Vitamin a and Interstitial Retinol-Binding Protein in an Eye with Recessive Retiniris Pigmentosa

Vitamin A and Interstitial Retinol-Binding Protein in an Eye with Recessive Retiniris Pigmentosa

C. D. D. Bridges,* Stephen O'Gorman.y Shao-Ling Fong,* Richard A. Alvarez,* and Eliot Bersony

The composition and amount of vitamin A stored in the retinal pigment epithelium and choroid (RPE- Ch) was evaluated in postmortem donor eyes from a patient with retinitis pigmentosa that was probably inherited by an autosomal recessive mode. Additionally, the soluble proteins in the neural retina and RPE-Ch cytosols and interphotoreceptor matrix were examined collectively for the presence of interstitial retinol-binding protein (IRBP). Although there was depletion of the amount of vitamin A stored in the RPE, this was commensurate with the histopathologic findings on the RPE extent and thickness. No evidence was found for an accumulation of free retinol. Nearly all of the vitamin A stored in the RPE was esterified. As in normal eyes, the retinyl esters consisted mainly of palmitate mixed with a small proportion of stearate. Eleven-cis retinyl esters were present, although their proportion was lower than that reported for normals. IRBP could not be detected in stained gels of the soluble proteins, or by autoradiography of these gels after treatment with 125I-concanavalin A. These findings suggest that depletion of stored vitamin A, accumulation of free retinol, oir deficiency of 11-cis isomer are unlikely to be causative factors in the retinal degeneration examined here. Although the depletion of IRBP seen at this advanced stage might be secondary to the advanced loss of photoreceptors, the authors cannot rule out the possibility that a relative deficiency or abnormality in this protein at earlier disease stages may contribute to the pathogenesis of retinitis pigmentosa. Invest Ophthalmol Vis Sci 26:684-691, 1985

Vitamin A has been established as an essential transport.78 In early stages of this disease, photore- dietary factor for the preservation of normal photo- ceptor cell dysfunction can apparently be restored to receptor function and structure.1"3 Because of simi- normal with large doses of vitamin A9 and stabilized larities between the structural alterations of the retina over longer time periods with vitamin A and E in vitamin A deficient rodents and the alterations therapy.10 seen in histopathologic studies of retinitis pigmen- In the more typical forms of retinitis pigmentosa, tosa,4'5 there have been several attempts to establish however, serum retinol and retinol binding protein a link between the disease and anomalies in the (RBP) levels, and some properties of the RBP present, absorption, transport, delivery, or utilization of vita- have been reported to be comparable to those of min A. For example, a photoreceptor cell degeneration normal individuals."12 These findings, coupled with with some features in common with retinitis pigmen- failure to reverse the functional deficits of the disease tosa has been documented histologically in advanced, by vitamin A therapy,1314 make it unlikely that untreated stages of abetalipoproteinemia,6 a condition defects in the absorption of vitamin A or its delivery associated with inadequate chylomicron formation to the retinal and choroidal vasculature contribute to and consequent impairment of fat-soluble vitamin the pathogenesis of retinitis pigmentosa. Although electroretinographic15 and fundus reflectometry16 findings in humans with serum vitamin A deficiencies From the Cullen Eye Institute *Department of Ophthalmology, associated with, respectively, alcoholism or malab- Baylor College of Medicine, Houston, Texas and Berman-Gund sorption, differ from those seen in typical cases of Laboratory for the Study of Retinal Degenerations,! Harvard retinitis pigmentosa, there is no evidence to rule out Medical School, Massachusetts Eye and Ear Infirmary, Boston, the possibility that abnormalities in storage, metabo- Massachusetts. Supported by the Retina Research Foundation of Houston, lism, or transport of vitamin A within ocular tissues National Eye Institute grants EY-02014, EY-02489, EY-02520, contribute to the development of the pathologic al- National Retinitis Pigmentosa Foundation, Baltimore, Maryland, terations seen in retinitis pigmentosa. Indeed, in one and an unrestricted departmental grant from Research to Prevent eye from an individual with hereditary chorioretinal Blindness. degeneration similar to sector retinitis pigmentosa, a Submitted for publication: August 31, 1984. selective loss of 11-cis retinyl ester in the vitamin A Reprint requests: Dr. C. David Bridges, Department of Ophthal- 17 mology, Baylor College of Medicine, Houston, TX 77030. stores of the pigment epithelium was reported.

