Metabolomic Profiling of Recombinant Erythropoietin (rHuEpo) in Trained Caucasian Athletes.

OBJECTIVE: Recombinant human erythropoietin (rHuEpo) is prohibited by the World Anti-Doping Agency but remains the drug of choice for many cheating athletes wishing to evade detection using current methods. The aim of this study was to identify a robust metabolomics signature of rHuEpo using an untargeted approach in blood (plasma and serum) and urine. DESIGN: Longitudinal study. SETTING: University of Glasgow. PARTICIPANTS: Eighteen male participants regularly engaged in predominantly endurance-based activities, such as running, cycling, swimming, triathlon, and team sports, were recruited. INTERVENTIONS: Each participant received 50 IU·kg -1 body mass of rHuEpo subcutaneously every 2 days for 4 weeks. Samples were collected at baseline, during rHuEpo administration (over 4 weeks) and after rHuEpo administration (week 7-10). The samples were analyzed using hydrophilic interaction liquid chromatography mass spectrometry. MAIN OUTCOME MEASURES: Significant metabolic signatures of rHuEpo administration were identified in all biofluids tested in this study. RESULTS: Regarding metabolomics data, 488 plasma metabolites, 694 serum metabolites, and 1628 urinary metabolites were identified. Reproducible signatures of rHuEpo administration across all biofluids included alterations of pyrimidine metabolism (orotate and dihydroorotate) and acyl-carnitines (palmitoyl-carnitine and elaidic carnitine), metabolic pathways that are associated with erythropoiesis or erythrocyte membrane function, respectively. CONCLUSIONS: Preliminary metabolic signatures of rHuEpo administration were identified. Future studies will be required to validate these encouraging results in independent cohorts and with orthogonal techniques, such as integration of our data with signatures derived from other "omics" analyses of rHuEpo administration (eg, transcriptomics).

Improved detection methods significantly increase the detection window for EPO microdoses.

To reproduce a potential doping scenario, a 2 week administration of recombinant erythropoietin (rEPO) microdoses alone or in combination with growth hormone (GH) microdoses (three times a week) was performed on healthy and athletic male subjects. The aim of this study was to evaluate the identification capability of rEPO in samples obtained during and post treatment. Detection was tested in urine and blood using the antidoping techniques for rEPO detection (iso-electric focusing (IEF)-, sodium-dodecyl-sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and for some urine samples the sarcosyl (SAR)-PAGE method) with some improvements: for blood samples, instead of a simple concentration step, immuno-extraction of EPO was performed for all urines to limit protein contamination that can affect migration. In addition, elution buffer modifications also improved the quality of migration. The use of a recently validated biotinylated anti-EPO antibody simplified the protocols, allowing a single transfer step instead of a double-blot even by IEF with a lowered background. The criteria for suspicious blood and urine samples by IEF were also re-evaluated. While endogenous EPO was not decreased over the course of the study, EPO microdoses were detectable in blood and urine between 24 h and 72 h after an administration. Detection in urine in combination with SDS-PAGE was the most sensitive combination for prolonged detection (100% identification after 48 h, 91% after 72 h), slightly better than IEF. Urine samples also tested by SAR-PAGE indicated a similar sensitivity of detection to SDS-PAGE. GH co-administration had no impact on rEPO elimination/detection.

The analytical approach for detection of carbamylated erythropoietin for doping control purposes.

Erythropoietin (EPO) has protective effects in several tissues and could be used for therapeutic purposes, but the doses of EPO that can be beneficial in case of hypoxic-ischemic conditions due to overinduced erythropoiesis could be detrimental in treated patients. Carbamylation of erythropoietin maintains the tissue-protective effects of EPO but without erythropoietic effects. Carbamylated EPO (CEPO) is listed in WADA Prohibited List in class S2 as "Innate repair receptor agonists." The CEPO was synthesized using the method described previously. Digestion with endoproteinase Lys-C was used to distinguish rhEPO from CEPO. The digested samples containing recombinant EPO, urinary EPO (uEPO), or CEPO were analyzed by the SAR-PAGE method (sarcosyl polyacrylamide gel electrophoresis-PAGE). Endoproteinase Lys-C breaks the peptide chains of lysine. Lysine residues, converted to homocitrulline by carbamylation, cannot be cleaved by endoproteinase Lys-C. Therefore, the CEPO protein chain remained unchanged in contrast to rhEPO and uEPO, which allows for easily differentiation of them.

Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation.

The detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue, and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were not detected by direct application but were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except for PEG-bound epoetin ß pegol. The 22 kDa band from an anemic patient's urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo. Severe hypoxia (7% O2, 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules. These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney but not the liver is the main site of Epo production in control and severe hypoxia. Our method will make the tests for Epo doping and detection easy.

Research on spiking rat EPO as internal standard in doping control samples for detection of EPO using SAR-PAGE analysis with biotinylated primary antibody.

According to the current Technical Document (TD) for erythropoietin (EPO), SAR-PAGE is the most commonly applied method for both screening and confirmation procedures. Although this method is effective and robust, it lacks an internal standard (IS) to monitor the efficiency of analysis for each sample covering every step of the whole procedure, including preparation, immunopurification, and western blotting. This internal standard needs to be recognized by both anti-EPO antibodies used for immunopurification and western blotting, respectively. Besides that, the band of IS could not be allowed to interfere with the recognition of all types of targeted EPO and analogs. To meet these two principles, rat EPO was selected. In this study, rat EPO was used to spike both urine and blood samples at the beginning of analysis. After preparation and immunopurification, single blotting was performed with biotinylated AE7A5 as the primary antibody, followed by incubation with streptavidin-coupled HRP. Based on the comparison of different immunopurification methods, the AB-286-NA antibody coupled to M-280 magnetic beads was the better choice for urine samples, whereas the MAIIA column was suitable for blood samples. All these methods were validated for selectivity, repeatability, and sensitivity. The modified method in this study could not only eliminate the cross-reactivity between antibodies but also monitor the whole procedure of the analysis of EPO with spiked rat EPO. Besides that, rat EPO could also be used as an indicator for monitoring the presence of protease(s) in urine samples.

Comparison and optimization of SAR-PAGE tests for erythropoietins in doping analysis.

Erythropoietins (EPOs) are substances listed in S2 of the World Anti-Doping Agency (WADA) Prohibited List and are used commonly by athletes to increase endurance performance. According to the current WADA Technical Documents, sarcosyl-polyacrylamide gel electrophoresis (SAR-PAGE) followed by western blotting to differentiate erythropoietins based on their molecular weights is the only method that can be used for both screening and confirmation of all types of erythropoietins. The efficiency of immunopurification and protein transfer is crucial for ensuring the selectivity and sensitivity of erythropoietin detection. Several comparisons and optimization of the SAR-PAGE tests were conducted in this study. We optimized the first blotting conditions and then compared different immunopurification methods based on their selectivity, repeatability, and sensitivity for both urine and blood analysis. Additionally, rapid procedures for both urine and blood analysis were established and compared. The two-step procedure at 1.0 mA/cm2 for 60 min followed by 1.56 mA/cm2 for 20 min increased the blotting efficiency compared with the commonly used constant current approach. Comparison of immunopurification revealed no significant difference in selectivity and sensitivity between the different methods. For other factors, such as operation complexity, time and cost, a StemCell® purification kit followed by single blotting and magnetic beads followed by double blotting are recommended for urine screening and confirmation, respectively. While magnetic beads and a MAIIA® kit followed by double blotting are recommended for both screening and confirmation of blood samples, respectively. To ensure high sensitivity and selectivity, double blotting is recommended for a rapid procedure for both urine and blood analysis.

A simple method to immunopurify erthyropoiesis stimulating agents from urine, aiming to optimize erythropoietin screening by SAR-PAGE.

