Data from a microdosed recombinant human erythropoietin administration study applying the new biotinylated clone AE7A5 antibody and a further optimized sarcosyl polyacrylamide gel electrophoresis protocol.

Erythropoietin (EPO) is a hormone, which stimulates the production of red blood cells. Due to its performance-enhancing effect, it is prohibited by the World Anti-Doping Agency (WADA). In order to reduce the detection window of EPO doping, athletes have been applying low doses of recombinant EPO (e.g., <10 IU/kg body weight, daily or every second day) instead of larger doses twice or more per week (e.g., 30 IU/kg). Microdoses of Retacrit (epoetin zeta), an EPO biosimilar, were administered intravenously and subcutaneously to human males and females. Urine and serum samples were collected and analysed applying the new biotinylated clone AE7A5 EPO antibody and a further optimized sarcosyl polyacrylamide gel electrophoresis (SAR-PAGE) protocol. With the improved protocol, microdosed Retacrit (7.5 IU/kg body weight [BW]) was detectable for at least 52 h after intravenous administration. Detection windows were approximately the same for serum and urine and doubled after subcutaneous administration (~104 h). Previous studies applying different electrophoretic techniques and the not further optimized SAR-PAGE protocol revealed considerably shorter detection windows for recombinant human erythropoietin (rhEPO) microdoses. Because the new biotinylated antibody performed significantly more sensitive than the nonbiotinylated version, the new protocol will improve the sensitivity and hence detectability of recombinant EPO in doping control.

Urine manipulation with liquid soap: A case report.

Among the various strategies to enhance performance by the use of doping substances or methods, sample manipulation to cheat common detection strategies is prohibited by the World Antidoping Agency. Two cases of urine sample adulteration with surfactants are presented in this article, as well as an easy strategy for the detection of this sort of sample manipulation.

Synthesis and identification of hydroxylated metabolites of the anti-estrogenic agent cyclofenil.

The detection of metabolites of the anti-estrogenic substance cyclofenil, listed on the World Anti-Doping Agency (WADA) Prohibited List since 2004 is described. Target substances are hydroxylated metabolites, bearing an aliphatic hydroxyl group either in the 2-, 3- or 4-position of the aliphatic ring, in addition to the phenolic functions on the aromatic rings. Structural identification used NMR as well as high-resolution mass spectrometry after nano-electrospray ionisation (ESI). Unambiguous detection of all three synthesised cyclofenil metabolites M1-M3 was done using gas chromatography for separation and electron ionisation mass spectrometry for detection of the per-silylated compounds in comparison with a reference urine deriving from an excretion study within the WADA 2007 Educational Programme.

Unambiguous identification and characterization of a long-term human metabolite of dehydrochloromethyltestosterone.

In doping control analysis, the characterization of urinary steroid metabolites is of high interest for a targeted and long-term detection of prohibited anabolic androgenic steroids (AAS). In this work, the structure of a long-term metabolite of dehydrochloromethyltestosterone (DHCMT) was elucidated. Altogether, 8 possible metabolites with a 17α-methyl-17ß-hydroxymethyl - structures were synthesized and compared to a major DHCMT long-term metabolite detected in reference urine excretion samples. The confirmed structure of the metabolite was 4α-chloro-18-nor-17ß-hydroxymethyl-17α-methyl-5α-androst-13-en-3α-ol.

Metabolomic signatures of low birthweight: Pathways to insulin resistance and oxidative stress.

Several studies suggest that low birthweight resulting from restricted intrauterine growth can leave a metabolic footprint which may persist into adulthood. To investigate this, we performed metabolomic profiling on 5036 female twins, aged 18-80, with weight at birth information available from the TwinsUK cohort and performed independent replication in two additional cohorts. Out of 422 compounds tested, 25 metabolites associated with birthweight in these twins, replicated in 1951 men and women from the Hertfordshire Cohort Study (HCS, aged 66) and in 2391 men and women from the North Finland Birth 1986 cohort (NFBC, aged 16). We found distinct heterogeneity between sexes and, after adjusting for multiple tests and heterogeneity, two metabolites were reproducible overall (propionylcarnitine and 3-4-hydroxyphenyllactate). Testing women only, we found other metabolites associated with lower birthweight from the meta-analysis of the three cohorts (2-hydroxy-butyric acid and γ-glutamylleucine). Higher levels of all these metabolites can be linked to insulin resistance, oxidative stress or a dysfunction of energy metabolism, suggesting that low birthweight in both twins and singletons are having an impact on these pathways in adulthood.

