OBJECTIVE

Anabolic steroids are synthetic derivatives of testosterone, modified to enhance its anabolic actions (promotion of protein synthesis and muscle growth). They have numerous side effects, and are on the International Olympic Committee's list of banned substances. Gas chromatography-mass spectrometry allows identification and characterisation of steroids and their metabolites in the urine but may not distinguish between pharmaceutical and natural testosterone. Indirect methods to detect doping include determination of the testosterone/epitestosterone glucuronide ratio with suitable cut-off values. Direct evidence may be obtained with a method based on the determination of the carbon isotope ratio of the urinary steroids. This paper aims to give an overview of the use of anabolic-androgenic steroids in sport and methods used in anti-doping laboratories for their detection in urine, with special emphasis on doping with testosterone.

METHODS

Review of the recent literature of anabolic steroid testing, athletic use, and adverse effects of anabolic-androgenic steroids.

RESULTS

Procedures used for detection of doping with endogenous steroids are outlined. The World Anti-Doping Agency provided a guide in August 2004 to ensure that laboratories can report, in a uniform way, the presence of abnormal profiles of urinary steroids resulting from the administration of testosterone or its precursors, androstenediol, androstenedione, dehydroepiandrosterone or a testosterone metabolite, dihydrotestosterone, or a masking agent, epitestosterone.

CONCLUSIONS

Technology developed for detection of testosterone in urine samples appears suitable when the substance has been administered intramuscularly. Oral administration leads to rapid pharmacokinetics, so urine samples need to be collected in the initial hours after intake. Thus there is a need to find specific biomarkers in urine or plasma to enable detection of long term oral administration of testosterone.

BACKGROUND

The conspicuous interindividual differences in metabolism and urinary excretion of testosterone and its metabolites make it challenging to reveal testosterone doping. The variation in testosterone glucuronide excretion is strongly associated with a deletion polymorphism in the uridine diphosphate-glucuronosyltranferase (UGT) 2B17 gene.

OBJECTIVE

The objective of the study was to identify additional biomarkers to detect testosterone abuse and to elucidate alternative pathways for testosterone elimination in individuals devoid of the UGT2B17 enzyme. For this purpose a new ultraperformance liquid chromatographic tandem mass spectrometric method for simultaneous determination of 10 different sulfo- and glucuronide-conjugated steroids was developed.

METHODS

Fifty-four healthy male volunteers with two, one, or no allele (ins/ins, ins/del, or del/del) of the UGT2B17 gene participated in the study.

METHODS

Intervention included a single im dose of 500 mg testosterone enanthate.

METHODS

Urinary sulfo- and glucuronide-conjugated steroids were measured.

RESULTS

Testosterone sulfate levels decreased in all individuals after the dose. The individual differences in the excretion of all sulfated metabolites were large. Thus, these metabolites will not serve as appropriate biomarkers for testosterone abuse. However, androsterone glucuronide excretion increased in all of our study subjects after the testosterone dose. Etiocholanolone sulfate was excreted at significantly higher levels in UGT2B17 del/del individuals.

CONCLUSIONS

We propose that the androsterone glucuronide to epitestosterone glucuronide ratio may serve as a complementary biomarker to reveal testosterone abuse.

Anabolic-androgenic steroids (AASs) are frequently misused. To determine causes of death, characteristics, toxicology, and pathology of AAS positive cases, all cases (n = 24) presenting to the New South Wales Department of Forensic Medicine (1995-2012) were retrieved. All were male, and the mean age was 31.7 years. Deaths were mainly due to accidental drug toxicity (62.5%), then suicide (16.7%) and homicide (12.5%). Abnormal testosterone/epitestosterone ratios were reported in 62.5%, followed by metabolites of nandrolone (58.3%), stanozolol (33.3%), and methandienone (20.8%). In 23 of 24 cases, substances other than steroids were detected, most commonly psychostimulants (66.7%). In nearly half, testicular atrophy was noted, as was testicular fibrosis and arrested spermatogenesis. Left ventricular hypertrophy was noted in 30.4%, and moderate to severe narrowing of the coronary arteries in 26.1%. To summarize, the typical case was a male polydrug user aged in their thirties, with death due to drug toxicity. Extensive cardiovascular disease was particularly notable.

