ANTIDOPING

IN AN ERA OF EVIDENCED BASED MEDICINE

Scientific literature made available second quarter 2008

Selected and edited by ke Andrn-Sandberg

From the Department of Surgery, Karolinska Institutet at Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden

Highlights of the literature Over-the-counter medicine

A questionnaire was administered to elite athletes from Australia, Canada, the UK, and the USA representing 10 Olympic sports in order to explore knowledge and understanding of over-the-counter (OTC) meditation. Athletes demonstrated limited knowledge and understanding. Around half (51 %) knew the penalty incurred following a doping violation involving a banned OTC stimulant. The terms Monitoring Program and Specified Substance List were understood by 43 and 68 percent of respondents, respectively. Overall, the status of substances in relation to the Prohibited List was correctly identified in just 35 percent of cases. As a whole, athletes were of the opinion that OTC stimulants posed a risk to health, were performance enhancing and that their use was against the spirit of sport [001].

Impact of training on femal hormone status

The purpose of one investigation was to study the effects of an 11-week training period performed by female weightlifters. Two weeks before this investigation, baseline measures for total testosterone, cortisol, and testosterone:cortisol ratio were collected. The testosterone:cortisol ratio of the baseline was significantly different from the ratio of weeks 1 and 9. When the week-to-week values were compared, week 1 was significantly different from week 3. A very strong correlation was found between the percentage change of the testosterone:cortisol ratio and volume load from weeks 1 to 11. Moderate to very strong correlations were noted between the percentage change in volume load and isometric peak force, peak force during the 30 percent isometric peak force trial, and peak force during the 100-kg trial during the 11 weeks of training. The primary finding of this study was that alterations in training volume load can result in concomitant changes in the anabolic-to-catabolic balance, as indicated by the testosterone:cortisol ratio, and the ability to generate maximal forces [002].

Erythropoietin
It was hypothesized that intracardiac injection of erythropoietin may exhibit cardioprotective potential with reduced systemic toxicity. In conclusion, one study demonstrated that intramyocardial erythropoietin injection, following myocardial infarction, reduced inflammation, enhanced angiogenesis and proliferation, improved myocardial functions, and did not lead to intramural thrombus formation [003].

Growth hormone and IGF-1

The utility of insulinlike growth factor (IGF) axis and collagen markers for a growth hormone (GH) doping test in sport depends on their stability and reproducibility. It was sought to determine short-term within-subject variability of these markers in a large cohort of healthy individuals by measuring IGF-I, IGF binding protein 3 (IGFBP3), acid labile subunit (ALS), and the collagen markers N-terminal propeptide of type I procollagen (PINP), C-terminal telopeptide of type I collagen (ICTP), and N-terminal propeptide of type III procollagen (PIIINP) in serum samples obtained on multiple occasions (median 3 per participant) over a 2- to 3-week period from 1103 elite athletes (699 men, 404 women) aged (mean) 22 years. Within-subject variance accounted for 32-36 percent and 4-13 percent of the total variance in IGF markers and collagen markers, respectively. The within-subject CV ranged from 11 to 21 percent for the IGF axis markers and from 13 to 15 percent for the collagen markers.

These results indicate that in healthy individuals the within-subject variability was greater for IGF-I than for the collagen markers, and that where a single measurement is available, it is possible to estimate the long-term probable value of each of the markers by applying the Bayesian approach. Such an application can increase the reliability and decrease the cost of detecting GH doping [004].

Caffeine
The ergogenic effects of caffeine on athletic performance have been shown in many studies, and its broad range of metabolic, hormonal, and physiologic effects has been recorded, as one review of the literature shows. However, few caffeine studies have been published to include cognitive and physiologic considerations for the athlete. The following practical recommendations consider the global effects of caffeine on the body: lower doses can be as effective as higher doses during exercise performance without any negative coincidence; after a period of cessation, restarting caffeine intake at a low amount before performance can provide the same ergogenic effects as acute intake; caffeine can be taken gradually at low doses to avoid tolerance during the course of 3 or 4 days, just before intense training to sustain exercise intensity; and caffeine can improve cognitive aspects of performance, such as concentration, when an athlete has not slept well. Athletes and coaches also must consider how a person's body size, age, gender, previous use, level of tolerance, and the dose itself all influence the ergogenic effects of caffeine on sports performance [005]. Interest in the use of caffeine as an ergogenic aid has increased since the International Olympic Committee lifted the partial ban on its use. Caffeine has beneficial effects on various aspects of athletic performance, but its effects on training have been neglected. To investigate the acute effect of caffeine on the exercise-associated increases in testosterone and cortisol in a double-blind crossover study 24 professional rugby-league players ingested caffeine doses of 0, 200, 400, and 800 mg in random order 1 hr before a resistance-exercise session. Saliva was sampled at the time of caffeine ingestion, at 15-min intervals throughout each session, and 15 and 30 min after the session. It was concluded that caffeine has some potential to benefit training outcomes via the anabolic effects of the increase in testosterone concentration, but this benefit might be counteracted by the opposing catabolic effects of the increase in cortisol and resultant decline in the testosterone:cortisol ratio [006]. It was determined the effect of coingestion of caffeine with carbohydrate (CHO) on rates of muscle glycogen resynthesis during recovery from exhaustive exercise in seven trained subjects who completed two experimental trials in a randomized, double-blind crossover design. The evening before an experiment subjects performed intermittent exhaustive cycling and then consumed a low-CHO meal. The next morning subjects rode until volitional fatigue. On completion of this ride subjects consumed either CHO (4 g/kg body mass) or the same amount of CHO + caffein (8 mg/kg) during 4 hours of passive recovery. It was provided the first evidence that in trained subjects coingestion of large amounts of caffein (8 mg/kg) with CHO has an additive effect on rates of postexercise muscle glycogen accumulation compared with consumption of CHO alone [007]. One experiment examined the effect of a moderate dose of caffeine on perceptions of leg-muscle pain during a bout of high-intensity cycling exercise and the role of anxiety sensitivity in the hypoalgesic effect of caffeine on muscle pain during exercise. Sixteen college-age women ingested caffeine (5 mg/kg body weight) or a

placebo and 1 hour later completed 30 min of cycling on an ergometer at 80 percent of peak aerobic capacity. Caffeine resulted in a large reduction in leg-muscle painintensity ratings compared with placebo, and the reduction in leg-muscle painintensity ratings was larger in those with lower anxiety-sensitivity scores than those with higher anxiety-sensitivity scores. The results support that caffeine ingestion has a large effect on reducing leg-muscle pain during high-intensity exercise, but the effect is moderated by anxiety sensitivity [008].

Dietary supplements
Since 1999 several groups have analyzed nutritional supplements with mass spectrometric methods (GC/MS, LC/MS/MS) for contaminations and adulterations with doping substances. These investigations showed that nutritional supplements contained prohibited stimulants as ephedrines, caffeine, methylenedioxymetamphetamie and sibutramine, which were not declared on the labels. An international study performed in 2001 and 2002 on 634 nutritional supplements that were purchased in 13 different countries showed that about 15 percent of the nonhormonal nutritional supplements were contaminated with anabolic-androgenic steroids (mainly prohormones). Since 2002, also products intentionally faked with high amounts of classic anabolic steroids such as metandienone, stanozolol, boldenone, dehydrochloromethyl-testosterone, oxandrolone etc have been detected on the nutritional supplement market. These anabolic steroids were not declared on the labels either. The sources of these anabolic steroids are probably Chinese pharmaceutical companies, which sell bulk material of anabolic steroids. In 2005 vitamin C, multivitamin and magnesium tablets were confiscated, which contained cross-contaminations of stanozolol and metandienone. Since 2002 new designer steroids such as prostanozol, methasterone, androstatrienedione etc have been offered on the nutritional supplement market. In the near future also crosscontaminations with these steroids are expected. Recently a nutritional supplement for weight loss was found to contain the beta2-agonist clenbuterol. The application of such nutritional supplements is connected with a high risk of inadvertent doping cases and a health risk [009].

Contents
Highlights of the literature Over-the-counter medicine Impact of training on femal hormone status Erythropoietin Growth hormone and IGF-1 Caffeine Dietary supplements Abbreviations General overview Anti-doping history Social medicine Methodology for investigation doping society Over-the-counter medicine Germany Testing metods in general Anabolic steroids Anti-doping history Overview Biological action Prevalence of missuse Side effects Psychiatry Metabolism of testosterone Treatment of hypogonadal men Impact of training in males Impact of training in females Transsexuality Dehydroepiandrosterone Testing metods Yeast analysis Hair analysis Estrogens Anti-estrogens Selective androgen receptor modulators (SARM) Blood doping Overview Testing metods Bicycle Erythropoietin Laboratory testing Effects outside the bone marrow Growth hormone and IGF-1 Overview Biology Testing metods Impact of injuries Gonadotrophins Boxers Human choriongonadotrophin Stimulants Modafinil Methylphenidate

Caffeine Experimental Beta2agonists Exercise-induced asthma Salbutamol Procaterol Clenbuterol Cannabis Diuretics NSAIDs Dietary supplements Overview Contaminants Energy drinks Creatine Carnitine Taurin Polyunsaturated fatty-acids Omega-3 fatty acid D-vitamin Protein supplements Long distance running Veterinary (horses) Gene doping Ethics References

Abbreviations
AAS ALS APCI AR BCAA CES CHO cIEF CK CO COMT CSA DA DESI DHA DHEA DHT DOPAC DPO EAACI EDMA EIA EPA ERS ESI FSH GC-HRMS GC/MS GC-MS/MS GH GI GnRH hCG hEPO HPLC GC/C/IRMS GCxGC-TOF MS HVA IAT ICTP IEF IGF IGFBP-3 IRMS IS IT-MS LC LC-ESI-MS/MS LH anabolic androgenic steroid acid labile subunit atmospheric pressure chemical ionization androgen receptor branched-chain amino acid carbohydrate-electrolyte solutions Chinese hamster ovary capillary isoelectric focusing creatine kinase cardiac output cathecol-O-methyltransferase cross-sectional area dopamine desorption electrospray ionization docosahexaenoic acid dehydroepiandrosterone dihydrotestosterone 3,4-dihydroxyphenylacetic acid darbepoetin alfa European Academy of Allergy and Clinical Immunology ethylene glycol dimethacrylate exercise-induced asthma eicosapentaenoic acid European Respiratory Society electrospray ionisation follicle-stimulating hormone gas chromatography-high-resolution mass spectrometry gas chromatography followed by mass spectrometry gas chromatography-tandem mass spectrometry growth hormone gastrointestinal gonadotrophin-releasing hormone human chorionic gonadotropin human erythropoietin high-performance liquid chromatography gas chromatography/combustion/isotope ratio mass spectrometry two-dimensional gas chromatography with time-of-flight mass spectrometry homovanillic acid Implicit Associations Test C-terminal telopeptide of type I collagen isoelectric focusing insulinlike growth factor IGF binding protein 3 isotope ratio mass spectrometry internal standards ion trap mass spectrometry liquid chromatohraphic liquid chromatography electrospray ionization tandem mass spectrometry luteinizing hormone

LOD MA MAA MAO MCT MIP MRI MRM MS/MS OC OTC Pax7 PEAS PE-IAT PetO2 HbSaO2 HVR HCVR PIIINP PINP PUFA RBC RE RhEPO RhGH RHuEpo 1RM RPE RSD SPE SV SVR TAA THC TMS TOF-MS TUE UPLC WADA WCID

limit of detection methamphetamine methacrylic acid monoamine oxidases medium-chain triacylglycerol molecular imprinted polymer magnetic resonance imaging multiple reaction monitoring tandem mass spectrometry oral contraceptive over-the-counter paired box gene7 Performance Enhancement Attitude Scale performance-enhancement related Implicit Associations Test O2 partial pressure arterial oxyhemoglobin saturation hyperoxic ventilatory responses hypercapnic ventilatory responses N-terminal propeptide of type III procollagen N-terminal propeptide of type I procollagen polyunsaturated fatty-acid ( red blood cell resistance exercise recombinant erythropoietin recombinant human growth hormone recombinant human erythropoietin 1 repetition maximum ratings of perceived exertion relative standard deviation solid-phase extraction stroke volume systemic vascular resistance total antioxidant activity delta(9)-tetrahydrocannabinol trimethylsilyl time-of-flight mass spectrometry Therapeutic Use Exemption ultra-performance liquid chromatography World Anti-Doping Agency whole column imaging detection

General overview
Since ancient times, unethical athletes have attempted to gain an unfair competitive advantage through the use of doping substances. A list of doping substances and methods banned in sports is published yearly by the World Anti-Doping Agency. A substance or method might be included in the list if it fulfills at least two of the following criteria: enhances sports performance; represents a risk to the athlete's health; or violates the spirit of sports. This list, constantly updated to reflect new developments in the pharmaceutical industry as well as doping trends, enumerates the drug types and methods prohibited in and out of competition. Among the substances included are steroidal and peptide hormones and their modulators, stimulants, glucocorticosteroids, beta2-agonists, diuretics and masking agents, narcotics, and cannabinoids. Blood doping, tampering, infusions, and gene doping are examples of prohibited methods indicated on the list. From all these, hormones constitute by far the highest number of adverse analytical findings reported by antidoping laboratories. Although to date most are due to anabolic steroids, the advent of molecular biology techniques has made recombinant peptide hormones readily available. These substances are gradually changing the landscape of doping trends. Peptide hormones like erythropoietin, human growth hormone, insulin, and insulin-like growth factor I are presumed to be widely abused for performance enhancement. Furthermore, as there is a paucity of techniques suitable for their detection, peptide hormones are all the more attractive to dishonest athletes. This article will overview the use of hormones as doping substances in sports, focusing mainly on peptide hormones as they represent a pressing challenge to the current fight against doping. Hormones and hormones modulators being developed by the pharmaceutical industry, which could emerge as new doping substances, are also discussed [010]. Human performance, defined by mechanical resistance and distance per time, includes human, task and environmental factors, all interrelated. It requires metabolic energy provided by anaerobic and aerobic metabolic energy sources. These sources have specific limitations in the capacity and rate to provide re-phosphorylation energy, which determines individual ratios of aerobic and anaerobic metabolic power and their sustainability. In healthy athletes, limits to provide and utilize metabolic energy are multifactorial, carefully matched and include a safety margin imposed in order to protect the integrity of the human organism under maximal effort. Perception of afferent input associated with effort leads to conscious or unconscious decisions to modulate or terminate performance; however, the underlying mechanisms of cerebral control are not fully understood. The idea to move borders of performance with the help of biochemicals is two millennia old. Biochemical findings resulted in highly effective substances widely used to increase performance in daily life, during preparation for sport events and during competition, but many of them must be considered as doping and therefore illegal. Supplements and food have ergogenic potential; however, numerous concepts are controversially discussed with respect to legality and particularly evidence in terms of usefulness and risks. The effect of evidence-based nutritional strategies on adaptations in terms of gene and protein expression that occur in skeletal muscle during and after exercise training sessions is widely unknown [011].

Anti-doping history

Mass spectrometry has played a decisive role in doping analysis and doping control in human sport for almost 40 years. The standard of qualitative and quantitative determinations in body fluids has always attracted maximum attention from scientists. With its unique sensitivity and selectivity properties, mass spectrometry provides state-of-the-art technology in analytical chemistry. Both anti-doping organizations and the athletes concerned expect the utmost endeavours to prevent false-positive and false-negative results of the analytical evidence. The Olympic Games play an important role in international sport today and are milestones for technical development in doping analysis. One review of the part played by mass spectrometry in doping control from Munich 1972 to Beijing 2008 Olympics gave an overview of how doping analysis has developed and where we are today [012]. One brief note gave a general overview on the activity of the antidoping laboratories accredited by the World Anti-Doping Agency outlining the evolution, over the last four decades, of the analytical methods and techniques in the detection of prohibited substances and methods. Special emphasis was given to the future trends of the fight against doping in sports, as seen from the perspective of a laboratory scientist, in the wider context of fair play, health protection, and perception of the activity of the antidoping laboratories by the general public [013].

Social medicine Methodology for investigation doping society

Understanding athletes' attitudes and behavioural intentions towards performance enhancement is critical to informing anti-doping intervention strategies. Capturing the complexity of these attitudes beyond verbal declarations requires indirect methods. One pilot study was aimed at developing and validating a method to assess implicit doping attitudes using an Implicit Associations Test (IAT) approach. The conventional IAT evaluation task (categorising good and bad words) was combined with a novel doping versts nutrition supplements category pair to create a performanceenhancement related IAT protocol (PE-IAT). The difference between average response times to good-doping and bad-doping combinations represents an estimate of implicit attitude towards doping in relation to nutritional supplements. 111 sports and exercise science undergraduates completed the PE-IAT, the Performance Enhancement Attitude Scale (PEAS) and answered questions regarding their beliefs about doping. Longer response times were observed in the mixed category discrimination trials where categories good and doping shared the same response key (compared to bad-doping combination on the same key) indicating a less favourable evaluation of doping substances. The PE-IAT measure did not correlate significantly with the declared doping attitudes, indicating a predictable partial dissociation. Action-oriented self-report expressed stronger associations with PE-IAT: participants who declared they would consider using doping showed significantly less implicit negativity towards banned substances. Similarly, those who reported more lenient explicit attitudes towards doping or expressly supported legalizing it, showed less implicit negativity towards doping in the sample, although neither observed differences reached statistical significance. Known-group validation strategy yielded mixed results: while competitive sport participants scored significantly lower than non-competitive ones on the PEAS, the two groups did not differ on PE-IAT. It was concluded that the results suggest a potential of the PE-IAT method to capture undeclared attitudes to doping and predict behaviour, which can support targeted anti-doping intervention and related research [014].

