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Trends in Food Science & Technology 49 (2016) 74e84 Contents lists available at ScienceDirect Trends in Food Science & Technology journal homepage: http://www.journals.elsevier.com/trends-in-food-scienceand-technology Review Lutein and zeaxanthin: Production technology, bioavailability, mechanisms of action, visual function, and health claim status Ifeanyi D. Nwachukwu a, Chibuike C. Udenigwe b, Rotimi E. Aluko a, * a Department of Human Nutritional Sciences, and the Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba, Winnipeg, R3T 3N2 MB, Canada b Department of Environmental Sciences, Faculty of Agriculture, Dalhousie University, Truro, B2N 5E3 NS, Canada a r t i c l e i n f o a b s t r a c t Article history: Received 13 October 2015 Received in revised form 3 December 2015 Accepted 27 December 2015 Available online 30 December 2015 The xanthophylls, lutein and zeaxanthin have been demonstrated to act as protective shields against high energy blue light, key contributors to central vision as well as to high visual acuity, and antioxidants that repair photo-induced oxidative damage. Like other phytochemicals, the sundry techniques for extraction, purification, structural characterization and identification of lutein and zeaxanthin have undergone considerable refinement. Supercritical CO2 extraction, apart from being quicker and more eco-friendly than traditional organic solvent extraction, offers the advantages of higher yield and absence of solvent residues in the extracted material. Improved industrial lutein extraction and purification procedures have translated to increased efficiency in generating lutein supplements used in large scale human clinical trials. This paper reviews recent studies on the vision-enhancing potentials of lutein, and concludes that even in the absence of health claims formally and explicitly advising the public on the benefits of diets rich in lutein and zeaxanthin, both xanthophylls have the potential to substantially contribute to eye health if regularly consumed as part of a healthy diet. © 2015 Elsevier Ltd. All rights reserved. Keywords: Age-related macular degeneration (AMD) Bioavailability Cataract Supercritical carbon-dioxide extraction Meso-zeaxanthin Oxycarotenoids Safety Transport Xanthophylls Carotenoids 1. Introduction The xanthophyll lutein (b,ε-carotene-3,30 -diol) generally coexists in nature with its stereoisomer zeaxanthin (b,b-carotene3,30 -diol) (Shegokar & Mitri, 2012) and occurs in abundance in green leafy vegetables such as kale and spinach, where its yelloworange colour is masked by the dominant green colour of chlorophyll (Reif et al., 2012; Shahidi, Chandrasekara, & Zhong, 2011). While up to 40 mg lutein þ zeaxanthin can be found in 100 g of a dark, green leafy vegetable like (raw) kale where the xanthophylls mainly occur in their pure crystalline forms, only < 1 mg lutein þ zeaxanthin was shown to be present in 100 g of the edible portion of cooked and raw foods with a yellow-orange colour such as baby carrots, peaches, corn, papaya, and raw oranges (Holden et al., 1999). Other fruits and vegetables with high amounts of lutein and zeaxanthin include collards, turnip greens, broccoli, * Corresponding author. E-mail address: [email protected] (R.E. Aluko). http://dx.doi.org/10.1016/j.tifs.2015.12.005 0924-2244/© 2015 Elsevier Ltd. All rights reserved. Japanese persimmons, peaches and olives (Holden et al., 1999). While the amount of lutein and lutein esters in wheat and wheat products such as whole wheat bread is low (Ziegler et al., 2015) in comparison to its relative abundance in fruits and vegetables, the consumption of foods and food products made from wheat flour as a staple in many regions of the world positions this grain as an important source of carotenoids. According to data made available by the Agricultural Research Service of the US Department of Agriculture, among poultry and dairy foods and food products, the highest amounts of lutein and zeaxanthin are found in egg yolk, chicken (broilers) and cheese (USDA, 2015). Importantly, the lipiddissolved physical state of the xanthophylls in these animal sources makes them highly bioavailable (Schweiggert & Carle, 2015). Although there is currently no recommended dietary allowance for lutein and zeaxanthin, the amounts of the xanthophylls in 100 g of the afore-mentioned foods exceed the approximate dose of 6 mg/ d that has been linked with improvements in visual function (Rasmussen & Johnson, 2013). To our knowledge, there are no data on the most abundant source of zeaxanthin alone in nature but fruits and vegetables such I.D. Nwachukwu et al. / Trends in Food Science & Technology 49 (2016) 74e84 as wolfberries, collards, Capsicum annuum, yellow corn and spinach have been reported to contain some of the highest concentrations of the xanthophyll (Sajilata, Singhal, & Kamat, 2008). In nature, lutein is most abundant in the flower petals of yellow Marigold (Tagetes erecta L.) which contain lutein chemically bound to fatty acids including lauric and palmitic acids (Khalil et al., 2012). Consequently, this source is used in the commercial extraction of lutein in industries using supercritical CO2 extraction due to its eco friendly and time-saving advantages (Hojnik, Skerget, & Knez, 2008). For a detailed discussion of the carotenoid content of about 215 raw, cooked, boiled and/or processed foods, see the USDA-NCC Carotenoid Database (Holden et al., 1999). Decades of research have established the role of lutein and zeaxanthin as potent filters of high energy blue light in both plants and animals e a role that results directly in their function as formidable antioxidants, which quench and scavenge photoinduced reactive oxygen species, ROS (Bian et al., 2012). Although as shown in Fig. 1, the xanthophylls play critical roles in the promotion of other aspects of health and well-being not directly related to vision, they are best known for their contribution to visual health (Alves-Rodrigues & Shao, 2004; Johnson, 2014). This is not surprising given that lutein and zeaxanthin are the only carotenoids present in both the macula and lens of the human eye (Ma & Lin, 2010), the two ocular tissues critical for vision that are among the most vulnerable to oxidative damage as a result of frequent exposure to intense light (Chalam, Khetpal, Rusovici, & Balaiya, 2011). The most recent data available from the WHO estimate that 285 million people in the world are visually-impaired while 39 million Fig. 1. Some non-vision related properties of lutein. Studies have shown that lutein which acts as an antioxidant is important for maintaining a healthy skin by protecting the skin from photo-damage and erythema caused by exposure to ultraviolet radiation (Shegokar & Mitri, 2012; Stahl & Sies, 2003). A recent study which found that dietary lutein and zeaxanthin consumption significantly reduced the rate of pancreatic cancer development in diabetics with a mean age of 65.8 and 67 (for the control and test subjects respectively) has also spawned discussions on the anticancer potentials of lutein (Jansen et al., 2013). Additionally, in a recent human intervention trial using lutein supplements, lutein was found to increase verbal fluency, memory and overall cognitive function in unimpaired subjects (Johnson et al., 2008). Finally, lutein seems to contribute to the reduction of coronary heart disease (Dwyer et al., 2001; Howard et al., 1996), and has been linked to the reduction of adhesion molecules present on the surface of endothelial cells (Alves-Rodrigues & Shao, 2004). The expression of adhesion molecules on endothelial cell surfaces is recognized as a biomarker for disease progression in atherosclerotic tissues. 