Chapter 6 - Hemoglobin PDF

6/28/2024 CHAPTER 6 Erythrocytes: Hemoglobin PREAMBLE PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. PowerPoints DO NOT cover the details need...

6/28/2024 CHAPTER 6 Erythrocytes: Hemoglobin PREAMBLE PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. PowerPoints DO NOT cover the details needed for the Unit exam Each student is responsible for READING the TEXTBOOK for details to answer the UNIT OBJECTIVES Unit Objectives are your study guide (not this PowerPoint) Test questions cover the details of UNIT OBJECTIVES found only in your Textbook! 2 1  6/28/2024 CHARACTERISTICS AND BIOSYNTHESIS OF HEMOGLOBIN Genetic inheritance of hemoglobin Normal adult hemoglobin A is inherited in simple mendelian fashion. Four polypeptide chains (574 amino acids) Two alpha and two beta chains Each has an attached heme group Genotype for normal hemoglobin is A/A. There are approximately 350 variant types; hemoglobin defects are mostly due to either amino acid substitution (hemoglobinopathies) or diminished production of the polypeptide chains (thalassemias). 3 CHEMICAL COMPOSITION AND CONFIGURATION OF HEMOGLOBIN #2 4 2  6/28/2024 HEMOGLOBIN FUNCTION #1 The role of 2,3-diphosphoglycerate The oxygen affinity of the hemoglobin molecule is associated with the spatial rearrangement of the molecule and is regulated by the concentration of phosphates, particularly 2,3- diphosphoglycerate (2,3-DPG) in the erythrocyte. The manner in which 2,3-DPG binding to reduced hemoglobin (deoxyhemoglobin) affects oxygen affinity is complex. Basically, 2,3-DPG combines with the beta chains of deoxyhemoglobin and diminishes the molecule’s affinity for oxygen. 5 OXYGEN DISSOCIATION AND ALTERATIONS 2,3,-Diphosphoglycerate combines with beta chains of deoxyhemoglobin and diminishes the molecule’s affinity for oxygen Heme groups unload oxygen in tissues and beta chains are pulled apart and 2,3-DPG form salt bridges Results in lower affinity of oxygen 6 3  6/28/2024 OXYGEN DISSOCIATION AND ALTERATIONS Oxygen uptake in lungs causes salt bridges to be broken and 2,3-DPG to be expelled In cases of tissue hypoxia, oxygen moves from hemoglobin to tissues and amount of deoxyhemoglobin increases in RBC Produces binding of 2,3-DPG which lowers hemoglobin affinity of oxygen If hypoxia persists, depletion of free 2,3- DPG leads to increased production of more 2,3-DPG and a persistently lowered affinity of the hemoglobin molecule for oxygen Add a Footer 77 OXYGEN DISSOCIATION Changes in oxygen affinity of the molecule are responsible for the ease with which hemoglobin can be loaded with oxygen in the lungs and unloaded in tissue Shape and position of the oxyhemoglobin dissociation curve graphically describe the relationship between oxygen content (% saturation) and partial pressure of oxygen (PO2) P50 value is defined as the partial pressure of oxygen required to produce half saturation of hemoglobin when the deoxyhemoglobin concentration equals the oxyhemoglobin concentration at a constant pH and temperature Humans have a pH of 7.4 and temperature of 37.50C 8 4  6/28/2024 OXYGEN DISSOCIATION A decrease in DPG causes an increase in oxygen affinity is demonstrated by a shift to the left An increase is DPG causes a decrease in oxygen affinity is represented by a shift to the right Add a Footer 99 CARBON DIOXIDE TRANSPORT Carbon dioxide can be carried to the lungs in three ways Indirectly by erythrocytes Directly by erythrocytes In plasma 10 5  6/28/2024 CARBON DIOXIDE TRANSPORT In the predominant indirect erythrocyte mechanism, approximately three fourths of the activity for removing carbon dioxide, carbon dioxide diffuses into the erythrocytes, is catalyzed by the enzyme carbonic anhydrase, and is transformed into carbonic acid. H2O + CO2 --------- H2CO3 The hydrogen ion of carbonic acid is accepted by the alkaline deoxyhemoglobin, and the bicarbonate ion diffuses back into the plasma. H2CO3 ----------- H+ + HCO3- 11 CHLORIDE SHIFT Free bicarbonate diffuses out of RBC into plasma Plasma chloride diffuses into cell In the lungs bicarbonate is converted back into carbon dioxide and water and eliminate from lungs through respiration 12 6  6/28/2024 CARBON DIOXIDE TRANSPORT ¼ of the carbon dioxide is directly bound with deoxyhemoglobin forming carbamino hemoglobin Approximately 