A comparison of the physiological response to simulated altitude exposure and r-HuEpo administration.

Concerns have been raised about the morality of using simulated altitude facilities in an attempt to improve athletic performance. One assumption that has been influential in this debate is the belief that altitude houses simply mimic the physiological effects of illegal recombinant human erythropoietin (r-HuEpo) doping. To test the validity of this assumption, the haematological and physiological responses of 23 well-trained athletes exposed to a simulated altitude of 2650-3000 m for 11-23 nights were contrasted with those of healthy volunteers receiving a low dose (150 IU x kg(-1) per week) of r-HuEpo for 25 days. Serial blood samples were analysed for serum erythropoietin and percent reticulocytes; maximal oxygen uptake (VO2max) was assessed before and after r-HuEpo administration or simulated altitude exposure. The group mean increase in serum erythropoietin (422% for r-HuEpo vs 59% for simulated altitude), percent reticulocytes (89% vs 30%) and VO2max (6.6% vs -2.0%) indicated that simulated altitude did not induce the changes obtained with r-HuEpo administration. Based on the disparity of these responses, we conclude that simulated altitude facilities should not be considered unethical based solely on the tenet that they provide an alternative means of obtaining the benefits sought by illegal r-HuEpo doping.

Detection of recombinant human erythropoietin abuse in athletes utilizing markers of altered erythropoiesis.

BACKGROUND AND OBJECTIVES: The detection of recombinant human erythropoietin (r-HuEPO) abuse by athletes remains problematic. The main aim of this study was to demonstrate that the five indirect markers of altered erythropoiesis identified in our earlier work were reliable evidence of current or recently discontinued r-HuEPO use. A subsidiary aim was to refine weightings of the five markers in the initial model using a much larger data set than in the pilot study. A final aim was to verify that the hematologic response to r-HuEPO did not differ between Caucasian and Asiatic subjects. DESIGN AND METHODS: Recreational athletes resident in Sydney, Australia (Sydney, n = 49; 16 women, 33 men) or Beijing, China (Beijing, n=24; 12 women, 12 men) were randomly assigned to r-HuEPO or placebo groups prior to a 25 day administration phase. Injections of r-HuEPO (or saline) were administered double-blind at a dose of 50 IU/kg three times per week, with oral iron (105 mg) or placebo supplements taken daily by all subjects. Blood profiles were monitored during and for 4 weeks after drug administration for hematocrit (Hct), reticulocyte hematocrit (RetHct), percent macrocytes (%Macro), serum erythropoietin (EPO) and soluble transferrin receptor (sTfr), since we had previously shown that these five variables were indicative of r-HuEPO use. RESULTS: The changes in Hct, RetHct, %Macro, EPO and sTfr in the Sydney trial were qualitatively very similar to the changes noted in our previous administration trial involving recreational athletes of similar genetic origin. Statistical models developed from Fisher's discriminant analysis were able to categorize the user and placebo groups correctly. The same hematologic response was demonstrated in Beijing athletes also administered r-HuEPO. INTERPRETATION AND CONCLUSIONS: This paper confirms that r-HuEPO administration causes a predictable and reproducible hematologic response. These markers are disturbed both during and for several weeks following r-HuEPO administration. This work establishes an indirect blood test which offers a useful means of detecting and deterring r-HuEPO abuse. Ethnicity did not influence the markers identified as being able to detect athletes who abuse r-HuEPO.

Reticulocyte parameters as potential discriminators of recombinant human erythropoietin abuse in elite athletes.

