Download HEMOGLOBIN AND PORPHYRINS

HEMOGLOBIN
AND
PORPHYRINS
HEME-CONTAINING PROTEINS
• Hemoglobin
• Myoglobin
• Cytochromes
• Catalase
• Some peroxidases
STRUCTURE OF HEMOGLOBIN
„
Globular proteins belong to hemeproteins
Heme proteins
Heme
The prosthetic group
function dictated by
the protein part
+
Protein
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Hemoglobin (Hb) is the red blood pigment, found in
ERYTHROCYTES (greek:- erythrose – red; kytose – a hollow
vessel).
Normal level of Hb in blood in males is 14-16g/dl and in
females 13-15g/dl.
The adult hemoglobin (HbA) has 2 α chains & 2β chains.
Mol.wt of HbA is 67,000.
Hb is a conjugated protein, containing GLOBIN- the
apoprotein part & the HEME – the non – protein part
(prosthetic group).
Hb is a tetrameric allosteric protein,
Each gram of Hb contains 3.4 mg of iron.
STRUCTURE OF GLOBIN
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Globin consists of 4 polypeptide chains of two different
monomeric units. 2 α chains & 2β chains.
Each α –chain contains 141 a.a.
Each β- chain contains 146 a.a.
Thus HbA1 contain 574 a.a.
The 4 subunits of Hb are held together by non-covalent
interactions. ( hydrophobic, ionic & H-bonds)
Each subunit contain a Heme group.
α -chain gene is on chromosome 16, while β,γ &δ- chains are
on chromosome 11.
Structure of heme
Characteristic red colour of blood
HEME
Organic component + central iron atom
„
Protophorphyrin IX
4 pyrrole rings linked by
methenyl bridges to form
tetra pyrrole
„ 4 methyl groups, 2 vinyl
groups, 2 propionate
side chains are attached
„
Central iron atom
Iron atom is in Ferrous oxidation state (Fe2+)
„ Iron has 6 valencies
„
4 bonded to pyrrole N2
Other two
5th coordinated site
linked to
Imidazole
N2 of histidine
deoxy-Hb
oxy Hb & Myoglobin
6th coordinated site
deoxy-Hb un occupied
oxy Hb &
Myoglobin – O2
6th
5th
Structure of HbA
„ 38 histidine residues – buffering action
„ 58th residue in α-chain- distal histidine
„ 87th residue in α-chain- proximal histidine
„ Each α and β sub units has stretches of
α-helix and heme binding pocket
Interaction of heme with goblin:
„ 4 heme residues per Hb molecule, 1 for
each subunit in Hb
„ The heme is located in a cleft between the
E and F helices of Hb chain
„ Heme group accounts for 4% of whole
mass of Hb
Quaternary structure of Hb
„ Hb tetramer can be viewed as being composed
of two identical dimers (αβ)1, (αβ)2
„ 2 polypeptide chains within the dimer are held
primarily by strong hydrophobic interactions
„ The two dimers move with respective each
other, being held primarily by weaker ionic and
Hydrogen bonds
The movement produces two different
structures of Hb namely a “T,” or taut,
structure of deoxy-Hb and “R,” or relaxed,
structure of Oxy-Hb
“T” form
„ Deoxy form of Hb
„ Taut or tense form
„ The two dimers interact through a
network of ionic and hydrogen bonds that
constrain the movement of the
polypeptide chain
„ Low O2 affinity form of Hb
“R” form
„ Oxygen bound form of hemoglobin
„ Relaxed form of Hb
„ Binding of O2 causes rupture of some of ionic
and hydrogen bonds between the dimers and
hence freedom of movement.
„ High O2 affinity form of Hb
™ “T”
and “R” form interchange during loading
and unloading of O2 with Hb
Structure and function of myoglobin
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Myoglobin – present in heart and skeletal
muscle
Single polypeptide chain of M.wt 17000
153 A.A residues
Single polypeptide chain structurally similar to
individual subunit polypeptide chains of Hb
Compact molecule, 80% of it folded to 8 stretches
of alpha helix (A - H)
Mb has higher affinity for O2 than Hb. Bohr effect,
co-operative effect & 2,3-BPG effects are absent.
