Abstract
Blood rheology and hemodynamic parameters have never been explored together during acclimatization to altitude. This study aimed to investigate changes in blood rheology parameters and pulmonary hemodynamics during the first days of real moderate altitude exposure.
Seventeen athletes were tested at sea-level, 20 hours after their arrival at 2,400 meters of altitude (H1) and five days later (H2). Blood was sampled to analyze red blood cell (RBC) aggregation, blood viscosity and hematocrit. Pulmonary arterial pressure (PAP), pulmonary capillary pressure (Pcap) and pulmonary vascular resistance (PVR) were assessed by echocardiography.
We observed a rise in hematocrit, blood viscosity, RBC aggregation, PAP, Pcap and PVR between sea-level and H1. In H2, RBC aggregation, hematocrit, PAP, Pcap and PVR remained different compared to sea-level and no difference was observed between H1 and H2. Blood viscosity decreased in H2 and returned to sea-level values.
Our results suggest that hemoconcentration occurring within the first hours of altitude exposure increased blood viscosity, which contributed to the changes in pulmonary hemodynamic. When blood viscosity decreased in H2, no change occurred in pulmonary hemodynamic parameters suggesting that hypoxic pulmonary vasoconstriction was still present. The elevated RBC aggregation observed after in H2 could participate in the increase of Pcap.
Keywords
Introduction
Altitude exposure is associated with a drop in atmospheric pressure and partial O2 pressure leading to a decrease in arterial oxygen content and an inadequate O2 supply. This stressful situation is accompanied by specific adaptations, which are dependent on the level of altitude and duration of exposure [1–3], such as hypoxic pulmonary vasoconstriction [4, 5]. Recent studies suggested that hypoxic pulmonary vasoconstriction could depend on mitochondrial O2 sensing [6, 7]. Hypoxic pulmonary vasoconstriction serves to optimize ventilation-perfusion matching in focal hypoxia to enhance pulmonary gas exchange [5, 8]. However, when hypoxia is generalized to the entire pulmonary vascular bed, like during altitude exposure, hypoxic pulmonary vasoconstriction may cause a rise in pulmonary vascular resistance (PVR) and pulmonary artery pressure (PAP). PAP and PVR increase within hours after arrival at high altitude above 3,000 m [9] and persist upon prolonged exposure [10, 11], which may ultimately lead to pulmonary vascular remodeling [5]. However, if these responses are well reported in high altitude, only few studies [12–15] focused on the effects of moderate altitude (2,000–3,000 m) despite it is an usual altitude for short and prolonged sojourns, particularly with the development of outdoor sport activities.
It is well established that pulmonary artery pressure, like systemic artery pressure, is highly dependent on vascular resistance. According to the Poiseuille Law [16], vascular resistance depends on vessel diameters but also on blood viscosity. Blood is a non-newtonian shear thinning fluid whose viscosity is highly dependent on hematocrit but also on red blood cell rheological properties [17]. Few studies investigated the effects of hypoxia on blood rheology. Grau et al. [18] reported a decrease of red blood cell deformability after 12 h of hypoxic stimulus (normobaric hypoxia; FiO2 = 12.35%). RBC aggregation and blood viscosity have been shown to increase after 18 hrs at 4,500 m but no change occurred in RBC deformability or plasma viscosity [19]. To the best of our knowledge, blood rheology responses to prolonged moderate altitude are still unknown. The contributions of blood viscosity to PAP and PVR increase in hypoxia have been previously took into account using equations [20, 21] but no direct measurement has been made in the same time than pulmonary hemodynamics at moderate altitude.
The aim of the present study was to investigate the effects of prolonged real moderate altitude on blood rheology and pulmonary hemodynamics during the first five days of moderate altitude exposure. We hypothesized that hypoxia could induce concomitant changes in blood rheology and pulmonary hemodynamics.
Material and methods
Subjects
Seventeen healthy male volunteers were included in the study. They were aged between 20 and 40 years old, non-smokers and did not suffer from any known cardiovascular, metabolic or pulmonary diseases. They trained at least 8 hours a week in the previous 5 years. All participants resided at sea level and did not stay above 500 m in the previous 3 months before the protocol. Approval for this study was obtained from local ethics committee conformed with the Declaration of Helsinki (German Trias I Pujol Hospital, Badalona, Spain for sea-level condition and Health Ministry of Andorra for altitude condition). All participants gave their written informed consent.
Protocol
Baseline measurements were made at sea level (SL) in Barcelone, a city located 10 m above SL. All subjects were driven by bus to moderate altitude at 2,400 m (Pas de la Casa, Enveigt, Andorra), where they stayed for five days. The different physiological/biological measurements were made after one night of exposure (H1); and at a later stage after five days and nights of exposure (H2). All subjects maintained similar physical activity levels during the study. To avoid temperature impact on hemodynamics analyses, ambient temperature was regulated to match with SL values at the time of physiological measurements.
