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Light and aging effects on vitamin E in the retina and retinal pigment epithelium
l’irion Res. Vol. 27, No. 11, pp. 18754879, 1987 Printed in Great Britain. AH rights resewed
0042-6989/87 S3.00 + 0.00
Copyright0 1987 Pergamon Journals Ltd
LIGHT AND AGING EFFECTS ON VITAMIN E IN THE RETINA AND RETINAL PIGMENT EPITHELIUM MARTINL. KATZ’ and W. GERALD ROBISON JR~ ‘Mason Institute of Ophthalmology, University of Missouri, School of Medicine, Columbia, MO 65212
and ‘National Eye Institute, National Institutes of Health, Bethesda, MD 20892, U.S.A. (Received 27 February 1987; in revised form 1 June 1987)
Abstract-Experiments were performed to determine the effects of senescence and light adaptation on vitamin E levels in the neural retina and RPE-choroid-sclera of pigmented rats. Aging multed in significant increases in a-tocopherol levels in both tissues, the effect being most pronounced in the RPE-choroid-sclera. The state of light adaptation had no influence on a-tocopherol kvels in the neural retina at any of the ages examined, whereas in the RPE-choroid-sclera, a-tocopherol levels were substantially higher in light-adapted than in dark-adapted animals at all three ages (12.22, and 32 months) at which they were measured. The effect of light adaptation on RPE-choroid-scleral a-tocopherol levels was most pronounced in the oldest age group. Vitamin E
Aging
Light adaptation
Retina
INTRODUCTION
Lipofuscin deposition in the retinal pigment epithclium (RPE) is one of the most prominent cellular changes to occur in the mammalian retina during senescence (Katz and Robison, 1984; Wing et al., 1978). Vitamin E deficiency induces a substantial acceleration in RPE lipoftin deposition rates (Robison et al., 1979, 1980; Katz et al., 1986b), suggesting that lipofuscin may contain products of autoxidation of cellular components. Other age-related changes in the retina, including photoreceptor cell loss and changes in vitamin A metabolism, are also accelerated by vitamin E deficiency (Katz et al., 1984, 1987a). It therefore appeared possible that senescent alterations in the neural retina and RPE could be secondary to decreases in the vitamin E contents of these tissues. To determine whether this was the case, vitamin E measurements were performed on eye tissues from pigmented rats of various ages. Because light exposure exerts such profound influences on various aspects of retinal metabolism, the vitamin E measurements were performed on animals in both the dark- and light-adapted states. MATERIALS AND METHODS
Experimental animals Male pigmented AC1 rats were purchased at 8 weeks of age from Harlan Sprague Dawley
Retina1 pigment epithelium
Rat
Inc. (Indianapolis, Ind.). Animals were housed in hanging wire cages in a room maintained at a constant temperature of 21°C. Illumination from General Electric F15T12-CW fluorescent tubes was provided for 12 hr each day followed by 12 hr of total darkness. During the light phase of the daily cycle, illumination at the fronts of the cages was a maximum of 100 lx, while at the backs of the cages, illumination was less than 10 lx. Animals were fed a natural ingredient open formula diet (NIH 07) (Hess et al., 1985) and given water ad libitum. The diet contained an average of 37 international units of vitamin E per kg. Vitamin E determinations At 12, 22, and 32 months of age, rats were dark adapted for a minimum of 13 hr, 12 hr of which was during the normal dark phase of their lighting cycle. At 1 to 2 hr after the light phase of the cycle would normally have begun, rats were anesthetized with CO2 and one eye from each animal was enucleated under dim red light. The eye socket was packed with Gelfoam (Upjohn) to retard bleeding. The rats recovered rapidly from CO, anesthesia after enucleation and showed no apparent signs of discomfort from the surgery. Immediately after enucleation, the hides, corneas, lenses and vitreous were dissected from each eye and discarded. The retina was then dissected from the RPEchoroid-scleral complex, and each of these two
1875
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MARTIN
L. KATZ and W. GERALD ROBIWN JR
