Bernard J. White ond Carl 1. Tipton Iowa Stote University Ames, 50010
Egg Yolk Lecithin
Mary Dressel
Winono state College Winono, Minnesota 55987
A biochemical laboratory project
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In the selection of a compound for 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 chnmatographic techniques have revolutionized lipid hmchemistrs and have ersentialls renlaced the older methods of analysis on lipid mixtures.-~ighlyspecific 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 he obtained. The final objective of the procedure is to determine the fatty acid composition a t positions 1 and 2 of lecithin by gas-liquid chromatography.
or a,the other primary glycerol carbon as 1 or a', and the secondary carbon as 2 or p. 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 substances. Phosphatides occur in biological membranes where thev interact in some fashion with oroteins. The resulting lipoprotein complex is essential in membrane transoort . processes. Phosohatides are ~ossiblvinvolved as activators in the blood ciotting system a n d i n the transnort of ~otassiumand sodium ions. Thev also function in the transport, storage, and metabolism of fatty acids. Lecithin is an abundant naturallv occurrine ohosphatide, comprising up to 50% of the ihospholipGiin some membranes. A freshly isolated lecithin mixture is a white, waxlike mass which darkens on exposure to air due to oxidation. Sovbean 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% phospbatidylcholine. In addition, lecithins ohtained from a natural product are not a chemically homogeneous substance, but usually contain a mixture of fatty acids. Rat liver lecithin, for example, contains 29.290 palmitic acid, 1% palmitoleic, 22.4% steaBackground ric, 7.9% oleic, 11.5% linoleic, 21.8% arachidonic and 6.1% (22:6) (1). It has been shown however that a majority of Glvcero~hosohatides are a class of comnlex linids which . may he considered as derivatives of phosphatidic acid in lecithin molecules contain one saturated and one unsaturated fatty acid. Generally the unsaturated fatty acids combination with a nitroeenous base. such as serine. ethaoccur a t the 2-position. nolamine, or choline, o; an alcohoi such as inositol or glycerol. The fatty acids of glycerophosphatides are generEee volk is a rich source of lioid., containine nhosp h ~ ~ d & h o l i n e phosphatidylethanolamine, , sphingomyelally 12-22 carbon atoms in length and may he either satuin, cholesterol. cholesterol esters, as well as various elvcrated or unsaturated. 0 0 erides. ~ e c i t h i ncomprises approximately 14% of t h e d r y H ll H I/ weight of the yolk (2). HCOCR HCOCR 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 t o 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 ~. (lecithin) ohtained by evaporation of the solvent. These phospholipPhosphatides containing choline are commonly called ids are susceptible to air oxidation, especially in the preslecithin; those containina serine or ethanolamine are ence of sunlight which catalyzes the reaction. It is necestermed cephalins. These common names are remnants of sary, therefore, that all concentrating and drying procean obsolete lipld clasitication svsrem based on solubilitv. dures involving these materials be performed in an inert Smtematicallv lecithin is denoted a i nhc~inhatidvlch~>line atmosphere. or choline phosphoglyceride. In naturally occurring phosThe phosphatides from egg yolk can be rapidly identiphatides the phosphate ester is always linked at a termified by small-scale thin-layer chromatography on Silica nal hydroxyl group of glycerol. Two conventions of lahelGel (3). The sample is developed on thin-layer plates in parallel with known phosphatide standards, and the saming the glycerol carbon chain persist. The carbon atple components determined by comparison of the distance tached to the phosphoryl choline may he designated as 3
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of migration. A sequence of different color developing sprays can also he used to verify the identification. Alumina column chromatography was first used t o fractionate egg yolk phosphatides by Hanahan, e t al., (4). This nrocedure proved a convenient means of isolating the lecithin since the choline phosphatides are eluted while the non-choline phosnhatides remain adsorbed on t h e column. T h e smali amount of sphingomyelin found in egg yolk lipids will he eluted along with the lecithin hut, since it is not hydrolyzed under the conditions used in analysis of the fatty acid composition of the lecithin, its presence does not affect t h e results. Cholesterol is a frequent contaminant in the crude phosphatide mixture, and it must he absent from t h e material placed on the column as alumina cannot easily differentiate between lecithins and steroids. T h e Liehermann-Burchard reaction is routinely applied t o detect the presence of cholesterol and related compounds. Any cholesterol contamination in phosphatides can he removed by dissolving the product in chlorofonn and renrecioitatine the ~ h o s ~ h o l i o i with d s acetone. . T h e phospholipases are phospiatidk degrading enzymes found in manv tissues. These enzvmes are hiahlv - -specific . and much of the present structural information on phosphatides has been ohtained 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 hydrolvsis a t one or more of the susceptible honds of the phGphatide. Emzymes of the phospholipase A type hydrolyze one ester linkage of phosphatides t o liberate a fatty acid. They are present in the venoms of snakes, bees, wasps, scorpions, and in the pancreas, liver, kidney, and