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The steroid module of the Athlete Biological Passport (ABP) is based on the analysis of six endogenous steroids in urine samples and a Bayesian statistical approach. However, the urinary steroid concentrations may be affected by confounders like microbial degradation, possible co-administration of diuretics as masking agents, insufficient conjugate hydrolysis or UGT2B17 gene polymorphisms affecting glucuronidation. Therefore, it can be helpful to use other matrices (ABP blood and serum samples) to quantify steroids and thereby support noticeable deviations in the Athlete Biological Passport, for example, abnormally increased urinary testosterone/epitestosterone (T/E) ratios. Aim of the study was to investigate the feasibility to re-use plasma obtained from athlete ABP blood samples for measuring a steroid profile. Therefore, testosterone, androstenedione, cortisol and cortisone were quantified in 36 intra-individual matching ABP blood and serum samples. The steroid levels measured in both matrices showed a high agreement indicating a good stability uninfluenced by storage temperature and duration. Our results pointed out the possibility to expand the athlete ABP blood analysis for steroid profiling., (© 2022 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

The World Anti-Doping Agency (WADA) has introduced an Athlete Biological Passport (ABP) with a steroidal module, which is intended for the monitoring of longitudinal profiles of an athlete's steroid variables in urine to identify endogenous anabolic androgenic steroids that are administered exogenously. It has been in use since 2014. The prevalence of UGT2B17 gene deletion with relatively low levels of testosterone (T) glucuronide in urine is high in the Asian region. There are cases in which urinary T is below the detection limit in specific urine samples, for example, diluted urine, urine collected from females, or urine collected from UGT2B17 deletion individuals. Additional steroid markers T, 4-androstenedione (A4), and the T/A4 ratio in serum were newly added to the ABP steroidal module by WADA in 2023 to compensate for the urine steroid profile. In this study, populations of blood steroid markers in Asians (n = 510) were investigated and classified according to UGT2B17 polymorphism to confirm the effectiveness of blood steroid markers in monitoring Asian athletes. No significant difference in the T/A4 ratio was observed between the genotypes. Furthermore, an administration study of T enanthate in females (n = 10) who were classified according to UGT2B17 genotypes was performed. The concentration of T and the T/A4 ratio were found to be significantly increased in all post-administration samples until 15 days after administration (p < 0.01). The overall results supported the high effectiveness of subject-based monitoring for serum T and T/A4 ratio for recently identified shortcomings in the detection of T abuse in Asians., (© 2023 John Wiley & Sons Ltd.)

In this work, male rats were exposed to multiple phthalate esters (MIXPs) in a long-term low-dose model for the early evaluation of reproductive toxicity. An ananlysis method with better sensitivity, accuracy and precision was established to determine the five sex hormones (androstenedione, testosterone, dehydroepiandrosterone, dihydrotestosterone, and estrone) in collected urine samples. The results showed that all the analytes in the MIXPs treated group changed in a time-dependent manner. Specifically, estrone significantly decreased from the 30th day and the other four changed from the 30th day and then significantly increased on the 60th day, while no obvious changes were found in the control group. Therefore, a possible way was provided for the early evaluation of male reproductive toxicity induced by Phthalate esters (PEs) . The reliability of judgment was improved by observing the changes of five target hormones simultaneously. Furthermore, good compliance was predicted for the practical application due to the noninvasive and convenient urine sample collection.

Selective estrogen receptor modulators (SERMs), anti-estrogens and aromatase inhibitors are prohibited in human sports doping. However, they also present a risk of being used illegally in animal husbandry for fattening purposes. A method was developed and validated using UHPLC-MS/MS for the determination and confirmation of SERMs, anti-estrogens and aromatase inhibiters in bovine and porcine urine. This method was used in a survey of more than 200 bovine and porcine urine samples from Dutch farms. In 18 out of 103 porcine urine samples (17%) and two out of 114 bovine samples (2%) formestane, an aromatase inhibitor, was detected. None of the other compounds was detected. From human doping control it is known that formestane can, in some cases, be of natural origin. Analyses of reference samples from untreated bovine and porcine animals demonstrated the presence of formestane in bovine animals, but not yet in porcine animals. Future research will focus on whether the detected formestane in porcine and bovine urine is from endogenous or exogenous origin, using GC-c-IRMS.

Chronic and heavy alcohol consumption disrupts lipid metabolism and hormonal balance including testosterone levels. However, studies doubt the relationship between moderate alcohol intake and sex hormone levels. Therefore, the aim of the present investigation was to establish the direct impact of chronic and moderate alcohol intake on cholesterol homeostasis and steroid hormone synthesis. Asymptomatic chronic and moderate alcoholics (n=12) without chronic liver disease and healthy volunteers (n=14) were selected for the study. Furthermore, effects of standardized water extract of Tinospora cordifolia (Willd) Mier. (Menispermaceae) (TCJ), a well reported anti-alcoholic herbal drug, on urinary steroids was studied. This study included four groups, i.e. a) healthy; b) healthy+TCJ; c) alcoholic; d) alcoholic+TCJ. The blood and urine samples from each group were collected on day 0 and 14 of the post-treatment with TCJ and analyzed. Alcoholic blood samples showed the significantly higher values of traditional biomarkers γ-GT and MCV along with cholesterol, LDL, TGL and urinary methylglucuronide compared to healthy. Qualitative analysis of steroids showed that moderate alcohol intake in a chronic manner increased the cholesterol synthesis and directed its flow toward C-21 steroids; shown by increased levels of corticosterone (2.456 fold) and cortisol (3.7 fold). Moreover, alcohol intake also increased the synthesis of estradiol and clearance rate of other steroids through the formation of glucuronides. Therefore, it decreased the synthesis and increased the clearance rate of testosterone (T) and androstenedione (A). Quantitative analysis confirmed decreased T/A ratio from 2.31 to 1.59 in plasma and 2.47 to 1.51 in urine samples of alcoholics. TCJ intervention normalized the levels of steroids and significantly improved the T:A ratio to 2.0 and 2.12 in plasma and urine. The study revealed that TCJ modulated lipid metabolism by inhibiting cholesterol and glucuronides synthesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Members of the order Carnivora rely on urinary scent signaling, particularly for communicating about reproductive parameters. Here, we describe reproductive endocrine patterns in relation to urinary olfactory cues in a vulnerable and relatively unknown viverrid--the binturong (Arctictis binturong). Female binturongs are larger than and dominate males, and both sexes engage in glandular and urinary scent marking. Using a large (n = 33), captive population, we collected serum samples to measure circulating sex steroids via enzyme immunoassay and urine samples to assay volatile chemicals via gas chromatography-mass spectrometry. Male binturongs had expectedly greater androgen concentrations than did females but, more unusually, had equal estrogen concentrations, which may be linked to male deference. Males also expressed a significantly richer array of volatile chemical compounds than did females. A subset of these volatile chemicals resisted decay at ambient temperatures, potentially indicating their importance as long-lasting semiochemicals. Among these compounds was 2-acetyl-1-pyrroline (2-AP), which is typically produced at high temperatures by the Maillard reaction and is likely to be responsible for the binturong's characteristic popcorn aroma. 2-AP, the only compound expressed by all of the subjects, was found in greater abundance in males than females and was significantly and positively related to circulating androstenedione concentrations in both sexes. This unusual compound may have a more significant role in mammalian semiochemistry than previously appreciated. Based on these novel data, we suggest that hormonal action and potentially complex chemical reactions mediate communication of the binturong's signature scent and convey information about sex and reproductive state.

Traditionally, steroids other than testosterone are considered to be synthetic, anabolic steroids. Nevertheless, in stallions, it has been shown that β-Bol can originate from naturally present testosterone. Other precursors, including phytosterols from feed, have been put forward to explain the prevalence of low levels of steroids (including β-Bol and ADD) in urine of mares and geldings. However, the possible biotransformation and identification of the precursors has thus far not been investigated in horses. To study the possible endogenous digestive transformation, in vitro simulations of the horse hindgut were set up, using fecal inocula obtained from eight different horses. The functionality of the in vitro model was confirmed by monitoring the formation of short-chain fatty acids and the consumption of amino acids and carbohydrates throughout the digestion process. In vitro digestion samples were analyzed with a validated UHPLC-MS/MS method. The addition of β-Bol gave rise to the formation of ADD (androsta-1,4-diene-3,17-dione) or αT. Upon addition of ADD to the in vitro digestions, the transformation of ADD to β-Bol was observed and this for all eight horses' inocula, in line with previously obtained in vivo results, again confirming the functionality of the in vitro model. The transformation ratio proved to be inoculum and thus horse dependent. The addition of pure phytosterols (50% β-sitosterol) or phytosterol-rich herbal supplements on the other hand, did not induce the detection of β-Bol, only low concentrations of AED, a testosterone precursor, could be found (0.1 ng/mL). As such, the digestive transformation of ADD could be linked to the detection of β-Bol, and the consumption of phytosterols to low concentrations of AED, but there is no direct link between phytosterols and β-Bol., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Formestane (4-hydroxy-androstenedione) is an aromatase inhibitor prohibited in sports and included, since 2004, in the list of prohibited substances updated yearly by the World Anti-Doping Agency (WADA). Since the endogenous production of formestane has been described, it is mandatory for the anti-doping laboratories to use isotope ratio mass spectrometry (IRMS) to establish the exogenous origin before issuing an adverse analytical finding. The described IRMS methods for formestane detection are time-consuming, requiring usually two consecutive liquid chromatographic sample purifications in order to have final extracts of adequate purity before the mass spectrometric analysis. After establishing a procedure for the determination of the origin of formestane by IRMS without the need of derivatization, and integrated in the overall analytical strategy of the laboratory for pseudo-endogenous steroids, a mass spectrometric analysis by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) of formestane metabolites was carried out in order to investigate whether other biomarkers of formestane abuse could be integrated in order to avoid time-consuming and expensive IRMS confirmations for formestane. From the metabolic studies performed, the inclusion of 3β,4α-dihydroxy-5α-androstan-17-one (4α-hydroxy-epiandosterone) in the routine GC-MS procedures has demonstrated to be diagnostic in order to reduce the number of unnecessary confirmations of the endogenous origin of formestane., (Copyright © 2014 John Wiley & Sons, Ltd.)

