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

摘要

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 mu m Zn, 1 mL) of enriched stable isotope tracers of Zn in the form of sulfate and/or gluconate. Supplementations with (ZnSO4)-Zn-t68 or Zn-t70-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. Zn-nat, Zn-t68, and Zn-t70 molar fraction images obtained from cells and cell aggregates allowed confirming the uptake of exogenous Zn by HRPEsv cells, being Zn-t68 and Zn-t70 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.