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News feature, October 11, 2006

Beating the cheaters

Anti-doping labs search for test for EPO masking agent

Recent news reports reveal that scientists from the Lausanne anti-doping laboratory think they've detected attempts by athletes to mask the presence of the blood-boosting drug erythropoietin (EPO). They hypothesize that a protease enzyme was introduced into the urine while the athlete was giving the sample in order to destroy traces of the drug. Is this possible, and can the scientists develop a test to detect this method? Laura Weislo reports.


Erythropoietin (EPO) is naturally produced by the kidneys, and this peptide hormone stimulates the production of red blood cells in the bone marrow. As a drug, synthetic EPO is used to treat severe anaemia. Endurance athletes use it illegally to boost the production of red blood cells as high as they can within the legal limits. The increased oxygen carrying capacity generated by the extra red blood cells allows muscles to get more oxygen, and therefore do more work for longer periods of time before fatigue.

EPO is a small protein, and as a protein it can easily be destroyed by a class of enzymes known as proteases.

What is a protease?

Proteases are enzymes that occur naturally in all living organisms. These enzymes work in a variety of physiological processes, from digestion of food to complex physiological functions such as blood clotting. Proteases can break either specific peptide bonds, depending on the amino acid sequence of a protein, or break down a complete protein into its component parts, amino acids.

Proteases are big business. For hundreds of years, protein-degrading enzymes such as bromelain (from pineapple) have been used as meat tenderisers. Bromelain can also be used for medicinal purposes, from home remedies for bee stings to use as an anti-inflammatory agent for athletic injuries, digestive problems and arthritis.

In modern industry, proteases perform a variety of functions in diverse applications. They represent a huge share of industrial enzymes, and are used in detergents, leather-making, food processing, medicine and bio-remediation. The most popular application of proteases is in laundry detergents, where they are employed as gentle stain removers which act specifically on protein-based stains. They are also used in bio-pharmaceutical applications such as contact-lens enzyme cleaners and wound care products.

EPO, protease and the test

The urine test for EPO relies on the presence of the EPO protein and the sugars, or oligosaccharides, that are naturally attached to it in the body. Normal urine contains a certain amount of EPO, and in fact, the first therapeutic doses were purified from human urine.

The EPO synthesized by the human body has a slightly different pattern of oligosaccharides than those of the manufactured hormone, which is made in cultures of animal cells, making the drug distinguishable from normal EPO by the test.

A person who has injected synthetic EPO would show both normal human EPO and synthetic EPO "isoforms" on the test.

Since most proteases digest proteins indiscriminately, addition of the enzyme to a urine sample would destroy both the normal human EPO and the synthetic form, completely negating the test.


Athletes go to tremendous lengths to beat the EPO tests. Are they now using protease as a masking agent?
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When EPO was introduced as a therapeutic agent for anaemia in the late 1980's, it was a miracle drug for many patients suffering from chronic renal failure or those in chemotherapy. But the drug's darker side, the abuse by endurance athletes to boost the oxygen carrying capacity of their blood, has led to many sporting scandals in recent years.

EPO is so effective at improving performance that athletes have continued to risk scandal, sanctions or even death in order to gain its benefits. The introduction of an EPO urine test in 2001 should have reduced the prevalence of the abuse of the drug, but methods for avoiding positive tests have always stayed ahead of the anti-doping laboratories.

When the test was first introduced, it could only detect EPO within a few days of its administration. Since the drug's effects are strongest several weeks after the dose, the drug was used well in advance of races where the athletes would be subjected to anti-doping controls, and undetectable by that time.

As the test was made more sensitive, athletes switched from using the normal therapeutic doses to "micro-dosing." Using this method, the drug is only detectable within a day of its use. But as the test continues to be refined, several high-profile EPO positives may be inspiring athletes to find new ways to continue to use the drug without being caught.

Scientists in Switzerland suspect that the latest method to elude the test involves the use of a powder that destroys all traces of EPO, natural or synthetic. Martial Saugy, head of the Swiss anti-doping laboratory said "There has been a significant increase in the number of samples in which there is no EPO detected at all, leading us to believe they are being manipulated. We have no proof so far, but there are indications that a powder exists."

Matthias Kamber, of the Swiss federal sports office, was reportedly the first to detect the problem, and helped put forth the theory that the powder was protease, an enzyme that destroys other proteins. "It's possible that the absence of EPO in the probes have a natural cause, but it also feeds the suspicion of manipulation."

Proteases are widely available as stain removers, meat tenderisers, or dietary supplements, and would be needed only in small amounts to completely destroy the protein in a urine sample. It has been speculated that riders put a bit on their fingers and then urinated across the powder to add it to their sample. The use of such a product seems fairly obvious, so why hasn't this been detected before?

Dr. Don Catlin, director of the UCLA Olympic Analytical Laboratory in Los Angeles, California, tells Cyclingnews that they've been on the lookout for proteases in urine samples since 2002. His lab, like the Swiss lab, has seen a number of samples since 2002 that have no detectable EPO. But Catlin says, "It is not a total absence of proteins," indicating that whatever the masking agent is, it is more specific than the proteases that exist in products such as stain removers, as these enzymes would break down all the proteins in a sample.

Both the UCLA and Swiss laboratories say that the search for the protease is not simple because the enzyme breaks down very quickly, making detection difficult. Until the scientists can come up with a way to definitively identify the masking agent, if it exists, it is unlikely that riders can be sanctioned as a result of a blank EPO test. There are some medical conditions that can result in the lack of EPO production naturally, so it is possible that the blank results are not the result of an attempt to cheat the test. A blank test can also be the result of a lab error, so prosecution of an athlete over a blank test would be difficult.

So the race is on to come up with a test to detect this suspected method of cheating the EPO test. USADA lawyer Travis Tygart said that the agency is "aware of the efforts [of the laboratories]" and that under the WADA code, "masking agents are prohibited," indicating that once a method of detecting the substance is developed, the anti-doping authorities will have legal recourse to sanction riders based on a masking agent-positive result. Until then, one strategy for anti-doping efforts is to simply reduce the opportunity for athletes to get the substance into their urine. Tygart says "USADA already requires athletes to wash their hands" prior to giving their urine samples.

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