tissues was placed in 350 ~1 of cold 10 mM HEPES [4-(2-hydroxyethyl)- 1-piperazine ethane sulfonic acid], pH 7.4, containing 150 mM NaCl, and 5 mM diethylenetriamine pentaacetic acid to which 0.2% pyrogallol was added just prior to use. Microscopic examination indicated that the dissections produced very clean separations between the neural retinas and RPEs (Katz et al., 1987a). The tissues were dissociated by sonication under dim red light while being cooled in an ice-water bath. Sonication completely dissociated the retinas, RPEs, and choroids, as well as the connective tissue cells of the sclera, but left the collagenous extracellular matrix of the sclera relatively intact. After sonication, a-tocopherol was extracted from aliquots of the samples with hexane-ethanol. A 200 ~1 aliquot of each sample was transferred to a vial with a teflon-lined screw cap. To each sample was then added 200 ~1 of ethanol, and the mixture was vortexed for about 15 sec. Exactly 1.Oml of hexane, containing 4.0 nmol tocol per ml as an internal standard, was then added to each sample, and the mixture was vortexed again for 1.5 min. An additional 200 ~1 of ethanol and 400 ~1 of water were then added, and the sample was vortexed for 15 sec. The phases were separated by centrifugation at 500 g for 5 min, and the organic phase was collected and filtered through a 0.20 pm pore-size Millipore type FG filter. Approximately l.O’ml of hexane was then added to the sample, with care being taken not to disturb the phase boundary. The hexane phase was again collected and filtered and was pooled with the first hexane phase. Finally, the filter apparatus was rinsed with another 1.Oml of hexane, which was then added to the first two filtrates. The hexane was removed from the extracts by evaporation under a stream of argon. After they were dried, the samples were immediately dissolved in approximately 35 ~1 of methanol. Quantitation of a-tocopherol was performed with high performance liquid chromatography (HPLC). The chromatographic system consisted of Gilson model 302 pumps, a Rheodyne model 7161 injector with a 20~1 sample loop, a Brownlee 4.6 mm x 3 cm guard column with an RP-18 1Opm spherical absorbant, a 4.6 mm x 25 cm LiChrosorb analytical column with a 10pm RP-18 adsorbant (Alltech, Deerfield, Ill.), and a Kratos Spectroflow 773 absorbance detector equipped with a 12 ~1 flow cell. Absorbance was monitored at 295 nm. Chromatograms were run isocratically with the
mobile phase consisting of methanol : water (97: 3, v/v) at a flow rate of 2.0 ml/min. Quantitation of a-tocopherol was performed by determining the peak area of this compound relative to the peak area of the internal standard. Analysis of a -tocopherol standards indicated that the peak area ratios for these compounds were linear over the range found in our samples. Experiments in which known amounts of a-tocopherol were added to tissue samples demonstrated that with the methods employed, recovery of these compounds was complete relative to internal standard recoveries. Samples from dark-adapted rats were handled under dim red light until they were mixed with an equal volume of ethanol. All subsequent steps of the extraction and analyses were performed under dim incandescent illumination. One hour after initiation of the light phase of the light cycle, the day after the above measurements were performed, the pupil of the remaining eye was dilated with 1% Mydriacil. The rats were then placed in clear plastic cages that were illuminated with Sylvania F15T8-CW fluorescent tubes. Average illumination at the bottoms of the cages was 16001x. After 1 to 1.25 hr, when at least 95% of the visual pigment had been bleached (Katz and Robison, 1986b; Katz et al., 1987b), the rats were anesthetized with CO* and each had its remaining eye enucleated. Sonicates of the retinas and RPEchoroid-scleral complexes were prepared as described above for the dark-adapted eyes, except that the sonicate preparation was performed under bright fluoresecent lights. The sonicates were extracted with hexane-ethanol under dim incandescent lighting and the extracts were analyzed as described for the dark-adapted eyes. Efects of mydriasis and contralateral enucleation on vitamin E levels Experiments were performed in order to determine whether mydriasis alone influenced retina and RPE vitamin E levels. Mydriacil (1%) was applied to one eye each of five lightadapted lZmonth-old rats. The animals were immediately placed in total darkness. After 1.5 hr, the rats were anesthetized with CO, and their eyes enucleated. The neural retina and RPE a-tocopherol levels were then determined on each eye as described above. All enucleations were performed 2.5-3 h after the start of the light phase of the normal lighting cycle.
