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1.
Drug Test Anal ; 15(11-12): 1371-1381, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37749856

RESUMO

The Athlete Biological Passport (ABP) is a longitudinal tool used in anti-doping to monitor biological parameters known to change with performance-enhancing drug use. The ABP consists of multiple modules, including two aimed at detecting the use of endogenous anabolic androgenic steroids: the urinary and serum steroid modules. Human chorionic gonadotropin (hCG) is a protein hormone potentially abused by male athletes to increase the production of endogenous testosterone. To date, no studies have investigated the impact of extended hCG administration on the urinary and serum steroid modules of the ABP. The goal of this study was to identify the impact of multiple hCG administrations on the parameters tracked as part of the urinary and serum steroid modules of the ABP. Ten recreationally active, healthy male individuals self-administered seven 250 µg hCG injections over 3 weeks. Serum and urine samples were collected before, during, and 2 weeks following the final injection. All ABP parameters were quantified in the respective matrix, and steroid profiles were created with Anti-Doping Administration and Management System adaptive model upper and lower limits for both matrices. In both serum and urine profiles, testosterone increased; however, the testosterone/epitestosterone ratio in urine and the testosterone/androstenedione ratio in serum showed minimal changes. Additionally, serum luteinizing hormone (LH) was quantified using an immunoassay, and a serum testosterone/LH ratio was generated. Serum LH values decreased during administration causing large increases in the serum T/LH ratio, indicating this ratio may be a more sensitive parameter for detecting hCG abuse than urinary testosterone/epitestosterone or serum testosterone/androstenedione.


Assuntos
Doping nos Esportes , Epitestosterona , Humanos , Masculino , Epitestosterona/urina , Androstenodiona , Testosterona/urina , Atletas , Esteroides/urina , Hormônio Luteinizante/urina , Gonadotropina Coriônica/urina , Detecção do Abuso de Substâncias
2.
Drug Test Anal ; 14(10): 1795-1799, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35964965

RESUMO

INTRODUCTION: A young male was found dead on the bed of a hotel room. He was expected to take part in a bodybuilding competition the day after. During the site inspection, drugs of different types were found. The next day, an autopsy was performed. The evidence of cardiomegaly with organ congestion involving lung, liver, kidneys, adrenal glands, spleen and brain was confirmed by both the autoptic and the histopathological exam. However, the cause of death needed to be investigated. METHODS: A thorough toxicological investigation was undertaken by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-high resolution mass spectrometry (LC-HRMS) and liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) on samples of urine, blood and hair. RESULTS AND DISCUSSION: Clenbuterol, a long-acting selective beta2 agonist, was found in both blood (1 ng/ml) and urine (1 ng/ml), and evidence of its use was provided by the analysis of the 3-cm hair (25 pg/mg). The main metabolite of drostanolone (2 alpha-methyl-androsterone), an anabolic steroid, was found in the urine (202 ng/ml), where an increased ratio of testosterone/epitestosterone (T/E = 11) emerged. Due to the results of the hair analysis, a long-term use of various anabolic steroids was supposed. The integrated analysis of the results and the absence of other possible causes (such as trauma or cardiac conduction anomalies) led to the identification of the abuse of doping substances as the underlying cause of death. CONCLUSION: Hair analysis has proven to be crucial in identifying drug misuse and the contributing cause of death.


Assuntos
Anabolizantes , Clembuterol , Doping nos Esportes , Anabolizantes/urina , Androsterona , Autopsia , Cromatografia Líquida , Clembuterol/análise , Epitestosterona , Humanos , Masculino , Detecção do Abuso de Substâncias , Espectrometria de Massas em Tandem/métodos , Congêneres da Testosterona
3.
Drug Test Anal ; 14(4): 653-666, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34811948

RESUMO

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,17ß-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.


Assuntos
Doping nos Esportes , Testosterona , Androgênios/urina , Di-Hidrotestosterona , Epitestosterona/urina , Feminino , Humanos , Esteroides/urina , Testosterona/urina
4.
Drug Test Anal ; 14(5): 833-850, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-33817997

RESUMO

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,17ß-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.


