Methods to increase the delivery of oxygen

By Juan Manuel Alomso

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    The delivery of oxygen to the muscles is of paramount importance in aerobic exercise and oxygen transport is a limiting factor for endurance sports. People involved in sport have tried different methods to increase oxygen transfer to working muscles and thereby improve performance. Some of these, including altitude training and hypoxic devices, are ethically acceptable. Others, including several with justified and accepted indications in clinical settings, are illegal in the sports environment. In this article, the Chairman of the IAAF Medical and Anti-Doping Commission reviews the various methods for increasing the oxygen content of the blood currently in use or in development. He details their development and appearance in sport, their associated risks and, in the case of prohibited substances and techniques, the means of detection developed by scientists and sports authorities. He concludes by supporting current anti-doping regulations and condemning the use of banned substances in sport.



    The delivery of oxygen to the muscles is of paramount importance in submaximal and maximal endurance exercise, and oxygen transport is a limiting factor in muscle cell work. The transfer of oxygen to working muscles is a function of the muscle blood flow and the oxygen content of the blood. Oxygen is delivered in two ways: either diffused in plasma (3%) or linked to Hb (haemoglobin) (97%).
    It is possible to increase the oxygen content of the blood by: (i) raising the Hb concentration, or modifying the capacity of the Hb to deliver oxygen using allosteric effectors of Hb; or (ii) using oxygen carriers to fill the role of Hb. Once the availability of oxygen in the blood is increased by one or more of these means, its delivery to the muscles improves, allowing the enhancement of aerobic performance.
    The attention of athletes, coaches and others in sport interested in the improvement of endurance capacity is drawn by methods to increase the oxygen content of the blood. Some of these methods are ethically acceptable but others have been forbidden by athletics and by the rest of the sports world. The author of this paper firmly supports current anti-doping rules and condemns the use of banned drugs or other methods to artificially enhance performance.

    The aim of this paper is to review a series of methods and drugs that can increase the oxygen content of the blood. It describes the development of the methods, their efficacy and potential for use in doping and their associated health risks. It is hoped that the provision of information in this form will contribute to the fight against doping by assisting anyone involved in sport to make informed and ethically sound decisions.

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1.     Direct action on the haemoglobin (Hb)

    Improved oxygen delivery by direct action on Hb can be achieved either by increasing the Hb via raising the number of red blood cells (RBC) or modifying the capacity of the Hb to deliver oxygen using allosteric effectors of Hb.