Aging and light effects on vitamin E
1877
much greater. At 32 months of age, the a-tocopherol content of this tissue complex was almost 48% higher than it was at 12 months of age (P < 0.001). At all three ages examined, light adaptation resulted in significant increases in the a-tocopherol contents of the RPE-choroid-sclera complexes (Table 1). An 11% increase in mean a-tocopherol content was induced by light adaptation in animals 12 months of age (P < 0.01). At 22 months, light adaptation resulted in a 7.6% increase in the vitamin E content of this tissue complex (P < O.Ol),while at 32 months of age, the ant-indu~ increase amounted to about 36% (P < 0.001). Assessments of the significance of aging and The differences in RPE-choroid-sclera vitalight effects on the various measured parameters min E levels between the light-adapted and were performed using analysis of variance. dark-adapted eyes did not appear to result Comparisons between individual treatment from the effects of mydriasis or trauma due to groups were performed using the Newman- enucleation of the contralateral eye one day Keuls procedure (Wirier, 1971). previously. In control experiments on 12month-old rats, neither retinal nor RPEchoroid-scleral vitamin E levels were affected by RESULTS mydriasis or by prior enucleation of the contraIn all tissues examined, vitamin E appeared to lateral eye. Under all four conditions used in the occur exclusively as a-tocopherol. Neither j?- control experiments, vitamin E levels were nor y$ocopherol were detectable in any of the essentially the same as those found in darksamples. Aging resulted in moderate increases in adapted eyes of the IZminth-old animals used the a-tocopherol contents of the neural retinas. in the aging study (Table 1). Between 12 and 32 months of age, retinal vitamin E content increased an average of 12% DSCUSSION (P < 0.05) in the dark-adapted state, and 9% The fact that lipofuscin accumulation can be (P < 0.05) in the light-adapted state (Table 1). The state of light adaptation had no significant greatly accelerated by antioxidant nutrient influence on the a-tocopherol content of the deficiency serves as strong evidence that autoxidative mechanisms are involved in age-related neural retina at any age examined. lipofuscin deposition (Katz and Robison, Senescence had a more pronoun~d effect on 1986a). The progressive age-related increase in the a-tocopherol content of the RPE-choroidsclera complex. In the dark-adapted state, RPE lipofuscin content that occurs in various tissues a-tocopherol content increased an average of suggests that senescence may be accompanied 21% (P < 0.001) between 12 and 32 months of by a decline in antioxidant protective mechage (Table 1). In the light-adapted state, this anisms. Indeed, several antioxidant defense age-related increase in a-tocopherol content was mechanisms have been shown to decline during Experiments were also cod&Wed to determine whether the trauma of enucleation of one eye could influence the vitamin E levels in the contralateral eye enucleated a day later. Rats were dark-adapted for 13-14 hours and the neural retina and RPE vitamin E levels were then measured in one eye from each animal as described earlier. The rats were then placed in the light until the end of their normal daily light cycle. After dark adapting the rats overnight (13-14 hr), the other eye of each animal was enucleated and neural retina and RPE a-tocopherol levels were again determined.
Table 1. Influences of age and light on u-tocopherol content of the retina and RF’E-choroid-sclera a-Tocuphcrol content @mol@ye) Light-adapted Dark-adapt& Atzc _ (months) . 12 22 32
Retina
RPE-C-S
911 f21b(10)E 869&34(10) 931 f 33 (11) 935 f 26 (11) 1021 f 57 (6) 1051f 56 (6)
lRPE-choroid+ciera.
bvafues exprased as mean f SE. ‘Number of eyes analyzed in parentheses.
Retina
RPE-C-S
944*25(13) 937f36(11) 1029f 74 (7)
%7&42(13) 1006f36(13) 1429f 93 (7)
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MARTIN L. KATZ and W. GERALD ROBIWN JR
