Total BilirubinDefinitionBilirubin (formerly referred to as hematoidin) is the yellow breakdownproduct of normal heme catabolism. Heme is formed from hemoglobin, aprincipal component of red blood cells. Bilirubin is excreted in bile, andits levels are elevated in certain diseases. It is responsible for the yellowcolour of bruises and the yellow discolouration in jaundice.FunctionBilirubin is created by the activity of biliverdin reductase on biliverdin.Bilirubin, when oxidized, reverts to become biliverdin once again. Thiscycle, in addition to the demonstration of the potent antioxidant activityof bilirubin, has led to the hypothesis that bilirubins main physiologicrole is as a cellular antioxidant.SourceThe majority of bilirubin (80%) is produced from: a) Degradation of hemoglobin from erythrocytes undergoing normal (removal of aged or effete cells) b) Abnormal destruction (i.e. intravascular or extravascular hemolysis) within mononuclear phagocytes (principally splenic, hepatic and bone marrow macrophages).A small percentage (20%) is derived from: a) Catabolism of various hepatic hemoproteins (myoglobin, cytochrome P450) b) Overproduction of heme from ineffective erythropoiesis in the bone marrow.Metabolism Within macrophages, a free heme group (iron + porphyrin ring) is oxidized by microsomal heme oxygenase into biliverdin and the iron is released (the iron is then stored as ferritin or released into plasma, where it is bound to the transport protein, transferrin). Biliverdin reductase then reduces the green water-soluble biliverdin into unconjugated bilirubin. Heme oxygenase is also located in renal and hepatic parenchyma, enabling these tissues to take up heme and convert it to bilirubin.
Birds lack biliverdin reductase, thus they excrete heme breakdown products as biliverdin rather than bilirubin. Unconjugated or free bilirubin is then released into plasma where it binds to albumin. Uptake of unconjugated bilirubin occurs in the liver and is carrier-mediated. The carrier-mediated uptake is shared with unconjugated bile acids and dyes such as BSP. Once within the hepatocyte, unconjugated bilirubin is transported with ligand (Y protein) or other proteins (e.g. Z protein) and the majority is conjugated to glucuronic acid by glucuronyl transferase. The remainder is conjugated to a variety of neutral glycosides (, xylose). In the horse, the majority of bilirubin is conjugated to glucose. Bilirubin must be conjugated before it can be excreted into bile (conjugation makes bilirubin water soluble). Bilirubin is secreted into the intestine. In the intestine, bacterial degrade it to urobilinogen. Urobilinogen is reabsorbed (about 10%) or broken down (90%) into urobilin and stercobilin (both of which are excreted in the feces). Of the resorbed urobilinogen, most is taken up by the liver (enterohepatic circulation, i.e. the urobilinogen is absorbed into the portal vein, taken up by the liver and re-excreted into bile, whilst the rest bypasses the liver and is excreted into the urine.
Fig.1 Conjugated bilirubin is not normally found in the urine of domesticanimals, although small to (1+) amounts of conjugated bilirubin may beseen in concentrated urine from dogs (particularly males), due to the lowcanine renal threshold for bilirubin. In all species (but dogs, in particular), bilirubinuria may precede anincrease in serum bilirubin in cholestatic disorders. Remember, onlyconjugated bilirubin can be excreted in urine as it is water soluble.Circulating bilirubin exists in two main forms as determined by the Vanden Bergh reaction, which differentiates bilirubin into conjugated (direct)and unconjugated (indirect) forms.
Bilirubin (in blood) is in one of two forms:(I) Direct-reacting (conjugated) Bilirubin.(II) Indirect-reacting (unconjugated) Bilirubin.(I) Direct-reacting (conjugated) BilirubinConjugated bilirubin (direct-reacting). This form reacts in the diazoreaction without the addition of alcohol.Increases in conjugated bilirubin occur with: Hemolysis. Liver disease. Cholestasis.N.B. increased conjugated bilirubin in blood will produce bilirubinuria,which in all species, excluding the dog, is diagnostic for cholestasis.In horses, if conjugated bilirubin comprises > 25% of total bilirubinvalues, cholestasis likely exists (a concurrent bilirubinuria will bepresent). Extrahepatic bile duct obstruction produces the most markedincreases in total bilirubin (20-30 mg/dL).(II) Indirect-reacting (unconjugated) BilirubinFree bilirubin (indirect-reacting or unconjugated). This is a relativelyinsoluble, nonpolar form requiring the addition of alcohol in the diazoreaction to allow color formation.Increases in unconjugated bilirubin occur with: Hemolysis. Liver disease. Cholestasis. Fasting in horses.
