The in vivo metabolism of Furazadrol in greyhounds.

Samples of the 'dietary supplement' Furazadrol sourced through the internet have been reported to contain the designer anabolic androgenic steroids [1',2']isoxazolo[4',5':2,3]-5α-androstan-17ß-ol (furazadrol F) and [1',2']isoxazolo[4',3':2,3]-5α-androstan-17ß-ol (isofurazadrol IF). These steroids contain an isoxazole fused to the A-ring and were designed to offer anabolic activity while evading detection, raising concerns over the potential for abuse of this preparation in sports. The metabolism of Furazadrol (F:IF, 10:1) was studied by in vivo methods in greyhounds. Urinary phase II Furazadrol metabolites were detected as glucuronides after a controlled administration. These phase II metabolites were subjected to enzymatic hydrolysis by Escherichia coli ß-glucuronidase to afford the corresponding phase I metabolites. Using a library of synthetically derived reference materials, the identities of seven urinary Furazadrol metabolites were confirmed. Major confirmed metabolites were isofurazadrol IF, 4α-hydroxyfurazadrol 4α-HF and 16α-hydroxy oxidised furazadrol 16α-HOF, whereas the minor confirmed metabolites were furazadrol F, 4ß-hydroxyfurazadrol 4ß-HF, 16ß-hydroxyfurazadrol 16ß-HF and 16ß-hydroxy oxidised furazadrol 16ß-HOF. One major hydroxyfurazadrol and two dihydroxyfurazadrol metabolites remained unidentified. Qualitative excretion profiles, limits of detection and extraction recoveries were established for furazadrol F and major confirmed metabolites. These investigations identify the key urinary metabolites of Furazadrol following oral administration, which can be incorporated into routine screening by anti-doping laboratories to aid the regulation of greyhound racing.

Steroids excreted in urine by neonates with 21-hydroxylase deficiency. 3. Characterization, using GC-MS and GC-MS/MS, of androstanes and androstenes.

Urine from neonates with 21-hydroxylase deficiency contains a large range of androstane(ene)s, many of which have not been previously described. We present their characterization as the third part of a comprehensive study of urinary steroids, aiming to enhance the diagnosis of this disorder and to further elucidate steroid metabolism in neonates. Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra were used together to determine the structure of hitherto undescribed androstane(ene)s. GC-MS/MS was pivotal for the structural characterization of 2-hydroxylated androstenediones but GC-MS was generally more informative for androstane(ene)s, in contrast to 17-hydroxylated pregnane(ene)s. Parallels were found between the GC-MS and GC-MS/MS characteristics of structurally similar androstenediones and progesterones without a substituent on the D-ring, but not with those of 17-hydroxylated progesterones. Assignment of 5α(ß) orientation, based on GC-MS characteristics, was possible for 11-oxo-androstanes. The major endogenous 3ß-hydroxy-5-enes in 21-hydroxylase deficiency did not differ from those in unaffected neonates. The key qualitative and quantitative differences encompassed 5α(ß)-androstanes and 3-oxo-androst-4-enes. Major positions of hydroxylation in these were C(2), C(6), C(11), C(16) and C(18). Additional oxo-groups were common at C(6), C(11) and C(16). We conclude that the presence of multiple further oxygenated metabolites of androstenedione in urine from neonates with 21-hydroxylase deficiency and their pattern indicate a predominance of the classical pathway of androgen synthesis and reflect an increased demand for clearance. The positions of oxygenation in androstane(ene)s are dependent on the configuration at C(3)-C(5).

A pilot study on subject-based comprehensive steroid profiling: novel biomarkers to detect testosterone misuse in sports.