1.1    Blood transfusion

    The idea of using the blood transfusions to increase oxygen delivery emerged in the 1970's. The interest of the lay press in "blood doping" stems from alleged use in distance running, cycling, cross-country skiing and biathlon events starting with the 1972 Olympic Games. In 1976, the Medical Commission of the International Olympic Committee (lOC) formally condemned the practice of blood transfusion for athletes in good health but the practice continued. Only after the revelation, made by the US Olympic Committee, that seven members of the US team at the 1984 Olympic Games had received transfusions did the IOC ban the procedure.
    Blood doping is defined in the World Anti- Doping Code 2004 List as "the use of autologous, homologous or heterologous blood or red blood cell products of any origin, other than for legitimate medical treatment".
    In the case of autologous infusion, several units of blood (~ 450ml each) are removed by phlebotomy. The RBCs are then harvested, stored, and later reinfused. Storage techniques for autologous erythrocytes permit a shelf life of 35-42 days if stored at 4°C or up to 10 years if stored at -65°C in glycerol. If storage at 4°C is chosen, the RBCs must be harvested within 42 days of the targeted championships, or at least 4 to 8 weeks prior to the event at which the athlete would like to have a benefit. Using this method, an athlete will experience reduced aerobic capacity during the several weeks the bone marrow requires to replenish the harvested cells. From an exercise physiology perspective, this does not constitute an ideal preparation for a major competition. However, the alternative of freezing erythrocytes at -65°C and then thawing them for reinfusion is time consuming and the cost of equipment, materials and skilled technologist's time are considerable. Therefore, each phlebotomy is spaced by several weeks so that the normal hematocrit (Hct) can be re-established prior to the next phlebotomy or reinfusion. Usually reinfusion, after preparation of the blood unit, is carried out 1 to 7 days prior to the target event.
    Autologous infusion of RBC is not perfectly safe. Clerical error, mislabeling and mishandling of the blood products are the most common causes of serious infused related morbidity. Persons receiving autologous infusions also face the risk of bacterial infections from mishandled blood products.
    In the case of homologous transfusions, refrigeration techniques may be used for short-term storage; however, RBCs will progressively degrade and the maximum storage period is about 42 days. As a result, athletes wishing to receive a homologous transfusion must seek a medical centre or blood bank that can provide them with blood units less than 42 days old. This is a difficult task in some countries, as blood availability is limited and patients with haematological conditions obviously have priority.
    There are also several recognised risks with homologous infusion or transfusion. There is the possibility of acquiring the Human Immunodeficiency Virus (HIV), Hepatitis B or Hepatitis C, although the overall risk of these types of infection is low. Other risks from banked blood include major transfusion reactions (due to blood type incompatibility, usually the result of clerical error), minor transfusion reactions including fever and body aches, transfusion-related acute lung injury, and bacterial infection2.
    With regard to the detection of blood doping, we can say that there has been some progress. Although the research has not been published, it now seems possible to identify transfusions of homologous blood using sophisticated haematological methods and DNA techniques. Promising results have recently been shown in experiments utilizing cytometry, a method based on the use of anti-bodies to identify different populations of RBCs. Autologous transfusions, however, cannot be detected unless multiple blood samples are obtained before and after reinfusion. It is clear, therefore, that research on the detection of blood doping is still necessary.
    It should be mentioned that the combination of two illegal methods, autologous blood infusion and the use of recombinant human erythopoietin (rHuEPO), can increase their respective efficacy and reduce the chances of detection. This technique may have been used in elite sports for the first time at the 2002 Winter Olympic Games in Salt lake City. Athletes could also be experimenting with a variation of the well-established presurgical practice of rHuEPO enhanced autologous transfusion (EEAT). This modified EEAT could permit healthy young subjects to collect and store two or three units of their own blood to be transfused to themselves immediately before important competitions. However, full exploitation of EEAT would require illicit skilled medical support.

1.2    Endogenous stimulation of sed blood cell production
    Natural stimulation of the proliferation and differentiation of erythroid progenitor cells in bone marrow is caused by EPO, whose production is in turn regulated by oxygenation. Therefore, tissue hypoxia is a stimulus for endogenous EPO synthesis. There are several methods for using this physiological concept to increase EPO and RBC. The, increase of RBC can also be achieved using rHuEPO as well as related products like encapsulated EPO or EPO mimetics, which are available on the market.

