Haemoglobin

& porphyrins
 FUNCTIONS OF BLOOD
• Respiration: Transport of Oxygen
from lungs to tissues and that of
CO2 from tissues to lungs

• Nutrition: Transport of absorbed

food material
FUNCTIONS OF BLOOD
• Maintenance of normal
acid-base balance
• Defense against
infection by white
blood cells and
circulating antibodies
• Excretion: Transport of
metabolic wastes
 FUNCTIONS OF BLOOD
• Regulation of water balance through the
effect of blood on exchange of water
between circulating fluids and tissue fluid
FUNCTIONS OF BLOOD
• Transport of hormones
and regulation of
metabolism
• Transport of metabolites
• Coagulation
HEMEPROTEINS
 HEMEPROTEINS
• Specialized proteins that contain heme as a
tightly bound prosthetic group
• Role of heme group depends on
3-dimensional structure of protein
e.g. heme group of a cytochrome
functions as an electron carrier that is
alternately oxidized and reduced
 HEMEPROTEINS
• Heme gp of catalase
enzyme is part of active
site of the enzyme that
catalyses breakdown of
hydrogen peroxide
• In hemo globin and
myoglobin, heme gp
serves to reversibly bind
oxygen
 EXAMPLES OF SOME IMPORTANT
HUMAN AND ANIMAL HEMOPROTEINS
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 xenobiotics.
Catalase Degradation of Hydrogen
Peroxide
Tryptophan pyrrolase Oxidation of Tryptophan
 Structure of heme
• Heme is a complex of
protoporphyrin IX and
ferrous iron (Fe2+)
• Iron is held in center
of heme molecule by
bonds to 4 N of
porphyrin ring
 Structure of heme
• Fe2+ can form 2 additional bonds, one
on each side of planar porphyrin ring
• In myo globin and Hb, one of these
positions is coordinated to side chain of a
histidine residue of globin molecule, the
other position binds O2
Myoglobin

• A hemeprotein present in heart and

skeletal muscle
• Functions both as a reservoir & carrier
for O2
• Increases the rate of transport of O2
within the muscle cell
 Myoglobin
• A single polypeptide chain, structurally similar
to individual subunit polypeptide chains of Hb
• Heme group sits in a crevice in the molecule
• A proximal histidine (F8) binds directly to iron
of heme , distal histidine (E7), helps stabilize
binding of O2 to ferrous iron
Myoglobin
• Globin, portion of myoglobin favours
reversible binding of heme with one O2
molecule
HEMOGLOBIN
• Found exclusively in RBCs

• A conjugated protein having heme as a

prosthetic group with protein: Globin
 HEMOGLOBIN A

• Hb A, is the major Hb in adults

• It is composed of 4 polypeptide chains:2α,
2 β chains, held together by noncovalent
interactions
• Each subunit has stretches of α-helical
structure, and a heme-binding pocket
Hemoglobin Structure
• Tetrameric Hb can transport H+ and
CO2 from tissues to lungs, and can
carry 4 molecules of O2 from lungs to
cells
• Hb is required because O2 is only
slightly soluble in aqueous solutions
such as blood
Quaternary structure of Hb
• Hb tetramer consists of 2 identical dimers:
(αβ)1 and (αβ)2
• Two polypeptide chains within each dimer are
held tightly together, by hydrophobic
interactions
• Interchain hydrophobic interactions form
strong associations between α-subunits and β-
subunits in the dimer
 Structure of hemoglobin
Ionic and H- bonds occur between
members of dimer
Two dimers are able to move with respect
to each other, being held together by polar
bonds
Weaker interactions between these mobile
dimers result in two dimers occupying
different relative positions in deoxy-
hemoglobin as compared with oxy-
hemoglobin
Structural changes resulting from oxygenation and
deoxygenation of hemoglobin
Structure of hemoglobin
• Binding of O2 to heme iron pulls iron
into the plane of heme
• As iron is also linked to proximal
histidine (F8), there is movement of
globin chains that changes the interface
between the αβ dimers
 T & R forms of hemoglobin
• Deoxy form of Hb is called the “T,” or
taut (tense) form, low oxygen-affinity
state
• Two αβ dimers interact through ionic
and H bonds that constrain the
movement of polypeptide chains
T & R forms of hemoglobin
• Binding of O2 to Hb causes rupture of
some of ionic and hydrogen bonds
between αβ dimers
• This leads to “R” or relaxed form, in which
polypeptide chains have more
freedom of movement
• R form is high oxygen-affinity form of Hb
Binding of oxygen to myoglobin and Hb
• Myoglobin can bind only 1 molecule of
O2, because it contains only 1 heme gp,
Hb can bind 4 oxygen molecules
• Degree of saturation (Y) of these O2-
binding sites on all myoglobin or Hb
molecules can vary between zero (all sites
are empty) and 100% (all sites are full)
O2 dissociation curves for myoglobin & Hb
Oxygen dissociation curve
• A plot of Y measured at different partial
pressures of oxygen (pO2) is called
oxygen dissociation curve. The curves
for myoglobin and Hb show important
differences
• Graph shows that myoglobin has higher
O2 affinity at all pO2 values than Hb
 Oxygen dissociation curve
• Partial pressure of O2 needed to achieve half-
saturation of binding sites (P50) is approximately
1 mm Hg for myoglobin and 26 mm Hg for Hb
• The higher the O2 affinity ( the more tightly O2
binds), the lower the P50
• Oxygen dissociation curve for myoglobin has a
hyperbolic shape
 Oxygen dissociation curve
• Myoglobin reversibly binds a single molecule
of O2
• Oxygenated (MbO2) and deoxygenated (Mb)
myoglobin exist in a simple equilibrium
• Mb + O2 MbO2 Equilibrium is shifted
to right or to left as O2 is added to or
removed from the system
Myoglobin & O2
• Myoglobin is designed to bind O2
released by Hb at low pO2 found in
muscle
• Myoglobin, in turn, releases O2 within
the muscle cell in response to O2
demand
Hemoglobin Binding
• Oxygen dissociation curve for Hb is
sigmoidal in shape, indicating that the
subunits cooperate in binding O2
• Cooperative binding of O2 by 4 subunits of
Hb means that binding of an O2 molecule
at one heme group increases the O2 affinity
of remaining heme gps in the same Hb
molecule
Hb binding
• This effect is referred to as heme-heme
interaction
• It is more difficult for first O2
molecule to bind to Hb, the subsequent
binding of O2 occurs with high affinity,
as shown by steep upward curve in
region near 20–30 mm Hg
Hb binds oxygen with increasing affinity
Allosteric effecters
• Their interaction at one site on Hb
molecule, affects binding of O2 to heme
gps, at other locations on the molecule
• Binding of O2 to myoglobin is not
influenced by allosteric effectors
Transport of O2 and CO2 by Hb
Allosteric effects
• The ability of Hb to reversibly bind O2 is
affected by:
1. pO2 through heme-heme interactions
2. pH of environment
3. Partial pressure of CO2 ( pCO2)
4. Availability of 2,3-bisphosphoglycerate
pO2 through Heme-heme interactions
• Sigmoidal O2 dissociation curve shows
specific structural changes that start
occurring at one heme gp, and are
transmitted to other heme gps in Hb
tetramer
• Net effect is that the affinity of Hb for
last O2 molecules bound, is approx. 300
times greater than its affinity for first
O2 bound
 Loading and unloading of O2

