Uptake of carotenoids and their antioxidant action in ARPE–19 cells in culture

Abstract

Abstract: : Purpose: Lutein, mezo–zeaxanthin and zeaxanthin accumulate in the retina, however the mechanism responsible for their preferential uptake remains unclear. The aim of this study was to test a hypothesis that retinal pigment epithelium (RPE) plays a role in the uptake of carotenoids, and their incorporation results in diminished susceptibility to the oxidative damage to the RPE cells. Methods: Confluent ARPE–19 cells in culture were fed for up to three weeks with the following carotenoids: lutein, dehydrolutein, meso–zeaxanthin, zeaxanthin, or ß–carotene solubilized at a concentration of 1µM in the culture medium containing 10% fetal calf serum. Cell viability and accumulation of carotenoids were monitored by MTT and propidium iodite assays, and UV–visible absorption spectroscopy. Sub–fractionation of cells followed by HPLC/UV–visible absorption spectroscopy and TEM was used to determine the localisation of carotenoids in the cells. To test whether the accumulated carotenoids can protect against photosensitized damage, the cells were exposed to rose bengal and visible light for up to 60 min. To compare the antioxidant potential of the carotenoids studied, liposomes consisting of unsaturated lipids in the presence and absence of carotenoids were exposed to rose bengal/visible light and their susceptibility to photo oxidation was determined by oximetry, and HPLC analysis of products of cholesterol oxidation. Results: ARPE–19 preferentially accumulated lutein, meso–zeaxanthin, and zeaxanthin over dehydrolutein and ß–carotene. Accumulated carotenoids were present mainly in the phagosomal fractions of the cells. Even though all carotenoids tested proved highly protective against photo–oxidative damage in the liposomal system, no protection was offered against photosensitized damage to the cells, unless the cells had carotenoids present in their plasma membranes. Conclusions: RPE may be involved in the preferential accumulation of lutein, meso–zeaxanthin and zeaxanthin in the retina. Accumulated carotenoids provide no protection against photosensitized damage to their plasma membranes. It remains to be determined whether they protect against photosensitized damage generated inside the cells.

Keywords: antioxidants • carotenoids/carotenoid binding proteins • oxidation/oxidative or free radical damage