Oxygen transport, diving and high altitude

Oxygen dissolves poorly in water, and therefore in plasma. Human plasma at a normal arterial PO₂ of 100 mmHg holds only about 3 mL of oxygen per litre. To supply a resting metabolic rate of about 250 mL of oxygen per minute, the heart would have to pump more than 80 litres of plasma per minute, far beyond its capacity. Respiratory pigments, proteins that bind oxygen reversibly, solve the problem: human blood carries about 200 mL of oxygen per litre, almost all of it bound to hemoglobin. This lesson explains how respiratory pigments load oxygen at the gas exchanger and unload it in the tissues, how their properties are tuned to each animal's needs, and how animals cope with two extreme challenges, breath-hold diving and life at high altitude.

Respiratory pigments

Animals use three families of respiratory pigments. Hemoglobins, which contain iron in a heme group, are found in vertebrates and scattered across invertebrate groups, including some annelids, insects such as chironomid midge larvae, and crustaceans. Vertebrate hemoglobin is a tetramer of two α and two β chains, each binding one oxygen molecule, packed inside red blood cells. Hemocyanins, which bind oxygen between two copper atoms and turn blue when oxygenated, are dissolved in the hemolymph of many molluscs and arthropods, including octopuses, snails, crabs, lobsters and horseshoe crabs. Hemerythrins, also iron-based, occur in a few marine worms. Myoglobin, a single-chain relative of hemoglobin in muscle, stores oxygen and helps it diffuse within muscle fibres.

Packaging hemoglobin in red cells rather than dissolving it in plasma has advantages. A high concentration of a dissolved protein would raise the viscosity and oncotic pressure of the blood, and the red cell provides a controlled environment in which organic phosphates and enzymes such as carbonic anhydrase can regulate oxygen binding and carbon dioxide transport. The hemocyanin of an octopus, dissolved in plasma, is a huge molecule, which keeps its contribution to oncotic pressure manageable, but octopus blood still carries only about a quarter as much oxygen per litre as human blood.

The oxygen equilibrium curve

The relationship between PO₂ and the percentage saturation of a pigment is the oxygen equilibrium curve, often called the oxygen dissociation curve. For myoglobin, with a single binding site, the curve is hyperbolic. For vertebrate hemoglobin it is sigmoid, because binding is cooperative: the binding of oxygen to one subunit increases the affinity of the others, by shifting the tetramer from a low-affinity tense (T) state to a high-affinity relaxed (R) state. The curve is described empirically by the Hill equation, in which the Hill coefficient n measures cooperativity (about 2.7 to 3 for human hemoglobin, 1 for myoglobin) and the P50, the PO₂ at which the pigment is half saturated, measures affinity. A low P50 means high affinity.

The sigmoid shape is exactly what an oxygen carrier needs. On the flat upper part of the curve, around the PO₂ of the lungs, saturation changes little even if PO₂ falls somewhat, so loading is secure at altitude or in mild lung disease. On the steep middle part, around the PO₂ of active tissues, a small fall in PO₂ releases a large amount of oxygen. Cooperative binding therefore makes hemoglobin a better carrier than a non-cooperative pigment with the same P50.

The Bohr effect and other modulators

Hemoglobin's affinity for oxygen is not fixed. In 1904 Christian Bohr, father of the physicist Niels Bohr, found that carbon dioxide reduces the affinity of hemoglobin for oxygen. The Bohr effect, a rightward shift of the curve caused mainly by falling pH, helps unloading in active tissues, where carbon dioxide and acid accumulate, and helps loading in the lungs, where carbon dioxide is removed. Rising temperature also shifts the curve to the right, so warm, working muscles receive more oxygen. Inside mammalian red cells, 2,3-bisphosphoglycerate (2,3-BPG) binds to deoxygenated hemoglobin and lowers its affinity; birds use inositol pentaphosphate and many fish use ATP and GTP for the same purpose. Changing the concentration of these organic phosphates lets an animal adjust its blood oxygen affinity within hours to days.

