Lipid Extraction 41

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From: Methods in Molecular Biology, vol. 313: Yeast Protocols: Second EditionEdited by: W. Xiao © Humana Press Inc., Totowa, NJ

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Extraction of Yeast Lipids

Roger Schneiter and Günther Daum

SummaryQuantitative extraction of lipids from the tissue or microorganism of choice is key to

their subsequent analysis. In this chapter, we describe a simple and rapid protocol thatrelies on glass bead disruption in the presence of organic solvents to quantitatively extractlipids from yeast cells.

Key Words: Lipids; phospholipids; fatty acids; yeast; thin-layer chromatography.

1. IntroductionThe aim of the present chapter is to consider practical aspects of the isola-

tion of lipids from yeast cells and from isolated subcellular fractions. Becauselipids are water-insoluble, their extraction requires a combination of polar andnonpolar organic solvents. The goal of the extraction procedure generally is aquantitative recovery of all the different lipid classes.

Three methods for the liquid–liquid extraction of lipids widely cited in theliterature are those of Folch, Lees, and Stanley (1); Bligh and Dyer (2); andWays and Hanahan (3). All three methods use chloroform/methanol (2:1, v/v)as the extracting solvent. These protocols can be adapted for the extraction oflipids from whole yeast cells by including a step to break open the yeast cellwall, which is usually done by disintegrating the cells in the presence of glassbeads. Glass beads can be omitted for the extraction of lipids from isolatedsubcellular fractions.

The Folch et al. procedure (1) described herein is the most common methodused. It employs 20 volumes of chloroform/methanol (2:1; v/v) per volume oftissue or membrane pellet to yield a single homogenous suspension. If the ratiois smaller (12–15 volumes), two phases will be formed. The single phase ofsolvent, however, provides a better interaction of polar and nonpolar solvents

42 Schneiter and Daum

with membrane lipids. A higher ratio is probably harmless to some extent, buttoo high a ratio of solvent will lower the water content, making polar lipidextraction incomplete.

Lipid extracts have the tendency to trap water-soluble, nonlipid material,such as sugars, amino acids, and salts, within lipid micelles. These can beremoved simply by washing the extract with 0.2 volume of water or varioussalt solutions. Addition of the wash medium results in the formation of a two-phase system: the lower phase will consist of chloroform/methanol/water(86:14:1; per vol.) while the upper phase will contain the same solvents in aratio of 3:48:47 (per vol.). The lower phase, which comprises approx two-thirdsof the total volume, contains the lipids. The upper phase, which retains thenonlipid contaminants, is discarded.

After the lipid extract has been washed to remove any water-soluble, nonlipidcomponents, the organic phase is evaporated off by using either a rotary vaporor, in case the volume is small, by drying under a stream of nitrogen.

Taken together, the preparation of a lipid extract includes the following basicsteps:

1. Homogenization of the cells in the presence of organic solvents and glass beads.2. Extraction of the lipids with chloroform/methanol (2:1; v/v).3. Removal of nonlipid contaminants by washing the extract with aqueous salt solu-

tions.4. Drying of the extract by removal of the organic solvent.

2. Materials2.1. Isolation of Whole Cell Lipids

1. Medium for growing yeast (e.g., YEPD): 1% (w/v) yeast extract, 1% (w/v)bactopeptone, 2% (w/v) glucose.

2. Glass beads: 0.25–0.3 mm diameter (e.g., Braun Melsungen, Melsungen, Germany).3. Merkenschlager (Braun Melsungen) cell homogenizer with fitting glass bottles.4. The organic solvent used, i.e., chloroform and methanol, are of analytical grade.

All solvent manipulations are to be carried out in a fume cupboard while wearingprotective clothing, because chloroform is listed as harmful, and methanol is toxic.

5. Sintered glass funnel.6. Wash solutions: 0.034% MgCl2; 2 N KCl/methanol (4:1; v/v); artificial upper

phase: chloroform/methanol/water (3:48:47; per vol.).7. Table-top centrifuge with appropriate glass tubes.8. Rotary evaporator.9. 12-mL Pyrex glass vials with Teflon liner caps.

Lipid Extraction 43

3. Methods3.1. Isolation of Whole Cell Lipids

1. Grow yeast in a suitable medium (e.g., YEPD) so as to obtain 50–200 mg dry cellweight (0.5–2 g wet weight); cell densities between 5 × 106 and 2 × 108 cell/mL,in 100 mL medium, should be adequate.

2. Harvest cells by centrifugation at 300g for 10 min. Pour off supernatant and dis-card.

3. Wash the cells with deionized water.4. Mix the harvested cells (1.5 mL aqueous suspension) with 10 mL methanol and

transfer to a 70-mL Merckenschlager glass bottle.5. Add 20 g (15 mL) of glass beads and disrupt the cells at 1700 rpm in a

Merckenschlager under CO2 cooling by shaking for four periods of 30 s with 30-scooling intervals.

6. Add 20 mL chloroform to the suspension to give a ratio of chloroform/metha-nol of 2:1 (v/v) and stir the suspension for 1 h on a flat-bed stirrer at roomtemperature.

7. Filter the extract through a sintered glass funnel and wash the glass beads with 10mL chloroform/methanol (2:1; v/v).

8. Transfer the extract to a 250-mL glass beaker, add 10 mL (0.2 volume) of 0.034%MgCl2 solution, and stir for 10 min.

9. Centrifuge the extract in appropriate glass vials at 3000 rpm in a table-top centri-fuge for 5 min.

10. Aspirate off the upper aqueous layer and wash the organic phase with 10 mL of 2 NKCl/methanol (4:1; v/v).

11. Centrifuge again at 3000 rpm in a table-top centrifuge for 5 min.12. Aspirate off the upper aqueous layer and wash the organic phase with 10 mL of

artificial upper phase (chloroform/methanol/water; 3:48:47; per vol.).13. Centrifuge again at 3000 rpm in a table-top centrifuge for 5 min.14. Aspirate off the upper aqueous layer, including the protein layer that formed at

the phase boundary, and repeatedly wash the organic phase with 10 mL of theartificial upper phase until the phase boundary becomes clear.