Heme
Arrangement of A.A residues
„ Interior – non polar A.A stabilized by hydrophobic
interaction
„ Surface – charged A.A forming hydrogen bonds with
each other and water
„ the heme group of myoglobin sits in crevice lined by
non polar A.A, except for two histidine residues
„ 1, the proximal histidine bind directly to the 5th
coordinated site
„ 2nd, distal histidine does not interact directly with heme
but helps to stabilize the binding of O2 to Fe ion
Function of Myoglobin
Reservoir for O2
„ O2 carrier
„
red muscles
increases the rate
of transport of O2
with in the muscle cell
Biomedical importance of
Myoglobin
Myoglobinuria
follows massive crush injury
„ Myocardial infarction
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FUNCTIONS OF Hb
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Transport of O2 from lungs to tissues .
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Transport of CO2and protons from tissues
to lungs .
Transport of O2 by Hb
Each Hb molecule can bind 4 molecules of O2
one at each of its 4 heme groups the degree
of saturation of all Hb molecules can vary
from 0-100%
O2 dissociation curve (ODC) Hb
„ Plot of degree of saturation (Y-axis)
measured at different PO2 (X-axis).
„ ODC for Hb is sigmoid
„ Explains the ability of Hb to load and unload
O2 at physiological PO2
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PO2 of inspired air 158mmHg,
PO2 of alveolar air 100mmHg(Hb 97% sat)
PO2 of tissue capillaries 40mmHg(Hb 60% sat)
40% of bound O2 released at the tissue level
ODC-MYOGLOBIN
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Hyperbola
At po2in lungs –myoglobin-100% saturated
At po2in tissues-almost same
so at physiological po2 – myoglobin does not deliver
it bound oxygen
However ,at po2of 5mmHg (exist during sternous
physical exercise)-myoglobin readily release O2
ODC OF Hb
1.Sigmoid
2.Binds 4mol of O2
3.Delivers its O2 to
tissues at physiological
pO2
ODC OF MYOGLOBIN
1. Hyperbola
2.Binds only one mol
of O2
3.Functions mainly in
storage.Delivers its
bound O2 only
during sternous
physical exercise
Heme-Heme interaction and co-operativity
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ODC for Hb- sigmoidal
Indicates that the subunits co-operate in
binding O2 which is called co-operativity
It means that binding of an O2 molecule at one
heme increases the O2 affinity of remaining
heme groups in the same Hb molecule this is
called heme-heme interaction
What is the advantage of Heme-Heme
interaction ?
1. Higher affinity
Although it is more difficult for first O2
molecule to bind to hemoglobin,
subsequent binding of O2 occurs with
higher affinity
Net effect of this- Last O2 bound has
approximately 300times > affinity than for
the first
2. Easy loading and unloading of O2
„ Co-operative binding of O2 allow Hb to
deliver more O2 to the tissues in response
to relatively small changes in P O2
Conformational changes
accompany Oxygenation of Hb
1.Rupture of salt bonds between the
carboxyl terminal residues of all 4
subunits
2.One pair of rigid subunit dimer (αβ)2
rotates through 15 relative to the
other rigid pair(αβ)1
(T-R change takes place)
3.Iron atom of deoxyhemoglobin which
lies about 0.06nm beyond the plane
of heme ring moves into the plane of
heme ring this is transmitted to
proximal histidine and also to the
residues attached
Factors affecting ODC
1. P O2
2. pH
3. PC O2
4. 2,3-bisphospho glycerate(BPG)
5. Temperature
6. Bohr effect
7. Chloride shift
Allosteric
effectors
pH and pCO2
„ Tissues- formation of metabolic acids like
lactate
pH & pC O2
affinity of Hb for O2 (ODC shift to right)
R-T change takes place in Hb
O2 released to tissues
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Lungs:
pO2, pCO2 & pH
Affinity of Hb for O2(ODC shift to left)
T-R change takes place in Hb
Bohr effect
The influence of pH and pCO2 to facilitate
oxygenation of Hb in lungs and
deoxygenation at the tissues is known as
Bohr effect
„ Bohr effect causes a shift in the ODC to the
right.