1.2.1    Altitude and other hypoxic approaches
    It is well known that hypoxia stimulates erythropoiesis, thus increasing Hb mass and red cell volume while tending to decrease plasma volume. Since the 1968 Olympic Games in Mexico City, many studies have been published to support the use of altitude training as an ergogenic aid for aerobic performance. However, there is still much controversy about the precise altitude required for training to optimise endurance performance at sea level. Also, it is difficult to quantify the benefits of altitude training and therefore not easy to determine whether physiological changes that occur after altitude training can be attributed to an improvement in physical condition or to the additive effects of hypoxia itself.
    Levine et al introduced in 1991 the concept of "live high-train low", in which athletes live at 2000-2700m and train at 1000m or less. It is believed that living at relatively high altitude brings about increases in serum EPO levels, RBC mass and haemoglobin. This approach allows improving haematology and training at similar intensities to sea level. The latter can induce the necessary neuromuscular adaptations via sufficient stimuli. Both physiological adaptations may lead to enhancement of sea level maximal oxygen uptake (VO2max) and endurance performance
    In recent years endurance athletes have begun to utilise several new devices and modalities that can be used in conjunction with the "live high-train low" approach to altitude training including: (i) normobaric hypoxia via nitrogen dilution (hypoxic apartment); (ii) supplemental oxygen; (iii) hypoxic sleeping devices; and (iv) intermittent hypoxic exposure (lHE)
    The use of "altitude houses" is an approach developed in Finland in the 1990s and then spread to other countries including Australia. A hypoxic apartment is a normobaric hypoxic living environment that simulates altitudes of 2000 to 3000m allowing the athlete to follow the "live high-train low" method. Several scientific data suggest that this method may produce changes in serum EPO, reticulocyte count and RBC mass. However, other studies have failed to show changes in the erythropoiesis indices resulting from normobaric hypoxic exposure". A limited number of studies have suggested that anaerobic capacity and performance are enhanced through the use of an hypoxic apartment.
    Supplemental oxygen has been used for simulating either normoxic (sea level) or hypoxic conditions during high intensity workouts conducted at altitude. Use of supplemental oxygen in this manner is a modification of the "high-low" strategy in that athletes live in a natural terrestrial altitude environment but train at "sea level" with the aid of supplemental oxygen. Although limited, scientific data regarding the efficacy of hyperoxic training suggest that high-intensity workouts at moderate altitude (1800m) and endurance performance at sea level may be enhanced through the use of supplemental oxygen. There is a need of further research in this field.
    Endurance athletes have started to use hypoxic sleeping devices as part of their altitude training programmes. There are several modalities available on the market. These devices can simulate altitudes up to 4000m. Currently no studies have been published on the effects of these devices on RBC, VO2max or aerobic performance.
    The use of IHE for the purpose of enhancing athletic performance is based on the fact that brief exposures to hypoxia (1.5-2 hours) stimulate the release of EPO. Athletes use IHE while at rest or in conjunction with training sessions. The latter is referred to Intermittent Hypoxic Training (lHT). It is unclear whether IHE or IHT lead to improvements of RBC, Hb or Hct despite increments of serum EPO. There are minimal data to support the claim that IHT or IHE enhances VO2max and performance in well-trained athletes. However, preliminary data suggests that anaerobic power and anaerobic capacity may be improved as a result of I Hr. Further research is also needed in this area.
    Some have objected the use of hypoxic devices on ethical grounds. In fact, use of simulated altitude devices by athletes living in the Olympic Village was prohibited by the organisers of the 2000 Olympic Games in Sydney. Nevertheless. arguments against these devices seem to be unfounded. The Norwegian Olympic Committee has come forward with a position statement supporting the use of altitude houses and stating that utilising theses devices falls within the ethical norms which sport follows.


