III.

Methodology against cholesterol concentration to obtain the

standard curve.
A. Blood serum preparation
C. Measurement of total serum cholesterol of
To measure total blood serum cholesterol, two
blood sample
5.0mL blood samples were drawn from two different
sources and were placed in a red capped vacutainer To measure the total cholesterol in the blood
tubes having no anticoagulant to make sure that the sample, 0.5 mL of the serum sample mixed with 4.95
blood can clot. The blood sample was allowed to clot mL of ferric chloride reagent in a test tube, and was
at room temperature for 15-30 minutes, and was allowed to stand for 15 minutes to react. This was
then placed in cooler at approximately 4˚C to prevent also done in triplicates. Precipitates were removed
degradation. When the sample was ready for by centrifuging the solution at 2000xg and the
processing, it was centrifuged at 1550xg for 10 supernate was collected and placed in another test
minutes to separate the clot from the serum. The tube. Three mL of Sulfuric acid was then added to
serum was then aspirated and placed in a 15-mL the serum solution and was incubated for 30
falcon tube without any presence of cells and minutes. The absorbance of each solution was
turbidity to make sure that the sample is pure serum. measured using a UV-Vis spectrophotometer at 560
Excess serum was stored in microcentrifuge tubes nm, to determine the concentration of cholesterol in
and stored in the freezer at -80˚C. the serum. The absorbance were recorded and the
total serum cholesterol concentration is computed
B. Generation of cholesterol standard
using the obtained equation of the standard curve.
calibration curve
For the basis of the colorimetric quantification of total
serum cholesterol, a standard curve was established
using stock cholesterol. The 1mg/ mL stock
cholesterol was prepared by dissolving 25 mg of
pure cholesterol in 25 mL chloroform. Ferric chloride
solution, the coloring agent, was prepared by diluting
1 mL of Kiliani’s reagent to 100 mL with glacial acetic
acid. Varying concentrations of cholesterol stock in
ferric chloride solution was prepared in a test tube
and the concentration (in mg/dL) of each were
computed (see table 1). This was done in triplicates.
Table 1. Cholesterol standards preparation

Cholesterol Ferric chloride

Standard No.
stock (mL) solution (mL)
1 Blank 5.0
2 0.05 4.95
3 0.1 4.90
4 0.2 4.80
5 0.3 4.70
6 0.4 4.60

The mixture was allowed to stand for 15 minutes with

occasional mixing. Precipitates were removed by
centrifuging it at 2000xg and the supernate was
collected and placed in a test tube. Three mL of
Sulfuric acid was then added and incubated for 30
minutes. The absorbance of each solution was
measured using a UV-Vis spectrophotometer at 560
nm. The absorbance were recorded was and plotted