Projets de recherche

Code: 10C24ZN

Detection of autologous blood transfusion is probably currently the greatest challenge in the doping control. For the reasons of convenience, for its considerable security and the difficulty of detecting it, autologous transfusion has become the method chosen by many athletes to illegally increase their oxygen delivery capacity. This project aims is to study the proteome changes of erythrocytes during storage, prior to (re) infusion of the blood to the same athlete.

For the project were selected proteins associated with the erythrocyte membrane, because they are easily available and have been well characterized in previous studies with methodologies such as 2D electrophoresis, DIGE, liquid chromatography coupled with mass spectrometry, and other combinations of the above.

The studies made so far can be classified broadly as protein-centered or peptide-centered. In the first case, the proteomics approach is based on the separation of membrane proteins from the sample by 2D electrophoresis; in the second case, the most powerful among those described is the isobaric tagging (iTRAQ), which allows quantitative change detection in multiple samples.

The results of our preliminary studies, as well as those available from other authors, identified proteins which can be grouped as follows:

• cytoskeletal proteins,

• transmembrane proteins

• other proteins related to our research findings by other researchers. To continue our study, we consider efforts in two directions:

First, to validate the results obtained, confirm the same with the largest number of subjects,

Second, to verify by independent technical changes detected in the membrane by proteomic methods, we use Western blot and/or flow cytometry. Ultimately, the goal is to develop a reliable test for the analysis of doping by autologous transfusion, using appropriate markers from membrane proteins or the cytoskeleton of erythrocytes, whose levels vary due to storage of blood in standard transfusion conditions.

Main Findings

Autologous blood transfusion doping (ABT) is defined as the transfusion of stored red blood cells (RBCs) from the same individual. The World Anti-Doping Agency (WADA) includes ABT in the List of Prohibited Substances but no official method exists yet to directly detect it.

In this project the focus was directed towards quantification of erythrocyte membrane protein changes after transfusion. Previously described data showed that some proteins translocate from cytosol to the membrane during erythrocyte storage under standard blood banking conditions. Based on this data, we have selected a panel of proteins to be further characterized. The list included peroxiredoxin-2, glyceraldehyde-3-phosphate dehydrogenase, catalase, tropomodulin-1, sorcin and annexin-7. Using immunoblotting we have observed that in general these proteins were already present in fresh blood samples and their levels increased on the membrane of the erythrocyte during in storage. Additionally, when stored RBC concentrates were mixed in vitro with fresh blood simulating a transfusion procedure, an increase of the marker proteins in the mixtures was observed. However, when blood from recently transfused patients was analyzed, there was no a clear trend in the increment or decrement of the marker proteins. On the other hand, we have observed that the training for endurance disciplines as compared to non-endurance disciplines (both in aquatic sports) seem to influence the basal concentrations of some of the marker proteins, especially those linked with the oxidation protection of RBC.

Based on all the above considerations, it is doubtful whether the changes in marker RBC membrane proteins by translocation from cytosol to membrane may afford useful methodology for detecting blood transfusion in antidoping control by analyzing blood collected from a transfused athlete. However, the analysis of a transmembrane protein, glycophorin A, unraveled modifications that modify protein molecular weight during blood bag preparation or storage conditions, suggesting that characteristic changes on erythrocyte surface are occurring. Therefore, the characterization of the time sequence line of these modifications produced on integral membrane proteins could represent a useful approach to detect autologous blood transfusion.

