Isolation and Characterization of Complex Lipids from Fish Brain (Microscale method)

Angelo Alexan Tanguio, Justin C. Tumanan*, Venn-Irene M. Vallester and Chester Aldwin Yap Group 11, 3BIO-6 Department of Biological Sciences, College of Science, University of Santo Tomas, Manila Abstract With the use of organic solvents and filtration, the gradual separation of the lipids namely, cholesterol, glycerophosphatide and sphingolipid (sphingosine phosphatide) from the other components of the tissue resulted. Such lipids are selectively solubilized by the solvents acetone, hexane and 95% ethanol; with cholesterol being soluble in acetone, glycerophosphatide in hexane and sphingolipid in boiling 95% ethanol. After isolation, the lipids were subjected to color reaction tests to characterize the composition of the lipids and that of its similarity to the standards: lecithin, cholesterol and galactocerebroside. In the Liebermann-Burchard and Salkowski tests, only the isolated cholesterol gave the positive visible result thus, the presence of sterols. While in Ninhydrin and Krauts test, the standard lecithin and the sphingolipid yielded positive indicating presence of -amino acids and choline. In the Molisch test, lecithin, galactocerebroside, sphingolipid, glycerophosphatide were positive thus, the presence of a carbohydrate moiety. Lastly, lecithin and galactocerebroside yielded positive in the test for phosphate.

I. Terminology 1. Cerebroside is the common name for a group of glycosphingolipids called monoglycosylceramides which are important components in animal muscle and nerve cell membranes. 2. Deamination removal of the amino radical from an amino acid or other amino compound; enzymes which catalyse this reaction are called deaminases. 3. Glycerophosphatide are common constituents of cellular membranes. They have a glycerol backbone. The hydroxyls at C1 & C2 of glycerol are esterified to fatty acids. 4. Lecithin is a generic term to designate any group of yellow-brownish fatty substances occurring in animal and plant tissues, and in egg yolk, composed of phosphoric acid, choline, fatty acids, glycerol, glycolipids, triglycerides, and phospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol). 5. Sphingolipid(s) are a class of lipids derived from the aliphatic amino alcohol sphingosine and these compounds play important roles in signal transmission and cell recognition.

II. Introduction

The lipids are a large and diverse group of naturally occurring organic compounds that are related by their solubility in nonpolar organic solvents (e.g. ether, chloroform, acetone & benzene) and general insolubility in water. The word lipid is derived from the Greek word lipos meaning fat. Fats supply over twice as much energy per unit weight as proteins or carbohydrates. Lipids are anhydrous due to non-polar nature and represent more energy than carbohydrates which are heavily hydrated due to polar nature. Lipids occur in plants and animals as storage and structural components. Structural lipids present in animals and plants are in the form of meat and vegetables respectively. Storage fats occur in milk and adipose tissue of farm animals and in seed oils. Lipids also supply the essential fatty acids which are not synthesized in human beings but are essential for growth. This statement was taken from Lehninger verbatim [1]. Simple lipids are defined as those that on hydrolysis yield at most two types of primary product per mole. Complex lipids, on the other hand, yield three or more primary hydrolysis products per mole. Alternatively, the terms "neutral" and "polar" lipids respectively are used to define these groups, but are less exact. The complex lipids for many purposes are best considered in terms of either the glycerophospholipids (or simply if less accurately as phospholipids), which contain a polar phosphorus moiety and a glycerol backbone, or the glycolipids (both glycoglycerolipids and glycosphingolipids), which contain a polar carbohydrate moiety, since these are more easily analysed separately. The picture is further complicated by the existence of phosphoglycolipids and sphingophospholipids (e.g. sphingomyelin). Egg yolk and brain yield lecithin with arachidonic acid as one of its component fatty acid. The physiological importance of this fatty acid has been indicated in connection with the essential fatty acids. It is believed that lecithin formation is as intermediary stage in the metabolism of fatty acids. In the experiment, the brain from the fish Oreochromis sp. (perch or locally, tilapia) was used and the lipids isolated were cholesterol, sphingolipid and glycerophosphatide. Since lipids constitute about one-half of brain tissue dry weight, it is not surprising that lipid biochemistry and neurochemistry have evolved together. The brain contains many complex lipids, including gangliosides, cerebrosides, sulfatides and phosphoinositides, which were first discovered in brain, where they are highly enriched compared to other tissues. Phospholipids account for the high total phosphorus content of brain, which led to an alchemical mystique in the nineteenth century that associated phosphorescence with thought and to the apocryphal claim that fish

