TITLE:

Determination of blood cholesterol

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

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 7. Add 4 ml acetone, to the pellet. Mix thoroughly.
8. Centrifuge and discard the supernatant.
9. Dry the residue by placing the tube in a boiling water-bath.
10. Then, add 6 ml glacial acetic acid and solubilize the residue by placing the
tube in a boiling water-bath for 5 min.
c. Cholesterol standards & Blank
1. Prepare a series of cholesterol standards at a concentration range of 0 - 0.3
mmol/L.
Notes: Dilute with glacial acetic acid.
2. The final volume should be 6 ml.

FERRIC
GLACIAL TOTAL
CONCENTRATIO GLUCOSE CHLORIDE
TUBES ACETIC mL PER
N (mg/dL) STANDARD (mL) SULPHURIC
(mL) TUBE
(mL)
Glacial
M1V1 : M2V2 Acetic Acid
1 0 (0.3)V1: 0 (6.0 ) = 6.0 ml - 6.0 4.0
V1 : 0 mL 0.0 mL
= 6.0 mL
Glacial
M1V1 : M2V2 Acetic Acid
2 0.05 (0.3)V1: 0.05 (6.0 ) = 6.0 ml - 6.0 4.0
V1 : 1 mL 1.0 mL
= 5.0 mL
Glacial
M1V1 : M2V2 Acetic Acid
3 0.10 (0.3)V1: 0.10 (6.0 ) = 6.0 ml - 6.0 4.0
V1 : 2 mL 2.0 mL
= 4.0 mL
Glacial
M1V1 : M2V2 Acetic Acid
4 0.15 (0.3)V1: 0.15 (6.0 ) = 6.0 ml - .0 6.0 4.0
V1 : 3 mL mL
= 3.0 mL
5 0.20 M1V1 : M2V2 Glacial 6.0 4.0
(0.3)V1: 0.20 (6.0 ) Acetic Acid
V1 : 4 mL = 6.0 ml -

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 4.0 mL
= 2.0 mL
M1V1 : M2V2 Glacial
(0.3)V1: 0.25 (6.0 ) Acetic Acid
6 0.25 V1 : 5 mL = 6.0 ml - 6.0 4.0
1.0 mL
= 5.0 mL
M1V1 : M2V2 Glacial
(0.3)V1: 0.30 (6.0 ) Acetic Acid
7 0.30 V1 : 6 mL = 6.0 ml - 6.0 4.0
6.0 mL
= 0.0 mL

BLANK - - 6.0 mL 6.0 4.0

d. Blank : 6 ml glacial acetic acid

e. Spectrophotometric measurements
1. Add 4 ml ferric chloride-sulphuric acid reagent to all the test, standard, and
blank tubes.
2. Mix thoroughly and measure the optical density at 570 nm.
RESULTS:

TUBES 1 2 3 4 5 6 7

ABSORBANCE 0.0 0.212 0.449 0.764 1.013 1.359 1.498

TEST ABSORBANCE

TOTAL
0.424
CHOLESTROL
FREE
CHOLESTERO 0.021
L

CALCULATION:
TOTAL CHOLESTEROL • From graph: • Total Cholesterol in 10 ml
= 0.080 mmol/L (In 1000ml) solution (0.5 ml + 9.5 ml
• 1 ml solution; Acetone Alcohol).

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 o 0.080/1000
• 10 ml solution;
o 0.080/1000 X 10
= 8.0 X 10-4 mmol Cholesterol
• This equivalent to Total
Cholesterol in 2 ml
Supernatant is used.
• From graph:
= 0.025 mmol/L (In 1000ml)
• 1 ml solution;
o 0.025/1000
• 10 ml solution;
FREE CHOLESTROL
o 0.025/1000 X 10
= 2.5 X 10-4 mmol Cholesterol
CONCENTRATION OF PERCENTAGE OF
ESTERIFIED UNESTERIFIED
CHOLESTEROL CHOLESTEROL (SERUM)
• Total Cholesterol –Free • Free Cholesterol/Total
Cholesterol Cholesterol X 100%
= 8.0 – 1.0 = 1.0/8.0 X 100%
= 7.0 mmol/L = 12.5%
DISCUSSION:
In this experiment, acetone alcohol is used for extraction process of cholesterol. This done
by separating the cholesterol from serum by centrifugation process. The cholesterol extract can
= 10 (8.0 X 10-4 ) / 2
= 4.0 X 10-4 mmol Cholesterol
• This equivalent to amount
of Cholesterol in 0.5 ml
serum.
• Total Cholesterol in serum;
= 4.0 X 10-3 X (1000/0.5)
= 8.0 mmol/L
• Supernatant is being used:
5ml.
• Free Cholesterol (in 10
mL)
= 10 (2.5 X 10-4 ) / 5
= 5.0 X 10-4 mmol/L
• Free Cholesterol (in
serum)
= 5.0 X 10-4 / (1000/0.5)
= 1.0 mmol/L
PERCENTAGE OF
ESTERIFIED CHOLESTEROL
(SERUM)
• 100% - 12.5 %
= 87.5%
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used for to determine the esterified and unesterified cholesterol.
Acetone alcohols vaporized and leave behind the cholesterol. Free cholesterol is easily
determined by direct precipitation. Digitonin is a substrate to differentiate free cholesterol from
total cholesterol and esterified cholesterol. In digitonin precipitation method, alcoholic solution of
cholesterol is mixed with an alcoholic solution of digitonin, which formed cholesterol digitonide.
This complex has a low solubility and making the test is more sensitive.
After precipitation is completed, digitonide is separate into its component. Ester and
digitonide give a purple colour of equal intensity. Cholesterol digitonide is insoluble in water but
soluble in glacial acetic acid. Ferric chloride-sulphuric acid is added to the tube when measuring
the density for cholesterol standard as the radiators.
Below are the information ranges for the serum cholesterol:
3.6-5.2 mmol/L (normal)
5.3 -6.2 mmol/L (borderline high)
> 6.2 mmol/L (hypercholesterolemic)
The serum cholesterol that obtained from this experiment is 7.0 mmol/L. This means the
serum sample is hypercholesterolemic. The free cholesterol level is 1.0 mmol/L and indicated low
than normal ranges. This is because of vaporizations process of acetone alcohol, which means by
overheat it.
CONCLUSION:
1. Concentration of the total cholesterol is 7.0 mmol/L.
2. Whereby, the free cholesterol is 1.0 mmol/L.