Hemoglobin-oxygen affinity in high-altitude vertebrates: is there evidence for an adaptive trend?

Abstract

In air-breathing vertebrates at high altitude, fine-tuned adjustments in hemoglobin (Hb)-O₂ affinity provide an energetically efficient means of mitigating the effects of arterial hypoxemia. However, it is not always clear whether an increased or decreased Hb-O₂ affinity should be expected to improve tissue O₂ delivery under different degrees of hypoxia, due to the inherent trade-off between arterial O₂ loading and peripheral O₂ unloading. Theoretical results indicate that the optimal Hb-O₂ affinity varies as a non-linear function of environmental O₂ availability, and the threshold elevation at which an increased Hb-O₂ affinity becomes advantageous depends on the magnitude of diffusion limitation (the extent to which O₂ equilibration at the blood-gas interface is limited by the kinetics of O₂ exchange). This body of theory provides a framework for interpreting the possible adaptive significance of evolved changes in Hb-O₂ affinity in vertebrates that have colonized high-altitude environments. To evaluate the evidence for an empirical generalization and to test theoretical predictions, I synthesized comparative data in a phylogenetic framework to assess the strength of the relationship between Hb-O₂ affinity and native elevation in mammals and birds. Evidence for a general trend in mammals is equivocal, but there is a remarkably strong positive relationship between Hb-O₂ affinity and native elevation in birds. Evolved changes in Hb function in high-altitude birds provide one of the most compelling examples of convergent biochemical adaptation in vertebrates.

Keywords: Biochemical adaptation; Blood oxygen transport; Hemoglobin; High-altitude adaptation; Hypoxia; Physiological adaptation.

Fig. 1.

The allosteric regulation of hemoglobin (Hb)–O₂ affinity. (A) The oxygenation reaction of tetrameric Hb (α₂β₂) involves an allosteric transition in quaternary structure from the low-affinity T-state to the high-affinity R-state. The oxygenation-induced T→R transition entails a breakage of salt bridges and hydrogen bonds within and between subunits (open squares), dissociation of allosterically bound organic phosphates (OPHs), Cl⁻ ions and protons, and the release of heat (heme oxygenation is an exothermic reaction). Oxygenation-linked proton binding occurs at multiple residues in the α- and β-chains, Cl⁻ binding mainly occurs at the N-terminal α-amino groups of the α- and β-chains in addition to other residues in both chains, and phosphate binding occurs between the β-chains in the central cavity of the Hb tetramer. (B) O₂ equilibrium curves for purified Hb in the absence of allosteric effectors (Stripped) and in the presence of chloride ions (+Cl⁻) and organic phosphates (+OPH). The preferential binding of allosteric effectors to deoxyHb stabilizes the T-state, thereby shifting the allosteric equilibrium in favour of the low-affinity quaternary structure. The O₂ equilibrium curves are therefore right-shifted (Hb–O₂ affinity is reduced) in the presence of such effectors. Hb–O₂ affinity is indexed by the P₅₀ value (dashed grey lines) – the P_O2 at which Hb is half-saturated. The sigmoidal shape of the O₂ equilibrium curves reflects cooperative O₂ binding, involving a P_O2-dependent shift from low- to high-affinity conformations.

Fig. 2.

Schematic illustration of blood O₂ transport. (A) An O₂ equilibrium curve under the physicochemical conditions prevailing in arterial blood (a, continuous curve, open symbol) and venous blood (v, dashed curve, closed symbol). The curve is a plot of blood O₂ content (y-axis) versus P_O2 (x-axis), with paired values for arterial and venous blood connected by a continuous line. Ca_O2−Cv_O2 denotes the arterial–venous difference in blood O₂ content, Pa_O2−Pv_O2 denotes the corresponding difference in P_O2, βb_O2 denotes the blood O₂ capacitance coefficient (see text for details), denotes cardiac output, and denotes the rate of O₂ consumption. On the right-hand side of the graph, the area of the rectangle is proportional to total O₂ consumption, which can be enhanced by increasing and/or by increasing βb_O2. Increases in βb_O2 produce a corresponding increase in Ca_O2−Cv_O2 through shifts in the shape or position of the O₂ equilibrium curve. (B) O₂ equilibrium curves showing the effect of changes in Hb–O₂ affinity on tissue O₂ delivery under conditions of moderate hypoxia (open symbols) and severe hypoxia (filled symbols). For each pair of arterial and venous points, the P_O2 for venous blood (Pv_O2) is marked by a vertical grey line that extends to the x-axis. The sigmoid O₂ equilibrium curves are shown for high, intermediate and low Hb–O₂ affinities; P₅₀, the P_O2 at which Hb is 50% saturated. Each change in Hb–O₂ affinity produces a shift in Pv_O2, but the P_O2 of arterial blood (Pa_O2) is assumed to remain constant. Note that under conditions of moderate hypoxia the right-shifted curve maximizes βb_O2 and preserves a higher Pv_O2 (an overall index of tissue oxygenation). Under severe hypoxia, by contrast, the left-shifted curve maximizes βb_O2 and preserves a higher Pv_O2 relative to the right-shifted curve. When the kinetics of O₂ transfer across the alveolar gas–blood barrier is a limiting step (diffusion limitation), a left-shifted O₂ equilibrium curve may also be advantageous under less severe hypoxia (Bencowitz et al., 1982).

