Identication and Quantitation of Carotenoids and their Metabolites

in the Tissues of the Human Eye

PAUL S. BERNSTEI N
a
*, FREDERI CK KHACHI K
b
, LORENA S. CARVALHO
b
,
GARTH J. MUI R
a
, DA-YOU ZHAO
a
AND NI KI TA B. KATZ
a
a
Department of Ophthalmology and Visual Sciences, Moran Eye Center, University of Utah School of
Medicine, Salt Lake City, UT 84132, U.S.A. and
b
Department of Chemistry and Biochemistry, Joint Institute
for Food Safety and Applied Nutrition (JIFSAN), University of Maryland, College Park, MD 20742, U.S.A.
(Received Washington 11 July 2000, accepted in revised form 18 October 2000 and published
electronically 9 January 2001)
There is increasing evidence that the macular pigment carotenoids, lutein and zeaxanthin, may play an
important role in the prevention of age-related macular degeneration, cataract, and other blinding
disorders. Although it is well known that the retina and lens are enriched in these carotenoids, relatively
little is known about carotenoid levels in the uveal tract and in other ocular tissues. Also, the oxidative
metabolism and physiological functions of the ocular carotenoids are not fully understood. Thus, we
have set out to identify and quantify the complete spectrum of dietary carotenoids and their oxidative
metabolites in a systematic manner in all tissues of the human eye in order to gain better insight into
their ocular physiology.
Human donor eyes were dissected, and carotenoid extracts from ocular tissues [retinal pigment
epithelium/choroid (RPE/choroid), macula, peripheral retina, ciliary body, iris, lens, vitreous, cornea,
and sclera] were analysed by high-performance liquid chromatography (HPLC). Carotenoids were
identied and quantied by comparing their chromatographic and spectral proles with those of
authentic standards.
Nearly all ocular structures examined with the exception of vitreous, cornea, and sclera had
quantiable levels of dietary (3R,3
0
R,6
0
R)-lutein, zeaxanthin, their geometrical (E/Z) isomers, as well as
their metabolites, (3R,3
0
S,6
0
R)-lutein (3
0
-epilutein) and 3-hydroxy-b,E-caroten-3
0
-one. In addition,
human ciliary body revealed the presence of monohydroxycarotenoids and hydrocarbon carotenoids,
while only the latter group was detected in human RPE/choroid. Uveal structures (iris, ciliary body, and
RPE/choroid) account for 50% of the eye's total carotenoids and 30% of the lutein and zeaxanthin.
In the iris, these pigments are likely to play a role in ltering out phototoxic short-wavelength visible
light, while they are more likely to act as antioxidants in the ciliary body. Both mechanisms, light
screening and antioxidant, may be operative in the RPE/choroid in addition to a possible function of this
tissue in the transport of dihydroxycarotenoids from the circulating blood to the retina. This report lends
further support for the critical role of lutein, zeaxanthin, and other ocular carotenoids in protecting the
eye from light-induced oxidative damage and aging. # 2001 Academic Press
Key words: carotenoid; lutein; zeaxanthin; age-related macular degeneration; cataract; oxidation
products; metabolites; uvea; macular pigment; antioxidants.
1. Introduction
It is well established that the human retina and lens in
general and the macular region in particular are
highly enriched in lutein [(3R,3
0
R,6
0
R)-b,E-carotene-
3,3
0
-diol] and two diastereomers of zeaxanthin,
[(3R,3
0
R)-b,b-carotene-3,3
0
-diol and (3R,3
0
S,meso)-
b,b-carotene-3,3
0
-diol] (Bone, Landrum and Tarsis,
1985; Bone et al., 1993). While (3R,3
0
R,6
0
R)-lutein is
one of the major carotenoids in most fruits and
vegetables, dietary sources of (3R,3
0
R)-zeaxanthin are
limited to greens and certain yellow/orange fruits and
vegetables such as corn, nectarines, oranges, papaya,
and squash, and (3R,3
0
S,meso)-zeaxanthin is not part
of the normal human diet. In 1998, the United States
Department of Agriculture (USDA) updated their
Carotenoid Database for U.S. foods to include the
concentrations of lutein, zeaxanthin and other caro-
tenoids in some of the most common fruits and vege-
tables consumed in the United States {http://
www.nal.usda.gov/fnic/foodcomp/Data/car98/
car98.html}.
Despite its limited dietary source, zeaxanthin pre-
dominates over lutein in the foveal region of the
human eye by a ratio of greater than 2 : 1 (Bone et al.,
1988), and most of the carotenoids are concentrated
in the Henle ber layer and in the inner plexiform layer
(Snodderly, Auran and Delori, 1984). The foveal caro-
tenoids may also be present in Mu ller glial cells (Gass,
Exp. Eye Res. (2001) 72, 215223
doi:10.1006/exer.2000.0954, available online at http://www.idealibrary.com on
0014-4835/01/03021509 $35.00/0 # 2001 Academic Press
Presented in part at the annual meeting of the Association for
Research in Vision and Ophthalmology (ARVO), Fort Lauderdale,
Florida, May, 1998 and at the 12th International Carotenoid
Symposium, Cairns, Australia, July, 1999.
