Projets de recherche

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.

Code: 09E6DH

This study evaluates the threat for sports doping of a class of hormones called gonadotrophin releasing hormone (GnRH) analogs.  These are synthetic, small peptide superactive analogs of the natural hypothalamic hormone GnRH, a decapeptide which is a major regulator of the reproductive system. GnRH stimulates pituitary LH and testicular testosterone (T) secretion but exogenous use of both LH and T are banned in sports. Consequently, GnRH and its analogs are also prohibited in sports for their potential to act as doping and/or masking agents. 
Used as intended for prolonged periods, GnRH analogs suppress gonadal function in hormone dependent diseases like breast or prostate cancers. However, when used for short periods they stimulate gonadal function, creating a so-called “flare” reaction, before suppression sets in. This “flare” reaction could be used by athletes for indirect androgen doping by stimulating the body’s own LH and testosterone secretion to unnaturally high levels. So far, although GnRH analogs are banned and their use by athletes is suspected, there has been no detailed evaluation of how GnRH analogs affect sports doping tests, when to suspect their use and how to detect them in urine specimens. 
This study will examine in detail the hormonal effects of a superactive GnRH analog (leuprolide) on serum and urine LH and T when used for short periods, and when repeated with a drug-free interval and well as with an androgen-suppressed gonadal axis. Our preliminary evidence proves major hormonal effects are produced so a detection test for this GnRH analog is required. We will develop suitable LC/MS/MS methodology, apply it to the clinical study and to a set of urine samples from athletes with high-normal urine testosterone but normal T/E ratio where the suspicion of GnRH analog use might be highest. 

Main Findings:

Non-steroidal drugs that increase endogenous testosterone may be used to exploit ergogenic effects of androgens in power sports. While superactive GnRH analog use is suspected, neither screening nor detection tests are developed.  
Objective: To determine if (a) stimulation for 5 days by leuprolide of serum and urine steroids and urine LH is reproducible at a 2 week interval, (b) nandrolone decanoate (ND) co-administration masks responses to leuprolide administration, (c) performance of urine measurement of leuprolide and M1, its major metabolite, as a detection test.  
Healthy men randomized into a 4 week parallel group, open label clinical study. Leuprolide (1mg) was injected sc daily for 4 days in 1st & 3rd week with hormone-free 2nd & 4th weeks. In the 3rd week, men were randomized to either N decanoate injections or no extra treatment. Serum and urine steroids and urine leuprolide and M1 and LH.  
Results: Leuprolide stimulated striking, reproducible increases in serum and urine LH and steroids (serum T, DHT, 3α diol; urine T, E & A). ND suppressed basal serum T, E2, 3α diol, and urinary E but did not mask or change the magnitude of responses to leuprolide. Urine leuprolide and M1 measurement had 100% sensitivity and specificity in detecting leuprolide administration up to one day after cessation of injections with the detection window between 1 to 3 days after last dose. Screening using urine steroid and LH measurements, optimally by urinary log10(LH x T), correctly classified 82% of urine samples.  
Conclusions: Leuprolide stimulation of endogenous testosterone is reproducible after a 10 day interval, is not masked by ND and is reliably detected by urine leuprolide or M1 measurement for up to 1 to 3 days after administration. 
Publications 
Detection and effects on serum and urine steroid and LH of repeated GnRH analog (leuprolide) stimulation. Handelsman DJ, Idan A, Grainger J, Goebel C, Turner L, Conway AJ. J Steroid Biochem Mol Biol. 2014 May; 141:113-20. doi: 10.1016/j.jsbmb.2014.01.011. Epub 2014 Feb 2.

Code: 09A15PV

Gas chromatography mass spectrometry (GC-MS) is a technique routinely used for screening of doping control samples on the misuse of anabolic steroids. The current project would use a novel type of instrument, namely a triple quadrupole tandem mass spectrometer(GCQqQ-MSn) to develop a method which is faster and is capable of detecting lower concentrations of prohibited anabolic steroids. This would allow for longer detection times of steroid misuse and higher sample throughput (i.e. faster reporting times). 