Based on aqueous two-phase systems (ATPS) consisting of 1-butyl-3-methylimidazolium chloride, a hydrophilic ionic liquid (IL), and K2HPO4, a new and simple extraction technique, coupled with a reversed-phase high performance liquid chromatography (RP-HPLC), was developed for the simultaneous concentration and analysis of testosterone (T) and epitestosterone (ET) in human urine. Under the optimal conditions, the extraction efficiencies for both analytes were 80-90% in a one-step extraction. The method required only 3.0 mL of urine and a single hydrolysis/deproteinization/extraction step followed by direct injection of the IL-rich upper phase into HPLC system for analysis. The method has been satisfactorily applied to the analysis of T and ET in human urine with detection limits of 1 ng/mL and linear ranges of 10-500 ng/mL for both compounds. Compared with conventional liquid-liquid extraction or solid phase extraction, this new method is much "greener" due to no use of volatile organic solvent and low consumption of IL. The proposed extraction technique opens up new possibilities in the separation of other drugs.

Tribulus terrestris is an herbal nutritional supplement that is promoted to produce large gains in strength and lean muscle mass in 5-28 days (15, 18). Although some manufacturers claim T. terrestris will not lead to a positive drug test, others have suggested that T. terrestris may increase the urinary testosterone/epitestosterone (T/E) ratio, which may place athletes at risk of a positive drug test. The purpose of the study was to determine the effect of T. terrestris on strength, fat free mass, and the urinary T/E ratio during 5 weeks of preseason training in elite rugby league players. Twenty-two Australian elite male rugby league players (mean +/- SD; age = 19.8 +/- 2.9 years; weight = 88.0 +/- 9.5 kg) were match-paired and randomly assigned in a double-blind manner to either a T. terrestris (n = 11) or placebo (n = 11) group. All subjects performed structured heavy resistance training as part of the club's preseason preparations. A T. terrestris extract (450 mg.d(-1)) or placebo capsules were consumed once daily for 5 weeks. Muscular strength, body composition, and the urinary T/E ratio were monitored prior to and after supplementation. After 5 weeks of training, strength and fat free mass increased significantly without any between-group differences. No between-group differences were noted in the urinary T/E ratio. It was concluded that T. terrestris did not produce the large gains in strength or lean muscle mass that many manufacturers claim can be experienced within 5-28 days. Furthermore, T. terrestris did not alter the urinary T/E ratio and would not place an athlete at risk of testing positive based on the World Anti-Doping Agency's urinary T/E ratio limit of 4:1.

The assessment of steroidal hormones in the environment requires sensitive and selective analytical techniques suitable for sample matrices. This paper reports a simple method to analyze simultaneously six corticosteroids (triamcinolone, cortisol, dexamethasone, flumethasone, prednisolone, triamcinolone acetonide), four androgens (boldenone, epitestosterone, methyltestosterone, nortestosterone), and progesterone in river and drinking water sources. The developed method is based on a single solid-phase extraction (SPE) followed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) with atmospheric pressure chemical ionization (APCI). The main advantage of this method over other methods includes the use of a single SPE with a low volume cartridge for sample preparation, separation of steroids on alkyl-amide stationary phase with no matrix interferences by LC-MS/MS analysis, and simultaneous analysis of more than two groups of steroids. The method was characterized by generally good performance, analyzing three groups of steroids using 100 and 1000mL samples with average recovery ranges of 80-109% and 68-126%, respectively at a level of 1ngL(-1). The limit of detection (LOD) ranged from 0.06 to 0.2 and 0.01-0.21ngL(-1) for 100mL and 1000mL samples volumes, respectively. Sixty samples of Danube River and drinking water sources from different regions of Hungary were collected to analyze target steroids in two sampling periods in 2008 and 2009. Steroids, except cortisol, dexamethasone, flumethasone, prednisolone, epitestosterone and progesterone were below detection limits. Endogenous steroids (cortisol, epitestosterone, progesterone) were present in the concentration range of 0.08-2.67ngL(-1) while synthetic corticosteroids (dexamethasone, flumethasone, and prednisolone) varied from 0.064 to 1.43ngL(-1). Steroids were present in river water, except progesterone, which was present only in ground water. Levels of steroids are compared with other rivers in the world and were briefly discussed.

