Phospholipid extraction

Although extraction of lipids from membranes can be induced in atomic force apparatus (Leckband et al., 1994) and biomembrane force probe (Evans et al., 1991) experiments, spontaneous dissociation of a lipid from a membrane occurs very rarely because it involves an energy barrier of about 20 kcal/mol (Cevc and Marsh, 1987). However, lipids are known to be extracted from membranes by various enzymes. One such enzyme is phospholipase A2 (PLA2), which complexes with membrane surfaces, destabilizes a phospholipid, extracts it from the membrane, and catalyzes the hydrolysis reaction of the srir2-acyl chain of the lipid, producing lysophospholipids and fatty acids (Slotboom et al., 1982 Dennis, 1983 Jain et al., 1995). SMD simulations were employed to investigate the extraction of a lipid molecule from a DLPE monolayer by human synovial PLA2 (see Eig. 6b), and to compare this process to the extraction of a lipid from a lipid monolayer into the aqueous phase (Stepaniants et al., 1997). 

Black, G. E. Snyder, A. Heroux, K. Chemotaxonomic differentiation between the Bacillus cereus group and Bacillus subtilis by phospholipid extracts analyzed with electospray tandem mass spectrometry. J. Microbiol. Meth. 1997, 28, 187-190. 

Several cell lines were used to inveshgate the role of PS oxidahon in apoptosis. Preferenhal oxidahon of PS was observed in human leukemia HL-60 cells (Fabisiak et al, 1998, 2000 Kawai et al, 2000), and normal human keratinocytes (Shvedova et al, 2001). Similarly, in pheochromocytoma PC 12 cells exposed to a radical-generating anhneoplashc dmg, neocarzinostatin, extemalizahon of PS was potentiated by its selechve oxidation in whole cells (Schor et al, 1999). In contrast, this selechve PS oxidahon did not occur in liposomes prepared from mixtures of PnA-labeled phospholipids extracted from the ceUs and exposed to oxidants imder the same conditions (Fabisiak et al, 1998 Kagan et al, 2000 Shvedova et al,... 

C.1.1 Modified Bligh and Dyers Method for Phospholipid Extraction... 

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). 

Miller, N.G., Hill, M.W. and Smith, M.W. (1976). Positional and species analysis of membrane phospholipids extracted from goldfish adapted to different environmental temperatures. Biochimica et BiophysicaActa 55,644-654. 

Calfactant is purified surfactant phospholipids extracted from calf lungs and purified by gel permeation chromatography. Calfactant include phospholipids, neutral lipides, and hydrophobic surfactant - three biophysically active proteins SP-A, SP-B, and SP-C. It contained no preservatives. 

The observations on the aromas from cysteine + ribose reaction mixtures have been extended to compare the effect of different lipids triglycerides and phospholipids extracted from beef, and commercial egg lecithin (phosphatidylcholine) and egg cephalin (phosphatidylethanolamine) (L.J. Salter D.S Mottram, unpublished data). The inclusion of the beef triglycerides (TG) did not appear to have any effect on the aroma of the cysteine + ribose reaction mixture, which was sulfurous with an underlying meatiness. However, when beef phospholipids (FL) were used the meaty aroma increased markedly. Similarily, addition of egg lecithin (LEC) or egg cephalin (CEPH) to the cysteine + ribose reaction mixture gave increased meatiness, with the cephalin-containing mixture being judged to have the most meaty character. 

Bills et al. (1963) used pre-treated Amberlite resin dispersed in hexane to isolate FFAs from milk. Fat was removed from the resin using hexane, absolute ethanol and methanol and the FFAs were esterified prior to analysis by GC. Needs et al. (1983) extracted lipids from milk by using ether and the FFAs were isolated using a strong basic anion exchange resin (Amberlyst 26, BDH Ltd, Poole Dorset, UK). The FFAs were methylated and resolved by GC. McNeill et al. (1986) also used Amberlyst resin to isolate FFAs in conjunction with silicic acid to remove phospholipids. Extracted FFAs were then analyzed by GC. This method was used by McNeill and Connolly (1989) to quantify FFAs in a number of semi-hard cheeses. 

Phospholipids (Figure 3) are constituents of membranes and are only minor components of oils and fats, sometimes responsible for cloudiness. They are usually removed during degumming, the residue from soybean oil processing being a source of phospholipids used as food emulsifiers. The term lecithin is used very loosely for such material, and it may variously mean phosphatidylcholine, mixed glycerophospholipids, or cmde phospholipid extracts from various sources. Where possible, more specific nomenclature or the source and purity should be used (14). 

