Archives of Biochemistry and Biophysics
Vol. 385, No. 1, January 1, pp. 28 – 40, 2001
doi:10.1006/abbi.2000.2171, available online at http://www.idealibrary.com on
MINIREVIEW
Lutein, Zeaxanthin, and the Macular Pigment
John T. Landrum* ,1 and Richard A. Bone†
*Department of Chemistry and †Department of Physics, Florida International University, Miami, Florida 33199
Received September 11, 2000, and in revised form October 18, 2000; published online December 7, 2000
The predominant carotenoids of the macular pig-
ment are lutein, zeaxanthin, and meso-zeaxanthin.
The regular distribution pattern of these carotenoids
within the human macula indicates that their deposi-
tion is actively controlled in this tissue. The chemical,
structural, and optical characteristics of these carote-
noids are described. Evidence for the presence of mi-
nor carotenoids in the retina is cited. Studies of the
dietary intake and serum levels of the xanthophylls
are discussed. Increased macular carotenoid levels re-
sult from supplementation of humans with lutein and
zeaxanthin. A functional role for the macular pigment
in protection against light-induced retinal damage
and age-related macular degeneration is discussed.
Prospects for future research in the study of macular
pigment require new initiatives that will probe more
accurately into the localization of these carotenoids in
the retina, identify possible transport proteins and
mechanisms, and prove the veracity of the photopro-
tection hypothesis for the macular pigments. © 2001
Academic Press
Key Words: macular pigment; carotenoid; lutein; ze-
axanthin; meso-zeaxanthin; photoprotection.
The macular pigment (MP) 2 of the human retina is
visibly discernible as a yellow spot (macula lutea) in
the central retina (Fig. 1) (1– 4). The most striking
characteristic of the MP is its ability to absorb and
attenuate blue light striking the retina (5). One func-
tional benefit of its presence is that it reduces chro-
matic aberration in the eye (6). The well-known psy-
1
To whom correspondence should be addressed. Fax: (305) 348-
3772. E-mail: landrumj@fiu.edu.
2
Abbreviations used: MP, macular pigment; MPOD, macular pig-
ment optical density; HFP, heterochromatic flicker photometry;
AMD, age-related macular degeneration.
28
chophysical phenomena, Haidinger’s brushes and
Maxwell’s spot, are the result of blue light absorption
by the MP (7–12). Haidinger’s brushes are explained by
the dichroic properties of the conjugated polyene caro-
tenoids that compose the MP (13). The prevalent, but
by no means universal, view of the MP is that its
primary purpose is to function in a photoprotection role
within the retina (14, 15). The extent to which MP
serves this photoprotective role remains to be firmly
established, though several lines of investigation sup-
port this idea. Carotenoids are well known to serve an
antioxidant role in natural systems, especially those
where light and oxygen are simultaneously present, as
in plants (16 –18). This too is a role that may be played
by the macular carotenoids. The retina is a tissue that
is abundantly illuminated and has large respiratory
demands for oxygen (19).
What are the distinguishing characteristics of the
macular carotenoids, lutein and zeaxanthin, that ac-
count for their virtually exclusive accumulation within
the primate macula? What makes them functionally
unique? A preface to this discussion is warranted. The
suggested functions of the MP mentioned above have
not yet been proven. Undoubtedly, the function of the
MP must explain why lutein and zeaxanthin, as op-
posed to ␤-carotene or any of the other abundant caro-
tenoids, are present in the macula. From a simplistic
view, function in the chemical world is dependent upon
structure. The chemical structure embodies the physi-
cal shape, charge distribution, and energy levels of a
molecule. In the biological world, function manifests
itself in anatomy, both macroscopic and microscopic.
Biological function is the complex behavior of the bio-
chemical systems that result from the spatial relation-
ships of the anatomy that limit or promote biochemical
processes. To recognize the function of the MP we must
understand the anatomy, chemistry, and physiology of
the retinal carotenoids. In this minireview we will fo-
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 MINIREVIEW
FIG. 1. A fundus view of the human retina centered at the fovea
and extending to just beyond the optic disk. Shading at the center
represents the visible MP. Carotenoids are present throughout the
retina at levels below detection by visual inspection.
cus on each of these in turn. Our objective here will not
be to prove the function of the macular carotenoids, so
much as to describe what is known, and how it provides
the foundation for hypothetical function. In doing this
we hope to identify what new information is needed to
expand our knowledge in each of these areas and to
help identify new and informative paths for investiga-
tion.
FIG. 2. The structures of the major carotenoid components found in the human macula.
29
THE CHEMICAL IDENTITY AND STRUCTURE OF THE
MACULAR PIGMENT COMPONENTS
Structure
The macular pigment is composed principally of
three isomeric carotenoids, lutein, zeaxanthin, and
meso-zeaxanthin (20 –24). They represent roughly 36,
18, and 18% of the total carotenoid content of the
retina. (See below for a discussion of the minor carot-
enoid components of the MP.) Lutein and zeaxanthin
share the carbon skeleton and bonding framework of ␣-
and ␤-carotene, respectively. As seen in Fig. 2 the
bonding frameworks of these two carotenoids may ap-
pear, at first glance, to be identical. The chemical for-
mulas of lutein and zeaxanthin are chemically distin-
guished from one another in important ways. Zeaxan-
thin exists in three stereoisomeric forms, the result of
the two stereocenters at carbons 3 and 3⬘ the sites of
the secondary hydroxyl groups (25). Lutein can exist in
eight stereoisomeric forms as a result of the presence of
three stereocenters at the 3, 3⬘, and 6⬘ carbon atoms. In
addition, the hydroxyl group at carbon 3⬘ of lutein is
allylic (25).
