Yamane M, Abe A
Department of Biochemistry, Tokyo Medical College, Japan.
J Chromatogr B Biomed Appl. 1996 Apr 12;678(2):339-43. doi: 10.1016/0378-4347(95)00519-6.
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.
我们开发了一种在组织匀浆中分析5,6 - 二羟基二十碳三烯酸 - 1,5 - 内酯（5,6 - DiHETriE - δ - 内酯）的方法，该组织匀浆中添加了烟酰胺腺嘌呤二核苷酸磷酸（NADPH）和花生四烯酸[20:4(n - 6)]作为底物。在孵育和提取过程中，大部分5,6 - 环氧二十碳三烯酸（5,6 - EpETriE）转化为5,6 - 二羟基二十碳三烯酸（5,6 - DiHETriE），并且大部分5,6 - DiHETriE转化为5,6 - DiHETriE - δ - 内酯。因此，孵育混合物中5,6 - EpETriE和5,6 - DiHETriE的主要降解产物是5,6 - DiHETriE - δ - 内酯。利用反相高效液相色谱 - 热喷雾质谱法获得的离子分析结果表明，与[20:4(n - 6)]相对应的5,6 - DiHETriE - δ - 内酯具有高强度的准分子离子（MH⁺和MNH₄⁺）。在5,6 - DiHETriE - δ - 内酯的选择离子监测（SIM）色谱图上，以氘标记的15(S) - 羟基二十碳四烯酸作为内标，峰面积比与5,6 - DiHETriE - δ - 内酯量的回归方程为y = 12.2x + 0.7（r = 0.9996）。5,6 - 环氧合酶活性表示为孵育混合物中30分钟后每毫克蛋白质中5,6 - DiHETriE - δ - 内酯、5,6 - EpETriE和5,6 - DiHETriE量的总和。大鼠脑匀浆的活性随着大鼠的生长而显著降低。
