Addis Ababa University

College of Health Sciences

School of Medicine

Department of Biochemistry

Laboratory Report for Lipid profile test (3)

Prepared by: Wondimnew Walle

ID : UGR/2899/12

August 5,2021

Total cholesterol Determination

Objective
 The lipid profile test is a group of tests that are often ordered
together to determine the risk of coronary heart disease. This test
was done to identify whether or not the subject from whom the sample was
taken has normal cholesterol level in his/her blood and determine the
presence of associated metabolic disorders.

Principle
The measurement of total cholesterol in serum involves the use of three enzyme: cholesterol esterase
(CE), cholesterol oxidase (CO) and peroxidase (POD). Cholesterol esters are hydrolyzed into cholesterol
and fatty acids in the presence of cholesterol esterase.
Cholestryl Esters CE Cholesterol + Fatty Acids
The amount of cholesterol produced in the reaction is proportional to the cholesterol esters. Then
cholesterol will be oxidized to give cholestenone and hydrogen peroxide by the enzyme cholesterol
oxidase.
Cholesterol + O2 CO Cholestenone + H2O2
In the presence of peroxidase the mixture of phenol and 4-aminoantipyrine (4-AA) are condensed by
hydrogen peroxide to form a quinonimine dye proportional to the concentration of cholesterol in the
sample. When4-aminoantipyrine is oxidized to form quinoneimine, the color changes, i.e.
compound quinoneimine absorbs light, so that its concentration can be measured at a specific
wavelength in a spectrophotometer. The absorption of light is, therefore, proportional to the
concentration of cholesterol in the original sample of blood.

4-AA +phenol + H2O2 POD Quinoneimine + 4H2O

Material used

 Spectrophotometer adjusted at 340 nm

 Water bath
 Micropipettes
 Micropipette tips
 Test tubes
 Test tube racks
 Cuvettes with light path of 1cm water path

1
Procedure

1. Three test tubes were labeled as B(blank), S(standard), and T( test sample).
2. 1000µl of R1 reagent was added to each test tube.
3. 10µl of cholesterol standard, 10µl of sample and 10µl of distilled water was added to test
tube S, T and B respectively.
4. The solutions in all three cuvettes were incubated in water bath of 370c for 5 minutes.
5. The spectrophotometer was calibrated with the Blank Solution.
6.The solutions S and T were inserted in the spectrophotometer and the absorbance values
were read.
“B” “S” “T”
Reagent 1000 1000 1000
Distilled water 10 - -
CAL - 10 -
Sample - - 10

Results
The results obtained when the cuvettes were placed in the spectrophotometer were:

Test Tube Abs

“S” Test Tube 0.311

“T” Test Tube 0.612

Calculation
C = 200 X (AT/ AS) mg/dl
= 200 x (0.612/0.311) mg/dl
= 393.57 mg/dl

Discussion
Updated clinical values of total cholesterol used to classify risk groups

2
 Total Risk classification
cholesterol
<200 mg/dl Desirable
200-239 mg/dl Borderline high
>240 mg/dl High

The result obtained (393.57 mg/dl) was not in the normal range i.e. it lies in the high risk classification
group. But, in order to be reliable on the assay as cholesterol in serum or plasma is not stable more than 5
days at 2-80c, the sample have to be taken within the time limit and the method have to be protected from
interference from substances that compete in the oxidase and peroxidase reactions like bilirubin,
hemoglobin, ascorbic acid and other drugs.

Conclusion
A cholesterol test can help determine your risk of the buildup of fatty deposits (plaques) in your
arteries that can lead to narrowed or blocked arteries throughout your body (atherosclerosis).

From the results of our test, the sampled individual has high cholesterol level. This means he/she
is affected by disorders of high plasma lipoproteins called hypolipoproteinemia, which include
coronary heart disease and atherosclerosis and also other diseases such as obstructive jaundice,
diabetes, hypothyroidism, and certain types of kidney diseases.

Reference:
https://www.healthline.com/health/cholesterol-test
https://www.mayoclinic.org/tests-procedures/cholesterol-test/about/