BACKGROUND

Epitestosterone is prohibited by sport authorities because its administration will lower the urinary testosterone/epitestosterone ratio, a marker of testosterone administration. A definitive method for detecting epitestosterone administration is needed.

METHODS

We developed a gas chromatography-combustion-isotope ratio mass spectrometry method for measuring the delta(13)C values for urinary epitestosterone. Sample preparation included deconjugation with beta-glucuronidase, solid-phase extraction, and semipreparative HPLC. Epitestosterone concentrations were determined by gas chromatography-mass spectrometry for urines obtained from a control group of 456 healthy males. Epitestosterone delta(13)C values were determined for 43 control urines with epitestosterone concentrations>> or =40 microg/L (139 nmol/L) and 10 athletes' urines with epitestosterone concentrations>> or =180 microg/L (624 nmol/L), respectively.

RESULTS

The log epitestosterone concentration distribution was gaussian [mean, 3.30; SD, 0.706; geometric mean, 27.0 microg/L (93.6 nmol/L)]. The delta(13)C values for four synthetic epitestosterones were low (less than or equal to -30.3 per thousand) and differed significantly (P <0.0001). The SDs of between-assay precision studies were low (< or =0.73 per thousand). The mean delta(13)C values for urine samples obtained from 43 healthy males was -23.8 per thousand (SD, 0.93 per thousand). Nine of 10 athletes' urine samples with epitestosterone concentrations >180 microg/L (624 nmol/L) had delta(13)C values within +/- 3 SD of the control group. The delta(13)C value of epitestosterone in one sample was -32.6 per thousand (z-score, 9.4), suggesting that epitestosterone was administered. In addition, the likelihood of simultaneous testosterone administration was supported by low delta(13)C values for androsterone and etiocholanolone.

CONCLUSIONS

Determining delta(13)C values for urinary epitestosterone is useful for detecting cases of epitestosterone administration because the mean delta(13)C values for a control group is high (-23.8 per thousand) compared with the delta(13)C values for synthetic epitestosterones.

This investigation gives an overview of the concentrations of naturally occurring androgens, progestogens, corticosteroids, and their precursors and metabolites in meat from bulls and steers. A recently developed gas chromatography-mass spectrometry IGC-MS) method with improved sensitivity for steroid analysis was used. Eighty-two beef samples were analyzed using the GC-MS method. Beef from bulls contained higher concentrations of testosterone, which is an anabolic androgen, and its metabolite epitestosterone (P < .01) and the androgen precursor dehydroepiandrosterone (P < .05) than beef from steers. Beef from steers contained higher (P < .05) concentrations of the basic hormone precursor pregnenolone and cortisol, which is a catabolic corticosteroid, than beef from bulls. A classification of an unknown beef sample to one of the categories (bull or steer) was possible in most cases (>90%) using a masculinity index (MI) that was calculated using the concentrations of testosterone, epitestosterone, and pregnenolone. Because the hormonal status of beef cattle is related to meat quality characteristics, such as tenderness or fat and protein distribution, the MI may contribute to meat quality assessment and meat quality control.

5 alpha-reductase activity was investigated in scalp hair roots in 5 patients with 5 alpha-reductase deficiency and in 10 normal volunteers (5 men and 5 women) who served as controls. Metabolism of [3H]testosterone and [3H]epitestosterone was measured under standardized conditions in 1-4 growing (anagen) hair roots. When the mean values of the 5 alpha-reduced metabolites from testosterone (androstanedione and dihydrotestosterone) or from epitestosterone (epidihydrotesterone) were compared in both groups, similar values were observed. However, since most of the hair roots were incubated singly, it could be demonstrated that in two patients 20 respectively 24% of the hair roots showed neither dihydrotestosterone nor epidihydrotestestosterone formation. This did not influence the mean values determined for the whole group perceptibly. Our results support the concept that 5 alpha-reductase deficiency is a genetically heterogenous disorder by demonstrating that the enzyme defect is variously expressed in hair roots of affected individuals.