aging in a variety of species (Sohal and Allen, 1986). A number of experiments have demonstrated the importance of vitamin E in protecting against autoxidative damage to the neural retina and against excessive lipofuscin accumulation in the RPE (Robison et al., 1979, 1980; Katz et al., 1986a; Riis et al., 1981). Retinal photoreceptor cells play a central role in RPE lipofuscin deposition, and it is likely that autoxidized photoreceptor outer segment components are incorporated into RPE lipofuscin granules (Robison et al., 1982; Katz et al., 1986a, 1987~; Katz and Robison, 1986a). It therefore appeared possible that the age-related accumulation of lipofuscin in the RPE was due to declining levels of vitamin E in the neural retina. The current experiments indicate, however, that this was not the case. It is possible that levels of other antioxidants in the retina, such as glutathione or protective enzymes such as superoxide dismutase, do decline during senescence, leading in turn to an increased rate of lipofuscin formation. Alternatively, the progressive accumulation of lipofuscin in the RPE and other age-related changes in the retina may result from autoxidative damage occurring at a constant rate throughout the lifespan. Lipofuscin deposition could reflect a failure of retinal tissues to remove the products of autoxidation at a rate matching their rate of formation. It is not currently possible to measure total RPE a-tocopherol content alone due to the difficulty of quantitatively isolating the RPE from the underlying choroid-sclera complex. However, it is likely that the overall increase in RPE-choroid-scleral a-tocopherol levels during aging is indicative of a similar increase in the RPE alone. It is improbable that RPE vitamin E levels decline due to an age-related shift in vitamin E stores from the RPE to the choroid and sclera. On the contrary, the apparent agerelated increase in RPE lipid droplet content (Katz er al., 1984) suggests that a-tocopherol levels probably increase to a greater relative extent in the RPE than in the tissue complex as a whole, since there is an age-related increase in the lipophilic environment within the RPE into which vitamin E can partition. The data presented probably understate the effect of senescence in increasing a-tocopherol levels, at least in the neural retina. There is an age-related loss of cells from all layers of the neural retina (Katz and Robison, 1986b). Thus, if the data were expressed on a per-cell basis, the influence of senescence on retinal a-tocopherol
levels would have appeared substantially greater. Organisciak and Feeney-Bums (personal communication) have recently measured vitamin E levels in both the neural retinas and RPEs from human donor eyes. Vitamin E levels were found to increase substantially with increasing donor age in both tissues. The finding of similar increases in rats maintained under controlled environmental and dietary conditions suggests that the observed changes in vitamin E levels may be fundamentally associated with the process of senescence. The light-induced increase in the a-tocopherol content of the RPE-choroid-scleral complex was unexpected, and the mechanism for this effect is unclear. The increase does not appear to be due to a greater engorgement of the choroidal capillaries with blood in the lightadapted state, since retinol levels in the RPE-choroid-scleral complex were not affected by light adaptation (Katz et al., 1987a). Light adaptation does not appear to induce a transfer of a-tocopherol from the neural retina to the RPE, since retinal a-tocopherol levels were the same in both the light- and dark-adapted states. It therefore appears likely that light exposure induces uptake of a-tocopherol from the choroidal circulation into the RPE-choroid-sclera. This light-induced uptake of vitamin E may serve an important physiological function, such as enhancing protection against light-mediated autoxidation (Hunt et al., 1984). Acknowledgemenrs-The authors wish to thank A. B. Groome and C. M. Drca for their expert technical assistance and Dr G. E. Eldred for his helpful comments on the manuscript. Tocol was a generous gift from Hoffmann-La Roche, Inc. The investigations reported in this paper were supported in part by U.S. Public Health Service Grant EYO6458 and by a grant from Research to Prevent Blindness, Inc. REFERENCES
Hess H. H., Knapka J. J., Newsome D. A., Westney I. V. and Wartofsky L. (1985) Dietary prevention of cataracts in the pink-eyed RCS rat. Imb. A&r. Sci. 35, 47-53. Hunt D. F., Grganisciak D. T., Wang H. M. and Wu R. L. C. (1984) a-Tocopherol in the developing rat retina: a high pressure liquid chromatographic analysis. Curr. Eye Res. 3, 1281-1288. Katz M. L., Drea C. M., Eldred 0. E., Hess H. H. and Robison W. G. Jr (1986a) InIIuence of early photoreceptor degeneration on lipofuscin in the retinal pigment epithelium. Expl Eye Res. 43, 561-573. Katz M. L., Drea C. M. and Robin W. G. Jr (1986b) Relationship between dietary retinal and lipofuscin in the retina1 pigment epithelium. Me&. Age. Deu. 35.291-305.
