Transport of Oxygen and Carbon Dioxide in the Blood

The amount of O ₂ dissolved in blood is far too small to meet the metabolic demands of the body

Blood carries oxygen in two forms. More than 98% of the O ₂ normally binds to hemoglobin within the erythrocytes, also known as red blood cells or RBCs (see pp. 434–435 ). A tiny fraction physically dissolves in the aqueous phases of both blood plasma and the cytoplasm of blood cells (predominantly RBCs). What is the significance of the O ₂ that is bound to hemoglobin?

Imagine that we expose a liter of blood plasma, initially free of O ₂ , to an atmosphere having the same as alveolar air—100 mm Hg. Oxygen will move from the atmosphere to the plasma until an equilibrium is established, at which time the concentration of dissolved O ₂ ([O ₂ ] _dis ) in the blood obeys Henry's law (see p. 593 ):

If we express
in units of mm Hg at 37°C and [O ₂ ] _dis in units of (mL of O ₂ gas)/(dL of blood), then the solubility
is ~0.003 mL O ₂ /(dL of blood · mm Hg). For arterial blood,

Is such an O ₂ -carrying capacity adequate for supplying O ₂ to the systemic tissues? If these tissues could extract all the O ₂ dissolved in arterial blood so that no O ₂ remained in venous blood, and if cardiac output (see p. 414 ) were 5000 mL/min, then—according to the Fick principle (see p. 423 )—the delivery of dissolved O ₂ to the tissues would be

However, the average 70-kg human at rest consumes O ₂ at the rate of ~250 mL/min. Dissolved O ₂ could supply the body's metabolic demands only if cardiac output increased by a factor of 250/15, or nearly 17-fold! Thus, the body cannot rely on dissolved O ₂ as a mechanism for O ₂ carriage.

Hemoglobin consists of two α and two β subunits, each of which has an iron-containing “heme” and a polypeptide “globin”

Normal adult hemoglobin (Hb) is a tetramer having a molecular weight of ~68 kDa, each monomer consisting of a heme and a globin ( Fig. 29-1 A ). The heme is a porphyrin compound coordinated to a single iron atom. The globin is a polypeptide, either an α chain (141 amino acids) or a β chain (146 amino acids). The homology between the α and β chains is sufficient that they have similar conformations, a series of seven helices enveloping a single heme. Thus, the complete Hb molecule has the stoichiometry [α(heme)] ₂ [β(heme)] ₂ and can bind as many as four O ₂ molecules, one for each iron atom. The erythroblasts (see pp. 431–433 ) that synthesize Hb closely coordinate the production of α chains, β chains, and heme.

Heme is a general term for a metal ion chelated to a porphyrin ring. In the case of Hb, the metal is iron in the Fe ²⁺ or ferrous state (see Fig. 29-1 B ). The porphyrin consists of four linked pyrrole rings that, through their nitrogen atoms, coordinate a single, centrally located Fe ²⁺ . Because the iron-porphyrin complex is rich in conjugated double bonds, it absorbs photons of relatively low energy (i.e., light in the visible range). The interaction among O _2, Fe ²⁺ , and porphyrin causes the complex to have a red color when fully saturated with O ₂ (e.g., arterial blood) and a purple color when devoid of O ₂ (e.g., venous blood).

Hb can bind O ₂ only when the iron is in the Fe ²⁺ state. The Fe ²⁺ in Hb can become oxidized to ferric iron (Fe ³⁺ ), either spontaneously or under the influence of compounds such as nitrites or sulfonamides. The result of such an oxidation is methemoglobin (metHb), which is incapable of binding O ₂ . Inside the RBC, a heme-containing enzyme methemoglobin reductase uses the reduced form of nicotinamide adenine dinucleotide (NADH) to reduce metHb back to Hb, so that only about 1.5% of total Hb is in the metHb state. In the rare case in which a genetic defect results in a deficiency of this enzyme, N29-1 metHb may represent 25% or more of the total Hb. Such a deficiency results in a decreased O ₂ -carrying capacity, leading to tissue hypoxia.

