Degradation of Hemoglobin, Jaundice and case studies

1. Haemoglobin
Degradation
Akanksha Dubey

2. Erythrocytes have a life span of 120 days in
adults(60-90 days in neonates , 35-50 days in pre-
mature neonates). At the end of this period, they
are removed from the circulation. Erythrocytes
are taken up and degraded by the macrophages of
the reticulo-endothelial (RE) system in the spleen
and liver.

3. The haemoglobin is cleaved to the protein
part globin and non-protein heme.
 About 6 g of haemoglobin per day is
broken down, and resynthesized in an
adult man.

4. Fate of globin : The globin may be reutilized
as such for the formation of haemoglobin
or degraded to the individual amino acids.
The Iatter undergo their own metabolism,
including participation in fresh globin
synthesis.

5. Sources of heme : 80% of the heme is
derived from the erythrocytes and the rest
(2O%) comes from immature RBC,
myoglobin and cytochromes.

6. Heme oxygenase is present in
microsomes of spleen, liver and
bone marrow
Heme oxygenase cleaves
methylene bridges b/w I and II
pyrrole rings.
Iron is used for recycling into
Iron pool

7. Biliverdin is green colored bile
pigment which is excretory
product in birds and amphibians
but in humans it is further
degraded.
Biliverdin is linear tetrapyrrole
compound
An enzyme Biliverdin
Reductase cleaves methylene
bridges of III and Iv pyrrole rings
to form Yellow colored pigment
Bilirubin.
1 gm of Hb is converted to 35
mg of Bilirubin, 250-350 mg of
Bilirubin is produced everyday

8. Bilirubin formed is insoluble in water
hence it is bound to albumin
100 ml of plasma can carry 25 mg of
bilirubin-albumin complex beyond this
bilirubin is loosely bound to albumin and
hence can be easily released into circulation.
Certain drugs like aspirin, sulfa-antibiotics
salicylates compete with bilirubin to bind to
albumin , which makes free bilirubin to cross
blood brain barrier .
As the albumin-bilirubin complex enters
the liver, bilirubin dissociates and is taken up
by sinusoidal surface of the hepatocytes by
a carrier mediated active transport.
The transport system has a very high
capacity and therefore is not a limitation for
further metabolism of bilirubin. Inside the
hepatocytes, bilirubin binds to a specific
intracellular protein namely Ligandin.

9. Conjugation in Liver
i. Inside the liver cell, the bilirubin is conjugated with
Glucuronic Acid, to make it water
soluble(conjugation disturbs internal hydrogen bonding
that limits solubility of bilirubin)
ii. The first carbon of glucuronic acid is combined with the
carboxyl group of the propionic acid side chains of the
bilirubin molecule. About 80% molecules are in the
diglucuronide form, while 20% are monoglucuronides.

10. iii. Drugs like primaquine, novobiocin,
chloramphenicol, and androgens may interfere
in this conjugation process and may cause
jaundice.

11. The water soluble conjugated bilirubin is
excreted into the bile by an active process
and this occurs against a concentration
gradient.
This is the ra t e l i m i t i n g s t e p i n
t h e c a t a b o l i s m o f h e m e . It is
induced by phenobarbitone.
Excretion of conjugated bilirubin into bile
is mediated by an ATP binding cassette
protein which is called Multispecific organic
anion transporter (MOAT), located in the
plasma membrane of the biliary canaliculi.
.The conjugated bilirubin reaches the
intestine through the bile.
Intestinal bacteria deconjugate the
conjugated bilirubin. This free bilirubin is
further reduced to a Colorless
Tetrapyrrole Urobilinogen (UBG)
 Further reduction of the vinyl substituent
groups of UBG leads to formation of
stercobilinogen (SBG)
The SBG is mostly excreted through feces
(250-300 mg/day)

12. Enterohepatic Circulation
20% of the UBG is reabsorbed from the intestine
and returned to the liver by portal blood.
The UBG is again re-excreted (enterohepatic
circulation). Since the UBG is passed through
blood, a small fraction is excreted in urine (less
than 4 mg/day)

14. Fate of Bilirubin:
Bilirubin Glucuronides are hydrolysed in the intestine by
specific bacterial enzymes namely Beta-glucuronidases to
liberate bilirubin.
The latter is then converted to urobilinogen (colourless
compound), a small part of which may be reabsorbed into
the circulation. Urobilinogen be converted to urobilin (an
yellow color compound) in the kidney and excreted. The
characteristic colour of urine is due to urobilin

15. A major part of urobilinogen is converted
by intestinal bacteria to stercobilin which is
excreted along with feces.
The characteristic brown colour of feces is
due to stercobilin.

