Improvement of analytical conditions of mineral Caco-2 cell uptake assays

Caco-2 cell mineral uptake assays are used to estimate the bioavailability of minerals from foods. The uptake of minerals by Caco-2 cells can be affected by several factors - particularly the conditions of the in vitro digestion process and the growth conditions of the cell culture. Therefore, a standardisation of the assays conditions is required to obtain reproducible results. This work determined the effect of enzyme demineralisation, the inactivation of the proteolytic activity of the digest and the replacement of distilled-deionised water by cell culture degree water. Treatment with Chelex-100 resin was applied to remove calcium, iron and zinc from pepsin and pancreatin-bile salts. The treatment was efficient (>90%) for removing calcium, iron and zinc from pepsin and calcium from pancreatin-bile salts, though the efficiency was lower (less than or equal to50%) in the case of iron and zinc from pancreatin-bile salts. The use of demineralised enzymes in the digestion resulted in lower (p < 0.01) calcium and zinc contents in the soluble fraction than when non-demineralised enzymes were used. Inactivation of proteolytic enzymes at 100 degrees C for 4, 10 and 15 min was tested, and it was found that 4 min sufficed to maintain the integrity of the cell monolayer during the assay. The replacement of distilled-deionised water by cell culture degree water improved the viability of the cell culture.