Clin Chem Lab Med 2021; 59(6): 1025–1033

Mini Review

Mercy Thomas*, Winita Hardikar, Ronda F. Greaves, David G. Tingay, Tze Ping Loh,
Vera Ignjatovic, Fiona Newall and Anushi E. Rajapaksa

Mechanism of bilirubin elimination in urine:

insights and prospects for neonatal jaundice
https://doi.org/10.1515/cclm-2020-1759
Received November 26, 2020; accepted December 22, 2020;
published online January 15, 2021
Abstract: Despite a century of research, bilirubin meta-
bolism and the transport mechanisms responsible for ho-
meostasis of bilirubin in serum remain controversial.
*Corresponding author: Mercy Thomas, RN RM MSN, New Vaccines,
Murdoch Children’s Research Institute, Melbourne, Victoria,
Australia; Department of Paediatrics, University of Melbourne,
Melbourne, Australia; Newborn Research Centre, Royal Women’s
Hospital, Melbourne, Victoria, Australia; Department of Nursing, Royal
Children’s Hospital, Melbourne, Victoria, Australia; and Murdoch
Children’s Research Institute, The Royal Children’s Hospital 50
Flemington Rd, Parkville, Victoria 3052, Australia,
E-mail: mercy.thomas@mcri.edu.au
Winita Hardikar, Department of Paediatrics, University of Melbourne,
Melbourne, Australia; Department of Gastroenterology, Royal
Children’s Hospital, Melbourne, Victoria, Australia; and Clinical
Sciences, Murdoch Children’s Research Institute, Melbourne,
Victoria, Australia
Ronda F. Greaves, Department of Paediatrics, University of
Melbourne, Melbourne, Australia; School of Health and Biomedical
Sciences, RMIT University, Melbourne, Victoria, Australia; and
Victorian Clinical Genetics Services, Melbourne, Victoria, Australia.
https://orcid.org/0000-0001-7823-8797
David G. Tingay, Department of Paediatrics, University of Melbourne,
Melbourne, Australia; Newborn Research Centre, Royal Women’s
Hospital, Melbourne, Victoria, Australia; Neonatal Research, Murdoch
Children’s Research Institute, Melbourne, Victoria, Australia; and
Department of Neonatology, Royal Children’s Hospital, Melbourne,
Victoria, Australia
Tze Ping Loh, Department of Laboratory Medicine, National University
Hospital, Singapore, Singapore
Vera Ignjatovic, Department of Paediatrics, University of Melbourne,
Melbourne, Australia; and Haematology Research, Murdoch
Children’s Research Institute, Melbourne, Victoria, Australia
Fiona Newall, Department of Paediatrics, University of Melbourne,
Melbourne, Australia; and Department of Nursing, Royal Children’s
Hospital, Melbourne, Victoria, Australia
Anushi E. Rajapaksa, New Vaccines, Murdoch Children’s Research
Institute, Melbourne, Victoria, Australia; Department of Paediatrics,
University of Melbourne, Melbourne, Australia; and Newborn
Research Centre, Royal Women’s Hospital, Melbourne, Victoria,
Australia
Emerging evidence on the hepatic membrane transporters
and inherited disorders of bilirubin metabolism have
contributed to a greater understanding of the various steps
involved in bilirubin homeostasis and its associated
excretory pathways. We discuss these recent research
findings on hepatic membrane transporters and evaluate
their significance on the newborn bilirubin metabolism
and excretion. New insights gained speculate that a pro-
portion of conjugated bilirubin is excreted via the renal
system, as an alternative to the intestinal excretion, even in
normal physiological jaundice with no associated patho-
logical concerns. Finally, this paper discusses the clinical
relevance of targeting the altered renal excretory pathway,
as bilirubin in urine may hold diagnostic importance in
screening for neonatal jaundice.
Keywords: bilirubin metabolism; bilirubin – urine; conju-
gated bilirubin; hepatic membrane transporters; neonatal
jaundice; unconjugated bilirubin.
Introduction
Neonatal jaundice has been a significant topic of interest in
medicine for centuries [1]. Bilirubin, the key biomarker
responsible for jaundice peaks in the first three to four
days of neonatal life before gradually decreasing, and is
considered a transient physiological phenomenon [2]. Of
particular concern is when total serum bilirubin concen-
tration remains elevated, and at times if undetected can
lead to neurological complications such as acute bilirubin
encephalopathy and kernicterus [3].
Bilirubin metabolism is a complex process that in-
volves several organs, multiple membrane transport sys-
tems and numerous enzymes. A large body of research over
the last few decades describes the complex mechanisms
involved in newborn bilirubin metabolism and the detri-
mental pathophysiological impact of altered bilirubin
metabolism [4–7]. Recent related studies have provided a
greater understanding of the hepatic membrane trans-
porters (OPATP1B1/OATP1B3, MRP2 and MRP3) responsible
1026 Thomas et al.: Bilirubin elimination in newborns
for the transportation and clearance of bilirubin [8–13].
Analysis of the age-related differences in hepatic membrane
transporters demonstrated low expression of these trans-
porters in newborns compared to adults [14]. This new
knowledge on age related differences in these hepatic
membrane transporters combined with the emerging
research on these transporters led to the speculation that, a
proportion of conjugated bilirubin is excreted via the renal
system as an alternative to intestinal excretion, even in
normal physiological jaundice with no associated patho-
logical concerns [10, 12, 15].