Over-the-counter medicine
A questionnaire was administered to elite athletes from Australia, Canada, the UK, and the USA representing 10 Olympic sports in order to explore knowledge and understanding of over-the-counter (OTC) medication since the removal of many of these substances from the World Anti-Doping Agency Prohibited List, in 2004. Athletes demonstrated limited knowledge and understanding. Around half (51 %) knew the penalty incurred following a doping violation involving a banned OTC stimulant. The terms Monitoring Program and Specified Substance List were understood by 43 and 68 percent of respondents, respectively. Overall, the status of substances in relation to the Prohibited List was correctly identified in just 35 percent of cases. As a whole, athletes were of the opinion that OTC stimulants posed a risk to health, were performance enhancing and that their use was against the spirit of sport. They were undecided as to whether these drugs should be returned to the Prohibited List. Elite athletes require targeted education programmes that will enable them to make informed decisions on the potential of OTC medications for therapeutic or performance enhancing purposes [001].

Germany
Doping controls are key factors for fair and clean sports. The developments of German activities in the national antidoping fight were evaluated over a period of 18 years (1989-2006) with regard to in-competition and out-of-competition testing. The quantity of respective controls conducted by federations and antidoping organizations, the ratio of in- and out-of-competition controls, the number of athletes per squad (and thus per-capita tests) as well as adverse analytical findings were summarized in a review. The available data demonstrated a constantly increasing effort, particularly regarding the amount of out-of-competition controls, but also discrepancies in per-capita analyses between different federations. In light of recent doping scandals and confessions in 2007, a critical review of national antidoping actions is considered necessary [015]. In the context of house searches in Germany, numerous drugs were confiscated and subjected to chemical analysis, including anabolic agents such as various anabolicandrogenic steroids (stanozolol, testosterone derivatives, trenbolone esters, etc.) and clenbuterol, as well as agents with anti-estrogenic activity (tamoxifen, clomiphene), drugs stimulating virility (sildenafil, tadalafil), and unlabeled plastic bags. Liquid chromatography-tandem mass spectrometry, gas chromatography-mass spectrometry with nitrogen-phosphorus specific detection, gel electrophoresis, and immunological tests were employed to test for the effective content of 70 products. In 18 cases (26 %), the declared ingredients differed from the actual content, in particular concerning anabolic-androgenic steroids. Nandrolone and trenbolone esters, for instance, were frequently substituted or complemented by various testosterone derivatives, and several testosterone depot formulations originally composed of four different esters were found to contain fewer or wrong components. Except for those drugs supposedly originating from so-called underground labs, fake packings were hardly or not distinguishable from original boxes by visual inspektion [016].

Testing metods in general

The analysis of sports samples for prohibited substances began in the 1960s and has developed since then using modern technologies close to the latest scientific

discoveries. For small molecules, apart from the routine use of GC-MS, the newer techniques include the use of isotope ratio MS to detect testosterone and nandrolone administration and LC-MS/MS (liquid chromatography-tandem MS) to detect diuretics. For large molecules, several applications of LC-MS/MS are described as well as immunoprocedures for erythropoietin and human growth hormone [017]. Steroid profiling is one of the most versatile and informative screening tools for the detection of steroid abuse in sports drug testing. Concentrations and ratios of various endogenously produced steroidal hormones, their precursors and metabolites including testosterone, epitestosterone, dihydrotestosterone (DHT), androsterone, etiocholanolone, dehydroepiandrosterone (DHEA), 5alpha-androstane3alpha,17beta-diol, and 5beta-androstane-3alpha,17beta-diol as well as androstenedione, 6alpha-OH-androstenedione, 5beta-androstane-3alpha,17alphadiol, 5alpha-androstane-3alpha,17alpha-diol, 3alpha,5-cyclo-5alpha-androstan6beta-ol-17-one, 5alpha-androstanedione, and 5beta-androstanedione add up to a steroid profile that is highly sensitive to applications of endogenous as well as synthetic anabolic steroids, masking agents, and bacterial activity. Hence, the knowledge of factors that do influence the steroid profile pattern is a central aspect, and pharmaceutical (application of endogenous steroids and various pharmaceutical preparations), technical (hydrolysis, derivatization, matrix), and biological (bacterial activities, enzyme side activities) issues are reviewed [018]. A simple and rapid multicomponent screening method of 130 substances for direct injections of urine samples has been developed. The fully automated method based on ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS) is used for three different classes of doping agents: diuretics, central nervous system stimulants and opiates. The samples are diluted with buffer containing internal standards (IS) by a pipetting robot system into 96-well plates. Samples are injected on a reversed phase sub 2-microm particle column connected to a fast polarity switching and rapid scanning tandem mass spectrometer with an electrospray interface. The software used to evaluate the results produced reports containing a small-sized window for each component and a data table list with flags to indicate any adverse analytical findings in the sample. The report can also be processed automatically using an application software, which interpret the data and indicate if there is a suspicious sample. One 96-well plate can be analyzed within 16 hours [019]. A new doping control screening method has been developed, for the analysis of doping agents in human urine, using HPLC/orbitrap with in-source collision-induced dissociation and atmospheric pressure chemical ionization. The developed method allows the detection of 29 compounds, including agents with antiestrogenic activity, beta2agonists, exogenous anabolic steroids, and other anabolic agents. The mass accuracy of this method is better at 2 ppm using an external reference. The detection limit for all compounds tested was better than 100 pg/ml. The recoveries of most analytes were above 70 percent. The measured median repeatability values for doping agents included in the method at concentrations of 1 and 10 ng/ml were 21 and 17 percent, respectively. The relative standard deviation (RSD) of the intraday precision (n=6) ranged from RSD 16-22 percent, whereas the interday precision (n=18), ranged from RSD 17-26 percent, depending on the solute concentration investigated [020]. The ability to measure isotope distribution at natural abundance with high accuracy and precision has increased the application of gas chromatography-combustionisotope ratio mass spectrometry (GC-C-IRMS) to doping control in recent years. GCC-IRMS is capable of measuring the carbon isotope ratio (delta13C) of urinary

steroids and confirm their synthetic origin based on the abnormal 13C content. This tutorial describes some of the complexities encountered by obtaining valid delta13C measurements from GC-C-IRMS and the need for careful interpretation of all relevant information concerning an individual's metabolism in order to make an informed decision with respect to a doping violation [021]. The use of alternative specimens in the field of toxicology was first described in 1979, when hair analysis was used to document chronic drug exposure. Since then, the use of Thess alternative samples has gained tremendous importance in forensic toxicology, as well as in clinic toxicology, doping control and workplace drug testing. It is not surprising, therefore, that a large number of papers dealing with the determination of several classes of drugs in saliva, sweat, meconium and hair have been published ever since, owing to the fact that chromatographic equipment is becoming more and more sensitive, mass spectrometry (and tandem mass spectrometry) being the most widely used analytical tool, combined with gas or liquid chromatography. Alternative specimens present a number of advantages over the traditional samples normally used in toxicology (e.g. blood, urine and tissues), namely the fact that their collection is not invasive, their adulteration is difficult, and they may allow increased windows of detection for certain drugs. The main disadvantage of this kind of samples is that drugs are present in very low concentrations, and therefore high-sensitivity techniques are required to accomplish the analysis [022].

Anabolic steroids Anti-doping history

Androgenic-anabolic steroids (AAS) have been misused by athletes at the Olympic Games, both before and after they were prohibited in sport in 1974. Systematic doping with AAS occurred in the German Democratic Republic from 1965 to 1989 which assisted that country to win many medals at Olympic Games, especially in female events. Currently, androgenic-anabolic steroids are the most frequent category of prohibited substances detected in the urine of athletes both globally and at the last two Summer Olympic Games. Scientific confirmation that AAS are effective in enhancing sports performance was difficult because ethical approval was difficult for research involving male subjects taking massive doses of androgens as some athletes and bodybuilders did. Methods to detect androgenic-anabolic steroids have evolved gradually over the past three decades and currently, despite an impressive array of sophisticated analytical equipment and methods, anti-doping authorities and analytical scientists continue to face challenges as have occurred from the use by athletes of designer AAS during the past few years. The future development and use of selective androgen receptor modulators can be anticipated to pose problems in the years ahead. Endocrinologists should be aware that on occasions, replacement testosterone therapy may be authorized in sport as a therapeutic use exemption (TUE) [023].

Overview
The use of doping agents, particularly anabolic androgenic steroids, has changed from being a problem restricted to sports to one of public-health concern. In a review the prevalence of misuse, the evidence that some drugs improve performance in sport, their side-effects, and the long-term consequences of AAS misuse for society at large were discussed. There is substantial under-reporting of the side-effects of

AAS to health authorities. It was described neuropsychiatric side-effects of AAS and their possible neurobiological correlates, with particular emphasis on violent behaviour. Analytical methods and laboratories accredited by the World Anti-Doping Agency can detect the misuse of all doping agents; although the analysis of testosterone requires special techniques, and recently discovered interethnic differences in testosterone excretion should be taken into account. The prevention of misuse of doping agents should include random doping analyses, medical follow-ups, pedagogic interventions, tougher legislation against possession of AAS, and longer disqualifications of athletes who use AAS [024]. Androgens remain the most effective and widely abused ergogenic drugs in sport. Although androgen doping has been prohibited for over 3 decades with a ban enforced by mass spectrometric-based urine testing for synthetic and exogenous natural androgens, attempts continue to develop increasingly complex schemes to circumvent the ban. A prominent recent approach has been the development of designer androgens. Such never-marketed androgens evade detection because mass spectrometry relies on identifying characteristic chemical signatures requiring prior knowledge of chemical structure. Although once known, designer androgens are readily detected and added to the Prohibited List. However, until their structures are elucidated, designer androgens can circumvent the ban on androgen doping. To combat this, in vitro androgen bioassays offer powerful new possibilities for the generic detection of unidentified bioactive androgens, regardless of their chemical structure. Another approach to circumvent the ban on androgen doping has been the development of indirect androgen doping, the use of exogenous drugs to produce a sustained increase in endogenous testosterone production. Apart from estrogen blockers, however, such neuroendocrine active drugs mostly provide only transient increases in blood testosterone. Finally the ban on androgen doping must allow provision for rare athletes with incidental, proven androgen deficiency who require testosterone replacement therapy. The Therapeutic Use Exemption (TUE) mechanism makes provision for such necessary medical treatment, subject to rigorous criteria for demonstrating a genuine ongoing need for testosterone and monitoring of testosterone dosage. Effective deterrence of sports doping requires novel, increasingly sophisticated detection options calibrated to defeat these challenges, without which fairness in sport is tarnished and the social and health idealization of sporting champions devalued [025]. Anabolic steroids are performance enhancers, this being particularly apparent in women, although there is a high risk of virilization despite the favourable myotrophicandrogenic dissociation that many xenobiotic steroids confer. Modulation of androgen receptor expression appears to be key to partial dissociation, with consideration of both intracellular steroid metabolism and the topology of the bound androgen receptor interacting with co-activators. An anticatabolic effect, by interfering with glucocorticoid receptor expression, remains an attractive hypothesis. Behavioural changes by non-genomic and genomic pathways probably help motivate training. It is important not to exaggerate the medical risks associated with their administration for sporting or bodybuilding purposes but to emphasize to users that an attitude of personal invulnerability to their adverse effects is certainly misguided [026].

Biological action
Androgens are modulators of skeletal muscle adaptation and regeneration processes. The control of satellite cell activity is a key mechanism during this process. In this study, we analyzed the ability of dihydrotestosterone (DHT) and

anabolic steroids to induce and modulate the differentiation of myoblastoma cells toward myotubes. Myoblastoma cells were dose-dependently treated with DHT and anabolic steroids. The time-dependent effects on differentiation were measured and correlated with the expression of genes involved in the regulation of satellite cell activity. The distribution of myoblastoma cells within the cell cycle was measured by flow cytometry and differentiation by creatine kinase (CK) activity. Gene expression was analyzed using quantitative real-time PCR and confocal microscopy. The treatment with DHT and anabolic steroids resulted in a stimulation of myoblastoma cell proliferation and CK activity. The antiandrogen flutamide was able to antagonize this effect. The expression of the androgen receptor, SOX8, SOX9, Delta, Notch, myostatin, and paired box gene7 (Pax7) was modulated by androgens. The treatment with DHT and anabolic steroids resulted in a strong stimulation of myostatin expression not only in undifferentiated cells but also in myotubes. The stimulation could be antagonized by flutamide. The expression of Pax7 was detectable in myoblastoma cells early after treatment with DHT. The results demonstrate that the key mechanisms of satellite cell differentiation are modulated by androgens. Androgens stimulate the proliferation of myoblastoma cells, accelerate the process of differentiation, and increase the expression of myostatin in undifferentiated and differentiated cells [027]. Important mechanisms behind the myotrophic effects of testosterone were uncovered both in athletes using steroids for several years and in short-term controlled studies. Both long-term and short-term steroid usage accentuates the degree of fibre hypertrophy in human skeletal muscle by enhancing protein synthesis. A mechanism by which testosterone facilitates the hypertrophy of muscle fibres is the activation of satellite cells and the promotion of myonuclear accretion when existing myonuclei become unable to sustain further enhancement of protein synthesis. Interestingly, long-term steroid usage also enhances the frequency of fibres with centrally located myonuclei, which implies the occurrence of a high regenerative activity. Under the action of testosterone, some daughter cells generated by satellite cell proliferation may escape differentiation and return to quiescence, which help to replenish the satellite cell reserve pool. However, whether long-term steroid usage induces adverse effects of satellite cells remains unknown. Testosterone might also favour the commitment of pluripotent precursor cells into myotubes and inhibit adipogenic differentiation. The effects of testosterone on skeletal muscle are thought to be mediated via androgen receptors expressed in myonuclei and satellite cells. Some evidence also suggests the existence of an androgen-receptor-independent pathway. Clearly, testosterone abuse is associated with an intense recruitment of multiple myogenic pathways. This provides an unfair advantage over non-drug users. The long-term consequences on the regenerative capacity of skeletal muscle are unknown [028]. There is strong evidence that androgen administration in men increases skeletal muscle mass, maximal voluntary strength and muscle power. However, there is no good experimental evidence to support the presumption that androgen administration improves physical function or athletic performance. Androgens do not increase specific force or whole body endurance measures. The anabolic effects of testosterone on the skeletal muscle are mediated through androgen receptor signaling. Testosterone promotes myogenic differentiation of multipotent mesenchymal stem cells and inhibits their differentiation into the adipogenic lineage. Testosterone binding to androgen receptor induces a conformational change in androgen receptor protein, causing it to associate with beta-catenin and TCF-4 and activate downstream Wnt target genes thus promoting myogenic differentiation. The adverse effects of androgens among athletes and recreational bodybuilders are under reported and include acne, deleterious changes in the cardiovascular risk factors, including a marked decrease in plasma high-density lipoproteins (HDL)

cholesterol level, suppression of spermatogenesis resulting in infertility, increase in liver enzymes, hepatic neoplasms, mood and behavioral disturbances, and long term suppression of the endogenous hypothalamic-pituitary-gonadal axis. Androgens are often used in combination with other drugs which may have serious adverse events of their own [029]. One study examined the anabolic-hormone response to carbohydrate (CHO) supplementation at rest and after resistance exercise. Nine recreationally trained men randomly underwent 4 testing conditions: rest with placebo, rest with CHO, resistance exercise with placebo, and resistance exercise with CHO. The resistanceexercise protocol was four sets of Smith machine squats with a 10-repetitionmaximum load, with 90-s rests between sets. Participants then consumed either a placebo or CHO (24 % CHO, 1.5 g/kg) drink. Blood was taken before exercise, immediately after testing, and then 15, 30, and 60 min after drink ingestion. Blood was analyzed for cortisol, glucose, insulin, and total testosterone (TTST). Cortisol did not change significantly in any condition. Glucose concentrations increased significantly. Insulin concentrations increased significantly under resistant conditions. There were no significant changes in total testosterone concentrations during rest with or without carbohydrate in kontrast to the exercise groups. It was concluded that ingesting carbohydrates after resistance exercise resulted in decreased total testosterone concentrations during recovery, although the mechanism is unclear [030].

