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NCBI Book sh elf. A ser v ice of t h e Na t ion a l Libr a r y of Medicin e, Na t ion a l In st it u t es of Hea lt h .

StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-.

Physiology, Bilirubin
Authors

Aditya Kalakonda1; Bianca A. Jenkins 2; Savio John3.

Affiliations
1 Baystate Medical Center
2 Central Michigan University College of Medicine
3 SUNY Upstate Medical University

Last Update: December 21, 2020.

Introduction
Bilirubin is an important metabolite of heme (ferroprotoporphyrin IX), a coordination complex that serves to coordinate iron in
various proteins. It is a potentially toxic substance. However, the body has developed mechanisms for its safe detoxification
and disposition.  Bilirubin and its metabolites also provide the distinctive yellow color to bile and stool and a lesser degree,
urine. This article will summarize the mechanism of heme metabolism and bilirubin synthesis.[1][2][3]

Formation of Bilirubin

Bilirubin is derived from two main sources. Roughly, 80% of bilirubin is made from the breakdown of hemoglobin in senescent
red blood cells, and prematurely destroyed erythroid cells in the bone marrow. The remainder originates from the turnover of
various heme-containing proteins found in other tissues, primarily the liver and muscles. These proteins include myoglobin,
cytochromes, catalase, peroxidase, and tryptophan pyrrolase.[4][5] About 4 mg/kg body weight of bilirubin is produced daily.

Cellular
Cellular Heme Metabolism

Heme is a ring of four pyrroles joined by carbon bridges and a central iron atom. Bilirubin is generated by a two-stage
sequential catalytic degradation reaction that primarily takes place in the cells of the reticuloendothelial system, notably the
spleen. Other cells include phagocytes and the Kupffer cells of the liver. Heme is taken up into These cells take up the heme,
and enzyme heme oxygenase acts on them. The enzyme liberates the chelated iron by catalyzing the oxidation of the alpha
carbon bridge. This reaction produces an equimolar amount of carbon monoxide which is excreted by the lungs and leads to
the formation of the green pigment, biliverdin. This green pigment is acted upon further by the nicotinamide adenine
dinucleotide phosphate (NADPH) dependent enzyme, biliverdin reductase. This process releases an orange-yellow pigment
known as bilirubin. Heme oxygenase as mentioned above is present in high concentrations in the Kupffer cells of the liver and
the cells of the reticuloendothelial system. Heme oxygenase is the rate-limiting factor in bilirubin production.

The final structure is highly compacted by hydrogen bonding rendering the molecule essentially insoluble in aqueous solutions at
neutral pH.  The fully bonded structure of bilirubin is designated as bilirubin IX-alpha-ZZ. Bilirubin, being insoluble in an
aqueous solution, is carried in circulation bound to albumin which is a reversible and covalent type of bonding.

Development
Metabolism of Bilirubin

Albumin binding: Once bilirubin is released into the plasma, it is taken up by albumin which serves as its transporter
throughout the body. The binding affinity for albumin to bilirubin is extremely high, and under ideal conditions, no free
(non-albumin bound) unconjugated bilirubin is seen in the plasma. To a lesser degree, especially in states of
hypoalbuminemia, binding also occurs with high-density lipoprotein. The binding of albumin limits the escape of bilirubin
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from the vascular space minimizes glomerular filtration and prevents its precipitation and deposition in tissues. When the
albumin-bilirubin complex reaches the liver, the highly permeable hepatic circulation allows the complex to reach the
sinusoidal surface of the hepatocyte. This allows the pigment to disassociate from the albumin and enter the liver. This
process is relatively inefficient with the first pass clearance of bilirubin being approximately 20%. This inefficient process
allows for always having the ability to measure a concentration of unconjugated bilirubin bound to albumin in the venous
circulation. The binding of albumin to bilirubin is reversible.

Hepatic transport mechanisms: Bilirubin is taken up into the hepatocytes from the liver sinusoids by two different
mechanisms:  passive diffusion and receptor-mediated endocytosis. The process of passive diffusion is not energy-
consuming and as a result, follows a concentration gradient making the flow bi-directional. Active transporter uptake of
unconjugated bilirubin from the hepatic sinusoids is mediated by carrier proteins that are not well understood. A
majority of the unconjugated bilirubin entering the hepatocytes is extracted in the periportal region. A fraction of
conjugated and unconjugated bilirubin within the hepatocyte is transported back into the sinusoidal space, and this
fraction is once again taken up downstream to the sinusoidal flow. The uptake is mediated by the 1A and 1B members
of the organic anion transporting polypeptide family (OATP). These polypeptides are encoded by the genes: SLCO1B1
and SLCO1B3. Conjugated bilirubin that escapes reuptake into the hepatocyte is excreted in the urine. Bilirubin binding
to glutathione S-transferases, which by itself increases net uptake, minimizes the efflux of internalized bilirubin.

Hepatocyte Conjugation

Conjugation is mandatory to render bilirubin aqueous soluble and facilitate its secretion across the canalicular membrane and
excretion into bile. Bilirubin is conjugated within the hepatocyte to glucuronic acid by a family of enzymes, termed uridine-
diphosphoglucuronic glucuronosyltransferase (UDPGT). The process of glucuronidation is one of the many crucial
detoxification mechanisms of the human body. Many different isoforms of UDPGT exist, but the physiologically important
isoform in bilirubin glucuronidation is UDPGT1A1. The enzyme esterifies two glucuronide moieties to the propionic acid side
chains of bilirubin. Under normal conditions, bilirubin diglucuronide is the predominant molecule synthesized. However, if the
conjugation system is overwhelmed under conditions of excessive bilirubin synthesis, the majority of bilirubin may be
conjugated as bilirubin monoglucuronide. The ratio of mono-conjugated to the dis-conjugated pigment in bile is 1:4.
Conjugation of bilirubin to the water-soluble form involves the disruption of the hydrogen bonds, an essential process for its
elimination by the liver and kidney. This is achieved by glucuronic acid conjugation of the propionic acid side chains of
bilirubin.

