Short-term stability of testosterone and epitestosterone conjugates in urine samples: quantification by liquid chromatography-linear ion trap mass spectrometry

A simple method using liquid chromatography-linear ion trap mass spectrometry for simultaneous determination of testosterone glucuronide (TG), testosterone sulfate (TS), epitestosterone glucuronide (EG) and epitestosterone sulfate (ES) in urine samples was developed. For validation purposes, a urine containing no detectable amount of TG, TS and EG was selected and fortified with steroid conjugate standards. Quantification was performed using deuterated testosterone conjugates to correct for ion suppression/enhancement during ESI. Assay validation was performed in terms of lower limit of detection (1-3ng/mL), recovery (89-101%), intraday precision (2.0-6.8%), interday precision (3.4-9.6%) and accuracy (101-103%). Application of the method to short-term stability testing of urine samples at temperature ranging from 4 to 37 degrees C during a time-storage of a week lead to the conclusion that addition of sodium azide (10mg/mL) is required for preservation of the analytes.     

Changes in androgenic steroid profile due to urine contamination by microorganisms: a prospective study in the context of doping control

Urine contamination by microorganisms may affect the interpretation of urinalysis in different areas of clinical diagnosis. This is particularly relevant in doping control. A prospective study was designed to assess the effects of urine contamination by selected pathogens on the endogenous androgenic steroid profile. Pooled urine from a healthy male volunteer with standard steroid profile compared with reference values for the Caucasian population was sterilized by filtration and stored in sterile glass tubes. Aliquots were inoculated with known amounts of 15 different organisms (bacteria, fungi, and moulds) and incubated at 37 degrees C for 2 weeks. Different markers of urine contamination, such as pH, deconjugation of steroids, and metabolic by-products, were determined. Alkalization of urinary pH was not a reliable indicator of urine contamination as several organisms grew in this medium and no alteration of this parameter was found. In uncontaminated urine, less than 10% of steroid glucuronide conjugates were spontaneously hydrolyzed. Higher rates of hydrolysis for sulfate conjugates were found. An unconjugated fraction higher than 10% of the total amount of testosterone was a reliable indicator of urine contamination. However, microbial production of testosterone or epitestosterone was not detected. In contrast, a few organisms were able to synthesize 5alpha-androstanedione, 5beta-androstanedione, and androstenedione using endogenous steroids as substrates.     

Plasma and urinary markers of oral testosterone undecanoate misuse.

Orally administered testosterone undecanoate (TU), an anabolic, androgenic steroid, can potentially be abused by athletes. Indirect evidence for detecting oral TU intake could be deduced from the changes in steroid profile post-administration. Direct evidence could be obtained by detection of unchanged TU in plasma. To this end, both urinary and plasma steroid profiles of six healthy male subjects given a single oral dose of 120 mg of TU were studied by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/tandem mass spectrometry (GC/MS/MS). The increased concentration of glucuronidated testosterone in plasma appears to be the most characteristic sign of oral TU intake. The testosterone glucuronide (TG)/nonconjugated testosterone (T) ratio, TG/17-hydroxyprogesterone (17OHP) ratio, and TG/luteinizing hormone (LH) ratio were observed to be significantly elevated above their basal levels for 10 h, 10 h, and 6 h, respectively. Urinary ratios of TG/epitestosterone glucuronide (EG) were found to be higher than the cut-off value of 6 for the period 4 approximately 8 h post-administration, but only in three subjects. One subject failed to respond with respect to all of the above-mentioned indirect markers, as TG was not significantly increased in either plasma or urine. Unchanged TU was directly detected in plasma of all six subjects from 1 approximately 1.5 h to 4 approximately 6 h after oral TU intake by GC/MS/MS, providing unequivocal proof of exogenous testosterone intake. Distinct and complementary markers for detecting oral TU intake could be obtained from plasma and urine, respectively.     

