A procedure for fractionation of sphingolipid classes by solid-phase extraction on aminopropyl cartridges

Abstract

Solid-phase extraction (SPE) methods are easy, rapid, and reliable. Their growing popularity is in part due to their operational simplicity and cost reduction in solvents, and partly because they are easier to automate. Sphingolipids are implicated in various cellular events such as growth, differentiation, and apoptosis. However, their separation by small SPE cartridges has attracted limited attention. Here we describe an SPE procedure on aminopropyl cartridges that by sequential elution allows the separation of a lipid mixture into free ceramides, neutral glycosphingolipids, neutral phospholipids (sphingomyelin), and a fraction containing the acidic phospholipids and phosphorylated sphingoid bases, phosphoceramides and sulfatides. Individual components are obtained in high yield and purity. We applied the procedure to obtain data on separation of [3H]myristic acid-labeled sphingolipids from fish gills, and from human melanoma tumor tissue. Individual lipids in the SPE fractions were identified by chromatography on several high-performance thin-layer chromatography (HPTLC) systems. The chromatographic behavior of free sphingoid bases is also reported. —Bodennec, J., O. Koul, I. Aguado, G. Brichon, G. Zwingelstein, and J. Portoukalian. A procedure for fractionation of sphingolipid classes by solid-phase extraction on aminopropyl cartridges. J. Lipid Res. 2000. 41: 1524–1531. Solid-phase extraction (SPE) methods are easy, rapid, and reliable. Their growing popularity is in part due to their operational simplicity and cost reduction in solvents, and partly because they are easier to automate. Sphingolipids are implicated in various cellular events such as growth, differentiation, and apoptosis. However, their separation by small SPE cartridges has attracted limited attention. Here we describe an SPE procedure on aminopropyl cartridges that by sequential elution allows the separation of a lipid mixture into free ceramides, neutral glycosphingolipids, neutral phospholipids (sphingomyelin), and a fraction containing the acidic phospholipids and phosphorylated sphingoid bases, phosphoceramides and sulfatides. Individual components are obtained in high yield and purity. We applied the procedure to obtain data on separation of [3H]myristic acid-labeled sphingolipids from fish gills, and from human melanoma tumor tissue. Individual lipids in the SPE fractions were identified by chromatography on several high-performance thin-layer chromatography (HPTLC) systems. The chromatographic behavior of free sphingoid bases is also reported. —Bodennec, J., O. Koul, I. Aguado, G. Brichon, G. Zwingelstein, and J. Portoukalian. A procedure for fractionation of sphingolipid classes by solid-phase extraction on aminopropyl cartridges. J. Lipid Res. 2000. 41: 1524–1531. Sphingolipids, ceramides, and sphingoid bases are involved in numerous cellular events such as differentiation, growth, and apoptosis (1Hannun Y.A. Functions of ceramide in coordinating cellular responses to stress.Science. 1996; 274: 1855-1859Google Scholar, 2Spiegel S. Merrill Jr., A.H. Sphingolipid metabolism and cell growth regulation.FASEB J. 1996; 10: 1388-1397Google Scholar, 3Riboni L. Viani P. Bassi R. Prinetti A. Tettamanti G. The role of sphingolipids in the process of signal transduction.Prog. Lipid Res. 1997; 36: 153-195Google Scholar, 4Igarashi Y. Functional roles of sphingosine, sphingosine-1-phosphate, and methylsphingosines: in regard to membrane sphingolipid signalling pathways.J. Biochem. 1997; 122: 1080-1087Google Scholar, 5Gomez-Munoz A. Modulation of cell signalling by ceramides.Biochim. Biophys. Acta. 1998; 1391: 92-109Google Scholar, 6Hannun Y.A. The role of ceramide in cell signalling.Biochim. Biophys. Acta. 1998; 1436: 233-243Google Scholar, 7Ariga T. Jarvis W.D. Yu R.K. Role of sphingolipid-mediated cell death in neurodegenerative diseases.J. Lipid Res. 1998; 39: 1-16Google Scholar). Studies with these compounds usually require tedious methods that often involve preparative thin-layer chromatography or liquid–solid cartridge chromatography or both. Solid-phase extraction (SPE) is a technique that has elicited growing interest because it is rapid and reliable, requires less solvent, and can be easily automated (8Thurman E.M. Mills M.S. Overview of solid phase extraction.in: Winefordner J.D. Solid-Phase Extraction. Principles and Practice. 