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Monitoring recombinant human erythropoietin abuse among athletes
... 20 Since recombinant human erythropoietin (rHuEPO) became available in the 1980s, 3 its use in sports has increased considerably, leading to its ban in sports in official listings in the early 1990s. 19,20 The use of EPO and ESAs has been reported in different sports, such as the well-known cases in cycling, 21 athletics, 22 and more recently mixed martial arts (MMA). 23 Growth hormone (GH) is a polypeptide hormone secreted by the pituitary gland. ...
Peptides are very diverse molecules that can participate in a wide variety of biological processes. In this way, peptides are attractive for doping, since these molecules can activate or trigger biological processes that can improve the sports performance of athletes. Peptide molecules are found in the official World Anti-Doping Agency lists, mainly in sections S2, S4, and S5. In most cases, these molecules have a very short half-life in the body and/or are identical to natural molecules in the body, making it difficult to analyze them as performance-enhancing drugs. This article reviews the role of peptides in doping, with special emphasis on the peptides used as reference materials, the pretreatment of samples in biological matrices, the instrumentation, and the validation of analytical methodologies for the analysis of peptides used in doping. The growing need to characterize and quantify these molecules, especially in complex biological matrices, has generated the need to search for robust strategies that allow for obtaining sensitive and conclusive results. In this sense, strategies such as solid phase peptide synthesis (SPPS), seeking to obtain specific peptides, metabolites, or isotopically labeled analogs, is a key tool for adequate quantification of different peptide molecules in biological matrices. This, together with the use of optimal methodologies for sample pretreatment (e.g., SPE or protein precipitation), and for subsequent analysis by high-resolution techniques (mainly hyphenated LC-HRMS techniques), have become the preferred instrumentation to meet the analytical challenge involved in the analysis of peptides in complex matrices.
... Nevertheless, if aptamer tissue penetration is lacking, conjugating the aptamer with large molecular weight compounds such as cholesterol will increase the size of the affinity ligand and prevents it from being filtered through the glomerulus thereby retaining it in the circulation for much longer [109,110]. ...
The blood-brain barrier (BBB) is a highly specialised network of blood vessels that effectively separates the brain environment from the circulatory system. While there are benefits, in terms of keeping pathogens from entering the brain, the BBB also complicates treatments of brain pathologies by preventing efficient delivery of macromolecular drugs to diseased brain tissue. Although current non-invasive strategies of therapeutics delivery into the brain, such as focused ultrasound and nanoparticle-mediated delivery have shown various levels of successes, they still come with risks and limitations. This review discusses the current approaches of therapeutic delivery into the brain, with a specific focus on non-invasive methods. It also discusses the potential for aptamers as alternative delivery systems and several reported aptamers with promising preliminary results.
... Interestingly, the plasma cortisol level was recently shown to be positively correlated to the erythropoietin (EPO) level in healthy human volunteers [13]. EPO is a glycoprotein controlling early events of erythropoiesis in the bone marrow thereby increasing the number of red blood cells [14]. In this context, it could be interesting to measure serum EPO levels in piglets from both populations. ...
Background
The sustainability of farming and animal welfare requires the reconsideration of current selection schemes. In particular, implementation of new selection criteria related to animal health and welfare should help to produce more robust animals and to reduce anti-microbial use. The hypothalamo-pituitary-adrenocortical (HPA) axis plays a major role in metabolic regulation and adaptation processes and its activity is strongly influenced by genetic factors. A positive association between HPA axis activity and robustness was recently described. To explore whether selecting pigs upon HPA axis activity could increase their robustness, a divergent selection experiment was carried out in the Large White pig breed. This allowed the generation of low (HPAlo) and high (HPAhi) responders to adrenocorticotropic hormone administration.
Results
In this study, we compared 23 hematologic and immune parameters of 6-week-old, HPAlo and HPAhi piglets and analysed their response to a low dose of lipopolysaccharide (LPS) two weeks later. At six weeks of age, HPAhi piglets displayed greater red blood cell and leucocyte number including CD8α⁺ γδ cells, cytotoxic T lymphocytes, naive T helper (Th) cells and B lymphocytes as compared to HPAlo individuals. The ability of blood cells to secrete TNFα in response to LPS ex vivo was higher for HPAhi pigs. At week eight, the inflammatory response to the LPS in vivo challenge was poorly affected by the HPA axis activity.
Conclusions
Divergent selection upon HPA axis activity modulated hematologic and immune parameters in 6-week-old pigs, which may confer an advantage to HPAhi pigs at weaning. However, HPAlo and HPAhi piglets did not exhibit major differences in the parameters analysed two weeks later, i. e. in 8-week-old pigs. In conclusion, chronic exposure to high cortisol levels in HPAhi pigs does not negatively impact immunity.
... Recombinant human erythropoietin (epoetin alfa and darbepoetin) can also be used to increase the oxygen-carrying capacity of blood and to boost the performance in sports such as running, cycling, and skiing where endurance is the key for performance. [23] Human chorionic gonadotropin is a glycoprotein hormone secreted by placenta and responsible for the development of sexual glands. It is used to raise gonadal testosterone synthesis during and after self-administration of testosterone or anabolic steroids. ...
Abuse of substances or methods to enhance the performance is becoming very common in the sports, which often destroys the spirit of competition. The regulatory bodies for sports have reported rates ranging from 5% to 31% for the use of performance-enhancing substances among athletes. Athletes can have serious injuries and morbidities, leading to poor health with the use of such substances. Commonly abused agents in sports include anabolic–androgenic steroids and its analogs, blood, erythropoietin, growth hormone and its derivatives, nutritional supplements, creatine, amphetamines, beta-hydroxy-beta-methylbutyrate (HMB), stimulants, and analgesics. Health-care professionals need to be careful while prescribing medicines to sportspersons. Knowledge of exercise physiology, pharmacology of the commonly used agents for sports-related injuries, and agents used for doping could help the sportspersons and health-care professionals to avoid the embarrassment arising because of misuse of these agents. Sports pharmacology includes study of the various aspects of the drug use and abuse in sports and treatment of sports-related injuries. Focusing on sports pharmacology in the medical curriculum can help the upcoming health-care professionals to support the sportspersons to improve the quality of their life by using various drugs and other substances within the standardized limits and avoid embarrassment of doping.
... Although all the proteins that were identified in the presented case-reports (see Table 2) were identified by elucidation of their primary structure, multiple (therapeutic) proteins contain PTMs (e.g., glycosylations) which may be essential or may alter their biological functionality. Therefore, analytical methods have been proposed in literature for hCG [85], and for EPO in biological fluids regarding its use as doping agent [86]. Specifically, in the case of EPO, electrophoretic techniques, immunoassays and the analysis of glycans with IEC post selective enzymatic cleavage, were used to distinguish recombinant versions from endogenous EPO. ...
... The direct method mainly concerns the identification of the use of recombinant human erythropoietin (rHuEPO), which stimulates red blood cell production. The rHuEPO detection methods currently exist, including measurement of hematologic parameters, use of peptide markers, isoelectric focusing (IEF) double immunoblotting, aptamer/antibody based methods [76]. The indirect methods are used in order to highlight e.g. the autologous blood transfusion. ...
Eukaryotic genomes transcribe up to 90% of the genomic DNA but only 1-2% of these transcripts encode for proteins, whereas the vast majority are transcribed as non-coding RNAs (ncRNAs). They are divided into short ncRNA, particularly micro-RNA (miRNA) and small interference RNA (siRNA), and long ncRNAs. Noteworthy, they are unexpectedly stable since they are protected from degradation through different mechanisms: package in exosomes/microvesicles structures, in apoptotic bodies, in HDL lipoprotein, or by RNA binding proteins. For several years already, biomarkers have been used to detect biological disease; in the last years, a requirement appeared to find some of them to unearth the signs of doping. The potential of ncRNAs as a biological candidate is strongly debated and it seems to have become the right tool in the anti-doping hands. In the recent years, the next-generation sequencing (NGS) technology was used by the World Anti-Doping Agency to draft the athlete biological passport (ABP), measuring the circulating miRNAs and applying these new biomarkers in anti-doping. NGS technology does not require any prior knowledge of ncRNAs, but the limit to employ this biomarker to detect performance-enhancing drug use must consider the intrinsic and extrinsic factors that might affect measurements.
... L'hGH aumenta la massa corporea magra dopo settimane di somministrazione; ma la maggior parte del cambiamento avviene nel comparto cellulare acquoso e non nella massa cellulare corporea. L'hGH di solito non è somministrato singolarmente ma spesso in combinazione con androgeni e gli effetti avversi sono dose-correlati sia negli uomini che nelle donne (Hoffman et al., 2009 (Citartan et al., 2015). ...
