Unit:
Department of BiologyLibrary of the School of Science
Author:
Sakellariou Panagiotis
Dissertation committee:
Ουρανία Τσιτσιλώνη, Καθηγήτρια, Τμήμα Βιολογίας, Ε.Κ.Π.Α.
Ανδρέας Σκορίλας, Καθηγητής, Τμήμα Βιολογίας, Ε.Κ.Π.Α.
Μιχαήλ Κουππάρης, Ομότιμος Καθηγητής, Τμήμα Χημείας, Ε.Κ.Π.Α.
Διαμάντης Σίδερης, Αναπληρωτής Καθηγητής, Τμήμα Βιολογίας, Ε.Κ.Π.Α.
Δημήτρης Στραβοπόδης, Αναπληρωτής Καθηγητής, Τμήμα Βιολογίας, Ε.Κ.Π.Α.
Ευάγγελος Μπακέας, Αναπληρωτής Καθηγητής, Τμήμα Χημείας, Ε.Κ.Π.Α.
Δημήτρης Κλέτσας, Διευθυντής Ερευνών, Ινστιτούτο Βιοεπιστημών και Εφαρμογών, Ε.Κ.Ε.Φ.Ε. “Δημόκριτος”
Original Title:
Συγκριτική μελέτη μεθόδων έμμεσης ανίχνευσης αυτόλογης μετάγγισης αίματος και διέγερσης ερυθροποίησης με εντοπισμό βιοδεικτών σε βιολογικά δείγματα για τον έλεγχο ντόπινγκ αθλητών
Translated title:
Comparative study of methods for indirect detection of autologous blood transfusion and erythropoiesis stimulation by biomarkers detection in biological samples for the athletes' doping control
Summary:
Despite being prohibited by the World Anti-Doping Agency (WADA), blood doping through Erythropoiesis Stimulating Agents (ESAs) misuse and blood transfusion, is used by athletes who want to gain an unfair advantage by increasing red blood cell production and hence, their aerobic capacity. Methods of direct detection can be used to detect ESAs and homologous blood transfusion in athletes’ samples. The absence of a method for the direct detection of autologous blood transfusion, the inability to detect new banned substances, and the difficulty in detecting microdoses of recombinant erythropoietins have led anti-doping science to develop methods of indirect detection. The Athlete Biological Passport (ABP), is an important achievement, as ABP is a strategy for blood doping detection through the long-term monitoring of hematological parameter levels and endogenous androgen concentrations of an individual athlete. However, new approaches to blood doping create new analytical challenges for doping control and there is a need for the enforcement of this excellent detection tool. The combination of the Athlete Biological Passport with other indirect biomarkers, such as monitoring of iron metabolism protein levels and metabolites, is believed to enhance the effectiveness of blood doping tests.
The present study focused on the identification of indirect biomarkers for the detection of banned substances and/or methods included in WADA’s Prohibited List and associated with blood doping. Special attention was given to the possible effect of their abuse in erythropoiesis, the changes that may cause in the parameter of the hematological and steroid module of ABP, as well as to the estimation of iron metabolism proteins and erythropoietin expression levels.
An in vivo metabolic study was performed following a single administration of 10 mg methylnortestosterone to a volunteer. Urine samples were analyzed by gas chromatography-tandem mass spectrometry (GC-MS/MS). Seven metabolites were identified (M1-M4 and S1-S3). Phase II glucuronide metabolites M2, M3, M4 and sulfate metabolites S2 and S3, were detected until the end of the excretion study (192 h), while M1 and S1 were detected for 72 h. The two glucuronide metabolites M3 and M4 and the three sulfate metabolites S1, S2, S3 are detected for the first time. It has also been observed that single administration of methylnortestosterone alters steroid profile parameters at least 48 h after administration, while not affecting urinary erythropoietin levels as shown following analysis by electrophoresis.
An in vivo metabolic study was performed following a single administration of 10 mg LGD-4033 to a volunteer. Urine samples were analyzed by liquid chromatography-mass spectrometry (LC-MS). Six phase I metabolites M1-M6 were identified, with M5 metabolite having the largest detection window (20,5 days). The Μ5 metabolite is suggested as the most suitable biomarker for LGD-4033 detection in hydrolyzed urine samples. The M6 metabolite was detected for first time in human urine. Furthermore, after dilute-and-inject preparation of urine samples, the parent compound and the M1, M2, M3 metabolites were detected as gluco-conjugated phase II metabolites. Gluco-conjugated parent LGD-4033 can be used as a biomarker for LGD-4033 detection up to 2,5 days after administration to samples analyzed after dilution. The LGD-4033 administration did not alter steroid profile or urinary erythropoietin levels.
The blood doping detection, by analyzing athletes’ blood samples for changes in the ABP hematological parameters, can be performed in the Doping Control Laboratory of Athens after the evaluation of the Sysmex XT-2000i and Sysmex XN-1000 hematological analyzers.
Analysis of urine samples by LC-MS, after multiple administration of inhaled salmeterol to a volunteer at 100 μg / 12 h, demonstrated bioaccumulation of the parent substance and the α-hydroxysalmeterol metabolite, with increasing urinary concentrations up to 3,5 ng/ml for salmeterol and up to 11,11 ng/mL for its metabolite. In addition, blood samples analysis showed that salmeterol bioaccumulation may cause changes in the ABP hematological parameters and iron metabolism proteins’ level, while it also affects the urinary erythropoietin levels, as shown after analysis by electrophoresis. These results support the view that β2-adrenergic receptors are involved in the early stages of erythropoiesis in which progenitor cells do not express erythropoietin receptors.
Main subject category:
Science
Keywords:
blood doping, methylnortestosterone, LGD-4033, salmeterol, Athlete Biological Passport, metabolites, erythropoietin
Number of references:
159
