Abstract
Even though exercise hemorheology at hypoxic condition has been considered as a good tool to understand clinical hemorheology, there have been limited studies reported. Previous researches showed that hemorheological variables are closely correlated with oxygen delivery capacity during exercise. The present study investigated hypoxic responses including RBC deformability and aggregation, metabolic parameters and complete blood cell counts at various hypoxic conditions during cycling exercise and recovery. Eleven Korean healthy male subjects performed submaximal bike exercise at sea level (20.9% O2) and under various hypoxic conditions (16.5% O2, 14.5% O2, 12.8% O2, and 11.2% O2) in a random order. The submaximal bike exercise intensity of the subjects was 70% maximum heart rate at sea level. All variables were measured at rest, during exercise and recovery 30-minute, respectively. As oxygen partial pressure decreased, arterial blood oxygen saturation decreased but oxygen uptake did not change much. Heart rate and lactate concentration during exercise increased when oxygen partial pressure is less than or equal to 14.5% O2 condition. Red blood cell (RBC) counts, hemoglobin counts, and hematocrit level were not apparently altered with hypoxic conditions. RBC deformability showed significant alterations at 11.2% O2 conditions compared with other hypoxic conditions during exercise or recovery, except at 10 minutes recovery. However, decreases in oxygen partial pressure did not affect red blood cell aggregation. Therefore, we conclude that alterations in RBC deformability may reduce aerobic capabilities at hypoxic condition.
Introduction
Acute exposure to high altitudes can decrease maximal and submaximal aerobic exercise capacity. Numerous factors can affect aerobic performance including oxygen partial pressure, arterial blood oxygen saturation (% SaO2), artery-vein oxygen difference, blood lactate concentration, cardiovascular capacity, acid-base balance, maximal oxygen uptake (VO2 max), and oxygenated or deoxygenated hemoglobin (Hb). However, there is no comprehensive consensus on the effect of these factors on aerobic exercise capacity [6, 38]. Furthermore, the effects of high altitude on such variables and their subsequent effects on aerobic capacity have not been fully elucidated.
Previous studies suggested that hemorheological variables would affect the oxygen delivery capacity of the blood [8, 34]. Blood flowing through a capillary which diameter is smaller than RBC is accompanied with Red blood cell (RBC) deformation. The deformation is an energy-consuming and energy-dependent process that alters RBC morphology and deformable healthy RBC are correlated with oxygen delivering capacity [15, 33]. RBC aggregation refers to a binding between two RBCs such as one-dimensional rouleaux formation. In general, heathy bloods yield moderate aggregation tendency. Elevated RBC aggregation is frequently observed in vascular diseases and rheumatoid arthritis. The hyper-aggregation of RBCs may cause flow resistance in microcirculation and yield poor oxygen delivery [5, 30].
Hemorheological variables have been studied previously to evaluate the oxygen delivery capacity of the blood in various subjects [13, 30], for different exercise methods [41] and exercise intensities [15], and between genders [28]. In addition, a few studies have evaluated changes in hemorheological variables induced by high altitudes [21, 37]. Doyle and Walker [16] reported that acute exposure to high altitudes decreased the maximum rate of oxygen consumption (VO2 max), exercise capacity, and artery-vein oxygen difference in non-athletes. Moreover, they found that such exposure damaged the RBC membrane, negatively affecting RBC deformability and aggregation to reduce aerobic exercise capacity. Cheng et al. [12] found that those chronically exposed to high altitudes, such as hillside residents, had higher hematocrit (Hct) levels and RBC deformability than those living at sea level. Grau et al. [21] reported that activation of RBC-NOS decreased with increasing hypoxia and RBC deformability, which is influenced by RBC-NOS activation, decreased under mild hypoxia, but surprisingly increased at severe hypoxia in vivo and in vitro. Rohm et al. [37] reported that exposure to hypobaric hypoxia in high altitude, 3000 m, led to a significant decrease in the participants’ oxygen saturation, and an increase in the breathing frequency whereas blood pressure and heart rate were not significant altered.
