A review of erythropoietin and its use in doping

By Anaya Sirothia

Upon hearing the term ‘doping’ in sports, many recall the drug scandal involving Lance Armstrong in 2012, or perhaps the disqualification of Festina from 1998’s Tour de France.1 As the news coverage focused primarily on the athletes themselves and the implications of the doping on their respective sports, the science behind these controversies was somewhat sidelined. Doping remains an issue in many sports, with usage rates varying between 5% to 31%.2 This article aims to provide a brief summary of natural and recombinant erythropoietin, including its synthesis and effects in the body, medical and illicit use, side effects, and detection. 

In order to understand the significance of the production of recombinant erythropoietin (used in doping), the complexity and importance of erythropoietin should be discussed. Erythropoietin, commonly referred to as EPO, is a glycoprotein hormone that acts as a cytokine. It is used by the body to regulate erythrocyte production through the stimulation of a signalling cascade.3 

Endogenously, EPO is produced as a polypeptide that undergoes several modifications including the removal of terminal amino acids and glycosylation.4 The primary site of EPO production in adults is in the peritubular capillary bed of the renal cortex, inside interstitial cells.5 Extrarenal sites of EPO production include the liver and brain, albeit in much smaller quantities.6 The body triggers EPO production when undergoing hypoxic stress, i.e., when tissues have insufficient oxygen to function normally and thus undergo hypoxia.2 Hypoxic stress is commonly caused by the body detecting low oxygen levels due to either low levels of oxygen in the blood or a low blood supply to the tissues being affected.4 Hypoxia increases the production of protein HIF-1 (through the production and subsequent dimerization of proteins HIF-1α and HIF-1β). HIF-1 binds to a target region in the EPO gene, promoting cellular transcription of EPO.4

The plasma membranes of erythrocyte progenitor cells contain embedded EPO receptors. These bind to EPO, leading to the stimulation of JAK2, a receptor-associated tyrosine kinase, which phosphorylates STAT5, a pair of proteins involved in the activation of transcription and signal transduction.3,6 Once STAT5 is activated via phosphorylation, it is relocated to the nucleus, allowing it to bind to specific DNA sequences where it promotes the transcription (and therefore the expression) of its target genes.3 Ultimately, this process allows the body to regulate the haematocrit, through increased production of erythrocytes in the bone marrow.1-3 Other pathways involving EPO also produce similar outcomes. Studies have shown that erythropoietin promotes erythroid progenitor cell survival by maintaining its viability. In fact, EPO is required to prevent the DNA cleavage of certain progenitor cells, such as CFU-E, thus enabling their survival and maturation.6 Without the presence of EPO, CFU-E cells wouldn’t be able to differentiate further.

Recent developments in biomedicine have allowed synthetic EPO to be mass produced using recombinant DNA technology, often using E. coli as a host.4,6 The resultant recombinant human erythropoietin (rHuEPO) promotes increased expression of EPO-encoding genes through the previously summarised mechanism. Medically, rHuEPO is administered to people suffering from anaemia of chronic disease, chronic kidney disease, or chronic lung disease leading to hypoxia.4,7 The aim is to increase patient haematocrit.1 Some studies have found rHuEPO administration to significantly increase platelet count and megakaryocyte progenitors in patients with advanced renal disease, suggesting an alternative medical use for the hormone.6

Synthetic EPO is well-known for its illegal administration in doping to increase athletic endurance due to the role it plays in the body. The abundance of rHuEPO-related doping scandals in cycling, boxing, and athletics demonstrates how widely this hormone has been misused. Athletes training at high altitudes or in hypoxic tents have been shown to adapt to the decrease in atmospheric oxygen by producing more erythropoietin.7 Whilst these natural methods of increasing EPO levels exist, the potential for rHuEPO to dramatically increase aerobic capacity provides an incentive for some to illicitly improve their performance.7 Several studies investigating the quantifiable effect of rHuEPO treatment on exercise performance have found increases in the oxygen content in arteries and maximal aerobic power.8 These improvements showed a 9% increase in maximal aerobic power and a 54% increase in exercise performance (alongside other improvements) when administering rHuEPO to subjects over a period of 12 weeks.8 These significant changes lasted for prolonged periods after stopping rHuEPO administration, as compared to the immediate decrease in haemoglobin concentration measured following natural methods of increasing EPO.8 

