Détection de l'administration de l'hormone de croissance équine recombinante (application au contrôle antidopage dans les courses hippiques)

L hormone de croissance est une molécule protéique à activité anabolisante dont lespropriétés peuvent être mises à profit pour améliorer les performances sportives des chevaux de courses et accroître la rentabilité de production des animaux d élevage. Cependant, la législation est très stricte car l utilisation de cette molécule ou de sa forme recombinante est interdite par le code des courses et les directives européennes en vigueur. Le problèmeest qu il n existe aucune méthode directe fiable de détection de cette molécule présente à l état de trace dans le plasma ( g.L-1) pour appliquer la réglementation. Dans ce contexte, les travaux de thèse ont permis d aboutir à la mise au point de deux méthodes, l une directe et l autre indirecte, de détection de l administration frauduleuse d hormone de croissance équine recombinante (reGH) à des chevaux. La méthode de détection directe de la reGH a été obtenue avec la mise au point d une nouvelle stratégie analytique basée sur la LC-ESI-(+)-MS/MS en identifiant par spectrométrie de masse le peptide N-terminal caractéristique de la forme recombinante. La méthode de détection indirecte a quant à elle été développée par ELISA afin de mettre en évidence la production d anticorps anti-reGH consécutive à un traitement avec cette molécule. Les résultats obtenus, à savoir la validation de la méthode directe de détection dans le plasma à 5 g.L-1 et la détection d anticorps anti-reGH pendant plus de 5 mois après traitement, permettent d envisager des applications immédiates à la lutte antidopage des animauxThe growth hormone is a peptidic molecule with anabolic activity potentially used to improve horse athletic efficiency and to increase the production profitability of farm animals. However, the legislation is very strict and the use of this molecule or its recombinant form is strictly forbidden by the races code and the European directives in force. The problem is that no reliable direct method exists to detect this molecule present at trace levels in plasma ( g.L-1) in order to comply the regulation. In this context, this PhD thesis work enabled the development of two methods, one direct and one indirect, for the detection of recombinant equine growth hormone (reGH) misuse in horses. The direct detection method of reGH was obtained with the development of a new analytical strategy based on LC-ESI-(+)-MS/MS through the identification by mass spectrometry of the N-terminal peptide, characteristic of the recombinant form. The indirect method was developed by ELISA to detect the production of anti-reGH antibodies consecutive to a treatment with reGH. The results obtained, namely the direct detection of reGH in plasma at 1 g.L-1 and the anti-reGH antibodies detection up to five months after treatment, allow to consider an immediate application in illegal administration of this molecule to race horses and food producing animalsNANTES-BU Sciences (441092104) / SudocSudocFranceF

In vitro simulation of the hindgut as a tool to study the influence of phytosterol consumption on the excretion of anabolic-androgenic steroids in horses

Introduction: In race horses zero tolerance is held for most anabolic steroids (AAS). Nevertheless, some natural precursors, including phytosterols from grains, have been put forward as a possible explanation for the endogenous prevalence of low concentrations of steroids, with β-Bol as the most illicit candidate. Purpose: This study aimed to evaluate the use of in vitro digestions as a tool to simulate in vivo hindgut fermentation and to study the possible biotransformation from phytosterols or other androgenic precursors to forbidden AAS. Methods: To investigate the possible endogenous biotransformation, in vitro simulations of the horse hindgut were set up, using fecal inocula obtained from eight different horses. In vitro digestion samples were analyzed with a fully validated UHPLC-MS/MS method. Results: The digestive transformation of ADD could be directly linked to the detection of βBol, and the consumption of phytosterols to low levels of AED, a testosterone precursor, but no direct link between phytosterol consumption and βBol detection was observed. Significance: The results are in line with previously described in vivo results, proving that in vitro digestions are a useful tool to study the fermentation reactions that take place in the horse’s hindgut, offering new insights in the detection of, endogenous, anabolic steroids

