Quantification of major classes of Xenopus phospholipids by high performance liquid chromatography with evaporative light scattering detection

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. —Stith, B. J., J. Hall, P. Ayres, L. Waggoner, J. D. Moore, and W. A. Shaw. Quantification of major classes of Xenopus phospholipids by high performance liquid chromatography with evaporative light scattering detection. J. Lipid Res. 2000. 41: 1448–1454. 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. —Stith, B. J., J. Hall, P. Ayres, L. Waggoner, J. D. Moore, and W. A. Shaw. Quantification of major classes of Xenopus phospholipids by high performance liquid chromatography with evaporative light scattering detection. J. Lipid Res. 2000. 41: 1448–1454. Major phospholipids vary greatly in their polarity and this has made separation of the major classes difficult (1Myher J.J. Kuksis A. General strategies in chromatographic analysis of lipids.J. Chromatogr. 1995; 671: 3-33Google Scholar). 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 (2Stith B.J. Woronoff K. Espinoza R. Smart T. sn-1,2-Diacylglycerol and choline increase after fertilization in Xenopus laevis.Mol. Biol. Cell. 1997; 8: 755-765Google Scholar). Phosphatidylserine (PS) acts a cofactor for protein kinase C (3Newton A.C. Johnson J.E. Protein kinase C: a paradigm for regulation of protein function by two membrane-targeting modules.Biochim. Biophys. Acta. 1998; 1376: 155-172Google Scholar), phosphatidylinositol (PI) is a precursor for two lipids known to be important in lipid signaling (e.g., phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 3′-phosphate) (4Anderson R.A. Boronenkov I.V. Doughman S.D. Kunz J. Loijens J.C. Phosphatidylinositol phosphate kinases, a multifaceted family of signaling enzymes.J. Biol. Chem. 1999; 274: 9907-9910Google Scholar). Sphingomyelin (SM) is a substrate for sphingomyelinase and this enzyme has been shown to be involved in cell regulation (5Liu G. Kleine L. Herbert R.L. Advances in the signal transduction of ceramide and related sphingolipids.Crit. Rev. Clin. Lab. Sci. 1999; 36: 511-573Google Scholar). The method described below separates PC, PE, PS, PI, and SM. We also report resolution of ceramide, triglycerides (TG), cholesterol (CH), free fatty acids (FA), phosphatidylglycerol (PG), cardiolipin (CA), monoacyl phosphatidylethanolamine (LPE), and monoacyl phosphatidylcholine (LPC). Most solvent systems used for separation of lipids begin with either n-hexane–2-propanol–water (6Christie W.W. Advances in Lipid Methodology—One. The Oily Press, Ayr, Scotland1992: 239-271Google Scholar) or chloroform–methanol (7Becart J. Chavalier C. Biesse J.P. Quantitative analysis of phospholipids by HPLC with light scattering detector: application to raw materials for cosmetic use.J. High Resolut. Chromatogr. 1990; 13: 126-129Google Scholar). Even with improvements of the hexane–2-propanol solvent system, there are problems with solvent viscosity and separation of PI, PS, and SM [see summary in ref. 8Caboni M.F. Menotta S. Lercker G. High-performance liquid chromatography separation and light scattering detection of phospholipids from cooked beef.J. Chromatogr. A. 1994; 683: 59-65Google Scholar; note the lack of sphingomyelin peak in ref. 9Homan R. Anderson M.K. Rapid separation and quantification of combined neutral and polar lipid classes by high-performance liquid chromatography and evaporative light-scattering mass detection.J. Chromatogr. B. 1998; 708: 21-26Google Scholar; negligible amounts of PS or sphingomyelin are reported for salmon eggs (10Silversand C. Haux C. Improved high-performance liquid chromatographic method for the separation and quantification of lipid classes: application to fish lipids.J. Chromatogr. B. 1997; 703: 7-14Google Scholar)]. Modifications include use of dangerous solvents (6Christie W.W. Advances in Lipid Methodology—One. The Oily Press, Ayr, Scotland1992: 239-271Google Scholar) or tetrahydrofuran [Christie and Urwin (11Christie W.W. Urwin R.A. Separation of lipid classes from plant tissues by high performance liquid chromatography on chemically bonded stationary phases.J. High Resolut. Chromatogr. 1995; 18: 97-100Google Scholar) and others recommend against its use because of oxidation]. Taking these criticisms into consideration, we chose the chloroform–methanol solvent system. However, the laboratories at the University of Colorado-Denver and at Avanti Polar Lipids (Alabaster, AL) independently found that the method of Becart, Chavalier, and Biesse (7Becart J. Chavalier C. Biesse J.P. Quantitative analysis of phospholipids by HPLC with light scattering detector: application to raw materials for cosmetic use.J. High Resolut. Chromatogr. 