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We present here some recent studies on vitamin A and its putative interphotoreceptor matrix transport protein (IRBP)18"25 in the eye from a donor with retinitis pigmentosa. Preliminary reports of some of this work have been published.26'27

Materials and Methods

The morphologic and biochemical analyses described in this report were conducted on eyes from a 69-year-old white man with retinitis pigmentosa. His condition was probably inherited by an autosomal recessive mode because the family history revealed that the donor's maternal and paternal grandmothers were sisters. There was no history of retinitis pigmentosa in other members of the pedigree. The patient was first examined in the Electroreti- Fig. 1. Utilization of tissue from left eye (RP 179): see Materials nography Service of the Massachusetts Eye and Ear and Methods for description of how each numbered sector was Infirmary at age 58. He reported that symptoms of handled. night-blindness had developed at about 25 years of age, and at that time a diagnosis of retinitis pigmentosa 20/40 to 20/50 in the left eye. His dark-adaptation was first established. Between the ages of 37 and 54, threshold showed further elevation to 3.5 log units his visual acuity declined from 20/20 OD and 20/25 above normal in the right eye and 4.0 log units above OS to 20/40 OU, and at age 47, classic ring scotomas normal in the left eye. Over this period his central were first noted. The patient was aware of loss of visual field narrowed to the 8° isopter and the pe- peripheral vision but denied any difficulty with hearing ripheral crescents of field, though still present, were or history of polydactyly. diminished in extent as monitored with confrontation When first examined at the Massachusetts Eye and testing. Because he experienced difficulty with reading, Ear Infirmary, his best corrected visual acuity was his right cataract was removed without complication 20/50 OU. His refractive error was OD +6.00 -1.50 3 months prior to his death. Postoperatively, his X 165° and OS +6.00 -1.50 X 176°. Kinetic visual vision was reported to be 20/40 in that eye. field testing with a Goldman perimeter revealed a The donor died of liver failure due to metastatic concentric constriction to the 10° isopter with a IV- carcinoma. During the 2 years preceding his death 4e white test light OU, and a constriction to the 5° he was maintained on a chemotherapeutic regime isopter with a I-4e white test light. Large midperipheral that consisted of six series, lasting 5-8 weeks each, of ring scotomas were observed. The patient retained an weekly intravenous injections of 5-fluorouracil. During inferior crescent of field, approximately 10° in di- his final hospitalization his medications included ameter, both inferonasally and inferotemporally in indomethacin, oxazepam, cimetidine, prochloroper- each eye with a IV-4e white test light. Slit-lamp azine, furosemide, and oxycodon hydrochloride. Both examination revealed a slight nuclear sclerosis and eyes were enucleated approximately 45 min after small central posterior subcapsular cataracts OU. death. The right eye, designated as Berman-Gund Intraocular tensions by applanation were normal. Laboratory Specimen H-91, was immediately fixed Fundus examination revealed slight waxy pallor of in 1% formaldehyde and 2% glutaraldehyde in 0.1 M each disc, granularity of each macula, attenuation of sodium phosphate buffer pH 7.4 and was subsequently retinal vessels, and moderately heavy intraretinal postfixed in 2% osmium tetroxide in the same buffer, bone-spicule pigment distributed 360° around the dehydrated in graded ethanols, and embedded in midperiphery. Dark-adaptation testing with an 11 ° Epon (Ladd Research Industries, Inc; Burlington, white test light, centrally fixated, revealed a threshold VT). All quadrants of the eye were systematically elevated 2.5 log units above normal after 45 min of surveyed in semithin (1 /um-thick) sections stained dark-adaptation. Conventionally recorded, full-field with toluidine blue. Particular attention was paid to electroretinograms to single flashes of white light those areas that corresponded to the tissues of the under dark-adapted conditions and to 30 Hz white companion eye that were used for the biochemical flicker were not detectable (ie, less than 5 /iV). studies of this report. Thin sections from the fovea Over the next 10 years, the patient's visual acuity and from the midperipheral retina just superior to decreased to 20/70 in the right eye and remained at the temporal horizontal meridian were prepared and

