Longitudinal evaluation of multiple biomarkers for the detection of testosterone gel administration in women with normal menstrual cycle

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-day 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.     

Effects of the administration of miconazole by different routes on the biomarkers of the “steroidal module” of the Athlete Biological Passport

This article reports the results obtained from the investigation of the influence of miconazole administration on the physiological fluctuation of the markers of the steroid profile included in the “steroidal module” of the Athlete Biological Passport. Urines collected from male Caucasian subjects before, during, and after either systemic (i.e., oral and buccal) or topical (i.e., dermal) treatment with miconazole were analyzed according to validated procedures based on gas chromatography coupled to tandem mass spectrometry (GC–MS/MS) (to determine the markers of the steroid profile) or liquid chromatography coupled to MS/MS (LC–MS/MS) (to determine miconazole urinary levels). The results indicate that only after systemic administration, the markers of the steroid profile were significantly altered. After oral and buccal administration, we have registered (i) a significant increase of the 5α-androstane-3α,17β-diol/5β-androstane-3α,17β-diol ratio and (ii) a significant decrease of the concentration of androsterone, etiocholanolone, 5β-androstane-3α,17β-diol, and 5α-androstane-3α,17β-diol and of the androsterone/etiocholanolone, androsterone/testosterone, and 5α-androstane-3α,17β-diol/epitestosterone ratios. Limited effects were instead measured after dermal intake. Indeed, the levels of miconazole after systemic administration were in the range of 0.1–12.5 μg/ml, whereas after dermal administration were below the limit of quantification (50 ng/ml). Significant alteration started to be registered at concentrations of miconazole higher than 0.5 μg/ml. These findings were primarily explained by the ability of miconazole in altering the kinetic/efficacy of deglucuronidation of the endogenous steroids by the enzyme β-glucuronidase during the sample preparation process. The increase of both incubation time and amount of β-glucuronidase was demonstrated to be effective countermeasures in the presence of miconazole to reduce the risk of uncorrected interpretation of the results.     

Detection of dihydrotestosterone gel, oral dehydroepiandrosterone, and testosterone gel misuse through the quantification of testosterone metabolites released after alkaline treatment

The natural occurrence of endogenous anabolic steroids together with their availability in different administration forms makes the detection of their misuse a great challenge for doping control laboratories. Nowadays, the detection of endogenous steroids abuse is performed by the analysis of the steroid profile. Recently, androst-1,4-dien-3,17-dione (1,4-AD), androst-4,6-dien-3,17-dione (4,6-AD), 17β-hydroxy-androst-4,6-dien-3-one (6-T), and androst-15-en-3,17-dione (15-AD) have been described as testosterone (T) metabolites released after basic treatment of the urine. In the present work, the usefulness of these metabolites has been evaluated detecting the use of three different forms of endogenous steroids in a single dose: dihydrotestosterone gel (DHT), oral dehydroepiandrosterone (DHEA), and T gel. After the independent administration of these endogenous steroids, a rise in the value of several of the ratios calculated between the tested metabolites was noticed. For DHT, a small increase was observed for the ratios 1,4-AD/15-AD, 6-T/15-AD and 4,6-AD/15-AD although only for one volunteer. Better results were obtained for oral DHEA and T gel where an increase was observed in all volunteers for several of the tested ratios. The detection time in which the misuse can be detected (DT) has been evaluated using two different approaches: (1) comparison with population based reference limits, and (2) comparison with individual threshold levels. The obtained DTs were compared with the results of previously published markers for the misuse of such substances. When using basic released metabolites, shorter DTs were obtained for DHT, similar DTs for DHEA, and the detectability was substantially improved for T gel.     