Characterization
The identity of lutein and zeaxanthin found in the
macula has been well established (20 –24, 26 –29). Sev-
eral complementary chemical methods have unambig-
uously established the identities of these two carote-
noids: mass spectrometry (22), UV-visible spectrome-
try (21, 28), match of retention times by co-elution with
authentic standards on multiple chromatographic sta-
30 MINIREVIEW
tionary phases (21, 22), and chemical derivatization
(21, 22).
Stereochemistry
The stereochemical isomerism possible for both lu-
tein and zeaxanthin poses important chemical ques-
tions. Are the stereochemical structural differences
significant to any separate and distinct functional role
these molecules may individually fulfill? Derivatiza-
tion of lutein and zeaxanthin with benzoic anhydride to
form dibenzoates or with (S)-(⫹)-1-(1-napthyl) ethyl
isocyanate to form dicarbamates enables the stereoiso-
mers to be separated chromatographically (22, 23, 30,
31). For example, zeaxnathin dibenzoates elute on
chiral phase HPLC in the order RS, RR, then SS, while
the zeaxanthin dicarbamates elute on normal phase
HPLC in the order, SS, RS, then RR (22, 27). Using
these two methods, Bone and Landrum (22) have
shown which stereoisomers of lutein and zeaxanthin
are present in the retina (see Fig. 2). Retinal lutein is
composed solely of the most abundant natural stereo-
isomer, (3R,3⬘R,6⬘R)-␤,⑀-carotene-3,3⬘-diol. Zeaxan-
thin within the retina was demonstrated to be primar-
ily composed of two stereoisomers. R,R-zeaxanthin,
(3R,3⬘R)-␤,␤-carotene-3,3⬘-diol, is the most abundant
natural form and is approximately 50% of the zeaxan-
thin present. Most of the remaining retinal zeaxanthin
was shown to be chromatographically indistinguish-
able from R,S-zeaxanthin. R,S-meso-zeaxanthin,
(3R,3⬘S)-meso-␤,␤-carotene-3,3⬘-diol, is not a natu-
rally abundant form of zeaxanthin and its presence
poses interesting questions (25, 30). Where does retinal
meso-zeaxanthin originate? Is it present in the diet or
serum? Where is meso-zeaxanthin formed in the body?
By what biochemical process? The anatomical distribu-
tion of the macular components provides several clues
to these questions (see The Anatomy of the Macular
Pigment, below). Data show that small quantities of
the S,S-zeaxanthin, (3S,3⬘S)-␤,␤-carotene-3,3⬘-diol,
stereoisomer, ca. 7% of the zeaxanthin, are also
present. This observation is based on UV-visible spec-
troscopy and on the correct position of the chromato-
gram peaks relative to RR and RS peaks regardless of
which derivative, dibenzoate or dicarbamate, is used.
S,S-zeaxanthin, like meso-zeaxanthin, is not naturally
abundant (25, 30).
Identification of carotenoid stereoisomers is often as-
sisted by determination of the circular dichroism spec-
tra (32). Because of sample size requirements and tech-
nical challenges required to separate the individual
isomers, circular dichroism spectra have not yet been
reported for either R,R-zeaxanthin or lutein purified
from the human macula. These would be welcome ad-
ditions to the characterization of the macular carote-
noids (32). Circular dichroism is not of significant util-
FIG. 3. Chromatograms of the retinal extracts obtained from por-
tions of a single human retina; disk 0 –5°, L : Z ⫽ 1 : 1.6; medial
annulus 5–19°, L : Z ⫽ 1.4 : 1; outer annulus 19 –38°, L : Z ⫽ 2 : 1. L ⫽
lutein, Z ⫽ zeaxanthin, IS ⫽ internal standard (monohexyllutein ether).
ity for the characterization of meso-zeaxanthin because
it is achiral and consequently lacks optical activity.
The consequences of these stereochemical and geo-
metrical differences between the macular carotenoids
may be significant to our understanding of the function
of the MP physiology. A major question that we would
hope to answer is this: are these stereochemical struc-
tural differences functionally significant? It has been
demonstrated that lutein and zeaxanthin differ in their
behavior in model membrane systems (33, 34). Lutein,
in which carbon 6⬘ at the junction between the polyene
chain and the ⑀-ring is tetrahedral, appears to have a
different preferred orientation in membrane systems
as compared to zeaxanthin (35). Zeaxanthin tends to
span the bilipid layer occupying a site that lies perpen-
dicular to the membrane surface. Lutein can appar-
ently insert itself into the membrane in a nonorthogo-
nal manner.
Minor Carotenoids
In addition to lutein and zeaxanthin, several minor
peaks have been observed in HPLC of retinal extracts
(see Fig. 3). Khachik has reported the identities of
several of these components to be carotenoids (24). Of
these, the Z- or cis-isomers are present in only minute
amounts. The presence of 9- and 13-Z isomers of both
lutein and zeaxanthin is indicated by peaks having
elution times slightly longer than those of the all-E
isomers (24, 36). The occurrence of these Z isomers in
small quantities is also a common feature of human
tissues (36). It is hard to postulate a functional signif-
icance to the observation of Z-isomers in the retina;
however, their membrane behavior is distinct from
that of the all-E carotenoids (33). Z-carotenoids are
unable to span bilipid membranes because of their
 MINIREVIEW 31

double bond (42). Such a description exaggerates the

role played by the ␤-ring double bond.
