doping with artificial oxygen carriers

Sports Med 2004; 34 (3): 141-150LEADING ARTICLE 0112-1642/04/0003-0141/$31.00/0

© 2004 Adis Data Information BV. All rights reserved.

Doping with ArtificialOxygen CarriersAn Update

Yorck Olaf Schumacher1 and Michael Ashenden2

1 Department of Sports Medicine, University of Freiburg, Freiburg, Germany2 SIAB – Science and Industry Against Blood Doping Research Consortium, Surfers Paradise,

Queensland, Australia

There is a long history of science seeking to develop artificial substitutes forAbstractbody parts damaged by disease or trauma. While defective teeth and limbs arecommonly replaced by imitations without major loss of functionality, the develop-ment of a substitute for red blood cells has proved elusive.

There is a permanent shortage of donor blood in western societies. Neverthe-less, despite whole blood transfusions carrying measurable risks due to immuno-genicity and the transmission of blood-borne infectious diseases, red blood cellsare still relatively inexpensive, well tolerated and widely available. Researchersseeking to develop products that are able to meet and perhaps exceed these criteriahave responded to this difficult challenge by adopting many different approaches.Work has focussed on two classes of substances: modified haemoglobin solutionsand perfluorocarbon emulsions. Other approaches include the creation of artificialred cells, where haemoglobin and supporting enzyme systems are encapsulatedinto liposomes.

Haemoglobin is ideally suited to oxygen transport when encased by the red cellmembrane; however, once removed, it rapidly dissociates into dimers and iscleared by the kidney. Therefore, it must be stabilised before it can be safelyre-infused into humans. Modifications concomitantly alter the vascular half-life,oxygen affinity and hypertensive characteristics of raw haemoglobin, which canbe sourced from outdated blood stores, genetically-engineered Escherichia coli oreven bovine herds. In contrast, perfluorocarbons are entirely synthetic moleculesthat are capable of dissolving oxygen but biologically inert. Since they dissolverather than bind oxygen, their capacity to serve as a blood substitute is determinedprincipally by the oxygen pressure gradients in the lung and at the target tissue.

Blood substitutes have important potential areas of clinical application includ-ing red cell replacement during surgery, emergency resuscitation of traumaticblood loss, oxygen therapeutic applications in radiography (oxygenation oftumour cells is beneficial to the effect of certain chemotherapeutic agents), othermedical applications such as organ preservation, and finally to meet the require-ments of patients who cannot receive donor blood because of religious beliefs.

Given the elite athlete’s historical propensity to experiment with novel dopingstrategies, it is likely that the burgeoning field of artificial oxygen carriers has

142 Schumacher & Ashenden

already attracted their attention. Scientific data concerning the performancebenefits associated with blood substitutes are virtually nonexistent; however,international sporting federations have been commendably proactive in addingthis category to their banned substance lists. The current situation is vulnerable toexploitation by immoral athletes since there is still no accepted methodology totest for the presence of artificial oxygen carriers.

Since the advent of competitive sports, athletes blood transfusion (which even under controlled clin-have sought to gain an edge over their opponent ical settings gives rise to a quantifiable risk ofthrough the intake of performance-enhancing prod- haemolytic reaction associated with mismatcheducts. According to contemporary sources, bull’s tes- blood, or sepsis due to bacterial contamination of theticles and stimulating herbal potions were common- transfused blood). The recognition of blood-bornely used by Olympic athletes in ancient Greece to diseases such as AIDS and viral hepatitis during theimprove their strength and stamina. Doping, a term 1980s immediately preceded the commercial availa-derived from a stimulative South African beverage, bility of recombinant human erythropoietin (rHuE-has become increasingly sophisticated over time, PO). Subsequently, rHuEPO proved an irresistibledriven at least in part by knowledge derived from lure for athletes seeking to obtain the same surrepti-pharmacological interventions during wartime. The tious performance advantage of blood transfusionuse of amphetamines (developed to increase alert- without the associated health risks. Rumours ofness of soldiers) and anabolic steroids (used to rHuEPO use surfaced at the 1988 Calgary Winterroborate prisoners of war) in post-World War II Olympic Games, and although rHuEPO was addedsport became so pervasive that it was considered to the IOC’s list of forbidden substances in 1994, itprudent to start searching for evidence of such com- took almost 10 years for international sporting fed-pounds in the urine of Olympic athletes during the erations to come to grips with the widespread use of1960s. this drug by elite athletes. The international publici-