Degradation of urine samples and its influence on the ¹³C/¹²C ratios of excreted steroids

The degradation processes in deficiently stored urine samples are well investigated regarding steroid concentrations and diagnostic ratios, such as the quotient of testosterone divided by epitestosterone. In contrast, nothing is known about the influence on carbon isotope ratios (CIR) by inappropriate storage conditions. In general, it is assumed that degradation, i.e. deconjugation or dehydrogenation, does not change CIR and thus CIR can be used in cases where the steroid profile turns out to be invalid. Therefore, the CIR of urinary steroids was investigated in different urine samples during the course of degradation over a time period of six months. Several steroids excreted as glucuronides (androsterone (A), etiocholanolone (E), testosterone, pregnanediol (PD) and 5α- and 5β-androstane-3α,17β-diol) or sulfo-conjugated (A, E and androst-5-ene-3β,17β-diol (5EN17b)) were investigated together with their unconjugated correspondents (A, E, PD and 5EN17b) and the main dehydrogenation products (5α- and 5β-androstane-3,17-dion and androst-4-ene-3,17-dion). For this purpose, the exiting methods for CIR determination were extended and validated. In addition, the urinary concentrations of all investigated steroids were monitored. Particular attention was paid to dehydroepiandrosterone conjugated and unconjugated together with its degradation product 3α,5-cyclo-5α-androstan-6β-ol-17-one as here the strongest influence on CIR was expected.     

Investigations on changes in ¹³C/¹²C ratios of endogenous urinary steroids after pregnenolone administration

For the detection of possible misuse of naturally occurring anabolic androgenic steroids like testosterone (T), anti-doping laboratories use a combination of two techniques. One is molecular steroid profiling to evaluate urinary steroid concentrations and normal diagnostic ratios. The other is isotope ratio mass spectrometry (IRMS), in which the 13C/12C ratios of target analytes like T are compared to the 13C/12C ratios of endogenous reference compounds (ERCs). The 13C/12C of the most commonly used ERC, pregnanediol (5β-pregnane-3α,20α-diol, PD), can be influenced by administration of pregnenolone (3β-hydroxy-pregn-5-en-20-one, PREG). Therefore PREG administration bears the potential to circumvent IRMS testing for doping control samples. In order to investigate the influence of PREG on PD and on other urinary excreted steroids, administration studies with oral and transdermal application of PREG were carried out. The influence of PREG administration on concentrations and 13C/12C ratios of all investigated target analytes was negligible. Only PD and 5β-pregnan-3α-ol-20-one (3aP) showed significant depletion in both their glucuronidated and sulfated steroids. The results suggest that appropriate alternative ERCs are: 11β-hydroxy-androsterone/etiocholanolone, 5β-pregnane-3α,17,20α-triol, pregn-5-ene-3β,17,20α-triol and cholesterol. Due to its properties to disguise the misuse of anabolic steroids by influencing the 13C/12C ratio of PD, PREG should be considered to be added to the World Anti-Doping Agency (WADA) list of prohibited substances as a masking agent.     

5α-androstane-3β, 17β-diol binds to androgen and estrogen receptors without activating copulatory behavior in female rats

The ability of the androgen metabolite 5α-androstane-3β, 17β-diol (3β-A-diol) to facilitate copulatory behavior was assessed directly in adult ovariectomized rats. Neither the highest dosage of 5 mg/day for three days, nor 2 mg/day for 15 days could induce lordosis behavior in females that displayed typically high lordosis quotients with low dosages of estradiol (E). Furthermore, prolonged administration of 5α-dihydrotesterone (DHT) induced a low but significant level of male-typical mounting behavior in females, whereas 3β-A-diol administered for 20 days (2 mg/day) had no effect on mounting behavior. However, this reduced androgen metabolite did compete moderately well for DHT and E binding sites on androgen and estrogen receptors respectively in hypothalamic cytosol preparations. We conclude that in spite of its ability to bind to these receptors in the brain 3β-A-diol, a major metabolite of DHT, is totally inert with respect to sexual behavior.     

