LAB - 4

Fospholipids, steroids and fat-soluble vitamins

Aim of the class: preparation of phospholipids, study on phospholipid composition, detection of some steroids and vitamins. Phospholipids Phospholipids exist mainly in biological membranes. Glycerol or more composed alcohol sphingosine - constitutes a skeleton of phospholipid molecules. Derivatives of glycerol, phospholipids, are named glycerophospholipids. One of them is phosphatydylcholine (lecithin), which contains glycerol, two chains of fatty acids, phosphate and choline. Large amounts of lecithin exist in the yolk of hen egg. It may be extracted with a mixture of chloroform with methanol (ratio 2:1). Lecithin is hardly soluble in anhydrated acetone. The presence of hydrophilic phosphocholine group makes possible to form a stable suspension (emulsion) of lecithin in water. Heating lecithin with hydrosulphate (VI) results in dehydration of glycerol molecule and formation of unsaturated aldehyde acrolein a substance of unpleasant, irritating smell. It may be detected with a reductive test, e.g. with potassium chromate (VI). Acrolein reduces an orange potassium chromate (VI) to green chromium (III) sulphate. Steroids Steroids are derivatives of cyclopentanoperhydrophenantrene. They may be classified into several classes, dependently on functional groups, attached to cyclopentanoperhydrophenantrene rings. The mains of them are alcohols (e.g. cholesterol), alcoholoacids (e.g. bile acids), phenols and ketones (e.g. steroid hormones).

Cholesterol
Cholesterol is a representative of animal sterols. It is a component of cell membranes and plasma lipoproteins. Furthermore, cholesterol is a precursor of bile acids, cholecalciferol (a precursor of vitamin D3) and steroid hormones. The presence of a double bond in one of cholesterol rings is responsible for its ability to form colour products in the presence of concentrated inorganic acids. The action of concentrated sulphuric acid results in dehydration of cholesterol molecule (Salkowski test) with a formation of a red bicholestadien disulphonate, which in the presence of acetate anhydride forms a green colour bicholestadien monosulphonate (Lieberman-Burchard test). Traces of water make this reaction impossible.

Bile acids
Bile acids are steroid compounds, which are synthesised from cholesterol in the liver. Human bile contains mainly cholic and deoxycholoic acids. A part of these acids interact with glycine or taurine forming glicocholic and taurocholic acids. The mechanism of this interaction is similar to a peptide bond formation. The carboxyl groups of bile acids interact with amino group of glycine or taurine. The hydrocarbon character of both the ring structures and the side chains, as well as the presence of polar OH groups and electrically charged groups, -COO-, -SO3H-, cause that the bile acids demonstrate character of strong detergents. They reduce the surface tension of alimentary lipids contained in the intestine. The decrease in surface tension may be detected by a Hay test. If a powdered sulphur (in laboratory dialect it is referred to as a sulphur flower) is applied to a test tube (containing emulsion of olive oil in water) the sulphur floats on the surface of this suspension. If bile acids are present in such a
1

