Publication
Optimized Sample Handling Strategy for Metabolic Profiling of Human Feces
Publisher:
American Chemical Society (ACS)
Date:
21-04-2016
DOI:
10.1021/ACS.ANALCHEM.5B04159
Abstract: Fecal metabolites are being increasingly studied to unravel the host-gut microbial metabolic interactions. However, there are currently no guidelines for fecal s le collection and storage based on a systematic evaluation of the effect of time, storage temperature, storage duration, and s ling strategy. Here we derive an optimized protocol for fecal s le handling with the aim of maximizing metabolic stability and minimizing s le degradation. S les obtained from five healthy in iduals 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 (1)H NMR spectroscopy-based metabolic profiling approach. Interin idual variation was much greater than that attributable to storage conditions. In idual stool s les were found to be heterogeneous and spot s ling resulted in a high degree of metabolic variation. Crude fecal s les 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 s les stored at room temperature and this fermentative process was reduced when stored at 4 °C. Crude fecal s les frozen at -20 °C manifested elevated amino acids and nicotinate and depleted short chain fatty acids compared to crude fecal control s les. The relative concentrations of branched-chain and aromatic amino acids significantly increased in the freeze-thawed crude fecal s les, suggesting a release of microbial intracellular contents. The metabolic profiles of fecal water s les were more stable compared to crude s les. Our recommendation is that intact fecal s les 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 s les should be extracted from a representative amount (∼15 g) of homogenized stool s le, aliquoted, and stored at <-20 °C, avoiding further freeze-thaw cycles.