Cell differentiation increases UDP-glucuronosyltransferase (UGT) gene expression in Caco-2 cells. Glucuronidation of 13 UGT substrates, 1-naphthol, diclofenac, epitestosterone, estradiol, ethinylestradiol, indomethacin, oxazepam, R- and S-propranolol, propofol, testosterone, trifluoperazine, and zidovudine, were studied to derive a broad view on the effect of cell differentiation on the glucuronidation activities of different human UGTs. In parallel, the glucuronidation of these compounds in human liver microsomes (HLM) and human intestinal microsomes (HIM) was analyzed. Because many of the substrates are highly lipophilic, the effects of dimethyl sulfoxide (DMSO) concentrations in the reaction mixture on glucuronidation rates were tested, as well as the effect of alamethicin, a pore-forming peptide. Large differences were observed in the effects of DMSO and alamethicin between recombinant UGTs and Caco-2 cells and HLM and HIM, and, therefore, the activity assays were performed under multiple conditions. Regardless of the assay conditions, however, the results clearly indicated that although differentiation increases glucuronidation activity, the rates in Caco-2 cells are mostly very low, much lower than those in either HLM or HIM. One clear exception was observed: substrates of UGT1A6, such as 1-naphthol, were glucuronidated at very high rates in both undifferentiated and differentiated Caco-2 cells. It may thus be concluded that Caco-2 cells, even differentiated ones, do not provide a good model system to assess first-pass drug glucuronidation in the intestine.

The effect of temperature on the seasonal production of testicular androgens, in vitro, was examined in the scincid lizard Tiliqua (Trachydosaurus) rugosa. Testicular tissue was incubated, in vitro, at various temperatures (18, 25, 32 and 37 degrees C). Endogenous androgens, testosterone and epitestosterone, were measured by radioimmunoassay. Epitestosterone production was maximal at 37 degrees C and minimal at 18 degrees C. There was no consistent effect of incubation temperature on testosterone production. Incubation temperature had no effect on the seasonal pattern of androgen production. The results suggest that temperature may play a part in the regulation of androgen biosynthesis in T. rugosa.

1. Steroids interact with bovine plasma albumin at a binding region that involves tryptophanyl, tyrosyl, arginyl and lysyl residues. The function of the tryptophanyl residues is demonstrated by: (a) the decrease of albumin binding affinity after modification of one tryptophanyl with 2-nitrophenylsulfenyl chloride; (b) steroid quenching of albumin tryptophanyl fluorescence; and (c) steroid quenching of 1-anilinonaphth-alene-8-sulfonate fluorescence, when it is excited by energy transfer from excited tryptophanyls. The function of tyrosyl residues is demonstrated by the decrease of albumin binding affinity after nitration of 30% tyrosyls with tetranitromethane, or deprotonation of tyrosyls by variation of pH. The function of arginyl and lysyl residues is demonstrated by the decrease of binding affinity after modification of these residues with glyoxal, formaldehyde or acetic anhydride. The presence of both apolar (Trp, Tyr and Lys (deprotonated)) and polar (Arg and Lys(protonated)) residues at the steroid binding site fits in well with the site relative apolarity, when expressed on the Kosower scale (Kosower, E.M. (1958) J. Am. Chem. Soc. 80, 3253-3260). 2. The contribution of specific amino acid residues to steroid binding depends to some extent on the steroid structure, as exemplified by the quantitatively different role of arginyl (or lysyl) residues in albumin interaction with testosterone acetate and epitestosterone, respectively, or that of tyrosyl residues in albumin interaction with 11-deoxycorticosterone and epitestosterone, respectively. 3. The concerted action of polar and apolar amino acid residues is an essential requirement for steroid binding, since unfolding of albumin polypeptide chain by guanidine-HC1, urea, or by reduction of disulfide bridges with 2-mercaptoethanol, strongly decreases steroid binding to albumin while, conversely, reoxidation and refolding of the unfolded polypeptide chain restore albumin affinity for steroids. 4. Parallel determinations of steroid binding constants by equilibrium dialysis and fluorimetric titration, as well as the general pattern of the pH and temperature effects on steroid quenching of albumin fluorescence, confirm the validity of the fluorescence quenching titration as an effective method for measuring albumin-steroid molecular interactions.