Aging and light effects on vitamin E Katz M. L., Drea C. M. and Robison W. G. Jr (1987a) Age-related alterations in vitamin A t&t&bolism in the rat retina. Expi Eye Res. 44, 939-949. Katz M. L,, Drea C. M. and Robison W. G. Jr (1987b) Dietary vitamins A and E influence retinyl ester composition and content of the retinal pigment epithelium. Biochim. biophys. Acta. 924, 432-l. Katz M. L.; Eldred G. E. and Robison W. 0. Jr (1987~) Lipofuscin autofluorescence: evidence for vitamin A involvement in the retina. Mech. Age. Dee. 39, 81-90. Katz M. L. and Robison W. G. Jr (1984) Age-related changes in the retinal pigment epithelium of pigmented rats. Expi Eye Res. 38, 137-151. Katz M. L. and Robison W. G. Jr (1986a) Nutritional influences on autoxidation, lipofuscin accumulation, and aging. In Free Radicals, Aging, end Degenerative Bisecses (Edited by Johnson J. E., Walford R., Harman D. and Miquel J.), pp. 221-259. Alan R. Liss, New York. Katz M. L. and Robison W. G. Jr (1986b) Evidence of cell loss from the rat retina during senescence. Expi Eye Res. 42,293-304. Katz M. L., Robison W. G. Jr and Dram E. A. (1984) Potential role of autoxidation in age changes of the retina and retinal pigment epithelium of the eye. In Free Radicals in Molecular Biology, Aging, mul LXseae (Edited by
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Armstrong D., Sohal R. S,, cutler R. G. and Slater T. S.), pp. 163-180. Raven Press, fitcw York. Riis R. C., Sheffy B. E., Loew E., Kern T. J. and Smith J. S. (1981) Vitamin E deficiency retinopathy in dogs. Am. J. Vet. Res. 42, 74-86. Robison W. G. Jr, Kuwabara T. and Bieri J. G. (1979) Vitamin E deficiency and the retina: photoreceptor and pigment epithelial changes. Innest. Ophthal. otkuaf Sci. 18, 683-690. Robison W. G. Jr, Kuwabara 1. and Bieri J. G. (1980) Deficiencies in vitamins E and A in the rat. Retinal damage and lipofuscin accumulation. Jnvest. Ophthal. visual Sci. 19, 1030-1037. Robison W. G. Jr, Kuwabara T. and Bieri J. G. (1982) The roles of vitamins E and unsaturated fatty acids in the visual process. Retina 2, 263-281. Sohal R. S. and Allen R. G. (f986) ~lation~p between oxygm metabolism, aging and development. A&. Free Bad. Biol. Med. 2, 117-160. Winer B. J. (1971) Statistical Principles in Experimental Des&t, 2nd edn. McGraw-Hill, New York. Wing G. L., Blanchard 0. C. and Weiter J. J. (1978) The topography and age relationship of lipofuscin concentration in the retinal pigment epithelium. Znuesr. OphrhaL visual Sci. 17,601607.
0042-6989/87 S3.00 + 0.00
Copyright0 1987 Pergamon Journals Ltd
LIGHT AND AGING EFFECTS ON VITAMIN E IN THE RETINA AND RETINAL PIGMENT EPITHELIUM MARTINL. KATZ’ and W. GERALD ROBISON JR~ ‘Mason Institute of Ophthalmology, University of Missouri, School of Medicine, Columbia, MO 65212
and ‘National Eye Institute, National Institutes of Health, Bethesda, MD 20892, U.S.A. (Received 27 February 1987; in revised form 1 June 1987)
Abstract-Experiments were performed to determine the effects of senescence and light adaptation on vitamin E levels in the neural retina and RPE-choroid-sclera of pigmented rats. Aging multed in significant increases in a-tocopherol levels in both tissues, the effect being most pronounced in the RPE-choroid-sclera. The state of light adaptation had no influence on a-tocopherol kvels in the neural retina at any of the ages examined, whereas in the RPE-choroid-sclera, a-tocopherol levels were substantially higher in light-adapted than in dark-adapted animals at all three ages (12.22, and 32 months) at which they were measured. The effect of light adaptation on RPE-choroid-scleral a-tocopherol levels was most pronounced in the oldest age group. Vitamin E
Aging
Light adaptation
Retina
INTRODUCTION
Lipofuscin deposition in the retinal pigment epithclium (RPE) is one of the most prominent cellular changes to occur in the mammalian retina during senescence (Katz and Robison, 1984; Wing et al., 1978). Vitamin E deficiency induces a substantial acceleration in RPE lipoftin deposition rates (Robison et al., 1979, 1980; Katz et al., 1986b), suggesting that lipofuscin may contain products of autoxidation of cellular components. Other age-related changes in the retina, including photoreceptor cell loss and changes in vitamin A metabolism, are also accelerated by vitamin E deficiency (Katz et al., 1984, 1987a). It therefore appeared possible that senescent alterations in the neural retina and RPE could be secondary to decreases in the vitamin E contents of these tissues. To determine whether this was the case, vitamin E measurements were performed on eye tissues from pigmented rats of various ages. Because light exposure exerts such profound influences on various aspects of retinal metabolism, the vitamin E measurements were performed on animals in both the dark- and light-adapted states. MATERIALS AND METHODS
