A comprehensive presentation on haemoglobin metabolism and its clinical application for MBBS,BDS, B.Tech,B.Pharma students for self study.

1. Hemoglobin metabolism and
its clinical applications
Dr. Rohini C Sane

6. Functions of Hemoglobin molecule in oxygen transport

7. Functions of Hemoglobin molecule in carbon dioxide transport

9. Degradation of Heme to bile pigments
Macrophages of reticuloendothelial (RE )
in spleen /liver /bone marrow-Hb
RBC à 12Odays
Non protein ‘Heme ’Globin
6 gm /day heme broken down /resynthesisà Bilirubin ( 300 mg /day ) = 250 mg/day Hb +
50 mg/day myoglobin

13. Fate globin (reutilization )
• Formation of Hb ( re synthesis )
• Degraded to amino acids –metabolism including formation of Hb

17. Sources of Heme
Heme
20% immature RBC /Myoglobin /globin /cytochrome /peroxidase
/catalase Trp pyrrolase
80% RBC Hb

18. ( GREEN –excreted by birds & amphibians )→
Transferrin
←αGlobin chains
←βGlobin chains
RBC ( Aged erythrocytes )with Hb
phagocytosis by macrophages →
specificity for α methylene bridge ←
Presence in mammals ←
Yellow with 36H←
→Bilirubin- Albumin complex in Blood
+

19. Heme oxygenase
1. Microsomal enzymes
2. NADPH ,O₂ , Methylene bridges between two pyrrole ringsà
biliverdin
3. Fe ²⁺ à Fe³⁺
Heme oxygenase
Heme Biliverdin+ Fe³⁺+ CO
• Biliverdin à excreted by birds ,amphibians & mammals

20. Degradation of Heme to bile pigments
Biliverdin reductase
Biliverdin (green ) Bilirubin(non functional therefore excreted)
1gm hemoglobin *à 35 mg Bilirubin
Biliverdin + Bilirubin à Heme derivatives
• (* sources- 80% RBC + 10% ineffective erythropoiesis + 10% Myoglobin )
• 6 gm /day heme broken down /resynthesisà Bilirubin ( 300 mg /day ) = 250
mg/day Hb + 50 mg/day myoglobin

21. Transport of Bilirubin to Liver
• Bilirubin- ( lipophilic –insoluble in water)
• Bilirubin + Albumin à Transported in plasma
• Per 100ml plasma à 25 mg Bilirubin bound tight to Albumin
• Bilirubin has low affinity for Albumin à easily detached & enter
tissue

23. Drugs : sulphonomides /salicylates / Penicillin displace
Bilirubin from Albumin binding sites
Bilirubin enters CNS
Damage to neurons à Kernicterus
Degradation of Heme to bile pigments

26. Conjugation 0f Bilirubin Liver
Albumin –Bilirubin complex
I Uptake carrier mediated active transport
Sinusoidal surface of hepatocytes
Bilirubin + protein ( Ligandin )
2 UDP Glucoronate
II Conjugation UDP Endoplasmic-reticulum
(hepatic microosomes ) Bilirubin Glucuronyltransferase (chromosome 2 )
Bilirubin + 2 molecules of Glucuronate
80% Bilirubin Diglucuronide+ 20% Bilirubin monoglucuronide
(MOAT –multiple specific organic ion transport )-
present in canalculi
III secretion
Bilirubin Oligonucleotide
Bile salts
Fate of Bilirubin in human body

29. Factors affecting conjugation of Bilirubin in liver
1. Drugs like Primaquine/Novobiocin/Chloramphenicol,/Androgen/Pregnanediol
interfere with conjugation process may cause jaundice.
2. Decrease concentration of UDP-Glucuronyl Transferase
3. Phenobarbital induces UDP-Glucuronyl Transferase

30. Production & excretion of Bilirubin
Retiiculoendothelial system ( RES )
Hb
↓
Biliverdin (38 H )
↓
Bilirubin ( 36 H )
Liver
Bilirubin
Diglucuronide
BilirubinDiglucuronide
↓ Deconjugation
Free Bilirubin (36 H)
↓ reduction
Urobilinogen (44H ) by E coli
(colorless )
Reduction of vinyl group of
stercobilinogen (48H )
↓
stercoblin(46H )
DARK BROWN /200 MG/DAY
BLOOD
UROBILINOGEN
KIDNEY
UROBILINOGEN
UROBILLINOGEN (44H )
↓
UROBILIN IN URINE (42H )
( < 4 GM/DAY )
BILE DUCT→
←PORTAL
CIRCULATION
EHC
↓
←SMALL PORTION
EHC –Entero hepatic circulation

