Bilirubin Metabolism Rajendra

BILIRUBIN METABOLISM
Rajendra Dev Bhatt
Clinical Biochemist & Lecturer
Dhulikhel Hospital-Kathmandu University Hospital
3/1/2018 Rajendra Dev Bhatt

Haemoglobin
•Haemoglobin is a conjugated protein
made up of four subunits
•Each subunit contains a haem group and
a polypeptide chain
•The haem group is made up of
porphyrin and a ferrous ion
•Haemoglobin can reversibly
combine with oxygen, and transports
oxygen in the body

Catabolism of Haemoglobin
 When life-span of RBCs is over, they are broken
down in reticulo-endothelial system
 Haem and globin are separated.
 Globin is broken down into amino acids.
 The iron liberated from heme is reutilized.
 The porphyrin ring is broken down in reticuloendothelial
(RE) cells of liver, spleen and bone marrow to bile
pigments, mainly bilirubin
1.Generation of Bilirubin

1. Generation of Bilirubin Cont…
 About 6 g of Hb is broken down per day, from which
about 250 mg of bilirubin is formed
 From myoglobin and other heme containing proteins,
another 50 mg of bilirubin is formed.
 Approximately 35 mg of bilirubin is formed from 1 g
of Hb.
 A total of 300 mg of bilirubin is formed everyday; of
which 80% is from destruction of old RBCs, 10% from
ineffective erythropoiesis and the rest 10% from
degradation of myoglobin and other heme containing
proteins.3/1/2018 Rajendra Dev Bhatt

 Heme is degraded primarily by a microsomal
enzyme system; heme oxygenase. It requires
molecular oxygen and NADPH
 The oxygenase enzyme specifically catalyzes the
cleavage of the alpha methenyl bridge, which is
linking the pyrrole rings I and II.
 The linear tetrapyrrole formed is biliverdin which is
green in color.
 In mammals it is further reduced to bilirubin, a red-
yellow pigment, by an NADPH dependent biliverdin
reductase.3/1/2018 Rajendra Dev Bhatt

In the first reaction, a
bridging methylene group
is cleaved by heme
oxygenase to form Linear
Biliverdin from Cyclic
H e m e m o l e c u l e .
Fe 2+ is released from the
ring in this process.

RBCs Breakdown
Hemoglobin Produces & Breakdown
Heme
Biliverdin
Bilirubin
Heme
Oxygenase
Biliverdin
Reductase

2. Transport to Liver
 The bilirubin formed in the reticuloendothelial cells is
insoluble in water.
 The lipophilic bilirubin is therefore transported in plasma bound to
albumin.
 One molecule of albumin can bind 2 molecules of bilirubin.
 Albumin binds bilirubin in loose combination. So when present in
excess, bilirubin can easily dissociate from albumin.
 The binding sites for bilirubin on albumin can be occupied by
aspirin, penicillin, etc. Such drugs can, therefore, displace bilirubin
from albumin. Hence, care should be taken while administering such
drugsto newborn babies to avoid kernicterus.
 When the albumin–bilirubin complex reaches the sinusoidal surface
of the liver, the bilirubin is taken up.

2. Transport to Liver
Bilirubin is not very water-soluble, so most of it is carried to the liver bound to albumin.

3. Conjugation in Liver
 Inside the liver cell, the bilirubin is conjugated
with glucuronic acid, to make it water soluble.
 The first carbon of glucuronic acid is combined with the
carboxyl group of the propionic acid side chains of the
bilirubin molecule. About 80% molecules are in the
diglucuronide form, while 20% are monoglucuronides.
 Drugs like primaquine, novobiocin, chloramphenicol,
androgens and pregnanediol may interfere in this
conjugation process and may cause jaundice.

3. Conjugation in Liver

Unconjugated bilirubin:
Bilirubin that are not conjugated with gluconic
acid , also called hemobilirubin, indirect
bilirubin.
conjugated bilirubin:
Bilirubin that are conjugated with gluconic acid,
also called hepatic bilirubin, direct bilirubin.