The environment provided by the globin portion of Hb is crucial for the O ₂ -heme interaction. To be useful, this interaction must be fully reversible under physiological conditions, allowing repetitive capture and release of O ₂ . The interaction of O ₂ with free Fe ²⁺ normally produces Fe ³⁺ , the simplest example of which is rust. Even with isolated heme, O ₂ irreversibly oxidizes Fe ²⁺ to Fe ³⁺ . However, when heme is part of Hb, interactions with ~20 amino acids cradle the heme in the globin, so that O ₂ loosely and reversibly binds to Fe ²⁺ . The crucial residue is a histidine that bonds to the Fe ²⁺ and donates negative charge that stabilizes the Fe ²⁺ -O ₂ complex. This histidine is also crucial for transmitting, to the rest of the Hb tetramer, the information that an O ₂ molecule is or is not bound to the Fe ²⁺ . When all four hemes are devoid of O ₂ , each of the four histidines pulls its Fe ²⁺ above the plane of its porphyrin ring by ~0.06 nm (the blue con­formation in Fig. 29-1 C ), distorting the porphyrin ring. Thus, the Fe ²⁺ -histidine bond is under strain in deoxyhemoglobin, a strain that it transmits to the rest of the α or β subunit, and thence to the rest of the Hb molecule. The various components of the Hb tetramer are so tightly interlinked, as if by a snugly fitting system of levers and joints, that no one subunit can leave this tensed (T) state unless they all leave it together. Because the shape of the heme in the T state sterically inhibits the approach of O ₂ , empty Hb has a very low affinity for O ₂ .

When one O ₂ binds to one of the Fe ²⁺ atoms, the Fe ²⁺ tends to move down into the plane of the porphyrin ring. If the Fe ²⁺ actually could move, it would flatten the ring and relieve the strain on the Fe ²⁺ -histidine bond. When enough O ₂ molecules bind, enough energy builds up and all four subunits of the Hb simultaneously snap into the relaxed (R) state, whether or not they are bound to O ₂ . In this R state, with its flattened heme, the Hb molecule has an O ₂ affinity that is ~150-fold greater than that in the T state. Thus, when is zero, all Hb molecules are in the T state and have a low O ₂ affinity. When is very high, all Hb molecules are in the R state and have a high O ₂ affinity. At intermediate values, an equilibrium exists between Hb molecules in the T and R states.

Myoglobin (Mb) is another heme-containing, O ₂ -binding protein that is specific for muscle (see p. 249 ). The globin portion of Mb arose in a gene duplication event from a primordial globin. Additional duplications along the non-Mb branch of the globin family led first to the α and β chains of Hb, and then to other α-like and β-like chains ( Box 29-1 ). Mb functions as a monomer, homologous to either an α or a β chain of Hb. Although capable of binding only a single O ₂ molecule, Mb has a much higher O ₂ affinity than Hb. In the capillaries, Hb can thus hand off O ₂ to an Mb inside a muscle cell; this Mb then transfers its O ₂ to the next Mb, and so on, which speeds diffusion of O ₂ through the muscle cell. Because of the low solubility of O ₂ , this action is essential: There is insufficient dissolved O ₂ , by itself, to establish an intracellular O ₂ gradient large enough to deliver adequate O ₂ to mitochondria.

The Hb-O ₂ dissociation curve has a sigmoidal shape because of cooperativity among the four subunits of the Hb molecule

Imagine that we expose whole blood (see pp. 429–431 ) to a gas phase with a that we can set at any one of several values ( Fig. 29-2 ). For example, we could incubate the blood with a of 40 mm Hg, typical of mixed-venous blood, and centrifuge a sample to separate plasma from erythrocytes, as one would for determining hematocrit (see pp. 102 and 429 ). Next, we could individually determine the O ₂ content of the plasma (i.e., dissolved O ₂ ) and packed RBCs. If we know how much water is inside the RBCs, we can subtract the amount of O ₂ dissolved in this water from the total O ₂ , arriving at the amount of O ₂ bound to Hb.

