Hereditary Jaundice Cap125

Chapter 125: Hereditary Jaundice and Disorders of Bilirubin Metabolism
PART 13: PORPHYRINS

Jayanta Roy Chowdhury, Allan W. Wolkoff, Namita Roy Chowdhury, Irwin M. Arias
Abstract
1. Bilirubin is an orange pigment derived from the degradation of the heme moiety of hemoproteins,
particularly the hemoglobin of mature circulating erythrocytes.
2. Bilirubin is a potentially toxic waste product that is normally rendered harmless by binding to serum
albumin, conjugation in the liver, and efficient excretion into bile by the liver. Bilirubin is an
antioxidant, and a protective role of bilirubin against oxidant damage has been suggested. On the
other hand, patients with profound unconjugated hyperbilirubinemia are at risk for bilirubin
encephalopathy (kernicterus). Accumulation of bilirubin in plasma and tissues results in jaundice,
which has attracted the attention of patients and clinicians since antiquity.
3. Following formation in the reticuloendothelial system, bilirubin is released into the circulation, where it
avidly binds to serum albumin and is rapidly cleared by the liver. Extraction of bilirubin from the
circulation is a specific hepatic function involving facilitated diffusion. Within the hepatocyte, bilirubin
binds to cytosolic proteins, primarily to glutathione-S-transferases. The water-insoluble bilirubin
molecule is transformed into polar bilirubin monoglucuronide and diglucuronide by the action of
bilirubin-UDP-glucuronosyltransferase (bilirubin-UGT) and is excreted into the bile canaliculus against
a concentration gradient by energy-consuming mechanisms.
4. Inherited disorders of bilirubin metabolism result in hyperbilirubinemia. These include disorders
resulting in predominantly unconjugated hyperbilirubinemia (Crigler-Najjar syndrome types I and II,
and Gilbert syndrome) and those resulting in predominantly conjugated hyperbilirubinemia
(Dubin-Johnson syndrome, Rotor syndrome, and benign recurrent intrahepatic cholestasis).
5. Bilirubin-UGT (protein/gene for UDP-glucuronosyltransferase 1, family member 1A equivalent to
bilirubin-UGT (UGT1A1)) and several additional isoforms of the UGT1A subfamily that mediate the
glucuronidation of other aglycone substrates are expressed from the locus UGT1A, located on
chromosome 2q37. Genetic lesions in any of the five exons that encode UGT1A1 may lead to
complete absence (Crigler-Najjar syndrome type I; MIM 218800) or incomplete deficiency
(Crigler-Najjar syndrome type II) of bilirubin glucuronidation. In contrast, Gilbert syndrome (MIM
143500) is associated with an abnormality of the TATAA box within the promoter region upstream to
exon 1 of UGT1A1 that results in reduced expression of structurally normal UGT1A1. Dubin-Johnson
syndrome (MIM 237500) is caused by a genetic abnormality of bile canalicular multispecific organic
anion transporter, which is involved in the excretion of many non–bile salt organic anions by an
adenosine triphosphate (ATP)-requiring active process. Dubin-Johnson syndrome is associated with
a characteristic accumulation of pigments in the liver and an abnormality of porphyrin metabolism in
which over 80 percent of urinary coproporphyrin is coproporphyrin I, as compared with less than 35
percent in normal individuals. Rotor syndrome (MIM 237450) is primarily a disorder of hepatic storage
and differs from Dubin-Johnson syndrome by the lack of hepatic pigmentation, urinary coproporphyrin
excretion pattern, and hepatic sulfobromophthalein (BSP) metabolism. Dubin-Johnson syndrome is
caused by genetic lesions of the gene MRP2 (also termed cMOAT). Other genetic abnormalities can
cause hyperbilirubinemia, secondary to structural abnormalities of the biliary system or derangement
of specific excretory functions of the bile canaliculus. These include progressive familial intrahepatic
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cholestasis, type I (MIM 211600) and benign recurrent intrahepatic cholestasis (MIM 243300), both of
which are associated with mutations of the protein/gene for familial intrahepatic cholestasis-1 (FIC1)
gene on chromosome 18q21. Progressive familial intrahepatic cholestasis type II (MIM 601847) is
characterized by abnormality of bile salt excretion and is associated with mutations of the gene
SPGP, which is located on chromosome 2q24. A third type of progressive familial intrahepatic
cholestasis (MIM 602347) involves mutations of the MDR3 gene, the products of which are needed
for phospolipid excretion in bile. Several heritable developmental disorders of the biliary system have
been described. Of these, Alagille syndrome (MIM 118450) has been found to be caused by lesions of
JAG1, a gene located on chromosome 20p12.
6. Crigler-Najjar syndrome types I and II have an autosomal-recessive pattern of inheritance. Patients
with Gilbert syndrome are homozygous for the specific promoter abnormality, but all subjects homozygous
for this genotype do not exhibit the clinical picture of Gilbert syndrome. Studies of urinary coproporphyrin
excretion reveal autosomal-recessive patterns of inheritance for Dubin-Johnson and Rotor syndromes.
7. Several animal models of inherited disorders of bilirubin metabolism are important in understanding
the pathophysiology of their human counterparts. These models include the Gunn rat (Crigler-Najjar
syndrome type I); the Bolivian population of squirrel monkeys (Gilbert syndrome); and mutant albino rats
with organic anion excretion defect (TR −\−), golden lion tamarin monkeys, and mutant Corriedale
sheep (Dubin-Johnson syndrome).
Bilirubin is an orange pigment derived from the degradation of heme proteins, particularly the hemoglobin
of mature circulating erythrocytes and hepatic hemoproteins. Bilirubin is a potentially toxic waste product
that is generally harmless because of binding to serum albumin. However, patients with profound
unconjugated hyperbilirubinemia are at risk for bilirubin encephalopathy (kernicterus).
Studies of bilirubin chemistry, synthesis, transport, metabolism, distribution, and excretion have attracted
the attention of generations of chemists, biologists, and clinical investigators. Because bilirubin is an
organic anion of limited aqueous solubility, it has proved to be a model for the study of the transport,
metabolism, and excretion of other biologically important organic anions. Defects in bilirubin formation or
disposal are usually manifested by hyperbilirubinemia and jaundice. A number of inherited disorders
affecting these pathways have been described in both humans and animals. Study of these disorders has
provided important information regarding normal and abnormal metabolic pathways.
Crigler-Najjar syndrome type I, a potentially lethal disorder, requires liver transplantation as a definitive
treatment, and transplantation of isolated hepatocytes also has been used. Development of strategies for
noninvasive therapy for this condition remains a therapeutic challenge and continues to stimulate
research.
BILIRUBIN
Formation of Bilirubin
Bilirubin is exclusively derived from heme. In humans, 250 to 400 mg of bilirubin is formed daily by the
breakdown of hemoglobin, other hemoproteins, and free heme. 1 Approximately 80 percent is derived from
the hemoglobin of senescent erythrocytes. 2 After injection of radiolabeled porphyrin precursors (glycine or
δ-aminolevulinic acid) in humans or rats, radioactivity is incorporated into bile pigments in two peaks 3
(Fig. 125-1). The first peak (early-labeled peak of bilirubin, ELB) appears within 3 days and contains an
initial component and a slow later phase. The initial component comprises two thirds of the ELB in
humans and is largely derived from hepatic hemoproteins such as cytochromes, catalase, peroxidase,
and tryptophan pyrrolase. 4 The labeled bilirubin that appears in bile within 15 min after administration of
the precursor may be derived from a rapidly turning over pool of free heme in the cytosol of hepatocytes
that is degraded without incorporation into hemoproteins. 5 Myoglobin has a relatively long half-life and is
an unlikely source. Induction of hepatic cytochrome P450 enhances the ELB. 6 The slower phase of the
ELB is derived from both erythroid and nonerythroid sources and is enhanced in conditions associated
with ineffective erythropoiesis, such as congenital dyserythropoietic anemias, megaloblastic anemias,
iron-deficiency anemia, and lead poisoning. 4 The ELB is also increased in erythropoietic porphyria 3 but
not in porphyria cutanea tarda 7 or acute intermittent porphyria. 8 The erythroid phase is increased in
accelerated erythropoiesis, probably because of intramedullary destruction of normoblasts, destruction of
reticulocytes in the peripheral circulation, and injury to reticulocytes during maturation. 9 δ-Aminolevulinic
acid is preferentially incorporated into hepatic hemoproteins. 10 A late-labeled peak appears at
approximately 50 days in rats and by 110 days in humans and is derived from the hemoglobin of
senescent erythrocytes.
Labeling of plasma bilirubin in UDP-glucuronosyltransferase–deficient rats (Gunn strain) after the injection
of [14C]glycine. The early (0 to 3 days) peak has an initial “sharp” and a slower component. [Reprinted
with permission from Robinson SH: In Stohlman F Jr (ed): Hemopoietic Cellular Proliferation. New York:
Grune & Stratton, 1970, p 180.]
In the liver, heme derived from exogenously administered hemoglobin is quantitatively converted to
bilirubin. 11 A portion of heme associated with hepatic hemoproteins may not be converted to bilirubin. 12
This suggests that exogenous heme and hepatocellular heme may be processed differently by the liver.
Mechanism of Opening of the Heme Ring.

Ferroprotoporphyrin IX is the heme prosthetic group (Fig. 125-2) in vertebrate hemoproteins. The
porphyrin ring is selectively cleaved at the α-methene bridge. The first step is catalyzed by microsomal
heme oxygenase and requires an electrophilic attack at Fe(II) by a reducing agent, such as NADPH and
oxygen. This reaction results in formation of α-oxyheme (Fig. 125-2). 13 Heme oxygenase activity is
highest in the spleen, which is involved in sequestration of senescent erythrocytes, and is enhanced in
hemolytic states. Within the liver, hepatocytes and Kupffer cells have heme oxygenase activity. In Kupffer
cells, the enzyme activity is comparable with that in the spleen. 14 Heme stimulates the synthesis of heme
oxygenase, 15 which enhances heme degradation, 16 suggesting that heme oxygenase is rate limiting in
heme oxidation and bilirubin formation. 16 However, evidence also has been presented supporting a
contrasting view that heme oxygenase is present in excess as compared with its substrate. 17 Heme
oxygenase has been purified from microsomal fractions of pig 18 and bovine 19 spleen and rat liver. 20 The
binding of purified heme oxygenase to heme requires heme iron and the propionic acid substituents in the
C6 and C7 positions. Protoporphyrins that contain other metals, such as tin, bind with even greater
affinity 19 but are not degraded by heme oxygenase. Thus, noniron hemes may serve as dead-end
inhibitors of heme oxygenase. 19
Mechanism of heme ring opening and subsequent reduction of biliverdin to bilirubin.
The second step in opening the heme ring involves oxidation by molecular oxygen and probably occurs
nonenzymatically. 21 Carbons at the angular positions of the porphyrin ring neighboring the α-methene
bridge are oxidized, and CO is eliminated. During this step, two oxygen atoms, derived from two different
oxygen molecules, are added. 22 These oxygen atoms appear as the lactam oxygens of biliverdin and
bilirubin. Release of iron occurs after addition of electrons, suggesting that conversion of ferric to ferrous
iron is required. 23 The resulting green pigment is biliverdin.
Conversion of Biliverdin to Bilirubin.

Biliverdin is readily excreted by the liver and is the major bile pigment in many amphibian, avian, and fish
species. However, in most mammals, biliverdin is converted to bilirubin, which requires several
energy-consuming metabolic steps for biliary excretion. The physiologic benefits of conversion of biliverdin
to bilirubin are not clear. Bilirubin is less polar than is biliverdin and crosses the placental membranes
more readily than does biliverdin. 24 However, some placentate animals, such as nutria and rabbits,
excrete biliverdin as the main bile pigment, 25 whereas many nonplacentate vertebrates, such as some
species of fish, excrete predominantly bilirubin in bile. 26 Bilirubin is an antioxidant and is thought to play
an important antioxidant defense for the body. 27–29 The antioxidant activity of bilirubin may be particularly
important during the neonatal period, when concentrations of other antioxidants are low in body fluids.
Conversion of biliverdin to bilirubin is catalyzed by a cytosolic enzyme, biliverdin reductase, which requires
NADH or NADPH for activity. 30, 31 Guinea pig liver biliverdin reductase is a 70-kDa protein. 32 Three
interconverting molecular forms of biliverdin reductase have been described in rat liver. 33
Because of the specificity of heme oxygenase for the α-carbon bridge, the most abundant bile pigment is
bilirubin IXα, and only minute amounts of non-α isomers (Fig. 125-3) have been detected in human and
animal bile. 34
Nonenzymatic cleavage of heme in vitro results in the formation of four isomeric forms of biliverdin owing
to the nonequivalence of the four methene bridge positions (α, β, γ, and δ). P, CH2CH2COOH.
Quantification of Bilirubin Production.

Bilirubin production reflects turnover of biologically important hemoproteins. At a steady-state condition of
blood hemoglobin levels, the rate of bilirubin production approximates the rate of heme synthesis. For
these reasons, the quantification of bilirubin production is important in clinical and physiologic
investigations. 35 Normally, bilirubin is almost quantitatively excreted in bile; therefore, bilirubin production
can be quantified in biliary excretion in animals, but this is not practical in humans. Bilirubin is converted to
urobilinogen by bacteria in the gastrointestinal tract, and fecal urobilinogen excretion approximates daily
bilirubin production, 36 although conversion to urobilinogen may not be quantitative.
In humans, bilirubin production is conveniently quantified from the turnover of radioisotopically labeled
bilirubin. Radiolabeled bilirubin bound to albumin is injected intravenously, blood samples are collected at
frequent intervals, and plasma bilirubin concentration and radioactivity are measured. 37 Plasma bilirubin
clearance (the fraction of plasma from which bilirubin is irreversibly extracted) is proportional to the
reciprocal of the area under the radiobilirubin disappearance curve. 38 Bilirubin removal is quantified as
the product of plasma bilirubin concentration and clearance. When plasma bilirubin concentrations remain
constant, removal of bilirubin equals the amount of newly synthesized bilirubin entering the plasma pool.
This method does not take into account a small portion of bilirubin that is produced in the liver and
excreted directly into bile without appearing in the circulation and, therefore, slightly underestimates
bilirubin production.
Bilirubin formation also can be quantified from carbon monoxide production. The subject is placed in a
closed rebreathing system to prevent CO excretion. CO production is calculated from the CO
concentration in the breathing chamber or from an increment in blood carboxyhemoglobin saturation. 39
This method assumes that body CO stores rapidly equilibrate, blood carboxyhemoglobin reflects total
body CO, and metabolism of CO is insignificant compared with its rate of production. However, under
certain circumstances, such as anoxia, assumption of a steady equilibrium of body stores of CO with
blood carboxyhemoglobin may not be correct. 40 CO production exceeds plasma bilirubin turnover by 12
to 18 percent. This discrepancy is partly due to a small portion of bilirubin produced in the liver and
excreted into bile without appearing in serum. A portion of CO in expired air may be produced from
nonheme sources, such as halogenated methane 41 and polyphenolic compounds, including
catecholamines. 42 A small fraction of the CO may be formed by intestinal bacteria. 43
Pharmacologic Inhibition of Bilirubin Production.

Administration of nonmetabolized dead-end inhibitors of heme oxygenase, such as tin-protoporphyrin or
tin-mesoporphyrin, results in marked inhibition of the enzyme activity in various organs. 19 A single dose of
tin-mesoporphyrin administered in neonates, shortly after birth, resulted in an average of 76 percent
reduction of serum bilirubin levels and abolished the need for phototherapy. 44, 45
Chemistry of Bilirubin
The systemic name given to bilirubin IXα is
1,8-dioxo-1,3,6,7-tetramethyl-2,8-divinylbiladiene-a,c-dipropionic acid. 4, 5 The gross chemical structure
(Fig. 125-3) assigned to bilirubin by Fischer and Plieninger 46 has been confirmed by x-ray diffraction
analysis (Fig. 125-4). 47 The bonds between pyrrolenone rings A and B (C4 to C5) and C and D (C15 to
C16) are in the Z or trans-configuration. The oxygen attached to the outer pyrrolenone ring is in a lactam
rather than lactim configuration. Titration of bilirubin in aqueous solutions suggests a pK value of 7.0 to
8.0. 48 Because bilirubin tends to form insoluble aggregates below pH 8.0, determination of pK by titration
of aqueous solutions of bilirubin may be misleading. 49 Studies using 13 C NMR spectra and potentiometric
and spectrophotometric titrations in aqueous solutions indicate that bilirubin has four acidic groups. The
pK value of the two carboxyl groups is 4.4 and that of the two lactam groups is 13.0. 49
X-ray crystallographic structure of bilirubin showing a ridge-tile configuration caused by internal hydrogen
bonding of the propionic acid carboxyls to the amino groups and the lactam oxygen of the pyrrolenone
rings of the opposite half of the molecule. The bonds between pyrrolenone rings A, B, C, and D are in the
Z (trans) configuration.
Physical Conformation and Solubility of Bilirubin IXα.

Crystallized bilirubin IXα-ZZ with two protonated carboxyl groups is virtually insoluble in water. At acidic,
neutral, or mildly alkaline pH, bilirubin can be extracted from aqueous solutions into water-immiscible
solvents such as chloroform, ethyl acetate, or methylethyl ketone. After intravenous injection, bilirubin
stains phospholipid membranes of brain and those covering adipose tissues. However, bilirubin is not truly
lipophilic. Determination of solubility in progressively nonpolar solvents indicates that bilirubin is readily
soluble in polar solvents, provided the intramolecular hydrogen bonds can be interrupted. 50 Bilirubin and
polar ligands, such as sulfonamides, share a binding site within a polar domain of albumin. 49, 51 Thus,
despite its insolubility in water at physiologic pH, bilirubin should be considered a relatively polar
substance, and its mechanism of toxicity may differ from that of truly lipid-soluble toxins, such as DDT. 51
Although both bilirubin and biliverdin have two propionic acid side chains, bilirubin IXα is less polar than is
biliverdin IXα at physiologic pH (Fig. 125-2). An explanation for this was suggested initially by Fog and
Jellum 52 and Kuenzle et al., 53 who proposed that bilirubin IXα may be internally stabilized by hydrogen
bonding between the carboxyl and the two external pyrrolenone rings (Fig. 125-5). X-ray diffraction
studies of crystalline bilirubin confirm hydrogen bonding between each propionic acid side chain and the
pyrrolic and lactam sites in the opposite half of the molecule. 47 The molecule takes the form of a ridge tile
in which the two dipyrrolic halves of the molecule lie in two different planes with an interplanar angle of 98
to 100 degrees (Fig. 125-4). The integrity of the hydrogen bonded structure requires the interpyrrolic
bridges at the 5 and 15 position of bilirubin to be in trans- or Z configuration. In nonpolar solvents, the
structure of unconjugated bilirubin oscillates between that shown in Fig. 125-5 and its mirror image. 54 A
similar conformation has been proposed for bilirubin dianions in aqueous solutions. 55 The hydrogen
bonded structure of bilirubin may explain many of its physicochemical properties. The two carboxylic
groups, all four NH groups, and the two lactam oxygens are engaged by hydrogen bonding, making the
molecule insoluble in water. Addition of methanol, ethanol, or 6 M urea disrupts the hydrogen bonds,
thereby making bilirubin water soluble and more labile. 56 The central methene bridge becomes accessible
to diazo reagents after disruption of hydrogen bonds, making bilirubin readily reactive to these reagents.
Ionic species of bilirubin. A. Internally hydrogen bonded form. B. Bilirubin acid with hydrogen bonds
disrupted. C. Bilirubin dianion.
Ultraviolet/Visible Absorption Spectra.

The position of the main absorption band (λ max ) of bilirubin depends on the bile pigment and the solvent.
Unconjugated bilirubin IXα has a λ max of 450 to 474 nm in most organic solvents (Fig. 125-2) and an
extinction coefficient (E max ) of 48.0 to 63.4/mM. In alkaline aqueous solutions, there is a 10- to 30-nm shift
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of the λ max toward shorter wavelengths (hypsochromic shift) and a weaker absorption band at 280 to
300 nm.
Helical Conformation of Bilirubin and Biliverdin.