75 are legally blind (Pascolini & Mariotti, 2012; WHO, 2014). Several studies have suggested that lutein and zeaxanthin play critical roles in delaying the onset and reducing the risk of cataract and agerelated macular degeneration (AMD), both of which are responsible for 56% of all cases of blindness globally (Bone, Landrum, Cao, Howard, & Alvarez-Calderon, 2007; Bone & Landrum, 2010; Ma et al., 2012b; Murray et al., 2013; Pascolini & Mariotti, 2012). Since 80% of all visual impairments are avoidable or curable (WHO, 2014), it has become pertinent to undertake a comprehensive review of the contributions of dietary lutein and zeaxanthin to visual health with a view to highlighting the position of these xanthophylls as critical players in reducing the incidence of ocular abnormalities. Furthermore, given heightened consumer interest in health, Dumais, Chao, & promoting foods in recent years (L'Abbe Junkins, 2008), and the growing number of studies linking diet and functional foods to human wellness (Aluko, 2015), the pendulum for efficient disease control and health promotion is tipped towards prevention rather than treatment. Thus nutrientbased strategies could prove useful in reducing the incidence of impaired vision considering that 90% of visually-impaired persons worldwide live in developing countries (WHO, 2014) where access to adequate healthcare is often limited. 2. Structure and occurrence Named for its characteristic yellow-orange colour, pure lutein typically appears as a yellow-orange crystalline, water-insoluble, lipophilic solid with a melting point of 190 C and a molecular mass of 568.87 g/mol (Shegokar & Mitri, 2012). Although lutein is thought to have been first isolated from the human corpus luteum (the Latin “luteum” stands for “yellow” or “egg yolk”), the oxycarotenoid like all other carotenoids is only synthesized de novo by plants, certain bacteria and fungi, as well as photosynthetic ndez, 2012). Therefore, microalga (Delgado-Pelayo & Hornero-Me humans and lower animals must consume plant-based diets as ndez, 2012). The sources of lutein (Delgado-Pelayo & Hornero-Me ionone rings of free lutein and zeaxanthin contain a hydroxyl group, although the esterified forms contain fatty acids attached at either or both ends of their structures (Fig. 2). Lutein can exist in 8 possible stereoisomeric forms because of its 3 chiral centers. However, it naturally exists mainly in the Z (cis)-form (R,R,R) (Abdel-Aal, Akhtar, Zaheer, & Ali, 2013). Chemically, as shown in Fig. 2, lutein contains the basic C40 isoprenoid structure characteristic of carotenoids as well as 10 conjugated double bonds (9 conjugated double bonds in the polyene chain and a single double bond in the b-ionone ring) (Sparrow & Kim, 2010). Comparatively, in addition to its C40 isoprenoid structure, zeaxanthin contains 11 conjugated double bonds comprising of 9 conjugated double bonds in the polyene chain and 2 double bonds in the b-ionone rings (Sparrow & Kim, 2010). 3. Extraction, purification, identification and structural characterization Supercritical carbon-dioxide (SCeCO2) extraction, illustrated in Fig. 3, has become the method of choice for extracting biologically active materials from plant matter especially in industries because it is quicker, more efficient and more eco-friendly than traditional organic solvent processing in addition to offering higher target product yield (Careri et al., 2001). Furthermore, as a supercritical fluid, CO2 is cheap, non-flammable, non-toxic, and readily available at high purity, while its low critical temperature and pressure make it an ideal solvent for the isolation of heat-sensitive compounds like carotenoids (Abbas, Mohamed, Abdulamir, & Abas, 2008; Brunner, ndi, & Wang, 2009). Although the 2005; Gao, Nagy, Liu, Sima 76 I.D. Nwachukwu et al. / Trends in Food Science & Technology 49 (2016) 74e84 Fig. 2. Structures of free and esterified forms of lutein and zeaxanthin. (A) and (B): The free forms of Lutein and Zeaxanthin. R1 ¼ laurate, C12H24O2; R2 ¼ myristate, CH3(CH2)12COOH; R3 ¼ palmitate, CH3(CH2)14COOH; and R4 ¼ stearate, C17H35COOH. It has been reported that the free form of lutein predominates in the diet and tends to occur in large amounts in green leafy vegetables (Aleman et al., 2001), while lutein esters are less prevalent in the diet (Alves-Rodrigues & Shao, 2004) often occurring in little quantities in yellow-orange fruits (Sommerburg et al., 1998; Su, Rowley, Itsiopoulos, & O'Dea, 2002). The highest molar percentage of zeaxanthin has been reported to be present in orange pepper (37%) while it occurs in very low amounts (0e3%) in most green leafy vegetables (Sommerburg et al., 1998). (C) and (D): The monoesters of lutein and zeaxanthin have been ndez, 2012), and red orange, Citrus sinensis (Dugo et al., 2008). found in plants as diverse as the berries of rough bindweed (Smilax aspera L.) (Delgado-Pelayo & Hornero-Me Monoesters of both oxycarotenoids have also been obtained as de-esterification products following the incomplete enzymatic saponification of their diesters as has been shown in the partial hydrolysis of lutein diesters using a lipase from the fungus Candida rugosa (Breithaupt, Wirt, & Bamedi, 2002). (E) and (F): Xanthophyll diesters like lutein dipalmitate and zeaxanthin laurate myristate occur naturally in many plants such as the flowers of Tagetes erecta (Hojnik et al., 2008) and Crocus sativus (Goupy et al., 2013). The esterified forms of both xanthophylls can occur as mixed diesters (zeaxanthin laurate myristate) or as homogeneous diesters (lutein dipalmitate). (G): The carotenoid, meso-zeaxanthin. Fig. 3. Schematic diagram depicting supercritical CO2 extraction of lutein. Ground Marigold flower petals are packed into 7 and CO2 is pumped from 1 through 3 (where it is compressed/pressurized and cooled to a temperature 5 C), before