Talanta 178 (2018) 222–230
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Quantitative study of zinc and metallothioneins in the human retina and RPE cells by mass spectrometry-based methodologies
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Sara Rodríguez-Menéndeza,b, Beatriz Fernándeza,b, , Montserrat Garcíab,c, Lydia Álvarezb, Maria Luisa Fernándeza, Alfredo Sanz-Medela, Miguel Coca-Pradosb,d, Rosario Pereiroa,b, ⁎ Héctor González-Iglesiasb,c, a
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 c Instituto Oftalmológico Fernández-Vega, Avda. Dres, Fernández-Vega, 34 33012 Oviedo, Spain d Department of Ophthalmology and Visual Science, Yale University School of Medicine, 300 George St, 8100A, New Haven, CT 06510, USA b
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A B S T R A C T
Keywords: Laser ablation inductively coupled plasma mass spectrometry Elemental bio-imaging Quantitative speciation Zn-MTs redox cycle Aged-related macular degeneration
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 25 µm, 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., 68ZnSO4), 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.
1. Introduction One of the main hallmarks of age-related macular degeneration (AMD), a progressive neurodegenerative eye disease, is the formation of extracellular deposits, between the retinal pigment epithelium (RPE) and the Bruch´s membrane (called drusen) [1,2]. The composition of these sub-RPE deposits is very complex, containing proteins, lipids and a high accumulation of Zn, essential element that may play an important role in drusen formation [3]. During ageing, oxidative damage to retina and RPE and inflammatory-mediated processes occurs, contributing to the development and progression of AMD [4]. The inner ocular tissues contain a wide range of antioxidant enzymes, including the Zn-metallothionein redox complex, which is involved in the protection against oxidative damage and inflammatory processes within
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the eye [5]. Metallothioneins (MTs) are low molecular mass (6–7 kDa) cysteine- and metal-rich proteins, which can bound up to 7 divalent metals atoms [6]. MTs play pivotal roles in Zn and Cu homeostasis, and the Zn-MT complex captures and neutralize free radicals through cysteine sulfur ligands, releasing Zn in a redox dependent fashion [5,7]. Conventional methodologies based on molecular biology and optical microscopy are not enough sensitive and specific to study the roles of Zn and MTs in drusen formation during AMD. Therefore, the use of powerful hybrid mass spectrometry-based techniques, such as HPLC and laser ablation (LA) coupled to inductively coupled plasma mass spectrometry (ICP-MS), could improve the understanding of the role of the Zn-MT system in AMD. Bio-imaging analytical techniques with spatial resolution in the low micrometer range are today of crucial interest in life science studies to
Corresponding authors at: Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, Julian Clavería, 8, 33006 Oviedo, Spain. E-mail addresses: fernandezbeatriz@uniovi.es (B. Fernández), h.gonzalez@fio.as (H. González-Iglesias).
http://dx.doi.org/10.1016/j.talanta.2017.09.024 Received 20 June 2017; Received in revised form 6 September 2017; Accepted 10 September 2017 Available online 13 September 2017 0039-9140/ © 2017 Elsevier B.V. All rights reserved.