Among patients with rheumatoid arthritis, we recently observed strong and consistent associations between vitamin B6 status and several indicators of inflammation, including disease-related disability, morning stiffness, the degree of pain, C - reactive protein levels, serum albumin levels, and erythrocyte sedimentation rate. Suboptimal vitamin B6 status has been reported in several inflammatory conditions. However, despite that several clinical trials and epidemiological studies demonstrated the associations between vitamin B6 and inflammatory diseases, the cause-effect relationship between these two has not been elucidated. In the present study we hypothesize that inflammatory response results in tissue specific vitamin B6 depletion, which further impairs homocysteine catabolism. The accumulated homocysteine may drive the metabolic flux towards remethylation, and subsequently alter s-adenosyl methionine synthesis. Aberrant DNA methylation has been observed in rheumatic diseases and systemic lupus erythematosus. As the universal methyl donor, altered s-adenosyl methionine may indirectly influence DNA methylation patterns in these animals. We propose to use an adjuvant arthritis model to study the interactions between inflammatory response, vitamin B6 status, homocysteine metabolism, methyl group kinetics, and DNA methylation. The combination of stable isotopic tracers and GC/MS is a powerful tool for studying the kinetics and regulation of many metabolic pathways in vivo. These approaches may reveal regulatory mechanisms by which inflammatory diseases results in metabolic changes and possibly alters DNA methylation status in vivo.

本研究室近年研究發現人類類風溼性關節炎中多種發炎反應指標與與血中維生素B6

呈現負相關。近來亦有多項研究報告顯示疾病中的發炎反應和維生素B6缺乏有關。 但此二者間的關係多限於關聯性研究，以流行病學或是臨床研究形式為主。至本研 究以前尚無證據顯示其間有因果關係，相關的機制尚無定論。本計畫將以活體動物 模型探討發炎反應對維生素B6狀態直接及間接的影響。除了希望闡明其因果關係， 並以獨特的方式定量代謝路徑，深入探討營養狀況、發炎反應與轉甲基反應之間的 交互作用。我們發現發炎反應會引發特定組織的維生素B6 缺乏，由於依賴維生素 B6之 cystathionineβsynthase 為同半胱氨酸降解過程中的關鍵酵素，因此發炎反 應可能降低cystathionineβsynthase 活性並導致同半胱氨酸堆積。而升高同半胱氨酸 可能會促進其再甲基化反應及甲硫氨酸生合成，因而間接影響s-腺核苷甲硫 胺酸的供應甚DNA 甲基化。維生素B6 缺乏亦可能影響組織中seirine hydroxylmethyl transferase 活性，進而影響同半胱氨酸、甲硫氨酸、S-腺核苷甲硫胺 酸及嘧啶合成代謝路徑的平衡。本計畫將進行一系列實驗追蹤各代謝途徑，希望進 一步了解發炎反應、維生素B6 營養狀態及甲基族代謝路徑之間的關係。相信本計 畫可為自己和他人的研究提供疾病模式、營養及代謝等多層面的重要資訊，研究成 果將兼具學術與實用的價值。