Assuntos
Doping nos Esportes , Epitestosterona , Biomarcadores/urina , Di-Hidrotestosterona , Feminino , Humanos , Ciclo Menstrual , Esteroides/urina , Detecção do Abuso de Substâncias/métodos , Testosterona/urina , Congêneres da Testosterona
5.
Drug Test Anal ; 13(10): 1735-1742, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34228890

RESUMO

At the Swedish national forensic toxicology laboratory, a measured testosterone/epitestosterone (T/E) ratio ≥ 12 together with testosterone/luteinizing hormone (T/LH) in urine > 400 nmol/IU is considered as a proof of exogenous testosterone administration. However, according to the rules of the World Anti-Doping Agency (WADA), samples with T/E ratio > 4 are considered suspicious and shall be further analysed by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) to confirm the origin of testosterone and its metabolites. The aim of this study was to investigate the possibility of false negative results and to estimate the frequency of negative results using the current criteria for detection of abuse of testosterone in forensic investigations. Urine and serum samples were collected by the police at suspected infringement of the doping law in Sweden. Fifty-eight male subjects were included in the study. Urinary testosterone was determined by gas chromatography-mass spectrometry (GC-MS), serum testosterone and LH-by immunoassay. The origin of testosterone and its metabolites was confirmed by means of GC-C-IRMS. Twenty-six of the 57 analysed subjects tested positive for exogenous testosterone using the criteria T/E ≥ 12 combined with T/LH > 400 nmol/IU. The IRMS analyses confirmed 47 positives; thus, 21 were considered false negatives. Negative predictive value was 32% (95% confidence interval [CI]: 16%-50%) and sensitivity 55%. No false positive subjects were found. The number of false negative cases using the current criteria for the detection of testosterone abuse and hence the low sensitivity indicates a need to discuss introduction of new strategies in forensic doping investigations.


Assuntos
Doping nos Esportes/prevenção & controle , Epitestosterona/urina , Hormônio Luteinizante/urina , Testosterona/urina , Adulto , Epitestosterona/análise , Reações Falso-Negativas , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Hormônio Luteinizante/análise , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Detecção do Abuso de Substâncias/métodos , Suécia , Testosterona/análise , Adulto Jovem
6.
Drug Test Anal ; 13(8): 1576-1579, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33864421

RESUMO

To detect doping with pseudo-endogenous anabolic steroids in sports, a urinary steroid profile with glucuronidated plus unconjugated androgens is used. In addition to analyze androgen glucuronide metabolites, it can be of interest to also include sulfate metabolites in the urinary steroid profile. The combined ratios of epitestosterone sulfate/epitestosterone glucuronide to the ratios of testosterone sulfate/testosterone glucuronide ((ES/EG)/(TS/TG)) have previously been investigated as a complementary biomarker for testosterone doping. In this restudy, the aim was to evaluate this biomarker in a larger study sample population. A single dose of 500-mg testosterone enanthate was administered to 54 healthy male volunteers. Urine was collected prior to (Day 0) administration and throughout 15 days and analyzed for the sulfate and glucuronide conjugates of testosterone and epitestosterone. The results show that the combined ratio increased to a larger extent than the traditional T/E ratio in all subjects. This increase was independent on UGT2B17 gene polymorphism. Moreover, a delayed peak of the combined ratio was observed in ~60% of the participants. The results confirm that complementary analyses of the sulfate metabolites may be a useful approach to detect testosterone doping in men.


Assuntos
Epitestosterona/análise , Testosterona/análogos & derivados , Testosterona/urina , Adolescente , Adulto , Biomarcadores , Doping nos Esportes , Glucuronosiltransferase/genética , Humanos , Injeções Intramusculares , Masculino , Pessoa de Meia-Idade , Antígenos de Histocompatibilidade Menor/genética , Polimorfismo Genético , Reprodutibilidade dos Testes , Detecção do Abuso de Substâncias/métodos , Testosterona/análise , Congêneres da Testosterona , Adulto Jovem
7.
Drug Test Anal ; 13(3): 550-557, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33142032

RESUMO

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.


