Detection of doping substances in paired dried blood spots and urine samples collected during doping controls in Danish fitness centers.

The use of dried blood spot (DBS) in anti-doping can be advantageous in terms of collection, transportation, and storage compared with the traditional anti-doping testing matrices urine and venous blood. There could, nonetheless, be disadvantages such as shorter detection windows for some substances compared with urine, but real-life comparison of the detectability of prohibited substances in DBS and urine is lacking. Herein, we present a liquid chromatography-high resolution mass spectrometry (LC-HRMS)-based screening method for simultaneous detection of 19 target analytes from the doping substance categories S1-S5 in a single spot. Ninety-eight urine and upper-arm DBS (Tasso-M20) sample pairs were collected from fitness centers customers notified for doping control by Anti Doping Denmark, and three sample pairs were collected from active steroid users undergoing clinical evaluation and treatment at a Danish hospital. The analytical findings were cross compared to evaluate the applicability of the developed DBS testing menu in terms of feasibility and analytical performance. To our knowledge, this is the first study to compare the detectability of prohibited substances in DBS and urine samples collected in a doping control setting. Twenty-seven of the urine samples and 23 DBS samples were positive, and we observed a very high concordance (95%) in the overall analytical results (i.e., positive or negative samples for both urine and DBS). Collectively, these results are very promising, and DBS seems suitable as a stand-alone matrix in doping control in fitness centers likely because of the high analyte concentration levels in these samples.

Standardization of reticulocyte counts in the athlete biological passport: A practical update.

INTRODUCTION: The athlete biological passport monitors blood variables over time to uncover blood doping. With the phasing in of a new series of blood analyzers, the Sysmex XN series, it was necessary to examine the comparability of results with the previously employed XT/XE series. A previous comparison between XN and XT/XE series suggested a small but significant bias between the two instruments in the measurements of RET%. Here, we examined the comparability of RET% on the XN and XT/XE platform using data collected over the first year since the transition. METHODS: The comparability of results obtained from XN and XT/XE instruments was assessed using three datasets: (i) 767 blood samples measured on both instrument series in 22 WADA-accredited laboratories, (ii) 27 323 samples measured on either instrument across 31 laboratories, and (iii) 119 clinical samples and 110 anti-doping samples measured on both instruments in a single laboratory. RESULTS: Analysis of the three datasets confirms the previous observation of a bias toward higher RET% values for samples measured on Sysmex XN instruments compared with the XT/XE series. Using data across a larger number of XN instruments and a larger athlete population, the current work suggests that the bias is proportional and slightly higher than previously observed across most of the range RET% values. CONCLUSION: A model is proposed for the comparison of data across XN and XT/XE technologies whereby the instrument bias increases proportionally with RET% measured on Sysmex XN Series, but where the rate of increase is negatively related to IRF%.

Stability and detectability of testosterone esters in dried blood spots after intramuscular injections.

While misuse of testosterone esters is widespread in elite and recreational sports, direct detection of intact testosterone esters in doping control samples is hampered by the rapid hydrolysis by esterases present in the blood. With dried blood spot (DBS) as sample matrix, continued degradation of the esters is avoided due to inactivation of the hydrolase enzymes in dried blood. Here, we have developed the method further for detection of testosterone esters in DBS with focus on robustness and applicability in doping control. To demonstrate the method's feasibility, DBS samples from men receiving two intramuscular injections of Sustanon® 250 (n = 9) or placebo (n = 10) were collected, transported, and stored prior to analysis, to mimic a doping control scenario. The presented nanoLC-HRMS/MS method appeared reliable and suitable for direct detection of four testosterone esters (testosterone decanoate, isocaproate, phenylpropionate, and propionate) after extraction from DBS. Sustanon® was detected in all subjects for at least 5 days, with detection window up to 14 days for three of the esters. Evaluation of analyte stability showed that while storage at room temperature is tolerated well for a few days, testosterone esters are highly stable (>18 months) in DBS when stored in frozen conditions. Collectively, these findings demonstrate the applicability of DBS sampling in doping control for detection of steroid esters. The fast collection and reduced shipment costs of DBS compared with urine and standard blood samples, respectively, will allow more frequent and/or large-scale testing to increase detection and deterrence.

