A Three-Week Traditional Altitude Training Increases Hemoglobin Mass and Red Cell Volume in Elite Biathlon Athletes Physiology & Biochemistry 350
Abstract It is well known that altitude training stimulates erythropoiesis, but only few data are available concerning the direct altitude effect on red blood cell volume (RCV) in world class endurance athletes during exposure to continued hypoxia. The purpose of this study was to evaluate the impact of three weeks of traditional altitude training at 2050 m on total hemoglobin mass (tHb), RCV and erythropoietic activity in highly-trained endurance athletes. Total hemoglobin mass, RCV, plasma volume (PV), and blood volume (BV) from 6 males and 4 females, all members of a world class biathlon team, were determined on days 1 and 20 during their stay at altitude as well as 16 days after returning to sea-level conditions (800 m, only males) by using the CO-rebreathing method. In males tHb (14.0 0.2 to 15.3 1.0 g/kg, p < 0.05) and RCV (38.9 1.5 to 43.5 3.9 ml/kg, p < 0.05) increased at altitude and returned to near sea-level values 16 days after descent. Similarly in females, tHb (13.0 1.0 to 14.2 1.3 g/kg, p < 0.05) and RCV (37.3 3.3 to 42.2 4.1 ml/kg, p < 0.05) increased. Compared
Introduction Traditional altitude training is a frequently used method for endurance athletes to improve exercise performance. A key component of this performance improvement is primarily a hypoxia-in-
K. Heinicke1,3 I. Heinicke2,3 W. Schmidt3* B. Wolfarth4*
to their sea-level values, the BV of male and female athletes showed a tendency to increase at the end of the altitude training period, whereas PV was not altered. In male athletes, plasma erythropoietin concentration increased up to day 4 at altitude (11.8 5.0 to 20.8 6.0 mU/ml, p < 0.05) and the plasma concentration of the soluble transferrin receptor was elevated by about 11% during the second part of the altitude training period, both parameters indicating enhanced erythropoietic activity. In conclusion, we show for the first time that a three-week traditional altitude training increases erythropoietic activity even in world class endurance athletes leading to elevated tHb and RCV. Considering the relatively fast return of tHb and RCV to sea-level values after hypoxic exposure, our data suggest to precisely schedule training at altitude and competition at sea level. Key words Hypoxia ´ blood volume ´ plasma volume ´ erythropoietin ´ transferrin receptor
duced increase in plasma erythropoietin (Epo) levels leading to elevated red cell volume (RCV) [22] and VÇO2max. Training at a moderate altitude (1700 ± 3000 m) improves exercise performance at moderate altitude due to the resulting acclimatization effects [6]. Nevertheless, studies concerning the impact of altitude
Affiliation 1 Division of Physiology, Department of Medicine, University of California, San Diego, La Jolla, California, USA 2 Institute of Veterinary Physiology, University of Zurich, Switzerland 3 Department of Sports Medicine/Sports Physiology, University of Bayreuth, Germany 4 Department of Preventive and Rehabilitative Sports Medicine, TU Munich, Klinikum rechts der Isar, Munich, Germany * The contribution of both senior authors was equivalent. Correspondence K. Heinicke ´ Division of Physiology, Department of Medicine, University of California, San Diego ´ 9500 Gilman Drive ´ La Jolla, CA 92093-0623 A ´ USA ´ Phone: + 185 85 34 4713 ´ Fax: + 185 85 34 4812 ´ E-mail:
[email protected] Accepted after revision: April 28, 2004 Bibliography Int J Sports Med 2005; 26: 350 ± 355 Georg Thieme Verlag KG ´ Stuttgart ´ New York ´ DOI 10.1055/s-2004-821052 ´ Published online on November 8, 2004 ´ ISSN 0172-4622
There are little data available concerning the direct effect on RCV in world class endurance athletes during continued hypoxic exposure. Therefore, the purpose of this study was to evaluate the impact of a three-week traditional training camp at moderate altitude (2050 m) on tHb, RCV, and erythropoietic activity in highly-trained endurance athletes.
