Bernard J. White and Carl L. Tipton Iowa State University
Egg
Ames, 50010
Mary Dressel Winona State College Winona, Minnesota 55987
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In the selection of
a
compound for
A biochemical laboratory project
use
in the study of
lipid structure and properties, lecithin is an ideal choice. Familiar because of its frequent use in food products, lecithin is easily isolated by techniques which are fundamental to lipid studies. It is the most prevalent member of the
phosphatide class of lipids in animals and is known to have a role in numerous important cellular activities. Improved methods of separation and identification of these compounds are now available and can be expected to stimulate further studies which will provide more complete information on the biological functions of phosphatides. Modern chromatographic techniques have revolutionized lipid biochemistry and have essentially replaced the older methods of analysis on lipid mixtures. Highly specific enzymes have also found increasing use in determining the details of lipid structure. We present here an undergraduate laboratory project involving lecithin which integrates these two general aspects of lipid methodology: chromatographic techniques and the use of enzyme specificity to obtain structural information. The lecithins (choline phosphoglycerides) of egg yolk are first identified and isolated by thin-layer and column chromatography. Then, by sequential hydrolysis involving snake venom enzyme and base, the constituent fatty acids of the lecithin can be obtained. The final objective of the procedure is to determine the fatty acid composition at positions 1 and 2 of lecithin by gas-liquid chromatography.
Background Glycerophosphatides are a class of complex lipids which may be considered as derivatives of phosphatidic acid in combination with a nitrogenous base, such as serine, ethanolamine, or choline, or an alcohol such as inositol or glycerol. The fatty acids of glycerophosphatides are generally 12-22 carbon atoms in length and may be either saturated or unsaturated. o H I! HCOCR O |
o H
II
HCOCR O I I!
R'COCH
HCOPOH
R'COCH
HCOPOCHXH.NWCH,), H
I
OH
Phosphatidic acid
Yolk Lecithin
Phosphatidylcholine
(lecithin)
Phosphatides containing choline are commonly called lecithin; those containing serine or ethanolamine are are remnants of termed cephalins. These common names an obsolete lipid classification system based on solubility. Systematically lecithin is denoted as phosphatidylcholine or choline phosphoglyceride. In naturally occurring phosphatides the phosphate ester is always linked at a terminal hydroxyl group of glycerol. Two conventions of labeling the glycerol carbon chain persist. The carbon attached to the phosphoryl choline may be designated as 3
a,the other primary glycerol carbon as 1 or a', and the secondary carbon as 2 or fi. Phosphatides are widespread and occur in all plant and animal cells. Several biochemical roles have been proposed for these phospholipids. Some of these functions are clearly associated with the fact that phosphatides possess both hydrophilic and hydrophobic groups and can therefore interact strongly with both polar and nonpolar subin biological membranes stances. Phosphatides occur where they interact in some fashion with proteins. The resulting lipoprotein complex is essential in membrane transport processes. Phosphatides are possibly involved as activators in the blood clotting system and in the transport of potassium and sodium ions. They also function in the transport, storage, and metabolism of fatty acids. Lecithin is an abundant naturally occurring phosphatide, comprising up to 50% of the phospholipids in some membranes. A freshly isolated lecithin mixture is a white, waxlike mass which darkens on exposure to air due to oxidation, Soybean and corn extracts contain phospholipids which are commonly used as food additives because of their emulsifying properties. Food products labeled as containing “lecithin” would suggest the presence of a single pure chemical substance but in fact the “lecithin” may contain less than 50% phosphatidylcholine. In addior