147. John Wiley & Sons, New York1998: 1-23Google Scholar). This technique has been used for extraction of lipid classes such as gangliosides (9Williams M.A. McCluer R.H. The use of Sep-Pak C18 cartridges during the isolation of gangliosides.J. Neurochem. 1980; 35: 26-269Google Scholar), prostaglandins, and related eicosanoids (10Powell W.S. Rapid extraction of arachidonic acid metabolites from biological samples using octadecylsilyl silica.Methods Enzymol. 1982; 86: 467-477Google Scholar) from biological samples. SPE with silica gel and aminopropyl-bonded matrix has been used in the fractionation of complex lipid mixtures, specifically for purification of neutral lipids (reviewed in refs. 11Ebeler S.E Shibamoto T. Overview and recent developments in solid-phase extraction for separation of lipid classes.in: Shibamoto T. Lipid Chromatographic Analysis. Marcel Dekker, New York1994: 1-49Google Scholar, 12Ebeler S.E. Ebeler J.D. SPE methodologies for the separation of lipids.Inform. 1996; 7: 1094-1103Google Scholar, 13Kaluzny M.A. Duncan L.A. Merritt M.V. Epps D.E. Rapid separation of lipid classes in high yield and purity using bonded phase cartridges.J. Lipid Res. 1985; 26: 135-140Google Scholar) and for recovery of some phospholipid classes (14Kim H.Y Salem Jr., N. Separation of lipid classes by solid phase extraction.J. Lipid Res. 1990; 31: 2285-2289Google Scholar, 15Suzuki E. Sano A. Kuriki T. Miki T. Improved separation and determination of phospholipids in animal tissues employing solid phase extraction.Biol. Pharm. Bull. 1997; 20: 299-303Google Scholar). Despite a growing interest in the study of sphingolipid metabolism, however, no detailed SPE procedure is available to sequentially separate free ceramides, neutral glycosphingolipids, and sphingophospholipids in a given sample. The sequential separation of different sphingolipids in the same sample is particularly important because their intracellular concentrations are interdependent, and they share structural, metabolic, and regulatory relationships (6Hannun Y.A. The role of ceramide in cell signalling.Biochim. Biophys. Acta. 1998; 1436: 233-243Google Scholar, 16Nikolova-Karakashian M. Morgan E.T. Alexander C. Liotta D.C. Merrill Jr., A.H. Bimodal regulation of ceramidase by interleukin-1β. Implications for the regulation of cytochrome P450 2C11 (CYP2C11).J. Biol. Chem. 1997; 272: 18718-18724Google Scholar, 17Komori H. Ichikawa S. Hirabayashi Y. Ito M. Regulation of intracellular ceramide content in B16 melanoma cells. Biological implications of ceramide glycosylation.J. Biol. Chem. 1999; 274: 8981-8987Google Scholar, 18Auge N. Nikolova-Karakashian M. Carpentier S. Parthasarathy S. Neger-Salvayre A. Salvayre R. Merrill Jr., A.H. Levade T. Role of sphingosine-1-phosphate in the mitogenesis induced by oxidized low-density lipoprotein in smooth muscle cells via activation of sphingomyelinase, ceramidase, and sphingosine kinase.J. Biol. Chem. 1999; 274: 21533-21538Google Scholar). We describe a procedure that allows simultaneous separation of these sphingolipids in a sample without any loss of resolution or recovery. Other molecules of interest in the lipid mixture, neutral lipids and phospholipids, are also recovered. The efficacy and the degree of separation were determined in two ways: a) by monitoring radioactivity in lipids labeled with [3H]myristate and b) by visualization of lipid spots on high-performance thin-layer chromatography (HPTLC) plates. The solid-phase extraction procedure was applied to separate lipids extracted from fish gills, and to purify free ceramides, neutral glycosphingolipids, and sphingomyelin from human melanoma tumors. All solvents were of analytical grade and were purchased from SdS (Peypin, France) or Carlo Erba (Milan, Italy). Lipid standards (cholesterol, cholesteryl oleate, methyl oleate, oleic acid, α-hydroxy palmitic acid, monoolein, diolein, triolein, oleyl alcohol, ceramides type III with sphingosine and normal fatty acids, ceramides type IV containing sphingosine and α-hydroxy fatty acids), sphingoid bases (sphingosine, phytosphingosine, dl-erythro-dihydrosphingosine), sphingosine 1-phosphate, ceramide 1-phosphate, cerebroside sulfate, and bovine serum albumin (fatty acid free) were from Sigma (Saint Quentin Fallavier, France). Ceramides containing phytosphingosine and normal fatty acid were from Coletica (Lyon, France). Mono-, di-, and trihexosylceramide and globoside standards were isolated from melanoma cells according to the procedure of Saïto and Hakomori (19Saïto S. Hakomori S. Quantitative determination of total glycosphingolipids from