Non-steroidal anti-inflammatory drugs interact with testosterone glucuronidation

Testosterone and epitestosterone are secreted mainly as glucuronide metabolites and the urinary ratio of testosterone glucuronide to epitestosterone glucuronide, often called T/E, serves as a marker for possible anabolic steroids abuse by athletes. UDP-glucuronosyltransferase (UGT) 2B17 is the most important catalyst of testosterone glucuronidation. The T/E might be affected by drugs that interact with UGT2B17, or other enzymes that contribute to testosterone glucuronidation. Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used by sportsmen and we have examined the effect of two NSAIDs, diclofenac and ibuprofen, on testosterone and epitestosterone glucuronidation in human liver microsomes. In parallel, we have studied the inhibitory effect of these NSAIDs on recombinant UGT2B17 and UGT2B15, as well as other human hepatic UGTs that revealed low but detectable testosterone glucuronidation activity, namely UGT1A3, UGT1A4, UGT1A9 and UGT2B7. Both diclofenac and ibuprofen inhibited testosterone glucuronidation in microsomes, as well as UGT2B15 and UGT2B17. Interestingly, UGT2B15 was more sensitive than UGT2B17 to the two drugs, particularly to ibuprofen. Human liver microsomes lacking functional UGT2B17 exhibited significantly higher sensitivity to ibuprofen, suggesting that UGT2B15 plays a major role in the residual testosterone glucuronidation activity in UGT2B17-deficient individuals. Nonetheless, a minor contribution of other UGTs, particularly UGT1A9, to testosterone glucuronidation in such individuals cannot be ruled out at this stage. The epitestosterone glucuronidation activity of human liver microsomes was largely insensitive to ibuprofen and diclofenac. Taken together, the results highlight potential interactions between NSAIDs and androgen glucuronidation with possible implications for the validity of doping tests.     

Combined profile of androgen glucuro- and sulfoconjugates in post-competition urine of sportsmen: a simple screening procedure using gas chromatography-mass spectrometry

An analytical screening procedure has been developed for the estimation of total androgen conjugates in post-competition urine, using gas chromatography-mass spectrometry with computerized data acquisition and concentration calculation. Rapid acid-catalyzed methanolysis is a key feature of the method, which allows simulatenous cleavage of glucuronides and sulfates. Analytical data generated by this method for testosterone and epitestosterone are in accordance with our previous results obtained by more accurate isotope dilution mass spectrometry. The usefulness of the ratio of testosterone glucuronide-total epitestosterone as an aid for a better discrimination between physiologically high and pharmacologically high ratios of testosterone glucuronide-epitestosterone glucuronide, which was demonstrated previously, has been confirmed here.     

Substantial advantage of a combined Bayesian and genotyping approach in testosterone doping tests

Testosterone abuse is conventionally assessed by the urinary testosterone/epitestosterone (T/E) ratio, levels above 4.0 being considered suspicious. A deletion polymorphism in the gene coding for UGT2B17 is strongly associated with reduced testosterone glucuronide (TG) levels in urine. Many of the individuals devoid of the gene would not reach a T/E ratio of 4.0 after testosterone intake.Future test programs will most likely shift from population based- to individual-based T/E cut-off ratios using Bayesian inference. A longitudinal analysis is dependent on an individual's true negative baseline T/E ratio.The aim was to investigate whether it is possible to increase the sensitivity and specificity of the T/E test by addition of UGT2B17 genotype information in a Bayesian framework.A single intramuscular dose of 500 mg testosterone enanthate was given to 55 healthy male volunteers with either two, one or no allele (ins/ins, ins/del or del/del) of the UGT2B17 gene.Urinary excretion of TG and the T/E ratio was measured during 15 days.The Bayesian analysis was conducted to calculate the individual T/E cut-off ratio.When adding the genotype information, the program returned lower individual cut-off ratios in all del/del subjects increasing the sensitivity of the test considerably.It will be difficult, if not impossible, to discriminate between a true negative baseline T/E value and a false negative one without knowledge of the UGT2B17 genotype.UGT2B17 genotype information is crucial, both to decide which initial cut-off ratio to use for an individual, and for increasing the sensitivity of the Bayesian analysis.     

