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.

Advertisements

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.

 

 

 

Disseminated Intravascular Coagulation

Disseminated intravascular coagulation (DIC) is a generalized activation of homeostasis secondary to a systemic disease. There are multiple different diseases that can activate different homeostatic factors that can contribute to DIC. Conditions such as physical trauma and endothelial cell damage exposing tissue factor that finds its way into circulation; or being exposed to tissue factor through vasodilation from hypovolemic shock, malignant hypertension or even heat stroke. There is a long list of secondary conditions that can set off an array of events that can lead to DIC. DIC involves all aspects of homeostasis; the vascular intima, platelets, leukocytes, coagulation, coagulation regulation, and fibrinolysis. DIC is often called a consumptive coagulopathy as it is consuming platelets at a rapid rate that form fibrin microthrombin that partially occlude small vessels. These thrombi that form are small and ineffective so systemic hemorrhage occurs which is often the first sign of DIC or one of the first signs prevalent.

To understand the pathophysiology of DIC its important to have a handle on normal primary and secondary homeostasis in the body. Normal physiological coagulation initiation begins on tissue-bearing cells such as fibroblasts and the subendothelial cells. This is called the extrinsic tenase complex which is composed of tissue factor, factor VIIa, and calcium. When tissue factor that is released from damaged subendothelial cells comes into contact with coagulation factor VII it activates it and that complex produces factors Xa, and IX and miniscual amounts of thrombin. There is also a minute amount of factor VIIa that is circulating in the blood that is resistant to breakdown from tissue factor pathway inhibitor (TFPI) and can bind to tissue factor to start coagulation. The initiation phase and the small amount of thrombin produced starts the initial fibrin formation by splitting the fibrinogen peptides A and B from fibrinogen and activates platelets through cleavage of protease activated receptors PAR-1 and Par-4, cofactors, factor Va released from the platelet alpha granules, factor VIIIa to be released by vWF, and procoagulants such as factor IX to be used further in propagation.

Coagulation Cascade

Propagation is where more than 95% of the thrombin is generated and occurs on the surface of the platelets. Initiation attracts a copious amount of platelets to adhere to the site of the injury from both the low-level thrombin released and exposed collagen. These initial platelets are sometimes called COAT-platelets or platelets partially activated by collagen and thrombin. The COAT-platelets have a higher level of procoagulant activity than platelets activated by collagen alone. These platelets also provide a surface for the intrinsic and prothrombinase tenases to form. Factors Va and VIIIa that were activated by thrombin in initiation bind to platelet surfaces and become receptors for factors IXa and Xa. Factor IXa binds to VIIIa and forms in the intrinsic complex. The intrinsic complex then activates factor Xa, which binds to factor Va that forms the prothrombinase complex. The prothrombinase complex activates prothrombin which generates thrombin. Thrombin activates factor XIII to stabilize the fibrin clot by covalently cross-linking the fibrin polymers initiated by the extrinsic tenase, binds to its cofactor thrombomodulin to activate the protein C pathway and also activates thrombin activatable fibrinolysis inhibitor (TAFI) to inhibit fibrinolysis to protect the formation of the fibrin clot.

Platelets as mentioned above have an important role in homeostasis. Platelet activation occurs once the platelet binds to collagen or the vWF that is present on the surface of the damaged endothelial cells. Adhesion occurs through the integrin GP IX V platelet receptor. Upon binding they secrete their primary granules that secretes molecules such as ADP, epinephrine, serotonin, and calcium. Calcium and other molecules like ADP activate phospholipase A2 through GCPRs otherwise known as 7 transmembrane receptors. Thomboxane A2 (TXA2) is then synthesized by thromboxane synthase in multitude of events. TXA2 generates secondary messengers DAG and IP3. DAG helps mediate actin contraction for shape conformational changes and IP3 binds to the IP3 receptors in the dense tubular system that opens calcium channels to allow release of more calcium. The activation of DAG and IP3 induces a conformational change that activates the fibrinogen receptor GP IIb/IIIa which allows adjacent platelets to aggregate and form the initial platelet plug. Platelets are also important in that they allow a surface for propagation of coagulation to occur.