animal cells.J. Lipid Res. 1971; 12: 257-259Google Scholar) and purified by high-performance liquid chromatography (HPLC) (20Kannagi R. Hakomori S. and purification of glycosphingolipids by high-performance liquid Enzymol. Scholar). and silica gel without were from E. silica gel cartridges and were from or acid was purchased from and was from France). acid was in to bovine serum albumin according to A. for the of free fatty acid to Biochem. Scholar). from fish labeled with [3H]myristic acid were used to obtain data on the lipid recovery in fraction because that a high of free ceramide containing phytosphingosine and α-hydroxy fatty was J. G. J. G. Improved methods for the simultaneous separation of free from ceramides containing phytosphingosine and sphingosine bases with and fatty 1999; Scholar). these lipids were labeled with [3H]myristic acid J. G. O. M. G. A thin-layer chromatography procedure for simultaneous separation of ceramide and Lipid Res. 1997; Scholar), a with obtain data on lipid recovery with lipids from human melanoma in phytosphingosine is in The were obtained from and labeled as J. G. O. M. G. A thin-layer chromatography procedure for simultaneous separation of ceramide and Lipid Res. 1997; Scholar). were their and in were for in in of containing of [3H]myristic acid to bovine serum albumin A. for the of free fatty acid to Biochem. Scholar). the of were with and the lipids were extracted according to the procedure of J. M. A for the isolation and purification of total lipids from animal Biol. Chem. Scholar). The lipid were according to S. G. R. G. The of on the phospholipid metabolism of an Scholar) the was to and in to part of sphingosine in the The phase was used for separation of lipids by the SPE procedure and the fractions by different of the lipid from fish was with an lipid standards mixture containing of of the and ceramide type ceramide type ceramide with phytosphingosine to normal fatty acid, cholesteryl oleate, oleic acid, methyl oleate, oleyl alcohol, α-hydroxy palmitic acid, neutral glycosphingolipids of ceramide ceramide ceramide and phytosphingosine, and The mixture was and in of and on a cartridge with of were with of The sample was to to the matrix by the cartridge by The cartridge was with A of of a of by The cartridges were in of the same This was to be the to elution of However, be to a any was with the lipid in the can be used and are particularly high of lipids on the cartridges to of A neutral lipids in a fraction without ceramides, and free fatty and α-hydroxy fatty The was with and for of free ceramides and the A of free sphingosine and was also with The mixture applied was of and for of acid normal and α-hydroxy free fatty and of free sphingoid bases in fraction The was with and for of neutral glycosphingolipids and the free sphingoid bases in fraction their phosphorylated to the phospholipids were from the cartridge with and for of of sphingosine 1-phosphate, ceramide 1-phosphate, and were obtained by the cartridge with for of fraction The fractions from the aminopropyl cartridge were and in of was to the radioactivity in the The radioactivity in the cartridge was determined by the silica gel matrix from the it in a with of and of the N. A for liquid of lipids separation by thin-layer Acta. Scholar). The radioactivity was in a as J. G. O. M. G. A thin-layer chromatography procedure for simultaneous separation of ceramide and Lipid Res. 1997; Scholar). The radioactivity was as a of total radioactivity the The procedure was with cartridges from or and the were in in a Quentin Fallavier, France) with of the sample on the the different were sequentially applied to different lipid were used for cartridges with an of as detailed The melanoma were isolated from human and for lipid extraction according to J. M. A for the isolation and purification of total lipids from animal Biol. Chem. Scholar). The total phase was to and to to the phase was and the was in of and for the of of was and The phase obtained was and the phase was with phase by (8Thurman E.M. Mills M.S. Overview of solid phase extraction.in: Winefordner J.D. Solid-Phase Extraction. Principles and Practice. 