Unconjugated VS Conjugated Bilirubin
FEATURE Unconjugated Conjugated
Normal serum level More Less (less than 0.2mg/dl)
Water solubility Absent Present
Affinity to lipids (alcohol
solubilty)
Present Absent
Serum albumin binding High Low
Van den Bergh reaction Indirect (Total minus
direct)
Direct
Reanal excretion Absent Present
Affinity to brain tissue Present (kernicterus) Absent

4. Excretion of Bilirubin to Bile
 The water soluble conjugated bilirubin is excreted
into the bile by an active process and this
occurs against a concentration gradient.
 This is the rate limiting step in the catabolism of
heme. It is induced by phenobarbitone.
 Excretion of conjugated bilirubin into bile is mediated
by an ATP binding cassette protein which is called
Multispecific organic anion transporter (MOAT),
located in the plasma membrane of the biliary
canaliculi.

4. Excretion of Bilirubin to Bile Cont..

5.Fate of Conjugated Bilirubin in Intestine
 The conjugated bilirubin reaches the intestine through the bile.
 In the small intestine, conjugated bilirubin is poorly reabsorbed
and partly hydrolyzed back to unconjugated bilirubin by catalytic
action of bacterial ß-glucuronidases.
 In the distal ileum and colon, anaerobic flora mediate further catabolism of
bile pigments.
a) hydrolysis of conjugated bilirubin to unconjugated bilirubin by
bacterial β-glucuronidases;
b) multistep hydrogenation (reduction) of unconjugated bilirubin to
form colorless urobilinogens (44 Hydrogen)
c) Further reduction of UBG leads to formation of stercobilinogen
(SBG,48 hydrogen)
 The SBG is mostly excreted through feces (250-300 mg/day).
 Upto 20 % of urobilinogen produced daily is reabsorbed from the intestine
& enters the entero-hepatic circulation.

5.Fate of Conjugated Bilirubin in Intestine

6. Final Excretion
UBG and SBG are both colorless compounds
but are oxidized to colored products, urobilin
(42 hydrogen) or stercobilin (46 hydrogen)
respectively by atmospheric oxidation.
Black color is seen in constipation. If intestinal
flora is decreased by prolonged administration
of antibiotics, bilirubin is not reduced to
bilinogens, and in the large gut, it is re-oxidized
by O2 to form biliverdin. Then green tinged feces
is seen, especially in children.

BLOOD
CELLS
LIVER
Bilirubin diglucuronide
(water-soluble)
2 UDP-glucuronic acid
via bile duct to intestines
Stercobilin
excreted in feces
Urobilinogen
formed by bacteria KIDNEY
Urobilin
excreted
in urine
CO
Biliverdin
Heme oxygenase
O2
Bilirubin
(water-insoluble)
NADP+
NADPH
Biliverdin
reductase
Heme
Globin
Hemoglobin
reabsorbed
into blood
Bilirubin
(water-insoluble)
via blood
to the liver
INTESTINE
Catabolism of hemoglobin
unconjugated

Summary of bilirubin metabolism
Old red cells are major source of hemeproteins
Breakdown of heme to bilirubin occur in macrophage of reticuloendithelial
system ( tissue macrophages, spleen and liver).
Unconjugated bilirubin is transported through blood ( complex to albumin) to liver.
Bilirubin is taken into liver and conjugate with glucuronic acid.
Bile is secreted into intestine where glucuronic acid is removed and the resulting
bilirubin is converted to urobilinogen.
A portion of urobilinogen is reabsorbed into blood, where it is converted to the
yellow urobilin and excreted by kidneys.
Urobilinogen is oxidized by intestinal bacteria to the brown stercobilin.

