Projets de recherche

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Code: R15M02RC

This project will assist in establishing an analytical laboratory for testing nutritional supplements to detect prohibited substances such as steroids, stimulants and other performance-enhancing drugs. The project will provide the necessary financial assistance to fund a full time instrument technician for a period of one year and a contribution towards the cost of method validation and laboratory accreditation. The funding provided by this project will supplement a substantial, ongoing (LIVE) investment by the University of Hertfordshire to acquire state-of-the-art analytical instrumentation.

Main findings

A simple and straightforward approach for determining MRPL for the detection and identification of prohibited substances in dietary supplements has been followed. A keyword search for recommended doses of prohibited substances was conducted, followed by the collection of recommended daily intake of a range of dietary supplements. These data were then used to calculate MRPL from which the limit of detection (LOD) for each prohibited substance was derived. The corresponding MRPL-derived LOD were found to be three orders of magnitude greater than those determined experimentally from the in-house, multiplex assay. From these data, it has been possible to estimate an LOD that would be anticipated as an absolute maximum for each substance intended to be used as part of our validation [9]. It also facilitates the considerations and parameters necessary for the reporting of findings from any future quantitative methods that will be developed. Currently, substances administered via routes other than oral have been included; further considerations are required to determine whether there is potential for their inclusion in solid dietary supplements before eliminating them.

Code: 15C13MZ 

According to WADA, the issue of beta-2 agonists will continue to be a focus of its research activity in order to ensure that the administration of large doses of these substances is prevented and prohibited, but the appropriate care and treatment of asthmatic athletes is facilitated. In this project we will conduct a human pharmacological study to investigate dosing and combinatory beta-2 agonist effects. Single vs. combinatory threshold doses of non-prohibited, short-acting (salbutamol) and long-acting (formoterol) beta-2 agonists will be administered by inhalation and potential additive effects will be investigated by measuring skeletal muscle metabolic and hypertrophic signaling, endurance performance (10 min cycling time trial) and cardiopulmonary function (cardiac output and VO2max). The main objective of the proposed study is to investigate dose-dependent additive/synergistic effects of short- and long-acting beta-2 agonists in terms of skeletal muscle metabolism/hypertrophy, endocrine regulation, cardiopulmonary function and endurance performance.  

Main Findings: 

Results: All medication combinations were reliably detected in the urine samples by LC-MS/MS meeting WADA standards. None of the samples collected after application of verum medication resulted in concentrations exceeding the threshold concentrations set for doping control analysis. Mean Power Output during TT was not different between the different study arms. There was a treatment effect regarding lung function observable without any influence on performance or health. There was a treatment effect on myocardial contractility measured by Echocardiographic Longitudinal Strain which increased for both strains and there was a marked effect of combined treatment. 

Microarray subsample analysis revealed no significant treatment effect on gene expression of NR4A1 or NR4A3, but an effect was observable for NR4A2 with the most significant difference between Placebo and salbutamol+formoterol. The β2-combination influenced up- and downregulation of differently expressed genes most compared to the other study arms. Muscle analysis did not show any treatment effect on NR4A protein and NR4A1/NR4A3 gene expression, whereas a whole group treatment effect was observable for NR4A2. Further pathway analysis with gene expression software TACx and linked WikiPathways revealed treatment effects in energy metabolism related genes ATF3 (e.g. Hypertrophy model; TGF-beta signaling pathway), PDK4 (e.g. Estrogen receptor pathway; nuclear receptors meta-pathway), LPL (e.g. Metabolic pathway of LDL, HDL and TG; PPAR signaling pathway), CREM (e.g. mBDNF and proBDNF regulation of GABA neurotransmission), and ATP1B3/ATPase (e.g Calcium regulation in cardiac cells).

Noradrenaline, adrenaline and TGF-β concentrations in blood were not affected by treatment or gender, whereas IGF concentrations showed a treatment effect 24h Post compared to Pre for women. CO data determined by Clearsight® device were not reliably reproduced in all measurements due to technical artefacts. 

