SPECIAL PROCEDURES

Extraction of all lipids with a mixture of hexane/isopropanol.


To reduce the danger of toxicity of chloroform, Hara et al. (Anal Biochem 1978, 90, 420) described an efficient extraction procedure particularly adapted to nervous tissues.
The tissue sample is homogenized with 18 volumes of a mixture of hexane/2-propanol (3/2) for 1 minute, the suspension is filtered and the filter rinsed with 3 x 2 vol of the same solvent. As the content of non-lipids is very low (proteins, pigments, small molecules), the whole liquid phase is evaporated and the dried extract dissolved (Eder K et al. Clin Chim Acta 1993, 219, 93).

An adaptation of the Hara's method was demonstrated to be the most efficient procedure for the extraction of plant sphingolipids (Markham JE et al., J Biol Chem 2006, 281, 22684).
Briefly, to the frozen tissue, 5 ml of the lower phase of isopropanol/hexane/water, 55/20/25 (v/v) were added. The tissue was disrupted in a glass homogenizer and transferred to a glass tube which is capped and incubated at 60°C for 15 min with occasional shaking. After centrifugation, the pellet was extracted twice more, each time with 5 ml of solvent and the supernatants were combined. A 98% recovery was obtained with leaf tissue of Arabidopsis, tomato, and soybean.

Dry-column method


An alternative to the traditional Folch method was described using solvent elution of a dry column composed of a tissue sample, anhydrous sodium sulfate, and Celite diatomaceous earth ground together (Marmer WN et al. Lipids 1981, 16, 365). Alternatively, lipids may be isolated and simultaneously separated into neutral and polar fractions by a sequential elution procedure. Analyses of muscle and adipose tissues demonstrated that results were similar to those obtained with chloroform/methanol methods.
A less time-consuming dry-column method was scaled down and adapted to 1-g samples (liver and muscle tissues) (Elmer-Frohlich K et al., JAOCS 1992, 69, 243).

Procedure: The sample (about 1 g) is ground for 30 sec. in an ice-chilled mortar with 4 g anhydrous Na₂SO₄ and 0.1 ml BHT (20 mg/l dichloromethane). Celite 545 (3 g) (Fisher Scientific) is added and the mixture is ground for an additional 30 sec. to obtain a fine homogenized powder.
The powder is poured into a 16 mm X 30 cm glass column packed with glass wool and 2g of CaHPO₄/Celite 545 (1/9 w/w) at its tip. A slight compression was accomplished at the top with a glass rod. Mortar, pestle, and glass rod are rinsed with 15 ml of dichloromethane/methanol (9/1, v/v) which are transferred into the column. In addition to these 15 ml, 50 ml of solvent mixture are added to elute lipids which are isolated, weighed and analyzed after evaporation of the solvent under nitrogen flushing.
Multiple columns may be run simultaneously.

A patented process, first reported in 1989 (Barker SA et al., J Chromatogr 1989, 475, 353), known as "matrix solid-phase dispersion" was described to conduct simultaneously disruption and extraction of solid and semi-solid samples. Thus, a highly viscous, semi-solid or solid sample can be placed in a mortar containing a bonded-phase solid support material (C18 bonded silica) and mechanically blended to perform a complete disruption and dispersal of the sample. This blend is sufficiently dry to transfer and pack into a column for more classical application of solid-phase extraction to the isolation of sample components. This technique has been most frequently applied to the isolation of drugs, herbicides, pesticides and other pollutants from animal tissues, fruits and vegetables (review in : Barker SA, J Chromatogr A 2000, 885, 115).

Bligh and Dyer Method


Bligh and Dyer (Can J Biochem Physiol 1959, 37, 911) introduced a method where extraction and partitioning are simultaneous, the precipitated proteins are isolated between the two liquid phases. This method is particularly suitable for lipid extraction of incubation medium, tissue homogenates or cell suspensions. The extraction can be carried out in a single tube where previous studies took place.

It must be pointed out that this extraction method was shown to give significantly lower estimates of lipid content in samples containing more than 2% lipid (mainly triacylglycerols) and this underestimation increased with increasing lipid content of the sample (Iverson SJ et al. Lipids 2001, 36, 1283). Thus, the total lipid content of fatty samples are accurately determined using the Folch extraction method.

Procedure: To a sample containing 1 ml water (1 ml cell suspension, homogenized tissue, plasma...), add 3.75 ml of a mixture chloroform/methanol (1/2) and vortex during 10-15 min, then add 1.25 ml chloroform with mixing 1 min and 1.25 ml water with mixing another minute before centrifugation. Discard the upper phase and collect the lower phase through the protein disk with a Pasteur pipette. For large volumes of liquid, it is advisable to filter the mixture to remove the insoluble parts of the sample and to centrifuge the liquid phase to allow the formation of the two liquid phases. After evaporation, the lipid extract (lower phase) will be redissolved in a small volume of chloroform/methanol (2/1).

