the corneal and conjunctival surface in vitamin a deficiency: a

The corneal and conjunctival surface invitamin A deficiency:

a scanning electron microscope studyRoswell R. Pfister and Mark E. Renner

A model system of acute vitamin A deficiency in the guinea pig was used to investigate changesin corneal and conjunctival morphology. Hypovitaminosis A was induced by feeding experi-mental animals a purified gel diet deficient in vitamin A, while a control group received avitamin A enriched diet. By the fifth week experimental animals exhibited a significant decreasein plasma vitamin A levels compared to controls. Animals were sacrificed at intervals from 5 to9 weeks, and the corneas and conjunctivae were examined histologically and by scanningelectron microscopy. Conjunctivae from control animals showed large numbers of goblet cells,whereas 16 of 18 experimental conjunctivae were completely devoid of goblet cells. Tarsal,palpebral, and cul-de-sac epithelia from deficient animals all showed many superficial squa-mous cells uplifted from the surface. Also, rugae were notably absent from the palpebral andcul-de-sac conjunctivae. The corneas of vitamin A-deficient animals exhibited varying degreesof superficial epithelial cell desquamation and keratinization. In addition, reduced numbers ofmicroprojections and membrane breakdown were observed. These morphological and clinicalfindings are correlated with the possible role of vitamin A in cellular physiology.

Key words: vitamin A deficiency, cornea, conjunctiva, guinea pigs,scanning electron microscopy

A the major histological characteris-tics of vitamin A deficiency in the externaleye are corneal and conjunctival keratiniza-tion,1"3 with disappearance of conjunctivalgoblet cells.1* 4> 5 Decreases or absence ofsurface microprojections have been shownwith transmission electron microscopy.3 Allthese findings have depended on the evalua-tion of limited numbers and sizes of sectionsobtained from small pieces of tissue. The ac-curacy of light and transmission electron mi-croscopy studies alone is contingent upon the

From the Combined Program in Ophthalmology, Uni-versity of Alabama in Birmingham-Eye FoundationHospital, Birmingham, Ala.

Supported by National Eye Institute grants EY 01913-01, EY 02018-01 and Ellen Gregg Ingalls Eye Re-search Institute, Birmingham, Ala.

Submitted for publication Dec. 30, 1977.Reprint requests: Roswell R. Pfister, M.D., 1720—8th

Ave., South, Birmingham, Ala. 35233.

uniformity of the disease process. It is impor-tant to gain an over-all perspective of the ex-ternal ocular surfaces in vitamin A deficiencyby a technique capable of examining the en-tire cornea and conjunctival epithelium.

It is the purpose of this study to describethe scanning electron microscopy (SEM) ap-pearance of the corneal and conjunctival sur-faces in an established acute vitamin Adeficiency model in the guinea p ig 6 Addi-tional determinations of plasma vitamin Alevels and light microscopy help to evaluatethe results.

Materials and methods

Fourteen healthy, weanling, male, albino Hart-ley guinea pigs, weighing 175 to 250 gm, wereplaced on various dietary regimens. The experi-mental group of 10 animals was fed a purified geldiet7 deficient in vitamin A. Two control groups ofanimals were utilized. Two animals were fed a pur-ified gel diet, with vitamin A, 28.5 mg/kg, and two

874 0146-0404/78/0917-0874801.00/0 © 1978 Assoc. for Res. in Vis. and Ophthal., Inc.

Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933080/ on 04/12/2018

Volume 17Number 9 Corneal, conjunctival surface, vitamin A deficiency 875

CONTROLS

EXPERI MENTALS

TIME (WEEKS)

lo .

0 0

n-IOxn-4

CONTROLS

EXPERI MENTALS __.

X ^1 1 1 1

n - 3

n-7

TIME (WEEKS)

Fig. 1. Comparisons of animals fed vitamin A-deficient diet and control diets. Upper: Themean weights of the animals fed the vitamin A-deficient diet are slightly less than those of thecontrols, although the differences are statistically insignificant. Lower: In contrast, the meanserum vitamin A levels for the group fed the vitamin A-deficient diet are significantly differentfrom the control group at weeks 4 and 6. The differences in the initial vitamin A levels areinsignificant. Circle represents the mean, brackets indicate standard error of the mean, n - 1weighted.

animals were fed a standard laboratory feed(Purina Guinea Pig Chow) which contained vita-min A at an approximate concentration of 10.3mg/kg. The amount of vitamin A in the standardfeed is only approximate because of the variationof vitamin A in the natural ingredients, the age ofthe feed, and other factors.

