EPO-Fc proteins have been under investigation as a potential drug for treating anaemia and have shown larger half-life values than other erythropoiesis-stimulating agents (ESAs). Sodium dodecyl sulfate/sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SDS/SAR-PAGE) methods and subsequent immunoblotting are used for routine anti-doping analysis. This paper reports that EPO-Fc fusion proteins can be detected in serum samples by isoelectric focusing-polyacrylamide gel electrophoresis (IEF-PAGE) in carrier ampholyte-based gels with a pH 2-6 gradient after removing the Fc part via site-specific IdeS protease cleavage. The IdeS-digested EPO-Fc protein yields three fragments: two Fc fragments and one dimeric EPO-hinge fragment. After IEF-PAGE was followed by double Western blotting with chemiluminescent detection, the dimeric EPO-hinge fragment showed a unique isoelectric pattern, which differed from those of any other currently known analogue of EPO. We observed that the removal of the Fc fragment from EPO-Fc reduced the apparent molecular weight of entire fusion protein and increased its electrophoretic mobility. As a result, the band for the EPO-hinge fragment was located in a region between the rEPO and NESP standards, at which lower amounts of serum proteins are present. Simple and selective protocols for determining the EPO-Fc protein in human serum were developed to extend the methodological anti-doping arsenal. This protocol has been characterized. The limit of detection (LOD) of the IEF-PAGE method was 20 pg, and that of SDS/SAR-PAGE was 15 pg.
Erythropoietin Fc (EPO-Fc) fusion proteins are potential drug candidates that have been designed for the treatment of anemia in humans by stimulating erythrocyte production. Such compounds can be considered performance-enhancing agents that may be used by athletes in endurance sports. This study describes the primary structure of commercially available EPO-Fc based on comprehensive liquid chromatography coupled with mass spectrometry (LC-MS) analysis. A bottom-up approach and the intact molecular weight (MW) measurement of deglycosylated protein and its IdeS proteolytic fractions was used to determine the amino acid sequence of EPO-Fc. Using multiple proteases, peptides covering unknown fusion breakpoints (spacer peptides) were identified. We demonstrated that "spacer peptides" can be used in the determination of EPO-Fc fusion proteins in biological samples using common LC-tandem MS methods.
Background: The analysis of individual microRNAs (miRNAs) as a diagnostic and prognostic tool for the effective treatment of various diseases has aroused particular interest in the scientific community. The determination of circulating miRNAs makes it possible to assess biological changes associated with nutritional processes, the intake of dietary supplements and drugs, etc. The profile of circulating miRNAs reflects the individual adaptation of the organism to the effect of specific environmental conditions. Objective: to systematize the data and show the importance of circulating miRNAs as new potential biomarkers of the organism's response to the intake of various dietary supplements, drugs, and consider the possibility of their use in doping control. Method: a systematic analysis of scientific publications (ncbi.nlm.nih.gov) on the miRNA expression profile in response to the intake of dietary supplements and drugs most often used by athletes, and supposed their role as potential markers in modern doping control was carried out. Results: the profile of circulating miRNAs is highly dependent on the intake of a particular drug, and, therefore, may be used as a marker of the effects of biologically active supplements and drugs including the substances from the Prohibited List of the World Anti-Doping Agency (WADA). Conclusion: monitoring of circulating miRNAs can serve as a high-precision marker for detecting doping abuse in elite sports. However, it is necessary to conduct additional studies on the effect of complex drugs on the profile of circulating miRNAs and individual circulating miRNAs on a particular biological process.
Objectives. Over the last decade, hematopoietic stimulants have grown increasingly popular in elite sports. This is supported by the growing number of high-profile doping scandals linked to their use. A group of these stimulants includes cobalt salts, which cause an increase in the oxygen capacity of the blood as well as a powerful stimulation of metabolic processes, resulting innoticeable competitive advantages. The use of cobalt salts is regulated according to the Prohibited List of the World Anti-Doping Agency (WADA). Currently, only a few works have been dedicated to solving the problem of detecting the abuse of cobalt salts in anti-doping control. Only a few laboratories have included cobalt salt determination in their methodological bases. The purpose of this review is to attract the attention of the scientific community to the toxicity of cobalt compounds, consequences of their intake, and pharmacokinetics, as well as the problems in their detection methods due to their widespread availability in the modern market and the growing number of abuse cases.Results. The main biological functions of cobalt, cellular levels of exposure, toxicity, and symptoms of cobalt salt poisoning are presented in detail in this review article. The data from the literature on the main methods for detecting cobalt as a doping agent have been generalized and systematized. There is a major focus on the amount of cobalt in dietary supplements that could cause an athlete to test positive for cobalt when they are consumed.Conclusions. After analyzing promising cobalt detection approaches and methods, it was determined that high-performance liquid chromatography in combination with inductively coupled plasma mass spectrometry has an undeniable advantage for detecting cobalt as a doping agent. The lack of explicit WADA requirements for detection methods and the lack of its obligation to determine cobalt make it tempting for unscrupulous athletes to use its salts. Therefore, antidoping laboratories must implement the abovementioned method as soon as possible.
