BLOCK

XVIII | Module 01 | Lesson 4 MODULE 1

IRON, HEMOGLOBIN AND BILIRUBIN METABOLISM
Dr. Fred Guillergan
January 8, 2018

OUTLINE BIOMEDICAL IMPORTANCE
I. Biomedical Importance
II. Iron Metabolism • Knowledge of the biochemical and molecular
A. Absorption of Iron properties of Hb and its degradation products
B. Transport of Iron from Enterocytes
provide understanding of:
C. Changes in the Amount of Iron during Transport
D. Regulation of Iron Metabolism: action of Hepcidin o Structure and function
E. Ferroportin-Hepcidin Mechanism o Relationship between structure and function
F. Regulation of Iron Metabolism: Other Molecules
G. Results of Abnormalities in Iron Metabolism o Function of hemoproteins
H. Summary • Knowledge of the biosynthesis and degradation of
III. Myoglobin Structure and Function
IV. Hemoglobin Structure and Function
Hb and its derivatives will provide understanding of:
A. States and Affinities 1) various diseases like sickle cell, porphyrias,
B. Heme Transporter of Oxygen thalassemias;
C. CO Binding Affinity in Heme
D. Oxygen Affinities 2) clinical conditions like jaundice
E. Synthesis of Hemoglobin • Understand the effects of toxins or poisons that can
F. Quantity of Hemoglobin in the Cells
G. Other Hemoproteins cause disturbance in the function of Hb
H. Globin Synthesis • Provide knowledge for diagnosis and prognosis of
I. Alpha Globin Chain
J. Beta Globin Chain
diseases
V. Synthesis of Heme
VI. Heme Catabolism IRON METABOLISM
& Harper’s Illustrated Biochemistry, 30e. Ch. 6 & 31
Absorption of Iron and Transport from Enterocytes
! Audio notes
" https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3999603/
" http://sickle.bwh.harvard.edu/hbsynthesis.html
# Previous (upperclass) notes

SCOPE

• Iron absorption and regulation, transport, and

utilization
• Hemoglobin synthesis, metabolism and
regulation
• Disorders associated with Iron and Hemoglobin
metabolism

OBJECTIVES Iron Absorption and Transport
• Discuss the biomedical importance of the
biochemistry & physiology of hemoglobin,
porphyrins, iron and bile pigments
• Discuss the Globin & Heme synthesis
o Globin gene expression
o Porphyrin structure & synthesis
o Iron metabolism
• Discuss Hemoglobin (Hb) Degradation
• Discuss abnormalities of hemoglobin
o Qualitative & Quantitative abnormalities of
Hemoglobin synthesis
o Abnormalities of Hemoglobin degradation ! There are more or less 3-5 grams of iron in the
body
33 | Talidong, Tariq, Tirador Page 1 of 18

BLOCK XVIII | Module 01 | Lesson 2
IRON, HEMOGLOBIN AND BILIRUBIN METABOLISM

! 80% of these iron is found in the erythrocytes ! In the intestines, the reduction of the Ferric iron to
! 20% are stored in the liver and macrophages Ferrous iron is affected by the following factors:
! Iron is stored in ferric form that is in complex • Ascorbic – used as part in the preparation of
with the storage protein which is ferritin the supplements
& Ferric iron (Fe3+ ) is converted to Ferrous (Fe2+) by • pH of the duodenum where it is absorbed
ferric reductase. ! this is to allow the action by the plasma
! In the lumen, it is absorbed the form of ferric membrane ferroreductase
iron (since this is the most stable form of iron)
will be reduced in ferrous so that it can be
absorb in the enterocytes.
& Fe2+ is transported into the enterocyte by the apical
membrane iron transporter Divalent Metal
Transporter (DMT)1.
& Heme is transported into the enterocyte by a
separate heme transporter (HT) and heme oxidase
(HO)
& Transport mechanism only transport ferrous iron
! The least oxidized form of iron is ferrous and the
most oxidized is the ferric form
! Before transportation the ferric form must be
reduce. This is done by the ferric reductase or
duodenal cytochrome B.
! Electron source in the reduction is dependent on
the ascorbic acid.
& Action of the HO causes the releases Fe2+ from the
heme.
! Some of the intracellular Fe2+ is converted to
Fe3+ and bound by ferritin.
& The remainder binds to the basolateral Fe2+
transporter ferroportin (FP), aided by hephaestin
(HP), and is transported into the bloodstream
! Hephaestin is an oxidoreductase. It oxidizes the
ferrous form as it is being transported by the FP
forming the ferric iron (oxidized form).
! What regulates the transfer from enterocytes to
the plasma is the ferroportin. In macrophages, it
is still involved as well as in the liver. If ever we
have plenty of iron, we have plugging of
ferroportin through hepcidin. It causes the
internalization of the ferroportin that will signal
its degradation.
& In plasma, Fe3+ is bound to the iron transport
protein transferrin (TF), and Fe3+ is transferred to
different areas of the body
! Hepcidin is produced in response to iron
level to regulate the transport of iron from
the erythrocytes to the circulation.

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IRON, HEMOGLOBIN AND BILIRUBIN METABOLISM

! Iron per se does not have any excretion mechanism.
It`s good for the women, they can get rid of excess
iron through menstruation. But they are also subject
for deficiency anemia.