CONTEXT: Until now, the testosterone/epitestosterone (T/E) ratio is the main marker for the detection of testosterone (T) misuse in athletes. As this marker can be influenced by a number of confounding factors, additional steroid profile parameters indicating T misuse can provide substantiating evidence of doping with endogenous steroids. The evaluation of a steroid profile is currently based upon population statistics. As large inter-individual variations exist, a paradigm shift towards subject-based references is ongoing in doping analysis. OBJECTIVE: Proposition of new biomarkers for the detection of testosterone in sports using extensive steroid profiling and an adaptive model based upon Bayesian inference. SUBJECTS: Six healthy male volunteers were administered with testosterone undecanoate. Population statistics were performed upon steroid profiles from 2014 male Caucasian athletes participating in official sport competition. DESIGN: An extended search for new biomarkers in a comprehensive steroid profile combined with Bayesian inference techniques as used in the athlete biological passport resulted in a selection of additional biomarkers that may improve detection of testosterone misuse in sports. RESULTS: Apart from T/E, 4 other steroid ratios (6α-OH-androstenedione/16α-OH-dehydroepiandrostenedione, 4-OH-androstenedione/16α-OH-androstenedione, 7α-OH-testosterone/7ß-OH-dehydro-epiandrostenedione and dihydrotestosterone/5ß-androstane-3α,17ß-diol) were identified as sensitive urinary biomarkers for T misuse. These new biomarkers were rated according to relative response, parameter stability, detection time and discriminative power. CONCLUSION: Newly selected biomarkers were found suitable for individual referencing within the concept of the Athlete's Biological Passport. The parameters showed improved detection time and discriminative power compared to the T/E ratio. Such biomarkers can support the evidence of doping with small oral doses of testosterone.

The potential of urinary androstdiene markers to identify 4-androstenediol (4-ADIOL) administration in athletes.

Doping control laboratories accredited by the World Anti-Doping Agency (WADA) require criteria that allow endogenous steroids to be distinguished from their synthetic analogues in urine. Methodology based on "looking outside the metabolic box" was used in this study to identify diagnostic urinary markers of 4-androstenediol (4-ADIOL) administration. Androst-2,4-diene-17-one and androst-3,5-diene-17-one are proposed to be formed in urine from acid-catalyzed hydrolysis of 4-ADIOL sulfoconjugate, a major phase II metabolic product of 4-ADIOL. The presence of these markers in the routine gas chromatography-mass spectrometry (GC-MS) steroid screen was suitable to identify samples requiring confirmation by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) - to measure the carbon isotope ratio (δ(13)C) of the androstdiene markers and confirm their likely synthetic origin based on depleted (13)C content.

Concentration compared with total urinary excretion of 11,17-DOA in cynomolgus monkey urine.

Strees sensitive molecules exhibit great variation in concentration in the circulation and it may often be advantageous to quantify these in urine or feces rather than in serum or plasma. We advocate that all urine-or feces-should be collected, and that excretion of stress sensitive molecules should be expressed as amounts excreted per time unit per kg body-weight, rather than being expressed as concentrations in samples. Urine and feces excretion varies significantly within and between animals over time, which may render simple concentration measures of molecules of little biological relevance.

Urinary and fecal immunoglobulin A, cortisol and 11-17 dioxoandrostanes, and serum cortisol in metabolic cage housed female cynomolgus monkeys (Macaca fascicularis).

BACKGROUND AND METHODS: Quantitative enzyme-immunoassays of urinary and fecal immunoglobulin A (IgA), cortisol and 11-17-dioxoandrostanes (11,17-DOA), and serum cortisol in eight metabolic-cage-housed female cynomolgus monkeys were performed. The monkeys were divided into two groups, B and NB. Group B animals were blood sampled every 6 hours, whereas Group NB animals were not handled/blood sampled. RESULTS: No differences were recorded between the amounts of feces and urine excreted by the two groups. Group B animals excreted more urinary cortisol than did Group NB animals indicating that restraint-blood sampling resulted in a stress response. Excreted amounts of IgA and 11,17-DOA (urine and feces) did not differ between the groups. CONCLUSIONS: Urinary cortisol was a reliable marker of the stress associated with repeated blood sampling. Declining amounts of excreted urinary cortisol indicated that cynomolgus monkeys acclimated quickly to repeated blood sampling in metabolism cages. Within and between animal variation in amounts of feces voided demonstrated the importance of expressing fecal markers as 'amounts excreted per time unit per kg body weight' rather than just measuring the concentrations in fecal samples.

