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BIOCHEMISTRY 601 LABORATORY FORMAL REPORT

Isolation and Characterization of Complex Lipids from Egg Yolks


Peafuerte, Noel S., *Pimentel, Maria Danica B. Group 20 Department of Chemistry, College of Science University of Santo Tomas, Espaa, Manila 1008

Abstract. In this experiment, the lipids found in the egg yolks of chicken egg were isolated with organic solvents. The isolated lipids are then separated into two classes: phosphorylated and non-phosphorylated with the use of acetone. The two classes of lipids solution were characterized with various chemical tests: Libermann-Burchard, Krauts, Salkowski, test for phosphate, ninhydrin and Molisch test. The isolation of lipid was successful however the separation into two classes is not that successful since there were a lot errors observed in the various chemical test.

INTRODUCTION Lipid ,from the Greek word lipos which means fat, are low molecular weight biomolecules and nonpolar chemical substances that can be extracted from plant, microbial, and animal tissues by organic solvent. It can be found in most cells and tissues, but rarely exists as free or uncombined state (Boyer, 2000). They are usually bounded to proteins and polysaccharides of tissues in complexes of widely varying degrees of stability. Because of their association with proteins and carbohydrates, it is complicated to extract and to identify the structure of lipids. Lipids have a wide variety of molecular structures and involved in various biological functions but have similar properties. Lipids are insoluble in water and ordinary solvents but soluble in organic solvents. Most lipids are ionic or polar derivatives of hydrocarbon belonging to the amphiphiles. They have ionic or polar groups which are hydrophilic and nonpolar hydrocarbons which are hydrophobic. The more polar the lipid is the stronger amphiphiles it is (Cabatit, 1988) One source of lipid is food. Food contains fats, complex lipids and steroids. Fats are triglycerides, esters of fatty acids and glycerol. Complex lipids also contain fatty acids but their

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alcohol may be either glycerol or sphingosine. The fatty acids also contain other constituents such as phosphate choline or mono- to oligo- saccharides (Bettelheim, 2001). Complex lipids are usually more stable than those from simple lipids because of the ionic and polar attractions involved (Clark, 1977). Sterols are derivatives of a cyclopentanorphenanthrene nucleus or simple sterid nucleus. The sterid nucleus is a combination of cyclopentane and perhydrophenanthrene rings (Figure 1). If the compound has one or more hydroxyl groups and no carbonyl or carboxyl group then it is a sterol. On the other hand, if it has one carbonyl or carboxyl group then it is a steroid (Cabatit, 1988).

Figure 1. Structure of cyclopentanoperhydrophenanthrene In this experiment the source of lipids is a chicken egg. In an egg, about 11% by weight is made up of lipids found in the egg yolk (Todd, 1979). The lipids in the egg yolk are lipoprotein in the native state (Clark, 1979). Chicken eggs have a consistent composition of its lipids. The little variation is due to the strain and diet the chicken have. The eggs lipid composition have approximately 62% if triglycerides, 33% of phospholipids, and 5% cholesterol. The cholesterol is 84% exists as free cholesterol and 16% as cholesterol ester. The phospholipids are 65% lecithin, 20% cephalin and various minor components (Todd, 1979). Egg lipids can be divided into two general classes: non-phosphorylated and phosphorylated or phospholipids.