males). PV and BV were calculated for sea-level values (day ± 1) by blood samples taken before starting the training camp using hemoglobin concentration ([Hb]) and hematocrit (Hct), assuming that tHb and RCV did not changed within 24 hours at altitude. The values of [Hb], Hct as well as the plasma concentrations of erythropoietin (Epo), soluble transferrin receptor (TfR), serumiron, ferritin, and transferrin were determined at sea-level before starting the training camp on days 1, 2, 4, 10, 20 at altitude, and after 16 days at sea-level conditions (only males). General methods Total hemoglobin mass (tHb) was determined using the established CO-rebreathing method as described earlier [19]. In brief, after 15 min in a sitting position the subject was connected to a Krogh spirometer filled with a mixture of oxygen (5 l) and carbon monoxide (60 ml). If necessary, the oxygen was refilled. The percentage of hemoglobin ligated with CO (HbCO) was measured in heparinized cubital-venous blood (10 ± 15 ml blood per test) by the ABL 520 blood gas system (Radiometer, Copenhagen, Denmark). Blood samples were taken and HbCO was determined before and every 2 min during inspiration of the gas mixture for about 15 min until a plateau with maximum HbCO-values were observed. Red cell volume (RCV), blood volume (BV), and plasma volume (PV) were calculated as follows:
Physiology & Biochemistry
training on sea-level performance are controversial. Relevant parameters for an increase of RCV seem to be normal iron stores, a sufficient altitude (2000 ± 3000 m), and the duration of hypoxic exposure e.g. three to four weeks, 12 ± 16 hours per day [10, 25]. Levine et al. (1997) reported an increase in RCV and VÇO2max but these changes did not correspond with an improvement of running performance at sea level in well-trained runners living and training at moderate altitude for four weeks. Others found no effects on hemoglobin mass (tHb) and VÇO2max in highly-trained athletes [1,16]. Some authors speculate that highly-trained subjects do not increase both parameters after altitude training due to initial values being close to the natural physiological limit with little scope for further change [16]. Stray-Gundersen and coworkers (2001) showed increased erythropoietic activity, VÇO2max, and improved running performance at sea level in highly-trained athletes during a ªliving high-training lowº protocol. This study provided evidence that even for athletes who may have achieved near maximal oxygen transport capacity, the mechanism and magnitude of altitude acclimatization appear similar to that observed in athletes with less exercise performance.
(1) RCV = tHb/MCHC ´ 100 (2) BV = RCV ´ 100/Hct
Materials and Methods (3) PV = BV ± RCV Subjects and protocol Six healthy males and four females of a world class biathlon team were investigated during a three-week training camp at moderate altitude (2050 m). The athletes entered the study after giving written consent and all experiments were performed with the permission of a German ethical committee. Male and female biathletes followed a similar but not identical training program. The daily training time was approximately 4 ± 6 hours at an altitude between 1850 ± 2600 m with one day per week rest. At the time the tests were carried out, all athletes were within their annual preparation period. The athletes received a 1 100 mg elementary iron supplement daily for three weeks in advance and during their entire stay at the altitude training camp. The anthropometric data of the athletes were 26.0 3.5 yrs, 181.5 5.1 cm, 80.1 6.4 kg, and BMI 24.3 1.2 kg/m2 in males, and 25.5 1.8 yrs, 169.3 3.9 cm, 59.8 1.5 kg, and BMI 20.9 0.6 kg/m2 in females. The mean body mass was stable over the entire study. The values of mean VÇO2max were 81.7 2.7 ml/ kg ´ min in males and 66.8 0.9 ml/kg ´ min in females indicating two groups of highly-trained endurance athletes. VÇO2max was determined by a discipline specific treadmill test independently from the altitude training camp at a different time point at sea level. Total hemoglobin mass (tHb), red cell volume (RCV), plasma volume (PV), and blood volume (BV) were measured by using the CO-rebreathing-method [9] on days 1 and 20 at altitude, and also at day 16 after returning to sea-level conditions (800 m, only
