Abstract
BackgroundStool metabolites provide essential insights into the function of the gut microbiome. The current gold standard for storage of stool samples for metabolomics is flash-freezing at − 80 °C which can be inconvenient and expensive. Ambient temperature storage 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 storage and stabilization of feces for metabolomics; we aimed to test the performance of this kit vs. flash-freezing. To do this stool was 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).ResultsPaired fecal samples (flash-frozen and ambient temperature) from 16 infants were collected at 2 time points (32 individual samples, 64 aliquots). 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 storage 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 storage 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 (p < 0.05) at timepoint 2 relative to timepoint 1 was similar in flash-frozen versus ambient temperature storage. Changes in microbiota modified metabolites over time were also consistent across both methodologies.ConclusionAmbient temperature storage 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) changes in metabolites over time that are plausibly microbiota-induced. This method potentially provides a more convenient, less expensive home collection and storage option for stool metabolomic analysis.
Highlights
Stool metabolites provide essential insights into the function of the gut microbiome
Attention in the microbiome field is increasingly turning to metabolomics – the discipline of measuring metabolites in a comprehensive and non-targeted manner [7] – as a powerful tool to elucidate how the gut microbiome exerts its effects on human health and disease [8,9,10]
Sixty-two metabolites from 7 super-pathways and 38 sub-pathways were only found in frozen samples; 43 metabolites from 8 super-pathways and 24 subpathways were only found in ambient temperature samples (Supplemental Table 1)
Summary
Stool metabolites provide essential insights into the function of the gut microbiome. Attention in the microbiome field is increasingly turning to metabolomics – the discipline of measuring metabolites in a comprehensive and non-targeted manner [7] – as a powerful tool to elucidate how the gut microbiome exerts its effects on human health and disease [8,9,10] Despite this growing interest, the practical challenge of collecting fecal samples from human volunteers has constrained the application of metabolomics in gut microbiome research. Storage and shipping of frozen at-home collected samples can be inconvenient for participants and prohibitively expensive for researchers, pushing the need for ambient-temperature storage options To fill this need, several research groups have recently sought to repurpose currently available sampling devices, such as DNA-stabilizing tubes and fecal immunochemical test (FIT) tubes [12, 13], for metabolomic analysis. Use of these devices significantly distorted the metabolomic profile of stool samples relative to the goldstandard flash-freezing methodology [12, 13]
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