- Email: [email protected]
Blood doping: Then and now. A narrative review of the history, science and efficacy of blood doping in elite sport
Journal Pre-proof Blood doping: Then and now. A narrative review of the history, science and efficacy of blood doping in elite sport
Thomas S. Atkinson, Marc J. Kahn PII:
S0268-960X(19)30146-8
DOI:
https://doi.org/10.1016/j.blre.2019.100632
Reference:
YBLRE 100632
To appear in:
Blood Reviews
Please cite this article as: T.S. Atkinson and M.J. Kahn, Blood doping: Then and now. A narrative review of the history, science and efficacy of blood doping in elite sport, Blood Reviews(2019), https://doi.org/10.1016/j.blre.2019.100632
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
Journal Pre-proof
1
Blood Doping: Then and Now. A narrative review of the history, science and efficacy of blood doping in elite sport Thomas S. Atkinson1,2, MD; Marc J. Kahn1,* [email protected], MD, MBA, MACP, FRCP 1Tulane
of
University School of Medicine, Department of Medicine, Section of Hematology/Medical Oncology. New Orleans, LA 70112, USA 2Southeast Louisiana Veterans Health Care Systems, Department of Medicine, Section of Hematology/Medical Oncology. New Orleans, LA 70119, USA
T.S. ATKINSON. ORCID: 0000-0002-6699-7152 *Corresponding
ro
M.J. KAHN. ORCID: 0000-0002-9781-9316
-p
author at: 1430 Tulane Ave, #8010, Tulane University School of Medicine, New Orleans, LA 70112, USA.
re
Keywords: blood doping, erythropoietin, sports hematology, oxygen delivery.
lP
Abstract
As the line between excellent and exceptional athletic performance narrows and as the
na
financial remuneration for exceptional performance increases, unscrupulous athletes
ur
and their trainers will strive to enhance performance regardless of cost. One method of performance enhancement is by the augmentation of the oxygen-carrying capacity of
Jo
the blood through blood doping. We discuss the science behind erythropoiesis and means by which these processes can be exploited to the potential advantage of the athlete. These include pre-sport transfusion practices as well as supplemental recombinant human erythropoietin (rHuEpo) and the use of newer erythropoietic agents, many of which have not received FDA approval. Finally, we discuss the data behind the efficacy of blood doping in an attempt to discern whether or not the practice actually works to improve athletic and competitive performance. Introduction
Journal Pre-proof
2
As the pressure to improve athletic performance intensifies due to everincreasing financial remuneration and the allure of prestige, unscrupulous athletes will seek ways to beat the system. Blood doping has been intertwined with elite endurance athletics for roughly the past half-century. In recent memory, no single event stands out more than the 2018 Seoul Olympics where the entire Russian team was barred from participation due to systematic doping.
of
The term doping refers to a multitude of practices which seek to artificially
ro
improve athletic performance through augmentation of the oxygen-carrying capacity of
-p
the blood. Blood doping, broadly defined by the World Anti-Doping Agency (WADA) as “any form of intravascular manipulation of the blood or blood components by physical or
re
chemical means,1” includes such practices as transfusion, recombinant human
lP
erythropoietin (rHuEpo) administration, and administration of artificial stimulants of
na
erythropoiesis.
Aside from blood transfusion, the use of rHuEpo is perhaps the most notorious
ur
and widely-studied method of blood doping. Athletes also have at their disposal other,
Jo
more novel ways of blood doping: hypoxia inducible factor (HIF) stabilizers, and the use of artificial hemoglobin (i.e. “synthetic blood”) are other examples of means to increase oxygen carrying capacity and thus increase aerobic exercise capacity. Despite the intent of dopers to improve athletic performance, the true effects on athletic performance are unknown. In what is inarguably the most notorious case of blood doping, Lance Armstrong was stripped of his seven Tour de France titles in 2012 after exhaustive investigation showed that he had been blood doping since at least 1999. His methods of doping were
Journal Pre-proof
3
extensive and included not only the use of anabolic steroids but also the use of rHuEpo and its derivatives, in combination with autotransfusion. The true incidence of blood doping, which athletes go to great lengths to conceal, is unknown. One study estimates the prevalence of blood doping in endurance athletes to be between 15%-22% based on results of testing and direct inquiry.2 In addition to potential adverse health consequences of doping, doping also carries the risk of an
of
athlete being suspended or banned from competition. When considering the
ro
seriousness of these consequences, it is reasonable to pose the question: does blood
-p
doping actually work to improve sporting performance?
re
As governing bodies have instituted new rules and new means of detecting the use of performance-enhancing drugs, athletes and their trainers have devised ever-
lP
more sophisticated means of blood doping and strategies to avoid detection.
na
This paper will review blood doping techniques, blood doping detection methods,
Physiology
ur
and will review the data regarding the effects blood doping has on performance.
Jo
Hemoglobin plays a central role in transporting oxygen throughout the body by increasing the oxygen carrying-capacity of the blood by approximately 70-fold.3 Oxygen binds to hemoglobin under conditions of high oxygen concentration in the lungs and delivers oxygen to tissues where oxygen concentration is low. Oxygen unloading is promoted by decreased pH, which allows for increased oxygen delivery to metabolically active tissues. Alterations in the oxygen affinity of hemoglobin can have negative ramifications for aerobic performance if the affinity for oxygen by hemoglobin is increased, which results in decreased oxygen delivery to active tissues and is therefore
Journal Pre-proof
4
not benefit the endurance athlete. Free plasma hemoglobin has a host of toxic effects to the brain and kidney. Red blood cells, containing hemoglobin, are there essential for the safe delivery of oxygen to metabolically active cells. Consequently, an increase in red blood cell mass may be of benefit to the endurance athlete, for whom prolonged aerobic exercise requires efficient delivery of oxygen to muscle tissues.
of
Hypoxia is responsible for increased levels of erythropoietin production by the
ro
renal Epo-producing cells of the kidney.4 In the athlete, periods of intense exercise
-p
decrease renal blood flow via splanchnic vasoconstriction,5 which may also be
re
responsible for increased Epo production and secretion. Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors that are
lP
responsible for inducing expression of the Epo gene under hypoxic conditions. In the
na
absence of hypoxia, the HIF-alpha subunit is ubiquitinated by the von Hippel-Lindau/E3 ligase complex and subsequently rapidly degraded in the proteasome of Epo-producing
ur
cells.6 However, when oxygen tension is low, this degradation ceases. The stable HIF
Jo
dimer is then able to enter the nucleus, where it binds to promoter regions on DNA and results in increased transcription and translation of Epo.7 Increased circulating erythropoietin from HIF expression results in increased binding of Epo to its receptor EPOR. EPOR is highly expressed on hematopoietic stem cells in the bone marrow. The Epo-EPOR interaction leads to intracellular signaling along the JAK2 and STAT5 pathways, to induce stem cell differentiation along the erythroid lineage. Early erythropoiesis occurs within the bone marrow. Reticulocytes are then released into the circulating blood where they shortly become mature erythrocytes.
Journal Pre-proof
5
The hematocrit has been observed to decrease with vigorous exercise, but the phenomenon of "sports anemia" is a misnomer. To the contrary, athletes experience an increase in erythropoiesis as a result of vigorous exercise. However, rapid expansion of the plasma volume also follows heavy training.8 This expansion of plasma volume and resultant hemodilution is responsible for the laboratory appearance of a decreased hematocrit and hemoglobin concentration. Thus, the total red cell mass is a more
of
accurate indicator of total body hemoglobin in athletes.9
ro
Early History of Blood Doping
-p
The study of altitude physiology and the story of blood doping are closely
re
connected. The various effects of high altitude on human physiology have been described since antiquity.10 However, the first detailed account of "mountain sickness"
lP
due to low inhaled partial pressure of oxygen is commonly accepted to have been first
na
described in 1590, when the Spanish missionary Joseph de Acosta described the symptoms of shortness of breath, chest pain, cough, and vomiting that he experienced
ur
while crossing the Andes of Peru.11
Jo
Almost three hundred years later, the French physician Denis Jourdanet, who traveled for nearly two decades in the mountainous regions of Mexico, hypothesized that it was the hypoxemia of high elevation (as opposed to low barometric pressure) that was responsible for an increased blood viscosity of subjects living at altitude.12 These observations were then advanced by his countryman, Francois-Gilbert Viault, who correlated the increased viscosity to increased hematocrit by measuring an increase in his own red cell count after living for 23 days at altitude. Viault concluded: “So it seems that one of the first effects of a period spent by man in the high mountains consists of an
Journal Pre-proof
6
increase in the normal function of hemapoiesis.”13 This astute observation, published in 1890, presciently anticipated the widespread adoption of high-altitude training roughly three-quarters of a century before it became clear that such practices conferred benefits to athletes, as demonstrated at the 1968 Mexico City Olympics, where athletes, from high-altitude areas won most of the endurance races.14 In the early 20th century, the widespread practice of canine experimentation led
of
to a vast expansion in our understanding of exercise physiology. A 1927 article by
ro
Barcroft and Stevens reported a series of experiments demonstrating that dogs
-p
increased their hematocrit by contracting their spleens prior to exercise, in effect
re
autotransfusing in preparation for increased aerobic demand.15 Natural experiments like this outlined a theoretical framework for the practice of autotransfusion that would come
lP
into practice among elite athletes several decades later. Humans, however, incapable of
na
such convenient means of autotransfusion, would initially rely upon allogeneic blood transfusions to gain a competitive advantage.
ur
In 1945, Hurtado et al. demonstrated that the hypoxia of altitude (rather than
Jo
decreased barometric pressure) triggered increased hematopoiesis, supporting the notion that increased red cell mass could improve oxygen delivery.16 This idea was given practical application when Pace proved that allogenic blood transfusion increased exercise tolerance under conditions of hypoxia.17 Nearly 50 years after Barcroft and Stevens described canine splenic autotransfusion, the practice of autologous red cell transfusion to improve aerobic exercise efficiency in humans was demonstrated in the scientific literature. In August 1972, Ekblom et al. reported a series of experiments in which healthy subjects were
Journal Pre-proof
7
phlebotomized and then later reinfused with their own blood prior to controlled exercise experiments. These results demonstrated that, all other things being (roughly) equal, increasing hemoglobin concentration does improve measures of aerobic capacity.18 It was not until 1981 that the Finnish athlete Mikko Juhani Ala-Leppilampi was the first athlete to go on record admitting to having received a transfusion prior to competing in the 3000-meter steeplechase at the 1972 Munich Olympics. Although unplaced in his
of
event,19 Ala-Leppilampi’s more renowned teammate Lasse Virén won two gold medals
ro
and set two world records at the same Games, and has been long-rumored to have also
-p
benefited from pre-event transfusions at those same Games.
re
Blood doping reached mainstream attention in 1984 after it was revealed that seven members of the American men's cycling team, four of whom had won medals,
lP
had been transfused before competing in that year’s Olympics. This resulted in public
na
outcry, including an editorial in the New England Journal of Medicine that unequivocally decried the practice.20 Public and medical opinion notwithstanding, the practice of
ur
seeking a competitive edge at the most elite levels of sport continued and expanded
Transfusion
Jo
with the FDA approval of recombinant human erythropoietin (rHuEpo) in 1989.