34. Excretion of Bilirubin into bile
Conjugated Bilirubin
Active transport* ( against concentration gradient )
Rate limiting step
Bile
> 98% Bilirubin enters bile in conjugated form
*Induced by Phenobarbitone

36. Production & excretion of Bilirubin
• Excretion of Biliverdin à green color stool( children & prolonged
antibiotics therapy )
• Black color stool during constipation
• Schlesinger test : Stercobilin (SB ) & Urobilin (UB)+Zn ²⁺ à green
fluroscent

37. Fouchet’s Diagnostic test for Bilirubin in urine

39. Bilirubin –yellow ,Biliverdin –green ,Bilinofuschin –Red , Bilicyanin -violet

40. ←URINE→
Gmelin’s Test
←CONC HNO₃→
Bilirubin –yellow ,Biliverdin –green ,Bilifuschin –Red , Bilicyanin -violet

44. Fate of Bilirubin
Bilirubin Glucuronides
Hydrolysis ↓ bacterial enzyme (β Glucuronidase )
Bilirubin
↓ small portion reabsorbed
Urobilinogen(colorless )à SBG ( Erhlich +ve )
↙ ↘
Stercobilin Urobilin
(excreted in stool -) (excreted in urine by kidney )
↓ ↓
Brown color of stool pale yellow color of urine
Serum Bilirubin à Van Der Berg Test, Urine Bilirubin à Fouchet’s Test ,Gmelin Test

49. Conjugated and unconjugated bilirubin ( Direct and indirect bilirubin )

50. Stercobilin and Urobilin

51. Formation of conjugated bilirubin in Hepatocytes

52. Types of Bilirubin
Free bilirubin Conjugated bilirubin
1 In H₂ O insoluble soluble
2 In alcohol soluble soluble
3 Normal plasma levels 0.2 mg /dl 0.2 -0.8 mg /dl
4 In bile Absent present
5 In urine Always absent Normally absent ( present in
hemolytic & Obstructive jaundice )
6 Absorption from GIT Absorbed Not Absorbed
7 Diffusion in tissue Diffuses to cause yellow Dose not diffuse
8 Van der Bergh Test Indirect positive direct positive

53. Bilirubin & its reduction products
number of H atoms color
Bilirubin (BR ) 36 Red yellow
Meso bilirubin (MB) 40 yellow
Urobillinogen (UBG ) 44 colorless
Stercobilinogen ( SBG ) 48 colorless
Urobilin(UB ) 42 Orange brown
Stercobillin( SB ) 46 Dark brown

54. Jaundice- Hyperbilirubinemia
Physiological – Total Bilirubin concentration in serum = ( 0.2- 0.8mg/dl )
0.2 -0.6 mg/dl ( unconjugated ) 0- 0.2 mg/dl ( conjugated )
Yellow color sclera & skin ( deposition of bilirubin )-conc of serum bilirubin > 2mg/dl
q Conjugated Hyper bilirubinemia
q Unconjugated Hyper bilirubinemia
vSymptoms
1. Nausea
2. Vomiting
3. Appetite
Ø Latent Jaundice (1-2 mg/dl )

55. *Diagnostic laboratory to set up own quality controls for reference ranges

56. Pre hepatic ,Hepatic and post hepatic jaundice – a classification based on site (cause of of Hyperbilirubinemia)

57. Classification of Jaundice based on type of Bilirubin

58. Intra hepatic jaundice (Genetic /inherited causes )

59. Cause
• Hemolytic Jaundice
• Causes – Hemolysis
Malaria blood
transfusion sickle cell
anemia
• Liver fails to conjugate
excess of Bilirubin
• Therefore
↑unconjugated bilirubin
↑Urobilinogen
↑ stercobilinogen
(brown color stool )
• SGPT / ALP -Normal
• Hepatic Jaundice
• Causes –dysfunction of
Liver Hepatitis
al infection
poisons/toxins
cirrhosis/CCF
• ↑unconjugated
bilirubin ↑conjugated
bilirubin
↑Urobilinogen
↑ stercobilinogen
(brown color stool )
↑SGPT / ALP
• Obstructive Jaundice
• Gall stone
stool contains fat
unavailability of bile salts
• ↑conjugated bilirubin
↑Urobilinogen
↓ stercobilinogen (pale
color stool )
↑SGPT / ALP
Comparison of Hemolytic / hepatic and obstructive Jaundice