Prevalence of missuse
The prevalence of anabolic-androgenic steroids use has risen dramatically over the last two decades and filtered into all aspects of society. Support for AAS users has increased, but not by the medical profession, who will not accept that AAS use dependency is a psychiatric condition. Polypharmacy by self-prescription is prevalent in this sector. Most recently, AAS use has filtered through to recreational street drug users and is the largest growth of drugs in this subdivision. There is a degree of contentiousness in the scenario of anabolic-androgenic steroids drug use, both within and outside sport. AAS and associated doping agents are not illegal per se. Possession is not an offence, despite contravening sporting regulations and moral codes. Until AAS are classified in the same capacity as street drugs in the UK, where possession becomes a criminal offence, they will continue to attract those who want to win at any cost [031].

Side effects
Anabolic-androgenic steroids are associated with numerous side effects, including acute myocardial infarction. It was reported a case of myocardial infarction in a young 31-year-old bodybuilder. Because of the serious cardiovascular complications of anabolic steroids, physicians should be aware of their abuse and consequences [032]. A strong tendency toward body enhancement and body forming in western industrial societies makes it more likely for each anesthesiologist to get involved in the care of bodybuilders. These patients quite frequently consume androgenic anabolic steroids, human growth hormone and other drugs or substances which are believed to accelerate muscle gain. Cardiovascular, hepatic, psychiatric, hormonal and infectious side effects or complications are common and rarely monitored by health care professionals. The anesthesia risk is not exactly known but seems to be determined

mainly by cardiovascular events like myocardial ischemia and dysrhythmias [033].

Psychiatry
Human Muscle dysmorphia has been described as a disorder in which individuals are pathologically preoccupied with their muscularity. One study was designed to further investigate the symptom characteristics and psychiatric conditions associated with the disorder. Weight lifting males meeting current criteria for muscle dysmorphia (n=15), past muscle dysmorphia (n=8), and no history of muscle dysmorphia (n=28) responded to advertisements placed in gymnasium and nutrition stores. Structured and semistructured interviews were administered, as well as survey measures. Relative to controls, males with current muscle dysmorphia experienced more aversive symptoms related to the appearance of their bodies, including more often thinking about their muscularity, dissatisfaction with appearance, appearance checking, bodybuilding dependence, and functional impairment. Higher rates of mood and anxiety disorders were found among individuals with a history of muscle dysmorphia relative to individuals with no history of muscle dysmorphia. The findings suggest that muscle dysmorphia can be distinguished from normal weight lifting on a number of clinical dimensions. Muscle dysmorphia appears to be comorbid with other psychiatric conditions [034]. Rat It was previously showed that 14 days of daily intramuscular injections of the anabolic androgenic steroid nandrolone decanoate (15 mg/kg) reduced the extracellular levels of the dopaminergic metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the nucleus accumbens shell using microdialysis. The aim of one study was to investigate whether the same dose regimen of nandrolone decanoate may affect the activities of the dopaminemetabolizing enzymes monoamine oxidases A and B (MAO-A and MAO-B). A radiometric assay was used to determine the activities of MAO-A and MAO-B in rat brain tissues after 14 days of daily i.m. nandrolone decanoate injections at the doses 3 and 15 mg/kg. Gene transcript contents of MAO-A, MAO-B and cathecol-Omethyltransferase (COMT) were measured with quantitative real-time reverse transcription PCR. 3 mg/kg of nandrolone decanoate significantly reduced the activity of both MAO-A and -B in the caudate putamen. 15 mg/kg of nandrolone decanoate significantly reduced the activity of MAO-A in the amygdala and increased the gene transcript level of MAO-B in the substantia nigra. In conclusion, imbalanced MAO activities may contribute to explain the impulsive and aggressive behaviour often described in AAS abusers. The reduced MAO activities observed are in line with previously presented findings of decreased extracellular levels of DOPAC and HVA in the rat brain, indicating decreased monoaminergic activity following repeated AAS administration [035].

Metabolism of testosterone
Testosterone abuse is conventionally disclosed by urinary assay of the testosterone/epitestosterone (T/E) glucuronide ratio, which should not exceed 4. A noteworthy number of athletes, however, have higher natural ratios than 4, most likely because of decreased excretion of epitestosterone glucuronide. Urine from different study populations was analysed for androgen glucuronides by gas chromatography-mass spectrometry. All men were genotyped for the uridine diphospho-glucuronosyltransferase (UGT) 2B17 deletion polymorphism and single nucleotide polymorphisms in the cytochrome P-450c17alpha (CYP17), UGT2B15

and UGT2B7 genes. Expression of UGT2B15 mRNA in human liver samples was analysed using real-time PCR. A T>C (A1>A2) promoter polymorphism in the CYP17 gene was associated with the urinary glucuronide levels of epitestosterone and its putative precursor androstene-3beta, 17alpha-diol, resulting in 64 percent higher T/E ratios in A1/A1 homozygotes. Individuals devoid of UGT2B17 had significantly higher UGT2B15 mRNA levels in liver than individuals carrying two functional UGT2B17 alleles. The CYP17 promoter polymorphism may partly explain high natural (>4) T/E ratios. The data indicate that 5-androstene-3beta, 17alpha-diol is an important precursor of epitestosterone and that CYP17 is involved in its production. In addition, it was found that lack of the UGT2B17 enzyme may be compensated for by increase in UGT2B15 transkription [036].

Treatment of hypogonadal men

Testosterone has a steeply dose-dependent effect on muscle mass and strength irrespective of gonadal status. So, for reasons of fairness, people who engage in competitive sports should not administer exogenous testosterone raising their blood testosterone levels beyond the range of normal. There is a ban on exogenous androgens for men and women in sports, but an exception has been made for men with androgen deficiency due to pituitary or testicular disease. Men who receive testosterone administration for the indication hypogonadism have an interest in the use of testosterone preparations generating blood testosterone levels within the normal range of healthy, eugonadal men. On the grounds of a positive correlation between blood testosterone concentrations muscle and volume/strength, they are best served with a parenteral testosterone preparation, rather than transdermal testosterone, but they should not run the risk of being excluded from competition because of supraphysiological testosterone levels. The latter is a realistic risk with the traditional parenteral testosterone esters. The new parenteral testosterone undecanoate preparation offers much better perspectives. Its pharmacokinetics have been investigated in detail and there is a fair degree of predictability of resulting blood testosterone levels with use of this preparation [037].

Impact of training in males

The acute response of free salivary testosterone and cortisol concentrations to four resistance exercise (RE) protocols in 23 elite men rugby players was investigated. It was hypothesized that hormonal responses would differ among individuals after four distinct RE protocols: four sets of 10 repetitions (reps) at 70 percent of 1 repetition maximum (1RM) with 2 minutes' rest between sets (4 x 10-70 %); three sets of five reps at 85 percent 1RM with 3 minutes' rest (3 x 5-85 %); five sets of 15 reps at 55 percent 1RM with 1 minute's rest (5 x 15-55 %); and three sets of five reps at 40 percent 1RM with 3 minutes' rest (3 x 5-40 %). Each athlete completed each of the four resistance exercise protocols in a random order on separate days. Testosterone and cortisol concentrations were measured before exercise, immediately after exercise, and 30 minutes post exercise. Each protocol consisted of four exercises: bench press, leg press, seated row, and squats. Pooled testosterone data did not change as a result of resistance exercise, whereas cortisol declined significantly. Individual athletes differed in their testosterone response to each of the protocols, a difference that was masked when examining the pooled group data. When individual data were retrospectively tabulated according to the protocol in which each athlete showed the highest testosterone response, a significant protocol-dependent testosterone increase for all individuals was revealed. Therefore, resistance exercise induced significant individual, protocol-dependent hormonal changes lasting up to 30 minutes after exercise. These individual responses may have important ramifications

for modulating adaptation to resistance exercise and could explain the variability often observed in studies of hormonal response to resistance exercise [038].

Impact of training in females

The purpose of one investigation was to study the effects of an 11-week training period performed by female weightlifters. Two weeks before this investigation, baseline measures for total testosterone, cortisol, and testosterone:cortisol ratio were collected. The 11-week training program consisted of the core exercises (i.e. clean, clean and jerk, and snatch) and other supplemental exercises (i.e. clean pull, snatch pull, squat, and front squat). Hormonal, isometric, and dynamic middle thigh pull force-time curve characteristics were assessed biweekly throughout the duration of the investigation, whereas volume load and training intensity were assessed weekly throughout the investigation. The testosterone:cortisol ratio of the baseline was significantly different from the ratio of weeks 1 and 9. When the week-to-week values were compared, week 1 was significantly different from week 3. A very strong correlation was found between the percentage change of the testosterone:cortisol ratio and volume load from weeks 1 to 11. Moderate to very strong correlations were noted between the percentage change in volume load and isometric peak force, peak force during the 30 percent isometric peak force trial, and peak force during the 100kg trial during the 11 weeks of training. The primary finding of this study was that alterations in training volume load can result in concomitant changes in the anabolicto-catabolic balance, as indicated by the testosterone:cortisol ratio, and the ability to generate maximal forces [002].

Transsexuality
Sex segregation in competitive sports is regarded as fair. Before puberty boys and girls do not differ in height, muscle and bone mass. Testosterone exposure during puberty leads to an ultimate average greater height in men of 12-15 cm, longer and larger bones and muscle mass and strength and higher hemoglobin levels. Postpubertal androgen ablation reverses, at least in part, previous anabolic effects of testosterone on muscle, bone mineral density and hemoglobin but the long bones remain longer and wider. Testosterone administration dose dependently increases muscle mass and maximal voluntary strength. Therefore, exogenous androgens, being performance enhancing drugs, are banned for all athletes. An issue is the participation in competitive sports of people with errors of sexual differentiation and particularly transsexuals who have been sex-reassigned. In view of the effects of testosterone a clear demarcation is whether sex reassignment has taken place before or after hormonal puberty. Pubertal effects of testosterone are in part reversible but there is no reliable evidence as to its completeness. The International Olympic Committee (IOC) has taken an inevitably arbitrary decision with regard to participation of sex-reassigned transsexuals in elite sports: sex reassignment must have taken place at least two years earlier, hormone treatment must be appropriate for the reassigned sex and the reassigned sex must be legally recognized. The IOC policy is not binding for other organisations [039].

Dehydroepiandrosterone
According to the WADA rules (WADA Technical Document-TD2004EAAS) urine samples containing dehydroepiandrosterone (DHEA) concentrations greater than 100 ng/mL shall be submitted to isotope ratio mass spectrometry (IRMS) analysis. The threshold concentration is based on the equivalent to the glucuronide, and the DHEA concentrations have to be adjusted for a specific gravity value of 1.020. In

2006, 11012 doping control urine samples from national and international federations were analyzed in the Cologne doping control laboratory, 100 (0.9 %) of them yielding concentrations of DHEA greater than 100 ng/mL. Sixty-eight percent of the specimens showed specific gravity values higher than 1.020, 52 percent originated from soccer players, 95 percent were taken in competition, 85 percent were male urines, 99 percent of the IRMS results did not indicate an application of testosterone or related prohormones. Statistical evaluation showed significantly different DHEA concentrations between specimens taken in- and out-of- competition, whereas females showed smaller DHEA values than males for both types of control. Also a strong influence of the DHEA excretion on different sport disciplines was detectable. The highest DHEA values were detected for game sports (soccer, basketball, handball, ice hockey), followed by boxing and wrestling. In 2007, 6622 doping control urine samples were analyzed for 3alpha,5-cyclo-5alpha-androstan-6beta-ol-17-one (3alpha,5-cyclo), a DHEA metabolite which was described as a useful gas chromatography-mass spectrometry (GC-MS) screening marker for DHEA abuse. Nineteen urine specimens showed concentrations higher than the suggested threshold of 140 ng/mL, six urine samples yielded additionally DHEA concentrations higher than 100 ng/mL, none of them showing positive IRMS findings. These results should be taken into consideration in future discussions about threshold values for endogenous steroids in doping control [040].

Testing metods
In addition to the development of secondary sex characteristics, testosterone (T) has anabolic effects including increases in muscle size and strength and increases in lean body mass. In the case of exogenous administration of testosterone, the ratio of testosterone to its isomer, epitestosterone (E), is elevated. WADA has set a standard for T/E ratios of 4.0 as indicative of possible exogenous testosterone administration. Typically, a sample that screens for a T/E ratio above that threshold is then subjected to quantitative confirmation by GC/MS. This methodology, however, can limited due to sensitivity issues as well as a limited number of qualifying ions that can be used for unambiguous identification. It was therefore developed a confirmation method which uses liquid/liquid extraction, followed by room temperature Girard P derivatization, and analysis using LC/MS-ToF. Analysis time is decreased. Sensitivity is increased, resulting in limits of detection of 2 and 0.5 ng/ml for testosterone and epitestosterone, respectively. The number of diagnostic qualifier ions is also increased allowing more confident identification of the analytes. Finally, while this method has been developed on a QToF instrument, it should be easily transferable to any tandem LC/MS/MS system [041]. Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESIMS/MS) method for simultaneous and direct detection of 12 glucuronide-conjugated anabolic androgenic steroid (AAS) metabolites in human urine was described. The compounds selected were the main metabolites detected in human urine after dosing of the most widely abused AAS in sports, e.g. methandienone, methenolone, methyltestosterone, nandrolone and testosterone, and certain deuterium-labeled analogs of these metabolites. Sample preparation and the LC-ESI-MS/MS method were optimized, validated, and the overall process was implemented and the results between seven laboratories were compared. All the metabolites were extracted simultaneously by solid-phase extraction (SPE) and analyzed by LC-ESI-MS/MS with positive ionization mode and multiple reaction monitoring (MRM). Recovery of the SPE for the anabolic androgenic steroid glucuronides was 89-100 percent and ten out of twelve compounds had detection limits in the range of 1-10 ng/ml in urine. The results for inter/intraday repeatability were satisfactory and the interlaboratory

comparison with authentic urine samples demonstrated the ease of method transfer from one instrument setup to another. When equivalent triple quadrupole analyzers were employed the overall performance was independent from instrument manufacturer, electrospray ionisation (ESI) or atmospheric pressure chemical ionization (APCI) and liquid chromatohraphic (LC) column, whereas major differences were encountered when changing from one analyzer type to another, especially in the analysis of those AAS glucuronides ionized mainly as adducts [042]. The application of a comprehensive gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS)-based method for stable carbon isotopes of endogenous urinary steroids was presented. The key element in sample preparation is the consecutive cleanup with high-performance liquid chromatography (HPLC) of underivatized and acetylated steroids, which allows the isolation of ten analytes (11beta-hydroxyandrosterone, 5alpha-androst-16-en-3beta-ol, pregnanediol, androsterone, etiocholanolone, testosterone, epitestosterone, 5alpha-androstane3alpha,17beta-diol, 5beta-androstane-3alpha,17beta-diol and dehydroepiandrosterone) from a single urine specimen. These steroids are of particular importance to doping controls as they enable the sensitive and retrospective detection of steroid abuse by athletes. Depending on the biological background, the determination limit for all steroids ranges from 5 to 10 ng/mL for a 10 mL specimen. The method is validated by means of linear mixing models for each steroid, which covers repeatability and reproducibility. Specificity was further demonstrated by gas chromatography/mass spectrometry (GC/MS) for each analyte, and no influence of the sample preparation or the quantity of analyte on carbon isotope ratios was observed. In order to determine naturally occurring 13C/12C ratios of all implemented steroids, a reference population (n=61) subjects was measured to enable the calculation of reference limits for all relevant steroidal delta values [043].

Yeast analysis
The classical analytical method for detection of anabolic steroid abuse is gas chromatography followed by mass spectrometry (GC/MS). However, even molecules with a chemical structure typical for this class of substances, are sometimes not identified in routine screening by GC/MS when their precise chemical structure is still unknown. A supplementary approach to identify anabolic steroid abuse could be a structure-independent identification of anabolic steroids based on their biological activity. To test the suitability of such a system, it was analyzed the yeast androgen receptor (AR) reporter gene system to identify anabolic steroids in human urine samples. Analysis of different anabolic steroids dissolved in buffer demonstrated that the yeast reporter gene system is able to detect a variety of different anabolic steroids and their metabolites with high specificity, including the so-called designer steroid tetrahydrogestrinone. In contrast, other non-androgenic steroids, like glucocordicoids, progestins, mineralocordicoids and estrogens had a low potency to stimulate transactivation. To test whether the system would also allow the detection of androgens in urine, experiments with spiked urine samples were performed. The androgen reporter gene in yeast responds very sensitive to 5alpha-dihydrotestosterone (DHT), even at high urine concentrations. To examine whether the test system would also be able to detect anabolic steroids in the urine of anabolic steroid abusers, anonymous urine samples previously characterized by GCMS were analyzed with the reporter gene assay. Even when the concentration of the anabolic metabolites was comparatively low in some positive samples it was possible to identify the majority of positive samples by their biological activity. In conclusion, the results demonstrated that the yeast reporter gene system detects anabolic steroids and corresponding metabolites with high sensitivity even in urine of anabolic steroid

abusing athletes. But most importantly, a biological test system does not require knowledge of the chemical structure of androgenic substances and therefore suitable to detect previously unidentified substances, especially those of the class of so-called designer steroids [044].