No pain, just gain: Painless, easy, and fast dried blood spot collection from fingertip and upper arm in doping control.

This study aimed to determine and compare the perception, painfulness, and usability of the minimally invasive dried blood spot (DBS) collections from fingertip versus upper arm from different athlete populations: males and females representing sports dependent on hand/arm, sports less dependent on hand/arm and para-athletes. To accomplish this, 108 national level athletes from Denmark were recruited (♀ = 49, ♂ = 59, 25 ± 6 years; mean ± SD) and 11 Doping Control Officers (DCOs) collected manual fingerprick DBS (HemaSpot HF) and automated upper-arm DBS (Tasso-M20) from each athlete. Athletes and DCOs responded to questionnaires regarding the perception of sample collection procedures. On a 0-10 scale, the athletes reported a low pain score and a very good general experience for both sampling sites, but following upper-arm DBS collection, the associated pain was rated lower (-0.4 ± 1.6, p < 0.05), and the general experience rated better (+0.6 ± 2.3, p ≤ 0.001) than after the fingerprick DBS collection. The DCOs rated the general experience with the upper-arm DBS collection better (+1.6 ± 1.1, p ≤ 0.01) than the fingerprick DBS collection, partly because problems occurred more frequently during the DBS collection from the fingertip (28%) than from the upper arm (6%). In conclusion, it appears that DBS sampling is affiliated with minimal sensation of pain and is preferred by both DCOs and athletes, independent of gender and discipline, over conventional sample collection methods. Collection of DBS from the upper arm was preferred over fingerprick by both athletes and DCOs.

Pharmacokinetics of salmeterol and its main metabolite α-hydroxysalmeterol after acute and chronic dry powder inhalation in exercising endurance-trained men: Implications for doping control.

As of 2020, use of beta2 -agonist salmeterol is restricted by the World Anti-Doping Agency (WADA) and is only permitted by inhalation at therapeutic doses not exceeding 200 µg in 24 h. In contrast to beta2 -agonists salbutamol and formoterol, WADA has not established a urine threshold for salmeterol despite its muscle hypertrophic actions observed in animals. Herein, we investigated plasma (0-4 h) and urine (0-24 h) concentrations (by ultra-high-performance liquid chromatography-tandem mass spectrometry [UHPLC-MS/MS]) of salmeterol and α-hydroxysalmeterol after dry powder inhalation at supratherapeutic (400 µg) and high therapeutic (200 µg) doses, and after seven consecutive days of therapeutic inhalation (200 µg × day-1 ) in 11 healthy endurance-trained men. During each trial, participants inhaled salmeterol before 1½ h moderate-intensity cycling. Mean ± SD maximum urine concentrations of salmeterol unadjusted for specific gravity reached 4.0 ± 1.6, 2.1 ± 1.5, and 2.2 ± 1.1 ng × ml-1 for 400 µg, 200 µg, and seven consecutive days of 200 µg, respectively, with corresponding maximum urine concentrations of α-hydroxysalmeterol being 11.6 ± 6.1, 5.7 ± 4.6, and 6.5 ± 2.6 ng × ml-1 . Within the relevant window for doping control (first 6 h post-inhalation), the present data (119 samples), along with 64 biobank urine samples, showed that a combined salmeterol and α-hydroxysalmeterol urine threshold with equal cut-offs of 3.3 ng × ml-1 was superior to a salmeterol-only threshold to discriminate therapeutic (200 µg) from supratherapeutic use (400 µg) with a sensitivity of 24% with 0% false positives when applying the WADA technical document (TD2019DL.v2) method of specific gravity adjustment. Thus, a combination of urine salmeterol and α-hydroxysalmeterol concentrations may be suitable for discriminating between therapeutic and supratherapeutic prohibited inhalation of salmeterol.

An Intramuscular Injection of Mixed Testosterone Esters Does Not Acutely Enhance Strength and Power in Recreationally Active Young Men.

