CYAN EXHIBIT 1027
`
`Retinal degeneration in monkeys induced by
`
`deficiencies of Vitamin E or A
`
`K. C. Hayes
`
`The effects of vitamin E or vitamin A deficiency on the retina were assessed in monkeys for
`as long as two and three-quarters years. A macular degeneration developed after two years
`in monkeys fed vitamin E-deficient diets. The lesion was characterized by focal, massive disrup-
`tion of photoreceptor outer segments attributed to lipid peroxidation of these lipoprotein
`structures containing highly unsaturated fatty acids. The focal nature of the lesion precluded
`any evidence of clinical blindness. Vitamin A deficiency was typical of that described by
`others, and was accompanied by xerophthalmia, keratomalacia, and clinically impaired vision.
`Anatomic evidence suggested that
`the structural disruption of photoreceptors was more
`advanced in cones and was most pronounced in the macula with a lesser involvement of the
`peripheral retina.
`
`Key words: vitamin A, vitamin E, retina, blindness, macular degeneration.
`
`Experimental vitamin E deficiency has
`
`elicited a multitude of pathologic entities
`in a variety of species. In terms of ocular
`pathology, cataract formation has been re-
`ported" ‘—' but retinal degeneration has not
`been previously described. A group of dogs
`found deficient
`in vitamin E at necropsy
`was found to have advanced retinal de-
`
`its etiology and patho-
`generation,3 but
`genesis were unclear. Children afllicted
`with abetalipoproteinemia suffer from vis-
`ual
`impairment thought to be related to
`a failure in absorption of fat and the fat-
`soluble vitamins,4 and the onset of neuronal
`
`From the Department of Nutrition, Harvard School
`of Public Health, Boston, Mass. 02115.
`This work was supported in part by United States
`Public Health Service Research Grants EY-00631
`and HL-10098, and the Fund for Research and
`Teaching, Department of Nutn'tion, Harvard
`School of Public Health.
`
`Submitted fer publication Ian. 21, 1974.
`
`(Batten’s disease), a
`ceroid-lipofuscinosis
`syndrome resembling the ceroid accumula-
`tion of chronic vitamin E deficiency,5 is
`usually accompanied by impaired vision
`and progressive blindness of undetermined
`pathogenesis.” These conditions and the ob-
`servation that vitamin E is concentrated in
`
`(OS)7
`the photoreceptor outer segments
`suggest that tocopherol may have a spe-
`cific function in the retina.
`
`The present study in primates provides
`details of a retinal degeneration in the
`macula of two species of monkeys follow-
`ing long-term deprivation of vitamin E.
`In addition,
`the lesion is compared with
`,that produced by vitamin A deficiency in
`one of these species and difierences be-
`tween the deficiencies are discussed.
`
`Methods
`
`The complete description of the animals, diets,
`and procedures for the vitamin E study has been
`reported elsewhere.S Two species of monkeys ap-
`
`499
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`