# Oxygen delivery with the Hill equation and the Bohr effect (approximate human values)
def sat(P, P50, n=2.7):
    return P ** n / (P ** n + P50 ** n)

cap = 200.0              # oxygen capacity, mL O2 per L blood when fully saturated
art = 100.0              # arterial PO2, mmHg
cases = [("rest, pH 7.40", 27.0, 40.0),
         ("exercise, pH 7.40", 27.0, 20.0),
         ("exercise, pH 7.20 (Bohr shift)", 33.0, 20.0)]
print("condition                         Sa     Sv    O2 released (mL/L)")
for name, p50, tissue in cases:
    sa, sv = sat(art, p50), sat(tissue, p50)
    print(f"{name:32s} {sa*100:5.1f}  {sv*100:5.1f}   {cap * (sa - sv):6.1f}")

# a non-cooperative pigment with the same P50 gives less release over the same range
sa, sv = sat(art, 27.0, n=1.0), sat(40.0, 27.0, n=1.0)
print(f"{'non-cooperative, P50 27, rest':32s} {sa*100:5.1f}  {sv*100:5.1f}   {cap * (sa - sv):6.1f}")

condition                         Sa     Sv    O2 released (mL/L)
rest, pH 7.40                     97.2   74.3     45.8
exercise, pH 7.40                 97.2   30.8    132.8
exercise, pH 7.20 (Bohr shift)    95.2   20.6    149.3
non-cooperative, P50 27, rest     78.7   59.7     38.1

At rest, blood gives up only about a quarter of its oxygen, about 50 mL per litre, leaving a large venous reserve. During exercise, the fall in tissue PO₂ alone nearly triples the oxygen released, and the Bohr shift adds substantially more, so that most of the oxygen is unloaded. A non-cooperative pigment with the same P50 does worse at both ends: it would be only about 79 per cent saturated in the lungs and would release less oxygen even at rest, and the gap widens in exercise, because its curve is neither flat at high PO₂ nor steep in the range where tissues work.

In many fish, acid has a more drastic effect, the Root effect: low pH not only reduces affinity but also reduces the maximum saturation, so that hemoglobin cannot be fully saturated even at very high PO₂. Fish use the Root effect to deliver oxygen against large gradients. In the swim bladder, gas gland cells secrete lactic acid into the blood, driving oxygen off hemoglobin; the countercurrent rete mirabile (lesson 9) multiplies the resulting rise in PO₂ until it exceeds the pressure in the bladder, which can be many atmospheres in deep-sea fish. A similar system, the choroid rete, supplies the avascular retina of many fish with oxygen at very high partial pressures.

Carbon dioxide transport

Carbon dioxide is carried in blood in three forms: dissolved, bound to hemoglobin as carbamino compounds, and, mostly, as bicarbonate. In red cells, the enzyme carbonic anhydrase speeds the reaction of carbon dioxide with water to form carbonic acid, which dissociates into bicarbonate and hydrogen ions. Bicarbonate leaves the red cell in exchange for chloride, the chloride shift, and hydrogen ions are buffered by hemoglobin. Deoxygenated hemoglobin is a better buffer and binds more carbon dioxide than oxygenated hemoglobin, the Haldane effect, so unloading oxygen in the tissues helps blood pick up carbon dioxide, and loading oxygen in the lungs helps release it. The same reactions link breathing to acid–base balance, as the Henderson–Hasselbalch equation shows.

Matching affinity to the environment

The P50 of hemoglobin varies widely among species, and the variation makes adaptive sense. Small mammals, with high mass-specific metabolic rates, tend to have higher P50 values, lower affinity, which favours unloading in their tissues. Animals that live where oxygen is scarce, such as burrowing mammals, fish in hypoxic waters and animals at high altitude, tend to have lower P50 values, higher affinity, which favours loading. Fetal mammals have hemoglobin, or red cell conditions, with higher affinity than maternal blood, so oxygen passes from mother to fetus across the placenta.

Some of the best evidence that changes in hemoglobin are adaptive comes from studies of North American deer mice. Jay Storz and colleagues have shown that deer mice living above 3,000 m in the Rocky Mountains carry hemoglobin variants with higher oxygen affinity than lowland populations, caused by a small number of amino acid substitutions in the α and β chains, and that these variants improve aerobic performance under hypoxia. Similar changes have evolved independently in high-altitude birds, including the bar-headed goose and Andean geese and hummingbirds, often through different amino acid substitutions, a case of convergent evolution at the molecular level.