1990; 13: 126-129Google Scholar) produces a varying baseline and does not separate certain phospholipids well (e.g., PI or PS from PC). We have modified the Becart, Chavalier, and Biesse (7Becart J. Chavalier C. Biesse J.P. Quantitative analysis of phospholipids by HPLC with light scattering detector: application to raw materials for cosmetic use.J. High Resolut. Chromatogr. 1990; 13: 126-129Google Scholar) procedure to analyze lipids from Xenopus laevis cells. In addition, while many methods separate small amounts of phospholipid standards, cellular samples demand greater separation of elution times. That is, because large PE and PC peaks can obscure small PI and PS peaks (8 and 12), we altered the elution times of phospholipids so that PI and PS peaks are separated by 4 min or more from the larger PC and PE. The new method also corrects the varying baseline that occurs with the Becart, Chavalier, and Biesse (7Becart J. Chavalier C. Biesse J.P. Quantitative analysis of phospholipids by HPLC with light scattering detector: application to raw materials for cosmetic use.J. High Resolut. Chromatogr. 1990; 13: 126-129Google Scholar) method. Evaporative light scattering detectors (ELSD) are universal detectors because they quantify any solutes less volatile than the solvents (6Christie W.W. Advances in Lipid Methodology—One. The Oily Press, Ayr, Scotland1992: 239-271Google Scholar, 13McNabb T.J. Cremesti A.E. Brown P.R. Fischl A. High performance liquid chromatography/evaporative light-scattering detector techniques for neutral, polar, and acidic lipid classes: a review of methods and detector models.Semin. Food Anal. 1999; 4: 53-70Google Scholar). The use of the ELSD eliminates lipid derivatization, produces a quantitative response (6Christie W.W. Advances in Lipid Methodology—One. The Oily Press, Ayr, Scotland1992: 239-271Google Scholar, 7Becart J. Chavalier C. Biesse J.P. Quantitative analysis of phospholipids by HPLC with light scattering detector: application to raw materials for cosmetic use.J. High Resolut. Chromatogr. 1990; 13: 126-129Google Scholar), and allows the use of chloroform (this nonpolar solvent improves lipid solubility, especially that of sphingomyelin). The ELSD is not sensitive to solvent flow rate or ambient temperature (6Christie W.W. Advances in Lipid Methodology—One. The Oily Press, Ayr, Scotland1992: 239-271Google Scholar). In contrast, thin-layer chromatography (TLC) separation is sensitive to loading, has a relatively narrow linear response, and many have found that even the use of a scanner did not produce sufficient “accuracy„ (7Becart J. Chavalier C. Biesse J.P. Quantitative analysis of phospholipids by HPLC with light scattering detector: application to raw materials for cosmetic use.J. High Resolut. Chromatogr. 1990; 13: 126-129Google Scholar). Refractive index detectors are less sensitive, and their baseline changes with variation in temperature, pressure, and flow rate and with different solvents. Because of the latter problem, gradient separation with these detectors is restricted to a few pairs of solvents that have virtually identical refractive indices. UV absorption (typically, at 203–214 nm) is contraindicated because chloroform absorbs below about 245 nm. In addition, UV absorption is largely dependent on the number of double bonds present in the lipid (absorbance by naturally occurring functional groups in lipids is negligible; refs. 14Jungalwala F.B. Evans J.E. McCluer R.H. High-performance liquid chromatography of phosphatidylcholine and sphingomyelin with detection in the region of 200 nm.Biochem. J. 1976; 155: 55-60Google Scholar and 15Mawatari S. Murakami K. Analysis of membrane phospholipid peroxidation by isocratic high-performance liquid chromatography with ultraviolet detection.Anal. Biochem. 1998; 264: 118-123Google Scholar). Thus distearate phosphatidylcholine (no double bonds) has an extinction coefficient (m−1 cm−1) of 200 whereas that of dilinoleate phosphatidylcholine (4 double bonds) is 12,900 m−1 cm−1 (16Stolyhwo A. Martin M. Guiochon G. Analysis of lipid classes by HPLC with the evaporative light scattering detector.J. Liquid Chromatogr. 