IRON AND HEME

METABOLISM
Dr. Göksemin Fatma
ŞENGÜL
 IRON
Iron is an essential and vital element for every kind of living
organism including bacteria, plants, animals and humans.

Iron takes part in oxygen transport, energy production, DNA, RNA

and protein synthesis with its ability to gain and lose electrons.

Iron is required in the structure and is required for the correct

functioning of many enzymes.

Iron is also important in neurological development and is required

for neurological functions.
2
IRON IN THE LIVING ORGANIMS
• Iron is present mainly in hemoglobin, myoglobin and
cytochromes, where they are distributed in various
tissues of the living organisms.
• Hemoglobin is, also known as blood globulin, a
porphyrin derivative protein, which gives blood its
red color and is responsible for oxygen transport to
the tissues.
• Myoglobin, is found extensively in muscle tissue and
serve as a carrier and storage site for oxygen in
muscle cells.

3
IRON IN THE LIVING ORGANIMS
• The total amount of iron in the body is 4-5 g.
– 65 % of iron binds to hemoglobin,
– 4 % of iron binds to myoglobin,
– 1 % of iron binds to various heme-containing compounds,
– 0.1 % of iron binds to transferrin in plasma.
• Transferrin, is a plasma protein, is responsible for the
transport of iron to relevant tissues.
• The remaining 15-30 % of iron is stored in the form
of ferritin in reticuloendothelial system and in liver
parenchymal cells (hepatocytes).
4
 SOURCES OF IRON
• The most important sources of iron, in order of
importance, are meat, liver (offal food), eggs, kidney
(offal food), molasses, dried fruits and green leafy
vegetables.
• The best source of iron is the liver (offal food) which
contains 12 mg of iron in 150 g of it.
• That much of iron can be obtained either from 8 eggs or
from 500 g of meat.
• Additionally, the fermentation of food grains leads to
reduction of phytates and increases the use of iron.
• On the other hands, drinking tea with meals reduces the
absorption of iron.

5
 IRON METABOLISM
• The loss of iron from the body is not high
in adults.
–However, the loss of iron in women is two
times more than men due to menstrual
bleeding (0.6 mg/day in men, 1.3 mg/day in
women).
6
 IRON METABOLISM

• The loss of iron from the body is not high in

adults, but iron malabsorption is the most

common nutritional deficiency on world.

7
 IRON METABOLISM
• The absorption of iron is difficult because iron tend to
form salts with anions such as hydroxide, phosphate and
bicarbonate.

• Iron can also form complex with phytates, tannins and

grain fibers in foods which inhibit their absorption.

• Gastric acid is important for the dissolution of iron from

these complexes. That is why, iron deficiency anemia
occurs after gastrectomy surgeries.
8
 IRON
• Iron can be primarily found in two oxidized
forms either in ferrous (FeII) or ferric
(FeIII) states in cells and body fluids while
fulfilling its different biological functions, or
during its transport and storage.

9
 IRON
• Although these electron exchange and redox
potential of iron are necessary for many
biological processes, excessive free iron serves
as pro-oxidants and leads to the formation of
free oxygen radicals.

10
 IRON
• Iron-catalyzed Haber-Weiss/Fenton reactions leads to the
formation of free oxygen species specifically hydroxyl radicals.
• When these reactive oxygen species are not adequately
detoxified by antioxidants, they would be extremely harmful and
toxic to cells.
• Iron is not left free in the cell.
Otherwise, it can damage the cells
by catalyzing the formation of free
oxygen species.
• Therefore, iron is transported by
transferrin and stored as ferritin
or hemosiderin. Furthermore, the
concentration of iron in the body is
strictly controlled.
11
 IRON METABOLISM
• In contrast to iron uptake and recycling, there is no
physiologic regulatory mechanism for excreting iron from
the body.
• People lose a small, but steady amount of iron by
gastrointestinal bleeding, sweating and by shedding cells of
the skin and the mucosal lining of the gastrointestinal tract.
• Excess of iron in the body is toxic. The systemic balance of
this element is maintained entirely by the control of its
absorption. 12
 IRON METABOLISM
• Vitamin C (ascorbate), increases the absorption
of iron by forming soluble compounds with it.
• Ascorbate also reduces Fe+++ (ferric iron) to Fe++
(ferrous iron) state.
• It is difficult to absorb ferric form of iron
because it tends to form insoluble compounds.
• Additionally, the iron reductase enzyme in the
brush border of erythrocytes also contributes
to the formation of Fe++ (ferrous iron) form.