This study investigated using reticulocyte (retic) parameters as indirect markers of human recombinant erythropoietin (r-HuEPO) abuse in elite athletes. Absolute reticulocyte count (# retic), the per cell haemoglobin content of reticulocytes (CHr), reticulocyte haemoglobin mass per litre of blood (RetHb) and red blood cell:reticulocyte haemoglobin (RBCHb:RetHb) ratio were assessed using flow cytometry. Venous blood was drawn from 155 elite athletes from six sports during regular training to establish reference ranges (95% confidence interval) for these parameters. The reference ranges were compared with those of a non-athletic population (n = 23), four groups of athletes (n = 24) before and after exposure to simulated altitudes (2,500-3,000 m for 11-23 nights), two groups of elite cyclists (n = 13) before and after four weeks of training at natural altitude (1,780 and 2,690 m), and with those of non-athletic subjects from a separate study (n =24) before and 1-2 days after they were injected with 1,200 U x kg(-1) r-HuEPO over a 9-10 day period. Generally the changes induced by r-HuEPO injection exceeded by approximately 100% the magnitude of the changes associated with natural altitude exposure. Simulated altitude exposure did not significantly alter the reticulocyte parameters. From the sample of 155 non-users and 24 r-HuEPO users, the population mean and variance, as well as the 95% confidence limits for the population mean and population variance, were estimated. Relative to arbitrarily chosen cut-off levels, the confidence limits for the rate of true positives and rate of true negatives were also calculated. Based on the lowest rate of false positives and highest rate of true positives, the best discriminator between r-HuEPO users and non-users was # retic, marginally superior to RBCHb: RetHb ratio and RetHb. At a cut-off for # retic of 221 x 10(9)x L(-1) we could be 95% sure that we would find no more than 7 false positives in every 100,000 tests. We would expect to pick up 51.8% of users, and could be 95% sure of picking up at least 38% of current or recent users. This result highlights the potential power of retic parameters for detecting r-HuEPO abuse among athletes. However, the efficacy of these cut-offs for detecting r-HuEPO abuse is unknown if an athlete is a chronic user or stops using r-HuEPO several weeks before being tested.

A novel method utilising markers of altered erythropoiesis for the detection of recombinant human erythropoietin abuse in athletes.

BACKGROUND AND OBJECTIVE: The use of recombinant human erythropoietin (r-HuEPO) to enhance athletic performance is prohibited. Existing tests cannot readily differentiate between exogenous and endogenous EPO. Therefore the aim of our study was to investigate possible indirect detection of r-HuEPO use via blood markers of altered erythropoiesis. DESIGN AND METHODS: Twenty-seven recreational athletes were assigned to three groups prior to a 25 day drug administration phase, with the following protocols: EPO+IM group (n = 10), 50 Ukg(-1) r-HuEPO at a frequency of 3wk(-1), 100 mg intramuscular (IM) iron 1wk(-1) and a sham iron tablet daily; EPO+OR group (n = 8), 50 U.kg(-1) r-HuEPO 3wk(-1), sham iron injection 1wk(-1) and 105 mg of oral elemental iron daily; placebo group (n = 9), sham r-HuEPO injections 3wk(-1), sham iron injections 1wk(-1) and sham iron tablets daily. Each group was monitored during and for 4 weeks after drug administration. RESULTS: Models incorporating combinations of the variables reticulocyte hematocrit (RetHct), serum EPO, soluble transferrin receptor, hematocrit (Hct) and % macrocytes were analyzed by logistic regression. One model (ON-model) repeatedly identified 94-100% of r-HuEPO group members during the final 2 wk of the r-HuEPO administration phase. One false positive was recorded from a possible 189. Another model (OFF-model) incorporating RetHct, EPO and Hct was applied during the wash-out phase and, during the period of 12-21 days after the last r-HuEPO injection, it repeatedly identified 67-72% of recent users with no false positives. INTERPRETATION AND CONCLUSIONS: Multiple indirect hematologic and biochemical markers used simultaneously are potentially effective for identifying current or recent users of r-HuEPO.

Simulated moderate altitude elevates serum erythropoietin but does not increase reticulocyte production in well-trained runners.

