Hemoglobin Mass and Peak Oxygen Uptake in Untrained and Trained Residents of Moderate Altitude

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1 572 Physiology and Biochemistry Hemoglobin Mass and Peak Oxygen Uptake in Untrained and Trained Residents of Moderate Altitude D. Böning 1, J. Rojas 2, M. Serrato 3, C. Ulloa 3, L. Coy 2, M. Mora 2, J. Gomez 2, M. Hütler 1 1 Institute of Sports Medicine, Free University Berlin, Berlin, Germany 2 Centro de Fisiologia de Ejercicio, Universidad Nacional de Colombia, Bogotµ, Colombia 3 Centro de Servicios Biomedicos, Coldeportes Colombia, Bogotµ, Colombia Böning D, Rojas J, Serrato M, Ulloa C, Coy L, Mora M, Gomez J, Hütler M. Hemoglobin Mass and Peak Oxygen Uptake in Untrained and Trained Residents of Moderate Altitude. Int J Sports Med 2001; 22: 572 ± 578 Accepted after revision: August 22, 2001 nnnn Blood composition, hemoglobin mass (CO rebreathing method) and VÇO 2 peak were measured in 15 untrained (UT-Bogotµ) and 14 trained males (TR-Bogotµ) living at 2600 m of altitude, and in 14 untrained lowlanders (UT-Berlin). [Hb] amounted to (SE) g/dl in UT-Berlin, g/dl in UT-Bogotµ and g/dl in TR-Bogotµ. Hb mass was significantly higher in UT-Bogotµ ( g/kg, P < 0.01) and in TR-Bogotµ ( g/kg, P < 0.001) than in UT-Berlin ( g/kg). In TR-Bogotµ also plasma volume was expanded. Erythropoietin concentrations in UT-Bogotµ and TR-Bogotµ were not significantly increased. There was a positive correlation between blood volume and VÇO 2 peak for the pooled values of all subjects, if the oxygen uptake of UT-Berlin was corrected for an ascent to 2600 m. For the Hb mass - VÇO 2 peak relation two groups are indicated pointing to two types of altitude acclimatization with different Hb mass increases but similar distribution of aerobic performance capacity. We suggest that different genetic properties in a population of mixed ethnic origin might play a role. n Key words: Erythropoietin, exercise, hypoxia, iron, transferrin receptor, doping. Introduction It has been a well-known fact for many decades that altitude hypoxia stimulates erythropoiesis thus increasing hemoglobin (Hb) mass and red cell volume, whereas plasma volume tends to decrease [20]. Most measurements have been done in sojourners and residents acclimatized to more than 3000 m above sea level (e. g. [25,26, 29,40], for a review see [31]). Astonishingly publications on blood volume in residents of the densely populated mountains between 2000 and 3000 m above sea level do not seem to exist. A threshold for erythropoietic stimulation at 2500 m in residents has only been estimated by interpolation from measurements at 1600 m and 3100 m [40]. Endurance training at sea level also increases the red cell volume, but more the plasma volume [9,11,13, 31, 35]. Thus, in contrast to the effect of environmental hypoxia, there is a tendency for a decrease in Hb concentration and hematocrit (Hct) value. Endurance training of lowlanders at altitude produces similar effects like a sojourn without systematic physical activity (increase of red cell volume and Hb concentration, decrease of plasma volume) with a threshold for erythropoietic stimulation not higher than 2500 m [7,13,24,31, 39]. To our knowledge nothing is known about the influence of regular exercise on erythropoiesis and blood volume in altitude residents. Does it exert an additional increase of hemoglobin mass similar to that at sea level? Is the plasma volume affected, too? Measurements of hemoglobin concentration alone do not give sufficient information because a rise (altitude reaction) and a decline from an increase in plasma volume (training reaction) might cancel each other. The purpose of this investigation was to measure hemoglobin mass in untrained and trained highlanders at moderate altitude (2600 m) and to study possible causes of changes as well as effects on performance. Especially the following questions were addressed: 1. Is there really an erythropoietic threshold at approximately 2500 m above sea level for altitude residents? 2. Is the effect of physical training on hemoglobin mass and plasma volume in altitude residents different from that at sea level and what importance do possible changes have for performance in this environment? 3. Are possible effects related to erythropoietin (EPO) secretion? Int J Sports Med 2001; 22: 572±578 Georg Thieme Verlag Stuttgart New York ISSN