„ Bohr effect is responsible for the release of
O2 from oxyHb to the tissues.
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Mechanism of bohr effect
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Bohr effect is caused by binding of H+ and
CO2 to Hb.
O
CH2
C
N
+
O----------HN
Asp 94
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HbO2 + H+
His 146
Hb H+ + O2
Effect of 2,3BPG
2,3BPG
„ Intermediate in glycolytic pathway
„ Most abundant organic phosphate in RBC
„ One molecule of 2,3BPG binds to a pocket formed
by 2 beta globin chains (pocket contains +vely
charged A.A that form ionic bonds with –vely
charged phosphate of 2,3BPG)
„ 2,3BPG is expelled from Hb on oxygenation
„ Normal level 15+1.5 mg/g Hb.
„ High O2 affinity of HbF is due to inability of γ-chain
to bind 2,3- BPG
Binding of
2,3-BPG
Shift of ODC
2,3-BPG preferentially binds to the deoxy Hb and
not to oxyhemoglobin
Stabilizes the taut conformation of
deoxyhemoglobin
Reduces the affinity of Hb for O2
Shift ODC to right
O2 released from Hb efficiently at the partial
pressure found in tissues
CLINICAL SIGNIFICANCE
Increased 2,3-BPG in RBC
1. Chronic hypoxia:- for high O2 supply. Include
high altitude, obstructive pulmonary
emphysema.
„ Chronic anemia
In these conditions 2,3-BPG lowers the O2
affinity of Hb permitting grater unloading of
O2 in the capillaries of the tissues
Decreased 2,3-BPG in RBC
„ In transfused blood 2,3-BPG in RBCs
results in abnormally high O2 affinity and
fails to unload its bound O2 to tissues.
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This can be prevented by adding inosine
Chloride Shift
In Tissues:
Pco2
Co2+H2o
H2Co3
H+ OxyHb
Hco3HHb+o2 released to cells
Diffuses into plasma and Chloride Shifts from plasma into
RBC to maintain equilibrium.This shift of Chloride is
Chloride shift.
***
Reversal of Chloride Shift
In Lungs
O2+Hb
Hbo2
Inhaled
H++Hco3
HCO3
H2co3
H2o+Co2
Exhaled
As Hco3- levels falls inside the RBC’S more and more
Hco3- gets in and chloride ion diffuses out and it is
called Reversal of chloride Shift
SHIFT OF ODC
PO2
PH
PCO2
2,3BPG
Temperature
RIGHT
LEFT
Transport of Co2
„ Most
of CO2 is hydrated and is
transported as bicarbonate ion.
At rest, about 200ml of CO2 is produced
per minute in tissues.
„ In aerobic metabolism, for every molecule
of O2 utilized, one molecule of CO2 is
liberated.
„ About 15% of CO2 carried in blood directly
binds with Hb.
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Carriage as carbamino - hemoglobi
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Some CO2 is carried as carbamate bound to the
uncharged α amino group of hemoglobin (Carbaminohemoglobin). Hb-NH2 + CO
Hb-NH-COO- + H+
2
Binding of CO2
stabilizes the T form of hemoglobin,
decrease in its affinity for oxygen .
In lungs, CO2 dissociates from hemoglobin and is
released in the breath.
Dissolved form
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About 10% of CO2 is transported as dissolved
form.
CO2 + H2O
H2CO3
HCO3- + H+
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The H+ thus generated, are buffered by the
buffer system of plasma.