Code: 10D7GW

In 2002 The International Olympic Committee (IOC) established the requirement for athletes to present evidence of current asthma, exercise induced asthma (EIA), exercise-induced bronchoconstriction (EIB) or airway hyperresponsiveness (AHR) through the therapeutic use exemptions (TUE) process. The World Anti-Doping Agency (WADA) introduced the IOC policy on inhaled short acting 2-agonists in January, 2009. However, they have since abandoned the requirement of an objective airway challenge for inhaled salbutamol and salmeterol since January, 2010. The inclusion of inhaled 2-agonists is based upon health concerns rather than anti-doping concerns however; evidence is only available for the performance enhancing effect of inhaled short acting 2-agonists in endurance sports at low to moderate doses and based on athletes taking a one off dose of inhaled 2-agonists. The vast majority of this work has been undertaken in Caucasian, male athletes with limited knowledge of the ergogenic effect or pharmacokinetics in females or other races. The current research team currently has a WADA grant investigating one-off high doses of short-acting 2-agonists on performance in endurance running and football specific trials across race and gender. At present there are no investigations examining the chronic use of short or long acting inhaled 2-agonists on performance.

Main Findings

Between 2002 and 2010 The World Anti-Doping Authority (WADA) and The International Olympic Committee (IOC) established the requirement for athletes to present evidence of current asthma, exercise induced bronchoconstriction (EIB) or airway hyper responsiveness (AHR) through the Therapeutic Use Exemption (TUE) process. These regulations, guided by the IOC Medical Commission (IOC-MC), were based on health not doping (performance enhancing) concerns for athletes in light of a marked increase in the notification by athletes for the use of inhaled short acting β2-agonist from 3.7% at the Atlanta Olympic Games, 1996, to 5.7% at the Sydney Olympic Games. Our group along with others have demonstrated that the requirement of demonstrable evidence through the TUE process improves the quality of care for athletes and has improved the diagnostic sensitivity and specificity of asthma, EIB or AHR. Whilst there appears to be no ergogenic effect from acute, single-dose inhaled salbutamol, no study has investigated the impact of chronic inhalation of the WADA daily upper limit of 1600 µg (~16 inhalations of a standard salbutamol inhaler) in association with endurance and strength and power performance. Sixteen trained male athletes provided written consent and agreed to take part in the study (mean + SD: age 20.1 ± 1.6 years; height 179.9 ± 8.2 cm; weight 74.6 ± 9.1 kg). Participants entered into a 6-week, 4 times per week training study having been assigned to one of two groups in a double blind design. Group 1 (n=8) inhaled 4 x100 μg of placebo, via pocket chamber, 4 times per day for 6 weeks (PLA). Group 2 (n=8) inhaled 4 x100 μg of Salbutamol, via pocket chamber, 4 times per day for 6 weeks (SAL). Pre- and post-training endurance, power and strength and body composition was assessed. Results demonstrated an improvement in O2peak; 3 km running time-trial performance; 1 RM bench press and leg press; and peak extension and flexion torque. Body composition remained unchanged across the study period. Of note, no difference in any endurance; strength and power; or body composition measure was noted between Salbutamol and Placebo groups pre-, during, or post-intervention. In conclusion, there was no improvement in endurance, or strength and power performance following the inhalation of 1600 µg of salbutamol per day for six weeks in non-asthmatic males. This would suggest that the current WADA recommendations, which allow athletes to inhale up to 1600 µg per day is sufficient to avoid an ergogenic impact on endurance and, strength and power performance. Future studies in long term use of inhaled salbutamol focusing on strength and power performance with a greater training volume may be required. Data from this study will assist WADA in the implementation of regulations on the use of inhaled short acting β2-agonist and assist in the resolution of contested doping violations.

Code: 10D9MM

Cytochrome P450 (CYP450) enzymes are essential for the metabolism of many drugs. Although this class has more than 50 enzymes, six of them metabolize 90% of known drugs, with the two most significant enzymes being CYP3A4 and CYP2D6. Genetic variability in these enzymes may influence the individual response to commonly prescribed drug classes. Moreover, CYP450 enzymes can be inhibited by several drugs, resulting in clinically significant drug-drug interactions that can cause alteration in metabolic routes of absorption and elimination with consequent pharmacological effect or toxic reactions. The extent to which a CYP450 inhibitor affects the metabolism of a drug depends on different factors such as the dose and the ability of the inhibitor to bind to the enzyme.

