Analysis of macular carotenoids in the developing macaque retina: The timeline of macular pigment development.

In the mature retina, the components of the macular pigment, lutein (L), R,R-zeaxanthin (RRZ), R,S-zeaxanthin (RSZ, meso-zeaxanthin) are most concentrated in the central macula. L and RRZ are of dietary origin but RSZ is produced in situ from L. The relative proportions of L and Z isomers vary across the retina with eccentricity in the adult retina. Early reports have shown that during development, the proportions of L and Z isomers undergo changes as the total pigment levels increase. The methods described here demonstrate the unique utility of chiral phase HPLC to measure the amounts of L, RRZ, and RSZ, discriminating between the two zeaxanthin stereoisomers. In three concentric retinal sections of macaque retinas chiral phase HPLC has been employed to document the developmental changes in the distribution of each L, RSZ, and RRZ during the period just prior to full term gestation through 19 months after birth. The net rate of accumulation of carotenoids within the central retina during the first 20 months is quasi-linear and fit by a linear regression. During development, the rate of transport of L (0.12 (±0.033)ngmm-2mo-1 (SE)) into the central 2mm of the retina is double that of RRZ (0.062 (±0.02)ngmm-2mo-1 (SE)). The rate of accumulation of RSZ (0.06 (±0.01)ngmm-2mo-1 (SE)) is comparable to that of RRZ. In the peripheral retina, the rates of accumulation of L and RRZ are not correlated with increasing age, whereas accumulation of RSZ does correlate with age. The changing proportions of L to Z isomers in the central retina during development are explained by the rates for carotenoid accumulation within the central retina. At birth, the macular pigment in the central retina is dominated by L and RRZ, 0.35±0.11 and 0.21±0.054ngmm-2. In the central retina, RSZ was rarely detected in the youngest tissues analyzed. It can be estimated to represent 6% of the total macular pigment (0.033±0.11ngmm-2) at birth based on extrapolation from measurements in the peripheral retina and the ratio of L/(RRZ+RSZ) is ≈1.5. At maturity, the concentrations for L, RRZ, and RSZ in the central macaque retina are estimated to be 1.7, 1.8 and 1.08ngmm-2, with L/(RRZ+RSZ) being 0.6.

Comparison of Antioxidant Properties of Dehydrolutein with Lutein and Zeaxanthin, and their Effects on Cultured Retinal Pigment Epithelial Cells.

Dehydrolutein accumulates in substantial concentrations in the retina. The aim of this study was to compare antioxidant properties of dehydrolutein with other retinal carotenoids, lutein, and zeaxanthin, and their effects on ARPE-19 cells. The time-resolved detection of characteristic singlet oxygen phosphorescence was used to compare the singlet oxygen quenching rate constants of dehydrolutein, lutein, and zeaxanthin. The effects of these carotenoids on photosensitized oxidation were tested in liposomes, where photo-oxidation was induced by light in the presence of photosensitizers, and monitored by oximetry. To compare the uptake of dehydrolutein, lutein, and zeaxanthin, ARPE-19 cells were incubated with carotenoids for up to 19 days, and carotenoid contents were determined by spectrophotometry in cell extracts. To investigate the effects of carotenoids on photocytotoxicity, cells were exposed to light in the presence of rose bengal or all-trans-retinal. The results demonstrate that the rate constants for singlet oxygen quenching are 0.77 × 1010, 0.55 × 1010, and 1.23 × 1010 M-1s-1 for dehydrolutein, lutein, and zeaxanthin, respectively. Overall, dehydrolutein is similar to lutein or zeaxanthin in the protection of lipids against photosensitized oxidation. ARPE-19 cells accumulate substantial amounts of both zeaxanthin and lutein, but no detectable amounts of dehydrolutein. Cells pre-incubated with carotenoids are equally susceptible to photosensitized damage as cells without carotenoids. Carotenoids provided to cells together with the extracellular photosensitizers offer partial protection against photodamage. In conclusion, the antioxidant properties of dehydrolutein are similar to lutein and zeaxanthin. The mechanism responsible for its lack of accumulation in ARPE-19 cells deserves further investigation.

