These topics are closely related, because heme is synthesized

from porphyrins and iron, and the products of degradation of

heme are the bile pigments and iron.

Knowledge of the biochemistry of the porphyrins and of heme

is basic to understanding the varied functions of
hemoproteins in the body.

The porphyrias are a group of diseases caused by

abnormalities in the pathway of biosynthesis of porphyrins.

Jaundice, due to elevation of bilirubin in the plasma

METALLOPORPHYRINS
& HEMOPROTEINS ARE
IMPORTANT IN NATURE
Hemeproteins have diverse biological functions including:
1. Oxygen transport:

hemoglobin
myoglobin
neuroglobin
cytoglobin
leghemoglobin
2. Catalysis
Cytochrome P450s
cytochrome c oxidase
ligninases
peroxidases
3. Electron transfer/transport
cyctochrome a
Cytochrome b
cytochrome c

4. Sensory
FixL (Oxygen sensor)
Soluble guanylyl cyclase
CooA (CO sensor)

5. Defence
catalase
Quaternary structure of deoxy- and
oxyhemoglobin

T-state R-state
The visible
absorption spectra
for hemoglobin
The red color arises from the
differences between the
energy levels of the d orbitals
around the Ferrous atom.
There is an energy difference
between them, which
determines the size of the
wavelength of the maximal
absorbance band.
Fe(II) = d6 electron
configuration: Low spin state
Binding of oxygen
rearranges the electronic
distribution and alters the
d orbital energy.
This causes a difference
in the absorption spectra.
Bluish for deoxy Hb
Redish for Oxy Hb
Measuring the absorption
at 578 nM allows an easy
method to determine the
percent of Oxygen bound
to hemoglobin.
 Function of the globin

Protoporphyrin binds oxygen to the sixth

ligand of Fe(II) out of the plane of the heme.
The fifth ligand is a Histidine, F8 on the side
across the heme plane.
His F8 binds to the proximal side and the
oxygen binds to the distal side.
The heme alone interacts with oxygen such that
the Fe(II) becomes oxidized to Fe(III) and no
longer binds oxygen.
It is estimated that 1 g of hemoglobin yields 35 mg of bilirubin. The
daily bilirubin formation in human adults is approximately 250350 mg,
deriving mainly from hemoglobin but also from ineffective
erythropoiesis and from various other heme proteins such as
cytochrome P450.

The chemical conversion of heme to bilirubin by reticuloendothelial

cells can be observed in vivo as the purple color of the heme in a
hematoma is slowly converted to the yellow pigment of bilirubin.
Bilirubin formed in peripheral tissues is transported to the liver by
plasma albumin.

The further metabolism of bilirubin occurs primarily in the liver. It can

be divided into three processes: (1) uptake of bilirubin by liver
parenchymal cells, (2) conjugation of bilirubin with glucuronate in the
endoplasmic reticulum, and (3) secretion of conjugated bilirubin into
the bile. Each of these processes will be considered separately.
 THE LIVER TAKES UP BILIRUBIN

In 100 mL of plasma, approximately 25 mg of bilirubin can be

tightly bound to albumin at its high-affinity site.
Bilirubin in excess of this quantity can be bound only loosely and
thus can easily be detached and diffuse into tissues.

A number of compounds such as antibiotics and other drugs

compete with bilirubin for the highaffinity binding site on albumin
Thus, these compounds can displace bilirubin from albumin and
have significant clinical effects.
In the liver, the bilirubin is removed from albumin and taken
up at the sinusoidal surface of the hepatocytes by a carrier-
mediated saturable system.

This facilitated transport system has a very large capacity, so

that even under pathologic conditions the system does not
appear to be rate-limiting in the metabolism of bilirubin.

Once bilirubin enters the hepatocytes, it can bind to certain

cytosolic proteins, which help to keep it solubilized prior to
conjugation.

Ligandin (a member of the family of glutathione S

transferases) and protein Y are the involved proteins. They
may also help to prevent efflux of bilirubin back into the blood
stream.
Conjugation of Bilirubin with Glucuronic Acid
Occurs in the Liver

