The influence of chronic L-carnitine supplementation on the formation of preneoplastic and atherosclerotic lesions in the colon and aorta of male F344 rats.

L-Carnitine, a key component of fatty acid oxidation, is nowadays being extensively used as a nutritional supplement with allegedly "fat burning" and performance-enhancing properties, although to date there are no conclusive data supporting these claims. Furthermore, there is an inverse relationship between exogenous supplementation and bioavailability, i.e., fairly high oral doses are not fully absorbed and thus a significant amount of carnitine remains in the gut. Human and rat enterobacteria can degrade unabsorbed L-carnitine to trimethylamine or trimethylamine-N-oxide, which, under certain conditions, may be transformed to the known carcinogen N-nitrosodimethylamine. Recent findings indicate that trimethylamine-N-oxide might also be involved in the development of atherosclerotic lesions. We therefore investigated whether a 1-year administration of different L-carnitine concentrations (0, 1, 2 and 5 g/l) via drinking water leads to an increased incidence of preneoplastic lesions (so-called aberrant crypt foci) in the colon of Fischer 344 rats as well as to the appearance of atherosclerotic lesions in the aorta of these animals. No significant difference between the test groups regarding the formation of lesions in the colon and aorta of the rats was observed, suggesting that, under the given experimental conditions, L-carnitine up to a concentration of 5 g/l in the drinking water does not have adverse effects on the gastrointestinal and vascular system of Fischer 344 rats.

Endocrine characterization of the designer steroid methyl-1-testosterone: investigations on tissue-specific anabolic-androgenic potency, side effects, and metabolism.

Various products containing rarely characterized anabolic steroids are nowadays marketed as dietary supplements. Herein, the designer steroid methyl-1-testosterone (M1T) (17ß-hydroxy-17α-methyl-5α-androst-1-en-3-one) was identified, and its biological activity, potential adverse effects, and metabolism were investigated. The affinity of M1T toward the androgen receptor (AR) was tested in vitro using a yeast AR transactivation assay. Its tissue-specific androgenic and anabolic potency and potential adverse effects were studied in a Hershberger assay (sc or oral), and tissue weights and selected molecular markers were investigated. Determination of M1T and its metabolites was performed by gas chromatography mass spectrometry. In the yeast AR transactivation assay, M1T was characterized as potent androgen. In rats, M1T dose-dependently stimulated prostate and levator ani muscle weight after sc administration. Oral administration had no effect but stimulated proliferation in the prostate and modulated IGF-I and AR expression in the gastrocnemius muscle in a dose-dependent manner. Analysis of tyrosine aminotransferase expression provided evidence for a strong activity of M1T in the liver (much higher after oral administration). In rat urine, 17α-methyl-5α-androstane-3α,17ß-diol, M1T, and a hydroxylated metabolite were identified. In humans, M1T was confirmed in urine in addition to its main metabolites 17α-methyl-5α-androst-1-ene-3α,17ß-diol and 17α-methyl-5α-androstane-3α,17ß-diol. Additionally, the corresponding 17-epimers as well as 17ß-hydroxymethyl-17α-methyl-18-nor-5α-androsta-1,13-dien-3-one and its 17-epimer were detected, and their elimination kinetics was monitored. It was demonstrated that M1T is a potent androgenic and anabolic steroid after oral and sc administration. Obviously, this substance shows no selective AR modulator characteristics and might exhibit liver toxicity, especially after oral administration.

Seized designer supplement named "1-Androsterone": identification as 3ß-hydroxy-5α-androst-1-en-17-one and its urinary elimination.

