Biochemistry of blood bds

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Biochemistry of blood bds

  1. 1. Biochemistry ofHaemoglobin and Myoglobin
  2. 2. Hemoprotein (Cytochrome C)Structure of Heme
  3. 3. A case presentation• A 21 year old black African male student of dentistry presented to the medical department complaining of fever and generalized body aches for two days accompanied by JAUNDICE.• He told that jaundice was on and off for the last 2 years and was associated with dark urine.
  4. 4. • There was no history of nausea ,vomiting , bleeding or change in bowel habits. He had history of exchange transfusions.• On physical examination he was febrile and deeply jaundiced with pulse rate of 90/ minute(regular) and blood pressure of 110/70 mmHg. No chronic liver disease. Other systemic examination were normal.
  5. 5. • His laboratory examinations showed ; Hb 11.3 mg/dl , total bilirubin :425 μmol/L (< 7 μmol/L) , direct bilirubin : 234 μ mol/L ,ALP-632 IU/L• Urine had bilirubin and urobilinogen +ive• An abdominal ultrasound was normal apart from one single large stone in the gall bladder with no CBD dilatation.
  6. 6. • He was admitted to medical ward where I.V fluids ,cefotoxime and metronidazole were initiated.• Hb electrophoresis revealed Hb –S 35% and he was diagnosed a patient of sickle cell anemia.
  7. 7. TOPICS OF STUDY(HEME METABOLISM) A.HAEMOGLOBIN (Hb) B.MYOGLOBIN (Mb)
  8. 8. HEMOGLOBIN
  9. 9. HAEMOGLOBIN AND MYOGLOBINa) STRUCTURE / FUNCTIONS [ Oxygen binding ]b) TYPES OF HAEMOGLOBIN ,ITS DERIVATIVESd) CATABOLISM OF HAEMOGLOBIN (PORPHYRIN DEGRADATION OR BILE PIGMENT FORMATION) JAUNDICE
  10. 10. e) HAEMOGLOBINOPATHIES i) Sickle Cell Disease [SCD] ii) Thalassemias [ α & β ]
  11. 11. Specific Learning Objectives (SLO’s)• Students may be able to learn:• Heme synthesis and its inborn errors of enzymes leading to PORPHYRIAS.• Structure Function relationship of Proteins (Hb & Mb) i.e. Sickle Cell disease.• O2 transport by Hb and Mb , also its regulation by allosteric effectors• Degradation / Catabolism of Hb or Formation of BILIRUBIN (Bile Pigment)
  12. 12. 5. Transport , Conjugation and Excretion of bilirubin6.Types of bilirubin and how they differ from each other?6. What are Haemoglobinpathies and their biochemical basis ?7. How BILIRUBIN metabolism helps in diagnosis of various types of jaundice and liver function.
  13. 13. BIOMEDICAL IMPORTANCEA. HAEMOPROTEINS WHICH CONTAINS PORPHYRINS WITH IRON [HEME] – HAEMOGLOBIN (IRON) – MYOGLOBIN (RESPIRATORY PIGMENT IN MUSCLE)(IRO – ERYTHROCRUORIN (INVERTEBRATES) – CYTOCHROME P-450 (IRON) – CYTOCHROME – C (IRON) – CATALASE (IRON) – TRYPTOPHANE PYRROLASE (IRON) – NITRIC OXIDE SYNTHASE – PEROXIDASE
  14. 14. BIOMEDICAL IMPORTANCE (Contd)B. JAUNDICEC. HAEMOGLOBIN AND MYOGLOBIN BOTH ILLUSTRATE – PROTEIN STRUCTURE & FUNCTION RELATIONSHIPS – MOLECULAR BASIS OF GENETIC DISEASES, LIKE “SICKLE CELL DISEASE” AND “THALASSEMIAS”
  15. 15. Protein FunctionHemoglobin Transport of oxygen in bloodMyoglobin Storage of oxygen in muscleCytochrome c Involvement in electron transport chainCytochrome P450 Hydroxylation of xenobioticCatalase Degradation of hydrogen peroxideTryptophan pyrrolase Oxidation of tryptophan Some important Human and Animal Hemoproteins
  16. 16. GLOBIN HEMEHEMOGLOBIN
  17. 17. Fe
  18. 18. Uroporphyrin III (asymmetric) A (acetate) = -CH2COOH P (propionate) = -CH2CH2COOHFischer’s Short Hand formula
  19. 19. Uroporphyrins and coproporphyrins. (A , acetate, P, propionate; M, methyl)
  20. 20. FACTORS AFFECTING THE SYNTHESIS OF Hb1. METALS = Fe++ , CU, COBALT, Mn, Zn++2. PROTEIN DIET (ESSENTIAL AMINO ACIDS)3. VITAMINS – VIT B12 (METHYLCOBALAMINE) – VIT C (ASCORBIC ACID) – PANTOTHENIC ACID – NICOTINIC ACID – PYRIDOXINE [ACTIVATE GLYCINE]
  21. 21. FACTORS AFFECTING THE SYNTHESIS OF Hb4.HORMONES – GH, THYROXINE, TESTOSTERON, ESTROGEN5.HYPOXIA6.ERYTHROPOITIN (Renal erythropoitic factor) – ERYTHROPOISES
  22. 22. In mitochondria Step-I Zn+Step-II Lead In cytosol Biosynthesis of Porphobilinogen
  23. 23. Step-III Conversion of 4 molecules ofporphobilinogen touroporphyrinogen (in cytosol)
  24. 24. Step-IV(in cytosol)
  25. 25. Step-V Formation of Protoporphyronogen III• Coproporphyrinogen enters the mitochondria.• The enzyme is coproporphyrinogen III oxidase• This enzyme only acts on type-III coproporphyronogen.• Decarboxylation and oxidation of two- propionates of pyrrole rings I&II to form two vinyl (V) groups.