Accurate and sensitive determination of recombinant glycoproteins is in great demand for the treatment of anemia-induced chronic kidney disease and the illegal use of doping agents in sports. In this study, an antibody and enzyme-free electrochemical method for the detection of recombinant glycoproteins was proposed via the sequential chemical recognition of hexahistidine (His6) tag and glycan residue on the target protein under the cooperation interaction of nitrilotriacetic acid (NTA)-Ni2+complex and boronic acid, respectively. Specifically, NTA-Ni2+ complex-modified magnetic beads (MBs-NTA-Ni2+) are employed to selectively capture the recombinant glycoprotein through the coordination interaction between His6 tag and NTA-Ni2+ complex. Then, boronic acid-modified Cu-based metal-organic frameworks (Cu-MOFs) were recruited by glycans on the glycoprotein via the formation of reversible boronate ester bonds. MOFs with abundant Cu2+ ions acted as efficient electroactive labels to directly produce amplified electrochemical signals. By using recombinant human erythropoietin as a model analyte, this method showed a wide linear detection range from 0.01 to 50 ng/mL and a low detection limit of 5.3 pg/mL. With the benefits from the simple operation and low cost, the stepwise chemical recognition-based method shows great promise in the determination of recombinant glycoproteins in the fields of biopharmaceutical research, anti-doping analysis and clinical diagnosis.
The rules of fair play in sport generally prohibit the use of performance-enhancing drugs (PEDs). The World Anti-Doping Agency (WADA) oversees global antidoping regulations and testing for elite athletes participating in Olympic sports. Efforts to enforce antidoping policies are complicated by the diverse and evolving compounds and strategies employed by athletes to gain a competitive edge. Now between the uniquely proximate 2021 Tokyo and 2022 Beijing Olympic Games, we discuss WADA's efforts to prevent PED use during the modern Olympic Games. Then, we review the major PED classes with a focus on pathophysiology, complexities of antidoping testing, and relevant toxicities. Providers from diverse practice environments are likely to care for patients using PEDs for a variety of reasons and levels of sport; these providers should be aware of common PED classes and their risks.
Abstract
Recombinant human Erythropoietin (rHuEPO) is an important hormone drug that is used to treat several medical conditions. It is also frequently abused by athletes as a performance enhancing agent at sporting events. The time window of the rHuEPO in blood is short. Therefore, the rapid detection of rHuEPO use/abuse at points of care and in sports requires selective analytical method and a sensitive sensor. Herein, we present a highly selective method for the rapid detection of rHuEPO in human blood plasma by a sensitive optical sensor. rHuEPO is selectively extracted from human blood plasma by a target-specific extractor chip and converted into a biothiol by reducing its disulfide bond structure. The formed biothiol reacts with a water soluble (E)-1-((6-methoxybenzo[d]thiazole-2-yl)diazenyl)naphthalene-2,6-diolHg (II) (BAN-Hg) optical sensor and causes its rapid decomposition. This leads to a rapid change in the sensor color from blue to pink that can be observed by the naked eye. The optical sensor was used to quantify rHuEPO in the concentration range 1×10-8 M to 1×10-12M by UV-Vis spectroscopy. For the screening of blood plasma, an EPO-specific extractor chip was synthesized and used to selectively extract the protein from the biological matrix prior to its conversion into biothiol and quantification by the optical sensor. Since many proteins have disulfide bond structure, the new method has strong potential for their rapid sensitive and selective detection by the BAN-Hg sensor and UV-Vis spectroscopy.
This review is the eighth update of the original article published in 1999 on the application of Matrix‐assisted laser desorption/ionization mass spectrometry (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2014. Topics covered in the first part of the review include general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation, and arrays. The second part of the review is devoted to applications to various structural types such as oligo‐ and poly‐ saccharides, glycoproteins, glycolipids, glycosides, and biopharmaceuticals. Much of this material is presented in tabular form. The third part of the review covers medical and industrial applications of the technique, studies of enzyme reactions, and applications to chemical synthesis.
Performance-enhancing substances (PESs) have unfortunately become ubiquitous in numerous sports, often tarnishing the spirit of competition. Reported rates of PES use among athletes are variable and range from 5 to 31 %. More importantly, some of these substances pose a serious threat to the health and well-being of athletes. Common PESs include anabolic-androgenic steroids, human growth hormone, creatine, erythropoietin and blood doping, amphetamines and stimulants, and beta-hydroxy-beta-methylbutyrate. With recent advances in technology, gene doping is also becoming more conceivable. Sports medicine physicians are often unfamiliar with these substances and thus do not routinely broach the topic of PESs with their patients. However, to effect positive change in the sports community, physicians must educate themselves about the physiology, performance benefits, adverse effects, and testing methods. In turn, physicians can then educate athletes at all levels and prevent the use of potentially dangerous PESs.
Carbon nanomaterials were usually exploited as nanocarriers in electrochemical immunosensor, but rarely acted as redox nano-probes. Herein, our motivation is to adequately utilize the inner redox activity of fullerene (C60) to obtain a new type of redox nano-probe based on a hydrophilic C60 nanomaterial. Firstly, C60 nanoparticles (C60NPs) were prepared by phase-transfer method and functionalized with amino-terminated polyamidoamine (PAMAM) to obtain the PAMAM decorated C60NPs (PAMAM-C60NPs) which have better hydrophilicity compared to that of unmodified C60NPs and possesses abundant amine groups for further modification. Following that, gold nanoparticles (nano-Au) were absorbed on PAMAM-C60NPs surface, and the resultant Au-PAMAM-C60NPs were employed as a new type of redox nano-probe and nanocarrier to label detection antibodies (Ab2). Doping control has become the biggest problem facing international sport. Erythropoietin (EPO) as a blood doping agent has been a hotspot in doping control. After sandwich-type immunoreaction between EPO (as a model) and Ab2-labeled Au-PAMAM-C60NPs, the resultant immunosensor was further incubated with a drop of tetraoctylammonium bromide (TOAB) which acts as booster to arouse the inner redox activity of Au-PAMAM-C60NPs, thus a pair of reversible redox peaks is observed. As a result, the proposed immunosensor shows a wide linear range and a relatively low detection limit for EPO. This strategy paves a new avenue for exploring the redox nano-probe based on carbon nanomaterials in electrochemical biosensor filed.
Advances in recombinant DNA technology have created one of the most powerful weapons in the current doping arsenal: recombinant proteins [Sweeney HL. Gene doping. Sci Am 2004;291:62-9; Unal M, Ozer Unal D. Gene doping in sports. Sports Med 2004;34:357-62]. Recombinant erythropoietin (EPO) and human growth hormone (hGH) are currently being abused but are fortunately detectable either directly by employing isoelectric focusing and immunoassays or indirectly by assessing changes in selected hematopoietic parameters. The detection is technically demanding due to the extent of similarity between the recombinant proteins and their endogenous counterparts. Another issue facing detection efforts is the speed and conditions at which blood samples are collected and analyzed in a sports setting. Recently, gene doping, which stemmed out of legitimate gene therapy trials, has emerged as the next level of doping. Erythropoietin (EPO), human growth hormone (hGH), insulin-like growth factor-1 (IGF-1), peroxisome proliferator-activated receptor-delta (PPAR delta), and myostatin inhibitor genes have been identified as primary targets for doping. Sports clinical scientists today are racing against the clock because assuring the continued integrity of sports competition depends on their ability to outpace the efforts of dopers by developing new detection strategies.
We evaluated the safety and efficacy of darbepoetin alfa (DA), an attractive alternative to recombinant human erythropoietin (rHuEPO) in managing renal anemia, in Japanese children with chronic kidney disease (CKD) on peritoneal dialysis (PD) and hemodialysis (HD), and not on dialysis (ND).
A total of 31 pediatric CKD patients (13 PD, 2 HD, and 16 ND) were enrolled. DA was administered bi-weekly intravenously (IV) or subcutaneously (SC) for PD or ND patients, and weekly IV for HD patients for 24 weeks. The target Hb was defined as 11.0 to ≤13.0 g/dl. In patients receiving rHuEPO, the initial DA dose was calculated at 1 μg DA for 200 IU rHuEPO. The initial DA dose for naïve patients was determined by body weight, and intended not to exceed 0.5 μg/kg per administration. For some PD or ND patients, the dosing frequency was subsequently changed to once every 4 weeks.
Mean Hb values increased from 10.5 ± 1.1 to 11.1 ± 1.1 g/dl after 4 weeks of DA treatment. The target Hb was achieved in all patients, 64.5 % of whom maintained the value at completion of the study. Hb responses were similar between IV and SC. The dosing frequency was extended to once every 4 weeks in 37.9 % of PD or ND patients. Eighty-seven adverse events were noted in 27 (87.1 %) of 31 patients, none of which were associated with DA.
These results suggest that IV or SC administration of DA is an effective and safe treatment for renal anemia in Japanese children with CKD.
Purpose:
Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells.
Methods:
For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. 5 ml ascitic fluid was harvested from each mouse in two times. Evaluation of mAb titration was assessed by ELISA method. The ascitic fluid was examined for class and subclasses by ELISA mouse mAb isotyping Kit. mAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose. Purity of monoclonal antibody was monitored by SDS -PAGE and the purified monoclonal antibody was conjugated with FITC.
Results:
Monoclonal antibodies with high specificity and sensitivity against human CD34 by hybridoma technology were prepared. The subclass of antibody was IgG1 and its light chain was kappa.
Conclusion:
The conjugated monoclonal antibody could be a useful tool for isolation, purification and characterization of human hematopoietic stem cells.
