Any non-lipid contaminants that are co-extracted must then be eliminated from the recovered lipids by washing or other solvent partition procedures, before the sample can be subjected to detailed analysis. At every stage, precautions must be taken to minimize the risk of hydrolysis of lipids or of autoxidation of unsaturated fatty acids. The choice of extraction procedure will depend on the nature of the tissue matrix, and for example, whether the sample is of animal, plant or microbial origin.
Another factor is the amount of information required from the sample; many simple extraction procedures can be used for triacylglycerol-rich tissues such as adipose tissue or oil seeds if the main lipid class only is required for analysis. On the other hand, if a detailed knowledge of every minor lipid class is required, no short cuts are possible. Particular care is necessary for the more polar near-water-soluble lipids such as gangliosides or polyphosphoinositides. This review describes the principles and good practice of tissue handling, lipid extraction and elimination of non-lipid contaminants and the many potential pitfalls.
The topic has been reviewed in general terms elsewhere [17,47,,85,,], and in addition there exist many specialist reviews that deal with the extraction of specific lipid classes such as gangliosides or inositides see the appropriate sections below. All tissues, whatever their origin, should ideally be extracted immediately after removal from the living organism, so that there is little opportunity for changes to occur to the lipid components.
It is of course essential that plasma or tissue samples be taken with the minimum of stress or trauma, otherwise lipolysis will occur in vivo. Discussion of appropriate surgical or collection procedures is outwith the scope of this review, but it has been dealt with in relation to plasma elsewhere [73,74]. With plant and heart or brain tissues, say, where tissue enzymes are especially active, rapid extraction is essential. The process of freezing tissues will damage them irreversibly, because the osmotic shock together with formation of ice crystals disrupts the cell membranes.
When the original environment of the tissue lipids is altered in this way, they encounter enzymes from which they are normally protected. Similar results have been presented for plasma [66,71,72,87] and shrimp [92]. Slow thawing can have a devastating effect on the lipids of tissues. Phenomena of the same kind have been observed in many plant and animal tissues on storage. The presence of large amounts of unesterified fatty acids, diacylglycerols, phosphatidic acid or lysophospholipids in lipid extracts must be an indication that some permanent damage to the tissues and thence to the lipids has occurred.
Such lipids are powerful surfactants and have been found to have disturbing effects on enzymes and membrane functions in vitro and in vivo , so the high concentrations sometimes reported in the literature are clearly incorrect. In plant tissues in particular, the enzyme phospholipase D is released and can attack phospholipids, so that there is an appreciable accumulation of phosphatidic acid and related compounds.
For example, phosphatidylmethanol was found to be produced by phospholipase D-catalysed transphosphatidylation during extraction of developing soybean seeds with chloroform-methanol [89]. All the acyl lipids in potatoes were hydrolysed in minutes upon homogenization [28]. Other alterations to lipids can occur that are subtler and so are discerned less easily. For example, losses of galactolipids can take place without any obvious accumulation of partially hydrolysed intermediates [28,93]. Lipoxygenases can cause artefactual formation of oxygenated fatty acids, and autoxidation can be troublesome.
Problems in stabilizing plant membranes for lipid compositional analyses have been reviewed [14].
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Often these changes are marginal in their overall importance, since alterations to the main lipid components may be small. On the other hand, they can make a crucial difference to the concentrations of some important lipid metabolites. The precise free fatty acid and 1,2-diacylglycerol concentrations of tissues are recognized to be key metabolic parameters. With the latter, autolysis was presumed to occur during extraction.
The levels of diacylglycerols were also threefold higher when the latter technique was used, a finding later confirmed by others [1]. Similarly, lysophosphatidylcholine, which had earlier been reported to be a major constituent of chromaffin granules in the adrenal gland, was found to be absent when the tissues were frozen in liquid nitrogen immediately after dissection [4]. It has also been demonstrated that enzymic oxidation can cause losses not only of unsaturated fatty acids, but apparently of intact lipids [81].
Hydroperoxide groups of oxidized lipids apparently reacted to form covalent bonds with the proteins of membranes, from which they were released only on treatment with bacterial proteases. Presumably, similar effects would be seen with autoxidized lipids in tissues. Various pre-treatments have been suggested for de-activating enzymes so that tissues can be stored for longer periods. The lipases in small samples of plant [36] or animal [29] origin have been denatured by plunging into boiling water for short periods, and the shelf life of samples treated in this way was reportedly prolonged to a considerable extent.