  26. 26. Step-VI Formation of Protoporphyrin III• Enzyme protoporphyrinogen -III oxidase in mitochondria.• Requires molecular oxygen & removes six hydrogen atoms• All bonds of protoporphyrin i.e. alpha, beta, gamma and delta are converted into methyne bridges (=HC--)• Porphyronogens are colourless compounds• Porphyrins are coloured compounds
  27. 27. Step-VII LeadFormation of Heme involves incorporation of iron into Protoporphyrin III (IX)
  28. 28. Steps of biosynthesis ofporphyrin derivatives from porphobilinogen
  29. 29. ALA Synthase Is The Key Regulatory Enzyme In Hepatic Biosynthesis Of Heme• ALAS1 is present in the liver• Heme through aporepressor molecule acts as a negative regulator of this enzyme• Heme induces this enzyme and transfers it from cytosol to mitochondria• Drugs metabolized by cytochrome P-450 derepress this enzyme and precipitate attack of Porphyrias• Glucose and hematin can prevent derepression of this enzyme and decreases heme synthesis
  30. 30. ALAS-2 erythroid form of thisenzyme occurs in bone marrow• This enzyme is not induced by drugs• It does not undergo feed back regulation by heme.85% heme synthesized in RBC,s• Erythropoisis is regulated by ERYTHROPOITIN which is produced by the kidney• Hypoxia stimulates the production of this enzyme
  31. 31. PORPHYRINS ARE COLORED AND FLUORESCE• Porphyrinogens are colorless and porphyrins are all-colored• Porphyrins have characteristic absorption spectrum near 400 nm called Soret band• This photodydamic property is used for cancer phototherapy• Spectrophotometery is used to test for Porphyrins and their precursors• The above properties are due to double bonds joining the pyrrole rings.
  32. 32. Absorption spectrum of hematoporphyrin (0.01% solution in 5% HCl)
  33. 33. (1)(2)
  34. 34. PORPHYRIAS• The porphyrias are genetic or acquired disorders of Heme metabolism with heterogenous mutations• Inherited in an autosomal dominant manner except congenital erythropoitic porphyria(reccessive)• Signs and Symptoms result due to defficiency of metabolic products beyond the enzyme block or an accumulation of metabolites behind the block• Can express clinically as Acute abdominal pain, neuropsychiatric problems and photosensitive dermatitis
  35. 35. CLINICAL IMPORTANCEPorphyrias though not prevalent but should be considered in differential diagnosis in the following conditions:2.Abdominal pain3.Neuropsychiatric problems4.Dermatitis (Photosensitive)
  36. 36. Classification of PorphyriasI. Based on clinical featuresb) Abdominal pain, neuropsychiatric problems and no photosensitivity Due to accumulation of delta ALA and PBG in the tissues and urine Acute intermittent porphyriae) All other five pophyrias are accompanied with photosensitivity to light (dermatitis) Due to accumulation of porphyrinogens and porphyrins in the tissues
  37. 37. Biochemical causes of the major signs and symptoms of the porphyrias
  38. 38. Steps of biosynthesis ofporphyrin derivatives from porphobilinogen
  39. 39. II. Based on the organs involved which are mainly responsible for the synthesis of heme – Erythropoitic – Hepatic – Hepatic and erythropoiticA. Hepatic porphyria can be acute or chronic
  40. 40. (a) Acute Hepatic Porphyria (Hepatic) – Acute intermittent porphyria (uroporphyrinogen 1 synthase enzyme also called PBG deaminase or HMB synthase) – Hereditary coproporphyria (coproporphyrinogen oxidase) – Varigate porphyria (protoporphyrinogen oxidase)
  41. 41. Clinical features – Acute episodes of intestinal, neurologic, psychiatric and cardiovascular symptoms – Increased ALA and PBG cause abdominal pain and neuropsychiatric symptoms – ALA inhibit ATPase in nervous tissue – or taken up by brain and cause conduction paralysis – Precipitated by drugs barbiturates and ethanol due to increased activity of ALA-synthase
  42. 42. (b) Chronic Porphyria (Porphyria Cutanea Tarda)• It is hepatic and erythropoitic porphyria• Enzyme: Uroporphyrinogen decarboxylase Precipitating factors – Hepatic iron overload – Exposure to sunlight – Hepatitis B, C and HIV infections• The most common Porphyria
  43. 43. Clinical features – Skin eruptions due to photosensitivity – Damage of membranes by ROS, and enzymes released from lysosomes – Urine turns red to brown in the natural light and pink to red in fluorescent light
  44. 44. Skin eruptions in a patient
