Received Date : 05-Dec-2016

Accepted Article
Accepted Date : 17-Dec-2016

Article type : Reviews

Handling Editor: Mario Mondelli

The molecular basis of jaundice: An old symptom revisited

Silvia Gazzin1, Flora Masutti1,2, Libor Vitek3, Claudio Tiribelli1*

1
Liver Research Center, Italian Liver Foundation - ONLUS, AREA Science Park, Trieste,

Italy.
2
Center for Liver Diseases, Department of Medical Sciences, University of Trieste, Trieste,

Italy.
3
1st Faculty of Medicine, Charles University, Prague, Czech Republic.

* Correspondence

Claudio Tiribelli
Italian Liver Foundation – ONLUS
AREA Science park, Bldg Q
Ss 14, Km 163,7
Trieste, 34149
Italy
Tel: 0039 040 375 7840
Fax: 0039 040 375 7832
e-mail: ctliver@fegato.it

This article has been accepted for publication and undergone full peer review but has not been through
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between this version and the Version of Record. Please cite this article as doi: 10.1111/liv.13351
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 List of the abbreviations
Accepted Article
TSB: total serum bilirubin; UCB: unconjugated bilirubin; CB: conjugated bilirubin; HMOX:

heme oxygenase; BV: biliverdin; BLVR: biliverdin reductase; Bf: free UCB (UCB not bound to

serum albumin); OATP: organic anion transporting polypeptides; UGT1A1: glucoronosyl-

transferase 1A1; ABC: ATP Binding Cassette transporters; CN: Crigler-Najjar syndrome;

HMG-CoA: 3-hydroxy-3-methylglutaryl-coenzyme A; ROS: reactive oxygen species; CVD:

cardiovascular disease; LDL: low-density lipoproteins; Chol: cholesterol; Apo: apolipoprotein;

VLDL: very low-density lipoprotein; IL: interleukin; IFN: interferon; TNF: tumor necrosis factor;

NADPH oxidase: nicotinamide adenine dinucleotide phosphate–oxidase; VCAM: vascular cell

adhesion protein; iNOS: inducible nitric oxide synthases; ANG II: angiotensin II; MAPK:

mitogen activated protein kinases; PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase; Ahr:

aryl hydrocarbon receptor; Nrf2: nuclear factor (erythroid-derived 2)-like 2; PKC: protein

kinase C; CYPs: cytochrome P450 monooxygenases; NTCP: Na+-taurocholate co-

transporting polypeptide; HPV: hepatic portal vein; HA: hepatic arteries; CLV: centrolobular

vein; BS: Bile salts; gtPBREM: phenobarbital –responsive enhancer module of UGT1A1;

PXR: pregane X receptor; GR: glucocorticoid responsive element; Nrf2: nuclear factor

(erythroid-derived 2)-like 2; AhR: aryl hydrocarbon receptor; PPARα: peroxisome proliferator-

activated receptor alpha; HNF: hepatocyte nuclear factor; CAR: constitutive androstane

receptor; RXR: retinoic X receptor; RAR: retinoid acid receptor; FXR: farnesoid X receptor;

SXR: steroid X receptor; SP1: specificity protein 1; AP: activator protein, ERE: estrogen

responsive elements; HRE: hypoxia responsive elements; HIF1: hypoxia inducible factor 1;

HFN: hepatic nuclear factor; ER: endoplasmic reticulum; cAMP: cyclic adenosine mono

phosphate; CaM: Ca calmodulin dependent serine-threonine phosphatase; ERK: extracellular

signal regulated kinases.

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 Conflict of Interest:
Accepted Article
No conflict of interest to be declared.

Financial support:

SG was supported by an internal research grant from Italian Liver Foundation - ONLUS. LV

was supported by AZV 15-28895A from the Czech Ministry of Education, and RVO

VFN64165 from the Czech Ministry of Health.

Abstract

Increased serum bilirubin level is a widely used diagnostic marker for hepatic illnesses.

Nevertheless, mild elevation of unconjugated serum bilirubin (such as in Gilbert syndrome)

has been recently demonstrated to correlate with low risk of chronic pro-inflammatory and/or

oxidative stress-mediated diseases. In accord, a low serum bilirubin level has emerged as an

important predisposing factor or a biomarker of these pathologic conditions including

cardiovascular, tumor, and possibly neurodegenerative diseases. Bilirubin possesses multiple

biological actions with interaction in a complex network of enzymatic and signaling pathways.

The fact that the liver is the main organ controlling for the bioavailability of bilirubin in the body

emphasizes the central role of this organ in human health.