147. John Wiley & Sons, New York1998: 1-23Google Scholar). The phase containing the sphingolipids was to of lipids in of were a The lipids were with the different solvents for SPE The of solvents were the given in the SPE procedure and in to with such high of solvents, an separation and recovery of lipids be The fractions were and on and the were in for separation of free ceramides, or in for separation of neutral The phospholipid was in and the spots were with and for the isolation and purification of total lipids from animal Biol. Chem. Scholar). The were with to neutral glycosphingolipids and with and by for to free ceramides The and of the SPE procedure were by chromatography of fraction on plates. standards were the samples. and were used to separate were used to the of of recovery and of fractions by use of the of the different lipids from fish were applied to cartridges and as in the procedure The fractions were with lipid and into neutral and lipids lipids were by the procedure by J. G. J. G. Improved methods for the simultaneous separation of free from ceramides containing phytosphingosine and sphingosine bases with and fatty 1999; Scholar). This allows the separation of different ceramide free fatty fatty alcohol, and methyl fatty acids, and cholesteryl from lipids and neutral and free sphingoid bases the The are according to a different procedure J. R. G. Improved for thin-layer chromatographic of lipids on silica gel Scholar), phospholipids from and from neutral and ceramide and were from by in A. A. L. H. R. are of and cell by Scholar). This procedure was in the the extraction of sphingolipids from melanoma of fraction to can with these sphingolipids the in the used a that in some in the determination of radioactivity in the fractions were to sphingolipids in the phase that were plates. the were and to or with and for visualization of and sphingomyelin for the isolation and purification of total lipids from animal Biol. Chem. Scholar). The lipid spots identified on the were into for determination of radioactivity J. G. O. M. G. A thin-layer chromatography procedure for simultaneous separation of ceramide and Lipid Res. 1997; Scholar). radioactivity in given spots of lipid the fractions was determined and used as the for that lipid The radioactivity in a given lipid in fraction as a of the for that This is the of the that of the lipid is in This is for lipid as in lipid from fraction in lipid spots in were for the lipid the of SPE the silica gel was from the cartridge to the radioactivity in the and data were as a of the total radioactivity the of of the in a fraction to the elution of free However, a small of is in the fraction containing free ceramides We a study of in the ceramide A of was with of The were separate SPE cartridges and according to the procedure The radioactivity in fraction was determined to the recovery of in ceramide and fractions was as a of the total radioactivity of in the different fractions of sphingoid recovery. free sphingoid bases were to in different fractions an of the recovery of sphingosine, and phytosphingosine in of these fractions was of these different sphingoid bases was a cartridge and as The fractions were on an and in McCluer R.H. chromatographic separation of sphingosine and related Lipid Res. Scholar). was used to the by with a of sphingoid bases was also by of their according to the procedure by Scholar). the was for a in a of in The was for and the spots were into containing in The were and to for The were and the was used to of a sphingoid was as the of the total of that sphingoid in the different We the SPE procedure with [3H]myristic fish lipids with standards to separate lipid components on an cartridge obtained from the cartridge were on and into components as in that neutral lipids were in the fraction with cholesteryl methyl fatty acids, and and are in the However, a small of is in fraction This is also in The data obtained from that of is in fraction as a to neutral free fatty and are in fraction are on the The fatty are from the cartridge with acid in fraction elution and recovery of ceramides in fraction is by the that different of free ceramides are in fraction and are in fractions as by the and were in the fraction containing the different neutral glycosphingolipids and are in fraction as in and of these glycosphingolipids was in any fractions and an recovery of glycosphingolipids with All sphingomyelin was in fraction as determined by with and is a fraction containing the phosphorylated and with and as in of were with sphingomyelin such as the and phase during the extraction were also to in fraction and the different free sphingoid bases sphingosine is with free ceramides in fraction the part of phytosphingosine is with neutral glycosphingolipids in fraction is these of free sphingoid bases were to compounds sphingosine on This be by using purified of lipids in the different fatty from