AND HEMOGLOBINOPATHIES

LECTURE 01 FATE OF RBCS AND

LECTURE 02 & 03 HEMOGLOBIN

FIRST YEAR MBBS 2020 Features of a Mature RBC

• Biconcave disc • Mean Diameter 7.8 um • Can deform easily. • Bag of fluid with dissolved substances and hemoglobin • No sub cellular particles • Metabolism – Anaerobic respiration- Glycolysis – Pentose phosphate pathway. RBC Count

• Remains remarkably constant although there

are some variations.

• MALE : 5.2 ± 0.3 x 106 /uL.

• FEMALE : 4.7 ± 0.3 x 106 /uL.

• Life span : 120 Days HEMOGLOBIN -CONTAINING PROTEINS

 Hemoglobin

 Catalase

 Some peroxidases

HEMOGLOBIN

• Metallo-conjugate protein • Molecular weight is 64,500 • One hemoglobin molecule is composed of four heme groups (subunits) attached with (having four polypeptide chains) • One hemoglobin molecule binds with it four molecules (eight oxygen atoms) • 1 gm hemoglobin carries 1.34 ml of oxygen • Heme synthesis occurs in the mitochondria of bone marrow erythroblasts HEMOGLOBIN FORMATION 2 succinyl-CoA + 2 glycine Pyrrole 4 pyrrole Protoporphyrin IX Protoporphyrin IX + Fe++ Heme Heme + Polypeptide

Hemoglobin chain (α β)

2 α chains + 2 β chains

Hemoglobin A Types of Hemoglobin • Variations in Hb subunit chains – W.r.t amino acid composition of polypeptide portion – Alpha, beta, gamma, delta

Type of Hb Chain Fraction of total composition Hb

HbA α2 β2 90%

HbF α2 γ2 < 2%

HbA2 α2 δ2 2-5 %

HbA1C α2 β2 - Glucose 3-9% Embryonic

• Gower 1 Two zeta & two epsilon chains

• Gower 2 Two alpha & two epsilon chains

• Portland Two zeta & two gamma chains Embryonic/Minor Hemoglobins Organization of Hemoglobin Genes Developmental changes in Hb

Hemoglobin F

• of the human fetus normally contains • Its structure is similar to that of except that the β chains are replaced by γ chains • hemoglobin F is α2γ2. The γ chains have 37 amino acid residues that differ from those in the β chain. • Fetal hemoglobin is normally replaced by adult hemoglobin soon after birth Hemoglobin F

• In certain individuals, it fails to disappear and persists throughout life. • In the fetal body, its O2 content at a given PO 2 is greater than that of adult hemoglobin because it binds 2,3-BPG less avidly. • Hemoglobin F is therefore critical to facilitate movement of O2 from the maternal to the fetal circulation, particularly at later stages of gestation where oxygen demand increases . • In young embryos there are, in addition, ζ and ε chains, forming Gower 1 hemoglobin (ζ2ε2) and Gower 2 hemoglobin (α2ε2). • Switching from one form of hemoglobin to another during development seems to be regulated largely by oxygen availability, with relative hypoxia favoring the production of hemoglobin F both via direct effects on globin gene expression, as well as upregulated production of erythropoietin. Fetal Hb Abnormalities of hemoglobin formation

Hb Type Chain Position Replacement Hemoglobinopathies Qualitative defects Glutamic acid by valine HbS Beta 6 Glutamic acid by lysine HbC Beta 6 Glutamic acid by lysine HbE Beta 26 Hb Bart’s Four gamma chains Hb H Four beta chains Thalassemia Quantative defects

Beta thalassemia beta chain Inadequate synthesis of beta chains Alpha thalassemia alpha chain Inadequate synthesis of alpha chains

Sickle cell anemia Sickle-Cell Anemia: A Base-Pair Substitution

1 Normal amino acid sequence at the start of the hemoglobin beta chain.

2 One amino acid substitution results in the abnormal beta chain of sickle hemoglobin (HbS). The sixth amino acid in such chains is valine, not glutamic acid.

3 Glutamic acid carries an overall negative charge; valine carries no charge. This difference causes the protein to behave differently. At low oxygen levels, HbS molecules stick together and form rod-shaped clumps that distort normally round red blood cells into sickle shapes. (A sickle is a farm tool with a crescent-shaped blade.)