Efficacy of Diacetate Esters of Macular Carotenoids: Effect of Supplementation on Macular Pigment.

The accumulation of the carotenoids lutein, zeaxanthin, and mesozeaxanthin in the center of the human retina, and known as the macula lutea or macular pigment, is believed to protect the retina from age-related macular degeneration. Since the macular pigment is of dietary origin, supplements containing the relevant carotenoids are readily available. In this study, we compared the changes in macular pigment over a 24-week supplementation period for two groups of 24 subjects each assigned to either of two supplement formulations, 20 mg/day of lutein or 20 mg equivalent free carotenoids of a combination of diacetate esters of the macular carotenoids. The latter group responded with a larger increase (0.0666 ± 0.0481) in macular pigment optical density than the former group (0.0398 ± 0.0430), driven largely by the older subjects. The difference was statistically significant (p=0.0287). There was a general trend towards smaller increases in macular pigment for those subjects whose baseline value was high. However, the trend was only significant (p < 0.05) for subjects in the diacetate group. No differences in response could be attributed to the gender of the subjects. We also observed no indication that the use of statin drugs by a few of the older subjects influenced their responses.

Direct observation of differences of carotenoid polyene chain cis/trans isomers resulting from structural topology.

In the present paper, trapped ion mobility spectrometry (TIMS) and theoretical calculations have been used to study carotenoid geometrical motifs generated by photoisomerization from the all-trans geometry. Multiple geometric isomers of the carotenoids lutein and zeaxanthin were separated using TIMS (R > 110) for [M](+), [M + H](+), and [M - 18](+) molecular species. Comparison of observed cross sections with those obtained from molecular dynamics calculations showed that the number of cis double bonds and s-cis single bonds in the polyene chain determine the topology space of the carotenoid. The intensities of IMS signals are correlated with the relative stability of these geometric isomers.1,2 The most stable isomer is the all-trans geometry regardless of the ionization state ([M - 18](+), [M](+), and [M + H](+)), and structural stability decreases with the increasing number of cis and/or s-cis bonds in the polyene chain.

Dose-dependent response of serum lutein and macular pigment optical density to supplementation with lutein esters.

We conducted a study to determine the effect of different doses of a lutein supplement on serum lutein concentration and macular pigment optical density (MPOD). Lutein is one of the major components of human macular pigment. Eighty-seven subjects received daily doses of 5, 10, or 20 mg of lutein, or a placebo, over a 140 day period. Serum lutein concentration was determined by HPLC and MPOD by heterochromatic flicker photometry (HFP). Serum lutein responded positively, except in the placebo group, reaching a plateau that, averaged for each dosage group, was linearly dependent on dose. Likewise MPOD, on average, increased at a rate that varied linearly with dose. For subjects deemed more proficient at HFP, approximately 29% of the variability in MPOD response could be attributed to a linear dependence on the fractional change in serum lutein concentration. We did not detect any significant influence of age on serum lutein uptake or MPOD response.

The conformation of end-groups is one determinant of carotenoid topology suitable for high fidelity molecular recognition: a study of beta- and epsilon-end-groups.

Conformation affects a carotenoid's ability to bind selectively to proteins. We calculated adiabatic energy profiles for rotating the ring end-groups around the C6C7 bond and for flexing of the ring with respect to the polyene chain. The choice of computational methods is important. A low, 4.2 kcal/mol barrier to rotation exists for a beta-ring. An 8.3 kcal/mol barrier exists for rotation of an epsilon-ring. Rotation of the epsilon-ring is sensitive to substitution at C3. In the absence of external forces neither beta- nor epsilon-rings are rotationally constrained. The nearly parallel alignment of the beta-ring to the C6C7 bond axis contrasts to the more perpendicular orientation of the epsilon-ring. Flexion of a beta-ring to the minimized epsilon-ring conformation requires approximately 23 kcal/mol; extension of the epsilon-ring to the minimized beta-ring conformation requires approximately 8 kcal/mol. Selectivity associated with beta- versus epsilon-rings is dominated by the inability of the beta-ring to flex to minimize protein/ring steric interactions and maximize van der Waal's attractions with the binding site.