In anti-doping testing, the knowledge of the pharmacokinetics of a drug/class of drugs is a key component of the analytical strategies for the detection of banned drug administration. Athletes may intentionally take advantage of CYP450 inhibition by co-administrating different drugs to obtain an alteration of the metabolism of the banned drug(s), making more complicated the detection by the anti-doping laboratories. This project is designed to provide information on the role that inhibitors of the most abundant CYP450 enzymes may have in doping scenarios. Specifically, the metabolic profile of selected banned compounds, with special emphasis on threshold compounds and to compounds that are excreted in urine mainly as CYP450 metabolites will be assessed individually and in the presence of selected CYP inhibition agents in vitro, in order to establish if the co-administration of CYP inhibitors with doping agents could be used by athletes as masking strategy.

Main Findings

This project was designed to investigate the role that inhibitors of the most abundant CYP450 enzymes may play in doping scenarios. The in vitro metabolic profile of some representative banned compounds was investigated, with special emphasis to compounds that are excreted in urine mainly as CYP450 metabolites, specifically evaluating the effect of selected, non-banned drugs that are also CYP450 inhibitors, with the aim of verifying whether the co-administration of CYP inhibitors with doping agents could be used by athletes as a masking strategy.

In the first part of this project the in vitro metabolism protocol using either pooled human liver microsomes or recombinant human CYP450 isoenzymes (CYP3A4, CYP2D6, CYP2C9 and CYP2C19) was optimized and validated with five representative banned compounds (the selective oestrogen receptor modulator toremifene, the anabolic agents stanozolol, methandienone and the glucocorticoids ciclesonide and deflazacort) in order to obtain a good correlation with the metabolism reported in humans. The optimized in vitro model was subsequently utilized in presence of non banned medicaments commonly used by athletes (primarily among them non antifungals, antiacid and antidepressant agents) to investigate their effects on the in vitro metabolic profile and on the activity of the CYP450 isoforms involved in the phase I metabolism of the selected banned agents.

The in vitro model set up in this study showed good correlation with the previously described metabolism in humans. The CYP450 isoforms involved in the phase I metabolism of the selected banned compounds are the CYP2C9, the CYP2C19, the CYP2D6 and the CYP3A4 isoforms.

On the basis of our results i) the co-administration of banned compounds with antifungals or antidepressants could lead to an incorrect interpretation of the analytical results, producing a masking effect based on the alteration of the phase I metabolic pathways; ii) the number of the markers of drug abuse currently selected during routine analyses should be expanded as much as possible to include also the parent compounds and the possible additional metabolites produced by alternative routes; iii) being the inclusion in our normal routine screening method by LC-MS/MS of the CYPs inhibitors considered in this study very straightforward, if so allowed by the WADA rules, a monitoring study on the real occurrence of CYP inhibitors in the urine samples analyzed by the WADA laboratories would markedly enhance the statistical and epidemiological relevance of our in vitro observations.

Code: R09B2JD

In addition to the increasing number of different recombinant erythropoietins (rhEPO) that are present on the market at the present time, peptide-mimetics of these hormones producing identical effects but presenting unrelated molecular structures will be soon commercialized. Due to this difference in structure, such mimetics cannot be detected by the test used for anti-doping control of rhEPO. Specific methods have to be developed.

One of these peptide mimetics, peginesatide (formerly known as Hematide™), produced by the biopharmaceutical company Affymax is now investigated in Phase 3 trials for the potential treatment of anemia associated with chronic kidney disease (CKD). This drug figures on the WADA list of prohibited substances in sport.

Affymax Company is collaborating with WADA in order that anti-doping laboratories have a test to detect Hematide at their disposal.

Affymax has developed different antibodies directed against Hematide and has tested these antibodies in two different methods, ELISA and immunoblot following SDS electrophoresis.

This project was a collaborative study including Affymax and the anti-doping laboratories of Lausanne (Switzerland) and Châtenay-Malabry (France) and was intended to validate the two different methods proposed by Affymax in two different laboratories.