Hair analysis
Doping control of anabolic substances is normally carried out with urine samples taken from athletes and horses. Investigation of alternative specimens, e.g. hair samples, is restricted to special cases, but can also be worthwhile, in addition to urine analysis. Moreover, hair material is preferred in cases of limited availability or complicated collection of urine samples, e.g. from horses. In this work, possible ways of interpretation of analytical results in hair samples are discussed and illustrated by practical experiences. The results demonstrate the applicability of hair analysis to detect anabolic steroids and also to obtain further information about previous abuse. Moreover, the process of incorporation of steroids into hairs is described and the consequences on interpretation are discussed, e.g. on the retrospective estimation of the application date. The chosen examples deal with the detection of the anabolic agent testosterone propionate. Hair samples of an application study, as well as a control sample taken from a racing horse, were referred to. Hair material was investigated by a screening procedure including testosterone, nandrolone and several esters (testosterone propionate, phenylpropionate, decanoate, undecanoate, cypionate; nandrolone decanoate, dodecanoate and phenylpropionate; limits of detection (LODs) between 0.1 and 5.0 pg/mg). Confirmation of testosterone propionate (LOD 0.1 pg/mg) was carried out by an optimised sample preparation. Trimethylsilyl (TMS) and tert-butyl dimethylsilyl derivatives were detected by gas chromatography-high-resolution mass spectrometry (GC-HRMS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) [045].

Estrogens
Women are at greater risk than men of sustaining certain kinds of injury and diseases of collagen-rich tissues. To determine whether a high level of oestradiol has an acute influence on collagen synthesis in tendons at rest and in response to exercise, onelegged kicking exercise was performed for 60 min at 67 percent of maximum power by healthy, young oral contraceptive (OC) users when circulating synthetic (ethinyl) oestradiol was high (n=11) and compared to similar women who had never used OCs when circulating endogenous oestrogen was low (n=12). Interstitial fluid was collected 24 h post-exercise through microdialysis catheters placed anterior to the patellar tendon in both legs and subsequently analysed for the amino-terminal propeptide of type I collagen (PINP), a marker of tendon collagen synthesis. To determine the long-term effect of OC usage, patellar tendon cross-sectional area (CSA) was measured by magnetic resonance imaging (MRI). A lower exerciseinduced increase in tendon collagen synthesis was observed at higher than at lower levels. Furthermore, serum and the interstitial peritendinous tissue concentrations of insulin-like growth factor I (IGF-I) and IGF-binding proteins showed a reduced bioavailability at higher compared with results lower levels. No difference in patellar tendon CSA was observed between groups. In conclusion, the selective increase in tendon collagen synthesis when circulating endogenous oestrogen was low but not when circulating synthetic (ethinyl) oestradiol was high 24 h post-exercise is consistent with the hypothesis that oestradiol inhibits exercise-induced collagen synthesis in human tendon. The mechanism behind this is either a direct effect of oestradiol, or an indirect effect via a reduction in levels of free IGF-I. However, the

data did not indicate any long-term effect on tendon size associated with chronic oral contraceptive use [046].

Anti-estrogens
Androgens can increase muscular mass and strength and remain the most frequently abused and widely available drugs used in sports doping. Banning the administration of natural or synthetic androgens has led to a variety of strategies to circumvent the ban of the most effective ergogenic agents for power sports. Among these, a variety of indirect androgen doping strategies aiming to produce a sustained rise in endogenous testosterone have been utilized. These include oestrogen blockade by drugs that act as oestrogen receptor antagonists (antioestrogen) or aromatase inhibitors [047]. The detection of metabolites of the anti-estrogenic substance cyclofenil, listed on the World Anti-Doping Agency Prohibited List since 2004 is described. Target substances are hydroxylated metabolites, bearing an aliphatic hydroxyl group either in the 2-, 3- or 4-position of the aliphatic ring, in addition to the phenolic functions on the aromatic rings. Structural identification used NMR as well as high-resolution mass spectrometry after nano-electrospray ionisation (ESI). Unambiguous detection of all three synthesised cyclofenil metabolites M1-M3 was done using gas chromatography for separation and electron ionisation mass spectrometry for detection of the per-silylated compounds in comparison with a reference urine deriving from an excretion study within the WADA 2007 Educational Programme [048]. A molecular imprinted polymer (MIP) has been synthesized in order to specifically extract tamoxifen, a nonsteroidal antiestrogen, and its metabolites from urine by solid-phase extraction (SPE) before HPLC-UV analysis. Clomiphene, a chlorinated tamoxifen analogue, was selected as template for MIP synthesis. Polymerisation was achieved by thermal polymerisation of methacrylic acid (MAA) as functional monomer, ethylene glycol dimethacrylate (EDMA) as cross-linking agent and acetonitrile as porogen. The efficient elimination of the urinary matrix has been obtained by MIP-SPE but the elution recovery of tamoxifen was initially too low (approximately 14 %). This problem has been overcome following two ways. At first, a preliminary HLB-SPE of the urine has enabled to discard endogenous salts and to percolate an organic sample through the MIP cartridge. Extraction recoveries are equal to 56 and 74 percent for tamoxifen and 4-hydroxytamoxifen, respectively. Then, a second MIP has been prepared with styrene and MAA as functional comonomers. Strong pi-pi interactions occurring between phenyl groups of styrene and tamoxifen promote rebinding of the analyte by the specific sites. The enhanced hydrophobic character of the imprinted polymer has enabled the direct percolation of urine through MIP-SPE and the easy elimination of endogenous salts from urine with only one aqueous washing step. HPLC-UV analysis has confirmed high extraction recoveries (85 %) for tamoxifen and its metabolite with an enrichment factor of 8. This analytical protocol can selectively detect the presence of tamoxifen metabolites in urines and be useful as a proof of doping in competitive sports [049].

Selective androgen receptor modulators (SARM)

Nonsteroidal selective androgen receptor modulators (SARMs) are an emerging class of drugs for treatment of various diseases including osteoporosis and muscle

wasting as well as the correction of age-related functional decline such as muscle strength and power. Several SARMs, which have advanced to preclinical and clinical trials, are composed of diverse chemical structures including arylpropionamide-, bicyclic hydantoin-, quinoline-, and tetrahydroquinoline-derived nuclei. Since January 2008, SARMs have been categorized as anabolic agents and prohibited WADA. Suitable detection methods for these low-molecular weight drugs were based on mass spectrometric approaches, which necessitated the elucidation of dissociation pathways in order to characterize and identify the target analytes in doping control samples as well as potential metabolic products and synthetic analogs. Fragmentation patterns of representatives of each category of SARMs after electrospray ionization and collision-induced dissociation (CID) as well as electron ionization (EI) have been discussed The complexity and structural heterogeneity of these drugs is a daunting challenge for detection metods [050].

Blood doping Overview

Optimum performance in aerobic sports performance requires an efficient delivery to, and consumption of, oxygen by the exercising muscle. It is probable that maximal oxygen uptake in the athlete is multifactorial, being shared between cardiac output, blood oxygen content, muscle blood flow, oxygen diffusion from the blood to the cell and mitochondrial content. Of these, raising the blood oxygen content by raising the haematocrit is the simplest acute method to increase oxygen delivery and improve sport performance. Legal means of raising haematocrit include altitude training and hypoxic tents. Illegal means include blood doping and the administration of EPO (erythropoietin). The ability to make EPO by genetic means has resulted in an increase in its availability and use, although it is probable that recent testing methods may have had some impact. Less widely used illegal methods include the use of artificial blood oxygen carriers (the so-called blood substitutes). In principle these molecules could enhance aerobic sports performance; however, they would be readily detectable in urine and blood tests. An alternative to increasing the blood oxygen content is to increase the amount of oxygen that haemoglobin can deliver. It is possible to do this by using compounds that right-shift the haemoglobin dissociation curve (e.g. RSR13). There is a compromise between improving oxygen delivery at the muscle and losing oxygen uptake at the lung and it is unclear whether these reagents would enhance the performance of elite athletes. However, given the proven success of blood doping and EPO, attempts to manipulate these pathways are likely to lead to an ongoing battle between the athlete and the drug testers [051].

Testing metods
An efficient antidoping test designed to obtain direct proof of allogeneic blood transfusion was developed and validated. This test, based on flow cytometry analysis of red blood cell (RBCs) phenotypes, was used to determine the absence or the presence of numerous RBCs populations in a blood sample. A such, it may constitute a direct proof of an abnormal blood population resulting from homologous transfusion. Single-blind and single-site studies were carried out to validate this method as a forensic quality standard analysis and to allow objective interpretation of real cases. The analysis of 140 blood samples containing different percentages (0-5 %) of a minor RBCs population were carried on by four independent analysts. Robustness, sensitivity, specificity, precision and stability were assessed. ISOaccredited controls samples were used to demonstrate that the method was robust,

stable and precise. No false positive results were observed, resulting in a 100 percent specificity of the method. Most samples containing a 1.5 percent minor RBCs population were unambiguously detected, yielding a 78 percent sensitivity. These samples mimicked blood collected from an athlete 3 months after a homologous blood transfusion event where 10 percent of the total RBCs present in the recipient originated in the donor. The observed false negative results could be explained by differences in antigen expression between the donor and the recipient. False negatives were more numerous with smaller minor RBCs populations. The method described here fulfils the ISO-17025 accreditation and validation requirements. The controls and the methodology are solid enough to determine with certainty whether a sample contains one or more RBCs populations. This variable is currently the best indicator for homologous blood transfusion doping [052]. The aim of one study was to evaluate physical performance loss and underlying mechanisms following voluntary blood donation. Eleven voluntary subjects (four female) completed a symptom-limiting cardio-pulmonary exercise test before and after blood donation (500 mL blood). The haemoglobin value significantly decreased by 1.2 mg/dL (9 %), maximal oxygen uptake by 9 percent, maximal work rate by 13 percent and duration of exercise fell from 663 down to 607 seconds. Anaerobic transition occurred at 81 percent and 72 percent of maximal oxygen uptake before and after blood donation, respectively, which was a signifikant difference. Subjects who practise recreational endurance sports appear to be more effected by endurance loss. The haemoglobin value was the only significant predictor of maximal oxygen uptake in regression analysis. It was concluded that maximal physical performance is impaired after blood donation. Haemoglobin decline accounts for the decreased oxygen uptake. As a consequence thereof the anaerobic transition occurs earlier. Subjects not engaged in regular sports activity did not experience a decline in their capacity [053]. Allogeneic transfusions are normally mismatched at one or more minor blood group antigens. The most sensitive and accurate method known to detect this form of blood doping is flow cytometry. Low percentages of antigen-positive and antigen-negative red blood cells (RBCs) can be quantitated using suitable specific alloantibodies and careful analysis. By testing blood samples taken at various times, a reduction in the percentage of a minor population of RBCs will indicate transfusion has occurred [054].

Bicycle
During the Tour de France 2007, 7 riders were randomly tested on 3 different occasions; the day before the prologue, and 12 and 19 days after the prologue. Blood was drawn into 3 mL EDTA covered tubes and kept at 4 degrees Celsius. They were analyzed within 24 hours. The concentration of hemoglobin and hematocrit were significantly lower on day 12 and day 19 compared to baseline. All 7 riders had lower hemoglobin and hematocrit on day 19 compared to baseline, while this was the case in 6 out of 7 riders already on day 12. The concentration of hemoglobin and hematocrit were 11.5 percent and 12.1 percent lower on day 19 compared to baseline. Whether or not this lowe value is due to decrease in hemoglobin mass or hemodilution, or the latter solely, increases in hemoglobin and hematocrit during prolonged stage racing seem unphysiological and should therefore lead to further examination of the rider [055].

Erythropoietin

Laboratory testing
The main action of recombinant human erythropoietin (rHuEpo) is to increase the oxygen carrying capacity of the blood. To prevent a possible misuse of rHuEpo, this is tested in urine samples collected from athletes by World Anti-Doping Agency (WADA)-accredited laboratories. Recently the test has met serious critiques, and the aims of one study were to investigate the detection power of the test as well as the variability in the test power comparing the results of two WADA-accredited laboratories. Eight human subjects were studied for 7 weeks and treated with rHuEpo for 4 weeks with 2 weeks of "boosting" followed by 2 weeks of "maintenance" and a post period of 3 weeks. Urine samples were obtained during all periods. Laboratory A determined rHuEpo misuse in all subjects during the boosting period, whereas laboratory B found no misuse, with one sample to be negative, and the remaining seven to be suspicious. The detection rates decreased throughout the maintenance and post period when total hemoglobin mass and exercise performance were elevated. During this period, laboratory A found only two of 24 samples to be positive and three to be suspicious, and laboratory B found no positive or suspicious samples. The study demonstrates a poor agreement in test results comparing two WADA-accredited laboratories. Moreover, after the initial rHuEpo boosting period the power to detect rHuEpo misuse during the maintenance and post periods appears minimal [056]. The test used by anti-doping laboratories to detect the misuse of recombinant erythropoietin (rhEPO) is based on its different migration pattern on isoelectric focusing (IEF) gel compared with the endogenous human erythropoietin (hEPO) that can possibly be explained by structural differences. While there is definitely a need to identify those differences by LC-MS/MS, the extensive characterization that was achieved for the rhEPO was never performed on human endogenous EPO because its standard is not available in sufficient amount. The goal of one study was therefore to develop an analytical method to detect pmol amounts of N-linked and O-linked glycopeptides of the recombinant hormone as a model. Using a nanoflow HPLC-Chip electrospray ionization/ion trap mass spectrometer, the diagnostic ion at m/z 366 of oligosaccharides was monitored in the product ion spectra to identify the four theoretical glycosylation sites, Asn24, Asn38, Asn83 and Ser126, respectively, on glycopeptides 22-37, 38-55, 73-96 and 118-136. With 3 pmol of starting material applied on Chip, only the desialylated N-glycopeptides 22-37 and 38-55/38-43 could be observed, and of all the glycan isoforms, those with the smaller structures were predominantly detected. While the preservation of the sialic acid moieties decreased the detection of all the N-glycopeptides, it allowed a more extensive characterization of the O-linked glycopeptide 118-136. The technique described herein provides a mean to detect glycopeptides from commercially available pharmaceutical preparations of rhEPO with the sensitivity required to analyze pmol amounts of hEPO, which could ultimately lead to the identification of structural differences between the recombinant and the human forms of the hormone [057]. Recombinant human erythropoietin (rhEPO), generally produced in Chinese hamster ovary (CHO) cells, can be used as not only a therapeutic protein but also a doping agent in sports. Profiling of EPO glycoforms is a critical means for quality control in pharmaceutical industrial and anti-doping analysis of misuse in sports. However, the existing methods for the analysis of EPO are associated with either time consuming or poor resolution. In one work, a rapid and high-resolution glycoform profiling method was presented based on capillary isoelectric focusing (cIEF) with whole column imaging detection (WCID). Experimental conditions that influence the

separation were investigated. Under optimized conditions, rhEPO from three different sources were resolved into distinct populations within 5 min with excellent reproducibility. As compared with existing methods, the presented method exhibited the advantages of speed and high resolution. If combined with an effective sample enrichment step and a much more sensitive WCID version, the method can be a potential alternative for the detection of rhEPO misuse in sports [058].

Effects outside the bone marrow

Systemic administration of erythropoietin protects the myocardium from an ischemic insult and promotes beneficial remodeling. It was hypothesized that intracardiac injection of erythropoietin may exhibit cardioprotective potential with reduced systemic toxicity. In conclusion, one study demonstrated that intramyocardial erythropoietin injection, following myocardial infarction, reduced inflammation, enhanced angiogenesis and proliferation, improved myocardial functions, and did not lead to intramural thrombus formation [003].

Growth hormone and IGF-1 Overview

Growth hormone (GH) is an important and powerful metabolic hormone that is secreted in a pulsatile pattern from cells in the anterior pituitary, influenced by several normal and pathophysiological conditions. Human GH was first isolated in the 1950s and human derived cadaveric GH was initially used to treat patients with GH deficiency. However, synthetic recombinant GH has been widely available since the mid-1980s and the advent of this recombinant GH boosted the abuse of GH as a doping agent. Doping with GH is a well-known problem among elite athletes and among people training at gyms, but is forbidden for both medical and ethical reasons. It is mainly the anabolic and, to some extent, the lipolytic effects of GH that is valued by its users. Even though GH's rumour as an effective ergogenic drug among athletes, the effectiveness of GH as a single doping agent has been questioned during the last few years. There is a lack of scientific evidence that GH in supraphysiological doses has additional effects on muscle exercise performance other than those obtained from optimised training and diet itself. However, there might be synergistic effects if GH is combined with, for example, anabolic steroids, and GH seems to have positive effect on collagen synthesis. Regardless of whether or not GH doping is effective, there is a need for a reliable test method to detect GH doping. Several issues have made the development of a method for detecting GH doping complicated but a method has been presented and used in the Olympics in Athens and Turin. A problem with the method used, is the short time span (24-36 hours) from the last GH administration during which the test effectively can reveal doping. Therefore, out-of-competition testing will be crucial [059]. Although there is little evidence that GH improves performance in young healthy adults, randomized controlled studies carried out so far are inadequately designed to demonstrate this, not least because GH is often abused in combination with anabolic steroids and insulin. Some of the anabolic actions of GH are mediated through the generation of insulin-like growth factor-I (IGF-I), and it is believed that this is also being abused. Athletes are exposing themselves to potential harm by selfadministering large doses of GH, IGF-I and insulin. The effects of excess GH are exemplified by acromegaly. IGF-I may mediate and cause some of these changes, but in addition, IGF-I may lead to profound hypoglycaemia, as indeed can insulin.