The significance of epigenetic alterations in autoimmune rheumatic diseases and degenerated joint diseases has drawn great attention among clinicians and researchers. Aberrant methylation status has been demonstrated in human chronic inflammation yet more efforts have focused on global and sequence-specific hypomethylation and overexpression of specific genes. Few studies investigated the regulation of S-adenosylmethionine homeostasis and regulation during inflammation. At present the relevance and regulation of the complex epigenetic profiles and their modifications among different tissues and organs during inflammation remain largely unknown. We propose to conduct a translational study on the regulation of S-adenosylmethionine synthesis and cellular methylation reactions during chronic inflammation. Development of in vitro cell models may reveal the regulatory mechanisms by which specific inflammatory mediators cause metabolic changes and alter DNA methylation status. Metabolic and pharmacological studies in the in vivo models will enable us to better understand the regulation of inter-organ homeostasis of S-adenosyl methionine and help identify tissue specific biomarkers for methylation and epigenetic modifications in different stage of chronic inflammation. The clinical study in human subjects will help distinguish the impacts of autoimmune rheumatic disease, degenerated joint disease, or specific medication use on significant clinical markers.We hope the present study can identify specific clinical markers for potential epigenetic changes in patients, which will contribute to better clinical management of these diseases in humans.
發炎作用對於單碳傳遞系統中之特定酵素的調控機制尚未被詳細闡明。過去研究 已知自體免疫病患DNA 整體甲基化有異常現象，但疾病發炎作用對於體內甲基 供應、消耗、平衡及其中詳細調控機制尚未被詳細研究。在本領域申請人至今已 累積多篇臨床轉譯或基礎研究論文並有相關專利申請中。本計畫結合醫學中心優 質的臨床經驗及龐大病患群與多年來基礎與臨床研究成果，運用即將建立的細胞 模式與追蹤系統全面性探討風濕性或退化性關節疾病發炎過程當中特定發炎媒 介分子對體內腺苷甲硫胺酸合成及平衡之調控。我們將逐一探討臨床慢性發炎反 應對腺苷甲硫胺酸生合成路徑的作用及其中調控機制，希望能由此闡明發炎過程 如何影響腺苷甲硫胺酸依賴隻甲基轉移代謝路徑、相關基因表現剖繪、並探討甲 基傳遞與甲基化，並將基礎研究成果與臨床發現相互印證。計畫所提出的內容基 於多項前驅研究數據，有充分證據顯示確實可行。除豐富經驗外，對於預計可能 遭遇之困難及解決途徑亦設想周全，已有相當完整準備。本計畫結合臨床與基礎 學者共同投入此項研究，期能將臨床上的發現與活體模式及細胞層級相互印證， 經由追蹤代謝路徑深入研究體內系統全面性發炎反應或特定發炎媒介分子對於 腺苷甲硫胺酸代謝與甲基轉移失衡之影響。臨床上的發現將以特定動物模式深入 探討，同時也將基礎研究延伸至臨床病患，例如探究抗發炎免疫調節藥物的使用 對於硫腺苷甲硫胺酸恆定之影響及臨床上如何改善腺苷甲硫胺酸平衡等。研究後 期會進行整合，有效串聯不同層面實驗結果，相信會在學術及臨床應用上都會有 相當好的貢獻。

Among patients with rheumatoid arthritis, we recently observed strong and consistent associations between vitamin B6 status and several indicators of inflammation, including disease-related disability, morning stiffness, the degree of pain, C - reactive protein levels, serum albumin levels, and erythrocyte sedimentation rate. Suboptimal vitamin

B6 status has been reported in several inflammatory conditions. However, despite that several clinical trials and epidemiological studies demonstrated the associations between vitamin B6 and inflammatory diseases, the cause-effect relationship between these two has not been elucidated. In the present study we hypothesize that inflammatory response results in tissue specific vitamin B6 depletion, which further impairs homocysteine catabolism. The accumulated homocysteine may drive the metabolic flux towards remethylation, and subsequently alter s-adenosyl methionine synthesis. Aberrant DNA methylation has been observed in rheumatic diseases and systemic lupus erythematosus. As the universal methyl donor, altered s-adenosyl methionine may indirectly influence DNA methylation patterns in these animals. We propose to use an adjuvant arthritis model to study the interactions between inflammatory response, vitamin B6 status, homocysteine metabolism, methyl group kinetics, and DNA methylation. The combination of stable isotopic tracers and GC/MS is a powerful tool for studying the kinetics and regulation of many metabolic pathways in vivo. These approaches may reveal regulatory mechanisms by which inflammatory diseases results in metabolic changes and possibly alters DNA methylation status in vivo.