The efficiency of the immunopurification step of urinary erythropoietin (EPO) and recombinant forms is important for their optimal detection in antidoping screening. Previous investigations of immunopurification techniques have been done for immunomagnetic beads, EPO Purification Kit (EPK) columns (MAIIA Diagnostics), and enzyme-linked immunosorbent assay (ELISA) microplates (Stemcell Technologies) conjugated/coated with anti-EPO antibodies. In this study, a new immunopurification technique using anti-EPO sepharose gel beads, developed by MAIIA Diagnostics, to simplify and minimize sample handling was evaluated. This EPO Purification Gel Kit (EPGK) was compared with our current routine EPK for limit of detection (LOD). Linearity, recovery, repeatability, sample incubation time, and sample volume were also evaluated for EPGK. The LODs and linearity for EPK and EPGK were comparable to each other and the recovery for BRP, NESP, CERA, and EPO-Fc were within the range of other studies, and concentration of the sample eluate improved the recovery results. Little variation was seen within days, between days, and between operators. A 90 minute incubation of the sample with the sepharose gel beads is sufficient for most of the erythropoiesis stimulating agents (ESAs) tested, with 10 mL being an optimal sample volume for EPGK. The improved sample handling, higher sample throughput and the reduced working time demonstrate that the EPGK is a better alternative to the current MAIIA EPK immunopurification method for urine. The EPO Purification Gel Kit (from MAIIA Diagnostics) was evaluated and validated for immunopurification of endogenous erythropoietin and exogenous erythropoiesis stimulating agents from urine samples. The kit was a better alternative to that currently used (EPO Purification Kit) in many antidoping laboratories because it improves sample handling and increases sample throughput.

Sensitivity and specificity of detection methods for erythropoietin doping in cyclists.

Recombinant human erythropoietin (rHuEPO) is used as doping a substance. Anti-doping efforts include urine and blood testing and monitoring the athlete biological passport (ABP). As data on the performance of these methods are incomplete, this study aimed to evaluate the performance of two common urine assays and the ABP. In a randomized, double-blinded, placebo-controlled trial, 48 trained cyclists received a mean dose of 6000 IU rHuEPO (epoetin ß) or placebo by weekly injection for eight weeks. Seven timed urine and blood samples were collected per subject. Urine samples were analyzed by sarcosyl-PAGE and isoelectric focusing methods in the accredited DoCoLab in Ghent. A selection of samples, including any with false presumptive findings, underwent a second sarcosyl-PAGE confirmation analysis. Hematological parameters were used to construct a module similar to the ABP and analyzed by two evaluators from an Athlete Passport Management Unit. Sensitivity of the sarcosyl-PAGE and isoelectric focusing assays for the detection of erythropoietin abuse were 63.8% and 58.6%, respectively, with a false presumptive finding rate of 4.3% and 6%. None of the false presumptive findings tested positive in the confirmation analysis. Sensitivity was highest between 2 and 6 days after dosing, and dropped rapidly outside this window. Sensitivity of the ABP was 91.3%. Specificity of the urine assays was high; however, the detection window of rHuEPO was narrow, leading to questionable sensitivity. The ABP, integrating longitudinal data, is more sensitive, but there are still subjects that evade detection. Combining these methods might improve performance, but will not resolve all observed shortcomings.

Droplet Digital PCR Detection of the Erythropoietin Transgene from Horse Plasma and Urine for Gene-Doping Control.

Indiscriminate genetic manipulation to improve athletic ability is a major threat to human sports and the horseracing industry, in which methods involving gene-doping, such as transgenesis, should be prohibited to ensure fairness. Therefore, development of methods to detect indiscriminate genetic manipulation are urgently needed. Here, we developed a highly sensitive method to detect horse erythropoietin (EPO) transgenes using droplet digital PCR (ddPCR). We designed two TaqMan probe/primer sets, and the EPO transgene was cloned into a plasmid for use as a model. We extracted the spiked EPO transgene from horse plasma and urine via magnetic beads, followed by ddPCR amplification for absolute quantification and transgene detection. The results indicated high recovery rates (at least ~60% and ~40% in plasma and urine, respectively), suggesting successful detection of the spiked transgene at concentrations of >130 and 200 copies/mL of plasma and urine, respectively. Additionally, successful detection was achieved following intramuscular injection of 20 mg of the EPO transgene. This represents the first study demonstrating a method for detecting the EPO transgene in horse plasma and urine, with our results demonstrating its efficacy for promoting the control of gene-doping in the horseracing industry.

Detection of ESAs in equine urine and blood by SAR-PAGE.