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Phospholipids contain a nitrogenous base and phosphoric acid. It is also called phosphatids or phosphorized fats. It is an important component of membrane lipids. Lipids can be classified as saponifiable and non-saponifiable lipids. Saponifiable lipids are triglycerides, waxes, phospholipids, and sphingolipids which are esters that can be hydrolyzed under basic conditions. These first types of lipids are derivatives of fatty acids. Nonsaponifiable lipids such as isoprenoids and eicosanoids belong to this type because they are not esters and cannot be hydrolyzed (Seager and Slabaugh, 2005). The chickens egg yolk contains saponifiable and non-saponifiable lipids. The saponifiable lipids presents are lecithin, cephalin, sphingomyelins, cerebrosides, palmitin, stearin, olein, and small amounts of linoleic acid. The only non-saponifiable and most abundant lipid in egg yolks is cholesterol. Vitamins A, D, and B complex are also present if the feed fed to the chicken contains these vitamins. The yolks also contain inorganic substances like sodium and potassium chlorides, iron, and few amounts of calcium and magnesium phosphates (EspinoCabatit, 1978). Two standard lipids were used in this experiment. They are lecithin and cholesterol. Lecithin is also known as phospatidyl choline. It is present in great quantities in egg yolk, liver and nervous tissues. Lecithin (Figure 2) is a phospholipid that has choline as a nitrogenous base. It can exists in alpha or beta form. The -lecithin is asymmetric while the -lecithin is symmetrical. Egg yolk yields lecithin with arachidonic acid as one of its component fatty acids (Cabatit, 1988). Its effects on the body are: decreases the blood pressure, slowing of the heart stimulation of gastric and intestinal peristalsis, and increase of salivary secretions. Cholesterol is an unsaturated alcohol (Figure 3). It is a sterol and is widely distributed in all cells in the body, especially the nervous tissues. It can be synthesized from small fragments like acetic acid. It

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serves as an insulator in the central nervous system where it exists in its free state. (Cabatit, 1988).

Figure 2. Structure of Lecithin

Figure 3. Structure of Cholesterol

In this experiment, the lipids in egg yolks is isolated and separated into phosphorylated and non-phosphorylated lipids. The isolated lipids are characterized with various chemical tests. These tests are Libermann-Burchard test, Salkowski test, test for Phosphate, Krauts test, Ninhydrin test and Molisch test. EXPERIMENTAL I. Isolation of Complex Lipids An egg was cracked and the egg yolk was separated from the egg white. The egg yolk was placed in a clean 250-mL beaker. The yolk was stirred with 80mL of a solvent mixture (CHCl3:CH3OH, 2:1, v/v). The mixture was allowed to stand for 10 minutes. The mixture was then filtrated through a filter paper. The filtrate was placed in a graduated cylinder to measure its volume. The filtrate is then placed in a separatory funnel and was extracted with equal volume of 1% NaCl solution. The organic layer (bottom layer) is separated from the aqueous layer. The aqueous layer was discarded. The organic layer is placed in a graduated cylinder and its volume measured. It was then placed in a

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separatory funnel and was extracted with equal volume of 1% NaCl solution. The organic layer is separated and dried with anhydrous Na2SO4. The mixture was filtered off into a clean Erlenmeyer flask. A pinch of hydroquinone was added and transferred into an evaporating dish. The solution was evaporated to dryness over a beaker of warm water in the hood. The sticky yellow residue was added with 15mL of acetone and cooled in an ice bath for about 15 minutes. The Acetone solution is carefully decanted through a filter paper and the filtrate was collected in a clean Erlenmeyer flask. The precipitate or residue was washed with 5mL of cold acetone. The solution was decanted and filtered. The residue is kept for used later on. The acetone solution was evaporated to dryness in a water bath in the hood. The residue is dissolved in 3mL of solvent mixture and added with a pinch of hydroquinone. The solution is transferred into a test tube and labeled as non-phosphorylated lipids (NPL). The residue kept earlier is dissolved in 3mL of solvent mixture and a pinch of hydroquinone was added. The solution is transferred into a test tube and labeled as phosphorylated lipids (PL). II. Characterization of the Isolated Complex Lipids The isolated lipids were characterized with the following tests. Cholesterol and lecithin also underwent the following tests to serve as standards. A. Libermann-Burchard test An amount of 0.5mL of each of the isolated lipid and standards were placed in a separate test tube. Ten drops of acetic anhydride was added to each test tube and was gently swirled. Four drops of concentrated sulfuric acid (H 2SO4) was carefully added to each test tube and was mixed well. The color produced was noted.