1987; 10: 1237-1253Google Scholar). Derivatization of lipids can result in higher levels of sensitivity than the ELSD but the procedure is subject to problems such as the use of “dry„ reagents, and varying levels of derivatization from preparation to preparation and from lipid to lipid (6Christie W.W. Advances in Lipid Methodology—One. The Oily Press, Ayr, Scotland1992: 239-271Google Scholar). Standards (all obtained from Avanti Polar Lipid; >99% chromatographically pure) were as follows: PA, 1,2-dioleoyl-sn-glycero-3-phosphatidic acid; TG, 1,2,3-trioleoylglycerol; CH, cholesterol; CA, 1,1′,2,2′-tetramyristoyl-acyl-cardiolipin; PG, 1,2-dimyristoyl-sn-glycero-3-phospho(rac-1-glycerol); LPE, 1-oleoyl-2-hydroxy-sn-glycero-3-phosphatidylethanolamine; LPC, 1-oleoyl-2-hydroxy-sn-glycero-3-phosphatidylcholine; 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-dioleoyl-sn-glycero-3-phosphoserine. All solvents were from J. T. Baker (Phillipsburg, NJ) or Fisher (Fairlawn, NJ) (high performance liquid chromatography [HPLC] grade) (choroform stabilized by 0.75% ethanol). 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). Excess liquid was removed and 80 Xenopus oocytes were rinsed and washed once with O-R2 (83 mm NaCl, 0.5 mm CaCl2, 1 mm MgCl2, and 10 mm HEPES, pH 7.9). After all excess O-R2 is removed, 1 ml of chloroform–methanol 1:2 was added and cells were homogenized with 20 strokes with a Dounce homogenizer filled with nitrogen. After transferring the extract to a test tube, the mortar and pestle were washed with 0.5 ml of chloroform followed by 0.5 ml of 1 m NaCl. Use of acid (instead of 1 m NaCl) to negate charge and drive phospholipids into the organic phase resulted in significant lyso formation (as detected by an enzymatic sn-1,2-diacylglycerol [sn-1,2-DAG] assay)(2Stith B.J. Woronoff K. Espinoza R. Smart T. sn-1,2-Diacylglycerol and choline increase after fertilization in Xenopus laevis.Mol. Biol. Cell. 1997; 8: 755-765Google Scholar). The extract and washes were combined and then centrifuged (clinical centrifuge; setting of high, 2 min) and the organic layer was removed and stored at −20°C. All lipid solutions were kept under nitrogen and were stored in chloroform to minimize oxidation and polymerization. Just before use, cell extracts were dried under nitrogen and reconstituted in chloroform–methanol 2:1. We examined whether this step resulted in loss of standards; less than 2% of PE, PI, PC, or SM was lost at this step but the loss of PS amounted to 14.6%. To separate lipids, the at Avanti Polar Lipids used an and an silica column mm mm to was 20 and no flow was the University of a HPLC was to a silica column mm These silica are the of bonded as the but bonded greater and more times. of the flow to a and to the ELSD were for this loss of of the of either 20 or 200 were In an to and this did not use a column heater a more silica or nitrogen for the and we modified the solvent gradient for a two-pump HPLC system. In there was a flow rate of 1 and column were about 1 as as 2 solvents with silica to silica loss from the Because the of a column can column can peak and Press, Scholar), and a column can a of were not In the ELSD was at a detector temperature of nitrogen flow pressure of to and a of of ELSD has a relatively low rate of nitrogen and can at without that the laboratories of Avanti Polar Lipids used grade nitrogen pure) whereas the University of Colorado-Denver used grade In the University of Colorado-Denver ELSD were and with the whereas Avanti laboratories used an to of detector the are not To phospholipid we altered solvent added a the amount of and the of by Becart, Chavalier, and Biesse (7Becart J. Chavalier C. Biesse J.P. Quantitative analysis of phospholipids by HPLC with light scattering detector: application to raw materials for cosmetic use.J. High Resolut. Chromatogr. 1990; 13: 126-129Google Scholar). The gradient used by Becart, Chavalier, and Biesse (7Becart J. Chavalier C. Biesse J.P. Quantitative analysis of phospholipids by HPLC with light scattering detector: application to raw materials for cosmetic use.J. High Resolut. Chromatogr. 1990; 13: 126-129Google Scholar) for phospholipid separation is a linear from to NJ) was kept in a and new are used as as the laboratories of Avanti Polar Lipids were to of a system, the method was for the more commonly two-pump HPLC as that at the University of a that the use of two different solvents by this procedure was used 1 is the solvent and is run for at min (this PS separation because a column between the 10 there is a linear gradient from 1 to 2 2 is run for min and then there is a linear gradient to has is run for To the HPLC for the a linear gradient to 2 on is then The on the to is so that 1 is and there is a linear gradient to B. the on is between and 20 and at in a new we the column with for 20 followed by for 20 The column was stored in begin with a of 1 was run the then a run (no was and a run with standards was after these a cell extract be The method of Becart, Chavalier, and Biesse (7Becart J. Chavalier C. Biesse J.P. Quantitative analysis of phospholipids by HPLC with light scattering detector: application to raw materials for cosmetic use.J. High Resolut. Chromatogr. 