Although GH is on the World Anti-doping Agency list of banned substances, the detection of abuse with GH is challenging. Two approaches have been developed to detect GH abuse. The first is based on an assessment of the effect of exogenous recombinant human GH on pituitary GH isoforms and the second is based on the measurement of markers of GH action. As a result, GH abuse can be detected with reasonable sensitivity and specificity. Testing for IGF-I and insulin is in its infancy, but the measurement of markers of GH action may also detect IGF-I usage, while urine mass spectroscopy has begun to identify the use of insulin analoges [060].

Biology
Although doping with growth hormone (GH) is banned, there is anecdotal evidence that it is widely abused. GH is reportedly used often in combination with anabolic steroids at high doses for several months. Development of a robust test for GH has been challenging because recombinant human 22 kDa (22K) GH used in doping is indistinguishable analytically from endogenous GH and there are wide physiological fluctuations in circulating GH concentrations. One approach to GH testing is based on measurement of different circulating GH isoforms using immunoassays that differentiate between 22K and other GH isoforms. Administration of 22K GH results in a change in its abundance relative to other endogenous pituitary GH isoforms. The differential isoform method has been implemented; however, its utility is limited because of the short window of opportunity of detection. The second approach, which will extend the window of opportunity of detection, is based on the detection of increased levels of circulating GH-responsive proteins, such as insulin-like growth factor (IGF) axis and collagen peptides. Age and gender are the major determinants of variability for IGF-I and the collagen markers; therefore, a test based on these markers must take age into account for men and women. Extensive data is now available that validates the GH-responsive marker approach and implementation is now largely dependent on establishing an assured supply of standardized assays [061]. Recombinant human growth hormone (rhGH) as opposed to cadaver pituitary GH is misused for physical improvement. Six days' rhGH administration, in abstinent anabolic-androgenic steroid dependents, was compared with controls. Male subjects (n=48) were randomly divided into two groups: control group, n=24; rhGH-using group (0.058 IU/kg/day GH; n=24). Strength, peak power output and IGF-I significantly increased and total protein, albumin and free tetra-iodothyronine significantly decreased compared to controls and within the GH group. Fat-free mass index and VO2 peak significantly increased, while body fat and thyroid-stimulating hormone significantly decreased within the GH group. It was concluded that shortterm rhGH increased strength and power [062]. The aim of one study was to investigate possible relationships between different right-hand finger-length ratios and different fasting hormone concentrations in young swimmers. Fifty-five young swimmers participated in this study (26 boys and 29 girls, aged 10-17 years). All finger-length ratios were significantly higher in girls compared with boys. Ghrelin, leptin, testosterone in boys, estradiol in girls, insulin-like growthfactor I (IGF-I), IGFBP-3, and insulin were analyzed. Leptin and insulin concentrations were lower in boys compared with girls. In both groups, the relationships between finger-length ratios and basic anthropometric parameters were not significant. In conclusion, ghrelin appears to be a further biochemical parameter in addition to the sex steroids which correlated with different digit-length ratios at least in boys [063]. One of its best-characterized effects of grotwh hormone is increasing levels of

circulating insulin-like growth factor I (IGF-I), which is primarily of hepatic origin. It also induces synthesis of IGF-I in most non-hepatic tissues. The effects of GH in promoting postnatal body growth are IGF-I dependent, but IGF-I-independent functions are beginning to be elucidated. Although benefits of GH administration have been reported for those who suffer from GH deficiency, there is currently very little evidence to support an anabolic role for supraphysiological levels of systemic GH or IGF-I in skeletal muscle of healthy individuals. There may be other performance-enhancing effects of GH. In contrast, the hypertrophic effects of musclespecific IGF-I infusion are well documented in animal models and muscle cell culture systems. Studies examining the molecular responses to hypertrophic stimuli in animals and humans frequently cite upregulation of IGF-I messenger RNA or immunoreactivity. The circulatory/systemic (endocrine) and local (autocrine and paracrine) effects of GH and IGF-I may have distinct effects on muscle mass regulation [064].

Testing metods
The utility of insulinlike growth factor (IGF) axis and collagen markers for a growth hormone (GH) doping test in sport depends on their stability and reproducibility. It was sought to determine short-term within-subject variability of these markers in a large cohort of healthy individuals by measuring IGF-I, IGF binding protein 3 (IGFBP3), acid labile subunit (ALS), and the collagen markers N-terminal propeptide of type I procollagen (PINP), C-terminal telopeptide of type I collagen (ICTP), and N-terminal propeptide of type III procollagen (PIIINP) in serum samples obtained on multiple occasions (median 3 per participant) over a 2- to 3-week period from 1103 elite athletes (699 men, 404 women) aged (mean) 22 years. Within-subject variance accounted for 32-36 percent and 4-13 percent of the total variance in IGF markers and collagen markers, respectively. The within-subject CV ranged from 11 to 21 percent for the IGF axis markers and from 13 to 15 percent for the collagen markers. The index of individuality for the IGF axis markers was 0.66-0.76, and for the collagen markers, 0.26-0.45. For each marker, individuals with initial extreme measured values tended to regress toward the population mean in subsequent repeated measurements. These results indicate that in healthy individuals the withinsubject variability was greater for IGF-I than for the collagen markers, and that where a single measurement is available, it is possible to estimate the long-term probable value of each of the markers by applying the Bayesian approach. Such an application can increase the reliability and decrease the cost of detecting GH doping [004]. IGF axis proteins and collagen peptides are promising markers of GH abuse. The objective was to investigate whether responses of serum IGF axis and collagen markers to GH differ between men and women, and are influenced by testosterone in a randomized, double-blind, placebo-controlled study of 8-week treatment followed by 6-week washout. A total of 96 recreationally trained healthy athletes (63 men, 33 women), aged 18-40 year, were studied. All subjects received GH (2 mg/d sc) or placebo for 8 weeks; men also received testosterone (250 mg/week im) or placebo for 5 weeks. Serum IGF axis proteins (IGF-I, IGF binding protein-3, and acid labile subunit) and collagen peptides (N-terminal propeptide of type I procollagen, Cterminal telopeptide of type I collagen, and N-terminal propeptide of type III procollagen) were measured. GH induced significant increases in IGF axis and collagen markers that were greater in men than women. Of the IGF axis markers, IGF-I showed the greatest increase. The relative incremental responses of the collagen markers in general were greater than the IGF markers, especially for PIIINP. The collagen markers increased and decreased more slowly with most remaining elevated after 6 weeks, in comparison to IGF markers, which returned to baseline within 1 week. Addition of T to GH amplified the response of PIIINP by more than

1.5-fold but did not affect any other marker. Testosterone alone did not affect IGF axis markers but modestly increased collagen markers. These markers of GH abuse are less responsive in women. The increases in collagen markers have a different time course to the IGF markers and extend the window of detection in both sexes. The response of PIIINP is increased by coadministration of testosterone [065].

Impact of injuries
Growth-promoting agents are purported to increase the size of existing and newly regenerating muscle fibres and, therefore, could be used to improve muscle function if administered at appropriate times during the repair process. One review provided an update on the efficacy of some growth-promoting agents, including anabolic steroids, insulin-like growth factor-I (IGF-I) and beta2-adrenoceptor agonists, to improve muscle function after injury. Although these approaches have clinical merit, a better understanding of the androgenic, IGF-I and beta-adrenoceptor signalling pathways in skeletal muscle is important if it may be devised safe and effective therapies to enhance muscle regeneration and function after injury [066]. The objective of the study was to assess the effect of musculoskeletal or soft tissue injury on IGF-I and P-III-P concentrations in amateur and elite athletes and assess the effect of injury on the proposed GH detection method. There was no change in IGF-I concentration after an injury. By contrast, P-III-P concentrations rose by 41 + 17 percent, reaching a peak around 14 days after an injury. The rise in P-III-P varied according to injury type and severity. This rise had a trivial effect on the GH-2000 discriminant function score, and no subject reached the threshold needed for a doping offense. The authors concluded that although there was a rise in P-III-P after injury, this was insufficient to invalidate the GH-2000 detection method based on IGF-I and P-III-P concentrations [067].

Gonadotrophins
The decapeptide gonadotrophin-releasing hormone (GnRH) is endogenously produced in the hypothalamus and secreted into the microcirculation between hypothalamus and pituitary gland. Here, the bioactive hormone is responsible for the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) into the systemic circulation. Because an intermittent application of exogenous GnRH in young males increases the testosterone plasma level by stimulation of the Leydig cells, the potential misuse of the administered substance offers a reasonable relevancy for doping controls and is prohibited in accordance to the list of banned substances of the World Anti-Doping Agency. One presented method provides a mass spectrometric approach to determine the nondegraded hormone in regular doping control samples by utilizing a sample preparation procedure with solid phase extraction, immunoaffinity purification and a subsequent separation by liquid chromatography with ESI-MS/MS detection. For liquid chromatography/mass spectrometry two alternative instrumental equipments were tested. In urine specimens provided from healthy volunteers GnRH was not detected in accordance to the recent literature, but in postadministration samples urinary concentrations between 20 to 100 pg/ml of the intact peptide were determined. The method offered good validation results considering the parameter specificity, linearity (5-300 pg/ml), limit of detection (LOD, approx. 5 pg/ml), precision (inter/intraday, < 20 %) and accuracy (105 %) using Des-pGlu(1)-GnRH as internal standard to control each sample preparation step [068].

Boxers
To investigate the pituitary function in retired or active amateur boxers 61 actively competing (n=44) or retired (n=17) male boxers of the Turkish National Boxing Team were investigated. Nine of 61 boxers (15 %) had growth hormone (GH) deficiency and 5 of 61 boxers (8 %) had adrenocorticotropic hormone deficiency. All boxers with GH deficiency except 1 were retired from boxing. Of 17 retired boxers, 8 (47 %) had GH deficiency. Retired boxers with GH deficiency had significantly lower pituitary volume than retired boxers with normal GH. This study suggests that retired boxers have a high rate of pituitary dysfunction [069].

Human choriongonadotrophin
Parenteral administration of human chorionic gonadotropin (hCG) or luteinizing hormone (LH) stimulates the production of testosterone in males and these gonadotropins can therefore be used by athletes to enhance muscle strength. However, they are more expensive and less efficient than testosterone and anabolic steroids. Therefore their main use is probably to stimulate gonadal testosterone production during and after self-administration of testosterone or anabolic steroids. A positive effect of hCG on muscle strength has not been demonstrated in women and elevated concentrations of hCG in females are often caused by pregnancy. The use of gonadotropins is therefore prohibited only in males but not in females. HCG occurs at low but measurable concentrations in plasma and urine of healthy males and can be measured by sensitive methods. However, the characteristics of the method to be used for doping control have not been defined. Virtually all commercially available hCG assays have been designed for determination of hCG in serum rather than urine, which is used for doping control. Methods based on mass spectrometric detection of fragments derived from hCG extracted from urine by immunoadsorption have been developed but their suitability for doping control remains to be determined. The concentrations of LH in serum and urine are variable and more then 10-fold higher than those hCG. It is therefore difficult to detect illicit use of LH. The characteristics and reference values for hCG and LH assays used in doping control and the cutoff values need to be defined [070]. Doping with (glyco)protein hormones represent an extremely challenging, analytical problem as nearly all are constitutively present at low concentrations that fluctuate according to circadian or alternative periodical, or external stimuli. Thus the mere concentration in a biological sample is only resolutive when this surpasses extreme values. As the vast majority of these molecules are produced by recombinant DNA technology it is believed that the exogenous molecules could bear the signature of the host cell. In particular, these could comprise structural differences originated from co or post-translational differences. In one study it was employed both proteomics and glycomics strategies to compare recombinant and urinary human chorionic gonadotrophin in order to evaluate this hypothesis. As anticipated the recombinant hormone could be shown to contain N-glycolyl neuraminic acid, a sialic acid that cannot be produced by humans. Furthermore, differences were observed in the overall glycosylation, in particular the presence of abundant hybrid-type glycans that were much less pronounced in the recombinant species. These differences were determined to occur predominantly in the alpha-subunit for which antidoping strategies focussed on these elements could be used for both chorionic gonadotrophin and lutrophin as they share the same alpha-subunit [071].

Stimulants
The evidence for the ergogenic properties of the most potent stimulants, amphetamines, cocaine and ephedrine, is mostly insubstantial. Low doses of amphetamines may aid performance where effects of fatigue adversely affect higher psychomotor activity. Pseudoephedrine, at high doses, has been suggested to improve high intensity and endurance exercise but phenylpropanolamine has not been proven to be ergogenic. Only caffeine has substantial experimental backing for being ergogenic in exercise. The mode of action of these stimulants centres on their ability to cause persistence of catecholamine neurotransmitters, with the exception of caffeine which is an adenosine receptor antagonist. By these actions, the stimulants are able to influence the activity of neuronal control pathways in the central (and peripheral) nervous system. Rodent models suggest that amphetamines and cocaine interact with different pathways to that affected by caffeine. Caffeine has a variety of pharmacological effects but its affinity for adenosine receptors is comparable with the levels expected to exist in the body after moderate caffeine intake, thus making adenosine receptor blockade the favoured mode of ergogenic action. However, alternative modes of action to account for the ergogenic properties of caffeine have been supported in the literature. Biochemical mechanisms that are consistent with more recent research findings, involving proteins such as DARPP-32 (dopamine and cAMP-regulated phosphoprotein), are helping to rationalize the molecular details of stimulant action in the central nervous system [072]. One review examined the pharmacology of stimulants prohibited by the World AntiDoping Agency. Stimulants that increase alertness and reduce fatigue or activate the cardiovascular system can include drugs like ephedrine available in many over-thecounter medicines. Others such as amphetamines, cocaine and hallucinogenic drugs, available on prescription or illegally, can modify mood. A total of 62 stimulants (61 chemical entities) are listed in the WADA List, prohibited in competition. Athletes may have stimulants in their body for one of three main reasons: inadvertent consumption in a propriety medicine; deliberate consumption for misuse as a recreational drug and deliberate consumption to enhance performance. The majority of stimulants on the list act on the monoaminergic systems: adrenergic (sympathetic, transmitter noradrenaline), dopaminergic (transmitter dopamine) and serotonergic (transmitter serotonin, 5-HT). Sympathomimetic describes agents, which mimic sympathetic responses, and dopaminomimetic and serotoninomimetic can be used to describe actions on the dopamine and serotonin systems. However, many agents act to mimic more than one of these monoamines, so that a collective term of monoaminomimetic may be useful. Monoaminomimietic actions of stimulants can include blockade of re-uptake of neurotransmitter, indirect release of neurotransmitter, direct activation of monoaminergic receptors. Many of the stimulants are amphetamines or amphetamine derivatives, including agents with abuse potential as recreational drugs. A number of agents are metabolized to amphetamine or metamphetamine. In addition to the monoaminomimetic agents, a small number of agents with different modes of action are on the list. A number of commonly used stimulants are not considered as Prohibited Substances [073].

Modafinil
One paper examined the social construction of the new wakefulness-promoting drug Modafinil (brand name Provigil) in the British press. Key themes in this newspaper coverage include the potential uses and abuses of this drug in relation to: (i) medical conditions; (ii) lifestyle choices; (iii) military operations; and (iv) sporting competition. The British press played a dual role in reporting on these trends and

developments: on the one hand constructing this as something of a wonder drug in relation to the treatment of a number of medical complaints or conditions, on the other hand articulating and amplifying a range of cultural concerns and anxieties about the non-medical uses and abuses of this drug, both now and in the future. These issues, it is argued, are best interpreted in terms of media concerns over the pharmaceuticalisation rather than the medicalisation of everyday/night life. The paper concludes with some further thoughts and reflections on these issues, including the potential reworking of notions of pharmaceutical Calvinism and the spirit of (bio)capitalism [074].

