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FIMS International Perspectives

Altitude Training and Recombinant Human Erythropoietin: Considerations for Doping Detection

Sutehall, Shaun BSc1; Muniz-Pardos, Borja MSc2; Lima, Giscard MSc3,4; Wang, Guan PhD3; Malinsky, Fernanda Rossell PhD3; Bosch, Andrew PhD1; Zelenkova, Irina MD, PhD5; Tanisawa, Kumpei PhD6; Pigozzi, Fabio MD, PhD4; Borrione, Paolo MD4; Pitsiladis, Yannis PhD3

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Current Sports Medicine Reports: April 2019 - Volume 18 - Issue 4 - p 97-104
doi: 10.1249/JSR.0000000000000577
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Abstract

Introduction

The dominance of East African athletes in endurance events is well known and illustrated by the marathon world record holders over the last 15 years being solely Kenyan or Ethiopian (1). A high maximal oxygen uptake (V˙O2max), good running economy, high fractional utilization of V˙O2max, and low body mass have all been proposed to explain this remarkable success of East African endurance athletes (2). Notably, the most successful distance runners live and train at moderate to high altitude (3). The disparity between Caucasian and East African athletes in elite endurance performance has led to many investigations into the use of altitude exposure to enhance performance in sea level competitions.

Altitude exposure elicits unique stress on the human cardiovascular system through a linear decrease in pressure of inspired oxygen (PiO2) with increasing altitude. A reduced PiO2 leads to a decreased arterial blood saturation, impaired oxygen (O2) delivery, and significantly inhibited oxidative metabolism (4). To minimize the homeostatic disruption and attenuate the decline in performance caused by altitude exposure, a number of short- and long-term physiological adaptions take place. The initial exposure to altitude of 2000 m or more above sea level triggers several acute adaptations including: 1) a reduction in plasma volume that increases the transit time of each red blood cell across the alveoli; 2) an increase in heart rate that enhances delivery of O2 to the active muscle; and 3) an increase in ventilation rate with a concurrent decrease in bicarbonate stores, enhancing O2 delivery without the typically associated alkalosis (5). Within hours after arrival at altitude, endogenous erythropoietin (Epo) production begins to increase, stimulating the production of red blood cells (6) and subsequently elevating total hemoglobin mass (tHb-mass) by a process controlled primarily by the regulatory protein hypoxia-inducible factor (7). Increasing tHb-mass facilitates a greater O2 delivery to active muscles during exercise and partially counteracts the altitude-related impairment in V˙O2max. Following sufficient adaptation, performance at altitude improves (8), but the magnitude of the performance enhancement at sea level remains a source of debate.

More than 20 years have passed since the seminal study that found significant improvements in sea level 5000 m time trial performance in athletes that lived at moderate altitude (2400 m) while completing training at low altitude (LHTL) during a 4-wk training camp, compared to athletes who both lived and trained at moderate altitude (LHTH) or in the sea level control group (9). This study led to many investigations with the aim of determining the optimum method of altitude training to improve sea level performance. A meta-analysis concluded that tHb-mass will increase by 1.1%/100 h of altitude exposure of LHTL (approximately 3000 m) and LHTH (>2100 m) when the altitude exposure is more than 10 h ·d−1 and as little as 2 wk could confer significant enhancement in tHb-mass (10), irrespective of the individual’s initial tHb-mass value (11). Despite these findings, there is concern that a large proportion of the changes in tHb-mass and performance may be due to the increased training stimulus of the “camp” setting. Given that athletes were nonelite, this increased training stimulus would have caused the plasma volume to expand and thus greater hematological changes are observed (12). This has led some to conclude that altitude training does not improve exercise performance and should not be recommended for athletes (13).

