Lta4 methyl ester

Preparation of Substrate: LTA4 was prepared through the hydrolysis of LTA4 methyl ester (Cayman Chemical) in a degassed solution of 50 mM NaOH (20%, v/v) in cold acetone under an inert atmosphere of nitrogen at 25 °C for 60 min. The resulting LTA4 solution was directly diluted using a freshly prepared buffer (10 mM sodium phosphate, pH 7.4, 4 mg/mL BSA, 2.5% v/v DMSO). The LTA4 solution was freshly prepared prior to use.
Epoxide Hydrolase Assay: To determine the effect of the selected compounds on the epoxide hydrolase activity of LTA₄H, 300 ng of enzyme was incubated with test compounds (final concentration of 25 µM) in 180 µL of reaction buffer (10 mM sodium phosphate, pH 7.4, 4 mg/mL BSA, 2.5% v/v DMSO) for 15 min at 37 °C. Then 20 µL of the LTA4 was added (final concentration of 150 nM, final volume of 200 µL) and incubated for another 10 min at 37 °C. Then the assay was terminated by diluting 20-fold in assay buffer. The amount of LTB4 produced was quantified in the diluted samples by a commercially available LTB4 ELISA kit (Cayman Chemical) [49 (link)].

To investigate the effect of LCMs on LTC₄S activity, we used microsomal membranes from HEK-293 cells stably expressing LTC₄S^{70 (link)}. Membranes (2.5 µg total protein) were treated with LCMs for 10 min at 4 °C in potassium phosphate buffer (0.1 M, pH 7.4) containing 5 mM glutathione. The conversion of LTA₄ to LTC₄ methyl esters was initiated by addition of LTA₄ methyl ester (Cayman Chemicals; 1 µM). After 10 min at 4 °C, the reaction was terminated by ice-cold methanol (one volume), and LTC-d5 methyl ester (5 ng; Cayman Chemicals) was added as internal standard. LTC₄ was extracted and analyzed by UPLC-MS/MS as described for lipid mediators. Vehicle (DMSO)-treated microsomal membranes produced 7.1 ± 0.5 nmol LTC₄. The LTC₄ reference inhibitor MK-886 (Cayman Chemicals, 10 µM) suppressed LTC₄ formation by 95.6 ± 0.9%.