Lec 03 & Lec 04:

Assist. Prof. Dr. Mudhir S. Shekha

Erythrocyte membrane

• RBC plasmalemma is trilaminar– 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

• ankyrin

• membrane proteinsFunction of plasmalemma

– 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 of sodium and potassium

content

Erythrocyte membrane

• Cell membrane selectively permeable Na and K• Potassium prevents escape of Hb and other cell

material• RBC contain→ 60% H20, 40% conjugated proteins

globin and heme pigment (4%) to form (hemoglobin)• RBC membrane is permeable to H20, electrolytes,

polysaccharides impermeable to Hb.

• osmolarity the concentration of a solution

expressed as the total number of solute particles per litre.

• Hb Osmolarity in RBC is = to that of plasma (isotonic), water absorption for plasma or RBC

• The osmotic fragility test (OFT) is used to measure erythrocyte resistance to hemolysis

• RBC is subjected to decreasing salt concentration until it hemolyses→fragility value

• RBC fragility is a diagnostic test for certain anemias.

Erythrocyte Function• Erythrocytes are adopted to

respiratory gas transport• Hemoglobin→oxygen in the

blood is bound to hemoglobin

• Hemoglobin is composed of the protein globin, made up of two alpha and two betachains, each bound to a heme group

• Each heme group bears an atom of iron, which can bind to one oxygen molecule

• Each hemoglobin molecule can transport four molecules of oxygen

Importance of Hb within RBC

• Intra RBC environment is a little more acidicthan plasma for capable respiration

• Hb is removed from metabolic pool, preventing its rapid turn over.

• Half-Life for Hb in RBC is months,

• Half-Life for Hb in plasma is only 3 hrs.

• Hb chemical state is required for O2 transport absolutely.

• Each Saturated gram of Hb can carries 1.3 ml oxygen

Metabolism of erythrocytes

• Tricarboxylic acid (TCA) cycle or Krebs cycle • Embden – Meyerhof (EM) pathway or glycolysis • Pentose cycle (Hexose monophosphate shunt) or HMS

mature erythrocyte lacks TCA cycle and capacity for oxidative phosphorylation because of lack of organelles mitochondria, ribosomes and endoplasmic reticulum

Abnormalities in RBC metabolism

• Deficiency of glucose – 6 – phosphate dehydrogenase (G6PD)

• Deficiency in pyruvate kinase

• Deficiency of NADH – methaemoglobin reductase

Destruction of erythrocytes

Cell decrease deformability in microcirculation is

associated with;

1. increase in red cell rigidity

2. increase in blood viscosity,

3. impeded blood flow

4. cell fragmentation

The changes in deformability depends on

Maintenance of cell geometry or biconcave shape

Normal internal or hemoglobin fluidity Intrinsic

membrane deformability or visco-elastic properties

• When red blood cells are delivered from the bone marrow into the circulatory system, they normally circulate an average of 120 days before being destroyed.

• Even though mature red cells do not have a nucleus, mitochondria, or endoplasmic reticulum, they do have cytoplasmic enzymes that are capable of metabolizing glucose and forming small amounts of adenosine triphosphate.

• These enzymes also:

• (1) maintain pliability of the cell membrane.

• (2) maintain membrane transport of ions.

• (3) keep the iron of the cells’ hemoglobin in the ferrous form rather than ferric form (Which causes the formation of methemoglobin that will not carry oxygen)

• (4) prevent oxidation of the proteins in the red cells.

Lec 04:Hemoglobin

Structure, Function, Synthesis & Destruction

Assist. Prof. Dr. Mudhir S. Shekha

• Molecules of hemoglobin account for 95% of the proteins in RBCs

• Hemoglobin is a globular protein, formed from two pairs of polypeptide subunits

– Each subunit contains a molecule of heme which reversibly binds an oxygen molecule

• Damaged or dead RBCs are recycled by phagocytes

Hemoglobin

Structure of Hemoglobin◼ 4 polypeptide

Subunits

◼ Heme group

◼ Porphyrin ring

◼ Ferrous iron

◼ Globin chain

◼ 2 Alpha Chains

◼ 2 Beta chains

Hemoglobin synthesis

• Although hem & globin synthesis separately within developing red cell precursors their rate of synthesis are carefully coordinated to ensure optimal efficiency of hemoglobin assembly.

– Occurs in the mitochondria of developing red cells as they mature in the bone marrow.