Detection of testosterone esters in blood.

Injections of synthetic esters of testosterone are among the most common forms of testosterone application. In doping control, the detection of an intact ester of testosterone in blood gives unequivocal proof of the administration of exogenous testosterone. The aim of the current project was to investigate the detection window for injected testosterone esters as a mixed substance preparation and as a single substance preparation in serum and plasma. Furthermore, the suitability of different types of blood collection devices was evaluated. Collection tubes with stabilizing additives, as well as non-stabilized serum separation tubes, were tested. A clinical study with six participants was carried out, comprising a single intramuscular injection of either 1000 mg testosterone undecanoate (Nebido(®)) or a mixture of 30 mg testosterone propionate, 60 mg testosterone phenylpropionate, 60 mg testosterone isocaproate, and 100 mg testosterone decanoate (Sustanon(®)). Blood was collected throughout a testing period of 60 days. The applied analytical method for blood analysis included liquid-liquid extraction and preparation of oxime derivatives, prior to TLX-sample clean-up and liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection. All investigated testosterone esters could be detected in post-administration blood samples. The detection time depended on the type of ester administered. Furthermore, results from the study show that measured blood concentrations of especially short-chained testosterone esters are influenced by the type of blood collection device applied. The testosterone ester detection window, however, was comparable.

Metabolomic identification of a novel pathway of blood pressure regulation involving hexadecanedioate.

High blood pressure is a major contributor to the global burden of disease and discovering novel causal pathways of blood pressure regulation has been challenging. We tested blood pressure associations with 280 fasting blood metabolites in 3980 TwinsUK females. Survival analysis for all-cause mortality was performed on significant independent metabolites (P<8.9×10(-5)). Replication was conducted in 2 independent cohorts KORA (n=1494) and Hertfordshire (n=1515). Three independent animal experiments were performed to establish causality: (1) blood pressure change after increasing circulating metabolite levels in Wistar-Kyoto rats; (2) circulating metabolite change after salt-induced blood pressure elevation in spontaneously hypertensive stroke-prone rats; and (3) mesenteric artery response to noradrenaline and carbachol in metabolite treated and control rats. Of the15 metabolites that showed an independent significant association with blood pressure, only hexadecanedioate, a dicarboxylic acid, showed concordant association with blood pressure (systolic BP: ß [95% confidence interval], 1.31 [0.83-1.78], P=6.81×10(-8); diastolic BP: 0.81 [0.5-1.11], P=2.96×10(-7)) and mortality (hazard ratio [95% confidence interval], 1.49 [1.08-2.05]; P=0.02) in TwinsUK. The blood pressure association was replicated in KORA and Hertfordshire. In the animal experiments, we showed that oral hexadecanedioate increased both circulating hexadecanedioate and blood pressure in Wistar-Kyoto rats, whereas blood pressure elevation with oral sodium chloride in hypertensive rats did not affect hexadecanedioate levels. Vascular reactivity to noradrenaline was significantly increased in mesenteric resistance arteries from hexadecanedioate-treated rats compared with controls, indicated by the shift to the left of the concentration-response curve (P=0.013). Relaxation to carbachol did not show any difference. Our findings indicate that hexadecanedioate is causally associated with blood pressure regulation through a novel pathway that merits further investigation.

The interference of heparin on IEF-PAGE of erythropoietins.

Because of the risk of suffering a stroke or heart attack, some athletes and their medical supervisors admitted having used anticoagulants (e.g. acetylsalicylic acid) in combination with doping with recombinant erythropoietins (rhEPO). Heparin is one of the oldest and cheapest anticoagulants. The anticoagulative effect of heparin is a result of the binding of heparin to the plasma protein antithrombin III and the subsequent inactivation of blood clotting factors (e.g. factor IIa, IXa, Xa, XIa, XIIa). Heparin-a polyanion-is known to interact with carrier ampholytes used in IEF-PAGE. Two different types of heparin pharmaceuticals are used for medical purposes: unfractionated heparins (UFH) and low molecular weight heparins (LMWH). Their influence on IEF- and SDS-PAGE was investigated. Only UFH had a profound impact on IEF-PAGE, leading to excessive smearing or complete abolishment of the EPO IEF-profile and shifting of acidic EPO-isoforms in the endogenous region of the gel. No such effect was observable for SDS-PAGE. Remedies include immunoaffinity purification of EPO before IEF-PAGE or the treatment of the urinary retentate with solid urea. A combined usage of IEF- and SDS-PAGE is recommended for confirming the presence of rhEPO in urine and for further analysis of smearing (and therefore suspicious) samples. This two-method approach is already in accordance with the technical document on EPO-analysis (TD2009EPO) of the World Anti-Doping Agency (WADA).

