Article hematology
Neonatal Jaundice: Bilirubin
Physiology and Clinical Chemistry
Ronald J. Wong, MD,*
David K. Stevenson, MD,*
Charles E. Ahlfors, CE,*
Hendrik J. Vreman, MD*
Author Disclosure
Drs Wong, Stevenson,
and Vreman and
Mr Ahlfors did not
disclose any financial
relationships relevant
to this article.
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Objectives After completing this article, readers should be able to:
1. Identify the sources and chemical forms of bilirubin in the body.
2. Delineate the normal metabolic pathways for bilirubin production.
3. Describe how bilirubin is transported in the body.
4. Delineate the normal metabolic pathway for bilirubin excretion.
5. Differentiate and appreciate the limitations of the various methods used to measure
bilirubin.
Abstract
Cleavage of the alpha-methene bridge of heme by membrane-bound heme oxygenase
yields equimolar amounts of biliverdin, carbon monoxide, and reduced iron. Biliver-
din is catalyzed by biliverdin reductase to bilirubin. The process occurs in all nucleated
cells except mature anucleated red blood cells. Neonates in whom bilirubin produc-
tion is increased tend to have higher bilirubin concentrations, and excessive bilirubin
production or impairment of elimination causes dramatic deviations from the hour-
specific nomogram that can be seen as “jumping” percentile tracks early in the
postnatal period or later in the first week after birth. After formation, bilirubin diffuses
into the circulation. In the absence of conjugates, the total bilirubin concentration in
plasma is the sum of bilirubin bound to albumin plus a minimal amount of free
bilirubin. Bilirubin is excreted more slowly in newborns than in adults. Although no
clinical tests can measure bilirubin uptake and conjugation by the liver, an elevated
hour-specific total bilirubin value when bilirubin production is normal or decreasing is
a sign of impaired or abnormally delayed bilirubin excretion. The accuracy and
precision of clinical laboratory total bilirubin measurements are a concern, and studies
are underway to assess whether measurements of free bilirubin, the bilirubin-binding
constant, the bilirubin:albumin ratio, or albumin binding capacity might improve the
ability to identify infants at greater risk for bilirubin-induced neuroinjury rather than
simply those at greater risk for having a higher bilirubin concentration.
Introduction
Neonatal jaundice is one of the most common clinical phenomena encountered in
newborns. It is caused by the accumulation in the skin and sclerae of the yellow-orange
pigment bilirubin (4Z,15Z-bilirubin-IX-alpha), the predominant isomer that is formed
naturally from the degradation of heme in mammals. Hyperbilirubinemia causes jaundice
and, in the newborn, reflects the relative lack of bilirubin glucuronidation by the immature
liver during the transitional period after birth. With increasing conjugation in the maturing
infant (or in cholestatic states), the blood contains not only unconjugated bilirubin bound
to albumin and a small amount of free bilirubin, but also bilirubin glucuronides and
biliproteins (delta-bilirubin), the latter of which results from the covalent bonding
between conjugated bilirubin and albumin. (1) Because hyperbilirubinemia in the new-
born is largely due to unconjugated bilirubin, this type of jaundice is practically uncom-
plicated in most infants in the first several days after birth. An elevated “direct” fraction of
bilirubin, however, signals the need for an expanded differential diagnosis and other
*Department of Pediatrics, Division of Neonatal and Developmental Medicine, Stanford University School of Medicine, Stanford,
Calif.