The desire to increase tHb-mass beyond levels that can be attained through altitude training (14) has led some athletes to use recombinant human erythropetin (rHuEpo). Within our group, rHuEpo has been shown to increase an individual’s V˙O2max by approximately 6% and endurance performance by approximately 8% (15), with similar improvements in V˙O2max reported by others (16,17). The current method to detect rHuEpo involves longitudinal monitoring of an athlete’s hematological parameters, known as the Athlete Biological Passport (ABP), to detect changes that may indicate rHuEpo abuse (18). An augmented tHb-mass is considered the primary mechanism responsible for the ergogenic effects of both rHuEpo use and altitude exposure (14). It is plausible that an athlete could administer rHuEpo to enhance the tHb-mass while attending an altitude training camp and attempt to explain any suspicious hematological change as a response to altitude. This tactic has previously been employed by an elite-level athlete, but it did not succeed as the height of the altitude (800–1600 m) was insufficient to elicit a significant hematological response (19). The aim of this perspective is to highlight the pitfalls of current altitude training guidelines and summarize the difficulty antidoping agencies face in detecting rHuEpo abuse in athletes training at altitude.

Altitude Training

Early investigations into the effect of LHTH yielded mixed results, with some demonstrating an ~10% increase in sea level V˙O2max and significantly faster time trial performance (20) while others reported no effects (21). Although numerous elite athletes have incorporated this method of altitude exposure into their training program, the lack of well-controlled studies investigating the effects of LHTH leaves the efficacy of this method unclear (22,23). The modest to no effect of altitude training on either V˙O2max or performance as reported in some studies (21,24) has been attributed to several factors, such as the insufficient elevation and duration of stay at altitude, and the different training intensities (25,26).

Levine and Stray-Gundersen (9) were the first to suggest that the well-documented reduction in V˙O2max at altitude elicits a lower relative training intensity and subsequently a degree of deconditioning. To overcome this, these investigators developed the concept of LHTL (living at approximately 2500 m while training at approximately 1250 m) to counter the attenuation in training intensity (i.e., running speed) seen with LHTH. Their study found that both LHTL and LHTH improved sea level V˙O2max and hemoglobin concentration, but only the LHTL led to a significant increase in sea level 5000 m time trial performance. It was concluded that relative training intensity is crucial in the improvement in sea level performance following an altitude training camp.

This seminal study led to additional investigations focusing on the LHTL model (27–32), with some data suggesting it is the most efficient and effective method of altitude training to improve endurance performance (33,34). A refinement of the original LHTL design was a model in which athletes live high (2500 m) and perform most of their training at that altitude, but perform the high-intensity interval running sessions at a lower altitude (approximately 1250 m) (35). The positive effects of this training paradigm also have been demonstrated in elite runners (30).

Siebenmann et al. studied double-blinded, placebo-controlled groups of highly trained endurance cyclists spending 16 h ·d−1 for 4 wk in chambers flushed with either normal air (control group) or normobaric hypoxia simulating 3000 m above sea level (36). The hypoxia group did not improve tHb-mass, V˙O2max, or 26 km cycling time trial performance at sea level, when compared to the control group. A recent review (23) suggests a minimum of 22 h ·d−1 at altitude is required to optimize the hypoxic exposure and 16 h ·d−1 of normobaric hypoxia could be insufficient to elicit hematological changes. In contrast, a more recent meta-analysis suggests altitude exposure of as little as 10 h ·d−1 was sufficient to increase tHb-mass in well-trained athletes (10).

Robach et al. (37) have recently shown that 4 wk of LHTL using hypobaric hypoxia (i.e., terrestrial altitude) in trained cross-country skiers, did not elicit additional hematological, ventilatory, or performance improvements compared with a sea level control group. It could be that the physiological and performance gains previously seen with the LHTL model may be related to training stimulus and not to altitude acclimatization, evidenced by the similar improvements in tHb-mass in the control and LHTL groups (~3%) (37). Studies that do not include a sea level control group when assessing the effect of altitude on performance must be considered with caution.