Searching for new markers of endogenous steroid administration in athletes: "looking outside the metabolic box".

A simple means of detecting the abuse of steroids that also occur naturally is a problem facing doping control laboratories. Specific markers are required to allow the detection of the administration of these steroids. These markers are commonly measured using a set of data obtained from the screening of samples by gas chromatography-mass spectrometry (GC-MS). Doping control laboratories further need to confirm identified abuse using techniques such as gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). An interesting urinary species was found while following the pharmacokinetics and changes to the steroid profile from single and multiple oral doses of the International Olympic Committee/World Anti Doping Agency (IOC/WADA) prohibited substance, dehydroepiandrosterone (DHEA). The urine samples collected from the administration studies were subject to GC-MS and GC-C-IRMS steroid analysis following cleanup by solid phase extraction techniques. A useful urinary product of DHEA administration was detected in the urine samples from each of the administration studies and was identified by GC-MS experiments to be 3alpha,5-cyclo-5alpha-androstan-6beta-ol-17-one (3alpha,5-cyclo). This compound occurs naturally but the concentrations of 3alpha,5-cyclo were elevated following both the single DHEA administration (up to 385 ng/mL) and multiple DHEA administrations (up to 1240 ng/mL), in relation to those observed prior to these administrations (70 and 80 ng/mL, respectively). A reference distribution of urine samples collected from elite athletes (n = 632) enabled the natural concentration range of 3alpha,5-cyclo to be established (0-280 ng/mL), with a mean concentration of 22 ng/mL. Based on this an upper 3alpha,5-cyclo concentration limit of 140 ng/mL is proposed as a GC-MS screening marker of DHEA abuse in athletes. GC-C-IRMS analysis revealed significant 13C depletion of 3alpha,5-cyclo following DHEA administration. In the single administration study, the delta13C value of 3alpha,5-cyclo changed from -24.3 per thousand to a minimum value of -31.1 per thousand at 9 h post-administration, before returning to its original value after 48 h. The multiple administration study had a minimum delta13C 3alpha,5-cyclo of -33.9 per thousand during the administration phase in contrast to the initial value of -24.2 per thousand. Preliminary studies have shown 3alpha,5-cyclo to most likely be produced from DHEA sulfate found at high levels in urine. The complementary use of GC-MS and GC-C-IRMS to identify new markers of steroid abuse and the application of screening criteria incorporating such markers could also be adapted by doping control laboratories to detect metabolites of androstenedione, testosterone and dihydrotestosterone abuse.

Screening of free 17-alkyl-substituted anabolic steroids in human urine by liquid chromatography-electrospray ionization tandem mass spectrometry.

A qualitative liquid chromatography-electrospray ionization tandem mass spectrometry method was developed for screening of the abuse of 4-chlorodehydromethyltestosterone, danazol, fluoxymesterone, formebolone, metandienone, oxandrolone, and stanozolol. The introduced method measures simultaneously nine different 17-alkyl-substituted anabolic androgenic steroids or their unconjugated metabolites in human urine, using methyltestosterone as an internal standard. Sample preparation involved one-step liquid extraction. Liquid chromatographic separation was achieved on a reversed-phase column with methanol-water gradient containing 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid. Compounds were ionized in the positive mode and detected by multiple reaction monitoring. All steroids within the study could be selectively detected in urine with detection limits of 0.1-2.0 ng/ml. The method showed good linearity up to 250 ng/ml with correlation coefficients higher than 0.9947. With simple and fast sample preparation, low limits of detection, and high selectivity and precision, the developed method provides advantages over the present testing methods and has the potential for routine qualitative screening method of unconjugated 17-alkyl-substituted anabolic steroids in human urine.

Comparative urinary androstanes in the great apes.

