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Structure and Conformation of the Carotenoids in Human Retinal Macular Pigment.

Arteni AA, Fradot M, Galzerano D, Mendes-Pinto MM, Sahel JA, Picaud S, Robert B, Pascal AA - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrative Biology of the Cell (I2BC) & Institut de Biologie et de Technologies de Saclay, CEA, UMR 8221 CNRS, Université Paris Saclay, Gif-sur-Yvette, France.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0135779.g003: 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.

Mentions: Ex vivo retina samples including the macular region were mounted for direct comparison of their resonance Raman spectra with those of lutein and zeaxanthin in vitro, and these spectra were measured at different excitation wavelengths (Fig 3). The spectra are broadly similar both to each other and to those of the isolated carotenoids. In the right-hand inset to Fig 3, a close-up of the ν1 region is shown. The ν1 band is clearly wider for the macular tissue than for the isolated carotenoids, particularly at 488 and 501.7 nm (half-width ~20 cm-1 compared to ~15 cm-1 for isolated pigments; see Fig 2). This indicates that both lutein and zeaxanthin contribute at all wavelengths, as expected from their small difference in absorption position. However, when shifting the excitation to higher wavelengths, the ν1 position shifts from 1525 cm-1 at 488 nm to 1521.5 cm-1 at 514.5 nm (Fig 3, right inset; see also Fig 4). This downshift in ν1 position indicates that at least two different carotenoid populations can indeed be distinguished in the macula, dominating alternately at different wavelengths. Given this shift in ν1 frequency and comparing with the spectra for the isolated carotenoids, we conclude that the lutein molecules present dominate at 488 nm, whereas zeaxanthin enters more into resonance as the excitation wavelength is increased. Indeed, given the increase in conjugation length for zeaxanthin, this carotenoid is expected to absorb slightly more to the red relative to lutein (see above) [17]. Note, in addition, that in vitro reconstitution of the hypothesised xanthophyll-binding protein for each macular carotenoid indicates a (0,0) absorption peak around 482 and 510 nm for protein-bound lutein and zeaxanthin, respectively [22,23]. Therefore we can indeed distinguish between the two carotenoids in ex vivo macular tissue by choosing the appropriate excitation wavelength, taking advantage of their different absorption properties.


Structure and Conformation of the Carotenoids in Human Retinal Macular Pigment.

Arteni AA, Fradot M, Galzerano D, Mendes-Pinto MM, Sahel JA, Picaud S, Robert B, Pascal AA - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4552419&req=5

pone.0135779.g003: 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.
Mentions: Ex vivo retina samples including the macular region were mounted for direct comparison of their resonance Raman spectra with those of lutein and zeaxanthin in vitro, and these spectra were measured at different excitation wavelengths (Fig 3). The spectra are broadly similar both to each other and to those of the isolated carotenoids. In the right-hand inset to Fig 3, a close-up of the ν1 region is shown. The ν1 band is clearly wider for the macular tissue than for the isolated carotenoids, particularly at 488 and 501.7 nm (half-width ~20 cm-1 compared to ~15 cm-1 for isolated pigments; see Fig 2). This indicates that both lutein and zeaxanthin contribute at all wavelengths, as expected from their small difference in absorption position. However, when shifting the excitation to higher wavelengths, the ν1 position shifts from 1525 cm-1 at 488 nm to 1521.5 cm-1 at 514.5 nm (Fig 3, right inset; see also Fig 4). This downshift in ν1 position indicates that at least two different carotenoid populations can indeed be distinguished in the macula, dominating alternately at different wavelengths. Given this shift in ν1 frequency and comparing with the spectra for the isolated carotenoids, we conclude that the lutein molecules present dominate at 488 nm, whereas zeaxanthin enters more into resonance as the excitation wavelength is increased. Indeed, given the increase in conjugation length for zeaxanthin, this carotenoid is expected to absorb slightly more to the red relative to lutein (see above) [17]. Note, in addition, that in vitro reconstitution of the hypothesised xanthophyll-binding protein for each macular carotenoid indicates a (0,0) absorption peak around 482 and 510 nm for protein-bound lutein and zeaxanthin, respectively [22,23]. Therefore we can indeed distinguish between the two carotenoids in ex vivo macular tissue by choosing the appropriate excitation wavelength, taking advantage of their different absorption properties.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrative Biology of the Cell (I2BC) & Institut de Biologie et de Technologies de Saclay, CEA, UMR 8221 CNRS, Université Paris Saclay, Gif-sur-Yvette, France.

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.

No MeSH data available.


Related in: MedlinePlus