Download pract_cholesterol

King Saud University
College of science
Biochemistry department
Title of the experiment:
Cholesterol estimation
Objectives:
To determine serum cholesterol level
Introduction:
Cholesterol is an unsaturated steroid alcohol; it exists in both esterified and non
esterified forms which depend on the presence of fatty acids at carbon # 3.
Around 80 % of total plasma cholesterol is in the esterified form.
The main use of cholesterol testing is to detect disorders of blood lipids and to
evaluate risk potential for atherosclerosis related to coronary heart disease.
The test should be carried out in patients who are more susceptible to CHD, mainly
hypertensive , smokers, hyperglycemic and obese patients, it should be carried out in
either plasma or serum NOT whole blood because blood contains haemoglobin which
interferes with the reaction as it has maximum reading at the same area of the
spectrum.
Cholesterol has two major sources:
a) Exogenous: from diet such as egg yolk, red meat and dairy products, it is
present in abundance in animal fat.
b) Endogenous: it can be synthesized in liver and intestines.
Cholesterol is a lipid, as it is insoluble in water so it has to be carried in blood by a
carrier protein ( lipoproteins):
LDL: transports 60 – 75 % of cholesterol from liver to cells, it is referred to as bad
cholesterol, high levels of LDL are related to CHD.
HDL: transports 15 – 35 % of cholesterol from cells to liver for its excretion and is
considered as good cholesterol.
Cholesterol can be eliminated through conversion to salts of bile acids which is
performed by the liver, these salts pass to the intestines and excreted as bile acids and
coprosterol in fecus, part of the excreted cholesterol is reabsorbed and returns to the
liver( interhepatic circulation )
Cholesterol normal levels:
Normal levels varies with age, diet and from one location to another, however, the
average is 150 – 250 mg/dl.
High levels of cholesterol will lead to narrowing of the arteries by the formation of an
atherosclerotic plaque; this will decrease blood flow and cause CHD.
Principle of the test:
The reaction depends on the colorimetric determination of a green colored compound
resulting from the reaction of cholesterol with acetic anhydride-sulphuric acid ( strong
dehydrating and oxidizing agents), the absorbance of the green compound is then
measured at 610 nm.
Materials:
1.
2.
3.
4.
5.
6.
7.
8.
9.
Cholesterol reagent (Acetic anhydride + acetic acid)
Sulphuric acid 95 – 97 %
Standard cholesterol ( 300 mg/dl)
Samples
Test tubes
Pipettes
cuvettes
spectrophotometer
Water bath
Method:
Label 7 test tubes for test 1 ( A,B), test 2 ( C,D), standard ( E,F), and blank( B),
pipette the following:
Sample 1
Sample 2
Standard
cholesterol
Distilled water
Cholesterol
reagent
(A,B)
0.1 ml
(C,D)
(E,F)
(B)
0.1 ml
0.1 ml
4 ml
4 ml
4 ml
0.1 ml
4 ml
Stand for 20 minutes, pipette 0.5 ml of sulphuric acid into each tube running the acid
cautiously down the side of the tube, place the tube in a water bath at room
temperature, after around 5 minutes remove from the water bath and shake vigorously
to remove any adhered proteins to the walls of the tubes , stand for 10 minutes then
measure the absorbance of the samples against the blank at 610 nm.
Results:
Absorbance of samples X 300 = -------------- mg/dl
Absorbance of standard
Comment on your results comparing them with normal values