moving under high pressure to 6. The CO2 is then heated in 6 prior to entering 7 to avoid excessive cooling (Gao et al., 2009). Following extraction, lutein is identified, quantified and characterized using LC-MS systems. The HPLC retention times for lutein and zeaxanthin are approximately 7.7 and 8.1 min, respectively (Chan et al., 2013). [See (Gao et al., 2009) for a detailed and excellent comparison of SC-CO2 extraction procedures with and without ultrasound; (Chan ndez, 2012; Taylor et al., 2006) for comprehensive descriptions of relevant HPLC procedures, as well as (Fu et al., 2012; Goupy et al., 2013; et al., 2013; Mellado-Ortega & Hornero-Me Kopec et al., 2013) for comprehensive MS structural characterization methodologies]. Fig. 2 is reproduced from (Gao et al., 2009) with permission from Elsevier. I.D. Nwachukwu et al. / Trends in Food Science & Technology 49 (2016) 74e84 isolation of carotenoids by means of solvent extraction is still a common practice, concerns with residual solvents in the product, the toxicity of certain solvents, and the presence of unwanted plant matter such as chlorophyll in the extracted material render this method less safe and attractive (Gao et al., 2009; Mendes, 2007; Sajilata et al., 2008). Large scale commercial lutein and zeaxanthin SC-CO2 extraction procedures generally involve flashfreezing of the plant source such as Marigold flower petals in liquid nitrogen, freeze-drying and grinding them into fine powder, before packing the powder (solid matrix) into the extractor unit of the SC-CO2 apparatus (Gao et al., 2009; Hojnik et al., 2008; Taylor, Brackenridge, Vivier, & Oberholster, 2006). It has been reported that the preliminary stage of lutein SC-CO2 extraction from ground Marigold flower petals was accomplished by sub-cooling the solvent in the refrigerant compressor unit of the SC-CO2 apparatus to about 5 C (below its critical temperature of 31.1 C) where it then exists as a liquid, in order to prevent the formation of gas bubbles (cavitation) (Gao et al., 2009; Martínez & Vance, 2007). The solvent is then pumped as a liquid at a pressure of about 50 bar (below its critical pressure of 73.8 bar) into the extractor (7 in Fig. 3.), which contains the solid matrix (Martínez & Vance, 2007). Prior to being pumped into the extractor, the temperature of the supercritical fluid is raised above its critical temperature to the extraction temperature in order to prevent excessive cooling, while the temperature of the extractor is maintained at the extraction temperature with the aid of an electrically- or water-heated jacket (10 in Fig. 3). Once inside the extraction vessel, the solvent rapidly diffuses into the solid matrix while the xanthophyll diffuses out of the matrix into the solvent. The low viscosity of CO2 and the absence of surface tension ensure a high level of selectivity as well as the penetration of the solvent into spaces otherwise inaccessible to non-supercritical fluids/conventional extraction solvents as the xanthophyll which is now dissolved in the solvent is carried through a pressure reduction valve towards the separator (Durante, Lenucci, & Mita, 2014; Martínez & Vance, 2007). The drop in pressure of the supercritical fluid as it leaves the extracting vessel results in a decrease in its solvent power leading to the precipitation of the oxycarotenoid (Martínez & Vance, 2007). In order to ensure complete precipitation of the extracted carotenoid, an increase in the temperature of the supercritical fluid above the saturation temperature is initiated, thereby transforming the liquid CO2 to the gaseous state and facilitating the collection of the specific xanthophyll, as the CO2 exiting the separator is either recycled and re-used or released into the atmosphere (Martínez & Vance, 2007). Variations of the described extraction protocol have been employed by various investigators in trying to optimize SC-CO2 extraction procedures. For instance, an ultrasound-assisted SC-CO2 technique with extraction temperature of 55 C, flow rate of 167 g/ min and pressure of 32.5 MPa was found to give the maximum yield of lutein esters in a study that compared various extraction parameters and conditions with and without ultrasound (Gao et al., 2009). Additionally, another study reported optimal lutein and zeaxanthin yields from daylily (Hemerocallis disticha) flower petals following SC-CO2 extraction at 80 C and 600 bar (Hsu, Tsai, Chen, Ho, & Lu, 2011). In this case, the extraction was preceded by freezing the fresh flower petals at 80 C for 24 h and freeze-drying at 42 C for another 48 h. Given the exponential growth rate of microalgae, their significantly higher free lutein content and yield, and the possibility of their continuous production in bioreactors, there is a growing interest in the commercial production of lutein from algae (Lin et al., 2015), thus suggesting an end to the sole reliance on Marigold flower as the industrial source of lutein. SC-CO2 extraction yields a lower total volume of extracted material but a higher concentration and purity of the target compound in comparison to traditional solvent extraction because of the poor 77 selectivity of the latter. In addition to the afore-stated drawbacks of solvent extraction, extracting the relatively polar xanthophylls by the traditional method with non-polar solvents such as hexane and petroleum ether has been shown to be inefficient given their poor solubility (particularly for zeaxanthin) in such solvents (Sajilata et al., 2008). While similar solubility challenges have been encountered with the use of non-polar carbon-dioxide as a solvent in SC-CO2 extraction, such difficulties could be tackled by the addition of ethanol as a modifier in SC-CO2 extraction techniques (Reverchon & De Marco, 2006). Apart from its GRAS (generally recognized as safe) status, ethanol is only present in the extracted material in trace quantities post SC-CO2 extraction, making it an ideal co-solvent in the extraction of lutein and zeaxanthin for nutraceutical formulations (Radzali, Baharin, Othman, Markom, & Rahman, 2014; Reverchon & De Marco, 2006). A study which examined the effect of seven different co-solvents on carotenoid supercritical fluid extraction found that the technique which included ethanol as a co-solvent produced the highest carotenoids yield (Radzali et al., 2014). However, Kitada and colleagues showed that although the yield of lutein increased from 0.5 mg/g to 3 mg/g following the use of ethanol as a co-solvent in a SC-CO2 procedure, about 9 mg/g of cholorophyll was also co-extracted when the modifier was used (Kitada et al., 2009). Thus, the use of a co-solvent comes with the unappealing prospect of a compromise in product purity. Nevertheless, such trade-offs are often preferable to the lower quality of extracts from