BILIRUBIN METABOLISM 1-Bilirubin production. 2-Transport in blood. 3-Hepatocellular uptake. 4-Intracellular transport in hepatocytes. 5-Conjugation with glucuronic acid. 6-Secretion into bile ducts. 7- Intestinal metabolism. 8- Renal excretion of bilirubin 9- Renal excretion of urobilinogen
1-BILIRUBIN PRODUCTION • Bilirubin is the terminal product of heme metabolism. Heme is present in hemoglobin and in other oxidative compounds such as hepatic mitochondrial and microsomal cytochromes (P-450). • Thus plasma bilirubin is part erythropoietic and part non-erythropoietic. • Approximately, 85 % erythropoietic and 15% non-erythropoietic.
Cont .. • The erythropoietic fraction originates from two sources: the circulating normal aging red cells and the immature defective red cells of the bone marrow. • The daily production of bilirubin is 250 to 350 mg. • Shunt bilirubin is called that portion that does not originate from circulating red cells but originates from immature and defective red cells (7%) and from non- hemoglobin heme compounds, particularly from hepatic cytochromes and from myoglobin.
Cont.. • pathologic states: sideroblastic anemia, megaloblastic anemia, erythroleukemia, lead poisoning and a congenital disorder called "idiopathic dyserythropoietic jaundice". • The patients affected by this condition do not have hemolysis. They have hyperbilirubinemia and jaundice. • The hyprbilirubinemia is due to shunt bilirubin. )
Cont.. • Bilirubin from erythropoietic heme is produced by monocytic macrophages, reticulo-endothelium, in every organ but especially in the spleen, liver and bone marrow. • The bilirubin from non-erythropoietic hepatic heme is produced in the hepatocytes. HEME +Heme oxygenase = OXY- HEME ( closed tetrapyrrolic ring with iron) OXY- HEME + heme reductase = BILIVERDIN (open tetrapyrrolic ring without iron) BILIVERDIN + biliverdin reductase = BILIRUBIN (unconjugated)
Pathology of bilirubin production • Hyperbilirubinimia with jaundice occurs in increased destruction of red blood cells namely: hemolysis. • It occurs in : - • 1)congenital disorders of red cells (sickle cells, thalassemia, spherocytosis), • 2) immune hemolysis (erythroblastosis fetalis) • 3) acquired diseases of red cells (dyserythropoiesis), etc. • In the adult, even a marked hemolysis does not produce significant increase of serum bilirubin if the hepatic bilirubin clearance is normal. • In the newborn, a marked hemolysis will be catastrophic. At levels of 20mg/dl of serum bilirubin the infant will be deeply jaundiced and will develop kernicterus (Nuclear jaundice: a grave form of yellow staining and degeneration of intracranial gray matter especially of lenticular nucleus and subthalamic area).
2-BILIRUBIN TRANSPORT IN BLOOD • Bilirubin is toxic to tissues, therefore, it is transported in the blood bound to albumin. Only a minute amount of free form is present in the blood. • Pathology of bilirubin transport in blood. • If the free fraction increases, bilirubin will invade and damage the tissues. It will cross the blood -brain barrier and cause kernicterus in the neonate. • Free plasma bilirubin can increase in the following pathologic conditions: • -1- overproduction. • -2- defective conjugation in the hepatocyte. • -3- presence of substances interfering with bilirubin-albumin binding: sulphonamides
3-HEPATOCELLULAR UPTAKE OF BILIRUBIN • Bilirubin is taken up by hepatocytes at their sinusoidal surface. The albumin-bilirubin bond is broken. Albumin remains in the plasma. The free molecule of bilirubin enters the hepatocyte.This uptake is very rapid. • Pathology of bilirubin uptake by hepatocytes. • The impairment of uptake will result in unconjugated hyperbilirubinemia.
4-INTRACELLULAR TRANSPORT OF BILIRUBIN IN HEPATOCYTES • In the hepatocye bilirubin is bound tocytoplasmic proteins: ligandins and Z protein. • Ligandins are a group of enzymes that represent 2% of cytosolic proteins. • Z proteins bind fatty acids. • The primary function of these proteins is that of avoiding the reflux of free bilirubin into the blood. • Pathology of intracellular transport. • No hyperbilirubinemia and jaundice is known due to deficiency of ligandins.