ty associated with the doping scandal at the 1997Those sports where competitive success is heavi-Tour de France served as a stimulus to revitalisely influenced by a physiological characteristic areanti-doping efforts. Investigators were stymied bymost prone to doping. Endurance-oriented sportsthe inability to distinguish between the recombinanthave most often been linked with rumours of bloodand endogenous forms of erythropoietin; however, adoping, which involves transfusing red blood cellscombination of blood and urine tests was eventuallyfrom a donor to increase the amount of haemoglobinapproved by the IOC for detecting rHuEPO use atavailable to transport oxygen to muscle (and therebythe 2000 Sydney Olympic Games.[4,5]improve aerobic performance). Although rumours

persist that prominent endurance athletes from the Historically, athletes quickly progress to novel1960s were blood doping, use of this practice was doping strategies when a test is introduced that isfinally confirmed at the 1984 Los Angeles Olympic capable of detecting contemporary drugs. FacedGames when some cyclists from the US admitted to with the continued refinement and optimisation oftransfusing blood prior to competition. This practice indirect and direct methods to identify the 1990swas immediately banned by the International ‘drug of choice’, rHuEPO, it is likely that athletesOlympic Committee (IOC). have already begun to experiment with another sur-

reptitious means of improving oxygen transport,Although blood doping is demonstrably effectiveso-called artificial oxygen carriers.in improving athletic performance,[1-3] it entails in-

vasive procedures and the risk that transfusion Artificial oxygen carriers or ‘blood substitutes’materials will be detected by anti-doping authorities. are being developed to serve as a temporary replace-Furthermore, there is a health risk associated with ment for transfused red blood cells, and these agents

© 2004 Adis Data Information BV. All rights reserved. Sports Med 2004; 34 (3)

Doping with Artificial Oxygen Carriers 143

are either based upon modified haemoglobin or per- molecule can bind an oxygen molecule at each offluorocarbon emulsions. Several products are in ad- four specific iron-containing regions (Haeme), per-vanced clinical trials; however, knowledge garnered mitting each gram of haemoglobin to bind 1.34mLduring these trials and parallel research has revealed of oxygen per 100mL of blood.unexpected sequelae of the administration of blood Although it is relatively straightforward to ex-substitutes, such as hypertension, gastrointestinal tract and purify haemoglobin from within red bloodirritability and impaired oxygen delivery to tis- cells, haemoglobin is an inherently unstable mole-sues.[6] Furthermore, the literature describing these cule and will become dysfunctional because theproducts is confusing and often contradictory.[7] oxygen binding characteristics of haemoglobin areNevertheless, tremendous effort and resources have dependent on the conformational arrangement of thebeen focussed on developing a product to address molecule which are extremely difficult to preservethe worldwide shortage of donor blood, resulting in when isolating haemoglobin from red cells.[8] It isthe refinement of existing products, as well as explo- dangerous to infuse even autologous-derived freeration of second- and third-generation products that haemoglobin since the tetramers rapidly dissociateseek to address many of the concerns plaguing the into dimers, which are filtered by the kidney andcurrent crop of artificial oxygen carriers. One com- cause rapid acute renal tubular necrosis, leading topany already has haemoglobin-based products ap- chronic renal failure.proved for both veterinary and human use. Interna- Furthermore, serum oncotic pressure is increasedtional sporting federations have reacted to the immi- by free haemoglobin. Once outside the protectivenent commercial arrival of artificial oxygen carriers environment of the red cell membrane, the absenceby including this class of pharmaceutical on their of allosteric effectors such as 2,3-diphosphoglycer-banned substance lists. ate increases the oxygen affinity of haemoglobin.

Because the development of blood substitutes is Nevertheless, the affinity of haemoglobin for nitricunambiguously devoted to clinical scenarios involv- oxide (NO) is 3000 times higher than for oxygen.ing very sick patients or surgical candidates, it is With NO widely present in vessel walls throughoutperhaps not surprising that there has been very little the body, free haemoglobin results in a markedresearch devoted to understanding the performance- vasoconstrictor effect (so-called ‘NO scaveng-enhancing effects of these products. Therefore, in ing’).[9,10] Free haemoglobin is rapidly bound bythe present article, we review the current knowledge haptoglobin and metabolised in the liver. Aboveon artificial oxygen carriers, focussing on the likely plasma haemoglobin concentrations of 200 mg/dL,impact on performance as well as potential dangers haemoglobin is directly excreted through the kid-and adverse effects. We address the potential impact ney.that blood substitutes will have on doping in sport. In order to address these issues, biochemical

modifications have been undertaken in an attempt to1. Artificial Oxygen Carriers develop safe, effective and tolerable haemoglobin-

based products. The major goals have been to:

• decrease oxygen affinity of free haemoglobin;1.1 Haemoglobin-Based Products• prolong intravascular retention;

• prevent breakdown and renal toxicity;1.1.1 Physiology and Development• decrease oncotic pressure mediated by haemo-Haemoglobin is a tetrameric protein responsible

globin solutions;for the transport of oxygen from the lung to the• avoid immunogenic effects.tissue. Normal haemoglobin contains four protein

subunits (two α- and two β-chains), has a molecular Strategies differ not only by the chosen source ofweight of 64 kDa and constitutes around 95% of the the raw protein material but the method used todry weight of a red blood cell. Each haemoglobin transform the molecules into functional units.



Several haemoglobin-based oxygen carriers blood flow away from the muscle, despite overalloxygen transport capacity being improved.(HBOCs) are currently under development in North

Two separate views on the mechanism throughAmerica and readers are directed to comprehensivewhich HBOCs influence vasoconstriction can bereviews of this field.[11,12] Three principle approach-located in the literature: (i) NO scavenging; or (ii)es have been used to stabilise and modify tetramericautoregulation. Arguments suggesting that NOhaemoglobin: polymerisation using polyaldehydes,scavenging has primacy have evolved from theconjugation of polymers to the surface of thelong-recognised affinity of haemoglobin for NO, ahaemoglobin, or crosslinking the α- and β-dimers ofpotent vasoconstrictor that is released from endothe-the protein. Both products that have been commer-lial cells into the vessel wall (NO causes the smoothcially released, Hemopure™1 (human use) and Ox-muscle of the vessel wall to relax). Ordinarily, cell-yglobin™ (veterinary use), use polymerised bovinebound haemoglobin is unable to access the intersti-haemoglobin. Researchers are also seeking to createtial space; however, tetrameric haemoglobin, unfet-artificial red cells by encapsulating haemoglobintered by the red cell membrane, can leave the vesselsinto liposomes which contain complex enzyme sys-and bind NO. It is logical that a reduction in localtems to prevent reperfusion injury.[13]

NO concentration could be associated with vaso-constriction; however, research has shown that the1.1.2 Effects on Tissue OxygenationNO affinity of model haemoglobins does not corre-and Performancelate with their effect on mean arterial blood pressure

Many investigations have studied the impact ofin rats[27] suggesting that this mechanism is not yet

HBOCs on tissue oxygenation in animal and humanfully understood.

models. These studies have reported an increase inWhilst NO scavenging seems important, it is

tissue oxygenation under various experimental set-neither necessary nor sufficient to cause the noted

tings (readers are directed to Creteur et al.[14] for a increase in blood pressure.[7] An alternative viewreview of clinical studies). Tissue oxygenation has has been proposed that draws attention to the sophis-been maintained via HBOCs, even when normal ticated mechanisms in place to regulate the amountblood flow was reduced (through artificial stenosis, of oxygen that reaches local tissues. This view ar-haemodilution or anaemia). The principle therapeu- gues that there is a local mechanism within thetic applications for HBOCs include avoidance or microcirculation that matches perfusion to local ox-reduction of blood transfusions in trauma patients ygen requirements, and deduces that oxygen deliv-and patients undergoing elective surgery, treatment ery to the precapillary regulatory arterioles ulti-of septic shock by excess-NO scavenging, and en- mately holds the key to the availability of oxygen tohancement of tumour sensitivity to radiother- the tissue.[6]

apy.[15-25] However, several scientists have raised Only recently have the first reports concerningconcerns about the vasoactivity of the substances the administration of HBOCs in clinical practicethat might alter blood distribution in the body. It has (outside of experimental settings) been published.been demonstrated that during application of Mullon et al.[28] describe the case of a 21-year-oldHBOCs in humans, the vasoconstriction caused by female with severe autoimmune haemolytic anae-free haemoglobin is not distributed evenly through- mia. In the presence of Hemopure™, no signs ofout the body; the effect is more pronounced in cardiac ischaemia were noted at a haematocrit ofmuscle-supplying vessels, almost absent in the mes- 4.4%, suggesting an improved oxygen delivery withenteric vascular system and manifests as vasodilata- this product. Vasoactivity was not observed in thistion in the coronaries.[26] This might result in im- case, but might have been masked by other disease-paired oxygen delivery with a redistribution of the related effects on the circulation.

