Blood is the fluid circulating in a closed system of blood vessels and the chambers of the heart

It is the medium which transports substances from one part of the body to the other Blood is composed of

Plasma Platelets Cells  WBCs  RBCs (Erythrocytes)

Hemoglobin (Hb) is red , oxygen carrying pigment present exclusively in erythrocytes A conjugated protein containing Globin Protein part ( 4 polypeptide chains- ) 96% of the total Hb mass Varies from species to species( species specificity)  Non protein (prosthetic group) Red colour Iron containing derivative  4% of the total Hb mass Reversibly binds Oxygen Structure of Heme An Iron –porphyrin (Protoporphrin IX) compound with tetrapyrrole structure Protoporphyrin IX consists of 4 rings combined through — CH= bridges (methyne bridges) The methyne bridges are referred as α,β,γ, and δ. The 2 Hydrogen atoms in the –NH groups pyrrole rings (II & IV) are replaced by Ferrous( Fe++)

. The four pyrrole rings present in the porphyrin molecule are designated as I,II,III & IV

. Each of these four rings has 2 groups attached to them

M = Methyl –CH3

V = Vinyl – CH=CH2

P = Propionyl - CH2 - CH2 - COOH . The Fe++ can form 2 additional bonds .One of these position is linked internally (5th linkage ) to of imidazole ring of Histidine of the Globin polypeptide chains

. Other position is available to bind Oxygen

Heme is the most prevalent metalloporphyrin in humans Common prosthetic group in

 Hemoglobin — Transport of O2 in blood Myoglobin — Storage of O2 in muscles Cytochromes — Part of electron transport chain

Catalase — Degradation of H2O2 Tryptophan pyrolase — Oxidation of Tryptophan Cytochrome P450 — Hydroxylation of Xenobiotics HEME SYNTHESIS Major sites  Liver  Erythrocyte producing cells of bone marrow

 Rate of heme synthesis in liver is highly variable & depends upon size of heme pool while it is relatively constant in in bone marrow is relatively constant

Mature RBC lack mitochondria and are unable to synthesize heme. Initial & last 3 reactions occur in mitochondria Intermediate steps takes place in cytosol

1. Formation of δ-amino levulinic acid

Glycine (non- essential A.A) Succinyl CoA (intermediate in citric acid cycle)

Glycine & succinyl CoA condense to form ALA- δ Amino levulinic acid

. Enzyme is ALA synthase

. Co-enzyme is pyridoxal PO4 . An intermediate compound α-amino β-keto- adipic acid is formed

which on losing CO2 gives rise to ALA . ALA synthase activity is the rate-limiting factor in the synthesis of ALA Synthase activity is increased by  Barbiturates  Sulfonylureas

& decreased by  Heme/ hemin  High carbohydrate diet End product inhibition by hemin: When porphyrin production exceeds the availability of globin or other apoproteins heme accumulates & is converted to hemin by oxidation of Fe2+ to Fe3+

Hemin decreases the activity of ALA synthase by decreasing the synthesis of enzyme, through inhibition of mRNA synthesis. Effect of drugs on ALA synthase Administration of certain drugs increase hepatic ALA synthase activity eg. . Griseofulvin – antifungal . Hydantions . Phenobarbitols- (anti convulsants used in t/m of epilepsy) These drugs are metabolized by microsomal cytochrome p450 monooxygenase system – a hemprotein oxidase system found in liver. In response to these drugs synthesis of cytochrome p450 increases leading to an increased consumption of heme - a component of cytochrome p450

This in turn decreases the heme conc in liver cells , leading to synthesis of ALA synthase resulting in increase in ALA synthesis. 2.Formation of

The condensation of 2 molecules of ALA takes place to give rise to porphobilinogen- mono pyrrole

 This reaction takes place in cytosol. o Enzyme – ALA dehydratase ( Zinc containing enzyme ) oIt needs reduced glutathione oALA dehydratase is extremely sensitive to inhibition by heavy metal ions like lead oThis inhibition is responsible for the elevated ALA and anemia seen in lead poisoning 3.Formation of . Porphobilinogen is converted to hydroxy methylbilane by the cytosolic enzyme I synthase . Hydroxy methyl bilane is linear tetrapyrrole 4.Formation of uroporphyrinogen III

Hydroxymethylbilane can spontaneously change into uroporphyrinogin I Uroporphyrinogin is the first cyclic tetra pyrrole derivative Hydroxymethylbilane is converted in to uroporphyrinogen III Enzyme –Uroporphyrinogen co-synthase are colourless compounds

In porphyrinogens four pyrrole rings are attached to each other by methylene

(-CH2-) bridges & N of each pyrrole ring has one H attached

Porphyrinogens are readily converted by auto-oxidation to their respective porphyrins which are coloured compounds 5.Formation of coproporphyrinogen III

. Occurs in cytosol

. Acetate groups of uroporphyrinogen III

lose CO2 & are changed to methyl groups

. Enzyme – Uroporphyrinogen III decarboxylase

6.Formation of protoporphyrinogen IX

Coproporphyrinogen III enters the mitochondria & its 2 propionate side chains are decarboxylated to vinyl groups forming protophophyrinogin IX Enzyme : Coprophyrinogen III oxidase oxidation & decarboxylation of 2 propionate chains 7.Formation of protoporphyrin IX

Enzyme: Protoporphyrinogen IX oxidase. 8.Formation of Heme Introduction of iron into protoporphyrin occurs spontaneously but rate is increased by enzyme ferochelatase Reduced glutathione is needed for this reaction Enzyme – Ferochelatase This enzyme is inhibited by lead

Heme containing Proteins

 Myoglobin  Hemoglobin Myoglobin

A heme protein present in heart & skeletal muscles functions as

A reservoir for oxygen An oxygen carrier that increase the rate of transport of oxygen within the muscle cell Myoglobin consists of a single polypeptide chain 1. α Helical Content Myoglobin is a compact molecule with approximately 80% of its polypeptide chains folded into 8 stretches of α-Helix labeled as A to H. These α helix regions are terminated by