Excretion of bilirubin in urine is not considered as a
normal physiological phenomenon, traditionally being
associated with pathological problems of bilirubin meta-
bolism. The focus of this paper is to describe the possible
mechanisms responsible for the elimination of bilirubin in
urine in neonates and its associated clinical significance.
In the following sections, we have provided details related
to bilirubin formation and metabolism of bilirubin in ne-
onates with the new insights gained on hepatic membrane
transporters in the excretion of conjugated bilirubin, and
the speculations inferred from these new findings.
Figure 1: Bilirubin metabolism – a schematic presentation on the production, transportation, conjugation and clearance of bilirubin.
Newborn differences are highlighted with additional index.
Bilirubin formation and clinical
implications
Bilirubin is a by-product of haemoglobin metabolism,
where the haem component of haemoglobin is oxidised by
haem oxygenase and converts the haem into a green
pigment known as biliverdin (Figure 1). An enzyme bili-
verdin reductase reduces biliverdin into a water-insoluble
yellow pigment known as unconjugated bilirubin [16–18].
The average concentration of bilirubin production in neo-
nates ranges between 6 and 8 mg/kg/day, of which 80% is
produced from RBC destruction [17, 19, 20]. The remaining
20% is derived from ineffective erythropoiesis and turnover
from other haemoproteins such as myoglobin, cyto-
chromes and catalases [19].
Unconjugated bilirubin production is high in neo-
nates, at approximately double the rate in adults, due to
the rapid breakdown of foetal haemoglobin, higher hae-
moglobin mass and a shorter RBC life span [7, 21]. In utero,
the foetus uses the high concentration of foetal haemo-
globin to more efficiently obtain oxygen from maternal

haemoglobin through the placenta [22]. Post-birth,
once breathing is established, neonates receive ample ox-
ygen, thus rapidly reducing the need for high foetal hae-
moglobin concentration. Subsequently, there is increased
red blood cell destruction and haemoglobin metabolism
leading to increased production of unconjugated bilirubin
in neonates [22].
The chemistry of unconjugated bilirubin (ZZ mole-
cule – the natural bilirubin) is such that, the extremely
compacted intra-molecular hydrogen bonding makes the
bilirubin poorly soluble in water [23, 24]. Therefore, the
ZZ-natural bilirubin requires biotransformation to become
more water-soluble for excretion. This unconjugated bili-
rubin is processed in the liver to become more water-
soluble bilirubin (conjugated bilirubin) for excretion in the
bile [25]. The unconjugated bilirubin also when exposed to
light, isomerisation of ZZ bilirubin occurs and results in the
formation of EZ, ZE and EE isomers. This isomerisation
results in less intra-molecular hydrogen bonding, making
the bilirubin more water-soluble for excretion, a key factor
responsible for the outcome of phototherapy. Hence the E
isomers of bilirubin are efficiently excreted from the cir-
culation via urine, although the major pathway for excre-
tion of bilirubin is still through bile [24, 26, 27].
Knowledge of this bilirubin chemistry is essential in
understanding the different forms of bilirubin that could be
excreted via bile as well as through urine. This paper will
focus on the two major forms of bilirubin, conjugated and
unconjugated bilirubin.
Bilirubin induced complications
Total serum bilirubin concentration, an average of 86–
103 μmol/L is common among neonates in the first few days
of life and is considered to be physiological [16]. One in 10
healthy term neonates without any other risk factors, still
progress to clinically significant hyperbilirubinemia,
where the total serum bilirubin(TSB) concentration ex-
ceeds 250 μmol/L requiring monitoring and timely
interventions [28, 29]. The rise in serum bilirubin can also
be intensified in preterm and other high-risk group neo-
nates including neonates with erythroblastosis fetalis or Rh
incompatibility [30].
Unconjugated bilirubin, being hydrophobic as well
highly lipophilic, tends to accumulate in tissues and can
cross the neonatal blood-brain barrier [31]. Physiologically,
unconjugated bilirubin is bound to albumin in serum, and
this albumin-bilirubin complex limits the movement of
bilirubin to tissues [4, 5, 23]. Conversely, with increased
bilirubin overload among certain neonates, there is
Thomas et al.: Bilirubin elimination in newborns 1027
insufficient bilirubin binding sites on albumin, resulting in
high proportion of unbound bilirubin (free unconjugated
bilirubin) in the systemic circulation [32, 33]. The binding
capacity of bilirubin to albumin could also be affected in
altered pH circumstances including respiratory and meta-
bolic acidosis that are commonly observed among pre-
mature neonates, resulting in high unbound bilirubin
among premature neonates [32].