  45. 45. (c) Erythropoitic porphyrias Types 1.Congenital erythropoitic porphyria. Enzyme uroporphyrinogen III synthase 2.Erythropoitic protoporphyria – Enzyme ferrochelatase – Clinical features – Uroporphyrinogen I & coproporphyrinogen-I accumulates in urine. Proteprophyrin accumulates in erythrocytes and bone marrow – Photosensitive - skin rashes and blisters during childhood
  46. 46. 2. Increased ALA Synthase Activitya. Heme level is decreased in all porphyriasb. Derepression of ALA synthasec. Increased synthesis of intermediates prior to genetic blockd. Accumulation of toxic intermediates in tissue is the main biochemical basis of disease
  47. 47. 3. Diagnosisa. Family History and Clinical Featuresb. Appropriate laboratory tests • Urine test for different intermediates • Enzymes assay in erythrocytes and hepatocytes in suspected case of porphyria • Spectrophotometery is used to test for porphyrins in urine and faecesc. Prenatal diagnosis by using appropriate gene probes
  48. 48. 4. Treatmenta. To avoid drugs which precipitate attackb. Symptomatic treatment by analgesics, high glucose intake, intravenous hemin to repress the ALA synthase activityc. Administration of B carotene to combat free radicals and sunscreens to avoid photosensitivityd. Gene therapy in future to replace enzymes
  49. 49. STRUCTURES AND FUNCTIONS OFMYOGLOBIN & HEMOGLOBIN
  50. 50. MYOGLOBIN
  51. 51. Structure and Functions of Myoglobin Present in heart and skeletal muscles Reservoir and carrier of O2 that increases the rate of O2 transport in muscles Single polypeptide chain and resembles to the individual subunit of Hb This is a useful model for interpreting some of the more complex properties of Hb
  52. 52. 1-α- Helical content• Compact molecule• 80 % of polypeptide chain folded into 8 stretches α- helix ; labeled A to H.• One polypeptide chain contains 153 aminoacids• These are terminated by proline or joined by β bends / loops stabilized by hydrogen and ionic bonds
  53. 53. 2-Location of polar and non polar amino acid residue• Interior of the Mb contain non- polar amino acids (hydrophobic amino acids)• These are closely packed together forming a structure stabilized by hydrophobic interactions• Charged amino acids on outer surface of Mb ;form H- bonds both with each other and with water
  54. 54. 3-Binding of the Heme group• Heme group of Mb sits in the crevice in the molecule which is lined by the non polar amino acids• Exceptions are two proline• One the proximal histidine (F8) binds directly to Fe of heme• Second do not bind directly to iron but stabilizes binding of O2 to the ferrous iron
  55. 55. Model Of Myoglobin showing the Heme and Oxygen
  56. 56. 4- Oxygen dissociation curve of Mb is hyperbolic shape Mb can bind only one molecule of O2 as this has only one heme This is a reversible binding of O2 Oxygenated and deoxygenated Mb exist in a simple equilibrium This equilibrium is shifted to the right or to the right when O2 is added to or removed from Mb respectively
  57. 57. Oxygen dissociation Curve ofMyoglobin is Hyperbolic andHb has sigmoid i.e Steepest dissociation curve
  58. 58. 5- Binding of O2 to Mb is notinfluenced by allosteric effectors• There is only one chain and one heme• No heme/heme interactions• Mb binds to oxygen released by Hb at the low pO2 found in muscle.
  59. 59. • Mb in turn releases oxygen within the muscle cell in response to oxygen demand.• pH , pO2 ,pCo2 and 2,3 BPG do not effect oxygen binding
  60. 60. Hemoglobin (Hb)
  61. 61. Structure and function of Hb• Exclusively present in erythrocytes• Contains four polypeptide chains (tetramer)• Hb-A has two α -chains and two β- chains and it is a major Hb in adults• α and β chains are held together by non- covalent interactions(hydrophobic interactions)
  62. 62. • Transport H+ ions and Co2 from tissues to lungs• Transports four molecules of oxygen from lungs to the body cells• Oxygen binding properties of Hb are regulated by interaction with allosteric effectors pH , pCo2 and pO2 Concentration of 2,3 BPG in erythrocytes
  63. 63. Quaternary Structure of Hb• It is tetrameric protein• Composed of two identical dimers ,(αβ)1 and (αβ)2 ; called dimer one and dimer two• Two polypeptide chains within each dimer are held tightly together by hydrophobic interactions• Ionic and hydrogen bonds between the members of the each dimer
