Investigation of the differences between the Tibetan and Han populations in the hemoglobin–oxygen affinity of red blood cells and in the adaptation to high-altitude environments

ABSTRACT

Objective: High altitude is characterized by low oxygen pressure, resulting in multiple adaptive responses. Tibetans who have lived in the plateau for thousands of years have developed unique phenotypes, such as downregulation of the HIF pathway through EPAS1 and EGLN1 gene mutation. However, the changes of hemoglobin–oxygen affinity under hypoxia environment remain elusive.

Methods: A blood cell analyzer and a blood oxygen analyzer were used to conduct routine blood tests and measure the oxygen affinity P₅₀ in in the Han population that rapidly entered the plateau (for 3–7 days), the plateau-acclimatized Han population (residing for 30 days on the plateau), the plateau Han population (more than 10 years on the plateau), and the Tibetan population.

Results: The Han population that rapidly entered the plateau had increasing higher P₅₀ values, RBCs counts and hemoglobin (HGB) levels, while the acclimatized Han population, the plateau Han population and Tibetan all had significantly lower P₅₀ values. However, there were no significant differences in the RBCs counts and HGB levels between the plateau Han, Tibetan populations and the Han population of the plains.

Discussion: The adaptability of the Tibetan and plateau Han populations to the plateau was mainly due to the strong affinity of HGB for oxygen, which provided sufficient oxygen for tissues and organs.

Conclusions: The change of P₅₀ could be a feature of the adaptation to the plateau and to avoid altitude sickness, such as high-altitude polycythemia and dyspnea.

KEYWORDS:

• High altitude
• RBCs
• HGB
• oxygen affinity
• P₅₀

Introduction

The Qinghai-Tibet Plateau (>3500 m) is known for its abundant sunshine, strong ultraviolet light, dry climate and thin air, and its oxygen content is only 60% of that of the plains [1]. The low oxygen pressure has a huge impact on the physiological function of populations who have lived on the plains for generations and recently entered the plateau, but Tibetans who have inhabited locations at an altitude of approximately 4000 m for generations present strong adaptability to the plateau [2]. When people from the plains rapidly enter the plateau, a series of physiological changes will occur in the body, including increased red blood cell (RBCs) counts, hematocrit (Hct), hemoglobin (HGB), breathing rate and depth of breathing, lung ventilation in the respiratory system and decreased arterial oxygen saturation (SaO₂) in the blood system [3]. In contrast, Tibetans inhabiting the plateau do not show increased RBCs, Hct, or HGB concentrations, and the SaO₂ and pulmonary arterial blood pressure of Tibetans are almost comparable to those of the who reside in the plains, suggesting that the good plateau adaptation mechanism in Tibetans involves evolutionary pressures and genetic changes [4].

The oxygen affinity P₅₀ (P_O2 at which HGB is 50% saturated) is an important indicator evaluating the capability to adapt to low oxygen at high altitude. The P₅₀ value is the partial pressure of oxygen required to achieve 50% HGB saturation measured under the standard condition (whole blood with a normal level of 2,3-diphosphoglycerate (2,3-DPG), 37°C, pH 7.40), and the average P₅₀ is 26 ± 2 mmHg in the normal population of the plains [5]. However, we have no idea on whether P₅₀ could change during entering high-altitude environment. In the present study, we aimed to systematically compare the ability of RBCs to transport and release oxygen between the Han population of the plains, the Han population that rapidly entered the plateau (within 3–7 days of entering the plateau), the plateau-acclimatized Han population (30 days after entering the plateau), the plateau Han population (living on the plateau for more than 10 years), and the plateau Tibetan population to provide a basis for exploring the mechanism underlying the adaptive changes of RBCs in transporting and releasing oxygen as a response to high-altitude hypoxia in different populations.

