A fluorescent assay for ceramide synthase activity

Abstract

The sphingolipids are a diverse family of lipids with important roles in membrane compartmentalization, intracellular signaling, and cell-cell recognition. The central sphingolipid metabolite is ceramide, formed by the transfer of a variable length fatty acid from coenzyme A to a sphingoid base, generally sphingosine or dihydrosphingosine (sphinganine) in mammals. This reaction is catalyzed by a family of six ceramide synthases (CerS1-6). CerS activity is usually assayed using either radioactive substrates or LC-MS/MS. We describe a CerS assay with fluorescent, NBD-labeled sphinganine as substrate. The assay is readily able to detect endogenous CerS activity when using amounts of cell or tissue homogenate protein that are lower than those reported for the radioactive assay, and the Michaelis-Menten constant was essentially the same for NBD-sphinganine and unlabeled sphinganine, indicating that NBD-sphinganine is a good substrate for these enzymes. Using our assay, we confirm that the new clinical immunosuppressant FTY720 is a competitive inhibitor of CerS activity, and show that inhibition requires the compound's lipid tail and amine headgroup. In summary, we describe a fluorescent assay for CerS activity that circumvents the need to use radioactive substrates, while being more accessible and cheaper than LC-MS based assays. The sphingolipids are a diverse family of lipids with important roles in membrane compartmentalization, intracellular signaling, and cell-cell recognition. The central sphingolipid metabolite is ceramide, formed by the transfer of a variable length fatty acid from coenzyme A to a sphingoid base, generally sphingosine or dihydrosphingosine (sphinganine) in mammals. This reaction is catalyzed by a family of six ceramide synthases (CerS1-6). CerS activity is usually assayed using either radioactive substrates or LC-MS/MS. We describe a CerS assay with fluorescent, NBD-labeled sphinganine as substrate. The assay is readily able to detect endogenous CerS activity when using amounts of cell or tissue homogenate protein that are lower than those reported for the radioactive assay, and the Michaelis-Menten constant was essentially the same for NBD-sphinganine and unlabeled sphinganine, indicating that NBD-sphinganine is a good substrate for these enzymes. Using our assay, we confirm that the new clinical immunosuppressant FTY720 is a competitive inhibitor of CerS activity, and show that inhibition requires the compound's lipid tail and amine headgroup. In summary, we describe a fluorescent assay for CerS activity that circumvents the need to use radioactive substrates, while being more accessible and cheaper than LC-MS based assays. ceramide synthase dhCer (2S,3R)-2-amino-18-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)octadecane-1,3-diol polyethylenimine The sphingolipids are a diverse family of lipids and glycolipids that are essential for compartmentalization of cell membranes as signaling metabolites and as membrane anchors for carbohydrate patterning of the extracellular surface (1Hannun Y.A. Obeid L.M. Principles of bioactive lipid signalling: lessons from sphingolipids.Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2454) Google Scholar, 2Lopez P.H. Schnaar R.L. Gangliosides in cell recognition and membrane protein regulation.Curr. Opin. Struct. Biol. 2009; 19: 549-557Crossref PubMed Scopus (237) Google Scholar). The lipid ceramide is the central metabolite of the sphingolipid family and can be modified on its primary hydroxyl headgroup through the addition of a phosphocholine group forming sphingomyelin, a phosphate group forming ceramide 1-phosphate, or a hexose group forming glucosyl- or galactosylceramide. Sequential addition of galactose, N-acetylgalactosamine, and neuraminic acid groups to glucosylceramide gives rise to a diverse array of glycolipids termed globosides and gangliosides, which pattern the cell surface and are critically important mediators of higher order mammalian physiology (2Lopez P.H. Schnaar R.L. Gangliosides in cell recognition and membrane protein regulation.Curr. Opin. Struct. Biol. 2009; 19: 549-557Crossref PubMed Scopus (237) Google Scholar). In de novo sphingolipid synthesis, dihydroceramide