  64. 64. • Two dimers can move with respect to each other being held together by polar bonds (hydrogen bonds/ionic bonds or called salt bridges)• The weaker interactions between these mobile dimers make them to acquire two different positions in deoxyhemoglobin (taut) and oxyhemoglobin (relaxed )forms of Hb
  65. 65. Structural changes resulting from oxygenation(R-form) and deoxygenation (T-form) of hemoglobin
  66. 66. BINDING OF OXYGEN TO MYOGLOBIN AND HEMOGLOBINMYOGLOBIN = ONE MOLECULE OF O2 WITH ONE MYOGLOBIN AT ONE HEME GROUPHEMOGLOBIN = FOUR MOL OF O2 WITH ONE Hb ONE AT EACH OF ITS FOUR HEME GROUPS
  67. 67. OXYGEN DISSOCIATION CURVE• DEFINITION:• A PLOT(Y) AXIS IS %SATURATION OF BOTH PROTEINS MEASURED AT DIFFERENT PRESSURES OF (PO2) at (X) AXIS• AND PLOTTED AGAINST Y AND X- AXIS
  68. 68. • MYOGLOBIN HAS HIGH O2 AFFINITY• PARTIAL PRESSURE REQUIRED FOR 50% SATURATION WITH O2 MYOGLOBIN = 1mm Hg (HYPERBOLIC CURVE) HEMOGLOBIN = 26 mm Hg (SIGMOIDAL CURVE)
  69. 69. ALLOSTERIC EFFECTORS MODIFYING THE BINDIG OF O2 WITH HEMOGLOBINII. Heme Heme interactiomIV. pO2VI. pHVIII. pCO2X. 2 , 3 BPG
  70. 70. 1-HEME-HEME INTERACTIONSa. LOADING AND UNLOADING OXYGENc. SIGNIFICANCE OF SIGMOIDAL OXYGEN DISSOCIATION CURVE
  71. 71. SEEP RISE OFCURVE AT 30 mmHg
  72. 72. a-Loading and Unloading Oxygen• In lungs it is saturated with oxygen due to high pO2 in the alveoli and LOADED• In peripheral tissues oxyhemoglobin releases or unloads maximum oxygen for use in oxidative metabolism
  73. 73. b- Significance of sigmoidal oxygen dissociation curve• The steep slope of the curve between high (lungs) to sites of low pO2 (tissues) permits Hb to carry and deliver O2 very efficiently• Good degree of O2 release with in this range of small changes in partial pressure of O2 in peripheral tissues• Permits O2 delivery to respond to small changes in pO2
  74. 74. Oxygen dissociation Curve ofMyoglobin is Hyperbolic andHb has sigmoid i.e Steepest dissociation curve
  75. 75. 2-BINDING OF CO2(CARBAMATE FORM (15%)Hb-NH2+CO2 → Hb-NH2-COO+H+• BINDING OF CO2 STABILIZE THE “T” STATE OF Hb (DEOXYGENATED FORM)• DECREASE O2 AFFINITY OF Hb & SHIFT THE CURVE TO THE RIGHT• PROMOTE UNLOADING OXYGEN
  76. 76. 3-BOHR EFFECT(p H)– INCREASE H+ ION AND DECREASE Ph FAVOURS DISSOCIATION– INCREASE PCO2 ALSO – BOTH SHIFT THE CURVE TO RIGHT– CHANGE IN OXYGEN BIDING WITH Hb BY H+(pH) & Co2 IS CALLED BOHR EFECT
  77. 77. 4-BINDING OF CO (Hb CO)– BINDS TIGHTLY TO THE Hb IRON BUT REVERSIBLY– INCREASES AFFINITY OF O2 BINDING [R-STATE]– SHIFT THE CURVE TO LEFT SIDE / (HYYPERBOLIC)– CO HAS 220 TIMES MORE AFFINITY TO FORM CARBON MONOXYHEMOGLOBIN– > 60% CO IS FATAL
  78. 78. Nitric oxide gas(NO) transport• Hb can carry NO• Potent vasodilator• It can be released from RBCs• NO influences the vessel diameter
  79. 79. 5-EFFECT OF 2,3 BPG ON OXYGEN AFFINITY• IMPORTANT REGULATOR OF BINIDNG OF O2 TO Hb• [ HIGH CONCENTRATION IN RBC’S UPTO 280 MILLION]• SYNTHESIZED FROM AN INTERMEDIATE OF GLYCOLYSIS
  80. 80. a-BINDING OF 2,3 BPG TO DEOXYHEMOGLOBIN:• PREFERENTIALLY BIND TO DEOXYGENATED FORM AND STABILIZES THE TAUT CONFORMATION OF Hb• DECREASES AFFINITY OF O2 WITH Hb
  81. 81. b- BINDING SITE OF 2,3 BPG• BIND TO A POCKET FORMED BY THE TWO β- CHAINS HAVING +VE CHARGES OF HYDROPHOBIC AMINO ACIDS• 2,3 BPG IS EXPELLED ON OXYGEATION OF THE Hb
  82. 82. c-Shift of oxygen the dissociation curve• Hb without 2,3 BPG has high affinity for O2• 2,3BPG ‘s presence in RBCs significantly reduces the O2 affinity & Hb releases O2 efficiently at tissue level• Curve is shifted to the right
  83. 83. d-Response of 2,3BPG levels to chronic hypoxia or anemia1-Chronic obstructive pulmonary diseases Chronic bronchitis Emphysema2-High altitude3-Anemia Delivery of maximum O2 to tissues
  84. 84. 5-ROLE OF 2,3 BPG IN TRANSFUSED BLOOD.∀ ↓ 2,3 PBG IN Hb WILL LEAD∀ ↑ AFFINITY OF O2 WITH Hb• Hb WILL TRAP O2 , RBCs CAN RESTORE 2,3 BPG IN 6- 24 HRs OF STRIPPED BLOOD• CURVE SHIFTED TO THE LEFT
  85. 85. TYPES OF HAEMOGLOBINSNORMAL AND ABNORMAL
  86. 86. TYPES of Hb [NORMAL]1. All are tetrameric.2 . Composed of two α-globin like polypeptides & two β-globin like polypeptide chains3. Present in various ages of life
  87. 87. 4. HbA (adult Hb) is the major one & all others are minor– HbA2 is synthesized in adults (12-weeks after birth)– Modified by addition of hexoses (glucose) i.e. HbA1C7. Hb-F is also present < 2%
  88. 88. TYPES of CHAINS IN HbThese are classified due the presence ofsix types of chains α-β,γ,δ,ε,z • α - chain same in all (with 141 amino acids) • β- chain differ in each in respect of amino acid sequence [146-amino acids] • • Hb A (Adult Hb) [α 2β 2] 97%
