Hemoglobin Metabolism - MIDTERM EXAM PPT PDF

Hemoglobin Metabolism Mary Rose M. Apuyan, RMT, DTA  Hemoglobin Structure First protein whose structure was described using x-ray crystallography. The hemoglobin molecule is a globular protein consisting of two different pairs of polypeptide chains and four heme groups, with on...

Hemoglobin Metabolism Mary Rose M. Apuyan, RMT, DTA  Hemoglobin Structure First protein whose structure was described using x-ray crystallography. The hemoglobin molecule is a globular protein consisting of two different pairs of polypeptide chains and four heme groups, with one heme group imbedded in each of the four polypeptide chains. Hemoglobin is an oxygen-transporting protein contained within erythrocytes. The heme portion of hemoglobin gives erythrocytes their characteristic red color.  Heme Structure Consists of a ring of carbon, hydrogen, and nitrogen atoms called protoporphyrin IX, with a central atom of divalent ferrous iron (Fe2+). Each of the four heme groups is positioned in a pocket of the polypeptide chain near the surface of the hemoglobin molecule. The ferrous iron in each heme molecule reversibly combines with one oxygen molecule. When the ferrous irons are oxidized to the ferric state (Fe3+), they no longer can bind oxygen. Methemoglobin = oxidized hemoglobin  Globin Structure The four globin chains comprising each hemoglobin molecule consist of two identical pairs of unlike polypeptide chains, 141 to 146 amino acids each. Variations in amino acid sequences give rise to different types of polypeptide chains. Each chain is designated by a Greek letter.  Globin Structure Each globin chain is divided into eight helices separated by seven non-helical segments. The helices, designated A to H, contain subgroup numberings for the sequence of the amino acids in each helix and are relatively rigid and linear. Flexible nonhelical segments connect the helices, as reflected by their designations: NA for the sequence between the N-terminus and the A helix, AB between the A and B helices, and so forth, with BC, CD, DE, EF, FG, GH, and finally HC between the H helix and the C-terminus.  Hemoglobin Molecule Primary structure- amino acid sequence of the polypeptide chains. Secondary structure - chain arrangements in helices and non- helices. Tertiary structure - arrangement of the helices into a pretzel-like configuration. Globin chains loop to form a cleft pocket for heme. Each chain contains a heme group that is suspended between the E and F helices of the polypeptide chain.  Hemoglobin Molecule Globin chain amino acids in the cleft are hydrophobic, whereas amino acids on the outside are hydrophilic, which renders the molecule water soluble. This arrangement also helps iron remain in its divalent ferrous form regardless of whether it is oxygenated (carrying an oxygen molecule) or deoxygenated (not carrying an oxygen molecule). Quaternary structure - a tetramer, the complete hemoglobin molecule. The complete hemoglobin molecule is spherical, has four heme groups attached to four polypeptide chains, and may carry up to four molecules of oxygen.  Hemoglobin Biosynthesis 65% hemoglobin synthesis occurs in immature nRBCs. 35% hemoglobin synthesis occurs in reticulocytes. Heme synthesis occurs in the mitochondria of normoblasts and is dependent on glycine, succinyl coenzyme A, aminolevulinic acid synthetase, and vitamin B6 (pyridoxine). Globin synthesis occurs in the ribosomes, and it is controlled on chromosome 16 for alpha chains and chromosome 11 for all other chains. Each globin chain binds to a heme molecule in the cytoplasm of the immature RBC.  Heme Synthesis Begins in the mitochondria with the condensation of glycine and succinyl coenzyme A (CoA) catalyzed by aminolevulinate synthase to form aminolevulinic acid (ALA). In the cytoplasm, aminolevulinic acid dehydratase (also known as porphobilinogen synthase) converts ALA to porphobilinogen (PBG). PBG undergoes several transformations in the cytoplasm from hydroxylmethylbilane to coproporphyrinogen III. This pathway then continues in the mitochondria until, in the final step of production of heme, Fe2+ combines with protoporphyrin IX in the presence of ferrochelatase (heme synthase) to make heme.  Heme Synthesis Transferrin - a plasma protein, carries iron in the ferric form to developing erythroid cells; binds to transferrin receptors on erythroid precursor cell membranes and the receptors and transferrin (with bound iron) are brought into the cell in an endosome. Acidification of the endosome releases the iron from transferrin.  