Fig. 3.

Blood P₅₀ and Hill's cooperativity coefficient, n, influence blood O₂ transport (indexed by the difference in arterial and venous O₂ saturation). (A) Normoxia; (B) hypoxia. In these three-dimensional plots, the difference in arterial and venous O₂ saturation (Sa_O2−Sv_O2) is indicated by the height of the projection above the reference plane. The higher the projection, the greater the difference in O₂ saturation.

Fig. 4.

Isobars showing predicted values of venous P_O2 (an index of tissue oxygenation) as a function of arterial P_O2 at different values of blood P₅₀, assuming 50% tissue O₂ extraction and constant cardiac output. At normal or moderately reduced Pa_O2, a higher P₅₀ results in a higher Pv_O2 (and hence improved tissue oxygenation). Under more severe hypoxemia, by contrast, a lower P₅₀ results in a higher Pv_O2 while still maintaining constant O₂ extraction.

Fig. 5.

Phylogenetic relationships of 14 mammalian taxa and 58 avian taxa used in comparative analyses of Hb function. Rows corresponding to high-altitude taxa are shaded. (A) In the mammalian phylogeny, branches in bold connect pairs of high- and low-altitude taxa that were used to test for a relationship between Hb–O₂ affinity and native elevation. As there are no overlaps in the paths of descent connecting each designated pair of high- and low-altitude taxa, the seven pairwise comparisons are statistically independent. For information regarding elevational ranges, see Storz et al. (2009, 2010a), Revsbech et al. (2013), Janecka et al. (2015), Natarajan et al. (2015a) and Tufts et al. (2015). (B) In the avian phylogeny, branches in bold connect pairs of high- and low-altitude taxa that were used to test for a relationship between Hb–O₂ affinity and native elevation. As in the case with the mammals, the 29 pairwise comparisons are phylogenetically independent. For information regarding elevational ranges, see Projecto-Garcia et al. (2013), Cheviron et al. (2014), Galen et al. (2015) and Natarajan et al. (2015b, 2016).

Fig. 6.

O₂ affinities of purified Hbs from representative pairs of high- and low-altitude mammals. O₂ equilibria were measured at pH 7.40, 37°C, in the presence and absence of allosteric effectors ([Cl⁻], 0.10 mol l⁻¹; [Hepes], 0.1 mol l⁻¹; DPG:tetrameric Hb ratio, 2.0: [heme], 0.2–0.3 mmol l⁻¹). For each taxon, P₅₀ values (±s.e.m.) are reported for stripped Hbs in the absence of added anions, in the presence of Cl⁻ alone (added as KCl), in the presence of DPG alone, and in the presence of both anions combined. This latter ‘KCl+DPG’ treatment is most relevant to in vivo conditions in mammalian red blood cells, but measurements of O₂ affinity under each of the four standardized treatments can provide insights into the functional mechanism responsible for observed differences in P_50(KCl+DPG). (A) Comparison between Hb variants of high- and low-altitude deer mice, Peromyscus maniculatus, from the Rocky Mountains and Great Plains, respectively [data from Natarajan et al., 2015a; for additional details, see Storz et al. (2009, 2010a); Jensen et al. (2016)]. (B) Comparison between Hbs of the high-altitude American pika (Ochotona princeps) and the low-altitude collared pika (O. collaris) (data from Tufts et al., 2015). (C) Comparison between Hbs of the high-altitude yellow-bellied marmot (Marmota flaviventris) and the low-altitude hoary marmot (M. caligata) (data from Revsbech et al., 2013). (D) Comparison between Hbs of the high-altitude snow leopard (Panthera uncia) and the low-altitude African lion (P. leo) (data from Janecka et al., 2015). For both cat species, P₅₀ is shown as the mean value for two co-expressed isoforms, HbA and HbB, which are present at roughly equimolar concentrations (Janecka et al., 2015).

Fig. 7.