Changes in the Amount of Iron during Transport

! Different cell types that are the sources of iron:
endothelial cell (absorbed iron in the gut),
hepatocyte, macrophages (iron from the
erythrocytes that was engulfed by the
macrophages)
! Most of the iron is utilized in the RBC

IRON METABOLISM

! A portion of iron that is directed to the different
parts of the body. Dietary iron contains 10-20mg
then it is absorbed and transported by the
transferrin. 1-2mg gone through desquamation of
epithelia.
! Most of the iron are used for erythropoeisis, some
are stored in iron-ferritin complex, some are used in
other processes like enzymes-containing iron in
Fig. The transfer of iron and its uses to form different electron transport chain. If no regulation is at play,
molecules in the body. the body will just accumulate these excess irons.
! About 0.6mg daily of iron is excreted. In people
! Iron can be taken in by the protomers of mostly, they can get rid of iron through sloughing of
erythrocytes which are the ones that produces the cells that contains iron. Mostly in the skin and
hemoglobin because mature erythrocytes do not intestines.
produce hemoglobin. No necessary organelles that
will do the job or replace its function. Regulation of Iron Metabolism: Action of Hepcidin
! Later, these RBC will be destroyed or become
senescent and will be taken up by macrophages. • Hepcidin – down regulates,
These macrophages will store the iron from the RBC. o Intestinal iron absorption
Again it is subject to regulation of when it will give o Placental iron transfer
out the iron or when it will store the iron. o Release of iron from macrophages
! Lastly, it could also be transformed into bilirubin ! Hepcidin is mostly synthesized in the liver and the
and then excreted. primary regulator of the iron.

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IRON, HEMOGLOBIN AND BILIRUBIN METABOLISM

! It decreases the iron stores. It is increased in
inflammation and during increase in iron stores. It
acts through blocking the transport in the
ferroportin.
! In the macrophage, iron cannot be transported. In
the intestines, it cannot be transported towards the
blood. Same also in the liver.

Ferroportin-Hepcidin Mechanism
! Iron deficiency – decrease Hb synthesis:
‒ Causes: (1) blood loss due to menstruation or
undetected bleeding (GIT), (2) diet poor in iron,
(3) impaired intestinal absorption of iron
! Iron excess – high tissue levels of iron correlate with
MI (increase reactive O2 radicals favors oxidation of
LDL)
! Also a result of impaired signaling
• Hemochromatosis 1o or 2o– excessive
accumulation of iron in tissues causing damage
• Lab tests: RBC ct.; Hb det.; TIBC; % transferrin sat.;
! In iron overload, the liver produces Hepcidin and ferritin det by radioimmunoassay; prussian blue
block the ferroportin. stain; tissue biopsy (det iron in tissues)
! With hepcidin, once it blocked, it causes a
transformational change that will internalize the Summary
ferroportin-hepcidin complex and cause its • Ingested as heme or non-heme iron
degradation. It decreases the available ferroportin • Ingestion is tightly regulated
in the cells thus preventing the transport of iron. • Enterocytes of the proximal duodenum absorbs iron
! If there is an increase in the need for iron in the • Ferrous iron is absorbed; ferric iron is reduced to by
body, the concentration of hepcidin is regulated. ferrireductase, other reducing agents i.e (Vit. C)
favor iron absorption
Regulation of Iron Metabolism: Other Molecules • Divalent Metal transporter (DMT1) transports iron
inside enterocytes ( found in the apical membrane)
• Hemojuvelin – modulates expression of hepcidin • Iron Regulatory Protein (IRP1) interacts with
but mechanism is unknown Hephaestin in the basolateral membrane to allow
• Ferroportin&Hephaestin – basolateral transporter iron to pass out
of iron ! Daily dietary habit contains 10-20 mL of iron
& Ferroportin interact with the copper-containing
! About 1-2 mL iron is absorbed per day
protein hephaestin
! Carried by these transport proteins to different
& Hephaestin is thought to have a ferroxidase activity,
tissues and 75-80% of this is utilized for
which is important in the release of iron from cells.
hematopoiesis, 10-20% is in the form of storage
Thus, Fe2+ is converted back to Fe3+, the form in
which it is transported in the plasma by transferrin. iron, the remaining goes to different enzymes and
hemoproteins aside from hemoglobin.
Results of Abnormalities in Iron Metabolism ! No physiologic excretion of iron
& IRP 1 and IRP 2 sense cytosolic iron. Binds to iron
responsive element in the mRNA messenger ferritin.

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Increase in iron uptake and decreases The α-helical regions are designated A through H. The distal (E7) and
proximal (F8) histidine residues are highlighted in blue and orange,
sequestration.
respectively. The polar propionate substituents (Pr) project out of the
heme toward solvent.