Minor Hemoglobins
HbA2
„ Fetal Hb (HbF)
„ Embryonic Hb
„ HbA1C
„
MINOR HEMOGLOBINS
TYPE
% OF Hb
Hb A2
COMPOSITION
& SYMBOL
α2δ2
Hb F
α2γ2
<2%
Hb A1c
Α2β2-glucose
<5%
<5%
HbA2
2% of total normal adult Hb
„ Appears about 12 weeks after birth
„ 2α & 2δ chains
„ Moves slower on electrophoresis compared
to the normal Hb
„ As a compensation HbA2 levels are increased
in β-thalassemia
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Fetal Hb
Tetrameric protein consisting of 2α& 2γ
chains
„ Alpha chain is identical to those found in HbA
„ γ chain has 146 A.A (36 A.A differ from
those of β)
„ Major Hb found in fetus and newborn
„ Synthesis starts from 7th week of gestation.
At birth about 80% of Hb is HbF. During the
first 6months of life it decreases to about 5%
of total
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Fetal Hb cont..
Differentiating features;
„ Decreased interaction with 2,3-BPG
„ Increased solubility of deoxyHbF
„ Slow electrophoretic mobility
„ Increased resistance of Hbf to alkali
denaturation
Embryonic Hemoglobin
Normal during fetal life
„ Hb Gower1 & Hb Gower 2
„ Synthesized during 3rd to 8th week of
gestation
„ Hb Gower2 - 2α & 2ε
Hb Gower1 - 2ζ & 2ε
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Hb derivatives
Formed by the combination of different
ligands with the heme part or by the
change in the oxidation state of iron
1. Carboxy hemoglobin
2. Met-hemoglobin
3. Sulf hemoglobin
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Carboxy hemoglobin:
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co combines with Hb
Affinity of co to Hb is 200 times more than that of O2
Binding of co:
co binds to one or more of 4 heme sites
T-R changes takes place in Hb
O2 binds to other heme sites
ODC shift to left (sigmoid ODC to Hyperbola)
Decreased O2 release of the affected Hb to tissues
CO poisoning
causes
„ Major occupation hazards in mines
„ Breathing the automobile exhausts in closed
space- commonest cause
Normal level
„ <1% (0.16%)
Smokers
„ >4%
Clinical symptoms
„ ≥20%
„ Nausea, vomiting, head ache, breathlessness,
pain in chest
„ 40-60% death
Treatment
„ 100% O2
Met-Hemoglobin
Iron in heme is in ferric state (Fe3+)
Causes
„ Mutations in α or β globin chains
„ Free radicals
„ Congenital deficiency of enzymes
NADH met-hemoglobin reductase
NADPH met-hemoglobin reductase
Glutathione met-Hemoglobin reductase
„ Intake of certain drugs
Nitrates, Acetaminophen, Phenacetin
„
Normal level
„ < 1%
Clinical symptom
Met-Hb – has got only 5 valencies
does not bind O2
does not involve in O2 transport
Manifested as cyanosis
Chocolate color blood due to dark
colored met-Hb called as chocolate cyanosis
Treatment
Oral administration of methylene blue or
ascorbic acid decreases met-Hb levels &
reverses cyanosis
Sulfhemoglobin
Sulfhemoglobin results from the union of
hemoglobin with medications such as
phenacetin or sulfonamides. This form of
hemoglobin is unable to transport oxygen,
and is untreatable. The only solution is to
wait until the affected red blood cells are
destroyed as part of their normal life
cycle.
Hemoglobinopathies
Production of structurally abnormal Hb or
synthesis of insufficient quantities of
normal Hb or rarely both
„ Includes
1. Sickle cell disease (HbS)
2. Hemoglobin C disease (HbC)
3. Hemoglobin SC disease(HbSC)
4. Thalassemias
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Sickle cell disease
Genetic disorder of Hb caused by a point mutation
in the β-globin gene
„ Homozygous recessive disorder
Pathology:
Point mutation
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Glutamic acid replaced by valine in 6th position of β
chain of HbA
Sticky patch on surface of Hb
Sequence of events leading to cell death
Sickle cell disease cont..
