Nutritional Supplement Use and Age-Related Macular Degeneration

Oxidative stress and the cellular damages caused by reactive oxygen species (ROS) has been implicated in aging and age-related eye diseases 2. Most intracellular ROS are derived from the mitochondria in the electron transport chain. During fuel metabolism, oxygen consumption and ATP synthesis in the mitochondria, electrons are shuffled in sequential reduction and oxidation reactions in the electron transport chain. Yet, these reactions are not 100% efficient; electrons may “leak” out and result in the formation of ROS. ROS are highly reactive and unstable oxygen-containing atoms, ions, or molecules such as hydroxyl radical (OH), superoxide anion (O2-) and hydrogen peroxide (H2O2). Due to the presence of the “unpaired” electron in the outer shell, ROS are very unstable. In trying to achieve stability, ROS will then participate in further reduction and oxidation reactions, oxidizing target molecules and resulting in generation of other free radicals by chain reaction.


Introduction
Age-related macular degeneration (AMD) is a leading cause of irreversible visual impairment and blindness in the aging population 1 .Yet, individuals with AMD have limited treatment options.Given the high prevalence and considerable public health burden, it is essential to understand the etiology and pathogenesis of AMD.
AMD is a multifactorial disease, with complex genetics and confounding environmental risk factors.The etiology of AMD still remains unknown, but oxidative stress to the retina and the retinal pigment epithelium (RPE) is one of the leading hypotheses in AMD pathogenesis.

Oxidative stress and AMD
Oxidative stress and the cellular damages caused by reactive oxygen species (ROS) has been implicated in aging and age-related eye diseases 2 .Most intracellular ROS are derived from the mitochondria in the electron transport chain.During fuel metabolism, oxygen consumption and ATP synthesis in the mitochondria, electrons are shuffled in sequential reduction and oxidation reactions in the electron transport chain.Yet, these reactions are not 100% efficient; electrons may "leak" out and result in the formation of ROS.ROS are highly reactive and unstable oxygen-containing atoms, ions, or molecules such as hydroxyl radical (OH), superoxide anion (O 2 -) and hydrogen peroxide (H 2 O 2 ).Due to the presence of the "unpaired" electron in the outer shell, ROS are very unstable.In trying to achieve stability, ROS will then participate in further reduction and oxidation reactions, oxidizing target molecules and resulting in generation of other free radicals by chain reaction.
Oxidative damages by ROS affect DNA and lipids inside the cell.Earlier senescence, which may be related to shortening of telomeric DNA, occurs after oxidative damage [3][4][5][6] .Oxidative damage also results in point mutations and deletions in mitochondrial DNA 7 .In fact, mitochondrial DNA is more susceptible to ROS-induced damage than nuclear DNA 8 .As for lipids, ROS causes oxidation of lipid in a process called lipid peroxidation.The polyunsaturated fatty acids, a common and significant component of cell membrane, are particularly vulnerable to oxidation by ROS as a result of their many conjugated double bonds.Oxidation of polyunsaturated fatty acids results in the formation of reactive aldehyde intermediates which are toxic to the cell 9 .
The retina is a structure that is particularly susceptible to oxidative damage by ROS.Firstly, the retina has the highest oxygen consumption in the body 10 .In addition, constant exposure to incoming light in the retina can lead to photo-oxidation.The high oxygen consumption and high light exposure in the retina may in turn generate ROS.Moreover, the retina has a high lipid content, with abundant polyunsaturated fatty acids in the photoreceptor outer segments which are most prone to lipid peroxidation.In the neighborhood of photoreceptors are the RPE cells.Besides providing metabolic support to the photoreceptors, they also phagocytose the constantly shed parts of the photoreceptor outer segments.All these factors contribute to the susceptibility of the retina and RPE to oxidative stress.
With age, the susceptibility to oxidative damage in the retina increases.Aged rat retina showed decreased GSH-Px and catalase activities, which are related to increased lipid peroxidation with age 11 .In particular, RPE cells accumulate lipofuscin granules during life.Lipofuscin granules are lysosomal residual bodies containing undigested end products from phagocytosis of photoreceptor outer segments 12 .It was estimated that lipofuscin can occupy up to 19% of RPE cytoplasmic volume by the age of 80 when compared with only 1 % during the first decade of life 13 .Lipofuscin has been shown to contain toxic substances, such as retinoids (products of the visual cycle) and oxidized proteins 14 .Lipofuscin was also able to reduce RPE lysosomal and antioxidant activity 15 .In vitro studies using porcine RPE cells showed that visible light irradiation can degrade RPE melanosomes, reduce melanin amount and increase ROS production, changes that also occur in human RPE melanosomes with aging 16 .

Nutritional supplements and AMD
Oxidative stress has a recognized role in aging and AMD; treatments for AMD are therefore aimed at reducing oxidative stress-induced damage within the retina and RPE cells.This can be approached in two ways, either by decreasing the source of oxidative stress or by increasing the defense against oxidative stress.Among them, a tempting measure in lowering oxidative damage would be by dietary antioxidant supplementation.Data from observational studies have supported a link between nutritional factors with antioxidant properties and risks of AMD 17,18 .Carotenoids, vitamin C and vitamin E with their antioxidant properties have been identified as having a potentially protective role.Other nutrients such as zinc and omega-3 fatty acids have been shown to be associated with reduced risk of AMD.Recently, B vitamins (folic acid, B 6 and B 12 ) have also been proposed to provide protection by a non-oxidative mechanism.Another nutritional supplement that has gained interest recently is the extracts from a group of fruit, berries.