The aim of the project was to investigate, eventually improve and provide a full validation of the two methods in order that they can be used for detection of the drug in blood samples as screening(ELISA) and confirmation (SDS electrophoresis) tests.

Investigations of ELISA and SDS electrophoresis for eventual improvements were especially performed by the Swiss and French laboratories respectively. Validation of both methods was performed by both laboratories.

Main findings

After some modifications of the SDS electrophoresis method, our laboratory has fully validated both this method and the ELISA method as transmitted by the laboratory of Lausanne.

Our investigations have shown that both methods are specific and robust and can thus be applied for anti-doping control of Hematide using either plasma or serum samples.

Their limit of detection are similar (0.1 ng/mL for ELISA and <0.25 ng/mL for SDS electrophoresis). They have been used in a blind test performed on blood samples from a clinical trial and provided by Affymax to our laboratory. Neither false negative nor false positive results have been observed.

The window of detection was 21 to 28 days following an injection of 0.05 mg/kg (IV).

Code: 09A26WS 

Stimulants generally belong to the substances prohibited in Sports. Among these there are several compounds that are easily available as over-the-counter drugs, that may be present in certain foodstuffs, or that may be produced in the human body from precursors in the diet. This gives rise to the possibility of accidental doping offenses on the one hand. But on the other hand, it also enables deceptive athletes to fraudulently relate a positive test result to dietary habits. 
Hence, in case of a positive test for stimulants, it will be most advantageous to be able assign the relevant compounds to defined sources. Also the possibility to exclude certain sources will be most useful. 
Problems like this can be addressed by stable isotope analysis. The elements hydrogen, carbon, nitrogen, and oxygen make up stimulants and related substances. These elements feature more than one isotope, atoms with slightly differing masses.  The ratios of these isotopes depend on the primary source of the molecule. But they also may be changed during biosynthesis or degradation. 
The project aims to exploit the information present in what is often called "isotope fingerprint". By stable isotope analysis we hope to be able to detect accidental application of prohibited substances as well as pretended pleas. 

Main Findings: 

• Methods have been developed for the 13C=12C and 15N=14N analysis of pure and urinary ephedra alkaloids.
• Comparably large amounts of the compounds are required especially for 15N/14N analysis. Valid 15N/14N analysis roughly requires 3 µg of any single ephedra alkaloid compound to be injected into the GC-C-IRMS system.  In practice, this will be rarely achievable, in particular for the most interesting compounds NE and NPE when formed during metabolism.
• The methodology has been developed also to analyze synephrine and octopamine.  Purity and in particular recovery are however far too poor for stable isotope analysis. This has therefore not been tested for synephrine and related compounds.• 15N/14N analysis of ephedra alkaloids is fundamentally not suited for the purposes intended here. First of all, metabolism induces excessive nitrogen isotope fractionation. The fractionation is time dependent.   After already short periods the 15N=14N ratios of the excreted drug will not correspond to that of the administered compound at all. In addition, there seems to be significant variation in the 15N=14N signatures of various drugs and pharmaceuticals.  Eventually, no sufficiently definite 15N=14N isotopic link between drug and metabolite can be established. This would have to be evaluated much differently if valid 15N=14N analysis of ephedrine metabolites was possible.
• 15N/14N isotopic fractionation during metabolism of ephedra alkaloids appears to be inverse. This means that the parent compound becomes 15N depleted. By contrast, “normal” isotope effects result in heavy isotope enrichment of the reagent because the isotopically lighter species tend to react faster. Inverse isotope effects have been rarely observed. This is therefore highly interesting in basic research but beyond the scope of the study.
• If at all, source assignment of urinary ephedra alkaloids can be based on 13C/12C analysis. Fractionation of carbon isotopes appears comparably small. In addition 13C/12C analysis in practice is much less demanding. In respect to ephedra alkaloids and similar compounds the amount required for valid analysis is ca. 20 less as compared to 15N/14N analysis.
• Combined 15N/14N and 13C/12C isotope fingerprinting appears promising for source assignment of unmetabolized ephedrine pharmaceuticals and preparations. This, however, is beyond the scope of the study.