Circular dichroism spectroscopy shows that bilirubin preferentially adopts a plus-helicity when bound to
human serum albumin, whereas biliverdin prefers a minus helicity. 57
Fluorescence.
Pure bilirubin does not fluoresce. When it is dissolved in detergent, albumin solution, or alkaline methanol,
an intense fluorescence is observed at 510 to 530 nm. 58 Determination of fluorescence of bilirubin can be
used for rapid quantification of blood bilirubin concentrations and the unsaturated bilirubin binding capacity
of albumin.
Geometric Isomerization and Cyclization.

As mentioned earlier (Fig. 125-3), the 5 and 15 bridges of bilirubin are in a trans- or Z configuration.
Exposure of circulating bilirubin to light changes the configuration of one or both of the interpyrrolic
bridges at the 5 and 15 positions to an E or cis-configuration. The resulting ZE, EZ, or EE isomers lack
one or more internal hydrogen bonds, are more polar than is bilirubin IXα-ZZ, and can be excreted in bile
without conjugation. 59 The vinyl substituent in the endovinyl half of bilirubin IXα-EZ is subsequently
cyclized with the methyl substituent on the internal pyrrole ring, forming the structural isomer
E-cyclobilirubin. 60 Although cyclization of bilirubin occurs at a slower rate than formation of configurational
isomers, because of the relative stability of cyclobilirubin, this form may be quantitatively more important in
phototherapy of neonatal jaundice. 60
Photooxidation and Degradation.

Whether in aqueous solution or bound to proteins or lipids, bilirubin undergoes gradual bleaching in light
and oxygen. 61 Bleaching results in formation of colorless fragments, chiefly maleimides and
propentdyopent adducts, owing to a self-sensitized reaction involving singlet oxygen. A small amount of
biliverdin is also formed by mechanisms that are not established. 61
Dipyrrolic Scrambling.
When bilirubin IXα is irradiated in deoxygenated aqueous solution, free radical disproportionation results
in formation of bilirubin IIIα and bilirubin XIIIα (Fig. 125-6), which are nonphysiologic symmetrical isomers
of bilirubin. 61 The reaction is faster in the presence of oxygen and is catalyzed by acid 61 and inhibited by
ascorbic acid.
Isomerization of unconjugated and conjugated bilirubin. Upper panel. Geometric isomerization: The bond
between pyrrolenone rings A and B or C and D can change into an E (cis) configuration, as shown here
on the left half of the bilirubin molecule, resulting in the EZ, ZE, or EE isomers. E configuration of the bond
between the pyrrolenone rings interferes with hydrogen bonding and renders the molecule relatively polar.
Middle panel. Nonenzyma...
Toxicity of Bilirubin
A toxic effect of bilirubin on the brain of neonates has been known for at least five centuries. 62 Yellow
discoloration of basal ganglia in babies with intense jaundice was described in 1847, and the term
kernicterus was coined to describe these changes in 1903. 63, 64
Biochemical Mechanisms of Bilirubin Toxicity.

Bilirubin inhibits DNA synthesis in a mouse neuroblastoma cell line. 65 Bilirubin also may uncouple
oxidative phosphorylation and inhibit adenosine triphosphatase (ATPase) activity of brain mitochondria. 66
Studies in vitro reveal that bilirubin inhibits hydrolytic enzymes, 67 dehydrogenases, 68 and enzymes
involved in electron transport. 69 In mutant rats (Gunn strain) with congenital nonhemolytic
hyperbilirubinemia, bilirubin inhibited RNA synthesis, protein synthesis, and carbohydrate metabolism in
brain and inhibited protein synthesis in liver. 70, 71 Bilirubin also decreased respiration of isolated brain
mitochondria, uncoupled oxidative phosphorylation, inhibited ATPase activity, and induced swelling in the
brain. 72–74 Bilirubin has been shown to inhibit protein kinase-mediated phosphorylation of neural
proteins. 75, 76 In a cell-free system, bilirubin irreversibly inhibited Ca 2+ -activated, phospholipid-dependent
protein kinase (protein kinase C) activity and cyclic adenosine monophosphate (cAMP)-dependent protein
kinase activity. 77 All toxic effects of bilirubin are reduced or reversed by albumin in vivo and in vitro.
Increased plasma concentrations of bilirubin increase the risk of bilirubin encephalopathy in newborn
babies. A serum concentration of 20 mg/dl is often quoted as the highest limit of safety, although
kernicterus can occur at lower serum bilirubin concentrations. 78 Individual differences in the susceptibility
to bilirubin encephalopathy is underscored by the finding that Gunn rats bred against different normal
background strains have similar serum bilirubin levels but markedly different incidences of kernicterus and
mortality. 79 Serum albumin concentrations, pH, and substances that compete for albumin binding are
important in the pathogenesis of bilirubin encephalopathy. 80
Clinical Features of Bilirubin Encephalopathy.

Kernicterus occurs in infants with severe unconjugated hyperbilirubinemia and in young adults with high
serum unconjugated bilirubin levels due to severe inherited deficiency of bilirubin-UGT activity.
Kernicterus usually presents between the third and sixth days of life, with poor feeding and feeble suck
reflex, high-pitched cry, hypertonia or hypotonia, reflex opisthotonus in response to a startling stimulus,
convulsion, incomplete Moro reflex, and instability of thermal regulation (hypothermia or hyperthermia). 78
This may progress to lethargy, atonia, and death. Long-term sequelae include delay in motor
development, chorioathetosis, asymmetric spasticity, sensorineural hearing loss, paralysis of upward
gaze, dental dysplasia, cognitive dysfunction, and mental retardation. The cochlear nucleus is particularly
sensitive to bilirubin-induced damage. The affected areas in the cochlear nucleus and superior olive are
innervated by large axosomatic end bulbs or caliceal endings. Cells receiving synaptic input from end
bulbs or calices appear to be early targets of bilirubin toxicity in the auditory system. 81 Brainstem auditory
evoked responses correlate well with plasma free bilirubin levels in humans 82 and with morphologic
changes in the cochlear nucleus in Gunn rat pups. 83 There is prolongation of interval between the
auditory stimulus and appearance of wave I (latency), indicating delayed auditory nerve conduction. The
intervals between wave I and wave III (reflecting the superior olive) and wave I and wave V (reflecting
inferior colliculus) are also prolonged (interpeak latency). This test may be made more sensitive by
recording binaural difference waves obtained by subtracting the sum of two monaural brainstem auditory
evoked potentials from a binaural brainstem auditory evoked potential. 84 Moderately high serum
unconjugated bilirubin levels may result in a higher incidence of impaired neurologic or intellectual
performance in later life. 85, 86 In some children with Crigler-Najjar syndrome type I there may be late
clinical presentation of bilirubin encephalopathy with cerebellar symptoms as the presenting feature. 87
Magnetic resonance imaging of the brain provides a sensitive means of evaluating kernicterus. 86–90
Abnormally high intensity signals are observed over the globus pallidus, particularly over the
posteromedial border. In many cases, increased intensity is also seen over the thalamus, internal capsule,
and hippocampi. These magnetic resonance imaging abnormalities are also found in several other types
of metabolic encephalopathy and must be interpreted in the context of the clinical presentation. Bilirubin
staining of the hippocampus, basal ganglia, and nuclei of the cerebellum and brainstem is observed in
infants who die from acute kernicterus. 91 In infants who die within 72 h after the onset of kernicterus,
there may be no cellular damage of the brain seen by light microscopy. Early histologic changes occur
after this period and include cytoplasmic degeneration, loss of Nissl substance, and fine vacuolation and
swelling of nuclear chromatin. 92 Evidence of cell death may be present. In children who die in the chronic
stage of the disorder, bilirubin staining is not found in the brain, 91 but focal necrosis of neurons and glia
are found. Gliosis of the affected areas occurs in later cases. 92
Role of the Blood–Brain Barrier.

Tight junctions between capillary endothelial cells and foot processes of astroglial cells restrict the
exchange of water-soluble substances and proteins between blood and brain. 93 In addition, specific
transport processes for ions, water, and nutrients from plasma to brain may provide a functional
blood–brain barrier. Conventionally, the immaturity of the blood–brain barrier in neonates has been
thought to contribute to kernicterus. However, a more rapid passage of labeled markers 94 or lipophilic
substances 95 into the immature brain has been difficult to confirm, and there is little evidence to support
the concept of immaturity of the blood–brain barrier in the neonate. Moreover, opening of the blood–brain
barrier is expected to permit both bilirubin and albumin to enter the brain. Because albumin binding
neutralizes the toxic effects of bilirubin, this should not result in increased bilirubin toxicity. Current
evidence indicates that the non–albumin-bound (free) fraction of bilirubin enters the brain, and such entry
is independent of the intactness of the blood–brain barrier. A special proclivity of the neonatal brain cells
to bind bilirubin may facilitate its retention in the brain of newborns with severe unconjugated
hyperbilirubinemia. Hyperosmolarity-associated shrinkage of capillary endothelial cells results in
temporary and reversible opening of the tight junctions. When the blood–brain barrier is opened in
newborn rats by infusion of hypertonic urea 96 or arabinose, 93 intravenously administered
albumin–bilirubin complex rapidly enters the brain. Following reversal of opening of the blood–brain
barrier, bilirubin is rapidly cleared from the brain in parallel with clearance from the serum, suggesting that
bilirubin is cleared by diffusion or transport back into the general circulation. 97 However, damaged and
edematous brain may bind bilirubin, 98 may be unable to clear it rapidly, and may be more vulnerable to
bilirubin toxicity for that reason.
Binding of Bilirubin to Albumin

In the circulation, bilirubin is tightly but reversibly bound to albumin. Albumin binding plays a critical role in
the disposition of bilirubin by the body. It keeps bilirubin in solution and transports the pigment from its
sites of production, primarily the spleen and the bone marrow, to its site of excretion, the liver. The tight
binding of unconjugated bilirubin to albumin prevents its excretion by the kidney, except during
albuminuria. Conjugated bilirubin is bound much less tightly to albumin, and the relatively larger unbound
fraction undergoes glomerular filtration and is excreted in the urine. During disease states that are
associated with prolonged increase in plasma-conjugated bilirubin levels, a fraction of the pigment
becomes irreversibly bound to albumin. 99 This fraction, termed δ-bilirubin, is not excreted in the bile or
urine and disappears slowly, reflecting the long half-life of albumin.
Albumin-binding protects against all known toxic effects of bilirubin, and a small unbound fraction of
bilirubin is thought to be responsible for its toxicity. Coadministration of equimolar amounts of albumin
protects against otherwise lethal effects of unconjugated bilirubin following intravenous injection in
puppies. 100
In normal plasma, bilirubin is bound to a primary binding site on albumin, almost exclusively as the
dianion. Normal molar concentration of albumin (500–700 µM) exceeds that of bilirubin (upper limit
17 µM). However, during neonatal jaundice, in patients with Crigler-Najjar syndrome, and occasionally in
acquired liver diseases, the molar ratio of unconjugated bilirubin to albumin can exceed 1. The molar
excess of bilirubin can be accentuated by reduction of serum albumin levels due to inflammatory states,
chronic malnutrition, or liver diseases. In these circumstances, bilirubin also can bind weakly to a second,
third, and a fourth site. However, such binding is much weaker, so the unbound fraction of bilirubin
increases sharply. Use of sulfonamides in newborn babies enhances bilirubin encephalopathy, 101 as a
result of dissociation of bilirubin by sulfonamide from its binding to albumin. 102 Infusion of albumin
increases the plasma bilirubin concentration because of transfer of bilirubin from tissues to plasma. 103
Because of the clinical importance of estimation of the unbound fraction of unconjugated bilirubin, the
binding of bilirubin to albumin has been evaluated by separating bound from free bilirubin by ultrafiltration,
ultracentrifugation, gel chromatography, affinity chromatography on albumin agarose polymers, dialysis,
and electrophoresis. Unbound bilirubin is rapidly destroyed by treatment with H 2 O 2 and horseradish
peroxidase, as compared with bound bilirubin. Binding of bilirubin to albumin induces bilirubin
fluorescence, circular dichroism, quenching of protein fluorescence, and a shift in the absorbance spectra.
In most studies, the primary binding constant at physiologic pH and temperature is slightly below 10 8 M −1 .
The binding constant for the secondary site is believed to be lower by one order of magnitude. 49, 104
Enzymatic hydrolysis and analysis of albumin covalently bound to bilirubin indicate that bilirubin binds to
lysine 240 in human albumin and to lysine 238 in bovine serum albumin. 105 Binding of other ligands to
albumin plays a major role in determining bilirubin binding capacity. The other ligand may bind at the
same site as does bilirubin, resulting in competitive displacement, or it may bind noncompetitively at a
different site. Noncompetitive binding may not affect bilirubin binding or may produce conformational
changes that enhance (cooperative binding) or decrease (anticooperative) bilirubin binding. Sulfonamides,
antiinflammatory drugs, and contrast media used for cholangiography displace bilirubin competitively from
albumin and increase the risk of kernicterus in jaundiced newborn babies. 106 Some benzodiazepine drugs
and long-chain fatty acids in low concentration bind to human albumin without affecting bilirubin
binding. 107, 108 Albumin binding of medium-chain fatty acids, such as laureate and myristate, increase the
binding constant for bilirubin. 109 Short-chain fatty acids bind to albumin anticooperatively with bilirubin. 110
When large amounts of fatty acid bind to albumin, major conformational changes occur that generally
decrease the binding of other ligands, including bilirubin. Acidosis increases the risk of brain damage in
neonatal jaundice 111, 112 but does not influence bilirubin binding to the primary site of albumin. The
increased risk of kernicterus may result from enhanced transport of bilirubin from plasma to selected
areas of the central nervous system. 49 Because of the influence of many metabolites and drugs on
albumin binding of bilirubin and on its transfer from plasma to the central nervous system, measurement
of plasma bilirubin concentration does not accurately estimate the risk of brain damage from unconjugated
bilirubin. It is generally believed, although it has not been verified, that unbound bilirubin is transferred
from plasma to the central nervous system. 102 Efforts have been made to quantify unbound bilirubin in
serum by gel chromatography, 113 peroxidase treatment, 114 electrophoresis on cellulose acetate, 115 and
fluorimetry of serum with or without detergent treatment. 116
Free bilirubin concentration is determined from the equilibrium equation
where [F] is the free bilirubin concentration, [B] is albumin-bound bilirubin concentration, [RA] is the
concentration of reserve bilirubin binding sites on albumin, and K is the association constant for bilirubin.
Equilibrium between free and bound bilirubin is assumed, and binding of bilirubin to tissues and secondary
binding sites on albumin are ignored. The numerical values for binding constants, as determined from
experiments with pure albumin and bilirubin, are assumed to be valid in serum. These assumptions may
not be valid with icteric serum, so it is not possible to calculate reliably the concentration of unbound
bilirubin. The alternative approach is to determine the amount of unoccupied bilirubin binding sites on
albumin. Titration of serum with bilirubin or a dye that binds to albumin has been used to estimate
unoccupied bilirubin binding sites.
Binding to secondary binding sites begins before primary sites are saturated, and some dyes bind at sites
other than the bilirubin site. Binding of bilirubin to erythrocytes depends on the albumin:bilirubin ratio in
serum and indirectly reflects reserve bilirubin binding sites on albumin. 117 Competitive binding by a
14 C-labeled ligand (monoacetyl-4,4′-diaminodiphenyl sulfone) 118 or a spin-labeled ligand [1-N-(2,2,6,6,
tetramethyl-1-oxyl-4-piperidinyl)5-N-(1-aspartate)-2,4,-dinitrobenzene] 119 has been used to determine

reserve binding capacity. A fluorimetric method has been described for determination of bound albumin
and reserve bilirubin binding capacity. 116 Despite inaccuracies, several empirical tests for determination
of reserve bilirubin binding capacity of serum albumin correlate clinically with brain damage 120 and may
be useful in assessing the risk of bilirubin toxicity.
Uptake of Bilirubin by the Liver