  89. 89. • Hb A2 2.5% [α 2 δ 2] appear 12 week after birth• Hb-F [fetal Hb] [α 2 γ 2] < 2%• Hb A1-C α 2β 2 glucose 3-9%• Hb-c• Hb Gower - I (Embryonic Hb z2 e2)• Hb Gower- II (α 2 e2)
  90. 90. FOETAL HAEMOGLOBIN Hb-F (α 2γ 2)• Differ at 37.amino acid in as compared with β chain• Major Hb found in fetus and newborn• It has more affinity for O2 as compared to HbA• This property facilitates transfer O2 from maternal circulation to fetal RBC
  91. 91. • Binding of 2,3 BPG to HbF is very weak therefore O2 bind with Hb F more strongly• HbF-γ-chains lack some positively charged A-Acids• P50 of Hb F is 20mmHg as compared to HbA i.e. 26 mmHg which favours to get more O2 from mother
  92. 92. Developmental Changes in Hemoglobin during fetal &post natal life
  93. 93. Hemoglobin –A2 (Hb A2)• Minor component of normal adult Hb i.e 2% of total Hb• Composed of two α- globin chains and two δ- globin chains (α2 δ2)
  94. 94. Hb – A1C. (3-9% of total normal Hb)• This results due to slow and non enzymic glycosylation of NH2 groups of the N- terminal valines ,ε – amino group of lysine residue in the β globin chains• Level depends upon plasma levels of respective hexose (glucose) for long periods (7 months)• This glycosylation is irreversible
  95. 95. • This persists for life span of RBC’s• This is increased in diabetes mellitus about 2 to 3 fold• Hb A-I-C provides a measure of how well treatment has normalized blood glucose in diabetes( in mean blood glucose i.e 150mg dl = 7% A1 - C of total Hb)• Included in WHO criteria for diagnosis of diabetes mellitus
  96. 96. Nonenzymicaddition of glucose to hemoglobin
  97. 97. ABNORMAL HAEMOGLOBINS(Hemoglobinopathies)
  98. 98. Organization of the Globin Gene• Knowledge of structural organization of gene families explains• How the genes are expressed ?• How genetic alterations in the structure and synthesis of globin chains lead to haemoglobinopathies ?
  99. 99. α Gene Family• 2 α genes α1& α2 on each chromosome-16 for α globin chains• 1 ζ (zeta) gene expressed in embryonic stage• A number of globin like genes that are not expressed are called Pseudogenes
  100. 100. β Gene Family• A single gene for β globin chain is present on each chromosome-11• Additional four β globin like genes : epsilon (ε), two γ genes and one δ gene for adult Hb A2Alteration of globin gene expression(one geneto an other) during development is called Hb switching .This is regulated byTranscription factors by binding to promoter region on DNA
  101. 101. Synthesis of Globin Chains
  102. 102. HAEMOGLOBINOPATHIES (globin part is defective)• Family of disorders: Inherited due to gene mutation which compromises bilogical functions of Hb• There are 900 mutations but are extremely rare& benign
  103. 103. • Classified as: – Qualitative abnormal Hb: (Structural defect of β chain i.e altered amino acid sequence) Sickle Cell anemia Hb-S Hb –C & Hb- SC II.Quantitative abnormal Hb (Insufficient synthesis of α or β chains) (Thallasemias)
  104. 104. I. Sickle Cell Anemia (Hb-S) (α 2S2)a. Sickle cell disease 1:500 ratio (100% HbS) – Homozygous, two gene mutation [chromosome 11] Recessive disorder, one gene from each parent. Black African Americans are more affected – More severe anemia, infections & poor circulation – Acute chest syndrome, stroke splenic & renal infarcts
  105. 105. a. Sickle cell trait : 1:10 ratio • Heterozygous • One mutant gene & one normal gene • Not serious & do not show clinical features if there is no hypoxia • 60% Hb-A [α 2β 2] • 40% Hb-S [α 2β 2]
  106. 106. CHARACTERISTICS OF SICKLE CELL ANEMIA• Hbs have α2β2 S2
  107. 107. Sicklling of the cells & clinical effects• Valine is a non polar amino acid, on β chain• It results in (↓) solubility in deoxygenated blood due to sticky patch• Molecules of Hbs form fibers and precipitate in RBC & give the shape of sickle to RBC• These block the flow of blood in capillaries• Pain due to ischaemia in respective tissue.• Episodes of severe pain (crises) & infections• Ultimately death of tissue due to lack of oxygen
  108. 108. The sticky patch on Hb-S and receptor on deoxyhemoglobin-A and deoxyhemoglobin-S. The complementary surfaces will allow deoxy Hb-S to polymerise into fibrous structure. Deoxy Hb-A willterminate the polymerization due to lack of sticky patch.