Effect of oxidizing adulterants on human urinary steroid profiles

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

Direct detection of boldenone sulfate and glucuronide conjugates in horse urine by ion trap liquid chromatography-mass spectrometry

A study of the equine phase II metabolism of the anabolic agent boldenone is reported. Boldenone sulfate, boldenone glucuronide and their C17-epimers were synthesised as reference standards in our lab and a method was developed for their detection in a horse urine matrix. Solid phase extraction was used to purify the analytes, which were then detected by ion trap LC/MS. Negative and positive ionisation mode MS(2) were used for the detection of sulfate and glucuronide conjugates, respectively. Boldenone sulfate and 17-epiboldenone glucuronide were detected as the major and minor phase II metabolites, respectively, in horse urine samples collected following the administration of boldenone undecylenate by intramuscular injection.     

Dietary green and white teas suppress UDP-glucuronosyltransferase UGT2B17 mediated testosterone glucuronidation

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.     

Bioavailability of (-)-epicatechin upon intake of chocolate and cocoa in human volunteers

We evaluated the levels of (-)-epicatechin (EC) and its metabolites in plasma and urine after intake of chocolate or cocoa by male volunteers. EC metabolites were analyzed by HPLC and LC/MS after glucuronidase and/or sulfatase treatment. The maximum levels of total EC metabolites in plasma were reached 2 hours after either chocolate or cocoa intake. Sulfate, glucuronide, and sulfoglucuronide (mixture of sulfate and glucuronide) conjugates of nonmethylated EC were the main metabolites present in plasma rather than methylated forms. Urinary excretion of total EC metabolites within 24 hours after chocolate or cocoa intake was 29.8 ± 5.3% and 25.3 ± 8.1% of total EC intake. EC in chocolate and cocoa was partly absorbed and was found to be present as a component of various conjugates in plasma, and these were rapidly excreted in urine.     

Bioavailability of (-)-epicatechin upon intake of chocolate and cocoa in human volunteers

We evaluated the levels of (-)-epicatechin (EC) and its metabolites in plasma and urine after intake of chocolate or cocoa by male volunteers. EC metabolites were analyzed by HPLC and LC/MS after glucuronidase and/or sulfatase treatment. The maximum levels of total EC metabolites in plasma were reached 2 hours after either chocolate or cocoa intake. Sulfate, glucuronide, and sulfoglucuronide (mixture of sulfate and glucuronide) conjugates of nonmethylated EC were the main metabolites present in plasma rather than methylated forms. Urinary excretion of total EC metabolites within 24 hours after chocolate or cocoa intake was 29.8 ± 5.3% and 25.3 ± 8.1% of total EC intake. EC in chocolate and cocoa was partly absorbed and was found to be present as a component of various conjugates in plasma, and these were rapidly excreted in urine.     

Differential metabolism of dehydroepiandrosterone sulfate and estrogen conjugates by normal or malignant AXC/SSh rat prostate cells and effects of these steroid conjugates on cancer cell proliferation in vitro

Normal AXC/SSh rat ventral prostate and clonally derived AXC/SSh rat prostate cancer cells were evaluated for ability to metabolize estrone sulfate (E1S), estrone glucuronide (E1G), or dehydroepiandrosterone sulfate (DHEAS). Both normal and malignant prostate cells converted E1S to estrone. Neither normal nor malignant prostate cells had significant ability to metabolize DHEAS to DHEA, indicating differential specificity of prostate sulfatases(s) for estrogen and androgen sulfates. Both normal and neoplastic prostate cells possess beta-glucuronidase which hydrolyzed E1G to estrone. To assess potential physiologic consequences of these enzymatic activities, we determined the effect of steroid conjugates on in vitro proliferation of selected clonal lines of AXC/SSh rat prostate cancer cells. DHEAS, 10(-6) to 10(-9) M in decade intervals, did not affect in vitro proliferation of AXC/SSh prostate cancer cells; however, 10(-5) M DHEAS decreased in vitro proliferation of these cells. Neither E1S nor E1G, 10(-5) to 10(-9) M in decade intervals, affected in vitro proliferation of AXC/SSh prostate cancer cells. These findings suggest that low residual levels of steroid conjugates, which are not removed by charcoal stripping of serum, do not affect demonstrated in vitro androgen modulation of AXC/SSh rat prostate cancer cell proliferation (Cancer Res. 46, 3775-3781, 1986).     

