**4.3. Basic research**

to 1 year. MPOD increased significantly after 8 months of supplementation, and plasma concentration of lutein increased by 1.8-fold to 7.6-fold compared to the baseline values. In addition, visual acuity in lutein group remained stable while the declined visual acuity was exhibited in the placebo group, indicating that stabilization of visual acuity was probably maintained by the elevated MPs level. These results were in accord with the study carried out by Ma et al. [13, 59], who demonstrated the significant improved responses of multifocal electroretinogram (mfERG) in lutein group and in lutein plus zeaxanthin group, and tended

In a randomized controlled clinical trial (known as the Carotenoids with Coantioxidants in Age-Related Macular Degeneration [CARMA] study), 433 patients who were identified to be at highest risk of progression to advanced AMD received a daily supplementation of lutein, zeaxanthin, vitamin C, vitamin E, zinc, and copper at the duration of 12–36 months [60]. Visual acuity was increased in the intervention groups at 12 months, but not statistically significant until 24 months. Contrast sensitivity was slightly improved without significance. Level of MPs declined steadily in the placebo group, while MPs was increased in the supplemented groups throughout the whole trial. A rise of plasma concentration of all contents in the supplementation, especially lutein and zeaxanthin, was observed after 6 months. Although the increase of all antioxidants in blood was not associated with VA improvement, higher serum level of lutein slowed the progression of AMD. Fewer eyes progressed to severe state in the

The Age-Related Eye Disease Study 2 (AREDS2) was a randomized, placebo-controlled, double-masked trial conducted in the USA from 2006 to 2012 [61]. The participants involved in AREDS2 were subjects aged 50–85 years at risk for progression to advanced AMD with bilateral large drusen or large drusen in one eye and advanced AMD in the fellow eye. The main objective of AREDS2 was to evaluate the effects of lutein, zeaxanthin, and omega-3 long-chain polyunsaturated fatty acids adding into the AREDS formulation, which was composed of vitamin C (500 mg), vitamin E (400IU), β-carotene (15 mg), and zinc (80 mg zinc oxide) with copper (2 mg cupric oxide). After the follow-up of 6.5 years on average, the AREDS supplements was proved to significantly decrease the development to advanced AMD, and an approximately 25% reduction in risk of progressing to late AMD was observed at 5 years [62]. Moreover, the beneficial effects of this AREDS formula were found to persist for 5 more years of followup after the end of this trial [63]. However, supplementation of β-carotene may lead to the increased risk of lung cancer in cigarette smokers [64, 65]. In addition, 80 mg/day zinc is out of tolerance for individuals and high amount of zinc was associated with increased genitourinary complications [62, 66]. Therefore, AREDS2 supplementation was changed as follows: the primary randomization was composed of AREDS formulation with (1) lutein (10 mg) + zeaxanthin (2 mg), (2) fish oil (350 mg DHA + 650 mg EPA), (3) lutein + zeaxanthin + EPA + DHA, and (4) placebo; the secondary randomization included (1) AREDS formulation, (2) AREDS formulation with low zinc (25 mg), (3) AREDS formulation without β-carotene, and (4) AREDS formulation with low zinc (25 mg) and without β-carotene. Former and current smokers are randomly assigned to the groups without β-carotene. In the primary analysis, no further reduced risk of developing advanced AMD was observed when comparing each of the treatment groups with placebo group [61]. Although the preconceived goal of 25% incremental

to be related to the increase of MPOD.

182 Progress in Carotenoid Research

intervention group than in the placebo group (15.3 vs. 18%).

Several animal models that mimic the pathological changes in AMD have been adapted to further study effects of lutein and zeaxanthin on AMD in human. Apolipoprotein E-deficient mice (apoE-/-), a well-established genetic mouse model of hypercholesterolemia, exhibited deposits on the basal laminar, vacuoles in RPE cells, and increased Bruch's membrane thickness, which are similar to the retinal changes in human AMD. These alterations were associated with the elevation of retinal lipid peroxidation and VEGF expression [68]. Administration of lutein alone could partially prevent the retinal alterations, and decrease expression level of VEGF but with no statistical significance was observed in comparison with controls. However, the combination of lutein and multivitamin and glutathione complex ameliorated all the morphological changes observed in retina and decreased VEGF levels significantly [68, 69]. In the mouse models that show similar retinal changes in human dry AMD, AREDS2 formulation prevented accumulation of liposomes and lipofuscin in RPE, loss of photoreceptors, and increased ONL thickness. In molecular level, mRNA expression levels of pro-inflammatory factors including inducible nitric oxide synthase (iNos), tumor necrosis factor-α(TNF-α), Cox-2, IL-1β, and angiogenic factors such as VEGF was significantly lower in AREDS2-treated group than control groups [70]. Furthermore, supplementation of lutein and zeaxanthin from grapes or marigold extract attenuated a reduction of a-wave amplitude in ERG, suggesting protective effects on photoreceptor functions [71]. Mouse model for the wet form of AMD is induced by laser photocoagulation, characterized by the formation of CNV. It has been reported that pretreatment of lutein significantly inhibited macrophage infiltration in CNV and expression of pro-inflammatory molecules such as NF-κB that subsequently resulted in significant suppression of CNV development [72].

Data from in vitro studies were also consistent with findings from animal experiments. Addition of lutein and other antioxidants (zeaxanthin, lycopene, or α-tocopherol) led to a significant decrease in formation of lipofuscin in RPE cells from bovine and rabbit under hypoxia condition [73]. Oxidative damages in ARPE-19 cells (a human RPE-derived cell line) were induced by the challenge of H2 O2 , leading to decreased cell viability, increased cell apoptosis, and ROS generation. Pretreatment of lutein protected ARPE-19 cells from these oxidative injuries and accumulation of Alu RNA, which is related to the pathogenesis of AMD [74, 75]. In addition, G2/M phase arrest triggered by oxidative damage was reversed by lutein in a dose-dependent manner [75].