Needless to say, the numerous ‘benefits’ rHuEPO provides to athletic performance do not arise without posing significant health risks. Side effects of prolonged use include hypertension, a rise in blood viscosity and erythrocyte count leading to increased risks of thromboembolism.2 Repeated abuse may result in the production of anti-EPO antibodies, causing anaemia.1

The search for effective rHuEPO testing since the early 2000s has led to various techniques being used in conjunction with each other to increase the likelihood of rHuEPO detection. Direct testing (such as urine testing) is used to identify rHuEPO itself, whilst indirect blood testing identifies the effects of rHuEPO (or similar erythropoiesis-increasing substance) use over a prolonged period. Direct testing includes the use of SDS-PAGE and isoelectric focusing to separate proteins by their respective size and charge. Despite their widespread use, such methods are often unable to differentiate between endogenous and recombinant human EPO.4 Research into indirect testing involving genetic expression, peptide markers, and changes in haematologic parameters related to rHuEPO use shows the potential to identify exogenous EPO with near-complete accuracy.1,2 Such developments would enable a thorough investigation into doping allegations, levelling the playing field and preventing misuse of such powerful hormones.

This review aimed to shed light on the importance of erythropoietin as well as its uses as a treatment and performance-enhancing hormone. EPO has been shown to play a crucial part in the maintenance and proliferation of erythrocytes, with life-saving applications in the treatment of chronic illnesses affecting the haematocrit. The misuse of this hormone provides significant advantages in athletic endurance whilst posing numerous health risks.

References:

  1. Berthold, E. Erythropoietin (EPO). https://www.science.org.au/curious/people-medicine/erythropoietin-epo. [Accessed 10th June 2022].
  2. Momaya A, Fawal M, Estes R. Performance-Enhancing Substances in Sports: A Review of the Literature. Sports Med. 2015;45:517–531. doi: 10.1007/s40279-015-0308-9. 
  3. Middleton SA, Barbone FP, Johnson DL, Thurmond RL, You Y, McMahon FJ, et al. Shared and Unique Determinants of the Erythropoietin (EPO) Receptor Are Important for Binding EPO and EPO Mimetic Peptide. Journal of Biological Chemistry. 1999;274(20): 14163-14169. doi:10.1074/jbc.274.20.14163. 
  4. Citartan M, Gopinath SCB, Chen Y, Thangavel L, Tang TH. Monitoring recombinant human erythropoietin abuse among athletes. Biosensors & bioelectronics. 2016;63:86-98. doi:10.1016/j.bios.2014.06.068. 
  5. Fisher JW, Koury S, Ducy T, Mendel S. Erythropoietin production by interstitial cells of hypoxic monkey kidneys. British Journal of HaematologyJournal of Haematology. 1996;95(1):27-32. doi:10.1046/j.1365-2141.1996.d01-1864.x. 
  6. Krantz, SB. Erythropoietin. Blood. 1991;77(3): 419-434. doi: 10.1182/blood.v77.3.419.419. 
  7. Wedro M. What Is Blood Doping? Risks, Side Effects, EPO, Lance Armstrong. https://www.medicinenet.com/blood_doping/views.htm. [Accessed 10th June 2022].
  8. Thomsen JJ, Rentsch RL, Robach P, et al. Prolonged administration of recombinant human erythropoietin increases submaximal performance more than maximal aerobic capacity. Eur J Appl Physiol. 2007;101:481–486. doi:10.1007/s00421-007-0522-8.

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