INVESTIGATION OF 1-DEOXYDIHYDROCERAMIDE BIOSYNTHESIS BY MAMMALIAN CELLS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 最近在对培养物和动物中的哺乳动物细胞的研究中发现了一种新的鞘氨醇碱1-脱氧鞘氨醇及其N-酰基代谢物（Zitomer et al.，284：4786，2009），并且代表了鞘脂的新的新类别。1-脱氧鞘氨醇被证明是由L-丙氨酸与棕榈酰辅酶A在培养中的细胞缩合而产生的，并且大多数被代谢为1-脱氧二氢神经酰胺，而不是作为游离鞘氨醇碱积累。在这些化合物的预期利益，如他们可能在临床相关的样品中的海拔，我们现在已经开发了一种高通量和结构特异性的方法，用于分析1-脱氧鞘脂的高效液相色谱耦合到电喷雾电离（ESI）QSTAR脉冲星i飞行时间（TOF）质谱仪，结合，速度，灵敏度，高质量精度和高分辨率。1-从稳定过表达丝氨酸棕榈酰转移酶的Hek 293细胞系中提取脱氧二氢神经酰胺（SPT 1/2细胞），其与不同的稳定同位素标记的氨基酸如[3- 13 C]-L-丝氨酸、[2，3 - 13 C2]-L-丙氨酸和[3-D3]-L-丙氨酸一起孵育，并通过LC-ESI MS/MS进行检测。这些氨基酸的表征表明，新的1-脱氧鞘脂来自丙氨酸与棕榈酰的缩合。CoA，如通过培养物中的细胞将[2，3 - 13 C2]-L-丙氨酸和[3-D3]-L-丙氨酸分别掺入13 C2 -1-脱氧二氢神经酰胺和D3-1-脱氧二氢神经酰胺中所证明。结果表明，这些未被充分重视的类别的生物活性鞘氨醇碱和“神经酰胺”，可能发挥重要作用的细胞调控，可以分离和检测开发的LC MS/MS方法。

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.