• Cooperative binding of O2 allows Hb to

deliver more O2 to tissues in response to
relatively small changes in pO2
• This way pO2 in alveoli and capillaries
of tissues is maintained
Loading and unloading of O2
• In lungs, conc. of O2 is high, Hb
becomes “loaded” with O2
• In peripheral tissues, oxyhemoglobin
“unloads” much of its O2 for use in
oxidative metabolism of tissues
 Significance of sigmoidal O2
dissociation curve
• Steep slope of oxygen dissociation
curve, permits Hb to deliver O2
efficiently from sites of high to low
pO2
Significance of sigmoidal oxygen
dissociation curve
• Myoglobin can not achieve same
degree of O2 release within this
range of pO2, like Hb does
• Myoglobin has max affinity for O2
throughout this O2 pressure range
and, will deliver no O2 to tissues
 Bohr effect
• Release of O2 from Hb is Increased
when pH is lower or pCO2 increases
• Both result in: decreased O2 affinity of
Hb , a shift to right in O2 dissociation
curve, stabilization of T state
• This change in O2 binding is called
Bohr effect
Bohr effect
• Conversely, raising pH or lowering
conc. of CO2 results in greater affinity
for O2, a shift to left in O2
dissociation curve, stabilization of R
state
Source of protons that lowers pH:
• Conc. of both CO2 & H+ in capillaries
of tissues is higher than alveolar
capillaries, where CO2 is released in
expiration
• In tissues, CO2 is converted by
carbonic anhydrase to carbonic acid:
• CO2 + H2O H2CO3
which loses a proton, becoming
bicarbonate
H2CO3 HCO3– + H+CO2
 Effects of low pH
• H+ produced , lowers pH in tissues
• Differential gradient in pH i.e high in
lungs,low in tissues, favours unloading of
O2 in peripheral tissues & loading of O2
in lungs
• O2 affinity of Hb responds to small shifts
in pH between lungs and tissues, making
Hb an efficient transporter
 Mechanism of Bohr effect
• Deoxy Hb has greater affinity for protons than
oxyHb because of ionizable gps, like histidine
side chains that have higher pKa in deoxy Hb
than in oxy Hb
• Increased conc of protons causes them to
become protonated & they form ionic bonds
which stabilize deoxy form & decrease O2
affinity
Bohr effect can be represented as:

HbO2 + H+ HbH + O2

•Increase in protons shifts equilibrium to

right, favoring deoxyhemoglobin,
whereas a decrease in protons shifts
equilibrium to left
Effect of 2,3-bisphosphoglycerate on O2
affinity
• 2,3-BPG is a regulator of O2 binding to
Hb
• It is most abundant organic phosphate
in RBC, with a conc., approximately
equal to Hb
• Synthesized from an intermediate of
glycolysis
Synthesis of 2,3,BPG
 Binding of 2,3-BPG to
deoxyhemoglobin
• 2,3-BPG decreases O2 affinity of Hb by
binding to deoxy Hb but not to oxy Hb This
stabilizes taut conformation of deoxy Hb
• Effect of binding 2,3-BPG can be
represented schematically as:
HbO2 + 2,3-BPG Hb–2,3-BPG +O2
 Binding site of 2,3-BPG
• One molecule of 2,3-BPG binds to a
pocket, formed by two β-globin chains, in
center of deoxyHb tetramer
• This pocket contains positively charged
amino acids that form ionic bonds with -
ively charged phosphate groups of 2,3-BPG
• 2,3-BPG is expelled on oxygenation of Hb
Binding of 2,3-BPG with deoxy Hb
Effect of 2,3BPG on O2 dissociation curve
• In RBC, presence of 2,3-BPG reduces
affinity of Hb for O2, shifting O2
dissociation curve to right
• Reduced affinity enables Hb to release
O2 efficiently at partial pressures found
in tissues
Effect of 2,3-BPG levels to chronic
hypoxia & anemia:
• Conc. of 2,3-BPG in RBC increases in
response to chronic hypoxia,e.g. in
COPD, emphysema, or at high altitudes,
where Hb has insufficient O2 & in
chronic anemia
• Elevated 2,3-BPG lowers O2 affinity
of Hb
Role of 2,3-BPG in transfused blood
• Storing blood in currently available
media results in a decrease in 2,3-PBG
Stored blood displays an abnormally
high O2 affinity, and fails to unload its
bound O2 in tissues
• Hb deficient in 2,3-BPG acts as an O2
“trap”
 Role of 2,3-BPG in transfused blood
• Transfused RBCs are able to restore their
depleted supplies of 2,3-BPG in 6–24 hours
• Severely ill patients may be compromised
if transfused with large quantities of such
2,3-BPG–“stripped” blood
• Max. storage time for red cells is 21 to 42
days
 Binding of CO2
• CO2 from metabolism is transported as HCO3
• Some CO2 is carried as carbamate bound to N-
terminal amino gp of Hb ,forming
carbaminohemoglobin
Hb –NH2 +CO2 Hb – NH –COO– + H+
• Binding of CO2 stabilizesT form,decreased affinity for
O2 ,right shift in O2 dissociation In lungs,CO2
dissociates , released in breath
 Binding of CO
• Carbon monoxide binds tightly (reversibly)
to Hb iron, forming carboxyhemoglobin
• When CO binds to one or more of the four
heme sites, Hb shifts to relaxed
conformation, causing remaining heme sites
to bind O2 with high affinity
 Binding of CO
• This shifts O2 dissociation curve to left,
changes sigmoidal shape to hyperbola
• Affected Hb is unable to release O2 to
tissues
• Affinity of Hb for CO is 220 times
greater than for O2
• Even minute conc. of CO in environment
can produce toxic conc. of carbon
monoxyhemoglobin in blood
 Binding of CO
• Increased levels of CO are found in
blood of tobacco smokers
• CO toxicity results from combined
tissue hypoxia and direct CO-mediated
damage at the cellular level
• CO poisoning is treated with 100% O2
at high pressure which facilitates
dissociation of CO from Hb
Binding of CO
• CO inhibits Complex IV of the ETC
• In addition to O2, CO2, CO, nitric
oxide gas is also carried by Hb
• NO is a potent vasodilator and can be
taken up/released by RBCs with NO
regulating vessel diameter
• Minor Hemoglobins
Minor hemoglobins

• ‘Hemoglobins’ is a family of proteins

• Hb A is one of the members of this
family
• Each of these proteins is a tetramer,
composed of two α-globin and two β-
globin polypeptides
Minor Hemoglobins
Hemoglobin A
• Hb A synthesis starts in the bone
marrow at about the eighth month of
pregnancy and gradually replaces Hb F
Hemoglobin A2 (Hb A2)
• Hb A2 is a minor component of normal
adult Hb
• Synthesized in adults, with levels lower
than Hb A
• First appears shortly before birth
Hemoglobin A2
• Later in life,it constitutes about 2% of
total Hb
• Contains two α-globin chains and two
δ-globin chains (α2δ2)
Hemoglobin A1c
• Under physiological conditions, Hb A is
slowly and nonenzymically
glycosylated
• Extent of glycosylation is dependent on
plasma concentration of a particular
hexose
Hemoglobin A1c
• Most abundant form of glycosylated
Hb, is Hb A1c
• It has glucose residues attached to NH2
gps of N-terminal valines of β-globin
chains
• Modification is done by covalent
addition of glucose
Hemoglobin A1c
• Increased amounts of Hb A1c are found in
RBCs of patients with diabetes mellitus,
because their Hb A has contact with higher
glucose concentrations during 120-day
lifetime
• This phenomenon is used in assessing
average blood glucose levels in persons with
diabetes
Hemoglobin A1c
Fetal Hemoglobin (Hb F):
• Normally synthesized only during fetal
development
• A tetramer consisting of two α chains,
two γ chains
• γ chains are members of β globin gene
family, and are less positively charged
Hemoglobin F
• It represents less than 1% of Hb in most
adults, and is concentrated in RBCs known
as F-cells
Hb F synthesis during development
• Eembryonic Hb: Hb Gower 1, consisting of
two α-like zeta (ζ) chains & two β-like
epsilon (ε) chains (ζ2ε2), are synthesized by
embryonic yolk sac in first month of
development
• In fifth week , site of globin synthesis
shifts, first to liver and then to marrow,
primary product being Hb F
Hemoglobin F
• Hb F is major Hb found in fetus and
newborn, accounting for about 60% of
total Hb in erythrocytes during last
months of fetal life
 TYPES OF HEMOGLOBINS
Embryonic Fetal Adult hemoglobins
hemoglobins hemoglobins
Gower 1 Hemoglobin F Hemoglobin A
Zeta(2), epsilon(2) Alpha(2), Alpha(2), beta(2)
gamma(2)

