k19- biokima kelainan eritrosit

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Production of Erythrocytes: Erythropoiesis

Figure 17.5



An Erythrocyte (RBC)

Normal Values

RBCs, male 4.7-6.1 x

106/µL

female 4.2-5.4 x

106/µL

Hb, male 13.0-16.0 g/dL

female 12.0-15.0 g/dL

Hct, male 42-53%

female 37-47%

MCH 29±2 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 FeRBC x 3 = Hb

Hb x 3 = Hct

Microcytic < 81 fL

Macrocytic > 94 fL



Erythrocytes (RBCs)

Erythrocytes are anexample of the

complementarity ofstructure and function

Structuralcharacteristics

contribute to its gastransport function

Biconcave shape thathas a huge surface

area relative tovolume

Discounting watercontent, erythrocytes

are more than 97%hemoglobin

ATP is generatedanaerobically, so the

erythrocytes do notconsume the oxygen

they transport



They lackorganelles

no ATP productionin oxidative

phosphorylation

no ability to replacedamaged lipids and

proteins (lowmetabolic activities,

with no ability to

synthesize newproteins or lipids)

Erythrocyte

exceptions



Free radicalsexposure

haemoglobinautoxidation(O2

•- release)

a cellmembrane rich

inpolyunsaturated

fatty acids(susceptible to

lipidperoxidation)

deformation intiny capillaries;catalytic ions

leakage (causeof lipid

peroxidation)

Erythrocyte exceptions



The erythrocyte membrane

50% l ip id bi layer (phosp hol ip id s, cholesterol)

50% pro teins

SDS-PAGE: separation of proteins (band 1-7)

isolation and analysis (10 main proteins)

Integral: Anion exchanger protein, Glycophorin A, B, CPeripheral: 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 membraneagainst 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 Provide deformability, elasticity

Allows for passage through microvessels

Provides permeability

Allows water and electrolytes to exchange

RBC controls volume and H2O content primarily through control ofsodium and potassium content



Erythrocyte Membrane Composition

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



Spectrin: the most prominent component (two isoforms α,β; 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.

The erythrocyte membrane

St t f H l bi



Structure of Hemoglobin

Figure 17.4



Hemoglobin Structure Changes

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



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

Hemoglobin Genes and Gene Products



http://www.blackwellpublishing.com/korfgenetics/figure.asp?chap=13&fig=Fig13-1

http://www.embryology.ch/anglais/qblood/blut03.html

Hemoglobin Genes and Gene Products

HbF: 2 and 2 HbA1: 2 and 2

HbA2: 2 and 2HbE: 2 and 2

Umbilical vesicle

liver

spleen

Bone marrow



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

Hemoglobin Gene Product Production

HbF: 2 and 2

HbA1: 2 and 2

HbA2: 2 and 2HbE: 2 and 2

Yolk sac Liver Spleen Bone marrow



The Erythrocytemetabolism



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



• 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

Fe3+

, 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 plasmaconcentration as a morker of hemolysis

• 5-10% of glucose diverted to the pentose phosphate pathway, whichproduces NADPH

• Glucose uptake is not insulin-dependent, intracellular glucose metabolism isaffected by insulin

• Erythrocytes do not synthesize glycogene, fatty acids, proteins

Erythrocyte metabolism

Glucose source of energy



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 maintainingthe physical characteristics of the RBC.

Embden-Meyerhof glycolytic pathway Generates 90% of energy needed by RBC’s

Glucose is metabolized and generates two molecules of ATP (energy).

Hexose monophosphate shunt 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-phosphatedehydrogenase (G-6PD).

If the pathway is deficient, intracellular oxidants can’t be neutralized andglobin denatures then precipitates. The precipitates are referred to as Heinz bodies. (Must use supravital stain to visualize them.)



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. Leubering-Rapaport shunt

Allows the RBC to regulate oxygen transport during conditions ofhypoxia or acid-base imbalance.

Permits the accumulation of 2,3-DPG which is essential formaintaining normal oxygen tension, regulating hemoglobin affinity



E r

y t h r o c y t e

m e t a b o l i s m



Anaerobic glycolysis and 2,3-BPG

• In erythrocytes, one of the glycolytic

reactions (which produces ATP) can bebypassed

– 2,3-bisfosfoglycerate is produced as anintermediary product

– energy is dissipated as heat

• This shunt enables glycolysis to run evenwhen energy requirements are low

2 3 bisphosphoglycerate



2,3-bisphosphoglycerate

• Allosteric effector of haemoglobin:

– binds to deoxyhaemoglobin (a central cavity capable of binding 2,3-BPG)

– decreases haemoglobin‘s 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

• DeoxyHb affinity to bind 2,3-BPG is 100-foldhigher that that of OxyHb

• Binding of 2,3-BPG to Hb decreases Hbaffinity to bind O2 → right shift of the curve