traditional solvent extraction. For instance, a SC-CO2 procedure was reported to be 18 times more selective for carotenoids than a traditional extraction method with methanol even when the latter included the use of ultrasound (Macías-S anchez et al., 2009). Since extracted carotenoids often contain their esterified forms, the extracted material must be purified prior to identifying, quantifying and structurally characterizing the xanthophylls. The solvent residue in the extract is evaporated under an inert gas such as nitrogen or argon and saponification is carried out using any of a number of reagents such as 10% or 20% (w/v) KOH-methanol mixture (Goupy, Vian, Chemat, & Caris-Veyrat, 2013; Melladondez, 2012). Recent studies have examined Ortega & Hornero-Me the effect of varying saponification conditions and specific cosolvents on xanthophyll yield and purity (Palumpitag, Prasitchoke, Goto, & Shotipruk, 2011; Vechpanich & Shotipruk, 2011). Although saponification is critical to the removal and degradation of undesirable materials including chlorophyll from the extract as well as the isolation of free xanthophylls, it could also alter the structure of the extracted carotenoid. Thus HPLC methods represent a better alternative for the isolation and separation of the extract mixture as they assure highly accurate and unaltered profiles of the extracted xanthophyll (Schweiggert, Kammerer, Carle, & Schieber, 2005). Additionally, due to its poor selectivity, extracts obtained by means of organic solvent extraction typically contain a mixture of substances, which could be separated by RP-HPLC (Taylor et al., 2006). The xanthophylls are identified by comparing their retention times and visible spectra with those of authentic standards and with data available in the literature while the calibration curves generated with the pure standards at different dilution levels are used for quantification (Kopec, Schweiggert, Riedl, Carle, & Schwartz, 2013; Nimalaratne, Sun, Wu, Curtis, & Schieber, 2014; Taylor et al., 2006). Lutein can also be detected ttir, using UV-spectrophotometry at 450 nm (Fu, Magnúsdo lfson, Palsson, & Paglia, 2012). The structural characterizaBrynjo tion of xanthophylls are routinely conducted using several LC/MS methods including the highly sensitive atmospheric pressure chemical ionization technique (Goupy et al., 2013; Kopec et al., 2013; Liu, Chen, Kao, & Shiau, 2014; Schweiggert et al., 2005), which is preferred to electrospray ionization due to its detector 78 I.D. Nwachukwu et al. / Trends in Food Science & Technology 49 (2016) 74e84 response's greater linearity (Nimalaratne et al., 2014; Schweiggert et al., 2005). For instance, a HPLC-DAD-MS system was recently employed to analyze and identify different regio-isomers of lutein esters on the basis of their unique mass spectral behaviour e precisely the differing fragmentation patterns of the b- and ε-rings (Ziegler et al., 2015). Given the absence of commercial lutein ester standards (Ziegler et al., 2015), this strategy highlights the benefits of such technologies. Additionally, a study which investigated the effects of various dopants on the ionization of several analytes including lutein and zeaxanthin found that while the use of dopants generally improved the xanthophylls' signal strength, greater enhancement in signal strength was observed with less polar ca, & Canela, 2011). rotenoids such as carotenes (Rivera, Vilaro While a detailed account of the chemical syntheses of both lutein (3R, 30 R, 60 R) and zeaxanthin (3R, 30 R) is beyond the scope of this work, it is instructive to note that the synthetic production of both xanthophylls has been achieved via the widely applicable Wittig reaction (Khachik & Chang, 2009; Widmer et al., 1990). Importantly, chemical synthesis facilitates the isotopic labelling of xanthophylls and their stereoisomers for use in clinical studies such as the Age-Related Eye Disease Study 2, AREDS2 (Chew et al., 2013; Khachik & Chang, 2009). 4. Transport, metabolism and bioavailability The uptake of lutein from the intestinal lumen is thought to be partly mediated by the cholesterol membrane influx transporters NPC1L1 (Niemann-Pick C1 Like 1) and SR-B1 (Scavenger Receptor Class B type I) with no involvement of such efflux membrane transporters as ABC (ATP Binding Cassette) transporters (Sato et al., 2012). Using simulated physiological lutein-rich mixed micelles and Caco-2 TC-7 cellular monolayers, Reboul and colleagues showed that the specific transporter SR-B1 is involved in lutein transport (Reboul et al., 2005). However, this finding does not support transport by simple diffusion as had been earlier suggested for lutein and other carotenoids (Sugawara et al., 2001). The mechanism by which lutein and zeaxanthin are selectively taken up in the presence of other carotenoids by the retina has also been studied. While the exact mechanisms for the accumulation of lutein in the retina remain unclear, StARD3 (a member of the steroidogenic acute regulatory domain) has been identified as the lutein-binding protein in the human retina (Li, Vachali, Frederick, & Bernstein, 2011). Conversely, the retinal capture of zeaxanthin in humans is thought to be mediated by specific xanthophylls-binding proteins (XBP) especially the p isoform of glutathione S-transferase (GSTP1), (Bhosale et al., 2004; Loane et al., 2008). Compared to other xanthophyll carrier proteins such as tubulin, LDL, HDL, blactoglobulin and albumin, GSTP1 purified from the human macula showed the highest affinity for zeaxanthin, but did not display any high affinity binding towards lutein, in ligand binding studies (Bhosale et al., 2004). Dietary lutein is known to be transported, upon absorption, from the lumen of the enterocytes to their serosal surface, from where they would be subsequently attached to lipoproteins (specifically chylomicrons) before being transported into the circulation through the posterior vena cava. Following uptake by hepatocytes, lutein is absorbed into HDL and thereafter transported into the circulatory system (Yeum & Russell, 2002). Since only the free forms of the xanthophylls can be absorbed into the circulation, lutein and zeaxanthin esters present in the diet must be hydrolyzed by lipases and esterases in the gut and enterocytes prior to their uptake (Alves-Rodrigues & Shao, 2004; Chitchumroonchokchai & Failla, 2006), although esterification may not limit the bioavailability of lutein (Bowen, Herbst-Espinosa, Hussain, & StacewiczSapuntzakis, 2002). Using marigold-derived esterified and free lutein formulations packed into gelatin capsules in a 17-day clinical trial with 19 healthy adults, esterified lutein was found to be more bioavailable than the free oxycarotenoid (Bowen et al., 2002). Based on the study, bioavailability of lutein appeared