Allotransfusion was the earliest reported method of blood doping. While relatively safe when done under appropriate conditions, allotransfusion is not without risk, and can cause volume overload or transfusion reactions in the setting of antigen mismatch between donor and recipient. Because of antigenic differences between donor and host, attempts at blood doping with homologous erythrocytes are relatively easy to detect via flow cytometric analysis, which can easily identify very low levels of homologous
Journal Pre-proof
8
antigens. As such, this practice is uncommon, with a recent article reporting that few athletes have tested positive for homologous blood.21 By contrast, autologous transfusion is both rampantly widespread and far more difficult to detect. And while considerably safer than allotransfusion when carried out in medical clinics, there have been reports of severe morbidity associated with autotransfusion in sport. In 2011, the Italian cyclist Riccardo Ricco fell critically ill
of
following a transfusion of autologous blood he had kept in his own refrigerator. This
ro
highlights the covert and unsafe practices that accompany blood doping and illustrate
-p
how the necessity to keep the practice covert leads to significant safety concerns.
re
No single test is uniformly available to directly identify the presence of autologously transfused blood, however several biomarkers are under investigation for
lP
this purpose. Plasticizers involved in manufacture of the bags and tubes used in the
na
blood donation and storage process can be detected in the urine after re-transfusion, and can suggest recent doping.22 Additional emerging detection methods include
ur
analysis of circulating micro-RNA,23 changes in hepcidin (aka "ironomics"),24 changes in
Jo
transcriptomic biomarkers,25 and the analysis of RBC-microparticles.26 While promising, these methods remain too premature for routine use, but if nothing else the number of analytical methods under development for detection of autologous transfusion highlights an area of critical need in the crusade against doping. rHuEpo and ESAs In 1985, the first erythropoietin stimulating agent (ESA) was synthesized, changing the world of blood doping from that point forward. Erythropoietin alfa (Epogen, Procrit) was FDA-approved for the treatment of anemia associated with kidney disease
Journal Pre-proof
9
in 1989. It was subsequently banned by the International Olympic Committee the following year.27 Despite the ban, it took a decade to develop a method for the detection of rHuEpo in athletes.28 Erythropoietin (Epo) is a glycoprotein and its glycosylation pattern varies depending on the cells in which it is cultured.29 Recombinant human erythropoietin (rHuEpo) was initially transfected and cloned using Chinese hamster ovary cells, and subsequently produced in baby hamster kidney cells. 30 By exploiting
of
the differences in glycosylation patterns between endogenous human Epo and rHuEpo,
ro
a method of separation based on charge using isoelectric focusing was developed. This
-p
assay can detect nanogram amounts of rHuEpo in the urine.31 Further refinements have
erythropoietin stimulating agents.32
re
been made which allow for detection of newer-generations of rHuEpo as well as other
lP
The first-generation rHuEpo agents have a short half-life of 8-24hrs, requiring
na
frequent dosing schedules. Darbepoetin alfa (Aranesp®) is a next-generation erythropoiesis-stimulating protein which was FDA-approved in 2011. Darbepoetin-alfa is
ur
a modified version of Epo with significantly increased molecular mass. This increased
Jo
mass allows for a three-fold increase in half-life, as well as increased potency. Due to its increased half-life, darbepoetin alfa also has a longer detection window, making it less appealing as an agent for use in blood doping. Nonetheless, three athletes tested positive for the drug at the 2002 Salt Lake City Winter Olympics; the drug's creator Amgen aided in the development of detection methods for the drug during those Games.33 A PEGylated rHuEpo with an even longer half-life of approximately 130 hours (Mircera®) was FDA-approved in 2007.34 Since it has the longest window of detection of
Journal Pre-proof
10
all the ESAs, it is also considered to have a low risk of abuse in blood doping, and the drug's developer provided an ELISA for detection of the drug that was provided to WADA before the drug was even released.27 Despite the assumption of the drug’s lowrisk status, in 2008 multiple athletes tested positive for it nonetheless, including Bernard Kohl, the third overall finisher of that year’s Tour de France. The use of ESAs is associated with multiple risks. Elevated hematocrit from ESA
of
use results in increased blood viscosity, which increases the risk of thromboembolic
ro
events such as stroke and MI.35 ESA use also reduces endogenous Epo production via
-p
negative feedback, and it has been reported that this negative feedback, and resultant
re
anemia, may persist long after ESA withdrawal. Due to antibody formation to ESA with potential cross-reactivity to endogenous Epo, although rare, ESA abuse also has the
lP
ominous potential to induce pure red cell aplasia.36 Finally, it has long been recognized
na
that ESAs are associated with elevations in blood pressure. A recent systematic review by Sgrò, et al. evaluated a series of clinical trials
ur
dating from 1991 to 2016 investigating the effects of Epo on exercise performance.
Jo
They concluded that erythropoietin used in clinical dosages can lead to relative improvements of maximal aerobic power by 6%-8%,37 with positive effects on submaximal exercise performance as well. There seems little question at this point that Epo, by means of raising the red cell volume, improves aerobic exercise capacity. But whether this improved aerobic capacity translates to improved sporting performance remains a contentious topic. A well-designed and executed, albeit controversial research article published in 2017, suggests not. Heuberger et al. carried out a double-blind, randomized placebo-controlled trial of well-trained amateur cyclists
Journal Pre-proof
11
to evaluate the effects of rHuEpo on maximal, submaximal, and race performance. 38 Fourty-eight participants were randomized equally to receive either rHuEpo or placebo over the course of eight weeks. Tests of maximal and submaximal exercise were obtained at baseline and periodically thereafter. Finally, at the end of the trial, all the participants competed in a race to the summit of Mont Ventoux in France. Not surprisingly, the group receiving rHuEpo had significantly larger increases in
of
their hemoglobin concentrations over the course of the study compared to the placebo
ro
arm. This study also showed significant increases in multiple variables associated with
-p
maximal exercise performance in the treatment arm. However, there were no significant
re
differences between the treatment group and the placebo group with measures of submaximal exercise performance. Most notably, there were no differences between
lP
the two groups in the average race times. It is also worth noting that participants were
study.
na
unable to correctly guess if they had received rHuEpo or placebo in either arm of the
ur
Although limited by a relatively small study population, the Heuberger study
Jo
supports decades of literature that have demonstrated the correlation between hemoglobin concentration and maximal exercise performance. However, the study raises questions about the effects of Epo on submaximal exercise capacity and its benefits to competitive performance. Unlike other studies with less stringent design, this paper did not demonstrate a difference between the two groups in submaximal exercise performance.39-41 Part of these differences between studies may be due to differences in the methodology of how "submaximal" exercise was conducted. Heuberger et al conducted submaximal exercise tests of 80% of maximal power lasting 45 minutes for
Journal Pre-proof
12
their study, in order to more closely mimic the conditions of a race; other studies carried out shorter tests lasting from 3-30 minutes, arguably more closely mimicking tests of exhaustion than endurance (according to Heuberger). Professional athletes could not participate in the Heuberger study for ethical reasons and so the study did not demonstrate to what effect blood doping modulates the performance of the most elite athletes. However, this study is perhaps the most comprehensive study to assess the
of
effects of rHuEpo on actual sporting performance, and the evidence presented suggests
ro
the effect is not significant.
-p
HIF stabilizers
re
Hypoxia-inducible factor-1 (HIF-1) is a dimeric molecule that is expressed in numerous tissues of the body. It is composed of an alpha subunit and a beta subunit.
lP
Within kidney cells, the alpha-subunit is rapidly degraded under conditions of normoxia,
na
but under conditions of hypoxia, the dimer is stabilized and acts as a transcription factor to facilitate the production of erythropoietin. There exist several small-molecule agents
ur
which act as HIF stabilizers. By stabilizing HIF-1-alpha, renal cells are
Jo
pharmacologically prompted to increase Epo production, resulting in increased hematocrit. Although none of the HIF stabilizers have FDA approval, some are in clinical trials. WADA added HIF stabilizers to the list of banned substances in 2011, suggesting that they have been available on the black market for some time. The first athlete to test positive for a HIF stabilizer was reported in 2016.42 Early clinical trial outcomes suggest these drugs may be safe for treatment of anemia in chronic kidney disease (CKD), however the study populations have generally not included healthy subjects without anemia. As such, the risks of HIF-stabilizers in the
Journal Pre-proof
13
athlete is largely unknown. It would seem reasonable to conclude that any means of increasing the Epo level would share similar risks in the healthy, non-anemic patient, namely increased blood viscocity, hypertension, and cardiovascular adverse events. However the early trials in CKD appear to suggest that these risks are actually lower compared to exogenous Epo administration.43 Furthermore, the role of HIF-1 in tumorigenesis has been recognized since at
of
least 1999,44 and there are theoretical concerns that increased activation of HIF via HIF
ro
stabilizers could potentially increase the incidence of various cancers.45-46
-p
Cobalt acts as an elemental HIF-alpha stabilizer and acts to prevents
re
ubiquitination of the HIF-alpha subunit.47 Cobalt has been used in the distant past for the purposes of stimulating erythropoiesis, however it is associated with numerous side
lP
effects including severe organ damage such as cardiotoxicity, which limit its use as a
na
blood doping agent.48
Xenon gas also increases the production of HIF-1a49 with downstream effect of
ur
increasing Epo levels.50 Xenon was added to the WADA list of banned substances in
Jo
2014. There have been widely-circulated claims that members of the Russian Winter Olympics team used xenon at the Sochi Olympics in 2014.51 Synthetic Oxygen Carriers Free hemoglobin not encapsulated by the red blood cell (RBC) membrane is unstable and has a host of toxic effects, from vasoconstriction resulting from scavenging of free nitric oxide, to acute tubular necrosis of the kidneys leading to renal failure. Hemoglobin-based Oxygen Carriers (HBOCs) are a class of therapeutic agents which utilize modified forms of hemoglobin to circumvent these toxicities with the aim of
Journal Pre-proof
14
improving oxygen delivery in the anemic patient. There are no currently FDA-approved HBOCs. Hemopure™ is a bovine-derived, crosslinked acellular form of hemoglobin. Trials designed to evaluate the use of Hemopure™ to augment athletic performance have failed to demonstrate a significant benefit,52 concluding that the adverse cardiovascular effects of hypertension potentially negate the benefits of improved oxygen delivery to
of
tissues.53
ro
In addition to HBOCs, perfluorocarbon emulsions (PFCs), which are halogen-
-p
substituted carbon nonpolar oils, can carry and deliver oxygen. PFCs require a large
re
concentration gradient to drive oxygen dissolution, which suggests that PFCs are only of benefit in the presence of supplemental oxygen. This would seem to limit the utility of
lP
PFCs in the realm of endurance sports.54
na
Both HBOCs and perfluorocarbon-based compounds have been banned by WADA, and tests for their detection have been available since 2004.55
ur
The Athlete Biological Passport
Jo
At present, the only officially sanctioned method for detection of transfusion is the Athlete Biological Passport (ABP). The ABP is a program developed by WADA to detect doping by means of measuring changes in an athlete's unique biometric values over time, rather than from the detection of any particular substance itself. The program, implemented in 2009, contains multiple modules intended to detect doping in its numerous forms. The operating guidelines are regularly updated and are currently in the sixth version.56 Currently WADA can sanction athletes based on suspicious changes in the ABP alone.
