革新體外代謝分析技術：用於兒童脂肪肝病的早期檢測與機制探討

Abstract

本論文針對脂肪變性肝病（SLD）的診斷與管理中的關鍵缺口進行探討，該疾病在肥胖兒童中尤為普遍。當前的診斷方法具侵入性或對早期偵測敏感度不足，而有效的藥物治療亦相當有限。本研究致力於開發一種新穎的非侵入性診斷方法，並探討腸道微生物群在SLD發病機制中的作用。

第一部分 介紹了一種pH依賴性的核磁共振（NMR）尿液代謝體學方法，可直接測定原始尿液中的代謝物，無需調整pH值，從而提高對肥胖相關代謝疾病的診斷準確性。本方法建立了pH 依賴性的化學位移資料庫，並鑑定出尿液濃度校準物及pH探針，以增強訊號識別能力並提高樣本穩定性。

第二部分 驗證了pH依賴性NMR方法在三個獨立兒童SLD隊列中的臨床應用價值，發現異丁酸與纈氨酸在非空腹尿液中可作為高度準確的早期生物標誌物（AUC 0.955），強調非空腹尿液採集的重要性。隨後，在臨床相關的小鼠SLD模型中進行驗證，進一步確認該方法的診斷潛力。此外，在飲食逆轉的小鼠SLD模型中，發現靛基硫酸（indoxylsulfuric acid）可作為監測SLD改善的新型腸道微生物標誌物。

第三部分 研究了腸道微生物群失調與 SLD 發病機制之間的關聯。在小鼠模型中進行16S rRNA定序，鑑定出SLD相關的致病菌Faecalibaculum rodentium與Olsenella massiliensis，以及具有益生菌特性的Duncaniella freteri。進一步的體內外研究顯示，這些致病菌促進肝臟脂肪變性，而D. freteri能有效減少肝臟脂質累積，其效果優於常見的Lactobacillus rhamnosus GG。這突顯了益生菌治療在SLD管理中的潛力，以及腸道菌群在疾病進展與逆轉中的關鍵作用。

本論文提供了一種創新的非侵入性尿液代謝體學平台，用於SLD的早期診斷與監測，並闡明了腸道微生物群失調在SLD進展中的作用。研究成果為診斷與治療SLD提供了新策略，深化了對腸–肝互動的理解，並有助於改善兒童SLD的臨床管理。

This dissertation addresses critical gaps in the diagnosis and management of steatotic liver disease (SLD), a prevalent condition, especially in obese children. Current screening methods are invasive or lack sensitivity for early detection, and effective pharmacological treatments are limited. This research focused on developing a non-invasive, novel diagnostic approach and exploring the role of gut microbiota in SLD pathogenesis.

Part I introduces a pH-dependent nuclear magnetic resonance (NMR) urine metabolomics method that quantifies metabolites in native urine, eliminating the need for pH adjustments and improving diagnostic accuracy for obesity-associated metabolic disorders. This method establishes a pH-dependent chemical shift database and identifies a urine concentration calibrator and a pH probe, enhancing signal recognition and sample stability.

Part II demonstrated the clinical utility of the pH-dependent NMR method for pediatric SLD detection across three independent cohorts, revealing isobutyric acid and valine as highly accurate early-stage biomarkers (AUC 0.955) in non-fasting urine, underscoring the importance of non-fasting collection. Subsequent validation in a clinically relevant murine SLD model confirmed the method's diagnostic potential. Additionally, a diet-reversal murine SLD model identified indoxylsulfuric acid as a novel microbial biomarker for monitoring SLD regression.

Part III investigates the mechanistic link between gut microbiota dysbiosis and SLD pathogenesis in the murine models. 16S rRNA sequencing identified Faecalibaculum rodentium and Olsenella massiliensis as SLD-associated pathobionts and Duncaniella freteri as a beneficial probiotic. In vitro and in vivo studies demonstrated that the pathobionts promote hepatic steatosis while D. freteri effectively reduces lipid accumulation, surpassing commonly used Lactobacillus rhamnosus GG. This highlights the potential of probiotic interventions for SLD management and the role of gut microbiota in both disease progression and regression.

This dissertation provides a novel, noninvasive urine metabolomics platform for early SLD detection and monitoring and elucidates the role of gut dysbiosis in SLD progression. The findings offer innovative diagnostic and therapeutic strategies, advancing our understanding of gut–liver interactions and improving pediatric SLD management.