Toxicological Centre, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium; Natural Products and Food - Research & Analysis (NatuRA), University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium.
Toxicological Centre, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium.
J Pharm Biomed Anal. 2019 Oct 25;175:112768. doi: 10.1016/j.jpba.2019.07.016. Epub 2019 Jul 10.
Recent data clearly show that the gut microbiota plays a significant role in the biotransformation of many endogenous molecules and xenobiotics, leading to a potential influence of this microbiotic metabolism on activation, inactivation and possible toxicity of these compounds. To study the colonic biotransformation of xenobiotics by the gut microbiome, in vitro models are often used as they allow dynamic and multiple sampling overtime. However, the pre-analytical phase should be carefully optimized to enable biotransformation product identification representative for the in vivo situation. During this study, chlorogenic acid was used as a model compound to optimize a ready-to-use gut microbiome biotransformation platform using an in vitro gastrointestinal dialysis-model with colon phase together with an instrumental platform using liquid chromatography coupled to high resolution mass spectrometry (LC-QTOF-MS). Identification of the biotransformation products of chlorogenic acid was performed using complementary suspect and non-targeted data analysis approaches (MZmine + R and MPP workflow). Concerning the pre-analytical phase, (i) the influence of different incubation media (Wilkins-Chalgren Anaerobic Broth (WCB) and (versus) phosphate buffer) and different incubation times (prior to implementation in the colonic stage of the dialysis model) on fecal bacterial composition and concentration were investigated and (ii) four different sample preparation methods (centrifugation, extraction, sonication and freeze-drying) were evaluated targeting colonic biotransformation of chlorogenic acid. WCB as incubation medium showed to introduce substantial variation in the bacterial composition of the fecal samples, while the sterile phosphate buffer guaranteed a closer resemblance to the in vivo composition. Furthermore, incubation during 24 h in sterile phosphate buffer as medium showed no significant increase or decrease in anaerobic bacterial concentration, concluding that incubation prior to the colonic stage is not needed. Concerning sample preparation, centrifugation, sonication and extraction gave similar results, while freeze-drying appeared to be inferior. The extraction method was selected as an optimal sample preparation method given the quick execution together with a good instrumental sensitivity. This study optimized a ready-to-use platform to investigate colonic biotransformation of xenobiotics by using chlorogenic acid as a model compound. This platform can be used in the future to study differences in colonic biotransformation of xenobiotics using fecal samples of different patient groups.
最近的数据清楚地表明，肠道微生物群在许多内源性分子和外源性化合物的生物转化中发挥着重要作用，这导致这种微生物代谢可能会影响这些化合物的激活、失活和潜在毒性。为了研究肠道微生物群对外源化合物在结肠中的生物转化，通常使用体外模型，因为它们允许随时间进行动态和多次采样。然而，应仔细优化分析前阶段，以实现对体内情况具有代表性的生物转化产物鉴定。在这项研究中，使用绿原酸作为模型化合物，使用带有结肠阶段的体外胃肠道透析模型和使用液相色谱与高分辨率质谱（LC-QTOF-MS）联用的仪器平台，优化了一种即用型肠道微生物群生物转化平台。使用互补的可疑和非靶向数据分析方法（MZmine+R 和 MPP 工作流程）对绿原酸的生物转化产物进行了鉴定。关于分析前阶段，（i）研究了不同孵育培养基（威尔金斯-查尔格伦厌氧肉汤（WCB）和（与）磷酸盐缓冲液）和不同孵育时间（在结肠阶段透析模型之前）对粪便细菌组成和浓度的影响，（ii）评估了四种不同的样品制备方法（离心、提取、超声和冷冻干燥），旨在靶向绿原酸的结肠生物转化。作为孵育培养基的 WCB 显示出对粪便样本中细菌组成的实质性变化，而无菌磷酸盐缓冲液保证了与体内组成更接近。此外，在无菌磷酸盐缓冲液中孵育 24 小时并没有导致厌氧细菌浓度的显著增加或减少，这表明在结肠阶段之前不需要孵育。关于样品制备，离心、超声和提取给出了相似的结果，而冷冻干燥似乎较差。由于快速执行和良好的仪器灵敏度，选择提取方法作为最佳的样品制备方法。本研究优化了一种即用型平台，使用绿原酸作为模型化合物来研究外源化合物在结肠中的生物转化。该平台可用于未来使用不同患者组的粪便样本研究外源化合物在结肠中的生物转化差异。