Journal Pre-proof
15
The blood module of the ABP (Blood ABP) is built on years of work aimed at correlating changes in detectable serum markers of erythropoiesis and iron homeostasis to the administration of rHuEpo and other blood doping techniques. Early work around the turn of the new millennium demonstrated significant and predictable changes in hematocrit, reticulocyte%, erythropoietin, and serum soluble transferrin receptor levels that resulted from rHuEpo use.57-58
of
For example, recent Epo use is expected to result in an increase in hematocrit
ro
and reticulocyte count with an early spike in EPO levels (from exogenous
-p
administration), which is then expected to fall below the normal baseline in response to
re
the increased RBC mass. Changes from an athlete’s baseline for these parameters can raise the suspicion of blood doping even in absence of any detectable contraband
lP
substance. However, small changes in these values may be (mis)attributed to other
remains uncertain.27
na
causes, and the sensitivity and specificity of such changes in detecting autotransfusion
ur
Limitations of the early system led to refinements that both simplified detection
Jo
and improved sensitivity of the algorithm, leading to the development of the so-called OFF-score (OFF-S),59 which has proven valuable for its ability to detect the effects of rHuEpo beyond the window of detection of the actual doping substance itself. The OFFS can be considered an “index of stimulation” that suggests recent Epo use even in absence of significantly elevated Epo levels, and is calculated by the formula: OFF-S = Hb (g/L) - 60√(Reticulocyte%), with a normal range that falls between 85-95.60 Further work in this area led to the development of the Athlete Abnormal Blood Profile Score (ABPS), which is a multi-parametric test similarly designed to predict
Journal Pre-proof
16
rHuEpo administration as well as blood transfusion.61 The ABPS and the OFF-score compose the framework for the blood module of the ABP.62 By following a strict protocol for blood collection, transportation, storage, and analysis, a set of eight values available on routine CBC measurements such as hemoglobin, hematocrit, MCV, RDW, and reticulocyte indices, the ABP is able to track changes in an athlete's hematologic parameters over time. Bayesian Network analysis
of
is then applied to detect abnormal values which may require additional testing or follow-
ro
up.63
-p
The Blood ABP represents the most powerful tool available at present to combat
re
doping. In 2017, the most recent year for which records have been made available, over 29,000 ABP Blood Module samples were analyzed, marking an increase of 3% over the
lP
previous year.64
na
Discussion
The Heuberger study suggests that Epo administration alone does not
ur
significantly improve submaximal sporting performance in well-trained athletes, even
Jo
though it does improve some measures of exercise capacity. It might be easy to imagine that an argument could be made to use this study to decriminalize the use of rHuEpo. However, in any randomized controlled trial, the clinical endpoints stand as the most clinically relevant factors—in other words, a study is only as useful as its endpoints. In practical terms, the benefit of illicit Epo abuse would best be shown by its ability to lead to victory in sporting performance. Other less important endpoints such as physiologic variables are less clinically relevant. What good is improving oxygen delivery if it doesn’t help you win? Additionally, both the medical (i.e. thrombosis) risks
Journal Pre-proof
17
and professional (i.e. expulsion from competition) risks, being as high as they are, cast doubt on the use of Epo to improve athletic performance, even if aerobic exercise is marginally improved. In other words: the proven benefit of Epo abuse—a 6%-8% improvement in maximal aerobic capacity—simply is not enough to justify its multitudinous drawbacks. But if Epo doesn’t improve sporting performance, why is its abuse in endurance
of
sports so widespread? Epo abuse is only one ingredient in the recipe for blood doping.
ro
And the most important ingredient is the athlete themself. Due to ethical considerations,
-p
elite athletes have not been included in trials related to Epo abuse. And there have
re
been no clinical trials that evaluate the rather elaborate blood doping regimens that are routinely employed by elite athletes. It is not unlikely that Epo, when used by an elite
lP
athlete in combination with additional doping methods, can confer an actual
na
performance benefit, and that scant difference in performance between top elite athletes hinges upon the slightest of advantages. Even if that advantage is largely, or even
ur
entirely based on the placebo effect, if the athlete believes it can help them perform, the
Jo
practice will persist and it will be difficult to eliminate blood doping from sport. It seems unlikely that any clinical trial will ever—or could ever—be designed to test this hypothesis. While the practice of blood doping is sure to continue long into the future, the utility and efficiency of the practice has only relatively recently begun to gain rigorous scientific and clinical investigation. Cynically, it may be logical to conclude that as long as competitive sports exist, athletes will seek to gain an advantage; unscrupulous athletes will continue to go to illicit measures to gain that advantage; and the crusade to
Journal Pre-proof
18
ensure a level playing field will continue. Athletes and their trainers are frequently ahead of the curve when it comes to exploiting the science of performance enhancement and evading detection, and this trend seems unlikely to change. Future Considerations The science behind athletic performance enhancement remains in its infancy. Despite this fact, elite athletes will undoubtedly continue to seek to gain a competitive
of
edge over their rivals, even if the methods employed are not the subject of rigorous
ro
scientific scrutiny. As novel methods of blood doping emerge, novel methods of
-p
detection will be necessary to combat them. Better understanding the limits of human
re
athletic performance could lead to better understanding of the normal aging process and could assist with injury and recovery.
na
ur
Erythropoietin is capable of increasing the red blood cell volume and thus the aerobic capacity of athletes, however this increase comes with potential drawbacks such as cardiovascular and thromboembolic complications (e.g. hypertension, heart attack, and stroke). HIF stabilizers have the potential to increase Epo levels and hematocrit. These agents are not FDA approved and their safety in elite athletes is largely unknown.
Jo
lP
Practice Points:
Research Agenda:
Analytical detection methods for autologous blood transfusion remain an unmet need in the battle against blood doping.
Conflict of Interest Neither of the authors have any financial or other conflicts of interest to report. The work is original and has not been published elsewhere. Both authors have contributed substantially to the work. Funding Source
Journal Pre-proof
19
None. References
Jo
ur
na
lP
re
-p
ro
of
1. WADA: World Anti-Doping Agency [Internet]. Montreal: WADA; c2019. Manipulation of blood and blood products; 2019 [cited 2019 Feb 18]; [about 1 screen] Available from: https://www.wada-ama.org/en/content/what-isprohibited/prohibited-at-all-times/manipulation-of-blood-and-blood-components 2. Sottas PE, Robinson N, Fischetto G, Dollé G, Alonso JM, Saugy M. Prevalence of blood doping in samples collected from elite track and field athletes. Clin Chem. 2011;57(5):762-9. 3. Mairbäurl H. Red blood cells in sports: effects of exercise and training on oxygen supply by red blood cells. Front Physiol. 2013;4. 4. Souma T, Suzuki N, Yamamoto M. Renal erythropoietin-producing cells in health and disease. Front Physiol. 2015;6. 5. McAllister RM, Adaptations in control of blood flow with training: splanchnic and renal blood flows. Med Sci Sports Exerc. 1998;30:375-81. 6. Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ, et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science. 2001;292:468-472. 7. Kallio PJ, Okamoto K, O'Brien S, Carrero P, Makino Y, Tanaka H, et al. Signal transduction in hypoxic cells: inducible nuclear translocation and recruitment of the CBP/p300 coactivator by the hypoxia-inducible factor-1alpha. EMBO J. 1998;17(22):6573-86. 8. Green HJ, Sutton JR, Coates G, Ali M, Jones S. Response of red cell and plasma volume to prolonged training in humans. J Appl Physiol (1985). 1991;70(4):1810-5. 9. Otto JM, Plumb JOM, Clissold E, Kumar SB, Wakeham DJ, Schmidt W, et al. Hemoglobin concentration, total hemoglobin mass and plasma volume in patients: implications for anemia. Haematologica. 2017;102(9):1477-85. 10. Ward, Michael, James S. Milledge, and John B. West, editors. High Altitude Medicine and Physiology. 3rd ed. London: Arnold, 2000. 448p. 11. Gilbert DL. The first documented description of mountain sickness: the Andean or Pariacaca story. Respir Physiol. 1983;52(3):327-347. 12. West JB, Richalet JP. Denis Jourdanet (1815-1892) and the early recognition of the role of hypoxia at high altitude. Am J Physiol Lung Cell Mol Physiol. 2013;305(5):L333-340. 13. West JB. High altitude physiology. Stroudsburg, Pa. New York: Hutchinson Ross; Distributed by Academic Press; 1981. xvi, 461 p.p. 14. Shaskey DJ, Green GA, Sports haematology. Sports Med. 2000;29:27-38. 15. Barcroft J, Stephens JG. Observations upon the size of the spleen. J Physiol 1927;64:1-22. 16. Hurtado A, Merino C, Delgado E. Influence of Anoxemia on the Hemopoietic Activity. Arch Int Med 1945;75:284-323.