Repeating this exercise over a range of values, we obtain the red curve in Figure 29-3 . The right-hand y-axis gives the O ₂ bound to Hb in the units (mL O ₂ )/(dL blood). The left-hand y-axis gives the same data in terms of percent O ₂ saturation of Hb ( or “Sat”). To compute , we need to know the maximal amount of O ₂ that can bind to Hb at extremely high values. Expressed in terms of grams of Hb protein, this O ₂ capacity is ~1.39 mL O ₂ /g Hb—assuming that no metHb is present. In real life, the O ₂ capacity may be closer to 1.35 mL O ₂ /g Hb because O ₂ cannot bind to Hb that either is in the Fe ³⁺ state (e.g., MetHb) or, as discussed below, is bound to carbon monoxide. We can translate this O ₂ capacity to a value for the maximal amount of O ₂ that can bind to Hb in 100 mL of blood. If the Hb content is 15 g Hb/dL blood (i.e., normal for an adult male), then

The percent saturation of Hb is

Notice that the curve in Figure 29-3 is sigmoidal or S-shaped, owing to the cooperativity among the four O ₂ -binding sites on the Hb molecule. At low values, increases in produce relatively small increases in O ₂ binding, which reflects the relatively low O ₂ affinity of Hb in the T state. At moderate values, the amount of bound O ₂ increases more steeply with increases in , which reflects the increased O ₂ affinity as more Hb molecules shift to the R state. The at which the Hb is half saturated is known as the P ₅₀ . Finally, the Hb-O ₂ versus curve flattens at high values as the Hb saturates. The difference in Hb saturation at low versus high values is the basis for an important clinical tool, the pulse oximeter ( Box 29-2 ).

At the prevailing in normal arterial blood ( )—approximately 100 mm Hg—the Hb saturation ( ) is ~97.5% or 19.7 mL O ₂ /dL bound to Hb ( Table 29-3 ). The dissolved O ₂ (purple curve in Fig. 29-3 ) would add an additional 0.3 mL O ₂ /dL for a total O ₂ content of 20.0 mL O ₂ /dL (point a on the brown curve in Fig. 29-3 ). In mixed-venous blood, in which ( ) is ~40 mm Hg, the Hb saturation ( ) is ~75% or 15.2 mL O ₂ /dL bound to Hb (see Table 29-3 ). The dissolved O ₂ would add 0.1 mL O ₂ /dL for a total of 15.3 mL O ₂ /dL (point in Fig. 29-3 ). The difference in total O ₂ content between points a and , the a- difference, is the amount of O ₂ that the lungs add to the blood in the pulmonary capillaries, which is the same amount that all the tissues extract from the blood in the systemic capillaries:

TABLE 29-3

Summary of a-
Difference in O ₂ Composition of Blood

	(mm Hg)	Hb SATURATION ( )	O 2 BOUND TO Hb (mL/dL) *	O 2 DISSOLVED (mL/dL)	TOTAL O 2 CONTENT (mL/dL)
a	100	97.5%	19.7	0.3	20.0
	40	75%	15.2	~0.1	15.3
a- difference	60	22.5%	4.5	~0.2	4.7

* Assuming an Hb content of 15 g Hb/dL blood and an O ₂ capacity of 1.35 mL O ₂ /g Hb. Here, fully saturated Hb would carry 20.3 mL O ₂ /dL blood.

Of the total a- difference of 4.7 mL O ₂ /dL, Hb provides 4.5 mL O ₂ /dL or nearly 96% of the O ₂ that the lungs add and the systemic tissues extract from blood (see Table 29-3 ). Is this a- difference in O ₂ content enough to satisfy the metabolic demands of the body (i.e., ~250 mL O ₂ /min)? Using the Fick principle, as we did in Equation 29-3 , we see that the combination of a cardiac output of 5 L/min and an a- difference of 4.7 mL/dL would be nearly adequate:

By either increasing cardiac output by ~6% or decreasing the
of mixed-venous blood, the body could meet a demand of 250 mL O ₂ /min. We spend our lives moving endlessly from point a in Figure 29-3 to point
(as we deliver O ₂ to the tissues) and then back to point a (as we take up more O ₂ from alveolar air).

