INVESTIGATION OF 1-DEOXYDIHYDROCERAMIDE BIOSYNTHESIS BY MAMMALIAN CELLS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. A novel sphingoid base, 1-deoxysphinganine, and its N-acyl-metabolites were recently discovered in studies of mammalian cells in culture and animals (Zitomer et al., J. Biol. Chem. 284:4786, 2009) and represent a novel new category of sphingolipid. 1-Deoxysphinganine was shown to arise from condensation of L-alanine with palmitoyl-CoA by cells in culture, and most is metabolized to 1-deoxydihydroceramides, rather than accumulating as the free sphingoid base. In anticipation of interest in these compounds, such as their possible elevation in clinically relevant samples, we have now developed a high-throughput and structure-specific method for analysis of 1-deoxysphingolipids by high performance liquid chromatography coupled to an electrospray ionization (ESI) QSTAR Pulsar i time-of-flight (TOF) mass spectrometer which combines, speed, sensitivity, high mass accuracy and high resolution. 1-Deoxydihydroceramides were extracted from a Hek293 cell line stably overexpressing serine palmitoyltransferase (SPT1/2 cells) which was incubated with different stable isotope labeled amino acids, such as [3-13C]-L-Serine, [2,3-13C2]-L-Alanine, and [3-D3]-L-Alanine, and examined by LC-ESI MS/MS. These amino acid supplementations show that the novel 1-deoxysphingolipid arises from condensation of alanine with palmitoyl-CoA as evidenced by incorporation of [2,3-13C2]-L-alanine and [3-D3]-L-Alanine into 13C2-1-deoxydihydroceramides and D3-1-deoxydihydroceramides, respectively, by cells in culture. The results show that these underappreciated categories of bioactive sphingoid bases and "ceramides" that are likely to play important roles in cell regulation can be separated and detected by developed LC MS/MS methods.