Glycolytic and HMP pathway enzyme deficiencies

1. Metabolism of RBC &
RBC Enzymopathies
Presented by:
Pradeep Singh
M.Sc. Medical Biochemistry 2nd Year
HIMSR, Jamia Hamdard
08/04/2019
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2. Introduction
• RBCs are anucleated and devoid of ribosomes and mitochondria.
• Solely depends on continuous supply of glucose for their metabolic needs.
• RBCs must sustain active metabolism to transport and deliver oxygen to
tissue and maintain flexibility and integrity of red cell membrane.
• This is achieved by metabolic pathways: anaerobic glycolysis, hexose
monophosphate shunt, glutathione metabolism, and nucleotide salvage
pathway.
• Glucose is transported through RBC membrane by facilitated diffusion
through glucose transporters (GLUT-1).
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3. Metabolism of RBC
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4. Metabolic
Pathways of
RBC
Glucose is metabolized by
two major pathways:
a) Glycolytic or “energy-
producing” pathway –
Provides ATP
b) Hexose
Monophosphate
Shunt or “protective
pathway” – Provides
NADPH
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5. Glycolysis
Importance of glycolysis in red blood cells:
• Energy production: It is the only
pathway that supplies ATP to the RBC
• Reduction of methemoglobin: Glycolysis
provides NADH for reduction of metHb
by NADH-cyto b5 reductase.
• In RBC 2,3-bisphosphoglycerate binds to
Hb, decresing its affinity for O2 and helps
in its availability to tissues.
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6. Utilization of ATP in RBC:
• Phosphorylation of sugars and proteins
• ATPase driven ion pumps
• Maintenance of membrane asymmetry
• Maintenance of red cell shape and deformability using ATP
dependent cytoskeleton.
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7. Pentose Phosphate Pathway
• Production of NADPH – ‘reducing
power’
• NADPH is required by glutathione
reductase which reduces GSSG to
GSH
• Glutathione is needed in reduced
form for:
- Elimination of peroxide
- Protection of proteins –SH groups
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8. 8

9. RBC Enzymopathies
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10. RBC Enzyme Disorders
• A red blood cell enzyme disorder should be assumed in cases of
persistent normocytic haemolytic anemia in which haemoglobin
abnormalities and antiglobulin reactions have been excluded,
Spherocytes are absent and osmotic fragility is normal.
• Red Blood Cell enzymopathies cause hereditary nonspherocytic
haemolytic anemia (HNSHA)
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11. Red Blood Cell Enzymes
• Glucose-6-phosphate dehydrogenase*
• Glutathione peroxidase
• Glutathione reductase
• Glutathione synthetase
• Glutathione-S-transferase
• Glyceraldehyde-3-phosphate dehydrogenase
• Glyoxalase I
• Hexokinase*
• Hypoxanthine-guanine phosphoribosyl
transferase
• ITPase
• Lactate dehydrogenase
• NADPH diaphorase
• Phosphofructokinase
• Phosphoglucomutase
• Phosphoglycerate Kinase
• Pyrimidine-5’-nucleotidase*
• Pyruvate kinase*
• Triosephosphate isomerase
• 6-Phosphogluconate dehydrogenase
• 6-Phosphogluconolactonase
• Acetylcholinesterase
• Adenine phosphoribosyl transferase
• Adenosine deaminase
• Adenylate kinase
• Aldolase
• AMP deaminase
• Bisphosphoglycerate mutase
• Carbonic anhydrase I
• Carbonic anhydrase II
• Catalase
• Cytochrome b5 reductase
• δ-ALA dehydratase
• Enolase
• Galactokinase
• Galactose-1-P-uridyltransferase
• γ-Glutamylcysteine sunthetase
• Glucose phosphate isomerase
*Enzymatic activity is strongly age-dependent 11

12. Enzymes With Clear Cause Effect Relationship
• Glucose-6-phosphate dehydrogenase*
• Glutathione peroxidase
• Glutathione reductase
• Glutathione synthetase
• Glutathione-S-transferase
• Glyceraldehyde-3-phosphate dehydrogenase
• Glyoxalase I
• Hexokinase*
• Hypoxanthine-guanine phosphoribosyl
transferase
• ITPase
• Lactate dehydrogenase
• NADPH diaphorase
• Phosphofructokinase
• Phosphoglucomutase
• Phosphoglycerate Kinase
• Pyrimidine-5’-nucleotidase*
• Pyruvate kinase*
• Triosephosphate isomerase
• 6-Phosphogluconate dehydrogenase
• 6-Phosphogluconolactonase
• Acetylcholinesterase
• Adenine phosphoribosyl transferase
• Adenosine deaminase (hyperactivity)
• Adenylate kinase
• Aldolase
• AMP deaminase
• Bisphosphoglycerate mutase
• Carbonic anhydrase I
• Carbonic anhydrase II
• Catalase
• Cytochrome b5 reductase
• δ-ALA dehydratase
• Enolase
• Galactokinase
• Galactose-1-P-uridyltransferase
• γ-Glutamylcysteine sunthetase
• Glucose phosphate isomerase
*Enzymatic activity is strongly age-dependent 12