 The presence of Proline whose 5- membered ring can not be accommodated in an α helix or β-bends and loops stabilized by hydrogen bonds and ionic bonds Location of polar and non polar Amino acid residues

Interior consists almost entirely of nonpolar residues

Exterior consists of charged, polar and nonpolar residues

Histidine is found in the interior of the protein which play a critical role in the binding of iron and oxygen

The polar amino acids can form hydrogen bonds with each other and with water Binding of the heme groups The heme group of myoglobin sits in crevice in the molecule which is lined with non polar amino acids

Two histidine residues are very important . Proximal histidine (F helix) binds directly to the iron of heme . Distal histidine (E helix) helps to stabilize the binding of oxygen to the ferrous iron

Molecular wt. — 17,000

Combines with O2 to form oxymyoglobin & serves as O 2 reservoir within muscle cells

The O2 binding power of myoglobin is much more so oxygenated myoglobin cannot readily provide its O2 to tissue

When pO2 of tissues falls to 5 mm of Hg or below oxy Mb dissociates to provide O2 to tissue mitochondria for ATP formation. HAEMOGLOBIN Haemoglobin — a conjugated protein Heme — prosthetic group Globin — apo protein part 4 polypeptide chains of 2 different monomeric units ( α and β chains ) Each α chain contains 141 amino acids Each β chains 146 amino acids Each sub unit contains a heme group Found exclusively in RBCs Mol. wt.— 64458

Main function is to

 transport O2 from lungs to the tissues transport of CO2 from tissues to lungs

Hb A

Major adult Hb is composed of four polypeptide chains 2 α chains 2 β chains These 4 chains are held together by non- covalent interactions. Each subunit has stretches of α-helical structure & a heme binding pocket similar to that of myoglobin

 The tetrameric Hb molecule is structurally & functionally more complex than myoglobin

+ Hb can transport H and CO2 from tissues to lungs and can carry 4 molecules of O2 from the lungs to cells of the body

. Oxygen binding properties of Hb are regulated by interaction with allosteric effectors Quaternary structure of Hb The Hb tetramer is composed of 2 identical diamers (α β)1 and (α β)2

The two polypeptide chains in each diamer are held tightly together primarily by hydrophobic interactions . Hydrophobic amino acid residues are localized in the interior & also on the surface of the each subunit

. Interchain hydrophobic interactions form strong associations between α and β sub units in the diamers

. Ionic and H+ bonds also occur between the members of a diamer The two diamers are able to move with respect to each other being held together primarily by polar bonds

The weaker interactions between these mobile diamers result in the 2 diamers occupying different relative positions in deoxy Hb as compared with oxy Hb

The binding of O2 to heme iron pulls the iron into the plane of heme The iron is also linked to the proximal histidine (F8), there is movement of globin chains that alters the interface between α β dimers

Hemoglobin exists in 2 forms

T form ( Taut /Tense form) R form ( Relaxed form)

T form

The deoxy from of Hb is called the T or Taut (Tense) form In T form the two α β diamers interact through a network of ionic bonds & H+ bonds that constrain the movement of polypeptide chains

The T form is the low O2 affinity form of Hb R form

The binding of O2 to Hb causes the rupture of some of the ionic bonds & H+ bonds between α β diamers

This leads to a structure called R or relaxed form in which polypeptide chains have more freedom of movement

R form is high O2 affinity form of Hb

Binding of O2 to Myoglobin & Hb

Myoglobin can bind one molecule of O2 (one heme group)

Hemoglobin can bind 4 O2 (4 heme groups)

The degree of saturation (Y ) of these O2 binding sites on all myoglobin or hemoglobin molecules can vary between Zero (all sites are empty) and 100%(all sites are full) O2 Dissociation curve The OxyHb dissociation curve describes the relation between partial pressures of

O2 ( X axis) & the Oxygen saturation (Y axis)

This graph shows that myoglobin has higher oxygen affintiy at all pO2 values as compared to Hb

The pressure of O2 needed to achieve half saturation of binding sites (P50) is approximately 1mm of Hg for myoglobin and 26 mm of Hg for hemoglobin

The higher the oxygen affinity the lower

P50 as O2 binds more tightly Myoglobin

The O2 dissocation curve for myoglobin has hyperbolic shape

Myoglobin reversibly binds a single molecule of O2 Oxygenated and deoxygenated myoglobin exists in a simple equilibrium The equilibrium is shifted to the right or left as O2 is added to or removed from the system

Myoglobin is designed to bind oxygen released by hemoglobin at the low pO2 found in muscle

Myoglobin in turn releases O2 within the muscle cell in response to oxygen demand Hemoglobin

The O2 dissociation curve for Hb is sigmodal in shape

Cooperative binding of O2 by the four subunits of Hb means that the binding of an oxygen molecule at one heme group

increases the O2 affinity of the remaining of the heme groups in the same Hb molecule This effect is referred as heme- heme interaction

Although it is more difficult for the first

O2 molecule to bind with hemoglobin, the subsequent binding of O2 occurs with high affinity Allosteric effect

 The ability of Hb to reversibly bind O2 is affected by a) pO2 b) pH c) pCO2 d) 2-3 BPG 1.Heme-Heme interaction The sigmoidal oxygen –binding curve reflects specific structural changes that are initiated at one heme group & transmitted to other heme groups in hemoglobin tetramer

The net effect is that the affinity of Hb for last O2 bound to it is 300 times greater than its affinity for the first oxygen bound

Loading and unloading oxygen

. The cooperative binding of oxygen allows Hb to deliver more oxygen to the tissues in response to relatively small change in the

pO2

. The pO2 is different in lungs and tissues .In lungs the pO2 is high

.Hb saturated (loaded) with O2

.In peripheral tissue pO2 is less oxy Hb releases its O2 (unloads) Significance of sigmoidal O2 dissociation curve

