Phospholipid analysis

Supercritical fluid chromatography (SFC) has also been used in phospholipid analysis. According to Lafosse et al., phospholipid classes can be separated by SFC using a simple isocratic solvent consisting of 78.4/21.6 (w/w) mixture of carbon dioxide and a mixture of methanol, water, and triethylamine (95/4.95/0.05) in combination with a Zorbax Sil stationary phase detection was performed by evaporative light-scattering (20). 

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). 

J De Kock. The European Analytical Subgroup of ILPS—a joint effort to clarify lecithin and phospholipid analysis. Fat Sci Technol 95 352-355,1993. 

P Van der Meeren, J Vanderdeelen, L Baert. Phospholipid analysis by HPLC. In LML Nollet, ed. Food Analysis by HPLC. New York Marcel Dekker, 1992, pp 241-258. 

Van den Berg, J.J.M., Winterbourn, C.C., Kuypers, F.A. 1993. Hypochlorus acid-mediated modification of cholesterol and phospholipids analysis of reaction products by gas chromatography-mass spectrometry. J. Lipid Res. 34, 2005-2012. 

Fig. 11.4 shows separately curve 1 from Fig. 11.3 which is the dependence of W on the DPPC concentrations in the AF. The W(C) curves allow to determine the threshold concentration C i.e. the minimum phospholipid concentration at which there is a 100% probability of observation of black films (see Eq. (3.130)). At concentrations lower than C, NBFs are no more observed, since W sharply decreases to zero (films rupture). At concentrations higher than C, (W = 1), NBFs always form. Special studies with phospholipid analysis of amniotic fluid indicate that of all phospholipids in the AF, it is the DPPC that stabilises the foam bilayers. This analysis gives grounds to conclude that the concentration of each phospholipid (except DPPC) in the native AF is of an order lower than the corresponding... 

It has long been proposed that cytochrome c oxidase contains various phospholipids as intrinsic constituents and that two or three cardiolipins are essential for its catalytic activity (Robinson et al., 1980 Robinson, 1982 Sedlak and Robinson, 1999). However, the composition and structures of phospholipids in the enzyme are still matters of controversy. No experimental evidence has been obtained to discount the proposal that all phospholipids suggested are not intrinsic components of the enzyme. Thus, a careful phospholipid analysis of the crystalline enzyme and an X-ray structure determination are indispensable for the determination of the phospholipid composition. 

Modern phospholipid analysis is typically accomplished by high-performance liquid chromatography (HPLC) methods (80) or by nuclear magnetic resonance (NMR) techniques (81). 

Vaskovsky VE, Kostetsky EY, Vasendin IM (1975) A universal reagent for phospholipid analysis. J Chromatogr 114 129-141... 

Neerhof MG, Haney El, Silver RK, Ashwood ER, Lee IS, Piazze JJ. Lamellar body counts compared with traditional phospholipid analysis as an assay for evaluating fetal lung maturity. Obstet Gynecol 2001 97 305-9. 

Rutters, H., Sass, H., Cypionka, H. and Rullkotter, J. (2002b) Phospholipid analysis as a tool to study complex microbial communities in marine sediments. Journal of Microbiological Methods 48,... 

J De Kock, (1993) The European Analytical Subgroup of 1 L P S - A Joint Effort to Clarify Lecithin and Phospholipid Analysis Fat Sci Technol 95 (9), 352-355 J De Kock, (1995) The European Analytical Subgroup of 1 L P S In Characterization, Metabolism and Novel Biological Applications, pp 22-28 Edited by G Cevc F Paltauf Champaign AOCS Press L L Olson, A P Cheung, JPharm Biomed Anal 8, 725-728 (1990)... 

R.F. Jack, D.B. Ringelberg, D.C. White. Differential corrosion rates of carbon steel by combinations of a Bacillus sp., Hafnia alvei and Desulfovibrio gigas established by phospholipid analysis of electrode biofilm. Corrosion Science, Vol. 33, No. 12, pp. 1843-1853, 1992. 

Some recent references to phospholipid analysis will be found in various chapters of Hawthorne and Ansell (1982). 

Detailed analysis of phospholipids, as well as neutral lipids and nonesterified fatty acids, has become progressively more important as these molecules have come to represent intercellular messenger molecules that are involved in a number of disease states. The analysis of lipid patterns of blood and lipoproteins may therefore constitute a useful diagnostic tool. As noted in the previous edition, suitable methods for phospholipid analysis are often time consuming and tedious, since most of them include several separation and derivatization steps. 

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