Context: Chronic supraphysiological glucocorticoid therapy controls the androgen excess of 21-hydroxylase deficiency (21OHD) but contributes to the high prevalence of obesity, glucose intolerance, and reduced bone mass in these patients. Abiraterone acetate (AA) is a prodrug for abiraterone, a potent CYP17A1 inhibitor used to suppress androgens in the treatment of prostate cancer., Objective: The objective of the study was to test the hypothesis that AA added to physiological hydrocortisone and 9α-fludrocortisone acetate corrects androgen excess in women with 21OHD without causing hypertension or hypokalemia., Design: This was a phase 1 dose-escalation study., Setting: The study was conducted at university clinical research centers., Participants: We screened 14 women with classic 21OHD taking hydrocortisone 12.5-20 mg/d to enroll six participants with serum androstenedione greater than 345 ng/dL (>12 nmol/L)., Intervention: AA was administered for 6 days at 100 or 250 mg every morning with 20 mg/d hydrocortisone and 9α-fludrocortisone acetate., Main Outcome Measure: The primary endpoint was normalization of mean predose androstenedione on days 6 and 7 (< 230 ng/dL [<8 nmol/L)] in greater than 80% of participants. Secondary end points included serum 17-hydroxyprogesterone and testosterone (T), electrolytes, plasma renin activity, and urine androsterone and etiocholanolone glucuronides., Results: With 100 mg/d AA, mean predose androstenedione fell from 764 to 254 ng/dL (26.7-8.9 nmol/L). At 250 mg/d AA, mean androstenedione normalized in five participants (83%) and decreased from 664 to 126 ng/dL (23.2-4.4 nmol/L), meeting the primary end point. Mean androstenedione declined further during day 6 to 66 and 38 ng/dL (2.3 and 1.3 nmol/L) at 100 and 250 mg/d, respectively. Serum T and urinary metabolites declined similarly. Abiraterone exposure was strongly negatively correlated with mean androstenedione. Hypertension and hypokalemia were not observed., Conclusion: AA 100-250 mg/d added to replacement hydrocortisone normalized several measures of androgen excess in women with classic 21OHD and elevated serum androstenedione.

Context: Polycystic ovary syndrome (PCOS) is a triad of anovulation, insulin resistance, and hyperandrogenism. Androgen excess may correlate with metabolic risk and PCOS consensus criteria define androgen excess on the basis of serum T. Here we studied the utility of the androgen precursor serum androstenedione (A) in conjunction with serum T for predicting metabolic dysfunction in PCOS., Patients and Methods: Eighty-six PCOS patients fulfilling Rotterdam diagnostic consensus criteria and 43 age- and body mass index-matched controls underwent measurement of serum androgens by tandem mass spectrometry and an oral glucose tolerance test with homeostatic model assessment of insulin resistance and insulin sensitivity index calculation. We analyzed 24-hour urine androgen excretion by gas chromatography/mass spectrometry., Results: PCOS patients had higher levels of serum androgens and urinary androgen metabolites than controls (all P < .001). Within the PCOS cohort, both serum A and T were positively correlated with the free androgen index (T × 100/SHBG) and total androgen metabolite excretion (all P < .001). All subjects with T above the normal reference range [high T (HT)] also had high A (HA/HT group, n = 56). However, the remaining 30 patients had normal T levels, either in the presence of HA (HA/NT; n = 20) or normal A (NA/NT; n = 10). The groups did not differ in age or BMI. The HA/HT and HA/NT groups had higher total androgen excretion than NA/NT (P < .01 and P < .05, respectively). Multiple linear regression showed a strong negative association between serum androstenedione and insulin sensitivity. The incidence of dysglycemia according to an oral glucose tolerance test increased with the severity of androgen phenotype (NA/NT, 0%; HA/NT, 14%; HA/HT, 25%, P = .03)., Conclusion: Simultaneous measurement of serum T and A represents a useful tool for predicting metabolic risk in PCOS women. HA levels are a sensitive indicator of PCOS-related androgen excess.

Alterations in the maternal excretion of steroids during pregnancy are not restricted to the production of progesterone and estriol by the fetoplacental unit. Although there is a lack of longitudinal data on urinary androgen concentrations during pregnancy, some studies revealed that modifications in the excretions of androgens might be significant. Recently, several testosterone metabolites excreted as cysteine conjugates have been reported in human urine. We conducted a longitudinal study on androgens conjugated with cysteine and major androgens and estrogens excreted as glucuronides in three pregnant women by mass spectrometric techniques. The urinary concentrations obtained in samples weekly collected during each of the three trimesters and samples collected before pregnancy were compared. Results showed a significant increase in urinary estrogens and norandrosterone and a moderate decrease in the urinary concentrations for most of the androgens. The most significant exception to this behavior was the rise observed for epitestosterone glucuronide when comparing basal levels with the first trimester. Cysteinyl conjugates of testosterone metabolites showed a different behavior. Whereas 4,6-androstanedione remained almost constant through the three trimesters, and Δ(6)-testosterone decreased as the majority of androgens, the excretion profile of 1,4-androstanedione notably increased, reaching a maximum at the third trimester. Alterations in the steroid profile are used in doping control analysis for the screening of endogenous anabolic androgenic steroid misuse. In this study, the main parameters proposed for doping control have been determined for basal samples and samples collected in the first trimester and they have been compared. In spite of the limited number of cases, significant variations have been found in all pregnancies studied. These alterations have to be taken into consideration if anabolic steroids are included into the Athlete Biological Passport. This article is part of a Special Issue entitled 'Pregnancy and Steroids'., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Formestane (F, androst-4-en-4-ol-3,17-dione) is an irreversible aromatase inhibitor with the ability to suppress the estrogen production from anabolic steroids. Consequently, F is mentioned on the World Anti-Doping Agency (WADA) prohibited list and because studies have shown that F is produced endogenously in small amounts, a threshold for urinary excreted F of 150 ng/mL was introduced. Lower concentrations could be due to endogenous production and need further investigation to prove the exact origin through determination of the carbon isotope ratio. However, because the current screening methods are a lot more sensitive, F is detected in practically every urine sample. A strict implementation of this WADA rule would imply that almost every urine sample needs additional investigation to verify an exogenous or endogenous origin. The main aim of this study was to propose and introduce a lower concentration limit of 25 ng/mL beneath which the detected F is considered as being endogenous and no further investigation is needed. The data presented in this paper suggests that this threshold provides a good balance between a sufficiently large detection window and not having to perform isotope ratio mass spectrometry (IRMS) analyses on negative urine samples., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The steroidogenic enzyme 21-hydroxylase is necessary for the synthesis of both glucocorticoids and mineralocorticoids. 21-hydroxylase is a cytochrome P-450 enzyme and is encoded by the gene CYP21A2. Here we report a 68-year-old phenotypically 'male' but genetically female patient with 21-hydroxylase deficiency (21OHD) and the concomitant virilizing adrenocortical carcinoma. This patient grew up as a male and has not encountered any episodes of adrenal insufficiency without glucocorticoid replacement in his lifetime. A chromosome test at admission, however, identified the 46, XX karyotype, and serum 17-hydroxyprogesterone and urine pregnanetriolone and 11β-hydroxyandrostendione were all elevated, consistent with 21OHD. Moreover, serum testosterone was 1.90 ng/ml, much higher than the female standard levels, and serum cortisol was 5.7 µg/ml, slightly lower than standard levels. Genetic analysis identified the patient as a heterozygote of the two pathogenic mutations in the CYP21A2 gene: IVS2-13C(A)>G and R356W. Magnetic resonance imaging (MRI) revealed the presence of left adrenal tumor measuring 6 cm, which was subsequently diagnosed as adrenocortical carcinoma based on the criteria of Weiss. Immunohistochemical analysis of the tumor specimens revealed the expression of various enzymes involved in testosterone production, including 3β-hydroxysteroid dehydrogenase, 17α-hydroxylase/17,20-lyase, and 17β-hydroxysteroid dehydrogenase. Importantly, the expression of immunoreactive 21-hydroxylase was detected in these tumor cells. The levels of adrenal tumor-derived steroid metabolites were all markedly decreased following the surgery. This is the first report on a virilized 21OHD patient associated with the adrenocortical tumor that produces testosterone. Moreover, the concomitant adrenocortical tumor may ameliorate adrenocortical insufficiency by producing cortisol.