is formed by transfer of a fatty acid chain from fatty acid-CoA to the amino group of sphinganine. This reaction is catalyzed by a family of six ceramide synthases (CerS1-6; also referred to as LASS1-6), in the endoplasmic reticulum. Dihydroceramide is then enzymatically desaturated to form ceramide. Sphingosine may also be N-acylated by CerS enzymes, directly forming ceramide through what is known as the sphingolipid salvage pathway. The fatty acids transferred to sphinganine or sphingosine vary in length from 14 to 28 carbons, are most commonly saturated or mono-unsaturated, and may be hydroxylated (1Hannun Y.A. Obeid L.M. Principles of bioactive lipid signalling: lessons from sphingolipids.Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2454) Google Scholar, 3Christie, W. W., . The Lipid Library. Available from: http://www.lipidlibrary.aocs.org. Accessed February 15, 2012.Google Scholar). Each CerS enzyme exhibits a preference for fatty acids of different length, for example CerS1 preferentially catalyses the addition of 18-carbon (C18) fatty acids to sphinganine (4Venkataraman K. Riebeling C. Bodennec J. Riezman H. Allegood J.C. Sullards M.C. Merrill Jr., A.H. Futerman A.H. Upstream of growth and differentiation factor 1 (uog1), a mammalian homolog of the yeast longevity assurance gene 1 (LAG1), regulates N-stearoyl-sphinganine (C18-(dihydro)ceramide) synthesis in a fumonisin B1-independent manner in mammalian cells.J. Biol. Chem. 2002; 277: 35642-35649Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar), whereas CerS2 preferentially adds long chain (C22–C26) fatty acids (5Laviad E.L. Albee L. Pankova-Kholmyansky I. Epstein S. Park H. Merrill Jr., A.H. Futerman A.H. Characterization of ceramide synthase 2: tissue distribution, substrate specificity, and inhibition by sphingosine 1-phosphate.J. Biol. Chem. 2008; 283: 5677-5684Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). The different acyl chain lengths of the ceramides appear to confer distinct functional roles; for example, long chain ceramides synthesized by CerS2 are essential constituents in myelin (6Ben-David O. Pewzner-Jung Y. Brenner O. Laviad E.L. Kogot-Levin A. Weissberg I. Biton I.E. Pienik R. Wang E. Kelly S. et al.Encephalopathy caused by ablation of very long acyl chain ceramide synthesis may be largely due to reduced galactosylceramide levels.J. Biol. Chem. 2011; 286: 30022-30033Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), C18 ceramide is an important pro-apoptotic mediator (7Koybasi S. Senkal C.E. Sundararaj K. Spassieva S. Bielawski J. Osta W. Day T.A. Jiang J.C. Jazwinski S.M. Hannun Y.A. et al.Defects in cell growth regulation by C18:0-ceramide and longevity assurance gene 1 in human head and neck squamous cell carcinomas.J. Biol. Chem. 2004; 279: 44311-44319Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 8Senkal C.E. Ponnusamy S. Bielawski J. Hannun Y.A. Ogretmen B. Antiapoptotic roles of ceramide-synthase-6-generated C16-ceramide via selective regulation of the ATF6/CHOP arm of ER-stress-response pathways.FASEB J. 2010; 24: 296-308Crossref PubMed Scopus (195) Google Scholar), and very long chain ceramides synthesized by CerS3 are critically important for skin barrier function (9Jennemann R. Rabionet M. Gorgas K. Epstein S. Dalpke A. Rothermel U. Bayerle A. van der Hoeven F. Imgrund S. Kirsch J. et al.Loss of ceramide synthase 3 causes lethal skin barrier disruption.Hum. Mol. Genet. 2012; 21: 586-608Crossref PubMed Scopus (202) Google Scholar). There are two methods that are regularly used for assaying ceramide synthase activity. One method uses tritiated sphinganine or sphingosine and resolution of ceramide products using TLC (10Lahiri S. Lee H. Mesicek J. Fuks Z. Haimovitz-Friedman A. Kolesnick R.N. Futerman A.H. Kinetic characterization of mammalian ceramide synthases: determination of K(m) values towards sphinganine.FEBS Lett. 2007; 581: 5289-5294Crossref PubMed Scopus (64) Google Scholar, 11Mizutani Y. Kihara A. Igarashi Y. Mammalian Lass6 and its related family members regulate synthesis of specific ceramides.Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (304) Google Scholar). Tritiated chemicals, although safe if used properly, are dangerous if ingested. Unlike other common radiochemicals, tritium is difficult to monitor. In most countries, work with radiochemicals involves extra paperwork whereas work with fluorescent substrates is safer and involves much less bureaucracy. An alternative radioactive assay with 14C-labeled fatty acid substrate has also been described (12Bose R. Kolesnick R. Measurement of ceramide synthase activity.Methods Enzymol. 2000; 322: 378-382Crossref PubMed Google Scholar). Another approach is to quantify ceramide formation using LC-MS/MS (13Berdyshev E.V. Gorshkova I. Skobeleva A. Bittman R. Lu X. Dudek S.M. Mirzapoiazova T. Garcia J.G. Natarajan V. FTY720 inhibits ceramide synthases and up-regulates dihydrosphingosine 1-phosphate formation in human lung endothelial cells.J. Biol. Chem. 2009; 284: 5467-5477Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Although highly accurate and sensitive, this approach requires lengthy access to highly specialized equipment and specific expertise. The HPLC takes 15 min per sample. For a relatively small experiment involving, for example, eight samples in triplicate and external standards at the start and end of the run, plus regular blanks, the machine time required is at least a full working day and usually an overnight booking. This method is therefore somewhat impractical when equipment is shared or centralized and regular CerS assays are needed. It is also associated with considerable costs when access or cost-recovery fees are charged, as is commonly the case with expensive core equipment. Herein, we describe a simple fluorescent assay for CerS activity in cultured cell or tissue homogenates, which uses commercially available sphinganine labeled with an NBD fluorescent group (NBD-sphinganine; (2S,3R)-2-amino-18-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)octadecane-1,3-diol). We show that this substrate is efficiently utilized by CerS enzymes, and that the assay can be used to screen for inhibitors of CerS activity. NBD-sphinganine, nervonoyl (C24:1)-CoA, sphinganine, C16:0 dihydroceramide, and C24:1 dihydroceramide were purchased from Avanti Polar Lipids (Alabaster, AL). Palmitoyl (C16:0) and stearoyl (C18:0)-CoA, and Fumonisin B1 were from Sigma Aldrich (Castle Hill, NSW, Australia). Silica Gel 60 TLC plates were from Merck (Kilsyth, VIC, Australia). All cell culture media reagents were from Invitrogen (Mulgrave, VIC, Australia). Human CerS2 and CerS5 cDNAs were purchased as full-length TrueClone cDNAs from OriGene (Rockville, MD) in vector pCMV-XL6 FTY720 was purchased from Cayman Chemical (Ann Arbor, MI). AAL(S) was synthesized following the procedure reported by Hinterding et al. (14Hinterding K. R. S. synthesis of of the immunosuppressant Lett. 2002; Scopus Google Scholar). The synthesis of AAL(S) and be described in in a were in with and were in with polyethylenimine Lu W. A and for protein in mammalian Biol. PubMed Scopus Google Scholar). Each of was with and of a 1 of and were for min to the and were of cell or tissue was essentially as described by et (10Lahiri S. Lee H. Mesicek J. Fuks Z. Haimovitz-Friedman A. Kolesnick R.N. Futerman A.H. Kinetic characterization of mammalian ceramide synthases: determination of K(m) values towards sphinganine.FEBS Lett. 2007; 581: 5289-5294Crossref PubMed Scopus (64) Google Scholar). of were using a cell then and in 1 and inhibitor The were on with to of a small a to that were The was then at for min to and The cell was and at The protein was by acid assay to tissue homogenates, were in as The assay was essentially as described (10Lahiri S. Lee H. Mesicek J. Fuks Z. Haimovitz-Friedman A. Kolesnick R.N. Futerman A.H. Kinetic characterization of mammalian ceramide synthases: determination of K(m) values towards sphinganine.FEBS Lett. 2007; 581: 5289-5294Crossref PubMed Scopus (64) Google using NBD-sphinganine in of unlabeled sphinganine with tritiated sphinganine. was fatty NBD-sphinganine, and fatty For was in the reaction to in reaction different samples due to different of homogenate in different that the of the reaction The were with the addition of homogenate protein and with at for were with the addition of The were at for 1 min to the and the lower was transferred to a The was as and the two were then in a in with Silica Gel 60 TLC and in and by Enzymol. 