Keywords:

Bilirubin; jaundice; chronic diseases; human health; oxidative stress; Gilbert syndrome.

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 Key point box:
Accepted Article
 While increased total serum bilirubin [TSB] is a known marker of liver diseases, low TSB

has recently emerged as an important risk factor for development of multiple chronic

diseases.

 The role of gender on the correlation between serum bilirubin levels and the risk of

developing chronic diseases is emerging.

 The intricate network of enzymes, metabolic products and signaling pathways involved in

bilirubin metabolism determines the beneficial or detrimental biological impact of this bile

pigment.

The central role of liver in human health has been recognized since the antiquity - the

Prometheus’s Myth, the consideration of the liver as the core of bravery in the Middle East,

with the bile representing the anger from Plato until current days in Latin culture. By

controlling more than 500 different biological functions and possessing the high expression of

enzymes involved in bilirubin and drug metabolism and bile, liver is the main site of body

detoxification. Liver dysfunctions/pathologies often lead to alteration of bilirubin metabolism

resulting in jaundice. For this reason, bilirubin is a well-known diagnostic marker of liver

function, as acknowledged already by Hippocrates. The multiple biological actions of bilirubin

on different organs are the main topic of this review.

A review of bilirubin metabolism

Physiological total serum bilirubin [TSB] level varies in the range of 0.2-1 mg/dL (3.4-

17.1 µmol/L), with unconjugated bilirubin (UCB) forming a predominant fraction; the

conjugated bilirubin (CB) being a negligible part. The classical definition of jaundice is an
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 elevation of serum bilirubin level above 2.5 mg/dL (43 µmol/L) manifested with a yellow
Accepted Article
coloration of the sclera, mucosal surfaces and skin.

Pre-hepatic phase of bilirubin metabolism

Bilirubin is a breakdown product of the heme tetrapyrrolic ring, originating usually

from senescent red blood cells (80%, plus the remaining 20 percent resulting from ineffective

erythropoiesis and breakdown of muscle myoglobin and liver cytochromes). The successive

action of heme oxygenase (HMOX), producing the hydrophilic biliverdin (BV, plus CO and

ferrous iron), and biliverdin reductase (BLVR) (Fig 1), converts the heme group in the

lipophilic UCB in a rate of about 4 mg/kg per day) (1).

Since UCB is highly insoluble in water (the solubility threshold in plasma is as low as

70 nmol/L = 0.004 mg/dL (2)), it must be transported in plasma bound to serum albumin.

Under physiological conditions, only a very marginal part (less than 0.1% (3)) of serum UCB is

not bound to albumin and is termed “free UCB” [Bf]. UCB enters the hepatocyte (and all

tissues) largely by passive diffusion and in a minimal part by an active transport mediated by

organic anion transporting polypeptides (OATP) 1B1 and 1B3 (OMIM*604843 & *605495) (4,

5) (Fig 1). The intracellular fraction of UCB plays relevant biomolecular actions in the control

of inflammatory and redox tissue homeostasis (see later) (6).

Intra-hepatic phase of bilirubin metabolism

Once in the hepatocyte, the fat soluble UCB is conjugated to 1-2 molecules of

glucuronic acid by the action of the UDP-glucuronosyl transferase 1A1 isoform (UGT1A1,

OMIM*191740; EC2.4.1.17), forming the water soluble conjugated bilirubin (CB). UGT1A1

expression is almost undetectable at birth (<1% of adult levels) and reaches the adult levels

in 3 month (7). UGT1A1 activity is under hormonal control, with progesterone enhancing and

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 testosterone inhibiting its activity (8). This accounts for the mean value of TSB in females
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being lower by approximately 30% as compared to males (9).

Post-hepatic phase of bilirubin metabolism

Once water soluble, CB is actively secreted into the bile canaliculi by the (apical) ATP

binding cassette (ABC) C2 (OMIM*601107) and, possibly by ABCG2 (OMIM*603756)

multidrug transporters (Fig 2) (10). The transporters export bilirubin glucuronosides into the

biliary ducts. As for UGT1A1, the hepatic expression of ABCC2 is low at birth , but

surprisingly also n 12-month children compared to adults (200 and 100-fold lower,

respectively) (11).

The sinusoidal liver-to-blood loop (Fig 2).