60. Differential diagnosis Jaundice /Icterus

61. Hemolytic Disease of Adults à unconjugated Hyperbilirubinemia
1. increase in unconjugated bilirubin in blood
2. Absence of bilirubin in urine
3. Excretion of urobilinogen à ( Ehrlich Test positive )
4. Excretion of stercobilinogen in faeces
vDiseases associated with
a) congenital spherocytosis
b) G6PD deficiency
c) Autoimmune hemolytic anemia
d) Toxin carbon tetrachloride

62. Hemolytic Disease of Adults à unconjugated Hyperbilirubinemia
Incompatibility between maternal & fetal blood groups
Anti antibodies ABO
IgM type cannot be transferred to placenta
vRh incompatibility
Fetus mother
Rh ( +ve ) Rh ( -ve )
RBC RBC elicit immune response Anti-D ( IgG )
Destruction Anti-D( IgG )
Of RBC ← Placenta
Second pregnancy ( before birth –destruction of RBC ) à CHILD born with severe hemolytic disease à
Erythroblastosis fetalis

63. Erythroblastosis Fetalis
• Serum Bilirubin à > 20 mg/dl à no more bound to Albumin
• Bilirubin Brain (Kernicterus-deposition of bilirubin in brain )
• Basal ganglia à mental retardation
• ↓ ATPase mitochondria à fits ,spasticity ,toxic encephalitis
• Treatment : (1) phototherapy before age < 1 year à
isomerization ZZ à ZE
(2 ) Blood transfusion

72. Hepatocellular Jaundice
1. Viral Hepatitis (viruses A ,B,C,D or G )à pure hepatocellular
diseases
2. ↓ conjugated bilirubin therefore ↑free Bilirubin
3. Inflammatory odema of cells à intracellular canalculi compressed
à obstruction ( at site of bile formation )
4. Increase obstruction à ↑ obstructionà ↑conjugated Bilirubin
therefore Biphasic
5. Bilirubinuria
6. UrobilinogenàNORMAL or decreased in hepatocellular Jaundice

73. Obstructive Jaundice à conjugated Hyperbilirubinemia
1. ↑ conjugated bilirubin
2. Bilirubin in urine
3. ↓ urobilinogen ( nil if obstructive is complete )
4. Faeces clay color (↓ stercobilinogen )
5. Absence of bile salts à Stetorrhoea may result
6. Causes I Intrahepatic Cholestasis
a) Active hepatitis
b) Biliary cirrhosis
c) Lymphomas
d) Hepatoma
e) Viral hepatitis

74. Obstructive Jaundice à conjugated Hyperbilirubinemia
• Causes II Extra hepatic Cholestasis
1. Stones in biliary tract
2. Stones in gall bladder
3. Biliary atresia
4. Carcinoma head of pancreasà lymph glands enlarged
5. Porta hepatitis

75. Acquired hyperbilinogen( unconjugated hyper bilinogen)
vPhysiological Jaundice of newborn ( neonates )
1. After second day of life ,transient hyper bilinogen –Jaundice
2. ↑ rate of destruction of RBC ( transient )
3. à immature hepatic system of conjugation
4. à Bilirubin donot exceed 5mg/dl
5. à 2 weeks phototherapy + barbitone to induce conjugation( ZZ
bilirubin à ZE + EE à excreted without conjugation in urine )
6. Undue prolongation à crenism
7. Breast milk jaundice ( estrogen à ↓ enzyme Glucuronyl transferase)

77. Bile pigment Diagnostic Test
Bilirubin Van Der Bergh (serum ) ,Fouchet’s Test & Gmelin ‘s test
in Urine
Urobilinogen ( UBG ) Ehrlich’s Test
Urobilin Schlesinger’s test

78. DIRECT BILIRUBIIN - Azo pigment (p H 5 ) àPURPLE COLOR-
↓
INDIRECT REACTION –FREE BILIRUBIN ( H2O insoluble , alcohol soluble )

80. *Diagnostic laboratory to set up own quality controls for reference ranges
Enzyme estimations help in differential diagnosis of Jaundice