Life at high altitude

At 4,000 m, where communities live on the Tibetan Plateau and in the Andes, barometric pressure is about 60 per cent of sea level and inspired PO₂ about 85 mmHg. Lowlanders who travel to high altitude respond in stages. Within minutes, peripheral chemoreceptors detect the low arterial PO₂ and increase ventilation, which raises alveolar PO₂ but lowers PCO₂ and causes respiratory alkalosis. Over days, the kidneys excrete bicarbonate to correct the pH, allowing ventilation to rise further, and 2,3-BPG levels increase. Over weeks, the kidneys secrete more erythropoietin, which stimulates red cell production and raises hemoglobin concentration. Some people develop acute mountain sickness, and rarely life-threatening high-altitude pulmonary or cerebral edema.

Populations that have lived at high altitude for thousands of years show different patterns. Many Andean highlanders have high hemoglobin concentrations, sometimes excessively high, which can lead to chronic mountain sickness. Tibetan highlanders, by contrast, typically have hemoglobin concentrations near sea-level values, with higher ventilation and blood flow and higher levels of nitric oxide in their breath. Genome studies have found that variants of the EPAS1 gene, which encodes a transcription factor in the hypoxia-sensing pathway, are common in Tibetans and associated with lower hemoglobin levels. Emilia Huerta-Sánchez and colleagues reported in 2014 that the Tibetan EPAS1 haplotype was introduced into the ancestors of Tibetans by interbreeding with Denisovans, an extinct archaic human group, an example of adaptive introgression discussed in BIOL-321.

Birds are the champions of high-altitude flight. Bar-headed geese migrate between South and Central Asia, crossing the Himalayas. Satellite and logger studies by Lucy Hawkes, Charles Bishop and colleagues showed that most geese fly through passes and valleys at 5,000 to 6,000 m rather than over the highest peaks, but they do so by flapping flight, often at night when air is cooler and denser, and occasional records exceed 7,000 m. Their success depends on several features: hemoglobin with high oxygen affinity, a large lung and efficient crosscurrent gas exchange (lesson 9), a strong ventilatory response to hypoxia without disabling alkalosis, more capillaries in flight muscle and mitochondria positioned close to the capillaries.

import math

# Inspired PO2 versus altitude, using an approximate barometric formula
# P = 760 * exp(-h / 8000) (simplified isothermal atmosphere, h in m)
def baro(h):
    return 760.0 * math.exp(-h / 8000.0)

print("altitude (m)   barometric (mmHg)   inspired PO2 (mmHg)   place")
for h, place in ((0, "sea level"), (1600, "Denver, Colorado"),
                 (3000, "high Rocky Mountain valleys"), (4300, "Pikes Peak summit"),
                 (5500, "Himalayan passes (bar-headed geese)"), (8849, "Everest summit")):
    pb = baro(h)
    pio2 = 0.2095 * (pb - 47.0)     # humidified at 37 degC (saturated water at 47 mmHg)
    print(f"{h:8d}         {pb:7.0f}             {pio2:7.0f}            {place}")

altitude (m)   barometric (mmHg)   inspired PO2 (mmHg)   place
       0             760                 149            sea level
    1600             622                 121            Denver, Colorado
    3000             522                 100            high Rocky Mountain valleys
    4300             444                  83            Pikes Peak summit
    5500             382                  70            Himalayan passes (bar-headed geese)
    8849             251                  43            Everest summit

For Everest the simple formula happens to agree closely with measurements made on the summit, a barometric pressure of about 250 mmHg and an inspired PO₂ of about 43 mmHg, but in general real pressures depend on air temperature, latitude and season, and standard aviation atmospheres give somewhat lower values at the same altitude. Even so, the trend is clear, and it explains why the summit of Everest is near the limit of human tolerance without supplementary oxygen.