Neosporosis is a disease caused by the apicomplexan parasite Neospora caninum, which is closely related to Toxoplasma gondii. N. caninum infection represents an important cause of reproductive failure in sheep, goats, horses, and cattle worldwide. The diagnosis of neosporosis is based on the detection of pathogen-specific antibodies in animal sera or the presence of tissue cysts. However, morphological similarities and serological cross-reactivity between N. caninum and T. gondii can result in the misdiagnosis. In this study, the N. caninum tachyzoite surface protein Ncp-43 was expressed in a recombinant form to elicit polyclonal antibodies (pAb) response. The pAb was purified and conjugated to horseradish peroxidase (HRP) or fluorescein isothiocyanate (FITC) to detect the recombinant and native Ncp-43 proteins, respectively. The pAb and pAb/HRP were able to recognize rNcp-43 by dot blot and ELISA, and pAb/FITC immunolabeled the apical complex of tachyzoites. A blocking enzyme-linked immunosorbent assay (b-ELISA) was performed to evaluate pAb/HRP as a diagnostic tool. The mean percent inhibition for the positive and negative serum samples from cattle with neosporosis was significantly different (P < 0.0001). These results suggest that the pAb may bind to the same epitopes of Ncp-43 as anti-N. caninum antibodies in the positive samples tested. The b-ELISA using the pAb/HRP can facilitate diagnostic testing for neosporosis, since fewer steps are involved, and cross-reactivity with secondary antibodies is avoided. In summary, this report describes the production of antibodies against N. caninum, and evaluates the potential of these tools for the development of new diagnostic tests for neosporosis.
Glycoscience is a research field focusing on complex carbohydrates (otherwise known as glycans)1, which can, for example, serve as "switches" that toggle between different functions of a glycoprotein or glycolipid. Due to the advancement of glycomics technologies that are used to characterize glycan structures, many glycomics databases are now publicly available and provide useful information for glycoscience research. However, these databases have almost no link to other life science databases.
In order to implement support for the Semantic Web most efficiently for glycomics research, the developers of major glycomics databases agreed on a minimal standard for representing glycan structure and annotation information using RDF (Resource Description Framework). Moreover, all of the participants implemented this standard prototype and generated preliminary RDF versions of their data. To test the utility of the converted data, all of the data sets were uploaded into a Virtuoso triple store, and several SPARQL queries were tested as "proofs-of-concept" to illustrate the utility of the Semantic Web in querying across databases which were originally difficult to implement.
We were able to successfully retrieve information by linking UniCarbKB, GlycomeDB and JCGGDB in a single SPARQL query to obtain our target information. We also tested queries linking UniProt with GlycoEpitope as well as lectin data with GlycomeDB through PDB. As a result, we have been able to link proteomics data with glycomics data through the implementation of Semantic Web technologies, allowing for more flexible queries across these domains.
The transcriptional networks that regulate gene expression and modifications to this network are at the core of the cancer phenotype. MicroRNAs, a well-studied species of small non-coding RNA molecules, have been shown to have a central role in regulating gene expression as part of this transcriptional network. Further, microRNA deregulation is associated with cancer development and with tumor progression. Glioblastoma Multiform (GBM) is the most common, aggressive and malignant primary tumor of the brain and is associated with one of the worst 5-year survival rates among all human cancers. To study the transcriptional network and its modifications in GBM, we utilized gene expression, microRNA sequencing, whole genome sequencing and clinical data from hundreds of patients from different datasets. Using these data and a novel microRNA-gene association approach we introduce, we have identified unique microRNAs and their associated genes. This unique behavior is composed of the ability of the quantifiable association of the microRNA and the gene expression levels, which we show stratify patients into clinical subgroups of high statistical significance. Importantly, this stratification goes unobserved by other methods and is not affiliated by other subsets or phenotypes within the data. To investigate the robustness of the introduced approach, we demonstrate, in unrelated datasets, robustness of findings. Among the set of identified microRNA-gene associations, we closely study the example of MAF and hsa-miR-330-3p, and show how their co-behavior stratifies patients into prognosis clinical groups and how whole genome sequences tells us more about a specific genomic variation as a possible basis for patient variances. We argue that these identified associations may indicate previously unexplored specific disease control mechanisms and may be used as basis for further study and for possible therapeutic intervention.
Diagnosis of schizophrenia does not have a clear objective test at present, so we aimed to identify the potential biomarkers for the diagnosis of schizophrenia by comparison of serum protein profiling between first-episode schizophrenia patients and healthy controls. The combination of a magnetic bead separation system with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS) was used to analyze the serum protein spectra of 286 first-episode patients with schizophrenia, 41 chronic disease patients and 304 healthy controls. FlexAnlysis 3.0 and ClinProTools(TM) 2.1 software was used to establish a diagnostic model for schizophrenia. The results demonstrated that 10 fragmented peptides demonstrated an optimal discriminatory performance. Among these fragmented peptides, the peptide with m/z 1206.58 was identified as a fragment of fibrinopeptide A. Receiver operating characteristic analysis for m/z 1206.58 showed that the area under the curve was 0.981 for schizophrenia vs healthy controls, and 0.999 for schizophrenia vs other chronic disease controls. From our result, we consider that the analysis of serum protein spectrum using the magnetic bead separation system and MALDI-TOF/TOF-MS is an objective diagnostic tool. We conclude that fibrinopeptide A has the potential to be a biomarker for diagnosis of schizophrenia. This protein may also help to elucidate schizophrenia disease pathogenesis. Copyright © 2013 John Wiley & Sons, Ltd.
We performed a pilot study using human peripheral blood lymphocytes (PBL) as a novel system to identify new biomarkers of dihydrotestosterone (DHT) and insulin-like growth factor-1 (IGF-1) abuse in sport. First, to obtain a gene signature, we treated cultures of lymphocytes from sedentary males with three doses of 0.237 microg/ml DHT, each of which is 80-fold the physiological concentration in young adult male serum, at days 0, 2 and 4, or with a single dose of 1.25 microg/ml IGF-1, which is 5-fold the physiological concentration in young adult male serum. We then used the Human Genome U133 Plus 2.0 microarray to identify a gene signature related to DHT or IGF-1 administration. Gene expression was evaluated after 7 and 21 days of DHT treatment, and after 24 h, 72 h and 7 days of IGF-1 treatment. Microarray analysis yielded a list of genes whose expression was altered after DHT or IGF-1 treatment. Among these we selected the genes that are most representative of the pathways associated with skeletal and muscular disorders using the IPA bioinformatics tool. We identified six (IDO1, CXCL13, CCL1, GZMB, VDR and IL2RA) and two (FN1 and RAB31) genes that were up-regulated in lymphocytes from sedentary subjects after 7 days of DHT and IGF-1 treatment, respectively. The expression of these genes in lymphocytes from differently trained athletes was either down-regulated or similar to that in lymphocytes from sedentary subjects. This finding suggests that up-regulation was due to the drug and not to physical exercise. In conclusion, we demonstrate that PBL can be useful in anti-doping checks, and we describe new biomarkers of DHT and IGF-1 abuse which can be included in the Athletes Biological Passport.
Aptamers are single-stranded oligonucleotides that fold into well-defined three-dimensional shapes, allowing them to bind their targets with high affinity and specificity. They can be generated through an in vitro process called "Systemic Evolution of Ligands by Exponential Enrichment" and applied for specific detection, inhibition, and characterization of various targets like small organic and inorganic molecules, proteins, and whole cells. Aptamers have also been called chemical antibodies because of their synthetic origin and their similar modes of action to antibodies. They exhibit significant advantages over antibodies in terms of their small size, synthetic accessibility, and ability to be chemically modified and thus endowed with new properties. The first generation of aptamer drug "Macugen" was available for public use within 25 years of the discovery of aptamers. With others in the pipeline for clinical trials, this emerging field of medical biotechnology is raising significant interest. However, aptamers pose different problems for their development than for antibodies that need to be addressed to achieve practical applications. It is likely that current developments in aptamer engineering will be the basis for the evolution of improved future bioanalytical and biomedical applications. The present review discusses the development of aptamers for therapeutics, drug delivery, target validation and imaging, and reviews some of the challenges to fully realizing the promise of aptamers in biomedical applications.
Detection of the Mycobacterium tuberculosis cell wall antigen lipoarabinomannan (LAM) in urine permits diagnoses of tuberculosis (TB) to be made in HIV-infected patients with advanced immunodeficiency. This can be achieved at the point-of-care within just 30 minutes using the Determine TB-LAM, which is a commercially available, lateral-flow urine 'strip test' assay. The assay has been shown to have useful diagnostic accuracy in patients enrolling in antiretroviral treatment services or in HIV-infected patients requiring admission to hospital medical wards in sub-Saharan Africa. Such patients have high mortality risk and have most to gain from rapid diagnosis of TB and immediate initiation of treatment. However, few studies using this assay have yet been reported and many questions remain concerning the correct use of the assay, interpretation of results, the role of the assay as an add-on test within existing diagnostic algorithms and the types of further studies needed. In this paper we address a series of questions with the aim of informing the design, conduct and interpretation of future studies. Specifically, we clarify which clinical populations are most likely to derive benefit from use of this assay and how patients enrolled in such studies might best be characterised. We describe the importance of employing a rigorous microbiological diagnostic reference standard in studies of diagnostic accuracy and discuss issues surrounding the specificity of the assay in different geographical areas and potential cross-reactivity with non-tuberculous mycobacteria and other organisms. We highlight the importance of careful procedures for urine collection and storage and the critical issue of how to read and interpret the test strips. Finally, we consider how the assay could be used in combination with other assays and outline the types of studies that are required to build the evidence base concerning its use.