other tissues. The venom of Crotalus adamanteus (Eastern Diamondback Rattlesnake) contains a phospholipase, requiring CaZ+ for activity, which is known to hydrolyze the ester linkage a t the 2-position exclusively. Considerable confusion exists in the literature as to specificity of this enzyme, referred t o now as phospholipase Az type. Early workers observed t h a t this enzyme acted on egg or liver lecithins to release unsaturated acids and concluded t h a t the enzyme was specific for unsaturated fatty acids. Other reports show the enzyme hydrolysis to he specific only for the l-position; still other sources indicate hydrolysis a t hoth the 1- and 2-positions. The experiments of Tattrie (5) and those of d e Haas and van Deenen (6) firmly estahlished the enzyme to he specific for the 2-position hydrolysis only. The phospholipase A from other sources has been shown to attack the l-ester position of phosphatides. Enzymes of this type are designated phospholipase AI. The lysosomes of bovine adrenal medula, for example, possess hoth phospholipase A1 and phospholipase A? activity, the two enzymes being distinguished by differentpH optima (7). Lecithin is hydrolyzed in the presence of phospholipase
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'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 studentslhr. If rotary evaporators are not available, the flask can he connected to a water aspirator, using a trap. Bath temperature could he increased to 60'C. ZThe solvent must not be reused because the chloroform is highly volatile and its evaporation leaves a solvent too rich in methanol. 3D~~gendorf reagent: A) 1.7 g basic bismuth nitrate, BiON03, 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. Ninhydrin spray reagent: 0.2% ninhydrin in 95% ethanol (wtl "01). 5 Aluminum oxide neutral, Woelm, activity grade 1. 534 / Journal of Chemical Education
A2 t o yield free fatty acids from the 2-position and monoacyl derivatives referred t o as lysolecithin. T h e enzymatic hydrolysis is most frequently performed in ether since the lysolecithin is insoluble in this solvent. T h e 2-position fatty acids are easily isolated by removing and drying the ether phase. The l-position fatty acids of the original lecithin are ohtained by base hydrolysis of the lysolecithin residue. Fatty acid fractions ohtained by hydrolysis may he characterized by analysis of the methyl esters using gasliquid chromatography. By comparison with known fatty acid methvl esters. the individual fatty acids of the hydrolysis fractions can he identified and their relative ahundances determined. This procedure is hoth rapid and sufficiently sensitive so t h a t even minor components can he detected. This analysis provides hoth qualitative and quantitative information on fatty acid samples. For laboratories where a aas-liquid chromatogaph is not available, a n alternate method-for the analysis of the fatty acid esters is thin-layer chromatography. Mixtures of methyl esters of fatty acids can he separated on Silica Gel plates impregnated with silver nitrate, again providing identification of fatty acids present in t h e original lecithin sample (8,9). Experimental Part I. isolation of the Phosphatide Mixture Place the separated yolk of an egg into a 50-ml glass or polyethylene centrifuge tuhe. 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 40.1111 portions of ehloroform-methanol 2:l ("1") 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.' 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 wouldserve well here has been described by Fisk (10). The composition of the solid lipid material can he 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 chloraform. (Save 1 ml to be used in tle.) Place 2 ml of the chloroform solution in a test tuhe, add 1 ml of acetic anhydride, then cautiously add, hy pouring dawn the side of the tube, 2 ml of concd 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 ehloroform-methanol-water (65:25:4 v/v/v) solvent is placed in a small screw top jar.2 Plates spotted with a capillary tuhe are placed in the solvent and removed just before the solvent reaches the top. Sample components can he detected on thoroughly dry plates by spraying lightly with an appropriate color producing reagent: a) 6 M HzSOI. 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.' Spray lightly. Heat at 105°C for 3 mi". Compounds containing primary amines react to give reddish-purple colors. Standards of phosphatidylcholine, phasphatidylethanolamine, and cholesterol can be used to confirm the identity of the spots on the tlc plates. Chromatography should be repeated until good separation of phasphatidylcholine, phosphatidylserine, phasphatidylethanolamine, and cholesterol is ohtained, since this method will be used to determine the purity of the lecithin fraction ohtained from the column chromatography in the next section of the experiment. Part 11. Isolation of Lecithin by Adsorption Column Chromatography Deactivate 50 g of alumina5 hy 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. C h m e 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 CHC4methanol 1 : l ("1"). The remainder of the adsorbent is then slurried in CHCls-methanol (1:l) 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 phasphatide mixture (dissolve the solid pmduct from Part I in a minimum volume of chl~roform)~ to the column and elute with 100 ml of CHCIJmethanol and collect the eluate in 25-ml fractions. Using tlc, compare the eluate fractions with a sample of the phosphatide mixture before the column separation and with the phasphatidylchaline 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.
