EPO transgene detection in dried blood spots for antidoping application

The modification of gene expression to treat diseases is a field of research with exponential growth. As doping in sport closely follows emerging therapies, a surveillance of the modification of gene expression to enhance performance is needed. The gene coding for erythropoietin (EPO) is one target of interest. Since 2010, several protocols have been proposed to identify EPO gene doping by focusing on the presence in blood of a transgene that differ in size from the endogenous gene sequence, normally found in the human DNA. In this work, our aim was to validate an easily applicable method for EPO gene doping detection in dried blood spots (DBS). We evaluated the detection of EPO transgene in 20-μl DBS after the spike of a plasmid carrying the EPO transgene in whole blood. Three different DBS were compared: Nucleic-Card™, Whatman® 903, and the volumetric 20-μl VAMS™. Detection was performed with real-time polymerase chain reaction (PCR) and validated with two Taqman assays (one commercial and one custom) specific for the EPO transgene. The initial testing procedure could be done using one assay (custom) and the confirmation using the second one (commercial Taqman) with a final check of the size of the PCR product. Starting from 20-μl dried blood, 1000 copies of EPO transgene could efficiently be detected with the three types of DBS, VAMS showing a slightly better sensitivity. No loss of sensitivity was observed after 1-month storage of DBS at room temperature. This method could be applied to DBS collected during doping controls and allows reanalysis.     

促红细胞生成素（EPO）联合化疗治疗癌症相关性贫血

目的 :恶性肿瘤化疗的同时联合应用促红细胞生成素 (EPO) ,观察对癌症相关性贫血 (MTA)临床症状的改善效果。方法 :观察组在化疗前 1周开始给予EPO ,对照组只进行化疗不用EPO ,其他辅助治疗两组相同 (包括营养支持、粒细胞刺激因子应用、出血者止血药物的应用、重要脏器的保护等 )。结果与结论 :化疗联合用EPO能预防和治疗MTA。对靠输血提升血红蛋白后化疗的患者可明显减少输血量 ,甚至可停止输血。EPO对MTA的治疗和与化疗所致贫血的预防有肯定的疗效     

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.     

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. Copyright © 2009 John Wiley & Sons, Ltd.     

口服多糖铁复合物联合EPO对血液透析患者贫血的临床疗效观察

目的 观察口服多糖铁复合物（商品名：红源达）联合促红细胞生成素（EPO）对维持性血液透析患者的临床效果。方法 选取2016年1月至12月招远市人民医院收治的40例血液透析患者为研究对象,患者使用EPO的同时服用多糖铁复合物（红源达）,于3月份、7月份和11月份分别对患者进行3次采样收集所需信息。结果 ①口服多糖铁复合物联合EPO干预,血液透析患者三次数据血红蛋白达标率分别为30%、63%、72.5%,Hb≥110 g·L^-1的比例6个月增长了42.5%,从3月份的30%,增长到11月份 72.5%。②口服多糖铁复合物联合EPO干预后,血液透析患者3次数据血清铁蛋白维持在100~500μg·L^-1患者比例分别为17.5%,25%,25%,增长了7.5%,且SF>500μg·L^-1的患者比例由5%下降到0%。结论 口服多糖铁复合物（红源达）联合EPO 治疗维持性血液透析患者,可显著增加患者血红蛋白达标率,值得在临床上推广应用。     

Discovery of prolyl hydroxylase 2 inhibitors with new chemical scaffolds as in vivo active erythropoietin inducers through a combined virtual screening strategy

Hypoxia‐inducible factor (HIF) is identified to be a promising target to mediate the response to hypoxia. Its stability and activation are negatively controlled by prolyl hydroxylase 2 (PHD2). Thus, PHD2 inhibition has been perceived as a promising anti‐anemia therapy. In this study, we carried out a structure‐based virtual screening followed by in vitro and in vivo biological validation, with the goal to identify novel PHD2 inhibitors. As a result, a set of hits with new chemical scaffolds were revealed to be active in vitro for PHD2 inhibition. Compounds 2 and 3 were revealed to be capable of stabilizing HIF‐α and stimulating erythropoietin (EPO) expression in cell‐based assays. Notably, further in vivo assays revealed that 2 was capable of elevating the EPO plasma levels in C57BL/6 mice model. These findings provide new chemical scaffolds for further development of PHD2 inhibitors.     

