Red Blood Cell Metabolism: Objectives
1. What is the role of erythropoietin in red blood cell development and how is it regulated in
response to oxygen?
a. EPO’s role:
i. Stimulates the growth of specific red blood cell precursors
ii. Suppresses apoptosis of erythroid precursors
iii. Stimulates globin synthesis
b. EPO is regulated by:
i. A mechanism which is sensitive to oxygen
1. At low oxygen, EPO is stimulated (thus red blood cell production
increases)
2. How is glutathione synthesized?
a. Glutamate + Cysteine + ATP gamma-Glutamyl Cysteine
i. Enzyme: Glutamyl Cysteine Synthetase
b. Gamma-Glutamyl Cysteine + Glycine + ATP  gamma-glutamylcysteinyl
glycine (AKA Glutathione)
i. Enzyme: Glutathione Synthetase
3. What are the functions of glutathione?
a. Detoxification of hydrogen peroxide (H2O2)
b. Reduction of oxidized protein thiols (sulfate reduced)
c. Reduction of MetHB (Fe+3) to Hb (Fe+2)
4. How is reduced glutathione produced enzymatic-ly from oxidized glutathione?
Regeneration of Reduced Glutathione (GSH)
NADPH + H+
NADP+
Glutathione
reductase
GSSG
2GSH
Glutathione
peroxidase
H2 O
H2O2
a.
5. How is 2,3-DPG synthesized and how does its concentration change in response to
hypoxia in erythrocytes?
a. 2,3-DPG is synthesized by:
i. Glyceraldehyde-3-phosphate  1,3-diphosphoglycerate 2,3-DPG
1. Enzyme to 2,3-DPG: Diphosphoglycerate mutase
2. It can go back into the glycolysis pathway with the enzyme: 2,3diphosphoglycerate phosphatase
ii. Increase in 2,3-DPG will inhibit Diphosphoglycerate mutase
b. 2,3-DPG’s concentration during hypoxia (like high altitude, heart failure, anemia,
obstructive pulmonary disease, cystic fibrosis, congenital heart disease)
i. Increases (so as to increase the oxygen release into tissues)
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Red Blood Cell Metabolism: Objectives
6. What is the role of 2,3-DPG in erythrocyte function?
a. Effector for hemoglobin that promotes the release of O2
b. Prevents the excessive accumulation of ATP
i. By acting as an effector for phosphofructokinase
1. Which prevents the inhibition of glycolysis
c. Regulates its own synthesis
i. Inhibited by excess 2,3-DPG…inhibits enzyme: diphosphoglycerate
mutase
d. Can constitute 70% of the organic phosphate in red blood cells
7. What genetic diseases alter the concentration of 2,3-DPG?
a. Hexokinase deficiency:
i. With decreased flux through glycolysis, the concentration of 2,3-DPG is
REDUCED
b. Pyruvate Kinase deficiency:
i. A reduction in the rate of conversion of phosphoenol pyruvate (PEP) to
pyruvate results in HIGHER concentrations of 2,3-DPG
8. What pathways are utilized in erythrocytes for energy production? How does the energy
production in erythrocytes differ from other cell types?
a. Pathways utilized in RBCs for E production:
i. Glycolysis (90-95% of E)
1. Produces: ATP, NADPH, and 2,3-DPG
ii. Hexose Monophosphate Pathway (5-10% of E)
1. Produces: NADPH
b. E production differs in RBCS by:
i. Oxygen is not involved with ATP production (lacks mitochondria)
ii. Thus, all E production is anaerobic
9. What is the major function of the hexose monophosphate pathway in erythrocytes? What
steps are required for the reduction of NADP to NADPH?
a. Major function of PPP:
i. To produce NADPH
1. Keeps Fe+2 form; prevents oxidative damage to the RBCs
ii. Also, reduces glutathione
b. NADP NADPH…steps required include:
i. Glucose-6-Phosphate  6-P-gluconolactone
1. Enzyme: Glucose-6-P dehydrogenase
2. NADP+  NADPH
ii. 6-P-gluconolacton + H2O  6-P-gluconate
1. Enzyme: Gluconolactase
iii. 6-P-gluconate  3-keto-6-phosphogluconate
1. Enzyme: 6-P-gluconate dehydrogenase
2. NADP+  NADPH
iv. Thus, 2 NADPH are formed from 1 molecule of Glucose-6-P
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Red Blood Cell Metabolism: Objectives
10. What reactions are responsible for converting methemoglobin to hemoglobin? What
conditions can increase the concentration of methemoglobin?