Gower 2 Hemoglobin A2
Alpha(2),epsilon(2) Alpha(2), delta(2)
Portland Carboxy Hemoglobin
Zeta(2), gamma(2) Met Hemoglobin
Binding of 2,3-BPG to Hb F
• Under physiologic conditions, Hb F has
a higher affinity for O2 than does Hb A
• In adult RBCs, 2,3,BPG decreases
affinity of Hb for O2
Binding of 2,3-BPG to Hb F
• 2,3-BPG is also present in fetal RBCs,
but interacts less efficiently with HbF
than adult, due to a change in a single
amino acid 143 found in 2,3-BPG
'binding pocket‘, from Histidine to
Serine, which gives rise to greater O2
affinity
Binding of Hb F to 2,3,BPG
Greater affinity for O2 is because histidine is
positively charged and interacts well with the
negative charges found on surface of 2,3-
BPG, serine has a neutrally charged side
chain at physiological pH, and interacts less
well
This change results in less binding of 2,3-
BPG to HbF, and as a result O2 will bind to it
with higher affinity than adult Hb
Binding of 2,3-BPG to Hb F
• If both Hb A and Hb F are stripped of
their 2,3-BPG, they then have a similar
affinity for O2
Significance of HbF O2 affinity
Higher O2 affinity of Hb F facilitates the
transfer of O2 from maternal circulation
across the placenta to RBCs of the fetus
Organization of the globin genes
Organization of globin genes
• Diseases result from genetic alterations
in structure of Hb
• Normal hemoglobin genes, direct
synthesis of different globin chains, that
are structurally organized into gene
families and then are expressed
Organization of globin genes
• α-Gene family: Genes coding for α-
globin-like & β-globin-like subunits of
Hb chains occur in two separate gene
families located on two different
chromosomes
•
Organization of globin gene
• α-gene family on chromosome 16 contains
two genes for α-globin chains
• It also contains ζ gene that is expressed early
in development as a component of embryonic
Hb
• Globin gene famillies also contain globin-like
genes that are not expressed, called
pseudogenes
β-Gene family
• A single gene for β-globin chain is
located on chromosome 11
• There are additional 4 β-globin-like
genes: the ε gene, expressed early in
embryonic development, 2 γ genes, and
δ gene, coding for globin chain found in
Hb A2
Steps in globin chain synthesis
• Expression of a globin gene begins in
nucleus of red cell precursors, where the
DNA sequence encoding gene is
transcribed
β-Gene family
• RNA produced by transcription is a
precursor of mRNA
• Two noncoding stretches of RNA (introns)
must be removed from mRNA and
remaining three fragments (exons) joined
• Finalform of RNA is used as a template
for synthesis of a globin chain
Synthesis of globin chains.
β-Gene family
• The resulting mature mRNA enters
cytosol, gets translated & produces a
globin chain
Porphyrin metabolism
Porphyrin metabolism
• Porphyrins are cyclic compounds that
readily bind metal ions Fe2+/Fe3+
• Metalloporphyrin heme is rapidly
synthesized/degraded, 6-7 g of Hb are
synthesized/day to replace loss through
normal RBC turnover, simultaneous
turnover of porphyrins,& recycling of
bound Iron ions
. Structure of porphyrins
• Porphyrins are cyclic molecules with
linkage of 4 pyrrole rings through
methenyl bridges
Porphyrins
• Glycine and Succinyl Coenzyme A
provide carbon atoms to form 4 Pyrrole
rings
Porphyrins
• Side chains, attached to each pyrrole
rings vary
• Uroporphyrin: has acetate (–CH2–
COO), propionate(–CH2–CH2–COO–)
side chains
Structure of porphyrins
• Coproporphyrin: contains:
methyl (–CH3) & propionate groups
• Protoporphyrin IX (and heme) contain
vinyl (–CH=CH2), methyl & propionate
groups
Distribution of side chains
• Side chains are arranged around
tetrapyrrole nucleus in 4 different ways
• Only Type III porphyrins, containing an
asymmetric substitution on ring D are
important in humans
Biosynthesis of heme
 Starting Material

•Amino acid taking part in Heme

synthesis is a non essential amino
acid : Glycine
Starting material
• Succinyl Coenzyme A
A Citric Acid Cycle Intermediate
 Sites of heme biosynthesis
• Liver: rate of heme synthesis is highly
variable depending upon fluctuating
demand
• Erythrocyte producing cells of bone
marrow, where rate of heme synthesis is
relatively constant
Location of reaction inside the cell

