Nasso, R., et al.: sport training and erythropoiesis Sport Science 12 (2019) 1: 11-15

ALTITUDE SPORT TRAINING AND ERYTHROPOIESIS

Rosarita Nasso1,2,Valentina Pagliara1, Martina Simonetti2, Rosaria Arcone1 and Mariorosario Masullo1

1University of Naples “Parthenope”, Naples, Italy 2University of Naples Federico II, Naples, Italy

Review paper

Abstract In this paper we report the effect of altitude training on blood levels. Over 1500 meters of altitude, athlete training and competing undergoes acclimatization to environmental which, in turn, induces metabolic and cardiorespiratory adaptations. Acclimatization improves endurance performance which is accompanied by an increase (3%) of maximal uptake (VO2 max)which preserve skeletal muscle structure and function. Studies on molecular mechanisms underlying this adaptation demonstrated that, after 3 weeks at altitude, either circulating erythropoietin and the soluble transferrin receptor levels were significantly increased compared to the values measured at sea level. In addition, concentration also increases indicating anerythropoiesis stimulation. These results demonstrated that improvements in endurance sport performance by altitude training (4 weeks) involves erythropoietin mediated stimulation of erythropoiesis which enhances maximal oxygen transport capacity in athletes.

Key words: altitude training, erythropoietin, hemoglobin, maximal oxygen uptake.

Introduction

A sport physical activity can be considered carried the VO2max decreases by 10% every 1000 m of out at high when it takes place over 1500 additional altitude (Smith et al.,1984). Thus, the meters, also considering the finding that at lower further you are away from the sea level, the more altitudes no significant changes in the performance the respiratory muscle strength decreases (Levin et have been noted. Acclimatization to environmental al., 1992). Furthermore, under stress condition, a hypoxia initiates a series of metabolic and similar behaviour applies to the diaphragm cardiorespiratory muscle adaptations that influence movement (Levin et al., 1992). Both cases are a the transport and use of oxygen within the consequence of hypoxia and hyperventilation organism (Chapman et al., 1985). Although on caused by altitude, since the lowering of oxygen altitude training there are discordant supply decreases the energy supply to the muscles opinions,including the finding that the performance (Yamamoto et al., 1993). As pO2 decreases, does not always improve at sea level, elite athletes hemoglobin saturation is also reduced (from 98% continue to devote considerable time and resources at sea level to 92% if we are at 2,439 m). While to these types of training (Stray-Gundersen et al., the arterial pO2 decreases with altitude, the tissue 2001). In recent years, scientific research has pO2 remainsalmost unchanged, at least up to focused on optimizing the theoretically beneficial reasonable heights (about 2,500 m), so the aspects of acclimatization caused by altitude. difference between these two conditions (pressure Regardless of the altitude, while the composition of gradients) is drastically reduced. The passage of the breathing air is similar (its composition varies oxygen from the blood to the tissues depends above 13,500 m), the partial pressure of the precisely on this pressure gradient. Lowering the individual gas changes. Moreover, every 150 arterial pO2 is one of the main causes of the meters, the temperature decreases by 1°C. These decline in VO2max at high altitudes (Płoszczyca K., conditions, can lead to dryness and irritation of the et al. 2018). The decrease in VO2max starts to be mucous membranes and the walls of the alveoli. relevant after 1,500-1,600m, when atmospheric After three weeks of "adaptation" the body pO2 falls below 125 mmHg. Several studies showed develops defence systems, improving the that staying at certain altitudes can induce the vascularization of the mucous membranes. Even for body to develop a certain tolerance to hypoxia the above mentioned reasons, athletes training and (Wolski et al., 1996; Young et al., 1988). After a competing at high altitudes have a greater need for period of between 18 and 57 days, subjects who in water to compensate for the huge water losses of the past had been exposed to similar conditions, their bodies, especially for endurance sports. The after the initial drop in VO2max, showed a fairly partial pressure of oxygen (pO2) decreases with good improvement of this parameter, without increasing altitude, resulting in less oxygen level in changing their aerobic capacity. However, even the pulmonary alveoli, and in lower oxygen with a certain acclimatization, the maximum saturation of the arterial blood. These findings lead O2uptake at altitude will never be comparable to to a decrease in VO2max (maximum oxygen that near the sea level. To adapt to changes in the uptake), which affects aerobic endurance respiratory system, the cardiovascular system also performance. At an altitude of more than 1,500 m, undergoes modifications.