• Myoglobin is a 153-aminoacyl residue polypeptide

(MW 17,000)
• Folds into a compact shape that measures 4.5 x 3.5
x 2.5 nm

• 75% of the residues are present in eight right-
handed, 7–20 residue α helices (termed A-H)
• The surface of myoglobin is rich in amino acids
bearing polar and potentially charged side chains
while the interior contains only nonpolar residues
such as Leu, Val, Phe, and Met.
• Exceptions are His E7 and His F8, the seventh and
! Mature red cells do not contain any organelles. In eighth residues in helices E and F, which lie close to
immature forms there are many different the heme iron, where they function in O2 binding.
organelles and they have this mechanism by • Heme of myoglobin lies in between helices E and F
which they can synthesize hemoproteins and oriented with its polar propionate groups facing the
enzymes that incorporate the iron. They can form surface of the globin and the remainder resides in
the nonpolar interior.
cytochrome C and even mature red blood cells
• The iron of unoxygenated myoglobin lies 0.03 nm
! Synthesis of hemoglobin can ONLY occur in the (0.3 Å) outsidethe plane of the heme ring, toward
immature cells containing these organelles His F8. Consequently,the heme “puckers” slightly.
(nucleus, mitochondria and polyribosomes) not in When O2 occupies the sixth coordinationposition,
mature forms the iron moves to within 0.01 nm (0.1 Å) of the
! There are drugs that can interfere with the levels plane of the heme ring. Oxygenation of myoglobin
of heme in red blood cells. These xenobiotics are thus isaccompanied by motion of the iron, of His F8,
and of residues linked to His F8.
metabolized in the liver by cytochromes
(hemoprotein). These cytochromes contain heme ! Has only one unit. Not suited for transport of
moiety thereby using them for its production. oxygen but rather for storage
This makes the heme level low.
MYOGLOBIN STRUCTURE AND FUNCTION HEMOGLOBIN STRUCTURE AND FUNCTION

Fig. Hemoglobin. Shown is the three-dimensional structure of
deoxyhemoglobin with a molecule of 2,3-bisphosphoglycerate (dark
blue) bound. The two subunits are colored in the darker shades of
green and blue, the two b subunits in the lighter shades of green and
blue, and the hemeprosthetic groups in red.

Fig. Three-dimensional structure of myoglobin. Shown is a ribbon SUMMARY
diagram tracing the polypeptide backbone of myoglobin. The color of • Two heme proteins mediate oxygen transport
the polypeptide chain is graded along the visible spectrum from blue (Myoglobin&Hemoglobin)
(N-terminal) to tan (C-terminal). The heme prosthetic group is red.
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BLOCK XVIII | Module 01 | Lesson 2
IRON, HEMOGLOBIN AND BILIRUBIN METABOLISM

• Myoglobin is monomeric ring from a position about 0.04 nm beyond it (Figure
• Hemoglobins are tetramers composed of pairs of 6–8). This motion is transmitted to the proximal (F8)
two differentpolypeptide subunits. histidine and to the residues attached thereto,
• Myoglobin occurs in muscles. which in turn causes the rupture of salt bridges
• Hemoglobin has four polypeptide chains. HbA is the between the carboxyl terminal residues of all four
major type in adults. subunits. As a result, one pair of α/b subunits
• The subunit composition of the principal rotates 15° with respect to the other, compacting
hemoglobins are α2b2 (HbA; normal adult the tetramer (Figure 6–9). Profound changes in
hemoglobin),α2γ2 (HbF; fetal hemoglobin), α2bS2 secondary, tertiary, and quaternary structures
(HbS; sickle cellhemoglobin), and α2δ2 (HbA2; a accompany the O2-induced transition of
minor adult hemoglobin). hemoglobin from the low-affinity T (taut) state to
the high-affinity R (relaxed) state. These changes
BIOMEDICAL IMPORTANCE OF MYOGLOBIN AND significantly increase the affinity of the remaining
HEMOGLOBIN unoxygenated hemes for O2, as subsequent binding
• The heme proteins myoglobin and hemoglobin events require the rupture of fewer salt bridges
maintain a supply of oxygen essential for oxidative (Figure 6–10). The terms T and R also are used to
metabolism. Myoglobin, a monomeric protein of red refer to the low-affinity and high-affinity
muscle, stores oxygen as a reserve against oxygen conformations of allosteric enzymes, respectively.
deprivation. Hemoglobin, a tetrameric protein of
erythrocytes, transports O2 to the tissues and
returns CO2 and protons to the lungs. Cyanide and
carbon monoxide kill because they disrupt the
physiologic function of the heme proteins
cytochrome oxidase and hemoglobin, respectively.
The secondary- tertiary structure of the subunits of
hemoglobin resembles myoglobin. However, the
tetrameric structure of hemoglobin permits
cooperative interactions that are central to its
function. For example, 2,3-bisphosphoglycerate
(BPG) promotes the efficient release of O2 by
stabilizing the quaternary structure of
deoxyhemoglobin. Hemoglobin and myoglobin
illustrate both protein structure-function
relationships and the molecular basis of genetic
diseases such as sickle cell disease and the
thalassemia.

Hemoglobin States and Affinities
• Hemoglobin binds four molecules of O2 per
tetramer, one per heme. A molecule of O2 binds to
a hemoglobin tetramer more readily if other O2
molecules are already bound. Termed cooperative
binding, this phenomenon permits hemoglobin to
maximize both the quantity of O2 loaded at the Po2
of the lungs and the quantity of O2 released at the
Po2 of the peripheral tissues. Cooperative
interactions, an exclusive property of multimeric
proteins, are critically important to aerobic life.
• The binding of the first O2 molecule to deoxyHb
shifts the heme iron toward the plane of the heme

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BLOCK XVIII | Module 01 | Lesson 2
IRON, HEMOGLOBIN AND BILIRUBIN METABOLISM

HEME STRUCTURE










• In addition to transporting O2 from the lungs to
peripheral tissues, hemoglobin transports CO2, the
byproduct of respiration, and protons from
peripheral tissues to the lungs. Hemoglobin carries
! Composed of 4 pyrrole rings and methine bridges CO2 as carbamates formed with the amino terminal
with iron at the center along with other nitrogens of the polypeptide chains:
substituents, which may be acetate, propyl, APAP.


