Departments of Chemistry, Vanderbilt University, Nashville, TN; the Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN.
Pharmacology, Vanderbilt University, Nashville, TN; the Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN.
J Lipid Res. 2013 Mar;54(3):859-868. doi: 10.1194/jlr.D033332. Epub 2013 Jan 10.
Monitoring lipid distribution and metabolism in cells and biological fluids poses many challenges because of the many molecular species and metabolic pathways that exist. This study describes the synthesis and study of molecules that contain an alkyne functional group as surrogates for natural lipids in cultured cells. Thus, hexadec-15-ynoic and hexadec-7-ynoic acids were readily incorporated into RAW 264.7 cells, principally as phosphocholine esters; the alkyne was used as a "tag" that could be transformed to a stable dicobalt-hexacarbonyl complex; and the complex could then be detected by HPLC/MS or HPLC/UV(349nm). The 349 nm absorbance of the cobalt complexes was used to provide qualitative and quantitative information about the distribution and cellular concentrations of the alkyne lipids. The alkyne group could also be used as an affinity tag for the lipids by a catch-and-release strategy on phosphine-coated silica beads. Lipid extracts were enriched in the tagged lipids in this way, making the approach of potential utility to study lipid transformations in cell culture. Both terminal alkynes and internal alkynes were used in this affinity "pull-down" strategy. This method facilitates measuring lipid species that might otherwise fall below limits of detection.
监测细胞和生物体液中的脂质分布和代谢存在许多挑战，因为存在许多分子种类和代谢途径。本研究描述了合成和研究含有炔基官能团的分子，这些分子可作为培养细胞中天然脂质的替代品。因此，十六-15-炔酸和十六-7-炔酸很容易被 RAW 264.7 细胞吸收，主要作为磷酸胆碱酯；炔基可用作“标记物”，可转化为稳定的二羰基六羰基钴配合物；然后可以通过 HPLC/MS 或 HPLC/UV（349nm）检测配合物。钴配合物的 349nm 吸光度可用于提供关于炔脂分布和细胞浓度的定性和定量信息。炔基还可以通过膦涂层硅胶珠上的捕获和释放策略用作脂质的亲和标记。通过这种方式，脂质提取物中富含标记的脂质，这使得该方法有可能用于研究细胞培养中的脂质转化。在这种亲和“下拉”策略中，既使用末端炔基又使用内部炔基。该方法有助于测量可能低于检测限的脂质种类。