In many instances, if unconjugated bilirubin dominates, hemolysis (or inthe case of horses, fasting) is the likely cause of the icterus. WaterAbb. Name(s) Reaction Soluble? Reacts quickly when dyes are Yes (bound to "Conjugated" or added to the blood specimen to"BC" glucuronic "Direct bilirubin" produce azobilirubin "Direct acid) bilirubin" Reacts more slowly. Still produces azobilirubin. Ethanol "Unconjugated" or No, but makes all bilirubin react"BU" "Indirect bilirubin" fat soluble promptly then calc: Indirect bilirubin = Total bilirubin - Direct bilirubinTotal bilirubin measures both BU and BC. Total and direct bilirubinlevels can be measured from the blood, but indirect bilirubin is calculatedfrom the total and direct bilirubin.Measurement methodOriginally the Van den Bergh reaction was used for a qualitative estimateof bilirubin.Causes of hyperbilirubinemiaClinical icterus is observed when total bilirubin values exceed 1.5mg/dL. 1. Artifact. 4. Cholestasis. 2. Hemolysis. 5. Physiologic. 3. Liver disease. 6. Inherited. (1) Artifact:
Hemolysis (destruction of red cells, whether through extravascular or intravascular hemolysis will increase the production of unconjugated bilirubin) and lipemia (even mild) will cause artifactually high bilirubin values. As bilirubin is unstable in light, samples stored for several days, in the presence of light, may have falsely reduced bilirubin values. (3) Liver disease: Hepatic disease may cause increases in both unconjugated and conjugated bilirubin.(4) Cholestasis:This is defined as decreased bile flow due to: a. Physical obstruction of bile flow. b. Functional defects in the transporters that deliver bile salts or bilirubin into the biliary system.a) Physical obstructions to bile flow can be: 1. Intrahepatic (hepatocyte swelling due to hepatic lipidosis in cats). 2. Extrahepatic (bile duct obstruction from pancreatic neoplasia, cholelithiasis, Fasciola hepatica in cattle).b) Functional defects in bile salt or bilirubin transporters 1. Secondary to inflammatory cytokines (endotoxemia) and drugs. 2. Defects in these transporters also occur with physical obstructions to bile flow.Cholestasis will result in bilirubinemia with a higher conjugated thanunconjugated bilirubin. There is often a concurrent bilirubinuria (excessconjugated bilirubin in blood is excreted into the urine, because it iswater soluble).(5) Physiologic: a. Fasting. b. Neonatal. b.
a) Fasting: In horses, fasting will produce a hyperbilirubinemia due to unconjugated bilirubin.b) Neonatal: Young animals, especially foals, often have jaundice (dueprimarily to unconjugated bilirubin). This is due to multifactorial causes,including: 1. Hemolysis of fetal red blood cells. 2. Decreased liver uptake of bilirubin. 3. Immaturity of hepatic conjugation mechanisms. 4. Poor albumin binding.
(6) Inherited:Inherited defects in hepatic uptake, conjugation and excretion ofbilirubin occur in monkeys, sheep, and rats..
Intravascular hemolysis Fig.2 Intravascular hemolysis results from the rupture or lysis of red blood cells within the circulation, and the release of their hemoglobin into the plasma. Haptoglobin binds the liberated free hemoglobin. If intravascular hemolysis continues, the hemoglobin is present in excess amount (>20 mg/dL) resulted in hemoglobinemia and hemoglobinuria. The remaining hemoglobin is oxidized to met-hemoglobin, which disassociates into a free heme and globin chains.