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100,000 X g. The supernatant, which contained retinal 325 cytosol, interphotoreceptor matrix, and RPE-Ch cy- 0.002r tosol, was treated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) on 5-20% gradient gels according to Liou et al.19 The binding of 125I-concanavalin A to these gels 28 0L was carried out as described by Bridges and Fong. For comparison, sectors of similar area and location were cut from a normal donor eye and treated ^—U^~> identically. Sectors 2 and 3 consisted only of RPE- Ch and sclera (total sclera area, 3.0 cm2). The RPE- Ch was peeled away from the sclera and the vitamin 0 15 30 A extracted with acetone and quantitated by the Carr-Price reaction as described by Bridges et al.29 time (min) High-performance liquid chromatography (HPLC) of this extract was carried out using the equipment and Fig. 2. Occurrence of 11-cis retinyl ester in left eye (RPE-Ch, 30 sector 3, Fig. 1). Peak 1, 11-cis retinyl palmitate (peak) and stearate procedures described by Bridges and Alvarez. Data (shoulder); peak 2, all-trans retinyl palmitate (peak) and stearate from this analysis were compared with our published (shoulder). Columns, two 4.6-mm diameter, 25-cm long Ultrasphere data for normal donor eyes.17'29 The quantities of 11- 17 Si60 and Spherisorb CN in series ; mobile phase, 0.4% diethyl cis and all-trans esters were obtained by measuring ether in n-hexane; flow rate, 1.0 ml/min. The equipment was as the areas under the HPLC peaks (Fig. 2) and com- previously described.29 paring them with those obtained from standard mix- tures of authentic 11-cis and all-trans retinyl stearates and palmitates. Sectors 4 and 5 and the neural retina examined with a JEOL 100C electron microscope from sectors 2 and 3 are still being studied at the (Jeol; Tokyo, Japan). Berman-Gund Laboratory, with specific reference to The left eye was opened and dissected according a comparison of the in vitro cyclic nucleotide phos- to the plan presented in Figure 1. The tissues used phodiesterase activities and the protein synthetic ca- for the present study included the retina, RPE, choroid pacities of postmortem retinal tissues of normal donors and sclera of sector 1, and the RPE, choroid and and donors with retinitis pigmeniosa. sclera of sectors 2 and 3. These were frozen at —80°C within an hour of enucleation and shipped on dry Results ice to the Cullen Eye Institute, where they were designated RP 179; the biochemical analyses described Histopathology of the Right Eye here were initiated within 12 hr of the donor's death. The results of histologic examination of the right Sector 1 consisted of retina, RPE-Ch, and sclera eye were consistent with the clinical diagnosis of (scleral area, 0.86 cm2). The sclera was removed and advanced retinitis pigmentosa. The only substantive its area measured on a Zeiss Videoplan (Carl Zeiss, population of surviving photoreceptor cells was the Inc; New York, NY). The remaining combined tissues foveal cones (Fig. 3A). No photoreceptor cells could were homogenized in 0.15 M NaCl, 0.01 M Na be positively identified by morphologic criteria in the phosphate (pH 7.4; PBS) and centrifuged for 1 hr at near and midperiphery (Fig. 3B, C), and in the far

