Center for Digestive and Gut Health, Institute of Global Health Innovation, Imperial College London , South Kensington, London SW7 2AZ, U.K.
School of Biosciences, Cardiff University , Cardiff CF10 3XQ, U.K.
Anal Chem. 2016 May 3;88(9):4661-8. doi: 10.1021/acs.analchem.5b04159. Epub 2016 Apr 21.
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 (1)H 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.
粪便代谢物正被越来越多地用于揭示宿主-肠道微生物代谢相互作用。然而，目前还没有基于对时间、储存温度、储存时间和采样策略的影响进行系统评估的粪便样本采集和储存指南。在这里，我们制定了一个优化的粪便样本处理方案，旨在最大限度地提高代谢稳定性，最大限度地减少样本降解。分析了来自五名健康个体的样本，以评估粪便的地形同质性，并使用（1）H NMR 光谱代谢组学方法评估储存时间、温度和冻融循环对粗粪便和粪便水的代谢变化。个体间的变异比储存条件的变异大得多。发现个体粪便样本不均匀，点样导致代谢变化程度很高。粗粪便样本随时间的推移极不稳定，在不同的储存温度下表现出不同的代谢谱。在室温下储存时，微生物发酵是粪便样本中观察到的时间相关变化的主要驱动因素，而在 4°C 下储存时，发酵过程减少。与粗粪便对照样本相比，-20°C 冷冻的粗粪便样本表现出升高的氨基酸和烟酸，以及减少的短链脂肪酸。在冻融的粗粪便样本中，支链和芳香族氨基酸的相对浓度显著增加，表明微生物细胞内物质的释放。与粗样本相比，粪便水样本的代谢谱更稳定。我们的建议是，应采集完整的粪便样本，在运输过程中保持在 4°C 或冰上，并在采集后 1 小时内提取，最长不超过 24 小时。应从均匀化的粪便样本中提取代表量（约 15 g）的粪便水样本，等分并储存在<-20°C 下，避免进一步的冻融循环。