Purpose: Limited data are available on the acute performance-enhancing effects of single-dose administration of testosterone in healthy humans. Studies of testosterone administrations to healthy humans are rare due to the difficult nature and necessity of close clinical monitoring. However, our unique physiological experimental facilities combined with close endocrinological collaboration have allowed us to safely complete such a study. We tested the hypothesis that an intramuscular injection of 250 mg mixed testosterone esters (TEs) enhances physical performance in strength and power exercises acutely, measured 24 h after injection. Additionally, we investigated whether the basal serum testosterone concentration influences the performance in countermovement jump (CMJ), 30-s all out cycle sprint, and one-arm isometric elbow flexion. Methods: In a randomized, double-blind, placebo-controlled design, 19 eugonadal men received either a TE (n = 9, 23 ± 1 years, 183 ± 7 cm, 83 ± 10 kg) or a PLA (n = 10, 25 ± 2 years, 186 ± 6 cm, 82 ± 14 kg) injection. Hormonal levels and the performance in CMJ, 30-s all out cycle sprint, and one-arm isometric elbow flexion were measured before and 24 h after injection. Results: Firstly, an intramuscular injection of 250 mg mixed TEs did not enhance the vertical jump height in a CMJ test, peak power, mean power, and fatigue index in a 30-s all-out cycle sprint or rate of force development and maximal voluntary contraction in a one-arm isometric elbow flexion 24 h post-injection. Secondly, baseline testosterone levels appeared not to influence performance in strength and power exercises to a large extent in healthy, recreationally active young men. Conclusion: A single intramuscular injection of 250 mg mixed TEs has no acute ergogenic effects on strength and power performance in recreationally active, young men. This novel information has implication for basic physiological understanding. Whether the same applies to an elite athlete population remains to be determined. If so, this would have implications for anti-doping efforts aiming to determine the most cost-efficient testing programs.

Single-dose administration of clenbuterol is detectable in dried blood spots.

Clenbuterol is a ß2 -agonist prescribed for asthmatic patients in some countries. Based on its anabolic and lipolytic effects observed in studies on rodents and in livestock destined for food production, clenbuterol is abused by bodybuilders and athletes seeking leanness. Urinary clenbuterol analysis is part of routine doping analysis. However, the collection of urine samples is time-consuming and can be intimidating for athletes. Dried blood spot (DBS) appears attractive as an alternative matrix, but the detectability of clenbuterol in humans through DBS has not been investigated. This study evaluated if clenbuterol could be detected in DBS and urine collected from six healthy men after oral intake of 80 µg clenbuterol. The DBS and urine samples were collected at 0, 3, 8, 24, and 72 h post-ingestion, with additional urine collections on days 7 and 10. Using LC-MS/MS, it was shown that clenbuterol could be detected in all DBS samples for 24 h post-ingestion and with 50% sensitivity 3 days after ingestion. The DBS method was 100% specific. Evaluation of analyte stability showed that clenbuterol is stable in DBS for at least 365 days at room temperature when using desiccant and avoiding light exposure. In urine, clenbuterol was detectable for at least 7-10 days after ingestion. Urinary clenbuterol concentrations below 5 ng/mL were present in some subjects 24 h after administration. Collectively, these data indicate that DBS are suitable for routine doping control analysis of clenbuterol with a detection window of at least 3 days after oral administration of 80 µg.

Changes in blood parameters after intramuscular testosterone ester injections - Implications for anti-doping.

Testosterone treatment stimulates the production of red blood cells and alters iron homeostasis. Thus, we investigated whether the 'haematological module' of the athlete biological passport (ABP) used by the World Anti-Doping Agency can be used to indicate misuse of testosterone. Nineteen eugonadal men received intramuscular injections of either 250 mg Sustanon®, a blend of four testosterone esters, or placebo on days 0 and 21 in a randomized, placebo-controlleddouble-blind design. Urine samples and blood samples were collected twice pre-treatment, at least 5 days apart, and on days 1, 3, 5, 10 and 14 post-injections to assess steroidal and haematological biomarkers of the ABP. The steroidal profile was flagged suspicious in all Sustanon®-treated subjects, whereas the haematological profile was flagged suspicious in six out of nine subjects. When both sensitivity and specificity were considered, reticulocyte percentage (RET%) appeared as the best marker of the haematological module for implying testosterone ester misuse. Atypical blood passport samples were used to select time points for further isotope-ratio mass spectrometry (IRMS) analysis of testosterone and its metabolites in simultaneously collected urine. In addition to the testosterone (T) to epitestosterone (E) ratio, the RET% and OFF-Score could help identify suspicious samples for more targeted IRMS testing. The results demonstrate that unexpected fluctuations in RET% can indicate testosterone doping if samples are collected 3-10 days after injection. From an anti-doping perspective, the haematological and steroidal modules of the ABP should complement each other when planning targeted follow-up testing and substantiating likely misuse of testosterone.