All animals received water ad lib, supplement-ed with ascorbic acid (0.5 gm/L) throughout theexperiment. The animals were weighed and exam-ined at weekly intervals for up to 9 weeks to ascer-

tain general health and ocular appearance by slit-lamp microscopy. Plasma vitamin A levels weremeasured on days 7, 30, and 43. The assay forvitamin A employed the silicic acid chromatog-raphy and spectrofluorometric method of Garry etal.8 Blood samples were collected in heparinizedcapillary tubes according to the method of Lopezand Navia.9

Animals were sacrificed during weeks 6, 7, 8,and 9. One guinea pig on the deficiency diet wasfound dead in his cage in week 5 and was dis-


876 Pfister and RennerInvest. Ophthalmol. Visual Set.

September 1978

Fig. 2. A, Mucocutaneous junction of lids from guinea pigs in the control group shows minimalnumbers of keratinized, desquamating cells (arrows). Tarsal conjunctiva is normally smoothand flat; palpebral and cul-de-sac conjunctiva demonstrates large rugae. All conjunctival sur-faces show minimal surface cell desquamation. B, Mucocutaneous junction of a lid from a6-week vitamin A-deficient guinea pig showed large collections of keratinized epithelial cells(arrows). Loss of rugae in palpebral and cul-de-sac conjunctivae is accompanied by largenumbers of keratinized desquamating epithelial cells from all conjunctival surfaces. T, Tarsalconjunctiva; P, palpebral conjunctiva; C, cul-de-sac conjunctiva.

carded from the experiment. A second animal inthe deficient group was in a cachectic state whenkilled. Animals showing the extremes of clinicalappearance at given time periods were taken sothat the entire range of the clinical spectrum wasobtained at a given sampling. For example, twoanimals in generally poor health and one animal ingood health were taken in weeks 6 and 7.

Prior to death, each eye was examined by slit-lamp microscopy. Guinea pigs were sacrificed byan intraperitoneal injection of sodium pentobarbi-tal. The corneas and conjunctivae were im-mediately irrigated with 4% glutaraldehyde in0.1M Sorensen's phosphate buffer at room tem-perature. The lids were sutured closed, the orbitexenterated, and the globe slit open equatorially.The tissues were immersed in 4% glutaraldehydefor 1 hr and were dissected to remove upper lids,lower lids, and cornea. Pieces of these tissueswere then processed for conventional histology.The bulk of the tissue was examined for surface

appearance with the scanning electron microscope.Subsequent processing techniques for electronmicroscopy have been previously described.10

SEM was performed on an ETEC Autoscan.

Results

General clinical. All animals exhibitednormal health, full coats, and similar weightgains over a period of 4 weeks. Althoughweight gain in the vitamin A-deficient groupwas less than in the control group from 4 to 7weeks, this difference was not statisticallysignificant (p > 0.5 at week 6, t test compari-son of the means) (Fig, 1). No hair loss oc-curred in control animals, but five of ninevitamin A-deficient animals demonstratedalopecia (the tenth animal died in week 5 andwas omitted from the study). Two deficientanimals had periocular hair loss (spectacle



Fig. 3. A, Control comea. A smooth, continuous sheet of flat polygonal epithelial cells is found.B, Seven-week vitamin A-deficient cornea. The entire surface layer of cells shows extensiveseparation and desquamation from the epithelial sheet. C, Control conjunctiva. Numerousgoblet cells (arrowheads), in various stages of maturation, stud the surfaces of palpebral andcul-de-sac conjunctiva. D, Six-week vitamin A-deficient conjunctiva. Keratinized epithelialcells show extensive separations and desquamation frotn the surface epithelial sheet. The totalabsence of goblet cells is a striking finding.

eye). Narrowing of the interpalpebral fissures(squinting) developed in five of nine deficientanimals. Rectal prolapse was present in threedeficient animals, of which one developedbloody diarrhea.

Ocular examinations. The control eyeswere normal throughout the experiment.