The basic procedure was improved to increase the yield of lipids. One of the most common modifications is to replace water by 1M NaCl. This addition blocked the binding of some acidic lipids to denatured lipids. If necessary, the addition of 0.2 M phosphoric acid to the salt solution is possible (Hajra, lipids, 1974, 9, 502) to improve their recovery. In this case, plasmalogens are converted to lyso lipids.
If an exhaustive extraction is necessary, an extraction with two steps can be used.
Similarly, it was described that the addition of acetic acid (0.5% v/v) in the water phase significantly increased the recovery of acidic phospholipids (Weerheim AM et al., Anal Biochem 2002, 302, 191).


Two-step Bligh and Dyer method


To a sample containing 1 ml water (1.25 g tissue, 1 ml cell suspension, homogenized tissue, plasma...), add 3.75 ml of a mixture chloroform/methanol (1/2) and vortex during 10-15 min, then add 1.25 ml chloroform with mixing 1 min and 1.25 ml water with mixing another minute before centrifugation. Collect the lower phase in another glass tube. 
Add 1.88 ml of chloroform to the non-lipid residue, vortex, centrifuge. Mix the lower phase to the first chloroform phase in the glass tube. After evaporation, the lipid extract will be dissolved in a small volume of chloroform/methanol (2/1).


Extraction of bacteria


An efficient modification of the Bligh and Dyer method, given below, was proposed for the extraction of lipids from unicellular organisms. Several parameters were optimized to improve the fatty acid recovery (Lewis T et al., J Microbiol Meth 2000, 43, 107). Thus, it was shown that the total amount of recovered fatty acids  increased by about 30% by adding solvents to the biomass in order of increasing, as opposed to decreasing, polarity.

Cells were harvested by centrifugation at high speed for 15 min. The supernatant was discarded, the cell pellet re-suspended in 100 ml 1.0% NaCl (w/v), and re-centrifuged. The second supernatant was discarded and the cell pellet frozen overnight at -30°C. Frozen biomass was freeze dried for 15 h and subsequently stored in a sealed glass container at -30°C. 
To freeze-dried cells (about 100 mg) to which a total of 114 ml solvents were added in the sequence: chloroform, methanol, water to achieve a final chloroform/methanol/water ratio of 1/2/0.8 (v/v/v). Samples were shaken for 15 s immediately following the addition of each solvent, and allowed to stand for about 18 h, with occasional shaking by hand.
Phase separation of the biomass-solvent mixtures in the separatory funnels was achieved by adding chloroform and water to obtain a final chloroform/methanol/water ratio of 1/1/0.9 (v/v/v). A known portion of each total lipid extract recovered from the lower chloroform phase was used for further analysis.

Comparing various extraction procedures, it has been shown that a modified (miniaturized) Bligh and Dyer extraction technique was the most efficient with an oleaginous bacteria Thraustochytrium sp (Burja AM et al., J Agric Food Chem 2007, 55, 4795). If only fatty acid determination is required, a direct saponification using KOH in ethanol was almost as efficient as the previous one.

An hexane-isopropanol solvent system was found to be effective for the bacterium Pseudomonas atlantica (Guckert J B et al., J Microbiol Methods 1988, 8, 131), this system gaving a lower recovery of lipids in the green alga Chlorella sp. (Guckert J B et al., J Microbiol Methods 1988, 8, 139).

Extraction of highly polar lipids


When tissues are rich in highly polar lipids such as gangliosides, a reliable extraction method is needed to prevent their loss while extracting all other lipid classes. All the previously described techniques brings gangliosides and likely a part of other very polar lipids into the water-rich layer. A method, recently described for nervous tissues (Dreyfus et al., Anal Biochem 1977, 249, 67-78), prevents these drawbacks.
Small tissue samples corresponding up to 10 mg protein suspended in 0.5 ml water are mixed with 5 ml chloroform/methanol mixture (1/1) for 30 min. The pellet obtained by centrifugation is extracted again with successively 3 ml of the same solvent, 3 ml of a mixture chloroform/methanol (1/2) and 3 ml of a mixture chloroform/methanol/water (60/30/4.5). The four lipid extracts are combined and evaporated and the dry residue dissolved for further purification.

