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From the Department of Ophthalmology and Visual Sciences, Moran Eye Center, University of Utah School of Medicine, Salt Lake City, Utah.
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Abstract |
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METHODS. Lenses and circular punches of macula (4-mm diameter) and equatorial peripheral retina (8-mm diameter) were dissected from donor eyes free of ocular disease procured from the local eye bank. The amounts of lutein, zeaxanthin, meso-zeaxanthin, and 3'-oxolutein were determined by HPLC with photodiode array and mass spectral detection.
RESULTS. Eighteen percent of eyes from donors age 48 and older had unusually high levels (66.3 ± 15.1 ng) of macular carotenoids that were three times the rest of the older population’s mean level (23.0 + 12.1 ng; P < 0.001). Carotenoid levels in these outliers were also unusually high in the lens and in the peripheral retina. Similar outliers were not present in donors younger than 48. Most of these outliers regularly consumed high-dose lutein supplements before death. Lutein supplementation was uncommon in older donors whose macular carotenoids were in the normal range.
CONCLUSIONS. The presence of unusually high levels of macular carotenoids in older donors who were regularly consuming high-dose lutein supplements supports the hypothesis that long-term lutein supplementation can raise levels of macular pigment. Elevated carotenoid levels in the peripheral retina and lens in these same donors could have important implications for understanding why some clinical methods of macular pigment measurement have had difficulty detecting robust and consistent responses in carotenoid supplementation trials.
Carotenoid supplementation strategies for AMD rely on the assumption that consumption of these supplements will lead to clinically significant increases in the macular carotenoid pigment, but published and unpublished studies have reported unexpectedly modest and variable responses despite consistent increases in serum levels.15 16 17 18 19 20 21 22 23 Possible explanations of these results include inadequate study length, saturation of ocular xanthophyll-binding proteins, and inherent limitations of the noninvasive measurement methods. The most commonly used noninvasive measurement methods, heterochromatic flicker photometry and autofluorescence imaging, operate under the premise that carotenoid levels at a peripheral retinal reference point are so low that they are optically undetectable.24 25 26 27 28 29 This assumption has been well validated by studies on primate cadaver eyes that demonstrated the concentration of macular carotenoids per unit area measured by absorption spectroscopy, resonance Raman spectroscopy, and high-pressure liquid chromatography (HPLC) is 10 to 100 times higher in the macula than it is in the peripheral retina.9 10 11 30 31 32 33 34 It should be noted, however, that this assumption cannot be simply extrapolated to the supplemented situation because these autopsy studies were conducted before the lutein supplementation era. It is possible that the pharmacologic doses of lutein currently consumed by many elderly persons could lead to substantial increases in peripheral lutein levels that would artifactually alter noninvasively measured macular responses. To assess this possibility, we measured ocular carotenoid levels by HPLC in a large number of human donor eyes now that high-dose lutein supplementation has become a common practice among older adults in Utah.
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Methods |
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Preparation of Macular, Retinal, and Lens Tissue Samples
Human donor eyes were obtained between January 2004 and April 2005 from the Utah Lions Eye Bank within 24 hours of death after corneas had been harvested for transplantation. Tissue procurement and distribution complied with the tenets of the Declaration of Helsinki. All eyecups were visually inspected to exclude the presence of obvious ocular disease. After carefully removing overlying vitreous, macular tissue was excised with a 4-mm–diameter circular trephine centered on the fovea, and peripheral equatorial retinal tissue was excised with an 8-mm–diameter circular trephine, making certain that the nearest edge of the punch was at least 10 mm from the fovea. Tissue samples were extracted three times with tetrahydrofuran (THF) containing 0.1% BHT by sonication at 5°C to 10°C for 30 minutes each time. Whole lenses were homogenized and then extracted in the same manner. Combined extracts were evaporated to dryness under vacuum at room temperature. The dried residue was re-dissolved in 1 mL HPLC mobile phase and centrifuged at approximately 2000g for 10 minutes to remove the minor amounts of insoluble solid particles.
HPLC Conditions
The extracts were analyzed on two HPLC systems on a gradient HPLC (Thermo Separations, San Jose, CA) equipped with a high-sensitivity UV6000 photodiode array detector.
System 1: Routine Carotenoid Separation.
