QUANTITATIVE DETERMINATION OF CHOLESTEROL BY ENZYMATIC
By Matthias Heyner
Group 2 Pair 3
The biosynthesis of cholesterol predominantly takes place in the liver and in the intestinal
mucosa, but almost all cells synthesize it. It is a constituent of many membranes, it is also
essential in the synthesis of bile acids and steroid hormones.
4-cholesten-3-one and H2O2 are formed from cholesterol by cholesterol oxidase (ChOD). A
measurable red quinone imine derivative is formed from hydrogen peroxide and 4-amino-
antipyrine in the presence of phenol and peroxidase (POD).
Cholesterol + O2 4-cholesten-3-one + H2O2
2 H2O2 + phenol + 4-aminoantipyrine red quinone + 4 H2O
- cholesterol stock solution (50µg/100µL)
- REAGENT having the following composition:
PIPES buffer pH=6.7, phenol, cholesterol oxidase, peroxidase, 4-aminoantipyrine, sodium
Prepare a calibration series of solutions from a stock solution (50µg/100µL) by dilution with
ethanol according to the following table:
Volume of stock Volume of solvent
1. 0.5 mL 2.0 mL 10µg/100µL
2. 1.0 mL 1.5 mL 20µg/100µL
3. 1.5 mL 1.0 mL 30µg/100µL
4. 2.0 mL 0.5 mL 40µg/100µL
Prepare the mixtures shown in the following table:
It is advisable to measure first 3 mL REAGENT into 7 tubes, and then add the different
concentration cholesterol solutions and distilled water to them. The tubes must be covered and
shaken, then let them stay for 20 minutes at room temperature to form the color.
Reference Tube1 Tube2 Tube3 Tube4 Tube5 Unknown Absorbance
Distilled water 0.5 mL
10 μg/100μL 0,21
20 μg/100μL 0,45
30 μg/100μL 0,67
40 μg/100μL 0,87
50 μg/100μL 1,14
cholesterol sln. 0.5 mL
Unknown 0.5 mL
REAGENT 3 mL 3 mL 3 mL 3 mL 3 mL 3 mL 3 mL
Measure the absorbance of the known concentration and the unknown samples.
Making an absorbance with BIOCHROM WPA CO 7500 Colorimeter:
1./ Switch the instrument on by pressing the ON/OFF button
2./ Select the 520 nm wavelength by turning the thumbwheel at the side of the instrument.
The wavelength selected is displayed in the window above the cuvette compartment.
Note: two of the locations are empty.
3./ Select Abs (absorbance) mode
4./ Transfer the volume of reference into plastic vial. Place the Vialreference into the cuvette
compartment and press the R (reference) button. The display will show 0.00 Abs.
5./ Transfer the volume of Tube1 into plastic Vial1. Remove the reference sample and
replace with Vial1 sample. Press the T (test) button. The result is displayed in
Multiple samples can be compared with the same reference by placing Vial 2-Vial5-
Vialunknown samples in the cuvette chamber and making measurements for each other.
It is recommended to re-reference with the reference solution every 10 to 15 minutes to
avoid any slow instrument drift. If in doubt, always re-reference.
Note: at high absorbance the time taken for the measurement will be longer (up to 10
seconds) as light intensity is proportionally lower.
Collect the Abs values and draw a calibration curve (Abs-μg/100μL). Determine the
concentration of the unknown sample.
The light absorbance measured for the unknown sample was 0.93.
From the linear average of the points in the graph we get the formula: y = 0.022*x - 0.007
x = (y + 0.007) / 0.022
x = (0.93 + 0.007) / 0.022
x = 42,5909091
The concentration of the unknown sample was 42.59 µg/µl.