Eicosanoids analysis

Analysis of individual lipid species of a class in the Lipid MAPS protocols was largely developed based on reversed-phase LC-MS. For example, in the protocol for eicosanoid analysis, a C18 column (250 X 2.1 mm) was employed and a specific gradient from mobile phase A (water-acetonitrile-formic acid, 63 37 0.02, v/v/v) to B (acetonitrile-isopropanol, 50 50, v/v) was applied [71]. Individual eicosanoid species were detected by using the MRM approach and quantified in comparison to the relevant internal standard. 

ESI for eicosanoid analysis has been in the negative-ion mode [18]. In this section, a few examples of CID mass spectra of eicosanoid species are given, whereas general description of these CID spectra is referred to an excellent review article written by Dr Murphy and colleagues [18],... 

After more than 30 years of intensive research on the eicosanoids, it is not surprising that a vast collection of analytical techniques are available to the investigator. Exhaustive reviews dealing with various methodological aspects of eicosanoid analysis may be found in the literature. In the next pages, I will give a few examples of these methods and provide a series of select references to serve as an introductory guide. 

The absolute stereochemistries for both compounds were determined by CD analysis of the p-bromobenzoate derivatives. Two other eicosanoids were also isolated from whole animals [198], The trihydroxylated oxylipin 8jR,11S,12R-trihydroxyeicosa-5Z,9 ,14Z,17Z-tetraenoic acid (trioxilin A4,150) was detected in, or isolated from, five starfish species P. miniata, Dermasterias imbricata, Pycnopodia helianthoides, Culcita novaeguinea, and Nardoa tubercolata. Its structure was determined by H and 13C NMR and FAB-MS analyses of the natural product and of an acetonide derivative. The co6 analog of this compound (151) was isolated only from P. helianthoides. The relative stereochemistry in these metabolites (150, 151) was established from a comparison of NMR shifts with malyngic acid, a trihydroxylated C18 compound isolated from Lyngbya majuscula, while the absolute stereochemistry was proposed on the basis of the earlier isolation of 8R-HETE from P. miniata. 

Terms in bold are defined i neuroendocrine system 882 radioimmunoassay (RIA) 884 Scatchard analysis 884 endocrine glands 886 paracrine 886 autocrine 886 insulin 887 epinephrine 888 norepinephrine 888 catecholamines 888 eicosanoid... 

Brash, A.R. and Hawkins, D.J. 1990. High-performance liquid chromatography for chiral analysis of eicosanoids. Methods Enzymol. 187 187-195. 

The first identified cannabinoid receptor subtype, CB was cloned and demonstrated to have an amino acid sequence consistent with a tertiary structure typical of the seven transmembrane-spanning proteins that are coupled to G proteins. In addition to being found in the central nervous system, mRNA for CB has also been identified in testes. The central nervous system responses to cannabinoid compounds are believed to be mediated exclusively by CB, inasmuch as CB2 transcripts could not be found in brain tissue by either Northern analysis or in situ hybridization studies. CBj transduces signals in response to central-nervous-system-active constituents of C. sativa as well as synthetic bicyclic and tricyclic cannabinoid analogs, aminoalkylindole, and eicosanoid cannabimimetic compounds. CB is coupled to G, to inhibit adenylate cyclase activity and to a pertussis-sensitive G protein to regulate Ca2+ currents. 

Global lipid profiling methods typically cover molecular lipid species from the major classes of lipids such as cholesteryl esters, ceramides, mono-(MG), di- (DG), and TGs, and membrane PLs, for example, sphingomyelins (SMs), PCs, phosphatidylethanolamines (PEs), PSs, and lysophospholipids. In targeted lipid analysis, specific lipid classes that are poorly covered by the global profiling methods are usually analyzed. These lipids include steroids, sterols, bile acids, fatty acids, signaling lipids such as eicosanoids, and ceramides, as well as polar lipids and inositol lipids. 

The LC-MS analysis of these compounds is briefly reviewed in this section. Balazy [33] reviewed the analysis of eicosanoids in terms of targeted lipidomics. 

Griffiths et al. [36] described the analysis of HETE and diHETE by ESI-MS-MS of [M-H] on a sector-time-of-flight hybrid instrument equipped with a focal-plane detector. The HETE and diHETE could be analysed in underivatized form at the pmol level. The MS-MS spectra allow differentiation of isomeric eicosanoids. 

Next to these more fundamental studies on the structural characterization of eicosanoids, LC-MS was applied in the qualitative and/or quantitative analysis of eicosanoids in biological systems. Some selected examples are briefly reviewed. 

Margalit et al. [48] reported the simultaneous quantitative analysis of 14 eicosanoids in biological samples using column-bypass negative-ion ESI-MS in SRM mode. Detection limits ranged from 0.5 pg for TXBj to 10 pg for 6-keto PGF, . 

Eicosanoid Protocols, Q itQdhy Elias A. Lianas, 1999 119. Chromatin Protocols, edited by. 8. Becker, 1999 118. RNA CProtein Interaction Protocols, edited by Susan R. Haynes, 1999 117. Electron Microscopy Methods and Protocols, edited by M A. Nasser Hajibagheri, 1999 116. Protein Lipidation Protocols, edited by Michael H. Gelb, 1999 115. Immunocytochemical Methods and Protocols (2nd ed.), edited by Lorette C. Javois, 1999 114. Calcium Signaling Protocols, edited by David G. Lambert, 1999 113. DNA Repair Protocols Eukaryotic Systems, edited by Daryl S. Henderson, 1999 112. 2-D Proteome Analysis Protocols, edited by Andrew J. Link, 1999 111. Plant Cell Culture Protocols, edited by Robert D. Hall, 1999 110. Lipoprotein Protocols, edited by M Ordovas, 1998 109. Lipase and Phospholipase Protocols, edited by Mark H Doolittle and Karen Reue, 1999 108. Free Radical and Antioxidant Protocols, edited by Donald Armstrong, 1998... 

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). 

Mesaros C, Lee SH, Blair I A. Targeted quantitative analysis of eicosanoid lipids in biological samples using liquid chromatography—tandem mass spectrometry. J Chromatogr B 2009 877 2736-2745. 

The newer MS techniques, such as FAB/MS, TS/MS and ES/MS, allow the analysis of non-derivatized products. The combination of these techniques with MS/MS has also facilitated the identification of eicosanoids. 

Dumlao, D.S., Buczynski, M.W., Norris, P.C., Harkewicz, R., and Dennis, E.A. (2011) High-throughput lipidomic analysis of fatty acid derived eicosanoids and N-acylethanolamines. Biochim. Bio-phys. Acta, 1811 (11), 724-736. doi 10.1016/j.bbalip.2011.06.005... 

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