Macular pigment response to a supplement containing meso-zeaxanthin, lutein and zeaxanthin.

BACKGROUND: Age-related macular degeneration (AMD) is a disease with multiple risk factors, many of which appear to involve oxidative stress. Macular pigment, with its antioxidant and light-screening properties, is thought to be protective against AMD. A result has been the appearance of dietary supplements containing the macular carotenoids, lutein and zeaxanthin. More recently, a supplement has been marketed containing, in addition, the third major carotenoid of the macular pigment, meso-zeaxanthin. The purpose of the study was to determine the effectiveness of such a supplement in raising macular pigment density in human subjects. METHODS: A 120 day supplementation study was conducted in which 10 subjects were given gel-caps that provided 20 mg/day of predominantly meso-zeaxanthin, with smaller amounts of lutein and zeaxanthin. A second group of 9 subjects were given gel caps containing a placebo for the same 120 day period. Prior to and during the supplementation period, blood serum samples were analyzed by high performance liquid chromatography for carotenoid content. Similarly, macular pigment optical density was measured by heterochromatic flicker photometry. Differences in response between the supplementation and placebo groups were tested for significance using a student's t-test. RESULTS: During supplementation with the carotenoids, blood samples revealed the presence of all three carotenoids. Macular pigment optical density, measured at 460 nm, rose at an average rate of 0.59 +/- 0.79 milli-absorbance unit/day in the 10 supplemented subjects. This was significantly different from the placebo group (9 subjects) for whom the average rate was -0.17 +/- 0.42 milli-absorbance units/day. CONCLUSION: We have shown for the first time that meso-zeaxanthin is absorbed into the serum following ingestion. The data indicate that a supplement containing predominantly meso-zeaxanthin is generally effective at raising macular pigment density, and may turn out to be a useful addition to the defenses against AMD.

Dose-ranging study of lutein supplementation in persons aged 60 years or older.

PURPOSE: To examine the dose-response relationship between oral lutein supplementation and serum lutein concentrations in persons aged 60 years and older, with or without age-related macular degeneration (AMD). METHODS: Forty-five participants with no AMD, large drusen, or advanced AMD, were randomized to receive one of three doses (2.5, 5, or 10 mg) of lutein for 6 months and to be observed for 6 additional months after the cessation of lutein supplementation. RESULTS: The mean age of the participants (33 women) was 71 years (range: 60-91). The serum lutein concentrations of each dose group were similar before supplementation, increased at 1 month, and peaked by 3 months. Median serum concentrations of the 2.5-, 5-, and 10-mg groups from baseline to month 6 increased from 18.7 to 35.1 microg/dL (2-fold increase), from 17.8 to 59.2 microg/dL (2.9-fold increase), and from 15.1 to 66.8 microg/dL (4-fold increase), respectively (all P < 0.001). The increases in lutein serum concentrations did not vary with AMD disease severity (P = 0.98). No toxicity was observed with any dose of lutein. No significant changes were detected in visual acuity or visual field tests. CONCLUSIONS: Increasing doses of lutein supplements significantly increased the serum levels of lutein and zeaxanthin, and doses up to 10 mg were safely administered. A long-term large clinical trial is necessary to investigate the safety and efficacy of lutein in reducing the risk of the development of advanced AMD.

Macular pigment and the edge hypothesis of flicker photometry.

