Glutathione (GSH) is the most prevalent low-molecular weight thiol in mammalian cells. It plays an important role in the detoxification of a variety of electrophilic compounds and peroxides via catalysis by glutathione S-transferases and glutathione peroxidises, and is crucial for antioxidant defense. GSH deficiency contributes to oxidative stress, and, therefore, may play a key role in aging and the pathogenesis of many diseases. However, little is known regarding GSH transport across human intestinal epithelial cells. The objective of this work was to examine whether intact GSH can be transported across intestinal epithelial cell (Caco-2 and HT-29) monolayers, and characterize its uptake. The intact GSH was detected in the basolateral transport chamber of the cell monolayers using a GSH/GSSG enzyme assay and the structure of intact GSH was confirmed by mass spectrometry (LC/TOF-MS). There was no significant difference between the concentration of GSH detected in the presence or absence of the inhibitors Acivicin AT-125 or buthionine sulfoximine BSO, indicating that the GSH present in the basolateral chamber was likely due to the uptake/transport of intact GSH across the Caco-2/HT-29 monolayers, and not GSH re-synthesis. There was no difference in the permeability (Papp) of GSH in the presence or absence of either inhibitor. Moreover, no products of GSH degradation (e.g. cysteine, cysteinyl-glycine) were observed in the transported samples using LC/MS analysis. To further characterize GSH uptake across the Caco-2 monolayers, transport was carried out in the presence of the inhibitor Probenecid (PB), an inhibitor of GSH transport in basolateral membrane vesicles. The amount of GSH detected in the basolateral solution of PB-treated cells was significantly reduced when compared to all other samples. We further confirmed that GSH uptake is not proton-dependent. In conclusion, this is the first study which demonstrated that intact GSH can be transported across intestinal epithelial cell.

Transport of intact glutathione across human intestinal epithelial cell monolayers using Caco-2 and HT-29 models

248th National Meeting (2014)

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