4. 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.
5. • 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.
6. • 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.
7. • 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.
11. HAEMOGLOBIN AND MYOGLOBIN
a) STRUCTURE / FUNCTIONS
[ Oxygen binding ]
b) TYPES OF HAEMOGLOBIN ,ITS
DERIVATIVES
d) CATABOLISM OF
HAEMOGLOBIN (PORPHYRIN
DEGRADATION OR BILE
PIGMENT FORMATION)
JAUNDICE
13. 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)
14. 5. Transport , Conjugation and Excretion
of bilirubin
6.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.
15. BIOMEDICAL IMPORTANCE
A. 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
16. BIOMEDICAL IMPORTANCE
(Contd)
B. JAUNDICE
C. HAEMOGLOBIN AND MYOGLOBIN
BOTH ILLUSTRATE
– PROTEIN STRUCTURE & FUNCTION
RELATIONSHIPS
– MOLECULAR BASIS OF GENETIC
DISEASES, LIKE “SICKLE CELL
DISEASE” AND “THALASSEMIAS”
17. Protein Function
Hemoglobin Transport of oxygen in blood
Myoglobin Storage of oxygen in muscle
Cytochrome c Involvement in electron transport chain
Cytochrome P450 Hydroxylation of xenobiotic
Catalase Degradation of hydrogen peroxide
Tryptophan pyrrolase Oxidation of tryptophan
Some important Human and Animal Hemoproteins
28. 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.
29. 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
30. Step-VII
Lead
Formation of Heme involves incorporation
of iron into Protoporphyrin III (IX)
33. 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
34. ALAS-2 erythroid form of this
enzyme 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
35.
36. 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.
39. 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
40. CLINICAL IMPORTANCE
Porphyrias though not prevalent but should
be considered in differential diagnosis in
the following conditions:
2.Abdominal pain
3.Neuropsychiatric problems
4.Dermatitis (Photosensitive)
41. Classification of Porphyrias
I. Based on clinical features
b) Abdominal pain, neuropsychiatric problems
and no photosensitivity
Due to accumulation of delta ALA and PBG in
the tissues and urine
Acute intermittent porphyria
e) All other five pophyrias are accompanied with
photosensitivity to light (dermatitis)
Due to accumulation of porphyrinogens and
porphyrins in the tissues
44. II. Based on the organs involved which are
mainly responsible for the synthesis of
heme
– Erythropoitic
– Hepatic
– Hepatic and erythropoitic
A. Hepatic porphyria can be acute or
chronic
46. 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
47. (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
48. 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
50. (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
51. 2. Increased ALA Synthase Activity
a. Heme level is decreased in all porphyrias
b. Derepression of ALA synthase
c. Increased synthesis of intermediates prior
to genetic block
d. Accumulation of toxic intermediates in
tissue is the main biochemical basis of
disease
52. 3. Diagnosis
a. Family History and Clinical Features
b. 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 faeces
c. Prenatal diagnosis by using appropriate
gene probes
53. 4. Treatment
a. To avoid drugs which precipitate attack
b. Symptomatic treatment by analgesics,
high glucose intake, intravenous hemin to
repress the ALA synthase activity
c. Administration of B carotene to combat
free radicals and sunscreens to avoid
photosensitivity
d. Gene therapy in future to replace enzymes
59. 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
60. 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
61. 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
62. 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
65. 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
66. Oxygen dissociation Curve of
Myoglobin is Hyperbolic and
Hb has sigmoid i.e Steepest
dissociation curve
67. 5- Binding of O2 to Mb is not
influenced 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.
68. • 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
71. 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)
72. • 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
73.
74.
75. 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
76. • 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
79. BINDING OF OXYGEN TO
MYOGLOBIN AND
HEMOGLOBIN
MYOGLOBIN =
ONE MOLECULE OF O2 WITH
ONE MYOGLOBIN AT ONE
HEME GROUP
HEMOGLOBIN =
FOUR MOL OF O2 WITH ONE Hb
ONE AT EACH OF ITS FOUR
HEME GROUPS
80. 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
81. • MYOGLOBIN HAS HIGH O2
AFFINITY
• PARTIAL PRESSURE REQUIRED
FOR 50% SATURATION WITH O2
MYOGLOBIN = 1mm Hg
(HYPERBOLIC CURVE)
HEMOGLOBIN = 26 mm Hg
(SIGMOIDAL CURVE)
82.
83. ALLOSTERIC EFFECTORS
MODIFYING THE BINDIG OF O2
WITH HEMOGLOBIN
II. Heme Heme interactiom
IV. pO2
VI. pH
VIII. pCO2
X. 2 , 3 BPG
84. 1-HEME-HEME
INTERACTIONS
a. LOADING AND UNLOADING
OXYGEN
c. SIGNIFICANCE OF SIGMOIDAL
OXYGEN DISSOCIATION CURVE
87. 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
88. 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
89. Oxygen dissociation Curve of
Myoglobin is Hyperbolic and
Hb has sigmoid i.e Steepest
dissociation curve
90. 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
91.
92.
93. 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
94.
95. 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
96. Nitric oxide gas(NO) transport
• Hb can carry NO
• Potent vasodilator
• It can be released from RBCs
• NO influences the vessel diameter
97. 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
98.
