Dear Shaima,

The following publications cover the answer to your question.

The first publication describes the method for extraction. Please for the following link for more details. The second publication is a review article on the estimation of lipids.

1- LIPID EXTRACTION

GENERAL METHODOLOGIES

 Several extraction procedures may be found in books and articles aiming at the improvement of lipid recovery from any kind of organisms, tissues or cell types. After the first famous studies of Chevreul on the dissolution of lipid materials in various solvents, it is necessary to jump to 1879 when Franz von Soxhlet described the first method based on an automatic solvent extraction (diethyl ether) for milk lipids (Die gewichtsanalytische Bestimmung des Milchfettes, Dingler's Polytechnisches J 1879, 232, 461). A further improvement was made in 1914 when a mixture of ethanol/ether (3/1) was used for lipid extraction (Bloor WR, J Biol Chem 1914, 17, 377). 

Despite the early use of chloroform in extracting lipids (Bornmann JH, Assoc Off Agric Chem 1931, 14, 489), the greatest improvement of the extraction of polar lipids from animal tissues was made when Folch described in 1957 his classical extraction procedure (see Folch biography). This procedure remains one of the best described and the most commonly used by lipidologists around the world. Some other procedures were proposed by Bligh and Dyer (1959) and Sheppard (1963), which also used solvent mixtures made of chloroform/methanol and ethanol/diethyl ether, respectively. More recently, a simple methyl tert-butyl ether-based extraction has been proposed for the simultaneous extraction of lipophillic metabolites and lipidome from small amounts of tissue sample (Chen S et al., J chromatogr A 2013, 1298, 9).

The estimation of the fat content in foods by measuring the amount of acyl lipids is of common practice but the choice of an extraction procedure needs to be evaluated to get reliable results. As a guide, a comparison of several methods to measure the oil contents in oilseeds may be consulted (Barthet VJ et al., J Oleo Sci 2002, 51, 589). A simplified method for chloroform/methanol extraction and gravimetric determination of total fat was tested on several foods and food stuffs (Phillips KM et al., J Food Lipids 2008, 15, 309). 

A critical discussion of the various problems arisen in determining the content and chemical nature of lipids in feedstuffs has been done in the paper of Palmquist DL (Palmquist DL et al., J Anim Sci 2003, 81, 3250). The authors have shown clearly that the preferred method of fat analysis in feedstuffs is the determination of the total fatty acid concentration by converting fatty acid salts, as well as the acyl components in all lipid classes, such as triacylglycerols, phospholipids, and sphingolipids, to methyl esters using a simple, direct one-step esterification procedure. Fatty acid methyl esters are then quantified by GLC, which provides information on both fatty acid quantity and profile in a single analysis (Sukhija PS et al., J Agric Food Chem 1988, 36, 1202). 

All tissues must be sampled freshly before lipid extraction to prevent any hydrolysis or oxidation before its dispersion in the solvent mixture. The presence of lysophospholipids, phosphatidic acid, monoacylglycerols, diacylglycerols or free fatty acids should indicate a possible degradation before extraction. In some special cases, special precautions are necessary to minimize rapid degradation of lipid constituents. Thus, plant tissues need to be processed immediately after collection by immersion in hot 2-propanol or water. Plant phospholipase D is known to be active even in some solvents !!

Any tissue which cannot be extracted immediately should be frozen as rapidly as possible in dry ice or better in liquid nitrogen and stored in sealed glass containers at -70°C.

Fresh or frozen tissues should be homogenized in the chosen solvent mixture and agitated during a fixed time before elimination of the solid part. Soft tissues may be minced with scissors in the cold and homogenized in solvent with an Ultraturax type device, a Warring blender or a glass-Teflon Potter. Hard tissues (muscle, vessels, bone...) are best pulverized in a mortar filled with liquid nitrogen.

A special programmed and motorized device (Freezer Mill 6800 from Fisher Bioblock Scientific) may be used to pulverize finely and easily soft or hard tissues in one or two minutes in liquid nitrogen.

The powder is then rapidly weighed and poured in a glass tube where the extraction solvent is added. We have adopted this methodology for analyzing very labile compounds such as free fatty acids, diacylglycerol and phosphoinositides. Liquid medium (plasma, blood, culture medium) are directly extracted by addition of the required solvent and strong mixing.