Journal Pre-proof
20
Jo
ur
na
lP
re
-p
ro
of
17. Pace N, Lozner EL, Consolazio WV, Pitts CP, Pecora LJ. The increase in hypoxia tolerance of normal men accompanying the polycythemia induced by transfusion of erythrocytes. Am J Physiol 1947;148:152-163. 18. Ekblom B, Goldbarg AN, Gullbring B. Response to exercise after blood loss and reinfusion. J Appl Physiol 1972;33:175-180. 19. Fitch KD. Blood doping at the Olympic Games. J Sports Med Phys Fitness 2017;57:1526-1532. 20. Klein HG. Blood transfusion and athletics. Games people play. N Engl J Med 1985;312:854-856. 21. Mørkeberg J. Blood manipulation: current challenges from an anti-doping perspective. Hematology Am Soc Hematol Educ Program 2013;2013:627-631. 22. Leuenberger N, Barras L, Nicoli R, Robinson N, Baume N, Lion N., et al. Urinary di-(2-ethylhexyl) phthalate metabolites for detecting transfusion of autologous blood stored in plasticizer-free bags. Transfusion 2016;56:571-578. 23. Leuenberger N, Schumacher YO, Pradervand S, Sander T, Saugy M, Pottgiesser T. Circulating microRNAs as biomarkers for detection of autologous blood transfusion. PLoS One 2013;8:e66309. 24. Leuenberger N, Barras L, Nicoli R, Robinson N, Baume N, Lion N, et al. Hepcidin as a new biomarker for detecting autologous blood transfusion. Am J Hematol 2016;91:467-472. 25. Salamin O, Mignot J, Kuuranne T, Saugy M, Leuenberger N. Transcriptomic biomarkers of altered erythropoiesis to detect autologous blood transfusion. Drug Test Anal 2017;10:604-608.. 26. Voss SC, Jaganjac M, Al-Thani AM, Grivel JC, Raynaud CM Al-Jaber H, et al. Analysis of RBC-microparticles in stored whole blood bags - a promising marker to detect blood doping in sports? Drug Test Anal 2017;9:1794-1798. 27. Salamin O, Kuuranne T, Saugy M, Leuenberger N. Erythropoietin as a performance-enhancing drug: Its mechanistic basis, detection, and potential adverse effects. Mol Cell Endocrinol 2017;464:75-87. 28. Lasne F, de Ceaurriz J. Recombinant erythropoietin in urine. Nature 2000;405:635. 29. Skibeli V, Nissen-Lie G, Torjesen P. Sugar profiling proves that human serum erythropoietin differs from recombinant human erythropoietin. Blood 2001;98:3626-3634. 30. Lin FK, Suggs S, Lin CH, Browne JK, Smalling R, Egrie JC, et al. Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci U S A 1985;82:7580-7584. 31. Lasne F, Martin L, Crepin N, de Ceaurriz J. Detection of isoelectric profiles of erythropoietin in urine: differentiation of natural and administered recombinant hormones. Anal Biochem 2002;311:119-126. 32. Leuenberger N, Reichel C, Lasne F. Detection of erythropoiesis-stimulating agents in human anti-doping control: past, present and future. Bioanalysis 2012;4:1565-1575.
Journal Pre-proof
21
Jo
ur
na
lP
re
-p
ro
of
33. Catlin DH, Breidbach A, Elliott S, Glaspy J. Comparison of the isoelectric focusing patterns of darbepoetin alfa, recombinant human erythropoietin, and endogenous erythropoietin from human urine. Clin Chem 2002;48:2057-2059. 34. Curran MP, McCormack PL. Methoxy polyethylene glycol-epoetin beta: a review of its use in the management of anaemia associated with chronic kidney disease. Drugs 2008;68:1139-1156. 35. Pope HG, Wood RI, Rogol A, Nyberg F, Bowers L, Bhasin S. Adverse health consequences of performance-enhancing drugs: an Endocrine Society scientific statement. Endocr Rev 2014;35:341-375 36. Casadevall N, Eckardt KU, Rossert J. Epoetin-induced autoimmune pure red cell aplasia. J Am Soc Nephrol 2005;16 Suppl 1:S67-69. 37. Sgrò P, Sansone M, Sansone A, Romanelli F, Di Luigi L. Effects of erythropoietin abuse on exercise performance. Phys Sportsmed. 2018;46(1):105-115. 38. Heuberger J, Rotmans JI, Gal P, Stuurman FE, van 't Westende J, Post TE, et al. Effects of erythropoietin on cycling performance of well trained cyclists: a doubleblind, randomised, placebo-controlled trial. Lancet Haematol. 2017;4(8):e374e86. 39. Annaheim S, Jacob M, Krafft A, Breymann C, Rehm M, Boutellier U. RhEPO improves time to exhaustion by non-hematopoietic factors in humans. Eur J Appl Physiol 2016;116:623-633. 40. Wilkerson DP, Rittweger J, Berger NJ, Naish PF, Jones AM. Influence of recombinant human erythropoietin treatment on pulmonary O2 uptake kinetics during exercise in humans. J Physiol 2005;568:639-652. 41. Thomsen JJ, Rentsch RL, Robach P, Calbet JAL, Boushel R, Rasmussen 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. 42. Buisson C, Marchand A, Bailloux I, et al. Detection by LC-MS/MS of HIF stabilizer FG-4592 used as a new doping agent: Investigation on a positive case. J Pharm Biomed Anal 2016;121:181-187. 43. Hasegawa, S, Tanaka, T, Nangaku, M. Hypoxia-inducible factor stabilizers for treating anemia of chronic kidney disease. Curr Opin Nephrol Hypertens. 2018;27:331-338. 44. Soni S, Padwad YS. HIF-1 in cancer therapy: two decade long story of a transcription factor. Acta Oncol 2017;56:503-515. 45. Pergola PE, Spinowitz BS, Hartman CS, Maroni BJ, Haase VH. Vadadustat, a novel oral HIF stabilizer, provides effective anemia treatment in nondialysisdependent chronic kidney disease. Kidney International 2016;90:1115-1122. 46. Keith B, Johnson RS, Simon MC. HIF1alpha and HIF2alpha: sibling rivalry in hypoxic tumour growth and progression. Nat Rev Cancer 2011;12:9-22. 47. Yuan Y, Hilliard G, Ferguson T, Millhorn DE. Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von Hippel-Lindau protein by direct binding to hypoxia-inducible factor-alpha. J Biol Chem 2003;278:15911-15916.
Journal Pre-proof
22
Jo
ur
na
lP
re
-p
ro
of
48. Ebert B, Jelkmann W. Intolerability of cobalt salt as erythropoietic agent. Drug Test Anal 2014;6:185-189 49. Ma D, Lim T, Xu J, Tang H, Wan Y, Zhao H, et al. Xenon preconditioning protects against renal ischemic-reperfusion injury via HIF-1alpha activation. J Am Soc Nephrol 2009;20:713-720 50. Stoppe C, Ney J, Brenke M, Goetzenich A, Emontzphol C, Schälte G, et al. Subanesthetic Xenon Increases Erythropoietin Levels in Humans: A Randomized Controlled Trial. Sports Med 2016;46:1753-1766. 51. Bennetts M. Russian athletes admit taking banned substances supplied by Rodchenkov. The Guardian. Nov 9, 2017. 52. Hughes GS, Yancey EP, Albrecht R, Locker PK, Francom SF, Orringer EP, et al. Hemoglobin-based oxygen carrier preserves submaximal exercise capacity in humans. Clin Pharmacol Ther 1995;58:434-443. 53. Ashenden MJ, Schumacher YO, Sharpe K, Varlet-Marie E, Audran M. Effects of Hemopure on maximal oxygen uptake and endurance performance in healthy humans. Int J Sports Med 2007;28:381-385 54. Borrione P, Mastrone A, Salvo RA, Spaccamiglio A, Grasso L, Angeli A. Oxygen delivery enhancers: past, present, and future. J Endocrinol Invest 2008;31:185192. 55. WADA: World Anti-Doping Agency. Q&A: Blood Doping; 2019. World Anti-Doping Agency website. https://www.wada-ama.org/en/questions-answers/blood-doping Updated 2019. Accessed Feb 18, 2019. 56. WADA: World Anti-Doping Agency. Athlete Biological Passport Operating Guidelines & Compilation of Required Elements Version 6.1; July 2018. World Anti-Doping Agency website. https://www.wadaama.org/sites/default/files/resources/files/guidelines_abp_v61_2018_jul_en.pdf. Accessed Feb 18, 2019. 57. Parisotto R, Gore CJ, Emslie KR, Ashenden MJ, Brugnara C, Howe C, et al. A novel method utilising markers of altered erythropoiesis for the detection of recombinant human erythropoietin abuse in athletes. Haematologica 2000;85:564-572. 58. Parisotto R, Wu M, Ashenden MJ, Emslie KR, Gore CJ, Howe C, et al. Detection of recombinant human erythropoietin abuse in athletes utilizing markers of altered erythropoiesis. Haematologica 2001;86:128-137. 59. Gore CJ, Parisotto R, Ashenden MJ, Stray-Gundersen J, Sharpe K, Hopkins W, et al. Second-generation blood tests to detect erythropoietin abuse by athletes. Haematologica 2003;88:333-344. 60. Jelkmann W, Lundby C. Blood doping and its detection. Blood. 2011;118:23952404. 61. Sottas PE, Robinson N, Giraud S, Taroni F, Kamber M, Mangin P, et al. Statistical Classification of Abnormal Blood Profiles in Athletes. Int J Biostat 2006;2. 62. Sottas PE, Robinson N, Rabin O, Saugy M. The athlete biological passport. Clin Chem 2011;57:969-976.
Journal Pre-proof
Jo
ur
na
lP
re
-p
ro
of
63. Sottas PE, Robinson N, Saugy M. The athlete's biological passport and indirect markers of blood doping. Handb Exp Pharmacol 2010:305-326. 64. WADA: World Anti-Doping Agency. 2017 Anti-Doping Testing Figures. World Anti-Doping Agency website. https://www.wadaama.org/sites/default/files/resources/files/2017_antidoping_testing_figures_en_0.pdf Accessed Feb 18, 2019.
