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From the Cullen Eye Institute, Departments of Ophthalmology and Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas.
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Abstract |
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METHODS. Retinas from human donor eyes were dissected to obtain two regions: an annular ring of 1.5- to 4-mm eccentricity representing the area centralis excluding the fovea (perifoveal retina) and the remaining retina outside this region (peripheral retina). ROS and residual (ROS-depleted) retinal membranes were isolated from these regions by differential centrifugation and their purity checked by polyacrylamide gel electrophoresis and fatty acid analysis. Lutein and zeaxanthin were analyzed by high-performance liquid chromatography and their concentrations expressed relative to membrane protein. Preparation of membranes and analysis of carotenoids were performed in parallel on bovine retinas for comparison to a nonprimate species. Carotenoid concentrations were also determined for retinal pigment epithelium harvested from human eyes.
RESULTS. ROS membranes prepared from perifoveal and peripheral regions of human retina were found to be of high purity as indicated by the presence of a dense opsin band on protein gels. Fatty acid analysis of human ROS membranes showed a characteristic enrichment of docosahexaenoic acid relative to residual membranes. Membranes prepared from bovine retinas had protein profiles and fatty acid composition similar to those from human retinas. Carotenoid analysis showed that lutein and zeaxanthin were present in ROS and residual human retinal membranes. The combined concentration of lutein plus zeaxanthin was 70% higher in human ROS than in residual membranes. Lutein plus zeaxanthin in human ROS membranes was 2.7 times more concentrated in the perifoveal than the peripheral retinal region. Lutein and zeaxanthin were consistently detected in human retinal pigment epithelium at relatively low concentrations.
CONCLUSIONS. The presence of lutein and zeaxanthin in human ROS membranes raises the possibility that they function as antioxidants in this cell compartment. The finding of a higher concentration of these carotenoids in ROS of the perifoveal retina lends support to their proposed protective role in age-related macular degeneration.
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Introduction |
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In addition to its role as an optical filter, macular pigment has been widely hypothesized to have an antioxidant function in the human retina.2 5 8 10 11 12 13 14 In vitro studies have shown that lutein and zeaxanthin are highly effective antioxidants capable of scavenging peroxyl radicals and quenching reactive oxygen species.15 16 However, a specific requirement for antioxidant protection would not be predicted for the plexiform layers of the fovea where macular pigment density is highest. On the other hand, photoreceptor outer segments are the primary target of oxidative insult in the retina due to their unusually high content of long-chain polyunsaturated fatty acids and the relatively high oxygen tension in which they exist.17 18 To function as lipophilic antioxidants in vivo, lutein and zeaxanthin would be expected to be located within or in close proximity to the membranes they are protecting. For these reasons, the photoreceptor outer segment membrane is the most likely site for an antioxidant function of lutein and zeaxan-thin, if it occurs in the retina.
The spatial distribution of lutein and zeaxanthin in the extrafoveal regions of the human retina has been carefully mapped by Bone et al.2 19 using high-performance liquid chromatography (HPLC). The amount of macular pigment declines rapidly with distance from the foveal center and reaches a plateau in the periphery. Nonetheless, a substantial amount of the total retinal macular pigment is present within the perifoveal macula and peripheral retina because of the greater retinal expanse represented by these regions compared with the fovea.2 5 6 19 Zeaxanthin is the predominant pigment in the fovea, but its level declines more rapidly than lutein as a function of eccentricity. For this reason, the mass ratio of lutein to zeaxanthin ranges from approximately 0.4:1 at the foveal center to over 2:1 in the periphery.19
Information remains limited on the subcellular localization of macular pigment in different regions of the retina. Bernstein et al.20 showed that carotenoids bind tubulin in retinal homogenates and proposed that tubulin was the site for macular pigment binding in the plexiform layers of the fovea. However, macular pigment has also been detected in the photoreceptor outer segment layer in the central fovea, even though the microtubules (which contain tubulin) do not reach this layer.7 This implies that macular pigment may be an integral component of the outer segment membranes or that it may be bound to membrane-associated proteins other than tubulin. Concerning retinal regions outside of the fovea, there is scant information about the cellular and subcellular distribution of lutein and zeaxanthin. Recently, Sommerburg and colleagues21 were the first to show that lutein and zeaxanthin are associated with rod outer segments (ROS) isolated from whole human retinas. However, lutein and zeaxanthin in ROS were expressed as their total amounts in the retina, and their concentrations in ROS membranes were not determined. Furthermore, no attempt was made to examine lutein and zeaxanthin concentrations in ROS from specific regions of the retina or in other membrane fractions.
