第一部分:開發化學衍生化搭配LC-MS/MS定量腸道菌代謝物的分析方法 第二部分:探討腸道菌代謝苯丙胺酸與苯乙醯谷氨醯胺的生成

Part I: Development of a chemical derivatization-based LC-MS/MS method for quantifying gut microbial metabolites Part II: Investigation on gut microbial metabolism of phenylalanine and phenylacetylglutamine production

廖馨瑜(Hsin-Yu Liao)

指導教授 ： 郭錦樺

共同指導教授 ： 吳偉愷(Wei-Kai Wu)

國立臺灣大學/醫學院/藥學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202102608

摘要

人體內的微生物是由細菌和真菌群落所組成，複雜度更甚於人體之基因。與相對恆定的人類基因不同，人體的微生物組成是相對動態的，會隨著飲食、使用的抗生素和生活方式等許多因素而變化。有越來越多的研究顯示，人類的腸道微生物可以藉由其代謝物調節宿主免疫、防禦病原體並影響宿主的心理或行為。這些腸道菌代謝物（Gut microbiota-derived metabolites, GMs）不僅存在於腸道中，還存在於宿主循環中。而循環系統裡的GMs在許多疾病進展中常常扮演著重要的角色。
在本論文的第一部分，我們開發了一種使用液相層析串聯式質譜儀(Liquid Chromatography Tandem Mass Spectrometry, LC-MS/MS) 廣泛定量人體血漿內的GMs。在各種 GMs 中，短鏈脂肪酸 (Short-chain fatty acids, SCFAs)、膽酸 (Bile acids, BAs) 和芳香族胺基酸 (Aromatic amino acids, AAAs) 及其代謝物是最常被討論的。在本研究中，我們開發同時分析 SCFAs、BAs 和色氨酸 (Tryptophan, TRP) 代謝物的方法。 LC-MS/MS 在定量上具有良好的靈敏度和選擇性，然而，SCFAs游離化效率差、具高極性等問題皆增加其用LC-MS/MS進行分析的困難度，尤其當需要分析血中低濃度的SCFAs。此外，在串聯質譜儀上不易有非共軛膽酸的特徵碎片離子且TRP和其代謝物與膽酸需在不同離子模式下被偵測，這些問題，都使得GMs分析既複雜又耗時。為了克服這些問題，我們開發了一種結合 LC-MS/MS 的衍生化方法，以提高偵測GMs的靈敏度和增加代謝物在管柱上的滯留。藉由 3-硝基苯肼 (3- nitrophenylhydrazine, 3-NPH) 進行衍生，以C18管柱分離，7種 SCFAs、9種 BAs和6種TRP代謝物可以在14分鐘完成分析。本研究使用13C6-3NPH 和GMs標準品的衍生物作為一對一的內標，用於準確定量。經過一系列的方法優化和確校，我們進一步應用此方法來探討心血管疾病患者的腸道微生物代謝物組成。這項研究提供了一種靈敏且有效的 LC-MS/MS 方法，可同時定量人血漿中22種腸道微生物代謝物，將有益於未來的腸道微生物之相關研究。
在第二部分，我們提出了一種口服苯丙氨耐量試驗 (Oral phenylalanine challenge test, OPCT) ，結合代謝物、食物頻率和腸道菌分析，來研究宿主-飲食-微生物群與苯丙胺酸(Phenylalanine, PHE)代謝之相互關係。PHE屬於芳香族氨基酸，有兩種由腸道微生物群代謝途徑（還原性和氧化性）。苯乙醯谷氨醯胺（Phenylacetylglutamine, PAGln）是PHE氧化途徑中的代謝物之一，已被報導為CVD的危險因子。本研究應用 OPCT 來研究人體血液中的 PHE 和 PHE代謝物濃度，用以評估標的代謝物之間的相關性。研究結果顯示還原途徑的代謝物與氧化途徑代謝物呈顯著負相關。有趣的是，我們還發現 Defluviitaleaceae_UCG-011 和 Subdoligranulum 的豐度都與PHE氧化代謝顯著正相關，而與PHE還原代謝物呈負相關。本研究另外將受試者分為高PAGln生產者和低PAGln 生產者，結果發現這兩組的腸道微生物群組成不同，高PAGln生產者的Chao1指數更高。飲食習慣分析顯示，高PAGln生產者的SFA (8:0)和SFA (12:0)較高，而低PAGln生產者的MUFA(14:1) 較高。
總結本論文開發了一種同時測量22種GM的分析方法，預計其可被廣泛應用於各種臨床研究；本論文並進一步採用OPCT的策略了解宿主-飲食-微生物的相互關係。