5-CONJUGATION WITH GLUCURONIC ACID • One way for cells to neutralize unwanted compounds is to conjugate them with a modified sugar, a glycosyl. The sugars used for this reaction are xylose, glucose or glucuronic acid. • Glucose is normally present in the cell , xylose and glucuronic acid are formed from glucose by UDP-glucose dehydrogenase. Xylosidation is predominant in plants, glucosidation in bacteria and glucuronidation in mammals. • Unconjugated bilirubinin is lipophilic. Its conjugation with glucuronic acid renders it hydrophilic, thus, it can be eliminated in the bile. • Many other agents are eliminated by conjugation with glucuronic acid: steroids, thyroid hormone, catecholamines, estradiol, testosterone, bile acids, phenols, morphine, which can be conjugated by other cells besides hepatocytes
glucuronidation of bile proceeds in two steps: • The first glucuronic acid (GA) is synthesized from cytosolic glucose that is complexed with uridinediphosphate (UDP) and forms udpglucuronic acid (UDPGA). From this compound, the glucuronic acid is transferred to blirubin. The first reaction is catalyzed by a UP- glucose dehydrogenate, • the second reaction is catalyzed by bilirubin-- transferase that is synthesized by microsomes. Any deficiency of these two enzymes will result in defective conjugation and elimination of bilirubin. • On the other end, administration of microsomal enzyme inducers such as phenobarbital, glutethimide and antipyrine favour bilirubin conjugation and elimination by increasing blirubin transferase activity.
Cont.. • Conjugation occurs in the endoplasmic reticulum and consists of forming an ester between glucuronic acid and one or both propionic side-chains of bilirubin. The result will be formation of bilirubin mono and di-glucuronides. In general, about 80% of the di and less than 20% of the mono are formed. • Human bile contains also small amounts of unconjugated bilirubin. • GLUCOSE + UDP-Glucose-dehydrogenase = UDP-GLUCURONIC ACID (UDPGA) • UDPGA + BILIRUBIN + Glucuronyl transferase = BILIRUBIN MONO & • DI- GLUCURONIDES
Pathology of bilirubin conjugation • GILBERT�SYNDROME • Is due to a very mild deficiency of glucuronyl transferase. It affects 5 to 7% of the general population. More common in males. It consists of mild fluctuating jaundice due to non- hemolytic unconjugated hyperbilirubinemia in the range of 5 to 7mg/dl or rarely higher. • The liver is morphologically normal. State of health and life-span are normal. Hemolysis, low caloric diet, nicotinic acid will increase the jaundice. • A lipid diet will decrease the jaundice. • Phenobarbital and other enzyme inducing agents are beneficial.
Cont.. • CRYGLER-NAJJAR SYNDROME, TYPE I • Is due to a severe deficiency of glucuronyl tranferase. Deep jaundice develops at birth, High serum unconjugated hyperbilirubinemia, >20 mg/dl., not responding to phenobarbital. Absent formation of diglucuronides. Death usually in the first year or two with kernicterus. Phototherapy, plasmaferesis and albumin exchange are beneficial. Liver transplantation may be life-saving. The liver is histologically normal. • It is apparently a hereditary autosomal recessive trait.
CRYGLER- NAJJAR SYNDROME TYPE II • Is due to a moderate deficiency of glucuronyl transferase. Milder unconjugated hyperbilirubinemia responding to enzyme inducing agents: phenobarbital, gltethimide, phenazone, chlorpromazine. Both, mono and di-glucuronides are formed. Patients develop normally but some may suffer bilirubin encephalopathy, kernicterus. They will have unremitting jaundice for the whole life. It is a familial disorder. • defect of bilirubin uptake by hepatocytes.
PHYSIOLOGICAL JAUNDICE OF THE NEWBORN It is due to a very transient insufficiency of glucuronyl transferase. During the first few days of life there is an overproduction of bilirubin and an underdeveloped mechanism of the liver to dispose of bilirubin. • Together with deficient conjugation, bilirubin production, blood transport, hepatic uptake and secretion are all deficient. Sometimes extrahepatic factors exist to aggravate the situation: infections, drugs competing for binding sites of bilirubin and breast feeding. The long chains of fatty acids of the breast milk interfere with bilirubin-albumin binding sites.
6- BILE SECRETION FROM HEPATOCYTES • The liver is an endocrine and an exocrine gland. It secretes synthesized products internally into the blood through the sinusoidal surface such as blood proteins, coagulation factors etc. and secretes external into the biliary tract and the intestine bile and many other substances, the terminal products of detoxifying function. • The mechanism of this external secretion is the least clear in the physiology of the liver. • It seems that many cellular organelles are involved in this process: vesicles, Golgi complexes, lysosomes, plasma membranes, mitochondria, cytoskeleton, plasma membranes, canalicular villi.
