A Mechanistic Review of β-Carotene, Lutein, and Zeaxanthin in Eye Health and Disease

Carotenoids are natural lipid-soluble antioxidants abundantly found as colorful pigments in fruits and vegetables. At least 600 carotenoids occur naturally, although about 20 of them, including β-carotene, α-carotene, lycopene, lutein, zeaxanthin, meso-zeaxanthin, and cryptoxanthin, are detectable in the human blood. They have distinct physiological and pathophysiological functions ranging from fetal development to adult homeostasis. β-carotene is a precursor of vitamin A that essentially functions in many biological processes including vision. The human macula lutea and eye lens are rich in lutein, zeaxanthin, and meso-zeaxanthin, collectively known as macular xanthophylls, which help maintain eye health and prevent ophthalmic diseases. Ocular carotenoids absorb light from the visible region (400–500 nm wavelength), enabling them to protect the retina and lens from potential photochemical damage induced by light exposure. These natural antioxidants also aid in quenching free radicals produced by complex physiological reactions and, consequently, protect the eye from oxidative stress, apoptosis, mitochondrial dysfunction, and inflammation. This review discusses the protective mechanisms of macular xanthophylls in preventing eye diseases such as cataract, age-related macular degeneration, and diabetic retinopathy. Moreover, some preclinical animal studies and some clinical trials are discussed briefly to understand carotenoid safety and efficacy.


Introduction
The major causes of progressive and irreversible loss of vision include various ophthalmic diseases such as cataract, age-related macular degeneration (AMD), glaucoma, and diabetic retinopathy. Initiation and progression of these disorders involve oxidative stress, apoptosis, mitochondrial dysfunction, and inflammation [1,2]. For instance, increased oxidative stress of retinal cells damages the mitochondrial DNA in diabetic retinopathy, one of the most deleterious eye-related complications of diabetes [3]. Oxidative stress is also a significant contributor to the pathophysiology of age-related cataract, a leading cause of blindness globally [2,4]. A growing body of evidence indicates that dietary antioxidants can prevent and treat many ophthalmic disorders associated with oxidative stress. Lutein, zeaxanthin, and meso-zeaxanthin (synthesized from lutein in the retina) are dipolar, terminally dihydroxylated carotenoids, also known as macular xanthophylls, and are obtained from dietary sources [5,6]. Macula lutea of the eye, also known as the yellow spot, contains high concentrations of macular xanthophylls. The peak concentrations of lutein and zeaxanthin appear at the center of the fovea [7]. Lutein and zeaxanthin are also found in the lens; however, β-carotene and lycopene have not been detected [8]. macular xanthophylls has been proposed [5]. Macular xanthophylls located transversely in the lipid bi-layer of the retinal membrane are able to prevent AMD and protect the retina against peroxidation and photo-damage by acting as antioxidants that quench free radicals and ROS [5]. They also prevent blue light exposure to fovea's photoreceptors significantly [25] (Figure 1).  Figure 1. Schematic diagram showing the mechanisms of action of carotenoids to prevent age-related macular degeneration (AMD). HR-LBP: human retinal lutein-binding protein; GSTP1: glutathione S-transferase Pi 1; R: free radical (symbolic representation). Bhosale et al. (2004) isolated and purified a membrane-associated xanthophyll-binding protein from human macula using ion-exchange chromatography and gel-exclusion chromatography. This protein is a Pi isoform of human glutathione S-transferase (GSTP1) to which zeaxanthin displayed the highest affinity. Uptake, metabolism, and stabilization of zeaxanthin in the retina were found to be mediated by this xanthophyll-binding protein [27]. The HR-LBP, a membrane-associated human retinal lutein-binding protein, displayed a saturable and specific binding toward lutein [28].
Once incorporated in the lipid bilayer, macular xanthophylls help quench singlet oxygen and other free radicals and thus prevent lipid peroxidation in the retina [29][30][31][32][33]. Carotenoids also protect against oxidative damage by repairing α-tocopherol and acting synergistically with vitamin C [23]. Figure 1 illustrates the mechanisms of action of ocular carotenoids to prevent AMD.

