Targeted metallomic study of a retinal pigment epithelium cellular model under inflammatory conditions using mass spectrometry Ana Álvarez Barrios 1,2 , Lydia Alvarez 2 , Rosario Pereiro 1 , Hector Gonzalez-Iglesias 3 1 University of Oviedo, Department of Physical and Analytical Chemistry, Oviedo (Spain), 2 Fundación de Investigación Oftalmológica, Oviedo (Spain), 3 Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Villaviciosa (Spain) To preserve homeostasis, cells respond to foreign or damaged material with inflammation. Prolonged low levels of inflammation occur during ageing and may increase the risk of disease [1]. In fact, inflammation has been linked with age-related macular degeneration (AMD), one of the leading causes of irreversible blindness in the elderly. AMD affects the retinal pigment epithelium (RPE) and surrounding vasculature of the macula, causing the degeneration of photoreceptors and the loss of central vision [2]. Inflammation, oxidative stress, and metal dyshomeostasis have been described as potential damaging processes in AMD. Biologically active metals such as Zn, Cu and Fe are structural components and cofactors of enzymes involved in multiple cellular pathways that, when altered, can have detrimental effects on essential biological functions. Zn and Cu homeostasis is partly carried out by metallothioneins (MTs), which have shown changes with age [3]. Alterations of metal homeostasis during ageing could increase the risk of AMD onset and progression, being related to inflammatory damage [4]. Here, we intend to delve into the link between inflammation and metal dyshomeostasis in a cellular model of RPE exposed to a proinflammatory environment induced by interleukin-1α. In this work, targeted metallome analysis is conducted using elemental mass spectrometry and two different methodological approaches. First, a quantification of the total content of biologically relevant metals (e.g., Zn, Cu, and Fe) in the cytosol and membranes of the cell cultures is conducted by inductively coupled plasma mass spectrometry (ICP-MS). Secondly, we carry out a species-specific quantification of Zn, Cu and S using a combination of size exclusion- based high-performance liquid chromatography (SEC-HPLC), isotope dilution and ICP-MS detection to determine Zn and Cu bind to MTs and their stoichiometry (i.e., S). This analytical approach allowed to explore the relationship between inflammatory damage and metal homeostasis in the RPE. References 1. Franceschi C. et al. (2018) Rev. Endocorinol. 14(10), 576-590 2. Kauppinen A. et al. (2016) Mol. Life Sci. 73(9), 1765-1786 3. Ugarte M. et al. (2013) Eye Res. 58(6), 585-609Gilbert R. et al. (2019) Nutr. Food Res. 63(15), 1-16
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