Conjugation of bilirubin with glucuronic acid. The glucuronate donor, UDP-

glucuronic acid, is formed from UDP-glucose as depicted. The UDP-
glucuronosyltransferase is also called bilirubin-UGT.
Bilirubin Is Secreted into Bile
Secretion of conjugated bilirubin into the bile occurs by an
active transport mechanism, which is probably rate-limiting for
the entire process of hepatic bilirubin metabolism.
The protein involved is MRP-2 (multidrugresistance-like
protein 2), also called multispecific organic anion transporter (MOAT)
It is located in the plasma membrane of the bile canalicular
membrane and handles a number of organic anions.
It is a member of the family of ATP-binding cassette (ABC)
transporters. The hepatic transport of conjugated bilirubin into the
bile is inducible by those same drugs that are capable of
inducing the conjugation of bilirubin. Thus, the
conjugation and excretion systems for bilirubin behave as a
coordinated functional unit.
Diagrammatic representation of the three major processes (uptake, conjugation, and secretion)
involved in the transfer of bilirubin from blood to bile. Certain proteins of hepatocytes, such as
ligandin (a member of the glutathione S transferase family of enzymes) and Y protein, bind
intracellular bilirubin and may prevent its efflux into the blood stream. The process affected in a
number of conditions causing jaundice is also shown
 Conjugated Bilirubin Is Reduced to
Urobilinogen by Intestinal Bacteria
As the conjugated bilirubin reaches the terminal ileum and the large intestine,
the glucuronides are removed by specific bacterial enzymes ( -glucuronidases),
and the pigment is subsequently reduced by the fecal flora to a group of
colorless tetrapyrrolic compounds called urobilinogens.

In the terminal ileum and large intestine, a small fraction of the urobilinogens is
reabsorbed and reexcreted through the liver to constitute the enterohepatic
urobilinogen cycle. Under abnormal conditions, particularly when
excessive bile pigment is formed or liver disease interferes with this
intrahepatic cycle, urobilinogen may also be excreted in the urine.

Normally, most of the colorless urobilinogens formed in the colon by the fecal
flora are oxidized there to urobilins (colored compounds) and are excreted in the
feces. Darkening of feces upon standing in air is due to the oxidation of
residual urobilinogens to urobilins.
HYPERBILIRUBINEMIA CAUSES JAUNDICE
When bilirubin in the blood exceeds 1 mg/dL (17.1 mol/L),
hyperbilirubinemia exists.

Hyperbilirubinemia may be due to the production of more

bilirubin than the normal liver can excrete, or it may result from
the failure of a damaged liver to excrete bilirubin produced in
normal amounts. In the absence of hepatic damage, obstruction
of the excretory ducts of the liverby preventing the excretion of
bilirubinwill also cause hyperbilirubinemia. In all these
situations, bilirubin accumulates in the blood, and when it
reaches a certain concentration (approximately 22.5 mg/dL), it
diffuses into the tissues, which then become yellow. That
condition is called jaundice or icterus.
A method for quantitatively assaying the bilirubin content of
the serum was first devised by van den Bergh by application
of Ehrlich's test for bilirubin in urine.

Bilirubin that would react without the addition of methanol was thus
termed "direct-reacting." It was then found that this same direct
reaction would also occur in serum from cases of jaundice due to
biliary obstruction.

It was still necessary to add methanol to detect bilirubin in normal

serum or that which was present in excess in serum from cases of
hemolytic jaundice where no evidence of obstruction was to be
found. To that form of bilirubin which could be measured only after
the addition of methanol, the term indirect-reacting" was applied.
Depending on the type of bilirubin present in plasmaie,
unconjugated or conjugatedhyperbilirubinemia may be classified
as: retention hyper b.u, due to overproduction, or regurgitation
hyper b.u, due to reflux into the bloodstream because of
biliary obstruction.
Separation & quanti of unconjugated bilirubin and the conjugated
species can be performed using HPLC. Because of its
hydrophobicity, only unconjugated bilirubin can cross the blood-
brain barrier into the CNS, thus, encephalopathy due to
hyperbilirubinemia (kernicterus) can occur only in connection
with unconjugated bilirubin, as found in retention hyper b.u. On the
other hand, because of its watersolubility, only conjugated bilirubin
can appear in urine. Accordingly, choluric jaundice (choluria is the
presence of bile pigments in the urine) occurs only in regurgitation
hyperbilirubinemia, and acholuric jaundice
occurs only in the presence of an excess of unconjugated bilirubin.
Elevated Amounts of Unconjugated Bilirubin in
Blood Occur in a Number of Conditions
HEMOLYTIC ANEMIAS
NEONATAL "PHYSIOLOGIC JAUNDICE
Type I & II CriglerNajjar syndrome. mutasi UGT
GILBERT SYNDROME
TOXIC HYPERBILIRUBINEMIA

Obstruction in the Biliary Tree Is the Most Common

Cause of Conjugated Hyperbilirubinemia
DUBINJOHNSON SYNDROME ..mutasi MRP2
ROTOR SYNDROME . ?
Some Conjugated Bilirubin Can Bind Covalently to Albumin
Urobilinogen & Bilirubin in Urine Are Clinical Indicators
BILE
Bile acids as signalling molecules
The nuclear hormone receptors (NHR) belong to a family of proteins that, upon
binding an appropriate ligand, can activate or suppress gene expression.

