Oxygen dissociation curve

The honor of plotting the first oxygen and carbon dioxide dissociation curves apparently belongs to Paul Bert. In his monumental book La Pression Barométrique he reported the relationships between partial pressure and blood gas concentration for both oxygen and carbon dioxide as experimental animals were exposed to lower and lower barometric pressures, or as they were gradually asphyxiated by rebreathing in a closed space (Bert 1943). However, he did not discover the S-shaped curve for oxygen because he did not reduce the PO₂ far enough. The first oxygen dissociation curve over its whole range was published by Christian Bohr in 1885. The measurements were made on dilute solutions of hemoglobin and showed precise hyperbolas (Bohr 1885). They were clearly not compatible with the data obtained by Bert in experimental animals, although Bohr did not comment on this. Hüfner (1890) published similar curves for hemoglobin solutions and argued that a hyperbolic shape would be expected from the simple equation:

An important advance was made by Bohr when he used whole blood rather than hemoglobin solutions; this led him to the discovery of the now-familiar S-shaped curve. In the following year, he showed in collaboration with Hasselbalch and Krogh, that the dissociation curve was shifted to the right when the PCO₂ of the blood was increased, a phenomenon which came to be known as the Bohr effect (Bohr et al. 1904). A few years later, Barcroft found that the addition of acid displaced the dissociation curve to the right (Barcroft and Orbeli 1910), and also that an increase in temperature had the same effect (Barcroft and King 1909). This work has been reviewed in detail by Astrup and Severinghaus (1986).

Soon after these important modulators of the oxygen affinity of hemoglobin were discovered, physiologists wondered about their importance at high altitude. For example, when Barcroft accompanied the first International High Altitude Expedition to Tenerife in 1910, he made a special study of the position of the oxygen dissociation curve, expecting it to be displaced to the left by the low arterial PCO₂. In the event, he found that the oxygen dissociation curves of some members of the expedition at 2130 m and 3000 m were shifted to the right when measured at the normal sea level PCO₂ of 40 mmHg. However, when he repeated the equilibrations at the subjects’ actual PCO₂ at altitude, the positions of the curves were essentially the same as at sea level (Barcroft 1911). He concluded that the decrease in carbonic acid in the blood was compensated for by an increase in some other acid, possibly lactic acid. One year later, Barcroft went to Mosso’s laboratory, the Capanna Regina Margherita on Monte Rosa (4559 m), and reported a slight excess acidity of the blood at that altitude (Barcroft et al. 1914).

Some 10 years later, during the 1921–22 International High Altitude Expedition to Cerro de Pasco in Peru, Barcroft and his colleagues found an increased oxygen affinity in acclimatized lowlanders as a result of the increased alkalinity of the blood. It also appeared that the increase in affinity was greater than could be explained by the change in acid–base status (Barcroft et al. 1923).

The question of oxygen affinity of hemoglobin was examined again on the International High Altitude Expedition to Chile in 1935. It was found that the “physiological” dissociation curves (that is, measured at a subject’s own PCO₂) were displaced slightly to the left of the sea-level values up to about 4270 m, but above that altitude, the curves were displaced increasingly to the right of the sea-level positions (Keys et al. 1936). Measurements of oxygen affinity of the hemoglobin were also made at constant pH and these showed a uniform tendency to a decreased affinity. The investigators argued that this rightward shift of the curve might be advantageous at high altitude because it would facilitate oxygen unloading to the tissues.

An important discovery was made in 1967 by two groups working independently (Benesch and Benesch 1967; Chanutin and Curnish 1967) that a fourth factor (in addition to PCO₂, pH, and temperature) had an important effect on the oxygen affinity of hemoglobin. This was the concentration of 2,3-diphosphoglycerate (2,3-DPG) within the red cells. This unexpected development raised doubts about much of the earlier work where this important factor had not been controlled, and it was shown that 2,3-DPG was depleted when blood was stored. It was subsequently shown that 2,3-DPG increased at high altitude (Lenfant et al. 1968), after which it was argued that the resulting decrease in oxygen affinity, which facilitated unloading of oxygen in the tissues, was an important part of the adaptation process (Lenfant and Sullivan 1971).