Formestane (4-hydroxyandrost-4-ene-3,17-dione) is an irreversible steroidal aromatase inhibitor with reported abuse in human sports. In 2011, our laboratory identified the presence of formestane in a horse urine sample from an overseas jurisdiction. This was the first reported case of formestane in a racehorse. The metabolism of formestane in humans has been reported previously; however, little is known about its metabolic fate in horses. This paper describes the in vitro and in vivo metabolic studies of formestane in horses, with the objective of identifying the target metabolite with the longest detection time for controlling formestane abuse. In vitro metabolic studies of formestane were performed using homogenized horse liver. Seven in vitro metabolites, namely 4-hydroxytestosterone (M1), 3β,4α-dihydroxy-5β-androstan-17-one (M2a), 3β,4β-dihydroxy-5β-androstan-17-one (M2b), 3β,4α-dihydroxy-5α-androstan-17-one (M2c), androst-4-ene-3α,4,17β-triol (M3a), androst-4-ene-3β,4,17β-triol (M3b), and 5β-androstane-3β,4β,17β-triol (M4) were identified. For the in vivo studies, two thoroughbred geldings were each administered with 800 mg of formestane (32 capsules of Formadex) by stomach tubing. The results revealed that the parent drug and seven metabolites were detected in post-administration urine. The six in vitro metabolites (M1, M2a, M2b, M2c, M3a, and M3b) identified earlier were all detected in post-administration urine samples. In addition, 3α,4α-dihydroxy-5α-androstan-17-one (M2d), a stereoisomer of M2a/M2b/M2c, was also identified. This study has shown that the detection of formestane administration would be best achieved by monitoring 4-hydroxytestosterone (M1) in the glucuronide-conjugated fraction. M1 could be detected for up to 34 h post-administration. In blood samples, the parent drug could be detected for up to 34 h post administration., (Copyright © 2013 John Wiley & Sons, Ltd.)

This study describes an on-line stacking CE approach by sweeping with whole capillary sample filling for analyzing five anabolic androgenic steroids in urine samples. The five anabolic steroids for detection were androstenedione, testosterone, epitestosterone, boldenone, and clostebol. Anabolic androgenic steroids are abused in sport doping because they can promote muscle growth. Therefore, a sensitive detection method is imperatively required for monitoring the urine samples of athletes. In this research, an interesting and reliable stacking capillary electrophoresis method was established for analysis of anabolic steroids in urine. After liquid-liquid extraction by n-hexane, the supernatant was dried and reconstituted with 30 mM phosphate buffer (pH 5.00) and loaded into the capillary by hydrodynamic injection (10 psi, 99.9 s). The stacking and separation were simultaneously accomplished at -20 kV in phosphate buffer (30 mM, pH 5.0) containing 100 mM sodium dodecyl sulfate and 40 % methanol. During the method validation, calibration curves were linear (r≥0.990) over a range of 50-1,000 ng/mL for the five analytes. In the evaluation of precision and accuracy for this method, the absolute values of the RSD and the RE in the intra-day (n=3) and inter-day (n=5) analyses were all less than 6.6 %. The limit of detection for the five analytes was 30 ng/mL (S/N=5, sampling 99.9 s at 10 psi). Compared with simple MECK, this stacking method possessed a 108- to 175-fold increase in sensitivity. This simple and sensitive stacking method could be used as a powerful tool for monitoring the illegal use of doping.

The aromatase inhibitor formestane (4-hydroxy-androst-4-ene-3,17-dione, F) is prohibited in sports by the World Anti-Doping Agency (WADA). F possesses only weak androgenic properties and is presumed to be employed in order to suppress estrogen production during the illicit intake of anabolic steroids by athletes. Former studies additionally showed that F is an endogenous steroid produced in low amounts. According to the regulations of WADA, urinary concentrations above 100 ng/ml are assumed to be due to ingestion of F. To distinguish between endogenous or exogenous sources of urinary F, isotope ratio mass spectrometry (IRMS) is the method of choice. Therefore, a method to determine the carbon isotope ratio (CIR) of F in urine samples was developed and validated. Routine samples (n = 42) showing concentrations of F above 5 ng/ml were investigated and enabled elucidation of the CIR of endogenous F and subsequent the calculation of a reference limit. A reference population encompassing n = 90 males and females was investigated regarding endogenous concentrations of F. An excretion study with one male volunteer was conducted to test and validate the developed method and to identify possible impact of F administration on other endogenous steroids. By CIR determination of F it is clearly possible to elucidate its endogenous or exogenous source. Taking into account the CIR of other target analytes like testosterone, a differentiation between F and androstenedione intake is possible. In 2011, the first exogenous F below the WADA threshold could be detected by means of the developed IRMS method., (Copyright © 2012 John Wiley & Sons, Ltd.)

19-Norandrostenedione (NAED) and nandrolone are anabolic-androgenic steroids (AASs). Nandrolone was regarded solely as a synthetic AAS until the 1980s when trace concentrations of apparently endogenous nandrolone were detected in urine samples obtained from intact male horses (stallions). Since then, its endogenous origin has been reported in boars and bulls; endogenous NAED and nandrolone have been identified in plasma and urine samples collected from stallions. More recently, however, it was suggested that NAED and nandrolone detected in urine samples from stallions are primarily artifacts due to the analytical procedure. The present study was undertaken to determine whether NAED and nandrolone detected in plasma and urine samples collected from stallions are truly endogenous or artifacts from sample processing. To answer this question, fresh plasma and urine samples from ≥8 stallions were analyzed for the two AASs, soon after collection, by liquid chromatography hyphenated to tandem mass spectrometry (LC-MS/MS). NAED and nandrolone were not detected in fresh plasma samples but detected in the same samples post storage. Concentrations of both AASs increased with storage time, and the increases were greater at a higher storage temperature (37°C versus 4°C, and ambient temperature versus 4°C). Although NAED was detected in some fresh stallion urine samples, its concentration (<407 pg/mL) was far lower (<0.4%) than that in the same samples post storage (at ambient temperature for 15 days). Nandrolone was not detected in most of fresh urine samples but detected in the same samples post storage. Based on these results, it is concluded that all NAED and nandrolone detected in stored plasma samples of stallions and most of them in the stored urine samples are not from endogenous origins but spontaneously generated during sample storage, most likely from spontaneous decarboxylation of androstenedione-19-oic acid and testosterone-19-oic acid. To our knowledge, it is the first time that all NAED and nandrolone detected in plasma of stallions and most of them detected in the urine have been shown to be spontaneously generated in vitro during sample storage. This finding would have significant implications with regard to the regulation of the two steroids in horse racing., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The use of anabolic steroids as growth promoters for meat-producing animals is banned within the European Union. However, screening for the illegal use of natural steroid hormones still represents a difficult challenge because of the high interindividual and physiological variability of the endogenous concentration levels in animals. In this context, the development of untargeted profiling approaches for identifying new relevant biomarkers of exposure and/or effect has been emerging for a couple of years. The present study deals with an untargeted metabolomics approach on the basis of GC-MS aiming to reveal potential biomarkers signing a fraudulent administration of 4-androstenedione (AED), an anabolic androgenic steroid chosen as template. After a sample preparation based on microextraction by packed sorbent, urinary profiles of the free and deglucurono-conjugates urinary metabolites were acquired by GC-MS in the full-scan acquisition mode. Data processing and chemometric procedures highlighted 125 ions, allowing discrimination between samples collected before and after an administration of 4-AED. After a first evaluation of the signal robustness using additional and independent non-compliant samples, 17 steroid-like metabolites were pointed out as relevant candidate biomarkers. All these metabolites were then monitored using a targeted GC-MS/MS method for an additional assessment of their capacity to be used as biomarkers. Finally, two steroids, namely 5α-androstane-3β,17α-diol and 5α-androst-2-en-17-one, were concluded to be compatible with such a definition and which could be finally usable for screening purpose of AED abuse in cattle., (Copyright © 2012 John Wiley & Sons, Ltd.)

