Blood doping

Blood doping is a form of doping in which the number of red blood cells in the bloodstream is boosted in order to enhance athletic performance. Because such blood cells carry oxygen from the lungs to the muscles, a higher concentration in the blood can improve an athlete's aerobic capacity and endurance. Blood doping can be achieved by making the body produce more red blood cells itself using drugs, giving blood transfusions either from another person or back to the same individual, or by using blood substitutes.
Many methods of blood doping are illegal, particularly in professional sports where it is considered to give an artificial advantage to the competitor. Anti-doping agencies use tests to try to identify individuals who have been blood doping using a number of methods, typically by analyzing blood samples from the competitors.

History

Blood doping is defined as the use of illicit products, darbepoetin-alfa, hypoxia-inducible factor and methods in order to enhance the O₂ transport of the body to the muscles.
The body undergoes aerobic respiration in order to provide sufficient delivery of O₂ to the exercising skeletal muscles and the main determining factors are shown in figure 1. The rate of maximum O₂ uptake depends on cardiac output, O₂ extraction and hemoglobin mass. The cardiac output of an athlete is difficult to manipulate during competitions and the distribution of cardiac output is at the maximum rate during competitions. In addition, the O₂ extraction is approximately 90% at maximal exercise. Therefore, the only method to enhance the physical performance left is to increase the O₂ content in the artery by enhancing the hemoglobin mass. In other words, hemoglobin concentration and blood volume contribute to hemoglobin mass.

Methods

Drug treatments

Many forms of blood doping stem from the misuse of pharmaceuticals. These drug treatments have been created for clinical use to increase the oxygen delivery when the human body is not able to do so naturally.

Erythropoietin

is a glycoprotein hormone produced by the interstitial fibroblasts in the kidney that signal for erythropoiesis in bone marrow. The increased activity of a hemocytoblast allows the blood to have a greater carrying capacity for oxygen. EPO was first developed to counteract the effects of chemotherapy and radiation therapy for cancer patients. EPO also stimulates increased wound healing. Because of its physiological side effects, particularly increased hematocrit, EPO has become a drug with abuse potential by professional and amateur cyclists.

Hypoxia-inducible factor (HIF) stabilizer

Hypoxia-inducible factor stabilizer is a pharmaceutical used to treat chronic kidney disease. Like most transcription factors, the HIF transcription factor is responsible for the expression of a protein. The HIF stabilizer activates the activity of EPO due to anemia-induced hypoxia, metabolic stress, and vasculogenesis. HIF stabilizers as used by cyclists in combination with cobalt chloride/desferrioxamine to stimulate and de-regulate the natural production of erythropoietin hormone. At physiologically low PaO₂ around 40 mmHg, EPO is released from the kidneys to increase hemoglobin transportation. The combination of drugs consistently releases EPO due to increased transcription at the cellular level. The effect wears off when the HIF stabilizers, cobalt chloride/desferrioxamine is excreted and/or decayed by the body.

Myo-inositol trispyrophosphate (ITPP)

, also known as compound number OXY111A, is an allosteric effector of hemoglobin which causes a rightward shift in the oxygen–hemoglobin dissociation curve, increasing the amount of oxygen released from red blood cells into surrounding tissue during each passage through the cardiovascular system. ITPP has been a subject of anti-doping research in both humans and racehorses.

Blood transfusion

Blood transfusions can be traditionally classified as autologous, where the blood donor and transfusion recipient are the same, or as allogeneic/homologous, where the blood is transfused into someone other than the donor. Blood transfusion begins by the withdrawal of 1 to 4 units of blood several weeks before competition. The blood is centrifuged, the plasma components are immediately reinfused, and the corpuscular elements, principally red blood cells, are stored refrigerated at 4 °C or frozen at −80 °C. As blood stored by refrigeration displays a steady decline in the number of RBCs, a substantial percentage, up to 40%, of the stored RBCs may not be viable. The freezing process, conversely, limits the aging of the cells, allowing the storage of the blood for up to 10 years with a 10% to 15% loss of RBCs.

Blood substitutes

Biochemical and biotechnological development has allowed novel approaches to this issue, in the form of engineered O₂ carriers, widely known as "blood substitutes". The blood substitutes currently available are chiefly polymerized haemoglobin solutions or haemoglobin-based oxygen carriers and perfluorocarbons.