Unraveling the anabolic-androgenic steroid profile of untreated horses by UHPLC-MS/MS

Clean sport is a priority for the regulatory organs of both the horse racing industry (IFHA, International Federation of Horseracing Authorities) and the Equestrian sports (FEI, Fédération Equestre Internationale). Urinary and blood samples are screened for the presence of hundreds of forbidden substances including anabolic-androgenic steroids (AAS). Because of their importance as ergogenic drugs of abuse and their involvement in equine reproduction, AAS remain an important field of investigation. Consequently, based on the suspected endogenous origin of some forbidden AAS (ß-boldenone as the most elicit one) this study focused on non-treated, out of competition horses, to improve the knowledge on the natural endogenous AAS profile of horses in general. In total, 105 guaranteed non-treated horses (47 geldings, 53 mares and 5 stallions serving as a control) were screened for ß-boldenone and five related steroids: ADD (androstadienedione), AED (androstenedione), αT (alpha-testosterone), ßT (ß-testosterone) and P (progesterone) via UHPLC-MS/MS (Triple Quadripole). The extraction and detection method was validated according to EC guidelines for ß-boldenone, ADD, AED, αT and ßT. Correlations between the occurrences of these AAS were calculated and hypotheses for the alleged endogenous traces were formulated. By using in vitro digestive simulations we were able to test different hypotheses

Does the consumption of moulded feed affect the excretion of anabolic-androgenic steroids in horses?

Background: To ensure fair competition, sport horses have to compete on their own merits, without any unfair advantage that might follow the use of drugs. Therefore, the FEI (Fédération Equestre Internationale) lists all substances that are not allowed in competition. Many steroids are part of this list as the administration of steroids increases nitrogen retention, protein synthesis and the release of erythropoietin in the kidneys, making them very popular as drugs of abuse. Hypothesis/Objectives: Some feed-related moulds can transform plant phytosterols into (anabolic) steroids. What would happen if a horse consumes feed contaminated with these moulds? Due to the strict anti-doping policies applied by the FEI, the excretion of steroids originating from the feed could have major consequences. Methods: A rapid UHPLC-MS/MS analytical method was developed and successfully and thoroughly validated. Multiple spontaneously moulded feed samples were tested for the presence of forbidden and related natural steroids (including boldenone and testosterone). The effect of the consumption of these feeds was tested by in vitro simulation of the horse’s hindgut, in static batch incubations. Samples were taken after 0, 12, 24, 48, and 72h and the incubations were executed in triplicate. The results were compared to a blank in vitro digestion and the digestion of unmoulded feed. Results: In most feed samples no steroids were detected, even when the products were moulded. However, moulded corn contained 1.8 ± 0.5 ng/g AED (4-andostradienedione, a testosterone precursor), and this concentration increased when corn was digested in vitro. The addition of phytosterols to the in vitro digestion led to a further increase of AED (up to 2.9 ± 0.4 ng/g feed). Additionally, one of the phytosterol-rich herbal supplements contained α-testosterone as well (up to 40 ng/g)

Blood cells RNA biomarkers as a first long-term detection strategy for EPO abuse in horseracing

International audienc

A validated UHPLC-MS/MS method to quantify low levels of anabolic-androgenic steroids naturally present in urine of untreated horses