However, there have been limited studies of altitude-induced hemorheological changes during exercise and recovery. Understanding of hemorheological changes with various hypoxic conditions could provide further information on reduced aerobic exercise capacity caused by acute high altitude exposure. Therefore, we investigated the effect of simulated altitudes on hemorheological variables, and how they might affect aerobic exercise capacity. In the present study, RBC deformability, RBC aggregation, metabolic parameters and complete blood cell counts were measured at rest, and during exercise and recovery at various hypoxic conditions.
Methods
Study participants
Eleven healthy male volunteers, who have not participated in any planned exercise program or consumed any type of dietary supplements for 1-year, were included in this study. All participants received information about the purpose and process of the study and submitted informed consent prior to commencement of the study. This study was approved by the Institutional Review Board of Kyung Hee University (KHSIRB 2015-024) in Korea and was conducted according to the declaration of Helsinki.
Study design
All subjects performed a physical work capacity (PWC) 70% heart rate (HR)max test [29] to determine their cycle exercise intensity at sea-level (∼20.9% O2). The Aerobike 75XL II (Combi Corporation, Tokyo, Japan) was used to measure PWC70% HRmax. The power at 70% HRmax was recorded, and the individual power was used to determine exercise intensity (relative fixed intensity). The PWC70% HRmax was observed to vary among participants (85∼166 W) and the total average of the PWC70% HRmax was 121.2±24.1 W/minute.
We measured arterial oxygen saturation (% SpO2), rate of oxygen consumption (VO2), blood lactate concentration (mmol/L), heart rate (HR), RBC count, Hb count, hematocrit (Hct), RBC deformability, and RBC aggregation at rest, during cycle exercise (30 minutes; PWC70% HR max, 60 rpm), and during recovery (30 minutes, seated), at sea level (20.9% O2) and under various hypoxic conditions. Hypoxic conditions included 11.2% O2, 12.8% O2, 14.5% O2, and 16.5% O2 . An interval of 6 days was applied between assessments at each exposure level. A nitrogen generator (SFET Co., Siheung-si, Gyeonggi-do, Korea) was used to produce normobaric hypoxic environments. The various normobaric hypoxic conditions were simulated by introducing nitrogen into the environmental chamber(width 6.5 m×length 7.5 m×high 3 m), using a nitrogen generator (Separation & Filter Energy Technology Cooperation, Siheung, Korea) with the capacity to simulate normobaric hypoxic conditions for altitudes of up to 6000 m. The temperature within the environmental chamber was maintained at 20±2°C and the humidity was maintained at 60±2% for all the conditions.
Blood sampling
Baskurt et al. [5] recommended that blood samples should be taken between 8:00 and 10:00 am following a day of fasting to conduct RBC deformability and aggregation analysis. However, we were unable to limit the blood sampling time because the blood had to be collected at rest, during exercise, and recovery. Moreover, the participants were asked to fast for 4 hours prior to blood sample collection. A 20 G catheter (Sewoonmedical Co. Ltd., Cheonan-si, Chungcheongnam-do, Korea) was inserted in the forearm vein and connected using a 3-way extension line (Sewoonmedical Co. Ltd.). Blood (6 mL) was collected in 2 K3EDTA tubes (Greiner bio-one Ltd., Chon Buri, Thailand) at rest, during exercise (5, 10, 15, and 30 minutes), and recovery (10 and 30 minutes). We refrained from using a tourniquet as much as possible because the use of a tourniquet during venous blood sampling can decrease RBC deformability and increase RBC aggregation [11]. However, if necessary, the use of the tourniquet was limited to 5 seconds.
Height and body composition measurements
All participants fasted overnight prior to measurement of body composition (i.e., height, weight, free fat mass, and body fat percentage). They wore lightweight clothing and were asked to remove any metal items. An X-SCAN PLUS (Jawon medical, Gyeongsan-si, Gyeongsangbuk-do, Korea) was used to measure height and body composition.
Pulse oximetry and oxygen saturation measurement
Subjects were seated on the cycle ergometer for 30 minutes and their index finger was placed on the % SPO2 measurement sensor. The % SPO2 was measured at rest, during exercise (5, 10, 15, and 30 minutes), and during recovery (10 and 30 minutes).