– Processes necessary for normal synthesis

• Adequate iron supply & delivery

• Adequate synthesis of protoporphyrins

• Adequate globin synthesis

Heme Synthesis• Chain of Events

– Iron delivery & supply

• Iron is delivered to the reticulocyte by transferrin

– Synthesis of protoporphyrins

• Occurs in the mitochondria of RBC precursors

• Mediated by EPO and vitamin B6

– Protoporphyrin+iron =heme

Globin synthesis

• Humans normally carry 8 functional globin genes, arranged in two duplicate gene clusters:

• The β-like cluster on the short arm of chromosome 11.

• The α-like cluster on the short arm of chromosome 16.

• These genes code for 6 different types of globin chains: Alpha α, beta β, delta δ, epsilon ε, gamma γ, zetaζ

Globin synthesis• Two distinct globin chains+Hem→ combine to form

hemoglobin. • One of the chains is designated alpha. The second chain is

called "non-alpha". With the exception of the very first weeks of embryogenesis, one of the globin chains is always alpha.

• The fetus has a distinct non-alpha chain called gamma→hemoglobin F.

• After birth ( 10 to 12 ), a different non-alpha globin chain, called beta, pairs with the alpha chain→ hemoglobin A (18 to 24 weeks of birth).

• The combination of two alpha chains and two non-alpha chains produces a complete hemoglobin molecule.

Globin synthesis

• one alpha + one non-alpha →hemoglobin dimer(two chains)→ ( not efficiently deliver oxygen, however).

• Two dimers combine to form a hemoglobin tetramer, which is the functional form of hemoglobin.

• Complex biophysical characteristics of the hemoglobin tetramer permit the attractive →control of oxygen uptake in the lungs and release in the tissues that is necessary to sustain life.

Globin synthesis

Ontogeny of globin synthesis

Type of HbType of Globin GeneRegionTime

Hb Gawer1 ζ ε)2)ζ & εYolk Sac 3 weeks of

Gestation

Hb Portland(ζ γ)2

Hb GawerII (αε)2

γ&αYolk Sac 5 weeks of

Gestation

Hb F (α γ)2α & γ & βLiver & spleen 6-30 weeksof

Gestation

Hb A2 (α δ 2(δLiver 30 weeks of

Gestation

HbA(α β)2___B.MAt Birth

Alpha α, beta β, delta δ, epsilon ε, gamma γ, zetaζ

Factors controlling Haemoglobin formation

• Role Of Proteins – First class proteins provide amino acids.

• Most imp – food of animal origin, liver, spleen, kidney & heart

• Intermediate value – muscles

• Least – cereals, dairy products, veg & fruits.

ROLE OF IRON

• Important for formation of Heme part of Hemoglobin.

• Sources of iron – Dietary iron

• Other sources – Iron released from degradation of RBC.

Role of other metals

• Copper – Promotes Absorption, Mobilization & Utilization of iron.

• Cobalt – Increases production of Erythropoietin.

• Calcium – conserve iron & subsequent utilization.

• Role of vitamins.

– Vit B12, Folic acid help in synthesis of nucleic acid.

– vit C helps in absorption of iron from gut. (Fe3+ to Fe2+)

• Role of bile salts.

– Imp for proper absorption of copper & nickel.

Adult hemoglobin

Hb A Hb A2 Hb F

structure a2b2 a2d2 a2g2

Normal % 96-98 % 1.5-3.2 % 0.5-0.8 %

Heme has a variety of functions. As a cofactor

• Oxygen transport in hemoglobin

• Storage in myoglobin

• A prosthetic group for cytochrome p450 enzymes

• A reservoir of iron

• Electron shuttle of enzymes in the electron transport chain

• Cellular respiration

• Signal transduction-heme regulates the antioxidant response to circadian rhythms, microRNA processing

• Cellular differentiation and proliferation

Difference between oxygenation

and oxidation of Hemoglobin

OXYGENATION

• IRON(Fe +2) IN

FERROUS STATE

• CARRIER OF OXYGEN

OXIDATION

• IRON(Fe +3) IN FERRIC

STATE

• OXYGEN CARRYING CAPACITY IS LOST

Functions of Hemoglobin

• Oxygen delivery to the tissues

• Reaction of Hb & oxygen

• Oxygenation not oxidation

• One Hb can bind to four O2 molecules

• Less than 0.01 sec required for oxygenation

• When oxygenated 2,3-DPG is pushed out

Normal Hemoglobin Function

• When fully saturated, each gram of hemoglobin binds 1.34 ml of oxygen.

• The relation between oxygen tension and hemoglobin oxygen saturation is described by the oxygen-dissociation curve of hemoglobin.