Androstenedione (4-androstene-3,17-dione) is banned by the World Anti-Doping Agency (WADA) as an endogenous steroid. The official method to confirm androstenedione abuse is isotope ratio mass spectrometry (IRMS). According to the guidance published by WADA, atypical steroid profiles are required to trigger IRMS analysis. However, in some situations, steroid profile parameters are not effective enough to suspect the misuse of endogenous steroids. The aim of this study was to investigate the atypical steroid profile induced by androstenedione administration and the detection of androstenedione doping using IRMS. Ingestion of androstenedione resulted in changes in urinary steroid profile, including increased concentrations of androsterone (An), etiocholanolone (Etio), 5α-androstane-3α,17β-diol (5α-diol), and 5β-androstane-3α,17β-diol (5β-diol) in all of the subjects. Nevertheless, the testosterone/epitestosterone (T/E) ratio was elevated only in some of the subjects. The rapid increases in the concentrations of An and Etio, as well as in T/E ratio for some subjects could provide indicators for initiating IRMS analysis only for a short time period, 2-22h post-administration. However, IRMS could provide positive determinations for up to 55h post-administration. This study demonstrated that, 5β-diol concentration or Etio/An ratio could be utilized as useful indicators for initiating IRMS analysis during 2-36h post-administration. Lastly, Etio, with slower clearance, could be more effectively used than An for the confirmation of androstenedione doping using IRMS., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Because of their perceived and reported effects on self-image, muscle development, performance, and similar factors, anabolic-androgenic steroids (AAS) and their precursors are among the most abused substances by professional, amateur, and recreational athletes. However, AAS abuse is not limited to athletes, but is also prevalent in the workplace, especially those professions in which image, strength, and endurance are coveted attributes. The detection of many steroids in biological specimens is analogous to the detection of an abused drug such as cocaine. Identification of the parent drug or its characteristic metabolite(s) in a donor's sample with a drug screening technique and confirmation of the drug/metabolite with a suitable alternative technology provides evidence of use. These analyses and subsequent interpretive scenarios become far more complex when the ingested AAS is an endogenous compound such as dehydroepiandrosterone (DHEA), androstenedione (Adione), or dihydrotestosterone (DHT). These compounds and their metabolites are present in specimens such as urine as a course of our natural endocrine function. Therefore, it becomes much more challenging for the laboratory to establish testing and interpretative paradigms that can distinguish "normal" urinary profiles of these steroids and their metabolites from profiles indicative of exogenous use. Distinguishing "normal" from "abnormal" urine profiles is particularly challenging during screening when literally tens of steroids and their metabolites may be tested simultaneously in a single chromatographic analysis. The purpose of this paper is to review the relevant literature about DHEA, Adione, and DHT administration, detection, and interpretation specifically as it relates to changes in the urinary AAS profile that may be identified during the routine laboratory screening of donor urine specimens.

The use of anabolic agents in food producing animals is prohibited within the European Union since 1988. The illegal use of natural steroid hormones control is however still a current challenge, especially regarding the limitations of existing screening methods. In this context, the present study aimed to develop a new screening approach based on the emerging 'untargeted profiling' concept, but with a special emphasis on steroids phase II conjugated metabolites, in the scope of revealing potential biomarkers signing a fraudulent administration of 4-androstenedione. After extraction and separation of the urinary glucuronide and sulfate steroid fractions, each one was analyzed separately by UPLC-MS/MS using the precursor ion scan acquisition mode. This approach was carried out in order to monitor product ion characteristic of sulfate (m/z 97) and glucuronide (m/z 113) functional groups, and then to fish for any potential conjugated steroid leading to these ionic species after fragmentation. After statistical analysis, 86 metabolites (33 from steroid compounds and 53 from other unknown substances) were highlighted as potential biomarkers of 4-androstenedione abuse. After application of several robustness criteria, 26 metabolites (whom 5 were unambiguously structurally identified), were finally selected to build a statistical model which could be used as new diagnostic tool for screening purposes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The use of steroid hormones as growth promoters in cattle is banned within the European Union since 1988 but can still be fraudulently employed in animal breeding farms for anabolic purposes. While efficient targeted confirmatory methods have been implemented in control laboratories for many years, fast and reliable screening methods are still required, especially in the case of natural hormones abuse, but more globally for new "fishing" strategies allowing to reveal the use of even unknown anabolic agents. The development of focused profiling or untargeted metabolomic approaches is thus emerging in this context. The present study was a focused profiling study using steroids phase II metabolites, with the aim to get a better understanding of the steroid metabolism disruptions after exogenous administration of androstenedione and finally reveal potential biomarkers signing its administration. A sample preparation procedure was first developed, based on a separation of 31 glucuronide and sulphate conjugate compounds using an anion exchange SPE system. Each fraction was then analysed by UPLC-MS/MS in MRM mode showing a rapid (between 4h and 4 days after treatment) and huge excretion of several direct metabolites of androstenedione such as etiocholanolone-glucuronide or epiandrosterone-sulphate., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: To evaluate the metabolic changes in urinary steroids in pre- and post-menopausal women and men with papillary thyroid carcinoma (PTC)., Methods: Quantitative steroid profiling combined with gas chromatography-mass spectrometry was used to measure the urinary concentrations of 84 steroids in both pre- (n = 21, age: 36.95 ± 7.19 yr) and post-menopausal female (n = 19, age: 52.79 ± 7.66 yr), and male (n = 16, age: 41.88 ± 8.48 yr) patients with PTC. After comparing the quantitative data of the patients with their corresponding controls (pre-menopause women: n = 24, age: 33.21 ± 10.48 yr, post-menopause women: n = 16, age: 49.67 ± 8.94 yr, male: n = 20, age: 42.75 ± 4.22 yr), the levels of steroids in the patients were normalized to the mean concentration of the controls to exclude gender and menopausal variations., Results: Many urinary steroids were up-regulated in all PTC patients compared to the controls. Among them, the levels of three active androgens, androstenedione, androstenediol and 16α-hydroxy DHEA, were significantly higher in the pre-menopausal women and men with PTC. The corticoid levels were increased slightly in the PTC men, while progestins were not altered in the post-menopausal PTC women. Estrogens were up-regulated in all PTC patients but 2-hydroxyestrone and 2-hydroxy-17β-estradiol were remarkably changed in both pre-menopausal women and men with PTC. For both menopausal and gender differences, the 2-hydroxylation, 4-hydroxylation, 2-methoxylation, and 4-methoxylation of estrogens and 16α-hydroxylation of DHEA were differentiated between pre- and post-menopausal PTC women (P < 0.001). In particular, the metabolic ratio of 2-hydroxyestrone to 2-hydroxy-17β-estradiol, which could reveal the enzyme activity of 17β-hydroxysteroid dehydrogenase, showed gender differences in PTC patients (P < 1 × 10-7)., Conclusions: These results are expected be helpful for better understanding the pathogenic differences in PTC according to gender and menopausal conditions.

The use of steroid hormones as growth promoters in cattle has been banned within the European Union since 1988 but can still be fraudulently employed in animal breeding farms for anabolic purposes. If an efficient monitoring of synthetic compounds (screening and confirmation) is ensured today by many laboratories, pointing out suspicious samples from a natural steroids abuse remains a tricky challenge due to the difficulty to set relevant threshold levels for these endogenous compounds. The development of focused profiling or untargeted metabolomic approaches is then emerging in this context, with the objective to reveal potential biomarkers signing an exogenous administration of such natural steroids. This study aimed to assess sample preparation procedures based on microextraction and adapt them to high throughput urinary profiling or metabolomic analyses based on gas chromatography-mass spectrometry measurement. Two techniques have been tested and optimised, namely solid phase microextraction (SPME) and microextraction by packed sorbent (MEPS), using five model steroid metabolites (16α-hydroxyandrosterone, 2α-hydroxytestosterone, 11-keto,5β-androstanedione, 6α-hydroxyestradiol and 7β-hydroxypregnenolone). The considered performance criteria included not only the absolute response of the targeted compounds but also the robustness of the materials, and the global aspect of the diagnostic ion chromatograms obtained. After only five successive urinary extractions, a clear degradation of the SPME fiber was observed which led to discard this method as a relevant technique for profiling, whereas no degradation was observed on MEPS sorbent. Repeatability and recovery yields were calculated from urine samples fortified at 500 μg L⁻¹ and extracted by MEPS. They were found respectively below 11% and above 60% for all model compounds. Detection limits were in the 5-15 μg L⁻¹ range depending on the compounds, and a good linearity was observed on the 10-75 μg L⁻¹ range (R² > 0.99). This methodology was applied on urine samples collected from control versus androstenedione-treated bovines, revealing a significant concentration increase for several well-known metabolites such as etiocholanolone, 5α-androstane-3β,17α-diol, 5β-androstane-3α,17α-diol and 5-androstene-3β,17α-diol. Finally, these results allowed to confirm the suitability of the developed strategy and give to this new MEPS application a promising interest in the field of GC-MS based steroid profiling and metabolomic., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Congenital adrenal hyperplasia is a group of disorders caused by defects in the adrenal steroidogenic pathways. In its most common form, 21-hydroxylase deficiency, patients develop varying degrees of glucocorticoid and mineralocorticoid deficiency as well as androgen excess. Therapy is guided by monitoring clinical parameters as well as adrenal hormone and metabolite concentrations., Content: We review the evidence for clinical and biochemical parameters used in monitoring therapy for congenital adrenal hyperplasia. We discuss the utility of 24-h urine collections for pregnanetriol and 17-ketosteroids as well as serum measurements of 17-hydroxyprogesterone, androstenedione, and testosterone. In addition, we examine the added value of daily hormonal profiles obtained from salivary or blood-spot samples and discuss the limitations of the various assays., Summary: Clinical parameters such as growth velocity and bone age remain the gold standard for monitoring the adequacy of therapy in congenital adrenal hyperplasia. The use of 24-h urine collections for pregnanetriol and 17-ketosteroid may offer an integrated view of adrenal hormone production but target concentrations must be better defined. Random serum hormone measurements are of little value and fluctuate with time of day and timing relative to glucocorticoid administration. Assays of daily hormonal profiles from saliva or blood spots offer a more detailed assessment of therapeutic control, although salivary assays have variable quality.

