Intramyocellular diacylglycerol concentrations and [U-13C]palmitate isotopic enrichment measured by LC/MS/MS
Intramyocellular diacylglycerol concentrations and [U-13C]palmitate isotopic enrichment measured by LC/MS/MS
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DOI：
10.1194/jlr.d035006
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发表时间：
2013-06-01
影响因子：
6.5
通讯作者：
Jensen, Michael D.
中科院分区：
文献类型：
--
作者：
Blachnio-Zabielska, Agnieszka U.;Zabielski, Piotr;Jensen, Michael D.
二酰基甘油（DAG）是一种重要的脂质代谢产物，被认为在过量存在时会诱导肌肉胰岛素抵抗;它们可以从血浆游离脂肪酸（FFA）重新合成或通过水解预先存在的细胞内脂质产生。我们提出了一种新的方法，同时测量肌细胞内的浓度和[U-C-13]棕榈酸酯从静脉输注到个别DAG物种的掺入。使用异丙醇：水：乙酸乙酯（35：5：60; v：v：v）从粉碎的肌肉样品中提取DAG。在反相柱上以二元梯度进行色谱分离，使用1.5 mM甲酸铵、0.1%甲酸的水溶液作为溶剂A，使用2 mM甲酸铵、0.15%甲酸的甲醇溶液作为溶剂B。我们使用UPLC-ESI+-MS/MS在多反应监测（MRM）模式下从样品中分离感兴趣的离子。由于DAG是中性脂质类，因此将其作为铵加合物[M+ NH 4]（+）进行监测。为了测量同位素富集（对于（13）C16：0/16：0-DAG和（13）C16：0/C18：1-DAG），我们将碱性离子监测为[M+2+ NH 4]（+），将富集的化合物监测为[M+16+ NH 4]（+）。我们能够使用从接受[U-C-13]棕榈酸酯连续输注的大鼠获得的20 mg骨骼肌样品测量浓度和富集。将该方法应用于生物肌肉样品，证明该方法灵敏、准确、高效。
Diacylglycerols (DAG) are important lipid metabolites thought to induce muscle insulin resistance when present in excess; they can be synthesized de novo from plasma free fatty acids (FFA) or generated by hydrolysis of preexisting intracellular lipids. We present a new method to simultaneously measure intramyocellular concentrations of and the incorporation of [U-C-13] palmitate from an intravenous infusion into individual DAG species. DAG were extracted from pulverized muscle samples using isopropanol: water: ethyl acetate (35:5:60; v:v:v). Chromatographic separation was conducted on reverse-phase column in binary gradient using 1.5 mM ammonium formate, 0.1% formic acid in water as solvent A, and 2 mM ammonium formate, 0.15% formic acid in methanol as solvent B. We used UPLC-ESI+-MS/MS in the multiple reaction monitoring (MRM) mode to separate the ions of interest from sample. Because DAG are a neutral lipid class, they were monitored as an ammonium adduct [M+NH4](+). To measure isotopic enrichment (for (13)C16:0/16:0-DAG and (13)C16:0/C18:1-DAG), we monitored the basic ions as [M+2+NH4](+) and the enriched compounds as [M+16+NH4](+). We were able to measure concentration and enrichment using 20 mg of skeletal muscle samples obtained from rats receiving a continuous infusion of [U-C-13] palmitate. Applying this protocol to biological muscle samples proves that the method is sensitive, accurate, and efficient.