13
 IRON METABOLISM
• There is also a very serious iron economy in the
organism.
• 1- 2 mg/day of iron, corresponding to 10 % of
dietary iron, is absorbed through duodenum while
1-2 mg/day of iron is excreted with feces.
• The absorption of iron and maintaining iron balance
in the body are highly popular subjects that have
been researched in recent years.

14
 IRON ABSORPTION
• Dietary iron can be found in two forms: organic heme
iron, which originates from hemoglobin and myoglobin
of animal food products and inorganic iron, which is
supplied from non-meat sources.
• The absorption pathways of organic heme iron, taken
from meat dishes, and inorganic iron, originates from
non-meat sources, are completely different from each
other. 15
 HEME IRON ABSORPTION
• Heme iron absorption plays an important role in the provision of
iron storage in carnivore-diet following people.
• Maximum of 50 % of orally taken heme can be absorbed in the
body.
• Hemoglobin and myoglobin proteins found in meat products are
broken down into heme and globulin by intestinal enzymes.
Furthermore, heme iron and inorganic iron are maintained in a
soluble form by globin degradation products so that iron remains
available for absorption.
16
 HEME IRON ABSORPTION
• Contrary to non-heme (inorganic) iron, the
absorption of heme iron does not require low-
duodenal pH, ascorbic acid and/or citric acid.

• The absorption of heme iron is also not affected by

iron binders that exist in foods.

• Only calcium ion has been shown to negatively

regulate the absorption of heme iron. 17
 HEME IRON ABSORPTION
• To be absorbed, iron must be in the ferrous (FeII)
Intestinal Enterocyte Blood
Blood
state or bound by a protein such as hemoglobin. lumen

• Heme iron is also found in the ferrous (FeII) form.

• Iron deficiency increases the absorption of heme iron Heme Heme

e
2-3 times. HCP1

hephaestin
Heme
• Heme is transported to the duodenal enterocyte via Ferric oxygenase
Fe+++
a special transporter protein, which is called as heme
reductase

ceruloplasmin
carrier protein 1 (HCP1).

• Both heme iron and inorganic iron use the same

pathway while they are transported to the ferroportin
bloodstream from the enterocyte.
18
NON-HEME (INORGANIC) IRON
ABSORPTION:
Enterocyte Blood
• Nonheme iron in the diet is
Intestinal
lumen

primarily in the ferric (FeIII) iron

form. Heme Heme
e
HCP1

• The solubility and absorption of Heme

hephaestin

Ferric oxygenase
non-heme iron from the lumen into reductase
Fe+++

ceruloplasmin
the duodenal enterocyte require
low-intraluminal pH, which is
ensured by gastric acid.
ferroportin

19
NON-HEME (INORGANIC) IRON
ABSORPTION:
• The absorption of inorganic iron is Intestinal Enterocyte Blood

tightly controlled by complex molecular lumen

systems.
• Ascorbate-dependent Heme
HCP1
Heme
e

duodenal cytochrome b (DCytb), Heme

hephaestin

Ferric oxygenase
is identified as mammalian transplasma reductase
Fe+++

ceruloplasmin
ferric reductase, that catalyzes the
reduction of ferric iron (FeIII) to
ferrous iron (FeII) form in the first ferroportin

step of inorganic iron absorption. 20

NON-HEME (INORGANIC) IRON
ABSORPTION:
• Ferrous (FeII) iron is taken into the enterocyte by Intestinal Enterocyte Blood
lumen
divalent metal transporter 1 (DMT-1), which is located
on the lumen-facing surface of the mature
enterocyte. Heme Heme
e
HCP1
• DMT-1 is the most important protein that takes role hephaestin
Heme
in the non-heme iron uptake into the enterocyte. Ferric oxygenase
reductase
Fe+++
• The synthesis of both DCytb and DMT-1 increases

ceruloplasmin
due to iron deficiency.

• Some part of the iron taken into the enterocyte are

stored as ferritin and is excreted during duodenal cell ferroportin

renewal (exfoliation) process.

21
NON-HEME (INORGANIC) IRON
ABSORPTION:
• If iron is needed to be taken into the organism, it
Intestinal Enterocyte Blood
is transported to the basolateral side of the lumen

enterocyte after it is absorbed.