We have reported previously that 17 beta-estradiol (E2) inhibits selectively the cAMP response to human (h) PTH and PTH-related protein (hPTHrP), but not to vasoactive intestinal peptide, in human osteoblast-like cells (SaOS-2). We have now extended these studies to investigate the actions of androgens on hPTH-stimulated accumulation of cAMP, and on the roles of new protein synthesis and pertussis toxin (PTox) substrates in the actions of steroid hormones on SaOS-2 cells. Pretreatment with testosterone (T) or 5 alpha-dihydrotestosterone (5 alpha-DHT) for 4-12 h at concentrations of 10(-12) to 10(-8) M inhibited significantly the cAMP response to hPTH by up to 50-70% of control. Like E2, the actions of T and 5 alpha-DHT were selective for hPTH or hPTHrP; there was no inhibition of the stimulatory action of vasoactive intestinal peptide. Two related steroids, 5 beta-DHT and 17 alpha-epitestosterone, did not inhibit the action of hPTH. Pretreatment of cells with cycloheximide, under conditions which inhibited protein synthesis by greater than 90%, reduced the cAMP response to hPTH but did not block the further inhibitory actions of E2, T, or 5 alpha-DHT. Pretreamtent of cells with PTox (100 ng/ml) for 24 h, enhanced the accumulation of cAMP stimulated by hPTH consistent with an action of PTox on Gi; however, the inhibitory actions of E2, T, and 5 alpha-DHT on PTH-stimulated cAMP accumulation were not attenuated by PTox. We conclude that androgens, as well as estrogens, act directly on human bone cells to modulate selectively an early effect of hPTH. The inhibitory actions of these steroid hormones do not appear to depend on new protein synthesis and may not involve a functionally active PTox substrate, presumably Gi.

OBJECTIVE

Premature androgenic alopecia has been suggested as a feature of the male equivalent of the syndrome of polycystic ovary. However, the hormonal pattern of men with premature balding has been investigated in only a few studies with inconsistent results.

METHODS

We examined 37 men with premature balding (defined as frontoparietal and vertex hair loss before the age of 30 years with alopecia defined as grade 3 vertex or more on the alopecia classification scale of Hamilton with Norwood modification). The plasma concentrations of total testosterone, dihydrotestosterone, epitestosterone, androstenedione, cortisol, 17-OH-progesterone (17OHP), estradiol, LH, FSH, prolactin, SHBG and TSH and free thyroxine were measured.

RESULTS

The frequency of subnormal values in SHBG, FSH, testosterone and epitestosterone (but not in free androgen index) was significant in the balding men. A borderline significant trend was recorded with respect to increased levels in 17OH-P and prolactin.

CONCLUSIONS

The hormonal pattern of a substantial number of men with premature balding resembles in some respects the hormonal pattern of women with polycystic ovary syndrome.

BACKGROUND

Carbon isotope ratio methods are used in doping control to determine whether urinary steroids are endogenous or pharmaceutical.

METHODS

Gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) was used to determine the delta(13)C values for 5 beta-androstane-3 alpha,17 beta-diyl diacetate (5 beta A), 5 alpha-androstane-3 alpha,17 beta-diyl diacetate (5 alpha A), and 5 beta-pregnane-3 alpha,20 alpha-diyl diacetate (5 beta P) in a control group of 73 healthy males and 6 athletes with testosterone/epitestosterone ratios (T/E) >6.

RESULTS

The within-assay precision SDs for 5 beta A, 5 alpha A, and 5 beta P were +/- 0.27 per thousand, +/- 0.38 per thousand, and +/- 0.28 per thousand, respectively. The between-assay precision SDs ranged from +/- 0.40 per thousand to +/- 0.52 per thousand. The system suitability and batch acceptance scheme is based on SDs. For the control group, the mean delta(13)C (SD) values were -25.69 per thousand (+/- 0.92 per thousand), -26.35 per thousand (+/- 0.68 per thousand), and -24.26 per thousand (+/- 0.70 per thousand), for 5 beta A, 5 alpha A, and 5 beta P, respectively. 5 beta P was greater than 5 beta A and 5 alpha A (P <0.01), and 5 beta A was greater than 5 alpha A (P <0.01). The means - 3 SD were -28.46 per thousand, -28.39 per thousand, and -26.37 per thousand for 5 beta A, 5 alpha A, and 5 beta P, respectively. The maximum difference between 5 beta P and 5 beta A was 3.2 per thousand, and the maximum 5 beta A/5 beta P was 1.13. Three athletes with chronically elevated T/Es had delta(13)C values consistent with testosterone administration and three did not.

