The red blood cell (RBC, erythrocyte) transports oxygen from the lungs to tissues and organs and helps to transport carbon dioxide to the lungs to be exhaled. The mammalian RBC has evolved to do this quite efficiently but has sacrificed greatly for this purpose by losing its nucleus.2,3 The resulting RBC shape facilitates oxygen acquisition and release. However, the cell is left without the ability to synthesize replacement proteins for vital pathways of energy production and membrane integrity, among others. As a result, its life span is limited to about 120 days because these processes inevitably fail. Mechanisms have evolved to maximize oxygen exchange during the life span of the cell and to counter the natural chemical processes that oxidize heme iron, which would leave it nonfunctional for oxygen transport. This chapter examines the energy production pathways of the RBC, discusses mechanisms for maintaining reduced heme iron and facilitating oxygen delivery, and explores the features of the RBC membrane that contribute to effective oxygen delivery for RBCs.

Energy Production—Anaerobic Glycolysis

At the time the RBC ejects its nucleus, the cellular organelles are also packaged and ejected from the cell. Without mitochondria, the RBC is left to rely on glycolysis for energy production. Fortunately, oxygen exchange and oxygen binding to heme iron do not require energy.⁴ Other RBC metabolic processes do require energy, however, and they are listed in Box 9-1. Loss of energy leads to RBC death. In fact, a hereditary deficiency of nearly every enzyme involved in glycolysis has been identified. A common result of these deficiencies is shortened RBC survival, known as hereditary nonspherocytic hemolytic anemia.

BOX 9-1 Erythrocyte Metabolic Processes Requiring Energy

Maintenance of intracellular cationic electrochemical gradients

Maintenance of membrane phospholipid

Maintenance of skeletal protein plasticity

Maintenance of functional ferrous hemoglobin

Protection of cell proteins from oxidative denaturation

Initiation and maintenance of glycolysis

Synthesis of glutathione

Mediation of nucleotide salvage reactions

Transmembranous cation gradient concentration proteins, called cation pumps, provide a particular example of how energy failure, even in otherwise normal cells, leads to cell death. The pumps maintain high concentrations of intracellular potassium (K⁺), low concentrations of sodium (Na⁺), and very low concentrations of calcium (Ca²⁺) in contrast to extracellular concentrations of low K⁺ and high levels of Na⁺ and Ca²⁺. The pumps consume approximately 15% of RBC adenosine triphosphate (ATP) production as they prevent adverse accumulations of intracellular sodium and calcium. As energy-producing enzymes degrade over time, the pumps fail, Na⁺, Ca²⁺, and water flow in, and the RBC swells and is destroyed. This process is accelerated in hereditary nonspherocytic anemias in which the cells begin their life in the circulation with diminished enzyme concentrations.

Plasma glucose enters the RBC through a facilitated membrane transport system.⁵ Anaerobic glycolysis, the Embden-Meyerhof pathway (EMP; Figure 9-1), requires glucose to generate ATP, a high-energy phosphate source that is the greatest reservoir of energy in the RBC. Energy reserves like glycogen are not available in RBCs, so the RBCs rely mostly on external glucose for glycolysis-generated ATP. Via the EMP, glucose is catabolized to pyruvate, consuming two molecules of ATP per molecule of glucose and maximally generating four molecules of ATP per molecule of glucose, for a net gain of two molecules of ATP.

Figure 9-1 Glucose metabolism in the erythrocyte. *ADP,* adenosine diphosphate; *ATP,* adenosine triphosphate; *G6PD,* glucose-6-phosphate dehydrogenase; *NAD,* nicotinamide adenine dinucleotide; *NADH,* nicotinamide adenine dinucleotide (reduced form); *NADP,* nicotinamide adenine dinucleotide phosphate (oxidized form); *NADPH,* nicotinamide adenine dinucleotide phosphate (reduced form).

The sequential list of biochemical intermediates involved in glucose catabolism, with corresponding enzymes, is given in Figure 9-1. Tables 9-1 through 9-3 organize this information into three phases for better comprehension.

TABLE 9-1

Glucose Catabolism: First Phase

Substrates	Enzyme	Products
Glucose, ATP	Hexokinase	G6P, ADP
G6P	Glucose phosphate isomerase	F6P
F6P, ATP	Phosphofructokinase	F-1,6-P; ADP
F-1,6-P	Fructodiphosphate adolase	DHAP, G3P

ADP, Adenosine diphosphate; ATP, adenosine triphosphate; DHAP, dihydroxyacetone phosphate; F-1,6-P, fructose 1,6-bisphosphate; F6P, fructose 6-phosphate; G3P, glucose 3-phosphate; G6P, glucose 6-phosphate.

TABLE 9-2

Glucose Catabolism: Second Phase

Substrates	Enzyme	Product
G3P	Glyceraldehyde-3-phosphate dehydrogenase	1,3-BPG
1,3-BPG, ADP	Phosphoglycerate kinase	3-PG, ATP
1,3-BPG	Bisphosphoglyceromutase	2,3-BPG
2,3-BPG	Bisphosphoglycerate phosphatase	3-PG

1,3-BPG, 1,3 Bisphosphoglycerate; 2,3-BPG, 2,3 bisphosphoglycerate; 3-PG, 3-phosphoglycerate; ADP, adenosine diphosphate; ATP, adenosine triphosphate; G3P, glucose 3-phosphate.

