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Enzyme Deficiencies in Anemia
Terms in this set (34)
Erythrocyte Composition in Blood
- Erythrocytes are 40% of blood volume
- 5 types of Leukocytes
- Enzymes present in the cytoplasm of erythrocytes that play important roles
Formation of Erythrocytes
- Starts in bone marrow
- Stimulated by erythropoetin
Lifespan is 60-120 days
- Every 120 days you get a whole new set of RBCs
Red Cell Indices
- Hemoglobin - concentration of oxygen transporting metaloprotien
- Hematocrit - number of RBCs found in blood volume
- Hemoglobin an hematocrit are the two parameters assessed for anemia
- Reticulocyte - immature RBCs found in blood volume
Clinical Features of Anemia
- Pale skin color
- Decreased O2 content within cells - can lead to tissue hypoxia
- Leads to heart issues, liver issues
- Rapid heart rate
- Palpitation, etc
- Kidney - hypoperfusion of kidneys, decrease in urine output
Classifications of Anemia
- Blood loss - trauma
- Increased red cell destruction (hemolysis)
- Decreased red cell production - decrease in folate or vitamin B12
Red Cell Morphology
- Red cell size: normocytic (normal), microcytic (small), macrocytic (large)
- Hemoglobinization (color): normochromic (normal), hypochromic (less colorful)
Definition, classification and clinical features of anemia
- Anemia is a reduction of the total circulating red cell mass below normal limits that reduces the oxygen-carrying capacity of the blood.
- Red cells, or erythrocytes, represent 40-45% of blood volume and >90% of the formed elements in blood. Mature erythrocytes do not have nuclei or other internal organelles. Their red color is due to the eosinophilia of hemoglobin.
- Erythrocyte formation is triggered by erythropoietin released from the kidney in response to reduced oxygenation
- Erythroid progenitors develop in the marrow and the progeny are released into the peripheral blood. Hemoglobin is synthesized in erythrocyte precursor cells. The main functions of erythrocytes are to transport O2 and remove CO2 and H+ ions. Since erythrocytes lack organelles and cannot synthesize protein and repair themselves, they have a finite life span of 60-120 days before degradation in the spleen.
Mechanism of Anemia: Increased Red Cell Destruction
Causes of increased red cell destruction/hemolysis are extrinsic (E) or intrinsic (I)
- Antibody-mediated destruction (E)
- Mechanical trauma (I)
- Infections of red cells (E)
- Toxic or chemical injury
- Membrane lipid abnormalities (I)
- Acquired genetic defects (I)
- Inherited genetic defects (I)
Normoblasts are immature RBC that still have a nucleus
- Presents of normoblasts is an indication of hemolytic anemia
Based upon their etiology or red cell morphology, anemias are most often classified according to their:
• Etiology/Mechanism o Blood loss
o Increased red cell destruction (hemolysis)
o Decreased red cell production
• Red Cell Morphology
o Red cell size: normocytic, microcytic, macrocytic
o Hemoglobinization: normochromic, hypochromic o Shape
The causes of hemolysis
Increased red cell destruction, or hemolysis, is one classification of anemia. The causes of hemolysis are either extrinsic or intrinsic. They are:
• Antibody-mediated destruction
• Mechanical trauma
• Infections of red cells
• Toxic or chemical injury
• Membrane lipid abnormalities
• Acquired genetic defects
• Inherited genetic defects
Inherited Genetic Defects of Hemolytic Anemia
Red cell membrane disorders - may effect shape
- Can become very rigid (RBCs need to be flexible to move through small capillaries)
- Sequestered by the spleen and tagged for degradation
- Sickle cell anemia
- Hexose monophosphate shunt/pentose phosphate pathway
- Glucose-6-phosphate dehydrogenase (G6PD)
- Produce 5 carbon sugars and NADPH
- Pyruvate kinase
- Sole source of ATP production in the RBC
Among the various causes of hemolysis are the inherited genetic defects. They fall into 3 categories:
• Red cell membrane disorders
• Hemoglobin abnormalities
• Enzyme deficiencies
o Hexose monophosphate shunt/pentose phosphate pathway
• Glucose-6-phosphate dehydrogenase (G6PD)
- Pyruvate kinase
Major Metabolic Pathways of RBCs
Hexose monophosphate shunt/Pentose phosphate pathway (PPP)
- Ribose-5-P- important for nucleotide biosynthesis
- NADPH - helps protect cells from oxydative injuries
- 2,3-BPG - important for binding to hemoglobin - reduce hemoglobin affinity for oxygen - important for oxygen release to tissues
Major metabolic pathways in red blood cells
There are several major metabolic pathways that converge in the red blood cell. These pathways include glycolysis, hexose monophosphate shunt/pentose phosphate pathway (PPP), glutathione synthesis and adenosine salvage/metabolism. The purpose of these pathways is to:
(a) generate ATP to supply the Na-K-ATPase pump with energy to transport ions across the cell membrane in order to maintain the flexible, biconcave disc shape of the red blood cell.