Hb carry & deliver O2 efficiently from sites of high to sites of low pO2 (Sigmoidal curve)

Mb has maximum affinity for O2 through out this O2 pressure & can not achieve the same degree of O2 release within this range of pO2 ( Hyperbolic curve)

Bohr effect

 Release of O2 from Hb is increased (in peripheral tissues) . pH↓

. pCO2 ↑ Results in a

1. ↓ O2 affinity of Hb 2.Shift to the right ( O2 dissociation curve ) 3. Stabilize the T state

This change in O2 binding is known as Bohr effect  Binding of O2 with Hb is increased (In lungs) . ↑Ph

. ↓ pCO2 Results in

1.↑ O2 affinity of Hb

2.shift to the left ( O2 dissociation curve) 3. Promoting the R State a. Sources of protons that lower pH + The conc of CO2 and H in capillaries of metabolically active tissues is higher than

in alveolar capillaries of lungs where CO2 is released in expired air Lungs Tissues pO2 pCO2 pH In the tissues CO2 is produced by .

It combines with H2O and is converted into H2 CO3 Reaction is reversible.

CO2 + H2O H2 CO3 Enzyme :-Carbonic anhydrase H2 CO3 spontaneously loses a proton - and forms HCO 3 - + H2 CO3 HCO 3 + H

The H+ produced by this reaction decreases the pH. This differential pH gradient (Lungs having higher pH and tissue a lower pH) favours the unloading of oxygen in the peripheral tissue and the loading of O2 in the lungs. So the O 2 affinity of Hb molecule responds to small shifts of pH between lungs and tissues making Hb a more efficient

transporter of O 2. b.Mechanism of Bohr Effect

The bohr effect shows that the deoxy Hb has a greater affinity for protons than does oxy Hb. This effect is due to ionizable groups such as N-Terminal α- amino groups and specific histidine side chains that have higher pkas in dexoy Hb than oxy Hb so an increase in concentration of protons resulting in decrease in pH causes these groups to become protonated(charged) and able to form ionic bonds also known as salt bridges. These bonds stabilize the deoxy Hb

producing a decrease in O2 affinity Bohr effect is represented as

+ HbO2 + H HbH + O2

Oxy Hb Deoxy Hb An increase in Protons and decrease in pO2 shifts equilibrium to right favours deoxy Hb as in Tissues

An increase in pO2 and decrease in protons shifts equilibrium to left favours oxy Hb as in lungs .

3. Effect of 2, 3 BPG on O2 affinity 2,3- Bisphosphoglycreate (2,3BPG) is an important regulator of the

binding of O2 to Hb.

It is most abundant organic PO4 in RBC where it’s concentration is approximately equal to that of Hb. 2,3-BPG is synthesized from an intermediate of glycolytic pathway. Glycolysis Glucose

1,3-BPG 2,3-BPG

H2O

PO4 3, Phosphoglycreate

Pyruvate

Lactate Binding of 2,3-BPG to dexoy Hb.

2,3-BPG decreases the O2 affinity of Hb by binding to deoxy Hb. This binding stabilizes the T- taut conformation of deoxy Hb.

HbO2 +2,3 BPG Hb-2,3-BPG + O2 Oxy Hb Deoxy Hb.

So it facilitates O2 release from Hb O2 b. Binding site of 2,3- BPG

One molecue of 2,3- BPG binds to a pocket formed by the two β-globin chains in the centre of deoxy Hb tetramer. This pocket contains several + vely charged A.A that forms ionic bonds with –vely

charged PO4 groups of 2,3-BPG. 2,3-BPG is expelled on oxygenation of Hb.

c. Shift of O2 dissociation curve Hb from which 2,3-BPG has been removed has a high affinity for O2. However the presence of 2,3 BPG in RBC significantly the affinity of Hb for O2 shifting the O2 dissociation curve to the right. This decreased affinity helps Hb to release O2 efficiently at the pO2 found in tissues. d. Response of 2,3-BPG to chronic hypoxia or anemia. The conc. of 2,3 BPG in RBC increases in response to chronic hypoxia. eg as in (i) Obstructive pulmonary emphysema (ii) At high altitude In these conditions circulating Hb may

have difficulty in receiving sufficient O2 Intracellular levels of 2,3-BPG are also elevated in chronic anemia. In chronic anemia less than normal RBC

are available to supply O2 to the body . Increased 2,3-BPG levels decreases the

O2 affinity of Hb permitting greater unloading of O2 in the tissue capillaries. e. Role of 2,3-BGP in transfused Blood

2,3 BPG is essential for normal O2 transport function of Hb. Storing blood in Acid -Citrate- Dextrose leads to a decreased 2,3 BPG in RBC. Such blood has an abnormally high oxygen affinity and fails to unload O2 properly in the tissues . Hb deficient in 2,3 BPG thus act as an

O2 trap rather than O2 transport system. Transfused RBC take 24-48 hours to restore their 2,3 BPG but severely ill

patients need blood with proper O2 transport system. This decrease in 2,3BPG can be prevented by adding substrates such as inosine to the storage medium. Inosine is Hypoxanthine and Ribose . It is an uncharged molecule. It can enter RBC where its ribose is released , phosphorylated and enters the HMP pathway and eventually being converted to 2,3-BPG. 4. Binding of CO2

Most of CO2 produced is hydrated and - transported as HCo 3.

Some of CO2 is carried as Carbamate bound to uncharged α –Amino groups of Hb – Carbamino-Hb.

- Hb-NH2+ CO2 Hb-NH-COO + H+ The binding of CO2 stabilizes the T or deoxy form of Hemoglobin resulting in

a decrease in its affinity for O2 .