Hemoglobin-based oxygen carriers (HBOCs)

are intra/ inter-molecularly engineered human or animal hemoglobins, only optimized for oxygen delivery and longer intravascular circulation. The presence of 2,3-diphosphoglycerate within erythrocytes maintains the normal affinity of hemoglobin for oxygen. HBOCs do not contain erythrocytes and lose this interaction, thus, unmodified human HBOC solutions have a very high oxygen affinity which compromises their function. Chemical methods developed to overcome this problem have resulted in carriers that effectively release oxygen at the physiological pO₂ of peripheral tissues.
A common feature of all HBOCs is their resistance to dissociate when dissolved in media, which contrasts hemoglobin of natural dissociation under non-physiological conditions. HBOCs may hypothetically supply greater benefits to athletes than those provided by the equivalent hemoglobin in traditional RBC infusion. Recent developments have shown that HBOCs are not only simple RBC substitutes, but highly effective O₂ donors in terms of tissue oxygenation. Additional effects include increases in blood serum iron, ferritin, and Epo; up to 20% increased diffusion of oxygen and improved exercise capacity; increased CO₂ production; and lower lactic acid generation in anaerobic activity. HBOCs have been shown in trials to be extremely dangerous in humans. Because HBOCs increase both the risk of death and risk of myocardial infarction clinical trials were ended. They are not commercially available in the US or Europe and there is no approved use for them.

Perfluorocarbons (PFCs)

PFCs, also known as fluorocarbons, are inert, water-insoluble, synthetic compounds, consisting primarily of carbon and fluorine atoms bonded together in strong C–F bonds. PFCs are substantially clear and colorless liquid emulsions that are heterogeneous in molecular weight, surface area, electronic charge, and viscosity; their high content of electron-dense fluorine atoms results in little intramolecular interaction and low surface tension, making such substances excellent solvents for gases, especially oxygen and carbon dioxide. Some of these molecules can dissolve 100 times more oxygen than plasma. PFCs are naturally hydrophobic and need to be emulsified to be injected intravenously. Since PFCs dissolve rather than bind oxygen, their capacity to serve as a blood substitute is determined principally by the pO₂ gradients in the lung and at the target tissue. Therefore, their oxygen transport properties differ substantially from those of whole blood and, especially, from those of RBCs. At a conventional ambient pO₂ of 135 mmHg, the oxygen content of 900 mL/L perfluorocarbon is less than 50 mL/L, whereas an optimal oxygen content of 160 mL/L, which is still lower than that of whole blood in normal conditions, can be achieved only by a pO₂ greater than 500 mmHg. In practice, at a conventional alveolar pO₂ of 135 mmHg, PFCs will not be able to provide sufficient oxygenation to peripheral tissues.
Due to their small size, PFCs are able to permeate circulation where erythrocytes may not flow. In tiny capillaries, PFCs produce the greatest benefit, as they increase local oxygen delivery much more efficiently than would be expected from the increase in oxygen content in larger arteries. In addition, as gases are in the dissolved state within PFCs, it pO₂ promotes efficient oxygen delivery to peripheral tissues. Since the mid-1980s, improvements in both oxygen capacity and emulsion properties of PFCs have led to the development of second-generation PFC-based oxygen carriers; two PFC products are currently being tested in phase III clinical trials.

Cobalt chloride administration

Transition metal complexes are widely known to play important roles in erythropoiesis; as such, inorganic supplementation is proving to be an emerging technique in blood doping. Particularly of note is the cobalt complex, cobalamin commonly used as a dietary supplement. Cobalamin is an important complex used in the manufacture of red blood cells and thus was of interest for potential use in blood doping. Experimental evidence, however, has shown that cobalamin has no effect on erythropoiesis in the absence of a red blood cell/oxygen deficiency. These results seem to confirm much of what is already known about the functioning of cobalamin. The signaling pathway that induces erythropoietin secretion and subsequently red blood cell manufacture using cobalamin is O₂ dependent. Erythropoietin is only secreted in the kidneys when there is an O₂ deficiency, as such, RBC manufacture is independent of the amount of cobalamin administered when there is no O₂ deficiency. Accordingly, cobalamin is of little to no value in blood doping.
More potent for use in blood doping is Co²⁺. Cobalt chloride has been known to be useful in treating anemic patients. Recent experimental evidence has proved the efficacy of cobalt chloride in blood doping. Studies into the action of this species have shown that Co²⁺ induces hypoxia like responses, the most relevant response being erythropoiesis. Co²⁺ induces this response by binding to the N-terminus of the Hypoxia inducing transcription factors HIF-1α and HIF-2α, and thus stabilizes these protein complexes. Under normal O₂ conditions, HIFs are destabilized as proline and asparagine residues are hydroxylated by HIF-α hydroxylases, these unstable HIFs are subsequently degraded following a ubiquitin-proteosome pathway, as such, they cannot then bind and activate transcription of genes encoding Erythropoietin. With Co²⁺ stabilization, degradation is prevented and genes encoding EPO can then be activated. The mechanism for this Co²⁺ N terminus stabilization is not yet fully understood. In addition to N-terminus binding, it has also been hypothesized that replacement of Fe²⁺ by Co²⁺ in the hydroxylase active site could be a contributing factor to the stabilizing action of Co²⁺. It is understood however, is that Co²⁺ binding permits Ubiquitin binding but prevents proteosomal degradation.