Material and methods

Populations and samples

Thirty healthy male volunteers were selected as the subjects for each group and the baseline characteristics of each group were shown in Table 1. The subjects that were enrolled at the same time were divided into the Han population of the plains (inhabiting an altitude 500 m above sea level), the Han population that rapidly entered the plateau (on the 3rd and 7th days after entering the plateau at an altitude of 4300 m above sea level), the plateau-acclimatized Han population (30 days after entering the plateau an altitude of 3700 m above sea level), the plateau Han population (living on the plateau for more than 10 years at an altitude of 3700 m above sea level), and the plateau Tibetan population (inhabiting at an altitude of 3700 m above sea level; Figure 1). Peripheral blood samples with anticoagulant were collected before and after they entering plateau. This study was approved by the Institutional Review Board of General Hospital of Air Force, PLA and all volunteers signed a written informed consent.

Figure 1. Schematic representation of the altitude in each group.

Table 1. The baseline characteristics of each group.

	Age	BMI	Smoking
Plain	24.17 ± 4.73	23.00 ± 1.74	No
Han entered plateau 3rd day	23.40 ± 4.30	23.49 ± 2.55	No
Han entered plateau 7th day	25.20 ± 3.97	22.73 ± 2.14	No
Plateau-acclimatized Han population	21.77 ± 1.28	22.68 ± 1.80	No
The plateau Han population (>10years living in plateau)	41.27 ± 6.92	22.28 ± 2.54	No
Tibetan	32.03 ± 9.71	22.99 ± 2.75	No

Sample processing

EDTA-anticoagulated whole blood were collected and packed in EP tubes for RBCs and HGB concentration detection. After centrifugation, the precipitated RBCs were collected and then mixed with compound glycerol solution (Beijing Bode Sangte, batch number: H11022503) at a 1:1 ratio for P₅₀ detection. Aliquots were stored at –80°C, and tests were conducted within 1 week.

RBCs counts and HGB concentration

The RBCs and HGB concentration were measured by the automatic blood cell analyzer (China Tekang Technology, model TEK3600).

Determination of oxygen affinity P₅₀

The P₅₀ values of each sample were measured at 37°C and pH 7.4 using the HEMOX™ blood oxygen analyzer (TCS Scientific Corp, Southampton, PA, U.S.A.).

Statistical analysis

The RBCs, HGB concentration results and P₅₀ values were statistically analyzed using SPSS (V.20) statistical software (SPSS Inc., Chicago, IL, U.S.A.). Significant differences were calculated by one-way analysis of variance (ANOVA) followed by the Bonferroni test when performing multiple comparisons between groups and P < 0.05 indicated a statistically significant difference.

Results

RBCs counts and HGB concentration in the Tibetan and Han populations

Compared to the Han population of the plains, the Han population of the plains on the 3rd day after entering the plateau (RBCs 4.8–6.2 × 10¹²/l, median 5.4 × 10¹²/l; HGB 153–188 g/l, median 170 g/l), the Han population of the plains on the 7th day after entering the plateau (RBCs 4.8–6.4 × 10¹²/l, median 5.4 × 10¹²/l; HGB 150–194 g/l, median 170 g/l), and the plateau-acclimatized Han population (RBCs 4.7–6.6 × 10¹²/l, median 5.5 × 10¹²/l; HGB 150–201 g/l, median 176 g/l) showed significantly increased RBCs and HGB levels (P < 0.05; Figure 2(A,B)). There were a slightly increasing in the RBCs and HGB levels of the Han population who living in plateau for more than 10 years (RBCs 3.9–7.8 × 10¹²/l, median 5.6 × 10¹²/l; HGB 124–240 g/l, median 172 g/l), while a decreasing in the RBCs and HGB levels of the plateau Tibetan population (RBCs 3.5–5.2 × 10¹²/l, median 4.5 × 10¹²/l; HGB 99–157 g/l, median 141 g/l) were detected when compared to the Han population of the plains (Figure 2(C,D)).

Figure 2. RBCs count and HGB concentration in different population. Whole blood was collected from the volunteers before and after entering the plateau, as well as Tibetan and Han population who lived in plateau for at least 10 years. RBCs count (A, C) and HGB concentration (B, D) were detected within 8 hours after blood collection. *P < 0.05, **P < 0.01 or ***P < 0.001 for subjects versus plain group.

