798 Reports Investigative October 1974

normal lenses than in cataractous lenses indicates 6. van Kleef, F. S. M., and Hoenders, H. J.: that it does represent a real change in protein Population character and variety in subunit structure. Conceivably, such protein in the intact structure of high-molecular weight proteins may be composed of even larger macromolec- from the bovine eye lens, Eur. J. Biochem. ular aggregates than the HMWP fraction and 40: 549, 1973. 7. Jedziniak, J. A., Kinoshita, J. H., Yates, E. would, therefore, also contribute to the scattering M., et al.: On the presence and mechanism of of light. The transformation to insoluble protein formation of heavy molecular weight aggre- may be accelerated in the developing and gates in human normal and cataractous lenses, thus partially account for the observation of only Exp. Eye Res. 15: 185, 1973. modest increments in HMWP in such lenses. 8. Maraini, C, and Mangili, R.: in The human It is of interest to note that in the cortical lens in relation to cataract, Ciba Foundation region the light scattering cannot be explained Symposium 19 (new series), Elliott, K., and simply on the basis of the appearance of HMWP. Fitzsimons, D., editors. Amsterdam, 1973, Association of Scientific Publishers, p. 80. However, recent experiments of Kuwabara's (T. 9. Spector, S., Stauffer, J., and Sigelman, J.: Kuwabara: private communication) suggest a pos- Preliminary observations upon the proteins of sible explanation for such scattering. Scanning human lens, in: Human Lens in Relation to electron microscopy of the cortical region of nor- Cataract, Ellis, H. C, and Fitzsimmons, D. mal lens indiates highly irregular interdigitating W., editors. Ciba Foundation Symposium, No. fiber surfaces which may cause light scatter. The 19 (n.s.). Amsterdam, 1973, Elsevier, p. 185. increase in scattering from this region with aging 10. Benedek, G. B.: Theory of transparency of may be due prinicpally to the increase in size the eye, Appl. Optics 10: 459, 1971. of the cortical region. In contrast, the nuclear re- 11. Jedziniak, J. A., Kinoshita, J. H., Yates, E. M., gion contains highly regular interdigitating fiber et al.: Calcium-induced aggregation of bovine boundaries which would not be expected to cause lens alpha-crystallin, INVEST. OPHTHALMOL. 11: 905, 1972. significant scattering. Thus in the aging process, 12. Perry, A. J., and Koenig, V. L.: Some physio- molecular changes may cause most nuclear scat- chemical properties of the water-soluble pro- tering and normal morphological structure, cortical teins of the bovine eye lens, Biochim. Bio- scattering. phys. Acta 46: 413, 1961. 13. Huggert, A.: On the iso-indicial surfaces of The skilled technical assistance of Stoimen the human crystalline lens, Acta Ophthalmol. Djalazov is appreciatively noted. We thank Dr. Suppl. 30: 1, 1948. Emil Wirostko for permitting us to use his Zeiss 14. Sigelman, J., Trokel, S. L., and Spector, A..: slit lamp camera. Quantitative biomicroscopy of lens light back- From the Department of Ophthalmology, Col- scatter in aging and opacification, Arch. Oph- lege of Physicians and Surgeons, Columbia Uni- thalmol. In press. versity, New York, N. Y. 10032. Supported by 15. Trokel, S.: The physical basis for the trans- grants from the National Eye Institute. Submitted parency of the lens, INVEST. OPHTHALMOL. 1: tor publication April 26, 1974. "Dedicated to 493, 1962. David G. Cogan, who created a free, stimulating 16. van Heyningen, R.: The human lens. III. intellectual atmosphere at the Howe Laboratory, Some observations on the postmortem lens, Harvard University, which led to an unprecedented Exp. Eye Res. 13: 155, 1972. era of scientific accomplishment. * "Fight for Sight 17. Stauffer, J., Rothschild, C, Wandel, T., et Fellow. al.: Transformation of alpha-crystallin poly- peptide chains with aging, INVEST. OPHTHAL- Key words: lens, high molecular weight protein, MOL. 13: 135, 1974. aging, light back-scatter, nucleus, cortex. REFERENCES 1. Spector, A., Freund, T., Li, L-K., et al.: in pigmentosa: evidence Age-dependent changes in the structure of that retinol is in normal concentration alpha-crystallin, INVEST. OPHTHALMOL. 10: 677, 1971. in serum and the retinol-binding protein 2. Spector, A.: Aggregation of alpha-crystallin complex displays unaltered fluorescence and its possible relationship to cataract for- properties. SIDNEY FUTTERMAN, DAVID mation, Isr. J. Med. Sci. 8: 1577, 1972. SWANSON, AND ROBERT E. KALINA. 3. Spector, A.: The aging of alpha-crystallin: a review, Exp. Eye Res. 16: 115, 1973. The concentration of retinol in serum in retinitis 4. Spector, A.: The purification and characteri- pigmentosa was determined by a reliable, con- zation of the highly labeled protein fraction ventional fiuorometric procedure involving extrac- from calf lens, INVEST. OPHTHALMOL. 7: 179, tion and column chromatography, and by a new 1968. procedure that measures directly the fluorescence 5. Spector, A., Li, L-K., Augusteyn, R., et al.: Alpha-crystallin, the isolation and characteri- of the retinol-binding protein complex in serum. zation of distinct macromolecular fractions, The mean values for 15 serum samples analyzed Biochem. J. 124: 337, 1971. by the two methods were, respectivtly, 66 and 67