The highest yield of carotenoids from a microalga (Neochloris oleoabundans) was obtained using freezing–thawing and cryogenic grinding to break the cell walls using a CryoMill mixer mill (Retsch, Haan, Germany) similar to that described above (Castro-Puyana M et al., Anal Bioanal Chem 2013, 405, 4607).

Neutral lipids may be extracted by non-polar solvents (hexane, diethyl ether) and thus separated from more polar lipids. Membrane associated lipids and lipoproteins require polar solvents to separate lipids from proteins (mixtures of hexane, alcohols and/or chloroform).

First, we give details on the classical Folch's procedure but several other procedures are described below taking into account the tissues or biological preparations used in more specialized studies. 

GENERAL PROCEDURE (Folch method)

SPECIAL PROCEDURES FOR:

ALL SAMPLES :

        - Hexane/isopropanol extraction

       - Methyl-tert-butyl ether extraction

       - Dry-column method

       - Soxhlet extraction

LIQUID SAMPLES (Bligh and Dyer)

HIGHLY POLAR LIPIDS

PHOSPHOINOSITIDES

PLASMA LIPIDS

DELIPIDATION OF PLASMA

PLANT MATERIALS

BACTERIA

OILSEED PROCESSING

DRY MATERIALS (MICROWAVE EXTRACTION)

VERY SMALL SAMPLES

MINERALIZED SAMPLES

SKIN SURFACE

EXTRACTION WITH SPECIFIC INSTRUMENTS

MICROWAVE OVEN

SUPERCRITICAL FLUID EXTRACTION

PRESSURIZED FLUID EXTRACTION

SOXHLET-TYPE EXTRACTION

ULTRASOUND-ASSISTED EXTRACTION

ROBOT AUTOMATED EXTRACTION

http://www.cyberlipid.org/extract/extr0002.htm

2-Comparison of Different Procedures for the Lipid Extraction from HL-60 Cells:

A MALDI-TOF Mass Spectrometric Study

Marijana Petkovic´*

, Andreas Vocks, Matthias Müller, Jürgen Schiller, and

Jürgen Arnhold

Institute of Medical Physics and Biophysics, Medical Faculty, University of Leipzig,

Liebigstraße 27, D-04103 Leipzig, Germany. Fax: +49 34 19 71 57 09.

E-mail: [email protected]

* Author for correspondence and reprint requests

Z. Naturforsch. 60 c, 143Ð151 (2005); received August 13/September 17, 2004

A human leukaemia cell line Ð HL-60 Ð can be differentiated into neutrophils or macrophages and both differentiation processes are accompanied by changes of the lipid composition. Various methods were described for the extraction of lipids from cellular systems, but only two of them were applied to the HL-60 cell line so far. In this study we compared five selected extraction methods for the lipid extraction from HL-60 cells with regard to their qualitative analysis by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS): chloroform/methanol at volume ratios 2:1 and 1:2, isopropanol/ chloroform, isopropanol/hexane and butanol. In addition, the cholesterol and phospholipid

concentrations in organic extracts were measured by colorimetric assays. Results can be summarized as follows: For the analysis of polar phospholipids obtained from HL-60 cells by MALDI-TOF MS, a chlorofom/methanol (1:2) or isopropanol/chloroform mixture or butanol can be applied as extraction systems. On the other hand, if one would like to analyze changes in triacylglycerols, then chloroform/methanol (2:1) would be the method of choice.

Key words: HL-60 Cells, Lipid Composition, MALDI-TOF MS

Cell culture

HL-60 cells were maintained in the continuous cell culture (0.5 ¥ 106 cells/ml) in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 nm l-glutamine, 10 mg/ml streptomycine and 100 U/ml penicillin. The cell viability was determined

by the trypan blue exclusion test prior to the lipid extraction. Afterwards, the cells were washed twice with 10 mm phosphate buffered saline (PBS), pH 7.4, followed by centrifugation.

