Modified lipids analysis

Three modifications of TLC are in use for lipid analysis 1) separation on unmodified silica gel layer, silica gel TLC 2) separation on a layer impregnated with silver ions, silver ion-TLC (Ag-TLC) and 3) separation on a layer modified with silanes or long-chain hydrocarbons to give a nonpolar stationary phase, reverse-phase TLC (RP-TLC). 

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). 

Packed columns. In this technique separation is by virtue of selectivity. A number of different types of modified silica particles developed for HPLC are applicable. In addition, there are a number of in situ modified packings that are of interest in connection with lipid analysis (Demirbiiker and Blomberg, 1991, 1992 Demirbiiker, Hagglund and Blomberg, 1990 Hag-glund, Demirbiiker and Blomberg, 1994)... 

The universal TLC facilities are utilized plates, adsorbents, microcapillaries, or micropipettes for sample application, development tanks, detection spray reagents, devices for spraying, and densitometers for quantification. Plates are either commercially precoated or handmade. Silica gel G (G, for gypsum as a binding substance), silica gel H (no binding substance) and, rarely, alumina and kieselguhr, form the thin-layer stationary phases. Complete sets of devices necessary for the preparation of handmade plates are commercially available. After the silica gel slurry is spread on the plates, they are left to dry in the air for at least 24 hr and shortly in an oven at 110°C. The plates are then ready for either direct use or for modification of the layer. From the great variety of precoated plates, which are commercially available and preferred nowadays, silica gel plates and plates with layers modified with carbon chains from C2 to C18 are of interest in lipid analysis. Understandably, precoated plates for Ag-TLC are not commercially available. Preparative TLC is performed mostly on 20 X 20 cm plates with layer thickness of... 

Turini, M.E., and Martin, J.-C. (2001) Sources, Functions, and Analysis of Conjugated Linoleic Acid and Its Metabolites, in Structured and Modified Lipids, (Gunstone, F.D., ed.) pp. 251-284, Marcel Dekker, New York. 

Modified Gas-Liquid Chromatography Method in Amniotic Fluid Lipids Analysis Used as the Index of Fetal Lung Maturation Aust. J. Med. Technol. 7(2) 58-63 (1976) CA 86 39568s... 

Hyperlipidemia has not clearly been established as a risk factor for stroke, although it is a modifiable risk factor for coronary heart disease. Recent studies show that statin use may reduce the incidence of a first stroke in high-risk patients (e.g., hypertension, coronary heart disease, or diabetes) including patients with normal lipid levels. A recent meta-analysis showed a 25% risk reduction for fatal and non-fatal strokes with statin use.4 Patients with a history of MI, elevated lipid levels, diabetes, and... 

A first approach to study the interaction of posttranslational modified Ras proteins with membranes was the analysis of binding and exchange of isoprenyl-ated peptides with and between lipid vesicles utilizing a fluorescent bimanyl label. Studies with K-Ras peptides revealed that a single isoprenyl group is sufficient for membrane association only if supported by carboxymethylation of the C-terminal cysteine [227,228]. 

New insights into the analysis of hydrophobically post-translational modified proteins could be achieved by the construction of lipidated proteins in a combination of bioorganic synthesis of activated lipopeptides and bacterial expression of the protein backbone (Fig. 19). The physico-chemical properties of such artificial lipoproteins differ substantially from those of the corresponding lipopeptides. The pronounced dominance of the hydrophilic protein moiety (e.g., for the Ras protein 181 amino acids) over a short lipopeptide with one or two hydrophobic modifications provides solubility up to 10 4 mol/1, while the biotinylated or fluorescence labeled lipopeptides exhibit low solubility in aqueous solutions and can be applied in the biophysical experiments only in vesicle integrated form or dissolved in organic solvent. 

A few years ago, we began a research program to develop methods of analysis which would involve the use of FAB and a high performance tandem mass spectrometer. The tandem instrument was the first triple sector mass spectrometer to be designed and built by a commercial instrument company (Kratos of Manchester, U.K.). The first mass spectrometer of the combination is a double focussing Kratos MS-50 which is coupled to a low resolution electrostatic analyzer, which serves as the second mass spectrometer U). This FAB MS-MS combination has been used to verify the structures of an unknown cyclic peptide (2), a new amino acid modified by diphtheria toxin (3), and an ornithine-containing lipid (4). A number of methods have also been worked out which rely on this instrumentation. They Include the structural determination of cyclic peptides (5), nucleosides and nucleotides (6), and unsaturated fatty acids (7) and the analysis of mixtures of both anionic (8) and cationic surfactants (9). 

Selected examples of analytical methods used for the determination of global profiling of lipids are listed in Table 5. Extraction is usually based on simple liquid extraction, using modified Folch or Blight and Dyer extraction (4,5). For more acidic lipids, such as PSs and phosphatidic acids, adjustment of the pH in the aqueous phase is required. The analysis is most typically performed with LC-MS in RPLC mode, with the UHPLC methods gradually replacing the conventional HPLC methods. HRMS systems, such... 

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