Biochemistry practice lesson 16

Liver functions:
Bile formation

Heme degradation
Bilirubin formation
 Bile formation.
Bile acids and salts,
their biological role.
 BIOMEDICAL IMPORTANCE
Bile salts are synthesized by the liver
and secreted, together with
cholesterol and phospholipids, into
the bile ducts to form bile.
Bile salts solubilize lipids in the
intestinal lumen and thus enhance
their digestion. Bile salts also form
mixed micelles with lipids and thus
facilitate their absorption.

Insufficient production of bile acids

causes lipid malabsorption.
If the liver produces an insufficient
amount of bile salts or secretes too
Biliary system. Bile is formed in the liver (L), much cholesterol, cholesterol
stored in the gallbladder (GB), and secreted gallstones may form in the
into the duodenum (D). CD, cystic duct;
gallbladder or bile ducts.
CBD, common bile duct; P, pancreas.
 Bile Acids Are Formed from Cholesterol

The liver hydroxylates cholesterol to produce the primary bile acids

chenodeoxycholic acid and cholic acid; these bile acids are then conjugated with
the amino acids glycine or taurine to yield primary bile salts.

In the lumen of the ileum and colon, bacteria can remove the 7α-hydroxyl group
from primary bile salts to yield secondary bile salts.
 Biosynthesis and
degradation of bile
acids

*Catalyzed by
microbial enzymes.
 Biosynthesis of
bile acids
The primary bile acids are
synthesized in the liver from
cholesterol.
These are cholic acid (found in
the largest amount in most
mammals) and
chenodeoxycholic acid.
The 7α-hydroxylation of cholesterol is the first and principal regulatory step in the
biosynthesis of bile acids and is catalyzed by cholesterol 7`-hydroxylase, a microsomal
cytochrome P450 enzyme designated CYP7A1.
A typical monooxygenase, it requires oxygen, NADPH, and cyt P450.
Subsequent hydroxylation steps are also catalyzed by monooxygenases.
The primary bile acids enter the bile as glycine or taurine conjugates. Conjugation
takes place in liver peroxisomes. In humans, the ratio of the glycine to the taurine
conjugates is normally 3:1. In the alkaline bile (pH 7.6-8.4), the bile acids and their
conjugates are assumed to be in a salt form—hence the term “bile salts.”
Primary bile acids are further metabolized in the intestine by the activity of the
intestinal bacteria. Thus, deconjugation and 7α-dehydroxylation occur, producing the
secondary bile acids, deoxycholic acid, and lithocholic acid.
 Bile composition
Bile contains micelles of bile salts,
cholesterol, and the phospholipid
phosphatidylcholine
(also called lecithin).
The secreted bile salts are
principally glycocholic,
glycochenodeoxycholic, taurocholic
and taurochenodeoxycholic acids.

Cholic acid has one more hydroxyl group than chenodeoxycholic acid and is
therefore more soluble in water. Conjugation of bile acids with glycine or taurine
further increases the solubility in water.
The amount of bile salts in the bile is an important determinant of the amount of
water in bile and thus the flow of bile out of the liver. The gallbladder then
concentrates bile by removing water from it.
The composition of the micelles in bile is determined by the relative rates of export
of bile salts, cholesterol, and phospholipids, each of which has its own export system
from the liver.
Bile functions
 Bile acids assist the digestion and absorption of
dietary fat
The secretion of bile from the liver and the emptying of the gallbladder are
controlled by the gastrointestinal hormones hepatocrinin and
cholecystokinin, respectively.
They are released when partially digested food passes from the stomach
to the duodenum.
Once secreted into the intestine, the bile acids act as detergents (they
possess polar carboxyl and hydroxyl groups), assisting the emulsification of
ingested lipids.
Bile acids cover triacylglycerol drops interface thus allowing colipase -
pancreatic lipase complex to anchor itself to the water-lipid interface.
These aid the enzymatic digestion of dietary fat.

