2. G. Wald: Human Vision and the Spectrum. Sci-
`ence 101: 653-658. 1945
`
`3. V.M. Reading and RA. Weale: Macular Pig-
`ment and Chromatic Aberration. J. Opt. Soc.
`Am. 64: 231-234, 1974
`
`4. MR. Malinow, L. Feeney-Burns. L.H. Peterson.
`M.L. Klein and M. Neuringer: Diet-Flelated Ma-
`cular Anomalies in Monkeys. Invest. Ophthal-
`mol. Vis. Sci. 19: 857-863, 1980
`
`5. L. Feeney-Burns, M.Fl. Malinow, L. Peterson.
`M. Klein and M. Neuringer: Macular Hyper-
`fluorescence in Monkeys. Presented at West-
`
`CYAN EXHIBIT 1029
`
`ern Section Regional Meeting. Association for
`Research in Vision and Ophthalmology. Seal-
`tle, October, 1978
`
`6. B.S. Fine and RP. Kwapien: Pigment Epithelial
`Windows and Drusen: An Animal Model. In-
`
`vest. Ophthalmol. Vis. Sci.
`1978
`
`17: 1059-1068.
`
`7. M. Neuringer, D. Denney and J. Sturman: Re-
`duced Plasma Taurine Concentration and
`
`Cone Electroretinogram Amplitude in Monkeys
`Fed a Protein-Deficient. Semi-Purified Diet.
`Am. J. Clin. Nutr. 32: xxvi, 1979
`
`THE EFFECT OF DEFICIENCY OF
`
`VITAMINS E AND A
`
`ON THE RETINA
`
`Vitamin E deficiency causes an extensive accumulation of lipofuscin in the pigment
`epithelium of the retina. Rats deficient in vitamins E and A, had less
`retinal lipofuscin than rats deficient in vitamin E only.
`
`Key Words: vitamin E, vitamin A, retina, rod outer
`segment, retinal pigment epithelium, photoreceptor
`cells, antioxidant, peroxidation. lipofuscin
`
`The membranes of the rod outer segments
`(ROS) of the retina have served as models for
`the elucidation of the structure of membranes
`
`since Brown‘ showed that rhodopsin rotates
`freely in the lipid environment of the ROS mem-
`brane. Flecently, the antioxidant theory of the
`function of vitamin E in membranes received
`
`direct and unexpected support from the work of
`Robison, Kuwabara and Bieri.2~3 Again, the
`membranes of the rods of the retina served as a
`
`model. The outer segment of the vertebrate rod
`is made up of a stack of disk membranes con-
`taining an exceptionally high concentration of
`polyunsaturated fatty acids (nearly one-half of
`the ROS fatty acids contain six double bonds)!
`Since the peroxidizability of unsaturated fatty
`acids is directly proportional to the number of
`double bonds they contain,5 the ROS is particu-
`larly susceptible to lipid peroxidation. To aggra-
`vate this situation, the retina also is served with a
`
`336
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`NUTRITION REVIEWS/VOL. 39. NO. HINOVEMBER 1980
`
`plentiful supply of oxygen since it has an unusu-
`ally high rate of oxidative metabolism. To make
`matters worse, light is known to enhance lipid
`peroxidation in the retina.6
`Vitamin E, a free-radical scavenger, is thought
`to protect membrane lipids from peroxidation.
`The concentration of vitamin E is exceptionally
`high in ROS, 1 mole of a-tocopherol for each 36
`moles of rhodopsin.7 A seasonal dietary decline
`in vitamin E intake in cattle was found reflected in
`
`increased lipid oxidation products in retina in
`vitro, together with some destruction of ROS
`membrane structures.’ In vivo, Hayes‘ observed
`degeneration of the macular region of retina
`when monkeys were fed a vitamin E-deficient
`diet for two years.