16. Summary of bilirubin metabolism in liver showing its transfer from blood
to bile involving three processes - uptake, conjugation and secretion.
( = represents blockage at the respective steps showing the diseases
that occur due to the blockage).
SUMMARY OF BILIRUBIN METABOLISM IN LIVER

17. Plasma Bilirubin
 Normal plasma bilirubin level ranges from 0.2–
0.8 mg/dl.
The Unconjugated bilirubin is about 0.2–0.6 mg/dl
The Conjugated bilirubin is only 0–0.2 mg/dl.
If the plasma bilirubin level exceeds 1 mg/dl, the
condition is called Hyperbilirubinemia.
Levels between 1- 2 mg/dl are indicative of latent
jaundice(without any visible symptoms)

18. When the bilirubin level exceeds 2 mg/dl, it
diffuses into tissues producing yellowish
discoloration of sclera, conjunctiva, skin and
mucous membrane resulting in JAUNDICE.
Icterus is the Greek term for jaundice

19. Van den Bergh reaction
This is a specific reaction to identify the increase in serum
bilirubin. Normal serum gives a negative van den Bergh
reaction.
Mechanism of the reaction : van den Bergh reagent is a
mixture of equal volumes of sulfanilic acid (in dilute HCI)
and sodium nitrite.
The principle of the reaction is that diazotised sulfanilic acid
reacts with bilirubin to form a purple coloured azobilirubin.

20. Direct and indirect reactions : Bilirubin as such is insoluble
in water while the conjugated bilirubin is soluble. van den
Bergh reagent reacts with conjugated bilirubin and gives a
purple colour immediately (normally within 30 seconds).
This is referred to as a direct positive van den Bergh
reaction. Addition of methanol (or alcohol) dissolves the
unconjugated bilirubin which then gives the van den Bergh
reaction (normally within 30 minutes) positive and this is
referred to as indirect positive.

21. lf the serum contains both unconjugated and
conjugated bilirubin in high concentration, the
purple colour is produced immediately (direct
positive) which is further intensified by the
addition of alcohol (indirect positive).
This type of reaction is known as Biphasic.

22. van den Bergh reaction and jaundice : This reaction
is highly useful in understanding the nature of
jaundice. This is due to the fact that the type of
jaundice is characterized by increased serum
concentration of unconjugated bilirubin
(hemolytic), conjugated bilirubin (obstructive) or
both of them (hepatic).

23. Therefore, the response of van den Bergh
reaction can differentiate the jaundice as
follows
Indirect positive - Hemolytic jaundice
Direct positive - Obstructive jaundice
Biphasic - Hepatic jaundice.

24. HYPERBILIRUBINEMIAS
• Depending on the nature of the bilirubin
elevated, the condition may be grouped into
conjugated or unconjugated hyperbilirubinemia .
• Based on the cause it may also be classified into
congenital and acquired.

25. Congenital
Hyperbilirubinemias

26. Crigler-Najjar Syndrome
Here the defect is in conjugation.
In Type 1 (Congenital non-hemolytic jaundice), there
is severe deficiency of UDP Glucuronyl Transferase.
The disease is often fatal and the children die before
the age of 2.
Jaundice usually appears within the first 24 hours of
life. Unconjugated bilirubin level increases to more
than 20 mg/dl, and hence Kernicterus results.

