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Code: ISF16E11FP 

The variable effects of anabolic androgenic steroids (AAS) on increasing skeletal muscle mass and strength has been well documented, as is the misuse of AAS in sport. Recent evidence suggest either a long- or short-term exposure to AAS might have a sustained effect on muscle morphological changes, for example, with increased muscle mass, capillary per fibre, muscle fibre size and myonuclei density, leading to improved performance. A positive correlation between the number of myonuclei and training response
following exposure to AAS in a mouse model seems to suggest a link between the formation of extra myonuclei and the extent of “muscle memory”; an idea that requires further investigation in humans.

Strength training can also increase the number of nuclei in muscle fibres. Adaptations in muscle mass by strength training are significantly enhanced in previously trained individuals despite a prolonged detraining period. Given the persistence of muscle nuclei, the use of AAS combined with training will have a greater impact on muscle hypertrophy than either training or steroid use alone. To detect the long-term effect of AAS and training (even after drugs are no longer detectable in the human system), abnormal changes in
skeletal muscle morphology illustrated by specific gene markers in response to the stimuli exist and will allow these molecular signals to be picked up by modern gene screening methods. In the proposed project, gene expression profiling of skeletal muscle in response to AAS exposure will be carried out using total RNA-seq. The molecular, histological and training response markers will be integrated for the detection of short- and long-term effects of AAS and will be incorporated into the steroid module of the Athlete Biological
Passport for improved validity and reliability.
 

Main Findings:

Fat Free Mass (FFM) of RP2-5, who ceased AAS usage ≤2 weeks prior to visit one with 19-28 weeks between visits, decreased by 3.9-4.7 kg. FFM of RP1, who ceased AAS usage 34 weeks prior to visit one with 28 weeks between visits, decreased by 0.9 kg. Fibre CSA decreased for RP1 and RP2 between visits (7566 vs 6629 µm²; 7854 vs 5677 µm²) whilst myonuclei per fibre remained similar (3.5 vs 3.4; 2.5 vs 2.6). Fibre CSA (7167 vs 7889 µm²) and myonuclei per fibre (2.6 vs 3.3) increased for RP3 between visits. Mean fibre CSA was significantly higher in RT-AS (n=17) (8160 ± 1769 µm²) compared to C (n=5) (6477 ± 1271 µm², p=0.028). There were no significant differences between C, RT (n=15), RT-AS & PREV (n=6) for myonuclei per fibre. Myonuclei per fibre and CSA for all biopsied participants (n=43) was significantly correlated (r=0.8, p<0.001). 
All whole blood samples (n=60) were subjected to RNA-Seq as they had purified total RNA that was of sufficient concentration, purity, and integrity. In comparison with RT participants who ceased AAS exposure <1 week ago (n=10), 1-2 weeks ago (n=5), 10-50 weeks ago (n=4), and >52 weeks ago (n=7), had 612, 464, 173 and 188 genes differentially expressed, respectively. RP2-5 had 33 differentially expressed genes between visits which were mainly associated with the interferon signalling pathway linked to the immune system. RNA-Seq of corresponding muscle samples is currently underway. The finding of comparable myonuclei per fibre numbers despite decrements in fibre CSA post AAS usage is consistent with the “muscle memory” mechanism. RNA-Seq identified 33 genes that could provide novel beneficial biomarkers. 
Further research, particularly the longitudinal monitoring of AAS users post usage is required to confirm these intriguing findings.

Code: 16A11AC 

Urine, as a biological waste material, is an extremely complex fluid counting thousands of components belonging to more than 200 different chemical classes. It is conventionally adopted for investigating the endogenous steroids profile in athletes to prevent doping. Isotopic Ratio Mass Spectrometry (IRMS) is the technique able to distinguish the source of the steroid via carbon isotopic measurements (13C/12C), differentiating the exogenous from the endogenous ones. Reliable IRMS determinations strongly depend on adequate purity of the investigated steroids. This demand is guaranteed by labor-intensive and time consuming preliminary steps (i.e. sample preparation, derivatization, liquid chromatography fractionation).
Multidimensional gas chromatography (MDGC) is a consolidated technique for the separation of complex matrices as well as the investigation of target compounds. In such approach, two columns are arranged in a series (e.g. non-polar stationary phase in the first dimension, followed by a polar stationary phase in the second dimension). The principle is to select the peak of interest in the first dimension and send it (heart-cut) into the second - ideally orthogonal - dimension for further separation.

