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.
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).
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.
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:
EXTRACTION WITH SPECIFIC INSTRUMENTS