Erythropoiesis-stimulating agents (ESAs) have been used in horses for doping purposes to increase the performance of these animals in endurance sports. Currently, enzyme-linked immunosorbent assay (ELISA) and mass spectrometry methods are used to detect ESA abuse in equines. However, the sarcosyl polyacrylamide gel-electrophoresis (SAR-PAGE) technique could also be used, since its application in human doping control is well established and has proven to be more sensitive. In this work, the SAR-PAGE method was used to detect recombinant human erythropoietin (rHuEPO), novel erythropoiesis stimulating protein (NESP), continuous erythropoietin receptor activator (CERA), and fusion protein of erythropoietin with human immunoglobulin heavy chain Fc region (EPO-Fc) in horse blood and urine. The purification technique for human blood using MAIIA kits worked well for horse samples. The major challenge was horse urine immunopurification, which proved difficult due to filter clogging, but heating and cooling of the horse urine followed by filtration in 30-kDa molecular weight cut-off filters solved this problem. The limits of detection (LODs) of 1.3, 1.6, 6.6, and 13.3 pg/mL for rHuEPO, NESP, CERA, and EPO-Fc, respectively, obtained in spiked urine and 40, 100, 80, and 400 pg/mL for rHuEPO, NESP, CERA, and EPO-Fc, respectively, acquired in spiked blood are lower than the LODs reported in the literature using liquid chromatography-mass spectrometry (LC-MS) methods. In addition, the presence of ESAs was detected up to 9 days after the administration of microdoses of Hemax (rHuEPO), NESP, and CERA in horse blood and urine. SAR-PAGE may be implemented in the routine analysis of horse doping control laboratories for screening and confirmation of ESA abuse, mainly due to its high sensitivity for both matrices compared to published mass spectrometric methods.

SARCOSYL-PAGE: Optimized Protocols for the Separation and Immunological Detection of PEGylated Proteins.

PEGylation of recombinant proteins and synthetic peptides aims to generate biopharmaceuticals with altered physical properties. The modification may lead to a prolonged serum half-life caused by decreased receptor-mediated endocytosis and/or delay in renal clearance caused by the increased hydrodynamic volume of the pharmaceutical. MIRCERA, a PEGylated recombinant erythropoietin (rhEPO) used in the treatment of anemia due to chronic kidney disease, has also been abused by athletes as performance-enhancing drug. While it can be detected by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, the sensitivity of the test is significantly lower compared to other epoetins. By replacing SDS with sarcosyl in the sample and running buffers, the interaction between SDS and the PEG group of the protein no longer reduces the affinity of the monoclonal anti-EPO antibody (clone AE7A5) to the protein chain. Contrary to SDS, sarcosyl only binds to the amino acid chain of the PEGylated protein and thus leads to a sharper electrophoretic band and enhanced antibody binding. While the method was originally developed for anti-doping purposes, it may also be useful for the electrophoretic separation and immunological detection of other PEGylated proteins. Protocols for urine and serum are presented. They are also applicable for the general detection of EPO-based erythropoiesis-stimulating agents (ESA) in these matrices.

Detection of erythropoiesis stimulating agents in urine samples using a capillary Western system.

The presence of erythropoiesis stimulating agents (ESAs) in the urine samples collected from athletes is detected using traditional Western blotting following either size-based separation (SDS/SAR-PAGE) or isoelectric focusing (IEF). Although there is an important testing effort, there is little doubt that ESAs are still abused in sports and that reducing the costs of the tests might increase the number of tests and improve deterrence. The capillary electrophoresis system developed by Protein Simple may be useful to this end. This platform is fully automated and could be easily implemented in anti-doping laboratories, which would contribute to the improvement of the overall assay performance and standardization of the method. Such an automated system could be of interest during major sports events, such as the Olympic Games, where a high number of samples needs to be analyzed in a short period of time. From the experiments conducted so far, we conclude that the technique is promising, with the sensitivity and reproducibility needed to screen ESAs in human urine samples.

El síndrome de apneas-hipoapneas del sueño (SAHS) grave incrementa la excreción urinaria de eritropoyetina. Efecto del tratamiento con CPAP / Increased Urinary Erythropoietin Excretion in Severe Sleep Apnea-Hipoapnea Syndrome: The Effect of CPAP