A recent review (23) suggests that athletes should live at 2000 to 2500 m for at least 4 wk and for greater than 22 h ·d−1 to ensure an adequate hypoxic exposure that would favor an increase in sea level performance. This recommendation however, is limited by the lack of studies examining the effects of living at altitude over an extended period of time (e.g., >10 wk) on the acclimatization of sea level athletes. Studies by Brothers et al. (38,39) suggest that a complete physiological acclimatization to moderate altitude by individuals relocating from sea level may require 7 to 15 months. It has been hypothesized that for athletes acclimatizing to moderate altitude, the relative importance of training low in the LHTL model will decrease or even vanish (22), although there are no long-term studies to support this hypothesis. Therefore, additional research is required to determine the effect of several high-intensity sessions performed at lower altitude when applied to a group of elite level, altitude-acclimatized athletes.

An additional variable to consider in the physiological response to altitude is the large and unpredictable variation among individuals (40). For example, elite athletes with a higher tHb-mass prior to the altitude exposure might have a reduced physiological response to altitude compared to athletes with a lower tHb-mass (22). After 24 h of acute exposure to 3000 m, a previous study has demonstrated that blood Epo concentration can range from a 40% decrease to a 400% increase compared with sea level baselines (6). This large variability may influence the effects of altitude on athletic performance and account for some of the differences observed in studies that are otherwise well-controlled and well-designed.

While neither LHTH nor LHTL have received unequivocal support, both are likely to improve performance in some athletes. However, the scientific literature is not adequate to make universal recommendations (8,22,41). Comparisons between altitude studies are difficult due to the disparity in certain variables such as living and training altitudes, total duration and minimum daily dose of altitude exposure, performance testing protocols, fitness status at study entry, individual physiological variation, and lack of sea level control groups. These factors fuel the debate on the optimum elevation, duration, and method of altitude exposure to elicit both maximal adaptation to altitude and sea level performance enhancement.

Doping

With the benefits of altitude training unresolved, some athletes may be searching for alternatives with greater efficacy to enhance endurance performance through various forms of doping. In 2009, the World Anti-Doping Agency (WADA) introduced an indirect method of monitoring blood manipulation to detect possible blood and rHuEpo doping. The ABP longitudinally monitors various hematological parameters for each athlete and detects fluctuations exceeding a pre-determined threshold deemed “suspicious.” This approach has had some successes (42–44) but also has several inherent weaknesses such as a short analytical window (e.g., 36 h between blood sample collection and analysis (45)) and a limited sensitivity (42). The global antidoping testing figures published by WADA (46) reveal that the number of ABP blood tests has increased from 6082 in 2009 to 29,130 in 2017, whereas the number of overall adverse analytical findings has increased by just 0.35%. This small increase in the number of adverse analytical findings suggests that: 1) the ABP could detect the majority of those doping in 2009, 2) the sensitivity of the ABP has not increased despite a significant investment of financial resources, 3) the number of athletes doping has not changed significantly in 8 years, or 4) the current implementation of the ABP does not have the specificity required to detect current doping practices. The latter explanation is supported by an anonymous survey in 2017 which found a 45% to 49% prevalence of doping, many times greater than that estimated from the ABP (47).

The hematological and functional adaptations to altitude exposure are complex and attempts to account for these augmentations in the ABP have not been successful (48). Tissue oxygenation in the kidneys and liver regulates Epo hormone production and local hypoxia accelerates this process (49). The activation of Epo receptors in the bone marrow results in the formation of new red blood cells, a primary adaption of altitude acclimatization (49,50). Differentiating altitude-induced polycythemia from rHuEpo administration is currently the largest challenge facing the ABP. Biomarkers that differentiate changes induced by hypoxia and from those induced by Epo administration must be explored to improve the current ABP.