23
Thomas S. Atkinson, Marc J. Kahn PII:
S0268-960X(19)30146-8
DOI:
https://doi.org/10.1016/j.blre.2019.100632
Reference:
YBLRE 100632
To appear in:
Blood Reviews
Please cite this article as: T.S. Atkinson and M.J. Kahn, Blood doping: Then and now. A narrative review of the history, science and efficacy of blood doping in elite sport, Blood Reviews(2019), https://doi.org/10.1016/j.blre.2019.100632
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
Journal Pre-proof
1
Blood Doping: Then and Now. A narrative review of the history, science and efficacy of blood doping in elite sport Thomas S. Atkinson1,2, MD; Marc J. Kahn1,* [email protected], MD, MBA, MACP, FRCP 1Tulane
of
University School of Medicine, Department of Medicine, Section of Hematology/Medical Oncology. New Orleans, LA 70112, USA 2Southeast Louisiana Veterans Health Care Systems, Department of Medicine, Section of Hematology/Medical Oncology. New Orleans, LA 70119, USA
T.S. ATKINSON. ORCID: 0000-0002-6699-7152 *Corresponding
ro
M.J. KAHN. ORCID: 0000-0002-9781-9316
-p
author at: 1430 Tulane Ave, #8010, Tulane University School of Medicine, New Orleans, LA 70112, USA.
re
Keywords: blood doping, erythropoietin, sports hematology, oxygen delivery.
lP
Abstract
As the line between excellent and exceptional athletic performance narrows and as the
na
financial remuneration for exceptional performance increases, unscrupulous athletes
ur
and their trainers will strive to enhance performance regardless of cost. One method of performance enhancement is by the augmentation of the oxygen-carrying capacity of
Jo
the blood through blood doping. We discuss the science behind erythropoiesis and means by which these processes can be exploited to the potential advantage of the athlete. These include pre-sport transfusion practices as well as supplemental recombinant human erythropoietin (rHuEpo) and the use of newer erythropoietic agents, many of which have not received FDA approval. Finally, we discuss the data behind the efficacy of blood doping in an attempt to discern whether or not the practice actually works to improve athletic and competitive performance. Introduction
Journal Pre-proof
2
As the pressure to improve athletic performance intensifies due to everincreasing financial remuneration and the allure of prestige, unscrupulous athletes will seek ways to beat the system. Blood doping has been intertwined with elite endurance athletics for roughly the past half-century. In recent memory, no single event stands out more than the 2018 Seoul Olympics where the entire Russian team was barred from participation due to systematic doping.
of
The term doping refers to a multitude of practices which seek to artificially
ro
improve athletic performance through augmentation of the oxygen-carrying capacity of
-p
the blood. Blood doping, broadly defined by the World Anti-Doping Agency (WADA) as “any form of intravascular manipulation of the blood or blood components by physical or
re
chemical means,1” includes such practices as transfusion, recombinant human
lP
erythropoietin (rHuEpo) administration, and administration of artificial stimulants of
na
erythropoiesis.
Aside from blood transfusion, the use of rHuEpo is perhaps the most notorious
ur
and widely-studied method of blood doping. Athletes also have at their disposal other,
Jo
more novel ways of blood doping: hypoxia inducible factor (HIF) stabilizers, and the use of artificial hemoglobin (i.e. “synthetic blood”) are other examples of means to increase oxygen carrying capacity and thus increase aerobic exercise capacity. Despite the intent of dopers to improve athletic performance, the true effects on athletic performance are unknown. In what is inarguably the most notorious case of blood doping, Lance Armstrong was stripped of his seven Tour de France titles in 2012 after exhaustive investigation showed that he had been blood doping since at least 1999. His methods of doping were
Journal Pre-proof
3
extensive and included not only the use of anabolic steroids but also the use of rHuEpo and its derivatives, in combination with autotransfusion. The true incidence of blood doping, which athletes go to great lengths to conceal, is unknown. One study estimates the prevalence of blood doping in endurance athletes to be between 15%-22% based on results of testing and direct inquiry.2 In addition to potential adverse health consequences of doping, doping also carries the risk of an
of
athlete being suspended or banned from competition. When considering the
ro
seriousness of these consequences, it is reasonable to pose the question: does blood
-p
doping actually work to improve sporting performance?
re
As governing bodies have instituted new rules and new means of detecting the use of performance-enhancing drugs, athletes and their trainers have devised ever-
lP
more sophisticated means of blood doping and strategies to avoid detection.
na
This paper will review blood doping techniques, blood doping detection methods,
Physiology
ur
and will review the data regarding the effects blood doping has on performance.
Jo
Hemoglobin plays a central role in transporting oxygen throughout the body by increasing the oxygen carrying-capacity of the blood by approximately 70-fold.3 Oxygen binds to hemoglobin under conditions of high oxygen concentration in the lungs and delivers oxygen to tissues where oxygen concentration is low. Oxygen unloading is promoted by decreased pH, which allows for increased oxygen delivery to metabolically active tissues. Alterations in the oxygen affinity of hemoglobin can have negative ramifications for aerobic performance if the affinity for oxygen by hemoglobin is increased, which results in decreased oxygen delivery to active tissues and is therefore
Journal Pre-proof
4
not benefit the endurance athlete. Free plasma hemoglobin has a host of toxic effects to the brain and kidney. Red blood cells, containing hemoglobin, are there essential for the safe delivery of oxygen to metabolically active cells. Consequently, an increase in red blood cell mass may be of benefit to the endurance athlete, for whom prolonged aerobic exercise requires efficient delivery of oxygen to muscle tissues.
of
Hypoxia is responsible for increased levels of erythropoietin production by the
ro
renal Epo-producing cells of the kidney.4 In the athlete, periods of intense exercise
-p
decrease renal blood flow via splanchnic vasoconstriction,5 which may also be
re
responsible for increased Epo production and secretion. Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors that are
lP
responsible for inducing expression of the Epo gene under hypoxic conditions. In the
na
absence of hypoxia, the HIF-alpha subunit is ubiquitinated by the von Hippel-Lindau/E3 ligase complex and subsequently rapidly degraded in the proteasome of Epo-producing
ur
cells.6 However, when oxygen tension is low, this degradation ceases. The stable HIF
Jo
dimer is then able to enter the nucleus, where it binds to promoter regions on DNA and results in increased transcription and translation of Epo.7 Increased circulating erythropoietin from HIF expression results in increased binding of Epo to its receptor EPOR. EPOR is highly expressed on hematopoietic stem cells in the bone marrow. The Epo-EPOR interaction leads to intracellular signaling along the JAK2 and STAT5 pathways, to induce stem cell differentiation along the erythroid lineage. Early erythropoiesis occurs within the bone marrow. Reticulocytes are then released into the circulating blood where they shortly become mature erythrocytes.
Journal Pre-proof
5
The hematocrit has been observed to decrease with vigorous exercise, but the phenomenon of "sports anemia" is a misnomer. To the contrary, athletes experience an increase in erythropoiesis as a result of vigorous exercise. However, rapid expansion of the plasma volume also follows heavy training.8 This expansion of plasma volume and resultant hemodilution is responsible for the laboratory appearance of a decreased hematocrit and hemoglobin concentration. Thus, the total red cell mass is a more
of
accurate indicator of total body hemoglobin in athletes.9
ro
Early History of Blood Doping
-p
The study of altitude physiology and the story of blood doping are closely
re
connected. The various effects of high altitude on human physiology have been described since antiquity.10 However, the first detailed account of "mountain sickness"
lP
due to low inhaled partial pressure of oxygen is commonly accepted to have been first
na
described in 1590, when the Spanish missionary Joseph de Acosta described the symptoms of shortness of breath, chest pain, cough, and vomiting that he experienced
ur
while crossing the Andes of Peru.11
Jo
Almost three hundred years later, the French physician Denis Jourdanet, who traveled for nearly two decades in the mountainous regions of Mexico, hypothesized that it was the hypoxemia of high elevation (as opposed to low barometric pressure) that was responsible for an increased blood viscosity of subjects living at altitude.12 These observations were then advanced by his countryman, Francois-Gilbert Viault, who correlated the increased viscosity to increased hematocrit by measuring an increase in his own red cell count after living for 23 days at altitude. Viault concluded: “So it seems that one of the first effects of a period spent by man in the high mountains consists of an
Journal Pre-proof
6
increase in the normal function of hemapoiesis.”13 This astute observation, published in 1890, presciently anticipated the widespread adoption of high-altitude training roughly three-quarters of a century before it became clear that such practices conferred benefits to athletes, as demonstrated at the 1968 Mexico City Olympics, where athletes, from high-altitude areas won most of the endurance races.14 In the early 20th century, the widespread practice of canine experimentation led
of
to a vast expansion in our understanding of exercise physiology. A 1927 article by
ro
Barcroft and Stevens reported a series of experiments demonstrating that dogs
-p
increased their hematocrit by contracting their spleens prior to exercise, in effect
re
autotransfusing in preparation for increased aerobic demand.15 Natural experiments like this outlined a theoretical framework for the practice of autotransfusion that would come
lP
into practice among elite athletes several decades later. Humans, however, incapable of
na
such convenient means of autotransfusion, would initially rely upon allogeneic blood transfusions to gain a competitive advantage.
ur
In 1945, Hurtado et al. demonstrated that the hypoxia of altitude (rather than
Jo
decreased barometric pressure) triggered increased hematopoiesis, supporting the notion that increased red cell mass could improve oxygen delivery.16 This idea was given practical application when Pace proved that allogenic blood transfusion increased exercise tolerance under conditions of hypoxia.17 Nearly 50 years after Barcroft and Stevens described canine splenic autotransfusion, the practice of autologous red cell transfusion to improve aerobic exercise efficiency in humans was demonstrated in the scientific literature. In August 1972, Ekblom et al. reported a series of experiments in which healthy subjects were
Journal Pre-proof
7
phlebotomized and then later reinfused with their own blood prior to controlled exercise experiments. These results demonstrated that, all other things being (roughly) equal, increasing hemoglobin concentration does improve measures of aerobic capacity.18 It was not until 1981 that the Finnish athlete Mikko Juhani Ala-Leppilampi was the first athlete to go on record admitting to having received a transfusion prior to competing in the 3000-meter steeplechase at the 1972 Munich Olympics. Although unplaced in his
of
event,19 Ala-Leppilampi’s more renowned teammate Lasse Virén won two gold medals
ro
and set two world records at the same Games, and has been long-rumored to have also
-p
benefited from pre-event transfusions at those same Games.
re
Blood doping reached mainstream attention in 1984 after it was revealed that seven members of the American men's cycling team, four of whom had won medals,
lP
had been transfused before competing in that year’s Olympics. This resulted in public
na
outcry, including an editorial in the New England Journal of Medicine that unequivocally decried the practice.20 Public and medical opinion notwithstanding, the practice of
ur
seeking a competitive edge at the most elite levels of sport continued and expanded
Transfusion
Jo
with the FDA approval of recombinant human erythropoietin (rHuEpo) in 1989.