Both β2-agonists stimulated hypertrophy in a dose dependent manner compared to negative control in C2C12 myotubes. Diameters relative to control were increased for all β2-agonists treatments, but an additive effect were clearly observed for salbutamol+formoterol compared to control or the respective β2-agonists alone.

Discussion: There is presumably no performance enhancing effect in this study design with the used doses of β2-agonists either alone (salbutamol or formoterol) or in combination (salbutamol+formoterol) compared to Placebo, whereas it was shown that in cell culture, β2-agonists may indeed have a strong hypertrophic effect and exert their effects in an additive manner that can be relevant for human in vivo pharmacologic kinetics.

An acute effect on the lung function was observable without side effects and with presumably no impact on exercise performance capacity in healthy subjects. Acute effects were observable for heart contractility but without objective impact on aerobic performance capacity or health.The impact of chronic β2-application in healthy and asthmatic subjects on TT performance of 
longer duration, which simulates real life competition even closer, has still to be determined.

Code: ISF15E10YP 

The current research on the molecular signature of rHuEPO doping has, so far, provided some evidence that “omics” technologies such as transcriptomics have the potential to significantly strengthen the current ABP approach and contribute to other traditional anti-doping tests. This approach if successful can in the future be applied to the detection of other doping substances and methods difficult to detect such a recombinant human growth hormone and blood transfusions. There is also the interesting possibility that an "omics"-based approach could help reduce the pressure on the anti-doping obligations of athletes such as the “athletes whereabouts”. In order to confirm that an integrative “omics” approach is a possible solution to improve rHuEPO detection, it is of paramount importance to precisely determine normal gene expression reference values as well as to carefully assess the potential effects of external factors on blood gene expression profiles, such as prior training, altitude including different hypoxic “dose” and protocols, sport discipline, level of competition, gender, ethnicity and age. The investigations proposed are necessary before including the promising blood gene biomarkers in the ABP and/or the development of a stand alone test to reveal doping or identify suspicious samples for targeting purposes.

Code: 15A31JP 

A project (acronym GOpep) was approved by WADA with the objective of detecting a Neu5Gc containing EPO O-glycopeptide using latest generation MS instruments (i.e. AB Sciex Qtrap 6500).

The EPO O-glycopeptide shows the lowest glycan heterogeneity, thus being the best choice to reach the necessary MS sensitivity. Results obtained showed that the glycopeptide isoform containing Neu5AcNeu5Gc was found to be the most abundant form with its triply charge species at m/z 810.3 giving the best signal. A limit of detection of around 2 IU EPO/L, from a standard preparation was achieved, compatible with the expected concentrations in human urine. An antibody against the peptide was also developed and initial results show that it also recognizes the glycopeptide. However, matrix effect when spiking real samples at very low concentrations,
as well instrumental conditions to speed-up the analysis are still to be solved.
The hypothesis of this project is that MS sensitivity has reached a status in which EPO glycopeptides, particularly O-glycopeptides as they present lower heterogeneity, are detectable in urine or blood samples. Using a proper combination of specific peptide immunopurification with other desalting techniques, matrix effects can be avoided and the required sensitivity for the EPO O-glycopeptide containing the non-human tag (Neu5Gc) reached.

Objectives:
1.- To improve sample clean-up by using the already developed polyclonal antibody against the peptide backbone and other desalting techniques.
2.-. To improve the nanoLC-MS/MS set-up using monolithic columns for high throughput and on-line sample clean-up.
3.- To develop monoclonal antibody for future use of the methodology if the use of the polyclonal proves successful.
4.- To validate the procedure in urine and serum samples

Main Findings:

The main objective of the project was to develop an MS-based analytical procedure for the detection of an EPO O-glycopeptide containing the non-human monosaccharide N-glycolyl-neuraminic acid (Neu5Gc) as an unambiguous proof of the exogenous origin of the hormone (i.e. rEPO or analogues). The trypsin released EPO O-glycopeptide T13: (E117-R131) shows the lowest glycan heterogeneity, thus maximizing signal sensitivity while the peptide backbone will make it unique for EPO.
Through the method development, it was found that the formation of ubiquitous ammonium adducts was unavoidable, even under conditions where no ammonium salts were used.

