**4.2. Clinical trials**

#### *4.2.1. Observational studies (dietary intake and serum concentrations of lutein)*

Initially, the relationship between dietary consumption of lutein plus zeaxanthin and AMD has attracted much attention from researchers. Although the results of these studies were not consistent, most of them have demonstrated that a high dietary intake of lutein and zeaxanthin is correlated with lower risk of AMD. A systematic review and meta-analysis was performed to analyze six longitudinal cohort studies and found that intake of lutein and zeaxanthin had different effects on early and late AMD [45]. Consumption of these dietary xanthophylls was strongly associated with the reduced risk of late AMD (relative risk [RR] 0.74; 95% confidence interval [CI] 0.57, 0.97) and neovascular AMD (RR 0.68; 95%CI 0.51, 0.92). However, an inverse relation was not observed between dietary intake of lutein plus zeaxanthin and the risk of early stage AMD. In the Age-Related Eye Disease Study (AREDS) report No. 22, 4519 subjects aged 60–80 years were included for the analysis of association between dietary lutein plus zeaxanthin and AMD status. Compared with the lowest quintiles of dietary lutein and zeaxanthin intake, there were a 55, 35, and 27% lower probability to develop geographic atrophy, neovascular AMD, and large or extensive intermediate drusen, respectively [6]. Similarly, in the Blue Mountains Eye Study, Tan and colleagues [46] evaluated dietary intake of different antioxidants in relation to the long-term risk of incident AMD in Australia, and indicated a 65% reduction in neovascular AMD between the individuals having highest and lowest uptake of lutein and zeaxanthin. The data from Rotterdam Study further revealed the influence of both genetic and environmental risk factors on AMD, demonstrating a protective role of high intake of dietary antioxidants including lutein and zeaxanthin, β carotene, omega-3 fatty acids, and zinc, in AMD at early stage [47].

As early as 1993, Eye Disease Case-Control Study (EDCCS) has reported the direct correlation between serum levels of lutein plus zeaxanthin and AMD risk, demonstrating a distinct risk reduction of neovascular AMD to one-third in subjects with highest serum concentration of lutein and zeaxanthin when compared to those in the lowest group [48]. The research performed by Delcourt et al. [49] has further confirmed that AMD was significantly related with plasma lutein and zeaxanthin and tended to be associated with plasma lutein. A recent study carried out in an Irish population-based sample was in accord with the results discussed above, presenting a lower plasma concentration of lutein in AMD patients no matter whether they were aware of their suffering from AMD or not [50].

#### *4.2.2. Observational studies (MPs levels in the retina)*

VEGF in the development of CNV. Although it has been proved that anti-VEGF compounds can restrict growth of abnormal blood vessels, therefore making vision stabilized or even improved, the cost of each injection is relatively high and monthly intravitreal injection may be required for some patients. In contrast, the current treatments for non-exudative AMD are very limited. Hitherto, no medicine has yet approved for dry AMD in the world. Hence, strategies to delay the onset of this severe visual loss have been focused on the decrease of modified risk factors. Among these modified risk factors, oxidative stress is recognized as one of the major contributing factors in AMD. Since lutein is a powerful antioxidant that is highly concentrated in the retina, the effects of lutein on AMD have been widely investigated.

Initially, the relationship between dietary consumption of lutein plus zeaxanthin and AMD has attracted much attention from researchers. Although the results of these studies were not consistent, most of them have demonstrated that a high dietary intake of lutein and zeaxanthin is correlated with lower risk of AMD. A systematic review and meta-analysis was performed to analyze six longitudinal cohort studies and found that intake of lutein and zeaxanthin had different effects on early and late AMD [45]. Consumption of these dietary xanthophylls was strongly associated with the reduced risk of late AMD (relative risk [RR] 0.74; 95% confidence interval [CI] 0.57, 0.97) and neovascular AMD (RR 0.68; 95%CI 0.51, 0.92). However, an inverse relation was not observed between dietary intake of lutein plus zeaxanthin and the risk of early stage AMD. In the Age-Related Eye Disease Study (AREDS) report No. 22, 4519 subjects aged 60–80 years were included for the analysis of association between dietary lutein plus zeaxanthin and AMD status. Compared with the lowest quintiles of dietary lutein and zeaxanthin intake, there were a 55, 35, and 27% lower probability to develop geographic atrophy, neovascular AMD, and large or extensive intermediate drusen, respectively [6]. Similarly, in the Blue Mountains Eye Study, Tan and colleagues [46] evaluated dietary intake of different antioxidants in relation to the long-term risk of incident AMD in Australia, and indicated a 65% reduction in neovascular AMD between the individuals having highest and lowest uptake of lutein and zeaxanthin. The data from Rotterdam Study further revealed the influence of both genetic and environmental risk factors on AMD, demonstrating a protective role of high intake of dietary antioxidants including lutein and zeaxanthin, β carotene,

As early as 1993, Eye Disease Case-Control Study (EDCCS) has reported the direct correlation between serum levels of lutein plus zeaxanthin and AMD risk, demonstrating a distinct risk reduction of neovascular AMD to one-third in subjects with highest serum concentration of lutein and zeaxanthin when compared to those in the lowest group [48]. The research performed by Delcourt et al. [49] has further confirmed that AMD was significantly related with plasma lutein and zeaxanthin and tended to be associated with plasma lutein. A recent study carried out in an Irish population-based sample was in accord with the results discussed above, presenting a lower plasma concentration of lutein in AMD patients no matter whether

*4.2.1. Observational studies (dietary intake and serum concentrations of lutein)*

omega-3 fatty acids, and zinc, in AMD at early stage [47].

they were aware of their suffering from AMD or not [50].

