Download Bilirubin

Molecular Biochemistry II
HEME DEGRADATION
AND JAUNDICE
xiaoli
RBCs last 120 days, degraded by
reticuloendothelial (RE) system
Under
physiologic conditions in the
human adult, 1~2×108 erythrocytes are
destroyed per hour. Thus, in 1 day, a 70kg
human turns over approximately 6g of
hemoglobin.
When hemoglobin is destroyed in
the body, globin is degraded to its
constituent amino acids, which are
iron + Bile pigments
reused, and the iron of heme enters
the iron pool, also for reuse.
most heme from RBCs (85%) - rest
from turnover of cytochromes, p450s,
immature erythrocytes
Amino acids
Heme
globin
iron
Bile pigments
Bile pigments
Any of several coloured compounds
derived from porphyrin that are found in
bile; principally bilirubin and biliverdin.
Bilirubin Metabolism

Bilirubin formation

Transport of bilirubin in plasma

Hepatic bilirubin transport


Hepatic uptake

Conjugation

Biliary excretion
Excrect through intestine system
Bilirubin formation
microsomal

cytosol
The iron-free porphyrin portion of heme is also
degraded, mainly in the reticuloendothelial cells of the
liver, spleen, and bone marrow.
The first step
Heme oxygenase
Heme oxygenase (HO) is an enzyme that
catalyzes the degradation of heme. This
produces biliverdin, iron, and carbon
monoxide.
Heme oxygenase
There are three known isoforms of heme oxygenase.

Heme oxygenase 1 (HO-1) is an inducible isoform in
response to stress such as oxidative stress, hypoxia, heavy
metals, cytokines, etc. Its activity is induced by its substrate
heme and by various nonheme substances.

Heme oxygenase 2 (HO-2) is a constitutive isoform which
is expressed under homeostatic conditions. Both HO-1 and
HO-2 are ubiquitously expressed and catalytically active.

