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Experiment 5. Qualitative reaction to lecithin with cadmium chlorideLabwork 2. Cholesterol. Phospholipids. Experiment 1. Cholesterol isolation from brain tissue Put 3 g of brain tissue dried out with gypsum into a clean dry test tube and add 6 ml of chloroform alcohol. Shake for 10 minutes. Filter the extract into a dry test tube.
Experiment 2. Qualitative reactions to cholesterol 2.1. The Schiff test. Take a test tube with 1 ml of chloroform cholesterol solution and carefully underlay 1 ml of concentrated sulfuric acid by the side of the tube. A red ring is to appear on the boundary of the liquids. 2.2. The Salkowski reaction. Shake the liquid after the Schiff test. After the delamination the upper layer becomes red; the lower one is of a yellow-orange color with a green fluorescence. 2.3. The Liebermann-Burchard reaction. Add 0,5 ml of acetic anhydride and 2 drops of concentrated sulfuric acid into a test tube with1 ml of chloroform cholesterol solution. Mix well. A red coloring changing into purple and then blue and green is to be observed. The green coloring can be instantly observed due to the little amount of cholesterol. Color reactions to cholesterol are determined by dehydration and oxidation of cholesterol under the action of concentrated sulfuric acid with the following condensation of the reaction products and the formation of compounds with conjugated double bonds, which give different color derivatives to sulfuric acid.
Experiment 3. Quantitative evaluation of total cholesterol in blood serum by direct method according to Zlatkis-Zak color reaction The method is based on the reaction of cholesterol to acetic and sulfuric acids with ferric chloride. Add 3 ml of concentrated acetic acid and 2 ml of ferric chloride process solution to 0.1 ml of blood serum and mix. Prepare the control by adding 3 ml of concentrated acetic acid and 2 ml of ferric chloride process solution to 0.1 ml of distilled water. Leave for 15 minutes. Use the photoelectric colorimeter to measure optical density against the control at the wavelength of 510-560 nm (green filter) in a optical cell with a layer thickness of 1 cm. Prepare dilutions from the standard cholesterol solution according to Table 1.
Table 1. Cholesterol solution preparation for calibration graph
Photometer the samples against the control in 20 minutes. Draw a calibration graph according to the results. Use the graph to determine the cholesterol content in blood serum. The norm of cholesterol in blood serum is 120-250 mg/l (3,12-6,50 mmol/l).
Experiment 4. Lecithin isolation from brain tissue Put 5 g of brain tissue dried out with gypsum into a flask and add 10 ml of alcohol. Attach the backflow condenser and place it into a water bath (700?) for 15-20 minutes. Shake from time to time. If a significant amount of the alcohol is evaporated, add some more. At the end of the extraction cool the flask and filter the extract.
Experiment 5. Qualitative reaction to lecithin with cadmium chloride Add 1 ml of saturated alcoholic solution of cadmium chloride to 2 ml of alcoholic solution of lecithin (experiment 1). A white flake-like residue of the complex compound of lecithin with cadmium chloride is observed. Cholesterol and vegetable oil solutions do not produce this reaction.
Test Questions 1. Why is hypercholesterolemia commoner than hypocholesterolemia? 2. What change will the cholesterol synthesis undergo on a vegetable food diet? Why? 3. What organs synthesize cholesterol for export? 4. How is cholesterol excreted from the organism? Date: 2016-06-13; view: 6
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