Experimental animals Male pigmented AC1 rats were purchased at 8 weeks of age from Harlan Sprague Dawley
Retina1 pigment epithelium
Rat
Inc. (Indianapolis, Ind.). Animals were housed in hanging wire cages in a room maintained at a constant temperature of 21°C. Illumination from General Electric F15T12-CW fluorescent tubes was provided for 12 hr each day followed by 12 hr of total darkness. During the light phase of the daily cycle, illumination at the fronts of the cages was a maximum of 100 lx, while at the backs of the cages, illumination was less than 10 lx. Animals were fed a natural ingredient open formula diet (NIH 07) (Hess et al., 1985) and given water ad libitum. The diet contained an average of 37 international units of vitamin E per kg. Vitamin E determinations At 12, 22, and 32 months of age, rats were dark adapted for a minimum of 13 hr, 12 hr of which was during the normal dark phase of their lighting cycle. At 1 to 2 hr after the light phase of the cycle would normally have begun, rats were anesthetized with CO2 and one eye from each animal was enucleated under dim red light. The eye socket was packed with Gelfoam (Upjohn) to retard bleeding. The rats recovered rapidly from CO, anesthesia after enucleation and showed no apparent signs of discomfort from the surgery. Immediately after enucleation, the hides, corneas, lenses and vitreous were dissected from each eye and discarded. The retina was then dissected from the RPEchoroid-scleral complex, and each of these two
1875
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MARTIN
L. KATZ and W. GERALD ROBIWN JR
tissues was placed in 350 ~1 of cold 10 mM HEPES [4-(2-hydroxyethyl)- 1-piperazine ethane sulfonic acid], pH 7.4, containing 150 mM NaCl, and 5 mM diethylenetriamine pentaacetic acid to which 0.2% pyrogallol was added just prior to use. Microscopic examination indicated that the dissections produced very clean separations between the neural retinas and RPEs (Katz et al., 1987a). The tissues were dissociated by sonication under dim red light while being cooled in an ice-water bath. Sonication completely dissociated the retinas, RPEs, and choroids, as well as the connective tissue cells of the sclera, but left the collagenous extracellular matrix of the sclera relatively intact. After sonication, a-tocopherol was extracted from aliquots of the samples with hexane-ethanol. A 200 ~1 aliquot of each sample was transferred to a vial with a teflon-lined screw cap. To each sample was then added 200 ~1 of ethanol, and the mixture was vortexed for about 15 sec. Exactly 1.Oml of hexane, containing 4.0 nmol tocol per ml as an internal standard, was then added to each sample, and the mixture was vortexed again for 1.5 min. An additional 200 ~1 of ethanol and 400 ~1 of water were then added, and the sample was vortexed for 15 sec. The phases were separated by centrifugation at 500 g for 5 min, and the organic phase was collected and filtered through a 0.20 pm pore-size Millipore type FG filter. Approximately l.O’ml of hexane was then added to the sample, with care being taken not to disturb the phase boundary. The hexane phase was again collected and filtered and was pooled with the first hexane phase. Finally, the filter apparatus was rinsed with another 1.Oml of hexane, which was then added to the first two filtrates. The hexane was removed from the extracts by evaporation under a stream of argon. After they were dried, the samples were immediately dissolved in approximately 35 ~1 of methanol. Quantitation of a-tocopherol was performed with high performance liquid chromatography (HPLC). The chromatographic system consisted of Gilson model 302 pumps, a Rheodyne model 7161 injector with a 20~1 sample loop, a Brownlee 4.6 mm x 3 cm guard column with an RP-18 1Opm spherical absorbant, a 4.6 mm x 25 cm LiChrosorb analytical column with a 10pm RP-18 adsorbant (Alltech, Deerfield, Ill.), and a Kratos Spectroflow 773 absorbance detector equipped with a 12 ~1 flow cell. Absorbance was monitored at 295 nm. Chromatograms were run isocratically with the
mobile phase consisting of methanol : water (97: 3, v/v) at a flow rate of 2.0 ml/min. Quantitation of a-tocopherol was performed by determining the peak area of this compound relative to the peak area of the internal standard. Analysis of a -tocopherol standards indicated that the peak area ratios for these compounds were linear over the range found in our samples. Experiments in which known amounts of a-tocopherol were added to tissue samples demonstrated that with the methods employed, recovery of these compounds was complete relative to internal standard recoveries. Samples from dark-adapted rats were handled under dim red light until they were mixed with an equal volume of ethanol. All subsequent steps of the extraction and analyses were performed under dim incandescent illumination. One hour after initiation of the light phase of the light cycle, the day after the above