Lutein and Zeaxanthin—Food Sources, Bioavailability and Dietary Variety in Age-Related Macular Degeneration Protection

Lutein and zeaxanthin (L/Z) are the predominant carotenoids which accumulate in the retina of the eye. The impact of L/Z intake on the risk and progression of age-related macular degeneration (AMD), a leading cause of blindness in the developed world, has been investigated in cohort studies and clinical trials. The aims of this review were to critically examine the literature and evaluate the current evidence relating to L/Z intake and AMD, and describe important food sources and factors that increase the bioavailability of L/Z, to inform dietary models. Cohort studies generally assessed L/Z from dietary sources, while clinical trials focused on providing L/Z as a supplement. Important considerations to take into account in relation to dietary L/Z include: nutrient-rich sources of L/Z, cooking methods, diet variety and the use of healthy fats. Dietary models include examples of how suggested effective levels of L/Z can be achieved through diet alone, with values of 5 mg and 10 mg per day described. These diet models depict a variety of food sources, not only from dark green leafy vegetables, but also include pistachio nuts and other highly bioavailable sources of L/Z such as eggs. This review and the diet models outlined provide information about the importance of diet variety among people at high risk of AMD or with early signs and symptoms of AMD.


Introduction
Lutein and zeaxanthin (L/Z) are two fat-soluble antioxidants belonging to the class of carotenoids called xanthophylls. Along with their conversion isomer meso-zeaxanthin, they are the major constituents of macular pigment, a compound concentrated in the macula region of the retina that is responsible for fine-feature vision. Given their accumulation in the retina, the role L/Z play in eye health has been investigated with a particular focus on how consumption of these carotenoids may prevent and/or slow the progression of age-related macular degeneration (AMD), the leading cause of blindness in older adults [1]. Recently published prevalence rates for early AMD in Irish adults over 50 years and New Zealand adults 45−85 years are 7.2% [2] and 10.3% [3], respectively. Furthermore, as the leading cause of severe vision impairment in Australians over 40 years of age, AMD continues to be an important target for the development of effective preventative therapies to ease the medical and economic burden of this disease in the developed world [4]. US research has forecast that the number of patients with AMD is likely to double between 2010 to 2050 and this disease is becoming a crucial public health issue [5]. Recent review articles have detailed the evidence linking L/Z and AMD with an emphasis on L/Z supplements, the transport and storage of L/Z in the macula as well as the potential mechanisms of action they have in AMD [6][7][8][9]. These papers provide limited details regarding the potential role of dietary sources of L/Z in AMD. The aim of this paper is to summarise the current available evidence regarding L/Z's role in AMD and, specifically, to hypothesise and model a likely suitable amount of lutein and zeaxanthin to obtain from dietary sources that may reduce the risk of AMD (progression and prevention), taking into account common food sources, such as vegetables and eggs, and factors affecting their absorption and bioavailability in the body.

Dietary Carotenoids
Around 700 carotenoids have been characterised in nature; approximately 20−30 have been identified in human blood and tissues, but only lutein and zeaxanthin are found in the eye [9,10]. L/Z (with the isomer meso-zeaxanthin) accumulate in the retina of the eye and form the retinal macular pigment (MP) [11]. MP has several functions in improving visual performance and protecting against the damaging effects of light, and MP levels are used as a proxy for macular health-specifically to predict the likelihood of developing AMD [11]. In the retina, these three compounds (lutein, zeaxanthin and meso-zeaxanthin) exhibit regional dominance with meso-zeaxanthin being the dominant carotenoid at the epicentre, zeaxanthin at the mid-periphery and lutein at the periphery of the macula [12].

Age-Related Macular Degeneration (AMD)
AMD is one of the leading causes of blindness in older adults in the developed world [13]. AMD increases with advancing age and appears to be more likely in men than women [14], although this differs between countries [15]. Table 1  Synergistic associations between diet, lifestyle and genes and risk of AMD have been shown, with unhealthy lifestyles associated with an increased AMD risk regardless of AMD risk genotype [17]. A number of dietary measures have been associated with slowing the progression of AMD. These include a diet rich in omega-3 fatty acids [18], a diet with a lower dietary glycemic index [19,20] and intake of nutrients including vitamins C and E, beta-carotene, zinc, selenium, B vitamins, folate and vitamin B12 and lutein and zeaxanthin [21][22][23][24].
Their unique presence in the centre of the macula implies an important role for L/Z in visual performance [7]. Antioxidant and blue light-filtering properties of L/Z for short wavelengths are hypothesised to protect the eye against AMD [9]. The filtration of blue light reduces chromatic aberration which can enhance visual acuity and sensitivity [25]. The following review focuses on L/Z and includes a summary of the observational evidence obtained from cohort and case-control studies as well as data obtained from randomised controlled trials which have investigated the relationship between dietary and supplemental L/Z and AMD development and/or progression.

