Heme degradation and occurance of jaundice. Direct vs indirect and other normal process in the metabolism of bilirubin

1. HEME DEGRADATION
AND JAUNDICE
xiaoli
Molecular Biochemistry II

2. RBCs last 120 days, degraded by
reticuloendothelial (RE) system
most heme from RBCs (85%) - rest
from turnover of cytochromes, p450s,
immature erythrocytes
Under physiologic conditions in the
human adult, 1~2×108 erythrocytes are
destroyed per hour. Thus, in 1 day, a 70kg
human turns over approximately 6g of
hemoglobin.
When hemoglobin is destroyed in
the body, globin is degraded to its
constituent amino acids, which are
reused, and the iron of heme enters
the iron pool, also for reuse.
globin
Heme
Amino acids
iron + Bile pigments

3. iron Bile pigments

4. Bile pigments
Any of several coloured compounds
derived from porphyrin that are found in
bile; principally bilirubin and biliverdin.

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

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

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

8. Heme oxygenase
There are three known isoforms of heme oxygenase.
 Heme oxygenase 1 (HO-1) is an inducible isoform in
response to stress such as oxidative stress, hypoxia, heavy
metals, cytokines, etc. Its activity is induced by its substrate
heme and by various nonheme substances.
 Heme oxygenase 2 (HO-2) is a constitutive isoform which
is expressed under homeostatic conditions. Both HO-1 and
HO-2 are ubiquitously expressed and catalytically active.
 A third heme oxygenase (HO-3) is not catalytically active,
but is thought to work in oxygen sensing.

9. 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.

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

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

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

13. Transport of Bilirubin in Plasma
can be replaced by
Other organic anions
PH UB
Molar
Ratio
Bilirubin
H affinity binding sites
2:1
Bilirubin
Plasma protein
Albumin
L affinity binding sites
>2:1
Albumin has two binding sites for bilirubin---a high
affinity site and a low affinity site.
Albumin per 1L of plasma can bind about 342～
427.5μmol bilirubin, but 1.7～17.1μ bilirubin per 1L .

14. blood
[bilirubin-albumin]
Other
antibiotics
[albumin]↑
[bilirubin]
membrance cytosol
[bilirubin-lipids]
[bilirubin]
Certain drugs as sulfonamides and salicylates compete with
bilirubin for albumin binding and displace bilirubin to enter
into the brain in neonates and increase the risk of
kernicterus (a type of brain damage that can result from high
levels of bilirubin in a baby’s blood. It can cause athetoid
cerebral palsy and hearing loss) .

15. Hepatic phase
On coming in contact with the hepatocyte surface,
unconjugated bilirubin is preferentially
metabolized which involved 3 steps:
Hepatic uptake
Conjuation
Secretion in bile

16. Hepatic uptake of Bilirubin
Bilirubin ~ Albumin Complex
Albumin
Bilirubin
(lipid soluble)
carrier protein
Bilirubin
transfer
ER
ligation (Y protein, Z protein)
(be) taken up by membrane of the liver
(be) bound to

17. Conjugation of bilirubin
Conjugation occur in endoplasmic reticulum UDP-
glucuronosyl transferase,
Product: mono or diglucuronides.
The process of conjugation can be induced by drugs
phenobarbital.
Bilirubin +UDP-gluconic acid
Bilirubin
monoglucuronides + UDP
UDPGT
UDPGT
Bilirubin diglucuronide + UDP
UDP-gluconic acid

18. CH2
CH2
HOOC
N
H
CH
O
N
H
CH
2
N
H
CH
N
H
O
M M M M
V V
CH2
CH2
COOH
H H H
CH2
CH2
C
O
N
CH
O
N
CH2 N
H
CH
N
O
M M M M
V V
CH
2
CH2
CO
O
O
OH
HO
COOH
OH
O
OH
OH
HOOC
OH
O bilirubin diglucuronides
conjugated bilirubin
★Increase the solubility of whole molecule
★ delete the toxicity of bilirubin.