LC-MS/MS characterization of the urinary excretion profile of the mycotoxin deoxynivalenol in human and rat

The understanding of mycotoxins transfer to biological fluids is challenged by the difficulties in performing and replicating in vivo experiments as well as the lack of suitable methods of analysis to detect simultaneously a range of chemically different metabolites at trace levels. LC-MS/MS has been used herein to study the urinary excretion profile of the mycotoxin deoxynivalenol in human and Wistar rat. Deoxynivalenol and deoxynivalenol glucuronide were found in both human and rat urines, whereas de-epoxydeoxynivalenol and its glucuronide conjugate were only detected in rat urine. The presence of two deoxynivalenol glucuronide isomers in Wistar rat urine has been shown for the first time. Structure confirmation of the detected metabolites was provided by the analysis of fragmentation patterns. A solid phase extraction clean up procedure allowing recoveries in the range 72-102% for deoxynivalenol, de-epoxydeoxynivalenol, and their glucuronide conjugates was optimized. A multiple reaction monitoring method for the simultaneous determination of all investigated metabolites was elaborated allowing the direct detection of deoxynivalenol metabolites without the hydrolysis step. Deoxynivalenol urinary levels in the range 0.003-0.008 μg/ml were detected in healthy human subjects, whereas deoxynivalenol and de-epoxynivalenol levels between 1.9-4.9 μg/ml and 1.6-5.9 μg/ml, respectively were found in administered rat urine. These findings emphasize the relevance of the highly selective and sensitive LC-MS/MS technique for the direct detection and characterization of deoxynivalenol metabolites in complex biological matrices.     

Quantification of the alpha- and gamma-tocopherol metabolites 2,5,7, 8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman and 2,7, 8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman in human serum.

alpha- and gamma-tocopherol are the major vitamin E compounds found in human blood and tissues. The metabolites are 2,5,7, 8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman (alpha-CEHC) and 2,7,8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman (gamma-CEHC, LLU-alpha), respectively. alpha-CEHC is excreted mainly as glucuronide or sulfate conjugates in the urine. Here we describe a sensitive and reliable method to analyze alpha- and gamma-CEHC in human serum. The concentration of alpha-CEHC in human serum is in the range of 5-10 pmol/ml but increases significantly up to 200 pmol/ml upon supplementation with RRR-alpha-tocopherol. About one-third of the alpha-CEHC circulating in the blood is present as a glucuronide conjugate. Baseline levels of gamma-CEHC are about 50 to 85 pmol/ml.     

Enzyme-assisted synthesis and characterization of glucuronide conjugates of neuroactive steroids

Synthesis of reference standards is needed to determine the presence and function of steroid glucuronides in the brain or other tissues, because commercial sources of steroid glucuronide standards are limited or unavailable. In the present study porcine, rat, and bovine liver microsomes were tested to evaluate their ability to glucuronidate eight neurosteroids and neuroactive steroids of various types: dehydroepiandrosterone, pregnenolone, isopregnanolone, 5alpha-tetrahydrodeoxycorticosterone, corticosterone, cortisol, beta-estradiol, and testosterone. In general, the glucuronidation efficiency of rat liver was rather poor compared with that of bovine and porcine liver microsomes. Since porcine liver apparently has a relatively large amount of dehydrogenase, its microsomes also produced dehydrogenated steroids and their glucuronides, as well as various regioisomers in which the site of glucuronidation varied. In contrast, bovine liver microsomes produced mainly a single major glucuronidation product and few dehydrogenation products and gave the best overall yield for two-third of the steroids tested. The enzymatic synthesis of five glucuronides of four steroids was carried out and the conditions, purification, and analytical methods for the glucuronidation products were optimized. The steroid glucuronides synthesized were characterized by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography-mass spectrometry (LC-MS). The stereochemically pure steroid glucuronide conjugates were recovered in milligram amounts (yield 10-78%) and good purity (>85-90%), which is sufficient for LC-MS/MS method development and analyses of steroid glucuronides in biological matrices such as brain, urine, or plasma.     

