1. Erythrocyte metabolism
Alice Skoumalová

2. Erythrocytes
deliver oxygen to body tissues and remove carbon dioxide and protons
biconcave 7.7μm
lack cell organelles
120 days
women 4,2-5,4 million/μl, men 4,6-6,2 million/μl

3. The erythrocyte membrane
50% lipid bilayer (phospholipids, cholesterol)
50% proteins
SDS-PAGE: separation of proteins (band 1-7)
isolation and analysis (10 main proteins)
Integral: Anion exchanger protein, Glycophorin A, B, C
Peripheral: Spectrin, Ankyrin, Actin

4. Spectrin: the most prominent component (two isoforms α,β; a tetramer; a meshwork )
fixed to the membrane- ankyrin
binding sites for several other proteins (glycophorin C, actin, band 4.1, adducin)
This organization keeps the erythrocyte shape.

5. Hereditary spherocytosis
autosomal dominant
a deficiency in a spectrin amount and its abnormalities
the presence of spherocytes in the blood
the spleen‘s hemolysis

6. 4 protein chains + 4 haem groups
Haemoglobin
4 protein chains + 4 haem groups

7. Haem

8. Movements of the heme and the F helix during the T – R transition in hemoglobin:

10. Hemoglobin saturation curves:

11. Hem - Fe2+- O2 Hem - Fe3+ - O2•-
Hemoglobin autooxidation
the side effect - every so often a molecule of oxyhaemoglobin undergoes decomposition and release superoxide
Hem - Fe2+- O2 Hem - Fe3+ - O2•-
3% of the haemoglobin undergoes oxidation every day
Methemoglobin (Fe3+) is unable to bind O2
(methaemoglobin reductase - converts methaemoglobin back to ferrous haemoglobin to permit continued O2 transport)
Methaemoglobinemia
1. Congenital type
methaemoglobin reductase deficiency (AR)
variant haemoglobin M (HbM)- mutation; tend to be oxidized to methaemoglobin
2. Acquired type- drugs or chemicals (sulphonamides, aniline)
Visual indicator- a blue tint to the skin (10% of metHb)
Treated- reductants (methylene blue, ascorbic acid)

12. Erythrocyte exceptions
They lack organelles
no ATP production in oxidative phosphorylation
no ability to replace damaged lipids and proteins (low metabolic activities, with no ability to synthesize new proteins or lipids)
Free radicals exposure
haemoglobin autoxidation (O2•- release)
a cell membrane rich in polyunsaturated fatty acids (susceptible to lipid peroxidation)
deformation in tiny capillaries; catalytic ions leakage (cause of lipid peroxidation)

13. Erythrocyte metabolism
Glucose as a source of energy
Glycolysis generates ATP and 2,3-bisphosphoglycerate
The pentose phosphate pathway produces NADPH
Glutathione synthesis- the antioxidant defence system

15. Glucose- source of energy Glycolysis in erythrocytes
Glucose transporter:
integral membrane protein (12 membrane-spanning helices)
a channel for the glucose transport
insulin-independent transporter
Glycolysis in erythrocytes
1. Source of ATP
Lactate- the end product
Cover energy requirement
2. Generate 2,3-bisphosphoglycerate (2,3-BPG)
a major reaction pathway for the consumption of glucose in erythrocytes
the specific binding of 2,3-BPG to deoxyhemoglobin decreases the oxygen affinity of hemoglobin and facilites oxygen release in tissues

16. 2,3-bisphosphoglycerate
Allosteric effector of haemoglobin:
binds to deoxyhaemoglobin (a central cavity capable of binding 2,3-BPG)
decreases haemoglobin‘s O2 affinity
Clinical aspects:
In people with high-altitude adaptation or smokers the concentration of 2,3-BPG in the blood is increased (low oxygen supply)
Fetal haemoglobin has low BPG affinity - the higher O2 affinity - facilitates the transfer of O2 to the fetus via the placenta

