Erythropoietin, a glycoprotein hormone, increases the number of circulating red blood cells, which consequently increases tissue oxygenation. Shortly after production of recombinant erythropoietin athletes began to abuse the product as a way of doping. Both indirect and direct methods exist for the detection of erythropoietin. Improvements in detection of recombinant erythropoietin are needed as it is suspected that athletes are finding ways to avoid detection. EPO gene doping is expected to soon be a threat to sporting federations and no method of detection is currently available.
Erythropoiesis is the production of red blood cells (RBCs) almost exclusively in the red bone marrow (Diagram 1). A RBCs life span is ~120 days demonstrating the need for replacement. Erythropoietin (EPO), a glycoprotein hormone, regulates the rate of erythropoiesis. Consequently, this increases tissue oxygenation, resulting in increased endurance. EPO stimulates growth and prevents apoptosis of developing RBCs. The renal peritubular interstitial cells synthesise 90% of EPO, although the brain and liver are capable of producing small quantities. EPO is produced in response to low tissue oxygenation (hypoxia) (Diagram 2). "EPO synthesis is mediated by the transcription activator hypoxia-inducible factor-1(HIF-1)". HIF-1 binds to the EPO gene activating transcription and therefore production of EPO.
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In 1985, the human erythropoietin gene was successfully cloned. This led to large scale production of recombinant human EPO (rHuEPO). rHuEPO has enormous therapeutic value and is used to treat a range of conditions, such as, severe anaemia. rHuEPO is currently being researched extensively as a potential therapeutic agent for a variety of diseases. Very shortly after production, rHuEPO became a preferred way of doping. It was considered more effective and an easier method of doping, leading to the diminishment of blood transfusion doping. A study showed that 3-7% of athletes competing in endurance competitions were abusing rHuEPO. Doping with rHuEPO puts the athlete at risk of serious adverse side effects, predominantly related to the cardiovascular system. Unfortunately, despite the risks athletes are still willing to abuse with rHuEPO. The financial gain, social status and performance enhancement is merely too much to resist for some athletes.
The majority of international sports federations have banned the use of EPO. The World Anti-Doping Agency (WADA) "promotes, coordinates, and monitors the fight against doping in sport in all its forms". A variety of detection methods aimed at preventing EPO abuse in athletes are used. The development of various novel recombinant EPO analogues and their easy accessibility has led to the widespread difficulty in controlling doping. Current methods of detection of rHuEPO approved by WADA have questionable efficacy. EPO doping damages the fundamental principles that make up the ethical framework surrounding sport. Dopers make competitions "unfair", by breaking the rule of equal opportunity for all those competing, as they possess a competitive advantage.
Google Scholar, Metalib, Pubmed and Science Direct were used to gather articles used within this report. Search terms used were: "EPO detection"â€¦
In 1987, the availability of rHuEPO soon became a method of doping within sports. EPO was banned in 1990 by the International Olympics Committee, following the suspected deaths of at least 17 European cyclists doping with rHuEPO. In order to enforce the widespread ban of EPO doping in sporting federations, detection strategies needed to be implemented. Two philosophies were developed for the detection of rHuEPO doping - Indirect and Direct Methods. [EPO and Blood doping]
Indirect methods of detection
Indirect approaches to detection involve monitoring markers of enhanced or reduced erythropoiesis.
A trial was carried out in preparation for the Sydney Olympic Games in 2000 to establish blood parameters for the indirect method. The trial involved 30 club level athletes who were administered 50 IU kg-1 rHuEPO three times a week, for duration of four weeks. The results provided a wide range of blood parameters. After statistical analysis, two indirect models were produced. The "ON" model was capable of detecting athletes for up to a few days after rHuEPO administration. The "OFF" model was most effective for detection in the washout phase. The "ON" model included reticulocyte haematocrit, soluble transferrin receptor, haematocrit, serum EPO and % macrocytes. It identified 94-100% of rHuEPO dopers and one false-positive was recorded. The "OFF" model incorporated serum EPO, haematocrit, and reticulocyte haematocrit. It was effective with no false-positives recorded.
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Refined "second generation" models were soon developed based on previous work. Follow-up studies were completed using varying doses of rHuEPO, a larger subject group and different ethnicities. Easier to measure parameters were included in the models, such as, haemoglobin concentration and % reticulocytes. It proved a more effective way of detecting rHuEPO abuse after injections had ceased. The "OFF" model is still the only means of detecting athletes who have ceased EPO doping at least a week before the competition. The models main disadvantage is the inability to distinguish between dopers and athletes with naturally elevated blood parameters. Therefore a third generation model was developed. It involves a longitudinal follow-up of an athlete's individual haematological values, rather than using population derived thresholds. It forms the concept being the "Biological Passport" detection method adopted by the International Cycling Union (UCI), and has proven effective. It is likely other sporting federations will soon incorporate this approach into their detection strategies.
Although these models are incapable of conclusively proving rHuEPO abuse they are strong indicators and can be used in conjunction with the direct testing procedure. At present "models are included into target-testing, exclusion from competitions and the concepts of the "Biological Passport".