AREDS and AREDS-type formulation
The Age-Related Eye Disease Study (AREDS) was a clinical trial sponsored by the National Eye Institute 19 .This was to date the largest prospective randomized controlled trial to investigate the effect of an active supplement formula on the risk of development of AMD.The dosages of the supplements were at a high-than-normal level, because it was considered a form of active treatment, instead of a simple supplement pill.There were a total of 3,640 subjects, being monitored for an average of 6.3 years.Each subject was given either the AREDS formula, or placebo, to be taken on a twice-daily basis.Main components of the AREDS formula are vitamin A, vitamin C, vitamin E, and zinc.These were chosen because of their anti-oxidative abilities [20][21][22][23][24][25] .When compared with the Dietary Reference Intake (DRI) issued by the Institute of Medicine, US National Academy, the dosage of ingredients in the AREDS formula was at a much higher level 26 .For instance, the dosage of vitamin C in the AREDS formula was 500 mg/day, while that of the DRI was only 90mg per day.As far as vitamin C was concerned, one has to take at least 7 to 8 oranges per day, just to match up with what is provided by the AREDS pill 27 .A comparison of the dosage in AREDS formulation with common fruits is given in Table 3.  28 .Hence, the protection offered by the AREDS formula may not be applicable to all.Therefore, it was only recommended to high-risk individuals (those under category 3 or 4).
Risk associated with regular intake of the AREDS formula was of particular concern, mainly because it was meant for long-term use.In particular, the risk of regular intake of such a high level of vitamins and minerals was unknown.However, observations from the AREDS cohort failed to show any statistically significant serious side effects as mentioned above.Documented minor side effects included 1) increased genitourinary symptoms; 2) increased self-reported anemia; and 3) yellow discoloration of skin due to high level of vitamin A. Self-reported anemia was not correlated with any genuine reduction in blood hematocrit level.Smokers in the AREDS were discouraged from smoking, therefore whether the risk of lung cancer was increased was not being addressed.However, this has already been confirmed in two other trials 29,30 .Hence, all smokers should be discouraged from smoking before the commencement of the AREDS formula.If he or she is not willing to quit smoking, the risk of having lung cancer may outweigh the potential benefit in AMD protection.
In general, the AREDS formula was deemed safe and effective, in selected high-risk individuals 31 .Inadequacy of the AREDS formula was that it did not include other potential ingredients such as lutein, zeaxanthin, and omega-3 fatty acid, which are also of particular interest due to their antioxidant abilities.In view of this, the National Eye Institute has launched the Age-Related Eye Disease Study 2 (AREDS2) in 2006, in hope to fill up the knowledge in this gap 32,33 .In the AREDS2 formula, lutein, zeaxanthin, and omega-3 fatty acid have been added to the existing AREDS formula, and vitamin A was removed, mainly due to the potential risk associated with lung cancer.Results of the AREDS2 are expected to be available in 2012.Until then, the AREDS formula remains the only evidenced-based formula to reducing the risk of development of advanced AMD.

