Rymantas Kazlauskas

Medication use in athletes selected for doping control at the Sydney Olympics (2000).

During the Olympic Games held in Sydney in September, 2000 Doping Control was undertaken as specified in the International Olympic Code. During this process information about the medications taken by athletes was collected as a routine and formed part of the paperwork associated with a urine test. In their Post Games Report the World Anti-Doping Agency (WADA) recommended that the information about medications be collated with a view to assessing their use by athletes.Mandatory doping control for winners of events as well as random selection of athletes both during competition and out of competition allowed data to be collected about medications and supplements used by athletes. At the Doping Control Stations all competitors selected for a test, after providing a urine sample for analysis, were asked the same question: “what medications have you taken in the past three days?” The answer was to include all prescription drugs, over-the-counter medications, any other substances taken by mouth, injection, inhalation, ointment or by suppository, as well as vitamins, minerals, and all other supplements.This paper reviews the data from the 2758 Declaration Forms obtained at doping control. The prevalence of use of medications, the number used by an individual, and the pattern of use by these elite sports people were examined.The trends seen in this survey point to a dangerous overuse of nonsteroidal anti-inflammatory agents and an unnecessary overuse of vitamins in this population, while pointing out the increased prevalence of asthma and the dangers of drug interactions. ObjectiveThe main objective here is to review some of the medications used by athletes in the Olympic Games in Sydney 2000. Data SourcesDuring these Games Doping Control was undertaken as specified by the International Olympic Committee. As well as a urine test, information about medications routinely taken was collected. Mandatory doping control for winners of events as well as random selection of athletes both during competition and out of competition required data to be collected about medications and supplements used by athletes as part of the sample collection protocol. At the Doping Control Stations all competitors selected for a test, after providing a urine sample for analysis, were asked the same question: “what medications have you taken in the past three days?” The answer was to include all prescription drugs, over-the-counter medications, any other substances taken by mouth, injection, inhalation, ointment or by suppository, as well as vitamins, minerals, and all other supplements. Data SelectionIn this article we review the data from the laboratory copy of the 2758 Declaration Forms obtained at doping control. The cut down version of the Declaration Form submitted to the laboratory had all information identifying the athlete removed. Thus all information used in this article is completely anonymous. The prevalence of use of medications, the number used by an individual, and the pattern of use by these elite sports people were examined at the request of the IOC. ConclusionsIn their Post-Games Report, the World Anti-Doping Agency (WADA) acting as independent observers of the anti-doping process recommended to the IOC that the information obtained in the Athlete Declaration Forms concerning medications be collated with a view to assessing their use by athletes. The trends in their use seen in this survey point to an overuse of supplements as well as a dangerous overuse of drugs such as nonsteroidal anti-inflammatory agents together with multiple drug use emphasising the dangers of drug interactions and points out the increased prevalence of asthma in this population.

Carbon isotope ratio (δ13C) values of urinary steroids for doping control in sport

The detection of steroids originating from synthetic precursors in relation to their chemically identical natural analogues has proven to be a significant challenge for doping control laboratories accredited by the World Anti-Doping Agency (WADA). Endogenous steroid abuse may be confirmed by utilising the atomic specificity of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) that enables the precise measurement of differences in stable isotope ratios that arise as a result of fractionation patterns inherent in the source of steroids. A comprehensive carbon isotope ratio (delta(13)C) profiling study (n=1262) of urinary ketosteroids is reported that demonstrates the inter-individual variation that can be expected from factors such as diet, ethnicity, gender and age within and between different populations (13 countries). This delta(13)C distribution is shown by principal component analysis (PCA) to provide a statistical comparison to delta(13)C values observed following administration of testosterone enanthate. A limited collection of steroid diol data (n=100; consisting of three countries) is also presented with comparison to delta(13)C values of excreted testosterone to validate criteria for WADA accredited laboratories to prove doping offences.

Pharmacodynamics of growth hormone abuse biomarkers and the influence of gender and testosterone: a randomized double-blind placebo-controlled study in young recreational athletes.

CONTEXT IGF axis proteins and collagen peptides are promising markers of GH abuse. OBJECTIVE Our 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 (T). DESIGN This was a randomized, double-blind, placebo-controlled study of 8-wk treatment followed by 6-wk washout. SETTING The study was performed at a clinical research facility. PARTICIPANTS A total of 96 recreationally trained healthy athletes (63 men, 33 women), aged 18-40 yr, were studied. INTERVENTION All subjects received GH (2 mg/d sc) or placebo for 8 wk; men also received T (250 mg/wk im) or placebo for 5 wk. MAIN OUTCOME MEASURES Serum IGF axis proteins (IGF-I, IGF binding protein-3, and acid labile subunit) and collagen peptides (N-terminal propeptide of type I procollagen, C-terminal telopeptide of type I collagen, and N-terminal propeptide of type III procollagen) were measured. RESULTS GH induced significant increases in IGF axis and collagen markers that were greater in men than women (P < 0.001). 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 (P < 0.01) after 6 wk, in comparison to IGF markers, which returned to baseline within 1 wk. Addition of T to GH amplified the response of PIIINP by more than 1.5-fold but did not affect any other marker. T alone did not affect IGF axis markers but modestly increased collagen markers. CONCLUSIONS 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 T.