Heme Synthesis Iron is transported out of the endosome and into the mitochondria where it is reduced to the ferrous state, and is united with protoporphyrin IX to make heme. Heme leaves the mitochondria and is joined to the globin chains in the cytoplasm.  Globin Synthesis The production of globin chains takes place in erythroid precursors from the pronormoblast through the circulating polychromatic erythrocyte, but not in the mature erythrocyte. Transcription of the globin genes to messenger ribonucleic acid (mRNA) occurs in the nucleus, and translation of mRNA to the globin polypeptide chain occurs on ribosomes in the cytoplasm. After translation is complete, the chains are released from the ribosomes in the cytoplasm.  Hemoglobin Ontogeny The z- and e-globin chains normally appear only during the first 3 months of embryonic development. These two chains, when paired with the a and g chains, form the embryonic hemoglobins. During the second and third trimesters of fetal life and at birth, Hb F (a2g2) is the predominant hemoglobin. By 6 months of age and through adulthood, Hb A (a2b2) is the predominant hemoglobin, with small amounts of Hb A2 (a2d2) and Hb F.  Types of Hemoglobin Fetal Hemoglobin - Hgb F contains two alpha- and two gamma-globin chains. - Hgb F functions in a reduced oxygen environment. - Hgb F predominates at birth (80%). Gamma chain production switches over to beta chain production and is complete by 6 months of age. a. Laboratory: Alkali denaturation test and Kleihauer-Betke acid elution stain (Hgb F is resistant to denaturation/elution), column chromatography, radial immunodiffusion b. Hgb F is a compensatory hemoglobin and can be increased in homozygous hemoglobinopathies and beta-thalassemia major.  Types of Hemoglobin Adult a. Hgb A contains two alpha- and two beta-globin chains. 1) Hgb A is subdivided into glycosylated fractions. 2) Alc fraction reflects glucose levels in the blood and is used to monitor individuals with diabetes mellitus. b. Hgb A2 contains two alpha- and two delta-globin chains. c. Reference range for a normal adult is 97% Hb A, 2% Hb A2, and 1% Hb F.  Types of Hemoglobin Embryonic Hemoglobin Gower 1 – composed of 2 zeta & 2 epsilon chains Gower 2 – composed of 2 alpha & 2 epsilon chains Portland – composed of 2 alpha & 2 gamma chains  Hemoglobin/Erythrocyte Breakdown 1. Intravascular hemolysis (10%) a. Occurs when hemoglobin breaks down in the blood and free hemoglobin is released into plasma b. Free hemoglobin binds to haptoglobin (major free hemoglobin transport protein), hemopexin, and albumin, and it is phagocytized by liver macrophages. c. Laboratory: Increased plasma hemoglobin, serum bilirubin, serum LD, and urine urobilinogen; hemoglobinuria and hemosiderinuria present; decreased serum haptoglobin  Intravascular Hemolysis Hemoglobin, hematocrit, and red cell count are low. Serum bilirubin is elevated. Serum haptoglobin is low. Hemoglobinemia (free hemoglobin in plasma) may be present. Hemoglobinuria (hemoglobin in urine) may be present. Reticulocyte count is elevated. Lactate dehydrogenase (LDH) is elevated.  Hemoglobin/Erythrocyte Breakdown 2. Extravascular hemolysis (90%) a. Occurs when senescent/old RBCs are phagocytized by macrophages in the liver or spleen b. Protoporphyrin ring metabolized to bilirubin and urobilinogen; excreted in urine and feces c. Globin chains are recycled into the amino acid pool for protein synthesis. d. Iron binds to transferrin and is transported to bone marrow for new RBC production, or it is stored for future use in the form of ferritin or hemosiderin.  Extravascular Hemolysis Hemoglobin, hematocrit, and red cell count are low. Serum bilirubin is elevated. Serum haptoglobin is low. Hemoglobinemia (free hemoglobin in plasma) may be present. Hemoglobinuria (hemoglobin in urine) may be present. Reticulocyte count is elevated. Lactate dehydrogenase (LDH) is elevated.  Hemoglobin and Iron 1. Most iron in the body is in hemoglobin and must be in the ferrous state to be used. Fe2+ binds to oxygen for transport to lungs and body tissues. 2. Ferric iron (Fe3+) is not able to bind to hemoglobin, but does bind to transferrin. Iron is an essential mineral and is not produced by the body. a. Serum iron measures the amount of Fe3+ bound to transferrin. b. Total iron-binding capacity (TIBC) measures the total amount of iron that transferrin can bind when fully saturated. c. Serum ferritin is an indirect measurement of storage iron in tissues and bone marrow.  