Although tightly bound to albumin, bilirubin is rapidly removed from the circulation by the liver (Fig. 125-7).
Kinetic studies of bilirubin uptake in isolated perfused livers of dogs 121 and rats 122 and in intact rats in
vivo 123 reveal that the process is saturable. Following an intravenous loading dose of bilirubin, the plasma
disappearance of a subsequent tracer dose of [ 3 H]-bilirubin is enhanced, 123 suggesting a
carrier-mediated facilitated diffusion. Countertransport of bilirubin (i.e., efflux of radiolabeled ligand from
liver after subsequent infusion with unlabeled ligand) has been claimed, 123 but the data also could be
interpreted to represent efflux of ligand from intracellular binding sites. Mutual competition for hepatic
uptake in vivo has been described with respect to bilirubin and other organic anions, such as indocyanine
green (ICG), 123, 124 sulfobromophthalein (BSP), 123, 124 and conjugated bilirubin. 124 Bile acids do not
compete with these compounds for hepatic uptake. 123, 125
Summary of hepatic metabolism of bilirubin (B). Bilirubin is strongly bound to albumin in the circulation.
This complex dissociates, and bilirubin enters hepatocytes by a specific uptake mechanism (1). A fraction
of the bilirubin is also derived from catabolism of hepatocellular heme proteins. Within the hepatocyte,
bilirubin binds to a group of cytosolic proteins, termed ligandins; this inhibits the efflux of bilirubin from the
cell. UDP-gl...
Bilirubin dissociates from albumin before entering the hepatocyte, and albumin does not accompany the
pigment into the hepatocyte. Five minutes after intravenous injection of a mixture of [ 3 H]-bilirubin and
131 I-labeled albumin into rats, approximately 60 percent of injected bilirubin is internalized by the liver,
whereas only 10 percent of the injected albumin is present in the liver, probably in the vascular space. 122
In isolated perfused rat and dog livers, simultaneous injection of [ 125 I]-albumin and [ 3 H]-bilirubin discloses
rapid bilirubin uptake, with no removal of albumin from the perfusate. 121, 126 It is not clear whether free or
albumin-bound bilirubin interacts with the hepatocyte. Early studies suggested that the unbound fraction of
bilirubin is taken up by the hepatocyte. 127 This view was challenged by investigators who found that
uptake of albumin-bound ligands by the perfused rat liver correlated poorly with the expected
concentration of unbound ligand. 128, 129 For example, increasing the albumin concentration tenfold from
0.5 g/dl to 5 g/dl reduced the concentration of free taurocholate by a factor of five, but reduced uptake by
only 50 percent. 129 Similar results were found for uptake of fatty acids, bilirubin, and BSP. 128, 130 In
addition, uptake of a 1:1 complex of one of these ligands with albumin was saturable and competitively
inhibited by albumin. 128 Based on these observations, it was suggested that albumin mediated hepatic
uptake of these ligands, 129 and the presence of a receptor for albumin on the liver cell surface was
postulated. 128 However, an alternative hypothesis to explain albumin receptor-like kinetics suggests that
the unbound ligand interacts with the liver cell plasma membrane, but the rate of dissociation of the ligand
from albumin may limit uptake. 131, 132 This new model of dissociation-limited uptake of bilirubin is
compatible with the existence of an albumin receptor and adequately describes BSP uptake in perfused
elasmobranch liver. 133
To evaluate the role of albumin binding on solute distribution within the zones of the hepatic acinus,
Gumucio et al. studied BSP transport in isolated rat liver perfused with 0.01 to 1.0 mM BSP with or without
albumin. 134 When steady-state conditions of BSP excretion were established, the liver was frozen and
relative BSP concentrations in various zones of the liver acinus were estimated. Without albumin, there
was 95 percent extraction of BSP in a single pass; a decreasing concentration gradient from zone 1
(periportal) to zone 3 (pericentral) was observed. Inclusion of 4.5 percent or 1 percent albumin in the
perfusate resulted in single-pass extraction of only 8 to 22 percent of BSP, and the zonal gradient of BSP
content was abolished. The results demonstrate that albumin binding produces more homogeneous
distribution of organic anions within the liver acinus. When the liver was perfused retrogradely (through the
hepatic vein) in the absence of albumin, BSP was taken up predominantly by hepatocytes of zone 3 and a
decreasing gradient from zone 3 to zone 1 was produced. BSP-glutathione conjugates appeared in bile
during antegrade and retrograde perfusion, indicating that hepatocytes of both zones have the ability to
conjugate and excrete BSP.
To elucidate the mechanism and driving forces responsible for hepatic organic anion uptake, a number of
studies have been performed on isolated rat hepatocytes 135–138 or liver sinusoidal vesicles. 139 These
experiments, conducted in the absence of albumin, suggested temperature-dependent,
sodium-independent uptake. Studies of [ 35 S]-BSP uptake in short-term cultured rat hepatocytes,
performed in the presence of a molar excess of bovine albumin, revealed linear uptake of BSP over at
least 15 min with little formation of its glutathione (GSH) conjugate over this time. 140 The initial uptake of
[ 35 S]-BSP was depressed by isosmotic substitution of NaCl by sucrose. A specific cation requirement for
BSP uptake is unlikely because uptake was unaffected by substitution for NaCl by KCl or LiCl. However,
substitution of Cl − by HCO 3 − or gluconate − markedly inhibited BSP uptake. Similar observations were
made for the uptake of [ 3 H]-bilirubin glucuronides. 141 Studies in rat liver perfused with NaCl − or Na
gluconate-substituted mediums revealed similar inhibition of bilirubin influx. 140 The mechanism by which
hepatocyte organic anion transport is stimulated by inorganic anions does not appear to be related to
transport of the inorganic anion in or out of the cell. 142 Studies using 36 Cl revealed that in short-term
cultured rat hepatocytes, BSP uptake requires external Cl − and is not stimulated by unidirectional Cl −
gradients. 142 Thus, there was no evidence for linkage of chloride transport with organic anion transport.
Studies of binding of [ 35 S]-BSP to hepatocytes at 4°C revealed an approximately tenfold higher affinity in
the presence of Cl − as compared with its absence. 142 Whether this is the complete explanation for
Cl − -dependent kinetics of BSP transport remains to be determined. In the transfer of organic anions from
the space of Disse to the hepatocyte, the liver cell plasma membrane is the first barrier to entry into the
cell and presumably is the site of interaction with organic anions that results in carrier-mediated uptake
kinetics. Although kinetic evidence has been presented that bilirubin has rapid transit across lipid
bilayers, 132, 143 the cellular specificity and driving forces of this event suggest a hepatocyte transporter for
this ligand.
Several studies of organic anion interaction with liver plasma membrane preparations have been
performed in an attempt to describe the nature of the putative carrier. A number of early studies
demonstrated saturable binding of BSP to liver plasma membrane. 143–145 Several putative plasma
membrane organic binding proteins were isolated. One isolated by Berk, Stremmel, and colleagues was
termed a “BSP/bilirubin binding protein.” 143, 146 These investigators reported that polyclonal antibodies to
this protein inhibited uptake of BSP and bilirubin by isolated rat hepatocytes. 147 However, interpretation of
these studies is difficult because a relatively large amount of antibody was needed for even a partial
inhibition of BSP uptake. A second protein termed bilitranslocase was isolated by Tiribelli, Sottocasa, and
colleagues. 148 Bilitranslocase is a 170-kDa protein composed of 37-kDa and 35-kDa subunits and has
been reported to reconstitute BSP transport in liposomes 148 and in erythrocyte membrane vesicles. 149 In
studies by Wolkoff and Chung, a photoaffinity probe was devised in which [ 35 S]-BSP was covalently
bound to liver cell plasma membrane after exposure to ultraviolet light. 144 Subsequent sodium dodecyl
sulfate polyacrylamide gel electrophoresis and fluorography revealed radioactivity predominantly
associated with a single 55-kDa protein. 150 This organic anion-binding protein was purified and
characterized immunologically. 144 It appears to differ from the BSP/bilirubin binding protein and
bilitranslocase. Using an antibody against this organic acid- binding protein to screen a rat liver λgt11
expression library, a 1550-bp complementary DNA (cDNA) was cloned. 151 On further characterization, it
was found that this cDNA encoded the β subunit of mitochondrial F 1 -ATPase.
Based on the characteristics of [ 35 S]-BSP extraction from albumin by hepatocytes, 140 an assay was
devised that enabled detection of transport in Xenopus laevis oocytes that had been injected with rat liver
poly(A)+ RNA. 152 These oocytes were able to extract BSP from albumin in a chloride-dependent fashion.
Subsequently, a single complementary RNA (cRNA) was isolated that when injected into oocytes resulted
in marked enrichment of BSP transport activity. The corresponding cDNA encodes a rat liver protein that
has been named organic anion-transporting polypeptide (oatp). The oatp cDNA contains an open reading
frame of 2010 nucleotides and suggests that oatp is a hydrophobic protein. The best computer-generated
model of oatp predicts that the protein has 12 transmembrane domains and three potential
N-glycosylation sites. Northern blot analysis revealed that oatp is expressed in the liver and kidney.
Recent data indicate that it is also present in the choroid plexus, a tissue that also expresses several
otherwise liver-specific proteins. At low stringency, oatp cDNA also hybridizes with messenger RNA
(mRNA) extracted from other organs, including lung, skeletal muscle, and proximal colon. Studies using
HeLa cells stably transfected with oatp indicate that oatp-mediated taurocholate transport is Na +
independent, saturable, and associated with HCO 3 − exchange. 153 However, the role of oatp or one of the
related proteins in bilirubin transport has not been directly established, and is being investigated. Oatp
appears to be the first member of a family of sodium-independent plasma membrane transport proteins.
Other members include transporters for prostaglandins, 154 digoxin, and folic acid.
Intrahepatocellular Storage of Bilirubin

Fifteen minutes following intravenous injection of [ 3 H]-bilirubin into rats, over 90 percent disappeared from
plasma and 25 to 30 percent of the injected dose remained in liver. 155 Radioactivity does not appear in
bile until 3 to 4 min after injection and subsequently appears at a rate of approximately 3 percent of the
injected dose per minute. 156 Thus, from the time bilirubin is cleared from plasma and subsequently
excreted into bile, it is stored within hepatocytes. At all times after intravenous injection, a large proportion
of [ 3 H]-bilirubin is associated with the cytosolic fraction of liver homogenates. 155, 156 Because the water
solubility of unconjugated bilirubin is very low at physiologic pH, it is kept in solution by binding to cytosolic
proteins. Gel filtration of cytosol containing [ 3 H]-bilirubin or [ 35 S]-BSP reveals that radioactivity is
associated with two protein peaks that were originally termed Y and Z (Fig. 125-8). 157 Tracer quantities of
anions bind almost exclusively to the Y protein; with larger amounts, binding to the Z protein becomes
apparent. 157 This suggests that, under physiologic conditions, Y protein is the principal cytoplasmic
protein to which organic anions bind. Purified Y protein was shown to bind many compounds, including
drugs, hormones, and organic anions. 157, 158 Similar proteins with the ability to bind a cortisol
metabolite 159 and an azo-dye carcinogen 160 were identified by various laboratories. These proteins were
found to be immunologically cross-reactive and were termed ligandin. Ligandin accounts for 5 percent of
liver cytosol proteins. 161 Subsequently, ligandin was found to be a family of proteins, identical to the α
class of glutathione-S-transferases in the rat liver. 162, 163 There are corresponding proteins in human
hepatocytes as well. These proteins bind bilirubin and a wide variety of organic anions as nonsubstrate
ligands. 163 The high affinity of these proteins for organic anions suggests that they may play a role in
transport by the liver. The role of ligandin in transport of bilirubin was studied in isolated perfused liver
from rats in which ligandin levels were increased following thyroidectomy or treatment with
phenobarbital. 164 There was no correlation between hepatic ligandin concentration and the influx rate of
bilirubin. However, the efflux rate of bilirubin from liver back to plasma varied inversely with hepatic
ligandin concentration. Thus, intracellular protein binding of bilirubin appears to play no role in its
extraction from serum albumin and subsequent influx into the hepatocyte. However, binding to ligandin
increases the net uptake of bilirubin by reducing efflux from the hepatocyte. With respect to organic anion
transport, ligandin may function within the hepatocyte much as albumin does in the circulation, binding
bilirubin and preventing efflux from the hepatocyte back into the circulation and nonspecific diffusion of
bilirubin into compartments of the hepatocyte in which it may do harm. This hypothesis is supported by the
finding that bilirubin inhibits mitochondrial respiration in vitro, an effect that is prevented by ligandin. 165
Binding of bilirubin to cytosolic proteins. Sephadex-G75 gel chromatography of 110,000 × g rat liver
supernatant to which [14C]bilirubin has been added reveals association of radioactivity with two protein
peaks, Y and Z. Y protein was determined to be quantitatively more important in organic anion binding
and was named ligandin. Subsequently, ligandin was found to consist of several proteins belonging to the
glutathione...
Conjugation of Bilirubin
Bilirubin Conjugates.
Efficient excretion of bilirubin across the bile canaliculus requires its conversion to polar conjugates by
esterification of the propionic acid carboxyl groups. Esterification of one or both propionic acid side chains
forms monoconjugates or diconjugates, respectively. Glucuronic acid is by far the major conjugating group
in normal mammalian bile pigments, 166 although smaller amounts of glucosyl and xylosyl conjugates are
also found. 167 Bilirubin glucuronides are present as monoconjugates and diconjugates (Fig. 125-9). 168
Bilirubin IXα is asymmetrical; therefore, bilirubin IXα monoglucuronide exists as two isomers, depending
on where the glucuronosyl group is attached. 168–171 Bilirubin diglucuronide is the major pigment in normal
human bile. 169–171
Bilirubin glucuronides. Both propionic acid side chains are glucuronidated in bilirubin diglucuronide.
Bilirubin monoglucuronide can exist as two molecular species, depending on whether the C12 or C8
propionic acid is conjugated.
Enzyme-Catalyzed Glucuronidation of Bilirubin.

Conjugation of bilirubin with glucuronic acid is catalyzed by a specific form of uridine
diphosphoglucuronate glucuronosyltransferase, abbreviated as UDP-glucuronosyltransferase or UGT.
Protein isolation 172–177 and molecular cloning studies 178–184 revealed that UGTs are a family of
enzymes 185 present in the endoplasmic reticulum and nuclear envelope of hepatocytes and many other
cells. 186 All UGT isoforms accept uridine diphosphoglucuronic acid as the donor substrate and use a
broad range of aglycone acceptor substrates; they catalyze the transfer of the glucuronic acid moiety from
uridinediphosphoglucuronic acid (UDP-glucuronic acid), forming ether, ester, thiol, and N-glucuronides. 187
Aglycone substrates of UGTs are diverse, including steroid hormones, thyroid hormones, bile salts,
neurotransmitters, bilirubin, and various exogenous substrates, such as drugs, environmental toxins, and
laboratory xenobiotics. UGTs are integral membrane proteins that require specific membrane lipids for
their function. 188, 189 Delipidation results in loss of enzyme activity, which is restored upon addition of
appropriate phospholipids. 188, 189 UGT activity in microsomal vesicles is partially latent. Membrane
perturbation by detergent treatment, sonication, or brief incubation with phospholipase A removes the
latency, and full activity of the enzyme is expressed. 188–191 UGT activity in native microsomes is also
enhanced by low concentrations of UDP-N-acetylglucosamine, which may be a physiologic activator of the

transferase. Two mechanisms have been proposed to explain this activation. In the compartmental model,
microsomal lipid membranes are thought to pose a partial barrier between UDP-glucuronic acid and the
catalytic site of UGT, which is thought to be located within the endoplasmic reticular lumen. The amino
acid sequences of UGTs have been interpreted to indicate a single transmembrane domain of the protein.
Transport of UDP-glucuronic acid into the endoplasmic reticulum cisternae by specific mechanisms has
been proposed. 192 This model also postulates the presence of a transporter in the microsomal
membranes, which facilitates access of UDP-glucuronic acid to the catalytic site of the transferase.
UDP-N-acetylglucosamine is envisioned as an activator of the permease. Membrane perturbation is
thought to enhance enzyme activity in vitro by increasing the permeability of the lipid membranes to
UDP-glucuronic acid. 193 The alternative hypothesis, termed the allosteric model, postulates that the
enzyme activity is constrained by the membrane. Activating agents release the enzyme from constraint
and thereby enhance enzyme activity. 194
Structure and Expression of UGT Isoforms.

UGT isoforms are expressed from multiple loci. The C-terminal domain of UGT isoforms have a high
degree of homology and is responsible for binding of the common donor substrate, UDP-glucuronic
acid, 195 whereas the N-terminal domains are more heterogeneous and account for the aglycone substrate
specificity of individual isoforms. 196 Based on the degree of homology of the mRNA sequences, UGT
genes have been classified among multiple families, and each family into several subfamilies. 185 Only
one UGT isoform is physiologically relevant in bilirubin glucuronidation. 196 The locus that expresses
bilirubin-UGT is termed UGT1A (Fig. 125-10). 197 This locus expresses several UGT isoforms and
contains four consecutive exons (exons 2–5) at the 3′ end that are used in all mRNAs expressed from this
locus. Upstream from these four common region exons are a series of unique exons, each preceded by a
separate promoter. Depending on which promoter is used, transcripts of various sizes are generated. The
unique exon, present at the 5′ end of the transcript, is spliced to exon 2, and the intervening sequence is
spliced out. Thus, all isoforms expressed from the UGT1A locus have identical C-terminal domains
(encoded by exons 2–5) but variable N-terminal domains (encoded by a single unique exon). Within the
UGT1A locus, genes encoding individual isoforms are named according to the unique exon used in the
expressed mRNA. Thus, the gene for the bilirubin-UGT mRNA, which consists of the first unique region
exon of the UGT1A locus (plus exons 2–5), is termed UGT1A1 according to current terminology, and the
enzyme is termed UGT1A1. Another isoform that catalyzes phenolic substrates is encoded by the unique
region exon 6 (plus exons 2–5); therefore, this gene is termed protein/gene for
UDP-glucuronosyltransferase 1, family member A6 (UGT1A6), and the expressed isoform is termed
UGT1A6.
Schematic representation of the human UGT1A locus, located at 2q37. This locus contains multiple genes
that express bilirubin-UGT and several other UGT isoforms. Exons 2, 3, 4, and 5, located at the 3′ end of
UGT1A, encode the identical C-terminal domains of all UGT isoforms expressed from this locus.
Upstream to these common region exons are a series of unique exons (exons 1A1 through 1A12), each of
which encodes the variable N-termi...
Differential Expression of UGT Isoforms.

The presence of a separate promoter upstream from each unique region exon (Fig. 125-10) permits
differential regulation of individual UGT isoforms during ontogenic development 198 and enzyme
induction. 199, 200 Enzyme activity toward 4-nitrophenol and other simple phenolic substrates (catalyzed by
UGT1A6) develops in late fetal life, whereas UGT activity toward bilirubin (UGT1A1) develops after
birth. 198 Treatment of rats with 3-methylcholanthrene induces UGT1A6. 199, 200 In contrast, UGT activity
toward bilirubin is specifically induced by clofibrate. 199 Treatment of rats with triiodothyronine results in a
threefold increase in transferase activity toward 4-nitrophenol, whereas activity toward bilirubin is
decreased by 80 percent. 198
Quantification of Bilirubin and Its Conjugates

Bile pigments are quantified in body fluids as native or derivatized tetrapyrroles, or after conversion to
azoderivatives. Total bilirubin also can be quantified indirectly by quantification of the intensity of yellow
discoloration of the skin.
Methods Involving Conversion of Bilirubin to azo Derivatives.

Conversion to azodipyrroles by reaction with diazo reagents is used commonly for determination of serum
bilirubin levels for clinical purposes. Reaction of bilirubin with diazo reagents begins with electrophilic
attack by a diazonium ion at the 9 and 11 positions of bilirubin 201 and converts 1 mole of the tetrapyrrole
to 2 moles of diazotized azodipyrrole and 1 mole of formaldehyde (Fig. 125-11). The azoderivatives are
more stable than is bilirubin, and its conjugates and can be quantified colorimetrically. Unconjugated
bilirubin is converted to two unconjugated dipyrroles. Bilirubin diconjugates form two conjugated
azodipyrroles, and bilirubin monoconjugates form one conjugated and one unconjugated azodipyrrole. In
1916, van den Bergh and Muller 202 showed that, on the basis of diazo reaction, serum bile pigments can
be classified into a direct and an indirect reacting species. The direct reaction occurs within minutes, and
the indirect reaction occurs rapidly only in the presence of accelerator substances such as methanol or
caffeine. Subsequently, the direct and indirect reacting components were identified as conjugated and
unconjugated bilirubin, respectively. 203 The basis of the direct and indirect van den Bergh reactions can
be understood from the crystal structure of bilirubin. Because of internal hydrogen bonding, the central
methene bridge of bilirubin is not readily accessible to diazo reagents. Addition of accelerators results in
disruption of the hydrogen bonds, and completes the reaction. Bilirubin glucuronides lack some or all of
the hydrogen bonds and, therefore, react immediately with diazo reagents, without requiring the addition
of accelerators. For confirmation, the conjugated and unconjugated azodipyrroles can be extracted and
analyzed by thin-layer chromatography 204 or high-pressure liquid chromatography (HPLC) 205 (Fig.
125-12).
Reaction of bilirubin tetrapyrrole with the diazonium salt of ethylanthranilate results in the formation of
equimolar amounts of two azodipyrroles. The central methenyl bridge carbon is converted to
formaldehyde. GA, glucuronic acid.
Separation of ethylanthranilate azodipyrroles by HPLC. Wistar rat bile was diazotized with
ethylanthranilate diazo reagent, azodipyrroles were extracted, 208 organic solvents were eliminated in
reduced pressure, and the pigments were dissolved in methanol and separated by reverse-phase HPLC
(µ-Bondapak C-18 column, Waters Associates) using a concave gradient (dashed line) of methanol (80 to
100 percent) in sodiu...
Because 10 to 15 percent of unconjugated bilirubin may give direct diazo reaction, the direct-reacting
fraction slightly overestimates the levels of conjugated bilirubin. In addition, the irreversibly albumin-bound
fraction of serum bilirubin, which is formed in the serum of patients with prolonged conjugated
hyperbilirubinemia, exhibits direct diazo reaction. 99 Because irreversibly protein-bound bilirubin is cleared
slowly, it persists in serum for a relatively long period after correction of biliary obstruction. Finding of
direct-reacting bilirubin during this period may give a false impression of continued biliary obstruction. In
patients with renal failure, indican accumulates in serum and may interfere with the diazo reaction of
bilirubin. 206 Finally, the diazo method cannot be applied to separately quantify bilirubin monoconjugates
and diconjugates when they are present in a complex mixture. The latter requires analysis of intact
bilirubin tetrapyrroles.
Separation and Quantification of Intact Bilirubin Tetrapyrroles.