27. • Type 2 disease is a milder form; the process of
conjugation is partly impaired(addition of second
glucoronide is defective). When barbiturates are
given, some response is seen and jaundice
improves.
• Bilirubin level in blood exceeds 20 mg/dl in
Crigler-Najjar syndrome Type 1 and does not
exceed 20 mg/ dl in Crigler-Najjar syndrome Type
2.

28. The gene encoding Bilirubin-UDP Glucuronyl
Transferase is a part of large gene complex
present in chromosome number 2.
This contains 13 substrate specific exons, each
with its own promoter.
 Exon A1 is involved with conjugation of
bilirubin.

29. Gilbert's Disease
It is inherited as an autosomal dominant trait
affecting mostly males
The defect is in the uptake of bilirubin by the liver
and activity of UDP-Glucoronyl Transferase is partly
defective hence conjugation is affected. Even bilirubin
secretion in bile is also affected.
Bilirubin level is usually around 3 mg/dl, and patient
is asymptomatic, except for the presence of mild
jaundice(Unconjugated)

30. Dubin-Johnson Syndrome
 It is an autosomal recessive trait leading to defective excretion
of conjugated bilirubin; so conjugated bilirubin in blood is
increased.
 The disease results from the defective ATP dependent organic
anion transport in bile canaliculi. There is a mutation in the
MRP-2 (Multidrug Resistance Associated Protein) protein which
is responsible for transport of conjugated bilirubin into bile. The
bilirubin gets deposited in the liver and the liver appears black.

32.  The condition is referred to as Black liver jaundice.
 In normal persons, when 250 mg BSP (Bromosulphthalein)
is given intravenously, blood contains only 5% of original
value at 45 minutes, and only 2% at 2 hr. BSP is taken up by
hepatocytes, conjugated and then excreted through liver.
In Dubin-Johnson's syndrome, the BSP level in serum at 2
hours is more than the value at 45 min. This is because by
45 min, BSP is taken by hepatocytes, and hence blood level
is reduced. But excretory defect for conjugated BSP leads
to regurgitation into plasma. Hence higher value at 2 hr.

33. Rotor Syndrome
It is a similar condition, but the exact defect is
not identified.
Bilirubin excretion is defective, but there is no
staining of the liver.
 It is an autosomal recessive condition.

34. CONGENITAL HYPERBILIRUBINEMIAS

35. Acquired
Hyperbilirubinemias
or
Jaundice

36. Jaundice

37. Physiological Jaundice
It is also called as Neonatal Hyperbilirubinemia.
 In all newborn infants after the 2nd day of life,
mild jaundice appears. This transient
hyperbilirubinemia is due to an accelerated rate of
destruction of RBCs and also because of the
immature hepatic system of conjugation of
bilirubin(activity of enzyme UDP Glucoronyl
Transferase is low).

38. In such cases, bilirubin does not increase
above 5 mg/dl.
It disappears by the second week of life.

39. However in some cases Bilirubin level increases
above 25 mg/dl , which crosses blood brain
barrier and causes Hyperbilirubinemic Toxic
Encephalopathy or Kernicterus which leads to
mental retardation.
Drug phenobarbital is used for treatment becoz it
can induce conjugation
Above 5 mg/dl bilirubin is treated by
Phototherapy

40. Phototherapy : Bilirubin can absorb blue light
(420-470 nm) maximally. Phototherapy deals with
the exposure of the jaundiced neonates to blue
light. By a process called photoisomerization , the
toxic native unconjugated bilirubin gets converted
into a non-toxic isomer namely lumirubin.

41. Lumirubin can be easily excreted by the kidneys
in the unconjugated form (in contrast to bilirubin
which cannot be excreted).
Serum bilirubin is monitored every 12-24 hours,
and phototherapy is continuously carried out till
the serum bilirubin becomes normal (< 1 mg/dl).

42. Breast milk jaundice
In some breast-fed infants, unconjugated
hyperbilirubinemia occurs. This is because of the
presence of enzyme Beta Glucoronidase in breast
milk which de-conjugates bilirubin in the
intestine.