* Address correspondence to: Paul S. Bernstein, Department of
Ophthalmology and Visual Sciences, Moran Eye Center, University
of Utah School of Medicine, 50 North Medical Drive, Salt Lake City,
Utah 84132, U.S.A. E-mail: paul.bernstein@hsc.utah.edu
1999). Retinal carotenoid content falls dramatically
with increasing eccentricity from the fovea; macular
pigment optical density drops by a factor of nearly 100
(Hammond, Wooten and Snodderly, 1997b), and the
zeaxanthin to lutein ratio reverses to approximately
1 : 2 (Bone et al., 1988). A portion of the peripheral
carotenoids appears to be localized to the photo-
receptor outer segments and to the RPE (Sommerburg
et al., 1999; Rapp, Maple and Choi, 2000). Nearly
half of the zeaxanthin in the macula is dietary
(3R,3
0
R)-zeaxanthin, and the other half is
(3R,3
0
S,meso)-zeaxanthin which constitutes 51% of
the total zeaxanthin found in human blood (Bone
et al., 1993; Khachik et al., 1999a). In the human
lens, lutein and zeaxanthin are found in higher
concentrations in the epithelial and cortical layers
relative to the nucleus (Yeum et al., 1995, 1999).
Although to date as many as 25 dietary carotenoids
and nine of their metabolites have been identied in
the human serum, the uptake of lutein and zeax-
anthin into the retina and lens is remarkably specic.
This is in view of the fact that many other prominent
dietary carotenoids such as lycopene, b-carotene, b,E-
carotene (a-carotene), b-cryptoxanthin, g-carotene,
phytouene, and phytoene which are found in
relatively high concentrations in human serum and
other tissues, have not been detected in the retina and
lens in more than trace amounts (Handelman et al.,
1992; Khachik, Bernstein and Garland, 1997a;
Khachik et al., 1997b; Yeum et al., 1999).
Lutein and zeaxanthin appear to play a signicant
protective role against visual loss from age-related
macular degeneration (AMD). Nutritional studies
have demonstrated that increased consumption of
foods or supplements rich in lutein and zeaxanthin
elevates serum levels of these carotenoids and in
many cases can result in increased macular pigment
density (Hammond et al., 1997a; Landrum et al.,
1997a; Landrum, Bone and Kilburn, 1997b). A large
case-control study reported that individuals with high
dietary intakes and high serum levels of lutein and
zeaxanthin have a much lower rate of exudative AMD
(Eye Disease Case Control Study Group, 1993; Seddon
et al., 1994). Meanwhile, an autopsy study has
claimed that maculae from patients with a history of
AMD have lower concentrations of the macular
carotenoid pigments relative to control eyes from
patients without a known history of AMD (Landrum
et al., 1997b). Also, various epidemiological studies
have indicated that lutein and zeaxanthin may be
protective against age-related cataract (Brown et al.,
1999; Chasan-Taber et al., 1999; Lyle et al., 1999).
The two most common hypotheses for the protective
role of lutein and zeaxanthin are based on the ability
of these carotenoids to lter out phototoxic short-
wavelength visible light and on their efcient
capacities to quench light-induced free radicals such
as singlet oxygen (Snodderly, 1995; Schalch,
Dayhaw-Barker and Barker, 1999).
While considerable attention has been directed
toward elucidating the functions of lutein and
zeaxanthin in the retina and lens, there have been
no detailed literature reports on the identication and
quantitation of the full spectrum of dietary carotenoids
and their oxidation products in other structures of the
human eye. In 1997, we reported the isolation and
identication of several oxidation products of lutein
and zeaxanthin in human and monkey retinas; this
nding has lent further support to the hypothesis that
these carotenoids may protect the retina from light-
induced oxidation by serving as antioxidants (Khachik
et al., 1997a). Here, we have extended our studies on
identication of carotenoids and their oxidation
products to the other tissues of the human eye and
have demonstrated that tissues of the uveal tractthe
retinal pigment epithelium/choroid (RPE/choroid), the
ciliary body, and the irisare enriched in lutein,
zeaxanthin, and their metabolites. In addition, a wide
range of dietary carotenoids that are absent in the
human retina and lens have now been identied and
quantied in the ciliary body and RPE/choroid. The
possible physiological roles of carotenoids in these
ocular structures will be discussed.
2. Materials and Methods
Dissection of Human Eyes and Selection of Experimental
Samples
Human donor globes were obtained from the Utah
Lions Eye Bank within 24 hr after death. Tissue
procurement and distribution complied with the
tenets of the Declaration of Helsinki. In virtually all
cases, the cornea had already been removed for
transplantation by Eye Bank personnel. Eyes with
any discernable ocular pathology except for pseudo-
phakia were excluded. Several corneas rejected for
transplantation on the basis of serum antigenicity of
the donor were also made available for this research.