Fig. 3. Histopathologic features of the right eye. A, Fovea: photoreceptor cell loss reduced the thickness of the outer nuclear layer to a depth of two cell bodies. The inner segments of these cells were shorter than those present in normal tissue, and only short fragments of vesiculated outer segment material were present in the subretinal space. A thick preretinal membrane extended across the whole of the fovea, its depth at two sites in this micrograph is demarcated by the pairs of arrowheads. B, Midperipheral nasal retina: illustrating typical alterations of retinal structure in areas homologous to the tissue of the left eye that was used for the biochemical analyses. By morphologic criteria, photoreceptors could not be identified. The apical surface of the pigment epithelium had a highly irregular contour, but in other respects it was relatively normal in appearance. Both Bruch's membrane and the choriocapillaris were normal in appearance. C, Midperipheral nasal retina: intraretinal, bone-spicule pigment (P) was present throughout the posterior and midperipheral retina. In several areas of the midperiphery there were small gaps in the pigment epithelium (to left of arrow) where the neural retina directly abutted Bruch's membrane. D, Peripheral retina: the low density of atrophic photoreceptor cells is shown. Some of these cells are indicated by straight arrows. All inner segments of peripheral photoreceptors (curved arrows) were severely shortened and no outer segment material could be discerned. A layer of drusen-like material was present between Bruch's membrane and the basal surface of the pigment epithelium. Scale bar (all micrographs) = 100 urn.

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Table 1. Amount and composition of vitamin A where the outer limiting membrane of the neural stored in the RPE-Ch (sectors 2, 3) of the left eye retina directly abutted Bruch's membrane (Fig. 3C). (RP 179) compared with normal controls However, such areas constituted no more than 10% of the length of RPE present in any one section. In Normals* RP 179 the far periphery the RPE was continuous but thin, Vitamin A and a confluent layer of drusen-like material was nmol/cm2 0.55 0.28 present between the basal surface of the RPE and nmol/eye 7.9 ±4.5 (2.5)f Bruch's membrane (Fig. 3D). The choriocapillaris Fraction esterified 0.98 ± 0.26 1.0 All-trans stearate 18:0/all-trans appeared to be normal throughout the eye. palmitate 16:0 0.25 0.3 11-cis/all-trans 1.52 ±0.48 0.33 Vitamin A Content and Composition

* See Bridges et at.29 and Bridges and Alvarez.17 As shown by Table 1, vitamin A was present in t Based on an estimated total area. the RPE-Ch from sectors 2 and 3 of the left eye. The quantity per unit area was approximately half that of normal donor eyes, and the estimated amount for periphery the few atrophic photoreceptor cells present the whole eye was more than one standard deviation were separated from each other by large gaps in the lower than normal. Virtually all of the vitamin A was outer nuclear layer (Fig. 3D). At the center of the esterified, and the esters consisted mainly of retinyl foveal pit the outer nuclear layer was approximately stearate and palmitate in proportions similar to those two nuclei deep. The inner segments of these surviving found in normal eyes. No accumulation of free cones appeared to be shorter and broader than normal retinol was apparent. foveal cones and were tipped by short (1-7 nm long) Unlike the eye examined by Bridges and Alvarez,17 fragments of disorganized and vesiculated outer seg- there was evidence for only mild depletion of the 11- ment material. Photoreceptor cell density declined cis isomer. The presence of 11-cis retinyl stearate and abruptly along the foveal slope; at 1.0 mm peripheral palmitate in the RPE-Ch is shown in Fig. 2, using an to the foveal center the outer nuclear layer was HPLC system that was optimized for isomer separa- reduced to a single discontinuous row of photoreceptor tion. Two major peaks are evident. Peak 1 corresponds cell nuclei, the broadened inner segments of which to 11-cis retinyl palmitate: the shoulder on the leading barely protruded beyond the outer limiting membrane. edge is 11-cis retinyl stearate. Peak 2 corresponds to No outer segment material could be discerned at the all-trans retinyl palmitate: the presence of the all- light microscopic level. Recognizable photoreceptor trans retinyl stearate is apparent from the shoulder cells were completely absent from an area extending on the rising phase of peak 2. from 2.2 mm eccentric to the fovea to within 4.5 mm of the ora serrata throughout the superior hemisphere (Fig. 3B, C). Within this zone the outer margin Interstitial Retinol-Binding Protein (IRBP) of the neural retina was formed by what appeared to Figure 4 compares the SDS-gel pattern for the be Miiller cell processes and cells of the inner nuclear soluble proteins from the combined retina and RPE- layer. Anterior to this region, widely scattered pho- Ch (including any interphotoreceptor matrix) from toreceptor cells in advanced stages of atrophy were RP 179 with that from a comparable tissue sample present. Only a few of these cells had inner segments from a normal eye. While several proteins appear to that protruded beyond the outer limiting membrane, be depleted or absent in the sample from the dys- and there was no evidence for outer segment material trophic eye, the most noticeable is the 135K IRBP in the subretinal space (Fig. 3D). that has been purified and characterized from normal The appearance of the retinal pigment epithelium human interphotoreceptor matrix by Fong et al.2324 (RPE) paralleled the condition of the overlying pho- At the time these studies were carried out, antibovine 25 toreceptors. The foveal RPE was relatively normal in IRBP antiserum was not available. However, human appearance. The cells measured approximately 15 IRBP is a glycoprotein with a strong affinity for 24 nm in height and contained large numbers of lipo- concanavalin A, and we utilized this property to fuscin, melanin, and melanolysosome granules, and develop a sensitive technique for detecting its presence. The same gel was incubated with l25I-labeled conca- a few phagosomes. In the near periphery and mid- 19 periphery the RPE was hypopigmented and thinned navalin A. The autoradiogram showed that there (averaging approximately 7 /xm in height; Fig. 3B, was prominent binding of the lectin at the 135K C). In some sections through the superior hemisphere position in the track from the normal eye: none was there were small gaps in the pigment epithelium, evident in the track from the dystrophic tissue (data