Under physiological conditions, prior its secretion into bile a part of CB is transported

back to the blood by the action of the sinusoidal (basolateral) ABCC3 (OMIM*604323) and

subsequently reuptaken into the hepatocyte by the OATP1B1/3 (4, 5). The level of expression

of the CB transporters (OATP1B1/3) at the sinusoidal face increases from the portal to the

centrolobular space. This differential expression, paralleling the blood flow, creates an intra-

lobular transport system (the sinusoidal liver-to-blood loop), possibly protecting the periportal

hepatocytes from the excessive bilirubin and xenobiotics accumulation (1). As for the other

actors of bilirubin metabolism, the OATP1B1/3 expression is also developmentally regulated,

being around 500-fold and 100-fold lower in newborns and 12-month old children,

respectively, compared to adults (11).

Within the intestinal lumen (Fig 1), a very small portion of bilirubin is excreted in the

stool, while the rest is metabolized by the beta-glucuronidase expressed by the intestinal

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 bacteria (12) and enterocytes. Beta-glucuronidase first deconjugates CB to UCB, which is
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then reduced to urobilinogen and stercobilinogen by the microbiota, being almost absent at

birth and needing 6-12 month to stably populate the intestine (13-15). After oxidation,

stercobilin and urobilin are excreted via feces. Physiologically, only a small fraction of

urobilinogen undergoes enterohepatic/enterosystemic circulation and is then filtered by the

kidney and excreted in the urine (16). The same process is also true for UCB, which can be,

under certain conditions, also reabsorbed A small portion of UCB is reabsorbed in the colon

and delivered back to liver by portal circulation(12, 13).

Abnormalities and pathology of the bilirubin metabolism

TSB levels may be altered in a large number of conditions (Table 1-3).

Bilirubin overproduction

Increased production of UCB may significantly increase the serum TSB level resulting

in pre-hepatic unconjugated hyperbilirubinemia (Table 1) (17, 18). Unconjugated

hyperbilirubinemia is very frequent in newborns (neonatal jaundice, peaking at around 2-4

days after birth). Considered physiological and even beneficial due to the antioxidant

properties of UCB (birth represents severe pro-oxidative challenge), neonatal, non-hemolytic

unconjugated hyperbilirubinemia is due to a combination of the immaturity of the hepatic

UGT1A1 and transporting systems, the hemolysis of fetal red blood cells, and the absence of

intestinal microbiota reducing bilirubin. Bilirubin may enter the brain and damage specific

areas of the central nervous system with possible permanent (kernicterus) or even lethal

consequences (19). Neonatal hyperbilirubinemia may also appear around the 3rd week after

birth, due to the presence of both pregnane-3α, 20β diol and -glucuronidase in the breast

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 milk (breast milk jaundice), inhibiting the UGT1A1 activity (20) and suggested a infant
Accepted Article
phenotype of Gilbert syndrome (21).

Deficient conjugation activity

Well known genetic causes of severe unconjugated hyperbilirubinemia are the

Crigler-Najjar I (CN I) (22) and Crigler-Najjar type II (CN II) syndromes (23). These rare

diseases are due to mutations in the UGT1A1 gene (chromosome 2q37). The gene spans

218 kb, and contains 13 alternative exons 1 (9 transcribes into a functional, 4 in non-

functional enzyme), coding for the substrate specificity (N-terminal part of the enzyme) (Fig

3). Each of the 9 of the alternative transcripts of exon 1 is preceded by a promoter region

containing a TATA box that allows independent expression of each of these 9 different

UGT1A isoforms. Indeed, the promoter contains several binding domains for transcription

factors making it highly responsive to a variety of stimuli (24). The four common exons (exons

2 – 5), present in all the UGTs, codes for the C-terminal portion of the enzymes, containing

the functional (conjugating) site, and the membrane-spanning region. Among the UGTs, the

1A1 isoform is the only capable of handling bilirubin (25).

Mutations causing the CN syndrome may affect all the exons, as well the regulatory

or splicing sites, modulating the severity of the resulting syndrome. CN I results from

mutations altering or deleting a large number of amino acid residues (premature stop codon

formation, reading frameshift mutations). As consequence, the enzyme is inactive and not

susceptible to induction. Currently, 35 mutations have been identified for CN I (25-27), mainly

in exons 2 and 5. For this reason, all UGT1A isoforms are affected, with broad consequences

on UGTs´ substrate conjugation. If mutations involve specifically the exon 1, only bilirubin

metabolism is affected (type Ib CN) (20). Mutations leading to the substitution of a single

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 amino acid in the translated enzyme substantially reduce but not abolish the activity, and
Accepted Article
result in CN II; 18 such mutations have been identified in CN II so far (26, 27).