Cataracts
A cataract is a visualization problem in which the lens develops opacity, and age-related cataract is a leading cause of blindness. Depending on the morphology, cataract is classified into different types. The outer section of the tissue becomes opaque in cortical cataracts, the inner core in nuclear cataracts, and the superficial region below the capsule on the posterior side in posterior subcapsular cataracts [34]. In western countries, cataract surgery is most frequently done in people aged 65 years or older [35]. This is one of the most common surgical procedures among the general population, and the prevalence is increasing each year [36]. In the United States, 3.38 million cataract surgeries were performed in 2017 [37]. Although cataract is mainly an age-related phenomenon, socioeconomic and lifestyle factors (smoking, diet, intake of nutrients, alcohol consumption, etc.) also influence cataract initiation and progression [35,38].
The main constituents of an eye lens are crystallins (90%), and cytoskeletal and membrane proteins. Crystallins have a high refractive index and form a complex protein solution in the cytoplasm of lens fibers, conferring transparency. With age, this protein slowly leaves the soluble phase. Subsequently, disulfide bond formation and non-enzymatic glycation alter attractive forces between lens proteins [34] ( Figure 2). Masters et al. (1977) observed aspartic acid racemization during aging and cataract formation on a D/L enantiomeric analysis of control human lenses and cataracts [39]. The insoluble fraction of D-aspartic acid becomes less abundant in cataractous lenses [40]. Thus, crystallins may undergo various post-translational modifications such as oxidation, glycation, proteolysis, transamidation, carbamylation, and phosphorylation [41]. These changes result in aggregation of proteins, disruption of healthy lens cell structure, and opacification.
Ocular oxidative stress may result from an imbalance between the generation of reactive oxygen species (ROS) and the cellular antioxidant defense mechanisms and subsequently initiate lens opacification [42]. ROS, such as hydrogen peroxide, superoxide, and hydroxyl radicals, negatively modifies the lens, whereas antioxidants, including glutathione (GSH), ascorbate, and catalase, rescue the lens proteins against ROS [43,44]. Hydrogen peroxide, the primary oxidant in the pathogenesis of cataract, is eliminated by catalase and glutathione through enzymatic reactions. A decreased level of reduced glutathione in older lenses' nucleus promotes cataract formation [43,45]. An imbalance in redox reactions can also initiate lipid peroxidation, promoting cataractogenesis. Spector (1995) mentioned that the massive oxidation of thiol to protein and mixed disulfides, cysteic acid, and methionine sulfoxide and cataract-extensive methionine sulfoxide formation are common in older lens [34]. In the nucleus of nuclear cataracts, covalently linked disulfide bonds containing polypeptides and in cortical cataracts, high molecular weight disulfide-linked aggregates were found [46]. Thus, oxidation of crucial sulfhydryl groups of enzymes and membrane proteins and the peroxidation of lenticular plasma membrane lipids also contribute to cataract pathogenesis [47].
Carotenoids' roles as antioxidants are known for many decades. β-carotene was found to markedly inhibit lipid peroxidation induced by xanthine oxidase in a pioneering study by Kellogg III and Fridovich [48]. Chemical antioxidants (e.g., α-tocopherol, β-carotene, ascorbate, and GSH) and structural antioxidants (e.g., cholesterol and membrane protein) are implicated in preventing oxidative damage of the ocular tissues [49]. Christen (1994) reviewed antioxidants' protective effects in cataract and macular degeneration and found that animal studies invariably advocated in favor of dietary antioxidants, although results from epidemiological analyses were inconclusive [50]. The mechanisms of preventive functions of carotenoids in cataract formation are shown in Figure 2.
Human lens contains lutein and zeaxanthin but not β-carotene [51]. It has been suggested that antioxidants lutein and zeaxanthin are delivered continuously from the body pool to the epithelial/cortical layer of the lens, where they scavenge ROS by up-regulating GSH, catalase and SOD activities [52]. Gao et al. (2011) reported that lutein and zeaxanthin could reduce the risk for senile cataract by protecting lens protein, lipid, and DNA from oxidative damage. They incubated human lens epithelial cells with or without 5 µM lutein, zeaxanthin, or α-tocopherol for 48 h. Then the cells were exposed to 100 µM H 2 O 2 for 1 h to induce oxidative stress. By using a battery of in vitro analyses, the authors observed that the levels of H 2 O 2 -induced protein carbonyl, MDA, and DNA damage were significantly reduced by lutein and zeaxanthin [53]. Interestingly, cataract patients exhibited increased serum levels of pro-oxidants and decreased levels of antioxidants. Serum level of MDA was significantly higher, and levels of superoxide dismutase (SOD) and glutathione peroxidase (GPX) were substantially lower in age-related cataract patients compared to healthy volunteers [54,55].
Antioxidants 2020, 9, x FOR PEER REVIEW 5 of 22 exhibited increased serum levels of pro-oxidants and decreased levels of antioxidants. Serum level of MDA was significantly higher, and levels of superoxide dismutase (SOD) and glutathione peroxidase (GPX) were substantially lower in age-related cataract patients compared to healthy volunteers [54,55].

