Abstract
Lipid signaling has become a major research area of cell biology and there is a need for methods that accurately and easily measure substrates and products of lipases involved in cell signaling. In this report, we provide new methodology for separation of more than 10 lipids in one chromatographic run by high pressure liquid chromatography (HPLC) and detection with an evaporative light scattering detector (ELSD). There is no significant loss of sphingomyelin and no large baseline change, no peak obscures another, and acidic phospholipids are cleanly separated. We have optimized the procedure for a two-pump HPLC, an inexpensive silica column without the use of a column heater jacket and for low grade nitrogen. An application of the procedure separates lipids from Xenopus laevis cells. These cells are commonly used in the study of various lipid signaling paths in cell division, fertilization, and after expression of exogenous membrane receptors.
Highlights
Lipid signaling has become a major research area of cell biology and there is a need for methods that accurately and measure substrates and products of lipases involved in cell signaling
Because phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are substrates for phospholipase D and phosphatidic acid (PA) is a product, separation techniques for these lipids are of interest (2)
Phosphatidylserine (PS) acts a cofactor for protein kinase C (3), phosphatidylinositol (PI) is a precursor for two lipids known to be important in lipid signaling (4)
Summary
Standards (all obtained from Avanti Polar Lipid; Ͼ99% chromatographically pure) were as follows: PA, 1,2-dioleoyl-sn-glycero-3phosphatidic acid; TG, 1,2,3-trioleoylglycerol; CH, cholesterol; CA, 1,1Ј,2,2Ј-tetramyristoyl-acyl-cardiolipin; PG, 1,2-dimyristoyl-sn-glycero-3-phospho(rac-1-glycerol); LPE, 1-oleoyl-2-hydroxy-sn-glycero-3phosphatidylethanolamine; LPC, 1-oleoyl-2-hydroxy-sn-glycero-3phosphatidylcholine; PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; PC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; SM, sphingomyelin from egg (percentage of molecular species: 78% for 16:0, 7% 18:0, 2% 20:0, 4% 22:0, 4% 24:1, 3% 24:0, 2% 22:6); PI, bovine liver phosphatidylinositol (2.7% 16:0; 48.4% 18:0; 14.5% 18:1; 8.8% 18:2, 9.2% 20:3; 13.4% 20:4); soy PI (29.5% 16:0, 8.18% 18:0, 5.7% 18:1, 47.26% 18:2, 7.18% 18:3), PS, 1,2-dioleoylsn -glycero-3-phosphoserine. It is important to obtain solvents with the lowest particulate value (“ppm”) because the ELSD can detect these particulates. The relationship between the amount of standards and the ELSD peak size was examined by linear or polynomial regression analysis, using Sigmaplot 4.0 for Windows (SPSS, Chicago, IL)
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