The UV-visible spectra of lutein and zeaxanthin, in
ethanol, are qualitatively the same (see Fig. 5). They
have a typical carotenoid signature possessing a cen-
tral maximum flanked at longer wavelengths by a sec-
ondary maximum and a distinct shoulder at shorter
wavelengths. These transitions are numbered I, II, and
III from short wavelength to long wavelength (41). The
presence of the modest added interaction of the second
FIG. 4. Conformation of the ␤-ring end-group showing the ca. 40° ␤-ring double bond in zeaxanthin with the extended
angle between the plane of the polyene chain and the ring double conjugation of the polyene chain very slightly lowers
bond. the energy separation between the ground state and
excited state. This small effect results in a roughly
6-nm red-shift in the absorption maximum of zeaxan-
nonlinear geometry. When incorporated into membrane
thin (␭ max ⫽ 451 nm in ethanol) when compared to that
systems they disrupt the packing order of the alkyl
of lutein (␭ max ⫽ 445 nm) (Fig. 5). The other optically
chains and alter membrane fluidity. Oxo-lutein, (3-hy-
significant difference in these two carotenoids is the
droxy-␤,⑀-carotene-3⬘-one), epilutein, and ⑀,⑀⫺carotene-
spacing between peaks II and III, illustrated by the
3,3⬘-dione were also reported by Khachik to be present in
intervening minimum, and the ratio of intensities that
the retina (and also serum, see below) (24, 36). When
results. For zeaxanthin, peaks II and III are both some-
retinal carotenoid extracts are analyzed by HPLC two
what broader and the spacing between them is smaller,
peaks are often observed to preelute lutein on C-18 re-
resulting in a less distinct maximum for III with a
versed-phase columns. See peaks labeled M1 and M2
more shallow minimum between peaks II and III. For
in Fig. 3. Peak M2 elutes with a retention time that is
zeaxanthin, ␭ III–␭ II is 26 nm and the II/III ratio is 38%.
consistent with that of authentic oxo-lutein prepared
The corresponding spacing for lutein is 29 nm and a
by partial synthesis in our laboratory (37). Together
distinct minimum between II and III is observed in
these minor MP components may approach 20% of the
well-purified samples. The lutein II/III ratio is 60%.
total carotenoid present within a given portion of the
For lutein, peak I is a fully distinct maximum, not a
retina. There are two components in the serum that
shoulder. Oxo-lutein (M2 in Fig. 3) has a spectrum that
chromatographically match M1 and M2 (36 –38). They
is virtually indistinguishable from that of lutein. The
have been shown to be metabolites of lutein (39). More
visible spectrum of the MP peaks at ⬃460 nm roughly
information on this topic is combined with a broader
10 nm red-shifted relative to that of a comparable
discussion of MP metabolism, and follows description
mixture of lutein and zeaxanthin (28). This red-shift is
of the anatomical distribution of the MP components.
reproduced by incorporation of lutein and zeaxanthin
into phosphatidyl lipid liposomes (13).
SPECTROSCOPIC PROPERTIES OF THE MACULAR
CAROTENOIDS
The most obvious characteristic of all carotenoids is
their intense coloration (40 – 42). This is the result of
the extensive conjugation in the polyene chain. The
color differences of carotenoids arise from the differ-
ences in the number of conjugated bonds. Lutein and
zeaxanthin differ very slightly in color. Purified zeax-
anthin typically has a rosy appearance not observed in
lutein. In both of these carotenoids the number, n, of
fully conjugated double bonds in the polyene chain is 9.
The two ␤-end group double bonds of zeaxanthin are
partially, but not fully, conjugated due to the steric
hindrance of the methyl substituents on the ring.
Steric hindrance constrains the dihedral angle be-
tween the plane of these double bonds and the rest of
the polyene chain to about 40° (see Fig. 4). Many
sources describe the extent of conjugation of zeaxan- FIG. 5. Spectra of lutein and zeaxanthin, in ethanol, illustrate the
thin as consisting of 11 double bonds and that of lutein characteristic differences in the absorption properties of the two
as 10 double bonds including the influence of the ␤-ring carotenoids.
32 MINIREVIEW
CHEMICAL REACTIVITY
Carotenoids are frequently cited for their ability to
function as antioxidants in biological systems (16, 17,
43). This function is proposed to occur by at least two
different mechanisms and is dependent upon the na-
ture of the oxidant species. Singlet oxygen is known to
be quenched by energy transfer to the carotenoid, gen-
erating a triplet state carotenoid that is able to harm-
lessly relax through vibrational transitions and colli-
sion without destructive bond breaking (18, 44 – 46).
Radical species, once generated, are capable of oxidiz-
ing an organic molecule via a mechanism that either
occurs with hydrogen atom extraction or by direct ad-
dition. Such mechanisms are chain reactions that re-
generate an active radical capable of further destruc-
tive reactivity. Carotenoids may interfere with the
chain reaction by either reducing the rate of chain
propagation or by participating in a chain terminating
chemical event (43). Carotenoids are believed to react
directly with peroxy radicals that are characteristic of
lipid autooxidation, producing a highly resonance-sta-
bilized carotenoid radical in which the unpaired elec-
tron is no longer oxygen-centered, rather it is delocal-
ized over the conjugated polyene. The enhanced stabil-
ity of this radical bestows on it a very long lifetime,
providing ample opportunity to react with one of the
many naturally abundant reductants present within
the tissues. These may include tocopherol and/or ascor-
bic acid (47). Both lutein and zeaxanthin have been
demonstrated to function well as antioxidants with
similar protective ability to that of other carotenoids
(48, 49).