New analogues of androgens that had never been available as approved drugs are marketed as "dietary supplement" recently. They are mainly advertised to promote muscle mass and are considered by the governmental authorities in various countries, as well as by the World Anti-doping Agency for sport, as being pharmacologically and/or chemically related to anabolic steroids. In the present study, we report the detection of a steroid in a product seized by the State Bureau of Criminal Investigation Schleswig-Holstein, Germany. The product "1-Androsterone" of the brand name "Advanced Muscle Science" was labeled to contain 100mg of "1-Androstene-3b-ol,17-one" per capsule. The product was analyzed underivatized and as bis-TMS derivative by GC-MS. The steroid was identified by comparison with chemically synthesized 3ß-hydroxy-5α-androst-1-en-17-one, prepared by reduction of 5α-androst-1-ene-3,17-dione with LS-Selectride (Lithium tris-isoamylborohydride), and by nuclear magnetic resonance. Semi-quantitation revealed an amount of 3ß-hydroxy-5α-androst-1-en-17-one in the capsules as labeled. Following oral administration to a male volunteer, the main urinary metabolites were monitored. 1-Testosterone (17ß-hydroxy-5α-androst-1-en-3-one), 1-androstenedione (5α-androst-1-ene-3,17-dione), 3α-hydroxy-5α-androst-1-en-17-one, 5α-androst-1-ene-3α,17ß-diol, and 5α-androst-1-ene-3ß,17ß-diol were detected besides the parent compound and two more metabolites (up to now not finally identified but most likely C-18 and C-19 hydroxylated 5α-androst-1-ene-3,17-diones). Additionally, common steroids of the urinary steroid profile were altered after the administration of "1-Androsterone". Especially the ratios of androsterone/etiocholanolone and 5α-/5ß-androstane-3α,17ß-diol and the concentration of 5α-dihydrotestosterone were influenced. 3α-Hydroxy-5α-androst-1-en-17-one appears to be suitable for the long-term detection of the steroid (ab-)use, as this characteristic metabolite was detectable in screening up to nine days after a single administration of one capsule.

Detection of Δ6-methyltestosterone in a "dietary supplement" and GC-MS/MS investigations on its urinary metabolism.

Since a few years more and more products have appeared on the market for dietary supplements containing steroids that had never been marketed as approved drugs, mostly without proper labeling of the contents. Syntheses and few data on pharmacological effects are available dated back mainly to the 1950s or 1960s. Only little knowledge exists about effects and side effects of these steroids in humans. The present study reports the identification of Δ6-methyltestosterone in a product named "Jungle Warfare", which was obtained from a web-based supplement store. The main urinary metabolites, 17α-hydroxy-17ß-methylandrosta-4,6-dien-3-one (Δ6-epimethyl-testosterone), 17α-methyl-5ß-androstane-3α,17ß-diol (3α,5ß-THMT), and 17ß-methyl-5ß-androstane-3α,17α-diol, as well as the parent compound excreted after a single oral administration were monitored by GC-MS/MS. Δ6-Epimethyltestosterone and 3α,5ß-THMT served for long-term detection (still present in the 181-189 h urine). 17α-Methyltestosterone and its 17-epimer were not detected in the urines (LOD 0.3ng/mL). The highest concentrations were found in the 14-20.5h urine for Δ6-epimethyltestosterone (600 ng/mL), and 3α,5ß-THMT (240 ng/mL) and in the 36-44.5h urine for 17ß-methyl-5ß-androstane-3α,17α-diol (7 ng/mL). For reference methyltestosterone and epimethyltestosterone were dehydrogenated with chloranil. The characterization of the products was performed by GC-MS(/MS) and NMR.

Sports-related issues and biochemistry of natural and synthetic anabolic substances.

Testosterone is the principal male sex hormone. As with all natural steroids, it is biosynthesized from cholesterol. Phase I metabolism employs some very specific enzymes and pathways. Phase II metabolism and excretion follow more general patterns. The effects of testosterone are twofold: anabolic and androgenic. Because of its anabolic effects, testosterone is frequently abused in sports. Because of its endogenous nature, testosterone doping is difficult to detect. The standard procedure is based on the evaluation of the urinary steroid profile. Conspicuous samples then are submitted to compound-specific (13)C/(12)C analysis. Synthetic and endogenous steroids differ in this measure. Numerous xenobiotic compounds have been derived from testosterone. The modifications typically aim at a reduction of the androgenic properties while maintaining the anabolic potential. Most of these compounds have been withdrawn from the legal market. However, they are found to be illicitly added to otherwise inefficient nutritional supplements. These products represent a major problem to doping control. Recently, clinical trials with selective androgen receptor modulators have been started.

