Abstract
The presence of proteolytic enzymes in urine samples, coming from exogenous or endogenous sources, enhances the cleavage of human chorionic gonadotropin (hCG). Moreover, elevated temperatures occurring occasionally during the delayed transportation of sport urine samples, favor the nicking of the hCG molecule. The aim of the current study, funded by the World Anti-Doping Agency (WADA), was the application of a stabilization mixture in athletes’ urine samples to chemically inactivate proteolytic enzymes coming from exogenous or endogenous sources so as to prevent the degradation of hCG. The stabilization mixture applied, already tested for the stabilization of endogenous steroids and recombinant erythropoietin (rEPO), was a combination of antibiotics, antimycotic substances, and protease inhibitors. Incubation experiments were conducted in the presence or absence of the stabilization mixture in urine aliquots spiked with six proteases (first series of experiments) and one microorganism associated with urinary tract infections (UTI) (second series of experiments). Intact hCG levels were evaluated by using the EIAgen Total hCG kit. In the first series of experiments, hCG levels were reduced in the untreated aliquots following incubation at 37 °C. The addition of the chemical stabilization mixture prevented degradation of hCG induced by four of the proteases applied. In the second series of experiments, no significant difference was found in urine inoculated with E. coli, between aliquots treated with chemical mixture and the untreated aliquots. The addition of the proposed chemical stabilization mixture improves the quality of athletes’ urine samples against possible deterioration due to high temperatures or attempts of proteolytic manipulation.
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Notes
hCG-H, hyperglycosylated hCG; hCGn, nicked form of hCG; hCGCTP, hCG missing the C-terminal peptide; hCGβ, β-subunit of hCG; hCGβCTP, β-subunit of hCG missing the C-terminal peptide; hCGβcf, β-core fragment of hCGβ, according to [15] and to the nomenclature proposed by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) [16].
hCGα, α-subunit of hCG; hCGβn, nicked β-subunit of hCG.
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Acknowledgments
The current work was supported by a research grant awarded by the World Anti-Doping Agency (05D6CG). We are grateful to Ms. C. Papameletiou (Laboratory of Microbiology, Department of Agricultural Biotechnology, Agricultural University of Athens) for her technical assistance in microbiological preparations. The cooperation of J. Papaparaskevas (Department of Microbiology, Medical School of the University of Athens) in the provision of E. coli strains is highly acknowledged.
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Tsivou, M., Dimopoulou, H.A., Georgakopoulos, D.G. et al. Stabilization of human urine doping control samples: IV. Human chorionic gonadotropin. Anal Bioanal Chem 398, 1313–1318 (2010). https://doi.org/10.1007/s00216-010-4033-9
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DOI: https://doi.org/10.1007/s00216-010-4033-9