Erythrocyte Metabolism and Function

• Erythrocytes rely on specific metabolic pathways for energy and function
• Red blood cell metabolism, membrane structure, and function are essential for their role in oxygen transport

Erythrocyte Metabolism

• RBCs are produced through normoblastic proliferation in the bone marrow
• Maturing normoblasts have a nucleus, which is extruded before entering circulation
• Additional organelles like ribosomes and mitochondria degrade within 24-48 hours of release
• RBCs lack mitochondria and rely on anaerobic glycolysis (Embden-Meyerhof pathway) for ATP production
• ATP is essential for maintaining hemoglobin function and membrane integrity
• Oxidation gradually impairs these functions, leading to RBC aging
• Red blood cell lifespan is limited due to oxidative damage and lack of organelles for repair
• Exchange of O2 and CO2 activity are passive processes from high to low partial pressure
• Maintaining hemoglobin iron in the active ferrous (Fe2+) state requires energy (ATP)
• ATP is needed to drive the cation pump for intracellular sodium (Na+) and potassium (K+) concentration maintenance
• ATP is needed to maintain membrane integrity

Important Metabolic Pathways in RBCs

• Glycolytic (Embden Meyerhof) Pathway
• Hexose Monophosphate Pathway
• Luebering Rapoport Pathway or Shunt
• Methemoglobin Reductase Pathway

Glycolytic Pathway

• This is also know as the Embden Meyerhof pathway
• Red blood cells lack internal energy stores and rely on plasma glucose for ATP generation
• Anaerobic glycolysis (Embden-Meyerhof pathway) serves as the primary source of ATP
• Anaerobic glycolysis is a process that generates ATP from glucose without oxygen
• The majority, 90-95%, of glucose entering a cell is metabolized via this pathway

Glycolytic Pathway: Phase 1 - Preparation (Investment Stage)

• Glucose phosphorylation leads to Fructose 1,6-bisphosphate
• Enzymes involved are Hexokinase which consumes ATP, Glucose-6-phosphate isomerase, and Phosphofructokinase (PFK) which is the rate-limiting step and consumes ATP
• Outcome: Converts glucose into F1,6-BP for cleavage

Glycolytic Pathway: Phase 2 - Energy Generation (Payoff Stage)

• Glyceraldehyde-3-phosphate (G3P) is converted to 3-phosphoglycerate (3-PG)
• G3P dehydrogenase (oxidation, NADH production) and Phosphoglycerate kinase (generates ATP) are the enzymes involved
• 2 ATP molecules are generated per glucose molecule

Glycolytic Pathway: Phase 3 - ATP Formation and Pyruvate Production

• The process is 3-PG converted to Pyruvate via intermediates 2-PG & PEP
• Phosphoglycerate mutase (3-PG to 2-PG), Enolase (2-PG to PEP), and Pyruvate kinase (PK) are the enzymes involved
• Two ATP are generated
• Pyruvate is formed, which can exit the RBC or convert to lactate
• The net ATP gain is 2 ATP, as a total of 4 ATP molecules are produced, but 2 are used in the first phase

Erythrocyte Metabolism Alternative Pathways

• Three alternative pathways branch from glycolysis
• These are the Hexose Monophosphate Pathway (HMP), Methemoglobin Reductase Pathway, and Rapoport-Luebering Pathway

Hexose Monophosphate Pathway or HMP

• Approximately 5-10% of glucose entering the cell is metabolized this way
• It is also called Pentose Phosphate Shunt
• It detoxifies oxidative compounds
• The key enzyme is Glucose-6-phosphate dehydrogenase (G6PD)
• This plays an oxidative role in the first step of HMP
• Converts glucose-6-phosphate (G6P) to 6-phosphogluconate (6-PG)
• Generates NADPH, which reduces glutathione
• NADPH protects the cell from oxidative injury
• It protects the RBC membrane and hemoglobin from oxidative damage, so the RBC can safely carry O2
• G6PD Deficiency leads to oxidative stress and anemia
• Heinz bodies, which are denatured hemoglobin, are present

Methemoglobin Reductase Pathway

• It is responsible for maintaining iron in its reduced state (Fe2+)
• H2O2 oxidizes heme iron (Fe2+ to Fe3+), forming nonfunctional methemoglobin that cannot carry O2
• The solution is a reduction of Fe3+ back to Fe2+
• The key enzyme is Cytochrome-b5 reductase (Methemoglobin reductase)
• It reduces ferric iron (Fe3+) back to ferrous state
• It uses NADH from glycolysis as an electron donor
• It converts methemoglobin back to functional hemoglobin
• Approximately 2% of hemoglobin produced daily is in the form of methemoglobin
• Deficiency in this pathway/enzyme leads to methemoglobinemia
• Methemoglobin carries ferric iron and cannot bind oxygen, resulting in cyanosis
• Cyanosis is due to increased hemoglobin not carrying oxygen

Rapoport-Luebering Pathway

• This pathway is stimulated during hypoxia
• It produces 2,3-bisphosphoglycerate (2,3-BPG), which is its function
• Bisphosphoglycerate mutase is the key enzyme
• Effect on Hemoglobin: 2,3-BPG binds to hemoglobin, stabilizing the deoxygenated form
• This shifts the O2 dissociation curve to the right, enhancing O2 delivery to tissues
• ATP Trade-Off: It generates less ATP but is important as it enhances oxygen delivery to tissues
• Sacrifices 2 ATP molecules to form 2,3-BPG
• Balances oxygen release versus ATP production