Urinary androstanes from seven species of male great apes (human, bonobo, chimpanzee, lowland gorilla, mountain gorilla, Bornean orangutan, and Sumatran orangutan) were separated by HPLC and detected by RIA using two testosterone antibodies. All animals examined showed the presence of testosterone and six additional immunoreactive peaks. Although testosterone was the dominant peak (85%) in human urine, its proportion in urine was much less in the other apes, ranging from a high of 59% in the bonobo and chimpanzee to a low of 24% in the mountain gorilla. Urinary androstanes were also directly visualized using nano-spray mass spectrometry (nanoESI-MS). Although the RIA can qualitatively produce a strong signal for testosterone in unchromatographed urine, it is quantitatively present only as a trace metabolite, as demonstrated by nanoESI-MS. The combination of the two techniques showed large differences in androstane metabolism between the seven species. A previously undescribed testosterone metabolite (tentatively identified as either delta1- or delta6-testosterone sulfate) was present in significant proportions in all of the non-human apes examined. We conclude that in the great apes, testosterone is only a trace metabolite in urine, and as a consequence, its measurement may not produce results that parallel the levels of serum testosterone. The RIA measurement of urinary testosterone in part records additional androstane metabolites, which vary even between closely related genera, making the results neither equivalent with nor comparable to different species.

Detection of exogenous intake of natural corticosteroids by gas chromatography/combustion/isotope ratio mass spectrometry: application to misuse in sport.

A detailed procedure for the analysis of exogenous hydrocortisone and cortisone in urine by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is proposed. As urinary levels of hydrocortisone are rather low for GC/C/IRMS analysis, the focus is on the main corticosteroid metabolites, tetrahydrocortisone (THE) and tetrahydrocortisol (THF). Following different solid phase extraction purifications, THE and THF are oxidized to 5beta-androstanetrione before analysis by GC/C/IRMS. Significant differences in delta(13)C per thousand values of synthetic natural corticosteroids and endogenous human corticosteroids have been observed. Therefore, a positive criterion, to detect exogenous administration of synthetic corticosteroids in anti-doping control, is proposed.

[Plasma dehydroepiandrosterone sulfate and urine steroid excretion in hepatic cirrhosis]. / Il deidroepiandrosterone solfato plasmatico e la escrezione steroidea urinaria nella cirrosi epatica.

In the present study we evaluated the alterations of plasma levels of dehydroepiandrosterone sulphate (DHEA-S) and of steroid-derivatives urinary excretion in cirrhotic patients compared with normal controls. 107 patients have been studied: 45 affected by liver cirrhosis (22 males and 23 females) both in compensated and in ascitic state, and 62 healthy controls (32 males and 30 females). In all patients we assayed plasma DHEA-S by RIA as well as common liver function tests; moreover gaschromatographic profile of adrenal steroid excretion was performed in daily urine. Our findings show a significantly lower DHEA-S in cirrhotic patients compared with healthy controls, both males (451.7 SD 440.3 ng/ml vs 1438.3 SD 604.8, p less than 0.001) and females (225.5 SD 152.6 ng/ml vs 974.0 SD 405.7, p less than 0.001). Variance analysis confirmed that the difference was significant both for compensated and uncompensated ones. We also found some interesting relationships between DHEA-S and liver function tests, cholesterol and age. Urinary steroids were markedly low in both sexes, particularly adrenal sexual fraction. Our data show clear disfunction in DHEA-S metabolism in liver cirrhosis, partially related with the severity of the disease.

[Analysis of the high risk factors of endometrial adenocarcinoma--abnormal excretion in urinary steroids in androgen sterilized rats].

We experimentally produced spontaneous endometrial carcinoma in anovulatory sterilized Sprague-Dawley rats (ASR) after 500 days of age and compared the 24 hour urinary steroid excretion patterns in ASR with those in normal control rats (NR). The results obtained were as follows: The 24 hours urinary 17-ketosteroid, 11-deoxy-17-ketosteroid and 11-oxy-17-ketosteroid levels of 500-day-old ASR were 127.5 micrograms/day, 73.4 micrograms/day and 54.1 micrograms/day which were 1/3, 2/5 and 1/3 of the same aged NR, respectively. Although the 24 hour urinary androsterone, etiocholanolone and dehydroepiandrosterone of 100-day-old ASR were significantly higher than those of the same aged NR, the seven fractionated 17-ketosteroid levels of 500-day-old ASR were lower than those of NR. Moreover, the urinary androsterone, etiocholanolone and 11-ketoetiocholanolone of 500-day-old ASR were significantly lower than those of NR. The total excretion of tetrahydrocortisol, tetrahydrocortisone and tetrahydrocorticosterone of 100- and 500-day-old ASR were 318 micrograms/day and 244 micrograms/day which were slightly higher levels than those of NR. And the urinary tetrahydrocortisol level of ASR was found to be higher than that of NR. The present results suggested that an abnormality in androgen metabolism of ASR relative to NR was one of the high risk factors in endometrial carcinogenesis in ASR.

