Institute of Nutrition and Functional Foods (INAF), Faculty of Agriculture and Food Sciences, Laval University, Québec, QC, Canada.
Nutrition, Health and Society Centre (NUTRISS), INAF, Laval University, Québec, QC, Canada.
Food Funct. 2022 Oct 31;13(21):10895-10911. doi: 10.1039/d2fo02229j.
Many strategies are used to quantify microbial (poly)phenol metabolites (MPMs) in urine. Currently, to obtain accurate results, the use of phase II conjugate analytical standards is deemed to be the gold standard. However, these standards are expensive or commercially unavailable. Quantification using an affordable and commercially available unconjugated analytical standard following hydrolysis with the crude preparation from containing arylsulfatase and β-glucuronidase was once considered to be an alternative, but previous studies have shown poor hydrolysis efficiency for conjugated MPMs. In this work, we evaluated the efficiency of purified recombinant enzymes and compared them with the preparation from using 75 urine samples. 38 conjugated MPMs were identified before hydrolysis, associated with 17 unconjugated MPMs. Rapid chemical synthesis of sulfated compounds was carried out to increase the confidence level for the identification of 13 sulfated MPMs. Recombinant enzymes had a mean hydrolysis efficiency of over 95% for 36 out of 38 conjugated MPMs with a hydrolysis time of 30 min. In comparison, the preparation from achieved similar efficiency for only 28 conjugated MPMs after 6 h of hydrolysis. When comparing the concentration of unconjugated MPMs released after enzymatic hydrolysis, recombinant enzymes were more or as effective for almost every MPM. These results demonstrate that accurate quantification of MPMs in urine can be quickly achieved using purified recombinant enzymes and represent an affordable alternative to the use of conjugated analytical standards, improving access to the analysis of the metabolism of (poly)phenols by the gut microbiota.
许多策略被用于定量尿液中的微生物（多）酚代谢物（MPMs）。目前，为了获得准确的结果，使用 II 相共轭分析标准品被认为是金标准。然而，这些标准品昂贵或商业上不可用。使用价格合理且商业上可获得的未共轭分析标准品进行定量，然后用含有芳基硫酸酯酶和β-葡萄糖醛酸酶的粗制剂进行水解，曾经被认为是一种替代方法，但以前的研究表明，共轭 MPMs 的水解效率较差。在这项工作中，我们评估了纯化重组酶的效率，并将其与使用 75 个尿液样本的 进行比较。在水解前鉴定出 38 种共轭 MPMs，与 17 种未共轭 MPMs相关。进行了快速的硫酸化化合物化学合成，以提高对 13 种硫酸化 MPMs 的鉴定置信度。重组酶对 38 种共轭 MPMs 中的 36 种具有超过 95%的水解效率，水解时间为 30 分钟。相比之下，在用 水解 6 小时后，来自 的制剂仅对 28 种共轭 MPMs 达到相似的效率。当比较酶解后释放的未共轭 MPMs 的浓度时，重组酶对几乎每种 MPM 的效果都更好或相同。这些结果表明，使用纯化的重组酶可以快速准确地定量尿液中的 MPMs，为使用共轭分析标准品提供了一种经济实惠的替代方法，从而改善了对肠道微生物群代谢（多）酚的分析。
