1. 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

2. 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.

3. 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

5. 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

6. 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

8. 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)

9. 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.

11. 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

12. 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

13. 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.

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

15. Bilirubin
Biologically active end product of heme metabolism

16. 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.

17. 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

18. 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

19. 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

20. 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

21. 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

22. 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

23. 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

24. 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.

25. 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)

27. 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.

28. 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.

29. 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.

32. 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

33. 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

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

35. Treatment of Indirect Hyperbilirubinemia:
Phototherapy:

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Volume withdrawn/infused based on weight

37. 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).

38. 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

39. 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

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

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

42. 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.

43. 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

44. 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 .

45. 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