Boiling with dilute acetic acid solution appeared to have a similar effect [82,83]. However, there is a need for practical re-evaluation of these procedures in the light of modern knowledge before they can be recommended. Boiling plant tissues before extraction certainly de-activated lipoxygenases and increased the recovery of linoleic and linolenic acids []. Conventional freeze-drying and perchloric acid pre-extraction of tissues were found to produce artefacts from phospholipids; acetone desiccation did not, but this would cause losses of simple lipids [62].
The use of appropriate solvents for extraction per se can also limit enzymic degradation of plant lipids see Section C and Section F. Endogenous tissue antioxidants generally provide sufficient protection against oxidation under these conditions, although this may not be true for serum [71,91].
Plastic bags, vials or other containers should be avoided scrupulously for storage purposes. As soon as possible, tissues should be homogenized and extracted with solvent at the lowest temperature practicable and certainly without being allowed to thaw. Safe storage of plasma lipoproteins, where both the protein and lipid components are potentially unstable, is a major topic in its own right and cannot be discussed here.
Similar precautions must be taken for the storage of lipids, after they have been extracted from tissues. In this circumstance, the principle danger is a loss of unsaturated fatty acid components through autoxidation. For example, rapid autoxidation of cardiolipin was found to occur on storage in chloroform [80]. Lipid extracts should therefore be stored at the lowest practical temperature, in an inert atmosphere, in an apolar solvent and in the presence of antioxidants [17].
In addition to storage considerations, there are two main facets to any practical procedure for extracting lipids from tissues, i. They are discussed in this and the next section respectively. Many different solvents will dissolve pure single lipid classes, but they are only suitable for extracting lipids from tissues if they can overcome the strong forces of association between tissue lipids and other cellular constituents, such as proteins and polysaccharides. However, even polar complex lipids, which do not normally dissolve easily in non-polar solvents, can sometimes be extracted with these when they are in the presence of large amounts of simple lipids such as triacylglycerols.
Therefore, the behaviour of a given solvent as a lipid extractant for a specific tissue cannot always be predicted. In order to release all lipids from their association with cell membranes or with lipoproteins, the ideal solvent or solvent mixture must be fairly polar.
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Yet, it must not be so polar that it reacts chemically with the lipids nor that triacylglycerols and other non-polar simple lipids do not dissolve and are left adhering to the tissues. If chosen carefully, the extracting solvent may have a function in preventing any enzymatic hydrolysis, but vice versa it should not stimulate any side reactions. There is an increasing awareness of the potential toxicity of solvents to analysts, and this is another factor that must be taken into consideration when selecting a solvent mixture, especially if the laboratory is not adequately equipped with fume hoods or other ventilation.
No solvent is completely safe. Those factors affecting the extractability of lipids by solvents have been reviewed comprehensively by Zahler and Niggli []. The two main structural features of lipids controlling their solubility in organic solvents are the hydrophobic hydrocarbon chains of the fatty acid or other aliphatic moieties and any polar functional groups, such as phosphate or sugar residues, which are markedly hydrophilic. Any lipids lacking polar groups, for example triacylglycerols or cholesterol esters, are very soluble in hydrocarbons such as hexane, toluene or cyclohexane and also in moderately polar solvents such as diethyl ether or chloroform.
In contrast, they are rather insoluble in a polar solvent such as methanol. The solubility of such lipids in alcohols increases with the chain length of the hydrocarbon moiety of the alcohol, so they tend to be more soluble in ethanol and completely soluble in butanol. Similarly, lipids with fatty acyl residues of shorter chain length tend to be more soluble in more polar solvents; tripalmitin is virtually insoluble in methanol but tributyrin dissolves readily.
Unless solubilized by the presence of other lipids, polar lipids, such as phospholipids and glycosphingolipids, are only slightly soluble in hydrocarbons, but they dissolve readily in more polar solvents like methanol, ethanol or chloroform. Such solvents with high dielectric constants and polarity are required to overcome ion-dipole interactions and hydrogen bonding.
Tabulated data on the solubilities of a limited range of "typical" lipids are available [98]. Analysts should be aware that water is also a solvent for lipids, contrary to some definitions of the term, and water in tissues or that used to wash lipid extracts, for example, can alter the properties of organic solvents markedly. Most complex lipids are slightly soluble in water and at least form micellar solutions, and lipids such as gangliosides, polyphosphoinositides, lysophospholipids, acyl-carnitines and coenzyme A esters are especially soluble see Section G.
Lipids exist in tissues in many different physical forms. The simple lipids are often part of large aggregates in storage tissues, such as oil bodies or adipose tissue, from which they are extracted with relative ease.