Diabetic Retinopathy
Glycemic control, diabetes duration, hypertension, hyperlipidemia, smoking, age, and genetic factors are responsible for developing microvascular complications like diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy [56]. Diabetic retinopathy is prevalent in people with Type 1 and Type 2 diabetes mellitus. Glycated hemoglobin (HbA1c), a measure of mean glycemia, has been identified as a risk factor for the progression of diabetic retinopathy [57,58]. Carotenoids enhance insulin sensitivity and have a protective effect against diabetes-related infectious diseases [59].
In diabetes, the high glucose level present in the microvasculature of the retina compromises the electron transport chain system, produces superoxides, damages mitochondrial DNA and decreases proteins encoded by its DNA, and thus, causes metabolic, structural, and functional changes in the retina [60]. Hyperglycemia can initiate many biochemical changes in the retinal microvasculature, including increased oxidative stress in the polyol pathway, protein kinase C (PKC) activation, and advanced glycation end-product formation [61] (Figure 3). Rat retinal endothelial cells exposed to high glucose (HG) showed a down-regulation of the protein kinase B (also known as AKT) pathway and increased apoptosis [62]. HG was also found to increase mitochondrial fragmentation and proapoptotic cytochrome c levels in vascular cells of rat retinal capillaries [63,64]. Increased oxidative

