Phosphatidylcholine from "Healthful" Egg Yolk Varieties An Organic Laboratory Experience Linda C. Hodges Agnes Scott College, Decatur, GA 30030 One of the traditional laboratory exercises in biochemistry is the isolation of phosphatidylcholine (PC) from some convenient source such as egg yolk (1) and spinach (2). The oooularitv of this laboratorv exercise is due in large meas;re to its-illustration of three of the most usefulchromatographic methods-
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column chromatography using silica gel, thin-layer chromatography (TLC),and gas liquid chromatography (GLC).
PC is extracted usina conventional methods. The extract is purified further h;chromatography on silicic acid, a common method for the separation of phospholipids, and the column fractions are assayed for the presence and puritv of PC bv TLC. Fractions containing pure PC are and the concentrated sample is hydrolyzed and esterified to obtain the fatty acid methyl esters (FAME). FAMEs are separated and identified by GLC. Although this procedure allows students to become familiar with a variety of analytical methods useful in biochemistry and organic chemistry, the experiment a s it is usually performed lacks the investigative component. Teaching science the way that we do science is becoming a rallvine crv in the chemistrv oedaaoeical communitv. and s t d i e i s u p p o r t the idea tha"t ;stud&& are attracted to science hv oarticioatine in the discoverv "asoect . of the discipline 6,h). The new emohasis in societv on healthier foods orovides a convenient vehicle for adding a n investigative element to this classic experiment. In our area of the country, grocery stores now carry several alternate brands of eggs that come from chickens raised on diets lacking animal products. These chickens presumably eat a diet low in Baturated fat. The nutritional advantage to the consumer is a n egg slightly, hut not substantially, rower in cholesterol. Additionally, a t least one manufacturer claims that studies shuw th,~tthcsc eggs, althouqh nor lower in choleitcrol, do not r a s e serum cholesterol. Pwsumnbly, less saturated fat in the diet ofthr chickoni could be rellccred in the firtv acid composition of various classes of biological lipids, including phospholipids, in the eggs from these chickens. Comparing FAMEs derived from PC of yolks of regular eggs to those obtained from the other special brands adds a novel twist to the students'work, and the results provoke some discussion on the connections between structure and function of these biologically significant molecules. I have adapted this experiment for use in our sophomore organic chemistry laboratories. Students typical1;perform experiments using silica gel column chromatography or TLC and GLC a t this level, and the separation of organic molecules like PC from a biological sample often sparks student interest. In addition, the larger numbers of students enrolled in organic chemistry theoretically allows for better statistical treatment of data and for teamwork, another important element of the scientific system.
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'Eggland's Best-radio commercial.
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Journal of Chemical Education
Materials and Methods Students were divided into teams of two, and each team was assigned the task of extracting PC from regular eggs, which serve as "control", or one of two alternate brands of eggs. often referred to a s "healthier". that are available in our area. All the eggs are treated identically. The procedure that we use is essentiallv that of Devor (2)adaoted for egg yolks rather than spinach using the method ofegg extraction in Clark and Switzer (1).Additional details may he found in these references. Two egg yolks are stirred in 100 mL of cold acetone for 10-15 min, vacuum-filtered, and reextracted with a second 100-mL portion of cold acetone. The mix is again vacuumfiltered, and the liquid is discarded. I have found that the extractions can be done successfullv " bv " mixing on a maguetic stirrer, and this modification alleviates the flammahilitv oroblem in the orieinal exoeriments associated with blending egg yolks in organic solvents. The solids are extracted with 50 mL of cold methanol-chloroform (1:l vlv) and stirred in a n ice bath for 15 min. The mixture is vacuum-filtered, and the residue is reextracted with another 50 mL of methanol-chloroform. The two liquid filtrates from these two extractions are combined and rotary-evaporated to about 5-10 mL. Column chromatography is performed using Bio-Sil A (Bio-Rad)that has been heat-activated a t 110 "C for 16 h. Students prepare a 1.8- x 35-cm column with the resin (about 35 g ) in chloroform. About 1-2 mL of the yolk extract is applied to the packed column, and the column is eluted sequentially with 175 mL of chloroform-methanol (8:2 vlv), 100 mL chloroform-methanol (1:l vlv), and 150 mL methanol. The first 250 mL of eluent is discarded, and 20 7-mL fractions are then collected. Alternate fractions are evaporated to about 0.5 