This work presents the validation study of the comprehensive two-dimensional gas chromatography (GC x GC)-time-of-flight mass spectrometry method performance in the analysis of the key World Anti-Doping Agency (WADA) anabolic agents in doping control. The relative abundance ratio, retention time, identification and other method performance criteria have been tested in the GC x GC format to confirm that they comply with those set by WADA. Furthermore, tens of other components were identified with an average similarity of >920 (on the 0-999 scale), including 10 other endogenous sterols, and full mass spectra of 5,000+ compounds were retained. The testosterone/epitestosterone ratio was obtained from the same run. A new dimension in doping analysis has been implemented by addressing separation improvement. Instead of increasing the method sensitivity, which is accompanied by making the detector increasingly "blind" to the matrix (as represented by selected ion monitoring mode, high-resolution mass spectrometry (MS) and tandem MS), the method capabilities have been improved by adding a new "separation" dimension while retaining full mass spectral scan information. Apart from the requirement for the mass spectral domain that a minimum of three diagnostic ions with relative abundance of 5% or higher in the MS spectra, all other WADA criteria are satisfied by GC x GC operation. The minimum of three diagnostic ions arises from the need to add some degree of specificity to the acquired mass spectrometry data; however, under the proposed full MS scan method, the high MS similarity to the reference compounds offers more than the required three diagnostic ions for an unambiguous identification. This should be viewed as an extension of the present criteria to a full-scan MS method.

Anabolic and androgenic steroids (AASs) are synthetic substances related to the primary male sex hormone, testosterone. AASs can be abused in both human and equine sports and, thus, are banned by the International Olympic Committee and the Association of Racing Commissioners International (ARCI). Enforcement of the ban on the use of AASs in racehorses during competition requires a defensible and robust method of analysis. To address this requirement, a high-throughput ultra high-performance liquid chromatography-mass spectrometric (UHPLC-MS) method was developed for the detection, quantification and confirmation of 55 AASs in equine plasma. AASs were recovered from equine plasma samples by liquid-liquid extraction with methyl tert-butyl ether (MTBE). Analytes were chromatographically separated on a sub-2 µm particle size C(18) column with a mobile phase gradient elution and detected by selected-reaction monitoring (SRM) on a triple quadrupole mass spectrometer. AASs with isobaric precursor ions were either chromatographically resolved or mass spectrometrically differentiated by unique precursor-to-product ion transitions. A few of them that could not be resolved by both approaches were differentiated by intensity ratios of three major product ions. All the epimer pairs, testosterone and epitestosterone, boldenone and epiboldenone, nandrolone and epinandrolone, were chromatographically base-line separated. The limit of detection and that of quantification was 50 pg/ml for most of the AASs, and the limit of confirmation was 100-500 pg/ml. Full product ion spectra of AASs at concentrations as low as 100-500 pg/ml in equine plasma were obtained using the triple quadrupole instrument, to provide complementary evidentiary data for confirmation. The method is sensitive and selective for the detection, quantification and confirmation of multiple AASs in a single analysis and will be useful in the fight against doping of racehorses with AASs.

OBJECTIVE

A regular and intense physical exercise significantly modifies hormonal metabolism and there are many reports of a change in urine steroid levels accompanying the practice of sport. The aim of this study was to compare the urinary steroid profile between highly trained cyclists and untrained subjects.

METHODS

Urine levels of testosterone (T), epitestosterone (Epit), androstenedione, dehydroepiandrosterone (DHEA), androsterone (A), etiocholanolone (E), beta-estradiol (E2), estrone (E1) and the most abundant urine metabolites of cortisol and cortisone, tetrahydrocortisone (THE) and tetrahydrocortisol (THF) were determined by gas chromatography-mass spectrometry in urine samples from a group of professional cyclists (n=15) submitted to maximum level training for several years and compared with urine samples from sedentary subjects (n=15). The relationships between T/Epit, A+E/ THE, A+E/ THF, DHEA/THE and DHEA/THF were also studied.

RESULTS

Cyclists showed lower urine levels of T, A, E and E2 and higher urine levels of androstenedione and E1 than sedentary individuals. A+E/THE and A+E/ THF ratios were higher in sedentary subjects than in cyclists.

CONCLUSIONS

We conclude that cyclists showed a urinary steroid profile different from sedentary individuals, probably due to an adaptation to regular and intense physical training .

Context: Little is known about how exogenous testosterone (T) affects the steroid profile in women. More knowledge would give the anti-doping community keys on how to interpret tests and detect doping.

Objective: To investigate the steroid profile in serum and urine in young healthy women after testosterone administration.

