Epo in Sport
Competitive athletes are constantly in search of ways to get better, seeking a slight edge over their closest competition. They are willing to practice for countless hours, put themselves through rigorous training and follow a very strict diet. Those who are passionate about their sport are willing to do just about anything to improve performance, but just how far are athletes willing to go? With recent advancements in sports science, it has become possible to alter some elements of human physiology.
The human body has been meticulously studied over the years, and as a result we are able to comprehend how complex systems function enabling the human body to perform simple everyday functions, as well as, impressive athletic performances. Science has discovered there are ways to improve the physiology of the human body to enhance athletic performance. By carefully tailoring specific functions to enhance a specific task an athlete will most likely be able to get the “one up” on the competition.
Science has also discovered there are dangers associated with tampering with these complex systems that keep the human body alive and well. Unfortunately, some athletes ignore the physiological risks/ professional repercussions and indulge in enhancing some physiological processes in order to gain a slight edge against the competition. Today, as well as in the past, various sporting organizations have had to deal with performance-enhancing issues through testing of their athletes, yet these people continue to seek out ways to sneak under the wire, undetected.
One example of athletes trying to beat the system is that of the recently publicized performance-enhancing dispute with blood doping in the sport of cycling, namely the use of recombinant human erythropoietin (Robinson, Mangin, and Saugy 2003). The following will discuss the function of erythropoietin, its uses in medicine and athletics, the benefits and risks of artificial along with testing methods for detection of illegal use. In order to perform in endurance sports, efficient oxygen delivery from lungs to muscles is crucial.
The cells responsible for oxygen delivery are erythrocytes, or red blood cells. The functional portion of the red blood cell that acts as an oxygen carrier is the protein molecule hemoglobin (Kraene, Fleck and Deschenes 2012). Hemoglobin is a four part haem-iron containing protein, with two alpha and two beta subunits associated with each molecule. Hemoglobin accounts for 99% of the protein composition of an erythrocyte (Lippi, Franchini, Salvengo et al). Circulating blood contains approximately 40-45% red blood cells in its composition (Kraene, Fleck and Deschenes 2012).
The hemoglobin associated with each red blood cell has a specific mechanism for pick-up and delivery of oxygen. This mechanism depends on varying physiological body conditions during which oxygen has differing affinity for the hemoglobin molecule. The conditions at which affinity for oxygen is high include lower body temperature, low carbon dioxide, and low 2,3-diphosphoglyerate (Elliott 2008). As these are the conditions found in the lungs, plentiful oxygen will bind to the hemoglobin for transport to the tissue cells in the body.
In the tissue where carbon dioxide concentrations are high, body temperature increases, higher hydrogen ion and ,2,3-disphosphoglycerate concentrations, oxygen affinity for hemoglobin is reduced, resulting in the delivery of oxygen to tissues (Elliott 2008). During physical exercise, the body’s consumption of oxygen is increased due to the demand of working muscles. As a result of this process, the carrying capacity of hemoglobin is adjusted automatically to deliver adequate oxygen to the muscle tissues (Lippi, Franchini, Salvango et al 2006).
Applying this principle of supply and demand, to an endurance sport, one can see how an athlete’s aerobic training regime aims to peak the efficiency of the process of oxygen delivery from lungs to muscle tissue. To maximize the process of oxygen delivery, a high number of circulating erythrocytes is desired, resulting in more available hemoglobin and therefore more oxygen can be delivered to working muscles. Red blood cell (RBC) production, called erythropoiesis, is carefully controlled and monitored by the body (Lippi, Franchini, Salvango et al 2006).
This monitoring system involves oxygen-sensing cells to detect hypoxia (low oxygen concentration) in the body. During oxygen deprivation, a nerurosecretory mechanism is activated through chemoreceptors found in the carotid body ( in the carotid artery found in the neck) and in the lungs. If out of balance, the body undergoes rapid cardiopulmonary adjustments to compensate for the current stress of hypoxia (Kraene, Fleck and Deschenes 2012). One of the factors present during hypoxia is the hypoxia inducible factor, HIF-1.
This molecule acts as a transcription factor for controlling several genes (Lippi, Franchini, Salvango et al 2006). When oxygen levels are low, the enzymes that normally inhibit HIF-1 cease their activity. The HIF-1a molecule becomes available is now capable of binding with HIF-b to cross the nuclear membrane of the cell and promotes gene transcription (Lippi, Franchini, Salvango et al 2006). One of the main coding events that occurs as a result of the gene transcription is production of erythropoietin (Epo).