There are several studies that have explored bilirubin-
albumin binding ratio and the significance of free bilirubin
in management of jaundice [32, 34, 35]. Currently, there are
no commercial methods available in determining free
bilirubin. Instead, the measurement of bilirubin-albumin
ratio is considered as an alternate parameter in measuring
free bilirubin [32]. However, current laboratory methods in
measuring bilirubin and albumin need better stand-
ardisation before bilirubin-albumin ratio can be more
consistently applied clinically [36]
Free unconjugated bilirubin is potentially toxic to
brain tissue, especially in high concentrations (severe
hyperbilirubinemia) [33, 37]. Unconjugated bilirubin gets
deposited in the basal ganglia and various brainstem
nuclei of the brain leading to athetoid cerebral palsy,
impairment of upward gaze, deafness or hearing loss and
corresponds to the pathologic lesions in the brain result-
ing from the amount and duration of exposure to free
bilirubin deposition [37, 38]. “Kernicterus Spectrum Dis-
orders (KSDs)” is described as a spectrum of neurological
sequelae associated with extreme hyperbilirubinemia in
neonates [39, 40].
Conjugated serum bilirubin concentration of approxi-
mately 6–12 μmol/L is considered to be normal [41], and
any concentration above 17.1 μmol/L requires further in-
vestigations to explore the underlying pathology [42–44].
Conjugated hyperbilirubinemia indicates an obstruction of
the biliary tract, reduced hepatobiliary excretory function
or defective hepatic transporter protiens available for the
clearance of bilirubin [43, 45].
An understanding of the types of bilirubin and their
concentration in various biological fluids helps to explain
the underlying abnormality in bilirubin metabolism.
Current paradigms of bilirubin
metabolism in newborns
This section will briefly discuss four critical steps involved
in the process of bilirubin metabolism in newborns incor-
porating new knowledge in the various stages of bilirubin
uptake through to its eventual excretion (Figure 1).
1028 Thomas et al.: Bilirubin elimination in newborns
Bilirubin uptake by the liver
Several studies have examined the transport mechanisms
responsible for hepatocyte uptake of bilirubin; however,
the transporters accountable for this uptake are not fully
understood [4, 46]. Once the albumin-bound unconjugated
bilirubin reaches the microcirculation of the liver sinu-
soids, it separates from albumin at the space of Disse and
enters the hepatocyte, postulated to be partly mediated by
a diffusion process and partly by an unknown transporter
[5, 12]. Once, inside the hepatocyte, the unconjugated
bilirubin binds to ligandin, a protein that belongs to
glutathione S-transferase (GST) family for transportation
to the endoplasmic reticulum of the hepatocyte [4]. Bind-
ing to ligandin keeps the bilirubin in the cytosol of hepa-
tocyte and reduces the efflux of this molecule from the cell
[12, 46].
Recent research exploring the age-related differences
in hepatic membrane transporters revealed the fraction of
GST is 1.5 to four times lower in newborns compared to
adults [11]. Consequently, the binding of unconjugated
bilirubin to GST is limited and besides with increased
bilirubin production, a proportion of free unconjugated
bilirubin remains within the hepatocytes [7, 12]. Kaplan
(2002) also demonstrated that there is a bidirectional
movement of unconjugated bilirubin across the sinusoidal
membrane of the hepatocytes and hence, the unbound
unconjugated bilirubin, rather than accumulating in he-
patocytes, may transfer back into the blood through the
sinusoidal membrane [47]. This transfer of unconjugated
bilirubin back into the sinusoidal blood is one of the factors
contributing to the rise in serum unconjugated bilirubin in
newborns [10, 47].
Bilirubin conjugation
Conjugation is a critical step in bilirubin metabolism as it
converts unconjugated bilirubin into bilirubin glucuro-
nides, a water-soluble form for subsequent excretion.
Conjugation is a complex two-step process mediated by
enzyme uridine diphosphate-glucuronosyltransferase
(UGT1A1) present in the endoplasmic reticulum of the he-
patocyte [4, 5, 7, 23, 47, 48]. Firstly, the UGT1A1 enzyme
catalyses the transfer of one molecule of glucuronic acid to
one of the two carboxyl groups on bilirubin and forms
monoglucuronide (BMG). Further, diglucuronide forma-
tion (BDG) occurs at the canalicular region of hepatocyte
when another molecule of glucuronic acid is attached to
the second carboxyl group on bilirubin [5, 23, 48]. Conju-
gation of bilirubin with glucuronic acid disrupts the
internal hydrogen bond, and the resultant BMG and BDG
are highly soluble in water for easy excretion [48].
The UGT1A1 enzyme activity is also observed to be very
low at birth progressing from no activity during foetal
period to a plateau similar to adult values by four months
of age [49]. Due to the relative insufficiency of the UGT1A1
enzyme in the early postnatal age, a significant amount of
bilirubin remains unconjugated in the hepatocyte [5]. The
unconjugated bilirubin with the capability of moving
across the sinusoidal surface easily leaks back into the
blood and contributes to the high serum unconjugated
bilirubin in newborns [47].
The relative insufficiency of the UGT1A1 enzyme also
contributes to the increased mono-conjugated bilirubin
formation compared to di-conjugates in the first few days
of newborn life and consequently an increase in the ratio of
BMG is noted in bile secretions of neonates [50, 51]. The
serum conjugated bilirubin in neonates is also predomi-
nantly mono-conjugated bilirubin in the first 24–48 h,
followed by the appearance of di-conjugated bilirubin from
the third day of life [47, 51, 52] demonstrating progress in
the maturation of the UGT1A1 enzyme and conjugation
capacity of the liver which occurs in the first few weeks of
life [4, 8].