The metabolism in primary human hepatocyte cultures often deviates from that in clinical studies, which in turn are hampered by ethical constraints. Here the use of urokinase-type plasminogen activator-severe combined immunodeficiency [uPA(+/+)-SCID] mice transplanted with human hepatocytes was investigated as a model for in vivo metabolic studies. The urinary excretion profile after oral administration of 4-androstene-3,17-dione (AD) in chimeric mice was investigated by using gas chromatography-mass spectrometry detection and was compared with previously reported metabolites of AD in humans and cell cultures. Chimeric mice exhibited an AD metabolic profile similar to that of humans, showing androsterone and etiocholanolone as major metabolites. Several hydroxylated steroids were detected as minor metabolites in the chimeric mice compared with hepatocyte cultures. A significant correlation between the extent of liver replacement and the relative abundances of human-type metabolites was established. The results for AD showed that humanized liver uPA-SCID mice can serve as an alternative model for in vivo metabolism studies in humans. In the future, this model could possibly be used for other steroids or pharmaceutical compounds.

Boldenone (17-hydroxy-androsta-1,4-diene-3-one, Bol) and boldione (androst-1,4-diene-3,17-dione, ADD), are currently listed as exogenous anabolic steroids by the World Anti-Doping Agency. However, it has been reported that these analytes can be produced endogenously. Interestingly, only for Bol a comment is included in the list on its potential endogenous origin. In this study, the endogenous origin of ADD in human urine was investigated, and the potential influence of phytosterol consumption was evaluated. We carried out a 5-week in vivo trial with both men (n=6) and women (n=6) and measured alpha-boldenone, beta-boldenone, boldione, androstenedione, beta-testosterone and alpha-testosterone in their urine using gas chromatography coupled to multiple mass spectrometry (GC-MS-MS). The results demonstrate that endogenous ADD is sporadically produced at concentrations ranging from 0.751 ng mL(-1) to 1.73 ng mL(-1), whereas endogenous Bol could not be proven. We also tested the effect of the daily consumption of a commercially available phytosterol-enriched yogurt drink on the presence of these analytes in human urine. Results from this study could not indicate a relation of ADD-excretion with the consumption of phytosterols at the recommended dose. The correlations between ADD and other steroids were consistently stronger for volunteers consuming phytosterols (test) than for those refraining from phytosterol consumption (control). Excretion of AED, bT and aT did not appear to be dependent on the consumption of phytosterols. This preliminary in vivo trial indicates the endogenous origin of boldione or ADD in human urine, independent on the presence of any structural related analytes such as phytosterols.

A new solid phase microextraction (SPME) method coupled with gas chromatography-mass spectrometry (GC-MS) was developed for rapid determination of four anabolic steroids such as 3alpha-hydroxy-5alpha-androstane-17-one (HA), dihydrotestosterone (DHT), androstenedione (AD) and methyltestosterone (MT) in pig urine. SPME was used to extract the four anabolic compounds directly without derivatization. The optimum SPME sampling conditions were based on the home-made carbowax-divinylbenzene (CW-DVB) fiber coating during extraction at 40 degrees C for 50 min with 0.18 g/mL NaCl solution and 750 rpm stirring speed. The linear ranges of the proposed method were in the range of 8-640 pg/mL for HA and DHT and 16-510 pg/mL for AD and MT, respectively. The detection limits (S/N=3) were from 2 to 8 pg/mL for the four anabolic steroids. This SPME method provided very high enrichment factors for the four anabolic steroids, which were 1063-fold and 965-fold for HA and DHT at the concentration of 8 pg/mL and 207-fold and 451-fold for AD and MT at the concentration of 16 pg/mL, respectively. The recoveries ranged from 71.3 to 121%, and the RSDs were lower than 12.9%. The method was sensitive and reliable for determination of trace anabolic steroids in biological samples.

Introduction: Quantities of various anabolic/androgenic steroids have been found in dietary supplements without their presence being disclosed on the label. The aim of this study was to quantify the excretion patterns of the diagnostic metabolites, 19-norandrosterone (19-NA), and 19-noretiocholanolone (19-NE) after ingestion of small doses of 19-nor-4-androstene-3,17-dione (19-norandrostenedione)., Methods: Eleven males and nine females entered the laboratory in the morning after an overnight fast. An initial urine sample was collected, and volunteers then ingested 500 mL of water containing 5 g of creatine monohydrate and 1.0, 2.5, or 5.0 microg of 19-norandrostenedione. The volume of each urine void was measured, and an aliquot was taken. Samples were analyzed for the metabolites 19-NA and 19-NE by GCMS., Results: Baseline urinary 19-NA concentrations were 0.19 +/- 0.14 ng x mL. Ingestion of the supplement resulted in peak mean urinary 19-NA concentrations of 0.68 +/- 0.36, 1.56 +/- 0.86, and 3.89 +/- 3.11 ng.mL in the 1.0-, 2.5-, or 5.0-microg trials, respectively. Under current WADA regulations, ingestion of the 1.0-microg dose produced 0 positive doping tests, 5 subjects (20%) tested positive in the 2.5-microg trial, and 15 subjects (75%) had urinary 19-NA concentrations exceeding 2 ng x mL after ingesting creatine containing 5.0 microg of the steroid. The recovery of the ingested dose was highly variable between individuals, with values ranging from 11% to 84% (mean +/- SD = 47% +/- 18%)., Conclusions: Ingestion of trace amounts of 19-norandrostenedione can result in transient elevations of urinary 19-NA and 19-NE concentrations. The addition of as little as 2.5 microg of 19-norandrostenedione to a supplement (0.00005% contamination) appears sufficient to result in a doping violation in some individuals.

Studies have shown that the administration of androstenedione (ADIONE) significantly increases the urinary ratio of testosterone glucuronide to epitestosterone glucuronide (T/E) - measured by gas chromatography/mass spectrometry (GC/MS) - in subjects with a normal ( approximately 1) or naturally high (>1) initial values. However, the urinary T/E ratio has been shown not to increase in subjects with naturally low (<1) initial values. Such cases then rely on the detection of C(6)-hydroxylated metabolites shown to be indicative of ADIONE administration. While these markers may be measured in the routine GC/MS steroid profile, their relatively low urinary excretion limits the use of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to specifically confirm ADIONE administration based on depleted (13)C content. A mass spectrometry strategy was used in this study to identify metabolites of ADIONE with the potential to provide compound-specific detection. C(4)-hydroxylation was subsequently shown to be a major metabolic pathway following ADIONE administration, thereby resulting in urinary excretion of 4-hydroxyandrostenedione (4OH-ADIONE). Complementary analysis of 4OH-ADIONE by GC/MS and GC/C/IRMS was used to confirm ADIONE administration., (Copyright 2008 Commonwealth of Australia. Published by John Wiley & Sons, Ltd.)

Efficient control of the illegal use of anabolic steroids must both take into account metabolic patterns and associated kinetics of elimination; in this context, an extensive animal experiment involving 24 calves and consisting of three administrations of 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate esters was carried out over 50 days. Urine samples were regularly collected during the experiment from all treated and non-treated calves. For sample preparation, a single step high throughput protocol based on 96-well C(18) SPE was developed and validated according to the European Decision 2002/657/EC requirements. Decision limits (CCalpha) for steroids were below 0.1 microg L(-1), except for 19-norandrosterone (CCalpha=0.7 microg L(-1)) and estrone (CCalpha=0.3 microg L(-1)). Kinetics of elimination of the administered 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate were established by monitoring 17beta-estradiol, 17alpha-estradiol, estrone and 17beta-nandrolone, 17alpha-nandrolone, 19-noretiocholanolone, 19-norandrostenedione, respectively. All animals demonstrated homogeneous patterns of elimination both from a qualitative (metabolite profile) and quantitative point of view (elimination kinetics in urine). Most abundant metabolites were 17alpha-estradiol and 17alpha-nandrolone (>20 and 2 mg L(-1), respectively after 17beta-estradiol 3-benzoate and 17beta-nandrolone laureate administration) whereas 17beta-estradiol, estrone, 17beta-nandrolone, 19-noretiocholanolone and 19-norandrostenedione were found as secondary metabolites at concentration values up to the microg L(-1) level. No significant difference was observed between male and female animals. The effect of several consecutive injections on elimination profiles was studied and revealed a tendency toward a decrease in the biotransformation of administered steroid 17beta form.

