The impact of apical and basolateral albumin on intestinal zinc resorption in the Caco-2/HT-29-MTX co-culture model

Abstract

The molecular mechanisms of intestinal zinc resorption and its regulation are still topics of ongoing research. To this end, the application of suitable in vitro intestinal models, optimized with regard to their cellular composition and medium constituents, is of crucial importance. As one vital aspect, the impact of cell culture media or buffer compounds, respectively, on the speciation and cellular availability of zinc has to be considered when investigating zinc resorption. Thus, the present study aims to investigate the impact of serum, and in particular its main constituent serum albumin, on zinc uptake and toxicity in the intestinal cell line Caco-2. Furthermore, the impact of serum albumin on zinc resorption is analyzed using a co-culture of Caco-2 cells and the mucin-producing goblet cell line HT-29-MTX. Apically added albumin significantly impaired zinc uptake into enterocytes and buffered its cytotoxicity. Yet, undigested albumin does not occur in the intestinal lumen in vivo and impairment of zinc uptake was abrogated by digestion of albumin. Interestingly, zinc uptake, as well as gene expression studies of mt1a and selected intestinal zinc transporters after zinc incubation for 24 h, did not show significant differences between 0 and 10% serum. Importantly, the basolateral application of serum in a transport study significantly enhanced fractional apical zinc resorption, suggesting that the occurrence of a zinc acceptor in the plasma considerably affects intestinal zinc resorption. This study demonstrates that the apical and basolateral medium composition is crucial when investigating zinc, particularly its intestinal resorption, using in vitro cell culture.

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

“…In vitro basolateral zinc excretion of intestinal cells is enhanced by basolateral albumin, whereas cellular zinc uptake from the apical side seems to be unaffected by this zinc acceptor [102]. This also reiterates previous knowledge on intestinal zinc uptake and transport kinetics.…”

“…Detailed processes of cellular distribution of zinc into enterocytes and its transfer through the cells after its absorption are not yet completely understood. Examinations of free zinc (pools) in enterocytes in vitro with the eCalwy biosensor [101] and the fluorescent zinc probe Zinpyr-1 [102,103] document that enterocytes contain at least two different free zinc pools that are involved in the maintenance of zinc homeostasis during zinc absorption: cytoplasmic-free zinc and vesicular zinc [101][102][103]. Nevertheless, these processes have to be further scrutinized.…”

“…Three main zinc pools in enterocytes have been described: (i) cytoplasmic-free zinc, which is only complexed by low molecular weight ligands, (ii) protein-bound zinc, depicted here as metallothionein (MT)-bound zinc, and (iii) free zinc stored in vesicles [104]. The vesicular [102,103] and cytoplasmic-free zinc pools [101] are recognized to be involved in zinc absorption by enterocytes [105]. Cellular zinc homeostasis is maintained by three main groups of proteins: the zinc transporter (ZnT)-and the Zrt-, Irt-like protein (ZIP)-family as well as the zinc-binding metallothioneins [99].…”

“…Presumably, these increase luminal solubility of the metal, and, consequently, enhance its availability to enterocytes [154,155]. Serum albumin is an important serosal factor, acting as a basolateral zinc-acceptor and enhancing enterocytic zinc release into the blood circulation [102]. Additionally, systemic humoral factors, such as hepcidin, seem to influence ZnT-1-mediated export of zinc by intestinal cells [156], which indicates that the liver might play an important role in secreting humoral factors regulating intestinal zinc absorption.…”

“…Human blood contains about 30-50 mg mL −1 human serum albumin (HSA) [220], where it is the main zinc binding and transporting protein [23] and has high zinc binding affinity (molecular ratio of the albumin/zinc-complex 1:2, K d (site A) = 100 nM) [221]. Bovine serum albumin (BSA) acts as a basolateral zinc acceptor, which enhances serosal zinc export on the basolateral side of a three dimensional in vitro Caco-2/HT-19-MTX co-culture model [102]. This remains to be confirmed using HSA, as interspecies differences in the structure of this protein [222] might affect their zinc-binding properties and, consequently, their role as a basolateral zinc acceptor.…”