關鍵字

腸道代謝物 ； 短鏈脂肪酸 ； 膽酸 ； 衍生化 ； 液相層析質譜儀 ； 心血管疾病 ； 苯乙醯谷氨醯胺 ； 口服苯丙胺酸耐量試驗 ； 腸道菌

並列摘要

The human microbiome is composed of communities of bacteria and fungi that have greater complexity than the human genome itself. Unlike the human genome, which is relatively constant, the microbiome is dynamic and changes with many factors such as diet, the antibiotics used, and lifestyle. More and more researches reveal that the human gut microbiome can modulate host immunity, defense against pathogens, and impact our mental or behavior by their metabolites. These gut microbiota-derived metabolites (GMs) not only exist in the intestinal but also in host circulation. GMs in circulation has also been reported to cause disease progression.
In the first part of this thesis, we developed a comprehensive and quantitative LC-MS/MS method to measure GMs in human plasma samples. Among various GMs, short-chain fatty acids (SCFAs), bile acids (BAs), and aromatic amino acids (AAA) derivatives are the most frequently discussed gut metabolites. In this study, we aim to simultaneously analyze SCFAs, BAs, and tryptophan (TRP) metabolites. LC-MS/MS shows advantages in quantifying metabolites with good sensitivity and selectivity; however, the poor ionization efficiency and polar characteristics of SCFAs make their analysis challenging, especially when analyzing plasma samples with low SCFA concentrations. Moreover, without characteristic fragment ions for unconjugated BAs and different detection ion modes for TRP metabolites and BAs, GM analysis is complex and time consuming. To overcome these problems, we developed a derivatization method combined with LC-MS/MS to enhance the sensitivity and LC retention of GMs. Through derivatization with 3-nitrophenylhydrazine (3-NPH), 7 SCFAs, 9 bile acids, and 6 tryptophan metabolites can be simultaneously analyzed via separation within 14 min on a reversed-phase C18 column. For accurate quantification, 13C6-3NPH-labeled standards were used as one-to-one internal standards. This derivatization approach was optimized and then validated. We further applied this method to investigate the targeted GM profile in patients with cardiovascular disease (CVD). In summary, this work provides a sensitive and effective LC-MS/MS method for simultaneously quantifying gut microbiota-related metabolites in human plasma, which could benefit various future gut microbiota-related studies.
In the second part, we proposed an oral phenylalanine challenge test (OPCT) strategy combined with metabolites, food frequency, and gut microbiota analysis to investigate the PHE metabolism through host-diet-microbiota interaction. PHE belongs to aromatic amino acids, which has two metabolic pathways (reductive and oxidative) modulated by our gut microbiota. Phenylacetylglutamine (PAGln) was one of the PHE-derived metabolites in the PHE oxidative pathway, which has been reported as a risk factor for CVD. OPCT was applied in this study to study PHE and PHE-derived metabolites levels and their associations in human circulation. Analytical results revealed the metabolites of the reductive pathway showed negative correlation with the metabolites in oxidative pathway. Interestingly, we also found that the abundance of Defluviitaleaceae_UCG-011 and Subdoligranulum showed positive-correlated with the PHE oxidative metabolism, while negatively correlated with the PHE reductive metabolism. We additionally classified our study subjects into high and low PAGln producers. The gut microbiota profiles were different between these two groups, in which Chao 1 index was higher in the high PAGln producers. Moreover, food frequency analysis revealed diets containing SFA (8:0) and SFA (14:0) were higher in the high PAGln producers, while MUFA(14:1) was higher in the low PAGln producers.
In conclusion, this study developed an analytical method for simultaneous quantifying 22 GMs, which was anticipated to broaden their applications to various clinical studies. Furthermore, the OPCT approach was successfully used to study host-diet-microbiota interaction.

並列關鍵字

gut metabolites ； SCFAs ； bile acids ； derivatization ； LC−MS ； cardiovascular disease ； phenylacetylglutamine (PAGln) ； oral phenylalanine challenge test ； gut microbiota

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