KOISTINEN, P. O., H. RUSKO, K. IRJALA, A. RAJAMÄKI, K. PENTTINEN, V-P. SARPARANTA, J. KARPAKKA, and J. LEPPÄLUOTO. EPO, red cells, and serum transferrin receptor in continuous and intermittent hypoxia. Med. Sci. Sports Exerc., Vol. 32, No. 4, pp. 800–804, 2000.
Purpose: Erythropoietic response in 10 healthy nonsmoking volunteers exposed to normobaric hypoxia continuously or intermittently 12 h daily for 7 d was evaluated in a randomized cross-over study.
Methods: An oxygen content of 15.4% corresponding to an altitude of 2500 m was created by adding nitrogen into room air in a flat. Venous blood samples for hemoglobin (Hb), hematocrit (Hct), reticulocytes, serum erythropoietin (S-EPO), red cell 2,3-diphosphoglycerate (2,3-DPG), serum ferritin (S-Ferrit), and serum soluble transferrin receptor (S-TransfR) were drawn at 8:00 a.m.
Results: S-EPO was increased from baseline values of 22.9 ± 9.6 and 20.5 ± 10.1 U·L−1 to 40.7 ± 12.9 (P < 0.05) and 35 ± 14.3 U·L−1 (P < 0.05) after the first night in continuous and intermittent hypoxia, respectively, and remained elevated throughout both exposures. Hb and Hct values did not show any significant changes. Red cell 2,3-DPG rose from baseline a value of 5.0 ± 0.8 to 5.9 ± 0.7 mmol·L−1 (P < 0.05) after the first day in continuous hypoxia and from 5.2 ± 0.7 mmol·L−1 to 6.1 ± 0.5 mmol·L−1 on day 3 (P < 0.05) during intermittent hypoxia. The reticulocyte count rose significantly (P < 0.05) after 5 d in both experiments. S-transferrin receptor level rose significantly from 2.2 ± 0.4 and 2.1 ± 0.5 mg·L−1 to 2.6 ± 0.5 mg·L−1 and 2.3 ± 0.6 mg·L−1 on day 5 (P < 0.05), to 2.7 ± 0.5 mg·L−1 and 2.5 ± 0.6 mg·L−1 on day 7 (P < 0.05) under continuous and intermittent hypoxia, respectively.
Conclusions: We suggest that intermittent exposure to moderate normobaric hypoxia 12 h daily for 1 wk induces a similar stimulation of erythropoiesis as continuous exposure.
Exposure to hypoxic environment results in an acceleration of erythropoiesis that depends on the intensity and duration of the hypoxic exposure (3,4,7). A significant increase in plasma erythropoietin levels has been observed already after a 114-min exposure to normobaric or hypobaric hypoxia (FIO2 0.105 and 460–520 Torr), respectively (7,19). The rate of change from normoxic to hypoxic conditions also plays an important role in physiological responses to acute environmental hypoxia (12). Both clinical and experimental data show that even intermittent hypoxia stimulates erythropoietin production in humans (19,31) and rats (26). It has been proposed that “living high, training low” would be the best strategy to improve physical performance, by avoiding the detrimental effects of reduced training intensity at hypoxic conditions while retaining the benefits of altitude acclimatization (22). Intermittent normobaric hypoxia has recently been used by endurance athletes to stimulate erythropoiesis and to improve physical performance (28,29).
When training is performed under normoxic conditions, it is crucial that the intervening hypoxic exposures stimulate erythropoiesis to such an extent that the number of red cells increases (29). There are, however, no studies comparing the stimulation of erythropoiesis in continuous and intermittent normobaric hypoxia in man. Serum soluble transferrin receptor reflects the rate of erythropoiesis (2,15) in healthy iron-repleted subjects. The measurement of serum transferrin receptor gives a feasible noninvasive method for the estimation of erythropoietic activity in bone marrow. This is further strengthened by simultaneous measurements of serum erythropoietin (2).