Bile acids are able to facilitate formation of micelles, which can be

absorbed.
This aids the absorption of dietary fat.
Bile acids recirculate via the enterohepatic circulation
Up to 30 g of bile acids pass from the bile duct
into the intestine each day but only 2% of this
(approximately 0.5 g) is lost with the feces.
Most are deconjugated and reabsorbed. Their
passive reabsorption occurs in the jejunum
and colon but they are mostly taken up by
active transport in the ileum.
Reabsorbed bile acids are transported back to
the liver via the portal vein, being
noncovalently bound to albumin, and are
re-secreted into the bile. The process is known
as the enterohepatic circulation.
This recirculation also explains why bile
contains both primary and secondary bile
acids. The total bile acid pool is only 3 g and
therefore they have to recirculate 5–10 times a
day.
The bile acid flux also contributes to the
control of bile acid synthesis; 7α-hydroxylase is
subject to feedback inhibition by the bile acids
returning to the liver through the portal vein.
 Diseases of Bile Metabolism
For bile to be free of crystals and stones,
cholesterol must constitute less than 10% of the
total moles of cholesterol, bile salts, and lecithin;
furthermore, bile salts must be greater than 40%
and lecithin less than 60% of this total. On a
molar basis, normal bile contains approximately
6% cholesterol, 74% bile salts, and 20% lecithin.
Gallstones can form in case of:
• excessive rate of cholesterol secretion (may
occur in patients who take one of the fibrate
drugs (e.g., gemfibrozil or fenofibrate) to
lower triglycerides in the blood);
Phase diagram for a model system of cholesterol • decreased rate of bile salt secretion
in micelles in bile. The green area represents the (decreased reabsorption of bile salts from the
combinations of solutes that give rise to a single intestine in patients with diseases that affect
phase of micelles. The composition of bile of
healthy persons is typically well within the green
the ileum; decreased synthesis of bile salts
area, while that of persons with cholesterol due to liver dysfunction);
gallstones is close to the border of this area or • bile gets highly concentrated in the
clearly outside of it. The fractions refer to the total gallbladder (the gallbladder empties at a low
moles of solute in a solution that is 90% water and
10% solute (cholesterol + lecithin + bile salts). rate, or when the bile system has low
motility).
 Diseases of Bile Metabolism: Gallstones
The presence of gallstones in the gallbladder
(cholelithiasis) or bile duct
(choledocholithiasis) is quite common and
usually does not cause symptoms.
However, gallstones that block the cystic duct
(which connects the gallbladder to the
common bile duct) or the common bile duct
can cause pain. Pain is especially common
after a fatty meal because the fat in the
duodenum elicits the secretion of the
hormone CCK, which stimulates the
contraction of the gallbladder.

Gallstones can lead to:

•cholecystitis (inflammation of the gallbladder),
•cholangitis (inflammation of the bile duct),
• jaundice (due to impaired bilirubin excretion),
•pancreatitis (due to blockage of the flow of enzymes from the pancreatic duct
into the common bile duct).
Heme degradation.
Bilirubin formation.
Types of Jaundices.
 BIOMEDICAL IMPORTANCE
When red blood cells are degraded, the iron in heme is
recycled, while the porphyrin moiety is degraded to bilirubin.
Bilirubin is metabolized by the hepatocytes and excreted in
bile.

An excessive amount of bilirubin in the blood indicates

reduced survival of red blood cells and/or insufficient
excretion of bilirubin.

