Selective detection phospholipids

Figure 10 shows the NMR spectrum of sonicated POPC-TTC vesicles at room temperature and selected pressures. Already the one-dimensional NMR spectra exhibit some interesting features. With increasing pressure, the signal intensity of the acyl-chain protons at 0.85 and 1.24 ppm decrease due to the pressure-induced rigidization of the acyl-chains, as it is also observed for pure phospholipid samples. At pressures above the fluid-gel main transition, which is detected at a pressure of about 1200 bar at 20 °C in pure POPC dispersions, the acyl-chain signals of pure lipid samples disappear completely, whereas in the spectra of the POPC-TTC system considerable signal intensities remain even up to pressures of 2800 bar. Furthermore, we observe for the... 

Pressure was applied in this study to fine tune the lipid chain-lengths and conformation and to select specific lamellar phases. For example, the phospholipid bilayer thickness increases by 1 A/kbar in the liquid-crystalline phase, and up to six gel phases have been found in fully hydrated DPPC dispersions in the pressure-temperature phase space up to 15 kbar and 80 °C, respectively. NMR spectral parameters were used to detect structural and dynamic changes upon incorporation of the polypeptide into the lipid bilayers. 

The hydroperoxides obtained on thermal oxidation of cholesteryl acetate (191e) can be selectively separated by SPE and elution with a polar solvent. After reduction to the corresponding alcohols by NaBH4 and further derivatization to the trimethylsilyl ether, the products can be subjected to GLC with ion-trap MS detection. It can be thus demonstrated with the aid of standards that under the oxidation conditions (160 °C for 90 min) only the 7-position is attacked, leading to the la- and 7/3-hydroperoxy derivatives, while the plausible 4-position remains unscathed . Treatment of erythrocite ghosts with t-BuOOH causes a manyfold content increase of 5-hydroxyeicosatetraenoic acid (5-HETE), 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and 5-oxoeicosatetraenoic acid (5-oxo-ETE) residues of phospholipids. These acids can be separated by HPLC, identified and quantitized by tandem MS . ... 

Many HPLC methods for phospholipids have been developed, but chromatographic resolution and dynamics of detection are not always satisfactory. For each source of phospholipids, special standards are needed due to the different distribution of fatty acids. These standards are expensive and in some cases are not available. Another problem is represented by the analysis of phospholipids in complex matrices. In many cases, separation is impossible or very difficult, not least due to the surface activity, which is desired in the application of phospholipids, but which complicates the analysis of these compounds. Therefore, a method is needed which is selective in the detection of phospholipids in order to avoid a separation from the matrix. The P NMR spectroscopy of phospholipids meets these requirements. The I.L.P.S. (Internationa Lecithin and Phospholipid Society) has chosen the P NMR method as the reference method [62],[63],[64]. It has been tested world-wide by round robin tests in comparison to various HPLC and TLC methods. With triphenylphosphate as internal standard, a pulse angle of 15°, 10-s relaxation delay, and 32-256 accumulations, the method has a precision of <0,5%. 

In the analysis of phospholipids from brain tissue section, over 30 experiments were performed on a single tissue sample before re-application of matrix. Performing multiple experiments on a single tissue section provided the opportunity to analyze multiple MS/MS spectra collected from the entire tissue section these data showed that at several m/z-values, three different phospholipid species were identified (PC, phosphtidylethanolamine (PE), and PS). Furthermore, at every single wt/z-value selected for an MS/MS experiment, a DHB cluster ion was detected (primarily based on the loss of 136 Da). 

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