We undertook this randomized cross-over study in order to evaluate stimulation of erythropoiesis at normobaric hypoxia under prolonged continuous or repetitive intermittent exposures. Serum soluble transferrin receptor and erythropoietin measurements were used as indicators of erythropoietic activity.
Health Center Hospital of Oulu, FINLAND; Research Institute for Olympic Sports, Jyväskylä, FINLAND; Department of Clinical Chemistry and Hematology, University of Turku, FINLAND; and Vuokatti Sports and Training Center, FINLAND; Department of Sports Medicine, Deaconess Institute, Oulu, FINLAND; Kainuu Central Hospital, Kajaani, FINLAND; and Department of Physiology, University of Oulu, FINLAND
Submitted for publication August 1997.
Accepted for publication August 1998.
Address for correspondence: Dr. Pentti Koistinen, Health Center Hospital of Oulu, Box 8, SF-90015 Oulu, Finland; E-mail: Pentti.Koistinen@ouka.fi.
Ten nonsmoking, healthy volunteers aged from 21 to 34 yr of age (five women, five men) gave their written informed consent to participate in the study after having been informed about the aim and design of the experiment. The study protocol was approved by the Ethics Review Board of the Medical Faculty, University of Oulu, Finland. All the subjects participating in this study were physiotherapy students, who continued to practice approximately 2 h daily also during both study periods. They were advised to reduce working intensity in hypoxia, so that the self-estimated strain of the work was equal to normoxic conditions.
The study was carried out at Vuokatti Sports Institute, where a low-oxygen facility has been created by adding nitrogen into room air in a flat of ca. 100 m2. The oxygen content of room air was adjusted to 15.4%, corresponding to an altitude of 2500 m. The oxygen content in the air-tightened apartments was carefully monitored by a double-control system without interruption. The subjects were randomly exposed to continuous or intermittent normobaric hypoxia for 7 d. During continuous exposure to normobaric hypoxia, the subjects did not leave the low-oxygen apartment on any occasion, even the meals were served there. During the intermittent episode, the participants slept in the low-oxygen apartment and spent additional time there in the evening, so that they were exposed to normobaric hypoxia for 12 h daily. The experiment was repeated after a wash-out period of 2 months in reversed cross-over fashion. All the subjects were sea-level residents and unacclimatized to altitude. The female participants were advised to take supplemental iron corresponding to a daily dose of 100-mg ferrous sulfate during the hypoxic exposures.
Venous blood samples for hemoglobin (Hb), hematocrit (Hct), serum erythropoietin, red cell 2,3-diphosphoglycerate, serum ferritin, and serum soluble transferrin receptor were drawn at 8:00 a.m. Serum ferritin values were determined by Delfia fluoroimmunoassay kits (Wallac, Finland). For the analyses of red cell 2,3-diphosphoglycerate, blood was taken into heparinized test tubes, centrifuged, and deproteinized immediately by perchloric acid. The supernatants were assayed by kits provided by Boehringer (Ingelheim, Germany). The individual concentrations of 2,3-diphosphoglycerate are expressed proportional to the number of blood erythrocytes per L. Serum erythropoietin was measured before and during the altitude training by a commercial kit provided by Ramco Laboratories, Inc. (Houston TX). Serum soluble transferrin receptor was measured by a quantitative immunoenzymometric assay from Orion Diagnostica (Finland).
The data were analyzed using two-way analysis of variance with repeated measures with duration of hypoxia and exposures (12 h vs 24 h) as factors, followed by Student-Newman-Keuls tests for multiple comparison.
