Metabolism of Red Blood Cell
&
Membrane Stability
Prof Dr Kwan Teck Kim
Department of Biochemistry
Faculty of Medicine
Mahsa University College
Learning outcomes
On completion of this topic, the student will be
able to:
 Discuss the role of glycolysis in erythrocytes with
respect to ATP production, supply of intermediates
for Rapoport-Luebering glycolytic shunt and
Hexose monophosphate pathway.
 Explain the role of NADPH in glutathione
metabolism
 Explain the role of ATP and glutathione in the
maintenance of erythrocyte membrane stability
 Explain the molecular basis of hemolytic anemias
Red Blood Cell
• The red blood cell is simple in terms of its structure
and function, consisting principally of a concentrated
solution of hemoglobin surrounded by a membrane.
The erythrocyte membrane is flexible because of its
cytoskeletal structure.
• The red cell contains a battery of cytosolic enzymes,
such as suproxide dismutase, catalase, and glutathione
peroxidase, to dispose of powerful oxidants (ROS,
reactive oxygen species) generated during its
metabolism.
• Genetically determined deficiency of the activity of
glucose-6-phosphate dehydrogenase, which produces
NADPH, is an important cause of hemolytic anemia.
Glycolysis Pathway
Rapoport-Luebering
glycolytic shunt
 To generate 2,3Bisphosphoglycerate
(2,3-BPG)
• 2,3-BPG is important
regulator of binding of O2
to Hb.
 HbO2 + 2,3-BPG ↔
Hb-2,3-BPG + O2
(deoxyhemoglobin)
2,3-Bisphosphoglycerate
shunt
ADP
ATP
2-
2,3-Bisphosphoglycerate mutase/phosphatase
• This enzyme is bifunctional, serving as mutase for
the formation of 2,3-bisphosphoglycerate and also
as a phosphatase.
• Phosphatase hydrolyzes 2,3-bisphosphoglycerate to 3-
phosphoglycerate and Pi .
• Erythrocytes contain very high concentrations of
2,3-bisphosphoglycerate, which functions as important
negative allosteric regulator of the binding of oxygen
to hemoglobin.
2,3-Bisphosphoglycerate mutase/phosphatase (contd)
• 15-25% of glucose converted to lactate in red blood
cells goes by way of the “BPG shunt”for the synthesis of
2,3-bisphosphoglycerate.
• No net production of ATP occurs when glucose is
converted to lactate by the BPG shunt as it bypasses
the phosphoglycerate kinase step.
• However, it does serve to provide 2,3-BPG, which binds
to Hb, decreasing its affinity for O2, and so making O2
more readily available to tissues.
2,3-Bisphosphoglycerate (BPG) in Red Blood Cells
Modulates Oxygen release from Hemoglobin
• BPG dissociates as deoxy-Hb is converted to oxy-Hb.
H+BPG + Hb + 4O2 ↔ Hb(O2)4 + BPG + nH+
• Increased concns of BPG force the equilibrium to the
left, and they correspondingly shift the saturation plot
to the right with an increase in P50 (see Fig. 9.34).
• In contrast, lowered concns of BPG force this
equilibrium to the right, & correspondingly shift the
saturation plot to the left with a lower P50 .
2,3-Bisphosphoglycerate (BPG) in Red Blood Cells
Modulates Oxygen release from Hemoglobin (contd 1)
• BPG is formed in a minor pathway for glucose
metabolism & is present in small amounts in all cells.
• In rbc, this pathway is highly active, & BPG concns are
approx. equimolar to that of Hb.
• BPG binds to the deoxy (T) but not to oxy (R)
conformation.
• Binding of BPG to Hb stabilizes the T conformation &
increases its concn relative to the R conformation.
2,3-Bisphosphoglycerate (BPG) in Red Blood Cells
Modulates Oxygen release from Hemoglobin (contd 2)
• Conditions that cause hypoxia (deficiency of oxygen)
such as anemia, smoking, and high altitude increase
BPG levels in rbc.
• In turn, conditions leading to hyperoxia result in lower
levels of BPG.
• Changes in rbc levels of BPG are slow & occur over
hours and days to compensate for chronic changes in
pO2 levels.
Glucose is translocated by Passive Transport
Glucose transport & metabolism
• Depending on the concn gradient, erythrocyte GLUT 1
(glucose transporter) facilitates transport of glucose in
both directions
• Glucose is metabolized mainly by glycolysis in
erythrocytes.
• Since erythrocytes lack mitochondria, the end product
of glycolysis is lactic acid, which is released into the
blood plasma.
