High-performance liquid chromatography-thermospray mass spectrometry of 5,6-dihydroxyeicosatrienoate-1,5-lactone from tissue homogenates

Abstract

We have developed a method for the analysis of 5,6-dihydroxyeicosatrienoate-1,5-lactone (5,6-DiHETriE-δ-lactone) in tissue homogenates, supplemented with NADPH and arachidonic acid [20:4( n−6)] as a substrate. During the incubation and the extraction, most of the 5,6-epoxyeicosatrienoic acid (5,6-EpETriE) was converted to 5,6-dihydroxyeicosatrienoic acid (5,6-DiHETriE), and most of the 5,6-DiHETriE was converted to 5,6-DiHETriE-δ-lactone. Consequently, the chief degradation product of 5,6-EpETriE and 5,6-DiHETriE in the incubation mixture was 5,6-DiHETriE-δ-lactone. 5,6-DiHETriE-δ-lactone, corresponding to [20:4( n−6)], was shown to be characterized by a high intensity of quasimolecular ions (MH + and MNH 4 +), using ion analysis obtained by reversed-phase HPLC-thermospray MS. On selected-ion monitoring (SIM) chromatograms of 5,6-DiHETriE-δ-lactone and with deuterium-labeled 15( s)-hydroxyeicosatetraenoic acid as the internal standard, the regression equation of the peak-area ratio and the amount of 5,6-DiHETriE-δ-lactone was y = 12.2 x + 0.7 ( r = 0.9996). 5,6-Epoxygenase activity was represented as the sum of the amount of 5,6-DiHETriE-δ-lactone, 5,6-EpETriE and 5,6-DiHETriE per mg protein, after 30 min in an incubation mixture. The activity from rat brain homogenate decreased considerably with growth of the rat.