Values for serum erythropoietin, Hb, red cell 2,3-DPG, serum transferrin receptor, and reticulocytes are shown in Figures 1 and 2. S-EPO increased from baseline values of 22.9 ± 9.6 and 20.5 ± 10.1 U·L−1 to 40.7 ± 12.9 (P < 0.05) and 35 ± 14.3 U·L−1 (P < 0.05) during the first night in continuous and intermittent exposures, respectively. The rapidity of the onset of hypoxic exposure could have caused the sharp rise in S-EPO during the first night. As judged from ANOVA or a multiple comparison test, there was no statistically significant difference on any of the days in S-EPO values between continuous and intermittent exposures. Hb and Hct values did not show any statistical changes during hypoxic exposures either. Compared with values at normoxia, 2,3-DPG rose from 5.0 ± 0.8 to 5.9 ± 0.7 mmol·L−1 (P > 0.05) on the first morning in continuous hypoxia and remained on that significantly higher level through the rest of the exposure. Under intermittent normobaric hypoxia 2,3-DPG showed a statistically significant rise from a baseline value of 5.2 ± 0.7 mmol·L−1 to 6.1 ± 0.5 mmol·L−1 from day 3 (P < 0.05) throughout the exposure. No statistically significant differences were observed between the 2,3-DPG values measured during continuous and intermittent hypoxic exposures. Serum soluble transferrin receptor was measured before the experiments and on day 5 and day 7 under hypoxia. Serum transferrin receptor concentration rose significantly from 2.2 ± 0.4 mg·L−1 (baseline) to 2.6 ± 0.5 mg·L−1 on day 5 (P < 0.05) and to 2.7 ± 0.5 mg·L−1 on day 7 (P < 0.05) under continuous hypoxia. Correspondingly, serum transferrin receptor level rose from 2.1 ± 0.5 mg·L−1 to 2.3 ± 0.6 mg·L−1 on day 5 (P < 0.05) and to 2.5 ± 0.6 mg·L−1 on day 7 during intermittent exposure to normobaric hypoxia. Reticulocytes showed a statistically significant rise on day 5 and 7 in both continuous and intermittent hypoxia (P < 0.05). Serum ferritin was measured before each of the two experimental periods. Serum ferritin was 47.2 ± 34 μg·L−1 (mean ± SD) before the continuous and 37.9 ± 20.3 μg·L−1 before the intermittent hypoxic exposure. The values did not differ significantly. All ferritin measurements were within the normal limits given by the laboratory.
We observed here that serum EPO increased after the first day by 78% in continuous and by 71% in intermittent normobaric hypoxia corresponding to the altitude of 2500 m. This increase seems to be greater than reported earlier at moderate altitude (3,18) and after short-term intermittent normobaric hypoxia (19,32), evidently due to differences between hypoxic stresses and rates of hypoxic exposures. After the initial peak, serum EPO decreased slightly but remained elevated (P < 0.05) during all days under both continuous and intermittent normobaric hypoxia. It has been documented that circulating levels of erythropoietin increase in relation to the severity of ambient hypoxia and duration of the hypoxic stimulus (1,7,19). Under exposures to both high and moderate altitudes serum erythropoietin has been shown to peak from day 1 to day 5 after the ascent and to decline thereafter toward baseline levels (1,3,8,19). Several additional factors facilitate physiological responses to acute hypoxia, such as the rate of shift from normoxic to hypoxic conditions (12). Repeated abrupt changes from normoxic to hypoxic conditions are probably the feature in this experiment that could explain the delayed stimulation of erythropoietin during the intermittent hypoxic exposures. Acid-base balance and work performed under hypoxia also influence the secretion of erythropoietin at hypoxia (9,23,32). Neither maximal nor submaximal exercise has an immediate effect on erythropoietin secretion at normoxia or hypoxia. Submaximal exercise has been reported to cause a late-onset rise in serum erythropoietin. This is in contrast to maximal exercise that seemed to hamper erythropoietin secretion or even to decrease serum erythropoietin (32). In the present study, the light work performed under continuous hypoxia should have further stimulated erythropoietin secretion compared with intermittent exposure, where the subjects worked during daytime in normoxic conditions but were rather sedentary under hypoxic exposure. The results in serum erythropoietin measurements did, however, show quite different figures. Serum erythropoietin peaked after the first night up to a significantly higher level than the prestudy value or the values obtained during consequent measurements during continuous hypoxia. All the measurements of serum erythropoietin under intermittent exposure were statistically significantly higher than the prestudy value but were more evenly distributed, so that no statistical differences were observed between measurements during the intermittent exposure.