This endogenous Epo is then produced in the body, specifically in the peritubular capillary-lining cells of the renal cortex of the kidneys, with minute amounts produced in the liver and brain (Kraene, Fleck and Deschenes 2012). When the Epo molecule is synthesized, the composition is initially a 193 amino acid molecule that eventually is released as a 165 amino acid protein with much of the total molecule composed of carbohydrate (Lippi, Franchini, Salvango et al 2006). The release of Epo from the kidney to the blood then stimulates erythropoiesis in the bone marrow (Kraene, Fleck and Deschenes 2012).
Science advancements in the 1980’s have led to a synthetic form of Epo known as recombinant human Epo (rHuEpo) (Spedding and Spedding 2008). It was first introduced by a team of researchers at the Northwest Kidney Centres, who conducted clinical trials that resulted in the first successful artificial form of this hormone (Eichner 2007). The production of rHuEpo, from mammalian cells to treat anemic patients was approved by the U. S. Food and DrugAdministration in 1989 (Elliott 2008). Today human recombinant erythropoietin is available in a variety of forms.
It is synthesized with an amino acid sequence identical to that of endogenous erythropoietin, with slight differences in composition of carbohydrate portions of the molecule (glycosylation) (Lippi, Franchini, Salvango et al 2006). Alpha and beta erythropoietin are produced from Chinese hamster ovary cells with the only differences being a slightly longer half-life and slight difference in molecular weight (Lippi, Franchini, Salvango et al 2006). Another form of Epo is Erythropoietin gamma. It is produced from a different host cell and as a result has a different glycosylation pattern (Lippi, Franchini, Salvango et al 2006).
Erythropoietin delta, yet another variation of the synthetic hormone, is the most recently introduced form. This type is produced from human cells, and has identical amino acid and glycosylation patterns as endogenous Epo, with a longer half-life of 18-20 hours compared to the 7-12 hour range of alpha and beta forms (Lippi, Franchini, Salvango et al 2006). The current research is clinically testing a protein called Continuous Erythropoietin Receptor Activator (CERA). This protein has a half-life of 133-137 hours, which equates to less frequent dosing.
CERA unlike other synthetic forms of this hormone, has very mild side-effects and has yet to produce any serious adverse effects (Lippi, Franchini, Salvango et al 2006). This type of synthetic Epo may be the best option available for patients who require treatment for anemia (low hemoglobin levels). Unfortunately, some people suffer anemia due to various medical issues such as kidney disease, chemotherapy for cancer, HIV, blood loss, et cetera (Kraene, Fleck and Deschenes 2012). The body’s demand for Epo becomes more significant when such medical conditions arise.
Often times Epo needs to be artificially supplemented to compensate for the lowered hemoglobin production/ hemoglobin loss (Catlin, Fitch and Ljungqvist 2008). Originally, recombinant human erythropoietin was developed as a substitute for endogenous Epo for those who suffered from abnormal blood conditions. It is highly effective in increasing hemoglobin levels, and as a result has numerous benefits such as, reduction in required blood transfusions, restoring energy levels, increase in exercise capacity, improves cognitive function and overall quality of life improvement (Elliott 2008).
When administering this hormone, the dose, frequency of administration, the rate of rise of hemoglobin and target hemoglobin levels are strictly controlled (between 10-12g per 100mL), slightly lower than the range for normal range of 13-15g per 100mL. The lower range is maintained in order to keep the risks and side effects of the rHuEpo minimal (Lippi, Franchini, Salvango et al 2006). Careful monitoring and control is used to maximize the benefits for patients while minimizing the risks.
Recombinant Epo not only benefits those who are suffering a blood condition but it has significant benefits to athletic performance (Elliott 2008). It is used illegally as an ergogenic aid primarily in endurance sports, such as cross-country skiing, track, swimming, and most notoriously, cycling (Bento, Damasceno, Neto 2003). One study, as noted in Exercise Physiology (Kraene, Fleck and Deschenes 2012), that involved well-trained male endurance athletes administered recombinant human erythropoietin 3 times a week for 30 days or until hematocrit levels reached 50%.