5% of carbon dioxide is carried in solution in the plasma to the lungs 13 BIOSYNTHESIS OF HEMOGLOBIN #1 Formation of Heme from Porphyrin Hemoglobin is synthesized during most of the erythrocytic maturation process 65% of cytoplasmic hemoglobin is synthesized before the nucleus is extruded; 35% is synthesized in the early reticulocyte 14 7  6/28/2024 BIOSYNTHESIS OF HEMOGLOBIN #2 Formation of Heme from Porphyrin Heme is an erythrocyte precursor Formed mainly in the red bone marrow and liver Heme produced in the erythroid precursors is chemically identical to that in the cytochromes and myoglobin 15 THE ROLE OF IRON IN HEMOGLOBIN SYNTHESIS Iron is delivered by transport protein, transferrin, to the membrane of immature cell Iron in the ferric form (Fe3+) affixes to cell membrane and transferrin is released Iron enters cell and proceeds to mitochondrion Inserted into the protoporphyrin ring to form heme 16 8  6/28/2024 THE ROLE OF IRON IN HEMOGLOBIN SYNTHESIS #2 Excess iron accumulates as ferritin aggregates in the cytoplasm of immature erythrocytes Amount of non-heme iron deposited depends on the ratio between the plasma iron level and the iron required by the cell 17 GLOBIN STRUCTURE AND SYNTHESIS #1 Globin structure and production are governed by genetics. Rate of globin chain synthesis is a function of the rate at which the DNA code is transcribed into mRNA. 18 9  6/28/2024 GLOBIN STRUCTURE AND SYNTHESIS #2 Alpha Globin Locus: Chromosome 16 governs alpha chain production in adults and zeta chain production in the fetus. Beta Globin Locus: Chromosome 11 governs the beta globin chains, which include beta, gamma, delta, and epsilon. Production of functional hemoglobin pairs two alpha globin chains and two beta globin chains together. 19 GLOBIN CHAINS AND HEMOGLOBIN COMPOSITION Normal Hemoglobin Types Hemoglobin A Subfraction A1, A2 Fetal hemoglobin Embryonic hemoglobin Each has a distinctive composition of polypeptide chains Many other types of variant (abnormal) hemoglobin's 20 10  6/28/2024 DISORDERS RELATED TO HEMOGLOBIN (PORPHYRIN) BIOSYNTHESIS 21 DISORDERS OF HEME (PORPHYRIN) SYNTHESIS Porphyrias are disorders in the synthesis of porphyrin. Can be inherited or acquired Inherited: Rare autosomal recessive condition; Congenital erythropoietic porphyria Acquired: Lead poisoning which inhibits synthesis at several points by inhibiting enzymes 22 11  6/28/2024 DISORDERS OF HEME (PORPHYRIN) SYNTHESIS Porphyrias can be classified based on various characteristics: Clinical presentation (acute versus chronic) Source of enzyme deficiency Site of enzyme deficiency in the heme biosynthetic pathway 23 DISORDERS OF IRON METABOLISM #1 Iron deficiency Genetic defect of iron Iron overload Sideroblastic anemia 24 12  6/28/2024 DISORDERS OF IRON METABOLISM #2 Iron deficiency anemia pathogenesis: Iron is distributed in three different compartments: Storage: primarily as ferritin in bone marrow macrophages and liver cells Transport: with serum transferrin Functional: as hemoglobin, myoglobin, and cytochromes Normal iron status continues as iron intake lags behind iron loss, but eventually the loss will be too great for the intake to keep up  depletion of iron stores  iron deficiency anemia. 25 DISORDERS OF IRON METABOLISM #3 Causes of sideroblastic anemia include the following: Congenital defect: hereditary sex-linked (primarily males); autosomal Acquired defect: primary (one of the myelodysplastic syndromes); may evolve into acute myelogenous leukemia Association with malignant marrow disorders: acute myelogenous leukemia, polycythemia vera, myeloma, myelodysplastic syndromes Secondary to drugs: isoniazid (INH), chloramphenicol; after chemotherapy Toxins, including alcohol, and chronic lead poisoning 26 13  6/28/2024 DISORDERS OF IRON METABOLISM #4 Hereditary hemochromatosis HFE gene related (type 1). Different mutations of the HJV gene are responsible for juvenile hemochromatosis (or type 2 hemochromatosis). Type 3 hemochromatosis is a different form of the disease that usually appears in midlife. Type 4 is related to the SLC40A1 gene that encodes for ferroportin. 