MCHC: mean corpuscular hemoglobin concentration, Hct: hematocrit corrected for trapped plasma by 0.96 [11], and whole body hematocrit by the cell factor 0.91 according to Fricke [13]. For all tests the identical medical equipment was used by the same investigator. After the subject had rested in a sitting position for 15 min, cubital venous blood samples were taken before the CO-rebreathing period was started. Determinations of [Hb] by the ABL 520 (Radiometer, Copenhagen, Denmark) and Hct by microhematocrit centrifugation (Rapid/K, Hettich, Tuttlingen, Germany) at 10 000 rpm were immediately performed. Every blood sampling for the kinetic analysis (e.g. Epo) was taken between 8 and 9 a. m.. The blood samples were centrifuged and the plasma was immediately frozen at ± 20 8C until tested. The plasma Epo concentration was measured by a chemiluminescence immunoassay (Nichols Institute, San Juan Capistrano, CA, USA) using a mouse monoclonal and a sheep polyclonal antibody. The intra-assay coefficient of variation (CV) was 5.4 %, the inter-assay CV 9.7 %. The sensitivity was 1.4 mU/ml. The determination of the plasma concentration of the soluble transferrin receptor TfR was based on a sandwich enzyme immunoassay using two different monoclonal antibodies (R & D Systems, Minneapolis, MN, USA). The CV of this test kit was 5.7 % and 5.9 %, respectively. The sensitivity was 0.5 nmol/l.
Heinicke K et al. Training of Elite Biathletes at Altitude ¼ Int J Sports Med 2005; 26: 350 ± 355
351
The parameters of the iron status were determined by routinely used test kits: serum-iron concentration (SI) photometrically (Roche Diagnostics, Mannheim, Germany), ferritin concentration by a chemiluminescence immunoassay (Chiron Diagnostics Cooperation, East Walpole, MA, USA), and transferrin concentration (Tf) by a nephelometric method (Behring, Marburg, Germany).
Physiology & Biochemistry 352
Statistics We assume a Gaussian distribution of the overall population of the physiological values measured in this study. All data are presented as the arithmetic mean value standard deviation. Statistical comparisons were performed by a one-way analysis of variance (ANOVA) with repeated measurements. The paired t-test was used as a post-hoc test to determine differences between the sea-level values and the different days at altitude. Significance was accepted at the 0.05 confidence level.
Results In male biathletes [Hb] (14.9 0.7 to 15.9 0.5 g/dl, p £ 0.01) and Hct (43.8 2.0 to 46.8 1.6 %, p £ 0.05, Fig. 1) were significantly increased at the end of the altitude training period (day 20) compared to sea-level values. Most likely due to the low number of female biathletes investigated in this study we found only a tendency to elevated [Hb] (14.2 0.2 to 14.6 0.3 g/dl) and Hct (41.9 0.9 to 45.1 1.8 %, Fig. 1) values at the end of the altitude training period compared to sea-level conditions. However, on day 4 we observed a decrease in [Hb] and Hct values in males. This was most likely due to a resting day with no training that allowed PV compensation that in turn reduced both values. tHb (14.0 0.2 to 15.3 1.0 g/kg, p £ 0.05) and RCV (38.9 1.5 to 43.5 3.9 ml/kg, p £ 0.05) increased clearly at altitude and returned to near sea-level values after 16 days in males (Fig. 2). This observation is in line with a significant increase in tHb (13.0 1.0 to 14.2 1.3 g/kg, p £ 0.05) and RCV (37.3 3.3 to 42.2 4.1 ml/kg, p £ 0.05) in females (Fig. 2). In both groups, BV showed a trend to increase at the end of training at altitude compared to sea-level values, whereas PV remained unchanged (Fig. 2). The absolute values for tHb and the blood volumes are presented in Table 1. The Epo plasma concentration significantly increased up to day 4 at altitude compared to sea-level values (11.8 5.0 to 20.8 6.0 mU/ml, p £ 0.05) and stayed at a slightly elevated level thereafter in males (Fig. 1). 16 days after returning from altitude, no significant differences in Epo were observed compared to pre-altitude values. A similar tendency regarding the Epo plasma concentration was observed in females (Fig. 1). In male biathletes the mean plasma concentration of the soluble transferrin receptor was elevated by about 11% during the second part of the altitude training program (p £ 0.05, Fig. 1). Ferritin showed lower levels during the altitude training compared to sea-level values (107.3 24.9 to 91.0 18.2 ng/ml, p £ 0.05, Table 2), whereas serum iron and transferrin were not altered and remained within the normal range (Table 2). Except from an increased plasma transferrin level at day 1 at altitude compared to sea-level values female biathletes showed no significant changes in the iron status and TfR level (Table 2 and Fig. 1).