2 mmol/l

4 mmol/l

6 mmol/l

H b s a t u r a

t i o n

pO2

t i s s u e s

l u n g s

Lungs

Only a small decreaseof oxygen loading

Tissues

Significant increase

of oxygen

unloading



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

Factors Affecting Binding of O2

pH_

BPG or T; right shift

pH

BPG or T_; left shift

The pentose phosphate pathway in erythrocytes



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



OxyhaemoglobinO



Haemoglobin

Methaemoglobin

Methaemoglobin reductase

O2

Superoxide

Superoxide dismutase

H2O2

Catalase

½ O2+H2O

Glutathione peroxidase

H2O

GSH

GSSG

Glutathione reductase

NADPH

NADP+

Pentose phosphate

pathway

GSH-reduced form; GSSG-oxidized form of glutathione



Hb oxygen saturation and erythrocyte metabolism

• 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 2,3-

BPG is produced → oxygen binding

curve shifts to the right, more oxygen

released

High oxygen saturation Low oxygen saturation



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- → O2

•-

- Fenton reaction: Fe2+ + H2 O2 → Fe3+ + OH + OH•-

- Haber-Weiss reaction: O2•- + H2O2 → OH + OH•-

- Superoxiddismutase (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

Reactive oxygen species



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 (γ-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-6-

phosphatedehydrogenase (G6PD)

• G6PD deficiency

– Probably the most frequent enzymopathy (~100 million people, 300 geneticvariants of the enzyme)

– Frequent in mediterranean countries, tropical Africa, Asia

– Results in hemolytic anemia, worsened by some foods (broad beans) or drugswhich act as oxidants

– Hemolysis is caused by oxidative impairment of membrane lipids (peroxidation),proteins (oxidation of thiol groups to disulfides)

M h l bi i



Methemoglobinemia

• About 3% of Hb is converted to metHb and back in 24 hours, butmetHb 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 andvegetables)

• 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)

I h it d ethemoglobinemia



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

Inherited methemoglobinemia

Glutathione synthesis in erythrocytes



GlutathioneEli i ti f H O d i h d id



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

Gly

Cys

Glu

Gly

Cys

Glu

Gly

Cys SH

Glu

S S

Oxidized form of glutathione

(dimer, disulphide)

Reduced form of glutathione

(monomer)

Glutathione peroxidase


+ R-O-O-H

+ H2O

+ NADPH

Haemoglobin autoxidation• 3% of the haemoglobin undergoes oxidation every day



• a constant flux of O2•-

Hem - 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)

Methaemoglobinemia1. Congeni tal type

– methaemoglobin reductase deficiency (AR)

– variant haemoglobin M (HbM)- mutation; tend to be oxidized tomethaemoglobin

2. Ac quired type - drugs or chemicals (sulphonamides, aniline)

Visual indicator- a blue tint to the skin (10% of metHb)Treated- reductants (methylene blue, ascorbic acid)



2. Glutathione peroxidase

H O b li it d ti t H O ith id ti f d d



• removes H2O2 by coupling its reduction to H2O with oxidation of reducedglutathione (GSH)

H2O2+2GSH GSSG+2H2O


• 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



α-tocopherol (vitamin E)

• Present in the erythrocyte membrane

• Prevents lipid peroxidation (chain-breaking antioxidant)

α-TocH+LO2• α-Toc•+LO2H

Ascorbic acid (vitamin C)

• Present in the cytoplasm• Recycles α-tocopherol

• Dehydroascorbate reductase (GSH-dependent) regeneratesascorbate

Haemoglobinopathy• abnormal structure of the haemoglobin (mutation)



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 α or β-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- α or β-chains are not produced (α or β-thalassemia)

Sickle Cell Disease



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

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

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

HbS beta-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



Haemoglobin S (sickle-cell)

• Causes a sickle cell anemia



• 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

ti l l ti ( l ti ) b di t t i



• 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 previousweeks; in individuals with poorly controlled diabetes, increases in the quantities ofthese glycated hemoglobins are noted (patients monitoring)

Sugar CHO + NH2 CH2 Protein

Sugar CH N CH2 Protein

Sugar CH2 NH CH2 Protein

Schiff base

Glycosylated protein

Amadori reaction



Checkpoint Which erythrocyte metabolic pathway is

responsible for providing the majority ofcellular energy?

For regulating oxygen affinity?

For maintaining hemoglobin in a reducedstate?

Summary

Erythrocytes lack cell organelles; their membranes are rich in

polyunsaturated fatty acids and proteins (fluidity and elasticity)



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 glutathione

and NADPH

There is a large number of haemoglobin mutations; some of them are

pathological (haemoglobinopathy)

k19- biokima kelainan eritrosit

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