The following resulted: an average hematocrit increase of 18. 9% (range of 42. 7-50. 8%), cycling time to exhaustion had increased 9. 4% (12. 8-14. 0 minutes longer), and cycling VO2 peak had increased 7% (range of 63. 8-68. 1 ml/kg/min). Another study also noted in Exercise Physiology (Kraene, Fleck and Deschenes 2012) gave low-dose subcutaneous injections of rHuEpo over a 6 week period to moderately to well-trained athletes and what resulted was a 6-8% increase in VO2 peak, time to exhaustion on a treadmill increased 13-17%, and hemoglobin concentration and hematocrit both increased by approximately 10% each.
The use of recombinant human erythropoietin is found to have clear benefits in athletic performance, with higher trained individuals exhibiting enhanced results. At an elite level, where competition is so close, it is tempting for athletes to gain an edge over their competition though the use of rHuEpo. There is a certain amount of pressure on athletes in cycling to use ergogenic aids due to the fact that so many of the sport’s top competitors are using it to boost performance (Vogel 2004). In cycling, the abuse of this ergogenic aid has recently come to light in the media.
Although many benefits can be reaped in athletic performance from recombinant erythropoietin, it is not without risks. When synthetic forms were first introduced, many of the risks were unknown to athletes and use was not medically monitored as would be the case with an anemic patient. As a result, sudden heart attacks occurred that led to more than a dozen deaths of Dutch and Belgian cyclists (Vogel 2004). Their deaths were connected to inappropriate administration of rHuEpo. This form of Epo had not yet been clinically studied from an athletic perspective.
The combination effect of increasing hemoglobin to well above normal range along with other factors associated with endurance sports, makes tampering with the body’s natural blood physiology dangerous and potentially deadly (Robinson, Magin and Saugy 2003). Myocardial infarction, cerebrovascular disease, transient ischemic attack and venous thromboembolism were all found to be potential events associated with the misuse of rHuEpo (Catlin, Fitch and Ljungqvist 2008). Due to the increase of red blood cells, the blood becomes more viscous and leads to an increased frequency risk of thrombotic events.
There have also been proven reports of increased risk for migratory thrombophlebitis, microvascular thrombosis and thrombosis of cerebral sinuses, retinal artery, and temporal veins. The increased blood viscosity also increases systolic blood pressure during sub-maximal exercise and increases platelet reactivity resulting in risk of more blood clotting (Bento, Damasceno, and Neto 2003). One of the most serious risks found to be associated is that of red cell aplasia in which red blood cell formation ceases. Although rare but ife-threatening, this condition was found to be linked to the use of subcutaneous alpha-Epo (Lippi, Franchini, Salvango et al 2006). Anemia may also develop in individuals who mis-use rHuEpo after they discontinue the hormone, as it causes progressive erythroid marrow exhaustion due to prolonged periods of use. Some other risks and side effects include headache, muscle cramps, incomplete deviation of red blood cells, convulsion, and upper respiratory tract infections (Kraemer, Fleck and Deschenes 2012). The risks of using rHuEpo are more significant for athletes than average patients who are using for treatment.
Athletes pushing to increase hemoglobin outside of a normal range run the risk of life-threatening circulatory/blood abnormalities. Testing for the use of banned erythropoietin in sports has been an ongoing challenge. As quickly as testing laboratories can produce testing methods for banned substances, new ways to slide under detection are being found (Cazzola 2000). It is difficult to directly identify rHuEpo as it has a relatively short half-life in most forms, for example an administration of 50 IU/kg given subcutaneously has a half-life of approximately 35. hours, and intravenous administration has a half-life ranging from 4 to 7 hours (Lippi, Franchini, Salvango et al 2006). Athletes could selectively time the administration of Epo and combined with concealing strategies to slip under the wire. As a result, laboratories are required to look at specific biomarkers that indicate past or current use of rHuEpo (Delanghe, Bollen and Beullens 2007). Human recombinant erythropoietin was initially a challenge to detect as various forms are extremely similar to that of endogenous Epo (Skibeli, Nissen-Lie and Torjesen 2001).
As it is a rising issue in sport, laboratories are required to find better ways to detect the illegal use of rHuEpo. Initially as a measure to deter doping and identify usage, cutoff levels of hematocrit (the percentage of red blood cells in the blood) were established in some sports (Adamson and Vapnek 1991). For example, the International Cycling Union established cutoff hematocrit levels of 47% for women and 50% for men. This method was flawed, as it sometimes produced false positive results in athletes with naturally high hematocrit levels (Casoni, Ricci, Ballarin et al 1993).