27 DISORDERS OF GLOBIN SYNTHESIS Globin synthesis is highly coordinated with porphyrin synthesis. When globin synthesis is impaired, protoporphyrin synthesis is correspondingly reduced. Similarly, when porphyrin synthesis is impaired, excess globin is not produced. There is no such fine regulation of iron uptake with impairment of either protoporphyrin or globin synthesis. When globin production is deficient, iron accumulates in the cytoplasm of cells as ferritin aggregates. Defects of globin synthesis are manifested in the thalassemias. 28 14  6/28/2024 ONTOGENY OF HEMOGLOBIN #1 Embryonic hemoglobins Embryonic hemoglobins are primitive hemoglobins formed by immature erythrocytes in the yolk sac. These hemoglobins include Gower I, Gower II, and Portland types. They are found in the human embryo and persist until approximately 12 weeks of gestation. 29 ONTOGENY OF HEMOGLOBIN #2 Fetal hemoglobin Fetal hemoglobin (hemoglobin F) is the predominant hemoglobin variety in the fetus and the newborn. This hemoglobin type has two alpha and two gamma chains. Fetal hemoglobin appears by the 5th week of gestation and persists for several months after birth. Diminishes to low levels, but constant levels throughout adulthood 30 15  6/28/2024 ONTOGENY OF HEMOGLOBIN #3 Hemoglobin A Although adult hemoglobin is predominantly of the A variety (95% to 97%), the A2 type is also found in small quantities (2% to 3%). Hemoglobin A is made up of two alpha (a) and two beta (b) chains. Hemoglobin A2 is made up of two alpha (a) and two delta (d) chains. A is produced in utero in small concentrations and converts to high concentrations 3 to 6 months after birth. A2 production begins shortly before birth and continues at a slow rate throughout the person’s life. 31 GLYCOSYLATED HEMOGLOBIN (HEMOGLOBIN A1) A subfraction of normal hemoglobin A is hemoglobin A1. This subfraction can be termed glycosylated hemoglobin and includes the separate hemoglobin fractions A1a, A1b, and A1c. Formed during RBC maturation. Proteins are vulnerable to modification after being synthesized by the ribosomes. This modification takes the forms of glycosylation of hemoglobin in patients with hyperglycemia. This is a long-term monitoring tool for diabetes control. Glycosylated hemoglobin is a stable hemoglobin and is structurally the same as hemoglobin A except for the addition of a carbohydrate group at the terminal valine of the beta chain. The concentration of hemoglobin A1 is 3% to 6% in normal persons and 6% to 12% in both insulin- dependent and non–insulin-dependent diabetics. 32 16  6/28/2024 VARIANT FORMS OF NORMAL HEMOGLOBIN Carboxyhemoglobin: Hemoglobin has a higher affinity for carbon monoxide (200×) than oxygen does. This results in oxygen deprivation and tissue necrosis if left untreated. Sulfhemoglobin: The binding of hemoglobin to hydrogen sulfide produces an irreversible change in the polypeptide chains This causes denaturation and precipitation of Heinz bodies Methemoglobin: Hemoglobin with iron in the ferric state, instead of the ferrous state. Results in poor delivery of oxygen. Cyanosis occurs with methemoglobin levels at greater than 10% and hypoxia at greater than 60%. 33 ABNORMAL HEMOGLOBIN MOLECULES Abnormal hemoglobin molecules such as those seen in sickle cell anemia result from mutant, codominant genes. Mutant genes may be homozygous (such as SS in sickle cell disease) or heterozygous (such as SA in Sickle cell trait) Sickle gene may occur with C, E or D giving rise to SC, SE or SD disease. The defective S molecule has the amino acid valine at the 6th position of the beta globin chain instead of glutamic acid 34 17  6/28/2024 ANALYSIS OF HEMOGLOBIN #1 Hemoglobincyanide (cyanmethemoglobin) (Defunct) Alkaline electrophoresis Citrate agar electrophoresis Denaturation procedures Chromatography Molecular testing 35 ANALYSIS OF HEMOGLOBIN #2 Alkaline electrophoresis Screening procedure that separates Hgbs A, F, S, and C. Principle: Hemoglobin molecules in an alkaline solution have a net negative charge and move toward the anode Fast hemoglobins are those that move furthest from the point of application. Include Hgb A, F, Bart’s, H, and I Slow hemoglobins are those that move close to the application point. Include Hgb C, E, O, and A2 36 18  6/28/2024 ANALYSIS OF HEMOGLOBIN #3 Citrate agar electrophoresis In this method, hemoglobins are separated on the basis of a complex interaction between hemoglobin, agar, and citrate buffer ions. Citrate agar separates hemoglobin fractions that migrate