The purpose of this study was to investigate whether the modest increases in serum erythropoietin (sEpo) experienced after brief sojourns at simulated altitude are sufficient to stimulate reticulocyte production. Six well-trained middle-distance runners (HIGH, mean maximum oxygen uptake, VO2max = 70.2 ml x kg(-1) x min(-1) spent 8-11 h per night for 5 nights in a nitrogen house that simulated an altitude of 2650 m. Five squad members (CONTROL, mean VO2max= 68.9 ml x kg(-1) x min(-1) undertook the same training, which was conducted under near-sea-level conditions (600 m altitude), and slept in dormitory-style accommodation also at 600 m altitude. For both groups, this 5-night protocol was undertaken on three occasions, with a 3-night interim between successive exposures. Venous blood samples were measured for sEpo after 1 and 5 nights of hypoxia on each occasion. The percentage of reticulocytes was measured, along with a range of reticulocyte parameters that are sensitive to changes in erythropoiesis. Mean serum erythropoietin levels increased significantly (P < 0.01) above baseline values [mean (SD) 7.9 (2.4) mU x ml(-1)] in the HIGH group after the 1st night [11.8 (1.9) mU x ml(-1), 57%], and were also higher on the 5th night [10.7 (2.2) mU x ml(-1), 42%] compared with the CONTROL group, whose erythropoietin levels did not change. After athletes spent 3 nights at near sea level, the change in sEpo during subsequent hypoxic exposures was markedly attenuated (13% and -4% change during the second exposure; 26% and 14% change during the third exposure; 1st and 5th nights of each block, respectively). The increase in sEpo was insufficient to stimulate reticulocyte production at any time point. We conclude that when daily training loads are controlled, the modest increases in sEpo known to occur following brief exposure to a simulated altitude of 2650 m are insufficient to stimulate reticulocyte production.

Can reticulocyte parameters be of use in detecting iron deficient erythropoiesis in female athletes?

BACKGROUND: The purpose of this study was to investigate whether monitoring reticulocyte profiles, which are known to respond to iron store depletion in sedentary populations, could also be utilised with intensely training athletes. METHODS: A retrospective study of blood samples from 134 national level athletes (61 males, 73 females) at the Australian Institute of Sport were analysed, from which reference ranges were calculated. To ascertain the stability of reticulocyte profiles during periods of intense physical training, the intra-individual variation of these parameters in 12 iron-replete female athletes over a four month period of training was documented. The precision with which the analyzer measured these parameters was also determined using duplicate samples from 37 female athletes. To establish whether reticulocyte parameters were sensitive to iron deficient erythropoiesis in athletes, reticulocyte profiles of five female athletes diagnosed by medical personnel as having depleted iron stores were compared before and after iron therapy to seven controls. RESULTS: Corpuscular hemoglobin concentration mean (CHCMr) and mean corpuscular volume (MCVr) showed little variation over time in iron-replete females, with 95% of all fluctuations being within 5.8% and 4.3% of original values, respectively. Iron supplementation in athletes with depleted iron stores elicited an increase in CHCMr (p = 0.01), and a decrease in the distributions of reticulocyte volume (RDWr, p = 0.01) and cell hemoglobin concentration (HDWr, p < 0.01). The ratios of reticulocyte to mature cell MCV (p < 0.01) and CHCM (p < 0.01) also changed following iron therapy. No such changes occurred in non-supplemented controls with normal iron stores. CONCLUSIONS: These data lend support to the thesis that monitoring of reticulocyte parameters can be of use in detecting iron deficient erythropoiesis in female athletes.

Altitude training at 2690m does not increase total haemoglobin mass or sea level VO2max in world champion track cyclists.

Haemoglobin mass (Hb mass), maximum oxygen consumption (VO2max), simulated 4000 m individual pursuit cycling performance (IP4000), and haematological markers of red blood cell (RBC) turnover were measured in 8 male cyclists before and after (A) 31 d of altitude training at 2690 m. The dependent variables were measured serially after altitude on d A3-4, A8-9 and A20-21. There was no significant change in Hb mass over the course of the study and VO2max at d A9 was significantly lower than the baseline value (79.3 +/- 0.7 versus 81.4 +/- 0.6 ml x kg(-1) x min(-1), respectively). No increase in Hb mass or VO2max was probably due to initial values being close to the natural physiological limit with little scope for further change. When the IP4000 was analysed as a function of the best score on any of the three test days after altitude training there was a 4% improvement that was not reflected in a corresponding change in VO2max or Hb mass. RBC creatine concentration was significantly reduced after altitude training, suggesting a decrease in the average age of the RBC population. However, measurement of reticulocyte number and serum concentrations of erythropoietin, haptoglobin and bilirubin before and after altitude provided no evidence of increased RBC turnover. The data suggest that for these elite cyclists any benefit of altitude training was not from changes in VO2max or Hb mass, although this does not exclude the possibility of improved anaerobic capacity.