`The formation of a yellow autofluorescent
`pigment termed lipofuscin is correlated with
`polyunsaturated fatty acid oxidation and ac-
`cumulates as the end product of lipid peroxida-
`tion. This pigment is found in many tissues with
`advancing age and especially in the pigment
`epithelium of
`the retina. Physiological
`mechanisms that prevent lipid auto-oxidation
`also decrease lipofuscin accumulation. Thus,
`
`

`
`vitamin E protects membrane lipids from auto-
`oxidation and at the same time retards accumu-
`
`lation of lipofuscin.
`Katz et al.9 showed that rats maintained on a
`
`diet deficient in vitamin E accumulated large
`amounts of lipofuscin in the pigment epithelium
`of the retina (RPE), especially in diets also high
`in polyunsaturated fatty acids. The RPE seemed
`to be especially sensitive to vitamin E deficiency,
`since the lipofuscin accumulation in other tis-
`sues was less severe. it is known that the RPE
`
`absorbs the material released during breakdown
`and turnover of ROS disk membranes. The old-
`
`est disks are shed from the ROS tips, absorbed
`and catabolized by the RPE.‘° In fact, the only
`other part of the retina in which Katz et al.9 de-
`tected a small amount of lipofuscin was at the
`ROS tips.
`Robison, Kuwabara and Bieriz investigated
`the effect of vitamin E deficiency together with
`normal or marginal vitamin A intake in the retina
`of the rat. The reason for considering marginal
`vitamin A in conjunction with vitamin E deficiency
`is that vitamin A depletion is known“ to result in
`deterioration of the ROS disk structure, due to
`loss of rhodopsin. Female rats were given a
`vitamin E-deficient diet. One-half received
`
`adequate vitamin A (8.0 mg retinol per kilogram
`diet), -E+A, and one-half received a vitamin
`A-marginal diet (0.8 mg per kilogram), -E-A.
`Control rats received adequate vitamin E (250
`mg dl-a-tocopheryl acetate per kilogram diet),
`with normal or marginal vitamin A (+E+A and
`+ E-A). Tissue vitamin A levels were affected by
`vitamin E deficiency. After five months, the -E-A
`group had one-tenth the liver concentration of
`retinol compared to the -E+A group. The RPE
`cells of all the -E rats had five times the number
`
`of lipofuscin granules (identified by their
`characteristic fluorescence and PAS staining).
`Electron microscopy revealed more secondary
`lysosomes in the RPE in the -E rats. Clearly, the
`great accumulation of lipofuscin in RPE and dis-
`ruption of the rods in all -E rats is the end resultof
`the oxidation of polyunsaturated fatty acids in the
`absence of antioxidant protection. The simul-
`taneous breakdown of the rods and accumula-
`
`tion of lipofuscin suggests that the breakdown
`products of the rods contribute to the substance
`of the lipofuscin granules. The RPE merely con-
`tinues its normal function of phagocytizing and
`
`degrading pieces of ROS membrane disks and
`thus accumulates the end products of lipid
`peroxidation.
`Surprisingly, there was a 46 percent loss of
`rod nuclei in the -E-A group,whereas the-E+A
`group lost none, compared to controls. There-
`fore, the normal level of dietary vitamin A was
`essential for preserving the number of rod cells.
`The marginal vitamin A intake had no effect on
`the rod cells of the + E rats, whereas a marginal
`vitamin A intake caused a great loss of these
`cells in the -E group. The animals on the -E-A
`diet received some dietary vitamin A, and their
`plasma vitamin A level, though lower than that of
`the -E+A rats, was still appreciable. Hence, the
`authors postulate the occurrence of local tissue
`deficiencies of vitamin A, caused by the lack of
`antioxidant in the microenvironment of the re-
`tina. The decline in vitamin A level in the RPE
`
`and the ROS may be such as to mimick the effect
`of a frank vitamin A deficiency in the whole ani-
`mal, and thus disrupt the ROS disk structure.
