Abstract
Ceramide-1-phosphate (C1P) is a bioactive sphingolipid with roles in several biological processes. Currently, high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC ESI-MS/MS) offers the most efficient method of quantifying C1P. However, the published protocols have several drawbacks causing overestimations and carryovers. Here, the reported overestimation of C1P was shown to be due to incomplete neutralization of base hydrolyzed lipid extracts leading to the hydrolysis of SM to C1P. Actual quantity of C1P in cells (6 pmols/10(6) cells) was much lower than previously reported. Also, the major species of C1P produced by ceramide kinase (CERK) was found to be d(18:1/16:0) with a minority of d(18:1/24:1) and d(18:1/24:0). The artifactual production of C1P from SM was used for generating C1Ps as retention time markers. Elimination of carryovers between samples and a 2-fold enhancement in the signal strength was achieved by heating the chromatographic column to 60 (degrees) C. The role of ceramide transport protein (CERT) in supplying substrate to CERK was also revalidated using this new assay. Finally, our results demonstrate the presence of additional pathway(s) for generation of the C1P subspecies, d(18:1/18:0) C1P, as well as a significant portion of d(18:1/16:0), d(18:1/24:1), and d(18:1/24:0). In conclusion, this study introduces a much improved and validated method for detection of C1P by mass spectrometry and demonstrates specific changes in the C1P subspecies profiles upon downregulation of CERK and CERT.
Highlights
Ceramide-1-phosphate (C1P) is a bioactive sphingolipid with roles in several biological processes
The first report of a biological effect for C1P was by Gomez-Muñoz et al [1], who demonstrated that short chain C1P induced DNA synthesis in Rat-1 fibroblasts [1]
These results, coupled with the previous findings that ceramide kinase (CERK)/C1P pathway is required for cPLA2␣ activation in response to calcium ionophore and inflammatory cytokines [7], demonstrated that C1P was a major regulator of the eicosanoid synthetic pathway
Summary
Ceramide-1-phosphate (C1P) is a bioactive sphingolipid with roles in several biological processes. We introduce a modified and validated method for quantifying cellular levels of C1P by reverse phase HPLC ESI-MS/MS based on the previously reported protocol by Merrill et al [12].
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