Code: T09E3MT 

The Influence of orally avallable drugs on the productivity of selected genes has been demonstrated manifold In the past. and an endogenoue substance termed AICAR has bean found to be of considerable Interest ln the treatment of the metabolic syndrome associated with type-II diabetes, obesity, etc. The stimulation of fat utillzatlon and the Increased production of mitochondria generated conslderabla concerns whether this substance might be misused in sports also since laboratory rodents demonstrated significantly enhanced endurance performance arter a 4-weaks treabnant with AICAR, and the Worid Anti-Doping Agency has banned Its use since January 2009. Dua to the natural occurrence of AICAR as a (by-)product in purine biosynthesis, a quantitative determination of natural urinary AICAR values In healthy Individuals Is necessary to provide a means to uncover the llllclt administration of this drug, which should increase the renal allmlnatlon and, thus, the urine concentration of AICAR, slgnlflcantly. 
Prellmlnary data have demonstrated that urinary AICAR levels vary consldarably depending on the studied population (athletes, healthy lndlvlduals, vitamin B12 or follc acid daflclent persons, etc.), and AICAR concentrations higher than 7,500 ng/mL ware detected In athletes' doping control samples. Since It can not be excluded that some athletes misuse AICAR already today, urinary AICAR levels of healthy Individuals shall be determined to obtain reliable reference values. Within the planned project. a cohort of approxlmately 500 sport students shall be analyzed for urinary AICAR levels including pre- and post-exercise samples, males and females, as well aa different sport dlsclpllnaa (endurance, strength, and game sporta) to allow a statlstlcal evaluation of the obtained results and enable the consideration of threshold value(s) that are Indicative for AICAR misuse ln sporta. Future projects might further elucldate the option to differentiate endogenous AICAR from the synthetically derived analog by Isotope-ratio mass spectrometry. 

Main Findings: 

The adenosine monophosphate activated protein kinase (AMPK) activator 5-amino-4-imidazolecarboxyamide ribonucleoside (AICAR) was found to significantly enhance the endurance of rodents even under sedentary conditions. Thus, usage of this substance was classified as gene doping and AICAR was added to the list of prohibited substances of the World Anti-Doping Agency (WADA). Due to the endogenous production of AICAR in healthy humans, considerable amounts are present in the circulation and, thus, are excreted into urine. Considering these facts, the present study was initiated to establish reference values of renally cleared AICAR in elite athletes. Therefore a quantitative analytical method by means of isotope-dilution liquid chromatography coupled to tandem mass spectrometry, following a sample preparation consisting of a gentle dilution of native urine, was developed. Doping control samples of 500 healthy volunteers were analysed and AICAR concentrations in urine were determined. The statistical evaluation showed a significantly better distribution (normality for log-transformed values) for creatinine corrected data compared to density correction. Data evaluation of the analysis of 500 urine samples yielded a mean concentration of 863 ng/mL with sd=462 ng/mL (corrected via density) or 552 ng/mg with sd=290 ng/mg (corrected via creatinine). Computing the 99.99% reference intervals values for the creatinine corrected amounts of 3361 ng/mg were obtained and these calculations for the present dataset suggest that amounts of urinary AICAR higher than 3500 ng/mg are not consistent with an endogenous production in healthy humans.