The application of molecular biology may improve the current antidoping discriminatory capacity through the discovery and application of transcriptomic biomarkers of doping (51). Five genes significantly expressed in response to darbepoetin alpha have been identified for both high and microdoses of rHuEpo using serial analysis of gene expression and validation (51). Follow-up investigations of the altered global gene expression patterns as a result of doping have been conducted (52) which specifically targeted the genes that are switched on and off in response to the use of a doping substance or compared to hypoxia. Our group has carried out whole-blood gene expression profiling using a DNA microarray-based approach to detect rHuEpo doping (50 IU ·kg−1 body mass every 2 d for 4 wk). Two independent groups further validated the microarray analysis of 34 transcripts on a different quantification platform (53). Subsequently, we identified 9 of the 34 transcripts in a randomized, double-blind, placebo-controlled cross-over rHuEpo microdosing study (54). These gene transcripts could be incorporated into the ABP to potentially improve its sensitivity and specificity. Other proteomic and metabolomic signatures should be explored as potential markers of doping to enhance the current detection methods.

Altitude Training and Doping

The WADA guidelines (version 6.1, July 2018) (55) require more comprehensive information from the athlete to incorporate and account for physiological adaptations to altitude exposure into the algorithm for doping control. This information requires disclosure of the specific conditions of altitude exposure, use of simulating devices (e.g., hypoxic tent), blood transfusions, or significant blood loss incidents. Athletes also must report “whereabouts” data to their regional antidoping agency. An expert panel is charged with performing analysis on data which has been flagged as “suspicious” and make conclusions on the likelihood that illicit blood manipulation has occurred. Despite significant progress in the ABP, the detection of blood doping remains inadequate (56,57).

A recent narrative review (58) addressed the increase in Epo production following altitude training. These authors showed that blood Epo concentration increases during continuous altitude exposure reaching a peak in 1 to 3 d and subsequently begins to gradually fall. They also stated that the rate at which blood Epo decreases is influenced by the degree of hypoxia, not the type of training. Although hypoxic training at altitudes ranging from 1800 to 2000 m might be sufficient to stimulate Epo production within the first day of exposure, Chapman et al. observed that blood Epo concentration decreased after 72 h of exposure in the 1780 m training group compared to the 2085 m, 2454 m, and 2800 m training groups (59). While blood Epo concentration decreased in the lower altitude group, it remained above the baseline values for 3 d (59). A recent meta-analysis comparing the hematological variations observed during and following altitude exposure highlighted the complexity associated with effects of altitude and the resulting hematological responses on the application of the ABP (48). This illustrates the urgent need for further examination of the effects of both rHuEpo and altitude training on the hematological responses of elite athletes.

We performed two studies with rHuEpo administration (60) and altitude training to highlight the limitations of the ABP; summarized below.

Altitude study

Fourteen endurance-trained runners living and training at sea level in Cape Town (South Africa) trained for 27 d in Suluta (Ethiopia) at approximately 2800 m above sea level. All athletes provided blood samples 4 d before departure and at 1 d, 9 d, 16 d, and 24 d after arrival at altitude. Upon completion of the altitude camp, the athletes returned to sea level and blood samples were taken at 1 d, 6 d, 13 d, and 27 d. The percentage of reticulocytes (RET%) within the blood sample could not be assessed 24 h after arrival at altitude, so this time point was excluded in the ABP analysis.

rHuEpo study

Twenty well-trained Kenyan endurance runners from the Kalenjin tribe living and training in Eldoret (Kenya) at 2150 m above sea level were recruited (60). Each athlete self-injected (under supervision) 50 IU ·kg−1 body mass of rHuEpo subcutaneously every second day for 4 wk. Blood samples were taken at baseline over a period of 2 wk (three samples), during rHuEpo administration (10 samples) and for 4 wk after rHuEpo administration (seven samples).

Current observations

Analyses of the hematological responses from the two studies were completed using the current WADA ABP Operating Guidelines (55). The results were indicative of doping when the hematological dynamics tested (blue line) surpassed the borderline or reference line values (upper and lower red lines) for hemoglobin (HGB), RET%, OFF-Score (OFFS) and Abnormal Blood Profile Score (ABPS) in Figures 1, 2, 3, and 4. Overall, 14 athletes from the altitude study and 18 from the rHuEpo study were analyzed using the ABP analysis software. Among the 14 altitude athletes, 5 did not surpass the ABP threshold in any of the parameters evaluated, and 9 surpassed the ABP threshold in one or more blood markers (see a representative example in Fig. 1); with 9 (64%) of 14 athletes were declared as suspicious for at least one parameter.