Detection of Δ6-methyltestosterone in a "dietary supplement" and GC-MS/MS investigations on its urinary metabolism

Since a few years more and more products have appeared on the market for dietary supplements containing steroids that had never been marketed as approved drugs, mostly without proper labeling of the contents. Syntheses and few data on pharmacological effects are available dated back mainly to the 1950s or 1960s. Only little knowledge exists about effects and side effects of these steroids in humans.The present study reports the identification of Δ6-methyltestosterone in a product named “Jungle Warfare”, which was obtained from a web-based supplement store.The main urinary metabolites, 17α-hydroxy-17β-methylandrosta-4,6-dien-3-one (Δ6-epimethyl-testosterone), 17α-methyl-5β-androstane-3α,17β-diol (3α,5β-THMT), and 17β-methyl-5β-androstane-3α,17α-diol, as well as the parent compound excreted after a single oral administration were monitored by GC-MS/MS. Δ6-Epimethyltestosterone and 3α,5β-THMT served for long-term detection (still present in the 181-189 h urine). 17α-Methyltestosterone and its 17-epimer were not detected in the urines (LOD 0.3 ng/mL). The highest concentrations were found in the 14-20.5 h urine for Δ6-epimethyltestosterone (600 ng/mL), and 3α,5β-THMT (240 ng/mL) and in the 36-44.5 h urine for 17β-methyl-5β-androstane-3α,17α-diol (7 ng/mL).For reference methyltestosterone and epimethyltestosterone were dehydrogenated with chloranil. The characterization of the products was performed by GC-MS(/MS) and NMR.     

Pregnancy greatly affects the steroidal module of the Athlete Biological Passport

Concentrations of urinary steroids are measured in anti‐doping test programs to detect doping with endogenous steroids. These concentrations are combined into ratios and followed over time in the steroidal module of the Athlete Biological Passport (ABP). The most important ratio in the ABP is the testosterone/epitestosterone (T/E) ratio but this ratio is subject to intra‐individual variations, especially large in women, which complicates interpretation. In addition, there are other factors affecting T/E. Pregnancy, for example, is known to affect the urinary excretion rate of epitestosterone and hence the T/E ratio. However, the extent of this variation and how pregnancy affect other ratios has not been fully evaluated. Here we have studied the urinary steroid profile, including 19‐norandrosterone (19‐NA), in 67 pregnant women and compared to postpartum. Epitestosterone was higher and, consequently, the T/E and 5αAdiol/E ratios were lower in the pregnant women. Androsterone/etiocholanolone (A/Etio) and 5αAdiol/5βAdiol, on the other hand, were higher in the first trimester as compared to postpartum (p<0.0001 and p=0.0396, respectively). There was no difference in A/T during pregnancy or after. 19‐NA was present in 90.5% of the urine samples collected from pregnant women. In this study, we have shown that the steroid profile of the ABP is affected by pregnancy, and hence can cause atypical passport findings. These atypical findings would lead to unnecessary confirmation procedures, if the patterns of pregnancy are not recognized by the ABP management units.     

Inter‐individual variation of the urinary steroid profiles in Swedish and Norwegian athletes

The steroidal module of the Athlete Biological Passport (ABP) aims to detect doping with endogenous steroids, e.g. testosterone (T), by longitudinally monitoring several biomarkers. These biomarkers are ratios combined into urinary concentrations of testosterone and metabolically related steroids. However, it is evident after 5 years of monitoring steroid passports that there are large variations in the steroid ratios complicating its interpretation. In this study, we used over 11000 urinary steroid profiles from Swedish and Norwegian athletes to determine both the inter‐ and intra‐individual variations of all steroids and ratios in the steroidal passport. Furthermore, we investigated if the inter‐individual variations could be associated with factors such as gender, type of sport, age, time of day, time of year, and if the urine was collected in or out of competition. We show that there are factors reported in today's doping tests that significantly affect the steroid profiles. The factors with the largest influence on the steroid profile were the type of sport classification that the athlete belonged to as well as whether the urine was collected in or out of competition. There were also significant differences based on what time of day and time of year the urine sample was collected. Whether these significant changes are relevant when longitudinally monitoring athletes in the steroidal module of the ABP should be evaluated further.     