Methods: In a randomized, double-blind, placebo-controlled study, 48 healthy young women, were assigned to treatment with T cream (10 mg) or placebo (1:1) for 10 weeks. Urine and blood were collected before and at end of treatment. Serum steroids were analyzed with LC-MS/MS, and urine levels of T, epitestosterone (E) and metabolites included in the Athlete Biological Passport (ABP) were analyzed with GC-MS/MS.

Results: In serum, T and dihydrotestosterone levels increased, whereas sex-hormone-binding globulin and 17-hydroxyprogesterone decreased after T treatment as compared to placebo. In urine, T and 5α-androstanediol increased in the T group. The median T increase in serum was 5.0-fold (range 1.2-18.2) and correlated to a 2.2-fold (range 0.4-14.4) median increase in T/E in urine (rs=0.76). Only two of the 24 women receiving T reached the T/E cut-off ratio of 4, while when the results were added to the ABP, six of 15 subjects showed atypically high T/E (40%). In comparison, 22/24 women in the T group increased serum T more than 99.9% of the upper confidence limit of non-treated values.

Conclusion: It seems as T/E ratio is not sufficient to detect exogenous testosterone in women. Serum total T concentrations could serve as a complementary marker of doping.

Keywords: athlete biological passport; dihydrotestosterone; doping; steroid profile; testosterone; testosterone/epitestosterone.

A novel steroid-inducible 17 alpha-hydroxysteroid dehydrogenase (17 alpha-HSDH) has been purified over 850-fold from an intestinal Eubacterium sp. VPI 12708. The purified protein has a subunit molecular mass of 42,000 daltons and a native molecular weight (M(r)) of 160,000 as estimated by gel filtration chromatography. Enzyme activity was induced by growth in the presence of androstenedione or cholic acid, but not deoxycholic acid. Enzymatic activity required anaerobic conditions and was highly specific for NADP+ and the 17 alpha-hydroxy group of C-19 steroids. Estimated Km values were 31 microM and 70 microM for androstenedione and epitestosterone, respectively. Vmax values were estimated to be 3250 nmol/min per mg protein and 1800 nmol/min per mg protein for the reductive and oxidative reactions, respectively. The pH optima for both the oxidative and reductive reactions ranged between 5.5 and 7.0. Treatment with EDTA completely inactivated 17 alpha-HSDH activity but activity was partially restored by the addition of either Mg2+ (1 mM) or Zn2+ (10 mM). The N-terminal amino acid sequence analysis of purified enzyme suggests that 17 alpha-HSDH may belong to a disulfide reductase gene family.

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

Manipulation of urine sampling in sports drug testing is considered a violation of anti-doping rules and is consequently sanctioned by regulatory authorities. In 2003, three identical urine specimens were provided by three different athletes, and the identity of all urine samples was detected and substantiated using numerous analytical strategies including gas chromatography-mass spectrometry with steroid and metabolite profiling, gas chromatography-nitrogen/phosphorus detector analysis, high-performance liquid chromatography-UV fingerprinting, and DNA-STR (short tandem repeat) analysis. None of the respective athletes was the donor of the urine provided for doping analysis, which proved to be a urine sample collected from other unidentified individual(s). Samples were considered suspicious based on identical steroid profiles, one of the most important parameters for specimen individualization in sports drug testing. A database containing 14,224 urinary steroid profiles of athletes was screened for specific values of 4 characteristic parameters (ratios of testosterone/epitestosterone, androsterone/etiocholanolone, androsterone/testosterone, and 5alpha-androstane-3alpha,17beta-diol/5beta-androstane-3alpha,17beta-diol) and only the three suspicious samples matched all criteria. Further metabolite profiling regarding indicated medications and high-performance liquid chromatography-UV fingerprinting substantiated the assumption of manipulation. DNA-STR analyses unequivocally confirmed that the 3 urine samples were from the same individual and not from the athletes who provided DNA from either buccal cell material or blood specimens. This supportive evidence led to punishment of all three athletes according to the rules of the World Anti-Doping Agency. Application of a new multidisciplinary strategy employing common and new doping control assays enables the detection of urine substitution in sports drug testing.

