Department of Biochemistry

Production of Erythrocytes: Erythropoiesis

Figure 17.5

An Erythrocyte (RBC)

Normal Values RBCs, male 4.7-6.1 x 106/L female 4.2-5.4 x 106/L Hb, male 13.0-16.0 g/dL female 12.0-15.0 g/dL Hct, male 42-53% female 37-47% MCH 292 pg MCV 81-94 fL MCHC 32-37.5%

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 < 81 fL Macrocytic > 94 fL

Erythrocytes (RBCs)

Erythrocytes are an example of the complementarity of structure and function

Structural characteristics contribute to its gas transport function

Biconcave shape that has a huge surface area relative to volume

Discounting water content, erythrocytes are more than 97% hemoglobin

ATP is generated anaerobically, so the erythrocytes do not consume the oxygen they transport

Erythrocyte exceptions

They lack organelles

no ATP production in oxidative phosphorylation

no ability to replace damaged lipids and proteins (low metabolic activities, with no ability to synthesize new proteins or lipids)

Erythrocyte exceptions

Free radicals exposure

haemoglobin autoxidation (O2- release)

a cell membrane rich in polyunsaturated fatty acids (susceptible to lipid peroxidation)

deformation in tiny capillaries; catalytic ions leakage (cause of lipid peroxidation)

The erythrocyte membrane

50% lipid bilayer (phospholipids, cholesterol) 50% proteins SDS-PAGE: separation of proteins (band 1-7) isolation and analysis (10 main proteins) Integral: Anion exchanger protein, Glycophorin A, B, C Peripheral: Spectrin, Ankyrin, Actin

Red Blood Cell Membrane

Development

Trilaminar, three-dimensional structure Outermost layer: glycolipids, glycoproteins Central layer: cholesterol, phospholipids Inner layer: cytoskeleton

spectrin
Composed of alpha & beta chains Join to form a matrix which strengthens the membrane against sheer force and controls biconcave shape

ankrin membrane proteins

Red Blood Cell Membrane

Function

Shape

Provides the optimum surface to volume ratio for respiratory exchange AND is essential to deformability
Allows for passage through microvessels

Provide deformability, elasticity

Provides permeability

Allows water and electrolytes to exchange RBC controls volume and H2O content primarily through control of sodium and potassium content

Spectrin: the most prominent component (two isoforms ,; a tetramer; a meshwork )

fixed to the membrane- ankyrin binding sites for several other proteins (glycophorin C, actin, band 4.1, adducin) This organization keeps the erythrocyte shape.

Erythrocyte Membrane Composition

http://www.ruf.rice.edu/~bioslabs/studies/sds-page/rbcmembrane.html

The erythrocyte membrane

Spectrin: the most prominent component (two isoforms ,; a tetramer; a meshwork )

fixed to the membrane- ankyrin binding sites for several other proteins (glycophorin C, actin, band 4.1, adducin) This organization keeps the erythrocyte shape.

Structure of Hemoglobin

Figure 17.4

Hemoglobin Structure Changes

http://www.mfi.ku.dk/PPaulev/chapter8/images/8-3.jpg

Hemoglobin Genes and Gene Products

http://www.mun.ca/biology/desmid/brian/BIOL3530/DB_Ch09/fig9_24.jpg

Hemoglobin Genes and Gene Products

liver Umbilical vesicle Bone marrow

spleen

HbF: 2 and 2

HbA1: 2 and 2

HbE: 2 and 2

HbA2: 2 and 2

http://www.blackwellpublishing.com/korfgenetics/figure.asp?chap=13&fig=Fig13-1 http://www.embryology.ch/anglais/qblood/blut03.html

Hemoglobin Gene Product Production

Yolk sac Liver Spleen Bone marrow

HbF: 2 and 2 HbA1: 2 and 2

HbE: 2 and 2

HbA2: 2 and 2

Mehta, A. B., and A. V. Hoffbrand. 2000. Haematology at a glance, Blackwell Science, Malden, Mass.