Liquid chromatography-mass spectrometry (LC-MS/MS) offers superior analytical specificity compared to immunoassays, but is not available in many regions and hospital due to expensive instrumentation and tedious sample preparation. Thus, we developed an automated, high-throughput LC-MS/MS assay for simultaneous quantification of 10 endogenous and synthetic steroids targeting diseases of the hypothalamo-pituitary-adrenal-axis and gonads.
Deuterated internal standards were added to 85µl serum and processed by liquid-liquid-extraction. Cortisol, cortisone, prednisolone, prednisone, 11-deoxycortisol, dexamethasone, testosterone, androstenedione and progesterone were resolved by ultra-high pressure chromatography on a reversed-phase column in 6.1 minutes, and detected by triple-quadrupole MS. The method was used to assess steroid profiles in women with Addison's disease (AD, n=156) and blood donors (BD, n=102).
Precisions ranged 4.5-10.1% RSD, accuracies 95-108%, and extraction recoveries 60-84%. The method was practically free of matrix effects and robust to individual differences in serum composition. Most postmenopausal AD women had extremely low androstenedione below 0.14 nmol/L and median testosterone 0.15 nmol/L [interquartile range 0.00-0.41], considerably lower than postmenopausal BD (1.28 nmol/L [0.96-1.64] and 0.65 nmol/L [0.56-1.10], respectively). AD women in fertile years had androstenedione 1.18 nmol/L [0.71-1.76] and testosterone 0.44 nmol/L [0.22-0.63], approximately half of levels found in BD of corresponding age.
This LC-MS/MS assay provides highly sensitive and specific assessments of glucocorticoids and androgens with low sample volumes, and is suitable for endocrine laboratories and research. Its utility was demonstrated in a large cohort of women with AD, and the data suggest that women with AD are particularly androgen deficient after menopause.
Iron is an essential component in the structure of certain molecules such as hemoglobin (Hb), myoglobin, cytochrome C and some enzymes. The iron gateway to cells is transferrin receptor (TfR). Soluble transferrin receptor (sTfR) is a product of the TfR that circulates in plasma, its concentration therefore, is proportional to the total concentration of cellular TfR. Expression of TfR is elevated in anemia. The aim of this study was to evaluate the efficacy of sTfR determination in the diagnosis of iron deficiency in thalassemia carriers. In this cross-sectional study, 168 cases were examined. The subdivision of cases included: iron deficiency, concurrent thalassemia and iron deficiency, severe α-thalassemia (α-thal) (α-thal-1), mild α-thal (α-thal-2) and β-thal minor. Hematological and biochemical variables were analyzed by the Statistical Package for Social Sciences (SPSS) version 16 statistical software. Analysis of variance was carried out using the non parametric Kruskal-Wallis test. Significant differences were observed in median values in sTfR concentration and sTfR/log ferritin (sTfR-F index) iron deficient groups, compared to thalassemia groups (p value <0.001), with both variables having higher values in iron deficient groups. In this study it was demonstrated that in iron deficient thalassemic patients, high sTfR can be a good indicator of iron deficiency anemia. The result of the sTfR test can be used for calculation of the sTfR-log ferritin index (sTfR-F index) (obtained by division of sTfR into log ferritin). This is a more sensitive parameter for iron deficiency diagnosis because in its calculation, two valuable amounts of sTfR and ferritin were taken into consideration.
Abstract Peptide hormones have been abused in sports to improve physical performances since recombinant products which are the exact replicate of their natural models became available. Both human growth hormone and erythropoietin are now available in large quantities on the unofficial market for those who want to use them for applications lying outside the accepted medical applications Such abuse calls for strict controls, because non-medical abuses of these powerful products can produce dangerous side-effects. The duration time of both peptides in the body is so short that direct detection of the recombinant products is impossible. The actual tests available for their detection rely on immunological assays which are not considered good enough for determining their abuse in sports. All possible secondary markers are now being sought for adoption as a screening test which still requires confirmation by the direct measurement of the hormones themselves. Blood samples might well appear to be more useful for the comparison of quantitative data obtained from carefully selected secondary markers of the abuse of these drugs although large population studies on their variations are not yet accomplished. The final proof of the abuse of such products is sought through the use of the newest analytical techniques like liquid chromatography coupled with mass spectrometry. The state of the art in this field is analysed in view of the specific problems that any doping control laboratory is faced with, including the ethical and legal requirements
This protocol shows how to obtain a detailed glycan compositional and structural profile from purified glycoproteins or protein mixtures, and it can be used to distinguish different isobaric glycan isomers. Glycoproteins are immobilized on PVDF membranes before the N-glycans are enzymatically released by PNGase F, isolated and reduced. Subsequently, O-glycans are chemically released from the same protein spot by reductive β-elimination. After desalting with cation exchange microcolumns, the glycans are separated and analyzed by porous graphitized carbon liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Optionally, the glycans can be treated with sialidases or other specific exoglycosidases to yield more detailed structural information. The sample preparation takes approximately 4 d, with a heavier workload on days 2 and 3, and a lighter load on days 1 and 4. The time for data interpretation depends on the complexity of the samples analyzed. This method can be used in conjunction with the analysis of enriched glycopeptides by capillary/nanoLC-ESI-MS/MS, which together provide detailed information regarding the site heterogeneity of glycosylation.
Erythropoietin (EPO) is the main hormonal regulator of red blood cell production. Recombinant EPO has become the leading drug for treatment of anaemia from a variety of causes; however, it is sometimes misused in sport with the aim of improving performance and endurance. This paper presents an introductory overview of EPO, its receptor, and a variety of recombinant human EPOs/erythropoiesis stimulating agents (ESAs) available on the market (e.g. epoetins and their long acting analogs - darbepoetin alfa and continuous erythropoiesis receptor activator). Recent efforts to improve on EPO's pharmaceutical properties and to develop novel replacement products are also presented. In most cases, these efforts have emphasized a reduction in frequency of injections or complete elimination of intravenous or subcutaneous injections of the hormone (biosimilars, EPO mimetic peptides, fusion proteins, endogenous EPO gene activators and gene doping). Isoelectric focusing (IEF) combined with double immunoblotting can detect the subtle differences in glycosylation/sialylation, enabling differentiation among endogenous and recombinant EPO analogues. This method, using the highly sensitive anti-EPO monoclonal antibody AE7A5, has been accepted internationally as one of the methods for detecting misuse of ESAs in sport. Copyright © 2012 John Wiley & Sons, Ltd.
We present here a novel conjugated aptamer-gold nanoparticle (Apt-AuNPs) fluorescent probe and its application for specific detection of recombinant human erythropoietin-α (rHuEPO-α). In this nanobiosensor, 12 nm AuNPs function as both a nano-scaffold and a nano-quencher (fluorescent energy acceptor), on the surface of which the complementary sequences are linked (as cODN-AuNPs) and pre-hybridized with carboxymethylfluorescein (FAM)-labeled anti-rHuEPO-α aptamers. Upon target protein binding, the aptamers can be released from the AuNP surface and the fluorescence signal is restored. Key variables such as the length of linker, the hybridization site and length have been designed and optimized. Full performance evaluation including sensitivity, linear range and interference substances are also described. This nanobiosensor provides a promising approach for a simple and direct quantification of rHuEPO-α concentrations as low as 0.92 nM within a few hours.
Erythropoietin (EPO) is a 30.4 kDa glycoprotein produced by the kidney, and is mostly well-known for its physiological function in regulating red blood cell production in the bone marrow. Accumulating evidence, however, suggests that EPO has additional organ protective effects, which may be useful in the prevention or treatment of acute kidney injury. These protective mechanisms are multifactorial in nature and include inhibition of apoptotic cell death, stimulation of cellular regeneration, inhibition of deleterious pathways, and promotion of recovery.
In this article, we review the physiology of EPO, assess previous work that supports the role of EPO as a general tissue protective agent, and explain the mechanisms by which it may achieve this tissue protective effect. We then focus on experimental and clinical data that suggest that EPO has a kidney protective effect.
In elite sports, the growing availability of doping substances identical to those naturally produced by the human body seriously limits the ability of drug-testing regimes to ensure fairness and protection of health.
The Athlete Biological Passport (ABP), the new paradigm in testing based on the personalized monitoring of biomarkers of doping, offers the enormous advantage of being independent of this endless pharmaceutical race. Doping triggers physiological changes that provide physiological enhancements. In the same way that disease-related biomarkers are invaluable tools that assist physicians in the diagnosis of pathology, specifically selected biomarkers can be used to detect doping.
