Blackburn M L, Ketterer B, Meyer D J, Juett A M, Bull A W
Department of Chemistry, Oakland University, Rochester, Michigan 48309-4401, USA.
Chem Res Toxicol. 1997 Dec;10(12):1364-71. doi: 10.1021/tx970087a.
The enzymatic oxygenation of linoleic acid leads to the production of 13-hydroxyoctadecadienoic acid (13-HODE). Subsequent dehydrogenation of 13-HODE by the NAD(+)-dependent 13-HODE dehydrogenase results in the formation of the 2,4-dienone 13-oxooctadecadienoic acid (13-OXO). These oxidized derivatives of linoleic acid have been shown to be involved in several cellular regulatory processes. In the present study, we have examined the enzymatic and nonenzymatic reaction of 13-OXO with glutathione (GSH) and N-acetylcysteine (N-AcCySH). Nonenzymatic reaction rates were determined spectrophotometrically and exhibited a pH optimum of 9.0 which is consistent with attack of a thiolate anion. Product formation was evaluated by reverse-phase HPLC which showed formation of one major product upon reaction with either GSH or N-AcCySH. The HPLC-purified products were examined by FAB MS as well as one- and two-dimensional NMR. The products, with either GSH or N-AcCySH, were found to consist of an equal mixture of two diastereomers arising from addition of a thiolate to the 9 position of 13-OXO. Using GSH as the thiol, the reaction was also shown to be catalyzed by rat glutathione transferase 8-8. In the case of the enzymatic reaction there is stereoselective product formation. Furthermore, submicromolar concentrations of the 13-OXO-GSH conjugate were shown to significantly inhibit glutathione transferase activity in HT-29 homogenates. These investigations provide insight into the potential metabolic disposition of linoleate oxygenation products.
亚油酸的酶促氧化作用会生成13-羟基十八碳二烯酸（13-HODE）。随后，依赖烟酰胺腺嘌呤二核苷酸（NAD(+)）的13-HODE脱氢酶会使13-HODE发生脱氢反应，生成2,4-二烯酮13-氧代十八碳二烯酸（13-OXO）。这些亚油酸的氧化衍生物已被证明参与了多种细胞调节过程。在本研究中，我们检测了13-OXO与谷胱甘肽（GSH）和N-乙酰半胱氨酸（N-AcCySH）的酶促反应和非酶促反应。通过分光光度法测定非酶促反应速率，其最适pH值为9.0，这与硫醇盐阴离子的攻击相一致。通过反相高效液相色谱法（HPLC）评估产物形成情况，结果表明与GSH或N-AcCySH反应时均形成一种主要产物。通过快原子轰击质谱（FAB MS）以及一维和二维核磁共振（NMR）对经HPLC纯化的产物进行检测。发现与GSH或N-AcCySH反应生成的产物由两种非对映异构体的等量混合物组成，这两种非对映异构体是硫醇盐加成到13-OXO的9位上形成的。以GSH作为硫醇时，该反应还被证明可由大鼠谷胱甘肽转移酶8-8催化。在酶促反应中存在立体选择性产物形成。此外，亚微摩尔浓度的13-OXO-GSH缀合物可显著抑制HT-29匀浆中的谷胱甘肽转移酶活性。这些研究为亚油酸氧化产物的潜在代谢途径提供了深入了解。