Chromatographic separation of unmodified bile pigments was attempted by Cole et al. as early as in
1954. 207 Subsequently, Heirwegh and associates developed highly resolving thin-layer chromatographic
systems for separation of bilirubin and its conjugates. 208 Higher resolution and quantitative recovery of
bile pigments is possible by using HPLC. Methyl esters formed by alkaline methanolysis of bilirubin
monoconjugates and diconjugates have been separated and quantified by HPLC. 209 Because the
conjugating sugars are replaced by methyl groups, the pigments cannot be separated on the basis of their
conjugating moieties. Therefore, methods for separation and quantitation of underivatized bilirubin
tetrapyrroles by HPLC have been developed 169–171, 210 (Fig. 125-13). Reverse-phase HPLC of
incompletely deproteinated serum has been used to quantify simultaneously irreversibly protein-bound
and other bilirubin fractions. 99 These studies indicate that the irreversibly protein-bound serum bilirubin
fraction is present in conditions associated with conjugated hyperbilirubinemia. After successful surgical
correction of biliary obstruction, the reversibly protein-bound fraction of serum bilirubin is rapidly excreted
in bile; this results in an increase in the proportion of the irreversibly protein-bound fraction of serum
bilirubin. If biliary obstruction persists, both reversibly protein-bound and irreversibly protein-bound
fractions are retained, and no increase in the proportion of the latter is observed.
HPLC of intact underivatized bilirubin tetrapyrroles in human bile from a normal individual (A) and from
patients with Crigler-Najjar syndrome type I (B), Crigler-Najjar syndrome type II (Arias syndrome) (C),
Gilbert syndrome (D), and Dubin-Johnson syndrome (E). Bile pigments were separated by reverse-phase
chromatography. Absorbance at 436 nm (ordinate) and retention time (abscissa) are shown. Peaks are as
follows: 1, bilirubin diglu...
Slide Tests.
Two slide tests have been introduced for determination of conjugated, unconjugated, and irreversibly
protein-bound bilirubin. One slide (Ektachem TIBL) is used for measurement of total bilirubin by a diazo
technique. 211 The other slide has a special coating that allows only the free and reversibly protein-bound
bilirubins to come in contact with the diazo reagent; conjugated and unconjugated bilirubin are separately
quantified by reflectometric measurements at two wavelengths. 212 The difference between total bilirubin
and the sum of conjugated and unconjugated bilirubin gives the value for irreversibly protein-bound
bilirubin. These results have been verified by HPLC and indicate that the results obtained by the
Ektachem slide tests are consistent and reliable.
Transcutaneous Bilirubinometry.
The current emphasis on early discharge of neonates makes it imperative to assess the risk of severe
neonatal hyperbilirubinemia by evaluating the rate of increase of serum bilirubin levels during the first 24
to 48 hours of life. This requires repeated measurement of serum bilirubin levels, which is painful and
expensive. Measurement of the yellow color of the skin by analysis of reflected light provides a
noninvasive and relatively inexpensive method for estimating serum bilirubin levels without drawing
samples. 213, 214 The analyzers use on-board computers programmed to measure the yellow color without
interference by underlying skin pigmentation or degree of erythema. In 900 term and premature infants of
various races, bilirubin levels estimated by transcutaneous bilirubinometry correlated well with serum
billirubin concentrations measured by a standard diazo method. 215
Fluorimetric Analysis.
Fluorescence characteristics of bilirubin have been used in the development of a method for determination
of total bilirubin, albumin-bound bilirubin, and reserve bilirubin-binding capacity from as little as 0.1 ml of
whole blood. Bilirubin bound to the high-affinity site of albumin has a fluorescence peak at 520 nm when
excited at 430 nm. Unbound bilirubin and bilirubin bound to other proteins have negligible fluorescence.
Addition of a saturating amount of bilirubin to blood results in maximum fluorescence, allowing
determination of total bilirubin-binding capacity. Addition of a detergent, dodecylmethylamine oxide, to
whole blood results in hemolysis and quantitative incorporation of bilirubin into the detergent micelles.
Fluorescence of detergent-bound bilirubin is used for quantitation of total bilirubin. These parameters can
be readily determined using a digital hematofluorometer. Because fluorescence also depends on
hemoglobin concentration, hemoglobin values are independently determined by the hematofluorometer
and taken into account in calculation of displayed values for total bilirubin and albumin-bound bilirubin and
reserve bilirubin binding capacity. 216
Biliary Secretion of Bilirubin

In mammals, conjugation is essential for the secretion of bilirubin acoss the bile canaliculus. Gunn rats
and patients with Crigler-Najjar syndrome type 1 lack bilirubin UGT activity and manifest lifelong
unconjugated hyperbilirubinemia. Their bile contains only a small amount of bilirubin. Normally, the
conjugating capacity and biliary excretory capacity of bilirubin are closely matched. Thus, drugs and
chemicals that reduce the maximal secretory capacity for bilirubin cause the retention of conjugated
bilirubin. On the other hand, when bilirubin-UGT activity is partially deficient, conjugation becomes rate
limiting in bilirubin secretion. 217 In rats there is approximately a 15-fold functional reserve in the ability to
secrete conjugated bilirubin into bile.
Prior to 1991, the major driving force for the transport of bilirubin conjugates from the hepatocyte into the
bile was thought to be the electrochemical gradient of −35 mV. 218 Relative intracellular negativity is
generated by the sodium pump, which tranports three molecules of sodium out of the cell while
transporting two molecules of potassium into the cell. The sodium pump is functional mainly in the
basolateral domain of the hepatocyte plasma membrane, and is present but inactive in the canalicular
domain. The observed potential difference is too small to account for the large bilirubin glucuronide
concentration gradients between the hepatocyte and bile, which may be as high as 150-fold. The energy
for this uphill transport is provided by an ATP-dependent system in the canalicular membranes that is
specific for non–bile acid organic anions, including bilirubin and other glucuronides and glutathione
conjugates. 218–223 In contrast to the bidirectional transport at the sinusoidal aspect of the hepatocyte,
canalicular transport of organic anions is unidirectional from the cytoplasmic aspect of the hepatocyte into
the bile. In addition to the energy-consuming process, the canalicular transport may be assisted by the
membrane potential, but the contribution of membrane potential in organic anion transport has not been
quantified. The membrane potential–driven canalicular transport of non–bile acid organic anions is distinct
from that driven by ATP hydrolysis. 223 Mutant animals that lack ATP-dependent canalicular transport of
non–bile acid organic anions retain normal activity with respect to potential-driven canalicular transport of
non–bile acid organic anions, including bilirubin glucuronides. 224, 225 The benign phenotype of the defect
in human and animal mutants may reflect persistence of the potential-driven transport system in the bile
canaliculus.
The ATP-dependent canalicular non–bile acid organic anion transporter (also called MOAT, multiorganic
anion transporter) is functionally distinct from other canalicular ATP-dependent transporters for organic
cations, 226–228 bile acids, 229, 230 and phospholipids. 231 Attempts to purify the ATP-dependent transporter
for bilirubin glucuronide were unsuccessful. Its identification by cloning followed the serendipitous
observation that a drug-resistant cancer cell line expressed an ATP-dependent multidrug resistance–like
protein (MRP) that transports various organic cations. 232 The MRP family of ATP-dependent membrane
transporters differs from the previously described multidrug resistance (MDR) family of ATP-dependent
membrane proteins in amino acid sequence, molecular weight, hydropathy plots, and substrates. Both
have multiple transmembrane domains and two nucleotide binding sites that are separated by distinct
linker domains. Whereas MRP1 is present in the plasma membrane of many cell types, a family member,
MRP2 (initially termed multispecific organic anion transporter, MOAT), is restricted to the bile canalicular
membrane, where it is responsible for ATP-dependent transport of a wide variety of non-bile acid organic
anions, which are primarily glucuronides and glutathione conjugates. 233
Patients with the Dubin-Johnson syndrome, 234, 235 mutant Corriedale sheep, 224 transport-deficient mutant
rat strain (TR) and Eisai hyperbilirubinemic rat (EHBR) rats 225 and mutant golden lion tamarin
monkeys 236 manifest conjugated hyperbilirubinemia that is transmitted as an autosomal-recessive trait.
These mutants share defective capacity to transport bilirubin glucuronide, other organic anionic
metabolites (including leukotriene C4 and metanephrine glucuronide), and other organic anions, such as
BSP, ICG, iopanoic acid, and phylloerythrin, from hepatocytes into the bile. Affected patients and mutant
animals have a normal transport maximum for infused bile acids. 235, 237 These observations
demonstrated that there are at least two mechanisms for organic anion secretion by the liver, one for bile
acids and another for other organic anions. In canalicular membrane vesicles, these processes were
functionally distinct and required ATP hydrolysis. 230 The major, and possibly only, canalicular bile acid
transporter has been demonstrated to be sister-p-glycoprotein (SPGP), a member of the MDR family of
transmembrane transporters. 238
The predominant member (approximately 90 percent) of MDR family gene products in the canalicular
membrane is MDR3 (the human homologue of murine mdr2). MDR3 couples ATP hydrolysis to the
selective transfer of phosphatidylcholine from the inner to the outer leaflet of the bile canalicular
membrane. 239 Phospholipids are required to form mixed micelles with bile acids, which protects small bile
ducts from the detergent action of bile acids.
Maximal bilirubin secretory capacity (T max ) depends on bile flow. Flow is increased by infusion of bile
acids 240 or by phenobarbital treatment, which enhances bile flow rate by a non–bile acid dependent
mechanism. 241 The T max of bilirubin is enhanced in both cases. Several other bile acid–independent
choleretics increase bile flow but not the T max for organic anions. 242 The maximal ATP-dependent biliary
secretion of bile acids and non–bile acid organic anions greatly exceeds the apparent capacity and
amount of SPGP and MRP2 present in the canalicular membrane under basal conditions. 243 Bile acids
increase the transfer of MRP2, SPGP, and MDR3 from the Golgi to the apical domain, thereby more than
doubling the amount of each transporter in the canalicular membrane. 244 Recruitment of canalicular
ATP-dependent transporters involves microtubular-dependent vesicular trafficking, which requires
association with and activity of phosphoinositide 3-kinase and its lipid products.
A small amount of unconjugated bilirubin (up to 3% of total bile pigments) is found in normal bile. This may
be explained by hydrolysis of bilirubin glucuronide by biliary β-glucuronidase, canalicular ATP-dependent
transport of bilirubin, or self-aggregation and incorporation of bilirubin in mixed micelles.
Although bilirubin glucuronides, bile acids, and phospholipids are transported across the canalicular
membrane by different ATP-dependent transporters, heritable defects in bile acid or phospholipid
transport produce bile secretory failure (i.e., cholestasis), which is manifested by conjugated
hyperbilirubinemia. Molecular defects in heritable cholestatic disorders remained unknown until the
mechanisms responsible for biliary secretion of bilirubin glucuronide, bile acids, and phospholipids were
identified. The discovery of bile canalicular ATP-dependent transporters for bile acids (SPGP), non–bile
acid organic anions (MRP2), and phospholipids (MDR3) enabled the molecular characterization of several
heritable hyperbilirubinemias, cholestatic or not.
Fate of Bilirubin in the Gastrointestinal Tract

Bilirubin reaches the intestinal tract mainly conjugated and is not substantially absorbed. 245 In some
circumstances, there may be enhanced excretion of unconjugated bilirubin into the intestine. Absorption of
unconjugated bilirubin from the intestine may contribute to neonatal hyperbilirubinemia. 246 Milk inhibits
intestinal absorption of unconjugated bilirubin. However, human milk inhibits this reabsorption less than
does infant milk formula. 247 These observations suggest that intestinal absorption of bilirubin may
contribute to jaundice associated with breast-feeding. Absorption of bilirubin from the gallbladder occurs in
animals. 248 Bilirubin is degraded by intestinal bacteria into a series of urobilinogen and related
products. 249 The specific products may relate to strains of bacteria present in the intestine. 250
Urobilinogens are present in the deconjugated state. It is not known whether deconjugation precedes or
follows bilirubin degradation, but bacterial β-glucuronidase plays a role in the deconjugation. 246, 251 Most
of the urobilinogen reabsorbed from the intestine is reexcreted in the bile. A small fraction is excreted by
the kidney. Enhanced tubular absorption and instability of the pigment in acid urine makes urobilinogen
excretion in urine an unreliable indicator of the status of bilirubin metabolism. Absence of urobilinogen in
stool and urine indicates complete obstruction of the bile duct. In liver disease and states of increased
bilirubin production, urinary urobilinogen excretion is increased. Urobilinogen is colorless. Oxidation leads
to formation of urobilin, which contributes to the color of normal urine and stool.
Alternative Pathways of Bilirubin Elimination

After injection of labeled unconjugated bilirubin, only 3 percent of radioactivity is normally excreted by the
kidney in humans. Even in the presence of marked hyperbilirubinemia, bile remains the main route of
bilirubin excretion. In patients with Crigler-Najjar syndrome and in Gunn rats, a small amount of
unconjugated bilirubin is secreted in bile. Additional unconjugated bilirubin may reach the intestinal lumen
by passage across the intestinal wall or by desquamation of intestinal epithelial cells. 252 Ambient light or
phototherapy forms geometric isomers of bilirubin (EE, EZ, or ZE forms), which are excreted in
unconjugated forms and converted to bilirubin IXα-ZZ in the bile. 253 Considerable amounts of bilirubin are
degraded to polar diazo-negative compounds, which are excreted in both bile and urine. 252
The presence of hydroxylated products of bilirubin in Gunn rat bile suggests a role of enzyme-catalyzed
oxidation in the disposition of bilirubin. 254 Induction of a specific isoform of microsomal P450s by
administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in Gunn rats results in a sevenfold increase
in the fractional turnover of bilirubin and reduction of the bilirubin pool. 255 A mitochondrial bilirubin oxidase
found in rat liver, 256 intestine, 257 and kidney consumes 1 to 1.5 moles of oxygen per mole of bilirubin and
forms propentdyopents. The enzyme does not require NADP, NAD, or ATP and is inhibited by potassium
cyanide (KCN), thiol reagents, NADH, and albumin. 256 Enzyme-mediated oxidation of bilirubin also has
been reported to occur in mitochondrial fractions of brain, lung, heart, and skeletal muscle.
Disposition of Bilirubin by the Kidney.

In intrahepatic or extrahepatic cholestasis, the plasma-conjugated bilirubin concentration increases. After
injection of radiolabeled bilirubin in animals with experimentally ligated bile ducts 258 and in children with
biliary atresia, 259 50 to 90 percent of injected radioactivity is excreted in urine. In total biliary obstruction,
urinary excretion becomes the major pathway of bilirubin excretion. 260 Renal excretion of conjugated
bilirubin depends on glomerular filtration of a small non–protein-bound fraction of conjugated
bilirubin. 260, 261 There is evidence for tubular reabsorption but none for tubular secretion of bilirubin. 261
Antioxidative Property of Bilirubin

Until recently, bilirubin has been considered a waste product with no known physiologic utility. However,
both unconjugated 262 and conjugated 263 bilirubin have been shown to be inhibitors of lipid peroxidation.
This property of bilirubin depends on its ability to donate hydrogen ions. 264 Bilirubin bound to hepatic
cytosolic proteins has a greater free-radical scavenging effect than does the bilirubin–albumin complex. It
has been postulated that bilirubin is converted to its peroxidative product, biliverdin, thereby sparing
hepatic endoplasmic reticulum membranes of lipid peroxidation. 265 Regeneration of bilirubin by the action
of cytosolic biliverdin reductase may enhance the protective effect of bilirubin. Albumin binding increases
the cytoprotective activity of bilirubin, 265 suggesting a possible beneficial role of bilirubin on tissues other
than the liver.
DISORDERS OF BILIRUBIN METABOLISM RESULTING IN UNCONJUGATED

HYPERBILIRUBINEMIA
General Considerations
The hepatic transport of bilirubin involves four distinct but probably interrelated stages: (1) uptake from the
circulation; (2) intracellular binding or storage; (3) conjugation, largely with glucuronic acid; and (4) biliary
excretion. Abnormalities in any of these processes may result in hyperbilirubinemia. Complex clinical
disorders, such as hepatitis or cirrhosis, may affect multiple processes. In several inherited disorders, the
transfer of bilirubin from blood to bile is disrupted at a specific step. Study of these disorders has permitted
better understanding of bilirubin metabolism in health and disease. Each disorder is characterized by
varied degrees of hyperbilirubinemia of the unconjugated or conjugated type.
Neonatal Jaundice
By adult standards, every newborn baby has hyperbilirubinemia, and about half of all neonates become
clinically jaundiced during the first 5 days of life. Serum bilirubin is predominantly unconjugated.
Exaggeration of this physiologic jaundice can result in marked hyperbilirubinemia, with an attendant risk of
kernicterus (see earlier section on Toxicity of Bilirubin). In 4000 consecutive infants, 16 percent had
maximal serum bilirubin concentrations of 10 mg/dl or above, and in 5 percent, bilirubin concentrations
exceeded 15 mg/dl. 266 In the normal, full-term human neonate, the serum bilirubin concentration
increases rapidly from 1 to 2 to 5 to 6 mg/dl in approximately 72 h and subsequently decreases until
normal levels are attained in 7 to 10 days. 267 Physiologic jaundice of the newborn appears to result from
a combination of increased bilirubin production and delayed maturation in the capability of the liver to
dispose of bilirubin. Severe neonatal unconjugated hyperbilirubinemia results from exaggeration in one or
more of the regularly occurring developmental restrictions that are characteristic of the newborn period or
from superimposition of additional mechanisms. Although the incidence of cerebral toxicity from neonatal
jaundice had decreased markedly after the introduction of immunoglobulin therapy for maternal–fetal Rh
blood group incompatibility, the incidence of neonatal kernicterus may be on the increase again. This may
be partly related to early discharge after delivery, which is being practiced in a majority of hospitals in the
United States and elsewhere. This issue and the current concepts of neonatal jaundice have been
reviewed elsewhere. 78 The physiologic basis of neonatal hyperbilirubinemia and mechanisms of its
exaggeration to potentially harmful states are discussed briefly below.
Increased bilirubin production in the newborn period is evidenced by increased endogenous carbon
monoxide production, 268 increased early-labeled peak from erythroid and nonerythroid sources, and
decreased erythrocyte half-life. 269 Meconium contains unconjugated bilirubin derived primarily from
hydrolysis of conjugated bilirubin by intestinal β-glucuronidase. 270 As compared with adults, newborns
lack intestinal bacteria that degrade bilirubin to urobilinogen and have a greater surface-to-volume ratio of
the bowel. As a result, intestinal absorption of unconjugated bilirubin in neonates may be increased. 270
Hemolytic diseases of the fetus increase bilirubin production and may lead to severe neonatal
unconjugated hyperbilirubinemia. Rh incompatibility between mother and fetus was formerly a common
cause of severe neonatal unconjugated hyperbilirubinemia and kernicterus (erythroblastosis fetalis). This
disease can be prevented by treatment of the mother with anti-Rh immunoglobulins. 271 Major blood group
(ABO) incompatibility remains a common cause of exaggerated neonatal hyperbilirubinemia that often
requires treatment. 272
Hepatic Bilirubin Uptake During the Neonatal Period.

Cumulative hepatic bilirubin uptake capacity is reduced during the first 25 h of life in the rhesus monkey.
Relative hepatic uptake deficiency extends beyond the second day of life and correlates with maturation of
hepatic ligandin, 273 which influences the net hepatic uptake of bilirubin (see earlier section on Uptake of
Bilirubin by the Liver). Delayed closure of the ductus venosus may permit portal blood, which is enriched
in unconjugated bilirubin from the intestine, to bypass the liver. Reduced caloric intake, which reduces
hepatic bilirubin clearance in adults (see later section on Gilbert Syndrome), may have a similar effect in
neonates.
Postnatal Development of Bilirubin-Glucuronidating Activity.

In many mammals, including humans, UGT activity toward bilirubin is deficient in fetal liver and rapidly
develops to adult levels during the first few days of life. 274 Deficiency of UGT activity may be prolonged
and exaggerated in some inherited disorders due to inhibitory factor(s) in maternal milk or serum (see
later section on Transient Familial Neonatal Hyperbilirubinemia).
Recently, a variant TATAA element within the promoter region of UGT1A1 has been found to be
associated with Gilbert syndrome. 275 This variant promoter reduces the expression of bilirubin-UGT
(UGT1A1). The Gilbert genotype has been found to accentuate neonatal hyperbilirubinemia 276 and
prolong the duration of neonatal jaundice. 277
Inhibition of Bilirubin Glucuronidation by Maternal Milk.