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Sticky patch- present on oxyHbS & deoxyHbs
but not on HbA
complement to sticky patch
- present on the surface of
deoxyHbA & deoxyHbS but not on oxyHbA &
oxyHbS
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Harper slide
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Events leading to sickle cell crisis
Sticky patch on deoxy Hbs bind to complement site
on another deoxy Hbs
Polymerization of deoxyHbS
Long fibrous precipitates
Intracellular fibers of HbS distort the erythrocyte
Elongated erythrocytes occlude blood flow in
capillaries
Microinfarcts produce tissue anoxia, resulting severe
pain
Defective haemoglobins bind together, forming long rods that
stretch the red blood cell into a crescent. These “sickled” red
blood cells cannot fit through small blood vessels.
**
Factors which increase sickling
„ Decreased O2 tension
„ Increased pCO2
„ Decreased pH
„ Increased conc of 2,3-BPG in RBC
Cause more
deoxyHb
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Diagnosis of sickle-cell anemia
normal
Sickle-cell
trait
Sickle-cell
anemia
-
origin
Hb S
Hb A
+
Electrophoresis of hemoglobin's
Hemoglobin C
Point mutation gene level changing the
Amino acid at 6th position of β globin chain
„ Glutamic acid replaced by Lysine (Basic)
„ Heterozygous state- No Clinical symptoms
„ Homozygous state- Chronic hemolytic
anemia
„ No infarction crises and no specific
theraphy
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Hemoglobin SC
Compound heterozygote state
„ Some β globins have sickle cell mutation
Some β globulins have Hbc mutation
„ Painful crises beginning in childhood
remain normal until they suffer an Crises.
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Hemoglobin D
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Caused by the substitution of glutamine in
place of glutamate in the 121st position.
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On electrophoresis moves along with HbS
Hemoglobin E
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Replacement of glutamate by lysine at
26th position.
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No clinical symptoms
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In India, it is prevalent in west Bengal.
THALASSEMIAS
Hereditary hemolytic disease due to an
imbalance in the synthesis of globin chains
„ Synthesis of either the alpha or beta chain is
defective
„ Caused by variety of mutations including gene
deletions or gene substitutions or deletion of 1
to many neucleotides in the DNA
„ α0 or β0 – no globin chains are produced
α+ or β+ - some chains but at reduced rate
„
α- Thalassemia
Silent carrier:- loss of one α-globin gene
2. α- Thalassemia trait:- loss of two genes
3. Hemoglobin H disease:- loss of three
genes
4. Hydrops fetalis:- loss of all 4 genes.
Most sever form.
1.
β - Thalassemia
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Commonest
Synthesis of β – globin chains is decreased or
absent.
α - globin chains synthesized normally
α - globin chains precipitate forming
intracellular precipitates or inclussion bodies lead
to membrane damage and destruction of RBC.
As a compensation γ or δ chain synthesis is
increased.
Cont …..
Homozygous state
„ Clinical manifestations are severe and they
are called Thalassemia major includes
- Anemia
- Hyper splenism
- Hepato Splenomegaly
Cont ….
Heterozygous State
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Clinical Signs and symptoms are minimal
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Called as Thalassemia minor
BIOSYNTHESIS OF
HEME
Structure of Porphyrins
most common porphyrin in humans is heme
- cyclic, with 4 pyrrole rings attached by methenyl bridges
- side chains may vary
- uroporphyrin has acetate and propionate chains
- coproporphyrin has methyl and propionate chains
- order of chains define subgroups of porphyrins: only
Type III porphyrins are normally important for humans
„
Synthesis takes place in liver &
erythrocyte-producing cells of bone
marrow.
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The pathway is partly cytoplasmic & partly
mitochondrian.
CH3
CH3
S
HC
CH2 protein
N
H3C
CH3
N
−
OOC
CH2 CH2
Fe
N
CH
N
S
CH2
protein
CH3
CH2
CH3
CH2
COO−
Heme c
O
−
OOC
CH2
CH2
C
S-CoA +
succinyl-CoA
δ-Aminolevulinic
Acid Synthase
−
OOC
CH2
NH3+
glycine
H+
CoA-SH
CO2
O
−
OOC
CH2
CH2
C
CH2
NH3+
δ-aminolevulinate (ALA)
Heme synthesis begins with condensation
of glycine & succinyl-CoA, with
decarboxylation, to form δ-aminolevulinic
acid (ALA).
