Cholesterol Determination PDF

1. Determination of Serum Cholesterol Objective: 1. To determine the concentration of cholesterol in a serum patient sample using the Lieberman- Burchard reagent. 2. To evaluate whether the determined patient’s cholesterol is normal or abnormal. Theor...

1. Determination of Serum Cholesterol Objective: 1. To determine the concentration of cholesterol in a serum patient sample using the Lieberman- Burchard reagent. 2. To evaluate whether the determined patient’s cholesterol is normal or abnormal. Theory: The determination of serum cholesterol is very important because of its implication in the development of atherosclerosis and heart disease. Blood is commonly analyzed for changes in the normal concentrations of its many chemical constituents. Such analyses have been the mainstays in the diagnosis of many clinical disorders. The quantitative analyses of the chemical constituents of blood can be done on a) whole blood sample, b) serum, and c) protein-free filtrate. Whole blood is prepared by preventing blood from freefiltrate clotting at the time shortly after withdrawal by adding an anticoagulant (e.g., oxalate, citrate, EDTA etc.) to the tube in which the blood is delivered. When whole blood is centrifuged, the liquid portion is called plasma, where plasma contains coagulation proteins. Serum is the liquid portion obtained after coagulation of freshly drawn blood, and thus, in contrast to plasma, serum does not contain coagulation proteins. Serum and plasma, when not in used should be stored frozen, so that its enzyme constituents will remain stable for several days. In the following procedure, the Lieberman-Burchard reagent, containing acetic acid, acetic anhydride, and sulfuric acid, reacts directly with all cholesterol in the serum to produce an unstable green chromophore which is measured spectrophotometrically. Structure of this chromophore is shown in figure. 15: ofvarious a  HO Cholesterol L-B reagent H2SO4/HOAc HOO2S 8 08 Cholestahexaene sulfonic acid max = 620 nm Figure. 15: The chromophore produced by the reaction of cholesterol with Lieberman- Burchard reagent This is a direct reaction without the need for extraction or precipitation of proteins. This reaction is not specific to cholesterol but interferences in serum are very minimal. Procedure: ne A. Preparation of the cholesterol color reagent This reagent was prepared as follows: 1. Place 350 ml of glacial acetic acid into a 2-liter Erlenmeyer flask. Slowly add 550 ml of acetic anhydride and mix. 2. Seal the top of the flask with parafilm to prevent exposure to atmospheric moisture, refrigerate overnight or pack in an ice bath and allow to cool for at least an hour. 3. Cool 200 ml of concentrated sulfuric acid in an ice bath for at least an hour before mixing with the acetic acid-acetic anhydride solution. 4. Place the acetic acid-acetic anhydride mixture onto a magnetic mixer and add the pre- cooled sulfuric acid slowly to the stirring mixture. 5. Add 20 g. of anhydrous sodium sulfate and continue stirring until the salt is dissolved. Do not use if the solution becomes brown. B. Preparation of the Cholesterol standard 2.5 mg / ml stock cholesterol (MW 386.65) solution was prepared in 100 ml volumetric flask by dissolving 250 mg of cholesterol in 100 ml glacial acetic acid. C. Measurements: 1. Before starting, clean and dry the required number of test tubes as water interferes with the color reaction. 2. After numbering the dry test tubes (see test tube no. listed in table below), place the following in total volumes of 5.5 ml: Test tube no. Serum Cholesterol Glacial Acetic Cholesterol (patient) (ml) (ml) acid (ml) Color reagent (ml) 1 - - 0.5 5.0 2 - 0.1 0.4 5.0 3 - 0.2 0.3 5.0 4 - 0.3 0.2 5.0 5 - 0.4 0.1 5.0 6 - 0.5 0.0 5.0 7 0.1 - 0.4 5.0 3. Mix on a vortex mixer. 4. Place all tubes in 37oC water bath for 10 minutes. Be sure that the level of water in the bath is high enough so that the mixture in the tubes is beneath the water level in the bath. 5. Allow all tubes to cool by placing them in an ice bath or cold-water bath for 10 minutes. 6. Transfer a portion of the contents of the tubes to cuvettes. Be sure to wipe off the water from the outside of the test tubes. 7. Read the absorbance at 620 nm against the blank. 8. Establish a standard curve of cholesterol by plotting standard concentrations (mg / ml) against corresponding absorbances. 9. Determine cholesterol concentration (mg / ml) of the patient serum from the established standard curve. 10. Convert the determined cholesterol concentration of patient serum to mmoles/L. 11. According to your results, interpret whether this patient has High, normal, or low cholesterol level (Cholesterol level ranges between 3.2 - 5.2 mmol/L is desirable).  Report Sheet Determination of Serum Cholesterol Student name: _____________________ Student Id no: _________________ Student name: _____________________ Student Id no: _________________ Group NO. Unknown: _________________ Test tube Absorbance Concentration Concentration no. Reading (mg/ml) mmoles/L 1 2 3 4 5 6 7 What does the patient cholesterol value reflect?