diagnostic tests. Thus, cholestatic jaundice of the new-
born, although it can be seen in the context of hemolysis
and phototherapy, is beyond the scope of this article.
Bilirubin Production
The source of bilirubin is singular, involving a phyloge-
netically ancient enzyme, heme oxygenase (HO), which
initiates the first of a two-step heme (ferriprotoporphyrin
IX) degradation process in mammals. (2) The process is
nearly ubiquitous, occurring in all nucleated cells except
mature anucleated red blood cells (RBCs), which lack
this enzymatic system for degrading heme. The reticu-
loendothelial system, in particular the spleen, is impor-
tant in degrading heme from hemoglobin-containing,
senescing RBCs. Thus, continuous culling of aged RBCs
by the spleen is a major contributor to the production of
bilirubin in the body under normal conditions. In fact,
approximately 80% of heme is derived from this source.
The remainder is derived from the processes of ineffec-
tive erythropoiesis and the turnover of nonhemoglobin
hemoproteins (eg, myoglobin, catalase, nitric oxide syn-
thase, peroxidases, and cytochromes).
Isotopic labeling studies have aided in our under-
standing of the sources of heme that eventually yield
bilirubin. Two pools of 14C-labeled bilirubin (an “early-
labeled” or “shunt” bilirubin pool and a “late-labeled”
pool) appear when labeled heme precursors (14C-labeled
glycine- and delta-amino-levulinic acid) are administered
to animals. (3) The early-labeled pool is produced from
the turnover of nonerythropoietic heme precursors, he-
moprotein turnover, and ineffective erythropoiesis. The
late-labeled pool appears when RBCs containing labeled
heme become senescent and are degraded.
The schematized results in Figure 1 were obtained
using fetal and adult RBCs, which have shorter and
longer life spans, respectively, in various animal models.
Although these two pools may differ quantitatively in the
various animal models compared with humans, because
of the differences in the relative life spans of the fetal and
adult RBCs in the various species, qualitatively the hu-
man circumstance is likely to be comparable because
human fetal RBCs also have a shorter life span (approx-
imately 70 d) compared with human adult RBCs (ap-
proximately 120 d).
The concentration of the two well-described HO
isozymes, HO-1 and HO-2, (4) varies between tissues.
For example, HO-1 predominates in spleen and HO-2
predominates in the brain. (5) HO-2 is considered the
constitutive (or “housekeeping”) enzyme; it is develop-
mentally regulated, and its expression appears to be
influenced only by steroids. In contrast, HO-1 is induc-
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hematology bilirubin physiology
Figure 1. A scheme for the origin of bilirubin. The major
portion of bilirubin appears at 100 days from the degradation
of heme of senescent RBCs. At 3 to 5 days, bilirubin arises
from the heme loss from ineffective erythropoiesis. The
components present throughout the first 24 hours are asso-
ciated with the turnover of major tissue hemoproteins (II),
while the earliest fraction (I) is derived from a rapid turnover
of the pool of hemoproteins or heme precursor. Modified from
Israels et al. (3) and reprinted with permission.
ible by its substrate heme and various other nonheme
substances and stress conditions, (5) reflecting a very
complex set of biologic roles. A putative isoenzyme,
HO-3, also has been reported as largely homologous
with HO-2, but with uncertain activity and an unknown
role. (6)
Although the role of HO as the initial and rate-
limiting step in the production of bilirubin is relevant to
understanding neonatal jaundice, it is important to rec-
ognize that the biology of HO is old and probably
essential to much life on this planet because of the
physical and chemical exigencies of light, oxygen, and
plentiful iron. In particular, the role of HO in the evolu-
tion of the human species invites intriguing speculation
about the influences of pathogens on the human ge-
nome, such as the protozoan Plasmodium falciparum,
which causes malaria. (7) Infection with this agent cer-
tainly would involve increased expression of HO-1 in
response to the heme loads caused by hemolysis. Al-
though the role in the actual pathogenesis of the infec-
tion remains speculative, the fact that there is great
variation in HO-1 expression in humans related to (GT)n
polymorphism of the HO-1 gene promoter, with long
(GT)n repeats influencing basal activity or inducibility of
NeoReviews Vol.8 No.2 February 2007 e59
hematology bilirubin physiology
Figure 2. Heme catabolic pathway. COⴝcarbon monoxide,
Feⴝiron, NADPHⴝnicotinamide adenine dinucleotide phos-
phate (reduced form). Reprinted with permission from Steven-
son DK. American Pediatric Society Presidential Address 2006:
science on the edge with life in the balance. Pediatr Res.
2006;60:630 – 635.
the HO-1 gene promoter, and, in particular, promoter
activity decreasing with increasing (GT) repeat numbers,
is intriguing in this context. (8)(9) Perhaps host differ-
ences in HO-1 expression contribute to the individual
vulnerability to certain infections, such as malaria, (7)
and the predilection for pregnancy disorders. (10) More-
over, HO-1 expression may be generally important in
antioxidant defense, and it may have roles in other con-
ditions that involve oxidant stress, such as hypoxia, heat
shock, and disorders of aging, including cardiovascular,
neurologic, and renal diseases. (7)
Cleavage of the alpha-methene bridge of heme by
membrane-bound HO is the initiating event in heme
catabolism (Fig. 2) and ultimately yields equimolar
amounts of green-colored biliverdin, carbon monoxide
(CO), and reduced iron (Fe2⫹). (2) The reaction mech-
anism is complex and requires three molecules of oxygen
and at least seven electrons donated from the nicotin-
amide adenine dinucleotide phosphate (NADPH)-
cytochrome-P-450 reductase system. The actual cleav-
age of the tetrapyrrole macrocycle containing iron is
autocatalytic in the sense that the bound heme serves
both as a prosthetic group and a substrate. Biliverdin is
reduced rapidly in the cytoplasm, catalyzed by biliverdin
reductase to bilirubin, thus linking CO production and
bilirubin production. (11)
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The stoichiometry of the reaction presents the oppor-
tunity to use CO production as an index of biliverdin/
bilirubin production. CO, an important signaling mole-
cule in its own right, (12) has the potential for interacting
with other heme-containing compounds, including
many important enzymes. CO diffuses easily into blood,
binding with the hemoglobin in RBCs to form carboxy-
hemoglobin (COHb). In the lung, the high concentra-
tion of oxygen displaces CO from COHb, allowing it to
diffuse into the alveoli and be exhaled continuously in
breath. The reduced iron is rendered harmless by binding
to ferritin, the major iron storage protein. On a body
weight basis, a newborn produces about two to three
times as much bilirubin as an adult, thus setting the stage
for neonatal jaundice because the capacity to eliminate
the pigment is temporarily diminished after birth com-
pared with an adult. (13)
In the human newborn, many pathologic conditions
increase bilirubin production, and neonates in whom
bilirubin production is increased are inclined to have
higher bilirubin concentrations, depending on their ca-
pacity to eliminate the pigment. Even the hour-specific
nomogram (see the accompanying article in this issue by
Keren and Bhutani) is informed by bilirubin production,
with infants tracking in the high quartiles having, on the
average, higher bilirubin production because all infants
have relatively impaired elimination. (14) Excessive bili-
rubin production or impairment of elimination causes
dramatic deviations from the nomogram that can be
observed easily as “jumping” percentile tracks early in the
postnatal period (typically related to increased bilirubin
production) or later in the first week after birth (typically
related to impaired elimination) (Fig. 3). Excessive bili-
rubin production has many causes, but one of the more
serious and relatively common ones is maternofetal
blood group incompatibility, with a positive Coombs
test contributing to increased RBC degradation and
bilirubin production.
Although the hour-specific bilirubin nomogram is a
convenient method for early detection of babies who
have increased bilirubin production (because such in-
fants “jump” to higher percentile tracks), a more specific
method for detecting increased bilirubin production is to
measure CO in exhaled air. (15)(16) Neonates who have
abnormally high rates of bilirubin production have been
shown to have abnormally high CO excretion rates or
end-tidal breath CO values, corrected for ambient CO.
Increased bilirubin production sets the stage not only
for transitional bilirubin physiology and pathophysiol-
ogy, but increases the bilirubin load that must distribute
into a variety of body compartments, including the cir-