Allotransfusion was the earliest reported method of blood doping. While relatively safe when done under appropriate conditions, allotransfusion is not without risk, and can cause volume overload or transfusion reactions in the setting of antigen mismatch between donor and recipient. Because of antigenic differences between donor and host, attempts at blood doping with homologous erythrocytes are relatively easy to detect via flow cytometric analysis, which can easily identify very low levels of homologous
Journal Pre-proof
8
antigens. As such, this practice is uncommon, with a recent article reporting that few athletes have tested positive for homologous blood.21 By contrast, autologous transfusion is both rampantly widespread and far more difficult to detect. And while considerably safer than allotransfusion when carried out in medical clinics, there have been reports of severe morbidity associated with autotransfusion in sport. In 2011, the Italian cyclist Riccardo Ricco fell critically ill
of
following a transfusion of autologous blood he had kept in his own refrigerator. This
ro
highlights the covert and unsafe practices that accompany blood doping and illustrate
-p
how the necessity to keep the practice covert leads to significant safety concerns.
re
No single test is uniformly available to directly identify the presence of autologously transfused blood, however several biomarkers are under investigation for
lP
this purpose. Plasticizers involved in manufacture of the bags and tubes used in the
na
blood donation and storage process can be detected in the urine after re-transfusion, and can suggest recent doping.22 Additional emerging detection methods include
ur
analysis of circulating micro-RNA,23 changes in hepcidin (aka "ironomics"),24 changes in
Jo
transcriptomic biomarkers,25 and the analysis of RBC-microparticles.26 While promising, these methods remain too premature for routine use, but if nothing else the number of analytical methods under development for detection of autologous transfusion highlights an area of critical need in the crusade against doping. rHuEpo and ESAs In 1985, the first erythropoietin stimulating agent (ESA) was synthesized, changing the world of blood doping from that point forward. Erythropoietin alfa (Epogen, Procrit) was FDA-approved for the treatment of anemia associated with kidney disease
Journal Pre-proof
9
in 1989. It was subsequently banned by the International Olympic Committee the following year.27 Despite the ban, it took a decade to develop a method for the detection of rHuEpo in athletes.28 Erythropoietin (Epo) is a glycoprotein and its glycosylation pattern varies depending on the cells in which it is cultured.29 Recombinant human erythropoietin (rHuEpo) was initially transfected and cloned using Chinese hamster ovary cells, and subsequently produced in baby hamster kidney cells. 30 By exploiting
of
the differences in glycosylation patterns between endogenous human Epo and rHuEpo,
ro
a method of separation based on charge using isoelectric focusing was developed. This
-p
assay can detect nanogram amounts of rHuEpo in the urine.31 Further refinements have
erythropoietin stimulating agents.32
re
been made which allow for detection of newer-generations of rHuEpo as well as other
lP
The first-generation rHuEpo agents have a short half-life of 8-24hrs, requiring
na
frequent dosing schedules. Darbepoetin alfa (Aranesp®) is a next-generation erythropoiesis-stimulating protein which was FDA-approved in 2011. Darbepoetin-alfa is
ur
a modified version of Epo with significantly increased molecular mass. This increased
Jo
mass allows for a three-fold increase in half-life, as well as increased potency. Due to its increased half-life, darbepoetin alfa also has a longer detection window, making it less appealing as an agent for use in blood doping. Nonetheless, three athletes tested positive for the drug at the 2002 Salt Lake City Winter Olympics; the drug's creator Amgen aided in the development of detection methods for the drug during those Games.33 A PEGylated rHuEpo with an even longer half-life of approximately 130 hours (Mircera®) was FDA-approved in 2007.34 Since it has the longest window of detection of
Journal Pre-proof
10
all the ESAs, it is also considered to have a low risk of abuse in blood doping, and the drug's developer provided an ELISA for detection of the drug that was provided to WADA before the drug was even released.27 Despite the assumption of the drug’s lowrisk status, in 2008 multiple athletes tested positive for it nonetheless, including Bernard Kohl, the third overall finisher of that year’s Tour de France. The use of ESAs is associated with multiple risks. Elevated hematocrit from ESA
of
use results in increased blood viscosity, which increases the risk of thromboembolic
ro
events such as stroke and MI.35 ESA use also reduces endogenous Epo production via
-p
negative feedback, and it has been reported that this negative feedback, and resultant
re
anemia, may persist long after ESA withdrawal. Due to antibody formation to ESA with potential cross-reactivity to endogenous Epo, although rare, ESA abuse also has the
lP
ominous potential to induce pure red cell aplasia.36 Finally, it has long been recognized
na
that ESAs are associated with elevations in blood pressure. A recent systematic review by Sgrò, et al. evaluated a series of clinical trials
ur
dating from 1991 to 2016 investigating the effects of Epo on exercise performance.
Jo
They concluded that erythropoietin used in clinical dosages can lead to relative improvements of maximal aerobic power by 6%-8%,37 with positive effects on submaximal exercise performance as well. There seems little question at this point that Epo, by means of raising the red cell volume, improves aerobic exercise capacity. But whether this improved aerobic capacity translates to improved sporting performance remains a contentious topic. A well-designed and executed, albeit controversial research article published in 2017, suggests not. Heuberger et al. carried out a double-blind, randomized placebo-controlled trial of well-trained amateur cyclists
Journal Pre-proof
11
to evaluate the effects of rHuEpo on maximal, submaximal, and race performance. 38 Fourty-eight participants were randomized equally to receive either rHuEpo or placebo over the course of eight weeks. Tests of maximal and submaximal exercise were obtained at baseline and periodically thereafter. Finally, at the end of the trial, all the participants competed in a race to the summit of Mont Ventoux in France. Not surprisingly, the group receiving rHuEpo had significantly larger increases in
of
their hemoglobin concentrations over the course of the study compared to the placebo
ro
arm. This study also showed significant increases in multiple variables associated with
-p
maximal exercise performance in the treatment arm. However, there were no significant
re
differences between the treatment group and the placebo group with measures of submaximal exercise performance. Most notably, there were no differences between
lP
the two groups in the average race times. It is also worth noting that participants were
study.
na
unable to correctly guess if they had received rHuEpo or placebo in either arm of the
ur
Although limited by a relatively small study population, the Heuberger study
Jo
supports decades of literature that have demonstrated the correlation between hemoglobin concentration and maximal exercise performance. However, the study raises questions about the effects of Epo on submaximal exercise capacity and its benefits to competitive performance. Unlike other studies with less stringent design, this paper did not demonstrate a difference between the two groups in submaximal exercise performance.39-41 Part of these differences between studies may be due to differences in the methodology of how "submaximal" exercise was conducted. Heuberger et al conducted submaximal exercise tests of 80% of maximal power lasting 45 minutes for
Journal Pre-proof
12
their study, in order to more closely mimic the conditions of a race; other studies carried out shorter tests lasting from 3-30 minutes, arguably more closely mimicking tests of exhaustion than endurance (according to Heuberger). Professional athletes could not participate in the Heuberger study for ethical reasons and so the study did not demonstrate to what effect blood doping modulates the performance of the most elite athletes. However, this study is perhaps the most comprehensive study to assess the
of
effects of rHuEpo on actual sporting performance, and the evidence presented suggests
ro
the effect is not significant.
-p
HIF stabilizers
re
Hypoxia-inducible factor-1 (HIF-1) is a dimeric molecule that is expressed in numerous tissues of the body. It is composed of an alpha subunit and a beta subunit.
lP
Within kidney cells, the alpha-subunit is rapidly degraded under conditions of normoxia,
na
but under conditions of hypoxia, the dimer is stabilized and acts as a transcription factor to facilitate the production of erythropoietin. There exist several small-molecule agents
ur
which act as HIF stabilizers. By stabilizing HIF-1-alpha, renal cells are
Jo
pharmacologically prompted to increase Epo production, resulting in increased hematocrit. Although none of the HIF stabilizers have FDA approval, some are in clinical trials. WADA added HIF stabilizers to the list of banned substances in 2011, suggesting that they have been available on the black market for some time. The first athlete to test positive for a HIF stabilizer was reported in 2016.42 Early clinical trial outcomes suggest these drugs may be safe for treatment of anemia in chronic kidney disease (CKD), however the study populations have generally not included healthy subjects without anemia. As such, the risks of HIF-stabilizers in the
Journal Pre-proof
13
athlete is largely unknown. It would seem reasonable to conclude that any means of increasing the Epo level would share similar risks in the healthy, non-anemic patient, namely increased blood viscocity, hypertension, and cardiovascular adverse events. However the early trials in CKD appear to suggest that these risks are actually lower compared to exogenous Epo administration.43 Furthermore, the role of HIF-1 in tumorigenesis has been recognized since at
of
least 1999,44 and there are theoretical concerns that increased activation of HIF via HIF
ro
stabilizers could potentially increase the incidence of various cancers.45-46
-p
Cobalt acts as an elemental HIF-alpha stabilizer and acts to prevents
re
ubiquitination of the HIF-alpha subunit.47 Cobalt has been used in the distant past for the purposes of stimulating erythropoiesis, however it is associated with numerous side
lP
effects including severe organ damage such as cardiotoxicity, which limit its use as a
na
blood doping agent.48
Xenon gas also increases the production of HIF-1a49 with downstream effect of
ur
increasing Epo levels.50 Xenon was added to the WADA list of banned substances in
Jo
2014. There have been widely-circulated claims that members of the Russian Winter Olympics team used xenon at the Sochi Olympics in 2014.51 Synthetic Oxygen Carriers Free hemoglobin not encapsulated by the red blood cell (RBC) membrane is unstable and has a host of toxic effects, from vasoconstriction resulting from scavenging of free nitric oxide, to acute tubular necrosis of the kidneys leading to renal failure. Hemoglobin-based Oxygen Carriers (HBOCs) are a class of therapeutic agents which utilize modified forms of hemoglobin to circumvent these toxicities with the aim of
Journal Pre-proof
14
improving oxygen delivery in the anemic patient. There are no currently FDA-approved HBOCs. Hemopure™ is a bovine-derived, crosslinked acellular form of hemoglobin. Trials designed to evaluate the use of Hemopure™ to augment athletic performance have failed to demonstrate a significant benefit,52 concluding that the adverse cardiovascular effects of hypertension potentially negate the benefits of improved oxygen delivery to
of
tissues.53
ro
In addition to HBOCs, perfluorocarbon emulsions (PFCs), which are halogen-
-p
substituted carbon nonpolar oils, can carry and deliver oxygen. PFCs require a large
re
concentration gradient to drive oxygen dissolution, which suggests that PFCs are only of benefit in the presence of supplemental oxygen. This would seem to limit the utility of
lP
PFCs in the realm of endurance sports.54
na
Both HBOCs and perfluorocarbon-based compounds have been banned by WADA, and tests for their detection have been available since 2004.55
ur
The Athlete Biological Passport
Jo
At present, the only officially sanctioned method for detection of transfusion is the Athlete Biological Passport (ABP). The ABP is a program developed by WADA to detect doping by means of measuring changes in an athlete's unique biometric values over time, rather than from the detection of any particular substance itself. The program, implemented in 2009, contains multiple modules intended to detect doping in its numerous forms. The operating guidelines are regularly updated and are currently in the sixth version.56 Currently WADA can sanction athletes based on suspicious changes in the ABP alone.