Currently, there is no foolproof testing method to detect the use of recombinant human erythropoietin. A combination of indirect and direct testing is currently the most effective method to identify blood dopers (Cazzola 2000). Indirect testing uses a blood sample and is based on the analysis of hematological parameters, including measures of hemoglobin, hematocrit, soluble transferrin receptors, serum Epo, percent reticulocytes, and macrocytes (Delanghe, Bollen and Beullens 2007).
Changes observed in the above measures are often a result of introducing recombinant Epo to the body and can be used as an indirect marker to detect the substance (Skibeli, Nissen-Lie and Torjesen 2001). There is a reference range of parameters set for this form of testing, one indicating current use of Epo while the other can indicate recently discontinued use of Epo (Parisotto, Wu, Ashenden et al 2001). Indirect testing has the advantage of being able to detect Epo use several weeks after it has been administered, however the disadvantage of possibly producing false-positive results (Delanghe, Bollen and Beullens 2007).
Changes in the measuring parameters used in indirect testing can also be the result of the body’s natural modifications from training methods such as altitude training (increasing RBC levels due to lower oxygen at higher altitude, a naturally occurring body compensation) (Kraemer, Fleck and Deschenes 2012). Indirect testing is useful in being a primary indication of recombinant erythropoietin use, yet it is not completely reliable.
If use of Epo is suspected after using indirect testing methods, direct testing will follow to confirm or deny the results (Birkeland and Hemmersbach 1999). Direct testing for recombinant Epo involves the collection of a urine sample. The urine sample needs to be fairly large (20ml) and strongly concentrated (between 700-1000 fold) (Elliott 2008). The approved test that uses the direct approach is based on differences in glycosylation between endogenous Epo and artificial forms (Elliott 2008).
The recombinant and endogenous forms of erythropoietin have varying isoelectric points (pI). Using isoelectric focusing (IEF), the isoelectric points can be determined (Skibeli, Nissen-Lie and Torjesen 2001). The normal range for the pI of endogenous Epo is 3. 7-4. 7, while alpha and beta Epo have a slightly higher range of 4. 4-5. 1. The Aransep form of Epo has 2 extra N-glycosylaton sites in order to increase its stability, resulting in a pI range of 3. 7-4 (Parisotto, Wu, Ashenden et al 2001).
In order to see the isoforms of Epo, double immunoblotting is used in combination with monoclonal anti-Epo antibodies. The interaction of the antibodies with the recombinant forms of Epo shows if illegal forms are present in the urine (Skibeli, Nissen-Lie and Torjesen 2001). The purpose of the double immunoblotting technique is to avoid secondary antibodies interacting with proteins in urine and affect the test. A technique known as chemiluminescence is used on the blot to image the Epo (Skibeli, Nissen-Lie and Torjesen 2001).
Direct testing can detect most forms synthetic Epo. When a test is found to be positive for an illegal form of Epo, a second test is performed due to the fact that occasionally enzyme activity causes a shift in the electrophoretic banding pattern of the molecule (Parisotto, Wu, Ashenden et al 2001). Additional stability testing is performed where the urine sample is incubated overnight in an acetate buffer and rHuEpo. If a banding shift is observed during the isoelectric focusing, it can be determined that the sample is negative for rHuEpo (Parisotto, Wu, Ashenden et al 2001).
The direct testing method is currently the most reliable and approved approach and can be used during competition and off-competition periods (Elliott 2008). The development of recombinant human erythropoietin was originally an approach to treat low hemoglobin levels in anemic patients. The athletic gains that can be exhibited through introducing rHuEpo have caused abuse at the elite level in many sports. Other than disqualification and loss of credibility as an honest athlete, there are also medical risks associated with tampering with the blood’s physiology in artificial ways.
A combination of testing methods is currently used to identify those using rHuEpo as an ergogenic aid, as there is no single test that can clearly deny of confirm use. New ways to slip under the wire with testing are being discovered and used by athletes and laboratories are constantly working to keep up. The use of recombinant human erythropoietin is a serious issue of misconduct in sport and needs to be ended in order to keep competition ethical and fair.