1. Heme Degradation & Bilirubin Metabolism
Dr. Shumaila Asim
Lecture # 7

2. FATE OF RED BLOOD CELLS  Life span in blood stream is 60-120 days
 RBCs are phagocytosed and/or lysed
Normally, lysis occurs extravascularly in the reticuloendothelial system subsequent to RBC phagocytosis
Lysis can also occur intravascularly (in blood stream)

3. Extravascular Pathway for RBC Destruction
(Liver, Bone marrow,
& Spleen)
Phagocytosis & Lysis
Hemoglobin
Globin
Heme
Bilirubin
Amino acids
Fe2+
Amino acid pool
Excreted

4. Handling of Free (Intravascular) Hemoglobin
Purposes: 1. Scavenge iron
2. Prevent major iron losses
3. Complex free heme (very toxic)
Haptoglobin: hemoglobin-haptoglobin complex is readily metabolized in the liver and spleen forming an iron-globin complex and bilirubin. Prevents loss of iron in urine.
Hemopexin: binds free heme. The heme-hemopexin complex is taken up by the liver and the iron is stored bound to ferritin.
Methemalbumin: complex of oxidized heme and albumin.

5. Bilirubin Metabolism Bilirubin formation
Transport of bilirubin in plasma
Hepatic bilirubin transport
Hepatic uptake
Conjugation
Biliary excretion
Excrete through intestine system

6. Bilirubin is the terminal product of heme metabolism
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.

7. 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.

8. 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

9. Bilirubin from erythropoietic heme is produced by monocytic macrophages, reticulo-endothelium, in every organ but especially in the spleen, liver and bone marrow in order of importance.
The bilirubin from non-erythropoietic hepatic heme is produced in the hepatocytes.

10. Bilirubin formation
microsomal
cytosol
The iron-free porphyrin portion of heme is also degraded, mainly in the reticuloendothelial cells of the liver, spleen, and bone marrow.

11. Heme oxygenase The first step
Heme oxygenase (HO) is an enzyme that catalyzes the degradation of heme. This produces biliverdin, iron, and carbon monoxide.

12. The tetrapyrrolic ring of heme is broken by an oxygenase at the alpha bridge, the bond between the two carbons opposite to the gamma bridge which is between the two carbons carrying the two propionic acids.
The tetrapyrrolic molecule from a ring is transformed into a tetrapyrrolic chain without iron

13. In mammalian cells Heme oxygenase (HO1) has two basic functions:
1. it recycles iron supplies within the cell to maintain homeostasis.
2. biliverdin and biliruben (its reduced form), are powerful antioxidants believed to aid in the prevention of oxidative cell damage.

14. M：-CH2
V：-CH=CH2
CH2
HOOC N H CH O M V
COOH

15. The building of intermolecular hydrogen bonds by the NH and COOH groups is spatially hided.
Bilirubin is lipophilic and therefore insoluble in aqueous solution.the solubility in water is less.

16. 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)

17. Definition of bilirubin
Bilirubin is the water insoluble breakdown product of normal heme catabolism
It’s a yellow pigment present in bile ( a fluid made by the liver) , urine and feces .
Heme is found in hemoglobin, a principal component of RBCs [Heme: iron + organic compound “porphyrin”].
Heme source in body:
80% from hemoglobin
20% other hemo-protein: cytochrome, myoglobin)

18. Heme and bilirubin
Heme four pyrrols rings connected together to form (porphyrin).
Bilirubin consists of open chain of four pyrrols-like rings

19. 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

20. 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.

21. Transport of Bilirubin in Plasma
Molar
Ratio
Bilirubin
H affinity binding sites
2:1
Bilirubin
Plasma protein
Albumin
L affinity binding sites
>2:1
can be replaced by
Other organic anions
PH
UB
Albumin has two binding sites for bilirubin---a high affinity site and a low affinity site.

22. 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

24. 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

25. 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

27. 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)

28. 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.

30. 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

31. 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

32. 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.