Analytical Methods and Steps to Sample Preparation for Determination of Molecular Species of Fatty A

The generation of the lipid-signal molecules diacylglycerol (DAG) and phosphatidic acid (PA) from phospholipids like phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositols (PIs) has been implicated in the transduction events essential for proliferation and differentiation in many cells. If the DAG or PA produced by cells is being generated by hydrolysis of a given phospholipid, then the fatty acid molecular species (MSFA) profile of the lipid-signal molecule should be nearly identical to that of the hydrolyzed phospholipid. Therefore, it is possible to find the source of stimulated DAG or PA by comparing the molecular species profile of the cellular DAGs or PAs to those of parent phospholipids (1 ,2 ). Lipids can be characterized by their fatty acids that differ in chain length, degree of unsaturation, configuration, and position of the double bonds, and the presence of other functionalities (see Note 1 ). High-performance liquid chromatography (HPLC) methods have been developed and are now routinely used to resolve most of the molecular species, either with or without derivatization. The nonderivatized HPLC procedures are fast and simple but relatively large amounts of lipids are required and the resolution is sometimes questionable. The derivatized HPLC procedures are more complex, but have a good resolution and are essential when diacyl, alkylacyl, and alkenylacyl subclasses have to be separated.