Hemoglobin saturation
Hemoglobin saturation was evaluated at rest in a sitting position through peripheral capillary oxygen saturation (SpO2) with a pulse oximeter probe (Nonin Medical Inc., Plymouth, MN, USA) placed on the ear lobe. The PalmSAT® technology used in the Nonin Medical pulse oximeter yields a measurement accuracy of±2.1% relative to the gold standard (CO-oximetry analysis of arterial blood samples) [22].
Measurements of hemodynamic parameters
Echocardiography measurements were performed by a cardiologist with a portable system (Vivid-I, General electric, Boston, MA, USA) in lying position. Stroke volume (SV) was derived from the following equation SV = 3.14×(LVOT diameter/2)2×TVI where LVOT is the left ventricular outflow tract cross sectional area measured at rest and TVI the pulsed Doppler time-velocity integral measurements [23]. Cardiac output (Qc) was estimated as the product of SV and heart rate (HR). Systolic pulmonary arterial pressure (sPAP) was calculated from the maximum velocity of the continuous-Doppler tricuspid regurgitation jet and mean pulmonary arterial pressure (mPAP) was calculated as mPAP = 0.61*sPAP + 2, as previously reported [24]. Pulmonary vascular resistance (PVR) was defined as the ratio of the difference between mPAP and left atrial pressure (LAP) divided by Qc [25]. LAP was estimated from the ratio of Doppler mitral early flow-velocity wave (E) and tissue Doppler mitral annulus early diastolic velocity (E’) with the following equation: LAP = 1.9 + (1.24*E/E’) [26]. Pulmonary capillary pressure (Pcap) was calculated as 0.4×(mPAP-LAP) + LAP.
Hemorheological parameters
Blood samples were collected in the same lying position from the right antecubital vein in EDTA tubes (Becton Dickinson). Hemorheological analyses were performed within thirty minutes after blood sampling to avoid any blood alteration. Hematocrit (Hct) level was determined using a photometer (Hemocontrol, EKF Diagnostics, Cardiff, UK). Blood viscosity was measured after complete blood oxygenation, at native hematocrit and several shear rates (22.5, 45, 90 and 225 s–1) using a cone/plate viscometer (Brookfield DVII+ with CPE40 spindle, Brookfield Engineering Labs, Natick, MA) [27]. Red blood cell aggregation was assessed by light transmission with the Myrenne aggregometer after shearing the suspension to 600 s-1 to dissociate pre-existing aggregates [28]. As recommended, the hematocrit of the suspension was standardized at 40% before the measurement to avoid any influence of the concentration of red blood cells on aggregation properties [27].
Statistical analysis
Data are reported as the mean±standard deviation (SD). All statistical analyses were performed using SPSS software (Ver 17.0). The effect of condition (SL, H1, H2) was analyzed by using a One-Way ANOVA completed with LSD Post-Hoc with a threshold for statistical significance set to p < 0.05.
Results
The participants’ characteristics (height, body mass, age, body fat) are shown in Table 1. Table 2 shows the variation of SpO2, pulmonary hemodynamics, cardiac parameters and hematocrit in sea-level and altitude conditions. SpO2 decreased in H1 and H2 compared to SL. sPAP, mPAP, Pcap and PVR were higher in H1 and in H2 compared to SL. HR was higher in H1 and H2 compared to SL while SV remained unchanged. Cardiac output was not significantly different between the three conditions.
Subject characteristics
Subject characteristics
Physiological, cardiac and hemodynamic variations during exposure to moderate hypoxia
Post hoc differences compared to normoxia: *p < 0.05.
Hematocrit and HR were higher in H1 and H2 compared to SL (Table 2). The impact of altitude exposure on blood viscosity is presented in Fig. 1. Blood viscosity increased above SL values in H1 at all shear rates and returned to SL levels in H2, except for viscosity at 225s-1 which only tended to return SL level (p = 0.09). Changes in RBC aggregation are shown in Fig. 2. RBC aggregation index was higher in H1 compared to SL and remained higher in H2.
Effect of moderate hypoxia exposure on blood viscosity. *Difference compared to normoxia, †difference compared to H1.
Effect of moderate hypoxia exposure on red blood cell aggregation. *Difference compared to normoxia.
The major finding of this study is that moderate altitude exposure (2,400 m) induced changes in blood viscosity, RBC aggregation and pulmonary hemodynamics. Despite blood viscosity was restored to sea-level values after five days of exposure to hypoxia, pulmonary pressure, Hct and RBC aggregation remained higher under prolonged hypoxia.