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fig per 100 ml. The results indicate that neither the extractahle retinol nor the retinol-binding pro- tein itself are present in reduced amounts. No alteration in the fluorescence properties of the transport complex of retinol in serum could be found. The findings strongly suggest that in retini- tis pigmentosa, retinol transport in the general circulation is unimpaired.

Defective absorption or transport of retinol as in a /3-apoproteinemia1 or cystic fibrosis,2' 3 or impaired cleavage of /3-carotene occurring with an exclusively vegetarian diet4 can lead to night blindness and characteristic retinal changes.5 These disorders occurring in man as well as experimen- tally produced deficiency states in ani- mals" share certain physiologic and morphologic 7 features with , a prevalent 20 40 60 retinal degeneration of man. Conventionol method, retinol The , unlike other tissues requiring vita- min A, cannot utilize retinoic acid (vitamin A Fig. 1. Comparison of range of values of retinol in serum in retinitis pigmentosa patients (RP) acid). Since both intestinal mucosa and liver can 1 convert retinal (vitamin A aldehyde) to retinoic and normal control subjects (C). The correlation 8 !) coefficient for analyses by the two methods was acid, - it is reasonable to postulate that one vari- 0.87 for the retinitis pigmentosa group and 0.85 ety of retinitis pigmentosa might stem from a de- for the normal group. fect in the transport of retinol from liver to target tissues. While the photoreceptors were experienc- ing a selective deficiency, the systemic vitamin A tion yielding dilated fundus examination evidence requirements could be met adequately by circu- of advanced pigmentary and nar- lating retinoic acid derived by the transformation rowed retinal blood vessels. Some, but not all, of into retinoic acid of a small fraction of the retinol the subjects also demonstrated immature , formed during the course of intestinal cleavage of waxy optic discs, or pigmentary macular changes. dietary /3-carotene. It is, therefore, of great in- Goldmann perimetry confirmed characteristic pe- terest that a low concentration of retinol in blood ripheral visual field loss in patients with retained has been found in 91 per cent of one group of vision. Affected relatives of subjects were identified adult patients with retinitis pigmentosa.10 How- by history but were not examined. Subjects were ever, this observation has not been confirmed in questioned in order to exclude recent supplemental other laboratories.11 "13 More recently, it has been vitamin A intake. reported that the concentration of circulating Analytic methods used for the determination retinol-binding protein is reduced in retinitis pig- of retinol in serum are described in the preceding mentosa.14 paper.15 One possible mechanism that has not yet been Results. The concentration of retinol in serum investigated is that the retinol transport complex samples from retinitis pigmentosa patients (Table might be present in normal concentration but have I) fell largtely (Fig. 1), although not entirely altered properties that reduce its effectiveness. A within the range of samples from normal subjects significantly altered retinol-binding protein might previously analyzed by these methods. The mean be expected to display altered fluorescence excita- values were not significantly in excess of the mean tion or emission maxima or altered fluorescence values of 60.1 and 60.8 ng per 100 ml. found for enhancement.15 The present study was undertaken normal subjects.15 These findings indicate that both to explore these ideas and to re-examine the the range of fluorescence enhancement that is question of the concentration of retinol in blood observed for retinol in its native complex in serum in retinitis pigmentosa with a comparison of results samples from retinitis pigmentosa patients does obtained by two different assay methods,15 one not differ significantly from the range of fluores- giving a measure of the native retinol-binding pro- cence enhancement found for normal subjects. tein, and the other determining retinol recovered Fluorescence spectra were obtained for each after extraction and chromatographic separation. of the diluted serum samples. All gave excitation Methods. Subjects were selected because of a and emission maxima at 335 nm. and 458 nm. history of decreased night vision, progressive loss for the retinol peak, and showed no deviations of visual field, and a previously established diag- from normal spectra.15 The height ratios of the nosis of retinitis pigmentosa. The diagnosis was retinol (335 nm.) to "protein" (285 nm.) peaks confirmed in all cases by a complete eye examina- also were all within the normal range.15