• First reaction (ALA synthesis) and last 3

reactions occur in mitochondria
• Others in cytoplasm
• Mature RBCs are unable to synthesize
heme as they lack mitochondria
 STEP 1
• Site: mitochondria
Condensation reaction

• Enzyme: ALA
synthase
(rate limting enzyme)
• CoEnzyme:
Pyridoxalphosphate

• Inhibitor : heme,Iron
• Product: amino
δ-Aminolevulinic acid
 ALA Synthase 1 & 2
• Two Isoforms of enzyme ALAS
 Only ALAS 2 is produced by Erythroid
tissue and is controlled by intracellular
Iron
 Its gene is present on X chromosome
• Loss of function mutation in gene can
cause Sideroblastic Anaemia
Sideroblastic Anaemia
• Iron is present in cells but cannot be
incorporated into Hb
• Bone marrow produces ringed
sideroblasts in place of RBCs
Sideroblastic Anaemia
Endproduct inhibition of ALAS1 by
Hemin

• When porphyrin production exceeds

availability of (globin) apoproteins,
heme accumulates
• Its Fe2+ is oxidized to Fe3+ and it is
then called as Hemin
 End product inhibition of ALAS1
by hemin
• Hemin decreases synthesis of hepatic
ALAS1 by
1. inhibiting mRNA synthesis
2. inhibiting mitochondrial import of
enzyme
Effect of drugs on ALAS 1
• Drugs are metabolized
by microsomal
cytochrome P450
monooxygenase system
(a heme protein oxidase
system found in liver)
Effect of drugs on ALAS 1
• In response to taking a drug, synthesis
of cytochrome P450 proteins increases,
increasing consumption of heme, a
component of cytochrome P450
proteins
Effect of drugs on ALAS 1
• A decrease in heme conc, in liver
occurs
• This leads to an increase in synthesis of
ALAS1 and a corresponding increase
in ALA synthesis
•
STEP 2:
Reactants: 2 molecules of
δ-ALA condense

Site: cytosol

Enzyme: Zinc containing

δ-ALA acid dehydratase

Inhibitor: lead
Products:
porphobilinogen,
2 water molecules
Inhibition of ALA dehydratase
• ALA dehydratase ( porphobilinogen
synthase ) is extremely sensitive to
inhibition by heavy metal ions e.g. lead
that replaces the zinc
• This inhibition is responsible for raised
ALA and anemia seen in lead poisoning
STEP 3:
Reactants: 4 molecules of
porphobilinogen condense
Enzyme: hydroxymethyl
bilane synthase
Products: Linear
tetrapyrrole,
hydroxymethylbilane
 STEP 4:
• Enzyme:
uroporphyrinogen III
synthase
• Reaction:
Isomerization &
cyclization
• Product
Produc s:
uroporphyrinogen III
(asymmetric)
 STEP 5:
Enzyme:
UroporphyrinogenIII
Decarboxylase
Product:
Coproporphyrinogen
III, which enters
mitochondria
STEP 6:
Site: mitochondria
Enzyme: Coproporphyrinogen
oxidase
Product: Protoporphyrinogen
IX
STEP 7:

•Site: Mitochondria
•Enzyme:
Protoporphyrinogen
oxidase
•Products:
Protoporphyrin IX
 Introduction of Fe2+ into
protoporphyrin IX

• Occurs spontaneously, rate is enhanced

by ferro chelatase , an enzyme, that is
inhibited by lead
• STEP 8:
• Ferrous iron introduced
into
Protoporphyrin IX
• Site: Mitochondria
• Enzyme: Ferrochelatase
• Inhibitor: lead
• Product: Heme
PORPHYRIAS
Porphyrias

These are a group of inherited or acquired

disorders caused by defects in heme
synthesis leading to excessive accumulation
of porphyrins or porphyrin precursors and
their consequent excretion in urine
Porphyrias
• Inherited as autosomal dominant disorders,
with few exceptions
• Mutations causing them, are heterogenous
Every affected family has its own mutation
Porphyrias
• Each porphyria has accumulation of a
unique pattern of intermediates due to
deficiency of an enzyme in heme
synthesis
• “Porphyria” refers to purple color
caused by pigment-like porphyrins in
urine of some patients
Porphyrias
Porphyrias are classified as:
• Erythropoietic: due to deficiency of
enzymes in the erythropoietic cells
• Hepatic: due to deficiency of enzymes
in the liver cells
• Acute
• Chronic
Clinical manifestations
• Individuals with an enzyme defect prior
to synthesis of tetrapyrroles, have
abdominal and neuro psychiatric signs
• Patients having enzyme defects, leading
to accumulation of tetrapyrrole
intermediates have photosensitivity
Clinical manifestations
• Photosensitivity leads to skin itching and
burns (pruritus) when exposed to visible light
• It is due to oxidation of colorless
porphyrinogens to colored porphyrins, which
are photosensitizing molecules that cause
formation of superoxide radicals from O2
Clinical manifestations
• The reactive oxygen species (ROS) can
oxidatively damage cell membranes,
and cause release of destructive
enzymes from lysosomes
 Chronic hepatic porphyria
Porphyria Cutanea Tarda
• Most common porphyria, chronic disease of
liver
• Deficiency of uroporphyrinogen decarboxylase
• Effected by hepatic iron overload, exposure to
sunlight, alcohol ingestion,hepatitis B, C, HIV
infections
Chronic hepatic porphyria
Porphyria cutanea tarda
• Clinical onset during 4th/5th decade of
life
• Porphyrin accumulation leads to
cutaneous symptoms
• Urine is red to brown in natural light
and pink to red in fluorescent light
Porphyria cutanea tarda
Porphyria cutanea tarda
Porphyria cutanea tarda
Porphyria cutanea tarda
Porphyria cutanea tarda
 Acute Hepatic Porphyrias
Include:
•ALA dehydratase deficiency
•Acute intermittent porphyria
•Hereditary coproporphyria,
•Variegate porphyria
Symptoms: acute attacks of GIT, neuro psychiatric,
motor symptoms with/without photosensitivity
Acute Hepatic Porphyrias
Often precipitated by drugs
Barbiturates/ethanol, induce synthesis of
heme containing cytochrome P450
microsomal drug oxidation system
This decreases available heme, which, in
turn, promotes increased synthesis of
ALAS1
Acute Hepatic Porphyrias
Acute intermittent porphyria cause
accumulation of ALA& porphobilinogen,
which cause abdominal pain and neuro
psychiatric disturbances e.g. anxiety,
delirium
 Erythropoietic Porphyrias