Lyso derivatives (N-deacylated) of glycosphingolipids  are not efficiently recovered from cell extracts because of their high polarity. The glycosylsphingosine are found in  tissues from patients and in animal models. In normal conditions, their concentration are very low and needs very efficient techniques to be evaluated. It was shown that a second extraction with water-saturated butanol of the upper aqueous phase obtained after a Folch extraction was necessary to recover up to 98% of the tissue lysoglycosphingolipids (Bodennec J et al., J Lipid Res 2003, 44, 218).


Extraction of plasma total lipids


For the extraction of plasma lipids we used a very rapid and efficient method.
To 0.2 ml plasma add 0.3 ml 0.5 M KH₂PO₄ , 1.5 ml chloroform and 0.5 ml methanol. After vortexing 2 minutes and centrifugation, the lower phase is collected with a Pasteur pipette through the protein disk and evaporated.

A procedure using the detergent TritonX-114 was shown to be very efficient for the extraction of plasma lipids, while sparing the protein fraction for further use (Ferraz TPL et al., J Biochem Biophys Meth 2004, 58, 187).

Delipidation of plasma, serum or plant seeds


When plasma proteins, including the apolipoproteins, must be preserved from denaturation during the extraction of lipids a specific solvent system must be used (Cham BE et al., J Lipid Res 1976, 17, 176).
The most common procedure used for delipidation of plasma, protein solutions or cell culture medium involves the extraction of all kinds of lipids with a mixture of butanol and di-isopropyl ether. The proteins remain in solution in the aqueous phase, while the organic phase contains the dissolved lipids.

Procedure : One volume of serum or plasma containing 0.1 mg/ml of ethylenediamine tetraacetate (EDTA) is added to 2 volumes of a mixture of butanol/di-isopropyl ether (40/60, v/v). The vials are tightly closed and fastened on a mechanical rotator providing end-over-end rotation at about 30 rpm for 0.5 h.
After extraction, the mixture is centrifuged at low speed (2000 rpm) for 2 min to separate the aqueous and organic phase. The aqueous phase containing the delipidated proteins is removed by careful suction with needle and pump or syringe.
Traces of butanol remaining in the aqueous solution may be removed if necessary by washing that phase with 2 volumes of di-isopropyl ether. Residual solvent may be removed by an extraction with a water pump aspirator at 37°C for some minutes.

When proteins from plant oilseeds are studied by electrophoresis, lipids must be removed to prevent important interferences and thus to obtain good resolution. It was demonstrated that, in the presence of chloroform methanol, lipid contaminants can be thoroughly removed by the combination of two precipitation steps (10% TCA/acetone and acetone) and aqueous TCA wash steps (Wang W et al., Anal Biochem 2004, 329, 139). 


Extraction of plasma fatty acids and acylglycerols


A reliable extraction procedure was described by Dole (J Clin Invest 1956, 37, 350) for the extraction of free fatty acids and non polar acylglycerols.
To 1 ml plasma (or aqueous solution) add 5 ml of Dole reagent (40 ml isopropanol, 10 ml heptane and 1 ml 1M H₂SO₄), 3 ml heptane and 2 ml water.
After vortexing and centrifugation, the upper phase is collected. Residual phospholipids may be removed by the addition of 200 mg silica gel (vortex and centrifuge) before evaporation under nitrogen.


Phosphoinositides extraction


Several procedures were described to extract the polyphosphoinositides since they are known to bind strongly to proteins during their denaturation. To improve the recovery, the use of an acidic solution is necessary. We used the procedure of Honeyman (Biochem J 1983, 212, 489) which is a modification of the Lloyd's method (Br J Haematol 1972, 23, 571-585).

This is the one-step method of Bligh and Dyer modified by the inclusion of HCl to improve recovery of acidic phospholipids :

1 ml of cell suspension is mixed with 3.75 ml of chloroform/methanol/12N HCl (2/4/0.1, v/v). After thorough mixing, 1.25 ml of chloroform is added with vortexing 30 sec followed by 1.25 ml of water with similar mixing. After centrifugation 10 min at low speed, the lower chloroform layer is removed and transferred to a glass tube for evaporation.


Method used to extract platelet phosphoinositides


Stop incubation of the cell suspension by adding 1 ml of chloroform/methanol (1/1) to 1.5 ml of aqueous medium and vortex some seconds. Transfer the mixture with a Pasteur pipette into a 15 ml Falcon polypropylene tube.
Add to the mixture 4 ml of chloroform/methanol (1/1), then 0.4 ml 10N HCl and then 0.5 ml water. Vortex during 5 min to extract lipids and centrifuge the plastic tube at low speed 10 min at 4°C.
Transfer the lower phase into a second Falcon tube by sampling through the proteinaceous disk with a Pasteur pipette. Add to this solution 2.5 ml methanol, 2.1 ml water and 0.4 ml 10N HCl. Vortex some seconds and centrifuge at low speed in the cold.
Transfer the lower phase as previously in a glass tube, evaporate with the help of nitrogen and dissolve the residue with a small volume of chloroform/methanol (1/1).