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Table I. Retinol content (micrograms per 100 ml.) of serum in retinitis pigmentosa patients as determined by a new, direct fluorometric method and by conventional method involving extraction and chromatography

Retinitis Retinol determination pigmentosa Conventional Carotenoids Protein serum sample Age and sex New method method (fig/100 ml.) (Gm./lOO ml.) 1 21, M 83 63 203 7.4 2 24, M 62 56 68 9.3 3 26, F 47 53 140 7.6 4 28, M 43 41 128 9.3 5 30, M 83 70 126 7.3 6 35, F 46 46 93 6.6 7 39, M 55 65 84 6.5 8 40, M 67 75 64 7.6 9 48, F 56 66 72 8.6 10 50, M 87 92 77 7.1 11 52, M 52 55 86 6.9 12 52, M 89 104 108 7.5 13 58, F 97 95 145 7.5 14 64, M 56 58 106 7.3 15 76, F 66 67 92 6.9 Mean 65.9 67.1 106.1 7.56 S.E. 4.3 4.6 9.6 0.22 P* NS NS NS NS "Significance of difference from normal subjects.15

Table II. Distribution (per cent) among component fractions of total fluorescence of serum extract recovered after chromatography on alumina

Hydrocarbon Serum samples fraction Retinyl esters Retinol Retinitis pigmentosa (15) 17.9 ± 2.6 9.1 ± 0.9 73.0 ± 3.2 Control subjects (21)15 24.2 ± 1.4 7.3 ± 0.4 68.5 ± 1.5 Significance of difference P < 0.05 NS NS Values are means ± S.E. for data obtained from analyses of retinol by the conventional method.15 Average recovery of total fluorescence in the column fractions was the same (94 per cent) for the two groups.

In the assay of retinol by the conventional sive inheritance pattern also had normal serum procedure, retinyl esters, and a fluorescent hydro- values as did two patients from probable autosomal carbon fraction are separated chromatographically dominant pedigrees. Eleven patients had either from retinol. The relative proportion of retinyl no family history or a history of retinitis pigmen- esters in serum samples in retinitis pigmentosa tosa suggesting autosomal recessive inheritance. did not differ significantly from the proportion in Those eleven patients had a mean serum retinol normal subjects (Table II). The fraction of the level greater than the other five patients (p < total fluorescence attributable to the hydrocarbon 0.05) and greater than the normal subjects (p fraction was somewhat reduced in retinitis pig- < 0.01). However, the significance of these mentosa, but in view of the known dietary origin results is reduced both by the small number of of phytofluene11! as the major component of this patients and the older average age of the eleven fraction, it is unlikely that this difference is attrib- patients. utable to the disease. The carotenoid concentra- Discussion. The concentration of retinol in blood tion did not differ significantly from the value below which night blindness becomes manifest obtained for normal subjects. in human volunteer subjects17 can be calculated The mean serum retinol and carotenoid levels to be about 15 Mg per 100 ml. All retinitis pig- of several subgroups of patients were compared mentosa serum samples examined in this study with the other patients and with normal subjects. had retinol concentrations higher at least by about Seven patients with waxy optic discs and two pa- threefold. tients with late onset of symptoms had serum Both the concentration of retinol in serum and values not significantly different from the other the fluorescence properties of the retinol-binding patients or from normal subjects. Three patients protein complex in retinitis pigmentosa patients with family histories suggesting an x-linked reces- are indistinguishable from those of normal sub-