It has been generally accepted for 20 years that the growth of Shionogi carcinoma 115 (SC115) is stimulated only by androgen. However, we recently found that the growth of SC115 cells is also stimulated by pharmacological doses of estrogen in vivo but not in cell culture. In the present study, the growth-stimulatory effect of glucocorticoid on SC115 cells was examined. In castrated mice, daily injections of high doses of dexamethasone (100 micrograms/mouse) markedly stimulated the tumor growth, and the growth approached that found in normal males. However, daily injections of physiological doses of dexamethasone (4 micrograms/mouse) or high doses of epitestosterone, progesterone, or cholesterol (200-5000 micrograms/mouse) did not enhance the tumor growth in castrated mice. The androgen dependency, growth speed, steroid receptors, and histological type of the tumors grown by pharmacological doses of glucocorticoid were not significantly different from those of the original SC115 tumors grown by androgen. In a serum-free medium [Ham's F-12:Eagle's minimum essential medium (1:1, v/v) containing 0.1% bovine serum albumin], the proliferation of SC-3 cells (a cloned cell line from SC115 cells) was markedly (by up to 25-fold) stimulated by 10(-10)-10(-8) M testosterone, whereas the proliferation was only slightly but significantly (by up to 3.3-fold) stimulated by 10(-8)-10(-5) M dexamethasone. The present findings demonstrate that the growth of SC115 cells in vivo and in cell culture is significantly stimulated by physiological doses of androgen or pharmacological doses of glucocorticoid.

For the first time in Europe hair and urine testing have been applied to assess drugs of abuse consumption in couples undergoing assisted reproductive technology and the eventual association of toxic habits with other lifestyle, health status and sociodemographic factors was also investigated. Couples attending five assisted reproduction centers in Rome were invited to join the study. When they presented at the Centre for the visit, they were asked to answer a structured questionnaire concerning sociodemographic characteristics and lifestyle habits, and at the same time to provide hair and urine samples. Hair and urine testing for drugs of abuse, urinary profile of principal endogenous steroids involved in fertility process (testosterone, epitestosterone, androsterone, etiocholanolone and dehydroepiandrosterone) and of alcohol and tobacco smoke biomarkers were performed with validated methodologies. Of the 594 enrolled individuals (297 couples), 352 (164 couples and 24 single individuals from the couple) completed the questionnaire and gave both hair and urine samples, apart from 3 bald men, who only gave urine samples. Urine testing showed an overall 4.8% (17 individuals) positivity to drugs of abuse: 4.2% to cannabinoids, 1.4% to cocaine and 0.85% to both drugs. Results of 4cm segment hair samples testing matched those from urine samples. Thus, taking together, results of urine and hair testing confirmed repeated use of cannabis, cocaine and both drugs in 3.7, 0.85 and 0.57% examined individuals, respectively. Drug consumers were in a statistically higher percentage active smokers and alcohol drinkers, less prone to physical activity and with a trend towards higher weight than non consumers. Finally, repeated drug consumption was associated with significant lower concentration of urinary testosterone in males and of urinary dehydroepiandrosterone in females. The findings of the present study confirm the suitability of urine testing to disclose recent drugs of abuse consumption and of hair analysis to verify repeated consumption. Association between different toxic habits and sedentary lifestyle is also substantiated by the obtained results in our cohort of couples attending assisted reproduction centers.

Within the scope of the National Plan for Hormone Control in The Netherlands, a study was performed to develop a system for control of the illegal use of three naturally occurring hormones [oestradiol-17 beta (E2-17 beta), testosterone (T), progesterone (P)] for fattening purposes in animal production. Using a specific high-performance liquid chromatographic-radioimmunoassay method, reference values were established for concentrations of E2-17 beta, T and P and some of their metabolites in blood plasma and urine from untreated male and female veal calves. E2-17 beta levels of both male and female calves were less than 0.01 microgram/l in blood plasma and less than 0.2 microgram/l in urine. For male veal calves levels of T and epitestosterone (epiT) in blood plasma and urine varied widely. The P levels were less than 0.1-0.3 micrograms/l in blood plasma and less than 0.6-10 micrograms/l in urine from both male and female calves. To investigate the effect of anabolic treatment on the hormone levels in plasma and excreta, male veal calves were injected, subcutaneously into the dewlap, with a solution containing 20 mg of E2-17 beta benzoate and 200 mg of T propionate in 5 ml of arachis oil. Only the levels of E2-17 beta and E2-17 alpha in blood plasma and excreta were elevated until about one week after injection, compared with the untreated control calves and the reference values. T and epiT levels were similar in plasma and excreta from both untreated and treated animals.

