Red Cell Metabolism and Enzyme Deficiencies Flashcards
Write about red cell metabolism
RBCs contain no mitochondria so they cannot carry out respiration, no citric acid cycle, no oxidation of fatty acids or ketone bodies
Energy in the form of ATP is obtained only from the glycolytic breakdown of glucose with the production of lactate (anaerobic glycolysis)
ATP produced is being used for keeping the biconcave shape of RBCs and in the regulation of transport of ions and water in and out of the red blood cells
Three areas of RBC metabolism are crucial for RBC survival and function:
- RBC membrane
- Haemoglobin structure and function
- RBC metabolic pathways = cellular energy
How does the RBC obtain ATP
Energy in the form of ATP is obtained only from the glycolytic breakdown of glucose with the production of lactate (anaerobic glycolysis)
What does the RBC do with the ATP produced
ATP produced is being used for keeping the biconcave shape of RBCs and in the regulation of transport of ions and water in and out of the red blood cells
What three areas of RBC metabolism are crucial for RBC survival
- RBC membrane
- Haemoglobin structure and function
- RBC metabolic pathways = cellular energy
What are the functions of ATP in RBCs
Protection against oxidative stress
Initiation of glycolysis
Glutathione synthesis
Reduction of haem iron to ferrous form
Na/K exchanger
Red Cell Biconcave shape
What are some requirements of the red cell
Must be able to pass repeatedly through the microcirculation in order to carry Hb into close contact with tissues and for successful gaseous exchange
The RBCs total journey throughout its 120 day lifespan has been estimated to be 480km
Hb must be maintained in a reduced (ferrous) state
Write about the biconcave feature of red blood cells
(3)
Flexible
With an ability to generate energy as ATP by an anaerobic glycolytic pathways (anaerobic glycolysis) and to generate reducing power as NADH by this pathway
Generates energy as reduced NADH by the Pentose Phosphate pathway
What are the two main metabolic pathways in the mature RBC
- Anaerobic glycolysis (90%)
- Pentose Phosphate Pathway (10%)
What are the main metabolic intermediates of anaerobic glycolysis
ATP: the main energy compound of the RBC
NADH: an essential reducing agent cofactor
What are the main metabolic intermediate of the pentose phosphate pathway
Reduced NADPH
Write about anaerobic glycolysis
The pathway consists of a highly complex series of metabolic reactions whereby energy (in the form of ATP, NADH and NADPH) is generated from glucose
An early step in this pathway is the generation of glucose-6-phosphate
Most of the glucose-6-phosphate proceeds along the glycolytic pathway to form two molecules of glyceraldehyde-3-phosphate
For each molecule of glucose used, two molecules of ATP and thus two high-energy phosphate bonds are generated
10% of the G6P is diverted to a sub-pathway, the pentose phosphate pathway, where an atom of hydrogen is transferred to NADP, to generate the hydrogen carrier NADPH
What happens to 90% of the G6P in anaerobic glycolysis?
It proceeds along the glycolytic pathway to form two molecules of glyceraldehyde-3-phosphate
What does the RBC do with the ATP produced in anaerobic glycolysis
ATP provides energy for RC volume, shape and flexibility
What happens to 10% of the G6P in anaerobic glycolysis
10% is diverted to the pentose-phosphate pathway
Here an atom of hydrogen is transferred to NADP to generate the hydrogen carrier NADPH
What three energy molecules are produced by anaerobic glycolysis
ATP
NADH
NADPH
What is the methemoglobin reductase pathway
(3)
Anaerobic glycolysis produces NADH which is used in the methemoglobin reductase pathway
NADH is used to reduce functionally dead methaemoglobin (oxidised Hb) containing ferric iron to functionally active, reduced Hb
What is methaemoglobin
Oxidised Hb
How is ferric iron produced
Produced by oxidation of approximately 3% of Hb each day
What is the function of the methemoglobin reductase pathway?
Maintains iron in the ferrous state (Fe+++)
In the absence of the enzyme (methemoglobin reductase), methemoglobin accumulates and it cannot carry oxygen
What is 2,3-DPG
2, 3-diphosphoglycerate
What does 2,3-DPG do
Competes for oxygen for binding to Hb, in lung and tissue
It decreases affinity of Hb for oxygen so it helps oxyhaemoglobin to unload oxygen
Storing blood results in a decrease of 2,3-DPG leading to high oxygen affinity Hb. This leads to oxygen trap
6-24 hours are needed to restore the depleted 2,3 DPG
Maximum storage time for RBC’s is 21-42 days
In people with high-altitude adaptation or smokers the concentration of 2,3-DPG in the blood is increased (low oxygen supply)
Foetal Hb has a low DPG affinity: the higher the O2 affinity facilitates the transfer of O2 to the foetus via the placenta
When is increased DPG concentration seen
High-altitude adaptation
smokers
What form of Hb has a low affinity for DPG and why
Foetal Hb
The higher O2 affinity facilitates the transfer of O2 to the foetus via the placenta
Write about the Pentose Phosphate Pathway
(7)
Approximately 10% of glucose metabolism occurs by this oxidative pathway in which G6P is converted to ribulose-5-phosphatase
NADPH is the main intermediate of the pathway and is required for the reduction of glutathione
Reduced glutathione is essential for the protection of Hb and for the reduction of cellular intermediates that can cause cellular damage
This pathway protects the RBC from oxidative injury
This pathway can be increased up to 30% in times of oxidant stress
Oxidant stress can arise due to natural metabolism or can be drug induced. It can cause damage to Hb, membrane proteins and intracellular enzymes
If the pathway is deficient, intracellular oxidants can’t be neutralised and globin denatures and then precipitates. The precipitates are referred to as Heinz bodies
What happens to G6P in the pentose phosphate pathway
Approximately 10% of glucose metabolism occurs by this oxidative pathway in which G6P is converted to ribulose-5-phosphatase
What is the main intermediate in the pentose phosphate pathway?
