High-performance liquid chromatography-thermospray mass spectrometry of 5,6-dihydroxyeicosatrienoate-1,5-lactone from tissue homogenates.
We have developed a method for the analysis of 5,6-dihydroxyeicosatrienoate-1,5-lactone (5,6-DiHETriE-delta-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-delta-lactone. Consequently, the chief degradation product of 5,6-EpETriE and 5,6-DiHETriE in the incubation mixture was 5,6-DiHETriE-delta-lactone. 5,6-DiHETriE-delta-lactone, corresponding to [20:4(n-6)], was shown to be characterized by a high intensity of quasimolecular ions (MH+ and MNH4+), using ion analysis obtained by reversed-phase HPLC-thermospray MS. On selected-ion monitoring (SIM) chromatograms of 5,6-DiHETriE-delta-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-delta-lactone was y = 12.2x + 0.7 (r = 0.9996). 5,6-Epoxygenase activity was represented as the sum of the amount of 5,6-DiHETriE-delta-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.
Yamane
M
M
Department of Biochemistry, Tokyo Medical College, Japan.
Abe
A
A
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D000090762:Cytochrome P-450 CYP2J2; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D006893:Hydroxyeicosatetraenoic Acids / Q000032:analysis* / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D009249:NADP / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012044:Regression Analysis
C103232:5,6-dihydroxyeicosatrienoate-1,5-lactone; D006893:Hydroxyeicosatetraenoic Acids; D016718:Arachidonic Acid; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D000090762:Cytochrome P-450 CYP2J2