Class II NHRs function as heterodimers in which :

the common retinoid X receptor RXR complexes with a partner
that can be farnesoid X receptor (FXR),
constitutive androstane receptor (CAR),
pregnane X receptor (PXR),
the liver X receptor (LXR),
the retinoic acid receptor (RAR),
the peroxisomal proliferatoractivated receptor (PPAR) or
the vitamin D receptor (VDR)

Drugs, bile acids and intermediates of bile acid biosynthesis,

the oxysterols, are major ligands for these NHRs.
LIVER
 FUNCTIONS OF LIVER
Storage, metabolism and release of nutrients and some
vitamins.

Detoxification and elimination of toxins, drugs and

metabolites.

Synthesis of biologically important protein such as

albumin, clotting factors, apolipoproteins.

Synthesis and secretion of bile important for lipid

digestion and absorption.

Role in immune function and clearance of intestinally

absorbed bacteria.
Cellular Components of the Liver
Hepatocytes
Stellate or Ito Cells
Kupfer Cells (macrophages)
Sinusoidal Endothelial Cells
Bile Ductular Epithelial Cells
(Cholangiocytes)
Anatomical Relationships in Liver
blood sinusoid

paracellular
space of Disse
pathway

bile tight
canaliculus junctions

hepatocyte

blood sinusoid
Components of Bile

Water ~ 1 liter/day
Bile Acids - major organic constituents
Phospholipids
Cholesterol
Bile pigments - bilirubin
Metabolites of Hormones, Drugs
Inorganic ions - HCO3 from duct cells
Transport of Biliary Components by Hepatocytes

Sinusoid Hepatocyte Canaliculus

Bilirubin Bilirubin
OATP Glucuronide
Na +
MRP2
Bile Salts
Bile
Salts
BSEP
NTC
Na +

P Bile Salts
K+ ABC5/8 Cholesterol
Phospholipid
Cholesterol
Na+ Bilirubin
Electrolytes
Water
MDR3

Na+

.
 MOLECULAR COMPONENTS
OF BILE SECRETION
1. Uptake of bile salts by Na+ coupled co-transporter (NTCP)

2. Basolateral membrane also contains several organic anion

transport proteins (OATP)

3. Bile salts excreted into bile by the Bile Salt Export Protein
(BSEP) an ATP binding cassette protein which belongs to
multidrug resistance (MDR) gene family

4. Other apical proteins are MRP2 which transport a number

of drugs, ABC 5/8 involved in cholesterol transport and
MDR3 a phospholipid transporter (flipase)

5. Gene expression of many of above transporters is

regulated by bile salts through the Bile Acid
Receptor/Farnesoid X Receptor (FXR)
 Function of Bile Ducts

1. Bile ducts are lined by cholangiocytes, columnar epithelial cells

specialized to modify bile.

2. Ductules are freely permeable to water so bile rapidly becomes

isotonic.

3. Ductules scavenge solutes such as glucose and amino acids that

entered leaky canaliculus.

4. Cholangiocytes secrete HCO3- in response to secretin (bile slightly

alkaline)

5. Ductules secrete IgA molecules into bile

Chemistry of Bile Acids

17

27

Cholesterol

12 24

3 7

Bile acid
(Cholic acid)

John Williams
 BILE SALT SYNTHESIS

Bile acids synthesized in the liver from

cholesterol

In the classical pathway the first and most

important regulated step is 7 hydroxylation
by 7 hydroxylase (CYP7A1)

Next 12 hydroxylation is followed by several

steps leading to cholic acid

The alternative pathway starts with initial

formation of oxysterols and leads to
chenodeoxycholic acid
 Primary Bile Acids

12 24 24

3 7 3 7

Cholic acid Chenodeoxycholic acid

(3 OH groups) (2 OH groups)

7-dehydroxylation
by gut bacteria

Secondary Bile Acids

12 24 24

3 3

Deoxycholic acid Lithocholic acid

(2 OH groups) (1 OH group)

John Williams
 Bile Acid
Conjugation
Cholic acid

Glycine
Bile Acids + OR

Taurine
Conjugation lowers the pKa
so bile acids exist in the more
soluble dissociated form
OR