The oxidized free heme (met-heme) binds to hemopexin and the met-heme and hemopexin complex (met-heme/Hpx) is taken up by hepatocytes and macrophages within the spleen, liver and bone marrow (only hepatocyte uptake is illustrated in the image above). Similarly, the hemoglobin/haptoglobin complex is taken up by hepatocytes and macrophages (to a lesser extent). Within these cells, the hemoglobin disassociates into heme and globin chains. The globins are broken down to amino acids, which are then used for protein synthesis. The heme is oxidized by heme oxygenase forming biliverdin and releasing iron. The iron can be transferred to apotransferrin (the iron transport protein) in plasma or can be stored within cells as ferritin (i.e. the iron is bound to the storage protein, apoferritin). The remaining porphyrin ring (biliverdin) is degraded to unconjugated bilirubin by biliverdin reductase. If the hemoglobin/haptoglobin complex is internalized by macrophages, the unconjugated bilirubin is released into the plasma, where it binds to albumin (to render it water-soluble) and is taken up by hepatocytes. Thus, with intravascular hemolysis, increases in bilirubin are usually due to unconjugated bilirubin (indirect) and are likely of macrophage (rather than hepatocyte) origin. The intravascular hemolysis is usually accompanied by extravascular hemolysis which is the source of most of the unconjugated bilirubin observed in hemolytic anemia. Because haptoglobin is consumed during intravascular hemolysis, serum values of this protein usually decline with intravascular hemolytic anemias or when hemoglobin is liberated into plasma by artifactual lysis of red cells in vitro. Since heme oxygenase is also present in renal tubular cells, the renal epithelium is capable of converting hemoglobin to bilirubin. However, this only occurs when there is intravascular hemolysis with hemoglobinuria (i.e. the renal epithelium does not take up unconjugated bilirubin or hemoglobin from blood!). The renal epithelium absorbs the filtered hemoglobin from the urine, converting it to unconjugated bilirubin and then conjugating it for excretion into the urine (see fig.3). This may be responsible for some of the bilirubinuria seen in animals with intravascular hemolysis, however in most of these animals, there is concurrent cholestasis that is responsible for the bilirubinuria (which is conjugated).
Fig.3Note that red cells can also lyse or rupture in vitro (either in the bloodcollection tube or during collection). When this occurs, the hemolysis isconsidered an artifact and does not indicate the animal has a hemolyticanemia.
Extravascular hemolysis Fig.4 Extravascular hemolysis occurs when RBCs are phagocytized by macrophages in the spleen, liver and bone marrow. Extravascular hemolysis is the most common form of hemolytic anemia in animals. It usually occurs alone (without intravascular hemolysis), but will always (to some extent) accompany intravascular hemolysis. Note that during the normal aging of red cells in the circulation, effete red cells are destroyed by macrophages, i.e. extravascular
hemolysis is always occurring to some degree. However, this is a physiologic process and does not result in anemia or excessive unconjugated bilirubin production. With extravascular hemolysis, the erythrocytes are degraded within macrophages, so hemoglobin is not released free into the cytoplasm.Thus, we do not see hemoglobinemia or hemoglobinuria with extravascular hemolysis alone, unless it is accompanying intravascular hemolysis. Within macrophages, the hemoglobin is broken down into its constituents, i.e. the heme ring and globin chains. The globins are broken down to amino acids, which are then used for protein synthesis. The porphyrin ring of heme is oxidized by microsomal heme oxygenase, producing biliverdin and releasing the iron. The iron can then be exported into plasma through iron channels, where it binds to apotransferrin forming transferrin or can be stored within cells as ferritin, with time, ferritin becomes oxidized and degrades to form hemosiderin. Hemosiderin can be visualized within macrophages as a dusky blue-gray pigment and can be definitively stained with Prussian blue (which turns hemosiderin blue). Biliverdin is reduced by biliverdin reductase to unconjugated bilirubin (water insoluble). The unconjugated bilirubin is released into the plasma, where it binds to albumin (to render it water-soluble) and is taken up by hepatocytes.
