ERYTHROPOIESIS (RBC PRODUCTION) • Mature erythrocytes are derived from committed erythroid proginator cells through a series of mitotic divisions and maturation phases. • Erythropoietin, a humoral agent produced mainly by the kidneys stimulates erythropoiesis by acting oncommitted stem cells to induce proliferation and differentiation of erythrocytes in the bone marrow.
ERYTHROPOIESIS • Tissue hypoxia (lack of oxygen) is the main stimulus for erythropoietin production. • Nucleated red cell precursors in the bone marrow are collectivelycalled normoblasts or erythroblasts. • RBCs that have matured to the non-nucleated stage gain entry to the peripheral blood. • Once the cells have lost their nuclei, they are called erythrocytes.
ERYTHROPOIESIS • Young erythrocytes that contain residual RNA are called reticulocytes. • Bone marrow normoblast proliferation and maturation occurs in anorderly and well defined sequence. • The process involves: • a gradual decrease in cell size • condensation and eventual expulsion of the nucleus • an increase in cytoplasm and hemoglobin production
BASIC BLOOD CELL MATURATION • Nearly all hematopoietic cells mature in the manner shown below. For RBCs the nucleus is eventually extruded and the cytoplasm increase correlates with hemoglobin increase.
ERYTHROPOIESIS • For red cell production to be efficient , 85% or more ofthe erythroid activity must have a balanced incorporation of hemeand globin to form hemoglobin. • The immature, nucleated RBC must have an adequate supply of iron‚ as well as normal production of porphyrin and globin polypeptide chains‚ for adequate synthesis ofhemoglobin. • Folic acid and vitamin B12‚ are also needed in adequate amounts to maintain proliferation and differentiation. • Defects may occur at any stage of development and these defects will lead to the death of the cell.
ERYTHROPOIESIS • Normally 1-15% of the RBCs die during maturation. • Ineffective erythropoiesis occurs when, despite an increase in RBC production, there is a failure to deliver the appropriate number of erythrocytes to the peripheralblood. • Normoblasts normally spend 4-7 days proliferating and maturing in the bone marrow. • The stages of maturation from the most immature to the most mature are:
ERYTHROPOIESIS (Metarubricyte) (Rubricyte) (Prorubricyte) (Ribriblast)
ERYTHROPOIESIS • Reticulocytes are released from the bone marrow into the peripheral blood where they mature into erythrocytes , usually within 24 hours. • It is rare to see more than 1% reticulocytes in the peripheral smear from an adult, but common in healthy newborns. • They can be visualized more easily by staining with new methylene blue which allows for visualization of the remnants of the ribosomes on the endoplasmic reticulum.
ERYTHROPOIESIS • Mature RBCs have a lifespan of 100-120 days and senescent RBCs are removed by the spleen. • 3 areas of RBC structure/metabolism are crucial for normal erythrocyte maturation, survival and function: • The RBC membrane • Hemoglobin structure and function • Cellular energetics
ERYTHROPOIESIS • Defects or problems associated with any of these will result in impaired RBC survival. • The RBC must be flexible in order to squeeze through the capillaries of the spleen. • Flexibility is a property of the membrane and the fluidity of the cell’s content. • Any decrease in flexibility results in a decrease in RBC deformability and a decrease in RBC survival in passage through the spleen.
THE RBC MEMBRANE • The RBC membrane is a semi-permeable lipid bilayer supported by a protein cytoskeleton (contains both integral and peripheral proteins). • Since the mature cells lack enzymes and cellular organelles necessary to synthesize new lipid or protein, extensive damage cannot be repaired and the cell will be culled in the spleen.
THE RBC MEMBRANE • The constituents of the RBC membrane include: • Phospholipids- exchange between phospholipids in the membrane and the plasma may occur. • Since the fatty acid content of the diet and the plasma are correlated, changes in the diet may have an effect on the fatty acid composition of the phospholipids in the RBC membrane which can adversely effect the flexibility of the RBC and may result in an RBC with a decreased survival time.
