應用極致高效能液相層析搭配四極柱串聯飛行時間質譜儀探討氧化鋅微粒暴露後大鼠血漿之脂質體改變

Using UHPLC-QTOF/MS based lipidomics to study lipid effects of ZnO particle exposure on the rat plasma

陳怡儒(Yi-Ru Chen)

指導教授 ： 林靖愉

國立臺灣大學/公共衛生學院/環境衛生研究所/碩士(2018年)

https://doi.org/10.6342/NTU201803129

摘要

脂質體學為代謝體學其中一個分支，其針對細胞、組織或生物液等樣本內之脂質進行全面性的分析，藉以了解外在環境或刺激對於生物體內脂質造成的影響。過去研究中，氧化鋅微粒的暴露被證實與人、實驗動物產生金屬煙燻熱及肺部發炎、肝臟傷害等不良健康效應有關。此外，本實驗室過去的研究也發現吸入氧化鋅微粒的大鼠，其肺部沖洗液和肺部組織中代謝物及脂質產生擾動。然而，有關於氧化鋅微粒造成系統性脂質改變的研究有限，本研究應用極致高效能液相層析搭配四極柱串聯飛行時間質譜儀，對於大鼠血漿中脂質進行非標的脂質體分析，藉以了解氧化鋅微粒暴露對於大鼠血漿中脂質產生的擾動。
多種類別脂質標準品用於分析方法的測試，包含甘油脂質、甘油磷脂質、鞘脂質等能在系統中有效偵測，且經由二級質譜圖確認。系統確認後，應用該系統分析呼吸暴露於250 nm氧化鋅微粒之七週大SD大鼠血漿與吸入過濾後乾淨空氣之控制組大鼠血漿，進行進一步的脂質分析。經圖譜處理後，血漿中脂質有超過1000筆特徵訊號被保留，80%之訊號其相對標準差小於20%，顯示該系統在分析上存在良好穩定性，能夠進行後續的統計分析及深入探討。
本研究發現大鼠血漿中脂質組成主要以甘油脂質及甘油磷脂質為主。在多變量及單變量統計結果中發現250 nm氧化鋅微粒暴露之暴露組和控制組之大鼠血漿脂質存在明顯差異，磷脂醯膽鹼及三酸甘油脂為最主要顯著改變之脂質類別。本研究發現的脂質改變可能與氧化鋅微粒暴露引發的肺部、肝臟相關傷害有關。
本研究成功利用極致高效能液相層析搭配四極柱串聯飛行時間質譜儀系統對大鼠血漿中的脂質成分進行了解，並進一步應用於探討氧化鋅微粒暴露對脂質產生的效應。將來該系統可應用於了解不同器官或生物液之脂質成分。此外，本研究的發現也為開發氧化鋅顆粒暴露的脂質生物標誌物篩選提供了進一步了解。

關鍵字

氧化鋅微粒 ； 脂質體學 ； 質譜儀 ； 毒性 ； 血漿

並列摘要

Lipidomics, a subset of metabolomics, is the comprehensive measurement of the lipids in cells, tissues, biological fluids, and so on, in order to link lipid changes with gene modification or environment stress. ZnO exposure is linked to metal fume fever and pulmonary inflammation symptoms in the human and experiment animals. Besides, the perturbation of metabolites and lipids from bronchoalveolar lavage fluid and lung tissues of ZnO particles inhaled rats were observed in our recent studies. However, the knowledge of systemic lipids changes after ZnO particles exposure was limited.
The objectives of present study were utilized an untargeted lipidomic approach by ultra-high-performance liquid chromatography (UHPLC) combined with quadrupole-time of flight (Q-TOF) mass spectrometry (MS) and applied to investigate changes of lipidome from the plasma of rats inhaled ZnO particles.
Several classes of lipid standards used to test the analytical method, including lysophospholipids, monoacylglycerols, phospholipids, sphingomyelins, ceramides, diacylglycerols, triacylglycerols were successfully detected in this system. Then, plasma lipids profiling on plasma samples from 7-week male SD rats treated with 250 nm ZnO particles by inhalation for 24 hours and the filtered control were analyzed by the UHPLC-QTOF/MS system. After data processing, more than 1000 features of the lipid in the plasma were retained, 80% signal showed the relative standard deviation was less than 20%, indicating the good stability in the system, providing the data which can be further investigated and discussion.
In the plasma of male SD rat, glycerolipids and glycerolphospholipids were the most dominant lipid categories. Multivariate and univariate statistical analysis were used to examine the differences of lipidome from the ZnO particles exposure and control rats. In this study, glycerolphosphocholines and triacylglycerols represented the major significant changed lipid subclasses. These findings were possibly related to lung and liver injury induced by ZnO particles exposure.
This study successfully utilized the UHPLC-QTOF/MS system to understand the lipid composition in the rat plasma and further applied it to examine the lipid effects of ZnO particle exposure. In the future, the system can be used to explore the lipid changes in different organs or biological fluids. The findings in the study also provide a better insight for developing the potential lipid biomarker for ZnO particles exposure.

並列關鍵字

ZnO particles ； Lipidomics ； Q-TOF/MS ； Toxicity ； Plasma

參考文獻

1. Osmond MJ, Mccall MJ: Zinc oxide nanoparticles in modern sunscreens: an analysis of potential exposure and hazard. Nanotoxicology 2010, 4(1):15-41.

2. Gordon T, Fine JM: Metal fume fever. Occupational medicine (Philadelphia, Pa) 1993, 8(3):504-517.

3. Fine JM, Gordon T, Chen LC, Kinney P, Falcone G, Beckett WS: Metal fume fever: characterization of clinical and plasma IL-6 responses in controlled human exposures to zinc oxide fume at and below the threshold limit value. Journal of occupational and environmental medicine 1997, 39(8):722-726.

4. Jacobsen NR, Stoeger T, van den Brule S, Saber AT, Beyerle A, Vietti G, Mortensen A, Szarek J, Budtz HC, Kermanizadeh A et al: Acute and subacute pulmonary toxicity and mortality in mice after intratracheal instillation of ZnO nanoparticles in three laboratories. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 2015, 85:84-95.

5. Morimoto Y, Izumi H, Yoshiura Y, Tomonaga T, Oyabu T, Myojo T, Kawai K, Yatera K, Shimada M, Kubo M: Evaluation of pulmonary toxicity of zinc oxide nanoparticles following inhalation and intratracheal instillation. International journal of molecular sciences 2016, 17(8):1241.