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- Material and methods
The purpose of this study was to analyze the composition of 103 dietary supplements bought on the internet. The supplements were dispatched in four different categories according to their announced contents [creatine, prohormones, “mental enhancers” and branched chain amino acids (BCAA)]. All the supplements were screened for the presence of stimulants and main anabolic steroids parent compounds. At the same time, the research was focused on the precursors and metabolites of testosterone and nandrolone.
The study pointed out three products containing an anabolic steroid, metandienone, in a very high amount. The ingestion of such products induced a high quantity of metandienone metabolites in urines that would be considered as a positive antidoping test. The results have also shown that one creatine product and three “mental enhancers” contained traces of hormones or prohormones not claimed on the labels and 14 prohormone products contained substances other than those indicated by the manufacturer. The oral intake of the creatine product revealed the presence of the two main nandrolone metabolites (19-norandrosterone and 19-noretiocholanolone) in urine.
In the last few years, dietary supplements have been widely used by elite athletes who believe that products like creatine, prohormones, amino acids and “mental enhancers” would boost their physical and psychological abilities (Ekblom, 1996; Engelhardt et al., 1998; Appelgate, 1999; Di Luigi et al., 1999; Terjung, 2000; van Gammeren et al., 2001; Ziegenfuss et al., 2002; Sundgot-Borgen et al., 2003). Although a meta-analysis of available literature has recently demonstrated ergogenic effects of creatine and HMB (Nissen & Sharp, 2003), the beneficial effects of most of these products are not clearly established even if many studies have been carried out in this field (King et al., 1999; Leder et al., 2000; Graham, 2001; Lawrence & Kirby, 2002; Powers, 2002; van Gammeren et al., 2002). The widespread accessibility is also the cause of the expansion of the use of dietary supplements. Indeed, the industry specialized in supplement manufacture generates annual seals estimated at US $12 billion in 1999 (Pipe & Ayotte, 2002).
At the same time, positive cases appeared with very small urinary concentrations of forbidden substances like 19-norandrosterone. All these reasons drove some antidoping laboratories to investigate the real composition of the over-the-counter (OTC) supplements available on the internet, in shops or in fitness clubs (Geyer et al., 2000; De Cock et al., 2001; Green et al., 2001; Kamber et al., 2001). Many of these investigations pointed out that both hormonal and non-hormonal dietary supplements are mislabelled and may contain anabolic androgenic steroids or prohormones that could be metabolized to compounds that are produced by the metabolism of banned anabolic steroids like 19-nortestosterone (nandrolone) (Uralets & Gillette, 1999; Catlin et al., 2000; Ayotte et al., 2001; Colker et al., 2001; Geyer et al., 2001). It has also been demonstrated that some supplements may be contaminated with other substances like stimulants (caffeine, which is no more present on the World Anti-Doping Agency (WADA) prohibited list from 2004 and ephedrine) (Gurley et al., 2000).
Therefore, there is an evident risk for unintentional doping among the dietary supplement users. This danger is not well understood by the athletes and a regular education of athletes, coaches and medical staff would be necessary to decrease the abuse of the dietary supplements and its related risks.
In 2000, the Swiss Antidoping Laboratory and the Swiss Federal Office of Sports performed a first study on the dietary supplements. From the obtained results, athletes were informed about the findings and warned of products bought from unknown sources. With this new study, the authors first wanted to know whether the situation has improved (or worsened) on the market and second to focus on products that are widely used and easily accessible in Switzerland. In our country, most people who consume dietary supplements buy the products through different internet sites. Indeed, not all the products are accessible at the markets or in drugstores. Therefore, the aim of this study was to check the real compositions of 103 OTC dietary supplements. The selection of the supplements was carried out randomly, except for the few Swiss manufacturers products, which were the most popular among the athletes. We screened the supplements for contaminations with major anabolic steroid parent compounds, stimulants and traces of testosterone, nandrolone and their precursors.
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- Material and methods
The purpose of this investigation was to determine the content and the purity of 103 OTC dietary supplements. All the supplements were ordered on different web sites in Europe and in America. The received products were separated into four categories: prohormones, creatine, BCAAs and “mental enhancers” (Table 2).
Table 2. The four categories in which the 103 complements were dispatched
|Class of products||Number of analyzed products||Main claimed compounds||Claimed effects of the products|
|Prohormone||37||DHEA, androstenediol, androstenedione, norandrostenediol and norandrostenedione||Increase in muscle mass, muscle strength are believed to occur via its conversion to testosterone and other steroids androgenic properties (e.g., as on mood, sexual behavior)|
|Creatine||42||Creatine monohydrate (pure or conjugated)||Assistance in providing muscle fibers with the energy needed to facilitate quick and forceful movements. More energy storage, increased stamina, strength, protein synthesis and muscle mass|
|BCAA||12||l-valine, l-leucine, l-isoleucine||Increase glycogen storage and neoglucogenese; increase protein anabolism and decrease catabolism⇒muscle; accelerate recovery; prevent over-training|
|“Mental enhancers”||12||Ephedrine, caffeine, Ma Huang, Guarana, Synephrin||Increase blood circulation (brain); help maintain concentration, improve memory and mood; increase metabolic rate and caloric expenditure; fat loss|
No stimulant contaminations were found in the analyzed dietary supplements but high amounts of stimulants like caffeine, ephedrine or synephrin were detected in the “mental enhancers” products (data not shown). This are not considered as contaminants as these substances were declared on the labels.