Figure 3. Black lines on the hour-specific bilirubin nomogram represent infants “jump- in addition to total bilirubin has the
ing” percentile tracks for each risk group. Modified from Bhutani et al (14) and reprinted potential to provide useful informa-
with permission.
culation and body tissues, based on biochemical princi-
ples and a variety of other influences on this biochemis-
try. Nonetheless, the more bilirubin that is produced, the
more bilirubin that potentially can distribute within the
body of an individual and the more risk for a toxic
reaction, depending on the vulnerability of the exposed
tissues. Bilirubin toxicity also is beyond the scope of this
article, but must be understood as a potential adverse
effect of the pathologic accumulation of bilirubin that
exceeds the very much lower concentrations in cells,
tissues, or circulation and contributes to antioxidant
defense. (17)(18)(19)
Bilirubin Transport
After bilirubin is formed, it diffuses into the circulation,
where it binds reversibly to albumin (bilirubin:albumin).
The equilibrium concentration of the nonalbumin-
bound or “free” bilirubin is about three orders of mag-
nitude less than the bilirubin:albumin concentration. In
the absence of conjugates, the total bilirubin concentra-
tion in plasma is the sum of bilirubin:albumin plus a
minimal amount of free bilirubin concentrations. Al-
though the bilirubin:albumin complex may be thought
of as a reservoir of bilirubin that potentially can leave the
circulation and enter tissues, free bilirubin governs the
speed with which bilirubin in circulation actually enters
the tissues and the steady-state distribution of bilirubin
between the circulation and the tissues. Because albumin
concentrations and bilirubin-albumin binding constants
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hematology bilirubin physiology
vary considerably among babies,
the free bilirubin at any given bili-
rubin concentration in circulation
can vary greatly, making it difficult
to assess bilirubin movement into
tissues. (20)(21)(22)(23)(24)
The bilirubin concentration in
circulation, measured as serum or
plasma total bilirubin, is the con-
ventional clinical laboratory mea-
surement on which the treatment
of newborn jaundice is based be-
cause most newborns have nominal
concentrations of direct-reacting
bilirubin, which includes all biliru-
bin moieties except bilirubin bound
to albumin and free bilirubin.
However, measuring free bilirubin
tion about the relative amount of
bilirubin that is outside the circula-
tion in tissues, in particular, the brain. Importantly,
“low” total bilirubin concentrations are not always due
to lower-than-normal concentrations of bilirubin in the
body (that is, the miscible bilirubin pool, which is the
bilirubin in circulation plus the bilirubin in tissue).
Rather, they could be due to weaker-than-normal
bilirubin-albumin binding, with a higher proportion of
bilirubin residing outside the circulation in tissue, includ-
ing the brain.
Measuring the free bilirubin in addition to the hour-
specific total bilirubin and estimating bilirubin produc-
tion using CO measurements can be helpful in under-
standing the risk faced by some babies because they have
a large bilirubin load distributed in part outside the
circulation. Thus, it is possible that some babies who
have “low” total bilirubin values are at risk and others
who have “high” total bilirubin values are not, depend-
ing on the load and distribution of that load. Routine
measurement of free bilirubin is presently not available,
but measurements of albumin and the total bilirubin:
albumin ratio can assist in identifying newborns who may
have compromised bilirubin transport. The total
bilirubin/albumin molar ratio at which intervention is
recommended increases with increasing gestational age,
but in otherwise well term newborns, it should be below
0.8 (about 7 mg of bilirubin per gram of albumin).
Although the free bilirubin concentration determines
the movement of bilirubin from the circulation into
tissue, less is known about the in vivo variables that affect
NeoReviews Vol.8 No.2 February 2007 e61
hematology bilirubin physiology
exposure of the brain to bilirubin, including the various
transporters involved with pumping bilirubin out of the
central nervous system. Other relevant factors include
the intracellular metabolism of bilirubin (eg, bilirubin
oxidase catalyzed oxidation of intracellular bilirubin to
nontoxic biliverdin, colorless diazo-negative com-
pounds, and monopyrrole propentdyopents). Because
such factors are not accessible clinically, bilirubin toxicity
is determined by clinical means, including physical exam-
ination (eg, lethargy, irritability, and opisthotonus), au-
ditory brainstem response, and magnetic resonance im-
aging, which may show changes in the basal ganglia and
hippocampus.
Bilirubin Excretion
Bilirubin is excreted more slowly in the newborn com-
pared with the adult because of a transiently slower rate
of hepatic uptake of free bilirubin from the blood and
decreased concentrations of uridine diphosphogluc-
uronate glucuronosyltransferase (UGT), the enzyme
that catalyzes conjugation of bilirubin with glucuronic
acid to form water-soluble, nonneurotoxic bilirubin
mono- and diglucuronides, which are required for bili-
rubin excretion. Although excretion can occur with mono-
glucuronides, diglucuronides facilitate bilirubin excre-
tion further. As free bilirubin uptake and conjugation
improve over the first 2 weeks after birth, increasing
amounts of conjugated bilirubin enter the bile ducts and
are excreted with the bile into the intestinal tract. In the
distal segments of the intestinal tract, primarily in the
colon, bacterial flora progressively hydrogenate bilirubin
to urobilinogen, urobilins, and stercobilins. (25) Biliru-
bin oxidase is also present in the liver and intestinal
mucosa, but whether bilirubin oxidation by bilirubin
oxidase contributes significantly to bilirubin excretion is
unknown.
Bilirubin monoglucuronide and bilirubin digluc-
uronide may be deconjugated in the gut by beta-
glucuronidase to bilirubin, which then is reabsorbed into
the bloodstream. This process, known as the “entero-
hepatic bilirubin circulation,” is common in newborns
and is affected by breastfeeding and infant nutritional
status. The meconium accumulating in the gut in utero
also contains about 100 to 200 mg of bilirubin per 100 g
of meconium at birth, of which 50% or more is uncon-
jugated. (25) Thus, the miscible pool of bilirubin is
affected not only by the relative rates of bilirubin produc-
tion and removal from the circulation by the liver, but
also by the rates of enterohepatic circulation and meco-
nium excretion.
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No clinical tests are available to measure bilirubin
uptake and conjugation by the liver, but an elevated
hour-specific total bilirubin value when bilirubin produc-
tion is normal or even decreasing, as it should be after
birth, is a sign of abnormally delayed bilirubin excretion.
Although it is tempting to ascribe abnormally high hour-
specific total bilirubin in the presence of a normal biliru-
bin production rate to impaired excretion or a normal
total bilirubin in the presence of increased bilirubin pro-
duction to enhanced bilirubin excretion, the effect of
bilirubin-albumin binding on the total bilirubin should
not be overlooked. For example, an abnormally high
hour-specific bilirubin might occur when both bilirubin
production and excretion are normal if bilirubin-albumin
binding is very strong.
Two genetic conditions (Crigler-Najjar disease and
Gilbert syndrome) are associated with abnormal bilirubin
excretion, (26)(27)(28)(29) and the risk of affected in-
fants developing neonatal hyperbilirubinemia. (30)(31)
In Gilbert disease, known polymorphisms involve the
UGT1A1 promoter sequence. (32) There is also a poly-
morphism (G71R) that is more common in Asians and
results in deficient conjugation. (33)(34) However,
there are also variants in the gene polymorphism for the
organic anion transporting protein (OATP2), which can
impair uptake into the liver. (35) Interactions of these
genetic predispositions may contribute to the hyperbil-
irubinemia seen in certain well-known syndromes, such
as human milk jaundice, as well as a variety of other
conditions of the newborn that involve increased biliru-
bin production, (36) most of which are equivalent, but
some of which are genetic in origin, such as the natural
variations in HO-1 expression.
Clearly, the interaction of genes that impede bilirubin
elimination from the body combined with any factors
that increase bilirubin production creates the most dan-
gerous circumstance for the neonate. Some gene inter-
actions present serious challenges, such as the com-
bined presence of glucose-6-phosphate dehydrogenase
deficiency and Gilbert syndrome. (32)(37)(38) Other
genetic variants can affect bilirubin excretion and are
less likely to complicate the neonatal syndrome of
indirect-reacting hyperbilirubinemia, but still are factors
in the overall excretion of bilirubin. Such deficiencies
include the canalicular multispecific organic anion
transporter (cMOAT), also known as the multidrug
resistance-associated protein 2 (MRP-2), which is asso-
ciated with cholestasis in the Dubin-Johnson syn-
drome. (39)
 hematology bilirubin physiology

