Digestion of fats. Influence of bile salts upon pancreatic lipase activity.

Laboratory work 10. Lipid metabolism

Emulsification of fats.

Emulsifiers are surface-active materials as proteins, bile salts, salts of higher fatty acids. They adsorb on the surface of fat droplets forming thin layer. Hydrophilic groups of emulsifiers are turned to the aqueous phase, and hydrophobic ones – to the fat. It results in the decrease of the surface tension on the border of two phases (water|fat), and fat droplets decompose to smaller droplets.

Emulsification of fats is very important for fat digestion in the intestine by lipase enzyme. It increases the contact surface of fat and facilitates lipase action.

Take 5 test tubes. Pour 1 ml of distilled water in the first tube, 1 ml of diluted bile in the second, 1 ml of protein solution to the third, 1 ml of sodium oleate 1% solution to the forth, 1 ml of sodium carbonate 1% solution to the fifth. Add 3-4 drops of vegetable oil into each test tube. Shake all tubes at the same time and leave them in the tube rack. Analyze the stability of resulting emulsions, the degree of dispersion.

The function of the serum albumin in the transport of higher fatty acids in the blood.

Blood always contains some amount of free higher fatty acids, which are formed as a result of enzymatic breakdown of lipids. Higher fatty acids are insoluble in water. They are transported in the blood mainly in complexes with albumins. There is a cavity formed by hydrophobic radicals of amino acids in albumin molecule. Hydrocarbon chain of fatty acids can be located in this cavity:

Pour 0.5 ml of 1% sodium oleate solution in two test tubes. Add 1.5 ml of blood serum to the first tube and 1.5 ml of water to the second. Add HCl solution to the second test tube (with water) drop by drop until precipitation of higher fatty acids. Add the same number of acid drops to the first test tube (with serum).

Digestion of fats. Influence of bile salts upon pancreatic lipase activity.

The breakdown of fats in the intestine occurs mainly with the participation of pancreatic lipase. Triacylglycerols are broken down into β-monoacyglycerol and fatty acids. Fatty acids can be detected by titration with alkali. Bile salts emulsify fats, increasing their contact surface with lipase, as well as activate lipase.

Pour 20 ml of milk in the flask, add 2 drops of phenolphthalein and neutralize by adding 10% sodium hydroxide solution drop by drop, until you see pale pink color. Using neutralized milk prepare experimental samples according to the table 1. Pancreatin drug is used as a source of lipase. The samples are left at room temperature.

Table 1. Composition of reaction mixtures

№	V of milk, ml	Mass of pancreatin, mg	V of bile, ml	V of water, ml
			-
				-
		-		-

In every 15 min from the start of the experiment, the samples are titrated with 0.1 n NaOH solution to the appearance of pale pink color. The volume of alkali solution is fixed in the table. The alkali solution volume is proportional to the amount of fatty acids which are formed from milk fat during hydrolysis by lipase in a given time (15 minutes).

Write the equation of triacylglycerols splitting by lipase. Depict graphically the dynamics of the fat splitting by lipase for each sample by plotting on the X-axis time in minutes, and the Y-axis – volume of 0.1 n solution of NaOH (in ml).

Calculate the lipase activity as the concentration of carboxyl groups of fatty acids formed in 100 ml of milk during experiment, using the following formula:

, where

C is the concentration of COOH-groups, resulting in 100 ml of milk (g/100 ml);

m_e – equivalent mass of the carboxylic group, g|mole-eq.;

0,1 is normality of a NaOH solution;

V is the volume of 0.1n solution of NaOH, used for the titration of 5 ml of milk during the experiment (the sum of all titrations), ml;

5 is the volume of milk in the titrated sample, ml.

On the basis of the curves on the graph and the received data of the concentration of carboxyl groups give a conclusion about the bile salts effect on the activity of pancreatic lipase.

Table 2. Results of titration

Sample number	Results of titration (in ml) of 0.1n. NaOH solution at intervals, min:

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