A third heme oxygenase (HO-3) is not catalytically active,
but is thought to work in oxygen sensing.
In mammalian cells Heme oxygenase (HO1) has two
basic functions:
1. it recycles iron supplies within the cell to
maintain homeostasis.
2. biliverdin and biliruben (its reduced form), are
powerful antioxidants believed to aid in the prevention
of oxidative cell damage.
HOOC
M
V
M
CH2
CH2
CH
O
N
H
CH2
CH2 M
CH2
N
H
M：-CH2
V：-CH=CH2
M V
COOH
N
H
O
CH
N
H
The building of
intermolecular
hydrogen bonds by the
NH and COOH groups
is spatially hided.
Bilirubin is lipophilic
and therefore insoluble
in aqueous solution.the
solubility in water is
less.
Transport of Bilirubin in Plasma
Bilirubin on release from macrophages
circulates as unconjugated bilirubin in plasma
tightly bound to albumin.
Albumin + free Bilirubn
Why bound to albumin?
Bilirubin ~ Albumin Complex
unconjugated bilirubin
Significance:
★Increase
★ Prevent
the solubility of whole molecule
unconjugated bilirubin freely
come into other tissue, cause damage.
Transport of Bilirubin in Plasma
H affinity binding sites
2:1
Bilirubin
Molar
Ratio
>2:1
L affinity binding sites
Plasma protein
Albumin
Bilirubin
Other organic anions
can be replaced by
PH
UB
Albumin has two binding sites for bilirubin---a high
affinity site and a low affinity site.
Albumin per 1L of plasma can bind about 342～
427.5μmol bilirubin, but 1.7～17.1μ bilirubin per 1L .
blood
membrance
[bilirubin-albumin]
Other
antibiotics
cytosol
[bilirubin-lipids]
[albumin]↑
[bilirubin]
[bilirubin]
Certain drugs as sulfonamides and salicylates compete with
bilirubin for albumin binding and displace bilirubin to enter
into the brain in neonates and increase the risk of
kernicterus (a type of brain damage that can result from high
levels of bilirubin in a baby’s blood. It can cause athetoid
cerebral palsy and hearing loss) .
Hepatic phase
On coming in contact with the hepatocyte surface,
unconjugated bilirubin is preferentially
metabolized which involved 3 steps:
Hepatic uptake
Conjuation
Secretion in bile
Hepatic uptake of Bilirubin
Bilirubin ~ Albumin Complex
Albumin
Bilirubin
(lipid soluble)
(be) taken up by membrane of the liver
Bilirubin
carrier protein
(be) bound to
transfer
ER
ligation (Y protein, Z protein)
Conjugation of bilirubin
UDPGT Bilirubin
Bilirubin +UDP-gluconic acid
monoglucuronides + UDP
UDPGT
UDP-gluconic acid
Bilirubin diglucuronide
+ UDP
Conjugation occur in endoplasmic reticulum UDPglucuronosyl transferase,
Product: mono or diglucuronides.
The process of conjugation can be induced by drugs
phenobarbital.
HOOC
M V
CH2
CH2
CH
O
O
M
COOH
CH2
CH2 M
CH
N
H
N
H
2
N
H
O
CH
N
H
OH
COOH
O
HO
OH
M
M V
O
O
OH
N
H
MV
C
CH
O2
CH2
CO
CH2
CH M
OHHOOC
O
OH
bilirubin diglucuronides
conjugated bilirubin
M V
2
CH
N
H
CH2
N
H
O
CH
N
H
★Increase the solubility of whole molecule
★ delete the toxicity of bilirubin.
Unconjugated bilirubin:
Bilirubin that are not conjugated with
gluconic acid , also called hemobilirubin,
indirected bilirubin.
conjugated bilirubin:
Bilirubin that are conjugated with gluconic
acid, also called hepatic bilirubin, directed
bilirubin.
Major differences between unconjugated and
conjugated bilirubin
FEATURE
Unconjugated
bilirubin
CONJUGATED
BILIRUBIN
Normal serum level
More
Less (less than 0.25mg/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
Excretion of bilirubin into bile
Bilirubin diglucuronide is actively
transported against a concentration gradient
into bile duct.
 This energy-dependent, rate –limiting step
is susceptible to impairment in liver disease.
Uncojugated bilirubin is normally not
excreated.

Formation of urobilins in the intestine



Bilirubin diglucuronide is hydrolysed and reduced by
bacteria in the gut to yield urobilinogen, a colorless
compound.
Most of the urobilinogens of the feces are oxidized
by intestinal bacteria to stercobilin, which gives
stools their characteristic brown color.
Some urobilinogen is reabsorbed from the gut into
the portal blood and transported to the kidney, where
it is converted to the yellow urobilin and excreted,
giving urine its characteristic color. (bilinogen
enterohepatic circulation)
urobilin
Bilirubin diglucuronide
urobilinogen
bilin
stercobilin
Conjugated
bilirubin
blood
0.5～4mg/day
urobilin
bilinogen
prototype
Portal
vein
reabsorbed
Sterobilin
excrection
10 ～ 20%
80%～90%
bilinogen
bilinogen enterohepatic
circulation
BLOOD
CELLS
Stercobilin
excreted in feces
Urobilin
excreted in urine
Hemoglobin
Globin
Heme
O2
Heme oxygenase
Urobilinogen
formed by bacteria
INTESTINE
KIDNEY
reabsorbed
into blood
CO
Biliverdin IX
via bile duct to intestines
NADPH
Bilirubin diglucuronide
(water-soluble)
Biliverdin
reductase
NADP+
Bilirubin
(water-insoluble)
unconjugated
Catabolism of hemoglobin
2 UDP-glucuronic acid
via blood
to the liver
Bilirubin
(water-insoluble)
LIVER
Summary of bilirubin metabolism
Senescent 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.
＊normal range of bilirubin:
1～16mol/l (0.1 ～1mg/dl)
4/5 are unconjugated bilirubin, others
are conjugated bilirubin.
<1mg/dl
1-2mg/dl
>2mg/ dl
normal
occult
jaundice
Hyperbilirubinemia
Hyperbilirubinemia: the concentration of blood bilirubin
are more than 1mg/dl.
Occult: the concentration of blood bilirubin are increased ,
but have no clinic sympotom, normally 1-2mg/dl.
Jaundice : ( also called icterus) refers to the yellow color of the
skin and scleare caused by deposition of bilirubin, secondry to
increased bilirubin levels in the blood.
Although not a disease itself, jaundice is usually a symptom
of an underlying disorder.
Based on pathophysiology, jaundice may result
from one or more of the following mechanism:
1. Increased bilirubin production ( excessive red cell
destruction)
2. Decreased hepatic uptake ( ligandin, drug, prolonged
starvation, and sepsis)
Decreased hepatic conjugation (enzyme,drugs, cirrhossis)
3. Decreeased excretion of bilirubin into bile ( gallstone,
tumour)
Simple Classification of jaundice

