Rapid quantitation of a large scope of eicosanoids in two models of inflammation: development of an electrospray and tandem mass spectrometry method and application to biological studies.
Assessment of eicosanoid levels in biological systems is important for understanding their role in cell function and pathophysiological events. Current methods of eicosanoid quantitation are limited by sensitivity, scope, or throughput. The development of a new method for eicosanoid assessment in biological samples by electrospray and tandem mass spectrometry (MS/MS) in the multiple reaction monitoring mode is described here. In this study, 14 biologically significant eicosanoids were quantitated in a single sample. Complete sample analysis required two repeated injections of 5 microliters with an analysis time of 1.5 min/injection. Limits of detection ranged from 0.5 pg for thromboxane B2 (TxB2) to 10 pg for 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha). The reliability, reproducibility, sensitivity, and cross-detection of the method is also described. The MS/MS method was used to explore eicosanoid production in two inflammation models: lipopolysaccharide (LPS)-stimulated human whole blood and carrageenan-challenged rat air pouch. The most abundant metabolites in LPS-stimulated whole blood were prostaglandin E2 (PGE2), TxB2, and 6-keto PGF1 alpha; prostaglandins E1, D2, and F2 alpha and leukotrienes B4 and C4 were detected in lower amounts. Eicosanoid levels determined by MS/MS were similar to those obtained by immunoassay and GC-MS. The most abundant metabolites detected in carrageenan-challenged rat air pouch were PGE2, 6-keto PGF1 alpha, and TxB2. The method described in this work is accurate and rapid and should greatly aid in evaluating the role of multiple eicosanoids in future biological studies.
Margalit
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A
Department of Inflammatory Diseases, Searle Research and Development, St. Louis, Missouri 63198, USA.
Duffin
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KL
Isakson
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PC
D000818:Animals; D015777:Eicosanoids / Q000097:blood / Q000378:metabolism*; D006801:Humans; D007249:Inflammation / Q000097:blood / Q000378:metabolism*; D018836:Inflammation Mediators / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D013058:Mass Spectrometry / Q000379:methods*; D051381:Rats; D011917:Rats, Inbred Lew; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D015777:Eicosanoids; D018836:Inflammation Mediators; D008070:Lipopolysaccharides