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中华普通外科学文献(电子版)

中华普通外科学文献(电子版) ›› 2019, Vol. 13 ›› Issue (05) : 345 -349. doi: 10.3877/cma.j.issn.1674-0793.2019.05.003

所属专题： 文献；

论著

气相色谱质谱联用仪定量检测内源性大麻素花生四烯乙醇胺及2-花生四烯酸甘油在胆道恶性肿瘤中的表达规律
袁锡裕1, 谭炳炎2, 黄力3,†()   
  1. 1. 523059　东莞市人民医院普外科
    2. 510080　广州，中山大学公共卫生学院营养学系
    3. 510080　广州，中山大学附属第一医院胆胰外科
  • 收稿日期:2019-05-22 出版日期:2019-10-01
  • 通信作者: 黄力
  • 基金资助:
    国家自然科学基金资助项目(81201919); 广东省自然科学基金资助项目(2017A030313495); 东莞市社会科技发展项目(2016105101269)

Quantification of endocannabinoid arachidonoyl ethanolamide and 2-arachidonoylglycerol by gas chromatography/mass spectrometry in biliary tract cancer

Xiyu Yuan1, Bingyan Tan2, Li Huang3,†()   

  1. 1. Department of General Surgery, Dongguan People’s Hospital, Dongguan 523059, China
    2. Department ofNutrition, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China
    3. Department of Pancreatobiliary Surgery, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China
  • Received:2019-05-22 Published:2019-10-01
  • Corresponding author: Li Huang
  • About author:
    Corresponding author: Huang Li, Email:
    Yuan Xiyu and Tan Bingyan are the first authors who contributed equally to the article
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袁锡裕, 谭炳炎, 黄力. 气相色谱质谱联用仪定量检测内源性大麻素花生四烯乙醇胺及2-花生四烯酸甘油在胆道恶性肿瘤中的表达规律[J/OL]. 中华普通外科学文献(电子版), 2019, 13(05): 345-349.

Xiyu Yuan, Bingyan Tan, Li Huang. Quantification of endocannabinoid arachidonoyl ethanolamide and 2-arachidonoylglycerol by gas chromatography/mass spectrometry in biliary tract cancer[J/OL]. Chinese Archives of General Surgery(Electronic Edition), 2019, 13(05): 345-349.

目的

探讨两种主要的内源性大麻素花生四烯乙醇胺（AEA）及2-花生四烯酸甘油（2-AG）在胆道恶性肿瘤（BTC）患者体液及肿瘤组织中的表达规律。

方法

采集中山大学附属第一医院2013年12月至2014年9月收治的22例BTC患者及8例良性胆道疾病患者的外周血及胆汁，其中15例BTC患者同时收集癌巢组织及癌旁正常组织，利用氯仿/甲醇抽提法配合固相萃取，分离患者体液及组织样本中的脂质，以二甲基异丙基硅烷-咪唑对脂质进行硅烷化处理，气相色谱质谱联用仪测定AEA及2-AG的浓度。

结果

与良性胆道疾病患者相比，BTC患者血浆中2-AG显著升高[180.01（140.72~283.84）nmol/L vs 42.33（25.61~148.93）nmol/L，P<0.05]，AEA浓度降低但差异无统计学意义；胆汁中AEA浓度显著降低[1.80（0.50~5.00）nmol/L vs 10.15（2.68~17.49）nmol/L，P<0.05]，2-AG浓度升高但差异无统计学意义。BTC癌巢组织与癌旁正常组织相比，AEA浓度显著降低[22.01（16.55~53.61）pmol/g vs 58.68（25.36~68.97）pmol/g，P<0.05]，2-AG显著升高[1.97（1.44~5.43）nmol/g vs 1.10（0.36~1.47）nmol/g，P<0.05]。

结论

本研究建立了在BTC中定量检测AEA和2-AG两种内源性大麻素浓度的有效方法，测定AEA及2-AG在BTC患者体液及肿瘤组织中的异常表达，可作为BTC的辅助诊断。

关键词: 胆管肿瘤, 内源性大麻素, 气相色谱-质谱法, 花生四烯乙醇胺, 2-花生四烯酸甘油
Objective

To investigate the expression of endocannabinoid (EC), arachidonoyl ethanolamide (AEA) and 2-arachidonoylglycerol (2-AG) in biliary tract cancer (BTC) patients’ samples.

