Scott, Joshua MD; Phillips, George C. MD, FAAP*
Address *Department of Pediatrics, Children's Hospital of Iowa, 200 Hawkins Drive, Iowa City, IA 52242, USA. E-mail: firstname.lastname@example.org
Erythropoietin (EPO) is found naturally in the human body and has been commercially available as recombinant human EPO (rEPO) for almost 20 years. Although the positive benefits of rEPO cannot be denied for those who suffer from anemia, athletes have also used the drug as an ergogenic aid. It has replaced conventional “blood doping” as the drug of choice to improve performance in contests requiring aerobic potential. Testing for the drug in the past has proven difficult due to several factors. The similarities in structure and metabolism of EPO and rEPO allow those who choose to abuse rEPO to avoid detection. Due to the inherent danger of abusing rEPO and its analogues, the various governing bodies of international sports continue to improve testing methods for this illegal substance.
Erythropoietin (EPO) has long been known to be an important protein in maintaining the human body's erythrocyte production. When recombinant human erythropoietin (rEPO) was made available in the late 1980s, it did not take long for the athletic world to recognize the positive effects of the drug. Use of rEPO as a performance-enhancing drug became prevalent in sports requiring aerobic potential. There have been multiple international incidents in the past two decades, particularly in cycling and cross country skiing, in which athletes either admitted to taking or tested positive for rEPO. The female winner of the 2004 Ironman triathlon tested positive for rEPO and was stripped of her crown . Bernard Lagat, Olympic bronze medalist in the 1500m distance run, tested positive for rEPO in 2003 . The scandals involving the 1998 Tour de France Festina team being caught with enormous amounts of rEPO, and the 2002 winter Olympic cross country skiers found using darbepoietin, an analogue of rEPO, are just some of the more recent examples [3,4]. Athletes' repeated use of the drug in previous years was only encouraged by the inability to reliably test for an otherwise “natural” body protein.
By the early 1900s, scientists realized that the body's ability to perform well in endurance sports was directly related to maximal oxygen uptake (VO2max). Over the next few decades, the equation was proved to fact that the increases in total body hemoglobin improved VO2max . The benefits of high-altitude training came to the forefront after the 1968 Olympics in Mexico City. Athletes from countries who trained and lived at higher altitudes performed better in distance events. By the 1970s, athletes that wanted to artificially improve their performance in distance races started “blood doping.” Blood doping consists of either autologous or homologous transfusions, whereby extracted blood was spun to filter out erythrocytes that were later injected days before an event [6••]. Although effective, the process was cumbersome and required transfusion equipment. In addition, the ability to store the packed cells was needed while the body naturally replenished its supply. These athletes now shared a common desire with patients suffering from severe anemia: a method of increasing total erythrocyte counts without the complications of transfusion. In 1977, the isolation of pure EPO from the urine of anemic patients by Miyake et al.  lead to the identification of the amino acid sequence of the protein and ultimately its synthesis.
Erythropoietin is a relatively small but complex glycoprotein made of 165 amino acids. EPO production occurs primarily in peritubular cells within the renal cortex of healthy adults. The liver manufactures a small amount as well. The liver controls fetal production of EPO until shortly after birth, when the kidneys become responsible for synthesis [8•]. The average adult must maintain a production of approximately 200 billion erythrocytes daily due to natural cell apoptosis that occurs approximately every 120 days. Regulation of EPO is controlled by a gene in chromosome 7, specifically in the region of 7q21 . The transcription of this gene is controlled by hypoxia-inducible factor (HIF). HIF is upregulated by decreased oxygen tension of tissue. This low oxygen tension can take the form of either decreased ambient oxygen as found at higher altitudes, lower blood oxygen carrying capacity (ie, anemia), or an increase in hemoglobin affinity for oxygen as found in hemoglobinopathies producing polycythemia [8•,10]. It has been proposed that the same peritubular cells that produce EPO in the kidney also sense oxygen tension.
Like all cells undergoing hematopoiesis, erythrocytes begin as pluripotential stem cells that differentiate into burst-forming units, then colony-forming units (CFU), and ultimately reticulocytes. EPO has its largest effect on the outcome of CFUs. Thus, larger circulating levels of EPO stimulate erythrocyte production in the bone marrow by increasing the number of CFUs that undergo differentiation into erythroblasts [8•,10]. Without erythropoietin, the stem cells committed to the erythroid pathway will die by apoptosis. As circulating erythrocytes become more prolific, oxygen is more readily supplied to the body's tissues inducing a negative feedback loop on EPO production.