In lung CO2 dissociates from Hb and is released in the breath. 5 Binding of CO CO binds tightly but reversibly to Hb iron forming Carbon monoxy Hb or Carboxy Hb. When CO binds to one or more of the heme sites Hb shifts to relaxed conformation causing the remaining

heme sites to bind O2 with high affinity .

This shifts O2 saturation curve to left and changes the normal sigmoidal shape towards a hyperbola. As a result affected Hb is unable to

release O2 to the tissues. The affinity of Hb for CO is 220 times

greater than O2 so even minute amounts of CO in the environment can produce toxic concentrations of Carbon monoxy Hb in the blood. eg; Increased levels of CO are found in the blood of tobacco smokers. CO toxicity appears to result from a combination of tissue hypoxia and direct CO-mediated damage at the cellular level.

CO poisoning is treated with 100% O2 therapy, which facilitates the dissociation of CO from the Hb. Minor Hemoglobins form family of proteins functionally and structurally related to each other .

Each of these O2 carrying proteins is a tetramer, composed of 2 α- globin polypeptides and 2-β- globin or globin like polypeptides Certain Hemoglobins are normally synthesized only during certain period. Fetal life eg HbF

Adults eg HbA Very low level HbA2

Diabetics eg HbA1c Form Chain Fraction of composition Total Hb

HbA α2 β2 90%

HbF α2 γ2 2%

HbA2 α2 δ2 2-5%

HbA1c α2 β2 glucose 3-9 % Normal adult Hb 1.Fetal hemoglobin (HbF)

HbF is a tetramer consisting of 2 α chains identical to those found in

HbA, plus two γ chains α2 γ2 The γ chains are members of β-globin gene family. a. HbF synthesis during development In the first few weeks after conception embryonic Hb (Hb gower1) composed of two zeta chains and two epsilon chains

( ς2 ε2 ) is synthesized by the embryonic yolk sac. Within few weeks the fetal liver begins to synthesize HbF in the developing bone marrow. HbF is the major Hb found in the fetus and newborn accounting for about sixty percent of the total Hb in the erythrocytes during the last months of fetal life.

HbA synthesis starts in the bone marrow at about the eighth month of pregnancy and gradually replaces HbF. b. Binding of 2,3- BPG to HbF Under physiologic conditions HbF has

a higher affinity of O2 than does HbA as a result of HbF’s binding only weakly to 2,3, BPG . The γ - globin chains of HbF lack some of +vely charged amino acids that are responsible for binding 2,3-BPG in the β- globin chains. As 2,3-BPG decreases the affinity of Hb for O2 the weaker interaction between 2,3-BPG and HbF results in a higher O2 affinity for HbF relative to HbA. When free , HbA and HbF have similar affinity for O2. The higher O2 affinity of HbF facilitates the transfer of O2 from the maternal circulation across the placenta to RBC of the fetus. 2. Hemoglobin A2 ( HbA2)

HbA2 is a minor component of normal adult Hb. It first appears about 12 weeks after birth. Ultimately it constitutes about 2% of the total Hb. It is composed of 2 α- globin chains and

2 δ–globin chains (α2 δ2 ) 3. Hemoglobin A1c (HbA1c) Under physiologic conditions HbA is slowly and non enzymically glycosylated. The extent of glycosylation being dependent on the plasma concentration of a particular hexose. The most abundant form of

glycosylated Hb is HbA1c . It has glucose residues attached predominantly to the NH2 groups of the N-terminal valines of the β globin chains.

Increased amounts of HbA1c are found in RBC of patients with diabetes mellitus, because their HbA has contact with higher glucose concentrations during the 120-days life time of these cells This is used for assessing average blood glucose levels of diabetic patients .

Organization of globin genes Hemoglobin genes direct the synthesis of different globin chains. They are structurally organized into gene families Alterations in these gene families result in changes in structure and function of Hb. Gene families are divided into . α- gene family β - gene family α- Gene family The genes coding for α globin like subunits of the Hb chains occur as a separate cluster. The α cluster is located on chromosome No 16 . It has two genes for α globin chains. It also contains the (i) Zeta ς gene that is expressed early in the development of embryo as a component of embryonic Hb. (ii) A number of globin like genes that are not expressed . These are known as pseudo genes. β-Gene family

A single gene for β-globin chain is located on chromosome No11. There are additional β-globin like genes : (i) ε epsilon gene expressed in embryonic development

(ii) 2γ genes (Gγ and A γ ) expressed in HbF

(iii) δ gene present in HbA2

Steps in globin chain synthesis Expression of a globin gene begins in the nucleus of red cell precursors where the DNA Sequence encoding the gene is transcribed . The RNA produced by transcription is actually a precursor of the mRNA – messenger RNA that is used as a template for the synthesis of a globin chain. Before it starts functioning two non coding regions of RNA(introns) must be removed and the remaining 3 fragments (exons-coding regions) are reattached in a linear manner. The resulting mature mRNA enters cytosol where it’s genetic information is translated producing a globin chain.

Hemoglobinopathies Hemoglobinopathies are defined as a family of genetic disorders caused by production of a • Structurally abnormal Hb molecule • Synthesis of insufficient quantities of normal Hb • Rarely both Imp hemoglobinopathies include. • Sickle cell anemia (HbS) qualitative. • Hemoglobin C disease (HbC)qualitative. • Thalassemia syndromes quantitative. Qualitative hemoglobinopathies Due to production of Hb with an altered amino acid sequence.

