Bilirubin Production and Metabolism
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Transcript of Bilirubin Production and Metabolism
8/2/2019 Bilirubin Production and Metabolism
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BILIRUBIN METABOLISM
BILIRUBIN PRODUCTION AND METABOLISM
WHY SHOULD YOU BE INTERSTED IN BILIRUBIN METABOLISM?
1. bilirubin is a toxic metabolite of haem degradation and must be excreted from
the body
2. it is metabolized in the same way that many other compounds and xenobiotics,
or pharmaceuticals, are metabolized and eliminated from the body
3. bilirubin is a coloured compound making it easily measured and therefore a
good indicator of disturbances in the different steps of its metabolism and
excretion
BILIRUBIN PRODUCTION AND METABOLISM
Bilirubin is generated from haem metabolism, with the majority from
haemolysis of red blood cells and haemoglobin breakdown. Haemoglobin
contains iron, and once the iron moiety is extracted what is left is the haem
molecule. The metabolism of haem results in the generation of bilirubin . A
minor component of bilirubin comes from other haem-containing proteins such
as the respiratory chain cytochromes and drug-metabolizing cytochrome P450s
Bilirubin and its metabolites are differently coloured compounds (this
distinction can be seen in the figure above). Once iron is removed from the red
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haem, a green coloured compound results; this is biliverdin. Formation of
biliverdin occurs within the reticuloendothelial systems of the body, mainly
within the Kupffer cell. The bilirubin is then released from the Kupffer c ell and
transported to the hepatocytes. Bilirubin is a very non-polar compound and as
such, is water insoluble. Since blood is an aqueous medium, in order to
transport bilirubin from the Kupffer cell, through the capillaries of the liver, orsinusoids, to the hepatocyte, it must be carried in a way that makes it water
soluble. This is done by complexing the bilirubin with serum albumin so that it
can then be delivered to the hepatocyte. Note that at this point, bilirubin is
unconjugated and you will see that under normal conditions, the blood mainly
contains unconjugated bilirubin (UCB). The metabolic reactions within the
hepatocyte result in the production of conjugated bilirubin (CB).
The hepatocytes (the major epithelial cell of the liver) perform all of the
metabolic reactions necessary to permit the excretion of bilirubin into the bile
and eventually out of the body. At the surface of the hepatocyte, bilirubin is
taken up or absorbed into the cell and within the hepatocyte, it is conjugated.
Conjugation is a way of making bilirubin more water soluble. Once bilirubin is
conjugated it is more hydrophilic and therefore cannot cross the plasma
membrane of the hepatocyte. Instead, it must be transported across the
membrane into the bile.
Once in the bile, bilirubin is delivered to the intestine where it can be further
metabolized by bacteria and at that point, the majority becomes a component
of feces (endowing the later with its normal colour), and is eliminated from the
body. A minor portion can be reabsorbed in to the blood and excreted as CB by
the kidneys.
BILIRUBIN STRUCTURE
this is the structure of
bilirubin in its non-polar
state. Many xenobioitcs
must undergo the same
metabolic processes for thesame reason as bilirubin -
they are not very soluble.
Bilirubin is a planar, rigid
compound. From this
illustration you can see that
there are six intramolecular
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hydrogen and oxygen atoms within the molecule. In fact, if you were to unfold
this molecule it would be water soluble, but its normal structure, on its own, is
to fold in on itself forming a very rigid compound with all the atoms that could
normally interact with water interacting , instead, with each other. When
bilirubin binds with albumin these oxygens and hydrogens now interact with
groups on the serum protein. In 2008 a crystal structure of the bilirubin-albumin complex was synthesized, hence we know its real structure.
The lower scheme shows how bilirubin unfolds upon complexing with albumin
(note that the albumin molecule has been removed so that you can see how
the bilirubin molecule has rotated around the three carbon atoms indicated by
the red stars). Thus, the groups that can form hydrogen bonds are now
exposed and can interact with groups within the protein. The reason this is
being focused on is because the same thing happens in the hepatocyte, i.e.,
the conjugation of bilirubin forces the molecule to unfold and interact with the
aqueous environment of the cell
CRYSTALLIZED BILIRUBIN AND ALBUMIN
Note the blue Ns and the red Os of (non-rigid) bilirubin extending to the
periphery of the compound and next, with part of the protein molecule inmagenta surrounding the grey coloured bilirubin.