JaundiceDefinition Jaundice known as icterus, is a yellowish discoloration of the skin, theconjunctival membranes over the sclerae, and other mucous membranes.Jaundice is most frequently caused by an increase of bilirubin in thecirculation, although it can be caused by other substances such ascarotene or certain drugs. Conjugated bilirubin causes more jaundice thanunconjugated bilirubin because of its higher water solubility and easierabsorption into tissues. General circulation .Fig. 5. Normal enterohepatic circulation of bile pigments
Classification of JaundiceJaundice is classified into three categories, depending on which part ofthe physiological mechanism the pathology affects. The three categoriesare: • (I) Pre-hepatic: The pathology is occurring prior the liver. • (II) Hepatic: The pathology is located within the liver. • (III) Post-hepatic: The pathology is located after the conjugation of bilirubin in the liver. In both pre-hepatic and post-hepatic jaundice types, the function of theliver itself is not impaired. In many of these situations, the liver is, in fact,functioning at its maximum capacity in a compensatory effort to alleviatethe problems caused by other factors. This is not the case with hepaticjaundice where the abnormalities are caused by an intrinsic liver defect ordisease.
(I) Pre-hepatic (Hemolytic Jaundice)Pre-hepatic jaundice is caused by an increased production and release ofbilirubin most commonly due to: 1- Hemolytic process. 2- Ineffective erythropoiesis.Increased hemolysis may be due to: a. Variety of hemolytic anemias. b. Exposure to chemicals. c. Hemolytic antigen antibody reactions. d. Disease such as some cancers. e. Drugs coating red blood cells.Ineffective erythropoiesis Is a pathologic process where a very low proportion of red cellsformed in the bone marrow enter the circulation and those remaining inthe bone marrow are prematurely destroyed. An increase in the amount ofbilirubin released from the bone marrow results and is called earlylabeled bilirubin since it has not been circulating within the red bloodcells for 120 days. The rate of hemolysis and the ability of the liver to transport,conjugate, and excrete bilirubin will determine the degree of jaundice in apatient. In most cases of pre-hepatic jaundice, the production of bilirubinis well below the capacity of the liver to conjugate and excrete it. Serumbilirubin levels may still be essentially normal when there is a 50%reduction in red cell survival as long as liver function is normal. Liverfunction tests are helpful in the diagnosis of pre-hepatic jaundice. Theincrease in bilirubin is the most obvious abnormality, being primarily ofthe unconjugated type. Depending on the degree of hemolysis, varyingamounts of bilirubin enter the liver and corresponding amounts ofconjugated bilirubin are found in the intestine. This causes an increasedformation of urobilinogen in the gut (that is excreted in the feces orabsorbed into the enterohepatic circulation and ultimately excreted in theurine.
There should be no bilirubin found in the urine because the increase isof the unconjugated type, which is not filtered by the glomerulus of thekidney. Liver enzyme assays should be normal in this condition except inconditions where there is hemolysis. In these situations, lacticdehydrogenase (LD) will be increased due to the high concentration ofLD found within red cells that is now released into the plasma. It isoccurred as a result of excessive destruction of RBCs. General circulationFig. 6, Hemolytic crisis. Note the increase in the quantities ofunconjugated bilirubin (indirect reacting) in the serum (unable to pass therenal filter), stercobilin in the stool (imparting a darker color to the stool),and urinary urobilinogen. Increased urinary urobilinogen may be partlydue to secondary liver damage (less re-excreted into the bile and hencelost to the serum and urine) in addition to the increased quantity of bilepigments metabolized owing to erythrocyte hemolysis. If secondary liverdamage is extensive from hemosiderosis or bile pigment overload, somebilirubin glucuronide may be regurgitated and lost to the urine (not indiagram). RE, Reticuloendothelium