Heterochromatic flicker photometry is commonly used to measure macular pigment optical density (MPOD) in the human retina. It has been proposed, and accepted by many, that the MPOD so measured represents the value at a retinal location corresponding to the edge of the flickering, circular stimulus. We have investigated this proposal by using a series of annular stimuli to determine the MPOD distribution in the central 1.5 degrees of the retina for both eyes of 10 subjects. The MPOD obtained using a 1.5 degrees circular stimulus matched the MPOD distribution at a retinal eccentricity that was always less than the stimulus radius, and averaged, for the 10 subjects, 51% of the stimulus radius. Similar results were obtained using a 1 degrees stimulus. Thus the edge hypothesis is inconsistent with our data. We suggest that involuntary eye movements may be responsible for an apparent edge effect.

Heterochromatic flicker photometry.

Measurement of the macular pigment optical density (MPOD) by heterochromatic flicker photometry (HFP) is accomplished by viewing a small circular stimulus that alternates between a test wavelength that is absorbed by the MP (typically--blue, 460 nm) and a reference wavelength that is not absorbed (typically-green, 540 nm). Flicker observed by the subject is reduced to a null point by adjusting the intensity of the former while viewing the stimulus centrally, and then peripherally. A higher intensity, I, of the blue component of the stimulus is needed under central viewing conditions owing to attenuation by the MP. The MPOD at the test wavelength is given by log (Icentral/Iperipheral). Variation of the test wavelength has been used to measure the MPOD spectrum. This in vitro MPOD spectrum matches that of the carotenoids present in the macular region of the retina and demonstrates the validity and specificity of this methodology. The distribution of MPOD in the retina can be determined with HFP using a series of annular stimuli of different diameters.

Lutein and zeaxanthin dietary supplements raise macular pigment density and serum concentrations of these carotenoids in humans.

Age-related macular degeneration (AMD) is thought to be the result of a lifetime of oxidative insult that results in photoreceptor death within the macula. Increased risk of AMD may result from low levels of lutein and zeaxanthin (macular pigment) in the diet, serum or retina, and excessive exposure to blue light. Through its light-screening capacity and antioxidant activity, macular pigment may reduce photooxidation in the central retina. Lutein supplements, at 30 mg/d, were shown previously to increase serum lutein and macular pigment density in two subjects. In this study, we compared the effects of a range of lutein doses (2.4- 30 mg/d), as well as a high zeaxanthin dose (30 mg/d), on the serum and macular pigment in a series of experiments. Serum carotenoids were quantified by HPLC. Macular pigment densities were determined psychophysically. Serum lutein concentrations in each subject reached a plateau that was correlated with the dose (r = 0.82, P < 0.001). Plateau concentrations ranged from 2.8 x 10(-7) to 2.7 x 10(-6) mol/L. Zeaxanthin was less well absorbed than an equal lutein dose, resulting in plateaus of approximately 5 x 10(-7) mol/L. The rate of increase in macular pigment optical density was correlated with the plateau concentration of carotenoids in the serum (r = 0.58, P < 0.001), but not with the presupplementation optical density (r = 0.13, P = 0.21). The mean rate of increase was (3.42 +/- 0.80) x 10(5) mAU/d per unit concentration (mol/L) of carotenoids in the serum. It remains to be demonstrated whether lutein or zeaxanthin dietary supplements reduce the incidence of AMD.

Biologic mechanisms of the protective role of lutein and zeaxanthin in the eye.

The macular region of the primate retina is yellow in color due to the presence of the macular pigment, composed of two dietary xanthophylls, lutein and zeaxanthin, and another xanthophyll, meso-zeaxanthin. The latter is presumably formed from either lutein or zeaxanthin in the retina. By absorbing blue-light, the macular pigment protects the underlying photoreceptor cell layer from light damage, possibly initiated by the formation of reactive oxygen species during a photosensitized reaction. There is ample epidemiological evidence that the amount of macular pigment is inversely associated with the incidence of age-related macular degeneration, an irreversible process that is the major cause of blindness in the elderly. The macular pigment can be increased in primates by either increasing the intake of foods that are rich in lutein and zeaxanthin, such as dark-green leafy vegetables, or by supplementation with lutein or zeaxanthin. Although increasing the intake of lutein or zeaxanthin might prove to be protective against the development of age-related macular degeneration, a causative relationship has yet to be experimentally demonstrated.