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Isotopically Enriched Tracers and Inductively Coupled Plasma Mass Spectrometry Methodologies to Study Zinc Supplementation in Single-Cells of Retinal Pigment Epithelium in Vitro

Rodríguez-Menéndez, Sara, Fernández, Beatriz, González-Iglesias, Héctor, García, Montserrat, Álvarez, Lydia, García Alonso, José Ignacio, Pereiro, Rosario
Analytical chemistry 2019 v.91 no.7 pp. 4488-4495
atomic absorption spectrometry, cell aggregates, cell nucleus, cryopreservation, cultured cells, dietary mineral supplements, epithelium, gluconates, isotope labeling, mass spectrometry, mineralization, models, mole fraction, stable isotopes, sulfates, zinc
Mass spectrometry-based techniques, such as inductively coupled plasma mass spectrometry (ICPMS) and laser ablation (LA) ICPMS, combined with an isotope pattern deconvolution mathematical tool are proposed for a better understanding of supplementation studies in cultured cells. An in vitro model of human retinal pigment epithelium (HRPEsv) cells was treated with different concentrations (0–150 μm Zn, 1 mL) of enriched stable isotope tracers of Zn in the form of sulfate and/or gluconate. Supplementations with ᵗ⁶⁸ZnSO₄ or ᵗ⁷⁰Zn-gluconate alone and in combination (1:1 molar ratio) were investigated to evaluate the exogenous contribution and distribution of Zn in the treated cells. In order to obtain not only the Zn concentration for a cell population (mineralized cells) but also single cell information about the contribution of exogenous Zn and their distribution within micrometer cells structures, LA-ICPMS was employed to directly analyze cryopreserved cells. ⁿᵃᵗZn, ᵗ⁶⁸Zn, and ᵗ⁷⁰Zn molar fraction images obtained from cells and cell aggregates allowed confirming the uptake of exogenous Zn by HRPEsv cells, being ᵗ⁶⁸Zn and ᵗ⁷⁰Zn molar fractions close to 1 in the cell nuclei. Under the selected experimental conditions tested (24 h treatments), no significant differences were obtained in the Zn distribution depending on its chemical form.