JAUNDICE
Jaundice is a yellow discoloration of the skin, mucous
membranes, and sclera caused by increased amounts of
bilirubin in the blood. Jaundice is a sign of an underlying
disease process.
Jaundice results from the accumulation of bilirubin.
Hyperbilirubinemia may be due to abnormalities in the formation,
transport, metabolism, and excretion of bilirubin. Total serum
bilirubin is normally 0.2–1.2 mg/dL and jaundice may
not be recognizable until levels are about 3 mg/dL.
In the normal adult the rate of systemic bilirubin production is equal
to the rates of hepatic uptake, conjugation, and biliary excretion.
Jaundice occurs (bilirubin levels may reach 30-40 mg/dL in severe
disease) when the equilibrium between bilirubin production and
clearance is disturbed by one or more of the following
mechanisms.

JAUNDICE
(1) Excessive production of bilirubin,
(2) Reduced hepatic uptake,
(3) Impaired conjugation,
(4) Decreased hepatocellular excretion, and
(5)Impaired bile flow (both intra hepatic and extra hepatic).
The first three mechanisms produce unconjugated
hyperbilirubinemia, and the latter two produce
predominantly conjugated hyperbilirubinemia.
More than one mechanism may operate to produce
jaundice, especially in hepatitis, which may produce
Unconjugated and conjugated hyperbilirubinemia.

Classification of jaundice
1. Pre-hepatic (before bile is made in the liver)
Jaundice in these cases is caused by rapid increase in
the breakdown and destruction of the red blood cells
(hemolysis), overwhelming the liver’s ability to
adequately remove the increased levels of bilirubin
from the blood.
Jaundice in these cases is caused by rapid increase in
destruction of the red blood cells (hemolysis),
overwhelming the liver’s ability to adequately remove
the increased levels of bilirubin from the blood.

1.Pre-hepatic Jaundice Cont…
Examples of conditions with increased
breakdown of red blood cells include:
-Malaria,
-Sickle cell crisis,
-Spherocytosis,
-Thalassemia,
-Glucose-6-phosphate dehydrogenase deficiency
-Drugs or other toxins, and
–Autoimmune disorders.

2.Hepatic (the problem arises within the liver)
Jaundice in these cases is caused by the liver’s
inability to properly metabolize and excrete
bilirubin.
Causes
–Viral – hepatitis A, B, or C, yellow fever, Bacterial
sepsis, tuberculosis,
–Alcohol, Drugs e.g. estrogens, contraceptive pills
and Pregnancy
–Carcinoma: metastases. Lymphoma.
Adenocarcinoma of kidney
-Chronic active hepatitis

3.Post-hepatic (after bile has been made in the liver)
Jaundice in these cases, also termed obstructive
jaundice, is caused by conditions which interrupt the
normal drainage of conjugated bilirubin in the form
of bile from the liver into the intestines. The obstruction
may be intrahepatic or extra hepatic.
• Causes of obstructive jaundice include:
a) Intra hepatic Obstruction
-Biliary atresia
-Primary Biliary Cirrhosis
-Malignant infiltration of ducts

3.Post-hepatic Jaundice Cont…
• Causes of obstructive jaundice include:
b) Extra hepatic obstruction
-Gallstones in the bile ducts,
-cancer (pancreatic and gallbladder/bile duct
carcinoma),
-strictures of the bile ducts,
-Pressure on the common bile duct from enlarged
lymph nodes,
-cholangitis,
-congenital malformations,
-pancreatitis,

Congenital conditions that may cause jaundice
1. Crigler-Najjar syndrome: An inherited condition that may
lead to severe Unconjugated hyperbilirubinemia
(high bilirubin concentrations); a gene mutation leads to a
deficiency in an enzyme necessary for bilirubin conjugation.
Unconjugated bilirubin Conjugates in the liver in the presence of an
enzyme UDP-glucuronosyltransferase. In Crigler-Najjar patients
the enzyme is either inactive (type I) or severely reduced (type II).
Therefore bilirubin cannot be excreted into the bile and remains in
the blood.
The high plasma level of Unconjugated bilirubin leads to jaundice
and may lead to kernicterus (bilirubin encephalopathy) .