The results of the screening of the 103 products for the presence of steroids parent compounds revealed the presence of metandienone in three different dietary supplements. Similar results have been obtained by two other antidoping laboratories (Gmeiner, 2002; Geyer et al., 2003) (Table 3). Excretion studies were carried out with an oral administration of the recommended dosage of each of these three contaminated supplements. The urine following the intake of 40 mg of the prohormone 34 was collected during 1 week. High urinary concentrations of the metandienone metabolites were detected and traces of these metabolites were found in urine 1 week after the administration (Fig. 3).
Table 3. Quantification of the metandienone contaminations
|Product||Amount of metandienone (mg/g)|
Figure 3. Urinary excretion of metabolites 2 (m/z 517) and 3 (m/z 358) of metandienone after the intake of the recommended dosage (40 mg/day) of the prohormone 34 contaminated product.
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No contamination with other steroid parent compound was pointed out by the GC–MS analyses.
Research of the precursors and metabolites of testosterone and nandrolone revealed that 18 products were contaminated. A macro programmation allowed a rapid identification of the substances included in the supplement composition (Fig. 2). The number of mislabelled supplements represents 18% of the 103 products analyzed. Fourteen of these supplements were in the prohormones, three in the “mental enhancers” and one in the creatine category. The “mental enhancer 8” product was in this category because it was mainly composed of ephedrine, caffeine and synephrin but it also contained androstenediol. The most frequent contaminant was the testosterone parent molecule and the amounts were smaller in the creatine and “mental enhancers” than in the prohormone products (Table 4).
Table 4. Summary of the 18 contaminated products
|Product||Weight/cap (g)||Declared compounds||Contaminants||Concentration (…/cap)|
|Creatine 02||0.60||Creatine pyruvate||Adione||390 ng|
| || ||DHEA||4900 ng|
| || ||Adiol||2500 ng|
| ||Magnesium stearate||NorAdione||1200 ng|
| || ||Testosterone||45 ng|
|“Mental enhancers” 06||0.50||Ephedrine|| || |
| ||Caffeine||Adiol||600 ng|
|“Mental enhancers” 07||0.60||Caffeine anhydrous||NorAdione||600 ng|
|“Mental enhancers” 08||0.55||Adiol||Testosterone||300 μg|
|Prohormone 01||0.69||Adione, Adiol||Testosterone||2000 μg|
|Prohormone 02||0.72||19-NorAdione||Adione||2000 μg|
|Prohormone 05||1.30||19-NorAdione, Adiol, Adione||Testosterone||50 000 μg|
|Prohormone 06||0.30||Adiol||Testosterone||15 000 μg|
| || ||Testosterone||100 μg|
|Prohormone 07||0.33||19-NorAdiol||DHEA||30 μg|
| || ||Nandrolone||1500 μg|
|Prohormone 11||0.66||Adione||Testosterone||400 μg|
|Prohormone 13||0.70||19-NorAdiol||Nandrolone||2000 μg|
| || ||DHEA||80 μg|
|Prohormone 15||0.32||Adiol||Testosterone||6500 μg|
|Prohormone 17||0.59||Adiol, 19-NorAdione||Testosterone||550 μg|
|Prohormone 18||0.35||Adiol, 19-NorAdione||Testosterone||700 μg|
| || ||Testosterone||1100 μg|
|Prohormone 20||0.36||Adiol||NorAdione||800 μg|
|Prohormone 21||0.30||Adiol||DHEA||250 μg|
|Prohormone 27||0.70||Adiol, 19-NorAdione||Testosterone||650 μg|
| || ||Adione||200 000 μg|
|Prohormone 31||0.20||Adiol||Testosterone||500 μg|
The contaminated supplements, except those of the prohormone category, were orally administered to volunteers. The urine was collected after the intake in order to detect the presence of one or more substances, like nandrolone metabolites, that could generate a positive antidoping test. The most interesting excretion study was carried out with the creatine product. Indeed, the administration of the recommended dosage (5.25 g) led to the presence of noticeable urinary concentrations of 19-norandrosterone and 19-noretiocholanolone (Fig. 4). The concentrations were close to the WADA limit of 2 mg/L defined in the international standards (TD2004MRPL –WADA, 2004).
Figure 4. Urinary excretion of 19-norandrosterone and 19-noretiocholnolone (m/z 405) after the intake of the recommended dosage (7 caps/day) of the creatine contaminated product. The arrows indicate the intake of the 3 or 4 caps, and the International Olympics committee limit of 2 ng/mL is shown by the bold line.