Probably the most important distinction between lu-
tein and zeaxanthin is that the allylic hydroxyl of lu-
tein is much more easily oxidized than the secondary
hydroxyl groups present in zeaxanthin. We should em-
phasize the distinct difference that is involved in the
2e ⫺ oxidation of the hydroxyl groups in the xantho-
phylls as compared to the one electron oxidation which
is a characteristic reaction of all hydrocarbon carote-
noids. The 1e ⫺ oxidation of carotenoids results in the
formation of a resonance-stabilized radical cation (50,
51). Chemical oxidation of the 3⬘ hydroxy group of
lutein with MnO 2 produces 3⬘-oxo-lutein, 3R,6⬘R-3-
hydroxy-␤,⑀-carotene-3⬘-one, in an 80% yield (52). Ze-
axanthin upon reaction with MnO 2 also is oxidized but
the reaction proceeds only sluggishly and results in
oxidation at both end groups and extension of the con-
jugation of the alkene system (53). This 6e ⫺ oxidation
produces rhodoxanthin, a retro-carotenoid, in yields of
5–10% with considerable cleavage of the starting xan-
thophyll. Zeaxanthin secondary hydroxyl groups are
more resistant to oxidation than the allylic hydroxyl
group of lutein. In lutein the allylic hydroxyl may serve
a protective function preventing oxidative cleavage of
FIG. 6. Graphical variation of the absorbance of the macular pig-
ment with eccentricity in the macaque monkey as measured by
microspectrophotometric methods (adapted from Snodderly et al.
IOVS 32, 268 –279 (1991)).
the polyene. These chemical processes are not suffi-
ciently well understood and kinetic as well as thermo-
dynamic factors may be important to biological mech-
anisms of oxidation. With extended exposure to strong
oxidizing agents such as MnO 2, both lutein and zeax-
anthin are oxidatively cleaved and bleached. In the
retina the ratio of lutein to meso-zeaxanthin suggests
that lutein is the primary source of meso-zeaxanthin
within the retina. If this is so, oxo-lutein may be an
intermediate in the conversion mechanism.
THE ANATOMY OF THE MACULAR PIGMENT
A broad picture of the anatomy of the macular pig-
ment has now been well described in the literature
(19). The concentration of the macular pigment rises
remarkably to almost 1 mM within the central macula
(38). For perspective, this corresponds to more that 3
orders of magnitude above that in normal serum (54,
55). Using microspectrophotometry on thin retinal sec-
tions, Snodderly and co-workers have described the
spatial distribution of the carotenoids in the central
few millimeters of the retina (29, 56, 57). Their results
show that in the macaque retina the concentration of
the macular carotenoids reaches a peak at the center of
the fovea, increasing dramatically over the space of
only ⬃2 mm (see Fig. 6) (29, 57). This is completely
consistent with measurements of the concentration
profile determined by HPLC (23, 26). Snodderly et al.
demonstrated with microspectrophotometry that the
carotenoids are asymmetrically distributed across the
depth of the retina and are found in the greatest con-
centrations in the inner retinal layers (Fig. 7) (56).
Until very recently it has been hypothesized, but un-
proven, that substantial quantities of carotenoids
would be found in the photoreceptors. Solid evidence
has recently been obtained that the macular carote-
noids are present in the rod outer segments (58, 59). An
important question that must now be answered is what
 MINIREVIEW 33

FIG. 7. Cross-sections through the macaque macula show the asymmetric distribution of the macular pigment (dark region, top). Contours
(middle) show the variation in absorbance graphically superimposed on the cross-section outline. Regions of isodensity are indicated and
illustrate the anatomic fine structure of the macular pigment distribution (Bottom) (Snodderly et al. IOVS, 25, 674 – 685. (1984)).

is the absolute concentration of the carotenoids found
in the photoreceptors and precisely where are they
within the photoreceptors? It has not yet been demon-
strated that cones, like the rods, have a significant
carotenoid content. The dramatic, almost exponential,
increase in concentration that is observed as the foveal
center is approached is accompanied by a surprising
inversion in the ratio of lutein to zeaxanthin (see Figs.
3 and 8) (23, 26). The chromatograms in Fig. 3 are of
the MP carotenoids obtained from three concentric sec-
tions of the retina consisting of an inner disk covering
the range of visual angles 0 to 5° and two concentric
annuli (5 to 19° and 19 to 38°). Zeaxanthin is the
dominant component in the inner disk whereas lutein
dominates in the outer annulus. This trend has been
observed both in the whole retina and in the purified
rod outer segment membranes (59). In Fig. 8 it is seen
that the decrease with decreasing eccentricity in the
abundance of lutein relative to zeaxanthin is accompa-
nied by a corresponding increase in total zeaxanthin.
Meso-zeaxanthin, a carotenoid not found in the normal
human diet, is observed to reach its maximum in the
central macula at the same point where the lutein to
zeaxanthin ratio reaches a minimum (22, 23, 29). The
amounts of the oxidative metabolites, labeled M1 and
M2 in Fig. 3, are also seen to increase in proportion
within the central macula. They represent 22% of the
macular carotenoids in the inner disc. The striking
observation that the proportion of lutein in the MP
decreases with increasing proportion of meso-zeaxan-
thin and that the meso-zeaxanthin is maximal in the
central retina suggests a functional relationship. One
hypothesis to explain the lutein/zeaxanthin ratio and
the presence of meso-zeaxanthin is that zeaxanthin may
34 MINIREVIEW
FIG. 8. Graphical plot of the concentration of the macular pigment
in the human retina as calculated from HPLC measurements (solid
line). The lutein:zeaxanthin ratio varies by a factor of more than 4
over this narrow range of eccentricity (dotted line). The lutein:zeax-
anthin ratio reaches a minimum in the central macula where meso-
zeaxanthin reaches it highest levels and is approximately 50% of the
total zeaxanthin present.
be more effective than lutein at some essential role in the
central macula but is unneeded in the peripheral retina.