Assuntos
Epitestosterona/urina , Hormônios/urina , Ciclo Menstrual/urina , Esteroides/urina , Adulto , Atletas , Biomarcadores/sangue , Biomarcadores/urina , Doping nos Esportes/prevenção & controle , Epitestosterona/sangue , Feminino , Hormônios/sangue , Humanos , Ciclo Menstrual/sangue , Ciclo Menstrual/fisiologia , Esteroides/sangue , Detecção do Abuso de Substâncias/métodos
8.
Steroids ; 161: 108691, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32603755

RESUMO

A novel method for determining the testosterone/epitestosterone concentration ratio in human urine was established by capillary electrophoresis with diode-array detector. The urine samples were firstly purified by the solid extraction. The optimal experimental conditions were: running buffer pH = 4.74, 15.0 mmol L-1 HAc-NaAc, separation voltage 25 kV, temperature 25 °C, sample injection pressure 3.43 × 103 Pa, and duration 10 s. The testosterone and epitestosterone linear range were determined as 8.0-960.0 ng mL-1, respectively. The testosterone and epitestosterone detection limits were determined as 4.6 and 4.5 ng mL-1, respectively. The relative standard deviation was less than 0.36%.


Assuntos
Eletroforese Capilar , Epitestosterona/urina , Testosterona/urina , Urinálise/métodos , Soluções Tampão , Humanos , Concentração de Íons de Hidrogênio , Limite de Detecção , Modelos Lineares , Temperatura
9.
Mol Cell Endocrinol ; 511: 110850, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32387527

RESUMO

Testosterone (T) and its 17-α epimer, epitestosterone (EpiT), are described as having non-classical effects in addition to their classical androgen actions via the intracellular androgen receptor (iAR). The actions of these androgens play an essential role in triggering factors that shift Sertoli cells from the proliferation phase to the maturation phase. This process is essential for successful spermatogenesis and normal fertility. The aim of this work was to investigate the difference between T and EpiT effects in normal and in chemically castrated Wistar rats. We also tested the effects of these hormones when the iAR-dependent pathways were inhibited by the antiandrogen flutamide. Rats were chemically castrated on postnatal day (pnd) 5 using EDS, a cytotoxic agent that promotes apoptosis of Leydig cells, reducing androgen levels. Then, animals received replacement with T or EpiT and were treated or not with flutamide from pnd 6 to pnd 13 or 20 and were euthanized on pnd 14 and 21. Animals treated with EpiT and flutamide had lower body weight overall. Epididymis weight was also reduced in animals treated with EpiT and flutamide. Flutamide per se reduced epididymis weight at both ages (pnd 14 and 21). Testicular weight and the testicular/body weight ratio were reduced in EDS animals, and flutamide further reduced this weight in animals which received T replacement. EDS administration reduced mRNA levels of both AMH (anti-Müllerian hormone) and its receptor, AMHR2, at pnd 14. In the testes of flutamide-treated animals, EpiT reduced AMH, and both T and EpiT replacement diminished AMHR2 mRNA expression also on pnd 14. EDS decreased iAR expression, and androgen replacement did not change this effect on pnd 21. In rats receiving flutamide, only those also receiving T and EpiT replacement exhibited decreased iAR expression. An increase in connexin 43 expression was observed in animals treated with EpiT without flutamide, whereas in rats treated with flutamide, both hormones were ineffective to increase connexin 43 expression reduced by EDS. Our results suggest that EpiT has an antiandrogen effect on androgen-sensitive tissues such as the epididymis. Nonetheless, the effects of T and EpiT on testicular development parameters are similar. Both hormones may act through their iAR-independent non-classical pathway, regulating AMH and AMHR2, as well as iAR expression.