Table. Bilirubin Nomenclature and Chemical Behavior

Terms
Unconjugated Bilirubin or Indirect Bilirubin
Conjugated Bilirubin or Direct Bilirubin
Albumin-bound Bilirubin or Bound Bilirubin
Unbound Bilirubin or Free Bilirubin or
Nonalbumin-bound Bilirubin
Delta Bilirubin
Configurational Bilirubin Photoisomers
Lumirubin; Structural Bilirubin Photoisomer
Description
Produced by heme degradation by heme oxygenase; indirect bilirubin
requires an “accelerator” to react with diazo reagents
Bilirubin conjugated with one or two glucuronic acid molecules; direct
bilirubin forms diazo derivatives immediately after the addition of diazo
reagents
Usually refers to bilirubin bound to albumin, but bilirubin
monoglucuronide or bilirubin diglucuronides also bind to albumin
Usually refers to bilirubin not bound to albumin but also includes bilirubin
monoglucuronide or bilirubin diglucuronides not bound to albumin
Bilirubin covalently bound to albumin (reacts with direct bilirubin fraction
of the diazo test)
Photolabile isomers of bilirubin occurring on exposure to light of
wavelength 400 to 525 nm
Photolabile isomers of bilirubin occurring on exposure to light of
wavelength 400 to 525 nm