are good brain food since fish, too, is rich in phosphorus. The standards used are lecithin, cholesterol and galactocerebroside. Phospholipids are the main constituents of cell membranes. They resemble the triglycerides in being ester or amide derivatives of glycerol or sphingosine with fatty acids and phosphoric acid. The phosphate moiety of the resulting phosphatidic acid is further esterified with ethanolamine, choline or serine in the phospholipid itself. The following diagram shows the structures of some of these components. Phosphatidylcholine or "lecithin", although the term is now used more often for the mixed phospholipid by-products of seed oil refining) is usually the most abundant lipid in the membranes of animal tissues, and it is often a major lipid component of plant membranes, but only rarely of bacteria. With the other choline-containing phospholipid, sphingomyelin, it is a key structural component and constitutes much of the lipid in the external monolayer of the plasma membrane of animal cells especially. For the qualitative analysis of the complex lipids, specific reactions are used namely Liebermann-Burchard and Salkowskis test, which are both specific tests for sterols (cholesterol).Also, tests for the detection of glycerophosphatides and sphingolipids include Krauts, Ninhydrin, Molisch and test for Phosphate. The experimentation or test has the objective of isolating non-saponifiable lipids from the fish brain using the microscale method. Moreover, it has the aim of obtaining information and characterizing the nature and composition of the isolated lipids through color reaction tests.

III. Experimental

For the isolation of the complex lipids from the fish brain, the following steps were performed: Two pieces of medium-sized tilapia brain with 10.0 milliliters acetone were triturated using sand to aid in the disruption of the cells. The system was then transferred to a 25-mL vial and 10.0 mL of acetone was added to the solution. The vial was covered to ensure that no possible evaporation will take place. The solution was allowed to stand overnight in the refrigerator (this has been done two days before the extraction). Cholesterol For the extraction, the extracts was filtered and washed with 10.0 mL acetone. The residue was saved for the preparation of glycerophosphatides and other lipids. The filtrate, on the other hand, was evaporated over a steam bath. From the solution, cholesterol was crystallized. The remaining liquid was pipetted out and the crude product was collected. The cholesterol was recrystallized by dissolving it in 5.0 mL of hot 95% ethanol, filtering the solution while it is hot. The filtrate was then cooled over an ice bath allowing the cholesterol to recrystallize. The crystals were collected and dissolved in 5.0 mL of methanolchloroform mixture and characterized. Glycerophosphatide The residue from the filtrated extracts discussed earlier was transferred to another 25mL vial and extracted with 15.0 mL hexane. The solution was allowed to stand 30 minutes with occasional shaking. The product was then filtered and the residue was saved for the isolation of sphingosine phosphatides. The extract was concentrated over a steam bath in the fume hood and the concentrated extract was then poured into 10.0 mL acetone and stirred. The solution was decanted and the supernatant was discarded. The precipitate was then dissolved in 5.0 mL of methanolchloroform mixture and characterized. Sphingosine phosphatides The residue from the filtrated solution discussed earlier was transferred to a 50-mL Erlenmeyer flask and extracted with 20.0 mL of boiling ethanol. The mixture was boiled for qo minutes over a steam bath. Then, the mixture was filtered while hot and the residue was discarded.

