應用基於質譜儀的脂質體學探討萘對小鼠血清中脂質的影響

Abstract

萘是一種廣泛存在於環境中的空氣汙染物，其最主要的來源是各種形式的不完全燃燒，例如二手煙、交通廢氣及工廠排放。萘產生物種及器官特定的毒性，文獻指出萘的急性暴露造成小鼠呼吸系統傷害，特別在小鼠肺部的克氏細胞出現腫脹、空泡化，甚至壞死等現象，且具有劑量效應關係。脂質是組成細胞膜的主要成分，同時也參與了包含細胞間訊息傳遞等多項生理功能，過去本實驗室的研究也觀察到萘暴露造成小鼠肺臟與肺部灌洗液中的含磷酸膽鹼脂質擾動。本研究應用高效能液相層析搭配四極柱串聯飛行時間質譜儀為基礎的脂質體學，以非標的分析方式觀察經不同劑量之萘處理後的小鼠血清，嘗試進一步篩查更多脂質類別的擾動情形來了解萘所引發的毒性反應。 為了優化本實驗室目前使用的分析平台，本研究的第一部分測試了管柱長度、溫度、層析梯度設定以及一級質譜掃描頻率等儀器設定條件，並成功地在維持大量特徵訊號的同時大幅縮短了分析時間。標準品表現顯示此系統能有效偵測甘油脂、甘油磷脂、鞘脂等多種脂質，超過90%特徵訊號的相對標準差小於20%也顯示此分析系統具有良好穩定性。 第二部分則應用優化後的分析平台對萘暴露小鼠血清中的脂質體進行篩查。使用七週大的雄性ICR小鼠，分成對照組、低劑量與高劑量暴露組，分別以腹腔注射施予以橄欖油為載體、劑量為0、100、200 mg/kg body weight的萘。小鼠於暴露後24小時犧牲，抽取血清樣品並萃取脂質後上機分析。偏最小平方判別法結果顯示三組間有明顯的分群。磷脂醯膽鹼在萘引起的小鼠血清顯著改變脂質中所占比例最高；而改變幅度最大的則是磷脂醯絲氨酸。此外，本研究也觀察到包含磷脂醯乙醇胺、神經醯胺、三酸甘油脂在內等多種脂質擾動，變動方向與幅度依據之質類別不盡相同，並可能與萘所造成的傷害或生物體對發炎反應的調控相關。 此優化後的分析平台未來能應用於不同器官或生物液樣本的脂質成分研究。本研究對萘暴露小鼠血清中的多類別脂質進行了廣範圍篩查，能夠更全面的了解萘所造成的脂質效應，幫助我們進一步了解其背後的可能毒性機制，同時應用於生物標誌物的開發。

Naphthalene, an air pollutant widely present in the environment, came from various forms of incomplete combustion, such as second-hand smoke, traffic exhaust and factory emissions. Toxicity induced by naphthalene is species- and organ-specific. Literatures pointed out that acute exposure to naphthalene caused respiratory toxicity in mice, especially swelling, vacuolation, and necrosis of Clara cells in mouse lungs with dose-response effects. Lipids are the main components of cell membranes, and also participating in various physiological functions. Previously, our group had also observed naphthalene-induced perturbation of phosphocholine-containing lipids in the mouse lungs and bronchoalveolar lavage fluid. Ultra-high-performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometer based lipidomics was applied in this study to examine serum from mice treated with different doses of naphthalene. We aim to profile the perturbation of more lipid classes to understand the molecular events of naphthalene. The first part of present study tested the instrumental conditions such as column length, temperature, gradient settings, and mass spectrum acquisition rate to optimize the analysis platform currently used in our group. We successfully shortened the analysis time while maintaining a large feature amount. Lipid standard performance showed that this system could effectively detect various lipids such as glycerolipids, glycerophospholipids, and sphingolipids. More than 90% of features showed the coefficient of variation less than 20%, indicating well-stability in the system. In the second part, the optimized analytical platform was applied to profile the lipids in the serum of naphthalene-treated mice. 7-week-old male ICR mice were divided into control group, low-dose, and high-dose exposure group. 0, 100, and 200 mg/kg body weight of naphthalene was treated with olive oil as a carrier by intraperitoneal injection. Mice were sacrificed 24 hours after exposure and serum samples were taken for lipid extraction and analysis. The score plot of partial least squares discriminant analysis showed that three groups separated clearly. Glycerophosphocholines accounted for the highest proportion of naphthalene induced significantly changed lipids in mouse serum; and the most dramatic changed lipids were glycerophosphoserines. Meanwhile, the disturbances of various lipid classes including glycerophosphoethanolamines, ceramides, and triacylglycerols were observed. These disturbances might be related to the naphthalene induced damage or the organism's regulation of the inflammatory responses. This optimized analytical platform can be applied to investigate the lipid composition and changes in different organs or biological fluids in the future. Present study profiled a wide range of lipid classes in naphthalene-treated mouse serum to provide a more comprehensive understanding of naphthalene induced lipid effects. These results may help us further explore the possible toxicity mechanisms of naphthalene, and also provide knowledge for biomarker development.