Main Findings: 

Distinguishing endogenous anabolic steroids from their synthetic copies in urine samples from athletes requires a specific analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS ,IRMS). Such analysis is mandatory to be implemented in a doping control laboratory, since many of the adverse analytical findings involved endogenous steroids whose administration cannot be characterized by other techniques.

In order to obtain suitable and consistent results by IRMS, a laborious and time-consuming sample treatment is required due to both the natural presence of the target analytes and the high comlexity of the matrix, ensuring the convenient purification of the steroids prior to the analysis. Many steps are involved, including one or more preparative high performance liquid chromatography (HPLC) to isolate the steroids from matrix interference. Besides a long time, preparative HPLC necessitates many sample transfer steps, which represents potential losses of the amonut of steroids to be analyzed and possible contribution to the measurement uncertainties.

In order to accelerate the sample preparation and increase the automation of the process, the use of multidimensional gas chromatography (MDGC) prior to IRMS experiments has been investigated as an alternative to preparative HPLC. After preliminary studies already published, a full validation was perfomred in two doping control laboratories accredited by the World Anti-Doping Agency (WADA) to conclude about the advantages in replacing preparative HPLC with MDGC in the routine confirmatory analysis. A well-established instrumental configuration based on two independent GC ovens and one heart-cutting device was used. The first dimension (1D) separation was obtained by a non-polar colum which assured high efficiency and good loading capacity, while the second dimension (2D), based on a mid-polar stationary phase, provided good selectivity. The assembled MDGC set-up was applied for measuring testosterone, 5α- and 5β-androstanediol, androsterone and etiochlanolone as target compounds, and pregnanediol, 11-ketoetiocholanolone and 16-androstenol as endogenous reference compounds. One additional solid phase extraction was included at the end of the conventional sample treatment already implemented in the two laboratories, at which two fractions were obtained to be analyzed, minimizing matrix effects and column overloads. Following WADA regulations, the experiments comprised linearity of the instrument, repeatibility, linear mixing models, method performance assessment and limit quanitification.

Code: 16A19MP

In the fight against doping the laboratories are confronted with an increasing number of substances to screen on. Thus, a comprehensive screening for different classes of substances using dilute-and-inject methods in anti-doping screening is desirable. As lots of xenobiotics are excreted as conjugates a detection of the intact conjugates is performed by this approach. While chemical synthesis of phase-II metabolites works efficiently for compounds 
having only one potential conjugation site, several analogous compounds could not be chemically synthesized effectively, due to their more complex chemical structure. For the synthesis of the phase-II metabolites (glucuronides and sulfates) of these compounds a biotechnological production will be implemented. Fission yeast strains, that enable the biotechnological production of glucuronides and sulfates that cannot be synthesised efficiently via classical chemical synthesis will be generated and used to produce the relevant human conjugates. The produced reference material can be used for method set-up for direct detection. If laboratories still rely on hydrolysis of the conjugates, these reference compounds may
serve as control for hydrolysis efficiency and quality assurance.

As proof of concept the use of the generated fission yeast strains will be demonstrated by generation of salbutamol-sulfate, salbutamol-glucuronide, fenoterol-sulfate and 4-hydroxy-DHEA-sulfate within the project.

Main Findings: 

A complete set of recombinant fission yeast strains each expressing one of the human sulfotransferases hSULT1A1, hSULT1A2, hSULT1A3, hSULT1B1, hSULT1C2, hSULT1C3a, hSULT1C3d, hSULT1C4, hSULT1E1, SULT2A1, hSULT2B1a, hSULT2B1b, SULT4A1, or hSULT6B1, respectively, was successfully generated. For each hSULT two strains were generated, one integrating the sequence in the leu1 gene of the cells, and an additional with a second expression unit in the pREP1-plasmid. Conjugation efficiency of the strains for the sulfonation of one test substrate (known from literature for the respective isoforms) for each SULT isoenzymes was successfully demonstrated. Ten out of twelve enzymes for which substrates are known were already shown to produce the respective sulfoconjugates. Analysis of the two remaining strains is in progress. These results prove that the intracellular production of the cofactor PAPS necessary for the SULT activity in fission yeast is sufficiently high to support metabolite production by whole-cell biotransformation. We also developed a new and convenient SULT activity assay based on the sulfonation of a proluciferin compound and established enzyme bag assays for SULTs. Taken together, we have developed the technology to systematically generate conjugates relevant for doping control by human SULTs.