The filtrate was cooled and the precipate formed was collected by filtering. The precipitate was then dissolved in 5.0 mL of methanol-chloroform mixture and characterized. The succeeding steps to be performed were that of the color reactions. Liebermann-Burchard test In a separate small test tube, 0.5mL of each lipid solution was added. 10 drops of acetic anhdrirde were added to each lipid solution and were gently swirled. 4 drops of conc. H2SO4 were added down side of the test tube and it was mixed well. The color produced was noted. Salkowski test In a small test tube, 10 drops of the lipid solution were added. 20 drops of conc. H2SO4 were added down side of the test tube. The 2 layers were not mixed and then the color at the interface was noted. Test for Phosphate In a crucible, 0.5mL of the lipid was mixed with the fusion mixture. It was ignited over a free flame until all organic matter is burned away and the mixture turns to a grayish or colorless fluid or a white gray ash was obtained. It was cooled and dissolved in a 3mL of warm water. The contents were transferred to a test tube and were acidified with 3M HNO3. The solution was heated to 65C.3mL of 2.5% ammonium molybdate and warmed the tube and then the color of the precipitate and color of the solution produced was noted. Krauts test 10 drops of the lipid solution were placed in a small test tube. The tube was put in a boiling water bath in the fume hood to evaporate off the solvent from the lipid solution. The dried lipid was suspended in 10 drops of distilled water. 15 drops of Krauts reagent were added. The tube was warmed for 1-2 minutes and the colour of the solution and of the precipitate was noted. Ninhydrin test 10 drops of the lipid solution were placed in a small test tube then 5 drops of nihydrin with ethanol were added. The tube was warmed for 1-2 minutes and the color of the solution was noted. Molisch test In a small test tube, 10 drops of the lipid solution were added. The tube was put in a boiling water bath in the fume hood to evaporate off the solvent from the lipid solution. The lipid was suspended in 20 drops of distilled water. 2 drops of Molisch reagent was added and the solution was mixed well. 20 drops of conc. H2SO4 were added down the side of the tube and 2 layers formed were not mixed. The color of the interphase was noted.

IV. Results and Discussion The following figures, which are presented in tabular form, show the results or sets of data obtained in such experiment.

Color Reaction Tests

Visible Results

Standard Lipid: Lecithin 1. LiebermannBurchard test 2. Salkowski test 3. Krauts test 4. Ninhydrin test 5. Molisch test

Cholesterol

Glycerolipid (Glycerophosphatide) Light yellow or yellowish solution Orange Solution Dark red Solution Yellow Solution Upper layer: Pink solution Interface: Violet ring Bottom layer: Colorless solution Light yellow precipitate in colorless Solution

Sphingolipid

Red solution Light red solution Red precipitate in orange solution Blue-violet solution Upper layer: Cloudy white solution Interface: Violet ring Bottom layer: Red violet solution Fine yellow Precipitate in colorless solution

Deep green or Blue green solution Cherry red solution Dark red solution Dark red solution Upper layer: Pink solution Bottom layer: Colorless solution Light yellow precipitate in colorless solution

Yellow solution Red interface in orange solution Red precipitate in orange solution Blue-violet solution Upper layer: Pink solution Interface: Violet ring Bottom layer: Colorless solution

6. Phosphate test

Colorless solution

Fig. 1 Visible results for the color reaction tests of the Standard Lipid (Lecithin) and Complex Lipids extracted from the Fish Brain

Color Reaction Tests

Visible Results

1. LiebermannBurchard test 2. Salkowski test

Cholesterol Deep green or Blue green solution Cherry red solution

Galactocerebroside Colorless turbid liquid solution Upper layer: Yellow solution Bottom layer: Colorless solution Light red solution with black precipitate

3. Krauts test

Black precipitate in dark red solution

4. Ninhydrin test 5. Molisch test

Light pink solution Pink solution with red precipitate

Colorless solution Upper layer: Pink solution Interface: Violet ring Bottom layer: Colorless solution Colorless Solution

6. Phosphate test

Fine yellow precipitate

Fig. 2 Visible results for the color reaction tests of the other Standard Lipids Cholesterol and Galactocerebroside