Relevance of the selective oestrogen receptor modulators tamoxifen, toremifene and clomiphene in doping field: endogenous steroids urinary profile after multiple oral doses

The present study was performed to investigate the influence of the intake of selective oestrogen receptor modulators on the urinary endogenous steroids profile. For this purpose the circadian variability of luteinizing hormone, follicle-stimulating hormone, testosterone, 5α-androstan-3α,17β-diol, 5β-androstan-3α,17β-diol, epitestosterone, 4-androstenedione, androsterone and etiocholanolone were measured on eight subjects (four males and four females) by gas chromatography–mass spectrometry and chemiluminescent immunometric assay techniques before and after oral administration of multiple doses of either tamoxifen (80 mg for 2 days) or toremifene (120 mg for 2 days) or clomiphene (100 mg for 2 days). The individual baseline variability of the steroids studied was set up by collecting the urine samples every 3 h, for 3 days prior to the treatment; whereas the evaluation of the effects of the oral administration of multiple doses of selective oestrogen receptor modulators on the steroid urinary profile was assessed by collecting urine samples every three hours for at least five days from the first administration.The results of our measurements showed that, only in male subjects, the relative urinary concentrations of testosterone, epitestosterone and 4-androstenedione were significantly altered generally after the second day of drug administration. While no significant effects were recorded in both sexes on the luteinizing hormone, follicle-stimulating hormone, androsterone, etiocholanolone, 5α-androstan-3α,17β-diol and 5β-androstan-3α,17β-diol urinary levels and on testosterone/epitestosterone, 5α-androstan-3α,17β-diol/5β-androstan-3α,17β-diol and androsterone/etiocholanolone ratios.     

Sex steroids in scleractinian coral,Euphyllia ancora: implication in mass spawning

The objectives of this study were to investigate the presence and annual cycle of sex steroids in scleractinian coral, Euphyllia ancora. The free and conjugated forms of sex steroids in coral and spawning seawater were investigated, and aromatase activity in the coral tissue was identified. Polyps collected from corals and seawater were extracted with diethyl ether, and purified by alumina column and reversed-phase HPLC; testosterone and estradiol-17beta (E2) was measured by a validated RIA. E2 and testosterone in their free and glucuronide forms were consistently detected in coral tissue throughout the year. Peak concentrations of free E2, E2 glucuronide, and testosterone glucuronide were obtained in the coral tissue just prior to spawning. The presence of specific aromatase activity was demonstrated in coral tissue. Free E2 and E2 glucuronide concentrations were higher than androgen (testosterone and testosterone glucuronide) in coral tissue and spawning seawater. Higher concentrations of free E2 than E2 glucuronide were detected in coral tissues throughout the year. In contrast, higher concentrations of E2 glucuronide than free E2 and testosterone glucuronide were found in seawater during mass coral spawning. No steroid sulfate could be detected in the coral tissue and seawater. We suggest that the release of E2 glucuronide may play an important role in coral mass spawning.     