to be dependent on distribution and solubilization in micelles (dissolution) rather than ester hydrolysis (Bowen et al., 2002). The bioavailability of carotenoids in general depends on their bioaccessibility which is in turn influenced by factors such as the incorporation of additional lipids during or after processing, structural barriers including food matrix characteristics, dietary fiber content, as well as processing treatments like heating and bioencapsulation (Aschoff et al., 2015b; Rodriguez-Amaya, 2015; Saini, Nile, & Park, 2015). For instance, the addition of extra oil is known to improve the bioavailability of carotenes while it hampers that of the polar xanthophylls. Additionally, recent studies have demonstrated that the bioavailability of lutein and zeaxanthin among other carotenoids is decreased by the presence of dietary fibers, while thermal processing (pasteurization) was shown to increase the bioavailability of lutein but not that of zeaxanthin (Aschoff et al., 2015a, 2015b). Recent genetic evidence suggests that the transport and delivery of carotenoids in silkworms may be tissue-specific, and that certain membrane transporters could distinguish between the chemical structure of individual carotenoid molecules (Tsuchida & Sakudoh, 2015). This prospect of selective carotenoid transportation could deepen current understanding and provide intriguing insight into the transport of xanthophylls in humans. 5. Safety The designation of lutein as GRAS in 2001 was a recognition of its safety in the human diet (Kruger, Murphy, DeFreitas, Pfannkuch, & Heimbach, 2002). The path to GRAS status usually involves a thorough review of efficacy, pharmacokinetics, safety/toxicity, metabolism, and related sundry data, and for lutein, the safety upper limit of consumption has been set to 20 mg/day (AlvesRodrigues & Shao, 2004; Yao et al., 2013). There have been no reports of adverse effects following long-term supplementation with dietary lutein: supplementations of 30 mg/day for 120 days (Wenzel et al., 2007), 20 mg/day for about 48 weeks (Ma et al., 2012b; Yao et al., 2013), and 40 mg/day for > 8 weeks (Dagnelie, Zorge, & McDonald, 2000), were not linked to any adverse effects in humans. Similarly, studies conducted using animal subjects have shown no safety concerns for lutein and zeaxanthin in mammals such as rats and monkeys (Fatani et al., 2015; Vishwanathan, Neuringer, Max Snodderly, Schalch, & Johnson, 2013). Although high and frequent consumption of fruits and vegetables (both of which contain carotenoids including lutein) is associated with lower risks of developing lung cancer (Chew et al., 2013), very few studies have examined the relationship between specific carotenoids and lung cancer (Michaud et al., 2000; Satia, Littman, Slatore, Galanko, & White, 2009). However, unlike b-carotene which was reported to exert a potentially pro-carcinogenic effect in ferret models because of its provitamin A activity (Wang et al., 1999; Wolf, 2002) and to actually increase the risk of developing lung cancer in high-risk groups (Woodside, McGrath, Lyner, & McKinley, 2015), no such pro-carcinogenic property has been associated with lutein. Although antioxidants including carotenoids are thought to have prooxidative effects that could result in oxidative DNA damage, lutein is not only non-mutagenic at high doses, but has also been shown to possess dose-dependent anti-mutagenic effects (Wang et al., 2006). The lutein oxidative metabolites in the macula and in human skin are known to be safe and to possess antioxidant function, and thus are able to provide photo-protection to both the tissue and the organ (Alves-Rodrigues & Shao, 2004). I.D. Nwachukwu et al. / Trends in Food Science & Technology 49 (2016) 74e84 Conversely, ezitimibe, the selective intestinal NPC1L1 and cholesterol absorption inhibitor, which is a major component of such cholesterol-lowering-drugs as Zetia® (also marketed as Ezetrol®), limits lutein absorption in a dose-dependent manner, thus raising serious concerns for hyperlipidemic individuals who are also visually-challenged (Sato et al., 2012). The link between obesity and impaired vision, which includes AMD, cataract and diabetic retinopathy (Seddon, 2013), raises serious concerns for obese individuals using medications like ezitimibe as treatment for hyperlipidemia. 6. Lutein and visual health Lutein has been shown to play a central role in reducing the incidence of eye diseases such as AMD, cataract, and retinitis pigmentosa (Aleman et al., 2001; Olmedilla, Granado, Blanco, & Vaquero, 2003). A frequently used parameter in determining the condition of the retina especially while diagnosing AMD is the macular pigment density (MPD), which is an indirect measurement of the concentration of lutein and zeaxanthin in the macula (AlvesRodrigues & Shao, 2004). The macular pigment, which principally absorbs harmful blue light (at 440 nm) and shields sensitive photoreceptors from damaging UV rays is entirely composed of lutein, its metabolite meso-zeaxanthin, and zeaxanthin, and is thought to be critical in preserving visual health (Alves-Rodrigues & Shao, 2004; Johnson, 2014). High levels of MPD have been positively correlated with lower rates of AMD and retinitis pigmentosa (Dagnelie et al., 2000; Phelan & Bok, 2000; Richer et al., 2002). For cataract, a similar parameter is the lens optical density (Hammond, Wooten, & Snodderly, 1997) but unlike the directly proportional relationship between MPD and incidence of AMD, increasing lens optical density is correlated with decreasing lens function or health (Trumbo & Ellwood, 2006). A number of studies between 1987 and 1999 suggested that dietary carotenoids (lutein, zeaxanthin, lycopene, a-carotene and bcarotene) from fruits and vegetables may play a role in reducing the risk of eye diseases (Goldberg, Flowerdew, Smith, Brody, & Tso, 1988; Sommerburg, Keunen, Bird, & Van Kuijk, 1998; Yannuzzi et al., 1993). Although it was relatively straightforward to hypothesize that lutein and zeaxanthin could have a role in visual health given the particularly high concentrations and exclusive presence of both xanthophylls in certain ocular tissues (Alves-Rodrigues & Shao, 2004), demonstrating their contributions to vision improvement or in delaying the onset of ocular diseases was a more labyrinthine exercise. First, through the means of various observational studies, the following were established: (a) depending on the outcome assessed, dietary intake of lutein significantly reduced the risk of AMD (Seddon et al., 1994), or reduced the risk of cataract extraction (Brown et al., 1999; Chasan-Taber et al., 1999; Hankinson et al., 1992); (b) pure crystalline lutein supplementation (Bernstein et al., 2002) as well as dietary intake (Curran-Celentano et al., 2001) resulted in