Studies of methylhexaneamine in supplements and geranium oil

A number of supplements are now available which are sold as fat burners or pre-workout boosters and contain stimulants which are banned in sport. Many contain methylhexaneamine under one of many pseudonyms including Geranamine, geranium oil or extract, or a number of chemical names such as 1,3-dimethylpentylamine. This has resulted in many athletes returning an adverse finding and having sanctions imposed. Other stimulants such as caffeine, phenpromethamine, synefrine, and phenethylamines are also to be found in supplements. This communication shows that geranium oils do not contain methylhexaneamine and that products labelled as containing geranium oil but which contain methylhexaneamine can only arise from the addition of synthetic material. Since the usual dose of methylhexaneamine is large, the drug is excreted at relatively high amounts for more than 29 h, the time for which the excretion was studied.

Evaluation of androgenic activity of nutraceutical-derived steroids using mammalian and yeast in vitro androgen bioassays.

Androgenic steroids marketed online as nutraceuticals are a growing concern in sport doping. The inability of conventional mass spectrometry (MS)-based techniques to detect structurally novel androgens has led to the development of in vitro androgen bioassays to identify such designer androgens by their bioactivity. The objective of this study was to determine the androgenic bioactivity of novel steroidal compounds isolated from nutraceuticals using both yeast and mammalian cell-based androgen bioassays. We developed two new in vitro androgen bioassays by stably transfecting HEK293 and HuH7 cells with the human androgen receptor (hAR) expression plasmid together with a novel reporter gene vector (enhancer/ARE/SEAP). The yeast β-galactosidase androgen bioassay was used for comparison. Our new bioassay featuring the enhancer/ARE/SEAP construct (-S) displayed simpler assay format and higher specificity with lower sensitivity compared with the commonly used mouse mammary tumour virus (MMTV)-luciferase. The relative potencies (RP), defined as [EC(50)] of testosterone/[EC(50)] of steroid, of nutraceutical extracts in the yeast, HEK293-S, and HuH7-S, were 34, 333, and 80,000 for Hemapolin; 208, 250, and 80 for Furazadrol; 0.38, 10, and 106 for Oxyguno; 2.7, 0.28, and 15 for Trena; and 4.5, 0.1, and 0.4 for Formadrol, respectively. The wide discrepancies in rank RP of these compounds was reconciled into a consistent potency ranking when the cells were treated with meclofenamic acid, a nonselective inhibitor of steroid metabolizing enzymes. These findings indicate that steroids extracted from nutraceuticals can be converted in vitro into more or less potent androgens in mammalian but not in yeast cells. We conclude that the putative androgenic bioactivity of a new compound may depend on the bioassay cellular format and that mammalian cell bioassays may have an added benefit in screening for proandrogens but sacrifice specificity for sensitivity in quantitation.

The detection of androstenedione abuse in sport: a mass spectrometry strategy to identify the 4-hydroxyandrostenedione metabolite.

Studies have shown that the administration of androstenedione (ADIONE) significantly increases the urinary ratio of testosterone glucuronide to epitestosterone glucuronide (T/E) - measured by gas chromatography/mass spectrometry (GC/MS) - in subjects with a normal ( approximately 1) or naturally high (>1) initial values. However, the urinary T/E ratio has been shown not to increase in subjects with naturally low (<1) initial values. Such cases then rely on the detection of C(6)-hydroxylated metabolites shown to be indicative of ADIONE administration. While these markers may be measured in the routine GC/MS steroid profile, their relatively low urinary excretion limits the use of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to specifically confirm ADIONE administration based on depleted (13)C content. A mass spectrometry strategy was used in this study to identify metabolites of ADIONE with the potential to provide compound-specific detection. C(4)-hydroxylation was subsequently shown to be a major metabolic pathway following ADIONE administration, thereby resulting in urinary excretion of 4-hydroxyandrostenedione (4OH-ADIONE). Complementary analysis of 4OH-ADIONE by GC/MS and GC/C/IRMS was used to confirm ADIONE administration.