A growing body of evidence from epidemiologic and experimental studies has implicated a role for macular pigment in protection against age-related macular degeneration (AMD).10 14 The most direct evidence comes from the Eye Disease Case-Control Study, which found a markedly reduced risk for neovascular AMD in persons with high serum levels of carotenoids and lutein/zeaxanthin specifically.22 High dietary consumption of foods containing lutein/zeaxanthin was also associated with a significant reduction in AMD risk.23 In considering the role of macular pigment in AMD, it should be recognized that AMD is a heterogeneous disease thought to be caused by a complex interaction of age-related, genetic, and extrinsic factors. Among other contributing factors, the likely involvement of oxidative stress in the etiology of AMD has received much attention in recent years.8 24 Although the mechanisms of AMD remain poorly understood, there is sufficiently compelling evidence to warrant a more complete understanding of the purported antioxidant function and protective value of macular pigment in the human retina.
The present study sought to determine whether lutein and zeaxanthin are components of ROS membranes in the human retina. Experiments were performed to evaluate the concentrations of lutein and zeaxanthin relative to membrane protein in ROS compared with residual (i.e., ROS-depleted) membrane fractions, and in the perifoveal compared with peripheral regions of the retina. We observed that outside of the fovea lutein and zeaxanthin occurred at their highest concentration in ROS membranes in the perifoveal region of the retina. These findings place lutein and zeaxanthin at sites in the human retina where they would most likely be required to function as antioxidants.
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Methods |
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Each dissected region was immediately placed in a sealed tube previously filled with argon in which the corresponding regions from the left and right eyes of each donor were combined and then frozen at -70°C. RPE was then recovered as small sheets, which usually had begun to detach from the eyecups after remaining in cold buffer in the dark for approximately 1 to 2 hours. The detachment was aided by gentle trituration or by gently scraping with the blunt end of a scalpel blade. Samples of the RPE isolated in this manner were examined by phase contrast microscopy, which confirmed that the preparation consisted almost exclusively of RPE. However, the possibility that some ROS tips adhered to the RPE cells could not be excluded. RPE from the entire eyecup was collected with buffer into a tube and washed twice with low-speed centrifugation, and the pellet was covered with argon and frozen at -70°C. Subsequent analysis of frozen samples usually took place within a few weeks.
Dark-adapted bovine retinas were obtained from the W. L. Lawson Co. (Lincoln, NE). They were obtained only during the months November through March, because retinas harvested during other times of the year could be adversely affected by higher ambient temperatures. They were shipped on dry ice to the laboratory by overnight mail in packages of 50 retinas. The frozen retinas were stored at -70°C before analysis.