COMPLETE BILIRUBIN-ALBUMIN COMPLEX
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The bilirubin is almost
completed surrounded by the
serum protein making it
"invisible" to the aqueous
environment of the blood and
hence making it possible totransport through the vascular
compartment to the
hepatocyte.
Another discovery made from
this study is that each albumin
molecule can only bind one
bilirubin molecule. The significance of this is that transport of bilirubin and
xenobiotics through the blood is limited by the amount of albumin present.
Hence, saturation of this site by drugs could result in a decreased capacity of
the body for bilirubin and drug excretion
COVERSION FROM INSOLUBLE TO SOLUBLE BILIRUBIN
The result of this
metabolism is an
unfolded molecule that
can interact with
cellular water so that it
can be excreted via thebile. This is
accomplished by the
addition of bulky, acidic
sugar molecules,
specifically glucuronic
acid. One or two
glucuronic acid
molecules are added in
an enzyme-catalyzed
mono- or diglucuronidation reaction.
This conjugation reaction is catalyzed by the UDP-glucuronyl transferases
(UGTs). There exist a number of UGT isoforms of which one, UGT1A1, is the
major enzyme invovled in bilirubin metabolism. Clinically, this is important
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because up to 15% of the population has mutations in this isoenzyme, resulting
in a mild unconjugated hyperbilirubinemia called Gilbert's syndrome.
This scheme shows how the addition of the two bulky glucuronic acid
molecules causes bilirubin to rotate around the three carbon atoms denoted by
the red stars, opening it up so that the hydrogen atoms and additional carboxylgroups can interact with water. The end result is a much more hydrophilic
compound that is soluble in the aqueous environment of the cell, bile, and
blood.
TRANSPORT AND EXCRETION OF BILIRUBIN
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Transport, metabolism and excretion of bilirubin requires the following steps
1. solubilization and transport through the blood stream
the first step is the transport of the hydrophobic bilirubin as a
complex with the soluble serum protein albumin to the hepatocyte
surface
2. delivery to the hepatocyte
UCB is transported across the sinusoidal plasma in two distinct
ways
• Firstly, because it is hydrophobic, it can simply dissolved
into the phospholipid bilayer
• Secondly, there are a number of different transporters that
can mediate the passage of bilirubin and other xenobiotics
across the plasma membrane.
Once across the bilayer, the bilirubin again needs to be carried asa complex with a hydrophilic protein to the site of conjugation. The
carrier protein is referred to as "ligandin" or "glutathione S-transferase,
also an important protein in the metabolism of many drugs.
The ligandin-UCB complex moves through the cytosol to the
endoplasmic reticulum, where the conjugating enzymes, the UGTs,
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reside. The UGTs catalyze the addition of one or two glucuronic acid
molecules to bilirubin to form bilirubin mono- or diglucuronide.
3. metabolism and excretion into the bile
The glucuronide-conjugated bilirubin is now quite water soluble
and hence requires as transport protein to move it from the cytosol,
across the canalicular membrane and into the bile. Canalicular
transport is mediated by the canalicular multi-organic anion
transported or cMOAT. This transporter was identified in the mid '90s
as a multidrug resistance-associated protein (MRP2). Note that the
conjugated bilirubin is normally transported only across the canalicular
membrane, and not the sinusoidal membrane. This occurs because of
the polarized expression of specific uptake and efflux transporters on
the sinusoidal and canalicular membranes respectively
This scheme shows the polarized hepatocyte epithelium. Each hepatocyte has
a blood-facing, or sinusoidal surface, and a biliary lumen-facing, or canalicular
surface. The goal is to deliver bilirubin and any other xenobiotics from the
blood, through the hepatocyte and into the bile
IMBALANCES IN BILIRUBIN
This scheme
illustrates
situations that
result in
imbalances in
bilirubin handling
by the body,
resulting in
jaundice. Jaundice
can result from
impairment of any
of the steps of
bilirubin handling
that we havediscussed, either
before the liver
(prehepatic
jaundince), within the liver (hepatic jaundice), or after the liver (post-hepatic
jaundice).
