title: Influence of Freezing and Storage Procedure on Human Urine Samples in NMR-Based Metabolomics.
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.
authors: Department of Physiology and Biochemistry of Nutrition, Max Rubner-Institut (MRI), Haid-und-Neu-Str. 9, Karlsruhe 76131, Germany. Manuela.Rist@mri.bund.de.
authors: Manuela J
authors: MJ
authors: Rist
authors: Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 6, Karlsruhe 76131, Germany. Claudia.Muhle@kit.edu.
authors: Claudia
authors: C
authors: Muhle-Goll
authors: Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 6, Karlsruhe 76131, Germany. Benjamin.Goerling@kit.edu.
authors: Benjamin
authors: B
authors: Görling
authors: Department of Physiology and Biochemistry of Nutrition, Max Rubner-Institut (MRI), Haid-und-Neu-Str. 9, Karlsruhe 76131, Germany. Achim.Bub@mri.bund.de.
authors: Achim
authors: A
authors: Bub
authors: Institute of Functional Interfaces, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, Eggenstein-Leopoldshafen 76344, Germany. Stefan.Heissler@kit.edu.
authors: Stefan
authors: S
authors: Heissler
authors: Department of Physiology and Biochemistry of Nutrition, Max Rubner-Institut (MRI), Haid-und-Neu-Str. 9, Karlsruhe 76131, Germany. Bernhard.Watzl@mri.bund.de.
authors: Bernhard
authors: B
authors: Watzl
authors: Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 6, Karlsruhe 76131, Germany. Burkhard.Luy@kit.edu.
authors: Burkhard
authors: B
authors: Luy