The human faecal metabolome is complex, but rich in information and allows investigation of the host metabolism as a function of diet and health. The faecal metabolome is still much less explored than the plasma and urine metabolome, and in order to generate comparable data across laboratories and cohorts, standard operating procedures are required. This study evaluates 10 protocols, using different extraction solvents and sample processing methods for measuring the human faecal metabolome using proton nuclear magnetic resonance (¹H NMR) spectroscopy. Three solvents: water, methanol, and dimethyl sulfoxide (DMSO) were investigated at varying concentrations for their ability to extract metabolites directly from faecal slurry or after freeze-drying. The protocols were evaluated on four different pools of human feces. The study also demonstrates a novel signature mapping (SigMa) method for rapid and unbiased processing of complex NMR spectra applied for the first time to human faecal metabolomics. The method is provided with a library containing the chemical shift ranges of 81 common faecal metabolites for future unambiguous and rapid faecal metabolite annotations. The result from the 10 faecal extraction protocols were investigated in terms of reproducibility, coverage, and ability to extract low concentration metabolites. The solvent type was shown to induce the highest variation in the data (45.7%) and the water based extractions allowed detection of the greatest number of metabolites and resulted in the highest reproducibility. Direct extraction of faecal slurry was proved to be more reproducible than freeze-drying. In addition, freeze-drying caused a relative loss of short chain fatty acids (SCFA). DMSO was used for the first time to extract faecal metabolites and enabled the detection of certain bile acids. Some derivatives of SCFA were only detected using methanol as solvent.

中文翻译：

---

人粪便1H NMR代谢组学：溶剂和样品处理对特征代谢物的覆盖率和再现性的评估。

人的粪便代谢组很复杂，但是信息丰富，可以根据饮食和健康状况研究宿主的新陈代谢。与血浆和尿液代谢组相比，粪便代谢组的探索性仍然要低得多，并且为了在实验室和队列中生成可比数据，需要标准的操作程序。这项研究评估了10种方案，使用不同的萃取溶剂和样品处理方法通过质子核磁共振测量人粪便代谢组（¹1 H NMR）光谱。研究了三种溶剂（水，甲醇和二甲基亚砜（DMSO））在不同浓度下的直接从粪便浆液中或冷冻干燥后提取代谢产物的能力。在四个不同的人类粪便库上评估了方案。这项研究还证明了一种新颖的特征图谱（SigMa）方法，可快速，无偏地处理首次应用于人类粪便代谢组学的复杂NMR谱图。该方法配有一个库，该库包含81种常见粪便代谢物的化学位移范围，可用于将来明确和快速的粪便代谢物注释。从重现性，覆盖率和提取低浓度代谢物的能力方面研究了10种粪便提取方案的结果。结果表明，溶剂类型引起的数据变化最大（45.7％），水基提取物可检测到最大数量的代谢物，并具有最高的重现性。事实证明，直接提取粪便比冷冻干燥具有更高的重现性。另外，冷冻干燥引起短链脂肪酸（SCFA）的相对损失。DMSO首次用于提取粪便代谢产物，并能够检测某些胆汁酸。仅使用甲醇作为溶剂才能检测到一些SCFA衍生物。冷冻干燥导致短链脂肪酸（SCFA）的相对损失。DMSO首次用于提取粪便代谢产物，并能够检测某些胆汁酸。仅使用甲醇作为溶剂才能检测到一些SCFA衍生物。冷冻干燥导致短链脂肪酸（SCFA）的相对损失。DMSO首次用于提取粪便代谢产物，并能够检测某些胆汁酸。仅使用甲醇作为溶剂才能检测到一些SCFA衍生物。