Folch extraction procedure

Prior to phospholipid analysis, it is imperative to extract the lipids from their matrix and free them of any nonlipid contaminants. Phospholipids are generally contained within the lipid fraction, which may be recovered by the traditional Bligh and Dyer or Folch extraction procedure (9,22). In any phospholipid extraction method it is recommended to include a rather polar solvent in addition to a solvent with high solubility for lipids. The former is needed to break down lipid-protein complexes that prevent the extraction of the lipids in the organic phase. Traditionally, mixtures of chloroform and methanol (especially 2 1, v/v) have been recommended. These are washed with water or aqueous saline to remove nonlipid contaminants. Comparing the recovery of phospholipids, Shaikh found that the neutral phospholipids PC, PE, SPH as well as DPG were nearly quantitatively extracted by all solvent systems studied (Table 1), although Bligh and Dyer, in which the lower phase was removed only once, was somewhat worse (23). 

In the Folch extraction procedure ground or homogenized tissue is shaken with a 2 1 mixture of chloroform and methanol and the organic extract is subsequently partitioned with aqueous potassium chloride solution. It is important that the ratio of chloroform, medianol and water in the final mixture be 8 4 3. The extracted lipids are in the (lower) chloroform layer. 

The extraction procedure used to isolate lipids from biological tissue depends on the class of lipid desired and the nature of the biological source (animal tissue, plant leaf, plant seed, bacteria, cell membranes, etc.). Because lipids are generally less polar than other cell constituents, they may be selectively extracted with the use of organic solvents. Early studies of lipids used ether, acetone, hexane, and other organic solvents for extraction however, these solvents extract only lipids bound in a nonpolar or hydrophobic manner. In the 1950s, Folch s group reported the use of chloroform and methanol (2 1) in... 

In order to prevent the formation of a stable emulsion at any stage of the extraction procedure, the water content of the hydrated WPC has to be controlled so as not to obtain a biphasic solvent system during extraction with mixtures of chloroform and methanol. Besides, nonlipid contaminants are removed from the extract by gel filtration on nonlipophilic Sephadex G-25 instead of traditional aqueous washing total lipids were eluted with a 19/1 (v/v) mixture of chlo-roform/methanol, saturated with water, whereas a 1/1 (v/v) mixture of water and methanol eluted nonlipid contaminants. The method yields a similar total lipid content to the Folch method, but it is about four times faster (24). 

The preparation of a lipid sample includes extraction of the lipid material from the examined object (seeds, tissues, food, etc.), choosing among the several widely accepted procedures. The extraction with chloroform-methanol (2 1) (the Folch extraction) is the most popular. A solution of known concentration in hexane or di-chloroethane is prepared and a suitable aliquot is applied on the plate as a small spot or, better, as a narrow band. Two, three, or more solvents, mixed in different proportions, give the mobile phase. Development is performed in common tanks (Desaga type, for example), in the ascending mode. For fine separations, cylindrical or sandwich-type tanks provide better results. 

Metabolism of CLN in Intestinal Epithelial Cells. Caco-2 cells (5 X 10 cells/dish) were precultivated in a 55-cm tissue culture dish for 24 h. Then, c9,tll,tl3-CLN was added to Ae culture medium as 10% AmeAyl sulfoxide (DMSO) solution. The final concentration of DMSO never exceeded 0.1% (v/v). After incubation, cells were trypsinized and washed three times with phosphate buffered saline. The TL of cells was extracted by Ae Folch s procedure (23). 

Extraction in 20 vol. of ethanol as described above is likely to give the most complete recovery of all types of bile acids. Alkaline conditions favor extraction. Folch extraction has been used to get a simultaneous purification, and the conjugated bile acids are then found in the upper phase (20). A different type of extraction is that described by Hofmann, who used a liquid anion exchanger, tetraheptylammonium chloride (21). This was added to bile and the salts formed with bile acids could be quantitatively extracted with ethyl acetate. The use of this solvent makes the evaporation of bile extracts simple. The amine can be removed with a cation exchanger, a procedure first described by Anderson (22). 

Intestinal contents can be extracted with ethanol or chloroform/ methanol (1, 20). Shioda et al. (20) used a Folch extraction for duodenal bile and recovered the conjugated bile acids in the top water phase. Free bile acids are often present in intestinal contents (usually distally) and may be partly lost in this procedure. 

Numerous extraction procedures are available for lipids, and this subject has been reviewed by Phillips and Privett (1979), Christie (1984), and Fried (1993). Of all extraction procedures, that of Folch et al. (1957) is used most frequently. This procedure involves extracting fresh animal or plant material with chloroform-methanol (2 1), usually in a volume ratio of 20 parts of the solvent to 1 part tissue or fluid. There are many variations of the Folch et al. (1957) procedure, and for descriptions of these variations, see Christie (1982, 1984). Experiment 4 involves the use of the Folch et al. (1957) procedure on 100 mg of animal tissue and 100 pi of serum. The Folch et al. (1957) procedure extracts nonlipid material along with lipids. Nonlipids are usually removed by an aqueous wash with either water or a dilute salt solution. Phillips and Privett (1979) devised an extraction procedure for brain tissue in which nonlipid material is first removed with dilute acetic acid. The brain tissue is then treated with chloroform-methanol essentially as described in Folch et al. (1957) to get a relatively pure lipid fraction. Because so many different lipid extraction procedures have been used prior to TLC analysis, different results for a given separation may reflect technique differences rather than acmal differences in lipid constituents of a sample. 

