Optimized metabolite extraction from blood serum for 1H nuclear magnetic resonance spectroscopy

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Abstract

Blood serum is commonly used for clinical diagnostics because its protein composition bears a wealth of information about the health of an organism. More recently the analysis of the small molecule composition, the metabolome, has received increased attention because the metabolite composition is influenced by many diseases, by the administration of drugs and toxins, and by the diet and life style of an individual. When nuclear magnetic resonance spectroscopy is used as an analytical tool it is often preferable to remove catalytically active proteins, in particular for longer measurements, because metabolite concentrations are otherwise in constant flux. Here we have compared different protocols for the separation of proteins and metabolites, including precipitation methods and ultrafiltration. Whereas most extraction methods involving protein precipitation deplete some metabolites, ultrafiltration is superior in retaining metabolite concentrations and offers excellent reproducibility. We also describe a new method to recover the hydrophobic fraction for ultrafiltration with good reproducibility.

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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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