suspension, the emulsion of olive oil in water becomes more stable and the sulphur flower falls down to the bottom of test tube. The chemical structure of bile acids resembles some other compounds, which contain both ring components and several OH groups, like resorcin or -naphtol. For this reason the bile acids are able to condense with hydroxymethylenfurfural, which is produced by the action of concentrated sulphuric acid on saccharose. An appearance of red colour product of condensation process indicates the presence of bile acids. Fat-soluble vitamins Main forms of vitamin D are ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3). They are synthesised in human body from precursors, which are unsaturated sterols. Vitamin D2 is produced in human skin from ergosterol under the action of UV-light (which is a component of sun light). Vitamin D3 exists in cod-liver oil. It is synthesised in human liver from 7-dehydrocholesterol. Vitamins A demonstrate isoprene structure. They exist exclusively in animal tissues in two main forms, A1 (retinol-1) and A2 (retinol-2). Both of them are alcohols containing 6-carbon ring, with a long side chain composed of two isoprene units. Vitamin A alcohol form is oxidised in human body to aldehyde. Many plants contain a group of substances, refereed to as carotene, which are polyunsaturated derivatives of isoprene. The high number of conjugated double bonds in carotene structure makes them colour. In human body the carotenes: , and play a role of provitamins A. One molecule of -carotene is converted into 2 molecules of vitamin A, whereas and are transformed with a release of one molecule of vitamin A. The presence of conjugated double bonds, both in vitamin A and D, allows them to form characteristic colour complexes with antimony chloride (SbCl3). Vitamin A1 gives a product of a blue colour, whereas vitamin D3 gives a product of a red-violet colour. Laboratory procedures 1. Extraction of lecithin from the yolk of hen egg Grind about 2-3 g of dried yolk of hen egg in a mortar in 10 ml of solvent containing chloroform and methanol (mixed in a ratio 2:1). Filter the extract into a dry test tube through a paper moistened with the same solvent. Evaporate the solvent in a boiling water bath. Observe an appearance of a dark-brown, oillooking substance, which appears vaseline-like consistency after cooling. This is a lecithin contaminated with other constituents of yolk compounds. 2. The solubility of lecithin Transfer about 3 drops of lecithin into 2 test tubes. Supplement one of the samples with 2 ml of H2O and heat it for a few seconds in a boiling water bath. Lecithin does not dissolve in water but forms a stable turbid suspension. Supplement the second sample with 1 ml of chloroform and heat it in a boiling water bath for a few seconds. Notice that lecithin dissolves in chloroform. An addition of 2 ml of acetone results in precipitation of lecithin from the solution. 3. The chemical composition of lecithin a. detection of glycerol - acrolein test Transfer about 3 drops of lecithin into a dry test tube. Add 1.5 ml of orange colour potassium dichromate (K2Cr2O7) and acidify it with 3 drops of concentrated sulphuric acid. Put the test tube into boiling water bath until green colour of solution appears. Notice a change of an orange potassium dichromate (K2Cr2O7) into a green chromium sulphate (Cr2 (SO4)3) .
2

b. detection of fatty acids saponification reaction Transfer about 3 drops of lecithin into a test tube and supplement it with 3 ml of 10% KOH alcoholic solution. Heat the mixture in a boiling water bath for a few minutes. This procedure results in hydrolytic cleavage of lecithin. The released fatty acids are converted into potassium salts (soaps). Add 5 ml of distilled water and shake the tube. Because the presence of soap the solution covers with foam. This indicates the presence of fatty acids in lecithin. c. detection of choline Prepare a test tube containing about 3 drops of lecithin and add 2 ml of 20% NaOH. Heat the sample in a boiling water bath for 5 minutes. The action of strongly alkaline medium and high temperature results in hydrolysis of ester bond between choline and phosphate residue, contained in lecithin, with a release of free choline. The released choline decomposes with a release of ethylene glycol and trimethylamine, which demonstrates a characteristic bad smell. d. detection of phosphorus Prepare a test tube containing about 3 drops of lecithin and add 0.5 ml of 20% NaOH. Heat the sample in a boiling water bath for 2 minutes and then add 2 ml of ammonium molybdate solution. Observe an appearance of yellow colour ammonium phosphomolybdate solution. 4. Detection of cholesterol a. Salkowski reaction Transfer 0.5 ml of cholesterol (dissolved in chloroform) into a dry test tube and then introduce gently on the test tube wall about 0.5 ml of concentrated sulphuric acid (H2SO4). Two-phase system is formed. The upper (chloroform-containing) layer stains red, whereas the lower (H2SO4- containing) layer fluoresces green. b. Lieberman-Burchard reaction Transfer 1 ml of cholesterol solution (in chloroform) into a dry test tube, add 3 drops of acetate anhydride and introduce gently 2 drops of concentrated H2SO4. The solution stains red and then changes its colour into blue and finally into green. 5. Detection of bile acids a. Hay test Prepare 2 test tubes containing 3 ml of distilled water. Supplement one of them with a drop of bile. Introduce a few grains of powdered sulphur to each sample. The sulphur grains float on the surface of water, whereas in a sample containing the bile they fall down to the bottom of the test tube. b. emulsifying action of bile acids Prepare 2 test tubes containing 3 ml of distilled water and add 3 drops of olive oil to each of them. Supplement one of the samples with a few drops of bile. Shake intensively both tubes. The oil does not dissolve in water. The water-oil mixture separates into two phases. The oil locates on the top and water drops down to the bottom. A phase border is visible. In contrast to that in the sample containing water, olive oil and bile (bile acids) forms a stable emulsion.
3

6. Detection of fat-soluble vitamins Prepare 1 ml of saturated solution of antimony chloride (SbCl3) in chloroform and add 2 drops of pharmacological preparation of vitamins A+D3. The blue colour, which appears immediately after mixing, indicates the presence of vitamin A. The change of colour into a red-violet indicates the presence of vitamin D3.

Laboratory task Check, if the tested solution contains cholesterol or bile acids?