Nishiumi Shin, Shima Keisuke, Azuma Takeshi, Yoshida Masaru
Division of Gastroenterology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan.
Global Application Development Center, Analytical & Measuring Instruments Division, Shimadzu Corporation, 1 Nishinokyo Kuwabara-cho, Nakagyo-ku, Kyoto 604-8511, Japan.
J Biosci Bioeng. 2017 Jun;123(6):754-759. doi: 10.1016/j.jbiosc.2017.01.015. Epub 2017 Mar 11.
Metabolomics has recently been developed, and there have been a considerable number of metabolomics-based biomarker studies in the medical research field. Therefore, as a first step toward the practical use of metabolite biomarkers, a simple and quick sample preparation method involving metabolite extraction, metabolite measurement, and data analysis needs to be developed. In this study, we evaluated whether the use of simpler metabolite extraction methods would facilitate the stable analysis of hydrophilic blood metabolites during liquid chromatography/triple quadrupole mass spectrometry (LC/QqQMS)-based metabolome analysis. As a result, the anion and cation metabolites in plasma were stably analyzed via a methanol-based extraction procedure followed by ultrafiltration, and it was also confirmed that a lyophilization step was not necessary. When extraction was performed without a lyophilization step, approximately >50% and >80% of the detected metabolites had relative standard deviation values of <20% during LC/QqQMS-based anion and cation analyses, respectively. In addition, the plasma levels of l-valine, l-leucine, l-isoleucine, l-tyrosine, and l-phenylalanine were quantitatively measured using the corresponding stable isotopes; the SCLAM-2000, a fully automatic pre-treatment system for LC/MS that can be connected online to an LC/MS device; and an extraction procedure based on the simple procedure that we developed. Our findings suggest that simpler pretreatment procedures can be employed during LC/QqQMS-based metabolomics and might aid searches for metabolite biomarker candidates, the validation of metabolite biomarker candidates, and the practical use of metabolite biomarkers.
代谢组学是近年来发展起来的，在医学研究领域已经有大量基于代谢组学的生物标志物研究。因此，作为迈向代谢物生物标志物实际应用的第一步，需要开发一种简单快速的样品制备方法，包括代谢物提取、代谢物测量和数据分析。在本研究中，我们评估了使用更简单的代谢物提取方法是否有助于在基于液相色谱/三重四极杆质谱（LC/QqQMS）的代谢组分析中对亲水性血液代谢物进行稳定分析。结果表明，通过基于甲醇的提取程序随后进行超滤，可以稳定地分析血浆中的阴离子和阳离子代谢物，并且还证实不需要冻干步骤。在不进行冻干步骤的情况下进行提取时，在基于LC/QqQMS的阴离子和阳离子分析过程中，分别约有>50%和>80%的检测到的代谢物的相对标准偏差值<20%。此外，使用相应的稳定同位素、SCLAM-2000（一种可在线连接到LC/MS设备的用于LC/MS的全自动预处理系统）以及基于我们开发的简单程序的提取程序，对l-缬氨酸、l-亮氨酸、l-异亮氨酸、l-酪氨酸和l-苯丙氨酸的血浆水平进行了定量测量。我们的研究结果表明，在基于LC/QqQMS的代谢组学中可以采用更简单的预处理程序，这可能有助于寻找代谢物生物标志物候选物、验证代谢物生物标志物候选物以及代谢物生物标志物的实际应用。