mL in preparation for spotting . . samples onto con\.ention:il 20 r 20 crn silica gt.1 T ~ ~ ~ p l a t r . - ~ l'ht. waporation of fr;tctinns l ~ t h tTLC ! spotting is t i m e consuming. Ideally, fractions should be evaporated under nitrogen to avoid oxidation of the fatty acids. I have had reasonable success, however, by allbwing fractions to evaporate a t room temperature between lab sessions. The &of the lab d~ctates;"hen this expediency i s necessary. About 50-100 JL oFaltt!rnate fr:rcti(mi is sputted on tht, TLC plate a s weil as 20-30 pL of a standard-l mg1mL PC solution in acetone. The plate is developed in chloroformmethanol-water (65:35:5'v/vlv), and s p k s are visualized with iodine vapors. Fractions containing pure PC are combined along with the appropriate alternate fractions that were not spotted. The oooled fractions are evaoorated under nitrogen to about i m ~and , a 0.2-mL sample is transferred to a screw-caooed test tube and dried under nitrogen. Two milliliters'df 5% sulfuric acid in methanol (vlv) and 100 pg of the internal standard methylheptadecanoate a s a 1mg/mL solution in methanol are added. The tube is nureed with nitroeen and heated a t 70 "C for 1h. The tube " is cooled, and the solution is then extracted three times with 2 mL of hexane. The combined hexane layers are evaporated under nitrogen, the residue is taken up in
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about 100 FL of HPLC grade methylene chloride, and about 1FL is injected into the GLC. FAMEs obtained can be analyzed by a number of different columns. A wide-bore cadllarv column from S u ~ e l c o (30 x 0.75 mm ID, 1.0 mm fik, ~ u i e l c o w a x10) is reasonablv" affordable and gives excellent s e ~ a r a t i o nfor the ease of uict. Usually FAMIKs can be separated isothermally at 225 'C or with a tttnluerarure oromam bwlnninrat 210 "C -~~ then to 240 "C a t 4 "C per minute. The isothermal separation requires about 15 min or less per sample run. Students identify their FAMEs by comparison to standards. Matreya sells a reasonable priced sample of FAMEs from rapeseed oil that contains most of the common FAMEs. Students auantitate t h e FAMEs based on the internal standard i d d e d during the esterification assuming a n equal detector response for all FAMEs.
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Results and Discussion
The experiment a s we perform i t takes four 2-3-hour laboratory periods. Students are able to calculate the number of micrograms of each type of fatty acid in their sample, the percentage of each type of the total, and consequently, a percentage of saturated to unsaturated fatty acids in the sample. This exercise may be used every year without losing its investigative value because actual composition of e variabilitv the PC can varv from vear to vear d e ~ e n d i n on in chickens, actual formulation of their diets, and various brands of alternate eggs available. We devoted one additional laboratory session to the discussion and comparison . year's m o u of ~ students actnof results. For e x a m ~ l elast ally found that the totai percentageof saturated fats i n the PC was higher i n the extracts from the alternate egg brands compared to the regular eggs (Table). This result sparked discussion on both reliability of data based on limited determinations, as well a s possible biochemical and nutritional implications. Instructors obviously can vary the format of the laboratory by involving larger teams with each team extracting both control and alternate egg varieties and by including
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Percent Fatty Acid Composition of Phosphatidylcholinefrom ~ g g s ~
Fatty Acid
Control
Variety 1
Variety 2
C 16:O
41
46
38
C 180
12
18
22
C 18:l
40
27
33
C 182
4
6
7
C 20:1
3
3
0
Total Unsaturated
47
36
40
Total Saturated
53
64
60
'Percentages represent averages from GLC analyses of FAMEs obtained by two teams of students. Control eggs were a store brand. "Healthful' variety 1 was 'Eggland's Best', and variety 2 was ''Four Grains".
different varieties of eggs that may be available i n other regions of the country In rural areas, instructors may be able to collaborate with local poultry farmers to investigate the consequence of other experimental diets. This iu'nd of experience generates @-eat& student interest and involvement in both the collection and the interpretation of data than a simple isolation of a biological compound alone evokes. Students recognize that real science has no "right answers", only conclusions based on statistically valid data obtained in the context of the experimental design and its variables. Literature Cited 1. Clark. J. M.;Switeer R. L.Erprrimentoi Biochrmidry, 2nd ed.; W. H. Freeman: Sen Fran"sco. 1977; p 187. -
2. Deuor, K J. Chem. Edue. 1919.56.758-759. 3. Tobias, S. Reuitulking Undergraduot~Science Research Corporation: Tucson,AZ, 1992. 4. w h a t Works: Building Natural science
communitiesx,Val. 1; project Kaleidoscop:
Washington, DC, 1991.
Volume 72 Number I 2 December 1995
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