Lutein and Zeaxanthin and AMD-Epidemiological and Intervention Studies
Evidence of an association between the consumption of fruits and vegetables and the risk of AMD was reported in 1988 [26] when data obtained from the first US National Health and Nutrition Examination Survey was published. Following this, a number of cohort and case-control studies have examined associations between dietary intake of L/Z and risk of AMD [21,[27][28][29][30][31]. These studies are summarised in Table 2.
A 2012 systematic review and meta-analysis of six longitudinal cohort studies observed that the protective effects of high intakes of dietary L/Z may be confined to late AMD [32]. Researchers calculated a pooled relative risk (RR) for early AMD, comparing the highest with the lowest category of L/Z intake, 0.96 (95% confidence interval (CI) 0.78, 1.17), whereas dietary intake of these carotenoids was significantly related with a reduction in risk of late AMD (RR 0.74; 95% CI 0.57, 0.97). Furthermore, the study found a 32% risk reduction of neovascular AMD (RR 0.68; 95% CI 0.51, 0.92) [32] among those people who consumed the highest category of L/Z compared to those who consumed the lowest.
Variation is seen in the results from cohort and case-control studies available to date. Overall, the evidence suggests a potential protective effect of L/Z consumption particularly in relation to younger cohorts, late AMD and those at high genetic risk of AMD. Abbreviations: FFQ, food frequency questionnaire; OR, odds ratio; CVD, cardiovascular disease; SD, standard deviation.
Further evidence that L/Z intake may provide protection against the development of AMD comes from the Age-Related Eye Disease Study 2 (AREDS2)-a major clinical trial conducted in the US. In the original Age-Related Eye Disease Study (AREDS1), researchers found that supplementation with vitamins C (500 mg) and E (400 IU), β-carotene (15 mg), zinc (80 mg) and copper (2 mg) at levels well above the recommended daily allowances reduced the risk of progression to a more advanced AMD by about 25% [23]. Later, AREDS2 incorporated lutein (10 mg) and zeaxanthin (2 mg) into the formulation being used in the study among people with early AMD [38]. In secondary analysis, L/Z supplements on top of the AREDS supplement lowered the progression to advanced AMD, though this only occurred in persons with low dietary L/Z [38]. A reduction in risk was seen for individuals in quintile 1 who had a median intake of 696 µg L/Z per 1000 cal per day (interquartile range 552−823 µg/1000 cal per day) with a hazard ratio (HR) of 0.74 (95% CI, 0.59−0.94) when compared with no L/Z intake. For those with a median intake of 1134 µg per 1000 cal per day (interquartile range 1030−1244 µg/1000 cal per day) the HR was 0.94 (95% CI, 0.74−1.21) showing that the supplement had no additional impact of progression of the disease when the background diet was sufficient in L/Z. This implies that dietary intakes of at least 2268 µg of L/Z (with a range of 2060-2488 µg, assuming an average daily energy intake of 2000 calories) may provide some level of protection against late AMD, but this needs to be confirmed in other dietary trials of L/Z among people with AMD, or at high risk of AMD. The overall findings from the AREDS2 and other studies suggest that L/Z is likely to be more appropriate and safer than beta-carotene in the original AREDS-type supplements [39,40]. The findings also point to the potential beneficial effects sufficient dietary intake of L/Z can have on AMD progression. Table 3 provides a summary of the findings from the AREDS2 study.
Evidence also suggests greater L/Z intake may particularly protect against AMD in persons with high genetic risk based on two major AMD genes [41]. Using data from the Blue Mountains Eye Study (BMES) and Rotterdam Study (RS), among participants with high genetic risk, the highest tertile of dietary intake of L/Z was associated with more than 20% reduced risk of early AMD (p = 0.0009). No similar association was, however, found for those with low genetic risk [41]. These findings are similar to those documented in Ho et al. [36] which is summarised as part of Table 2.
Randomised controlled trials with L/Z supplements have been conducted evaluating how L/Z supplementation affects visual performance in individuals with established AMD [42][43][44][45][46][47][48][49]. A meta-analysis of these seven studies was conducted and published in 2015 [6]. Results of this meta-analysis suggest that L/Z supplementation is associated with significant improvements in visual acuity and contrast sensitivity in a dose-response manner [6]. Furthermore, a linear association was indicated between these improvements and macular pigment optical density (MPOD) increase [6].
MPOD, a measurement of the attenuation of blue light by the macular pigment, is linearly related to the amount of L/Z in the macula. Precise roles and mechanisms are not fully understood; however, it is thought that MPOD levels, particularly in the central retina, may impact AMD [50].
The majority of studies have shown an age-dependent decrease in MPOD levels and a lack of MPOD in AMD compared to healthy controls [51]. A 2016 meta-analysis of 20 randomised controlled trials found that lutein, zeaxanthin and meso-zeaxanthin supplementation improves MPOD in both healthy subjects and in AMD patients in a dose-response manner [52]. These improvements were also positively associated with increases in serum L/Z levels. Further research supports the consumption of dietary sources of L/Z to improve MPOD, particularly for individuals with low MPOD at baseline. An example of this is a study in healthy adults which compared the effect of L/Z-rich foods and supplements on MPOD as well as serological markers of endothelial activation, inflammation and oxidation. The eight-week intervention [53] which used spinach powder as the L/Z-rich food found improvements in MPL in the highest serum responders and in those with initially low MPL, supporting observational evidence to date. Another study showed the consumption of six eggs per week significantly increased the concentration of macular carotenoids (as measured by MPOD) as well as serum zeaxanthin levels; however, serum lutein levels remained unchanged [54]. Further evidence is warranted to better ascertain how different L/Z-containing foods may impact MPOD and which individuals would benefit from L/Z intake given there is evidence which suggests that the structural components responsible for the storage and availability of L/Z in the eye are genetically determined [50].