In September 2005, our laboratory detected the presence of 4-androstene-3,17-dione and androsterone in a standard steroid screen of a post-race gelding urine sample received from an overseas authority. All other urine samples from the same batch tested negative. Subsequent gas chromatography/mass spectrometry (GC/MS) confirmatory analyses, however, repeatedly failed to detect any amount of 4-androstene-3,17-dione and androsterone in the suspicious sample. On the other hand, identical results were obtained when the initial GC/MS screening method was repeated on the suspicious sample as well as on the other samples of the same batch, showing the presence of 4-androstene-3,17-dione and androsterone only in the suspicious sample. These unusual and contradictory findings between the screening and confirmatory procedures were investigated, leading to the unequivocal conclusion that the 4-androstene-3,17-dione and androsterone observed during screening were artefacts from the internal standards, [16,16,17-d3]-testosterone and [16,16,17-d3]-5alpha-androstane-3alpha,17beta-diol. The two deuterated internal standards were thought to have undergone first an enzymatic oxidation of the 17beta-hydroxyl group to a 17-keto function by the enzyme 17beta-hydroxysteroid dehydrogenase; complete deuterium-hydrogen exchange at C16 during the methanolysis deconjugation step would then produce the two artefacts. The findings from this study highlight the potential problem of using internal standards in qualitative confirmatory analyses, which may lead to undesirable false positive results.

ACE is a popular technique for evaluating association constants between drugs and proteins. However, ACE has not previously been applied to study the association between electrically neutral biomolecules and plasma proteins. We studied the affinity between human and bovine serum albumins (HSA and BSA, respectively) and three neutral endogenous steroid hormones (testosterone, epitestosterone and androstenedione) and two synthetic analogues (methyltestosterone and fluoxymesterone) by applying the partial-filling technique in ACE (PF-ACE). From the endocrinological point of view, the distribution of endogenous steroids among plasma components is of great interest. Strong interactions with albumins suppress the biological activity of steroids. Notable differences in the association constants were observed. In the case of the endogenous steroids, the interactions between testosterone and the albumins were strongest, and those between androstenedione and the albumins were substantially weaker. The association constants, K(b), for testosterone, epitestosterone and androstenedione and HSA at 37 degrees C were 32 100 +/- 3600, 21 600 +/- 1500 and 13 300 +/- 1300 M(-1), respectively, while the corresponding values for the steroids and BSA were 18 800 +/- 1500, 14 000 +/- 400 and 7800 +/- 900 M(-1). Methyltestosterone was bound even more strongly than testosterone, while fluoxymesterone was only weakly bound by the albumins. Finally, the steroids were separated by PF-ACE with HSA and BSA used as resolving components.

4-Hydroxyandrost-4-ene-3,17-dione is a second generation, irreversible aromatase inhibitor and commonly used as anti breast cancer medication for postmenopausal women. 4-Hydroxytestosterone is advertised as anabolic steroid and does not have any therapeutic indication. Both substances are prohibited in sports by the World Anti-Doping Agency, and, due to a considerable increase of structurally related steroids with anabolic effects offered via the internet, the metabolism of two representative candidates was investigated. Excretion studies were conducted with oral applications of 100mg of 4-hydroxyandrostenedione or 200mg of 4-hydroxytestosterone to healthy male volunteers. Urine samples were analyzed for metabolic products using conventional gas chromatography-mass spectrometry approaches, and the identification of urinary metabolites was based on reference substances, which were synthesized and structurally characterized by nuclear magnetic resonance spectroscopy and high resolution/high accuracy mass spectrometry. Identified phase-I as well as phase-II metabolites were identical for both substances. Regarding phase-I metabolism 4-hydroxyandrostenedione (1) and its reduction products 3beta-hydroxy-5alpha-androstane-4,17-dione (2) and 3alpha-hydroxy-5beta-androstane-4,17-dione (3) were detected. Further reductive conversion led to all possible isomers of 3xi,4xi-dihydroxy-5xi-androstan-17-one (4, 6-11) except 3alpha,4alpha-dihydroxy-5beta-androstan-17-one (5). Out of the 17beta-hydroxylated analogs 4-hydroxytestosterone (18), 3beta,17beta-dihydroxy-5alpha-androstan-4-one (19), 3alpha,17beta-dihydroxy-5beta-androstan-4-one (20), 5alpha-androstane-3beta,4beta,17beta-triol (21), 5alpha-androstane-3alpha,4beta,17beta-triol (26) and 5alpha-androstane-3alpha,4alpha,17beta-triol (28) were identified in the post administration urine specimens. Furthermore 4-hydroxyandrosta-4,6-diene-3,17-dione (29) and 4-hydroxyandrosta-1,4-diene-3,17-dione (30) were determined as oxidation products. Conjugation was diverse and included glucuronidation and sulfatation.

Separation of anabolic and androgenic steroids by micellar electrokinetic chromatography (MEKC) has been little studied. Simultaneous separation of the endogenous alpha-epimers testosterone and epitestosterone has not been achieved with any electroseparation technique. Here, a partial filling micellar electrokinetic chromatographic (PF-MEKC) method is described for the analysis of three endogenous steroid hormones (androstenedione, testosterone, epitestosterone) and two synthetic anabolic steroids (fluoxymesterone, methyltestosterone). The resolution efficiency of single-isomer sulphated gamma-cyclodextrins and the surfactants sodium dodecyl sulphate and sodium taurocholate was exploited. The method is based on the sequential introduction of short plugs of two different pseudostationary phases into the capillary. The separation was completed in less than 10 min. The method can be used in quantitative analysis. Linear correlation was obtained between concentration and peak area of 0.996 or better. The repeatability (RSD) of the compound peak areas ranged from 3.6% (methyltestosterone) to 6.2% (androstenedione). Limits of detection were between 73 microg/L (testosterone) and 160 microg/L (fluoxymesterone). As a demonstration of the method, androstenedione, testosterone and epitestosterone were determined in a spiked urine sample.

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 17alpha- and 17beta-boldenone (alpha and beta BOL); 1,4-androstadiene-3,17-dione (ADD); 4-androstene-3,17-dione (AED), testosterone (T) and epitestosterone (ET). Urine contained no alpha BOL, beta 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, alpha BOL, ADD, AED and ET levels were extremely high (much higher than accounted for by increases due to drying), and beta BOL and T were absent. It is concluded that de novo synthesis of alpha BOL and metabolites occurs naturally in bovine faeces and only uncontaminated urine should be analysed for illegal boldenone.

Unlabelled: Adrenocortical tumours (ACT) are a rare but important cause of virilisation in infancy and childhood. Four cases of virilising ACT are presented. Two girls (age 0.9 years and 3.9 years) and two boys (age 6.2 years and 6.4 years) had symptoms and signs of virilisation before the age of 6 years. Diagnosis of a virilising adrenal tumour was confirmed by laboratory tests, diagnostic imaging and histology. However, one female patient was misdiagnosed and treated for 3 months as atypical congenital adrenal hyperplasia. Ultrasonography of the adrenal region could not visualise the tumour in three out of four cases. The most sensitive method of diagnostic imaging was MRI. In all cases, treatment consisted of complete surgical resection of the adrenal tumour by open abdominal surgery. Immunohistochemistry was performed in all patients and in two patients there was an overexpression of p53, indicating p53 mutation and in three cases the ki67 proliferation index was greater than 5%. The classification of ACT in childhood is extremely difficult. Histology scores adapted from adrenal tumours in adults and molecular markers are under investigation, but there is still not enough clinical experience since ACT are so rare., Conclusion: Long-term follow-up is mandatory not only because of the uncertainty in classification of adrenocortical tumours, but also for observation of growth and pubertal development.

Progesterone (P) is a strong mineralocorticoid receptor (MR) antagonist in vitro. The high P concentrations seen in normal pregnancy only moderately increase renin and aldosterone concentrations. In previous in vitro studies we hypothesized that this may be explained by intrarenal conversion of P to less potent metabolites. To investigate the in vivo anti-MR potency of P, we performed an infusion study in patients with adrenal insufficiency (n = 8). They omitted 9alpha-fluorocortisol for 4 d and hydrocortisone for 0.5 d before a continuous iv infusion of aldosterone for 8.5 h, with an additional iv P infusion commenced at 4 h. During aldosterone infusions the initially elevated urinary sodium to potassium ratio decreased significantly. Despite the 1000-fold excess of P over aldosterone, the urinary sodium to potassium ratio and urinary sodium excretion increased only slightly after 3 h of P infusion. We detected inhibition of renal 11beta-hydroxysteroid dehydrogenase type 2 by P, thus giving cortisol/prednisolone access to the MR. Urinary and plasma concentrations of 17alpha-hydroxyprogesterone, a major metabolite of renal P metabolism, and those of serum androstenedione and deoxycorticosterone, a mineralocorticoid itself, increased significantly during P infusion. This supports the hypothesis of an effective protection of the MR from P by efficient extraadrenal downstream conversion of P.