2000; PubMed Google Scholar). was directly on the TLC plates using a and fluorescent was using the associated NSW, Australia). NBD-sphinganine standards of different were the TLC in order to a for the of and Fumonisin B1 on enzyme the cell homogenate was for min with then to an of reaction the substrates to start the that the inhibitor was NBD-sphinganine, or and or cell were for at The reaction was and on TLC as described which were due to were of the TLC and the with The were and in then on an NSW, using a The was with a in the on the day of The were for on of using The was in using the at a resolution of at were a and the were the was in full 60 and using the in ceramide formation using the assay was essentially as described that sphinganine was used as the substrate in of that for these the sphinganine from to while other were Each was with of ceramide as an for and to the with were then and as described and in HPLC of A acid in acid in Lipids were on a 3 at a of using a 15 min at to 1 at for then to for 1 Lipids were on a in and used to reaction products were as ceramide, C16:0 dihydroceramide, C24:1 dihydroceramide, were using C16:0 and C24:1 dihydroceramide external standards the The of external to was used to and the of dihydroceramide in samples (13Berdyshev E.V. Gorshkova I. Skobeleva A. Bittman R. Lu X. Dudek S.M. Mirzapoiazova T. Garcia J.G. Natarajan V. FTY720 inhibits ceramide synthases and up-regulates dihydrosphingosine 1-phosphate formation in human lung endothelial cells.J. Biol. Chem. 2009; 284: 5467-5477Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). All were using which directly and from the Michaelis-Menten of was as the by the as a We NBD-sphinganine be as a substrate for CerS enzymes. were from or human CerS2 or and CerS activity was using or as the fatty acid substrate. was formed in the of cell 3 and or NBD-sphinganine and a was in the of fatty acid-CoA substrate The of formed was on the CerS enzyme and with to protein are in with that CerS5 preferentially fatty acid substrates whereas CerS2 preferentially substrates (C22–C26) Y. Kihara A. Igarashi Y. Mammalian Lass6 and its related family members regulate synthesis of specific ceramides.Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (304) Google and indicating that CerS5 in a much higher specific activity than CerS2 (10Lahiri S. Lee H. Mesicek J. Fuks Z. Haimovitz-Friedman A. Kolesnick R.N. Futerman A.H. Kinetic characterization of mammalian ceramide synthases: determination of K(m) values towards sphinganine.FEBS Lett. 2007; 581: 5289-5294Crossref PubMed Scopus (64) Google Scholar). formation was with to time in which more than of the NBD-sphinganine substrate was to this of substrate for as for the reaction in The of the products formed was using a C16:0 and C24:1 were using the fatty acid-CoA substrates and of CerS5 (C16:0) or CerS2 and from TLC plates as described in and values of and were for C16:0 and C24:1 indicating that the products been in C16:0 more on TLC than C24:1 using the and by Enzymol. 2000; PubMed Google Scholar). It was also to detect CerS activity and substrates in tissue Using amounts of the homogenate much more C24:1 CerS activity, and higher C16:0 CerS activity, whereas CerS activity was if higher in This pattern is in with the that CerS2 is at a much higher in the (5Laviad E.L. Albee L. Pankova-Kholmyansky I. Epstein S. Park H. Merrill Jr., A.H. Futerman A.H. Characterization of ceramide synthase 2: tissue distribution, substrate specificity, and inhibition by sphingosine 1-phosphate.J. Biol. Chem. 2008; 283: 5677-5684Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 11Mizutani Y. Kihara A. Igarashi Y. Mammalian Lass6 and its related family members regulate synthesis of specific ceramides.Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (304) Google Scholar). CerS1 is in the and we therefore higher CerS activity in This was of the CerS1 and other that C18 CerS activity, and or regulation of It is also that lipids in the homogenate somewhat with the CerS Using our assay, is to detect of reaction on the TLC values for triplicate in the in different the of on the TLC the that this of was by and the on TLC distinct from the of using a and is therefore a of the of resolution for the assay with cell NBD-sphinganine to the substrate in of enzyme the for NBD-sphinganine, with using TLC and a was with that with unlabeled sphinganine, in which the was using LC-MS/MS. In with as the fatty acid a of was for This is very to the of for sphinganine using the LC-MS C24:1 is utilized by different CerS with C16:0 we also the of CerS for NBD-sphinganine using C24:1 as the fatty acid an of was with NBD-sphinganine, and with sphinganine. A experiment for is in that NBD-sphinganine is a good substrate for ceramide with very to the substrate. from inhibition of CerS activity by the new for of FTY720 was reported to endogenous and CerS activity of human cell S. Park H. Laviad E.L. Lu X. Bittman R. Futerman A.H. synthesis is by the sphingosine FTY720 via a of and inhibition in an chain Biol. Chem. 2009; 284: Full Text Full Text PDF PubMed Scopus Google as as primary human endothelial (13Berdyshev E.V. Gorshkova I. Skobeleva A. Bittman R. Lu X. Dudek S.M. Mirzapoiazova T. Garcia J.G. Natarajan V. FTY720 inhibits ceramide synthases and up-regulates dihydrosphingosine 1-phosphate formation in human lung endothelial cells.J. Biol. Chem. 2009; 284: 5467-5477Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). We the FTY720 AAL(S) C. R.L. E. H. for sphingosine in a sphingolipid by FTY720 Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, V. C.E. R. S. R. C. E. T. et FTY720 sphingosine 1-phosphate Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google C16:0 CerS activity in cell C16:0 CerS activity was with by AAL(S) when with FTY720 The of inhibition for FTY720 and AAL(S) was competitive with to NBD-sphinganine as the for NBD-sphinganine from in the to or whereas the In with for the sphinganine inhibition was with to with in the a in the from protein in the to and for FTY720 and We two of are required for CerS the acyl lipid tail and the amine group the amine group in and the lipid tail in were essential for inhibition of C16:0 dihydroceramide synthesis, and the lipid tail by reduced the of inhibition at from to to the C16:0 CerS activity in our assay was also by Fumonisin B1 a CerS inhibitor Jr., A.H. van Wang E. K. Fumonisin B1 inhibits sphingosine (sphinganine) and de novo sphingolipid in cultured in Biol. Chem. Full Text PDF PubMed Google Scholar). was competitive with to NBD-sphinganine from to FTY720 and inhibition with the pattern of inhibition with to and The has that NBD-sphinganine may be used in of sphinganine as a substrate for CerS activity assays. The assay with endogenous or CerS enzymes, and cell or tissue on of the Michaelis-Menten the substrate to show an enzyme to the sphinganine. values reported 1 and for C16:0 and 3 and for C24:1 CerS are very to those reported in a assay was (10Lahiri S. Lee H. Mesicek J. Fuks Z. Haimovitz-Friedman A. Kolesnick R.N. Futerman A.H. Kinetic characterization of mammalian ceramide synthases: determination of K(m) values towards sphinganine.FEBS Lett. 2007; 581: 5289-5294Crossref PubMed Scopus (64) Google Scholar, R. Kolesnick R. Measurement of ceramide synthase activity.Methods Enzymol. 2000; 322: 378-382Crossref PubMed Google Scholar). by et al. (10Lahiri S. Lee H. Mesicek J. Fuks Z. Haimovitz-Friedman A. Kolesnick R.N. Futerman A.H. Kinetic characterization of mammalian ceramide synthases: determination of K(m) values towards sphinganine.FEBS Lett. 2007; 581: 5289-5294Crossref PubMed Scopus (64) Google Scholar), using different assay much higher et al. (13Berdyshev E.V. Gorshkova I. Skobeleva A. Bittman R. Lu X. Dudek S.M. Mirzapoiazova T. Garcia J.G. Natarajan V. FTY720 inhibits ceramide synthases and up-regulates dihydrosphingosine 1-phosphate formation in human lung endothelial cells.J. Biol. Chem. 2009; 284: 5467-5477Abstract Full Text Full Text PDF PubMed Scopus (136) Google reported a of for that is the a to the CerS enzymes. CerS activity was to substrate inhibition at sphinganine or NBD-sphinganine, the inhibition is for and at M.C. A. The of substrate a with diverse 2010; PubMed Scopus Google Scholar). is difficult to if substrate inhibition of CerS is of the in the sphinganine in the endoplasmic substrate inhibition be a the synthesis of ceramides in the of sphinganine. The assay was by that C16:0 CerS activity is by We competitive inhibition with to NBD-sphinganine, in