THE RBC MEMBRANE • Cholesterol- membrane cholesterol exists in free equilibrium with plasma cholesterol. • Therefore, an increase in free plasma cholesterol results in an accumulation of cholesterol in the RBC membrane. • RBCs with increased cholesterol appear distorted and the increased cholesterol results in the formation of target cells, and acanthocytes. • An increase in the cholesterol to phospholipid ratio results in a cell membrane that is less deformable and therefore, the RBC has a decreased survival time.
THE RBC MEMBRANE • RBC membrane proteins- 10 major and over 200 minor proteins are asymmetrically organized in the RBC membrane. • Integral proteins- many carry RBC antigens and act as receptors or are transport proteins. Glycophorins are the major integral membrane proteins in the RBC. • Located in the membrane are proteins that function as cationic pumps. • The RBC maintains its volume and water homeostasis by controlling the intracellular concentrations of Na+ and K+ via these cationic pumps which require ATP. • ATP is also required in the Ca++ pump system that prevents excessive intracellular build-up of Ca++. • In ATP depleted cells there is an intracellular build-up of Na+ and Ca++ and a loss of K+ and water. This leads to dehydrated, rigid cells that are culled by the spleen.
THE RBC MEMBRANE • Any abnormality that increases membrane permeability or alters cationic transport may lead to decreased RBC survival. • The major peripheral protein is spectrin and it binds with other peripheral proteins such as actin to form a skeleton of microfilaments on the inner surface of the membrane. This strengthens the membrane and gives it its elastic properties. • For spectrin to participate in this interaction, it must be phosphorylated by a protein kinase that requires ATP. • Thus, a decrease in ATP leads to decreased phosphorylation of spectrin. • Unphosphorylated spectrin can no longer bind to actin to give the membrane its elastic properties. • This then leads to a loss in membrane deformability and a decreased RBC survival time.
HEMOGLOBIN STRUCTURE AND FUNCTION • Hemoglobin occupies 33% of the RBC volume and 90-95% of the dry weight. • 65% of the hemoglobin synthesis occurs in the nucleated stages of RBC maturation and 35% during the reticulocyte stage. • Normal hemoglobin consists of 4 heme groups, which contain a protoporphyrin ring plus iron, and globin, which is a tetramer of 2 pairs of polypeptide chains.
HEMOGLOBIN STRUCTURE AND FUNCTION • Normal hemoglobin production is dependent upon 3 processes: Adequate iron delivery and supply, adequate synthesis of protoporphyrins and adequate globin synthesis. • Iron delivery and supply: • Iron is delivered to the RBC precursor by transferrin. It goes to the mitochondria where it is inserted into protoporphyrin to form heme. • Synthesis of protoporphyrin: • Begins in the mitochondria where glycine + succinyl CoA delta aminolevulenic acid ( ALA). This is the rate limiting step. • In the cytoplasm 2 ALA prophobilinogen (PBG)
HEMOGLOBIN STRUCTURE AND FUNCTION • 4 prophobilinogen (PBG) uroporphyrinogen I and III (UPG I and III). Only type III is used. Type I represents a dead-end pathway. PBG deaminase and UPG cosynthase are both required for UPG III synthesis. UPG I synthesis requires only PBG deaminase. In the absence of UPG cosynthase large amounts of UPG I accumulate in the RBCs , bone marrow, and urine causing a condition called congenital erythropoietic porphyria (more on this later). • Decarboxylation of UPG III coproporphyrinogen III (CPG III). This moves to the mitochondria. • In the mitochondria CPG III protoporphyrin IX • Fe is added to form ferroprotoporphyrin IX= HEME