Introducción: La hipoxia tisular estimula la producción de eritropoyetina (EPO) que tiene como principal función estimular la eritropoyesis. El SAHS es una entidad caracterizada por la presencia de episodios repetidos de hipoxemia durante el sueño. Objetivo: Analizar si dicha hipoxemia es un estímulo suficiente para incrementar la excreción urinaria de EPO. Si la respuesta fuera positiva, valorar si el tratamiento con presión continua positiva de la vía aérea (CPAP) la inhibiría. Métodos: Se han estudiado 25 sujetos con sospecha de SAHS, a los que se les realizó un estudio polisomnográfico. En todos ellos se determinaron los niveles de EPO en la primera orina de la mañana (uEPO), así como los niveles de creatinina y hemoglobina en sangre. En los pacientes con SAHS grave se repitieron las mismas determinaciones tras el tratamiento con CPAP. Resultados: Doce sujetos fueron diagnosticados de SAHS grave (media ± SD, IAH de 53,1 ± 22,7). La creatinina y la hemoglobina fueron normales en todos los sujetos. La uEPO fue cuatro veces superior en el grupo SAHS respecto a los controles (1,32 ± 0,83 vs. 0,32 ± 0,35 IU/l, p < 0,002). El tratamiento con CPAP descendió la uEPO hasta 0,61 ± 0,49 IU/l (p < 0,02), acercándose al valor de los sujetos sanos. No se observó una relación dosis-respuesta entre la gravedad de las alteraciones de la PSG y los valores de uEPO. Conclusiones: Los pacientes con SAHS grave muestran un incremento en su excreción de uEPO, que se normaliza tras el tratamiento con CPAP

Introduction: Tissue hypoxia stimulates the production of erythropoietin (EPO), the main effect of which is, in turn, to stimulate erythropoiesis. Sleep apnea-hypopnea syndrome (SAHS) is an entity characterized by repeated episodes of hypoxemia during sleep. Objective: To analyze whether hypoxemia stimulated increased urinary excretion of EPO, and if so, to evaluate if treatment with continuous positive airway pressure (CPAP) can inhibit this phenomenon. Methods: We studied 25 subjects with suspected SAHS who underwent a polysomnography study (PSG). EPO levels in first morning urine (uEPO) and blood creatinine and hemoglobin were determined in all patients. Patients with severe SAHS repeated the same determinations after CPAP treatment. Results: Twelve subjects were diagnosed with severe SAHS (mean ± SD, AHI 53.1 ± 22.7). Creatinine and hemoglobin levels were normal in all subjects. uEPO was 4 times higher in the SAHS group than in the control group (1.32 ± 0.83 vs. 0.32 ± 0.35 UI/l, p <.002). CPAP treatment reduced uEPO to 0.61 ± 0.9 UI/l (p <.02), levels close to those observed in healthy subjects. No dose-response relationship was observed between severity of PSG changes and uEPO values. Conclusions: Patients with severe SAHS show increased uEPO excretion, but this normalizes after treatment with CPAP

Increased Urinary Erythropoietin Excretion in Severe Sleep Apnea-Hipoapnea Syndrome: The Effect of CPAP. / El síndrome de apneas-hipoapneas del sueño (SAHS) grave incrementa la excreción urinaria de eritropoyetina. Efecto del tratamiento con CPAP.

INTRODUCTION: Tissue hypoxia stimulates the production of erythropoietin (EPO), the main effect of which is, in turn, to stimulate erythropoiesis. Sleep apnea-hypopnea syndrome (SAHS) is an entity characterized by repeated episodes of hypoxemia during sleep. OBJECTIVE: To analyze whether hypoxemia stimulated increased urinary excretion of EPO, and if so, to evaluate if treatment with continuous positive airway pressure (CPAP) can inhibit this phenomenon. METHODS: We studied 25 subjects with suspected SAHS who underwent a polysomnography study (PSG). EPO levels in first morning urine (uEPO) and blood creatinine and hemoglobin were determined in all patients. Patients with severe SAHS repeated the same determinations after CPAP treatment. RESULTS: Twelve subjects were diagnosed with severe SAHS (mean ± SD, AHI 53.1 ± 22.7). Creatinine and hemoglobin levels were normal in all subjects. uEPO was 4 times higher in the SAHS group than in the control group (1.32 ± 0.83 vs. 0.32 ± 0.35 UI/l, p <.002). CPAP treatment reduced uEPO to 0.61 ± 0.9 UI/l (p <.02), levels close to those observed in healthy subjects. No dose-response relationship was observed between severity of PSG changes and uEPO values. CONCLUSIONS: Patients with severe SAHS show increased uEPO excretion, but this normalizes after treatment with CPAP.

Residual renal function in chronic dialysis is not associated with reduced erythropoietin-stimulating agent dose requirements: a cross-sectional study.