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not shown) confirming its absence in the stained pattern. -3

Discussion Given the clinical evidence for symmetric disease 200 progression in this donor (see Materials and Methods), it is likely that the histologic findings in the right eye represent an accurate measure of the structural alterations present in the left eye. Assuming that this is the case, then the retina and RPE-Ch sample of sector 1 from the left eye (Fig. 1), used for biochemical 116 analyses, contained in its most peripheral segments a small population of photoreceptor cells that either 93 lacked or had severely shortened inner segments, but had no outer segment membranes. Sectors 2 and 3 were largely composed of thinned and hypopigmented 66 RPE from the near and midperipheral regions, to- gether with a smaller amount of better preserved RPE from the far periphery. Because the RPE was thinned in areas of the companion eye homologous to sectors 2 and 3 of the left eye, the total RPE cell volume per 45 unit of retinal surface area in these sectors was probably substantially reduced in comparison with that found in normal eyes. Considered in the light of the histopathologic findings, the reduction of total vitamin A stores in this 31 tissue per unit retinal surface area (Table I) would approximate what one would expect were there normal vitamin A concentrations in the remaining volume of RPE. It further appears that the end stages of 22 retinal disease in this individual were not associated with abnormalities of the forms of vitamin A stored within the RPE-Ch. Previous observations on the 14 Royal College of Surgeons (RCS) rat model of hereditary retinal dystrophy31 prompted the suggestion that in retinitis pigmentosa there might be a defect in the ability of the RPE to esterify retinol with consequent damage to photoreceptor cells by release of lysosomal enzymes32 (see Berman et al33 for further discussion of this point). However, the present results, since they Fig. 4. Absence of IRBP in the soluble proteins of left eye. Lane demonstrate that the vitamin A stored in the RPE of I, supernatant fraction from a centrifuged homogenate of retina this patient consisted almost entirely of retinyl esters and RPE-Ch combined (sector 1, Fig. I); lane 2, supernatant rather than retinol, do not support this hypothesis. A fraction from the combined retina and RPE-Ch from a comparable possible failure to isomerize all-trans to 11-cis retinoid sector obtained from a normal eye; lanes S, molecular weight standards. Arrow at Mr 135K on the right-hand side denotes the was first explored in a clinical trial by Chatzinoff et position of IRBP, which appears as a prominent band in lane 2. al.14 Eleven-cis vitamin A (the retinoid used was not reported) was administered intramuscularly to retinitis pigmentosa patients over a 3-year period. This group for review) casts doubt on the likelihood that in this was compared with a parallel group of patients that study 11-cis retinol would have survived to be deliv- had received the all-trans isomer. No beneficial effect ered to the RPE. More recently, this question was of 11-cis retinoid was found. However, the ease with raised again. Bridges and Alvarez17 found that while which the 11-cis isomer is converted back to all-trans the vitamin A in the RPE of a patient with a retinal 27 when dispersed in tissue preparations (see Bridges degeneration resembling sector retinitis pigmentosa34-35