1.2.2     Recombinant human erythropoietin (rHuEPO)
    Endogenous EPO is the principal hormone that regulates mammalian erythrocyte and Hb production. It is a 166 amino acid glycoprotein hormone and is generated mainly in the kidneys, although up to 10% may be produced in the liver. EPO has some heterogeneity, as there are several isoforms. EPO stimulates the proliferation and differentiation of erythroid progenitor cells in bone marrow towards functional erythroblasts. EPO production is regulated by hypoxia. The serum levels go from 2 UI/L to 24 UI/L though 95% of subjects are inside the range from 6 to 10 UI/L. The maturation process from EPO liberation and action on erythroid progenitor cells in bone marrow to the appearance of mature adult erythrocytes on blood stream requires from 5 to 9 days under normal physiologic conditions.
    The development of recombinant DNA techniques has facilitated the pharmaceutical production of rHuEPO. In 1985, the human EPO gene was cloned. Within in a few years rHuEPO was commercialised in Europe (in 1987) and then in the USA (in 1989. In a clinical setting, rHuEPO is prescribed commonly to patients with renal disease, mainly patients suffering from anaemia related to renal failure. It is also used for patients with HIV-related anaemia, individuals who have lost significant amounts of blood due to major surgery, prevention of anaemia in surgical patients and the prevention or treatment of cancer and chemotherapy related anaemia. Over 500,000 patients throughout the world suffering from different conditions are now receiving rHuEPO.
    There are several kinds of rHuEPO currently available on the market or under research. First commercialised was rHuEPO-α, with rHuEPO-β introduced later. Both products are obtained by expressing a human EPO gene introduced to Chinese hamster ovary cells. An alternative to both is rHuEPO-ω, which is isolated from baby hamster kidney cells. Recently, investigators have succeeded in producing EPO through human cells using a slightly different approach. This process of protein production resulting from upregulation of an inactive endogenous gene in human cells is called "gene activation". The EPO derived from this method is called Gene-Activated Erythropoietin (GA-EPO) and has recently ended the clinical trial stage. The novel erythropoiesis-stimulating protein (NESP) or darbepoietin is an EPO derivative, which results from mutations that have been intentionally introduced into the EPO gene. As compared to EPO, NESP has more sugar side chains (increased carbohydrate content) that leads to increased serum half-life and enhanced biological activity. This implies the clinical advantage of less frequent dosing and patients may successfully switch from 2-3 times weekly with rHuEPO to once weekly or every other week with NESP.
    It seems that rHuEPO made its appearance on the sport environment at the 1988 Winter Olympic Games in Calgary. The illegal use of rHuEPO for doping purposes is by subcutaneous or intravenous injection of 200 to 250 UI/kg of body weight two to three times a week over a 4 to 6 week period. This could be accompanied by some use of exogenous iron administration (intravenous, intramuscular or oral). The illegal treatment can stand for some more weeks at lower dose and with 1 to 2 injections per week. Recently, it has been speculated that athletes could be switching to lower doses of rHuEPO, but taking them continuously.
    It is difficult to objectively quantify the prevalence of rHuEPO use among athletes. Although lay sports literature has reported conspicuous use of rHuEPO by international calibre athletes, there are minimal scientific data to support this claim. Scarpino et al interviewed Italian male and female athletes regarding the prevalence of blood doping (RBC reinfusion and/or rHuEPO injection). Seven percent of the athletes reported they were regular users, whereas 25% said they were occasional users. Anecdotal estimates by international drug control personnel suggest that 3 to 6% of top endurance athletes have used rHuEPO at some point at their career. Thus, owing to ethical factors and lack of detection data, it is difficult to accurately quantify the prevalence of rHuEPO use among athletes.
    There is speculation that blood doping with rHuEPO may have been involved in the deaths of professional cyclists from the Netherlands in the early 1990s. At that time, rHuEPO abuse was largely uncontrolled and Hct values in excess of 60% were purportedly achieved. These polycythemic conditions compounded by dehydration during exercise readily predisposed athletes to thromboembolic complications. Nowadays, rHuEPO abuse is undoubtedly more finely tuned, However, the medical risks associated with rHuEPO used are still considerable.
    Hyperviscosity (Hct > 52% and 55 % for females and males respectively) is a documented side effect of rHuEPO, The use of rHuEPO markedly increases the risk of thromboembolic complications, Although only a minority of athletes abusing rHuEPO will develop a thromboembolic disease, the unlucky ones might experience serious handicaps for the rest of their life or even die from it. Administration of rHuEPO also involves an increase in the systolic blood pressure during submaximal exercise. Hypertension should be considered a risk factor for rHuEPO use in athletes. Related to it, cerebral convulsion and hypertensive encephalopathy have been reported, Other side effects associated with rHuEPO use have included influenza-like syndrome and increased potassium plasma levels (Hyperkaliemia). Additionally, primary observations suggest that the abuse of rHuEPO might involve a risk of blunted endogenous EPO production, including severe anaemia. In particular, these individuals would be unable to develop an adequate erythropoietic response to stress conditions". There has also been the revelation that long-term rHuEPO use can lead to the development of antibodies. In fact, a severe anaemia, called Pure-Red Cell Aplasia, secondary to the virtual absence of red blood cell precursors in the bone marrow due to the presence of antierythopoietin antibodies, has been consistently reported on the scientific literature. There has been a significant increase in the number of patients suffering from Pure Red-Cell Aplasia and despite the very low risk, it has terrible consequences as patients become dependent on blood transfusions to maintain an acceptable level of haemoglobin. On the other hand, it has to be stated that we still do not know the effects of long-term treatment with haematopoietic growth factors, but observations in animals suggest that there may be a risk of development of myeloproliferative disorders. A further risk is iron overload comparable to that of patients with genetic haemochromatosis, with ferritin levels often in excess of normal due to the misuse of iron - oral, intramuscularly or intravenously - by athletes abusing rHuEPO. Sports cheaters using rHuEPO are exposing themselves to unjustified health risks, providing that they are healthy individuals that do not need this kind of treatment.
    The IOC officially prohibited the use of rHuEPO in 1989 when the IOC Medical Commission introduced the new doping class of peptide hormones and analogues. This class includes a series of natural endogenous hormones, rHuEPO among others, their mimetics, analogues and releasing factors.
    Since rHuEPO was banned, a number of methods have been proposed for detecting its use in athletes. There are several different indirect methods, which are based on the analysis of various markers on blood samples. There is also a direct method that allows the differentiation between endogenous EPO and exogenous rHuEPO.


