探討人體腸道菌代謝肉鹼與生成氧化三甲胺產能之研究

Abstract

近期研究發現腸道菌在人體內所產生氧化三甲胺(TMAO)在心血管疾病上扮演了重要角色。本論文第一章藉由藥物動力學的測試，並招募葷食與素食受試者進行確效實驗，建立一個具臨床實用性的肉鹼耐量試驗(oral carnitine challenge test, OCTT)，藉此測量飲食-腸道菌-宿主間交互作用產生TMAO的能力。結果顯 示葷食者的腸道菌比素食者有較高的TMAO生成能力，而OCCT相較空腹TMAO對於辨別腸道菌生成TMAO能力有顯著較佳效果。而在相同時間採集的血液中與尿液中的TMAO濃度，呈現非常顯著的正相關(r = 0.92, p<0.0001)，有助於提升OCCT臨床應用的可行性，未來此檢測有機會作為是否建議減少紅肉攝取的個人化營養參考依據。在第二章中，我們讓招募的葷素食者受試著在不改變原本飲食習慣的情況下，連續每日服用肉鹼補充錠一個月，並且在介入前後進行OCCT，觀察腸道菌TMAO產能是否有所改變，並採集服用肉鹼一個月前後的糞便進行菌相分析。結果發現，服用一個月肉鹼補充錠後，葷食者空腹 TMAO上升的程度較素食者明顯。不過，素食者腸道菌生成TMAO的產能(OCCT TMAOAUC and TMAOMAX)增加的情形則較葷食者顯著。我們發亦現原先腸道菌產能較低的TMAO低產能者(TMAOMAX <10 μM)，在服用肉鹼補充劑一個月後，其 TMAO的產能有顯著上升(p=0.0003)的情形，並且有23%的TMAO低產能者變成了TMAO高產能者，但對TMAO高產能者來說，服用肉鹼補充錠一個月對腸道菌TMAO生成能力並無明顯影響。此外，我們發現血中肉鹼低於20 μM時，腎臟會傾向將 肉鹼留在身體內，且素食者與TMAO低產能者有較高的肉鹼生物利用率。在菌相分析部分，我們發現TMAO高產能與低產能者有微幅顯著的差異，不過介入肉鹼補充劑一個月後對腸道菌相並沒有造成顯著改變，以16S rRNA片段分析則發現 Ruminococcaceae與Eubacterium這兩群細菌，在TMAO高產能者有顯著增加的情形。本論文第三章的部分，我們參考克里夫蘭Stanley Hazen團隊2019年發表Emergencia timonesis能將gamma- butyrobetaine(gBB)代謝成TMA，並可能是決定人體腸道菌TMAO高產能者重要菌種的發現，針對我們的研究數據進行分析。發現Emergencia timonesis僅能解釋四分之一的TMAO高產能者，且目前將gBB轉換成TMA的基因表現與蛋白質合成仍未清楚。因此，我們利用我們所開發的OCCT與相關菌相數據，以多體學的方式搭配仿人體腸道菌鼠模型進行研究，嘗試找出TMAO高產能者將gBB轉換成TMA的腸道菌種並進行分離培養，希望能找出相對應的基因序列與酵素，作為開發抑制腸道菌減少TMA合成的新藥標的。

Recently, a gut microbiota-generated metabolite, the trimethylamine N-oxide (TMAO), was proved to play important role in cardiovascular disease. In the first chapter of this thesis, we developed a practical oral carnitine challenge test (OCCT) in clinical scenario based on pharmacokinetics study and a validation in a cohort composed of omnivores and vegetarians. The OCCT well demonstrate the interactions between diet, gut microbiota, and the host in producing TMAO in human body. Our results showed the omnivores had stronger TMAO producing capacity as compared with vegetarians and the OCCT also had better discriminative power than fasting TMAO in identifying the gut microbial function of TMAO production. Besides, we found very significant correlation between plasma and urine TMAO (r = 0.92, p<0.0001) and may promote the feasibility of this test in clinical use. The OCCT may also serve as a reference of personalized nutrition in guiding the control of red meat consumption. In the second chapter, these recruited omnivores and vegetarians were supplemented with carnitine tablet once daily for 1 month without change of original diet habit. The OCCT was performed before and after the period of carnitine supplementation for each individual to know whether the TMAO producing capacity from gut microbiota would be changed. Stool samples were also collected during each OCCT. The omnivores demonstrated higher fasting TMAO after carnitine supplementation as compared with vegetarians. However, the vegetarians exhibited more significant increased TMAO producing capacity (OCCT TMAOAUC and TMAOMAX) as compared with omnivores. We also noticed that the TMAO production capacity in TMAO low producer (TMAOMAX <10 μM) could be enhanced significantly after carnitine supplementation, and there were about 23% TMAO low producer shifting to TMAO high producer after 1-month carnitine supplementation. In contrast, the TMAO production capacity in TMAO high producers was not altered during the intervention. Besides, we found the kidney tend to preserve the carnitine in the body as while excrete TMAO as wastes. The vegetarians and TMAO low producers also had better bioavailability of carnitine. For the microbiome analysis, we found significant but mild difference of gut microbial profiles between TMAO high and low producers, but no significant alteration of compositional profile was found for the carnitine supplementation. We also found the groups of Ruminococcacea and Eubacterium were enriched in TMAO high producers. In the third chapter, we reanalyze our microbiome data according the recent discovery from the team of Stanley Hazen that Emergencia timonesis can convert gamma- butyrobetaine (gBB) into TMA and may be a key in TMAO production in vivo. However, we only detected this microbe in the samples from 25% of our TMAO high producer subjects. Since the genes and protein responsible for converting gBB into TMA remains unknown, we attempted to find solution by using multi-omic approach and humanized gnotobiotic model in order to elucidate the relevant gene sequences and enzyme. By doing so, we hope we can find a new target for reducing TMA synthesis in the gut and for drug development.