Methods

Lipids of patients’ plasma and bile (22 BTC patients and 8 benign biliary disease patients) and of paired BTC nest and adjacent normal tissue (15 of the above 22 BTC patients) were extracted by the Bligh and Dyer method (chloroform containing internal standards and methanol were added to above samples) and solid-phase extraction. Derivatisation of the hydroxyl groups of ECs was performed with the silylating agent dimethylisopropylsilyl imidazole. Lipid samples were quantitated by gas chromatography/electron ionization (EI)-mass spectrometry.

Results

Level of 2-AG was upregulated in BTC patients’ plasma [180.01 (140.72-283.84) nmol/L vs 42.33 (25.61-148.93) nmol/L, P<0.05], while level of AEA appeared decreasing trend without significant difference. Level of AEA was lower in BTC patients’ bile [1.80 (0.50-5.00) nmol/Lvs 10.15 (2.68-17.49) nmol/L, P<0.05], while level of 2-AG appeared increasing trend without significant difference. Level of AEA was lower [22.01 (16.55-53.61) pmol/g vs 58.68 (25.36-68.97) pmol/g, P<0.05], while level of 2-AG was upregulated [1.97 (1.44-5.43) nmol/g vs 1.10 (0.36-1.47) nmol/g, P<0.05] in BTC cancer nest than in adjacent normal tissues.

Conclusions

The present study develops a sufficient and reliable method for quantitative determination of AEA and 2-AG in BTC. The dysregulated AEA and 2-AG in body fluids and tumor tissues of patients with BTC can be used as assistant diagnosis of BTC.

Key words: Bile duct neoplasms, Endocannabinoid, Gas chromatography-mass spectrometry, Arachidonoyl ethanolamide, 2-Arachidonoylglycerol
表1 胆道恶性肿瘤患者及良性胆管疾病患者临床病理资料比较
组别 例数 性别(男/女,例) 年龄(<65/≥65岁,例) 总胆红素(μmol/L) 丙氨酸转氨酶(U/L) γ-谷氨酰转移酶(U/L) 碱性磷酸酶(U/L)
良性组 8 6/2 4/4 67.6(43.1~105.5) 82(42~138) 89(60~242) 175(106~439)
BTC组 22 8/14 12/10 82.2(63.6~161.5) 90(51~265) 103(71~285) 144(92~381)
统计值 ? χ2=0.330 χ2=0.047 Z=-1.641 Z=-0.085 Z=-0.623 Z=-0.776
P值 ? 0.566 0.828 0.092 0.930 0.524 0.426
表2 气质联用仪定量检测患者血浆及胆汁中AEA及2-AG的参数
EC 特征离子峰质荷比(m/z) 保留时间(min)
AEA 404 16.95
AEA-d4 408
2-AG 535 18.35
2-AG-d5 540
1-AG 535 18.70
1-AG-d5 540
图1 气质联用仪检测花生四烯乙醇胺（AEA）和2-花生四烯酸甘油（2-AG）及相应的氘化内参标准品的特征离子峰　A为特征离子峰总图；B为AEA特征离子峰；C为AEA-d4特征离子峰；D为2-AG特征离子峰；E为2-AG-d5特征离子峰
图3 胆道恶性肿瘤（BTC）患者癌巢组织与癌旁正常组织中花生四烯乙醇胺（AEA）与2-花生四烯酸甘油（2-AG）浓度的比较* P<0.05；A为BTC组织AEA浓度；B为BTC组织2-AG浓度
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