• Iron is exported from enterocyte to plasma via Heme Heme
e
ferroportin and then, it is loaded onto HCP1

hephaestin
transferrin. Heme
Ferric oxygenase
Fe+++
• However, transferrin can bind only to ferric reductase

ceruloplasmin
(FeIII) iron. Therefore, hephaestin, a
ceruloplasmin homolog protein and ferroxidase
protein and ceruloplasmin, a copper-containing ferroportin
oxidase, catalyze the oxidation of ferrous iron
(FeII) to ferric (FeIII) iron form. 22
 IRON ABSORPTION
• The absorption of dietary iron mainly occurs in
Intestinal Enterocyte Blood
duodenum. lumen

• Heme iron is transported via HCP-1 while inorganic

iron is transported by DMT-1 transporter protein.Heme Heme
e

• Some part of the iron is stored as ferritin in

HCP1

hephaestin
Heme
enterocytes while the remaining part is exported Ferric oxygenase
Fe+++
reductase
from the basolateral side of enterocyte to

ceruloplasmin
bloodstream via ferroportin carrier protein.

• Hephaestin is not an iron-transporter protein, but it

facilitates iron transport from basolateral side to ferroportin

the plasma and it is also a ferroxidase protein. 23

THE ABSORPTION and TRANSPORT of IRON
Intestinal Enterocyte Blood
• When Fe++ is transported to the plasma, it lumen

is converted to Fe+++ by ceruloplasmin

protein and then, Fe+++ can bind to Heme Heme
e
HCP1

transferrin. hephaestin
Heme
Ferric oxygenase
• Transferrin is a 1-globulin with two iron reductase
Fe+++

ceruloplasmin
binding sites.

• Normally, 35 % of transferrin is saturated

with iron. ferroportin

24
THE UPTAKE OF IRON BY OTHER CELLS
• Iron is transported in the plasma by transferrin
Intestinal Enterocyte Blood
protein, which is a glycoprotein synthesized in the lumen

liver.

• Ferrous iron is exported from the basolateral side Heme Heme

e
of enterocytes to plasma via ferroportin and it can
HCP1

hephaestin
Heme
bind to transferrin after it is oxidized to ferric Ferric oxygenase
reductase
Fe+++
(FeIII) iron by hephaestin on integral membrane of

ceruloplasmin
enterocyte or by ceruloplasmin in the plasma.

• Transferrin-bound iron is transported from the

portal vein to the cells, most of which are ferroportin

erythrocyte precursor cells in the bone marrow. 25

IRON METABOLISM
• Hemopexin: binds to circulatory HEME ring and
enables the reusage of iron.
• Transferrin: allows iron to be transported in the
circulation in the ferric (Fe3+) form.
• Ferritin: provides storage of iron in the ferric
(Fe3+) form.

26
IRON METABOLISM
• Total iron binding capacity (TIBC), is the
measurement of all proteins that can bind to mobile
iron.
– Transferrin is the most abundant iron-binding protein.
Therefore, TIBC test can be accepted as an indirect
measurement of transferrin.

– Ferritin stored iron is not included into TIBC test because

ferritin only binds to storage iron.
27
IRON METABOLISM
• Apoferritin is the protein part of the ferritin. Apoferritin is Iron free form of Ferritin

– The amount of apoferritin determines the size of the iron

storage pool.
• A small amount of iron (the remaining iron after binding to
apoferritin) is stored as hemosiderin.
• The separation of iron from hemosiderin is difficult because
hemosiderin is water-insoluble and thermally denatured but,
ferritin is water-soluble and heat-resistant up to 75 0C.
Hemosiderin is iron sorage complex formed within the tissue when iron is found in excess and Ferritin becomes overloaded

28
IRON METABOLISM
• Hemosiderin, is partially digested form of ferritin
protein and it releases iron slowly.
– Apoferritin remains insufficient to bind iron in diseases
characterized by iron overload due to repeated blood
transfusions. Thus, iron is stored in hemosiderin granules in
the tissues.

29
HEMOSIDEROSIS

30
1998 MSE APRIL
Which of the following intracellular proteins
is formed due to overloading of iron in a
patient with hemolytic anemia who has
received repeated blood transfusions?
• A) Hemopexin
• B) Hemosiderin
• C) Transferrin
• D) Ferritin
• E) Ferroportin Answer: B
31
HEMOCHROMATOSIS

32
IRON METABOLISM
• While iron deficiency causes anemia, excessive
iron leads to hemosiderosis.

• The large amounts of hemosiderin accumulation

in the tissues causes a condition called as
hemochromatosis. Hemochromatosis because of increased iron absorption can cause
irreversible damage of organ, few cases of hemochromatosis is managable

33
IRON METABOLISM
• Darkening of the skin, diabetes due to pancreatic
damage (bronze diabetes), cirrhosis, increased
incidence of liver carcinoma and gonad atrophy
can be encountered in patients with
hemochromatosis.

