High Altitude
Environmental Physical Agents

Sherpas' adaptation at high altitude: a matter of genes

Lorenzo Tua

Introduction

Sherpa is a Tibetan word that means “eastern people” and refers to the specific ethnic group of Nepales inhabitants who lives in the Himalayan range. The Sherpa people migrated from the Tibetan plateau to the actual location (eastern and central Nepal) over 500 years ago, carrying along the Buddhist tradition, until then unknown to the nepalese people, and developing their own language. There are currently over 150 000 Sherpas in Nepal and many others who immigrated to the west ( New York City has the largest Sherpa Community in the U.S.A with a number of 2500).
With the developing success of mountaineering in the Himalayan region by western people, Sherpas became gradually fundamental as an active part of the expeditions. They work as carriers, using their tremendous strength, resistance and adaptation to the environment as tools who simplify the tasks required to the climbers and trekkers. Sherpas are known for being so well-adapted, that little mountain sickness occur in them. Over the years, studies have been conducted to determine where this remarkable adaptation comes from and it finally came out that the origin of it is within Sherpas’ genes. It has been found that there are at least 10 genes which allow this people not to suffer from the main problem that a normal person faces at a similar altitude: hypoxia(medical).

Background of physiology at high altitude

Barometric pressure decreases with increasing altitude, as a result of the air being attracted to the ground by the force of gravity. Since air is compressible, proceeding to high altitude it becomes more rarified, although its gases' concentration does not vary significantly. Until 11000 m of altitude oxygen remains the 21% of the total barometric pressure. What instead decreases it’s the amount of oxygen, since the 21% of less total air means less total oxygen. This is what causes hypoxia in an organism that is not adapted to such a deprivation of oxygen. In addition, the humidification of inspired air by adding aqueous vapor continues regardless of the external pressure and this lowers even more alveolar partial pressure of oxygen.
On the summit of Mt. Everest (8848 m), barometric pressure is 253 mmHg. Subtracting to this value the partial pressure of aqueous vapor (47 mmHg) which remains constant, we obtain a pressure of 206 mmHg. The 21% of this values equals 43,26 mmHg: this is the partial pressure of inspired oxygen.
Deprivation of oxygen affects all cells, but mostly the nervous cells, which strictly rely on glucose and oxygen for their metabolism. This causes a condition known as Acute Mountain Sickness, characterized by headache, dizziness, dyspnea, weakness, nausea, anorexia, sweating, blurring of vision, hypoacusia, insomnia. All these symptoms are the result of different processes that occur in the body exposed to an hypoxic environment, the main of which are:

• low oxygen to the brain
• hypocapnia due to hyperventilation, which is in turn a result of hypoxia itself
• respiratory alkalosis, a consequence of hypocapnia
• cerebral edema, due to vasodilation in order to contrast the decreased oxygen supply

How the body reacts

The first mechanism is carried by the chemoreceptors in aortic and carotid sinuses, that signal to the regulatory centres of the respiration to increase ventilation; this causes almost immediately a depletion of alveolar CO2, which results in respiratory alkalosis. As soon as central chemoreceptors in the brain stem sense the decreased CO2 partial pressure, this same gas diffuses out of the cerebrospinal fluid (CSF) causing a local alcalosis which depresses chemoceptors. Thus there are 2 stimuli that act in opposite ways.
As far as the mechanics of respiration the rhythm increases, together with depth and work of the single respiratory acts.
The cardiovascular system tries to provide a better distribution of the blood by increasing the cardiac output, the cardiac frequency and the sympathetic stimulation of blood vessels; this causes an increased blood pressure with better perfusion, mostly of the lungs. Recruiment of vessels previously closed provide a larger diffusion surface. If this condition is exaggerated, this may lead to pulmonary edema.
Hemoglobin levels increase in the first 2 days at high altitude but, interestingly, not for an increased erythropoiesis, but as a result of the shift of liquids from red blood cells to the extravascular compartment.

Acclimatization

After a few days, the kidney begins to contrast respiratory alcalosis excreting bases and holding acids. Erythropoiesis begins after 3-5 days and this causes an elevation of oxygen in the blood, while not an elevation of the oxygen partial pressure.
2,3 DPG, which facilitates release of oxygen in tissues can sometimes increase.
The ventilatory response to CO2 curve moves to the left which means that for any given PCO2 the response is improved after a couple of days.
At the same time pH of the CSF returns to physiological values causing the progressive disappearance of all nervous symptoms. This is caused both by net movement of HCO3- outside the CSF and a decreased production of nitric oxide.
Among the heart’s responses, they all go back to normality except for hypoxic pulmonary vasoconstriction and hypertension; these two, together with the increased viscosity of the blood due to the enhanced erythropoiesis, may lead to hypertrophy of the right ventricle and sometimes ventricular insufficiency.

