I Kenneth A. Devor California State University Los Angeles. CA 90032
I
Isolation and Characterization of
I
An undergraduate biochemistry experimeni
Phasphatidyl Choline from Spinach Leaves
T h e isolation a n d characterization of phosphatidyl choline from spinach leaves is a relatively inexpensive but informative experiment which introduces t h e s t u d e n t to m a n y of t h e techniques which a r e used t o d a y in lipid hiochemistry. T h e experimental procedures introduce t h e s t u d e n t to t h e techniques of lipid extraction, separation of lipids b y silicic acid column chromatography, identification of t h e isolated phosohatidvl choline bv t h i n laver chromatography, a n d identikcation of f a t t y acids utilizing gas chromatography. T h e experiment is designed such t h a t three 3-hr periods a r e needed to complete the experiment.
Experimental Spinach may be obtained a t local markets. The spinach leaves are washed with distilled water and cut into 1in. squares after removal of the center vein from the leaves. The extraction procedure described is carried out by the instructor. For a class of twelve students, 1kg of chopped leaves is weighed out. The leaves are then put into a chilled Waring blender in 5 portions of 200 g each with 300 ml methanol: chloroform 21 vlv. The mixture is homogenized for 2 min and then 100 ml chloroform is added and homogenization is continued for 30 see.' The combined homogenates are next filtered through 4 layers of cheesecloth using a Buchner funnel and filter flask connected to a vacuum line. The filtrate which is now two phases is powed into 50 ml polypropylene or polyethylene centrifuge tubes and centrifuged for 5 min a t 10,000 rpm in a Sorvall SS-34rotor or the equivalent to enhance the phase separation. After centrifugation, the top layer, which contains water and methanol, is removed by using a disposable pipet connected to a filter flask and a vacuum line. The lower phase containing the extracted lipid is removed with a disposable pipet and bulb and is put into a 1000-ml round-bottomed flask. The extract is concentrated to a volume of 25 ml by flash evaporation. In the first laboratory period, each student weighs 15 gof heat activated silicic acid (llO0C, 16 hr.) (Bia-Sil A, 100-200mesh, Bio Rad Laboratories, Richmond, CA) which is subsequently slurried in chloroform and powed into a 1.8 X 35 cm glass column with a Teflonm stopcock and a glass wool plug a t the bottom. Two milliliters of the lipid extract is added by each student, to his or her column. After the sample has entered the silicic acid completely, 175 ml of chloroform:methanol 8 9 vlv is used to elute the column. Next, 100 ml chloraform:methanoll:l v/v is added and last 150 ml methanol is used t o elute the column. One should be careful to allow each solvent to go completely into the silicic acid before adding the next more polar solvent to prevent solvent mixing. After 250 ml totaleluant has been collected, twenty 7-ml fractions are collected in testtubes. The fractions are evaporated, preferably under nitrogen t o 0.5 ml using a 60°C waterbath. Approximately 100 p1 of every other fraction, plus 20-30 pg of phosphatidyl choline as standard (Sigma Chemical Co., St. Louis, MO) are spotted on a preeoated silica gel 60 sheet (E. Merck, A.G. Darmstadt, Germany) 2.5 em from the base of the sheet. The sheet is developed with chloroform:metbanol:water 65:35:5 vlvlv (development time is ahout 1hr). The sheet is sprayed with 1%iadine in methanol to detectphosphatidyl choline and any lipids which might be contaminants. The fractions containing only phosphatidyl choline are combined and evaporated to 2 ml, again preferably under nitrogen to prevent the oxidation of unsaturated fatty acids. The silica gel plate
758 1 Journal of Chemical Education
Gas chromalographcel.t!on profileof lsny acid methyl esters obtalneo horn phosphstdyl cho ne pbr I ed lrom spmach leares ( ) nexane peak (Solvent!I1 C l e o Ill C,,. (Internal Stanaarol IV Ciao V C l e , VI C , R ?V C W JmoW 85-