1990; 13: 126-129Google Scholar) was used to separate phospholipids from a Xenopus egg extract Separation was but these were the the to separate PI and PS, and that the elution times were relatively that large peaks of PE and PC to new a separation of phospholipid standards at the University of Colorado-Denver separation of column jacket is PS analysis is to be because there is a in PS peak size and peak using a column at temperature elution of PI, PC, and PE was not by column temperature to Separation of many different standards is shown in a from Avanti Polar Lipids that they not use the min of the run described in lipids (e.g., ceramide, and cholesterol Standards the of 10 to a lipid with a molecular of this be about to and these were used in the analysis of cellular using and did not the regression (6Christie W.W. Advances in Lipid Methodology—One. The Oily Press, Ayr, Scotland1992: 239-271Google Scholar, W.W. Urwin R.A. Separation of lipid classes from plant tissues by high performance liquid chromatography on chemically bonded stationary phases.J. High Resolut. Chromatogr. 1995; 18: 97-100Google Scholar) reported a linear ELSD response There are of ELSD of L. B. J. new method for phospholipid separation by high-performance liquid chromatography with light-scattering detection.J. Chromatogr. Scholar) or C. High-performance liquid chromatography method with light-scattering detection for of lipid analysis of from Chromatogr. B. Scholar). Because of of the solvent and the (16Stolyhwo A. Martin M. Guiochon G. Analysis of lipid classes by HPLC with the evaporative light scattering detector.J. Liquid Chromatogr. 1987; 10: 1237-1253Google Scholar), the ELSD produces different for the for In of the use of different solvent systems or different laboratories that acidic phospholipids (e.g., PI, PS, and produce detector Most ELSD detectors before about with of less than (6Christie W.W. Advances in Lipid Methodology—One. The Oily Press, Ayr, Scotland1992: 239-271Google Scholar, L. B. J. new method for phospholipid separation by high-performance liquid chromatography with light-scattering detection.J. Chromatogr. Scholar, An method for the and quantification of lipids by HPLC using light-scattering detection.J. Lipid Res. 1990; of of and Urwin and Haux and Anderson are of variation in from one to be to phospholipid or the amount of and Urwin (11Christie W.W. Urwin R.A. Separation of lipid classes from plant tissues by high performance liquid chromatography on chemically bonded stationary phases.J. High Resolut. Chromatogr. 1995; 18: 97-100Google Scholar) used a and A.E. Quantitative high-performance liquid chromatography analysis of plant phospholipids and using light-scattering Scholar) used a whereas and Haux (10Silversand C. Haux C. Improved high-performance liquid chromatographic method for the separation and quantification of lipid classes: application to fish lipids.J. Chromatogr. B. 1997; 703: 7-14Google Scholar) used a (all these laboratories used and and Anderson R. Anderson M.K. Rapid separation and quantification of combined neutral and polar lipid classes by high-performance liquid chromatography and evaporative light-scattering mass detection.J. Chromatogr. B. 1998; 708: 21-26Google Scholar) used a from their they used an acid In higher the by and Anderson R. Anderson M.K. Rapid separation and quantification of combined neutral and polar lipid classes by high-performance liquid chromatography and evaporative light-scattering mass detection.J. Chromatogr. B. 1998; 708: 21-26Google Scholar) to be to in the present no in a new These are of variation in from one to be to phospholipid or the amount of and Urwin (11Christie W.W. Urwin R.A. Separation of lipid classes from plant tissues by high performance liquid chromatography on chemically bonded stationary phases.J. High Resolut. Chromatogr. 