Hepatic clearance of conjugated bilirubin
The conjugated bilirubin glucuronides being water-soluble
is easily transported through the lumen of biliary canaliculi
into the bile, and further through the bile duct into the
intestine for excretion [5]. Multi-drug resistance protein-2
(MRP2) based at the canalicular surface of hepatocyte,
plays a crucial role in the active transportation of conju-
gated bilirubin from the hepatocyte into the biliary canal-
iculi [5, 12].
Recent findings suggest that MRP2 transporters in
newborns are significantly lower, approximately 200 fold
lower compared to that of adults, and compromises the
efficient transportation of conjugated bilirubin glucuro-
nides into the biliary canal [4, 5, 8, 11, 53]. Hence, whenever
there is increased bilirubin load, biliary excretion of con-
jugated bilirubin is compromised by insufficient MRP2
transporters [12]. Consequently, a substantial portion of
conjugated bilirubin is directed back to the sinusoidal
surface of the hepatocyte and secreted into the sinusoidal
blood [13]
At the sinusoidal surface of hepatocyte, MRP3 trans-
porters (multi-drug resistance protein-3) are upregulated
under conditions of MRP2 deficiency [54]. The expressions
of these MRP3 transporters are comparable among

neonates and adults [54] and successfully mediate the
hepatic clearance of conjugated bilirubin into the sinu-
soidal blood [55]. Further clearance of this conjugated
bilirubin from the sinusoidal blood is discussed in detail in
Alternate excretory pathways of bilirubin in newborns.
Bilirubin excretion
Several factors play a role in the efficient excretion of
bilirubin. Once conjugated bilirubin glucuronides reach
the newborn intestine via the bile duct, it is then converted
further to its derivates such as urobilinogen and stercobi-
linogen by bacterial flora in the large intestine for easy
elimination through faeces. A certain percentage of uro-
bilinogen is reabsorbed back from the intestine, and is
transported back into portal circulation for further excre-
tion; however, a small amount is excreted through urine
[7, 56].
In neonates, due to reduced bacterial flora present in
the intestine, there is limited conversion of conjugated
bilirubin to its derivatives for elimination [3, 47, 57].
Besides, the high beta-glucuronidase enzyme activity in
the intestine results in a significant proportion of con-
jugated bilirubin being deconjugated back to its un-
conjugated form. The longer the conjugated bilirubin
stays in the intestines, the further the deconjugation
process may proceed. Deconjugated bilirubin makes its
way back to the liver through the portal circulation,
adding burden on the liver for further conjugation and
excretion [23, 57]. The resulting increased enterohepatic
circulation of unconjugated bilirubin supplements the
already exaggerated concentration of unconjugated
bilirubin in serum [23, 58]. Hence, the predominant form
of bilirubin responsible for jaundice among neonates is
unconjugated bilirubin.
Alternate excretory pathways of
bilirubin in newborns
Bilirubin excretion was traditionally considered a one-
way pathway, where conjugated bilirubin is actively
transported from the hepatocytes to the biliary canal
and then via the bile duct into the intestine for excre-
tion [10, 23]. Until recently, it was believed that only if
there is an obstruction at the biliary duct, would con-
jugated bilirubin be transported back from the hepato-
cytes into the sinusoidal blood and then excreted in the
urine.
Thomas et al.: Bilirubin elimination in newborns 1029
Conjugated bilirubin in urine
Advancement in understanding the role of hepatic mem-
brane transporters (MRP3 transporters) predicts that even
in the normal physiological state, a significant amount of
conjugated bilirubin effluxes from the hepatocyte into si-
nusoidal blood and is excreted in urine [4, 5]. Due to the
low expression of MRP2 transporters and the resultant
upregulation of MRP3 transporters in newborns, more
conjugated bilirubin is likely to be transported back into
the sinusoidal blood from the hepatocytes [4, 12, 55, 59].
Interestingly, conjugated bilirubin concentration in
neonates is maintained at a ratio of 2% total serum bili-
rubin, even in conditions where there is increased total
serum bilirubin concentration [10, 15, 51]. Recent research
on patients with Rotor syndrome (deficiency of OATP1B1/
B3 transporters) advocates a new paradigm for conjugated
bilirubin transportation and provides insights into this
serum conjugated bilirubin homeostasis. There is evidence
that OATP1B1/B3 transporters, based at the sinusoidal
surface of the hepatocyte enables reuptake of conjugated
bilirubin back into the hepatocyte and maintains excretion
of conjugated bilirubin through the biliary canal (Figure 2)
[4, 8, 10, 12, 53].
However, these OATP1B1/B3 transporters are signifi-
cantly low in newborns in the first two weeks of life,
estimated to be approximately 500 fold lower compared
to adults [5, 11]. The substantial decrease of OATP trans-
porters, thereby impacts the reuptake of conjugated
bilirubin back into the hepatocytes for biliary excretion in
newborns [13]. The ability to sustain serum conjugated
bilirubin homeostasis with remarkably low OATP trans-
porters presents the possibilities of alternate bilirubin
excretory pathways. This leads to our speculation that
there is potentially renal excretion of conjugated bilirubin
in healthy term newborns as seen in cases of biliary
obstruction.
Unconjugated bilirubin in urine
Traditionally it has been noted that unconjugated bilirubin
is insoluble in water at neutral pH, cannot pass through the
glomerular membrane of the kidneys, and remains unde-
tectable in the urine in patients with jaundice [10, 60].
