Lutein, Zeaxanthin, and the Macular Pigment

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 pigment are lutein, zeaxanthin, and meso-zeaxanthin. The regular distribution pattern of these carotenoids within the human macula indicates that their deposition is actively controlled in this tissue. The chemical, structural, and optical characteristics of these carotenoids are described. Evidence for the presence of minor carotenoids in the retina is cited. Studies of the dietary intake and serum levels of the xanthophylls are discussed. Increased macular carotenoid levels result 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 photoprotection hypothesis for the macular pigments. © 2001 Academic Press

Key Words: macular pigment; carotenoid; lutein; zeaxanthin; 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 functional benefit of its presence is that it reduces chromatic aberration in the eye (6). The well-known psy1

To whom correspondence should be addressed. Fax: (305) 3483772. E-mail: [email protected]. 2 Abbreviations used: MP, macular pigment; MPOD, macular pigment 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 carotenoids 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 support 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 account 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 opposed to ␤-carotene or any of the other abundant carotenoids, are present in the macula. From a simplistic view, function in the chemical world is dependent upon structure. The chemical structure embodies the physical 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 biochemical systems that result from the spatial relationships 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 fo0003-9861/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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THE CHEMICAL IDENTITY AND STRUCTURE OF THE MACULAR PIGMENT COMPONENTS

Structure

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 investigation.

FIG. 2.

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 carotenoid 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 appear, at first glance, to be identical. The chemical formulas of lutein and zeaxanthin are chemically distinguished from one another in important ways. Zeaxanthin 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). Several complementary chemical methods have unambiguously established the identities of these two carotenoids: mass spectrometry (22), UV-visible spectrometry (21, 28), match of retention times by co-elution with authentic standards on multiple chromatographic sta-

The structures of the major carotenoid components found in the human macula.

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tionary phases (21, 22), and chemical derivatization (21, 22). Stereochemistry The stereochemical isomerism possible for both lutein and zeaxanthin poses important chemical questions. Are the stereochemical structural differences significant to any separate and distinct functional role these molecules may individually fulfill? Derivatization of lutein and zeaxanthin with benzoic anhydride to form dibenzoates or with (S)-(⫹)-1-(1-napthyl) ethyl isocyanate to form dicarbamates enables the stereoisomers 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 stereoisomer, (3R,3⬘R,6⬘R)-␤,⑀-carotene-3,3⬘-diol. Zeaxanthin within the retina was demonstrated to be primarily 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 zeaxanthin present. Most of the remaining retinal zeaxanthin was shown to be chromatographically indistinguishable from R,S-zeaxanthin. R,S-meso-zeaxanthin, (3R,3⬘S)-meso-␤,␤-carotene-3,3⬘-diol, is not a naturally 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 distribution 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 spectroscopy and on the correct position of the chromatogram 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 assisted by determination of the circular dichroism spectra (32). Because of sample size requirements and technical 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 additions to the characterization of the macular carotenoids (32). Circular dichroism is not of significant util-

FIG. 3. Chromatograms of the retinal extracts obtained from portions 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 geometrical 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 structural 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 perpendicular to the membrane surface. Lutein can apparently insert itself into the membrane in a nonorthogonal 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 significance 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

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FIG. 4. Conformation of the ␤-ring end-group showing the ca. 40° angle between the plane of the polyene chain and the ring double bond.

nonlinear geometry. When incorporated into membrane systems they disrupt the packing order of the alkyl chains and alter membrane fluidity. Oxo-lutein, (3-hydroxy-␤,⑀-carotene-3⬘-one), epilutein, and ⑀,⑀⫺carotene3,3⬘-dione were also reported by Khachik to be present in the retina (and also serum, see below) (24, 36). When retinal carotenoid extracts are analyzed by HPLC two peaks are often observed to preelute lutein on C-18 reversed-phase columns. See peaks labeled M1 and M2 in Fig. 3. Peak M2 elutes with a retention time that is consistent with that of authentic oxo-lutein prepared by partial synthesis in our laboratory (37). Together these minor MP components may approach 20% of the total carotenoid present within a given portion of the retina. There are two components in the serum that chromatographically match M1 and M2 (36 –38). They have been shown to be metabolites of lutein (39). More information on this topic is combined with a broader discussion of MP metabolism, and follows description of the anatomical distribution of the MP components. 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 differences in the number of conjugated bonds. Lutein and zeaxanthin differ very slightly in color. Purified zeaxanthin 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 between 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 zeaxanthin as consisting of 11 double bonds and that of lutein as 10 double bonds including the influence of the ␤-ring