2.Dubin-Johnson syndrome: an inherited disorder that
causes the retention of conjugated bilirubin (and other
compounds that turn the liver black) in liver cells;
patients may have intermittent jaundice.
This is an autosomal recessive disorder in which patients have an
increase in conjugated bilirubin in the blood. This is caused by a
defect in secretion of bilirubin glucuronides (already conjugated)
across the canalicular membrane (patients are missing a
canalicular protein that transports bilirubin glucuronides into
bile).
The protein was originally termed the canalicular multiple organic
anion transporter (cMOAT) but is also known as multidrug
resistance protein 2 (MRP2).

3.Rotor’s syndrome: Is another autosomal
recessive disorder in which patients have an
increase in conjugated bilirubin in the blood.
The exact molecular defect is unknown – but it
seems these patients have multiple defects in
hepatocyte uptake and excretion of bilirubin
pigments. The liver looks normal, and as in
Dubin-Johnson syndrome, most patients are
asymptomatic.

4. Gilbert syndrome: This syndrome is common
which is estimated around 5-10% of the population
has it. In this disorder, patients have a decreased
activity of UGT1A1. It sounds just like type II
CNS. However, Gilbert syndrome (which is an
autosomal recessive syndrome) has a UGT1A1
activity level of about 30% of normal, which is
quite a bit higher than the amount of activity we see
in CNS. Patients usually have only mild
Unconjugated hyperbilirubinemia.

Physiological Neonatal Jaundice
 Most infants (around 60%) develop visible jaundice
due to elevation of unconjugated bilirubin concentration
during their first week. This common condition is called
Physiological Neonatal Jaundice.
 Bilirubin produced by the fetus is cleared by the placenta
and eliminated by the maternal liver.
 Immediately after birth, the neonatal liver must assume
responsibility for bilirubin clearance and excretion.
However, many hepatic physiologic processes are
incompletely developed at birth.3/1/2018 Rajendra Dev Bhatt

MECHANISM OF NEONATAL JAUNDICE
1.More bilirubin produced
 Destruction of HbF
 Shorter life span of fetal red blood cells
2.The low capability of albumin on unconjugated bilirubin
transportation
3.The low capability of hepatocytes
4.High workload of the hepato-enteric circulation
 Less bacterial (low conversion of bilirubin to urobilinogen by
the intestinal flora)
 Low enzymatic activity in intestine

 The liver is the largest organ in the
body.
 It is located below the diaphragm in
the right upper quadrant of the
abdominal cavity and extended
approximately from the right 5th
rib to the lower border of the rib
cage.
 The working cells of the liver are
known as hepatocytes.
The Liver

Functions of Liver
1.Synthetic function
a.Synthesis of plasma proteins (albumin,
coagulation factors, many globulins)
b.Synthesis of cholesterol
c.Synthesis of Triacylglycerol
d.Lipoprotein synthesis

Functions of Liver
2. Metabolic function
a. Carbohydrates : Glycolysis; glycogen
synthesis; glycogen breakdown;
Gluconeogenesis
b. Ketogenesis; fatty acid synthesis and breakdown
c. Protein catabolism
d. Citric acid cycle, production of ATP

Functions of Liver
3. Detoxification and excretion
a. Ammonia to urea
b. Bilirubin (bile pigment)
c. Cholesterol
d. Drug metabolites

Functions of Liver
4. Homeostasis: Blood glucose regulation
5. Storage function : Vitamin A, D, K, B12
6. Production of bile salts; help in digestion

Liver Function Tests (LFT)
• Biochemical tests are of immense value in diagnosis and
monitoring of liver diseases. These tests are usually referred to
as “liver function tests” (LFT). LFTs are the most widely
performed biochemical tests in the laboratory.
• Number of tests are required to assess hepatic function
• All the tests need not be performed in every patient
• The tests should be selected according to the clinical
symptoms and signs
• Liver function tests are performed usually to
 Detect hepatocellular damage
 Evaluate the functional status of liver
 Distinguish between different types of jaundice

Liver Function Tests (LFT)
Indications for Liver Function Tests
1. Jaundice
2. Suspected liver metastasis
3. Alcoholic liver disease
4. Any undiagnosed chronic illness
5. Annual check up of diabetic patients
6. Coagulation disorders
7. Therapy with statins to check hepatotoxicity