(b) produce 2,3 bisphosphoglycerate (BPG) to facilitate O2 release in tissues due to its ability to reduce the O2 affinity for hemoglobin.
(c) generate the reducing equivalent, NADPH, which will be used to maintain glutathione in its reduced state.
(d) synthesize glutathione which can protect the RBCs against a range of oxidative stressors and chemical insults.
Erythrocyte Shape: Optimized for Maximal Performance
- RBC structure is optimized for gas exchange and the transport of ions and water across the membrane.
- RBCs uses most of the ATP to power the Na-K-ATPase pump to transport ions and water across the cell membrane and, thus, maintain shape.
- Biconcave shape is important for gas exchange and helpful to transport water and ions across cell membrane
- Needs to be very flexible and able to deform
- Loses ability to conform - sequested by spleen for degradation
- Na/K pump pumps out ions but allows water in to maintain its shape
The small, biconcave disc shape of the RBC is optimized for maximal performance. The cell's shape facilitates gas exchange across the membrane because it increases surface:volume ratio. The composition of proteins in the red cell membrane allow it to deform when traversing through small capillaries and through the elliptical passages of the spleen roughly 120 times per day. Damaged RBCs that lose their flexibility are no longer to deform. They become trapped in the spleen and destroyed by macrophages. The red blood cells also travel through the hypertonic environment of the kidney where they shrink and expand. The ATP generated in the RBCs is used to power the Na-K-ATPase pump in order to maintain the proper electrochemical and ion gradients across the cell membrane when traveling through the kidneys.
Formation and elimination of reactive oxygen species.
During metabolism, powerful reactive oxygen species (ROS) are produced in blood cells and other cells. These include: superoxide, hydrogen peroxide, peroxyl radicals and hydroxyl radicals. A major source of superoxide (O2-) is the electron transport chain. Reduced coenzyme Q, which accepts one electron at a time occasionally transfers one electron to O2 rather than to the Fe- S center associated with cytochrome b-c1. These reactive oxygen intermediates can cause damage to DNA, proteins and unsaturated lipids and lead to cell death. ROS have been implicated in reperfusion injury, cancer, inflammatory disease and aging. They cause auto-oxidation of iron in hemoglobin from the ferrous (Fe2+) to ferric (Fe3+) state to convert it to methemoglobin.
Reactive Oxygen Species I
- Reactive oxygen species (ROS) form during metabolism in RBCs and other cells, and contribute to cell injury.
- Hb-Fe2+ ---> Hb-Fe3+
- Hemoglobin ---> Methemoglobin
- Hydrogen peroxide
- Hydroxyl radical
- Reactive oxygen species are things that the RBC needs to be protected against
- They contribute to cancer, inflammation, aschemia
- Superoxide - converts hemoglobin to methemoglobin which is bad cuz it is unable to bind oxygen
- Oxidizes Hb ferrous to Hb ferric - not reactive
Reactive Oxygen Species II
- Important enzymes: superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase
- Important products: glutathione, NADPH
- Glutathione gets reduced and then protects the cell from oxidative injury
- Gets reduced by accepting an H from NADPH
Our cells utilize protective mechanisms to minimize the damaging effects of these ROS.
Our cells utilize protective mechanisms to minimize the damaging effects of these ROS. If left unguarded in the RBCs, these ROS will react with the lipids in the cell membrane causing them to become rigid. RBCs that are unable to deform when passing through small blood vessels and the spleen will be targeted for destruction by macrophages. Enzymes, such as superoxide dismutase and catalase, catalyze the conversion of these ROS into lesser ROS intermediates or H2O. In addition, glutathione peroxidase will detoxify H2O2 by acting on reduced glutathione (GSH) and H2O2 to produce oxidized glutathione (GSSG) and H2O. GSSG will be converted to its reduced state (GSH) by the enzyme glutathione reductase. The reducing equivalent, NADPH, donates a H+ ion to glutathione. Glutathione is a tripeptide synthesized from 3 amino acids: glutamine, cysteine and glycine.
- a tri-peptide (not encoded by mRNA)
- synthesized from 3 amino acids: glutamate, cysteine and glycine
ROS and Methemoglobin
- Oxidation of heme iron in hemoglobin from ferrous (Fe2+) to ferric (Fe3+) state forms methemoglobin.
- Methemoglobin cannot bind or transport oxygen.