Plasma bilirubin concentrations tend to be higher in breast-fed infants as compared with formula-fed
babies 278 and occasionally rise to maximum concentrations of 15 to 24 mg/dl within 10 to 19 days of life.
This transient nonhemolytic, unconjugated hyperbilirubinemia is promptly ameliorated by discontinuation
of breast-feeding; otherwise, the hyperbilirubinemia may take up to a month to disappear. Kernicterus is
rare, but isolated cases have been reported. 279 Neonatal unconjugated hyperbilirubinemia related to
breast-feeding is associated with an inhibitor of UGT activity in maternal milk but not maternal serum. 280
A progestational steroid, 3α,20β-pregnanediol, was isolated from the milk of mothers of infants who had
the syndrome. The steroid inhibited bilirubin glucuronidation by rat and rabbit liver, 280 but activity with
human liver was not inhibited. 281 Experimental feeding of the steroid to healthy infants yielded
contradictory results. 282, 283 The free fatty acid concentration in maternal milk correlates positively with its
inhibitory effect on human hepatic bilirubin-UGT activity, and free fatty acids inhibit UGT activity in vitro in
proportion to the number of double bonds in unsaturated fatty acids and in inverse proportion to the chain
lengths of saturated fatty acids (C10 to C18). 284 Odievre and associates have postulated that a lipolytic
enzyme, which is present in some maternal milk samples, may be responsible for the increased
concentration of free fatty acids in the milk. 284 The inhibitory effect of maternal milk on UGT increases on
storage and is destroyed by heating at 56°C.
Inhibition of Bilirubin Glucuronidation by Factors Derived from Maternal Plasma.

Transient familial neonatal hyperbilirubinemia (Lucey-Driscoll syndrome) was described by Lucey and
associates in 1961. 285, 286 This syndrome is characterized by jaundice occurring within the first 4 days of
life. In 24 infants, 286 peak serum bilirubin concentrations of 8.9 to 65 mg/dl were reached within 7 days.
An unidentified inhibitor of UGT was found in the serum of mothers of these infants. One infant died at
36 h. This condition is clinically distinguished from maternal milk jaundice by earlier onset of severe
hyperbilirubinemia, a more prolonged course, and occasional kernicterus.
Immaturity of Canalicular Excretion.

During the late newborn period, hepatic bilirubin uptake, conjugation, and canalicular excretion attain adult
levels, even though the bilirubin load remains increased. In this period of life, canalicular excretion may be
rate limiting in the hepatic disposition of bilirubin. Consequently, when the bilirubin load is further
increased, conjugated bilirubin accumulates in serum. 287
Management of Neonatal Unconjugated Hyperbilirubinemia.

In the great majority of cases, neonatal hyperbilirubinemia is innocuous, and indeed may provide an
important antioxidant defense for the newborn. However, it is critical to be vigilantly prepared for the
occasional case in which neonatal jaundice can reach levels that may cause cerebral toxicity. 78 Although
a plasma bilirubin concentration of 20 mg/dl is usually considered dangerous, cerebral toxicity can occur
at lower concentrations of bilirubin (see earlier section on Toxicity of Bilirubin). The goal of treatment is to
decrease serum bilirubin concentrations to an acceptable level until the capacity of the liver to dispose of
bilirubin matures. Phototherapy is the most common treatment modality used. In severe cases, exchange
transfusion is used to reduce serum bilirubin levels rapidly (see later section on Treatment of
Crigler-Najjar Syndrome Type I). Although phototherapy is useful and safe, concern persists about its
potential side effects. Ingestion of agar to bind unconjugated bilirubin in the intestine also decreases
serum bilirubin concentrations, 270 but the efficacy of this treatment is not certain. 269 Inhibition of heme
oxygenase activity by the administration of tin-mesoporphyrin at birth has been shown to prevent the
development of significant levels of neonatal jaundice, thereby abolishing the need for phototherapy or
exchange transfusion. 44, 45 However, the routine use of this compound in all newborns is not generally
recommended.
Hyperbilirubinemia Due to Bilirubin Overproduction

Hyperbilirubinemia in the presence of normal liver function often occurs in disorders associated with
increased bilirubin production. The serum bilirubin is unconjugated and rarely exceeds 3 to 4 mg/dl.
Higher levels usually indicate hepatobiliary dysfunction in addition to bilirubin overproduction. 4 The most
common cause of increased bilirubin production is hemolysis such as occurs in sickle cell anemia,
hereditary spherocytosis, and toxic or idiosyncratic drug reactions in susceptible individuals. These
disorders are associated with premature destruction of erythrocytes; red cell morphology and life span are
often abnormal. In the absence of any hepatobiliary disorder, a small amount of conjugated bilirubin,
mainly bilirubin monoglucuronide, may accumulate in the serum, in addition to unconjugated bilirubin. In
these cases, the proportion of conjugated bilirubin does not usually exceed the normal limits
(approximately 4 percent of total bilirubin). The conjugated bilirubin probably appears in the serum by
diffusion out of the hepatocyte. In contrast to previous interpretation, 288 this phenomenon does not
necessarily indicate that the rate of bilirubin production has exceeded the excretory transport maximum for
biliary excretion of conjugated bilirubin. Ineffective erythropoiesis occurs in thalassemia and other
hematologic disorders and is often associated with hyperbilirubinemia. 289 Congenital dyserythropoietic
anemias are a group of rare hereditary anemias characterized by ineffective erythropoiesis, intramedullary
normoblastic hyperplasia, secondary hemochromatosis, and unconjugated hyperbilirubinemia. 290–293
Crigler-Najjar Syndrome Type I
Clinical Findings.
Crigler-Najjar syndrome type I is a rare disorder in which hepatic bilirubin-UGT activity is absent or barely
detectable (Table 125-1). The syndrome was described by Crigler and Najjar in 1952 in six infants in three
families. 294 All infants manifested severe nonhemolytic icterus within the first few days of life. Jaundice
was characterized by increased plasma concentration of indirect-reacting bilirubin and was lifelong. Five
of the six infants died of kernicterus by the age of 15 months. Although icteric, the single surviving infant
was free of neurologic disease until 15 years of age, when kernicterus suddenly developed, and he died 6
months later. 295 A female cousin also had Crigler-Najjar syndrome; she developed neurologic symptoms
at 18 years of age and died at the age of 24. 296 This family had increased consanguinity and several
other recessively inherited traits, such as Morquio syndrome, homocystinuria, metachromatic
leukodystrophy, and bird-headed dwarfism. 297 However, association with these disorders was not
observed in the subsequently reported cases of Crigler-Najjar syndrome type I. The syndrome occurs in
all races, is transmitted as an autosomal-recessive trait (Fig. 125-14), 295–298 and is associated with
known consanguinity in some, but not all, cases. Until the introduction of phototherapy, almost all patients
died with kernicterus during the first 18 months of life. 299 Several individuals have survived only to
succumb to kernicterus later in life. 291, 294, 296, 297 With the advent of phototherapy and intermittent
plasmapheresis, survival until puberty, without significant brain damage, is not unusual. However, the risk
of bilirubin encephalopathy persists, and kernicterus is common around the time of adolescence, when
phototherapy becomes less effective. Orthotopic liver transplantation or auxiliary transplantation of a
single liver lobe has resulted in long-term survival in several cases. 300 As discussed below, isolated
hepatocyte transplantation has been used in a single case with partial amelioration of hyperbilirubinemia.
Table 125-1: Principal Differential Characteristics of Inherited Unconjugated Hyperbilirubinemia
Crigler-Najjar
Crigler-Najjar
Syndrome Type Gilbert Syndrome
Syndrome Type II
I
Histology of liver Normal Normal Normal
Serum bilirubin concentration 20–50 mg/dl <20 mg/dl Usually <3 mg/dl
Routine liver function test

Normal Normal Normal
results
Usually normal; may be

45-min plasma BSP retention Normal Normal elevated in some
patients
Usually pale;
Increased
contains small Increased proportion of
proportion of
Bile amounts of bilirubin
bilirubin
unconjugated monoglucuronide
monoglucuronide
bilirubin
Hepatic bilirubin
UDP-glucuronosyltransferase Absent Markedly reduced Reduced
activity
Effect of phenobarbital on
None Reduction Reduction
serum bilirubin
Autosomal Autosomal
Mode of inheritance Autosomal recessive
recessive recessive
Common (~9% of
caucasians are
homozygous for a
Prevalence Rare Rare
variant TATAA box;
4–5% have
hyperbilirubinemia)
Kernicterus,
Usually benign,
unless
Prognosis kernicterus occurs Benign
vigorously
rarely
treated
Homozygous Bolivian squirrel

Animal model —
Gunn rat monkey
Inheritance of Crigler-Najjar syndrome type I. This family, originally described by Crigler and Najjar in
1962, is unique in that two cousins, J.D.H. and M.E.H., escaped kernicterus in infancy only to die at ages
16 and 24, respectively. (Reprinted with permission from Blaschke et al. 363 )
Laboratory Tests.
Laboratory test results in Crigler-Najjar syndrome type 1 are normal except for the serum bilirubin level,
which is usually 20 to 25 mg/dl, but may be as high as 50 mg/dl. 294–297, 299 Virtually all the serum bilirubin
is unconjugated, and no serum conjugated bilirubin has been found. There is no bilirubinuria, but the urine
may be yellow due to a chloroform-soluble pigment of unknown structure. 300 The level of icterus in a
given patient varies—it is lower in summer and on exposure to sun and higher during intercurrent
illness. 301 Stool color is normal, but fecal urobilinogen excretion is reduced. 294–301 Bilirubin production,
hematocrit, bone marrow morphology, and red cell survival are normal. 250, 302 Results of routine liver
function tests are normal, including studies of plasma disappearance of BSP and ICG. 294, 303 Because the
canalicular excretion mechanism is normal in these patients, radiologic visualization of the biliary tree by
cholecystographic agents is normal. Jaundice and occasional neurologic impairment are the only
abnormal physical findings. Liver biopsy reveals normal histology. In several patients, pigment plugs were
observed in bile canaliculi and bile ducts (Fig. 125-15). 294, 301, 303 Pigment stones have been found in
several cases. The pigment plugs and stones probably result from biliary excretion of unconjugated
bilirubin as an effect of long-term phototherapy. Electron microscopy of the liver reveals no specific
pathologic change. 304
High-power view (hematoxylin and eosin; magnification ×650) of a liver biopsy obtained from a patient
with Crigler-Najjar syndrome type I during pregnancy. A portal area is shown with portal vein (PV) and a
bile ductule (B) containing amorphous material, which appeared to be bilirubin. (Reprinted with permission
from Wolkoff et al. 299 )
Molecular Defect in Crigler-Najjar Syndrome Type 1.

Of the numerous isoforms of UGT, UGT1A1 (bilirubin-UGT1) is the only isoform that contributes
significantly to bilirubin metabolism in humans (GenBank AJ005162). 196 Genetic lesions of exons
constituting the UGT1A1 gene that cause Crigler-Najjar syndrome type 1 were first described in
1992. 196, 305–307 Subsequently, several laboratories have reported the genetic lesions in over 60 patients
with Crigler-Najjar syndrome type 1 by polymerase chain reaction–based DNA sequence analysis. These
genetic lesions and their relationship to Crigler-Najjar syndrome have been reviewed. 308 The genetic
lesions can be deletions, insertions, missense mutations, or premature stop codons, and can be located in
any of the five exons comprising the UGT1A1 mRNA (Fig. 125-16). 308 In one case the mutation is located
in the hydrophobic signal peptide that is cleaved off from the enzyme during synthesis. 309 In two other
cases, the mutation was located within the intronic regions of the gene at a splice donor and a splice
acceptor region, respectively. 310 These mutations result in the utilization of a cryptic splice site within an
exon, which leads to the splicing out of a part of the exon. In most families, with or without known
consanguinity, both alleles carry identical mutations, although different genetic lesions on the two alleles
are not rare. 308 A 13-nucleotide deletion in exon 2 of UGT1A1 has been observed in three unrelated
families of different ethnic origins. Crigler-Najjar syndrome type 1 is relatively common among the Amish
and Mennonite communities of Lancaster County, Pennsylvania. 311 All Crigler-Najjar syndrome type 1
patients within these related communities carry identical mutations in exon 1, indicating a founder effect.
Similarly, four apparently unrelated families, originating from Punjab, India, have been fround to carry an
identical mutation (unpublished observation, J. Roy Chowdhury and N. Roy Chowdhury). In cases where
the genetic lesion is located in exon 1, only bilirubin-UGT (UGT1A1) activity is expected to be abnormal,
whereas when the mutation affects one of the common region exons (exons 2 to 5), all UGT isoforms
expressed from the UGT1A locus are expected to be abnormal.
Genetic lesions causing Crigler-Najjar syndrome type I, Crigler-Najjar syndrome type II, and Gilbert
syndrome. Crigler-Najjar syndrome type I is produced by mutations, deletions, or insertions within the five
exons that constitute the UGT1A1 mRNA. These genetic lesions may cause premature stop codons or
substitution of a single amino acid. In two cases, there were mutations in the splice donor sequences on
intron 1 and splice acceptor region...
Diagnosis.
The combination of very high levels of serum unconjugated bilirubin and the absence of any other
abnormality of routine liver function tests is diagnostic of Crigler-Najjar syndrome type 1. Hemolysis alone
does not increase serum bilirubin levels beyond 6 to 8 mg/dl. Differential diagnosis includes Crigler-Najjar
syndrome type 2, with or without coexisting hemolysis. Although serum bilirubin levels are relatively lower
in Crigler-Najjar syndrome type 2, ranges of bilirubin concentration in the two disorders overlap. In most
cases, serum bilirubin concentrations are reduced by more than 25 percent after phenobarbital
administration (60 to 120 mg for 14 days) in Crigler-Najjar syndrome type 2, but not in type 1. 301 The two
types of Crigler-Najjar syndrome can be differentiated conveniently by chromatographic analysis of bile
collected from the duodenum through a perorally placed duodenal catheter or an upper gastrointestinal
endoscope. In bile from patients with Crigler-Najjar syndrome type 1, bilirubin glucuronides are absent or
are present in traces only (in concentrations less than that of unconjugated bilirubin). In contrast, in
Crigler-Najjar syndrome type 2 significant amounts of conjugated bilirubin are excreted in bile, although
the proportion of bilirubin diglucuronide is reduced (see below in Crigler-Najjar syndrome type 2). Liver
biopsy is not necessary for diagnosis, unless a coexisting liver disease is suspected. If a biopsy is
performed, UGT activity toward bilirubin can be determined, and is expected to be undetectable. The
diagnosis can be made also on the basis of genetic analysis of DNA extracted from blood, buccal
scrapings, or any tissue. The five exons of the UGT1A1 gene, and the flanking intronic sequences are
amplified by polymerase chain reaction and the nucleotide sequences are determined. 305 If the genetic
lesion matches one of the previously identified lesions associated with Crigler-Najjar syndrome type 1, the
diagnosis is established. If a new mutation is found that predicts the truncation of a major portion of the
enzyme or causes a frame-shift, the diagnosis is also established. When a novel mutation predicts the
substitution of a single amino acid residue, the mutation can be generated in an expression plasmid by
site-directed mutagenesis and the effect of the mutation can be determined after transfection of the
plasmid into African Green monkey kidney cell lines (COS cells). 196 Genetic analysis can be utilized to
detect heterozygous carriers of one Crigler-Najjar syndrome type 1 allele. Genetic analysis of chorionic
villus samples has been used successfully for prenatal identification of the Crigler-Najjar syndrome type 1
genotype in four fetuses in three families (J. Roy Chowdhury and N. Roy Chowdhury, unpublished
observation).
Animal Model for Crigler-Najjar Syndrome Type 1: The Gunn Rat.

The description by Gunn in 1938 of mutant Wistar rats with nonhemolytic unconjugated
hyperbilirubinemia 312 and the wisdom of the late Professor William E. Castle, Emeritus Professor of
Genetics at Berkeley, who maintained the mutants for over 15 years, have resulted in major advances in
understanding bilirubin metabolism, transport, and encephalopathy. 313, 314 Jaundice in these animals is
inherited as an autosomal recessive trait. 312 Heterozygotes are anicteric. Depending on the background
strain against which a Gunn rat colony is maintained, homozygous Gunn rats have bilirubin levels that
range from 3 to 20 mg/dl. Serum bilirubin is all unconjugated, 313 there is no bilirubinuria, and bile is light
yellow in color due to the excretion of small amounts of unconjugated bilirubin. 313 Liver histology is
normal. 315
Neurologic Lesions.
Homozygous Gunn rats are prototypes of Crigler-Najjar syndrome type I and frequently develop
kernicterus. 299, 315–317 The Gunn rat is the only experimental model in which endogenously produced
bilirubin results in neuropathologic lesions and neurologic deficits. Cytoplasmic neuronal changes develop
in these rats on the third day of life, and by 2 weeks, degeneration of Purkinje cells and other neurons
occurs. The degenerative changes begin by enlargement of mitochondria and formation of membranous
cytoplasmic bodies. By 8 days of age, many mitochondria contain glycogen. 316, 317 When a clinically
healthy Gunn rat is killed and rapidly perfused with saline or formalin, the brain does not show yellow
staining. Administration of sulfadimethoxine, a drug that competes with bilirubin for binding to albumin, to
14-day-old animals results in neurologic deterioration and yellow staining in the brain. 318
Urinary Concentration Defect.

Gunn rats cannot concentrate urine and do not tolerate water deprivation. 319 The renal medullary bilirubin
concentration is high and interferes with sodium and water transport. 319 Occasionally, renal papillary
necrosis occurs. 320 Treatment of rats with agents or methods designed to lower serum bilirubin, such as
cholestyramine, agar, or phototherapy, may ameliorate the renal lesion. 319 Similar concentrating
problems have not been described in patients with Crigler-Najjar syndrome type I, although bilirubin is
deposited in the kidney. 321
Abnormalities of UGT Activity and Their Relationship to Genetic Lesions.

The rat UGT1A locus closely resembles its human counterpart in exon organization. 322 The genetic lesion
in Gunn rats consists of the deletion of a single guanosine residue in the common region exon, exon 4,
which creates a frameshift and deletes a large segment of the C-terminal domain of all UGT isoforms that
are expressed from this locus. 322–324 Consequently, not only bilirubin glucuronidation is absent in this
strain, other UGT isoforms expressed from the UGT1A locus are also truncated and nonfunctional. 325 As
in patients with Crigler-Najjar syndrome type 1, Gunn rats lack bilirubin conjugates in bile. 252, 313, 315 Gunn
rat livers lack UGT activity toward digitoxigenin monodigitoxoside. 326 The transferase activity toward
4-nitrophenol is present at a lower level, 327 suggesting that this activity is partly provided by UGT isoforms
expressed from other loci. Several functionally normal forms of UGT have been isolated from the Gunn rat
liver, 328 indicating that UGT isoforms expressed from genes that do not belong to the UGT1A locus are
normal. UGT activities for aniline, 329 steroid substrates, 330 and thyroid hormone 331 are normal in Gunn
rat liver. Biliary excretion of substances that do not require glucuronidation, such as BSP, 313 phenol
red, 332 and exogenously administered conjugated bilirubin, 125 are normal in Gunn rats.
Treatment of Crigler-Najjar Syndrome Type 1.

