Basic Concepts in Clinical Biochemistry-A Practical Guide.7z

4-aminophenazone to form red quinone imine dye. The color produced is measured
at 520 nm and compared with that of cholesterol standard.

35.2.2 Reagents

1.Cholesterol reagent composition

Phosphate buffer (100 mM, pH 6.5)
4-aminophenazone (0.25 mM)
Phenol (5 mM)
Peroxidase (>5 KU/L)
Cholesterol esterase (>150 U/L)
Cholesterol oxidase (>100 U/L)
Sodium azide (0.05%)

2.Cholesterol standard–200 mg/dl

35.2.3 Procedure

Take three test tubes, and label as blank, standard (S), and test (T). Pipette 1 ml
cholesterol reagent to all tubes, and add 20μl serum sample in the tube T. Then add
20 μl of 200 mg % cholesterol standard into the tube labelled as“S.”Mix and
incubate tubes at 37C for 5 min and measure absorbance at 530 nm against blank.
Calculate cholesterol amount in given sample.

35.2.4 Calculations

Calculate total cholesterol amount in serum sample by using equation

Total cholesterol mgðÞ¼=dlserum

ODof testamount of standard 100

ODof standardvolume of sample mlðÞ

35.2.5 Precautions

1. Do not use lipemic or grossly lysed serum samples for the investigation.


2. Fasting blood sample is preferable.


3. If cholesterol value is more than 750 mg/dl, dilute the sample, and repeat the test.


4. Store the reagents at 2– 8 C.

140 35 To Measure Lipid Profile in Serum Sample