Molecular Recognition Research Center, Korea Institute of Science and Technology, Seoul 136-791, Republic of Korea; College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea.
Department of Clinical Pharmacology and Therapeutics, Seoul National University College of Medicine, Seoul National University Hospital, Seoul 110-744, Republic of Korea.
J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Jan 1;944:157-65. doi: 10.1016/j.jchromb.2013.10.029. Epub 2013 Oct 26.
A simple and fast methodology to detect and identify multiple classes of lipid from human plasma is developed utilizing ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF) as lipidomics platform. All the conditions for the sample preparation and analytical instruments were optimized in detail to detect nine lipid classes (phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), triacylglyceride (TG), phosphatidylinositol (PI), lysophosphatidylcholine (LysoPC), lysophosphatidic acid (LysoPA), and sphingomyelin (SM)), which are the most important biologically active lipids but have different characteristics. Finally, the plasma was prepared after a liquid-liquid extraction with a mixture of chloroform/methanol (1:2v/v) including salting out by adding 0.15M of NaCl and the residue after evaporation was reconstituted with a mixture of chloroform/methanol (1:1v/v) to dissolve all lipids which have different polarity. The chromatographic conditions were set up such that mobile phase (A) comprised 10mM ammonium acetate in 40% acetonitrile and mobile phase (B) comprised 10mM ammonium acetate in acetonitrile:isopropanol=10:90(v/v) with ACQUITY BEH C18 as the stationary phase. In particular, a retention time index of PC was constructed by analyzing known standards to confirm each variant of PC without the use of any additional standards in every experiment. The lipidomic methodology and the retention time index of PC were applied to analyze the lipidomic profiling of human plasma from rosuvastatin (lipid lowering drug) treated subjects. In the developed lipidomic platform, all lipids were successfully analyzed within 16min and PCs could be confirmed with the PC retention time index. In rosuvastatin treatment, the lipid profiling was changed in all the eight lipid classes. The level of SM, TG, PI and PE decrease significantly but LysoPCs and PCs were whether decreased or increased. Those results indicated that the plasma level of overall lipids decreased by drug response, however, the changes in the lipids which are important components for biological membrane such as LysoPC and PC were more complicated, and it could be related to the side effect of rousuvastatin. In conclusion, it was found that our lipidomic methodology and the PC retention time index provided not only overall lipidomic information but also profiled specific information of drug response.
建立了一种利用超高效液相色谱-四极杆飞行时间质谱（UPLC-QTOF）作为脂质组学平台来检测和鉴定人血浆中多种脂质类别的简单快速方法。详细优化了样品制备和分析仪器的所有条件，以检测九种脂质类（磷脂酰丝氨酸（PS）、磷脂酰甘油（PG）、磷脂酰乙醇胺（PE）、磷脂酰胆碱（PC）、三酰基甘油（TG）、磷脂酰肌醇（PI）、溶血磷脂酰胆碱（LysoPC）、溶血磷脂酸（LysoPA）和鞘磷脂（SM）），它们是最重要的生物活性脂质，但具有不同的特性。最后，采用氯仿/甲醇（1:2v/v）混合液进行液-液提取，并加入 0.15M 的 NaCl 盐析，将血浆制备好，蒸发后的残渣用氯仿/甲醇（1:1v/v）混合液重新溶解，以溶解所有具有不同极性的脂质。色谱条件设定为：流动相（A）为含 10mM 乙酸铵的 40%乙腈溶液，流动相（B）为含 10mM 乙酸铵的乙腈：异丙醇=10:90（v/v）溶液，以 ACQUITY BEH C18 为固定相。特别是，通过分析已知标准品构建了 PC 的保留时间指数，以确认每个 PC 变体，而无需在每次实验中使用任何其他标准品。脂质组学方法和 PC 的保留时间指数被应用于分析瑞舒伐他汀（降脂药物）治疗患者的人血浆脂质组学谱。在开发的脂质组学平台中，所有脂质都在 16 分钟内成功分析，并且可以使用 PC 保留时间指数确认 PC。在瑞舒伐他汀治疗中，所有八种脂质类别的脂质谱都发生了变化。SM、TG、PI 和 PE 的水平显著降低，但 LysoPCs 和 PCs 的水平则是降低或升高。这些结果表明，药物反应使整体血浆脂质水平降低，但对生物膜重要组成部分（如 LysoPC 和 PC）的脂质变化则更为复杂，这可能与瑞舒伐他汀的副作用有关。总之，研究发现我们的脂质组学方法和 PC 保留时间指数不仅提供了整体脂质组学信息，还提供了药物反应的特定信息。