Breath-hold diving

Diving mammals and birds face the opposite problem from high-altitude animals: they cannot breathe at all while submerged, sometimes for over an hour. Weddell seals in the Antarctic routinely dive for 10 to 20 minutes and can stay down for more than an hour; northern elephant seals off California dive repeatedly for 20 minutes to depths of 400 to 600 m, spending almost 90 per cent of months at sea under water; and in 2020 Nicola Quick and colleagues reported a Cuvier's beaked whale off North Carolina that stayed submerged for 222 minutes. Diving mammals succeed by storing more oxygen and using it more slowly.

Diving mammals have large blood volumes, high hemoglobin concentrations and very high muscle myoglobin concentrations, often ten times those of terrestrial mammals, which colours their muscles almost black. Myoglobin of diving mammals carries a higher positive surface charge than that of land mammals, which, as Scott Mirceta and colleagues showed in 2013, lets it accumulate to high concentrations without aggregating. Seals store much of their oxygen in blood and muscle rather than in the lungs. They exhale before diving, and their lungs collapse at depth, which reduces the uptake of nitrogen into the blood and the risk of decompression sickness. Diving birds such as penguins and murres, by contrast, dive with air in their lungs and air sacs.

The dive response, first studied in detail by Per Scholander and Laurence Irving in the 1930s and 1940s, reduces oxygen use during a dive. Heart rate falls sharply, a diving bradycardia, and peripheral blood vessels constrict, so blood flow is reserved mainly for the brain and heart. Muscles, cut off from blood flow, rely on their myoglobin stores and then on anaerobic glycolysis, accumulating lactate that is washed into the circulation after surfacing. Gerald Kooyman's studies of Weddell seals in the Antarctic in the 1970s and 1980s showed that most natural dives are shorter than the aerobic dive limit, the longest dive possible without a rise in blood lactate after surfacing, which for adult Weddell seals is about 20 minutes. Dives beyond the limit are possible but require long recovery periods at the surface, so seals usually stay within it.

# Oxygen stores and aerobic dive limit (ADL) estimates (approximate values per kg body mass)
def stores(mass, blood_l_per_kg, hb_g_per_l, mb_g_per_kg_muscle, muscle_frac, lung_ml_per_kg):
    blood_o2 = mass * blood_l_per_kg * hb_g_per_l * 1.34 * 0.8      # mL O2; 1.34 mL per g Hb, 80% usable
    muscle_o2 = mass * muscle_frac * mb_g_per_kg_muscle * 1.34       # mL O2 bound to myoglobin
    lung_o2 = mass * lung_ml_per_kg * 0.15                            # 15% O2 usable from lung air
    return blood_o2, muscle_o2, lung_o2

animals = {
    #                      mass  blood Hb  Mb   muscle lung  diving VO2 (mL/kg/min)
    "Weddell seal":       (400, 0.15, 230, 50,  0.35,  20,   3.2),
    "human free-diver":   (75,  0.07, 150, 5,   0.40,  80,   4.5),
}
print("animal              blood   muscle   lung    total O2 (L)  ADL (min)")
for name, (m, bl, hb, mb, mf, lung, vo2) in animals.items():
    b, mu, lu = stores(m, bl, hb, mb, mf, lung)
    total = b + mu + lu
    print(f"{name:18s} {b/1000:6.1f}  {mu/1000:6.1f}  {lu/1000:6.2f}    {total/1000:6.1f}       {total / (m * vo2):5.1f}")

animal              blood   muscle   lung    total O2 (L)  ADL (min)
Weddell seal         14.8     9.4    1.20      25.4        19.8
human free-diver      0.8     0.2    0.90       1.9         5.8

Per kilogram, the seal stores two to three times as much oxygen as a human, and most of it is in blood and muscle rather than in the lungs. Using a diving metabolic rate somewhat below the seal's resting rate on land, as measured in freely diving Weddell seals, the estimated aerobic dive limit comes out near the 20 minutes measured by Kooyman; the human's is a few minutes. Trained human free-divers can hold their breath far longer than this estimate while resting, because their metabolic rate falls and they tolerate low oxygen, but they do so at real risk of blackout. The calculation shows why storage, not tolerance alone, is the key to long dives.