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. 684 Downloaded from iovs.arvojournals.org on 10/02/2021 No. 5 VITAMIN A AND IRDP IN RETINITIS PIGMENTOSA / Bridges er ol. 685 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 de- scribed 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 pigmen- tosa 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 Downloaded from iovs.arvojournals.org on 10/02/2021 686 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Moy 1985 Vol. 26 100,000 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).