Liver Function tests Based on
Laboratory Findings
Group I (Tests of hepatic excretory function)
i. Serum Bilirubin; total, conjugated, & unconjugated
ii. Urine Bile pigments, bile salts and urobilinogen
Group II: Liver enzyme panel
(markers of liver injury/cholestasis)
i. Alanine amino transferase (ALT/SGPT)
ii. Aspartate amino transferase (AST/SGOT)
iii. Alkaline phosphatase (ALP)
iv. Gamma glutamyl transferase (GGT)

Liver Function tests Based on
Laboratory Findings
Group III: Plasma proteins (synthetic function)
i. Total proteins
ii. Serum albumin, globulins, A/G ratio
iii. Prothrombin time
Group IV: Special tests
i. Ceruloplasmin
ii. Ferritin
iii. Alpha-1-antitrypsin
iv. Alpha-fetoprotein

Liver Function tests Cont…
• Measurement of total, unconjugated and
conjugated bilirubin in serum helps in:
• Detection of jaundice and differentiate types of
jaundice
• In haemolytic jaundice, total and unconjugated
bilirubin are raised
• In hepatocellular jaundice,total,unconjugated and
conjugated bilirubin are raised
• In obstructive jaundice, total and conjugated bilirubin
are raised

• Bilirubin is the major bile pigment in human
beings
• Normally, it is not present in urine
• Only conjugated bilirubinis soluble in water,
and can be excreted in urine
• Conjugated bilirubin in serum is raised in
hepatocellular and obstructive jaundice
• Therefore, bilirubin is present in urine in
hepatocellular and obstructive jaundice
Bile pigments in urine

Urobilinogen in urine
• Urobilinogen is formed from bilirubin in
the intestine
• It is absorbed into portal circulation, and is
mostly re-excreted by the liver in bile
• Some of it escapes from liver into systemic
circulation, and is excreted in urine
• Normal urine contains a very small amount of
urobilinogen

• In haemolytic jaundice,formation of bilirubin
is increased
• Hence, there is increased formation of urobilinogen
• Consequently, urinary urobilinogen is increased
• In obstructive jaundice, bilirubin doesn’t reach the
intestine
• Hence, no urobilinogen is formed in the intestine
• Therefore, no urobilinogen is present in urine
• In hepatocellular jaundice, urinary urobilinogen is:
Normal

Bile salts in urine
• Bile salts are formed in the liver from cholesterol
• They are excreted in bile
• In obstructivejaundice, bile salts cannot reach
intestine due to biliary obstruction
• They are regurgitated from liver into systemic
circulation
• Since they are water-
soluble, they are excreted in urine

• Inhepatocellular jaundice, swollen liver
cells compress biliary canaliculi
• Hence, there is intrahepatic obstruction of
biliary canaliculi
• Bilesalts cannot reach intestine, and appear in
urine
• In haemolytic jaundice, there is no obstruction to
the flow of bile therefore, bile salts are not
present in urine

Haemolytic
jaundice
Hepatic
jaundice
Obstructive
jaundice
Serum bilirubin
(total)
Raised Raised Raised
Serum bilirubin
(unconjugated)
Raised Raised Normal
Serum bilirubin
(conjugated)
Normal Raised Raised
Bile pigments in
urine
Absent Present Present
Urobilinogen in
urine
Raised Normal Absent
Bile salts in
urine
Absent Present Present

Serum proteins and albumin: globulin
ratio
• Liver is the only site of albumin synthesis
• Albumin synthesis decreases in liver
disease
• Hence, serum albumin is level is decreased
• Globulin synthesis may be increased
specially in infective diseases

Serum enzymes
• In viral hepatitis, there acute necrosis of liver cells
• Enzymes present in hepatic cells are released in blood
• This raises the concentration of several enzymes in serum
• The rise in serum GPT is generally greater than that in GOT in
liver disease
• Serum -glutamyl transpeptidase (GGT) is increased in many
liver diseases
• Rise in serum GGT is a sensitive indicator of alcoholic
hepatitis
• 5’-Nucleotidase is also raised in many liver diseases

