Projets de recherche

Code: 13D13JD 

This research project will contribute to the understanding of inhaling high therapeutic doses of asthma medication (long acting Beta-2-agonists) over a sustained period of time on athletic performance. The majority of research undertaken with inhaled Beta-2-agonists involves acute doses of short acting Beta-2-agonists in male athletes. Investigating acute doses of Beta-2-agonists does not necessarily replicate real life situations, where athletes prescribed Beta-2-agonists will use them on a daily basis for a prolonged period of the time.

The potential performance enhancement from inhaled Beta-2-agonists focuses on increases in skeletal muscle protein, leading to increased muscle mass.  Increased muscle mass is associated with increased muscle force production.  In addition the long term use of Beta-2-agonists may also decrease body fat.  No study has investigated the potential for these performance gains to occur when athletes inhale long acting Beta-2-agonists over a prolonged period of time.

There is limited ecologically valid data available that demonstrates whether long acting Beta-2-agonists, taken over a prolonged period of time is performance enhancing. Further to this there is no data available that details the movement of Beta-2-agonists through the body following long-term use of high therapeutic doses of inhaled long-acting Beta-2-agonists.

This research project will investigate the impact of inhaling the long acting Beta-2-agonists Salmeterol or Formoterol twice daily over a 12 week period on sprint performance, strength, power, recovery and body composition in male and female athletes. In addition the movement of the inhaled long acting Beta-2-agonists through the body will be analysed at 6 weeks and 12 weeks.  The results of this study will provide data to inform the use of inhaled Beta-2-agonists by elite athletes. In particular they will provide data to assist in the implementation of regulations on the use of inhaled Salmeterol and Formoterol and assist in the resolution of contested doping violations.
 

Main Findings: 

The purpose of this study was to contribute to the understanding of inhaling high therapeutic doses of long acting β2-agonists over a sustained period of time on athletic performance. In particular this study investigated the impact of inhaling Salmeterol or Formoterol twice daily over a ten week period on strength, power, recovery and body composition in male and female athletes.
To carry out the investigation into the potential ergogenic effect of long term use of long acting β2-agonists we recruited 38 participants (23 male and 15 female) who had no history of asthma. All participants underwent a eucapnic voluntary hyperpnoea challenge to confirm absence of asthma. Participants completed baseline assessments which included measuring strength and power performance as well as assessing body composition. Participants were randomly assigned to one of three groups: placebo inhaler, salmeterol inhaler (100 µg twice daily) or formoterol inhaler (12 µg twice daily). The participants undertook a 10 week training protocol with a repeat of the baseline assessments taking place at five and ten weeks. Over the course of the training periods participants took their assigned inhaled dose of β2-agonists twice daily.  During the training weeks participants attended three training sessions a week that focused specifically on developing strength, power and sprint performance. Assessments of recovery and mood were recorded via questionnaires during the training period. 
All 38 participants completed all data collection between weeks 0 and 5, however only the male participants completed all 10 weeks. Between week zero and week five 30 m sprint time improved in both the formoterol (–0.29 ± 0.11 s; P<0.049) and salmeterol (– 0.35 ± 0.05 s; P<0.04) groups when compared to the placebo group (+0.01 ± 0.11 s).  However, there were no changes in other strength and power measurements, recovery, mood or sum of 4 skin folds between groups over the course of the 5 weeks between groups. Between weeks 5 and 10 decreases in sprint time in salmeterol and formoterol groups were similar to placebo group in the male participants. 
Our results suggest that large daily doses of salmeterol and formoterol may lead to improvements in sprint performance over a 5 week period. However the improvements in sprint performance are not sustained over a longer period of time.  Future work investigating the mechanisms will provide an understanding how daily use of long acting β2-agonists may influence sprint performance over a sustained period of time.

Code: 12A5CG

The number of peptide hormones, growth hormones and related substances which are required to be tested for are consistently increasing. The main catalyst for this fact is the marketing of products which have had limited data published in medical journals and are then produced and sold via internet websites. This makes them easily accessible and a concern for fair play in sport as well as the health of the athlete as the products may not have therapeutic use approval in humans and can be of poor quality.

In general the analysis of peptide hormones has been addressed one by one with many of the techniques requiring large volumes of urine and complicated extraction protocols. Recently several publications have multiplex methods to include a selection of peptides of interest to antidoping laboratories. These methods have involved either a direct analysis of urine samples for compounds expected at high concentrations or used antibodies for purification to enable lower detection limits.