Quantitative hemoglobinopathies Due to the decreased production of normal Hb. a. Sickle cell disease Also known as Hb S disease . It is a genetic disorder of blood caused by a single nucleotide alteration -point mutation in the β- globin gene. It is most common inherited blood disorder in the united states affecting 80,000 Americans. It primarily occurs in African – Amercian population ( Infants) in the united states. It is homozygous recessive disorder. It occurs in individuals who have inherited 2 mutant genes (one from each parent ) that code for synthesis of the β-chains of the globin molecules. The β-mutant gene is designated as βs and s resulting Hb as α2 β 2 referred as HbS. An infant does not begin showing symptoms of the disease until sufficient HbF has been replaced by HbS so that the sickling can occur. Sickle cell disease is characterized by life long episodes of Pain (crises) Chronic hemolytic anemia Susceptibility to infections usually beginning in early childhood The life time of an erythrocyte in sickle cell disease is less than 20 days as compaired to 120 days of normal RBC, hence causing anemia. Other symptoms include  Acute chest syndrome  Stroke  Splenic dysfunction  Renal dysfunction  Bone changes due to marrow hyperplasia . Heterozygotes represents 1 in 12 African –Americans. They have 1 normal and 1 sickle cell gene Blood cells of these heterozyotes contain both HbA and HbS. These individuals have sickle cell trait They usually do not show clinical symptoms and can have normal life. Amino acid substitution in HbS β Chain A molecule of HbS contains 2 normal α- globin chains and two mutant β-globin chains (βs ) in which glutamate at position 6 has been replaced by valine. During electrophoresis at alkaline pH HbS migrates more slowly towards the anode ( +Ve) electrode than does HbA. This altered mobility of HbS is a result of absence of –vely charged glutamate residues in two β- chains making HbS less-ve than HbA. Electrophoresis of Hb obtained from lyzed RBC is routinely used in the diagnosis of sickle cell trait and sickle cell disease. Effect of sickling -Tissue Anoxia The substitution of non polar valine for a charged glutamate residue forms protrusion on the β- globin chain that fits into a complementary site on the α- chain of another Hb molecule in the cell.

At low O2 tension deoxy HbS polymerizes inside the RBC first forming a gel, then subsequently assembling into a net work of fibrous polymers that stiffen and distort the cell, producing rigid and mis shaper erythrocytes. Such sickle cells frequently block flow of blood in narrow capillaries .

This interruption in the supply of O2 leads to localized anoxia in tissues causing pain and eventually death (infarction) of cells in the vicinity of blockage. The normal diameter of RBC is 7.5 µm and that of micro vessels is 3 to 4 µm normally RBC pass through them by squeezing. As sickled cells have a ability to deform and so have difficulty in moving through small vessels.

Variables that increase sickling The extent of sickling and severity of disease is enhanced by any factor that increase the proportion of HbS in the deoxy state and reduces the affinity of

HbS for O2. These factors include

• Decreased O2 tension as a i Result of high altitude ii Flying in non pressurized plane

• Increased pCO2 • Decreased pH • Dehydration • Increased Concentration of 2,3,- BPG in RBC Treatment T/M involves • Adequate hydration • Analgesics • Aggressive antibiotic therapy if infection • Transfusion in patients at high risk for fatal occlusion of blood vessels. Intermittent transfusions with packed red cells decreases the risk of stroke but benefits must be weighed against the complications of transfusion like  Iron over load (hemosidrosis)  Blood born infections  Immunologic complications • Hydroxy urea, an antitumor drug is therapeutically useful because it increses circulating levels of HbF, which decreses RBC sickling. • This leads to decreased frequency of painful crises and mortality. Advantage of hetrozygous state The high frequency of the HbS gene amoung black African despite its damaging effect in the homozygous state suggests a selective advantage for hetrozygous individuals. e.g. heterozygotes for the sickle cell gene are less susceptible to malaria caused by the parasite Plasmodium falciparum. This organism spends an obligatory part of its life cycle in the RBC. Because these cells in individuals hetrozygous for HbS like those in homozygous have a shorter life span than normal, the parasite can not complete the intracellular stage of its development. This fact may provide a selective advantage to hetrozygotes living in regions where malaria is major cause of death. The morbidity and mortality associated with sickle cell disease has led screening panels to allow prophylactic antibiotic therapy to begin soon after the birth of an affected child. Hemoglobin C disease HbC is the Hb that has a single amino acid substitution in the 6th position of the β-globin chain. In HbC a lysine is substituted for the glutamate. This substitution causes HbC to move more slowly toward the anode than HbA or HbS does.

Patients homozygous for HbC generally have relatively mild, chronic hemolytic anemia. These patients do not suffer from infarctive crises. No specific therapy is required. Hemoglobin SC disease In this disease some β-globin chains have the sickle cell mutation and other β- globin chains carry the mutation found in HbC disease. Patients with HbSC disease are doubly heterozygous (compound heterozygous) because both of their β globin genes are abnormal, although different from each other. Hb levels are higher in HbSC disease than in sickle cell disease and may be at the low end of normal range. The patients with HbSC disease remain well and undiagnosed until they suffer an infarctive crises. This crises often follows child birth or surgery and may be fatal. Met Hemoglobinemias Oxidation of heme component of Hb to ferric (Fe3+)) state forms met Hb which

can not bind O2. The extra valence of ferric iron serves to bind a hydroxyl ion or other –ve group. Met Hb is dark brown in colour and its excess in blood produces a picture of cynosis. The presence of 1.5 grams of met- Hb/dl of blood results in the same degree of cyanosis as by 5 gm of deoxy Hb/dl of blood. Under normal circumstance a small fraction of the blood Hb gets converted to met Hb. The met Hb normally formed is reduced by met -Hb reductase. Formation of met-Hb takes place by the dissociation of super oxide anion from oxy-Hb. 2+ 3+ - Hb +O2 Hb +O2 The met-Hb so formed is reduced by the following reaction. Hb3+ +cyt b Hb2+ + cyt b (reduced ) (oxidized) Cytb (oxidized) is reconverted into its reduced form by the reaction.