Preparation of ROS Membranes
ROS membranes were isolated according to the procedures described
by Papermaster and Dreyer,26
with minor modifications to
accommodate the smaller amounts of tissue in this study. Human retinal
tissue to be used for ROS preparations consisted of either the
perifoveal or the peripheral regions from a particular donor (tissue
from corresponding regions of the left and right eyes was pooled as
described above). Portions of frozen bovine retina were thawed in the
dark and approximately 1.0 to 1.2 g wet weight was used for ROS
membrane preparation. Samples were suspended by vigorous vortexing for
2 minutes in 2 ml of a homogenizing medium of 34% sucrose (density of
1.15 g/ml) containing 65 mM NaCl, 2 mM MgCl2, and
5 mM Tris buffer (pH 7.4). This step sheared off most of the ROS into
the homogenizing medium, and the remaining retina was pelleted by
centrifugation at 5000 rpm for 5 minutes. After collecting the
supernatant (crude ROS), the pellet was resuspended with 2 ml of fresh
sucrose homogenizing medium, and the steps to obtain crude ROS were
repeated to maximize their yield. The supernatants of crude ROS were
then combined and centrifuged at 15,000 rpm for 30 minutes with twice
the volume of 10 mM Tris buffer (pH 7.4). This pellet was suspended in
2 ml of sucrose, density of 1.100 g/ml, containing 1 mM
MgCl2 and 10 mM Tris buffer, pH 7.4, and layered
on top of a discontinuous sucrose gradient consisting of incremental 2
ml steps of 1.150, 1.130, and 1.110 g/ml, buffered with 10 mM Tris
buffer (pH 7.4) and 1 mM MgCl2. The resultant
gradient was centrifuged at 25,000 rpm for 1 hour, and the band
containing purified ROS, which appeared at the 1.110/1.130 g/ml
interface, was collected. All the remaining bands were combined in a
manner similar to that described by Fliesler et al.27
and
were in turn combined with the membrane pellet from the earlier crude
ROS isolation steps. This represented the "residual retina"
membrane fraction. The results of other studies (see discussion in Ref. 28
) suggest that cone outer segment membranes are enriched at the
1.130/1.15 g/ml interface rather than at 1.110/1.130 g/ml (containing
the ROS membranes) and would therefore represent a small fraction of
the residual membranes. The ROS and residual retina membrane fractions
were then washed by adding approximately 10 ml of 10 mM Tris buffer (pH
7.4) and centrifuging at 15,000 rpm for 30 minutes. The resulting
pellets were reconstituted in 0.5 to 2 ml of 10 mM Tris buffer (pH 7.4)
and frozen at -70°C for subsequent analysis. Tissue processed in
this manner yielded approximately 540 to 660 µg of ROS membrane
protein, and approximately 5.8 to 7.6 mg of residual, per pair of human
retinas. For the samples from the perifoveal retinal region,
corresponding yields were approximately 32 to 53 µg of ROS, and
approximately 420 to 610 µg of residual retinal membrane protein. The
purity of membrane preparations was evaluated routinely by sodium
dodecyl sulfate—polyacrylamide gel electrophoresis (SDS–PAGE)
according to the method of Laemmli,29
and protein yields
were determined by the BCA protein assay (Pierce Chemical, Rockford,
IL).
Lutein and Zeaxanthin Analysis by Normal-Phase HPLC
The methods for HPLC analysis of lutein and zeaxanthin and the
synthesis of 3'-ethoxylutein to be used as the internal standard were
adapted from those reported by Khachik et al.3
Photo-isomerization and degradation of the carotenoids were minimized
by conducting all procedures under yellow light (Kodak OO safelight
filter) or occasionally under very low levels of indirect room light.
Aliquots of known protein amounts of retinal membranes or RPE, or the
3-mm retinal area corresponding to the macula lutea plus immediate
surrounding area, were homogenized in 0.5 ml of 10 mM Tris buffer. The
homogenate was then combined with 0.5 ml ethanol containing 2.5 ng of
internal standard and 0.1% BHT. Two milliliters of hexane was then
added, followed by vortexing for 180 seconds. Phase separation was
accomplished by centrifuging at 1900 rpm, and the upper hexane layer
was transferred to a clean vial. Two milliliters of hexane was added to
the remaining sample, and the extraction steps were repeated. The
extracts were combined, dried under argon, and resuspended in 50 µl
of mobile phase. Separations were carried out on a silica-based,
nitrile-bonded (normal-phase) column (5 µm particle size, 250 x
4.6 mm ID; Rainin, Woburn, MA). The mobile phase consisted of an
isocratic mixture of 80% hexane/19.4% dichloromethane/0.5%
methanol/0.1% N,N –diisopropylethylamine, and
column flow rate was set at 0.7 ml/min. The monitoring wavelength of
the eluent was 450 nm. Using these methods, the internal standard
eluted at approximately 7 minutes and therefore did not interfere with
any of the carotenoid peaks, the first of which appeared approximately
8 minutes later. Lutein and zeaxanthin were quantified as previously
described,3
by determining their peak areas relative to
that of the internal standard and converting to nanograms, using
standard curves derived from external standards of lutein (Sigma) and
zeaxanthin (generously donated by Hoffmann–LaRoche, Nutley, NJ) that
were injected daily on the column. Analytic recovery of lutein and
zeaxanthin determined by adding known amounts of external standard to
rat ROS (which we established have no endogenous lutein or zeaxanthin)
was greater than 98%. Sensitivity of the assay was estimated at 0.1 ng
for both carotenoids.
