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Chemical derivatization-based LC-MS/MS method for quantitation of gut microbial short-chain fatty acids
Journal of Industrial and Engineering Chemistry ( IF 6.1 ) Pub Date : 2020-03-01 , DOI: 10.1016/j.jiec.2019.12.001
Won-Suk Song , Han-Gyu Park , Seong-Min Kim , Sung-Hyun Jo , Byung-Gee Kim , Ashleigh B. Theberge , Yun-Gon Kim

Abstract Short chain fatty acids (SCFAs) are end products of fermentation by anaerobic gut microbiota. They can be used as beneficial metabolites to regulate the host’s physiological processes. Despite their importance, SCFAs are difficult to analyze with mass spectrometric technologies due to their poor ionization efficiency and susceptibility to water loss during ionization of low molecular weight organic acids. Here, we developed a sensitive and reliable method to quantify SCFAs by liquid chromatography tandem mass spectrometry (LC–MS/MS) in multiple reaction monitoring (MRM) mode. SCFAs were chemically derivatized by Girard’s reagent T (GT), providing a permanent cationic charge. This technique demonstrated an excellent quantitative capacity, showing good linearity (R2 > 0.99) and limit of quantification (femtomole levels) for five SCFAs (i.e., acetate, propionate, butyrate, valerate, and caproate). Next, we applied this derivatization method to quantitate SCFAs from a small volume of total extracellular metabolites produced by Eubacterium rectale (E. rectale), one of main butyrate-producing gut bacteria. GT-labeled SCFAs were quantitated well in small volumes of culture medium (5, 10, 15, and 20 μL), with the amount of SCFA measured being proportional to the volume of culture medium, as expected. We also investigated plant-derived polysaccharides as prebiotics that could enhance the production of butyrate by E. rectale. Finally, the production of butyrate was successfully monitored in a co-culture system for E. rectale and Bifidobacterium longum (B. longum) by analyzing GT-labeled butyrate. Taken together, our results suggest that this highly sensitive method would be useful for quantifying SCFAs extracted from stool in an aqueous solution to monitor gut health.

中文翻译:

基于化学衍生化的 LC-MS/MS 方法定量肠道微生物短链脂肪酸

摘要 短链脂肪酸 (SCFA) 是厌氧肠道微生物群发酵的终产物。它们可以作为有益代谢物来调节宿主的生理过程。尽管它们很重要,但 SCFA 很难用质谱技术进行分析,因为它们的电离效率很差,并且在低分子量有机酸的电离过程中容易失水。在这里,我们开发了一种灵敏可靠的方法,通过液相色谱串联质谱 (LC-MS/MS) 在多反应监测 (MRM) 模式下定量 SCFA。SCFA 由 Girard 试剂 T (GT) 化学衍生,提供永久的阳离子电荷。该技术展示了出色的定量能力,显示出良好的线性 (R2 > 0.99) 和五种 SCFAs (i. 例如,乙酸盐、丙酸盐、丁酸盐、戊酸盐和己酸盐)。接下来,我们应用这种衍生化方法从由直肠真杆菌(E. rectale)产生的少量总细胞外代谢物中定量 SCFA,直肠真杆菌是一种主要的产生丁酸盐的肠道细菌。正如预期的那样,在小体积培养基(5、10、15 和 20 μL)中对 GT 标记的 SCFA 进行了很好的定量,测量的 SCFA 量与培养基的体积成正比。我们还研究了植物来源的多糖作为益生元,可以提高 E. rectale 的丁酸产量。最后,通过分析 GT 标记的丁酸盐,在 E. rectale 和长双歧杆菌 (B. longum) 的共培养系统中成功监测了丁酸盐的产生。综合起来,
更新日期:2020-03-01
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