Excretion may be inadequate due to impaired conjugation

with glucuronic acid in the liver or impaired secretion of
conjugated bilirubin from the liver into the intestine.
 CATABOLISM OF HEME
PRODUCES BILIRUBIN
Human adults normally destroy about 200
billion erythrocytes per day. Thus, a 70-kg
human turns over approximately 6 g of
hemoglobin daily.
The catabolism of heme from all heme proteins
is carried out in the microsomal fraction of cells
in the liver, spleen, and bone marrow.
After the protein part (globin) has been
removed, the tetrapyrrole ring of heme is
2+
oxidatively cleaved between rings A and B by
heme oxygenase. This reaction requires
molecular oxygen and NADPH+H+, and
produces green biliverdin, as well as CO (carbon
monoxide) and Fe2+, which remains available
for further use. CO production from heme
degradation is normally too small to cause
appreciable toxicity.

Biliverdin reductase reduces the central

methylene bridge of biliverdin to a methyl
group, producing the yellow pigment bilirubin.
Conversion of heme to bilirubin
Conversion of heme to bilirubin
by reticuloendothelial cells can
be observed visually as the purple
color of the heme in a hematoma
slowly converts to the yellow
pigment of bilirubin.
Not Conjugated Bilirubin
In water, bilirubin forms internal hydrogen
bonds that conceal the hydrophilic groups
from water, and thus render it poorly water
soluble.
Hence, in the bloodstream, bilirubin binds to
albumin. This complex does not pass
through the glomerular membrane in
the kidneys nor is it excreted into the bile.

Bilirubin is an abundant fat-soluble

antioxidant.
It protects polyunsaturated fatty acids from
peroxyl radicals and proteins from hydroxyl
radicals.

However, besides this beneficial role,

bilirubin at a high concentration is also
neurotoxic (kernicterus and Crigler-Najjar
syndrome).
Bilirubin Is Transported to the Liver
Bound to Serum Albumin

Bilirubin bound to serum albumin is readily

transported to the liver.
Albumin appears to have both a high-affinity site and
a low-affinity site for bilirubin.
More loosely bound bilirubin can readily be
detached and diffuse into tissues, and antibiotics and
certain other drugs can compete with and displace
bilirubin from albumin's high-affinity site.
Further Metabolism of Bilirubin
Occurs in the Liver
Hepatic catabolism of
bilirubin takes place in
three stages:
•uptake by the liver,
•conjugation with
glucuronic acid,
•secretion in the bile.
Hepatic catabolism of Bilirubin: Uptake by the liver

Bilirubin is removed from albumin and taken up at the sinusoidal

surface of hepatocytes by high-affinity facilitated transport system.

Once internalized, bilirubin binds to cytosolic proteins such as

glutathione S-transferase, previously known as a ligandin, to
prevent bilirubin from reentering the blood stream.
Hepatic catabolism of Bilirubin:
Conjugation With Glucuronate
Bilirubin is nonpolar, and would
persist in cells (eg, bound to
lipids) if not converted to a more
water-soluble form.

Bilirubin is converted to a more

polar molecule by conjugation
with glucuronic acid.
A bilirubin-specific UDP-glucosyl
transferase of the endoplasmic
reticulum catalyzes stepwise
transfer to bilirubin of two
glucosyl moieties from UDP-
glucuronate.
 Hepatic catabolism of Bilirubin:
Secretion into the Bile
Secretion of conjugated bilirubin into the bile occurs by an active
transport mechanism via an ATP-binding multispecific organic anion
transporter (MOAT) located in the plasma membrane of the bile
canaliculi.
The hepatic transport of conjugated bilirubin into the bile is inducible
by the same drugs that can induce the conjugation of bilirubin
(phenobarbital).
Conjugation and excretion of bilirubin thus constitute a coordinated
functional unit. Glucuronide synthesis and excretion is the rate-
determining step.
Most of the bilirubin excreted in the bile of mammals is bilirubin
diglucuronide. However, when bilirubin conjugates exist abnormally
in human plasma (eg, in obstructive jaundice), they are predominantly
monoglucuronides.
 Intestinal Bacteria Reduce Conjugated
Bilirubin to Urobilinogen
When conjugated bilirubin reaches the terminal ileum and the large
intestine, the glucuronosyl moieties are removed by specific bacterial β-
glucuronidases.
The bilirubin released is then reduced further by the fecal flora to form a
group of colorless tetrapyrroles called urobilinogens.