11 Nasso, R., et al.: Altitude sport training and erythropoiesis Sport Science 12 (2019) 1: 11-15

In fact, in the first 24-48 h of permanence at intrinsic O2 affinity for Hb as well as changes in the certain altitudes, there is a 25% reduction in responsiveness to allosteric effectors. Both plasma volume due to a certain loss of water properties can be related to modifications through breathing. However, the body performs a attributable to amino acid replacements in the α- series of adaptations that lead to an increase in and/or β-type subunits (Weber, 2007; Bellelli et al., blood volume, which at least partially compensates 2006; Storz and Moriyama, 2008). The reversibility for the reduction in pO2. After a stay of about 6 of oxygen-affinity changes can be achieved either months at an altitude of 4,000 m, the blood volume by a Hb-isoform composition (Hoffmann et al., increases by about 9-10%, requiring a greater 2012; Storz et al., 2011a, 2013; Storz, 2016b) or production of red blood cells and an expansion of through changes in the composition/concentration the plasma volume. Moreover, hypoxia also affects of the medium in which the protein carries out its the functions of the Central Nervous System (CNS), function (Nikinmaa, 2001; Jensen, 2009).The causing a reduction in the capacity for analytical relationship between the adaptation of affinity of thinking, the capacity for decision making and Hb towards oxygen and the altitude depended on judgment, an increase in nervousness, a worsening the reduced O2 availability. In fact, the reduced of sensory performance (which may depend on a pO2 of inspired air leadsto a corresponding decrease in visual accuracy caused by hypoxia), a reduction in the pO2 of arterial blood. Therefore, decrease in coordination capacity, and an increase under these conditions, a reduction in O2 flux from in sleep disturbance. The most favourable altitude the capillary blood to the cells of perfused tissue for training camps is between 2,000 and 3,000 m, will occur and,in turn, a variation in the blood which corresponds to a reduction in the available pressure is required for best adaptation. Finally, it O2 from 16 to 24% (Gledhill, et al., 1985). At an can be concluded that a reduced affinity of Hb altitude of less than 1,800 m, physiological towards O2 is generally helpful under moderate adaptations are too weak to allow a tangible hypoxia; vice versa, an increased affinity is useful improvement in sports performance. On the other under severe hypoxia (Turek et al., 1973; West and hand, at higher altitudes (beyond 3,000 m) air Wagner, 1980; Bencowitz et al., 1982; Willford et conditions become extreme, not allowing good al., 1982; Samaja et al., 2003; Scott and Milsom, physiological adaptations that compromise sports 2006). performance. The bulk of adaptations occur within two weeks of arrival at high altitude, so a stay of 2- Altitude adaptation and erythropoietin 3 weeks, combined with the right workouts, is considered optimal for achieving an improvement in The organism adaptationto the altitudeandthe sports performance (Gledhill et al., 1985). In fact, corresponding physiological responses vary from from the 22nd day of stay onwards the athletes' person to person. Under this regard, two categories performances start to have some significant drops of subjects, namely responders and non- (Ekblomet al., 1991). Furthermore, at least responders, can be identified: the former react regarding to endurance activities, repeating a positively to hypoxia due to high altitude, the training period at altitude during the year gives others do not. From a metabolic point of view, this better results than a single training camp at too finding can be correlated to the levels of long a level. Erythropoietin (EPO) (Metra et al., 1991). In fact, a studyperformed on a group of 48 athletes (32 men Altitude adaptation of hemoglobin-oxygen affinity and 16 women) showed that in the first 24 hours of training at 2,800 m, changes in plasma levels of In whole human body, all the oxygen is transported EPO are very different from person to person bound to hemoglobin (Hb) in erythrocytes(Perutz, (Celsing et al., 1987). The cause of this discrepancy 1970; Baldwin and Chothia, 1979), that derived would seem to be genetically determined and due from precursor stem cells. Oxygen affinity of Hbhas to individual polymorphisms of the EPO gene or the been vastly studied. Hb is a tetrameric protein EPO receptor gene. EPO is the major growth factor exhibiting a heterodimeric quaternary structure involved in erythropoiesis, a complex physiological composed of two α-and two β-chainssubunits process that controls of oxygen levels paired, in a semi-rigid αβ dimers. Each subunit in the body (Calabrò et al., 2008). EPO is a contains a heme group that can reversibly bind a , belonging to the large class 1 cytokine single O2 molecule. The α2β2 tetramer, undergoes family. EPO is mainly produced by interstitial cells a symmetrical transition during the oxygenation of the and to a lesser extent by the liver and process, in which the quaternary structure the brain (Elliott et al., 2008); once released into oscillates between a high-affinity relaxed and the bloodstream, it is transported to the bone oxygenated state (R) and a low-affinity tense and marrow where it acts as a promoter of proliferation deoxygenated state (T). The conformational and differentiation of the progenitor cells of the transition T<–>R is based on a homotropic erythroblasts (Calabrò et al., 2008). EPOconsists of allosteric effect exerted by oxygen binding itself a single polypeptide chain lasting 165 amino acids and an heterotropic allosteric effect exerted by with a molecular mass of 34 kDa. The other ligands, such as proton, CO2 and 2,3- erythropoietin gene encodes a protein of 193 amino bisphosphoglycerate (BPG),involved in the Bohr acids and the removal of 27 amino acids generates effect, bound even at remote sites from the heme a mature protein that undergoes a post- pocket (Perutz, 1970; Baldwin and Chothia, 1979). translational glycosylation. In addition, it contains Hb-O2 affinity changes can include variation in the two disulfide bridges essential for the biological

