第一部分:以液相層析質譜儀結合基質校正定量採血卡上的TMAO以及其前驅物和衍生物 第二部分：建立多重標記衍生法以分析尿液中之羧酸代謝物

Abstract

代謝組學為細胞、組織和體液中的代謝物廣泛性研究。透過代謝組學的研究，可瞭解個體的基因型態、飲食模式、受環境和腸道微生物影響之因子。代謝體量化分析提供了研究從正常生理到多種疾病狀態的機制資訊。因此有效率的分析方法對闡述代謝物的功能和人體健康非常重要。 本論文第一部分欲建立一個定量方法以分析採血卡上的TMAO與其前驅物質和衍生物，近年來人們對於腸道菌對許多疾病如心血管疾病和代謝異常的影響深感興趣，而氧化三甲胺（Trimethylamine-N-oxide，TMAO）被認為是影響心血管疾病的重要因子。採血卡是一種新型的採樣技術，被應用於許多臨床研究，且具有許多優點。藉由分析整體血點樣品可以降低血比容造成的定量誤差，但需要測量血液體積藉以校正濃度。我們開發了運用液相層析質譜儀結合基質效應導致的離子抑制現象（matrix-induced ion suppression，MIIS）測量血點體積並定量採血卡上的TMAO、膽鹼（choline）、肉鹼 （carnitine）和乙醯肉鹼 （acetylcarnitine）。MIIS方法為測量不同血點體積對離子抑制指示劑（ion suppression indicator，ISI）造成的訊號抑制程度。結果顯示利用MIIS的方法評估血點體積之準確度介於91.7~109.7 %。我們將MIIS方法結合液相層析串聯質譜儀做定量方法確校，包含線性、精準度與準確度評估。每個分析物的最低定量極限濃度之準確度小於119 %﹔方法確校之定量準確度均介於91.2~113.2 %之間，且精密度的相對標準差皆小於8.0 %。安定性實驗中，採血卡檢體中之分析物在所有測試的溫度下均穩定至少30天。最後我們將確校後的方法用於定量60個採血卡檢體。此方法可有效地運用在測量採血卡上的代謝物濃度，並有助於了解腸胃菌在人體中扮演的角色。 生物體內廣泛存在著大量的含有羧酸的代謝產物，包括有機酸、氨基酸和三羧酸循環產物。有機酸的變化與先天性代謝異常疾病（inborn errors of metabolism，IEM）相關，未及時治療可能會導致腦損傷，甚至死亡。觀察代謝物濃度在不同時間的變化有益於追蹤病程。利用多重分析的策略，除了改善層析分離還能達到高通量的分析方法。多重化學衍生法過去已用於代謝組學研究，但仍存在一些限制，例如需要昂貴的標記試劑與高解析儀器。因此，本論第二部分為利用多重標記法衍生尿液中之羧酸代謝物，並利用液相層析串聯質譜定量丁基衍生物D0-、D3-、D5-、D7-和D9-羧酸丁酯（carboxylic acid butyl ester，CABE）。結果顯示多重標記法之準確度在85.22~115.16 %之間。接著將此分析方法進行確校，包含線性、精密度和準確性。結果顯示除了兩種分析物外，其它分析物的定量準確度均在79.1~116.6 %之間，且精確度小於15 %。最後利用已確校的多重標記法定量四個尿液樣本中的羧酸代謝物，研究不同時間的羧酸代謝產物之變化。此創新的多重標記法被認為有助於IEM的研究並縮短臨床診斷時間，有益於未來之臨床研究。 總結本論文開發的分析方法可準確定量採血卡上的TMAO與其前驅物質和衍生物，以及同時定量不同時間點的尿液中羧酸代謝物。預期此兩種定量方法可廣泛被運用至許多臨床研究。

Metabolomics is the comprehensive study of the metabolome in cells, tissues, and body fluids. The metabolome reflects what has been encoded by the genome, and modified by diet, environmental factors, and the gut microbiome. The metabolic profile provides a quantifiable result from normal physiology to diverse disease status. Effective analytical methods are essential to delineate the function of metabolites and human health. In the first part of this thesis, we have developed a quantitative method to measure TMAO and its precursors and derivatives in DBS samples. Recently, there has been significant interest in the influences of the human gut microbiota on many diseases, such as cardiovascular disease (CVD) and metabolic disorders. Trimethylamine N-oxide (TMAO) is one of the most frequently discussed gut-derived metabolites. Dried blood spot (DBS) sampling has been regarded as an attractive alternative sampling strategy for clinical studies and offers many advantages. For DBS sample processing, whole-spot analysis could minimize hematocrit-related bias, but it requires blood volume calibration. This study developed a method combining matrix-induced ion suppression (MIIS) with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) to estimate blood volume and quantify TMAO and its precursors and derivatives, including choline, carnitine and acetylcarnitine, in DBSs. The MIIS method used an ion suppression indicator (ISI) to measure the extent of ion suppression caused by the blood matrix, which was related to the blood volume. The results showed that the volume estimation accuracy of the MIIS method was within 91.7-109.7%. The combined MIIS and LC-MS/MS method for quantifying TMAO, choline, carnitine and acetylcarnitine was validated in terms of linearity, precision and accuracy. The quantification accuracy was within 91.2-113.2% (with LLOQ <119%), and the precision was below 8.0% for all analytes. A stability study showed that the analytes in DBSs were stable at all evaluated temperatures for at least 30 days. The validated method was applied to quantify DBS samples (n=60). Successful application of the new method demonstrated the potential of this method for real-world DBS samples and to facilitate our understanding of the gut microbiota in human health. In the second part, a vast number of metabolites containing carboxyl moieties exist widely in living organism including organic acids, amino acids, and TCA cycle intermediates. Changes in these carboxylic acid profiles are sometimes associated with inborn errors of metabolism (IEM), which could cause severe damages and be lethal without treatment in time. As a result, the follow-up on relevant carboxylic acids with time-series analysis will be beneficial to study the disease progression. However, it is challenging to speed up analytical procedure with chromatographic techniques to achieve high-throughput analysis, unless innovative multiplex strategy is utilized. Chemical derivatization for multiplex approach has been used in studying metabolomics, but there are still some limitations such as expensive labeling tag and requiring high-resolution instruments. This study developed a multiplex method combined with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to quantify essential carboxylic acids with different butanol isotopes to form butyl-derivatives D0-, D3-, D5-, D7- and D9- carboxylic acid butyl ester (CABE) in urine. The results show the differential mixture estimation accuracy of the multiplex method was within 85.22% to 115.16%. The LC-MS/MS for quantified the carboxylic acids was validated in terms of linearity, precision and accuracy. The quantification accuracies for all analytes were ranged from 79.1-116.6%, and precisions were below 15%, except for two compounds. The validated multiplex method was applied to quantified analytes in four urine samples for simulated time-series study. In summary, the innovative multiplex approach is believed to contribute for studying IEM and shorten time for clinical diagnosis that would be greatly benefit clinical researches in the near future. In conclusion, the developed analytical methods were demonstrated to be accurate and precise. These two efficient methods are anticipated to broaden their applications to various clinical studies.