Small portions of urobilinogens are reabsorbed in the terminal ileum and

large intestine and subsequently are reexcreted via the enterohepatic
urobilinogen cycle.
Most of the colorless urobilinogens formed in the colon are oxidized there to
colored urobilins (yellow-colored stercobilin) and excreted in the feces.

Under abnormal conditions, particularly when excessive bile pigment is

formed or when liver disease disrupts this intrahepatic cycle, urobilinogen
may also be excreted in the urine, where oxidative processes darken it to
form urobilin.
 Measurement of Bilirubin in Serum
Normally, 95% or more of the bilirubin in the
blood is unconjugated and 5% or less is
conjugated. Most of the conjugated bilirubin
is bilirubin diglucuronide. In the laboratory, bilirubin is measured as
reddish-purple colored product formed when
bilirubin reacts with diazotized sulfanilic acid.
An assay conducted in the water solution
measures “direct bilirubin,” this fraction contains
all of the conjugated and some of the
unconjugated bilirubin: In water, the diazo dye
reacts with both conjugated and unconjugated
bilirubin, but it reacts more quickly with
conjugated (water-soluble) bilirubin.

An assay conducted in the presence of added methanol measures total bilirubin: in

alcohol, the diazo dye reacts with all of the bilirubin (conjugated and unconjugated).

The difference between total bilirubin and direct bilirubin is known as “indirect
bilirubin,” and it is unconjugated bilirubin.
 Bilirubin Lab Assays Interpretation
A normal value for the total bilirubin is 0.1 to 1.0 mg/dL; for the direct bilirubin, it is
0.3 mg/dL or less.
The direct bilirubin is always smaller than the total bilirubin in healthy person.

The lack of specificity of the direct bilirubin assay for conjugated bilirubin complicates
the interpretation of abnormalities.
Only a major elevation in conjugated bilirubin generates a lopsided direct bilirubin to
total bilirubin ratio.

There is a convenient 15% rule of thumb to interpret bilirubin measurements:

• If a patient has marked hyperbilirubinemia (e.g., >3 mg/dL), and if the direct
bilirubin is 15% or more of the total bilirubin, the patient is said to have a direct
hyperbilirubinemia, which is most likely due to cholestasis (i.e., the conjugated
bilirubin cannot properly get out of the liver and into the duodenum).
• A patient who has marked hyperbilirubinemia (e.g., >3 mg/dL) and a direct
bilirubin of 15% or less of the total bilirubin is said to have an indirect
hyperbilirubinemia. Such a patient either produces too much bilirubin or has a
problem conjugating bilirubin.

Note that the 15% rule of thumb does not apply to patients who have a normal or only
mildly elevated total bilirubin.
 HYPERBILIRUBINEMIA
CAUSES JAUNDICE
Hyperbilirubinemia – is a state when blood level of bilirubin exceeds 1 mg per
dL (17 μmol/L).

In such situation, when the blood concentration reaches 2 to 2.5 mg of

bilirubin per dL, it diffuses into the tissues, which turn yellow, a condition
termed jaundice or icterus. Jaundice is visible due to a deposition of bilirubin
in the sclerae and skin.

Jaundice is an indication of hyperbilirubinemia.

Bilirubin, at a high concentration, is neurotoxic.