Specific estimation of fifteen unconjugated, non-metabolized steroid hormones in human urine.

A method for the specific determination of 15 free, non-metabolized steroid hormones in human urine is described. The steroids progesterone (P), androstenedione (AD), pregnenolone (PL), 5 alpha-dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), testosterone (T), 11-deoxycorticosterone (DOC), 17-OH-progesterone (17-OHP), 17-OH-pregnenolone (17-PL), 11-deoxycortisol (S), 18-OH-11-deoxycorticosterone (18-OH-DOC), corticosterone (B), aldosterone (Aldo), cortisol (F), and 18-OH-corticosterone (18-OH-B) were extracted from 2 ml urine samples by a solid-phase technique, subjected to automatic high performance liquid chromatography (HPLC) and finally quantitated by radioimmunoassay (RIA). The combination of HPLC and RIA provides a high specificity of steroid estimates. The automatic mode of HPLC renders the method quite suitable for series analyses in research or for routine purposes. Precision and accuracy are satisfactory, and comparable with the level commonly achieved in RIA techniques. The mean values (nmol/24 h) of reference ranges established from 32 normal males were as follows: P: 0.36; AD: 9.76; PL: 0.90; DHT: 0.61; DHEA: 6.76; T: 1.43; DOC: 0.35; 17-OHP: 1.03; 17-PL: 0.20; S: 0.24; 18-OH-DOC: 2.11; B: 1.49; Aldo: 0.46; F: 68.3; 18-OH-B: 5.41. This highly practicable method may be particularly useful for the further investigation of the physiological or diagnostic significance of the non-conjugated, non-metabolized fraction of steroid hormones in urine.

The diagnosis of 21-hydroxylase deficiency in a prematurely born infant on the basis of the urinary steroid excretion pattern.

The urine of a 6-day-old prematurely born female infant (birth weight 1060 g) suspected of having a 21-OH-deficiency showed no steroid abnormalities on capillary GLC analysis. Using GC-MS tetrahydrocortisone (THE) and also 3 alpha, 17 alpha-dihydroxy-5 beta-pregnane-20-one (17-OH-Polone) were absent, but two androstanetriolone peaks were observed. In the urine collected on day 9 THE was absent, but a large amount of 3 alpha, 11 beta-dihydroxy-5-alpha-androstane-17-one (11-HA) was found by GC-MS to be contaminated by a small amount of 17-OH-Polone. The next urine specimen collected on the 22nd day while the child received cortisol therapeutically showed the characteristic steroid profile for the diagnosis 21-OH deficiency, large peaks of 17-OH-Polone, pregnanetriol (P3) and 11-keto-pregnanetriol (11-keto-P3). Over the next few weeks two other compounds were found to have been excreted in relatively large amounts, 3 xi, 16 xi, 17 xi, 20 xi-pregnanetetrol (16-OH-P3) and surprisingly also a 21-hydroxylated compound, namely 3 xi, 20 alpha, 21-trihydroxy-5-pregnene. These same two compounds were also found in the urine of another infant with suspected 21-OH deficiency. The urinary steroid excretion patterns characteristic for 21-OH deficiency are dependent on the maturity and age of the infant. In the prematurely born infant androstanetriolones appear in the urine before 17-OH-Polone. The occurrence of these different steroid excretion patterns is tentatively explained.

Anovulation and increased androgenic activity as breast cancer risk in women with fibrocystic disease of the breast.