Fatty Acid Composition
Samples of frozen ROS and residual retinal membranes were shipped
on dry ice by overnight mail to the University of Oklahoma Health
Sciences Center in Oklahoma City, OK, where gas-liquid chromatographic
analysis (GLC) of fatty acids was performed by Maureen Maude
(laboratory of R. E. Anderson) according to previously reported
methods.30
In brief, lipids from thawed samples were
extracted in 2:1 chloroform/methanol, and a portion of the extract was
applied to a silica gel plate and developed in hexane/ethyl
ether/glacial acetic acid. The region of the plate containing the total
phospholipids was scraped into a tube and methyl esters prepared with
BF3-methanol. Their masses were quantitated by
GLC using 17:0, 19:0, and 21:0 as internal standards.
Statistical Analysis
A three-way ANOVA was applied to the data for lutein and
zeaxanthin concentrations (nanograms per milligram of protein) in
human retinal membranes, examining factors of carotenoid (lutein versus
zeaxanthin), region (perifoveal versus peripheral), and membrane
fraction (ROS versus residual). A two-way ANOVA was applied to the data
obtained on human ROS membranes, specifically examining factors of
carotenoid (lutein versus zeaxanthin) and region (perifoveal versus
peripheral). A two-way ANOVA was applied to the data for the
lutein-to-zeaxanthin concentration ratio, examining factors of region
(perifoveal versus peripheral) and membrane fraction (ROS versus
residual). The level of significance for all tests was 0.05.
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Results |
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As an additional test of ROS membrane purity, fatty acid composition
was determined on ROS and residual membranes from bovine and human
retinas. A distinguishing characteristic of ROS membranes is their
enrichment in long-chain polyunsaturated fatty acids, and in particular
docosahexaenoic acid (DHA; 22:6
3), relative to total retinal
membranes or other neural tissue.18
In the bovine retinal
membrane preparations in this study, DHA represented 29.1 mol%
of the total ROS membrane fatty acids and only 14.5 mol% of the total
residual membrane fatty acids. A similar relationship was seen in the
human ROS and residual retinal membranes, where DHA represented 33.5
and 12.2 mol% of the total fatty acids, respectively (Table 1)
. This finding of enriched DHA in ROS preparations confirmed
the purity of the membranes to be used for lutein and zeaxanthin
analyses.
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Because the RPE was isolated as intact cells and analyzed without any further isolation of its membranes, lutein and zeaxanthin concentrations were expressed on the basis of total protein (membrane plus soluble). Lutein and zeaxanthin were detected in all samples (n = 6 donors) of human RPE (Table 3) . As in retinal membranes, lutein was the predominant carotenoid in the RPE. Three unidentified peaks consistently appeared in RPE chromatograms in addition to lutein and zeaxanthin (Fig. 2E) . The unidentified peak eluting just before lutein was also seen in most of the retinal membrane preparations but was always more prominent in the RPE samples.
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The relationship between the carotenoid concentration in ROS membranes and the carotenoid level in the central disks was determined for donor eyes in which both measurements were made (Fig. 4) . Among donors, the lutein plus zeaxanthin concentration of the perifoveal ROS was highly correlated with the carotenoid level in the central disks (P < 0.001; r = 0.983). A slightly weaker but still significant association was observed between the carotenoid concentration in the peripheral ROS and the level in the central disks (P < 0.05; r = 0.823).