The mobile phase contained hexane/dichloromethane/methanol/N,N'-di-isopropylethylamine (80:19.2:0.7:0.1 vol/vol). HPLC separation was carried out at a flow rate of 1.0 mL/min on a cyano column (Microsorb, 25-cm length x 4.6-mm inner diameter [ID]; Rainin Instrument Co., Woburn, MA). The column was maintained at room temperature, and the HPLC detector was operated at 450 nm. Peak identities were confirmed by photodiode-array (PDA) spectra, by coelution with authentic standards, and by mass spectrometry. Calibration curves for peak quantitation were performed by injection of known amounts of carotenoid standards. We do not routinely include an internal standard because it can mask the presence of low abundance carotenoid metabolites, especially when samples are split and run on multiple HPLC columns.35 We periodically perform quality control runs to confirm that our extraction and injection efficiencies exceed 95%.
System 2: Chiral Carotenoid Separation.
The mobile phase contained hexane/isopropanol (95:5 vol/vol). HPLC separation was carried out at a flow rate of 0.7 mL/min on a chiral column (ChiralPak AD, 25-cm length x 4.6-mm ID; Chiral Technologies, Exton, PA) using the detection methods described.
Mass Spectrometry Equipment and Conditions
Levels of ocular carotenoids and their metabolites were sometimes so low that reliable quantitation by absorbance at 450 nm was not possible. In these situations, we used in-line mass spectrometry analysis because it is approximately 100 times more sensitive.36 HPLC-MS was performed (Thermo Electron MSQ, San Jose, CA) with a single quadrupole mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) source after a 50% split of the column eluant. The molecular ions were initially acquired in full scan mode from 200 to 1000 Da with 0.2-step size and 2-ms dwell time. Selected ion monitoring (SIM) was performed using a dwell time of 200 ms for each channel. In SIM mode, the m/z channels 551 ± 1 (M–H2O+) and 569 ±1 (M+) were used for lutein, 569 ±1 for zeaxanthin (M+), and 567 ± 1 for 3'-oxolutein (M+). Typical conditions were corona discharge current, 5 µA; RF lens bias voltage 0.1 V; cone voltage, 80 V; and heater temperature, 550°C. The ion source and tuning lens parameters were optimized by infusing standards through the built-in injector. For routine quantitative analysis, we used software (ThemoElectron MSQ Quan Browser; XCaliber Systems, Brazil, IN) that provided a peak integration of the ion intensity for each of the programmed mass ranges against standard curves generated from injection of authentic carotenoid standards. Automated analysis was followed by manual confirmation of in-source fragmentation patterns.
Statistical Analysis
Statistical analysis was performed (Origin version 6.0; Microcal, Northampton, MA). In most cases a two-population (independent) t test was performed with a significance level set at 0.05. All reported values are mean ± SD.
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Both eyes were available for analysis in 15 of the 21 outlying donors. The concordance rate (>50 ng in both eyes) of these outliers was 53% (8 /15 pairs). The average level of carotenoids in the fellow eyes of discordant outliers (39.8 ± 4.4 ng; n = 7) was 45% less than that of the outlier eyes themselves (72.1 ± 25.2; n = 7; P < 0.001) but was significantly higher than the mean value of the rest of the "normal" older eyes (21.3 ± 11.3 ng; n = 124; P < 0.001).
Retrospective nutritional supplement and dietary histories were obtained from the families of 17 of the 21 outliers and from a random sample of 20 of the rest of the older donors. Eighty-two percent (14/17) of the outliers’ families reported that the donor regularly consumed a high-dose (2.5–10 mg) lutein supplement daily, and the rest of the outliers’ families reported that before death, the donor routinely consumed an unusually carotenoid-rich diet daily including spinach, corn, broccoli, and orange juice. None of the 20 nonoutliers’ families surveyed reported regular lutein supplementation or consumption of a carotenoid-rich diet (P < 0.001).
None consumed supplements that contained high percentages of zeaxanthin, though it should be noted that commercial lutein supplements typically contain 6% zeaxanthin, which would supply 0.36 to 1.2 mg of zeaxanthin from a 6- to 20-mg lutein supplement. The family of a 52-year-old outlier reported a diet unusually rich in zeaxanthin (daily servings of corn), and this donor had the highest dietary zeaxanthin level in the macula of any eye analyzed (Fig. 1D) .
