Abstract
IntroductionGlobal metabolomics analyses using body fluids provide valuable results for the understanding and prediction of diseases. However, the mechanism of a disease is often tissue-based and it is advantageous to analyze metabolomic changes directly in the tissue. Metabolomics from tissue samples faces many challenges like tissue collection, homogenization, and metabolite extraction.ObjectivesWe aimed to establish a metabolite extraction protocol optimized for tissue metabolite quantification by the targeted metabolomics AbsoluteIDQ™ p180 Kit (Biocrates). The extraction method should be non-selective, applicable to different kinds and amounts of tissues, monophasic, reproducible, and amenable to high throughput.MethodsWe quantified metabolites in samples of eleven murine tissues after extraction with three solvents (methanol, phosphate buffer, ethanol/phosphate buffer mixture) in two tissue to solvent ratios and analyzed the extraction yield, ionization efficiency, and reproducibility.ResultsWe found methanol and ethanol/phosphate buffer to be superior to phosphate buffer in regard to extraction yield, reproducibility, and ionization efficiency for all metabolites measured. Phosphate buffer, however, outperformed both organic solvents for amino acids and biogenic amines but yielded unsatisfactory results for lipids. The observed matrix effects of tissue extracts were smaller or in a similar range compared to those of human plasma.ConclusionWe provide for each murine tissue type an optimized high-throughput metabolite extraction protocol, which yields the best results for extraction, reproducibility, and quantification of metabolites in the p180 kit. Although the performance of the extraction protocol was monitored by the p180 kit, the protocol can be applicable to other targeted metabolomics assays.
Highlights
Global metabolomics analyses using body fluids provide valuable results for the understanding and prediction of diseases
We especially aimed to provide for each tissue type an optimized high-throughput compatible metabolite extraction protocol, which yields the best results with regard to extractability of metabolites as well as reproducibility in subsequent metabolite quantification with the p180 kit
Since the extraction efficiency depends on the chemical nature of the metabolites, we tested metabolite extraction from the tissues with three extraction solvents of different polarity [methanol (MeOH) as a non-polar, phosphate buffer (PB) as a polar, and a mixture of ethanol and phosphate buffer (EtOH/PB) as a mixed-polarity solvent]
Summary
Global metabolomics analyses using body fluids provide valuable results for the understanding and prediction of diseases. Methods We quantified metabolites in samples of eleven murine tissues after extraction with three solvents (methanol, phosphate buffer, ethanol/phosphate buffer mixture) in two tissue to solvent ratios and analyzed the extraction yield, ionization efficiency, and reproducibility. Conclusion We provide for each murine tissue type an optimized high-throughput metabolite extraction protocol, which yields the best results for extraction, reproducibility, and quantification of metabolites in the p180 kit. The application of metabolomics in studies related to human health, in which mostly body fluids were analyzed, becomes more and more frequent and has provided already valuable results and biomarkers for disease outcomes (Adamski 2016; Fan et al 2016; Roberts et al 2014). Many different sample preparation protocols and metabolomics measurements from tissue samples were developed previously [reviewed in Naz et al (2014)]
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