Quantification of phosphatidic acid and lysophosphatidic acid by HPLC with evaporative light-scattering detection

Abstract

Phosphatidic acid (PA) and lysophosphatidic acid (LPA) are lipids that regulate cellular processes. PA stimulates kinases and may play a role in exocytosis and membrane fusion. LPA can induce cell proliferation, platelet aggregation, and microfilament formation. Due to the growing interest in these lipids, rapid purification and quantification of these lipids is desirable. We now describe a method that utilizes one HPLC run to separate trace amounts of PA and LPA from large amounts of lipids found in cellular extracts. A two-pump HPLC with a solvent system consisting of chloroform, methanol, water, and ammonium hydroxide was employed to produce a reliable, efficient purification of the two lipids. Lipid mass was quantified by a sensitive evaporative light-scattering detector. Using this new method, insulin addition increased both PA (87%) and LPA (217%) mass in Xenopus laevis oocytes.