Hyperbilirubinemia is commonly seen in newborns and in patients who have a

hemolytic anemia, Gilbert syndrome, liver disease, or blockage of the bile
system.
Alternative pathways of bilirubin excretion
In patients with severe hyperbilirubinemia, alternative
pathways of bilirubin excretion become appreciable:
• Unconjugated bilirubin can spontaneously oxidize to
less hydrophobic products that can be excreted into the
bile.
• Unconjugated bilirubin can also photoisomerize to a
more water-soluble diastereomer, which is excreted in
the urine (neonatal jaundice).
• Conjugated bilirubin can be filtered in the glomeruli and
lost into the urine, thereby markedly darkening the
urine.
Hyperbilirubinemia Due to Impaired
Excretion of Conjugated Bilirubin
If conjugated bilirubin is not
adequately secreted from
the liver into the bile system
and from there into the
intestine, it may spill into the
bloodstream, thereby giving
rise to jaundice and a direct
bilirubin that is more than
15% of the total bilirubin.
 Hyperbilirubinemia Due to Impaired Excretion of
Conjugated Bilirubin: Hepatic jaundice

Hepatic jaundice is linked with damages of hepatocytes (in acute

viral infections, chronic and toxic hepatitis).

The liver cells undergo degradation, the excretion of conjugated bilirubin in

the bile capillaries is disturbed, and this bilirubin is excreted directly in the
blood stream:
•In blood both direct and indirect bilirubins are increased.
•Bilirubin is detected in the urine.
•The amount of stercobilin in feces is decreased.
 Hyperbilirubinemia Due to Impaired Excretion of
Conjugated Bilirubin: Cholestasis (Obstructive jaundice)
Intrahepatic cholestasis may be due to:
•primary biliary cirrhosis, an autoimmune disease seen in about 0.1% of women
over 40 years of age;
•primary sclerosing cholangitis (suspected to be of autoimmune origin) that causes
persistent inflammation and stricturing of bile ducts.

Extrahepatic cholestasis
is due to the physical
obstruction of the
common bile duct. The
obstruction may be
created by gallstones or
tumors (e.g., pancreatic
head cancer, carcinoma
of the ampulla of Vater)
that obstruct the bile
ducts.
 Hyperbilirubinemia Due to Impaired Excretion of
Conjugated Bilirubin: Dubin-Johnson Syndrome
Dubin-Johnson syndrome is due to a hereditary deficiency in the
transporter that excretes conjugated bilirubin into the bile canaliculi.

This transporter is variably called the multidrug resistance-associated

protein 2
(MRP2) or the ATP-binding cassette subfamily C member 2.

Dubin-Johnson syndrome is inherited in an autosomal recessive fashion.

Prevalence is unclear but is thought to be much less than 1 in 1,000.

It is reasonable to suspect Dubin-Johnson syndrome (or another heritable

problem of bilirubin excretion) when laboratory data show direct
hyperbilirubinemia but no abnormalities in other parameters that reflect
liver function (e.g., enzymes, coagulation factors, or albumin).
 Hyperbilirubinemia Due to Increased
Degradation of Heme or Due to Inadequate
Conjugation of Bilirubin
Excess production of bilirubin
(BR) and inadequate conjugation
of bilirubin can each lead to
indirect hyperbilirubinemia
in which the direct bilirubin is
less than 15% of the total
bilirubin.
 Hyperbilirubinemia Due to Increased
Degradation of Heme: Hemolytic jaundice
Increased degradation of heme and subsequent increased production of
bilirubin can be due to ineffective erythropoiesis or hemolytic disease.

Ineffective erythropoiesis is associated with the destruction of heme-

containing red blood cell precursors (i.e., polychromatophilic
erythroblasts and reticulocytes).
It is seen in:
• lead poisoning,
• congenital erythropoietic porphyria
• thalassemia major (due to a problem with globin synthesis),
• megaloblastic anemia (due to a problem with DNA replication).