Cyt b +NADH +H+ Cyt b + NAD+ (oxidized) (reduced)

This reaction is catalyzed by the enzyme cytochrome b reductase also known as met-Hb reductase. Ascorbic acid and Glutathione also function to reduce met-Hb . The amount of met-Hb in human blood is on an average 1.7% of total blood Hb. The absence of met-Hb reductase is responsible for Hereditary Methemoglobinemia Another cause of hereditary met hemo- globinemia is HbM disease. This Hb has abnormal α or β chains . The individuals with α- chain variants (HbM boston and Iwate) are cyanotic at birth . The individuals with β-chains variants (HbM Saskatoon, Hyde park and Milwankee) do not show cyanosis until they reach 4 to 6 months of age. HbM can be easily oxidized to met-Hb but the normal met Hb reducing enzyme systems fail to reduce it thus giving rise to met hemoglobinemia. These inherited disorders are clinically mild and except for cyanosis are asymptomatic Several drugs and toxins can oxidize Hb to met-Hb. These include

Chlorates Phenacitin Peroxides Sulfonamides Hydroquinone Procaine Pyrogallol Lidocaine Iodine Aniline Met hemoglobinemia occurs mostly in workers who work with aromatic nitro or amino compounds or in patients taking sulfonamides Sometimes it proves fatal. Death is due to hypoxia resulting from the following factors.

1. Met Hb like CO-Hb can not act as O2 carrier . 2. The presence of met Hb in blood also inhibits the dissociation of the remaining oxy-Hb. This aggravates hypoxia still further. This lead to cyanosis and if met-Hb level is high then dyspnea also occur . Methylene blue and Ascorbic acid are useful in treatment of met-Hbemia as they are good reducing agents. It’s cyanosis is known as Chocolate cyanosis - brown blue discolouration of skin and membrane. Blood is also dark coloured due to dark colour of met Hb- Chocolate blood . Anxiety and headache may also present. One very imp. property of met. Hb is it’s combination with cyanide to form cyanmet –Hb. This property is used for T/M of Cyanide poisioning. Nitrites are given to produce met Hb which then takes up cyanide to form cyan met-Hb. In this way cyanide ions are removed from circulation and they cannot exert their poisonous effect on the Cytochrome oxidase. Later on the cyanide is gradually detoxified to Thiocyanate and met Hb is converted to Hb by the reducing system present in RBC. Quantitative abnormalities

Thalassemias In these congenital disorders there is defect in the synthesis of one or more globin sub units of Hb They are the most common single gene disorders in humans. The RBC are hypochromic and microcytic The severty of disease varies from mild to life threatening degrees. In thalassemias there is quantitative abnormalities of polypeptide chain synthesis. In thalassemias synthesis of either α or β-globin chains is defective . A thalassemia can be caused by a variety of mutations including Entire gene deletions Substitutions and deletions of one or more nucleotides in DNA. According to decreased synthesis of α and β globin chains, the disease may be either α or β thalassemias Thalassemias can be differentiated by 1.RBC morphology 2. Hb electrophoresis 3. Globin chain synthesis in reliculocytes in vitro 4. Clinical severity.

A definite diagnosis can only be made by analysis of globin gene structure. α-Thalassemia The synthesis of α globin chains is decreased or absent as a result of gene deletional mutation There are 4 α globin genes in each individual . Severity of disease depends upon number of α genes defected Silent carrier α thalassemia trait Hb H disease Bart Hb disease Silent carrier If one of 4 genes is defective no physical manifestations of the disease occur and individual is termed silent carrier of disease. α Thalassemia trait Two α-globin genes are defective .They have little clinical effect. Also known as α-thalassemia minor.

Hb H disease Deletion of 3 globin genes result in Hb H disease Clinical symptoms include Hemolytic anemia Microcytic and hypochromic anemia RBC show Heinz bodies due to precipitated Hb H

Hb H has high affinity for O2 and delivers O2 poorly to tissues. Hb H is unstable and oxidatively denatured in Infections or upon exposure to oxidative drugs like Sulfonamides. Hb bart disease It is due to deletion of 4 genes. It is incompatible with life. Fetus dies in utero by hydrops fetalis. Hb is bart type (gamma 4)

β- Thalassemia Synthesis of β- globin chains is decreased or defective or absent as a result of point mutation that effect production of functional mRNA In most of cases the synthesis of β- globin chains my be defective due to . Premature β-chain termination Defect at a post- transcription stage i.e wrong splicing up of mRNA.

β- thalassemias are divided into

i β- thalassemia minor -trait ii β- thalassemia major –cooley’s anemia. β- thalassemia minor It is present in heterozygotes. It is relatively mild form and no T/M is required. β- thalassemia major It is seen in homozygotes Both β genes are defective. Most severe form of congenital hemolyitic anemia. Severe anemia appears at the age of 4 to 6 months when gamma chain formation is normally replaced by β chain formation which in this case does not take place. The ineffective erythropiosis is increased and there is compensatory increase in the synthesis of gamma and to a lesser extent delta chains. In typical cases 3 types of Hb A, A2 and F are zero, 4 to 10 and 90 to 96% respectively. Blood film shows abnormal RBC morphology i.e Anisocytosis Poikilocytosis (tear drop and cigar shaped cells) Target cell Stippled cell Excessive hemolysis of abnormal RBC takes place as excess of α-chains in the presence of decreased β- chains are unstable and precipitate . This leads to anemia of hemolytic type and . Infant shows a combination of pallor , icterus and excessive melanin in skin. Cardiomegaly Splenomegaly Hepatomegaly Skeletal lesions are seen due to compensatory hyperplasia of erythroid bone marrow. Enlargment of malar bones may produce chipmunk faces Mal-occlusion of teeth Growth is retarted Short life expectancy Treatment Needs frequent Blood transfusions which result in iron load, infections and alloimmunization Bone marrow transplant Splenectomy Folic acid adminstration.

