An optimized sample handling strategy for metabolic profiling of human feces

File Description SizeFormat 
Revised manuscript_Gratton et al_ac-2015-04159z_R2_clean version_11042016.pdfAccepted version1.06 MBAdobe PDFView/Open
Revised Supplementary Information_Gratton et al_R2.docxSupporting information3.37 MBMicrosoft WordView/Open
Title: An optimized sample handling strategy for metabolic profiling of human feces
Authors: Gratton, J
Phetcharaburanin, J
Mullish, BH
Williams, HRT
Thursz, M
Nicholson, JK
Holmes, E
Marchesi, JR
Li, J
Item Type: Journal Article
Abstract: © 2016 American Chemical Society.Fecal metabolites are being increasingly studied to unravel the host-gut microbial metabolic interactions. However, there are currently no guidelines for fecal sample collection and storage based on a systematic evaluation of the effect of time, storage temperature, storage duration, and sampling strategy. Here we derive an optimized protocol for fecal sample handling with the aim of maximizing metabolic stability and minimizing sample degradation. Samples obtained from five healthy individuals were analyzed to assess topographical homogeneity of feces and to evaluate storage duration-, temperature-, and freeze-thaw cycle-induced metabolic changes in crude stool and fecal water using a 1H NMR spectroscopy-based metabolic profiling approach. Interindividual variation was much greater than that attributable to storage conditions. Individual stool samples were found to be heterogeneous and spot sampling resulted in a high degree of metabolic variation. Crude fecal samples were remarkably unstable over time and exhibited distinct metabolic profiles at different storage temperatures. Microbial fermentation was the dominant driver in time-related changes observed in fecal samples stored at room temperature and this fermentative process was reduced when stored at 4 °C. Crude fecal samples frozen at -20 °C manifested elevated amino acids and nicotinate and depleted short chain fatty acids compared to crude fecal control samples. The relative concentrations of branched-chain and aromatic amino acids significantly increased in the freeze-thawed crude fecal samples, suggesting a release of microbial intracellular contents. The metabolic profiles of fecal water samples were more stable compared to crude samples. Our recommendation is that intact fecal samples should be collected, kept at 4 °C or on ice during transportation, and extracted ideally within 1 h of collection, or a maximum of 24 h. Fecal water samples should be extracted from a representative amount (∼15 g) of homogenized stool sample, aliquoted, and stored at <-20 °C, avoiding further freeze-thaw cycles.
Issue Date: 11-Apr-2016
Date of Acceptance: 11-Apr-2016
ISSN: 0003-2700
Publisher: ACS Publications
Start Page: 4661
End Page: 4668
Journal / Book Title: Analytical Chemistry
Volume: 88
Issue: 9
Copyright Statement: This document is the Accepted Manuscript version of a Published Work that appeared in final form in Analytical Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see
Sponsor/Funder: Medical Research Council (MRC)
Funder's Grant Number: MC_PC_12025
Keywords: Analytical Chemistry
0301 Analytical Chemistry
0904 Chemical Engineering
0399 Other Chemical Sciences
Publication Status: Published
Appears in Collections:Division of Surgery
Department of Medicine
Faculty of Medicine

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

Creative Commons