Degradation of tetrapyrroles – heme, and its disorders. Intravascular and extravascular decomposition of erythrocytes

Degradation

heme breakdown

After about 120 days in circulation, erythrocytes are captured and degraded by the reticuloendothelial system, mainly in the liver and spleen. Approximately 85% of the heme destined for degradation comes from old erythrocytes and 15% from the turnover of immature erythrocytes and cytochromes from non-erythroid cells.

The first step in heme degradation is catalyzed by the microsomal heme oxidase system of reticuloendothelial cells. In the presence of NADPH and O2, the enzyme adds a hydroxyl group to the methenyl bridge between the two pyrrole nuclei, simultaneously oxidizing the heme iron to Fe3+.

A second oxidation by the same enzyme system leads to the cleavage of the porphyrin cycle.

This produces the green pigment biliverdin with the simultaneous release of iron and CO (CO may have signaling effects).

Biliverdin is subsequently reduced to the orange pigment bilirubin. Bilirubin and its derivatives are collectively called bile pigments.

• Bilirubin, which is found only in mammalian organisms, can have an antioxidant function. As an antioxidant, it is oxidized to biliverdin and then reduced by biliverdin reductase back to bilirubin.

Bilirubin is only slightly soluble in plasma and is therefore transported to the liver in a non-covalent bond to albumin. After being unbound from albumin, it enters the hepatocyte by facilitated diffusion, where it binds to an intracellular protein, especially the ligandin molecule.

In the hepatocyte, the solubility of bilirubin increases by the addition of two molecules of glucuronic acid (a process called conjugation)

• The reaction is catalyzed by microsomal bilirubin glucuronyltransferase, which uses UDP-glucuronic acid as a source of glucuronate.

• Varying degrees of deficiency of this enzyme cause Crigler-Najjar syndrome type 1 or 2 or Gilbert's syndrome, with Crigler-Najjar syndrome type 1 having the worst enzyme deficiency.

Bilirubin diglucuronate is hydrolyzed and reduced by bacteria in the intestine to colorless urobilinogen.

Most urobilinogen is oxidized by intestinal bacteria to stercobilin, which gives the stool its characteristic brown color. However, some urobilinogen is reabsorbed from the intestine and enters the portal circulation. Some of these urobilinogen particles are taken up by the liver in the enterohepatic circulation and subsequently excreted again in the bile.

The rest of the urobilinogen is transported by the blood to the kidneys, where it is converted to yellow urobilin, which is then excreted in the urine. Urobilin gives urine its characteristic yellow color.

Hemolysis

Intravascular hemolysis

The breakdown of blood cells in the blood vessels can be caused by:

• deficits in erythrocytes = (intra)corpuscular hemolysis
• external factors = extracorpuscular hemolysis

Hemoglobin released from destroyed erythrocytes binds to haptoglobin and forms a complaex that does not pass through the glomerular filter (the function of haptoglobin formed in the liver is to prevent both kidney damage and iron loss). The complex is then broken down by macrophages to bilirubin and ferritin or hemosiderin.

With increased hemolysis, icterus and hemosiderosis occur.  If the released hemoglobin is more than haptoglobin can bind (especially in acute hemolysis, the concentration of haptoglobin in the serum is reduced by consumption (norm 0.3-2 g/l)), hemoglobinemia occurs and the excess hemoglobin passes through the glomerular filter into the renal tubules - hemoglobinuria and hemosiderinuria occur (part of the hemoglobin molecules is taken up by the cells of the proximal tubule and converted into hemosiderin, which is then gradually  releases into the urine) - the precipitation of hemoglobin then causes kidney damage known as hemoglobinuric nephrosis (a similar impairment is found in crush syndrome - myoglobinuric nephrosis).

Extravascular hemolysis

Outside the blood vessels, erythrocytes break down quickly. Hemoglobin released from them (or whole erythrocytes) is broken down by tissue macrophages.