`500 Hayes
`
`Investigative Ophthalmology
`July 1974
`
`Table I. Composition of semi-purified
`diet low in vitamin A
`
`Grams per
`.100 grams
`Ingredients
`20.0
`Casein, vitamin-free
`29.6
`Dextrin
`10.0
`Sucrose
`10.0
`Cottonseed-soybean oil
`23.8
`Cellulose“
`4.0
`Salt mix’r
`2.2
`Vitamin mixt
`0.3
`Choline chloride
`0.1
`Vitamin D3 (1,250 IU per gram)
`“Alphacel, General Biochemicals, Chagrin Falls, Ohio.
`iHegsted, D. M., et 81.: J. Biol. Chem. 135: ‘459, 1941.
`tThe vitamins added to 1,854 grams of dextrin were (in
`grams):
`a-tocopherol
`succinate, 10; ascorbic acid, 90;
`inositol, 10; menadione, 4.5- p-aminobenzoic acid, 10;
`nicotinic acid 9;
`riboflavin, 2; pyridoxine~ HCl,_ 2;
`thia-
`mine-HCl
`2;
`calcium pantothenate
`6; biotin, 0.04;
`folic acid, 0.18; and cyanocobalamine, 0.005.
`
`proximately 14 months of age and born. and
`raised in our primate colony were used. These
`included 12 New World cebus (Cebus albifrons
`and apella) and 14 Old World cynomolgus (Macaca
`fascicularis) monkeys. Animals were fed- a. semi-
`purified diet containing either stripped safflower
`oil
`(tocopherol removed) or coconut oilwith or
`without a vitamin E supplement for as long as two
`and three-quarters years. Three deficient monkeys
`died unexpectedly whereas all others were killed
`at the end of the experimental period. Eyes were
`either perfused in situ via the left ventricle ‘of
`the heart using a 1 per cent formaldehyde:l.25
`per cent glutaraldehyde fixative buffered to pH 7.4
`with 0.1 M phosphate buffer, or
`the eye was
`removed from the anesthetized monkey and the
`posterior hemisphere immersed in 2 per .cent
`osmium tetroxide in 0.1 M phosphate buffer.
`Representative eyes were fixed in formalin and
`embedded in paraffin for routine hematoxylin'and
`eosin (H 6: E) sections.
`1‘
`Six adult female cebus monkeys weighing 1,400
`to 1,700 grams were used for the study of vitamin
`A deficiency. They were fed an agar cake diet
`lacking in vitamin A (four monkeys) Or the same
`diet with added vitamin A (two monkeys)
`(Table I)
`for as
`long as 21 months. Periodic
`serum samples were taken for vitamin A analysis.”
`Although the entire retina was available foi- light
`microscopy, only paramacular and mid-peripheral
`retina was examined by electron microscopy in the
`vitamin A-deficient monkeys.
`two anesthetized
`In both experiments one or
`monkeys were visually screened along with the
`control animals
`for
`their b-wave threshold in
`response to a
`full-field (Ganzfeld) white-light
`stimulus.10 All
`the monkeys were housed under
`identical
`lighting and environmental conditions
`and were cared for by the same personnel. Cyclic
`
`light (12-hour periods) from overhead fluorescent
`lamps provided an approximate cage illumination
`of 8-foot candles.
`
`Vitamin E deficiency. The general pathophysiol-
`ogy of vitamin E deficiency in these monkeys5
`as well as the details of the anemia that devel-
`
`In sum-
`oped8 have been previously described.
`mary, deficient monkeys fed polyunsaturated saf-
`flower oil were most severely affected, developing
`severe hemolytic anemia, muscle degeneration,
`testicular degeneration, and marked ceroid ac-
`cumulation in several tissues. Retinal degeneration
`occurred in seven of
`the eleven monkeys
`fed
`either safflower oil or coconut oil without vitamin
`E. None of the vitamin E—supplemented monkeys
`developed retinal damage, and none of thOSe fed
`coconut oil became anemic. Blindness was never
`
`apparent clinically. The full-field electroretinogram
`(ERG) b-wave threshold was considered normal
`in two deficient monkeys tested on two occasions,
`later found to have histologic degeneration of the
`macula. No fundus abnormality was detected with
`the indirect ophthalmoscope. There was no evi-
`dence of xerophthalmia and the monkeys appeared
`healthy and alert. The plasma vitamin E concen-
`tration was generally less
`than 100 #g/dl
`in
`advanced deficiency with both dietary oils. Con-
`trol values were in excess of 600 flg/dl.3
`The plasma vitamin A levels varied with the
`species and dietary treatment, being lower in the
`cebus than the cynomolgus monkeys and lowest
`in the vitamin E-deficient monkeys fed safflower
`oil.5 However,
`the most extensive degeneration
`was observed in a cebus monkey fed coconut oil
`for the longest period of time (33 months), and
`the vitamin A concentration was among the highest
`recorded for the cebus (20 [lg/d1).
`Light microscopy of the retina revealed a focal
`disruption of the photoreceptor OS layer that was
`restricted to the macula (Fig. 1).
`The minimal
`change detected by electron
`microscopy was vacuolization and disorientation
`of OS lamellae among isolated photoreceptors of
`the macula. Adjacent 05 were often peculiarly
`spared in these areas of minimal damage (Fig. 2).
`This alteration most often involved the distal
`third, of the OS and included both rods and cones.
`:Inner' segments of these photoreceptors appeared
`normal (Fig. 3). In more advanced degeneration
`the entire OS was markedly swollen and lamellae
`were increasingly disrupted, often broken into
`short
`tubules or empty vesicles (Figs. 4 and 5).
`Other tubules and vesicles were packed with an
`electron-dense, finely granular debris (Figs. 5 and
`6)
`similar
`to the material seen in liver mito-
`chondria of
`these monkeys5
`and thought
`to
`represent peroxidized lipid. Adjacent OS often
`remained intact
`(Fig. 4). In other areas of the
`macula the OS were totally disrupted and had
`formed dense clusters of
`lamellar debris with
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`

