Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julian Clavería, 8, 33006 Oviedo, Spain; Instituto Universitario Fernández-Vega (Fundación de Investigación Oftalmológica, Universidad de Oviedo), Spain.
Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julian Clavería, 8, 33006 Oviedo, Spain; Instituto Universitario Fernández-Vega (Fundación de Investigación Oftalmológica, Universidad de Oviedo), Spain.
Talanta. 2018 Feb 1;178:222-230. doi: 10.1016/j.talanta.2017.09.024. Epub 2017 Sep 13.
The retina contains the highest concentration of zinc in the human eye and it is primarily associated with the retinal pigment epithelium (RPE). Metallothioneins (MTs) are the main cytosolic zinc-ion-binding proteins, exerting a tight control in the number of atoms of Zn-bound to the MTs related with their antioxidant and neuroprotective functions. In order to study the Zn-MT system in retina and RPE, we have implemented mass spectrometry (MS)-based technologies: two complementary element detection methodologies (HPLC- and laser ablation (LA)-ICP-MS) have been successfully employed to study metal content in the human eye as well as to perform speciation studies of Zn-MTs. First, Zn-elemental distribution was studied on cryogenic ocular sections by LA-ICP-MS. Quantitative images of Zn along RPE cell layer and the retina were obtained with a laser beam diameter of 25µm, showing a preferential distribution in the RPE. We carried out then the quantitative speciation of Zn, Fe, and Cu in the water-soluble protein fractions of RPE and retina to study their protein binding profile using HPLC-ICP-MS, where Zn is mainly associated to low molecular mass proteins (i.e., MTs). Finally, the effect of addition of different inductors, such as metal (i.e., ZnSO), dexamethasone (DEX) and erythropoietin, was investigated in an in vitro cellular model of human RPE cells (HRPEsv), again using HPLC-ICP-MS in combination with stable isotopes and mathematical calculations based on isotope dilution and isotope pattern deconvolution. Exogenous Zn and DEX were found to increase MT proteins synthesis and exerted a stoichiometric transition in MT proteins in HRPEsv cells.
视网膜含有人体内眼睛中最高浓度的锌，并且主要与视网膜色素上皮 (RPE) 相关。金属硫蛋白 (MTs) 是细胞溶质中主要的锌离子结合蛋白，通过控制与抗氧化和神经保护功能相关的 MT 结合的 Zn 原子数量来发挥紧密的调节作用。为了研究视网膜和 RPE 中的 Zn-MT 系统，我们采用了基于质谱 (MS) 的技术：两种互补的元素检测方法（HPLC 和激光烧蚀 (LA)-ICP-MS）已成功用于研究人眼的金属含量以及进行 Zn-MTs 的形态研究。首先，通过 LA-ICP-MS 研究了冷冻眼部切片中的 Zn 元素分布。使用 25µm 的激光束直径获得了 RPE 细胞层和视网膜中沿 Zn 元素的定量图像，显示出在 RPE 中的优先分布。然后，我们进行了 RPE 和视网膜中水溶性蛋白质部分的 Zn、Fe 和 Cu 的定量形态研究，使用 HPLC-ICP-MS 研究它们的蛋白质结合特性，其中 Zn 主要与低分子量蛋白质（即 MTs）结合。最后，在人 RPE 细胞 (HRPEsv) 的体外细胞模型中，通过添加不同的诱导剂（例如金属（即 ZnSO）、地塞米松 (DEX) 和促红细胞生成素），再次使用 HPLC-ICP-MS 结合稳定同位素和基于同位素稀释和同位素模式反卷积的数学计算，研究了其对蛋白质结合特性的影响。发现外源性 Zn 和 DEX 增加了 MT 蛋白的合成，并使 HRPEsv 细胞中的 MT 蛋白发生化学计量过渡。
