complementary approach to the 'urinary module' and underlines the importance of gathering bundle of evidence to support the scenario of an endogenous prohibited substance administration.

Keywords: Athlete Biological Passport; menstrual cycle; steroid profile; testosterone gel; women.

A method of gas chromatography-mass spectrometry (GC-MS) for the simultaneous determination of nine sex hormone residues, such as hexestrol, diethylstilbestrol, dienestrol, etiocholan-3alpha-ol-17-one, epitestosterone, estrone, estradiol, ethinylestradiol and estriol, in animal tissues was developed. The sex hormones were extracted with acetonitrile, then cleaned-up with a C18 solid-phase extraction (SPE) column. The microwave-assisted derivatization of the target components with N,O-bis( trimethylsilyl) trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS) (99:1, v/v) using pyridine as solvent was performed, and then the derivatives were analyzed by GC-MS. The limits of detection were 0.1-1 microg/kg for all hormones, and the limits of quantification were 0.2-2 microg/kg. The average recoveries of sex hormones were 68.8%-93.1%. The relative standard deviations (RSDs) of inter- and intra-assay were 4.1%-22.3% and 3.1%-17.9%, respectively. The real sample tests showed this method can be used for the sensitive and accurate determination of multi-sex hormones residues in biological samples.

Epididymal 5alpha reductase activity was found distitributed in the crude nuclear fraction (44 percent) and microsomal fraction (41 percent). Spermatozoa contaminating the nuclear preparation accounted for only 3 percent of its activity. There were no regional differences in the distribution of total 5alpha reductase activity. However, the nuclear enzyme was more active in caput than in other regions. Maximal activity was found at pH 6.2 and at 32 degrees C. Both enzymes had an absolute requirement of reduced dinucleotides. The microsomal preparation could only us NADPH while the nuclear enzyme could use NADPH and NADH. The apparent Km for the microsomal preparation was 0.62 +/- 0.05 X 10(-6)M and Vmax was 555 +/- 38 pmoles/mg protein/hour. The nuclear enzyme presented similar values. The reaction was not inhibited by accumulation of product in the medium, but other steroids such as progesterone, epitestosterone (17alpha-hydroxy-4-androsten-3-one) and 3-oxo-4-androstene-17beta-carboxylic acid were potent competitive inhibitors. The reaction was strongly inhibited by Hg, Zn and Cu. The properties of the epididymal reductase are similar to those of the prostatic enzyme.

The reduction of 17-ketosteroid estrone or androstenedione to corresponding 17alpha- and 17beta-estradiol or testosterone and epitestosterone has been performed with Saccharomyces cerevisiae. In the analysis of the cell culture, the solid-phase extraction (SPE) method was on-line coupled to high-performance liquid chromatography electrospray-ionization/mass spectrometry (HPLC-ESI/MS) for sample pretreatment to eliminate the complicated matrix interference and preconcentrate of the analytes before chromatographic separation. A novel quantification method with the continuous postcolumn infusion of internal standard was developed for the determination of substrate and products. This novel quantitative method can stabilize and enhance the ionization of all analytes during analysis. The HPLC-ESI/MS analysis of estrone, 17alpha-, and 17beta-estradiol was operated with a negative ion mode and the analysis of androstenedione, testosterone, and epitestosterone was operated with a positive ion mode. The optimal concentration of the internal standard progesterone with the continuous postcolumn infusion technique was 3 microg mL(-1) for estrogen analysis and 1 ng mL(-1) for androgen analysis and both were at a constant infusion rate of 0.5 microL min(-1). All of the linear correlation coefficients of the standard calibration curves were over 0.99 and had a linear range from 0 to 50 ng mL(-1). The limit of detections (LODs) and the limit of quantitations (LOQs) for steroids analyzed were from 0.12 to 0.36 ng mL(-1) and from 0.4 to 1.2 ng mL(-1), respectively. The analysis accuracies and precisions were better than 94% and lower than 8.8% R.S.D., respectively. The developed method for the analysis of steroids in the cell culture was successful.

Controversial results have been reported in the literature regarding the behavior of two testosterone (T) metabolites (3α-glucuronide-6β-hydroxyandrosterone and 3α-glucuronide-6β-hydroxyetiocholanolone) excreted after T administration. Due to their potential as biomarkers of T misuse, a UHPLC-MS/MS method for the direct quantification of these glucuronides was developed and validated. In addition, the main phase II metabolites of T that compose the steroid profile used for doping control purposes (glucuronides of T, epitestosterone, androsterone and etiocholanolone) were included. The method was found to be linear and with suitable LODs and LOQs for all metabolites. The average accuracies were between 86% and 120%, the RSDs for the intra- and inter-day precision were below 15% and 25% respectively. The method showed low matrix effect. Samples obtained before and after the administration of T were analyzed by both the developed UHPLC-MS/MS method and the GC-MS/MS method currently used by anti-doping laboratories. Relevant disagreements between the results obtained for 3α-glucuronide-6β-hydroxyandrosterone and 3α-glucuronide-6β-hydroxyetiocholanolone quantitation were observed. These markers seemed to be more suitable for the screening of T misuse when detected by UHPLC-MS/MS. These discrepancies were further investigated in 50 urine samples from healthy volunteers. The two methods gave highly correlated results for all metabolites that are currently included in the athlete's steroid profile confirming the reliability of the UHPLC-MS/MS method. However, the quantification of 3α-glucuronide-6β-hydroxyandrosterone and 3α-glucuronide-6β-hydroxyetiocholanolone, was only possible by using the UHPLC-MS/MS method since three interfering compounds were observed when performing the GC-MS/MS analysis with the most intense ion transitions. These results confirm the potential of the resistant glucuronides as biomarkers of T misuse. Additionally, they suggest that previously reported reference ranges for these metabolites should be reevaluated.

