Download Lutein and zeaxanthin: Production technology, bioavailability

Trends in Food Science & Technology 49 (2016) 74e84
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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
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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.
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