Retinal Pigment Epithelial Lipofuscin and Melanin and Choroidal Melanin in Human Eyes

John J. Weirer,*t Francois C. Delori,*t Glenn L. Wing,t and Karbrra A. Fitch*

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 > 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. Invest Ophthalmol Vis Sci 27:145-152, 1986

The human retinal pigment epithelium (RPE) con- Lipofuscin is thought to represent lysosomal residual tains two classes of pigment, melanin and lipofuscin, bodies containing end-products of oxidative damage that are quite different in terms of development and to lipids6 and in the RPE is related to photoreceptor function.1 The RPE melanosomes are synthesized in phagocytosis and digestion. Light damage mediated utero and remain virtually unchanged thereafter. through photosensitized oxidations has been postulated Rarely, premelanosomes are found in normal retina, to result in lipid peroxides7 and probably in the ac- suggesting a possible slow turnover of melanosomes in cumulation of RPE lipofuscin.8 This suggests that there mature RPE, although not to a significant degree.2 Ma- could possibly be a relation between melanin concen- ture melanosomes appear to be degraded by incorpo- tration and lipofuscin accumulation in the RPE. ration within lysosomes to form melanolysosomes and We have previously reported preliminary studies on melanolipofuscin; the process, however, is slow and the intracellular and topographical distribution of incomplete because of the inherent stability of the mel- melanin and lipofuscin in Caucasian RPE as a function anin granules.3 of age.9 An inverse relationship between melanin and The exact biologic role of melanin beyond light ab- lipofuscin was found in human RPE. The present study sorption is still controversial, but several important extends that work to include blacks, as well as choroidal properties include scavaging free radicals and excited melanin measurements in both whites and blacks. molecules, electron transfer, and affinity for drugs and metals.4 In contrast to melanin, lipofuscin in the RPE Materials and Methods 5 develops after birth and continues to increase with age. Subjects Optical measurements of melanin and lipofuscin From the Eye Research Institute of Retina Foundation* and Har- concentration were made on 38 eyes that had no known vard Medical School,f Boston, Massachusetts, and Fort Myers,t ocular pathology. The donors were 19 whites from 2 Florida. wk old to 88 yr old (average age of 42) and 16 American Supported in part by grant EY-03699 from the National Eye In- blacks from 6.5 yr to 90 yr (average age of 52). Mea- stitute. Submitted for publication: October 8, 1984. surements on both eyes in three subjects were averaged Reprint requests: John J. Weiter, MD, PhD, Eye Research Institute and counted as one. All eyes were obtained less than of Retina Foundation, 20 Staniford St, Boston, MA 02114. 12 hr after death. Eyes were fixed in formalin and pro-

