Fatty acid amides comprise a class of bioactive lipids that are frequently found in mammalian systems at very low concentrations. Function and metabolism are largely unknown. We do not know all of the bioactive fatty acid amides that are part of mammalian neural function. We also do not know whether abnormal metabolism of fatty acid amides leads to disease state, but a number of these amides are involved in the serotonin, GABA, and dopamine systems. There is speculation that they may be involved in neurological disorders such as depression, anxiety, and bipolar disorder. This proposal aims to address needs in the analysis of fatty acid amides. We will develop a microchip-based method for directly extracting lipids from cell or tissue samples, and couple it directly to a two-dimensional capillary liquid chromatography system that matches the sample size with the sensitivity of modern electrospray mass spectrometry. The system will be a combination of microchip technology and commercial instrumentation. The outcome will be a system for lipid analysis that is analogous to two-dimensional gel electrophoresis that is widely used in proteomics-optimized, in this case, for the analysis of polar neutral lipids, a class that contains the fatty acid amides. Because sensitivity is critical, we will also develop a microchip-based microdroplet generation platform for the chemical derivatization of selected lipid classes (fatty amines, acids, and amides) for ultrasensitive laser-induced fluorescence detection. It will work directly with the 2DLC system in place of mass spectrometry. We anticipate the ability to derivatize and measure lipids at concentrations on the order of 10-12 M and at masses below 1 zmol (10-21 mol). We will test these systems by surveying cell lines that are known to produce primary fatty acid amides when grown in fatty acids. We will test the 2DLC/MS system by following the uptake and metabolism of the fatty acids as they are converted to fatty acid amides through their putative intermediates. We will also test the LIF system by surveying the cell lines to see whether they naturally produce amides in the absence of acids in the growth medium. The broader impact of this work is (1) we will gain new knowledge of an important class of neuromodulator;(2) we will develop a definitive system for the separation of lipids for the field of lipidomics;(3) we will develop an extremely sensitive and efficient system for fluorescence derivatization that will be applicable to a wide variety of experiments involving ultralow concentrations and numbers of molecules. We expect this work to lead to experiments involving a broader approach to the purpose and function of fatty acid amides in normal and diseased tissue. We also expect this work to lead to experiments involving the extracellular dynamics of bioactive lipids.
The proposed work will develop new methods for the measurement of fatty signaling molecules in mammalian systems. The chromatography and spectroscopy methods will be broadly applicable to a number of lipid measurement problems. Specifically, we will develop a versatile system to separate lipid molecules according to their structure so they can be analyzed in very small amounts by very powerful mass spectrometry and laser spectroscopy tools. These methods will essentially provide a """"""""molecular map"""""""" of the fatty molecules in any given biological system.