fish were labeled with [3H]myristic acid and lipids were extracted as in the The lipids were on an cartridge and in fractions of fraction were with lipid standards and to and thin-layer chromatography to and neutral lipid as in the The spots were and the radioactivity was The radioactivity in a given lipid in a fraction was as the of the total in that lipid the the data on are as of the total for The are of separate of less are by a were obtained with from were with lipids to visualization of lipid spots determination of in a of free sphingoid bases in different of mixture of sphingoid standards was applied to an cartridge and in fractions as in The fractions were and applied to an to different sphingoid The sphingoid spots were with and by or as in The are of for A and for The fraction are given in in a from fish were labeled with [3H]myristic acid and lipids were extracted as in the The lipids were on an cartridge and in fractions of fraction were with lipid standards and to and thin-layer chromatography to and neutral lipid as in the The spots were and the radioactivity was The radioactivity in a given lipid in a fraction was as the of the total in that lipid the the data on are as of the total for The are of separate of less are by a were obtained with from were with lipids to visualization of lipid spots determination of A mixture of sphingoid standards was applied to an cartridge and in fractions as in The fractions were and applied to an to different sphingoid The sphingoid spots were with and by or as in The are of for A and for The fraction are given in We also applied the SPE to separate sphingolipids from melanoma and were to purified neutral glycosphingolipids in fraction This was the fraction that was on the The that and are the neutral glycosphingolipids in these and spots with and globoside are ceramides and sphingomyelin from the tumor were obtained in fractions and as in and is in phase in the solvents are used S. of Biochem. 1999; 272: Scholar) and is in the SPE fractionation of the phase The of the two spots in fraction is the they be acidic of the total of radioactivity on the cartridge and was by the cartridge matrix the of the elution the high of SPE procedure for lipid recovery and The SPE procedure allows the separation of sphingolipid classes in a the free ceramide with and a of the can be by and the can be by fraction a cartridge has been used by M.A. Duncan L.A. Merritt M.V. Epps D.E. Rapid separation of lipid classes in high yield and purity using bonded phase cartridges.J. Lipid Res. 1985; 26: 135-140Google Scholar) for elution of from aminopropyl cartridges by their was by using a of the we used for elution of and neutral However, we were to with the of in also in the total elution of because of it was in the ceramide fraction and This can be from the fraction as The for the can be and in the process of SPE cartridge their and in the of various solvents of bonded silica from solid-phase extraction of with chromatographic Scholar). However, in cartridges from two different and The of A in and the of in the cartridge with ceramides in fraction The mixture by M.A. Duncan L.A. Merritt M.V. Epps D.E. Rapid separation of lipid classes in high yield and purity using bonded phase cartridges.J. Lipid Res. 1985; 26: 135-140Google Scholar) for the elution of neutral lipids free fatty from that has been used (14Kim H.Y Salem Jr., N. Separation of lipid classes by solid phase extraction.J. Lipid Res. 1990; 31: 2285-2289Google Scholar, A. Rapid separation of the phospholipid classes on a aminopropyl Scholar, and in 11Ebeler S.E Shibamoto T. Overview and recent developments in solid-phase extraction for separation of lipid classes.in: Shibamoto T. Lipid Chromatographic Analysis. Marcel Dekker, New York1994: 1-49Google Scholar). free ceramides been to be J. G. J. G. Improved methods for the simultaneous separation of free from ceramides containing phytosphingosine and sphingosine bases with and fatty 1999; Scholar, J. G. O. G. J. from sphingosine in fish and a of methyl from Biophys. Res. 1998; Scholar). with we that a of with free ceramides in fraction This was by of and the same an recovery of free ceramide we the mixture of a was by and Salem (14Kim H.Y Salem Jr., N. Separation of lipid classes by solid phase extraction.J. Lipid Res. 1990; 31: 2285-2289Google Scholar) using and their neutral lipids with of fatty and α-hydroxy fatty was by that acid the used by acid in to in their study M.A. Duncan L.A. Merritt M.V. Epps D.E. Rapid separation of lipid classes in