43. Jaundice is a yellow discoloration of the skin,
mucous membranes, and sclera caused by
increased amounts of bilirubin in the blood.
Jaundice is a sign of an underlying disease
process.
Jaundice results from the accumulation of
bilirubin. Hyperbilirubinemia may be due to
abnormalities in the formation, transport,
metabolism, and excretion of bilirubin.

44. • Jaundice results from one or more of the
following mechanisms:
(1) excessive production of bilirubin,
(2) reduced hepatic uptake,
(3) impaired conjugation,
(4) decreased hepatocellular excretion, and
(5) impaired bile flow (both intra hepatic and extra
hepatic).

45. Hemolytic Jaundice
 This condition results from increased hemolysis
of erythrocytes(Malaria, hemolytic anaemia,
Sickle cell anaemia, incompatible blood
transfusion, G6PD Deficiency, Toxins like carbon
tetrachloride)

46. This results in production of bilirubin beyond
the capability of liver to conjugate(however
liver can conjugate ten times more bilirubin
than normally 0.3mg/day)
More bilirubin in bile, more formation of
stercobilinogen and urobilinogen

47. However bilirubin is more but can’t appear in
urine (unconjugated is non-polar)
In hemolytic jaundice:
a) Increased serum uncojugated Bilirubin
b) Increased urine urobilinogen
c) Increased feces stercobilinogen(dark color
stool)

48. Hepatocellular Jaundice
This kind of Jaundice is caused by perenchymal liver
disease
The most common cause is viral hepatitis, chronic
alcohol abuse leading to cirrhosis , cystic fibrosis
As a result of damaged hepatocytes bilirubin
secretion in Bile is impaired (this can be anywhere
from uptake of uncojugated bilirubin to transport of
conjugated bilirubin into bile duct)

49. Hepatic jaundice is characterized by:
Increased levels of conjugated and unconjugated
bilirubin in the serum.
Dark coloured urine due to the excessive excretion of
bilirubin and urobilinogen
 lncreased activities of alanine transaminase (SGPT)
and aspartate transaminase (SGOT) released into
circulation due to damage to hepatocytes.

50. The patients pass pale, clay coloured stools due
to the absence of stercobilinogen.
The affected individuals experience nausea and
anorexia (loss of appetite)

51. Obstructive Jaundice
This occurs d/t obstruction of bile duct that prevents
the flow of bile from liver to the intestine.
 The common causes of obstructive jaundice may be
gallstones or tumours
Due to blockage in the bile duct conjugated bilirubin
enters into the circulation and slowly diffuses into
tissues producing jaundice.

52. Obstructive Jaundice is characterized by
Increased concentration of conjugated bilirubin
in serum.
Serum alkaline phosphatase is elevated as it is
released from the cells of the damaged bile duct.
Dark coloured urine due to elevated excretion of
bilirubin and clay coloured feces due to absence
of stercobilinogen.

53. Feces contain excess fat indicating impairment
in fat digestion and absorption in the absence
of bile (specifically bile salts)
The patients experience nausea and
gastrointestinal pain.

54. van den Bergh reaction and jaundice : This reaction
is highly useful in understanding the nature of
jaundice. This is due to the fact that the type of
jaundice is characterized by increased serum
concentration of unconjugated bilirubin
(hemolytic), conjugated bilirubin (obstructive) or
both of them (hepatic).

55. Therefore, the response of van den Bergh
reaction can differentiate the jaundice as
follows
Indirect positive - Hemolytic jaundice
Direct positive - Obstructive jaundice
Biphasic - Hepatic jaundice.

57. A 24 year old male suffering from Malaria was put on
Primaquine. He developed malaise, fatigue and
yellow discolouration of sclera and skin.
On examination – There was pallor- ++, icterus ++,
Pulse – 100/min.
Liver and spleen were palpable.
The investigation report was as follows
Hb-5gm% Serum bilirubin- 6 mg%
Van den Bergh- Indirect positive.
Urine- Hemoglobin + and Urobilinogen + The color of
the urine was brownish black
1. What is the probable diagnosis?
2. What is the relationship of primaquine intake and
the present manifestations?