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cessed through ethanol dehydration to xylene to par- same sites as for melanin measurement. However, the affin embedding. Pupil-optic nerve sections, 8 nm melanin was first bleached using potassium perman- thick, were cut through the fovea, deparaffinized, and ganate and oxalic acid before fluorescence measure- mounted unstained on slides in ultra-violet-inert im- ment. Transmission measurements of bleached RPE mersion oil. showed no residual absorption by melanin, ensuring thereby that fluorescence from lipofuscin is not affected Measurement Sites by melanin absorption. To obtain general topographic information on mel- Lipofuscin fluorescence measurements were made anin and lipofuscin distribution, measurements were using as excitation the HBO-100 high-pressure mer- first made at regular spacing between the fovea and cury lamp of the Zeiss Photometer in conjunction with the periphery on several eyes. From these results, five a 365-nm interference filter (bandwidth 10 nm). The sites per eye were selected for RPE measurements on excitation beam was restricted to a 30-/im circular area all eyes: the two equators, the fovea, and the two para- on the specimen. The emitted fluorescence of lipofuscin foveal sites. The latter are located at half the distance was detected through a narrow band filter centered at from fovea to disc, on either side of the fovea. The 470 nm and sampled using the same 30 X 3-jttm rect- parafoveal and equatorial measurements were aver- angular aperture. aged. Three sites were selected for choroidal melanin At each use, fluorescence measurements were cali- measurement: the two equators and the fovea. The two brated using a single specific ZnS-CdS grain, to adjust equators were averaged. The far periphery was not in- the fluorescence reading to 100 units. As for melanin, cluded in this study because of the marked variability fluorescence measurements were made in 5 neighbor- of the RPE. The far peripheral retina frequently had ing apical and basal areas at each site and the results changes such as pavingstone degeneration that would averaged to yield the relative concentration of lipofus- have invalidated the study. cin. Total lipofuscin content was calculated as the product of RPE cell height and the average of apical RPE Melanin and basal fluorescence intensities. Measurements of melanin concentration in the RPE Reproducibility of the lipofuscin fluorescence mea- were made using a Zeiss Photomicroscope III (Carl surements was found to be ±5% for the same spot Zeiss, Inc.; Oberlochen, West Germany) with micro- measured over months on a melanin-bleached speci- spectrophotometric capabilities. Transmission mea- men from an 88-yr-old white male. Repeated measurements of the 8-/im thick sections were made at surements of individual specimens showed that 30 800X magnification using a rectangular aperture of 30 readings of 0.1 sec did not decrease fluorescence at any X 3 microns. The effective spectral range of the mea- spot, so 5 readings were taken at each spot and averaged suring light, determined by the tungsten lamp of the to give the fluorescence intensity at that spot. microscope and detection sensitivity of the PMT, was The excitation and emission bands used in this and 500-600 nm. The system was calibrated to 100% a previous study5 were selected to match the excitation transmission at a location adjacent to the section. and emission maxima reported by Chio,10 and also be- Transmission measurements were made by aligning cause, in our spectrophotometric system, this combi- the sampling area to the basal and apical side of the nation yielded the most efficient fluorescence detection RPE cell. These measurements were repeated at each in melanin-bleached tissue. Our use of 470 nm as the site for five adjacent areas and averaged to reduce vari- most efficient wavelength results from the combined ability. The apical and basal transmissions (T) were effects of the emission spectra of melanin-bleached li- converted in extinction coefficient of melanin by (—Vfe) pofuscin and the spectral sensitivity characteristics of X logio T, and expressed in density units per micron. our monochromator and photomultiplier (S-5 pho- This extinction coefficient is proportional to the mel- tocathode, detection sensitivity of the system at 470 anin concentration, and is used here as the relative nm is 1.2 times that at 560 nm). It should be noted melanin concentration expressed in density units per that Eldred,11 using appropriate spectrophotometric micron. RPE heights at each site were also measured. corrections, showed that lipofuscin fluoresces between Total optical density of the RPE at each site for light 440 nm, and 700 nm, with a broad and flat maximum traveling in the apical-basal direction was calculated between 540 nm and 640 nm. as the product of the RPE height and the mean of the As a check on the validity of measuring lipofuscin apical and basal extinction coefficients. fluorescence at 470 nm, we compared fluorescence measured at 470 nm and at a wavelength within the RPE Lipofuscin broad emission peak of lipofuscin, eg 560 nm, on 5 Lipofuscin concentrations were measured by fluo- eyes (20 sites) of unbleached tissue. We found that flu- rescence measurement on the same sections and at the orescence measured at 470 nm was proportional to

