Resonance Raman measurement of macular carotenoids in normal subjects and in age-related macular degeneration patients

Abstract

Purpose: Dietary carotenoids lutein and zeaxanthin may play a protective role against visual loss from age-related macular degeneration (AMD) through antioxidant and light screening mechanisms. We used a novel noninvasive objective method to quantify lutein and zeaxanthin in the human macula using resonance Raman spectroscopy and compared macular pigment levels in AMD and normal subjects.

Design: Observational study of an ophthalmology clinic-based population.

Participants and controls: Ninety-three AMD eyes from 63 patients and 220 normal eyes from 138 subjects.

Methods: Macular carotenoid levels were quantified by illuminating the macula with a low-power argon laser spot and measuring Raman backscattered light using a spectrograph. This technique is sensitive, specific, and repeatable even in subjects with significant macular pathologic features.

Main outcome measure: Raman signal intensity at 1525 cm(-1) generated by the carbon-carbon double-bond vibrations of lutein and zeaxanthin.

Results: Carotenoid Raman signal intensity declined with age in normal eyes (P < 0.001). Average levels of lutein and zeaxanthin were 32% lower in AMD eyes versus normal elderly control eyes as long as the subjects were not consuming high-dose lutein supplements (P = 0.001). Patients who had begun to consume supplements containing high doses of lutein (> or =4 mg/day) regularly after their initial diagnosis of AMD had average macular pigment levels that were in the normal range (P = 0.829) and that were significantly higher than in AMD patients not consuming these supplements (P = 0.038).

Conclusions: These findings are consistent with the hypothesis that low levels of lutein and zeaxanthin in the human macula may represent a pathogenic risk factor for the development of AMD. Resonance Raman measurement of macular carotenoid pigments could play an important role in facilitating large-scale prospective clinical studies of lutein and zeaxanthin protection against AMD, and this technology may someday prove useful in the early detection of individuals at risk for visual loss from AMD.

Figure 1

Schematic diagram of resonance Raman device to measure macular carotenoid levels in the living human eye. After pharmacologic dilation to at least 6 mm, the patient fixates on an aiming beam superimposed on a collection fiber array illuminated with a red light-emitting diode. Then, 0.5-mW 488-nm laser light from a low-power argon laser is routed through the multimode fiber, laser line filter (LLF), and mirrors (M1 and M2) into the human eye and is projected onto the foveal area as a 1-mm spot for 0.5 second. Raman shifted backscattered light is directed to the spectrometer through M2, beam splitter (BS), a holographic notch filter (HNF), and a fiberoptic bundle. The spectrometer is interfaced to a cooled charge-coupled device camera that is controlled by a Windows-based (Microsoft, Redmond, WA) custom software package.

Figure 2

Typical resonance Raman spectra collected by the instrument shown in Figure 1. The top two panels are spectra collected from the dilated eye of a 26-year-old male (A) before and (B) after background subtraction. The middle two panels are spectra collected from a solution of zeaxanthin (Hoffmann-La Roche, Basel, Switzerland) dissolved in tetrahydrofuran (THF) (C) before and (D) after background subtraction. The bottom two panels are spectra collected from a solution of lutein (Kemin Foods, Des Moines, IA) dissolved in THF (E) before and (F) after background subtraction. The lutein and zeaxanthin solutions were measured through the use of a “model eye” in which the solutions were placed in a 1-mm path-length quartz cuvette at the focal point of a 53-diopter lens generating a 1-mm laser spot size identical to the laser spot projected onto the macula in vivo. The number listed by each spectral peak denotes the wavenumber in cm⁻¹ for the local maximum.

Figure 3

Repeatability of ocular resonance Raman measurements. Two individuals had five repeated sessions of resonance Raman measurement of macular carotenoids over a 2-week period through the fully dilated pupil of the same eye in each patient. The mean ± standard deviation (SD) of peak height at 1525 cm⁻¹ is given for the best three of five readings. The filled circles were obtained from a 26-year-old white male, and the open circles were obtained from a 37-year-old Asian male.

Figure 4

Calibration curves of the resonance Raman instrument. Optical density at 450 nm was measured on tetrahydrofuran solutions of lutein and zeaxanthin by an ultraviolet-visible spectrophotometer (Hitachi Instruments, San Jose, CA) in a 1-mm path length cuvette after appropriate dilution when necessary, and then a resonance Raman measurement was made using the model eye described in Figure 2. The mean ± standard deviation (SD) for five Raman signal intensity measurements at 1525 cm⁻¹ at each concentration are shown. Note that the repeatability of measurement of this model system is so high that the error bars are not even visible. The top axis of the upper panel is the calculated amount of carotenoid in the illuminated volume (a cylinder 1 mm in diameter and 1 mm in height) using published extinction coefficient values for lutein and zeaxanthin. Filled circles are for lutein, and open circles are for zeaxanthin. A, The calibration curve is shown in the linear response range of the detector (R = 1.0 for both lutein and zeaxanthin along the best-fit line). B, Saturation of detector response is observed at very high carotenoid concentrations.

Figure 5

Resonance Raman measurement of macular carotenoid levels in normal eyes at 1525 cm⁻¹. Two hundred twenty eyes from 138 normal subjects were measured by the ocular resonance Raman instrument using the protocol described in Methods. The best-fit curve is an exponential fit with R = 0.664. Open circles are phakic eyes, and filled circles are pseudophakic eyes. The mean ± standard deviation (SD) for the best three of five readings is shown.

Figure 6

Daily lutein supplement consumption reported by age-related macular degeneration patients enrolled in this study.