1. Metabolism of Red Blood Cells (RBCs) HMIM224

2. Objectives of the Lecture 1- Understanding the general structural & functional features of red blood cells (RBCs). 2- Recognizing the main metabolic pathways occurring in RBCs with reference to their relations to functions of RBCs. 3- Identifying some of the main & common diseases of RBCs as implication of defects of RBCs metabolism. 4- Understanding the relation of characteristic features of structure of membrane of RBCs. 5- Recognizing changes occurring in aging of RBCs.

3. An Erythrocyte (RBC) Reference Ranges RBCs, male4.3-5.9 x 10 6 /µL female3.5-5.5 x 10 6 /µL Hb, male13.5-17.5 g/dL female12.0-16.0 g/dL Hct, male41-53% female36-46% MCH25.4-34.6 pg MCV80-100 fL MCHC31-36 % Practical Values 65% of Fe in Hb 1 g Hb = 3.46 mg Fe 1 mL blood at 15 g/dL Hb = 0.5 mg Fe RBC x 3 = Hb Hb x 3 = Hct Microcytic < 80 fL Macrocytic > 100 fL

4. not The red blood cells (RBCs) are not true cells. no not RBCs contain no nucleus or nucleic acids, and thus, can not reproduce. no no no nono RBCs contain no cell organelles (as mitochondria, Golgi, ER or lysosomes) and thus possess no synthetic activities (no protein biosynthesis, no lipid synthesis & no carbohydrate synthesis). RBCs must be able to squeeze through some tight spots in microcirculation. For that RBCs must be easily & reversibly deformable Introduction to the Red Blood Cells (RBCs)

5. Red cells contain 35 % solids. Hemoglobin Hemoglobin, the chief protein of the red cells. Other proteins are present in combination with lipids and oligosaccharide chains, forming the stroma and cell membrane. Potassiummagnesiumzinc Potassium, magnesium, and zinc concentrations in red cells are much higher than in the plasma. Biochemical composition of the RBCs

6. Hemoglobin

7. Functions of RBCs simple functions RBCs have relatively simple functions as they have much simpler structure than most human cells. The major functions of RBCs are delivering oxygen to the tissues & disposal of carbon dioxide & protons formed by tissue metabolism. hemoglobin This function is carried out by hemoglobin.

8. Introduction Introduction: no no no no RBCs contain no mitochondria, so there is no respiratory chain, no citric acid cycle, and no oxidation of fatty acids or ketone bodies. ONLY Energy in the form of ATP is obtained ONLY from the glycolytic breakdown of glucose with the production of lactate (anaerobic glycolysis). ATP ATP produced being used for keeping the biconcave shape of RBCs & in the regulation of transport of ions & water in and out of RBCs. Metabolism of RBCs

9. Metabolism of RBCs Metabolism of RBCs (cont.) Glucose transport through RBC membrane 1- Glucose transport through RBC membrane: Glucose Glucose is transported through RBC membrane glucose by a GLUT-1 facilitated diffusion by glucose transporters (GLUT-1). independent Glucose transporters (GLUT-1) are independent on insulin i.e. insulin does not promote glucose transport to RBCS

10. RBC Metabolic Pathways 2,3-BPG BPG mutase 2,3-BPG phosphatase PPP NADPH 6PG 3-7 C metabolites (R5P, F6P, G3P) G6PDH lactonase 6PGDH CO 2 NADP + + H + GSH GSSG GR GP H2O2H2O2 H2OH2O Glc Pyr G6P 1,3-BPG 3PG HK PGI PK F6P G3P PFK aldolase F16BP DHAP 2PG PEP PGK PGM enolase G3PDH Glycolysis Lactate No O 2 LDH

11. Glycolysis 2- Glycolysis : anaerobic glycolysis Glucose is metabolized in RBCs through anaerobic glycolysis no no (that requires no mitochondria and no oxygen). 2 molecules of ATP One molecule of glucose yields 2 molecules of ATP by one anaerobic glycolytic pathway. 2 molecules of lactate In addition, 2 molecules of lactate are produced. Lactate Lactate is transported to blood & in the liver it is converted to glucose. Metabolism of RBCs Metabolism of RBCs (cont.)

13. Genetic defects in enzymes of glycolysis Genetic defects in enzymes of glycolysis : Genetic defects of one of the enzymes of glycolysis in RBCs results in a reduced rate of only glycolysis in RBCs & by this way will deprive RBCs of the only means for producing energy. hemolytic anemia As a result, hemolytic anemia will be a consequence as RBCs will not be able to keep the biconcave flexible shape which allows it to squeeze through narrow capillaries with an end result of hemolysis (destruction of RBCs). pyruvate kinase 95% of cases of genetic defects in glycolytic enzymes is caused by pyruvate kinase deficiency deficiency. phosphoglucose isomerase deficiency 4% is caused by phosphoglucose isomerase deficiency. Metabolism of RBCs Metabolism of RBCs (cont.)

