Ligand-binding characterization of xanthophyll carotenoids to solubilized membrane proteins derived from human retina.

The macula of the human retina contains extraordinarily high concentrations of lutein and zeaxanthin, xanthophyll carotenoids that appear to play an important role in protecting against age-related macular degeneration, the leading cause of blindness among the elderly. It is likely that the uptake and stabilization of these carotenoids is mediated by specific xanthophyll-binding proteins. In order to purify and characterize such a binding protein, a carotenoid-rich membrane fraction derived from human macula or peripheral retina was prepared by homogenization, differential centrifugation, and detergent solubilization. Further purification was carried out using ion-exchange chromatography and gel-filtration chromatography coupled with continuous photodiode-array monitoring for endogenously associated xanthophyll carotenoids. The most highly purified preparations contained two major protein bands at 25 and 55 kDa that consistently co-eluted with endogenous lutein and zeaxanthin. The visible absorbance spectrum of the binding protein preparation closely matches the spectral absorbance of the human macular pigment, and it is bathochromically shifted about 10 nm from the spectrum of lutein and zeaxanthin dissolved in organic solvents. Binding of exogenously added lutein and zeaxanthin is saturable and specific with an apparent Kd of approximately 1 microM. Canthaxanthin and beta-carotene exhibit no significant binding activity to solubilized retinal membrane proteins when assayed under identical conditions. Other potential mammalian xanthophyll-binding proteins such as albumin, tubulin, lactoglobulin and serum lipoproteins possess only weak non-specific binding affinity for carotenoids when assayed under the same stringent binding conditions. This investigation provides the first direct evidence for the existence of specific xanthophyll-binding protein(s) in the vertebrate retina and macula. The possible roles of xanthophyll-binding proteins in normal macular function and in the pathogenesis of age-related macular degeneration remain to be elucidated.

Identification and quantitation of carotenoids and their metabolites in the tissues of the human eye.

There is increasing evidence that the macular pigment carotenoids, lutein and zeaxanthin, may play an important role in the prevention of age-related macular degeneration, cataract, and other blinding disorders. Although it is well known that the retina and lens are enriched in these carotenoids, relatively little is known about carotenoid levels in the uveal tract and in other ocular tissues. Also, the oxidative metabolism and physiological functions of the ocular carotenoids are not fully understood. Thus, we have set out to identify and quantify the complete spectrum of dietary carotenoids and their oxidative metabolites in a systematic manner in all tissues of the human eye in order to gain better insight into their ocular physiology. Human donor eyes were dissected, and carotenoid extracts from ocular tissues [retinal pigment epithelium/choroid (RPE/choroid), macula, peripheral retina, ciliary body, iris, lens, vitreous, cornea, and sclera] were analysed by high-performance liquid chromatography (HPLC). Carotenoids were identified and quantified by comparing their chromatographic and spectral profiles with those of authentic standards. Nearly all ocular structures examined with the exception of vitreous, cornea, and sclera had quantifiable levels of dietary (3R,3'R,6'R)-lutein, zeaxanthin, their geometrical (E / Z) isomers, as well as their metabolites, (3R,3'S,6'R)-lutein (3'-epilutein) and 3-hydroxy-beta,epsilon-caroten-3'-one. In addition, human ciliary body revealed the presence of monohydroxycarotenoids and hydrocarbon carotenoids, while only the latter group was detected in human RPE/choroid. Uveal structures (iris, ciliary body, and RPE/choroid) account for approximately 50% of the eye's total carotenoids and approximately 30% of the lutein and zeaxanthin. In the iris, these pigments are likely to play a role in filtering out phototoxic short-wavelength visible light, while they are more likely to act as antioxidants in the ciliary body. Both mechanisms, light screening and antioxidant, may be operative in the RPE/choroid in addition to a possible function of this tissue in the transport of dihydroxycarotenoids from the circulating blood to the retina. This report lends further support for the critical role of lutein, zeaxanthin, and other ocular carotenoids in protecting the eye from light-induced oxidative damage and aging.

Raman detection of macular carotenoid pigments in intact human retina.

PURPOSE: To develop and test a novel noninvasive optical technique suitable for the objective measurement of macular carotenoid levels in human retina. METHODS: A resonance Raman scattering apparatus was constructed to measure carotenoid levels in flat-mounted human retinas and eyecups and in experimental animal eyes. Light from an argon laser was used to resonantly excite the electronic absorption of the carotenoid pigments, and scattered light was collected and analyzed by a Raman spectrometer. After carotenoid Raman measurements were completed on the retinal samples, macular carotenoid levels were determined by high-performance liquid chromatography (HPLC). RESULTS: Carotenoid resonance Raman scattering proved to be a highly sensitive and specific method for the noninvasive measurement of macular pigments in the human retina. Signal strength scaled linearly with actual macular carotenoid content as measured by HPLC. Our apparatus was also used to record resonance Raman signals from xanthophyll carotenoids stored in the retinal pigment epithelium of intact frog eyes. CONCLUSIONS: This new noninvasive optical method will facilitate studies of ocular carotenoid distributions and their role in degenerative diseases of the eye and may allow for the rapid screening of carotenoid levels in large populations at risk for vision loss from age-related macular degeneration, the leading cause of blindness in the elderly in the United States. A prototype clinical instrument is under development.