The ABP is a new testing paradigm with immense potential value in the current climate of rapid advancement in biomarker discovery. In addition to its original aim of providing proof of a doping offense, the ABP can also serve as a platform for a Rule of Sport, with the presentation before competition of the ABP to objectively demonstrate that the athlete will participate in a healthy physiological condition that is unaltered by performance-enhancing drugs. Finally, the decision-support system used today for the biological monitoring of world top-level athletes can also be advantageously transferred to other areas of clinical practice to reach the goal of personalized medicine.
Transportation of doping control urine samples from the collection sites to the World Anti-doping Agency (WADA) Accredited Laboratories is conducted under ambient temperatures. When sample delivery is not immediate, microbial contamination of urine, especially in summer, is a common phenomenon that may affect sample integrity and may result in misinterpretation of analytical data. Furthermore, the possibility of intentional contamination of sports samples during collection with proteolytic enzymes, masking the abuse of prohibited proteins such as erythropoietin (EPO) and peptide hormones, is a practice that has already been reported. Consequently, stabilization of urine samples with a suitable method in a way that protects samples' integrity is important. Currently, no stabilization method is applied in the sample collection equipment system in order to prevent degradation of urine compounds. The present work is an overview of a study, funded by WADA, on degradation and stabilization aspects of sports urine samples against the above threats of degradation. Extensive method development resulted in the creation of a mixture of chemical agents for the stabilization of urine. Evaluation of results demonstrated that the stabilization mixture could stabilize endogenous steroids, recombinant EPO, and human chorionic gonadotropin in almost the entire range of the experimental conditions tested.
Aptamers and the SELEX process were discovered over two decades ago. These discoveries have spawned a productive academic and commercial industry. The collective results provide insights into biology, past and present, through an in vitro evolutionary exploration of the nature of nucleic acids and their potential roles in ancient life. Aptamers have helped usher in an RNA renaissance. Here we explore some of the evolution of the aptamer field and the insights it has provided for conceptualizing an RNA world, from its nascence to our current endeavor employing aptamers in human proteomics to discover biomarkers of health and disease.
RNAs can be physically classified into poly(A)+ or poly(A)- transcripts according to the presence or absence of a poly(A) tail at their 3' ends. Current deep sequencing approaches largely depend on the enrichment of transcripts with a poly(A) tail, and therefore offer little insight into the nature and expression of transcripts that lack poly(A) tails.
We have used deep sequencing to explore the repertoire of both poly(A)+ and poly(A)- RNAs from HeLa cells and H9 human embryonic stem cells (hESCs). Using stringent criteria, we found that while the majority of transcripts are poly(A)+, a significant portion of transcripts are either poly(A)- or bimorphic, being found in both the poly(A)+ and poly(A)- populations. Further analyses revealed that many mRNAs may not contain classical long poly(A) tails and such messages are overrepresented in specific functional categories. In addition, we surprisingly found that a few excised introns accumulate in cells and thus constitute a new class of non-polyadenylated long non-coding RNAs. Finally, we have identified a specific subset of poly(A)- histone mRNAs, including two histone H1 variants, that are expressed in undifferentiated hESCs and are rapidly diminished upon differentiation; further, these same histone genes are induced upon reprogramming of fibroblasts to induced pluripotent stem cells.
We offer a rich source of data that allows a deeper exploration of the poly(A)- landscape of the eukaryotic transcriptome. The approach we present here also applies to the analysis of the poly(A)- transcriptomes of other organisms.
Recombinant human erythropoietin (rHuEPO) is one kind of important hematopoietic growth factor, which is widely used in anaemia treatment as well as sometimes abused by endurance athletes. Based on a set of anti-rHuEPO-α aptamers successfully in vitro isolated in our laboratory, we herein describe a novel magnetic beads-based aptameric real-time PCR assay for the accurate quantification of rHuEPO-α, which combined the specific recognition with amplification capability of aptamers. Two detection strategies, termed 'recognition-after-hybridization' and 'recognition-before-hybridization' respectively, were constructed and compared. Strategy B, i.e.'recognition-before-hybridization', was finally adopted as the preferred one to measure rHuEPO-α. A limit of detection (LOD) of 1 pmol/L rHuEPO-α and a wide dynamic range from 6 pmol/L to 100 nmol/L were obtained for physiological buffer. Furthermore, a LOD of 6 pmol/L was achieved for more complicated matrix-half diluted artificial urine. These results indicate that the anti-rHuEPO-α aptamer fits the high sensitive detection of rHuEPO-α very well. The use of the aptamer with magnetic beads-based real-time PCR allows a direct and novel assay for rHuEPO-α.
MicroRNAs (miRNAs) have been implicated to play key roles in normal physiological functions, and altered expression of specific miRNAs has been associated with a number of diseases. It is of great interest to understand their roles and a prerequisite for such study is the ability to comprehensively and accurately assess the levels of the entire repertoire of miRNAs in a given sample. It has been shown that some miRNAs frequently have sequence variations termed isomirs. To better understand the extent of miRNA sequence heterogeneity and its potential implications for miRNA function and measurement, we conducted a comprehensive survey of miRNA sequence variations from human and mouse samples using next generation sequencing platforms. Our results suggest that the process of generating this isomir spectrum might not be random and that heterogeneity at the ends of miRNA affects the consistency and accuracy of miRNA level measurement. In addition, we have constructed a database from our sequencing data that catalogs the entire repertoire of miRNA sequences (http://galas.systemsbiology.net/cgi-bin/isomir/find.pl). This enables users to determine the most abundant sequence and the degree of heterogeneity for each individual miRNA species. This information will be useful both to better understand the functions of isomirs and to improve probe or primer design for miRNA detection and measurement.
Endogenous erythropoietin (EPO) consists of a central polypeptide core covered by post-translationally linked carbohydrates. Three of the four currently available erythropoiesis stimulating agents (ESA) - epoetin-alpha, epoetin-beta and epoetin-omega - are composed of an identical amino acid sequence, but glycosylation varies as a result of type- and host cell-specific differences in the production process. Epoetin-alpha and epoetin-beta resemble each other with respect to molecular characteristics and pharmacokinetic data, although epoetin-beta has a higher molecular weight, a lower number of sialylated glycan residues and possibly slight pharmacokinetic advantages such as a longer terminal elimination half-life. A serious adverse effect of long-term administration of ESA is pure red cell aplasia. This effect has been observed predominantly with subcutaneous use of epoetin-alpha produced outside the US after albumin was removed from the formulation. In comparison with the intravenous route, subcutaneous administration of epoetin has been reported to have a dose-sparing effect in some studies. Epoetin-beta has been the subject of studies aimed at proving efficacy with a reduced administration frequency but results are not unequivocal. Epoetin-omega is produced in a different host cell than all other erythropoietic agents, hence glycosylation and pharmacokinetics are different. Small-scale clinical studies found epoetin-omega to be slightly more potent than epoetin-alpha. Epoetin-delta is a recently approved agent produced by human cells that are genetically engineered to transcribe and translate the EPO gene under the control of a newly introduced regulatory DNA sequence. However, epoetin-delta is not yet on the market and few data are available. The erythropoietin analogue darbepoetin-alpha carries two additional glycosylation sites that permit a higher degree of glycosylation. Consequently, in comparison with the other epoetins, darbepoetin-alpha has a longer serum half-life and a higher relative potency, which further increases with extension of the administration interval. Dosage requirements of darbepoetin-alpha do not appear to differ between the intravenous and subcutaneous routes of administration. The less frequent administration of darbepoetin-alpha in comparison to the other epoetins may reduce drug costs in the long term, but the variability in dosage or dosage frequency required within a single patient is high. Further studies should be aimed at defining predictors of the individual demand for erythropoietic agents, thereby allowing nephrologists to prescribe a cost-effective, individualised regimen.
Erythropoietin (EPO) increases the number of circulating erythrocytes and thus muscle oxygenation. The availability of the recombinant protein (rEPO) has increased the risk of its illegal use in sports, its detection being a difficult challenge. Five different hematopoietic parameters were initially chosen as indirect markers of rEPO abuse: concentration of serum EPO, concentration of serum-soluble transferrin receptors (sTFr), hematocrit, percentage of reticulocytes, and percentage ofmacrocytes. New models considering only hemoglobin, serum EPO concentration, and percentage of reticulocytes are simpler and seem to be more sensitive when low doses of rEPO are used. A more direct method of urine analysis (isoelectrofocusing, double blotting, and chemiluminescent detection) based on the charge differences between rEPO and endogenous EPO, related to their carbohydrate composition, provides proof of rEPO use. Furthermore, this approach permits the detection of darbepoetin, a direct analogue of EPO also known as NESP ("new erythropoiesis stimulating protein"). Recently a protein conjugate, "synthetic erythropoiesis protein" (SEP), containing precision-length, monodisperse, negatively charged polymers instead of oligosaccharides has been synthesized. Finally, EPO-mimetics are molecules capable of acting as EPO in dimerizing the EPO receptor. Two kinds of EPO-mimetics have been described: peptides and nonpeptides. The enhancement of oxygen availability to muscles by rEPO, analogues, and mimetics constitutes one of the main challenges to doping control. Major steps have already been developed for detection of rEPO and some analogues. In the near future, the transfection to an athlete's body of genes that code for erythropoietin might be an emerging doping issue, and sports authorities have incorporated "gene doping" among the prohibited practices.