Differences in oxygen affinity P₅₀ between the Tibetan and Han populations

The P₅₀ value (17.44–25.86 mmHg, median 22.46 mmHg) of the Han population of the plains was slightly lower than the normal range (26.00 ± 2.00 mmHg), and that of the plain Han population on the 3rd day after entering the plateau (14.47–33.45 mmHg, median 23.22 mmHg) showed a slight increase, with no statistically significant difference compared to the Han population of the plains. The P₅₀ value of the Han population of the plains on the 7th day after entering the plateau (16.13–37.15 mmHg, median 28.17 mmHg) was significantly higher than that of the Han population of the plains (P < 0.01), whereas that of the Han population of the plains that became acclimatized to the plateau (14.55–24.49 mmHg, median 17.08 mmHg) was significantly lower than that of the Han population of the plains (P < 0.01) (Figure 3). The P₅₀ values of the plateau Han population (P₅₀ 16.38–25.42 mmHg, median 18.10 mmHg) and the plateau Tibetan population (P₅₀ 15.62–22.72 mmHg, median 17.51 mmHg) were significantly lower than that of the Han population of the plains (P < 0.001) (Figure 3).

Figure 3. P₅₀ value changes in different populations. The P₅₀ was detected in Han population (A) or in Tibetan (B) using a blood oxygen analyzer.

Correlation between oxygen affinity P₅₀ and the RBCs counts and HGB concentration in the Han population

The values of P₅₀ increased as the RBCs counts (Figure 4(A)) and HGB concentration (Figure 4(B)) increased on the 3rd and 7th days after the Han population of the plains entered the plateau. The RBCs counts and HGB concentration continued to increase, while the variation trend in the P₅₀ value was opposite, with a significant decrease after the acclimation to the plateau (Figure 4).

Figure 4. Linear relationship between P₅₀ value and RBC, HGB in plain population. The RBCs count (A) and HGB concentration (B) versus P₅₀ values during Han population rapidly entering plateau.

Discussion

The extreme environmental conditions of the Qinghai and Tibet Plateau (altitude > 3500 m), such as low pressure and low oxygen, can pose serious threats to the physical and mental health of people entering the plateau and may cause headaches, insomnia, dyspnea, and other plateau reactions as well as a series of physiological changes. In contrast, the Tibetan population that has lived on the plateau for generations can adapt well to the plateau environment: First, the Tibetan populations of the plateau and plains have similar HGB concentrations and SaO₂, which are significantly lower than those of people from the plains who migrate to the plateau [6]. Second, Tibetan people have lower hypoxic pulmonary vasoconstriction than the population of the plains, and hypoxic pulmonary vasoconstriction can cause hypoxic pulmonary hypertension and lead to pulmonary edema [4]. Third, Tibetan people also have a faster blood flow velocity to ensure a normal oxygen supply to all tissues and organs [7]. In this study, we analyzed the oxygen affinity P₅₀, RBCs counts, and HGB concentration of the Han population of the plains, the Han population that rapidly entered the plateau (entered the plateau for 3 and 7 days), and the plateau-acclimatized Han population as well as the oxygen affinity P₅₀ of the plateau Han and Tibetan populations to determine the basis for different adaptation mechanisms of the Tibetan and Han populations to the plateau environment.