However, some evidence suggests that the kidneys may be
permeable to unconjugated bilirubin under some patho-
logical conditions [61, 62]. It is likely that the methods used
for estimation of bilirubin in urine were crude with poor
analytical quality and potentially have a high variability.
Hence interpretation of results requires caution. Other
1030 Thomas et al.: Bilirubin elimination in newborns
studies suggest there is glomerular filtration of unconju-
gated bilirubin; however, this is reabsorbed back into the
system by the renal tubules [10, 60, 62]. A small amount of
unconjugated bilirubin is also found in the urine of pa-
tients with jaundice in whom the unconjugated bilirubin to
albumin ratio is disproportionately high. Hence, a pro-
portion of unconjugated bilirubin not bound to albumin
has the potential to be excreted via urine [23]. This theory
provides an impetus to explore urinary excretion of bili-
rubin in neonates who may have high serum unbound
unconjugated bilirubin.
Limited literature exists that deals with bilirubin con-
centration in urine of newborns with jaundice. However, a
small series of studies during the 20th century demon-
strated the presence of bilirubin in urine among newborns
with jaundice [63–67]. Inadequate technologies in the past
could have been a constraint in detecting the accurate
concentration of bilirubin in urine. A schematic presenta-
tion of the transport, conjugation and bilirubin elimina-
tion, with emphasis on newborn differences supporting the
hypothesis of urinary excretion of bilirubin, is presented in
Figure 3.
Is bilirubin in urine a useful
predictor of neonatal jaundice?
Jaundice associated mortality continues to be in one of the
top 15 causes of neonatal mortality in low to middle-income
countries [68]. Unconjugated hyperbilirubinemia, the most
prevalent type of jaundice and its severe forms remains a
Figure 2: A schematic representation of
conjugated bilirubin transport in
hepatocytes.
Unconjugated bilirubin (UCB) is taken over
by hepatocyte, where it is glucuronidated
by UGT1A1 into BMG and BDG and is
excreted by MRP2 transporters through the
biliary canal. A proportion of these
glucuronides are transported back into the
sinusoidal blood mediated by MRP3
transporters. These glucuronides (BMG and
BDG) are taken back from the sinusoidal
blood into the hepatocytes by the OATP1B1/
B3 transporters for further excretion
through bile.
life-threatening condition worldwide. Delay in diagnosis is
the major contributing factor towards the mortality and
morbidity associated with neonatal jaundice [69]. It is
worthwhile to explore the notion of unbound unconju-
gated bilirubin excretion in urine among neonates who
may have high serum unconjugated bilirubin.
Delayed diagnosis of conjugated hyperbilirubinemia is
also a leading indication for liver transplantation world-
wide among biliary atresia patients [70]. Harpavet et al. [43]
observed infants who are diagnosed with biliary atresia
had a rise in serum conjugated bilirubin shortly after birth
and have recommended early screening of conjugated
bilirubin for early diagnosis and better outcomes for these
patients. It is vital to explore a correlation between the
conjugated bilirubin concentration in urine and total
serum bilirubin concentration. If this correlation occurs,
this, in turn, could facilitate the development of a new
screening method for the early detection and diagnosis of
conjugated hyperbilirubinemia.
A serum-based laboratory test is still the current gold
standard method to diagnose conjugated and unconju-
gated hyperbilirubinemia [71]. A global effort is gathering
momentum to develop point-of-care bilirubin measuring
devices to promote better survival for neonates and to
reduce disabilities associated with unconjugated hyper-
bilirubinemia [69, 72]. Exploring novel diagnostic urine
testing methods in neonates may allow for early diagnosis
of hyperbilirubinemia and improve clinical outcomes.
Further research is required to determine the bilirubin
concentration in urine in any case of clinically significant
jaundice. This information is currently unavailable. The
clinical reference intervals of bilirubin in urine and
 Thomas et al.: Bilirubin elimination in newborns 1031

Figure 3: A schematic presentation of the transport, conjugation and bilirubin elimination, with emphasis on newborn differences supporting
the hypothesis of urinary excretion.
The normal process of production, transportation, conjugation, hepatic clearance and excretion of bilirubin is represented in yellow boxes and
the movement is indicated with blue arrows. Altered metabolisms seen in newborns are represented in red boxes, and the impact of these
altered mechanisms is indicated with orange arrows. Speculation of conjugated bilirubin (CB) excretion through renal system is represented
with the bold green arrow.

correlation with serum bilirubin levels (both unconjugated
and conjugated) have also not yet been established.
Exploring urine-based diagnostic tools in neonates
may have the potential to screen for neonatal hyper-
bilirubinemia. Such development could be facilitated
through increasingly advanced laboratory-based tech-
niques which could determine urine bilirubin levels that
were previously undetectable. This combined knowledge
of improved lab-based methods and point-of-care methods
could advance screening for neonatal hyperbilirubinemia.
Conclusions
During the last 10 years, significant progress has been
made in understanding the role of hepatic membrane
transporters in hepatic clearance of bilirubin. New in-
sights into hepatic membrane transporter’s function in
neonates have established an understanding of the
mechanism for the serum conjugated bilirubin homeo-
stasis and subsequent excretion in urine. Renal clear-
ance of conjugated bilirubin has been postulated as a
critical factor in maintaining the homeostasis of serum
conjugated bilirubin in neonates. Future studies should
aim to examine if bilirubin (both unconjugated and
conjugated bilirubin) is detected in the urine of neonates
and whether this could further accelerate the develop-
ment of a screening method in early recognition of
neonatal jaundice.