Detection of testosterone microdosing in healthy females

The ready detectability of synthetic androgens by mass spectrometry (MS)-based antidoping tests has reoriented androgen doping to using testosterone (T), which must be distinguished from its endogenous counterpart making detection of exogenous T harder. We investigated urine and serum steroid and hematological profiling individually and combined to determine the optimal detection model for T administration in women. Twelve healthy females provided six paired blood and urine samples over 2 weeks prior to treatment consisting of 12.5-mg T in a topical transdermal gel applied daily for 7 days. Paired blood and urine samples were then obtained at the end of treatment and Days 1, 2, 4, 7, and 14 days later. Compliance with treatment and sampling was high, and no adverse effects were reported. T treatment significantly increased serum and urine T, serum dihydrotestosterone (DHT), urine 5α-androstane-3α,17β-diol (5α-diol) epitestosterone (E), and urine T/E ratio with a brief window of detection (2–4 days) as well as total and immature (medium and high fluorescence) reticulocytes that remained elevated over the full 14 posttreatment days. Carbon isotope ratio MS and the OFF score and Abnormal Blood Profile score (ABPS) were not discriminatory. The optimal multivariate model to identify T exposure combined serum T, urine T/E ratio with three hematological variables (% high fluorescence reticulocytes, mean corpuscular hemoglobin, and volume) with the five variables providing 93% correct classification (4% false positive, 10% false negatives). Hence, combining select serum and urine steroid MS variables with reticulocyte measures can achieve a high but imperfect detection of T administration to healthy females.     

Variability of the urinary and blood steroid profiles in healthy and physically active women with and without oral contraception

The steroidal module of the athlete biological passport (ABP) targets the use of pseudo-endogenous androgenous anabolic steroids in elite sport by monitoring urinary steroid profiles. Urine and blood samples were collected weekly during two consecutive oral contraceptive pill (OCP) cycles in 15 physically active women to investigate the low urinary steroid concentrations and putative confounding effect of OCP. In urine, testosterone (T) and epitestosterone (E) were below the limit of quantification of 1 ng/ml in 62% of the samples. Biomarkers' variability ranged between 31% and 41%, with a significantly lesser variability for ratios (except for T/E [41%]): 20% for androsterone/etiocholanolone (p < 0.001) and 25% for 5α-androstane-3α,17β-diol/5ß-androstane-3α,17β-diol (p < 0.001). In serum, markers' variability (testosterone: 24%, androstenedione: 23%, dihydrotestosterone: 19%, and T/A4: 16%) was significantly lower than in urine (p < 0.001). Urinary A/Etio increased by >18% after the first 2 weeks (p < 0.05) following withdrawal blood loss. In contrast, serum T (0.98 nmol/l during the first week) and T/A4 (0.34 the first week) decreased significantly by more than 25% and 17% (p < 0.05), respectively, in the following weeks. Our results outline steroidal variations during the OCP cycle, highlighting exogenous hormonal preparations as confounder for steroid concentrations in blood. Low steroid levels in urine samples have a clear negative impact on the subsequent interpretation of steroid profile of the ABP. With a greater analytical sensitivity and lesser variability for steroids in healthy active women, serum represents a complementary matrix to urine in the ABP steroidal module.     

5beta-Androstane-3alpha,17beta-diol: an endogenous substrate for rabbit liver 3-hydroxyhexobarbital dehydrogenase.

Dehydrogenation of 5β-androstane-3α,17β-diol to 5β-androstan-3α-ol-17-one was found lo be catalysed by rabbit liver 3-hydroxyhexobarbital dehydrogenase. Rabbit liver cytosol contained several enzyme activities for the dehydrogenation of 5β-androstane-3α, 17β-diol. One of the activities was not separable from 3-hydroxyhexobarbital dehydrogenase in the course of purification and on polyacrylamide gel disc electrophoresis. The activity of 3-hydroxyhexobarbital dehydrogenase was inhibited competitively by 5β-androstane-3α, 17β-diol. Results of a mixed substrate method, thermal inactivation and inhibition by p-chloromercuribenzoate also supported the interpretation that a single enzyme was responsible for the dehydrogenation of 3-hydroxyhexobarbital and 5β-androstane-3α, 17β-diol. It was shown that, in the rabbit liver, 3-hydroxyhexobarbital dehydrogenase was separate from testosterone 17β-dehydrogenase (NADP) (EC 1.1.1.64) by TEAE-cellulose column chromatography, although both enzymes were found to be identical in the case of guinea-pig liver.     