• Hemochromatosis can be genetically inherited,

or it can be acquired. 34
HEME AND PORPHYRINS
HEME METABOLISM
 HEMOPROTEINS
• Hemoglobin
• Myoglobin
• Cytochromes
• Catalyze
• Peroxidases
• NO synthase
• Prostaglandin synthase
• Tryptophan pyrrolase

37
37
 GLOBULAR HEMOPROTEINS

• Hemoproteins are groups of proteins carrying

non-protein, iron bearing heme as the
prosthetic group in their structure.

• Heme molecule is a porphyrin ring complexed

with ferrous iron (Fe2+) and protoporphyrin-IX.
38
 Structure of Heme
• Heme group consists of ferrous ion (Fe 2+) which
binds to four nitrogen atoms of the porphyrin ring.
Ferrous ion also covalently anchored to the imidazole
ring of the proximal and distal histidine in Hemoglobin
and Myoglobin.
• Ferrous ion can reversibly bind to an O2 molecule from
the site where distal histidine binds in O2 unbound
state. 39
Oxygen binding site of Myoglobin
Proximal
histidine
Oxygen
molecule (O2)

Heme
Distal
histidine

Polypeptide
40
chain
2018 AUGUST MSE
Which of the following amino acids
stabilizes the bond between ferrous ion
and oxygen in Myoglobin?
A) Valin
B) Serin
C) Histidine
D) Lysine
E) Glutamate
Answer: C 41
PORPHYRINS
 PORPHYRINS
• Porphyrins are cyclic compounds that can readily bind to metal ions,
specifically iron in the form of both ferrous (Fe+2) and ferric (Fe+3)
iron ions.
• The most abundant metalloporphyrin in human is ‘HEME’.
• HEME is an essential prosthetic group in proteins with diverse
functions like hemoglobin, myoglobin, cytochromes, catalase and
tryptophan pyrolase.
• Hemoproteins are rapidly synthesized and degraded. For example,
6-7 g of heme is synthesized to replace the heme molecule which
undergoes catabolic degradation every day.
• Together with the turnover of hemoproteins, the accompanying iron
containing porphyrins are also broken down and synthesized and iron
ions also enter a cycle. 43
 PORPHYRIN SYNTHESIS
Porphyrins incorporate heme structure which is found in hemoglobin,
myoglobin and cytochrome P450.
Heme is mainly synthesized in liver (where especially cytochrome
P450 is synthesized) and in bone marrow (which contain actively
hemoglobin synthesizing erythrocytes).
Heme synthesis starts with δ-aminolevulinate and continues with
the synthesis of porphobilinogen and by the series of reaction ends
up with the formation of heme.
Heme synthesis takes place partly in mitochondria and partly in
cytoplasm. The first step and last three steps of porphyrin
synthesis occurs in mitochondria while intermediates steps take
place in cytoplasm.
Mature erythrocytes cannot synthesize heme because they do not
contain mitochondria. 44
 HEME SYNTHESIS:
Heme is mainly synthesized in
liver (for the synthesis of
cytochrome P450) and bone
- marrow (erythropoietic tissue
Hemin Pb produces hemoglobin-
-
containing erythrocytes).
The first step and final three
- Pb steps of heme synthesis occur
IX in mitochondria while
remaining intermediate stages
take place in the cytosol.
IX
Mature erythrocytes cannot
4
synthesize heme because they
do not contain mitochondria.
46
δ-Aminolevulinate Synthesis
PORPHYRIN SYNTHESIS

Protoporphyrin IX Protoporphyrinogen IX

48
2012 APRIL MSE
• Which of the following amino acid is used
in the first reaction of porphyrin
synthesis?
A) Alanine
B) Methionine
C) Cysteine
D) Glycine
E) Tyrosine Answer: D
50
 EXAMPLE QUESTION?
• Which of the following two enzymes are inhibited by
lead in the heme synthesis reactions?
A) Aminolevulinic acid dehydratase and ferrochelatase
B) Aminolevulinic acid synthase and ferrochelatase
C) Prophobilinogen deaminase and ferrocheletase
D) Uroporphyrinogen decarboxylase and aminolevulinic acid
dehydratase
E) Aminolevulinic acid synthase and aminolevulinic acid
dehydratase