Unconjugated hyperbilirubinemia in Crigler-Najjar syndrome type 1 is usually associated with bilirubin
encephalopathy (kernicterus). Conventional treatment is designed to reduce serum bilirubin levels. Unlike
in patients with Crigler-Najjar syndrome type II and Gilbert syndrome, the serum bilirubin levels are not
significantly reduced by the administration of UGT enzyme inducing agents, such as phenobarbital. 301, 302
Phototherapy has received widespread acceptance. It is the major treatment for icteric newborns whose
serum bilirubin concentrations place them at risk for kernicterus. 253, 299, 333 Experience with phototherapy
in older children and adults is limited to patients with Crigler-Najjar syndrome type 1 and occasional cases
of Crigler-Najjar syndrome type 2. An array of 140-W fluorescent lamps with devices for shielding the eyes
has been used effectively. However, about the time of puberty, phototherapy becomes relatively less
effective because of thickening of the skin, increased skin pigmentation, and decreased surface area in
relation to body mass. 299 Phototherapy converts a fraction of bilirubin IXα-ZZ into geometric and
structural isomers, that are excreted in bile (see earlier section on Chemistry of Bilirubin). A portion of the
unconjugated bilirubin excreted in bile may be reabsorbed in the small intestine. Oral administration of
agar, cholestyramine, or calcium salts 333 enhances the effect of phototherapy, probably by inhibiting the
reabsorption of unconjugated bilirubin.
Plasmapheresis is the most efficient method for rapidly reducing serum bilirubin concentration during
crisis (Fig. 125-17). 296, 299, 303 This procedure takes advantage of the fact that bilirubin is tightly bound to
serum albumin and removal of albumin results in the removal of equimolar amounts of bilirubin.
Summary of the hospital course of a 19-year-old patient with Crigler-Najjar syndrome type I who was
admitted with acute bilirubin encephalopathy. Before hospitalization, the patient’s serum bilirubin ranged
between 35 and 45 mg/dl. After an initial course of plasmapheresis and maintenance phototherapy, serum
bilirubin was maintained between 10 and 15 mg/dl. (Reprinted with permission from Wolkoff et ...
Liver Transplantation.
Orthotopic or auxiliary liver transplantation rapidly normalizes serum bilirubin levels. Currently, liver
transplantation is considered the only definitive treatment for Crigler-Najjar syndrome type 1. 334 Although
these procedures are not without risk in these individuals, some investigtors have suggested prophylactic
liver transplantation in patients with Crigler-Najjar syndrome type 1 to avoid the risk of kernicterus, which
once established, may not be fully reversible. 334
Hepatocyte Transplantation.
Because the liver is structurally normal in Crigler-Najjar syndrome type 1 and in Gunn rats, alternatives to
the irreversible, expensive, and risky procedure of liver transplantation are being sought. Transplantation
of congeneic normal isolated hepatocytes into Gunn rats by infusion into the portal vein, 335 intrasplenic
injection, 336, 337 or intraperitoneal injection after attachment to microcarrier beads 338 has been used
successfully to provide partial correction of bilirubin-UGT deficiency in Gunn rats. It has been shown that
after intrasplenic injection, a great majority of the hepatocytes rapidly translocate to the liver, where, in the
absence of immune rejection, they exhibit long-term persistence. 336 The liver is the preferred site for
long-term survival and function of isolated hepatocytes. Following transplantation by intraportal infusion or
intrasplenic injection, hepatocytes migrate out of the sinusoidal space and integrate into the liver chords
within days. The transplanted cells survive and function for prolonged periods and respond to normal
proliferative stimuli. 337
Based on the extensive experience in Gunn rats, an 11-year-old girl with Crigler-Najjar syndrome was
transplanted with 7.5 billion isolated allogeneic primary human hepatocytes by infusion through a
percutaneously placed portal venous catheter. 339, 340 Tacrolimus was used for prevention of allograft
rejection. The procedure did not cause portal hypertension, and resulted in excretion of bilirubin
glucuronides in bile and led to approximately 50 percent reduction of serum bilirubin concentration over
the course of several months. The hypobilirubinemic effect persists to date 16 months after the
transplantation. Although the experience is limited to one case only, transplantation of isolated
hepatocytes appears to be a safe and relatively inexpensive alternative to liver transplantation in patients
with Crigler-Najjar syndrome type 1.
Degradation of Bilirubin by Bilirubin Oxidase.

Bilirubin oxidase from Myrothecium verrucaria catalyzes the oxidation of bilirubin with oxygen to a
colorless derivative. Perfusion of filters packed with bilirubin oxidase immobilized on agarose with rat or
human blood containing bilirubin resulted in the degradation of 90 percent of the pigment in a single
pass. 341 When blood from a Gunn rat was passed through the column and returned to the venous
circulation, the serum bilirubin level was reduced to 50 percent in 30 min. However, the use of such
columns may be associated with removal of formed elements of blood. As an alternative, systemic
administration of bilirubin oxidase has been considered. To circumvent the short half-life of bilirubin
oxidase in circulation (2.5 min), the enzyme has been covalently linked to polyethyleneglycol. 342 The
linked enzyme has a plasma half-life of 190 min in rats. A single intravenous injection of
polyethyleneglycol-conjugated bilirubin oxidase in Gunn rats resulted in substantial reduction of serum
bilirubin level for 3 h.
Induction of P450c.
As mentioned above, the induction of P450c with TCDD results in a decrease of serum bilirubin levels in
Gunn rats, presumably due to oxidation of bilirubin in the liver. This observation has stimulated the search
for more innocuous drugs for induction of this enzyme. Several naturally occurring indoles extracted from
cruciferous vegetables, such as cabbage, cauliflower, and brussels sprouts induce P4501A1 and
P4501A2 in rat liver and intestine. 343 Administration of indole-3-carbinol, an inducer of P4501A2, results
in a short-term reduction of serum bilirubin levels in children with Crigler-Najjar syndrome type 1. 343
Gene Therapy.
Because the metabolic defect in Gunn rats and in patients with Crigler-Najjar syndrome type I is caused
by molecular lesions of a single gene, introduction of a normal bilirubin-UGT would be an elegant potential
therapeutic method. Although there has been no clinical trial of gene therapy for this disease as yet,
significant progress has been made by experiments on Gunn rats. Introduction of a normal bilirubin-UGT
gene can be performed by ex vivo methods, introduction of the gene into the liver by in situ perfusion, or
systemic administration of vectors that are capable of carrying the gene to the liver. These approaches
are briefly mentioned below.
In ex vivo gene therapy, liver cells harvested from a mutant subject by partial hepatectomy are established
in primary culture and transduced with a therapeutic gene using a method that permits stable gene
expression. 344 These cells are then transplanted into the same mutant subject. This method has been
used in low-density lipoprotein (LDL) receptor-deficient rabbits (Watanabe heritable hyperlipidemic strain),
with long-term reduction of plasma LDL cholesterol concentration. Because the cells are autologous,
immunosuppression is not needed. However, the number of hepatocytes that can be harvested and
transduced is limited, and because surgical resection is required, this procedure cannot be readily
repeated. Therefore, for the treatment of Gunn rats, hepatocytes have been conditionally immortalized
before transduction with the bilirubin-UGT gene and transplantation. 345 In addition to improving gene
transduction, this strategy could potentially assure a long-term supply of phenotypically corrected
autologous hepatocytes. Additional research is required to assure that the transplanted cells should not
become transformed into malignant cells.
Methods are also being developed to directly introduce a normal bilirubin-UGT gene into the liver of
mutant Gunn rats, using viral or nonviral vectors. Recombinant murine leukemia viruses have been used
to transfer the gene into the liver by perfusing the liver with the recombinant vector after transiently
occluding inflow and outflow vessels. 346 Because gene transfer by the murine leukemia viruses requires
cell division, these vectors are not very efficient in transferring genes to the intact liver.
Adenoviral vectors have the advantage of spontaneously localizing to the liver after systemic
administration and the ability to transfer genes into nondividing cells with high efficiency. However, these
viruses are episomal and would require repeated administration for long-term gene therapy. Unfortunately,
the vectors evoke both cellular and humoral immunity in the host, precluding repeated injection. Several
approaches are being developed to tolerize the host to antigens contained in the vector. These include
administration in neonatal animals, 347 intrathymic inoculation, 348 or oral tolerization to adenoviral
antigens. 349, 350 Specific tolerization to the vector proteins permits long-term gene therapy in Gunn rats
using adenoviral vectors. Whether these methods would be effective in nonhuman primates and humans
remains to be examined. Newer viral vectors, such as recombinant SV40 and recombinant lentiviruses
that can transfer genes into nondividing cells are being developed for trial in Gunn rats.
Receptor-mediated gene delivery to the liver using carrier proteins that deliver systemically administered
DNA into the liver also have been used in the treatment of Gunn rats. 351 Transgenes introduced in this
manner are expressed transiently. The expression can be prolonged to several months by inducing cell
proliferation by partial hepatectomy 352 or by pharmacologic disruption of microtubules. 353
Site-directed gene conversion is a recently described strategy. RNA-DNA chimeric molecules have been
used in an effort to repair genetic mutations. 354 These chimeric molecules are designed to align with
specific sequences within the genome and to create a single mismatch. This triggers the host mismatch
repair system to correct the mutation. One study has reported that it may be possible to insert the missing
guanosine residue into the Gunn rat bilirubin-UGT (UGT1A1) gene by this method. 355
Crigler-Najjar Syndrome Type II (Arias Syndrome)
Clinical Findings.
Crigler-Najjar syndrome type II, otherwise known as Arias syndrome, is phenotypically similar to
Crigler-Najjar syndrome type I, except that the serum bilirubin concentration is usually below 20 mg/dl, the
prognosis is much less severe, serum bilirubin levels are usually reduced after administration of
bilirubin-UGT inducing agents, such as phenobarbital, and the bile contains significant amounts of bilirubin
glucuronides (Table 125-1). This disorder was first described by Arias in 1962 in a study of chronic
unconjugated hyperbilirubinemia in eight patients between 14 and 52 years of age. 356 Although half the
patients were icteric before the age of 1 year, one patient was 30 years old before jaundice was noted. In
these patients, serum bilirubin concentration ranged from 8 to 18 mg/dl. Each had reduced hepatic
glucuronosyltransferase activities using bilirubin, o-aminophenol, or 4-methylumbelliferone as glucuronide
acceptor. Survival of 51 Cr-labeled red cells was normal. 301 All patients were clinically normal, apart from
icterus, except for a 43-year-old woman with a neurologic syndrome resembling kernicterus. The patient
died at the age of 44. Autopsy revealed a histologically normal liver. The brain was small and lacked
bilirubin staining but demonstrated the typical histology of kernicterus.
Several other cases of neurologic abnormality in Crigler-Najjar syndrome type II have been described
subsequently. Three brothers had Crigler-Najjar syndrome type II for over 50 years. 357 Two were
neurologically normal. The third had a slight bilateral intention tremor and nonspecific abnormalities on
electroencephalogram. These nonspecific neurologic changes had not been noted previously. Another
patient was a 15-year-old boy who was icteric from the second day of life. 358 Total serum bilirubin was
24 mg/dl at 10 months and averaged approximately 15 mg/dl thereafter. Development was normal,
although psychological testing revealed a perceptual deficit and slightly subnormal intelligence. At age 13,
following surgery for acute appendicitis, the serum bilirubin increased to 40 mg/dl, and diplopia,
generalized seizures, confusion, and an abnormal electroencephalogram developed. He was treated for
hyperbilirubinemia and, after recovering from surgery, resumed a bilirubin level of 15 mg/dl. His neurologic
status returned to baseline and he has remained well.
Laboratory Tests.
As in Crigler-Najjar syndrome type I, results of laboratory examination are normal except for elevated
serum bilirubin, which is usually less than 20 mg/dl but may be as high as 40 mg/dl during fasting 357 or
intercurrent illness. 358 Serum bilirubin is unconjugated, and there is no bilirubinuria. The bile is pigmented,
although less than 50 percent of estimated daily bilirubin production is excreted into bile. 301, 358 Although
over 90 percent of conjugated bilirubin in normal bile is bilirubin diglucuronide, the major pigment in this
syndrome is bilirubin monoglucuronide. 358, 359 The liver has markedly reduced bilirubin-UGT activity. 358
Effect of Phenobarbital.
The reduced levels of hepatic bilirubin-UGT activity in Crigler-Najjar syndrome type II suggested that an
inducer of microsomal enzymes could ameliorate the hyperbilirubinemia. 356 Subsequent studies revealed
that serum bilirubin concentrations are reduced significantly (greater than 25 percent) following treatment
with phenobarbital (Fig. 125-18). 301 Similar results were obtained with other liver microsomal enzyme
inducers. 360–364 The response to phenobarbital treatment differentiates Crigler-Najjar syndrome type 1, in
which there is no response, from Crigler-Najjar syndrome type II (Fig. 125-19). 301 Although phenobarbital
has been used commonly for inducing hepatic bilirubin-UGT activity, clofibrate is equally effective and is
associated with fewer side effects. 364 In some patients, the differentiation from Crigler-Najjar syndrome
type 1 may be difficult on the basis of serum bilirubin levels and phenobarbital response. 363 In these
cases, the differentiation can be made on the basis of chromatographic analysis of pigments excreted in
bile.
Effect of phenobarbital administration on serum bilirubin concentration, menthol tolerance test, and fecal
urobilinogen excretion in a patient with Crigler-Najjar syndrome type II. (Reprinted with permission from
Arias et al. 299 )
Differentiation of types I and II Crigler-Najjar syndrome on the basis of response to phenobarbital. All
patients had chronic unconjugated hyperbilirubinemia and were treated for at least several weeks with
phenobarbital. (Reprinted with permission from Arias et al. 301 )
Inheritance.
Crigler-Najjar syndrome type II runs in families. 301, 356 There is no sex predilection. Although the pattern
of inheritance was not certain for many years, genetic analysis clearly establishes an autosomal-recessive
pattern of inheritance. In some heterozygous carriers, the coexistence with a variant promoter associated
with Gilbert syndrome may lead to intermediate levels of jaundice (see later section on Gilbert Syndrome),
which caused confusion about the mode of inheritance in the past.
Molecular Mechanism.
As in Crigler-Najjar syndrome type I, Crigler-Najjar syndrome type II is caused by mutations of one of the
five exons that encode bilirubin-UGT (UGT1A1) (Fig. 125-16). 308, 365 However, in Crigler-Najjar syndrome
type II, the genetic lesion for at least one allele always consists of point mutations that result in
substitution of a single amino acid. Such substitutions result in marked reduction, but not a total loss of
bilirubin-UGT activity. 365 Phenobarbital works presumably by induction of the residual bilirubin-UGT
activity.
Gilbert Syndrome
Clinical Findings.
The syndrome, described by Gilbert in 1901, also has been called constitutional hepatic dysfunction and
familial nonhemolytic jaundice. 366 It is characterized by mild, chronic, unconjugated hyperbilirubinemia
(Table 125-1). Familial occurrence is common, 367 although many patients present as isolated cases.
Typically, Gilbert syndrome is diagnosed in young adults, who present with mild, predominantly
unconjugated hyperbilirubinemia. Serum bilirubin levels are usually less than 3 mg/dl and fluctuate with
time. Bilirubin concentrations can increase during intercurrent illness, but can be normal at other times.
Aside from icterus, physical examination is normal. Some patients complain of vague constitutional
symptoms, including fatigue and abdominal discomfort 368 ; these symptoms are probably unrelated to
bilirubin metabolism and may be manifestations of anxiety. Newly presenting patients are rarely
symptomatic. Results of routine laboratory tests are normal except for elevated serum bilirubin
concentrations. There is no elevation of serum alkaline phosphatase or aminotransferase activities. Oral
cholecystography allows visualization of the gallbladder. Although percutaneous liver biopsy is not
routinely indicated in patients with Gilbert syndrome, liver histology is normal, except for a nonspecific
accumulation of lipofuscin pigment in the centrilobular zones. Electron microscopic studies have not
revealed consistent ultrastructural alterations. Hepatic bilirubin-UGT activity is reduced to approximately
30 percent of normal (Fig. 125-20). 369, 370
Hepatic bilirubin-UGT activity in patients with hepatitis, cirrhosis, and Gilbert syndrome. The hatched area
indicates the normal range. (Reprinted with permission from Black and Billing. 369 )
Organic Anion Transport.

Several studies of the disappearance of plasma bilirubin after intravenous injection into patients with
Gilbert syndrome have demonstrated reduced clearance (Fig. 125-21). Multicompartmental analysis
suggests that reduced plasma clearance results from reduction in hepatic bilirubin uptake as well as
bilirubin conjugation. 371 Goresky and associates determined the initial plasma disappearance of
radiolabeled bilirubin and then determined an initial space of distribution by dividing the injected dose by
the plasma volume as determined after radiolabeled albumin injection. 372 The initial plasma
disappearance of bilirubin was as rapid in patients with Gilbert syndrome as in normal subjects,
suggesting that bilirubin uptake is normal in Gilbert syndrome. Although plasma disappearance of organic
anions other than bilirubin is usually normal in Gilbert syndrome (Fig. 125-22), two subsets were
described in which BSP 373 and ICG 374 plasma disappearance is abnormal (Fig. 125-23). Because the
excretion of neither of these compounds depends on bilirubin-UGT activity, these organic anion clearance
abnormalities may not be related to the reduced bilirubin-UGT activity, which is a constant feature of
Gilbert syndrome.
Plasma disappearance of a trace dose of [3H]bilirubin after intravenous administration to patients with
Gilbert syndrome and to normal volunteers. There is no overlap between the two groups for the first 16 h
after injection. (Reprinted with permission from Berk et al. 371 )
Plasma concentration of BSP 45 min after intravenous administration of 5 mg/kg to normal individuals and
patients with Gilbert syndrome. In one subset of patients, BSP retention was elevated. (Reprinted with
permission from Berk et al. 373 )
Plasma BSP disappearance curves in three patients with Gilbert syndrome. The shaded area indicates
the normal range. Curve 1 is indistinguishable from normal. Curves 2 and 3 are representative of the two
subtypes of abnormal BSP disappearance seen in some patients with otherwise typical Gilbert syndrome.
(Reprinted with permission from Berk et al. 373 )
The serum bilirubin levels fluctuate in patients with Gilbert syndrome. Factors such as intercurrent illness,
physical exertion, and stress have been implicated, and a relationship to the menstrual cycle has been
reported in two women. 375 A 48-h fast exaggerates the unconjugated hyperbilirubinemia of Gilbert
syndrome. 376 Serum bilirubin levels in normal individuals 377 and in individuals with other hepatobiliary
disorders also increase with fasting. 378 Thus, the fasting test appears to be of limited use in the
differential diagnosis of Gilbert syndrome. The mechanism of fasting-induced hyperbilirubinemia is
unclear. Studies in normal rats revealed no change in hepatic bilirubin-UGT activity during fasting, 378
although there was reduced activity of UDP-glucose dehydrogenase resulting in reduced hepatic content
of UDP-glucuronic acid. 379 Fasting also must affect hepatic disposition of bilirubin at a step other than
conjugation, because fasting exacerbates hyperbilirubinemia in homozygous Gunn rats. 380 It may be a
result of several factors, and a role for increased serum nonesterified fatty acid concentration has been
suggested. 381
Intravenous administration of nicotinic acid also has been proposed as a provocative test for the diagnosis
of Gilbert syndrome. 382 Its diagnostic value is controversial, and it does not clearly separate patients with
Gilbert syndrome from normal subjects or those with hepatobiliary disease. Unconjugated
hyperbilirubinemia following nicotinic acid administration does not occur after splenectomy, 382 suggesting
that nicotinic acid–induced unconjugated hyperbilirubinemia may result from increased erythrocyte fragility
and enhanced splenic heme oxygenase activity, leading to augmentation of splenic bilirubin formation. 383
Conventionally, The diagnosis of Gilbert syndrome has been applied to individuals with mild unconjugated
hyperbilirubinemia without evidence of hemolysis or structural liver disease. However, the coexistence of
clinical or subclinical hemolysis may exacerbate the hyperbilirubinemia, thereby bringing the patient to the
attention of the physician. The relative content of bilirubin monoglucuronide and diglucuronide in bile is of
use in the diagnosis of Gilbert syndrome. Similar to findings in patients with Crigler-Najjar syndrome type
II and heterozygous Gunn rats, the proportion of bilirubin monoglucuronide is increased in the bile from
patients with Gilbert syndrome. Normally approximately 90 percent of bilirubin excreted in bile is in the
form of bilirubin diglucuronide, 7 percent is bilirubin monoglucuronide, and 4 percent is unconjugated
bilirubin. 359
The other significant pigment is a bilirubin monoglucoside-monoglucuronide diester. In Gilbert syndrome

the percentage of bilirubin monoglucuronide increases to 14 to 34 percent. 359
Molecular Mechanism of Gilbert Syndrome.