5α-reductase inhibitors: Evaluation of their potential confounding effect on GC-C-IRMS doping analysis

5α-reductase inhibitors (5-ARIs) are considered by the World Anti-doping Agency as potential confounding factors in evaluating the athlete steroid profile, since they may interfere with the urinary excretion of several diagnostic compounds. We herein investigated 5α-reductase inhibitors from a different perspective, by verifying their influence on the carbon isotopic composition of 5α- and 5β-reduced testosterone and nandrolone metabolites. The GC-C-IRMS analysis was performed on a set of urine samples collected from three male Caucasian volunteers after the acute and chronic administration of finasteride in combination with the intake of 19-norandrostenedione, a nandrolone precursor. The excretion and the isotopic profile of androsterone (A), etiocholanolone (Etio) 5α-androstane-3α,17β-diol (5αAdiol), and 5β-androstane-3α,17β-diol (5βAdiol) were determined as well as those of 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE). Pregnanediol (PD) and pregnanetriol (PT) were also measured as endogenous reference compounds to define the individual endogenous isotopic profile. Our results confirmed the impact of finasteride, especially if chronically administered, on the enzymatic pathway of testosterone and nandrolone, and pointed out the influence of 5-ARIs on δ¹³C values of the selected target compounds determined in the IRMS confirmation analysis.     

Urinary steroid profile in relation to the menstrual cycle

The interpretation of the steroidal module of the Athlete Biological Passport (ABP) in female athletes is complex due to the large variation of the endogenous urinary steroids. The menstrual cycle seems to be one of the largest confounders of the steroid profile. The duration of the different phases in the menstrual cycle differs between women and is difficult to predict only by counting days after menstruation. Here, we have determined the follicle, ovulation, and luteal phases, by assessing the menstrual hormones in serum samples collected from 17 healthy women with regular menses. Urine samples were collected three times per week during two consecutive cycles to measure the urinary steroid concentrations used in the ABP. The metabolite that was mostly affected by the menstrual phases was epitestosterone (E), where the median concentration was 133% higher in the ovulation phase compared to the follicle phase (p < 0.0001). The women with a large coefficient of variation (CV) in their first cycle also had a large CV in their second cycle and vice versa. The inter-individual difference was extensive with a range of 11%–230% difference between the lowest and the highest T/E ratio during a cycle. In conclusion, E and ratios with E as denominator are problematic biomarkers for doping in female athletes. The timing of the sample collection in the menstrual cycle will have a large influence on the steroid profile. The results of this study highlight the need to find additional biomarkers for T doping in females.     

Sensitivity of doping biomarkers after administration of a single dose testosterone gel

Micro‐doping with testosterone (T) is challenging to detect with the current doping tests. Today, the methods available to detect T are longitudinally monitoring of urine biomarkers in the Athlete Biological Passport (ABP) and measuring the isotopic composition of excreted biomarkers to distinguish the origin of the molecule. In this study, we investigated the detectability of a single dose of 100 mg T gel in 8 healthy male subjects. We also studied which biomarkers were most sensitive to T gel administration, including blood biomarkers. The ABP successfully detected T gel administration in all 8 subjects. The most sensitive ratio was 5αAdiol/E, however, all ratios showed atypical findings. Isotope ratio mass spectrometry (IRMS) was performed on 5 subjects and only 2 met all the criteria for a positive test according to the rules set by the World Anti‐Doping Agency (WADA). The other 3 showed inconclusive results. Other markers that were affected by T gel administration, not used for this detection today, were serum dihydrotestosterone (DHT) and T as well as reticulocyte count and percentage in whole blood. miRNA‐122 was not significantly affected by the single T dose. A single dose of 100 mg T gel is possible to detect with today's doping tests. Since a single dose of T gel has an impact on some hematological biomarkers, access to both modules of the ABP when evaluating the athletes' profiles will increase the possibility to detect micro‐doses of T. In addition, serum DHT and T may be a useful addition to the future endocrine module of the ABP.     