Carotenoids (β-carotene, lutein and zeaxanthin)
Carotenoids are organic pigments naturally occurring in plants as well as in some algae, fungus and bacteria.Animals generally cannot synthesize carotenoids; they have to obtain carotenoids in their diet.There are two classes of carotenoids, xanthophylls (which contain oxygen) and carotenes (which are purely hydrocarbons, and contain no oxygen) accounting for over 600 known carotenoids.A well known carotene is beta-carotene, the pigment that makes carrots orange.Interestingly, there are only two carotenoids that are present in the human retina 34,35 , namely lutein [(3R,3'R,6'R)-beta,epsilon-Carotene-3,3'-diol] and its stereoisomer, zeaxanthin [(3R,3'R)-beta,beta-Carotene-3,3'-diol].These carotenoids are enriched in the macula in high concentrations, thus giving the macula its yellowish color.
In human, four carotenoids including beta-carotene, alpha-carotene, gamma-carotene, and beta-cryptoxanthin can be converted into retinal, which is an important molecule in the photo-transduction pathway and therefore vision.Carotenoids can also absorb light and act as antioxidants by scavenging ROS such as .O2 and peroxyl radicals 36 .In particular, two xanthophylls, lutein and zeaxanthin, have been shown to absorb the damaging blue light 36 as well as protect the retina 37 and retinal ganglion cells 38 from oxidative damage in vitro.In animal studies, lutein protected the inner retina against acute retinal ischemia/reperfusion injury due to its antioxidant properties 39 .
Due to their antioxidant properties and blue light-filtering effects, the association of carotenoids with risk of AMD was explored.There have been conflicting results.Decreased risk of neovascular AMD has been found to be associated with higher levels of carotenoids in the serum samples 40 .In monkeys, feeding a xanphophyll-free diet has been shown to promote drusen formation 41 .In an early study based on National Health and Nutrition Examination Survey I data, an inverse association between the consumption of fruits and vegetables rich in pro-vitamin A carotenoids and the prevalence of AMD was demonstrated 22 .In the Beaver Dam Eye Study, VandenLangenberg et al also found a significant but modest inverse association between intake of pro-vitamin A carotenoids and the incidence of large drusen 23 .Later studies using the AREDS formulation suggested a beneficial effect of beta-carotene 19 .The Rotterdam population-based study also reported a high dietary intake of beta-carotene together with vitamins C and E and zinc reduced the risk of AMD in elderly individuals 42 .A 35% reduced risk of AMD was observed when an above-median intake of these 4 nutrients was given.
On the other hand, opposing results were obtained from other clinical trials and populationbased studies.The Alpha-Tocopherol and Beta-Carotene (ATBC) Study in Finland assessed the involvement of beta-carotene in occurrence of AMD among smoking males 43 .Over 29,000 smoking males aged 50 to 69 years were given alpha-tocopherol (50 mg/day), betacarotene (20 mg/day), both of these, or placebo randomly.After 5 to 8 years of supplementation, Teikari et al found no beneficial effect of long-term beta-carotene supplementation on the incidence of AMD.The Blue Mountains Eye Study also reported no associations between beta-carotene intake and 5-year incidence of AMD 44 .This is a population-based study including 1,989 individuals who finished a food frequency questionnaire.This questionnaire assessed the baseline intake of nutrients including alphacarotene, beta-carotene, beta-cryptoxanthin, lutein and zeaxanthin, lycopene, retinol, vitamin A, vitamin C, and zinc.For beta-carotene, Teikari et al suggested no evidence of protection by beta-carotene on the 5-year incidence of AMD.Further studies in the same population after 10-year of follow-up showed some interesting results.Instead of showing no effect of beta-carotene in AMD, Tan et al actually reported an increased risk of neovascular AMD with increasing beta-carotene intake 45 .The authors found that increasing beta-carotene intake, either from diet alone or diet plus supplementation, was associated with higher risk neovascular AMD.This association also existed when the smoking status of the individuals was adjusted.
I n f a c t , o n e h a s t o b e a r i n m i n d a b o u t t h e possible harmful effect of beta-carotene supplementation.Apart from the skin coloration, changes in scotopic b-wave during electroretinography and crystal formation have also been shown with long-term betacarotene use 46 .More importantly, daily supplementation of beta-carotene in smokers was associated with a higher mortality rate due to ischemic heart disease and lung cancer 29,30 .
Since smoking also increases the risk of AMD, beta-carotene supplementation should be avoided in smokers.Currently, no biological explanation has been offered to clarify the harmful effect of beta-carotene in human.
Lutein and zeaxanthin are the only two carotenoids that exist in the human retina 34,35 .They are particularly dense in the macula in humans, where they are referred to as macular pigment 34 .Macular pigment is thought to be protective against retinal damage.Three casecontrolled studies showed that there was an inverse association between the macular pigment density in the human retina and the risk of AMD [47][48][49] .In an early study investigating the effects of high dietary carotenoid intake, lutein and zeaxanthin were found to be the specific carotenoids that are most strongly associated with reduced risk of AMD 20 .This result was also supported by two other studies.The population-based Pathologies Oculaires Liees a l'Age (POLA) Study measured the plasma carotenoid levels by highperformance liquid chromatography (HPLC) in 899 subjects and correlated them with the risk of AMD 50 .It was shown that high plasma levels of lutein and zeaxanthin were associated with a significant reduced risk of AMD.Similarly, a study in U.K. involving men and women aged 66 to 75 found that subjects with the lowest plasma level of zeaxanthin has a two-fold increased risk when compared with those with the highest plasma zeaxanthin, supporting the view that zeaxanthin may protect against AMD 51 .
Other studies also provide evidence in the association of lutein and zeaxanthin with AMD risk.In the Blue Mountains Eye Study, Flood et al reported a possible association between baseline intake of lutein and zeaxanthin and the 5-year incidence of early AMD 44 .A longer, 10-year follow-up study reported that high dietary lutein and zeaxanthin intake (top tertile) was associated reduced risk of incident neovascular AMD 45 .Participants with above median intakes had a reduced risk of indistinct soft or reticular drusen.