• Carbamate formation changes the charge on amino
terminals from positive to negative, favoring salt
bridge formation between α and b chains.
• Hemoglobin carbamates account for about 15% of
the CO2 in venous blood. Much of the remaining
CO2 is carried as bicarbonate, which is formed in
erythrocytes by the hydration of CO2 to carbonic
acid (H2CO3), a process catalyzed bycarbonic
anhydrase. At the pH of venous blood, H2CO3
dissociates into bicarbonate and a proton.

• Deoxyhemoglobin binds one proton for every two
O2 molecules released, contributing significantly to
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BLOCK XVIII | Module 01 | Lesson 2
IRON, HEMOGLOBIN AND BILIRUBIN METABOLISM

the buffering capacity of blood. The somewhat • The activities of these enzymes, and hence the level
lower pH of peripheral tissues, aided by of BPG in erythrocytes, are sensitive to pH. As a
carbamation, stabilizes the T state and thus consequence, BPG concentration and binding are
enhances the delivery of O2. influenced by and, reinforce the impact of, the Bohr
• In lungs, the process reverses. As O2 binds to effect on O2 binding and delivery by hemoglobin.
deoxyhemoglobin, protons are released and • Physiologic changes that accompany prolonged
combine with bicarbonate to form carbonic acid. exposure to high altitude include increases in the
Dehydration of H2CO3, catalyzed by carbonic number of erythrocytes, the concentration of
anhydrase, forms CO2, which is exhaled. Binding of hemoglobin within them, and the synthesis of BPG.
oxygen thus drives the exhalation of CO2 (Fig 6–11). Elevated BPG lowers the affinity of HbA for O2
(increases P50), which enhances the release of O2 at
peripheral tissues.

! 2,3 DPG is produced by glycolysis

! Confirguration of heme once oxygen molecule is

• A low Po2 in peripheral tissues promotes the attached. There is an angle of 121° formed. E7
synthesis of 2,3-bisphosphoglycerate (BPG) in histidine blocks this area. Not bothered by
erythrocytes. The hemoglobin tetramer binds one histidine cause of angle formed
molecule of BPG in the central cavity formed by its
four subunits. However, the space between the H
helices of the b chains lining the cavity is sufficiently
wide to accommodate BPG only when hemoglobin
is in the T state.
• BPG therefore stabilizes deoxygenated (T-state)
hemoglobin by forming additional salt bridges that
must be broken prior to conversion to the R state.
• Synthesis of BPG from the glycolytic intermediate
1,3-bisphosphoglycerate is catalyzed by the
bifunctional enzyme 2,3-bisphosphogylcerate
synthase/2-phosphatase (BPGM). BPG is hydrolyzed
to 3-phosphoglycerate by the 2-phosphatase activity
of BPGM and to 2-phosphoglycerate by a second ! Connection of carbon monoxide to iron is not
strong enough to dislodge due to different angle
enzyme, multiple inositol polyphosphate
formed
phosphatase (MIPP).

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BLOCK XVIII | Module 01 | Lesson 2
IRON, HEMOGLOBIN AND BILIRUBIN METABOLISM

higher affinity for O2 limits the quantity of O2
delivered to the tissues.

THE OXYGEN DISSOCIATION CURVES FOR MYOGLOBIN

& HEMOGLOBIN SUIT THEIR PHYSIOLOGIC ROLES
• Why is myoglobin unsuitable as an O2 transport
protein but well suited for O2 storage? The
relationship between the concentration, or partial
pressure, of O2 (Po2) and the quantity of O2 bound
is expressed as an O2 saturation isotherm. The
oxygen-binding curve for myoglobin is hyperbolic.
Myoglobin therefore loads O2 readily at the Po2 of
the lung capillary bed (100 mm Hg). However, since
myoglobin releases only a small fraction of its bound
O2 at the Po2 values typically encountered in active
muscle (20 mm Hg) or other tissues (40 mm Hg), it
SUMMARY: represents an ineffective vehicle for delivery of O2.
When strenuous exercise lowers the Po2 of muscle
Myoglobin being a single unit is not for transport but tissue to about 5 mm Hg, myoglobin releases O2 for
storage in the tissues so when oxygen dips below/at mitochondrial synthesis of ATP, permitting
hypoxic levels the oxygen found in myoglobin can be continued muscular activity.
utilized
Synthesis of Hemoglobin
Hemoglobin Oxygen Affinities