Tramadol Does Not Improve Performance or Impair Motor Function in Trained Cyclists.

PURPOSE: To investigate the hypothesis that a therapeutic oral dose of Tramadol improves cycling time trial performance and compromises motor-cognitive performance in highly trained cyclists. METHODS: Following two familiarization trials, 16 highly trained cyclists completed a preloaded time trial (1 h at 60% of peak power followed by a 15-km time trial) after ingestion of 100 mg Tramadol or placebo in a double-blind placebo-controlled counterbalanced crossover design separated by at least 4 d washout. Visuomotor tracking and math tasks were completed during the preload (n = 10) to evaluate effects on cognition and fine motor performance. RESULTS: Time trial mean power output (298 ± 42 W vs 294 ± 44 W) and performance (1474 ± 77 s vs 1483 ± 85 s) were similar with Tramadol and placebo treatment, respectively. In addition, there were no differences in perceived exertion, reported pain, blood pH, lactate, or bicarbonate concentrations across trials. Heart rate was higher (P < 0.001) during the Tramadol time trial (171 ± 8 bpm) compared with placebo (167 ± 9 bpm). None of the combined motor-cognitive tasks were impaired by Tramadol ingestion, in fact fine motor performance was slightly improved (P < 0.05) in the Tramadol trial compared with placebo. CONCLUSIONS: In highly trained cyclists, ingestion of 100 mg Tramadol does not improve performance in a 15-km cycling time trial that was completed after a 1-h preload at 60% peak power. Additionally, a therapeutic dose of Tramadol does not compromise complex motor-cognitive or simple fine motor performances.

Analysis of new growth promoting black market products.

Detecting agents allegedly or evidently promoting growth such as human growth hormone (GH) or growth hormone releasing peptides (GHRP) in doping controls has represented a pressing issue for sports drug testing laboratories. While GH is a recombinant protein with a molecular weight of 22 kDa, the GHRPs are short (3-6 amino acids long) peptides with GH releasing properties. The endogenously produced GH (22 kDa isoform) consists of 191 amino acids and has a monoisotopic molecular mass of 22,124 Da. Within this study, a slightly modified form of GH was discovered consisting of 192 amino acids carrying an additional alanine at the N-terminus, leading to a monoisotopic mass of 22,195 Da. This was confirmed by top-down and bottom-up experiments using liquid chromatography coupled to high resolution/high accuracy mass spectrometry. Additionally, three analogues of GHRPs were identified as Gly-GHRP-6, Gly-GHRP-2 and Gly-Ipamorelin, representing the corresponding GHRP extended by a N-terminal glycine residue. The structure of these peptides was characterised by means of high resolution (tandem) mass spectrometry, and for Gly-Ipamorelin and Gly-GHRP-2 their identity was additionally confirmed by custom synthesis. Further, established in-vitro experiments provided preliminary information considering the potential metabolism after administration.

Evaluation of uncertainty sources in the determination of testosterone in urine by calibration-based and isotope dilution quantification using ultra high performance liquid chromatography tandem mass spectrometry.

Three quantification methodologies, namely calibration with internal standard (Cal-IS, non-weighted), weighted calibration with internal standard (wCal-IS) and isotope pattern deconvolution (IPD) have been used for the determination of testosterone in urine by LC-MS/MS. Uncertainty has been calculated and compared for the three methodologies through intra- and inter-laboratory reproducibility assays. IPD showed the best performance for the intra-laboratory reproducibility, with RSD and combined uncertainty values below 4% and 9% respectively. wCal-IS showed similar performance, while Cal-IS where not constant and clearly worse at the lowest concentration assayed (2ng/mL) reaching RSD values up to 16%. The inter-laboratory assay indicated similar results although wCal-IS RSD (20%) was higher than IPD (10%) and Cal-IS get worse with RSD higher than 40% for the lowest concentration level. Uncertainty budgets calculated for the three procedures revealed that intercept and slope were the most important factors contributing to uncertainty for Cal-IS. The main factors for wCal-IS and IPD were the volumes of sample and/or standard measured.