Blood indirect tests

    In the past, several authors have tried to use different markers of accelerated erythropoiesis such as reticulocyte percentage, Hb, Hct, macrocytic hypochromatic erythrocytes, serum soluble transferrin receptor (sTfr) and others as a method of detecting rHuEPO abuse, The most widely accepted method, and the only one scientifically validated, is that of Australian researchers Parisotto et al. The test, introduced for the 2000 Olympic Games in Sydney, is based on a statistical multiparametric analysis defined in two kinds of mathematical models, ON and OFF, reflecting respectively the accelerated erythropoiesis due to current use of rHuEPO or the decelerated erythropoiesis due to past use of rHuEPO stopped shortly beforehand. The first blood tests proposed in 2000 were recently greatly improved upon in a second generation". It the second generation tests, two ON and two OFF models were defined on combinations of the blood parameters Hb, serum EPO concentration, percent reticulocytes and sTfr. These second generation tests have an enhanced sensitivity to be able to detect the impact of rHuEPO some days after an injection with moderate to low doses of rHuEPO (ON model) and thereby to provide a strong indication for the performance of a urine rHuEPO detection analysis. They also have increased sensitivity permitting the detection of the impact or rHuEPO up to 3 weeks after the last injection (OFF model) such that athletes who recently ceased using rHuEPO can be recognised and referred for follow-up testing. The results of these tests are based on statistics. They give a probability of rHuEPO abuse, not direct evidence. There are factors, mostly the effect of altitude, which can influence the results. Caution should be exercised when interpreting blood results from athletes who have recently been exposed to either terrestrial or simulated altitude. Notwithstanding this, these indirect blood tests are a useful tool for identifying athletes who are currently injecting rHuEPO or those who have recently stopped doing so.


Urine direct tests

    Wide et al reported a lower negative median charge of rHuEPO and less electrophoretic mobility in comparison with the natural hormone. Based on this physical characteristic they evaluated the validity and reliability of the electrophoretic mobility for detecting rHuEPO in serum and urine samples, Because of its considerable practical difficulties, this method has never been applied in anti-doping laboratories. It is well known that both the natural and the recombinant form of EPO present extensive microheterogeneity that is mainly determined by the several sialic radicals of the protein molecule. These differences in the glycosylation are influenced by the nature of the cell that produces the protein (human kidney, Chinese hamster ovary or baby hamster kidney cells) and the environmental conditions that may affect the cell. Owing to the microheterogeneity in their structures, all EPO products comprise multiple isoforms that differ in charge and isoelectric point and can be separated by isoelectric focusing. Isoforms of rHuEPO are more alkaline than those of endogenous EPO. These differences can be used for the unambiguous identification of rHuEPO misuse. Furthermore, darbepoietin has more sugar side chains than EPO and is considerably more acidic than EPO and rHuEPO. Lasne et al introduced in 2000 a test based upon isoelectric focusing that can separate the different isoforms allowing the unequivocal differentiation of rHuEPO-α, rHuEPO-β, rHuEPO-ω and NESP from EPO, but they cannot yet differentiate GA-EPO. This method is expensive (400 per test) time consuming (2-3 days) and requires highly trained technicians and a well-equipped laboratory. Thus, it can only be performed in a few specialised laboratories. Recently the Executive Committee of the World Anti-Doping Agency (WADA) accepted the results of an independent report stating that the Lasne urine method can stand alone in detecting the presence of rHuEPO.
    The IAAF has adopted the policy of carrying out out-of-competition and pre-competition doping controls for rHuEPO taking both blood and urine samples. At competitions, urine-only doping test have already been conducted, as was the case at the 2003 IAAF World Championships in Athletics in Paris. It is likely that urine-only out-of-competition doping controls will soon be put in place.