Doping control is a main priority for regulatory bodies of both the horse racing industry and the equestrian sports. Urine and blood samples are screened for the presence of hundreds of forbidden substances including anabolic-androgenic steroids (AASs). Based on the suspected endogenous origin of some AASs, with β-boldenone as the most illicit candidate, this study aimed to improve the knowledge of the naturally present AAS in horse urine. To this extent, a novel ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated according to the Association of Official Racing Chemists (AORC) and European Commission (EC) guidelines, proving the power of this new method. Low limits of detection (0.2 ng/mL), good reproducibility (percentage of standard deviation (%RSD)0.99 and lack-of-fit analysis) were obtained for all included AASs. With this method, urine samples of 105 guaranteed untreated horses (47 geldings, 53 mares and 5 stallions serving as a control) were screened for β-boldenone and five related natural steroids: androstadienedione (ADD), androstenedione (AED), alpha-testosterone (αT), beta-testosterone (βT), and progesterone (P). Progesterone, β-testosterone, and α-testosterone were detected in more than half of the horses at low concentrations (<2 ng/mL). Occasionally, not only testosterone and progesterone but also low concentrations of AED, ADD, and boldenone (Bol) were found (0.5–5 ng/mL)

High‐throughput untargeted screening of biotherapeutic macromolecules in equine plasma by UHPLC‐HRMS/MS: Application to monoclonal antibodies and Fc‐fusion proteins for doping control

International audienceAbstract Many innovative biotherapeutics have been marketed in the last decade. Monoclonal antibodies (mAbs) and Fc‐fusion proteins (Fc‐proteins) have been developed for the treatment of diverse diseases (cancer, autoimmune diseases, and inflammatory disorders) and now represent an important part of targeted therapies. However, the ready availability of such biomolecules, sometimes characterized by their anabolic, anti‐inflammatory, or erythropoiesis‐stimulating properties, raises concerns about their potential misuse as performance enhancers for human and animal athletes. In equine doping control laboratories, a method has been reported to detect the administration of a specific human biotherapeutic in equine plasma; but no high‐throughput method has been described for the screening without any a priori knowledge of human or murine biotherapeutic. In this context, a new broad‐spectrum screening method involving UHPLC‐HRMS/MS has been developed for the untargeted analysis of murine or human mAbs and related macromolecules in equine plasma. This approach, consisting of a “pellet digestion” strategy performed in a 96‐well plate, demonstrates reliable performances at low concentrations (pmol/mL range) with high‐throughput capability (≈100 samples/day). Targeting species‐specific proteotypic peptides located within the constant parts of mAbs enables the “universal” detection of human biotherapeutics only by monitoring 10 peptides. As proof of principle, this strategy successfully detected different biotherapeutics in spiked plasma samples, and allowed, for the first time, the detection of a human mAb up to 10 days after a 0.12 mg/kg administration to a horse. This development will expand the analytical capabilities of horse doping control laboratories towards protein‐based biotherapeutics with adequate sensitivity, throughput, and cost‐effectiveness

Detection of secondary biomarker of met-eGH as a strategy to screen for somatotropin misuse in horseracing

International audienceSince the Australian commercialisation of the recombinant equine growth hormone (reGH) in 1998 (EquiGen-5), Bresagen), this reGH, which differs only from eGH by an additional methionine at the N-terminal end (met-eGH), is worldwide suspected to be administered to racehorses as a doping agent. Indeed, the use of this biological drug is considered as a threat to horseracing since it acts both on growth, development or reproductive functions, and on the improvement of performances. In this work, we describe two reliable techniques based on surface plasmon resonance biosensor immunoassay (SPR-BIA) and solid-phase enzyme-linked immunosorbent assay (ELISA) as new, rapid and efficient long-term screening methods applicable to horseracing antidoping analysis. The ELISA and SPR-BIA tests were applied to octanoic acid purified IgGs from serum/plasma samples collected on two thoroughbreds treated with recombinant equine growth hormone for a period of two weeks. The first kinetic study of serum/plasma antibodies raised as a consequence of recombinant equine growth hormone administrations, which allows the detection from eight days up to 200 days after the beginning of the treatment, was performed. In order to trace the occurrence of anti-reGH antibodies in routine analysis and to monitor the animal level exposure to this forbidden molecule, a random population study was conducted on 233 post-race horses

Lutte contre le dopage, des méthodes toujours plus performantes

Présentation des méthodes de dépistage du Laboratoire des Courses Hippiques (LCH)