Mutations affecting the promoter region (TATA box) modulate the level of

transcription of the UGT1A1 (28), leading to Gilbert syndrome (29). Till now, about 131

mutations have been characterized (30). The UGT1A1*28 allele (consisting in a TA insertion -

A(TA)7-, resulting in a 20% reduced activity), is the most frequent cause of Gilbert syndrome,

in particular in Caucasian population. UGT1A1*28 allele mutation is present in the 35-40% of

Caucasians, without any gender differences (26), but with regional variability (20).

UGT1A1*28 allele homozygosity, needed (but not sufficient) to develop the phenotype of mild

unconjugated hyperbilirubinemia, is present in about 16% of Caucasians, but the prevalence

of Gilbert syndrome is only half of that, due to low penetrance of this mutation (26). Gilbert

syndrome phenotype may also be caused by other common mutations such as in the alleles

UGT1A*6; UGT1A*7 (3), and A(TA)8 (9, 27), UGT1A1*27 and UGT1A1*62 (1). A clear

association between the genotype distribution of the alleles (TA)7/(TA)7, (TA)6/(TA)7 and

(TA)6/(TA)6 (12: 45: 43, respectively) and the TSB (20.5, 14 and 12 µmol/L, respectively)

have been reported by Borlak et al. (31). The UGT1A1*28 genotype affects also the

metabolism of atazanavir (32), as well as other drugs biotransformed by this enzyme and

having a narrow therapeutic spectrum (33). Specific haplotypes of UGT1A1 mutations

characteristic for Gilbert syndrome and other UGT1A (3,6,7) exon mutations might further

complicate the reduction in drug clearance (16). However, Gilbert syndrome is a generally

benign condition, having also a potent protective potential against a wide variety of civilization

diseases (6, 34) (see also below).

Rotor syndrome (OMIM*237450) and Dubin-Johnson syndrome (OMIM*237500) are genetic

disorders characterized by predominantly conjugated hyperbilirubinemia (Table 2). The Rotor

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 syndrome is due to the mutation of the OATP1B1 (OMIM*604843) and OATPB3
Accepted Article
(OMIM*605495) genes, both on chromosome 12p. The c.521T>C variant on the OATP1B1

gene sequence limits the bilirubin transport (35), reducing the reabsorption by the intralobular

transport system (the sinusoidal liver-to-blood loop, see also above) (20, 36), modifying the

pharmacokinetics of various drugs including the antidiabetic drug repaginate or HMG-CoA

reductase inhibitors (35). Other less characterized gene sequence variants have been

described, with variable allele frequency among populations. Mutations leading to Dubin-

Johnson phenotype usually involve exons coding for the retention of the transporter in the

subcellular compartments, or result in stop codons, avoiding transcription of ABCC2 gene

coding for multidrug resistance-related protein 2 (MRP2) (36). Melanin-like lysosomal pigment

accumulation in liver, without any pathological implications, is frequently observed in these

patients (24).

Conjugated hyperbilirubinemia results also in the obstruction of the duct system, while

mixed hyperbilirubinemia (Table 3) is usually seen in advanced liver diseases.

Systemic effects of hyperbilirubinemia.

Epidemiological studies have revealed the benefits of slightly elevated UCB serum

levels as in Gilbert syndrome (6, 34), mainly due to an enhanced antioxidant capacity (37). By

protecting transmembrane proteins and lipids from reactive oxygen species [ROS] attack,

moderate unconjugated hyperbilirubinemia might reduce the platelet hyper-reactivity and pro-

thrombotic phenotype that represent a relevant risk factor in cardiovascular diseases (CVD)

(38). Indeed, higher serum bilirubin is associated with lowering LDL cholesterol, ApoB/ApoA1,

and VLDL, as well as with increased HDL/LDL ratio and protection of these lipids from

oxidation. UCB might also ameliorate metabolic syndrome and diabetes (38-40), reduces

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 cytokine release (IL2, IFNγ and TNFα), the T cell response, and the complement activation
Accepted Article
(41). The anti-inflammatory action of UCB seems to be mediated via the inhibition of NADPH

oxidase (42), and inhibition of VCAM-1 and iNOS (43, 44) reducing the transendothelial

leukocyte migration as shown in animal models of inflammatory colitis (45) and allergen-

induced airway inflammation and asthma (46).