Diabetic Retinopathy
Glycemic control, diabetes duration, hypertension, hyperlipidemia, smoking, age, and genetic factors are responsible for developing microvascular complications like diabetic retinopathy, diabetic nephropathy, and diabetic neuropathy [56]. Diabetic retinopathy is prevalent in people with Type 1 and Type 2 diabetes mellitus. Glycated hemoglobin (HbA1c), a measure of mean glycemia, has been identified as a risk factor for the progression of diabetic retinopathy [57,58]. Carotenoids enhance insulin sensitivity and have a protective effect against diabetes-related infectious diseases [59].
In diabetes, the high glucose level present in the microvasculature of the retina compromises the electron transport chain system, produces superoxides, damages mitochondrial DNA and decreases proteins encoded by its DNA, and thus, causes metabolic, structural, and functional changes in the retina [60]. Hyperglycemia can initiate many biochemical changes in the retinal microvasculature, including increased oxidative stress in the polyol pathway, protein kinase C (PKC) activation, and advanced glycation end-product formation [61] (Figure 3). Rat retinal endothelial cells exposed to high glucose (HG) showed a down-regulation of the protein kinase B (also known as AKT) pathway and increased apoptosis [62]. HG was also found to increase mitochondrial fragmentation and pro-apoptotic cytochrome c levels in vascular cells of rat retinal capillaries [63,64]. Increased oxidative stress, elevated oxidatively modified DNA, and up-regulated nitrosylated proteins ensue an impairment in antioxidant defense enzymes, which eventually leads to increased retinal capillary cell apoptosis [65]. Further, mitochondrial metabolism generates ROS, such as superoxides and hydrogen peroxide, that can damage proteins, lipids, and DNA. The damage of proteins can be compensated because of continuous biosynthesis; however, DNA damage can be devastating if a fixed mutation occurs. If reactive oxygen species damage a portion of a single DNA strand (e.g., the addition of 8-oxo-2 -hydroxyguanine in DNA strand) and DNA polymerases copy that damaged templates during replication, then, this error becomes permanent [66][67][68] (Figure 3).
DNA double-strand can also be affected and broken by free radicals. This breakdown is usually repaired by ligating nonhomologous DNA ends, an error-prone repair system [68]. In an in vitro study, Santos, Tewari and Kowluru, (2012) observed that the damage caused by ROS was compensated by increased mitochondrial DNA biosynthesis and repair system in the early stages of diabetes (15 days to 2 months). At a stable diabetic condition (at 6 months of diabetes) with constant production of high ROS, mitochondrial DNA and electron transport chain (ETC) were damaged because repair/replication machinery became subnormal and mitochondrial DNA copy number was significantly decreased. An increase in apoptosis was also observed in the above study [69]. Compromised DNA repair machinery, decreased gene expressions of mitochondrial-encoded proteins, and increased mtDNA damage were observed at high glucose exposure of retinal endothelial cells [70]. Aso et al. (2000) observed a higher amount of advanced glycation end-products (non-enzymatic binding of glucose to free amino groups of an amino acid) in patients with retinopathy [71,72]. In hyperglycemic conditions, a high glucose level causes overproduction of a glycolytic metabolite glyceraldehyde-3-phosphate. Glyceraldehyde-3-phosphate can easily be converted into 1, 3 Diphosphoglycerate by converting nicotinamide adenine dinucleotide (NAD+) into to its reduced form (NADH) when ROS inhibits the overproduction of glyceraldehyde-3-phosphate dehydrogenase (GADPH). NADH facilitates the protein kinase C (PKC) pathway and the AGE pathway [73]. Advanced glycation end-products (AGEs) (glucosepane and methylglyoxal hydroimidazolone) were significantly associated with the progression of retinopathy [74].
Antioxidants such as ascorbate, tocopherol, and carotenoids protect ocular oxidative damage [75]. Carotenoids can quench free radicals, scavenge reactive oxygen species, modulate gene expression, reduce inflammation, and prevent diabetes-related microvascular complications, including diabetic retinopathy, nephropathy, and neuropathy [76]. Macular pigment (MP), including lutein, zeaxanthin and mesozeaxanthin also contributes to the protection of the retinal tissue by conferring potent antioxidant and anti-inflammatory effects in diabetes. It has been demonstrated that patients with type 2 diabetes have a lower level of MP as compared to healthy controls [77]. Lutein supplementation is known to prevent oxidative damage in the retina [78]. In a mouse model of early diabetic retinopathy, long-term lutein administration attenuated inflammation, and vascular damage of the retina [79]. Intriguingly, short-term lutein treatment also down-regulated reactive oxygen species and up-regulated superoxide dismutase (SOD), attenuating inflammation and protecting the photo-stressed retina from oxidative damage [80]. Several signaling pathways, including PKC, vascular endothelial growth factor (VEGF), nuclear factor erythroid 2-related factor 2 (Nrf2), and Rho/Rho-associated coiled-coil containing protein kinase (Rho/ROCK), have been implicated in carotenoid-mediated protection of the retina in diabetic retinopathy [81]. An in vitro study showed that co-administration of lutein and zeaxanthin attenuated VEGF-induced oxidative stress in the retinal endothelium [82]. Lutein was found to modulate the SIRT1 signaling and inhibit premature senescence in retinal pigment epithelium cells [83]. Recent studies indicate that carotenoids could exert therapeutic benefits in diabetic retinopathy through multiple cellular and molecular pathways. The mechanisms of action of carotenoids to prevent diabetic retinopathy discussed above are sketched in Figure 3.

Safety of Carotenoids
Several lines of evidence have demonstrated the safety profiles of carotenoids supplementation at different doses and duration in experimental animals. Table 1 summarizes some of these outcomes.

Safety of Carotenoids
Several lines of evidence have demonstrated the safety profiles of carotenoids supplementation at different doses and duration in experimental animals. Table 1 summarizes some of these outcomes.

