cholestrol determination in blood - (Jan/20/2012 )
can any one tell me about cholestrol determiantion in blood
When we measure cholesterol, we extract all the lipids in the cell using a bligh and dyer differential extraction (google it, its simple). In our lab, we use an Iatroscan TLC-FID (thin layer chromatography flame ionization detector), which separates the lipids and when compared to standards, quantifies them by detecting the ions formed when it is burnt).
in vitro assays based on cholesterol oxidase reaction
Cholesterol total (by Zagami)
Reagent 1: Hepes buffer 50 mM pH 7.0, 2.03 g/L magnesium chloride, 0.508 g/L 4-aminoantipyrine, 1500 U/L cholesterol esterase, 2000 U/L cholesterol oxidase, 10 ml/L of 0.5 g/L Tween-20, 1 g/L NaN3.
Reagent 2: Hepes buffer 50 mM pH 7.0, 1.47 g/L N-Ethyl-N-(3-sulfopropyl)-3-methoxyaniline sodium salt (Fluka cod. 04340), 3000 U/L peroxidase, 10 ml/L of 0.5 g/L Tween-20, 1 g/L NaN3.
Sample and Reagent 1 and 2 is: 16 µl: 1000 µl: 500 μl, setting a dominant wavelength of 505 nm, sub-wavelength of 660 nm, after mixing the sample and reagent 1, 37 °C incubation 3 – 5 min, read after each tube absorbance A1, reagent 2 was added the reaction mix 5 min, read the absorbance A2, the change in absorbance per tube ΔAbssample = A2 - A1, after deduction of the reagent blank sample to calculate the change in absorbance value of the standard ΔAbsstandard = A2 - A1, then for to calculate unknown sample concentration apply the following formula
total cholesterol (mg/dl) = ΔAbssample/ΔAbsstandardX Standard cholesterol