Resonance Raman Measurement of Macular Carotenoids in Retinal, Choroidal, and Macular Dystrophies

Background  It has been hypothesized that the macular carotenoid pigments lutein and zeaxanthin may protect against macular and retinal degenerations and dystrophies.

Objective  To test this hypothesis by objectively measuring lutein and zeaxanthin levels in a noninvasive manner in patients who have retinitis pigmentosa (RP), choroideremia (CHM), and Stargardt macular dystrophy and comparing them with an age-matched healthy control population.

Methods  Using resonance Raman spectroscopy, a novel objective noninvasive laser–optical technique, we measured macular carotenoid levels in 30 patients (54 eyes) who have RP, CHM, and Stargardt macular dystrophy and compared them with 76 age-matched subjects (129 eyes) who did not have macular pathologic conditions in a case-control study.

Results  As a group, patients with RP and CHM had the same macular carotenoid levels as age-matched healthy control subjects (P =.76, 2-way analysis of variance). Patients with Stargardt macular dystrophy tended to have levels of macular carotenoid pigments that, on average, were about 50% lower than healthy controls (P = .02, unpaired 2-tailed t test).

Conclusions  The patients with RP and CHM had normal levels of macular carotenoids, suggesting that nutritional supplementation with macular carotenoids such as lutein, zeaxanthin, or both will be unlikely to affect the clinical course of RP and CHM. Although the number of patients with Stargardt macular dystrophy examined was limited, their macular carotenoid levels were usually lower than those of subjects of a similar age with no macular pathologic condition.

RETINITIS PIGMENTOSA (RP) is the common name of a group of hereditary retinal degenerative diseases in which there is a slow and progressive loss of photoreceptors, especially in the peripheral retina.¹ Choroideremia(CHM) is an X-linked recessive disorder similar to RP, characterized by the progressive degeneration of the retinal pigment epithelium, neural retina, and choroid.² Patients who have RP and CHM generally initially have night blindness and constriction of the visual field early in life, followed by severe impairment in central visual acuity by the fifth to seventh decades of life. Stargardt macular dystrophy (STGD), however, is an inherited condition that leads to an early death of the macular photoreceptors and retinal pigment epithelium with relative preservation of the peripheral vision.³ Stargardt macular dystrophy usually occurs in the first decades of life and is characterized by a progressive decrease in central visual acuity. Loss of macular function is particularly devastating in all 3 disorders, leading to loss of ability to read and recognize faces. Thus, interventions that could preserve macular function could have substantial influence on the quality of life of these patients.

The macular carotenoid pigments lutein and zeaxanthin are thought to protect foveal photoreceptors by absorbing harmful blue light, and their antioxidant properties may inhibit free radical damage caused by light and oxygen.^4-10 They are specifically concentrated in the primate macula at very high levels, presumably through the action of specific high-affinity–binding proteins.¹¹ Derived from the diet, lutein and zeaxanthin can be found in dark green leafy vegetables and orange and yellow fruits and vegetables. Despite limited dietary sources, zeaxanthin as a mixture of its stereoisomers predominates over lutein in the foveal region of the human eye by a ratio of greater than 2:1.⁵ It has been suggested that lutein may be converted biochemically to meso-zeaxanthin in the human eye.^7,8

We have recently reported that elderly individuals who have age-related macular degeneration (AMD) who do not regularly consume high-dose (≥4-mg/d) lutein supplements have 32% lower levels of lutein and zeaxanthin in their maculae relative to a matched-control population.¹² Patients with AMD who have begun taking high-dose lutein supplements after their initial diagnosis have macular pigment levels in the normal range for their age as determined by Raman spectroscopy.¹² These results are consistent with the hypothesis that AMD is in part a manifestation of an ocular deficiency of lutein, zeaxanthin, or both and that higher macular levels of these carotenoids may protect against AMD. Similarly, it has been suggested that lutein supplements might protect against loss of cone and rod function in patients with RP.¹³ In fact, a lutein supplement called Adaptinol (Bayer Yakuhin Ltd, Osaka, Japan) has been marketed for the treatment of patients with RP in Japan for decades.¹⁴ More recently, a pilot, Internet-based, open-label study of patients with RP suggested that lutein supplements may be efficacious, ¹⁵ but a randomized placebo-controlled study will be required to confirm these initial results before definitive recommendations can be made. In the meantime, there has been interest in determining whether macular carotenoid levels are abnormal in patients with RP and CHM. Aleman et al¹⁶ and Duncan et al¹⁷ used heterochromatic flicker photometry and found that patients with RP and CHM have normal macular carotenoid levels, but since this is a subjective psychophysical test performed on subjects with significant ocular pathologic conditions, this finding needs to be confirmed with objective testing methods.