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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 central maximum flanked at longer wavelengths by a secondary 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 ␤-ring double bond in zeaxanthin with the extended conjugation of the polyene chain very slightly lowers 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 zeaxanthin (␭ max ⫽ 451 nm in ethanol) when compared to that of lutein (␭ max ⫽ 445 nm) (Fig. 5). The other optically significant difference in these two carotenoids is the spacing between peaks II and III, illustrated by the intervening minimum, and the ratio of intensities that results. For zeaxanthin, peaks II and III are both somewhat broader and the spacing between them is smaller, resulting in a less distinct maximum for III with a more shallow minimum between peaks II and III. For zeaxanthin, ␭ III–␭ II is 26 nm and the II/III ratio is 38%. The corresponding spacing for lutein is 29 nm and a distinct minimum between II and III is observed in well-purified samples. The lutein II/III ratio is 60%. For lutein, peak I is a fully distinct maximum, not a shoulder. Oxo-lutein (M2 in Fig. 3) has a spectrum that is virtually indistinguishable from that of lutein. The visible spectrum of the MP peaks at ⬃460 nm roughly 10 nm red-shifted relative to that of a comparable mixture of lutein and zeaxanthin (28). This red-shift is reproduced by incorporation of lutein and zeaxanthin into phosphatidyl lipid liposomes (13).

FIG. 5. Spectra of lutein and zeaxanthin, in ethanol, illustrate the characteristic differences in the absorption properties of the two carotenoids.

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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 nature of the oxidant species. Singlet oxygen is known to be quenched by energy transfer to the carotenoid, generating a triplet state carotenoid that is able to harmlessly relax through vibrational transitions and collision without destructive bond breaking (18, 44 – 46). Radical species, once generated, are capable of oxidizing an organic molecule via a mechanism that either occurs with hydrogen atom extraction or by direct addition. Such mechanisms are chain reactions that regenerate an active radical capable of further destructive 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-stabilized carotenoid radical in which the unpaired electron is no longer oxygen-centered, rather it is delocalized over the conjugated polyene. The enhanced stability 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 ascorbic 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 lutein and zeaxanthin is that the allylic hydroxyl of lutein is much more easily oxidized than the secondary hydroxyl groups present in zeaxanthin. We should emphasize the distinct difference that is involved in the 2e ⫺ oxidation of the hydroxyl groups in the xanthophylls as compared to the one electron oxidation which is a characteristic reaction of all hydrocarbon carotenoids. 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-3hydroxy-␤,⑀-carotene-3⬘-one, in an 80% yield (52). Zeaxanthin 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 conjugation of the alkene system (53). This 6e ⫺ oxidation produces rhodoxanthin, a retro-carotenoid, in yields of 5–10% with considerable cleavage of the starting xanthophyll. 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 pigment 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 sufficiently well understood and kinetic as well as thermodynamic factors may be important to biological mechanisms of oxidation. With extended exposure to strong oxidizing agents such as MnO 2, both lutein and zeaxanthin 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 pigment 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 sections, 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 concentrations in the inner retinal layers (Fig. 7) (56). Until very recently it has been hypothesized, but unproven, that substantial quantities of carotenoids would be found in the photoreceptors. Solid evidence has recently been obtained that the macular carotenoids are present in the rod outer segments (58, 59). An important question that must now be answered is what

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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 demonstrated 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 sections 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 accompanied 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-zeaxanthin 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

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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

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:zeaxanthin ratio reaches a minimum in the central macula where mesozeaxanthin 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 systematic variation in the lutein/zeaxanthin ratio points strongly to the existence of a specific biochemical pathway 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 Sommerburg 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 remains 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 tubulin 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

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 developing 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 measured MP optical density (MPOD) and found no change with increasing age (4). In a longitudinal study, Hammond 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 subjects 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 significant 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 significant influence on this observation including lifetime exposure to blue light, dietary intake of carotenoids, 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).