The urine of 26 otherwise healthy women with fibrocystic disease of the breast was assayed by gas chromatography for testosterone and androstanediol (5alpha-androstane-3alpha, 17beta-diol), the major metabolite of dihydrotestosterone. The mean values for both androgens were significantly higher than in 18 normal women in the same age range. Sixteen of the 26 fibrocystic disease patients also had endometrial hyperplasia. Since the endometrial specimen was obtained in the premenstrual period, the presence of hyperplasia proved that the menstrual cycle in over two-thirds of the fibrocystic disease patients was nonovulatory.

PIP: Urine of 26 otherwise healthy women with fibrocystic breast disease (FCD) was assayed by gas chromatography for testosterone (Tes) and androstanediol (Ans), the major metabolite of dihydrotestosterone. The 26 patients under study had been subjected 3-5 months previously to biopsies of breast lumps, diagnosed as FCD. The histological features varied from simple cystic formations with moderate epithelial proliferation in 11 women (Group 1) to pronounced intraductal epithelial hyperplasia accompanied by epithelial cell atypia in 15 women (Group 2). The urinary Tes and Ans difference in FCD patients and controls (18 normal women) was significant at the level of p .01 for both androgens. In controls the mean excretion levels of Tes and Ans were 6.5 and 35 mcg/24 hours, respectively. In FCD patients, the mean Tes and Ans values were 17.4 and 68.5 mcg/24 hours, respectively. Group 2 presented a higher urinary Tes level than patients in Group 1, but the difference was not significant. The Ans level of Group 1 patients was significantly above normal (p .01) and near significantly higher (p .08) than that of the Group 2 patients; whereas the Ans level of Group 2 patients did not differ significantly from the normal value. Endometrial specimens showed that 16/26 FCD patients had endometrial hyperplasia. Since the endometrial specimen was obtained in the premenstrual period (Days 20-22), the presence of hyperplasia proved that the menstrual cycle in over two-thirds of the FCD patients was nonovulatory.

Disposition of ORF 9326, a novel contragestational steroid, in animals.

The disposition of ORF 9326 [17BETA-acetoxy-2alpha-chloro-3(p-nitrophenoxy) imino-5-androstane], an O-aryl oxime of 2beta-chlorodihydrotestosterone acetate, was studied in rats, dogs, monkeys and rabbits. Intravenous administration of 3H-ORF 9326 dissolved in PEG-400 to rats, dogs and monkeys resulted in a rapid decline of radioactivity in blood followed by a terminal slope suggesting long retention of radioactivity. Apparent half lives of radioactivity in blood were calculated to be from 50--95 hours for the three species, which peak levels of radioactivity in whole blood occurring within 4--7 hours after administration of the compound. Tissue distribution studies in the rat and dog indicate that body fat is one of the major depot areas for the drug and/or its metabolites. The major route of excretion for ORF 9326 and/or its metabolites in dog and rat is biliary whereas in monkey and rabbit it appears to be renal. Greater than 90% of the radioactive compounds excreted in the urine of dogs and monkeys following intravenous administration of 3H-ORF 9326 appear to be in the form of conjugates.

Radioimmunoassay of 17-alpha-alkylated anabolic steroids.

A method for the detection of anabolic 17-alpha-alkylated androstane derivatives in both plasma and urine is described and evaluated. The goat and rabbit antisera against 17-alpha-Methyltestosterone-3-carboxymethyloxim-Rinder-serum albumin were raised and compared using [3H]methandrostenolone as a tracer. 22 Steroids including 10 potent synthetic anabolics were tested for their cross-reaction with these antisera.

[Urine steroid profiles of hirsute women (author's transl)]. / Harn-Steroidprofile hirsuter Frauen.

Steroid profiles of women suffering from idiopathic hirsutism show in more than 50% of the cases of 10--100 fold increase in the excretion of dehydroepiandrosterone (DHEA) compared with normal values. The excretion of DHEA was reduced much more than that of other 17-ketosteroids if the adrenals (NNR) were suppressed by dexamethasone (DXM). Within one week they reached values at the compound noise level of the gas chromatograms. If the ovaries were stimulated with human chorionic gonadotropin during continued suppression of the NNR with DXM no increase of DHEA could be detected.