Ocular changes were confined to the vitaminA-deficient group. At the time of death, 36%of these eyes showed an obviously dull, dryappearance to the corneal surface, with cellu-lar debris in the precorneal tear film and atthe lid margins. Of the eyes, 71% exhibitedpunctate corneal irregularities, with diffuse


878 Pfister and RentierInvest. Ophthalmol. Visual Sri.

September 1978

Fig. 4. A, Control conjunctiva. A fine carpet of microprojections, primarily microvilli, texturethe flat, polygonal cells of the epithelial sheet. The numerous microvilli obscure the junctions(arrowheads) between cells. The orifice (arrow) probably represents an opening to a restinggoblet cell. B, Six-week vitamin A-deficient conjunctiva. Smaller numbers of stubby,moundlike, irregular microprojections (arrows) are common on the surface cells. Broad, reticu-late microplicae (arrowheads) are common. The cell margins are readily apparent, often sepa-rated from contiguous cells.

nebular opacities developing in 21%. Athickened horizontal white band appeared inone eye. No Bitot spots were noted.

Vitamin A levels. At the beginning of theexperiment there were no statistically signif-icant differences between the control and vi-tamin A—deficient animals with respect toplasma vitamin A levels (Fig. 1). By weeks 4and 6, vitamin A-deficient animals showedstatistically significant decreases in plasma vi-tamin A levels compared to controls (p <0.01 at both 4 and 6 weeks, t test comparisonof the means).

SEM of the conjunctivaControls. A small amount of epithelial cel-

lular debris was present at the mucocutane-ous junction of the lid margin (Fig. 2). Tar-sal conjunctival epithelium appeared as asmooth, regular surface with a few des-quamating surface cells, but no goblet cells.

A prominent rugose appearance of palpebraland cul-de-sac conjunctiva was evident. Gob-let cells in various stages of maturation werepresent in great profusion in palpebral andcul-de-sac conjunctiva (Fig. 3). The polygonalsurface cells and goblet cells were tightly ad-joined to one another in a contiguous sheet.Fine microprojections, primarily microvilli,were present on surface epithelial cells, al-though they were shorter on tarsal epithelialcells {Fig. 4).

Vitamin A-deficient animals. Large ac-cumulations of keratinized epithelial cellsand debris were found attached to themucocutaneous junction, partially extendingposteriorly over the tarsal conjunctiva (Fig.2). Tarsal conjunctival epithelium showed ex-tensive cellular separations and desquama-tion (Figs. 3 and 4). Gross appearance of pal-pebral and cul-de-sac epithelium was that of a



Fig. 5. Distribution of presumed bacterial forms in 6-week vitamin A-deficient eyes. A,Conjunctiva. Cocci (arrowheads) in this specimen formed a netlike distribution in the valleys offine wrinkles of the surface. B, Cornea. Bacilli (arrowheads) in this specimen were largelyconfined to severely degenerating cells which were distributed randomly over the cornea.

flat surface with only fine wrinkling. Moststriking was the complete absence of any gob-let cells in the conjunctivae of 16 out of 18vitamin A-deficient eyes (Fig. 3). Con-junctivae from both eyes of one vitaminA-deficient animal showed a normal com-plement of goblet cells. This animal, sac-rificed at 4 weeks, showed the highest plasmavitamin A level in the entire vitamin A-deficient group, a level which was not sig-nificantly below the level in the controlgroup. Most surface cells showed smallernumbers of stubby, moundlike, irregularmicroprojections, some even with wide mi-croplicae (Fig. 4). Many spherules 1.2 fim indiameter, and rods 2 fim in length, pre-sumed to be bacteria, were widely dispersedon the surfaces of cells in five of 18 eyes (Fig.5). These spherules and rods were found asclusters or in netlike distributions in the de-pressions of fine wrinkles. No attempt wasmade to identify bacterial species. No clinicalinfections were noted.

SEM of the corneaControls. The typical surface appearance

was that of flat polygonal cells with very fewdesquamating cells (Fig. 3). Cellular micro-projections were primarily microvilli without

evidence of plasmalemmal disruption (Fig.6). Holes were present in some surfaceepithelial cells but with much less frequencythan that noted in the rabbit cornea.10 Inter-cellular junctions were obscured by numer-ous cellular microprojections. No bacteriawere found on the corneal surface of any con-trol eyes.