Epitestosterone (17 alpha-hydroxy-4-androsten-3-one), a normal constituent of human plasma and urine, prevents the testosterone induced changes in body weight and in organ weights of seminal vesicles and kidney of castrated male mice. It competes with methyltrienolone in the binding to rat prostate cytosol (Ki = 29.8 nmol.l-1. It inhibits also the activity of 5 alpha-reductase from rat prostate pellet (Ki = 1.2 mumols.l-1). Epitestosterone can be considered as a weak antiandrogen in the term of displacement of androgen from receptor binding and as an efficient inhibitor of 5 alpha-reductase.

Epitestosterone (EpiT) is the 17 alpha-hydroxy epimer of testosterone (T) and a natural steroid metabolite. It has previously been shown to be a 5 alpha-reductase inhibitor. We have studied EpiT as an antiandrogen using the hamster flank organ model. One-centimeter silastic capsules of crystalline T or dihydrotestosterone (DHT) were implanted subcutaneously in female Golden Syrian hamsters to provide continuous androgenic stimulation. After 3 weeks, the pigmented spot was measured and the flank organs were fixed for histologic sectioning. The maximum surface area (SA) from a central section of the sebaceous gland and the diameter of hair follicles were measured using a computerized digitizing tablet. Following T and DHT, respectively, there was a significant increase in pigmented spot size (656/382%), sebaceous gland SA (210/315%), and mean hair follicle diameter (80/56%). A 1-cm capsule of EpiT alone had no androgenic effect. Five- and ten-fold doses of EpiT were implanted with T or DHT. Epitestosterone significantly inhibited the T-dependent stimulation of pigment, sebaceous gland, and hair follicle at either 5- and/or 10-fold excess doses. Additionally, a 10-fold dose of EpiT also inhibited DHT-dependent stimulation of all 3 cutaneous structures. We conclude that EpiT was effective as an antiandrogen and had no intrinsic androgenic activity in the hamster flank organ. It probably functions both as a competitive inhibitor of the androgen receptor and as a 5 alpha-reductase inhibitor. Pigment and sebaceous gland growth were more sensitive than the hair follicle to androgen inhibition by EpiT at the time and doses tested.

Epitestosterone (ET) and testosterone (T), free and sulfoconjugated, as well as 5-androstene-3 beta,17 alpha-diol (5AD3 beta 17 alpha) and its 17 beta-epimer have been analyzed, by gas chromatography-mass spectrometry with stable isotope dilution, in peripheral and spermatic venous plasma of patients with left varicocele. All these androgens are secreted by the testis as evidenced by the significant concentration gradients between peripheral and spermatic venous plasma. Half of the daily ET production is ascribed to the testis, while 95% of T sulfate and roughly 70% of ET sulfate are also of testicular origin. Significant correlations between ET and 5AD3 beta 17 alpha are an indication that the 5-ene pathway is also operative for ET biosynthesis. High ratios of spermatic to peripheral venous plasma levels of ET and 5AD3 beta 17 alpha are also related to the high clearance rates of 17 alpha-hydroxy-androgens.

The legally defensible proof of the abuse of endogenous steroids in sports is currently based on carbon isotope ratio mass spectrometry (IRMS), i.e. a comparison between (13)C/(12)C ratios of diagnostic precursors and metabolites of testosterone. The application of this technique requires a chromatographic baseline separation of respective steroids prior to IRMS detection and hence laborious sample pre-processing of the urinary steroid extracts including clean up by solid-phase extraction and/or liquid chromatography. Consequently, an efficient pre-selection of suspicious control urine samples is essential for appropriate follow up confirmation by IRMS and effective doping control. Two single transdermal administration studies of testosterone (50 mg Testogel® and Testopatch® at 3.8 mg in 16 h, respectively) were conducted and resulting profiles of salivary testosterone and urinary steroid profiles and corresponding carbon isotope ratios were determined. Conventional doping control markers (testosterone/epitestosterone ratio, threshold concentrations of androsterone, etiocholanolone, or androstanediols) did not approach or exceed critical thresholds. In contrast to these moderate variations, the testosterone concentration in oral fluid increased from basal values (30-142 pg/mg) to peak concentrations above 1000 pg/mg. It is likely that this significant increase in oral fluid is due to a pulsatile elevation of free (protein unbound) circulating testosterone after transdermal administration and may be assumed to represent a more diagnostic marker for transdermal testosterone administration.