Particular areas have been identified as playing an important role for the development of a multi-residue analysis technique; extraction/concentration including the use of two dimensional (2D) liquid chromatography, availability of standards and metabolites of peptide hormones. The extraction/concentration of the samples will be examined include solid phase extraction (cartridges/pipette tips), magnetic beads with antibodies, molecular weight cut-off filtration and 2D liquid chromatography. Many of the peptides which are sold over the internet are not available from suppliers of standard materials which makes it more difficult for laboratories to provide testing methodologies. This project includes making available standards suitable for laboratories to develop method with for testing procedures. An in vitro enzyme assay will also be setup with kidney microsomes to be applied to new peptide based drugs to determine the metabolites that are likely to be found in urine.

Main Findings

For over a decade the Australian Sports Drug Testing Laboratory (ASDTL) has been analyzing black market seizures for the Australian Border Force. Many different peptide hormone products have been identified and include Gonadotropin Releasing Hormone (GnRH) and analogues (eg. triptorelin), Growth Hormone Releasing Hormone (GHRH) and analogues (eg. CJC-1295), Growth Hormone Secretagogues (GHS) and mimetics (eg. ipamorelin) and Growth Hormone Releasing Peptides (eg. GHRP-6). Many of these substances continue to be confiscated at the border which means the risk of abuse by athletes in Australia, as well as other in countries around the world remains. The detection in bodily matrices is important, not only for ensuring fairness in sport but also ensuring the health and wellbeing of athletes.

As instrument sensitivity improved, publications were emerging outlining ‘dilute and shoot’ methods for small molecules. Given the difficulties encountered using SPE with the increasing list of peptides being analysed, a ‘dilute and shoot’ method was developed and validated where diluted samples were passed through Agilent Captiva ND Lipid plate to remove interferences. This methodology was superseded with the implementation of ionKey/MS systems and SPE sample preparation which can analyse approximately 50 small peptides, metabolites and peptide mimetics. Limits of detection for the majority of sample range between 0.1-0.5 ng/mL with recoveries between 20-99%. The workflow for the small peptide should also allow the addition of conjugated small molecules and the development will be included in future planned work.

The most significant impact of this project has been the production of reference material and the availability of these to WADA Laboratories. Some of the materials are available from commercial providers but the identification and production of new materials in particular metabolites can be considerably more responsive when initiated by a WADA laboratory than a commercial provider. The ASDTL now has available 60 parent and metabolites peptides.

Code: 13A04JW 

Traditional Chinese Medicine (TCM) has been used in China for thousands of years. Some drugs of TCM contain endogenous steroids such as musk, penis and testes of ox, and oviductus ranae, which make it possible to abuse TCM as doping. In the 2011 FIFA Women's World Cup, adverse analytical findings about the exogenous source of etiocholanolone and other steroids were reported , and musk was regarded as the source of the steroidal preparation. Isotope ratio mass spectrum is applied in doping test to confirm the endogenous steroids abuse by determining the isotopic ratio of 13C/12C, whereas the carbon isotope ratios of steroids in animal materials used in TCM were seldom reported in literature. In view of the fact that TCM materials would be abused as doping, their influences on doping test should be further studied. 
In this project, musk, penis and testes of ox, and oviductus ranae will be collected for the detection of concentrations and carbon isotope ratios of endogenous steroids by GC-MSD and GC/C/IRMS. Excretion study will also be executed to demonstrate the influences of musk intake on the doping test. 
Based on those studies, the possibility of those TCMs abuse as doping will be estimated.

Main Findings: 

Traditional Chinese Medicine (TCM) has been used for a long time. Some TCMs contain prohibited steroids, which make it possible to abuse TCMs as doping. In this study, steroid contents and δ13C-values were analyzed in musk, penis-testes of ox, and forest frog’s oviduct. Excretion study was executed to demonstrate the influences of musk ingestion on doping control.  
  The analysis on 20 batches of musk illustrated that the contents of androgen were greater than that of estrogen and progestin in musk. Etio, 5α-3β17α-diol, 5β-diol, and An were main androgen in musk. IRMS analysis demonstrated that the androgen in wild deer musk possessed δ13C-values in the range of exogenous steroid values, while the steroid δ13C-values in domestic deer musk samples were within the normal range of human body.  
  In penis-testes of ox, An, Etio, DHEA, DHT, 4-AD, T, EpiA, and Preg were detected; Chol had average δ13C-value of -22.6 ‰. In forest frog’s oviduct, Preg and 5β-diol could be identified; Chol bore the average δ13C-value of -27.8 ‰.  
  In musk excretion study, 200 and 100 mg of wild and domestic deer musk samples were administrated by 29 subjects. The urinary profile showed changes, but the changes were inconsistent among subjects. The fluctuations might be proportional to the musk quality. In the IRMS test, the δ13C-values of Etio and 5β-diol were more sensitive than other markers, and AAFs were obtained. It is the first report about the AAFs in the musk administration study.