2 Hb Mass and Peak Oxygen Uptake at Altitude Int J Sports Med 2001; Methods Subjects Measurements were performed in 15 untrained (UT) and 14 trained (TR) non-smoking male subjects in Bogotµ/Columbia (2600 m above sea level) after informed written consent and considering the rules of the Helsinki declaration of The untrained group (age [SE] years, body mass kg, height cm, body mass index BMI kg/m 2 ) consisted of university students. Twelve were born and had been living since then in the town, 3 were born at lower altitude (1 at 1500 m, 2 at 400 m) but had been living between 5 ± 24 years in Bogotµ. The trained group (13 runners with 36 ± 80 km training distance per week, 1 Karateka performing regular endurance training, age years, body mass kg, height cm, BMI kg/m 2 ) were natives from Bogotµ except one (born at 500 m) who had been living there since 7 years. All subjects were clinically healthy. Three-day protocols served for evaluation of nutrition and physical activity considering Colombian eating habits [14, 22, 23]. Measurements of most quantities (except nutrition and physical activity) in 14 male untrained lowlanders living at 35 ± 60 m above sea level in Berlin served for comparison (UT-Berlin, age years, body mass kg, height cm, BMI kg/m 2 ). Blood measurements The hemoglobin mass was determined by the CO rebreathing method with blood sampling from the hyperemized ear lobe [21]. The volume of inspired CO was adapted to yield approximately 5 ± 6 % difference for HbCO. The time course of HbCO in blood at altitude was not different from that at sea level: the approximately constant values from minute 8 to 12 (after an initial peak because of mixing effects) were used for the calculation. [Hb], oxygen saturation (SO 2 ) and HbCO were determined by use of a 6-wave length-spectrophotometer (Oxygen Saturation Meter 3, Radiometer Copenhagen), Hct by the microcentrifugation method. Mean cellular hemoglobin concentration (MCHC) was calculated from [Hb] and Hct taking into account 2 % trapped plasma. Erythrocyte (EV) and plasma volume (PV) were calculated from Hb mass, [Hb] and Hct (after correction of body hematocrit by the factor 0.91 according to [18]. Venous blood was sampled into vacutainers during the morning and processed immediately as far as necessary. Reticulocyte number was counted in 1000 red cells using stained blood smears. Aliquots of whole blood, serum, plasma, and washed red cells were stored at ± 30 8C and later transported in dry ice by plane to Germany for measurements of aspartate-aminotransferase activity (ASAT, UV-test at 340 nm, 258, Bayer RA System for clinical chemistry) in hemolyzed erythrocytes as indicator of cell age [33], and EPO (chemoluminescence assay, Nichols Institute Diagnostics), iron (colorimetric test with Ferene-S, Bayer RA System), transferrin (immunturbidimetric test, Bayer RA System), ferritin (chemoluminescence assay, Nichols Institute Diagnostics), soluble transferrin receptor (stfr, immunturbidimetric test, Bayer RA System) and total plasma protein (biuret method, Bayer RA System) concentrations in serum. Exercise tests Peak oxygen uptake was determined in incremental tests on a bicycle ergometer (ER 900, Jaeger Wuerzburg, Germany, + 40 W every 3 minutes, beginning with 40 W in untrained and 80 W in trained subjects) up to subjective exhaustion, using a Quinton Metabolic Cart QMC-TM for gas measurements. Because of nonappearance and technical faults, valid data were obtained for only 12 UT-Bogotµ. Tests in Berlin, which had originally been planned for other purposes, were performed on a treadmill (Woodway Ergo XELG, Weil a. Rh., Germany) using an Oxycon Gamma apparatus (Mijnhardt B. V., AE Bunnik, Netherlands) for determination of oxygen uptake (starting velocity 2.6 m/s, increase by 0.4 m/s every 3 min with 30 s rest interval). Since peak oxygen uptake with the cycle ergometer tends to be lower than with the treadmill (5±10% according to [13]), we have substracted 5 % from the measured values for comparison with the highlanders. Statistical analysis Statistical evaluation (SPSS for Windows, Release 9.0.1, 1999) was performed through analysis of variance with subsequent t-test after Bonferroni correction and regression analysis. In the case of [EPO] which were not normally distributed we applied the Kruskall-Wallis test and the Jonckheere-Terpstra test. Results Peak oxygen uptake Mean peak oxygen uptake of the subjects in Berlin amounted to ml/kg min ±1, a typical value for untrained young males. For the altitude of Bogotµ one might expect a reduction by 12 % [15] to ml/kg min ±1 which is very similar to the result for UT-Bogotµ ( ml/kg min ±1 ). Peak oxygen uptake of TR Bogotµ was markedly higher ( ml/ kg min ±1, P < compared to both UT groups). Blood composition and volumes The results of the measurements are presented in Table 1. In UT-Bogotµ [Hb] and Hct were clearly higher than in the sealevel population corresponding to a 5 % decrease in