Terbutaline Accumulates in Blood and Urine after Daily Therapeutic Inhalation.

PURPOSE: This study investigated pharmacokinetics of terbutaline after single and seven consecutive days of inhalation in exercising trained men. METHODS: Twelve healthy trained men underwent two pharmacokinetic trials comparing single dose (2 mg) and seven consecutive days (2 mg·d) of inhaled terbutaline. After inhalation of terbutaline at each trial, subjects performed 90 min of bike ergometer exercise at 55%-65% of maximal oxygen consumption after which they stayed inactive. Blood and urine samples were collected before and after inhalation of terbutaline. Samples were analyzed by high-performance liquid chromatography-tandem mass spectrometry. RESULTS: Maximum serum concentration of terbutaline (Cmax) (6.4 ± 1.2 vs 4.9 ± 1.2 ng·mL, P = 0.01) (mean ± 95% confidence interval) and area under serum concentration-time curve from 0 to 4 h after inhalation (AUC0-4) (16 ± 3 vs 13 ± 2 ng·mL·h, P ≤ 0.005) were higher after 7 d of inhalation compared with the first day. Seven days of terbutaline inhalation resulted in accumulation of terbutaline in urine, in which total urine excretion of terbutaline was higher after 7 d of inhalation compared with the first day (274 ± 43 vs 194 ± 33 µg, P ≤ 0.001). These differences were partly attributed to systemic accumulation of terbutaline after consecutive days of inhalation, in that baseline serum and urine samples revealed incomplete elimination of terbutaline. CONCLUSION: Terbutaline accumulates in serum and urine after consecutive days of inhalation. For doping control purposes, these observations are of relevance if a urine threshold and decision limit is to be introduced for terbutaline on the World Anti-Doping Agency's list of prohibited substances because asthmatic athletes may use their bronchorelievers for consecutive days.

Black market products confiscated in Norway 2011-2014 compared to analytical findings in urine samples.

Doping agents are widely and illicitly distributed through the Internet. Analysis of these preparations is useful in order to monitor the availability of prohibited substances on the market, and more importantly to predict which substances are expected to be found in urine samples collected from athletes and to aid clinical and forensic investigations. Based on a close collaboration with the Norwegian police and the Norwegian custom authorities, the Norwegian Doping Control Laboratory has performed analyses of confiscated material suspected of containing doping agents. The analyses were performed using gas chromatography (GC) and liquid chromatography (LC) combined with mass spectrometry (MS). The majority (67%) of the analyzed black market products contained anabolic- androgenic steroids (AAS) as expected, whereas peptide- and protein-based doping substances were identified in 28% of the preparations. The Norwegian Doping Control Laboratory receives samples collected from recreational and elite athletes in addition to samples collected in clinical and forensic investigations. The findings in the seized material reflected the findings in the urine samples analyzed regarding the anabolic steroids. Thus, analyzing material seized in Norway may give a good indication of doping agents available on the local market.

MAIIA EPO SeLect-a rapid screening kit for the detection of recombinant EPO analogues in doping control: inter-laboratory prevalidation and normative study of athlete urine and plasma samples.

Recombinant analogues of erythropoietin (EPO), epoetins, have been misused by athletes due to their performance enhancing effect since the first pharmaceutical epoetin was launched in 1987. The current methods for screening urine and plasma samples for the presence of epoetins, IEF and SAR-PAGE, have high sensitivity but are time-consuming to carry out. In an effort to ease and speed up the screening procedure for EPO, MAIIA Diagnostics has developed a combined affinity chromatography and lateral flow immunoassay, MAIIA EPO SeLect, which determines the percentage of migrated isoforms (PMI) of EPO in a sample. The reproducibility of the kit was tested by analyzing a set of negative and positive urine and plasma samples in three different laboratories. All data were analyzed with both curve fit parameters from the individual assay runs, and with lot-specific predefined curve calibration. To get a measure of endogenous variation, a normative study with athlete urine and plasma samples was conducted. The average intra-laboratory variation was 6.7% while the inter-laboratory variation for all samples was calculated to 8.8%. The athlete samples yielded an average PMI and standard deviation of 71.4 ± 7.7 for urine and 83.1 ± 10.2 for plasma, respectively. There were no signs of deviating results from tested effort urines. The results also support the use of predefined curve parameters.