Measurement of Bilirubin Direct spectrophotometry, fluorimetry, and high-

Total bilirubin traditionally was measured in serum, but
it now is measured more commonly in plasma because
most blood samples are obtained and centrifuged in
tubes containing anticoagulants (eg, heparin, EDTA, or
oxalate). (40) In addition, gels often are used to separate
the serum or plasma from the RBCs and buffy coat, but
the concentrations of bilirubin and its derivatives do not
appear to be affected by the type of liquid matrix. For the
sake of procedural and analytical accuracy, however, the
proper matrix always should be identified.
More than a century ago, Ehrlich (41) discovered that
bilirubin reacts with diazotized p-nitrobenzoic acid to
form ruby-colored azo-bilirubin. Van den Bergh (42)
studied this reaction further and developed it into a
clinical assay still used today that allows identification and
quantitation of the unconjugated (indirect) and conju-
gated (direct) bilirubin fractions (Table).
The traditional method involves direct and indirect
reactions with the diazo reagent, yielding colored biliru-
bin derivatives, which can be quantitated spectrophoto-
metrically. Conjugated bilirubin (mono- and digluc-
uronides) and delta-bilirubin react directly with the diazo
reagent (the so-called “direct”-reacting bilirubin). When
an accelerant, such as caffeine or DMSO, is added to the
in vitro diazo reaction mixture, unconjugated bilirubin
(bilirubin:albumin and unbound bilirubin) is energized
to react with the diazo reagent as well to form similar
colored products (the so-called “indirect” reaction bili-
rubin). Thus, “indirect” bilirubin concentrations are de-
rived from the total bilirubin concentration minus the
direct bilirubin concentration. Total bilirubin is the sum
of all bilirubin derivatives in a sample.
pressure liquid chromatography (HPLC) also are used to
measure plasma bilirubin, its isomers, and derivatives.
Although HPLC has proven to be a powerful technique
for demonstrating and quantifying the various species of
bilirubin present, it is not suitable for routine clinical use.
The accuracy and precision of clinical laboratory total
bilirubin measurements are a continuing concern. Sev-
eral groups have studied interlaboratory variability of
total bilirubin measurements made with automated clin-
ical analyzers using artificial sera containing bilirubin and
have found room for improvement with better standard-
ization. (43)(44)(45) Appropriate clinical intervention
should be based only on accurate bilirubin fraction mea-
surements, but this is not always the case at present. (45)
Plasma (and serum) also may contain various photoprod-
ucts of bilirubin and glucuronides, which may interfere
with some or all of the methods used to measure bilirubin
fractions. (46) The relative proportions of these products
are age- and subject-dependent. In addition, the admin-
istration of drugs, (47) nutrients (human milk), and
treatments, such as phototherapy or exchange transfu-
sion, may affect the relative concentrations of bilirubin
compounds in circulation and tissues unpredictably. (46)
Several studies suggest that measuring free bilirubin
would be useful in assessing the jaundiced newborn.
(48)(49) However, free bilirubin quantitation is not
widely available from clinical laboratories, even though a
relatively simple, United States Food and Drug
Administration-approved method for its measurement
has been available for decades. (50) A newer, less labor-
intensive semi-automated device has been introduced
(51) and is being evaluated for clinical use. (52)

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hematology bilirubin physiology
Studies are underway to assess whether measurements
of free bilirubin, the bilirubin-binding constant, the bil-
irubin:albumin ratio, or albumin binding capacity might
improve the ability to identify individuals who are at
greater risk for bilirubin-mediated neuroinjury rather
than simply at greater risk for having a higher bilirubin
concentration, which is what the hour-specific total bili-
rubin nomogram does. (14) However, the relationship
between bilirubin values and bilirubin toxicity is variable
and influenced by other factors, such as binding of bili-
rubin to albumin. Unbound (or “free”) bilirubin is more
likely to cross the blood-brain barrier and cause brain
injury. Most bilirubin normally is bound to albumin,
resulting in low concentrations of unbound bilirubin.
For patients who have high bilirubin concentrations, the
capacity of albumin to bind bilirubin is exceeded, leading
to higher concentrations of unbound bilirubin. The ratio
of bilirubin to albumin (B/A) can be used as an approx-
imate surrogate for unbound bilirubin. B/A ratio
(ⱖ6.8) is used only in conjunction with measurements of
bilirubin to determine whether an exchange transfusion
should be initiated, as outlined in the 2004 American
Academy of Pediatrics guideline, according to the in-
fant’s age and risk factors. (53) Preterm and sick infants
often have decreased serum albumin concentrations and
a reduced binding capacity, resulting in a higher propor-
tion of unbound bilirubin for a given bilirubin value
compared with healthy term infants. As a result, in these
patients, a lower B/A ratio or bilirubin threshold is used
to initiate therapy. Drugs such as sulfisoxazole, moxalac-
tam, and ceftriaxone can displace bilirubin from albumin
and increase the risk of bilirubin toxicity. Acidosis in-
creases movement of bilirubin into tissues, including the
brain and, thus, can contribute to the development of
bilirubin-induced brain injury.
Although bilirubin can be measured in other materials
(eg, meconium, bile, stool, urine, and cerebrospinal
fluid) and could provide additional clinical information,
how such information might be used currently is un-
known.
Less than 2% of the miscible bilirubin pool is con-
tained in the skin of the congenitally jaundiced Gunn rat.
(54) The percentage of the miscible pool in the skin of
the jaundiced human newborn is probably similar. Re-
gardless, transcutaneous bilirubin measurements have
been reported by many investigators to correlate with
total bilirubin concentrations in plasma, even in pig-
mented newborns. (55) The noninvasive nature of the
technology makes it easier to perform serial measure-
ments than bilirubin measurements and monitor the rate
at which the total bilirubin is changing in an hour-
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specific manner over time. The measurement of yellow-
orange color in the skin is, at best, a surrogate for total
bilirubin in circulation. Although it may be technically
possible using optical technology to measure the latter,
current technology provides only an index of total bili-
rubin and should be used as an indication for appropriate
blood sampling and testing.
Riskin and associates (56)(57) suggested that the
visual assessment of jaundice by an experienced clinician
may be reasonably reliable and may diminish the need for
universal blood sampling. However, it is impossible to
standardize visual assessments for the many variables
encountered, such as lighting, skin color, color percep-
tion, clinical experience, and training, and this approach
should be used with extreme caution. (58)(59) The
bottom line is that visual inspection is inadequate for
estimating the total bilirubin in circulation once a child is
jaundiced from head to toe.
Conclusion
Bilirubin is not only a waste product, but also a versatile
molecule that has an essential role in cellular metabolism.
It is one product of a catabolic pathway that is essential
for life on this planet. Although variations in HO-1
expression occur, it is probable that mammalian life as
well as many other forms of life on this planet, including
plant life, would not be possible without such an enzy-
matic system, although it may come in different forms.
Without some mechanism to deal with oxygen, light, and
iron, life would literally “burn out.” Thus, bilirubin is an
important molecule to measure in living things, includ-
ing newborn babies. Its measurement is not trivial, and
further improvements in measurement are required.
More importantly, however, is the understanding of the
beneficial effect of bilirubin as an antioxidant as well as its
toxic effects. The latter are known to occur, but the
mechanisms involved are still being investigated. Any
strategy to prevent such bilirubin-related injury must
begin with a sound understanding of bilirubin physiol-
ogy and clinical chemistry. Understanding the cellular
biology of heme catabolism and defining the roles of this
ancient and elegant set of reactions in other developmen-
tal and pathological circumstances remains a scientific
challenge.
References
1. Wu TW. Bilirubin analysis – the state of the art and future
prospects. Clin Biochem. 1984;17:221–229
2. Tenhunen R, Marver HS, Schmid R. The enzymatic conversion
of heme to bilirubin by microsomal heme oxygenase. Proc Natl
Acad Sci USA. 1968;61:748 –755