12 Nasso, R., et al.: Altitude sport training and erythropoiesis Sport Science 12 (2019) 1: 11-15 activity. The tertiary structure is very similar to that increasing the secretion of EPO. Certain conditions, of other haematopoietic factors and contains four such as hypoxia caused by altitude, urge the kidney antiparallel helices joined together by loops of to increase the production of the hormone even up variable size. The synthesis of EPO depends on the to a hundred times (Calabrò et al., 2008). The developmental stage; in fact, in the foetal phase, mechanism is based on the presence of ahypoxia- the protein is produced mainly in the liver, whereas inducible heterodimeric transcription factor(HIF-1), in the adult the main site of expression is the consisting of the inducible HIF1α subunit, and the kidney (Ebert and Bunn, 1999). The moment in nuclear constitutive HIFβ subunit, which increases which this switch takes place is species-specific and the expression of the erythropoietin gene. HIF-1α is in spite of this, the liver always maintains the unstable in the presence of oxygen and is rapidly ability to synthesize a certain level of the protein. degraded by the prolylhydroxylase with the Under normal conditions, the ratio of protein contribution of the von Hippel-Lindau protein expression levels in the kidney and liver is 9:1, (Gnarra et al., 1996) an angiogenic factor working whereas under conditions of severe hypoxia it has in strict cooperation with other angiogenic factors, been estimated that the production of EPO in the such as vascular endothelial growth factor (VEGF) liver reaches about 33% of the total amount and metalloproteinases (MMPs) (Kim et al. 2018; (Rankin et al., 2007). Erythropoietin is involved in Pagliara et al., 2014, 2108; Di Maro et al. the processes leading to the formation of 2014.During hypoxia, prolylhydroxylase is inactive, erythrocytes, promoting the division and consequently HIF-1α accumulates by activating the maturation of the erythroid precursors, the pro- expression of the erythropoietin gene, which in turn herytroblasts (BFU-E, CFU-E) (Jelkmannet al., stimulates the rapid expansion of erythroid 2004) and inactivating their apoptotic pathway, progenitors (Jelkmann et al., 2004; Wang et al., which leads to programmed cell death (Kouryet al., 1995; Wang, et al., 1993). Although there is a 1992; De Maria et al., 1999); pro-erhytroblasts direct correlation between the concentration of EPO take about a week in the to in the serum and the production of red blood cells, differentiate, mature, incorporate hemoglobin and the variation in the speed/extent of erythropoiesis iron, and once released into the blood, acquire the is lower than that in EPO levels. In fact, it has been morphology of mature erythrocytes (Calabròet al., seen that the factor that most influences the 2008). increase in the number of erythrocytes is not the absolute quantity of EPO, rather the time over In physiological conditions, EPO is present in the which EPO concentration is kept constant (Elliott et blood in small quantities. It has been observed that al., 2008). serum concentrations, ranging from 10 to 25 mU/ml, maintain hemoglobin levels within the Conclusion optimal range of 12-17 g/dl (Erslev et al.,1991; Groopman et al., 1999). pO2 directly regulates EPO The potential benefits of altitude training have long levels as low gas pressure in creasesits production. been the subject of many studies. Numerous Indirectly, low hemoglobin levels stimulate EPO experimental evidences have shown that hypoxic production. The discovery of EPO and its environment affects endurance sport performance fundamental role in the oxygen supply to tissues, by mechanisms based on O2 transport by red blood dates back to studies carried out in a high altitude cells to exercising muscles. As reported above, environment around the middle of the nineteenth athlete training in altitude, at least 250 h at 2,200- century. Studying high altitude animals (4,000 m), 2,500 m, resulted in an oxygen dependent increase a higher concentration of red blood cells associated of total Hb which has been correlated to elevated with a better availability of oxygen to the tissues levels of EPO. The elevated EPO production in compared to low quote was found. In addition, the hypoxia is a key factor in modulation of finding that the erythropoiesis process is stimulated hematological variables. However, the increase of acutely when the amount of oxygen in the blood is hypoxia induced EPO levels does not guarantee the reduced, confirmed this hypothesis (Jelkmann hematological improvements after the return to the 2007).During altitude training, the EPO levels sea level. Furthermore, many other variables and increase significantly after the first to third days at factors released from exercising muscle cells such altitude and then, after reaching the maximal as H+, CO2, and lactate into blood capillaries can increase, they return to baseline levels (Płoszczyca promote the O2 release from Hb. Further scientific K. et al., 2018). Erythropoiesis is controlled by a research in this field will allow the understanding of very sensitive feedback mechanism, which uses a factors involved in hematologic variables in sensor at the renal level, and is able to identify athletes’ altitude training to improve sports alterations in tissue oxygenation and respond by performance.

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Received: April 25, 2019 Accepted: June 5, 2019 Correspondence to: Rosaria Arcone Parthenope University, Naples, Italy E-mail: [email protected]

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