The ammonium adducts of the doubly and triply charged species of the “endogenous” species (T13 O-2Neu5Ac) have masses identical (within 0.5 m/z) to the non-adduct forms of the “exogenous” species (T13 O-1Neu5Ac-1Neu5Gc) containing a 13C atom. The potential risk of having false positives forced the development of a method in which there was a complete chromatographic separation between these two very similar species.

The overall results demonstrated that the “exogenous” glycopeptide (T13 O-1Neu5Ac-1Neu5Gc) could be detected in rEPO using both a QTRAP6500 (low resolution) and an Orbitrap Fusion Lumos (high resolution). This approach includes the development of an MRM and PRM method respectively and the enrichment of the target EPO T13 O-1Neu5Ac1Neu1Gc using the anti-EPO T13 antibodies.

The method developed in this project is relatively straightforward, however the LOD using the most sensitive instrument in the market is still far from its applicability for real biological samples.

Still, as opposed to SAR-PAGE method, the proposed strategy may have the potential to unequivocally identify rEPO by detecting the 1-2% of T13 O-1NeuAc1NeuGc present in the sample using the signal of T13 O2Neu5Ac, which is present in all forms of EPO (exogenous and endogenous), as its own internal standard and quality control. The peptide chosen (T13) is unique for EPO, so it might allow the development of specific immunopurification techniques.

Code: ISF15D14ZG

The ability to increase oxygen carrying capacity to exercising skeletal muscles is a highly effective method for improving athletic performance. Unfortunately, some athletes seeking to gain an edge over their competition, have turned to artificially enhanced performance gains through blood transfusions, despite these methods being banned by the World Anti-Doping Agency. While methods such as flow cytometry can reveal heterogeneity in red blood cell (RBC) surface antigens and thereby detect homologous transfusions – or blood doping from a different person, there is currently no method available to detect autologous blood transfusions (ABT) with an athletes own blood. The lack of a direct detection method represents a significant problem for endurance sports, and the absence of a test means that this performance enhancing method is still widely utilized. There is evidence that biochemical changes occur in RBCs stored ex-vivo, including changes in their cell membrane that do not occur in a normal RBC population. One major obstacle to the development of a specific and reliable method for detecting ABT is then the lack of an available technique for detecting these age-related changes in circulation and at low concentration.  
The goal of this proposal is to develop the ability to quantify these modifications in red blood cell storage age using a combination of dielectrophoretic spectroscopy and a storage sensitive membrane cross-linking reaction, and to employ this approach to develop a simple and specific test for the detection of autologous blood transfusions in endurance athletes. The successful outcome of this project will lead to the development of an entirely new electrical approach to monitoring an athlete’s blood sample, and will lead to a new ABT indicator that is simple, rapid, require only a small droplet of blood, and capable of being integrated into an athlete's Biological Passport. 

Main Findings:

Background. The ability to increase oxygen carrying capacity to exercising skeletal muscles is an effective method for improving athletic performance. Unfortunately, some athletes have turned to artificially enhanced performance gains using blood transfusions, despite these methods being banned by the World Anti-Doping Agency (WADA). One main limitation in detecting autologous blood transfusions (ABT) is that there is no direct method capable of performing specific detection across a large transfusion regime, including detecting re-infusion with a small volume of blood under conditions when re-infusion has occurred multiple weeks prior to a competition. The significance of this project is based on this effort to develop a new method to overcome this problem using a combination of electrokinetics and microfluidics.

Results. We used electrodes to measure the electrical behavior of RBCs. To perform our experiments, RBCs were collected from healthy human volunteers and stored in storage buffer. We discovered that the electrical properties of RBCs change when cells are stored. We also developed an ABT assay that can quantify differences in mechanical elasticity of RBCs based on the ability for RBCs to deform in a microfluidic channel.

