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Evaluation of metabolite extraction strategies from tissue samples using NMR metabolomics

机译：使用NMR代谢组学评估组织样品中的代谢物提取策略

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摘要

Metabolomic analysis of tissue samples can be applied across multiple fields including medicine, toxicology, and environmental sciences. A thorough evaluation of several metabolite extraction procedures from tissues is therefore warranted. This has been achieved at two research laboratories using muscle and liver tissues from fish. Multiple replicates of homogenous tissues were extracted using the following solvent systems of varying polarities: perchloric acid, acetonitrile/water, methanol/water, and methanol/chloroform/water. Extraction of metabolites from ground wet tissue, ground dry tissue, and homogenized wet tissue was also compared. The hydrophilic metabolites were analyzed using 1-dimensional (1D) 1H nuclear magnetic resonance (NMR) spectroscopy and projections of 2-dimensional J-resolved (p-JRES) NMR, and the spectra evaluated using principal components analysis. Yield, reproducibility, ease, and speed were the criteria for assessing the quality of an extraction protocol for metabolomics. Both laboratories observed that the yields of low molecular weight metabolites were similar among the solvent extractions; however, acetonitrile-based extractions provided poorer fractionation and extracted lipids and macromolecules into the polar solvent. Extraction using perchloric acid produced the greatest variation between replicates due to peak shifts in the spectra, while acetonitrile-based extraction produced highest reproducibility. Spectra from extraction of ground wet tissues generated more macromolecules and lower reproducibility compared with other tissue disruption methods. The p-JRES NMR approach reduced peak congestion and yielded flatter baselines, and subsequently separated the metabolic fingerprints of different samples more clearly than by 1D NMR. Overall, single organic solvent extractions are quick and easy and produce reasonable results. However, considering both yield and reproducibility of the hydrophilic metabolites as well as recovery of the hydrophobic metabolites, we conclude that the methanol/chloroform/water extraction is the preferred method.
机译：组织样品的代谢组学分析可应用于多个领域，包括医学，毒理学和环境科学。因此，必须对几种从组织中提取的代谢物进行全面评估。这是在两个使用鱼类的肌肉和肝脏组织的研究实验室中实现的。使用以下极性不同的溶剂系统提取均质组织的多个副本：高氯酸，乙腈/水，甲醇/水和甲醇/氯仿/水。还比较了从地面湿组织，地面干组织和均质湿组织中提取代谢物。使用一维（1D）1H核磁共振（NMR）光谱和二维J分辨（p-JRES）NMR进行分析，分析了亲水性代谢物，并使用主成分分析评估了光谱。产量，可重复性，简便性和速度是评估代谢组学提取方案质量的标准。两个实验室都观察到，在溶剂萃取过程中，低分子量代谢物的收率相近。但是，基于乙腈的萃取分离效果较差，并且将脂质和大分子萃取到极性溶剂中。由于光谱中的峰移动，使用高氯酸萃取在两次重复之间产生了最大的差异，而基于乙腈的萃取则产生了最高的重现性。与其他组织破坏方法相比，提取地面湿组织的光谱产生了更多的大分子，并降低了重现性。 p-JRES NMR方法减少了峰拥塞并产生了更平坦的基线，随后比1D NMR更清楚地分离了不同样品的代谢指纹。总的来说，单一有机溶剂的提取是快速简便的，并且产生合理的结果。但是，考虑到亲水性代谢物的产率和重现性以及疏水性代谢物的回收率，我们得出结论，甲醇/氯仿/水萃取是优选的方法。

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