Main Findings: 

The use of performance enhancing drugs in sports is prohibited. For the detection of misuse of such substances gas chromatography or liquid chromatography coupled to mass spectrometry are the most frequently used detection techniques. In this work the development and validation of a fast gas chromatography tandem mass spectrometric method for the detection of a wide range of doping agents was developed and validated.  
The method is capable to detect quantitatively 13 endogenous steroids (the steroid profile), 19-norandrosterone, salbutamol and 11-nor-9-tetrahydrocannabinol.9carboxylic acid in the applicable ranges and to detect qualitatively over 140 substances in accordance with the minimum required performance levels of the World Anti-Doping Agency in 1 ml of urine. The classes of substances included in the method are anabolic steroids, β2-agonists, stimulants, narcotics, hormone antagonists and modulators and beta-blockers. At these levels the identification according to WADA’s criteria for identification using chromatography and mass spectrometry was achieved. 
 Moreover, by using a short capillary column and hydrogen as a carrier gas the run time of the method is less than 8 min. This means the method is –in general- three times faster than most methods used routinely for the analysis of doping control samples. Hence, the method allows for a high throughput allowing faster reporting times and reduced instrument costs. 
Hence, this method is capable of detecting and confirming a wide range of doping substances at very low concentration in a short time. Such improvements increase the efficiency in anti-doping laboratories and allow for faster reporting. Additionally, the use of multiple internal standards allows for the evaluation of the quality of every single step in the analytical methodology. 

Code: 09D8TM

Nicotine is widely reported to increase alertness, improve coordination and enhance cognitive performance, however, only one study has to our knowledge attempted to replicate these findings to exercise capacity. We previously observed an improved exercise duration by 17% with transdermal nicotine, and in the absence of any effect on peripheral markers concluded that nicotine prolongs endurance by a central mechanism. This finding, coupled with increased anecdotal evidence of nicotine ‘experimentation’ amongst competitive cyclists and the fact that as yet it is neither a banned substance nor is its access restricted, raises an ethical dilemma over athletes gaining an unfair advantage and/or serious concerns over the safety of its use during competitive exercise/sport. We are currently finishing a follow-up study to determine whether nicotine administration (patch, gum vs. placebo) can improve ~1h time-trial performance in trained cyclists. The present project proposes to analyze frozen human plasma samples this study. Samples at rest, prior to and following exercise will be analysed for nicotine and cotinine, its’ major metabolite, using reverse-phase HPLC. Results will shed light onto concentrations of nicotine and its major metabolite when taken in the quantities that are commonly available ‘over-the-counter’ in endurance-trained cyclists competing in a simulated event 

Main Findings: 

Nicotine is widely reported to increase alertness, improve coordination and enhance cognitive performance; however, only one study has attempted to replicate these findings to exercise capacity, observing an improved exercise duration by 17% with transdermal nicotine administration. This finding, coupled with increased anecdotal evidence of ‘experimentation’ amongst athletes and the fact that as yet it is neither a banned substance nor is its access restricted, raises an ethical dilemma over athletes gaining an unfair advantage and serious concerns over the safety of its use during competitive sport and exercise.  
Therefore, we conducted a follow-up study to determine whether nicotine administration can improve a 1h time-trial performance in trained cyclists, a more face-valid protocol. We recruited 10 competitive male cyclists who attended the laboratory on three occasions one week apart in a randomized order. A 48-hr period of dietary and exercise control preceded each visit, which comprised a work-dependent time-trial on a cycle ergometer to simulate 40km or about 1 hr. On one occasion they received a nicotine patch (PAT, 7 mg·24hr-1) the evening before, on another nicotine gum (GUM, 2 mg) 30 mins prior to exercise, and finally placebo gum and patch (PLA). Venous blood samples were taken at rest, prior to and following exercise and analysed for nicotine and its major metabolite, cotinine, in plasma using high-performance liquid chromatography (HPLC).  
GUM (-0.6 ± 4.4%) and PAT (-1.0 ± 4.8%) resulted in no significant improvement in performance time compared to PLA (62.9 ± 4.1, 62.6 ± 4.5 and 63.3 ± 4.1 min, respectively), with mean power outputs of 264 ± 31 (GUM), 265 ± 32 (PAT) and 263 ± 33 W (PLA), respectively. None of the variables measured (core body temperature, heart rate, ratings of perceived exertion) were different between trials. Despite concerted efforts we were unable to recover sufficient nicotine from plasma samples, most likely due to its tendency to fluctuate, relatively short half-life of about 2h, or perhaps sensitivity of the HPLC (cf. mass spectrometry). Recovery of cotinine - nicotine’s major metabolite (70%) and with a longer retention time of 18-20h - was 100% and lends itself better to accurately and sensitively assessing blood concentrations following nicotine administration. For the baseline samples, traces of cotinine were found in 2 subjects during PLA, 2 subjects during GUM, and unsurprisingly all 10 when they had been wearing PAT overnight. During PLA, negligible concentrations were found at baseline (0.3 ± 0.3 ng/ml), pre-exercise (0.5 ± 0.2 ng/ml) and post-exercise (1.0 ± 0.6 ng/ml). During GUM, negligible concentrations were found at baseline (0.3 ± 0.3 ng/ml), pre-exercise (1.3 ± 0.5 ng/ml) rising significantly post-exercise (3.2 ± 0.8 ng/ml). During PAT, concentrations were significantly elevated compared to GUM and PLA at baseline (34.2 ± 0.7 ng/ml), pre-exercise (38.3 ± 6.3 ng/ml) and post-exercise (43.5 ± 6.3 ng/ml) but did not change over time. 
On the basis of our results, i) it appears as though route and/or duration of administration affect absorption, ii) mass spectrometry is likely preferable over HPLC, where available, for sample analysis, and iii) we recommend future studies to incorporate urine analysis in addition to blood, and dose-dependent effects to be investigated.  