Concentration of red cell 2,3-DPG increased on the first day in continued hypoxia and remained elevated (P < 0.05) throughout the exposure. Under intermittent hypoxia, 2,3-DPG increased significantly on the third day and remained elevated (P < 0.05) during the rest of the exposure. These results are in accordance with earlier findings (18,25). Mairbäurl et al. (25) studied training male students at 2300 m and observed a significantly higher rise in 2,3-DPG in the exercising group than in controls. The greater physical activity under continuous hypoxia could partly account for the more rapid rise of 2,3-DPG under continuous hypoxia in this study. On the other hand, the coincidence of the rise in 2,3-DPG with increased erythropoietin secretion is in accordance with the earlier findings that the increase of red cell 2,3-DPG is one of the first reactions to acute hypoxia and reflects the severity of hypoxic stress (20,21,24). The reticulocyte number increased steadily during exposures to both continuous and intermittent hypoxia and reached in both cases a significant rise (P < 0.05) on days 5 and 7. The late increase in reticulocyte number only during the last 3 d of the 7-d hypoxic exposure explains why no significant changes in Hct or Hb were attained.
This is the first experiment where changes in serum soluble transferrin receptor prompted by ambient hypoxia have been studied. In the present study, the serum transferrin receptor showed a significant increase on days 5 and 7 (P < 0.05) both in continuous and intermittent exposures to normobaric hypoxia. The same magnitude in erythropoietin stimulation and rise of serum transferrin receptor indicates equal erythropoietic response during both continuous and intermittent normobaric hypoxia. The level of serum transferrin receptor in normal subjects reflects the rate of erythropoiesis except in iron deficiency, where an unproportional increase from 2- to 15-fold is observed (2,15). It has been suggested that normal iron stores may not always be sufficient for the highly accelerated erythropoiesis after administration of recombinant human erythropoietin (r-HuEPO), even in case of simultaneous oral iron supplementation (5,31). Studies with r-HuEPO has shown a pronounced increase in Hct and a 2–3 fold rise in serum transferrin receptor, which indicates a far more accelerated erythropoiesis than in our study, where Hb and Hct values did not show any significant changes during the 7-d study period. Normal values of serum ferritin and serum transferrin receptor before hypoxic interventions in our subjects indicate normal reticuloendothelial iron stores. Additionally, keeping in mind that our hypoxic stress was moderate, it can be assumed that iron delivery was sufficient to provide a normal erythropoietic response (13,14). This is further supported by the coincidence of the reticulocyte response and the rise in serum transferrin receptor, which thus seems to reflect an increased rate of erythropoiesis.
Erythrocythemia induced by exogenous erythropoietin or blood transfusion has been shown to benefit exercise performance both at sea level (10,11,33,34) and under hypoxia (28), which is the main reason why altitude training is so generally incorporated in endurance training. However, studies of the effects of training at moderate altitude on physical performance have yielded variable results (35) even with improved hematological parameters (16,18). This is probably due to the deleterious effects of a reduced training intensity at altitude on the neuromuscular system, which has been proposed to oppose or even override the benefits in oxygen transport gained by the acclimatization process (22,30,35). Our results are novel and important from the training point of view indicating that daily intermittent exposures to normobaric hypoxia at sea level can yield the same hematological effects as living at the corresponding altitude. It should be considered that S-EPO, S-transferrin receptor concentration, and reticulocyte number are indirect measures of red cell production. Interestingly, our findings support those of a very recent study, where the same increase in the red cell number was observed in subjects living and training at 2500 m as in those living at 2500 m and training at 1250 m (22). It should be considered, however, that their subjects were affected by a rather severe hypoxia during the training sessions at 1250 m, with oxyhemoglobin saturations down <80% (22).