81. Enzyme estimations help in differential diagnosis of Jaundice
*Diagnostic laboratory to set up own quality controls for reference ranges

82. Enzymes indicating Hepatocellular damage
v↑ ALT ↑AST
Viral Hepatitis
v↑ IHD
Hepatocellular necrosis
v ALT /ALT > 1
Alcohol liver disease
Obstructive liver disease: cholestasis /hepatic carcinoma/parenchymal cell
damage

83. Parameter Hemolytic Jaundice Hepatic
Jaundice
Obstructive
Jaundice
1 Blood free bilirubin ↑ ↑ normal
2 Conjugated bilirubin normal ↑ ↑
3 ALP normal ↑ ↑ ↑
4 Bile salts Nil Nil PRESENT
5 Urine Bilirubin Nil Nil PRESENT
6 Urine urobilinogen ↑ Nil Nil
7 Stool Dark brown Clay color
Differential Diagnosis of Jaundice

87. Fate of Bilirubin

90. Congenital Hyperbilirubinemia
vAbnormal uptake ,conjugation or excretion of bilirubin due to inherited
defects
• 1.Gilbert Disease –defect in uptake
• 2. Crigler Najjar syndrome –
defect in conjugation

91. Congenital Hyperbilirubinemia
v(1) Gilbert’s Disease
a) Autosomal dominant trait
b) Defect uptake of Bilirubin
c) Asymptomatic
d) Presence of jaundice( Bilirubin à 3mg/dl )

92. Congenital Hyperbilirubinemia
(2) Crigler Najjar syndrome :Defect in conjugation
Type I
a) Congenital non hemolytic Jaundice
b) Deficiency of UDP –Glucuronyl transferase
c) Fatal ( death before two years of age )
d) Jaundice appears within 24hrs of life
e) Unconjugated bilirubin ( >20 mg/dl )
f) Kernicterus
g) Barbiturates no effect ( enzyme no defect )

93. Congenital Hyperbilirubinemia
(2) Crigler Najjar syndrome :Defect in conjugation
Type II
a) Congenital non hemolytic Jaundice
b) Deficiency of UDP –Glucuronyl transferase
c) Disease runs more benign course
d) Bilirubin –mononucleotide present
e) Total bilirubin seldome rises :Unconjugated bilirubin ( >15 mg/dl )
f) No Kernicterus
g) Barbiturates use in treatmentà Jaundice improves

94. Management of Crigler Najjar Syndrome

95. Congenital Hyperbilirubinemia
vDubin Johnson’s syndrome
a) Autosomal recessive trait
b) ↑ conjugated bilirubin in blood
c) Defect in excretion of conjugated Bilirubin( ATP ase dependent
organic anion Transporter protein-MOAT mutation
d) Defect ATP dependent organic anion transport in bile canalculi
e) Bilirubin gets deposited in liver therefore Bilirubin gets deposited in
liver ( Black Liver Jaundice )
f) Diagnostic Test : Bromosulphthalein Test

96. Congenital Hyperbilirubinemia
vDubin Johnson’s syndrome
Diagnostic Test : Bromosulphthalein Test
250 mg Bromosulphthalein ( intravenous )
Normal ( dye remaining plasma ) Dubin Johnson’s syndrome ( dye remaining plasma )
45 min < 5 % > 2hrs levels (< 2 % )
2 hrs < 2 % < 45 min levels ( > 5 % )
( 2hr level more than 45min )
At 45 min BSP taken up by hepatocytes / therefore
decrease in blood levels
EXCRETORY DEFECT :BSP regurgatesin blood

97. Congenital Hyperbilirubinemia
vRotor syndrome
a) Cause not known ( autosomal recessive )
b) Bilirubin excretion defective
c) No deposition of pigment in liver
?