significantly higher MPD; and (c) there is a negative correlation between increasing lutein levels in the retina and the risk of developing AMD (Bone et al., 2001). The details of selected lutein and AMD observational studies are comprehensively presented in Table 1. Various observational studies have also examined the relationship between lutein consumption and cataract risk. Some key findings of those studies include: (a) subjects consuming luteinrich spinach at least 5 times a week had a 39% lower risk of cataract extraction compared to those whose spinach consumption was once a week (Hankinson et al., 1992); (b) subjects on a lutein supplementation of 1.3 mg/day had a 50% lower risk of developing nuclear cataract compared to those receiving 0.3 mg lutein per day (Lyle, Mares-Perlman, Klein, Klein, & Greger, 1999); and (c) the 79 prevalence of posterior subcapsular cataract was 50% lower in 66e75 year old subjects with a plasma lutein concentration greater than 0.20 mmol/L when compared to those whose lutein plasma levels were less than 0.14 mmol/L (Gale, Hall, Phillips, & Martyn, 2001). Given the delicateness of the eye and the invasive nature of strategies for determining and quantifying metabolic products in the retina and the lens, it is impractical to directly measure the effect of eye lutein concentration on the incidence of ocular diseases in living subjects. A key study reported direct measurement of the actual concentration of lutein and zeaxanthin in the macular pigment of donor eyes with and without AMD (Bone et al., 2001). The study concluded after examining 56 retinas each from AMD and control subjects that those control subjects with the highest amount of lutein are 82% less inclined to develop AMD than those with the lowest levels of the xanthophylls. Observational studies are sufficient for the purpose of demonstrating links between nutrient supplementation and tissue concentrations of a particular compound with disease risk, but fall short of establishing a direct cause-and-effect relationship between the consumption of a particular nutrient and a particular salutary advantage (AlvesRodrigues & Shao, 2004). Therefore, intervention studies are used to establish causality. For example, the Age-Related Eye Disease Study Research Group (AREDSRG) investigated the effect of lutein on AMD progression in nearly 4000 patients over a six-year period (Kassoff et al., 2001). In this work, which investigated the impact of diet supplements on ocular disease progression, an oral antioxidant supplement was used because lutein supplements were not yet commercially available at the time the study commenced. The study concluded that the antioxidant supplement, which contains lutein significantly delayed the progression of AMD in patients. Another study, the Lutein Antioxidant Supplementation Trial (LAST), modelled after the AREDSRG trial, administered a daily regimen of 10 mg lutein, 10 mg lutein plus a mixed antioxidant formula or placebo to 90 AMD patients for 1 year (Richer et al., 2002). The study results indicated that patients who received the pure lutein supplement showed marked improvements in several objective parameters of visual function including contrast sensitivity, visual acuity and glare recovery when compared to those on the placebo supplement. In a similar study, it was reported that among three distinct groups of cataract subjects receiving a daily dose of 100 mg a-tocopherol, 15 mg lutein, or placebo respectively for 24 months, significant improvements in glare sensitivity and visual acuity were recorded for the lutein supplementation group relative to the group which received either the placebo or atocopherol (Olmedilla et al., 2003). More recently, other intervention studies have established that (a) 10 mg daily lutein or zeaxanthin supplements over a 48-week period significantly improved retinal functions, as determined by means of multifocal electroretinograms, in early AMD patients (Ma et al., 2012a); (b) lutein supplementation significantly increased visual acuity and macular function as measured by microperimetry after a 6-month administration period (Weigert et al., 2011); (c) a 42 and 41% lower risk of developing age-related nuclear cataract was found for people in the highest tertiles of plasma lutein and zeaxanthin concentrations, respectively compared to subjects in the lowest tertiles of both xanthophylls (Karppi, Laukkanen, & Kurl, 2012); (d) 0.5 mg/kg body weight daily lutein supplementation reduced the incidence and severity of cataracts in diabetic male Wistar rats but not in the control that received a placebo (Arnal et al., 2009); and (e) diets rich in lutein and zeaxanthin moderately reduced the prevalence of cataract in women aged 50e79 in contrast to women of the same age range who were placed on diets not containing the oxycarotenoids (Moeller et al., 2008). Additionally, a human intervention study has demonstrated that lutein supplementation resulted in an increase in MPD in patients 80 I.D. Nwachukwu et al. / Trends in Food Science & Technology 49 (2016) 74e84 Table 1 Selected observational studies on the relationship between lutein and AMD. Parametre(s) measured & methodology Results References Association between lutein supplementation and MPD in early AMD patients using a flicker-based technique Effect of L þ Z supplementation on MPD in early AMD patients. MPD in early AMD patients receiving lutein supplements were significantly higher than in the control group on placebo. MPD significantly increased in a dose-dependent manner among patients receiving L þ Z supplementation unlike the control subjects receiving the placebo. Serum lutein level increased linearly with increasing doses of lutein supplement as did MPD, while there was no significant increase in both parameters in the control placebo-receiving group Increase in the density of the macular pigment was observed for the group receiving carotenoid supplements in contrast to the control group which received a placebo. This study also demonstrated for the first time that meso-zeaxanthin contributes to the density of the macular pigment, and thus may offer protection against ocular diseases. Average macular pigment levels substantially greater in patients with AMD receiving a L supplement (4 mg/day) compared to patients not receiving a supplement. Substantial directly proportional relationship between serum L, serum Z and adipose tissue L, and MPD in male subjects with a high consumption of fruits and vegetables. Substantial negative correlation between increasing L concentration in the central region of the retina and risk of developing AMD. After adjustments for age and sex, healthy subjects with the highest L þ Z levels were found to be at 82% lower risk of developing AMD compared to those with the least L þ Z