Fig. Oxygen-binding curves of both hemoglobin and myoglobin.
Arterial oxygen tension is about 100 mm Hg; mixed venous oxygen
tension is about 40 mm Hg; capillary (active muscle) oxygen tension is
about 20 mm Hg; and the minimum oxygen tension required for
cytochrome oxidase is about 5 mm Hg. Association of chains into a • Formation of Hb begins in the proerythroblast and
tetrameric structure (hemoglobin) results in much greater oxygen up to the reticulocyte stage
delivery than would be possible with single chains.
• Globin synthesis – a polypeptide chain formed in
• Hemoglobin – sigmoidal; tetrameric (subunits
ribosomes
shows cooperative binding)
• Heme synthesis (see later discussion)
• Myoglobin – hyperbolic; monomeric
o Porphyrin synthesis
• The quantity P50, a measure of O2 concentration, is
o Incorporation of Ferrous iron
the partial pressure of O2 at which a given
• Iron metabolism–incorporation of iron
hemoglobin reaches half saturation.

o HbA = 26 mm Hg
HGB CONCENTRATION IN RBC
o HbF = 20 mm Hg
• 34 g in 100 mL of cells – metabolic limit of
• In the placenta, this difference enables HbF to
Hb-forming mechanism.
extract oxygen from the HbA in the mother’s blood.
However, HbF is suboptimal postpartum since its
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• When hematocrit (40-45%) and Hb • α and β proteins are derived from gene clusters
content in RBC are normal, then the o Alpha globin genes – chromosome 16
quantity of Hb in whole blood/100 mL: o Beta globin genes – chromosome 11
o ~15 g for men • gene cluster expression occurs at different periods
o ~14 g for women of life
each g of Hb is capable of binding • Smooth switch from fetal to adult gene expression
1.34 mL of O2, thus, 100 mL of blood shortly before birth.
(15 g of Hb) can carry ~20 mL of O2
! Two distinct globin chains (each with its individual heme
& Quantity of Hemoglobin in the cells. Red blood cells molecule) combine to form hemoglobin. One of the
can concentrate hemoglobin in the cell fluid up to chains is designated alpha. The second chain is called
~34 grams per 100 mL of cells. When the hematocrit "non-alpha". With the exception of the very first weeks of
(the percentage of blood that is cells—normally, 40- embryogenesis, one of the globin chains is always alpha.
45%) and the quantity of hemoglobin in each A number of variables influence the nature of the non-
respective cell are normal, the whole blood of a alpha chain in the hemoglobin molecule. The fetus has a
man contains an average of 15 grams of hemoglobin distinct non-alpha chain called gamma. After birth, a
per 100 milliliters of cells; for women, it contains an different non-alpha globin chain, called beta, pairs with
average of 14 grams per 100 milliliters. In the alpha chain. The combination of two alpha chains and
connection with blood transport of oxygen, each two non-alpha chains produces a complete hemoglobin
gram of pure hemoglobin is capable of combining molecule (a total of four chains per molecule).
with 1.34 mL of oxygen. Therefore, in a normal man, ! The combination of two alpha chains and two gamma
a maximum of about 20 mL of oxygen can be carried chains form "fetal" hemoglobin, termed "hemoglobin F".
in combination with hemoglobin in each 100 mL of With the exception of the first 10 to 12 weeks after
blood, and in a normal woman, 19 mL of oxygen can conception, fetal hemoglobin is the primary hemoglobin
be carried. in the developing fetus. The combination of two alpha
chains and two beta chains form "adult" hemoglobin, also
Other Hemoproteins called "hemoglobin A"; it becomes the predominant
• A hemoprotein or heme protein hemoglobin within about 18 to 24 weeks of birth.
! The pairing of one alpha chain and one non-alpha chain
• is a metalloprotein containing a heme prosthetic
produces a hemoglobin dimer (two chains). The
group- a virginal compound that allows a protein to
hemoglobin dimer does not efficiently deliver oxygen,
carry out several functions that it cannot do alone.
however. Two dimers combine to form a hemoglobin
• Heme remains bound to the protein permanently,
tetramer, the functional form of hemoglobin. Complex
either covalently or noncovalently bound or both.
biophysical characteristics of the hemoglobin tetramer
• Cytochrome c Oxidase- is an enzyme embedded in permit the control of oxygen uptake in the lungs and
the inner membrane of mitochondria which mainly release in the tissues necessary to sustain life.
oxidizes the Cytochrome c protein. Cytochrome c ! The genes that encode the alpha globin chains are on
oxidase contains several metal active sites. chromosome 16. Those that encode the non-alpha
• Catalase- is a common enzyme found in most living globin chains are on chromosome 11. Multiple individual
genes are expressed at each site. Pseudogenes are also
organisms exposed to oxygen (e.g. bacteria, plants,
present at each location. The alpha complex is called the
and animals). It catalyzes the decomposition "alpha globin locus", while the non-alpha complex is
of hydrogen peroxide to water and oxygen. called the "beta globin locus". The expression of the
• Tryptophan pyrrolase- oxidation of tryptophan alpha and non-alpha genes is closely balanced by an
unknown mechanism. Balanced gene expression is
Cytochromes P450 (CYPs) are proteins of required for normal red cell function.
the superfamily containing heme. CYPs use small
and large molecules as substrates in enzymatic
reactions including hydroxylations of xenobiotics.