Vitamin A—deficient animals. All corneasshowed extensive areas of surface epitheilialcells lifting off the cornea. Many corneasexhibited cellular disruption over the entiresurface sheet. Most cell junctions were sepa-rated, liberating the cells from the sheet (Fig.3). The surface of epithelial cells demon-strated up to four major morphologies: (1)large, moundlike, irregular microprojectionsdistributed sparsely over the plasmalemma;(2) broad, netlike microplicae; (3) nearlysmooth plasmalemma; and/or (4) uncom-monly, normal microprojection patterns. Sixof 18 vitamin A—deficient corneas showedmany surface bacteria. Bacteria clustered al-most exclusively on or in disintegrating sur-face epithelial cells (Fig. 5). The most severeexample of corneal keratinization occurred inone animal presenting a bandlike keratopathy(Fig. 7). The irregular surface was composed


880 Pjister and RennerInvest. Ophthalmol. Visual Sci.

September 1978

Fig. 6. A, High-magnification view of the control corneal surface discloses the high density offine microprojections, tightness of intercellular junctions, and plasmalemmal integrity. B, Incomparison, this micrograph of a vitamin A-deficient cornea shows three abnormal surfacemorphologies. Sparse numbers of moundlike, misshapen microprojections (arrows) are notedon the irregular surfaces. Netlike, broad microplicae (arrowheads) or no surface microprojec-tions (asterisk) are present in two other cells. Surface cells frequently were separated from oneanother and lifting off the epithelial sheet.

of keratin, inflammatory cells, and amor-phous debris.

Light microscopyControl and vitamin A-deficient corneas.

Control corneas showed five to seven layersof cells with normal orientation and appear-ance of basal, wing, and squamous cells.Keratocytes populated the stroma lying be-tween tightly packed collagen bundles (Fig.8). The epithelium of vitamin A-deficientcorneas lacked the columnar basal cell ap-pearance, was slightly thinner than normal,but possessed superficially keratinized cells.A variety of lymphocytes, polymorphonu-clear leukocytes, macrophages> and buddingcapillaries populated the anterior stroma.

Control and vitamin A-deficient conjunc-tivae. The epithelium from control conjunc-tiva was comprised of three or four layers ofcells with many interspersed goblet cells.Thickening of the keratinized epithelium ofthe vitamin A-deficient conjunctivae waslargely attributable to the development of

Fig. 7. Low-magnification scanning micrograph ofthe temporal half of the cornea of one animal, sac-rificed after 7 weeks on vitamin A-deficient diet,reveals the bandlike distribution of keratin (ar-rows). Epithelial regions superior and inferior (as-terisks) to the bandlike region also show the cellu-lar separation and desquamation which are charac-teristic of vitamin A deficiency P, Palpebral con-junctiva.


Volume 17Number 9 Cornea!, conjunctival surface, vitamin A deficiency 881

Fig. 8. Light microscopy comparisons between control and vitamin A-deficient cornea andconjunctiva reveal changes in epithelial and stromal cytology and morphology. All micrographsrepresent the same magnification except where noted. A, Typical control cornea shows thenormal appearance of basal, wing, and squamous epithelial cells. Many keratocytes are foundin the anterior stroma. B, In the 6-week vitamin A-deficient corneas the columnar appearanceof the basal epithelium was lost, with keratinizing epithelium at the surface. Although mostepithelia were slightly thinner than normal, this specimen shows a large accumulation ofkeratin, inflammatory cells, and amorphous cellular debris portrayed in Fig. 7. Manyinflammatory cells are found in the anterior stroma. Two cysts (arrows) are located in thesuperficial epithelial layers. C, Epithelium from control conjunctiva shows three or four layersof cells with goblet cells (arrows) interspersed (see inset). D, Six-week vitamin A-deficientconjunctiva shows epithelium with superficial keratinization and rete peg formation (arrows)where the thickness is twice normal.

large, rounded rete pegs bulging down intothe underlying stroma.

Discussion

The low plasma vitamin A levels found inthe experimental group of guinea pigs con-form to an established vitamin A-deficientmodel.6 The dull, dry corneal surfaces notedclinically corresponded to the extensive epi-thelial disruption, lifting, and desquama-tion of surface epithelial cells noted by SEM.The appearance of these cells, with promi-

nent and often separated intercellular junc-tions, desquamation, membrane dehis-cences, and infrequent stubby surface micro-projections, was similar to that of keratinizedcells in normal and lichenified skin.11' 12 Thecorneal surface abnormalities were noted inall deficient animals, even the deficientguinea pig with a normal complement of gob-let cells and the highest plasma vitamin Alevel of that group. By chance, this one ani-mal was sacrificed at a time when the cornealchanges had already developed but no loss of


882 Pfister and RennerInvest. Ophthalmol. Visual Sci.

September 1978

conjunctival goblet cells had occurred. Thissuggests that corneal epithelial keratinizationand disruption precede conjunctival gobletcell loss in vitamin A deficiency. Light mi-croscopy confirmed the epithelial atrophyand keratinization present in the cornea dur-ing the early stages of the disease. Laterkeratin thickening in one case resulted in abandlike keratopathy. This study supportsthe contention that keratinization of epithe-lial tissues is the earliest primary finding invitamin A deficiency.1'2