levels. Significant direct proportional relationship between L þ Z consumption and serum L þ Z levels, respectively, and MPD (Murray et al., 2013) (Ma et al., 2012b) Effect of various doses of lutein on serum lutein level (measured by HPLC) and MPD (measured by heterochromatic flicker photometry) Effect of LþZþ meso-zeaxanthin supplementation on macular pigment optical density as measured by heterochromatic flicker photometry Effect of L supplementation on MPD in AMD patients using resonance Raman spectroscopy Associations between MPD and serum L, serum Z, and adipose tissue L by means of spectral fundus reflectance. Associations between L þ Z concentration in the retina (determined by HPLC) and MPD, and relationship between MPD and AMD risk. Relationship between dietary L þ Z intake vs serum L þ Z levels (determined via HPLC) vs MPD (Bone & Landrum, 2010) (Bone et al., 2007) (Bernstein et al., 2002) (Broekmans et al., 2002) (Bone et al., 2001) (CurranCelentano et al., 2001) Relationship between dietary intake of carotenoids and relative AMD Increasing dietary intake of L þ Z was most strongly associated with decreasing (Seddon et al., risk. risk of developing AMD. 1994) Abbreviations: L, lutein; Z, zeaxanthin; AMD, Age-related macular degeneration; MPD, Macular pigment density. with retinitis pigmentosa although the contribution of the oxycarotenoid to vision improvement in this particular study does not seem to be as significant as the results recorded for AMD and cataract (Aleman et al., 2001). It is important to mention that most of the observational and intervention investigations focused on lutein alone (due to its significantly higher amounts in the serum, ocular tissues and most food sources) or a combination of lutein and zeaxantin (Bone et al., 1997; Khachik et al., 1997). 6.1. Spotlight on meso-zeaxanthin Like lutein and zeaxanthin, the carotenoid meso-zeaxanthin, is believed to play a critical role in visual function (Johnson, 2014). Together, the three carotenoids make up the macular pigment with meso-zeaxanthin pre-dominating at the epicenter of the macula (Nolan, Meagher, Kashani, & Beatty, 2013). It has been recently suggested that meso-zeaxanthin could be an important tool in chemo-preventive strategies given its anti-inflammatory potential (Firdous, Kuttan, & Kuttan, 2015). The long held notion that mesozeaxanthin is ‘non-dietary’ or rarely present in the human diet as maintained by various investigators (Johnson, 2014; Rasmussen, Muzhingi, Eggert, & Johnson, 2012) has been recently challenged (Nolan et al., 2013, 2014). Although a 1986 study had detected the presence of meso-zeaxanthin in 21 edible fish species (Maoka, Arai, Shimizu, & Matsuno, 1986), another study which reported the presence of meso-zeaxanthin in Californian and Mexican hen eggs did not find the carotenoid in fish and seafood (Rasmussen et al., 2012). Apart from the on-going debate on the dietary origin of meso-zeaxanthin and its prevalence in the human food chain, the thinking that meso-zeaxanthin is only available in the retina as a consequence of its bioconversion from retinal lutein (Johnson, Neuringer, Russell, Schalch, & Snodderly, 2005; Johnson, 2014) has also become an issue of contention (Bernstein, Johnson, Neuringer, Schalch, & Schierle, 2014; Nolan et al., 2013). Given the importance of meso-zeaxanthin as an integral component of the macular pigment and the well-documented role of the macular pigment in promoting visual health, it is imperative that the questions surrounding its retinal origin be definitively answered. The issues raised concerning the validity of the methodologies employed by (Rasmussen et al., 2012) and (Johnson et al., 2005), as well as the possible contamination of the lutein supplement used in the influential rhesus monkey study (Johnson et al., 2005) need to be convincingly laid to rest. Similarly, the concerns (Rasmussen et al., 2012) about the accuracy of the data obtained by (Maoka et al., 1986) as a result of “problematic methods” (Bernstein et al., 2014) cannot be dismissed without more supportive data. The recent investigation (Nolan et al., 2014) which reported the presence of meso-zeaxanthin in the skin of various tested fish species but not in fruits and vegetables is welcome. However, more studies conclusively demonstrating the presence (or absence) of this carotenoid in other fruits, vegetables and other common components of the regular diet are warranted (Bernstein et al., 2014). 7. Mechanism of action and structure-function properties The mechanisms of action of lutein in eye health are considerably well elucidated when compared to most carotenoid functions. Owing to the exclusive presence of lutein and zeaxanthin in the macula and lens of the eye at concentrations several thousand-folds higher than those of their respective serum levels (Collins, Olmedilla, Southon, Granado, & Duthie, 1998), it was possible to establish a biologically plausible mechanism of action for the oxycarotenoids as photo-protectants and antioxidants during early studies of the xanthophylls. Firstly, lutein and zeaxanthin are coloured compounds and are able to absorb light in the visible region of the electromagnetic spectrum, thus protecting macular and lens photoreceptors from photochemical damage (Kijlstra, Tian, Kelly, & Berendschot, 2012; Krinsky, 2002). Secondly, the broad wavelength for absorption (400e475 nm) (Krinsky, 2002), which peaks around 450 nm (Junghans, Sies, & Stahl, 2001) enables the compounds to I.D. Nwachukwu et al. / Trends in Food Science & Technology 49 (2016) 74e84 attenuate light of short wavelengths (Krinsky, 2002; Sparrow & Kim, 2010). This property helps to reduce blue light-mediated chromatic aberration and scatter in the fovea (Krinsky, 2002) and is facilitated by (i) the conjugated double bonds in xanthophylls (Kijlstra et al., 2012), and (ii) the hydroxyl substituents on their ionone rings, which enable the compounds to assume a certain conformation in eye tissues by forming hydrogen bonds with polar head groups at the surfaces of ocular membranes (Bone & Landrum, 1984; Gabrielska & Gruszecki, 1996; Sujak et al., 1999). In separate studies with dichroic and 1H NMR spectroscopy, lutein and zeaxanthin have been found to adopt a unique orientation perpendicular to the plane of the bilayer membrane in ocular tissues, which as aforementioned is aided by the eOH groups, unlike other carotenoids such as b-carotene, lycopene and b-cryptoxanthin (Bone & Landrum, 1984; Gabrielska & Gruszecki, 1996; Sujak et al., 1999). Additionally, the conjugated double bonds of lutein and zeaxanthin play a critical part in their functional roles as photoprotective agents and antioxidants (Stahl et al., 1997). While the oxycarotenoids possess the capacity to scavenge free radicals in general, they preferentially and primarily quench photo-induced singlet oxygen, thus acting as antioxidants (Winkler, Boulton, Gottsch, & Sternberg, 1999) and consequently protecting tissues from light-initiated oxidative damage (Krinsky, 2002). Furthermore, the extended conjugation system of lutein and zeaxanthin (Fig. 2) encourages the delocalization of p electrons and may reduce the electron densities of the xanthophylls (Sparrow & Kim, 2010). Thus, the polyene chains of lutein and zeaxanthin hardly participate in electrophilic chemical reactions involving singlet oxygen but instead exert most of their photoprotective and antioxidant effects by physical quenching (direct energy transfer between the molecules) (Sparrow & Kim, 2010). 