Coenzyme Q - A recent review on biomedical aspects of coenzyme Q (55) has appeared. Earlier work had established that ubiquinones and bacterial phospholipid extracts of various sources enhance phagocytosis, stimulate non-specific host-resistancel l 142 and exert... 

An automated SFE option has been utilized by Montanari et al. (47) and Taylor et al. (37,48) to develop the conditions most amenable to isolating PPL concentrates from deoiled soy meal. In the former case, a set of conditions was tested by programming the microprocessor controller of the automated SFE to run a large number of extractions on common samples at various pressures, temperatures, and cosolvent (ethanol) levels. The results showed that at 70°C and 40.7 MPa with 10 mol% ethanol, phosphatidylcholine could be extracted preferentially relative to the other phospholipids in the soya meal. By utilizing higher pressures, it was found that the total amount of phospholipids extracted could be substantially increased at the slight reduction of the phosphatidylcholine content in the final extract. 

Using a solvent-reagent system gives narrow line-widths in NMR spectra of phospholipid extracts when applied to human amniotie fluid. Resolution of the major components is achieved by manipulating... 

Odham et al. (1988) compared plasmaspray with thermospray by using ammonium acetate as a buffer, where PSP required no addition of buffer prior to ionization. The total ion current obtained in PSP was significantly higher than previously reported for TS (filament-on). No or very few molecular ions were observed as a result of the harder ionization in PSP compared with TS. Diacylglycerol- and monoacylglycerol-derived fragments of phospholipids were obtained. Cation-exchange HPLC separated PI and PE in a phospholipid extract from bacterial cells Pseudomonas fluorescens). Furthermore, PC, PE, PA, PS, cardiolipin (CL) and polyphosphoinositides (PIP and PIP-2) were also studied. 

Valeur, Michelsen and Odham (1993) showed how diacylglycerol- and monoacylglycerol-derived fragments of phospholipids were obtained. PG and PE were found in a phospholipid extract from bacterial cells Pseudomonas fluorescens). They achieved class separation of the phospholipids but did not apparently separate molecular species (Fig. 9.6). Collision-induced dissociation (CID)-MS-MS analyses of PI from soybean were performed which revealed specific fatty acid compositions of the selected diacylglycerol-derived fragment. 

Figure 9.12 Direct electrospray ionization-mass spectrometry analysis of human erythrocyte plasma membrane phospholipids (A) A positive-ion electrospray ionization (ESI) mass spectrum of erythrocyte plasma membrane phospholipid extract showing 14 molecular species of glycerophospholipids and 4 molecular species of sphingomyelin (B) A negative-ion ESI mass spectrum of the same extract of plasma membrane phospholipids showing more than 25 molecular species of ethanolamine glycerophospholipids and 8 molecular species of serine and inositol glycerophospholipids. Reprinted with permission from Han, X. and Gross, R. W., Electrospray ionization mass spectroscopic analysis of human erythrocyte plasma membrane phospholipids, Proc. Natl Acad. Scl USA, 91(22), 10635-9. Copyright (1994) National Academy of Sciences, USA.

For this reason we have tried to improve the sensitivity of the approach by prior removal of an important part of less essential phospholipids. Extraction of lyophilized cattle brain Na-K ATPase with n-hexane leads to removal of all cholesterol and of 68% (SE 1.8, n=5) of the phospholipids (Fig. 1). There is relatively little difference in extractability of the individual phospholipids, varying from 77% (SE 4.0, n=5) for phosphatidylserine to 61%... 

FIGURE 7.8 Comparison of the (a) FAB, (b) plasma desorption, (c) liquid SIMS, and (d) IRLD mass spectra of the phospholipids extracted from E coli. The mass spectra in (c) and (d) were obtained by delayed extraction after 20 and 50 (is, respectively.The molecular ions are MH in the FAB (a) and liquid SIMS spectra (c), M-H+2Na in the plasma desorption mass spectrum, (b) and MK in the IRLD mass spectrum (d). (Reprinted with permission from reference 20). 

Further studies showed that the acyl compositions of the major phospholipids could be significantly altered by changes in growth temperature, with phospholipids extracted from 15°C grown yeast containing higher proportions of C 3 polyenoic fatty acids than... 

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