13. RBC Enzymopathies
In general are categorized in two groups:
1. Enzyme deficiencies of glycolysis and nucleotide metabolism
Continously impaired ATP generation Lack of sufficient energy
ATP is required for maintenance of:
- Glycolysis
- Electrolyte red/cell plasma gradient
- Glutathione synthesis
- Membrane phospholipid asymmetry
- Purine/pyrimidine metabolism
- Chronic haemolytic anaemia
Most common cause: Pyruvate Kinase (PK) deficiency
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14. RBC Enzymopathies
2. Enzyme deficiencies of the
hexose monophosphate shunt and
glutathione metabolism
Inadequate levels of reduced
glutathione (GSH):
Inability to withstand oxidative stress
GSH is required for:
- Protection of metabolic enzymes
and membrane proteins from
oxidative stress.
- Acute haemolytic crisis
induced by oxidant drugs, foods (favism), infection, physiological stress 14

15. RBC Enzymopathies
 Metabolic dysfunction depends on:
- Importance of the affected enzyme (rate-limiting enzyme)
- Functional abnormalities (kinetic properties) of the mutant enzymes
- Stability of the mutant enzyme (RBC lacks biosynthesis)
- Possibility of compensation for the deficiency by isoenzyme overexpression or
use of alternative pathways
 Most enzymopathies are associated with extravascular hemolysis
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16. RBC Enzymopathies
 Clinically highly heterogeneous
- Fully compensated hemolysis (without anemia) to severe haemolytic anemia
requiring regular transfusions
 Presentation at later age vs neonatal death (hydrops fetalis)
 No correlation between residual enzyme activity and clinical severity
 Exacerbation of hemolysis during infection
 Non-haematological symptoms are also seen in some cases:
E.g: myopathy in phosphofructokinase (PFK) deficiency,
Severe neuropathy in triosephosphate isomerase (TPI) deficiency
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17. Some common RBC enzymopathy
disorders
1. Pyruvate kinase deficiency
2. Glucose-6-phosphate dehydrogenase deficiency
3. Pyrimidine 5’ nucleotidase deficiency
4. Cytochrome b5 reductase deficiency
5. Familial 2,3-bisphosphoglycerate deficiency
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18. 1. Pyruvate Kinase Deficiency
Key enzyme of glycolysis: Sole
source of energy for the red
blood cell
Catalyses the irreversible
phosphoryl group transfer from
phosphoenolpyruvate to ADP,
yielding pyruvate + ATP
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19.  Mammals: 2 genes and 4 isoenzymes of PK
PKM PK-M1 (muscle, heart, brain)
PK-M2 (leukocytes, platelets, erythroblasts)
PKLR PK-L (Liver)
PK-R (Red Blood Cell)
 Clinical symptoms confined to the red blood
cells
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20.  Most common cause of hereditary chronic non-spherocytic
haemolytic anemia
 Rare, but most frequently occurring abnormality of glycolysis
Autosomal recessive disorder
Estimated Prevalence: 1:20,000 (White Population)
Worldwide distribution
Highly variable phenotypic expression
Severe diagnosed at birth (hydrops fetalis)
Mild fully compensated haemolytic diagnosed later in life 20

21. Effects of Pyruvate Kinase Deficiency
Biochemical features of the PK-R deficient red blood
cell:
- PK enzymatic activity usually decreased
- ATP levels usually decreased
- 2,3-DPG levels increased due to metabolic block at PK step
Red cell destruction
- Precise mechanism of red blood cell destruction is unknown
- ATP depletion initiates a series of events leading to premature
destruction of (young) PK-deficient red cells in the spleen
Premature destruction as well as apoptosis of RBC
- Progenitors may contribute to pathogenesis of PK-R deficiency
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22. 2. Glucose-6-Phosphate Dehydrogenase
(G6PD) Deficiency
• Most common metabolic disorder of RBC affecting more than 400
million people worldwide
• X-chromosome linked hereditary transmission
- Women (heterozygous or homozygous)
- Men (hemizygous)
• The HMP shunt pathway metabolizes 5% to 10% of glucose used by
the RBC, and this is critical for protecting red cells against oxidant
injury.
• Commonly characterized by acute hemolytic episodes in the setting of
oxidative triggers such as fava beans, infections and certain
medications. 22