Conclusions. We believe that these two ABT assays complement each other and speculate that the microfluidic deformability assay can be used as a rapid screen for athletes to detect potential doped subpopulations of RBCs. The electrokinetic assay could then be used as a secondary detection method to verify the presence of aged RBCs. We are looking forward to evaluating the performance of these assays on in future work from samples collected from doping volunteers. 

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Code: 15C18MP 

Increasing numbers of dietary supplements with ecdysteroids are marketed as “natural anabolic agents”. Their advertisings promise to increase strength and muscle mass during resistance training, to reduce fatigue and to ease recovery. Several studies have reported a wide range of pharmacological effects of ecdysteroids in mammals, most of them beneficial to the organism. The most active phytoecdysteroid, ecdysterone (a “Russian secret”), was already suspected to be used by Russian Olympic athletes since the 1980s. Extensive investigations on the possible growth-promoting effects of ecdysterone in various animal species (rats, mice, Japanese quail and cattle) were reported. 
Recent studies suggest that the anabolic effect of ecdysterone is mediated by estrogen receptor (ER) binding. In comparison to the prohibited anabolic agents (e.g. metandienone and others) ecdysterone revealed to be even more effective in a recent study. However, scientific studies in humans are very rarely accessible.  
Thus, our project aims at investigating the effects of ecdysterone containing products on human athletic performance. A 12-week intervention study in young man will be conducted including regular resistance training for all volunteers. Different doses of ecdysterone containing supplements will be administered during the study to evaluate the performance enhancing effect. Analysis of blood and urine samples for ecdysterone and potential biomarkers of performance enhancement will be conducted. 
To exclude underlying effects by contamination of the supplement or adulteration of the results by administration of other anabolic agents regular screening for prohibited compounds is included in the project. Furthermore, the administered supplements will be tested for the absence of anabolic steroid contaminations. 

Main Findings: 

Increasing numbers of dietary supplements with ecdysteroids are marketed as “natural anabolic agents”. Their advertisings promise to increase strength and muscle mass during resistance training, to reduce fatigue and to ease recovery. Several studies have reported a wide range of pharmacological effects of ecdysteroids in mammals, most of them beneficial to the organism. The most active phytoecdysteroid, ecdysterone (a “Russian secret”), was already suspected to be used by Russian Olympic athletes since the 1980s. Extensive investigations on the possible growth-promoting effects of ecdysterone in various animal species (rats, mice, Japanese quail and cattle) were reported.

Recent studies suggest that the anabolic effect of ecdysterone is mediated by estrogen receptor (ER) binding. In comparison to the prohibited anabolic agents (e.g. metandienone and others) ecdysterone revealed to be even more effective in a recent study performed in rats. However, scientific studies in humans are very rarely accessible.

Thus, our project aimed at investigating the effects of ecdysterone containing products on human athletic performance. A ten-week intervention study in young man has been conducted including regular resistance training for all volunteers. Different doses of ecdysterone containing supplements have been administered during the study to evaluate the performance enhancing effect. Analyses of blood and urine samples for ecdysterone and potential biomarkers of performance enhancement have been conducted.

To exclude underlying effects by contamination of the supplement or adulteration of the results by administration of other anabolic agents screening for prohibited compounds was also performed. Furthermore, the administered supplements have been tested for the absence of anabolic steroid contaminations prior to administration.

The ecdysterone administration led to increased serum IGF1 concentrations in comparison to the control group while thyroxin (T4) concentrations decreased.

Significantly higher increases in muscle mass were observed in those volunteers that were dosed with the ecdysterone supplements. Even more relevant with respect to sports performance, also significantly more pronounced increases in one-repetition bench press performance were observed.
These data underline the effectivity of an ecdysterone supplementation with respect to sports performance. We therefore strongly recommend to include ecdysterone in the list of prohibited substances and methods in sports to improve clean competition in the future. As the exact mechanism of action is not yet fully understood, we suggest to include it in class S1.2 “other anabolic agents”.