Fig. 1 Time course of hematocrit (Hct), plasma erythropoietin concentration (Epo), and soluble transferrin receptor concentration (TfR) in highly-trained male (filled circles) and female (empty circles) biathletes at sea-level conditions (800 m) and during the altitude training (2050 m). Data are presented as arithmetic mean values standard deviation. * indicates significant differences from sea-level conditions, p £ 0.05.
Discussion The major finding of this study is that acclimatization to moderate altitude combined with training results in increased erythropoietic activity leading to elevated total hemoglobin mass and red cell volume in highly-trained endurance athletes.
Heinicke K et al. Training of Elite Biathletes at Altitude ¼ Int J Sports Med 2005; 26: 350 ± 355
tion in RCV [19]. However, the relatively fast return to sea-level RCV values while maintaining the training load indicate that most likely hypoxia alone was responsible for the observed alterations.
Moreover, sea-level residents exposed to long-term intermittent hypoxia (alternating 11 days at 3550 m and 3 days at sea level for 6 months) showed increased TfR and Epo levels leading to similarly elevated tHb and RCV as observed in high altitude residents [18]. This observation obtained at a higher altitude than the one in the present study (2050 m) together with data from previous studies on trained athletes [10, 24, 30] are in agreement with our findings that continued hypobaric hypoxia leads to increased RCV. Analogous with earlier reports, in this study Epo was near doubled with peak values between the 2nd and 4th day [17,18] and remained elevated at a lower level until the end of the altitude training.
Fig. 2 Changes in total hemoglobin mass (tHb), red cell volume (RCV), plasma volume (PV), and blood volume (BV) calculated for kg body mass in highly-trained male (black bars) and female (gray bars) biathletes between sea-level conditions (800 m) and moderate altitude (2050 m). Data are presented as arithmetic mean values standard deviation. * indicates significant differences from sea-level conditions, p £ 0.05.
