Human macular pigment assessed by imaging fundus reflectometry.

A computerized, television-based, imaging fundus reflectometer was used to obtain estimates of the spatial distribution of macular pigment (xanthophylls) from seven normal subjects. Digitized images of the bleached macula of each subject were acquired at illuminating wavelengths from 462 to 697 nm. An analysis of spectral reflectances indicated that differences in short-wavelength reflectance between the foveal center and parafovea were influenced by spatial variations in melanin and oxyhemoglobin absorption as well as by the distribution of macular pigment. To provide an estimate of the spatial distribution of macular pigment alone, we have corrected fundus images obtained at 462 nm for the effect of melanin and oxyhemoglobin absorption. The spatial variation in macular pigment double density across the horizontal and vertical meridians of the retina was well described by Gaussian functions. The peak double densities for the individual subjects ranged from 0.22 to 0.45 and the standard deviations of the Gaussian functions averaged approx. 1 degree.

Analysis of the macular pigment by HPLC: retinal distribution and age study.

High performance liquid chromatography (HPCL) has been employed to study the distribution throughout the human retina of zeaxanthin and lutein, the two major components of the macular pigment. Differences between individuals have also been studied with a view to uncovering possible age-related effects. Both pigments were detected in prenatal eyes (approximately 20 weeks gestation) but did not form a visible yellow spot. Generally they were not easily discernible until about 6 months after birth. For 87 donors between the ages of 3 and 95, no dependence on age was observed in the quantity of either pigment. For approximately 90% of these, zeaxanthin was dominant. For the remaining 10%, as well as for the seven youngest donors, all below the age of 2, and in prenatal eyes, lutein was the major pigment. In individual retinas, the lutein:zeaxanthin ratio increased from an average of approximately 1:2.4 in the central 0-0.25 mm to over 2:1 in the periphery (8.7-12.2 mm). The variation in this ratio with eccentricity was linearly correlated with the corresponding rod:cone ratio. A selective mechanism of uptake, which results in cones and rods preferentially acquiring zeaxanthin and lutein, respectively, could explain this correlation.

Macular corneal dystrophy: immunochemical characterization using monoclonal antibodies.

Macular corneal dystrophy is an inherited corneal disease characterized by corneal opacities resulting from intra- and extracellular deposits within the corneal stroma. Several monoclonal antibodies developed against antigens of corneal fibroblasts were screened for their reactivity with these abnormal deposits in corneas with macular dystrophy using an indirect peroxidase-conjugated immunostaining technique. One of these monoclonal antibodies (designated 8F1-3) reacted very strongly with these abnormal deposits. Although the antigen recognized by this monoclonal antibody was present in the normal corneal stromal and endothelial cells, its concentration in the cells in the corneas with macular dystrophy appeared to be considerably higher, based on the intensity of the immunostaining reaction. Corneal fibroblasts grown in tissue culture were employed for further characterization of the antigen. After fixing with paraformaldehyde and permeabilizing with Triton X-100, immunofluorescent staining of the corneal fibroblasts using these monoclonal antibodies revealed a filamentous pattern of staining which resembled that seen for vimentin filaments. On treatment of corneal fibroblasts with colchicine, the filaments recognized by this antibody were withdrawn from their cytoplasmic array to form a perinuclear cap as also observed for vimentin-containing intermediate filaments. Immunoelectron microscopic studies using colloidal gold-conjugated antimouse IgG indicated that this monoclonal antibody recognized an antigen associated with intermediate-type filament. However, antivimentin antibody did not react with the abnormal deposits in the corneas with macular dystrophy, indicating that the antigen identified in the present study, although associated with intermediate filaments, was not vimentin. Analyses of cytoskeletal antigens by the immunoblotting technique further revealed that this monoclonal antibody recognized two polypeptides with Mr48,000 and 45,000, while antivimentin antibody reacted with 58,000 Mr polypeptide (vimentin).

Macular pigment and reduced foveal short-wavelength sensitivity in retinitis pigmentosa.

Some patients with retinitis pigmentosa (RP) show a reduced foveal short-wavelength sensitivity that cannot be attributed to a reduction in the sensitivity of the short-wavelength cone system. To determine whether an increased amount of macular pigment (xanthophyll) might account for this finding, we derived estimates of the two-way optical density of the macular pigment of five such RP patients as well as of five normals. The spectral reflectance of the foveal region of each subject was obtained from digitized images of the bleached fundus provided by a television-based reflectometer. The density spectra of the macular pigment, melanin, and oxygenated hemoglobin were fit by a least-squares procedure to the log of the ratio of parafoveal to foveal spectral reflectance in order to obtain a quantitative estimate of the contribution of each of these ocular pigments to foveal short-wavelength reflectance. By this analysis, the two-way densities of the macular pigment, melanin, and oxyhemoglobin of the RP patients were not significantly different from those of the normals. Therefore, the reduced foveal short-wavelength sensitivity of these patients was not due to an increased amount of macular pigment, but may result instead from morphological abnormalities in the foveal cones such that a normal amount of macular pigment screens the cones more effectively.

Retinal pigment epithelial lipofuscin and melanin and choroidal melanin in human eyes.