•Congenital erythropoietic porphyria and

Erythropoietic proto porphyria are
characterized by skin rashes and blisters
that appear in early childhood
•Diseases are complicated by cholestatic
liver cirrhosis and progressive hepatic
failure
 VAMPIRES AND WEREWOLVES

Cutaneous Porphyrias have been suggested as an

explanation for origin of vampire and werewolf
legends, based upon a number of similarities between
the condition and the folklore that was first speculated
upon by biochemist David Dolphin in 1985
Increased ALA synthase activity
One common feature of porphyrias is a
decreased synthesis of heme
In liver, heme acts as a repressor of
ALAS1gene
Absence of heme results in increased
synthesis of ALAS1 (derepression)
Increased ALA synthase activity
• There is an increased synthesis of
intermediates prior to genetic block
• Accumulation of these toxic
intermediates is the major
pathophysiology of the porphyrias
 Porphyrias
Early lesions: accumulation of ALA &
Porphobilinogen lead to abdominal pain &
neuropsychiatric disorders
Later lesions: accumulation of
porphobilinogens which are converted to
porphyrins lead to photosensitivity
Photosensitivity: exposure of porphyrins to
visible light excites them and free radicals
are formed
 Treatment:
• Acute porphyria patients need treatment for pain
and vomiting
• Severity of symptoms can be reduced by I/V
injection of hemin, which decreases synthesis of
ALAS1
• Avoidance of sunlight
• Ingestion of β-carotene (a free-radical scavenger)
are helpful in porphyrias with photosensitivity
Degradation of heme
Degradation of heme
• After 120 days in circulation, RBCs are
degraded by reticuloendothelial system,
in liver and spleen
• 85% of heme destined for degradation
comes from them
• 15% is from turnover of immature RBCs
& cytochromes
Pathway for heme degradation
Handling of free Intravascular Hb
To prevent losses ,2 proteins make complexes with heme
Haptoglobin: Hb-haptoglobin complex is metabolized in
liver/spleen forming an iron-globin complex & bilirubin.
Prevents loss of iron in urine
Hemopexin: binds free heme. heme-hemopexin
complex is taken up by liver and iron is stored ,bound to
ferritin