Answer: A
51
2021 MARCH MSE
• How many mole(s) of glycine is/are
required for the synthesis of one mole of
“heme”?
A) 1
B) 2
C) 4
D) 6
E) 8 Answer: E
52
 EFFECTS of DRUGS ON THE
ACTIVITY of ALA SYNTHASE
• Many drugs such as barbiturates, griseofulvin, sulfonamide,
hexachlorobenzene increase the activity of hepatic ALA
synthase.
• These drugs can be detoxified via microsomal cytochrome P450
monooxygenase system in the liver.
• Cytochrome P450 synthesis also increases in response to these
drugs.
• These drugs also increases the heme requirement for a new
cytochrome P450 synthesis.
• As a result, heme concentration decreases in the liver.
• Therefore, these drugs directly stimulates the synthases of ALA
synthase enzyme so that increases the synthesis of ALA.
54
PORPHYRIAS
1. X-linked sideroblastic anemia
2. ALA dehydratase deficiency
3. Acute intermittent porphyria
4. Congenital erythropoietic
porphyria)
5. Porphyria cutanea tarda
6. Hereditary coproporphyria
7. Variegate porphyria
8. Erythropoietic protoporphyria
 PORPHYRIAS
• X-linked sideroblastic anemia
– ALA synthase enzyme deficiency
– Anemia
– Decrease in the numbers of RBC and hemoglobin levels
– X-linked sideroblastic anemia IS NOT accepted a type of
porphyria.
• ALA dehydratase deficiency
– Abdominal pain and neuropsychiatric symptoms
– Increased ALA concentration in urine
– It is a rare type of porphyria.
56
 PORPHYRIAS
• Acute intermittent porphyria
– Deficiency of porphobilinogen deaminase
– Increased concentration of ALA and porphobilinogen in urine
– Air or light exposure makes urine darker.
– Abdominal pain and neuropsychiatric symptoms
– Acute intermittent porphyria patients are not sensitive to light.
• Congenital erythropoietic porphyria
– Deficiency of uroporphyrinogen III synthase
– Urine is positive for uroporphyrin and negative for porphobilinogen.
– Congenital erythropoietic porphyria patients are sensitive to light.

57
 PORPHYRIAS
• Porphyria Cutanea Tarda
– Deficiency of uroporphyrinogen decarboxylase enzyme
– Increased concentration of uroporphyrin in urine
– Urine is negative for porphobilinogen
– Porphyria cutanea tarda is the most common type of porphyria.
– Porphyria cutanea tarda patients exhibit photosensitivity.
• Hereditary coproporphyria
– Coproporphyrinogen oxidase deficiency
– Coproporphyrinogen III and other intermediates before
coproporphyrinogen step accumulate in urine.
– Abdominal pain and neuropsychiatric symptoms
– Hereditary coprophyria patients are sensitive to light.
58
 PORPHYRIAS
• Variegate porphyria
– Deficiency of protoporphyrinogen oxidase
– Abdominal pain and neuropsychiatric symptoms
– Urine is positive for porphobilinogen and uroporphyrin and feces
is positive for protoporphyrin.
– Variegate porphyria patients exhibit photosensitivity.
• Erythropoietic protoporphyria
– Ferrochelatase enzyme deficiency
– Feces and erythrocytes are positive for protoporphyrin.
– Erythropoietic protoporphyria patients are sensitive to light.

59
2013 APRIL MSE
Which of the following porphyrias DOES show low
light sensitivity and minimal skin-related symptoms?
A) Erythropoietic protoporphyria
B) Porphyria cutanea tarda
C) Congenital erythropoietic porphyria
D) Acute intermittent porphyria
E) Variegate porphyria
Answer: D

60
EXAMPLE QUESTION?
Which of the following defective enzymes associated
with heme synthesis causes increase in protoporphyrin
IX levels?
A) Aminolevulinic acid dehydratase
B) Ferrochelatase
C) Porphobilinogen deaminase
D) Uroporphyrinogen decarboxylase
E) Aminolevulinic acid synthase

Answer: B
61
EXAMPLE QUESTION?
Which of the following defective enzymes associated
with heme synthesis causes increase in ALA levels?
A) Aminolevulinic acid dehydratase
B) Ferrochelatase
C) Porphobilinogen deaminase
D) Uroporphyrinogen decarboxylase
E) Aminolevulinic acid synthase

Answer: A

62
2008 SEPTEMBER MSE
• Toxicity of which of the following metal ions causes
increase in protoporphyrin levels in erythrocytes?
– A) Cobalt
– B) Copper
– C) Lead
– D) Iron
– E) Mercury