The most dramatic change was the com-plete absence of goblet cells from con-junctivae in the entire group of deficientanimals after 7 weeks of diet and in all buttwo eyes of the one animal sacrificed after 4weeks of this diet. It suggests that the smallsections of these tissues, previously exam-ined by light microscopy, were an accuratereflection of the state of the entire tissue.1"5

Small biopsies from such generalized sys-temic conditions as pemphigoid and Stevens-Johnson syndrome also show severe de-creases in conjunctival goblet cell popula-tions. The loss of goblet cells from humanalkali-burned conjunctiva, the result of lo-calized chemical injury, may be more re-gional. 13

The presence of presumed bacteria on fiveof 18 corneas in vitamin A-deficient animals,but on none of eight controls, supports thehigher frequency of bacterial contaminationof the corneal surface.2' 3 Bacteria localized toseverely degenerated cellular areas may indi-cate a tendency for trapping in irregular cel-lular surfaces or in highly nutritive areasand/or ability to destroy cells with whichthey are in contact. In vitamin A deficiencycorneal ulcers and keratomalacia may resultfrom the presence of these bacteria, multiplebreaches in the surface epithelium, and coex-isting protein energy malnutrition (PEM).4'14

Although many factors are undoubtedlyinvolved, the presence of corneal ulcers andkeratomalacia in some rat studies2' 3> 5 but notin others1 could be accounted for on the basisof differing degrees of PEM. Species differ-ences between rat and guinea pigs, as well as

similar weight gains of the control and de-ficient guinea pigs in this study, might sug-gest very mild degrees of PEM in our ex-perimental animals. This could explain thelack of corneal ulceration and keratomalacianoted in this experiment. The changes in thisstudy noted on SEM probably representthose of vitamin A deficiency primarily andare consistent with the changes noted byWolbach and Howe1 and Mori.2

Animals fed a vitamin A-deficient diet andmaintaining good health exhibited cornealand conjunctival changes indistinguishablefrom those of animals in poor health. Thechanges shown in this study therefore appearto be specific and not related to the generalhealth of the animal. Surface epithelialchanges such as microvillous loss and plasmamembrane dehiscences, typical of drying ef-fects15' 16 as well as vitamin A deficiency,3

were present in this study. This study did notexamine blinking patterns or lagophthalmos,factors which could bear on the surface epi-thelial integrity.

Although the role of vitamin A in the visualexcitation process is well accepted, no suchunanimity of opinion exists for its otherbiochemical roles. Evidence now favors thedirect action of vitamin A as a mannose—carrying lipid intermediary in glycoproteinsynthesis,17 in the protein synthetic pro-cesses by rough endoplasmic reticulum,18 indirect membrane activity, and as an electrondonor in biochemical reactions.19 The markeddecrease in intestinal goblet cells18 and totalloss of goblet cells of conjunctiva noted in thisstudy as well as others5'20 are given biochem-ical support by the finding of diminution ofspecific glycopeptides from such tissues.21' 22

Although a wide variety of membrane effectshave been adduced, bilayer lipid models oflecithin and vitamin A suggest an interactionbetween polar groups sufficient to causestructural membrane deformation.23

Plasma membrane alterations and disrup-tion may therefore be caused indirectly by adecrease in glycoprotein synthesis and di-rectly by physical chemical membrane alter-ations. The altered membrane morphology



observed in this study may represent thesephysical chemical and biochemical conse-quences of vitamin A deficiency.

In vitamin A deficiency, mucus productionfrom goblet cells and complex mucus-secret-ing glands of the eye is severely reduced.Whether or not aqueous tear production isreduced in vitamin A deficiency is debat-able.4' 20 Taken together with the abnormalcorneal and conjunctival surfaces shown inthis study, the rapid tear film breakup notedin vitamin A-deficient patients is not sur-prising.

Finally, there is a serious problem of uni-formity in systems investigated for vitamin Adeficiency. We suggest that standard use ofNavia's defined diet7 (which can be modifiedfor rabbits and rats as well as guinea pigs) anda reliable vitamin A assay system will pro-mote both the accuracy and precision in vi-tamin A deficiency research.

We are grateful to Drs. Suzanne Harris, Hady Lopez,and Juan M. Navia for their expert advice and assistance.We also thank Ms. Patty Parimore for her assistance withthe manuscript, Ms. Ruth Burns for typing the drafts,and Mr. Craig Luce for preparing the graphs.