1995; 18: 97-100Google Scholar) used a and A.E. Quantitative high-performance liquid chromatography analysis of plant phospholipids and using light-scattering Scholar) used a whereas and Haux (10Silversand C. Haux C. Improved high-performance liquid chromatographic method for the separation and quantification of lipid classes: application to fish lipids.J. Chromatogr. B. 1997; 703: 7-14Google Scholar) used a (all these laboratories used and and Anderson R. Anderson M.K. Rapid separation and quantification of combined neutral and polar lipid classes by high-performance liquid chromatography and evaporative light-scattering mass detection.J. Chromatogr. B. 1998; 708: 21-26Google Scholar) used a from their they used an acid In higher the by and Anderson R. Anderson M.K. Rapid separation and quantification of combined neutral and polar lipid classes by high-performance liquid chromatography and evaporative light-scattering mass detection.J. Chromatogr. B. 1998; 708: 21-26Google Scholar) to be to in the present no To of we used PI for lipid was because produces a ELSD response, and Avanti Polar Lipids have found that is sensitive to oxidation and is at The use of this lipid be a test of lipid and of PI the ELSD response was double a regression that was at the was the regression was polynomial to be a for all but a regression the regression was in to the and a was not the were not a function a regression in to the polynomial but the was the function did not produce an of of various regression analysis by and Quantitative analysis of phospholipids by high-performance liquid chromatography and light-scattering Chromatogr. B. 1995; Scholar) a function with as the regression analysis of a of PE to resulted in a polynomial the was than a linear regression and a polynomial to of the in the amount of phospholipid a R. Anderson M.K. Rapid separation and quantification of combined neutral and polar lipid classes by high-performance liquid chromatography and evaporative light-scattering mass detection.J. Chromatogr. B. 1998; 708: 21-26Google Scholar, An method for the and quantification of lipids by HPLC using light-scattering detection.J. Lipid Res. 1990; Scholar, A.E. Quantitative high-performance liquid chromatography analysis of plant phospholipids and using light-scattering Scholar). a PI molecular of the of the PI to also found a of about 1 (6Christie W.W. Advances in Lipid Methodology—One. The Oily Press, Ayr, Scotland1992: 239-271Google Scholar, W.W. Urwin R.A. Separation of lipid classes from plant tissues by high performance liquid chromatography on chemically bonded stationary phases.J. High Resolut. Chromatogr. 1995; 18: 97-100Google Scholar). We did not need to ELSD for sensitivity or increase the on the of we that the of detection be about is to the of by and A.E. Quantitative high-performance liquid chromatography analysis of plant phospholipids and using light-scattering Scholar). lipids that produce higher ELSD Becart, Chavalier, and Biesse (7Becart J. Chavalier C. Biesse J.P. Quantitative analysis of phospholipids by HPLC with light scattering detector: application to raw materials for cosmetic use.J. High Resolut. Chromatogr. 1990; 13: 126-129Google Scholar) detection of about These of detection can be with UV is about 1 of phosphatidylcholine one double is present in the phospholipid F.B. Evans J.E. McCluer R.H. High-performance liquid chromatography of phosphatidylcholine and sphingomyelin with detection in the region of 200 nm.Biochem. J. 1976; 155: 55-60Google Scholar). of detection are after phospholipid derivatization and the use of UV or of phospholipids can be detected F.B. Evans J.E. McCluer R.H. analysis of and by high-performance liquid J. Scholar). a measure of PI from the silica column used at the University of Colorado-Denver at min size for various amounts of PI as shown in These obtained a of 1 with different phospholipid with different made solvent and with with one a small of of variation less than of ELSD of of in a new The test of a method is whether separation of a cellular extract produces extracts molecular of lipids are not standards, and the amount of phospholipid an extract from Xenopus separation was Becart, Chavalier, and Biesse (7Becart J. Chavalier C. Biesse J.P. Quantitative analysis of phospholipids by HPLC with light scattering detector: application to raw materials for cosmetic use.J. High Resolut. Chromatogr. 1990; 13: 126-129Google Scholar) that can be used as an of and at about the negligible peak that and did not result in The amounts of phospholipids obtained from Xenopus eggs are to obtained and separation of phospholipids using Xenopus oocytes B.J. C. S. and increase of phospholipids and mass in Xenopus 13: in a Xenopus for 14.5% loss and with PS for 14.5% loss and in a new of can be added as an with the but we found no between peak in cellular extracts that and that did We that the method described produces resolution of acidic phospholipids and for this was by the and by from the at the University of