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B. Salkowski test Ten drops of the lipid solutions and standards were placed separately in small test tubes. Twenty drops of concentrated H2SO4 was carefully added down the side of the tubes to the solutions. The color of the interphase was noted. C. Test for Phosphate In a crucible, 0.5mL of the phosphorylated lipids was mixed with fusion mixture (5 times its bulk). The mixture is ignited over a free flame until all the organic matter is burned away and the mixture turned into a grayish or colorless liquid or a white or gray ash is obtained. The mixture was allowed to cool and dissolved in 3mL of warm water. The solution is transferred to a test tube and acidified with 3M nitric acid. The solution was heated to 65C. An amount of 3mL of 2.5% ammonium molybdate was added and the solution was warmed. The color of the solution and precipitate was noted. The same procedures were done for non-phosphorylated lipid solution and the standards solutions. D. Krauts test Ten drops of the lipids solutions and standards were placed separately into small test tubes. The test tubes were placed in a boiling water bath in the fume hood to evaporate the solvent. The dried lipid is suspended in 10 drops of distilled water. In each of the test tube, fifteen drops of Krauts reagent was added. The test tubes were warmed for about 1 t0 2 minutes. The color of the solution and precipitate was noted.

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E. Ninhydrin test Ten drops of the lipids solution and standards were placed into separate small test tubes. Five drops of ninhydrin in ethanol was added to each of the test tube. The solutions were warmed for 1 to 2 minutes and the color of the solutions was notes. F. Molisch test Ten drops of the lipids solution and standards were placed into separate small test tubes. The test tubes were placed in a boiling water bath in the fume hood to evaporate of the solvent. The dried lipid is suspended in twenty drops of distilled water. Twenty drops of concentrated H2SO4 was slowly added to the side of the tube. The color of the interphase was noted.

RESULTS AND DISCUSSION Egg yolk contains lipids such as triglycerides, phospholipids, and cholesterol. The lipids in the egg are isolated with the use of a solvent mixture which is a mixture of chloroform and methanol (2:1). This solvent mixture is improvised by Folchs group. The lipids are hard to isolate since they do not occur as free molecules and are covalently bonded to proteins or carbohydrates. Lipids are generally less polar than other cell constituents. This is why organic solvent can be used to extract lipids. Lipids are also insoluble in water. The solvent mixture causes the non-lipid components to transfer to the solvent system partly by ionic interactions. This can also denature proteins. This solvent can extract most of all lipids found in the egg yolk. Some of the extracted lipids form lipid-proteins complexes (Clark, 1977). The solvent mixture dissociates the lipid-protein complexes in plasma membrane in the yolk but the solvent mixture

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has a tendency to dissolve some non-lipid molecules such as proteins (Boyer, 2000). Phospholipids are polar lipids which is why polar solvent is used to extract this. The solvent mixture is also chosen since it is inexpensive, have relatively low boiling point, non-toxic and nonflammable. When the solvent mixture is mixed with egg yolk, it was allowed to stand and filtered off. The residue is the denatured proteins while the filtrate contains the solvent mixture and the extracted lipids. The filtrate is placed in the separatory funnel which will be extracted with 1% sodium chloride solution (NaCl). Sodium chloride is an inorganic salt. Inorganic salts in aqueous solution extract the lipids that are non-covalently attached to proteins or carbohydrate. The sodium chloride solution extracts the non-lipid components present in the filtrate. This inorganic solution disturbs the noncovalent bond between proteins and lipids so that the lipids will be the only one to be extracted (Switzer and Garrity, 1999). Multiple extractions were done for more efficient extraction of lipids. If single extraction was done, some of the lipids may remain in the aqueous layer. The solvent mixture is less dense than water which is why it is in the bottom layer. The aqueous layer consists of the water soluble components in the sample while the organic contains the lipids. The organic layer is added with anhydrous sodium sulfate (Na 2SO4). The anhydrous sodium sulfate traps and removes water molecules present in the organic layer. The solution is filtered off to remove the hydrated sodium sulfate. The filtrate is added with hydroquinone. Hydroquinone is an anti-oxidant. It is necessary for an anti-oxidant to be added since lipids can auto-oxidize upon exposure to air or sunlight. If lipids are exposed to air or sunlight, the unsaturated fatty acid chain of lipid would react with oxygen and cleave double bonds forming aldehydes or if further exposed, it will form carboxylic acids (Scheve, 1984). Hydroquinone is first oxidized before the lipid is oxidized. Only a small