Glycocholic acid
OR
Taurocholic acid

John Williams
Amphipathic Nature of Bile Acids

Glycocholic acid

Polar groups
3 7 12
COOH

John Williams
Amphipathic Molecules

Lecithin

Cholesterol Bile Salt

Oil

Water

Exist in micelles when

concentration is greater
than the CMC (Critical
Micellar Concentration)
Micelle

Source Undetermined
 Mixed Micelle
cholesterol lecithin

bile
salt

Source Undetermined
 GALLBLADDER FUNCTION

The gallbladder concentrates bile and stores

much of the bodies bile salt pool during fasting

Epithelia absorbs Na+, Cl- and H2O

Isotonicity maintained as a result of micelles

having minimal osmotic activity

Postprandially the gallbladder contracts in

response to CCK (cholecystokinin)
Role of CCK in Bile Secretion
Duodenum

FATTY ACID

CCK SECRETION

PLASMA CCK

Gallbladder Sphincter of Oddi

CONTRACTION RELAXATION

BILE FLOW INTO BILE FLOW

COMMON BILE DUCT INTO DUODENUM

John Williams
ENTEROHEPATIC CIRCULATION OF BILE SALTS
Portal Vein

Bile Salt

Liver
Hepatocyte Cholesterol

Bile Salt

Gallbladder Enterohepatic
Circulation

Concentrated
Bile Bile Duct

Salt contract
& gallbladder
Water
relax sphincer
of Oddi
CCK
Jejunum Ileum

Intestine Na+
Bile Salt Bile Salt
Fat

John Williams
Metabolism of Bile Pigment (Bilirubin)
LIVER
SPLEEN
Senescencent
BR + UDPGlucuronic rbc destruction
Acid
Fe2+ + Globin
Hemoglobin
BR Glucuronide
Systemic Circulation Biliverdin
BR-Albumin Complex Bilirubin (BR)

BILE DUCT

COLON
SMALL INTESTINE
BR Urobilinogen Stercobilin

Stool
KIDNEY

Urine

John Williams
 The Liver Synthesizes a Variety of Plasma Proteins

Major Plasma Proteins Albumin, fetoprotein, 2-Microglobulin

Hemostasis Proteins Fibrinogen, clotting factors and inhibitors

Plasmin (fibrinolysis)
Complement C3

Binding Proteins Ceruloplasmin (copper)

Steroid binding proteins
Thyroid hormone binding globulin (TBG)
Transferrin

Prohormones Angiotensinogen

Apolipoproteins Apo A-I, -II, and IV

Apo B-100
Apo D, Apo E
 THE LIVER METABOLIZES AND EXCRETES
FOREIGN MOLECULES (Drugs, Xenobiotics)

1. Most often involves oxidation (hydroxylation) and/or conjugation

2. Most hydroxylation is by family of enzymes termed cytochromes P450

a. Now usually referred to as CYPs. Three main gene families

CYP1, CYP2, and CYP3

b. Genetic and nongenetic factors influence P450 activity

3. Conjugation is by UDP glucuronyltransferases (glucuronic acid),

glutathione S-Transferase (glutathione) and sulfotransferases
(sulfate)

4. Metabolites which are more water soluble are secreted into bile or
plasma where can be excreted by kidney.
 Bile acids are formed from
cholesterol:

About 1 gram of cholesterol is

eliminated from the body per day.
Approximately half is excreted in the
feces after conversion to bile acids.
The remainder is excreted as
cholesterol.
The primary bile acids are synthesized
in the liver from cholesterol. These are
cholic acid and chenodeoxycholic acid.
 CLINICAL ASPECTS:
Serum cholesterol is correlated with the incidence
of atherosclerosis and coronary heart disease.

Changes in diet play an important role in

reducing serum cholesterol: Substitution in the
diet of polyunsaturated and monosaturated fatty
acids for some of the saturated fatty acids is most
beneficial. Sunflower, cottonseed, corn, olive, and
soybean oil contain high concentration of
monounsaturated fatty acids. On the other hand,
butterfat, beef fat, and palm oil contain a high
proportion of saturated fatty acids.
Lifestyle affects the serum cholesterol level:
 When diet changes fail, hypolipidemic drugs will
reduce serum cholesterol and triacylglycerol:
Cholestyramine resins: Block reabsorption of bile
acids.
Sitosterols: acts by blocking the absorption of
cholesterol from the gastrointestinal tract.
Mevocore or lovastatin: inhibitors of HMG-CoA
reductase
Clofibrate or gemfibrozil: exert their effect by
diverting esterification to oxidation of free fatty
acids.
Probucol: increase LDL catabolism via receptor
independent pathway, prevents oxidation of LDL
Nicotinic acid: reduces the flux of FFA by inhibiting
adipose tissue lipolysis, thereby inhibiting VLDL
production by the liver.