The reduction in atmospheric pressure at moderate altitudes, i.e 567mmHg at 2,400 m, caused a decrease of about 25% in partial pressure of oxygen compared to SL, and thus a reduction in arterial pressure in O2 and SpO2. In agreement with previous studies conducted in human [13–15], this drop in SpO2 was sufficient to induce tachycardia and a rise in pulmonary vascular tone after only 1 day exposure. Previous studies indicated that hypoxic pulmonary vasoconstriction may occur when the PaO2 is reduced to a level where SpO2 would be less than 90% [29, 30]. Hypoxic pulmonary vasoconstriction could be due to changes in mitochondrial O2 sensing [6, 7] as well as inflammatory and endothelin responses [15]. However, others showed evidence of hypoxic pulmonary vasoconstriction at altitude as low as 1,600–2,400 m [13, 14]. The drop in oxygen gradient during acute hypobaric hypoxia exposure is also known to cause natriuresis, diuresis and fluid shift between intra and extravascular spaces [31] leading to a rapid rise in Hct [32], as shown in our study. This acute hemoconcentration is, at least in part, responsible for the rise in blood viscosity observed between SL and H1. Indeed, the acute rise in PVR observed after one day at moderate altitude could be due to both hypoxic pulmonary vasoconstriction and the increase in blood viscosity.
We also observed an increase in RBC aggregation after one day at moderate altitude. Very few studies investigated the changes in RBC aggregation caused by hypoxia/altitude and the results are not consistent. While Moon et al. [33] recently reported that acute normobaric hypoxia did not affect RBC aggregation, Zhang et al. [34] observed that only 5 min of acute normobaric hypoxia increased RBC aggregation. Kang et al. [35] found that both chronic intermittent and continuous normobaric hypoxia resulted in a rise in RBC aggregation. The reasons of these changes in RBC aggregation during acute or chronic normobaric hypoxia exposure are unknown and the present study is the first one to report a rise in RBC aggregation after 1 day of hypobaric hypoxia (moderate altitude) exposure. RBC aggregation depends on both cellular (RBC aggregability) and plasma factors, such as fibrinogen concentration. Fibrinogen level was not measured in the present study but several studies provided evidence that fibrinogen production could be stimulated by hypobaric hypoxic stimulus [36, 37]. Indeed, one could speculate that the rise in RBC aggregation could be attributed to an increase of plasma fibrinogen level after one day of moderate altitude exposure. RBC aggregation is known to affect both blood viscosity (at low shear rates) and blood flow structuring in microcirculation [38]. RBCs need to flow as single cells when they go through small capillaries. Indeed, any increase in RBC aggregation would affect blood flow at the entry of capillaries, as well as into the capillaries [38, 39]. We suspect that the increased RBC aggregation, in association with pulmonary hypoxic vasoconstriction and the increase in blood viscosity, could have played a role in the increase of pulmonary vascular resistance, pulmonary capillary pressure and mean pulmonary arterial pressure at day 1 of moderate altitude exposure.
After five days of moderate altitude exposure, SpO2 remained lower and HR higher than in SL indicating that acclimatization process was not fully completed. Surprisingly, despite Hct and RBC aggregation were still high in H2, blood viscosity returned to SL values. Indeed, the drop in blood viscosity between H1 and H2 could not be attributed to hemodilution. Unfortunately, it was not possible to investigate RBC deformability and plasma viscosity in this study and no study investigated the changes in these parameters after five days of moderate altitude exposure. It is highly possible that five days of hypobaric hypoxia could have also affected RBC deformability and/or plasma viscosity, which could have compensated the rise in Hct to normalize blood viscosity. Further studies are clearly needed to address these points.
Although blood viscosity returned to SL level in H2, sPAP, mPAP, PVR and Pcap remained higher in H2 compared to SL. Levine et al. [13] previously reported high values of sPAP and mPAP in elderly subject after 5 days at 2,500 m above sea level and Lichtenauer et al. [15] showed a rise in PVR and sPAP after 3 days at 2,900 m. These authors suggested that these results would be partly due to pulmonary arterial remodeling process and the persistence of hypoxic pulmonary vasoconstriction. The small muscular pulmonary arteries are known to be the major site of hypoxic pulmonary vasoconstriction and pulmonary vascular remodeling [40] which could explain why sPAP and mPAP remained elevated in H2. As previously discussed, the persistence of increased RBC aggregation could also participate in the increase in pulmonary capillary pressure. Indeed, it seems that impaired pulmonary hemodynamics after 5 days of moderate altitude exposure would not be due to increased blood viscosity and would be caused by hypoxic pulmonary vasoconstriction/vascular remodeling and increased RBC aggregation.
Our study investigated for the first time the evolution of hemorheological parameters and their relationship with pulmonary hemodynamics in human during the first five days of moderate altitude exposure. Hemoconcentration, pulmonary vasoconstriction, and increased RBC aggregation and blood viscosity seem to be involved in the increase of pulmonary vascular resistance at the onset of moderate altitude exposure. Surprisingly, after 5 days of hypobaric hypoxia, blood viscosity returned to sea level despite persistent hemoconcentration. Indeed, the increase of pulmonary vascular resistance during prolonged moderate altitude exposure would be mainly due to hypoxic vasoconstriction and increased RBC aggregation. Further studies are needed to investigate the role of RBC deformability and plasma viscosity in the hemodynamic response to moderate altitude.