Bilirubin is a yellow compound that is the product of the catabolism of ageing heme. It can be used to determine abnormality in liver functions as characterized by yellowing of skin and eyes. In this article, we will discuss about the biochemistry of bilirubin. Specifically, how the compound is synthesized and what are the important enzymes used in the synthesis of this biomolecule.
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Bilirubin is a yellowish compound that is produced in the catabolic pathway of the breakdown of heme in vertebrates. Catabolism of heme is needed to clear the body of the waste products due to the death of aging red blood cells. The structure of bilirubin consists of an open-chain of four pyrrole-like rings.


Image: “Chemical formula of Bilirubin.” by Stefcho2 – Own work. License: Public Domain

Bilirubin is produced by the breakdown of the red blood cells in the body. It travels to the liver and is secreted to the bile duct. It is eliminated eventually from the body in the stool. A typical red blood cell has a lifespan of about 120 days. The hemoglobin present in the RBCs gets broken down into bilirubin and other substances.

Bilirubin needs to be excreted from the body because of its potential toxicity. Excessive levels of bilirubin in the blood stream can cause the condition called kernicterus. Due to its ability to cross the blood-brain barrier, leading to conditions that can impair normal brain functioning. The conditions can be fatal if left untreated.

Accumulation of this substance in the blood causes jaundice, the yellow discoloration of the skin and eyes. This condition is a common symptom for liver disease.

Cellular Heme Metabolism

 Bilirubin is produced by a two-stage reaction that occurs in the reticulo-endothelial system of cells. These cells include phagocytes, Kupffer cells of the liver, and cells in the bone marrow and spleen. The catabolism of heme proteins occur in the microsomal fraction of cell by heme oxygenase. The action of this enzyme is substrate-inducible which means the heme serves both as the substrate and the cofactor for the reaction.

The amino acids from the protein component of hemoglobin are released from the structure and are catabolized or reused for protein synthesis. The heme portion, on the other hand, undergoes degradation starting with a mixed-function oxidase reaction that causes opening of the ring and the conversion of one of the methane bridge carbons to carbon monoxide.

The next step in the process is the release of the iron from the resulting linear tetrapyrrole. The iron is then transported to storage pools in the bone marrow to be reused in erythrocyte production. This iron atom that reaches the heme oxygenase is usually oxidized to its hemin or ferric form.


Image: “Structural formula of Biliverdin.” by NEUROtiker – Own work. License: Public Domain

After the release of the iron atom and the ring-opening of the heme group, Biliverdin is produced. Biliverdin is a green tetrapyrrolic bile pigment that is responsible for a greenish color during bruises.

The Biliverdin is reduced by biliverdin reductase. Biliverdin reductase is an enzyme found in all tissues, especially in reticulo-macrophages of the liver and spleen. This enzyme is responsible for converting the biliverdin to bilirubin by reducing the double bond between the second and third pyrrole ring to form a single bond. The reduction process of biliverdin requires NADH or NADPH as a cofactor.

The biliverdin reductase catalyzes the reaction through an overlap in the binding istes of the Lys18, Lys22, Lys179, Arg183, and Arg185 as key residues. This binding sites attaches to the biliverdin molecule causing the dissociation of the bilverdin from the heme oxygenase, eventually leading to reduction of the green biliverdin to the yellow bilirubin.

BVR mechanism

Image: “Reduction of biliverdin to bilirubin catalyzed by biliverdin reductase.” by Boghog2 – Own work. License: Public Domain

In the uptake to the liver, bilirubin is taken up at the sinusoidal surface of liver cells by a facilitated transport system. Once the bilirubin molecule enters the cell, it is bound to the cytosolic proteins like glutathione S-transferase, also called ligandin, to prevent its reentry to the bloodstream.

In the liver, reacting it with two glucuronic acid molecules solubilizes bilirubin. This is then as for bilirubin not to persist in cells because of its highly non-polar nature. The conjugation with glucuronic acids converts bilirubin into a more polar molecule.

The enzyme UDP-glucosyltransferase, a bilirubin-specific enzyme in the endoplasmic reticulum catalyzes the step-wise transfer of two glucosyl moieties from a UDP-glucuronate to the bilirubin molecule. The solubilized bilirubin, bilirubin diglucoronide is then secreted to the bile and finally excreted via the intestine.

Review Questions on Bilirubin

The correct answers can be found below the references.

  1. What is the enzyme used to convert biliverdin to bilirubin?
    1. Bilirubin reductase
    2. Biliverdin reductase
    3. Heme oxidase
    4. Glucosyltransferase
  2. What is the color of bilirubin?
    1. Yellow
    2. Orange
    3. Green
    4. Blue
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