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that measured at 560 nm (linear regression r2 = 0.84, 1200 P < 0.0001 and intercept not significantly different from zero). Similar results were found by comparing |_ looo- • RPE LIPOFUSCIN u the two wavelengths on melanin-bleached tissues (P 0 800 < 0.007). We also compared fluorescence at 470 nm R using melanin-bleached tissue vs fluorescence at 560 BOO- nm using unbleached tissue. In 20 eyes, using young 400" and old eyes of both races, there was a positive correlation between the fluorescence measured at 470 nm 200" • EQUATOR MACULA EQUATOR on melanin-bleached tissue and fluorescence on an ad- h- —I H 1 1 jacent unbleached tissue section at 560 nm (r2 = 0.5, 1.0T P< 0.0001). 0.8-' RPE MELANIN Choroidal Melanin 0.6" Choroidal melanin concentrations were measured on an adjacent section to that used for RPE optical 0.4- measurements. Transmission measurements in the same manner as for RPE melanin were made. How- 0.2-• ever, the sampling aperture was 300 ^m long and had a height H, adjusted to cover half of the choroidal 0.0 thickness. Since the choroid in vivo is a vascular com-

partment, with variable degrees of extracellular fluid, 0.8- • it is not possible from histological sections to determine choroidal volume, and hence choroidal melanin con- 0.6 • centrations. Rather, transmission measurements were

0.4- • converted in optical density by (- —) X logi0 T for

8 0.2 • both the inner choroid (RPE side) and the outer choroid EQUATOR MACULA EQUATOR (scleral side). Total optical density of the choroid was 0.0 h- —I H 1 obtained by adding inner and outer choroid densities. -20 -15 -10 -5 10 15 DISTANCE FROM OPTIC OISC (MM.) Morphometric Studies Fig. 1. Topographical distribution of pigments of the RPE and Ocular sections were studied at 2,000X magnifica- choroid in a 60-yr-old white man. This topographical distribution is tion under oil immersion to corroborate the optical representative of all of the eyes studied. Total lipofuscin content (in measurements. At this magnification, discrete lipofus- arbitrary units) and total melanin density of RPE and choroid are cin granules can be counted, and melanosomes differ- displayed as a function of distance from the optic disc. Note the inverse relation between lipofuscin and melanin content in the RPE. entiated from melanolipofuscin. On representative RPE sections, the optical measurements of lipofuscin content decreased from the equator to the posterior fluorescence and melanin density correlated with li- pole, with a consistent peak of RPE melanin content pofusin and melanosome granule counts (P < 0.001). at the macula. Choroidal melanin content increases This positive correlation was verified in various regions gradually from the equator to the posterior pole, with of the eye in young and old patients in both blacks and the highest degree of choroidal pigmentation being in whites. the submacular choroid. Results RPE Melanin and Lipofuscin Concentration Topographical Pigment Distribution The results of the RPE melanin and lipofuscin con- The topographical distribution of the pigments of centration measurements are summarized in Table 1. the retinal pigment epithelium and choroid in human Melanin and lipofuscin are distributed differently postmorten eyes demonstrated a consistent pattern as within the RPE cell: melanin has a higher concentration typified by Figure 1. As previously shown,512 RPE li- in the apical aspect of the cell, whereas lipofuscin is pofuscin content increases from the equator to the more concentrated basally. This polarity of the lipo- posterior pole, with a consistent dip in RPE lipofuscin fuscin and melanin intracellular distributions was more content in the macula. In contrast, the RPE melanin pronounced in younger eyes than older eyes.

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Table 1. Relative lipofuscin and melanin concentration in the RPE

Whites Blacks N= 19 N = 16 Site Mean ± SD Mean :tSD Racial difference

Lipofuscin* Macula Apical 44 ± 16 35 ± 12 P < 0.05 Basal 57 ±24 41 ± 14 P < 0.05 Paramacula Apical 54 ± 19 50 ± 13 NS Basal 69 ±28 64 ± 19 NS Equator Apical 41 ± 19 26 ± 10 P<0.01 Basal 52 ±20 42 ± 13 NS Melaninf Macula Apical 31 ± 13 30 ± 10 NS Basal 24 ± 14 26 ± 12 NS Paramacula Apical 22 ± 8 19 ± 6 NS Basal 17 ± 10 15 ± 7 NS Equator Apical 45 ± 18 45 ± 15 NS Basal 32 ± 17 29 ± 12 NS Cell height! Macula 10. 3± 2.8 10.2 ± 1.3 NS Paramacula 8.8 ± 2.1 9.2 ± 1.5 NS Equator 7.0 ± 1.8 6.5 ± 1.0 NS

* Lipofuscin in arbitrary fluorescence intensity units/micron, % Cell height in microns. t Melanin in milli-density units/micron.