(II) Hepatic (Toxic Jaundice)Jaundice of the hepatic type can be subdivided into two types: (1) Retention jaundice. (2) Regurgitation jaundice.(1) Retention jaundiceIt results from a defect in the transport of bilirubin into the hepatocyte.In this type of jaundice: Conjugated bilirubin is less than 0.2 mg/dl. Urine bilirubin negative. Urine urobilinogen is decreased or normal.(2) Regurgitation jaundiceIt occurs when the hepatic cell is damaged or defective or the excretion ofproducts from the hepatocyte is impaired.If there is a regurgitation type of jaundice present, uptake, conjugationand excretion impairment are present because of damaged liver cells.In this type of jaundice: Increased total bilirubin, conjugated bilirubin, and urine bilirubin levels. Urine urobilinogen level is increased because uptake is blocked. Fecal urobilinogen may be decreased. Stool color is lighter than usual. Conjugation enzyme deficiencies.Gilberts disease, and Crigler-Najjar syndrome are examples of causes ofretention jaundice, and Dubin-Johnson syndrome, Rotor syndrome, viralhepatitis, and neoplastic conditions are examples of regurgitationjaundice. Laboratory values will vary within the category of hepatic jaundice.Although the total bilirubin concentration will invariably be increased,the relative amounts of unconjugated and conjugated bilirubin varyaccording to the defect in the disease process. In general, a decreased
amount of bilirubin reaches the intestines because of the malfunctioningliver and results in a decreased amount of urobilinogen being formed andexcreted into the feces. This is reflected in less urobilinogen beingabsorbed into the enterohepatic circulation and a decreased amount ofurobilinogen being excreted into the urine. A very small amount ofurobilinogen is normally excreted in the urine so a lower than normalvalue is difficult to determine. If the conjugated bilirubin concentration isincreased, an increased urine bilirubin can also be expected. General circulationFig. 7, Hepatocellular pathology. Increased levels of bilirubin conjugates(direct reacting) can be present in the serum; lesser amounts ofunconjugated bilirubin may also be elevated in the serum owing to adecreased uptake of the pigment. During recovery from cholestasis,increased serum levels of direct-reacting covalently bound bilirubinconjugates (biliprotein) may persist without bilirubinuria. Observe thepresence of bilirubin glucuronide and increased amounts of urobilinogenin the urine. Increased urinary urobilinogen is due to the inability of the.altered hepatic cells to re-excrete this pigment into the bile
(III) Post-hepatic (Obstructive Jaundice) Post-hepatic jaundice is caused by a blockage of the flow of bile fromthe liver. Although the liver itself is not the cause of the problem, bileproduced by the liver cannot be released into the intestines and overflowsback into the blood. Although a complete blockage of the flow of bile isuncommon, partial and intermittent obstructions are likely, and thejaundice found in conjunction with this condition varies.The most common obstructions are: Stones within the common bile duct. Obstructing neoplasm of the pancreas or other organs in close proximity to the ducts. Strictures severe enough to cause a blockage.Stones are usually formed in the gallbladder and rarely cause symptomsuntil they travel through the small ducts and lodge there.In Post-hepatic jaundice The increase in bilirubin is almost entirely of the conjugated type. Because of the requisite obstruction, the quantity of bilirubin reaching the intestines is decreased, resulting in clay-colored feces. This color is due to the decreased formation of urobilinogen from bilirubin in the intestines and its decreased excretion. There should be little or no urobilinogen but large quantities of bilirubin in the urine. The kidney provides the only route of excretion for the increased levels of conjugated bilirubin in the plasma, and the yellow-orange urine color reflects this excretion of bilirubin. Often there is no correlation between the plasma concentration of conjugated bilirubin and the concentration of bilirubin excreted in the urine. Much of the conjugated bilirubin in obstructive conditions circulatescovalently bound to albumin and is called delta bilirubin. Since deltabilirubin is protein-bound, it cannot pass the glomerulus of the kidney,and therefore urinary bilirubin concentrations are less than expected whenthe serum concentrations of conjugated bilirubin are significantlyelevated.
Fig.8, Extrahepatic obstruction. Note regurgitation to the serum andsubsequently the urine of all bilirubin diglucuronides conjugated in theliver. Biliprotein may also be present in the serum during cholestasis.Urinary urobilinogen and fecal stercobilin are absent.