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Cholesterol esterase hydrolyses cholesterol esters into free cholesterol and fatty acids. In the second reaction cholesterol oxidase converts cholesterol to cholest-4-en-3-one and hydrogen peroxide. In presence of peroxidase, hydrogen peroxide oxidatively couples with 4 - aminoantipyrine and phenol to produce red quinoneimine dye which has absorbance maximum at 510 nm. (500 - 530). The intensity of the red colour is proportional to the amount of total cholesterol in the specimen.
Buffer, pH 6.8
Stabilizers / Surfactants
SPECIMEN COLLECTION & PRESERVATION
Blood should be collected in a clean dry container. Fasting blood is preferred for cholesterol assay. Cholesterol in the serum is stable for 7 days when stored at 2-8 Degree C and 60 days when stored at -20 Degree C.
1.0 ml procedure
Incubate the assay mixture for 5 minutes at 37 Degree C or 10 minutes at room temperature (25-30 Degree C). After incubation measure the absorbance of assay mixture against blank at 510 nm. The final colour is stable for two hours if not exposed to direct light.
Borderline High Cholesterol
200- 239 mg/dl
Expected range varies from population to population. It is recommended that each laboratory should establish its own normal range.