Summary of Metabolic Pathways:

• Embden-Meyerhof pathway maintains cellular energy by generating ATP
• Oxidative pathway or hexose monophosphate shunt prevents denaturation of globin of the hemoglobin molecule by oxidation
• Methemoglobin reductase pathway prevents oxidation of heme iron
• Luebering-Rapoport pathway regulates oxygen affinity of hemoglobin

Metabolic Deficiencies

• The lifespan of the erythrocyte is reduced and hemolysis results if metabolic pathways are blocked or inadequate
• Metabolic defects include failure to provide sufficient reduced glutathione and insufficient energy-providing coenzymes like NADH, NADPH, and ATP
• The two most common erythrocytic enzyme deficiencies, involving the Embden-Meyerhof glycolytic pathway, are deficiencies of Glucose-6-Phosphate Dehydrogenase (G6PD) and Pyruvate Kinase (PK).
• G6PD is responsible for converting glucose-6-phosphate (G6P) to 6-phosphogluconate (6PG) and PK is responsible for converting pyruvate (pyruvic acid) to lactic acid

Maturing Erythrocyte Membrane Characteristics

• As a reticulocyte matures into an erythrocyte, three major changes occur
• There is an increase in shear resistance which is how much the material resists against shearing
• An applied force causes an opposite, parallel sliding motion of the planes of an object
• This sliding motion can cause tissues and blood vessels to move and interrupt blood flow
• Loss of surface area because of membrane lipid loss
• Acquisition of a biconcave shape

Mature Erythrocyte Membrane Characteristics

• The shape of the erythrocyte constantly changes as it moves through the circulation and performs complex maneuvers
• The cellular membrane is composed of a protein lattice-lipid bilayer to which the membrane skeleton is attached by trans bilayer (peripheral) proteins
• The cell membrane is deformable and tolerant against mechanical stress and various pH and salt concentrations both in vivo and in vitro
• Cell shape changes reversibly depending on ATP level in the cell and intracellular calcium ion concentration

RBC Membrane Deformability

• It enables RBCs to stretch up to 2.5x their resting diameter
• Deformability depends on geometry and hemoglobin viscosity
• High MCHC (>36 g/dL) increases viscosity, reducing deformability and lifespan
• Aging RBCs lose surface area, increasing MCHC and causing splenic entrapment and destruction

RBC Membrane Lipids

• Membrane composition: 8% carbohydrates, 40% lipids, and 52% proteins
• Lipid bilayer consists of equal parts cholesterol and phospholipids
• Phospholipid arrangement supports fluidity and self-sealing ability, which is important
• It maintains osmotic pressure, cation concentration, and promotes gas exchange
• The role of Cholesterol in RBC Membrane assists with providing tensile strength and elasticity
• Phospholipid Asymmetry and Function: Outer layer- Phosphatidylcholine (PC) & Sphingomyelin (SP), Inner layer- Phosphatidylserine (PS) & Phosphatidylethanolamine (PE)
• Disruptions in phospholipid balance lead to RBC destruction

RBC Membrane Proteins

• Membrane structure: 52% protein by mass
• Major functions: transport, adhesion, signal transduction
• Key proteins: Band 3, Protein 4.1, Protein 4.2, and Spectrin
• Together these components form a complex meshwork tethered to the RBC membrane (cytoskeleton)
• RBC membrane protein complexes ensure structural integrity and biconcave shape retention
• Transmembrane Proteins serve as transport sites, adhesion sites, and signaling receptors
• They regulate osmotic balance and prevent RBC aggregation

RBC Membrane Proteins: Membrane Deformation and Disorders

• Spectrin is a key cytoskeletal protein
• Defect in spectrin causes hereditary elliptocytosis and hereditary spherocytosis
• These result in abnormal RBC shapes and hemolytic anemia
• Spectrin stabilizes RBC shape and allows membrane flexibility

Osmotic Balance and Permeability

• The RBC membrane is permeable to water, Cl-, and HCO3-
• This is regulated by Aquaporin-1 and ATP-dependent cation pumps
• Pump failure leads to water influx, swelling, and hemolysis
• Overhydrated stomatocytosis: excessive Na+/K+ permeability
• Dehydrated stomatocytosis: excessive K+ loss
• Sickle Cell Disease and Ion Permeability; Hemoglobin S polymerization increases Ca2+ permeability
• It alters Na+ and K+ balance, causing membrane damage, which leads to sickled shape and hemolysis

Importance of RBC Membrane Integrity

• Prevents fragmentation and vesiculation
• Maintains deformability for efficient circulation
• Essential for effective oxygen transport and metabolic function
• Imbalance in lipid/protein content leads to RBC lifespan reduction

The Aging Erythrocyte Membrane

• Age-related changes can be monitored using plasma membrane calcium (PMCA) and glycated hemoglobin (Hgb A1C)
• PMCA strength declines as the RBC ages and Hgb A1C increases as the RBC ages
• This causes densification of the RBC membrane, which contributes to the membrane instability seen in senescent (old) RBCs