The resulting bilirubin diffuses into the surroundings and affects the coloring of the surrounding tissues (local icterus – typically in a subcutaneous hematoma, bruise).  The icteric coloration later disappears and the rust coloration persists (hemosiderin - e.g. in cerebral hemorrhage foci).  Another pigment that is formed in this process is ceroid - a lipopigment arising from the polymerization of lipid oxidation products (lipids released from decayed erythrocytes), the mixture of ceroid with hemosiderin is called hemofuscin. 

Subsequently, the hematoma is organized by non-specific granulation tissue - siderophages and fibrin are present on the periphery, along which granulation tissue grows into the hematoma, penetrates it, and only a small scar remains from the hematoma.  If the hematoma is larger, then the central part may liquefy before the fibrin fibers have been replaced by granulation tissue, the surface layer of the hematoma acquires the properties of a semi-permeable membrane, through which fluid is sucked into the hematoma and the hematoma enlarges, forming a post-hemorrhagic pseudocyst, or  after the discoloration of the posthemorrhagic hygroma.

Accelerated extravascular hemolysis accompanies hypersplenism, some disorders of erythrocyte metabolism, malaria, etc. The level of unconjugated bilirubin may be elevated (above 12 μmol/l), subicterus or icterus may be clinically evident. The symptoms of hemolytic anemia are also present - increased amount of reticulocytes, hemoglobinuria, anemia (hemoglobin below 120 g/l), decreased number of erythrocytes in the blood picture, increased activity of lactate dehydrogenase.

Pathobiochemistry

scleral icterus

Icterus (jaundice) is a yellow discoloration of the skin, nail beds, and whites of the eyes, caused by the accumulation of bilirubin or an increased level of bilirubin in the blood. Although it is not a disease, jaundice is often a manifestation of a disease.

Jaundice can be divided into three basic types (hemolytic, hepatocellular, and obstructive). However, from a clinical point of view, jaundice is often more of a complex of these types. Accumulation of bilirubin can result from a defect in more than one step of its metabolism.

The liver has the capacity to conjugate and excrete more than 3000 mg of bilirubin per day, while normal bilirubin production is only 300 mg/day. This large capacity allows the liver to respond to increased heme degradation with subsequent increased conjugation and excretion of bilirubin diglucuronidate. However, massive hemolysis (e.g., in patients with sickle cell anemia, pyruvate kinase deficiency, or glucose-6-phosphate dehydrogenase deficiency) can cause accelerated bilirubin production and insufficient conjugation. The level of unconjugated bilirubin in the blood increases, causing hemolytic jaundice, which is manifested by increased levels of urobilinogen in the urine.

Damage to hepatocytes (e.g., due to cirrhosis or hepatitis) causes increased levels of unconjugated bilirubin in the blood due to decreased conjugation. Urobilinogen is increased in the urine because liver damage reduces the enterohepatic circulation of this compound, allowing increased excretion into the blood and subsequently into the urine. The urine thus darkens, and the stools, on the contrary, become paler. The levels of AST and ALT (question III/8) are also increased. This type of jaundice is called hepatocellular jaundice.

Obstructive jaundice is not caused by overproduction of bilirubin or insufficient conjugation, but occurs as a result of obstruction of the bile duct (extrahepatic cholestasis). For example, the presence of a tumor or gallstones that block the bile duct, preventing the flow of bilirubin into the intestine. Patients with obstructive jaundice experience pain in the GIT, nausea, and their stools are pale in color and the urine turns black over time. The liver secretes unconjugated bilirubin into the blood (hyperbilirubinemia). This substance is subsequently excreted in the urine. However, urobilinogen is not found in the urine.

Sources

MATOUŠ, Bohuslav, et al. Základy lékařské chemie a biochemie. 1. vydání. Praha: Galén, 2010. 540 s.

KITTNAR, Otomar, et al. Lékařská fyziologie. 1. vydání. Praha : Grada, 2011. 790 s.