Monitoring the endogenous steroid profile disruption in urine and blood upon nandrolone administration: An efficient and innovative strategy to screen for nandrolone abuse in entire male horses

Nandrolone (17β‐hydroxy‐4‐estren‐3‐one) is amongst the most misused endogenous steroid hormones in entire male horses. The detection of such a substance is challenging with regard to its endogenous presence. The current international threshold level for nandrolone misuse is based on the urinary concentration ratio of 5α‐estrane‐3β,17α‐diol (EAD) to 5(10)‐estrene‐3β,17α‐diol (EED). This ratio, however, can be influenced by a number of factors due to existing intra‐ and inter‐variability standing, respectively, for the variation occurring in endogenous steroids concentration levels in a single subject and the variation in those same concentration levels observed between different subjects. Targeting an efficient detection of nandrolone misuse in entire male horses, an analytical strategy was set up in order to profile a group of endogenous steroids in nandrolone‐treated and non‐treated equines. Experiment plasma and urine samples were steadily collected over more than three months from a stallion administered with nandrolone laurate (1 mg/kg). Control plasma and urine samples were collected monthly from seven non‐treated stallions over a one‐year period. A large panel of steroids of interest (n = 23) were extracted from equine urine and plasma samples using a C₁₈ cartridge. Following a methanolysis step, liquid‐liquid and solid‐phase extractions purifications were performed before derivatization and analysis on gas chromatography‐tandem mass spectrometry (GC‐MS/MS) for quantification. Statistical processing of the collected data permitted to establish statistical models capable of discriminating control samples from those collected during the three months following administration. Furthermore, these statistical models succeeded in predicting the compliance status of additional samples collected from racing horses. Copyright © 2013 John Wiley & Sons, Ltd.     

Attenuation of cytogenetic effects by erythropoietin in human lymphocytes in vitro and P388 ascites tumor cells in vivo treated with irinotecan (CPT-11)

Erythropoietin (EPO) is a protein widely used against drug induced anemia at cancer patients. Irinotecan (CPT-11) is a genotoxic topoisomerase I inhibitor. We investigated the genotoxic, cytostatic and cytotoxic effects of EPO in the presence and in the absence of CPT-11 in human lymphocytes in vitro and in ascites cells of P388 leukemia in vivo. The levels of genotoxicity, cytostaticity and cytotoxicity were evaluated in human lymphocytes in vitro, and in P388 ascites tumor cells in vivo. The results show that EPO is not genotoxic. Unlikely to EPO, CPT-11 caused severe genotoxic, cytostatic and cytotoxic effects by significantly increasing SCE levels and decreasing PRI and MI values in peripheral lymphocytes in vitro and in P388 ascites tumor cells in vivo. Adding EPO in human lymphocyte cultures in vitro and in P388 leukemia bearing mice in vivo in the presence of CPT-11 decreased SCEs levels and increased PRIs and MIs were observed compared with cells treated either in vitro or in vivo with CPT-11 alone, which shows that EPO protected cells from the toxic action of CPT-11. EPO’s protective action on human peripheral lymphocytes in vitro and P388 cells in vivo from the topoisomerase I inhibitor CPT-11, lead us to propose it as a geno- and cytoprotective agent.     

Worldwide distribution of blood values in elite track and field athletes: Biomarkers of altered erythropoiesis

For the first time, blood samples were collected in all athletes participating in a major sporting event of the International Association of Athletics Federations (IAAF) (Athletics World Championships 2011, Daegu, Korea). All variables obtained from blood analyses were incorporated into the individual blood profiles of each athlete for the so‐called athlete biological passport (ABP). This unprecedented data collection highlighted differences for a few blood biomarkers commonly measured and reported for the ABP on some group of athletes. Subsequently, blood tests analyses for all athletes were repeated during the following World Championships (2013, Moscow, Russia). Both sets of blood tests were then used to set up the distribution of blood values for track and field athletes considering potential confounding factors such as gender, age, discipline, origin of the athlete (continental classification), and time of blood collection. Implementation of well‐defined distribution of blood values will allow to improve the estimation of blood doping prevalence among a specific population of athletes in track and field.     