Corneas were frozen at 708C and cut into
1 1 mm fragments while still solid. Round 5 mm
sections of the sclera were obtained randomly in the
posterior part of the globe after uveal dissection was
complete and treated similarly to the cornea.
Dissection of the globes was performed under dim
light and on ice to prevent photo-oxidation of
carotenoids. Irises were removed, placed on glass
slides and inspected to identify the background color.
Ciliary body was dened as the area of the uvea
between the root of the iris and the ora serrata.
Dissected ciliary bodies were separated from the lens
and its zonules and gently rinsed in 10 ml of ice-cold
isotonic phosphate-buffered saline to remove blood
and vitreous.
The bulk of the vitreous was removed by manual
vitrectomy. Maculae and submacular RPE/choroid
were excised with a 5 mm circular trephine centered
on the fovea, after which the retinal tissues were
216 P. S. BERNSTEI N ET AL.
peeled from the underlying RPE/choroid and carefully
separated from adhering vitreous. Mid-peripheral
retina and RPE/choroid were excised using the same
5 mm circular trephine centered approximately
57 mm distal to the fovea or optic nerve. Four
samples: superior, inferior, nasal, and temporal mid-
peripheral retina were obtained, and the underlying
RPE/choroid from the superior and inferior quadrants
was peeled off and stored separately. All dissected
tissues were stored in the dark at 708C. Duplicate
samples of all tissues were subsequently shipped on
dry-ice to the University of Maryland (UMD) for
larger-scale detailed extraction and analysis of
carotenoids and their metabolites. Upon arrival at
UMD, the samples were immediately stored at 708C
until analysis.
Extraction of Ocular Carotenoids
All extractions and analyses were performed under
dim or yellow light to prevent photo-isomerization
and degradation of carotenoids. The small-scale
extractions of the individual tissues were rst
performed at the University of Utah, and the large-
scale (pooled tissues) extractions were subsequently
carried out at UMD. Both extraction procedures were
quite similar; these are described as follows.
Small-Scale Extraction of Individual Tissues
Small samples of tissue from retina and RPE/
choroid were sonicated on ice into 0
.
5 ml of distilled
water until a uniform suspension was formed to
which an equal volume of 0
.
1% butylated hydro-
xytoluene (BHT) in methanol was added. Highly
brous samples derived from cornea and sclera were
initially sonicated into 0
.
5% sodium dodecyl sulfate to
facilitate better dispersion. After homogenization,
0
.
5 ml of extraction solvent was added (0
.
5% BHT
in 30% dichloromethane/70 % hexane). The sample
was sonicated again and subjected to centrifugation
(14 000 g for 10 min at 48C), after which the organic
layer was removed and evaporated to dryness. The
extracts were reconstituted in an appropriate volume
of the HPLC eluent prior to analysis.
Large-Scale Extraction of Pooled Tissues
Pooled tissues were weighed and then placed in a
50 ml centrifuge tube containing sodium sulfate
(30% by weight of the tissues); 20 ml of tetra-
hydrofuran (THF, 0
.
1% BHT) was added, and the tube
was sonicated at 5108C for 30 min. The solution
was centrifuged at 20 000 g for 5 min, and the
extracts were transferred to a 100 ml round-bottom
ask. The tissues were re-extracted with THF twice
(2 20 ml) as above. The extracts were combined
and evaporated to dryness using a rotary evaporator.
The residue was dissolved in dichloromethane (4 ml)
and ltered through a 0
.
45 mm disposable Acrodisk
polyvinylidene uoride lter assembly (VWR Scientic
Products, McGaw Park, IL, U.S.A.) into a 5 ml micro-
sample vial. The solvent was evaporated under
nitrogen, and the residue was reconstituted in an
appropriate volume of the HPLC injection solvents.
The vial was centrifuged at 20 000 g to remove the
minor insoluble solid particles, and 50 ml samples
were injected onto the chromatography system under
conditions which will be described later in this text.
High Performance Liquid Chromatographic Systems
All separations in Maryland were conducted on a
Hewlett-Packard (HP) 1050 High Performance Liquid
Chromatography (HPLC) system equipped with a
rapid-scanning UV/visible photodiode array detector,
and an HP-1050 autosampler. The absorption spectra
of the carotenoids were recorded between 200 and
600 nm at a rate of 12 spectra min
1
. In Utah, a
comparable Waters HPLC system with a single
wavelength detector was used. Two sets of HPLC
conditions were employed.
HPLC Conditions (System One)
Separation of lutein, zeaxanthin, and their oxidation
products was accomplished by normal phase chroma-
tography. These separations were carried out on a
silica-based nitrile-bonded (25 cm length 4
.
6 mm
i.d.; 5 mm spherical particle) column (Regis Chemical
Co., Morton Grove, IL, U.S.A.), which was protected
with a Brownlee nitrile-bonded guard cartridge (3 cm
length 4
.