together on cellulose acetate—hemoglobins S, D, G, C, E, and O. Due to similar migration patterns on cellulose acetate, all hemoglobin specimens that show an abnormal electrophoretic pattern in alkaline media should undergo electrophoresis on acid citrate agar. 37 ANALYSIS OF HEMOGLOBIN #6 A procedure commonly used to determine the amount of fetal blood that has mixed with maternal blood following delivery is the Kleihauer-Betke. This is a denaturation test because we expose the specimen that denatures adult hemoglobin. Fetal hemoglobin (F) is resistant to acid lysis and therefore stays intact and stains pink with safranin. Adult hemoglobin (A) is susceptible to acid lysis and therefore will be faintly colored. The number of fetal cells to adult cells is counted and a percentage is determined, which is compared to a reference range. Clinical significance: Sensitization for HDN occurs when there is too much intermingling of fetal and maternal blood. 38 19  6/28/2024 ANALYSIS OF HEMOGLOBIN #8 Chromatography Quantitation of hemoglobin A1 can be accomplished by cation exchange minicolumn chromatography. The results of this technique can be affected by several types of hemoglobin in addition to hemoglobin A1. Cellulose acetate and citrate agar electrophoresis should be used in conjunction with cation exchange chromatography to eliminate the possibility of interference by hemoglobin variants. Other assay methods for glycosylated hemoglobin include high-pressure liquid chromatography (HPLC) and colorimetric methods. 39 ANALYSIS OF HEMOGLOBIN #9 Molecular testing Gene deletions and mutations causing thalassemias and hemoglobinopathies can be identified using molecular testing. Since hemoglobin (particularly the globin component) is maintained under genetic control, determining the genetic influence of hemoglobinopathies and thalassemias is beneficial. 40 20  6/28/2024 CATABOLISM OF ERYTHROCYTES As an erythrocyte ages, the following processes occur: The membrane becomes less flexible The concentration of cellular hemoglobin increases Enzyme activity, particularly glycolysis, diminishes 41 EXTRAVASCULAR CATABOLISM OF ERYTHROCYTES #1 When an erythrocyte is phagocytized and digested by the macrophages of the spleen, the hemoglobin molecule is disassembled. The resulting components are as follows: Iron Protoporphyrin Globin 42 21  6/28/2024 EXTRAVASCULAR CATABOLISM OF ERYTHROCYTES #2 Iron is transported in the plasma by transferrin to be recycled by the red bone marrow in the manufacture of new hemoglobin. Globin is catabolized in the liver into its constituent amino acids and enters the circulating amino acid pool. 43 INTRAVASCULAR CATABOLISM OF ERYTHROCYTES #1 Intravascular destruction is an alternate pathway for erythrocyte breakdown. This process normally accounts for less than 10% of erythrocytic destruction. As the result of intravascular destruction, hemoglobin is released directly into the bloodstream and undergoes dissociation into alpha and beta dimers, which are quickly bound to the plasma globulin haptoglobin. 44 22  6/28/2024 INTRAVASCULAR CATABOLISM OF ERYTHROCYTES #2 Haptoglobin binds to the alpha and beta dimers forming a haptoglobin-hemoglobin complex that prevents urinary excretion of plasma hemoglobin. The complex is removed by the liver and processed. Thus, in active intravascular hemolysis, the haptoglobin levels in plasma are low. Once the haptoglobin is consumed, the alpha and beta dimers are rapidly filtered by the glomeruli in the kidneys, reabsorbed and converted to hemosiderin. 45 INTRAVASCULAR CATABOLISM OF ERYTHROCYTES #3 The renal tubular uptake capacity is approximately 5 g per day of filtered hemoglobin Beyond this level, hemoglobin appears in the urine (hemoglobinuria) While in the urinary tract, hemoglobin gets oxidized into two pigments: Oxyhemoglobin (alkaline urine) Methemoglobin (acidic urine) Hemoglobin that isn’t processed by the kidneys nor bound to haptoglobin becomes methemoglobin in circulation Carrier: hemopexin Excess binds to albumin to form methemalbumin 46 23  6/28/2024 POSTAMBLE READ the TEXTBOOK for the details to answer the UNIT OBJECTIVES. USE THE UNIT OBJECTIVES AS A STUDY GUIDE All test questions come from detailed material found in the TEXTBOOK (Not this PowerPoint) and relate back to the Unit Objectives 47 24