SARCOSYL-PAGE: a new method for the detection of MIRCERA- and EPO-doping in blood.

The detection of doping with MIRCERA (the brand name for Continuous Erythropoietin Receptor Activator, or CERA) is hampered by the limited excretion of the rather large molecule (approximately 60 kDa) in urine. Blood (serum, plasma) in combination with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) appears to be the ideal matrix for detecting all forms of doping with erythropoiesis-stimulating agents (ESAs) because the apparent molecular masses of ESAs are different from the mass of human serum erythropoietin (shEPO). While SDS-PAGE has proven the most sensitive method for the detection of doping with Dynepo, the sensitivity of SDS-PAGE for MIRCERA is drastically decreased. By exchanging the SDS for SARCOSYL (SAR) in the sample and running buffers the sensitivity problem was solved. SARCOSYL, a methyl glycine-based anionic surfactant, is only binding to the protein-part of MIRCERA but not to its polyethylene glycol (PEG)-chain, while SDS binds to both parts. In consequence, the monoclonal anti-EPO antibody (clone AE7A5) no longer interacts with the fully SDS-solubilized MIRCERA molecules. Only those molecules that contain SDS bound to the protein-chain are detected. Due to the inability of SARCOSYL to solubilize PEG-molecules, MIRCERA can be detected on SARCOSYL-PAGE with the same sensitivity as non-PEGylated epoetins. In a typical SAR-PAGE experiment, 200 microL of serum are used, which allows the direct detection of MIRCERA, recombinant epoetins (such as NeoRecormon, Dynepo, NESP), and shEPO in a single experiment and with high (i.e. femtogram) sensitivity.

SDS-PAGE of recombinant and endogenous erythropoietins: benefits and limitations of the method for application in doping control.

Doping of athletes with recombinant and genetically modified erythropoietins (EPO) is currently detected by isoelectric focusing (IEF). The application of these drugs leads to a significant change in the isoform profile of endogenous urinary erythropoietin (uhEPO). Dynepo, MIRCERA, biosimilars with variable IEF-profiles as well as active urines and effort urines have made additional testing strategies necessary. The new generation of small molecule EPO-receptor stimulating agents like Hematide will also challenge the analytical concept of detecting the abuse of erythropoiesis stimulating agents (ESA). By determining their apparent molecular masses with SDS-PAGE a clear differentiation between endogenous and exogenous substances also concerning new EPO modifications is possible. Due to the orthogonal character of IEF- and SDS-PAGE both methods complement each other. The additional benefits of SDS-PAGE especially in relation to active and effort urines as well as the detection of Dynepo were investigated. Due to significant differences between the apparent molecular masses of uhEPO/serum EPO (shEPO) and recombinant, genetically or chemically modified erythropoietins the presence of active or effort urines was easily revealed. The characteristic band shape and apparent molecular mass of Dynepo on SDS-PAGE additionally evidenced the presence of this substance in urine. A protocol for the detection of EPO-doping in serum and plasma by SDS-PAGE was developed. Blood appears to be the ideal matrix for detecting all forms ESA-doping in the future.

Anti-doping testing at the 2008 European football championship.

Big sports events like the 2008 European Football Championship are a challenge for anti-doping activities, particularly when the sports event is hosted by two different countries and there are two laboratories accredited by the World Anti-Doping Agency. This challenges the logistics of sample collection as well as the chemical analyses, which must be carried out timeously. The following paper discusses the handling of whereabouts information for each athlete and the therapeutic use exemption system, experiences in sample collection and transportation of blood and urine samples, and the results of the chemical analysis in two different accredited laboratories. An overview of the analytical results of blood profiling and growth hormone testing in comparison with the distribution of the normal population is also presented.