In our athletes, the mean values of ferritin, iron, and transferrin were within the normal range, and excluded any iron-deprived state. As described by others [12, 21] the elevated plasma TfR level and the decreased plasma ferritin level observed during the second part of the altitude training program in male biathletes indicated enhanced erythropoietic activity. The found increase in RCV by about 12% in males and 13% in females is in agreement with the results from a previous study of well-trained athletes who increased RCV by 10 % after living and training at 2500 m for four weeks [24]. One has to keep in mind that the athletes in our study were not in their peak competition period and the training effect per se might cause in part an eleva-
At the cellular level, acclimatization to reduced oxygenation occurs by the stabilization of the a-subunit of the hypoxia-inducible factor-1 (HIF-1a) that after heterodimerization with its partner HIF-1b leads to an upregulation of HIF-1 target genes such as Epo and TfR [20]. Remarkably, when HeLa cells were exposed to hypoxia, HIF-1a was stabilized instantaneously [23]. In vivo, the kinetics of HIF-1a accumulation has been shown to vary among different organs [31]. In kidney and liver, HIF-1a reaches maximal values after 1 ± 2 h and gradually decreases to baseline levels after 4 h of continuous hypoxia (6% O2). Thus, the decrease in HIF-1a concentration to baseline values after a relatively short time of hypoxic exposure might explain the decreasing Epo levels after some days at altitude, even if the hypoxic exposure continues [17,18]. This observation strongly suggests the existence of a threshold level in PO2 below which hypoxia leads to increased HIF-1a stabilization that in turn induces Epo gene expression. Once a given Epo plasma level is reached, enhanced red cell production occurs. Altitudes between 2100 ± 2500 m seem to represent a threshold for a short-term, sustained Epo production in response to moderate altitude [28]. In our study the observed Epo level plateau that follows the Epo level peak at altitude is in line with previous findings of Chapman and colleagues [10]. These authors showed individual variation in response to altitude training and distinguished between responders and nonresponders to a ªliving high-training lowº al-
Heinicke K et al. Training of Elite Biathletes at Altitude ¼ Int J Sports Med 2005; 26: 350 ± 355
Physiology & Biochemistry
Some studies have reported no increase in tHb and/or RCV after defined periods of hypoxia. These studies included conditions of (i) brief intermittent exposure (8 ± 11 h per night for 5 or 12 days, and 3 weeks) at a simulated normobaric altitude of 2650 m/ 3000 m [3 ± 5], (ii) traditional altitude training for 3 weeks to one month at 2300 m/2690 m [1,16], and (iii) 18 days to 4 weeks exposure at a relatively low altitude of 1800 m/1740 m [14,15]. However, clearly elevated RCV has been observed in residents living at various high altitudes [8,18, 29, 32]. In acclimatized subjects with an arterial oxygen tension (PaO2) below 67 mmHg, corresponding to an altitude of about 1600 m, RCV increases linearly with decreasing PaO2 [32]. Furthermore, it has recently been shown that an altitude of 2600 m is sufficient to increase RCV by 11% in highly-trained endurance athletes born and living at this altitude when compared with athletes from sea level [29].
353
Table 1 Changes in hemoglobin mass and blood volumes Altitude/days
tHb (g)
RCV (ml)
BV (ml)
PV (ml)
male
female
male
female
male
female
male
female
sea-level c./ day ± 1
±
±
±
±
8288 883
6011 324
5126 665
3833 230
2050 m/day 1
1120 89
777 40
3117 286
2225 151
7972 912
5993 321
4855 666
3767 205
2050 ml/day 20
1221 131*
851 61
3465 443*
2524 199*
8492 1111
6401 324
5027 688
3877 268
sea-level c./ day + 16
1152 68
±
3198 171
±
8332 737
±
5135 572
±
Physiology & Biochemistry
Total hemoglobin mass (tHb), red cell volume (RCV), plasma volume (PV), and blood volume (BV). Data are presented as arithmetic mean values standard deviation. * indicates significant differences from sea-level conditions, p £ 0.05
Table 2 Iron status Altitude/days
Serum-iron ( g/dl) male
Ferritin (ng/ml) female
male 107.3 24.9
Transferrin (mg/dl) female
male
female
sea-level c./day ± 1
82.2 29.0
122.0 37.1
2050 m/day 1
65.0 16.9
113.0 40.4
94.9 22.7*
29.2 9.6
205.7 27.7
228.8 34.1
28.4 12.6
190.8 18.3
245.5 37.9*
day 2
82.8 17.8
84.8 21.1
97.5 19.0
31.2 13.5
205.0 34.4
244.3 23.1
day 4
64.5 29.8
72.5 8.7
91.0 18.2*
28.4 9.4
193.8 26.7
210.5 28.5
day 10
73.7 35.5
74.3 24.3
83.5 23.9
26.4 7.2
191.0 24.4
247.5 48.9
2050 m/day 20
65.2 19.5
96.3 34.0
77.3 20.5
28.1 9.0
204.8 28.2
218.5 49.2
