Qualitative reaction of lecithin with cadmium chloride.

Laboratory work 11. Cholesterol. Phospholipids

Cholesterol is a representative of steroids. The basis of cholesterol molecule is cyclopentane perhydrophenantren. The body is able to produce cholesterol; we also take it from food.

Cholesterol is a part of cell membranes; it serves as the predecessor in the synthesis of other steroids – bile acids, steroid hormones, vitamin D₃.

Phospholipids contain higher fatty acid residues, phosphoric acid, nitrogenous bases or some other compounds. Phospholipids are the most polar among lipids. They are important components of cell membranes.

Extraction of cholesterol from the brain tissue.

Put 2 g of brain tissue dried out with gypsum into a clean dry test tube and add 6 ml of chloroform. Shake for 10 minutes. Filter the extract into a dry test tube.

Color reactions on cholesterol are based on the dehydration and oxidation of cholesterol and following condensation of products with conjugated double-bond system which give colored derivatives with sulfuric acid.

Qualitative reactions of cholesterol.

Schiff test.

Pour 1 ml of chloroform cholesterol solution in the test tube and add 1 ml of concentrated sulfuric acid by the wall carefully.

Salkowski rtest.

Shake the liquid after Schiff test.

Lieberman-Burchard test.

Pour 1 ml of chloroform cholesterol solution in the test tube and add 0.5 ml of acetic anhydride and 2 drops of concentrated sulfuric acid. Mix well.

Extraction of phospholipids from brain tissue.

Put 2 g of brain tissue dried out with gypsum into a flask and add 10 ml of alcohol. Attach a reflux condenser and place for 15-20 minutes in the water bath at 70⁰C. 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.

Qualitative reaction of lecithin with cadmium chloride.

Pour 2 ml of alcoholic solution of lecithin (experiment 3) in the test tube and add 1 ml of saturated alcoholic solution of cadmium chloride.

5. Quantitative determination of total cholesterol in blood serum by Zlatkis – Zak reaction.

The method is based on the reaction of cholesterol with acetic and sulfuric acids in the presence of ferric chloride. Cholesterol is dehydrated and oxidized to form colored products.

Experimental sample: pour 0.1 ml of blood serum in the test tube, add 3 ml of concentrated acetic acid and 2 ml of ferric chloride process solution and mix.

Control sample: pour 0.1 ml of distilled water in the test tube, add 3 ml of concentrated acetic acid and 2 ml of ferric chloride process solution and mix.

Leave samples for 15 minutes.

Use the photoelectric colorimeter to measure the optical density against the control at the wavelength of 510-560 nm (green filter) in a cuvette with a layer thickness of 1 cm.

To construct the calibration plot prepare dilutions from the standard cholesterol solution according to Table.

Table. Preparation of cholesterol solutions for the calibration curve

№ of tubes	Cholesterol standard solution, ml	Conc. acetic acid, ml	Ferric chloride solution, ml	Cholesterol content in the sample, mg%
	0,1	3,0	2,0
	0,2	3,0	2,0
	0,3	3,0	2,0
	0,4	3,0	2,0
	0,5	3,0	2,0

In 20 minutes measure the optical density of the samples against the control. Build a calibration plot according to the results.

Use the plot to determine the cholesterol content in blood serum. Make the conclusion about cholesterol level in the blood (norm, hypo- or hypercholesterolemia).

The norm of cholesterol in blood serum is 120-250 mg% (3.12-6.50 mmol/l).

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