Globin Synthesis

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BLOCK XVIII | Module 01 | Lesson 2
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α-Globin gene family β-Globin gene family

! This shows the different clusters, family of genes in

Chromosome 16 and 11. Each gene expresses their

products at different periods of time. ! Alpha Globin Locus. Each chromosome 16 has two alpha
globin genes that are aligned one after the other on the
chromosome.
! The two alpha globin genes (termed alpha1 and alpha2)
are identical. Since each cell has two chromosomes 16, a
total of four alpha globin genes exist in each cell.
! Each of the four genes produces about one-quarter of the
alpha globin chains needed for hemoglobin synthesis. The
mechanism of this coordination is unknown.
! Promoter elements exist 5' to each alpha globin gene.
! A powerful enhancer region called the locus control
region (LCR) is required for optimal gene expression. The
LCR is many kilobases upstream of the alpha globin locus.
The mechanism by which DNA elements so distant from
the genes control their expression is still of investigation.
! 2 zeta and alpha genes are present in Chromosome
! The transiently expressed embryonic genes that
16. The expression of these genes should be substitute for alpha very early in development,
coordinated so there will be no excess in the gene designated zeta, are also in the alpha globin locus.
products.

! Beta Globin Locus. The genes in the beta globin locus are
arranged sequentially from 5' to 3' beginning with the
gene expressed in embryonic development (the first 12
weeks after conception; called episolon).
! The beta globin locus ends with the adult beta globin
gene.

Fig. Hemoglobin types at different periods of life
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! The sequence of the genes is: epsilon, gamma, delta, and
beta. There are two copies of the gamma gene on each
chromosome 11. The others are present in single copies.
Therefore, each cell has two beta globin genes, one on
each of the two chromosomes 11 in the cell.
! These two beta globin genes express their globin protein
in a quantity that precisely matches that of the four alpha
globin genes.

HEME METABOLISM
Heme Structure Structure of Porphyrin

Synthesis of Porphyrin
• Synthesized from Porphyrin
! First “ingredient” for porhyrin production is succinyl
CoA, which comes from citric acid cycle.
! This is a product of the oxidation of alpha
ketoglutarate, which utilizes 5 co factors: thiamine
diphosphate, lipoate, NAD, FAD, and CoA. These are ! Porphyrin structures are asymmetric: arrangement
also the cofactors used by pyruvate dehydrogenase. of acetate and proprionate are reversed in the
! Other “ingredient” used is glycine. There should be pyrrole form in the fourth ring.
condensation of glycine and succinyl coA. In order ALA Synthase: Rate-Limiting Step
for this to occur, a cofactor is required: pyridoxal
phosphate, which activates glycine, so it can be
acted upon by the ALA synthase.
• ALA synthase 1 – rate limiting step; inhibited by
heme or hemin
• Iron incorporation - Ferrous (Fe2+) iron (reduced ) is
the functional form in Heme
o Spontaneous reaction but enhanced by
• Catalyze the first step in Pophyrin synthesis
ferrochelatase (inhibited by lead)
• The committed step and major control point
Tetrapyrrole Basic Structure • Regulated by Heme and related cpds – negative
feedback
o heme affects translation
o blocks translocation (from the cytosol to
mitochondria
• occurs in both hepatic (ALAS1) and erythroid
(ALAS2) forms

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o The rate-limiting reaction in the synthesis of
7. Protoporphyrinogen
heme in liver is that catalyzed by ALAS1 oxidase – to add more
• Its synthesis is regulated by the heme concentration double bond via oxidative
decarboxylation of 2 of the
4 propionyl side chains
forming vinyl groups.
*occurs in mitochondria

Protoporphyrin III

8. Ferrochelatase
2+
Fe

Heme

Heme Synthesis ** See the last page to see the molecular structures of
Succinyl-CoA + Glycine substrates involve in the process.
1. ALA Synthase- committed & Enzymes 1, 6, 7, and 8 are located in mitochondria,
step. Reaction is sensitive
repression- to the reaction to vitamin
the others inthe cytosol.
derepression B6. Drugs that are & Mutations in the gene encoding enzyme 1 causes X-
mechanism mediated inhibitory to pyridoxal
phosphate will inhibit the linkedsideroblastic anemia.
by Heme and its
Aporepressor process like penecillamin, & Mutations in the genes encoding enzymes 2–8cause
isoniazid.
the porphyrias
*condensation occurs in mitochondria & Regulation of hepatic heme synthesis occursat ALA
ALA
synthase (ALAS1) by a repression-derepression
2. ALA Dehydratase –
mechanismmediated by heme and its hypothetical
contain Zn; inhibited by Lead aporepressor.

Porphyrias
*subsequent reactions occur in cytosol • Inherited or acquired (occasional) defects in heme
Porphobilinogen synthesis; def. of an enzyme in heme synthesis &
3. Uroporphyrinogen I accumulation of intermediates
synthase – PBG deaminase • All are genetically autosomal dominant disorder
or HMB synthase
except congenital erythropoietic porphyria
• Increased ALA synthase activity due to decreased
Hydroxymethylbilane heme resulting to accumulation of intermediates
4. Uroporphyrinogen III that are produced before the genetic block
synthase • Treatment involves injection of hemin, avoidance of
sunlight, and β-carotene
Uroporphyrinogen III

Uroporphyrinogen I-
accumulates in congenital 5. Uroporphyrinogen
erythropoeticporphyria decarboxylase


Coproporphyrinogen III
6. Coproporphyrinogen
oxidase *occurs in
mitochondria

Protoporphyrinogen III
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BLOCK XVIII | Module 01 | Lesson 2
IRON, HEMOGLOBIN AND BILIRUBIN METABOLISM