When the plain Han population rapidly entered the plateau, the 2,3-DPG level in the RBCs increased, the oxygen dissociation curve shifted to the right (increased P₅₀), the oxygen affinity of HGB was decreased to increase the oxygen unloading efficiency, and SaO₂ was therefore reduced [8]. The results of this study revealed that that on the 3rd day after the Han population of the plains entered the plateau, the P₅₀ value (23.22 mmHg) was slightly higher than that of the population of the plains (22.46 mmHg), and on the 7th day after the Han population of the plains entered the plateau, the P₅₀ value (28.17 mmHg) increased significantly (P < 0.01). This finding suggests that after the healthy Han population rapidly entered the plateau, the HGB oxygen affinity kept decreasing, improving the oxygen release efficiency in the tissue and thus ensuring a normal oxygen supply to the body. After 30 days of altitude acclimatization, the P₅₀ value (17.08 mmHg) of the Han population decreased significantly (P < 0.01), indicating increased HGB oxygen affinity, which helps the lungs bind more oxygen molecules, maintains arterial SaO₂, and promotes the plateau adaptation [9]. In conclusion, the RBCs counts and HGB concentration were significantly increased in the early stage after the Han population of the plains entered the plateau and after adaption to the plateau. However, the P₅₀ value first increased as the RBCs counts and HGB concentration increased during the early stage after the Han population of the plains entered the plateau but decreased significantly after adaptation. Therefore, the oxygen unloading efficiency was enhanced during the initial stage to ensure the oxygen supply to the organism, while the oxygen affinity of HGB was increased during the prolonged stay at the plateau, and the arterial SaO₂ gradually increased, leading to plateau acclimatization.

Balaban et al. [10] found that the P₅₀ values of the residents of the plains, the plateau-acclimatized population (plain population living at an altitude of 3600–4100 m for 2–3 weeks), and the plateau dwellers (living for generations in the Andean Plateau) were 30.8, 24.8, and 26.5 mmHg, respectively, indicating that the in vivo oxygen dissociation curve of the plateau dwellers was shifted to the left compared to that of the residents of the plains; this change was related to respiratory alkalosis because the three populations had a blood pH of 7.37, 7.46, and 7.43. Consistent with the results of this study, the P₅₀ values of the plateau-acclimatized Han population (17.08 mmHg), the plateau Han population (plateau residence > 10 years) (18.10 mmHg), and the plateau Tibetan population (17.51 mmHg) were significantly lower than that of the Han population of the plains (22.46 mmHg, P < 0.01). The reasons might be as follows: 1. The adaptation in plateau-acclimatized and long-term plateau-dwelling Han populations may be related to changes in the 2,3-DPG level and pH value. 2. The adaptation in plateau Tibetan people may be associated with unique genetic characteristics and evolutionary pressure. The HGB oxygen affinity P₅₀ values of the plateau-acclimatized and long-term plateau-dwelling Han populations are consistent with that of the plateau Tibetan population, and whether genetic factors participate in the HGB oxygen affinity difference should be further studied. Greater oxygen affinity is an important adaptive mechanism in plateau species. In this study, a lower HGB concentrations and P₅₀ values were founded in the Tibetan subjects [11,12]. Thus, it is conceivable that reduced HGB would be associated with a reduction in P₅₀, a trait shown to be associated with the genetic factors in Tibetan [13]. In consistent with Simonson’s research [13], it also showed that there was no correlation between P₅₀ and HGB in Tibetan subjects or when Tibetan and Han Chinese (resistant in plateau at least 2 years) data were analyzed together as a single group. However, whether lower P₅₀ results from genetically based changes in Tibetans remains unclear.

Conclusion

In summary, during the early stage after the Han population of the plains entered the plateau (within 3–7 days after entering the plateau), the RBCs counts, HGB concentration, and P₅₀ values were significantly increased; these changes are a stress compensatory response to maintain the normal tissue oxygen level. After adaptation to the plateau environment for 30 days, the RBCs counts and HGB concentration were maintained at a certain level, and the P₅₀ value was significantly decreased. This change is a feature of the adaptation to the plateau to avoid altitude sickness, such as high-altitude polycythemia and dyspnea. The P₅₀ values of the plateau Han and Tibetan populations were also significantly lower than that of the Han population of the plains, which is a long-term adaptation to the plateau.

Acknowledgments

The authors thank Fengyan Fan and Ying Wang for excellent technical assistance.

Disclosure statement

No potential conflict of interest was reported by the authors.

ORCID

Cuiying Li http://orcid.org/0000-0002-7045-0838

Additional information

Funding

This work was supported by the Application Fundamental Research Project Foundation of China (No. AWS13J004).