• Alkaline phosphatase (ALP) is synthesized in
liver, bones, intestine and placenta
• In liver, ALP is synthesized by parenchymal
cells as well as epithelial cells of biliary
canaliculi
• Serum ALP is mildly raised in viral hepatitis
due to necrosis of parenchymal cells
• A marked elevation in serum ALP occurs in
obstructive jaundice

Prothrombin time
• Prothrombin is synthesized in liver
• Prothrombin synthesis is decreased in hepato-
cellular disease
• This leads to a decrease in prothrombin
concentration in plasma which prolongs
prothrombin time
• Prothrombin time is prolonged in vitamin K
deficiency also. (Newly-synthesized prothrombin is inactive, Vitamin K
is required for the post -translational modification)

Special Tests
Ceruloplasmin: A globulin protein which transports
copper. This test is used to aid in the diagnosis of
Wilson’s disease, a hereditary syndrome in which
decreased levels of ceruloplasmin are manufactured
by the liver. Without ceruloplasmin to transport it,
copper accumulates in the tissues of the brain, eye,
kidney, and liver. One of the hallmarks of this
disease is the presence of copper deposits around
the iris of the eye, known as Kayser-Fleischer rings.

Ferritin: Ferritin is the primary protein in the
body which stores iron. Thus, measurement of
the ferritin level provides a good indication of
the size of the body’s available iron stores.
The ferritin level decreases before symptoms
of anemia occur. For example, in Stage 1 of
iron deficiency anemia, ferritin, and
hemosiderin stores are depleted. Level of
Ferritin increased in acute hepatitis and
inflammatory disease.

Alpha-1-antitrypsin: Is a single polypeptide
chain having 394 amino acids, also known as
α1- antiproteinase. It increases during:
• Inflammation- an acute phase protein
• Chronic hepatocellular diseases
• Biliary tract obstruction
And it decreases in:
• Nephrotic syndrome
• Emphysema
• Liver cirrhosis

Alpha-fetoprotein: Alpha-fetoprotein (AFP) is a
globulin protein formed in the yolk sac and liver
of the fetus. As the fetus develops, the level of AFP
found in the mother’s serum increases.
Only minute amounts of AFP remain in the
bloodstream after birth.
AFP is also considered a tumor marker for several
types of cancer. Cancers typically are characterized by
undifferentiated cells.
The higher the AFP level, the greater amount of tumor
present. Thus, AFP can also be used to assess response
to cancer treatment.3/1/2018 Rajendra Dev Bhatt

Some Uncommon Liver Function Tests
Galactose tolerance test:
• Liver converts the dietary galactose into glucose
• Capacity of liver to convert galactose into glucose is decreased in liver
disease
• Hence, blood galactose remains elevated for a long time after
ingestion of galactose
• Oral galactose tolerance test is preferable, and is done after an
overnight fast
• Forty gm of galactose, dissolved in water is given by mouth to the
subject
• Blood galactose is measured 60 minutes later
• A blood galactose level above 60 mg/dl indicates impairment of
hepatic function

Hippuric acid test:
• This is a test ofconjugating function of liver
• Benzoic acid is given to the subject
• Liver conjugates benzoic acid with Glycine to
form Hippuric acid
• Hippuric acid is excreted in urine
• If liver function is normal, all the benzoic
acid is conjugated to form Hippuric acid
• All the Hippuric acid is excreted in urine
• If liver function is impaired, formation and
excretion of Hippuric acid is decreased

Immunological tests:
• Detection of anti-mitochondrial antibody
helps in the diagnosis of primary biliary
cirrhosis.
• Detection of anti-nuclear and/or anti- smooth
muscle antibodies helps in the diagnosis of
autoimmune hepatitis.

Blood ammonia:
• In advanced liver disease, liver may fail to
convert ammonia into urea
• This can cause hepatic encephalopathy
• Measurement of blood ammonia helps in its
diagnosis and monitoring.

Thank You

Bilirubin Metabolism Rajendra

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