TABLE 9-3

Glucose Catabolism: Third Phase

Substrates	Enzyme	Product
3-PG	Monophosphoglyceromutase	2-PG
2-PG	Phosphopyruvate hydratase (enolase)	PEP
PEP, ADP	Pyruvate kinase	Pyruvate, ATP

2-PG, 2-Phosphoglycerate; 3-PG, 3-phosphoglycerate; ADP, adenosine diphosphate; ATP, adenosine triphosphate; PEP, phosphoenolpyruvate.

The first phase of glucose catabolism involves glucose phosphorylation, isomerization, and diphosphorylation to yield fructose 1,6-bisphosphate (F-1,6-P). F-1,6-P serves as the substrate for aldolase cleavage for the final product of phase 1 glycolysis: glyceraldehyde 3-phosphate (G3P; see Figure 9-1 and Table 9-1). Hexokinase and phosphofructokinase are rate-limiting in steady-state anaerobic glycolysis, even though hexokinase has the lowest activity of all the glycolytic enzymes.

The second phase of glucose catabolism converts G3P to 3-phosphoglycerate (3-PG). The substrates, enzymes, and products for this phase of glycolytic metabolism are summarized in Table 9-2. During the first reaction step, G3P is phosphorylated with a high-energy phosphate and oxidized to 1,3-bisphosphoglycerate (1,3-BPG) through the action of glyceraldehyde-3-phosphate dehydrogenase (G3PD). 1,3-BPG is dephosphorylated by phosphoglycerate kinase, which generates ATP and 3-PG.

The third phase of anaerobic glucose catabolism converts 3-PG to pyruvate with the generation of ATP. Substrates, enzymes, and products are listed in Table 9-3. The product 3-PG is isomerized by phosphoglyceromutase to 2-phosphoglycerate (2-PG). Enolase then converts 2-PG to phosphoenolpyruvate (PEP). Pyruvate kinase (PK) splits off the phosphates, forming ATP and pyruvate. PK activity is allosterically modulated by increased concentrations of F-1,6-P, which enhances the affinity of PK for PEP.⁵ Thus when the F-1,6-P is plentiful, increased activity of PK favors pyruvate production. Pyruvic acid may diffuse from the erythrocyte or may become a substrate for lactate dehydrogenase with regeneration of the oxidixed form of nicotinamide adenine dinucleotide (NAD⁺). The ratio of NAD⁺ to the reduced form (NADH) may modify the activity of this enzyme.

Glycolysis Diversion Pathways (Shunts)

Without organelles, RBCs take advantage of diversions or shunts from the glycolytic pathway to generate products that maximize oxygen delivery from several vantage points. The three diversions are the hexose monophosphate pathway (HMP) or aerobic glycolysis, the methemoglobin reductase pathway, and the Rapoport-Luebering pathway.

Hexose Monophosphate Pathway

Aerobic or oxidative glycolysis occurs through a diversion of glucose catabolism into the HMP, also known as the pentose phosphate shunt (see Figure 9-1). The HMP detoxifies accumulated peroxide, an agent that oxidizes heme iron, proteins, and lipids, especially those containing thiol groups. Peroxide arises spontaneously from the reduction of oxygen in the cell’s aqueous environment.⁵ By detoxifying peroxide, the HMP extends the functional life span of the RBC.

The HMP diverts glucose 6-phosphate (G6P) to pentose phosphate by the action of glucose-6-phosphate dehydrogenase (G6PD). In the process, oxidized nicotinamide adenine dinucleotide phosphate (NADP⁺) is converted to the reduced form (NADPH). NADPH is then available to reduce the oxidized form of glutathione (GSSG) to its reduced form (GSH) using glutathione reductase. As the “thio” indicates, glutathione is a sulfur-containing compound and is, in fact, a tripeptide containing cysteine. The designation GSH highlights the sulfur moiety. Once glutathione is reduced, it is available to reduce peroxide to water and oxygen via glutathione peroxidase.

During steady-state glycolysis, 5% to 10% of G6P is diverted to the HMP. After oxidative challenge, the activity of the HMP may increase twentyfold to thirtyfold.⁶ The HMP catabolizes G6P to ribulose 5-phosphate and carbon dioxide by oxidizing G6P at carbon 1. The substrates, enzymes, and products of the HMP are listed in Table 9-4.

TABLE 9-4

Glucose Catabolism: Hexose Monophosphate Pathway

Substrates	Enzyme	Product
G6P	Glucose-6-phosphate dehydrogenase	6-PG
6-PG	Phosphogluconolactonase Phosphogluconate dehydrogenase	R5P

6-PG, 6-Phosphogluconate; G6P, glucose 6-phosphate; R5P, ribulose 5-phosphate.