Lipofuscin concentration was greater, on average, 50, a more rapid increase in lipofuscin was again noted, at all sites measured, in whites than in blacks. Because much more marked in whites than blacks. This age- of the large variability though, this lipofuscin difference related increase in RPE lipofuscin is not linear but was statistically significant at the P < 0.05 level only shows a sigmoidal pattern as our laboratory has pre- at the macular and equatorial regions. When apical viously reported.5 and basal concentration measurements at all sites were The RPE melanin concentration at all sites was on grouped, then a significance level of 0.002 was reached average the same in blacks and in whites (Table 1). for the difference in RPE lipofuscin concentration be- RPE melanin concentrations showed a trend to de- tween whites and blacks. crease with age in whites and blacks. This decrease was As expected, lipofuscin increases with age. Linear most pronounced in whites after age 50. correlation of lipofuscin concentration with age for the It is to be stressed that our optical measurements do different sites showed significant increase in whites (r2 not differentiate between melanin and melanolipofus- = +0.45 to +0.69, P = 0.0001) but only a trend to cin (complex) granules. It is very obvious, when viewed increase for blacks. The rate of change is greatest in at 2,000X magnification, that by the age of 50 most of the first two decades of life and shows only moderate the melanin is incorporated into melanolipofuscin increase in the third through fifth decades. After age granules. This may account for some loss of cellular

Q O L I 120- P O D 0 F D U 100- s m c I N 80 c Fig. 2. RPE lipofuscin concentration expressed as a 0 function of RPE melanin concentration, measured apically or basally at the same sites. Included are both E blacks and whites.

R 10 A T I 0 20

MELANIN CONCENTRATION

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melanin and lipofuscin distribution polarity mentioned Table 2. Choroidal melanin content (density units) above. Whites Blacks Racial RPE cells were taller in the macular region and di- Site Mean ± SD Mean ± SD difference minished in height as one progressed from the macula to the equator (Table 1). There was a statistically sig- Macula Inner 0.24 ± 0.04 0.55 ± 0.34 P = 0.003 nificant increase in RPE height with age at the different Outer 0.86 ± 0.3 0.93 ± 0.44 P = 0.001 2 Equator Inner 0.18 ±0.13 0.25 ±0.13 P = 0.05 sites (linear correlation coefficient r = +0.28 to +0.50, Outer 0.24 ±0.16 0.44 ±0.21 P = 0.05 P = 0.001), most marked in white eyes after age 50. RPE cell height correlated significantly with RPE li- multiplying by cell height. The choroidal values are pofuscin concentration (r2 = +0.34, P < 0.0001 for obtained by summing the outer and inner optical den- all sites). sity values. RPE Lipofuscin and Melanin Correlation Selected Disease States The relationship between lipofuscin and melanin The present study excluded any eyes with known apical and basal concentrations is shown in Figure 2 ocular pathology. In order to explore further the sig- for all sites in all eyes measured in this study. An inverse nificance of lipofuscin accumulation, several diseased relationship exists for RPE melanin and lipofuscin. eyes were studied. Figure 3 shows the topographical Linear correlation of both concentrations was signifi- distribution of lipofuscin in a 70-yr-old white man with cant (r2 = +0.23, P = 0.0001). It is to be noted that central areolar atrophy, an entity generally considered this inverse relationship holds both when the RPE pig- to be a variant of senile macular degeneration (SMD). ments are compared on a site basis (Fig. 2 and Table The lipofuscin concentration at any site is several times 1) and on a topographical basis (Fig. 1). higher than normal for this age and is higher than ever found in a normal eye. The RPE cells at the posterior Choroidal Melanin Content pole have already degenerated. The remaining RPE Regional choroidal melanin content is shown in Ta- cells were swollen and contained diminished RPE ble 2. Blacks have a significantly greater total choroidal melanin. A second eye with senile macular degenera- melanin content than whites (P = 0.005). Choroidal tion, but with intact RPE in the posterior pole was also melanin content is on average 2.35 ± 0.97 times higher found to have increased lipofuscin but not as marked in the outer choroid half (adjacent to the sclera) than as in Figure 3. in the inner choroid half (adjacent to the RPE). No Eyes from two individuals who died in their third statistically significant change in total choroidal mel- and fourth decades from neoplastic disease were also anin density with age was found, although there is a studied. Both individuals received extensive antime- trend for the ratio of outer/inner choroidal melanin tabolite therapy, and in both cases the RPE lipofuscin density to increase with age. concentration was approximately twice normal.