6 mm i.d.; 5 mm particle size). For this
separation an isocratic mixture of hexane (75%) and
dichloromethane (25%) containing 0
.
25% methanol
and 0
.
1% of N,N-diisopropylethylamine (DIPEA) at a
column ow rate of 0
.
70 ml min
1
was employed.
The HPLC mobile phase was also employed as the
injection solvent. The HPLC runs were monitored at
446 nm.
HPLC Conditions (System Two)
Carotenoid extracts from various tissues were also
analysed by reversed-phase HPLC on a Microsorb
(25 cm length 4
.
6 mm i.d.) C
18
(5 mm spherical
particles) column (Rainin Instrument Co., Woburn,
MA, U.S.A.), which was protected with a Brownlee
guard cartridge (3 cm length 4
.
6 mm i.d.) packed
with Spheri-5-C
18
(5 mm particle size). A combination
of isocratic and gradient HPLC employing a two pump
solvent module was used with this eluent. Pump A
pumped a mixture of acetonitrile/methanol (9/1,
v : v), and pump B pumped a mixture of hexane/
dichloromethane/methanol/DIPEA(4
.
5/4
.
5/0
.
99/0
.
01,
v : v : v : v). At time zero, an isocratic mixture of 95%
pump A and 5% pump B was pumped for 10 min. After
10 min, a linear gradient was run for 30 min resulting
TI SSUE DI STRI BUTI ON OF OCULAR CAROTENOI DS 217
in a nal composition of 45% pump A and 55% pump
B. The column ow rate was 0
.
70 ml min
1
. The HPLC
injection solvent with this eluent consisted of a mixture
of acetonitrile (40%), dichloromethane (20%), hexane
(20%), and methanol (20%). At the end of the gradient,
the column was equilibrated under the initial isocratic
conditions for 15 min. HPLC runs were simultaneously
monitored at 446, 400, 350 and 290 nm.
Reference Samples of Carotenoids and Their Oxidation
Products
(3R,3
0
R,6
0
R)-Lutein was isolated from marigold
owers according to a published procedure (Khachik,
Steck and Pfander, 1999b). (3R,3
0
R)-Zeaxanthin and
(3R,3
0
S,meso)-zeaxanthin were gifts from Hoffmann-
La Roche (Basel, Switzerland). (3R,3
0
S,6
0
R)-Lutein
(3
0
-epilutein) and (3R,6
0
R)-3-hydroxy-b,E-caroten-3
0
-
one were prepared from lutein by partial synthesis
(Khachik et al., 1992a, 1997b). Geometrical isomers
of lutein and zeaxanthin were prepared according to a
published procedure (Khachik et al., 1992b). Lyco-
pene was a gift from LycoRed Natural Products
Industries (Beer Sheva, Israel). Neurosporene and
g-carotene were isolated from an extract of tomato
paste by ash column chromatography and were
further puried by preparative HPLC according to a
published procedure (Khachik et al., 1992c). Retinol,
retinyl palmitate, a-tocopherol, and g-tocopherol were
obtained from Sigma Chemical Co. (St. Louis, MO,
U.S.A.). A sample of retinyl palmitate was partially
converted to its Z-isomers by reuxing in THF in the
presence of a catalytic amount of iodine. Butylated
hydroxytoluene (BHT), and N,N-diisopropylethyla-
mine (DIPEA) were purchased from Aldrich Chemical
Co. (Milwaukee, WI, U.S.A.). Tetrahydrofuran (THF)
and HPLC-grade solvents, acetonitrile, dichloro-
methane, hexane, and methanol (VWR Scientic
Products, Bridgeport, NJ, U.S.A.) were used without
further purication.
3. Results
The main objective of this study was to identify the
complete carotenoid prole of human ocular tissues
with particular attention to those carotenoids which
have not been previously identied. Therefore,
because the use of an internal standard could possibly
interfere with the presence of unknown carotenoids,
no internal standard in the extraction of the ocular
tissues was employed, however, to monitor the
accuracy and reproducibility of the HPLC analysis of
carotenoids, a solution containing known concentra-
tions of lutein, zeaxanthin, 3
0
-epilutein, a-crypto-
xanthin, b-cryptoxanthin, lycopene, a-carotene, and
b-carotene was routinely analysed by HPLC System
One and Two. The recovery and reproducibility of the
HPLC analysis for carotenoids was shown to be
greater than 95%.
In many cases, ocular tissues were pooled in order to
obtain a sufcient quantity of carotenoids for reliable
HPLC identication by photodiode array detection.
