以質譜技術為基礎的脂質體學研究小鼠經萘暴露後不同器官之效應

Abstract

萘，為最常見的多環芳香烴類化合物，是一種普遍存在樟腦丸和石油產物中的環境污染物。過去，以核磁共振 (NMR) 為基礎的代謝體學研究結果顯示，小鼠經萘暴露後，一些親水性和疏水性代謝產物會受到其幹擾並產生影響。然而，核磁共振的低解析度限制了脂質等疏水性代謝物的結構辨識能力。因此，本研究開發以液相層析串聯質譜法 (LC-MS/MS) 為基礎的脂質體學方法平臺，並應用於分析萘暴露對不同器官脂質體造成的改變。為了探討在萘處理後不同器官中的脂質物種變化，以及對萘毒性可能之角色，我們開發了一套二維 (2D)液相層析系統，以提高脂質物種的分析。本研究所研發結合親水性交互作用（hydrophilic interaction liquid chromatography，HILIC）暨逆相之二維液相層析系統 (2D HILIC-RPLC) 可針對從生物檢體中所萃取出的神經醯胺(ceramide) 和磷脂醯膽鹼脂質 (phosphorylcholine-containing lipid) 做全面性的研究分析。本系統於第一階段利用Silica HILIC 層析管柱將脂質依種類進行分離，之後利用ACQUITY BEH C18 層析管柱針對同類脂質做進一步的分離。與傳統一維液相層析串聯質譜系統相比，本次開發之二維液相層析串聯質譜系統顯著降低基質效應，並且其偵測極限相對降低了二至三倍。我們證明本研究所建立的方法具有高精確度與再現性，統計結果顯示三個添加濃度之相對標準偏差小於百分之二十。新開發的系統可有效地應用在經過萘處理後實驗小鼠中的肺臟，肝臟與腎臟脂質體的分析。從化學計量學分析顯示，在小鼠中的肺臟和肝臟中磷脂醯膽鹼和神經醯胺量均有顯著改變，而腎臟只有磷脂醯膽鹼有變化。本研究結合以液相層析串聯質譜為基礎的脂質體學與以核磁共振為基礎的代謝體學方法平台分析萘在不同器官引發的變化。總結，小鼠暴露萘48 小時後,代謝體結果呈現，受傷的器官進行修復，在這個時間點脂質的變化與細胞膜修復機制有關。

Naphthalene, a polycyclic aromatic hydrocarbon, a ubiquitous environmental pollutant present in mothballs and petroleum products, has the capability of causing illness in human. Previously, nuclear magnetic resonance (NMR)-based metabolomics revealed that several hydrophilic and hydrophobic metabolites were perturbed in mice after naphthalene treatments. However, low NMR resolution limited the identification of the hydrophobic metabolites including lipid. Therefore, in this study, liquid chromatography-tandem mass spectrometry- (LC-MS/MS) based focus lipidomics was applied to profile lipid species changes in various organs after naphthalene treatment. In order to profile the lipid species changes in various organs after naphthalene treatment, two-dimensional (2D) LC system has been developed to enhance the profiling of lipid species. Concisely, a 2D hydrophilic interaction liquid chromatography (HILIC) and reverse-phase (RP) liquid chromatography (LC) system coupled with triple-quadrupole mass spectrometry (MS) was developed to comprehensively profile ceramides and phosphorylcholine containing lipids in extracted biological samples. Developed 2D HILIC-RPLC system used a silica HILIC column operated in the first dimension to distinguish the lipid classes and a BEH C18 column operated in the second dimension to separate the lipid species of the same class. The regression linearity of each lipid was satisfactory in both systems; however,the absolute matrix effect factor was reduced in 2D LC-MS/MS system. Limits of detection of 2D LC-MS/MS system were 2-3-fold lower compared toone-dimensional (1D) RPLC-MS/MS. To summarize, developed method was proven to be accurate and producible, as relative standard deviations remained smaller than 20% at three spiked levels.The efficiency of this newly developed system was applied to profile lipidome in the lung, liver and kidney of mice after naphthalene treatment to understand the mechanisms of naphthalene induced toxicities. Chemometric analysis revealed that phosphorylcholine-containing lipids and ceramides were significantly altered in the lung and liver, whereas, only phosphatidylcholines (PCs) was observed in kidney. Elevated number of unsaturated bonds and fatty acyl chains in both ceramide and PCs were deemed to reduce cellular membrane rigidity and facilitating the trafficking of recovery elements into the cell for rejuvenation. Coupling the MS-based focus lipidomics and NMR-based metabolomics enabled the characterization of naphthalene-induced changes in various organs. As conclusion, 48 h after naphthalene treatment, the injured organs underwent rejuvenation and the lipids changes at this time points majorly responsible for the cellular rejuvenations.