Journal Pre-proof
15
The blood module of the ABP (Blood ABP) is built on years of work aimed at correlating changes in detectable serum markers of erythropoiesis and iron homeostasis to the administration of rHuEpo and other blood doping techniques. Early work around the turn of the new millennium demonstrated significant and predictable changes in hematocrit, reticulocyte%, erythropoietin, and serum soluble transferrin receptor levels that resulted from rHuEpo use.57-58
of
For example, recent Epo use is expected to result in an increase in hematocrit
ro
and reticulocyte count with an early spike in EPO levels (from exogenous
-p
administration), which is then expected to fall below the normal baseline in response to
re
the increased RBC mass. Changes from an athlete’s baseline for these parameters can raise the suspicion of blood doping even in absence of any detectable contraband
lP
substance. However, small changes in these values may be (mis)attributed to other
remains uncertain.27
na
causes, and the sensitivity and specificity of such changes in detecting autotransfusion
ur
Limitations of the early system led to refinements that both simplified detection
Jo
and improved sensitivity of the algorithm, leading to the development of the so-called OFF-score (OFF-S),59 which has proven valuable for its ability to detect the effects of rHuEpo beyond the window of detection of the actual doping substance itself. The OFFS can be considered an “index of stimulation” that suggests recent Epo use even in absence of significantly elevated Epo levels, and is calculated by the formula: OFF-S = Hb (g/L) - 60√(Reticulocyte%), with a normal range that falls between 85-95.60 Further work in this area led to the development of the Athlete Abnormal Blood Profile Score (ABPS), which is a multi-parametric test similarly designed to predict
Journal Pre-proof
16
rHuEpo administration as well as blood transfusion.61 The ABPS and the OFF-score compose the framework for the blood module of the ABP.62 By following a strict protocol for blood collection, transportation, storage, and analysis, a set of eight values available on routine CBC measurements such as hemoglobin, hematocrit, MCV, RDW, and reticulocyte indices, the ABP is able to track changes in an athlete's hematologic parameters over time. Bayesian Network analysis
of
is then applied to detect abnormal values which may require additional testing or follow-
ro
up.63
-p
The Blood ABP represents the most powerful tool available at present to combat
re
doping. In 2017, the most recent year for which records have been made available, over 29,000 ABP Blood Module samples were analyzed, marking an increase of 3% over the
lP
previous year.64
na
Discussion
The Heuberger study suggests that Epo administration alone does not
ur
significantly improve submaximal sporting performance in well-trained athletes, even
Jo
though it does improve some measures of exercise capacity. It might be easy to imagine that an argument could be made to use this study to decriminalize the use of rHuEpo. However, in any randomized controlled trial, the clinical endpoints stand as the most clinically relevant factors—in other words, a study is only as useful as its endpoints. In practical terms, the benefit of illicit Epo abuse would best be shown by its ability to lead to victory in sporting performance. Other less important endpoints such as physiologic variables are less clinically relevant. What good is improving oxygen delivery if it doesn’t help you win? Additionally, both the medical (i.e. thrombosis) risks
Journal Pre-proof
17
and professional (i.e. expulsion from competition) risks, being as high as they are, cast doubt on the use of Epo to improve athletic performance, even if aerobic exercise is marginally improved. In other words: the proven benefit of Epo abuse—a 6%-8% improvement in maximal aerobic capacity—simply is not enough to justify its multitudinous drawbacks. But if Epo doesn’t improve sporting performance, why is its abuse in endurance
of
sports so widespread? Epo abuse is only one ingredient in the recipe for blood doping.
ro
And the most important ingredient is the athlete themself. Due to ethical considerations,
-p
elite athletes have not been included in trials related to Epo abuse. And there have
re
been no clinical trials that evaluate the rather elaborate blood doping regimens that are routinely employed by elite athletes. It is not unlikely that Epo, when used by an elite
lP
athlete in combination with additional doping methods, can confer an actual
na
performance benefit, and that scant difference in performance between top elite athletes hinges upon the slightest of advantages. Even if that advantage is largely, or even
ur
entirely based on the placebo effect, if the athlete believes it can help them perform, the
Jo
practice will persist and it will be difficult to eliminate blood doping from sport. It seems unlikely that any clinical trial will ever—or could ever—be designed to test this hypothesis. While the practice of blood doping is sure to continue long into the future, the utility and efficiency of the practice has only relatively recently begun to gain rigorous scientific and clinical investigation. Cynically, it may be logical to conclude that as long as competitive sports exist, athletes will seek to gain an advantage; unscrupulous athletes will continue to go to illicit measures to gain that advantage; and the crusade to
Journal Pre-proof
18
ensure a level playing field will continue. Athletes and their trainers are frequently ahead of the curve when it comes to exploiting the science of performance enhancement and evading detection, and this trend seems unlikely to change. Future Considerations The science behind athletic performance enhancement remains in its infancy. Despite this fact, elite athletes will undoubtedly continue to seek to gain a competitive
of
edge over their rivals, even if the methods employed are not the subject of rigorous
ro
scientific scrutiny. As novel methods of blood doping emerge, novel methods of
-p
detection will be necessary to combat them. Better understanding the limits of human
re
athletic performance could lead to better understanding of the normal aging process and could assist with injury and recovery.
na
ur
Erythropoietin is capable of increasing the red blood cell volume and thus the aerobic capacity of athletes, however this increase comes with potential drawbacks such as cardiovascular and thromboembolic complications (e.g. hypertension, heart attack, and stroke). HIF stabilizers have the potential to increase Epo levels and hematocrit. These agents are not FDA approved and their safety in elite athletes is largely unknown.
Jo
lP
Practice Points:
Research Agenda:
Analytical detection methods for autologous blood transfusion remain an unmet need in the battle against blood doping.
Conflict of Interest Neither of the authors have any financial or other conflicts of interest to report. The work is original and has not been published elsewhere. Both authors have contributed substantially to the work. Funding Source
Journal Pre-proof
19
None. References
Jo
ur
na
lP
re
-p
ro
of
1. WADA: World Anti-Doping Agency [Internet]. Montreal: WADA; c2019. Manipulation of blood and blood products; 2019 [cited 2019 Feb 18]; [about 1 screen] Available from: https://www.wada-ama.org/en/content/what-isprohibited/prohibited-at-all-times/manipulation-of-blood-and-blood-components 2. Sottas PE, Robinson N, Fischetto G, Dollé G, Alonso JM, Saugy M. Prevalence of blood doping in samples collected from elite track and field athletes. Clin Chem. 2011;57(5):762-9. 3. Mairbäurl H. Red blood cells in sports: effects of exercise and training on oxygen supply by red blood cells. Front Physiol. 2013;4. 4. Souma T, Suzuki N, Yamamoto M. Renal erythropoietin-producing cells in health and disease. Front Physiol. 2015;6. 5. McAllister RM, Adaptations in control of blood flow with training: splanchnic and renal blood flows. Med Sci Sports Exerc. 1998;30:375-81. 6. Jaakkola P, Mole DR, Tian YM, Wilson MI, Gielbert J, Gaskell SJ, et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science. 2001;292:468-472. 7. Kallio PJ, Okamoto K, O'Brien S, Carrero P, Makino Y, Tanaka H, et al. Signal transduction in hypoxic cells: inducible nuclear translocation and recruitment of the CBP/p300 coactivator by the hypoxia-inducible factor-1alpha. EMBO J. 1998;17(22):6573-86. 8. Green HJ, Sutton JR, Coates G, Ali M, Jones S. Response of red cell and plasma volume to prolonged training in humans. J Appl Physiol (1985). 1991;70(4):1810-5. 9. Otto JM, Plumb JOM, Clissold E, Kumar SB, Wakeham DJ, Schmidt W, et al. Hemoglobin concentration, total hemoglobin mass and plasma volume in patients: implications for anemia. Haematologica. 2017;102(9):1477-85. 10. Ward, Michael, James S. Milledge, and John B. West, editors. High Altitude Medicine and Physiology. 3rd ed. London: Arnold, 2000. 448p. 11. Gilbert DL. The first documented description of mountain sickness: the Andean or Pariacaca story. Respir Physiol. 1983;52(3):327-347. 12. West JB, Richalet JP. Denis Jourdanet (1815-1892) and the early recognition of the role of hypoxia at high altitude. Am J Physiol Lung Cell Mol Physiol. 2013;305(5):L333-340. 13. West JB. High altitude physiology. Stroudsburg, Pa. New York: Hutchinson Ross; Distributed by Academic Press; 1981. xvi, 461 p.p. 14. Shaskey DJ, Green GA, Sports haematology. Sports Med. 2000;29:27-38. 15. Barcroft J, Stephens JG. Observations upon the size of the spleen. J Physiol 1927;64:1-22. 16. Hurtado A, Merino C, Delgado E. Influence of Anoxemia on the Hemopoietic Activity. Arch Int Med 1945;75:284-323.