a. Methemoglobin(Fe+3)  Hemoglobin (Fe+2)
i. Methemoglobin reductase I:
1. Transfers electrons from NADH to Cytochrome b5
2. Cytochrome b5 then reduces methemoglobin
3. Accounts for 67% of all methemoglobin reduction
ii. Methemoglobin reductase II:
1. MetHb + NADPH  Hb + NADP+
2. Accounts for 5-10% of all methemoglobin reduction
iii. Nonenzymatic Reduction via Glutathione
1. Glutathione reduces methemoglobin
2. Accounts for ~12% of all methemoglobin reduction
b. Conditions which increase the concentration of methemoglobin:
i. Defects in methemoglobin reductase II
1. Relatively mild
2. MetHb can be induced by exposure to some drugs (oxidative
stress)
ii. Defects in cytochrome b5 reductase of methemoglobin reductase I
1. Can be severe or mild
a. Depends on the amount of reduction activity needed
iii. Deficiency in cytochrome b5 methemoglobin reductase I
1. Usually mild
iv. Hemoglobin Ms
1. Mutations in the alpha or beta subunits
a. Make the iron more prone to oxidation
b. This is NOT a defect in the enzymes which reduce MetHb!
11. Explain how superoxide dismutase, ascorbate, and vitamin E are involved in protection of
cells from the effects of reactive oxygen species.
a. Superoxide Dismutase: eliminates superoxide (a highly reactive oxygen species)
by accelerating a biomolecular reaction thru a 1 e- reduction
i. 2H+ + O2- + O2-  O2 + H2O2
b. Ascorbate & Vitamin E:
i. Is converted to a less reactive peroxyvitamin E, which is then reduced by
ascorbate
12. What are the consequences of a deficiency in erythrocyte glucose-6-phosphate
dehydrogenase? What drugs or chemicals can have pathological consequences for
someone with a deficiency in this enzyme?
a. Deficiency in RBCs enzyme G-6-P dhd:
i. G-6-P dehydrogenase is less active or has a shorter half-life
ii. More sensitive to oxidative stress
1. Especially when certain common drugs are used
iii. Jaundice, Acute Hemolytic Anemia
b. Drugs/Chemicals with pathological consequences for these people:
i. Anti-malarials; sulfonamide antibiotics; phenacetin, naphthalene,
methylene blue, doxorubicin, vitK, fava beans, infections
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Red Blood Cell Metabolism: Objectives
13. What is the qualitative phospholipid distribution between the inner and outer leaflets of
an erythrocyte membrane and how is this distribution maintained.
a. Phospholipid distribution between inner and outer leaflets
i. There is an overall negative charge on the outside of RBCs
b. The distribution is maintained by:
i. Flipases and Floppases
1. They exchange lipids between the 2 leaflets
14. Explain the distinction between intrinsic and extrinsic membrane proteins.
a. Intrinsic: elements of the protein structure which lie with the interior of the
membrane
i. Makes contact with hydrocarbon layer thru nonpolar AA side chains
ii. Can only remove via harsh conditions (harsh detergents)
1. Example: Band 3
b. Extrinsic: proteins which are associated with the membrane, but are not typically
associated with the hydrocarbon layer
i. Can be removed with mild conditions
1. Ex. spectrin
15. What are the roles of spectrin and ankryn in maintaining the shape of an erythrocyte?
Why is maintaining the shape and elasticity of red blood cells important in their function?
a. Spectrin: forms links between the band 3 (thru ankryn) and glycophorin c (thru
actin and band 4.1)
i. Forms an antiparallel, coiled-coil triple helix
b. Ankyrin: links spectrin to band 3
c. Importance: shape greatly affects function
i. Without spectrin and ankyrin, the RBC cannot deform and reform in tight
capillary spaces – causing blood problems (like spherocytosis)
16. Describe some genetic disorders in red blood cytoskeletal and membrane proteins that
alter red cell shape.
a. Hereditary Spherocytosis: RBCs are not as resilient and are deformed as they pass
thru capillaries and unable to reform. These cells are cleared more readily by the
spleen.
i. Beta-spectrin
ii. Alpha spectrin (occasionally)
iii. Ankyrin
1. These defects in interaction cause defects in the vertical
interactions in the cytoskeleton and causes the RBCs to assume a
more spherical shape
b. Hereditary Elliptocytosis: defects in spectrin that affect interaction within the
spectrin tetramer.