Acknowledgments: The authors wish to thank Mr Blake
D’Souza for his support in drawings of bilirubin meta-
bolism (Figure 1).
Research funding: This study is supported by a National
Health and Medical Research Council (NHMRC)
Development Grant (GNT1139340) and the Victorian
Government Operational Infrastructure Support Program
(Melbourne, Australia). AER is supported by an NHMRC
Early Career Fellowship (GNT1123030). MT is supported by
an RTP scholarship governed by the University of
Melbourne. DGT is supported by a National Health and
Medical Research Council Clinical Career Development
Fellowship (Grant ID 1053889).
Author contributions: MT and AER developed the concept
of the manuscript. MT prepared the first draft under the
supervision of AER and RG. WH and DT contributed their
expertise in hepatology and neonatology, respectively, in
reviewing the manuscript. TPL contributed his expertise in
1032 Thomas et al.: Bilirubin elimination in newborns
reviewing the clinical chemistry of bilirubin and overall
structure of the manuscript. MT reviewed and screened the
included references. All authors contributed to the re-
drafting of the manuscript.
Competing interests: None of the authors have any
financial interests or conflicts of interest to declare.
The members of the team are involved in the
development of a method of detecting jaundice in
neonates using a urine-based testing system.
References
1. Cashore W. A brief history of neonatal jaundice. Med Health R I
2010;93:154–5.
2. Kaplan M, Bromiker R, Hammerman C. Severe neonatal
hyperbilirubinemia and kernicterus: are these still problems in
the third millennium? Neonatology 2011;100:354–62.
3. Moncrieff G. Bilirubin in the newborn: physiology and
pathophysiology. Br J Midwifery 2018;26:362–70.
4. Erlinger S, Arias IM, Dhumeaux D. Inherited disorders of bilirubin
transport and conjugation: new insights into molecular
mechanisms and consequences. Gastroenterology 2014;146:
1625–38.
5. Memon N, Weinberger BI, Hegyi T, Aleksunes LM. Inherited
disorders of bilirubin clearance. Pediatr Res 2016;79:378–86.
6. Devgun MS, Chan MK, El-Nujumi AM, Abara R, Armbruster D,
Adeli K. Clinical decision limits for interpretation of direct
bilirubin—a CALIPER study of healthy multiethnic children and
case report reviews. Clin Biochem 2015;48:93–6.
7. Hansen TWR. Core concepts: bilirubin metabolism. NeoReviews
2010;11:316–22.
8. Cvorovic J, Passamonti S. Membrane transporters for bilirubin
and its conjugates: a systematic review. Front Pharmacol 2017;8:
887.
9. Jedlitschky G, Leier I, Buchholz U, Hummel-Eisenbeiss J,
Burchell B, Keppler D. ATP-dependent transport of bilirubin
glucuronides by the multidrug resistance protein MRP1 and its
hepatocyte canalicular isoform MRP2. Biochem J 1997;327:
305–10.
10. Levitt DG, Levitt MD. Quantitative assessment of the multiple
processes responsible for bilirubin homeostasis in health and
disease. Clin Exp Gastroenterol 2014;7:307–28.
11. Mooij MG, Schwarz UI, de Koning BA, Leeder JS, Gaedigk R,
Samsom JN, et al. Ontogeny of human hepatic and intestinal
transporter gene expression during childhood: age matters. Drug
Metab Dispos 2014;42:1268–74.
12. Sticova E, Jirsa M. New insights in bilirubin metabolism and
their clinical implications. World J Gastroenterol 2013;19:
6398–407.
13. van de Steeg E, Stránecký V, Hartmannová H, Nosková L,
Hřebíček M, Wagenaar E, et al. Complete OATP1B1 and OATP1B3
deficiency causes human Rotor syndrome by interrupting
conjugated bilirubin reuptake into the liver. J Clin Invest 2012;
122:519–28.
14. van Groen BD, van de Steeg E, Mooij MG, van Lipzig MM,
de Koning BA, Verdijk RM, et al. Proteomics of human liver
membrane transporters: a focus on fetuses and newborn infants.
Eur J Pharmaceut Sci 2018;124:217–27.
15. Fujiwara R, Haag M, Schaeffeler E, Nies AT, Zanger UM, Schwab M.
Systemic regulation of bilirubin homeostasis: potential benefits of
hyperbilirubinemia. Hepatology 2018;67:1609–19.
16. Dennery PA, Seidman DS, Stevenson DK. Neonatal
hyperbilirubinemia. N Engl J Med 2001;344:581–90.
17. Fanaroff JM, Fanaroff AA. Klaus and Fanaroff’s care of the high-
risk neonate e-book. Philadelphia: Elsevier Health Sciences;
2012.
18. Bing D, Wang DY, Lan L, Zhao LD, Yin ZF, Yu L, et al. Serum
bilirubin level as a potential marker for the hearing outcome in
severe-profound bilateral sudden deafness. Otol Neurotol 2019;
40:728–35.
