Hou Waner, Zhong Danmin, Zhang Peiting, Li Yemeng, Lin Manna, Liu Guanghui, Yao Meicun, Liao Qiongfeng, Xie Zhiyong
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China; College of Chinese Traditional Medicine, Guangzhou University of Chinese Medicine, Guangzhou, China.
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, China.
J Chromatogr A. 2016 Jan 15;1429:207-17. doi: 10.1016/j.chroma.2015.12.031. Epub 2015 Dec 14.
Microbiota-host co-metabolites are well-known to play important physiological roles, and their dysregulation has been found to be closely related to various diseases, including but not limited to inflammatory disorders. We developed herein an original and feasible method using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The method developed enables rapid quantification of 11 key gut microbiota-host co-metabolites spanning the succinate, phenylacetylglutamine, hippurate and trimethylamine metabolic pathways within 10 min. With this method, we were able to simultaneously monitor inflammation-induced alterations of these metabolites in rat serum, urine and feces matrices. The measured levels for this panel of endogenous metabolites ranged from 0.001 to 172.8 μg m L(-1). The intra- and inter-day precision of three analytes was less than 13.1% and the accuracy was between -13.0 to 11.2% for all QC levels. The extraction recoveries in serum ranged from 85.4 to 103.2%, while the RSD was 9.0% or less for all recoveries. In addition, extraction recoveries of 11 analytes in urine and feces samples were between 85.7% and 102.0% and RSD was less than 9.5%. The method developed here has been successfully applied to the analysis of real samples from 2,4,6-trinitrobenzenesulfonic acid-induced Crohn's disease in rats. All of these results suggest that the presently developed method is sufficiently sensitive and robust to simultaneously monitor co-metabolites with diverse properties and a range of different concentrations. Therefore, this method will be expected to be useful for comprehensive studies of the pathophysiological roles and mechanisms of these key microbiota-host co-metabolites, which reflect the function of the intestine, consequently offering novel opportunities for evaluating the occurrence, development and therapeutic effects of diseases related to microbiota disturbances.
微生物群-宿主共代谢物在发挥重要生理作用方面广为人知，并且已发现其失调与包括但不限于炎症性疾病在内的各种疾病密切相关。我们在此开发了一种使用超高效液相色谱-串联质谱法（UPLC-MS/MS）的原创且可行的方法。所开发的方法能够在10分钟内快速定量11种关键的肠道微生物群-宿主共代谢物，这些共代谢物涵盖琥珀酸、苯乙酰谷氨酰胺、马尿酸盐和三甲胺代谢途径。通过该方法，我们能够同时监测大鼠血清、尿液和粪便基质中这些代谢物的炎症诱导变化。该组内源性代谢物的测量水平范围为0.001至172.8μg mL(-1)。三种分析物的日内和日间精密度均小于13.1%，所有质量控制水平的准确度在-13.0%至11.2%之间。血清中的提取回收率范围为85.4%至103.2%，而所有回收率的相对标准偏差（RSD）为9.0%或更低。此外，尿液和粪便样品中11种分析物的提取回收率在85.7%至102.0%之间，RSD小于9.5%。此处开发的方法已成功应用于分析大鼠2,4,6-三硝基苯磺酸诱导的克罗恩病的实际样品。所有这些结果表明，目前开发的方法足够灵敏且稳健，能够同时监测具有不同性质和不同浓度范围的共代谢物。因此，该方法有望用于对这些关键微生物群-宿主共代谢物的病理生理作用和机制进行全面研究，这些共代谢物反映了肠道功能，从而为评估与微生物群紊乱相关疾病的发生、发展和治疗效果提供新的机会。
