Metabolism of formestane in humans: Identification of urinary biomarkers for antidoping analysis.

Formestane (4-hydroxyandrost-4-ene-3,17-dione, 4OH-AED) is an aromatase inhibitor prohibited in sports. In recent years, it has been demonstrated that it can also originate endogenously by the hydroxylation in C4 position of androstenedione. Thus, the use of isotope ratio mass spectrometry (IRMS) is mandatory according to the World Antidoping Agency (WADA) to discriminate endogenous from synthetic origin. In a previous work and after oral administrations of formestane (4OH-AED), the ratio between the main formestane metabolite (4α-hydroxyepiandrosterone; 4OH-EA) and formestane parent compound could help to identify the endogenous origin, avoiding unnecessary and costly IRMS confirmations. In the present work, we investigated whether the same criteria could also be applied after transdermal applications. Six volunteers were transdermally treated once with formestane. Urine samples were collected for 120 h postadministration and analyzed by gas chromatography coupled to mass spectrometry (GC-MS and GC-MS/MS). Formestane and its major metabolites were monitored. The kinetic profile of formestane and its main metabolites was found different between oral and transdermal application. A shift on the excretion of the metabolites compared to formestane itself that can be observed after the oral administration, is absent after the transdermal one. This makes that a simple criteria cannot be applied to differentiate the endogenous from the synthetic origin based on metabolic ratios. The ratio between 4-hydroxyepiandrosterone and 4-hydroxyandrosterone (4OH-A) can be used to differentiate the route of administration. Ratios higher than one (4OH-EA/4OH-A > 1) are diagnostic of an oral administration. This allows to correctly interpret the 4OH-EA/4OH-AED ratio as proposed in our previous investigation. The results of this work demonstrate that the use of appropriate biomarkers (metabolic ratios) helps to reach correct conclusions without using complex and costly instrumentation approaches.

Ecdysteroids: A novel class of anabolic agents?

Increasing numbers of dietary supplements with ecdysteroids are marketed as "natural anabolic agents". Results of recent studies suggested that their anabolic effect is mediated by estrogen receptor (ER) binding. Within this study the anabolic potency of ecdysterone was compared to well characterized anabolic substances. Effects on the fiber sizes of the soleus muscle in rats as well the diameter of C2C12 derived myotubes were used as biological readouts. Ecdysterone exhibited a strong hypertrophic effect on the fiber size of rat soleus muscle that was found even stronger compared to the test compounds metandienone (dianabol), estradienedione (trenbolox), and SARM S 1, all administered in the same dose (5 mg/kg body weight, for 21 days). In C2C12 myotubes ecdysterone (1 µM) induced a significant increase of the diameter comparable to dihydrotestosterone (1 µM) and IGF 1 (1.3 nM). Molecular docking experiments supported the ERß mediated action of ecdysterone. To clarify its status in sports, ecdysterone should be considered to be included in the class "S1.2 Other Anabolic Agents" of the list of prohibited substances of the World Anti-Doping Agency.

Anabolic and androgenic activity of 19-norandrostenedione after oral and subcutaneous administration--analysis of side effects and metabolism.

One of the most frequently misused steroid precursors (prohormones) is 19-norandrostenedione (estr-4-ene-3,17-dione, NOR). Recently we have show that NOR stimulates skeletal muscle growth after s.c. administration in a highly selective manner but exhibits only weak androgenic activity in rats. Because most abusers take NOR orally, the aim of this study was to compare the anabolic and androgenic potency of NOR between s.c. and oral application. Orchiectomised rats were treated with NOR either s.c. (1 mg/kg BW/day) or orally (0.1, 1 and 10 mg/kg BW/day). The tissue weights of the levator ani, the seminal vesicle and the prostate were analysed to determine the anabolic and androgenic activity. Heart and liver wet weights were examined to identify side effects. Serum concentrations of NOR and its metabolite nandrolone (NT) were determined. GCMC analysis revealed that free and glucuronidated NOR and NT were detectable in the serum after oral and s.c. administration and that NOR was converted to NT in comparable amounts independent of the route of administration. In agreement to our previous study s.c. application of NOR stimulates skeletal muscle growth but has only weak androgenic effects. In contrast, after oral administration of NOR neither stimulation of the prostate nor the levator ani could be observed in the doses administered in this study. Interestingly, and in contrast to s.c. treatment, oral administration of NOR resulted in a dose-dependent decrease of body weight. In summary, oral administration of NOR, at least in the rat, seems to be a very ineffective strategy for stimulating skeletal muscle mass increases but may be associated with side effects.

Serum testosterone and urinary excretion of steroid hormone metabolites after administration of a high-dose zinc supplement.

OBJECTIVES: To investigate whether the administration of the zinc-containing nutritional supplement ZMA causes an increase of serum testosterone levels, which is an often claimed effect in advertising for such products; to monitor the urinary excretion of testosterone and selected steroid hormone metabolites to detect potential changes in the excretion patterns of ZMA users. SUBJECTS: Fourteen healthy, regularly exercising men aged 22-33 years with a baseline zinc intake between 11.9 and 23.2 mg day(-1) prior to the study. RESULTS: Supplementation of ZMA significantly increased serum zinc (P=0.031) and urinary zinc excretion (P=0.035). Urinary pH (P=0.011) and urine flow (P=0.045) were also elevated in the subjects using ZMA. No significant changes in serum total and serum free testosterone were observed in response to ZMA use. Also, the urinary excretion pattern of testosterone metabolites was not significantly altered in ZMA users. CONCLUSIONS: The present data suggest that the use of ZMA has no significant effects regarding serum testosterone levels and the metabolism of testosterone in subjects who consume a zinc-sufficient diet.