CONCLUSIONS

This GC-C-IRMS assay of urine diols has low within- and between-assay SDs; therefore, analysis of one urine sample suffices for doping control. The means, SDs, +/-3 SDs, and ranges of delta(13)C values in a control group are established. In comparison, testosterone users have low 5 beta A and 5 alpha A, large differences between 5 beta A or 5 alpha A and 5 beta P, and high 5 beta A/5 beta P and 5 alpha A/5 beta P ratios.

Use of testosterone enanthate has been shown to significantly increase strength within 6-12 weeks of administration (2, 9), however, it is unclear if the ergogenic benefits are evident in less than 6 weeks. Testosterone enanthate is classified as a prohibited substance by the World Anti-Doping Agency (WADA) and its use may be detected by way of the urinary testosterone/epitestosterone (T/E) ratio (16). The two objectives of this study were to establish (a) if injection of 3.5 mg.kg(-1) testosterone enanthate once per week could increase muscular strength and cycle sprint performance in 3-6 weeks; and (b) if the WADA-imposed urinary T/E ratio of 4:1 could identify all subjects being administered 3.5 mg.kg(-1) testosterone enanthate. Sixteen healthy young men were match-paired and were assigned randomly in a double-blind manner to either a testosterone enanthate or a placebo group. All subjects performed a structured heavy resistance training program while receiving either testosterone enanthate (3.5 mg.kg(-1)) or saline injections once weekly for 6 weeks. One repetition maximum (1RM) strength measures and 10-second cycle sprint performance were monitored at the pre (week 0), mid (week 3), and post (week 6) time points. Body mass and the urinary T/E ratio were measured at the pre (week 0) and post (week 6) time points. When compared with baseline (pre), 1RM bench press strength and total work during the cycle sprint increased significantly at week 3 (p < 0.01) and week 6 (p < 0.01) in the testosterone enanthate group, but not in the placebo group. Body mass at week 6 was significantly greater than at baseline in the testosterone enanthate group (p < 0.01), but not in the placebo group. Despite the clear ergogenic effects of testosterone enanthate in as little as 3 weeks, 4 of the 9 subjects in the testosterone enanthate group ( approximately 44%) did not test positive to testosterone under current WADA urinary T/E ratio criteria.

Epitestosterone has been demonstrated to act at various levels as a weak antiandrogen. So far, its serum levels have been followed up only in males. Epitestosterone and its major circulating precursor pregnenolone sulfate and T were measured in serum from 211 healthy women and 386 men to find out whether serum concentrations of epitestosterone are sufficient to exert its antiandrogenic actions. In women, epitestosterone exhibited a maximum around 20 yr of age, followed by a continuous decline up to menopause and by a further increase in the postmenopause. In men, maximum epitestosterone levels were detected at around 35 yr of age, followed by a continuous decrease. Pregnenolone sulfate levels in women reached their maximum at about age 32 yr and then declined continuously, and in males the maximum was reached about 5 yr earlier and then remained nearly constant. Epitestosterone correlated with pregnenolone sulfate only in males. In both sexes a sharp decrease of the epitestosterone/T ratio around puberty occurred. In conclusion, concentrations of epitestosterone and pregnenolone sulfate are age dependent and, at least in prepubertal boys and girls, epitestosterone reaches or even exceeds the concentrations of T, thus supporting its role as an endogenous antiandrogen. The dissimilarities in the course of epitestosterone levels through the lifespan of men and women and its relation to pregnenolone sulfate concentrations raise the question of the contribution of the adrenals and gonads to the production of both steroids and even to the uniformity of the mechanism of epitestosterone formation.