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Pre -hepatic jaundice could result from increased rates of haemolysis, thus
saturating the liver's capacity to handle the larger amounts of UBC in the
blood, and leading to an unconjugated hyperbilirubinaemia
Hepatic jaundice may be caused by a decreased capacity to conjugate UCB, or
a reduction in canalicular CB transport activity. Decreased conjugating activitycould result from an inhibiting mutation in UGT (e.g. Gilbert syndrome),
depressed expression (neonate), complete absence of UGT (Criglar-Najjar
syndrome), or loss of functioning hepatocytes (hepatitis). In each case, UCB
would accumulate in the blood. Conversely, inactivating mutations in
cMOAT/MRP2 would result in a CB hyperbilirubinaemia due to regurgitation of
CB into the blood (the hepatocyte expresses a new MRP in the sinusoidal
membrane in response to the depressed canalicular transport activity).
Cholestasis is a much more common cause of jaundice and can be either
intrahepatic (bile ductular), or post-hepatic (gallstone disease). The post-
hepatocellular blockage will cause a regurgitation of CB and UCB into theblood, generating a mixed CB/UCB hyperbilirubinemia.
QUIZ
1) Which of the following is the primary source of bilirubin in the body?
a) breakdown of hemoglobin from red blood cells
b) respiratory chain cytochromes
c) breakdown of bile salts in the gallbladder
d) drug-metabolizing chytochrome P450s
2) Which of the following cells is the primary site of production of bilirubin?
a) Hepatocyte
b) Red blood cell
c) Kupffer cell
d) White blood cell
3) Bilirubin is water soluble
a) True
b) False
4) Under normal circumstances, most of the bilirubin in the blood is conjugated
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a) True
b) False
5) Which statement correctly describes the relationship of bilirubin to albumin in the
body.
a) the large albumin molecule serves to transport several bilirubin molecules in
the capillaries of the liver
b) albumin combines with bilirubin to create a rigid planar compound
c) albumin functions to transport bilirubin across the canalicular facing wall of the
hepatocyte
d) albumin complexes with bilirubin causing the latter to unfold and become
hydrophilic
6) In Gilbert's syndrome, abnormalities of UDP-glucuronyl transferases (UGTs) result
in:
a) increased levels of conjugated bilirubin in the blood
b) increased levels of unconjugated bilirubin in the blood
c) Decreased levels of conjugated bilirubin in the blood
d) Decreased levels of conjugated and unconjugated bilirubin in the blood
7) In a patient with obstructive gallstones, what changes do you anticipate finding in
the blood?
a) decreased levels of unconjugated bilirubin
b) decreased levels of conjugated bilirubin
c) increased levels of conjugated bilirubin
d) increased levels of conjugated and unconjugated bilirubin
8) Match the following conditions or disease states with the best answer
hemolysisGilbert syndromeHepatitiscMOAT dysfunctiongallstones
mild unconjugated hyperbilirubinaemiapre-hepatic jaundicecholestatic jaundiceabnormal canalicular bilirubin transporthepatic jaundice
CASE ONE
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Lily likely has increased amounts of
conjugated bilirubin in her blood
a) True
b) False
How would you classify Lily's jaundice?
a) pre-hepatic
b) hepatic
c) post-hepatic
CASE TWO
Investigations:
gallbladder not visualized on USS
HIDA scan - no excretion of bile
Diagnosis: Congenital biliary atresia
What type of bilirubin will predominate in thisinfant's blood sample?
a) Unconjugatedb) Conjugated
c) Both
How would you classify Gregor's jaundice?
a) pre-hepatic
b) hepatic
c) post-hepatic