• The characteristics of this curve are related to pH, temperature, ionic strength, and concentration of phosphorylatedcompounds, especially 2,3-diphosphoglycerate (2,3-DPG).

HBO2→ 98.5 %O2 Dissolved in

Plasma→ 1.5%

CO2 in plasma 5-7% bicarbonate buffer system→ 85%CO2 Dissolved in Plasma→ 10%

Hb-oxygen dissociation curve

is a curve that plots the proportion of hemoglobin in its saturated (oxygen-laden) form on the vertical axis against the prevailing oxygen tension on the horizontal axis

Hb-oxygen dissociation curve

• Right shift (easy oxygen delivery)

• High 2,3-DPG

• High H+

• High CO2

• HbS

• Left shift (give up oxygen less readily)• Low 2,3-DPG

• HbF

NORMAL VALUES OF HEMOGLOBIN

• 1 year – 10-12 gm/dl

• Males - 14 – 17 gm/100ml

• Females- 12 – 15 gm/100ml

• Replaced at a rate of approximately 3 million new blood cells entering the circulation per second.

• Replaced before they hemolyze• Components of hemoglobin individually recycled

– Heme stripped of iron and converted to biliverdin, then bilirubin

• Iron is recycled by being stored in phagocytes, or transported throughout the blood stream bound to transferrin

• RBC life span 120 days only, short because of the lack of nuclei

• RBC is soft colloid to change shape in various sized vessels

RBC life span and circulation

Red Blood Cell Turnover

Erythropoiesis

• A hemocytoblast is transformed into a committed cell called the proerythroblast

• Proerythroblasts develop into early erythroblasts

• The developmental pathway consists of three phases

– Phase 1 – ribosome synthesis in early erythroblasts

– Phase 2 – hemoglobin accumulation in late erythroblasts and normoblasts

– Phase 3 – ejection of the nucleus from normoblasts and formation of reticulocytes

• Reticulocytes then become mature erythrocytes

Erythropoiesis

Circulating erythrocytes – the number remains constant and reflects a balance between RBC production and destruction

– Too few RBC leads to tissue hypoxia

– Too many RBC causes undesirable blood viscosity

• Erythropoiesis is hormonally controlled and depends on adequate supplies of iron, amino acids, and B vitamins

Regulation and Requirements for Erythropoiesis

Hormonal Control of ErythropoiesisErythropoietin (EPO) release by the kidneys is triggered by:

– Hypoxia due to decreased RBCs– Decreased oxygen availability– Increased tissue demand for oxygen

Enhanced erythropoiesis increases the: – RBC count in circulating blood– Oxygen carrying ability of the blood

Erythropoietin Mechanism

Stimulus:

• Hypoxia due to

decreased RBC

count,

• decreased

availability of O2 to

blood,

• increased tissue

demands for O2

Start

burst forming unit-erythroid

Colony Forming Unit-erythroid

Erythropoietin receptor

Erythropoiesis requires:

– Proteins, lipids, and carbohydrates

– Iron, vitamin B12, and folic acid

– The body stores iron in Hb (65%), the liver, spleen, and bone marrow

– Intracellular iron is stored in protein-iron complexes such as ferritin and hemosiderin

– Circulating iron is loosely bound to the transport protein transferrin

Dietary Requirements of Erythropoiesis

Destruction of erythrocytes• Cell decrease deformability in microcirculation is

associated with;

• increase in red cell rigidity

• increase in blood viscosity,

• obstructed blood flow and cell fragmentation

The changes in deformability depends on

• Maintenance of cell geometry or biconcave shape

• Normal internal or hemoglobin fluidity

• Intrinsic membrane deformability or viscoelasticproperties fragmentation

Modes of erythrocyte destruction1. Change in membrane permeability

2. Phagocytosis where (Na+, K+) levels are altered leading to increased osmotic fragility and hemolysis

3. Macrophage phagocytosis is extra-vascular hemolysis with increased un-conjugated bilirubin

4. Fragmentation, within circulation is intravascularhemolysis, followed by hemoglobinemia and hemoglobunuria hemolysis

1. Ferritin is the primary iron storage form . It is water-soluble protein-ironcomplex and contains a spherical shell of apoprotein enclosing a core of hydrated ferric phosphate. A single ferritin molecule can hold upto4500 iron atoms.

2. Hemosiderin is a brown pigment that is present in reticuloendothelialcells of bone marrow, spleen and liver. And higher iron protein/iron ration than ferretin

3. Transferrin: A plasma protein that transports iron through the blood to the liver, spleen and bone marrow.