Evaluation of metabolite extraction strategies from tissue samples using NMR metabolomics |
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Authors: | Ching Yu Lin Huifeng Wu Ronald S Tjeerdema Mark R Viant |
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Institution: | (1) Department of Environmental Toxicology, University of California, Davis, CA 95616-8588, USA;(2) School of Biosciences, The University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK |
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Abstract: | 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.
C. Y. Lin and H. Wu contributed equally. |
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Keywords: | metabolomics NMR J-resolved metabolite extraction sample preparation tissue |
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