لخّصلي

Abstract Lipid decomposition studies in frozen fish have led to the development of a simple and rapid method for the extraction and purification of lipids from biological materials. The entire procedure can be carried out in approximately 10 minutes; it is efficient, reproducible, and free from deleterious manipulations. 'The wet tissue is homogenized with a mixture of chloroform and methanol in such proportions that a miscible system is formed with the water in the tissue. Dilution with chloroform and water separates the homogenate into two layers, the chloroform layer containing all the lipids and the methanolic layer containing all the non-lipids. A purified lipid extract is obtained merely by isolating the chloroform layer. The method has been applied to fish muscle and may easily be adapted to use with other tissues. Introduction In the course of investigations concerning the deterioration of lipids in frozen fish, the need arose for an efficient and rapid method of total lipid extraction and purification. Furthermore, due to the highly unsaturated nature of fish lipids, the method had to involve only mild treatment so as to minimize oxidative decomposition and the production of artifacts. Several existing methods were considered but none was entirely satisfactory. The methods of Dambergs (1) and Folch et al. (2) were too time-consuming for routine investiga tions and since the former method entailed heating and evaporation it was considered unsuitable for lipid composition studies. The recent method of Folch et al. (3), which was published while this study was in progress, was more rapid than their previous method but still had the disadvantage of employing large and inconvenient volumes of solvent. The method used by Dyer and Morton (4) was rapid but extracted only a fraction of the total lipid. The present paper describes a method whereby the lipids of biological materials can be extracted and purified in a single operation. Mixtures of chloroform and methanol have had wide use as lipid extractants and examination of the chloroform-methanol-water phase diagram (Fig. 1) led to the following hypothesis. Optimum lipid extraction should result when 'Manuscript received February 27, 1959. Contribution from the Fisheries Research Board of Canada, Technological Station, Halifax, Nova
Scotia. Can. J. Biochem. Physiol. Vol. 37 (1959) 912 CANADIAN JOURNAL OF BIOCHEMISTRY AND PHYSIOLOGY. VOL. 37. 1959 t the tissue is homogenized with a mixture of chloroform and methanol which, when mixed with the water in the tissue, would yield a monophasic solution. The resulting homogenate could then be diluted with water and/or chloroform to produce a biphasic system, the chloroform layer of which should contain the lipids and the methanol-water layer the non-lipids. Hence, a purified lipid extract should be obtained when the chloroform layer is isolated. Procedure Reagents Methanol, absolute, analytical reagent ; chloroform, analytical reagent. Lipid Extraction and Purification The following procedure applies to tissues like cod muscle that contain 80+ 1% water and about lYo lipid. Each 100-g sample of the fresh or frozen tissue is homogenized in a Waring Blendor for 2 minutes with a mixture of 100 ml chloroform and 200 ml methanol. To the mixture is then added 100 ml chloroform and after blending for 30 seconds, 100 ml distilled water is added and blending continued for another 30 seconds. The homogenate is filtered through Whatman No. 1 filter paper on a Coors No. 3 Buchner funnel with slight suction. Filtration is normally quite rapid and when the residue becomes dry, pressure is applied with the bottom of a beaker to ensure maximum re covery of solvent. The filtrate is transferred to a 500-ml graduated cylinder, and, after allowing a few minutes for complete separation and clarification, the volume of the chloroform layer (at least 150 ml) is recorded and the alcoholic layer removed by aspiration. A small volume of the chloroform layer is also removed to ensure complete removal of the top layer. The chloroform layer contains the purified lipid. For quantitative lipid extraction the lipid withheld in the tissue residue is recovered by blending the residue and filter paper with 100 ml chloroform. The mixture is filtered through the original Buchner funnel and the blendor jar and residue are rinsed with a total of 50 ml chloroform. This filtrate is mixed with the original filtrate prior to removal of the alcoholic layer. Adaptation to Other Materials The above procedure can be applied directly to any 3 00-g sample containing 80 g water. Many alterations of the procedure are permissible but it is impera tive that the volumes of chloroform, methanol, and water, before and after dilution, be kept in the proportions 3 :2 :0.8 and 2 :2 :1.8, respectively. These ratios represent the total volumes present in the ternary systems, including the water present in the sample. Thus, in the extraction of materials that do not contain 80Yo water, either the size of the samples can be adjusted so that they contain 80 g water, or 100-g samples can be used and the volumes of chloroform and methanol changed to give the correct proportions. In cases where the mois ture content is much less than 80% (e.g. fish meal), it is necessary to add distil led water. When material containing a large amount of lipid is used, or where the supply of material is limited, the size of sample can be reduced and the above solvent quantities scaled down to meet requirements. BLIGH AND DYER: LIPID EXTRACTION AND PURIFICATION Determination of