sea-level c./ day + 16
82.8 12.0
±
70.6 14.2*
±
181.3 17.9
±
Data are presented as arithmetic mean values standard deviation. * indicates significant differences from sea-level conditions, p £ 0.05
354
titude training, based on the improvement in sea level 5000 m run time. Although in both groups Epo increased after 24 h at 2500 m, the responders had a significantly higher increase. Moreover, the Epo level remained elevated after 2 weeks in the responders, while in nonresponders Epo returned to sea-level values. These different Epo responses caused an increase in RCV and VÇO2max in responders. In contrast, nonresponders showed no difference in both parameters after altitude training. Importantly, there is a marked individual variability in the erythropoietic response to altitude. A recent study showed that in a group of athletes the variation in Epo plasma levels after 24 h at 2800 m simulated altitude ranged from + 400% to ± 41 % [28]. These data support the notion that in some cases no enhancement in erythrocyte production occurred due to a less pronounced hypoxic stimulus that is caused by either an insufficient duration of daily hypoxic exposure [3 ± 5] or insufficient altitude [14,15]. Obviously, the high variability in the erythropoietic response to hypoxia has to be considered. Indeed, such an individual variability occurring within a group of athletes might mask an increase of erythropoietic activity in the total group [1,16]. In all probability the most important adaptation for improving sea-level performance is an increase in RCV. This enhances blood oxygen-carrying capacity and improves aerobic power [7]. It is tempting to speculate that the elevated RCV observed in our
study impacts on VÇO2max. Assuming that this is the case, this would be in line with observations by Levine et al. [24] obtained from well-trained athletes after a traditional altitude training program. However, no improvement of running performance at sea level was observed in their study. Highly-trained athletes revealed improved sea-level performance after a modified ªliving high-training lowº altitude training, where most of the training sessions occurred between 2000 ± 2800 m but high intensity training occurred at lower altitude [30]. Whether this improvement in sea-level performance could also apply to highly-trained athletes after traditional altitude training remains unclear. Further investigations in controlled studies with higher numbers of elite athletes are necessary to target the mechanisms involved in acclimatization to altitude training and to define the training approach with the greatest benefit for athletes. Considering the relatively fast return in tHb and RCV to sea level values after hypoxic exposure, a fine tuning between altitude training and the competition schedule following the altitude period seems to be necessary to achieve best performance. Based on our data we propose a very short re-acclimatization period following the altitude training of approximately one to three days before competition. Of note, the observed reduction in tHb and RCV is in agreement with data from others who described an about 10 % fall in RCV in altitude dwellers within 10 days after descent from altitude to sea level [27] as well as on space flights where through
Heinicke K et al. Training of Elite Biathletes at Altitude ¼ Int J Sports Med 2005; 26: 350 ± 355
microgravity, the BV in the extremities shifts centrally and the PV decreases, causing plethora [2, 26]. The authors showed that neocytolysis, a selective destruction of the youngest circulating red cells, occurs. In summary, three weeks of living and training at moderate altitude is sufficient to increase erythropoietic activity leading to enhanced tHb and RCV, thereby potentially increasing the oxygen-carrying capacity of the blood in highly-trained athletes.
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Physiology & Biochemistry
We acknowledge the willingness of the athletes of a world leading biathlon team and their coaches to participate and cooperate in this study. The authors are grateful to H. Gaisser, P. Winchenbach, B. Biermann, and R. Becher for excellent technical assistance, as well as D. Schwenke for hormone determination (Antidoping laboratory, Kreischa, Germany). This study was financially supported by the Deutsche Gesellschaft für Sportmedizin und Prävention (DGSP), by a grant of the Deutsche Telekom, the Swiss National Science Foundation (3100AO-100 214), and the Bundesinstitut für Sportwissenschaften (VF0408/1. 4. 2001). K.H. is recipient of a Deutsche Forschungsgemeinschaft fellowship (HE 3471/1 ± 1).
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