Figure 1
Figure 1:
HGB, OFFS, ABPS, and RET% parameters of an athlete attending an altitude training camp (~2800 m) for 27 d.
Figure 2
Figure 2:
HGB, OFFS, ABPS, and RET% parameters of an athlete over the course of an rHuEpo administration regimen.
Figure 3
Figure 3:
HGB, OFFS, ABPS, and RET% parameters of the less responsive individual to rHuEpo administration over the course of the rHuEpo regimen.
Figure 4
Figure 4:
HGB, OFFS, ABPS, and RET% parameters of the same athlete in Figure 2 over the course of an rHuEpo administration regimen with a reduced number of samples used for analysis.

Twelve of the 18 athletes in the rHuEpo study had abnormal ABP markers in at least three of the four parameters (see a representative example in Fig. 2). One athlete’s blood values seem to fall within the normal ranges of all four parameters, showing a potential false negative ABP profile (Fig. 3). This athlete may be less responsive to rHuEpo administration and therefore may not improve performance using rHuEpo (60). Five of 18 athletes from the rHuEpo study were analyzed without the red blood cell count variable due to complications in the sample collection. Among these five athletes, three exceeded the reference line for the HGB, RET%, and OFFS and the other two exceeded the OFFS and RET% limits. The overall ABP performance in the rHuEpo study was improved by using 20 assessments over 10 wk, which allowed a better understating of the athlete’s hematological profile. A previous study demonstrated that the ABP failed to detect any of the 10 athletes administered with 20 to 30 IU ·kg−1 body mass rHuEpo twice weekly for 8 wk (56); this poor performance by the ABP may reflect the lack of post-rHuEpo samples collected in this cohort.

The findings of our preliminary analysis demonstrate that 9 of 14 of the athletes who participated in a 27-d sojourn to moderate altitude were flagged by the ABP due to abnormal hematological fluctuations (false positives), which would require further examination by a panel of experts. On the other hand, 17 of 18 athletes were detected by the ABP following rHuEpo administration and would likely be identified as doping (e.g.,Fig. 2).

Upon initial examination, the ABP performed well and detected all those responders to rHuEpo. However, this analysis was based on an unrealistic number of samples per athlete (20 samples over a 10-wk period). Recently, an elite athlete was sanctioned following five suspicious blood samples taken over a 10-month period (19). If we applied a similar number of samples across those athletes administered with rHuEpo in our study, selecting three samples during rHuEpo administration and one postadministration, the magnitude of the changes seen in the original analysis are attenuated and make interpretation much more difficult (Fig. 4). It is foreseeable that an athlete in this situation would be more able to claim his or her hematological profile is in response to natural changes (e.g., training at altitude) rather than due to rHuEpo administration. It is readily apparent that the utility of the ABP is dependent on the number of samples taken per athlete over a given period, and by chance sampling the athlete’s blood during a change in a doping regimen. It also is clear from comparing Figure 1 and Figure 4, the difficulty the WADA expert panel face attempting to distinguish hematological profiles indicative of altitude exposure or rHuEpo administration, something which has been highlighted by others (48,61).

Conclusions

More research is required to understand the efficacy of the “East African” model of altitude training, specifically long-term adaption to altitude with training carried out at both high (>3,000 m) and low (<2,000 m) altitude followed by sea level performance assessments and to find the optimum “dose” of altitude exposure which elicits maximal performance in lowlanders. The ABP is somewhat effective in detecting rHuEpo administration and haematological responses to altitude with sufficient number of samples. Developing new methods of detecting blood manipulation in the antidoping field is critical for fair competition and emerging “omics” approaches may fill this role.

The authors declare no conflict of interest and do not have any financial disclosures.

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