Introduction to Erythrocyte Metabolism

1. Blood-Hematopoiesis-Lymphatics Introduction to Erythrocyte Metabolism William F. Kern, MD Director, Clinical Hematopathology william-kern@ouhsc.edu

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3. Characteristics of Erythrocytes • Anucleate • No ribosomes or organelles • No protein synthesis or repair • Energy generation by anaerobic metabolism • Lifespan ~ 120 days

4. Erythrocyte Functions • Gas exchange: • Oxygen • Carbon dioxide • Acid-base balance: Carbonic anhydrase • Clear immune complexes • Clear nitric oxide

5. Erythrocyte Problems • Maintain intracellular Na+ and K+ levels against concentration gradients: • Na-K-ATPase • Maintain hemoglobin in reduced state: • Reduce methemoglobin (Fe3+) to hemoglobin (Fe2+) • Resist oxidant stress: • Maintain glutathione in reduced state • Must resist complement attack

6. Hemoglobin • Oxygen carrier in RBCs • Two components: • Globin: Protein chain • Heme: Protoporphyrin ring with iron atom (Fe2+) • Hemoglobin molecule consists of two pairs of globin chains: • Two a chains, 2 non-a (b, g, d) chains • Each hemoglobin molecule can carry 4 molecules of oxygen

7. Hemoglobin Molecule

8. Hemoglobin Variants • Hemoglobin F (fetal) is the predominant hemoglobin in utero; reaches normal adult levels by ~6 months * OU Medical Center; varies slightly between different labs

9. Hemoglobin Switching Hb A a2b2

10. Hemoglobin Genes a-Gene Cluster: Chromosome 16 HS-40 z a2 a1 5’ 3’ b-Gene Cluster: Chromosome 11 LCR e g g d b 3’ 5’ Key: Total of 4 a-globin chains; only 2b-globin chains

11. Hemoglobin-Oxygen Affinity • Quarternary structure of hemoglobin molecule variable: • Relationship between four different subunits • Alters affinity of hemoglobin for oxygen: • Measured as P50: • Partial pressure of oxygen at which hemoglobin is 50% saturated with oxygen • Allows physiologic regulation of oxygen supply

12. Hemoglobin-Oxygen Affinity Curve Oxygen released Curve can be shifted right or left

13. Hemoglobin: Effects of Changes in O2 Affinity • Decreased O2 affinity increasesO2unloading to tissues: • Rightward shift in O2 saturation curve • Higher P50 • Increased O2 affinity decreases O2unloading: • Leftward shift in O2 saturation curve • Lower P50 You have to know this stuff, folks!!

14. Hemoglobin-Oxygen Affinity: Effects of Alterations Normal curve Normal curve Rightward Shift Leftward Shift Hillman RS, Finch CA: Red Cell Manual, 7th Ed.

15. Hemoglobin-Oxygen Affinity: Effects of Alterations Alkalosis Normal Release Normal Acidosis Rightward Shift Leftward Shift Hillman RS, Finch CA: Red Cell Manual, 7th Ed.

16. Regulation of Hemoglobin-Oxygen Affinity: 2,3-DPG • 2,3-DPG = 2,3-diphosphoglycerate • Primary regulatory of hemoglobin-oxygen affinity • Increased by increased deoxyhemoglobin concentration • Decreases O2 affinity: Increases tissue oxygen supply

17. Hemoglobin: Regulation of O2 Affinity • Acidosis: Decreases O2 affinity (“Bohr effect”) • Alkalosis: Increases O2 affinity • Increased temperature: Decreases O2 affinity Acidosis and increased temperature are associated with infection or stress; states of increased O2 need

18. Heme Synthesis Glycine + Succinyl-CoA Pyridoxal 5’-Phosphate ALA synthetase Mitochondria d-aminolevulinic acid (ALA) Porphobilinogen Negative feedback inhibition Cytoplasm Protoporphyrin IX Fe2+ Mitochondria Ferrochelatase Heme

19. Heme Synthesis • ALA synthetase (ALAS) is rate-limiting step: • RBC specific enzyme • Gene on X chromosome • Requires Pyridoxal 5’-phosphate • Deficiencies of enzymes in heme synthesis pathway cause sideroblastic anemiasand inherited porphyrias

