Phospholipids 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). 

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. 

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. 

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. 

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). 

Total phospholipids are quantitated by determination of the phosphorus content of lipid extracts from which non-lipid phosphorus has been removed by purification procedures. For this purpose chloroform-methanol extracts subjected to diffusion purification (Folch et al. 1951, Sperry 1955) are suitable. Lipid phosphorus is determined in aliquots of these extracts after digestion. Most procedures for determination of phosphorus are based on the method of Fiske and Subbarow (1925) which utilizes conversion of phosphate to phosphomolybdate and its subsequent reduction to molybdic blue. Modifications of this method were reviewed by Lindberg andERNSTER (1956). Avery convenient phosphorus assay was described by Bartlett (1959). Total phospholipids are calculated by multiplication of the lipid phosphorus values with 25. These values are only approximations since phosphorus does not represent exactly 4% of each phospholipid molecule. 

The Bligh and Dyer method [25] is a simple adaptation of the Folch, Lees, and Stanley procedure, developed to minimize solvent consumption. At first, this method was adopted to extract phospholipids from a fish muscle tissue it is more generally intended for the extraction of large samples, with a high proportion of endogenous water (typically. 

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