Experiment 2

TITLE:

Liver Function: Estimation of Total Serum Bilirubin

OBJECTIVE:

 To determine the total amount of bilirubin in the given unknown serum sample.

PRINCIPLE:

There are two forms of bilirubin which are conjugated, with glucuronic acid and non-conjugated
type. For conjugated bilirubin, it will reacts in aqueous solution while for non-conjugated bilirubin
presence of organic solvent is required to be bound to albumin. These bilirubin will produce azo-
bilirubin which is blue/purple in colour when reacted with diazotized sulphanilic acid. Amount of
azo-bilirubin in solution can measured at 560 nm using spectrophotometer. Intensity of colour
formed indicates concentration of azo-bilirubin. Increased levels of total bilirubin are associated
with various liver diseases.

INTRODUCTION:

Within the reticuloendothelial system, bilirubin will be produce when the haem component of the
red blood cells undergoes hydrolysis (Limdi, n.d.). There are two forms of bilirubin present which
are the conjugated and unconjugated. The unconjugated bilirubin is transported to the liver loosely
bound to albumin. Since it is water insoluble, it cannot be excreted out through urination. While
the conjugated bilirubin is water soluble, therefore it can be found in the urine. By measuring the
bilirubin in the urine tests, we can also determine for any liver diseases and the health of the liver.
One of the diseases known as Gilbert syndrome that able to cause the reduction for rate of bilirubin
conjugation, producing the unconjugated hyperbilirubinemia.
Laboratory assays for bilirubin usually involve its cleavage in the presence of diazotized sulfanilic
acid to generate a colored azodipyrrole that can be assayed spectrophotometrically at 560 nm.
Unconjugated bilirubin has limited aqueous solubility, therefore it reacts slowly in the absence of
an accelerator, such as ethanol, whereas conjugated bilirubin reacts rapidly.

MATERIALS:

Chemicals Preparation method

Bilirubin standard 10 mg dissolved in 50 ml of mixture

containing 50 mg of sodium carbonate and
0.5g of bovine serum albumin.

Reagent 1 53.6 mg of DL malic acid, 0.25 mg of sodium

nitrite, 4.8 mg of disodium hydrogen
phosphate, in 2ml of distilled water.

Reagent 2 33.6 mg of DL malic acid, 75 mg of

cetylpyridinium chloride, 1.2mg of sulfanilic
acid and 4.0 mg of disodium hydrogen
phosphate in 2.0ml of distilled water.

Buffer 212 mM malic acid and 15.4 mM disodium

hydrogen phosphate

Serum sample
METHODOLOGY:

Serum A Serum B Blank Standard

Buffer 2.0 ml 2.0 ml 2.0 ml 2.0 ml

Reagent 1 2.0 ml 2.0 ml 2.0 ml 2.0 ml

Reagent 2 2.0 ml 2.0 ml 2.0 ml 2.0 ml

Table 1: Preparation of mixture for tube (serum A, serum B, Blank and Standard).

Tubes are prepared and stood for 1 minute at room temperature (25̊c) based on table 1 above.

200 µl of samples A and B (serum), standard (Bilirubin), and water are added to test tube
labelled serum A, B , blank and standard respectively.

Solution is mix and incubated at 37 ̊c for 6 minutes.

Absorbance is read immediately at 560 nm or within 10 minutes of the end of the incubation
period.
RESULTS:

Tube Blank Standard Test 1 (T1) Test 2 (T2)

Absorbance
0.000 0.202 0.083 0.042
(560nm)

Table 2: Absorbance of Blank, T1, T2 & Standard at 560nm.

0.25

y = 1.01x

0.2
Absorbance at 560nm

0.15

0.1
0.083

0.05 0.042

0 Cocentration of Indirect Bilirubin (mg/ml)

0 0.05 0.1 0.15 0.2 0.25

Abs 560nm T1 T2 Linear (Abs 560nm)

Figure 1: Graph of Absorbance at 560nm vs Concentration of bilirubin

CALCULATIONS:

Concentration of bilirubin in Test 1,

From the equation in Figure 1:

y = 1.01x

0.083 = 1.01x

x = 0.0822 mg/ml

(Total amount of bilirubin – Amount of Indirect bilirubin = Amount of Direct bilirubin)

Direct bilirubin = (0.2 mg/ml - 0.0822 mg/ml)

= 0.1178 mg/ml

= 11.78 mg/dl

Therefore, the amount of direct bilirubin for Test 1 is 11.78 mg/dl.

Concentration of bilirubin in Test 2,

From the equation in Figure 1:

y = 1.01x

0.042 = 1.01x

x = 0.0416 mg/ml

(Total amount of bilirubin – Amount of Indirect bilirubin = Amount of Direct bilirubin)

Direct bilirubin = (0.2 mg/ml - 0.0416 mg/ml)

= 0.1584 mg/ml

= 15.84 mg/dl

Therefore, the amount of direct bilirubin for Test 2 is 15.84 mg/dl.