**4.2. Clinical trials**

180 Progress in Carotenoid Research

In addition to dietary intake and serum concentration of lutein and zeaxanthin, MPs level in the retina was also inversely associated with the risk of AMD. In a case-control study, the actual amounts of lutein and zeaxanthin in donor retinas with and without AMD were measured. Levels of lutein and zeaxanthin in three concentric areas (inner, medial, and outer) centered on the fovea were markedly lower in AMD donor retinas than these in control donor retinas, especially in the outer area, where logistic regression analysis suggested that donors in highest quartile of MPs levels had an 82% lower risk for AMD when compared with those in the lowest quartile after adjustment of age and sex [51]. This is the first report showing the decreased retinal levels of lutein and zeaxanthin in AMD patients, which was consistent with above findings concluded from diet and serum xanthophylls concentrations. Subsequently, MPOD, an indicator for MPs levels in retina in vivo, has been widely studied between healthy individuals and AMD patients. There was a MPOD decline in healthy eyes as the individuals aged, and MOPD in healthy eyes at high risk of AMD was significantly lower than those at no such risk [52, 53]. Moreover, Bernstein and his co-workers [54] evaluated MPs levels in relation to the incidence of AMD using noninvasive resonance Raman spectroscopy, and found 32% reduction of retinal lutein and zeaxanthin levels in AMD versus normal participants. However, it was notable that lower MPOD has also been linked with other risk factors for AMD, such as smoking and family history of this disease [55]. This result further supported the hypothesis that lutein and zeaxanthin could prevent or delay the development of AMD by increasing MPOD.

#### *4.2.3. Interventional studies (supplementation of lutein)*

Observational results in relation to AMD have triggered a mass of interests in assessing effects of lutein supplementation on the risk of AMD. The supplementation trial was first reported in the Lutein Antioxidant Supplementation Trial (LAST) study [56]. This was a prospective, double-masked, placebo-controlled, randomized study to evaluate supplementation of lutein alone or lutein with other antioxidants, vitamins, and minerals in 90 atrophic AMD patients. After 12 months, higher MPOD, improved visual acuity and contrast sensitivity were observed in both of these groups than in placebo group. However, longer duration of the study, larger number of samples, and both genders are needed to examine the long-term effects of lutein or the combination of lutein with other nutrients in the treatment of dry AMD. Three years later, LASTII was performed to further analyze the specific factors that affected MPOD, including age, baseline levels of MPs, and combination of lutein and other antioxidants [57]. There was an increase in MPOD with supplementation, while a moderate reduction of MPOD was observed without supplementation. Patients with lowest baseline MPOD value were most likely to have a dramatic increase in MPOD than those with medium to high baseline MPOD during one-year supplementation of lutein or lutein with other nutrients. The reason might be the saturation mechanism that had an impact on the retinal transportation and stabilization of MPs.

In the Combination of Lutein Effects in the Aging Retina (CLEAR) study, Murray et al. [58] supplemented the patients at early stage AMD with 10 mg lutein esters per day for up 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 to be related to the increase of MPOD.

improvement over the original effective AREDS formulation was not achieved, analyses of patients with lutein and zeaxanthin supplements versus those without lutein and zeaxanthin supplements demonstrated a 10% decrease in the risk of progression to advanced AMD in the group with lutein and zeaxanthin [67]. Furthermore, the analyses of comparing participants receiving lutein and zeaxanthin with those receiving β-carotene were performed. The risk of developing advanced and neovascular AMD was significantly decreased in the group with lutein and zeaxanthin. In analyses restricted to eyes with bilateral large drusen at baseline, protective effects of lutein and zeaxanthin were more prominent. Therefore, considering beneficial effects of lutein and zeaxanthin as well as harmful effects of β-carotene on smokers, replacement of β-carotene with lutein and zeaxanthin in AREDS2 formula is preferred.

Lutein and the Aging Eye

183

http://dx.doi.org/10.5772/intechopen.79604

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

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)

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

, leading to decreased cell viability, increased cell

O2

**4.3. Basic research**

significant suppression of CNV development [72].

were induced by the challenge of H2

lutein in a dose-dependent manner [75].

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

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 improvement over the original effective AREDS formulation was not achieved, analyses of patients with lutein and zeaxanthin supplements versus those without lutein and zeaxanthin supplements demonstrated a 10% decrease in the risk of progression to advanced AMD in the group with lutein and zeaxanthin [67]. Furthermore, the analyses of comparing participants receiving lutein and zeaxanthin with those receiving β-carotene were performed. The risk of developing advanced and neovascular AMD was significantly decreased in the group with lutein and zeaxanthin. In analyses restricted to eyes with bilateral large drusen at baseline, protective effects of lutein and zeaxanthin were more prominent. Therefore, considering beneficial effects of lutein and zeaxanthin as well as harmful effects of β-carotene on smokers, replacement of β-carotene with lutein and zeaxanthin in AREDS2 formula is preferred.
