While over 10% of the human metabolome is directly associated with the gut microbial metabolism, specific metabolites are largely uncharacterized. Therefore, methods for the identification and quantification of microbiota-associated metabolites in biological fluids such as urine or plasma are necessary in order to elucidate the molecular basis of host-microbiota interaction. In this study, we focused on the tryptophan metabolism, employing quantitative assays by ultra-high performance liquid chromatography (UHPLC) and tandem mass spectrometry, specifically selected reaction monitoring (SRM). Metabolite standards were utilized to generate SRM library for 16 intermediates of the tryptophan metabolism which were human endogenous as well as microbiota-associated based on the HMDB classification. Next, the SRM assays were utilized for screening in maternal urine samples and in dried urine specimens from neonates. The approach resulted in the discovery of microbiota-associated metabolites (methyl indole-3-acetate and methyl indol-3-propionate) previously unreported in urine samples and additionally in quantification of 8 intermediates of the tryptophan metabolism. To the best of our knowledge, this study represents the first attempt to explore previously unreported microbial metabolites in urine by UHPLC-SRM and novel methodology for simultaneous determination of microbiota-modulated component of Trp metabolism.

中文翻译：

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色氨酸的尿液中间体作为肠道微生物代谢的指标

虽然超过 10% 的人体代谢组与肠道微生物代谢直接相关，但特定代谢物在很大程度上是未表征的。因此，为了阐明宿主-微生物群相互作用的分子基础，有必要对尿液或血浆等生物体液中的微生物群相关代谢物进行鉴定和量化。在这项研究中，我们专注于色氨酸代谢，采用超高效液相色谱 (UHPLC) 和串联质谱法进行定量分析，特别是选择反应监测 (SRM)。基于 HMDB 分类，代谢物标准品用于生成 16 种色氨酸代谢中间体的 SRM 库，这些中间体是人类内源性的以及与微生物群相关的。下一个，SRM 检测用于筛查母体尿液样本和新生儿的干燥尿液样本。该方法导致发现了以前未在尿样中报告的微生物群相关代谢物（甲基吲哚-3-乙酸酯和甲基吲哚-3-丙酸酯），此外还对色氨酸代谢的 8 种中间体进行了量化。据我们所知，这项研究代表了首次尝试通过 UHPLC-SRM 和同时测定 Trp 代谢的微生物群调节成分的新方法探索尿液中以前未报告的微生物代谢物。该方法导致发现了以前未在尿样中报告的微生物群相关代谢物（甲基吲哚-3-乙酸酯和甲基吲哚-3-丙酸酯），此外还对色氨酸代谢的 8 种中间体进行了量化。据我们所知，这项研究代表了首次尝试通过 UHPLC-SRM 和同时测定 Trp 代谢的微生物群调节成分的新方法探索尿液中以前未报告的微生物代谢物。该方法导致发现了以前未在尿样中报告的微生物群相关代谢物（甲基吲哚-3-乙酸酯和甲基吲哚-3-丙酸酯），此外还对色氨酸代谢的 8 种中间体进行了量化。据我们所知，这项研究代表了首次尝试通过 UHPLC-SRM 和同时测定 Trp 代谢的微生物群调节成分的新方法探索尿液中以前未报告的微生物代谢物。