Detection of hemoglobin-based oxygen carriers in human serum for doping analysis: screening by electrophoresis

Abstract

Background: Hemoglobin-based oxygen carriers (HBOCs) have recently been included in the International Olympic Committee and World Anti-Doping Agency lists of substances and methods prohibited in sports. To enforce this rule and deter abuse of HBOCs in elite sports, it is necessary to develop HBOC-specific screening and confirmation tests that are the usual steps in antidoping control analysis.

Methods: We developed a screening method based on electrophoresis of serum samples cleared of haptoglobin (Hp). Four successive steps (immunoprecipitation of Hp, electrophoresis of the cleared serum, Western blotting of the separated proteins, and detection of hemoglobin-related molecules based on the peroxidase properties of the heme moiety), provided electropherograms that could be easily interpreted in terms of the presence of HBOCs. This method was tested with serum samples enriched with various types of HBOCs: polymerized, conjugated, and cross-linked hemoglobins. It was also applied to blood samples collected from 12 healthy volunteers who had been infused with either 30 or 45 g of Hemopure, a glutaraldehyde-polymerized bovine hemoglobin.

Results: The method clearly detected the presence in serum of the various types of HBOCs tested and demonstrated no possible confusion with endogenous hemoglobin that may be present in cases of hemolysis. The test was able to detect Hemopure for 4-5 days after administration of 45 g to healthy individuals.

Conclusions: The electrophoretic method is a simple, fast, and sensitive procedure that appears to fulfill the criteria of a screening test for the presence of HBOCs in antidoping control samples.