G6PD provides the only means of generating NADPH for glutathione reduction, and in its absence erythrocytes are particularly vulnerable to oxidative damage.⁷ Normal G6PD activity is able to detoxify oxidative compounds and safeguard hemoglobin, sulfhydryl-containing enzymes, and membrane thiols, allowing normally functioning RBCs to carry enormous quantities of oxygen safely. However, G6PD deficiency is the most common human RBC enzyme deficiency worldwide resulting in hereditary nonspherocytic anemia (see Chapter 23).

Methemoglobin Reductase Pathway

Heme iron is constantly exposed to oxygen, an oxidizing agent.⁸ In addition, the accumulation of peroxide oxidizes heme iron from the ferrous to the ferric state, forming methemoglobin. Although the HMP is able to prevent some hemoglobin oxidation by reducing peroxide, it is not able to reduce methemoglobin once it forms. NADPH is able to do so, but only slowly. The reduction of methemoglobin by NADPH is far more efficient in the presence of methemoglobin reductase, more properly called cytochrome b₅ reductase (cytob5r). Using H⁺ from NADH formed when G3P is converted to 1,3-BPG, soluble cytochrome b₅ reductase acts as an intermediate electron carrier, swiftly returning the oxidized iron to its ferrous, oxygen-carrying state. Cytochrome b₅ reductase accounts for more than 65% of the methemoglobin-reducing capacity within the RBC.⁸

Rapoport-Luebering Pathway

A third metabolic shunt generates 2,3-bisphosphoglycerate (2,3-BPG; also called 2,3-diphosphoglycerate or 2,3-DPG). 1,3-BPG is diverted by bisphosphoglycerate mutase to form 2,3-BPG. 2,3-BPG regulates oxygen delivery to tissues by essentially competing with oxygen for hemoglobin. When 2,3-BPG binds, oxygen is released, which enhances delivery of oxygen to the tissues.

2,3-BPG forms 3-PG by the action of bisphosphoglycerate phosphatase. This diversion of 1,3-BPG to form 2,3-BPG immediately sacrifices the production of two ATP molecules via glycolysis. However, there is the loss of another two molecules of ATP at the level of PK, because fewer molecules of PEP are formed. Because two ATP molecules were used to generate 1,3-BPG and production of 2,3-BPG eliminates the production of four molecules, the cell is put in ATP deficit by this diversion. The cell maintains a delicate balance between ATP generation to support the energy requirements of cell metabolism and the need to maintain the appropriate oxygenation/deoxygenation status of hemoglobin. Acidic pH and low concentrations of 3-PG and 2-PG inhibit the activity of bisphosphoglyceromutase, thus inhibiting the shunt and retaining 1,3-BPG in the EMP. These conditions and decreased ATP activate bisphosphoglycerate phosphatase, which returns 2,3-BPG to the mainstream of glycolysis. In summary, these conditions favor generation of ATP by causing the conversion of more 1,3-BPG directly to 3-PG and also returning some 2,3 BPG to 3-PG for ATP generation downstream by PK.

Rbc Membrane

RBC Deformability

RBCs are biconcave and average 90 fL in volume.⁹ Their average surface area is 140 µm², a 40% excess of surface area compared with a 90-fL sphere. This excess surface-to-volume ratio enables RBCs to stretch undamaged up to 2.5 times their resting diameter as they pass through narrow capillaries and through splenic pores 2 µm in diameter, a property called RBC deformability.¹⁰ The RBC plasma membrane, which is 5 µm thick, is 100 times more elastic than a comparable latex membrane, yet has tensile (lateral) strength greater than that of steel. The deformable RBC membrane provides the broad surface area and close tissue contact necessary to support the delivery of oxygen from lungs to body tissue and carbon dioxide from body tissue to lungs.

RBC deformability depends not only on RBC geometry but also on relative cytoplasmic (hemoglobin) viscosity. The normal mean cell hemoglobin concentration (MCHC) ranges from 32% to 36% (see Chapter 14 and inside front cover), and as hemoglobin concentration rises, viscosity rises.¹¹ Hemoglobin concentrations above 36% compromise deformability and shorten the RBC life span, because the more viscous cells cannot accommodate to narrow capillaries or splenic pores. As RBCs age, they lose membrane surface area while retaining hemoglobin. The hemoglobin becomes more and more concentrated, and eventually the RBC, unable to pass through the splenic pores, is destroyed by splenic macrophages. Refer to Chapter 8 for a more complete discussion of RBC senescence theories.

RBC Membrane Lipids

Membrane elasticity (pliancy) is the third property that contributes to deformability. The RBC membrane consists of approximately 8% carbohydrates, 52% proteins, and 40% lipids.¹² The lipid portion, equal parts of cholesterol and phospholipids, forms a bilayer universal to all animal cells (see Figure 13-9). Four main phospholipids form an impenetrable fluid barrier as their hydrophilic polar head groups are arrayed upon the membrane’s surfaces, oriented toward both the aqueous plasma and the cytoplasm, respectively.¹³ Their hydrophobic nonpolar acyl tails

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