Abstract
Erythropoietin (EPO) from sera obtained from anemic patients was successfully isolated using magnetic beads coated with a human EPO (hEPO)-specific antibody. Human serum EPO emerged as a broad band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight slightly smaller than that of recombinant hEPO (rhEPO). The bandwidth corresponded with microheterogeneity because of extensive glycosylation. Two-dimensional gel electrophoresis revealing several different glycoforms confirmed the heterogeneity of circulating hEPO. The immobilized anti-hEPO antibody was capable of binding a representative selection of rhEPO glycoforms. This was shown by comparing normal-phase high-performance liquid chromatography profiles of oligosaccharides released from rhEPO with oligosaccharides released from rhEPO after isolation with hEPO-specific magnetic beads. Charge analysis demonstrated that human serum EPO contained only mono-, di-, and tri-acidic oligosaccharides and lacked the tetra-acidic structures present in the glycans from rhEPO. Determination of charge state after treatment of human serum EPO with Arthrobacter ureafaciens sialidase showed that the acidity of the oligosaccharide structures was caused by sialic acids. The sugar profiles of human serum EPO, describing both neutral and charged sugar, appeared significantly different from the profiles of rhEPO. The detection of glycan structural discrepancies between human serum EPO and rhEPO by sugar profiling may be significant for diagnosing pathologic conditions, maintaining pharmaceutical quality control, and establishing a direct method to detect the misuse of rhEPO in sports.
The potential of CE with native fluorescence detection (Flu) for the profiling of the therapeutic protein erythropoietin (EPO) was studied. EPO is a highly heterogeneous glycoprotein comprising a large number of isoforms. CE was applied to induce separation among the various glycoforms. Native Flu of EPO provided high detection selectivity yielding good signal-to-noise ratios and stable baselines, particularly when compared to conventional UV absorbance detection. In order to enhance EPO isoform resolution, CE was performed using a capillary with a neutral coating in combination with a simple BGE of 2.0 M acetic acid (pH 2.1). CE-Flu analysis of the EPO biological reference preparation of the European Pharmacopeia resulted in a highly detailed glycoform profile. Migration time RSDs for selected EPO isoforms were less than 0.22% and 0.80% for intraday and interday repeatability, respectively. RSDs for relative peak intensity of the major EPO isoforms were less than 3%. The achieved resolution, migration time stability, and sensitivity allowed discrimination of different EPO products (EPO-α and EPO-β) based on the recorded glycoform pattern. The developed CE-Flu method is relatively straightforward, and shows potential for quality control in biopharmaceutical production.
Background
The aim was to compare the clinical efficacy of recombinant human erythropoietin (rHuEPO) and darbepoetin alpha (DA) in the treatment of anemia in children with chronic kidney disease (CKD). Method
Thirty-four (13 female, 21 male) CKD patients were enrolled in the study. Mean age was 11.42 ± 4.05 years. Nine patients were on hemodialysis, 18 were on peritoneal dialysis and seven patients were in CKD stage 4. ResultsSeventeen patients received rHuEPO and the remaining 17 patients received DA. Hemoglobin (Hb) was not significantly different between the two groups during monthly follow up and at the end of 6 months (P > 0.05), but there was a significant increase within each group at the end of 6 months (P = 0.01 for rHuEPO; P = 0.02 for DA). Hb was not different between the patients on and not on dialysis in both groups at the end of the study (P > 0.05). The efficacy of the s.c. and i.v. routes was similar within each group (P > 0.05). Systolic hypertension was observed in only one patient in the DA group, no other adverse effect was observed in either groups. ConclusionDA is a reasonable alternative to rHuEPO in the treatment of anemia in pediatric CKD patients, due to its clinical efficacy, convenience of use, patient compliance and tolerability.
Liquid chromatography quadrupole time-of-flight spectrometry (LC-Q-TOF-MS), equipped with electrospray ionization (ESI), was developed for the determination of the main metabolites of dipyrone - 4-aminoantipyrine (4-AA), 4-acetylaminoantipyrine (4-AAA), 4-formylaminoantipyrine (4-FAA) and 4-methylaminoantipyrine (4-MAA) in communal wastewater after reversed-phase solid phase extraction (SPE) in the low to several μg/l concentration range. Samples originated from conventional wastewater treatment plant (WWTP) using activated sewage sludge as well as from a pilot-scale WWTP operating in mixed mode (activated sewage sludge and cascade biofilms reactors with biofilms growing on fix beds and roots of greenhouse plants). Results of the present study confirmed the outcomes of our previous report according to which, 4-FAA was the most persistent metabolite, while 4-AAA and 4-MAA could be determined in the highest and lowest concentration, respectively. Moreover, the study of intraday variation of the concentration of these metabolites revealed that the concentration of 4-AA, 4-AAA and 4-FAA registered a 46%-75% increase in the samples collected at noon compared to those collected at 6 AM. Chlorination did not affect considerably the removal efficiency (about 15%) of these metabolites in samples collected for 3 months consecutively before and after disinfection. Both wastewater treatment techniques efficiently removed 4-AAA (between 80 and 96%). However, in the summer season, the removal efficiency of conventional WWTP using open-air aerated tanks is lower by 30%, (on average) than in the cold season. The concentration of the investigated metabolites showed increased concentrations in the winter season confirming the intake habits of the population from this popular analgesic and antipyretic drug.
Fast, sensitive and simple methods for quantitative analysis of disparities in glycan expression between different biological samples are essential for studies of protein glycosylation patterns (glycomics) and the search for disease glycan biomarkers. Relative quantitation of glycans based on stable isotope labeling combined with mass spectrometric detection represents an emerging and promising technique. However, this technique is undermined by the complexity of mass spectra of isotope-labeled glycans caused by the presence of multiple metal ion adduct signals, which result in a decrease of detection sensitivity and an increase of difficulties in data interpretation. Herein we report a simplified quantitative glycomics strategy, which features non-reductive isotopic labeling of reducing glycans with either non-deuterated (d0-) or deuterated (d5-) Girard's reagent P (GP) without salts introduced and simplified mass spectrometric profiles of d0- and d5-GP derivatives of neutral glycans as molecular ions without complex metal ion adducts, allowing rapid and sensitive quantitative comparison between different glycan samples. We have obtained optimized GP-labeling conditions and good quantitation linearity, reproducibility and accuracy data of the method. Its excellent applicability was validated by comparatively quantitative analysis of the neutral N-glycans released from bovine and porcine immunoglobulin G as well as of those from mouse and rat sera. Additionally, we have revealed the potential of this strategy for the high-sensitivity analysis of sialylated glycans as GP derivatives, which involves neutralization of the carboxyl group of sialic acid by chemical derivatization.
The use of recombinant protein for therapeutic applications has increased significantly in the last three decades. The heterogeneity of these proteins, often caused by the complex biosynthesis pathways and the subsequent post translational modifications pose a challenge for drug characterization to ensure its safety, quality, integrity and efficacy. Capillary electrophoresis (CE), with its simple instrumentation, superior separation efficiency, small sample consumption, and short analysis time, is a well suited analytical tool for therapeutic protein characterization. Different separation modes, including capillary isoelectric focusing, sodium dodecyl sulfate capillary gel electrophoresis, capillary zone electrophoresis, and capillary electrophoresis - mass spectrometry, provide complementary information of the proteins. The CE applications for recombinant therapeutic proteins from 2000 to June 2013 are reviewed and technical concerns are discussed in this article. This article is protected by copyright. All rights reserved.
Colorectal cancer (CRC) is the third most common type of cancer and is responsible for 9 % of cancer deaths in both men and women in the USA for 2013. It is a heterogenous disease, and its three classification types are microsatellite instability, chromosomal instability, and CpG island methylator phenotype. Biomarkers are molecules, which can be used as indicators of cancer. They have the potential to achieve great sensitivities and specificities in diagnosis and prognosis of CRC. DNA methylation biomarkers are epigenetic markers, more specifically genes that become silenced after aberrant methylation of their promoter in CRC. Some methylation biomarkers like SEPT9 (ColoVantage®) and vimentin (ColoSure(TM)) are already commercially available. Other blood and fecal-based biomarkers are currently under investigation and clinical studies so that they can be used in the near future. Biomarker panels are also currently being studied since they show great potential in diagnosis as they can combine robust biomarkers to achieve even greater sensitivities than single markers. Finally, methylation-sensitive microRNAs (miRNAs) are very promising markers, and their investigation as biomarkers, is only at primitive stage.
Helicobacter pylori infection is the most common human infection where approximately 50% of the world populations are infected. The diagnosis of such infection is mainly done by endoscopy where gastric biopsies are examined for the presence of H. pylori. Such invasive approach is costly, time consuming and generally requires more than one test to confirm the infection. Serology on the other hand is a non-invasive approach that can detect H. pylori exposure. The lateral flow immunoassays (LFIA) support the serological approach and have the advantage of being fast, economic and require no additional equipment or experience. In this review the principles, components of the LFIA, sensitivities and specificities of the commercially available H. pylori test strips were compared and discussed.
