Serum Total Cholesterol
SUMMERY:
Elevated level of serum cholesterol are associated with atherosclerosis, nephrosis, diabetes mellitus, obstructive jaundice & myxedema. Decreased levels are observed in hyperthyroidism, malabsorption & anemia.
Aim: – Estimations of Serum Total Cholesterol by Colorimetric (Watson) Method.
PRINCIPLE:
Cholesterol reacts with acetic anhydride in the presence of glacial acetic acid and conc. sulfuric acid to form green colored complex. Intensity of the color is proportional to the cholesterol concentration and can be measured at 575 nm (Green-Yellow filter: 520-580 nm).
REQUIREMENTS:
Three test-tube,
Colorimeter
Cholesterol Reagents,
Distilled water,
Incubator,
Cuvette,
Pipette etc. Serum (Fasting)
PROCEDURE:
Dispense in the tubes labeled as follows:
| Contents | Blank | Standard | Test |
|---|---|---|---|
| Cholesterol reagent 1 | 2.5 ml | 2.5 ml | 2.5 ml |
| Serum | 100 µl | – | – |
| Cholesterol standard | – | 100 µl | – |
| Distilled water | – | – | 100 µl |
Mix well and cool to room temperature by placing in a water bath (at room temperature). Add the following reagent:
| Reagent | Blank | Standard | Test |
|---|---|---|---|
| Cholesterol reagent 2 | 500 µl | 500 µl | 500 µl |
Mix thoroughly, keep in the water-bath at room temperature (25°C +5°C) for 10 min. Read the absorbance of test and standard against blank at 575 nm (green-yellow filter).
Calculation:-
Serum Cholesterol (mg/dl) = Optical Density of Test X Concentration of Standard
Optical Density of Standard Concentration of Glucose Standard =200
mg/dl After Testing, Optical Density of Test = 0.25 Optical Density of Standard = 0.26 Then, 0.25*200
0.26 Serum Cholesterol (mg/dl) = 192.3
Result: – = 192.3 mg/dl
Normal Value:- 150 –
250 mg/dl