A variant TATAA element within the promoter region upstream to exon 1 of the gene encoding bilirubin
UGT (UGT1A1) has been found to be associated with Gilbert syndrome. 384 Sequence of the TATAA
element in normal subjects is A[TA] 6 TAA, whereas patients with Gilbert syndrome are homozygous for a
longer TATAA element, A[TA] 7 TAA (Fig. 125-16). Although this variant promoter was found in all Gilbert
syndrome patients studied in the United States and Europe, all subjects who are homozygous for the
variant TATAA box do not exhibit hyperbilirubinemia. Expression of the Gilbert genotype appears to
require a relatively high level of bilirubin production, in addition to the reduced expression of bilirubin-UGT.
For example, most patients diagnosed to have Gilbert syndrome are men, probably because the daily
production of bilirubin is greater in men than in women. The incidence of the Gilbert genotype is common
in the United States and Europe, approximately 9 percent of the general population being homozygous for
the variant promoter, and approximately half the population carrying at least one copy of the variant
promoter.
Because of the frequency of the Gilbert type promoter, some heterozygous carriers of a Crigler-Najjar
syndrome type I or II mutation are likely to carry the variant TATAA box. If the Gilbert type TATAA element
is present on the structurally normal allele of a heterozygous carrier of a Crigler-Najjar syndrome type I or
II mutation, the expression of the only normal allele will be reduced to approximately 30 percent of normal,
resulting in an intermediate level of jaundice. 385 This explains the frequent finding of intermediate levels of
hyperbilirubinemia in the family members of patients with Crigler-Najjar syndrome types I and II.
Inheritance.
Subjects who are heterozygous for the Gilbert genotype have higher average serum bilirubin
concentrations than subjects who are homozygous for the normal TATAA box. However, all patients in the
United States and Europe who were clinically diagnosed to have Gilbert syndrome were homozygous for
the variant promoter. On the basis of this, Gilbert syndrome may be considered to have an
autosomal-recessive mode of transmission.
Animal Model of Gilbert Syndrome: The Bolivian Squirrel Monkey.

The Bolivian population of squirrel monkeys (Saimiri siureus ) have a higher postcibal serum
unconjugated bilirubin concentration and a greater degree of fasting hyperbilirubinemia than does a
closely related Brazilian population. 386 Compared with the Brazilian population, Bolivian monkeys have
slower plasma clearance of intravenously administered bilirubin, a lower level of hepatic bilirubin-UGT
activity, and an increased bilirubin monoglucuronide to diglucuronide ratio in bile. 386 The two populations
of squirrel monkeys have a comparable erythrocyte life span and hepatic glutathione-S-transferase
activity. 386 In these respects, the Bolivian squirrel monkeys are a model of human Gilbert syndrome.
Fasting hyperbilirubinemia is rapidly reversed by oral or intravenous administration of carbohydrates, but
not by lipid administration. 387
DISORDERS OF BILIRUBIN METABOLISM RESULTING IN PREDOMINANTLY CONJUGATED

HYPERBILIRUBINEMIA
Dubin-Johnson Syndrome
Clinical Findings.
In 1954, Dubin and Johnson 236 and Sprinz and Nelson 388 described patients with chronic nonhemolytic
jaundice. The liver was grossly black, but the histology was normal except for an unidentified pigment in
hepatocytes. Subsequently, this disorder has been described in both sexes in virtually all nationalities and
races. 381–391 Dubin-Johnson syndrome is rare, except in Jews of Middle Eastern origin. Most reports
consist of individual cases or small groups. The largest series of 101 cases 391 was reported from Israel
on the basis of hospital records from 1955 to 1969. Of these 101 cases, 74 came from families that
immigrated from Iran, Iraq, and Afghanistan; nine were of Moroccan origin, and seven were
European-Ashkenazim. Among Jews of Persian origin, the incidence is 1:1300. 391 In this population,
Dubin-Johnson syndrome is associated with clotting factor VII deficiency. 392
The syndrome is clinically characterized by mild, predominantly conjugated hyperbilirubinemia (Table

125-2). Except for jaundice, physical examination is normal. Occasionally a patient may have
hepatosplenomegaly. Mild constitutional complaints such as vague abdominal pains and weakness occur,
but for the most part patients are asymptomatic. 389–391 Pruritus is absent in Dubin-Johnson syndrome,
and serum bile acid levels are normal. 390, 393 The degree of icterus is increased by intercurrent illness,
oral contraceptives, and pregnancy (Fig. 125-24). 390 Dubin-Johnson syndrome is rarely detected before
puberty, although cases have been reported in neonates. 394, 395 Often the disorder is not noted until a
woman becomes pregnant or receives oral contraceptives with conversion of mild chemical
hyperbilirubinemia into overt jaundice. 390
Table 125-2: Principal Differential Characteristics of Inherited Chronic Conjugated

HyperbilirubinemiasSeparate Window
Dubin-Johnson Syndrome Rotor Syndrome
Appearance of liver Grossly black Normal
Dark pigment; predominantly in

Histology of liver Normal; no increase in pigmentation
centrilobular areas; otherwise normal
Usually 1.5–5 mg/dl, occasionally as Usually elevated, occasionally as

Serum bilirubin high as 20 mg/dl; predominantly direct high as 20 mg/dl; predominantly
reacting direct reacting
Routine liver
Normal except for bilirubin Normal except for bilirubin
function tests
45-min plasma BSP Normal or elevated; secondary rise at Elevated; no secondary increase at
retention 90 min 90 min
BSP infusion T max Virtually zero; S normal T max and S both reduced
studies
Oral Usually does not visualize the

Usually visualizes the gallbladder
cholecystogram gallbladder
Urinary Normal total; >80% as coproporphyrin Elevated total; elevated proportion

coproporphyrin I of coproporphyrin I but <80%
Mode of inheritance Autosomal recessive Autosomal recessive
Prevalence Uncommon (1:1300 in Persian Jews) Rare
Prognosis Benign Benign
Animal model Mutant TR – or EHBR rats None
Mutant Corriedale sheep
Mutant golden lion tamarind monkeys
Exacerbation of conjugated hyperbilirubinemia by oral contraceptive administration in a patient with

Dubin-Johnson syndrome. Similar findings may be seen in pregnancy.
Laboratory Tests.
Routine laboratory examination 389, 391 reveals normal complete blood count, serum albumin, cholesterol,
transaminases, alkaline phosphatase, and prothrombin time. Serum bilirubin is usually between 2 and
5 mg/dl but can be as high as 20 to 25 mg/dl. Bilirubinuria is frequent, and 50 percent or more of total
serum bilirubin is conjugated. The serum bilirubin level fluctuates, and frequently individual determinations
may be normal. Both unconjugated and conjugated bilirubin accumulate in serum, and more than half of
the bilirubin gives a direct van den Bergh reaction. Prolonged retention of bilirubin glucuronides in plasma
results in the formation of irreversible adducts of bilirubin with plasma proteins, particularly albumin. The
bilirubin-albumin adduct, termed δ-bilirubin, is found in the serum of patients with Dubin-Johnson
syndrome, and various hepatobiliary disorders that are associated with prolonged conjugated
hyperbilirubinemia. δ-Bilirubin is not excreted in bile and urine and gives a direct van den Bergh reaction.
Excretion of Dyes Used for Imaging of the Biliary System.

Oral cholecystography, even using a double dose of contrast material, usually does not allow visualization
of the gallbladder, although visualization may occur 4 to 6 h after intravenous injection of iodipamide. 396
Hepatic Pigmentation.
On direct inspection, the liver is black. Light microscopy reveals normal histology except for accumulation
of a dense pigment, which on electron microscopy appears to be contained within lysosomes. 397, 398
Histochemical staining characteristics and physicochemical properties of extracted pigment resemble
those of melanin. 399, 400 In the mutant Corriedale sheep, an animal model of Dubin-Johnson syndrome,
the hepatic pigment resembles melanin histochemically. Studies performed in mutant Corriedale sheep
infused with [ 3 H]-epinephrine revealed reduced biliary excretion of radioactivity and demonstrated
incorporation of the isotope into the hepatic pigment, 401 which is consistent with the pigment being a
melanin-like derivative. However, electron spin resonance spectroscopy suggests that the Dubin-Johnson
pigment differs from authentic melanin, and could be composed of polymers of epinephrine
metabolites. 402 The TR rat, another animal model for the Dubin-Johnson syndrome, has an identical
phenotype except for the absence of pigmentation in the liver. 225 Biliary excretion of [ 3 H]-epinephrine is
also disturbed in this rat. 403 When these animals were fed a diet enriched in aromatic amino acids
(phenylalanine, tyrosine, and tryptophan), lysosomal pigmentation developed, which was absent in normal
rats. Impaired excretion of anionic metabolites of tyrosine, phenylalanine, and tryptophan in the TR − liver
may result in their retention, oxidation, polymerization, and subsequent lysosomal accumulation. 403 One
study of computerized tomography of the liver revealed that attenuation values were significantly higher in
patients with Dubin-Johnson syndrome as compared with normal controls, although there was
considerable overlap between the two groups. 404 The possible relationship of the liver cell pigment to this
finding is not known. The degree of hepatic pigmentation may be variable in individuals with the
Dubin-Johnson syndrome. Some variability in pigmentation may be due to occurrence of coincidental
disease such as acute viral hepatitis, in which the pigment is cleared from the liver only to reaccumulate
slowly after recovery. 405
Organic Anion Transport Defect.

In Dubin-Johnson syndrome, initial plasma disappearance of
bilirubin, 406, 407 BSP, 391, 407, 408 dibromosulphthalein (DBSP), 408 ICG 407, 408 and 125 I-labeled rose
bengal 408 following intravenous administration are usually normal. Of diagnostic significance is that in
approximately 90 percent of patients, the plasma BSP concentration is higher 90 min after intravenous
administration than at 45 min (Fig. 125-25). 390, 391, 395 This is due to reflux of conjugated BSP from the
liver cell into the circulation. 409 This secondary increase is not seen following intravenous administration
of other organic anions such as DBSP, 125 I-labeled rose bengal, and ICG, which are not conjugated prior
to excretion by the hepatocytes. 407, 408 A similar secondary increase has been described following
intravenous administration of unconjugated bilirubin. 402, 407 Although the secondary rise of plasma BSP is
characteristic of Dubin-Johnson syndrome, it is not diagnostic and occurs in other hepatobiliary
disorders. 410
Typical BSP plasma disappearance curve in a patient with Dubin-Johnson syndrome. A secondary
increase occurs 45 min after the intravenous injection of the dye. (Reprinted with permission from Erlinger
et al. 408 )
Studies of BSP transport during constant intravenous infusion reveal that the T max is reduced to only 10
percent of normal, and the relative hepatic storage capacity is normal. 390, 391 This finding was also
demonstrated directly in a patient with Dubin-Johnson syndrome who had a biliary fistula. 237 In this
patient, dehydrocholate choleresis did not augment biliary BSP excretion. Similar studies of BSP transport
have been performed in phenotypically normal parents and children (i.e., carriers) of Dubin-Johnson
syndrome patients, and it was found to be normal. 390
Inheritance and Urinary Coproporphyrin Excretion.

The familial nature of Dubin-Johnson syndrome was noted in its initial descriptions, but its mode of
inheritance was unclear. 389 Subsequently, it was observed that the ratio of coproporphyrin I to
coproporphyrin III excreted in the urine of patients with Dubin-Johnson syndrome is greater than that
found in other hepatobiliary disorders. 411 Of the two isomeric forms of coproporphyrin, isomer III is the
precursor of heme, whereas isomer I is a metabolic by-product without known function. 412 Coproporphyrin
isomers I and III are normally found in urine, where approximately 75 percent of total urinary
coproporphyrin is coproporphyrin III. In Dubin-Johnson syndrome, total urinary coproporphyrin excretion is
normal, but over 80 percent is coproporphyrin I. 411, 413 Urinary coproporphyrin excretion has been
determined in phenotypically normal relatives of patients with Dubin-Johnson syndrome (Fig.
125-26). 413–415 In obligate heterozygotes (i.e., unaffected parents and children of patients with
Dubin-Johnson syndrome), total urinary coproporphyrin excretion was reduced by 40 percent as
compared with normal control subjects. 413–415 This was due to a 50 percent reduction in coproporphyrin
III excretion. The proportion of coproporphyrin I in urine was intermediate between results in controls and
in patients with Dubin-Johnson syndrome. Analysis of data from studies revealed that with respect to
urinary coproporphyrin excretion, Dubin-Johnson syndrome is inherited as an autosomal-recessive
characteristic (Fig. 125-26 and Table 125-2). 414 A similar mode of inheritance was determined in a study
of BSP and bilirubin metabolism in 173 sibs of 44 patients with Dubin-Johnson syndrome. 416 No other
hepatobiliary disorder or porphyria has been described in which total urinary coproporphyrin excretion is
normal, with over 80 percent of the total as coproporphyrin I. In the presence of a consistent history and
physical examination, urinary coproporphyrin excretion appears to be diagnostic of this disorder.
Pedigree of a family in which consanguinity resulted in three children with Dubin-Johnson syndrome
(generation V). Solid symbols indicate individuals with Dubin-Johnson syndrome. Partially filled symbols
indicate phenotypically normal individuals with urinary coproporphyrin excretion in the heterozygous
range. Open symbols represent phenotypically normal individuals with normal urinary coproporphyrin
excretion. NT, individuals who were not t...
The overlap of results in carriers with those in controls 413–415 makes determination of urinary
coproporphyrin excretion less useful in deciding whether an individual carries the gene for the syndrome.
However, this disorder is benign, and genetic counseling is rarely required. Urinary coproporphyrin
excretion proved useful in diagnosing Dubin-Johnson syndrome in two neonates. 394, 395 Although
neonates normally have elevated urinary content of coproporphryin I as compared with adults, levels are
not as high as seen in Dubin-Johnson syndrome. 417
The pathogenesis of the abnormal urinary coproporphyrin excretion in this syndrome is unknown, as is its
relationship to conjugated hyperbilirubinemia. In addition to being present in urine, coproporphyrins are
also found in bile, where isomer I constitutes approximately 65 percent of the total. 412 Normally, total daily
biliary coproporphyrin excretion is approximately three times that of total daily urinary excretion. In most
hepatobiliary disorders, including cholestasis, coproporphyrin levels are increased in urine. 418 In these
disorders, total urinary coproporphyrin excretion is elevated and the proportion of isomer I in urine is
usually less than 65 percent. Dubin-Johnson syndrome is unique in that total urinary coproporphyrin is
normal, but the proportion of isomer I is over 80 percent. It seems unlikely that the abnormal pattern of
coproporphyrin isomers seen in Dubin-Johnson syndrome results simply from reduced biliary excretion,
and an alteration in hepatic porphyrin biosynthesis has been postulated (Fig. 125-27). 414, 417 Reduced
coproporphyrin III formation could result from decreased activity of hepatic uroporphyrin III
cosynthetase. 414 Enzyme activity as determined in blood cells and liver from four patients did not differ
from normal. 419 Following an intravenous load of δ-aminolevulinic acid, coproporphyrin III content of urine
and bile changed very little in patients with Dubin-Johnson syndrome, as compared with results in normal
control subjects. 420 Further study of porphyrin biosynthesis is required to elucidate the mechanism of
abnormal coproporphyrin excretion and the relationship of the porphyrin abnormality to the conjugated
hyperbilirubinemia that characterizes the syndrome.
Pathway of porphyrin biosynthesis. δ-Aminolevulinic acid (δ-ALA) condenses to form porphobilinogen

(PBG). In the presence of uroporphyrinogen synthetase, PBG forms the isomer I porphyrins, which are
excretory products without known function. On addition of uroporphyrinogen cosynthetase, PBG forms the
isomer III porphyrins, which are precursors of heme. (Reprinted with permission from Wolkoff AW, Cohen
LE, Arias IM: New Engl J M...
The differential diagnosis of Dubin-Johnson syndrome includes Rotor syndrome, another benign inherited
disorder characterized by the accumulation of both conjugated and unconjugated bilirubin in plasma (see
later section on Rotor Syndrome).
Molecular Mechanism of Dubin-Johnson Syndrome.

As discussed before, the bile canalicular transport of bilirubin glucuronides occurs against a concentration
gradient via an energy-consuming mechanism that is shared with many other organic anions, except bile
acids. The functional characteristics of canalicular non–bile acid organic anion transporter, termed the
canalicular multispecific organic anion transporter (cMOAT), have been defined largely by studies in
mutant animal models with a transport defect for organic anions. 421 cMOAT is also termed MRP2
(GenBank NM_000392).
Animal Models.
The first animal model to be described was the mutant Corriedale sheep. The metabolic defect in this
strain closely resembles that found in Dubin-Johnson syndrome. Biliary excretion of a large number of
organic anions, including conjugated bilirubin, glutathione-conjugated BSP, iopanoic acid, and ICG is
decreased, whereas taurocholate transport is normal. 235, 422 Although the biliary excretion of the
glutathione conjugates of BSP is markedly reduced, the secretion of unconjugated BSP is unimpaired. 240
Serum bilirubin levels are mildly elevated, and 60 percent of the pigments in the serum is glucuronidated.
As in Dubin-Johnson syndrome, the liver is pigmented, and the histology is otherwise normal. 422 Total
urinary coproporhyrin excretion is normal with increased excretion of coproporphyrin isomer I and
decreased isomer III excretion.
The most important animal model for Dubin-Johnson syndrome is the TR rat, also known as GY
(Groningen yellow) rat. 421 These rats have been used extensively for elucidation of the mechanism of
canalicular excretion of conjugated bilirubin and other organic anions. As in Dubin-Johnson syndrome, the
biliary excretion of conjugated bilirubin and many other organic anions is impaired, and isomer I of
coproporphyrin constitutes the major fraction of porphyrins excreted in the urine. 421 Although the liver of
TR − rats maintained on standard laboratory chow does not contain black pigments, lysosomal pigment
accumulation occurs upon feeding a diet enriched in aromatic amino acids. 403 Breeding studies indicated
autosomal-recessive inheritance for this disorder and suggested that a single gene is responsible for the
defect. For organic anions, such as glutathione-conjugated leukotriene C 4 , the canalicular secretion
defect is nearly complete, whereas for bilirubin glucuronides, there is a residual transport activity (about
10 percent of normal). 422, 423 In contrast, secretion of the synthetic compound bilirubin ditaurate is nearly
normal. 422 These observations suggest the presence of additional transport mechanisms for organic
anions that may not be affected in TR − rats.
Energy requirements for cMOAT were investigated using dinitrophenylglutathione, the transport of which
is nearly absent in TR rats. 421 Dinitrophenylglutathione transport by isolated hepatocytes 421, 424 and rat
liver plasma membrane vesicles requires ATP. 224, 425, 426 Similarly, the transport of bilirubin
glucuronide, 223 BSP, 220 cysteinyl leukotrienes 221 and p-nitrophenylglucuronide 427 in liver canalicular
membrane vesicles is also stimulated by the addition of ATP.
Substrate Specificity of cMOAT.