Epoetin (recombinant human erythropoietin) is used by some endurance athletes to increase oxygen transport and aerobic power in an attempt to improve endurance capacity and recovery during training and competition. Although currently on the list of banned substances issued by the medical commission of the International Olympic Committee, the use of epoetin as an ergogenic agent remains uncontrollable using classical analytical techniques. In the present paper, after subcutaneous administration of repeated high doses (200 units/kg), the pharmacokinetics of epoetin were evaluated in 18 athletes. The mean elimination half-life was 42.0 hours. The total clearance/bioavailability (F) and the volume of distribution/F averaged 0.05 L/h/kg and 2.95 L/kg, respectively. Significant changes in ferritin (fr), soluble transferrin receptor (sTfR) and ratio (sTfR/fr) were observed after administration. A population sigmoidal asymptotic maximum expected effect concentration (e(max) model has been developed to assess and quantify the relationship between the changes in sTfR, fr and the ratio sTfR/fr secondary to the repeated administration of epoetin. The mean population parameters were as follows: e(max): 12.6 mg/L, 44.9 μg/L and 2313.5; the concentration producing 50% of the e(max)(C 50): 18.3, 18.1 and 37.7 IU/L; the sigmoidicity factor (gamma): 2.96, 4.17 and 5.15; and ke 0 (rate constant of transfer between central and effect compartment): 1.29 x 10 -3, 7.14 x 10 -3 and 3.47 x 10 -3/h for the 3 markers, respectively. Moreover, in this paper, we propose an appropriate statistical methodology based on the measurement of sTfR, fr and sTfR/fr from blood samples to decide if the observed values of these markers could be related, at a given probability risk, to the administration of epoetin. Values greater than 10 mg/L for sTfR and 403 for sTfR/fr indicate a probable intake of epoetin. Because of the large interindividual variability of fr, it has not been possible to define a threshold value for this parameter. The sTfR seems to be the most effective marker in doping control. An increased haematocrit with concomitant changes in sTfR and sTfR/fr values seems to supply a valuable diagnostic tool that avoids false-positive doping and allows the identification of epoetin abusers. Blood samples could be collected using capillary blood from the fingertip or earflap. These controls could be carried out during training because they are easy to perform and do not add substantially to total screening costs. At present, this is the only tool available for detecting epoetin abusers during competition.
Recombinant erythropoietin (rhEPO) has been misused for over two decades by athletes, mainly but not only in endurance sports. A direct rhEPO detection method in urine by isoelectric focusing (IEF) was introduced in 2000, but the emergence of third-generation erythropoiesis-stimulating agents and so-called biosimilar rhEPOs, together with the sensitivity of human endogenous EPO (huEPO) pattern to enzymatic activities and its modification following short strenuous exercise, prompted the development of a complementary test based on SDS-PAGE analysis. While Mircera and NESP are easily detected with the existing IEF and SDS-PAGE methods, some samples containing both epoetin-α/β and huEPO present profiles that are still difficult to interpret. As doping practices have moved to micro-dosing, these mixed patterns are more frequently observed. We investigated the impact of enzymatic desialylation on the urinary and serum EPO profiles obtained by SDS-PAGE with the aim of improving the separation of the bands in these mixed EPO populations. We observed that the removal with neuraminidase of the sialic acid moieties from the different EPOs studied reduced their apparent molecular weight (MW) and increased the migration distance between huEPO and rhEPO centroids, therefore eliminating the size overlaps between them and improving the detection of rhEPO. Copyright © 2013 John Wiley & Sons, Ltd.
Enzyme linked aptamer assay (ELAA) uses an aptamer as recognition element and enzyme as signal readout element for establishing different kinds of aptasensors. We reported herein a high-throughput colorimetric aptasensor based on ELAA only requiring a single aptamer sequence for cocaine detection. An anti-cocaine aptamer was cleaved into two fragments, one of which was immobilized on a DNA-BIND 96-well plate via 5'-labeled primary amine and the other one was biotin labeled. The presence of two aptamer fragments and the target molecule led to the formation of aptamer fragments/target complexes. Streptavidin-horseradish peroxidase (SA-HRP) was used to react with biotin in order to obtain quantitative signals. A linear response towards cocaine concentration in the range of 5-200μM and a detection limit down to 2.8μM (S/N=3) were achieved. The specificity and application in real sample were validated. Furthermore, a verification test of thrombin detection in the same strategy illustrated its feasibility for not only small molecule but also biomacromolecule. With the advantage of high-throughput, easy operation, high specificity, the colorimetric assay based on ELAA requiring a single aptamer sequence opens up a new approach for detecting different kinds of targets via specific affinity recognition among target and suitably cleaved aptamer fragments.
We assessed the possibility of using soluble transferrin receptor (sTfR) as an indicator of doping with recombinant erythropoietin (rhEPO).
A double-blind, placebo-controlled study was conducted with the administration of 5,000 U of rhEPO (N = 10) or placebo (N = 10) three times weekly (181-232 U x kg(-1) x wk-1) for 4 wk to male athletes. We measured hematocrit and the concentration of hemoglobin, sTfR, ferritin, EPO, and quantified the effects on performance by measuring time to exhaustion and maximal oxygen uptake (VO2max) on a cycle ergometer.
Hematocrit increased from 42.7 +/- 1.6% to 50.8 +/- 2.0% in the EPO group, and peaked 1 d after treatment was stopped. In the EPO group, there was an increase in sTfR (from 3.1 +/- 0.9 to 6.3 +/- 2.3 mg x L(-1) , P < 0.001) and in the ratio between sTfR and ferritin (sTfR-ferritin(-1)) (from 3.2 +/- 1.6 to 11.8 +/- 5.1, P < 0.001). The sTfR increase was significant after 1 wk of treatment and remained so for 1 wk posttreatment. Individual values for sTfR throughout the study period showed that 8 of 10 subjects receiving rhEPO, but none receiving placebo, had sTfR levels that exceeded the 95% confidence interval for all subjects at baseline (= 4.6 mg x L(-1)). VO2max increased from 63.6 +/- 4.5 mL x kg(-1) x min(-1) before to 68.1 +/- 5.4 mL x kg(-1) x min(-1) 2 d post rhEPO administration (7% increase, P = 0.001) in the EPO group. Hematocrit, sTfR, sTfR-ferritin(-1), and VO2max did not change in the placebo group.
Serum levels of sTfR may be used as an indirect marker of supranormal erythropoiesis up to 1 wk after the administration of rhEPO, but the effects on endurance performance outlast the increase in sTfR.
Since its first publication in 2000, the isoelectric focusing (IEF) method of erythropoietin (EPO) used in doping control has been considered a procedure with relatively small sample number capacity. To overcome this limitation, a variation of the current protocol was evaluated, which uses double-sized gels with 48-120 wells plus three electrodes and hence multiplies the capacity of the electrophoretic chamber. With this modification up to 120 samples and standards can be run on a single gel - thus, making IEF-PAGE of EPO a high-throughput method. The protocol is ideally suited for large-scale screening purposes. Copyright © 2012 John Wiley & Sons, Ltd.
Erythropoiesis-stimulating agents (ESAs) have frequently been confessed to be illicitly used in elite sports due to their endurance enhancing effects. Recently, peginesatide, the first representative of a new generation of ESAs, referred to as Erythropoietin (EPO)-mimetic peptides, obtained approval in the USA under the trade name Omontys(®) for the treatment of anaemic patients. Lacking sequence homology with EPO, it consists of a pegylated homodimeric peptide of approximately 45kDa, and thus, specific approaches for the determination of peginesatide in blood were developed as conventional detection assays for EPO do not allow for the analysis of the EPO-mimetic peptides. However, as urine specimens are the most frequently provided doping control samples and pharmacokinetic studies conducted in rats and monkeys revealed the excretion of the pegylated peptide into urine, a detection method for peginesatide in urine would be desirable. A mass spectrometric assay in human urine was developed consisting of protein precipitation with acetonitrile followed by proteolytic digestion after the removal of the acetonitrile fraction under reduced pressure. Purification and concentration of the resulting proteotypic target peptide was accomplished by means of solid-phase extraction on strong cation-exchange resin prior to liquid chromatographic-tandem mass spectrometric analysis. Method validation was performed for qualitative purposes and demonstrated specificity, precision, linearity as well as sufficient sensitivity (limit of detection: 0.5ng/ml) while proof-of-concept for the applicability of the assay for the determination of peginesatide in authentic urine samples was obtained by analyzing animal in vivo specimens collected after a single i.v. administration of peginesatide over a period of 4 days.
Standard treatments of collusion in intermediate microeconomics textbooks frequently involve a Cournot duopoly facing linear demand with constant marginal costs of production. These presentations leave students with the misunderstanding that firms jointly behaving like a single-firm monopolist and profit maximising collusion are one and the same. We present a simple and effective way for improving student comprehension of collusion; this exercise results in collusion where the duopolists produce more total output than that of a monopolist while enjoying greater joint profits. The exercise can be used to clarify and lead to a better understanding of collusion and profit maximisation.