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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 zeaxanthin 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)-␤,⑀-carotene3,3⬘-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 dietary 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

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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 metabolites of lutein were observed to increase with supplementation and similarly decreased upon discontinuation of supplementation. The half-lives for appearance 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 oxolutein (37, 39). Khachik et al. reported several carotenoids 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 zeaxanthin supplementation, we found that zeaxanthin had two metabolites, 13-Z-zeaxanthin and a compound which co-elutes with oxo-lutein but whose visible spectrum is decidedly zeaxanthin-like. Our current understanding can be summarized briefly as follows: serum levels of lutein and zeaxanthin are dependent upon dietary intake, serum levels associated with the normal diet are far below the maximal levels achieved with supplementation, both lutein and zeaxanthin are metabolized, and oxidized ketocarotenoids and Z-isomers increase in the serum when lutein or zeaxanthin supplements are consumed. Analysis of 20 normal serums demonstrates that a component, 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 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 average value of 2.46 ⫻ 10 ⫺4 mmol/L (93). The same subjects 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-

Effects on Retinal Concentration The effect on the MP of supplementation of the normal 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 increased 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

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by human subjects (54, 55, 93). They studied supplementation 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 supplementation. In a similar study with 30 mg/day of zeaxanthin two subjects both showed increases in MPOD with rates comparable to that observed with lutein. Low-dosage 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 appears 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 increases, especially at low dosages. The studies by Landrum 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 accumulated 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 result in accumulation and crystallization of this carotenoid 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 (4hydroxy-␤,␤-carotene-4⬘-one) and isozeaxanthin (␤,␤carotene-4,4⬘-diol) were present in the retinas of monkeys 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 demonstrates that the primates are able to reduce keto-carotenoids to the corresponding alcohols. Oxo-lutein, 3-hydroxy-␤,⑀-carotene-3⬘-one, and ⑀,⑀⫺carotene3,3⬘-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 process 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 accurate 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 instruments used by different research groups. As discussed below, absorption of blue light is a potentially significant 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 absorbed. 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 reproducibility 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 reflected light also affected by the absorbance of hemoglobin in the choriocapillaris. Fluorescence measurements on RPE lipofuscin have also been developed as a means of mapping MPOD (111). Excitation wavelengths are selected that are absorbed to different degrees by the MP. This technique may be problematic for those age-related macular degeneration (AMD) subjects who have large drops in the amount of RPE lipofuscin. Recently, Bernstein et al. reported the promising 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.

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As yet there is no standardized method for the unequivocal 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 damage 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 studies 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 reduced in areas where MP levels are highest (117). A clinical condition, known as Bull’s eye maculopathy, associated with photosensitizing drugs, is characterized 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 functional 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-oxidation of reactive unsaturated lipid components of photoreceptor 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 epithelium 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 presence 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 species (47). Martin et al. have recently shown that the xanthophylls are somewhat better antioxidants that hydrocarbon carotenoids such as ␤-carotene (122). They exhibit 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

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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). Photooxidation 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 published results showing that individuals who have a high dietary intake of lutein and zeaxanthin have a reduced risk of advanced neovascular AMD (131). Similarly, in another study, they found that elevated serum 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 cellular level locations within the retina using immunological methods. We recognize that the MP probably undergoes 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 environmental factors such as changing diet. Here too, the ability to use immunoassay techniques to quantify the presence of MP specific proteins during this developmental period might provide great insight relevant to the functional 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 intermediate species that are involved in the metabolic formation of meso-zeaxanthin. We have seen that keto-carotenoids are apparently reduced in the retina. It will be important to establish whether this process is a specific

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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 provide 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 macular 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 exceptional levels. These carotenoids have been demonstrated 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 hypothesized 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, longitudinal 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 adequate 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 measurement 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 enable clinicians and their patients to make appropriate 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 Interchange 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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