Conversely, several studies showed different results on the association of lutein and zeaxanthin.An early study in Beaver Dam (Beaver Dam Eye Study) reported no significant association between lutein and zeaxanthin and the risk of large drusen when 1,709 participants were followed up for 5 years 23 .In a prospective follow-up study of women in the Nurses' Health Study and men in the Health Professionals Follow-up Study, Cho et al followed 77,562 women and 40,866 men ≥50 years old for up to 18 years for women and up to 12 years for men.It was reported that lutein and zeaxanthin were not strongly related to either early or neovascular AMD risk 52 .The Carotenoids in Age-related Eye Disease Study (CAREDS), an ancillary study of the Women's Health Initiative, followed 1,787 female participants aged 50 to 79 for 4 to 7 years 53 and assessed their diet by a food frequency questionnaire.Subjects were divided according to their lutein and zeaxanthin intake, but there was no statistical difference between the amount of lutein and zeaxanthin intake and the prevalence of intermediate AMD.A later large prospective follow-up study also reported similar results 54 .Two cohorts, the Nurses' Health Study and the Health Professionals Follow-up Study which included 51,564 men and 71,494 women aged ≥50 years were followed up for up to 18 years.Cho et al reported that there was no association between lutein/zeaxanthin intake and the risk of self-reported early AMD.Yet, a nonsignificant and nonlinear inverse association between lutein/zeaxanthin intake and neovascular AMD risk was observed.
More recently, lutein itself has gained special interests.Two prospective randomized controlled trials have investigated the association of lutein supplementation and the incidence of AMD.The larger Veterans LAST study (Lutein Antioxidant Supplementation Trial) involved 90 subjects with atrophic AMD who were randomly divided into three groups: lutein (10mg) group, lutein (10mg) plus additional antioxidants and nutrients group, and maltodextrin placebo group 55 .Subjects were followed for 12 months and those who received lutein alone or lutein plus antioxidants and nutrients had improved visual acuity.Richer et al concluded that lutein alone or in combination with other nutritional supplements (including zinc, beta-carotene and vitamins C and E) is protective and slow down the progression of AMD.On the other hand, a smaller prospective trial measured the contrast sensitivity in 25 subjects after lutein supplementation (6mg) with vitamins and minerals or placebo over a 6-month period 56 .No statistical difference was observed between the lutein and placebo group, suggesting no significant association between lutein supplementation and AMD.However, one has to be careful about these findings.The sample sizes in both studies were fairly small and the follow-up periods were limited to 12 months or less.
More supportive evidence came from a recent study in which participants in AREDS were genotyped for the hepatic lipase (LIPC) gene 57 .Hepatic lipase is a protein in the highdensity lipoprotein cholesterol pathway and has been shown in a large genome-wide association study to be a novel locus for advanced AMD risk 58 .It was observed in the AREDS participants that lower dietary lutein intake was significantly associated with increased risk of advance AMD, after controlling for the LIPC genotype.This suggests that high dietary lutein intake may reduce the risk of advanced AMD, after adjusting for genetic variants.
Lutein is also a macular pigment.Due to lutein's antioxidant properties and blue-light filtering capacity 36 , it was hypothesized that macular pigment may provide protection against the development of AMD 59 .The first prospective follow-up study, Muenster Aging and Retina Study (MARS), recently investigated the determinants of macular pigment optical density and its relation to AMD 60 .Foveal macular pigment optical density was accessed in 369 participants including patients with different stages of AMD and healthy controls.In the 2.6-year follow-up study, it was observed that serum level of lutein, lutein supplementation in particular, was the strongest determinants of macular pigment optical density.However, the hypothetical protective effect of macular pigment in AMD could not be confirmed.On the other hand, a recent double-masked controlled study, Lutein Intervention Study Austria (LISA), investigated the association of 6-month lutein supplementation with macular pigment optical density and visual acuity in 126 AMD patients randomly assigned to lutein supplementation or placebo 61 .Weigert et al observed that lutein could significantly increase macular pigment optical density despite having no effect on mean differential light threshold or visual acuity.Interestingly, a significant correlation was found between the lutein-induced increase in macular pigment optical density and the change in mean differential light threshold and visual acuity.This finding suggests that patients who experience a pronounced increase in macular pigment optical density after lutein supplementation may benefit in terms of visual function.
As lutein and zeaxanthin were not ready for manufacturing as a research formula, neither of them was included in the AREDS formula 28 .The US Food and Drug Administration (FDA) has conducted an evidence-based review to evaluate the role of lutein and zeaxanthin in reducing the risk of AMD 62 .After reviewing a number of intervention and observational studies, the FDA denied a health claim about the intake of lutein or zeaxanthin (or both) and the risk of AMD in 2006.However, in view of the conflicting findings, the National Eye Institute (Bethesda, Maryland, USA) launched the Age-Related Eye Disease Study 2 (AREDS2) in 2006, hoping to resolve the link between carotenoids (lutein and zeaxanthin) intake and AMD protection 32,33 .The AREDS2, a large, multi-centered, randomized trial, is currently underway to address the effects of high dose lutein and zeaxanthin supplementation and/or omega-3 fatty acids on the progression of AMD.Beta-carotene, which increases the risk of lung cancer in smokers 29,30 , is removed from the AREDS2 formula.Another on-going, similar randomized controlled trial is the Carotenoids in Age-Related Maculopathy (CARMA) Study 63 .In this study, 433 participants with either early AMD features or any level of AMD in one eye and advanced AMD in the fellow eye were recruited.Either lutein and zeaxanthin, in combination with antioxidants (including vitamin C, vitamin E, zinc, and copper) or placebo was given.Again, beta-carotene was excluded in the preparation due to the increased risk of lung cancer in smokers 29,30 .
Although the beneficial effects have not been proven, lutein and zeaxanthin are included in daily supplements and food additives and can be obtained over the counter.Moreover, the addition of crystalline lutein into food and beverage products is considered GRAS (generally recognized as safe) and is approved by the FDA 64 .Lutein toxicity studies in animals using high doses of purified crystalline lutein revealed no unfavorable events 64 and no adverse events are reported for lutein and zeaxanthin at doses up to 40 mg/day in human for 2 months 65 .The risk profile of lutein was also recently reviewed in 2006 by the Council for Responsible Nutrition (CRN) in Washington, D.C.It was concluded that apart from the reversible skin discoloration, no other adverse effects were observed 66 .The CRN suggested an upper level of intake for lutein up to 20 mg/day.Currently, the average daily intake for lutein and zeaxanthin is 2.0-2.3 mg/day for men and 1.7-2.0mg/day for women in United States (Food and Nutrition Board, 2001).
In view of their potential benefits as well as minimal side effects, lutein and zeaxanthin may be recommended for those who are keen and at risk of AMD 27 .