20. Hemoglobin: Oxygenation versus Oxidation Oxyhemoglobin (Fe2+) Deoxyhemoglobin + O2 Spontaneous Methemoglobin(Fe3+) + O2- (Superoxide) • Methemoglobin degrades into hemichromes & Heinz bodies: • Damage cell membranes & proteins • O2- is also toxic to cell proteins & membrane: • Converted to H2O2 by superoxide dismutase • H2O2 must itself be reduced

21. Hemoglobin: Methemoglobin Reductase System Hemoglobin (Fe2+) NAD+ Methemoglobin Reductase Spontaneous NADH Methemoglobin (Fe3+) • Methemoglobin reductase system reduces methemoglobin (Fe3+) to hemoglobin (Fe2+) • Requires NADH

22. Detoxification of H2O2: Glutathione & Glutathione Reductase 2 GSH + H2 O2 GSSG + 2 H20 NADP+ NADPH Glutathione Reductase • Glutathione (GSH) is “reducing bank” for RBC • Oxidized glutathione (GSSG) reduced back to GSH by glutathione reductase • Requires NADPH: Generated by hexose monophosphate shunt

23. Embden-Meyerhof Pathway (Glycolysis) Glucose Hexokinase Glucose 6-P NAD+ Generates ATP and NADH NADH 1,3-diP-Glycerate 3P-Glycerate Phosphoenolpyruvate Pyruvate kinase Pyruvate Lactic dehydrogenase (LDH) Lactate

24. Hexose Monophosphate Shunt: Generation of NADPH Glucose Regenerate GSH NADP+ NADPH Glucose 6-P 6-Phosphogluconate G6PD NAD+ Pentose-P NADH Hexose Monophosphate Shunt 1,3-diP-Glycerate 3P-Glycerate Phosphoenolpyruvate G6PD = Glucose-6-phosphate dehydrogenase Pyruvate Lactate

25. Erythrocyte Metabolism: Generation of 2,3-DPG Glucose Rapoport -Luebering Pathway Glucose 6-P NAD+ NADH 1,3-diP-Glycerate 2,3-Diphosphoglycerate (2,3-DPG) 3P-Glycerate Phosphoenolpyruvate 2,3-DPG = Primary regulator of hemoglobin-O2 affinity Pyruvate Lactate

26. Erythrocyte Metabolism Glucose Regenerate GSH NADP+ NADPH Glucose 6-P 6-Phosphogluconate MetHb (Fe3+) G6PD NAD+ Pentose-P Hexose Monophosphate Shunt NADH 1,3-diP-Glycerate Hb (Fe2+) 2,3-Diphosphoglycerate 3P-Glycerate Rapoport -Luebering Pathway Phosphoenolpyruvate Pyruvate Lactate

27. Erythrocyte Life Cycle • Normal life span ~120 days • Most cells trapped and phagocytosed by reticuloendothelial system, predominantly in spleen: • “Extravascular hemolysis” • A few cells (<10%) normally destroyed within circulation: • “Intravascular hemolysis”

28. Extravascular Hemolysis • Hemoglobin dissociated into globin protein and heme molecule • Iron removed from heme ring: • Recycled back to marrow or stored within macrophage • Protoporphyrin ring opened; results in biliverdin and carbon monoxide (CO) • Biliverdin converted to bilirubin (unconjugated or “indirect” bilirubin)

29. Intravascular Hemolysis • Hemoglobin tetramer dissociates to a-b dimers • a-b dimers complex with haptoglobin; removed from circulation by hepatocytes • Excess hemoglobin filtered by glomerulus: • May be phagocytosed by renal tubular cells; • May appear in urine as free hemoglobin or methemoglobin

30. Reticulocytes • Young RBCs that contain RNA • Visualized with supravital dyes (new methylene blue) • Usually counted as % of RBCs • RBC is normally reticulocyte for 4 days: • 3 days in marrow • 1 day in blood • Normal reticulocyte count ~0.5-2.5% 1 day as reticulocyte 120 day life span = ~1%

31. Reticulocytes (new methylene blue stain)

32. Corrected Reticulocyte Count • Reticulocyte count = ratio of retics to total RBCs • In anemia, total RBC count is decreased • If the absolute reticulocyte count is unchanged, the ratio will increase – falsely suggests increased reticulocyte production • Reticulocyte count must be corrected for this factor

33. Corrected Reticulocyte Count:Correction for Anemia Corrected reticulocyte count: Patient hematocrit Normal hematocrit Patient retic count (%) x Example: Hematocrit = 15%; Retic count = 10% (male) 15% 45% Corrected retic count = 10% x = 3.3%