The carotenoids in human ocular tissues were
identied by comparison of their HPLC retention
times and UV/visible data with those of synthetic or
isolated carotenoids similar to published procedures by
Khachik et al. (1997a, b). Analysis of the pooled
extracts from human retinal pigment epithelium
(RPE), peripheral retina, ciliary body, iris, and lens
by HPLC on a nitrile-bonded column (System One)
showed the presence of (3R,3
0
R,6
0
R)-lutein, zeax-
anthin, and their (E/Z)-geometrical isomers. Particu-
larly interesting was the presence of (3R,3
0
S,6
0
R)-
lutein (3
0
-epilutein) and 3-hydroxy-b,e-caroten-3
0
-one
in all of the pooled extracts examined. A typical HPLC
prole of a pooled extract from human RPE/choroid on
a nitrile-bonded column is shown in Fig. 1. This HPLC
condition does not resolve dietary (3R,3
0
R)-zeaxanthin
from non-dietary (3R,3
0
S,meso)-zeaxanthin, and both
of these compounds when present co-elute and appear
as one HPLC peak. Therefore, while the dietary
zeaxanthin would be expected to be present in all the
samples examined, the presence of (3R,3
0
S,meso)-
zeaxanthin in various ocular tissues is not known at
present. Bone et al. (1993) have previously demon-
strated that nearly half of the zeaxanthin in the
human macula consists of (3R,3
0
S,meso)-zeaxanthin,
and the rest is dietary (3R,3
0
R)-zeaxanthin.
The quantitative data for carotenoids in various
ocular tissues are shown in Table I. The pooled
extracts from various tissues were also analysed by
HPLC on a C
18
-reversed-phase column (HPLC System
Two). Under these conditions, carotenol esters, mono-
hydroxycarotenoids, and hydrocarbon carotenoids
which are not separated on the nitrile-bonded column
(System One) are well separated. Among all the tissues
examined, only ciliary body and RPE/choroid revealed
the presence of dietary carotenoids besides lutein and
zeaxanthin. No carotenol esters were detected in any
ocular tissues. A typical reversed-phase HPLC prole of
a pooled extract from human ciliary body is shown in
Fig. 2, and the levels of the carotenoids identied are
shown in Table II. With regard to the distribution of
FIG. 1. A typical HPLC prole of a pooled extract from
human RPE/choroid on a silica-based nitrile-bonded
column employing HPLC System One and monitoring at
446 nm.
218 P. S. BERNSTEI N ET AL.
carotenoids in the ciliary body, lycopene was found
at a higher concentration relative to other
hydrocarbon carotenoids such as a-carotene and b-
carotene. The two prominent forms of vitamin E
(g- and a-tocopherol, l
max
292 nm) as well as all-E-
(trans)-vitamin Apalmitate (l
max
330 nm) and a Zh
(cis) isomer of this compound (l
max
328 nm) were
also identied in the pooled extracts from human
ciliary body. This Z-isomer of retinyl palmitate was also
the major isomer formed when a reference sample of
all-E-retinyl palmitate was isomerized in reuxing THF
in the presence of catalytic amounts of iodine. The
location of the Z-bond in vitamin Apalmitate identied
in the extracts from human ciliary body is not known
at present.
In addition to the pooled extracts described above,
individual samples from various ocular tissues were
also examined for lutein and zeaxanthin by HPLC on
a nitrile-bonded column, and the data are shown in
Table III. Since these samples are much smaller, levels
of carotenoid metabolites and Z-isomers could not be
assessed reliably. All samples derived from the human
retina, lens, and uveal tract contained detectable
levels of lutein and zeaxanthin. By contrast, only trace
amounts of carotenoids were present in the corneal
and scleral samples examined, and no carotenoids
were detected in vitreous. Corresponding punches of
mid-peripheral RPE/choroid usually had 3040% of
the lutein content and 2030% of the zeaxanthin
content of the overlying retina (Table III). Superior
retina and RPE/choroid had somewhat higher levels
of lutein and zeaxanthin relative to inferior tissues.
Submacular RPE/choroid had modestly higher levels
of lutein and zeaxanthin than the periphery, but these
differences were not nearly as large as the 4ten-fold
difference seen in overlying retina. The average
TABLE I
Levels of dietary lutein and zeaxanthin and their metabolites in pooled extracts from human ocular tissues
Ocular tissues (average wet
weight +S.D.) [n number of
pooled tissues analysed]
Levels of lutein, zeaxanthin and their metabolites (ng per tissue +S.D.)
all-E-(trans)-
Lutein (L)
all-E(trans)-
Zeaxanthin (Z)
Z-(cis)-
(L Z)
L/Z
Ratio* 3
0
-Epilutein
3-Hydroxy-b,E-
caroten-3
0
-one
RPE/Choroid (0
.
20 g +0
.
05)
[n 8, 10, 10, 11, 21, 33]
18
.
27+5
.
08 4
.
85+1
.
50 N.D.** 3
.
5 3
.
71+2
.
37 1
.
33+1
.
22
Peripheral retina (0
.
27 g +0
.
07)
[n 10, 11, 12, 12]
32
.
93+7
.
74 12
.
70+4
.
94 4
.
9+2
.
7 2
.
5 2
.
33+0
.
50 5
.
50+2
.
06
Ciliary body (0
.
20 g +0
.
05)
[n 5, 9, 10, 10, 10]
10
.