The Erythrocyte metabolism

Erythrocyte metabolism
Glucose as a source of energy Glycolysis generates ATP and 2,3-bisphosphoglycerate The pentose phosphate pathway produces NADPH Glutathione synthesis- the antioxidant defence system

Erythrocyte metabolism
Energy (ATP) is necessary to keep the erythrocytes in normal functional state
Shape and deformability (membrane skeleton) Membrane transport (Na/K ATP-ase, band 3, ) Protection against reactive oxygen species (which oxidate heme iron from Fe 2+ to Fe 3+ , initiate peroxidation of membrane lipids,

(anaerobic) glycolysis
Produces 2,3-BPG as a by-product, which influences Hb-oxygen binding The end-product - lactate is released to the bloodstream High activity of lactate dehydrogenase in erythrocytes, increased plasma concentration as a morker of hemolysis

5-10% of glucose diverted to the pentose phosphate pathway, which produces NADPH Glucose uptake is not insulin-dependent, intracellular glucose metabolism is affected by insulin Erythrocytes do not synthesize glycogene, fatty acids, proteins

Glucose- source of energy

Glucose transporter:
integral membrane protein (12 membrane-spanning helices) a channel for the glucose transport insulin-independent transporter

Glycolysis in erythrocytes
1. Source of ATP
Lactate- the end product Cover 90% of energy requirement

2. Generate 2,3-bisphosphoglycerate (2,3-BPG)

a major reaction pathway for the consumption of glucose in erythrocytes the specific binding of 2,3-BPG to deoxyhemoglobin decreases the oxygen affinity of hemoglobin and facilites oxygen release in tissues

Red Blood Cell Metabolism

Metabolism

These pathways are essential for oxygen transport and maintaining the physical characteristics of the RBC. Embden-Meyerhof glycolytic pathway

Generates 90% of energy needed by RBCs Glucose is metabolized and generates two molecules of ATP (energy). Metabolizes 5-10% of glucose. NADPH is end product Protects the RBC from oxidative injury. Most common defect is deficiency of the enzyme glucose-6-phosphate dehydrogenase (G-6PD). If the pathway is deficient, intracellular oxidants cant be neutralized and globin denatures then precipitates. The precipitates are referred to as Heinz bodies. (Must use supravital stain to visualize them.)

Hexose monophosphate shunt

Red Blood Cell Metabolism

Methemoglobin reductase pathway

Maintains iron in the ferrous (Fe2) state. In the absence of the enzyme (methemoglobin reductase), methemoglobin accumulates and it cannot carry oxygen. Allows the RBC to regulate oxygen transport during conditions of hypoxia or acid-base imbalance. Permits the accumulation of 2,3-DPG which is essential for maintaining normal oxygen tension, regulating hemoglobin affinity

Leubering-Rapaport shunt

Erythrocyte metabolism

Anaerobic glycolysis and 2,3-BPG

In erythrocytes, one of the glycolytic reactions (which produces ATP) can be bypassed
2,3-bisfosfoglycerate is produced as an intermediary product energy is dissipated as heat

This shunt enables glycolysis to run even when energy requirements are low

2,3-bisphosphoglycerate
Allosteric effector of haemoglobin:
binds to deoxyhaemoglobin (a central cavity capable of binding 2,3-BPG) decreases haemoglobins O2 affinity

Clinical aspects:
In people with high-altitude adaptation or smokers the concentration of 2,3-BPG in the blood is increased (low oxygen supply) Fetal haemoglobin has low BPG affinity - the higher O2 affinity - facilitates the transfer of O2 to the fetus via the placenta

2,3-BPG and hemoglobin-oxygen binding

2 mmol/l 4 mmol/l

6 mmol/l

DeoxyHb affinity to bind 2,3-BPG is 100-fold higher that that of OxyHb Binding of 2,3-BPG to Hb decreases Hb affinity to bind O2 right shift of the curve

tissues

Hb saturation

lungs

Lungs Only a small decrease of oxygen loading

Tissues Significant increase of oxygen unloading

pO2

Factors Affecting Binding of O2

Depends on pH ([H+]), CO2, BPG (DPG), Temp

pH BPG or T _; left shift

pH _ BPG or T ; right shift

The pentose phosphate pathway in erythrocytes

Generates NADPH - reduction of glutathione (eliminates H2O2 formed in erythrocytes)

Clinical aspect: Glucose-6-phosphate dehydrogenase deficiency

Causes hemolytic anemia (decreased production of NADPH - reduced protection against oxidative stress - haemoglobin oxidation and Heinz bodies formation, membrane lipid peroxidation and hemolysis) Hemolytic crises are evocated by drugs (primaquine, sulphonamide antibiotics) and foods (broad beans) The most common enzyme deficiency disease in the world (100 million people)

Pentose phosphate pathway

Alternative pathway of glucose metabolism

In several cycles, it leads to complete glucose oxidation

Oxidation accomplished through dehydrogenation (without O2) Produces CO2 (not produced by glycolysis) Does not produce ATP (glycolysis produces ATP) The acceptor of H+ is NADP (it is NAD in glycolysis)

