An Ambient-temperature Collection and Stabilization Device Performs Comparably to Flash-frozen Collection for Stool Metabolomics in Infants

Background: Stool metabolites provide essential insights into the function of the gut microbiome. The current gold standard for collection and storage of stool samples for metabolomics is flash-freezing at -80°C which can be inconvenient and expensive. Ambient temperature collection of stool is more practical, however no available methodologies adequately preserve the metabolomic profile of stool. A novel sampling kit (OMNImet.GUT; DNA Genotek, Inc.) was introduced for ambient temperature collection and stabilization of feces for metabolomics; we aimed to test the performance of this kit vs. flash-freezing.Methods: Stool collected from an infant’s diaper was divided into two aliquots: 1) flash-frozen and 2) stored in an OMNImet.GUT tube at ambient temperature for 3-4 days. Samples from the same infant were collected at 2 different time points to assess metabolite changes over time. Subsequently, all samples underwent metabolomic analysis by liquid chromatography – tandem mass spectrometry (LC-MS/MS). Results: Paired fecal samples (flash-frozen and ambient temperature) from 16 infants were collected at 2 time points (n= 64 samples). Similar numbers of metabolites were detected in both the frozen and ambient temperature samples (1126 in frozen, 1107 in ambient temperature, 1064 shared between sample types). Metabolite abundances were strongly correlated between collection methods (median Spearman correlation Rs=0.785 across metabolites). Hierarchical clustering analysis and principal component analysis showed that samples from the same individuals at a given time point clustered closely, regardless of the collection method. Repeat samples from the same individual were compared by paired t-test, separately for the frozen and OMNImet.GUT. The number of metabolites in each biochemical class that significantly changed (pConclusion: Ambient temperature collection and stabilization of stool in the OMNImet.GUT device yielded comparable metabolomic results to flash freezing in terms of 1) the identity and abundance of detected biochemicals 2) the distinct metabolomic profiles of subjects and 3) the biochemical signature of microbiome development over time. This method potentially provides a more convenient, less expensive home collection option for stool metabolomic analysis.