Doping is in the news again due to the Lance Armstrong scandal. However, the use of performance enhancing drugs is nothing new. Philostratus “The Athenian” warned about doctors helping the athletes in ancient Greece by cooking bread with opium derivatives from the poppy plant.1 Today, the analytical chemist plays a significant role in the challenge of detecting doping substances. Performance-enhancing substances can be taken in small amounts and can be found in concentrations as low as a few parts per billion in blood or urine (1 part per billion is 0.0000001%!). To complicate matters, many drugs are excreted or broken down by the body within a few hours, so only a small fraction of the drug remains unchanged in the body. Modern techniques such as mass spectrometry or solid-phase microextraction are sensitive enough to test for analytes at low concentration. As well, doping evidence can be provided by detection of by-products from metabolized substances.
One of the alleged substances consumed by Armstrong and his team was erythropoietin — better known as EPO. This hormone stimulates the production of red blood cells and improves the athlete’s muscle recovery and endurance by increasing the amount of oxygen carried within the blood. The use of synthetic EPO for “blood doping” was not analytically detectable until 2000 and even now the analysis is difficult. The main issue was how to differentiate between natural (endogenous, i.e., produced by the body) from recombinant (exogenous) EPO. Both forms are composed of the same amino acid sequences, but there is a difference in the attachment of carbohydrates.2
To detect the presence of recombinant EPO in urine samples, the National Anti-Doping Lab in France modified a protein detection method known as a western blot. Western blotting is conventionally done on protein mixtures separated by electrophoresis on the basis of size. Recombinant and natural EPO have the same amino acid sequence and thus the same size. However, the presence of a carbohydrate attachment changes the overall charge of the natural protein. The two proteins can be distinguished when electrophoresed through a gel with a pH gradient. An electric field is then applied to the gel causing the proteins to move based on their overall charge. As the proteins migrate through the gel, they are exposed to different pH. At some point, the protein will reach its isoelectric point, the pH at which the protein has no net charge, and it will stop moving. The proteins are transferred onto a membrane where both types of EPO can be detected using specific antibodies. If recombinant EPO is present in the sample, an additional, more basic band should appear on the membrane. This would likely result in further analysis of the sample.
Analytical methods have since been developed for many erythropoietin stimulating agents, such as peginesatride which can be detected by liquid chromatography and mass spectrometry. As detection methods are developed and new doping methods are devised, scientists will continue to play a role in maintaining the honesty and integrity of sport.
References
- Doping in Ancient and Modern Olympic Games, Panayiotis J. Papagelopoulos; Andreas F. Mavrogenis; Panayotis N. Soucacos, Orthopedics- Volume 27 Issue 12 , December 2004
- Emma Davies, Chemistry at the Olympics. http://www.rsc.org/chemistryworld/2012/06/chemistry-olympics
[Thanks to Susan Kelso, a University of Waterloo biochemistry student, who helped outline the western blot procedure.]