- Methemoglobinemia ---> "chocolate cyanosis" - skin color becomes brownish blue is color, brown blood
- Toxic compounds: primaquine, pamaquine, chloroquine
- Fava beans contain: vicine, divicine, covicine, isouramil
- Give patient ascorbic acid and methyline blue
Cytochrome b5 reductase (aka methemoglobin reductase) reduces Fe3+ of methemoglobin back to the Fe2+ state, and regenerates reduced cytochrome b5.
Methemoglobin and methemoglobin reductase.
The ferrous iron (Fe2+) in hemoglobin is susceptible to oxidation to ferric iron (Fe3+) by ROS, certain drugs (i.e., nitrates, sulfonamides) or metabolic products of nutrients (e.g., fava beans). Methemoglobin, which is the result of this oxidation, cannot bind or transport O2. Therefore, it is necessary to convert methemoglobin back to hemoglobin. This conversion is catalyzed by the enzyme methemoglobin reductase (aka cytochrome b5 reductase). Individuals with the disease methemoglobinemia have a deficiency in this enzyme. Methemoglobinemia is caused by either an acquired or inherited defect in methemoglobin reductase. It also occurs in individuals who have inherited hemoglobin M. Individuals with methemoglobinemia exhibit "chocolate cyanosis" because their skin and mucous membranes have a brownish-blue coloration and their blood is tinged brown. Other symptoms are related to the degree of tissue hypoxia, including anxiety, headaches, dyspnea, coma and death. Methylene blue or ascorbic acid are reducing agents that get oxidized as Fe3+ gets reduced. They are used to treat methemoglobinemia.
Glucose-6-Phosphate Dehydrogenase and NADPH
- G6PD catalyzes a reaction in pentose phosphate pathway to produce NADPH.
- NADPH electrons destined for reductive biosynthesis and detoxification reactions.
- electron donor
- glutathione reductase ---> H2O2
G6PD Deficiency: Part I
- is a hereditary, X-linked disease characterized by hemolytic anemia
- highly prevalent in Middle East, tropical Africa and Asia, and part of Mediterranean
- increases resistance to Plasmodium falciparum malaria.
- Commonly found abnormality in humans
G6PD Deficiency: Part II
- decreases cellular detoxification of free radical (Hydrogen peroxide)
- diminishes ability to form NADPH to maintain GSH pool.
- leads to formation of denatured proteins from -SH oxidation and attachment of insoluble Heinz bodies to RBC membranes that are plucked out by macrophages ("bite cells").
- causes RBC rigidity and removal by macrophages.
- Macrophages come along and pluck heinz bodies out of red blood cells = get "bite cells"
- Heinz bodies cause RBC to become rigid - less flexible
- Decrease in NADPH reduction - decreased ability to protect from oxidative stresses
- G6PD deficiency is usually asymptomatic except in response to an oxidant stress.
- Severe symptoms include Heinz bodies in RBCs, jaundice, hematuria and dark-colored urine.
- Sulfa drugs and anti malarial drugs, infections, can cause oxidant stress
- Fava beans can cause oxidant stress
- Formation of bilirubin that is not being conjugated as a reaction to oxidant stress - jaundice, hematuria (blood in the urine), dark colored urine
- Symptoms occur within 24 days of being born
Sometimes they are not symptomatic
Glucose-6-phosphate dehydrogenase enzyme and deficiency.
- Glucose-6-phosphate dehydrogenase (G6PD) catalyzes a reaction in the pentose phosphate pathway (PPP) to produce NADPH. The NADPH electrons are destined to be utilized by cells for reductive biosynthetic and detoxification reactions.
- NADPH acts as an electron donor and it catalyzes the reduction of glutathione in order to degrade H2O2, a ROS intermediate.
- Since the PPP is the only pathway to produce NADPH, the RBCs lacking G6PD become very susceptible to oxidative damage.
- Deficiencies in the G6PD enzyme cause a hereditary, X-linked disease that is characterized by hemolytic anemia.
- G6PD deficiencies (aka "Favism") are highly prevalent in the Middle East, tropical Africa and Asia and parts of the Mediterranean.
- Individuals who have this deficiency exhibit an increased resistance to Plasmodium falciparum malaria. Only some of these mutant protein variants cause clinical symptoms.
Decreased activity of G6PD has several detrimental effects, including:
(a) decreased cellular detoxification of free radicals,
(b) insufficient production of NADPH under oxidative stress leading to damage and hemolysis of red cells,
(c) diminished ability to form NADPH to maintain the pool of GSH,
(d) formation of denatured proteins from sulfhydryl oxidation and attachment of insoluble Heinz bodies (inset) to RBC membranes that are plucked out by macrophages ("bite cells"), and
(e) rigidity of RBCs that are removed by macrophages.