with Merrill et al. Jr., A.H. van Wang E. K. Fumonisin B1 inhibits sphingosine (sphinganine) and de novo sphingolipid in cultured in Biol. Chem. Full Text PDF PubMed Google Scholar). with to was in our assay whereas Merrill et al. reported inhibition with to is a sphinganine and our and that with the sphinganine substrate is the primary Jr., A.H. van Wang E. K. Fumonisin B1 inhibits sphingosine (sphinganine) and de novo sphingolipid in cultured in Biol. Chem. Full Text PDF PubMed Google Scholar). of endogenous C16:0 CerS activity by FTY720 and its related AAL(S) was also competitive with to NBD-sphinganine, in with the of et al. (13Berdyshev E.V. Gorshkova I. Skobeleva A. Bittman R. Lu X. Dudek S.M. Mirzapoiazova T. Garcia J.G. Natarajan V. FTY720 inhibits ceramide synthases and up-regulates dihydrosphingosine 1-phosphate formation in human lung endothelial cells.J. Biol. Chem. 2009; 284: 5467-5477Abstract Full Text Full Text PDF PubMed Scopus (136) Google from the by et al. S. Park H. Laviad E.L. Lu X. Bittman R. Futerman A.H. synthesis is by the sphingosine FTY720 via a of and inhibition in an chain Biol. Chem. 2009; 284: Full Text Full Text PDF PubMed Scopus Google Scholar). In of the of inhibition for the sphinganine C16:0 CerS activity was by either the lipid or the amine which are of sphinganine. Using we quantify endogenous CerS activity in cell or This with using tritiated sphinganine (10Lahiri S. Lee H. Mesicek J. Fuks Z. Haimovitz-Friedman A. Kolesnick R.N. Futerman A.H. Kinetic characterization of mammalian ceramide synthases: determination of K(m) values towards sphinganine.FEBS Lett. 2007; 581: 5289-5294Crossref PubMed Scopus (64) Google Scholar). Measurement of endogenous CerS activity in has been reported to of of protein per assay, which is more than an order of more than the homogenate used in our assay K. J. Futerman A.H. The sphingoid of sphingolipids is at the surface of the endoplasmic in J. PubMed Scopus Google Scholar). In a 14C-labeled membrane protein was therefore much more than is for our assay (12Bose R. Kolesnick R. Measurement of ceramide synthase activity.Methods Enzymol. 2000; 322: 378-382Crossref PubMed Google Scholar). of reaction were on the TLC plates with very One to of the 1 substrate to a for in activity different The fluorescent NBD-sphinganine substrate is commercially available and costs the same as the tritiated substrate per assay, that 1 NBD-sphinganine in a reaction is used for the fluorescent and tritiated sphinganine per reaction is used for the radioactive assay, as (10Lahiri S. Lee H. Mesicek J. Fuks Z. Haimovitz-Friedman A. Kolesnick R.N. Futerman A.H. Kinetic characterization of mammalian ceramide synthases: determination of K(m) values towards sphinganine.FEBS Lett. 2007; 581: 5289-5294Crossref PubMed Scopus (64) Google Scholar, 11Mizutani Y. Kihara A. Igarashi Y. Mammalian Lass6 and its related family members regulate synthesis of specific ceramides.Biochem. J. 2005; 390: 263-271Crossref PubMed Scopus (304) Google Scholar). Although costs are is much cheaper to the fluorescent assay amounts of labeled substrate can be purchased with the of tritiated substrate that can be can be reduced by the fluorescent assay more The LC-MS/MS based assay is very sensitive, with a of generally much more when equipment access or fees be An important with the fluorescent assay is that the fluorescent on the TLC can be using detect tritiated ceramide, either the ceramide from the TLC and quantify the by or the TLC to or a to its lower in to other can for to The radioactive assay also requires that radioactive be used in a to the use of which can in the use of radioactive is A with the fluorescent is that the can if are to the samples from methods are able to long chain from very long chain (C22–C26) ceramide and dihydroceramide C16:0 from or from C24:1 for example and by Enzymol. 2000; PubMed Google Scholar, L. S. B. of ceramides in the of the of 2010; PubMed Scopus Google Scholar). Although this is a with the of a in our assay is on of a fatty acid-CoA substrate dihydroceramide can be readily using which is an of this In summary, we a fluorescent alternative to tritium or LC-MS/MS for ceramide synthase activity. It is that this fluorescent assay the of CerS assays the