measurements were performed, the pupil of the remaining eye was dilated with 1% Mydriacil. The rats were then placed in clear plastic cages that were illuminated with Sylvania F15T8-CW fluorescent tubes. Average illumination at the bottoms of the cages was 16001x. After 1 to 1.25 hr, when at least 95% of the visual pigment had been bleached (Katz and Robison, 1986b; Katz et al., 1987b), the rats were anesthetized with CO* and each had its remaining eye enucleated. Sonicates of the retinas and RPEchoroid-scleral complexes were prepared as described above for the dark-adapted eyes, except that the sonicate preparation was performed under bright fluoresecent lights. The sonicates were extracted with hexane-ethanol under dim incandescent lighting and the extracts were analyzed as described for the dark-adapted eyes. Efects of mydriasis and contralateral enucleation on vitamin E levels Experiments were performed in order to determine whether mydriasis alone influenced retina and RPE vitamin E levels. Mydriacil (1%) was applied to one eye each of five lightadapted lZmonth-old rats. The animals were immediately placed in total darkness. After 1.5 hr, the rats were anesthetized with CO, and their eyes enucleated. The neural retina and RPE a-tocopherol levels were then determined on each eye as described above. All enucleations were performed 2.5-3 h after the start of the light phase of the normal lighting cycle.
Aging and light effects on vitamin E
1877
much greater. At 32 months of age, the a-tocopherol content of this tissue complex was almost 48% higher than it was at 12 months of age (P < 0.001). At all three ages examined, light adaptation resulted in significant increases in the a-tocopherol contents of the RPE-choroid-sclera complexes (Table 1). An 11% increase in mean a-tocopherol content was induced by light adaptation in animals 12 months of age (P < 0.01). At 22 months, light adaptation resulted in a 7.6% increase in the vitamin E content of this tissue complex (P < O.Ol),while at 32 months of age, the ant-indu~ increase amounted to about 36% (P < 0.001). Assessments of the significance of aging and The differences in RPE-choroid-sclera vitalight effects on the various measured parameters min E levels between the light-adapted and were performed using analysis of variance. dark-adapted eyes did not appear to result Comparisons between individual treatment from the effects of mydriasis or trauma due to groups were performed using the Newman- enucleation of the contralateral eye one day Keuls procedure (Wirier, 1971). previously. In control experiments on 12month-old rats, neither retinal nor RPEchoroid-scleral vitamin E levels were affected by RESULTS mydriasis or by prior enucleation of the contraIn all tissues examined, vitamin E appeared to lateral eye. Under all four conditions used in the occur exclusively as a-tocopherol. Neither j?- control experiments, vitamin E levels were nor y$ocopherol were detectable in any of the essentially the same as those found in darksamples. Aging resulted in moderate increases in adapted eyes of the IZminth-old animals used the a-tocopherol contents of the neural retinas. in the aging study (Table 1). Between 12 and 32 months of age, retinal vitamin E content increased an average of 12% DSCUSSION (P < 0.05) in the dark-adapted state, and 9% The fact that lipofuscin accumulation can be (P < 0.05) in the light-adapted state (Table 1). The state of light adaptation had no significant greatly accelerated by antioxidant nutrient influence on the a-tocopherol content of the deficiency serves as strong evidence that autoxidative mechanisms are involved in age-related neural retina at any age examined. lipofuscin deposition (Katz and Robison, Senescence had a more pronoun~d effect on 1986a). The progressive age-related increase in the a-tocopherol content of the RPE-choroidsclera complex. In the dark-adapted state, RPE lipofuscin content that occurs in various tissues a-tocopherol content increased an average of suggests that senescence may be accompanied 21% (P < 0.001) between 12 and 32 months of by a decline in antioxidant protective mechage (Table 1). In the light-adapted state, this anisms. Indeed, several antioxidant defense age-related increase in a-tocopherol content was mechanisms have been shown to decline during Experiments were also cod&Wed to determine whether the trauma of enucleation of one eye could influence the vitamin E levels in the contralateral eye enucleated a day later. Rats were dark-adapted for 13-14 hours and the neural retina and RPE vitamin E levels were then measured in one eye from each animal as described earlier. The rats were then placed in the light until the end of their normal daily light cycle. After dark adapting the rats overnight (13-14 hr), the other eye of each animal was enucleated and neural retina and RPE a-tocopherol levels were again determined.
