Institute for Clinical Chemistry and Laboratory Medicine , University Hospital Regensburg , Franz-Josef-Strauss-Allee 11 , 93053 Regensburg , Germany.
Anal Chem. 2018 Jul 17;90(14):8487-8494. doi: 10.1021/acs.analchem.8b01278. Epub 2018 Jul 3.
There has been an increasing interest during recent years in the role of the gut microbiome on health and disease. Therefore, metabolites in human feces related to microbial activity are attractive surrogate marker to track changes of microbiota induced by diet or disease. Such markers include 5α/β-stanols as microbiome-derived metabolites of sterols. Currently, reliable, robust, and fast methods to quantify fecal sterols and their related metabolites are missing. We developed a liquid chromatography-high-resolution mass spectrometry (LC-MS/HRMS) method for the quantification of sterols and their 5α/β-stanols in human fecal samples. Fecal sterols were extracted and derivatized to N, N-dimethylglycine esters. The method includes cholesterol, coprostanol, cholestanol and sitosterol, 5α/β-sitostanol, campesterol and 5α/β-campestanol. Application of a biphenyl column permits separation of isomeric 5α- and 5β-stanols. Sterols are detected in parallel reaction monitoring (PRM) mode and stanols in full scan mode. HRMS allows differentiation of isobaric β-stanols and the [M + 2] isotope peak of the coeluting sterol. Performance characteristics meet the criteria recommended by Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines. Analysis of fecal samples from healthy volunteers revealed high interindividual variability of sterol and stanol fractions. Interestingly, cholesterol and sitosterol showed similar fractions of mainly 5β-stanols. In contrast, campesterol is substantially converted to 5α-campestanol and might be a poorer substrate for bacterial metabolism. Robust and fast quantification of fecal sterols and their related stanols by LC-MS/HRMS offers great potential to find novel microbiome-related biomarker in large-scale studies.
近年来，人们对肠道微生物组在健康和疾病中的作用越来越感兴趣。因此，与微生物活性相关的人类粪便中的代谢物是一种很有吸引力的替代标志物，可以跟踪饮食或疾病引起的微生物群变化。这些标志物包括 5α/β-甾体作为固醇微生物组衍生的代谢物。目前，缺乏可靠、稳健、快速的方法来定量粪便固醇及其相关代谢物。我们开发了一种液相色谱-高分辨率质谱（LC-MS/HRMS）方法，用于定量人粪便样品中的固醇及其 5α/β-甾体。粪便固醇经过提取并衍生为 N，N-二甲基甘氨酸酯。该方法包括胆固醇、粪甾醇、胆甾醇和谷甾醇、5α/β-谷甾醇、菜油甾醇和 5α/β-菜油甾醇。使用联苯柱可实现 5α-和 5β-甾体的异构体分离。甾醇在平行反应监测（PRM）模式下检测，甾体在全扫描模式下检测。HRMS 允许区分等质量数的 β-甾体和共洗脱甾醇的 [M + 2]同位素峰。性能特征符合食品和药物管理局（FDA）和欧洲药品管理局（EMA）指南推荐的标准。对健康志愿者粪便样本的分析显示，甾醇和甾体醇的分数个体间差异很大。有趣的是，胆固醇和谷甾醇主要表现为 5β-甾体醇的分数相似。相比之下，菜油甾醇大量转化为 5α-菜油甾醇，可能是细菌代谢的较差底物。LC-MS/HRMS 对粪便甾醇及其相关甾体醇的快速定量具有很大的潜力，可以在大规模研究中发现新的与微生物组相关的生物标志物。