We determined which carotenoids were responsible for the extraordinarily high levels in the outliers’ maculae. The high levels of total macular carotenoids were driven primarily by lutein and, to a lesser extent, by its probable metabolite meso-zeaxanthin (Figures 1B-1E) . The average lutein levels were 27.7 ± 8.9 ng in outlier eyes (n = 29) and 9.3 ± 5.8 (n = 131) in the rest of the older eyes (P < 0.001), and the average total zeaxanthin in outliers was 24.0 ± 10.9 (n = 29) compared with 10.1 ± 6.8 (n = 131) in the rest of the older eyes (P < 0.001). We observed that the mean values for dietary (3R, 3'R)-zeaxanthin were not statistically significant (P = 0.38) for outlier eyes (13.3 ± 6.9 ng; n = 16) than they were for older normal eyes (10.1 ± 7.1 ng; n = 94). However, significantly higher levels of nondietary (3R, 3'S-meso)-zeaxanthin were observed in the outlier eyes (19.4 ± 14.1 ng; n = 16) than in older normal eyes (7.4 ± 9.6 ng; n = 107; P < 0.001). Similarly, the ratio of (3R,3'R)-zeaxanthin to (3R,3'S-meso)-zeaxanthin (Z/M ratio) was significantly lower in outlier eyes than in control eyes (Table 1) . 3'-Oxolutein was not significantly higher in outlier eyes than in age-matched normal eyes (1.9 ± 1.3 ng [n = 16] vs. 1.2 ± 1.8 ng [n = 60]; P > 0.05; Fig. 1F ).
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Discussion |
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We also found that, on average, donors with high levels of macular carotenoids had significantly elevated levels of carotenoids in lens and peripheral retina. Elevated lutein levels were consistently encountered in the lens (range, 2- to 10-fold above average), which may explain why some studies have implicated lutein as a protective nutritional factor against cataracts.37 38 They were more variable, however, in the peripheral retina (range, 1- to 10-fold above average). Increased carotenoid concentrations in the lens and in the peripheral retina in response to supplementation could have profound effects on the various methods used to quantify macular pigment in living human eyes.
Heterochromatic flicker photometry (HFP) is the most commonly used method to measure macular pigment in clinical research.17 18 24 26 27 39 40 A small spot of light projected onto the fovea rapidly alternates between blue, which is well absorbed by the macular carotenoids, and green, which is not. The subject varies the relative intensities of the two lights until the perception of flicker is minimized or eliminated, and the average energy of the blue light (Bfov) at minimal flicker from multiple tests is recorded. Because subjects vary markedly in the ratio of L and M cones that mediate color perception in HFP, the test must be repeated with an eccentric reference fixation point. To determine Bref, macular pigment optical density (MPOD) is assumed to be zero. Typical reference points are at 4° to 9° eccentricity (1.2–2.6 mm at the retinal surface). The MPOD is then calculated with the equation MPOD = log (Bfov/Bref). The zero MPOD assumption at approximately 7° is likely to be reasonable in an unsupplemented primate eye based on microspectrophotometry using absorbance and resonance Raman techniques in cadaver eyes,32 33 34 but our data in this study raised questions as to whether this assumption remains valid in the supplemented state. In some subjects, substantial increases in carotenoid content (up to 10-fold) are exhibited in the equatorial region, very far beyond the traditional reference point of approximately 7°. We calculated that the average optical density attributable to xanthophyll carotenoids in the equatorial retina in one of these donors would be up to 0.01 at 460 nm. This would introduce a 3% underestimate of foveal MPOD for a subject with a typical foveal MPOD of 0.3, which is not particularly problematic if the equatorial periphery were used for Bref. In HFP, however, Bref is less than 2.6 mm from the center of the fovea. It is not unreasonable to predict that MPOD at approximately 7° might be in the 0.1 range or even higher in a supplemented subject because of a steep gradient of xanthophyll concentration from the periphery to the fovea, as evidenced in a previous human retinal HPLC microdissection study demonstrating that macular carotenoid levels at this eccentricity are typically more than 10 times higher than at the equatorial periphery.11 This level of MPOD at Bref would introduce a very large and potentially variable error in the foveal MPOD calculation (up to a 30% underestimate for a typical foveal MPOD of 0.3 or a 10% underestimate for a very high foveal MPOD of 1.0). The best way to avoid this problem would be to place Bref as far as possible in the periphery. Unfortunately, reliable psychophysical performance of HFP much beyond 7° (i.e., >14°) is extraordinarily difficult.
