Evaluation of metabolite extraction strategies from tissue samples using NMR metabolomics
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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.
Keywordsmetabolomics NMR J-resolved metabolite extraction sample preparation tissue
University of California, Davis
University of Birmingham
nuclear magnetic resonance
principal components analysis
Funding was provided by the UK NERC Post Genomic and Proteomic (PGP) Directed Program (NE/C507661/1), the US NOAA/UNH Coastal Response Research Center (NA17OZ2607), the California Department of Fish and Game, Office of Spill Prevention and Response (PO475011), and California Department of Fish and Game’s Oil Spill Response Trust Fund through the Oiled Wildlife Care Network at the Wildlife Health Center, School of Veterinary Medicine, UCD (18091, 18092 and 18093). We thank B. Anderson (Dept. Environmental Toxicology, UCD) for providing salmon smolts, T. Taylor (School of Biosciences, UB) for chub samples, Chenomx Inc. for use of their NMR metabolomics software, and A. Peng (Dept. Environmental Toxicology, UCD) for manuscript review. MRV thanks the NERC for an Advanced Fellowship (NER/J/S/2002/00618).
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