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生物檢體中脂蛋白脂 ? 活性測定之螢光高效能液相層析法的開發

生物檢體中脂蛋白脂 ? 活性測定之螢光高效能液相層析法的開發.

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生物檢體中脂蛋白脂 ? 活性測定之螢光高效能液相層析法的開發

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  1. 生物檢體中脂蛋白脂?活性測定之螢光高效能液相層析法的開發生物檢體中脂蛋白脂?活性測定之螢光高效能液相層析法的開發 • 脂蛋白脂?（Lipoprotein lipase, LPL）是由組織中的薄壁細胞所合成，以硫酸乙醯肝素黏附於微血管內皮細胞上，主要負責分解血漿中之三酸甘油酯。LPL為脂蛋白代謝中不可或缺的一環，其活性高低與糖尿病、心血管疾病、血脂異常…等疾病有關。本研究係發展一套螢光高效能液相層析法，使用螢光衍生化試劑4-nitro-7-piperazino-2,1,3-benzoxadiazole （NBD-PZ），將LPL與受質作用所產生之游離脂肪酸衍生化後分析，藉此定量LPL的活性。透過本分析法，檢品不須經過萃取步驟，並能完全分離干擾物質、衍生化之脂肪酸、和參與反應的受質。研究選用triolein為受質，其飽和濃度為10 mM，我們發現酵素與受質的作用須選用適當的乳化劑，透過血清白蛋白媒介，才能有效進行，實驗結果顯示1%的Gum arabic、5%的bovine serum albumin、及acetonitrile的使用為酵素反應的最佳模式，三者息息相關，未來測定LPL活性時應審慎評估。此分析方法的回收率介於108.73% - 114.36%之間，同日與異日間的精密度試驗，其相對標準偏差值分別在1.28%和2.91%之內，而偵測極限為4.53 nM。本論文所建立的HPLC螢光偵測分析法，可成功應用於正常與糖尿病老鼠血漿中LPL活性之分析，我們進而發現糖尿病老鼠之血漿LPL活性和正常老鼠相比顯著降低了52.3%。未來希望此法能應用於不同生理與病理狀態下LPL活性的測定，進一步探討LPL活性和糖尿病病程及併發症的相關機制。

  2. Determination of lipoprotein lipase activity in biological samples by high performance liquid chromatography with fluorescence detection • Lipoprotein lipase (LPL) is associated with the luminal side of capillaries and arteries where it hydrolyzes triglycerides in circulating lipoproteins to produce free fatty acids. It was valuable to determine LPL activity which was shown to vary in diseases and metabolic disorders.This research aim to develop a highly sensitive HPLC method using the fluorescent reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ), for derivatization of oleic acid (OA) liberated after triolein being hydrolyzed by LPL without sample extraction. The derivatized fatty acids could be adequately separated from interfering peaks. The substrate for LPL was loaded in with a saturated concentration of 10 mM, and bovine serum albumin (BSA) was found to be a key factor in LPL reactions. Gum Arabic (GA) was chosen for the emulsifier, but the used concentration as 1% was critical. Optimum condition for measuring the LPL activity was found when the produced OA dissolved in acetonitrile (MeCN). The accuracy values for the determination of LPL activity in 10 μL of rat post heparin plasma were 108.73 - 114.36%, and the intra- and inter-day precision values were within 1.28% and 2.91%, respectively. The limit of detection was about 4.53 nM.The proposed HPLC method was successfully applied to determine LPL activity in the post heparin plasma samples of normal and streptozotocin-induced diabetic rats. The LPL activity of diabetic rat was reduced about 52.3% compared with control. The established assay system is envisioned to be useful for determining LPL activity in different physiological and pathological conditions to clarify the relationship between LPL activity and diabetes mellitus.

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