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amount of hydroquinone is needed. If too much is added, it may serve as impurities. The solution is then evaporated dryness in a evaporating dish in a warm water bath in the hood. This is done to remove or evaporate the solvents. This process is done under the hood since the solvent may be harmful if it is inhaled. The residue left from the evaporation of the solvent is added with cold acetone. Acetone is used to separate the phospholipids from the non-phosphorylated lipids. Acetone is provides a mild but rapid method of dehydrating tissues and as the water content decreases the acetone extracts fats, sterols and other simple lipids. Complex lipids are relatively insoluble to acetone and are converted to friable powder (Clark, 1977). Phospholipids are more polar than nonphosphorylated lipids. Acetone extracts the non-phosphorylated lipids since acetone extracts non-polar and hydrophobic lipids (Boyer, 2000). The solution is filtered off. The residue contains the phospholipids while the non-phosphorylated is in the filtrate. The residue is mixed with solvent mixture. It is done to make the lipid into a solution and hydroquinone was added so that the sample wont oxidize when it is kept. This serves as the phospholipids solution. To the filtrate, it is again evaporated in the hood then added with solvent mixture and hydroquinone. This solution serves as the non-phosphorylated lipid solution. The appearance of the isolated lipid solution was noted (Table 1).

Table 1. The appearances of the isolated lipid solutions obtained in the experiment. SAMPLE Non- Phosphorylated Lipids (NPL) Phospholipids (PL) OBSERVATION Orange- yellow solution Yellow solution

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The isolated lipid solution as well as two standards, namely cholesterol and lecithin, underwent various chemical tests to determine its properties. These chemical tests are Libermann-Burchard test, Salkowski test, test for Phosphate, Krauts test, Ninhydrin test and Molisch test. These tests are color reactions. The Libermann-Burchard test is a specific test for the detection of cholesterol, unsaturated steroids or sterol. It is also known as acetic anhydride test since this tests uses acetic anhydride. If cholesterol is present, a deep green color will be observed. The color begins as purplish-pink color and progresses through a light green then to a very dark green solution. The color is due to the hydroxyl group (-OH) of cholesterol reacting with the acetic anhydride and concentrated sulfuric acid and the increasing conjugation of the unsaturation in the adjacent fused ring (Bettelheim, 2001). The acetic anhydride is for the acetylation of the hydroxyl group of cholesterol located at c-3 then when it is reacted with concentrated sulfuric acid, it undergoes sulfonation and the addition of unsaturation yielding polyenes, aromatic steroids and rearrangement of cholesterol molecule. This gives the intense color observed in the experiment. If water is present, the test doesnt show the deep green color instead it shows a red to dark red solution (Espino-Cabatitt, 1978). This test is not only used as a qualitative test but also as a quantitative test. The concentration of cholesterol is determined by the intensity of the color. This test is performed in the standard and sample solutions and the color is noted (Table 2). In the experiment, the cholesterol produced a deep blue green solution which expected since this test is specifically for this. In the lecithin sample, red-violet solution is observed since some water maybe present in the sample. In the non-phosphorylated lipid, a gray-violet solution is observed that is a negative result. In the phosphorylated lipid, a dark brown solution is observed which is negative for this test but it indicates that water is present in the sample. There is an error