Extraction of lipids from HL-60 cells

Extraction of lipids was carried out in all cases from 10 ¥ 106 cells collected in the logarithmic growth phase. The following procedures were applied to extract lipids from the cells: a) Chloroform/methanol (2:1, v/v) was used according to the original procedure described by Folch et al. (1957). Briefly, 2 ml of the extraction mixture (chloroform/methanol, 2:1) was added directly to the cell pellet (10 ¥ 106 cells)

obtained after centrifugation. The suspension M. Petkovic´ et al. · MALDI-TOF MS of Lipids Extracted from HL-60 Cells 145 was vortexed and incubated at room temperature with agitation for 20 min. After addition of 0.9% NaCl (400 µl) the mixture was vortexed and centrifuged (500 ¥ g, 10 min) to separate the organic from the aqueous phase. The chloroform (lower) layer was collected for the analysis.

b) The second extraction was done as originally described by Bligh and Dyer (1959). The cell suspension (10 ¥ 106 cells/ml, 1 ml) in PBS was extracted by the addition of 3.75 ml of chloroform/methanol (1:2, v/v) and vortexed for 2 min. Chloroform (1.25 ml) was then added and the suspension vortexed for additional 30 s. After that, the mixture was washed with 1.25 ml of 1.5 m NaCl by vortexing for further 30 s. Centrifugation was done under the conditions described in the previous procedure. Also in this case, the chloroform layer was used for analysis.

c) The next procedure uses an isopropanol/chloroform (11:7, v/v) system and was usually applied for isolation of lipids from erythrocytes (Rose and Oklander, 1965). Isopropanol (11 ml) was applied to 1 ml of the cell suspension in PBS (10 ¥ 106 cells/ml) that was further incubated for 1 h at room temperature with agitation.

Agitation was continued after the addition of 7 ml of chloroform followed by centrifugation under the same conditions as described above. The chlorofom layer was then used for further analysis. d) An isopropanol/hexane mixture was also used

for the lipid extraction (Hara and Radin, 1978). A mixture of isopropanol/hexane (2:3, v/v) was added to the cell pellet (10 ¥ 106 cells/ml, 1.8 ml of the extraction mixture/cell pellet) that was further left with magnetic stirring at room temperature

for 1 h. The mixture was filtered and the pellet washed three times with the double

volume of the extraction mixture. After filtration the combined liquid phases were used for analysis. e) The last extraction procedure uses a single extraction

step with butanol (Bremer, 1963). The extraction was attempted as originally described (0.5 vol. of butanol/1 vol. of the cell suspension), but in this case a voluminous protein layer was formed preventing the harvesting of the organic phase. Therefore, the moiety of the extraction solvent was increased and at the end

it was twice the volume of the cell suspension. After centrifugation under the conditions described above, the upper butanol layer was taken for further analysis.

In all cases, the organic phase was evaporated to dryness in a vacuum evaporator and the residual lipid film was resuspended in 300 µl of chloroform. The lipid suspension was stored in the refrigerator (Ð 20 ∞C) until use.

Digestion of lipids with pancreatic phospholipase

A2 (PLA2) All lipid extracts obtained from HL-60 cells were subjected to the digestion with pancreatic PLA2. After chloroform removal, the residual

lipid film was resuspended in 10 mm PBS containing 1 mm CaCl2 (pH 7.4). After vigorous vortexing, PLA2 (1 mg/ml final concentration) was added to the suspension and the mixture was left at room temperature for 4 h. The reaction was terminated by the addition of chloroform/methanol (chloroform/methanol/water

phase 1:1:0.5, v/v/v) and after vortexing and centrifugation the chloroform

phase was used for MALDI-TOF MS.

Measurement of phospholipid concentration

The PL concentration was measured by a colorimetric assay as described by Stewart (1980). The chloroform solutions of lipids extracted from HL-60 cells were mixed with a solution containing 0.1 m FeCl3 ¥ 6 H2O and 0.4 m NH4SCN in an 1:1

ratio (v/v). After vortexing for 15 s, the mixture was centrifuged at 500 ¥ g for 10 min. The absorbance of the chloroform layer was measured at 485 nm with chloroform as blank. The PL concentration was calculated from the standard curve obtained

with PC 16:0, 16:0.

Measurement of cholesterol concentration

After removing the chloroform by vacuum evaporation the concentration of cholesterol in the lipid extracts of HL-60 cells was measured by the Amplex“ Red Cholesterol Assay kit, purchased from Molecular Probes (USA). The assay Ð except

a very few modifications Ð was performed according to the manufacturer’s instructions. 

Hoping this will be helpful,

Rafik

nice answer

Hi, I would just say that Stewart Assay (1980) is also a good method to quantify phospholipid via UV.

check:

https://www.researchgate.net/publication/282812596_Proliposome_Powders_Prepared_Using_A_Slurry_Method_For_The_Generation_of_Beclometasone_Dipropionate_Liposomes

Article Proliposome Powders Prepared Using A Slurry Method For The G...