Bilirubin Metabolism

Bilirubin is a metabolite of heme. It serves as a means to excrete unwanted heme, which is derived from various heme-containing proteins such as hemoglobin, myoglobin, and various P450 enzymes. Bilirubin is also notable for providing the color to bile, stool, and to a lesser extent the urine. Its produced by a two-stage reaction that occurs in cells of the RES (reticuloendothelial system). The RES includes the phagocytes, mainly being the macrophages, the Kupffer cells in the liver and the cells in the spleen and bone. Heme is taken up into these cells and acted on by the enzyme heme oxygenase, liberating the chelated iron from the heme structure and releasing carbon monoxide. The carbon monoxide is excreted via the lungs. The reaction yields a green pigment known as biliverdin. Biliverdin is then acted on by the enzyme biliverdin reductase which produces bilirubin. Bilirubin consists of a yellow pigment. Bilirubin is derived from two main sources. The majority, about 80% comes from heme which is released from senescent red blood cells. The other 20% originates from other heme-containing proteins found in the liver and muscles.

Synthesis

Bilirubin is toxic to tissues, therefore it is transported in the blood in its unconjugated form bound to albumin. For that reason, only a small amount of the free form is present in the blood. If the free fraction increases, bilirubin with invade and cause damage to the tissues. Excess unconjugated bilirubin can cross the blood-brain barrier and cause kernicterus in neonates. The unconjugated bilirubin is taken up by hepatocytes where the albumin bond is broken. Inside the hepatocyte, the bilirubin is bound to cytoplasmic proteins ligandins and Z proteins. The primary function of these proteins is too prevent the reflux of bilirubin back into the circulatory system. Unconjugated bilirubin is lipophilic. Its conjugation with glucuronic acid renders it hydrophilic, therefore it can be eliminated utilizing bile. Conjugated bilirubin synthesis occurs in a two step reaction. First glucuronic acid is synthesized from cytosolic glucose which then attaches to uridinediphosphate (UDP) via the enzyme UDP-glucose-dehydrogenase. This forms UDP-glucuronic acid. This compound has an affinity for bilirubin for which then the glucuronic acid is transferred to the bilirubin which is catalyzed by glucuronyl transferase. Conjugation of bilirubin takes place in the endoplasmic reticulum of the hepatocytes and the end result is an ester between the glurcuronic acid and one or both of the propionic side-chains of bilirubin.

Pathways in bilirubin metabolism

Metabolism

Once bilirubin is conjugated it is excreted with bile acid into the small intestine. The bile acid is reabsorbed in the terminal ileum for enterohepatic circulation, the conjugated bilirubin is not absorbed and instead passes into the colon. In the colon, the bacteria metabolize the bilirubin into urobilinogen, which can be oxidized to form urobilin, and stercobilin. Urobilin is excreted by the kidneys to give urine its yellow color and stercobilin is excreted in the feces giving stool its characteristic brown color. There can be traces levels of urobilinogen present in the blood.

Toxicity

Unconjugated hyperbilirubinemia in a neonate can lead to an accumulation of unconjugated bilirubin in the brain tissue. The neurological disorder is called kernicterus. The blood-brain barrier is not yet fully developed and bilirubin can freely pass into the brain interstitium. In cases of liver impairment, biliary drainage is blocked, and some of the conjugated bilirubin leaks into the urine, turning it a dark amber color. In cases of hemolytic anemia, there is increased hemolysis of red cells causing an increase in unconjugated bilirubin in the blood. In these cases, there is no problem with the livers mechanism to conjugate the bilirubin, and there will be an increase in urobilinogen in the urine. This is the difference between an increased urine bilirubin, and an increased urine urobilinogen.

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Thalassemias

PIT11

The major hemoglobin that is present in adults is hemoglobin A (HbA). This is a heterotetramer that consists of one pair of alpha-globin chains and one pair of beta-globin chains. Alpha-globin chains are encoded by two copies of the alpha gene present on chromosome 16. Beta-globin chains are driven by one gene on chromosome 11. Normally there is tight regulation of the production of alpha and beta-globin chains and the ratio of production, but sometimes that regulation can be interrupted. This offsets the balance of globin chains being produced. These types of hematological disorders are coined thalassemias. They are a quantitative defect characterized by reduced or absent production of one and rarely two of the globin chains.