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was essentially normal in amount, it was selectively Key words: retinitis pigmentosa, vitamin A, interstitial depleted in the 11-cis isomer. It could not be deter- retinol-binding protein, 11-cis isomer, esterification mined whether this depletion was a primary cause of degeneration or if it was secondary to loss of photo- Acknowledgments receptor cells, given the suggested involvement of photoreceptors in the formation of 11-cis isomer.36 The authors thank the Lions Eyes of Texas Eye Bank, E. J. Farge, and Bob Fort for providing normal donor eyes; The present findings, however, show in at least this Susan Y. Schmidt for preparing and shipping this tissue to one case of retinitis pigmentosa that, even when Houston; and William A. Flaherty for expert histologic photoreceptor cell degeneration has advanced to the assistance. point where only a small population of cones survive, there may be substantial amounts of 11-cis retinyl References ester in the RPE. 1. Dowling JE and Wald G: Vitamin A deficiency and night There is strong evidence that IRBP is involved in blindness. Proc Natl Acad Sci USA 44:648, 1958. the transport of retinol between the RPE and ret- 2. Dowling JE and Gibbons IR: The effect of vitamin A deficiency 18 2025 ina. " Deficiency or molecular alteration of IRBP on the fine structure of the retina. In The structure of the Eye, could therefore result in deficiencies of vitamin A Smelser GK, editor. New York, Academic Press, 1961, pp. within the neural retinal, despite the presence of 85-99. normal vitamin A stores in the RPE, with consequent 3. Berson EL: Nutrition and retinal degenerations: vitamin A, 12 taurine, ornithine, and phytanic acid. Retina 2:236, 1982. photoreceptor cell death. Alternatively, the defi- 4. Szamier RB and Berson EL: Retinal ultrastructure in advanced ciency of IRBP could result in the toxic accumulation retinitis pigmentosa. Invest Ophthalmol Vis Sci 16:947, 1977. of excessive free retinol in the photoreceptor layer 5. Kolb H and Gouras P: Electron microscopic observations of (rather than in the RPE), another mechanism that human retinitis pigmentosa, dominantly inherited. Invest might lead to photoreceptor cell death. It has been Ophthalmol 13:587, 1974. 6. von Sallmann L, Gelderman AH, and Laster L: Ocular histo- recently shown that IRBP can also bind a-tocoph- 25 pathologic changes in a case of abetailipoproteinemia (Bassen- erol. An impairment of vitamin E delivery to the Kornzweig syndrome). Doc Ophthalrnol 26:451, 1969. neural retina secondary to a deficiency of IRBP could 7. Salt HB, Wolff OH, Lloyd JK, Fosbrooke AS, Cameron AH, therefore deprive photoreceptors of adequate supplies and Hubble DV: On having no beta-lipoprotein: a syndrome of this important antioxidant.37 comprising a-beta-lipoproteinemia, acanthocytosis and steat- orrhea. Lancet 2:325, 1960. The involvement of IRBP in the pathogenesis of 8. DiGeorge AM, Mabry