3. Israels LG, Levitt M, Novak W, Zipursky A. The early bilirubin.
Medicine (Baltimore). 1966;45:517–521
4. Maines MD. Heme oxygenase: function, multiplicity, regulatory
mechanisms, and clinical applications. FASEB J. 1988;2:
2557–2268
5. Maines MD. The heme oxygenase system: a regulator of second
messenger gases. Annu Rev Pharmacol Toxicol. 1997;37:517–554
6. McCoubrey WK Jr, Huang TJ, Maines MD. Isolation and
characterization of a cDNA from the rat brain that encodes hemo-
protein heme oxygenase-3. Eur J Biochem. 1997;247:725–732
7. Shibahara S, Kitamuro T, Takahashi K. Heme degradation and
human disease: diversity is the soul of life. Antioxid Redox Signal.
2002;4:593– 602
8. Saikawa Y, Kaneda H, Yue L, et al. Structural evidence of
genomic exon-deletion mediated by Alu-Alu recombination in a
human case with heme oxygenase-1 deficiency. Hum Mutat. 2000;
16:178 –179
9. Yamada N, Yamaya M, Okinaga S, et al. Microsatellite polymor-
phism in the heme oxygenase-1 gene promoter is associated with
susceptibility to emphysema. Am J Hum Genet. 2000;66:187–195
10. Denschlag D, Marculescu R, Unfried G, et al. The size of a
microsatellite polymorphism of the haem oxygenase 1 gene is
associated with idiopathic recurrent miscarriage. Mol Hum Reprod.
2004;10:211–214
11. Vreman HJ, Wong RJ, Stevenson DK. Sources, sinks, and
measurements of carbon monoxide. In: Wang R, ed. Carbon Mon-
oxide and Cardiovascular Functions. Boca Raton, Fla: CRC Press;
2001:273–307
12. Marks GS, Brien JF, Nakatsu K, McLaughlin BE. Does carbon
monoxide have a physiological function? Trends Pharmacol Sci.
1991;12:185–188
13. Maisels MJ, Pathak A, Nelson NM, Nathan DG, Smith CA.
Endogenous production of carbon monoxide in normal and eryth-
roblastotic newborn infants. J Clin Invest. 1971;50:1– 8
14. Bhutani VK, Johnson L, Sivieri EM. Predictive ability of a
predischarge hour-specific serum bilirubin for subsequent signifi-
cant hyperbilirubinemia in healthy term and near-term newborns.
Pediatrics. 1999;103:6 –14
15. Bartoletti AL, Stevenson DK, Ostrander CR, Johnson JD.
Pulmonary excretion of carbon monoxide in the human infant as an
index of bilirubin production. I. Effects of gestational and postnatal
age and some common neonatal abnormalities. J Pediatr. 1979;94:
952–955
16. Vreman HJ, Baxter LM, Stone RT, Stevenson DK. Evaluation
of a fully automated end-tidal carbon monoxide instrument for
breath analysis. Clin Chem. 1996;42:50 –56
17. Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames
BN. Bilirubin is an antioxidant of possible physiological impor-
tance. Science. 1987;235:1043–1046
18. Vreman HJ, Wong RJ, Sanesi CA, Dennery PA, Stevenson
DK. Simultaneous production of carbon monoxide and thiobarbi-
turic acid reactive substances in rat tissue preparations by an iron-
ascorbate system. Can J Physiol Pharmacol. 1998;76:1057–1065
19. Dore S, Takahashi M, Ferris CD, et al. Bilirubin, formed by
activation of heme oxygenase-2, protects neurons against oxidative
stress injury. Proc Natl Acad Sci USA. 1999;96:2445–2450
20. Wennberg RP. The importance of free bilirubin acid salt in
bilirubin uptake by erythrocytes and mitochondria. Pediatr Res.
1988;23:443– 447
21. Brodersen R, Stern L. Deposition of bilirubin acid in the
Downloaded from http://neoreviews.aappublications.org/ by guest on May 2, 2018
hematology bilirubin physiology
central nervous system – a hypothesis for the development of
kernicterus. Acta Paediatr Scand. 1990;79:12–19
22. Bratlid D. Criteria for treatment of neonatal jaundice. J Peri-
natol. 2001;21 (suppl 1):S88 –S92
23. Wennberg RP, Ahlfors CE, Bhutani VK, Johnson LH, Shapiro
SM. Toward understanding kernicterus: a challenge to improve the
management of jaundiced newborns. Pediatrics. 2006;117:
474 – 485
24. McDonagh AF, Maisels MJ. Bilirubin unbound: deja vu all
over again? Pediatrics. 2006;117:523–525
25. Odell GB. Normal metabolism of bilirubin during neonatal
life. In: Neonatal Hyperbilirubinemia. New York, NY: Grune &
Stratton, Inc; 1980:42– 49
26. Beutler E, Gelbart T, Demina A. Racial variability in the
UDP-glucuronosyltransferase 1 (UGT1A1) promoter: a balanced
polymorphism for regulation of bilirubin metabolism? Proc Natl
Acad Sci USA. 1998;95:8170 – 8174
27. Bosma PJ, Chowdhury JR, Bakker C, et al. The genetic basis of
the reduced expression of bilirubin UDP-glucuronosyltransferase 1
in Gilbert’s syndrome. N Engl J Med. 1995;333:1171–1175
28. Koiwai O, Nishizawa M, Hasada K, et al. Gilbert’s syndrome is
caused by a heterozygous missense mutation in the gene for biliru-
bin UDP-glucuronosyltransferase. Hum Mol Genet. 1995;4:
1183–1186
29. Kadakol A, Ghosh SS, Sappal BS, Sharma G, Chowdhury JR,
Chowdhury NR. Genetic lesions of bilirubin uridine-diphospho-
glucuronate glucuronosyltransferase (UGT1A1) causing Crigler-
Najjar and Gilbert syndromes: correlation of genotype to phenotype.
Hum Mutat. 2000;16:297–306
30. Watchko JF, Daood MJ, Biniwale M. Understanding neonatal
hyperbilirubinaemia in the era of genomics. Semin Neonatol. 2002;
7:143–152
31. Kaplan M, Hammerman C, Maisels MJ. Bilirubin genetics for
the nongeneticist: hereditary defects of neonatal bilirubin conjuga-
tion. Pediatrics. 2003;111:886 – 893
32. Kaplan M, Hammerman C, Rubaltelli FF, et al. Hemolysis and
bilirubin conjugation in association with UDP-glucuronosyltrans-
ferase 1A1 promoter polymorphism. Hepatology. 2002;35:905–911
33. Yamamoto K, Sato H, Fujiyama Y, Doida Y, Bamba T. Con-
tribution of two missense mutations (G71R and Y486D) of the
bilirubin UDP glycosyltransferase (UGT1A1) gene to phenotypes
of Gilbert’s syndrome and Crigler-Najjar syndrome type II. Biochim
Biophys Acta. 1998;1406:267–273
34. Kanai M, Akaba K, Sasaki A, et al. Neonatal hyperbilirubinemia
in Japanese neonates: analysis of the heme oxygenase-1 gene and
fetal hemoglobin composition in cord blood. Pediatr Res. 2003;54:
165–171
35. Huang MJ, Kua KE, Teng HC, Tang KS, Weng HW, Huang
CS. Risk factors for severe hyperbilirubinemia in neonates. Pediatr
Res. 2004;56:682– 689
36. Watchko JF. Genetics and the risk of neonatal hyperbiliru-
binemia: commentary on the article by Huang et al. on page 682.
Pediatr Res. 2004;56:677– 678
37. Kaplan M, Hammerman C, Beutler E. Hyperbilirubinaemia,
glucose-6-phosphate dehydrogenase deficiency and Gilbert syn-
drome. Eur J Pediatr. 2001;160:195
38. Kaplan M, Muraca M, Hammerman C, et al. Imbalance be-
tween production and conjugation of bilirubin: a fundamental
concept in the mechanism of neonatal jaundice. Pediatrics. 2002;
110:e47. Available at: http://pediatrics.aappublications.org/cgi/
content/full/110/4/e47
NeoReviews Vol.8 No.2 February 2007 e65
hematology bilirubin physiology