NADPH is the main intermediate of the pathway and is required for the reduction of glutathione
What is reduced glutathione essential for
(2)
The protection of Hb and for the reduction of cellular intermediates that can cause cellular damage
This pathway protects the RBC from oxidative injury
What happens to the pentose phosphate pathway in times of oxidant stress
This pathway can be increased up to 30% in times of oxidant stress
What might cause oxidative stress
Oxidant stress can arise due to natural metabolism or can be drug induced. It can cause damage to Hb, membrane proteins and intracellular enzymes
What happens if the pentose phosphate pathway is deficient
If the pathway is deficient, intracellular oxidants can’t be neutralised and globin denatures and then precipitates.
The precipitates are referred to as Heinz bodies
What can cause oxidant stress
Favism - fava beans
Drugs
Infection
What does oxidative stress do
(4)
Damages cell components e.g. haemoglobin and lipids
Heinz bodies start to precipitate
Permeability of RC membrane and deformability reduced
Damaged RC removed by extravascular haemolysis
Write about the history of enzyme deficiencies
(4)
Haemolysis first noted in plantation workers who received anti-malarial drugs (quinine)
Anaemias were originally called hereditary non-spherocytic haemolytic anaemia
The patients were seen not to contain abnormal haemoglobin, the antiglobulin test was negative and the osmotic fragility test was normal
They were later identified to have arisen due to enzyme deficiencies in the RBC
What are the two main enzyme defects
PK deficiency
G6PD deficiency
What is PK deficiency
Deficiencies in glycolytic pathway which results in shortened red cell survival and chronic haemolytic anaemia
What is G6PD deficiency
Defects in PPP are associated with periodic acute haemolytic episodes due to oxidant stress (drugs, chemicals or infections)
Define G6PD
A genetic disorder that affects red blood cells and may contribute to anaemia
The sever form is called favism which causes more symptoms and poses more risks than other types of G6P deficiency
What are the signs and symptoms of G6PD
Yellow of the eyes
Confusion and difficulty concentrating
Higher risk for enlarged spleen
Sudden rise in blood temperature
Rapid heart rate
Darker urine colour
Fatigue and weakness
Write about G6PD Deficiency (PPP)
Most common enzyme defect
Found worldwide but high prevalence in central Africa, southern Europe, Indian subcontinent the Middle East and SE Asia
X linked -> more prevalent in males
Confers a relative resistance against plasmodium falciparum
400 variants
Majority of variants function normally and are differentiated by the electrophoretic mobility
Write about the pathology associated with G6PD Deficiency
GP6D deficiency renders the RC susceptible to oxidant stress
Over 50 mutations identified - nearly all point mutations
Deficiency account for over 90% of haemolytic anaemia due to enzyme deficiency
G6PD is a rate limiting step in the PPP
G6PD is responsible for removing the hydrogen from its substrate G6P and the eventual production of the crucial antioxidant glutathione (GSH)
What are the functions of G6PD
G6PD is a rate limiting step in the PPP
G6PD is responsible for removing the hydrogen from its substrate G6P and the eventual production of the crucial antioxidant glutathione (GSH)
How is G6PD deficiency classified
I
II
III
IV
V
Write about class I G6PD deficiency
Most severe
Less than 1% enzyme activity
Cant deal with any oxidative stress
Chronic haemolytic anaemia
Write about class II G6PD
Less than 10% activity
Acute haemolytic anaemia (fava bean and drug-dependent)
Write about class III G6PD deficiency
Between 10 and 60% activity
Occasional acute haemolytic anaemia
Very frequent in malaria areas -> coverage from malaria with minimal symptoms
Write about class IV G6PD deficiency
Between 60 and 90% activity
Asymptomatic
Write about class V deficiency
> 110% increased activity
Asymptomatic
What are the clinical presentations of G6PD
(3)
Acute haemolytic anaemia -> majority are asymptomatic but will develop acute haemolytic anaemia if exposed to oxidative stress
Neonatal jaundice -> higher risk in G6PD patients than in normal newborns due to reduced liver activity
Chronic non spherocytic haemolytic anaemia -> can be seen in G6PD patients when stimulated by factors that cause acute haemolytic anaemia
What is the laboratory diagnosis for G6PD deficiency
Hb low
MCV normal, MCH normal
Bite cells (very characteristic of G6PDD)
Hemighosts
Polychromasia
NRBC
Reticulocytes high
Heinz bodies
Haemoglobinuria
Haptoglobin low
LDH and bilirubin high
What test do we carry out to investigate G6PDD