The UDP Glucuronosyl Transferase (UGT) enzymes are important in the pharmacokinetics, and conjugation, of a variety of drugs including non-steroidal anti-inflammatory drugs (NSAIDs) as well as anabolic androgenic steroids (AAS). Testosterone glucuronidation capacity is strongly associated with a deletion polymorphism in the UGT2B17 gene. As the use of high doses of NSAIDs has been observed in athletes there is a risk for a drug-drug interaction that may influence the doping tests for AAS. In vitro studies show inhibitory potential on UGT2B7, 2B15, and 2B17 enzymes by NSAIDs. The aim of this study was to investigate if concomitant use of NSAIDs and a single dose of testosterone enanthate would affect the excretion rate of testosterone and epitestosterone glucuronide (TG and EG) as well as the T/E ratio, thereby affecting the outcome of the testosterone doping test. The study was designed as an open, randomized, cross-over study with subjects being their own control. The 23 male healthy volunteers, with either two, one or no allele (ins/ins, ins/del, or del/del) of the UGT2B17 gene, received the maximum recommended dose of NSAID (Ibuprofen or Diclofenac) for 6 days. On day three, 500 mg of testosterone enanthate was administered. Spot urine samples were collected for 17 days. After a wash-out period of 4 months the volunteers received 500 mg testosterone enanthate only, with subsequent spot urine collection for 14 days. The glucuronides of testosterone and epitestosterone were quantified. NSAIDs did not affect the excretion of TG or EG before the administration of testosterone. The concomitant use of NSAIDs and testosterone slightly increased the TG excretion while the EG excretion was less suppressed compared to testosterone use only. The effects of the NSAIDs on the TG and EG excretion did not differ between the UGT2B17 genotype groups. In conclusion, the outcome of testosterone doping tests does not seem to be affected by the use of NSAIDs.

The anabolic steroid testosterone can be used by athletes to enhance athletic performance and muscle growth. UDP-glucuronosyltransferase (UGT2B17) is the key enzyme involved in the glucuronidation of testosterone to testosterone glucuronide, which also serves as a marker for the testosterone/epitestosterone (T/E) ratio used to detect testosterone abuse in sport. Inhibitors of testosterone glucuronidation could have an impact on circulating testosterone levels, thus aiding performance, as well as potentially affecting the urinary T/E ratio and therefore masking testosterone abuse. Previous reports have revealed that non-steroidal, anti-inflammatory drugs, diclofenac and ibuprofen, inhibit the UGT2B17 enzyme. The aim of this study is to analyse dietary tea samples for inhibition of testosterone glucuronidation and, where inhibition is present, to identify the active compounds. Analysis of testosterone glucuronidation was conducted by performing UGT2B17 assays with detection of un-glucuronidated testosterone using high performance liquid chromatography. The results from this study showed that testosterone glucuronidation was inhibited by the green and white tea extracts, along with specific catechin compounds, notably: epicatechin, epigallocatechin gallate (EGCG) and catechin gallate. The IC50 inhibition value for EGCG was determined, using a Dixon plot, to be 64 μM, equalling the most active NSAID inhibitor diclofenac. Thus, common foodstuffs and their constituents, for the first time, have been identified as inhibitors of a key enzyme involved in testosterone glucuronidation. Whilst these common compounds are not substrates of the UGT2B17 enzyme, we showed that they inhibit testosterone glucuronidation which may have implications on current doping control in sport.

BACKGROUND

Increased levels of pregnanes have been reported in foals with neonatal maladjustment syndrome (NMS). These steroids may cross the blood-brain barrier and have depressive effects in the central nervous system leading to behavioural abnormalities and altered states of consciousness in affected foals.

OBJECTIVE

The aim of this study was to determine the pregnane profile of foals with NMS and compare it with that of healthy controls and sick, non-NMS foals.

METHODS

Prospective-clinical study.

METHODS

Thirty-two foals with a clinical diagnosis of NMS, 12 foals with other neonatal disorders and 10 healthy control foals were selected for the study. Heparinised blood samples were collected from each group of foals and pregnane and androgen concentrations determined using liquid chromatography mass spectrometry at 0, 24 and 48 h of age.

RESULTS

Healthy foals showed a significant decrease in pregnane concentrations over the first 48 h of life (P<0.01). Foals with NMS and sick, non-NMS foals had significantly increased progesterone, pregnenolone, androstenedione, dehydroepiandrosterone and epitestosterone concentrations compared with healthy foals (P<0.05). Progesterone and pregnenolone concentrations of sick, non-NMS foals decreased significantly over 48 h (P<0.05), whereas concentrations in NMS foals remained increased.

CONCLUSIONS

Pregnane concentrations of ill, neonatal foals remain increased following birth, reflecting a delayed, or interrupted, transition from intra- to extra-uterine life. Serial progesterone and pregnenolone measurement may be useful in aiding diagnosis of NMS.