tion, lecithins obtained from a natural product are not a chemically homogeneous substance, but usually contain a mixture of fatty acids. Rat liver lecithin, for example, contains 29.2% palmitic acid, 1% palmitoleic, 22.4% stearic, 7.9% oleic, 11.5% linoleic, 21.8% arachidonic and 6.1% (22:6) (1). It has been shown however that a majority of lecithin molecules contain one saturated and one unsaturated fatty acid. Generally the unsaturated fatty acids occur at the 2-position. Egg yolk is a rich source of lipid, containing phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, cholesterol, cholesterol esters, as well as various glycerides. Lecithin comprises approximately 14% of the dry weight of the yolk (2). The lipid components of egg yolk can be separated by solvent extraction since the desired phosphatides are quite insoluble in acetone. Initial extraction with acetone serves to remove the nonpolar cholesterol, cholesterol esters, glycerides, and most of the egg yolk pigments and water. The polar phosphatides are then extracted with a chloroform-methanol mixture. A crude phospholipid product is obtained by evaporation of the solvent. These phospholipids are susceptible to air oxidation, especially in the presence of sunlight which catalyzes the reaction. It is necessary, therefore, that all concentrating and drying procedures involving these materials be performed in an inert atmosphere. The phosphatides from egg yolk can be rapidly identified by small-scale thin-layer chromatography on Silica Gel (3). The sample is developed on thin-layer plates in parallel with known phosphatide standards, and the sample components determined by comparison of the distance Volume 51. Number 8. August 1974
j
533
of migration. A sequence of different color developing can also be used to verify the identification. Alumina column chromatography was first used to fractionate egg yolk phosphatides by Hanahan, et al., (4). This procedure proved a convenient means of isolating the lecithin since the choline phosphatides are eluted while the non-choline phosphatides remain adsorbed on the column. The small amount of sphingomyelin found in egg yolk lipids will be eluted along with the lecithin but, since it is not hydrolyzed under the conditions used in analysis of the fatty acid composition of the lecithin, its presence does not affect the results. Cholesterol is a frequent contaminant in the crude phosphatide mixture, and it must be absent from the material placed on the column as alumina cannot easily differentiate between lecithins and steroids. The Liebermann-Burchard reaction is routinely applied to detect the presence of cholesterol and related compounds. Any cholesterol contamination in phosphatides can be removed by dissolving the product in chloroform and reprecipitating the phospholipids with acetone. The phospholipases are phosphatide degrading enzymes found in many tissues. These enzymes are highly specific and much of the present structural information on phosphatides has been obtained by the judicious use of their action. In lecithin four bonds are potentially susceptible to hydrolytic cleavage. Phospholipases of types A, B. C, and D have been characterized, each type promoting hydrolysis at one or more of the susceptible bonds of the phosphatide. Emzymes of the phospholipase A type hydrolyze one ester linkage of phosphatides to liberate a fatty acid. They of snakes, bees, wasps, scorpiare present in the venoms ons, and in the pancreas, liver, kidney, and other tissues. The venom of Crotalus adamanteus (Eastern Diamondback Rattlesnake) contains a phospholipase, requiring Ca2+ for activity, which is known to hydrolyze the ester linkage at the 2-position exclusively. Considerable confusion exists in the literature as to specificity of this enzyme, referred to now as phospholipase A2 type. Early workers observed that this enzyme acted on egg or liver lecithins to release unsaturated acids and concluded that the enzyme was specific for unsaturated fatty acids. Other reports show the enzyme hydrolysis to be specific only for the 1-position; still other sources indicate hydrolysis at both the 1- and 2-positions. The experiments of Tattrie (5) and those of de Haas and van Deenen (6) firmly established the enzyme to be specific for the 2-position hydrolysis only. The phospholipase A from other sources has been shown to attack the 1-ester position of phosphatides. Enzymes of this type are designated phospholipase Ai. The lysosomes of bovine adrenal medula, for example, possess both phospholipase Ai and phospholipase A2 activity, the two enzymes being distinguished by different pH optima (7). Lecithin is hydrolyzed in the presence of phospholipase sprays
1 It takes about 10 min to concentrate 160 ml of chloroformmethanol extract on a rotary evaporator, with the sample in a 40°C water bath. Thus if students work in pairs, each evaporator can accommodate 12 students/hr. If rotary evaporators are not available, the flask can be connected to a water aspirator, using a trap. Rath temperature could be increased to 60°C. 2 The solvent must not be reused because the chloroform is highly volatile and its evaporation leaves a solvent too rich in methanol. 3Dragendorf reagent: A) 1.7 g basic bismuth nitrate, BiONCL, in 100 ml 20% acetic acid: B) 40 g KI in 100 ml water. Just before using mix 20 ml A with 5 ml B and 70 ml water. If a brown precipitate forms, filter and discard the precipitate. 4 Ninhydrin spray reagent: 0.2% ninhydrin in 95% ethanol (wt/ vol). 5 Aluminum oxide neutral, Woelm, activity grade 1.