15. Transfer the organic phase to a round-bottom flask and evaporate the solvent in arotary evaporator set to 55°C and 200 mBar.

16. Dissolve the lipid film in 5–6 mL chloroform/methanol (2:1, v/v) and store thelipid extract in a high-quality glass vial (e.g., Pyrex) tightly sealed with Teflonliner caps at –20°C. This solution is now ready for lipid analysis and quantitativeestimations of different lipid classes. It contains approx 0.1–1.0 mg lipids/mLand is composed mainly of phospholipids, sterols, steryl esters, andtriacylglycerols.

44 Schneiter and Daum

4. Notes1. The organic solvents used are toxic or even carcinogenic. Chloroform/methanol

mixtures will rapidly leach the skin lipids from hands. Further contact with thesolvents will give rise to irritation. It is therefore advisable to handle all solventswith care. If mixtures of chloroform and methanol spill over a part of the body,the body part should immediately be kept under cold running water to minimizethe burning sensation, which could last for about 10 min.

2. Owing to the large volume of organic solvent used for the extraction any non-volatile trace contaminants in these solvents will become enriched in the finallipid extract and may then interfere with subsequent characterization, e.g., by gasliquid chromatography (GLC) analysis. To avoid this, the purest possible sol-vents (preferably redistilled) should be used.

3. Plastic bottles or tips should not be used at any stage of the extraction procedure.They contain oxidants and low molecular-weight polymers, which will be dis-solved by the solvents and which might subsequently interfere with the lipidanalysis. Therefore, glass materials should be used throughout the procedure.

4. To extract the lipids from isolated membrane fraction (15–20 mg protein), theextraction procedure can be downscaled accordingly. In this case, glass beads arenot required for the extraction step.

5. The evaporation of the organic solvent should be accomplished at as low a tem-perature as possible. If residual water does not azeotrope off in the first evapora-tion, it may be necessary to add further portions of chloroform to the evaporationflask and to repeat the evaporation step.

6. The merits and disadvantages of the Folch method have been discussed in detailin the literature (for a comprehensive review, see ref. 4). The method gives ex-cellent recoveries for neutral lipids and diacylglycerophospholipids (95–99%),whereas lysophospholipids are only partly recovered. The efficiency of the wash-ing procedure depends on the presence of salts, which alter the distribution oflipids and practically eliminate them from the upper phase. In the absence ofsalts, substantial amounts of acidic lipids are present in the aqueous phase andwould be lost during washing.It is possible to depart from the procedure, but it is essential that, while washingthe extracts, the ratio of chloroform/methanol/water is 8:4:3 (per vol). In calcu-lating these proportions, it is important to remember that the extract contains allthe water from the cells, which in the original protocol by Folch et al. (1) isassumed to have a density of 1.0 g/L. In the modified Folch method describedhere, the tissue is assumed to have a density of approx 1.0 g/mL.

7. When large amounts of material have to be extracted and quantitative recovery ofthe different lipid classes is not essential, the method of Bligh and Dyer (2) ispreferable to the Folch procedure. Major advantages of this method are that muchsmaller amounts of solvent are required and that less nonlipid material is retainedin the extract. The quantities of chloroform/methanol (2:1; v/v) are again suchthat when mixed with the water in the tissue, a single-phase solution is formed.

Lipid Extraction 45

8. For a quantitative recovery of the very polar yeast sphingolipids, the use of aspecialized extraction procedure developed by Hanson and Lester (5) is recom-mended.

9. Because most lipids are typical surface-active substances, their extraction is fre-quently accompanied by the formation of stable emulsions. Unfortunately nogeneral recommendations for avoiding emulsion formation can be given. Usuallythe best way to break down emulsions is centrifugation, which mostly results incomplete phase separation. Phase separation frequently is accompanied by for-mation of an intermediate fluffy layer. In such cases, the upper phase is removedby suction, a small amount of the upper phase of the starting Folch system isadded, and the intermediate layer is washed by rotating the vessel. After separa-tion of the phases, the upper layer is again removed by suction and the wholeoperation is repeated several times. Finally, chloroform/methanol (2:1; v/v) isadded until the intermediate phase disappears. Any solid material remaining isthen removed by filtration.

References1. Folch, J., Lees, M., and Sloane-Stanley, G. H. (1957) A simple method for the

isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226,497–509.

2. Bligh, E. G. and Dyer, W. J. (1959) A rapid method of total lipid extraction andpurification. Can. J. Biochem. Physiol. 37, 911–917.

3. Ways, P. and Hanahan, D. J. (1964) Characterization and quantification of redcell lipids in normal man. J. Lipid Res. 5, 318–328.

4. Nelson, G. J. (1975) Isolation and purification of lipids from animal tissues, inAnalysis of Lipids and Lipoproteins (Perkins, E. G., ed.), Am. Oil Chem. Soc.,Champaign, IL, pp. 1–22.

5. Hanson, B. A. and Lester, R. L. (1980) The extraction of inositol-containing phos-pholipids and phosphatidylcholine from Saccharomyces cerevisiae and Neuro-spora crassa. J. Lipid Res. 21, 309–315.