The aim of all isolation or extraction procedures done in this experiment is to separate such complex non-saponifiable lipids from the other constituents, proteins, polysaccharides, small molecules but also to preserve these lipids for further analyses. There is a great diversity of methodologies because biological tissues are not similar when considering their structure, texture, sensitivities and lipid contents. Removing the nonlipids without losing some lipids is a complex challenge, extracting some specific lipids is not always reliable for other kinds of lipids. Hence, in the course of the performance of such experiment, certain errors could have been unintentionally committed. The high sensitivity of the analytical methods needed for low amounts of extracted lipids requires the use of very pure solvents and clean glassware. Furthermore, all lipids must be protected against degradation through oxidation by solvent, oxygen, enzymes in combination with temperature and light. This statement was taken from Campbell verbatim.[1] In such experiment, the source of the specific lipids to be isolated was from the tissues of the brain of the fish Oreochromis sp. (locally, tilapia). The isolated lipids from the brain were cholesterol, sphingolipid and glycerophosphatide. As pointed out earlier, lipids are related by their solubility in non-polar organic solvents (e.g. ether, chloroform, acetone & benzene). However, such specific lipids, considering structure and nature, differ in chemical composition. Moreover, their solubility and ability to be solubilized by certain solvents also exhibit some differences and with such differences, the selectivity of solvents by the lipids would separate or isolate each lipid from the others. With this principle, the individual use of the selective solvents and filtration was applied. The lipid compound that has been first solubilized by the solvent (e.g. acetone) means that this certain lipid is cholesterol.

In the triturating of the fish brain, 10 mL of acetone was used and also sand for the initial disruption of the cells. As the extracts of the brain are obtained, the filtration process is employed to the mixture composed of the brain extracts, sand and acetone. The mixture is then separated into filtrate and residue. With this procedure, the cholesterol component of the mixture has already been isolated as the filtrate. The selective solvent for which was then acetone that was used in the extraction discussed earlier. The solvent acetone solubilizes sterols such as cholesterol. For the crystallization of the cholesterol, the filtrate was evaporated. Recrystallization was done after the crude product was obtained with the use of 95% ethanol and cooling over an ice bath. This is to avoid immediate precipitation. For the isolation of the glycerophosphatide, the lipid was extracted from the residue of the brain extracts that were filtered. The residue was added with acetone to further solubilize or dissolve the cholesterol left in the residue. This was again filtered to separate the filtrate that has the remaining cholesterol. The resulting residue becomes the source of the glycerophosphatide and the lipid was extracted by adding its selective solvent which is hexane. This was again filtered and the resulting filtrate was concentrated over a steam bath, the resulting crystals of which were glycerophosphatides. For the isolation of the sphingolipid, the resulting residue from the previous filtration now residue becomes the source of the sphingolipid and the lipid was extracted by adding its selective solvent which is 95% ethanol. This was again filtered and the resulting filtrate is cooled and the sphingolipids are isolated. The isolated lipids from the fish brain were subjected into different characterization tests namely: Liebermann-Burchard, Salkowski, Krauts, Ninhydrin, Molisch and test for Phosphate.

Fig. 3 Structure of the lipid cholesterol

Cholesterol gives characteristic color reactions with the Salkowskis and LiebermannBurchard test. This statement was taken from Lehninger verbatim [3].

In the observation of visible color reaction of the lipids for the Liebermann-Burchard test, both the standard lipid cholesterol and the cholesterol extracted from the fish brain were observed to have given the positive reaction for such test. The Lieberman-Burchard or acetic anhydride test is performed for the detection of the presence of sterols. This statement was taken from Lehninger verbatim [1]. For the positive visible result, the formation of a green or blue green color as the solution is subjected to the reagents is considered.

Included reagents in the Lieberman-Burchard test are acetic anhydride and conc. H2SO4. The test is used in a colorimetric test to detect cholesterol, which gives a deep green color. This color begins as a purplish, pink color and progresses through to a light green then very dark green color. The color is due to the hydroxyl group (-OH) of cholesterol reacting with the reagents and increasing the conjugation of the un-saturation in the adjacent fused ring.