Signs and symptoms Sickle Cell Anemia & trait

• Patients with heterozygous genotype (Hgb AS) have sickle cell trait • Patients with homozygous genotype (Hgb S) have • Sickle Cell Trait (Hgb AS) • Signs & Symptoms • Clinically normal • Acute vasoocclusion occurs only under extreme conditions (vigorous exertion at high altitude) • Painless hematuria sometimes present in adolescent males • Diagnostic testing – CBC and PBS normal – Hemoglobin electrophoresis shows that Hgb S comprises ~40% of hemoglobin and Hgb A 60% • Treatment – No treatment necessary – Genetic counseling appropriate Sickle Cell Anemia (Hgb SS)

• Signs and Symptoms – Vary significantly – some pt are virtually asymptomatic while others suffer repeated crises requiring hospitalization – • Chronic hemolytic anemia produces • Jaundice • Pigment (calcium bilirubinate) • Splenomegaly (early childhood only) • Splenic Infarct and atrophy in adulthood or Splenectomy • Poorly healing ulcers over the lower tibia Types of sickle cell disease

1. Sickle cell anemia: Homozygous state for HbS (βS- βS) 2. Sickle cell trait : Heterozygous carrier state for HbS (βS -β) 3. If one parent has sickle cell anemia and other is normal , all children will have sickle cell trait. 4. If one parent has sickle cell anemia and other has sickle cell trait there is 50% chance of either with each pregnancy. 5. If both parents have sickle cell trait? AA-normal AS-sickle cell trait SS-sickle cell Anemia • Sickle cell – β thalassemia : Double heterozygote in which sickle cell gene is inherited from one parent and beta thalssemia gene from other parent . gene type (βsβo-βsβ+) • 4. Combination of Hbs with other abnormal hemoglobin (HbSD, HbSC, HbSO(arab disease),HbSE). Thalassemias Thalassemias

• In normal hemoglobin, number of α and β polypeptide chains is equal. • In thalassemia, the production of these chains become imbalanced because of defective synthesis of globin genes. • This causes the precipitation of the polypeptide chains in the immature RBCs, leading to disturbance in erythropoiesis. • The precipitation also occurs in mature red cells, resulting in . Thalassemias • α­thalassemia occurs in fetal life or infancy. In this α­chains are less, absent or abnormal. • In adults, β­chains are in excess and in children, γ­chains are in excess.This leads to defective erythropoiesis and hemolysis.The infants may be stillborn or may die immediately after birth. β-Thalassemia In β­thalassemia, β­chains are less in number, absent or abnormal with an excess of α­chains. The α­chains precipitate causing defective erythropoiesis and hemolysis

BETA-THALASSAEMIA

• Beta-thalassemias are a group of hereditary blood disorders characterized by anomalies in the synthesis of the beta chains of hemoglobin resulting in variable phenotypes ranging from severe anemia to clinically asymptomatic individuals. • The total annual incidence of symptomatic individuals is estimated at 1 in 100,000 throughout the world and 1 in 10,000 people in the European Union. 1.5% of the global population (80 to 90 million people) are carriers of beta thalassemia, with about 60,000 symptomatic individuals born annually, the great majority in the developing world. Structure Of Haemoglobin SITES OF GOBIN CHAIN SYNTHESIS

Globin chain synthesis Point Mutation : Substitution of a single DNA nucleotide base for another – can change the genetic code. Deletion : Absence of one or more nucleotiodes. Insertion : Addition of one or more nucleotides.

3 bases = 1 codon 1 codon= 1 Amino Acid Frame Shift, Sense, Nonsense. Disorders of Haemoglobin Qualitative Haemoglobinopathies Quantitative – Thalassaemias Functionally 1. No problem Goes undetected 2. Solubility Gel.Crystalize, Haemolysis 3. O2 Affinity Cyanosis 4. O2 Affinity Polycythaemia 5. Stability Hemolysis 6. Production Anaemia Thalassaemias - classification Genetic • α –Thalassaemia (deletions) • β –Thalassaemia (mutations)

– β 0 - Thalassaemia – β+ Thalassaemia

BETA THALASEMIA ;Clinical Features Anaemia Hepato Splenomegaly Skeletal Changes Iron Overload Growth Retardation

Clinical Classification

Thal Minor –trait, asymptomatic Thal Major –transfusion dependent Thal Intermedia – transfusion not required Hydrops Fetalis –death in utero, α –Thalassaemia

α-Thalassemia

Lab Diagnosis

Blood CP Hb Electrophoresis Alpha/Beta Chain Analysis DNA Analysis Prenatal Diagnosis Prevention

Glycosylated / (HbA 1C)

• Glucose is attached with terminal valine of each beta chain

• Normally upto 6% . Increases in diabetics Hemoglobin Levels

• Hemoglobin levels are measured

– In grams (gm) per deciliter (dl) of blood.

