化学
单酰甘油脂肪酶
色谱法
气相色谱-质谱法
质谱法
内大麻素系统
生物化学
受体
作者
Arslan Arinc Kayacelebi,Celina Schauerte,Katharina Maria Kling,Jan Herbers,Bibiana Beckmann,Stefan Engeli,Jens Jordan,Alexander A. Zoerner,Dimitrios Tsikas
标识
DOI:10.1016/j.jchromb.2016.08.004
摘要
2-Arachidonoyl glycerol (2AG) is an endocannabinoid that activates cannabinoid (CB) receptors CB1 and CB2. Monoacylglycerol lipase (MAGL) inactivates 2AG through hydrolysis to arachidonic acid (AA) and glycerol, thus modulating the activity at CB receptors. In the brain, AA released from 2AG by the action of MAGL serves as a substrate for cyclooxygenases which produce pro-inflammatory prostaglandins. Here we report stable-isotope GC-MS and LC-MS/MS assays for the reliable measurement of MAGL activity. The assays utilize deuterium-labeled 2AG (d8-2AG; 10μM) as the MAGL substrate and measure deuterium-labeled AA (d8-AA; range 0-1μM) as the MAGL product. Unlabelled AA (d0-AA, 1μM) serves as the internal standard. d8-AA and d0-AA are extracted from the aqueous buffered incubation mixtures by ethyl acetate. Upon solvent evaporation the residue is reconstituted in the mobile phase prior to LC-MS/MS analysis or in anhydrous acetonitrile for GC-MS analysis. LC-MS/MS analysis is performed in the negative electrospray ionization mode by selected-reaction monitoring the mass transitions [M-H]-→[M-H - CO2]-, i.e., m/z 311→m/z 267 for d8-AA and m/z 303→m/z 259 for d0-AA. Prior to GC-MS analysis d8-AA and d0-AA were converted to their pentafluorobenzyl (PFB) esters by means of PFB-Br. GC-MS analysis is performed in the electron-capture negative-ion chemical ionization mode by selected-ion monitoring the ions [M-PFB]-, i.e., m/z 311 for d8-AA and m/z 303 for d0-AA. The GC-MS and LC-MS/MS assays were cross-validated. Linear regression analysis between the concentration (range, 0-1μM) of d8-AA measured by LC-MS/MS (y) and that by GC-MS (x) revealed a straight line (r2=0.9848) with the regression equation y=0.003+0.898x, indicating a good agreement. In dog liver, we detected MAGL activity that was inhibitable by the MAGL inhibitor JZL-184. Exogenous eicosatetraynoic acid is suitable as internal standard for the quantitative determination of d8-AA produced from d8-2AG by hepatic MAGL activity. The formation of d8-prostaglandin E2 by the consecutive catalytic action of recombinant MAGL on d8-2AG and recombinant cyclooxygenase-2 (COX) on d8-AA was demonstrated by GC-MS/MS.
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