  109. 109. Factors which (↓) O2 pressure lead more and more sickling• (↑) Altitude, or flying in non pressurized plane• (↑) CO2 concentration• Decreased pH• High 2,3 BPG in RBC (anaerobic glycolysis) In all above sicklling occurs even at normal O2 pressure which promote deoxygenated form of Hb Both HbA &HbS contain a complementary sticky patch on their surfaces that is exposed only in the deoxygenated taut / tense (T) state
  110. 110. SELECTIVE ADVANTAGE OF Hb-S (SICKLE CELL TRAIT)• As this disease is more common in African American black population 1:10• These people are resistant to the development of malaria especially in sickle cell trait• Reason being by (↓) life span of RBC• (↓) life of RBC interrupt the intracellular life cycle of parasite• The most dangerous is (plasmodium falciparum)
  111. 111. TREATMENT• Hydration• Analgesics• Antibiotic Therapy• Intermediate packed cell transfusions(Hemosidrosis)• Drug Hydroxyurea induces HbF expression• Stem cell transplantaion• In future,GENE THERAPY
  112. 112. Haemoglobin - C Disease• Haemoglobin : C-Disease – Single substitution (point mutation) – Glutamic acid at position – 6 in β-globin replaced by lysine – In homozygous state have mild, chronic hemolytic anemia, no infarct, no special treatment
  113. 113. Structurally abnormalHb(β globin chains)with altered aminoacid sequence.A single nucleotidealteration leads to apoint mutation
  114. 114. Haemoglobin - M Disease• Hemoglobin – M (Hb M disease)• Histidin- F8 is replaced by tyrosine• Abnormal α or β-chain structures∀ α chain variants [Hb M-abston and / wate] individuals are cyanotic at birth• Individuals with β-chain variants [Hb-M saskatoon, hyde park and Milwaukee] do not show cyanosis until age of 4-6 month• These Hb can be easily oxidized to met-Hb but the normal met Hb reducing enzyme system fails to reduce it (NADH-CYTOCHROME b -5 reductase)
  115. 115. I. THALASEMIAS [Quantitative]• Hereditary hemolytic diseases• Imbalance synthesis of globin chains• Most common single gene disorder• Each thalassemia can be of no globin chain (αo-or βo thalassemia) or at reduced rate of synthesis (α+-or β+ thalassemia)
  116. 116. α THALASSEMIA [α Chain ↓ or Absent]• Each partner genome contains – 2 copies of the α - globin gene on chromosome – 16• If one of four is defective, individual is termed silent carrier• In α-thalassemia trait – 2 gene involvement
  117. 117. • Hb-H disease : 3 genes defective mild to moderate and severe hemolytic anemia• All four genes are defective [fetal death] γ tetramers in newborn (γ 4 Hb Bart) β tetramers (β 4 - Hb H) These tetramers have very high O2 affinity and are useless as O2 deliverer to the tissues
  118. 118. α-Globin genedeletions in theα-thalassemias
  119. 119. Hemoglobin tetramers formed in α- thalassemias
  120. 120. β - THALASEMIAS• Metabolic defects : synthesis of β - globin chain is decreased or absent – Only two copies of the β - globin gene on chromosome- 11of both partners – Either one gene (minor) or both gene (major) – Physical manifestations of β - thalassemia appear only after birth because β - gene is not expressed until late in fetal gestation
  121. 121. Hemoglobin tetramers formed in β- thalassemias
  122. 122. I. β - Thalassemia Minor• One gene of one individual is defective• Can make some β - chains because they are heterozygous• No treatment is required
  123. 123. II. β - Thalassemia Major (Cooly’s anaemia)• Homozygous gene mutation – no β -chain• Healthy at birth but later on severely anemic∀ α - globin chains can not form stable tetramers and precipitate and premature death of RBC’s• Becomes severely anemic due to hemolysis• TREATMENT is repeated blood transfusions• Hemosidrosis [death between 15 and 25 years]• Bone marrow replacement is the best choice
  124. 124. CATABOLISM OF HEME (PORPHYRIN-III) [BILE PIGMENT METABOLISM]• 1-2 x 108 Senile RBC lysed / hour• Recognition of RBCs suitable for degradation – Senile RBC (age 120 days) – Young RBC, structurally / functionally abnormal• Why old RBCs are lysed? – Changes in membrane structure or ↑ rigidity (flexibility) – Loss of activity of enzymes – Changes in Hb conformation – Abnormal metabolic intermediates – Changes in electrolyte conc – Reduced ATP conc lead to ↑ rigidity