Answer: C
63
2016 APRIL MSE
The deficiency of which of the following enzymes
associated with heme synthesis causes increase in
protoporphyrin IX levels in both erythrocytes and
stool?
A) Aminolevulinic acid dehydratase
B) Ferrochelatase
C) Porphobilinogen deaminase
D) Coproporphyrinogen oxidase
E) Aminolevulinic acid synthase
Answer: B
64
 HEME DEGRADATION
• After erythrocytes remain in the circulation for approximately
120 days, they are destroyed by reticuloendothelial system
(RES) cells which are particularly found in liver and spleen.
• Approximately 85 % of the heme moiety comes from
hemoglobin degradation of red blood cells and the remaining is
derived from ineffective erythropoiesis and the breakdown of
other hemoproteins such as cytochromes, myoglobin and
catalyze.

65
BILIRUBIN FORMATION
• Bilirubin is the catabolic product of heme
metabolism. Bilirubin and its derivatives
are called as bile pigment.
• The first step in heme degradation is
catalyzed in RES cells by microsomal
heme oxygenase (HO) enzyme. HO
catalyzes the degradation of heme to
CO, Fe2+ and biliverdin.
• In the presence of NADPH and O2
through series of reactions, HO
catalyzes the oxidation of heme to CO,
Fe3+ and to a green-colored bile pigment,
biliverdin.
• The reduction of biliverdin by biliverdin
UDP-glucuronyl
transferase
reductase enzyme leads to the formation
of yellow-red bile pigment, bilirubin.
66
2016 SEPTEMBER DSE
Which of the following molecules undergoes
breakdown to form bilirubin?
A) Steroid nucleus (core structure)
B) Purine base
C) Nicotinamide
D) Indole ring
E) Heme ring
Answer: E
67
EXAMPLE QUESTION?
• Which of the following enzymes catalyzes
the formation of biliverdin from heme?
–A) Biliverdin reductase
–B) Heme oxygenase
–C) Beta glucuronidase
–D) UDP-glucose dehydrogenase
–E) UDP-glucuronyl transferase

Answer: B
68
EXAMPLE QUESTION?
• Which of the following enzymes catalyzes
the formation of unconjugated bilirubin from
biliverdin?
– A) Biliverdin reductase
– B) Heme oxygenase
– C) Beta glucuronidase
– D) UDP-glucose dehydrogenase
– E) UDP-glucuronyl transferase

Answer: A 69
EXAMPLE QUESTION?
• Which of the following enzymes provides
Fe2+ and CO to the environment during heme
degradation?
– A) Biliverdin reductase
– B) Heme oxygenase
– C) Beta glucuronidase
– D) UDP-glucose dehydrogenase
– E) UDP-glucuronyl transferase
Answer: B
70
2015 APRIL MSE
Which of the following enzymes catalyzes
a reaction causes the removal of CO
during heme degradation?
A) Biliverdin reductase
B) Heme oxygenase
C) Beta glucuronidase
D) UDP-glucose dehydrogenase
E) UDP-glucuronyl transferase
Answer: B
71
THE UPTAKE OF BILIRUBIN BY LIVER
• Unconjugated bilirubin can slightly dissolve in
blood and can be transported to the liver by non- BLOOD Bilirubin-albumin
complex
covalently binding to albumin.
• Conjugated bilirubin can also bind to albumin
LIVER
although it is much weaker than unconjugated 2 UDP-glucuronic
Bilirubin acid
bilirubin. glucuronyl
transferase 2 UDP

nH
• Certain drugs such as sulfonamides and salicylic Bilirubin diglucuronide

ed’meH
acid can separate bilirubin from albumin.
• In these cases, bilirubin can pass through blood Bilirubin diglucuronide

niburilib
brain barrier and and causes neuronal damage in
CNS.
GALL BLADDER 72
 THE FORMATION OF BILIRUBIN
DIGLUCURONIC ACID
• Bilirubin separates from albumin in liver and enter BLOOD Bilirubin-albumin
complex
hepatocyte where it binds to intracellular proteins,
particularly protein Y (ligandin). Ligandin is also called cytoplasmic
protein or Glutathione S tranferase
LIVER
• Bilirubin-ligandin complex is transported to smooth 2 UDP-glucuronic
Bilirubin acid
endoplasmic reticulum where bilirubin is conjugated glucuronyl
transferase 2 UDP
to form water soluble firstly mono, then diglucuronide

enH
Bilirubin diglucuronide

ed’meH
by uridine diphosphate bilirubin glucuronosyl
transferase (UDP-glucouronosyl transferase) Bilirubin diglucuronide

niburilib
enzyme.