Autofluorescence (AF) measurement of macular pigment is based on the measurement of attenuation of lipofuscin’s inherent fluorescence by xanthophyll carotenoids.25 28 29 41 In its most typical form, blue monochromatic light excites lipofuscin’s main A2E fluorophore, which fluoresces at long wavelengths. Lutein and zeaxanthin are essentially nonfluorescent and absorb incident blue light, thereby attenuating A2E’s fluorescence in the fovea. This method is amenable to high-resolution imaging with a digital camera or a scanning laser ophthalmoscope (SLO). If performed with a single wavelength, such as 488 nm, an assumption of reasonably uniform lipofuscin distribution and zero MPOD in the periphery must be made. The problems related to carotenoid supplementation discussed for HFP would therefore apply. Fortunately, a more peripheral reference point (i.e., >14°) could be chosen with this optical method. Sometimes the investigators make a second AF image with a green wavelength, such as 514 or 532 nm, that is not absorbed by carotenoids and that can then be used for digital subtraction from the 488-nm image to correct for inhomogeneities of lipofuscin distribution and various abnormalities. Zero MPOD assumption is still generally required.
Dual-wavelength reflectometry has also been performed using an SLO platform at 488 and 514 nm and a zero reference point of 14°.22 42 This method’s performance in a supplementation study would be expected to be comparable to AF imaging given that the assumptions are similar. Reflectometry can also be implemented in a quantitative nonimaging mode in which the fovea is illuminated with a series of narrow-bandpass filters, and a sophisticated optical model is used to calculate the average spectral contribution of the absorbance of the macular carotenoids. This method should be unaffected by peripheral changes in carotenoid content. On the other hand, changes in optical density of the lens in response to carotenoid deposition (approximately 0.01 optical density units) should be incorporated into the model or an overestimate of foveal MPOD will result.
Resonance Raman spectroscopy (RRS) has been used as a specific optical method to measure macular carotenoid pigments in the living human eye.21 32 43 Light (488 nm) is briefly illuminated on a 1-mm–diameter region of the macula centered on the fovea, and wavelength-shifted light is analyzed on a Raman spectrometer. The intensity of the xanthophyll carotenoids’ C = C vibration at 1525 cm–1 is linearly associated with macular carotenoid content. In the originally described integral mode, no peripheral correction is required; thus, values would be unaffected be any changes in the periphery, but substantial lens opacities or increases in lens carotenoid content could alter the values. Absorbance of the incident 488-nm laser light by carotenoids in the lens would mildly attenuate the observed Raman-shifted signal causing an underestimate of the foveal carotenoid content, but this would be counteracted by an opposing increase in the measured Raman signal from the lens carotenoids themselves. In the future, carotenoid supplementation studies using RRS should include a sufficient number of pseudophakic subjects to assess the magnitude of this potential problem in phakic subjects.
HPLC is sometimes referred to as the "gold standard" of ocular carotenoid measurement, but this technique has its own limitations, too. Although it is the most chemically specific, its sensitivity and spatial resolution cannot match any of the optical detection methods. In the past, we have consistently observed an age-related decline of macular pigment in our Utah clinic population with a variety of techniques, including RRS, AF imaging, and HFP,21 40 42 but we did not observe such a decline in this HPLC study. Possible explanations include the fact that we used 4-mm tissue punches, which might blunt any age-related changes of foveal MPOD observed with methods with higher spatial resolution and which look at regions with diameters measuring 1 mm or less. In addition, all our studies in living humans specifically recruited clinic-based subjects who were not routinely consuming high-dose carotenoid supplements, whereas the present study accepted all donor eyes regardless of supplementation history.
The findings reported here of elevated lenticular and retinal levels of ocular carotenoids apply only to lutein supplements because high-dose zeaxanthin supplementation is still too rare in our population. Zeaxanthin is more selectively concentrated in the fovea, where its binding protein, GSTP1, is found in very high concentrations.44 Hence, peripheral retinal rises in response to supplementation may not be as high as they were with lutein. Lutein’s binding protein in the human retina is still not identified, but we have partially purified one from human and quail liver (Bhosale P et al. IOVS 2005;46:ARVO E-Abstract 1756). This lutein-binding protein induces a large red shift of its carotenoid ligand’s absorbance, which may also have important implications for the various noninvasive methods of carotenoid measurement.
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Acknowledgements |
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Footnotes |
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Submitted for publication May 20, 2006; revised July 7 and September 4, 2006; accepted December 5, 2006.
Disclosure: P. Bhosale, None; D.Y. Zhao, None; P.S. Bernstein, Kemin Health (C, R)
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Paul S. Bernstein, Department of Ophthalmology and Visual Sciences, Moran Eye Center, University of Utah School of Medicine, 65 Medical Drive, Salt Lake City, UT 84132; paul.bernstein{at}hsc.utah.edu.
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