**3. Lutein and the eye**

According to the National Health and Nutrition Examination Survey, the intake of lutein and zeaxanthin combined is approximately 1–2 mg/day in USA [11]. In addition, German adults consume 1.9 mg/day in average and 1.4 mg/day of lutein consumption was reported for Canadians [1]. No adverse effects were reported after the supplementation of dietary lutein up to 20 mg/day for 48 weeks, 30 mg/day for 120 days, and 40 mg/day for more than 8 weeks [12–14]. Animal studies have demonstrated similar results. For rat, uptake of lutein up to 35 mg/day for 8 weeks or 208 mg/kg/day for 13 weeks, or 639 mg/kg/day was not associated with any exposure-related toxicity and adverse events [15, 16]. Thus, lutein is recognized as Generally Recognized as Safe (GRAS) by FDA. Although there is no relationship between side effects and long term, high dose supplementation of lutein, the total intake should not exceed 20 mg/day according to the report from Council for Responsible Nutrition (CRN) in 2006 [17]. Generally, the recommendation dose of lutein supplements is 10 mg/day. A recent case report has demonstrated bilateral "foveal sparkles" in an Asian woman who has taken a 20 mg/day lutein supplements together with a high consumption of dietary lutein. After 7 months of discontinuous uptake of lutein supplements but insistence of her high-lutein diet, the crystal dissolved in the right eye, but still existed in the left eye [18]. However, it is worth noting that upon the population-based surveys, consumption of lutein has gradually declined in the USA and Europe. Therefore, actions should be taken to emphasize the importance of

adequate intake of carotenoid-rich food, especially from dark-green leafy vegetables.

Since lutein and zeaxanthin are soluble in the fat, the absorption of these compounds follows a similar path like other lipophilic nutrients. After uptake of carotenoid-rich foods, xanthophylls are released from the food matrix with the aid of a variety of enzymes (e.g. esterase) and disperse in the stomach. The free xanthophylls then form micelles by incorporating with biliary phospholipids, bile salts, or dietary fats, which makes them more easily absorbed into the mucosal cells in the small intestine. Subsequently, they are transported from intestinal tract to the liver in the form of chylomicrons, where xanthophylls such as lutein and zeaxanthin are repackaged, carried by the relevant lipoproteins and released into the systemic circulation. In the circulation system, lipoproteins are responsible for transporting hydrophobic lipid including fat, plasma lipid, carotenoids, retinoids, etc. There are four types of lipoproteins: ultra-low density lipoproteins (ULDL), also known as chylomicrons; very low density lipoproteins (VLDL); low density lipoproteins (LDL); and high density lipoproteins (HDL). Compared to the non-polar carotenes such as lycopene and β-carotene, which are loaded onto VLDL and LDL, lutein and zeaxanthin are primarily transported by HDL. Both lutein and zeaxanthin are distributed in a variety of human tissues, but the distribution of them is not balanced among different tissues and organs. Retina, especially the macula, is regarded as the region where lutein, zeaxanthin, and its metabolite meso-zeaxanthin are concentrated, accounting for 25% of total carotenoids. Therefore, lutein, zeaxanthin, and meso-zeaxanthin are known as macular pigments (MPs), which play an important role in maintaining the normal functions of the eye. Although lutein is richest in the retina, it is also absorbed and distributed in other tissues such as adipose tissue in human body. It has been estimated that level of lutein in the retina was affected in obesity group, suggesting adipose tissue may compete with retina in terms of xanthophylls uptake [19].

**2.3. Absorption, metabolism, and transport of lutein**

174 Progress in Carotenoid Research

#### **3.1. Lutein in the retina**

The eye is made up of three separate layers, including the cornea and the sclera forming the outer fibrous layer; the uveal tract, which consists of the iris, ciliary body and choroid, forming the middle vascular layer; and the retina forming the inner neural tunic (**Figure 2**). In the central and posterior part of retina, there is an oval-shaped yellow area (approximately 5–6 mm in diameter) known as macula, which contains the highest concentration of photoreceptors. It is characterized by the yellow pigments that are entirely composed of lutein and zeaxanthin. The fovea, in the center of macula, is a small pit which is in charge of central vision and high-resolution visual acuity as a result of closely assembled cone cells. In addition, the retina consists of 10 layers from the outermost to the innermost, including RPE, photoreceptor cell layer, external limiting membrane (ELM), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion cell layer (GCL), nerve fiber layer (NFL), and internal limiting membrane (ILM).