  125. 125. CATABOLISM OF HEME (PORPHYRIN-III)[BILE PIGMENT METABOLISM]FORMATION OF BILIRUBIN• Site of degradation – Recticulo endothelial system (RES) • Liver (kupffer cells) • Spleen • Bone marrow (ineffective erythropoisis) • 80% bilirubin from RBCs , 20% other sources – Daily : 250 – 350 mg of uncojugated bilirubin is formed and transported to liver, bound with albumin at high affinity site
  126. 126. Microsomal Hemeoxygenase system Hemoprotein Globin+Heme
  127. 127. Liver Takes up Unconjugated Bilirubin• Unconjugated bilirubin binds with albumin non-covalently• Displaced by aspirin, antibiotics and Fatty acids• Taken up by liver by facilitated transport system .Binds with Ligandin &Y proteins• Uptake of bilirubin is dependant upon removal of conjugated bilirubin from liver cells to bile ductules
  128. 128. CONJUGATION OF BILIRUBIN• Site : Hepatocyte (SER),• Enzyme : glucuronosyl transferase• Substrates : Glucuronic acid and unconjugated biliburin• Product : bilirubin diglucuronide• This process is induced by Phenobarbital
  129. 129. Transport ; Conjugation and Excretion of BilirubinFormation and enterohepatic circulation of Urobilinogen
  130. 130. Conjugated Bilirubin is secreted into the Bile• By active transport system• Rate limiting for entire process of bilirubin metabolism• Induced by Phenobarbital• Both conjugation and secretion processes of bilirubin are coordinated and function as one unit
  131. 131. CONJUGATED BILIRUBIN IS REDUCED TO UROBILINOGEN BY INTESTINAL BACTERIAS• Glucuronic Acid is removed by glucuronidase• All UROBILINOGENS are colorless• Enterohepatic urobilinogen cycle and its significance• Urobilinogens form stercobilins in feces which give dark color to feces• Urobilinogens form urobilins in urine• During total hepatic or extrahepatic blockage no urobilinogen is formed
  132. 132. Differences between un-conjugated and conjugated bilirubin Condition Un-conjugated ConjugatedVanden Bergh Reaction Indirect Direct Solubility Lipid soluble Water soluble Can cross blood brain barrier Excretion in urine No Yes Acholuric jaundice (always pathological) Choluric jaundice Deposition in brain Yes No (lead to kernictrus)Plasma level increased Prehepatic jaundice Hepatic and post (hemolytic jaundice) hepatic jaundice
  133. 133. Major Three Processes Responsible For THE Transfer Of BILIRUBIN From BLOOD To BILERotor,syndrome
  134. 134. BILIRUBIN FUNCTIONS AS AN ANTIOXIDANT.IT IS OXIDIZED TO BILIVERDINWHICH IS AGAIN REDUCEDBY BILIVERDIN REDUCTASE AND REGENERATES BILIRUBIN
  135. 135. Hyperbilirubinemias (Jaundice) Serum bilirubin• Normal : 0.3-1.1 1mg /dl (17.1 umol/L upper limit)• Jaundice appears = > 2-2.5 mg/dl• Hyper bilirubinemias result due to5. Overproduction of bilirubin{HEMOLYTIC}6. Conjugation defect –[ Congenital ] or acquired [Toxic]7. Obstruction of transport (extrahepatic or intrahepatic)
  136. 136. MEASUREMENT OFBILIRUBIN IN THE SERUM ISOF GREAT VALUE INCLINICAL STUDIES OFJAUNDICETHIS IS ONE OF THEPARAMETERSOF LIVER FUNCTION TESTS
  137. 137. TYPES OFJAUNDICE
  138. 138. Hyperbilirubinemias (Jaundice)• Two types : 1. Conjugated (direct) 2. Un-conjugated (indirect) Jaundice• Jaundice when level > 50 µmol/L• Latent jaundice : when level below 50 µmol/L• KERNICTERUS
  139. 139. KERNICTERUS• ONLY UNCONJUGATED BILIRUBIN DUE TO ITS HYDROPHOBICITY CAN CROSS THE BLOOD BRAIN BRRIER IN THE CENTRAL NERVOUS SYSTEM WHICH LEADS TO ENCEPHALOPATHY DUE TO SEVERE JAUNDICE[Unconjugated Bilirubin]• SEVERE HYPERBILIRUBINEMIAS IN NEONATES CAN RESULT IN TO KERNICTERUS
  140. 140. I. Causes of Unconjugated Hyperbilirubinemia• HEMOLYTIC ANEMIAS• Increased lysis of RBCs due to different causes – There is slight increase of bilirubin < 4mg/dl 2. Heriditory spherocytosis 3. Red cell enzyme defect [glucose – 6P – dehydrogenase &Pyruvate kinase deficiency] 4. Hemoglobinopathies [sickle cell disease & thallasemia] 5. Autoimmune diseases – Infections [malaria, clostridium wellchei] – Drugs, chemicals [primaquin in malaria]
  141. 141. I. Causes of Unconjugated HyperbilirubinemiaA. Neonatal “Physiological Jaundice”• Most Common – (Transient condition)• Metabolic defect 1. Rapid hemolysis 2. Immature hepatic system for uptake, conjugation and secretion of bilirubin 3. Low activity of glucuronosyl transferase 4. Reduced synthesis of UDP-glucuronic acid If > 20-25 mg/dl can cause KERNICTERUS