SO 2. Also Hb mass and red cell volume were increased (12 and 14 %, respectively). The plasma volume, however, was lowered; thus, total blood volume did not differ from sea level values. In TR-Bogotµ all quantities except SO 2 were significantly different compared to UT-Bogotµ: [Hb], Hct and MCHC being lower, all other quantities higher. Hb mass was 11% greater than in UT-Bogotµ and correspondingly EV 16% (resulting from the lower MCHC). But even higher increased was the PV (+ 25 %). Both effects combined changed blood volume by + 21 %. Erythropoiesis and nutrition Compared to UT-Berlin there was a rise in the number of reticulocytes in UT-Bogotµ; in TR-Bogotµ it was much more increased (threefold) but with values scattering between 0.9 and 4 % (Table 2). Also ASAT was elevated in the athletes pointing to a reduced red cell age. [EPO] was highest in TR-Bogotµ, but no significant difference existed among all 3 groups inde-

3 574 Int J Sports Med 2001; 22 Böning D et al Table 1 Blood composition and volumes for untrained and trained males at sea level and altitude SO 2 [Hb] Hct MCHC Hb mass EV PV BV N % g/dl % g/dl g/kg ml/kg ml/kg ml/kg UT-Berlin UT-Bogotµ aaaa 17.4 aaaa 51.9 aaaa aaa 38.5 aaa 44.5 aaa TR-Bogotµ aaaa 16.0 bbbb 49.7 aaaabb 32.8 abb 14.7 aaaab 44.6 aaaabbb 55.8 aaabbbb aaaabbbb Measurements in ear-lobe blood. Values are means SE. UT untrained, TR trained, SO 2 oxygen saturation, Hct hematocrit, MCHC mean cellular Hb concentration, EV, PV, BV erythrocyte, plasma and blood volume, respectively. Significance of differences: altitude versus sea level a P < 0.05, aa P < 0.02, aaa P < 0.01, aaaa P < 0.001, TR-Bogotµ versus UT-Bogotµ correspondingly marked by b. Table 2 Erythropoiesis and iron metabolism for untrained and trained males at sea level and altitude Reticulocytes ASAT EPO Iron Transferrin Ferritin stfr N % U/g Hb U/l mmol/l g/l ng/ml mg/l UT-Berlin UT-Bogotµ aaaa TR-Bogotµ aaaa 3.4 a bbb 3.77 aabbb Measurements in venous blood. Values are means SE. UT untrained, TR trained, ASAT aspartate aminotransferase activity. EPO erythropoietin concentration, stfr serum transferrin receptor concentration. Significance of differences: altitude versus sea level a P < 0.05, aa P < 0.02, aaa P < 0.01, aaaa P < 0.001, TR-Bogotµ versus UT- Bogotµ correspondingly marked by b. pendent of altitude and training status. In both UT-Berlin and TR-Bogotµ there was one outlier each (75 and 159 U/l); if they are excluded, means and standard errors decrease to and mu/l, respectively; the differences remain, however, insignificant. Interestingly, the athlete at altitude with the high [EPO] had the lowest red cell and blood volume of TR-Bogotµ. In this group the ferritin level was low and the stfr concentration was high, indicating relatively small iron stores. Indeed the mean daily iron intake of 5 TR-Bogotµ amounted to less than 90% of the calculated daily need while 3 athletes supplemented iron orally; total caloric intake and protein supply were low, too (91 8 and 87 8%, respectively, of the requirement). In contrast all UT-Bogotµ consumed sufficient iron and protein; also, average total caloric intake was high except for 4 subjects (average 117 9%). Total plasma protein concentrations were not related to the nutrition, both altitude groups showed increased values (P < and 0.05, respectively) compared to the sea level subjects (UT-Berlin , UT-Bogotµ , TR-Bogotµ g/dl). Blood composition and peak oxygen uptake The correlations between various blood properties and peak VÇ O 2 are demonstrated in the following figures. For the Hb mass-peak VÇ O 2 relationship (Fig.1) two clusters independent of training are indicated, which both show a positive correlation but at different ranges of Hb mass. A very good curve fit can be obtained for each separate cluster (linear correlation coefficient left 0.77, right 0.96). It can also clearly be seen that the subjects with the highest Hb mass did not have the highest Fig. 1 Relationship between hemoglobin (Hb ) mass and peak oxygen uptake (VÇO 2 peak) in untrained (UT) and trained (TR) residents in Berlin and Bogotµ (35 and 2600 m of altitude, respectively). oxygen uptake. The sea level subjects fall completely on the edge of the left cluster after correction of their oxygen uptake to the altitude of Bogotµ. However, for the relation BV-peak VÇ O 2 this cluster difference disappears; if all subjects are pooled, a highly significant linear dependence is calculated (P < 0.001; Fig. 2). There is also a weak positive relationship between Hct and peak VÇ O 2 in each, UT-Berlin, UT-Bogotµ, TR-Bogotµ, which approaches significance (P < 0.1) for the untrained subjects at altitude (Fig. 3).

