Bilirubin exchange

Bilirubin is the end product of heme breakdown. The bulk (80-85%) of bilirubin is formed from hemoglobin and only a small part is formed from other heme-containing proteins, such as cytochrome P450. Bilirubin is formed in the cells of the reticuloendothelial system. About 300 mg of bilirubin is formed daily.

The conversion of heme to bilirubin involves the microsomal enzyme heme oxygenase, which requires oxygen and NADPH for its function. The porphyrin ring is cleaved selectively at the methane group in position a. The carbon atom in the a-methane bridge is oxidized to carbon monoxide, and instead of the bridge, two double bonds are formed with oxygen molecules coming from outside. The resulting linear tetrapyrrole is structurally IX-alpha-biliverdin. It is then converted by biliverdin reductase, a cytosolic enzyme, to IX-alpha-bilirubin. Linear tetrapyrrole of this structure should be water-soluble, while bilirubin is a fat-soluble substance. Lipid solubility is determined by the structure of IX-alpha-bilirubin - the presence of 6 stable intramolecular hydrogen bonds. These bonds can be broken by alcohol in a diazo reaction (van den Bergh), in which unconjugated (indirect) bilirubin is converted to conjugated (direct). In vivo, stable hydrogen bonds are broken by esterification with glucuronic acid.

About 20% of circulating bilirubin is derived from sources other than the heme of mature red blood cells. A small amount comes from immature cells of the spleen and bone marrow. This amount increases with hemolysis. The remainder is formed in the liver from heme-containing proteins such as myoglobin, cytochromes, and other unspecified sources. This fraction is increased in pernicious anemia, erythropoietic uroporphyrin, and Crigler-Najjar syndrome.

Transport and conjugation of bilirubin in the liver

Unconjugated bilirubin in plasma is tightly bound to albumin. Only a very small proportion of bilirubin is dialyzable, but it can increase under the influence of substances that compete with bilirubin for binding to albumin (e.g. fatty acids or organic anions). This is of importance in neonates, in whom a number of drugs (e.g. sulfonamides and salicylates) can facilitate the diffusion of bilirubin into the brain and thus contribute to the development of kernicterus.

The liver secretes many organic anions, including fatty acids, bile acids, and other non-bile acid components of bile such as bilirubin (despite its tight binding to albumin). Studies have shown that bilirubin is separated from albumin in the sinusoids and diffuses through the aqueous layer on the hepatocyte surface. Previous suggestions that albumin receptors are present have not been confirmed. Bilirubin is transported across the plasma membrane into the hepatocyte by transport proteins such as organic anion transport protein and/or by a flip-flop mechanism. Bilirubin uptake is highly efficient because of its rapid metabolism in the liver by glucuronidation and secretion into bile, and because of the presence of cytosolic binding proteins such as ligandins (glutathione-8-transferase).

Unconjugated bilirubin is a non-polar (fat-soluble) substance. In the conjugation reaction, it is converted into a polar (water-soluble substance) and can therefore be excreted into bile. This reaction occurs with the help of the microsomal enzyme uridine diphosphate glucuronyl transferase (UDPGT), which converts unconjugated bilirubin into conjugated mono- and diglucuronide bilirubin. UPGT is one of several isoforms of the enzyme that provide conjugation of endogenous metabolites, hormones and neurotransmitters.

The UDPHT gene of bilirubin is located on the 2nd pair of chromosomes. The structure of the gene is complex. In all UDPHT isoforms, exons 2-5 at the 3' end of the gene DNA are constant components. For gene expression, the involvement of one of the first several exons is necessary. Thus, for the formation of bilirubin-UDFHT isoenzymes 1*1 and 1*2, the involvement of exons 1A and ID, respectively, is necessary. Isoenzyme 1*1 participates in the conjugation of almost all bilirubin, and isoenzyme 1*2 participates almost or not at all. Other exons (IF and 1G) encode phenol-UDFHT isoforms. Thus, the choice of one of the sequences of exon 1 determines the substrate specificity and properties of the enzymes.

Further expression of UDFGT 1*1 also depends on a promoter region at the 5' end associated with each of the first exons. The promoter region contains the sequence TATAA.

Details of the gene structure are important for understanding the pathogenesis of unconjugated hyperbilirubinemia (Gilbert and Crigler-Najjar syndromes), when the liver contains reduced or absent enzymes responsible for conjugation.

The activity of UDFGT in hepatocellular jaundice is maintained at a sufficient level, and even increases in cholestasis. In newborns, the activity of UDFGT is low.

In humans, bilirubin is mainly present in the bile as diglucuronide. The conversion of bilirubin to monoglucuronide and diglucuronide occurs in the same microsomal glucuronyl transferase system. When there is an overload of bilirubin, such as during hemolysis, monoglucuronide is predominantly formed, and when the bilirubin supply decreases or the enzyme is induced, the diglucuronide content increases.

Conjugation with glucuronic acid is most important, but a small amount of bilirubin is conjugated with sulfates, xylose, and glucose; these processes are enhanced in cholestasis.

In the late stages of cholestatic or hepatocellular jaundice, despite the high plasma bilirubin content, bilirubin is not detected in the urine. Apparently, the reason for this is the formation of bilirubin type III, monoconjugated, which is covalently bound to albumin. It is not filtered in the glomeruli and, therefore, does not appear in the urine. This reduces the practical significance of the tests used to determine the bilirubin content in urine.

Bilirubin excretion into the tubules occurs via a family of ATP-dependent multispecific organic anion transport proteins. The rate of bilirubin transport from plasma to bile is determined by the bilirubin glucuronide excretion step.

Bile acids are transported into bile by a different transport protein. The presence of different mechanisms of transport of bilirubin and bile acids can be illustrated by the example of Dubin-Johnson syndrome, in which the excretion of conjugated bilirubin is impaired, but normal excretion of bile acids is preserved. Most of the conjugated bilirubin in bile is in mixed micelles containing cholesterol, phospholipids and bile acids. The importance of the Golgi apparatus and microfilaments of the hepatocyte cytoskeleton for the intracellular transport of conjugated bilirubin has not yet been established.

Bilirubin diglucuronide, which is found in bile, is water-soluble (polar molecule), so it is not absorbed in the small intestine. In the large intestine, conjugated bilirubin is hydrolyzed by bacterial b-glucuronidases to form urobilinogens. In bacterial cholangitis, part of the bilirubin diglucuronide is hydrolyzed in the bile ducts with subsequent precipitation of bilirubin. This process may be important for the formation of bilirubin gallstones.

Urobilinogen, having a non-polar molecule, is well absorbed in the small intestine and in minimal quantities in the large intestine. A small amount of urobilinogen, which is normally absorbed, is re-excreted by the liver and kidneys (enterohepatic circulation). When the function of hepatocytes is impaired, hepatic re-excretion of urobilinogen is impaired and renal excretion increases. This mechanism explains urobilinogenuria in alcoholic liver disease, fever, heart failure, and in the early stages of viral hepatitis.