98. Comparison of Dubin Johnson ’s and Rotor ‘s syndrome

99. Comparison of Gilbert ‘s and Crigler- Najjar syndrome

102. Biosynthesis of Heme ( Porphyrin ring )
• Site à Liver /RBC producing cells of bone marrow ( erythroid cells)
/other tissue
• Excretion to rule à mature RBC lacking mitochondria
• I Formation of δ amino Levulinate (mitochondria )
Glycine + succinyl CoA
* Pyridoxal phosphate dependent
δ amino Levulinate synthtase
δ amino Levulinate (ALA )
* rate controlling step

103. Biosynthesis of Heme ( Porphyrin ring )
II Synthesis of Porphobilinogen
2. δ amino Levulinate (ALA )
ALA Dehydratase *
Porphobilinogen (PBG )
• Activity decreases by Pb /Hg & Zinc containing enzyme

104. Biosynthesis of Heme ( Porphyrin ring )
1. Succinyl CoA + Glycine → ALA
2. ALA + ALA à Porphobilinogen ( PBG ) + 2H₂O
3. 4 X ( PBG )
4. Uroporbilinogen III (UBG )
5. Coporphyrinogen III ( CPG III )+ CO₂
6. Protoporphyrinogen III ( PPGIII )
7. HEME

105. Biosynthesis of Heme ( Porphyrin ring )
Succinyl CoA + Glycine → ALA
ALA + ALA à Porphobilinogen ( PBG ) + 2H₂O
4
4 X ( PBG )
Uroporphyrinogen
CoporphyrinogenIII ( CPG III ))
ProtoporphyrinogenIII ( PPGIII )
HEME
HEMGLOBIN
1. ALA SYNTHTASE 2. ALA DEHYDRATASE 3. PBG DEAMINASE & UPG III COSYNTHTASE 4 .UROPORPHYRIN
DECARBOXYLASE 5 COPORPHYRINOGEN OXIDASE 6.Heme synthtase
→ 4CO₂
NADP+O₂ → → NADPH+H+ 2CO ₂
ACETYL à METHYL
PROPINYL àVINYL

106. ACETYL à METHYL ,4CO₂
PROPINYL à VINYL+ 2CO₂
Biosynthesis of Heme

110. Metallo- porphyrin
1. Uroporphyrin I & III
2. Coporphyrin I & III
3. PROTOPORPHYRIN IX & HEME
vHeme containing proteins- hemoglobin ,cytochromes ,catalase
,tryptophan pyrrolase

111. Heme synthesis
• Site : mitochondria of all mammalian tissue except RBC predominantly
in Liver & bone marrow
vStep I :
*ALA synthase PLP CO₂ CoASH
Succinyl CoA + Glycine δ ALA
• δ ALA :Delta amino Levullinic acid
• * Regulatory enzyme = rate limiting step
qINH treatment à PLP ↓ à ALA SYNTHESIS ↓

112. Heme synthesis
vStep II : Site –MITOCHONDRIA
* ALA DEHYDRATASE PLP 2H ₂O
2 Molecules of ALA Porphobilinogen( PBG )
* activity decreased by heavy metals

113. Heme synthesis
vStep III : Site –MITOCHONDRIA
*PBG DEAMINASE 4NH₃
4 Molecules of PBG Uroporphyrinogen(UBG )
• *Uroporphyrinogen I synthtase & Uroporphyrinogen III synthtase
• * deficiency of Uroporphyrinogen III synthtase leads to formation of
Type I UBG à Porphyria

114. Heme synthesis
vStep IV : Site –MITOCHONDRIA
*Uroporphyrinogen decarboxylase 4CO₂
• Uroporphyrinogen(UBG)Type III Coproporphyrinogen III
• *Acetyl à Methyl
• * deficiency of à Porphyria

115. Heme synthesis
vStep V : Site -CYTOSOLIC
*Coproporphyrinogen oxidase 2CO₂
CoproporphyrinogenIII ProtoporphyrinogenIII
O ₂
• * Methyl à Vinyl
• * deficiency of Coproporphyrinogen oxidase à Porphyria

116. Heme synthesis
vStep VI : Site -CYTOSOLIC
*Protoporphyrinogenoxidase 4H
ProtoporphyrinogenIII ProtoporphyrinIII ( IX )
Methylene ( CH2 )à Methenyl ( CH - )
• * deficiency of Protoporphyrinogen oxidase à Porphyria

117. Heme synthesis
vStep VII : Site -CYTOSOLIC
*Heme synthtase
Protoporphyrin III ( IX ) Heme
Fe²⁺
• Ferrochelatase
• Heme + Protein= HEMOGLOBIN

124. 1.Succinyl CoA
+ Glycineà
ALA
ALA
2 .ALA à PBG
3.PBG à UBG III 4.UBG à CPGIII
CPG III
CPG III
5. CPGIII àPPG III 6. PPGà PP 7.PPà HEME
CYTOSOLIC -2,3,4
MITOCHONDRIAL
HEME SYNTHESIS

