{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,21]],"date-time":"2025-10-21T00:30:28Z","timestamp":1761006628792,"version":"build-2065373602"},"reference-count":30,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2014,11,18]],"date-time":"2014-11-18T00:00:00Z","timestamp":1416268800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/501100000925","name":"National Health and Medical Research Council","doi-asserted-by":"publisher","award":["APP1024966"],"award-info":[{"award-number":["APP1024966"]}],"id":[{"id":"10.13039\/501100000925","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Lipids"],"published-print":{"date-parts":[[2015,1]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Ceramides are a family of signalling lipids with diverse physiological functions that include pro\u2010differentiative and pro\u2010apoptotic signalling. Ceramides and their derivatives are major constituents of myelin, maintaining neuronal conductivity. Ceramides are synthesized by ceramide synthases, of which there are six isoforms in mammals (CERS1\u20136). These enzymes catalyse the transfer of a variable length fatty acid to a sphingoid base, typically sphingosine or dihydrosphingosine. We previously reported a fluorescent thin\u2010layer chromatography assay for ceramide synthase activity. In this paper we describe an improved fluorescent assay, using HPLC to achieve clear resolution of closely related ceramide species and to facilitate easy quantification of both product and substrate. Our HPLC assay protocol eliminates the need for a chloroform extraction step. Instead a simple three\u2010step procedure is used: (1) reactions are run; (2) reactions are terminated with addition of methanol and centrifuged; (3) products are quantified with HPLC. HPLC resolution enables assays in which multiple fatty acid substrates are used in the same reaction. Using this approach, we show that CERS2 demonstrates a preference for the monounsaturated C24:1 fatty acid substrate compared to the saturated C24:0 substrate, potentially explaining why myelin is enriched in ceramides containing the monounsaturated form of very long chain fatty acids.<\/jats:p>","DOI":"10.1007\/s11745-014-3969-5","type":"journal-article","created":{"date-parts":[[2014,11,21]],"date-time":"2014-11-21T06:17:35Z","timestamp":1416550655000},"page":"101-109","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["A Three\u2010Step Assay for Ceramide Synthase Activity Using a Fluorescent Substrate and HPLC"],"prefix":"10.1002","volume":"50","author":[{"given":"Timothy A.","family":"Couttas","sequence":"first","affiliation":[{"name":"Prince of Wales Clinical School University of New South Wales Level 2, C25 Lowy Building Sydney NSW 2052 Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xin Y.","family":"Lim","sequence":"additional","affiliation":[{"name":"Prince of Wales Clinical School University of New South Wales Level 2, C25 Lowy Building Sydney NSW 2052 Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anthony S.","family":"Don","sequence":"additional","affiliation":[{"name":"Prince of Wales Clinical School University of New South Wales Level 2, C25 Lowy Building Sydney NSW 2052 Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2014,11,18]]},"reference":[{"key":"e_1_2_7_2_1","doi-asserted-by":"publisher","DOI":"10.1021\/cr2002917"},{"key":"e_1_2_7_3_1","doi-asserted-by":"publisher","DOI":"10.2217\/17460875.3.3.273"},{"key":"e_1_2_7_4_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.8456305"},{"key":"e_1_2_7_5_1","doi-asserted-by":"publisher","DOI":"10.1096\/fj.09-135087"},{"key":"e_1_2_7_6_1","doi-asserted-by":"publisher","DOI":"10.1038\/nchembio.1059"},{"key":"e_1_2_7_7_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.270.51.30701"},{"key":"e_1_2_7_8_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M111.261206"},{"key":"e_1_2_7_9_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M112.413500"},{"key":"e_1_2_7_10_1","doi-asserted-by":"publisher","DOI":"10.1093\/hmg\/ddr494"},{"key":"e_1_2_7_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cmet.2012.04.002"},{"key":"e_1_2_7_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbalip.2010.05.003"},{"key":"e_1_2_7_13_1","doi-asserted-by":"publisher","DOI":"10.3390\/biom4010315"},{"key":"e_1_2_7_14_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.R111.254359"},{"key":"e_1_2_7_15_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbalip.2013.08.019"},{"key":"e_1_2_7_16_1","doi-asserted-by":"publisher","DOI":"10.1042\/BJ20050291"},{"key":"e_1_2_7_17_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M707386200"},{"key":"e_1_2_7_18_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M205211200"},{"key":"e_1_2_7_19_1","doi-asserted-by":"publisher","DOI":"10.1194\/jlr.D025627"},{"key":"e_1_2_7_20_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M113.494740"},{"key":"e_1_2_7_21_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0021-9258(19)74249-5"},{"key":"e_1_2_7_22_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M109.077594"},{"key":"e_1_2_7_23_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.febslet.2007.10.018"},{"key":"e_1_2_7_24_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M805186200"},{"key":"e_1_2_7_25_1","doi-asserted-by":"publisher","DOI":"10.1194\/jlr.M009142"},{"key":"e_1_2_7_26_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0076-6879(00)12900-3"},{"key":"e_1_2_7_27_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbalip.2012.04.008"},{"key":"e_1_2_7_28_1","doi-asserted-by":"publisher","DOI":"10.1186\/2051-5960-2-9"},{"key":"e_1_2_7_29_1","doi-asserted-by":"publisher","DOI":"10.1042\/BJ20110566"},{"key":"e_1_2_7_30_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00418-007-0344-0"},{"key":"e_1_2_7_31_1","doi-asserted-by":"publisher","DOI":"10.1042\/bj2900751"}],"container-title":["Lipids"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/aocs.onlinelibrary.wiley.com\/doi\/pdf\/10.1007\/s11745-014-3969-5","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,20]],"date-time":"2025-10-20T09:45:40Z","timestamp":1760953540000},"score":1,"resource":{"primary":{"URL":"https:\/\/aocs.onlinelibrary.wiley.com\/doi\/10.1007\/s11745-014-3969-5"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2014,11,18]]},"references-count":30,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2015,1]]}},"alternative-id":["10.1007\/s11745-014-3969-5"],"URL":"https:\/\/doi.org\/10.1007\/s11745-014-3969-5","archive":["Portico"],"relation":{},"ISSN":["0024-4201","1558-9307"],"issn-type":[{"type":"print","value":"0024-4201"},{"type":"electronic","value":"1558-9307"}],"subject":[],"published":{"date-parts":[[2014,11,18]]}}}