利用介質輔助雷射脫附離子化質譜儀技術建立快速的方法來分析食品及生物樣品中之精胺及亞精胺

Establish a fast method to identify spermine and spermidine in biological samples and foods by matrix- assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF MS)

蘇慧欣(Huai-Hsin Su)

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(2011年)

摘要

多胺類含兩個或兩個以上的氨基結構，具脂溶性且含氮的特性，此類的有機物廣泛存在動植物中，並且在生物體中扮演重要角色。本研究中所探討的亞精胺(spermidine)和精胺(spermine)為多胺類的成員之ㄧ，其前驅物為鳥胺酸(ornithine)，來源是由食物攝取和腸道的常在菌分泌以及de novo biosynthesis所合成，已知spermidine和spermine在腸道健康以及器官發展和生長發育扮演重要角色，但目前許多文獻指出spermidine和spermine兩種多胺類物質在體液過量表現，可能跟組織增殖及一些癌症具有相關性。
現今檢測spermidine及spermine的方法有層析法(chromatography)、電泳分析(electrophoresis)、放射線免疫分析法(radioimmunoassay)以及酵素分析法(enzymatic assay)..等，本研究使用介質輔助雷射脫附離子化質譜儀(matrix- assisted laser desorption ionization time-of-flight mass spectrometer; MALDI-TOF MS)來偵測生物檢品及食品中spermidine和spermine之含量。
本實驗針對MALDI-TOF MS之方法進行條件探討，其結果顯示先將樣品處理飽和的NaOH再利用ethyl acetate當做萃取的步驟，其萃取出來的上層溶液具有濃縮待測物的效果，最後以(2-mercaptobenzothi- azole; 2-MBT)為基質，為最理想的實驗條件，利用此方法偵測spermidine和spermine的定量下限以及偵測極限分別為0.1μg/mL和0.02μg/mL，同日及異日的(relative standard deviation; RSD)與(relative error; RE)均小於19％，這也表示本實驗具有良好的精密度以及準確度，利用此方法求得標準品之線性值應用在食品中spermidine以及spermine的定量。除此之外，本實驗也廣泛應用於尿液以及血液檢品，並採用標準添加法的方式，血液和尿液檢品中、spermidine及spermine的 r值大於0.994，RSD均小於20％，証實應用此方法所得到的線性質皆為良好。
另一方面，在細胞層面中，已知過量的spermidine和spermine在胚胎滋養層細胞(SGHPL-4 cells)會造細胞凋亡，本文欲探討大鼠腎小管上皮細胞(NRK-52E cells)處理過量的spermidine和spermine，利用液相層析串聯氏質譜儀(liquid chromatography coupled with tandem mass spectrometry; LC-MS/MS)觀看其蛋白質的表現，並比較加入多胺類抑制劑(difluoromethylornithine; DFMO)後，觀看凋亡性相關的蛋白質。

關鍵字

亞精胺 ； 精胺

並列摘要

Polyamines consist of two or more primary amino structures, and are organic compounds containing aliphatic nitrogens. These organic compounds widely spread in animals and plants, and play an important role in the biological organisms. Spermidine and spermine are members of polyamine, their precursor is ornithine, derived from foods, intestinal normal flora, and de novo biosynthesis. Spermidine and spermine participate in the intestinal health and organ development. Currently, overexpression of spermidine and spermine had been reported associating to cell proliferation and tumor growth.
There are several methods to analyze polyamines such as chromatography, radioimmunoassay, electrophoresis and enzymatic assay. As we known, this study is the first to introduce MALDI-TOF MS for monitoring spermidine and spermine in biological samples and foods without derivatization reaction.
For sample preparation, saturated sodium hydroxide (NaOH) was used to alkalize the sample solution then ethyl acetate was utilized for spermine and spermidine extraction. Then the supernatants were spotted on the target plate and mixed well with the matrix, 2-mercaptobenzothiazole (2-MBT). The limit of quantitation (LOQ) and limit of detection (LOD) for spermidine and spermine analyses were 0.1μg/mL and 0.02μg/mL, the relative standard deviation (RSD) and relative error (RE) for intra- and inter-day analyses were below 20％. This proposed method was further applied to quantify spermidine and spermine in foods, human urine and blood samples.
Documented sdudies showed that excess spermidine and spermine in the embryonic trophoblast cells (SGHPL-4 cells) could lead to apoptosis. Hence, spermidine and spermine were used to treat NRK-52E cells (from rat, kidney) and difluoromethylornithine (DFMO) was used as the inhibitor. The differences of apoptosis-related proteins were detected and compared by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).

並列關鍵字

spermidine ； spermine

參考文獻

[1] Scalabrino G, Lorenzini EC, Ferioli ME: Polyamines and mammalian hormones. Part I: Biosynthesis, interconversion and hormone effects. Mol Cell Endocrinol 1991, 77:1-35.

[2] Teti D, Visalli M, McNair H: Analysis of polyamines as markers of (patho)physiological conditions. J Chromatogr B Analyt Technol Biomed Life Sci 2002, 781:107-149.

[3] Gerner EW, Meyskens FL, Jr: Polyamines and cancer: old molecules, new understanding. Nat Rev Cancer 2004, 4:781-792.

[4] Khuhawar MY, Qureshi GA: Polyamines as cancer markers: applicable separation methods. J Chromatogr B Biomed Sci Appl 2001, 764:385-407.

[5] Russell D, Snyder SH: Amine synthesis in rapidly growing tissues: ornithine decarboxylase activity in regenerating rat liver, chick embryo, and various tumors. Proc Natl Acad Sci U S A 1968, 60:1420-1427.