Code: 09A23CH

To facilitate the continued anti-doping efforts it is becoming important to make the best use of the limited analysis resources available for doping detection. The primary tools for doping analysis consist of equipment that requires highly skilled operators and the analyses are often expensive and/or time consuming. As such the number of samples that can be tested for any one event is limited; affording those doping a possibility of not being tested. To overcome this deficiency we propose a method to both increase the amount of testing and focus the high quality testing on those samples most likely to be actual doping cases. 
This will be accomplished through the use of highly rapid capillary electrophoretic separations; capable of identifying key indicators of blood transfusion and other methods of enhancing oxygen delivery in the blood. The benefits of capillary electrophoretic separations are numerous; the high speed of the separations, the capability of having portable instruments and the low cost of operation. Capillary electrophoresis based separations can act as frontline screening systems to identify those blood samples that present signs of doping; flagging them for further analysis, while eliminating the bulk of the clean samples from further required testing. The advantage in the size of the capillary electrophoretic systems is the ability to perform multiple analysis with as little as a single drop of blood from the athlete; a much less invasive and rapid sampling method. 

Main Findings:

The objective of this project is to investigate how effectively capillary electrophoresis (CE) can be applied to the analysis of athlete blood samples for the identification of blood dopants. This work has specifically targeted three blood dopants: autologous blood transfusions, hemoglobin based oxygen carriers (HBOCs), and perfluorocarbon emulsions (PFC). The benefits of working with CE include the ability to perform very rapid separations, and the versatility to handle both molecular samples (i.e. HBOCs) and cellular samples (i.e. red blood cells). Furthermore, the benefits of CE separations can be translated from bench-top instruments to lab-on-a-chip devices, for what is often termed “point of care” analyses. This could be highly advantageous in anti-doping analyses, where the samples could be rapidly tested with instrumentation brought to the site of the athletic competition, likely reducing the delay in detecting those using performance enhancing agents. 
For our work, we have focused on the use of traditional, bench-top, CE instruments, as they provide the greatest flexibility in method development. In investigating the three above mentioned blood dopants we have been able to successfully apply CE to two of the three dopant methods. The one method that has been found to be incompatible with CE analysis were the PFC; we found that their inability to remain suspended in a blood sample lead to difficulties in obtaining reproducible injections into the CE. Furthermore, their significantly greater density than red blood cells (RBCs) allows for their rapid, initial identification simply through centrifugation, rendering CE analysis superfluous. The remaining dopants, HBOCs and autologous blood transfusions, have shown much better success in detection through CE. In this past year our work on the detection of HBOCs, mixed into fresh blood samples in vitro, was published in Electrophoresis (DOI:10.1002/elps.201100506). In that work we were able to detect the presence of HBOCs at concentrations down to 5.5 g/L of whole blood, an amount below a 5% increase in total hemoglobin concentration. The CE method that we have developed for the detection of autologous blood transfusions continues to show great promise. This analysis is based on the different electrophoretic mobilities experienced by RBCs of different sizes. As circulating RBCs in athletes tend to be newer, larger cells, whereas RBCs taken from storage tend to be substantially smaller, we are able to exploit the differences in size and mobility of the RBCs to identify the presence of RBCs from storage.  

Code: 09A10GG 

Determining the origin of testosterone and other steroids in human urine is a major issue in doping control. Looking at the latest published laboratory statistics of 2007 from the World-Anti-Doping Agency (WADA), testosterone was by far the most frequent reported adverse analytical finding among the anabolic steroids. Since it is well known that natural outliers of a normal steroid profile exist, a further investigation is recommended after a finding of an elevated testosterone/epitestosterone ratio or high testosterone levels. Commonly, an isotope ratio mass spectrometric (IRMS) analysis is conducted. Of the samples submitted to IRMS analysis, however, surprisingly few are reported as positive findings. 
In the proposed project confiscated testosterone preparations will be investigated. Through a close collaboration with the Special Task Force “Doping” of the Austrian Ministry of Internal Affairs, the Doping Control Laboratory in Seibersdorf is in the unique position to have access to a significant amount of seized testosterone and other anabolic preparations. 
The aim is to determine whether these products have been manipulated, with respect to carbone isotope ratios, in order to prevent a positive IRMS finding. Additionally, it will be investigated to what extent the preparations contain the masking agent epitestosterone. Epitstosterone may be used as a measure to manipulate a doping control result, by lowering the testosterone/epitestosterone ratio in urine steroid profiles.  Thereby, a further investigation of the sample may not come into consideration. 