G6PD deficiency is usually asymptomatic except
G6PD deficiency is usually asymptomatic except in response to an oxidant stress which usually manifests as hemolytic anemia within 2-3 days after exposure. An oxidant stress appears in the form of certain drugs (anti-malarials, sulfa drugs), diet (fava beans) and infection. Severe symptoms include formation of Heinz bodies in RBCs, jaundice, hematuria and dark-colored urine. Heinz bodies form as a result of disulfide crosslinked aggregates of oxidized hemoglobin. Jaundice is due to the accumulation of bilirubin, a product of heme metabolism, in plasma and tissues. Very severe hemolysis causes hemoglobin to spill into the urine resulting in hematuria and dark-colored urine.
Pyruvate Kinase and Deficiency
- Enzyme catalyzes the last reaction in glucose metabolism.
- 2nd most common enzymatic cause of hemolytic anemia.
- Autosomal recessive.
- Most prevalent among European descent. (Old Order Amish, PA)
- Deficiency confers resistance to most severe forms of malaria.
- Varies in severity
- mild = little intervention
- severe = requires regular transfusions
- Catalyzes last step in glycolytic pathway
- One of the ATP generating steps - reduced ability to produce ATP
Pyruvate kinase enzyme and deficiency.
Pyruvate kinase is an enzyme that catalyzes the last reaction in glucose metabolism to convert phosphoenolpyruvate into pyruvate. Pyruvate kinase (PK) deficiency is the most common of the hemolytic anemias that result from a deficiency in a glycolytic enzyme. It is second only to G6PD deficiency as an enzymatic cause of hemolytic anemia. PK deficiency is an inherited disease that is autosomal recessive. It is most prevalent among people of European descent and is often diagnosed among the Old Order Amish population of Pennsylvania due to intermarriages within a closed community. PK deficiency confers resistance to most severe forms of malaria. The PK deficient phenotype varies in severity from mild (requires little to no intervention) to severe (requires regular transfusions).
Mature RBCs lack mitochondria, so they rely on glycolysis for ATP production to supply energy for the ion pumps that maintain cell flexibility and shape. PK deficient RBCs have decreased cellular ATP concentrations due to a reduced rate of glycolysis, and compensatory accumulation of 2,3 BPG. Thus, PK deficiency affects cellular integrity. The accumulation of 2,3 BPG binds to hemoglobin and decreases its affinity for O2 to promote delivery to tissues, the increased 2,3 BPG concentration is a compensatory mechanism to prevent tissue hypoxia. Symptoms of PK deficiency include jaundice and mild to moderate splenomegaly.
Symptoms of Pyruvate Kinase Deficiency
- Jaundice - unconjugated bilirubin
- Mild to moderate splenomegaly - number of cells that have become rigid and sequestered in spleen
- Decreased ATP concentration
- Inability to synthesize ATP required to maintain RBC metabolism, ion gradients and cell shape leads to hemolytic anemia.
- Increased 2,3-bisphosphoglycerate concentration. - compensatory raction, promote oxygen release to tissue to decrease hypoxia
- Compensatory accumulation of 2,3 BPG that binds Hb and decreases its affinity for O2 to promote delivery to tissues.
Hexokinase Enzyme and Deficiency
- Hexokinase catalyzes first reaction in glucose metabolism and has lowest activity of glycolytic enzymes.
- Short half life in RBCs
- In a blood transfusion the transfused cells have to be supplemented with hexokinase because the half life is so short
- Clinical features vary in severity.
- Deficiency is rare, but affects glycolysis and PPP.
- decreased ATP synthesis - Na-K ATPase pump to maintain ion gradient - RBCs become rigid
- decreased NADPH production - lack of glutathione synthesis; failure to inactivate H2O2
- decreased erythrocyte life span and premature destruction => chronic hemolytic anemia
Hexokinase catalyzes the first reaction in glucose metabolism and has the lowest activity of glycolytic enzymes. Hexokinase deficiencies are rare and the clinical manifestations range in severity. However, this enzymatic deficiency affects the glycolytic pathway and PPP. This results in: (a) decreased ATP synthesis which affects the Na-K-ATPase pump to maintain the ion gradient across the RBC membrane, and (b) decreased NADPH production which affects synthesis of glutathione and inactivation of the ROS intermediate H2O2. The lack of ATP generated in cells with hexokinase deficiencies shortens the RBC life span due to premature destruction. This results in chronic hemolytic anemia.
Summary of enzyme deficiencies in anemia
The RBC, which lacks mitochondria and the capability for oxidative metabolism, obtains all of its ATP through glycolysis. Glycolysis provides metabolites for branch points to numerous other metabolic pathways including the PPP. This pathway provides pentoses for synthesis of DNA, RNA in nucleated cells, and NADPH for biosynthetic reactions. NADPH is required for maintenance of reduced glutathione, which is an essential cofactor for antioxidant defense mechanisms that protect the cell against oxidative stress.
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