NADPH provides H+ for reduction of oxidated glutathione contribuing to protection against reactive oxygen species

Oxyhaemoglobin
O2
Superoxide dismutase

Haemoglobin

Superoxide

H2O2

Catalase

Methaemoglobin reductase

Methaemoglobin O2+H2O
Pentose phosphate pathway

GSH NADP+

Glutathione reductase

Glutathione peroxidase

NADPH

GSSG H2O

GSH-reduced form; GSSG-oxidized form of glutathione

Hb oxygen saturation and erythrocyte metabolism

High oxygen saturation Low oxygen saturation

Phosphofructokinase (PFK) binds to band 3 (B3P) Preferention of pentose phospate pathway higher production of NADPH higher antioxidant capacity

DeoxHb beats the competition of PFK in binding to B3P Preferention of glycolysis, more 2,3BPG is produced oxygen binding curve shifts to the right, more oxygen released

Reactive oxygen species

ROS = molecules, atoms or ions capable to react avidly with lipids (membranes), proteins, nucleic acids and impair their structure and function

Radicals: ROS with an unpaired electron

Increased ROS production and/or decreased degradation = oxidative stress Important reactions
Superoxide generation: O2 + e- O2Fenton reaction: Fe2+ + H2 O2 Fe3+ + OH + OHHaber-Weiss reaction: O2- + H2O2 OH + OHSuperoxiddismutase (SOD): O2- + O2- H2O2 + O2 Catalase (CAT): 2 H2O2 2 H2O + O2 Glutathionperoxidase (GPX): 2 GSH + R-O-OH GSSG + H2O + ROH 2 GSH + H2O2 GSSG + 2 H2O

ROS in the erythrocytes

High O2 content A transition metal: Fe (2+3+), contained in Hb Hb autooxidation:
HemFe2+ + O2 (Hem Fe2+~O2 HemFe3+~ O2-) HemFe3+ + O2-

Regeneration of Hb: methemoglobinreductase Erythrocytes are rich in antioxidant enzymes SOD, GPX, CAT Key role of glutathione (-glutamyl-cysteinyl-glycin, GSH)
Reaction of GPX Direct reaction with molecules modified by ROS

GSH regeneration by glutathionreductase

GSSG + NADPH + H+ 2 GSH + NADP+

Pentose phosphate pathway is the source of NADPH + H+:

NADPH and antioxidant protection

NADPH + H+ is produced in a reaction catalyzed by glucose-6phosphatedehydrogenase (G6PD) G6PD deficiency

Probably the most frequent enzymopathy (~100 million people, 300 genetic variants of the enzyme) Frequent in mediterranean countries, tropical Africa, Asia Results in hemolytic anemia, worsened by some foods (broad beans) or drugs which act as oxidants Hemolysis is caused by oxidative impairment of membrane lipids (peroxidation), proteins (oxidation of thiol groups to disulfides)

Methemoglobinemia
About 3% of Hb is converted to metHb and back in 24 hours, but metHb concentration is normally very low

Causes of increased metHb concentration

Inherited deficiency of methemoglobinreductase Increased susceptibility of abnormal Hb variants to autooxidation (HbM, HbS - sickle) toxic chemicals: aniline, sulfonamides, nitrites (conservants in food, produced by microbial reduction of nitrates contained in fruits and vegetables)

MetHb cannot bind oxygen hypoxia

Cyanosis: bluish colour of skin due to presence of metHb
becomes apparent at metHb levels above 15g metHb/l (about 50g/l deoxyHb is required to produce cynosis)

Inherited methemoglobinemia

The blue people of Troublesome Creek. Science 1982. Ilustrace Walt Spitzmiller.

Glutathione synthesis in erythrocytes

Glutathione
Elimination of H2O2 and organic hydroperoxides 1. Cofactor for the glutathione peroxidase (removes H2O2 formed in erythrocytes) 2. Involved in ascorbic acid metabolism 3. Prevents protein SH groups from oxidizing and cross-linking

Glutathione peroxidase

Gly + R-O-O-H Cys SH

Gly

Gly

Cys S S

Cys + H2O

Glu + NADPH
Glutathione reductase

Glu

Glu

Reduced form of glutathione (monomer)

Oxidized form of glutathione (dimer, disulphide)

Haemoglobin autoxidation
3% of the haemoglobin undergoes oxidation every day a constant flux of O2Hem - Fe2+- O2 Hem - Fe3+ - O2-

Methaemoglobin reductase
Converts methaemoglobin back to ferrous haemoglobin to permit continued O2 transport System containing FAD, cytochrome b5 and NADH (glycolysis)