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in this test since in the non-phosphorylated lipid solution it should be positive. Cholesterol is a non-phosphorylated lipid; it should have been present in the non-phosphorylated lipid solution. The second test performed is the Salkowski test. This test is similar to LibermannBurchard test since it is a specific test for cholesterol. This taste is named after Leopold Salkowski, a German physiological chemist. In this test, only sulfuric acid was added in a chloroform solution of the samples. In the presence of the acid, dehydration occurs forming a bisteroid which gives a red color interphase (Espino-Cabatit, 1988). The red interphase can also be blue. This interphase is observed in the chloroform layer while in the acid layer a green fluorescence. This test is done in all the samples and the color of interphase was noted (Table 2). In the cholesterol sample, a red interphase is observed which is expected. In the lecithin sample, a red interphase was also observed. In the non-phosphorylated and phosphorylated lipids, both showed dark-brown interphase which indicates that they are positive for cholesterol. In the lecithin, there was an error since it should have been negative. It is clear that lecithin is not cholesterol. In the phosphorylated lipid, there is also an error since it should have been negative. Cholesterol is a non-phosphorylated lipid. The third test done is Krauts test. Krauts test is a modification of Dragendroffs test which is a test for alkaloids, pseudo alkaloids, and false alkaloids. The reagent used in this experiment is Krauts reagent which is bismuth subnitrate with potassium iodide in 3M nitric acid. When Krauts reagent reacts with a phospholipid, complexation involving the phosphorylated lipid occurs. This reaction gives a brick red precipitate. It also determines the presence of choline. Choline with bismuth potassium iodide undergoes a complexation reaction which also gives a brick red precipitate. This test is done in the samples and the color of the solution and its precipitate is noted (Table 2). In the cholesterol standard, a red orange solution

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and brown precipitate is observed since cholesterol is a false alkaloid. The cholesterol standard is a positive result. In the lecithin sample, a red orange precipitate was observed which is negative for this test. In the non-phosphorylated sample and phosphorylated sample, a red orange solution is observed as well as brown precipitate. Both of these samples are positive for false alkaloids or cholesterol. There is an error in the phosphorylated sample since cholesterol should not be present in this sample. The fourth test is the test for phosphate. It is a test that determines the presence of phospholipids. The samples were mixed with fusion mixture, a mixture of potassium nitrate and sodium carbonate (3:1), and combusted under an open flame. This is done to remove all carbon components and retain the phosphate in addition with water. The solution is also acidified with 3M nitric acid. The solution is acidified so that the ammonium hydroxide is converted to phosphate since the nitrogen is liberated. The solution is heated till 65C. After heating, 2.5% ammonium molybdate is added which reacts with phosphate to form ammonium phosphate molybdate which gives a light yellow to yellow green solution. The color is due to the oxidation of phosphate forming yellow to green solution depending on the concentration (Espino-Cabatit, 1978). This test is done on the samples and standards. The color of the solutions was noted. In the sample of cholesterol and non-phosphorylated lipid, they are clear solution with black or brown precipitate, this precipitate maybe residue of carbon. These samples are negative for the presence of phosphate group or are not phospholipids. In the sample or lecithin and phosphorylated, a light yellow solution is observed which is a positive test and indicates that the samples contain a phosphate group or phospholipids. Ninhydrin test is a chemical test that detects ammonia, primary and secondary amines. This test indicates if an amino acid is attached to the lipids. Most amino acid reacts with