The article is devoted to develop of an approach for the identification of new stimulator of ematopoiesis, EPO-Fc fusion protein, which is banned by the World Anti-doping Agency (WADA) to use by athletes since it has become doping. Existing methods of qualitative determination of this substances in routine practice of antidoping laboratories such as polyacrylamide gelelectrophoresis in presence of sodium dodecylsulphate (SDS-PAGE) or lauroylsarcosinate (SARPAGE) are insufficiently specific. The article shows the principal possibility of identification of EPO-Fc fusion protein by means of IEF-PAGE in carrier ampholyte-based gels with a pH range 2-6 after Fc-fragment removal via fermentative hydrolysis.It has been shown that the removing of the crystallizable fragment leads to decrease of molecular weight of whole hybrid molecule and to increase its electrophoretic mobility that allows to detect this banned substances with high specificity by existing methods. During the study the enzyme for hydrolytic cleavage and optimum conditions of hydrolysis of EPO-Fc in serum samples were selected.
Objectives. Cobalt mimics the state of hypoxia to prevent degradation of the alpha subunit of hypoxia-inducible factor, resulting in an increase in blood oxygen capacity and endurance. Athletes can use this property to gain competitive advantage. Nowadays, direct methods of inductively coupled plasma mass spectrometry and liquid chromatography-tandem mass spectrometry are used to determine total cobalt levels in the body. However, the World Anti-Doping Agency is yet to establish a maximum allowable threshold concentration of this element in biofluids. The lack of clear identification criteria complicates the interpretation of the obtained results for the purposes of doping control. In this regard, the present work proposes a new approach for the indirect determination of possible cobalt abuse based on changes in the expression levels of miRNAs involved in the regulation of hypoxia signaling pathways. Here, the aim is to identify possible microRNA markers whose expression does not depend on exercise-induced hypoxia, but changes markedly when taking cobalt preparations.Methods. MicroRNA isolation was performed from blood plasma samples using the PAXgene Blood miRNA Kit. Quantitative real-time polymerase chain reaction (PCR) was performed on CFX96 Bio-Rad (USA) analyzer using miScript® SYBR® Green PCR Kits and panels for studying the expression profiles of mature microRNAs of the hypoxia signaling pathway miScript® miRNA PCR Array.Results. Based on the statistical analysis of the data, it was found that the expression of hsa-miR-15b-5p in the blood plasma of the subjects does not depend on physical activity, but increases when taking cobalt preparations.Conclusions. The difference in expression levels during anaerobic exercise-induced hypoxia and cobalt-induced hypoxia makes hsa-miR-15b-5p a potential candidate to be a marker of erythropoiesis-stimulating agent abuse.
In recent years, interest in the search for new potential molecular biomarkers to assess the effectiveness of the training process in elite sports, various pathologies caused by overload, and abuse of prohibited substances has reached an unprecedented level. MicroRNAs that regulate the processes of metabolism, differentiation, proliferation, and apoptosis of cells at the post-transcriptional level are the most valuable candidates for use in this regard. They are found in all biological fluids of the human body, are stable during long-term storage and resistant to changes in environmental conditions. It is known that changes in the miRNA expression profile are closely associated with physical activity, as well as with the administration of recombinant hormones, erythropoiesis-stimulating agents, and other substances and methods that improve sports performance, which are prohibited by the World Anti-Doping Agency (WADA).Objective: In this study, plasma samples from athletes involved in endurance sports (race walking) and non-athletic volunteers were analyzed using a hypoxia signaling pathway panel to identify hypoxia candidate markers.Materials and methods: Expression profiles of plasma circulating miRNAs were assessed by reverse transcription followed by real-time quantitative polymerase chain reaction (RT Q-PCR). The obtained data were subjected to statistical processing using the CFX Manager Software v3.1 program.Results: Three potential microRNA markers were identified: hsa-miR-210-3p, hsa-miR-320a and hsa-miR-935 (increased in athletes by 61.6 times, 51.8 and 41.0 times, respectively) — of physiological response to the training, load, which may be associated with the emergence of hypoxia during endurance training.Conclusion: We have obtained preliminary data on differences in the expression profiles of circulating miRNAs in healthy non-athletic volunteers and professional athletes in the out-of-competition period. In the future, it is planned to expand the sample of studied blood plasma samples and compare the expression profiles of circulating microRNAs in athletes in competition and out-of-competition periods, as well as to compare the expression profiles of circulating microRNAs during aerobic and anaerobic loads. In addition, it is of interest to analyze changes in the expression levels of circulating miRNAs when using hypoxia mimetics applied to improve sports performance.
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