HEMOGLOBIN STRUCTURE AND FUNCTION • Since porphyrinogens are readily oxidized to form porphyrins, excess formation of porphyrins can occur if any of the normal enzymatic steps in heme synthesis is blocked. Metabolic disorders in which this occurs are called porphyrias. There are 2 categories of porphyrias: inherited and acquired • Inherited Erythropoietic porphyria - results from excessive production of porphyrins in the bone marrow. Hepatic porphyria - results from excessive production of porphyrins in the liver. • Acquired Lead intoxication - interferes with protoporphyrin synthesis Chronic alcoholic liver disease
HEMOGLOBIN STRUCTURE AND FUNCTION • Globin Synthesis • In the yolk sac, the embryonic hemoglobins epsilon and zeta are produced. • In the fetus and the adult, 4 types of hemoglobin chains may be formed: alpha ( α), beta (β ), gamma ( γ), and delta ( δ). • Normal hemoglobin's contain 4 globin chains. • Hemoglobin (hgb) F= α2 γ2 and is the predominant hgb formed during liver and bone marrow erythropoiesis in the fetus. A normal, full term baby has 50-85% hgbF. • Near the end of the first year of life, normal adult hgb levels are reached. All adult normal hgbs are formed as tetramers containing 2 α chains + 2 non-α chains. Normal adult RBCs contain:
HEMOGLOBIN STRUCTURE AND FUNCTION • 92-95% hgb A=α2β2 • 3-5% hgb Ac= glycosylated α2β2 • 2-3% hgb A2= α2δ2 • 1-2% hgb F (fetal hgb)= α2γ2 • Each globin chain links with heme to form hgb= 4 globin + 4 heme. • The precise order of the amino acids is critical for hgb structure and function. • An adequate amount of globin synthesis is also important. • A decreased production in 1 chain results in thalassemia (discussed later).
HEMOGLOBIN STRUCTURE AND FUNCTION • Hemoglobin formation is regulated by several mechanisms: • The regulation of globin chain synthesis. The rate of globin synthesis is directly related to the rate of heme synthesis because heme stimulates globin synthesis by inactivating an inhibitor of globin translation. • Negative feedback of heme. High concentrations of heme prevent the mitochondrial import of the first enzyme in heme synthesis, ALA synthase ( ALAS). • The concentration of iron. An iron responsive element-binding protein (IRE-BP) binds to mRNA iron response elements (IRE) to affect the translation of the mRNA for ALAS, ferritin (discussed later), and transferrin receptors (discussed later).
HEMOGLOBIN STRUCTURE AND FUNCTION • The affinity of IRE-BP for IRE is determined by the amount of cellular iron. • When iron levels are low, there is a high binding affinity which acts to inhibit the translation of ALAS mRNA resulting in a decrease in heme synthesis. • When iron levels are sufficient, the binding affinity is low, thus allowing translation of ALAS mRNA and stimulation of heme synthesis.
HOW IRON LEVELS AFFECT HEME SYNTHESIS IRE-BP Sufficient cellular iron Depleted cellular iron IRE-BP has low affinity for RNA binding IRE-BP has a high affinity for RNA binding Translation of ferritinmRNA Decreased stability of transferrin receptor mRNA Translation of ALAS mRNA Inhibits translation of ferritin RNA Stabilizes transferrin receptor mRNA Inhibits translation of ALAS Increased ferritin Decreased transferrin receptors Heme synthesis stimulated Decreased ferritin Increased transferrin receptors Heme synthesis decreased
HEMOGLOBIN STRUCTURE AND FUNCTION • If either heme or globin synthesis is impaired, iron accumulates in the RBC. • An RBC with accumlated iron is then called a siderocyte and the iron can be visualized using a Prussian blue stain. • When protoporphyrin synthesis is impaired, mitochondria become encrusted with iron. • This is visible as a ring around the nucleus of the RBC precursor when stained with Prussian blue. • A precursor cell with a ring of iron around the nucleus is called a ringed sideroblast.