Alpha thalassemia is largely due to the inadequate production of alpha globin chains, which leads to an excessive production of either gamma-globin chains or beta-globin chains. In the fetus alpha thalassemia leads to excess gamma chains and in adults it largely leads to excess beta chains. In neonates the absence of alpha-globin chains is incompatible with life, leading to hydrops fetalis or hemoglobin Barts and absolute death after delivery. Hb Barts cannot deliver the oxygen to the tissues because its affinity to oxygen is too high. The hydronic state is reflected in the fetus by heart failure and massive total body edema. Excess beta-globin chains are capable of forming homotetramers and precipitate that leads to a variety of clinical manifestations.

Beta thalassemia is an inherited hemoglobinopathy in which production of beta-globin chains is impaired. There are different classifications corresponding to the degree of reduction in the beta chains. Beta thalassemia major is due to mutations that completely stop all production of beta-globin chains. These are individuals who are homozygous for the disease. They lose the ability to make HbA and because of this will experience severe manifestations and are transfusion-dependent for the rest of life. Symptoms typically begin during late infancy (6-12 months), but some newborns are asymptomatic because the major hemoglobin in newborns is HbF (4A:4G) which is constructed by gamma-globin chains and not beta-globin chains. Beta-thalassemia major presents with pallor, jaundice, and bilirubin in the urine which indicates hemolysis. Hepatosplenomegaly is present as well as heart failure. Failure to thrive and recurrent infections are also other signs. There is so much hemolysis because of the faulty hemoglobin present in the red cells that the bone marrow can’t keep up with production so extra medullary hematopoiesis occurs that results in skeletal abnormalities in the face and long bones. Iron overload is often a symptom of late untreated disease which can affect almost every organ in the body. Mortality is upwards of 85% by age five if untreated. If treated the survival rate is only 60 years of age if lucky.

Beta thalassemia major is also called transfusion-dependent beta thalassemia. There is also a subtype called non-transfusion-dependent beta thalassemia otherwise known as beta thalassemia intermedia. These individuals present with a less severe phenotype of the disease. There is significant variability with the clinical findings in individuals with beta thalassemia intermedia; from osteoporosis to thrombosis to diabetes mellitus. Some individuals will develop hepatosplenomegaly and extramedullary hematopoiesis and some won’t. Also some individuals will have to become transfusion-dependent, but that is typically in the late decades of life.

Anemia is a severe clinical manifestation of both alpha and beta thalassemia. The pathophysiology of beta thalassemia causes excess alpha-globin chains to precipitate in the developing erythrocytes in the bone marrow. This causes inclusion bodies. The inclusion bodies create oxidative stress and damages the cellular membranes. Apoptosis gets activated downstream and the red cell precursors are subsequently phagocytized and destroyed in the bone marrow by activated macrophages. This is also called ineffective erythropoiesis. The bone marrow in an effort to compensate releases these red cell precursors into the peripheral blood riddled with these inclusion bodies. These cells are subsequently sequestered by extravascular hemolysis by the RES which  further contributes to the anemia. The red cells that survive are microcytic and hypochromic and have a significantly shortened life span. Severe tissue hypoxia is seen due to the increased HbF as a compensatory mechanism. HbF has an increased affinity for oxygen and causes a shift to the left on the oxygen dissociation curve.

Typical laboratory findings for an individual with beta thalassemia is a slightly decreased red cell count and a marked decrease in hemoglobin of usually about 2-3 g/dL (12.5-16.5 g/dL). There will be marked anisocytosis (microcytosis) and poikilocytosis, target cells, basophilic stippling, slight increase in reticulocytes and nucleated red cells.

The pathophysiology for anemia associated with alpha thalassemia is associated with precipitation of HbH. Remember HbH is formed when there is decreased production of the alpha-globin chains so there is an excess of beta-globin chains. The precipitation of HbH creates inclusion bodies, typically called Heinz Bodies. These inclusions are recognized by the RES and remove the red cells via extravascular hemolysis.

Laboratory findings for an individual with alpha thalassemia is very similar to that of an individual with beta thalassemia. Decreased hemoglobin, marked anisocytosis (microcytosis) and poikilocytosis, target cells, basophilic stippling, and reticulocytes and NRBCs. The HbH inclusions can be see seen using a cresyl blue stain.

Thalassemias are a quantitative hemoglobinopathy meaning that there is a deficiency or an excess of production of globin chains leading to clinical manifestations. They are inherited and some subtypes can significantly elevate mortality. It is important to diagnose early and to treat early.