Assuntos
Hormônio Antimülleriano/metabolismo , Epitestosterona/farmacologia , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Testosterona/farmacologia , Androgênios/farmacologia , Animais , Hormônio Antimülleriano/genética , Barreira Hematotesticular/efeitos dos fármacos , Barreira Hematotesticular/metabolismo , Peso Corporal/efeitos dos fármacos , Conexina 43/metabolismo , Masculino , Tamanho do Órgão/efeitos dos fármacos , Especificidade de Órgãos/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Testículo/efeitos dos fármacos
10.
Drug Test Anal ; 12(8): 1019-1030, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32307878

RESUMO

Testosterone treatment stimulates the production of red blood cells and alters iron homeostasis. Thus, we investigated whether the 'haematological module' of the athlete biological passport (ABP) used by the World Anti-Doping Agency can be used to indicate misuse of testosterone. Nineteen eugonadal men received intramuscular injections of either 250 mg Sustanon®, a blend of four testosterone esters, or placebo on days 0 and 21 in a randomized, placebo-controlleddouble-blind design. Urine samples and blood samples were collected twice pre-treatment, at least 5 days apart, and on days 1, 3, 5, 10 and 14 post-injections to assess steroidal and haematological biomarkers of the ABP. The steroidal profile was flagged suspicious in all Sustanon®-treated subjects, whereas the haematological profile was flagged suspicious in six out of nine subjects. When both sensitivity and specificity were considered, reticulocyte percentage (RET%) appeared as the best marker of the haematological module for implying testosterone ester misuse. Atypical blood passport samples were used to select time points for further isotope-ratio mass spectrometry (IRMS) analysis of testosterone and its metabolites in simultaneously collected urine. In addition to the testosterone (T) to epitestosterone (E) ratio, the RET% and OFF-Score could help identify suspicious samples for more targeted IRMS testing. The results demonstrate that unexpected fluctuations in RET% can indicate testosterone doping if samples are collected 3-10 days after injection. From an anti-doping perspective, the haematological and steroidal modules of the ABP should complement each other when planning targeted follow-up testing and substantiating likely misuse of testosterone.


Assuntos
Doping nos Esportes/prevenção & controle , Espectrometria de Massas/métodos , Reticulócitos/citologia , Testosterona/administração & dosagem , Adulto , Atletas , Biomarcadores/análise , Método Duplo-Cego , Epitestosterona/análise , Humanos , Injeções Intramusculares , Masculino , Sensibilidade e Especificidade , Detecção do Abuso de Substâncias/métodos , Testosterona/análise , Testosterona/farmacologia , Fatores de Tempo , Adulto Jovem
11.
J Anal Toxicol ; 44(2): 192-199, 2020 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-31322674

RESUMO

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


Assuntos
Castração , Epitestosterona/urina , Testosterona/urina , Adulto , Cromatografia Líquida , Doping nos Esportes , Europa (Continente) , Humanos , República da Coreia , Espectrometria de Massas em Tandem
12.
J Steroid Biochem Mol Biol ; 197: 105518, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31704245

RESUMO

Several drug-metabolizing enzymes are known to control androgen homeostasis in humans. UDP-glucuronosyltransferases convert androgens to glucuronide conjugates in the liver and intestine, which enables subsequent elimination of these conjugated androgens via urine. The most important androgen is testosterone, while others are the testosterone metabolites androsterone and etiocholanolone, and the testosterone precursor dehydroepiandrosterone. Epitestosterone is another endogenous androgen, which is included as a crucial marker in urine doping tests. Since glucuronide conjugates are hydrophilic, efflux transporters mediate their excretion from tissues. In this study, we employed the membrane vesicle assay to identify the efflux transporters for glucuronides of androsterone, dehydroepiandrosterone, epitestosterone, etiocholanolone and testosterone. The human hepatic and intestinal transporters MRP2 (ABCC2), MRP3 (ABCC3), MRP4 (ABCC4), BCRP (ABCG2) and MDR1 (ABCB1) were studied in vitro. Of these transporters, only MRP2 and MRP3 transported the androgen glucuronides investigated. In kinetic analyses, MRP3 transported glucuronides of androsterone, epitestosterone and etiocholanolone at low Km values, between 0.4 and 4 µM, while the Km values for glucuronides of testosterone and dehydroepiandrosterone were 14 and 51 µM, respectively. MRP2 transported the glucuronides at lower affinity, as indicated by Km values over 100 µM. Interestingly, the MRP2-mediated transport of androsterone and epitestosterone glucuronides was best described by sigmoidal kinetics. The inability of BCRP to transport any of the androgen glucuronides investigated is drastically different from its highly active transport of several estrogen conjugates. Our results explain the transporter-mediated disposition of androgen glucuronides in humans, and shed light on differences between the human efflux transporters MRP2, MRP3, MRP4, BCRP and MDR1.