BACKGROUND: Anaemia is a very common problem in patients with end-stage kidney disease (ESKD) and the use of erythropoietin-stimulating agents (ESA) has revolutionised its treatment. Residual renal function (RRF) is associated with a reduction in ESA resistance and mortality in chronic dialysis. The primary aim was to establish whether RRF has an association with ESA dose requirements in ESKD patients receiving chronic dialysis. METHODS: A single center, cross-sectional study involving 100 chronic dialysis patients was conducted from December 2015 to May 2016. Participants were divided into two groups depending on presence of RRF, which was defined as a 24-h urine sample volume of ≥ 100 ml. Erythropoietin resistance index [ERI = total weekly ESA dose (IU)/weight (kg)/haemoglobin concentration (g/dL] was used as a measure of ESA dose requirements. RESULTS: There was no difference in ERI between those with RRF as compared to those without (9.5 versus 11.0, respectively; P = 0.45). Also, ERI did not differ between those receiving haemodialysis as compared with peritoneal dialysis (10.8 versus 10.2, respectively; P = 0.84) or in those using renin-angiotensin system (RAS) blockers as compared with no RAS blocker use (11.6 versus 9.2, respectively; P = 0.10). Lower ERI was evident for those with cystic kidney disease as compared to those with other causes of ESKD (6.9 versus 16.5, respectively; P = 0.32) although this did not reach statistical significance. Higher ERI was found in those with evidence of systemic inflammation as compared to those without (16.5 versus 9.5, respectively; P = 0.003). CONCLUSIONS: There was no association between RRF and ESA dose requirements, irrespective of dialysis modality, RAS blocker use, primary renal disease or hyperparathyroidism.

Liquid chromatography - high resolution mass spectrometry-based metabolomic approach for the detection of Continuous Erythropoiesis Receptor Activator effects in horse doping control.

Erythropoiesis Stimulating Agents (ESAs) were developed for therapeutic purposes to stimulate red blood cell (RBC) production. Consequently, tissue oxygenation is enhanced as athlete's endurance and ESAs misuse now benefits doping. Our hypothesis is that most of ESAs should have similar mechanisms and thus have the same effects on metabolism. Studying the metabolome variations could allow suspecting the use of any ESAs with a single method by targeting their effects. In this objective, a metabolomic study was carried out on 3 thoroughbred horses with a single administration of 4.2µg/kg of Mircera®, also called Continuous Erythropoiesis Receptor Activator (CERA). Blood and urine samples were collected from D-17 to D+74 and haematological parameters were followed throughout the study as plasmatic CERA concentration (ELISA). Urine and plasma metabolic fingerprints were recorded by Liquid Chromatography coupled to High Resolution Mass Spectrometry (LC-HRMS) in positive and negative mode. After preprocessing steps, normalized data were analyzed by multivariate statistics to build OPLS models. Hemoglobin concentration and hematocrit showed a significant increase after CERA administration unlike reticulocytes. CERA concentration showed a high intensity peak and then a slow decrease until becoming undetectable after D+31. Models built with multivariate statistics allow a discrimination between pre and post-administration plasma and urine samples until 74days after administration, i.e. 43days longer than ELISA method. By reducing and studying variables (ions), some potential candidate biomarkers were found.

Anemia in nephrotic syndrome: approach to evaluation and treatment.

Nephrotic syndrome is one of the most common glomerular diseases that affect in children. Complications may occur in nephrotic syndrome as a result of the disease itself as well as its treatment. Most of these complications result from excessive urinary protein losses, and control of proteinuria is the most effective treatment strategy. Anemia is one of the many complications seen in patients with persistent nephrotic syndrome and may occur as a result of excessive urinary losses of iron, transferrin, erythropoietin, transcobalamin and/or metals. This leads to a deficiency of substrates necessary for effective erythropoiesis, requiring supplementation in order to correct the anemia. Supplementation of iron and erythropoietin alone often does not lead to correction of the anemia, suggesting other possible mechanisms which need further investigation. A clear understanding of the pathophysiologic mechanisms of anemia in nephrotic syndrome is necessary to guide appropriate therapy, but only limited evidence is currently available on the precise etiologic mechanisms of anemia in nephrotic syndrome. In this review we focus on the current state of knowledge on the pathogenesis of anemia in nephrotic syndrome.

Doping control container for urine stabilization: a pilot study.