We did not measure red cell mass in this 7-d experiment because a detectable rise in red cell mass in moderate hypoxia would take a considerably longer time and should be established in the future studies (4). Twelve hours of daily intermittent hypoxia was thought to be the maximum dose that could easily be incorporated into training. The level of induced hypoxia of 2500 m was suggested to be sufficient for erythropoietic stimulation but still well tolerated by the participants. The effect of other levels of altitude and duration should certainly also be studied further.
The suggestion that living at normobaric hypoxia as described in the present paper and training at sea level provides an optimal setting for enhancing performance in endurance exercise remains to be established.
This study was supported by the Finnish Olympic Committee and the Ministry of Education (DNRO 145/722/96). We would like to thank Ms. Liisa Tiusanen, Ms. Maria Uusimäki, Ms. Seija Linnaluoto, Mr. Kari Kajaus, and Mr. Teuvo Ylikoski for their expert assistance.
1. Abbrecht, P. H., and J. K. Littell. Plasma erythropoietin in men and mice during acclimatization to different altitudes. J. Appl. Physiol. 32:54–58, 1972.
2. Beguin, Y., G. K. Clemons, P. Pootrakul, et al. Quantitative assessment of erythropoiesis and functional classification of anemia based on measurements of serum transferrin receptor and erythropoietin. Blood 81:1067–1076, 1993.
3. Berglund, B., S. J. Fleck, J. T. Kearney, et al. Serum erythropoietin in athletes at moderate altitude. Scand. J. Med. Sci. Sports 2:21–25, 1992.
4. Berglund, B. High-altitude training: aspects of hematological adaptation. Sports Med. 14:289–303, 1992.
5. Brugnara, C., L. A. Chambers, E. Malynn, et al. Red cell regeneration induced by subcutaneous recombinant erythropoietin: iron-deficient erythropoiesis in iron-replete subjects. Blood 81:956–964, 1993.
6. Cahan, C., M. Decker, G. Rabinowitz, et al. Hormonal and biochemical markers associated with sleep apnea (Abstract). Am. J. Respir. Dis. 139:A81, 1989.
7. Eckardt, K.-U., U. Boutellier, A. Kurtz, et al. Rate of erythropoietin formation in response to acute hypobaric hypoxia. J. Appl. Physiol. 66:1785–1788, 1989.
8. Eckardt, K.-U., J. Dittmer, R. Neumann, et al. Decline of erythropoietin formation at continous hypoxia is not due to feedback inhibition. Am. J. Physiol. 258:F1432–F1437, 1990.
9. Eckardt, K.-U., A. Kurtz, and C. Bauer. Triggering of erythropoietin production by hypoxia is inhibited by respiratory and metabolic acidosis. Am. J. Physiol. 258:R678–R683, 1990.
10. Ekblom, B., and B. Berglund. Effect of erythropoietin administration on maximal aerobic power. Scand. J. Med. Sci. Sports 1:88–93, 1991.
11. Ekblom, B., A. N. Goldbarg, and B. Gullbring. Response to exercise after blood loss and reinfusion. J. Appl. Physiol. 33:175–180, 1972.
12. Hackett, P. H., D. Rennie, and H. D. Levine. The incidence, importance and prophylaxis of acute mountain sickness. Lancet 2:1149–1154, 1976.
13. Hillman, R. S., and P. A. Henderson. Control of marrow production by the level of iron supply. J. Clin. Invest. 48:454–460, 1969.
14. Hornbein, T. F. Evaluation of iron stores as limiting high altitude polycythemia. J. Appl. Physiol. 17:243–245, 1962.
15. Huebers, H. A., Y. Beguin, P. Pootrakul, et al. Intact transferrin receptors in human plasma and their relation to erythropoiesis. Blood 75:102–107, 1990.
16. Ingjer, F., and K. Myhre. Physiological effects of altitude training on elite male cross-country skiers. J. Sports Sci. 10:37–47, 1992.
17. Klausen, T., U. Ghisler, T. Mohr, et al. Erythropoietin, 2,3-diphosphoglyserate and plasma volume during moderate-altitude training. Scand. J. Med. Sci. Sports 2:16–20, 1992.