For screening of a large number of samples for androgenic activity, a robust system with minimal handling is required. The coding sequence for human androgen receptor (AR) was inserted into expression plasmid YEpBUbi-FLAG1, resulting in the plasmid YEpBUbiFLAG-AR, and the estrogen response element (ERE) on the reporter vector YRpE2 was replaced by an androgen response element (ARE), resulting in the plasmid YRpE2-ARE. Thus, a fully functional transactivation assay system with beta-galactosidase as a reporter gene could be created. Furthermore, green fluorescent protein (GFP) was introduced as an alternative reporter gene that resulted in a simplification of the whole assay procedure. For evaluation of both reporter systems, seven steroidal compounds with known AR agonistic properties (5 alpha-dihydrotestosterone, testosterone, androstenedione, 17 alpha-methyltestosterone, progesterone, epitestosterone, and d-norgestrel) were tested, and their potencies obtained in the different assays were compared. Furthermore, potencies from the transactivation assays were compared with IC(50) values obtained in radioligand binding assays. The newly developed androgen receptor transactivation assay is a useful tool for characterizing compounds with androgenic activity.

Hormonally induced azoospermia induced by weekly im injections of testosterone enanthate provides effective and reversible male contraception, but more practical regimens are needed. Given our previous findings that six 200-mg pellets implanted subdermally produced more stable, physiological T levels and reduced the delivered T dose by more than 50% while maintaining equally effective suppression of sperm output with fewer metabolic side-effects than weekly 200-mg testosterone enanthate injections, we sought in this study to determine 1) whether further dose-sparing could be achieved by lower testosterone doses while maintaining efficacy and 2) the efficacy of adding a depot progestin to a suboptimally suppressive depot testosterone dose as a model depot progestin/androgen combination male contraceptive. Healthy volunteers were randomized into groups (n = 10) who received either of two lower T doses (two or four 200-mg T pellets) or four 200-mg T pellets plus a single im injection of 300 mg depot medroxyprogesterone acetate (DMPA). Two T pellets (400 mg, 3 mg/day) had a negligible effect on sperm output. Four T pellets (800 mg, 6 mg/day) suppressed sperm output between the second to fourth postimplant months; output returned to normal by the seventh postimplant month, although only 4 of 10 men became azoospermic or severely oligozoospermic (< 3 mol/L/mL). The addition of a depot progestin markedly increased the extent, but not the rate, of sperm output suppression, with 9 of 10 becoming azoospermic and 10 of 10 becoming severely oligozoospermic. There were no serious adverse effects during the study. Plasma total and free testosterone levels remained within the eugonadal range at all times with each treatment. Plasma epitestosterone was suppressed by all 3 regimens, consistent with a dose-dependent inhibition of endogenous Leydig cell steroidogenesis. Plasma LH and FSH measured by a two-site immunoassay were suppressed in a dose-dependent fashion by T and further suppressed by the addition of DMPA. Sex hormone-binding globulin levels were decreased by DMPA, but not by either T dose. Prostate-specific antigen and lipids (total, low or high density lipoprotein cholesterol, and triglycerides) were not significantly changed in any group. Thus, a depot testosterone preparation with zero order release must be delivered at between 6-9 mg/day to provide optimal (but not uniform) efficacy at inducing azoospermia. The addition of a single depot dose of a progestin to a suboptimal testosterone dose (6 mg/day) markedly enhances the extent, but not the rate, of spermatogenic suppression, with negligible biochemical androgenic side-effects. These findings provide a basis for the use of a progestin/androgen combination depot for hormonal male contraception.

The ratio of the concentration of testosterone glucuronide to the concentration of epitestosterone glucuronide (T/E ratio) as determined in urine is the most frequently used method to prove testosterone abuse by athletes. A T/E ratio higher than 6 has been considered as proof of abuse in the past; however, cases of naturally occurring higher T/E ratios have been described. Since the introduction of the T/E ratio in doping analysis, the parameters that may or may not influence the T/E ratio, possibly leading to false-positive results, have been debated. To achieve more insight on the influencing circumstances, an overview is given to obtain an objective view on the merits of the urinary T/E ratio. Relevant analytical aspects of the T/E ratio, potential parameters of endogenous and exogenous origins, as well as some alternative methods to determine testosterone abuse, such as the urinary testosterone/luteinizing hormone ratio, gas chromatography-combustion-isotope-ratio mass spectrometry, hair analysis, and high-performance liquid chromatography-mass spectrometry, are discussed.