NORMAL RANGES :

• Adult women: 12-16 gm/dl

• Adult males: 14-18 gm/dl

• 1 G Hb – 1.34 ml of Oxygen Characteristics of Hb-O2 Bond • Combination of Hb with Oxygen is Loose – Do not combine with positive bond of Iron – Loose bond – Binding reversible – Molecular Oxygen binds and releases • Heme Heme Interaction – Cooperative bonding – Affinity for last oxygen - 300 times more The affinity of hemoglobin for O2 is affected by 1. pH 2. Temperature 3. Concentration of 2,3-Bisphosphoglycerate (2,3-BPG). + • 2,3-BPG and H compete with O2 for binding to deoxygenated hemoglobin

Decrease the affinity of hemoglobin for O2 Binding of Oxygen with Hb Binding of Oxygen with Hb Oxygen dissociation curve Functions of Hemoglobin • Transport of Oxygen • Transport of – Carbaminohemoglobin (CO2Hgb). • Buffer function + – Carbonic acid H and CO2 – Hb combine with H+ • Binding with Nitric oxide (NO) – NO binds with Sulphur atom SNO – NO relaxes and dilates arterioles • pigments formation Requirements for Hemoglobin Synthesis

• Metals – Iron, Copper, Cobalt, Manganese. • Protein Diet • Vitamins

– Vit B12 , Folic acid, Ascorbic acid • Hormones – Thyroid • Metals • Iron • Copper – Ceruloplasmin – Necessary for Iron transfer from storage sites • Bone marrow, and Spleen • Cobalt

– Forms a part of • Nickel and Manganese • Proteins – Formation of Globin – Protein deficiency

• Hormones • GH, Testosterone , TH, Cortisol, ACTH

• Other factors QUIZ

A 10-years-old African American male presented to ER with complain of pain "all over his body.“ His mother brought him into the ED at 4 pm .She reported that the pain began early that morning and had "gotten worse." She reported that it was not relieved by his usual doses of ibuprofen. He was medicated with strong IM pain killer. He reported minimal pain relief after receiving the medication. He reported that the slight relief was short-lived, and he continued to complain of unbearable pain through the night.

His past history is significant with many such hospital admissions and history of repeated chest infections and a non healing ulcer on his right ankle.

Family History: History of similar episodes of pain crisis and chest infection in two of the 5 siblings. Examination: Pale appearing child in agony oriented in time, space and person having a chronic ulcer on right ankle. Cardiovascular System: Moderate tachycardia, grade II/VI systolic murmur heard best over the upper left sternal border. Gastrointestinal Tract: Abdomen: Moderate hepatosplenomegaly. Complete Blood Count: Hb: 5gm/dl, TLC: 12,000/ul, Platelet count: 150,000/ul. Reticulocyte Count: 12%. Peripheral Film: Moderate poikilocytosis, anisocytosis, hypochromia, polychromasia target cells, many fragmented and sickle red cells. Special investigations Sickle Screening Test: Positive HbS: 70%, HbF: 13%, HbA: 17% Sickle cell anemia FATE OF RBCs Steps involved in the fate of RBCs

. The life span of an erythrocyte is 100-120 days.

. Old RBCs become rigid & fragile and their Hb begins to degenerate.

. Dying RBCs are engulfed by macrophages.

. Heme & globin are separated and the iron is salvaged for reuse.

. . Heme is degraded to a green pigment .

. Biliverdin is converted to a yellow pigment called .

. The bilirubin is picked up by the liver and is secreted into the intestines as bile. . The intestines metabolize it into and . . These degraded pigments leave the body in feces and urine in a pigment called stercobilin and . . Globin is metabloized into aminoacids and is released into the circulation. . Hb released into the blood is captured by haptoglobin and phagocytized. • Lower levels in blood stimulate kidney to produce

erythropoietin.

• Erythropoietin levels rise in blood.

• Erythropoietin and necessary raw materials in the blood

promote erythropoiesis in red bone marrow.

• New erythrocytes enter blood stream, function about 120

days. • Aged and damaged red blood cells are

engulfed by macrophages of liver,spleen &

bone marrow, the hemoglobin is broken

down.

RBCs after 120 days

Fragile

Membranes of RBC rupture

Phagocytized by Reticulo endothelial system

Tissue macrophages » Kupffer cells » Spleen Hemoglobin split

Heme Globin Amino acid pool- reuse

Free Iron Straight chain of 4 pyrrole nuclei

Transported in blood by transferrin

Reused Straight chain of 4 pyrrole nuclei HEME OXYGENASE Biliverdin BILIVERDIN REDUCTASE Free Bilirubin (released by Macrophages into plasma)

Combination with plasma Albumin

Blood Interstitial fluids

Liver kidney Nil

Free Bilirubin Free Bilirubin in blood Within Hours Absorption in hepatic cell membrane

