11 12 eet d11

Five hundred micrograms of heart lysate (lysed as above but in ice-cold PBS) were mixed with ice-cold solutions containing 50 ng of 14,15-EET, 11,12-EET, 12,13-EpOME, 19,20-EpDPE, or 17,18-EpETE (Cayman Chemical) in a total of 100 μl PBS containing 0.1% BSA. Samples were incubated at 37 °C for 10 min before reactions were stopped by the addition of 0.9 ml ethyl acetate. Ten microliters of internal standard [30 ng each of PGE₂-d9, 11,12-EET-d11, and 11,12-DHET-d11 (Cayman)] was added to each sample. Samples were mixed, centrifuged at 1000g, and ethyl acetate was removed to tubes containing 6 μl of 30% glycerol in methanol. Samples were dried under vacuum centrifugation, covered with argon gas, and stored at −80 °C. Rates of hydrolysis were determined by the formation of corresponding diols (14,15-DHET, 11,12-DHET, 12,13-DiHOME, 19,20-DiHDPE, or 17,18-DiHETE).