Long-term stability study and evaluation of intact steroid conjugate ratios after the administration of endogenous steroids

The most frequently detected substances prohibited by the World Anti-Doping Agency (WADA) belong to the anabolic steroids class. The most challenging compounds among this class are the endogenous anabolic steroids, which are detected by quantitative measurement of testosterone (T) and its metabolites with a so-called “steroid profiling” method. The current steroid profile is based on the concentrations and ratios of the sum of free and glucuronidated steroids. Recently, our group developed a steroid profiling method for the detection of three free steroids and 14 intact steroid conjugates, including both the glucuronic acid conjugated and sulfated fraction. The study aimed at evaluating the long-term stability of steroid conjugate concentrations and ratios, and the influence of different endogenous steroids on this extended steroid profile. A single dose of oral T undecanoate (TU), topical T gel, topical dihydrotestosterone (DHT) gel, and oral dehydroepiandrosterone (DHEA) was administered to six healthy male volunteers. One additional volunteer with a homozygote deletion of the UGT2B17 gene (del/del genotype) received a single topical dose of T gel. An intramuscular dose of TU was administered to another volunteer. To avoid fluctuation of steroid concentrations caused by variations in urinary flow rates, steroid ratios were calculated and evaluated as possible biomarkers for the detection of endogenous steroid abuse with low doses. Overall, sulfates do not have substantial additional value in prolonging detection times for the investigated endogenous steroids and administration doses. The already monitored glucuronides were overall the best markers and were sufficient to detect the administered steroids.     

Effect of glucocorticoid administration on the steroid profile

The steroid profile (SP) is a powerful tool to detect the misuse of endogenous anabolic androgenic steroids in sports, and it is included in the Athlete Biological Passport (ABP). Glucocorticoids (GCs), which are widely prescribed in sports and only prohibited in competition by systemic routes, inhibit the hypothalamic‐pituitary‐adrenal axis. Since the metabolites monitored in the SP have a partial adrenal origin, their excretion in urine might be altered by GCs consumption. The aim of the present work was to investigate if GCs administered by either systemic or local routes could influence the SP parameters. Three of the most frequently detected GCs in sports (prednisolone, betamethasone, and triamcinolone acetonide) were administered to healthy male and female volunteers (n=40) using different administration routes (topical, oral, and intramuscular administration at different doses). In total, 66 administrations of GCs were performed. Urine samples were collected before and after GCs administration. The SP was measured using gas chromatography‐mass spectrometry. The excretion rates of the SP metabolites decreased after systemic GCs administration. This excretion decrease showed to be associated with the dose and the administration route. However, the individual evaluation of the SP ratios (T/E, A/T, A/Etio, 5αAdiol/5βAdiol, and 5αAdiol/E) led to normal sequences for all the conditions tested. Therefore, GCs administration did not produce misinterpretations on the ABP evaluation. According to these results, GCs administration should not distort the establishment of normal ranges of the SP ratios, and does not need to be considered a confounding factor in the SP evaluation.     

An in vitro assay approach to investigate the potential impact of different doping agents on the steroid profile