high yield and purity using bonded phase cartridges.J. Lipid Res. 1985; 26: 135-140Google Scholar, in 11Ebeler S.E Shibamoto T. Overview and recent developments in solid-phase extraction for separation of lipid classes.in: Shibamoto T. Lipid Chromatographic Analysis. Marcel Dekker, New York1994: 1-49Google Scholar), we to the of acid, because α-hydroxy free fatty acid in the and with with neutral However, the of acid to in elution and recovery of fatty acids, and of neutral glycosphingolipids on the cartridge and their recovery in a fraction with on the isolation of neutral glycosphingolipids with small SPE the by and Salem (14Kim H.Y Salem Jr., N. Separation of lipid classes by solid phase extraction.J. Lipid Res. 1990; 31: 2285-2289Google Scholar) were with neutral phospholipids in the same fraction on and no was of to separate neutral glycosphingolipids from on the purification of neutral glycosphingolipids is available of the to separate neutral glycosphingolipids from phospholipids by SPE used silica gel cartridges (reviewed in refs. 11Ebeler S.E Shibamoto T. Overview and recent developments in solid-phase extraction for separation of lipid classes.in: Shibamoto T. Lipid Chromatographic Analysis. Marcel Dekker, New York1994: 1-49Google and of complex lipids by high-performance thin-layer Biochem. 1985; Scholar, A. S. A of fatty in serum phospholipids using Sep-Pak Acta. Scholar, R. J. of and relationships in membrane by fatty acid Biophys. Acta. Scholar), and used or to neutral However, the recovery and purity of lipids in the fraction are often reported. We aminopropyl-bonded silica gel matrix to silica gel because of to free fatty on This a separation of free ceramides from fatty of as with the silica gel cartridges. This is particularly of lipid during free fatty are is also obtained in fraction by with neutral phospholipids such as and phospholipids can be easily by to the SPE or by neutral glycosphingolipids are in Sphingolipid and are on the matrix and are with acidic phospholipids such as acid and in This was for of the fractions in to the used by and Salem (14Kim H.Y Salem Jr., N. Separation of lipid classes by solid phase extraction.J. Lipid Res. 1990; 31: 2285-2289Google Scholar) to acidic phospholipids from their of acid containing acid was to to separate and from were However, isolation of because it into the phase S. of Biochem. 1999; 272: Scholar, Y. H. Hakomori S. Y. Quantitative of sphingosine-1-phosphate in biological samples by with Biochem. Scholar), that the of extraction and the in the process the degree of in the phase and an SPE procedure allows simultaneous separation of the sphingolipid classes in the from neutral lipid classes and This is an because the two classes can be by M.A. Duncan L.A. Merritt M.V. Epps D.E. Rapid separation of lipid classes in high yield and purity using bonded phase cartridges.J. Lipid Res. 1985; 26: 135-140Google Scholar, 15Suzuki E. Sano A. Kuriki T. Miki T. Improved separation and determination of phospholipids in animal tissues employing solid phase extraction.Biol. Pharm. Bull. 1997; 20: 299-303Google Scholar, A. Rapid separation of the phospholipid classes on a aminopropyl Scholar). with of the neutral lipids are in a fraction with A. fraction containing cholesteryl methyl fatty acids, and can be into components on an cartridge according to the procedure or on a silica gel SPE cartridge according to and Rapid separation of neutral free fatty and lipids using silica Sep-Pak Scholar). the phospholipids and on the aminopropyl cartridge elution of with can be from the cartridge by the procedure by and A. Rapid separation of the phospholipid classes on a aminopropyl Scholar). it is to phospholipids on a silica gel cartridge by the procedure by E. Sano A. Kuriki T. Miki T. Improved separation and determination of phospholipids in animal tissues employing solid phase extraction.Biol. Pharm. Bull. 1997; 20: 299-303Google Scholar), the of and be in to of total lipids be applied a cartridge without the different We used cartridges for separation of of lipids in to purify free ceramides, neutral glycosphingolipids, and for The cartridges are particularly for a of lipids as or from a small sample. We fish lipids were used on of the radioactivity the cartridge was in the and the to of lipid samples be on these cartridges. The procedure in can be a to for isolation of sphingolipids of the SPE procedure is and can be used by for isolation of or in with separation for lipid was by from the and The the of This was by a from the of of the