Until recently, relatively little information was available on the oxygen affinity of hemoglobin at extreme altitudes. A few measurements from the Silver Hut Expedition of 1960–61 showed that lowlanders who were well acclimatized to 5800 m had an almost fully compensated respiratory alkalosis (West 1962). Data above this altitude were lacking for a long time until studies performed as part of the 1981 American Medical Research Expedition to Everest showed that climbers near the summit (8848 m) apparently had an extreme degree of respiratory alkalosis, which greatly increased the oxygen affinity of their hemoglobin. The arterial pH of Pizzo on the Everest summit exceeded 7.7 as determined from the alveolar PCO₂ and base excess, both of which were measured. Direct measurements of pH in four climbers breathing air at 8400 m as part of the 2007 Caudwell Xtreme Everest expedition showed a lower pH than that measured in Pizzo (mean 7.53, range 7.45–7.60), but nevertheless showed that climbers were alkalemic at the extremes of elevation (Grocott et al. 2009). The main difference between these two later studies and the Silver Hut expedition was that the climbers had only been at 8400 m and above on Mount Everest for a short period of time, whereas the members of the Silver Hut expedition had resided at that elevation for many weeks. The climbers on the Xtreme Everest expedition also used supplemental oxygen for the duration of their climb, although they were breathing ambient air at the time the blood gas samples were drawn.

Acid–base balance

The early history of acid–base balance at high altitude overlaps considerably with the discussion of oxygen affinity of hemoglobin above. However, the reaction of the blood (as it was called) at high altitude created a great deal of interest in its own right. Indeed, the acid–base status of the blood played an important role in early theories of the control of breathing at high altitude (Kellogg 1980). As long ago as 1903, Galeotti studied various experimental animals taken to Mosso’s Capanna Margherita laboratory on the Monte Rosa, and found that the amount of acid needed to bring their hemolyzed blood to a standard pH (determined from litmus paper) was decreased compared with sea level (Galeotti 1904). He interpreted this decrease in titratable alkalinity to mean that there was an increase in some acid substance in the blood. It was known that hypoxia caused lactic acid production (Araki 1891) and that acidic blood stimulated breathing (Zuntz et al. 1906). It was therefore natural to conclude that this explained the hyperventilation of high altitude, and that the PCO₂ fell as a consequence (Boycott and Haldane 1908). Winterstein (1911) formulated what became known as the “reaction theory” of breathing, which stated that the effects of both hypoxia and carbon dioxide as stimuli of ventilation could be explained by the fact that they both acidified the blood.

The correct explanation of how hypoxia stimulates ventilation at high altitude had to wait for discovery of the peripheral chemoreceptors by Heymans and Heymans (1925). Meanwhile, Winterstein (1915) provided evidence against his own theory when he showed that, in acute hypoxia, the blood becomes alkaline rather than acid. A few years later, Henderson (1919) and Haldane et al. (1919) correctly explained the alkalinity as being secondary to the lowered PCO₂ caused by hyperventilation. Nevertheless, even today the control of ventilation during chronic hypoxia is a subject of intense research (Chapter 9) and interest still remains in the acid–base status of the extracellular fluid (ECF) that forms the environment of the central chemoreceptors.

Basic physiology

Figure 10.1 shows the oxygen dissociation curve of human whole blood and the four factors that shift the curve to the right, that is, decrease the affinity of oxygen for hemoglobin. These four factors are increases in PCO₂, hydrogen ion concentration, temperature, and the red blood cell concentration of 2,3-DPG. Increasing the ionic concentration of the plasma also reduces oxygen affinity.

Almost all of the change in oxygen affinity caused by PCO₂ can be ascribed to its effect on hydrogen ion concentration, although a change in PCO₂ has a small effect on its own (Margaria 1957). The mechanism of the alteration of oxygen affinity through hydrogen ion concentration (Bohr effect) is by a change in configuration of the hemoglobin molecule which makes the binding site less accessible to molecular oxygen as the hydrogen ion concentration is raised. The molecule exists in two forms: one in which the chemical subunits are maximally chemically bonded (T form), and another in which some bonds are ruptured and the structure is relaxed (R form). The R form has a higher affinity for oxygen because the molecule can more easily enter the region of the heme. The approximate magnitudes of the effects of change in PCO₂ and pH on the oxygen dissociation curve are shown in the right insets of Figure 10.1.