Code: 09C1JB

The primary problems associated with routine detection of illegal doping with recombinant human growth hormone (rhGH) have been the identical amino acid sequences of natural and rhGH and the extremely low levels of hGH in urine (0.1% to 1% of serum levels). 
Operational Technologies Corp. (OpTech) proposes to develop DNA aptamer-based assays capable of detecting and discriminating:  1) natural hGH,
2) 20kD, 22kD, and heavier glycosylated hGH isoforms, and
3) E. coli-modified rhGH “markers” which exist as ~ 2% “contaminants” of the total hGH in recombinant hormone preparations (Hepner et al., 2005). The development of aptamer reagents for detection and discrimination of hGH and rhGH or its E. coli-modified forms is novel and carries certain advantages vs. traditional immunoassays including potentially improved sensitivity and specificity, reduced cost (due to obviating of animal hosts and expensive antibody production and purification procedures), and an assured supply of identical reagent from lot-to-lot once the aptamer DNA sequences are identified. 
In addition to the potential advantages of DNA aptamers over antibodies, OpTech intends to couple its aptamers to tosyl-magnetic beads (MBs) to concentrate the hGH-associated analytes in serum or urine and use either quantum dots (QDs) or electrochemiluminescence (ECL) to produce assays with lower than femtogram detection limits. The combination of aptamers, MBs, and QDs or ECL may produce assays capable of detecting the ~ 2% modified marker forms of rhGH in urine to eliminate invasive blood draws and facilitate frequent testing of urine. If successful, the assays may even be performed on-site at sporting events with a handheld fluorometer such as the PicofluorTM or a portable ECL sensor. The preferred embodiment will use OpTech’s one step (homogeneous) aptamer plastic-adherent cuvette assay and handheld reader as shown on OpTech’s spin out biotech company’s website (www.pronucleotein.com) and recently published (Bruno JG, et al. J. Fluorescence 2008). 

Main Findings: 

Operational Technologies Corporation has conducted a successful pilot study for WADA in which 8 aptamers were identified that appear to distinguish recombinant from natural hGH. 
The rhGH used in these studies is not a pharmaceutical grade, but served to provide proof-of-concept suggesting that pharmaceutical targets such as Genotropin® could be detected by an aptamer-based ELISA-like plate assay. The aptamer assay may also be embodied in various formats including presumptive lateral flow test strips or dipsticks, fluorescence, chemiluminescence or ECL or other types of assays.