Ganglioside GM3 . sodium salt (bovine brain) - ALX - Enzo Life Sciences
In contrast, complex lipids are usually constituents of membranes, where they occur in a close association with such compounds as proteins and polysaccharides, with which they interact, and they are not extracted so readily. These interactions are only very rarely through covalent bonds, and in general weak hydrophobic or van der Waals forces, hydrogen bonds and ionic bonds are involved. For example, the hydrophobic aliphatic moieties of lipids interact with the non-polar regions of the amino acid constituents of proteins, such as valine, leucine and isoleucine, to form weak associations.
Hydroxyl, carboxyl and amino groups in lipid molecules, on the other hand, can interact more strongly with biopolymers via hydrogen bonds. Lipids such as the polyphosphoinositides are most likely bound to other cellular biopolymers by ionic bonds, and these are not easily disrupted by simple solvation with organic solvents. It is usually necessary to adjust the pH of the extraction medium to effect quantitative extraction in this instance.
In addition, purely mechanical factors can limit the extractability of lipids. The helical starch amylose molecules in cereals form inclusion complexes with lysophosphatidylcholine, for example, limiting its accessibility to solvents. Also, cell walls in some microorganisms are rather impermeable to solvents, especially in the absence of water, which must be added to cause swelling of cellular polysaccharides. Some fatty acid or other alkyl moieties may indeed be linked directly to proteins or polysaccharides by covalent bonds, and then the optimum isolation procedure is likely to be one more suited to the analysis of the biopolymer rather than of the lipid.
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In order to extract lipids from tissues, it is necessary to use solvents that not only dissolve the lipids readily but overcome the interactions between the lipids and the tissue matrix, and it is essential to perturb both the hydrophobic and polar interactions at the same time. As with much other interesting work, the method was not extended to other tissues, and there is some danger of transesterification occurring as a side reaction under these conditions see Section E below.
While there are limitations to its use and alternatives are frequently suggested, most lipid analysts have accepted that a mixture of chloroform and methanol in the ratio of 2: Since the publication of a classic paper on the subject by Folch, Lees and Stanley [26] in , this has become the standard against which other methods are judged although there are earlier applications of these solvents [25,]. Schmid [96] has addressed the problem of why chloroform-methanol 2: The capacity of chloroform to associate with water molecules, presumably by weak hydrogen bonds, is a key property.
Provided that the ratio of chloroform-methanol to tissue assumed to be mainly water is greater than In contrast, mixtures of methanol with carbon tetrachloride or tetrachlorethylene, which lack the active proton of chloroform, were only able to solubilize relatively small amounts of water. Many practical methods have been developed for chloroform-methanol extraction and these are discussed in Section F below. There are disadvantages, however. In addition to the toxicity problem, which is controllable in a well-ordered laboratory, the mixture is a potent irritant to skin.
Neither chloroform nor methanol is completely stable and both were found to generate acidic by-products, which could catalyse esterification of free fatty acids or transesterification of lipids see also Section E below [99]. Better recoveries of prostaglandins were reported with this mixture than with the Folch procedure [94], but it does not extract gangliosides quantitatively. Others used propanol-hexane Systematic studies of the solubilities of certain lipids in toluene-ethanol mixtures indicated that this combination might have superior properties to chloroform-methanol, but it does not appear to have been tested adequately with complex lipids or with samples of real biological interest [97].
Benzene-ethanol [61], benzene-methanol [] and propanol-benzene-water 2: Butanol saturated with water has been recommended for the extraction of cereals or wheat-flour [60,68], in which the lipids may be in close association with starch, some in the form of inclusion complexes. The structure of the starch granules appears to be the most important factor, however [67].
This solvent may have wider uses, for example for quantitative extraction of lipids that are relatively soluble in water, such as lysophospholipids [7,31] or acylcarnitines [56,69]; hexanol has even been recommended for the latter [69]. It is well known that diethyl ether or chloroform alone are good solvents for lipids, yet they are poor extractants of lipids from tissues. They can, however, have some practical value for the isolation of the non-polar lipids from triacylglycerol-rich tissues, such as oil seeds or adipose tissue, as they do not extract significant amounts of non-lipid contaminants at the same time.
When they are used to extract plant tissues, these solvents also enhance the action of phospholipase D [42] unfortunately, as does butanol [20]. Propanol and propanol strongly inhibit this reaction and the latter, which has the lower boiling point, has been recommended for use with plant tissues, as a preliminary extractant especially [43,77,78]. While simple lipids and glycolipids dissolve readily in acetone, it will not dissolve phospholipids readily and indeed is often used to precipitate them from solution in other solvents, in effect as a crude preparative procedure.
