CHOLESTEROL DETERMINATION Flashcards
LABORATORY EXPERIMENT
Enzyme in Primary Reaction
Cholesterol esterase (CE)
Enzyme in Secondary Reaction
Cholesterol oxidase (COx)
Enzyme in Tertiary Reaction
Peroxidase (POD)
Color Developers
4-aminophenazone
Phenol
End Product of Primary Reaction
Free cholesterol and fatty acids
End Product of Secondary Reaction
Hydrogen Peroxide (H2O2) and Cholestenone (Cholest-4-en-3-en-one)
End Product of Tertiary Reaction
Quinoeimine dye RED COLOR (Oxidized Chromogen) and H2O
Wavelength (absorption maxima)
500nm
Cholesterol Standard
200mg/dL
Chemical Method: Liebermann-Burchard Reaction
Cholesterol and H2SO4 and Acetic Anhydride
Chemical Method: Salkowski Reaction
Cholesterol + 2 H2SO4 + Fe3+
a standard reference method for estimation of total serum cholesterol involving saponification of cholesterol ester by hydroxide, extraction with petroleum ether, and color development with acetic anhydride-sulfuric acid; the method avoids interference by bilirubin, protein, and hemoglobin.
Abell Kendall Method (Former Reference Method)
Borderline
200-240mg/dL
High
Greater than or equal to 240 mg/dL
New Reference Method
Gas Chromatography - Mass Spectroscopy
Temperature before use of reagent and standard
20-25 degree Celsius (Room Temperature)
Reagent and Standard are stable at
2-8 degree Celsius (refrigeration temp)
Denaturation of Enzymes
56-60 degree Celsius
Optimal Temperature of Enzymes
37 degrees celsius
Inactivation of Enzymes
Room temperature
Fasting for Total Cholesterol
12HRS
Total Cholesterol and HDL are stable for
4 days at 2-8 degree Celsius
Extension of stability for Total Cholesterol
-20 degree Celsius for three months
Extension of stability for HDL
7-14 days