Another hypothesis is that lutein undergoes a chemical
oxidation in the central retina. Presumably this oxidation
would not be a functional process. Reduction then results
in conversion to meso-zeaxanthin (24, 37). The system-
atic variation in the lutein/zeaxanthin ratio points
strongly to the existence of a specific biochemical path-
way and the likely existence of enzymes responsible for
the conversion of lutein into meso-zeaxanthin.
We have seen that at the tissue level the geographic
distribution of the macular pigment is well known.
Some details have recently appeared at the cellular
level with analysis of the photoreceptors by Sommer-
burg et al. (58) and Rapp et al. (59). The subcellular
localization of the carotenoids within the nerve axons
of the inner retina and within the photoreceptors re-
mains unknown. Bernstein has suggested that the MP
is bound by a protein (60, 61) possibly tubulin within
the cell whereas Bone and Landrum have provided
evidence based on dichroic properties of the macular
pigment that is consistent with incorporation within
membrane bilayers (13, 28). Crabtree and Adler have
pursued the concept of carotenoid binding by tubulin
and recently reported (62) results of modeling studies
that are consistent with this hypothesis. Because tu-
bulin is oriented axially within the cell axons, binding
of carotenoids to tubulin could also potentially explain
the dichroic characteristics of the macular pigment. The
development of analytical techniques with sensitivity
suitable to enable analysis at the cellular level will be
essential to our achieving a complete explanation of the
function of these carotenoids. While there are geometric
consequences of the structural and stereoisomerism of
these carotenoids, this may or may not be significant to
their possible function. The stereochemical geometry
would be of considerable importance to the binding
strength in a protein– carotenoid interaction but may be
less critical in the more fluid membrane environment.
DEVELOPMENTAL CHARACTERISTICS
The MP is present in the fetal and neonatal retina
(26). The quantitative relationships present in the
adult eye are not found in newborns and do not appear
until about the age of 3 years (26). At birth lutein is the
dominant carotenoid present throughout the retina.
The lutein:zeaxanthin ratio of the newborn retina is
similar to that found in the peripheral retina of adult
eyes. A careful analysis of the maturation of the lutein
and zeaxanthin distribution characteristics of the de-
veloping postnatal retina may provide insight into the
chemical and physiological processes responsible for
this distribution.
Only a modest amount of data have been published
relating MP levels to age. In a study of postmortem
retinas by HPLC of 87 subjects, Bone et al. found no
significant difference in the average MP level between
the 2nd and 9th decade of life (26). Werner has mea-
sured MP optical density (MPOD) and found no change
with increasing age (4). In a longitudinal study, Ham-
mond et al. (63) showed that over a span of as great as
16 years no significant change in MPOD was seen in 2
subjects. In the same study the MPOD of 8 other sub-
jects showed little or no variation over periods ranging
from 1 to 5 years. More recently, Hammond et al. have
reported that MPOD shows a small, statistically sig-
nificant decrease with age in a study group of 217
individuals in the Southwest (64, 65). The Hammond
study shows that there are many individuals in the
senior population whose MP levels are normal or high
when compared to the mean MPOD values for subjects
of all ages. It would be informative if the differences
between low and high MP seniors were studied to
determine their origin. Many factors may have a sig-
nificant influence on this observation including life-
time exposure to blue light, dietary intake of carote-
noids, smoking, and genetic character.
In addition to age, several phenotypes have been
associated with low MP levels. Iris color and sex have
been correlated with MP levels (66, 67). Females and
subjects with light iris color have reduced levels of
macular pigmentation when compared to males and
individuals with dark irises. Smoking also correlates
with lowered MP levels (68). These same factors have
been identified as risk factors for age-related macular
degeneration (69).
 MINIREVIEW
DIETARY INTAKE BY HUMANS
Normal Diet
Carotenoids found in mammalian systems originate
exclusively in the diet. The carotenoids are synthesized
in plants, algae, and bacteria (70). Higher animals are
unable to synthesize carotenoids but make extensive
use of them transforming and transporting them to
serve a variety of functions (42). The normal Western
diet contains 1.3–3 mg/day of lutein and zeaxanthin
combined (71, 72). In the recently completed Canadian
dietary intake study, the consumption of lutein and ze-
axanthin was 1.3 mg/day with a standard deviation of
2.45 mg/day, indicative of the very wide range of dietary
variation in this population (K. Gray-Donald, personal
communication). There is essentially a single dominant
dietary stereoisomer of lutein, (3R,3⬘R,6⬘R)-␤,⑀-carotene-
3,3⬘-diol in the human diet. (So-called epilutein, the
(3R,3⬘S, 6⬘R)-form, is found fairly commonly as a flower
pigment but not in substantial amounts in common di-
etary plants.) Similarly, dietary zeaxanthin is composed
exclusively of the single (3R,3⬘R)-stereoisomer. We have
estimated that the ratio of lutein to zeaxanthin in the diet
ranges from about 7:1 to 4:1 (73).