Pathology of bile secretion • DUBIN-JOHNSON SYNDROME. • The syndrome consists of chronic benign jaundice due to conjugated hyperbilirubinemia without pruritus or elevation of serum alkaline phosphatase or histological evidence of cholestasis. • The hepatocytes contain an abundance of coarse dark-brown pigment similar to melanin . The liver is black but normal. Serum bilirubin ranges between 2 and 20mg/dl, 60% conjugated. • Jaundice appears in the first 3 decades of life and is intermittent. Sometimes the onset is acute,. The prognosis is excellent. The disease is inherited as autosomal recessive trait. • The diagnosis is made by needle biopsy.
7- INTESTINAL METABOLISM OF BILIRUBIN • Bilirubin in the intestine is reduced to urobilins:- BILIRUBIN GLUCURONIDE + bacterial or intestinal beta-glucuronidase = FREE BILIRUBIN • FREE BILIRUBIN + bacterial dehydrogenase = UROBILINOGEN (colorless) • UROBILINOGEN + dehydrogenase = UROBILIN (orange-yellow). • The bulk of bilirubin, urobilinogen and urobilin is excreted in the feces. • Small amounts of bilirubin and urobilinogen are reabsorbed by the intestine and return to the liver. • The bilirubin is recunjugated in the liver and re-excreted in the feces. The reabsorbed urobilinogen is excreted in the urine, about 4 mg/ day
Pathology of biliary excretion into the intestine • COMPLETE BILIARY OBSTRUCTION. • The bile does not reach the intestine therefore the feces are acholic. There is conjugated hyperbilirubinemia and bilirubinuria. Urobilinogen is not formed in the intestine and there is no urobilinogen in the urine. because since the bile does not reach the intestine, urolinogen is not formed. • PARTIAL BILIARY OBSTRUCTION. • Less bile reaches the intestine. Urobilinogen is formed but in smaller amounts. There is less conjugated hyperbilirubinemia, absent bilirubinuria and small amounts of urobilinogen in the urine. • HEMOLYSIS. • Hemolysis causes unconjugated hyperbilirubinemia. There is no bilirubinuria because unconjugated bilirubin is not hydrophilic and cannot be excreted in the urine. • There is increased urobilinogen in the urine because more bilrubin reaches the intestine and more urobilinogen is formed an reabsorbed.
8- RENAL EXCRETION OF BILIRUBIN • Only conjugated bilirubin (the direct fraction) is excreted in the urine when its level in the plasma is increased above normal. It not present in the urine of normal subjects and it is not eliminated in the urine in cases of unconjugated hyperbilirubinemia, such as in cases of hemolysis. • Only the small fraction of non-protein bound bilirubin in the plasma passes in the urine. • Some drugs and bile salts which compete for protein binding (salicylates, sofosoxazole) increase the threshold of elimination. • Conjugated bilirubin can be demonstrated in the proximal renal tubules.
9-RENAL EXCRETION OF UROBILINOGEN • Urobilinogen is formed by bacteria in the small intestine and in the colon. • It is then reabsorbed by the small intestine and the colon and re-excreted by the liver into the intestine almost entirely. A very small amount is therefore excreted into the urine: 0-4 mg/day. • This amount will increase when more urobilinogen is formed or when the liver is sick and unable to re-excrete it. • This amount will decrease when its formation in the intestine is decreased such as in the case of complete bile duct obstruction when the bile cannot flow to the intestine where urobilinogen is formed by the specific bacteria. • The urobilinogen formed by bacteria in the small intestine is re-absorbed better than that formed in the colon.
Neonatal jaundice • Neonatal jaundice is a yellowing of the skin and other tissues of a newborn infant. A bilirubin level of more than 5 mg/dL manifests clinical jaundice in neonates whereas in the adults 2 mg/dL would look icteric. • In newborns , jaundice is detected by blanching the skin with digital pressure so that it reveals underlying skin and subcutaneous tissue. Jaundice newborns have an apparent icteric sclera, and yellowing of the face, extending down onto the chest. • In neonates the dermal icterus is first noted in the face and as the bilirubin level rises proceeds caudal to the trunk and then to the extremities
Cont.. • In accurate rules of thumb have been applied to the physical exam of the jaundiced infant. Some include estimation of serum bilirubin based on appearance. • One such rule of thumb includes infants whose jaundice is restricted to the face and part of the trunk above the umbilicus, have the bilirubin less than 12 mg/dL (less dangerous level). • Infants whose palms and soles are yellow, have serum bilirubin level over 15 mg/dL (more serious level). • In infants jaundice can be measured using invasive or non-invasive methods. • In non invasive method Ingram icterometer and Transcutaneous bilirubinometer are used.