1 The use of trade names is for product identification purposes only and does not imply endorsement.



Exercise-related studies using HBOC are scarce. mediately re-infused with their own blood demon-strated reduced performance characteristics. It wasIn animal testing, Crago et al.[29] studied fivenot clear against which control scenario lactatehealthy, non-anaemic swine infused with 5 mL/kglevels were lower. Finally, no explanation was givenof a 10% diaspirin crosslinked haemoglobin solu-as to why the correction factor for diffusion capacitytion (DCLHb) during graded treadmill exercise.was different for the two groups prior to re-infusionThey found no detrimental haemodynamic or meta-of either Hemopure™ or autologous blood (bothbolic effects during exercise with DCLHb, and re-groups were phlebotomised to the same degree andported a higher oxygen extraction in the DCLHbreceived equal infusions of Ringers lactate). It isgroup accompanied by a decrease in cardiac output.hoped that additional research will sharpen our un-This effect was evident during both exercise and thederstanding of the HBOC-mediated physiologicalrecovery periods, and was interpreted to be indica-perturbations encountered during intense physicaltive of an enhanced capacity for oxygen deliveryexercise.mediated by the HBOC solution. However, it is also

possible to conclude the opposite – that increased1.1.3 Adverse Effectsoxygen extraction in the DCLHb group might beA universal problem that has yet to be completelynecessary to counter deleterious effects associated

solved is the short duration of action: half-liveswith decreased muscle blood flow and/or blood re-range between 12 hours for cross-linked haemo-distribution induced by the vasoactivity of the sub-globin to about 2 days for surface-modified prod-stance. Similarly, the reduced cardiac output mayucts, compared with a life span of 120 days for thehave been a consequence of higher vascular resis-erythrocyte.[6] The adverse effects of HBOC are stilltance. The latter view is supported by data showingnot completely understood, but appear mainly deter-that both peripheral and pulmonary arterial pres-mined by the properties of free haemoglobin in thesures were higher in the DCLHb group. Unfortu-circulation. In many trials, high peripheral and pul-nately, no further animal studies with a similar focusmonary pressures under HBOC administration wereare available to add to this discussion.reported,[31] and the cause of this hypertension ap-

To date, only one investigation has studied the pears multifactorial.[7] When oxygen concentrationeffect of HBOC on exercise performance in humans. in the tissue is high, a hyperoxic arteriolar reflexHughes et al.[30] first phlebotomised ~150g of might reduce the number and diameter of functionalhaemoglobin from six healthy males then infused capillaries to avoid oxidative tissue damage, thuseither the same volume of blood or 45g of increasing vascular resistance. Furthermore, re-Hemopure™ prior to a submaximal exercise proto- duced shear stress associated with low viscosity-col (up to ~60% maximal oxygen uptake [VO2max]). HBOC could result in an impaired release of wallDuring the study, the substance was well tolerated bound vasodilators (NO, prostacyclins), thus reduc-and no adverse effects were observed. The authors ing the vasodilatative effect of high blood flowconcluded that Hemopure™ resulted in greater oxy- velocity. Nevertheless, the most prominent issuegen uptake and lower lactate levels compared with leading to increased pressures under HBOC is theautologous transfusion. Although this paper pro- ‘NO scavenging’ function of free haemoglobin,vides a valuable insight into the physiological ef- which reduces the NO-mediated vasodilatation infects of HBOCs in healthy individuals, the confus- arterioles and capillaries.ing nomenclature hinders efforts to ratify their con- Other adverse effects are gastrointestinal mani-clusions. For example, the authors did not address festations with increased tone of the intestinalwhy re-infusion of 150g of endogenous haemo- sphincters, marked flatulent activity and meteorism.globin appeared only to raise total haemoglobin Renal toxicity, induced by filtration of haemoglobinlevels by the same amount as 45g of haemoglobin monomers and consecutive tubulus necrosis, repre-derived from Hemopure™, nor why individuals im- sents a potentially fatal adverse reaction of these



pharmaceuticals.[32-34] As is recognised with other organise as drop-like particles that are not capable ofblood products, HBOCs derived from haemoglobin leaking out of the circulatory system.[38] Although aof human or bovine origin might contain infective prerequisite for their infusion into the circulation,agents such as viruses or prions, or induce immuno- the first generation of emulsifying agents were latergenic effects in the recipient. However unlike red realised to be the cause of complement activation,blood cells, pasteurisation, ultrafiltration and chemi- and have been substituted with alternatives such ascal means can sterilise blood substitutes.[35] egg yolk lecithin which has solved this problem. The

interrelationship between oxygen carrying capacity,1.2 Perfluorocarbon Emulsions the emulsifying agent’s ability to maintain stable