Heinz body stains - precipitates of oxidatively denatured Hb stained by crystal violet or methylene blue
MetHb reduction test
A fluorescent spot screening test
G6PD enzyme assay
DNA analysis
What is the MetHb reduction test
Where G6PD erythrocytes fail to reduce metHb in the presence of methylene blue
What is the fluorescent spot screening test
G6P, NADP are mixed with the patients blood and spotted onto filter paper
G6PD should reduce the NADP to NADPH and fluorescence will occur on the conversion of NADP to NADPH
Write about controls and EQA for G6PD
2 samples received bi-monthly from UK NEQAs
G6PD control normal, control deficient and control intermediate
Controls ran alongside each test (2 tests)
What morphology is associated with G6PD deficiency
Blood film with bite cells
Heinz bodies
Hemighosts or bull’s eye cells
How is G6PD treated
Offending drug is stopped
Any underlying infection is treated
A high urine output is maintained
Blood transfusion is undertaken where necessary for sever anaemia
G6PD-deficient babies are prone to neonatal jaundice and in severe cases phototherapy and exchange transfusion may be needed
The jaundice is usually not caused by excess haemolysis but by deficiency of G6PD affecting neonatal liver function
Splenectomy if severe
Write about G6PD and plasmodium vivax
Treatment of plasmodium vivax with primaquine poses a potential risk of mild to severe acute haemolytic anaemia in G6PD deficient people
There remains a lack of consensus on the requirement for G6PD deficiency testing before prescribing primaquine radical cure regimens
Write about anaerobic glycolysis deficiencies
Deficiency of any of the enzymes involved results in haemolysis due to:
- reduced levels of ATP and NADH
- accumulation of intermediate substrates including 2,3-DPG and MetHb
What are the 7 main enzymes that cause anaerobic glycolysis deficiency
Pyruvate kinase (most common)
G6P isomerase (next most common)
Glyceraldehyde-3-phosphate-dehydrogenase
Phosphofructokinase
Triose phosphate isomerase
Hexokinase deficiency (very rare)
Methaemoglobin reductase
Write about pyruvate kinase deficiency
Most significant defect of anaerobic glycolysis
About 20 different mutations causing haemolytic anaemia
Inherited as an autosomal recessive condition
Worldwide in distribution
Severity of the haemolytic episodes varies from mild to severe depending on the properties of the mutant enzymes
What are some features of pyruvate kinase deficiency?
(4)
Severe neonatal jaundice and anaemia
Severe CNSHA requiring repeated transfusions
Moderate haemolysis with exacerbation during infection or pregnancy
Symptomless compensated haemolysis with only a minor apparent anaemia
Write about the pathogenesis of PK deficiency
A reduction of ATP and decrease in NADH
Extravascular haemolysis occurs mostly but intravascular haemolysis may occur under oxidative stress
PK deficiency causes an increase in 2, 3 DPG, this causes a decrease in oxygen affinity, due to a shift in the oxygen association curve to the right, and results in increased oxygen delivery to the tissues
This results in more efficient use of oxygen at a lower haemoglobin level
The reticulocyte count may be moderately raised
Write about the epidemiology of pyruvate kinase deficiency
(3)
Occurs worldwide but most cases have been reported in northern Europe, Japan and the USA. Many cases are found in the Amish population of Pennsylvania
The prevalence is estimated at 51 cases per million by gene frequency studies but the observed prevalence in one northern england region was found to be 3.3 cases per million
Family history consistent with autosomal recessive inheritance. More than 150 different causative mutations have been identified
How is PK Deficiency Treated
Treatment is supportive in mild-to-moderate cases, some transfusion-dependent patients have benefited from splenectomy but haemolysis still occurs.
BMT has been successful
What are the laboratory findings for PK deficiency
Hb low
MCV normal and MCH normal
Echinocytes
Polychromasia
NRBCs
Reticulocytes
LDH and bilirubin up
Haptoglobin down
How do we test for Pyruvate kinase deficiency?
Fluorescence screening test
PK enzyme assay
DNA analysis
What is the fluorescence screening test
In PK deficiency the fluorescence will persist beyond 30 minutes
How is PK deficiency treated?
(6)
Blood tranfusion
Folic acid supplements
Phototherapy and or change transfusion
Chelation therapy
Splenectomy
Partial splenectomy
Why is splenectomy rarely done for PK deficiency
Risk of thromboembolic disease post splenectomy
Some patients developed splenic and portal vein thrombosis
How long did it take a BM transplant patient to be cured of PKD
Took 3 year for PK levels to return to normal