Depletion
The MP was first demonstrated to be responsive to
diet by Malinow et al. in a study of macaque monkeys
whose diet was deficient in lutein and zeaxanthin (74).
Malinow et al. showed that the macula of macaques
consuming a xanthophyll-depleted diet lacked the
characteristic pigmentation of animals on a normal
diet. There are no reported examples of total depletion
of the MP in human subjects. The identification of
human populations or subpopulations with depleted
MP would be very informative and would provide an
opportunity to study the consequences of depletion.
SUPPLEMENTATION
Effects on Serum Concentration
Several studies have been conducted on the dietary
intake of lutein and zeaxanthin by humans and the
serum concentrations (75–96). In human serum, like
the diet, lutein dominates over zeaxanthin. The ratio of
lutein to zeaxanthin in serum is somewhat variable,
ranging from 2.7 to 4.5:1 and depends upon diet and
individual characteristics such as genetics and lifestyle
(37–39, 81, 93). In a recent study involving 20 subjects,
the normal levels of lutein in serum were found to
range from 1.02 to 4.47 ⫻ 10 ⫺4 mmol/L, with an aver-
age value of 2.46 ⫻ 10 ⫺4 mmol/L (93). The same sub-
jects were found to have zeaxanthin concentrations
that ranged from 0.546 to 1.76 ⫻ 10 ⫺4 mmol/L and
averaged 8.98 ⫻ 10 ⫺5 mmol/L. In a study of two sub-
35
jects taking 30 mg/day of lutein, dramatic increases
over baseline serum lutein levels were observed to
occur, with these levels reaching values ca. 2.1–2.6 ⫻
10 ⫺3 mmol/L (54, 55). Serum levels returned to normal
after the lutein supplement was discontinued following
a first-order decay. The decay half-life was determined
to be between 10 and 14 days (39). In addition to
increases in the lutein concentration, three serum me-
tabolites of lutein were observed to increase with sup-
plementation and similarly decreased upon discontin-
uation of supplementation. The half-lives for appear-
ance of these components were found to be somewhat
longer than lutein, suggestive of a conversion occurring
in the body after absorption. The identities of two of
these have been established as 13-Z-lutein and oxo-
lutein (37, 39). Khachik et al. reported several carote-
noids in human serum that they identified as lutein
and/or zeaxanthin metabolites, including oxo-lutein
and ⑀,⑀-carotene-3,3⬘-dione (24, 36). In studies of zeax-
anthin supplementation, we found that zeaxanthin
had two metabolites, 13-Z-zeaxanthin and a compound
which co-elutes with oxo-lutein but whose visible spec-
trum is decidedly zeaxanthin-like.
Our current understanding can be summarized
briefly as follows: serum levels of lutein and zeaxan-
thin are dependent upon dietary intake, serum levels
associated with the normal diet are far below the max-
imal levels achieved with supplementation, both lutein
and zeaxanthin are metabolized, and oxidized keto-
carotenoids and Z-isomers increase in the serum when
lutein or zeaxanthin supplements are consumed. Anal-
ysis of 20 normal serums demonstrates that a compo-
nent, which appears to match the retention times of
meso-zeaxanthin, was present with an upper limit less
than 7% of the R,R-isomer. The average level was 3%
and for one quarter of those tested it was 0% (97). It
has not been demonstrated whether these apparent
metabolites are accumulated by the macula from the
serum or are produced by oxidation of lutein within the
retina.
Effects on Retinal Concentration
The effect on the MP of supplementation of the nor-
mal diet with lutein or zeaxanthin has been studied in
both primates and humans (54, 55, 93, 96 –99). In one
study, increased levels of zeaxanthin were provided to
rhesus monkeys in the form of carotenoid extracts from
Gou Zi Qi berry (Lycium chinense) (99). Increases in
the amounts of retinal zeaxanthin were observed after
the supplementation period. Hammond and co-workers
observed that human subjects eating diets with in-
creased levels of lutein and zeaxanthin in the form of
corn and spinach had increases in the MP levels at the
end of 15 weeks (96). Landrum et al. studied the effect
of consumption of lutein and zeaxanthin supplements
36 MINIREVIEW
by human subjects (54, 55, 93). They studied supplemen-
tation of the normal diet with 30 mg/day of lutein or
zeaxanthin, and 2.4 mg/day of lutein. For two subjects
taking 30 mg/day of lutein, 20 – 40% increases in MPOD
levels were observed to result from 140 days of supple-
mentation. In a similar study with 30 mg/day of zeaxan-
thin two subjects both showed increases in MPOD with
rates comparable to that observed with lutein. Low-dos-
age supplementation with 2.4 mg/day of lutein for 6
months produced an average increase of 10% in MPOD in
a group of 20 subjects. The inescapable conclusion of
these studies is that MP is demonstrably modulated by
dietary intake of carotenoids. The rate of response ap-
pears to be related to the serum concentration. In all
cases the rate of increase is rather slow, taking extended
periods of supplementation to achieve significant in-
creases, especially at low dosages. The studies by Land-
rum et al. and by Hammond et al. are indicative that MP
increases remain stable for significant time periods,
many months to years, even after supplementation is
discontinued.
Canthaxanthin
While not a major dietary component in humans,
canthanxanthin, ␤,␤-carotene-4,4⬘-dione, is accumu-
lated in the retina. It is of interest to us because of its
metabolism there. Canthaxanthin was used as an oral
tanning agent during the 1970s and 1980s (100 –102).
