Phospholipid, detection

Phosphatidylcholine (PC) is the main phospholipid in all mammalian cells (40-50%) and lipoproteins. As a consequence, most oxidized phospholipids detected in mammalian tissues contain a choline head group. In addition to oxidized PC (oxPC), oxidized phosphatidylserine (oxPS) and phosphatidy-lethanolamine (oxPE) have been detected. The latter was found in the retina, which is a tissue containing high amounts of phosphatidylethanolamine (Gugiu et ah, 2006), while OxPS was reported to be present on the surface of apoptotic cells (Kagan et ah, 2002 Matsura et ah, 2005). 

Hyphenated TLC techniques. TLC has been coupled with other instrumental techniques to aid in the detection, qualitative identification and, occasionally, quantitation of separated samples, and these include the coupling of TLC with high-pressure liquid chromatography (HPLC/TLC), with Fourier transform infra-red (TLC/FTIR), with mass spectrometry (TLC/ MS), with nuclear magnetic resonance (TLC/NMR) and with Raman spectroscopy (TLC/RS). These techniques have been extensively reviewed by Busch (1996) and by Somsen, Morden and Wilson (1995). The chemistry of oils and fats and their TLC separation has been so well established that they seldom necessitate the use of these coupling techniques for their identification, although these techniques have been used for phospholipid detection. Kushi and Handa (1985) have used TLC in combination with secondary ion mass spectrometry for the analysis of lipids. Fast atom bombardment (FAB) has been used to detect the molecular species of phosphatidylcholine on silica based on the molecular ion obtained by mass spectrometry (Busch et al, 1990). 

Addition of 10 pM ABA in the culture medium for 2 or 10 min during a 10 min incubation in the presence of [ P] reduced significantly the labelling of total phospholipids. Moreover in cells cultivated during three days in the presence of 10 pM ABA before a 10 min [ P] incubation, the incorporation was very low, and the only labelled phospholipids detected were PIP and PA. 

Sherma, J., and Bennett, S. (1983). Comparison of reagents for lipid and phospholipid detection and densitometric quantification on silica gel and Cij reversed phase thin layers. J. Liq. Chromatogr. 6 1193-1211. 

Figure 6 Drawing of a thin-layer chromatogram of phospholipids separated from an extract of the digestive gland-gonad (DGG) complex of Biomphalaria glabrata snails. The silica gel G plate was developed in the first direction in chloroform-methanol-NH40H (65 35 5) and in the second direction in chloroform-methanol-water-acetic acid (65 25 4 1). Phospholipids were detected by using a variety of specific phospholipid detection reagents. Reproduced from Ref. 97 with the permission of Pergamon Press, Ltd.

Defourcq, J., and Faucon, J.F., 1978, Specific binding of a cardiotoxin fxomNaja mossambica mossam-bica to charged phospholipids detected by intrinsic fluorescence. Biochemistry 17 1170-1176. 

The presence of impurities like free fatty acids in egg or soybean phosphatidylcholine, or in the (semi)synthetic phosphatidylcholines (e.g., DMPC, DPPC, DSPC) can be detected by monitoring the electrophoretic behavior of liposome dispersions of these phospholipids in aqueous media with low ionic strength a negative charge will be found on these liposomes when free fatty acids are present in the bilayers. 

Adsorption chromatography using small particle silica or alumina has also been employed in the separation of biologically meaningful substances. Phospholipids, for example, have been separated on silica (38). One of the big problems for such substances is detection, since many of the compounds are not U.V. active. Generally, the refractive index detector is employed for isocratic operation, and the moving wire detector for gradient operation. Formation of U.V.-active derivatives is also possible (39). 

An excellent example of PLC applications in the indirect coupling version is provided by the works of Miwa et al. [12]. These researchers separated eight phospholipid standards and platelet phospholipids from the other lipids on a silica gel plate. The mobile phase was composed of methylacetate-propanol-chloro-form-methanol-0.2% (w/v) potassium chloride (25 30 20 10 10, v/v). After detection with iodine vapor (Figure 9.2), each phospholipid class was scraped off and extracted with 5 ml of methanol. The solvent was removed under a stream of nitrogen, and the fatty acids of each phospholipid class were analyzed (as their hydrazides) by HPLC. The aim of this study was to establish a standardized... 