Although MPs exhibit high concentration in the retina, the distribution varies in different regions of the retina. The highest concentration of MPs is observed in the fovea at about 0.1–1 mM, which is over 100-fold higher than the rest area of retina. Moreover, the ratio of lutein and zeaxanthin also differs in different parts of retina. In the peripheral retina, lutein is the major carotenoids and the ratio of lutein to zeaxanthin is 2:1, whereas the ratio is reversed to 1:2 in the fovea.

fluorescent dye which was excitable by blue light. Different carotenoids were incorporated with the lipophilic membrane, and fluorescence intensity was lower in carotenoid-containing liposomes than in control group when exposed to blue light, indicating a role of carotenoids as the blue light filter [27]. It is noted that lutein is more efficient in filtering blue light than zeaxanthin and meso-zeaxanthin because of the orientation in the biological membranes [27, 28].

A free radical is defined as a molecule, atom, or ion containing an unpaired electron. Because of the unpaired electron in the outer shell, the free radical is chemically highly reactive and unstable. Therefore, the free radical will react with other substances, even with themselves to reach steady state. Free radicals generated from oxygen are called reactive oxygen species

matic and non-enzymatic reactions, respectively [30]. Singlet oxygen, perhydroxyl radical, and hydroxyl radical are oxidants causing oxidation of protein, DNA as well as lipid peroxidation in cell membrane lipid bilayer, resulting in damages to the integrity of biological membrane and subsequently cell necrosis [29]. In physiological condition, production and detoxification of ROS are balanced in the body. However, when the balance is disrupted, no matter the increase in ROS generation or reduction of endogenous antioxidants, damages in

The retina is constantly exposed to ROS due to its high consumption of oxygen, conversion of light photons into electrochemical signals, and a number of mitochondria in rods. Massive blood supplies to the choroid in the retina make it the highest oxygen uptake tissue in the human body. Continuous exposure to the light photons, especially the blue light, triggers photo-oxidative reactions and damages DNA in RPE cells. Mitochondria, which are believed to be the major site for the generation of ROS, are rich in the inner segments of rod cells. It has been estimated that about 5% activated oxygen electrons in mitochondria could leak out as they go through the complicated electron transport chain, forming superoxide radicals [31]. Furthermore, a high content of polyunsaturated fatty acids in the outer segments of rods makes it more prone to peroxidation. In general, retina exhibits high susceptibility to ROS,

Depending on the unique structure of MPs, one of the major biological functions of MPs in the retina is the prevention from oxidative damages via either physical quenching of singlet oxygen or chemical scavenging of free radicals. In the process of quenching of non-radical compound, such as singlet oxygen, the energy of singlet oxygen is transferred to the molecules of MPs, leading to excited triplet state of MPs and ground state of oxygen. Subsequently, the MPs in the triplet state dissipate the energy and return into the ground state. Since it is a physical mechanism, the structure of MPs is not changed, thus can be reused in the quenching cycles. It has been estimated that among carotenoids, lutein can react with singlet oxygen more strongly [32]. In contrast to physical mechanism, scavenging of ROS is achieved through chemical reactions in two ways. First, ROS accepts the missing electrons from MPs in which

), and hydroxyl radical (OH) [29]. Superoxide can be inverted into only hydrogen

together with singlet oxygen (non-radical compound) by enzy-

—), perhydroxyl radical (also known as hydroperoxyl

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

*3.2.2. Antioxidant function*

radical, HO2

peroxide (H2

(ROS), including superoxide anion (O2

) and H2

resulting in irreversible oxidative damages.

O2

the body occur. Thus it has been defined as "oxidative stress".

O2

**Figure 2.** The human eye. (A) A schematic diagram demonstrating the anatomy of the human eye [23]. (B) A schematic image of optical coherence tomography (OCT) showing the vertical section of the center of the retina.

Of the 25 dietary carotenoids found in human tissues and blood, the selectively high rate of absorption and accumulation of lutein and zeaxanthin in human retina remained unclear until the discovery of specific macular carotenoid-binding proteins. Bernstein et al. have demonstrated that tubulin, a hydrosoluble protein, could bind to both lutein and zeaxanthin, and may be involved in the high distribution of MPs in the retina, but presented relatively low binding affinity and specificity. As a result, the research team continued to identify carotenoid-binding proteins with higher affinity and specificity. Subsequently, glutathione S-transferase P1 (GSTP1) was identified to bind zeaxanthin in the macula specifically compared to GSTM1 and GSTA1, the members of GST protein family [24]. GSTP1 was further confirmed to prevent lipid membrane from oxidation. In 2011, steroidogenic acute regulatory domain protein 3 (StARD3), one of lipid transfer-related protein family, was discovered as the lutein-binding protein [25]. Further studies need to be carried out to reveal more functions of StARD3. Generally, GSTP1 and StARD3 selectively bind zeaxanthin and lutein, respectively, leading to the high concentration and stabilization in human retina. In addition, the retinoid transporters including inter-photoreceptor retinoid-binding protein (IRBP) and retinol binding protein 4 (RBP4) are believed to be involved in the transport of MPs from circulation to retina [26].