DISCUSSIONS:

Bilirubin is an orange-yellow compound that occurs in the normal catabolic pathway which was
produced by the breakdown of heme. Heme is a component found in haemoglobin, in the red blood
cells. Bilirubin is an unwanted product produces from our body which in turn being excreted out
of the body through bile and urine. Bile is secreted by the liver to help in further digestion. Bile
also contains bilirubin from the process of breaking down old red blood cells. A healthy liver can
normally get rid of bilirubin. But when one has liver problems, bilirubin can build up in the body
in excess. When the bilirubin levels is too high in the blood, a person may be appear to be infected
by jaundice or yellowing of skin and eyes.

Test results may vary depending on the age, gender, health history, and the method used to carry
out the test (Donna 2006). This test is done by collecting some blood sample. A needle is used to
draw blood from a vein in your arm or hand. Having a blood test with a needle carries some risks.
These include bleeding, infection, bruising, and feeling lightheaded (Englert 2008). When the
needle pricks your arm or hand, you may feel a slight sting or pain (Englert 2008). Medicines and
herbal supplements can increase the bilirubin level in the blood. A pregnant woman or a person
that drinks alcohol often can also cause an increase of bilirubin in liver.

From this experiment, students from our group has been provide a small amount of blood as a
sample. The serum sample is being added to the reagent 1 and 2 with the buffer to allow the
reaction to occur. The bilirubin in the serum sample will react with cetylpyridinium chloride and
the diazotized sulfanilic acid in reagent 2 to form azobilirubin which is blue or purple in colour.
This test will measure the amount of bilirubin present in the serum blood sample which indicates
the normality of the donor’s liver.

Bilirubin results depend on the age, gender, and health. The normal values of direct bilirubin range
in the blood is stated from 0 to 0.4 mg/ml. However, the total bilirubin which includes the direct
and indirect bilirubin range from 0.2 to 1.2 mg/ml (M.D., 1933). Some laboratories will have a
higher values which the maximum range can go until 1.9 mg/ml (Ong, 2014). Adults with jaundice
may have bilirubin levels higher than 2.5 mg/ml (Gowda, 2009).

The concentration of bilirubin in test 1 is 0.0822mg/ml. After eliminate the amount of indirect
bilirubin, the concentration of direct bilirubin in test 1 is 11.78mg/ml. The concentration of
bilirubin in test 2 is 0.0416 mg/ml. The direct bilirubin left in test 2 is 15.84mg/ml. Therefore the
first test has a higher concentration of bilirubin than in test 2. Test 2 falls beyond the normal range
while test 1 go beyond the range.

There are some precautions needed to be take note before carrying out this experiment or test.
Donors may not allowed to eat or drink before the taking of the blood sample. Some medicines,
herbs, vitamins, and supplements that is being taken up as a healthcare purpose may affect the
bilirubin level (Kimm, 2009). This includes medicines that don't need a prescription and any illicit
drugs you may use (Kimm, 2009). Pregnant woman is not suggest to involve in the test as well as
the results may differ from a normal person. When handling the blood sample, goggles must be on
and hand gloves is very important as well.

CONCLUSION:

After graph plotting and simple calculation, it has been determined that the bilirubin concentration
in the test 1 is found to be is 0.0822mg/ml and test 2 is 0.0416mg/ml. Comparing the value obtained
for both test, it can be seen that the value for test 1 does not fall under normal range value. The
value is way higher than normal range indicationg that the patient of Test 1 might be having
complication in his/her liver. While for test 2 value, if fall in the acceptable range value of bilirubin
indicating the patient is free from any liver related disorders.

ACKNOWLEDGEMENT:

I would like to thank Ms Anto Cordelia for her guidance in completing this experiment. Also, I
would like to thank the lab officer that were in charge of preparing at the materials, reagent and
apparatus needed to perform this experiment. Not to forget, I am grateful to my group members,
whom I have had the pleasure to work during this experiment.
REFERENCES:

1. Emedicine.medscape.com. (n.d.). Conjugated Hyperbilirubinemia: Background,

Pathophysiology,Etiology. [Online]. Available at:
https://emedicine.medscape.com/article/178757-overview [Accessed 8 Feb. 2018].
2. Limdi, J. (n.d.). Evaluation of abnormal liver function tests.
3. Gowda, S., 2009. A review on laboratory liver function tests. The Pan African Medical
Journal, 3(17), pp. -.
4. Kimm, H., 2009. Low Serum Bilirubin Level as an Independent Predictor of Stroke
Incidence. A Prospective Study in Korean Men and Women, 40(11), pp. 3422-3427.
5. M.D., W. T. V., 1933. The serum bilirubin test. The Journal of Laboratory and Clinical
Medicine, 18(9), pp. 974-980.
6. Ong, K. L., 2014. The Relationship between Total Bilirubin Levels and Total Mortality in
Older Adults. The United States National Health and Nutrition Examination Survey
(NHANES) , pp. 1999-2004.