Released from Albumin

Conjugated

Glucoronic acid Sulfates Other substances

Bilirubin Glucoronide Bilirubin Sulfate Bilirubin Glucoronide Bilirubin Sulfate

Excreted from hepatocytes

ACTIVE TRANSPORT Bile canaliculi

Conjugated Bilirubin in intestines

Bacterial Action Urobilinogen Urobilinogen

Reabsorbed by intestinal mucosa

Blood Liver 5% Kidneys

Reexcreted Urobilinogen Into Gut Stercobilinogen

Urine Feces Oxidation Oxidation Urobilin Stercobilin Normal serum Bilirubin Conc

Serum Bilirubin

• Total: 0.3 to 1.0 mg/dl

• Conjugated: 0.1 to 0.4 mg/dl

• Unconjugated: 0.2 to 0.7 mg/dl

JAUNDICE

Unconjugated Bilirubin Conjugated bilirubin

1 Lipid soluble & water insoluble Water soluble & lipid insoluble

2 Cannot travel easily in plasma Can travel easily in plasma

3 Can cross glomerular capillary Cannot cross glomerular capillary membrane membrane

4 Can permeate through lipid Cannot permeate through lipid bilayer bilayer

5 Plasma level rises in pre-hepatic Plasma level rises in posthepatic (hemolytic) jaundice (obstructive) jaundice Van den Bergh Test (Reaction)

Diazo reagent

Sodium nitrite, Sulphalinic acid, Dilute hydrochloric acid. • Serum + Diazo Reagent Reddish-violet colour (Direct Reaction) Inference: Direct or Conjugated Bilirubin

• Serum + Diazo Reagent No colour • Add caffeine or alcohol Reddish-violet colour (Indirect Reaction) Inference: Indirect or Unconjugated or Free Bilirubin JAUNDICE (ICTERUS)

Yellowish discolouration of skin, sclera and mucous membrane due to excessive bilirubin accumulation. BILIRUBIN LEVELS

• Normal serum/plasma bilirubin level = < 1.0mg/dl

• Unconjugated bilirubin = upto 0.7mg/dl

• Conjugated bilirubin = upto 0.3mg/dl

• Jaundice = > 2.0 mg/dl bilirubin level CLINICAL TYPES OF JAUNDICE

• Latent (Biochemical) Jaundice Clinically invisible

• Overt (Clinical) Jaundice Clinically visible

TYPES OF JAUNDICE ON THE BASIS OF CAUSE . PRE-HEPATIC OR HEMOLYTIC OR ACHOLURIC JAUNDICE

. HEPATIC OR HEPATOCELLULAR JAUNDICE

. POST-HEPATIC OR OBSTRUCTIVE JAUNDICE

PHYSIOLOGICAL JAUNDICE

• Usually mild form of jaundice appears in some newborn children on the 2nd or 3rd day of life, called Jaundice of Newbont or . Causes 1. Excessive destruction of RBCs after birth causing increase in serum bilirubin. 2. Due to hepatic immaturity. During intrauterine life bilirubin formed is mainly eliminated via the placenta and to lesser extent by liver. Immediately after birth the liver has to eliminate all the bilirubin formed, therefore, liver is unable to deal adequately with this increase PHYSIOLOGICAL JAUNDICE

• Amount of bilirubin during the first 10 days of life causing development of jaundice. That is why serum bilirubin continues to rise after birth to a peak which is generally reached during the 1st week and then it declines. • It is more common and of greater severity in premature babies. • In infants, when serum bilirubin rises beyond 5 mg/dL, clinical jaundice appears. PHOTOTHERAPY

• Exposure of the skin to white light converts bilirubin to Lumm b which has a shorter life than bilirubin • It acts by photoisomerisation of bilirubin to soluble forms, which are easily excreted. • Therefore, phototherapy (exposure to light) is of value in treating infants with jaundice (irrespective of its cause) COMPARISON OF DIFF TYPES OF JAUNDICE LAB TESTS PRE-HEPATIC HEPATIC POST-HEPATIC Serum Bilirubin Both Unconjugated ↑ Conjugated ↑ (mainly direct) Urine urobilinogen ↑↑ ↑ ↓ (If complete obstruction) Urine bile salts Absent Present Present Urine bilirubin Absent Present Present COMPARISON OF DIFF TYPES OF JAUNDICE LAB TESTS PRE-HEPATIC HEPATIC POST-HEPATIC Fecal urobilinogen ↑ ↓ ↓or absent Fecal fat Absent ↑ ↑ Alk. Phosphatase Normal ↑ ↑↑ ↑ Plasma albumin Normal ↓ Normal or ↓ Gamma globulins Normal ↑ Normal Van den Bergh test Direct mainly Indirect Direct (Biphasic)