Pre- lab 5

Cholesterol
 Principle
• In three steps:
1/ cholesterol esters are enzymatically hydrolyzed by
cholesterol esterase (CE) to cholesterol and free fatty acids.
2/ free cholesterol ; is oxidized by cholesterol oxidase (CO) to
Cholest-4-en-3-one and hydrogen peroxide.
3/ hydrogen peroxide combines with phenol and 4 –amino-
antipyrine ( 4AAP) in presence of peroxidase (POD) to form

a chromophore (quinoneimine ).
 Method
blank Standard Sample

Reagent (ml) 1 1 1

Standard (ml) --- 0.01 ---

Sample (ml) ---- ----- 0.01

Calculation:

concentration of cholesterol (test) =

absorbance of unknown(test) x Concentration of standard (Cs)

Absorbance of standard (As)

= ----x----- ------- mg/dl

------
SI unit = --------- mmol/L