Optical measurements of the pigments of the retinal pigment epithelium (RPE) and choroid were made on 38 human autopsy eyes of both blacks and whites, varying in age between 2 wk and 90 yr old. Lipofuscin in melanin-bleached RPE was measured as fluorescence at 470 mm following excitation at 365 nm and was found to be proportional to fluorescence measured at 560 nm in unbleached tissue. Transmission measurements of RPE and choroidal melanin were converted and expressed as optical density units. The choroidal melanin content increased from the periphery to the posterior pole. RPE melanin concentration decreased from the periphery to the posterior pole with an increase in the macula. Conversely, the amount of RPE lipofuscin increased from the periphery to the posterior pole with a consistent dip at the fovea. There was an inverse relationship between RPE lipofuscin concentration and RPE melanin concentration. The RPE melanin content was similar between whites and blacks. Lipofuscin concentration was significantly greater (P = 0.002) in the RPE of whites compared to blacks; whereas blacks had a significantly greater (P = 0.005) choroidal melanin content than whites. The amounts of both choroidal and RPE melanin showed a trend of decreasing content with aging, whereas the amount RPE lipofuscin tended to increase (whites greater than blacks). Per fundus area, the amount of choroidal melanin was always greater than that in the RPE. There was a statistically significant (P = 0.001) increase in RPE height with age, most marked in eyes of whites after age 50 and correlated with the increase in lipofuscin concentration.

Foveal cone pigment density difference in the aging human eye.

Using fundus reflectometry, we have measured a decrease in the density difference of the foveal cone visual pigments with age in human subjects. This decrease is consistent with a loss of visual pigment in the retina with age. Fundus reflectance and normalized density difference spectra data are presented for these subjects. A decrease in cone pigment with age would be consistent with both anatomic studies, which indicate a loss and displacement of photoreceptors with age, and psychophysical studies, which demonstrate loss of photoreceptor function with age.

Preliminary identification of the human macular pigment.

The human macular pigment has been found to be composed to two chromatographically separable components, which are tentatively identified as lutein [(3R,3'R,6'R)-beta,epsilon-Carotene-3,3'-diol] and zeaxanthin [(3R,3'R)-beta,beta-Carotene-3,3'-diol]. Chromatograms of retinal extracts, obtained by HPLC on three different stationary phases, were found to match those obtained with mixtures of lutein and zeaxanthin standards. Identical retention times were confirmed by coinjection of each of the isolated components with the appropriate standard. U.V.-visible spectra of the purified components were identical in all respects with those of lutein and zeaxanthin. Further support for our identification was obtained by the preparation and chromatographic comparison of derivatives of the macular pigment and of the standards.

The macular pigment. I. Absorbance spectra, localization, and discrimination from other yellow pigments in primate retinas.

The nonbleaching yellow pigments of the primate fovea were studied by microspectrophotometry (MSP). Retinas fixed with glutaraldehyde/paraformaldehyde mixtures retained yellow pigments with absorbance spectra very similar to those recorded by MSP in fresh retinas. This allowed the authors to prepare retinal sections for localization of the pigments. The spectrum of the macular pigment in fixed tissue is shifted slightly (about 6 nm) toward longer wavelengths, with maximum absorbance at 460 nm. Two short-wavelength yellow pigments also have been identified, with absorbance maxima at 410 nm ( P410 ) and 435 nm ( P435 ), respectively. All three yellow pigments are present in the fovea. The short-wavelength pigments are detected more easily outside the central foveal region because the macular pigment does not obscure them there. They are especially apparent when the MSP beam is confined to the outer nuclear layer or the inner segment layer of retinal sections. The macular pigment is most dense in the fiber layers (receptor axon layer and inner plexiform layer); its density declines markedly with retinal eccentricity. The maximal absorbance of P410 and P435 is usually lower than that of the macular pigment in the central fovea, but their densities and relative proportions change more gradually with eccentricity. Consequently, their maximal absorbance is higher than that of the macular pigment outside the foveal center. The P410 and P435 pigments may be two different oxidation states of one or more respiratory hemoproteins. Commonly used procedures for estimating the absorbance spectrum of the macular pigment by comparing the foveal center with a parafoveal region may be influenced by the amounts and the oxidation states of the short-wavelength pigments in the living eye.

A study of macular pigmentation in human eyes

The existence of the macular pigment in the living, human eye is at the present time fairly well established, but opinions on its role and effects on vision are still divided. To rectify this situation, several experiments were performed. In the main experiment, the optical density curve of the pigment was estimated for 49 European and West Indian subjects by comparing their foveal and extra-foveal spectral sensitivities measured by the flicker technique. No significant differences were observed related to race, normal environment, age, sex, colour of skin or colour of eyes, but red haired subjects had on average a significantly higher density of macular pigment. The extinction coefficient of the pigment, which appeared to be lutein, was the same for all subjects and differences in density were consistent with variations in pigment concentration and/or thickness of absorbing layer. In a subsidiary experiment, macular pigment density was assessed for a small sample of the 49 subjects by retinal photography. The combined results of this and the first experiment correlated well and led to the conclusion that visual performance is quite definitely affected by the presence in the fovea of a blue absorbing, non-photosensitive, pre-receptor pigment. The relatively low sensitivity of this region to blue light could not be explained satisfactorily in terms of differences associated with the receptors themselves. Spectral sensitivity measurements also revealed that West Indian subjects had more absorbent lenses than Europeans, equivalent to a difference in age of about 10 years. The dichroic ratio of the macular pigment layer was found to follow a similar spectral trend to the optical density spectrum. Some of the pigment molecules appeared to be arranged symmetrically about the fovea. It was shown that the number of such molecules was approximately the same for all subjects (AU)