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. Phosphatidic acid (PA) is believed to play a critical role in exocytosis, intracellular vesicle formation, membrane fusion (1-3), and insulin action (4). New reports suggest that PA also acts as a regulator in plant cells (5).Lysophosphatidic acid (LPA) has been identified as an important regulator of platelets and may be used clinically to fight cancer (6-9). LPA binds to G-protein-coupled receptors to stimulate phospholipase C-␤ , induce cell proliferation, and activate platelet aggregation (7).Thus, there is a desire to rapidly and accurately quantify these bioactive lipids in cellular extracts. However, it has been difficult to separate PA and LPA using HPLC since the trace amounts of these lipids present in cells typically coelute with cellular phospholipids that occur in much greater amounts [e.g., phosphatidylcholine (PC) and sphingomyelin (SM)]. An alternate method, two-dimensional TLC, has problems associated with the exposure of lipids at the plate surface. Lipid detection on TLC plates, typically with nonspecific stains, is relatively insensitive, and the intensity of the stain is linear only over a small range.We have modified our HPLC method for separation of major phospholipid groups (10), and with evaporative light-scattering mass detection, we now report an improved method that utilizes one HPLC run to separate PA and LPA from the major phospholipids found in cellular extracts.For detection of lipids, a spectrophotometer was not used, since lipids show negligible absorbance above about 215 nm. Detection with wavelengths lower than 215 nm prohibits the use of better solvents, such as chloroform (which absorbs in this range). We also did not use radioactive lipid precursors to quantify lipids, since this method is dependent upon labeling of all precursor pools to near-equilibrium; this method rarely produces actual mass values. Instead, we used evaporative light-scattering detection (ELSD) for sensitive quantification of the mass of lipids (10). MATERIALS AND METHODS Standards and chemicalsStandards (Avanti Polar Lipids; Alabaster, AL) were as follows: PA (1,2-dioleoyl-sn -glycero-3-PA); LPA (1-stearoyl-2-hydroxy-snglycero-3-phosphate); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; PC (1,2-dioleoyl-sn -glycero-3-phosphocholine); SM from egg (percentage, fatty acid composition: 78% for 16:0, 7% 1...

There are many psychological risk factors for cardiovascular disease, and the ability to reduce mortality depends on an ability to integrate care of these risk factors with traditional Framingham cardiovascular risk and use them both in routine practice. The aim of this article is to provide an update of all the major emotional and behavioral cardiovascular risk factors along with a practical treatment model for implementation. First, we provide a review of major emotional and behavioral cardiovascular risk factors, the associated primary effect, and proposed mechanism of action. Second, we provide an office-based approach to cardiovascular risk factor reduction and methods of reducing barriers to implementation, called Prevention Oriented Primary Care-Abridged. The approach integrates several forms of detection, assessment using the 3As (ask, assess, assist), and Stages of Change approaches, and subsequent efficient and targeted treatment with either Motivational Interviewing or further office intervention. A case example is provided to help illustrate this process.