Chimie et Interdisciplinarité: Synthèse, Analyse, Modélisation (CEISAM) UMR 6230, Université de Nantes, CNRS, BP 92208, 2 rue de la Houssinière, 44322 Nantes Cedex 03, France.
Anal Bioanal Chem. 2011 Oct;401(7):2133-42. doi: 10.1007/s00216-011-5310-y. Epub 2011 Aug 13.
Metabolomic analysis of mammalian cells can be applied across multiple fields including medicine and toxicology. It requires the acquisition of reproducible, robust, reliable, and homogeneous biological data sets. Particular attention must be paid to the efficiency and reliability of the extraction procedure. Even though a number of recent studies have dealt with optimizing a particular protocol for specific matrices and analytical techniques, there is no universal method to allow the detection of the entire cellular metabolome. Here, we present a strategy for choosing extraction procedures from adherent mammalian cells for the global NMR analysis of the metabolome. After the quenching of cells, intracellular metabolites are extracted from the cells using one of the following solvent systems of varying polarities: perchloric acid, acetonitrile/water, methanol, methanol/water, and methanol/chloroform/water. The hydrophilic metabolite profiles are analysed using (1)H nuclear magnetic resonance (NMR) spectroscopy. We propose an original geometric representation of metabolites reflecting the efficiency of extraction methods. In the case of NMR-based analysis of mammalian cells, this methodology demonstrates that a higher portion of intracellular metabolites are extracted by using methanol or methanol/chloroform/water. The preferred method is evaluated in terms of biological variability for studying metabolic changes caused by the phenotype of four different human breast cancer cell lines, showing that the selected extraction procedure is a promising tool for metabolomic and metabonomic studies of mammalian cells. The strategy proposed in this paper to compare extraction procedures is applicable to NMR-based metabolomic studies of various systems.
哺乳动物细胞的代谢组学分析可应用于多个领域，包括医学和毒理学。它需要获得可重复、稳健、可靠和同质的生物数据集。特别需要注意提取程序的效率和可靠性。尽管最近有许多研究致力于针对特定基质和分析技术优化特定方案，但没有通用的方法可以检测整个细胞代谢组。在这里，我们提出了一种从贴壁哺乳动物细胞中选择提取程序的策略，用于代谢组的全局 NMR 分析。在细胞淬火后，使用以下溶剂系统之一从细胞中提取细胞内代谢物，这些溶剂系统的极性不同：高氯酸、乙腈/水、甲醇、甲醇/水和甲醇/氯仿/水。使用（1）H 核磁共振（NMR）光谱分析亲水性代谢物图谱。我们提出了一种反映提取方法效率的代谢物的原始几何表示。在基于 NMR 的哺乳动物细胞分析中，该方法证明，使用甲醇或甲醇/氯仿/水可以提取更多的细胞内代谢物。从四种不同的人乳腺癌细胞系的表型引起的代谢变化的研究方面，评估了优选方法的生物学可变性，表明所选的提取程序是哺乳动物细胞代谢组学和代谢组学研究的有前途的工具。本文提出的用于比较提取程序的策略适用于各种基于 NMR 的代谢组学研究。