Oxygen uptake measurement
Breath-by-breath measurements of VO2 were conducted using a Vmax229 auto breathing metabolism analyzer (Sensormedics, FL, USA). Measurements were conducted at rest, during exercise (5, 10, 15, and 30 minutes), and during recovery (10 and 30 minutes).
Blood lactate concentration measurement
We collected 80 μL of blood in a capillary tube using the fingertip method and the blood lactate concentration was measured using an YSI-1500 lactate analyzer (YSI incorporated, OH, USA). Measurements were conducted at rest, during exercise (5, 10, 15, and 30 minutes), and during recovery (10 and 30 minutes).
RBC, Hb, and Hct measurements
Measurements were conducted by the Green Cross Cooperation (Korea) using a XE2100D hematology analyzer (Sysmex, Kobe, Hyogo, Japan); RBC count and Hct were measured using the electrical impedance method and Hb count was measured using the free Hb spectrophotometry method. Measurements were conducted at rest, during exercise (5, 10, 15, and 30 minutes), and during recovery (10 and 30 minutes).
RBC deformability and aggregation measurements
Uyuklu et al. [40] recommended that RBC deformability and aggregation should be analyzed at 25°C within 4–6 hours after collecting blood, so all samples were analyzed within 30 minutes of collection at a room temperature of 25°C using a Rheoscan-D (Rheo Meditech Inc., Seongbuk-gu Seoul, Korea). Briefly, the blood samples were agitated for 10–30 minutes using an agitator. Subsequently, for RBC deformability, the sample was transferred to a 2 mL microfuge tube and then diluted in 700 μL of 5.5% polyvinylpyrrolidone (360 kDa) dissolved in 1 mmol phosphate buffered saline (pH 7.4; osmolality = 300 mOsmol/kg) in a K3EDTA tube (Greiner bio-one, Thailand). Then, 0.5 mL of this solution was analyzed using the D-test kit according to manufacturer’s instructions (Rheo Meditech Inc.). For RBC aggregation, 8 μL of the blood sample (direct whole blood analysis) was analyzed using the A-test kit according to manufacturer’s instructions (Rheo Meditech Inc.). Measurements were conducted at rest, during exercise (5, 10, 15, and 30 minutes), and during recovery (10 and 30 minutes).
Statistical analyses
All data from this study were analyzed using PASW Statistics version 20.0 (IBM corporation, Somers, NY, USA). All data were presented as means and standard deviations. One-way repeated analysis of variance (ANOVA) was applied to determine the differences between dependent variables at each time point for each simulated altitude. Because the purpose of this study was to evaluate the effect of normobaric hypoxia environments during exercise and recovery, least significant difference post-hoc analysis was also conducted.
We calculated the effect size (statistical power 80%) according to the methods of Nam and Sunoo [31], and set the level of significance as p < 0.05. The sample size was calculated based on major variables such as RBC deformability, blood lactate concentration, and % SpO2. Variable sample sizes according to values at sea-level and various normobaric hypoxic environments were used to determine a sample size of 20 individuals for RBC deformability, 7 for blood lactate concentration, and 3 for % SpO2, giving an average sample size of 10, to which we added one more person to account for any subject withdrawals.
Results
The participants’ characteristics (age, sex, height, weight, free fat mass, and body fat) are shown in Table 1. Table 2 shows the variations of arterial oxygen saturation (% SpO2) at rest, and during exercise, and during recovery at various hypoxic conditions The % SPO2 significantly decreased as the oxygen partial pressure decreased at rest, during exercise and recovery (p < 0.05). In Table 3, the HR was significantly higher at 14.5% O2, 12.8% O2, and 11.2% O2 than at sea level. Furthermore, the HR was significantly higher at 12.8% O2 and 11.2% O2 than at 16.5% O2 (p < 0.05).The blood lactate concentration was significantly higher at 14.5% O2, 12.8% O2, and 11.2% O2 than at sea level (20.9% O2) and 16.5% O2, and the blood lactate concentration was higher at 11.2% O2 than at all other oxygen levels (Table 4). VO2, RBC count, Hb count, Hct were not significant different at various hypoxic conditions (data not shown).
