Koscielniak A, Serafin M, Duda M, Oles T, Zadlo A, Broniec A, Berdeaux O, Gregoire S, Bretillon L, Sarna T, Pawlak A
Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
Faculty of Electrical Engineering, Automatics, Computer Science and Biomedical Engineering, AGH-University of Science and Technology, Kraków, Poland.
Cell Biochem Biophys. 2017 Dec;75(3-4):443-454. doi: 10.1007/s12013-017-0832-3. Epub 2017 Nov 2.
The mammalian retina contains a high level of polyunsaturated fatty acids, including docosahexaenoic acid (22:6) (DHA), which are highly susceptible to oxidation. It has been shown that one of the products of DHA oxidation-carboxyethylpyrrole (CEP), generated in situ, causes modifications of retinal proteins and induces inflammation response in the outer retina. These contributing factors may play a role in the development of age-related macular degeneration (AMD). It is also possible that some of the lipid oxidation products are photoreactive, and upon irradiation with blue light may generate reactive oxygen species. Therefore, in this work we analysed oxidation-induced changes in photoreactivity of lipids extracted from bovine neural retinas. Lipid composition of bovine neural retinas closely resembles that of human retinas making the bovine tissue a convenient model for studying the photoreactivity and potential phototoxicity of oxidized human retinal lipids. Lipid composition of bovine neural retinas Folch' extracts (BRex) was determined by gas chromatography (GC) and liquid chromatography coupled to an electrospray ionization source-mass spectrometer (LC-ESI-MS) analysis. Liposomes prepared from BRex, equilibrated with air, were oxidized in the dark at 37 °C for up to 400 h. The photoreactivity of BRex at different stages of oxidation was studied by EPR-oximetry and EPR-spin trapping. Photogeneration of singlet oxygen (O, Δ) by BRex was measured using time-resolved detection of the characteristic phosphorescence at 1270 nm. To establish contribution of lipid components to the analysed photoreactivity of Folch' extract of bovine retinas, a mixture of selected synthetic lipids in percent by weight (w/w %) ratio resembling that of the BRex has been also studied. Folch's extraction of bovine neural retinas was very susceptible to oxidation despite the presence of powerful endogenous antioxidants such as α-tocopherol and zeaxanthin. Non-oxidized and oxidized BRex photogenerated singlet oxygen with moderate quantum yield. Blue-light induced generation of superoxide anion by Folch' extract of bovine neural retinas strongly depended on the oxidation time. The observed photoreactivity of the studied extract gradually increased during its in vitro oxidation.
哺乳动物视网膜含有高水平的多不饱和脂肪酸，包括二十二碳六烯酸（22:6）（DHA），这些脂肪酸极易被氧化。研究表明，DHA氧化的一种产物——羧乙基吡咯（CEP）在原位生成，会导致视网膜蛋白的修饰，并在外层视网膜诱导炎症反应。这些促成因素可能在年龄相关性黄斑变性（AMD）的发展中起作用。也有可能一些脂质氧化产物具有光反应性，在蓝光照射下可能产生活性氧。因此，在这项工作中，我们分析了从牛神经视网膜提取的脂质氧化诱导的光反应性变化。牛神经视网膜的脂质组成与人类视网膜非常相似，这使得牛组织成为研究氧化的人类视网膜脂质的光反应性和潜在光毒性的便利模型。通过气相色谱（GC）和液相色谱-电喷雾电离源-质谱仪（LC-ESI-MS）分析确定了牛神经视网膜福尔克提取物（BRex）的脂质组成。由BRex制备的脂质体在37℃黑暗中氧化长达400小时，与空气平衡。通过电子顺磁共振血氧测定法（EPR-oximetry）和电子顺磁共振自旋捕获法（EPR-spin trapping）研究了BRex在不同氧化阶段的光反应性。使用1270nm特征磷光的时间分辨检测来测量BRex产生单线态氧（O，Δ）的情况。为了确定脂质成分对牛视网膜福尔克提取物分析的光反应性的贡献，还研究了按重量百分比（w/w%）比例类似于BRex的选定合成脂质混合物。尽管存在强大的内源性抗氧化剂如α-生育酚和玉米黄质，但牛神经视网膜的福尔克提取非常容易氧化。未氧化和氧化的BRex以中等量子产率产生单线态氧。蓝光诱导牛神经视网膜福尔克提取物产生超氧阴离子强烈依赖于氧化时间。在所研究的提取物的体外氧化过程中，观察到的光反应性逐渐增加。