• UDP-glucuronic acid serves as a glucoronate (glucronic GALL BLADDER 73

2013 SEPTEMBER MSE
Which of the following is the cytoplasmic
protein binding to indirect bilirubin in the
liver?
A) Albumin

B) Ubiquitin

C) Ligandin

D) Carnitine
Answer: C 74
2009 APRIL MSE (BMS II)
• Which of the following enzymes catalyzes
the formation of bilirubin?
–A) Microsomal heme oxygenase
–B) Biliverdin reductase
–C) UDP-glucuronyl transferase
–D) Glutathione-S-transferase
–E) Glutathione reductase

Answer: B, C
75
EXAMPLE QUESTION?
• Which of the following enzymes catalyzes
the formation of conjugated bilirubin from
unconjugated bilirubin?
– A) Biliverdin reductase
– B) Heme oxygenase
– C) Microsomal heme oxygenase
– D) UDP-glucose dehydrogenase
– E) UDP-glucuronyl transferase

Answer: E 76
2010 DECEMBER MSE
Which of the followings does not take a
direct role in bilirubin metabolism in the
liver?
A) Cytochrome P450
B) Ligandin
C) UTP
D) Glucuronic acid
E) Glucuronyl transferase
Answer: A 77
EXCRETION OF BILIRUBIN TO GALLBLADDER
• Bilirubin diglucuronide is actively
Bilirubin diglucuronide
transported against concentration
gradient firstly to bile ducts and from
3. SECRETION
there to gallbladder. Dubin-Johnson syndrome
Rotor syndrome
• This energy-requiring and rate-limiting
step in the excretion of bilirubin is
BILE DUCT
defected in liver diseases.

• Unconjugated bilirubin cannot be Bilirubin diglucuronide

excreted in gallbladder and in urine. 80

FORMATION OF UROBILINOGEN IN INTESTINES
• Bilirubin diglucuronide is hydrolyzed and reduced by gut bacteria
in intestines to form colorless compound, urobilinogen.
• Some part of urobilinogen is reabsorbed from the intestines and
enters bloodstream and returns back to liver where it is recycled
into gall bladder.
• A small amount of urobilinogen is converted into yellow urobilin,
which provides the color of urine, and excreted in the urine.
• Most of the urobilinogen is oxidized by intestinal bacteria to
stercobilin, giving brown color of the stool, and is excreted in stool.
81
81
The Formation of Bilirubin Diglucuronite
BLOOD Bilirubin-albumin
complex

Stercobilin Urobilin
(excreted in feces) (excreted in urine)
LIVER
2 UDP-glucuronic

Hem
Bilirubin acid
glucuronyl

Hem’den
transferase 2 UDP

bilir
Bilirubin diglucuronide

bilirubin
oluşu
Bilirubin diglucuronide Urobilinogen
(Conjugated bilirubin)

oluşumu
Intestinal bacteria

82
GALL BLADDER INTESTINES
EXAMPLE QUESTION?
• Where does the bilirubin diglucuronide is
converted to urobilinogen?
–A) Reticuloendothelial system
–B) Liver
–C) Kidney
–D) Bile duct
–E) Intestine
Answer: E
83
2016 SEPTEMBER MSE
Which of the following molecules IS NOT
formed as a result of heme catabolism?
A) Bilirubin
B) Porphobilinogen
C) Urobilinogen
D) Stercobilin
E) Biliverdin
Answer: B
84
 JAUNDICE
• Jaundice (icterus) is a yellowish or greenish pigmentation of the
skin and sclera due to high blood bilirubin levels.

• Jaundice in adults is typically a sign indicating the presence of

underlying diseases due to abnormal heme metabolism, liver
dysfunction or biliary-tract obstruction.

• Jaundice can be classified into three main types:

– Hemolytic jaundice:

– Obstructive jaundice:

– Hepatocellular jaundice: 85
 JAUNDICE IN NEWBORNS
• Due to low hepatic glucuronyl transferase activity at birth, the
bilirubin levels reach to adult levels in newborn babies within two
weeks.
• This causes bilirubin accumulation in newborns, especially in
premature babies.
• When bilirubin exceeds albumin binding capacity, unconjugated
bilirubin can enter basal ganglia and leads to toxic encephalopathy.
• Newborns with high bilirubin levels are treated with blue
fluorescent light to decrease their blood bilirubin to normal levels.
• Under blue fluorescent light, bilirubin can be converted into its
isomer which is more polar and more water-soluble. These photo
isomers of bilirubin can be transferred to gallbladder without
conjugating to glucuronic acid and eventually, can be excreted
in the urine.
86
MEASUREMENT OF BILIRUBIN LEVELS
• Blood bilirubin level is most commonly measured using Van den
Bergh reaction. This chemical reaction mostly determines the
amount of conjugated bilirubin in blood.