During exercise and recovery, RBC deformability was affected by changes in oxygen partial pressure as listed in Table 5. RBC deformability proportionally decreased as the oxygen partial pressure decrease. Statistical significances were observed for all exercise at the lowest oxygen partial pressure (11.2% O2) but not at rest. It is worthy to note that RBC deformability during recovery was more notably affected than that during exercise at most hypoxic condition. Meanwhile, RBC aggregation did not shown any apparent alterations with varying hypoxic conditions even though data is not shown.
Discussion
The lower atmospheric partial pressures of oxygen at high altitudes decrease partial pressure of oxygen in the blood, the % SpO2. A slight decrease in the % SpO2 results in decrease aerobic exercise capacity with a consequential decline in the artery-vein oxygen difference [6, 31]. Due to homeostasis, various physiological responses are accompanied such as increases of ventilation, breathing frequency, HR, cardiac output, and the anaerobic metabolism ratio. These responses are more apparently observed during aerobic exercise at high altitudes. [1, 37]. However, some of these responses occur immediately upon the acute exposure of hypoxic conditions and some do not. For instance, during acute exposure to high altitude, cardiac output initially decreases because of a decrease in venous return and plasma volume, so HR increases in order to compensate [1, 37].
Acute exposure to high altitudes can cause activation of the sympathetic nervous system by increasing catecholamine release [4, 35]. In addition, the oxygen saturation level of Hb decreases at high altitudes causing a decrease in the partial pressure of oxygen in the blood, therefore increasing anaerobic metabolism ratio, even though the intensity of exercise may not have changed [7, 22]. Furthermore, increases in blood lactate concentration, pH, and reactive oxygen species, because of increased anaerobic metabolism, can negatively affect rheological parameters such as RBC deformability and aggregation [2, 39]. This study investigated changes in hemorheological and metabolic parameters during aerobic exercise and recovery when subjects were exposed to acute hypoxic condition to evaluate compensatory physiological response. We did not observe any significant differences in VO2 between sea-level and various hypoxic conditions at rest, during exercise, and during recovery, this is likely because the exercise intensity was fixed at constant level across simulated environments resulting in the same amount of energy expenditure. Concordant with previous studies, increases in ventilation, breathing rate, HR, and cardiac output adapted to acute hypoxic environments were possible without an increase in VO2 [1, 34].
In the present study, decrease of the oxygen partial pressure (increase of simulated altitude) resulted in significantly decrease of % SPO2 levels. The decreases in % SPO2, occurred with concomitant increases in HR, as a compensatory mechanism, as described previously [3, 35]. Investigations of oxygen delivery capacity are generally conducted by an analysis of changes in complete blood cell counts such as RBC and Hb counts, and Hct levels. In agreement with most previously published studies, we did not find any significant differences in such counts during acute exposure to various hypoxic conditions at rest, during exercise, and recovery [6, 38].
Previous studies have shown that changes in hemorheological parameters can significantly affect oxygen delivery capacity of the blood [2, 39], so we evaluated RBC deformability and aggregation to evaluate if the hemorheological responses at rest, during exercise, and recovery were affected by various hypoxic conditions. We did not observe any changes in RBC aggregation in response to acute hypoxic challenge, although significant decreases in RBC deformability were apparent at lower partial pressures during exercise and recovery. Such changes occurred with a concomitant increase in blood lactate concentrations suggesting that the decrease in RBC deformability might be associated with a reduction in aerobic exercise capacity.
Limitations
This study has several limitations. All subjects were young healthy individuals, so a complete cross-section of the population was not represented. The sample size was too small for sufficient power for some variables. Moreover, not all possible variables that might affect blood oxygen delivery capability were investigated. A follow-up review with an increased sample size, including the same study design with the addition of other related factors is needed to further investigate the impact of high altitudes on exercise in relation to changes in rheological parameters.
Conclusions
RBC deformability decreased significantly at partial pressures of oxygen <14.5%, and was associated with a decrease in blood oxygen saturation, concomitant with a compensatory increase in HR, and a decrease in aerobic exercise capacity, reflected by an increase in blood lactate concentration.
Footnotes
Acknowledgments
The authors thank participants in this study and members of the Hypobaric Hypoxic Training Center in Kyung Hee University for their assistance in data collection. In this study, the hemorheological analysis kits were provided by Rheomeditech Inc.