There is no evidence for a significant elevational trend in the Hb–O₂ affinities of mammals. The plot shows measures of Hb–O₂ affinity in the presence of anionic effectors [P_50(KCl+IHP) (±s.e.m.)] for seven matched pairs of high- and low-altitude taxa. Data points that fall below the diagonal (x=y) denote cases in which the high-altitude member of a given taxon pair possesses a higher Hb–O₂ affinity (lower P₅₀). The paired-lineage design ensures that all data points are statistically independent (see text for details). Filled symbols denote comparisons between species, whereas the open symbol denotes a comparison between high- versus low-altitude populations of the same species (P. maniculatus).

Fig. 8.

Comparison of oxygenation properties of the major Hb isoform (HbA) between pairs of high- and low-altitude birds in the Andes. O₂ equilibria were measured at pH 7.40, 37°C in the presence and absence of allosteric effectors ([Cl⁻], 0.10 mol l⁻¹; [Hepes], 0.1 mol l⁻¹; IHP:tetrameric Hb ratio, 2.0: [heme], 0.3 mmol l⁻¹). For each taxon, P₅₀ values (±s.e.m.) are reported for stripped Hbs in the absence of added anions, in the presence of Cl⁻ alone (added as KCl), in the presence of IHP alone, and in the presence of both anions combined. As explained in the main text, the ‘KCl+IHP’ treatment is most relevant to in vivo conditions in avian red blood cells, but measurements of O₂ affinity under each of the four standardized treatments provide insights into the functional mechanism responsible for observed differences in P_50(KCl+IHP). In each pairwise comparison shown here, slight differences in intrinsic Hb–O₂ affinity (reflected by stripped P₅₀ values) become more pronounced in the presence of IHP. (A) Comparison of HbA O₂ affinities between high- and low-altitude hummingbirds in the Emeralds clade (Trochilidae: Apodiformes): the green-and-white hummingbird, Amazilia viridicauda, and the amazilia hummingbird, A. amazilia, respectively. (B) Comparison of HbA O₂ affinities between high- and low-altitude hummingbirds in the Coquettes clade (Trochilidae: Apodiformes): the Andean hillstar, Oreotrochilus estella, and the speckled hummingbird, Adelomyia melanogenys, respectively. (C) Comparison of HbA O₂ affinities between high- and low-altitude flowerpiercers (Thraupidae: Passeriformes): the black-throated flowerpiercer, Diglossa brunneiventris, and deep-blue flowerpiercer, D. glauca, respectively. (D) Comparison of HbA O₂ affinities between high- and low-altitude populations of the house wren, Troglodytes aedon (Troglodytidae: Passeriformes). (E) Comparison of HbA O₂ affinities between high- and low-altitude populations of the hooded siskin, Spinus magellanica (Fringillidae: Passeriformes). (F) Comparison of HbA O₂ affinities between high- and low-altitude nightjars (Caprimulgidae: Caprimulgiformes): the band-winged nightjar, Hydropsalis longirostris, and Tschudi's nightjar, H. decussata, respectively. Data from Projecto-Garcia et al. (2013), Galen et al. (2015), and Natarajan et al. (2016).

Fig. 9.

Convergent increases in Hb–O₂ affinity in high-altitude Andean birds. (A) Plot of P_50(KCl+IHP) (±s.e.m.) for HbA in 29 matched pairs of high- and low-altitude taxa. Data points that fall below the diagonal (x=y) denote cases in which the high-altitude member of a given taxon pair possesses a higher Hb–O₂ affinity (lower P₅₀). Comparisons involve phylogenetically replicated pairs of taxa, so all data points are statistically independent. (B) Plot of P_50(KCl+IHP) (±s.e.m.) for the minor HbD isoform in a subset of the same taxon pairs in which both members of the pair express HbD. Sample sizes are larger for HbA than for HbD because the two ground dove species (Metriopelia melanoptera and Columbina cruziana) expressed no trace of HbD, and several hummingbird species expressed HbD at exceedingly low levels (Projecto-Garcia et al., 2013; Natarajan et al., 2016). In such cases, sufficient quantities of HbD could not be purified for measures of O₂ equilibria. Filled symbols denote comparisons between species, whereas open symbols denote comparisons between high- versus low-altitude populations of the same species. Data from Natarajan et al. (2015b, 2016).

Fig. 10.

There is no evidence for altitude-related differences in the relative abundance of HbA and HbD isoforms in Andean birds. Phylogenetically independent comparisons involving 26 pairs of high- and low-altitude taxa revealed no systematic difference in the relative expression level of the minor HbD isoform (Wilcoxon signed-ranks test, Z=−0.775, P=0.441). The diagonal represents the line of equality (x=y). Filled symbols denote comparisons between species, whereas open symbols denote comparisons between high- versus low-altitude populations of the same species. Data from Natarajan et al. (2016).