19. Bartlett M, Gourley GR. Neonatal jaundice and disorders of
bilirubin metabolism. Liver disease in children, 4th ed. New York:
Cambridge University Press; 2011.
20. Maisels MJ. What’s in a name? Physiologic and pathologic
jaundice: the conundrum of defining normal bilirubin levels in the
newborn. Pediatrics 2006;118:805–7.
21. Valaes T. Bilirubin and red cell metabolism in relation to neonatal
jaundice. Postgrad Med 1969;45:86–106.
22. Sharma A, Ford S, Calvert J. Adaptation for life: a review of
neonatal physiology. Anaesth Intensive Care Med 2011;12:
85–90.
23. Fevery J. Bilirubin in clinical practice: a review. Liver Int 2008;28:
592–605.
24. Vitek L, Ostrow JD. Bilirubin chemistry and metabolism; harmful
and protective aspects. Curr Pharmaceut Des 2009;15:
2869–83.
25. Schwartz HP, Haberman BE, Ruddy RM. Hyperbilirubinemia:
current guidelines and emerging therapies. Pediatr Emerg Care
2011;27:884–9.
26. Ebbesen F, Bjerre JV, Vandborg PK. Relation between serum
bilirubin levels ≥ 450 μmol/L and bilirubin encephalopathy; a
Danish population‐based study. Acta Paediatr 2012;101:384–9.
27. Orkin SH, Nathan DG, Ginsburg D, Look AT, Fisher DE, Lux S.
Nathan and Oski’s hematology of infancy and childhood e-book.
Philadelphia: Elsevier Health Sciences; 2008.
28. American Academy of Pediatrics. Management of hyperbilirubinemia
in the newborn infant 35 or more weeks of gestation. Pediatrics
2004;114:297.
29. Olusanya BO, Kaplan M, Hansen TW. Neonatal
hyperbilirubinaemia: a global perspective. Lancet Child Adolesc
Health 2018;2:610–20.
30. Bhutani VK, Zipursky A, Blencowe H, Khanna R, Sgro M,
Ebbesen F, et al. Neonatal hyperbilirubinemia and Rhesus
disease of the newborn: incidence and impairment estimates for
2010 at regional and global levels. Pediatr Res 2013;74(1 Suppl):
86–100.
31. Amin SB, Lamola AA. Newborn jaundice technologies: unbound
bilirubin and bilirubin binding capacity in neonates. Semin
Perinatol 2011;35:134–40.
32. Hulzebos CV, Dijk PH. Bilirubin-albumin binding, bilirubin/
albumin ratios, and free bilirubin levels: where do we stand?
Semin Perinatol 2014;38:412–21.
33. Watchko JF, Tiribelli C. Bilirubin-induced neurologic damage—
mechanisms and management approaches. N Engl J Med 2013;
369:2021–30.

34. Ahlfors CE. Bilirubin-albumin binding and free bilirubin. J
Perinatol 2001;21(1 Suppl):S40–2.
35. Wang G, Wang J, Huang N, Yu F. The study on clinical value of the
detection about serum and unconjugated bilirubin in diagnosis of
neonatal jaundice. Pak J Pharm Sci 2016;29:339–41.
36. van Imhoff DE, Dijk PH, Weykamp CW, Cobbaert CM, Hulzebos CV,
Group BS. Measurements of neonatal bilirubin and albumin
concentrations: a need for improvement and quality control. Eur J
Pediatr 2011;170:977–82.
37. Hansen TW. Prevention of neurodevelopmental sequelae of
jaundice in the newborn. Dev Med Child Neurol 2011;53:24–8.
38. Cayabyab R, Ramanathan R. High unbound bilirubin for age: a
neurotoxin with major effects on the developing brain. Pediatr
Res 2019;85:183–90.
39. Pichon J-BL, Riordan SM, Watchko J, Shapiro SM. The
neurological sequelae of neonatal hyperbilirubinemia:
definitions, diagnosis and treatment of the kernicterus spectrum
disorders (KSDs). Curr Pediatr Rev 2017;13:199–209.
40. Riordan SM, Gazzin S. Where do we stand in the field of neonatal
jaundice? Commentary on the 2017 J. Donald Ostrow Trieste
Yellow Retreat. Pediatr Res 2018;83:1090–2.
41. Colantonio DA, Kyriakopoulou L, Chan MK, Daly CH, Brinc D,
Venner AA, et al. Closing the gaps in pediatric laboratory
reference intervals: a CALIPER database of 40 biochemical
markers in a healthy and multiethnic population of children. Clin
Chem 2012;58:854–68.
42. Harpavat S, Devaraj S, Finegold MJ. An infant with persistent
jaundice and a normal newborn direct bilirubin measurement.
Clin Chem 2015;61:330–3.
43. Harpavat S, Finegold MJ, Karpen SJ. Patients with biliary atresia
have elevated direct/conjugated bilirubin levels shortly after
birth. Pediatrics 2011;128:1428–33.
44. Weiss AK, Vora PV. Conjugated hyperbilirubinemia in the neonate
and young infant. Pediatr Emerg Care 2018;34:280–3.
45. Brumbaugh D, Mack C. Conjugated hyperbilirubinemia in
children. Pediatr Rev 2012;33:291–302.
46. Wang X, Chowdhury JR, Chowdhury NR. Bilirubin metabolism:
applied physiology. Curr Paediatr 2006;16:70–4.