The application of a comprehensive gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS)-based method for stable carbon isotopes of endogenous urinary steroids is presented. The key element in sample preparation is the consecutive cleanup with high-performance liquid chromatography (HPLC) of underivatized and acetylated steroids, which allows the isolation of ten analytes (11beta-hydroxyandrosterone, 5alpha-androst-16-en-3beta-ol, pregnanediol, androsterone, etiocholanolone, testosterone, epitestosterone, 5alpha-androstane-3alpha,17beta-diol, 5beta-androstane-3alpha,17beta-diol and dehydroepiandrosterone) from a single urine specimen. These steroids are of particular importance to doping controls as they enable the sensitive and retrospective detection of steroid abuse by athletes. Depending on the biological background, the determination limit for all steroids ranges from 5 to 10 ng/mL for a 10 mL specimen. The method is validated by means of linear mixing models for each steroid, which covers repeatability and reproducibility. Specificity was further demonstrated by gas chromatography/mass spectrometry (GC/MS) for each analyte, and no influence of the sample preparation or the quantity of analyte on carbon isotope ratios was observed. In order to determine naturally occurring (13)C/(12)C ratios of all implemented steroids, a reference population of n = 61 subjects was measured to enable the calculation of reference limits for all relevant steroidal Delta values.

Epitestosterone has been identified as a natural component of biological fluids of several mammals including man. For a long time it was believed that it is a metabolite without any hormonal activity and without any marked relationship to the hormonal state in health and disease. Neither the biosynthetic pathway nor the site of its formation in man have been unequivocally confirmed to date. It apparently parallels the formation of testosterone (T), but on the other hand its concentration is not influenced by exogenous administration of testosterone. This fact creates the basis of the present doping control of testosterone abuse. In 1989 an observation was presented in a dermatological study that epitestosterone exerts an effect counteracting the action of testosterone on flank organ of Syrian hamster. Further studies showed that a complex action consisting of competitive binding of epitestosterone to androgen receptor, of inhibition of testosterone biosynthesis and its reduction to dihydrotestosterone and of antigonadotropic activity could be demonstrated in rat, mice and human tissues. It can be presumed that epitestosterone as a natural hormone can contribute to the regulation of such androgen dependent events as, e.g. the control of prostate growth or body hair distribution.

Sustained anabolic-androgenic steroid (AAS) use has adverse behavioral consequences, including aggression, violence and impulsivity. Candidate mechanisms include disruptions of brain networks with high concentrations of androgen receptors and critically involved in emotional and cognitive regulation. Here, we tested the effects of AAS on resting-state functional brain connectivity in the largest sample of AAS-users to date. We collected resting-state functional magnetic resonance imaging (fMRI) data from 151 males engaged in heavy resistance strength training. 50 users tested positive for AAS based on the testosterone to epitestosterone (T/E) ratio and doping substances in urine. 16 previous users and 59 controls tested negative. We estimated brain network nodes and their time-series using ICA and dual regression and defined connectivity matrices as the between-node partial correlations. In line with the emotional and behavioral consequences of AAS, current users exhibited reduced functional connectivity between key nodes involved in emotional and cognitive regulation, in particular reduced connectivity between the amygdala and default-mode network (DMN) and between the dorsal attention network (DAN) and a frontal node encompassing the superior and inferior frontal gyri (SFG/IFG) and the anterior cingulate cortex (ACC), with further reductions as a function of dependency, lifetime exposure, and cycle state (on/off).

The large variation in disposition known for most drugs is also true for anabolic androgenic steroids. Genetic factors are probably the single most important cause of this variation. Further, there are reasons to believe that there is a corresponding variation in efficacy of doping agents. Doped individuals employ a large variety of doping strategies in respect of choice of substance, dose, dose interval, duration of treatment and use of other drugs for enforcement of effects or correction of side effects. Metabolic steps up-stream and down-stream of testosterone are genetically variable and contribute substantially to the variation in disposition of testosterone, the most common doping agent in sports and in society. Large inter- and intra-ethnic variation in testosterone glucuronidation and excretion is described as well as the pit-falls in evaluation of testosterone doping test results. The hydrolysis and bioactivation of testosterone enanthate is also genetically variable yielding a 2-3 fold variation in excretion rate and serum concentration, thereby implicating a substantial variation in 'efficacy' of testosterone. Given this situation it is logical to adopt the new findings in the doping control programme. The population based cut-off level for the testosterone : epitestosterone ratio should be replaced by a Bayesian interpretation of consecutive tests in the same individual. When combined with the above genetic information the sensitivity of the test is considerably improved. The combination of the three approaches should reduce the rate of falsely negative or positive results and the number of expensive follow-up tests, stipulated by the World Anti-Doping Agency.