4 Hb Mass and Peak Oxygen Uptake at Altitude Int J Sports Med 2001; Fig. 2 Relationship between blood volume (BV ) and peak oxygen uptake (VÇO 2 peak) in untrained (UT) and trained (TR) residents in Berlin and Bogotµ (35 and 2600 m of altitude, respectively). Linear regression equation for all subjects: y = 0.49 x + 1.8, r = 0.65, P < Fig. 3 Relationship between hematocrit value (Hct ) and peak oxygen uptake (VÇO 2 peak) in untrained (UT) and trained (TR) residents in Berlin and Bogotµ (35 and 260 m of altitude, respectively). The linear regression for the pooled values of all UT (line) is significant (y = 0.46 x , r = 0.40, P < 0.05). After combining UT-Berlin and UT-Bogotµ, the common correlation coefficient becomes significant (P < 0.05). TR-Bogotµ, however, lies clearly above the regression line for the UT. Discussion Blood composition and volumes Our results for the Hb mass and the volumes of blood components in untrained lowlanders practically coincide with those published by Dill et al. [13], Heinicke et al. [19] and, after correction for the different calculation (no body Hct factor), also with those of Brotherhood et al. [9]. Green et al. [17] using 51 Cr and 125 I labelling measured similar EV ( ml/kg) but smaller PV ( ml/kg) in a comparable untrained group; in their experiments venous and body Hct were not different in contrast to older investigations [18, 25]. The findings for UT-Bogotµ (increased [Hb], reticulocyte number and Hb mass) demonstrate that this town is situated above a hypoxic threshold and that the small reduction of arterial oxygen saturation is sufficient to stimulate erythropoiesis. One might suggest that part of the increase in the Hb mass is related to the lower BMI indicating possibly less fat and more muscle tissue and thus more blood in the highlanders than in the lowlanders, but the blood volume was not increased in UT- Bogotµ. In addition, the body fat percentage may be markedly underpredicted in non-caucasians by the BMI as was recently found in an Asiatic population [12]. The overlap in Hb mass between sea level and altitude residents and the large range of values in the latter (Fig. 1) suggests an individual variation in the hypoxic threshold. The physiological cause for the rise in erythropoiesis remains unexplained, since [EPO] in blood was not significantly higher at altitude than at sea level. This corresponds to various studies in either sojourners (e. g. [4, 8]) or residents (e. g. [36, 41]) where no clear increase was found except for acute ascents. Possibly small changes in [EPO] might be sufficient for long term effects. However, one cannot exclude that the concentration during a long-term stay at altitude is only increased at times not investigated until now; [EPO] varies during the day with a peak in the night when the arterial oxygen saturation is especially low (10). One may also suggest that there is an additional unknown erythropoetic factor for long term acclimatization or that erythropoietin receptors on stem cells are independently upregulated during chronic hypoxia. The central reaction seems to be the stimulation of mrna production by the Hypoxia-Inducible Factor 1 for various cytokines, transporters and enzymes [38]. Hb mass and red cell volume were higher in UT-Bogotµ than in UT-Berlin (12 ± 14 %) by a similar amount like in trained runners at sea level [9,13,17,19]. The increase of the red cell volume did, however, not result in an augmentation of the blood volume because of the concomitant plasma volume decrease. This caused an additional increase in the Hb concentration and the classical view that the change of this quantity partly compensates for the decrease in arterial saturation at altitude, seems to be confirmed. An additional effect is an improvement of the diffusion capacity by the enlarged red cell area in the lungs as well as in the oxygen consuming tissues (e.g. [42]). But there remains a bottleneck for oxygen transport since peak VÇ O 2 does not reach sea level values. The TR-Bogotµ reacted in a very similar form like athletes at sea level [9,13,17,19]. Red cell volume, but even more, plasma volume increased leading to a decrease of [Hb] compared to UT-Bogotµ. It is a striking fact that man does not react to endurance