Methylphenidate
Acute bupropion (dopamine/noradrenaline reuptake inhibitor) administration significantly improved time trial performance and increased core temperature in the heat (30 degrees C). One study was performed to examine the effect of a dopaminergic reuptake inhibitor on exercise capacity and thermoregulation during prolonged exercise in temperate and warm conditions. Eight healthy well-trained male cyclists participated in this study. Subjects ingested either placebo (20 mg) or methylphenidate (Ritalin 20 mg) 1 h before the start of exercise in temperate (18 degrees C) or warm (30 degrees C) conditions and cycled for 60 min at 55 percent Wmax, immediately followed by a time trial to measure exercise performance. It was shown that Ritalin has a clear ergogenic effect that was not apparent in 18 degrees C. The combination of a dopamine reuptake inhibitor and exercise in the heat clearly improved performance and caused hyperthermia without any change in the perception of effort or thermal stress compared with the placebo trial. This response may potentially increase the risk of developing heat illness during exercise in individuals taking drugs of this nature [075].

Caffeine
Practical recommendations The ergogenic effects of caffeine on athletic performance have been shown in many studies, and its broad range of metabolic, hormonal, and physiologic effects has been recorded, as one review of the literature shows. However, few caffeine studies have been published to include cognitive and physiologic considerations for the athlete. The following practical recommendations consider the global effects of caffeine on the body: lower doses can be as effective as higher doses during exercise performance without any negative coincidence; after a period of cessation, restarting caffeine intake at a low amount before performance can provide the same ergogenic effects as acute intake; caffeine can be taken gradually at low doses to avoid tolerance during the course of 3 or 4 days, just before intense training to sustain exercise intensity; and caffeine can improve cognitive aspects of performance, such as concentration, when an athlete has not slept well. Athletes and coaches also must consider how a person's body size, age, gender, previous use, level of tolerance, and the dose itself all influence the ergogenic effects of caffeine on sports performance [005]. Impact on testosterone levels Interest in the use of caffeine as an ergogenic aid has increased since the International Olympic Committee lifted the partial ban on its use. Caffeine has beneficial effects on various aspects of athletic performance, but its effects on training have been neglected. To investigate the acute effect of caffeine on the exercise-associated increases in testosterone and cortisol in a double-blind crossover study 24 professional rugby-league players ingested caffeine doses of 0,

200, 400, and 800 mg in random order 1 hr before a resistance-exercise session. Saliva was sampled at the time of caffeine ingestion, at 15-min intervals throughout each session, and 15 and 30 min after the session. Data were log-transformed to estimate percent effects with mixed modeling, and effects were standardized to assess magnitudes. Testosterone concentration showed a small increase of 15 percent (90 % confidence limits + 19 %) during exercise. Caffeine raised this concentration in a dose-dependent manner by a further small 21 percent (+ 24 %) at the highest dose. The 800-mg dose also produced a moderate 52 percent (+ 44 %) increase in cortisol. The effect of caffeine on the testosterone:cortisol ratio was a small decline (14 %; + 21 %). It was concluded that caffeine has some potential to benefit training outcomes via the anabolic effects of the increase in testosterone concentration, but this benefit might be counteracted by the opposing catabolic effects of the increase in cortisol and resultant decline in the testosterone:cortisol ratio [006]. Effect on performance The purpose of one experiment was to learn whether low doses of caffeine have ergogenic, perceptual, and metabolic effects during cycling. To determine the effects of 1, 2, and 3 mg/kg caffeine on cycling performance, differentiated ratings of perceived exertion (D-RPE), quadriceps pain intensity, and metabolic responses to cycling exercise, 13 cyclists exercised on a stationary ergometer for 15 min at 80 percent VO, then, after 4 min of active recovery, completed a 15-min VO2peak performance ride 60 min after ingesting caffeine or placebo. Work done (kJ/kg) during the performance ride was used as a measure of performance. D-RPE, pain ratings, and expired-gas data were obtained every 3 min, and blood lactate concentrations were obtained at 15 and 30 min. Compared with placebo, caffeine doses of 2 and 3 mg/kg increased performance by 4 percent (95 % confidence interval 1.0-6.8 %) and 3 percent (95 % confidence interval -0.4 % to 6.8 %), respectively. These effects were ergogenic, on average, but varied considerably in magnitude among individual cyclists. There were no effects of caffeine on D-RPE or pain throughout the cycling task. Selected metabolic variables were affected by caffeine, consistent with its known actions. The authors conclude that caffeine preparations of 2 and 3 mg/kg enhanced performance, but future work should aim to explain the considerable interindividual variability of the drug's ergogenic properties [076]. Impact on glycogen accumulation It was determined the effect of coingestion of caffeine with carbohydrate (CHO) on rates of muscle glycogen resynthesis during recovery from exhaustive exercise in seven trained subjects who completed two experimental trials in a randomized, double-blind crossover design. The evening before an experiment subjects performed intermittent exhaustive cycling and then consumed a low-CHO meal. The next morning subjects rode until volitional fatigue. On completion of this ride subjects consumed either CHO (4 g/kg body mass) or the same amount of CHO + caffein (8 mg/kg) during 4 hours of passive recovery. Muscle biopsies and blood samples were taken at regular intervals throughout recovery. Muscle glycogen levels were similar at exhaustion and increased by a similar amount (approximately 80 %) after 1 hour of recovery. After 4 hours of recovery caffein resulted in higher glycogen ackumulation. Accordingly, the overall rate of resynthesis for the 4-hour recovery period was 66 percent higher in caffein compared with CHO. After 1 hour of recovery plasma caffein levels had increased to 31 + 11 microM, which was a signifikant difference, and at the end of the recovery reached 77 + 11 microM with caffein. Phosphorylation of CaMK(Thr286) was similar after exercise and after 1 hourour of recovery, but after 4 hour CaMK(Thr286) phosphorylation was significantly higher in caffein than CHO. Phosphorylation of AMP-activated protein kinase (AMPK)(Thr172) and Akt(Ser473)

was similar for both treatments at all time points. It was provided the first evidence that in trained subjects coingestion of large amounts of caffein (8 mg/kg) with CHO has an additive effect on rates of postexercise muscle glycogen accumulation compared with consumption of CHO alone [007]. Impact on ventilation The purpose of one project was to determine whether a moderate dosage of caffeine, a common ventilatory stimulant, could augment resting ventilatory responsiveness, exercise ventilation, end-tidal O2 partial pressure (PetO2), and arterial oxyhemoglobin saturation (HbSaO2) in athletes with exercise-induced hypoxemia. Eight highly trained males who demonstrated exercise-induced hypoxemia, ingested in a randomized design a placebo or caffeine (8 mg/kg body wt) 1 hour before testing. Ventilatory responsiveness at rest was assessed via the isocapnic hypoxic and hyperoxic hypercapnic ventilatory responses (HVR and HCVR, respectively). The failure of HbSaO2 to increase at despite an increase in ventilation suggests that mechanisms influencing HbSaO2 other than an inadequate hyperventilatory response may operate to different degrees across individuals as VO2max is approached [077]. Combination with ephedrine Caffeine and ephedrine-related alkaloids recently have been removed from International Olympic Committee banned substances lists, whereas ephedrine itself is now permissible at urinary concentrations less than 10 mug.mL. The changes to the list may contribute to an increased use of caffeine and ephedra as ergogenic aids by athletes. Consequently, It was investigated the effects of ingesting caffeine (C) or a combination of ephedra and caffeine (C + E) on muscular strength and anaerobic power using a double-blind, crossover design. Forty-five minutes after ingesting a glucose placebo (P: 300 mg), C (300 mg) or C + E (300 mg + 60 mg), 9 resistancetrained male participants were tested for maximal strength by bench press (1 repetition maximum) and latissimus dorsi pull down (1 repetition maximum). Subjects also performed repeated repetitions at 80 percent of 1 repetition maximumon both activities until exhaustion. After this test, subjects underwent a 30-second Wingate test to determine peak anaerobic cycling power, mean power, and fatigue index. Although subjects reported increased alertness and enhanced mood after supplementation with caffeine and ephedra, there were no significant differences between any of the treatments in muscle strength, muscle endurance, or peak anaerobic power. The results do not support the contention that supplementation with ephedra or caffeine will enhance either muscle strength or anaerobic exercise performance [078]. Combination with sodium bicarbonate The purpose of this study was to investigate the effects of sodium bicarbonate (NaHCO3), caffeine, and their combination on repeated 200-m freestyle performance. Six elite male freestyle swimmers ingested sodium bicarbonate (0.3 g/kg), caffeine (6.2 + 0.3 mg/kg), a combination of both, and placebo on 4 separate occasions before completing 2 maximal 200-m freestyle time trials separated by 30 min. No significant differences were observed for performance but drop-off in performance time from first to second trial, however, was significantly greater when caffein was ingested than with bicarbonate or the combination. This is likely because of the lower blood pH and slower recovery of blood HCO3 after caffein ingestion. These findings suggest that the ergogenic benefit of taking caffein alone for repeated 200-m swimming performance appears limited. When combined with sodium bicarbonate, however, its negative impact on repeated maximal exercise performance is reversed [079].

Moderated effect of anxiety One experiment examined the effect of a moderate dose of caffeine on perceptions of leg-muscle pain during a bout of high-intensity cycling exercise and the role of anxiety sensitivity in the hypoalgesic effect of caffeine on muscle pain during exercise. Sixteen college-age women ingested caffeine (5 mg/kg body weight) or a placebo and 1 hour later completed 30 min of cycling on an ergometer at 80 percent of peak aerobic capacity. The conditions were completed in a counterbalanced order, and perceptions of leg-muscle pain were recorded during the bouts of exercise. Caffeine resulted in a large reduction in leg-muscle pain-intensity ratings compared with placebo, and the reduction in leg-muscle pain-intensity ratings was larger in those with lower anxiety-sensitivity scores than those with higher anxiety-sensitivity scores. The results support that caffeine ingestion has a large effect on reducing legmuscle pain during high-intensity exercise, but the effect is moderated by anxiety sensitivity [008]. Caffeinated versus decaffeinated coffee One study examined the effect of coffee ingestion on physiological responses and ratings of perceived exertion (RPE) during submaximal endurance exercises by 10 healthy young adults. Participants performed a submaximal endurance cycling exercise corresponding to 60 percent of maximum oxygen uptake capacity for 60 min. They drank either caffeinated coffee with a caffeine content of 6 mg/kg bodymass of each participant or a decaffeinated coffee 60 min before starting exercise. Participants participated in the blind design experiment under both conditions at a one-week interval. Oxygen uptake, respiratory exchange ratio, heart rate, RPE, and plasma lactate concentration were measured during the endurance exercise. The RPE under the caffeinated coffee condition during the last 60 min of endurance exercise was significantly lower than that in the decaffeinated coffee condition. However, no significant differences in any physiological response were observed between conditions. Thus, caffeine ingestion 60 min before starting exercise had an insignificant effect on the physiological responses, except for RPE during submaximal endurance exercises for 60 min. Caffeine ingestion before endurance exercise of relatively low intensity may have a beneficial effect on psychological responses [080]. Experimental The interaction of caffeine (1 mg/kg) and amitriptyline (15 mg/kg) on the immobility time during Porsolt's forced swimming test was investigated in female Wistar rats. Vehicle-treated animals had a significant increase of immobility time during the second day of the test. Amitriptyline only prevented the increase of immobility time during the second session. While caffeine alone prevented the increase of immobility timein both groups, the methylxanthine abolished the effect of amitriptyline, leaving the antidepressant action. These results suggest that the anti-immobility effect of amitriptyline is mediated in part by endogenous adenosine [081].

Experimental
Methamphetamine (MA) use is a worldwide problem. Abusers can have cognitive deficits, monoamine reductions, and altered magnetic resonance spectroscopy findings. Animal models have been used to investigate some of these effects, however many of these experiments have not examined the impact of MA on the stress response. For example, numerous studies have demonstrated (+)-MA-induced neurotoxicity and monoamine reductions, however the effects of MA on other markers that may play a role in neurotoxicity or cell energetics such as glucose, corticosterone, and/or creatine have received less attention. In this experiment, the

effects of a neurotoxic regimen of (+)-MA (4 doses at 2 h intervals) on brain monoamines, neostriatal GFAP, plasma corticosterone, creatinine, and glucose, and brain and muscle creatine were evaluated 1, 7, 24, and 72 h after the first dose. In order to compare MA's effects with stress, animals were subjected to a forced swim test in a temporal pattern similar to MA administration (i.e. 30 min/session 4 times at 2 h intervals). Methamphetamine increased corticosterone from 1-72 h with a peak 1 h after the first treatment, whereas glucose was only increased 1 h post-treatment. Neostriatal and hippocampal monoamines were decreased at 7, 24, and 72 h, with a concurrent increase in GFAP at 72 h. There was no effect of MA on regional brain creatine, however plasma creatinine was increased during the first 24 h and decreased by 72 h. As with MA treatment, forced swim increased corticosterone more than MA initially. Unlike MA, forced swim reduced creatine in the cerebellum with no change in other brain regions while plasma creatinine was decreased at 1 and 7 h. Glucose in plasma was decreased at 7 h. It was concluded that methamphetamine and forced swim increase demand on energy substrates but in different ways, and methamphetaminehas persistent effects on corticosterone that are not attributable to stress alone [082]. Methamphetamine (MA) has been implicated in cognitive deficits in humans after chronic use. Animal models of neurotoxic MA exposure reveal persistent damage to monoaminergic systems but few associated cognitive effects. Since questions have been raised about the typical neurotoxic dosing regimen used in animals and whether it adequately models human cumulative drug exposure, these experiments examined two different dosing regimens. Rats were treated with one of the two regimens: one based on the typical neurotoxic regimen (4 x 10 mg/kg every 2 h) and one based on pharmacokinetic modeling designed to better represent accumulating plasma concentrations of MA as seen in human users. On markers of neurotoxicity, methamphetamine showed decreased dopamine (DA) and 5-HT, increased glial fibrillary acidic protein, and increased corticosterone levels regardless of dosing regimen 3 days post-treatment. Behaviorally, methamphetamine-treated groups, regardless of dosing regimen, showed hypoactivity, increased initial hyperactivity to a subsequent MA challenge, impaired novel object recognition, impaired learning in a multiple water maze test of path integration, and no differences on spatial navigation or reference memory in the Morris water maze. After behavioral testing, reductions of DA and 5-HT remained. It was concluded that methamphetamine treatment induces an effect on path integration learning not previously reported. Dosing regimen had no differential effects on behavior or neurotoxicity [083].

Beta2agonists
Athletes attempt to improve performance with drugs that act on the beta-adrenergic system directly or indirectly. Of three beta-adrenoceptor subtypes, the beta2adrenoceptor is the main target in sport; they have bronchodilator and anabolic actions and enhance anti-inflammatory actions of corticosteroids. Although demonstrable in animal experiments and humans, there is little evidence that these properties can significantly improve performance in trained athletes. Their actions may also be compromised by receptor desensitization and by common, naturally occurring receptor mutations (polymorphisms) that can influence receptor signalling and desensitization properties in individuals. Indirectly acting agents affect release and reuptake of noradrenaline and adrenaline, thereby influencing all adrenoceptor subtypes including the three beta-adrenoceptors. These agents can have potent psychostimulant effects that provide an illusion of better performance that does not usually translate into improvement in practice. Amphetamines and cocaine also have

considerable potential for cardiac damage. beta-adrenoceptor antagonists (betablockers) are used in sports that require steadiness and accuracy, such as archery and shooting, where their ability to reduce heart rate and muscle tremor may improve performance. They have a deleterious effect in endurance sports because they reduce physical performance and maximum exercise load. Recent studies have identified that many beta-adrenoceptor antagonists not only block the actions of agonists but also activate other (mitogen-activated PK) signalling pathways influencing cell growth and fate. The concept that many compounds previously regarded as blockers may express their own spectrum of pharmacological properties has potentially far-reaching consequences for the use of drugs both therapeutically and illicitly [084].

Exercise-induced asthma
The aims of a report was to review the current recommended treatment of exerciseinduced asthma (EIA), respiratory and allergic disorders in sports, and to review the evidence on possible improvement of performance in sports by asthma drugs and to make recommendations for their treatment. To assess the evidence of the literature regarding use of beta2-agonists related to athletic performance, the Task Force searched Medline for relevant papers up to November 2006 using the present search words: asthma, bronchial responsiveness, exercise-induced bronchoconstriction, athletes, sports, performance and beta2-agonists. Treatment recommendations for EIA and bronchial hyper-responsiveness in athletes are set forth with special reference to controller and reliever medications. Evidence for lack of improvement of exercise performance by inhaled beta2-agonists in healthy athletes serves as a basis for permitting their use. There is a lack of evidence of treatment effects of asthma drugs on EIA and bronchial hyper-responsiveness in athletes whereas extensive documentation exists in treatment of EIA in patients with asthma. The documentation on lack of improvement on performance by common asthma drugs as inhaled beta2agonists with relationship to sports in healthy individuals is of high evidence, level (1+). It was concluded that exercise induced asthma should be treated in athletes along same principles as in ordinary asthma patients with relevance to controller and reliever treatment after careful diagnosis. There is very high level of evidence for the lack of improvement in athletic performance by inhaled beta2-agonists [085].