By scavenging ROS produced byangiotensin II (ANG-II)-induced NADPH activation,

moderate unconjugated hyperbilirubinemia reversed the vasoconstrictor effect of ANG-II, the

drop of glomerular filtration rate (47), the increase of renal blood flow and systolic blood

pressure, all the factors contributing to amelioration of the CVD risk (48, 49). Moreover, UCB

counteracted also the ANG-II-induced expression of vasoconstrictive pre-pro-endothelin, as

well as affected the intracellular Ca2+ imbalance (50). The general improvement of renal

hemodynamics might be one of the mechanisms by which moderate hyperbilirubinemia

lowers blood pressure, helping in preventing cardiac hypertrophy, coronary artery as well as

renal diseases. ANG-II induces HMOX (48), the first enzyme in the heme catabolic pathway,

possibly fostering the de novo intracellular synthesis UCB. As recently documented in several

cell lines, such as liver, kidney, breast, colon, epidermal and erythroleukemia cancer cells

(51), the de novo synthesis of UCB has improved the cell survival (51).

Increased serum bilirubin, typically seen in the Gilbert syndrome phenotype, was

associated with a significant lower prevalence of colorectal cancer (9), and a reduction in

CVD risk (34). Both effects may be linked to the anti-proliferative activity of UCB ton mitogen

activated protein kinases (MAPK) (52, 53). UCB is also involved in the complex network of

other signaling pathways, such as PI3K, Ahr, Nrf2, and PKC, as recently reviewed in detail in

Gazzin et al. (6).

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 On the other side, when the plasma level exceeds 20 mg/dL (340 µmol/L), UCB
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substantially increases the risk of neonatal kernicterus (25). UCB binds to cellular membranes

and modifies their fluidity, interferes with mitochondrial cellular respiration inducing a cellular

energetic crisis and production of ROS. Moreover, when highly elevated bilirubin activates

microglia and induces the release of pro-inflammatory molecules and glutamate, causing

calcium imbalance. Collectively, UCB affects neurotransmission cellular division and

differentiation, migration and myelination (54-57). UCB is a substrate for or inducer of several

molecules, such as CYPs (57-59), ABCB1/C1 (60, 61), and HMOX itself (62). Classical

signaling pathways act across a pool of transcription factors and DNA responsive elements,

commonly shared by enzymes, such as UGT1A1 and CYPs, as well as and ABCs, OATPs

transporters involved in the bilirubin metabolism (Fig 4).

Conjugated hyperbilirubinemia also affects the liver physiology. In patients with biliary

cirrhosis, expressions of sinusoidal OATP1B1/3, NTCP, ABCC3/B3 transporters are

decreased, while ABCB1 is up-regulated (1). An increased expressions of the sinusoidal

ABCC1,C3 and C4 homologues (Fig 2), have been described in cholestasis, possibly as an

adaptive response to the accumulation of metabolites in the hepatocyte due to disappearance

of ABCC2 at the canalicular site (1, 63). This apparent disappearance of ABCC2 at the

canalicular site represents a quick modulation of its activity by the re-allocation from the

plasma-membrane where they can perform their function (10, 63, 64), to the subcellular pool

of vesicles (Fig 4).

Trafficking of ABCs transporters in the liver has been documented in both

unconjugated and conjugated hyperbilirubinemia (10, 16, 60, 64, 65). Inflammation might be

one of the driving forces for ABCC2 retrieval from the canalicular membrane (without any

effect on mRNA and protein levels) in models of obstructive cholestasis (22, 64),

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 endotoxemia, and alcoholic, drug-induced or autoimmune hepatitis. It takes 20 minutes to 3
Accepted Article
hrs to reach the nadir of ABCC2 retrieval, followed by the transporter internalization to the

lysosomal compartment for degradation or its reinsertion in the plasma-membrane (10).

ABCC2 is also internalized in the intrahepatic cholestasis of pregnancy where

retrieval seems to be driven by oxidative stress and iNOS activation, enhancing Ca-mediated

PKC signaling pathway (Fig 4). Interestingly, also ABCG2, the other possible CB transporter

may traffic between subcellular compartments “on demand” via thePI3K/Akt signaling

pathway (54). Altogether, the changes in the expression and localization of bile pigment and

biliary lipid transporters represent an effort to prevent/reduce the accumulation of toxic

intermediates within hepatocytes. Since accumulated bile acids trigger apoptosis and

necrosis, possibly by inducing mitochondrial dysfunction and redox stress, the accumulation

of bilirubin during cholestasis protects from harmful effects of accumulated bile acids (66).

Both redox stress and hepatocyte apoptosis, are significantly reversed by both UCB and CB

(67).

Low bilirubin levels: a possible systemic risk factor?