125. Regulation of Heme synthesis
1. ↑ Glucoseà↑catabolism of CRP(repressor protein )à decrease ALA
( induction prevented ,Glucose administered for treatment of
porphyria
2.↓ ALA synthase by Hematin ,Excess of free Heme ( Fe²⁺ à Fe³⁺)
3. Compartmentization of enzymes à easier for regulation ,rate limiting
steps in mitochondria
4. Heme synthesis controlled by a) globin synthesis b) Drug like
barbiturates ( require cytochrome 450)
5 .lead /Mercury ↓ ALA Synthase ↓ Ferrochelatase

127. Regulation of Heme synthesis
Three mechanisms controlling activity of ALA synthase by Heme
/Hematin Fe³⁺
a. Feedback inhibition(Heme /Hematin Fe³⁺ )
b. Repression of ALA synthase
C. Inhibition of ALA synthase transport from cytosol to mitochondria ( the
site of action )
Heme synthesis site : liver ( ALA synthase à regulatory enzyme )

129. Effect of drugs on ALA synthase activity
1. *Phenobarbital
2. * Insecticides ↑ activity of ALA synthase
3. * Carcinogen
* Metabolized by HEME containing protein cytochrome 450
↓
Increased in corporation of Heme in cytochrome 450
↓
↓ cellular level of Heme
↓
↑ ALA synthase ( de repression ) to meet cellular demands

130. Regulation in the Erythroid cells
vALA synthase dose not regulate Heme synthesis in Erythroid cells
vUroporphyrinogen synthase & Ferrochelatase regulate Heme
synthesis
vCellular uptake of iron regulate Heme synthesis
vHeme stimulate globin synthesis

131. Regulation of Heme synthesis

132. Porphyrin & Bilirubin Metabolism
• Porphyrin : Group of compounds of 4 substituted pyrrole rings
linked by Methane bridges
• à formation of complexes with metal ions bound to N₂ atom of
pyrrole ring
IRON
Heme
Hb
MAGNASIUM
Chlorophyll
Photosynthetic pigment

133. Porphyrin & Bilirubin Metabolism
vIron Porphyrins :
1. Hemoglobin
2. Myooglobin
3. Cytochrome
4. Catalase & peroxidase
5. Tryptophan pyrrolase

135. Porphyrin Metabolism
• Porphyria's: Group of inborn errors of metabolism associated with
the biosynthesis of Heme
• Characteristics of Porphyrias :increase in production & excretion of
porphyrin & their porphyrin precursors ALA & PBG
• Autosomal dominant or Autosomal recessive
• Diagnosis of Porphyria's:
• 1. urine ( exoposure to UV à red fluorescence )
• 2. urine + CHCl₃ à extraction
• Aqueous layer ( PBG ) + Ehrlich reagent à pink color

138. Porphyria's:
• Types Porphyria's:
1. inherited
a) Erythropoietin : enzymes deficiency occurs in erythrocytes
b) Hepatic : enzymes deficiency lies in the liver
2. Acquired 💟

145. Autosomal recessive
Autosomal dominant
Erythrodontia
RBC -FLUORESCENCE
Liver ,skin fluorescence

146. Acute intermittent Porphyrias
• Deficiency of enzymes Uroporphyrinogen synthase I
Succinyl CoA + Glycine
δ aminolevilinate (ALA ) ↑
Porphobilinogen ↑
UroporphyrinogenIII
δ aminolevilinate synthase
ALA Dehydratase
Uroporphyrinogen synthase
Uroporphyrinogen I
↑ ALA
↑PORPHYRINOGEN

149. 1. Acute intermittent Porphyria
• Age : after puberty (Artist –Vincet , King George III –MAD )
• Deficient enzyme : Uroporphyrinogen synthase I ( ↑ ALA –no feed back
mechanism )
• No porphyria ,no photosensitivity
• Excretion in urine : δ aminolevilinate (ALA ) & porphobilinogen ( PBG )
• Exposure of porphobilinogen ( PBG ) to air darkens ( formation of
Porphobilin )
• Diagnosis : urinary conc of PBG (darkens on exposure to air )
• Symptoms : Abdominal pain ,Vomiting ,cardiovascular abnormalities
,neuropsychiatric disturbances ( therefore ↓ Trp pyrrolase , ↑ Trp,
5 ʹOH Tyramine
• Treatment : Hematin à↓ ALA , ↓ PBG
• Adverse effects : Barbiturate ( ppt of attack )à ↑ ALA synthase (as
cytochrome 450 )àPBG↑, increase with carbohydrate diet & menopause