Journal Pre-proof
20
Jo
ur
na
lP
re
-p
ro
of
17. Pace N, Lozner EL, Consolazio WV, Pitts CP, Pecora LJ. The increase in hypoxia tolerance of normal men accompanying the polycythemia induced by transfusion of erythrocytes. Am J Physiol 1947;148:152-163. 18. Ekblom B, Goldbarg AN, Gullbring B. Response to exercise after blood loss and reinfusion. J Appl Physiol 1972;33:175-180. 19. Fitch KD. Blood doping at the Olympic Games. J Sports Med Phys Fitness 2017;57:1526-1532. 20. Klein HG. Blood transfusion and athletics. Games people play. N Engl J Med 1985;312:854-856. 21. Mørkeberg J. Blood manipulation: current challenges from an anti-doping perspective. Hematology Am Soc Hematol Educ Program 2013;2013:627-631. 22. Leuenberger N, Barras L, Nicoli R, Robinson N, Baume N, Lion N., et al. Urinary di-(2-ethylhexyl) phthalate metabolites for detecting transfusion of autologous blood stored in plasticizer-free bags. Transfusion 2016;56:571-578. 23. Leuenberger N, Schumacher YO, Pradervand S, Sander T, Saugy M, Pottgiesser T. Circulating microRNAs as biomarkers for detection of autologous blood transfusion. PLoS One 2013;8:e66309. 24. Leuenberger N, Barras L, Nicoli R, Robinson N, Baume N, Lion N, et al. Hepcidin as a new biomarker for detecting autologous blood transfusion. Am J Hematol 2016;91:467-472. 25. Salamin O, Mignot J, Kuuranne T, Saugy M, Leuenberger N. Transcriptomic biomarkers of altered erythropoiesis to detect autologous blood transfusion. Drug Test Anal 2017;10:604-608.. 26. Voss SC, Jaganjac M, Al-Thani AM, Grivel JC, Raynaud CM Al-Jaber H, et al. Analysis of RBC-microparticles in stored whole blood bags - a promising marker to detect blood doping in sports? Drug Test Anal 2017;9:1794-1798. 27. Salamin O, Kuuranne T, Saugy M, Leuenberger N. Erythropoietin as a performance-enhancing drug: Its mechanistic basis, detection, and potential adverse effects. Mol Cell Endocrinol 2017;464:75-87. 28. Lasne F, de Ceaurriz J. Recombinant erythropoietin in urine. Nature 2000;405:635. 29. Skibeli V, Nissen-Lie G, Torjesen P. Sugar profiling proves that human serum erythropoietin differs from recombinant human erythropoietin. Blood 2001;98:3626-3634. 30. Lin FK, Suggs S, Lin CH, Browne JK, Smalling R, Egrie JC, et al. Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci U S A 1985;82:7580-7584. 31. Lasne F, Martin L, Crepin N, de Ceaurriz J. Detection of isoelectric profiles of erythropoietin in urine: differentiation of natural and administered recombinant hormones. Anal Biochem 2002;311:119-126. 32. Leuenberger N, Reichel C, Lasne F. Detection of erythropoiesis-stimulating agents in human anti-doping control: past, present and future. Bioanalysis 2012;4:1565-1575.
Journal Pre-proof
21
Jo
ur
na
lP
re
-p
ro
of
33. Catlin DH, Breidbach A, Elliott S, Glaspy J. Comparison of the isoelectric focusing patterns of darbepoetin alfa, recombinant human erythropoietin, and endogenous erythropoietin from human urine. Clin Chem 2002;48:2057-2059. 34. Curran MP, McCormack PL. Methoxy polyethylene glycol-epoetin beta: a review of its use in the management of anaemia associated with chronic kidney disease. Drugs 2008;68:1139-1156. 35. Pope HG, Wood RI, Rogol A, Nyberg F, Bowers L, Bhasin S. Adverse health consequences of performance-enhancing drugs: an Endocrine Society scientific statement. Endocr Rev 2014;35:341-375 36. Casadevall N, Eckardt KU, Rossert J. Epoetin-induced autoimmune pure red cell aplasia. J Am Soc Nephrol 2005;16 Suppl 1:S67-69. 37. Sgrò P, Sansone M, Sansone A, Romanelli F, Di Luigi L. Effects of erythropoietin abuse on exercise performance. Phys Sportsmed. 2018;46(1):105-115. 38. Heuberger J, Rotmans JI, Gal P, Stuurman FE, van 't Westende J, Post TE, et al. Effects of erythropoietin on cycling performance of well trained cyclists: a doubleblind, randomised, placebo-controlled trial. Lancet Haematol. 2017;4(8):e374e86. 39. Annaheim S, Jacob M, Krafft A, Breymann C, Rehm M, Boutellier U. RhEPO improves time to exhaustion by non-hematopoietic factors in humans. Eur J Appl Physiol 2016;116:623-633. 40. Wilkerson DP, Rittweger J, Berger NJ, Naish PF, Jones AM. Influence of recombinant human erythropoietin treatment on pulmonary O2 uptake kinetics during exercise in humans. J Physiol 2005;568:639-652. 41. Thomsen JJ, Rentsch RL, Robach P, Calbet JAL, Boushel R, Rasmussen 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. 42. Buisson C, Marchand A, Bailloux I, et al. Detection by LC-MS/MS of HIF stabilizer FG-4592 used as a new doping agent: Investigation on a positive case. J Pharm Biomed Anal 2016;121:181-187. 43. Hasegawa, S, Tanaka, T, Nangaku, M. Hypoxia-inducible factor stabilizers for treating anemia of chronic kidney disease. Curr Opin Nephrol Hypertens. 2018;27:331-338. 44. Soni S, Padwad YS. HIF-1 in cancer therapy: two decade long story of a transcription factor. Acta Oncol 2017;56:503-515. 45. Pergola PE, Spinowitz BS, Hartman CS, Maroni BJ, Haase VH. Vadadustat, a novel oral HIF stabilizer, provides effective anemia treatment in nondialysisdependent chronic kidney disease. Kidney International 2016;90:1115-1122. 46. Keith B, Johnson RS, Simon MC. HIF1alpha and HIF2alpha: sibling rivalry in hypoxic tumour growth and progression. Nat Rev Cancer 2011;12:9-22. 47. Yuan Y, Hilliard G, Ferguson T, Millhorn DE. Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von Hippel-Lindau protein by direct binding to hypoxia-inducible factor-alpha. J Biol Chem 2003;278:15911-15916.
Journal Pre-proof
22
Jo
ur
na
lP
re
-p
ro
of
48. Ebert B, Jelkmann W. Intolerability of cobalt salt as erythropoietic agent. Drug Test Anal 2014;6:185-189 49. Ma D, Lim T, Xu J, Tang H, Wan Y, Zhao H, et al. Xenon preconditioning protects against renal ischemic-reperfusion injury via HIF-1alpha activation. J Am Soc Nephrol 2009;20:713-720 50. Stoppe C, Ney J, Brenke M, Goetzenich A, Emontzphol C, Schälte G, et al. Subanesthetic Xenon Increases Erythropoietin Levels in Humans: A Randomized Controlled Trial. Sports Med 2016;46:1753-1766. 51. Bennetts M. Russian athletes admit taking banned substances supplied by Rodchenkov. The Guardian. Nov 9, 2017. 52. Hughes GS, Yancey EP, Albrecht R, Locker PK, Francom SF, Orringer EP, et al. Hemoglobin-based oxygen carrier preserves submaximal exercise capacity in humans. Clin Pharmacol Ther 1995;58:434-443. 53. Ashenden MJ, Schumacher YO, Sharpe K, Varlet-Marie E, Audran M. Effects of Hemopure on maximal oxygen uptake and endurance performance in healthy humans. Int J Sports Med 2007;28:381-385 54. Borrione P, Mastrone A, Salvo RA, Spaccamiglio A, Grasso L, Angeli A. Oxygen delivery enhancers: past, present, and future. J Endocrinol Invest 2008;31:185192. 55. WADA: World Anti-Doping Agency. Q&A: Blood Doping; 2019. World Anti-Doping Agency website. https://www.wada-ama.org/en/questions-answers/blood-doping Updated 2019. Accessed Feb 18, 2019. 56. WADA: World Anti-Doping Agency. Athlete Biological Passport Operating Guidelines & Compilation of Required Elements Version 6.1; July 2018. World Anti-Doping Agency website. https://www.wadaama.org/sites/default/files/resources/files/guidelines_abp_v61_2018_jul_en.pdf. Accessed Feb 18, 2019. 57. Parisotto R, Gore CJ, Emslie KR, Ashenden MJ, Brugnara C, Howe C, et al. A novel method utilising markers of altered erythropoiesis for the detection of recombinant human erythropoietin abuse in athletes. Haematologica 2000;85:564-572. 58. Parisotto R, Wu M, Ashenden MJ, Emslie KR, Gore CJ, Howe C, et al. Detection of recombinant human erythropoietin abuse in athletes utilizing markers of altered erythropoiesis. Haematologica 2001;86:128-137. 59. Gore CJ, Parisotto R, Ashenden MJ, Stray-Gundersen J, Sharpe K, Hopkins W, et al. Second-generation blood tests to detect erythropoietin abuse by athletes. Haematologica 2003;88:333-344. 60. Jelkmann W, Lundby C. Blood doping and its detection. Blood. 2011;118:23952404. 61. Sottas PE, Robinson N, Giraud S, Taroni F, Kamber M, Mangin P, et al. Statistical Classification of Abnormal Blood Profiles in Athletes. Int J Biostat 2006;2. 62. Sottas PE, Robinson N, Rabin O, Saugy M. The athlete biological passport. Clin Chem 2011;57:969-976.
Journal Pre-proof
Jo
ur
na
lP
re
-p
ro
of
63. Sottas PE, Robinson N, Saugy M. The athlete's biological passport and indirect markers of blood doping. Handb Exp Pharmacol 2010:305-326. 64. WADA: World Anti-Doping Agency. 2017 Anti-Doping Testing Figures. World Anti-Doping Agency website. https://www.wadaama.org/sites/default/files/resources/files/2017_antidoping_testing_figures_en_0.pdf Accessed Feb 18, 2019.
23