PFCs in the circulation, and the excretion rate of the1.2.1 Physiology and Development substance from the body, determines the physiologi-Perfluorocarbons (PFCs) are chemically synthe- cal characteristic of PFCs.[39] Circulating PFC is

sised compounds with fluorine atom backbones, absorbed by phagocytotic cells of the reticuloen-which are closely related to the commercial product dothelial system, then slowly re-dissolves in theTeflon. Although insoluble in water, PFCs are able bloodstream and finally leaves the body unalteredto dissolve large quantities of gases (the highest of through the lung.all known liquids, some products can dissolve more The principle product under current investigationthan 100 times more oxygen per volume than plas- is a 60% perflubron emulsion which has a meanma) and yet be completely biologically inert. These particle diameter of <0.2μm (Oxygent™). Althoughtransparent liquids of low molecular weight early PFC solutions were initially developed for the(450–500 kDa) can be produced in unlimited quanti- treatment of anaemia, recent research has focussedty and at low cost. Such properties led scientists to upon their application as a temporary tissue oxygen-investigate this class of substances as potential ation support (for example LiquiVent™ as a liquidblood substitutes, which was spectacularly high- ventilation). Further applications may include in-lighted in 1966 when it was shown that a rat totally traluminal organ preservation, maintenance of tissueimmersed in oxygen-saturated PFC solution could oxygenation in critical ischaemic situations (cardio-survive for hours.[36] pulmonary bypass surgery, neuroprotection during

Unlike haemoglobin, PFCs do not bind oxygen. cerebral infarction) or avoidance and reduction ofAt a given temperature, the amount of oxygen that blood use in situations of acute haemorrhage.[40-46]

can be carried in solution is directly proportional to The potential for PFCs to increase the oxygen con-the gas partial pressure, and this is probably the most tent of tumours and therefore increase their sensitiv-significant limitation for widespread use of PFCs as ity to radiation or chemotherapy is also currentlyartificial oxygen carriers. A high partial pressure under investigation.gradient is required to dissolve a large quantity ofoxygen in the PFCs, and such gradients are also 1.2.2 Effects on Tissue Oxygenation

and Performancenecessary between PFC and tissue in order toachieve a biologically-useful degree of oxygen un- Presently, no study has been performed to deter-loading (the saturation curve for PFC is linear, con- mine the potential performance-enhancing effects oftrasting the sigmoid curve of haemoglobin). It has PFCs in healthy, normovolaemic humans. However,been argued that this property essentially precludes many PFC solutions have been investigated to deter-their use in settings where supplemental oxygen is mine their ability to improve tissue oxygenation innot available.[37] various settings (different levels of oxygen satura-

These agents have a high viscosity, which limits tion, different concentrations of PFC). Althoughthe concentration that can be infused. Furthermore, direct clinical experience with PFC has been mix-since PFCs are hydrophobic, they must be emulsi- ed,[47] and despite the aforementioned difficultiesfied before administration. In this form, PFCs related to the physiological properties of PFC (hy-



drophobicity, low solubility of oxygen), PFC has suitable test for this banned drug made the systembeen shown to improve oxygen delivery to the tissue vulnerable to exploitation by immoral athletes. Ulti-under many conditions.[42-45] mately, this gave rise to arguably the most wide-

The consensus from investigators explaining spread and morale-sapping doping episode in thetheir findings centres upon the small size of these history of elite sport. Although artificial oxygenmolecules (about 1/35 that of an erythrocyte), argu- carriers are similarly banned by the IOC and interna-ing that PFCs are able to improve oxygen delivery to tional federations, currently there is no test availablesmall capillaries that are not usually penetrated by to detect the presence of these products. To ignorered blood cells. Furthermore, these agents may the obvious similarities between rHuEPO and artifi-‘bridge’ oxygen transport from red blood cells to the cial oxygen carriers, in the hope that the likely willtissue by reducing the diffusion distance between not become inevitable, is fraught with danger.the cell and vessel endothelium. Although specula- When athletes first began to experiment withtive, it can be postulated that these effects might rHuEPO, there were numerous reports of deathsimprove endurance performance through increased caused by the naive ‘more is better’ administrationtissue oxygenation. of rHuEPO. There are (potentially) equally serious