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Discussion |
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Based on the protein yields of the membrane preparations and the concentrations of lutein and zeaxanthin reported herein, the total amounts of lutein plus zeaxanthin are estimated to be 145 ng in the entire retina and 14.9 ng in the ROS membranes. Considering the possibility of loss of some ROS during their isolation, lutein plus zeaxanthin amounts in ROS membranes would therefore represent approximately 10% to 15% of their amounts in the entire retina. In another recent study,21 approximately 25% of the total retinal carotenoids were in found in the ROS. However, a direct comparison between the two studies is not possible because different methodology was used for the isolation of ROS and the testing of their purity.
The lutein–to–zeaxanthin concentration ratios determined for human retinal membranes in the present study (Table 2) are consistent with the mass ratios determined by Bone et al.19 In making comparisons to the intact (i.e., not undergoing fractionation) retina samples examined in previous studies, it is important to note that ROS membranes represented approximately 8% to 9% of the total retinal membranes and should therefore be weighted less heavily in these comparisons. Thus, the closer correspondence of the residual rather than ROS retinal membranes to the ratios reported by Bone et al.19 supports this reasoning. Of greater significance, however, is the finding that lutein–to–zeaxanthin ratios in ROS membranes alone showed regional variation. Previous theories addressing the increase in the lutein–to–zeaxanthin ratio with linear distance from the fovea have proposed an association of lutein with rods and zeaxanthin with cones,19 or a preferential association of particular lutein–zeaxanthin ratios with different cone phenotypes.11 Our findings show that central (perifoveal) to peripheral differences in lutein-to-zeaxanthin ratio can also occur in ROS membranes independent of cones. Because the basis for this difference cannot involve different photoreceptor types, alternative possibilities include a spatial dependence of uptake of lutein and zeaxanthin into the ROS, or in carotenoid metabolism. Bone et al.2 proposed a lutein to meso–zeaxanthin conversion mechanism in the human retina that, if spatially distributed, could explain decreasing levels of meso–zeaxanthin and increasing levels of lutein with increasing distance from the fovea. With the HPLC methodology of the present study, zeaxanthin and meso–zeaxanthin co-eluted in a single peak.3 Therefore, even if such a conversion mechanism existed in human ROS membranes and varied with retinal region, it would not affect the zeaxanthin concentration values in our study. However, the possible involvement of this mechanism in lowering lutein levels in the perifoveal versus peripheral ROS cannot be excluded and is worthy of further evaluation.
Low levels of macular pigment have been detected in human RPE21 (also Bernstein PA, personal communication, August 1999), but no data have been provided regarding its concentration in this tissue. Our findings showed that human RPE isolated from all regions had a mean lutein plus zeaxanthin concentration of 4.29 ng/mg total (membrane plus soluble) protein. Assuming membrane protein to represent roughly half the total in RPE, the concentration of lutein plus zeaxanthin relative to membrane protein would likely be no more than 8 to 10 ng/mg. For most regions of the retina, this represents less than half the concentration found in ROS membranes. The possibility exists that some of the lutein/zeaxanthin measured in RPE is attributable to outer segment phagosomes or tips of ROS that remained attached during dissection. However, an equally likely possibility is that the human RPE stores some macular pigment to replenish what would be lost in the ROS due to turnover. The peaks in human RPE chromatograms other than those of lutein and zeaxanthin could not be identified by the HPLC methodology of the present study. However, the mobile phase and normal-phase columns used in this study were essentially the same as those used by Khachik et al.3 and in that study, oxidation products or isomers of lutein and zeaxanthin were identified that may represent the unknown peaks in the present study. Further experiments are needed to evaluate the possible relevance of these minor carotenoids as metabolites or oxidation products of lutein and zeaxanthin.