Ocular resonance Raman spectroscopy is a novel, noninvasive, objective laser optical method that we have developed to detect macular carotenoid levels in the living eye even in the presence of a substantial macular pathologic condition as long as central fixation is preserved (≥20/80 visual acuity).^18-20 This technique was initially reported by us in 1998 as a way to measure the macular carotenoid levels in situ in the flat-mounted, human cadaver retina with excellent correlation to high-performance liquid chromatographic analytical methods.¹⁸ Recently, we used the instrument for the first time to measure macular pigment levels in a clinical setting.¹² We report herein the use of our device to quantify the levels of lutein and zeaxanthin in the maculae lutea of patients who have RP, CHM, or STGD with the intention of determining whether macular carotenoid levels are below normal. Low macular carotenoid levels would suggest that supplementation with lutein, zeaxanthin, or both might be an effective method to treat these patients.

The institutional review board of the University of Utah, Salt Lake City, approved the project design, and informed consent was obtained from all subjects. The tenets of the Declaration of Helsinki were followed. All subjects were recruited from the clinic of the Moran Eye Center, University of Utah School of Medicine. Patients were diagnosed as having RP and CHM based on the following criteria: (1) a history of night blindness, (2) typical fundoscopic changes (peripheral atrophy, bone spicules, and vascular attenuation), (3) electroretinographic abnormalities consistent with a retinal dystrophy, and(4) progressive visual field loss. Patients with CHM also had typical choroidal atrophy and a family pedigree compatible with X-linked inheritance. Patients with STGD had macular atrophy, flecks, and a dark choroid on fluorescein angiography. Control subjects underwent dilated eye examinations to confirm their healthy status.

Patients and controls meeting these criteria were invited to enroll in the study as long as they were not routinely consuming high-dose (≥4-mg/d) lutein supplements. Eyes with significant cataracts, previous cataract surgery, or with a best-corrected visual acuity worse than 20/80 were excluded from this study. A total of 19 patients (9 males and 10 females) with RP (35 eyes), 6 male patients with CHM (10 eyes), and 5 female patients with STGD (9 eyes) were enrolled. Likewise, 76 healthy subjects (40 males and 36 females) with a total of 129 eyes were measured in this study. All subjects were nonsmokers. Subjects' pupils were dilated using a combination of 1% tropicamide and 2.5% phenylephrine hydrochloride eyedrops to a pupil size greater than 6 mm in diameter (mean [SD], 8.1 [0.7] mm).

We used ocular resonance Raman spectroscopy to measure human macular carotenoid levels. Details of the instrumentation have been described elsewhere.^19,20 For a typical measurement, a 0.5-m Wargon laser light (488 nm) is directed as a 1-mm spot onto the subject's macula for 0.5 second through a series of lenses and filters. The subjects self-align by superimposing the blue-green pilot laser beam on an array of collection fibers illuminated with a red light–emitting diode. Raman backscattered light is collected by a fiberoptic collection bundle and analyzed by a Raman spectrograph. Raman carotenoid peaks identifying lutein, zeaxanthin, or both are obtained at 1159 cm⁻¹ and 1525 cm⁻¹ with a good signal-noise ratio when the laser excitation beam is aimed at the fovea. The peak height at the carotenoid carbon-carbon double-bond stretch frequency of 1525 cm⁻¹ is quantified after subtraction of background fluorescence by Windows-based computer software (Eye-C-Spec; Spectrotek, LC, Salt Lake City, Utah). The Raman signal intensity was expressed as photon counts. Because subjects occasionally blink or misalign, the mean (SD) of the highest 3 of 5 measurements was used for statistical analysis. Data are given as mean (SD).