Vitamin C (L-ascorbic acid)
Vitamin C is a water-soluble nutrient that is synthesized in almost all animals and plants.It is well known for its potent antioxidant activities 67,68 .It also acts as an important co-factor in mammals as in the synthesis of collagen; therefore vitamin C is used in the treatment and prevention of scurvy.In ophthalmology, there has not been any randomized controlled trial in assessing the efficacy of vitamin C as a single supplement in AMD.Yet, in other studies combining vitamin C with other supplements, data on the protective effects of vitamin C has been mixed.Vitamin C is shown to be beneficial in the AREDS study 19 .In two large prospective studies of 135 men and 329 women with up to 18 years of follow-up 52 , it was found that higher fruit intake was related to a reduced risk of neovascular age-related maculopathy but none of the vitamins (including vitamin C) or carotenoids examined was clearly related to the disease.In a population-based cohort study involving 1,586 middleaged and older adults, the researchers found no significant associations between the risk of large drusen and intake of vitamin C 23 .Another population-based cohort study even suggested that an increasing baseline vitamin C intake from diet and supplements was associated with an increased risk of incident early age-related maculopathy when compared with the lowest quintile 44

Vitamin E (α-tocopherol)
Vitamin E is a collective term for a group of natural lipid-soluble compounds containing the tocopherols (α-, β-, γand δ-) and tocotrienols (α-, β-, γand δ-) with antioxidant properties.Among them, α-tocopherol is the only form to meet human requirements.In the eye, αtocopherol can be found in the retina, RPE and choroid 69 .Its concentration in the retina increases after oral supplements 70 .
As an antioxidant and a nutritional factor, vitamin E has been explored in its association with prevention of AMD.Again, data for vitamin E have been mixed.Some studies reported that higher intake are associated with lower risks of AMD or signs 23,42,71 whereas some concluded no associations 45,52,72,73 .
In particular, three large randomized controlled trials have assessed vitamin E in the incidence of AMD.The Alpha-Tocopherol and Beta-Carotene (ATBC) Study involved over 29,000 smoking males aged 50 to 69 years who were randomly assigned to alphatocopherol (50 mg/day), beta-carotene (20 mg/day), both of these, or placebo 43 .Of these, an end-of-trial ophthalmological examination was performed in a random sample of 941 participants aged 65 years or more.No beneficial effect of long-term supplementation with alpha-tocopherol on the occurrence of AMD was detected among smoking males.In the Vitamin E Cataract and Age-related Maculopathy Trial (VECAT), 1,193 healthy volunteers aged between 55 and 80 years were randomly given either vitamin E (500IU = 335 mg) or placebo daily for 4 years 74 .In the study, the incidence of early AMD in those receiving vitamin E (8.6%) was similar to those on placebo (8.1%) whereas for late disease the incidence was 0.8% versus 0.6%.Again, daily vitamin E supplement does not prevent the development or progression of early or later stages of AMD.In the Women's Health Study (WHS) 75 , a large scale randomized trial of women, 39,876 healthy female health professionals were randomly assigned to receive with natural source vitamin E (600IU) or placebo on alternate days.There were 117 AMD cases in the vitamin E group versus 128 cases in the placebo group after 10 years of treatment and follow-up.Similar to other studies, no large beneficial or harmful effect on risk of AMD was observed in long term vitamin E supplementation.

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Nutritional Supplement Use and Age-Related Macular Degeneration 197 More importantly, a negative association between vitamin E and AMD was recently reported.In the Blue Mountains Eye Study involving an Australian population-based cohort, Tan et al reported that high vitamin E intake was associated with increased risk of late AMD, suggesting a harmful effect of dietary vitamin E on risk of AMD 45 .However, one has to be cautious about these results.There was a moderate loss of participants in this particular study, while the levels of vitamin E intake between participants followed up and not followed up were significantly different.The authors mentioned that this might affect the interpretation of the observed results.