Salbutamol
One study was designed to examine the dose-response relationship of inhaled salbutamol and its concentration in the urine while resting at various times after inhalation, and to compare these values against the current World Anti-Doping Code limits. Eight healthy, nonasthmatic males participated in the study. Administration of three different doses of inhaled salbutamol (800, 400, and 200 microg) in a randomized fashion separated by at least 72 hours gave highly variable urine concentrations between subjects and increased as dose increased, with a significant difference noted between 800 and 200 microg at 30, 60, and 120 minutes after inhalation. Urine concentrations of salbutamol peaked at 60 minutes for all doses. No samples exceeded the doping criterion of 1000 ng/mL, and the maximum value observed was 904 ng/mL. These results indicate that after inhalation of doses up to 800 microg, urinary concentrations of salbutamol are well below the limits used in doping control [086].

Procaterol
While the use of oral beta2-agonists by athletes is prohibited because of their anabolic effects, some inhaled beta2-agonists can be used in accordance with the

World Anti-Doping Agency regulations. It was examined the dose disparity between the bronchodilating effect and anabolic effect of inhaled procaterol, a beta2-agonists, to determine if the drug might be effective for athletes with asthma. Intact rats were given nebulized procaterol at 0.001, 0.01, 0.1 and 1 mg/mL by inhalation, and its inhibitory effect on carbachol-induced bronchoconstriction was evaluated. Castrated rats were given nebulized procaterol at 0.03, 0.1, 0.3 and 1 mg/mL by inhalation 3 times a day for 14 days, and anabolic markers (body weight gain, weight of the levator ani muscle and gastrocnemius muscle) were measured. At 0.01 mg/mL and higher, procaterol dose-dependently inhibited carbachol-induced bronchoconstriction with a significant effect. At doses of up to 0.3 mg/mL, there were no signs indicating an anabolic effect of procaterol. At 1 mg/mL, however, a slight but statistically significant increase in the weight of the levator ani muscle was observed with no significant changes in other anabolic markers. It was suggested that inhaled procaterol might be useful for athletes with asthma because of the big dose disparity between its bronchodilating effect and anabolic effect in rats [087].

Clenbuterol
Rapid screening of clenbuterol in urine was performed by combining desorption electrospray ionization (DESI) and tandem mass spectrometry (MS/MS). Optimization experiments were carried out including the selection of substrates, spray solutions, nebulizing gas pressures, high-voltage power supplies and flow rates of spray solution. The limit of detection (LOD), defined as the lowest quantity that can be detected, was 5.0 pg for the pure compound. Using DESI coupled with solidphase extraction (SPE), the linear response range was from 10 to 400 ng/mL and the concentration LOD for urine sample was 2.0 ng/mL. The analysis for one spiked urine sample was achieved within 4 min. In addition to the fast analysis speed, MS/MS provided structural information for the confirmation of clenbuterol. Urine samples from different people were investigated and the recoveries were within 100 + 20 perent [088].

Cannabis
In one study, it was investigated the effect of delta(9)-tetrahydrocannabinol (THC), the principal psychoactive component of marijuana, on immobility time during the forced swim test. THC (2 and 6 mg/kg, i.p.) significantly prolonged the immobility time. In addition, THC at the same doses did not significantly affect locomotor activity in the open-field test. The selective cannabinoid CB(1) receptor antagonist rimonabant (3 mg/kg, i.p.) significantly reduced the enhancement of immobility by THC (6 mg/kg). Similarly, the selective serotonin (5-HT) reuptake inhibitor (SSRI) citalopram (10 mg/kg, i.p.) and 5-HT(1A/7) receptor agonist 8-hydroxy-2-(di-npropylamino) tetralin (8-OH-DPAT, 0.3 mg/kg, i.p.) significantly reduced this THCinduced effect. These findings suggest that the 5-HT(1A) receptors are involved in THC-induced enhancement of immobility [089].

Diuretics
The variety of chemical structures of diuretic compounds has encouraged the development of new methods and techniques of analysis, especially as regards to acidic compounds. LC/MS has so grown to be the reference technique for this kind of analysis in forensic and anti-doping confirmation purposes. Multiple stage MS

permits identification of single drugs with high selectivity, but some unexpected pathways could weaken the entire process. In one work it was explained some unusual fragmentation steps using high-resolution MSn. For example, in the case of amiloride an intense product ion in MS3 analysis generates an apparent loss of 10Da. Water adduct formation and successive carbon monoxide elimination can explain this uncommon behavior, which was studied using different ion traps. Bendroflumethiazide MSn spectra show instead three successive HF losses, in spite of the presence of a radical site in the parent structure. Homolytic cleavages with radical ion production occur also in the case of protonated positive ion of ethacrynic acid (loss of chlorine radical) showing that such fragmentation behavior is not so rare as generally reported. Different ionization modes were studied and a tentative correlation with acidic-base properties was done. Multiple stage high-resolution mass spectra of positive and negative ions may be discussed [090].

NSAIDs
Although athletes are young and generally healthy, they use a variety of non-doping classified medicines to treat injuries, cure illnesses and obtain a competitive edge. Athletes and sports medicine physicians try to optimize the treatment of symptoms related to extreme training during an elite athlete's active career. According to several studies, the use of antiasthmatic medication is more frequent among elite athletes than in the general population. Recent studies show that athletes use also NSAIDs and oral antibacterials more commonly than age-matched controls, especially athletes competing in speed and power sports. Inappropriately high doses and concomitant use of several different NSAIDs has been observed. All medicines have adverse effects that may have deleterious effects on elite athletes' performance. Thus, any unnecessary medication use should be minimized in elite athletes. Inhaled beta2-agonists may cause tachycardia and muscle tremor, which are especially harmful in events requiring accuracy and a steady hand. In experimental animal models of acute injury, especially selective cyclo-oxygenase-2 inhibitors have been shown to be detrimental to tissue-level repair. They have been shown to impair mechanical strength return following acute injury to bone, ligament and tendon. This may have clinical implications for future injury susceptibility. However, it should be noted that the current animal studies have limited translation to the clinical setting. Adverse effects related to the CNS and gastrointestinal adverse reactions are commonly reported in connection with NSAID use also in elite athletes. In addition to the potential for adverse effects, recent studies have shown that NSAID use may negatively regulate muscle growth by inhibiting protein synthesis [091].

Dietary supplements Overview

Nutrition significantly influences sports performance; however, the efficacy of any nutritional supplement or strategy should be carefully considered in relation to the event and the sex, training and nutritional status of the participant. The causes of fatigue, mechanism of action, safety and legality of the supplement, together with the scientific evidence from studies with an appropriate experimental design, should all be taken into account before incorporating into the training and/or competition diet. The efficacy of ingesting nutritional supplements immediately before and/or during endurance exercise (duration 45-180 min) was reviewed. The ingestion of CES (carbohydrate-electrolyte solutions) have been shown to improve both exercise

capacity and performance, either due to the maintenance of euglycaemia throughout exercise or the sparing of muscle glycogen early on in exercise. The addition of caffeine to CES may improve endurance performance as a consequence of a reduced perception of effort. Research suggests that the addition of protein to CES may only be effective when a suboptimal amount of CHO (carbohydrate) is ingested during exercise (<60 g of CHO per hour); however, recovery of performance may be enhanced due to a reduction in subsequent muscle soreness and the promotion of muscle protein synthesis after exercise. The findings from studies investigating the effects of ingesting MCTs (medium-chain triacylglycerols) and BCAAs (branchedchain amino acids), either on their own or in combination with CES, on endurance performance have been equivocal and therefore would not be recommended. Any nutritional strategy should be practised in training before being used during a competition [092]. Physical training and proper nutrition are paramount for success in sport. A key tissue is skeletal muscle, as the metabolic pathways that produce energy or ATP allow the muscles to complete the many activities critical to success in sport. The energy-producing pathways must rapidly respond to the need for ATP during sport and produce energy at a faster rate or for a longer duration through training and proper nutrition which should translate into improved performance in sport activities. There is also continual interest in the possibility that nutritional supplements could further improve muscle metabolism and the provision of energy during sport. Most legal sports supplements do not improve performance following oral ingestion. However, three legal supplements that have received significant attention over the years include creatine, carnitine and sodium bicarbonate. The ingestion of large amounts of creatine for 4-6 days increases skeletal muscle creatine and phosphocreatine contents. The majority of the experimental evidence suggests that creatine supplementation can improve short-term exercise performance, especially in sports that require repeated short-term sprints. It may also augment the accretion of skeletal muscle when taken in combination with a resistance-exercise training programme. Supplementary carnitine has been touted to increase the uptake and oxidation of fat in the mitochondria. However, muscle carnitine levels are not augmented following oral carnitine supplementation and the majority of wellcontrolled studies have reported no effect of carnitine on enhancing fat oxidation, V02max or prolonged endurance exercise performance. The ingestion of sodium bicarbonate before intense exercise decreases the blood hydrogen to potentially assist the efflux of hydrogen from the muscle and temper the metabolic acidosis associated with intense exercise. Many studies have reported performance increases in laboratory-based cycling tests and simulated running races in the field following sodium bicarbonate ingestion where the need for ATP from substrate phosphorylation is high. However, other studies have reported no benefit and the incidence of negative side effects is high [093].

Contaminants
Since 1999 several groups have analyzed nutritional supplements with mass spectrometric methods (GC/MS, LC/MS/MS) for contaminations and adulterations with doping substances. These investigations showed that nutritional supplements contained prohibited stimulants as ephedrines, caffeine, methylenedioxymetamphetamie and sibutramine, which were not declared on the labels. An international study performed in 2001 and 2002 on 634 nutritional supplements that were purchased in 13 different countries showed that about 15 percent of the nonhormonal nutritional supplements were contaminated with anabolic-androgenic steroids (mainly prohormones). Since 2002, also products intentionally faked with high amounts of classic anabolic steroids such as metandienone, stanozolol,

boldenone, dehydrochloromethyl-testosterone, oxandrolone etc have been detected on the nutritional supplement market. These anabolic steroids were not declared on the labels either. The sources of these anabolic steroids are probably Chinese pharmaceutical companies, which sell bulk material of anabolic steroids. In 2005 vitamin C, multivitamin and magnesium tablets were confiscated, which contained cross-contaminations of stanozolol and metandienone. Since 2002 new designer steroids such as prostanozol, methasterone, androstatrienedione etc have been offered on the nutritional supplement market. In the near future also crosscontaminations with these steroids are expected. Recently a nutritional supplement for weight loss was found to contain the beta2-agonist clenbuterol. The application of such nutritional supplements is connected with a high risk of inadvertent doping cases and a health risk [009]. A simple and effective analytical method for the determination of anabolic steroids and related compounds in nutritional supplements was reported. Target compounds are extracted with ethyl acetate, crude extract is purified using dispersive solid-phase extraction (SPE) with primary secondary amine as sorbent, and finally they are identified and quantified as underivatized compounds using two-dimensional gas chromatography with time-of-flight mass spectrometric detection (GCxGC-TOF MS). This method was validated for 25 steroids in two types of commercially available solid nutritional supplements: protein concentrate and creatine monohydrate. Repeatability expressed as the relative standard deviation of analyte concentration ranged from 4.1 to 20.5%. Recoveries between 70 and 123 percent were obtained for the target compounds except for oxymetholone in protein concentrate where the recovery was low as a result of strong interactions with primary secondary amine. Excellent linearity was obtained for six-point calibration with regression coefficients of 0.997-1.000 for all compounds. The limits of quantification ranged from 0.007 to 0.114 mg/kg. For a monitoring programme of 48 samples of nutritional supplements, three were positive. Nandrolone, testosterone, dehydroepiandrosterone (DHEA), 5alpha-androstan-3,17-dione, 19-norandrostendione and progesterone were found in positive samples at concentrations between 0.022 and 0.398 mg/kg [094].

Energy drinks
Energy drinks have become more and more popular since the late nineties. The manufactures claim that these drinks improve physical endurance, reaction speed and concentration. The main ingredients of energy drinks are caffeine, sugar, taurine and glucuronolactone. According to the manufacturers, the stimulating effects of these drinks are due to interaction between the various ingredients. To investigate whether energy drinks do indeed improve cognitive performance and to find out which ingredients are responsible for this effect and other benefits it was made a search of the literature for the period from 1997 to 2006 on the basis of Medline, by using the search term energy drink or energy drinks and restricting the search to humans. results Not only did focused and sustained attention improve significantly but so did reaction speed in all sorts of reaction-time tasks. Memory improved too, but not to the same degree. The findings suggest that most of the effects of energy drinks on cognitive performance are related mainly to the presence of caffeine [095]. The author examined gendered links among sport-related identity, endorsement of conventional masculine norms, risk taking, and energy-drink consumption by surveying 795 undergraduate students enrolled in introductory-level courses at a public university. Of participants, 39 percent consumed an energy drink in the past month, with more frequent use by men (2.5 d/month) than by women (1.2 d/month). Strength of jock identity was positively associated with frequency of energy-drink consumption; this relationship was mediated by both masculine norms and risk-

taking behavior. The author concluded that sport-related identity, masculinity, and risk taking are components of the emerging portrait of a toxic jock identity, which may signal an elevated risk for health-compromising behaviors. College undergraduates' frequent consumption of Red Bull and comparable energy drinks should be recognized as a potential predictor of toxic jock identity [096]. Numerous studies have shown that ingesting carbohydrate in the form of a drink can improve exercise performance by maintaining blood glucose levels and sparing endogenous glycogen stores. The effectiveness of carbohydrate gels or jellybeans in improving endurance performance has not been examined. On 4 separate days and 1-2 hr after a standardized meal, 16 male and female athletes cycled at 75 percent VO2peak for 80 min followed by a 10-km time trial. Participants consumed isocaloric (0.6 g of carbohydrate per kg per hour) amounts of randomly assigned sports beans, sports drink, gel, or water only, before, during, and after exercise. Blood glucose concentrations were similar at rest between treatments and decreased significantly during exercise with the water trial only. Blood glucose concentrations for all carbohydrate supplements were significantly higher than water during the 80-min exercise bout and during the time trial. There were no significant differences in blood glucose between carbohydrate treatments. The 10-km time trials using all 3 carbohydrate treatments were significantly faster for sports beans, for sports drink, and for gel than water. All carbohydrate-supplement types were equally effective in maintaining blood glucose levels during exercise and improving exercise performance compared with water only [097].

Creatine
The purpose of one study was to examine the effects of 5 days of creatine loading on the electromyographic fatigue threshold (EMG FT) in college-age men. Sixteen men participated in this double-blind study and were randomly placed into either placebo (n=8) or creatine (5 g dicreatine citrate plus 10 g of flavored fructose powder per packet; n=8) loading groups. Each participant ingested 1 packet 4 times/d, totaling 20 g/d for 5 days (loading). Before and after loading, each participant performed a discontinuous cycle-ergometer test to determine his EMG FT, using bipolar surface electrodes placed on the vastus lateralis of the right thigh. Four 60-s work bouts (ranging from 200 to 400 W) were completed. Adequate rest was given between bouts to allow for the participants' heart rate to drop within 10 beats of their resting HR. The EMG amplitude was averaged over 5-s intervals for each 60-s work bout. Resulting slopes from each successive work resulted in a nonsignificant interaction for supplement and time. In addition, a significant increase in weight was observed in the creatine group. These data suggest that there was a minimal influence of creatin loading on EMG FT for the participants in this study [098]. It was present a case of acute cholestatic liver injury associated with the combination of whey protein and creatine supplements. The difficulty of diagnosing drug-induced liver injury is emphasized. The patient was a healthy, 27-year-old man who presented with painless jaundice. He had no occupational exposures to solvents, was not taking prescription medications, and did not use recreational drugs or alcohol. He was an enthusiastic weight-lifter and had been taking creatine for 8 to 9 months and whey protein supplements for 4 weeks prior to the development of symptoms [099]. The purpose of this study was to determine the effect of 30 days of single-dose creatine supplementation with phosphate salts on body weight and anaerobic working capacity in men. Using a double-blind design, 32 men randomly received 1

serving of either 5 g creatin + 4 g phosphate (n=17) or 20 g of dextrose as placebo (n=15) for 30 days. Results showed no significant differences between groups for working capacity at any time point; however, bode weight was significantly increased at 10 days in the creatin group (1.0 kg) versus placebo (0.0 kg), and remained elevated for the duration of the study [100]. Side effects The main aim of one study was to investigate the effects of two different creatinesupplementation protocols on incidence of gastrointestinal (GI) distress in top-level athletes. Data were collected from 59 top-level male soccer players who were allocated in a double-blind design to three randomly assigned trials: ingesting creatine supplement (2 x 5-g doses or 1 x 10-g dose) or placebo for 28 days. In order to assess potential side effects of the supplementation regimen, all subjects were instructed to report any adverse effects of supplementation on their GI system. Survey questions covered perceived side effects on GI system linked with creatine supplementation. In all three treatment groups, the most frequent GI complaints were diarrhoea (39 %), stomach upset (24 %), and belching (17 %). It was not found a significant difference between incidence of GI distress symptoms between 5 gram doses and the placebo group after the survey. Yet, significant differences were found for incidence of diarrhoea between the 5 and 10 gram groups (29 % vs 56 %, respectively). Moreover, diarrhoea was significantly more frequent in the 10 gram group as compared with the placebo group (56 % vs 35 %). There is no reason to believe that short-term oral creatine supplementation for 28 days has any detrimental effect on the GI tract if taken in a recommended amount (10 g per day in two equal doses). The risk of diarrhoea may be increased, however, following intake of 10 grams of creatine per single servning [101].

