Hemoglobin degrading and bilirubin formation Spleen Plasma Protein and a.a pool Iron pool TO LIVER Liver globin iron Heme Hemoglobin Bilirubin Binds with albumin Conjugation process

Transport of Bilirubin in Plasma Bilirubin on release from macrophages circulates as unconjugated bilirubin in plasma tightly bound to albumin. Albumin + free Bilirubn Bilirubin ~ Albumin Complex unconjugated bilirubin Why bound to albumin? Significance: ★Increase the solubility of whole molecule ★ Prevent unconjugated bilirubin freely come into other tissue, cause damage.

Bilirubin formed in peripheral tissues is transported to liver by albumin IN LIVER: 1)Uptake of bilirubin by liver paranchmal cells 2)conjugation of bilirubin with glucuronate in endoplasmic reticulum 3)secretion of conjugated bilirubin into bile

Uptake of bilirubin by liver Bilirubin is only sparingly soluble in water İts solubility in plasma is increased by noncovalent binding to albumin Albumin has one high affinity site and one low affinity site for bilirubin In 100 ml plasma = 25 mg bilirubin can be tightly bound to albumin at high affinity site

Conjugation of bilirubin with glucuronic acid Bilirubin is non-polar. Hepatocytes convert bilirubin to a polar form by adding glucuronic acid to it (conjugation) Enzyme: glucuronosyl transferase Location:endoplasmic reticulum Glucuronyl donor:UDP-GLUCURONIC ACID

Major differences between unconjugated and conjugated bilirubin FEATURE Unconjugated bilirubin CONJUGATED BILIRUBIN Normal serum level More Less (less than 0.25mg/dl) Water solubility Absent Present Affinity to lipids (alcohol solubilty) Serum albumin binding High Low Van den Bergh reaction Indirect (Total minus direct) Direct Reanal excretion Affinity to brain tissue Present (kernicterus)

Excretion of bilirubin into bile Bilirubin diglucuronide is actively transported against a concentration gradient into bile duct. This energy-dependent, rate –limiting step is susceptible to impairment in liver disease. Uncojugated bilirubin is normally not excreated.

Formation of urobilins in the intestine Bilirubin diglucuronide is hydrolysed and reduced by bacteria in the gut to yield urobilinogen, a colorless compound. Most of the urobilinogens of the feces are oxidized by intestinal bacteria to stercobilin, which gives stools their characteristic brown color. Some urobilinogen is reabsorbed from the gut into the portal blood and transported to the kidney, where it is converted to the yellow urobilin and excreted, giving urine its characteristic color. (bilinogen enterohepatic circulation) Bilirubin diglucuronide urobilinogen urobilin stercobilin bilin

Catabolism of hemoglobin BLOOD CELLS Stercobilin excreted in feces Urobilinogen formed by bacteria Urobilin excreted in urine Heme Globin Hemoglobin KIDNEY CO Biliverdin IX Heme oxygenase O2 reabsorbed into blood INTESTINE via bile duct to intestines Bilirubin (water-insoluble) NADP+ NADPH Biliverdin reductase Bilirubin diglucuronide (water-soluble) 2 UDP-glucuronic acid Bilirubin (water-insoluble) via blood to the liver LIVER unconjugated Catabolism of hemoglobin

Summary of bilirubin metabolism red cells are major source of hemeproteins Breakdown of heme to bilirubin occur in macrophage of reticuloendithelial system ( tissue macrophages, spleen and liver). Unconjugated bilirubin is transported through blood ( complex to albumin) to liver. Bilirubin is taken into liver and conjugate with glucuronic acid. Bile is secreted into intestine where glucuronic acid is removed and the resulting bilirubin is converted to urobilinogen. A portion of urobilinogen is reabsorbed into blood, where it is converted to the yellow urobilin and excreted by kidneys. Urobilinogen is oxidized by intestinal bacteria to the brown stercobilin.

Dr. Shumaila Asim Lecture # 9 Hyperbilirubinemias Dr. Shumaila Asim Lecture # 9

Bilirubin Biologically active end product of heme metabolism

Hyperbilirubinemia Imbalance of bilirubin production and elimination Hyperbilirubinemia: the concentration of blood bilirubin are more than 1mg/dl Jaundice : ( also called icterus) refers to the yellow color of the skin and sclera caused by deposition of bilirubin, secondary to increased bilirubin levels in the blood. Although not a disease itself, jaundice is usually a symptom of an underlying disorder.