Zinc
Zinc is an essential trace element for almost all organisms including plants, animals and microorganisms.It has a multitude of biological roles, playing a fundamental role in cellular metabolism.For example, it plays a structural role in a large number of transcription factors containing zinc fingers and similar structural motifs.Most importantly, it was first shown to be required for the catalytic activity of carbonic anhydrase 76 .Later studies showed that zinc has a catalytic or structural role in at least 300 zinc metalloenzymes [77][78][79] , influencing many metabolic reactions.In fact, approximately 10% of the human genome encodes for proteins that can bind zinc 80 .
In the human body, there are about 2-3 g of zinc, making it the second most abundant trace element 79,81 .In ocular tissues, the concentration of zinc is unusually high when compared with other tissues 82 .In the eye zinc is most abundant in the retina and choroid, followed by ciliary body, iris, optic nerve, sclera, cornea, and lens 83 .A number of functions of zinc in the retina have been suggested, including modulation of retinal synaptic transmission, modification of photoreceptor plasma membrane, involvement in retinal vitamin A metabolism, regulation of light-rhodopsin reaction within the photoreceptor, and antioxidant activity 84,85 .
There are subtle ocular manifestations associated with zinc deficiency.In a prospective, randomized, double-masked, placebo-controlled investigation of the effects of oral zinc administration on the visual acuity outcome in 151 subjects with drusen or macular degeneration, the treatment group had significantly less visual loss than the placebo group 24 .As elderly patients are found to be at higher risk of zinc deficiency 86 , this may suggest an increased risk of vision loss from AMD in elderly patients.
For the past three decades, there have been considerable interest and controversy related to zinc supplementation in AMD patients.To date, results on zinc supplementation and AMD have been mixed.As described above, Newsome et al reported significant reduction in visual loss in AMD patients when supplemented with oral zinc 24 .Moreover, Mares-Perlman et al reported a weak protective effect of dietary zinc on the development of some forms of early AMD 71 .In the large double-masked clinical trial, The Age-Related Eye Disease Study (AREDS), involving 11 centers, participants taking zinc alone demonstrated an odds reduction of 0.75 for the development of advanced AMD.Zinc significantly reduced the odds of developing advanced AMD in the higher-risk group.A population-based cohort study reported that high dietary zinc intake was associated with a lower risk of incident AMD 42 .In the Beaver Dam Eye Study, it was observed that there is a significant inverse association between zinc and the incidence of pigmentary abnormalities, but there was no relationship between zinc intake and incidence of early AMD 23 .In fact, an early study by the Eye Diseases Case-Control Study Group reported no association between serum zinc levels and risk of neovascular AMD 40 .In a 2-year, double-masked, randomized, placebocontrolled study, Stur et al reported that oral zinc substitution has no short-term effect in patients who have an exudative form of AMD in one eye 87 .Unfortunately, this study was prematurely terminated because of no beneficial effects found in first 40 patients at 24 months.In addition, two large prospective studies involving 66,572 women and 37,636 men do not support a lowered AMD risk associated with higher zinc intake 88 .The Blue Mountains Eye Study Group reported no significant association between baseline zinc intake from diet or supplements and the 5-year incidence of early Age-related maculopathy 44 .
A systematic review and meta-analysis involving four prospective cohort studies 23,42,44,88 reported that a pooled odds ratio of zinc for early AMD was 0.91 (95% CI 0.74 to 1.11).Another meta-analysis reported that zinc supplementation can slow down AMD progression (adjusted odds ratio = 0.77, 95% CI 0.62 to 0.96) 89 .
Although the evidence is conflicting, recent studies support a protective role of zinc in AMD progression.The AREDS study indicated that the beneficial effect of zinc supplementation was of a similar order to that of vitamin supplementation.Despite the 5-year findings by The Blue Mountains Eye Study Group 44 , later studies by the same group published the 10year data in which individuals with total zinc intake in the highest decile are less likely to develop early or any AMD 45 .
Zinc intake and the genetic risk of AMD has also been assessed.In the AREDS population, the single nucleotide polymorphism in the CFH (Y402H, rs1061170) and LOC387715/ARMS2 (A69S, rs10490924) genes of 876 participants who were considered at high risk was genotyped 90 .The findings suggest that there is an interaction between CFH genotype and treatment with antioxidant plus zinc when compared with placebo.Moreover, a recent study involving 2,167 individuals from the population-based Rotterdam Study at genetic risk of AMD assessed their dietary intake at baseline using a semi-quantitative food frequency questionnaire and determined the genetic variants using TaqMan assay 91 .In this nested case-control study, it was observed that there is a significant possibility of biological interaction between CFH Y402H and zinc as well as between LOC387715 A69S and zinc (p < 0.05).Moreover, individuals with homozygous CFH Y402H with dietary intake of zinc in the highest tertile reduced their hazard ratio of early AMD from 2.25 to 1.27.
Again, one has to be cautious about the risks of high dose supplementary intake of zinc.In the AREDS study, more people in the zinc group reported difficulty in swallowing the tablets (17.8% vs. 15.3%, p < 0.04) 19 .Circulatory adverse experiences were also more frequently reported in individuals receiving zinc.Hospitalizations due to genitourinary problems as well as mild or moderate symptoms are also more frequent in these participants.In fact, it was found that there is a significant increase in hospital admissions for urinary complications in patients with high zinc supplementation (11.1% vs 7.6%, p = 0.0003) 92 .The risk was greatest in male patients (RR 1.26, 95% CI 1.07-1.50,p = 0.008).Significant increase in urinary tract infections was also found (p = 0.004), especially in females.Another problem was gastrointestinal symptoms.Of 286 participants, 5/146 zinc-treated participants withdrew from the studies due to gastrointestinal symptoms when compared with 2/140 in the placebo group 24,87 .