Levels of neonatal jaundice I - Bilirubin quantity between 5 and 8 mg/dl. II - Bilirubin quantity between 8 and 10 mg/dl. III - Bilirubin quantity between 10 and 13 mg/dl. IV - Bilirubin quantity between 13 and 16 mg/dl. V - Bilirubin quantity arround 20 mg/dl. .
Physiological jaundice • Most infants develop visible jaundice due to elevation of unconjugated bilirubin concentration during their first week. This common condition is called physiological jaundice. • This pattern of hyperbilirubinemia has been classified into two functionally distinct periods. • Phase one • Term infants - jaundice lasts for about 5 days with a rapid rise of serum bilirubin up to 12 mg/dL. • Preterm infants: For preterm infants jaundice lasts for about a week, with a rapid rise of serum bilirubin up to 15 mg/dL. • Phase two - bilirubin levels decline about 2 mg/dL for 2 weeks. • Preterm infants - phase two can last more than 1 month. • In babies who receive exclusive breast feedings, phase two can last more than 1 month.
Causes • Possible mechanisms involved in Physiological jaundice • Increase bilirubin load on liver cells • Increased red blood cell (RBC) volume • Increased labeled bilirubin • Increased circulation of bilirubin in the liver • Decreased RBC survival • Defective hepatic uptake of bilirubin from blood plasma • Decreased ligadin (Y protein) • Increased binding of Y proteins by other anions • Decreased liver uptake • Defective billirubin conjugation • Decreased UDPG activity • Defective bilirubin excretion
Pathological Jaundice of Neonates(syn. Unconjugated pathological hyberbilirubinemia) Any of the following features characterizes pathological jaundice: • Clinical jaundice appearing in the first 24 hours. • Increases in the level of total bilirubin by more than 0.5 mg/dL per hour or 5 mg/dL per 24 hours. • Total bilirubin more than 19.5 mg/dL (hyperbilirubinemia). • Direct bilirubin ( conjugated ) more than 2.0 mg/dL.
Causes of Pathological Jaundice of Neonates • Increased production • Fetomaternal blood group incompatibility: Rh, ABO • Hereditary spherocytosis. • Non-spherocytic hemolytic anemia: G-6-PD deficiency, a-thalassemia, Vitamin K induced hemolysis, pyruvate kinase deficiency. • Sepsis. • Increased enterohepatic circulation: Pyloris stenosis, or large bowel obstruction. • Decreased clearance • Inborn errors of metabolism: Criggler-Najjar syndrome type I and II • Drugs and Hormones: Hypothryoidism, breast milk jaundice.
Differentiation between Physiological and Pathological jaundice • The sign which helps to differentiate pathological jaundice of neonates from physiological jaundice of neonates are presence of intrauterine retardation, stigma of intrauterine infections (e.g. cataracts, microcephaly, hepatosplenomegaly etc), cephalhematoma, • History of illness is significant. Suggestive of pathological jaundice in neonates. • Family history of jaundice and anemia, • family history of neonatal or early infant death due to liver disease, • maternal illness suggestive of viral infection (fever, rash or lymphadenopathy), • Maternal drugs (e.g. Sulphonamides, anti-malarials causing hemolysis in G-6-PD deficiency)
Causes of jaundice • In neonates, benign jaundice tends to develop because of two factors – • the breakdown of fetal hemoglobin as it is replaced with adult hemoglobin and the relatively immature hepatic metabolic pathways which are unable to conjugate and so excrete bilirubin as quickly as an adult. • This causes an accumulation of bilirubin in the blood (hyperbilirubinemia), leading to the symptoms of jaundice. • If the neonatal jaundice does not clear up with simple phototherapy, other causes such as biliary atresia, other pediatric liver diseases should be considered. • Severe neonatal jaundice may indicate the presence of other conditions contributing to the elevated bilirubin levels, of which there are a large variety of possibilities .These should be detected or excluded as part of the differential diagnosis to prevent the development of complications.
. • They can be grouped into the following categories: • Neonatal jaundice Unconjugated Conjugatedbilirubin • Hepatic Posthepatic- • PathologicPhysiological jaundice of Neonates • Hemolytic Nonhemolytic- • Intrinsic causes Extrinsic causes
Intrinsic causes of hemolysis • Membrane conditions • Spherocytosis • Hereditary elliptocytosis • Systemic conditions • Splenomegaly • Sepsis • Arteriovenous malformation • Enzyme conditions • Glucose-6-phosphate dehydrogenase deficiency (also called G6PD deficiency) • Pyruvate kinase deficiency • Globin synthesis defect • sickle cell disease • Alpha-thalassemia
Extrinsic causes of hemolysis • Alloimmunity (The neonatal or cord blood gives a positive direct Coombs test and the maternal blood gives a positive indirect Coombs test) • Hemolytic disease of the newborn (ABO) • Rh disease • Hemolytic disease of the newborn (anti-Rh) • Other blood type mismatches causing hemolytic disease of the newborn • Breast milk feeding.