ALA Synthase is the committed step of the heme
synthesis pathway, & is usually rate-limiting for the
overall pathway.
Regulation occurs through control of gene
transcription.
Heme functions as a feedback inhibitor,
repressing transcription of the ALA Synthase
gene in most cells.
A variant of ALA Synthase expressed only in
developing erythrocytes is regulated instead by
availability of iron in the form of iron-sulfur clusters.
COO−
COO−
CH2
CH2
CH2
CH2
C
+
O
CH2
NH3
C
PBG Synthase
2 H2O
O
CH2
+
NH3+
2 δ-aminolevulinate
(ALA)
H2C
NH3
COO−
COO−
CH2
CH2
CH2
C
C
C
CH
+
N
H
porphobilinogen
(PBG)
PBG Synthase (Porphobilinogen Synthase), also called
ALA Dehydratase, catalyzes condensation of two
molecules of δ-aminolevulinate to form the pyrrole ring
of porphobilinogen (PBG).
4 PBG
Urophorphyrinogen I synthase
&
Urophorphyrinogen III cosynthase
4NH4
hydroxymethylbilane
-
OOC
COO -
COO -
CH 2
COO -
CH 2
COO
CH 2
CH 2
CH 2
CH 2
CH 2
CH 2
NH
HN
NH
HN
COO -
CH 2
-
OOC
uroporphyrinogen
III
-
CH 2
NH
HN
NH
HN
CH 2
CH 2
CH 2
COO -
HO
C
C
CH 2
C
COO
-
-
OOC
CH 2
C
CH 2
CH 2 CH 2
CH 2
COO -COO -
CH 2
COO -
Uroporphyrinogen III
Synthase
CH 2
CH 2
CH 2
CH 2
COO -
COO -
Urophorphyrinogen III
COO -
4 CO2
Urophorphyrinogen
decarboxylation
Coproporphyrinogen III
2 CO2
Copropophyrinogen
oxidase
Protoporphyrinogen IX
4 H+
Protoporphyrinogen
oxidase
Porphyrias
caused by hereditary or acquired defects in heme synthesis
- accumulation and increased excretion of metabolic
precursors (each unique)
- all porphyrias are autosomal dominant, except congenital
erythropoietic porphyria, which is recessive
can be hepatic or erythropoietic
- hepatic can be acute or chronic
Not a ‘vampire’s’ disease
•Extreme sensitivity to sunlight
•Anemia
This idea has been discarded both for
scientific reasons:
•Porphyrias do not cause a craving for
blood.
•Drinking blood would not help a victim
of porphyria.
And for compasionate reasons:Porphyria is a rare, but
frightening condition: hard to diagnose and there is no cure.
1. Acute intermittent porphyria
(AIP)
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ENZYME DEFECT:Urophorphyrinogen I synthase
INHERITNACE:- autosomal
dominant
ALA & PBG levels are elevated in
blood & urine
Urine gets darkened on exposure
to air due to photo-oxidation
phorphobilinogen to porphobilin
and porphyrin.
It is usually expressed after
puberty.
King George III Mad King
George
He had been
Suffered with AIP
SYMPTOMS:• Acute abdominal pain
• Vomiting
• Cardio vascular abnormalities
• Neurological manifestations include,
sensory & motor disturbances, confusion
and agitation ( due to reduced activity
tryptophan pyrrolase)
• Symptoms are more severe after
administration of drugs barbiturates .
• Patients are not photosensitive
‰ TREATMEMT :- hematin which inhibit the
enzyme ALA synthase.
„
2. congenital erythropoietic
porphyria
„
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ENZYME DEFECT:-uroporphyrinogen III cosynthase
INHERITNACE:- autosomal recessive
The individual excrete uroporphyrinogen I &
coproporphyrinogen I which oxidize to
uroporphyrin I & coproporphyrin I (red
pigments)
Appearance of urine is port wine colour.