`The authors are careful to point out, however,
`that the effect on the ROS of the vitamin E defi-
`
`ciency in conjunction with marginal vitamin A,
`differs from that of a straight dietary vitamin A
`deficiency. In the latter, the rods are affected
`before the cones, whereas in the -E-A rats, rods
`
`and cones deteriorated simultaneously and
`equally.
`In their second paper, Robison, Kuwabara
`and Bieri3 investigated the effect of vitamin A
`deficiency on the rod destruction induced by vi-
`tamin deficiency. They used vitamin E and
`frankly (not marginally) vitamin A-deficient rats in
`the configuration: -E-A, -E+A, +E-A and
`+E+A. All -A rats received retinoic acid to
`
`maintain their general health (retinoic acid can-
`not reverse the effect of vitamin A deficiency in
`the retina). After 21 weeks there was, as ex-
`pected, an accumulation of lipofuscin, the vi-
`tamin E-deficient retinas (-E+A and-E-A. The
`+E-A group had no lipofuscin, but some disk
`membrane disruption and a 27 percent loss of
`rod nuclei. The -E-A group showed a 60 percent
`loss of rod nuclei. After 35 weeks, the vitamin
`E-deficient retinas showed that the accumula-
`
`tion of lipofuscin was two-fold over control in the
`-E-A retinas, and more surprisingly, the pre-
`sence of retinol in the -E+A retinas caused a
`
`five-fold increase in lipofuscin granules. The
`
`NUTRITION REVIEWS/VOL. 38, NO. 11/NOVEMBER 1980
`
`387
`
`

`
`yellow autofluorescence intensity (measuring
`Iipofuscin concentration) also was increased.
`Thus,
`the vitamin E-deficient, vitamin A-
`
`adequate group not only had the largest number
`of Iipofuscin granules, but also the highest con-
`centration of fluorescent pigment, higher than in
`the -E-A group. Unexpectedly,
`though the
`+E+A group had far fewer Iipofuscin granules
`than the -E-A group, their fluorescence intensity
`was greater. The fluorescence intensity in the
`+ E-A group was lowest of all, lower than in the
`+ E+A group.
`Thus, the massive accumulation of Iipofuscin
`in the RPE of the -E animals is most probably
`caused by greatly increased peroxidation, re-
`sulting in damaged membranes. These mem-
`branes would then be phagocytized by the RPE,
`digested and the “undigestible” remnants pack-
`aged as Iipofuscin granules. Further,
`though
`larger numbers of these granules were formed in
`vitamin E deficiency, they were less fluorescent
`in the absence of vitamin A. Possibly, vitamin A
`is involved in Iipofuscin formation in the retina by
`influencing the composition of the pigment. Al-
`ternatively, vitamin A deficiency may decrease
`the rate of rod phagocytosis‘ by RPEJ2 As a
`control, the authors compared the uteri of the
`-E+A and -E-A rats (the vitamin E-deficient
`uterus is rich in Iipofuscin granules). They found
`no difference in that
`tissue. Therefore,
`antioxidant-related pigment formation in tissues
`outside the retina is not influenced by vitamin A.
`Possibly, this may be connected to the fact that
`the uterus responds, but the retina does not
`respond, to the retinoic acid which was being fed
`to the -A animals. It should be remembered that
`
`retinal, derived from retinol but not from retinoic
`acid, is a component of the rhodopsin molecule,
`the principal protein of the rods.
`With regard to the structure of the retina, the
`vitamin E deficiency alone (-E+A), after 35
`weeks, caused a disruption of the disk mem-
`branes and a 20 percent loss of photoreceptor
`cells. A vitamin A deficiency superimposed on
`the vitamin E deficiency (-E-A) led to almost
`complete destruction of the ROS membranes
`and loss of more than 90 percent of the photo-
`receptor cells. As one would expect, vitamin A
`deficiency alone (+E-A) led to a greatly shor-
`tened ROS and an intermediate loss of cells (34
`percent). Thus, a -E-A diet produced a greatly
`
`388
`
`NUTRITION REVIEWS/VOL. 38, NO. 11/NOVEMBER 1980
`
`accelerated degeneration of the photoreceptor
`cells compared to a + E-A diet. While vitamin A
`deficiency leads to some damage of the retina,
`the disruption caused by a combined vitamin
`E-vitamin A deficiency is greatly accentuated,
`apparently more than can be accounted for by
`an additive effect. The authors are of the opinion
`that the vitamin E deficiency caused increased
`oxidation of stored vitamin A in liver and RPE,
`greatly accelerating the process of rod destruc-
`tion.