Omega-3 Long chain polyunsaturated fatty acids
The retina contains abundant fatty acids, about 30% of which are polyunsaturated fatty acids 93 .Polyunsaturated fatty acids are classified into 2 groups: ω-3 and ω-6 depending on the position of the first double bond from the methyl end of the molecule.Docosahexaenoic acid (DHA), an omega-3 fatty acid, is highly enriched in the retina, particularly in the disc membrane of photoreceptor outer segments 94 .DHA is the major polyunsaturated fatty acid in cerebral gray matter as well.Yet, the specific role of DHA in the eye is not clear.DHA has been shown to be important for photoreceptor survival [95][96][97][98] .DHA may have a role in modulating G protein-coupled signaling pathways that are involved in visual transduction 99 .DHA may also affect rhodopsin function during photoreception by influencing the membrane's biophysical properties 100,101 .In rhesus monkeys, dietary depletion of alphalinolenic acid, a dietary precursor of DHA, resulted in undetectable plasma DHA level and more importantly, abnormal retinogram and visual impairment 102,103 .Nonetheless, DHA supplementation is effective in improving retinal function in a patient with autosomal dominant Stargardt-like retinal dystrophy 104 .The importance of DHA in retinal function suggest a possible beneficial role of DHA in retinal disease such as AMD.
Another omega-3 fatty acid, eicosapentaenoic acid (EPA), is the precursor of eicosanoids in the body.It can act as a competitive inhibitor of arachidonic acid conversion to proinflammatory eicosanoids prostaglandin E(2) and leukotriene B(4) 105 .As inflammation plays a role in the pathogenesis of AMD, EPA may be one of the protective factors in AMD.
Supplementation of omega-3 fatty acids, DHA and EPA in particular, has received much interest in association with lowering the risk of AMD.Although DHA can be synthesized from alpha-linolenic acid in the body, the process is ineffective.DHA and EPA can readily be obtained from marine fish oils in the diet.Based on their roles in retinal function and inflammation, dietary modification and supplementation of omega-3 fatty acids have become attractive alternatives in lowering the risk of AMD.
Many studies have provided evidence for a protective role of omega-3 fatty acids supplementation in AMD risk 91,[106][107][108][109][110][111][112][113][114][115][116][117] .The first study evaluating the relationship between dietary fat and AMD was published by Mares-Perlman et al 106 .They reported that high intake of saturated fat and cholesterol was associated with increased risk for early AMD.Later, a prospective follow-up study of participants in the Nurses' Health Study and the Health Professionals Follow-up Study showed that total fat intake was positively associated with increased risk of AMD 108 .Yet, a cross-sectional study involving participants in the Third National Health and Nutrition Examination Survey found no association between dietary fat and AMD risk.However, this study assessed only one eye per patient, thereby may have decreased the observed AMD prevalence.
There are further investigations into the association of omega-3 fatty acids with AMD risk.As dietary omega-3 fatty acids are obtained from marine fish oils, fish intake was also investigated.Earlier study on fish intake was performed in the Blue Mountain Eye Study population.In this cross-sectional, population based study, Smith et al showed that a higher www.intechopen.com Age Related Macular Degeneration -The Recent Advances in Basic Research and Clinical Care 200 fish consumption was associated with decreased odds of late AMD 107 .After 5 years of follow-up Chua et al reported that fish consumption at least once a week was protective against early AMD, whereas fish consumption at least 3 times per week could reduce the incidence of late AMD 110 .After 10 years of follow up in the same cohort, Tan et al suggested that a regular weekly serving of fish was associated with a reduced risk of early AMD 116 .Interestingly, it was also noted that fish consumption of more than one serving per week did not have a significant protective effect in reducing AMD risk in this cohort, suggesting a threshold effect.These findings are supported by other studies as well.Seddon et al in a multicenter eye disease case-control study reported that higher intake of omega-3 fatty acids and fish was associated with a lower risk for AMD among individuals with low linoleic acid intake 109 .More evidence on the protective role of omega-3 fatty acid came from a recent US Twin Study of Age-Related Macular Degeneration.This study investigated the association between dietary fat intake and fish consumption and risks of AMD in 681 twins 111 and found that both omega-3 and fish intake reduced the risk of AMD.
Oily fish rich in omega-3 fatty acids are also found to be beneficial in two European studies.
The population-based POLANUT study from Southern France found that fatty fish intake was protective against AMD when comparing more than once a month and less than once a month and after multvariate adjustment 112 .Interestingly, total and white fish intake has no significant association with AMD risk.Another population-based study, EUREYE, showed that oily fish intake (at least once per week versus less than once per week) was associated with significant reduction of risk for neovascular AMD 114 .Similar findings were also observed for either DHA or EPA intake.
Among the AREDS participants, a prospective cohort of individuals with neovascular AMD and central geographic atrophy was also analyzed for the relationship of omega-3 fatty acids and AMD.It was observed that dietary total omega-3 fatty acids or DHA intake was inversely associated with neovascular AMD 113 .Similar findings were also observed with fish consumption.Further studies showed that dietary omega-3 fatty acids intake is associated with a decreased risk of progression from bilateral drusen to central geographic atrophy 115 .
In addition, the association between omega-3 fatty acids and genetic risk of AMD is investigated.In the Blue Mountains Study group, 1881 participants were genotyped for complement factor H (CFH) genetic variants 117 .Wang et al reported that AMD risk increased with each additional C allele.Also, weekly compared with less than weekly consumption of fish was associated with reduced late AMD risk in participants with the CC genotype but not the CT or TT genotypes.This study provided evidence that weekly consumption of fish is protective on the development of late AMD, but not early AMD, among individuals with genetic susceptibility to AMD due to the Y402H variant.On the other hand, the dietary intake of 2167 individuals was assessed at baseline in a recent population-based Rotterdam study 91 .Ho et al reported a possible interaction between EPA/DHA and either CFH Y402H or LOC387715 A69S.The authors also suggested that high dietary intake of omega-3 fatty acids may reduce the risk of early AMD in those who are at high genetic risk.
Taken together, much data suggests that dietary omega-3 fatty acids intake and fish consumption are protective against AMD.Results from a recent meta-analysis also www.intechopen.com Nutritional Supplement Use and Age-Related Macular Degeneration 201 supported the protective role of omega-3 fatty acids supplementation 118 .It was reported that dietary intake of omega-3 fatty acids was associated with reduced risk of late AMD while fish consumption (at least twice a week) was associated with reduced risk of both early and late AMD.However, the authors also cautioned that due to insufficient evidence, few prospective studies and no randomized clinical trials, recommendation for a routine omega-3 fatty acids supplementation and fish consumption for AMD prevention is not supported.A similar conclusion was also reached in another systematic review 119 .
Hopefully, more definite answers on the protective role of omega-3 fatty acids will be provided by the ongoing AREDS2 randomized, multi-center trial.