Conformational changes in dopamine transporter intracellular regions upon cocaine binding and dopamine translocation.

The dopamine transporter (DAT), a member of the neurotransmitter:sodium symporter family, mediates the reuptake of dopamine at the synaptic cleft. DAT is the primary target for psychostimulants such as cocaine and amphetamine. We previously demonstrated that cocaine binding and dopamine transport alter the accessibility of Cys342 in the third intracellular loop (IL3). To study the conformational changes associated with the functional mechanism of the transporter, we made cysteine substitution mutants, one at a time, from Phe332 to Ser351 in IL3 of the background DAT construct, X7C, in which 7 endogenous cysteines were mutated. The accessibility of the 20 engineered cysteines to polar charged sulfhydryl reagents was studied in the absence and presence of cocaine or dopamine. Of the 11 positions that reacted with methanethiosulfonate ethyl ammonium, as evidenced by inhibition of ligand binding, 5 were protected against this inhibition by cocaine and dopamine (S333C, S334C, N336C, M342C and T349C), indicating that reagent accessibility is affected by conformational changes associated with inhibitor and substrate binding. In some of the cysteine mutants, transport activity is disrupted, but can be rescued by the presence of zinc, most likely because the distribution between inward- and outward-facing conformations is restored by zinc binding. The experimental data were interpreted in the context of molecular models of DAT in both the inward- and outward-facing conformations. Differences in the solvent accessible surface area for individual IL3 residues calculated for these states correlate well with the experimental accessibility data, and suggest that protection by ligand binding results from the stabilization of the outward-facing configuration. Changes in the residue interaction networks observed from the molecular dynamics simulations also revealed the critical roles of several positions during the conformational transitions. We conclude that the IL3 region of DAT undergoes significant conformational changes in transitions necessary for both cocaine binding and substrate transport.

A novel glutamate transporter blocker, LL-TBOA, attenuates ischaemic injury in the isolated, perfused rat heart despite low transporter levels.

OBJECTIVES: Loss of glutamate from cardiomyocytes during ischaemia may aggravate ischaemia-reperfusion injury in open heart surgery. This may be due to reversal of excitatory amino acid transporters (EAATs). However, the expression of such transporters in cardiomyocytes is ambiguous and quantitative data are lacking. Our objective was to study whether EAATs were expressed in the rat heart and to study whether blocking of transporter operation during cardiac ischaemia could be beneficial. METHODS: We used TaqMan real-time PCR and immunoisolation followed by western blotting to unequivocally identify EAAT subtypes in rat hearts. We used a novel high-affinity non-transportable competitive inhibitor, named LL-TBOA [(2S,3S)-3-(3-(6-(6-(2-(2-(2-(2-(2-aminoethoxy)ethoxy)-ethoxy)ethoxy) acetamido)hexanamido)- hexanamido)-5-(4-(trifluoromethyl)benzamido)benzyloxy) aspartic acid], to block EAAT-mediated transport during global ischaemia and reperfusion of isolated rat hearts. RESULTS: Rat hearts expressed EAAT subtypes 1 and 3, while subtypes 2 and 4 were not detected. Hearts were isolated and perfused with 1.6 µM LL-TBOA for 5 min before 30 min of induced global ischaemia and 60 min of reperfusion (n = 8). Control hearts were perfused either with the solvent dimethylsulfoxide 3.5 mM (n = 7) or with no pretreatment (n = 8). Infarct size was evaluated by triphenyl tetrazolium chloride (TTC) staining. LL-TBOA reduced infarct size from 33 ± 14 to 20 ± 5% (mean ± SD) (P = 0.015). Dimethylsulfoxide alone had no effect (35 ± 2%). Reperfusion arrhythmias were reduced by LL-TBOA (P = 0.009), but not by dimethylsulfoxide alone. CONCLUSION: Rat hearts express EAAT1 and EAAT3, but the mRNA levels are, respectively, ∼ 25 and 200 times lower than in the brain. Addition of LL-TBOA has a beneficial effect against ischaemia-reperfusion injury.

Detection of recombinant EPO in blood and urine samples with EPO WGA MAIIA, IEF and SAR-PAGE after microdose injections.