Assuntos
Epitestosterona/metabolismo , Glucuronídeos/metabolismo , Fígado/metabolismo , Testosterona/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico , Humanos , Modelos Moleculares , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo
13.
Drug Test Anal ; 11(10): 1566-1571, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31454165

RESUMO

Testosterone doping remains a prevalent and potent form of drug cheating among elite athletes. In men, the urine testosterone (T) to epitestosterone (E) ratio (T/E ratio) can identify administration of exogenous T by its suppression of endogenous T production through strong negative feedback on endogenous T and E production as well as spill over into urine of extra testosterone. However, this mechanism may be partially inoperative in females whose much lower circulating T derives from three sources, none subject to powerful negative T feedback. Hence, additional methods to detect T doping in females are required. In this study we report two cases of elite female athletes who were sanctioned for T doping proven by measurement of serum T using liquid chromatography-mass spectrometry (LC-MS), when serial urine T and T/E ratio in one were not indicative of T doping, and in the other were nullified by incidental genetic inactivation of T glucuronidation through the uridine diphosphoglucuronosyl transferase 2B17 (UGT2B17) deletion genotype-phenotype. These findings indicate the potential for serum T measurement by LC-MS to detect T doping in female athletes, especially if implemented in the Bayesian format of an athlete biological passport.


Assuntos
Epitestosterona/urina , Detecção do Abuso de Substâncias/métodos , Testosterona/urina , Atletas , Cromatografia Líquida/métodos , Doping nos Esportes , Feminino , Humanos , Espectrometria de Massas/métodos , Testosterona/sangue
14.
Drug Test Anal ; 11(8): 1218-1230, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30932347

RESUMO

The introduction of alternative markers to the steroid profile can be an effective approach to improving the screening capabilities for the detection of testosterone (T) misuse. In this work, endogenous steroid sulfates were evaluated as potential markers to detect intramuscular (IM) T administration. Fourteen sulfate metabolites were quantified using mixed-mode solid-phase extraction and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine samples after a single IM injection (100 mg) of T cypionate to six Caucasian and six Asian healthy male volunteers were analyzed. Principal component analysis (PCA) was used to characterize the sample cohort and to obtain the most useful markers for discrimination between pre- and post-administration samples. For Caucasian volunteers, a separation between pre- and post-administration samples was observed in PCA, whereas for Asian volunteers no separation was obtained. Seventeen ratios between sulfate metabolites were selected and further considered. Detection times (DTs) of each marker were evaluated using individual thresholds for each volunteer. The best results were obtained using ratios involving T and epitestosterone (E) sulfates in the denominator. The best marker was the ratio androsterone sulfate/testosterone sulfate (A-S/T-S) which prolonged the DT 1.2-2.1 times in respect to those obtained using T/E ratio in all Caucasian volunteers and 1.3-1.5 times in two Asian volunteers. Other ratios between A-S or etiocholanolone sulfate and E-S, and sulfates of etiocholanolone, dehydroandrosterone or epiandrosterone, and T-S were also found adequate. These ratios improve the DT after IM T administration and their incorporation to complement the current steroid profile is recommended.