Lipid Content A portion of the lipid extract containing 100-200 mg lipid is evaporated to dryness in a tared flask and the weight of the lipid residue determined. Evaporation, facilitated by a stream of nitrogen, is carried out in a water bath at 40-50" C and the residue is dried over phosphoric anhydride in a vacuum desiccator. After weighing, a small volume of chloroforrn is added to each flask to detect the presence of non-lipid material (insoluble). If non-lipids are present, the chloroform is carefully decanted and the flask rinsed three times with chloroform. The dry weight of the residue is determined and subtracted from the initial weight. The lipid content of the sample is calculated as follows: weight of lipid in aliquot X volume of chloroform layer Total lipid = volume of aliquot Experimental Optimum Conditions .for Lipid Extraction The first objective was to find which of various mixtures of chloroform and methanol would yield quantitative extraction. The solvents were mixed in FIG. 1. Chloroform-methanol-water phase diagram, % iw/w) at 20' C (5). . . . . . "maximum chloroform tie-line" estimated from data for 0 C (6). - - - dilutions 914 CANADIAN JOURNAL OF BIOCHEMISTRY AND PHYSIOLOGY. VOL. 37, 1959 such proportions that, with the water of the samples, ternary systems were formed having compositions as marked on the phase diagram (Fig. 1, points A-J). Samples of minced cod muscle (100 g each) were homogenized with the extractants (Table I) and filtered. To the monophasic extracts, sufficient water TABLE I Lipid extracted from 100 g cod muscle by ternary mixtures of chloroform-methanol-water Initial extraction mixture Dilution solvents Ref. pt. in Chloro- Chloro- Total Lipid in phase diagram form. Methanol. Water.* form. Water, volume, chloroform (Fig. 1) ml ml ml ml ml ml layer, g *Including 80 ml from tissue. tNo additional solvents required to render system biphasic. and chloroform were added to render the systems biphasic. The final composi tions of these ternary mixtures are given by the points A', B', C', and D'E' in Fig. 1. The lipid contents of all chloroform layers were determined as above. The results (Table I) confirmed the initial hypothesis in that more lipid was extracted by mixtures of the monophasic area of the diagram than by those of the biphasic area. Mixtures C, D, and E gave the highest values; however, the volumes of chloroform required for D and E were large and inconvenient. Therefore, the area around point C was considered most favorable. A tie-line has been drawn in Fig. 1 which for the pilrposes of this paper has been called "the maximum chloroform tie-line". This tie-line was particularly significant in this study since the lower layers of ternary systems having com positions on or below it are practically 100yo chloroform (6). Systems having compositions above this line have chloroform layers contaminated with methanol and water. This explains why the chloroform layer of the system represented by point F in Fig. 1 was the only one found to contain non-lipid. Point P in the monophasic area of Fig. 1 was chosen as a convenient starting point for further investigation since the composition was equivalent with 100 ml chloroform, 200 ml methanol, and 80 ml water. The composition after adding another 100 ml chloroform is represented by point Q in the biphasic area of the diagram. Point R, which is below the maximum chloroform tie-line, is obtained by subsequent dilution with 100 ml water. This sequence was followed with cod muscle. The tissue (100 g) was homogenized with 100 ml chloroform and 200 ml methanol, filtered, and the residue rinsed with 100 ml chloroform in three portions. The pooled filtrates were mixed with 100 ml distilled water and the mixture was allowed to separate in a
500-ml graduated cylinder. Separation of the layers was rather slow but satisfactory. The BLIGH AND DYER: LIPID EXTRACTION AND PURIFICATION 915 chloroform layer contained 0.70 g lipid, the highest yield obtained for this lot of cod muscle (Table I). Further experimentation showed that the same result was obtained when dilution with chloroform and water preceded filtration. The chloroform had to be added before the water and each addition followed by blending. This resulted in a rapidity of separation of the layers and a yield of lipid which could not be obtained by adding the solvents in a
different order. The extraction of frozen samples did not significantly lower the final tempera ture of the extract; consequently it was unnecessary to thaw samples prior to extraction. Substitution of glass fiber filter paper for Whatman No. 1 filter paper showed that with cod flesh there was no significant loss of lipid with the latter due to adsorption. Sintered glass filters also had no advantage over the regular filter except perhaps with materials such as fish meal. Non-Lipid in Chloroform Layer It was shown by three methods: (a) the determination of chloroform insoluble material, (b) purification according to Folch et al.