151. 2. Congenital erythropoietin Porphyria
vCongenital
vDeficient enzyme : ↓ Uroporphyrinogen co synthtase III
• Excretion in urine : Uroporphyrinogen I & Coporphyrinogen Ià
↓ oxidation ↓
Uroporphyrin I & CoporphyrinI
Symptoms :
1.↑ Hemolysis
2. Erythrodontia ( teeth ) ,port wine color urine
3. Exposure OF SKIN à UV sensitization of porphyrin à absorbed & emit red
fluorescent light
4. Dermatitis ,scarring burning & itching of skin- ear & nose ( Leprosy like ),(as ROS ↑
à accumulation of Porphyrin )
5. port wine color urine (Uroporphyrin I & CoporphyrinI )

152. Congenital erythropoietin Porphyria
Photosensitivity
Erythrodontia( teeth)

153. Congenital erythropoietin Porphyria
Dermatitis ,scarring burning & itching of skin- ear & nose ( Leprosy like ,
(as ROS ↑ à accumulation of Porphyrins)

154. Dermatitis ,scarring burning & itching of skin- ear & nose ( Leprosy like )
(as ROS ↑ à accumulation of Porphyrins
Congenital erythropoietin Porphyria

156. 3.Porphyria Cutanea Tarda
• Cutaneous hepatic porphyria
• Deficient enzyme : ↓ Uroporphyrinogen decarboxylase
• Excretion in urine : Porphobilinogen & Uroporphyrin ( I &III ) rarely
• Symptoms :
1. Cutaneous
2. Photosensitivity
3. Liver exhibits fluorescence
Treatment : Hematin( ↓ ALA synthase, ↓ accumulation various
intermediate )

157. 4.Hereditary Coporphyria
• Deficient enzyme : ↓ Coporphyrinogen oxidase
• Excretion in urine : Uroporphyrinogen I & Coporphyrinogen Ià
↓ oxidation ↓
Uroporphyrin I & Coporphyrin I
• Urine dark color : presence of Uroporphyrin I , Coporphyrin I,ALA, PBG
• Treatment : Hematin( ↓ ALA synthase, ↓ accumulation various
intermediate )
•

159. 5.Variegate porphyria
• Deficient enzyme :Protoporphyrinogen oxidase
• Accumulation in plasma & excretion in urine of: Porphobilinogen
Uroporphyrinogen ,Uroporphyrin ,Coproporphyrinogen
,Coproporphyrin ,Protoporphyrin ( ↓Heme synthesis )
• Photosensitivity exhibited
• Plasma exhibits red fluorescence ( due presence of
Coproporphyrinogen
• Neuropsychiatric manifestation

161. 6.Herediatory Protophyria ( Erythropoietic protoporphyria )
• Deficient enzyme: Ferrochelatase
• Accumulation in plasma ( increase in concentration ) , excretion in
urine ( increase in concentration )& faeces (increase in concentration )
of: PROTOPORPHYRIN IX
• RBC ,skin exhibit red fluorescence
•

162. 7. Acquired (toxic ) Porphyrias
• Exposure of body to toxic compounds ( toys / Xerox ink )
Examples : Heavy metals ( Lead ) ↓ALA Dehydratase
Hexchlorobenzene ↓Uroporphyrin synthase I
Drugs ( Griseoflavin ) ↓ Ferrochelatase
Therefore ↓Heme ↑ALA synthase
Aquried porphyrias associated with anamia

165. Reactions of Heme synthesis

166. Pre natal diagnosis of Porphyrias
Enzyme estimations /PCR

167. Anemia
• 75 % Indian population suffer from anemia
• Hb concentration decreases in anemia <10gm%
( normal concentration of blood Hb – 14-16 mg% -male ,13-15 mg% -female )
vI Iron Deficiency anemia –most common
a) Nutritional
b) Lack of absorption of Iron ( Gastrectomy & Achlorhydria)
c) Hookworm infection ( 0.3ml/ day / hookworm )
d) Repeated pregnancy ( ↓hemoglobin -1gm per delivery )
e) Nephrosisà Glomerular filtration →proteinuria
f) Loss of Haptoglobin /Hemopexin / Transferrin
g) Heavy metal poisoning
h) Loss of blood ( menustrual cycle , piles , peptic ulcers, Uterine hemorrhages )