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.

A gas chromatography/mass spectrometry (GC/MS) method was developed for the determination of multi-residues of steroid anabolic hormones epitestosterone (ETS), testosterone 17-propionate (PTS), nandrolone (17beta-NT), 17alpha-methyltestosterone (MTS), 17beta-estradiol (17beta-ES), estriol ( EST), 17alpha-ethinylestradiol (EES), estrone (ESN) and 17beta-estradiol 3-benzoate (BES) in the muscle tissues of various animal species. Homogenized tissue samples were enzymatically digested in acetate buffer (pH 5.0). Consequently, methanol was added and the mixtures were extracted under ultrasonication incubation. Clean-up was carried out for at least two times with methyl tert-butyl ether (MTBE) liquid-liquid partitioning followed by a reversed-phase solid phase extraction (SPE) cartridge purification. The eluate with methanol was evaporated to dryness by N2 at 40 degrees C and derivatization was achieved with N-methyl-N-( trimethylsilyl) trifluoroacetamide/iodotrimethylsilane/dithioerythritol (MSTFA-TMIS-DTE) at 60 degrees C for 45 min. The reaction mixture was injected into a gas chromatograph with a DB-1 capillary column coupled with a mass spectrometer. The samples were tested by different selected ion monitoring modes with electron impact (EI) source for the androgens and estrogens. The limits of quantitation (LOQ) for the above 9 hormones were in the range of 1.0 - 2.0 microg/kg. At the 2.0 microg/kg LOQ spiked level, the mean recoveries were within 62.5% - 80.5%, and the relative standard deviations were within 12.5% - 26.8%. The real sample tests showed this method can be used for the sensitive and accurate determination of multi-steroid anabolic hormones residues in biological muscle samples.

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To investigate the excretion and conjugation profile of testosterone (T), Epitestosterone (EpiT), and other androgen metabolites in different phases of pregnancy and postpregnancy as a reflection of the "androgenic exposure."

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Consecutive recruitment of pregnant women.

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Maternity outpatient low-risk pregnancy clinic.

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Seventy-seven pregnant women.

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Collection of urine for analyses of sulfate (S) and glucuronide (G) conjugates and metabolic ratios of androgens and androgen metabolites using liquid chromatography-tandem mass spectrometry.

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Excretion profiles and metabolic ratios of G and S conjugates of T, EpiT, dehydroepiandrosterone (DHEA), androsterone (A), etiocholanolone (Etio), and dihydrotestosterone in relation to trimester and postpartum, body mass index, fetal sex, and ethnicity.

UNASSIGNED

T-S excretion increased significantly between the second and third trimester, whereas excretion of T-G did not change. In contrast, both conjugates of EpiT increased markedly, more so for the S-(17-fold) than the G-conjugate (1.6-fold). The preference for S over G conjugation was conspicuous for EpiT and DHEA (S/G ratio 2.1 and 4.7, respectively, in the third trimester), whereas the reverse was true for T, A, and Etio (S/G 0.6, 0.13, and 0.11, respectively).

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Pregnancy influences the androgen excretion profile, with the most profound change being an increase in EpiT excretion throughout the trimesters. EpiT may modulate the effect of T, but its exact role during pregnancy is not known. There were marked differences in the S/G conjugate ratios between androgens upstream and downstream from T in the metabolic network. These results are interesting to compare with the androgen disposition in women with endocrine disorders or abuse of steroids.

The study of the metabolism of drugs, in particular steroids, by both in vitro and in vivo methods has been carried out in the authors' laboratory for many years. For in vitro metabolic studies, the microsomal fraction isolated from horse liver is often used. However, the process of isolating liver microsomes is cumbersome and tedious. In addition, centrifugation at high speeds (over 100 000 g) may lead to loss of enzymes involved in phase I metabolism, which may account for the difference often observed between in vivo and in vitro results. We have therefore investigated the feasibility of using homogenized horse liver instead of liver microsomes with the aim of saving preparation time and improving the correlation between in vitro and in vivo results. Indeed, the preparation of the homogenized horse liver was very simple, needing only to homogenize the required amount of liver. Even though no further purification steps were performed before the homogenized liver was used, the cleanliness of the extracts obtained, based on gas chromatography-mass spectrometry (GC-MS) analysis, was similar to that for liver microsomes. Herein, the results of the in vitro experiments carried out using homogenized horse liver for five anabolic steroids-turinabol, methenolone acetate, androst-4-ene-3,6,17-trione, testosterone, and epitestosterone-are discussed. In addition to the previously reported in vitro metabolites, some additional known in vivo metabolites in the equine could also be detected. As far as we know, this is the first report of the successful use of homogenized liver in the horse for carrying out in vitro metabolism experiments. Copyright © 2011 John Wiley & Sons, Ltd.