`Volume 13
`Number 1
`
`Retinal degeneration in monkeys
`
`501
`
`
`
`has intact inner (IS)
`Fig. 1. The peripheral retina (A) from a vitamin E-delicient monkey
`and outer (05) segments of phmmptors that do not appur- dilfcrent from control sec
`tions, whereas those from the macula in the same monkey (B) are markedly disrupted (ar-
`rows). On the other hand, both peripheral retina (C) and macula (D) in the vitamin A-
`delicient monkey have degenerated outer segmen
`ts, the latter appearing much worse than the
`former. Thinning of the ONL has also occurred in the mania. (x200).
`Fig. 2. Early stage of macular degenoration in vitamin E deficiency. An essentially normal
`rod outer segment (top) is adjacent to one undergoing degeneration (below). Disoricntation
`and vesiculalion of lamellae are apparent. 0:27.550).
`
`whorls of myelin figures (Figs. 6 and 7). The
`pigment epithelial cells appeared to sustain phago-
`cytic activity, often being distended with lipo-
`fuscin-type lysosomes and larger aggregates of
`phagocytized OS debris (Figs. '1, 8, and 9). The
`inner layers of the retina were without visible
`change except for an occasional pyknotic nucleus in
`the outer nuclear layer (ONL).
`Vitamin A deficiency. Clinical manifestation of
`
`vitamin A deficiency occurred in three of the four
`depleted monkeys after 16, 19, and 20 months of
`study. Plasma vitamin A levels in the deficient
`monkeys were negligible after a year ( < 5 lag/d1,
`whereas control values were 15 to 20 pg/dl).
`After 12 to 15 months body weight loss became
`apparent. Xerophthnlmia with reddening of the»
`conjunctiva became evident as well. In the more
`advanced stage, lethal-y, anorexia, and inanition
`
`

`

`502 Hayes
`
`luneah'unlim- Ophthalmology
`July 1914
`
`new
`
`‘33 2“
`H:
`\%§ (‘33;
`
`merits (BIS). These apparently undamaged IS were observed in the macula where outer
`segments were degenerating. The 615 is distinguished by its width and dense aggregation
`of mitochondria. Intercellular junctions constituting the outer limiting membrane are visible
`(arrow). Portions of outer segments are sectioned tangentially below. (X45300).
`Fig. 4. Swelling of photoreceptor outer segments and vesiculation of lamellae were predomi-
`nant changes in moderately advanced stages of retinal degeneration in vitamin E deficiency.
`Portions of adjacent 05 appear normal. Clusters of electron-dense particles (arrow) were
`characteristic of
`this degeneration. The pigment epithelium (PE) contains cigar-shaped
`pigment granules and less dense lysosomal bodies presumed to represent an end-stage in the
`removal of the phagosomes (P). (x9500).
`
`

`

`Retinal degeneration in. monkeys 503
`
`
`
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`
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`granular.
`Fig. 5. Many degenerating lemellae form tubules and vesicles packed with fine,
`electron-dense particles (arrows) thought to represent peroxidized lipoprotein or lipoiuscin
`formation in vitamin E deficiency. (X41900).
`Fig. 6. Marked degeneration of outer segments is represented by swelling and disruption of
`outer segments, forming whorls of lamellar debris (d) and myelin-lilre figures (My). Electron-
`dense particles fill tubules of degenerating lanrellae (arrow). 615,900).
`
`
`
`were accompanied by advanced night blindness,
`evidenced by an inability to maneuver in a dimly
`lighted room. Xerosis and kcratinization of focal
`areas of the cornea were associated with forma-
`tion of corneal ulcers and the rapid development
`of keratomalecie (Fig. 10).
`The youngest and smallest. monkey was killed
`
`in an advanced state of deficiency after 18 months
`and one eye was taken for electron microscopy
`(Figs. 12, 13, and 14) and the other for light
`microscopy (Fig. 1). Two of the other three
`deficient monkeys presented similar clinical condi-
`tions after 19 and 20 months. One of
`these
`monkeys, progressing from night blindness
`to
`
`