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Journal of Chemical Education
fatty acids from the 2-position and monoacyl derivatives referred to as lysolecithin. The enzymatic hydrolysis is most frequently performed in ether since the lysolecithin is insoluble in this solvent. The 2-position fatty acids are easily isolated by removing and drying the ether phase. The 1-position fatty acids of the original lecithin are obtained by base hydrolysis of the lysolecithin residue. Fatty acid fractions obtained by hydrolysis may be characterized by analysis of the methyl esters using gasliquid chromatography. By comparison with known fatty acid methyl esters, the individual fatty acids of the hydrolysis fractions can be identified and their relative abundances determined. This procedure is both rapid and sufficiently sensitive so that even minor components can be detected. This analysis provides both qualitative and quantitative information on fatty acid samples. For laboratories where a gas-liquid chromatograph is not available, an alternate method for the analysis of the fatty acid esters is thin-layer chromatography. Mixtures of methyl esters of fatty acids can be separated on Silica Gel plates impregnated with silver nitrate, again providing identification of fatty acids present in the original lecithin sample (8, 9). A2 to yield free
Experimental Part I. Isolation of the Phosphatide Mixture Place the separated yolk of an egg into a 50-ml glass or polyethylene centrifuge tube. Add 40 ml of acetone, stir thoroughly, and separate the phases in a clinical centrifuge. Discard the supernatant. Repeat the acetone extraction three more times. Extract the acetone-insoluble material four times with 40-ml portions of chloroform-methanol 2:1 (v/v) in the same manner. Place the solvent extracts in a 500-ml round bottom flask. Concentrate the extract under vacuum on a rotary evaporator at 40°C to a final volume of about 30 ml.1 Transfer the concentrated solution to a small beaker, rinsing the flask with a small amount of chloroform to recover any remaining lipid. Hold the beaker in a 40°C water bath and evaporate the mixture to near dryness with a stream of nitrogen. Add 100 ml of acetone to precipitate the phosphatides. Stir, allow the solid to settle, and decant the acetone. Completely dry the product in a stream of nitrogen. A solvent evaporator which would serve well here has been described by Fisk (10). The composition of the solid lipid material can be determined with thin-layer chromatography and the presence of cholesterol detected by the Liebermann-Burchard test as follows. Dissolve 15 mg of product in 3 ml of chloroform. (Save 1 ml to be used in tic.) Place 2 ml of the chloroform solution in a test tube, add 1 ml of acetic anhydride, then cautiously add, by pouring down the side of the tube, 2 ml of coned H2SO4. A green color indicates the presence of cholesterol. Thin-layer chromatographic analysis of the mixture can be carried out on glass microscope slides coated with Silica Gel G in chloroform, prepared as described by Randerath (11). Five to ten milliters of a chloroform-methanol-water (65:25:4 v/v/v) solvent is placed in a small screw top jar.2 Plates spotted with a capillary tube are placed in the solvent and removed just before the solvent reaches the top. Sample components can be detected on thoroughly dry plates by spraying lightly with an appropriate color producing reagent: a) 6 M H2SO4. Spray lightly. Heat at 105°C for 5 min. Carbonaceous materials appear as brown or black spots, b) Dragendorf reagent.3 Spray heavily. Choline containing compounds appear orange on a yellow background, and c) Ninhydrin spray.4 Spray lightly. Heat at 105°C for 3 min. Compounds containing primary amines react to give reddish-purple colors. Standards of phosphatidylcholine, phosphatidylethanolamine, and cholesterol can be used to confirm the identity of the spots on the tic plates. Chromatography should be repeated until good separation of phosphatidylcholine, phosphatidvlserine, phosphatidylethanolamine, and cholesterol is obtained, since this method will be used to determine the purity of the lecithin fraction obtained from the column chromatography in the next section of the
experiment.
Part II. Isolation of Lecithin by Adsorption Column Chromatography
Deactivate 50 g of alumina5 by shaking with 3 ml of water in a stoppered flask until any lumps have disappeared. Allow this ma-
terial to stand at least
2
hr. Choose
a
column of approximately
15-mm i.d. and 200-mm length. A buret with a Teflon stopcock will serve well. Tap a small piece of glass wool into the bottom, then cover with a small layer of washed sand. Place about 2 cm of deactivated alumina in the column, then moisten with CHCI3methanol 1:1 (v/v). The remainder of the adsorbent is then slurried in CHCI3-methanol (1:1) and poured into the column. Do not allow the column to run dry once it has been poured. Rinse the column with about 30 ml of solvent. Apply the phosphatide mixture (dissolve the solid product from Part I in a minimum volume of chloroform)6 to the column and elute with 100 ml of CHCI3methanol and collect the eluate in 25-ml fractions. Using tic, compare the eluate fractions with a sample of the phosphatide mixture before the column separation and with the phosphatidylcholine standard. Combine the fractions containing high lecithin concentrations and evaporate under vacuum until nearly dry. Remove the final traces of solvent in a stream of nitrogen.