Here are the factors that have being studied the most through the year to try to explain the tremendous adaptation that have occurred in Tibetans and, above all, Sherpas.

HIF1A polymorphisms

Hypoxia inducible factor 1, alpha subunit is a protein that acts as a transcription factor at low oxygen concentration; it is not the only HIF, since there is a class which includes HIF2A and HIF3A. The alpha subunit is the one, between two, which transfers to the nucleus and expresses its function (the beta subunit is already in the nucleus bounded to an hypoxia responsive element, HRE). This specific hypoxia inducible factor activates the transcription of genes involved in energy metabolism, angiogenesis, apoptosis and genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia.

When oxygen is present, there is a continuous demolition of HIF1A, which nevertheless continues to be synthesized. The demolition occurs by the prolyl hydroxylase (PHD), which hydroxylates it only if iron, ascorbic acid and KG are present. This action makes HIF1A eligible to be bound to ubiquitin by Von Hippel Lindau factor and therefore destructed. When oxygen is not present (mild to severe hypoxia) HIF1A fails to be depleted and can act as a transcription factor; the main genes it activates are glycolysis enzymes, erythropoietin (EPO) and proteins for angiogenesis such as VEGF.
Recently, studies (Association of polymorphisms of 1772 and 1790 in HIF1A gene with hypoxia adaptation in high altitude in Sherpas, 2007) on HIF1A have been conducted to determine whether it varies in people who have adapted to high altitude such as Sherpas. Single nucleotide polymorphisms (SNPs) were found particularly in exon 12 of the HIF1A gene. Blood samples were collected from Sherpas living at high altitudes and from healthy people living in Guangdong province; afterward genomic DNA was extracted and analyzed. The SNPs in 1772(C/T) and in 1790(G/A) were examined by restriction fragment length polymorphism-polymerase reaction (RFLP-PCR). While no significant difference was found between the two ethnic groups on polymorphism in 1772, there is a significant difference in occurrence of genotypes GG and GA as far as SNP in 1790: GG is more frequent in Guangdong people, while GA in Sherpas.

Another research (Genetic variation in hypoxia-inducible factor 1alpha and its possible association with high altitude adaptation, 2003) conducted in the same manner found a polymorphism in intron 13 which highlighted the same difference between Sherpas and native lowlanders: one genotype was more frequent with statistical significance in one group and the other in the second group.

The meaning of the SNPs in Sherpas remains unclear. Nevertheless, since the activity of HIF1A is regulated in multiple steps, polymorphisms might vary the activity and the activation itself of this protein.

Brain glucose metabolism

It has been confirmed that some vertebrates are hypoxia-tolerant thank to the reduced ATP utilization they have while exposed to extreme oxygen limitations. Therefore studies (Sherpa brain glucose metabolism and defense adaptations against chronic hypoxia, 1996) were conducted to determine whether the same strategy might be used by populations adapted to high altitude and therefore hypoxia. Positron-emission tomography (PET:) was used to measure glucose metabolic rates in the brain of Quechuas and Sherpas. Sherpas were studied more closely because it is recognized they have been indigenous to high altitudes for longer than Andeans people.
Two metabolic states were analyzed: 1) the normal state (hypoxia-adaptated), monitored right after subjects left Himalayas and 2) the de-acclimated state, monitored after 3 weeks at low altitudes. PET measurements of marked glucose (2-deoxy-2-fluoro-D-glucose) indicated that Sherpas’ brain metabolism differed from Quechuas’ but was identical to that of lowlanders.

Angiotensin-converting Enzyme (ACE) polymorphism

Angiotensin-converting enzyme (ACE) converts Angiotensin I, produced by the liver, in Angiotensin II, a strong vasoconstrictor; it also degrades bradykinin, a vasodilator, as well as other vasoactive peptides. The enzyme is an exopeptidase produced by pulmonary and renal endothelial cells.

