Microsomal epoxide hydrolase from rat, mouse and human was expressed from pET20b(+)-based expression constructs in E. coli BL21AI as C-terminally His-tagged proteins and purified essentially as described earlier. Microsomes were prepared by differential centrifugation of homogenates from the livers of 6 week old male sEH -/- mice according to standard protocols.
For substrate turnover, 10–50 ng of purified mEH, 8 µg microsomal protein (epoxide hydrolase activity) or 400 ng microsomal protein (2-AG hydrolase activity) were pre-incubated in a 1.5 ml eppendorf cup with or without 5 mM TCPO or 50 µM MAFP in 10 µl reaction buffer (Tris-HCl, 20 mM, NaCl, 100 mM, gelatine, 0.1%, pH 7.4) for 5 min at room temperature. Thereafter, the reaction was initiated by the addition of substrate, either 11,12-EET (final concentration 4 µM) or 2-AG (final concentration 100 µM), in 40 µl reaction buffer and the incubation was continued for 10 min at 37°C. Thereafter, the reaction was terminated by the addition of 50 µl acetonitrile. After centrifugation at 13'000 rpm for 5 min at room temperature, 80 µl of the supernatant were transferred to a crimp top vial, sealed and subjected to LC-MS/MS analysis that was performed on a 4000 QTRAP mass spectrometer (AB Sciex) connected to an Agilent 1100 HPLC system.
HPLC separation of analytes
20 µl of sample were injected per run. Analytes were separated on a reverse phase column (Gemini 5 µM NX-C18, 110 Å, 150×2 mm), using HPLC grade water + 0.0125% NH3 (solvent A) and acetonitrile + 5% methanol + 0.0125% NH3 (solvent B) as the eluents at a flow rate of 350 µl/min, with one of the following protocols.
Procedure 1 (11,12-EET and 11,12-DHET quantification after turnover experiments with pure enzymes). 1) isocratic elution at 84% solvent A for 0.1 min; 2) linear gradient to 74% solvent A within 1.9 min; 3) linear gradient to 64% solvent A within 7 min; 4) linear gradient to 5% solvent A within 0.7 min; 5) isocratic flow at 5% solvent A for 1.6 min; 6) linear gradient to 84% solvent A within 0.1 min; 7) isocratic flow at 84% solvent A for 3.6 min. Under these conditions, the product 11,12-DHET eluted at around 7 min and the substrate 11,12-EET eluted at around 10 min.
Procedure 2 (11,12-EET, 11,12-DHET and arachidonic acid quantification after turnover experiments with microsomes) 1) isocratic elution at 84% solvent A for 0.1 min; 2) linear gradient to 74% solvent A within 1.9 min; 3) linear gradient to 64% solvent A within 16 min; 4) linear gradient to 5% solvent A within 0.7 min; 5) isocratic flow at 5% solvent A for 1.6 min; 6) linear gradient to 84% solvent A within 0.1 min; 7) isocratic flow at 84% solvent A for 4.6 min. Under these conditions, 11,12-DHET eluted at around 7 min, 11,12-EET eluted at around 10 min and arachidonic acid eluted at 15–16 min.
Procedure 3 (arachidonic acid and 2-AG quantification after turnover experiments with purified human mEH; produces higher signal intensities with arachidonic acid using pure enzyme but cannot be used for microsomal turnover experiments, due to a pronounced matrix effect obscuring the arachidonic acid signal). 1) isocratic elution at 30% solvent A for 0.1 min; 2) linear gradient to 20% solvent A within 1.9 min; 3) linear gradient to 1% solvent A within 8 min; 4) isocratic flow at 1% solvent A for 3 min; 5) linear gradient to 30% solvent A within 2 min; 6) isocratic flow at 30% solvent A for 2 min. Under these conditions, the substrate 2-AG eluted at around 6.4 min and the hydrolytic product arachidonic acid eluted at around 1.5 min. Under these conditions, a strong matrix effect was observed after 2-AG hydrolysis with microsomes that heavily masked the arachidonic acid signal while leaving the 2-AG signal unaffected. The respective samples were therefore in addition analysed with Procedure 2.
MS-based quantification of analytes
All analytes were detected and quantified by multiple reaction monitoring (MRM) in the negative mode on a 4000 QTRAP equipped with an ESI source. For 11,12-EET, the parental ion had an M/Z of 319.2. The fragments with an M/Z of 301.1 and 167.1 were used as qualifier and quantifier, respectively. For 11,12-DHET, parental ion, qualifier and quantifier had an M/Z of 319.2, 168.9 and 166.8, respectively. For arachidonic acid, parental ion, qualifier and quantifier had an M/Z of 303.2, 58.9 and 259.2, respectively. For the identification of 2-AG, the analytical parameters for arachidonic acid were used, because the significant decay of 2-AG to arachidonic acid under the ionisation conditions employed resulted in a much stronger signal for this decay product than for the parent compound itself which is difficult to ionise using ESI. Entrance potential (EP), declustering potential (DP), collision energy (CE) and collision exit potential (CXP) for the different analyses were the following: 11,12-EET qualifier (EP -10, DP -70, CE -16, CXP -7); 11,12-EET quantifier (EP -10, DP -65, CE -18, CXP -13); 11,12-DHET qualifier (EP -10, DP -70, CE -26, CXP -11); 11,12-DHET quantifier (EP -10, DP -70, CE -26, CXP -11); arachidonic acid qualifier (EP -10, DP -80, CE -28, CXP -13); arachidonic acid quantifier (EP -10, DP -80, CE -22, CXP -11).
Quantification was achieved by relating the signal intensities to those obtained with external standards of known concentrations.
Examples of all types of analyses are provided as supplementary material to demonstrate the sensitivity of the employed analytical procedures. Figure S2 shows the purity of recombinant rat mouse and human mEH, as well as representative ion chromatograms for the 2-AG turnover to arachidonic acid. Figure S3 shows representative chromatograms of purified human EH 2-AG and 11,12-EET turnover in the presence and absence of inhibitors while figure S4 shows the same type of analyses with mouse liver microsomes.
The effect of inhibitor treatment on the respective enzyme activities in the individual preparations was statistically evaluated by running student's t-tests (unpaired, two-tailed). All measurements were done in triplicates, resulting in a degree of freedom of 4 for each comparison. Because 7 individual comparisons were made (see below), the threshold for statistical significance was set to p <0.007 after Bonferroni correction.
Figure 1B, 11,12-EET hydrolysis with purified human mEH:
solvent <-> TCPO: t(4) = 19.23, p <0.0001
solvent <-> MAFP: t(4) = 0.093, p =0.9306
Figure 1C, 2-AG hydrolysis with purified human mEH:
solvent <-> TCPO: t(4) = 1.353, p =0.2475
Figure 1D, 11,12-EET hydrolysis in mouse liver microsomes:
solvent <-> TCPO: t(4) = 24.53, p <0.0001
solvent <-> MAFP: t(4) = 1.061, p =0.3848