以標的代謝體學方法結合親水性作用液相層析質譜儀進行臨床應用 第一部分：探討非酒精性脂肪肝炎患者之單獨丙麩胺酸轉移酶上升與氧化壓力的角色 第二部分：探討血漿中三羧酸循環代謝物與敗血症多重器官衰竭嚴重度之相關性

Using targeted metabolomics approach and hydrophilic interaction liquid chromatography coupled with mass spectrometry for clinical application Part I: Investigating the role of isolated γ-glutamyltransferase elevation and oxidative stress in patients with non-alcoholic steatohepatitis Part II: Investigating the association between plasma level of tricarboxylic acid cycle metabolites and the severity of multiple organ dysfunction in sepsis

陳易萱(Yi-Hsuan Chen)

指導教授 ： 郭錦樺

共同指導教授 ： 鐘桂彬(Kuei-Pin Chung)

國立臺灣大學/醫學院/藥學研究所/碩士(2022年)

https://doi.org/10.6342/NTU202202695

摘要

代謝體學是透過全盤分析生物系統（細胞、組織、器官、體液或生物體）中代謝物的科學研究，可代表體內複雜生物系統之間的整體相互作用，並更直接反映表現型。代謝體學在研究病理機制和辨別疾病的潛在生物標誌已成為潛力領域，其中標的代謝體學是對特定代謝物進行定量分析的方法。在本論文中，我們使用標的代謝體學，透過選擇測量不同代謝途徑中的數個代謝物來回答臨床問題。考量本研究標的分析物的極性，我們使用親水性作用液相層析結合質譜儀來分析。
在本論文的第一部分，我們探討非酒精性脂肪性肝炎（non-alcoholic steatohepatitis, NASH）患者單獨丙麩胺酸轉移酶（γ-glutamyl transferase, GGT）升高和氧化壓力的相關性。非酒精性脂肪肝炎是屬於一種嚴重的非酒精性脂肪肝疾病（non-alcoholic fatty liver disease, NAFLD），其特徵為肝內脂肪堆積合併壞死性發炎，可能惡化為嚴重纖維化與肝癌。氧化壓力被認為是 NASH 的致病原因，在 NASH 病人中經常觀察到GGT單獨上升的情形。GGT 已被提出作為 NASH 患者進展為纖維化的標誌。然而，分子機轉層面的機制尚未被清楚地了解。本研究開發了液相層析串聯質譜儀方法來測量人體血清中的氧化壓力代謝物，分析了 NASH 患者血清中的代謝物，並將 GGT 升高的 NASH 患者（n=60）與 GGT 正常的 NASH 患者（n=44）進行了比較。研究結果顯示，在 GGT 升高組中有較低的t-cysteinylglycine和較高的glutathione前驅物，包括glutamic acid、cystine和glycine。此外，glutamine與glutamate的比值在GGT上升組與GGT正常組相比顯著降低（p = 0.014），顯示出glutamine被轉換成為glutamate，以增加glutathione的生成來調節氧化壓力。這些結果顯示GGT升高的NASH患者體內確實有較高的氧化壓力，為 NASH 研究提供更深入的機制資訊。
在第二部分中，我們研究了血漿三羧酸循環（TCA cycle）相關代謝物濃度與敗血症多重器官衰竭嚴重程度之間的關係。敗血症是由於宿主對感染的反應失調而導致的急性器官衰竭的嚴重疾病。過去的研究提出，生物能量受到干擾後會導致與敗血症相關的多重器官衰竭。TCA cycle由多個單向酵素反應組成，是真核細胞產生能量的基礎。然而，目前尚不清楚TCA cycle受損是否影響敗血症相關器官衰竭進展。在本研究中，我們應用LC-MS/MS標的代謝體學方法來測量血漿中TCA cycle代謝物，並研究 TCA cycle代謝物的血中濃度是否與敗血症器官衰竭的嚴重度相關。我們的研究結果顯示，敗血症造成的器官衰竭與malate累積和下游代謝物包括citrate/isocitrate和cis-aconitate無改變有關。我們指出citrate synthase缺乏可能與敗血症器官衰竭的發展有關。這項研究提供了對敗血症器官衰竭的分子機轉資訊，並支持未來的機轉研究。
總結，本論文成功使用標的代謝體學方法研究 NASH患者的血清代謝物和敗血症患者血漿中的TCA cycle代謝物，提供了對這兩種疾病分子機制的重要資訊。