Factors affecting the synthesis of Hb Normally the bone marrow produces about 7gm of Hb each day. Various factors which affect the formation of Hb and RBC are as follows. 1.Metals These include iron , copper , cobalt and manganese. 2.Protein diet Good quality proteins are necessary to supply essential amino acids for synthesis of globin. 3.Vitamins

Vitamin B12, Folic acid , Ascorbic acid, Nicotinic acid and Pyridoxin are of special importance in Hb synthesis. 4.Hormones Growth hormone and thyroid hormone take active part in Hb formation. In males red cell mass is more due to testosterone. Estrogen depresses erythropoiesis. Cortisol stimulates erythropoiesis. 5.Hypoxia and Erythropoietin (EPO) The stimulating effect of hypoxia on erythropoiesis is brought about through an increased formation of a hormonal agent erythropoietin (EPO) produced mainly by kidney. 6.Cyclic AMP (cAMP) It increases erythropoiesis by a direct action on the bone marrow as well as increase in erythropoietin formation. Porphyrias Porphyrias are rare inherited or occasionally acquired defects in the pathway of heme biosynthesis resulting in the accumulation and increased excretion of porphyrins or porphrin precursors. Porphyrias are inherited as (i) Autosomal dominant disorders –All (ii) Recessive disorder – Congenital erythorpoietic porphyria The mutations that cause porphyrias are hetrogenous- not all are at the same DNA locus Nearly every affected family has its own mutation. Each porphyria results in the accumulation of a unique pattern of intermediates caused by the deficiency of an enzyme in the heme synthetic pathway. Porphyria refers to the purple colour caused by a pigment- like porphyrin in the urine of some patients with defect in heme synthesis. Porphyrias are classified according to the site of enzyme defect. 1. Erythropoietic 2. Hepatic

Hepatic porphyrias are further classified into. (a) Acute porphyria (b) Chronic porphyria Clinical manifestations Individuals with an enzyme defect leading to accumulation of tetra pyrrole intermediates show photosensitivity – skin itches and burns (pruritis) when exposed to visible light. These symptoms are due to formation of porphyrin mediated superoxide radicals from oxygen. The reactive O2 species can oxidatively damage membranes and cause the release of destructive enzymes from lysosomes . Destruction of cellular components leads to photosensitivity. Chronic porphyria Porphyria cutanea tarda is most common porphyria. It is chronic disease of liver and erythroid tissue. It is associated with deficiency of Uroporphyrinogen decarboxylase. Clinical expression of enzyme deficiency is influenced by various factors like Hepatic iron overload Exposure to sunlight Presence of hepatitis B, C or HIV Clinical onset is typical duing 4th or 5th decade of life. Porphyrin accumulation leads to Cutaneous symptoms –Rashes and skin eruptions. Urine- Red to brown in natural light Pink to red in fluorescent light. Acute hepatic porphyrias They include (i)Acute intermittent porphyria (ii) Hereditary coproporphyria (iii) Variegate porphyria It is characterized by acute attacks of gastrointestinal, neurologic, psychiatric and cardiovascular symptoms Porphyrias leading to accumulation of ALA and porprobilinogen such as Acute intermittent porphyria cause abdominal pain and neuropsychiatric disturbances Symptoms of acute heptic porphyrias are often ppt. by administration of drugs like barbiturates and ethanol These drugs induce synthesis of heme containing cytochrome p450 microsomal drug oxidation system This further decreases available heme which in turn promotes the synthesis of ALA synthase. Erythropoietic Porphyrias Erythropoietic porphyrias are 1. Congenital erythropoietic porphyria 2. Erythropoietic protoporpyria They are characterized by skin rashes and blisters that appear in early childhood. This disease is complicated by cholestatic liver cirrhosis and progressive hepatic failure Increased ALA synthase activity

One common feature of porphyrias is decreased synthesis of heme . In liver heme normally functions as a repressor of ALA synthase. Therefore absence of this end product results in an increase in the synthesis of ALA synthase. (derepression ) This causes an increased synthesis of intermediates that occur prior to genetic block .

The accumulation of these toxic intermediates is the major pathophysiology of porphyrias Treatment

During acute attack supportive T/M for pain and vomiting. Severity of attack can be decreased by I/V hemin which decreases synthesis of ALA synthase. Avoid sunlight.

Degradation of Heme After approximately 120days in circulation RBC are taken up by the reticuloendothelial cells particularly in liver and spleen and degraded. Approximately 85% of heme comes from degradation of RBC. 15% come from turnover of immature RBC and cytochromes. About 7 gm of Hb is released daily from these degraded RBC Hb molecule is broken down into 3 parts 1.The protein part (Globin ) It is utilized partly as such or along with other body proteins. 2.The Iron. It is stored in reticuloendothelial cells and is reused in synthesis of Hb and other iron containing substances of body. 3. The Porphyrin part. It is converted into bile pigment i.e which is secreted in bile Stages involved in synthesis of bile pigment and its fate is as follows. 1.Hb dissociates into heme and globin Hb dissociates into heme and globin. Heme occurs mostly in ferri heme form (iron as Fe3+) before it reaches the microsomal fraction of rediculoendothelial system where it is catabolized. Here it is converted into ferro heme (iron as Fe2+) with the help of NADPH+H+. 2.Heme loses CO and Fe3+ Heme loses one molecule of CO and one atom of iron as Fe3+ and is converted in to . CO Fe3+ Heme Biliverdin

Enzyme : Heme oxygenase Co-enzyme NADPH+H+ In this reaction the porphyrin ring is cleaved by oxidation of α- methenyl bridge between pyrrole rings I and II of the porphyrin. + The enzyme needs NADPH+H & O2. In addition an abbreviated electron transport system is utilized which include cytochrome P450 and a flavoprotein Heme oxygenase is induced by its substrate i.e heme 3.Biliverdin is reduced to bilirubin Biliverdin is first bile pigment. It is green in colour . It is reduced to bilirubin –yellow in colour –which is main bile pigment

Biliverdin NADPH+H+ NADP +Bilirubin

Enzyme : Biliverdin reductase Co-enzyme: NADPH+H+ 1 gm of Hb forms 25mg of bilirubin. Daily 250-350mg of bilirubin are synthesized. Bilirubin is non-polar lipid soluble and water insoluble.