i. Defects cause a defect in the horizontal interactions of spectrin
ii. Manifestation: elliptical shape of RBCs
iii. Ovalocytosis (oval shaped RBCs) are the result of mutation in band 3
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Red Blood Cell Metabolism: Objectives
17. What is the function of the anion exchanger (band 3) and why is this function important
in CO2 and proton transport?
a. Forms a channel for the exchange of Cl- and HCO3- through the membrane
i. Transport is electroneutral (exchanging a – for a - )
b. Essential for efficient transport of CO2 from tissues to the lungs
c. Structure:
i. Transmembrane glycoprotein with 12 membrane spanning helices
d. Dimeric protein with a cytoplasmic N-terminal domain that interacts directly with
ankryin and other cytoskeletal proteins
i. Links the cytoskeleton to the membrane
ii. Site of attachment of a number of cytoskeletal proteins
e. Binding sites on cytoplasmic N-terminal domain for many glycolytic enzymes
f. Carbonic anhydrase IV is associated with the C-terminal domain (intracellular
side)
18. Explain the physiological function of red blood cell carbonic anhydrase.
a. Exchanges bicarbonate and chloride in and out of erythrocyte thru band 3
b. Catalyzes the hydration of CO2
c. The most abundant protein in erythrocytes after hemoglobin
19. What are the functions of aquaporin and the glucose transporter (Glut1) in erythrocytes?
Why are such transporters necessary for small polar solutes? Explain the distinction
between a uniporter, a symporter, and an antiporter.
a. Aquaporin: water transport protein in RBCs
b. GLUT1: Passive transport of glucose’s mediator in RBCs
i. Does not require energy
ii. Is not stimulated by insulin
c. Transporters are necessary for small polar solutes because:
i. Ion must shed its hydration, enter the nonpolar HC layer, and rehydrate on
the other side
ii. Entering the HC layer is unfavorable, and requires a large activation E
iii. SO….solute transporters interact noncovalently with the molecule,
overcoming the energy barrier and provides a more favorable pathway for
the small polar solute
d. Uniporter: A single molecule is transported (not coupled with another); E is not
required
e. Symporter: Cotransport of 2 solutes in the same direction
f. Antiporter: Cotransport of 2 solutes in opposite directions (Example: Band 3)
20. Explain how the Na+/K+ ATPase works and how it maintains the ionic conditions inside
an erythrocyte.
a. Na+/K+ ATPase: pumps out 3 Na+ for every 2 K+ that come into the RBC
i. Required to maintain integrity of the RBC
ii. Requires ATP to pump the 3 Na+ in (via “phosphoenzyme”)
b. Maintains ionic conditions by:
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Red Blood Cell Metabolism: Objectives
21. How are old red cells cleared and what marks a red cell as senescent?
a. The old RBCs are cleared from the circulation by the spleen
b. Marker:
i. A partially unfolded Hb binds tightly to N-terminus of band 3, forming
hemichrom-band 3 aggregates
1. This aggregated form of band 3 generates an “epitope” that is
detected on the cell surface
2. Epitope binds to an autologous IgG, resulting in clearance by
macrophages
c. The damage to RBCs are the result of reactive oxygen species (oxidative damage)
22. What are glycophorins and what is their fuction?
a. Glycophorins are:
i. Membrane-spanning glycoproteins
ii. Have hydrophobic and hydrophilic regions
b. Their function is to:
i. Maintain the negative charge on the RBCs surface
ii. Also, is the site of determination for the MNS blood group antigens
iii. Serve as receptors for malarial parasites
23. What molecules contribute to the overall negative charge of the surface of a red cell and
why is it important that the surface be negatively charged?
a. Molecules which contribute to the overall negative charge of RBC surface:
i. Glycophorins (due specifically to sialic acid)
b. Importance of the negative charge:
i. Prevents the RBCs from adhering to each other and the walls of blood
vessels
24. What is the nature of the ABO blood group determinants? To what components of the
erythrocyte are they linked?
a. ABO blood group antigens:
i. Arise from oligosaccharides on the RBC surface, linked to acceptors:
1. Band 3 (80%)
2. Glucose Transporter
3. Other membrane proteins
4. Glycolipids (like serumide)
b. The designated “blood group” type reflects which antigen(s) are on the cell
surface
i. Example: Blood Type A: Antigen A; Serum antibodies: Anti B
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