Consumption of large doses of canthaxanthin can re-
sult in accumulation and crystallization of this carot-
enoid in the retina (100 –102). Like MP, canthaxanthin
has a long half-life in the retina and canthaxanthin
crystals disappear from the retina only after several
years (102). In addition to canthaxanthin, researchers
reported finding that both 4-hydroxy-echinenone (4-
hydroxy-␤,␤-carotene-4⬘-one) and isozeaxanthin (␤,␤-
carotene-4,4⬘-diol) were present in the retinas of mon-
keys fed canthaxanthin (100, 101). Reminiscent of the
variation in the distribution of lutein and zeaxanthin
with eccentricity, the highest amounts of the reduced
forms of canthaxanthin were found in the macula (101).
This study is particularly intriguing because it demon-
strates that the primates are able to reduce
keto-carotenoids to the corresponding alcohols. Oxo-lu-
tein, 3-hydroxy-␤,⑀-carotene-3⬘-one, and ⑀,⑀⫺carotene-
3,3⬘-dione might be expected to be similarly reduced
within the retina. Such reduction steps may be involved
in the formation of meso-zeaxanthin and or epilutein in
the retina. It remains to be proven if this reduction pro-
cess is actually occurring in the human retina to form
either lutein or zeaxanthin from the keto-carotenoids.
MEASUREMENT OF MACULAR PIGMENT
Several methods for the in vivo determination of
MPOD have been reported in the literature (103–111).
The most widely used method and one that is relatively
easy to apply is heterochromatic flicker photometry
(HFP). HFP is a psychophysical technique in which the
subject seeks to eliminate flicker in a visual stimulus
that alternates between two wavelengths, typically
from the blue and green portions of the spectrum. The
technique is dependent upon subject response for ac-
curate and reliable results, and an assumption of equal
spectral sensitivities of the receptors in the fovea and
periphery. This method, while well suited to controlled
laboratory conditions, has significant drawbacks for
application in the clinical environment. A certain
amount of training is required before the subject can be
expected to produce meaningful data. Good vision is a
prerequisite so that subjects with advanced forms of
AMD may find the psychophysical task difficult if not
impossible. The average MPOD at 460 nm as measured
by HFP using a 1° stimulus is between 0.2 and 0.4
absorbance units. Different stimulus sizes will result
in different measured values for the MPOD (4). There
appear to be systematic differences between instru-
ments used by different research groups. As discussed
below, absorption of blue light is a potentially signifi-
cant function of the MP. At normal levels, between 20
and 40% of light at 460 nm is being absorbed in the
macula. In individuals with above normal MPOD, as
much as 90% of light at this wavelength can be ab-
sorbed.
Other techniques that have been utilized, and that
are objective, include photographic measurement of
MP by comparison of fundus images obtained using
green and blue illumination (106). This method has not
been extensively adopted despite the availability of
fundus cameras. The result depends upon the repro-
ducibility of illuminating the eye through the dilated
pupil and is complicated by the differences in optical
clarity of the vitreous humor between adults. Fundus
reflectometry (105) has been utilized and adapted for
the scanning laser ophthalmoscope (108). For such
methods to yield accurate data, effects of absorbing
species, other than MP, present in the retina and the
optical path must be taken into account. These include
the lens and vitreous humor. The intensity of the re-
flected light also affected by the absorbance of hemo-
globin in the choriocapillaris. Fluorescence measure-
ments on RPE lipofuscin have also been developed as a
means of mapping MPOD (111). Excitation wave-
lengths are selected that are absorbed to different de-
grees by the MP. This technique may be problematic
for those age-related macular degeneration (AMD) sub-
jects who have large drops in the amount of RPE lipo-
fuscin. Recently, Bernstein et al. reported the promis-
ing application of resonance Raman spectroscopy for
measurement of MP levels (109). The Raman signal
from carotenoids is very weak and necessitates dilation
of the subject’s pupil and the use of sensitive detectors.
 MINIREVIEW
As yet there is no standardized method for the un-
equivocal determination of MPOD which can be regarded
as a reference standard. This is a substantial hurdle
preventing the acquisition and comparison of large data
sets of suitable quality for many clinical purposes.
PROTECTION AGAINST PHOTODAMAGE
It is well known that intense light can produce dam-
age in the retina (112–115). The action spectrum for
light-induced damage shows a distinct maximum at
wavelengths between 400 and 450 nm, consistent with
the absorption spectrum of the MP (116). Several stud-
ies show clear evidence that MP attenuates photic
damage in the human retina. Haegerstrom-Portnoy
has reported that the age-related decline of retinal
sensitivity of the short-wavelength (blue) cones is re-
duced in areas where MP levels are highest (117). A
clinical condition, known as Bull’s eye maculopathy,
associated with photosensitizing drugs, is character-
ized by retinal degeneration in an annular pattern
which surrounds but significantly spares the macula
(118, 119). Photic damage by the operating microscope
has also been reported to result in lesions, but the
damage is least in illuminated regions that overlap the
MP (120, 121).
MP protection of the retina from photic damage has
been postulated to occur through two different func-
tional roles. The first of these is through absorption of
blue light as it enters the inner retinal layers thereby
attenuating the intensity and potential for photo-oxi-
dation of reactive unsaturated lipid components of pho-
toreceptor disk membranes. This might be referred to
as the passive protection mode. Lutein and zeaxanthin
present in the inner retinal layers are distant from the
photoreceptors and the underlying retinal pigment ep-
ithelium where photic damage is believed to produce
pathological effects. The lutein and zeaxanthin in rod
outer segments are potentially intimately associated
with photo-sensitive components (58, 59). While 1O 2
has not been directly detected in the retina, its pres-
ence has been postulated based upon the sensitivity of
the retina to photic damage and the demonstrated
ability of natural heme to function as a sensitizer (113).