Causes: Increased bilirubin production Reduced bilirubin uptake by hepatic cells Disrupted intracellular conjugation Disrupted secretion of bilirubin into bile canaliculi Intra/extra-hepatic bile duct obstruction Lead to increases in free (unconj.) bilirubin Result in rise in conj. bilirubin levels

INCREASED BILIRUBIN PRODUCTION (unconj. Hyperbilirubinemia) Hemolysis Increased destruction of RBCs eg sickle cell anemia, thalassemia Drastic increase in the amount of bilirubin produced Unconj. bilirubin levels rise due to liver’s inability to catch up to the increased rate of RBC destruction Prolonged hemolysis may lead to precipitation of bilirubin salts in the gall bladder and biliary network result in formation of gallstones and conditions such as cholecystitis and biliary obstruction Other Degradation of Hb originating from areas of tissue infarctions and hematomas Ineffective erythropoiesis

DECREASED HEPATIC UPTAKE (unconj. Hyperbilirubinemia) Several drugs have been reported to inhibit bilirubin uptake by the liver e.g. novobiocin, flavopiridol Bile MRP2 B CB Plasma Hepatic cell Alb : sER + UDPGA

3) DISRUPTED INTRACELLULAR CONJUGATION (unconj. Hyperbilirubinemia) Neonatal jaundice occurs in 50% of newborns fetal bilirubin is eliminated by mother’s liver causes: hepatic mechanisms are not fully developed resulting in decreased ability to conjugate bilirubin rate of bilirubin production is increased due to shorter lifespan of RBCs Acquired disorders hepatitis, cirrhosis impaired liver function

3) DISRUPTED INTRACELLULAR CONJUGATION (unconj. Hyperbilirubinemia) Crigler-Najjar Syndrome, Type I (CN-I) recessive allele; mutation-induced loss of conjugating ability in the critical enzyme glucuronosyltransferase CN-II greatly reduced but detectable glucuronosyltransferase activity due to mutation (predominantly recessive); enzymatic activity can be induced by drugs Gilbert’s Syndrome glucuronosyl transferase activity reduced to 10-30% of normal; also accompanied by defective bilirubin uptake mechanism Plasma Hepatic cell Bile Alb B B MRP2 B + UDPGA CB Alb : B sER

4) DISRUPTED SECRETION OF BILIRUBIN INTO BILE CANALICULI (conj 4) DISRUPTED SECRETION OF BILIRUBIN INTO BILE CANALICULI (conj. Hyperbilirubinemia) Dubin–Johnson Syndrome mild conj. hyperbilirubinemia, but can increase with concurrent illness, pregnancy, and use of oral contraceptives; otherwise asymptomatic Inability of hepatocytes to secrete CB after it has formed Due to mutation in the MRP2 gene (autosomal recessive trait) Rotor Syndrome Autosomal recessive condition characterized by increased total bilirubin levels due to a rise in CB Caused by a defect in transport of bilirubin into bile Hepatic cell Bile Plasma Alb B B MRP2 B + UDPGA CB Alb : B sER

5) Intra/extra-hepatic bile duct obstruction Intra-hepatic Obstruction of bile canaliculi, bile ductules or hepatic ducts Extra-hepatic Obstruction of cystic duct or common bile duct Cholecystitis Obstruction causes backup and reabsorption of CB which results in increased blood levels of CB

Treatment & Therapeutic Considerations PHOTOTHERAPY Through absorption of the wavelengths at the blue end of the spectrum (blue, green and white light), bilirubin is converted into water-soluble photoisomers. This transformation enhances the molecule’s excretion into bile without conjugation. PHENOBARBITAL This drug is not approved by FDA for use in neither adult nor pediatric hyperbilirubinemia patients, due to possibility of significant systemic side-effects. Exact pathway is not known, but it is believed to act as an inducing agent on UDP-glucuronosyltransferase, thereby improving conjugation of bilirubin and its excretion.

Simple Classification of jaundice Accordingly, a simple classification of jaundice is to divided into 3 predominant type: Pre-hepatic (hemolytic jaundice) Hepatic jaundice Post – hepatic cholestatic (obstructive jaundice)

Hemolytic jaundice massive lysis of red blood cells (for example, in patients with sickle cell anemia or malaria) may produce bilirubin faster than the liver can conjugate it. More bilirubin is excreted into the bile, the amount of the urobilinogen entering the enterohepatic circulation is increased, and urinary urobilinogen is increased. Unconjugated bilirubin is elevated in blood.

Hepatocellular jaundice Damage to liver cells( for example in patient with cirrhosis or hepatitis) causes a decrease in both bilirubin uptake and production of conjugated bilirubin. Unconjugated bilirubin occur in the blood and increased urobilinogen in the urine. The urine is dark in color and stool are pale, clay color. Plasma level of AST and ALT are elevated and the patient experience nausea and anorexia.