Strategies for rhepo Detection in Sport

This article examines available strategies for the detection of recombinant erythropoietin (rhEPO) abuse in sport. RhEPO was quickly recognized as an effective but hazardous performance-enhancing agent. In the absence of a valid procedure to detect rhEPO doping, at-competition health checks were introduced, which excluded athletes from competition when their hemoglobin or hematocrit values exceeded an arbitrary limit. This limited the danger to athletes, but did nothing to eliminate the use of rhEPO.Through the last decade, both direct and indirect methods for detecting rhEPO were investigated. No single indirect marker was found that satisfactorily demonstrated rhEPO use. A combination of blood and urine tests together formed the procedure and strategy approved by the International Olympic Committee (IOC) for detecting rhEPO use at the Sydney Olympics.However strategies for testing for EPO are as important as the developed laboratory analytical procedures. The use of extensive out-of-competition testing and analysis within the IOC accredited laboratory system is critical to any testing program. At-competition blood tests have merit as true health checks and will also be needed to detect acutely useful agents such as hemoglobin-based oxygen carriers. However the persistence of the “health check” rationale for on-site at-competition rhEPO testing has led to much wasted testing effort, as rhEPO use by athletes will rarely occur near to or at the time of the competition for fear of detection. Thus, direct testing methods (such as the rhEPO urine test) especially will fail due to the completed metabolism and elimination of administered rhEPO before the test, unless the international sporting federations use the information gathered to assist in targeted out-of-competition testing. This article discusses the limitations of testing at competition and proposed strategies for dealing with various phases of EPO doping in detail, concluding that no one single currently used strategy will detect all users of rhEPO.The development of strategies to diagnose rhEPO abuse may serve as a model to detect other biological agents.

Structure-activity relationships of synthetic progestins in a yeast-based in vitro androgen bioassay

The recent identification of tetrahydrogestrinone (THG), a non-marketed designer androgen used for sports doping but previously undetectable by established mass spectrometry-based urine drug screens, and its production by a facile chemical modification of gestrinone has raised concerns about the risks of developing designer androgens from numerous marketed progestins. We therefore have used yeast-based in vitro androgen and progesterone bioassays to conduct a structure-activity study assessing the intrinsic androgenic potential of commercially available progestins and their derivatives, to identify those compounds or structures with the highest risk of forming a basis for such misapplication. Progestins had a wide range of androgenic bioactivity that was not reliably predicted for individual steroids by their progestin bioactivity. 17alpha-Hydroxyprogesterone and 19-norprogesterone derivatives with their bulky 17beta-substituents were strong progestins but generally weak androgens. 17alpha-Ethynylated derivatives of testosterone, 19-nortestosterone and 18-methyl-19-nortestosterone such as gestrinone, ethisterone, norethisterone and norgestrel had the most significant intrinsic androgenicity of all the commercially marketed progestins. Facile chemical modification of the 17alpha-ethynyl group of each of these progestins produces 17alpha-methyl, ethyl and allyl derivatives, including THG and norbolethone, which further enhanced androgenic bioactivity. Thus by using the rapid and sensitive yeast bioassay we have screened a comprehensive set of progestins and associated structures and identified the ethynylated testosterone, 19-nortestosterone and 18-methyl-19-nortestosterone derivatives as possessing the highest risk for abuse and potential for conversion to still more potent androgens. By contrast, modern progestins such as progesterone, 17alpha-hydroxyprogesterone and 19-norprogesterone derivatives had minimal androgenic bioactivity and pose low risk.

Effects of recombinant human LH and hCG on serum and urine LH and androgens in men

Context  The administration of gonadotrophins is prohibited in sport but the effect in men of recently available recombinant hCG and LH on serum and urine concentrations of gonadotrophins and androgens has not been systematically evaluated in the antidoping context.

Screening for testosterone abuse in male athletes using the measurement of urinary LH, a revision of the paradigm.

The primary screening method for the detection of doping by athletes using synthetic versions of endogenous steroids such as testosterone relies on measurement of the ratio of testosterone (T) to epitestosterone (E) in urine. In 2005 the World Anti-Doping Agency (WADA) lowered the T/E value at which samples undergo further investigation from six to four. This has resulted in a large increase in the number of athletes with naturally elevated T/E ratios undergoing investigation without a corresponding increase in the number of proven doping offences involving testosterone.Our objective was to develop a new simple screening protocol that can, with high probability, not only distinguish athletes whose natural T/E values exceed four from those whose T/E values have been elevated by testosterone doping but also detect those athletes with naturally low T/E values that do not exceed four despite being administered testosterone.Testosterone (250 mg Sustanon) was administered weekly to a group of 47 young adult males for five weeks in a double-blind placebo controlled study and urine samples collected. The samples were analysed for steroid concentrations using GC/MS and for luteinizing hormone (LH) by immunoassay.The elevation of T/E that occurred in all subjects was accompanied by a significant reduction in urinary LH concentrations to levels that are rare in normal subjects.The appropriate measurement of urinary LH, with the measurement of T/E values, can markedly improve the efficiency of detection of doping with testosterone by male athletes, particularly those who have low natural T/E ratios.