REFERENCES1. Wolbach, S. B., and Howe, P. R.: Tissue changes

following deprivation of fat-soluble A vitamin, J.Exp. Med. 42:753, 1925.

2. Mori, S.: Primary changes in eyes of rats whichresult from a deficiency of fat-soluble A in diet,J. A. M. A. 79:197, 1922.

3. Beitch, I.: The induction of keratinization in thecorneal epithelium, INVEST. OPHTHALMOL. 9:827,1970.

4. Sullivan, W. R., McCulley, J. P., and Dohlman,C. H.: Return of goblet cells after vitamin A therapyin xerosis of the conjunctiva, Am. J. Ophthalmol.75:720, 1973.

5. Jayaraj, A. P., Leela, R., and Rama Rao, P. B.:Studies on corneal mucous metaplasia in vitamin Adeficient rats, Exp. Eye Res. 12:1, 1971.

6. Harris, S. S., and Navia, J. M.: Effect of vitamin Adeficiency on calcium and glycosaminoglycan me-tabolism in guinea pig bone, J. Nutr. 107:2198,1977.

7. Navia, J. M., and Lopez H.: A purified gel diet forguinea pigs, Lab. Anim. Sci. 23:111, 1973.

8. Garry, P. J., Pollack, J. D., and Owen, G. M.:Plasma vitamin A assay by fluorometry and use of asilicic acid column technique, Clin. Chem. 16:766,1970.

9. Lopez, H., and Navia, J. M.: A technique for re-peated collection of blood from guinea pigs, Lab.Anim. Sci. 27:522, 1977.

10. Pfister, R. R.: The normal surface of corneal epithe-lium: A scanning electron microscopic study, IN-VEST. OPHTHALMOL. 12:654, 1973.

11. Brown, A. C , andGerdes, R. J.: Cell desquamationin inherited and acquired ichthyosis. In Johari, O.,and Corvin, I., editors: Scanning Electron Micros-copy, Chicago, 1974, ITT Research Institute, p.814.

12. Dawber, R. P. R., Marks, R., and Swift, J. A.:Scanning electron microscopy of the stratum cor-neum, Br. J. Dermatol. 86:272, 1972.

13. Ralph, R.: Conjunctival goblet cell density in normalsubjects and in dry eye syndromes, INVEST. OPH-THALMOL. 14:299, 1975.

14. Kuming, B. S., andPolitzer, W. M.: Xerophthalmiaand protein malnutrition in Bantu children, Br. J.Ophthalmol. 51:649, 1976.

15. Pfister, R. R., and Renner, M. E.: The histopathol-ogy of experimental dry spots and dellen in the rab-bit cornea: a light microscopy and scanning andtransmission electron microscopy study, INVEST.OPHTHALMOL. VISUAL SCI. 16:1025, 1977.

16. Levenson, J. E.: The effect of short-term drying onthe surface ultrastructure of the rabbit cornea: ascanning electron microscopic study, Ann. Oph-thalmol. 5:865, 1973.

17. DeLuca, L., Rosso, G., and Wolf, G.: The biosyn-thesis of a mannolipid that contains a polar metabo-lite of 15-14 C-retinol, Biochem. Biophys. Res.Commun. 4:615, 1970.

18. DeLuca, L., Little, E. P., and Wolf, G.: Vitamin Aand protein synthesis by rat intestinal mucosa, J.Biol. Chem. 244:701, 1969.

19. Wasserman, R. H., and Corradino, R. A.: Metabolicrole of vitamins A and D, Annu. Rev. Biochem.40:502, 1971.

20. Dohlman, C. H., and Kalevar, V.: Cornea inhypovitaminosis A and protein deficiency, Isr. J.Med. Sci. 8:1179, 1972.

21. Kim, Y. C. L., and Wolf, G.: Vitamin A deficiencyand the glycoproteins of rat corneal epithelium, J.Nutr. 104:710, 1974.

22. DeLuca, L., Schumacher, M., and Wolf, G.:Biosynthesis of a fucose-containing glycopeptidefrom rat small intestine in normal and vitamin Adeficient conditions, J. Biol. Chem. 245:4551, 1970.

23. Roels, O. A., and Shah, D. O.: Molecular interac-tions in lecithin-retinol monolayers, J. Colloid Inter-face Sci. 29:279, 1969.


the corneal and conjunctival surface in vitamin a deficiency: a

Documents