Methaemoglobinemia 1. Congenital type

methaemoglobin reductase deficiency (AR) variant haemoglobin M (HbM)- mutation; tend to be oxidized to methaemoglobin 2. Acquired type- drugs or chemicals (sulphonamides, aniline) Visual indicator- a blue tint to the skin (10% of metHb) Treated- reductants (methylene blue, ascorbic acid)

Superoxide dismutase (SOD)

a high concentration in erythrocytes accelerates the dismutation O2- to H2O2

H2O2 remove
1. Catalase 2. Glutathione peroxidase 1. Catalase
a ferric haem group bound to the active site catalyses decomposition of H2O2 to water and oxygen:

2H2O2

2H2O+O2

2. Glutathione peroxidase
removes H2O2 by coupling its reduction to H2O with oxidation of reduced glutathione (GSH) H2O2+2GSH GSSG+2H2O

Glutathione reductase reduces oxidized glutathione back to reduced GSSG+NADPH+H+ 2GSH+NADP+

NADPH- the pentose phosphate pathway (glucose-6-phosphate dehydrogenase)

Cooperation of glutathione peroxidase and catalase The concentration of H2O2 is raised- catalase becomes more important (high Km for H2O2)

Low-molecular mass antioxidants

-tocopherol (vitamin E) Present in the erythrocyte membrane Prevents lipid peroxidation (chain-breaking antioxidant)

-TocH+LO2

-Toc+LO2H

Ascorbic acid (vitamin C) Present in the cytoplasm Recycles -tocopherol Dehydroascorbate reductase (GSH-dependent) regenerates ascorbate

Haemoglobinopathy
abnormal structure of the haemoglobin (mutation) large number of haemoglobin mutations, a fraction has deleterious effects sickling, change in O2 affinity, heme loss or dissociation of tetramer haemoglobin M and S, and thalassemias

Haemoglobin M replacement of the histidine (E8 or F7) in or -chain by the tyrosine the iron in the heme group is in the Fe3+ state (methaemoglobin) stabilized by the tyrosine methaemoglobin can not bind oxygen

Thalassemias genetic defects- or -chains are not produced ( or -thalassemia)

Sickle Cell Disease

(>6 major genotypes) at least 1 sickle gene, hemoglobin S (HbS) 50% Hb present.

homozygotic HbSS (sickle cell anemia) - HbS = 100% Hb present

HbSbeta-0 thalassemia - Severe double heterozygote for HbS and beta-0 thalassemia; almost indistinguishable from sickle cell anemia phenotypically (MCV low)

HbSC disease - Double heterozygote for HbS and HbC, with intermediate clinical severity

HbS/hereditary persistence of fetal hemoglobin (S/HPHP) - Mild form or symptom free

HbS/HbE syndrome - Rare and generally mild clinical course

Rare combinations of HbS with HbD Los Angeles, HbO Arab, G-Philadelphia, among others

http://www.emedicine.com/ped/TOPIC2096.HTM

Effects of therapy with hydroxyurea

http://www.emedicine.com/ped/TOPIC2096.HTM

Haemoglobin S (sickle-cell)
Causes a sickle-cell anemia Erythrocytes adopt an elongated sickle shape due to the aggregation of the haemoglobin S

Glycosylated haemoglobin (HbA1C)

formed by hemoglobin's exposure to high plasma levels of glucose non-enzymatic glycolysation (glycation)- sugar bonding to a protein normal level HbA1- 5%; a buildup of HbA1- increased glucose concentration the HbA1 level is proportional to average blood glucose concentration over previous weeks; in individuals with poorly controlled diabetes, increases in the quantities of these glycated hemoglobins are noted (patients monitoring)

Sugar

CHO

NH2

CH2

Protein

Sugar

CH

CH2

Protein
Schiff base

Amadori reaction

Sugar

CH2

NH

CH2

Protein
Glycosylated protein

Checkpoint
Which erythrocyte metabolic pathway is responsible for providing the majority of cellular energy? For regulating oxygen affinity? For maintaining hemoglobin in a reduced state?

Summary
Erythrocytes lack cell organelles; their membranes are rich in polyunsaturated fatty acids and proteins (fluidity and elasticity) Glucose as a energy source Glycolysis generates ATP and 2,3-BPG; the pentose phosphate pathway produces NADPH Haemoglobin autoxidation forms free radicals Free radicals are removed by the antioxidant defence system with and NADPH glutathione

There is a large number of haemoglobin mutations; some of them are pathological (haemoglobinopathy)