Code: T09E2WS

According to the prohibited list 2010, glycerol is specifically named as masking agent. Oral and intravenous application of glycerol is prohibited. Therefore it is necessary to establish threshold values for urinary glycerol concentrations because glycerol may also be of endogenous origin or may be taken up from other exogenous sources.
In sports glycerol is typically used in high doses in combination with excess fluid in order to increase total body water. However, glycerol plasma concentrations can also be elevated during fasting and endurance exercise.
Furthermore, there are natural sources of glycerol such as foodstuff (e.g. wine) and over-the-counter drugs. The urinary excretion of glycerol following administration has not been studied systematically at rest and during exercise.  Also, little is known about urinary glycerol following prolonged fasting, endurance exercise or the intake of glycerol-rich foods or drugs.
A quantification method for urinary glycerol has been published recently but urinary threshold values for the detection of glycerol misuse are lacking.
For the development of urinary threshold levels we intend to perform a) application studies of glycerol at rest and during exercise, b) to assess urinary glycerol excretion due to fasting and exercise and c) to determine reference values for urinary glycerol concentrations in normal doping control samples. Additionally, an IRMS method for glycerol will be implemented.

Main Findings: 

Glycerol is prohibited by the World Anti-Doping Agency (WADA) as masking agent since 2010. Exogenously administered glycerol is excreted in the urine so that the detection of the misuse of glycerol is possible in theory. However, little is known about the urinary excretion pattern of glycerol. In addition, glycerol may also occur endogenously during increased lipolysis and it can also be ingested from foodstuff or drugs. In order to contribute to the development of urinary threshold levels for the misuse of glycerol, two placebo-controlled application studies (glycerol dose: 1 g/kg body weight) were performed at rest and in combination with exercise. There was a rapid increase in urinary glycerol and maximum concentrations (50000-60000 µg/mL) were observed 2.5 to 4 h after the administration. Urinary concentrations remained significantly elevated for 12-15 h (exercise) to 15-18 h (rest). Plasma volume expansion (+1.5 to 3%) and the reduction in hemoglobin (-0.2 to -0.3 g/dL) and haematocrit (-1%) after glycerol administration were rather small when compared to the administration of placebo and statistically significant only after 2.5 h. 
After 18 h of food deprivation followed by 90 minutes of endurance exercise, highly elevated plasma glycerol concentrations were observed but urinary glycerol concentrations were only slightly increased (maximum concentration: 50.5 µg/mL). In 516 randomly selected routine doping control samples, it was confirmed that urinary concentrations are slightly higher in in-competition than in out-of-competition samples, but in all samples urinary glycerol concentrations remained below 200 µg/mL.
Further, a suitable IRMS-method was developed to measure δ13C-values of glycerol in urine. Directly after glycerol administration, δ13C-values were similar to the isotope ratio of the administered glycerol.
In conclusion, the urinary threshold of 200 µg/mL as suggested by Thevis et al. (2008) can be used to identify athletes, who have misused glycerol in relevant amounts. As shown in the study, this threshold is sufficiently high to minimize the risk of false-positive results due to exercise- or fasting induced lipolysis and unintentional intake from other sources.

Code: 09A19WS

As per list of the World Anti-Doping Agency (WADA) 2009 Boldenone and Boldione are explicitly listed in group S1 “anabolic androgenic steroids”, and are therefore prohibited in sports, while Androsta-1,4,6-triene-3,17-dione is classified as aromatase inhibitor (class S4. Hormone Antagonists and Modulators, particularised class S4.1. Aromatase Inhibitors). All these three substances are reported to be excreted as Boldenone and/or Boldenone metabolite in the urine. As class S1 substances are considered as “Non-Specified Substances” while class S4.1. substances are judged as “Specified Substances” the assignment to the administered substances is particularly important for the valuation of the adverse analytical finding. Thus, the project aims to investigate the urinary metabolism and pharmacokinetics using mass spectrometric analyses. Isotope ratio mass spectrometry (GC-C-IRMS) will be applied to identify origin of Testosterone and other endogenously occuring steroids. Based on the analytical data criteria to trace the administered substance have to be established. 

Main Findings: 

As per list of the World anti-soping agency (WADA) 2009 Boldenone and Boldione are explicitly listed in group S1 "anabolic androgenic steroids", and are therefore prohibited in sports, while Androsta-1,4,6-treine-3,17-dione is classified as aromatase inhibitor (class S4. Hormone antagonits and modulators, particularised class S4.1 aromatase inhibitors). All these three substance are reported to be excreted as Boldenone and/or Boldenone metabolite in the urine. As class S1 substances are considered as  " non-specified substances" while class S4.1. substances are judged as " specified substances" the assignment to the administered substances is particulary important for the valuation of the adverse analytical finding. Thus, the project aims to investigate the urinary metabolism and pharmacokinetics using mass spectrometric analyses. Isotope ratio mass spectrometry (GTC-C-IRMS) will be applied to identify origin of Testosterone and other endogenously occuring steroids. Based on the analytical data criteria to trace the administered substance have to be established. 