CC, and Auerbach VH: A specific retinal degenerations in animal models of hereditary disorder of lipid transport (acanthocytosis): treatment with retinal degeneration is as yet unclear. During the first intravenous lipids. Am J Dis Chil 102:580, 1961. three postnatal weeks of life the amount of IRBP in 9. Gouras P, Carr RE, and Gunkel RD: Retinitis pigmentosa in abetalipoproteinemia: effects of vitamin A. Invest Ophthalmol RCS rats increases in parallel with the elongation of 10:784, 1971. rod outer segments, but subsequently (as demonstrated 10. Bishara S, Merin S, Cooper M, Azizi E, Delpre G, and by immunocytochemical and biochemical methods) Deckelbaum RJ: Combined vitamin A and E therapy prevents it declines rapidly in amount.38"40 The appearance of retinal electrophysiological deterioration in abetalipoprotein- pyknotic photoreceptor cell nuclei in the outer nuclear emia. Br J Ophthalmol 66:767, 1982. 11. Futterman S, Swanson D, and Kalina RE: Retinol in retinitis layer precedes detectable decrements in IRBP levels, pigmentosa: evidence that retinol is in normal concentration and there is thus no evidence that abnormalities of in serum and the retinol-binding protein complex displays IRBP are responsible for the initial stages of photo- unaltered fluorescence properties. Invest Ophthalmol 13:798, receptor cell degeneration in these dystrophic rats. 1974. However, in RCS rats the retina ceases to synthesize 12. Maraini G, Fadda G, and Gozzoli F: Serum levels of retinol- binding protein in different genetic types of retinitis pigmentosa. and secrete IRBP when the photoreceptors have Invest Ophthalmol 14:236, 1975. degenerated, suggesting both that these cells are its 13. Bergsma DR and Wolf ML: A therapeutic trial of vitamin A source (see also Hollyfield et al41) and that its loss in in patients with pigmentary retinal degeneration: a negative the diseased human eye of the present study and the study. In Retinitis Pigmentosa, Landers MA, Wolbarsht ML, absence of 7S "receptor" for exogenous retinol (pos- Laties AM, and Dowling JE, editors. New York, Plenum Press, 42 1976, pp. 197-209. sibly identical to IRBP) seen in an earlier study was 14. Chatzinoff A, Nelson E, Stahl N, and Clahane A: Eleven-cis secondary to the degeneration of photoreceptor cells. vitamin A in the treatment of retinitis pigmentosa: a negative Future measurement of IRBP in earlier stages (with study. Arch Ophthalmol 80:417, 1968. more photoreceptor cells present) with more sensitive 15. Sandberg MA, Rosen JB, and Berson EL: Cone and rod immunological techniques might resolve whether a function in vitamin deficiency with chronic alcoholism and in retinitis pigmentosa. Am J Ophthalmol 84:658, 1977. deficiency or modification of this protein contributes 16 Ripps H, Brin K.P, and Weale RA: Rhodopsin and visual to the pathogenesis of some forms of retinitis pig- threshold in retinitis pigmentosa. Invest Ophthalmol Vis Sci mentosa. 17:735, 1978.