關鍵字

代謝體學 ； 氧化壓力 ； 丙麩胺酸轉移酶 ； 非酒精性脂肪性肝炎 ； 敗血症 ； 三羧酸循環

並列摘要

Metabolomics is a scientific study through a comprehensive analysis of metabolites in a biological system (cell, tissue, organ, biological fluid, or organism). Metabolomics could represent the overall interactions between whole complex biosystems in the body and be more direct in reflecting the phenotype. Metabolomics has become a promising area for investigating pathological mechanism and identifying potential biomarkers of diseases. Targeted metabolomics is a quantitative approach to evaluate a set of selected metabolites. In this thesis, we used targeted metabolomics methods by selecting several metabolites in different metabolic pathways to answer the clinical problem. Considering the polar properties of target analytes, a hydrophilic interaction liquid chromatography coupled with mass spectrometry was used in this study.
In the first part of this thesis, we investigated the association of isolated γ-glutamyltransferase (GGT) elevation and oxidative stress in patients with nonalcoholic steatohepatitis (NASH). NASH, the inflammatory subtype of nonalcoholic fatty liver disease, may deteriorate into advanced fibrosis and liver cancer, leading to substantial medical burdens. Oxidative stress is considered the major pathogenic determinant of NASH, and isolated GGT elevation is frequently observed in NASH patients. GGT has been proposed as a marker for fibrosis progression in NASH patients. However, specific molecular mechanisms are not yet clearly understood. We developed LC-MS/MS methods to comprehensively analyze metabolites associated with oxidative stress in human serum. The serum metabolic patterns in NASH patients were analyzed, and NASH patients with isolated GGT elevation (n = 60) were compared with NASH patients with normal GGT (n = 44). This study showed a significantly lower t-cysteinylglycine and higher glutathione (GSH) precursors, including glutamic acid, cystine, and glycine, are present in the GGT elevation group. Moreover, a significantly lower glutamine-to-glutamate ratio (p = 0.014) in the GGT elevation group implies the transformation of glutamine to glutamate was also increased to produce more GSH to relieve oxidative stress. The findings uncover the oxidative stress in NASH patients with isolated GGT elevation and provide mechanism insights into NASH progression.
In the second part, we investigated the association between the plasma level of tricarboxylic acid cycle (TCA cycle)-related metabolites and the severity of multiple organ dysfunction in sepsis. Sepsis is a life-threatening disease featured by acute organ dysfunction due to dysregulated host responses to infection. Previous studies suggested that perturbed bioenergetics lead to sepsis-related multi-organ dysfunction. TCA cycle, constituted by multiple unidirectional enzymatic reactions, is responsible for energy production in eukaryotic cells. However, it is unclear whether impaired TCA cycle underlies the development of sepsis-related organ dysfunction. In this study, we applied an LC-MS/MS-based targeted metabolomics method to measure metabolites in the TCA cycle and investigated whether the levels of TCA cycle-related metabolites are associated with the severity of organ dysfunction in sepsis. Our results demonstrated that sepsis-related organ dysfunction is associated with malate accumulation and unaltered downstream metabolites, including citrate/isocitrate and cis-aconitate. In addition, we pointed out that deficiency of citrate synthase may be related to the development of organ dysfunction in sepsis. This study provides mechanistic insights into sepsis-related organ dysfunction and supports future mechanistic translational studies.
In conclusion, these two studies successfully used targeted metabolomics methods to investigate GGT elevation in NASH and TCA cycle-related metabolites in sepsis. The results provide mechanistic insights into these two diseases.

並列關鍵字

metabolomics ； oxidative stress ； GGT ； NASH ； sepsis ； TCA cycle

參考文獻

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