The lipid mixture is usually dissolved in diethyl ether and then four volumes of cold anhydrous acetone is added to precipitate the phospholipids [33]. On the other hand, endogenous water and the solubilizing effects of other lipid components may permit acetone to extract more phospholipids from animal or plant tissues than might be predicted from a knowledge of the solubility of lipid standards in the pure solvent. For example, ethyl acetate-acetone-water 2: Acetone has also been recommended as a preliminary extraction solvent, before conventional chloroform-methanol extraction [59].
A disadvantage is that it can react with certain lipids to produce artefacts see Section E below. As glycolipids are soluble in acetone, chromatographic solvents containing this solvent are frequently utilized in the separation of glycolipids from phospholipids. In recent years, supercritical fluids have been evaluated as extractants for lipids reviewed elsewhere [6,50].
While these appear to hold promise for selected simple lipids, there appears to be little prospect for more general use at the moment. As an alternative to conventional solvent extraction, the technology of column chromatography has been adapted to the purpose in specific circumstances see Section G. When polar organic solvents are used to extract lipids from tissues, they tend to co-extract appreciable amounts of natural non-lipid materials, such as amino acids, carbohydrates, urea and even salts, as contaminants. A variety of procedures have therefore been developed to eliminate these, ideally without causing losses of lipids.
For example, one of the simplest methods consists in evaporating the polar solvents, followed by dissolving the residual lipids in a small volume of a relatively non-polar solvent, such as hexane-chloroform 3: Such a clean-up is rarely complete so the procedure is little used, although it should not be overlooked when large numbers of similar samples have to be purified for routine analysis by less demanding techniques, such as thin-layer chromatography TLC.
Other procedures that have been tried, but with limited success only, include dialysis, adsorption and cellulose column chromatography, electrodialysis and electrophoresis.
Preparation of Lipid Extracts Tissues
Much of the contaminating material can be removed from chloroform-methanol 2: The solvents then partition into two layers or phases, the lower consisting of chloroform-methanol-water in the ratio Unfortunately, any gangliosides that may have been present also partition into the upper layer. These are minor compounds and their analysis is rather specialized, so a simple washing procedure of this kind yields satisfactory lipid samples for most purposes.
When they are required for further analysis, gangliosides can be recovered from the Folch upper phase by dialysis followed by lyophilization see Section G. It is not always recognized how important it is that the proportions of chloroform, methanol and water in the combined phases should be as close to 8: When it is necessary to wash the lower phase again to ensure the elimination of all the non-lipid contaminants, methanol-water 1: In a successful adaptation of the above method that is especially suited to large samples with a high water content, Bligh and Dyer [8] took into account the water already present in the samples when adding further water in the washing step.
As this procedure uses smaller volumes of chloroform and methanol, it is both economical and convenient.
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While this type of lipid purification procedure was first developed by Wells and Dittmer [], a modification described by Wuthier [] is simpler and thus more suitable for large numbers of samples. In brief, chloroform, methanol and water in the ratio 8: While lipids were eluted rapidly by further lower phase, contaminants of low molecular weight remained on the column. Complex disease in humans: Translational research from basic mycobacteriology to clinical medicine. Japanese Journal of Infectious Diseases Journal of Biological Chemistry Current Opinion in Plant Biology Nikaido H and Vaara M Molecular basis of bacterial outer membrane permeability.
Pathology in Research and Practice Rose AL , Farmer PM and Mitra N Clinical, pathologic, and neurochemical studies of an unusual case of neuronal storage disease with lamellar cytoplasmic inclusions: Journal of Child Neurology European Journal of Biochemistry Simons K and Ehehalt R Cholesterol, lipid rafts, and disease. Journal of Clinical Investigation Cold Spring Harbor Perspectives in Biology 3 Journal of Cell Science Warnecke D and Heinz E Recently discovered functions of glucosylceramides in plants and fungi.
Cellular and Molecular Life Sciences Brandenburg K and Wiese A Endotoxins: Relationship between structure, function, and activity. Current Topics in Medicinal Chemistry 4: Molecular Membrane Biology Structures, Relevance and Applications. Journal of Endotoxin Research 3: Journal of Membrane Biology Nikaido H Transport across the bacterial outer membrane.
Journal of Bioenergetics and Biomembranes Progress in Lipid Research Abstract Glycolipids are amphiphilic components of cell membranes, composed of a hydrophilic polar sugar headgroup backbone and a hydrophobic apolar lipid moiety anchoring the molecule in the membrane. Chemical structures of a glucosyldiacylglycerol, b galactosylceramide, c ganglioside GM 1 and d lipid part of bacterial deep rough mutant lipopolysaccharide from Escherichia coli.
Bathing solution consisted of Hepes buffer including 0. Structure of the backbone of glycopeptidolipids of M. Chemical structure of phenolic glycolipid from M. Chemical structure of a phytoglycolipid.