利用質譜儀建立快速定量丙戊酸與活性代謝物及蛋白質表現分析方法

Rapid quantitation of valproic acid and its reactive metabolite combined protein profiling assay by mass spectrometry

吳俊毅(Chun-Yi Wu)

指導教授 ： 呂濟宇

高雄醫學大學/醫學院/生物化學研究所/碩士(2013年)

摘要

丙戊酸(VPA)為臨床上常用的小分子脂肪酸藥物，廣泛用於治療各類癲癇症和做為情緒穩定用藥，神經醫學亦使用此藥物預防神經退化性疾病和穩定腦內神經傳導物質GABA濃度。臨床藥物使用上，VPA藥物劑量的使用不當造成嚴重副作用症產生，包含胎兒神經管缺陷、小兒胰臟炎與高氨血症等。代謝上，VPA透過肝臟進行代謝轉換，產生已知近50種VPA代謝物，其中代謝物4-ene VPA具有肝毒性，可進行二次代謝成(E)-2,4-diene VPA，引發肝臟損傷(DILI），嚴重時造成肝臟衰竭。因VPA為第一線抗癲癇藥物，並不斷被發現具有新醫療功能，且具成為抗癌藥物的潛力，故因應未來趨勢，須發展一適用臨床快速檢測監控VPA分析方法。
本篇研究方向有二，(一)為利用基質輔助脫附離子化飛行時間式質譜儀(MALDI-TOF MS)建立有效快速監測血液VPA方法，並同時可檢驗血中肝毒性代謝物含量。(二)利用人類肝臟細胞株Huh-7探討VPA在人類血中治療濃度時，胞內蛋白質表現的差異，以全細胞萃取蛋白質方式，利用液相層析串連式質譜儀(LC-MS/MS)分析細胞內蛋白質的表現，搭配生物資訊資料庫進行VPA生物代謝和毒性探討。
本方法已專利申請中，申請案號102129354

關鍵字

丙戊酸 ； 基質輔助雷射脫附離子化質譜儀 ； 液相層析串聯式質譜儀

並列摘要

VPA is a broad-spectrum antiepileptic drug wildly used in anticonvulsant treatment, mood disorder and neuronprotective function by modulation of gamma amino butyric acid (GABA) level. The five carbon short chain fatty acid also have shown anti-tumor proliferation in vivo/vitro because of its histone deacetylase inhibitor property inducing apoptosis in malignant cells. Pharmacokinetic of VPA dosage in plasma level is not relevant linear, primarily VPA's protein binding capacity especially in serum albumin. The plasma level of free VPA fraction may increase or decrease, strongly effect the metabolic pathway. That's the TDM of VPA to monitor plasma concentration in prevention of side effect and drug interaction. One object of this study is to create a fast clinical method monitor VPA and its reactive metabolite 4-ene VPA in human plasma with MALDI-TOF MS and screeing protein and profiling. VPA could not be determined directly by MALDI-TOF MS, futhermore our method based on chemical derivative to establish. We investigated the parameters affecting the derivation. The regression equations of VPA and 4-ene VPA derivative possessed good linearity. The relative standard deviation (RSD) and relative error (RE) values in intra- and inter- assay were below 8%. This proposed method could applied to monitor of VPA and 4-ene VPA in biological sample. It's under processing in patent number 102129354 (ROC).
Documented studies have shown strong hepatotoxicity in over VPA dose. Therefore, we studied minimum clinical VPA dose in hepatocyte and analyzed protein expression with bioinformatic databases. Present results show VPA decrease amino acid level after treatment, and lower the level of GSH for detoxicfication function. Proteins profiling have established including acetylation proteins because of HDACi property. In conclusion, it demonstrate protein analysis provides useful information about VPA and 4-ene VPA metabolism and a considered way to test pre-clinical drug.

並列關鍵字

valproic acid ； MALDI-TOF MS ； LC-MS/MS

參考文獻

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