While increased TSB is a well-known indicator of liver diseases, recent data

demonstrate that low TSB levels are a marker of cardiovascular diseases, certain tumors,

metabolic syndrome and possibly neurodegenerative diseases (6, 34). Data from the Belgium

BIRNH retrospective population study using 10-year follow-up, show that bilirubin levels

below the midpoint were associated with a significant increase of cardiovascular and cancer

mortality in 5,460 men (68). A similar correlation (low TSB levels – higher risk) was reported

also in respect to CVD/myocardial infarction events (69). The correlation was confirmed in the

Serbian population at bilirubin levels below 10.5 µmol/L (70). Low serum bilirubin levels have

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 been detected also in Alzheimer disease (71), dementia (72), schizophrenia (73),
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amyotrophic lateral sclerosis (74), multiple sclerosis (75, 76), cerebral infarction (77) and optic

neuritis (75). A major question remains to be answered, whether the low TSB might be the

predisposing factor or a biomarker of an underling pathology involving oxidative stress.

Bilirubin protection: a matter of gender?

Despite rarely considered, an important issue is the dimorphic effect of bilirubin in

men and women. In the Belgium BIRNH study, the significant decrease in cancer mortality at

the high TSB levels and the increased risk at lower bilirubin concentration were observed in

males but not female (68). Similarly, the correlation between TSB and the lower risk of

developing colorectal cancer is more relevant in women than in men (9) and associated with

a substantial reduction of the risk if carrying the TA(7/7) genotype in males than in females

(78). The UGT1A1 is under estrogen control, and vice versa UGT1A1 controls for the

estrogen bioavailability by their biotransformation. Thus, a lower UGT1A1 activity implies both

a higher TSB and a lower biliary estrogen disposal. Estrogens have been proposed to

account for the gender differences in the CVD risk (79), non-alcoholic steatohepatitis (80),

and are certainly involved in breast cancer carcinogenesis (81). Since TSB levels are gender-

related with males having a significantly higher concentration of bilirubin, further studies are

needed to unravel the correlation between TSB and estrogen levels, UGT1A1 activity and the

pathogenesis of oxidative stress-mediated diseases.

Concluding remarks

From a simple symptom of the liver diseases, TSB concentration emerges as a

disease biomarker and modulator of the health status, acting directly or via the modulation of

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 several enzymes and intracellular signalling pathways. Thus, bilirubin is gaining an
Accepted Article
increasingly important role in the body homeostasis, with the liver conserving its central role

in human health.

Figure and table legends

Fig 1: Bilirubin metabolism

HMOX: heme oxygenase; BV: biliverdin; BLVR: bilirubin reductase; UCB: unconjugated

bilirubin; OATP: organic anion polypeptide transporter; UGT: UDP-glucoronosyl transferase;

CB: conjugated bilirubin; ABC: ATP binding cassette transporters.

Fig 2: Bilirubin hepatic transporters and the sinusoidal liver-to-blood loop

Adapted from Jirsa et al. (1)

HPC: hepatic portal vein (75% of total blood supply, from gastrointestinal tract and spleen)

HA: hepatic arteries (oxygenated blood from lungs and heart). CLV: centrolobular vein

(flushing into the inferior vena cava). UCB: unconjugated bilirubin. OATP1B1/3: organic anion

polypeptide transporter; CB: conjugated bilirubin; BD: bile duct; ABC: ATP binding cassette

transporters; BS: bile salts; NTCP: Na/taurocholate transporter.

Fig 3: UGT1A1 gene structure and DNA responsive elements on the main UCB/CB

transporters. gtPBREM: phenobarbital –responsive enhancer module of UGT1A1; PXR:

pregane X receptor; GR: glucocorticoid responsive element; Nrf2: Nuclear factor (erythroid-

derived 2)-like 2; AhR: aryl hydrocarbon receptor; PPARα: peroxisome proliferator-activated

receptor alpha; HNF: hepatocyte nuclear factor; CAR: constitutive androstane receptor; RXR:

retinoic X receptor; RAR: retinoid acid receptor; FXR: farnesoid X receptor; SXR: steroid X

receptor; SP1: specificity protein 1; AP: activator protein, ERE: estrogen responsive

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 elements; HRE: hypoxia responsive elements; HIF1: hypoxia inducible factor 1; HFN: hepatic
Accepted Article
nuclear factor. CAR; PXR, FXR and PPARα (peroxisome proliferator-activated receptor

alpha) are also present on the CYPs (cytochrome P450 monooxygenase) gene promoter;

CAR, PXR and RXR on the ABCC4 gene promoter; CAR, PXR on the ABCC3 gene

promoter; FXR on the ABCB11 and ABCB4 gene promoters, the last displaying also PPARα.