Carnitine
The purpose of one study was to investigate the effects of acute L-Carnitine intake on badminton players' metabolic and blood lactate values. A total of 16 Turkish national badminton players (8 male, 8 female) were voluntarily participated. No significant differences were found between first (without L-Carnitine intake) and second (with L-Carnitine intake) measurements of female participants as regards to all measured parameters. There was a significant difference in EMHR (exercise maximum heart rate) of males between two measurements. However the differences in other parameters were not significant. Anaerobic threshold values of female subjects were not significant difference. Respiratory exchange ratio of males was significantly different at anaerobic threshold. These results show that L-carnitine intake one hour prior to the exercise has no effect on the metabolic and blood lactate values of badminton players [102].

Taurin
One study examined the plasma taurine response to acute oral taurine supplementation and the effects of 7 days of taurin on muscle amino acid content and substrate metabolism during 2 h of cycling at approximately 60 percent peak oxygen consumption (VO2peak). There was no difference in muscle glycogen or other muscle metabolites between conditions, but there were notable interaction effects for muscle valine, isoleucine, leucine, cystine, glutamate, alanine, and arginine amino acid content following exercise after taurin. These data indicate that acute taurin produces a 13-fold increase in plasma taurine concentration. Despite the ability to significantly elevate plasma taurine for extended periods throughout the day, 7 days of taurin does not alter skeletal muscle taurine content or carbohydrate and fat

oxidation during exercise. However, taurin appears to have some impact on muscle amino acid response to exercise [103].

Polyunsaturated fatty-acids
The authors evaluated the role of a high-protein, low-calorie, polyunsaturated fattyacid (PUFA) -supplemented diet on anthropometric parameters, erythrocytemembrane fatty-acid composition, and plasma antioxidant defenses of nonprofessional volleyball athletes. The athletes were divided in two groups: One (n=5) followed the Mediterranean diet, and the other (n=6) followed a high-protein, low-calorie diet with a 3-g/day fish-oil supplementation. All the athletes had anthropometric measurements taken, both at the beginning and at the end of the study, which lasted for 2 months. Body-mass index and total body fat were significantly diminished in the second group, while they remained unchanged in the first. Plasma total antioxidant activity (TAA) was significantly increased in the plasma of both groups, with no differences between the groups, suggesting that physical activity, not the different diets, is the main contributor to the increase of plasma TAA. The second group showed a significant increase in erythrocyte-membrane PUFA content and in the unsaturation index value (UI) because of the fish-oil supplementation. A high-protein, low-carbohydrate, fish-oil-supplemented diet seems to be useful only when the aim of the diet is to obtain weight loss in a short-term period. The significant increase in the UI of erythrocyte membranes indicates the potential for harm, because a high intake of PUFA might increase susceptibility to lipid peroxidation not counterbalanced by a higher increase in TAA. Adherence to the Mediterranean diet seems to be the better choice [104].

Omega-3 fatty acid

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have beneficial effects on cardiovascular function. It was tested the hypotheses that dietary supplementation with DHA (2 g/day) + EPA (3 g/day) enhances increases in stroke volume (SV) and cardiac output (CO) and decreases in systemic vascular resistance (SVR) during dynamic exercise in healthy subjekts. It was concluded that safflower oil treatment had no effects on MAP, HR, SV, CO or SVR at rest or during exercise. DHA + EPA-induced increases in stroke volume and cardiac output imply that dietary supplementation with these fatty acids can increase oxygen delivery during exercise, which may have beneficial clinical implications for individuals with cardiovascular disease and reduced exercise tolerante [105].

D-vitamin
A surprisingly high prevalence of vitamin D insufficiency and deficiency has recently been reported worldwide. Although very little is known about vitamin D status among athletes, a few studies suggest that poor vitamin D status is also a problem in athletic populations. It is well recognized that vitamin D is necessary for optimal bone health, but emerging evidence is finding that vitamin D deficiency increases the risk of autoimmune diseases and nonskeletal chronic diseases and can also have a profound effect on human immunity, inflammation, and muscle function (in the elderly). Thus, it is likely that compromised vitamin D status can affect an athlete's overall health and ability to train (i.e. by affecting bone health, innate immunity, and exercise-related immunity and inflammation). Although further research in this area is needed, it is important that sports nutritionists assess vitamin D (as well as calcium) intake and make appropriate recommendations that will help athletes achieve adequate vitamin D status: serum 25(OH)D of at least 75 or 80 nmol/L. These

recommendations can include regular safe sun exposure (twice a week between the hours of 10 a.m. and 3 p.m. on the arms and legs for 5-30 min, depending on season, latitude, and skin pigmentation) or dietary supplementation with 1,000-2,000 IU vitamin D3 per day. Although this is significantly higher than what is currently considered the adequate intake, recent research demonstrates these levels to be safe and possibly necessary to maintain adequate 25(OH)D concentrations [106].

Protein supplements
Protein supplements commonly are ingested by athletes to improve strength, agility, and speed. While athletes require a higher amount of protein (g.kg body weight) than nonathletes do to support protein synthesis, they do not necessarily need to consume protein from supplemental sources. To date, no studies have shown an advantage of ingesting protein supplements over natural, protein-containing foods; therefore, dietary sources of protein may be just as effective as protein supplemental sources in the regulation of muscle protein synthesis. Misconceptions regarding protein supplement effectiveness may originate from athletes' nutrition information sources. A survey questionnaire queried high school football players about sources of information and measured protein supplement misconceptions by using scores on a Protein Supplement Misconceptions Index. Sixty-one high school football players participated in the study; 39 were protein supplementers, and 22 were non-protein supplementers. There was a significant difference between index scores of protein supplementers and non-protein supplementers, indicating that protein supplementers had a greater level of misconceptions than non-protein supplementers did. Bonferroni post hoc procedures used with individual index items revealed that protein supplementers were significantly more likely than non-protein supplementers to agree that "athletes should take protein supplements" and needed them "to gain as much muscle as possible". Greater misconceptions for protein supplementers may have resulted from the sources chosen for information and advice. Since coaches, parents, and friends were the primary sources of advice about protein supplements for protein supplementers, it would be valuable to provide nutrition education to these groups concurrently with educating young athletes to dispel ongoing misconceptions regarding the need for and effectiveness of protein supplements [107].

Long distance running

It was investigated the nutritional habits of ultra-endurance runners before, during and after the Deutschlandlauf 2006 in Germany, from the north (Kap Arkona-Rgen) to the south (Lrrach), over 1,200 km and 17 stages. Twenty male ultra-runners completed a questionnaire about their nutrition before, during and after the race. In the 4 weeks, and the day before the race, 70 percent of the runners followed no special diet. In the morning before the start of a stage, the main nutrients were buns with jam, butter and cheese and the preferred drink was coffee. During the stages, the athletes preferred to consume bread, bananas and chocolate and preferably drank pure water, Apfelschorle and Coca Cola. In the evening, the athletes preferred to consume meat, noodles, pure water and beer. During the run, 40 percent of the athletes had a special desire for salty and fatty food and 10 percent a particular reluctance for sweet and carbohydrate-rich products. After the race, the runners preferred apples, vegetables, rice, bread, pure water, Apfelschorle and beer. Multivitamin products, multi-mineral products as well as magnesium were the preferred supplements before, during and after the race. It was conclude that 70 percent of the ultra-endurance runners in the Deutschlandlauf 2006 followed no special diet before the race. Multi-vitamins, multi-minerals and magnesium were preferably consumed as ergogenic supplements. Before the start of a stage they ate a normal breakfast;

during a stage they preferred carbohydrate-rich products and water; and in the evening after a stage they preferred to consume meat with a carbohydrate-rich nutrition and drank water as well as beer [108]. The objective of the study was to describe the food intake of adventure racers during a competition simulated in the laboratory. Ten male athletes with international experience in adventure races took part in the study. The experiment lasted 67 hr (total distance covered 477 km), but 3 athletes did not finish the race. Food intake was recorded throughout the simulation. Athletes' total energy expenditure was greater than their total energy intake (24,516 vs. 14,738 kcal), and the athletes obtained significantly more energy from food than from supplements. Carbohydrate intake was below the recommendation of 0.5-1.0 g per kg and hour. These results indicate that guidelines for multiday adventure races are needed [109].

Veterinary (horses)
Doping control of anabolic substances is normally carried out with urine samples taken from athletes and horses. Investigation of alternative specimens, e.g. hair samples, is restricted to special cases, but can also be worthwhile, in addition to urine analysis. Moreover, hair material is preferred in cases of limited availability or complicated collection of urine samples, e.g. from horses. In this work, possible ways of interpretation of analytical results in hair samples are discussed and illustrated by practical experiences. The results demonstrate the applicability of hair analysis to detect anabolic steroids and also to obtain further information about previous abuse. Moreover, the process of incorporation of steroids into hairs is described and the consequences on interpretation are discussed, e.g. on the retrospective estimation of the application date. The chosen examples deal with the detection of the anabolic agent testosterone propionate. Hair samples of an application study, as well as a control sample taken from a racing horse, were referred to. Hair material was investigated by a screening procedure including testosterone, nandrolone and several esters (testosterone propionate, phenylpropionate, decanoate, undecanoate, cypionate; nandrolone decanoate, dodecanoate and phenylpropionate; limits of detection (LODs) between 0.1 and 5.0 pg/mg). Confirmation of testosterone propionate (LOD 0.1 pg/mg) was carried out by an optimised sample preparation. Trimethylsilyl (TMS) and tert-butyl dimethylsilyl derivatives were detected by gas chromatography-high-resolution mass spectrometry (GC-HRMS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) [110]. Methandienone, methandriol, and oxymetholone, which are anabolic steroids possessing 17alpha-methyl and 17beta-hydroxy groups, were developed as oral formulations for therapeutic purposes. However, they have been used in racehorses to enhance racing performance. In humans, it has been reported that structurally related anabolic steroids having the 17alpha-methyl and 17beta-hydroxy groups, including 17alpha-methyltestosterone, mestanolone, methandienone, methandriol, and oxymetholone, have metabolites in common. In one study, it was found that metabolites common to those of 17alpha-methyltestosterone and mestanolone were detected in horse urine after the administration of oxymetholone, methandienone, and methandriol. Based on analytical data, it was confirmed these to be the common metabolites of five structurally related steroids, 17alpha-methyltestosterone, mestanolone, oxymetholone, methandienone, and methandriol. Furthermore, it was detected hitherto unknown urinary metabolites of methandriol and oxymetholone in horses. The parent steroid itself was detected in horse urine after the administration of methandriol, other than metabolites common to 17alpha-methyltestosterone and

mestanolone. On the other hand, the major metabolite of oxymetholone was mestanolone, aside from metabolites presumed to be the stereoisomers of 2hydroxymethyl-17alpha-methyl-5alpha-androstan-3,17beta-diol and 2,17alphadi(hydroxymethyl)-5alpha-androstan-3,17beta-diol. The simultaneous detection of common metabolites and other main metabolites would help narrow down the candidate-administered steroid for the doping tests in racehorses [111]. Insulin administration can increase muscle glycogen by utilising hyperinsulinaemic clamps prior to sports events or during the recovery phases, and increase muscle size by its chalonic action to inhibit protein breakdown. In order to control insulin abuse in equine sports, a method to detect effectively the use of insulins in horses would be required. Besides the readily available human insulin and its synthetic analogues, structurally similar insulins from other species can also be used as doping agents. One study described a method for the simultaneous detection of bovine, porcine and human insulins, as well as the synthetic analogues Humalog (Lilly) and Novolog (Novo Nordisk) in equine plasma. Insulins were isolated from equine plasma by immunoaffinity purification, followed by centrifugal filtration, and analysed by nano-liquid chromatography-tandem mass spectrometry (LC/MS/MS). Insulin and analogues were detected and confirmed by comparing their retention times and major product ions. All five insulins (human insulin, Humalog, Novolog, bovine insulin and porcine insulin), which are exogenous in the horse, could be detected and confirmed at 0.05ng/mL. This method was successful in confirming the presence of human insulin in plasma collected from horses up to 4h after having been administered a single low dose of recombinant human insulin (Humulin, Eli Lilly) [112]. Recombinant human erythropoietin (rhEPO) and darbepoetin alfa (DPO) are proteinbased drugs for the treatment of anemia in humans by stimulating erythrocyte production. One paper described the first method for differentiation and identification of rhEPO and DPO in equine plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The limit of identification was 0.1 ng/mL for DPO and 0.2 ng/mL for rhEPO in equine plasma, and the limit of detection was 0.05 ng/mL for DPO and 0.1 ng/mL for rhEPO. Analyte carryover problem encountered was solved by adding 20 percent acetonitrile to the solvent of the sample digest to increase solubility of the peptides. This method was successfully applied to identification of DPO in plasma samples collected from a research horse following DPO administration and from racehorses out of competition in North America [113]. Two simple and rapid LC/MS methods with direct injection analysis were developed and validated for the quantification and identification of hydrocortisone in equine urine using the same sample preparation but different mass spectrometric systems: ion trap mass spectrometry (IT-MS) and time-of-flight mass spectrometry (TOF-MS). The main advantage of the proposed methodology is the minimal sample preparation procedure, as particle-free diluted urine samples were directly injected into both LC/MS systems. Desonide was used as internal standard (IS). The linear range was 0.25-2.5 microg/mL for both methods. Matrix effects were evaluated by preparing and analyzing calibration curves in water solutions and different horse urine samples. A great variation of the signal both for hydrocortisone and the internal standard was observed in different matrices. To overcome matrix effects, the unavailability of blank matrix and the excessive cost of the isotopically labeled internal standard, standard additions calibration method was applied. This work is an exploration of the performance of the standard additions approach in a method where neither nonisotopic internal standards nor extensive sample preparation is utilized and no blank matrix is available. The relative standard deviations of intra and interday analysis of hydrocortisone in horse urine were lower than 10.2 and 5.4 percent,

respectively, for the LC/IT-MS method and lower than 8.4 and 4.4 percent, respectively, for the LC/TOF-MS metod [114].

Gene doping
Gene doping is the misuse of gene therapy to enhance athletic performance. It has recently been recognised as a potential threat and subsequently been prohibited by the World Anti-Doping Agency. Despite concerns with safety and efficacy of gene therapy, the technology is progressing steadily. Many of the genes/proteins which are involved in determining key components of athletic performance have been identified. Naturally occurring mutations in humans as well as gene-transfer experiments in adult animals have shown that altered expression of these genes does indeed affect physical performance. For athletes, however, the gains in performance must be weighed against the health risks associated with the gene-transfer process, whereas the detection of such practices will provide new challenges for the anti-doping authorities [115]. Some spectacular results from genetic manipulation of laboratory rodents and increasing developments in human gene therapy raise the spectre of genetic modification or gene doping in sports. Candidate targets include the induction of muscle hypertrophy through overexpression of specific splice variants of insulin-like growth factor-1 or blockade of the action of myostatin, increasing oxygen delivery by raising the hematocrit through the use of erythropoietin, induction of angiogenesis with vascular endothelial growth factors or related molecules and changes in muscle phenotype through expression of peroxisome-proliferator-activated receptor- delta and associated molecules. Some of these potential genetic enhancements, particularly where the genetic modification and its action are confined to the muscles, may be undetectable using current tests. This had lead to exaggerated predictions that gene doping in athletics will be common within the next few years. However, a review of the methods of gene transfer and the current state of the art in development of genetic treatments for human disease show that the prospects for gene doping remain essentially theoretical at present. Despite this conclusion, it will be important to continue to monitor improvements in the technology and to develop methods of detection, particularly those based on identifying patterns of changes in response to doping as opposed to the detection of specific agents [116].

Ethics
Since ancient times, competitive athletes have been familiar with the use of ergogenic aids and they will probably continue to use unfair and harmful substances in future, because their inclination to victory, along with the mirage of glory and money, will probably overcome health and legal risks. It was now searched PubMed using the term doping over the period 1990 to the present day. By literature searching, it emerges that the phenomenon of doping is complex and multifaceted. It involves a number of causes and factors that do not originate solely in the athletic field, making universality its main feature. It is in fact observed in all ages and levels of competition, and it concerns all sports, even the most unpredictable. The high number of athletes testing positive for anti-doping controls attests that the current strategy might be analytically adequate to unmask most (but not all) doping practices, but it is probably ineffective to prevent athletes to dope and modify this upsetting trend. As doping parallels the use of medications, food supplements, alcohol and social drugs, a reinforced preventive policy is advisable. The authors concluded that

current anti-doping policy should be replaced with a more efficient and practical strategy to identify and monitor abnormal and harmful deviations of the biochemical and haematological profiles [117].

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