Estimation of Serum Bilirubin Total Direct 1

Lab# 7
BCH 471
Estimation of Serum Bilirubin (Total & Direct)

Objectives:
1- To estimate the amount of bilirubin in serum.

Bilirubin
Bilirubin
is a yellow compound that occurs in the normal catabolic pathway that breaks
down heme
BILIRUBIN
3 phases of Bilirubin
 Direct bilirubin: Conjugated with glucoronic acid
 Indirect bilirubin: unconjugated, insoluble in water
 Total bilirubin: sum of the direct and indirect of bilirubin.
Notes:
-Disturbances in the powers of conjugated and/or excretion of the liver of this yellow
compound will lead to raised levels in serum.
Above about 2 mg/dl in the blood, leads to disease called Jaundice.
Bilirubin and jaundice
Jaundice is caused by a build-up of bilirubin (yellow color) in the blood and
tissues of the body.
Jaundice is the discoloration of skin and sclera of the eye caused by high
concentration of bilirubin.
The causes of jaundice may be classified as:
Types of jaundice
Pre-Hepatic Jaundice Hepatic Jaundice Post-Hepatic Jaundice
Hemolytic -Cirrhosis of the liver

Cholecystitis.
disease -Infective Hepatitis
-Neonatal Jaundice
1-PRE-HEPATIC JAUNDICE
Hemolytic disease (excess hemolysis)

The production of un-conjugated bilirubin may exceed the conjugating capacity of
the liver and hence the serum levels of indirect (and of total) bilirubin will be
raised and that of direct in the upper normal range or just a little elevated.
The other liver function tests will usually give normal results
2-HEPATIC JAUNDICE
Cirrhosis (in the absence of infection)
Destruction of liver cells will lead to a reduced conjugating capacity with a:
• Raised serum level of indirect (and of total) bilirubin,
• with a low level of direct bilirubin
• and an abnormally high release, into the blood, of the enzymes: AST,
ALT and ALP.
• Synthesizing power of liver will be diminished and hence low levels of
total protein, albumin and cholesterol
2-HEPATIC JAUNDICE
Hepatitis (in the presence of infection)
The conjugative capacity of the liver is approximately normal, but there is the inability to
transport the conjugated bilirubin from the liver cells to the biliary system, and it will be
regurgitated back into the blood.
 Hence:
• The serum level of unconjugated bilirubin will be normal
• and that of conjugated (and total) bilirubin will be raised.
• Synthesizing power is diminished leading to low serum levels of proteins but the raising
of antibodies to infection usually leads to raised total proteins level.
2-Hepatic Jaundice
Neonatal Jaundice
Conjugating enzymes in the liver are often absent at birth.
Hence:
• Raised serum level of indirect (and total) bilirubin is to be expected
• Low level of direct bilirubin.
• The other liver functions are normal.
The indirect bilirubin level will rise for the first few days after birth until the
conjugating enzymes begin to synthesize.
3-POST-HEPATIC JAUNDICE
Cholecystitis
The bile duct is blocked.
Hence:
• The indirect bilirubin level is normal but conjugated bilirubin is regurgitated into
the blood and excreted into the urine (raised conjugated and total bilirubin).
• Enzymes will be regurgitated into the blood giving raised levels.
• The other liver function tests are normal
Normal unconjugated Normal unconjugated
bilirubin (in bilirubin (in blood)
blood)  conjugated bilirubin
 conjugated bilirubin (in blood)
(in blood)
Hepatic Jaundice.
(in the presence of
Post-Hepatic Jaundice
infection)
Pre-Hepatic, Hemolytic disease (Cholecystitis)
Principle:
Bilirubin in serum is coupled with diazotized sulphanilic acid to form azobilirubin .
The water soluble conjugated bilirubin (direct bilirubin) reacts easily with reagents such as
diazotized sulphanilic acid.
while the water insoluble unconjugated bilirubin (indirect bilirubin) requires a solubilizing
reagent, such as Caffeine, in order to react with the diazotized sulphanilic acid.
In this experiment, the direct bilirubin is estimated in the absence of the solubilizing agent
and then further bilirubin estimation in the presence of the solubilizing agent will give the
total bilirubin level.
The indirect or unconjugated bilirubin is then found by difference.

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Lab# 7

Estimation of Serum Bilirubin (Total & Direct)

1- To estimate the amount of bilirubin in serum.

 Indirect bilirubin: unconjugated, insoluble in water

 Total bilirubin: sum of the direct and indirect of bilirubin.

Pre-Hepatic Jaundice Hepatic Jaundice Post-Hepatic Jaundice

Hemolytic -Cirrhosis of the liver

Hemolytic disease (excess hemolysis)

• Raised serum level of indirect (and of total) bilirubin,

• with a low level of direct bilirubin

• The serum level of unconjugated bilirubin will be normal

• and that of conjugated (and total) bilirubin will be raised.

• Raised serum level of indirect (and total) bilirubin is to be expected

• Low level of direct bilirubin.

• The other liver functions are normal.

The indirect or unconjugated bilirubin is then found by difference.

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