Abstract
It is consensus in the metabolomics community that standardized protocols should be followed for sample handling, storage and analysis, as it is of utmost importance to maintain constant measurement conditions to identify subtle biological differences. The aim of this work, therefore, was to systematically investigate the influence of freezing procedures and storage temperatures and their effect on NMR spectra as a potentially disturbing aspect for NMR-based metabolomics studies. Urine samples were collected from two healthy volunteers, centrifuged and divided into aliquots. Urine aliquots were frozen either at −20 °C, on dry ice, at −80 °C or in liquid nitrogen and then stored at −20 °C, −80 °C or in liquid nitrogen vapor phase for 1–5 weeks before NMR analysis. Results show spectral changes depending on the freezing procedure, with samples frozen on dry ice showing the largest deviations. The effect was found to be based on pH differences, which were caused by variations in CO2 concentrations introduced by the freezing procedure. Thus, we recommend that urine samples should be frozen at −20 °C and transferred to lower storage temperatures within one week and that freezing procedures should be part of the publication protocol.
Highlights
The general aim of metabolomics studies is the identification of mostly subtle differences in biological systems under defined conditions
Most publications in the field of metabolomics note only the storage temperature of the samples prior to analysis, but for practical reasons, the actual freezing procedure may require conditions that are different from long-term storage conditions
We have systematically screened different freezing conditions and storage temperatures of human urine samples and investigated their effects on the metabolic fingerprint of samples analyzed by NMR
Summary
The general aim of metabolomics studies is the identification of mostly subtle differences in biological systems under defined conditions. The detection of those subtle differences usually requires the maximum possible reproducibility of both sample preparation and measurement. All studies on biological systems involve freezing and storage steps, and freezing conditions may vary—even within a study—usually because of local conditions, like the accessibility or inaccessibility of a freezer in close proximity to the site where the samples are generated and the corresponding use of dry ice for freezing or intermediate storage at a certain floor, etc. Our aim was the detailed investigation of common freezing conditions and their effects on resulting NMR spectra using buffered urine as one of the most commonly studied biological matrices
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