The investigations proposed in Core E involve the development of state of the art protocols to apply mass spectrometry for the quantitative and qualitative analysis of glycerolipids present in RAW cells, peritoneal macrophages and in tissues as part of the coordinated studies of LIPID MAPS as well as in specialized studies specifically relevant to this Core. Methods will be improved to quantitate triacylglycerols (TAG) at the isobaric molecular species level, cholesteryl esters (CE) and diacylglycerols (DAG) at the molecular species level using LC/MS strategies. Monoalkyl ether diacylglycerols (MeDAG) will also be quantitated at the isobaric molecular species level as well as techniques developed to structurally characterize the specific acyl groups and alkyl groups contained within each molecular species. A normal phase LC/MS system will be developed to generate quantitative data for these glycerolipid species at higher throughput than currently possible. Experiments are proposed to explore derivatization of diacylglycerols (DAG) to increase their ionization cross section as well as reduce acyl group migration that will make them amenable to the normal phase LC/MS analytical approach. The behavior of various derivatives in tandem mass spectrometry will be explored. The derivatization strategy will also be explored as a means to increase the ionization cross section and therefore detectability of unknown glycerolipids that appear in atherosclerotic plaques as part of the LIPID MAPS consortium investigations. Stable isotope tracer studies and LC/MS/MS strategies will be developed to measure metabolic flux (cte novo synthesis, turnover, and recycling) of glycerolipids and cholesterol esters in RAW and peritoneal macrophage cells using uniformly labeled carbon-13 fatty acids (palmitate, arachidonate, and linoleate). These LC/MS/MS strategies will be based on neutral loss scanning to uniquely quantitate tracee and tracer molecular species even when complex mixtures of molecular species are present. This stable isotope tracer method will be directly applied to determine the pathway of MeDAG biosynthesis as well as turnover of individual fatty acyl groups testing the hypothesis that polyunsaturated fatty acids present in MeDAG may be incorporated into eicosanoids following cellular stimulation. In addition, this research will lead to a better understanding of how lipids are involved in disease processes and information and methods developed will be used to make new drugs to treat diseases such as atherosclerosis and diabetes.
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