Angiotensin belongs to a pathway called the Renin-Angiotensin-Aldosterone System. When blood volume to the glomerular capillaries in the kidney is low, juxtaglomerular cells secrete Renin in the circulation. This enhances the conversion of angiotensinogen in Angiotensin I, which subsequently reaches the lungs where it is converted in Angiotensin II by ACE. This constricts blood vessels, causing an increase in blood pressure. It also stimulates the adrenal cortex to produce aldosterone, an hormone which acts on the kidney by increasing sodium reabsorbtion and thus the blood pressure to rise. In hypoxia, blood pressure is already increased by several mechanisms: orthosympathetic stimulation, Hering-Breuer reflex, hypoxic pulmonary vasoconstriction; it is therefore important for the organism to maintain low levels of ACE not to further increase blood pressure.
As usual, Sherpas were enrolled together with non-sherpas Nepalese from the Kathmandu valley for studying adaptation (Adaptation to high altitude in Sherpas: association with the insertion/deletion polymorphism in the Angiotensun-converting enzyme gene, 2008). The genotype of the insertion/deletion (I/D) polymorphism in ACE gene was determined through polymerase chain reaction (PCR). At the same time, ACE activity was recorded. The frequencies of the different genotypes were significantly different between the 2 groups, showing a higher frequency of the dominant genotype (II and ID) and of the I allele alone in Sherpas. Nevertheless the ACE levels were similar, indicating a different activity instead of a different amount of the enzyme.

Nitric oxide synthase (NOS)

This enzyme catalyzes the formation of NO from L-Arginine. NO is an important signalling molecule involved in vascular and airway tone, insulin secretion, peristalsis and the development of the nervous system; it is a free radical with an unpaired electron. NOS actually composes a family of enzymes whose principal members, according to the latest international classifications, are: eNOS (endothelial NOS), nNOS (neuronal), iNOS (inducible or immune) and bNOS (bacterial). Each one has a different gene location and a different function within the organism. The reaction catalyzed is:
L-arginine + 3/2 NADPH + H+ + 2 O2 = citrulline + nitric oxide + 3/2 NADP+

For the reaction, 5 cofactors are required: NADPH, FAD, FMN, heme, tetrahydrobiopterine.
The isoenzyme that has been studied the most is eNOS, which is the main controller of the vascular tone. NO produced was found to be identical to the endothelial-derived relaxing factor (EDRF), which dilates blood vessels in response to shear from increased flow. Distension of the vessel occur as a result of the relaxion of the smooth muscle cells in the arteries' wall. NO activates guanylate cyclase, which produces cGMP. This causes:

• inhibition of calcium entry, thus reducing contraction
• increased activity of K+ channels, which leads ho hyperpolarization and relaxation
• activation of a specific kinase that enhance the activity of the myosin light chain phosphatase; this once again brings relaxation of the smooth muscle cell

By dilating blood vessels, NO causes more blood to reach certain districts of the body and might be thus involved in better perfusion during hypoxia. In 2006, a study (Genetic contribution of the endothelial nitric oxide synthase gene to high altitude adaptation in Sherpas, 2006) was conducted to determine polymorphisms of the eNOS gene, using the usual two groups: Sherpas and non-Sherpas living in the Kathmandu valley. A significant difference in occurrence was found as far as two polymorphisms: Glu298Asp and eNOS4b/a. The wild type (Glu298Glu and eNOS4b) was greater in Sherpas (66.7% versus 47,7% in non-Sherpas).

The result of this study is hard to read. While NO might be useful to increase blood flow and therefore provide more oxygenated blood during hypoxia, on the other hand in regions of the body particularly liable to edema, this may cause a rise in the risk of such a condition. Nevertheless a dilated artery may also mean more red blood cells delivered to a certain region in less time; this contrasts with what happens in a lowlander who transfers at high altitude: his body starts producing more red blood cells, causing polycythemia, with increased blood viscosity and increased work for the heart.

Erythropoietin (EPO)

EPO is a hormone that enhances erythropoiesis, or red blood cells production, acting directly on their precursors in the bone marrow. It is produced by interstitial fibroblasts in the kidney and in perisinusoidal Ito cells, in the liver (this site of synthesis predominates during fetal and perinatal period). Sometimes EPO is used as a performance-enhancing drug, but it can often be identified in the blood due to small differences with the endogenous hormone.
EPO is produced by peritubular capillary lining cells of the renal cortex. Its production is regulated by a feed-back mechanism that rely on blood oxygenation. HIF1A is probably the main factor of this oxygen dosage. Once into the blood stream, EPO reaches its targets by binding specific receptors and activates JAK-STAT cascade.
This hormone increases red blood cells by two means:

• it protects red blood cells from apoptosis therefore prolonging their lifetime
• it cooperates with growth factors involved in the development of the precursors of red blood cells. The Colony-forming unit erythroid is completely dependent on EPO.