Metabolism of androsta-1,4,6-triene-3,17-dione and detection by gas chromatography/mass spectrometry in doping control.

The urinary metabolism of the irreversible aromatase inhibitor androsta-1,4,6-triene-3,17-dione was investigated. It is mainly excreted unchanged and as its 17beta-hydroxy analogue. For confirmation, 17beta-hydroxyandrosta-1,4,6-trien-3-one was synthesized and characterized by nuclear magnetic resonance (NMR) in addition to the parent compound. In addition, several reduced metabolites were detected in the post-administration urines, namely 17beta-hydroxyandrosta-1,4-dien-3-one (boldenone), 17beta-hydroxy-5beta-androst-1-en-3-one (boldenone metabolite), 17beta-hydroxyandrosta-4,6-dien-3-one, and androsta-4,6-diene-3,17-dione. The identification was performed by comparison of the metabolites with reference material utilizing gas chromatography/mass spectrometry (GC/MS) of the underivatized compounds and GC/MS and GC/tandem mass spectrometry (MS/MS) of their trimethylsilyl (TMS) derivatives. Alterations in the steroid profile were also observed, most obviously in the androsterone/testosterone ratio. Even if not explicitly listed, androsta-1,4,6-triene-3,17-dione is classified as a prohibited substance in sports by the World Anti-Doping Agency (WADA) due to its aromatase-inhibiting properties. In 2006 three samples from human routine sports doping control tested positive for metabolites of androsta-1,4,6-triene-3,17-dione. The samples were initially found suspicious for the boldenone metabolite 17beta-hydroxy-5beta-androst-1-en-3-one. Since metabolites of androst-4-ene-3,6,17-trione were also present in the urine samples, it is presumed that these findings were due to the administration of a product like 'Novedex Xtreme', which could be easily obtained from the sport supplement market.

Clenbuterol marketed as dietary supplement.

In several studies it has been demonstrated that products containing pharmaceutically active ingredients are marketed as dietary supplements. Most of these products contain anabolic steroids. Recently products for weight loss containing active drugs have also appeared on the market. In the present case a healthy male ordered the product 'Anabolic burner' via the Internet. The product was received from a German dispatcher and paid by bank transfer to a German bank account. After ingesting one tablet he reported tremor and delivered a urine sample. This urine was found to contain 2 ng/mL of clenbuterol utilizing LC-MS/MS analysis. Additionally the product itself was analyzed with GC-MS for clenbuterol, yielding a content of about 30 microg per tablet. The beta-2 agonist clenbuterol is only legally available on prescription and is classified as prohibited doping substance in sports. The present case for the first time confirms the presence of clenbuterol in a dietary supplement. It again demonstrates the common problem with products on the supplement market, where non-licensed pharmaceuticals and doping substances are easily available. The ingestion of these products containing additions of therapeutic drugs can lead to side effects and/or interactions with conventional medicines.

High amounts of 17-methylated anabolic-androgenic steroids in effervescent tablets on the dietary supplement market.

In numerous studies it has been demonstrated that several nutritional supplements contain prohormones not declared on the label. In the current study two products (effervescent tablets) containing high amounts of the 17-methylated anabolic androgenic steroids metandienone (product 1: 16.8 mg/tablet) and stanozolol (product 2: 14.5 mg/tablet) were identified. Additionally in both products norandrostenedione was detected, in product 2 with minor amounts of several other steroids. The substances identified can cause enormous health risks. In addition, the use of the analyzed tablets can lead to positive doping results for metabolites of the respective steroids in sports. This study again shows the insufficient surveillance of the production and trade of dietary supplements. Consumers should be aware of the enormous health and doping risks connected with the use of such products. For GC-MS identification of the analytes the trimethylsilyl derivatives of the steroids and the mixed N-t-butyldimethylsilyl,O-trimethylsilyl derivatives were used. The quantitation of metandienone, norandrostenedione, and stanozolol was performed using HPLC-DAD.