Different Forms of Normal Hemoglobin Oxyhemoglobin: Hemoglobin with Fe2+ + O2; seen in arterial circulation Deoxyhemoglobin: Hemoglobin with Fe2+ but no O2; seen in venous circulation Carboxyhemoglobin: Hemoglobin with Fe2+ and carbon monoxide (CO); hemoglobin has 200 X more affinity for CO than O2 so CO is earned instead of O2; can result in death, but is reversible if given pure O2  Different Forms of Normal Hemoglobin Sulfhemoglobin: Hemoglobin with S; cannot transport O2; seldom reaches fatal levels; caused by drugs and chemicals; irreversible, not measured by the cyanmethemoglobin method Methemoglobin: Hemoglobin with Fe3+; cannot transport O2; increased levels cause cyanosis and anemia  Oxygen Dissociation Curve 1. Oxygen affinity is the ability of hemoglobin to bind or release oxygen. Expressed in terms of the oxygen tension at which hemoglobin is 50% saturated with oxygen. 2. The relationship between oxygen tension and hemoglobin saturation with oxygen is described by the oxygen dissociation curve.  Oxygen Dissociation Curve a. Right shift decreases oxygen affinity, more O2 release to the tissues— high 2,3-bisphosphoglycerate (formerly 2,3-diphosphoglycerate/2,3-DPG) level or increased body temperature; decreased body pH b. Left shift increases oxygen affinity, less O2 release to the tissues—low 2,3-bisphosphoglycerate (2,3-BPG) level or decreased body temperature; increased body pH Disorders related to Hemoglobin Biosynthesis  Disorders of Heme (Porphyrin) Synthesis Inherited defects include a rare autosomal recessive condition, congenital erythropoietic porphyria. Acquired defects include lead poisoning, which inhibits heme synthesis at several points. - In this defect, inhibition of several enzymes, including heme synthetase, impairs synthesis reactions at several points, including ALA to PBG and protoporphyrin to heme. Porphyria Defined as a disease of heme metabolism in which a primary abnormality in porphyrin biosynthesis leads to excessive accumulation and excretion of porphyrins or their precursors by the biliary and/or renal route. 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  Porphyria Clinically, patients with porphyria have either neurological complications or skin problems - Acute neurologic - Nonacute cutaneous Some patients have no symptoms. Porphyria Derived from the Greek word, porphyra, which means purple. The purple-red pigment (porphyrins) is responsible for the wine-red color characteristic of porphyric urine. PBG is normally excreted in small amounts in urine; however, it appears in significantly elevated amounts in acute intermittent porphyria, which may be detected by testing the urine with Ehrlich’s aldehyde reagent. Porphyria When porphyrin synthesis is impaired, the mitochondria become encrusted with iron, and some granules exist around the nucleus erythrocyte are visible if the cell is stained with a special stain, Prussian blue stain. The cells are referred to as sideroblasts.  Disorders of Iron Metabolism Genetic Defect of Iron Genetic variations affect iron plasma concentrations in persons not affected by overt genetic disorders of iron metabolism. Transmembrane protease serine 6 (TMPRSS6) gene - an enzyme that promotes iron absorption and recycling by inhibiting hepcidin antimicrobial peptide transcription. The allele associated with lower iron concentrations was also associated with lower hemoglobin levels, smaller red cells, and high red blood cell distribution width (RDW). An association of TMPRSS6 variants with iron level has been established as anemia-related phenotypes.  Disorders of Iron Metabolism Iron Overload Primary overload disorders - iron absorption from a normal diet is increased due to inherited alteration in factors that control iron uptake and retention. Secondary iron overload - may arise in patients with chronic disorders or erythropoiesis or hemolytic anemias. Ex: Sideroblastic anemia as a consequent of iron therapy, due to excessive dietary or supplement ingestion of iron or from multiple RBC transfusions.  Disorders of Iron Metabolism Sideroblastic Anemia Excess iron accumulates as ferritin aggregates in the cytoplasm of immature erythrocytes. The amount of nonheme iron deposited depends on the ratio between the plasma iron level and the iron required by the cell. Associated with mitochondrial iron loading in marrow erythroid precursors (ringed sideroblasts) and ineffective erythropoiesis.  