.cont.. • Non-hemolytic causes • Cephalohematoma • Polycythemia • Sepsis • Hypothyroidism • Gilbert's syndrome • Crigler-Najjar syndrome • Post-hepatic • Biliary atresia • Bile duct obstruction
Cont … • Hepatic causes • Infections • Hepatitis B • TORCH infections • Sepsis • Metabolic • Galactosemia • Alpha-1-antitrypsin deficiency • Cystic fibrosis • Drugs • Total parenteral nutrition • Idiopathic
Non-organic causes • Breast feeding jaundice • "Breastfeeding jaundice or "lack of breastfeeding jaundice," is caused by insufficient breast milk intake, resulting in inadequate quantities of bowel movements to remove bilirubin from the body. This can usually be ameliorated by frequent breastfeeding sessions of sufficient duration to stimulate adequate milk production. • Passage of the baby through the vagina during birth helps milk production in the mother's body, so infants born by cesarean section are at higher risk for this condition
Breast milk jaundice • Whereas breast feeding jaundice is a mechanical problem, breast milk jaundice is more of a biochemical problem. • The term applies to jaundice in a newborn baby who is exclusively breastfed and in whom other causes of jaundice have been ruled out. • The jaundice appears at the end of the first week of life and hence overlaps physiological jaundice. It can last for up to two months.
Cont.. • Several factors are thought to be responsible for this condition. • First:- • in exclusively breastfed babies the establishment of normal gut flora is delayed. The bacteria in the adult gut convert conjugated bilirubin to stercobilinogen which is then oxidized to stercobilin and excreted in the stool. • In the absence of sufficient bacteria the bilirubin is de-conjugated and reabsorbed. This process of re-absorption is called entero-hepatic circulation.
Cont.. • Second : - • the breast-milk of some women contains a metabolite of progesterone called 3-alpha-20-beta pregnanediol. This substance inhibits the action of the enzyme uridine diphosphoglucuronic acid (UDPGA) glucuronyl transferase responsible for conjugation and subsequent excretion of bilirubin. • Reduced conjugation of bilirubin leads to increased level of bilirubin in the blood.
Cont.. • Third : - • an enzyme in breast milk called lipoprotein lipase produces increased concentration of nonesterified free fatty acids that inhibit hepatic glucuronyl transferase which again leads to decreased conjugation and subsequent excretion of bilirubin. • Breast-milk jaundice does not usually cause any complication (like kernicterus) if the baby is otherwise healthy. • The serum bilirubin level rarely goes above 20 mg /dL. It is usually not necessary to discontinue breast-feeding as the condition resolves spontaneously. • Adequate hydration should be maintained by giving extra fluids if necessary.
Non-invasive measurement of jaundice • This method is more accurate and less subjective in estimating jaundice. • Ingram icterometer: In this method a piece of transparent plastic known as Ingram icterometer is used. Ingram icterometer is painted in five transverse strips of graded yellow lines. The instrument is pressed against the nose and the yellow colour of the blanched skin is matched with the graded yellow lines and biluribin level is assigned.
Transcutaneous bilirubinometer: This is hand held, portable and rechargable. When pressure is applied to the photoprobe, a xenon tube generates a light; and this light passes through the subcutaneous tissue. The reflected light returns through the second fiber optic bundle to the spectrophotometric module. The intensity of the yellow color in this light, after correcting for the hemoglobin, is measured and instantly displayed in units.
Treatment • Exchange Transfusion (ExTx) • INTRODUCTION: This procedure, used most commonly to treat severe unconjugated • hyperbilirubinemia, removes the infant’s circulating blood and replaces it with donorblood. The amount of blood exchanged is expressed as multiples of the infant’s blood
PROCEDURE: several possible methods Method and types of catheters: - A. Continuous Exchange is performed by two operators, one infuses blood and theother simultaneously withdraws it. The best method is : - • withdrawal from an umbilical arterial catheter (UAC) and infusion into an umbilical venous catheter (UVC) with tip in IVC or right atrium. • Flush withdrawal catheter with heparinized saline every 10-15 min to prevent clotting. • Alternatives are: • withdrawal from a peripheral arterial catheter and infusion into a centralvenous catheter. However, this is slow and the arterial catheter frequently clots. • withdrawal from a central venous catheter and infusion into a peripheral vein. • Flush the central catheter frequently to prevent clotting.