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Qualitative and quantitative analysis of platelet activator factor (PAF) in human plasma, employing high-performance liquid chromatography coupled to sequential mass spectrometry (HPLCMS /MS)

Grant number: 15/18824-5
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): October 01, 2015
Effective date (End): September 30, 2016
Field of knowledge:Biological Sciences - Immunology
Principal Investigator:Lúcia Helena Faccioli
Grantee:Sarah Regina Pereira Santangelo
Home Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Associated research grant:14/07125-6 - New functional aspects of eicosanoids, AP.TEM

Abstract

Lipid mediators such as prostaglandins (PGs), leukotrienes (LTs), lipoxins (LXs) and Platelet Activator Factor (PAF) are important in the development and/or resolution of the inflammatory response. The biosynthesis of eicosanoids (EIC) and PAF involves activation of the enzyme phospholipase A2 (PLA2) and subsequent release of arachidonic acid (AA) and lyso-PAF. Free AA is then oxygenated by lipoxygenase or cyclooxygenase to eicosanoids, while the lyso-PAF is acetylated and converted to PAF by acetyl-CoA/ lyso-PAF-acetyltransferase. As the EIC, the PAFs are potent lipid mediators and are involved in many physiological and pathophysiological functions, including platelet aggregation, activation of neutrophils, neuronal differentiation and inflammatory response. Even today, the enzyme-linked immunosorbent assay (ELISA) is widely used for the quantitative determination of PAF in biological samples. Although, ELISA is highly sensitive, that not distinguish between different isoforms, such as PAF-C16:0, PAF-C18:0 and PAF-C18:1. The selectivity in the identification of PAFs is an important for the understanding of events related to these lipids, as it is known that different isoforms may be associated with different biological conditions. On this context, the spectrometric tandem mass (MS/MS) can provide structural information of phospholipids from their fragmentation patterns in collision-induced dissociation experiments (CID), which enables the development of highly selective detection methods, based on MS/MS spectra of each characteristic compound. Coupled with high-performance liquid chromatography (HPLC) which allows separating the compounds by elution time in chromatography column, HPLC-MS/MS (high-performance liquid chromatography coupled to mass spectrometry) is an important analytical tool which results high sensitivity and selectivity, and have been applied successfully to the analysis of different phospholipids in biological matrices.