Code: 15A29OP 

Screening for endogenous androgenic anabolic steroid (EAAS) misuse remains one of the main challenges in doping control.  Currently, this challenge is addressed by quantification of the seven markers forming the steroid profile included in the steroid module of the Athlete Biological Passport (ABP). The common approach for this quantification involves enzymatic hydrolysis, TMS-derivatization and GC-MS(/MS) analysis. However, several markers might be either lost or underestimated by this approach. 
Preliminary experiments performed by the project team show the occurrence of EAAS excreted as bis-sulfates and highlight their potential usefulness for the detection of testosterone misuse.  
The BIs-Conjugates in the Endogenous Profile of Steroids (BICEPS) project aims to evaluate the potential of steroid bis-conjugates for the detection of EAAS misuse. For this purpose, the project will be divided in two parts. 
In Part I, the usefulness of bis-sulfates for the detection of EAAS misuse will be evaluated. Bis-sulfate reference materials will be quantitatively synthesized and characterized, and an analytical approach for the quantification of urinary EAAS bis-sulfates will be developed and validated. The validated method will be applied to several samples from available drug administration excretion studies. 
In Part II, the occurrence and usefulness of other EAAS bis-conjugates will be evaluated. Libraries of steroid bis-glucuronides and glucuronide-sulfates will be synthesized. From these available reference materials an open screening strategy will be developed based on MS behavior of the synthesized compounds enabling the detection of EAAS bis-conjugates. The detection of these bis-conjugates in selected postadministration samples will be evaluated. 
Taken together, the BICEPS project will reveal which bis-conjugate metabolites are useful for screening of EAAS misuse. Further, the project will deliver a range of characterised reference materials derived by chemical synthesis for their further study and quantification. 

Main Findings: 

Several important EAAS might remain undetectable with the current approach used for the determination of the steroid profile i.e. gas chromatography-mass spectrometry (GC-MS) analysis after an enzymatic hydrolysis with E. coli β-glucuronidase, and the silylation of the steroids. Among them steroid bis-conjugates remain unexplored. The main goal of BICEPS is to evaluate the potential of steroid bis-conjugates for the detection of EAAS misuse.

Firstly, we have characterized the MS behavior of steroid bis-sulfates and we have developed an open screening method for their detection in urine. We have quantitatively synthesized 12 steroid bis-sulfates and we have developed and validated a quantitative method for their determination in urine. The method has been applied to samples collected after oral administration of testosterone undecanoate. We have evaluated several ratios between the validated analytes and we found that they have limited applicability for doping control purposes. However, we found two additional steroid bis-sulfates which allowed for the screening of the misuse with promising results. We hypothesize that these markers are two isomeric forms of the compound 3,16-dihydroxy-5-androstane-17-one bis sulfate. Synthesis of reference material is required to confirm the identity. Using these two markers, we obtained results comparable with those obtained with the best retrospective markers for oral misuse (resistant glucuronides and cysteinyl conjugates).

In a second part of the project we have synthesized some steroid bis-glucuronides and steroid glucuronide-sulfates. We have demonstrated the occurrence of some of them in human urine samples. Preliminary results showed that one of them (5α-androstane-3β,17β-diol 3-sulfate 17-glucuronide) clearly increased after oral testosterone administration supporting its potential usefulness for doping control.

Taken together, the results of BICEPS provide the first evidence about the potential usefulness of bis-conjugates in the doping control field.

The main results of BICEPS have been published at :
1.- McLeod MD, Waller CC, Esquivel A, Balcells G, Ventura R, Segura J, Pozo OJ*. A constant ion loss method for the untargeted detection of bissulfate metabolites. Anal Chem 2017; 89(3): 1602-1609.
2.- Pranata A, Fitzgerald CC, Khymenets O, Westley E, Anderson NJ, Ma P, Pozo O J, McLeod MD*. Synthesis of Steroid Bisglucuronide and Sulfate Glucuronide Reference Materials: Unearthing Neglected Treasures of Steroid Metabolism. Steroids 2019: doi 10.1016/j.steroids.2018.11.017.
Some results have been presented at: 1.- “Steroid bis-sulfates: a forgotten minority” Oral presentation at SUPA2017, Targeting Steroid Sulfation Pathways, Birmingham April 2017