Patients are PHOTOSENSITIVE. ( itching &
burning of skin when exposed to sun light)
Increased hemolysis.
„
„
„
Repeated attacks of
dermatitis & scarring lead
to a typical facial
deformity often referred
to as ‘monkey face’.
Repeated ulceration &
scarring may cause
mutilation of nose, ear &
cartilage. This may mimic
leprosy.
When UV light reflected
on to teeth a red
fluorescence is seen,
called ERYTHRODONTIA.
3.porphyria cutanea tarda
Also called as CUTANEOUS HEPATIC
PORPHYRIA.
„ Most common porphyria.
„ Occurs in alcoholics & iron overload.
„ ENZYME DEFECT:- uroporphyrinogen
decarboxylase
„ Photosensitivity is seen
„ Liver exhibits fiuoresence.
„
4. Hereditary coproporphyria
ENZYME DEFECT:- coproporphyrinogen
oxidase
„ Coproporphyrinogen III, ALA & PBG are
excreted in urine and feces.
„ Patients are PHOTOSENSITIVE
„ Clinical manifestations are similar to AIP
„ Treatment:hematin(inhibits ALA synthase)
„
5.Variegate porphyria
ENZYME DEFECT:- protoporphyrinogen
oxidase
„ Protoporphyrin IX synthesis is blocked.
„ All intermediates of Heme synthesis
accumulate in the body and excreted in
urine and feces.
„ Colored urine & photosensitivity is seen.
„ Plasma shows red fluorescence due to
coproporphyrinogen.
„
6. Protoporphyria
(erythropoietic protoporphyria)
ENZYME DEFECT:- ferrochelatase
„ Protoporphyrin IX accumulates in tissues
and excreted in urine and feces
„ RBC and skin biopsy exhibit red
fluorescence.
„
The porphyrias.
Type
Enzyme
Involved
Major Symptoms
Laboratory tests
Acute intermittent
porphyria
Uroporphyrinogen
synthase
Abdominal pain
Neuropsychiatric
urinary porphobilinogen ⇑
Congenital
erythropoietic
porphyria
Uroporphyrinogen
cosynthase
Photosensitivity
urinary uroporphyrin
porphobilinogen
⇑
⇔
Porphyria cutanea
tarda
Decarboxylase
Photosensitivity
urinary uroporphyrin
porphobilinogen
⇑
⇔
Variegate porphyria
Oxidase
Photosensitivity
Abdominal pain
Neuropsychiatric
urinary uroporphyrin
fecal coproporphyrin
fecal protoporphyrin
⇑
⇑
⇑
Photosensitivity
fecal protoporphyrin
red cell protoporphyrin
⇑
⇑
Erythropoietic
protoporphyria
Ferrochelatase
CATABOLISM OF
HEME
„
The end products of heme catabolism are
bile pigments(blirubin & biliverdin)
„
Catabolism takes place in macrophages of
reticuloendothelial system of spleen and
liver.
„
6g of Hb is broken down per day, from
which 250mg of bilirubin is formed.