Fig 4: Signaling pathways involved in bilirubin transporter trafficking

Transporters trafficking from the endoplasmic reticulum (ER) to the membrane, and back to

endosomal vesicles for recycling needs ATP, cytoskeleton elements (microtubules,

microfilaments), and motor proteins (dynein, kinesin) activated and coordinated through the

several signaling pathways. cAMP: cyclic adenosine monophosphate; PI3K: phosphatidyl

inositol 3 kinase; PKC: protein kinase C; CaM: Ca calmodulin dependent serine-threonine

phosphatase; ERK: extracellular signal regulated kinases; MAPK: mitogen activated protein

kinases, NTCP: Na+-taurocholate co-transporting polypeptide; BSEP: bile salt export pump.

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Table 1: Unconjugated hyperbilirubinemia

Cause In
 Conditions causing the red blood cell membrane defects (hereditary spherocytosis, glucose-6-phosphate dehydrogenase
deficiency (G6PD), autoimmune diseases (autoimmune hemolytic anemia (AIHA) or secondary haemolytic anemias such
as from infectious or tumour diseases)

production

Hemolysis of transfused red blood cells

Increased

 Drug-induced haemolytic anemias administration (e.g.: cefalosporine; dapsone; levodopa; levofloxacin; methyldopa;
bilirubin

nitrofurantoin; NSAID; ibuprofen; penicillin; fenazopyridin; chinidin; ribavirin)

 Ineffective erythropoesis (sickle cell disease, thalassemias)
 TSB usually up to 5 mg/dL= 85.5 µmol/L
 Neonatal jaundice (60%): usually TSB of 5-6 mg/dL(86-103 µmol/L) at 48-20 hours of age. Resolving spontaneously or
and absence of

with short-term phototherapy.

Immaturity of

conjugation

 Severe neonatal jaundice: a minority of subjects, total serum >20 mg/dL (340 µmol/L). Intervention needed to avoid Acute
microbiota
intestinal

Bilirubin Encephalopathy or kernicterus, the most severe and permanent form of neurological damage.
hepatic

 CN I (OMIM*218800): Serum unconjugated bilirubin levels range from 20 - 50 mg/dL(340 - 850 µmol/L.Acute bilirubin
encephalopathy and kernicterus may occur in untreated subjects. Phototherapy efficacy decrease by time due to
increasing of skin thickness. Liver transplantation is the only available curative therapy.
 CN II (OMIM*606785): lower bilirubin concentrations than type I (5 - 20 mg/dL= 86 340 µmol/L). Induction of residual of
UGT1A1 activity by phenobarbital (25, 82) allows reduction of hyperbilirubinemia to safe levels (10 mg/dL = 171 µmol/L)
Genetic modification of UGT1A1

(16, 20).
 Gilbert syndrome, benign hyperbilirubinemia (OMIM*143500): mild decrease in the activity of UGT1A1 causing an
increase in the indirect fraction of serum bilirubin to 1 - 5mg/dL (17-86 µmol/L) (68). Further elevation of the bilirubin level
may occur during periods of stress, fasting, or illness (83).
Mutation analysis is recommended for a definitive diagnosis of CN syndrome: The diagnosis of CN I is confirmed by detection of
homozygosity for “CN class I mutation”, and CN II by detection of heterozygosity (one allele carrying a “CN I class mutation”, the
other a “CN II class mutation”) (ref). Novel mutations need to be correlated with the phenotype.
Neonatal jaundice: Genetics and familial risk: Incidence is higher in infants with siblings who had significant neonatal jaundice
and particularly in infants whose older siblings were treated for neonatal jaundice. Incidence is also higher in infants with
mutations/polymorphisms of the genes that code for enzymes and proteins involved in bilirubin metabolism, and in infants with
homozygous or heterozygous glucose-6-phosphatase dehydrogenase G-6-PD deficiency and other hereditary hemolytic anemias.
Combinations of such genetic variants appear to exacerbate neonatal jaundice. Some herbal remedies taken by the lactating
mother may apparently exacerbate jaundice in the infant.

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ccepted Article
metabolism of
Disorders of
intestinal

bilirubin



Absence of bilirubin-reducing intestinal microbiota, such as during neonatal period or due to use of wide-spread oral
antibiotic therapy
Breast milk jaundice. Bilirubin may increase to levels requiring phototherapy and the discontinuation of breastfeeding.
Incidence is higher in infants who are breastfed or who receive inadequate nutrition. The mechanisms for this conditions
are still not fully understood. However, when inadequate feeding volume is involved, increased enterohepatic circulation
of bilirubin probably contributes to prolonged jaundice.
 Fasting leads to enhanced enterohepatic circulation of bilirubin and may mildly elevate TSB, although the cause(s) are
still not fully understood.