Four studies were performed to test the hypothesis that gonadotrophic hormones, and particularly luteinizing hormone (LH) play a role in the pathogenesis of ferrets: (I) adrenal glands of ferrets with hyperadrenocorticism were studied immunohistochemically to detect LH-receptors (LH-R); (II) gonadotrophin-releasing hormone (GnRH) stimulation tests were performed in 10 neutered ferrets, with measurement of androstenedione, 17alpha-hydroxyprogesterone and cortisol as endpoints; (III) GnRH stimulation tests were performed in 15 ferrets of which 8 had hyperadrenocorticism, via puncture of the vena cava under anesthesia; and (IV) urinary corticoid/creatinine (C/C) ratios were measured at 2-week intervals for 1 year in the same ferrets as used in study II. Clear cells in hyperplastic or neoplastic adrenal glands of hyperadrenocorticoid ferrets stained positive with the LH-R antibody. Plasma androstenedione and 17alpha-hydroxyprogesterone concentrations increased after stimulation with GnRH in 7 out of 8 hyperadrenocorticoid ferrets but in only 1 out of 7 healthy ferrets. Hyperadrenocorticoid ferrets had elevated urinary C/C ratios during the breeding season. The observations support the hypothesis that gonadotrophic hormones play a role in the pathogenesis of hyperadrenocorticism in ferrets. This condition may be defined as a disease resulting from the expression of LH-R on sex steroid-producing adrenocortical cells.

Androstenedione is a steroid hormone sold over-the-counter to individuals who expect that it will enhance strength and athletic performance. Endogenous androstenedione is the immediate precursor of testosterone. To evaluate the metabolism of oral androstenedione, we randomly assigned 37 healthy men to receive 0 (group 1), 100 mg (group 2), or 300 mg (group 3) of androstenedione in a single daily dose for 7 days. Eight-hour urines were collected 1 day before the start of androstenedione, and on days 1 and 7. Using gas chromatography-mass spectrometry, we measured excretion rates of glucuronide-conjugated epitestosterone, its putative precursor (E-precursor), and metabolites (EM-1 and EM-2), and we evaluated possible markers of androstenedione administration. Day 1 and 7 rates were not different: the means were averaged. The means (microg/h) for groups 1, 2, and 3, respectively were, for epitestosterone 2.27, 7.74, and 18.0; for E-precursor, 2.9, 2.0, and 1.5; for EM-1/E-precursor 0.31, 1.25, and 2.88; for EM-2/E-precursor 0.14, 0.15, and 1.15; for testosterone/epitestosterone (T/E) 1.1, 3.5, and 3.2. Epitestosterone, EM-1, and EM-2 excretion was greater in groups 2 and 3 versus group 1 (0.0001 < P < 0.03), as were EM-1/E-precursor, EM-2/E-precursor, and T/E. E-precursor excretion was lower in groups 2 (P = 0.08) and 3 (P = 0.047) versus group 1. Androstenedione increases excretion of epitestosterone and its two metabolites, while decreasing that of its precursor. Elevated ratios of EM-1- and EM-2/E-precursor, and the presence of 6alpha-hydroxyandrostenedione are androstenedione administration markers.

The effect of intensive exercise training on urinary androgen and cortisol excretions in prepubertal girls of various ages was tested in competitive premenarchal gymnasts ( n=56) and in age-matched controls ( n=53). Both sexual maturation and bone age were assessed. Urinary excretions of androstenedione, dehydroepiandrosterone sulphate (DHEA-S), testosterone and cortisol were determined by radioimmunoassay procedures. The gymnasts were taller [mean (SD) 138.3 (9.0) cm vs 144.7 (8.3) cm; P<0.001] and lighter [32.0 (6.9) kg vs 36.7 (8.2) kg; P<0.001], and had a lower percentage of fat mass [16.8 (2.2)% vs 22.5 (6.6)%; P<0.0001] than the controls. There was no difference between groups in urinary testosterone, cortisol and DHEA-S levels, while androstenedione levels were significantly lower in gymnasts than in controls [mean (SEM) 187.10 (18.00) nmol/24 h vs 256.50 (15.00) nmol/24 h; P<0.01). There was no difference in the regression slopes between the two groups for all hormonal values. In conclusion, these results suggest that during the prepubertal period, intensive training alters only urinary androstenedione concentrations in girls.

The dietary supplements 19-norandrostenedione and 19-norandrostenediol are potential metabolic precursors of nandrolone. They are considered by law in the United States as prohormones without proven therapeutic, curative or diagnostic properties, and therefore available as over-the-counter drugs. Oral dosages of 0.1-1 mg/kg body weight were readily absorbed in the equine intestinal tract and thereby led to urinary excretion of drastically increased 5alpha-estrane-3beta,17alpha-diol conjugates, which are known to be final metabolites of nandrolone. The actual rules for detection of illicit nandrolone administration to the horse have been found applicable for the detection of surreptitious oral 19-norandrostenedione and 19-norandrostenediol supplementation. Secondary markers of these administrations were high-level excretions of conjugated nandrolone, epinandrolone, 19-noretiocholanolone and 19-norepiandrosterone. No significant increase of circulating, biologically active nandrolone could be firmly evidenced, and it is therefore unclear to what extent continuous long-term administrations may have anabolic action.

Dehydroepiandrosterone (DHEA) and androstenedione are weak androgens, which need conversion to more potent testosterone in order to enhance anabolic action. Consequences of oral dosing at 1 mg/kg on the urinary and plasma androgen profile of mare and gelding have been evaluated with an analytical method involving conjugate fractionation and selective hydrolysis, group separation, and quantitation by gas chromatography-mass spectrometry with selected ion monitoring of trimethylsilyl ethers. Peak levels of testosterone total conjugates in urine (range 300-6000 microg/L) were attained a few hours after dosing. Renal clearance was fast, so the testosterone detection period lasted only 20 to 33 h, the longest time being generated by androstenedione. The urinary testosterone/epitestosterone ratio for detection of exogenous testosterone in the mare was inoperative after DHEA administration because there was a concomitant increase of epitestosterone, which thereby acted as a masking agent. Androstanediols and androstenediols, as well as some 17-ketosteroids, were additional markers. A transient increase of circulating free testosterone has been evidenced, and this would support possible anabolic/androgenic action by supplementation with DHEA and androstenedione along the oral route.

Androstenedione is a steroid hormone and the major precursor to testosterone. It is available without prescription and taken with the expectation that it will be converted to testosterone endogenously and increase strength and athletic performance. The metabolism of orally administered testosterone has not been well studied. We randomly assigned 37 healthy men to receive 0, 100, or 300 mg oral androstenedione in a single daily dose for 7 d. Single 8-h urine collections were performed on the day before the start of the androstenedione administration and on d 1 and 7 to assess excretion rates of free and glucuronide- conjugated testosterone, androsterone, etiocholanolone, and dihydrotestosterone. Serum testosterone glucuronide concentrations were measured by frequent blood sampling over 8 h on d 1 in 16 subjects (5 each in the 0 and 100 mg group and 6 in the 300 mg group). In the control group, mean (+/-SE) d 1 and 7 excretion rates for testosterone, androsterone, etiocholanolone, and dihydrotestosterone were 3 +/- 1, 215 +/- 26, 175 +/- 26, and 0.4 +/- 0.1 microg/h, respectively. In the 100 mg group, mean d 1 and 7 excretion rates for testosterone, androsterone, etiocholanolone, and dihydrotestosterone were 47 +/- 11, 3,836 +/- 458, 4,306 +/- 458, and 1.6 +/- 0.2 microg/h, respectively. In the 300 mg group, mean d 1 and 7 excretion rates for testosterone, androsterone, etiocholanolone, and dihydrotestosterone were 115 +/- 39, 8,142 +/- 1,362, 10,070 +/- 1,999, and 7.7 +/- 1.5 microg/h, respectively. Urinary excretion rates of all metabolites were greater in both the 100 and 300 mg groups than in controls (P < 0.0001). Urinary excretion rates of testosterone (P = 0.007), androsterone (P = 0.009), etiocholanolone (P = 0.0005), and dihydrotestosterone (P < 0.0001) were greater in the subjects who received 300 mg androstenedione than in those who received 100 mg. In the treated groups, excretion of free testosterone accounted for less than 0.1% of the total excreted testosterone measured. Serum testosterone glucuronide levels increased significantly during frequent blood sampling in both the 100 and 300 mg groups compared with controls (P = 0.0005 for the 100 mg group; P < 0.0001 for the 300 mg group). The net mean changes in area under the curve for serum testosterone glucuronide were -18 +/- 25%, 579 +/- 572%, and 1267 +/- 1675% in the groups receiving 0, 100, and 300 mg/d androstenedione, respectively. We conclude that the administration of both 100 and 300 mg androstenedione increases the excretion rates of conjugated testosterone, androsterone, etiocholanolone, and dihydrotestosterone and the serum levels of testosterone glucuronide in men. The magnitude of these increases is much greater than the changes observed in serum total testosterone concentrations. These findings demonstrate that orally administered androstenedione is largely metabolized to testosterone glucuronide and other androgen metabolites before release into the general circulation.