Code: 16A18IA

The direct detection of phase II metabolites of doping substances suitable for publication on WADA's website and steroids using LC-ESI-MS/(MS) in particular, is a current trend in doping control, offering simplicity, sensitivity and time effectiveness. This approach enables the detection of a great range of phase II metabolites, as the conjugation with glucuronic and/or sulfuric acids clearly improves the ionization efficiency of steroids. Recently, the detection of abundant and/or long term sulfate metabolites has been reported for exogenous AAS using LC-ESI-MS/(MS). However, metabolite characterizations and/or confirmation procedures of sulfate metabolites rely mainly on GC-MS/(MS) methods. This is due to the fact that their analysis in positive ionization mode lacks sensitivity, whereas in negative ionization mode, Collision Induced Dissociation (CID) experiments show dominant product ions that are limited to sulfate moiety and lack structural information. Typically, steroid metabolites have additional functional groups to conjugation site, usually hydroxyls and keto groups, that are prone to derivatization. Chemical derivatization can enhance the ionization efficiency of Phase II metabolites in positive ionization mode, whereas CID product ion spectra are not limited to ions related to conjugated group, alter their fragmentation behavior and hence they can be used as a robust alternative confirmation procedure. This is a novel approach that may facilitate the confirmation of sulfate metabolites, since the laborious deconjugation step will be skipped.  The simultaneous confirmation of intact sulfate and glucuronide metabolites will be examined in specific cases as the proposed methodology permits the confirmation of both sulfo- and gluco-conjugated metabolites possessing specific structural elements like keto groups with a single run. The herein proposed methodology may also used in the future as a complementary tool for the structural elucidation of newly found Phase II metabolites by LC-ESI-MS/(MS). 

Code: 16A01MP

The identification of anabolic androgenic steroids (AAS) is a vital issue in doping control. Due to the performance enhancing properties of AAS, the World Anti-Doping Association (WADA) banned their use but according to the annual report of WADA, steroids are still very popular amongst athletes and are responsible for half of all adverse analytical findings. The search for metabolites with longer detection times remains an important task and the introduction of new long term metabolites for exogenous AAS such as for example stanozolol, methanedione and dehydrochloromethyltestosterone, led to a 4 - 80-fold increase of adverse analytical findings due to the prolonged detection time. 
This project aims at finding new long term metabolites for a number of AAS by application of our newly developed gas chromatography chemical ionization triple quadrupole mass spectrometry (GC-CI-MS/MS) protocol for metabolite detection and identification. Chemical ionization in combination with triple quadruple technology has proven to significantly increase the sensitivity for a wide range of compounds in comparison with electron impact (EI). In addition, GC-CI-MS/MS is characterized by AAS structure correlated fragmentation pathways. The combination of both factors allows the set up of a sensitive MRM method, designed to find previously unknown but expected metabolites by selection of theoretical transitions for expected metabolites. 

Main Findings:

In 2015, a new GC triple quadrupole MS method that used chemical ionization (CI), instead of EI was introduced. This new GC-CI-MS/MS method opened new possibilities in the search for new metabolites as CI is a soft ionization. The correlations between fragmentation behavior and AAS structure could be revealed and fragmentation pathwasy have been postulated. This enabled the search for previously unknown but expected metabolites by selection of their predicted transitions. The aim of the current project was to set up an efficient approach for searching new metabolites by application of these newly dscivered structure depended fragmentation pathways and to find new long term metabolites. The following AAS were selected: drostanolone, metenolone, mesterolone, oxymesterone, formebolone and methyltestosterone.

Novel long-term metabolites for oxymesterone and mesterolone were detected and characterized. This demonstrates that GC-CI-MC/MC is capable of detecting (and characterizing) metabolites that can be missed with other (more frequently) used techniques. For oxymesterone, the metabolite was identified as 18-nor-17β-hydroxymethyl-17α-methyl-4-hydroxy-androst-4,13-dien-3-one. It is primarily excreted as a glucuronide. For mesteroloone, the metabolite was identified as 1α-methyl-5α-androstan-3,6,16-triol-17-one and its sulfate form resulted in a prolonged detection time for mesterolone abuse.