Identification of recombinant human EPO variants in greyhound plasma and urine by ELISA,LC‐MS/MS and western blotting: a comparative study

The recombinant human erythropoietins epoetin alfa (Eprex®), darbepoetin (Aranesp®) and methoxy polyethylene glycol‐epoetin beta (Mircera®) were administered to greyhounds for 7, 10 and 14 days respectively. Blood and urine samples were collected and analysed for erythropoietin by ELISA, LC‐MS/MS and western blotting. Limits of confirmation in plasma for western blotting and LC‐MS/MS methods ranged from a low of 2.5mIU/mL, and closely matched the sensitivity of ELISA screening. Copyright © 2015 John Wiley & Sons, Ltd.     

促红细胞生成素治疗慢性心衰合并贫血的临床观察

目的:观察在慢性充血性心力衰竭合并贫血时标准治疗基础上加用促红细胞生成素(EPO)的临床疗效。方法:入选的57例伴有贫血的慢性充血性心力衰竭患者随机分为治疗组(30例)和对照组(27例)。对照组给予心衰的标准治疗,治疗组给予标准治疗同时给予EPO和铁剂,观察并比较两组治疗6个月前后Hb、心功能分级、LVEF及BNP。结果:治疗组Hb、心功能分级和LVEF较治疗前有显著改善(P<0.05);两组治疗后血浆BNP水平均下降,两组治疗后血浆BNP比较有差异(P<0.05)。结论:EPO治疗慢性充血性心力衰竭合并贫血患者有效,能改善心功能指标。     

Occupational airborne contamination in south Brazil: 1. Oxidative stress detected in the blood of coal miners

Reactive oxygen species and nitrogen species have been implicated in the pathogenesis of coal dust-induced toxicity. The present study investigated several oxidative stress biomarkers (Contents of lipoperoxidation = TBARS, reduced = GSH, oxidized = GSSG and total glutathione = TG, α-tocopherol, and the activities of glutathione S-transferase = GST, glutathione reductase = GR, glutathione peroxidase = GPx, catalase = CAT and superoxide dismutase = SOD), in the blood of three different groups (n = 20 each) exposed to airborne contamination associated with coal mining activities: underground workers directly exposed, surface workers indirectly exposed, residents indirectly exposed (subjects living near the mines), and controls (non-exposed subjects). Plasma TBARS were increased and whole blood TG and GSH levels were decreased in all groups compared to controls. Plasma α-tocopherol contents showed approximately half the values in underground workers compared to controls. GST activity was induced in workers and also in residents at the vicinity of the mining plant, whilst CAT activity was induced only in mine workers. SOD activity was decreased in all groups examined, while GPx activity showed decreased values only in underground miners, and GR did not show any differences among the groups. The results showed that subjects directly and indirectly exposed to coal dusts face an oxidative stress condition. They also indicate that people living in the vicinity of the mine plant are in health risk regarding coal mining-related diseases.     

Investigations into the analysis of intact drug conjugates in animal sport doping control – Development and assessment of a rapid and economical approach for screening greyhound urine

Animal sport doping control laboratories are constantly reviewing ways in which they can improve their service offering whilst ensuring that they remain economically viable. This paper describes the development and assessment of a rapid and economical method for the detection of intact glucuronide conjugates of three anabolic steroids and their metabolites along with three corticosteroids in canine urine. The analysis of intact drug conjugates for animal sport doping control is generally not performed routinely as it presents a number of analytical challenges, not least of which is the lack of availability of appropriate reference standards. Here, we report the development of a UHPLC–MS/MS method using APCI in the negative ion mode for the detection of intact phase II conjugates, including the importance of in vitro incubations in order to provide appropriate reference materials. Cross‐validation of the developed method demonstrated that the detection capability of the intact phase II conjugates of stanozolol, boldenone, nandrolone, and their metabolites along with the corticosteroids dexamethasone and methylprednisolone was equivalent to that achieved in routine race‐day screens. The new process has been in operation for approximately 2 years and has been used to analyze in excess of 13500 canine urine samples, resulting in a number of positive screening findings. To the best of our knowledge, this is the first reported use of a routine screen for intact drug conjugates within animal sport doping control.     