18. Klausen, T., T. Mohr, U. Ghisler, et al. Maximal oxygen uptake and erythropoietic responses after training at moderate altitude. Eur. J. Appl. Physiol. 62:376–379, 1991.
19. Knaupp, W., S. Khilnani, J. Sherwood, et al. Erythropoietin response to acute normobaric hypoxia in humans. J. Appl. Physiol. 73:837–840, 1992.
20. Lenfant, C., J. Torrance, and C. Reynafarje. Shift of the O2
-Hb dissociation curve at altitude: mechanism and effect. J. Appl. Physiol. 30:625–631, 1971.
21. Lenfant, C., J. Torrance, E. English, et al. Effect of altitude on oxygen binding by Hb and on organic phosphate levels. J. Clin. Invest. 47:2652–2656, 1968.
22. Levine, B. D., and J. Stray-Gundersen. “Living high-training low”: effect of moderate-altitude acclimatization with low-altitude training on performance. J. Appl. Physiol. 83:102–112, 1997.
23. Mairbäurl, H. Red cell function in hypoxia at altitude and exercise. Int. J. Sports Med. 15:51–63, 1994.
24. Mairbäurl, H., W. Schobersberger, O. Oelz, et al. Unchanged in vivo P50
at high altitude despite decreased erythrocyte age and elevated 2,3-diphosphoglycerate. J. Appl. Physiol. 68:1186–94, 1990.
25. Mairbäurl, H., W. Schobersberger, E. Humpeler, et al. Beneficial effects of exercising at moderate altitudes on red cell oxygen transport and on exercise performance. Pflügers Archiv. Eur. J. Physiol. 406:594–599, 1986.
26. Moore-Gillon, J. C., and I. R. Cameron. Right ventricular hypertrophy and polycytemia in rats after intermittent exposure to hypoxia. Clin. Sci. 69:595–599, 1985.
27. Punnonen, K., K. Irjala, and A. Rajamäki. Serum transferrin receptor and its ratio to serum ferritin in the diagnosis of iron deficiency. Blood 89:1052–1057, 1997.
28. Robertson, R. J., R. Gilcher, K. F. Metz, et al. Effect of induced erythrocythemia on hypoxia tolerance during physical exercise. J. Appl. Physiol. 53:490–495, 1982.
29. Rusko, H. K., J. T. T. Penttinen, P. O. I. Koistinen, et al. A new solution to simulate altitude and stimulate erythropoiesis at sea level athletes. In:The Way to Win. Proceedings of the international Congress on Applied Research in Sports, August 9–11, 1994
, J. T. Viitasalo and U. Kujala, (Eds.). Helsinki: The Finnish Society for Research in Sport and Physical Education, 1995, pp. 287–289.
30. Rusko, H. R. New aspects of altitude training. Am. J. Sports. Med. 24:S48–S52, 1996.
31. Rutherford, C. J., T. J. Schneider, H. Dempsey, et al. Efficacy of different dosing regimens for recombinant human erythropoietin in a simulated perisurgical setting: the importance of iron availability in optmimizing response. Am. J. Med. 96:139–145, 1994.
32. Schmidt, W., K. U. Eckardt, A. Hilgendorf, et al. Effects of maximal and submaximal exercise under normoxic and hypoxic conditions on serum erythropoietin level. Int. J. Sports Med. 12:457–461, 1991.
33. Spriet, L. L., N. Gledhill, A. B. Froese, et al. Effect of graded erythrocythemia on cardiovascular and metabolic response to exercise. J. Appl. Physiol. 61:1942–1948, 1986.
34. Thomson, J. M., J. A. Stone, A. D. Ginsburg, et al. O2
transport during exercise following blood reinfusion. J. Appl. Physiol. 53:1213–1219, 1982.
35. Wolski, L. A., D. C. McKenzie, and H. A. Wenger. Altitude training for improvements in sea level performance. Sports Med. 22:251–263, 1996.
Chair: L. Bruce Gladden