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



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



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



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



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



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,



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).



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



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



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

Bilirubin Production and Metabolism

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