! Hemoglobin would be degraded to heme and
globin. Globin would be further broken down to
amino acids. Heme will release carbon monoxide
and other products would be processed in the liver
! NADPH is a source of reducing equivalent to
produce biliverdin.
& Under physiologic conditions in the adult, 1–2 × 108
erythrocytes are destroyed per hour. Thus, in 1 day,
a 70-kg human turns over approximately 6 g of
hemoglobin.
& When hemoglobin is destroyed in the body, globin
is degraded to its constituent amino acids, which are
reused, and the iron of heme enters the iron pool,
also for reuse. The iron-free porphyrin portion of
heme is also degraded, mainly in the
reticuloendothelial cells of the liver, spleen, and
bone marrow.
& The catabolism of heme from all of the heme
*take note of enzymes involved and disease and what
proteins is carried out in the microsomal fractions of
substrates will accumulate in enzyme deficiency. May be
cells by a complex enzyme system called
asked for exam hemeoxygenase. When the heme derived from
heme proteins reaches the oxygenase system, the
iron has been oxidized to the ferric (Fe3+) form,
constituting hemin. The heme oxygenase system is
substrate-inducible.
HEME CATABOLISM & The hemin is reduced to heme with NADPH, and,
• RBC life span – 120 days (adult – 1-2 X 108 RBC with the aid of more NADPH, oxygen is added to the
destroyed/hr) α-methyne bridge between pyrroles I and II of the
• Hb is degraded to globin and heme porphyrin. The ferrous iron is again oxidized to the
• Apoprotein portion (globin) is degraded to amino ferric form. With the further addition of oxygen,
acids and is reused. ferric ion is released, carbon monoxide is produced,
• Iron enters the iron pool for reuse and an equimolar quantity of biliverdin results from
• Heme is degraded in the microsomal fraction of cells the splitting of the tetrapyrrole ring.
by hemeoxygenase. Oxidation of heme cause
release of ferric iron, biliverdin, Carbon Monoxide
(liver, spleen, bone marrow)
• Biliverdin are taken from the plasma by hepatocyes
for conversion to bilirubin by biliverdin reductase

& In birds and amphibia, the green biliverdin IX is
excreted; in mammals, a soluble enzyme biliverdin
Fig. Degradation of Hemoglobin
reductase reduces the methyne bridge between
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BLOCK XVIII | Module 01 | Lesson 2
IRON, HEMOGLOBIN AND BILIRUBIN METABOLISM

pyrrole III and pyrrole IV to a methylene group to & This facilitated transport system has a very large
produce bilirubin, a yellow pigment. capacity, so that even under pathologicconditions
& An estimated 1 g of hemoglobin yields 35 mg of the system does not appear to be rate-limiting inthe
bilirubin. The daily bilirubin formation in human metabolism of bilirubin.
adults is ~250–350 mg, mainly from hemoglobin but & Since this facilitated transport system allows the
also from ineffective erythropoiesis and from equilibriumof bilirubin across the sinusoidal
various other heme proteins e.g. cytochrome P450. membrane of the hepatocyte,the net uptake of
The chemical conversion of heme to bilirubin by bilirubin will be dependent upon the removal of
reticuloendothelial cells can be observed in vivo as bilirubin via subsequent metabolic pathways.
the purple color of the heme in a hematoma is • Bilirubin in hepatocytes binds with cytosolic
slowly converted to the yellow pigment of bilirubin. proteins (Ligandin, protein Y)
! In the hepatocytes, the non polar bilirubin should bind
with certain proteins, Ligandin, protein Y, to maintain its
solubility.

& Ligandin (a member of the family of glutathione S

transferases) and protein Y are the involved
proteins. They may also help to prevent efflux of
bilirubin back into the blood stream.

! Bilirubin in conjugated in the liver by glucoronic acid
thus becoming bilirubin diglucoronide. This
conjugated bilirubin is then excreted to the bile.
! Bilirubin is taken up by the hepatocyte in a
facilitated transport mechanism.
! Rate limiting step in transport of bilirubin is
production of conjugated bilirubin transported to
Fig. Conjugation of Bilirubin
the bile by multispecific organic anion transporter.
& Bilirubin formed in peripheral tissues is transported
to the liver by plasma albumin. Further metabolism
of bilirubin occurs primarily in the liver, divided into
three processes: (1) uptake of bilirubin by liver
parenchymal cells, (2) conjugation of bilirubin with
glucuronate in the endoplasmic reticulum, and (3)
secretion of conjugated bilirubin into the bile. Each
of these processes will be considered separately.