93+4
.
53 2
.
54+1
.
13 4
.
8+1
.
1 3
.
1 1
.
53+0
.
46 0
.
79+0
.
33
Iris (0
.
03 g +0
.
01)
[n 8, 19, 13, 25, 41]
3
.
58+0
.
93 1
.
13+0
.
32 N.D.** 3
.
0 0
.
59+0
.
56 0
.
41+0
.
10
Lens (0
.
22 g +0
.
04)
[n 20, 20, 20, 21, 33]
1
.
39+0
.
28 0
.
90+0
.
21 N.D.** 1
.
6 0
.
36+0
.
16 0
.
19+0
.
08
* The ratio of lutein to zeaxanthin includes their geometrical (all-E and Z) isomers.
** N.D. not detected.
FIG. 2. HPLC prole of a pooled extract from human ciliary body on a C
18
-reversed-phase column employing HPLC System
Two. Carotenoids, retinoids, and tocopherols were simultaneously monitored and quantied at multiple wavelengths as follows:
retinol and (E Z)-retinyl palmitate at 325 nm ( ); lutein, neurosporene, g-carotene, a-carotene, and b-carotene at
446 nm (); g- and a-tocopherol at 290 nm (). Lycopene was monitored at 470 nm.
TI SSUE DI STRI BUTI ON OF OCULAR CAROTENOI DS 219
lutein : zeaxanthin ratio for RPE/choroid (individual
and pooled samples) ranged from 2
.
0 : 1 (Table III) to
3
.
5 : 1 (Table I) while overlying nonmacular and
macular retina averaged 1
.
9 : 1 and 0
.
7 : 1, respect-
ively (Table III). Individual and pooled extracts from
human ciliary body contained substantial amounts of
lutein and zeaxanthin at an averaged ratio that
ranged from 2
.
1 : 1 (Table III) to 3
.
1 : 1 (Table I).
The average levels of lutein and zeaxanthin in
pooled (Table I) and individual (Table III) extracts
from iris were within the same range. Brown irises
had the same average levels of lutein and zeaxanthin
as blue irises.
Our average lutein and zeaxanthin levels for retina
and lens correlate well with values reported in the
literature from other laboratories (Handelman et al.,
1992; Yeum et al., 1995; Landrum et al., 1997b).
Although lutein and zeaxanthin have been previously
reported in human lens (Yeum et al., 1995, 1999), the
presence of 3
0
-epilutein and the direct oxidation
product of lutein, 3-hydroxy-b,E-caroten-3
0
-one, is
of particular interest and will be discussed later in
this article.
4. Discussion
With each passing year, the scientic community
and the general public are gaining greater apprecia-
tion of the important role of carotenoids in the
maintenance of ocular health. For decades it has
been known that vitamin A active carotenoids such as
b-carotene, a-carotene, g-carotene, and b-cryptox-
anthin are the major dietary precursors of the
retinoids which are critical for the visual cycle of
rhodopsin bleaching and regeneration. These vitamin
A precursors belong to a large family of over 600
carotenoids which have all been isolated and identied
from natural sources (Pfander et al., 1987). The
number of carotenoids in the typical human diet is
estimated in excess of 40, and among these, 12 all-E
(trans)-carotenoids and 13 of their geometrical Z-(cis)
isomers are routinely found in human blood (Khachik
et al., 1991, 1997b). Two dihydroxycarotenoids,
lutein and zeaxanthin, were identied as the major
carotenoids in the human macular pigment (Bone
TABLE II
Levels of dietary monohydroxycarotenoids and
hydrocarbon carotenoids in pooled extracts from
human ciliary body and RPE/choroid
Dietray carotenoids
Carotenoid level (ng per tissue)
Pooled extracts
from human
ciliary body
(n 30)
Pooled extracts
from human
RPE/choroid
(n 20)
Monohydroxycarotenoids
a-Cryptoxanthin 1
.
36 N.D.*
b-Cryptoxanthin 0
.
36 N.D.*
Hydrocarbon carotenoids
Neurosporene 4
.
50 N.D.*
g-Carotene 4
.
48 N.D.*
Lycopene 7
.
80 8
.
64
a-Carotene 1
.
60 2
.
97
b-Carotene 2
.
72 10
.
80
Total 22
.
82 22
.
41
* N.D. not detected.
TABLE III
Lutein and zeaxanthin levels in individual human ocular tissues
Ocular region
Eyes
examined
Area
(mm
2
)
Lutein (L)
(ng per tissue +S.D.)
Zeaxanthin (Z)
(ng per tissue +S.D.) L/Z ratio
Macular retina 14 20 13
.
98+3
.
58 19
.
06+4
.
50 0
.
7
Peripheral retina 19 1000* 64
.
18+30
.
10 34
.
11+16
.
83 1
.
9
Superior retina 78 20 1
.
68+0
.
88 0
.
80+0
.
62 2
.
1
Inferior retina 78 20 1
.
46+0
.