`
`The membranes of the rod disks are unusually
`sensitive to oxidative damage, yet they are in an
`environment rich in oxygen. Therefore, the au-
`thors suggest, vitamin E appears to be espe-
`cially necessary for protection of the retina. in
`particular because of the accentuation of lipid
`peroxidative damage by light. Moreover, the
`work reveals that in vitamin E deficiency there
`may be increased oxidative destruction of the
`vitamin A stores, leading to more rapid develop-
`ment of damage in the photoreceptor cells. In
`sum,
`to quote the authors? "normal dietary
`levels of both vitamin E and A are essential for
`
`the structural maintenance of the neural retina
`
`and for a normal pigment epithelium," a discov-
`ery of a vitamin interaction of great consequence
`for the maintenance of normal vision. El
`
`1. P.K. Brown: Rhodopsin Rotates in the Visual Re-
`ceptor Membrane. Nature 236: 35-38, 1972
`2. W.G. Robison, Jr., T. Kuwabara and JG. Bieri:
`Vitamin E Deficiency and the Retina: Photo-
`receptor and Pigment Epithelial Changes. Invest.
`Ophthalmol. Vis. Sci. 18: 683-690, 1979
`3. W.G. Robison, Jr., T. Kuwabara and J.G. Bieri:
`Deficiencies of Vitamins E and A in the Rat: Reti-
`
`nal Damage and Lipofuscin Accumulation. In-
`vest. Ophthalmol. Vis. Sci. 19: 1030-1037, 1980
`4. F.J.M. Daemen: Vertebrate Rod Outer Segment
`Membranes. Biochim. Biophys. Acta 300: 255-
`288, 1973
`
`5. L.A. Witting: Lipid Peroxidation In Vivo. J. Am. Oil
`Chem. Soc. 42: 908-913, 1965
`
`6. L. Feeney and E.R. Berrnan: Oxygen Toxicity:
`Membrane Damage by Free Radicals. Invest.
`Ophthalmol. 15: 789-792, 1976
`7. C.C. Famsworth and E.A. Dralz: oxidative Dam-
`
`age of Retinal Rod Outer Segment Membranes
`and the Role of Vitamin E. Biochim. Biophys.
`Acta 443: 556-570, 1976
`
`

`
`8. K.C. Hayes: Retinal Degeneration in Monkeys
`Induced by Deficiencies of Vitamin E or A. Invest.
`Ophthalmol. 13: 499-510, 1974
`
`9. M.L. Katz, W.L. Stone and E.A. Dratz: Fluores-
`
`cent Pigment Accumulation in Retinal Pigment
`Epithelium of Antioxidant-Deficient Rats. Invest.
`Ophthalmol. Vis. Sci. 17: 1049-1058, 1978
`
`10. R.W. Young: The Renewal of Rod and Cone
`
`Outer Segments in the Rhesus Monkey. J. Cell
`Biol. 49: 303-318, 1971
`1 1 . L. Carter-Dawson, T. Kuwabara, P.J. O'Brien and
`
`J.G. Bieri: Structural and Biochemical Changes in
`Vitamin A-Deficient Rat Retinas.
`Invest.
`
`Ophthalmol. Vis. Sci. 18: 437-446, 1979
`12. J.E. Dowling and I.R. Gibbons in The Structure of
`the Eye. G.K. Smelser, Editor, p. 85. Academic
`Press, New York, 1961
`
`NUTRITION REVIEWS/VOL. 38. NO. 11/NOVEMBER 1980
`
`389