17. Glutathione synthesis in erythrocytes

18. Elimination of H2O2 and organic hydroperoxides
Glutathione
Elimination of H2O2 and organic hydroperoxides
1. Cofactor for the glutathione peroxidase (removes H2O2 formed in erythrocytes)
2. Involved in ascorbic acid metabolism
3. Prevents protein –SH groups from oxidizing and cross-linking
Glutathione peroxidase
Gly
Cys
Glu
Gly
Cys
Glu
Gly
Cys SH
Glu
+ R-O-O-H
S S
+ H2O
+ NADPH
Glutathione reductase
Reduced form of glutathione
(monomer)
Oxidized form of glutathione
(dimer, disulphide)

19. The pentose phosphate pathway in erythrocytes
Generates NADPH - reduction of glutathione (eliminates H2O2 formed in erythrocytes)
Clinical apect:
Glucose-6-phosphate dehydrogenase deficiency
Causes hemolytic anemia (decreased production of NADPH - reduced protection against oxidative stress - haemoglobin oxidation and Heinz bodies formation, membrane lipid peroxidation and hemolysis)
Hemolytic crises are evocated by drugs (primaquine, sulphonamide antibiotics) and foods (broad beans)
The most common enzyme deficiency disease in the world (100 million people)

20. Oxyhaemoglobin O2 Haemoglobin Superoxide H2O2 Methaemoglobin ½ O2+H2O
Superoxide dismutase
Haemoglobin
Superoxide
H2O2
Catalase
Methaemoglobin reductase
Methaemoglobin
½ O2+H2O
Pentose phosphate pathway
GSH
NADP+
Glutathione reductase
Glutathione peroxidase
NADPH
GSSG
H2O
GSH-reduced form; GSSG-oxidized form of glutathione

21. Haemoglobinopathy abnormal structure of the haemoglobin (mutation)
large number of haemoglobin mutations, a fraction has deleterious effects
sickling, change in O2 affinity, heme loss or dissociation of tetramer
haemoglobin M and S, and thalassemias
Haemoglobin M
replacement of the histidine (E8 or F7) in α or β-chain by the tyrosine
the iron in the heme group is in the Fe3+ state (methaemoglobin) stabilized by the tyrosine
methaemoglobin can not bind oxygen
Thalassemias
genetic defects- α or β-chains are not produced (α or β-thalassemia)

22. Haemoglobin S (sickle-cell)
Causes a sickle-cell anemia
Erythrocytes adopt an elongated sickle shape due to the aggregation of the haemoglobin S

23. Hg proteins aggregate into a long rodlike helical fiber
Replacing Glu with the less polar amino acid Val - forming „an adhesive region“ of the β chain
Hg proteins aggregate into a long rodlike helical fiber
Cross section

24. Red blood cells adopt a sickle shape in a consequence of the forming haemoglobin S fibers
The high incidence of sickle-cell disease coincides with a high incidence of malaria
Individuals heterozygous in haemoglobin S have a higher resistance to malaria; the malarial parasite spends a portion of its life cycle in red cells, and the increased fragility of the sickled cells tends to interrupt this cycle
Scanning electron micrograph of a sickled erythrocyte. The haemoglobin S fibers can be seen within the distorted cell. The cell has ruptured and haemoglobin fibers are spilling out.

26. Hemoglobin switching:

27. Glycosylated haemoglobin (HbA1)
formed by hemoglobin's exposure to high plasma levels of glucose
non-enzymatic glycolysation (glycation)- sugar bonding to a protein
normal level HbA1- 5%; a buildup of HbA1- increased glucose concentration
the HbA1 level is proportional to average blood glucose concentration over previous weeks; in individuals with poorly controlled diabetes, increases in the quantities of these glycated hemoglobins are noted (patients monitoring)
Sugar CHO + NH2 CH2 Protein
Sugar CH N CH2 Protein
Sugar CH2 NH CH2 Protein
Schiff base
Amadori reaction
Glycosylated protein