本研究室近年研究發現人類類風溼性關節炎中多種發炎反應指標與與血中維生素B6

呈現負相關。近來亦有多項研究報告顯示疾病中的發炎反應和維生素B6缺乏有關。 但此二者間的關係多限於關聯性研究，以流行病學或是臨床研究形式為主。至本研 究以前尚無證據顯示其間有因果關係，相關的機制尚無定論。本計畫將以活體動物 模型探討發炎反應對維生素B6狀態直接及間接的影響。除了希望闡明其因果關係， 並以獨特的方式定量代謝路徑，深入探討營養狀況、發炎反應與轉甲基反應之間的 交互作用。我們發現發炎反應會引發特定組織的維生素B6 缺乏，由於依賴維生素 B6之 cystathionineβsynthase 為同半胱氨酸降解過程中的關鍵酵素，因此發炎反 應可能降低cystathionineβsynthase 活性並導致同半胱氨酸堆積。而升高同半胱氨酸 可能會促進其再甲基化反應及甲硫氨酸生合成，因而間接影響s-腺核苷甲硫 胺酸的供應甚DNA 甲基化。維生素B6 缺乏亦可能影響組織中seirine hydroxylmethyl transferase 活性，進而影響同半胱氨酸、甲硫氨酸、S-腺核苷甲硫胺 酸及嘧啶合成代謝路徑的平衡。本計畫將進行一系列實驗追蹤各代謝途徑，希望進 一步了解發炎反應、維生素B6 營養狀態及甲基族代謝路徑之間的關係。相信本計 畫可為自己和他人的研究提供疾病模式、營養及代謝等多層面的重要資訊，研究成 果將兼具學術與實用的價值。

Among patients with rheumatoid arthritis, we recently observed strong and consistent associations between vitamin B6 status and several indicators of inflammation, including disease-related disability, morning stiffness, the degree of pain, C - reactive protein levels, serum albumin levels, and erythrocyte sedimentation rate. Suboptimal vitamin

B6 status has been reported in several inflammatory conditions. However, despite that several clinical trials and epidemiological studies demonstrated the associations between vitamin B6 and inflammatory diseases, the cause-effect relationship between these two has not been elucidated. In the present study we hypothesize that inflammatory response results in tissue specific vitamin B6 depletion, which further impairs homocysteine catabolism. The accumulated homocysteine may drive the metabolic flux towards remethylation, and subsequently alter s-adenosyl methionine synthesis. Aberrant DNA methylation has been observed in rheumatic diseases and systemic lupus erythematosus. As the universal methyl donor, altered s-adenosyl methionine may indirectly influence DNA methylation patterns in these animals. We propose to use an adjuvant arthritis model to study the interactions between inflammatory response, vitamin B6 status, homocysteine metabolism, methyl group kinetics, and DNA methylation. The combination of stable isotopic tracers and GC/MS is a powerful tool for studying the kinetics and regulation of many metabolic pathways in vivo. These approaches may reveal regulatory mechanisms by which inflammatory diseases results in metabolic changes a nd possibly alters DNA methylation status in vivo.

本研究室近年研究發現人類類風溼性關節炎中多種發炎反應指標與與血中維生素B6

呈現負相關。近來亦有多項研究報告顯示疾病中的發炎反應和維生素B6缺乏有關。 但此二者間的關係多限於關聯性研究，以流行病學或是臨床研究形式為主。至本研 究以前尚無證據顯示其間有因果關係，相關的機制尚無定論。本計畫將以活體動物 模型探討發炎反應對維生素B6狀態直接及間接的影響。除了希望闡明其因果關係， 並以獨特的方式定量代謝路徑，深入探討營養狀況、發炎反應與轉甲基反應之間的 交互作用。我們發現發炎反應會引發特定組織的維生素B6 缺乏，由於依賴維生素 B6之 cystathionineβsynthase 為同半胱氨酸降解過程中的關鍵酵素，因此發炎反 應可能降低cystathionineβsynthase 活性並導致同半胱氨酸堆積。而升高同半胱氨酸 可能會促進其再甲基化反應及甲硫氨酸生合成，因而間接影響s-腺核苷甲硫 胺酸的供應甚DNA 甲基化。維生素B6 缺乏亦可能影響組織中seirine hydroxylmethyl transferase 活性，進而影響同半胱氨酸、甲硫氨酸、S-腺核苷甲硫胺 酸及嘧啶合成代謝路徑的平衡。本計畫將進行一系列實驗追蹤各代謝途徑，希望進 一步了解發炎反應、維生素B6 營養狀態及甲基族代謝路徑之間的關係。相信本計 畫可為自己和他人的研究提供疾病模式、營養及代謝等多層面的重要資訊，研究成 果將兼具學術與實用的價值。