With a basic background of primary and secondary homeostasis it will now be easier to understand what actually occurs during DIC. Triggering events may activate coagulation at any point in its pathway. Circulating thrombin that is released activates platelets, activates coagulation proteins that have positive feedback loops within the coagulation cascade and catalyzes fibrin formation. The fibrinolytic system enzymes such as plasminogen and TPA may become active subsequent to fibrin clot formation. Monocytes may also be induced to released tissue factor caused by inflammation in DIC. Normally thrombin cleaves fibrinogen creating fibrin monomers which spontaneously polymerize to from this insoluble gel which is strengthened through factor XIII. In DIC, a high percentage of the fibrin monomers fail to polymerize and just circulate in plasma as soluble monomers. These circulating monomers coat platelets and coagulation proteins which doesn’t allow any binding creating an anticoagulant effect. Plasmin, which is the activated form of plasminogen is a part of the fibrinolytic system. In normal homeostasis plasmin only cleaves the solid fibrin clot formed. Although in DIC, plasmin circulates in the plasma and degrades all forms of fibrin. It is because of this that fibrin degradation products, otherwise known as D-dimers become detectable in the plasma in concentrations commonly exceeding 20,000 ng/mL. The normal range for the D-dimer is 0-240 ng/mL. At the same time coagulation pathway control is lost as protein C, protein S, and anti-thrombin are consumed by the plasmin. Plasmin also digests factors V, VIII, IX, and XI. The platelets become enmeshed within the fibrin monomers and become exposed to thrombin which triggers platelet further platelet activation and consumption. Plasmin can also trigger complement which causes hemolysis and the kinin system which triggers inflammation and hypotension and as an end result shock.

It is important to diagnose DIC early and as a physician be aware of the early signs. A lot of times the symptoms of DIC are masked by the underlying disease and may be chronic or acute. The initial laboratory testing includes a platelet count, blood film examination, PT, aPTT, D-dimer and fibrinogen assay. The PT time is usually >14 seconds (Ref. 11-14) aPTT is usually >35 seconds (Ref. 25-35). These time intervals clue in that there is a coagulation issue as the PT tests the function of the extrinsic pathway and the aPTT tests the function of the intrinsic pathway. The platelet count is lower than 150,000 uL (Ref. 150,000-450,000 uL). The D-dimer as noted previously is significantly elevated. Although a D-dimer alone can’t diagnose DIC because a D-dimer is elevated in other conditions such as inflammation, pulmonary embolism and deep vein thrombosis. The fibrinogen levels may drop below 220 mg/dL (Ref. 220-498), but that provides little diagnostic information because in a lot of cases the fibrinogen level may not rise or may become elevated because of the level of inflammation occurring while the patient is in DIC. A peripheral blood smear confirms thrombocytopenia as well as the presence of schistocytes. Schistocytes are broken red blood cells because the microvessel walls are occluded they get shredded while passing through the blood vessels. Although not one test result can rule in DIC or rule it out, a panel of specialized tests can help in the diagnosis. It’s important to get the whole picture.

1502FeinsteinTablehttp://www.cancernetwork.com/oncology-journal/disseminated-intravascular-coagulation-patients-solid-tumors

Treatment of chronic DIC is to diagnose and treat the underlying condition. This may include surgery, anti-inflammatory agents, or antibiotics to stabilize homeostasis. Supportive therapy to maintain fluid and electrolyte balance is important in the treatment of chronic DIC. In acute DIC where there is multi organ failure from microthrombi and hemorrhagic bleeding therapies are targeted at slowing the clotting process and to replace the consumed coagulation factors and proteins. Unfractionated heparin is commonly used for its anti-thrombotic properties. Normally the aPTT is used to monitor heparin therapy, but in the case of DIC other assays must be used so it’s important to pay close attention to the patient when administering heparin as it can aggravate bleeding tendencies. Physicians may also order fresh frozen plasma (FFP), platelets, and red cell transfusions as needed. FFP will replace the coagulation factors and proteins. The platelets will correct for the thrombocytopenia and the red cells are transfused because of the resulting anemia. Cryoprecipitate can also be administered to replace the low levels of fibrinogen. A physician may use an INR to figure out the best way to treat the DIC as well as monitor the therapy. The INR or international normalized ratio is a way of standardizing the PT results, regardless of test methods and where the testing occurred. A normal INR should be between 2-3. An INR too low puts the patient as risk for blood clots, on the contrary and INR too high puts the patient as too high of a risk for bleeding. As the underlying condition begins to stabilize the DIC will begin to subside and patient will slowly recover.

There will be more covered about DIC and how it relates to different leukemias and solid tumor cancers. This article provides an overview and how it affects normal homeostasis.

-Caleb