1.2.3    Other erythropoietins, EPO peptides and EPO mimetics
    Treatment with rHuEPO as a peptide medicinal drug is currently limited to intravenous and subcutaneous administrations. These injections are painful, and an alternative route of administration would seem to be desirable. To avoid degradation of rHuEPO by the acidic pH of the stomach or by enzymes in the gastrointestinal tract after oral administration, rHuEPO encapsulated in liposomes (sterols spheres than that can carry drugs inside) has been studied. Interest in using liposomes as carriers of several cytokines has also been reported. To date, rHuEPO encapsulated in liposomes has only been investigated in rats. Potential use in humans is far away, but rHuE- POjliposomes may represent an interesting, future alternative to intravenous and subcutaneous routes of rHuEPO administration. The development of a method to detect this new rHuEPO is, of course, not yet available, but detection methods will probably depend on the nature of the EPO encapsulated.
    A variation of the above mentioned approach is encapsulated cell technology. Recent research has been published regarding the effects of systemic delivery of rHuEPO by implantation of engineered cells immunoprotected in membrane polymers. This technique could represent another future alternative to current treatments with rHuEPO. Detection methods will probably depend on the nature of the cells used for encapsulation.
    Over the past five years, several reports have been published demonstrating the feasibility of EPO-gene transfer in rodents and non-human primates. The two principal approaches are either direct transfer in vivo or ex vivo gene transfer into isolated cells, which are then transplanted into the recipient organism. A sustained elevation of Hb levels can be achieved with both strategies. For several reasons the former is preferable for consideration in humans. Its applicability in patients will depend on safety issues related to gene transfer in general and reliable techniques to control EPO secretion in vivo.
    The latest trend regarding EPO treatment technology is the development of molecules to interact with the EPO receptor (EPOR) and on the modulation of EPOR activity. Some investigators have been pursuing mimetics of EPO. A mimetic is defined on the World Anti-Doping Code 2004 List as "a substance with pharmaceutical effect similar to that of another substance, regardless of the fact that it has a different chemical structure. The search for small mimetic molecules of EPO has lead to a family of peptides that demonstrate EPO mimetic activity. A member of this peptide family, the EPO mimetic peptide 1(EMP1), was obtained through combinatorial peptide-screening techniques. It has a chain of 20 amino acids, of which 13 are necessary for activity. Another EPO mimetic peptide family under current research is the termed erythropoietin receptor binders (ERB) 1-7. An EPOR-derived peptide (ERP) is being investigated3. Nonpeptide mimetics of EPO have also been discovered. A further potential target for therapeutic intervention is the intracellular signal transduction cascade of EPOR. Haematopoietic cell phosphatase (HCP) is a very important molecule in this process acting as a negative regulator of EPO signalling cascade. Some researchers are looking for potential inhibitors of HCP in the search of an EPO enhancer. Although many open questions and technical hurdles remain, these developments may eventually lead to the availability of orally administered drugs that activate the EPOR.
    There is, today, an increasing interest in molecular EPO mimicry. In general, steps in this direction are aiming to identify active peptide domains of EPO, synthesise their derivatives, and to discover non peptide small mimetics with resistance to proteolytic digestion, good permeability and suitability for oral administration. EPO mimetics may be of interest to athletes wishing to artificially enhance their performance. However these products are exogenous and will probably be easy to detect3.