99. 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
100. 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
101.
102.
103. 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
104. d-Response of 2,3BPG levels to
chronic hypoxia or anemia
1-Chronic obstructive pulmonary diseases
Chronic bronchitis
Emphysema
2-High altitude
3-Anemia
Delivery of maximum O2 to tissues
105. 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
108. TYPES of Hb [NORMAL]
1. All are tetrameric.
2 . Composed of
two α-globin like polypeptides &
two β-globin like polypeptide chains
3. Present in various ages of life
109. 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. HbA1C
7. Hb-F is also present < 2%
110. TYPES of CHAINS IN Hb
These are classified due the presence of
six 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%
113. 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
114. • 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
116. 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)
117. 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
118. • 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
121. 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 ?
122. 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
123. 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 A2
Alteration of globin gene expression(one gene
to an other) during development is called Hb
switching .This is regulated byTranscription
factors by binding to promoter region on
DNA
126. 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
129. 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
130. 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]
133. 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
134. 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 will
terminate the polymerization due to lack of sticky patch.
135.
136.
137. 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
138. 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)
140. 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
141. Structurally abnormal
Hb(β globin chains)
with altered amino
acid sequence.
A single nucleotide
alteration leads to a
point mutation
142.
143. 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)
144.
145. 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)
146. 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
147. • 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
150. 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
153. I. β - Thalassemia Minor
• One gene of one individual is defective
• Can make some β - chains because they are
heterozygous
• No treatment is required
154. 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
155. 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
156. 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
159. 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
160. CONJUGATION OF BILIRUBIN
• Site : Hepatocyte (SER),
• Enzyme : glucuronosyl transferase
• Substrates : Glucuronic acid and
unconjugated biliburin
• Product : bilirubin diglucuronide
• This process is induced by Phenobarbital
161.
162. Transport ; Conjugation and Excretion of Bilirubin
Formation and enterohepatic circulation of Urobilinogen
163.
164.
165. 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
166.
167. 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
168.
169.
170. Differences between un-conjugated and
conjugated bilirubin
Condition Un-conjugated Conjugated
Vanden 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
171.
172. Major Three
Processes
Responsible For
THE
Transfer Of
BILIRUBIN
From
BLOOD
To
BILE
Rotor,syndrome
173. BILIRUBIN FUNCTIONS AS
AN ANTIOXIDANT.IT IS
OXIDIZED TO BILIVERDIN
WHICH IS AGAIN REDUCED
BY BILIVERDIN REDUCTASE
AND REGENERATES
BILIRUBIN
174. 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 to
5. Overproduction of bilirubin{HEMOLYTIC}
6. Conjugation defect –[ Congenital ] or
acquired [Toxic]
7. Obstruction of transport
(extrahepatic or intrahepatic)
175. MEASUREMENT OF
BILIRUBIN IN THE SERUM IS
OF GREAT VALUE IN
CLINICAL STUDIES OF
JAUNDICE
THIS IS ONE OF THE
PARAMETERS
OF LIVER FUNCTION TESTS
179. 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
180. 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]
181. I. Causes of Unconjugated
Hyperbilirubinemia
A. 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
182. I. Causes of Unconjugated
Hyperbilirubinemia
Treatment
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
183. I. Causes of unconjugated
hyperbilirubinemia
A. 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
184. I. Causes of Unconjugated
Hyperbilirubinemia
A. Crigler – Najjar Syndrome – type -II
Metabolic 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
185. I. Causes of Unconjugated
Hyperbilirubinemia
A. 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 harmless
Treatment
– Benign course and no treatment
186. 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 obstruction
Causes
• Drugs, (ccl4, chloroform, paracetamol )
• Viral hepatitis
• Cirrhosis
• Mushroom poisoning
• Infections
187. II. Causes of Conjugated
Hyperbilirubinemia
A. Obstruction in billary tree [Cholestatic
Jaundice] (Choluric Jaundice)
2. Intrahepatic microbstruction in
infectious viral hepatitis
3. Extra hepatic obstruction
• Hepatic duct
• Common bile duct stones
• Tumor of head of pancrease
188. II. Causes of Conjugated
Hyperbilirubinemia
A. 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)
189. II. Causes of Conjugated
Hyperbilirubinemia
A. ROTORS SYNDROME [congenital]
• Metabolic defect:
• Decreased transport of congugated
bilirubin into bile canaliculi
• Liver histology normal (no pigment)
• Benign and autosomal recessive
disorder
190. Some cojugated bilirubin can
bind covalently to Albumin
In 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?
191. Condition Serum bilirubin Urine Urine Fecal
urobilinogen bilirubin urobilinogen
Normal 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 Increased
anemia (unconjugated)
Hepatitis ↑ Direct and Decreases if micro Present if Decreased
indirect obstruction is micro
present obstruction
occurs
Obstructive ↑ Direct Absent Present & it is Trace to absent
jaundice (conjugated) conjugated
called
choluric
jaundice
Laboratory results in normal subjects and patients with three
different causes of jaundice
192. Differences between un-conjugated and
conjugated bilirubin
Condition Un-conjugated Conjugated
Vanden 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