Macular carotenoid levels within a 1-mm-diameter circle centered on the fovea were measured in 76 healthy subjects (40 males, 36 females) with natural clear lenses whose ages ranged from 22 to 59 years (40.3 [10.5] years). Healthy subjects aged older than this range were examined in separately reported studies.^12,20 We found that macular carotenoid levels decreased with age (Table 1). There were no significant differences between right and left eyes in subjects in whom both eyes were measured (P =.53, paired t test). No statistically significant differences were noted between men and women (P =.46) using 2-way age-adjusted analysis of variance.

Table 2 gives the RP, CHM, and STGD clinical data and Raman spectroscopy readings. A total of 30 subjects, 15 males and 15 females (54 eyes), aged from 22 to 59 years (38.4 [10.6] years) were measured by ocular resonance Raman spectroscopy. All enrolled eyes had preserved central fixation (≥20/80 visual acuity). An example of a resonance Raman spectrum obtained from an RP-affected eye is shown in Figure 1.

The mean of the Raman spectroscopy readings was 607 (472) photon counts in 19 patients with RP (35 eyes) and 650 (510) photon counts in 6 patients with CHM (10 eyes). There were no statistically significant differences in macular carotenoid readings between patients with RP and CHM using the unpaired 2-tailed t tests (P = .81). As a group, patients with RP and CHM had normal levels of macular carotenoid pigments (617 [475] photon counts) compared with an age-matched healthy population(684 [492] photon counts) using 2-way age-adjusted analysis of variance (P = .76). The macular carotenoid levels in patients with RP and CHM tend to decrease with age in the same manner as in healthy subjects(Figure 2). Five female patients with STGD (9 eyes) had about 50% lower macular carotenoid pigment levels (289[178] photon counts) compared with the healthy subjects (P = .02, unpaired 2-tailed t test).

Ocular resonance Raman spectroscopy is a noninvasive, objective, and accurate method to detect macular carotenoid levels in living primates.^12,19,20 The technology uses a form of vibrational spectroscopy in which the molecules of interest (carotenoids) scatter the excitation light in an inelastic manner. In this process the light transfers a portion of its energy to the molecules. This leads to a molecule-specific wavelength shift of the scattered light and, thus, provides a "fingerprint" vibrational spectrum of the molecules. Macular carotenoids are particularly strong Raman scatterers, and they have a characteristic Raman spectral fingerprint generated from vibrations of their long polyene backbone.^12,20,21 The chemical structures of lutein and zeaxanthin are similar in that the effective conjugation lengths of their backbones differ only by 1 carbon-carbon double bond in 1 end group. Therefore, Raman backscattering cannot be readily used to distinguish lutein from zeaxanthin. When we calibrated our device against defined concentrations of lutein or zeaxanthin in a tetrahydrofuran solution, the recorded Raman spectra were identical to those from the human subjects after subtracting the background originating from endogenous fluorescence.²⁰ Because zeaxanthin and lutein are the only 2 major carotenoids found in the macular area of the human retina, ⁸ ocular resonance Raman spectroscopy is a specific and sensitive method to quantify them.

Raman spectroscopy detection with our current instrument requires that the subject have a pharmacologically dilated pupil. Once the pupil size exceeds 6 mm in diameter, a maximum Raman signal is acquired.^12,20 Our previous work has shown that resonance Raman spectroscopy detection has a typical intersession and intrasession variation of 5% to 10% and that there were no significant differences in healthy subjects between males and females, right and left eyes, and different iris colors.^12,20 However, it has been reported that macular pigment levels are significantly lower in female subjects and in individuals with light-colored irises using heterochromatic flicker photometry as the measurement method, ^22,23 but another recent study using similar psychophysical techniques reported that there were no significant associations between iris color or sex and macular pigment levels.²⁴

Macular pigment levels measured by resonance Raman spectroscopy decline steadily with increasing age, reaching a stable low level once the population is older than 60 years.^12,20 This decline may be partially due to the increasing yellowing of the lens as subjects age, ²⁵ but this effect could account for no more than half of the more than 75% decrease in average Raman signal observed between young and elderly adults.^12,20 Our previous study also revealed that elderly subjects older than 60 years with clear prosthetic intraocular lenses after cataract surgery had macular pigment levels that were not significantly different from age-matched healthy subjects who still had natural crystalline lenses. The average level for elderly individuals with pseudophakia never approached the average level for young phakic adults.¹² Our findings of a decline of macular pigment levels with advancing age were consistent with some, but not all, of the recent psychophysical reports on healthy populations.^24,26-28 More recently, we have directly compared resonance Raman measurement of macular carotenoid levels with heterochromatic flicker photometry in a healthy population younger than 61 years; we found a statistically significant correlation between the 2 techniques (P<.002), and we observed similar trends of a decline of macular pigment with age.²⁹