4.Uptake of bilirubin by liver

Bilirubin is slightly soluble in plasma and is transported to the liver by binding non-covalently to albumin.

Many substances compete for receptor sites on plasma albumin and can displace bilirubin. These include

Heme Free fatty acid Caffeine H+ Sulfonamides Salicylates About 25mg of bilirubin can be tightly bound with albumin per dl of plasma . Bilirubin dissociates from the carrier albumin molecule and enters the hepatocytes where it binds to intracellular proteins – ligandin Bilirubin enters the hepatocytes at their sinusoidal surface through a facilitated transport system. 5.Formation of Bilirubin diglucuoronide In the hepatocytes solubility of bilirubin is increased by the addition of 2 molecule of glucuoronic acid- by process of conjugation

Bilirubin + 2UDP-Glucuronic acid +2UDP

Enzyme :- Microsomal bilirubin glucuronyl transferase 6.Secretion of bilirubin into bile The conjugated bilirubin is water soluble and is secreted by active transport against con. gradient into bile canaliculi larger bile duct gall bladder and stored in bile biliverdin (green colour) This biliverdin gives green colour to bile. From gall bladder bile enter the small intestines

7. Formation of in intestines Bilirubin diglucuronide is hydrolyzed and reduced by bacterial β-glucuronidase into bilirubin and glucuronic acid. Bilirubin is acted upon by intestinal bacteria and converted into colour less tetra pyrrole compounds . Urobilirogen is converted to stercobilinogen (yellow brown colour) . Some of is reabosbed from gut and enter portal blood. A portion of this urobiloneogen participate into enterohepatic circulation, remainder is transported to kidney and converted into yellow urobilin and excreted in urine and give it characteristic colour.

Jaundice

Jaundice also known as Icterus is yellow colour of skin, nail beds and sclera of eyes caused by the deposition of bilirubin secondary to biliribin level in blood.

Sclera is rich in elastic tissue which has high affinity for bilirubin so scleral involvement is very first sign of hyperbilirubinemia.

Depends upon cause of plasma bilirubin level.

Pre hepatic jaundice Hepatic jaundice Post hepatic jaundice Pre-hepatic jaundice

Also known as hemolytic jaundice. This type of jaundice is due to plasma level of unconjugated bilirubin.

It is due to an excessive break down of RBC which leads to an production of bilirubin. As liver is not able to conjugate this excessive load the plasma bilirubin level leading to jaundice.

This unconjugated bilirbin can not be filtered by renal glumeruli so this bilirubin does not appear in urine. Prehepatic jaundice is also seen in neonates –physiological jaundice especially in pre matures due to deficiency of enzyme UDP-glucuronyl transferase. All liver function tests are normal. Urinary and fecal urobilin exceretion is

Hepatic jaundice

Also know as hepato cellular jaundice. This is typically seen in viral hepatitis A,B, C, D, & E Liver cells are damaged , inflammation produces obstruction of bile canaliculi due to swelling around them. Conjugation rate of biliribin is so un conjugated bilirubin level in blood.

The bilirubin which is conjugated can not be secreted in bile and leaks into the blood stream and level . Liver function tests are impaired.

Urobilinogen in urine as hepatic damage entero hepatic circulation so urine become dark colourd .

Stool become pale clay coloured. Post hepatic jaundice

Also known as obstructive jaundice .

It is due to obstruction of bile duct.

Bile can not reach s. intestine Cause

Hepatic tumor Bile stones

Pt. presents with gastrointestinal pain and nausea and reports pale, clay coloured stools and urine darkens upon standing The liver regurgitates conjugated bilirubin into the blood.

This conjugated biliribin is excreted in urine.

Liver function tests will vary depending upon extent of obstruction.

Properties of unconjugated and conjugated bilirubin

Property Un conjugated conjugated Bilirubin Bilirubin solubility Lipids YES No Water No YES

Excretion in urine No YES

Deposition in YES NO Brain nuclei Plasma level increase Pre Hepatic Hepatic and post Jaundice hepatic jaundice Neonatal jaundice

Newborn infants, especially if premature often accumulate bilirubin As the activity of hepatic bilirubin glucuronyl tranferase is low at birth and it can not conjugate bilirubin so level increases. This enzyme reaches adult level in 4 weeks. Elevated bilirubin can diffuse into Basal ganglia and can cause toxic encephlopathy- kernicterus New borns with bilirubin level are treated with blue fluorescent light which converts bilirubin to more polar and water soluble isomers. These photoisomers can be excreted without conjugation into bile.

Determination of Bilirubin conc.

Bilirubin is most commonly determined by Van dan bergh reaction. In this test Diazo sulfanilic acid reacts with Bilirubin to form red Azodipyrroles that are measured colorimetrically. In aqueous solution water soluble Conjugated Bilirubin reacts rapidly with the reagent – with in one minute and is said to be Direct reacting Bilirubin. The unconjugated bilirubin which is much less soluble in aqueous solutions reacts more slowly . When reaction is carried out in Menthol both conjugated and unconcjugated Biliribin are soluble and react with reagent giving Total Bilirubin value. The Indirect-reacting Bilirubin value is obtained by subtracting the Direct- reacting Bilirubin from Total Bilirubin. Delta Bilirubin

In long standing cases of Biliary obstruction & Chronic active hepatitis conjugated Bilirubin can form an irreversible, covalent complex with serum albumin referred to as Delta Biliribin or Biliprotein. As this Bilirubin is not free so it can not be excreted in urine. Only when albumin is catabolized it become free. As ½ life of serum albumin is 15-20 days so this Bilirubin remains detectable up to several week after relief of Biliary obstruction or Hepatic disease.