Total Optical Density of Melanin Discussion Since the melanin of the RPE and choroid are su- This study used optical methods to measure the pig- perimposed upon each other in vivo when exposed to ments of the RPE and choroid in human autopsy eyes. light, it is useful to know their optical density in relation Several limitations are inherent in the techniques uti- to each other. Table 3 gives, for black and white subjects lized. The major problem is ensuring that fluorescence separately, the average values of the total optical density measurements at 470 nm following melanin bleaching of melanin in both the RPE and choroid. For each eye, is representative of lipofuscin. When viewed with flu- the optical density of RPE melanin is obtained by av- orescent microscopy, lipofuscin appears golden-yellow, eraging apical and basal melanin concentrations and consistent with an emission peak in the 540 to 640 nm

Table 3. Total optical density of melanin (density units) Retinal pigmented epithelium Choroid

Whites Blacks Racial Whites Blacks Racial Site Mean ± SD Mean ± SD difference Mean ± SD Mean ± SD difference

Macula 0.29 ±0.11 0.29 ±0.10 NS 0.70 ± 0.49 1.44 ±0.75 P = 0.001 Paramacula 0.17 ±0.05 0.16 ±0.06 NS Equator 0.27 ±0.10 0.24 ±0.11 NS 0.42 ± 0.26 0.69 ± 0.36 P = 0.01