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 a decreased receptor-mediated endocytosis and/or a delay in renal clearance caused by the increased hydrodynamic volume of the pharmaceutical. MIRCERA, a PEGylated recombinant erythropoietin (rhEpo) frequently used in the treatment of anemia due to chronic kidney disease, has been also abused by athletes as a performance-enhancing drug. While it can be detected by sodium dodecyl sulfate 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, thus leading to enhanced antibody binding and a sharper electrophoretic band. While the method was originally developed for anti-doping purposes, it may be also useful for other PEGylated proteins and their electrophoretic separation and immunological detection.
Recently a novel technology, referred to as the 'EPO WGA MAIIA' test, has been developed by Swedish researchers to discriminate between endogenous and recombinant human erythropoietin. In contrast to existing electrophoretic methods that are used by antidoping laboratories, this dipstick-based technique is simple and fast. Moreover it can be applied to either blood or urine specimens. These characteristics could prove advantageous if the test were adopted by antidoping authorities to determine blood doping in sport. We evaluated the sensitivity of EPO WGA MAIIA to detect the presence of recombinant human erythropoietin (rhEPO) in some archived plasma specimens which had been collected from healthy, active subjects either 72 h or 96 h after a 'microdose' intravenous injection of rhEPO. Under these conditions the test had modest sensitivity to discriminate rhEPO, with only two of nine subjects exceeding an arbitrary cut-off 3.09 SDs beyond the expected population mean. Sensitivity was improved to five out of six subjects if positivity was assessed according to the subject's own previous values rather than a population-based threshold. We conclude that, with further refinement, the dipstick test may supplement existing antidoping tests.
Gene doping - or the abuse of gene therapy - will continue to threaten the sports world. History has shown that progress in medical research is likely to be abused in order to enhance human performance. In this review, we critically discuss the progress and the risks associated with the field of erythropoietin (EPO) gene therapy and its applicability to EPO gene doping. We present typical vector systems that are employed in ex vivo and in vivo gene therapy trials. Due to associated risks, gene doping is not a feasible alternative to conventional EPO or blood doping at this time. Nevertheless, it is well described that about half of the elite athlete population is in principle willing to risk its health to gain a competitive advantage. This includes the use of technologies that lack safety approval. Sophisticated detection approaches are a prerequisite for prevention of unapproved and uncontrolled use of gene therapy technology. In this review, we present current detection approaches for EPO gene doping, with a focus on blood-based direct and indirect approaches. Gene doping is detectable in principle, and recent DNA-based detection strategies enable long-term detection of transgenic DNA (tDNA) following in vivo gene transfer. Copyright © 2012 John Wiley & Sons, Ltd.
The increase of the body's capacity to transport oxygen is a prime target for doping athletes in all endurance sports. For this pupose, blood transfusions or erythropoiesis stimulating agents (ESA), such as erythropoietin, NESP, and CERA are used. As direct detection of such manipulations is difficult, biomarkers that are connected to the haematopoietic system (haemoglobin concentration, reticulocytes) are monitored over time (Athlete Biological Passport (ABP)) and analyzed using mathematical models to identify patterns suspicious of doping. With this information, athletes can either be sanctioned directly based on their profile or targeted with conventional doping tests.
Key issues for the appropriate use of the ABP are correct targeting and use of all available information (e.g. whereabouts, cross sectional population data) in a forensic manner.
Future developments of the passport include the correction of all concentration-based variables for shifts in plasma volume, which might considerably increase sensitivity. New passport markers from the genomic, proteomic, and metabolomic level might add further information, but need to be validated before integration into the passport procedure.
A first assessment of blood data of federations that have implemented the passport show encouraging signs of a decreased blood-doping prevalence in their athletes, which adds scientific credibility to this innovative concept in the fight against ESA- and blood doping. Copyright
Aptamers are single stranded DNA or RNA oligonucleotides that have high affinity and specificity towards a wide range of target molecules. Aptamers have low molecular weight, amenable to chemical modifications and exhibit stability undeterred by repetitive denaturation and renaturation. Owing to these indispensable advantages, aptamers have been implemented as molecular recognition element as alternative to antibodies in various assays for diagnostics. By amalgamating with a number of methods that can provide information on the aptamer-target complex formation, aptamers have become the elemental tool for numerous biosensor developments. In this review, administration of aptamers in applications involving assays of fluorescence, electrochemistry, nano-label and nano-constructs are discussed. Although detection strategies are different for various aptamer-based assays, the core of the design strategies is similar towards reporting the presence of specific target binding to the corresponding aptamers. It is prognosticated that aptamers will find even broader applications with the development of new methods of transducing aptamer target binding.
A rapid and easy-to-use test kit, EPO WGA MAIIA, which can be used for distinguishing various endogenous human erythropoietins (hEPOs) and several recombinant hEPO and EPO analogues, has been evaluated. The test is based on chromatographic separation of the glycosylated isoforms of EPO using wheat germ agglutinin (WGA) and a sensitive immunoassay using anti-EPO carbon black nanostrings and image scanning for quantification. All of the reactions take place along the porous layer of a lateral flow microcolumn containing WGA and anti-EPO zones. The presence of molecules resembling hEPOs, such as Mircera, was detected by the aberrant affinity interaction with the antibody zone on the strip. It was possible to distinguish nine recombinant hEPOs expressed in hamster and human cell lines, as well as Aranesp and Mircera, from endogenous urine hEPO. The required amount of EPO in the samples, a few picograms, is very low compared with other methods for EPO isoform identification. This EPO isoform determination method opens the possibility to monitor recombinant EPO therapy for clinical research and seems to be a valuable candidate to the arsenal of EPO doping control tests.
C.E.R.A. (Continuous Erythropoietin Receptor Activator) is a new third-generation erythropoiesis-stimulating agent that has recently been linked with abuse in endurance sports. The anti-doping community rapidly reacted by releasing a high-throughput screening ELISA allowing the detection of C.E.R.A. doping in athletes' blood. In order to return adverse analytical findings, anti-doping laboratories, however, need, as far as possible, to confirm the presence of the drug in athletes' samples through orthogonal methods. This article focuses on the comparison of 2 proposed confirmation assays based on gel electrophoresis that were coupled with a new sample immunopurification method. IEF, the classical method used to target erythropoietin (EPO) and its recombinant analogues in athletes' samples, and SARKOSYL-PAGE were applied to the plasma samples of subjects having received a single injection of C.E.R.A. It was demonstrated that SARKOSYL-PAGE was at least 6 times more sensitive than IEF, with comparable specificity. A longer detection window coupled with easier interpretation criteria led us to recommend the use of SARKOSYL-PAGE to confirm C.E.R.A. presence in athletes' blood.
The Athlete Blood Passport is the most recent tool adopted by anti-doping authorities to detect athletes using performance-enhancing drugs such as recombinant human erythropoietin (rhEPO). This strategy relies on detecting abnormal variations in haematological variables caused by doping, against a background of biological and analytical variability. Ten subjects were given twice weekly intravenous injections of rhEPO for up to 12 weeks. Full blood counts were measured using a Sysmex XE-2100 automated haematology analyser, and total haemoglobin mass via a carbon monoxide rebreathing test. The sensitivity of the passport to flag abnormal deviations in blood values was evaluated using dedicated Athlete Blood Passport software. Our treatment regimen elicited a 10% increase in total haemoglobin mass equivalent to approximately two bags of reinfused blood. The passport software did not flag any subjects as being suspicious of doping whilst they were receiving rhEPO. We conclude that it is possible for athletes to use rhEPO without eliciting abnormal changes in the blood variables currently monitored by the Athlete Blood Passport.
A lectin-mediated affinity chromatographic SELEX technique was developed to generate functional ssDNA aptamers for recombinant human erythropoietin-α (rHuEPO-α), an important pharmaceutical glycoprotein for the first time. Secondary structure analysis of the aptamer clones from sequential 6th, 7th, and 8th rounds showed that a certain fragment 'CGAGAT' in the 3' primer region could be used to trace increasing evolution stringency from its hybridization at different locations, in which a specific hybridization with its complement in the 5' primer region (aptamer 807) was evolved as the prevalent one with the simplest and most common motif. Characteristics of the aptamers with lower Gibbs' free energies and K(d) values (nM) were investigated. For aptamer 813, the minimer 813-42nt formed by the random and primer regions was indispensable for the specific binding with rHuEPO-α. While for aptamer 807, only the random region, that is, 807-39nt, was the functional motif. Further experiments of methylation, site-directed mutation and length variation showed that the loop of aptamer 807-39nt was the key region for binding with rHuEPO-α, and the stem should be considered as a stabilizing part. Lower cross-reactivity of aptamer 807-39nt was observed with human normal urothelium tissues than the anti-EPO monoclonal antibody AE7A5. Aptamer 807-39nt also exhibited a specific recognition for human bladder carcinoma cells and human urothelium tumors, which might provide a novel way to probe such tumors with overexpressed EPOs.