58. Case Details- This is a case of primaquine induced
Hemolytic anemia, progressing to jaundice. Glucose-6-P
dehydrogenase deficiency seems to be the underlying
defect. High fever is due to malaria, while pallor and icterus
are due to hemolytic anemia and underlying jaundice as
apparent from low Hb and high bilirubin levels. Primaquine
being an oxidant drug precipitates the underlying defect to
induce hemolysis. However massive lysis of red blood cells,
as in Glucose-6 –phosphate dehydrogenase deficiency, may
produce bilirubin faster than it can be conjugated.

59. A 50 –year-old woman had 8 day history of loss of appetite, nausea and flu-
like symptoms.
She had noticed that her urine had been dark in color over the past two
days. On examination she had tenderness in the right upper quadrant.
Laboratory investigations showed; Serum Total bilirubin 4.5 mg%
Direct bilirubin 2.5 mg% Indirect bilirubin 2.0 mg%
Serum AST- 40 IU/L Serum ALT-115 IU/L
Serum ALP- 20 Units (KA)
1. What is the probable diagnosis?
2. What will be the observation regarding bile pigments in urine?

60. Case details
Flu like symptoms are indicative of viral hepatitis.
Damage to liver cells can cause unconjugated bilirubin to increase in
the blood as a result of decreased conjugation. The bilirubin that is
conjugated is not efficiently secreted in to the bile, but instead
diffuses in to the blood. Urobilinogen is increased in urine because
hepatic damage decreases the enterohepatic circulation of this
compound allowing more to enter blood, from which it is filtered in
to the urine. The urine thus becomes dark in color, whereas stools
are pale colored. Plasma levels of AST and ALT are elevated. This is a
case of hepatic jaundice.

61. Based on the following clinical laboratory data, give
the most probable diagnosis
Serum bilirubin 4 mg% (N- 0.2-0.8 mg%)
Direct bilirubin 0.2 mg% (N-0.1-0.4 mg%)
Serum Alkaline phosphatase 6 units( KA) (N-3-13
KAU/100 ml)
SGOT- 30 IU/L (N-5-35 IU/L)
SGPT- 26 IU/L (N- 7-21 IU/L)
Urine Bilirubin- Negative
Urine urobilinogen-Positive
Urine Bile Salts- Negative

62. Case details Normal enzyme profile,
Hyperbilirubinemia, absence of urinary bilirubin
and positive urobilinogen are indicative of
Hemolytic jaundice.

63. A 40 –year- old, fat female, presents with intolerance to
fatty foods, pain in the right side of abdomen,
yellowing of eyes and passage of clay colored stools.
Laboratory Investigations revealed
Serum
Total Bilirubin – 20 mg% Direct Bilirubin- 16 mg%
ALP- 800 U(KA) SGPT- 90 IU/L
Urine Color- deep yellow Bilirubin- ++
Urobilinogen- absent
Stools
Clay colored Stercobilnogen- absent
1. What is the likely diagnosis?

64. Case details This is a case of Obstructive Jaundice Due To Gall Stones. This patient fits
the “classic” criteria of gallbladder disease: female, middle-aged, overweight. Gallstones may
cause pain. Pain develops when the stones pass from the gallbladder into common bile duct
and block the duct..The pain is felt in the upper abdomen, usually on the right side.
In this instance jaundice is not due caused due to overproduction of bilirubin, but instead
results from obstruction of the bile duct from the gall stones. The liver regurgitates
conjugated bilirubin in to the blood (Hyperbilirubinemia)
High direct bilirubin (Conjugated hyperbilirubinemia), high alkaline phosphatase (marker of
cholestasis),slightly increased SGPT level are suggestive of post hepatic or obstructive
jaundice.
Furthermore the diagnosis is supported by the presence of bilirubin (since it is conjugated)
and absence of urobilinogen (Since there is obstruction to the out flow of bile) in urine. Due
to the same reason of obstruction stool is clay colored as stercobilnogen is absent.
Prolonged obstruction of the bile duct can lead to liver damage and a subsequent rise in
unconjugated hyperbilirubinemia and a rise in SGPT levels.