Main Findings:

In the present study, the content of a number of black marked testosterone products collected in Austria has been analyzed. Additionally, δ 13C‰ values were measured for testosterone in the products after cleavage of the testosterone ester. The aim was to determine whether these products had been manipulated, with respect to carbon isotope ratios, in order to prevent a positive isotopic ratio mass spectrometric (IRMS) finding in doping control. Moreover, it was investigated to what extent the preparations were containing the masking agent epitestosterone, in order to lower the testosterone/epitestosterone ratio in urine steroid profiles. Out of 30 analyzed products, the declared ingredients differed from the actual content in 10 cases. Epitestosterone, however, could not be found in any of the products. The products displayed δ 13C‰ values between –23.6 and —29.4. For more than half of these products, the δ 13C‰ values were above – 26 and within a range reported for endogenous urinary steroids. Hence, the the current study clearly shows the extent of availability of testosterone products with endogenous-like carbon isotope profiles on the black market. Consequently, the applicability of the IRMS – technique to detect the use of these products is currently reduced. It is therefore considered important to continue monitoring this development and intensify research on alternatives for the detection of testosterone misuse. 

Code: 09A12JM

Testosterone is an anabolic steroid that is naturally present in everyone at various concentrations. Therefore it is not a simple task to determine whether it has been used by athletes for doping. A level of testosterone greater than four times that of its close analogue epitestosterone can point to steroid abuse in many subjects, but others have naturally high or low T/E ratios. Samples with high T/E ratios need further investigation by carbon isotope ratio mass spectrometry (IRMS) to confirm whether the steroid is from a natural or synthetic origin. This however is a relatively complex and time consuming technique.

Long-term monitoring of the T/E ratio and concentrations of related steroids over time can reveal unusual changes in metabolite levels and/or an atypical T/E ratio for a particular athlete that may be indicative of doping. For such longitudinal studies to be effective it is imperative that the results being produced by different laboratories around the world are accurate and comparable over extended periods of time. Reference materials of urine with concentration values traceable to the international system of measurement units (SI) are an excellent tool to verify the comparability of laboratory results.

The NMI Australia, with a research grant from WADA, has already produced a certified reference material (CRM) of human urine (NMIA MX005) with accurately known values for the concentration of testosterone and epitestosterone and a T/E ratio close to 4. The certification of this material is currently being extended to a range of other natural steroids related to testosterone. In this project, a pre-existing freeze-dried urine CRM (NMIA MX002) will become the basis of a second urine CRM with a different T/E ratio and concentration of these steroids. This will assist labs to maintain accuracy and comparability of results over a range of concentrations and T/E ratios.

Main Findings

An existing freeze-dried urine certified reference material (NMIA MX002) with property values for the mass fraction and mass concentration of 19-norandrosterone glucuronide has been provided with further certification of the concentrations of testosterone and epitestosterone glucuronides, the testosterone/epitestosterone ratio and the concentrations of four important testosterone metabolites used in longitudinal profiling studies; 5α-androstane-3α,17β-diol glucuronide, 5β-androstane-3α,17β-diol glucuronide, androsterone glucuronide and etiocholanolone glucuronide. The provision of these additional property values traceable to the international measurement system will provide an unequivocal benchmark for key measurement parameters in the detection of testosterone abuse. The NMIA MX002 certified reference material is the second reference material certified at NMIA for testosterone, epitestosterone and the key metabolites of testosterone at a range of concentrations to assist laboratories in longitudinal profiling measurements and in the detection of testosterone abuse.

Two independent reference methods were employed to certify the mass fractions of 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol, androsterone, etiocholanolone, testosterone and epitestosterone glucuronide conjugates in the urine CRM. The two independent reference methods employ different high-efficiency, two-dimensional HPLC clean-up techniques with determination by the complementary techniques of GC with high resolution mass spectrometry (GC/HRMS) and liquid chromatography with tandem mass spectrometry (LC/MS/MS). In both cases the primary ratio technique of isotope dilution mass spectrometry (IDMS) was employed with an exact-matching calibration protocol to minimize bias in quantification.