168. Anemia
II -Impaired production of RBC
Defect in Heme
synthesis
• Nutritional
• Deficiency of iron
,Copper, Vitamin
B12 ,Vitamin C
• Lead poisoning
Deficiency of
ERYTHROPOITIN
• Physiological –kidney
cells synthesize
ERYTHROPOIETIN
↓
• ↑RBC synthesis
• Chronic renal failure à
no ERYTHROPOIETIN
Decrease in
stem cells
• APLASTIC
ANEMIA
• MALIGNANT
INFITRATION
• INFLECTION

170. Hemin crystals
• Fe²⁺ ↔ Fe³⁺ à Fe³⁺ + Cl⁻ à Hematin chloride or HEMIN
vMedical legal cases
• Blood + Blood stains + Nippe ’s fluid
( 1% KCL + KBr + KI + Glacial acetic acid à dark brown RHOMBIC crystal
à microscopic inspection
Heme part of Blood à Test positive

171. Hemin crystals

172. Hemoglobin estimation
vDrabkin’s Test for Hemoglobin estimation : Cyanmeth Hemoglobin Method
• Blood ( Hb ) + Drabkin’s reagent àCyanmeth Hemoglobin ( Absorbance -
540nm )à colorimetric estimation
• Hb + potassium ferricyanide à Meth- Hb

174. Sulph Hemoglobinemia
• Oxy –Hb + H₂ S à Sulph –Hb ( absorption peak at 620nm)
vFormation of Sulph –Hb by sulphonamide ,Phenacetin , Dapsone ,Acetone
vBasophilic striping of RBC
←Irreversible

176. Abnormal Hb or Hb variants
1.Sickle syndrome
a) 1. sickle cell trait ( AS )
b) 2. sickle cell disease with SS ,SC,SD ,SO ,Sβ Thalassemia
2.Unstable Haemoglobins
Congenital Heinz body anemia –Hb Zurich
3. Hb with abnormal oxygen affinity
A. High affinity à Polycythemia (familial ) à Hb Chesapeake ,Olympia
B.Low affinity à Cyanosis (familial ) à Hb M, Hb –Kansas, Hb- Hoppe
4. Structural variation leading to Thalassemia's phenotype
A. Alpha Thalassemia à Hb Constant spring ,Delta beta Thalassemia, Hb –
Lepore
B. Beta Thalassemia: Hb Quong
C. 5.Hemoglobin that donot produce any clinical symptoms: HbP HbQ ,HbJ

178. SICKLE CELL DISEASE
vCharacteristics of Sickle cell disease
a) Glutamic acid à Valine (6th position on beta chain)
b) Hydrophilic à Hydrophobic ( stickiness on surface of molecule )
c) Polymerization of Hb in RBC àdistortion of RBC into sickle shaped
d) Deoxy HbS has protrusion on one side & cavity on other sideà
many molecules adhere together

188. SICKLE CELL DISEASE

196. Signs and symptoms of Thalassemia major

200. Inheritance of Thalassemia

203. Abnormal Hb or Hb variants
III Hemolytic anaemias due to intra -corpuscular defect
a) Hemoglobinopathies : HbS ,Hbc , HbM
b) Thalessemias : major & minor
c) Abnormal shape spherocytosis & elliptocytosis
d) Enzyme deficiency à Glucose 6 Phospho dehydrogenase
IV Hemolytic anemia due extra corpuscular cause /defects
a) Infection –malarial parasites ,streptococcus
b) Autoimmune Hemolysis –RBC membrane component ,Syphilis,
Lympholenticular neoplasia
c) Isoimmune hemolysis à Rh incompatibility in Newborn
d) Hemolysis due to drug sensitization –Dopa quinone ( fixed on RBC )à
Abnormal antibodies against altered membrane

204. Abnormal Hb or Hb variants
V hemorrhages
a) Hematuria
b) Hematomesis
c) Hemoptysis
d) Peptic ulcers
e) Hemorrhoides
f) Thrombocytopenia
g) Menorrhagia
h) Bleeding tendencies