1.2.4     Allosteric effectors of Hb
Allosteric effectors of Hb bind reversibly to Hb in RBCs without damaging the cell membrane. Such effectors decrease the oxygen affinity of RBCs; the Hb-oxygen dissociation curve is shifted to the right, which leads to increased oxygen release to the tissues. In the 1980s, two antilipidaemic drugs, clofibrate and bezafibrate, were found to decrease the affinity of Hb for oxygen. Since then, several allosteric effectors derived from fibrates have been synthesised. RSR (effaproxiral sodium) is currently in phase III clinical trials as a radio-sensitising agent for metastatic brain cancer or for the treatment of glioblastoma multiforme brain tumours. The ability to amplify physiological tissue oxygenation indicates that RSR has potential application in clinical conditions characterised by tissue hypoxia, including oncology, cardiovascular and cerebrovascular conditions3. At present, there is no evidence that RSR has entered sports. However, it may soon interest, or perhaps already interests athletes as a means of increasing oxygen transport. The World Anti-Doping Code 2004 List includes the effaproxiral as a prohibited method of enhancing oxygen transport4.

2    Indirect increase of oxygen delivery

2.1    Hb-based oxygen carriers (HBOCs)
    An alternative means of immediately increasing circulating haemoglobin levels is to infuse a HBOC. These products have been the focus of intense research and development in recent years to serve as a blood substitute that may ease the burden on blood donor supplies required in surgical settings and transfusion emergencies.
    Hb can be easily extracted from RBCs, but it breaks down in the body and causes renal toxicity. HBOCs are obtained by chemical ster- ilisation of haemoglobin extracted from a variety of sources (bovine, expired donor, recombinant human and transgenic human Hbs). Biotechnological cross-linking, recombinant modifications and micro-encapsulation not only stabilize the Hb molecule but also provide a range of different blood substitutes with a variety of clinical benefits. Most of the HBOCs are currently in the later phases of clinical trials and a few are already marketed. A number of adverse effects have been reported, the main one among these is vasoconstriction with resultant hypertension. Oxidation is the second most important side effect.
    It is anticipated that HBOCs will represent a new doping threat in endurance sports and it may be that they have recently entered the sport arena. A few studies investigating the effects of artificial HBOCs and how they might improve aerobic performance have been published. The World Anti-Doping Code 2004 List includes the Hb based blood substitutes, microencapsulated Hb products as examples of prohibited methods of enhancing oxygen transport. HBOCs have a short half-life (12- 24 hours) and do not appear in the urine. To identify the abuse of this class of products blood samples are required. A visual exam of plasma will show a precise red colour that represent a good index of suspicion that could be confirmed by more sophisticated methods. During 2004 Olympic Games in Athens, blood samples will be obtained to analyse HBOCs.

2.2    Perfluorochemicals (PFCs)
    The perfluorocarbons or perfluorochemicals (PFCs) developed during the World War II were introduced to biomedicine in 1966. They are synthetic fluids in which oxygen can be dissolved. In fact, PFCs are the best known gas solvents, They are simply-constructed molecules in which all hydrogen atoms have been replaced by halogens (fluoride, bromide). They can release up to three times more oxygen than HB. Because they are insoluble in water, PFCs should be administered on intravenous emulsion. These products are not metabolised and not excreted in the urine. PFCs are removed from the blood stream by phagocytes, then retained in the reticulo-endothelial system and finally exhaled via the lungs. Due to their ability to easily release oxygen, PFCs have a lower capacity for carrying oxygen, so the patient must breathe an oxygen-rich air mixture.
    The benefits of PFCs that can be expected by athletes without oxygen supplementation are reduced but not negligible. Although no study performed on athletes using PFC emulsions has been published, PFCs are included in the World Anti-Doping Code 2004 List as a method of illegal enhancement of oxygen transfer. PFCs can be detected in expired air or blood.



    There are a number of means for increasing an athlete's capacity to deliver oxygen to his/her muscles, and thereby improve performance in endurance events, currently in use or in development. Though some are ethically acceptable, many, including ones that may have legitimate clinical uses for patients with severe conditions, are rightfully prohibited in sport. It is unfortunate that some athletes; possibly aided by coaches, physicians, physiologists, researchers and others; choose to debase their sport and put themselves at risk of serious and sometimes unknown side effects by using prohibited substances and techniques. The author of this paper firmly reiterates his condemnation of the use of all banned drugs and methods to artificially enhance performance and hopes that by making the above information more widely available he has contributed to the worldwide fight against doping in sport.


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