Maprotiline in the treatment of endogenous depression: Comparison of therapeutic effect with serotonin level in blood platelets

In blood platelets of untreated patients with endogenous depression a significantly lower concentration of serotonin (5-HT) was found than in healthy persons (P less than 0.05). The patients were afterwards divided into two groups: group I with mild symptoms of depression (total scores by Hamilton Depression Rating Scale below 40) and group II with severe depression (total scores above 40). The gradual diminishing of symptoms in both groups was accompanied by no change in 5-HT level in the first group and by progressive and well marked increases in platelet 5-HT content in the second one, attaining average normal values at the end of week 3 of treatment with maprotiline. In in vitro experiments maprotiline inhibited the uptake of 5-HT into blood platelets of healthy persons less than doxepin. After injection of maprotiline in rats (50 mg/kg IP) the turnover rate of 5-HT in the brain was only marginally affected if measured by 5-HT rise and 5-hydroxyindoleacetic acid (5-HIAA) decline after administration of the MAO inhibitor pargyline, or by estimation of 5-HIAA increase after blocking active transport with probenecid. However, after treatment with maprotiline alone, the 5-HIAA level in the rat brain was significantly increased, although the concentration of 5-HT was not altered. These results suggest: a) the possibility of an action of maprotiline on serotoninergic neurons and b) the suitability of blood platelets as a model for such an investigation.     

C.E.R.A.: pharmacodynamics,pharmacokinetics and efficacy in patients with chronic kidney disease

C.E.R.A., a continuous erythropoietin receptor activator, has been developed for the treatment of anaemia in patients with chronic kidney disease. Compared with other erythropoiesis-stimulating agents, C.E.R.A. has a unique pharmacological profile, including a longer elimination half-life and slower clearance rate. This allows C.E.R.A. to be administered at extended intervals up to once every month. Phase III clinical trials have shown that C.E.R.A. once every 2 weeks corrects anaemia in erythropoiesis-stimulating agent-naive patients who are on or are not on dialysis, whereas once-monthly C.E.R.A. maintains stable haemoglobin levels when patients are directly converted from more frequent epoetin or darbepoetin alfa administration. C.E.R.A. is well tolerated. This review summarises clinical data on C.E.R.A. and discusses the potential effect of this novel agent on clinical practice.     

Investigations into the Stabilization of Drugs by Sugar Glasses: III. The Influence of Various High-pH Buffers

Purpose. To study the effect of the high-pH buffers ammediol, borax, CHES, TRIS, and Tricine on the glass transition temperature of the freeze concentrated fraction (Tg) of trehalose/buffer and inulin/buffer solutions at pH 6.0 and pH 9.8. Also, the glass transition temperature (Tg) of sugar glasses obtained after freeze drying of these solutions was elucidated. Additionally, the effect occurring during the freezing process on the pH of the various buffers was investigated. Furthermore, the stability of alkaline phosphatase (AP) incorporated in these sugar glasses prepared from solutions at pH 9.8 was evaluated. Methods. The Tg and Tg were measured using differential scanning calorimetry (DSC), and the change of pH during freezing was estimated by using an indicator solution added to the respective solutions. The enzymatic activity of AP after freeze drying and storage at 60°C was evaluated by an enzymatic activity assay. Results. It was found that the Tg and Tg of the samples investigated are strongly influenced by the presence of the buffer. On freezing, only minor changes of the pH were observed. The samples with the lowest Tg and the samples containing buffers that formed complexes with the sugars showed the poorest stability of the AP. Conclusions. The stabilizing capacities of sugars that are currently recognized as excellent stabilizers for proteins during drying and storage can be completely lost if certain high-pH buffers such as ammediol, borax, and TRIS are used at high concentrations. Loss of stabilizing capacities can be ascribed to strong depression of the Tg and Tg or to complex formation.