The steroid profile, that is, the urinary concentrations and concentration ratios of selected steroids, is used in sports drug testing to detect the misuse of endogenous steroids such as testosterone. Since several years, not only population-based thresholds are applied but also the steroid profile is monitored via the Athlete Biological Passport whereby the individual reference ranges derived from multiple test results of the same athlete are compared to population-based thresholds. In order to maintain a high probative force of the passport, samples collected or analyzed under suboptimal conditions should not be included in the longitudinal review. This applies to biologically affected or degraded samples and to samples excluded owing to the presence of other substances potentially (or evidently) altering the steroid profile. Nineteen different doping agents comprising anabolic steroids, selective androgen receptor modulators, selective estrogen receptor modulators, ibutamoren, and tibolone were investigated for their effect on the steroid profile using an androgen receptor activation test, an androgen receptor binding assay, an aromatase assay, and a steroidogenesis assay. The in vitro tests were coupled with well-established liquid chromatography/mass spectrometry-based analytical approaches and for a subset of steroidal analytes by gas chromatography/mass spectrometry. The variety of tests employed should produce a comprehensive data set to better understand how a compound under investigation may impact the steroid profile. Although our data set may allow an estimate of whether or not a substance will have an impact on the overall steroid metabolism, predicting which parameter in particular may be influenced remains difficult.     

Evaluation of longitudinal steroid profiling with the ADAMS adaptive model for detection of transdermal,intramuscular, and subcutaneous testosterone administration

The steroidal module of the Athlete Biological Passport (ABP) has been used since 2014 for the longitudinal monitoring of urinary testosterone and its metabolites in order to identify samples suspicious for the use of synthetic forms of endogenous anabolic androgenic steroids (EAAS). Samples identified by the module may then be confirmed by isotope ratio mass spectrometry (IRMS) to establish clearly the exogenous origin of testosterone and/or metabolites in the sample. To examine the detection capability of the steroidal ABP model, testosterone administration studies were performed with various doses and three routes of administration – transdermal, intramuscular, and subcutaneous with 15 subjects for each route of administration. Urine samples were collected before, during, and after administration and steroid profiles were analyzed using the steroidal ABP module in ADAMS. A subset of samples from each mode of administration was also analyzed by IRMS. The steroidal ABP module was more sensitive to testosterone use than population‐based thresholds and with high dose administrations there was very good agreement between the IRMS results and samples flagged by the module. However, with low dose administration the ABP module was unable to identify samples where testosterone use was still detectable by IRMS analysis. The testosterone/epitestosterone (T/E) ratio was the most diagnostic parameter for longitudinal monitoring with the exception of low testosterone excretors for whom the 5α‐androstane‐3α, 17β‐diol/epitestosterone (5αAdiol/E) ratio may provide more sensitivity.     

17α-甲基睾丸素及其某些衍生物对去势大鼠和小鼠同化作用的观察

本文用去势大鼠和小鼠研究17α-甲基睾丸素(Ⅰ)及一些衍生物的同化和雄性素样作用。同化作用指标是观察大、小鼠提肛肌和小鼠腎脏重量的增加,雄性素样作用是以精囊重量的增加来表示。結果指出,I的双氫衍生物——17α-甲基-(5α-雄甾烷-17β-羥-3-酮(Ⅱ)和17α-甲基-(5α)-雄甾烷-3β,17β-二羥(Ⅲ)的口服剂量为30毫克/公斤时,在去势大鼠的同化和雄性素样作用与I无显著不同。剂量12.5毫克/公斤肌肉注射时,則Ⅱ对去势小鼠的同化作用比I显著增强,雄性素样作用与I无显著不同;而Ⅲ的同化作用和雄性素样作用均有减弱。17α-甲基-(5α)-雄甾烷-17β-羥(Ⅳ)系I的3位脫氧衍生物,当給去势大鼠口服30毫克/公斤时,其同化作用比I显著增强;給去势小鼠肌肉注射12.5毫克/公斤时,其同化作用与I大致相同,但雄性素样作用只有I的1/3,因之,同化与雄性素样作用比值高至3.63—4.50。睾丸素17位脫氧衍生物——5α-雄甾烷-3-羥(Ⅴ)和5α-雄甾烷-3-酮(Ⅵ)的剂量为30—40毫克/公斤时,无論口服或注射,在去势大鼠和小鼠均不出現同化和雄性素样作用。以上結果表明,将化合物I 4,5位氫化和3位脫氧,可使同化作用:雄性素样作用比值明显提高,17位脫氧則失去同化和雄性素样作用。