Hemolytic disease is due to the destruction of red blood cells:

• sickle cell anemia, Indirect bilirubin in blood is
• glucose 6-phosphate dehydrogenase deficiency, increased.
Urobilun in urine is enhanced.
• pyruvate kinase deficiency Stercobilin in feces is also
increased.
Bilirubin in urine is absent.
 Hyperbilirubinemia Due to Inadequate
Conjugation of Bilirubin: Neonatal Jaundice
Newborns (especially premature babies) often have
insufficient amounts of bilirubin UDP-glucuronosyl
transferase, since the liver starts synthesizing this
enzyme only around the time of birth.
As a result, these newborns have an abnormally high
concentration of bilirubin in the blood, and they
have neonatal jaundice.
Albumin acts as a buffer for (unconjugated)
bilirubin. For this reason, a total bilirubin
concentration in blood plasma that does not rise
beyond 20 mg/dL is usually safe. For comparison, a
healthy 3-day-old baby has a bilirubin concentration
of ~6 mg/dL.
Kernicterus (acute bilirubin encephalopathy) is a
severe form of jaundice in which aggregates of
(unconjugated) bilirubin and phospholipids form on
the membrane surfaces of neurons, predominantly
Kernicterus can occur at a high
concentration of unconjugated in the basal ganglia, which initiates an
bilirubin (generally >20 mg/dL). encephalopathy that may cause permanent brain
damage (especially to the auditory system) or death.
 Hyperbilirubinemia Due to Inadequate
Conjugation of Bilirubin: Neonatal Jaundice

In babies with hyperbilirubinemia, the physician must be

careful not to introduce drugs (e.g., sulfonamides) or fatty
acids that bind to albumin and thereby displace
(unconjugated) bilirubin.

The physician also must be careful not to introduce drugs

(e.g., steroids) that are detoxified by UDP-glucuronosyl
transferase, the enzyme that conjugates bilirubin.
 Hyperbilirubinemia Due to Inadequate
Conjugation of Bilirubin: Neonatal Jaundice
Newborns with very high
concentrations of (unconjugated)
bilirubin can be treated in three main
ways:
(1) exchange transfusion can be
used to remove bilirubin;
(2) an infusion of albumin can help
bind some bilirubin, but it may also
increase blood pressure;
(3) most importantly, phototherapy
(“bili-lights”) can be used to
isomerize bilirubin in the skin in a
water-soluble form.
 Hyperbilirubinemia Due to Inadequate
Conjugation of Bilirubin: Crigler-Najjar Syndrome
Crigler-Najjar syndrome is due to a very rare, inherited deficiency of bilirubin UDP-
glucuronosyl transferase.
In type I disease, the enzyme deficiency is nearly complete, and in the absence of
treatment, patients die from kernicterus within a few years of life. The
lifelong, extreme hyperbilirubinemia requires daily treatment with bili-lights.
Bili-lights are less effective in adults than in babies because of less translucent
skin and a smaller surface/volume ratio. Replacement of the patient’s liver
with a transplanted normal liver (i.e., orthotopic liver transplantation) can
restore normal bilirubin concentrations but is irreversible and requires
immunosuppression.
In type II disease, the deficiency is only partial, and the affected patients develop
kernicterus only episodically during trauma or sepsis. Early identification of
these patients helps prevent brain damage because bili-lights can be used
promptly.
 Hyperbilirubinemia Due to Inadequate
Conjugation of Bilirubin: Gilbert Syndrome
Gilbert syndrome, seen in about 9% of the population, is due to a hereditary,
marked deficiency of bilirubin UDPglucuronosyl-transferase.

The deficiency is much less severe than what is seen in Crigler-Najjar syndrome.

The disease reduces enzyme gene expression to approximately one-third of the

normal levels.

Neonates with Gilbert syndrome have more pronounced and longer-lasting

hyperbilirubinemia than neonates normally do.

Children and adults with Gilbert syndrome show hyperbilirubinemia in the

absence of liver disease (i.e., liver enzymes and coagulation are normal).
Hyperbilirubinemia develops predominantly during periods of high rates of
heme degradation (e.g., during illness or stress, or after alcohol consumption;
mostly in males, possibly due to higher turnover of hemoglobin).

Ongoing therapy is not necessary.