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- Material and methods
Through the daily routine work of an antidoping laboratory, objective information and facts have been pointed out. Indeed, the athletes' declarations in the media or in the antidoping control forms revealed that the dietary supplements are being used more and more by athletes. Considering this information and the fact that their contents are not clearly mentioned, it was in our interest to investigate the OTC supplements. The Council of Europe and the WADA also want governments to work for “safe” supplements. So studies like this one, focusing on the mislabelling of dietary supplements, are needed to evaluate the extent of the problem and to make better recommendations to athletes.
The steroid parent compounds targeted in this study (Table 1) are the most common steroids found in athletes' urine. Moreover, the prohormones frequently used in the manufacture of such products are androstenedione, androstenediol, DHEA, 19-norandrostenedione and 19-norandrostenediol (Ayotte et al., 2001; De Cock et al., 2001; Green et al., 2001; Pipe & Ayotte, 2002). These molecules are involved in the metabolism of testosterone or nandrolone (Figs 1a and b) and are now on the official list of forbidden substances produced by the WADA (2004). These are the reasons why we focused on the detection of the molecules related to testosterone and nandrolone listed in Table 1. The ions chosen for the identification were normally the most abundant and the most typical for each compound.
The findings of the presence of metandienone in three products were concomitant with the results published previously (Gmeiner, 2002; Geyer et al., 2003). Metandienone is an anabolic steroid that was widely used by athletes in the 1980s. The therapeutical dose for this drug is between 5 and 10 mg/day (Royal Pharmaceutical Society, 1996). Regarding the manufacturer's advice, the intake of the recommended day dosage (40 mg) for the prohormone 34 corresponds to the achievement of about 26 mg of metandienone. The same calculation for the prohormone 36 implies an intake of about 22 mg of metandienone. These high amounts of metandienone could be very harmful for the consumers. Moreover, the numerous adverse effects such as liver dysfunction, increased risk of cardiovascular disease, severe psychological and psychiatric disorders (Royal Pharmaceutical Society, 1996) could be amplified by the supra-therapeutical doses. The analyses of the urines collected following the intake of the supplements revealed the presence of metandienone metabolites (Fig. 3) (Schänzer et al., 1991). Traces of these metabolites were found in urine 1 week after the treatment. These results are very important for the antidoping laboratories and the athletes because by consuming a unique dose of contaminated dietary supplements like the prohormones 34, 35 or 36, the athletes would have trouble with antidoping tests.
The pharmacokinetics (Fig. 3) pointed out a cyclic urinary elimination of the metandienone metabolites. This aspect has never been observed before and further investigations must be conducted to understand this phenomenon.
Considering the contaminations of the supplements with testosterone and/or nandrolone-related substances, the proportion (18%) of mislabelled supplements is in the same range as the previous studies (IOC, 2002). It has to be pointed out that the majority of the contaminations were found in the prohormone category. The presence of substances that are not indicated by the manufacturers is naturally not allowed and a legal procedure may be carried out by the consumers. Nevertheless, people who use prohormones supplements are already exposed, in a conscious or unconscious manner, to the side effects of these substances. Thus, the presence of contaminants like androstenedione or norandrostenediol in the prohormone supplements is not so dangerous as in other supplement types. Indeed, a product such as creatine is usually taken to facilitate quick and forceful movements in providing muscle fibers with the energy needed (creatine-phosphate). Anabolic effects are not the claimed effect of the creatine supplementation. The fact that prohormones are incorporated in the composition of this product could lead to several and unintentional consequences on morphological appearance and behavior. Depending on the time period of the treatment, these psychological and physiological effects could be dangerous and irreversible for the consumer. Nowadays, in terms of doping, the contamination of dietary supplements is a real problem for athletes, sport federations and antidoping laboratories. It appears clearly, in this study, that the intake of the recommended daily dose during 3 days of creatine contaminated with norandrostenedione led to the presence of the two main metabolites of nandrolone in urine with concentrations close to the official limit of 2 ng/mL. With a longer treatment, the concentrations would certainly exceed the limit and the urine sample would be considered as positive for antidoping laboratories. As the creatine product also contained precursors of testosterone, the concentrations of the endogens steroids like androsterone or etiocholanololne were determined (data not shown). No significant change was observed for these steroids and the value of the testosterone/epitestosterone (T/E) ratio, which is commonly used for detection of doping with testosterone (Kicman et al., 1990; Catlin, 1992; Donike et al., 1993), did not vary in a significant manner. These urinary data showed that the hypothalamic–pituitary–gonadal axis is punctually not altered by the intake of contaminated dietary supplements. A prolonged intake of supplement could have more influence on these parameters.
A similar experience was also done with caffeine contaminated with norandrostenedione. Four caps were orally taken (recommended dose) and urine was collected during 3 days. 19-norandrosterone and 19-noretiocholanolone were detected but the concentrations were about 1 ng/mL (data not shown). Probably a longer treatment would have produced higher 19-nortestosterone metabolite urinary concentrations that could imply a positive antidoping test.