游離脂肪酸串聯式質譜分析方法的研發與臨床應用:建立運用串聯式質譜分析方法，偵測血液中游離脂肪酸中之二十碳四烯酸/二十碳五烯酸

Developmental and clinical practice of the tandem mass analytical method for free fatty acid: Establishment of the LC-MS/MS quantification method for serum free arachidonic and eicosapentaenoic acid

王敦仁(Tuan-Jen Wang)

指導教授 ： 楊沂淵

臺北醫學大學/醫學科技學院/醫學檢驗暨生物技術學系/碩士(2013年)

摘要

腦血管和心臟疾病分佔台灣地區十大死因之第二和第四位，凸顯這些慢性疾病對國人健康的嚴重衝擊和影響。若能早期確立罹病危險度的診斷，同時給予多面向的防治措施，可望有效控制罹病的人口數並且降低疾病的危害程度。心血管疾病的診斷，除了初期症狀表現之外，現階段第一線之臨床生化指標，例如三酸甘油脂、膽固醇、C-反應蛋白等，其靈敏度與特異性未能適時有效反應出罹病的危險性和疾病的嚴重度。
　　近年來，從多篇研究顯示，運用血液中二十碳四烯酸/二十碳五烯酸(AA/EPA)之??-6和??-3游離脂肪酸的定量檢測，以評估血液內AA/EPA比值，以作為潛在性發炎狀況（silent inflammation）之指標，使之成為臨床上更富診斷價值的早期參考憑據，藉此期望可以彌補現行第一線臨床生化指標僅能評估急性期之發炎反應，而無法評估可能發展成慢性疾病之風險。
　　本研究擬研發並且建立串聯式質譜游離脂肪酸的分析方法，藉以取代傳統氣相層析質譜分析方法既繁複又費時的操作步驟，提供自動化、快速、準確、和大作業量的檢查機制。本計劃選擇五十名正常成人，四十名C-反應蛋白異常（正常參考值：＜0.8mg/dl）病人，採集禁食12小時以上之血液樣本進行偵測。血液樣本將以血清檢體為主。先去蛋白後再以有機溶劑(正己烷 hexane)萃取檢體中之脂肪酸成份，經氮氣吹乾後，再加入50% ACN/0.2% TCA溶液回溶，勿需任何衍生反應，即可上機進行偵測。已知濃度之同位素標幟之內標準品將加入在檢體中作為定量評比之依據。本研究檢測人體血液檢體，並且評估二十碳四烯酸/二十碳五烯酸(AA/EPA)（??-6和??-3脂肪酸）的比值，以作為評比的憑據和参考。
　　針對此次方法所得知結果如下: 正常人族群(N=50)發現其AA/EPA 平均比值為2.21，而CRP(N=40)測定異常之族群其AA/EPA 平均比值為16.6；而針對此次分析的兩個不飽和脂肪酸之血液中脂濃度，其在對照組（健康人）分別為Arachidonic acid: 1.56-10.81 ug/ml、EPA: 0.28-5.26 ug/ml；而實驗組（High-CRP病人）分別為、Arachidonic acid: 0-10.01 ug/ml、EPA: 0-1.20 ug/ml、而此兩項脂肪酸之檢量線分別為、Arachidonic acid:y=0.15x、EPA:y=0.625 x其r值介於0.9950至0.9986之間，其回收率經6組不同樣品測試發現介於65%至90%；而within run and between run則是使用品管檢體(有Low和Medium和High三種level)，經連續三天測試，發現皆小於13.6%(CV)，符合美國FDA所規範的需小於15%(CV)。
　　AA/EPA比值在是否有發炎反應上有明顯的差異，但兩者之間的數值並無明顯相關，或許是因兩個項目都代表發炎，但AA/EPA比值代表是慢性發炎，而CRP數值代表急性發炎，故只能依數據推論出CRP數值異常，AA/EPA比值也會異常，符合當初實驗目的假設，此外，由於本實驗是利用串聯式質譜分析，可大幅減少檢體前處理以及上機時間，算是蠻符合成本的儀器與檢驗方式，至於未來可研究方向，可藉由研究設計定時定量攝食EPA，因 AA與EPA都會被環氧化酶（cyclooxygenase；COX）與脂氧酶（lipoxygenase；LOX）代謝，故EPA可藉由競爭酵素，進而抑制AA代謝成發炎性類花生酸（Eicosanoid）的反應之特性；選擇一特定疾病族群之病人，定時給予病人定量之EPA，再定期偵測病人血液中脂肪酸之濃度，藉以評估是否能有效降低AA/EPA比值，並進一步，隨著AA/EPA比值之降低，觀察該病人與疾病相關之臨床症狀、理學檢查之評估結果及發病頻率是否也隨之改善，用於評估攝食EPA對改善特定慢性疾病之成效。

關鍵字

串聯式質譜, 游離脂肪酸, 二十碳四烯酸, 二十碳五烯酸

並列摘要

Cerebrovascular and cardiovascular disease are the second and the forth most common causes of death in Taiwan, and both result in serious health injure and high mortality. The principle etiology of above diseases is arteriosclerosis which is caused by prolonged and slowly progressive inflammation on the vascular epithelium cells. Arachidonic acid (AA), Eicosapentaenoic acid (EPA) and their ratio in plasma are thought to be an accurate indication of the level of inflammation occurring within the body. In this study, a unique tandem mass spectrometry method that measures the ratio of Arachidonic acid (AA) to Eicosapentaenoic acid (EPA) in serum is proposed.
　　Blood samples were collected from 50 normal adults after 12-hour fasting, and from 40 patients with increased C-Reactive Protein (CRP; normal reference range: <0.8mg/dl) of suffering atherosclerotic event. Serum samples were pretreated by organic solvent and hexane extraction, and the extract was ready for LC/MS/MS analysis. Derivative was not necessary in this method. An AB 4000 Q TRAP LC-MS/MS system with multiple reaction monitoring (MRM) mode was applied.
　　The within-run and between-run precisions (CV %) and the linearity of AA and EPA based on the IS were good (both less than 13.6%). The recovery of AA and EPA by using LC-MS/MS method (n=6) was 78.9% and 66.8% in average, respectively. The mean AA and EPA was 6.19 (?b2.31) and 2.77 (?b1.25) μg/mL in normal control (n=50) and 4.31 (?b2.80), and 0.25 (?b0.22) μg/mL in CRP increased patients (n=40), respectively. The average AA/EPA ratio was 2.21 in normal control and 16.6 in patients with increased CRP.
　　The AA/EPA ratio is significantly elevated 7.5-fold in patients with high CRP value than that in normal control (p value < 0.001). Our results show that the AA and EPA quantitative analyses may provide valuable information for the monitoring chronic inflammation, such as arteriosclerosis. The LS-MS/MS is a specific, sensitive, validated, high throughput method and applicable for simultaneous quantification of AA and EPA in blood.

並列關鍵字

LC-MS/MS quantification method, free fatty acid, serum free arachidonic, serum free eicosapentaenoic acid

參考文獻

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