The broad objective of this grant application is to make available state-of-the-art electrospray tandem mass spectrometry to investigators at the University of Colorado Denver (School of Medicine) and National Jewish Health in order to facilitate studies of lipid mediator biosynthesis through accurate, precise, and sensitive quantitative analysis;and basic biochemical studies centered around phospholipid metabolism. In addition, lipid mediators that are involved in bacterial quorum sensing will be structurally elucidated and quantitated. The instrumentation requested is a state-of-the-art tandem quadrupole mass spectrometer with a linear ion trap that permits several mass spectrometric experiments to be carried out, including precursor ion scanning product ion scanning, neutral loss scanning, as well as multiple reaction monitoring, MSn analysis, and direct mass spectrometric analysis (MS analysis). Several specific research programs are currently being supported by mass spectrometric analysis including the detailed studies of transcellular biosynthesis of leukotrienes in transgenic and chimeric mice, analysis of lipid mediators generated in studies of macrophage response to fungal infections, and the role of cytosolic phospholipase A2. Structural characterization of biologically active phosphatidylserine and lysophosphatidylserine metabolites relevant to cellular apoptosis, and quantitation of metabolites of arachidonic acid generated in studies of coculture of various tumor cells with primary cultures of macrophages and macrophage cell lines. These studies have the primary goal of understanding the role of lipid mediators in health and disease, including cancer, and involve the quantitative measurement of 20-30 different metabolites of arachidonic acid in a single HPLC run in order to obtain a complete picture of the metabolism of arachidonic acid within a specific biological system. This could also be called eicosanomics. Studies are also proposed to support investigation of phospholipid biochemistry, including the role of phosphatidylglycerol in pulmonary surfactant during pulmonary infections as well as reacylation of fatty acids back into phospholipids in a biochemical pathway termed the """"""""Lands Pathway."""""""" The instrument requested has the capability of a large number of multiple reaction monitoring (MRM) experiments that will support not only the eicosanomics studies, but also the detailed phospholipid and lysophospholipid biochemical studies related to critical cellular processes as well as pulmonary disease. The instrument will be placed in our lipid mass spectrometry facility and devoted entirely to lipid biochemical investigations.
Metabolites of arachidonic acid are known to play important roles in health and disease through the formation of prostaglandins and leukotrienes. In order to quantitatively measure these molecules in biological systems, the approach using stable isotope dilution and tandem mass spectrometry is the method of choice. In addition to this, detailed biochemical studies of phospholipid and lysophospholipids can be pursued that will reveal basic biochemical mechanisms of lipid biochemistry not previously considered. These studies will lead to breakthroughs in our understanding of normal physiological as well as pathophysiologic processes.
|Wooding, Kerry M; Hankin, Joseph A; Johnson, Chris A et al. (2015) Measurement of estradiol, estrone, and testosterone in postmenopausal human serum by isotope dilution liquid chromatography tandem mass spectrometry without derivatization. Steroids 96:89-94|