Experiments with monkeys given intramuscular injections of a mineral oil emulsion with [l-14C] -hexa-decane tracer provide data illustrating that absorbed C-16 hydrocarbon (a major component of liquid petrolatum) is slowly metabolized to various classes of lipids (Bollinger 1970). Two days after injection, substantial portions of the radioactivity recovered in liver (30%), fat (42%), kidney (74%), spleen (81%), and ovary (90%) were unmetabolized -hexadecane. The remainder of the radioactivity was found as phospholipids, free fatty acids, triglycerides, and sterol esters. Essentially no radioactivity was found in the water-soluble or residue fractions. One or three months after injection, radioactivity still was detected only in the fat-soluble fractions of the various organs, but 80-98% of the detected radioactivity was found in non-hydrocarbon lipids. 

Clarke RJ (2001) The dipole potential of phospholipid membranes and methods for its detection. Adv Colloid Interfac Sci 89-90 263-281... 

To demonstrate an application of TIRF-FLIM, a FRET study of annexin A4 translocation and self-aggregation near the plasma membrane is shown in Fig. 9.4. This is a particularly useful application of TIRF-FLIM, since TIRF provides the spatial contrast of detecting only molecules immediately adjacent to the plasma membrane and the lifetime contrast reports on the aggregation state of annexin A4. Annexins are calcium-dependent lipid-binding domains with a different type of lipid binding domain compared to the common C2 domains (e.g., found in protein kinase C). Annex-ins consist of an N-terminal domain and a core domain binding calcium and phospholipids. The core domain is conserved in the... 

The tissue sections must be washed with organic solvents when the detection targets include peptides and proteins. Washing with organic solvents promotes the ionization of peptides and proteins mainly by removing phospholipids from the sections.14 Washing also flushes out salts that could interfere with the crystallization of the matrix. 

Peroxyl radicals are the species that propagate autoxidation of the unsaturated fatty acid residues of phospholipids (50). In addition, peroxyl radicals are intermediates in the metabolism of certain drugs such as phenylbutazone (51). Epoxidation of BP-7,8-dihydrodiol has been detected during lipid peroxidation induced in rat liver microsomes by ascorbate or NADPH and during the peroxidatic oxidation of phenylbutazone (52,53). These findings suggest that peroxyl radical-mediated epoxidation of BP-7,8-dihydrodiol is general and may serve as the prototype for similar epoxidations of other olefins in a variety of biochemical systems. In addition, peroxyl radical-dependent epoxidation of BP-7,8-dihydrodiol exhibits the same stereochemistry as the arachidonic acid-stimulated epoxidation by ram seminal vesicle microsomes. This not only provides additional... 

DCIA has been used to label numerous proteins and other biomolecules, including phospholipids (Silvius et al., 1987), to study the interaction of mRNA with the 30S ribosomal subunit (Czworkowski et al., 1991), in the investigation of cellular thiol components by flow cytometry (Durand and Olive, 1983), in the detection of carboxylate compounds using peroxyoxalate chemiluminescence (Grayeski and DeVasto, 1987), and for general sulfhydryl labeling (Sippel, 1981). 

T. Ohyashiki, M. Nunomura, and T. Katoh, Detection of superoxide anion radical in phospholipid liposomal membrane by fluorescence quenching method using 1,3-diphenylisobenzofuran. Biochim. Biophys. Acta. 1421, 131-139 (1999). 

The environment of a cell membrane is often modeled by a monolayer of phospholipid on the air-water interface. Our attempts to detect enantiomeric recognition in such films has largely involved dipalmitoylphosphatidyl choline (DPPC), which has a chiral headgroup situated at the junction of two 16-carbon unit chains. 

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