Numerous disease-modifying anti-rheumatic drugs are used to reduce inflammation in the treatment of rheumatoid

arthritis. We investigated whether long-term disease- modifying anti-rheumatic drugs treatment at the dose of treating inflammation can alter vitamin B(6) status. In this study, in vivo models were used. C57BL/6J mice received drug A and B regimens that reflected clinical uses in treating human inflammation. We found that drug A treatment increased concentrations of bioactive B6 vitamers and reduced the concentrations of the end product of this vitamin in the liver. It also increased plasma concentrations of bioactive vitamin B6. And drug B treatment increased concentrations of bioactive B6 vitamers and increased erythrocyte concentrations of bioactive vitamin B6. We conclude that long-term disease-modifying anti-rheumatic drugs A and B treatments may assist the activation and utilization of vitamin B6 in those who are on this therapy. The elevated liver B6 vitamers may be beneficial for those with a higher B6 demand during inflammation. Results from in vivo models suggested a potential advantage of clinical disease-modifying anti- rheumatic drugs A and B use in treating inflammation with respect to vitamin B(6) status.
本計劃在本年度探討不同免疫調節藥物對於維生素B6代謝之影響。 本研究利用兩個獨立動物模式證明長期使用特定之臨床生理劑量抗發炎免疫調節藥物能夠分別增加內源性維生素B6。基於本研究發現, 推論特定抗發炎免疫調節藥物具有做為須服用消炎劑或免疫抑制劑 缺乏維生素B6患者之維生素B6促進劑之潛力，亦可提供臨床上供用 於治療或預防由於疾病所造成之體內維生素B6缺乏症用藥之參考。 較早期之報導曾指出罹患類風濕性關節炎之患者有維生素B6缺乏現 象, 而本計畫發現長期服用特定臨床治療免疫調節藥物對於患者具 有可增加其體內維生素活化之額外助益, 共三項研究成果專利申請 中。

第二環氧酶抑制劑 (Cyclooxygenase II inhibitor, Cox-II inhibitor) 屬於非類固醇類抗發炎藥物的一種，其主要是抑制調控發炎作用和造成疼痛的前列腺素之合成。目前在台灣臨床上使用Cox-II 抑制劑止痛藥之最大族群之一是慢性關節炎病患，主要用於緩解發炎與疼痛。然而在2004年美國一項為期三年的的隨機對照臨床研究中卻發現服用 rofecoxib的患者其產生心血管不良反應的危險性明顯高於對照組，因而造成此藥全球性下市。至於另一被普遍使用之 Cox-II 抑制劑止痛藥 Celecoxib是否確實會直接增加心血管副作用的危險性仍未有定論。國內外多項研究顯示Cox-II 抑制劑對不同心血管副作用的相關危險因子所產生之影響有顯著不同。 由於心血管疾病常見終點事件的發生多為長期累積多元危險因子而成的歷程，針對心血管疾病和 Cox-II 抑制劑止痛藥之使用來評估藥物使用之安全性，進行長期且大規模的流行病學世代追蹤研究是必要的。臺灣之全民健保資料庫包含多年間全民心血管疾病其終點事件例如心肌梗塞、腦血管事件等醫師診斷，及心血管疾病患者與非患者其用藥細節與醫師處方等，提供了獨特而寶貴資訊。唯全民健保資料庫中尚無相關生化指標相關資訊; 此外長期之追蹤性研究需耗費相當的人力物力，往往在短期內尚無定論。若追蹤時間過短，可能會因為某些終點事件尚未出現而無法反映真實的結果，然若研究期程太長，又無法儘快排除國人在Cox-II 抑制劑止痛藥 Celecoxib使用安全上的疑慮，解決此問題可說是刻不容緩，值得各方專家投入研究