(2), and (c) purifica tion according to Shorland et al. (7), that the lipid extracted by the above pro cedure contained no significant amounts of non-lipid material. The amounts of impurities, if present at all, were below the sensitivity limit of each of these methods. Lipid in Methanol- Water Layer The methanol-water layer was quantitatively removed and evaporated to less than 50 ml by distillation under reduced pressure at 40-SO0 C. The coricentrate was quantitatively transferred to a separatory funnel and extracted four times with an equal volume of ethyl ether. Any emulsions were broken by centrifugation. The weight of lipid recovered from the pooled extracts was 8 mg. This loss, being only about 1% of the total lipid, was considered insigni ficant in most applications. Lipid Remaining in Tissue Residue Several procedures were employed to recover any lipid remaining in the extracted tissue. The most effective procedure involved re-blending the tissue residue and filter paper with 100 ml chloroform, followed by filtration and rinsing of the blendor jar and residue with a total of 50 ml chloroform. The recovered lipid weighed 40 mg, approximately 6% of the total extracted lipid. Re-extraction of the washed residue with chloroform-methanol-water did not yield any further amount of lipid. Thus, the initial extraction isolated approximately 94% of the extractable lipid. The washed residue was further treated with 200 ml 6 N hydrochloric acid at 100' C for 90 minutes to establish whether there was lipid remaining which was bound in such a way that chloroform-methanol would not remove it. The liberated "lipids" were extracted from the digest according to A.O.A.C. specifications (8). The yield was 47 mg (Table 11). At least part of this material may have originated from inositides (see (9)), the protein complexes 916 CANADIAN
JOURNAL OF BIOCHEMISTRY AND PHYSIOLOGY. VOL. 37, 1959 of which have been found to be resistant to chloroform-methanol extractiorl (2). Furthermore, the drastic acid treatment may have produced some fat-like material from non-lipid constituents of the muscle. TABLE I1 Grams of lipid extracted from 100 g cod muscle by four methods Method of I'resen t method Dambergs (1) A.O.A.C. (9) Folch et al. (3) Sample I 0.65 0.67 0.66 0.68 Sample I1 0.76 0.76 0. 05* *Lipid isolated from residue after digestion with hydrochloric acid. Comparison with Existing Methods Samples of well-mixed minced cod muscle (100 g each) were extracted using the described method and the methods of Dambergs (I), Folch et al. (3), and A.O.A.C. (8). The results are shown in Table 11. The average lipid content of sample I as determined by the present method was 0.666% (standard deviation, 0.013), which was higher than the values obtained using the A.O.A.C. method (8). For sample 1 I, which had a higher lipid content than sample I, the method of Folch et al. (3) and the present method gave yields which were not significantly different; however, the extract of the former method usually contained small amounts of non-lipid material. The values obtained using the method of Dambergs (1) were somewhat lower than those of the other methods. The flesh of several species of fish (100-g samples) with different fat contents, and two lots of fish meal (10-g samples) were analyzed by the described method TABLE I11 Grams of lipid extracted from various samples using the present method and the method of Dambergs (1) Present Method of Sample* method Dambergs (1) Halibut fillets Cod fillets (pre-rigor) Cod fillets (5 yr at - 10' F) Cod fillets (18 mo at +lo0 F) Swordfish steaks Rosefish fillets Plaice fillets Fish meal (A) Fish meal (B) *All samples except the fish meals were 100 g; of the meals 10 g was used per extraction. +Tissue residue was not washed BLIGH AND DYER: LIPID EXTRACTION AND PURIFICATION 917 and that of Dambergs (1). The results in Table I11 show that the new method for the most part extracted more lipid than the method of Dambergs (1). Discussion When the proper proportions of chloroform and/or water are added to a miscible chloroform-methanol-water mixture, the system separates into two layers. The proportions may be chosen in such a manner that the lower layer will be practically 100yo chloroform and the upper layer nearly all methanol water. This concept was applied to the extraction and purification of tissue lipids. It was found to be very effective; the miscible solvent mixture was an efficient lipid extractant which upon the addition of chloroform and water separated into two layers and in so doing separated the lipids from the non lipids. The method which was developed is simpler and less time-consuming than procedures hitherto reported and is well adapted to routine lipid analyses, and also to lipid composition studies since only mild treatment of the samples is involved. For practical purposes, total lipid extraction was complete; slightly more fatty material could be isolated onl~r after hydrochloric acid digestion. The separation of lipids and non-lipids was nearly quantitative. The results of lipid determinations on a sample of cod muscle were reproducible to within + 2y0 (standard deviation). In some applications it may be unnecessary to wash the tissue residue since the washings contained only 6y0 of the total lipid and the lipid composition of the washings, as shown by free fatty acid and phospholipid determinations, did not appear to differ from that of the original extract. Some chloroforn~ is evaporated during filtration of the homogenate but this is not serious provided the decrease in volume is not too great. References DAMBERGS. N. T. Fisheries Research Board Can. 13, 791 (1956). FOLCH, J., ASCOLI, I., LEES, M., MEATH, J. A., and LEBARON, F. N. J. Biol. Chern. 191, 833 (1951). FOLCH,
J., LEES, M., and STANLEY, G. H. S. J. Biol. Chern. 226, 497 (1957). DYER, W. J. arld MORTON, M.
L. J. Fisheries Research Board Can. 13, 129 (1956). BANCROFT, W. L). Phys. Iiev. 3, 21 (1895).
BONNER, W. D. J. Phys. Chern. 14, 738 (1910). SHORLAND, F.B., BRUCE, L. W., and JESSOP, A. S.
Biochern. J. 52, 400 (1952). Official methods of analysis. 8th ed. Assoc. Offic. Agr. Chemists, Washington, D. C. 1955. p. 311. GARCIA, M. D., LOVERN, J. A., and OLLEY, J. Biochern. J. 62, 99 (1956).