Assuntos
Anabolizantes/urina , Androgênios/urina , Epitestosterona/urina , Sulfatos/urina , Testosterona/urina , Anabolizantes/administração & dosagem , Anabolizantes/metabolismo , Androgênios/administração & dosagem , Androgênios/metabolismo , Povo Asiático , Cromatografia Líquida , Doping nos Esportes , Epitestosterona/administração & dosagem , Epitestosterona/metabolismo , Humanos , Injeções Intramusculares , Masculino , Detecção do Abuso de Substâncias , Sulfatos/metabolismo , Espectrometria de Massas em Tandem , Testosterona/administração & dosagem , Testosterona/metabolismo , População Branca
15.
J Steroid Biochem Mol Biol ; 185: 47-56, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30031148

RESUMO

Growth and development of an embryo or fetus during human pregnancy mainly depend on intact hormone biosynthesis and metabolism in maternal amniotic fluid (AF). We investigated the hormonal milieu in AF and developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of 14 sulfated and 6 unconjugated steroids in AF. 65 A F samples (male: female = 35: 30) of mid-gestation ranging from 16th week of gestation to 25th week of gestation were analyzed. Reference data of 20 steroid levels in AF of healthy women were provided. 13 sulfated and 3 unconjugated steroids were for the first time quantified in AF by LC-MS/MS. Highest concentrations were found for pregnenolone sulfate (PregS: mean ±â€¯SD, 8.6 ±â€¯3.7 ng/mL), 17α-hydroxypregnenolone sulfate (17OHPregS: 4.9 ±â€¯2.0 ng/mL), epitestosterone sulfate (eTS: 7.3 ±â€¯3.6 ng/mL), 16α-hydroxydehydroepiandrosterone sulfate (16OH-DHEAS: 21.5 ±â€¯10.7 ng/mL), androsterone sulfate (AnS: 9.2 ±â€¯7.4 ng/mL), estrone sulfate (E1S: 3.0 ±â€¯3.0 ng/mL), estriol 3-sulfate (E3S: 8.1 ±â€¯4.0 ng/mL) and estriol (E3: 1.2 ±â€¯0.4 ng/mL). Only testosterone (T) showed a significant sex difference (p < 0.0001). Correlations between AF steroids mirrored the steroid metabolism of the feto-placental unit, and not only confirmed the classical steroid pathway, but also pointed to a sulfated steroid pathway.


Assuntos
Líquido Amniótico/química , Segundo Trimestre da Gravidez/fisiologia , Esteroides/análise , 17-alfa-Hidroxipregnenolona/análise , Androsterona/análise , Cromatografia Líquida , Desidroepiandrosterona/análise , Epitestosterona/análise , Estriol/análogos & derivados , Estriol/análise , Estrona/análogos & derivados , Estrona/análise , Feminino , Idade Gestacional , Humanos , Masculino , Gravidez , Pregnenolona/análise , Espectrometria de Massas em Tandem
16.
Drug Test Anal ; 10(11-12): 1744-1754, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30315670

RESUMO

The impact of dehydroepiandrosterone (DHEA) administration has been widely studied for anti-doping purposes in men, whereas only a few studies have been performed in women. In the present study, the impact of DHEA on the steroid profile parameters and their carbon isotopic ratios was explored. Eleven healthy young women and 10 healthy young men received two treatments: One with 100 mg/day of DHEA for 28 days and one with a placebo according to a double-blind crossover protocol. Urine and saliva (only in females) samples were collected before and for 72 hours after each short-term treatment. In all female subjects, concentrations of the urinary parameters of the steroid profile were highly impacted by short-term DHEA administration including epitestosterone (E). Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis was performed and positive results were observed for E in the four female subjects where E concentration was adequate for such analysis, whereas men results remained negative for E. Last, the ability of the Anti-Doping Administration and Management System (ADAMS) software used for the athlete biological passport to identify such doping was assessed. Of the 11 passports generated for female subjects, 10 were automatically classified as an atypical passport finding (ATPF). For the remaining passport with normal status in one woman, the variability of the concentrations prevented the ADAMS software from adjusting individual limits. The most impacted markers in women were T/E and 5αAdiol/E, with a detection window of 36 hours for 5αAdiol/E. In addition, good correlations were observed for DHEA and T concentrations in urine and saliva in females.