An increase in temperature has a large effect on the oxygen affinity of hemoglobin, as shown in the top inset of Figure 10.1. The temperature effect follows from thermodynamic considerations: the combination of oxygen with hemoglobin is exothermic so that an increase in temperature favors the reverse reaction, that is, dissociation of the oxyhemoglobin.

The compound 2,3-DPG is a product of red-cell metabolism, as shown in Figure 10.2. An increased concentration of this material within the red cell reduces the oxygen affinity of the hemoglobin by increasing the chemical binding of the subunits and converting more hemoglobin to the low affinity T form.

A useful number to describe the oxygen affinity of hemoglobin is the P₅₀, that is, the PO₂ at which 50% of the binding sites are occupied by oxygen. The normal value for adult whole blood at a PCO₂ of 40 mmHg, pH 7.4, temperature 37°C, and normal 2,3-DPG concentration is 26–27 mmHg. Human fetal blood has a P₅₀ of about 19 mmHg, mainly because fetal hemoglobin has two gamma rather than two beta chains, reducing the affinity for 2,3-DPG. An increase of 2,3-DPG within the red cell increases the P₅₀ by about 0.5 mmHg mol⁻¹ of 2,3-DPG. The magnitude of the Bohr effect is usually given in terms of the increase in log P₅₀ per pH unit. The normal value for human blood is 0.4 at constant PCO₂. Note that although historically the “Bohr effect” referred to the change in affinity caused by PCO₂, in modern usage, the term is restricted to the effect of pH. The temperature effect is 0.24 for the change in log P₅₀ (mmHg °C⁻¹).

Much can be learned about the effect of changes in the oxygen affinity of hemoglobin on the physiology of high altitude by modeling the oxygen transport system using computer subroutines for the oxygen and carbon dioxide dissociation curves (Bencowitz et al. 1982). Kelman described useful subroutines for the oxygen dissociation curve (Kelman 1966a; Kelman 1966b) and the carbon dioxide dissociation curve (Kelman 1967). The practical use of these procedures has been described (West and Wagner 1977). These procedures are able to accommodate changes in PCO₂, pH, temperature, and 2,3-DPG concentration, and allow the investigator to answer questions about the interactions of these variables, which would otherwise be impossibly complicated.

Animals native to high altitude

The oxygen transport properties of vertebrate red blood cells exhibit a high degree of plasticity in adjusting to changes in metabolic demands and/or environmental oxygen availability. It has been known for many years that many—but not all—animals that live at high altitude tend to have an increased oxygen affinity of their hemoglobin. Figure 8.6 in Chapter 8 shows part of the oxygen dissociation curves of the vicuna and llama, which are native to high altitude in the South American Andes (Hall et al. 1936). The diagram also shows the range of dissociation curves for eight lowland animals, including humans, horse, dog, rabbit, pig, peccary, ox, and sheep. It can be seen that the hemoglobin of high altitude native animals has a substantially increased oxygen affinity. This adaptation to high altitude is of genetic origin, as is shown by the fact that a llama brought up in a zoo at sea level has the same high oxygen affinity.

High altitude birds also have high oxygen affinities for hemoglobin. During the 1935 International High Altitude Expedition to Chile, Hall and his colleagues (Hall et al. 1936) reported that the high altitude ostrich and Huallaga have higher oxygen affinities than a group of six lowland birds including pigeon, Muscovy duck, domestic goose, domestic duck, Chinese pheasant, and domestic fowl. A particularly interesting example is the bar-headed goose, which is known to fly over the Himalayan ranges as it migrates between its breeding grounds in Siberia and its wintering grounds in India. This remarkable bird has a blood P₅₀ about 10 mmHg lower than its close relatives from moderate altitudes (Black and Tenney 1980; Scott et al. 2011). Diving emperor penguins also have hemoglobin with an increased oxygen affinity (Meir and Ponganis 2009). Tibetan chick embryos have a higher oxygen affinity of hemoglobin and higher red-cell concentrations than lowland embryos (Liu et al. 2009; Zhang et al. 2007).