47. Kaplan M, Muraca M, Hammerman C, Rubaltelli FF, Vilei MT,
Vreman HJ, et al. Imbalance between production and conjugation
of bilirubin: a fundamental concept in the mechanism of neonatal
jaundice. Pediatrics 2002;110:e47.
48. Zhou J, Tracy TS, Remmel RP. Bilirubin glucuronidation
revisited: proper assay conditions to estimate enzyme kinetics
with recombinant UGT1A1. Drug Metab Dispos 2010;38:
1907–11.
49. Miyagi SJ, Collier AC. Pediatric development of glucuronidation:
the ontogeny of hepatic UGT1A4. Drug Metab Dispos 2007;35:
1587–92.
50. Muraca M, Fevery J, Blanckaert N. Relationships between serum
bilirubins and production and conjugation of bilirubin: studies in
Gilbert’s syndrome, Crigler-Najjar disease, hemolytic disorders,
and rat models. Gastroenterology 1987;92:309–17.
51. Muraca M, Rubaltelli FF, Blanckaert N, Fevery J. Unconjugated and
conjugated bilirubin pigments during perinatal development. II.
Studies on serum of healthy newborns and of neonates with
erythroblastosis fetalis. Biol Neonate 1990;57:1–9.
52. Rosenthal P, Blanckaert N, Kabra PM, Thaler MM. Formation of
bilirubin conjugates in human newborns. Pediatr Res 1986;20:
947–50.
Thomas et al.: Bilirubin elimination in newborns 1033
53. Iusuf D, van de Steeg E, Schinkel AH. Hepatocyte hopping of
OATP1B substrates contributes to efficient hepatic detoxification.
Clin Pharmacol Ther 2012;92:559–62.
54. Brouwer KL, Aleksunes LM, Brandys B, Giacoia GP, Knipp G,
Lukacova V, et al. Human ontogeny of drug transporters: review
and recommendations of the pediatric transporter working
group. Clin Pharmacol Ther 2015;98:266–87.
55. Keppler D. The roles of MRP2, MRP3, OATP1B1, and OATP1B3 in
conjugated hyperbilirubinemia. Drug Metab Dispos 2014;42:
561–5.
56. Mitra S, Rennie J. Neonatal jaundice: aetiology, diagnosis and
treatment. Br J Hosp Med 2017;78:699–704.
57. Jirsova V, Jirsa M, Mikova M. Transformation of bilirubin in
gastrointestinal tract and its relationship to neonatal icterus.
Czech Med 1982;5:157–64.
58. Cohen RS, Wong RJ, Stevenson DK. Understanding neonatal
jaundice: a perspective on causation. Pediatr Neonatol 2010;51:
143–8.
59. Kamisako T, Kobayashi Y, Takeuchi K, Ishihara T, Higuchi K,
Tanaka Y, et al. Recent advances in bilirubin metabolism
research: the molecular mechanism of hepatocyte bilirubin
transport and its clinical relevance. J Gastroenterol 2000;35:
659–64.
60. Mundt LA, Graff L, Shanahan K. Graff’s textbook of routine
urinalysis and body fluids, 2nd ed. Philadelphia: Wolters Kluwer/
Lippincott Williams & Wilkins Health; 2011.
61. Bensley EH. The renal threshold of bilirubin. II. J Biol Chem 1933;
103:71–9.
62. Vickers HE. The renal threshold for bilirubin. J Clin Pathol 1950;3:
271.
63. Fevery J, Blanckaert N, Leroy P, Michiels R, Heirwegh KP. Analysis
of bilirubins in biological fluids by extraction and thin-layer
chromatography of the intact tetrapyrroles: application to bile of
patients with Gilbert’s syndrome, hemolysis, or cholelithiasis.
Hepatology 1983;3:177–83.
64. Rabinowitch IM. The renal threshold of bilirubin. J Biol Chem
1932;97:163–75.
65. Ross S, Waugh T, Malloy H. The metabolism and excretion of
bile pigment in icterus neonatorum. J Pediatr 1937;11:
397–408.
66. Rudi S. Direct-reacting bilirubin, bilirubin glucuronide, in serum,
bile, and urine. Science 1956;124:76–7.
67. Ullrich D, Tischler T, Sieg A, Fevery J. Renal clearance of bilirubin
conjugates in newborns of different gestational age. Eur J Pediatr
1993;152:837–9.
68. Olusanya BO, Teeple S, Kassebaum NJ. The contribution of
neonatal jaundice to global child mortality: findings from the GBD
2016 study. Pediatrics 2018;141:e20171471.
69. Slusher TM, Zipursky A, Bhutani VK. A global need for affordable
neonatal jaundice technologies. Semin Perinatol 2011;35:
185–91.
70. Hartley JL, Davenport M, Kelly DA. Biliary atresia. Lancet 2009;
374:1704–13.
71. Raimondi F, Ferrara T, Borrelli AC, Schettino D, Parrella C,
Capasso L. Neonatal hyperbilirubinemia: a critical appraisal of
current guidelines and evidence. J Pediatr Neonatal
Individualized Med 2012;1:25–32.
72. World Health Organization. Every newborn: an action plan to end
preventable deaths. Geneva, Switzerland: World Health
Organisation; 2014.