2 Materials
2.1 Fecal Sample
Collection
- Collect at least 50 mg of feces for sample in a 1.5 ml Eppendorf
tube (seeNote 1).
2.2 Reagents
for Sample Preparation
- D 2 O, 99.9 atom % D.
- Phosphate-buffered saline (PBS) tablet.
- Sodium azide (NaN3).
- Sodium salt of 3-(trimethylsilyl) propionic-2,2,3,3-d 4 acid,
98 atom % D (TSP).
2.3 Equipment 1. 500 MHz spectrometer (Bruker BioSpin Corp., Billerica, MA,
USA or Agilent Technologies, Santa Clara, CA, USA or Jeol
Ltd., Akishima, Tokyo, Japan). However, 400 or 600 MHz
NMR instruments are commonly also used in metabolomic
studies.
- Analytical balance.
- 1.5 ml Eppendorf tubes.
- Spatula.
- Micropipettes and pipette tips.
2.4 Software for Data
Analysis
Several commercial and free licensed software packages are available
for NMR data processing, postprocessing, and statistical analysis.
Only the most widely used software is reported.- Software for processing NMR data: TopSpin (Bruker BioSpin
Corp., Billerica, MA, USA), VNMRJ (Agilent Technologies,
Santa Clara, CA, USA), MetaboLab [5] in the MATLAB pro-
gramming environment (MathWorks, Inc., Natick, MA). - Software for postprocessing NMR data: ACD/NMR processor
(Advanced Chemistry Development Inc. (ACD/Labs), Tor-
onto, ON, Canada), MetaboLab [8] in the MATLAB pro-
gramming environment (MathWorks, Inc., Natick, MA). - Software for multivariate data analysis: SIMCA-Pþ(Umetrics,
Umea ̊, Sweden), PLS-Toolbox (Eigenvector Research, Man-
son, WA) in MATLAB.
3 Methods
3.1 Phosphate-
Buffered Saline
for NMR Sample
Preparation
Prepare the 10 mM D 2 O (99.9 atom % D) phosphate-buffered
saline solution at pH 7.4 and 25C by mixing PBS tablets (as indi-
cated by the manufacturer), TSP 1 mM and NaN 3 10 mM.Metabolomics and Clinical Needs 329