Deer mice (Peromyscus maniculatus) show the same relationships. A study was carried out on 10 subspecies that live at altitudes from below sea level in Death Valley in California to the high mountains of the nearby Sierra Nevada (4350 m), and it was found that there was a strong correlation between the habitat altitude and the oxygen affinity of the blood. The genetic source of this relationship was proved by moving one subspecies to another location and showing that the oxygen affinity was unchanged. It was demonstrated that this relationship persisted in second-generation animals (Snyder et al. 1982). More recently, it has been reported that high altitude deer mice have evolved an adaptive increase in oxygen affinity for hemoglobin, but this is not associated with compensatory changes in sensitivity to changes in pH or temperature. Instead, it appears that the elevated oxygen affinity for hemoglobin in high altitude deer mice is compensated for by an associated increase in the tissue diffusion capacity of oxygen (via increased muscle capillarization), which promotes oxygen unloading (Jensen et al. 2016). By contrast, river otters living at an altitude of 2357 m have been shown to have an increased hemoglobin concentration but a normal oxygen affinity compared with those at sea level (Crait et al. 2012). Broadly consistent with these findings, it has been reported that the snow leopard (Panthera uncia)—an animal with an extraordinarily broad elevational distribution including elevations above 6000 m in the Himalayas—exhibited low oxygen affinities for hemoglobin and DPG sensitivities that were comparable to the African lion (Panthera leo) (Jenecka et al. 2015). Given the low oxygen affinity and reduced regulatory capacity of feline hemoglobins (and, indeed, in some other vertebrates), the extreme hypoxia tolerance of snow leopards must be attributable to compensatory modifications of other steps in the oxygen transport pathway. For further details on high altitude adaptations in vertebrate hemoglobins, see Weber (2007) and Storz (2016).

Animals in other oxygen-deprived environments

High altitude is just one of the oxygen-deprived environments in which animals are found, and it is interesting to consider the variety of strategies that have been adopted to mitigate the problems posed by oxygen deficiency. Table 8.1 in Chapter 8 shows examples of some of the strategies that have been adopted through genetic adaptation. The change in the globin chains of hemoglobin and the subsequent alteration in the affinity for 2,3-DPG in the human fetus has already been referred to. An alteration in globin chains also occurs in the bar-headed goose. The next two groups increase the oxygen affinity of their hemoglobin by decreasing the concentration of organic phosphates. This is done with 2,3-DPG in the fetus of the dog, horse and pig, and by decreasing the concentration of adenosine triphosphate (ATP), in the trout and eel.

Some species of tadpoles that frequently live in stagnant pools have a high oxygen affinity hemoglobin, whereas the adult frogs produce a different type of hemoglobin with a lower affinity that fits their higher oxygen environment. Note also that the tadpole blood shows a smaller Bohr effect. This is useful because low oxygen and high carbon dioxide pressures are likely to occur together in stagnant water, and a large Bohr effect would be disadvantageous because it would decrease the oxygen affinity of the blood when a high affinity was most needed.

As indicated earlier, the human fetus also has a high oxygen affinity by virtue of its fetal hemoglobin. This is essential because the arterial PO₂ of the fetus is less than 30 mmHg. Indeed, the human fetus and the adult climber on the summit of Mount Everest have some similar features in that in both cases the arterial PO₂ is extremely low, and the P₅₀ of the arterial blood (at the prevailing pH) is also very low.

A particularly interesting example of an unusual human hemoglobin was described by Hebbel et al. (1978). The authors studied a family in which two of the siblings had a mutant hemoglobin (Andrew-Minneapolis) with a P₅₀ of 17.1 mmHg. They showed that the siblings with the abnormal hemoglobin tolerated exercise at an altitude of 3100 m better than the normal siblings.

The last row in Table 8.1 refers to the climber at extreme altitude who has a marked respiratory alkalosis, which greatly increases the oxygen affinity of the hemoglobin. This is discussed in detail below.