39. Kartenbeck J, Leuschner U, Mayer R, Keppler D. Absence of
the canalicular isoform of the MRP gene-encoded conjugate export
pump from the hepatocytes in Dubin-Johnson syndrome. Hepatol-
ogy. 1996;23:1061–1066
40. Vreman HJ, Wong RJ, Stevenson DK. Phototherapy: current
methods and future directions. Semin Perinatol. 2004;28:326 –333
41. Ehrlich P. Sulfondiazolenzol am reagenz auf bilirubin. Centr
Klin Med. 1883;4:721
42. Van den Bergh AAH, Muller P. Uber eine directe und eine
indirekte diazoreaktion auf bilirubin. Biochem Z. 1916;77:90
43. Valaes T. Problems with prediction of neonatal hyperbiliru-
binemia. Pediatrics. 2001;108:175–177
44. Vreman HJ, Verter J, Oh W, et al. Interlaboratory variability of
bilirubin measurements. Clin Chem. 1996;42:869 – 873
45. Lo SF, Doumas BT, Ashwood ER. Bilirubin proficiency testing
using specimens containing unconjugated bilirubin and human
serum: results of a College of American Pathologists study. Arch
Pathol Lab Med. 2004;128:1219 –1223
46. McDonagh AF. Ex uno plures: the concealed complexity of
bilirubin species in neonatal blood samples. Pediatrics. 2006;118:
1185–1187
47. Strauss KA, Robinson DL, Vreman HJ, Puffenberger EG, Hart
G, Morton DH. Management of hyperbilirubinemia and preven-
tion of kernicterus in 20 patients with Crigler-Najjar disease. Eur
J Pediatr. 2006;165:306 –319
48. Ahlfors CE, Shapiro SM. Auditory brainstem response and
unbound bilirubin in jaundiced (jj) Gunn rat pups. Biol Neonate.
2001;80:158 –162
49. Ahlfors CE, Wennberg RP. Bilirubin-albumin binding and
neonatal jaundice. Semin Perinatol. 2004;28:334 –339
50. Nakamura H, Lee Y. Microdetermination of unbound biliru-
bin in icteric newborn sera: an enzymatic method employing per-
oxidase and glucose oxidase. Clin Chim Acta. 1977;79:411– 417
51. Ahlfors CE, Marshall GD, Wolcott DK, Olson DC, Van Over-
meire B. Measurement of unbound bilirubin by the peroxidase test
using Zone Fluidics. Clin Chim Acta. 2006;365:78 – 85
52. Ahlfors CE, Vreman HJ, Wong RJ, et al. Effects of sample
dilution, peroxidase concentration, and chloride ion on the mea-
surement of unbound bilirubin in premature newborns. Clin Bio-
chem. 2006; doi:10.1016/j.clinbiochem. Epublished ahead of print
53. American Academy of Pediatrics. Management of hyperbiliru-
binemia in the newborn infant 35 or more weeks of gestation.
Pediatrics. 2004;114:297–316
54. Bessard G, Chouraqui JP, Rambaud P. A spectrophotometric
assay of skin bilirubin in adult Gunn rats. Biol Neonate. 1982;42:
59 – 65
55. Rubaltelli FF, Gourley GR, Loskamp N, et al. Transcutaneous
bilirubin measurement: a multicenter evaluation of a new device.
Pediatrics. 2001;107:1264 –1271
56. Riskin A, Abend-Weinger M, Bader D. How accurate are
neonatologists in identifying clinical jaundice in newborns? Clin
Pediatr (Phila). 2003;42:153–158
57. Riskin A, Kugelman A, Abend-Weinger M, Green M, Hemo
M, Bader D. In the eye of the beholder: how accurate is clinical
estimation of jaundice in newborns? Acta Paediatr. 2003;92:574 –576
58. Stevenson DK, Wong RJ, Vreman HJ. Reduction in hospital
readmission rates for hyperbilirubinemia is associated with use of
transcutaneous bilirubin measurements. Clin Chem. 2005;51:
481– 482
59. Maisels MJ, Kring E. Transcutaneous bilirubin levels in the first
96 hours in a normal newborn population of ⱖ35 weeks’ gestation.
Pediatrics. 2006;117:1169 –1173