Glucose metabolism via PPP
• Glucose used by the Pentose Phosphate pathway in
r.b.c. provides NADPH to keep glutathione in the
reduced state, which has an important role in the
destruction of organic peroxides and H2O2.
• Peroxides cause irreversible damage to membranes,
DNA and other cellular components and must be
removed to prevent cell damage and death.
Erythrocyte and Pentose Phosphate Pathway
• Glucose 6-phosphate dehyrogenase deficiency
causes drug induced hemolytic anemia
• Patients administered the drug pamaquine
experienced drastic loss of Hb
• Role of NADPH in RBC’s is to reduce gluthathione
to sulfhydryl form
• Essential for maintaining structure of Hb (keeps Hb
in ferrous state)
[ G-S-S-G → 2G-SH ]
Energy generation
• Generates ATP for energy to maintain
RBC shape and flexibility
• Via anaerobic glycolytic pathway
• 2 ATP for each molecule glucose
Overview of erythrocyte
metabolism
g
Overview of erythrocyte metabolism
• Mature erythrocytes contain no intracellular organelles,
•
•
•
•
so metabolic enzymes of rbc are limited to those found
in cytoplasm.
Glycolysis is the major pathway, with branches for the
hexose monophosphate shunt (for protection against
oxidizing agents) and
Rapoport-Luebering shunt (which generates 2,3-BPG,
which moderates oxygen binding to Hb).
The NADH generated from glycolysis can be used to
reduce methemoglobin (Fe3+) to normal Hb (Fe2+) or
To convert pyruvate to lactate, so that NAD+ can be
regenerated and used for glycolysis.
RBC Membrane Integrity
• Glutathione is present in RBCs. This is used for
•
•
•
•
inactivation of free radicals formed inside RBC.
The enzyme used is glutathione peroxidase.
The glutathione is regenerated by an NADPH
dependent glutathione reductase.
The NADPH is derived from the glucose 6phosphate dehydrogenase shunt pathway.
The occurrence of hemolysis in G6PD
deficiency is attributed to the decreased
regeneration of reduced glutathione.
Pentose phosphate
pathway (Hexose
monophosphate
shunt)
6-Phosphoglucolactonase
C5 + C5 ↔ C3 + C7
Transketolase
C7 + C3 ↔ C4 + C6
Transaldolase
6-Phosphogluconate
dehydrogenase
Oxidative phase
←
C5 + C4 ↔ C3 + C6
Transketolase
Non-Oxidative
Phase
←
Transketolase & transaldolase reactions
 C5 + C5 <---------------> C3 + C7 (transketolase)
 C7 + C3 <---------------> C4 + C6 (transaldolase)
 C5 + C4 <----------------> C3 + C6 (transketolase)
•
Transketolase- transfers 2 carbon units.
•
Transaldolase – transfers 3 carbon units.
Pentose Phosphate Pathway
• The oxidative branch consists of 3 irreversible reacns.
• Glucose-6-phosphate dehydrogenase (G6PD), the rate-
limiting enzyme, converts glucose 6-phosphate to 6phosphogluconolactone, which is further converted in
a series of reacns to ribulose 5-phosphate.
• The 2 NADPH formed are used for reductive biosynthesis
and for maintaining the anti-oxidant glutathione in the
reduced form.
Pentose Phosphate Pathway (contd)
• All of the reacns of the non-oxidative branch are
reversible and produce ribose 5-phosphate for RNA
and DNA synthesis.
• Also produced are the intermediates fructose 6-
phosphate and glyceraldehde 3-phosphate for
glycolysis (fed state) or gluconeogenesis (fasting state).
Agents that affect oxygen binding
Effect of H+ on
oxygen binding by
Hb
Effect of H+ on oxygen binding by Hb
• A. In the tissues, CO2 is released. In rbc, this
CO2 forms carbonic acid, which releases
protons. The protons bind to Hb, causing it to
release oxygen to the tissues.
• B. In the lungs, the reacns are reversed. O2
binds to protonated Hb, causing the release of
protons. They bind to HCO3- , forming
carbonic acid, which is cleaved to water and
CO2 , which is exhaled.
References
• Lieberman, M. & Marks, A. (2009). Marks’ Basic
Medical Biochemistry: A clinical approach (3rd
edition), Lippincott, Williams & Wilkins,
Philadelphia.
 Harvey, R.A. & Ferrier, D.R. (2011). Lippincott’s
Illustrated Reviews: Biochemistry (5th edition),
Lippincott, Williams and Wilkins, Philadelphia.
 Vasudeven, D.M., Sreekumari, S. & Vaidyanathan, K.
(2011). Textbook of Biochemistry for Medical
Students (6th edition), Jaypee Brothrs Medical
Publishers Ltd, New Delhi.