Furthermore, the principle for such reactions involves the esterification of OH at carbon 3 with acetic anhydride and also the epimerization of double bond at carbon 5.This statement was taken from Boyer verbatim.[3]

Cholesterol is by far the most common member of a group of steroids in animal tissues; it has a tetracyclic ring system with a double bond in one of the rings and one free hydroxyl group. It is found both in the free state, where it has an essential role in maintaining membrane fluidity, and in esterified form, i.e. as cholesterol esters.

The Salkowski reaction is a test for cholesterol; when concentrated sulfuric acid is added to a chloroform solution of cholesterol, the chloroform layer shows a cherry red-colored solution and the acid layer shows a green fluorescence. The reaction is a specific test for the

presence of cholesterol as presented in the sets of data in the figure above. Such reaction could be explained by the principle involved which is the addition followed by condensation.

Only the lipid cholesterol gave a positive visible result that is a cherry red solution. The reagents used are CHCl3 (chloroform) and conc. H2SO4 (sulfuric acid).

Fig. 4 Structure of Phosphatidyl choline (Lecithin)

Fig. 5 Structure of Sphingomyelin (Sphingolipid)

Krauts test for lipids is positive for the standard lecithin used in the experiment and the sphingolipid isolated from the fish brain as presented in the figure 1 above. A red precipitate in orange or red orange solution was observed for the two samples. The reagents used are KI and bismuth subnitrate. The presence of choline gives a dark orange-colored solution or red precipitate. The principle involved would be that of the complexation reaction and it is positive for lecithin and sphingomyelin. This statement was taken from Boyer verbatim.[3] Lecithin (Phosphatidyl choline) is present in great quantities in the egg yolk, liver and nervous tissues.

The test for the presence of -amino acids is the Ninhydrin test. A blue-violet solution was observed for the standard lecithin used in the experiment and the sphingolipid isolated from the fish brain as presented in the figure above while the rest yielded other colors. This means that only the two were positive in the reaction.

The principle involved in this test is oxidative deamination followed by decarboxylation and/or condensation. The reagent used is triketohydrindene hydrate. Such test or reaction is positive for cephalins, lecithin and sphingomyelin.

Fig. 6 Structure of Galactocerebroside

In the Molisch test, the detection of the lipids with the carbohydrate moiety is performed. In the experiment, the standard lecithin gave the visible result of a cloudy white solution in the upper layer, a violet ring as an interface and the bottom layer was a red violet solution.

The standard galactocerebroside and the isolated lipids glycerophosphatide and sphingolipid also yielded a violet ring as an interface which is then the positive result for such test. The principle involved in this test is oxidative deamination followed by decarboxylation and/or condensation. The principle involved in this test is dehydration and condensation and also hydrolysis. The reagents used are alpha-naphtol, 95% EtOH (ethanol) and conc. H2SO4 (sulfuric acid).

In the test for phosphate(s), the presence of the free phosphate in acidic solution can be detected by adding a molybdate to the solution. The equation below illustrates the pertinent reaction between phosphate and ammonium molybdate solution in the presence of nitric acid (HNO3).

Fig. 7 Reaction between phosphate and ammonium molybdate solution in the presence of nitric acid (HNO3). Yellow precipitate results from the reaction in the mixture. When lipids containing phosphate groups in their structures are added to the strong acid solution such as the solution

used, the lipid hydrolyses, producing the free phosphate, forming a yellow precipitate. Based on the sets of data in the figure 1 above, the standard lecithin and galactocerebroside gave fine yellow precipitate as their visible result while isolated lipids cholesterol and glycerophospatide had the visible results of colorless solution with light yellow precipitate. These lipids are the ones that are positive for such test which indicates that such lipids has the presence of free phosphates in the acidic solution. The four lipids yielded positive because of the presence of the yellow precipitate but the visible result of a finer yellow precipitate is more precise as the basis of the presence of free phosphates.

V. References Boyer, Rodney (2006). Modern Experimental Biochemistry, 3rd edition. John Wiley & Sons: San Francisco, CA Campbell, Mary (2008). Biochemistry, 6th edition. Brooks/Crole: Canada. Lehninger, A.L. (2008). Principles of Biochemistry. W. H. Freeman: New York.