14. Production of 2,3 bisphosphoglycerate (2, 3 BPG) 3- Production of 2,3 bisphosphoglycerate (2, 3 BPG): In RBCs, some of glycolysis pathways are modified so that, 3 bisphosphoglycerate 2, 3 bisphosphoglycerate is formed (by bisphosphoglycerate mutase). 2, 3 bisphosphoglycerate 2, 3 bisphosphoglycerate decreases affinity of HB for oxygen. So, it helps oxyhemoglobin to unload oxygen. Storing Storing blood results in decrease of 2,3-BPG leading to high oxygen affinity Hb. This leads to oxygen trap. 6-24 hours are needed to restore the depleted 2,3 BPG Maximum storage time for RBCs is 21-42 days Production of 2,3 bisphosphoglycerate (2, 3 BPG) 3- Production of 2,3 bisphosphoglycerate (2, 3 BPG): In RBCs, some of glycolysis pathways are modified so that, 3 bisphosphoglycerate 2, 3 bisphosphoglycerate is formed (by bisphosphoglycerate mutase). 2, 3 bisphosphoglycerate 2, 3 bisphosphoglycerate decreases affinity of HB for oxygen. So, it helps oxyhemoglobin to unload oxygen. Storing Storing blood results in decrease of 2,3-BPG leading to high oxygen affinity Hb. This leads to oxygen trap. 6-24 hours are needed to restore the depleted 2,3 BPG Maximum storage time for RBCs is 21-42 days Metabolism of RBCs Metabolism of RBCs (cont.)

15. Pentose phosphate pathway 4- Pentose phosphate pathway : NADPH (PPP is the only source for NADPH in RBCs) RBCs contain an active pentose phosphate pathway (PPP) for glucose that supplies NADPH (PPP is the only source for NADPH in RBCs) NADPHglutathione NADPH is important in keeping glutathione in the reduced glutathione. Reduced glutathione Reduced glutathione plays a very important role in the survival of the red blood cells. (prevents oxidation of membrane) Glucose 6- phosphate dehydrogenase deficiency (G6PD Deficiency Glucose 6- phosphate dehydrogenase deficiency (G6PD Deficiency): Glucose 6-phosphate dehydrogenase is the first enzyme of pentose phosphate pathway & its deficiency leads to reduced production of NADPH ending in acute hemolytic anemia. Metabolism of RBCs Metabolism of RBCs (cont.)

16. RBC Metabolic Pathways 2,3-BPG BPG mutase 2,3-BPG phosphatase PPP NADPH 6PG 3-7 C metabolites (R5P, F6P, G3P) G6PDH lactonase 6PGDH CO 2 NADP + + H + GSH GSSG GR GP H2O2H2O2 H2OH2O Glc Pyr G6P 1,3-BPG 3PG HK PGI PK F6P G3P PFK aldolase F16BP DHAP 2PG PEP PGK PGM enolase G3PDH Glycolysis Lactate No O 2 LDH

18. carbonicanhydrase The erythrocytes contain carbonic anhydrase Carbon dioxide combines with water only after it enters the red cells where hemoglobin, the most important buffer for the resulting carbonic acid, is present. CO 2 + H 2 O  HCO 3 - + H + CO 2 + H 2 O  HCO 3 - + H + rhodanese The red cell also contain rhodanese enzyme responsible for the detoxication of cyanides. Metabolism of RBCs Metabolism of RBCs (cont.)

19. RBCs must be able to squeeze through some tight spots in microcirculation (capillaries). For that RBCs must be easily & reversibly deformable. fluidflexible Its membrane must be both fluid & flexible. About 50% of membrane is protein, 40% is fat & up to 10% is carbohydrate. lipid bilayer fluidityproteinsflexibility carbohydrates RBCs membrane comprises a lipid bilayer (which determine the membrane fluidity), proteins (which is responsible for flexibility) that are either peripheral or integral penetrating the lipid bilayer & carbohydrates that occur only on the external surface. RBCs membrane structure

21. spectrin ankyrinprotein 4.1 actin The membrane skeleton is four structural proteins that include  &  spectrin, ankyrin, protein 4.1 & actin. Spectrin Spectrin is major protein of the cytoskeleton & its two chains (  &  ) are aligned in an antiparallel manner  &  chains are loosely interconnected forming a dimer, one dimer interact with another, forming a head to head tetramer. Ankyrin spectrin band 3 Ankyrin binds spectrin & in turn binds tightly to band 3 securing attachment of spectrin to membrane. Band 3 is anion exchange protein permits exchanges of Cl - for HCO 3 +. Actin spectrin protein 4.1 Actin binds to the tail of spectrin & to protein 4.1 which in turn binds to integral proteins, glycophorins A, B & C. glycoproteins Glycophorins A,B,C are transmembrane glycoproteins; Defects of proteins may explain some of the abnormalities of shape of RBCs membrane as Defects of proteins may explain some of the abnormalities of shape of RBCs membrane as hereditary spherocytosis & elliptocytosis. hereditary spherocytosis & elliptocytosis.

22. Changes in RBCs due to aging Decreased in old cellsIncreased in old cells Bisphosphoglycerate (BPG)Glycosylated HbHb Sialic acid K + Lipids and Proteins Osmotic fragility Na + Binding of IgG Membrane G6PD Pyruvate dehydrogenase Others Enzymes Deformability Disc like shape Cell density SphericityGeneral

27. Assignments Pyruvate kinase deficiency Pyruvate kinase deficiency Hereditary sphercytosis Hereditary sphercytosis