Terbutaline sulfoconjugate: characterization and urinary excretion monitored by LC/ESI-MS/MS.

Terbutaline is a fast-acting beta(2)-adrenergic agonist used in the treatment of obstructive pulmonary diseases. Doping control for beta(2)-agonists, which are forbidden in sports by the World Anti-doping Agency (WADA), is performed in screening by liquid chromatography/mass spectrometry after hydrolysis of phase-II metabolites. In this study, the mono-sulfoconjugated phase-II metabolite of terbutaline was synthesized and the chemical structure was characterized by (1)H-nuclear magnetic resonance spectrometry and high resolution/high accuracy Orbitrap mass spectrometry. The metabolite was designated as the phenolic esterified compound, which has been mentioned in most literature reports but has not been verified so far. The benzylic esterified compound was also synthesized and characterized by high-resolution/high accuracy Orbitrap mass spectrometry but was not detectable in urine samples from an excretion study performed after a single application of one terbutaline capsule (7.5 mg terbutaline sulfate salt). The phenolic sulfate of terbutaline was detected for two to four days after administration, whereas the unchanged terbutaline was detected for four to five days. A glucuronidated, disulfated or trisulfated phase-II metabolite of terbutaline was not found. The measurement of phase-II metabolites is planned to be incorporated into existing screening procedures to allow a faster sample preparation.

Analytical strategies for the detection of non-labelled anabolic androgenic steroids in nutritional supplements.

Recent studies showed that non-hormonal supplements such as vitamins, minerals and amino acids can contain anabolic androgenic steroids not declared on the labels of the products. These undeclared substances (often prohormones of testosterone or 19-nortestosterone) can cause health risks to consumers and might lead to positive results in sports doping control, especially for the nandrolone metabolite norandrosterone. The analysis of nutritional supplements for anabolic steroids has proven to be rather difficult due to the different matrices in the various products. To conduct a broad-based analysis, a few robust methods capable of analysing various matrices are needed. To obtain a sensitive gas chromatography-mass spectrometry (GC-MS) analysis, a method including extraction and purification of the analytes followed by GC-MS analysis of the trimethylsilyl (TMS) derivatives of the steroids was developed. The limit of detection was improved by the addition of a mixture of 1-N,N-diisopropylamino-n-alkanes (DIPAs) to the final extract. In pure creatine monohydrate powder the limits of detection were demonstrated to be 0.1 ng microg (-1) for dehydroepiandrosterone (DHEA) and estr-4-ene-3beta,17beta-diol, 0.7 ng g(-1) for 5alpha-androstane-3beta,17beta-diol and androsta-1,4-diene-3,17-dione, 1 ng g(-1) for estr-5-ene-3beta,17beta-diol, estr-4-ene-3,17-dione, 19-nortestosterone, androst-4-ene-3, 17-dione and testosterone, and 2 ng g(-1) for androst-4-ene-3beta,17beta-diol and androst-5-ene-3beta,17beta-diol. The recovery (determined at 200 ng g(-1)) ranged from 32% for 19-nortestosterone to 92% for androst-5-ene-3beta,17beta-diol. During the investigation of different nutritional supplements, several analytical difficulties occurred. Aspects of homogenization, extraction, separation, derivatization and GC-MS measurement as well as strategies for the solution of problems arising were optimized. For quantitative measurements of the steroids in nutritional supplements, deuterated internal standards of the specific steroids or standard addition are necessary to compensate for matrix effects.

Analysis of non-hormonal nutritional supplements for anabolic-androgenic steroids - results of an international study.

Several recent studies have shown evidence of some nutritional supplements containing prohibited anabolic androgenic steroids, so-called prohormones, which were not declared on the label. Therefore, a broad-based investigation of the international nutritional supplement market was initiated to clarify the extent of this problem. From October 2000 until November 2001, 634 non-hormonal nutritional supplements were purchased in 13 countries from 215 different suppliers. Most supplements were bought in shops in the respective countries (578 samples = 91.2 %) and on the internet (52 samples = 8.2 %). 289 supplements were from prohormone-selling companies and 345 supplements came from companies which do not offer prohormones. After isolation from the supplement matrix 11 different anabolic androgenic steroids, mainly prohormones of testosterone and nandrolone, were analysed by gas-chromatography/mass spectrometry. Out of the 634 samples analysed 94 (14.8 %) contained anabolic androgenic steroids not declared on the label ("positive supplements"). We could not obtain reliable data for 66 samples (10.4 %) due to matrix effects. In relation to the total number of products purchased per country, most of the positive supplements were bought in the Netherlands (25.8 %), in Austria (22.7 %), in the UK (18.8 %) and the USA (18.8 %). According to the label, all positive supplements were from companies located in only five countries: the USA, the Netherlands, the UK, Italy and Germany. 21.1 % of the nutritional supplements from prohormone-selling companies contained anabolic androgenic steroids, whereas 9.6 % of the supplements from companies not selling prohormones were positive. The positive supplements showed anabolic androgenic steroid concentrations of 0.01 micro g/g up to 190 micro g/g. The administration of supplements containing nandrolone prohormones adding up to a total uptake of more than 1 micro g resulted in positive doping results for norandrosterone for several hours.