As described above, carotenoid antioxidant function
can be an active process that involves direct chemical
interaction between the carotenoid and the reactive spe-
cies (47). Martin et al. have recently shown that the
xanthophylls are somewhat better antioxidants that hy-
drocarbon carotenoids such as ␤-carotene (122). They ex-
hibit a smaller tendency toward pro-oxidant behavior.
Epidemiological Correlations Related to MP
AMD is the leading cause of blindness in Western
cultures (69, 123). Both genetic and environmental
37
factors are evident as contributing to the risk of AMD
(123). Ocular exposure to sunlight has been linked to
AMD in some investigations (112, 124 –126). Photooxi-
dation of polyunsaturated lipids might well impair the
normal cycle of lipid biochemistry in the RPE during
photoreceptor phagocytosis, leading to the damaging
buildup of drusen which characterizes AMD (127). One
component of lipofuscin, a constituent material present
in drusen, has been identified (128). This compound is
capable of promoting DNA damage in the RPE in the
presence of light and oxygen (129, 131).
The Eye Disease Case Control Study Group has pub-
lished results showing that individuals who have a
high dietary intake of lutein and zeaxanthin have a
reduced risk of advanced neovascular AMD (131). Sim-
ilarly, in another study, they found that elevated se-
rum levels of lutein and zeaxanthin are associated with
lower risk for neovascular AMD (132). The Beaver
Dam Study found slightly (though not significantly)
lower levels of plasma lutein and zeaxanthin among
individuals with exudative AMD compared to controls
(84, 133). In a study comparing postmortem retinas
from AMD and control donors we have seen that the
amounts of MP in the outer portions of the retina are
often lower for those diagnosed with AMD (38).
CONCLUSIONS
Our current understanding of the MP enables us to
ask a number of specific questions that should serve to
direct further investigations. We have a broad view of
the composition, distribution, and location of the MP in
the retina. It will be important to refine this view and
obtain a quantitative cellular perspective. Evidence
supports the hypothesis that the MP carotenoids are
specifically transported to the sites where they are
found in the retina. The identification of transport
proteins would enable visualization of the MP at cellu-
lar level locations within the retina using immunolog-
ical methods. We recognize that the MP probably un-
dergoes developmental changes during the first 2 or 3
years of life. It is not known if these changes are
evidence of major physiological transformations under
genetic control or if they are the result of environmen-
tal factors such as changing diet. Here too, the ability
to use immunoassay techniques to quantify the pres-
ence of MP specific proteins during this developmental
period might provide great insight relevant to the func-
tional role of the macular carotenoids.
Further evidence must be gathered to demonstrate
the origin of the minor carotenoid components of the
retina and to establish whether they may be interme-
diate species that are involved in the metabolic forma-
tion of meso-zeaxanthin. We have seen that keto-caro-
tenoids are apparently reduced in the retina. It will be
important to establish whether this process is a specific
38 MINIREVIEW
or nonspecific process and where it is occurring. It
would appear that the carotenoids of the MP may have
a long residence time within the retina. Information on
the reaction processes of the MP components will pro-
vide us a perspective on the dynamic, as opposed to
static, character of the MP.
Absorption from the diet, transport into the blood,
and metabolism of the MP carotenoids by the various
tissues of the body are clearly important aspects of our
understanding of these carotenoids. These factors have
implications for dietary supplementation with lutein
and zeaxanthin. Therapeutic manipulation of the mac-
ular pigment, if it is proven desirable and generally
feasible, is dependent upon these processes which are
at present poorly understood. The catabolism of these
carotenoids and the identity and physiological activity
of their intermediary metabolites must be elucidated.
Proof of the functionality of the MP is the most
pressing issue for researchers in this field. The MP is
located in a critical tissue and accumulated to excep-
tional levels. These carotenoids have been demon-
strated to reduce blue-light levels reaching the most
delicate of retinal structures. Blue light is known to be
capable of inducing retinal damage. We also know that
the risk of AMD appears to be reduced by diets rich in
xanthophylls, for those having high serum levels of
lutein and zeaxanthin, and those with high MP levels.
It is now known that the MP carotenoids are also
present in the rod outer segments where it is hypoth-
esized that they may function as antioxidants. In order
to investigate a possibly causal relationship between
high levels of MP and a reduced risk of AMD, longitu-
dinal studies using human subjects must ultimately be
undertaken. The design of these studies will be critical
to the success of this effort and must include an ade-
quate means of measurement of the in vivo MP level
for aging subjects. To this end more work must be done
to establish an accurate and efficient method for mea-
surement of MP, preferably one that is independent of
subject response.
The picture of a functional MP that has emerged
during the past 15 years is promising. It is essential
that a detailed understanding of this function be
carefully and completely proven so that we can en-
able clinicians and their patients to make appropri-
ate decisions about the role of carotenoids in ocular
health.
ACKNOWLEDGMENTS
The authors acknowledge support received from NIH, NIGMS,
Grant 08205, and the Rehnborg Center for Nutrition and Wellness,
Division of Amway. We thank the National Disease Research Inter-
change and the Florida Lion’s Eye Bank for their provision of the
human tissues that have been essential to the completion of our
research. We thank D. M. Snodderly for permission to incorporate
graphics from his publications.
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