PUHRANEE A/P AMURULNATHAN

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Practical 3:
Determination of serum bilirubin level

Objective:
To identify and determine the serum bilirubin level

Introduction:
Bilirubin is a yellowish pigment which is found in bile, a fluid made by the liver.
Besides, bilirubin is ultimately processed by the liver to allow it’s elimination from
the body. Further, bilirubin is a waste product produced by the normal breakdown of
heme. Heme is a component of hemoglobin which is found in red blood cells. Any
condition that accelerates the breakdown of red blood cells may cause an elevated
blood level. Moreover, there are two forms of bilirubin which are direct or conjugated
bilirubin and indirect or unconjugated bilirubin. Bilirubin which is formed in the liver
when sugars are attached to it is known as direct or conjugated bilirubin. It enters the
bile and passes from the liver to the small intestines and is eventually eliminated in
the stool. Bilirubin which is not attached to sugars is known as indirect or
unconjugated bilirubin. When heme is released from hemoglobin, it is converted to
unconjugated bilirubin. Then, it is carried by proteins to the liver. Small amounts of
unconjugated bilirubin may be present in the blood. All the bilirubin in the blood is
known as total bilirubin. In addition, sometimes the liver cannot process the bilirubin
in the body because of excess bilirubin, thus inflammation of liver occur. If there are
too much bilirubin in the body, the skin and the sclera will start to change yellow.
This condition is known as jaundice. A bilirubin blood test is done to get accurate
counts of all three bilirubin levels in the blood which are direct bilirubin, indirect
bilirubin, and total bilirubin.

The normal ranges for Total Bilirubin:

Male : 0 – 24 umol/L (0 – 1.40 mg/dL)
Female : 0 – 15 umol/L (0 – 0.88 mg/dL)

Principle:
Indirect bilirubin is liberated by the detergent. Total bilirubin is coupled with the 3,5 –
dichlorophenyl – diazonium - tetrafluoroborate (DPD) to yield the corresponding
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azobilirubin. The absorbance of this dye at 546nm is directly proportional to the total
bilirubin concentration in the sample.

Reagents:
Total Bilirubin
1. Reagent (R1)
Caffeine 30 mmol/L
Detergent
Preservative

2. Reagent (R2)
Hydrochloric acid 170 mmol/L
DPD 3 mmol/L
Preservative

Apparatus:
Centrifuge, Spectrophotometer, Sample tubes and cuvettes, Pipettes, Beaker, Water
bath (temperature: 37OC)

Materials:
Distilled Water, Biochemistry Control N; expiry date: 2017-01 (25.1 umol/L), Human
Blood Samples

Procedures:
1. The human blood samples were centrifuged at 4000rpm around 10 minutes.
2. The spectrophotometer was set at 550 (540 – 560) nm.
3. The solution was pipetted into cuvette as below:
Blank Standard Sample
Reagent 1 1ml 1ml
Distilled water 1ml
Standard 40ul
Sample 40ul

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4. The solution was mixed and incubated for 3 minutes, then the absorbance (A1) was
read.
5. 250ul of Reagent 2 was added into both sample and standard solutions.
6. The solution was mixed and incubated for 5 minutes, then the absorbance (A2) was
read.
Asample = A2 – A1
7. The spectrophotometer was adjusted to 0 using the blank.
8. The concentration of sample was calculated by using the formula below:

Asample ____ X C standard = C sample

A standard

A = Absorbance C = Concentration

Results:

No Sample Absorbance (OD Value) Final Concentration

Reading 1 Reading 2 Reading ( umol/L )
1 Blank 0 0 0 0
2 Standard 0.059 0.023 0.041 25.1
3 Sample A 0.133 0.151 0.142 87
4 Sample B 0.011 0.009 0.010 6

Sample A:
= A sample × 25.1
A standard
= 0.142 × 25.1
0.041
= 87 umol/L

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Sample B:
= A sample × 25.1
A standard
= 0.010 × 25.1
0.041
= 6 umol/L

Discussions:
1. The terminal product of heme metabolism is bilirubin. Moreover, heme is found in
hemoglobin and also in other oxidative compounds such as hepatic mitochondrial and
microsomal cytochromes. Therefore, plasma bilirubin is a part of erythropoietic and
non- erythropoietic. Approximately, 85 % of erythropoietic and 15% of non-
erythropoietic. The erythropoietic fraction comes from two sources which are the
circulating normal aging red cells and the immature defective red cells of the bone
marrow. Besides that, about 250 to 400 mg bilirubin are produced per day. The
bilirubin from erythropoietic heme is produced by monocytic macrophages, in every
organ especially in the spleen, liver and bone marrow. Meanwhile, the bilirubin from
non-erythropoietic hepatic heme is produced in the hepatocytes.

2. The three bilirubin fractions which together make up the total bilirubin
concentration are unconjugated bilirubin, conjugated bilirubin and total bilirubin.
Unconjugated bilirubin is an albumin bound in serum. Besides, it is measured as
indirect bilirubin and it does not present in urine. When heme is released from
hemoglobin, the unconjugated bilirubin is converted to conjugated bilirubin. Then, it
is carried by proteins to the liver. The conjugated bilirubin is an unbound albumin in
serum. Furthermore, it is also measured as direct bilirubin and it presents in urine. The
conjugated bilirubin is formed in the liver when sugars are attached to bilirubin. Then,
it enters the bile and passes from the liver to the small intestines and is eventually
eliminated in the feces or stool. The total bilirubin is a bilirubin test which carried out
to measure the amount of bilirubin in a blood sample. It is produced when the liver
breaks down old red blood cells. The bilirubin is then removed from the body through
the feces, thus makes the stool to normal colour.

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3. There are three general causes of an elevated serum bilirubin level which are
jaundice, liver disease and rapid destruction of red blood cells. An elevated serum
bilirubin level can cause jaundice due to high bilirubin production. This reflects
impairment of liver cells functions which failed to metabolize bilirubin. Besides, the
skin and sclera looks yellow, skin itches and dark-coloured urine occur. Further, an
elevated serum bilirubin level can cause liver disease. A liver disease can cause
cholestasis, a reduction or blockage of bile flow. When the bile flow is stopped, the
pigment bilirubin, a waste product formed during old or damaged red blood cells are
broken down will leaked out into the bloodstream and accumulates. An elevated
serum bilirubin level can cause a rapid destruction of red blood cells in the blood such
as sickle cell disease, and allergic reaction. Moreover, allergic reaction occurs when
the immune system failed to respond to attack the foreign particles. Therefore, this
causes the bilirubin to become high.

Conclusion:
In this laboratory practical, the serum bilirubin level was determine, calculated and
recorded. The sample A shows that the serum bilirubin level is higher than the normal
range which is 87 umol/L. Therefore, the patient is suspected to have jaundice. The
sample B shows that the serum bilirubin level is within the normal range, which is 6
umol/L. Thus, the patient is normal.

References:
Lecturer’s notes, http://www.webmd.com/digestive-disorders/bilirubin