71 0
.
63+0
.
26 2
.
3
Nasal retina 7 20 1
.
76+1
.
01 0
.
81+0
.
53 2
.
2
Temporal retina 7 20 1
.
42+0
.
90 0
.
65+0
.
42 2
.
2
RPE/choroid 17 Whole 11
.
58+5
.
99 5
.
89+4
.
13 2
.
0
Superior RPE/choroid 78 20 0
.
63+0
.
26 0
.
19+0
.
09 3
.
3
Inferior RPE/choroid 78 20 0
.
53+0
.
26 0
.
16+0
.
09 3
.
3
Submacular RPE/choroid 25 20 0
.
77+0
.
50 0
.
32+0
.
20 2
.
4
Ciliary body 20 Whole 12
.
72+7
.
90 5
.
98+3
.
50 2
.
1
Iris 21 Whole 4
.
03+1
.
98 1
.
54+0
.
98 2
.
7
Lens 18 Whole 1
.
66+1
.
09 1
.
43+1
.
20 1
.
2
Cornea 3 Whole Trace Trace
Sclera 5 20 Trace Trace
Vitreous 3 0
.
5 ml N.D.** N.D.**
*The calculated average surface area of the human retina is 1095 mm
2
(72% coverage of a sphere with an internal diameter of 22 mm)
(Michels, Wilkinson and Rice, 1990).
**N.D. not detected.
220 P. S. BERNSTEI N ET AL.
et al., 1985; Handelman et al., 1992; Bone et al.,
1993), and low levels of lutein and zeaxanthin were
also identied in the human lens (Yeum et al., 1995,
1999). These ndings, as well as epidemiological
studies that demonstrated an inverse correlation
between consumption of foods rich in lutein and
zeaxanthin and risk of exudative AMD and cataract,
suggested a possible protective role for these two
non-vitamin A active carotenoids (Eye Disease Case
Control Study Group, 1993; Seddon et al., 1994;
Brown et al., 1999; Chasan-Taber et al., 1999; Lyle
et al., 1999). In 1997, we reported on the identica-
tion of the oxidation products of lutein and zeaxanthin
in human and monkey retinas and provided pre-
liminary evidence in support of an antioxidant
mechanism of action by these carotenoids in the
prevention of AMD (Khachik et al., 1997a).
In light of these ndings, we have now established
the identity of carotenoids and their metabolites and
measured their content in all tissues of the human
eye. In a systematic approach, we initially focused on
the highly pigmented structures that comprise the
uveal tract since these tissues are subjected to photo-
oxidative stresses that can be quite comparable to
those encountered by the human macula. The human
RPE/choroid, peripheral retina, ciliary body, iris, and
lens as shown in Table I, contain not only
(3R,3
0
R,6
0
R)-lutein and zeaxanthin but also signi-
cant amounts of 3
0
-epilutein and 3-hydroxy-b,E-
caroten-3
0
-one. Both of these non-dietary carotenoids
as proposed in our earlier publication, can be formed
by a series of light-induced or enzymatically mediated
oxidation-reduction and double-bond isomerization
reactions from lutein and zeaxanthin (Khachik et al.,
1997a). Although these and other carotenoid metab-
olites have been previously identied in low amounts
in the human serum (Khachik et al., 1992a, 1997b),
it is likely that they are formed locally in the eye since
they are found in unusually high concentrations in
ocular tissues relative to ocular lutein and zeaxanthin
levels and relative to the serum concentrations of
these metabolites.
In addition to lutein, zeaxanthin, and their oxida-
tion products, the pooled extracts from human RPE/
choroid showed the presence of lycopene, a-carotene,
and b-carotene, and an even more diverse range of
dietary carotenoids was found in the human ciliary
body (Table II). The selective uptake of lutein and
zeaxanthin by retina from approximately 25 dietary
carotenoids routinely found in human serum may be
due to certain structural requirements such as the
presence of the hydroxyl groups in these compounds.
There is evidence to suggest that the transport,
stabilization, and metabolic interconversions of lutein
and zeaxanthin in human retina are mediated by
nonspecic protein interactions (Bernstein et al.,
1997) and by specic xanthophyll-binding proteins
(Yemelyanov, Katz and Bernstein, 2001). The fact that
human RPE/choroid contains a- and b-carotene,
while overlying retina does not, suggests that meta-
bolic cleavage of these carotenoids to vitamin A may
occur in one or both of these ocular tissues. The
presence of signicant levels of lycopene in human
RPE and ciliary body is particularly interesting since
we have previously identied an oxidative metabolite
of this carotene, 2,6-cyclolycopene-1,5-diol, in the
human and monkey retina (Khachik et al., 1997a).
For the proposed metabolic transformation of lycopene
in humans, see the publication by Khachik, Pfander
and Traber (1998).