The misuse of microdoses of performance enhancing drugs like erythropoietin (EPO) constitutes a major challenge in doping analysis. When injected intravenously, the half-life of recombinant human EPO (rhEPO) like epoetin alfa, beta, and zeta is only a few hours and hence, the window for direct detection of rhEPO in urine is small. In order to investigate the detection window for rhEPO directly in blood and urine with a combined affinity chromatography and lateral flow immunoassay (EPO WGA MAIIA), we recruited nine healthy people who each received six intravenously injected microdoses (7.5 IU/kg) of NeoRecormon (epoetin beta) over a period of three weeks. Blood and urine samples were collected in the days following the injections and analyzed with EPO WGA MAIIA as well as the current validated methods for rhEPO; isoelectric focusing (IEF) and sarcosyl polyacrylamide gel electrophoresis (SAR-PAGE). For samples collected 18 h after a microdose, the sensitivity of the EPO WGA MAIIA assay was 100% in plasma and 87.5% in urine samples at the respective 98% specificity threshold levels. In comparison, the sensitivity in plasma and urine was 75% and 100%, respectively, with IEF, and 87.5% in plasma and 100% in urine when analyzed with SAR-PAGE. We conclude that EPO WGA MAIIA is a sensitive assay for the detection of rhEPO, with the potential of being a fast, supplemental screening assay for use in doping analysis.

The density of EAAC1 (EAAT3) glutamate transporters expressed by neurons in the mammalian CNS.

The extracellular levels of excitatory amino acids are kept low by the action of the glutamate transporters. Glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) are the most abundant subtypes and are essential for the functioning of the mammalian CNS, but the contribution of the EAAC1 subtype in the clearance of synaptic glutamate has remained controversial, because the density of this transporter in different tissues has not been determined. We used purified EAAC1 protein as a standard during immunoblotting to measure the concentration of EAAC1 in different CNS regions. The highest EAAC1 levels were found in the young adult rat hippocampus. Here, the concentration of EAAC1 was ∼0.013 mg/g tissue (∼130 molecules µm⁻³), 100 times lower than that of GLT-1. Unlike GLT-1 expression, which increases in parallel with circuit formation, only minor changes in the concentration of EAAC1 were observed from E18 to adulthood. In hippocampal slices, photolysis of MNI-D-aspartate (4-methoxy-7-nitroindolinyl-D-aspartate) failed to elicit EAAC1-mediated transporter currents in CA1 pyramidal neurons, and D-aspartate uptake was not detected electron microscopically in spines. Using EAAC1 knock-out mice as negative controls to establish antibody specificity, we show that these relatively small amounts of EAAC1 protein are widely distributed in somata and dendrites of all hippocampal neurons. These findings raise new questions about how so few transporters can influence the activation of NMDA receptors at excitatory synapses.

Effect of single doses of methoxypolyethylene glycol-epoetin beta (CERA, Mircera™) and epoetin delta (Dynepo™) on isoelectric erythropoietin profiles and haematological parameters.

Erythropoietin (EPO) has been misused in sports for many years due to its performance-enhancing effect. In the last decade, detection of abuse has been possible with isoelectric focusing (IEF) based on the different isoform profiles of endogenous and recombinant EPO. The release of new EPOs on the market, such as the recombinant erythropoietin epoetin delta (Dynepo™) and the chemically modified EPO, CERA (Mircera™) potentially represents analytical challenges to the fight against doping. This study set out to investigate the possibility of and the time window for detecting the administration of a single dose of Dynepo™ and CERA. Our results are in agreement with earlier findings that detection of Dynepo™ is best achieved by combining IEF with SDS-PAGE. Haematological parameters were monitored for possible effects due to the long half-life (130 hours) of CERA in blood. Interestingly, although several haematological parameters were significantly changed after the injection of CERA, the endogenous EPO signal was still present in all collected samples. Due to the long half-life and the large size of the CERA molecule (about 60 kDa), it was uncertain whether CERA would be excreted into urine in detectable amounts unless urine collection was preceded by strenuous physical exercise. We find that CERA can be detected in urine without prior exercise in several, but not all, subjects. CERA is nevertheless best detected in serum with regard to both probability and length of detection, in addition to stability in matrix over time.