training with an increase of [Hb] neither at low nor at high altitude. In our subjects the relative small iron stores of some athletes, as indicated by the low ferritin and high stfr concentrations in plasma, may explain part of the large difference in [Hb] between UT-Bogotµ and TR-Bogotµ, which is greater than for sea level residents. But also the insufficient nutrition with other substances plays a role: in the athletes there exists a positive correlation between protein consumption (g per kg body mass) and Hb mass (r = 0.65, P < 0.05 ). If some of them adjust their caloric and protein (and thereby also mineral) intake, this will probably solve the problem more physiologically than supplying iron and should reduce the [Hb] decrease. Already in 1980 Yoshimura et al. [43] demonstrated a positive effect of a protein rich diet on anemia in athletes.

5 576 Int J Sports Med 2001; 22 Böning D et al The additional increase in Hb mass in TR-Bogotµ is difficult to explain since there was again no consistent rise in [EPO]. This corresponds to various sea level investigations where [EPO] was neither significantly increased during or shortly after acute exercise, nor by systematic physical training [4, 6, 34]. A small rise of secretion might, however, be hidden by dilution in the enlarged plasma volume. The increase of the latter amounts to 10 and 25 %, respectively, compared to UT-Berlin and UT-Bogotµ; as, however, the distribution volume of the small peptide molecule EPO is probably the whole extracellular space, the effect cannot be quantified. In residents in La Paz (3600 m) the hormone concentration even decreased during the first hours after an exhaustive ergometer test [36]. However, 43 hours later [EPO] had slightly increased possibly caused by the Hct decrease resulting from the postexercise plasma volume expansion; similar observations have been made at sea level [6,34,37]. Thus it is conceivable that temporary, hardly detectable rises of [EPO] stimulate erythropoiesis sufficiently. On the other hand, Heinicke et al. [19] have suggested an increased production of the stfr caused by an increased sensitivity of erythroblasts for EPO after training. In this case a high [stfr] would not point to iron deficiency. A rise of [stfr] and especially of the stfr-ferritin ratio (multiplied by 100) has also been suggested as indicator of EPO doping [5,16]. As the ratio was as high (11.6) in TR-Bogotµ (in whom because of altitude, athletic level and lacking financial resources misuse of the hormone is improbable) as after 3 weeks of EPO administration (12.0) in athletes [5], application in doping control cannot be recommended. The additional stimulation of erythropoiesis in TR is also visible in the increased reticulocyte count. The number is higher than in the UT only in part of the TR resulting from the fact that each bout of intensive exercise causes a temporary rise for 2 ± 3 days (e. g. [34, 36]). The low MCHC as well as the high ASAT activity are indicators for a reduced mean cell age (e.g. [33]). In a long-term view this is only possible if concomitant to the increased erythropoiesis the red cell life-time is reduced. For sea level training it is known that exercise induced hemolysis destroys especially old erythrocytes [27, 35]. Blood composition and peak VÇO 2 There is a weak correlation between Hct and peak VÇ O 2 in the UT groups. However, if the relationship is calculated between the changes (DHct and DVÇ O 2 peak), also TR-Bogotµ can be included and the significance is improved (DVÇ O 2 peak = 0.8DHct 0.1, r = 0.39, P < 0.02). Thus peak VÇ O 2 increases by roughly 1 ml/ kg min ±1 for 1 % rise in Hct (i.e. approx. 2% relatively) independent of alveolar oxygen pressure and fitness state. This change is similar to that observed after experimental EPO administration [1, 5]. Since [Hb] as well as Hct were lower in TR-Bogotµ than in UT- Bogotµ, the large difference in peak VÇ O 2 is clearly caused by other more important factors like training of heart and skeletal muscles. Also total blood volume seems to play a role: a rise of peak VÇ O 2 by 1 ml/kg min ±1 was accompanied by 2 ml/kg increase in blood volume (i. e. approx. 