19. Unconjugated bilirubin:
Bilirubin that are not conjugated with
gluconic acid , also called hemobilirubin,
indirected bilirubin.
conjugated bilirubin:
Bilirubin that are conjugated with gluconic
acid, also called hepatic bilirubin, directed
bilirubin.

20. 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)
Present Absent
Serum albumin binding High Low
Van den Bergh reaction Indirect (Total minus
direct)
Direct
Reanal excretion Absent Present
Affinity to brain tissue Present (kernicterus) Absent

21. 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.

22. 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

23. Portal
vein
10 ～ 20%
reabsorbed
bilinogen
Sterobilin
excrection
80%～90%
bilinogen
prototype
Conjugated
bilirubin
bilinogen enterohepatic
circulation
0.5～4mg/day
blood
urobilin

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

25. Summary of bilirubin metabolism
Senescent 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.

27. ＊normal range of bilirubin:
1～16mol/l (0.1 ～1mg/dl)
4/5 are unconjugated bilirubin, others
are conjugated bilirubin.
<1mg/dl normal
1-2mg/dl occult
>2mg/ dl jaundice
Hyperbilirubinemia

28. Occult: the concentration of blood bilirubin are increased ,
but have no clinic sympotom, normally 1-2mg/dl.
Jaundice : ( also called icterus) refers to the yellow color of the
skin and scleare caused by deposition of bilirubin, secondry to
increased bilirubin levels in the blood.
Although not a disease itself, jaundice is usually a symptom
of an underlying disorder.
Hyperbilirubinemia: the concentration of blood bilirubin
are more than 1mg/dl.

31. Based on pathophysiology, jaundice may result
from one or more of the following mechanism:
1. Increased bilirubin production ( excessive red cell
destruction)
2. Decreased hepatic uptake ( ligandin, drug, prolonged
starvation, and sepsis)
Decreased hepatic conjugation (enzyme,drugs, cirrhossis)
3. Decreeased excretion of bilirubin into bile ( gallstone,
tumour)

32. Simple Classification of jaundice
 Accordingly, a simple classification of jaundice
is to divided into 3 predominant type:
① Pre-hepatic (hemolytic jaundice)
② Hepatic jaundice
③ Post – hepatic cholestatic (obstructive
jaundice)

33. Hemolytic jaundice
massive lysis of red blood cells (for example, in
patients with sickel cell anemia or malaria) may
produce bilirubin faster than the liver can conjuagte 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.

34. Causes of hemolytic jaundice
 Malaria
 Side effects of certain drugs :antibiotic and anti-tuberculosis medicines,
levodopa,
 Certain drugs in combination with a hereditary enzyme deficiency known as
glucose-6-phosphate dehydrogenase (G6PD)
 Poisons Snake and spider venom, certain bacterial toxins, copper, and some
organic industrial chemicals directly attack the membranes of red blood cells
 Artificial heart valves
 Hereditary RBC disorders sickle cell disease
 Enlargement of the spleen
 Diseases of the small blood vessels
 Immune reactions to RBCs cancer
 Transfusions
 Kidney failure and other serious diseases
 Erythroblastosis fetalis

35. Hepatocellular jaundice
Damage to liver cells( for example in patient with
cirrhosis or hepatitis) causes a decrease in both
bilirubin uptake and production of conjuagted 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.

36. 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.

37. 65 year-old lady presenting with obstructive jaundice
Biliary stones

38. Sample Indices Normal Obstructive
Jaundice
Hemolytic
Jaundice
Hepatic
Jaundice
Serum Total Bil ＜1mg/dl ＞1mg/dl ＞1mg/dl ＞1mg/dl
Direct Bil 0～0.8mg/dl ↑↑ ↑
Indirect Bil ＜1mg/dl ↑↑
Urine Color normal deep deeper deep
Bilirubin — ＋＋ — ＋＋
Urobilinogen a little ↓ ↑ uncertain
Urobilin a little ↓ ↑ uncertain
Stool Color normal Argilous
(complete
obstruction)
deeper lighter or
normal

40. Diagnoses of Jaundice