  142. 142. I. Causes of Unconjugated HyperbilirubinemiaTreatment 1. Recovery with in 03 weeks and observe only 2. Drug like barbiturate(Phenobarbital) 3. Phototherapy – polar isomers of bilrubin or derivatives like maleimide fragments excretion in bile
  143. 143. I. Causes of unconjugated hyperbilirubinemiaA. Crigler – Najjar Syndrome Type – I(CN-I) (Congenital nonhemolytic jaundice)Metabolic defect:Absence of enzyme glucuronosyl transferase• Rare autosomal recessive disorder• Severe jaundice > 20 mg / dl• Usually fatal with in 15 days but few can go upto teenagers• Phototherapy and drugs not effective, some response to phototherapy may be there• Phenobarbital has no effect
  144. 144. I. Causes of Unconjugated HyperbilirubinemiaA. Crigler – Najjar Syndrome – type -IIMetabolic defect:(congenital disorder)• Mild defect of conjugation due to – Low activity of glucuronosyl transferase that add second UDP-glucuronic acid moity – Serum bilirubin does not exceeds > 20mg/dl – Bile contains bilirubin monoglucuronide – Respond to high doses of phenobarbitol – Has benign course
  145. 145. I. Causes of Unconjugated HyperbilirubinemiaA. Gilbert Syndrome (Harmless)Metabolic defect(congenital) 1. ↓ levels of glucuronosyl transferase- I,small expanded nucleotide repeats at promoter region of enzyme 2. ↓ uptake of bilirubin by hepatocytes 3. Mild hemolysis in some cases due to reduced RBCs survival 4. Entirely harmlessTreatment – Benign course and no treatment
  146. 146. I. Causes of Unconjugated Hyperbilirubinemia F-Toxic hyperbilirubinemia[mixed typejaundice]Metabolic defect• Liver dysfunction due to damage of hepatocytes, which can lead to: 1. Decreased conjugation 2. Intra hepatic biliary tree obstructionCauses• Drugs, (ccl4, chloroform, paracetamol )• Viral hepatitis• Cirrhosis• Mushroom poisoning• Infections
  147. 147. II. Causes of Conjugated HyperbilirubinemiaA. Obstruction in billary tree [Cholestatic Jaundice] (Choluric Jaundice)2. Intrahepatic microbstruction in infectious viral hepatitis3. Extra hepatic obstruction • Hepatic duct • Common bile duct stones • Tumor of head of pancrease
  148. 148. II. Causes of Conjugated HyperbilirubinemiaA. Chronic idiopathic jaundice (Dubin- Johnson Syndrome)[congenital] Rare: Disorder of childhood and adults, bilirubin range from 2 to 5 mg/dl.It can be in the normal range or as high as 20 mg/dl• Metabolic defect – Secretory defect of hepatocytes for bilirubin and other substances – Diagnosed on histopathology of liver which shows brown pigment in liver cells (Melanin)
  149. 149. II. Causes of Conjugated HyperbilirubinemiaA. ROTORS SYNDROME [congenital]• Metabolic defect:• Decreased transport of congugated bilirubin into bile canaliculi• Liver histology normal (no pigment)• Benign and autosomal recessive disorder
  150. 150. Some cojugated bilirubin can bind covalently to AlbuminIn prolonged conjugated hyperbilirubinemia bilirubin binds covalently toAlbumin and this fraction has a longer life• This bilirubin is called δ bilirubin and it remains elevated during the recovery phase• This explains why some pateints have jaundice inspite of normal levels of cnjugated bilirubin?
  151. 151. Condition Serum bilirubin Urine Urine Fecal urobilinogen bilirubin urobilinogenNormal Direct : 0.1- 0.4 mg/dl 0-4 mg/ 24 h Absent 40-280 mg/ 24 Hours (Conjugated ) Indirect : 0.2-0.7 mg/dl (Unconjugated)Hemolytic ↑Indirect Increased Absent Increasedanemia (unconjugated)Hepatitis ↑ Direct and Decreases if micro Present if Decreased indirect obstruction is micro present obstruction occursObstructive ↑ Direct Absent Present & it is Trace to absentjaundice (conjugated) conjugated called choluric jaundice Laboratory results in normal subjects and patients with three different causes of jaundice
  152. 152. Differences between un-conjugated and conjugated bilirubin Condition Un-conjugated ConjugatedVanden Bergh Reaction Indirect Direct Solubility Lipid soluble Water soluble Can cross blood brain barrier Excretion in urine No Yes Acholuric jaundice (always pathological) Choluric jaundice Deposition in brain Yes No (lead to kernictrus)Plasma level increased Prehepatic jaundice Hepatic and post (hemolytic jaundice) hepatic jaundice

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