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17. Bridges CDB and Alvarez RA: Selective loss of 11-cis vitamin eyes: amount, distribution and composition. Invest Ophthalmol in an eye with hereditary chorioretinal degeneration similar to Vis Sci 22:706, 1982. sector retinitis pigmentosa. Retina 2:256, 1982. 30. Bridges CDB and Alvarez RA: Measurement of the vitamin A 18. Liou GI, Bridges CDB, and Fong S-L: Vitamin A transport cycle: Methods Enzymol 81:463, 1982. between retina and pigment epithelium: an interphotoreceptor 31. Reading HW: Retinal and retinol metabolism in hereditary matrix protein carrying endogenous retinol (IRBP). ARVO degeneration of the retina. Biochem J 100:34, 1966. Abstracts. Invest Ophthalmol Vis Sci 22(Suppl):65, 1982. 32. Burden EM, Yates CM, Reading HW, Bitensky L, and Chayen 19. Liou GI, Bridges CDB, and Fong S-L: Vitamin A transport J: Investigation into the structural integrity of lysosomes in the between retina and pigment epithelium: an interstitial protein normal and dystrophic rat retina. Exp Eye Res 12:159, 1971. carrying endogenous retinol (interstitial retinol-binding protein). 33. Berman ER, Segal N, Photiou S, Rothman H, and Feeney- Vision Res 22:1457, 1982. Burns L: Inherited retinal dystrophy in RCS rats: a deficiency 20. Adler AJ and Martin K.J: Retinol-binding proteins in bovine in vitamin A esterification in pigment epithelium. Nature 293: interphotoreceptor matrix. Biochem Biophys Res Commun 217, 1981. 108:1601, 1982. 34. Cope LA, Teeters VW, Borda RP, and McCrary JA: Sector 21. Adler AJ and Evans CD: Rapid isolation of bovine interpho- retinitis pigmentosa with chronic disc edema. Documenta toreceptor retinol-binding protein. Biochim Biophys Acta 761: Ophthalmologica. Proceedings Series, International Symposium 217, 1983. on Fluorescein Angiography. The Hague, Dr W Junk bv 22. Pfeffer B, Wiggert B, Lee L, Zonnenberg B, Newsome D, and Publishers, 1976, pp. 431-437. Chader G: The presence of a soluble interphotoreceptor retinol- 35. Rayborn ME, Moorhead LC, and Hollyfield JG: A dominantly binding protein (IRBP) in the retinal interphotoreceptor space. inherited chorioretinal degeneration resembling sectoral retinitis J Cell Physiol 117:333, 1983. pigmentosa. Ophthalmology 89:1441, 1982. 23. Fong S-L, Liou GI, Landers RA, Gonzalez-Fernandez F, and 36. Bridges CDB: Vitamin A and the role of the pigment epithelium Bridges CDB: Purification and partial characterization of an during bleaching and regeneration of rhodopsin in the frog interstitial retinol-binding glycoprotein from bovine interpho- eye. Exp Eye Res 22:435, 1976. toreceptor matrix: presence of a similar protein in human. Fed 37. Robison WG, Kuwabara T, and Bieri JG: The roles of vitamin Proc 42:2179, 1983. E and unsaturated fatty acids in the visual process. Retina 2: 263, 1982. 24. Fong S-L, Liou GI, Landers RA, Alvarez RA, Gonzalez- 38. Gonzalez-Fernandez F, Fong S-L, Liou GI, Landers RA, Lam Fernandez F, Glazebrook PA, Lam DMK, and Bridges CDB: The characterization, localization and biosynthesis of an inter- DMK, Glazebrook P, and Bridges CDB: A retinol-binding stitial retinol-binding protein in the human retina. J Neurochem glycoprotein of the interphotoreceptor matrix: localization, 42:1667, 1984. synthesis and secretion in normal and dystrophic rat retinas. J Cell Biol 97:454a, 1983. 25. Fong S-L, Liou GI, Landers RA, Alvarez RA, and Bridges 39. Gonzalez-Fernandez F, Landers RA, Liou GI, Glazebrook P, CDB: Purification and characterization of a retinol-binding Lam DMK, and Bridges CDB: An extracellular retinol-binding glycoprotein synthesized and secreted by bovine neural retina. glycoprotein in the eyes of mutant rats with retinal dystrophy: J Biol Chem 259:6534, 1984. development, localization and biosynthesis. J Cell Biol 99: 26. Bridges CDB, Alvarez RA, and Fong S-L: Vitamin A storage, 2092. esterification and interstitial retinol-binding protein in two 40. Eisenfeld AJ, Bunt-Milam AH, and Saari JC: Immunocyto- cases of retinal degeneration, one with retinitis pigmentosa. chemistry of interphotoreceptor retinoid-binding protein in ARVO Abstracts. Invest Ophthalmol Vis Sci 24(Suppl):141, developing normal and RCS rats. ARVO Abstracts. Invest 1983. Ophthalmol Vis Sci 25(Suppl):276, 1984. 27. Bridges CDB: Retinoids in photosensitive systems. In The 41. Hollyfield JG, Fliesler SJ, Rayborn ME, Fong S-L, Landers Retinoids, Sporn MB, Roberts AB, and Goodman DS, editors. RA, and Bridges CDB: Synthesis and secretion of interstitial New York, Academic Press, 1984, pp. 125-176. retinol-binding protein by the human retina. Invest Ophthalmol 28. Bridges CDB and Fong S-L: Lectins as probes of glycoprotein Vis Sci 26:58, 1985. and glycolipid oligosaccharides in rods and cones. Neurochem- 42. Bergsma DR, Wiggert B, Funahashi M, Kuwabara T, and istry 1:255, 1980. Chader G: Vitamin A receptors in normal and dystrophic 29. Bridges CDB, Alvarez RA, and Fong S-L: Vitamin A in human human retina. Nature 265:66, 1977.

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