• This chemical reaction is based on the reaction of diazotized

sulfanilic acid with bilirubin to form purple-red colored
azobilirubin (azodipyrolle) which can be detected and measured
colorimetrically.

87
EXCESSIVE LEVELS OF
BILURIBIN OR
BILURIBIN
DIGLUCURONIDE
 BILURIBIN METABOLISM

89
Copyright © 2005 Lippincott William & Wilkins. 89
 Excessive levels of bilirubin or
bilirubin diglucuronide
• Excessive levels of bilirubin or bilirubin diglucuronide cause
jaundice in various tissues of the body, especially in the sclera.

• Jaundice can be caused by hemolytic anemia due to excessive

production of bilirubin, by liver diseases leading to inability of
liver to conjugate and excrete bilirubin and by obstruction of
bile duct preventing the transport of bilirubin diglucuronide
from the liver to the intestine.
90
 Excessive levels of bilirubin or
bilirubin diglucuronide
• Neonatal jaundice is frequently observed and is caused by
immaturity of liver enzymes involved in bilirubin metabolism.
Bilirubin levels drops to normal levels rapidly, when the
maturation of bilirubin metabolism enzymes occurs.

• Permanent damage of bilirubin on the nervous system (kern

icteus) can be prevented via phototherapy (exposure of skin to
light), which converts unconjugated bilirubin into water-soluble
isomers that can be excreted in urine. 91
 BLOOD
Bilirubin . Albumin

CONGENITAL
1. UPTAKE
Albumin Gilbert syndrome

DISEASES HEPOTOCYTE

CAUSE HIGH 2. CONJUGATION

SERUM
Neonatal jaundice
Toxic jaundice
Crigler-Najjar syndrome

BILIRUBIN Gilbert syndrome

LEVELS Bilirubin diglucuronide

3. SECRETION
Dubin-Johnson syndrome
Rotor syndrome

BILE DUCT
92
Bilirubin diglucuronide
GILBERT SYNDROME
• Gilbert syndrome is the frequently
encountered liver disease in which liver does
not properly process bilirubin. Gilbert
syndrome is encountered in 3-5 % of
populations.
• Increase in the levels of indirect bilirubin is
observed due to defects in the uptake of
bilirubin by the liver or due to bilirubin-UDP-
glucuronyltransferase enzyme deficiency.
93
 CRIGLER-NAJJAR SYNDROME
• Crigler-Najjar Syndrome is a rare autosomal inhered disorder and is
characterized by the absence or decreased activity of bilirubin-UDP-
glucuronyltransferase enzyme.
• The blood bilirubin levels exceeds 20 mg/dl (hyperbilirubinemia 20-40
mg/dl) in neonatal period in Crigler-Najjar Syndrome type 1 which is the
most severe type with almost complete absence of bilirubin-UDP-
glucuronyltransferase enzyme. Untreated infants usually die with severe
neurological problems within the first 15 months.
• Crigler-Najjar Syndrome type 2 is a less severe form with a reduced level
of bilirubin-UDP-glucuronyltransferase deficiency so that the lifespan of
affected individuals can be extended till adult periods.
94
DUBIN-JOHNSON SYNDROME
• Dubin-Johnson Syndrome is an autosomal recessive disorder and
is characterized by a secretion defect of conjugated bilirubin.

• Total bilirubin levels usually range between 2 and 5 mg/dl with

increased plasma conjugated bilirubin in patients with Dubin-
Johnson Syndrome. Furthermore, a dark pigment lipofuscin
accumulates in the liver, giving it a characteristic black color.

95
ROTOR SYNDROME
• Rotor Syndrome is an autosomal
recessive disease and is characterized
by non-hemolytic anemia due to chronic
increase in predominantly conjugated
bilirubin.
• Impairment in conjugated bilirubin
secretion occurs without liver
pigmentation.

96
2018 APRIL MSE
• Which of the following diseases can occur
due to increased levels of conjugated
bilirubin (conjugated hyperbilirubinemia) in
plasma?
–A) Crigler-Najjar Sydrome type 1
–B) Hemolytic anemia
–C) Rotor syndrome
–D) Newborn jaundice
–E) Gilbert syndrome Answer: C 97