The technique just described for standards along with specific phospholipid spray tests have been used by Fried and Shapiro (1979) to analyze phospholipids in a parasitic flatworm. Sample preparation employed the Folch et al. (1957) extraction procedure (Experiment 4), and aliquots of sample were spotted essentially as described for standards. The results of that study showed that phosphatidyl choline, phosphatidyl serine, and phosphatidyl ethanolamine were the major phospholipids in the flatworm. Some variation should be expected in phospholipid analysis of animal tissues, although phosphatidyl choline, phosphatidyl serine, and phosphatidyl ethanolamine are usually major phospholipids in animal tissues. The TLC procedure described here following Folch et al. (1957) extraction of tissue should serve as a guide for the TLC analysis of phospholipids in biological materials. 

Folch Extraction Many modifications of the basic extraction procedure... 

Extraction of Plant Sampks With plant tissues, it is necessary to extract first with isopropanol in order to deactivate the enzymes, and a procedure devised by Nichols is usually recommended [44]. The plant tissues are macerated with 100 parts by weight of isopropanol. The mixture is filtered, the solid is extracted again in a similar manner and finally is shaken overnight with 199 parts of chloroform-isopropanol (1 1, v/v). The combined filtrates are taken up in chloroform-methanol (2 1, v/v) and given a Folch wash as described earlier. The purified lipids are recovered from the lower layer as described in Folch extraction. 

Entenman and Chaikoff (28) could find little difference between the choline contents of alcohol-ether extracts of plasma when choline reineck-ate was precipitated from solutions at pH 7-8 or from strongly acid solutions (29)—indicating the absence of other nitrogenous compounds that form insoluble reineckates. A difference was observed, however, when the choline content of liver phospholipids was analyzed by the two methods. Taurog, Entenman, and Chaikoff (88) also compared the choline phosphorus ratio of human plasma purified by the colloidal-iron procedure of Folch and Van Slyke (33) with the ratio obtained by their direct extraction procedure. The choline rpho horus ratio by direct extraction was 0.98, and when purified with colloidal iron was 0.96. 

Chromatographic methods are employed to analyze and classify brain lipids. The lipids from brain are generally extracted with a mixture of chloroform and methanol using variations of a method originally described by Folch et al. In most procedures, the tissue or homogenate is treated with 19 volumes of a 2 1 (v/v) mixture of... 

Procedures for isolation and measurement of lipids in foods include exhaustive Soxhlet extraction with hexane or petroleum ether (AOAC, 1995 see Basic Protocol 1), chloro-form/methanol (Hanson and Olley, 1963 Ambrose, 1969), chloroform/methanol/water (Folch et al., 1957 Bligh and Dyer, 1959 see Basic Protocol 2 and Alternate Protocol 2), acid digestion followed by extraction (see Basic Protocol 4), or, for starchy material, extraction with n-propanol-water (e.g., Vasanthan and Hoover, 1992 see Basic Protocol 3). Each method has its own advantages and disadvantages and successful measurement of lipid content is often dictated by the type of sample and extraction medium employed. Commercial extraction and preparation of edible oils are explained in the literature (Williams, 1997). 

However, Shaikh demonstrated that the aforementioned traditional methods are inappropriate to recover completely lysophospholipids as well as acidic phospholipids classical Folch gave 85-90% recovery of LPC and LPE, whereas Bligh and Dyer yielded only 75-80% recovery. Extraction with a mixture of chloroform and methanol, on the other hand, provided nearly complete recovery of acidic and lysophospholipids, but up to 15% losses were observed during subsequent washing, according to Folch. These losses could be circumvented by purification of the crude extract on Sephadex G-25, but this column chromatographic procedure is quite time-consuming. 

No single satisfactory procedure has been discovered that avoids all these complications. Thus, extraction conditions must be adapted to the tissue under study and to the quantity and type of lipid desired. As a point of departure, the total lipids of a wet tissue can usually be extracted with chloroform-methanol mixtures according to the procedure of Folch (or one of several variants of... 

Experiment I. In a time-course experiment, mucosal PGE production and phospholipid fatty acid profile were assessed at d 0,4,8,12, and 16 of dietary treatment in formula-fed and naturally reared piglets (n = 5 piglets per time per dietary treatment). Mucosal cells were scraped from proximal ends of the small intestine and frozen at -80°C for later lipid analysis. Lipids were extracted by a modified Folch procedure (15). Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were separated by thin-layer chromatography (16), and fatty acids in each phospholipid fraction were analyzed by gas chromatography. For eicosanoid measures, fresh mucosal tissue was incubated in Kreb s Ringer bicarbonate buffer as described previously (17). PGE2 was extracted from the incubation media with ethyl acetate and quantified using a competitive enzyme-linked immunosorbent assay (Cayman Chemical, Ann Arbor, MI). 

Lipids are commonly characterized by their solubility properties, i.e., they can be extracted by the Folch or Bligh and Dyer procedures. The next stage in the analytical procedure for mixtures involves... 

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