Urine collection containers used in the doping control collection procedure do not provide a protective environment for urine, against degradation by microorganisms and proteolytic enzymes. An in-house chemical stabilization mixture was developed to tackle urine degradation problems encountered in human sport samples, in cases of microbial contamination or proteolytic activity. The mixture consists of antimicrobial substances and protease inhibitors for the simultaneous inactivation of a wide range of proteolytic enzymes. It has already been tested in lab-scale, as part of World Anti-Doping Agency's (WADA) funded research project, in terms of efficiency against microbial and proteolytic activity. The present work, funded also by WADA, is a follow-up study on the improvement of chemical stabilization mixture composition, application mode and limitation of interferences, using pilot urine collection containers, spray-coated in their internal surface with the chemical stabilization mixture. Urine in plastic stabilized collection containers have been gone through various incubation cycles to test for stabilization efficiency and analytical matrix interferences by three WADA accredited Laboratories (Athens, Ghent, and Rome). The spray-coated chemical stabilization mixture was tested against microorganism elimination and steroid glucuronide degradation, as well as enzymatic breakdown of proteins, such as intact hCG, recombinant erythropoietin and small peptides (GHRPs, ipamorelin), induced by proteolytic enzymes. Potential analytical interferences, observed in the presence of spray-coated chemical stabilization mixture, were recorded using routine screening procedures. The results of the current study support the application of the spray-coated plastic urine container, in the doping control collection procedure. Copyright © 2016 John Wiley & Sons, Ltd.

Surface-enhanced Raman probe for rapid nanoextraction and detection of erythropoietin in urine.

We present a surface-enhanced Raman probe (SERS) platform for the determination of a prohibited substance, recombinant erythropoietin (rEPO), in urine matrix, using nanoparticles as substrate. Rod-shaped gold nanoparticles (GNR) were modified with a Raman label and an antibody as SERS probe. We developed two SERS-based immunoassays for detection and quantification of rEPO in urine. In the first assay, rEPO was determined by a sandwich assay with gold surfaces and GNR. In the second assay, rEPO was extracted by using core shell-structured magnetic iron oxide gold nanoparticles, and again sandwich assay was performed by using GNR. We also demonstrated the ability of the proposed method to discriminate rEPO and urinary erythropoietin (uEPO). A good linear correlation was obtained between logarithms of rEPO concentrations in urine and Raman intensities within the range of 10-1-103 pg mL-1 rEPO concentrations. Detection limits which are smaller than 0.1 pg mL-1 levels were achieved owing to the high extractive performance of the nanoextraction techniques. Graphical Abstract Schematic represantation of surface-enhanced Raman probe for rapid nanoextraction and detection of erythropoietin.

Analytics of nonpeptidic erythropoietin mimetic agents in sports drug testing employing high-resolution/high-accuracy liquid chromatography-mass spectrometry.

Since its release as anti-anemic drug, recombinant erythropoietin (rEPO) gradually entered the illicit way to sports competitions as endurance-enhancing drug. Novel modifications biopharmaceutically introduced into the rEPO molecule in the form of carbohydrate or polyethylene glycol moieties made robust and sensitive test methods vital to doping controls in order to provide the necessary tools enabling the conviction of dishonest athletes. Modern protein analysis by means of gel electrophoretic separation and western blotting represents the status quo in rEPO anti-doping analysis. However, new therapeutically promising erythropoietin receptor activating compounds have been developed that exhibit cytokine hormone-mimicking properties but lack any protein structure. Progression to evade parenteral application and substitute for rEPO by low molecular mass and orally available compounds is still one of the major objectives in pharmaceutical research. In this approach, four promising in-house synthesized nonpeptidic erythropoietin mimetic agents, namely compound 129, compound 163, A1B10C1, and A5B10C4 were thoroughly evaluated by employing high-resolution/high-accuracy liquid chromatography tandem mass spectrometry experiments. Characteristic product ions were determined supporting the identification of these drugs and putative metabolites as well as related compounds in future doping controls. Test methods employing direct urine injection and receptor affinity purification strategies were assessed, which demonstrated that EPO receptor purification is of limited utility for nonpeptidic EPOR agonists while direct urine injection allowed for comprehensive method characterization. Thereby, achieved limits of detection were 1 ng/mL for compounds 129/163 and 5 ng/mL for A1B10C1/A5B10C4.