The susbstrate specificity of cMOAT was studied by comparison of the biliary excretion of various
compounds in normal and TR rats. From these studies a picture emerged of a transporter that recognizes
a wide variety of compounds. 421 The common denominator of these substrates is that they are anionic
amphipaths. The majority of substrates has more than one negative charge in the molecule. Among the
recognized substrates are glucuronide conjugates, like that of bilirubin and triiodothyronine, but also of
xenobiotics like naphtol. Glutathione conjugates (including its own conjugate, GSSG) are high-affinity
subtrates. The endogenous glutathione conjugate leukotriene C 4 is actually the substrate with the highest
affinity known (K m 0.25 µM) for cMOAT. Sulfate conjugates are also transported but have much lower
affinity. There are also a number of anionic substrates that are not metabolized or conjugated in the
hepatocyte and that are excreted via cMOAT. Examples of this class are dibromosulphophtalein and the
cephalosporin ceftriaxone. 421
Studies in patients with Dubin-Johnson syndrome and in related animal models (Corriedale sheep and TR
rats) have shown that bile acids are secreted by a mechanism that is distinct from that of other organic
anions. Although both types of compounds undergo primary active (ATP-dependent) transport across the
canalicular membrane, 230 their transport processes are clearly mediated by different gene products. The
mutant animals as well as patients with Dubin-Johnson syndrome excrete bile salts normally, whereas the
biliary excretion of a wide variety of other organic anions is severely impaired. Taurocholate does not
compete with the transport of bilirubin glucuronides or BSP in canalicular membrane preparations, further
indicating that these compounds use separate transport systems. 219, 220 An exception to this is the bile
salts that are conjugated at the 3-OH position. In contrast to normal bile salts, these 3-OH conjugated bile
acids have a double-negative charge and behave as non–bile acid organic anions. 225, 428 There is an
ATP-driven and a membrane potential-dependent component of bilirubin glucuronide transport by
canalicular plasma membrane vesicles. 223 The ATP-dependent mechanism is absent in membrane
vesicles from the livers of TR − rats, but the membrane potential-dependent mechanism provides the
residual transport.
A mutant strain of golden lion tamarins (Leontopitheous rosalia rosalia ) with Dubin-Johnson–like
syndrome has been described. 236
Molecular Genetics of cMOAT.

cMOAT is a member of the family of ATP-binding cassette (ABC) transporters, which are, typically, single
polypeptides with two similar halves, each containing at least four and usually six transmembrane helices
and an ABC that mediates ATP hydrolysis. 429 The cDNA for cMOAT has been cloned. 430 cMOAT is a
1541–amino acid (200-kDa) integral membrane protein, localized in the apical membrane of the
hepatocyte. The protein is highly expressed in the liver and to a much lesser extent in kidney, duodenum,
and ileum. Among the other mammalian members of the ABC transporter family, the P-glycoproteins are
best characterized. The P-glycoprotein encoded by the human MDR1 gene mediates ATP-driven
extrusion of hydrophobic compounds from cells. 431 Overexpression of this protein in tumor cells makes
them multidrug resistant. 432 Another P-glycoprotein, encoded by the MDR2 gene, is concentrated in the
canalicular domain of hepatocyte plasma membranes and is required for the translocation of
phospholipids across the canalicular membrane into the bile. 239 The multidrug resistance–related protein,
MRP1, is distantly related to the P-glycoproteins and also confers resistance against cytotoxic drugs. 232
MRP1 mediates the canalicular transport of organic anions, such as dinitrophenylglutathione and
leukotriene C 4 , the transport of which is impaired in TR rats. 233, 236 However, the expression of MRP1 in
the liver is extremely low, and this protein is localized in the basolateral domain of epithelial cell plasma
membranes, 433 suggesting that it is not involved in the canalicular transport of organic anions. Direct
evidence for the role of the cMOAT in hepatocanalicular organic anion transport came from the discovery
of a mutation in this gene in the TR − rat. 430 The deletion of a single nucleotide leads to a frameshift and a
premature stop codon, generating a truncated protein. This mutation also markedly reduces the mRNA
level. cMOAT was immunologically undetectable in the plasma membrane preparations of TR − rats.
More recently, the human cMOAT cDNA has been isolated (GenBank U49248) on the basis of homology
with the rat cMOAT, 434 and the gene has been located on chromosome 10q23-q24. 435 Human cMOAT is
a 1545–amino acid protein with 78 percent amino acid homology to the rat protein. The first mutation that
was described in a Dubin-Johnson syndrome patient was a stop codon, predicted to express a truncated
protein. 434 In this patient and a subsequently reported case, 436 immunohistochemical staining of a liver
section was negative. Two additional patients with single-base deletions and one with a missense
mutation of the cMOAT gene have been reported. 437 Because of its homology with MRP1, the cMOAT
gene is also referred to as MRP2.
Inheritance.
The male-to-female ratio in clinically diagnosed cases of Dubin-Johnson syndrome is 1.5:1, 389 perhaps
because of a greater daily bilirubin production in postpubertal males than in females. With respect to
urinary coproporphyrin excretion pattern, Dubin-Johnson syndrome is inherited as an autosomal-recessive
characteristic. 412, 414 In the TR rat, which has a mutation in the same gene, the disease is clearly inherited
in an autosomal-recessive manner. This pattern is also confirmed in several patients with Dubin-Johnson
syndrome, in whom the genetic lesions have been identified. 435, 436
Clotting Factor VII Deficiency.

In a report from Israel, 60 percent of the patients with Dubin-Johnson syndrome were found to have
reduced prothrombin activity due to lower levels of clotting Factor VII. 437 Although Factor VII deficiency
was most common among the Jews originating from Iran, Iraq, and the neighboring areas, this
abnormality was also found in some patients belonging to other Jewish communities. However, in some
families the two disorders were found to segregate independently, indicating that the combined disorder
may be coincidental and does not represent a true association. The gene for Factor VII is located on
chromosome 13, 438 whereas the cMOAT gene (MRP2) is on chromosome 10, 435 excluding a primary
linkage between the two defects.
Rotor Syndrome
Clinical Findings.
In 1948, Rotor, Manahan, and Florentin described several individuals from two families in whom there was
chronic predominantly conjugated hyperbilirubinemia without any evidence of hemolysis. 439 Serum
alkaline phosphatase and cholesterol values were normal. Plasma disappearance of BSP was greatly
delayed. Liver histology was normal. Although previously Rotor and Dubin-Johnson syndromes were
thought to be variants of a single pathophysiologic disorder, 440 now these disorders are known to be
different entities (Table 125-2). 441 Rotor syndrome is benign. The liver is normal on histologic examination
and does not have excess pigmentation. 440 Although it has been described in several nationalities and
races, Rotor syndrome is rare.
Organic Anion Excretion.

Oral cholecystographic agents usually do not visualize the gallbladder in the Dubin-Johnson syndrome,
whereas roentgenologic visualization usually is possible in Rotor syndrome. 440 Unlike the findings in
Dubin-Johnson syndrome, patients with Rotor syndrome exhibit marked retention of BSP at 45 min after
injection, but biphasic plasma BSP peaks are not found (Fig. 125-25) and conjugated BSP does not
appear in plasma (Fig. 125-28). 442 There is also marked plasma retention of intravenously administered
unconjugated bilirubin and ICG. 443
Plasma disappearance of BSP after intravenous injection of a 50 mg/kg dose into 11 patients with Rotor
syndrome, 11 phenotypically normal first-degree relatives defined as heterozygotes for the syndrome on
the basis of urinary coproporphyrin administration, and six normal controls. There was no secondary
increase of plasma BSP, and conjugated BSP was not found in plasma. (Reprinted with permission from
Wolpert et al. ...
With the use of a constant infusion technique, the transport maximum (T max ) for BSP and the relative
hepatic storage capacity have been determined in patients with Rotor and Dubin-Johnson syndromes
(Fig. 125-29). 442, 443 In Dubin-Johnson syndrome, the T max is virtually zero, whereas the hepatic storage
capacity is normal. In Rotor syndrome, the hepatic storage capacity was reduced by 75 to 90 percent and
T max was reduced by 50 percent. 442, 443 Determination of T max and relative hepatic storage capacity (S)
in phenotypically normal obligate heterozygotes revealed results intermediate between those in patients
with Rotor syndrome and controls. 442 The modest reduction in T max accompanied by a larger reduction in
hepatic storage is similar to observations in hepatic storage disease, a familial disorder manifested by
predominantly conjugated hyperbilirubinemia and normal liver histology. 444 Because there is little to
differentiate Rotor syndrome from hepatic storage disease, they may represent a single pathophysiologic
entity.
Hepatic relative storage capacity (S) and transport maximum (Tmax ) for BSP in six patients with Rotor
syndrome, five phenotypically normal heterozygotes, and six normal controls. (Reprinted with permission
from Wolpert et al. 442 )
Urinary Coproporphyrin Excretion.

Unlike results for Dubin-Johnson syndrome, total urinary coproporphyrin excretion is increased by 250 to
500 percent as compared with control subjects, and the proportion of coproporphyrin I in urine is
approximately 65 percent of total. 441, 445 In one report, however, two brothers with clinical Rotor syndrome
had over 80 percent of urinary coproporphyrins as isomer I. 446 These results are similar to those seen in
many other hepatobiliary disorders. 447 Phenotypically normal obligate heterozygotes have a
coproporphyrin excretory pattern that is intermediate between that of control subjects and patients with
Rotor syndrome. With respect to urinary coproporphyrin excretion, Rotor syndrome is inherited as an
autosomal-recessive characteristic and is distinct from Dubin-Johnson syndrome (Table 125-2, Figs.
125-30, 125-31). 445 The urinary coproporphyrin abnormality in Rotor syndrome, unlike that in
Dubin-Johnson syndrome, is most likely caused by a reduced biliary excretion of coproporphyrins, with
consequent increase in renal excretion. The nature of the organic anion transport defect in Rotor
syndrome is unknown.
Pedigree of a Philippine family originally described by Rotor in 1948. Solid symbols indicate individuals
with Rotor syndrome. Partial symbols indicate phenotypically normal individuals with urinary
coproporphyrin excretion in the heterozygote range. Open symbols represent phenotypically normal
individuals with normal urinary coproporphyrin excretion. NT, individuals who were not tested. (Reprinted
with permission from Wolkoff et al. ...
Urinary coproporphyrin excretion in Dubin-Johnson and Rotor syndromes. The shaded bars represent the
percentage of total urinary coproporphyrin excreted as coproporphyrin I. The open bars represent total
urinary coproporphyrin excretion. Vertical bars represent 1 SEM. Total urinary coproporphyrin excretion is
normal in Dubin-Johnson syndrome (DJS), with a markedly elevated proportion of coproporphyrin I
(greater than 80 percent). Both variab...
Progressive Familial Intrahepatic Cholestasis

Progressive familial intrahepatic cholestasis (PFIC) is a heterogeneous group of autosomal-recessive
inherited diseases in infants and children. All are characterized by cholestasis associated with elevated
plasma levels of bile acids, defective bile acid secretion in bile, growth failure, and progressive liver
damage initially manifested by conjugated hyperbilirubinemia. On the basis of molecular genetic studies,
PFIC has been divided into three categories. The availability of DNA expressed sequence tags (ESTs)
generated by the Human Genome Project enabled identification of large and frequently inbred families
with individual disorders. The findings of the molecular genetic studies overlapped temporally with the
discovery of specific ATP-dependent transporters in the bile canaliculus.
Progressive familial intrahepatic cholestasis type I (Byler disease) was described in an old order Amish
pedigree of seven generations descended from Jacob Byler. 448 The disease is manifested by variable
conjugated hyperbilirubinemia, growth retardation, and progressive, fatal cholestasis. In some patients,
intestinal lipid malabsorption is present and not completely ameliorated by liver transplantation. Plasma
bile acids are elevated, particularly lithocholate. No unique cholestatic bile acids or metabolites have been
demonstrated in blood or urine.
The defective gene was mapped to chromosome 18q21 by screening of the genome for chromosomal
sequences shared by patients from the original Byler kindred. Probability calculations indicate that such
sharing is unlikely to occur by chance. A mutated gene, designated FIC1, that codes for a P-type ATPase
was identified (GenBank AF038007). 449 Northern hybridization reveals expression of the normal gene in
intestine, liver, and other tissues. Its cellular and subcellular sites have not yet been identified. How
defective FIC1 results in cholestasis and abnormal bile acid transport is not known. Normal P-ATPases
couple hydrolyis of ATP to the translocation of acidic phospholipids (i.e., phosphatidylserine,
phosphatidylethanolamine) from the outer to the inner layer of the plasma membrane of many different
cells. Alterations in hepatic or intestinal membrane lipids may affect the function of ATP-dependent
tranporters for bile acids, conjugated bilirubin, and other ligands.
Benign Recurrent Intrahepatic Cholestasis (BRIC).

This rare disease was described in 1959 450 and is characterized by recurrent episodes of cholestasis
followed by complete return to normalcy clinically, biochemically, and by liver histology. 451–453 The
disorder may begin in infancy or middle age but predominantly is manifested in adolescence or early
adulthood. 452, 454 Episodes last from several weeks to months, during which time patients manifest
malaise, anorexia, pruritus, weight loss, malabsorption, conjugated hyperbilirubinemia, and biochemical
evidence of cholestasis without severe hepatocellular injury. 453–455 Intervals between attacks may last
from a few weeks to several years. In a given patient, recurrent attacks resemble each other in symptoms,
signs, and duration. Liver histology reveals noninflammatory intrahepatic cholestasis without fibrosis
regardless of the number and severity of attacks. During remission, liver histology returns to normal
whether examined by light or electron microscopy. 456
The pathogenesis of this rare disorder is unknown. Recessive inheritance has been demonstrated by
family studies in isolated populations in which the disorder is frequently observed. There is no specific
treatment to prevent the occurrence of cholestatic episodes or to shorten their duration. Liver
transplantation has not been performed because of the episodic and nonprogressive course of the
disease.
Unexpectedly, mutated FIC1 was identified in BRIC by searching for chromosomal sequences shared by
only three distantly related patients. 449 Although BRIC and PFIC I have many clinical features in common,
the former is progressive whereas the latter is intermittent. How the different phenotypes result from the
same genetic defect is unknown.
Progressive Familial Intrahepatic Cholestasis Type II.

A second form of PFIC resembles Byler disease clinically but occurs in non-Byler families, mainly in the
Middle East and Europe. Homozygosity mapping and genome search in unrelated pedigrees associated
the disorder with chromosome 2q24, from which the gene for SPGP (sister of P-glycoprotein) was cloned
(GenBank AF091582). 457 Remarkably, at almost the same time, SPGP was shown to be a canalicular
ATP-dependent bile acid transporter. 238 Over 20 different point mutations in SPGP have been described
in patients with PFIC of this type. Liver transplantation ameliorates all manifestations of the disease in
concordance with the finding that SPGP is expressed only in the liver.
Progressive Familial Intrahepatic Cholestasis Type III.

A third PFIC group involves mutations in the loci encoding class III multidrug resistance (MDR)
P-glycoproteins (mdr2 in mice and MDR3 in humans), which mediate ATP-dependent translocation of
phosphatidylcholine from the inner to the outer leaflet of the bile canalicular plasma membrane. 231
Removal of mdr2 by homologous recombination in mice results in absence of phosphatidylcholine from
bile, progressive destruction of small bile ducts, cholestasis characterized by conjugated
hyperbilirubinemia, and eventual biliary cirrhosis. In mdr2 −/− mice, bile acids are secreted normally by
SPGP but are not incorporated into mixed micelles and progressively damage small bile ducts. 430
Two different point mutations in MDR3 resulting in a nonfunctioning protein were described in two children
with PFIC associated with elevated serum levels of γ-glutamyl transpeptidase activity. 458 Virtual absence
of MDR3 mRNA occurs in Navajo Indian children with PFIC and dysmyelinating peripheral and central
neuropathy. 459 The relationship between MDR3 and neuropathy is unknown. Elevated serum γ-glutamyl
transpeptidase activity is proposed to result from small bile duct damage and distinguishes this PFIC
group from PFIC I and BRIC.
Alagille Syndrome
Many heritable developmental disorders have been described; however, the Alagille syndrome is the first
to be described at the molecular level. The Alagille syndrome is transmitted as an autosomal-recessive
inherited characteristic that includes paucity or absence of small bile ducts resulting in progressive
intrahepatic cholestasis, and abnormalities of the eye, heart, and vertebrae. Identification of rare patients
with cytogenic deletions permitted mapping of the gene to chromosome 20p12, from which JAG1 was
identified. 460 JAG1 encodes an unidentified ligand that binds to the notch receptor, which is crucial for cell
fate development in Drosophila and mammals. Analysis of patients with Alagille syndrome who do not
have chromosomal deletions (see Chap. 65) revealed several point mutations in JAG1 (GenBank
U73936), each of which abolishes expression of the altered allele. 460
ACKNOWLEDGMENT
The authors thank Dr. Ajit Kadakol for assistance in preparation of this review. This work was supported in
part by National Institutes of Health Grants DK-46057, DK-39137, DK-41296, DK-23026, DK-35652, and
DK-34926.
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Hereditary Jaundice Cap125

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Chapter 125: Hereditary Jaundice and Disorders of Bilirubin Metabolism

PART 13: PORPHYRINS

Chapter 125: Hereditary Jaundice and Disorders of Bilirubin Metabolism

Mechanism of Opening of the Heme Ring.

Mechanism of heme ring opening and subsequent reduction of biliverdin to bilirubin.

Conversion of Biliverdin to Bilirubin.

Quantification of Bilirubin Production.

Pharmacologic Inhibition of Bilirubin Production.

Physical Conformation and Solubility of Bilirubin IXα.

Ultraviolet/Visible Absorption Spectra.

Helical Conformation of Bilirubin and Biliverdin.

Geometric Isomerization and Cyclization.

Photooxidation and Degradation.

Biochemical Mechanisms of Bilirubin Toxicity.

Clinical Features of Bilirubin Encephalopathy.

Role of the Blood–Brain Barrier.

Binding of Bilirubin to Albumin

Free bilirubin concentration is determined from the equilibrium equation

tetramethyl-1-oxyl-4-piperidinyl)5-N-(1-aspartate)-2,4,-dinitrobenzene] 119 has been used to determine

Uptake of Bilirubin by the Liver

Intrahepatocellular Storage of Bilirubin

Enzyme-Catalyzed Glucuronidation of Bilirubin.

enhanced by low concentrations of UDP-N-acetylglucosamine, which may be a physiologic activator of the

Structure and Expression of UGT Isoforms.

Differential Expression of UGT Isoforms.

Quantification of Bilirubin and Its Conjugates

Methods Involving Conversion of Bilirubin to azo Derivatives.

Separation and Quantification of Intact Bilirubin Tetrapyrroles.

Biliary Secretion of Bilirubin

Fate of Bilirubin in the Gastrointestinal Tract

Alternative Pathways of Bilirubin Elimination

Disposition of Bilirubin by the Kidney.

Antioxidative Property of Bilirubin

DISORDERS OF BILIRUBIN METABOLISM RESULTING IN UNCONJUGATED

Hepatic Bilirubin Uptake During the Neonatal Period.

Postnatal Development of Bilirubin-Glucuronidating Activity.

Inhibition of Bilirubin Glucuronidation by Maternal Milk.

Inhibition of Bilirubin Glucuronidation by Factors Derived from Maternal Plasma.

Immaturity of Canalicular Excretion.

Management of Neonatal Unconjugated Hyperbilirubinemia.

Hyperbilirubinemia Due to Bilirubin Overproduction

Crigler-Najjar Syndrome Type I

Table 125-1: Principal Differential Characteristics of Inherited Unconjugated Hyperbilirubinemia

Histology of liver Normal Normal Normal

Routine liver function test

Usually normal; may be

Homozygous Bolivian squirrel

Molecular Defect in Crigler-Najjar Syndrome Type 1.

intron 1 and splice acceptor region...

Animal Model for Crigler-Najjar Syndrome Type 1: The Gunn Rat.

Urinary Concentration Defect.

Abnormalities of UGT Activity and Their Relationship to Genetic Lesions.

Treatment of Crigler-Najjar Syndrome Type 1.

Degradation of Bilirubin by Bilirubin Oxidase.

Crigler-Najjar Syndrome Type II (Arias Syndrome)

Organic Anion Transport.

The other significant pigment is a bilirubin monoglucoside-monoglucuronide diester. In Gilbert syndrome

Molecular Mechanism of Gilbert Syndrome.

Animal Model of Gilbert Syndrome: The Bolivian Squirrel Monkey.

DISORDERS OF BILIRUBIN METABOLISM RESULTING IN PREDOMINANTLY CONJUGATED

The syndrome is clinically characterized by mild, predominantly conjugated hyperbilirubinemia (Table

Table 125-2: Principal Differential Characteristics of Inherited Chronic Conjugated

Dubin-Johnson Syndrome Rotor Syndrome