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Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts

Abstract

Metabolic profiling, metabolomic and metabonomic studies mainly involve the multicomponent analysis of biological fluids, tissue and cell extracts using NMR spectroscopy and/or mass spectrometry (MS). We summarize the main NMR spectroscopic applications in modern metabolic research, and provide detailed protocols for biofluid (urine, serum/plasma) and tissue sample collection and preparation, including the extraction of polar and lipophilic metabolites from tissues. 1H NMR spectroscopic techniques such as standard 1D spectroscopy, relaxation-edited, diffusion-edited and 2D J-resolved pulse sequences are widely used at the analysis stage to monitor different groups of metabolites and are described here. They are often followed by more detailed statistical analysis or additional 2D NMR analysis for biomarker discovery. The standard acquisition time per sample is 4–5 min for a simple 1D spectrum, and both preparation and analysis can be automated to allow application to high-throughput screening for clinical diagnostic and toxicological studies, as well as molecular phenotyping and functional genomics.

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Figure 1: 600 MHz 1H NMR spectrum of control rat urine, displaying hundreds of resolved peaks.
Figure 2: 600 MHz 1H NMR spectrum of blood serum sample.
Figure 3: Example of edited spectra of a plasma sample [all were acquired with solvent (water) suppression].
Figure 4: Mean score trajectories of principal component analysis of urinary NMR spectral data for each dose group (control, low-dose, high-dose) showing progression of metabolic effects of hydrazine treatment.
Figure 5: Score (t) scatter plots for PC1 versus PC2 from a principal component analysis model using the data acquired at two different sites using the same protocols for the analysis of split aliquots in a study on hydrazine toxicity (triangles and circles represent the two different datasets, respectively).
Figure 6: Metabonomics identifies different responses from the urine profiles of animals treated with different toxins.

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We thank our academic and industrial collaborators for helpful discussions in the formulation of this paper, including those participating in the COMET project.

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Beckonert, O., Keun, H., Ebbels, T. et al. Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts. Nat Protoc 2, 2692–2703 (2007). https://doi.org/10.1038/nprot.2007.376

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