`

`Investigate: Ophthalmology
`lulu 1914
`
`
`
`rent in the macula of a
`Fig. 7. Massive disruption of photoreceptor outer segments is apps
`vitamin E-deficient
`monkey. Formation of dense myelin-like figures‘represents accumulation
`of extracellular debris.'l'he
`pigment epithelium contains masses of phagocyfized outer seg-
`ments, both loosely packed in the basal cytoplasm (LOS) and in more typical dense phago-
`somes (arrows). Increased Iysosomal grandeur and cigar-shaped pigment granules are visible
`in the apical cytoplasm (X4,700).
`
`impaired vision in a lighted room, was tested and
`found to have a nondetectable b-wave in the full-
`Beld ERG. One eye was enucleahed under anes-
`thesia and prepared for electron microscopy (Fig.
`15). All
`the monkeys were then refed com-
`mercial monkey chow. The monkey with the
`enudeated eye died two weeks later having devel»
`oped acute panophthalmitis of its remaining eye.
`
`The other two deficient monkeys regained their
`appetite, physical
`activity, and body weight.
`Xerophthalmia and keratomalaeia disappeared in
`the monkey so affected and Vision appeared to be
`adequate, although slight corneal opacity remained
`after several months.
`
`Light microscopy of major organs from the two
`vitamin A-deficient monkeys necropsied at 16 and
`
`

`

`Valume 13
`Number 7
`
`Retinal degeneration in monkeys 505
`
`
`
`Fig. 8. Pigment epithelial cells from a vitamin Iii-deficient monkey contains distinctly different
`stages of phagosome digestion (A, B, C, and D). The middle cell is packed with lysosomes
`and appears to be pulling away from Bruch’s membrane (arrow), presumably to migrate
`into the inner retina. (X5100).
`Fig. 9. Different
`inclusions are observed in this pigment epithelial cell from a vitamin E-
`deficient monkey. A large loosely packed portion of outer segment (A) is contrasted by a
`more densely packed phagosorne (B) and a lysosome (C). A cigar-shaped melanin granule
`is also visible. Mitochondria (M). (A3300).
`
`revealed squamous metaplasia and
`21 months
`keratinization of the trachea, conjunctiva, excretory
`ducts in the pnrotid gland, and transitional epi-
`thelium of
`the renal pelvis
`(Fig. 11}. Pyelo-
`nephritis was associated with the latter change in
`both monkeys.
`The one eye examined by light microscopy after
`16 months revealed a shortened, disrupted pattern
`
`among all photoreceptor OS in the mania and
`many in the paramacular retina. This degenera-
`tion was less apparent in the peripheral retina. The
`ONL was reduced in thickness and contained
`degenerating nuclei corresponding to the degree
`of OS degeneration (Fig.
`l). The supercnasnl
`quadrant of
`the retina was allected, but
`to a
`lesser degree. In the opposite eye only the mid-
`
`

`

`506 Hayes
`
`Investigative Ophthalmology
`Julv 1974
`
`
`
`Fig. 10. Classical kerntomoleeia with exudative conjunctivitis was produced after 16 months
`on a vitamin A-free diet in this adult monkey.
`Fig. 11. Keratinizntion of the renal pelvis transitional epithelium provides histologic evi-
`dence of vitamin A deficiency. The inflammatory cells visible in the renal papilla were asso-
`ciated with pyelonephritis. (x450).
`
`by electron
`examined
`retina was
`peripheral
`revealing selective damage of
`the
`micmscopy,
`cone 05. This OS degeneration was characterized
`by vesiculation, distention, disorientation, and loss
`of the Iamellar discs (Fig. 12). Adjacent rod 05
`were
`structurally intact or were nrtifactually
`distorted by olefin produced during in vivo perfu-
`
`sion fixation. In other cones, lamellae were inter-
`rupted by formation of vesicles and tubules con-
`taining irregularly granular dense particles within
`some of
`them (Fig. 13). The enuclented eye
`taken after 21 months from the second monkey
`with
`an
`extinguished
`b-wave
`revealed more
`advanced OS disruption with seemingly equal
`
`

`

`Volume 13
`Number 7
`
`Retinal degeneration in monkeys 507
`
`
`
`Fig. 12. A degenerating cone outer segment (COS) is visible in this section from the mid-
`peripheral
`retina of a vitamin A-deficient monkey. Numerous
`lamellae have developed
`vesiculation typical of this deficiency. Adjacent rod outer segments appear normal except
`for cleft: due to fixation artifact. [x9,800).
`Fig. 13. Another cone outer segment in the mid-peripheral retina from the same vitamin
`A—deficient monkey has lost most
`lemellae. The proximal portion contains vesicles packed
`with irregular granules. (x9500).
`
`electron
`in
`and cones
`rods
`involvement of
`the paramacular region
`microscopic sections of
`(Fig. 14). The pigment epithelium contained an
`extraordinary number of partially digested cyto-
`plasmic lipid droplets, but was otherwise normal
`(Fig. 15). Phagosomes were routinely observed,
`and melanin granules were located in their usual
`apical position. Panophthahnitls of the other eye
`
`in this monkey resulted in total dissolution of all
`retinal layers.
`
`Discussion
`
`These descriptions serve to emphasize
`that prolonged vitamin E deficiency in at
`least
`two species of monkeys can result
`
`