For metenolone, a metabolite, primarily excreted as sulfate, was found to have a slightly improved detection window in comparison with the currently monitored metabolites. Likely, the metabolite is 1β-methyl-5α-androstan-17-one-3ζ-sulphate and for the first time it is documented to provide the longest detection time.

In general, this study illustrates that sulfated steroids are becoming increasingly important for doping control analyses as they allow longer detection times for AAS adn can provide valuable information.

Code: 16E05AB 

Since 2003, gene doping has been included in WADA’s list of banned substances and methods. Extensive research by us and other investigators has led to the development of a method for gene doping detection that directly targets a doping gene using highly-specific and sensitive PCR assays. The efforts of our laboratory led to the development of a test for erythropoietin (EPO) gene doping and its recent implementation in the Australian Sports Drug Testing Laboratory (ASDTL). Based on the successful implementation of the test at ASDTL and in response to WADA’s recommendations, we now propose to evaluate approaches to further improve the test by increasing its sensitivity, reducing its cost and streamlining the test protocol.  
The outcomes of this project will improve the EPO gene doping test. Importantly, this work will allow the first gene doping test to be integrated into the arsenal currently used in doping control and to bolster the fight against doping in sport. 

Main Findings: 

As a result of this research, we propose several modifications to the EPO gene doping test that will enhance its sensitivity and make it cheaper and easier, while maintaining reliability. These include an alternative method to identify PCR false positives and a method to concentrate the genetic material extracted from samples prior to analysis. We have extended the test from doping material in plasma to doping genes that might be associated with blood cells and have demonstrated that blood samples kept frozen at -80°C for at least three months are suitable for testing. This broadened scope increases the utility of the test for gene doping detection. We found that extracting genetic material from whole blood rather than its fractions provides operational robustness for gene doping testing. These improvements to the test will facilitate its implementation and use in doping control.

Code: ISF16D02NL

Proteins involved in erythropoiesis and iron metabolism have been demonstrated as potential biomarkers to detect blood doping. Hepcidin, a peptide hormone that is a regulator of iron homeostasis, is reported to be regulated by recombinant human erythropoietin (rhEPO) administration and by the autologous blood transfusion. It was suggested that measurement of iron in ethylenediaminetetraacetic acid (EDTA) plasma may be a costeffective marker for the screening of blood transfusion use. Increased levels of EDTA plasma iron were also detectable using a fast automated method. Thus, EDTA plasma iron may provide further evidence of blood manipulation. In 2014, erythroferrone (ERFE) was identified as a novel erythroid regulator of iron metabolism in a mouse model. These observations suggest that ERFE may be a potential biomarker in the detection of blood doping. However, the ironomics strategy to detect blood doping could be impacted by iron injection performed by athletes and be considerated as an confounding factor for iron metabolism biomarkers. In this project a clinical study will be performed to investigate the potential confounding effect of iron injection on iron metabolism biomarkers.

Main Findings:

Proteins involved in iron metabolism, such as hepcidin, are potential blood doping biomarkers. Monitoring of these markers could offer a strategy to complement the actual longitudinal follow-up of hematological parameters measurement, such as reticulocyte percentage (Ret%), to detect blood doping. Iron injections, which are frequently supplied to athletes, could affect hepcidin concentration and therefore, present a confounding factor in hematological profiling for blood doping detection. In this project, urinary iron was tested as a novel biomarker to monitor iron injection. 

A randomized, single-blind, placebo-controlled trial was conducted in male volunteers who received a single intravenous injection of ferric carboxymaltose or placebo. The effects of iron injection on iron metabolism markers and hematological were investigated. Hepcidin concentration was measured in blood by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) and the urinary excretion kinetics of iron was quantified by inductively coupled plasma mass spectrometry (ICP-MS).

Intravenous iron supplementation increased Ret% significantly, and also serum hepcidin concentration was increased 16-fold relative to baseline. Interestingly, urinary iron concentration was increased by 12-fold at 3 h after iron injection, and it remains significantly elevated until day 1 after administration. A specificity of 100% and a sensitivity of 79.3% were achieved with a proposed threshold of 251 ng/mL for urinary iron.

Due to the impact on Ret%, iron injection is a cofounding factor in evaluation of the hematological parameters for the detection of blood doping. Furthermore, urinary iron quantification could offer a novel strategy to monitor intravenous iron injection in the doping control samples.