Recommended Intake Values for Lutein and Zeaxanthin
There are currently no official recommended dietary intake levels for L/Z. However, based on a paper published in 1994 [31], an intake of 6 mg of L/Z per day for men and women has been suggested as a dietary target to reduce the risk of age-related macular degeneration [55]. However, evidence from both the AREDS2 study and the BMES suggest that levels of intake of L/Z considerably less than 6 mg are associated with a decreased likelihood of AMD indicating lower levels of intake may be sufficient to provide some protection from progression of the disease, although further intervention studies to ascertain an appropriate target level, particularly for individuals at high risk of AMD, is needed.
Data on population L/Z intake is limited, but current evidence suggests the intake of carotenoids varies in different populations as well as by season [56]. For example, in Europe, the average daily intake of major carotenoids (including retinol, alpha-tocopherol, beta-carotene, alpha-carotene, beta-cryptoxanthin, lutein, zeaxanthin, and lycopene) ranges from 3.5 mg/day in the Spanish population [57] to 5.33 mg/day in the German population [58]. Fewer studies have specifically measured L/Z consumption in populations, although in an American study of older adults, daily consumption of L/Z was 2.7 mg for men and 3.09 mg for women [59]. In another study, it was estimated that American adults consume approximately 1−2 mg of lutein per day [60]. In an Australian study of older adults living in the Blue Mountains, it was found that the average intake of L/Z was 0.9 mg and that women had slightly higher intakes than men [61]. The main contributors to L/Z intakes were broccoli, green beans and oranges. No other Australian studies have assessed the intake of these antioxidants in other population sub-groups; however, based on these results, current intakes in older adults are lower than ideal.
In countries including Australia and the US, the national intakes of lutein may be declining. According to US National Health Interview Studies, there was a decrease in lutein consumption amongst different categories of people between 1987 and 1992 due to decreased intakes of green leafy vegetables [62]. In Australia, the National Nutrition Survey from 1983, 1985 and 1995 identified that between 1983 and 1995, the mean daily intake of fruit decreased by between 30 g and 50 g for men and women, respectively, and that the mean daily intake of vegetables decreased by 11 g for any population assessed [63]. Results from the latest Australian Health Survey indicated that only 7% of Australian adults are consuming the recommended five or more servings of vegetables per day [64]. Overall, the available data on current intakes suggests they are considerably below the current suggested intake of 6 mg per day.

