Optimized metabolite extraction from blood serum for 1H nuclear magnetic resonance spectroscopy
Section snippets
Materials
Extractions were carried out using heat-inactivated fetal bovine serum (Gibco-Invitrogen Cat. No. 10108). Acetonitrile and 100% methanol used for extractions were purchased from Fisher Chemical and chloroform from Acros Organic. For ultracentrifugation Nanosep Omega centrifugal devices with a 3 kDa cutoff were purchased from Pall Life Sciences.
Perchloric acid extraction
0.5 ml of bovine serum was cooled for 10 min in an ice bath, 50 μl of 4 M perchloric acid was added, and then the solution was vortexed for 1 min. Afterward,
Results
1H-NMR spectra of pure and filtered bovine serum along with deproteinized serum using solvent extractions and acid precipitation methods are shown in Fig. 1; expanded spectral regions are shown in Fig. 2. To compare different extraction methods we used a representative selection of metabolites found in spectra of bovine serum and including all the compounds that were positively identified. Table 1 summarizes the results of the extraction methods for different metabolites.
Acetonitrile extraction
Discussion
Deproteinization of serum samples has been used frequently to prepare samples for metabolomics [2], [5], [6], [7], [8] although some investigators preferred to use pure serum and suppress protein signals by CPMG pulse sequences [4]. However, the presence of catalytically active proteins causes constant metabolic flux in the sample which is a particular problem for longer NMR measurements. Otherwise deproteinization alters the samples, removes important components, and introduces unwanted
Acknowledgments
We thank the EU for supporting S.T., A.E., and A.L. in the context of the MOTET Marie Curie project and the NERC for an Advanced Fellowship to M.R.V. (NER/J/S/2002/00618). This work was performed at the HWB-NMR facility in Birmingham which is supported by the Wellcome Trust. We are indebted to Chenomx Inc. for use of their NMR metabolomics software.
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