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pofuscin). What the contribution of secondary lyso- RPE LIPOFUSCIN somes are to our fluorescent measurements is not known. Finally, the optical methods we used for measuring melanin in the RPE can not differentiate me- BOO lanosomes from complex granules (melanolysosomes 0 fl and melanolipofuscin). With fluorescence microscopy, E s 600 the melanolipofuscin granules are easily identified, c E having a fluorescent rim and a nonfluorescent melanin N «oo- core. Although not specifically quantified, the mela- C E nosomes were noted to decrease with age and the me- 800 lanolipofuscin granules increased, such that by age 50 melanolipofuscin granules typically outnumbered melanosomes. -10 -5 5 DISTANCE FROM OPTIC DISC (MM.) This study verified our previous study and those of others,1314 showing an increase in the lipofuscin con- Fig. 3. Topographical distribution of RPE lipofuscin concentration tent with age. As noted previously,5 the increase in li- in a 70-yr-old white man with central areolar atrophy. Note that lipofuscin fluorescence is absent centrally because of atrophy of the pofuscin with age is not linear but shows a sigmoidal RPE. Compare the RPE lipofuscin concentration in this diseased eye pattern. The increase is greatest in the first two decades with lipofuscin concentration of a normal eye as shown in Figure 1. of life and shows only moderate increase in the third through sixth decades. Beginning with the sixth decade range. Our measurements were performed at 470 nm a more rapid increase in lipofuscin was again noted, in order to be consistent with our previous study.5 Fur- much more pronounced in whites than blacks. This thermore, 470 nm was considered the emission peak rapid accumulation of lipofuscin in the first two de- for extracted lipofuscin.59 Recently, though, the 470- cades of life has been noted by others.15 We hypothesize nm emission maximum of extracts of lipofuscin-ladden that the rapid lipofuscin buildup in the first two decades cells was shown possibly to result from instrumental of life is related to the increased exposure of the retina bias.12 In order to link our histologic measurements to shorter UV wavelengths (295 to 400 nm), since the made at 470 nm to those at 560 nm, we compared crystalline lens develops a yellow pigment that absorbs values of 5 eyes at both wavelengths and found a pos- the shorter wavelengths on a time scale that matches itive correlation (r2 = 0.84, P = 0.0001). Also, to de- the RPE lipofuscin buildup.16 termine the effects of melanin bleaching on the tissue RPE melanin decreases from the equator to the pos- we compared fluorescence measurements at 470 nm terior pole, as noted by others,1517 with an increase in using melanin bleached tissue with fluorescence mea- the macular region. Interestingly, the topographical sured at 560 nm on unbleached tissue and found a distribution of melanin is the inverse of the topograph- 2 positive correlation (r = 0.5, P < 0.001). ical distribution of lipofuscin (Fig. 1). Indeed, if the The fluorescence measurements were calibrated us- concentration of lipofuscin to melanin is compared at ing a single specific ZnS-CdS grain, to adjust the flu- each site measured (Fig. 2), this same inverse relation orescence reading to 100 units. Lipofuscin fluorescence holds. It is tempting to relate the increase in lipofuscin measurements were made relative to this standard, with in whites during the sixth decade to the decrease in the assumption that lipofuscin correlates linearly with RPE melanin noted at this time. fluorescence. Internal quenching most likely causes Although there is a large variability of melanin from deviation from linearity. Furthermore, the assumption site to site in the RPE and from individual to individ- was made that all fluorescence was derived from li- ual, on average the RPE melanin content was the same pofuscin. Using fluorescence microscopy at 2000X in both blacks and whites (Tables 1 and 3). On the magnification, all of the fluorescence appeared to be other hand, choroidal melanin, on average, was ap- derived from rather uniform lipofuscin granules. Since proximately twice as large in blacks as in whites (Tables the photoreceptors are not fluorescent under these 2 and 3). This is consistent with a previous study of conditions, phagosomes are probably not being in- ocular melanin.1819 These tissue differences in melanin cluded in the measurements, but this can not be reflect the fact that melanin is derived from two distinct proven. The fluorescent granules in infant eyes are not regions embryologically, the neural crest and neuro- only smaller than those found in adult eyes, but also epithelial cells. The neurocrest is the origin for mela- have a light green autofluorescence in contrast to the nocytes that migrate and provide pigment for such sites golden yellow fluorescence of adult eyes.5 Feeney13 as skin, hair, and uvea. Such pigmentation shows suggests that these smaller fluorescent bodies could marked racial variability. Since the neuroepithelium represent secondary rather than tertiary lysosomes (li- provides melanin for pigmented structures of the cen-