`

`508 Hayes
`
`[medication Ophthalmology
`July 1974
`
`
`
`
`.Jd‘» "
`
`Fig. 14. In more advanced vitamin A deficiency rod outer segments were also disrupted as
`evidenced by these tips from three outer segments adjoining the pigment epithelium (PE)
`in the paramacular refine. (x12,700).
`Fig. 15. Pigment epithelium from the same vitamin A-deficient monkey contains an abnormal
`number of lipid-laden lymsomee. (x8,700).
`
`in retinal damage having certain similarities
`and dissimilarities to those produced with
`insufficient vitamin A. On the one hand,
`protracted vitamin A depletion in adult
`monkeys produced classical signs of de-
`ficiency including xerophthalmia and kera-
`
`tomalncia. Ru
`
`of the cornea resulted
`
`in destructive panophthalmitis in one mon-
`key. These monkeys demonstrated clinical
`evidence of impaired vision prior to ad-
`vanced ophthalmitis and the EEG b-wave
`from one of them was undetectable.
`In
`
`

`

`Volume 13
`Number 7
`
`Retinal degeneration in monkeys
`
`509
`
`the first case light microscopy revealed
`relatively moderate to minor disruption of
`OS, primarily restricted to photoreceptors
`in the macula and paramacular
`retina.
`Electron microscopy suggested that
`the
`earliest OS damage, in the mid-peripheral
`retina examined, predominated in cones.
`Both rods and cones appeared damaged
`in the macula and in the surrounding retina
`of the more advanced lesion. Degeneration
`of the ONL and numerous lipid-laden lyso-
`somes
`in the pigment epithelium were
`the only other changes observed.
`On the other hand, vitamin E deficiency
`resulted in no signs of deficiency disease
`except the malaise and pallor accompanying
`severe anemia, and this only in monkeys
`fed the polyunsaturated fat. Retinal de-
`generation, however, occurred in animals
`fed both types of fat following an extra-
`ordinarily long depletion period. This de-
`generation was not manifest by clinical
`evidence of blindness or a change in b-
`wave threshold. Like vitamin A deficiency,
`the initial lesion was obsered in the photo-
`receptor OS. Unlike vitamin A deficiency
`it was restricted to the macula and more
`
`totally disrupted the affected OS. The in-
`tact retina surrounding the macula could
`explain the finding of a normal full-field
`ERG b-wave threshold.
`
`These differences reveal something of
`the pathogenesis of
`the lesions. From
`knowledge of vitamin A function in vision,
`it is predictable that depletion should re-
`sult in uniform loss of the visual pigments,
`rhodopsin and iodopsin,
`throughout
`the
`retina and thereby disrupt
`the electro-
`physical aspects of vision followed by struc-
`tural dismantling of the OS themselves.11
`It is interesting that peripheral cones and
`the cone-rich macula appeared to degener-
`ate first, a finding previously reported in
`monkeys.” Even in the rod-dominant reti-
`nas of vitamin A-deficient rats and guinea
`pigs, the central area of the retina is first
`to degenerate“) 1" The observation that
`cone structure may be more critically af-
`fected by the lack of vitamin A than that
`of rods is surprising since cone visual pig-
`
`ments are synthesized more rapidly than
`those of rods,H and cones have a much
`
`greater affinity for retinaldehyde.15 Further-
`more, psychophysical testing in man sug-
`gests that the functional correlates of the
`two photoreceptors are equally sensitive
`to the night blindness of vitamin A de-
`pletion.“" 1’ Perhaps the structural integrity
`of the cone is more difficult
`to maintain
`due to its slower
`turnover
`rate.18 Cone
`function and structure have been found
`altered in the cone-dominant retina of the
`
`ground squirrel made vitamin A deficient.”
`The function of tocopherol in the retina
`is not known, but it has been isolated from
`bovine OS7 and might be required as a
`lipid antioxidant for the extraordinary con-
`centration of polyunsaturated fatty acids
`found in the OS.20 This hypothesis is en-
`hanced by the vulnerability of the visual
`cell to x-irradiation, oxygen poisoning, and
`excessive light, all of which are manifest
`by disruptive oxidizing reactions in this
`delicately balanced
`structure.“ 22 One
`should also consider
`the possibility that
`vitamin E may protect retinaldehyde in
`the retina from peroxidative destruction;
`however,
`if true, the retinal degeneration
`and visual impairment measured by ERG
`might be expected to resemble vitamin
`A deficiency more closely.
`The susceptibility of the macula in vita-
`min E deficiency would again emphasize
`the peculiar character of this area, perhaps
`as a function of its cone density and the
`metabolic or structural correlates specific
`to this photoreceptor. It was not determined
`whether cone damage preceded or was
`more extensive than rod disruption in this
`deficiency, as both appeared equally in-
`volved. The focal, massive degeneration
`of retinal OS in vitamin E deficiency was
`in contrast to the less disruptive disman-
`tling and progressive shortening of OS in
`vitamin A deficiency. The remarkable ac-
`cumulation of lipofuscin pigment
`in the
`pigment epithelium of the vitamin E-de-
`ficient monkeys is
`identical
`to that pre-
`viously described in dogs3 and suggests
`that
`the pigment epithelium is capable
`
`