The major need for recombinant human EPO was realized when so many patients with chronic renal failure were found to have concomitant anemia. The failing kidneys do not produce EPO in sufficient quantities, leading to decreased erythrocyte mass. Side effects include fatigue that is sometimes so severe it limits even daily activities. In June 1989, the first rEPO product, Epogen (Epoetin alfa; Amgen, Thousand Oaks, CA), was marketed in the United States. This product has exactly the same amino acid sequence as isolated natural erythropoietin, and, therefore, has the same biologic effects. Using recombinant DNA technology, Epogen is made from Chinese hamster ovary cells, then isolated and purified. Normal EPO levels are between 15 and 30 mU/mL and may increase by 100 to 1000 times during hypoxia or anemia [11,12]. The therapeutic range for Epogen is 50 to 300 U/kg three times weekly administered intravenously (IV) or subcutaneously (SC). IV and SC dosing have a mean half-life of 8.5 and 24 hours, respectively . IV dosing results in a shorter duration of peak plasma levels of rEPO, thus making SC administration easier and less expensive. Due to the length of time required for natural erythropoiesis to occur, administration of the drug for between 2 and 6 weeks may be necessary before therapeutic rises in hematocrit are observed [13,14]. The rate of rise of hematocrit is dose dependant but may also be affected by baseline hematocrit levels, iron stores, and other disease factors. In phase III clinical trials, over 97% of dialysis patients achieved a mean hematocrit of 35% within 18 weeks of Epogen dosing . With a way to recover from anemia, millions of dialysis patients could live more productive lives, and rEPO soon became a multi-billion dollar industry. The list of indications for rEPO therapy was subsequently expanded to include cancer patients receiving chemotherapy, HIV-positive patients on zidovudine, and surgical patients to reduce the numbers of needed blood transfusions.
Several studies have shown that exogenously administered rEPO will increase hemoglobin levels in healthy adults as well. Berglund and Ekblom  showed dose response increases in hemoglobin of approximately 11% after giving SC doses of rEPO. With increases in erythrocyte counts and hemoglobin shown in studies of healthy adults given rEPO, investigators began to explore the effects that rEPO had on VO2max. rEPO administration seemed to improve VO2max and exercise tolerance in both healthy as well as hemodialysis subjects . Birkeland et al.  demonstrated increases in hematocrit concomitant with an average 7% increase in VO2max following use of rEPO . Ekblom and Berglund  further demonstrated that administration of rEPO prolonged run time to exhaustion in recreational runners by 17%. These same studies reflect that response is not only dose dependant, but may also depend on individual variables of the athlete.
With the rise in hematocrit after blood doping or using rEPO, there can be many adverse effects that accompany the benefits of higher erythrocyte counts. Direct effects of the drug can include, but are not limited to fever, nausea, headache, anxiety, and lethargy [8•]. Hyperkalemia can be found in dialysis patients receiving rEPO therapy and is usually greatest at the initiation of treatment [6••,8•]. Hypertension is the most common side effect of the drug when used in patients on hemodialysis, and can be found in one quarter to one third of patients receiving rEPO. This is likely due to increases in plasma volume, cardiac output, and blood viscosity [8•]. Hyperviscocity associated with high hematocrit levels increases the risk of thrombotic events such as stroke and myocardial infarction. The unexplained death of 18 otherwise healthy cyclists between 1997 and 2000 have been linked to rEPO by some sources due to the temporal relationship of the deaths and the commercial release of rEPO. However, no concrete evidence has ever proved that rEPO abuse caused those deaths. Seizures have also been reported in 2% to 3% of patients in the first 90 days of therapy. Finally, an antibody-mediated form of pure red cell aplasia has been linked mainly to SC dosing of rEPO [8•].
Recombinant EPO is only available by a doctor's prescription in the United States. It is illegal to use in any sport and has been banned by the National Collegiate Athletic Association, International Olympic Committee, and US Olympic Committee. Because rEPO is almost identical in structure and metabolism to endogenous erythropoietin, testing for abuse in sports has proved to be difficult in the past. In 1997, specific sports federations such as the Union Cycliste Internationale put upper limits on blood hematocrit percentages drawn several hours before races. These limits, 50% for men and 47% for women, are based on normal values for adults. They certainly protect athletes from the risks of competing with elevated hematocrit levels, but do not take into account individual variability or dilution with IV saline. Most importantly, they do not test specifically for the illegal use of rEPO. Indirect hematopoietic markers were then chosen to identify rEPO abuse. These were serum EPO levels, hematocrit, percentage of reticulocytes and percentage of macrocytes [19•].