Studies have been conducted on the homogeneity and stability of the urine material during long-term storage, transport and use with respect to the mass fractions of testosterone, epitestosterone and the testosterone metabolites. Sample analysis was performed using both the GC/HRMS and LC/MS/MS reference methods to investigate possible measurement biases.

Code: 09E22RG

The aim of all kinds of blood manipulation is to increase the total haemoglobin mass (tHb-mass), which is directly correlated to maximum aerobic power and hence performance. When using the current doping tests it is not yet possible to detect autologous blood transfusions or the application of all kinds of erythropoiesis boosting stimulants.

To minimize these illegal practices we recommend monitoring tHb-mass of endurances athletes over time. If the individual profile deviates substantially from the expected, the athlete has to undergo further follow-ups testing. Serial measurements of tHb-mass can also be used to demonstrate objectively that an athlete has or has not used blood doping practices.

Practical experience demonstrates that the recently developed method (optimized CO-rebreathing method) is valid, very reproducible and suitable to measure routinely an athlete’s tHb-mass. The practicability and significance of the method was evaluated in two scientific projects by Prof. Schmidt (WADA 2006-2007 and 2008-2010).

Nevertheless, the acceptance of this method is low in federations and athletes due to the relatively high amount of CO applied during the CO-rebreathing test exceeding the internationally existing threshold limits. The use of another tracer instead of CO, i.e. the isotopically labelled nitric oxide, has several advantages compared to the established CO-rebreathing method. The amount of inhaled tracer gas can be 4000-fold reduced, avoiding a toxic load for the athlete. This reduction is a combination of the isotopic ratio of 15NO/14NO (300) and the high sensitivity of the used detection method (Faraday-Rotation-Spectroscopy) for the measurement of 15NO.

Furthermore, NO has a 200-fold higher affinity to hemoglobin reducing the influence of possible confounding factors. We expect the NO-rebreathing technique using 15NO as innovative tracer gas as an optimal method to determine tHb-mass. As a consequence tHb-mass can be introduced as a key parameter into the athlete’s biological blood passport. 

Main Findings

The direct transfer of the determination of tHb-mass changing only the applied tracer gas from carbon monoxide to nitric oxide is not possible, even if the experimentally derived NO number for the saturation value is used instead of Hüfner’s number. The formula assumes that the applied NO amount binds completely to HbNO. Reactions with other haemoglobin forms and different binding positions have to be quantified in order not to underestimate tHb-mass.

The estimated reaction of oxyHb with nitric oxide prevents high toxic concentrations of NO in the blood leading to metHb, which is converted back to haemoglobin with a certain time constant by the enzyme cytochrome b5-reductase. Therefore for the routine tHb-mass measurement increasing the inhaled NO amount is not useful, since it only increases the metHb and the nitrate concentration in the blood.

As a consequence the use of nitric oxide instead of carbon monoxide has an additional physiological advantage. The endogenous concentration of the target haemoglobin form (HbNO) for the routine method of tHb-mass detection is extremely small. The interaction of NO with other haemoglobin forms prevents an increase of the HbNO concentration leading to a physiological range where the HbNO is relevant. Unfortunately with the laser breakdown after the first project year we were not able to measure neither the endogenous nor the increased or even saturated HbNO concentration since it was below our sensitivity limit of the chemiluminescence sensor and the Faraday-Rotation-Spectrometer (FRS). The only way is the improvement of the 15NO sensitivity of the FRS.

Considering our measurements with NEM/EDTA, the HbNO concentrations should be measured directly after drawing the blood samples. Storing the samples at -80°C lead to a decrease in HbNO concentration compared to the native samples, even when stabilizing them with NEM measurements.