consequences associated with the naive use of1.2.3 Adverse EffectsHBOCs. In contrast to the uniform haematologicalPFCs are eliminated rapidly from the body in aresponse induced by different brands of rHuEPO,biphasic manner, with half-lives reported of 2–4the HBOC industry is notable for the heterogeneityhours. The majority of PFC is exhaled un-of products currently under development. In addi-metabolised from the lung; however, they are alsotion to their role as oxygen carriers, HBOCs are alsoremoved from circulation by the reticuloendothelialbeing developed to serve as NO scavengers, with thesystem.[48] Subsequently, PFC is excreted from theexpected haemodynamic outcome of increasingreticuloendothelial system and eventually expired

through the lung. Similar to the HBOCs, the clinical blood pressure (such products are being developedsignificance of adverse effects associated with PFCs for the treatment of shock associated with systemicare not yet fully understood. Adverse effects asso- inflammatory response syndrome.[50] Were a naiveciated with PFCs have been attributed to temporary athlete to mistakenly infuse a vasoactive productcytokine release from PFC-mediated macrophage (anecdotal information suggests black marketactivation, which induces mild flu-like symptoms sources remove drug labels to avoid detection) andwith fever and myalgias in recipients of PFCs. These undertake intense exercise they would unknowinglysymptoms are annoying but not dangerous and are expose themselves to serious risk. The danger ofreported to be fully reversible after 4–12 hours. inaction on the part of sporting federations to imple-More severe conditions, such as thrombocytopenia ment strategies to deter this scenario seems farhave been reported. Because PFC is biologically greater than the danger of preventive action now.inert, it can only be removed through the In theory, the detection of artificial oxygen carri-reticuloendothelial system, which in turn may lead ers is straightforward – in contrast to rHuEPO thatto hepatic or spleenal engorgement with consecutive mimics closely the endogenous hormone, bothorgan failure and impairment of immune defence HBOCs and PFCs are demonstrably nonhuman inmechanisms. In addition to PFC itself, the emulsify- origin. Therefore, presence of the banned substanceing agents, such as egg yolk phospholipids, may in the system of an athlete should be sufficientcause allergic reactions.[39,49]

evidence of doping. Since PFCs are exhaled unal-tered by the lungs after passage through the2. Perspective and Strategyreticuloendothelial system, they can be measured inexpired air with a simple device based on thermalEven though rHuEPO was added to the IOC’s list

of banned substances in 1994, the absence of a conductance and mass transfer of gas,[51] or in blood



samples using gas chromatography/mass spectrome- sporting community must provide the legal, scien-tific and financial background necessary to developtry.[52-54] HBOCs will be most simply detected byand implement testing procedures to deter athletesthe bright red discolouration of either a plasma orfrom experimenting with this novel class of pharma-serum sample, which appears similar to aceutical agents.haemolytic state.[55] Plasma levels can be easily

quantified using a portable photometric system[56] ora more elaborate laboratory-based approach,[57] and 3. Conclusionsexceed detection limits for several days after infu-

Artificial oxygen carriers have physiological insion.[58] In order for the presence of HBOC to bevitro capacities on oxygen delivery and tissue oxy-confirmed with scientific certainty, several ap-genation that might encourage athletes to misuseproaches are available to separate human and modi-these new drugs to improve sporting performance.fied haemoglobins.[59,60] Current research is compar-However, there is virtually no scientific evidenceing the efficacy of several techniques including, butthat these substances truly improve exercise capa-not limited to, size exclusion or ion-exchange chro-city in athletes. On the other hand, potential adversematography, electrophoresis and mass spectrometry.effects are severe and might expose abusers to seri-

Together with the refinement and implementa-ous health risks. For this reason, suitable detection

tion of detection methodologies, it is vital that anti-techniques must be developed and legal background

doping authorities revise testing strategies to ad-has to be provided to prevent this new doping threat.

dress the specific characteristics of artificial oxygencarriers. For example, although both rHuEPO and

Acknowledgementsartificial oxygen carriers only persist for a short timein the circulation, only the latter must be used by the Science and Industry Against Blood Doping Researchathlete at the time of competition in order to bestow Consortium (SIAB) is funded by the World Anti Dopinga performance advantage. Therefore, testing for arti- Agency (WADA). The authors wish to thank P. Sundstedt for

her assistance. The authors have no conflicts of interest thatficial oxygen carriers should focus on podium fin-are directly relevant to the content of this manuscript.ishers immediately after the event (not during the

out-of-competition season). It would be futile toanalyse urine samples in search of PFCs and most References

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