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Part 111. Selective Hydrolysis of Fatty Acids from Lecithin A. Hydrolysis with Phospholipase A. Dissolve the purified lecithin from Part I1 in 100 ml of diethyl ether. Dissolve 5 mg of snake venom7 (phaspholipase A) in 1 ml of 0.005 M CaCh. Add the enzyme-CaClz 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 acids. Save the gel for further hydrolysis, (Section B). Dry the combined ether washings over anhydrous NazSO,, 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 Na2S04, 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 l-position released in the KOH hydrolysis. In the other test tube place the fatty acids from the 2-pasition released by phospholipase A. In the hood add 1 ml of 14% BFI-methanol reagent to each tube and heat the tuhes in a steam bath for 2-3 min. Using about 30 ml of hexane, transfer each reaction mixture as completely as possible to a small separatory funnel. Add 20 ml of HzO, 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 nrrdrd iur ga*chromatocmphie analysis. Standnrdm th~.gas chrumntograph with solutions of known concrntrorion 01' merh\l l~nuleatr.methyl ulentc, methyl palmltate, methyl stearate, and methyl araehidonate in hexane. Using gas-chromatographic analysis of the product methyl esters, it is pogsihle to determine which fatty acids are present and the relative quantity of each in the 1- and 2-pasition of the lecithin sample. Discussion of Results
Part I If t h e extraction is thorough, t h e Liehermann-Burchard test for cholesterol will he negative. If a brown rather than green color appears, i t indicates t h e absence of cholesterol and t h e presence of phospholipids. T h e thin-layer chromatography should show cholesterol with a high R,, followed by phosphatidylethanolamine, phosphatidylcholine,
6 It is passihle to overload the alumina column. The capacity for this seoaration is 10 meof drv..nhasohatides/eof " . . alumina. 7 Snake venom, Crotolus adamanteus, is inexpensive and readily available from Sigma Chemical Company, St. Louis, Missouri.
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RELATIVE RETENTION TIME
Figure 1 . Gas chromatographic analysis of 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. stearlc, 4. arachidonic.
Figure 2. Methyl esters of fatty acids from position 1 at lecithin.
and phosphatidylserine. Lysolecithins, if present, appear near t h e origin. Both phosphatidylserine a n d phosphatidylethanolamine react with ninhydrin, so a standard of phosphatidylethanolamine is needed t o complete t h e identification. T o obtain good chromatograms glass slides must be scrupulously clean and care m u s t he taken to avoid CHC13 evaporation from the solvent. In all steps involving solvent evaporation, gentle heat and a stream of nitrogen gas is used. This is essential t o prevent oxidation or decomposition of t h e unsaturated fatty acids. Part I1 When alumina is moistened with CHCla-methanol, heat is generated. If the entire column is packed dry, t h e addition of solvent causes vapor pockets t o form in t h e column, t h u s slowing t h e flow rate. So we have chosen t o pack a small portion of t h e column dry and add t h e remainder as a slurry. An alternative method of dry-column separation using inexpensive Nylon columns has been used by Bohen e t al., (12). Flow rates should be about 1 m l / Volume 51. Number 8. August 1974 / 535
min. The first fraction off the column will contain small amounts of cholesterol, hut no lecithin. Fractions 2 and 3 should be pure lecithin, and fraction 4 may contain a mixture of phosphatides. Part 111
Phospholipase A (snake venom) must be dissolved in the aqueous CaClz 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 hut complete hydrolysis is not absolutely necessary. Part I V
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:O) is separated from oleic (18:l) 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; Nz 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 BFR-methanol reagent and are carried through the subsequent BFa-methanol should be fresh since the reagent decomposes with time. The standards for gas ch;omatography are made up a t 10 mg/ml in hexane. As little as 10 fig of methyl ester gives % to full scale deflection, so quantities as low as 1
536
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fig can be detected. The entire experiment can be made more 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 advanced 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 I1 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, tlc, 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. Literature Cited (1) A w i & m G.A. E . J . Lipid Re% 8. 574 119651. (21 Rhodos.D. N., andLen, C, H.,Biochem. J.. 65,526 119571. (3) Brinkman,U.A.Th.. end DeVri-, G.. J . CHEM. EDUC.. d9.545(19721. (4) Hanahan, D.J..Turner. M. B., andJayko, M . E.,J. B i d C h m , 192.823 119511. (8) Tartrie. N . H . . J . LipidRea., 1,Mi(19SYl. (6) Van Deenan. L. L. M., and de Hsar, G . H.. Ann. Re". Biacham.. 35.157 (1966). (7) winkier. H.. Smith. A. D., Duhois. F.. van don Bmeh, H., Biachem. J . 105. 38C 118671. (81 Randersfh. Kurt. ''Thin~LayorChmmatomsphy," 2nd Ed., Academic P-, he.,
NewYork. 1966. p30. . M., Kaplan. F. A . and h e v , 8.. J. C H E M EDUC., SO, 1121 ohe en. J . M., ~ o u l l i &M.