Disorders of Iron Metabolism Causes of sideroblastic anemia include 1. Congenital defect: hereditary sex linked (primarily males); autosomal 2. Acquired defect: primary (one of the myelodysplastic syndromes); may evolve into acute myelogenous leukemia 3. Association with malignant marrow disorders: acute myelogenous leukemia, polycythemia vera, myeloma, myelodysplastic syndromes 4. Secondary to drugs: isoniazid (INH), chloramphenicol; after chemotherapy 5. Toxins, including alcohol, and chronic lead poisoning Erythrocyte Production & Destruction Mary Rose M. Apuyan, RMT, DTA  General Characteristics of Erythrocyte Biconcave disk with a central pallor that occupies the middle one-third of the cell. In the mature cell, the respiratory protein, hemoglobin, performs the function of oxygen–carbon dioxide transport. As the cell ages, cytoplasmic enzymes are catabolized, leading to increased membrane rigidity (density), phagocytosis, and destruction.  General Characteristics of Erythrocyte 1. Oxygen transport, removal of metabolic waste 2. Loss of nucleus is required for function. 3. Normal life span is 120 days. Moves to the tissue capillaries and splenic circulation.  Erythropoiesis Process of red cell production Encompasses differentiation from the hematopoietic stem cell (HSC) through the mature erythrocyte. Epitomizes highly specialized cellular differentiation and gene expression. Regulated partially by the combined actions of cytokine signaling pathways and transcription factors. As cells progress through the stages of erythropoiesis, their potential to differentiate into lymphoid or other hematopoietic cell types is restricted. They are increasingly committed to differentiate into erythrocytes.  Erythropoietin Produced primarily by the kidneys. Peritubular cells are the probable site of synthesis in the kidneys. Extrarenal organs such as the liver also secrete this substance. 10-15% of erythropoietin production occurs in the liver, which is the primary source of erythropoietin in the unborn.  Erythropoietin Growth factor that stimulates erythrocyte production from myeloid progenitor cell; influences colony-forming unit-erythrocytes (CFU-Es) to differentiate into erythroblasts Erythropoietin - first human hematopoietic growth factor to be identified; Gene is located on chromosome 7  Criteria Used In Identification of Erythroid Precursor Morphologic identification of blood cells depends on a wellstained peripheral blood film or bone marrow smear Wright or Wright-Giemsa- commonly used modified Romanowsky stain The stage of maturation of any blood cell is determined by careful examination of the nucleus and the cytoplasm.  General Trends Affecting the RBC Morphology 1. The overall diameter of the cell decreases. 2. The diameter of the nucleus decreases more rapidly than does the size of the cell. As a result, the N:C ratio also decreases. 3. The nuclear chromatin pattern becomes coarser, clumped, and condensed. 4. Nucleoli disappear.  General Trends Affecting the RBC Morphology 5. The cytoplasm changes from blue to gray-blue to salmon pink. - Blueness or basophilia - due to acidic components that attract the basic stain, such as methylene blue; Correlates with the amount of ribosomal RNA. These organelles decline over the life of the developing RBC, and the blueness fades. - Pinkness (eosinophilia or acidophilia) - due to accumulation of more basic components that attract the acid stain eosin; Correlates with the accumulation of hemoglobin as the cell matures. MATURATION SEQUENCE  PRONORMOBLAST (RUBRIBLAST) a. Earliest RBC, size up to 20 um, with an N:C ratio of 8:1 b. 1-3 nucleoli, nucleus has dark areas of DNA c. Chromatin is fine and uniform, and stains intensely d. Deep blue cytoplasm with no granules  BASOPHILIC NORMOBLAST (PRORUBRICYTE) a. Size up to 16 um with an N:C ratio of 6:1 b. Centrally located nucleus with 0-1 nucleoli c. Chromatin is coarsening. d. Cytoplasm is less blue but intensely basophilic (RNA).  POLYCHROMATOPHILIC NORMOBLAST (RUBRICYTE) a. Size up to 12 um with an N:C ratio of 4:1 b. Eccentric nucleus with no nucleoli c. Chromatin shows significant clumping. d. Begins to produce hemoglobin, resulting in gray-blue cytoplasm  ORTHOCHROMIC NORMOBLAST (METARUBRICYTE) a. Size up to 10 um with an N:C ratio of 0.5:1 b. Eccentric nucleus with small, fully condensed (pyknotic) nucleus; no nucleoli c. Pale blue to salmon cytoplasm d. Hemoglobin synthesis decreases  POLYCHROMATIC ERYTHROCYTE (RETICULOCYTE) a. Size up to 10 um b. A reticulocyte contains no nucleus but has mitochondria and ribosomes. c. Last stage to synthesize hemoglobin d. Last stage in bone marrow before release to the blood e. Reference ranges are 0.5-1.5% for adults and 2.5-6.5% for newborns, with slightly increased ranges at higher altitudes.  