Determination of blood cholesterol
AIMS AND OBJECTIVES:
To learn to estimate cholesterol level in the serum
To learn how to extract blood cholesterol
To understand the concept of total, free and bound cholesterol
Cholesterol accounts for almost all of the sterol in plasma. It exists as a mixture ofunesterified (30 to 40 per cent) and esterified (60 to 70 per cent) forms. The proportion of thetwo forms is constant within and between normal individuals. Various adaptations of thecolorimetric and the enzymatic assays can use for cholesterol estimation. Most diagnosticand the research laboratories use the enzymatic assay method. However, the followingexperiment uses the chemical (colorimetric) method to determine the plasma cholesterollevel.
Add 0.5 ml plasma into a graduated centrifuge tube.
Add 9.5 ml of acetone-alcohol solvent.
Place the tube in a water-bath at 60 C for 15 min.
Notes: Do not allow the acetone-alcohol solvent to over-boil.
Cool the mixture and add appropriate amount of the acetone-alcohol so that the finalvolume is 10 ml.
Cover the tubes and centrifuge for 5 min at 3000 rpm.
The supernatant obtained is used for the following analysis:
Determination of total cholesterol
Pipette 2 ml of the supernatant (from above procedure) into a centrifuge tube.
Place it in a water-bath at 80-90 C in order to evaporate off the solution.3.Add 6ml of glacial acetic acid and solubilize the residue by placing the tube ina boiling water-bath for 5 min.
Cool to room temperature.
b.Estimation of free cholesterol
1.Pipette 5 ml of supernatant into a centrifuge and evaporate it to 1 ml.
Cool to room temperature.3.Then add 1 ml of digitonin.4.Mix well for 10 min.
Centrifuge at 3000 rpm for 10 min.
Discard the supernatant. (Air-dry the pellet.)