Code: 09E7FP 

Platelets are an intriguing autologous source of growth factors (GFs) which have been demonstrated to be able to modulate the recruitment, duplication, activation and differentiation of cells involved in bone- and soft-tissue healing. Doping related issues are still matter of debate when considering this therapeutic approach for the treatment of sport-related injuries in particular because of the IGF-1 content in the platelets alpha granules. Certainly, the use of PRP for the treatment of bone, tendon, cartilage and ligament injuries cannot be considered as a technique able of enhancing the physical performances but its muscle injection is still a matter of debate. Moreover, some authors suggested the hypothesis that the use of PRP for ameliorating muscle healing may lead to late muscle fibrosis. 
The aim of the present study is to analyse, in a murine model, the effect of the different techniques used for the preparation of platelets derived GFs [i.e. Platelet Rich Plasma (PRP) and Platelet Rich Fibrin (PRF)] on muscle repair processes. Muscle injuries, induced on animals, will be treated with the 2 different techniques. Morphological and morphometrical analysis as well as the evaluation of myonucleous with electron mycroscopy will be carried out on repaired muscles. The evaluation, both quantitative and qualitative, of the satellite cells will be performed with the immunohistochemistry technique combined with confocal microscopy. Functional evaluation will be performed with the grafting test. All of the analysis will be carried out with both internal and external controls represented by untreated muscles and muscles treated with recombinant growth factors as well as treated with adeno-associated-virus(AAV) mediated –VEGF gene transfer which has been demonstrated to be able to stimulate angiogenesis and repair processes when trasnfected into skeletal muscles. Long term follow up will be able to highlight eventual muscle fibrosis. 

Main Findings: 

Background Ample evidences suggest that growth factors (GFs) may play a key role in the healing process, especially in the early stages of the inflammatory phase. Despite the reported clinical successes, there is still a lack of knowledge when considering the biological mechanism at the basis of platelet-rich plasma (PRP) activity during the muscle healing process. The aim of the present study was to analyze the early effects of PRP in an easily reproducible animal model.  Materials and methods 102 Wistar male adult rats were used in the present study. The muscle lesion was performed by scalpel on the flexor sublimis muscles. PRP was administered immediately after surgery. Treated, untreated and contralateral muscles were tested by morphological analysis, immunohistochemistry, RT-PCR analysis and Western Blot assay.  
Results In the PRP treated muscles, the leukocyte infiltration was significantly higher when compared to both untreated and contralateral samples. The latter showed a higher leukocyte infiltration when compared to the untreated muscles. PRP treatment also modified the cellular composition of the leukocyte infiltration leading to an increased expression of the CD3, CD8, CD19 and CD68 and to a decreased CD4 antigen expression in both PRP treated and contralateral muscles. The analysis of the blood vessel density and the blood vessel diameters showed no statistically significant differences when comparing the three groups analyzed. At day 2 and 5 after PRP administration there was a significant expression of Pax7 and MyoD1. The analysis of pro- and anti-inflammatory cytokines expression showed that PRP induced a more pronounced and/or early inflammatory response by expression of IL-1β and TGF-1β.  
Discussion The results of the present study showed that PRP treatment increased the physiological early inflammatory response further to a muscle injury with a parallel modification of the pattern of cellular recruitment. Moreover, the local PRP treatment may exert, directly or, more plausibly, indirectly, a systemic effect when healing processes were concerned, at least limited to the very first inflammatory phase. The results of the present study might suggest the hypothesis that PRP promoted an early inflammatory response together with a more efficient production of pre-myogenic progenitor population, satellite cell and myoblasts activation/proliferation during muscle regeneration. Conversely, the terminal differentiation markers (i.e., myogenin, Mrf4) as well as others pro-inflammatory cytokines (i.e., IL-6, IL-10, TNFα) and VEGF-A were not additionally modulated by PRP treatment. Further experimental studies are needed to fully understand the local and systemic mechanism of action before apply PRP in routine clinical practice as well as in order to deeply understand possible systemic effects on muscle performance.