Researches ( Erythropoietin levels in lowlanders and high-altitude natives at 3450 m, 2007) have been made to study the fluctuations of EPO levels in 4 different groups:

• lowland residents at sea level during a 11 days stay at 3450 m of altitude (LLR-SL)
• lowland residents acclimatized to high altitude (LLR-accl)
• high altitude natives (HAN)
• patients of acute mountain sickness and high altitude pulmonary edema (AMS-HAPE)

In LLR-SL it was found that EPO levels rise after only 8 hours of stay, with a significant peak in the first day and thereafter decreasing. Consequently, high levels of both hematocrit and hemoglobin were found for the whole length of the study.
EPO levels in LLR-accl. were significantly higher than LLR-SL, but not different than HAN’s levels.
As far as patients of AMS and HAPE, AMS levels were not significantly different from LLR, while HAPE’s were found to be increased.
As the authors state in the conclusion, prolonged residency at high altitude is associated with increased secretion of EPO, which in turn causes polycythemia and may cause heart problems. High altitude natives such as Sherpas have low levels of EPO, which correlates with low hematocrit and hemoglobin. Their better resistance to hypoxia is due to an increased “quality” of transport of oxygen instead of a simple increased “quantity”.
In addition, a study taken in 1976 (Sherpas living permanently at high altitude; A new pattern of adaptation, 1976) demonstrated that Sherpas living at low altitude have a higher affinity of blood for oxygen and when they transfer at low altitude, this affinity decreases and makes them mildly “_anemic_”. This is due to the fact that, differently from what happens in Andeans people, these Sherpas do not have an increased Bohr effect: theirs is identical if not less than that of lowlanders. Interestingly, it was found that Sherpas living at high altitudes have oxygen dissociation curves shifted to the left, meaning increased affinity for oxygen, while Sherpas living in Kathmandu have curves shifted to the right, meaning decreased affinity. It can be argued that "more affinity" also means "less release", but the reason why this does not happen in Sherpas living at high altitude is that they have an increased 2,3 DPG.
Where does this higher affinity comes from? The hypothesis of an abnormal hemoglobin was excluded since curves obtained with diluted hemolysates are identical between Caucasians and Sherpas. What was undoubtely found are an increased level of glutathione and and a decreased 2,3-P2G buildup upon incubation with inosine, pyruvate, and inorganic phosphate in the absence of oxygen. Both aspects need further study and evaluation. Several differences were found in glycolitic enzymes and ATP, ADP and lactate levels; these have been found to be the same in Caucasians acclimatized and Sherpas permanently living at high altitude. Therefore this can be seen as a "congenital" and permanent acclimatization of the Sherpas

Oxygen diffusing capacity

High altitude adaptation also includes a progressive increase in arterial partial pressure of O2 (PaO2). There are two major ways by which this can occur:

• increased ventilation
• improvements of lung gas exchange

While the first mechanism rely on physiological responses to hypoxia (chemoreceptors in the aortic and carotid sinuses transmit the signal to the brain stem and force the organism to hyperventilate), the second is an aspect that can be studied in order to determine whether high altitude natives such as Sherpas have modified values. It is important to highlight that the term “diffusing capacity” is somehow inappropriate for what it represents, since the equation for its calculation also rely on the chemical combination of a given gas with hemoglobin and not just the diffusion across the alveolar-capillary membrane; moreover, the term “capacity” is not completely correct since the test is usually measured under submaximal conditions and therefore does not truly reflect a capacity. Nevertheless the term remains.

The diffusing capacity for oxygen (DLO2) is found by dividing the rate of oxygen uptake into the lung to the oxygen gradient between the capillary blood and the alveoli:

In 2011, an Italian team measured the diffusion capacity in lowlanders at sea level (Milan) and at Mt. Everest South Base Camp (5400 m of altitude) after a 9-day trek and 2 weeks residence at that altitude; subjects with mountain sickness were excluded (High-altitude exposure of three weeks duration increases lung diffusing capacity in humans, 2011).
After 2 weeks at 5400 m, hemoglobin oxygen saturation increased from 77.2 ± 6.0 to 85.3 ± 3.6%. Compared with sea level, there were increases in hemoglobin, lung diffusing capacity, membrane diffusion, and alveolar volume, while pulmonary capillary blood volume was unchanged. Membrane diffusion normalized for alveolar volume was also increased, which means a decreased resistance to diffusion. Reduction in alveolar-capillary barrier resistance is possibly mediated by an increase of sympathetic tone.
Altough this study was conducted on lowland residents, the improvement in the diffusing capacity might be one of the many mechanisms Sherpas have adopted through the centuries to survive at high altitude.

Summary

The correct and complete explanation of Sherpas' adaptation remains far from being found, but researches are moving in the right direction and approaching to the goal as the days go by. Together with the explanation of an interesting natural fact, finding the actual modifications that occur in Sherpas might also help to prevent Acute Mountain Sickness and heal other hypoxia-related diseases. Keeping on with the study of this amazing people is therefore fundamental.