It is interesting to note that in frogs, greater than
95% of ocular carotenoids are localized in the RPE/
choroid and that they are completely esteried with
fatty acids (Bernstein et al., 1998). By contrast,
human RPE/choroid contains 3040% of the lutein
and 2030% of the zeaxanthin relative to punches of
the overlying mid-peripheral retina, and there was no
evidence for the presence of carotenol fatty-acid esters
during normal- or reversed-phase chromatography.
Submacular RPE/choroid had only slightly higher
levels of lutein and zeaxanthin relative to mid-
peripheral RPE/choroid. Since the overlying retina is
a rather transparent tissue, the RPE/choroid is
subjected to comparable light exposure levels, and
carotenoids may play similar roles in the protection
against light-induced oxidative damage. Moreover,
the RPE/choroid may be an intermediate control and
transfer point for lutein and zeaxanthin uptake by the
neural retina from circulating blood. A similar
transfer and control role for the RPE/choroid is well
established for ocular retinoids which must cross the
interphotoreceptor space during the normal function
of the visual cycle of rhodopsin regeneration (Carlson
and Bok, 1992). Interestingly, both lutein and retinol
are found in substantial concentrations in subretinal
uid collected from humans with retinal detachments
(Chan et al., 1998).
Levels of carotenoids in the ciliary body were
unexpectedly high since this heavily pigmented tissue
is not exposed to particularly intense levels of light. It
is, however, a metabolically active tissue responsible
for aqueous humor formation. Enzymes of the carbonic
anhydrase family found in the ciliary body are known
to be susceptible to oxidative damage and inactivation
(Cabiscol and Levine, 1995); therefore, the presence of
a diverse range of carotenoids including lycopene
(Table III) in the human ciliary body suggests that
these carotenoids might function as antioxidants in
this tissue. Whether or not carotenoids could be useful
in the treatment of glaucoma remains to be explored.
Iris and lens, on the other hand, are exposed to
intense light levels and have a very low metabolic rate.
The levels of lutein and zeaxanthin in these tissues per
square millimeter are similar to that of peripheral
retina, and the presence of the oxidation products of
lutein and zeaxanthin in lens is consistent with their
hypothesized role in the prevention and treatment of
cataract. It has been reported that darker iris
TI SSUE DI STRI BUTI ON OF OCULAR CAROTENOI DS 221
coloration is directly correlated with macular pigment
optical density (Hammond, Fuld and Snodderly,
1996), but we did not nd signicant differences in
carotenoid content between dark and light irises.
Lutein and zeaxanthin levels in the macula can be
modied by alterations in dietary intake, and it is
commonly recommended to patients at risk for visual
loss from AMD that they should increase consumption
of foods rich in lutein and zeaxanthin. Based on
the diverse distribution and accumulation of caro-
tenoids in the various structures of the human eye, it
is reasonable to assume that dietary modications
could also result in an increase in the pigment density
of these compounds in other ocular tissues besides
retina. The health food industry is already promoting
lutein supplements to those at risk for AMD, and
zeaxanthin supplements are likely to follow. Prospec-
tive data documenting the efcacy of carotenoid
supplements against ocular disease is still lacking,
however (Mares-Perlman, 1999).
Although the uveal tract accounts for 50% of the
eye's total carotenoids and 30% of the eye's total
lutein and zeaxanthin, the physiological roles of lutein
and zeaxanthin and other carotenoids within the
uvea still remain to be dened fully. In the iris, they
could act as rst-line lters against phototoxic blue
light, especially under intense light conditions when
the pupil is constricted. In other tissues, such as the
ciliary body, they could act as antioxidants against
locally produced free-radicals. In the RPE/choroid and
in nonuveal tissues such as retina and lens, protection
may be provided by both mechanisms. The RPE/
choroid may also play a key role in the transport of
lutein and zeaxanthin from circulating blood to the
neural retina. In plants, carotenoids play a critical role
in protection against light-induced oxidative damage
of the light harvesting structures of the chloroplasts
(Demmig-Adams, Gilmore and Adams, 1996). It
appears that the human organism has adopted these
same pigments to perform similar functions in its own
`light harvesting' organ, the human eye. Many of the
tissues of the human eye, and the macula in
particular, exhibit enormous specicity for the uptake
and stabilization of these compounds. Biochemical
characterization of the mechanisms underlying the
selective uptake and stabilization of the ocular
carotenoids and a further understanding of their
physiological functions are certain to advance our
knowledge of the pathogenesis and treatment of age-
related macular degeneration, cataract, and other
ophthalmic disorders.
Acknowledgements
The authors thank the Utah Lions Eye Bank for supplying
postmortem human eyes. The author PSB acknowledges
partial support from the National Institutes of Health,
Bethesda, MA, U.S.A. (Grant EY-11600), by grants from
Research to Prevent Blindness, Inc., New York, U.S.A. and
by Kemin Foods, Des Moines, IA, U.S.A. The author
FK acknowledges partial support from the University of
Maryland, The U.S. Food and Drug Administration
(UM-FDA) and Nutrilite, Division of Amway Corporation,
Lakeview, CA, U.S.A.
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