8. Lutein health claims Recently, the European Food Safety Authority (EFSA) concluded that the evidence collectively submitted by certain stakeholders for the approval of a health claim for lutein fell short of the standard required “to establish a cause and effect relationship between lutein and maintenance of normal vision” (EFSA Panel on Dietetic Products, Nutrition and Allergies., 2011; EFSA Panel on Dietetic Products, Nutrition and Allergies., 2012). In response to the EFSA verdict, two interested industry stakeholders (Kemin and DSM Nutritional Products) decided to “seek further clarification” from EFSA since the language in the denied claim considerably differs from the claim application submitted to EFSA (Frederiksen, 2012). Similarly, about a decade ago, the US FDA denied a qualified health claim which sought to positively correlate lutein consumption with reduced risks of AMD and cataract formation (Schneeman, 2005). Conversely, the Brazilian Health Surveillance Agency (ANVISA) and the Natural Health Product unit of Health Canada have authorized an eye health-maintaining antioxidant claim for lutein (Frederiksen, 2012; Health Canada, 2014). It therefore seems to be a case of mixed fortunes for lutein health claim applications. In spite of the volume of studies demonstrating a relationship between lutein consumption and a reduction in the incidence of eye diseases, health claim applications submitted on the basis of these studies have not enjoyed ubiquitous success. While every effort is taken to make the procedure for health claims approval in various (trans-)national jurisdictions simple and straightforward, the process and claims language could prove quite complex and even confusing, if not entirely opaque for industry stakeholders. For instance, although Kemin and DSM Nutritional Products applied for the authorization of the following health claim for lutein: “Lutein, a constituent of the retina and the lens, contributes to protecting these tissues from oxidative damage”, the health claim application 81 was not authorized by EFSA because it failed “to establish a cause and effect relationship between lutein and maintenance of normal vision,” (EFSA Panel on Dietetic Products, Nutrition and Allergies., 2011; EFSA Panel on Dietetic Products, Nutrition and Allergies., 2012; Frederiksen, 2012). Nevertheless, if an inconsistency or impreciseness in health claim language was responsible for denying the lutein health claim in the EU, the same cannot be said for the US where the FDA decided that “there is no credible evidence to support qualified health claims for Xangold® lutein esters, lutein, or zeaxanthin and reduced risk of age-related macular degeneration or cataract formation” (Schneeman, 2005). The argument could be made, however that since the US regulatory body reached that conclusion in 2005, seven years before the EFSA decision, it did not have the same amount of data at its disposal that EFSA did. 9. Conclusion and future directions A number of recent studies evince a possible role for lutein and zeaxanthin in cognitive function (Feeney et al., 2013; Johnson et al., 2013; Vishwanathan et al., 2014). For instance, one of the investigations conducted as part of the Irish Longitudinal Study on Aging determined that older adults with higher macular pigment optical density (MPOD) had significantly better results in various indices of cognitive function compared to those with lower MPOD (Feeney et al., 2013). Although these early pointers to a role in cognitive function for the xanthophylls are encouraging, no direct cause-and-effect relationship has been established between their consumption and an improvement in cognitive performance, since most of the studies carried out to date are correlative (Johnson, 2014). More intervention studies are therefore needed to clearly establish the extent to which lutein and zeaxanthin influence cognitive function. Lutein's contributions to the promotion of eye health have been amply reported in several human observational and intervention studies, which demonstrated credible links between a consumption of the dietary carotenoid and reduced risks of eye diseases, particularly AMD and/or cataracts onset or progression. The abundance of lutein and zeaxanthin in commonly available green leafy vegetables makes the intake of both dietary carotenoids as part of a regular healthy diet an attractive option for avoiding or at least delaying the onset of these eye diseases. Furthermore, when it is considered that 90% of visually impaired people live in developing countries where functional healthcare systems are a rarity, it is easy to see the practicability of a diet-based approach to eye disease control, as one of the strategies for reducing the incidence of avoidable blindness worldwide. Furthermore, additional studies which would more clearly show how and to what extent lutein attenuates the onset and/or progression of eye diseases are needed. This should hopefully open the door to a reconsideration of lutein health claims by national health regulatory bodies. Even in the absence of formal health claims, it is difficult to completely ignore currently available data pointing to a link between lutein/zeaxanthin and the promotion of vision. Therefore, it is in the interest of the health of the general public for experts such as dietitians, nutrition counselors, educators and physicians to emphasize such benefits in their everyday interactions with the lay population. Acknowledgment CCU's Natural Science and Engineering Research Council of Canada (NSERC) Discovery Grant, IDN's Manitoba Health Research Council (Research Manitoba) Scholarship, and Graduate Enhancement of Tri-Council Stipends (GETS) from the University of Manitoba, and REA's NSERC Discovery Grant and Manitoba Agri-Food 82 I.D. Nwachukwu et al. / Trends in Food Science & Technology 49 (2016) 74e84 Research and Development Initiative (ARDI) funding are all gratefully acknowledged. References Abbas, K. A., Mohamed, A., Abdulamir, A. S., & Abas, H. A. (2008). 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