Obstructive jaundice In this instance jaundice is results from obstruction of the bile duct. For example, the presence of a hepatic tumor or bile stone may block the bile ducts, preventing passage of bilirubin into the intestine, patients with obstructive jaundice experience GI pain, nausea and produce stools that are a pale, clay color.

Neonatal Jaundice Common, particularly in premature infants Transient (resolves in the first 10 days) Due to immaturity of the enzymes involved in bilirubin conjugation High levels of Unconjugated bilirubin are toxic to the newborn – due to its hydrophobicity it can cross the blood-brain barrier and cause a type of mental retardation known as kernicterus If bilirubin levels are judged to be too high, then phototherapy with UV light is used to convert it to a water soluble, non-toxic form If necessary, exchange blood transfusion is used to remove excess bilirubin Jaundice within the first 24 hrs of life or which takes longer then 10 days to resolve is usually pathological and needs to be further investigated

Effects of hyperbilirubinemia Bilirubin toxicity Toxicity due to unbound (free) form Focal necrosis of neurons and glia Acute bilirubin encephalopathy Chronic= kernicterus Most often affects basal ganglia and brainstem nuclei Movement disorders Impaired upward gaze Auditory abnormalities

Bilirubin Neurotoxicity What is kernicterus? Yellow staining of the brain Neuronal necrosis microscopically * Bilirubin deposits typically in basal ganglia, hippocampus, substantia nigra, etc.

Treatment of Indirect Hyperbilirubinemia: Phototherapy:

Treatment of Indirect Hyperbilirubinemia: Exchange Transfusion: Double-volume exchange 2 x blood volume = 2 x 80 cc/kg = 160 cc/kg Takes about 1-1.5 hours Exchange at rate of ~5cc/kg/3 min Volume withdrawn/infused based on weight

Inherited disorders of bilirubin metabolism Inherited disorders of bilirubin metabolism result in hyperbilirubinemia. These include disorders resulting in predominantly unconjugated hyperbilirubinemia (Crigler-Najjar syndrome types I and II, and Gilbert syndrome) Those resulting in predominantly conjugated hyperbilirubinemia (Dubin-Johnson syndrome, Rotor syndrome, and benign recurrent intrahepatic cholestasis).

Crigler-Najjar Syndrome Bilirubin-UDP-glucuronosyltransferase mediate the glucuronidation of bilirubin Genetic lesions may lead to complete absence (Crigler-Najjar syndrome type I) or incomplete deficiency (Crigler-Najjar syndrome type II) of bilirubin glucuronidation

Crigler-Najjar Syndrome Autosomal recessive Extremely rare < 200 cases worldwide – gene frequency is < 1:1000 High incidence in the “plain people of Pennsylvania” (Amish and Mennonites) Characterized by a complete absence or marked reduction in bilirubin conjugation Present with a severe unconjugated hyperbilirubinemia that usually presents at birth Afflicted individuals are at a high risk for kernicterus Condition is fatal when the enzyme is completely absent Treated by phototherapy (10-12 hrs/day) and liver transplant by age 5

Crigler –Najjar Syndrome Type I (congenital nonhemolytic jaundice) Rare Autosomal recessive Severe congenital jaundice (bilirubin>20mg/dl) Fatal within 15 months of life Phototherapy

Crigler –Najjar Syndrome Type II Rare Inherited More benign than type I bilirubin<20mg/dl Phenobarbital

In contrast, Gilbert syndrome is associated with an abnormality that results in reduced expression of structurally normal UDP-glucuronosyltransferase Dubin-Johnson syndrome is caused by a genetic abnormality of bile canalicular multispecific organic anion transporter, which is involved in the excretion of many bile salt organic anions by an adenosine triphosphate (ATP)-requiring active process.

Congenitally inherited disorders of Hyperbilirubinemias Gilbert’s Syndrome Benign liver disorder ½ of the affected individuals inherited it 30% of enzyme (UDP glucuronyl transferase )activity is preserved Harmles , Characterized by mild, fluctuating increases in un conjugated bilirubin caused by decreased ability of the liver to conjugate bilirubin – often correlated with fasting or illness Males more frequently affected then females Onset of symptoms in teens, early 20’s or 30’s Can be treated with small doses of phenobarbital to stimulate UDP glucuronyl transferase activity

Dubin Johnson Syndrome Benign autosomal recessive disorders Conjugated hyperbilirubinemia in childhood or during adult life Mutations in the gene encoding MRP-2, the protein involved in the secretion of conjugated bilirubin into bile Centrilobuler hepatocytes contain abnormal black pigment derived from epinephrine .

Rotor Syndrome Rare benign condition Chronic conjugated hyperbilirubinemia Normal liver histology It seems that these patients have multiple defects in hepatocyte uptake and excretion of bilirubin pigment