B vitamins
B vitamins are a group of water-soluble compounds that are important in cell metabolism.
The members of interest in AMD studies are folic acid, vitamin B 6 (pyridoxine) and vitamin B 12 (cyanocobalamin) because of their ability to reduce homocysteine levels in intervention studies 120 .Homocysteine is an amino acid formed during the metabolism of methionine.It can either be recycled back into methionine or converted into cysteine with the help of Bvitamins.
It was therefore proposed that lowering blood homocysteine levels with folic acid, vitamin B 6 and vitamin B 12 supplementation may help to reduce the risk of AMD.
In the Women's Antioxidant and Folic Acid Cardiovascular Study (WAFACS), 5,442 female health professionals participated in this randomized, double-masked, placebo controlled trial 132 .Christen et al reported that daily supplementation with folic acid/B 6 /B 12 reduce the risk of AMD in this large cohort of females after an average of 7.3 years of treatment and follow-up.Yet, disease report in this study was done by self-report questionnaires or medical records while no ophthalmic examinations were performed.More evidence and further research in other groups are needed despite the interesting association between folic acid/B 6 /B 12 supplementation and AMD prevention.

Berry extracts
Diets rich in fruits, nuts, and vegetables have long been considered to be an excellent source of antioxidants.There has been growing interest on berry extracts due to their high antioxidant properties.Among the berries, blueberries have been of specific interest because of their high antioxidant capacity (in some cases as high as 40−50 μmol Trolox equivalents/g) 133 .Indeed, of all the fresh fruits and vegetables tested to date, data indicate that blueberries have the highest antioxidant capacity, as estimated using the average oxygen radical absorbance capacity (ORAC) values [133][134][135] .Polyphenols in blueberries, specifically the anthocyanins that give the fruit its blue color, are the major contributors to antioxidant activity 133 . www.intechopen.com Age Related Macular Degeneration -The Recent Advances in Basic Research and Clinical Care 202 Anthocyanin is a water-soluble pigment present in all plants and is richly concentrated in berries.It is a powerful antioxidant in vitro 136 .It can absorb blue-green light and protects the cells from light stress in plant studies 137 .In laboratory studies, anthocyanin may protect the eyes from degenerative diseases such as AMD [138][139][140] .Yet, the evidence for the potential health effects of anthocyanin is mostly laboratory-based 141 .
Another berry that recently received lots of interest is the fruit of Lycium barbaurm, also called wolfberry or Gouqizi, a commonly used herb in Chinese Traditional Medicine.It is also taken as food in Asian countries.It is well known for improving eye sight.Increasing lines of evidence showed that the polysaccharides in Lycium barbaurm can exhibit anti-aging 142 and anti-oxidative effects 143 .Other properties such as anti-tumor effects, cytoprotection, neuromodulation, and immune modulations have also been suggested 142,144 .Unfortunately, most evidence for its beneficial effects is limited to the laboratory level.
At this moment, there are no legal requirements for quality control in the preparation of these extracts.It is not obligatory to disclose the content and the production method.Moreover, the dosage and frequency are unclear while potential toxicity and long-term side effects remain to be investigated.A lot of investigation is needed before the potential of berry extracts in prevention of AMD can be hinted.Currently, berry extracts should not be recommended 27 .

Future directions
Observational studies have shown beneficial effects from dietary supplementation of lutein and zeaxanthin as well as omega-3 fatty acids in the development of AMD.They are currently tested in AREDS2, the multi-centered randomized clinical trial launched by the National Eye Institute in 2006.The association of oral formulations containing lutein and zeaxanthin, and/or DHA and EPA, with the progression of AMD is being assessed.In AREDS2 participants will be followed for 5 years.Hopefully, data will be available by the end of 2012.Similarly, the ongoing CARMA study will also provide invaluable data on the protective effects of lutein and zeaxanthin in combination with antioxidants (vitamin C, vitamin E and zinc) with the exclusion of DHA and EPA.

Conclusions
To date a large body of evidence has supported a protective role of nutritional supplements in the development and progression of AMD.In particular, strongest evidence is present for the protective effect of lutein, zeaxanthin, DHA, and EPA.On the other hand, beta-carotene and vitamin E may have detrimental effects.While awaiting a further proof of the effects of lutein, zeaxanthin, DHA, and EPA, the AREDS formulation remains the best recommendation so far, although not without risk and maybe only for high-risk individuals.One concern for the AREDS formulation is the higher risk of lung cancer in smokers with daily beta-carotene supplementation.Therefore, in offering nutritional supplements to patients, physicians should consider on a case-by-case basis and fully explain the potential side effects from a long-term regular intake.It is also important to remind the patients that even with the AREDS formulation, AMD can still occur.It is equally important to teach the patients self-monitoring methods such as usage of the Amsler grid.Regular fundal examinations by ophthalmologists should also be strongly encouraged.

Table 2 .
Studies investigating nutritional supplements in the progression of AMD . Studies investigating nutritional supplements in the prevention of AMD

Table 4 .
Categorization of AMD according to AREDS guidelines