Lecture 4 - RBC Physiology and Function

Question 1

Haemoglobin Production

Answer 1

Hb is the protein responsible for oxygen transport
Its formed from haeme and globin
- haeme is formed in the mitochondria of RBC precursors
- globin is formed in the cytoplasm of RBC

Question 2

Haeme Formation

Answer 2

Occurs in mitochondria of developing RBC
Formation of protoporphyrin IX which then combines with iron ion to form haeme
Transported to cytoplasm to combine with globin

Question 3

Globin Synthesis

Answer 3

Occurs in the cytoplasm within polyribosomes
Globin chains must be in correct proportions to haeme
Globin chain synthesis ratios
- alpha:beta (1:1)
- sometimes delta and gamma chains
Alpha globin gene: chromosome 16
Beta globin gene: chromosome 11

Question 4

Haemoglobin Protein Structure

Answer 4

Globin tetramer combines with haeme to make haemoglobin

Composition of globin chains within the tetramer defines haemoglobin type

Question 5

Types of Haemoglobin

Answer 5

A (97%) - HbA (α2β2)
A2 (2-3%) - HbA2 (α2δ2)
F (<1%) - HbF (α2γ2)
At birth HbF makes up 70-80%
Replaced slowly from birth
Adult haemoglobin by ~6 months

Question 6

Measurement of Haemoglobin

Answer 6

Total haemoglobin
- sample: EDTA blood
- method: cyanmethaemoglobin method
Specific haemoglobin variants
- haemoglobin electrophoresis
- high performance liquid chromatography (HPLC)

Question 7

Physiological Modifications of Hb Function

Answer 7

Physiological modifications of Hb function may affect affinity for O2
Examples:
Bohr effect
- increased H+ ions
- leads to decreased Hb affinity for O2
- therefore O2 released to tissue
2,3-biphosphoglycerate (2,3-BPG)
- decreased 2,3-BPG -> increased Hb affinity for O2
- increased 2,3-BPG -> decreased Hb affinity for O2
Temperature
- decreased temperature -> increased Hb affinity for O2
- increased temperature -> decreased Hb affinity for O2

Question 8

Changes on Oxygen Dissociation Curve due to Physiological Modifications for Hb Function

Answer 8

Shift left
- decreased H+ ions
- decreased temperature
- reduced 2,3-BPG
Shift right
- increased H+ ions
- increased temperature
- increased 2,3-BPG

Question 9

Thalassemias

Answer 9

Decreased Hb production leads to decreased amount of Hb
Genetic lesions in globin genes
Types:
- alpha 
- beta

Question 10

Haemoglobinopathies

Answer 10

Altered Hb structure and function
Many types
- e.g. Hb S, Hb C, Hb E

Question 11

Sickle Cell Anaemia

Answer 11

Most common form of haemoglobinopathy
Genetic disorder characterised by the production of HbS
HbS is formed by the replacement of glutamic acid with valine at the sixth position on the β chain
Sickle cells deformed by the precipitation of polymerised HbS
Polymerisation of HbS and sickling is promoted by low oxygen tension
Also low pH, 2,3-BPG, high cellular [Hb], loss of cell water and concurrent HbC

Question 12

Sickle Cell Anaemia Process

Answer 12

Sickle cells are formed from biconcave disks that upon deoxygenation, change shape to become crescent shaped
When sickled cells receive oxygen they return to their normal shape
Repeated cycles of sickling and unsickling lead to permanent damage
This process ends in haemolysis
Haemolysis leads to anaemia

Question 13

Mature RBC and Metabolic Processes

Answer 13

Mature RBC maintain several metabolic processes necessary for function

• Embden-Meyerhof glycolytic pathway
• hexose monophosphate shunt
• methaemoglobin reductase pathway
• Leubering-Rapaport shunt

Question 14

Embden-Meyerhof Glycolytic Pathway

Answer 14

Generates energy for cellular purposes
Glucose is the substrate
Uptake of glucose is independent of insulin
Net generation of 2 ATP per glucose molecule

Question 15

Hexose Monophosphate Shunt

Answer 15

Uses ~10% of glucose
Produces NADPH (from NADP)
In turn, used to generate reduced glutathione (GSSG)
- protects cells from oxidative damage
Key enzyme: glucose-6-phosphate dehydrogenase

Question 16

Oxidative Damage to RBC

Answer 16

When RBCs are exposed to overwhelming oxidative damage
Hb becomes denatured
- leads to formation of Heinz bodies
Clinical disorders
- congenital; glucose-6-phosphate dehydrogenase deficieny
- acquired; oxidation of Hb due to drugs, chemicals etc.

Question 17

Heinz Bodies

Answer 17

Denatured Hb -> Heinz body
May form with unstable Hb or oxidation
Look like small dots on RBC
Removed by extravascular haemolysis 
Can be observed with supravital stain
- e.g. methylene blue, crystal violet (incubate stain with living RBC)

Question 18

Methaemoglobin Reductase

Answer 18

Hb = Fe2+ (ferrous)
Methaemoglobin = Fe3+ (ferric)
NADH used to reduce Fe3+ to Fe2+ in haeme
- metHb to Hb

Question 19

Methaemoglobinaemia

Answer 19

Fe2+ is converted to Fe3+
- doesn’t bind O2
- hypoxia
Normally <2% of total Hb
Clinical disorders
- congenital; methaemoglobin reductase deficiency
- acquired; oxidation of Hb due to drugs, chemicals etc.

Question 20

Leubering-Rapaport Shunt

Answer 20

Formation of 2,3-BPG
Modifies Hb affinity for O2
Can also be used to generate ATP

Question 21

Enzyme Deficiences

Answer 21

Pyruvate kinase deficiency
- deficiency -> reduced cell energy
- cant effectively regulate cell ion content
- decreased RBC lifespan
Glucose-6-phosphate dehydrogenase deficiency
- deficiency -> decreased resistance to oxidative injury
- decreased RBC lifespan

Question 22

Compensated Disorders of RBC

Answer 22

Disorders of RBC that results in a decreased lifespan of RBC may be compensated for by increased erythropoiesis
Increased erythropoiesis is characterised by:
Blood
- reticulocytosis (supravital)
- increased proportion of polychromatophilic RBC (Romanowsky)
Bone marrow
- increased erythroid component
- decreased myeloid:erythroid ratio

Question 23

Reticulocytosis

Answer 23

Increase number of reticulocytes
- indicates increased erythropoiesis
Ongoing reticulocytosis reflects continued release of immature cells into circulation
Production requires sufficient nutrients e.g. Fe