The location of macular pigment in the retina provides important clues to its function and role in protecting against AMD. The physical properties of macular pigment (i.e., wavelengths it absorbs and orientation relative to the direction of incoming light), and its very high density in the prereceptoral fovea, indicate that macular pigment is an ideal filter for improving the visual image and for protecting against short-wavelength visible light damage. The involvement of light exposure as a risk factor in AMD is uncertain, because epidemiologic studies have provided equivocal evidence regarding this relationship.8 10 However, the annular pattern of certain macular retinal degenerations (including those resulting from photosensitizing drug toxicity and AMD) shows a close correspondence between the region where macular pigment absorbs most strongly and the region of central sparing in these degenerations.32
The concentrations of lutein and zeaxanthin in the ROS of the perifoveal retina provides additional compelling support for an evolving theory pertaining to the role of oxidative stress and antioxidant protection in AMD. ROS membranes are uniquely susceptible to peroxidative insult because of their high content of long-chain polyunsaturated fatty acids and the high oxygen tension of their microenvironment. For unknown reasons, accumulation of diffuse lipid deposits, thought to be linked to the pathogenesis of AMD, occurs preferentially in the posterior pole of the eye.33 34 35 36 Recent studies have shown that lipids in these deposits are of cellular rather than blood origin, a likely source being debris derived from incompletely digested photoreceptor membranes.34 35 Most of the photoreceptor debris would originate from rods, being that they have a much higher density than cones (ranging from 4–30 times greater) in the perifoveal macula.37 Very recently, it has been shown that Bruch’s membrane isolates from the human macula contain peroxidized lipids that were derived in part from long-chain polyunsaturated fatty acids, typical of those found in outer segment membranes.38 Taken together, this evidence provides a direct link between peroxidation of human ROS in the perifoveal retina and the accumulation of debris that is associated with AMD. Furthermore, the location of relatively high concentrations of lutein and zeaxanthin in the perifoveal ROS corresponds to that that would be predicted to have the greatest requirement for antioxidant protection in the human retina.
Vitamin E is thought to be an important antioxidant in biological membranes and is found in high levels in mammalian ROS.39 40 If carotenoids function as antioxidants in human retinal membranes, they must do so in addition to, or in synergy with, vitamin E. In liver microsomes, the inhibition of lipid peroxidation for vitamin E and ß-carotene combined was found to be greater than the sum of their individual inhibitions.41 The vitamin E concentrations determined for the human retina have varied among studies, but the midrange was approximately 1 nmol/mg protein. Converting our concentration values to nanomoles, there is approximately 0.06 to 0.1 nmol/mg protein of lutein plus zeaxanthin in the membranes of perifoveal retina. The possibility for antioxidant synergism in membranes where vitamin E is in higher concentration than carotenoid has been proposed.41
Although the location of lutein and zeaxanthin in the ROS of the human retina suggests an antioxidant function, there has as yet been no direct demonstration of this role. Further studies are needed to determine whether or not the concentrations of macular pigment in human ROS membranes are capable of inhibiting lipid peroxidation at rates that might be expected to occur in the human retina. A protective role of lutein and zeaxanthin in AMD has been indicated by epidemiologic studies.22 23 These observations have prompted the inclusion of lutein in several commercially available nutritional and vitamin supplements. Lutein and zeaxanthin in animals are derived exclusively from dietary sources (there is no de novo synthesis), and macular pigment density was shown to increase in human subjects taking lutein supplements.42 However, until the physiological role of macular pigment in the retina is more fully understood and long-term safety established, caution should be exercised in taking such supplements, which may provide excessive doses. At this time, a sensible approach to optimizing visual health and preventing age-related retinal disease would be to consume a balanced diet that is rich in fruits and vegetables that contain these carotenoids.
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Acknowledgements |
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Footnotes |
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Submitted for publication September 24, 1999; revised December 3, 1999; accepted December 20, 1999.
Commercial relationships policy: N.
Corresponding author: Laurence M. Rapp, Department of Ophthalmology, NC-205 Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. lrapp{at}bcm.tmc.edu
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