`

`510 Hayes
`
`Investigative Ophthalmology
`July 1 974
`
`of extreme phagocytic activity and lyso-
`somal digestion in this deficiency state.
`Whether the retinal degeneration induced
`by tocopherol deficiency has a counterpart
`in any other species is unknown. It is note-
`worthy that a previous description of pro-
`longed vitamin E deficiency in dogs was
`associated with extensive retinal degenera-
`tion3 and that children with abetalipopro-
`teinemia have remarkably low circulating
`levels of tocopherol. The retinal degenera-
`tion found in these children can be in-
`
`fluenced by vitamin A.”3 Whether vitamin
`E is
`limiting has not been determined.
`In any event,‘ further work is needed to
`explore the role of antioxidants in photo-
`receptor function and structure.
`
`Grateful acknowledgment is given Antonio Ruiz
`for his care of the animals. I am grateful to Tom
`Faherty, Barbara Burgess, and Claudia Starr for
`their diligent microscopy and photographic ex-
`pertise and to Arnold Rabin for his help with
`the electroretinographic measurements. Thanks are
`also due Ann Blanchard for preparation of
`the
`manuscript.
`
`REFERENCES
`
`1. Ferguson, T. M., Rigdon, R. H., and Couch,
`J. R.: Cataracts
`in vitamin E deficiency.
`An experimental study in the turkey embryo,
`Arch. Ophthalmol. 55: 346, 1956.
`2. Devi, A., Raina, P. L., and Singh, A.: Ab-
`normal protein and nucleic acid metabolism
`as a cause of cataract formation induced by
`nutritional deficiency in rabbits, Br. J. Oph-
`thalmol. 49: 271, 1965.
`3. Hayes, K. C., Rousseau, J. E., Jr., and Heg-
`sted, D. M.: Plasma tocopherol concentrations
`and vitamin E deficiency in dogs,
`J. Am.
`Vet. Med. Assoc. 157: 64, 1970.
`4. Kayden, H.
`J.: Abetalipoproteinemia. Ann.
`Rev. Med. 23: 285, 1972.
`5. Hayes, K. C.: Pathophysiology of vitamin E
`de ciency in monkeys, Am.
`J. Clin. Nutr.
`In press.
`6. Zeman, W.: Studies in the neuronal ceroid-
`lipofuscinosis.
`J. Neuropathol. Exp. Neurol.
`33: 1, 1974.
`7. Dilley, R. A., and McConnell, D. G.: Alpha-
`
`in the retinal outer segment of
`tooopherol
`bovine eyes, J. Mem. Biol. 2: 317, 1970.
`Ausman, L. M., and Hayes, K. C.: Vitamin
`E deficiency anemia in Old and New World
`monkeys, Am. J. Clin. Nutr. In press.
`J.: A
`Hansen, L. G., and Wamick, W.
`fluorometric micro—method for
`serum vita-
`min A, Am. J. Clin. Pathol. 50: 525, 1968.
`Rabin, A. R., Hayes, K. C., and
`erson, E.
`L.: Cone and rod responses in n tritionally
`induced retinal degeneration in the cat,
`IN-
`VEST. OPHTHALMOL. 12: 694, 1973.
`Dawling,
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