This document summarizes serum bilirubin, including its biochemistry, clinical significance, and analytical methods. Bilirubin is produced from the breakdown of hemoglobin in red blood cells. It exists in unconjugated and conjugated forms. The liver conjugates bilirubin so it can be excreted in bile and urine. Inherited disorders like Gilbert's syndrome and Crigler-Najjar syndrome affect bilirubin metabolism. Analytical methods to measure bilirubin include the diazo method and enzymatic methods. Total and direct bilirubin are measured to evaluate jaundice and liver function.

1. Serum bilirubin
Dr.Ghulam Murtaza
Resident Chemical Pathology
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2. Out lines
• Biochemistry
• Clinical significance
• Inherited disorder of bilirubin metabolism
• Analytical methods
• References
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3. Definition
• Bilirubin is the orange-yellow pigment derived from the
catabolism of hemoglobin (Heme)
In the circulation, there are 4 types (fractions) of bilirubin
1. Unconjugated bilirubin is also called indirect bilirubin or
α-bilirubin.
2. Monoglucuronide (monoconjugated) bilirubin, also
called β-bilirubin.
3. Diglucuronide bilirubin (diconjugated), also called direct
bilirubin or γ-bilirubin.
4. There is a fraction of the bilirubin irreversibly bound to
the protein called δ-bilirubin
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4. Pathophysiology Of The Bilirubin
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Most of this daily production is from senescent red blood
cells
(85%)
1.1 to 2 x 108 RBCs are destroyed per hour in the
human body.
2.A normal person weighing 70 kg will have a roughly
turnover of 6 grams of haemoglobin/day
3.1 gram of the haemoglobin = 35 mg of bilirubin.
4.The total daily production of the bilirubin is 250 to
300 mg/day.
5.Rest is from the hepatic hemoproteins and immature
destruction of the RBCs in the bone marrow (15%)

5. Metabolism of bilirubin
1.The bilirubin metabolism can be described
in three stages:
1.The first stage is RBCs’ breakdown, the formation of
heme and biliverdin, bilirubin IXα by the mononuclear
phagocytic system.
2.The second stage is the conjugation of the bilirubin,
and this takes place in the hepatocytes.
3.The third stage is the secretion of the bile into the gall
bladder and intestine.
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6. Stage one ;
1. Hemoglobin from RBC is released and gives rise to
Heme and Globin.
2. Heme from the RBC gives rise to protoporphyrin's.
3. Now protoporphyrin's by microsomal heme
oxygenase form Biliverdin IXα.
4. Biliverdin reductase enzyme transforms it into
bilirubin IXα.
5. Globin is degraded to its components like amino
acids, which are reused or recycled.
6. Microsomal heme-oxygenase enzyme degrades the
heme into iron Fe++, which will be oxidized to
Fe+++ form and is reutilized again.
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7. First stage:
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1. The plasma albumin takes
this unconjugated bilirubin to
the liver. Albumin acts as
the carrier protein.
2. Albumin delivers the bilirubin
into the hepatocytes and
goes back to blood
circulation.
3. Its solubility increases when
it combines with albumin
(noncovalent binding to the
albumin).
4. Albumin has one high-
affinity site and one low-
affinity site for bilirubin.
5. In the liver, bilirubin is
removed from the albumin by
the hepatocytes’ sinusoidal
side by a carrier-mediated

