Bilirubin metabolism, jaundice Practical – experimentally induced obstructive jaundice Ústav patologické fyziologie LF MU Overview of bilirubin metabolism Historical aspects of bilirubin metabolism • 1916 – van den Bergh • 2 different types of bilirubin • 1933 – Hans Fischer • bilirubin structure • 1956 – Edmund Talafant • bilirubin transformation in the liver • 1968 – Tenhunen • description of heme oxygenase • 1987 – Stocker • antioxidant properties of bilirubin • ikterus • a yellow bird (Greek) • Baltimore oriole (Icterus galbula) Heme synthesis • localization • bone marrow – hemoglobin production - erythropoiesis • liver - cytochrome P450 – metabolism of drugs and toxins • occurs partly in mitochondria and partly in the cytosol – 8 reactions • initial substances are succinyl-CoA (from citric acid cycle ) and glycine • rate limiting enzyme ALA syntetase • lead poisoning inhibits 3 enzymes of heme synthesis and leads to • insufficient heme synthesis and anemia • accumulation of byproducts • toxic Bilirubin metabolism • bilirubin is the final product of heme degradation • 85% from hemoglobin • 15% from myoglobin, cytochrome and premature destruction of RBC • in reticuloendothelial cells • spleen, liver, bone marrow • enzyme heme oxygenase • induced by raised heme level • biliverdin reductase • cytosolic • this type of bilirubin (=unconjugated, indirect) • insoluble in water • in plasma bilirubin binds to albumin Bilirubin metabolism • bilirubin is carried bound to albumin • competition with certain medicaments and fatty acids • albumin-free anion fraction • diffusion into tissues - injury • in physiologic conditions low bilirubin plasmatic concentration • can be replaced by some substances (e.g. salicylates) • important in nursing • in the liver • free bilirubin is released from the albumin and moves into hepatocytes • process with great capacity • in hepatocyte bilirubin undergoes conjugation • conversion into soluble conjugate which can be secreted into the bile Bilirubin metabolism in the liver • in hepatocytes • proteins Y and Z • enzyme uridin diphosphate-glucuronyltransferase (UGT1A1) • family of conjugating enzymes • steroid hormones, drugs • conjugation of bilirubin with glucuronic acid in endoplasmic reticulum generates mono- and diglucuronides • conjugated bilirubin • specific transporter (cMOAT=MRP2) for release of conjugated bilirubin from hepatocyte • rate-limiting step • conjugated bilirubin is secreted via the bile to the small intestine • highly efficient process Bilirubin metabolism • bilirubin passes through the bile ducts into the small intestine • deconjugation by bacterial enzyme β- glucuronidase • production of urobilinogen (colourless) • urobilinogen is • re-absorbed (enterohepatic circulation) • or degraded into coloured urobilins and excreted in the feces • most of the absorbed urobilinogen is returned to the liver to be re-excreted into the bile • small amount excreted in the urine Alternative pathways of bilirubin metabolism • cytochrome P-448 • low expression in the liver • increased in hyperbilirubinemia • can be induced • indol-3-carbinol • direct secretion • unconjugated bilirubin • through the gut wall • passive diffusion • enzyme bilirubinoxidase • low importance in humans • substitution? Summary Icterus/jaundice • yellow discoloration of skin, mucose membranes and sclera • occurs when bilirubin concentration > 30 – 50 umol/l • deposition of bilirubin in tissues rich in elastin • normal plasma level < 17 umol/l • subicterus – small increase (35 - 40 umol/l) • low-grade icteric condition, does not have to be obvious • hyperbilirubinemia • increased plasmatic bilirubin level • objective symptom • alert to presence of other problem • marked icterus in conjugated hyperbilirubinemia Prehepatic jaundice • excessive load of bilirubin • increased supply • increased amount in the gut • mostly due to excessive destruction of red blood cells • mild jaundice • exceeding of conjugating capacity • unconjugated bilirubin is elevated • urobilinogen in urine • bilirubin is absent in urine • hypercholic stool (Intra)hepatic jaundice • caused by disorders that affect liver • disturbed ability of the liver to remove bilirubin from the blood or conjugate it • disintegration of hepatocytes and release of bilirubin into the circulation • conjugated and/or unconjugated levels may be elevated • depends on type of disorder • urobilinogen and bilirubin are both in urine • liver damage • hepatitis, cirrhosis, drugs, chemicals • hereditary hyperbilirubinemias • damage of liver architecture • communication between liver and bile capillaries • hypocholic stool • lower production of bilirubin Hereditary disorders of bilirubin metabolism Gilbert‘s syndrome • genetically determined disorder • autosomal recessive • mutations in the promoter of UDPGT gene • decreease of enzyme activity by 70 % • responds to phenobarbital • common • 5 – 10 % prevalence • more common in males • benign • lifelong hyperbilirubinemia • Up to 100 μmol/l • without liver disease or hemolysis • manifestation during puberty • icterus commonly insignificant • may worsen with • stress • fasting • sleep deprivation • dehydration • illness (flu) • requires no treatment Posthepatic (cholestatic) jaundice • bile flow is obstructed between the liver and the intestine • cholestasis • intrahepatic – failure inside the liver • extrahepatic – obstruction of the large bile ducts • strictures of the bile ducts, gallstones, tumors of the bile duct • increased conjugated bilirubin • accumulation of bile pigment in the liver common to all types of cholestasis • if the obstruction is complete • only bilirubin is found in urine • acholic stool, urobilinogen is absent in urine Neonatal hyperbilirubinemia • physiological neonatal hyperbilirubinemia (icterus neonatorum) • increased erythrocytes destruction • immature liver conjugation and transport systems • increased bilirubin absorption and its lowered binding to albumin • unconjugated hyperbilirubinemia • peak in first five days, in half newborns • kernicterus • in preterm infants, with hemolytic anemia and with neonatal hepatitis • considerably increased plasmatic bilirubin level • passes through the hematoencephalic barrier, deposits and damages basal ganglia Phototherapy Bilirubin metabolism during phototherapy Protective effect of hemeoxygenase • cardiovascular system • inhibition of thrombocytes aggregation • smooth muscle relaxation • inflammation • ↑ IL-10 • ↓ TNF-α a IL-1β • cell proliferation • cyclins • direct anti-apoptotic effect • metabolism • ↑ insulin secretion • smokers • higher CO in the blood Animal experiment – part I • the aim of the practical is to • prepare a model of obstructive jaundice • part I • observe changes of bilirubin metabolism • part II • procedure – part I • general anesthesia • laparotomy • ligature of ductus choledochus Practical part II Practical part II - procedure • one week after ligature of ductus choledochus • general anaesthesia, weighing of the animal • observation of changes in animal coloration • laparotomy • urinary bladder puncture • test strips – presence of bilirubin and urobilinogen • heart puncture, taking of blood and liver • measurement of bilirubin concentration in the serum • weighing of the liver Bilirubin measurement • van den Bergh reaction • bilirubin reacts with sulphanilic acid to produce purple coloured azo bilirubin • photometric measurement • conjugated bilirubin reacts fast • unconjugated reacts slowly • acceleration by methanol • releases bilirubin from albumin • (pre)analytic phase • prevent hemolysis • protect from sunlight