Experiment on Lipids D.

INFLUENCE OF BILE ON THE ACTION OF LIPASE Objectives To determine the effect of lipase and bile in lipid digestion Materials 5 test tubes Olive oil Neutral pancreatic extract Distilled water Bile

Incubator Iron stand Biuret Phenolphthalein

Methodology Five test tubes were prepared and labeled 1 to 5. The following were added to the test tubes: Test tube 1 0.5 ml olive oil, 5 ml neutral pancreatic extract, 4.5 ml of water Test tube 2 0.5 ml olive oil and 9.5ml water Test tube 3 - 0.5 ml olive oil, 8.5 ml of water and 1 ml bile Test tube 4 - 0.5 ml olive oil, 5 ml neutral pancreatic extract, 3.5 ml water and 1 ml bile Test tube 5 5 ml neutral pancreatic extract, 3.5 ml water and 1 ml bile The solutions were shaken thoroughly and incubated for an hour. With phenolphthalein as an indicator, the 5 solutions were titrated with 0.5 NaOH until they reach a permanent pink solution. Results: Test Tube No 1 2 3 4 5 Discussion: Lipids in the diet are usually in the form of triglycerides which are not readily absorbed in the body. This form of macromolecule must first be broken down or digested into its monomeric form to be absorbed and utilized by the body. For lipid absorption to take place two processes must occur: emulsification and enzyme catalysis. In the experiment, the olive oil was used as the substrate. Olive oil is composed mainly of triacylglycerols, as well as small amounts of free fatty acids, glycerol and phosphatides. The absence of olive oil in the fifth tube explains why it required less titrant compared to fourth tube. A substrate, on which the enzyme will act on, is of course needed for digestion to take place. Emulsification is a process by which large aggregates of lipid in the diet are physically broken down into smaller droplets with the aid of Contents 0.5 ml olive oil, 5 ml neutral pancreatic extract, 4.5 ml of water 0.5 ml olive oil, 9.5ml water 0.5 ml olive oil, 8.5 ml of water, 1 ml bile 0.5 ml olive oil, 5 ml neutral pancreatic extract, 3.5 ml water, 1 ml bile 5 ml neutral pancreatic extract, 3.5 ml water, 1 ml bile ml of NaOH 0.2 0.4 2.0 4.0 2.0

Figure 1. Action of Bile

bile (or bile salts). It is needed for lipid digestion since lipase, the enzyme which catalyzes lipids, can only act on the surface of the substrate, hence, and requires a longer time for a complete digestion. To optimize the enzymes activity, bile salts are required to decrease the surface area of the substrate on which the enzyme can readily act upon. The amphipathic nature of bile allows it to perform this function (See action of bile in figure 1). Bile acids are synthesized in the liver as cholesterol derivatives, and are stored in the gall bladder. They are released in the presence of fat. In the experiment, the absence of bile was analyzed through comparing test tube 1 and 4 (only the latter contains bile). More alkaline solution was needed to produce a pink color in the solution with bile compared to that without (4mL for test tube 4 while only 0.2 mL for test tube 1). This means that more fatty acid was produced in the presence of bile, hence requiring more titrant to make the solution basic. The absence of bile reduced the rate of lipid digestion since emulsification cannot take place. However, it should be noted that despite the absence of bile, digestion can still take place, though very minimal since rd lipase is present. Theoretically, the 3 test tube with no lipase (but with bile) should require less titrant st than the 1 tube since no fatty acids can be produced without lipase. However since bile are acidic in nature (may be present as bile acids or bile salts), it may have contributed to the resulting pH of the solution. (This may be a limitation of the experiment since fatty acids should only be the only source of hydrogen ions.) After emulsification, the substrate is acted upon by lipase in the process of hydrolysis. Lipase is an enzyme that is specific for the catalysis of lipids or triacylglycerols. It is secreted by the salivary glands (lingual lipase), stomach (gastric lipase) and pancreas (pancreatic lipase). The experiment utilized extract from pigs pancreas as the source of the lipase. Pancreatic lipase is the most efficient among the three type of lipase, being responsible for digesting most (amost 80%) of the lipids in the diet. The enzyme hydrolyzes triacylglcerols into monoglycerides and free fatty acids in the following reaction: Triacylglycerol + H2O diacylglycerol + a fatty acid anion

The concept of the fatty acids production in lipid digestion was used to determine indirectly the presence or absence of the said process. In the experiment, titration was employed to determine the pH of the solution. Phenolphthalein (a color indicator that is colorless on acidic solution but turns pink in basic solution) was also added in the solution prior to titration to detect the changes in pH during titration. Since one of the final products of the process is fatty acid, its presence will cause a decrease in the pH of the solution which will be detected in the titration. Hence, the volume of base needed to neutralize and make the solution basic (turning it to pink) reflects the presence of the fatty acids, and in turn the lipid digestion. The higher the volume of titrant used means that there is a higher rate of digestion that took place in the solution. The action of lipase was observed indirectly in the experiment. The fourth tube produced a more acidic solution than the third one due to the presence of lipase. Theoretically, lipid digestion should not have occurred in solutions without lipase such as in the second and third tube, and should have the required the least amount of titrant. However, as mentioned the bile, which may be in acid form, may have affected the pH. Discrepancy in the result might have also been aggravated by instrumental error (especially the defective biuret) and human error (subjective interpretation of the color of the solutio) as well. Conclusion Lipid digestion requires the presence of the substrate (triacylglycerols), lipase (enzyme) and bile (emulsifier) to take place. References: Bowen, R. (2007, August 8). Absorption of Lipids. Retrieved December 5, 2011, from Hypertexts for Biomedical Sciences: http://www.vivo.colostate.edu/hbooks/pathphys/digestion/smallgut/absorb_lipids.html Koeppen, B. M., & Stanton, B. A. (2010). Berne and Levy Physiology, 6th Edition. Mosby.

Olive Oil Chemical Characteristics. (n.d.). Retrieved December 5, 2011, from Olive Oil Source: http://www.oliveoilsource.com/page/chemical-characteristics Pancreatic Lipase. (n.d.). Retrieved December 5, 2011, from Wikipedia: http://en.wikipedia.org/wiki/Pancreatic_lipase Phenolphthalein. (2011, November 20). Retrieved December 5, 2011, from Wikipedia: http://en.wikipedia.org/wiki/Phenolphthalein