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Mix the above solutions by swirling gently and leave to stand at room temperature for 20 minutes. Read absorbance at 580 nm. Using the working cholesterol solution, prepare a standard curve for cholesterol as follows. Tube 1 Solution Acetic acid Cholesterol standard Propan-2-ol Colour reagent (ml) 3.6 0.0 0.5 2.9 Tube 2 (ml) 3.6 0.1 0.4 2.9 Tube 3 (ml) 3.6 0.2 0.3 2.9 Tube 4 (ml) 3.6 0.3 0.2 2.9 Tube 5 (ml) 3.6 0.4 0.1 2.9 Tube 6 (ml) 3.6 0.5 0.0 2.9
MATERIALS
Phosphotungstic acid (8.0 g dodeca-Tungstophosphoric acid in water, adjust to pH 7.6 with NaOH and make up to 100 ml with water). Refrigerate. Magnesium chloride solution (Dissolve 9.6 g MgCl2 in water and make up to 100 ml) Just prior to the experiment, mix equal volumes of solutions A and B and use the mixture as the precipitating reagent. PROCEDURE Mix 1.0 ml serum with 1.0 ml of the precipitating reagent. Shake well and leave to stand at room temperature for 15 minutes. Centrifuge at 3500 rpm for 30 minutes. Remove the supernatant and proceed as in determination of total cholesterol.
RESULTS
From the calibration curve, determine the concentration of total cholesterol and HDL-cholesterol in the serum (mg/1000 ml). How do your values compare to the literature values? What is the role of HDL-cholesterol?