Clinical Trials
Clinical trials are the final step assessments of any drug before it is approved for regular human application. Twenty-six (26) important clinical trials are summarized in Table 2 to understand the efficacy of carotenoids as prophylactic and therapeutic uses in eye diseases.    Serum level increased linearly with increased dose. Group 3 showed the highest ratio of MPOD change, which was statistically significant (p = 0.021).   Preclinical study results listed in Table 1 have established the safety profile of several carotenoids. Results from different clinical trials listed in Table 2 confirm that an increased serum level of lutein was correlated with enhanced visual acuity [93,96,116]. An increase in macular pigment optical density (MPOD) was seen with lutein and zeaxanthin supplementation [95][96][97][98][99][100]. Several studies reported that lutein and zeaxanthin administration was associated with a reduced risk of cataract [101][102][103][104][105]114]. Nonetheless, some studies did not find statistically significant effects of lutein and zeaxanthin on prevention of eye diseases or enhancement of macular pigments [99,102,113,115]. In a study by Manayi et al. (2015), lutein and zeaxanthin were found in the lens, but β-carotene and lycopene were not detected [8]. Among six clinical trials mentioned in Table 2, which examined the effects of β-carotene on cataract, five found no significant impact [104,107,109,112], and one showed a small reduction in the progression of age-related cataract [111]. A study found that smokers, people with high BMI, a history of hypertension, diabetes, and high cholesterol developed cataracts even after taking antioxidants [101]. These studies suggest that β-carotene is not adequate for cataract prevention as the lens does not contain any β-carotene. On the contrary, in vivo studies on animals showed that lutein is safe even at a very high dose, and the LD 50 of lutein exceeded 10,000 mg/kg body weight [86]. Eight in vivo studies were mentioned in this review (Table 1), and none of these studies observed any significant adverse effect or toxicity. Xu et al. (2013) suggested a daily intake of 3 mg/kg/day meso-zeaxanthin for human [88]. Intake of up to 20 mg/day for lutein was found to be safe for humans [117].

Conclusions
In this review, we summarized the detrimental effects of ocular oxidative stress generated from the continuous exposure of ultraviolet and blue lights. Then we discussed the protective roles of carotenoids, namely β-carotene, lutein, and zeaxanthin, against three distinct eye diseases, highlighting the outcomes from the clinical trials. A considerable number of studies including preclinical and clinical trials demonstrated that β-carotene, lutein, and zeaxanthin can prevent the progression of eye diseases, mainly by quenching free radicals and preventing oxidative damage to the retina. According to study outcomes, it is obvious that β-carotene, lutein, and zeaxanthin can efficiently attenuate oxidative stress in vivo and confer protection to the eye.
The biological functions of different carotenoids in human are established. As the human body cannot synthesize this important class of molecules, they must be supplied as dietary intake or food/pharmaceutical supplement. Thus, the optimum levels of cellular concentrations of β-carotene, lutein, and zeaxanthin in eye tissue may help maintain eye health. It is noteworthy that carotenoids, particularly MP, can be assessed noninvasively in retina; such assessment may be useful to determine the average dietary intake of lutein and zeaxanthin to meet the regular need of these molecules [118]. It has been shown that MP attenuates oxidative stress and slows down the progression of apoptosis, mitochondrial dysfunction, and inflammation in diabetes, which can be improved by increasing dietary supplementation of lutein and zeaxanthin [77].
A long-term cohort study by Wu et al. (2015) found a remarkable 40% reduced risk of advanced AMD progression for predicted plasma lutein/zeaxanthin scores [119]. Nevertheless, carotenoids show a high degree of variability in bioavailability, which poses a challenge for finding suitable forms (as foods, supplements, or medicines) that can be administered to the patients with AMD. Gastrointestinal absorption and subsequent distribution to ocular tissues are influenced by dietary factors, formulations, gender, age, disease states, and individual genetic variations [120,121]. A recent study found a significantly higher absorption of zeaxanthin and meso-zeaxanthin from a diacetate micromicelle preparation than free carotenoid preparations [120]. Intriguingly, a nano-formulation of lutein-poly-(lactic-co-glycolic acid) (PLGA)-phospholipid (PL) showed a significantly elevated level of lutein in plasma when administered at a lower dose in mice [87]. Novel drug delivery systems and formulations thus could further be exploited to achieve favorable pharmacokinetic and pharmacodynamic profiles of macular xanthophylls in humans. In addition, long-term clinical trials with large numbers of populations may be undertaken to confirm the effects of these molecules. Future studies will substantiate the therapeutic potentials of different β-carotenoids.