23. • G6PD is a housekeeping enzyme
that plays an important role in:
1. Reduction of NADPH from NADP
2. Generation of five carbon sugars
• GPD activity decreases significantly
as erythrocytes age, with a half life
of approximately 60 days.
• Reticulocytes have five times
higher activity than the oldest
erythrocyte subpopulation.
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24. • More than 300 G6PD variants have been defined.
• Glutathione functions as an intracellular reducing agent that protect
RBCs against oxidative stress (i.e., superoxide anion and hydrogen
peroxide produced by drugs or infections)
• The result of oxidative damage to RBC is the production of rigid, non-
deformable erythrocytes that are susceptible to destruction by
reticuloendothelial system.
• Both extracellular and intracellular hemolysis occur in acute
haemolytic episodes in G6PD-deficient individuals.
• Patient develop a low haemoglobin, increased indirect bilirubin,
elevated LDH, free plasma haemoglobin, low haptoglobin and
hemoglobinuria.
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27. Glucose-6-phosphate dehydrogenase
deficiency and resistance to malaria
 G6PD frequently in Africans – protects them from malaria
Plasmodium falciparum is dependent on HMP shunt and reduced
glutathione for their optimum growth in RBC
One theory to explain this, is that cells infected with the Plasmodium
parasite are cleared more rapidly by the spleen.
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28. 3. Pyrimidine 5’ Nucleotidase Deficiency
• The third most common enzymatic deficiency causing hemolysis.
• Autosomal recessive disorder.
• The pyrimidine 5’ nucleotidase gene is localized to chromosome 7p15.
• Pyrimidine 5’ nucleotidase activity is much higher in reticulocytes than
mature red cells and its activity rapidly declines during the first few days of
red cell maturation.
• Pyrimidine 5’ nucleotidase participates in RNA degradation in reticulocytes.
• In Pyrimidine 5’ nucleotidase deficiency, there is accumulation of
pyrimidines in red cells and causes toxicity and hemolysis.
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29. Lead poisoning
• Lead can induce pyrimidine 5’ nucleotidase deficiency.
• Would expect hemolytic anemia and basophilic stippling on
peripheral smear
• This is treatable with chelator and stopping lead exposure.
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30. 4. Cytochrome b5 reductase deficiency
• Methemoglobin is a derivative of
haemoglobin in which ferrous (Fe2+) ions
are oxidized to the ferric (Fe3+) state.
• Mechanism of formation of
methemoglobin:
- During oxygenation, one electron is partially
transferred from iron to the bound oxygen,
forming a ferric-superoxide (Fe3+-o2
-) anion
complex.
- During deoxygenation, some of the oxygen
leaves as a superoxide (O2
-) radicle leaving the
iron in ferric state forming methemoglobin. 30

31. • The ferric (Fe3+) heme of methemioglobin are unable to bind oxygen.
• Several potential mechanisms exist to reduce methemoglobin back to
haemoglobin, only the NADH-dependent reaction catalysed by
cytochrome b5 reductase is physiologically important.
• Cytochrome b5 reductase, previously known as NADH diaphorase or
methemoglobin reductase.
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32. Structure of cytochrome b5 reductase:
• Cytochrome b5 reductase contains a noncovalently bound prosthetic
Flavin adenine dinucleotide (FAD) group that acts as electron
acceptor.
• This enzyme transfers two electrons from NADH to two molecules of
cytochrome b5 via an enzyme bound FAD.
• NADH and FAD are bound to two different branches of the enzyme.
• Cytochrome b5 transfers the electrons to a variety of acceptors.
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33. Cytochrome b5 Reductase Deficiency
• There are two types of cytochrome b5R deficiency:
- Type I - only red blood cells are affected
- Type II - affects all cell types
• Most of the patients with cytochrome b5 reductase deficiency do not have
symptoms.
• Patients can tolerate up Methemoglobin levels as high as 40% because of
physiological adaptations:
- Polycythemia
- Changes in concentration of 2, 3- DPG and pH
- Synthesis of globin chains
• However can decompensate when exposed to oxidizing agents.
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34. Cytochrome b5 Reductase Deficiency
Treatment
• Avoid oxidant drugs
• Can use methylene blue &/or ascorbic acid for cyanosis. In type II
patients this treatment will not do anything for neurological
abnormalities.
• Methylene blue is converted to leucomethylene blue, which results in
a nonenzymatic reduction of methemoglobin. Ascorbic acid directly
reduces methehemoglobin, but it works slowly.
• Alternative therapy: hyperbaric oxygen and exchange transfusions
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35. 5. Familial 2,3-Bisphosphoglycerate Deficiency
• Most polycythemias are acquired,
some are congenital.
• One rare cause of congenital
secondary polycythemia is familial
2,3-bisphosphoglycerate deficiency,
which results from deficiency of
the red blood cell enzyme
bisphosphoglyceratemutase.
• 2,3-BPG binds haemoglobin,
allosterically changing
haemoglobin conformation and
modulating its affinity to bind
hemoglobin
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37. • Due to deficiency of bisphosphoglyceromutase, decrease in 2,3-BPG
level shifts the haemoglobin oxygen dissociation curve to the left.
• Increasing haemoglobin affinity for oxygen, resulting in decreased
delivery of oxygen into the peripheral tissues.
• Results in compensatory polycythemia due to release of
erythropoietin.
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38. Thank You !!!
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