2.5 % in UT). One might suggest that also improvements by erythrocyte infusion or erythropoietin application [1, 5, 32] are not only caused by the [Hb] increase but to some amount by an enlarged blood volume. Volume effects might result from improved filling of heart and vessels whereas effects of a high blood oxygen capacity are likely to be compensated for in part by a reduction of cardiac output, resulting from the increase in viscosity. Also the suggested positive effects of altitude training on sea level performance cannot be attributed to a high [Hb] because this is rapidly normalized by hemodilution after return to sea level [7, 8,13]. Considering the relation between Hb mass and VÇ O 2peak, one might suggest that there is no continuum of hypoxic thresholds but that two groups with different hypoxic stimulation of erythropoiesis exist (independent of training). However, those who react less may compensate by increasing their plasma volume. Thus, for the relation between blood volume and VÇ O 2 peak a separation into two clusters can no longer be detected. As similar dependencies between iron supply (ferritin or stfr concentrations) and VÇ O 2 peak do not exist, the separation, possibly, has a genetic base. Various authors (e. g. [3, 41]) have shown that Sherpas and Tibetans have lower [Hb] and lower arterial oxygen saturation than high altitude Indians, all being well adapted to the hypoxic environment. Beall et al. [3] present evidence for a hereditary origin. To compensate for the increase in blood viscosity, the fibrinogen concentration seems to be decreased in Indians; allele frequencies of fibrinogen polymorphisms associated with high fibrinogen concentration are extremely low in Quechua from the Peruvian Altiplano [28]. In the Colombian subjects a genetic heterogeneity is conceivable. The population in Bogotµ descends from aborigines living at moderate altitude possibly since years and lowlanders who have immigrated from Western Europe and Africa over the last 500 years. The proportion of Amerindian heritage has been calculated as approximately 28 % for the nuclear [30] and 78 % for the mitochondrial part (G. Keyeux, personal communication). Interestingly 27 % of the altitude residents in our study belonged to the right shifted cluster in Fig. 1. To finally confirm a genetic influence on Hb mass, not only a larger number of experiments is necessary; in addition a responsible gene has to be detected. Possible candidates are the suggested genes for hemoglobin concentration and arterial oxgen saturation [2]. Conclusions At 2600 m above sea level a hypoxic threshold for erythropoietic stimulation in altitude residents has already been transgressed. There is, however, a large scattering which might have genetic causes. The effect of endurance training on hemoglobin mass and plasma volume as well as the importance of changes in these quantities for peak oxygen uptake is similar in highlanders and lowlanders. The role of EPO for long-term altitude acclimatization and for training effects on erythropoiesis remains questionable.

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7 578 Int J Sports Med 2001; 22 Böning D et al 39 Videman T, Lereim I, Hemmingsson P, Turner MS, Rousseau-Bianchi MP, Jenoure P, Raas E, Schönhuber H, Rusko H, Stray-Gundersen J. Changes in hemoglobin values in elite cross-country skiers from 1987 to Scand J Med Sci Sports 2000; 10: 98± Weil JV, Jamieson G, Brown DW, Grover RF. The red cell mass-arterial oxygen relationship in normal man. J Clin Invest 1968; 47: 1627± Winslow RM, Chapman KW, Gibson CC, Samaja M, Monge CC, Goldwasser E, Sherpa M, Blume FD, Santolaya R. Different hematologic responses to hypoxia in Sherpas and Quechua Indians. J Appl Physiol 1989; 66: 1561± Wu EY, Ramanathan M, Hsia CCW. Role of hematocrit in the recruitment of pulmonary diffusing capacity: comparison of human and dog. J Appl Physiol 1996; 80: 1014± Yoshimura H, Inone T, Yamada T, Shiraki K. Anemia during hard physical training (sports anemia) and its causal mechanism with special reference to protein nutrition. World Rev Nutr Diet 1980; 35: 1±86 Corresponding Author: Prof. Dr. D. Böning Institute of Sports Medicine University Hospital Benjamin Franklin, Free University Berlin Clayallee 225C Berlin Germany Phone: + 49 (30) Fax: + 49 (30) boening@zedat.fu-berlin.de