Formestane (Lentaron(R), 4-hydroxyandrostenedione) is a steroidal aromatase inhibitor used for treatment of advanced breast cancer. Clinically, it is administered as a depot form once fortnightly by intramuscular (i.m.) injection. To investigate the pharmacokinetics, bioavailability and metabolism of the drug, seven patients received single 250 mg i.m. doses of commercial formestane on Days 0, 21, 35, 49 and 63 of this trial. On Day 63, three of the patients received an additional single intravenous (i.v.) pulse dose of 1 mg of 14C-labelled formestane. The plasma kinetics after i.m. dosing confirmed a sustained release of formestane from the site of injection. Within 24-48 h of the first dose, the circulating drug reached a C(max) of 48.0+/-20.9 nmol/l (mean+/-S.D.; N=7). At the end of the dosing interval, after 14 days, the plasma concentration was still at 2.3+/-1.8 nmol/l. The kinetic variables did not significantly change during prolonged treatment. Intramuscular doses appear to be fully bioavailable. Following i.v. injection of 14C-formestane, the unchanged drug disappeared rapidly from plasma, the terminal elimination half-life being 18+/-2 min (N=3). Plasma clearance, CL was 4.2+/-1.3 l/(h kg) and the terminal distribution volume V(z) was 1.8+/-0.5 l/kg. The drug is mainly eliminated by metabolism, renal excretion of metabolites accounting for 95% of dose. The excretory balance of 14C-compounds in urine and faeces totals up to 98.9+/-0.8% of the i.v. dose after 168 h. The 14C-compounds in plasma and urine were separated by HPLC, and three major metabolites were submitted to structural analysis by MS, NMR and UV spectroscopy. One of the metabolites is the direct 4-O-glucuronide of formestane. The other two represent 3-O-sulfates of the exocons 3beta,4beta-dihydroxy-5alpha-androstane-17-one and 3alpha,4beta-dihydroxy-5alpha-androstane-17-one, their ratio being 7:3. These exocons are formed by stereoselective 3-keto reduction, accompanied by reduction of the 4,5-enol function. The exocons do not inhibit human placental aromatase activity in vitro.

The aim of this study was to investigate the effect of intensive exercise on bone turnover (as reflected by bone resorption) in young elite female gymnasts. Forty-five healthy girls including 24 gymnasts (11.9+/-2 yr) and 21 controls (12.3+/-1.4 yr) were studied. Body weight, height, bone age and body composition were measured. Bone mineral density (BMD) was assessed at the whole body, lumbar vertebrae, hip and radius by means of DXA. Volumetric density (BMAD) was calculated. Bone velocity (SOS) and attenuation (BUA) were measured by QUS at the calcaneus. Urinary androstenedione (delta4), dehydroepiandrosterone sulfate (DHEA-S), and CrossLaps (CTx) were measured. BMD and BMAD were significantly greater in the gymnasts at all sites except whole body. SOS was found significantly higher. Delta4 values were significantly lower in the gymnasts. The distribution of the subjects according to Tanner stages was not different between groups. CTx levels were significantly higher in the gymnasts (989.08+/-154.63 microg/mmol Cr.) vs controls (580.25+/-123.99 microg/mmol Cr., p=0.02). CTx values decreased from Tanner stage 1 to stage 4 in each group, the gymnasts' levels always being higher than those of the controls. In conclusion, gymnastics seems to stimulate bone resorption activity in highly-trained young females. The coexistence of bone hyperresorption and higher BMD in gymnasts suggests increased bone turnover resulting in increased bone density in these subjects.

Examining gonadal function in the small excitable cotton-top tamarin monkey (Saguinus oedipus) requires noninvasive sampling techniques. Two studies were performed to identify the quantifiable urinary metabolites of testosterone in cotton-top tamarins and which of the measurable metabolites would best reflect a gonadal source of testosterone secretion. In the first study, we injected unlabeled testosterone i.m. in males at either 500-ng or 1-microg levels. Urine samples were analyzed for androgens and estrogens. Testosterone and dihydrotestosterone (DHT) increased significantly following the injections in test males but not in control males. No significant increases in androstenedione occurred. Mean levels of estradiol and estrone did not consistently increase during the 5 days following injection. In the second study, a gonadotropin-releasing hormone antagonist, Antide, was used to block LH stimulation of gonadal steroidogenesis. Males given Antide at either a 6 mg/kg dose or an 18 mg/kg dose showed significantly lower levels of urinary LH than controls. At the higher Antide dose, testosterone levels were significantly reduced during weeks 1 and 2 posttreatment, whereas DHT levels significantly declined during the 2nd week posttreatment. Estradiol levels were highly variable prior to treatment but decreased significantly following treatment, whereas estrone levels remained variable throughout. These results indicate that measurement of urinary testosterone and possibly DHT reflect gonadal function in male cotton-top tamarins. Other sources of urinary estrogens may occur for the male cotton-top tamarin, but these data suggest that a substantial part of urinary estradiol is from gonadal sources, whereas urinary estrone appears to be mainly from extragonadal sources.

Since the appearance of 4-androsten-3,17-dione (I) as a nutritional supplement in early 1997, we have frequently observed a characteristic deterioration of endogenous steroid profiles in athletes' urine in routine anabolic steroid testing in which concentrations of major endogenous urinary steroids and testosterone exceed normal. Human excretion studies are performed with I and newer, over-the-counter "supplements" 4-androsten-3beta,17beta-diol (II) and 19-nor-4-androsten-3,17-dione (III). Endogenous urinary steroids affected by I and II are androsterone, etiocholanolone, their hydroxylated derivatives 5alpha- and 5beta-androstan-3alpha,17beta-diols, testosterone, and epitestosterone. Their concentrations briefly increase by one to two orders of magnitude and return to normal 24 h after oral administration of I and II. The average male may test positive for testosterone because testosterone concentration rises faster than that of epitestosterone, causing the testosterone/epitestosterone (T/E) ratio to rise above the positive cutoff of 6:1. A remarkable distinction in excretion patterns was observed in eastern Asian men, for whom I and II did not affect urinary concentrations of testosterone and did not increase the T/E ratio. First-pass metabolism deactivates most of the orally administered drugs I and II, rapidly converting them into inactive androsterone and etiocholanolone. Drug II is a more effective testosterone booster because of its different metabolic pathway. After the use of III, a precursor of the potent anabolic nandrolone, high concentrations of norandrosterone and noretiocholanolone appear in urine, similar to nandrolone. These are detectable in urine for 7-10 days after a single oral dose of III (50 mg).

Objective: The mechanism of adrenarche is controversial, and there have been competing claims that its origin is in the hypothalamo-pituitary axis or in the adrenal gland itself. ACTH is proposed inducer of adrenarche so patients with ACTH resistance due to be familial glucocorticoid deficiency syndrome provide a model to clarify the degree to which ACTH is involved in the regulation of adrenal androgen secretion during adrenarche., Design: Random analysis of plasma adrenal androgens and urinary adrenal androgen output in treated patients with familial glucocorticoid deficiency., Patients: Eleven patients (6 males and 5 females, aged 6.5-21.6 years, 4 prepubertal, minimum bone age 9 years) with familial glucocorticoid deficiency were studied. In 6, mutations, in the coding region of the ACTH receptor are the cause of their ACTH resistance. In the remaining 5 the receptor was normal., Measurements: Physical examination, basal serum cortisol, basal plasma ACTH, serum cortisol after stimulation with 250 micrograms ACTH(1-24), plasma dehydroepiandrosterone-sulphate (DHEAS) and plasma androstenedione (A4), total urinary androgen excretion and single-stranded sequencing of the coding region of the ACTH receptor., Results: DHEAS was undetectable in 8, and detectable but below the age-matched reference values in 3 patients. A4 was measured in 10 patients, and in 3 patients (2 in late puberty) was found to be subnormal whereas in the remaining 7 patients A4 was normal for age and pubertal stage consistent with the gonadal contribution to peripheral A4 levels. Significantly diminished output of urinary adrenal androgen metabolites in 3 patients confirmed the results found in serum. The lack of adrenarche was independent of the presence of a mutation within the ACTH receptor and of the severity of glucocorticoid deficiency. Despite adequate glucocorticoid replacement therapy ACTH levels remained elevated in 10 of the 11 patients., Conclusions: Although reduced adrenocortical inner zone cell number may contribute to the lack of adrenarche, in some patients there appears to be a discrepancy between partial glucocorticoid deficiency and significantly diminished adrenal androgen secretion. These data imply a significant contribution of ACTH to the regulation of adrenarche in normal children either by having a priming effect on the adrenal gland or by acting in concert with other adrenal androgen stimulating factors.