Table 2: Conjugated hyperbilirubinemia

 Rotor syndrome (OMIM*237450): TSB 2-5 mg/dL (34 – 86 µmol/L), benign prognosis.
Geneti

 Dubin-Johnson syndrome: Benign, mild, predominantly conjugated hyperbilirubinemia with manifestation in young adulthood (36).

Intrahepatic cholestasis


-
Intrahepatic gallstones, inflammatory causes (G sepsis, cholestatic forms of infectious hepatitis), primary biliary cholangitis, primary sclerosing

cholangitis, drug-induced cholestasis, intrahepatic cholestasis of pregnancy (usually non-icteric), contraceptive-induced cholestasis (CIC), alcohol-
Obstructive

induced cholestasis, inborn errors of biliary secretion of biliary lipids (progressive familial intrahepatic cholestasis, benign recurrent intrahepatic

cholestasis), inborn errors of bile acid synthesis.

Extrahepatic cholestasis

 Extrahepatic malignancy (bile ducts, gallbladder, ampuloma, pancreas, lymphoma); choledocholithiasis, pancreatitis, surgical strictures, infectious

cholangitis (parasitic infections, Cryptosporidium infection in patients with acquired immunodeficiency syndrome), iatrogenic causes.

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ccepted Article
Table 3: Mixed hyperbilirubinemia
and decreased
bilirubin load
increased



Post-operative jaundice, trauma, multiple transfusion
Transient hypotension and perioperative shock can cause acute liver necrosis in the zone 3, highlighted by a rapid and considerable
clearance
hepatic
increase in the activities of aminotransferases (often > 1000 U / l). Jaundice is usually mild.

 Alcohol abuse in witch mitochondrial dysfunction lead to liver damage. Liver cell necrosis is confirmed by highly elevated serum liver
transaminase activities; alcohol abuse leads to elevation of GGT activities and carbohydrate deficient transferrin (CDT) levels, as well as
increase of erythrocyte MCV.
 Epstein-Barr, hepatitis A and cytomegalovirus: usually self-limiting with the exception of transplanted patients and immunosuppressed
subject in case of cytomegalovirus infection.
Infections

 Hepatitis B and C: jaundice in chronic infection progressed to liver cirrhosis, major liver damage.

 Hepatitis E: is usually self-limiting but some cases may develop into fulminant hepatitis (acute liver failure) when hepatitis E occurs during
pregnancy. Pregnant women with hepatitis E, particularly those in the second or third trimester, are at an increased risk of acute liver
failure, fetal loss and mortality.

Fatty liver usually results in mild symptoms without jaundice. Jaundice may appear when simple steatosis progresses to steatohepatitis (NASH)
NAFLD

and fibrosis/cirrhosis leading to cholestasis. Liver cell necrosis is confirmed by highly elevated serum liver transaminase activities.

Acute icteric hepatitis, usually considered a disease affecting especially younger peoples, recently it has emerged as possible diagnosis also
Autoimmun

in older patients.
 Primary biliary cholangitis: rare progressive liver disease, occurring typically in middle-aged women.
e

 Primary sclerosing cholangitis (possibly leading to cholangiocarcinoma): affecting predominantly men, with the 70% of subjects
presenting also inflammatory bowel disease.

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ccepted Article Pregnancy

 Pre-eclamptic liver disease and HELLP; acute fatty liver of pregnancy; intrahepatic cholestasis of pregnancy; reactivation of autoimmune
hepatitis

Parenteral nutrition: Prolonged total parenteral nutrition (TPN), may leads in the postoperative period a progressive cholestatic syndrome,
usually with a component of hepatocellular inflammation (cholestasis from TPN). This syndrome is rarely observed before 3 weeks after
starting TPN. The risk increases with duration of TPN but; Infants are particularly susceptible. Liver biopsy usually shows a mixed appearance,
not specific, inflammatory cholestasis, sometimes with progressive fibrosis.
Other

Sarcoidosis: multi-systemic granulomatous disease of undetermined etiology and pathogenesis

DILI (Drug-induced liver injury):

 Drugs: acetaminophen, penicillin, oral contraceptives, chlorpromazine and estrogenic or anabolic steroids
 Herbal: several herbs only partially reported. Among them: Teucrium chamaedrys, Mentha pulegium, Symphytum officinale,
Germander, Greater celandine, Atractylis gummifera, Camellia sinensis, Mentha pulegium, Viscum album, valerian, assafetida,
scutellaria.

Dietary Supplements: vitamin A, Chinese remedies and teas, chaparral, Kava (for a detailed review see references -hepatotoxicity-due-to-
herbal-medications-and-dietary-supplements).

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