Because the plot of [O ₂ ] _dis versus is linear (see Fig. 29-3 , purple curve), the amount of O ₂ that can dissolve in blood plasma has no theoretical maximum. Thus, breathing 100% O ₂ would raise arterial by ~6-fold, so that ~1.8 mL of O ₂ would be dissolved in each deciliter of arterial blood. Although dissolved O ₂ would make a correspondingly greater contribution to overall O ₂ carriage under such unphysiological conditions, Hb would still carry the vast majority of O ₂ . Hence, a decrease in the Hb content of the blood—known as anemia —can markedly reduce O ₂ carriage. The body can compensate for decreased Hb content in the same two ways that, in the above example, we increased the from 235 to 250 mL O ₂ /min. First, it can increase cardiac output. Second, it can increase O ₂ extraction, thereby reducing mixed-venous O ₂ content. Anemia leads to pallor of the mucous membranes and skin, reflecting the decrease in the red Hb pigment. Impaired O ₂ delivery may cause lethargy and fatigue. The accompanying increase in cardiac output may manifest itself as palpitations and a systolic murmur. Shortness of breath may also be a part of the syndrome.

If decreased Hb levels are detrimental, then increasing Hb content should increase the maximal O ₂ content and thus provide a competitive advantage for athletes. Even in normal individuals, [Hb] in RBC cytoplasm is already extremely high (see p. 434 ). Hypoxia (e.g., adaptation to high altitude) leads to the increased production of erythropoietin (see pp. 431–433 ), N18-2 a hormone that somewhat increases the amount of Hb per erythrocyte, but especially increases their number. Indeed, a few instances have been highly publicized in the international press in which elite athletes have infused themselves with erythrocytes or injected themselves with recombinant erythropoietin. However, an excessive increase in hematocrit— polycythemia —has the adverse effect of increasing blood viscosity and thus vascular resistance (see pp. 437–439 ). The consequences include increased blood pressure in both the systemic and pulmonary circulations and a mismatch of ventilation to perfusion within the lung. Such a ventilation-perfusion mismatch leads to hypoxia (see p. 693 ), and thus desaturation of arterial Hb. Thus, the optimal hematocrit—presumably ~45%—is one that achieves a high maximal O ₂ content, but at a reasonable blood viscosity.

The purplish color of desaturated Hb produces the physical sign known as cyanosis, a purplish coloration of the skin and mucous membranes. Cyanosis results not from the absence of saturated or oxygenated Hb, but from the presence of desaturated Hb. Thus, an anemic patient with poorly saturated Hb might have too little unsaturated Hb for it to manifest as cyanosis. The physician's ability to detect cyanosis also depends on other factors, such as the subject's skin pigmentation and the lighting conditions for the physical examination.

Increases in temperature, [CO ₂ ], and [H ⁺ ], all of which are characteristic of metabolically active tissues, cause Hb to dump O ₂

Metabolically active tissues not only have a high demand for O ₂ , they also are warm, produce large amounts of CO ₂ , and are acidic. Indeed, high temperature, high , and low pH of metabolically active tissues all decrease the O ₂ affinity of Hb by acting at nonheme sites to shift the equilibrium between the T and R states of Hb more toward the low-affinity T state. The net effect is that metabolically active tissues can signal Hb in the systemic capillaries to release more O ₂ than usual, whereas less active tissues can signal Hb to release less. In the pulmonary capillaries—where temperature is lower than in active tissues, is relatively low and pH is high—these same properties promote O ₂ uptake by Hb.

Temperature

Increasing the temperature causes the Hb-O ₂ dissociation curve to shift to the right, whereas decreasing the temperature has the opposite effect ( Fig. 29-4 ). Comparing the three Hb-O ₂ dissociation curves in Figure 29-4 at the of mixed-venous blood (40 mm Hg), we see that the amount of O ₂ bound to Hb becomes progressively less at higher temperatures. In other words, high temperature decreases the O ₂ affinity of Hb and leads to release of O ₂ . One mechanism of this temperature effect may be small shifts in the p K values of various amino-acid side chains, which cause shifts in net charge and thus a conformational change.

The maximal temperature achieved in active muscle is ~40°C. Of course, very low temperatures can prevail in the skin of extremities exposed to extreme cold.

