ABSTRACT
Erythropoietin (EPO) has protective effects in several tissues and could be used for therapeutic purposes, but the doses of EPO that can be beneficial in case of hypoxic-ischemic conditions due to overinduced erythropoiesis could be detrimental in treated patients. Carbamylation of erythropoietin maintains the tissue-protective effects of EPO but without erythropoietic effects. Carbamylated EPO (CEPO) is listed in WADA Prohibited List in class S2 as "Innate repair receptor agonists." The CEPO was synthesized using the method described previously. Digestion with endoproteinase Lys-C was used to distinguish rhEPO from CEPO. The digested samples containing recombinant EPO, urinary EPO (uEPO), or CEPO were analyzed by the SAR-PAGE method (sarcosyl polyacrylamide gel electrophoresis-PAGE). Endoproteinase Lys-C breaks the peptide chains of lysine. Lysine residues, converted to homocitrulline by carbamylation, cannot be cleaved by endoproteinase Lys-C. Therefore, the CEPO protein chain remained unchanged in contrast to rhEPO and uEPO, which allows for easily differentiation of them.
Subject(s)
Doping in Sports/prevention & control , Erythropoietin/analogs & derivatives , Substance Abuse Detection/methods , Amino Acid Sequence , Cytokine Receptor Common beta Subunit/chemistry , Cytokine Receptor Common beta Subunit/genetics , Cytokine Receptor Common beta Subunit/metabolism , Doping in Sports/methods , Erythropoietin/chemistry , Erythropoietin/genetics , Erythropoietin/urine , Humans , Receptors, Erythropoietin/chemistry , Receptors, Erythropoietin/genetics , Receptors, Erythropoietin/metabolism , Substance Abuse Detection/standardsABSTRACT
According to the World Anti-Doping Agency (WADA) Prohibited List, glucocorticosteroids are prohibited in competition and only when administered by oral, intravenous, intramuscular or rectal routes. Up to now, in order to differentiate whether glucocorticosteroids were administered by one of the prohibited routes or not, a specific reporting limit for urinary concentrations of parent compounds and their metabolites was established at 30 ng/mL. Additionally, the new specific regulation starting from 1 September 2014 for budesonide have been introduced that the 6ß-hydroxybudesonide shall be targeted. Budesonide is a glucocorticosteroid used mainly by inhalation for asthma management. Interestingly, anti-doping laboratory statistics show that budesonide adverse analytical findings (AAF) constitute almost 50% of all reported glucocorticosteroid AAFs, even though budesonide possesses a very low systemic activity which may cause performance enhance effects. This work presents the results of five studies of controlled budesonide administration carried out on professional athletes. The samples were analyzed by using a quantitative HPLC/MS/MS method for 16α-hydroxy-prednisolone, the most abundant budesonide metabolite in urine. Our data clearly show that inhalation of budesonide at least 12 h before a competition at therapeutic doses leads to appearance of the main budesonide metabolite in concentrations exceeding prior reporting limit for this compound. Therefore, our work strongly supports recent WADA decision not to target the main budesonide metabolite using the same reporting limit as for other glucocorticosteroids.
