Evaluation of Metallothionein formation as a proxy for zinc absorption in an in vitro digestion/Caco‐2 Cell culture Model

Abstract

Caco-2 cell metallothionein (MT) formation was studied to determine if MT could be used as a proxy for zinc (Zn) bioavailability in an in vitro/cell culture model. The intracellular concentration of MT was determined using a cadmium/hemoglobin affinity assay. Total cellular Zn uptake was determined on acid treatment (5% HNO3) using inductively-coupled argon-plasma emission spectroscopy (ICPES). The effect of phytic acid (PA) on cellular MT formation was also studied. Caco-2 cells were treated with media containing 0, 2, 5, 10, 25, 50, and 75 μmol/L Zn (as ZnCl2). Some Zn absorption studies employed radio-Zn (65ZnCl2). The effect of varying Zn:PA molar ratios (i.e., 1:0, 1:1, 1:5, 1:10, and 1:20) on Zn uptake was determined. A positive correlation between Zn-media concentrations and cellular 65Zn uptake and cellular MT formation was observed. The concentrations of Zn and MT in cells treated with increasing levels of non-radioactive Zn (i.e., 25, 50 or 75 μmol/L Zn) increased as Zn concentrations in the media increased. Zn and MT concentrations in cells incubated with non-radioactive Zn (2, 5 or 10 μmol/L) did not differ from that of cells receiving no added Zn. The presence of PA significantly lowered the cellular MT concentration. When Zn:PA molar ratios were at or above 1:5, cellular MT concentrations were not different from that of cells receiving no added PA. The results of the study suggest that the measurements of cellular Zn and MT concentration have some limitations at or below 10 μmol/L Zn. The initial Zn status of cells may play a role in cellular MT synthesis. Further studies are required to validate the usefulness of using MT as a proxy for Zn absorption in Caco-2 cells. Funded by HarvestPlus.