ters. may be sprayed to detect phospholipid phosphate after the iodine has ' been removed by heating a t llODCfor 5-10 mim2 For transesterification and gas chromatographic determination of the fatty acid content of the phosphatidyl choline, 0.2 ml of the combined fractions (corresponding in a typical experiment to 0.5-1 pmoleof phosphatidyl cholinephosphate) is put intoasprewcapped tube and is brought to dryness, preferably under nitrogen. Next 2 ml 5%sulfuric acid in methanol vlv is added, as well as 100 pg methylbeptadecanoate as internal standard (Sigma Chemical Co., St. Louis, MO). The tube is purged with nitrogen and incubated in a waterbath a t 70°C for 1 hr. After the sample has cooled t o room temperature 2 ml hevane is added. After the two phases have separated the upper (herane) phase is removed and the extraction is repeated twice more. The combined hexane extracts are brought t o 0.1 ml under nitrogen and 10 pl is injected into the gas chromatograph (Hewlett-Packard model no. 5720A, flame ionization detector, attenuation a t 400). The column used was aluminum tubing 0.125 in. O.D. by 6 ft packed with 15%diethylene glycol succinate on chromosorb W 60-80 mesh (both from Varian Aerograph, Walnut Creek, CAI (3).
Resulls and Discussion As stated in the experimental section when silicic acid column chromatography is carried out to purify the phosphatidyl choline, twenty 7-ml fractions are collected. Typical results of a student show that when thin layer chromatography is performed oqthese fractions the pure phosphatidyl choline is found in fractions 6-12. If a phoshate analysis is carried out, a typical yield of phosphatidyl choline is 8-10 "miles. When transesterification and eas liquid chramatog'Bligh, E. G. and Dyer, W. S., Can. J. Biochern. Physiol., 37,911 (1959). 2Dittmer, J. C. and Lester, R. L., J.Lipid Res., 5,126 (1964). 3Devar, K. A. and Mudd, J. B., J. Lipid Res., 12,396 (1971).
raphy were performed on 1pmole of the phwphatidyl choline in the presence of 1M) pg methylheptadecanaate the pattern of fatty acid methyl esters obtained was as is shown in Figure 1.The percentage of the total for each fatty acid methyl ester is shown in the table. The percentages are in quite good agreement with published results; however, the percentage of methyl linolenate is somewhat lower (3). This lower percentage is probably due to oxidation of linolenic acid. To calculate the total amount of fatty acid in the sample of phosphatidyl eholine the area under peak I11 (methyl heptadecamate) was used. First, the peak area of 5 pg of methylheptadecanoate was determined hy the instructor. From this data the area which would be given if 100pg of methylheptadecanoate were injected could be calculated. By measuring the area of peak I11 in the figure, the micrograms of methylheptadecanoate injected into the gas chromatograph can be calculated and therefore the percentage of the entire sample injected can he determined where 100%is equivalent to 100 pg of methylheptadecanoate. Knowing the percent injected, one can then first determine the total micrograms of fatty acid methyl ester from the phosphatidyl choline injected and second one can determine the total micrmams of fattv acid dlethvl ester in the entire samole. Next. ~. assuming nn dwragr mcdertrlnr weight of:lOO 1)altonr im the fatty at id methvl rstrr,onerw calrulnw the total nurnbrrufpmolesofthefarty n r ~ dmethyl esters. Theorrttcally, tnis number shtmld he twice the number of pmoles of phosphatidyl choline in the total sample; therefore, the total yield of phosphatidyleholine can he determined as well.
Percent Composlllon of Fatty Adds lrom Phosphatldyf Chollne
Fatty Acid
% Obsewed
ctss
28.0 internal Standard 1.2 14.7 20.6
Cwo Cleo Cml Cw:r C*.."
3.5 4
Peak NO. horn Figure
II 111 iv V VI ,111
The experiment ay rle~crilmlhi19 proven t