Table 1. Influences of age and light on u-tocopherol content of the retina and RF’E-choroid-sclera a-Tocuphcrol content @mol@ye) Light-adapted Dark-adapt& Atzc _ (months) . 12 22 32
Retina
RPE-C-S
911 f21b(10)E 869&34(10) 931 f 33 (11) 935 f 26 (11) 1021 f 57 (6) 1051f 56 (6)
lRPE-choroid+ciera.
bvafues exprased as mean f SE. ‘Number of eyes analyzed in parentheses.
Retina
RPE-C-S
944*25(13) 937f36(11) 1029f 74 (7)
%7&42(13) 1006f36(13) 1429f 93 (7)
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MARTIN L. KATZ and W. GERALD ROBIWN JR
aging in a variety of species (Sohal and Allen, 1986). A number of experiments have demonstrated the importance of vitamin E in protecting against autoxidative damage to the neural retina and against excessive lipofuscin accumulation in the RPE (Robison et al., 1979, 1980; Katz et al., 1986a; Riis et al., 1981). Retinal photoreceptor cells play a central role in RPE lipofuscin deposition, and it is likely that autoxidized photoreceptor outer segment components are incorporated into RPE lipofuscin granules (Robison et al., 1982; Katz et al., 1986a, 1987~; Katz and Robison, 1986a). It therefore appeared possible that the age-related accumulation of lipofuscin in the RPE was due to declining levels of vitamin E in the neural retina. The current experiments indicate, however, that this was not the case. It is possible that levels of other antioxidants in the retina, such as glutathione or protective enzymes such as superoxide dismutase, do decline during senescence, leading in turn to an increased rate of lipofuscin formation. Alternatively, the progressive accumulation of lipofuscin in the RPE and other age-related changes in the retina may result from autoxidative damage occurring at a constant rate throughout the lifespan. Lipofuscin deposition could reflect a failure of retinal tissues to remove the products of autoxidation at a rate matching their rate of formation. It is not currently possible to measure total RPE a-tocopherol content alone due to the difficulty of quantitatively isolating the RPE from the underlying choroid-sclera complex. However, it is likely that the overall increase in RPE-choroid-scleral a-tocopherol levels during aging is indicative of a similar increase in the RPE alone. It is improbable that RPE vitamin E levels decline due to an age-related shift in vitamin E stores from the RPE to the choroid and sclera. On the contrary, the apparent agerelated increase in RPE lipid droplet content (Katz er al., 1984) suggests that a-tocopherol levels probably increase to a greater relative extent in the RPE than in the tissue complex as a whole, since there is an age-related increase in the lipophilic environment within the RPE into which vitamin E can partition. The data presented probably understate the effect of senescence in increasing a-tocopherol levels, at least in the neural retina. There is an age-related loss of cells from all layers of the neural retina (Katz and Robison, 1986b). Thus, if the data were expressed on a per-cell basis, the influence of senescence on retinal a-tocopherol
levels would have appeared substantially greater. Organisciak and Feeney-Bums (personal communication) have recently measured vitamin E levels in both the neural retinas and RPEs from human donor eyes. Vitamin E levels were found to increase substantially with increasing donor age in both tissues. The finding of similar increases in rats maintained under controlled environmental and dietary conditions suggests that the observed changes in vitamin E levels may be fundamentally associated with the process of senescence. The light-induced increase in the a-tocopherol content of the RPE-choroid-scleral complex was unexpected, and the mechanism for this effect is unclear. The increase does not appear to be due to a greater engorgement of the choroidal capillaries with blood in the lightadapted state, since retinol levels in the RPE-choroid-scleral complex were not affected by light adaptation (Katz et al., 1987a). Light adaptation does not appear to induce a transfer of a-tocopherol from the neural retina to the RPE, since retinal a-tocopherol levels were the same in both the light- and dark-adapted states. It therefore appears likely that light exposure induces uptake of a-tocopherol from the choroidal circulation into the RPE-choroid-sclera. This light-induced uptake of vitamin E may serve an important physiological function, such as enhancing protection against light-mediated autoxidation (Hunt et al., 1984). Acknowledgemenrs-The authors wish to thank A. B. Groome and C. M. Drca for their expert technical assistance and Dr G. E. Eldred for his helpful comments on the manuscript. Tocol was a generous gift from Hoffmann-La Roche, Inc. The investigations reported in this paper were supported in part by U.S. Public Health Service Grant EYO6458 and by a grant from Research to Prevent Blindness, Inc. REFERENCES
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