A Sensitive, Nonradioactive Assay for Zn(II) Uptake into Metazoan Cells

Abstract

Sensitive measurements of cellular Zn(II) uptake currently rely on quantitating radioactive emissions from cells treated with 65Zn(II). Here, we describe a straightforward and reliable method employing a stable isotope to sensitively monitor Zn(II) uptake by metazoan cells. First, biological media selectively depleted of natural abundance Zn(II) using A12-resin [Richardson, C. E. R. et al. J. Am. Chem. Soc. 2018, 140, 2413] is restored to physiological levels of Zn(II) by addition of a non-natural Zn(II) isotope distribution comprising 70% 70Zn(II). The resulting 70Zn(II)-enriched media facilitates quantitation of Zn(II) uptake using inductively coupled plasma-mass spectrometry (ICP-MS). This sensitive and reliable assay assesses Zn(II)-uptake kinetics at early timepoints and can be used to delineate how chemical and genetic perturbations influence Zn(II) uptake. Further, the use of ICP-MS in a Zn(II)-uptake assay permits simultaneous measurement of multiple metal ion concentrations. We used this capability to show that, across three cell lines, Zn(II) deficiency enhances selectivity for Zn(II) over Cd(II) uptake.

A Guide to Human Zinc Absorption: General Overview and Recent Advances of In Vitro Intestinal Models

“…Aside of (stable) isotope techniques, zinc is generally quantified with inductively coupled mass spectrometry (ICP-MS), inductively coupled plasma optical emission spectrometry (ICP-OES), or atomic absorption spectrometry (AAS) [319]. In addition to measuring the most abundant stable zinc isotopes, 64 Zn or 66 Zn, with ICP-MS, 70 Zn was recently used to determine cellular zinc uptake kinetics while simultaneously distinguishing between cellular basal zinc levels and zinc that was actually absorbed by the cells [320]. Hence, applying this method in in vitro intestinal models would provide a fruitful approach for scrutinizing enterocyte homeostasis of this micronutrient during zinc absorption.…”

Argon Enclosed Droplet Based 3D Microfluidic Device Online Coupled with Time-Resolved ICPMS for Determination of Cadmium and Zinc in Single Cells Exposed to Cadmium Ion

“…In contrast, defined as free, loosely bound, accessible, or chelatable Zn, the “labile zinc pool” is a small fraction of total Zn ranging from pmol L –1 to fmol L –1 . The intracellular “labile zinc pool” is on behalf of the significant Zn-buffering capacity for mediating Zn 2+ -dependent signaling events in diverse biological settings, such as immune responses, endocrine signaling and so forth . Therefore, after exposure to Cd, the variation of total Zn and labile Zn in cells both attract close attention.…”

“…18 The intracellular "labile zinc pool" is on behalf of the significant Znbuffering capacity 19 for mediating Zn 2+ -dependent signaling events in diverse biological settings, such as immune responses, endocrine signaling and so forth. 20 Therefore, after exposure to Cd, the variation of total Zn and labile Zn in cells both attract close attention. Since the proposed 3D W/G MFD-TRA-ICPMS system cannot provide the information of labile Zn in single cells, flow cytometry and fluorescence spectrometry were used to explore the variation of labile Zn in cells.…”

Albumin-mediated extracellular zinc speciation drives cellular zinc uptake

“…An early radio-chemical study determined 65 Zn 2+ influx rates into human and rat erythrocytes of 0.22 fg h À1 cell À1 , 37 while an ICP-MS study of 70 Zn 2+ influx into human HEK293T cells in the presence of Zn-depleted media determined rates of 23.5 fg h À1 cell À1 . 36 Plotting the influx rates vs. total [BSA] clearly shows that cellular zinc uptake rates are dependent on albumin concentration (Fig. 3(a)).…”

“…A recent study highlighted the merits of working with stable isotopes. 36 Before commencement of the isotope assay, immortalised HUVECs were first equilibrated to physiologically relevant conditions (600 mM BSA and 20 mM natural-abundance Zn 2+ (designated as ''pre-conditioning medium'', Fig. 2) for 24 h. After this time, cells were washed with phosphate-buffered saline and incubated in fresh medium containing 20 mM 68 Zn 2+ (499% 68 Zn; ESI, † Table S1) with different extracellular BSA concentrations (0-600 mM).…”