Structure and Conformation of the Carotenoids in Human Retinal Macular Pigment

Abstract

Human retinal macular pigment (MP) is formed by the carotenoids lutein and zeaxanthin (including the isomer meso-zeaxanthin). MP has several functions in improving visual performance and protecting against the damaging effects of light, and MP levels are used as a proxy for macular health-specifically, to predict the likelihood of developing age-related macular degeneration. While the roles of these carotenoids in retinal health have been the object of intense study in recent years, precise mechanistic details of their protective action remain elusive. We have measured the Raman signals originating from MP carotenoids in ex vivo human retinal tissue, in order to assess their structure and conformation. We show that it is possible to distinguish between lutein and zeaxanthin, by their excitation profile (related to their absorption spectra) and the position of their ν1 Raman mode. In addition, analysis of the ν4 Raman band indicates that these carotenoids are present in a specific, constrained conformation in situ, consistent with their binding to specific proteins as postulated in the literature. We discuss how these conclusions relate to the function of these pigments in macular protection. We also address the possibilities for a more accurate, consistent measurement of MP levels by Raman spectroscopy.

Fig 1. Chemical structures of lutein and zeaxanthin.

Fig 2. Resonance Raman spectra of MP carotenoids in vitro.

Room temperature spectra in the 900–1650 cm^-1 region are shown for zeaxanthin (red) and lutein (blue) in THF, excited at 488.0 nm. Inset: detail of the ν₁ region.

Fig 3. Resonance Raman spectra of human macula.

Room temperature spectra (900–1600 cm^-1) are shown for ex vivo human retina in the macular region, excited at 488.0, 501.7, 514.5 & 528.7 nm (blue, olive, black, red respectively). Details of the ν₁ & ν₄ regions are shown in the insets. Representative spectra are shown for a single macula, but were the same for all 8 subjects used in this study.

Fig 4. ν₁ region of macula resonance Raman spectra.

77 K (solid lines) and room temperature (dashed lines) spectra are shown for excitation at 488.0 & 514.5 nm (blue, black respectively). For 488 nm, additional spectra are shown for measurements scanning in towards the foveal centre (dotted lines).