Part III. Selective Hydrolysis of Fatty Acids from Lecithin A. Hydrolysis with Phospholipase A. Dissolve the purified lecithin from Part II in 100 ml of diethyl ether. Dissolve 5 mg of snake venom7 (phospholipase A) in 1 ml of 0.005 M CaCl2. Add the enzyme-CaCl2 to the ether solution, shake gently, seal tightly to avoid ether evaporation, and allow the enzyme-catalyzed hydrolysis to proceed for 10-15 hr at 30'C, shaking the mixture occasionally. The lysolecithin produced will appear as a separate gel-like layer. Decant the ether supernatant and carefully wash the gel with small portions of ether to recover all the fatty adds. Save the gel for further hydrolysis, (Section B). Dry the combined
Figure 1. Gas chromatographic analysis ot methyl esters of fatty acids from position 2 of lecithin. The numbers associated with the peaks indicate the fatty acid present. They are: 1. palmitic, 2. oleic and linoleic, 3. stearic, 4. arachidonic.
ether washings over anhydrous Na2S04, decant, and evaporate to dryness. Save the solid fatty acids for Part IV, B. Alkaline Hydrolysis of Lysolecithin. Dissolve the lysolecithin gel in a minimum volume of 1 N KOH. Allow hydrolysis to proceed for 24 hr at room temperature or for 2 hr at 37°C. Acidify the resulting solution to pH 1 with 6 N HC1 and extract the mixture three times with 15-ml portions of n-hexane. Wash the combined hexane extracts with water until the washings are pH 5 or above. Dry the hexane solution over anhydrous Na2SC>4, decant., and evaporate off the hexane on a 70°C water bath. Save the solid fatty acids for Part IV.
Part IV. Identification of Fatty Acids Derived from Lecithin Use two test tubes. In one, place the fatty acids from the 1-position released in the KOH hydrolysis. In the other test tube place the fatty acids from the 2-position released by phospholipase A. In the hood add 1 ml of 14% BF3-methanol reagent to each tube and heat the tubes in a steam bath for 2-3 min. Using about 30 mi of hexane, transfer each reaction mixture as completely as possible to a small separatory funnel. Add 20 ml of HjO, shake vigorously, and discard the water-methanol layer. Pass the hexane portion through filter paper into a small beaker, and evaporate the solvent at 70°C. Be careful not to let the samples go completely dry. Tightly seal the methyl esters until they are needed for gas chromatographic analysis. Standardize the gas chromatograph with solutions of known concentration of methyl linoleate, methyl oleate, methyl palmitate, methyl stearate, and methyl arachidonate in hexane. Using gas-chromatographic analysis of the product methyl esters, it is possible to determine which fatty acids are present and the relative quantity of each in the 1- and 2-position of the lecithin sam-
ple.
Discussion of Results Part I
If the extraction is thorough, the Liebermann-Burchard test for cholesterol will be negative. If a brown rather than green color appears, it indicates the absence of cholesterol and the presence of phospholipids. The thin-layer chromatography should show cholesterol with a high ft/, followed by phosphatidylethanolamine, phosphatidylcholine, It is possible to overload the alumina column. The capacity for this separation is 10 mg of dry phosphatides/g of alumina. 7 Snake venom, Crotalus adamanteus, is inexpensive and readily available from Sigma Chemical Company, St. Louis, Missouri. 6
Figure 2. Methyl esters of fatty acids from position
1
of lecithin.
and phosphatidylserine. Lysolecithins, if present, appear the origin. Both phosphatidylserine and phosphati-
near
dylethanolamine react with ninhydrin, so a standard of phosphatidylethanolamine is needed to complete the identification. To obtain good chromatograms glass slides must be scrupulously clean and care must be taken to avoid CHCI3 evaporation from the solvent. In all steps involving solvent evaporation, gentle heat and a stream of nitrogen gas is used. This is essential to prevent oxidation or decomposition of the unsaturated
fatty acids. Part I!