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tral nervous system such as the ganglion substantia are missing for complete degradation.25 Feeney- nigra, it is not surprising that the RPE, which is derived Burns15 has shown that the percent of cytoplasmic from neuroepithelium, does not show racial differences space decreases with increasing lipofuscin. We have in pigmentation. shown that RPE cell height increases with age and is The inverse relation between RPE melanin and li- correlated with the increase in RPE lipofuscin concen- pofuscin is intriguing and may suggest a protective tration. Similar findings have been noted by others in mechanism in the formation of lipofuscin. Lipofuscin the rat RPE.26 We hypothesize that, just as in lysosomal formation is a complex process, most likely involving storage disease, the accumulation of undigestible res- lipid peroxidation.8-20 Many factors such as light, ox- idue (lipofuscin) leads to compromise of cellular func- ygen, and nutrients probably play a role. Melanin could tion. Supportive evidence comes from examination of provide photoprotection not only by direct absorption diseased eyes in which lipofuscin accumulation is ex- of light but also by serving as a scavenger of light-in- cessive. Figure 3 demonstrates the topographical li- duced free radicals.3 It is unlikely, though, that this pofuscin profile in an eye with a variant of senile mac- scavenger effect is the sole mechanism, since whites ular degeneration. It is very tempting to speculate that have a much greater RPE lipofuscin content in spite these RPE cells became so engorged with lipofuscin of the fact that both whites and blacks have an equal that they "ruptured" or degenerated from cytoplasmic content of RPE melanin. crowding. Notwithstanding these speculations, the Light damage would be related to the total accu- mechanism of lipofuscin buildup and the role of mel- mulated light exposure during one's life. The difference anin in possible photoprotection remain complex and in RPE lipofuscin between whites and blacks is prob- not well understood. ably related to the differences in their choroidal pig- Key words: lipofuscin, melanin, retinal pigment epithelium, mentation. A photon of light that was not absorbed by choroid, aging, pigments the photoreceptor-RPE complex would have a greater possibility of being reflected by the deeper layers of the Acknowledgments fundus and having a second pass through the photo- The authors thank Eleanor Wilkinson and Freda Swan at receptors in whites than in blacks. Indeed, fundus re- Massachusetts Eye & Ear Infirmary Eye Pathology Labora- flectance (white light) in whites is approximately 5% tory, Dr. Richard Robb and Richard Sullivan at Children's versus 1% in blacks.19 Thus, the ratio of total photo- Hospital Eye Pathology Laboratory, and Dr. Thomas Freddo receptor light exposure between whites and blacks will at University Hospital Eye Pathology Laboratory for assis- tance in obtaining human autopsy eyes. be 105/101. This ratio would be increased by the ad- ditional contributions from intraocular light scatter and References light penetrance through the iris and sclera. Whether 1. Feeney-Burns L: The pigments of the retinal pigment epithelium. this small light exposure ratio can account for differ- In Current Topics in Eye Research, Vol 2, Zadunaisky JA and ences in RPE lipofuscin between blacks and whites is Davson H, editors. New York, Academic Press, 1980, pp. 119- an unanswered question. Indeed, we currently believe 178. that the dip in lipofuscin noted at the macula reflects 2. John K and Fitzpatrick TB: Ultrastructural and biochemical the photoprotection afforded by not only the increased studies of the formation of melanosomes in the embryonic chick retinal pigment epithelium. In Pigmentation. Its Genesis and melanin in the RPE and choroid in the macula (Fig. Biologic Control, Riley V, editor. New York, Appleton-Century- 21 22 1) but also by the neurosensory macular pigment. ' Crofts, 1972, pp. 125-141. (As we previously discussed,5 though, differences in 3. Barr FE, Saloma JS, and Buchele MJ: Melanin: the organizing phagosome production in the RPE of rod-rich vs cone- molecule. Med Hypotheses 11:1, 1983. rich areas may contribute to this dip and cannot be 4. Garcia RI, Szabo G, and Fitzpatrick TB: Molecular and cell biology of melanin. In The Retinal Pigment Epithelium, Zinn discounted.) Furthermore, fundus pigmentation was KM and Marmor MF, editors. Cambridge, MA, Harvard Uni- also found to be an important parameter in the inci- versity Press, 1979, pp. 124-147. dence of senile macular degeneration in a study of a 5. Wing GL, Blanchard GC, and Weiter JJ: The topography and Caucasian population.23'24 age relationship of lipofuscin concentration in the retinal pigment Finally, the question of whether lipofuscin accu- epithelium. Invest Ophthalmol Vis Sci 17:600, 1978. 6. Sohol RS: Age Pigments. Amsterdam, Elsevier/North-Holland, mulation is toxic to the RPE has not been decisively 1981, pp. 102-155,317-334. answered, although inferential evidence strongly sug- 7. Noell WK, Walker VS, and Kang BS: Retinal damage by light gests that it is.20 If lipofuscin is formed within lysosomes in rats. Invest Ophthalmol 5:450, 1966. after phagic sequestration of damaged or aged mem- 8. Feeney L and Berman ER: Oxygen toxicity: membrane damage branes and organelles and represents undigestible res- by free radicals. Invest Ophthalmol 15:789, 1976. 9. Wing GL, Delori FC, Weiter JJ, and Kunis K: The relation be- idue, then lipofuscin is analagous to the accumulation tween melanin and lipofuscin in human RPE. ARVO Abstracts. of undigestible residues in lysosomal storage diseases, Invest Ophthalmol Vis Sci 22(Suppl):173, 1982. since in both cases the appropriate catabolic enzymes 10. Chio KS and Tappel AL: Synthesis and characterization of the