Transport of Bilirbin into Hepatocytes
• Albumin binds noncovalently with bilirubin through

two sites (high and low affinity sites) Fig. Transfer process of Bilirubin from Blood to Bile
• 100 ml of plasma can tightly bind 25 mg of bilirubin. ! The conversion from a hydrophobic to a hydrophilic
Certain drugs can compete with bilirubin binding structure of bilirubin aids its excretion to the bile.
• Bilirubin is taken up at the sinusoidal surface of
hepatocytes through carrier-mediated saturable
system (large capacity)
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BLOCK XVIII | Module 01 | Lesson 2
IRON, HEMOGLOBIN AND BILIRUBIN METABOLISM

• “Indirect reacting determines unconjugated bilirubin
(Addition of methanol)

Three Basic Circumstances that can Result to Abnormal
Hemoglobin
1. Structural defects in the hemoglobin molecule
2. Diminished production of one of the two subunits of
the hemoglobin molecule
3. Abnormal associations of otherwise normal subunits

Hemoglobinopathies
• Qualitative abnormalities:
o e.g. sickle cell anemia HbS (missense mutation
Hyperbilirubinemia (A to T) in the codon amino acid 6 (GAG to GTG)
• Blood bilirubin > 1 mg/dL (17.1µmol/L) the change converts glutamate to valine.
• May Occur due to o HbM – mutation in α- or β-globin. oxidation of
o Increased production of bilirubin more Fe2+ to Fe3+ may be due to drugs or endogenous
than the liver can excrete products.
o Failure to excrete bilirubin in the liver • Quantitative abnormalities – Thalassemia – affect
and biliary tract both clusters
• Jaundice will manifest if bilirubin accumulates in the
blood (2-2.5 mg/dL) Types of Hemoglobin
• Normal Hemoglobin
Types of Jaundice o Hemoglobin A - a tetramer with two α chains
• Hemolytic – Massive RBC lysis: and two β chains (a2b2)
o Unconjugated hyperbilirubinemia o Hemoglobin A2 - a minor component of the Hb
o capacity to excrete bilirubin = 3000 mg/day found in red cells after birth and consists of two
o Normal bilirubin production = 300 mg/day α chains and two δ chains (a2d2)
• Obstructive – results from obstruction of bile ducts o Hemoglobin F - a tetramer of two α chains and
liver spills conjugated bilirubin into the blood; two γ chains (a2g2)
prolonged obstruction cause increased blood • Variant Hemoglobin
unconjugated bilirubin o Hemoglobin S - sickle cell disease
• Hepatocellular – results to decrease bilirubin liver o Hemoglobin C - (α2βc2)- mutation in the Hb beta
uptake and unconjugated bilirubin production; chain
increased unconjugated bilirubin in blood and o Hemoglobin E - mutation in the Hb beta chain
urobilinogen in urine o Hemoglobin Constant Spring - a mutation in the
alpha globin gene
Jaundice in the Newborn o Hemoglobin H - a tetramer composed of four
• low activity of glucuronyl transferase at birth beta globin chains
reaches adult levels in 2 weeks o Hemoglobin Barts - develops in fetuses with
• treatment with blue fluorescent light – promotes four-gene deletion alpha thalassemia
unconjugated bilirubin hepatic excretion by
converting bilirubin to polar forms Compound Heterozygous Conditions
• Hemoglobin SC disease - SC disease inherit a gene
Bilirubin Concentration Determination for hemoglobin S from one parent, and a gene for
• Van den Bergh reaction – diazotized sulfanilic acid + hemoglobin C from the other
bilirubin = azodipyrroles (red) • Sickle/beta-thalassemia - the patient has inherited
• “Direct reacting” bilirubin determines Conjugated a gene for hemoglobin S from one parent and a
bilirubin (without methanol) gene for beta-thalassemia from the other

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BLOCK XVIII | Module 01 | Lesson 2
IRON, HEMOGLOBIN AND BILIRUBIN METABOLISM

• Hemoglobin E/beta-thalassemia - combination of
hemoglobin E and beta-thalassemia produces a

condition more severe than is seen with either
hemoglobin E trait or beta-thalassemia trait
• Alpha thalassemia/Hemoglobin Constant Spring -
This syndrome is a compound heterozygous state of
the alpha globin gene cluster.

SUMMARY OF IMPORTANT ASPECTS OF THE
METABOLISM OF THE RED BLOOD CELL
• Glucose is the energy source of RBC through
glycolysis; nooxidative phosphorylation

• The RBC has a variety of transporters that maintain
ionic and water balance.
• Production of 2,3-bisphosphoglycerate, by reactions
closely associated with glycolysis, is important in
regulating the ability of Hb to transport oxygen.
• PPP produces NADPH in the RBC (5–10% of the total
flux of glucose); hemolytic anemia - deficiency G-6-PD
• Reduced glutathione (GSH) counteract the action of
potentially toxic peroxides; requires NADPH to return
G-S-S-G to G-S-H.
• The iron of Hb should be in the Fe2+ state; Fe3+ iron is
reduced to the Fe2+ state by an NADH-dependent
methemoglobin reductase system involving
cytochrome b5 reductase and cytochrome b5.
• Synthesis of glycogen, fatty acids, protein, and nucleic
acids does not occur in the RBC; however, some lipids
(eg, cholesterol) in the red cell membrane can
exchange with corresponding plasma lipids.
• The RBC contains certain enzymes of nucleotide
metabolism (eg, adenosine deaminase, pyrimidine
nucleotidase, and adenylyl kinase); deficiencies of
these enzymes are involved in some cases of
hemolytic anemia.RBC degradation: the globin is
degraded to amino acids, the iron is released from
heme, and the tetrapyrrole component of heme is
converted to bilirubin, which is mainly excreted into
the bowel via the bile.

**HEME SYNTHESIS

*Take note of the enzymes

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BLOCK XVIII | Module 01 | Lesson 2
IRON, HEMOGLOBIN AND BILIRUBIN METABOLISM

Page 18 of 18