Using modified heterochromatic flicker photometry to measure the macular pigment optical densities on patients with RP, CHM and Usher syndrome, Aleman et al¹⁶ and Duncan et al¹⁷ found that, as a group, macular pigment density in patients was not different from healthy subjects.^16,17 Similarly, Alexander et al³⁰ could not detect any significant differences between macular pigment levels in patients with RP and healthy controls using fundus reflectometry.

Flicker photometry is a subjective psychophysical method that may not be valid in subjects with a significant ocular pathologic condition, and fundus reflectance measurement is a nonspecific test that may be subject to numerous optical artifacts. Resonance Raman spectroscopic measurement of macular carotenoid levels, however, is sensitive, specific, and objective, and as such it has ideal characteristics for studying populations with a substantial ocular pathologic condition. We have shown using our objective method that macular carotenoid levels in patients with RP and CHM did not differ from the levels of age-matched healthy populations. The levels of macular carotenoid pigments have the same trend as healthy populations, showing a progressive decrease with age. However, we measured macular carotenoid levels in 5 patients with STGD whose visual acuities were still 20/80 or better in at least 1 eye; all had low macular carotenoid levels for their ages. It is difficult to draw definite conclusions regarding the role of macular carotenoids in STGD, however, since it is uncommon to encounter adult patients who have STGD and a visual acuity of 20/80 or better.

Does lutein supplementation help to improve visual function in patients with RP and CHM? Aleman et al¹⁶ and Duncan et al¹⁷ reported that central vision in their patients did not change over 6 months of lutein supplementation whether the patients showed increments in macular pigment density. In another study it was reported that in patients with RP, lutein supplements improved visual functions such as visual field and visual acuity, ¹⁵ but the study was not placebo controlled, and the testing was in a nonstandardized format, making it difficult to draw definite conclusions.

Our research work reported herein confirms the results of previous studies^16,17,30 in an objective and specific manner, and it reveals that patients with RP and CHM had normal levels of macular carotenoids, unlike patients with AMD who have 32% lower levels relative to age-matched healthy controls.¹² Based on the current data, we hypothesize that high-dose supplements of dietary carotenoids will be unlikely to exert beneficial effects in patients with RP and CHM. Patients with STGD, however, have low age-adjusted levels of macular carotenoids, but this is likely to be a manifestation of genetically mediated macular photoreceptor cell death, so it is unclear whether lutein or zeaxanthin supplementation or both could be beneficial for these patients. Positive results from prospective intervention trials with concurrent objective monitoring of macular pigment levels in patients with retinal, choroidal, and macular dystrophies will be required before high-dose lutein or zeaxanthin supplementation or both can be recommended to patients with RP, CHM, and STGD.

Submitted for publication October 1, 2002; final revision received January 24, 2003; accepted March 5, 2003.

This study was supported in part by Spectrotek, LC, Salt Lake City, Utah; grants R29-EY-11600 (Dr Bernstein) and STTR R41-EY-12324 (Dr Gellermann) from the National Eye Institute, Bethesda, Md; and Research to Prevent Blindness Inc, New York, NY. Dr Bernstein is a Research to Prevent Blindness Sybil B. Harrington Scholar in macular degeneration research.

This study was presented as poster sessions at the 13th International Carotenoid Symposium; January 7, 2002, Honolulu, Hawaii, and at the 74th Annual Meeting of the Association for Research in Vision and Ophthalmology; May 7, 2002, Fort Lauderdale, Fla.

Corresponding author and reprints: Paul S. Bernstein, MD, PhD, Department of Ophthalmology and Visual Sciences, Moran Eye Center, University of Utah School of Medicine, 50 N Medical Dr, Salt Lake City, UT 84132 (e-mail: paul.bernstein@hsc.utah.edu).