Comparison of Antioxidant Properties of Dehydrolutein with Lutein and Zeaxanthin, and their Effects on Cultured Retinal Pigment Epithelial Cells

Abstract

Dehydrolutein accumulates in substantial concentrations in the retina. The aim of this study was to compare antioxidant properties of dehydrolutein with other retinal carotenoids, lutein, and zeaxanthin, and their effects on ARPE-19 cells. The time-resolved detection of characteristic singlet oxygen phosphorescence was used to compare the singlet oxygen quenching rate constants of dehydrolutein, lutein, and zeaxanthin. The effects of these carotenoids on photosensitized oxidation were tested in liposomes, where photo-oxidation was induced by light in the presence of photosensitizers, and monitored by oximetry. To compare the uptake of dehydrolutein, lutein, and zeaxanthin, ARPE-19 cells were incubated with carotenoids for up to 19 days, and carotenoid contents were determined by spectrophotometry in cell extracts. To investigate the effects of carotenoids on photocytotoxicity, cells were exposed to light in the presence of rose bengal or all-trans-retinal. The results demonstrate that the rate constants for singlet oxygen quenching are 0.77 × 1010, 0.55 × 1010, and 1.23 × 1010 M−1s−1 for dehydrolutein, lutein, and zeaxanthin, respectively. Overall, dehydrolutein is similar to lutein or zeaxanthin in the protection of lipids against photosensitized oxidation. ARPE-19 cells accumulate substantial amounts of both zeaxanthin and lutein, but no detectable amounts of dehydrolutein. Cells pre-incubated with carotenoids are equally susceptible to photosensitized damage as cells without carotenoids. Carotenoids provided to cells together with the extracellular photosensitizers offer partial protection against photodamage. In conclusion, the antioxidant properties of dehydrolutein are similar to lutein and zeaxanthin. The mechanism responsible for its lack of accumulation in ARPE-19 cells deserves further investigation.

Is There an Optimal Combination of AREDS2 Antioxidants Zeaxanthin, Vitamin E and Vitamin C on Light-Induced Toxicity of Vitamin A Aldehyde to the Retina?

“…Multi-lamellar liposomes were prepared either from EYPC in the presence and absence of ATR, zeaxanthin and/or α-tocopherol [ 42 , 43 , 44 ]. The compounds dissolved in chloroform were mixed at required ratios in a round-bottom flask, and then the chloroform was evaporated under a stream of argon to form a lipid film, which was then kept under vacuum for at least 1 h. The lipid films were hydrated in nitrogen-saturated Dulbecco’s phosphate buffered saline with calcium and magnesium (PBS), aliquoted into cryovials and kept frozen until use.…”

“…To determine the effect of antioxidants on photosensitized oxidation mediated by ATR, electron spin resonance (ESR) oximetry was used as described previously [ 42 , 44 , 45 ]. In short, oxygen concentrations were monitored by spectral characteristics of mHCTPO.…”

“…The irradiance inside the resonant cavity, measured with a photodiode PD Irradiance Meter (Hamamatsu, Photonics, K.K., Hamamatsu City, Japan), was 6.2 mW/cm 2 . Other experimental conditions were as described previously [ 44 ].…”

“…Both lutein and zeaxanthin are excellent quenchers of excited states of photosensitizers and singlet oxygen. Zeaxanthin is more effective than lutein as a singlet oxygen quencher with the quenching rate constant of 12.6 × 10 9 M −1 s −1 , which is close to the diffusion-controlled limit [ 42 , 43 , 44 ]. The process of singlet oxygen quenching by xanthophylls is mainly physical, whereby carotenoids accept the excess of energy from singlet oxygen and deactivate to the ground state via internal conversion, with only a small fraction of interactions with singlet oxygen, less than 0.1%, leading to the oxidation of the carotenoid [ 45 , 46 , 47 ].…”

Photodegradation of Lipofuscin in Suspension and in ARPE-19 cells and the Similarity of Fluorescence of the Photodegradation Product with Oxidized Docosahexaenoate

“…Cell viability was assessed by monitoring the morphology of ARPE-19 cell monolayer by phase contrast microscopy using an Olympus IX70 inverted microscope and by MTT assay of reductive activity where cells were exposed for 1 h to cell culture medium containing 0.5 mg/ml of MTT as described previously [78,104]. Then cells were washed with PBS and MTT-derived formazan was solubilised by acidified isopropanol and absorbance at 570 nm was red using a Multiscan Ascent plate reader.…”