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ninhydrin except proline and hydroxyproline. It is an oxidative deamination and decarboxylation reaction of the free serine group with ninhydrin in ethanol upon heating would yield a blue violet solution (Espino-Cabatit, 1988). The reagent ninhydrin is a strong oxidizing agent and reacts with the -amino acids. The color produce is purple. This test is only positive for cephalin because it is the only lipid with the free serine group. The solution was heated to catalyze the reactions. This test is done on the samples and standards. The color of the solution was noted (Table 2). In the cholesterol sample, a colorless solution is observed. This indicates that cholesterol doesnt contain cephalin. In the lecithin test, a purple solution is observed which indicates the present of cephalin. In the non-phosphorylated lipids, a red solution was observed which indicates that no presence of cephalin. In the phosphorylated lipid, a purple solution was observed which indicates the presence of cephalin. Molisch test is chemical test that indicates the presence of carbohydrates or sugars. It is named after an Austrian botanist named Hans Molisch. This test is very sensitive and can detect the presence of carbohydrates in dilute solution as low as 0.001% that will give a definite positive result (Espino-Cabatit, 1975). It is the hydrolysis of the glycosidic bonds present in the sample to convert them into monosaccharides then it is converted to furfurals. Then the furfural or its derivatives is condensed with two molecule of phenol from the Molisch reagent. Molisch reagent is -naphthol (Figure 4) dissolved in ethanol. The final product would be red or purple colored interphases. This test was done in the sample and the color of the interphase was noted (Table 2). In the cholesterol sample, the interphase was colorless which indicates that cholesterol is not a carbohydrate or sugar is present. In the lecithin sample, a purple interphase was observed which means this sample contains carbohydrates or sugars. In the non-phosphorylated and

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phosphorylated sample, a brown interphase was observed. This could be a positive result since the color brown is near to the color red which a positive result is.

Figure 4 Structure of -naphthol. Table 2. The results in the various chemical tests performed in the standards and samples. OBSERVATION TESTS Liebermann - Burchard Salkowski Krauts STANDARDS Cholesterol Blue-green solution Red interphase Red-orange solution and brown precipitate Clear solution and brown precipitate Colorless solution Colorless interphase Lecithin Red-violet solution Red interphase Red-orange solution and orange precipitate Light yellow solution Purple solution Purple interphase SAMPLES Nonphosphorylated Gray-violet solution Dark-brown interphase Phosphorylated Dark brown solution Dark-brown interphase

Red-orange Red-orange solution and solution and brown precipitate brown precipitate Clear solution and black precipitate Red solution Brown interphase Light yellow solution Purple solution Brown interphase

Phosphate Ninhydrin Molisch

CONCLUSION The isolation of lipids from chickens egg yolk was successful. On the other hand, the separation into two classes was not that reliable. The two classes were not separately properly which is why some of the tests have errors. The possible sources of errors are human errors, technique, the reagent (e.g. the acetone might not be cold enough), time pressure, etc.

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REFERENCES Bettelheim, F. A. Laboratory experiments for organic and biochemistry. Forth Worth : Saunders College. (2001). Pages 175-176. Boyer, R. Modern experimental biochemistry. San Francisco : Addison-Wesley. (2000). Pages 303-305. Cabatit, B. E. Biochemistry. 9th Ed. Manila: UST Press.(1978). Cabatit, B. E. Biochemistry. Manila : University of Santo Tomas Press.(1988). Pages 89-123. Clark, J. M. Experimental biochemistry. San Francisco : W. H. Freeman, (1977). Pages 47-48. Karlson, P.(1963) .Introduction to modern biochemistry. New York : Academic Press Lehninger, A. L. Biochemistry : the molecular basis of cell structure and function. New York : Worth Publishers, (1975). Pages 287-290 McKee, T. and McKee, J.R. (2009). Biochemistry: The Molecular Basis of Life. 4th Ed. Madison Avenue, NY: Oxford University Press, Inc. McGilvery, R.W. Biochemical concepts. Philadelphia : Saunders. (1975). Page 499. Nelson, D. L. Lehninger principles of biochemistry. New York : Worth Publishers (2000). Pages 383-384. Scheve, L. G. (1984). Elements of Biochemistry. 7 Wells Avenue, Newton, Massachusetts: Allyn and Bacon, Inc. Seager, S.L. and Slabaugh, M.R. (2005). Organic and Biochemistry for Today. 5th Ed. Thomson Learning, Inc. Switzer, R. L. Experimental biochemistry. New York : W.H. Freeman. (1999). Pages 186-188. Todd, D. Experimental organic chemistry. Englewood Cliffs, N.J. : Prentice-Hall.(1979). Pages 62-63, 66-67. USA: 198

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