Assuntos
Desidroepiandrosterona/administração & dosagem , Cromatografia Gasosa-Espectrometria de Massas/métodos , Saliva/química , Esteroides/análise , Esteroides/urina , Detecção do Abuso de Substâncias/métodos , Adulto , Biomarcadores/análise , Biomarcadores/urina , Isótopos de Carbono/análise , Isótopos de Carbono/urina , Desidroepiandrosterona/análise , Desidroepiandrosterona/urina , Doping nos Esportes , Método Duplo-Cego , Epitestosterona/análise , Epitestosterona/urina , Feminino , Humanos , Masculino , Testosterona/análise , Testosterona/urina , Adulto Jovem
17.
Steroids ; 138: 82-90, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30033342

RESUMO

Conjugates of 17α-substituted testosterone (1 and 2) and 17ß-substituted epitestosterone (3 and 4) with pyropheophorbide a were synthesized. The scheme consisted of synthesis of 17α-hydroxy-3-oxopregn-4-en-21-oic and 17ß-hydroxy-3-oxopregn-4-en-21-oic acids, and their coupling with pyropheophorbide a by means of either ethylene diamine, or 1,5-diamino pentane linkers. Mutual influence of steroidal and macrocyclic fragments in conjugates molecules was dependent on configuration of C17 and length of linker, that was established by analysis of 1H NMR spectra and molecular models of conjugates. Studies of interaction of conjugates with prostate carcinoma cells revealed that their uptake and internalization were independent on the androgen receptor activity, but dependent on the structure of conjugates, decreasing in the following row: 3 > 4 ≥ 1 > 2. Conjugates significantly decreased the LNCaP and PC-3 cells growth at 96 h incubation. Epitestosterone derivatives 3 and 4 also showed superior anti-proliferative activity versus testosterone ones. Conformationally more rigid conjugates 1 and 3, comprising short linkers, were more active than those with long linkers; conjugate 3 was the most potent.


Assuntos
Antineoplásicos/química , Clorofila/análogos & derivados , Epitestosterona/química , Testosterona/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Clorofila/química , Humanos , Masculino , Células PC-3 , Neoplasias da Próstata/metabolismo , Relação Estrutura-Atividade
18.
Drug Test Anal ; 10(10): 1518-1527, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29797687

RESUMO

The use of testosterone and its pro-drugs, such as dehydroepiandrosterone (DHEA), is currently regulated in horseracing by the application of international testosterone thresholds. However, additional steroidomic approaches, such as steroid ratios, to distinguish overall adrenal stimulation from drug administrations and an equine biological passport for longitudinal steroid profiling of individual animals could be advantageous in equine doping testing. Thus, DHEA concentrations and related ratios (testosterone [T] to DHEA and DHEA to epitestosterone [E]) were assessed in the reference population by quantitative analysis of 200 post-race gelding urine samples using liquid chromatography-tandem mass spectrometry. DHEA concentrations ranged between 0.9 and 136.6 ng/mL (mean 12.8 ng/mL), T:DHEA ratios between 0.06 and 1.85 (mean 0.43), and DHEA:E ratios between 0.21 and 13.56 (mean 2.20). Based on the reference population statistical upper limits of 5.4 for T:DHEA ratio and 48.1 for DHEA:E ratio are proposed with a risk of 1 in 10 000 for a normal outlier exceeding the value. Analysis of post-administration urine samples collected following administrations of DHEA, Equi-Bolic® (a mix of DHEA and pregnenolone) and testosterone propionate to geldings showed that the upper limit for T:DHEA ratio was exceeded following testosterone propionate administration and DHEA:E ratio following DHEA administrations and thus these ratios could be used as additional biomarkers when determining the cause of an atypical testosterone concentration. Additionally, DHEA concentrations and ratios can be used as a starting point to establish reference ranges for an equine biological passport.