Influence of diet on plasma steroids and sex hormone-binding globulin levels in adult men

Several experimental studies have suggested that diet can alter the production and metabolism of steroids in men. The purpose of this study was to determine the levels of unconjugated steroids and steroid glucuronides as well as sex hormone-binding globulin (SHBG) among normal adult men who were either omnivorous or vegetarians. The participants were white volunteers ranging from 25-35 years of age and the blood samples were taken between 0900 h and 1000 h and between 1600 h and 1700 h for two consecutive days. No significant statistical change was found in plasma dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, dihydrotestosterone and estradiol levels. Vegetarian group showed a higher levels of sex hormone-binding globulin (SHBG) while the free androgen index (FAI; calculated by the ratio testosterone/SHBG) was lower in this group. Although the concentrations of androsterone glucuronide were higher in vegetarian group, the vegetarians had a 25-50% lower level of androstane-3 alpha, 17 beta-diol glucuronide and androstane-3 beta,17 beta-diol glucuronide. Our data further indicate that both, androstane-3 alpha,17 beta-diol glucuronide and androstane-3 beta,17 beta-diol glucuronide concentrations are significantly correlated with SHBG levels and with the FAI values. The increases in androstane-3 alpha,17 beta-diol glucuronide and androstane-3 beta,17 beta-diol glucuronide levels in the omnivorous group are probably a consequence of the elevation of the FAI. Our data suggest that in a vegetarian group, less testosterone is available for androgenic action.     

Alternative markers for the long-term detection of oral testosterone misuse

The screening of testosterone misuse in the doping control field is normally performed by the measurement of the ratio between the concentrations of testosterone and epitestosterone excreted as glucuronides (T/E). Despite the satisfactory results obtained with this approach, the measurement of T/E presents some limitations like the long-term detection of oral testosterone administration. Recently, several testosterone metabolites released after basic treatment of the urine have been reported (androsta-1,4-dien-3,17-dione, androsta-4,6-dien-3,17-dione, 17β-hydroxy-androsta-4,6-dien-3-one and 15-androsten-3,17-dione). In the present work, the usefulness of these metabolites for the detection of oral testosterone misuse has been evaluated and compared with the conventional T/E measurement. For this purpose, 173 urine samples collected from healthy volunteers were analysed in order to obtain reference concentrations for the four metabolites released after alkaline treatment. On the other hand, urine samples collected from five volunteers before and after testosterone undecanoate administration were also analysed. Concentrations of androsta-4,6-dien-3,17-dione and 17β-hydroxy-androsta-4,6-dien-3-one showed a similar behaviour as the T/E, allowing the detection of the misuse for several hours after administration. More promising results were obtained by quantifying androsta-1,4-dien-3,17-dione and 15-androsten-3,17-dione. The time in which the concentrations of these analytes could be differentiated from the basal level was between 3 and 6 times longer than the obtained with T/E, as a result, an improvement in the detection of testosterone abuse can be achieved. Moreover, several ratios between these compounds were evaluated. Some of them improved the detection of testosterone misuse when comparing with T/E. The best results were obtained with those ratios involving androsta-1,4-dien-3,17-dione.     

Increased urinary testosterone/epitestosterone ratios found in Swedish athletes in connection with a national control program Evaluation of 28 cases

In connection with a national anti-doping control program, including analysis of 8946 urine samples, 28 athletes were found to have delivered samples free from xenobiotic anabolic steroids but with an increased testosterone/epitestosterone (T/E)_ratio (>6). Unannounced testing of the above athletes produced 2–4 additional urine samples during the next 2–3 months. A low degree of variation of the T/E ratio, with a C.V. below 30% was found in 17 of the subjects whereas 10 had a C.V. varying from 31% to 43%. One subject with a high urinary T/E ratio (10.5) had a C.V. of this ratio of 126% and also an extremely high ratio between testosterone and LH in urine. It has been reported that non-users of testosterone have T/E/ ratios fluctuating around a mean with a C.V. that will not exceed 30%. We found that administration of testosterone to seven healthy volunteers resulted in urinary T/E ratios that varied with a C.V. ranging from 67% to 130% during the following 4 weeks. It is concluded that among the above 28 cases, only one can be regarded as a clear case of testosterone doping. Although the vast majority of Swedish athletes have urinary T/E ratios below six, there is a subfraction with a constant higher ratio, possibly due to genetic factors.