Highlanders

Aste-Salazar and Hurtado (1944) measured the oxygen dissociation curves of 17 healthy Peruvians in Lima at sea level and 12 other permanent residents of Morococha (4550 m) and subsequently extended these studies to a total of 40 subjects in Lima and 30 in Morococha (Hurtado 1964). The mean value of the P₅₀ at pH 7.4 was 24.7 mmHg at sea level and 26.9 mmHg at high altitude (Figure 10.3). It was argued that the rightward displacement of the curve would enhance the unloading of oxygen from the peripheral capillaries.

Winslow et al. (1981) reported oxygen dissociation curves on 46 native Peruvians in Morococha (4550 m, P_B 432 mmHg) and reported that at pH 7.4 the P₅₀ was significantly higher in the high altitude population than in the sea level controls (31.2 mmHg as opposed to 29.2 mmHg, p < 0.001). However, these investigators also found that the acid–base status of the high altitude subjects was that of a partially compensated respiratory alkalosis with a mean plasma pH of 7.44. When the P₅₀ values were corrected to the subjects’ actual plasma pH, the mean value of 30.1 mmHg could no longer be distinguished from that of the sea-level controls (Figure 10.4). The conclusion was that the small increase in P₅₀ resulting from the increased concentration of 2,3-DPG in the red cells was offset by the mild degree of respiratory alkalosis, with the net result that the position of the oxygen dissociation curve was essentially the same as that in sea level controls.

In a controversial study, Morpurgo et al. (1976) reported that Sherpas living permanently at an altitude of 4000 m in the Nepalese Himalayas had a substantially increased oxygen affinity at standard pH. However, a subsequent study by Samaja et al. (1979) failed to confirm this provocative finding. Samaja et al. also showed that the oxygen affinity could be completely accounted for by the known effectors of hemoglobin function: pH, PCO₂, 2,3-DPG, and temperature. More recently, Li et al. (2018) compared arterial blood gas-derived P₅₀ values in groups of Han Chinese on the plains (500 m) and over 30 days acclimatization to 4300 m. These values were also compared to both resident Han Chinese and Tibetan groups who have resided for >10 years on the plateau at 3700 m. The P₅₀ values are illustrated in Figure 10.5. There was an initial increase in the P₅₀ values at days 3 and 7, which returned to below baseline following 30 days acclimatization to 4300 m (Figure 10.5A); such changes were likely explained by the progressive increases in concentrations in red blood cells and hemoglobin over this time period. In contrast, in the Han and Tibetan residents, the P₅₀ values were similar to and lower than those values on the plains (Figure 10.5B). However, despite these similar P₅₀ values—likely indicative of an adaptive phenotype—both the concentrations in red blood cells and hemoglobin were lower in the Tibetan residents when compared to the Han residents.

Acclimatized lowlanders

Early measurements by Barcroft (1911), Barcroft et al. (1923), Keys et al. (1936), and Hall et al. (1936) showed somewhat conflicting results. Possible reasons for this were clarified when the role of 2,3-DPG in the red cell was appreciated (Benesch and Benesch 1967; Chanutin and Curnish 1967). It was shown that this normal product of red-cell metabolism reduced the oxygen affinity of hemoglobin, and it was then clear that many previous measurements were unreliable because of ignorance of this factor. Lenfant and his colleagues (Lenfant et al. 1968; Lenfant et al. 1969; Lenfant et al. 1971) showed that the concentration of 2,3-DPG was increased in lowlanders when they became acclimatized to high altitude. The primary cause of the increase in 2,3-DPG was the increase in plasma pH above the normal sea-level value as a result of the respiratory alkalosis. When subjects were made acidotic with acetazolamide there was no increase in plasma pH or red cell 2,3-DPG concentration at high altitude, and the oxygen dissociation curve did not shift to the right. It was argued that the increase in 2,3-DPG was an important feature of the acclimatization process of lowlanders and of the adaptation to high altitude of highlanders (Lenfant and Sullivan 1971).