Dietary Sources of Lutein and Zeaxanthin
One of the difficulties in understanding and deciphering the exact role of dietary L/Z in AMD prevention and/or progression is the challenges inherent in analyzing and quantifying the L/Z content of foods. Until recently, L/Z were measured together because analytical procedures did not permit separate quantification of these carotenoids in foods. Because of this, composition tables commonly report the L/Z content of foods as one figure; however, testing of the individual carotenoids has been conducted [65] to ascertain the differing amounts of the individual carotenoids in foods. Given that L/Z accumulate in different regions of the eye and contribute to different functions, it may be important for both carotenoids to be consumed as part of a dietary pattern [65]. Accurate assessment of L/Z individually is crucial in evaluating their relative roles in eye health [65]. As shown in Table 4, the majority of vegetable sources of L/Z contain only lutein, whereas corn and eggs contain both lutein and zeaxanthin. It is also now suggested that meso-zeaxanthin may not be solely derived from retinal lutein and that it may in fact be present in small amounts in the food supply [12]. For example, meso-zeaxanthin is reported to be commonly used in hen feed in Mexico to enhance the colouration of the egg yolk [12]. These findings suggest that it may be important for all three macular pigment carotenoids, (lutein, zeaxanthin and meso-zeaxanthin) to be quantified in food. Table 5 shows the lutein and zeaxanthin contents of a number of common foods. From a whole diet perspective, the availability of carotenoids from different types of foods i.e., vegetables, nuts and eggs, allows for a more varied and potentially more easily maintainable dietary pattern delivering higher amounts of L/Z. One concern with dietary interventions to increase levels of macular pigment has been the perception that the quantities of vegetables (in particular green leafy vegetables such as spinach) required to increase L/Z levels adequately are unrealistic; however, a careful, targeted selection of foods containing L/Z can still provide variety.
Dietary models demonstrating this variety as well as what dietary intakes of L/Z at various levels look like are outlined in Tables 6 and 7. Two L/Z intake target levels were chosen given that: (1) work has previously suggested a dietary target of 5−6 mg L/Z [31]; and (2) 10 mg represents an upper level of what is achievable through dietary intake and is close to the L/Z level used in the AREDS supplement study [38]. While the dietary models are based on these higher levels of intake, evidence from cohort studies suggests lower dietary intakes of approximately 2.5 mg may be high enough to offer some protection against AMD development [21,38].

Absorption and Bioavailability of Dietary Carotenoids
While the total amount and distribution of L/Z in the food supply is important, it is also necessary to take into account factors that affect the absorption and bioavailability of these carotenoids. For example, the consumption of fat (in the form of a salad dressing, cooking oil such as extra virgin olive oil, or whole eggs) at the same meal as carotenoid intake (e.g., a raw vegetable salad or cooked vegetables) has been shown to effectively increase the absorption of some carotenoids [67][68][69][70][71].
Bioavailability of carotenoids may be decreased due to competition for absorption between carotenoids when consumed within the same meal [72]. Furthermore, dietary fibre from plant sources, such as pectin and guar gum, has been shown to reduce carotenoid absorption [73] and the localisation of carotenoids within the chloroplasts and chromoplasts of plants may decrease bioavailability [74]. The available research investigating the impact of cooking on carotenoids in plant sources suggests that although the heat decreases the carotenoid content, it may nonetheless enhance the bioavailability of the carotenoids compared with uncooked sources [69].
A particular example of a whole food where the bioavailability of L/Z may be higher than that of other sources is eggs. As seen in Table 5, the amount of L/Z in eggs is considerably lower than most L/Z-containing vegetables; however, research suggests the bioavailability of these compounds from eggs is higher than from vegetable sources, most likely due to the fat content [75]. The consumption of one egg per day over five weeks has been shown to increase serum lutein levels by 26% and zeaxanthin levels by 38% [76]. Other research has found the daily intake of three eggs for 12 weeks increased serum lutein and zeaxanthin by 21% and 48%, respectively, in 20 adults with the metabolic syndrome by increasing plasma HDL cholesterol [77].
A number of studies have also assessed the bioavailability of L/Z from eggs including eggs enriched with higher amounts of L/Z compared to standard eggs. One study showed the consumption of lutein-or zeaxanthin-enriched eggs or a lutein-enriched egg-yolk-based buttermilk beverage increased serum lutein levels significantly by 76% and 77%, respectively (p < 0.001) [78]. A high increase in the serum zeaxanthin concentration was also observed in individuals receiving zeaxanthin-enriched eggs: 430% (p < 0.001). These serum increases are comparable with daily use of 5 mg supplements [78].

Conclusions
Current evidence suggests that higher dietary intakes of lutein and zeaxanthin are likely to play an important role in protecting against age-related macular degeneration (AMD). Improvements in understanding and quantifying lutein and zeaxanthin (L/Z) as well as meso-zeaxanthin are needed to determine a recommended target for these macular carotenoids, and this will also help in better understanding their distinct roles in eye health. A diet high in a variety of foods is important for achieving adequate dietary levels of L/Z (as well as other nutrients). Moreover, such a diet should include plenty of leafy green vegetables, in keeping with dietary guidelines. There is also value in including a range of other foods to increase variety and improve the bioavailability of L/Z, such as eggs and selected nuts as part of a healthy dietary pattern. Intervention trials investigating the effectiveness of specific dietary patterns aimed at increasing macular pigment and preventing or delaying the progression of AMD are warranted. In the meantime, prudent advice to increase consumption of lutein-and zeaxanthin-containing foods in the diet of those people at high risk of AMD or who already have AMD should be encouraged.