HEMOGLOBIN STRUCTURE AND FUNCTION • Hemoglobin function • The primary function of hgb is gas transport. • The hgb molecule is capable of a considerable amount of allosteric movement as it loads and unloads O2. • This is due to the multichain structure of the molecule. • In unloading of O2, the space between the chains widens and 2,3 diphosphoglycerate (DPG) binds. • This is the T (tense) form of hgb and it is called deoxyhgb. • It has a lower affinity for O2, so O2 unloads from the hbg. • When hgb loads O2, the chains are pulled together, expelling 2,3 DPG. • This is the R (relaxed) form of hgb and it is called oxyhgb. • It has a higher affinity for O2, so O2 binds to or loads onto the hgb.
HEMOGLOBIN STRUCTURE AND FUNCTION • Binding and dissociation of O2 are not directly proportional to the O2 concentration. Note the hgb-O2 dissociation curve below:
HEMOGLOBIN STRUCTURE AND FUNCTION • The sigmoid shape of the curve shows that a significant amount of O2 delivery will occur with a small drop in O2 tension. • O2 affinity of hgb is expressed as the partial (P) O2 pressure (in mm Hg) at which hgb is 50% saturated with O2. • Increased O2 affinity means that hgb does not readily give up itsO2. • Decreased O2 affinity means that hgb releases the O2 more readily. • Normally the partial O2 pressure in the lungs is 100 mm. and the hgb is 100 saturated with O2. • In tissues the partial pressure is 40mm. and the hgb is 75% saturated with O2. • Therefore 25% of the O2 is delivered to the tissues
HEMOGLOBIN STRUCTURE AND FUNCTION • In hypoxia there is a compensatory shift to the right in the dissociation curve. • This is mediated by an increase in 2,3 DPG and results in decreased hgb affinity for O2 and increased O2 delivery to the tissues. • Therefore the RBCs are more efficient in O2 delivery. • A patient suffering from anemia due to blood loss may compensate by shifting the O2 dissociation curve to the right. • A shift to the right also occurs in acidosis and when the body temperature is increased.
HEMOGLOBIN STRUCTURE AND FUNCTION • A shift to the left in the O2 dissociation curve results in decreased O2 delivery to the tissues. This occurs: • In alkalosis • When there are increased quantities of abnormal hemoglobins such as methgb and carboxyhgb • When there is an increase in hgb F which has a higher affinity for O2 than does hgb A or • When a patient has received multiple transfusions with 2,3 DPG depleted blood.
COMPARISON OF AN O2 DISSOCIATION CURVE AT NORMAL PH AND WITH ACIDOSIS OR ALKALOSIS
HEMOGLOBIN STRUCTURE AND FUNCTION • Inherited abnormalities in hgb may result in either type of shift and can have profound effects on the RBCs ability to provide the tissues with O2. • Acquired abnormal hgbs of clinical importance are those that have been altered post- translationally to produce hgbs that are unable to transport or deliver O2 and they include: • Carboxyhgb - CO replaces O2 and binds 200X tighter than O2. • This may be seen with heavy smokers • This may be reversed with high concentrations of O2 • Methgb - occurs when iron is oxidized to the +3 (ferric) state. In order for hgb to carry O2 the iron must be in the +2 (ferrous) state. In the body, normally~ 2% is formed and reducing systems prevent an increase beyond 2%. • Increases above 2% can occur with the ingestion of strong oxidant drugs or • As a result of enzyme deficiency.
HEMOGLOBIN STRUCTURE AND FUNCTION • Methgb can be reduced by treatment with methylene blue or ascorbic acid. • Sulfhgb - occurs when the sulfur content of the blood increases due to ingestion of sulfur containing drugs or to chronic constipation. • Unlike the formation of carboxyhgb and methgb, the formation of sulfhgb is an irreversible change of hgb.
CELLULAR ENERGETICS • Maintenance of hgb function requires active RBC metabolic pathways for ATP production. ATP is required for: • Maintaining hgb in the reduced form • Membrane integrity and deformability • Maintaining the RBC intracellular volume • Producing adequate amounts of NADH, NADPH, and GSH • RBCs generate energy almost exclusively from the anaerobic breakdown of glucose - 4 metabolic pathways are important for maintaining cellular energetics.