Methemalbumin: complex of oxidized heme and

albumin
Degradation of heme
Formation of bilirubin
• Degradation of heme is catalyzed by
microsomal heme oxygenase system of
reticuloendothelial cells
• In the presence of NADPH & O2, the
enzyme adds a hydroxyl group to methenyl
bridge between two pyrrole rings, with
oxidation of ferrous iron to Fe3+
 Formation of bilirubin
• A second oxidation by same enzyme results in
cleavage of porphyrin ring
• Green pigment biliverdin is produced and
ferric iron & CO are released
Formation of bilirubin
• Biliverdin is reduced, forming red
orange bilirubin,which alongwith its
derivatives are collectively termed bile
pigments
• Changing colors of a bruise reflect the
pattern of intermediates that occurs
during heme degradation
Uptake of bilirubin by the liver
• Bilirubin is only slightly soluble in
plasma and, is transported to liver by
binding noncovalently to albumin
• Anionic drugs, salicylates and
sulfonamides, can displace bilirubin
from albumin, permitting bilirubin to
enter central nervous system
Uptake of bilirubin by the liver
• This causes neural damage in infants
• Bilirubin dissociates from carrier
albumin molecule, enters a hepatocyte
by facilitated diffusion, and binds to
intracellular proteins ligandin
Formation of bilirubindiglucuronide
• In hepatocytes, solubility of bilirubin is
increased by addition of 2 molecules of
glucuronic acid : conjugation
• Reaction is catalyzed by microsomal
bilirubin glucuronyl transferase using
uridine diphosphate-glucuronic acid as
glucuronate donor
Bilirubin Glucuronyl Transferase
Deficiency
• Varying degrees of deficiency of this
enzyme result in Crigler-Najjar I & II
and Gilbert syndrome, Crigler-Najjar I
being the most severe deficiency
Secretion of bilirubin into bile
Bilirubin diglucuronideis actively
transported against a conc. gradient into
bile
This rate-limiting step is susceptible to
damage in liver disease
Secretion of bilirubin into bile
• A deficiency in protein required for
transport of conjugated bilirubin out of
liver results in Dubin-Johnson
syndrome
• Unconjugated bilirubin is normally not
secreted
 Formation of urobilins in intestine
• Bilirubin diglucuronide is hydrolyzed &
reduced by int. bacteria to urobilinogen, a
colorless compound
• Urobilinogen is oxidized by int. bacteria to
stercobilin, giving feces characteristic brown
color
• Some urobilinogen is reabsorbed from gut and
enters portal blood
Formation of urobilins in intestine
• A portion of this urobilinogen participates in
enterohepatic urobilinogen cycle in which it
is taken up by liver & resecreted into bile
• Remainder of urobilinogen goes to kidney,
via blood, converted to yellow urobilin and
excreted, giving urine its characteristic color
Jaundice
• Jaundice (icterus) is yellow
discoloration of skin, nail beds, and
sclerae caused by deposition of
bilirubin, increased because of
excessive bilirubin levels in blood
(hyperbilirubinemia)
Jaundice
• Jaundice can be classified into 3 major
forms
• Hemolytic jaundice (PreHepatic)
• Hepatocellular jaundice (Hepatic)
• Obstructive jaundice (Post Hepatic)
Hemolytic jaundice
• Liver’s capacity to conjugate and excrete
bilirubin is over 3,000 mg/day
• Normal production of bilirubin is 300
mg/day
• Liver corresponds to increased heme
degradation, with increased conjugation
& secretion of bilirubin diglucuronide
 Hemolytic jaundice
• Massive lysis of RBCs in sickle cell anemia, P.K or
G6PD deficiency, produce bilirubin faster than it
can be conjugated
• Unconjugated bilirubin levels in blood are ,causing
jaundice
• Conjugated bilirubin excreted into bile
• Urobilinogen entering enterohepatic circulation is
& Urinary urobilinogen
Hemolytic jaundice
 Hepatocellular jaundice
• Damaged liver cells in cirrhosis, hepatitis
cause unconjugated bilirubin in blood
due to conjugation Urobilinogen is in
urine because hepatic damage its
enterohepatic circulation , allowing more
to enter blood, and then urine
 Hepatocellular jaundice
• Urine darkens, stools are pale, clay coloured
• Serum AST & ALT are elevated
• If conjugated bilirubin is not efficiently
secreted from liver into bile (intrahepatic
cholestasis), it can diffuse (“leak”) into blood,
causing a conjugated hyperbilirubinemia
 Obstructive jaundice
• Results from obstruction of bile duct
(extrahepatic cholestasis) due to a tumor/bile
stones, preventing passage of bilirubin into
intestine
• Patients have GIT pain, nausea, pale clay
coloured stools, dark urine
• Liver “regurgitates” conjugated bilirubin into
blood, later excreted in urine
Obstructive jaundice
• Urinary urobiloinogen is absent
• Pro longed obstruction of bile duct can
lead to liver damage and a subsequent
rise in unconjugated bilirubin
 Jaundice in newborns
• In newborn infants,premature, often bilirubin
accumulates, because hepatic bilirubin
glucuronyltransferase activity is low at birth
• It reaches adult levels in about 4 weeks
• Elevated bilirubin, in excess of binding
capacity of albumin, can diffuse into basal
ganglia
Jaundice in newborns
 Jaundice in newborns
• This can cause toxic encephalopathy
(kernicterus)
• Newborns with significantly elevated
bilirubin levels are treated with blue
fluorescent light
• This converts bilirubin to more polar and,
water-soluble isomers
• These photoisomers can be excreted into
bile without conjugation to glucuronic acid
 Determination of bilirubin concentration
• Bilirubin is determined by van den Bergh
reaction, in which diazotized sulfanilic acid
reacts with bilirubin to form red azodipyrroles
that are measured colorimetrically
• In aqueous solution,watersoluble, conjugated
bilirubin reacts with reagent within one minute,
and is said to be “direct-reacting”
Determination of bilirubin concentration
• Unconjugated bilirubin,is less soluble in
aqueous solution, reacts slowly
• When reaction is carried out in
methanol, both conjugated &
unconjugated bilirubin are soluble and
react with reagent, providing total
bilirubin value
Determination of bilirubin concentration
• The “in direct-reacting” bilirubin, which
corresponds to unconjugated bilirubin, is
obtained by subtracting direct-reacting
bilirubin from total bilirubin
• In normal plasma, only about 4% of total
bilirubin is conjugated or direct-reacting,
because most is secreted into bile
 Congenital hyperbilirubinemias
• Gilbert’s syndrome:
• Mutation in gene encoding bilirubin-UGT
• 30% enzyme activity preserved (harmless)
• Crigler Najjar syndrome types I & II:
• Deficiency of hepatic bilirubin glucuronyl
transferase
• Type I more severe (bilirubin > 20 mg/dL),
• Type II less severe,some enzyme activity
preserved (bilirubin < 20 mg/dL)
• Dubin-Johnson syndrome:
• Benign and autosomal recessive type of
conjugated hyperbilirubinemia
• Mutation in gene involved in secretion of
conjugated bilirubin into bile