Excretion of conjugated bile: Conjugated bilirubin and other substances destined to be excreted in bile are actively
transported across the bile canalicular membrane of the hepatocyte. The concentration gradient is very high and can
reach 1:1000. There are at least four known canalicular transporters that participate in the excretion of conjugated
bilirubin. However, the multidrug resistance-associated protein 2 (MRP2) appears to play the dominant role in the
canalicular secretion of conjugated bilirubin. A portion of conjugated bilirubin is transported into the sinusoids and portal
circulation by MRP3, which can undergo hepatocyte reuptake via the sinusoidal proteins, organic anion transport
protein 1B1 and 1B3 (OATP1B1 and OATP1B3). Thus some conjugated and unconjugated bilirubin may escape the
hepatocyte cytosol into the plasma where it binds to albumin and gets transported around the body. However, only
conjugated bilirubin can enter the bile. The conjugated bilirubin is then actively secreted into canalicular bile and drains
into the small intestine. The rate-limiting step in bilirubin throughput is the hepatic excretory capacity of conjugated
bilirubin. Part of the conjugated bilirubin may accumulate in serum when the hepatic excretion of the conjugated bilirubin
is impaired as in prolonged biliary obstruction or intrahepatic cholestasis. This fraction of conjugated bilirubin gets
covalently bound to albumin and is called delta bilirubin or delta fraction or biliprotein. As the delta bilirubin is bound to
albumin, its clearance from serum takes about 12-14 days (equivalent to the half-life of albumin) in contrast to the usual
2 to 4 hours (half-life of bilirubin).

The process of conjugation alters the physicochemical properties of bilirubin giving it many special properties. Most
importantly, it makes the molecule water-soluble which allows it to be transported in bile without a protein carrier. Conjugation
also increases the size of the molecule. Conjugation prevents bilirubin from passively being reabsorbed by the intestinal mucosa

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due to its hydrophilicity and large molecular size. Thus, conjugation works to promote the elimination of potentially toxic
metabolic waste products. Furthermore, conjugation modestly decreases the affinity of bilirubin for albumin.

Degradation in the digestive tract: Conjugated bilirubin is not reabsorbed from the proximal intestine as mentioned
above; in comparison, unconjugated bilirubin is partially reabsorbed across the lipid membrane of the small intestinal
epithelium and undergoes enterohepatic circulation. Within the proximal small intestine, there is no additional metabolism
of bilirubin, and very little deconjugation takes place. In stark contrast, when the conjugated bilirubin reaches the distal
ileum and colon, it is rapidly reduced and deconjugated by colonic flora to a series of molecules termed urobilinogen.
The major urobilinoids seen in stool are known as urobilinogen and stercobilinogen, the nature and relative proportion
of which will depend on the presence and composition of the gut bacterial flora. These substances are colorless but turn
orange-yellow after oxidation to urobilin, giving stool its distinctive color.

Related Testing
Measurement of Serum Bilirubin

Serum bilirubin is measured spectrophotometrically when the molecule undergoes a reaction with diazo reagents causing the
breakdown of the tetrapyrrole to two azodipyrroles. This reaction is termed as the “Van den Bergh.”  Unconjugated bilirubin
reacts slowly with the diazo reagent as the central carbon bridge of bilirubin is buried within the hydrogen bonds. In contrast,
conjugated bilirubin lacks these hydrogen bonds, and the reaction occurs rapidly even in the absence of accelerators. The
addition of accelerators such as caffeine or methanol disrupts the hydrogen bonds, and the reaction is quickly completed
yielding the value of total bilirubin. Unconjugated bilirubin is measured by subtracting the direct-reacting fraction from total
bilirubin. Potential sources of error include plasma lipids, drugs such as propranolol, and several other endogenous substances.
These interfere with the diazo assay and can potentially produce an unreliable result.

Clinical Significance
As unconjugated bilirubin is always bound to albumin in serum, it cannot be filtered by the glomeruli (in the absence of
glomerular disease). Thus, unconjugated bilirubin is never found in urine even when there is an elevated level of unconjugated
bilirubin in circulation. Jaundice that occurs with unconjugated hyperbilirubinemia is termed acholuric because the urine is not
darkened. Dark urine, however, occurs when there is an excretion of an excess of water-soluble, conjugated bilirubin. This is
seen in conjugated hyperbilirubinemia and signifies the presence of either liver or biliary disease. Thus the presence of bilirubin
in urine will help identify subtle hepatobiliary dysfunction leading to conjugated hyperbilirubinemia, even when the measured
concentration of conjugated bilirubin in serum is only slightly elevated. An exception to this rule is when bilirubinuria is not
detected in a patient with prolonged cholestasis and marked jaundice. This is due to the formation of delta bilirubin or
conjugated bilirubin that is tightly bound to serum albumin. The absence of bilirubinuria in such patients should not cause any
difficulty in diagnosing conjugated hyperbilirubinemia, as the patient is clearly jaundiced and serum conjugated bilirubin is
markedly elevated in such cases.[6][7][8][9][2]

Continuing Education / Review Questions

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References
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4. Hinds TD, Stec DE. Bilirubin, a Cardiometabolic Signaling Molecule. Hypertension. 2018 Oct;72(4):788-795. [PMC free
article: PMC6205727] [PubMed: 30354722]
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therapeutics. J Am Acad Dermatol. 2019 Dec;81(6):1371-1378. [PMC free article: PMC7825249] [PubMed:
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