9. Conjugation
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11. • Jaundice : (icterus) :
characterized by
hyperbilirubinemia &
deposition of bile pigments
in skin ,mucous membrane
,& sclera with results yellow
appearance of patient
• Serum bilirubin > 2.5 mg/dl
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12. Isolated increase in serum bilirubin
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14. Gilbert syndrome :
Benign condition ,mild unconjugated bilirubinemia ,respond to
phenobarbital .
Hepatic glucouronyltransferase activity is low
( approx. 30% of normal activity) because of mutation in bilirubin
UDPGT gene located on chromosomes 2.
Serum concentration of bilirubin fluctuates between 1.5 -4 mg/dl
tends to increase with fasting
Distinguished from chronic hepatitis by absence of anemia ,& bilirubin
in urine & by normal LFTs.
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15. Crigler-Najjar-syndrome type -1 Crigler-Najjar-syndrome type -2
Complete absence of UDP-
glucouronyltransferase 1A
Partial deficiency of UDP-
glucouronyltransferase 1A
Because no glucuronidation occurs,
so conjugated bilirubin is not
detectable
High concentration of serum bilirubin
often exceeds > 20mg/dl
Unconjugated bilirubin is usually 5- 20
mg/dl
Most patient die within first year of
life due to kernicterus .
Normal life span is expected
Plasmapheresis & phlebotomy can
reduce serum bilirubin
Liver transplantation is only effective
therapy
Respond to phenobarbital
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17. Analytical methods :
• There are four analytical method
• Diazo method (m0st commonly used method)
• HPLC
• Enzymatic method
• Transcutaneous measurement of bilirubin
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18. Hx of bilirubin estimation
Ehrlich(1883)
Described the reaction of bilirubin with diazotized sulfanilic acid= DIAZO
REACTION
• Malloy and Evelyn (1937)
Diazo reaction with 50% methanol as an accelerator
• Jendrassik and Grof ( 1938)
Diazo reaction with caffeine-benzoate-acetate as accelerator – Increased
sensitivity
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19. Measured vs. Calculated
Measured Analytes
Total Bilirubin
Conjugated bilirubin (DIRECT)
Calculated Analytes
Unconjugated bilirubin (INDIRECT)
Direct bilirubin Reacts with diazotized sulfanilic acid without an accelerator
Indirect /unconjugated Reacts with diazotized sulfanilic acid with an accelerator
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20. Specimen Collection and Storage
Bilirubin is extremely photosensitive, care should be taken to protect
from both daylight & fluorescent light to avoid photodegradation .
Sample should be stored at 4centigrade & analyzed within 5 days .
Serum or plasma preferred
Avoid hemolytic & lipemic samples
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21. Jendrassik-Grof
Measures Total and Conjugated bilirubin –
Principle
• Bilirubin pigments in serum react with a diazo reagent which
results in the production of azobilirubin( a purple product).
Measured at 540 nm.
• Caffeine -benzoate accelerates the coupling of bilirubin with
the diazo reagent.
• Ascorbic acid stops the reaction.
• Alkaline tartrate converts the purple azobilirubin to a blue
azobilirubin.
• This product is measured spectrophotometrically @ 600
nm.
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22. ADVANTAGES :
• Not affected by pH changes
• Maintains optical sensitivity at low bilirubin
concentrations
• Insensitive to high protein concentrations
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23. DIAZO REACTION
• In this reaction
diazotized sulfanilic acid
react with bilirubin two
azido pyrroles(azopigments).
Which are reddish-
purple at neutral pH &
blue at low or high pH
• Modified by van de
Bergh & muller
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24. Method used in our deptt
Assay is based on chemical
oxidation method using
vanadate as a oxidizing agent
Principle of Procedure (DBil-)
The bilirubin is oxidized by
vanadate at about pH 3 to
produce Biliverdin , in the
presence of detergent &
vanadate.
Conjugated (direct) bilirubin is
oxidized.
This oxidation reaction causes
decrease in optical density of
the yellow color, which is
specific to bilirubin
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25. • The decrease in optical density at 451/545 nm is
proportional to direct bilirubin concentration
• The concentration is measured as end point reaction
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26. Total bilirubin
•
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• . Chemical oxidation method
• Using vanadate as a oxidizing agent
• The bilirubin is oxidized by vanadate at
about pH 2.9 to produce Biliverdin , in
the presence of detergent & vanadate
• Both conjugate(direct) & indirect are
oxidized
• Oxidation reaction causes the decrease
in optical density of yellow color, which
is specific to bilirubin
• Decrease in optical density at 451/545
is proportional to total bilirubin
concentration in sample

27. Direct bilirubin Total bilirubin
pH- 3 pH - 2.9
No accelerator is used SURFACTANT is used
Tartrate buffer act as detergent Citrate buffer act as detergent
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28. Thank you …
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29. References
• Chapter 38 ,Tietz Text book of clinical
chemistry volume-2 sixth edition
(hemoglobin, iron bilirubin analytes)
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