Assuntos
Desidroepiandrosterona/urina , Cavalos/urina , Detecção do Abuso de Substâncias/métodos , Animais , Cromatografia Líquida/métodos , Doping nos Esportes , Epitestosterona/urina , Limite de Detecção , Masculino , Espectrometria de Massas em Tandem/métodos , Testosterona/urina
19.
Mol Cell Endocrinol ; 461: 112-121, 2018 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-28870779

RESUMO

Epitestosterone is the 17α-epimer of testosterone and has been described as an anti-androgen, since it inhibits the effects produced by testosterone and dihydrotestosterone via the nuclear androgen receptor (nAR). However, epitestosterone also displays an effect which is similar to the non-classical effect of testosterone, depolarizing the membrane potential of Sertoli cells and inducing a rapid Ca2+ uptake. This study aimed to investigate the effects of a treatment with epitestosterone on developmental parameters of immature rats. Animals were chemically castrated by using the gonadotropin-releasing hormone (GnRH) antagonist cetrorelix and then received a replacement of 7 days with epitestosterone or testosterone. Replacement with either epitestosterone or testosterone restored the anogenital distance (AGD) and testicular weight which had been reduced by chemical castration. The immunocontent of nAR and the nAR-immunoreactivity were reduced by epitestosterone treatment in the testis of both castrated and non-castrated animals. Furthermore, testosterone was unable of changing the membrane potential of Sertoli cells through its non-classical action in the group of animals castrated and replaced with epitestosterone. In conclusion, in relation to the level of protein expression of nAR epitestosterone acts as an anti-androgen. However, it acts in the same way as testosterone when genital development parameters are evaluated. Moreover, in castrated rats epitestosterone suppressed the non-classical response of testosterone, changing the pattern of testosterone signalling via a membrane mechanism in Sertoli cells.


Assuntos
Castração , Epitestosterona/farmacologia , Terapia de Reposição Hormonal , Testículo/crescimento & desenvolvimento , Testosterona/farmacologia , Animais , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Hormônio Liberador de Gonadotropina/metabolismo , Hormônio Liberador de Gonadotropina/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Ratos Wistar , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Túbulos Seminíferos/efeitos dos fármacos , Túbulos Seminíferos/metabolismo , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/metabolismo , Testículo/efeitos dos fármacos
20.
Drug Test Anal ; 10(3): 575-583, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28671321

RESUMO

Endogenous steroid use can increase urinary testosterone/epitestosterone (T/E) values. In addition, ethanol in amounts >0.5 g per kg of body weight (g/kg) can also increase T/E values. However, the effect of smaller doses of ethanol on T/E values is unknown. The influence of 0.2 and 0.4 g/kg of ethanol on baseline T/E values in 20 men and 20 women with low and high baseline T/E values was investigated and correlated with ethyl glucuronide (EtG) and ethyl sulfate (EtS) concentrations. T/E values for 7 of the women were excluded from the study because of undetectable T concentrations or for other reasons. One man and 1 woman with a high T/E baseline value had a significant increase in their T/E value after ingestion of 0.2 g/kg of ethanol. One man and 2 women with a high T/E baseline, and 1 woman with a low T/E baseline had significantly increased T/E values after ingestion of 0.4 g/kg of ethanol. There was wide variability in peak EtG concentrations and a lack of correlation between ethanol dose and EtG concentrations. Interestingly, 1 man and 2 women with increased T/E values following ethanol ingestion had EtG concentrations below the World Anti-Doping Agency (WADA) cut-off of 5000 ng/mL. These findings demonstrate that small amounts of ethanol can elevate T/E values, with women being more susceptible. In addition, consideration should be given to the lowering of the WADA EtG cut-off to detect samples with elevated T/E values from ingestion of low doses of ethanol.


Assuntos
Consumo de Bebidas Alcoólicas/urina , Epitestosterona/urina , Detecção do Abuso de Substâncias/métodos , Testosterona/urina , Adulto , Doping nos Esportes , Feminino , Cromatografia Gasosa-Espectrometria de Massas/métodos , Glucuronatos/urina , Humanos , Limite de Detecção , Masculino , Ésteres do Ácido Sulfúrico/urina , Espectrometria de Massas em Tandem/métodos , Adulto Jovem
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