①
②
③
Accordingly, a simple classification of jaundice
is to divided into 3 predominant type:
Pre-hepatic (hemolytic jaundice)
Hepatic jaundice
Post – hepatic cholestatic (obstructive
jaundice)
Hemolytic jaundice
massive lysis of red blood cells (for example, in
patients with sickel cell anemia or malaria) may
produce bilirubin faster than the liver can conjuagte it.
More bilirubin is excreted into the bile, the amount of
the urobilinogen entering the enterohepatic
circulation is increased, and urinary urobilinogen is
increased.
Unconjugated bilirubin is elevated in blood.
Causes of hemolytic jaundice












Malaria
Side effects of certain drugs :antibiotic and anti-tuberculosis medicines,
levodopa,
Certain drugs in combination with a hereditary enzyme deficiency known as
glucose-6-phosphate dehydrogenase (G6PD)
Poisons Snake and spider venom, certain bacterial toxins, copper, and some
organic industrial chemicals directly attack the membranes of red blood cells
Artificial heart valves
Hereditary RBC disorders sickle cell disease
Enlargement of the spleen
Diseases of the small blood vessels
Immune reactions to RBCs cancer
Transfusions
Kidney failure and other serious diseases
Erythroblastosis fetalis
Hepatocellular jaundice
Damage to liver cells( for example in patient with
cirrhosis or hepatitis) causes a decrease in both
bilirubin uptake and production of conjuagted bilirubin.
Unconjugated bilirubin occur in the blood and
increased urobilinogen in the urine.
The urine is dark in color and stool are pale, clay
color.
Plasma level of AST and ALT are elevated and the
patient experience nausea and anorexia.
Obstructive jaundice
In this instance jaundice is results from obstruction
of the bile duct.
For example, the presence of a hepatic tumor or
bile stone may block the bile ducts, preventing
passage of bilirubin into the intestine, patients with
obstructive jaundice experience GI pain, nausea and
produce stools that are a pale, clay color.
65 year-old lady presenting with obstructive jaundice
Biliary stones
Sample
Indices
Normal
Serum
Total Bil
＜1mg/dl
＞1mg/dl
Direct Bil
0～0.8mg/dl
↑↑
Indirect Bil
＜1mg/dl
Color
normal
deep
deeper
deep
Bilirubin
—
＋＋
—
＋＋
Urine
Stool
Obstructive Hemolytic
Jaundice
Jaundice
＞1mg/dl
Hepatic
Jaundice
＞1mg/dl
↑
↑↑
Urobilinogen
a
little
↓
↑
uncertain
Urobilin
a
little
↓
↑
uncertain
Argilous
(complete
obstruction)
deeper
lighter or
normal
Color
normal
Diagnoses of Jaundice