Direct methods of detection
The direct test involves taking a urine sample and utilising IEF and immunoblotting to differentiate between rHuEPO and eEPO. The application of rHuEPO in a non-anaemic suppresses the production of eEPO. This results in a continuous shift from eEPO to rHuEPO IEF profile, although is dependent on serum half-life and amount of rHuEPO used. It is possible to separate the endogenous from the exogenous EPO based on the differences in the charge status of different sugars [Epo and blood doping]. These principles make direct testing feasible. WADA use this method routinely in their anti-doping laboratories, despite the controversy associated with it.
Endogenous uepo shows a much wider isoform distribution expanding to pI values far more acidic that any commercial repo tested.
A range of recombinant analogues exist on the market, which all have differing IEF patterns due to the variations in their production process. All known analogues are detectable using the IEF method.
The method involves the detection of rHuEPO in urine and consists of a few main steps (Diagram 3). Detectability decreases in proportion to time of last injection. After 3 days only 50% of dopers could be declared positive and none could be declared positive after 7 days. Criteria are used for identification. Initially criteria were based on the bands in the basic region needing to represent 80% of the total intensity, in order to have a positive result. This was not always effective. Scientists refined the criteria, requiring at least three consecutive bands with two-fold intensity in the basic region than those in the acidic region. This reduced false-positives recorded.
The method of detection relies on the recognition of all EPO's regardless of origin with a single monoclonal antibody. The recommended antibody is AE7A5 and is non-specific for EPO. The antibody is capable of cross-reacting with other human or bacterial proteins. These proteins can sometimes escape the purification phase due to their similar size to EPO. For example, Escherichia coli are present in human urine and are adherent to human skin in the genital region. E. coli produces a bacterial protein, thioredoxin reductase that has been shown to cross-react with the monoclonal antibody. The specificity of the monoclonal antibody is still considered controversial and has been criticised on numerous occasions.
Athletes' microdosing with rHuEPO are able to drastically reduce the effectiveness of the direct test. The window for detection can be reduced to 12-18 hours post-injection. The full implications of microdosing are unclear. It speculated athlete receiving microdoses for an extended period (>2-4 weeks), would have the reappearance of endogenous bands to a significant magnitude to "pass" any direct testing.
It was reported that 15% of athletes tested had undetectable EPO profiles. Proteases were suspected of being used to manipulate results. Proteases are readily available and destroy rHuEPO and eEPO in short time if added during collection period. They are subject to autolysis meaning their activity is lost over time. Proteases have a distinct pattern of degradation meaning a detection test could be developed. Another alternative strategy is coating the collection vessel with protease inhibitors to prevent proteolysis.
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Various other problems have been discussed related to the reliability of the test. These include: length of sample preparation, lack of interlaboratory standardisation, bacterial contamination during collection process and reliability of the recommended software analysing programmes. Various analogues of the initial recombinant product are likely to not always be easily detectable by standard IEF- methods.
The pharmokinetics of rHuEPO and the long lasting biological effect within the body mean that off-competition testing is the most effective way of detecting abuse with the current strategy. The problems associated with the techniques necessitate the need to develop alternative strategies for detection of rHuEPO [Table 1].
Gene doping is a process that involves introducing a transgene that will subsequently alter expression, or modulate existing gene's activity, in order to enhance performance. Animal trials have demonstrated the effectiveness of EPO gene doping and highlighted the potential serious health risks associated (Table 2). It has been shown that long-term expression of the EPO transgene can last for more than 450 days. It was determined that expression was proportional to the amount of transgene introduced.
Currently there are no tests available capable of detecting gene doping in athletes. The WADA is currently funding extensive research into developing effective detection strategies. Methods under investigation include detecting the transgenic protein or delivery vector, monitoring immune response, and profiling approaches. [Table 3?]
Conceptual and practical problems have made the development process challenging. A major difficulty resides in the fact that EPO produced by the foreign gene or genetically manipulated cell will be very similar if not identical to eEPO. This is further complicated as gene doping is likely to involve a local injection into muscle. Therefore, the products may not be present in significant quantities for detection in blood or urine. The only solution would be to obtain a muscle biopsy from the athlete. This would be considered unviable in a sports setting and demonstrates the need for a feasible detection procedure.
Sport Federations and the scientific community are burdened with scientific and ethical challenges associated with the detection of EPO abuse. A close partnership between sports and science is needed to achieve the most effective detection strategies.
The WADA should approve plans for incorporating the "Biological Passport" into its detection strategies. This will encourage sporting federations to follow. Suspicious results could then be passed on for further direct testing. The IEF-Immunoblotting method has been heavily criticised and there are various controversies surrounding its use. An alternative direct detection strategy is needed to be used in conjunction with or eventually replace the current method. The growing development of novel recombinant analogues reinforces the need for a reliable detection method that does not have questionable efficacy.
It is highly possible that the first gene doping athletes could be competing in the London 2012 Olympic Games. This threat should not be underestimated. Clinical scientists are under increasing pressure to develop a detection method that is feasible and reliable.
Sporting authorities need to educate athletes about the importance of testing and the risks associated with EPO doping. It is clear some athletes lack this knowledge. The athlete may provide insight that can be used as an effective deterrent to doping. Educational programmes would have the potential of reinforcing personal ethical values and if publicised by professional athletes could prove effective in preventing the growing rise in adolescent drug abuse in sports.