Acid

In 1904, Christian Bohr, a physiologist and father of atomic physicist Niels Bohr, observed that respiratory acidosis (see p. 633 ) shifts the Hb-O ₂ dissociation curve to the right ( Fig. 29-5 A ). This decrease in O ₂ affinity has come to be known as the Bohr effect. A mild respiratory acidosis occurs physiologically as erythrocytes enter the systemic capillaries. There, the increase in extracellular causes CO ₂ to enter erythrocytes, which leads to a fall in intracellular pH (see p. 646 ). Other acidic metabolites may also lower extracellular and, therefore, intracellular pH. Thus, this intracellular respiratory acidosis has two components—a decrease in pH and an increase in . We now appreciate that both contribute to the rightward shift of the Hb-O ₂ dissociation curve observed by Bohr.

The effect of acidosis per se on the Hb-O ₂ dissociation curve (see Fig. 29-5 B )—the pH-Bohr effect —accounts for most of the overall Bohr effect. One can readily demonstrate the pH-Bohr effect in a solution of Hb by imposing a metabolic acidosis (e.g., decreasing pH at a fixed ). It should not be surprising that Hb is sensitive to changes in pH because Hb is an outstanding H ⁺ buffer (see p. 630 ):

Although Hb has many titratable groups, the ones important here are those with p K values in the physiological pH range. N29-2 As we acidify the solution, raising the ratio [Hb-H ⁺ ]/[Hb] for susceptible groups, we change the conformation of the Hb molecule, thus lowering its O ₂ affinity:

N29-2

Interactions of H ⁺ with Hemoglobin

Like most proteins, Hb has many titratable groups. However, the ones that are most important physiologically are the ones whose p K values are near the physiological pH. For a general discussion of buffers, consult the passage beginning on pages 628–633 . Of particular interest is the passage dealing with buffers in a closed system (see pp. 630–633 ) and Figure 28-2 B .

Several titratable groups in the Hb molecule contribute to the pH-Bohr effect. The most important single group is the histidine at residue 146 of the β chains. When the Hb molecule is fully deoxygenated (tensed state), the protonated His-146 forms a salt bridge with the negatively charged aspartate group at position 94 of the same β chain. This salt bridge stabilizes the protonated form of His-146, so that it has a high affinity for H ⁺ (i.e., a relatively high p K value of ~8.0).

When the Hb becomes fully oxygenated (relaxed state), the twisting of the Hb molecule disrupts the salt bridge and thus destabilizes the protonated form of His-146, so that it has a low affinity for H ⁺ and thus a relatively low p K value of ~7.1. This p K is roughly the same as the pH inside the RBC. According to Equation 28-5 , the ratio [ ]/[R-NH ₂ ] must be 1 : 1 (so that in 50 out of every 100 Hb molecules, this group is protonated).

Now let us fully de oxygenate this Hb and return the p K of His-146 to where it was at the beginning of this example, ~8.0. The pH inside the erythrocyte remains at 7.1. Equation 28-5 tells us that the ratio [ ]/[R-NH ₂ ] will be 8 : 1. That is, in 89 of every 100 Hb molecules, this His-146 group is protonated. Thus, for every 100 Hb molecules, 39 H ⁺ ions have been taken up from the solution to titrate 39 R-NH ₂ groups to form 39 additional groups at His-146.

The above is an extreme example, because the Hb does not become fully deoxygenated in the systemic capillaries. It is also a simplified example, inasmuch as multiple residues in Hb contribute to the pH-Bohr effect. Nevertheless, the example illustrates how deoxygenated Hb is better able to buffer excess protons.

In the above paragraphs, we have been emphasizing one side of the physiological coin: deoxygenation makes Hb a weaker acid (and thus causes Hb to take up H ⁺ ). However, as summarized by the reaction in Equation 29-9 ,

the converse is also true: the protonation of Hb lowers Hb's affinity for O ₂ .

This is an extreme example in which we added enough H ⁺ to cause Hb to dump all of its O ₂ . Under more physiological conditions, the binding of ~0.7 mole of H ⁺ causes Hb to release 1 mole of O ₂ . This property is important in the systemic tissues, where [H ⁺ ] is high. The converse is also true: O ₂ binding causes a conformational change in the Hb molecule, which lowers the affinity of Hb for H ⁺ .