Subsequent measurements on lowlanders at high altitude have confirmed these changes, although there is still some uncertainty about whether acclimatized lowlanders develop complete metabolic compensation for their respiratory alkalosis (that is, whether the pH returns to 7.4). Certainly, this does not happen at extremely high altitudes. During the 1981 American Medical Research Expedition to Everest, Winslow et al. (1984) made an extensive series of measurements on acclimatized lowlanders at an altitude of 6300 m. They also obtained data on two subjects who reached the summit (8848 m). These measurements were made on venous blood samples taken at an altitude of 8050 m the morning after the summit climb. Winslow and his colleagues found that the red-cell concentration of 2,3-DPG increased with altitude (Figure 10.6) and that this was associated with a slightly increased P₅₀ value when expressed at pH 7.4. However, because the respiratory alkalosis was not fully compensated, the subjects’ in vivo P₅₀ at 6300 m (27.6 mmHg) was slightly less than at sea level (28.1 mmHg). The estimated in vivo P₅₀ was found to become progressively lower at 8050 m (24.9 mmHg), and on the summit at 8848 m, it was as low as 19.4 mmHg in one subject. Thus these data show that, at extreme altitudes, the blood oxygen dissociation curve shifts progressively leftward (increased oxygen affinity of hemoglobin) primarily because of the respiratory alkalosis. Indeed, this effect completely overwhelms the relatively small tendency for the curve to shift to the right because of the increase in red cell 2,3-DPG. The results obtained on Operation Everest II were generally in agreement with these data (Sutton et al. 1988) except that the PCO₂ values at extreme altitude were higher, and the blood pH values therefore lower. These differences can probably be explained by the smaller degree of acclimatization for reasons that are still not clear. The P₅₀ and 2,3-DPG concentrations were not measured as part of the Caudwell Xtreme Everest expedition’s analysis of blood gas results in 2009 (Grocott et al. 2009), but the subjects in this study had a milder degree of alkalemia than that seen in the American Medical Research Expedition to Everest.

Physiological effects of changes in oxygen affinity

There have been differences of opinion on whether a decreased or an increased oxygen affinity is beneficial at high altitude. Barcroft et al. (1923) found a slightly increased affinity and argued that this would enhance oxygen loading in the lung. However, Aste-Salazar and Hurtado (1944) reported a slight decrease in oxygen affinity in high altitude natives at Morococha and reasoned that this would enhance oxygen unloading in peripheral capillaries (Figure 10.3). The same argument was used by Lenfant and Sullivan (1971) when the influence of the increased red-cell concentration of 2,3-DPG on the oxygen dissociation curve was appreciated. They stated that the decreased oxygen affinity would help the peripheral unloading of oxygen, and that this was one of the many features both of acclimatization of lowlanders to high altitude and of the genetic adaptation of highlanders.

However, there is now strong evidence that an increased oxygen affinity (left-shifted oxygen dissociation curve) is beneficial, especially at higher altitudes, and particularly on exercise (Bencowitz et al. 1982). Indeed, this should not come as a surprise when it is appreciated that many animals increase the oxygen affinity of their blood in oxygen-deprived environments by a variety of strategies (Table 8.1). In addition, Eaton et al. (1974) reported that rats whose oxygen dissociation curve had been left-shifted by cyanate administration showed an increased survival when they were decompressed to a barometric pressure of 233 mmHg. The controls were rats with a normal oxygen affinity. Turek et al. (1978) also studied cyanate-treated rats and found that they maintained better oxygen transfer to tissues during severe hypoxia than normal animals. In addition, we have already referred to the studies of Hebbel et al. (1978), who found a family with two members who had a hemoglobin with a very high affinity (Hb Andrew-Minneapolis, P₅₀ 17.1 mmHg). These two members performed better during exercise at an altitude of 3100 m than two siblings with normal hemoglobin.

Theoretical studies show that a high oxygen affinity is beneficial at high altitude, especially on exercise (Bencowitz et al. 1982; Turek et al. 1973). In one study, oxygen transfer from air to tissues was modeled for a variety of altitudes and a range of oxygen uptakes (Bencowitz et al. 1982). The oxygen dissociation curve was shifted both to the left and right with P₅₀ of 16.8 mmHg (left-shifted), 26.8 mmHg (normal).

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