POLYCHROMATIC ERYTHROCYTE (RETICULOCYTE) f. A supravital stain is used to enumerate reticulocytes. g. Reticulocyte count is one of the best indicators of bone marrow function, h. Stress reticulocytes are young cells released from bone marrow after older reticulocytes have been released. This is a response to increased need. i. Hemoglobin continues to be produced by reticulocytes for approximately 24 hours after exiting the bone marrow.  MATURE ERYTHROCYTE a. Size range is 6-8 um b. Round, biconcave discocyte c. Salmon with central pallor (clearing in the center) when a blood smear is Wright's stained 1) Normal cells have a central pallor that is one-third the diameter of the cell. 2) Decreased central pallor is seen with spherocytic disorders, including thermal injury and liver disease. 3) Central pallor greater than one-third the diameter of the cell is seen in microcytic anemias.  MATURE ERYTHROCYTE d. RBC reference ranges in SI units: 1) Females 4.0-5.4 X 1012/L (conventional units 4.0-5.4 X 106/ul) 2) Males 4.6-6.0 X 1012/L (conventional units 4.6-6.0 X 106/ul)  MATURE ERYTHROCYTE e. Erythropoiesis is regulated by erythropoietin produced in the kidney. Additional regulation includes: 1) Hypoxia due to high altitudes, heart or lung dysfunction, anemia 2) Androgens (male hormones that appear to enhance the activity of erythropoietin) and hemolytic anemias (increased erythrocyte destruction) ERYTHROCYTE PHYSIOLOGY  Early RBCs get energy from oxidative phosphorylation. During maturation, the mitochondria are lost, and energy is derived from glycolysis. Erythrocytes need proper volume ratio for exchange of blood gases and flexibility to travel through capillaries. Accomplished by the cation pump, a mechanism that keeps sodium outside and potassium inside the cell. Erythrocyte membrane is 50-60% lipid (phospholipids, cholesterol, and glycolipids) and 40-50% protein.  SUBSTANCES NEEDED FOR ERYTHROPOIESIS 1. Iron: Must be in the ferrous state (Fe2+) to transport oxygen 2. Amino acids: Globin-chain synthesis 3. Folic acid/vitamin B12: DNA replication/cell division 4. Others: Erythropoietin, vitamin 65 (pyridoxine), trace minerals ERYTHROCYTE MORPHOLOGY & ASSOCIATED DISEASE  Normocytes (discocytes) are normal erythrocytes that are approximately the same size as the nucleus of a small lymphocyte. Macrocytes a. RBCs greater than 8 um in diameter; MCV greater than 100 fl b. Seen in megaloblastic anemias, such as B12/folate deficiency c. Seen in non-megaloblastic anemia of liver disease or accelerated erythropoiesis; also seen in normal newborns  Microcytes a. RBCs less than 6 um in diameter; MCV less than 80 fL b. Seen in iron-deficiency anemia, thalassemias, sideroblastic anemia, and anemia of chronic disease  ANISOCYTOSIS a. Variation in RBC size, indicating a heterogeneous RBC population (dimorphism) b. Correlates with RDW (red blood cell distribution width), especially when the RDW exceeds 15.0% c. Seen post-transfusion, post-treatment for a deficiency (e.g., iron), presence of two concurrent deficiencies (e.g., iron and vitamin B12), and idiopathic sideroblastic anemia  POIKILOCYTOSIS a. General term to describe variation in shape b. Associated with a variety of pathologic conditions  Echinocytes (include crenated & burr cells) Have short, scalloped, or spike-like projections that are regularly distributed around the cell membrane. Have evenly spaced round projections; central pallor area present The projections can vary in number and appearance. Crenation can occur as the result of the physical loss of intracorpuscular water. Caused by changes in osmotic pressure Seen in liver disease, uremia, heparin therapy, pyruvate kinase deficiency, or as artifact Acanthocytes (spur cells) Have unevenly spaced pointed projections; lack a central pallor area Associated with alcoholic liver disease, post-splenectomy, and abetalipoproteinemia Caused by excessive cholesterol in the membrane Target cells (codocytes or Mexican hat cells) Resemble a shooting target. A central red bull’s-eye is surrounded by a clear ring and then an outer red ring. Show a central area of hemoglobin surrounded by a colorless ring and a peripheral ring of hemoglobin; cells have an increased surface-to-volume ratio Seen in liver disease hemoglobinopathies, thalassemia, iron-deficiency anemia Caused by excessive cholesterol in the membrane or a hemoglobin distribution imbalance Spherocytes Disk-shaped cell with a smaller volume than a normal erythrocyte; cells have a decreased surface-to-volume ratio Lack a central pallor area Associated with defects of the red cell membrane proteins MCHC may be >37%; increased osmotic fragility Damaged RBC; seen in hereditary spherocytosis, G6PD deficiency, and immune hemolytic anemias Microspherocytes (37 g/dL) when using an automated cell counting instrument.