BLOOD
CELLS
Stercobilin
excreted in feces
Urobilin
excreted in urine
Hemoglobin
Globin
Heme
O2
Heme oxygenase
Urobilinogen
formed by bacteria
INTESTINE
KIDNEY
reabsorbed
into blood
CO
Biliverdin IXα
via bile duct to intestines
NADPH
Bilirubin diglucuronide
(water-soluble)
Biliverdin
reductase
NADP+
Bilirubin
(water-insoluble)
2 UDP-glucuronic acid
via blood
to the liver
Bilirubin
(water-insoluble)
LIVER
Figure 2. Catabolism of hemoglobin
HYPERBILIRUBINEMIAS
Jaundice (icterus)
hyperbilirubinemia
- causes yellow color of skin, nail beds and sclerae
- not a disease, but symptom of underlying disorders
Types of Jaundice
hemolytic jaundice
- liver can handle 3000 mg bilirubin/day - normal is 300
- massive hemolysis causes more than can be processed
- can’t be conjugated
- increased bilirubin excreted into bile, urobilinogen
is increased in blood, urine
- unconjugated bilirubin in blood increases = jaundice
obstructive jaundice
- obstruction of the bile duct
- tumor or bile stones
- gastrointestinal pain - nausea
- pale, clay-colored stools
- can lead to liver damage and increased
unconjugated bilirubin
Types of Jaundice
Hepatocellular Jaundice
- liver damage (cirrhosis or hepatitis) cause increased
bilirubin levels in blood due to decreased conjugation
- conjugated bilirubin not efficiently exported to bile
so diffuses into blood
- increased urobilinogen in enterohepatic circulation
- so urine is darker and stool is pale, clay-colored
- AST and ALT levels are elevated
- nausea and anorexia
Jaundice in Newborns
premature babies often accumulate bilirubin due to
late onset of expression of bilirubin glucuronyltransferase
- maximum expression (adult level) at ~ 4 weeks
- excess bilirubin can cause toxic encephalopathy
(kernicterus)
- treated with blue fluorescent light
- converts bilirubin to more polar compound
- can be excreted in bile without conjugation
- Crigler - Najjar syndrome is deficiency in bilirubin
glucuronyltransferase
Determination of bilirubin concentration
van den Bergh reaction (aqueous)
- conjugated bilirubin reacts readily - direct reaction
- unconjugated, hydrophobic, reacts slowly
- both conjuaged and unconjugated react same in
methanol - gives total bilirubin value
- subtraction of direct from total gives indirect
in normal serum - only 4% is conjugated -
SGOT, SGPT levels elevated in hepatic jaundice.
ALP levels elevated in obstructive jaundice
γ-GT levels elevated in chronic alcoholics
CONGENITAL
HYPERBILIRUBINEMIAS
1.
„
„
„
„
„
CRIGLER-NAJJAR SYNDROME TYPE-I
DEFECT IN CONJUGATION
ENZYME DEFECT:UDP-glucuronyltransferase
Jaundice appears within 24hr. Of life
Unconjugated bilirubin increases to more
than 20mg/dl.
Children die first two years of life.
2. CRIGLER-NAJJAR SYNDROME TYPE-II
„ Less sever than type-I
„ Defect in bilirubin conjugation
„ Bilirubin levels will be below 20mg/dl
3. GILBERT’S DISEASE
„ It is inherited as an autosomal dominant trait.
„ defect is in the uptake of bilirubin, impairment
in conjugation, decreased hepatic clearance of
bilirubin
„ Bilirubin level around 3mg/dl
„ It is asymptomatic condition.
4. DUBIN JOHNSON’S SYNDROME
„ It is an autosomal recessive trait
„ Defect in excretion of conjugated bilirubin
& increased conjugated bilirubin in blood.
„ Bilirubin deposited in liver & appears block
(black liver jaundice)
„
5. Rotor syndrome
„ Bilirubin excretion is defective, but no
deposition in liver.
ACQUIRED HYPERBILIRUBINEMIAS
NEONATAL-PHYSIOLOGICAL JAUNDICE:
„ It is caused by increased hemolysis.
„ UDP-glucuronyl transferase activity is low
„ Bilirubin level less than 5mg/dl
„ It disappears by 2nd week of life.
„
Table 2- Genetic Disorders of Bilirubin Metabolism
Condition
Defect
Bilirubin
Clinical
Findings
CriglerNajjar
syndrome
severely defective
UDPglucuronyltransferase
Unconjugated
bilirubin ⇑⇑⇑
Profound
jaundice
Gilberts
syndrome
reduced activity of
UDPglucuronyltransferase
Unconjugated
bilirubin ⇑
Very mild
jaundice during
illnesses
DubinJohnson
syndrome
abnormal transport of
conjugated bilirubin into
the biliary system
Conjugated
bilirubin ⇑⇑
Moderate
jaundice
THAN Q