When alumina is moistened with CHCIs-methanol, heat is generated. If the entire column is packed dry, the addition of solvent causes vapor pockets to form in the column, thus slowing the flow rate. So we have chosen to
a small portion of the column dry and add the remainder as a slurry. An alternative method of dry-column separation using inexpensive Nylon columns has been used by Bohen et al., (12). Flow rates should be about 1 ml/
pack
Volume 51. Number 8. August 1974
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535
be detected. The entire experiment can be made quantitative by weighing the lecithin when it is dried, and by determining the quantity of each fatty acid methyl ester by comparison with standards of known concentration. This experiment has been used in its entirety with ad-
min. The first fraction off the column will contain small amounts of cholesterol, but no lecithin. Fractions 2 and 3 should be pure lecithin, and fraction 4 may contain a mixture of phosphatides.
Hg can more
Part III
vanced undergraduates in a section of about 20 students and requires three 3-hr laboratory periods. The methyl esters of the fatty acids can be kept if tightly sealed, so that student products can be analyzed over a period of several weeks if necessary. An abbreviated version including Parts I and II has been used in an introductory class and is usually completed in two 3-hr lab sessions. A typical time schedule would be: Period (1) Extraction, tic, hydration of alumina, Period (2) Column chromatography, enzyme hydrolysis—base hydrolysis, and Period (3) Preparation of methyl esters, introduction to gas-liquid chromatography, identification of products. Throughout the more than four years this experiment has been used, student groups have never failed to obtain methyl ester products, and comparison of their results with the literature has produced many interesting discussions and excellent lab reports.
Phospholipase A (snake venom) must be dissolved in the aqueous CaCl2 before addition to the ether solution of lecithin. Usually the ether phase becomes cloudy within an hour after the addition of the enzyme because of the formation of lysolecithin. The separation of products is much easier if the gel is allowed to settle out from the ether suspension. There is no simple way to detect the progress of hydrolysis of lysolecithin in KOH but complete hydrolysis is not absolutely necessary. Part IV
A typical trace from the gas chromatograph of the fatty acid methyl esters from position 2 is shown in Figure 1 and the methyl esters from position 1 in Figure 2. The fatty acid esters of varying chain length are well separated, and the stearic (18:0) is separated from oleic (18:1) and linoleic (18:2). The presence of double bonds causes a fatty acid ester to be eluted before its saturated homolog when using a relatively nonpolar liquid phase such as silicone or Apiezon. The chromatography conditions we use are: column: SE-30 on chromosorb, 160°C; N2 gas, flow rate 40 ml/min; injector: 220°C; detector: 230°C; with a Wilkens-Aerograph Model A-90-P2 with thermal conductivity detection. Solvent blanks are prepared to verify the absence of fatty acids in the solvents. Both hexane and ether are treated with BF3-methanol reagent and are carried through the subsequent procedure. BF3-methanol should be fresh since the reagent decomposes with time. The standards for gas chromatography are made up at 10 mg/ml in hexane. As little as 10 ng of methyl ester gives % to full scale deflection, so quantities as low as 1
Literature Cited (1) (2) (3) (4) (5) (6) (7)
(1967). (8) Randerath. (9) (10) (11)
(12)
+
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Journal of Chemical Education
Arvidson, G. A. E.,J. Lipid Res., 6, 574 (1965). Rhodes, D. N., and Lea, C. H., Biochem. J., 65, 526 (1957). Brinkman, U. A. Th.. and De Vries, G.. J. CHEM. EDUC., 49,545 (1972). Hanahan, D. J.,Turner, M. B,, and Jayko, M. E., J. Biol. Chem., 192,623(1951). Tattrie, N.H..J. Lipid Res., 1,60(1959). Van Deenan, L. L. M.. and de Haas, G. H., Ann. Rev. Biochem., 35, 157 (1966). Winkler. H., Smith. A. D.. Dubois, F., van den Bosch, H.. Biochem. J., 105, 38C
Kurt, “Thin-Layer Chromatography," 2nd Ed.. Academic Press, Inc., New York. 1966, p 125. T. Morris, J., Chem. and Ind 1962, 1238(1962). Fisk, David P., J. CHEM. EDUC., 50,401 (1973). Randerath, Kurt, "Thin-Layer Chromatography,” 2nd Ed.. Academic Press, Inc., New York, 1966, p30. Bohen, J. M., Joullie, M. M., Kaplan, F. A., and Loev, B., J. CHEM. EDUC., 50, 367 (1973). .