Extraction of plant material


Plant tissues are difficult to extract because of active lipases which hydrolyze rapidly phospholipids glycolipids and increase the amount of free fatty acids in the extract. Thus, a solvent frequently used to inhibit these enzymes is isopropanol.
Nichols' method: Plant tissues are minced and macerated with 100 parts (w/w) of isopropanol. The mixture is filtered, the solid is extracted again with 200 parts of chloroform/isopropanol mixture (1/1, v/v). The combined filtrates are evaporated, dissolved in a small volume of chloroform/methanol (2/1, v/v) and, if necessary, washed according the Folch's procedure.

The extraction of algae is made with hot isopropanol (60°C) added to the cell suspension. It appears that unicellular algae (plankton) must be extracted rapidly with a minimum of preparative mechanical treatments (centrifugation, filtration). A preliminary small scale extraction is recommended to choose the procedure to be adopted.
The extraction of neutral lipids from microalgae has been efficiently done on lyophilized material (Fajardo AR et al., Eur J Lipid Sci Technol 2007, 109, 120). Briefly, first, 96% ethanol was used to extract the lipids from the dry biomass. Second, a biphasic system was formed by adding water and hexane to the extracted crude oil. Thus, the purified lipids were transferred to the hexane phase while most impurities remained in the aqueous phase.

Oilseeds may be analyzed for oil content by an exhaustive extraction with petroleum ether. A comparison of five methods to measure the oil contents in oilseeds may be studied before choosing a specific procedure (Barthet VJ et al., J Oleo Sci 2002, 51, 589).

A one-tep extraction has been described to study the composition of triglycerides in small piece of seed and is suitable for a large number of tissue samples should be examined as in selecting new plant varieties. This easy and reliable method is based on an incubation of samples (20-50 mg) without shaking in a mixture containing heptane / 0.17 M NaCl in methanol (66.6/33.3, v/v), for 2 h at 80°C. After cooling, the upper phase containing mainly triglycerides was transferred to a new test tube for further analysis. Even under incomplete triglyceride extraction (80% maximum) the triglyceride ecomposition is representative of the total triglycerides found in the tissue (Ruiz-Lopez N et al., Anal Biochem 2003, 317, 247).

The extraction of xanthophyll was shown to be improved using cellulolytic enzymes and highly competitive when compared to the traditional process of pigment extraction (Navarrete-Bolanos JL et al., J Agric Food Chem 2004, 52, 3394). These data may foster the development of new extraction procedures in plants based on previous enzymatic hydrolysis of cell membranes.

The extraction of flour can be made with hexane at room temperature but the efficiency of phospholipid recovery is dependent upon the temperature and the moisture content (Snyder HE, Inform 2004, 15, 575).

The reading of the review of various techniques of preparing plant material by Romanik G et al. is suggested before any plant extraction (Romanik G et al., J Biochem Biophys Methods 2007, 70, 253). 

Very small samples


When tissue samples are very tiny (1 mm or less), the analysis of their lipid content (neutral lipids or phospholipids) may be effected directly on the TLC plate without previous extraction (Lecomte M et al., Prostagl Leukot Essent Fatty Acids 1998, 59, 363).

Procedure:

The tiny piece of tissue is directly applied in a small hole or groove made in the concentration zone (kieselgur preadsorbent) of the TLC plate (cf Whatman plates). This plate is then rapidly applied onto a steel plate pre-cooled in liquid nitrogen. After one or two minute freezing, the plate is lyophilized one or two hours under vacuum. The plate is then rapidly submitted to a solvent elution adapted to the desired analysis. 


Mineralized samples

As some acidic phospholipids (mainly phosphatidylserine) are known to complex with calcium, particularly in the presence of Pi, a complete extraction of membrane lipids in mineralized tissues must be done after chemical demineralization (Wu L et al., J Biol Chem 2002, 277, 5126).

Procedure:

Small tissue samples are powdered in the cold (Freezer mill) and extracted with chloroform/methanol (2/1) mixture. After a short sonication (1-2 min), the suspension is centrifuged (3000 rpm, 12 min) and the extract collected. The pellet is demineralized with 0.5M Na salt EDTA for 20 min and sedimented after a short centrifugation. After removal of the supernatant, the decalcified residue (pellet) is reextracted using a chloroform/methanol/conc. HCl (200/100/1) mixture. The two lipid extracts are mixed, dried and washed with saline to remove non-lipid contaminants.