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fluorescent products derived from malonaldehyde and amino 19. Hunold W and Malessa P: Spectrophotometric determination acids. Biochemistry 8:2821, 1969. of the melanin pigmentation of the human ocular fundus in 11. Eldred GE, Miller GV, Stark WS, and Feeney-Burns L: Lipo- vivo. Ophthalmic Res 6:355, 1974. fuscin: resolution of discrepant fluorescence data. Science 216: 20. Feeney-Burns L, Berman ER, and Rothman H: Lipofuscin of 757, 1982. human retinal pigment epithelium. Am J Ophthalmol 90:783, 12. Weiter JJ, Delori FC, and Fitch K: Pigments of the retinal pigment 1980. epithelium and choroid in human eyes. ARVO Abstracts. Invest 21. Snodderly DM, Brown PK, Delori FC, and Auran JD: The mac- Ophthalmol Vis Sci 25(Suppl):247, 1984. ular pigment. I. Absorbance spectra, localization, and discrim- 13. Feeney L: Lipofuscin and melanin of human retinal pigment ination from other yellow pigments in primate retinas. Invest epithelium. Fluorescence, enzyme cytochemical and ultrastruc- Ophthalmol Vis Sci 25:660, 1984. tural studies. Invest Ophthalmol Vis Sci 17:583, 1978. 22. Snodderly DM, Brown PK, Delori FC, and Auran JD: The mac- 14. Streeten BW: The sudanophilic granules of the human retinal ular pigment. II. Spatial distribution is primate retinas. Invest pigment epithelium. Arch Ophthalmol 66:391, 1961. Ophthalmol Vis Sci 25:674, 1984. 15. Feeney-Burns L, Hilderbrand ES, and Eldridge S: Aging human 23. Wing GL, Weiter JJ, and Delori FC: Senile macular degeneration RPE: morphometric analysis of macular, equatorial and periph- and its relation to ocular pigmentation. ARVO Abstracts. Invest eral cells. Invest Ophthalmol Vis Sci 25:195, 1984. Ophthalmol Vis Sci 24(Suppl): 170, 1983. 16. Lerman S: Radiant Energy and the Eye. New York, Macmillan Publishing Co., 1980, p. 153. 24. Weiter JJ, Delori FC, Wing GL, and Fitch K: Relationship of 17. Peisch RD and Schmidt SY: Variations in melanin concentration senile macular degeneration to ocular pigmentation. Am J in the pigment epithelium of postmortem human eyes. ARVO Ophthalmol 99:85, 1985. Abstracts. Invest Ophthalmol Vis Sci 18(Suppl):20, 1979. 25. Hers HA and Van Hoof F, editors: Lysosomes and Storage Dis- 18. Geeraets WJ, Williams RC, and Chan G: The relative absorption eases. New York, Academic Press, 1973, p. 283. of thermal energy in retina and choroid. Invest Ophthalmol 1: 26. Katz ML and Robison WG Jr: Age-related changes in the retinal 340, 1962. pigment epithelium of pigmented rats. Exp Eye Res 38:137, 1984.

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