• Rotor syndrome:
• Benign and chronic type of conjugated
hyperbilirubinemia
• Exact cause is unknown
 Hemoglobinopathies
A family of genetic disorders caused by
production of structurally abnormal Hb
molecule, synthesis of insufficient
quantities of normal Hb, or both
• Sickle cell anemia (Hb S)
• Hb C disease (Hb C)
• Hb SC disease (Hb S + Hb C)
• Thalassemia syndromes
 Sickle cell anemia
•homozygous, recessive disorder
• mutant genes code for synthesis of β chains
of globin molecules ,resulting Hb, α2βS2, is
referred to as Hb S
•Symptoms appear when Hb F is replaced by
Hb S
•Sickling occurs, episodes of pain (“crises”),
chronic hemolytic anemia with
hyperbilirubinemia, increased susceptibility to
infections
 Sickle cell anemia
• RBC life in patient is less than 20 days
• Heterozygotes,contain both Hb S & Hb A ,
sickle cell trait
• A molecule of Hb S contains 2 normal α-
globin chains , 2 mutant β-globin chains (βS),
in which glutamate at position 6 has been
replaced with valine
Sickle cell anemia
• During electrophoresis at alkaline pH,
Hb S migrates slowly toward anode
than does Hb A This altered mobility is
due to absence of negatively charged
glutamate in two β chains, rendering Hb
S less negative than Hb A
• Electrophoresis is used for diagnosis
 Sickling and tissue anoxia
• Replacement of charged glutamate with
nonpolar valine forms a protrusion on β-globin
that fits into a complementary site on β chain of
another hemoglobin molecule in cell
• At low O2 tension, deoxyhemoglobin S
polymerizes inside RBC, forming fibrous
polymers, that stiffen and distort the cell,
producing rigid erythrocytes
 Sickle Cell Anemia
• Sickled cells block flow of blood in narrow
capillaries, causing O2 deprivation in
tissues, pain & eventually death due to
infarction
• Mean diameter of RBCs is 7.5 μm, whereas
that of microvasculature is 3–4 μm, sickled
cells can not squeeze through small vessels
 Treatment
• Therapy involves adequate hydration,
analgesics, antibiotic and transfusions
• Hydroxyurea, an antitumor drug
increases circulating levels of Hb F,
which decreases RBC sickling &
decreased frequency of painful crises
and mortality
 Malaria prevention
• Heterozygotes for sickle cell gene are less
susceptible to malaria, by Plasmodium falciparum,
which spends part of its life cycle in RBC
• RBCs have a shorter life span, the parasite cannot
complete intracellular stage
• A selective advantage to heterozygotes living in
regions where malaria is major cause of death
 Hb C and Hb SC
• Hb C: a single AA substitution in 6th position of
β-globin chain, by a lysine, substituted for
glutamate
• Patients homozygous for Hb C have a mild,
chronic hemolytic anemia
• Hb SC disease: Some β-globin chains have sickle
cell mutation, others have HbC mutations, Hb
levels tend to be higher
Thalassemias
• Hereditary hemolytic diseases with
most common single gene disorders
• Synthesis of either α- or β-globin chain
is defective due to mutations
 β-Thalassemias
• Synthesis of β-globin chains is
decreased/absent, due to point mutations
that affect functional mRNA
• α-globin chain synthesis is normal, but
unstable tetramers formed & precipitate,
causing premature death of cells
• Increase in α2γ2 (Hb F) and α2δ2 (Hb
A2) occurs
β-Thalassemias
β-globin gene
Individuals with β-globin gene defects
have either
β-thalassemia trait (β-thalassemia minor)
if they have only one defective β-globin
gene,
or β-thalassemia major (Cooley anemia)
Thalassemia Blood Smears
α thalassemias
• Defects in synthesis of α-globin
chains,which are decreased/absent, due to
deletional mutations
• Each individual’s genome contains 4
copies of α-globin gene (two on each
chromosome 16), there are several levels of
α-globin chain deficiencies & severe
hemolytic anemia
 α thalassemias
• If one of, 4 gene is defective: a silent carrier
• If two α-globin genes are defective: α-
thalassemia trait
• If three α-globin genes are defective: Hb H (β4)
disease ,a mild/moderate hemolytic anemia
• If all four α-globin genes are defective: Hb Bart
(γ4) disease, fetal death
Glucose 6-P dehydrogenase deficiency
X-linked disorder

Hemolytic anemia, if treated with:

• an oxidant drug
• Ingest fava beans
• Contract a severe infection
• Babies with G6PD deficiency may
experience neonatal jaundice
Rate limiting step in Pentose Reactive oxygen
Phosphate Pathway intermediates

Catalyzed by Damage to
G6PD erythrocyte cell
wall

Mutations in DNA
of G6PD gene Hemolysis

Enzyme activity
Hemolytic anemia

Production of
NADPH

Reduced
glutathione
Clinical manifestations:
Bleeding gums
Fatigue
Pallor
Rapid heart rate
Easy bruising
Nose bleeds
Shortness of breath with exercise