NeoReviews Quiz
1. Isotopic labeling studies have aided in our understanding of the sources of heme that yield bilirubin. Of the
following, the earliest fraction of bilirubin is derived from:
A. Heme degradation from senescent erythrocytes.
B. Ineffective erythropoiesis.
C. Turnover of heme precursors.
D. Turnover of nonhemoglobin hemoproteins.
E. Turnover of tissue enzymes.

2. Heme oxygenase (HO) is a phylogenetically ancient enzyme that initiates the first step in the degradation
of heme and the resultant production of bilirubin. Of the two HO isozymes, HO-1 is inducible and HO-2 is
constitutive. Of the following, the MOST predominant expression of the constitutive HO-2 isozyme occurs
in the:
A. Brain.
B. Heart.
C. Kidney.
D. Liver.
E. Spleen.

e66 NeoReviews Vol.8 No.2 February 2007

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 hematology bilirubin physiology

3. Although the measurement of free bilirubin is presently not available for routine clinical use, an alternative
measurement of the total bilirubin:albumin molar ratio can assist in identifying newborns who may have
compromised bilirubin transport. Of the following, the threshold for total bilirubin:albumin molar ratio in
jaundiced, but otherwise well term neonates is closest to:
A. 0.4.
B. 0.8.
C. 1.2.
D. 1.6.
E. 2.0.

4. Bilirubin conjugated in the liver and secreted into bile can be deconjugated in the gut, from where it then
is reabsorbed into the bloodstream, resulting in a process termed enterohepatic bilirubin circulation. Of the
following, the enzyme most responsible for contributing to enterohepatic bilirubin circulation is:
A. Beta-glucuronidase.
B. Bilirubin hydrogenase.
C. Bilirubin oxidase.
D. Biliverdin reductase.
E. Uridine diphosphoglucuronate glucuronosyltransferase.

NeoReviews Vol.8 No.2 February 2007 e67

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 Neonatal Jaundice: Bilirubin Physiology and Clinical Chemistry
Ronald J. Wong, David K. Stevenson, Charles E. Ahlfors and Hendrik J. Vreman
NeoReviews 2007;8;e58
DOI: 10.1542/neo.8-2-e58

Updated Information & including high resolution figures, can be found at:
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 Neonatal Jaundice: Bilirubin Physiology and Clinical Chemistry
Ronald J. Wong, David K. Stevenson, Charles E. Ahlfors and Hendrik J. Vreman
NeoReviews 2007;8;e58
DOI: 10.1542/neo.8-2-e58

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