More recent findings have led to detection strategies that offer better precision. Wide et al.  demonstrated the ability to detect the administration of rEPO by electrophoretic testing of urine and blood. rEPO has a less negative charge than endogenous EPO, which allows electrophoresis to discriminate between the two compounds. However, limitations to this method include a short testing window of 2 to 3 days postadministration of the drug. Birkland et al.  showed that serum levels of soluble transferrin receptor might be used as a marker for elevated erythropoiesis following exogenous EPO administration. A combination of urine and blood testing made up the procedure for detection of rEPO at the 2000 Olympics in Sydney, Australia .
Since the appearance of rEPO as an ergogenic aid in international sports almost 20 years ago, various governing bodies have made great efforts to screen for abuse of the drug. Using urine testing as well as serum blood markers, scientists can now determine if athletes have used rEPO recently before a competition. Whether using rEPO or blood doping by other means, athletes take a great risk at the expense of their health to illegally gain a competitive edge. The medical risks of using rEPO in sports have been proven and are amplified by the dehydration and stress associated with distance contests. For both ethical and medical reasons, it is obvious that rEPO must remain a banned substance for athletes. The medical community must continue to strive to improve methods to expose those who choose to use rEPO illegally.
References and Recommended Reading
Papers of particular interest, published recently, have been highlighted as: • Of importance, •• Of major importance
3. Kamber M: Fight against doping--national and international developments after Tour de France 1998. Ther Umsch
4. Leigh-Smith S: Blood boosting. Br J Sports Med
2004, 38: 99–101.
5. Joyner MJ: V02MAX, blood doping, and erythropoietin. Br J Sports Med
6.•• Sawka MN, Joyner MJ, Miles DS, et al.
: American College of Sports Medicine position stand: the use of blood doping as an ergogenic aid. Med Sci Sports Exerc
An excellent review of the literature on blood doping and EPO with several studies on ergogenic properties and methods of detection. This position stand by the American College of Sports Medicine discusses physiology, ergogenic benefits and risks of using EPO, and is a useful summary article.
7. Miyake T, Kung CK, Goldwasser E: Purification of human erythropoietin. J Biol Chem
A detailed article reviewing the history, physiology, and pharmacology of EPO and rEPO. This article is most helpful in reviewing erythropoiesis as well as the dosing and pharmacokinetics of rEPO.
10. Beris P: Erythropoietin and erythropoiesis. Haematologica
11. Reikes ST, Martin KJ: Renal failure. Textbook of Primary Care Medicine,
edn 3. Edited by Noble J. St. Louis: Mosby; 2001:1395–1402.
12. Graber SE, Krantz SB: Erythropoietin and the control of red cell production. Ann Rev Med
13. Eschbach JW, Egrie JC, Downing MR, et al.
: Correction of the anemia of end-stage renal disease with recombinant human erythropoietin. Results of a combined phase I and II clinical trial. N Engl J Med
14. Eschbach JW, Abdulhadi MH, Browne JK, et al.
: Recombinant human erythropoietin in anemic patients with end-stage renal disease. Results of a phase III multicenter clinical trial. Ann Intern Med
15. Berglund B, Ekblom B: Effect of recombinant human erythropoietin treatment on blood pressure and some haematological parameters in healthy men. J Intern Med
16. Lundin AP, Akerman MJ, Chesler RM, et al.
: Exercise in hemodialysis patients after treatment with recombinant human erythropoietin. Nephron
17. Birkeland KI, Stray-Gundersen J, Hemmersbach P, et al.
: Effect of rhEPO administration on serum levels of sTfR and cycling performance. Med Sci Sports Exer
18. Ekblom B, Berglund B: Effect of erythropoietin administration on maximal aerobic power. Scand J Med Sci Sports
1991, 1: 88–93.
19.• Pascual JA, Belalcazar V, de Bolos C, et al.
: Recombinant erythropoietin and analogues: a challenge for doping control. Ther Drug Monit
An interesting article reviewing recent approaches to rEPO detection with indirect serum markers, urine analysis, and future methods of testing for EPO and its analogues.
20. Wide L, Bengtsson C, Berglund B, Ekblom B: Detection in blood and urine of recombinant erythropoietin administered to healthy men. Med Sci Sports Exerc
21. Kazlauskas R, Howe C, Trout G: Strategies for rhEPO detection in sport. Clin J Sport Med