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jects. These findings constitute new evidence in 5. McLaren, D. S.: Nutritional aspects of the support of the view that the transport of vitamin eye, In: Biochemistry of the Eye, Graymore, A in retinitis pigmentosa is unimpaired, but do C, editor, New York, 1970, Academic Press, not rule out the possibility that the mechanism of pp. 519-561. release of retinol from retinol-binding protein may 6. Dowling, J. E., and Gibbons, I. R.: The effect of on the fine be abnormal. structure of the retina, In: The Structure of The well-known tendency of the retinol con- 2 17 the Eye, Smelser, G. K., editor, New York, centration in blood to increase with age - prob- 1961, Academic Press, pp. 85-99. ably reflects a gradual diet-related increase in the 7. Cogan, D.: Primary chorioretinal aberrations storage of retinyl esters in the liver that commonly with night blindness, Trans. Am. Acad. continues throughout life, but can be greatly ac- Ophthalmol. 54: 629, 1950. celerated by vitamin supplementation. It seems 8. Dmitrovskii, A. A.: Oxidation of vitamin A likely that the presence of a high concentration aldehyde to vitamin A acid catalyzed by alde- of retinyl esters in the liver might increase pro- hyde oxidase, Biokhimiya 26: 126, 1961. duction and release of retinol-binding protein into 9. Futterman, S.: Enzymatic oxidation of vita- min A aldehyde to vitamin A acid, J. Biol. the circulation. There was some indication that Chem. 237: 677, 1962. the retinol concentration in blood increased with 10. Campbell, D. A., and Tonks, E. L.: Bio- age (Table I) in the retinitis pigmentosa patients chemical findings in human retinitis pig- in this study as well as in the normal subjects mentosa with particular relation to vitamin previously examined.15 Although the retinitis pig- A deficiency, Br. J. Ophthalmol. 46: 151, mentosa patients and the normal subjects were not 1962. precisely matched by age, statistical analysis re- 11. Wagreich, H., Lasky, M. A., and Elkan, B.: veals no significant difference between the groups Some biochemical studies in retinitis pig- with respect to the concentration of retinol in the mentosa, Clin. Chem. 1: 143, 1961. circulation. It would appear that some uncontrolled 12. Mehra, K. S., and Khare, B. A.: Vitamin A factor of age, diet, or methodology may have in retinitis pigmentosa, Oriental Arch. Oph- thalmol. 3: 80, 1965. accounted for the reduced concentrations of retinol 10 14 13. Krachmer, J. H., Smith, J. L., and Tocci, in blood that have been reported ' in retinitis P. M.: Laboratory studies in retinitis pig- pigmentosa. mentosa, Arch. Ophthal. 75: 661, 1966. 14. Rahi, A. H. S.: Retinol-binding protein Excellent technical assistance was provided by (RBP) and pigmentary dystrophy of the Mrs. Martha H. Rollins and advice concerning retina, Br. J. Ophthalmol. 56: 647, 1972. statistical analysis of the data was obtained from 15. Futterman, S., Swanson, D., and Kalina, R. Ms. Barbara Campbell. E.: A new, rapid fluorometric determination From the Department of Ophthalmology, Uni- of retinol in blood, INVEST. OPHTHALMOL. versity of Washington School of Medicine, Seattle. In press. This study was supported by United States Public 16. Thompson, J. N., Erdody, P., Brien, R., et Health Service Research Grant No. EY 00343 and al.: Fluorometric determination of vitamin A Training Grant No. EY 00050 from the National in human blood and liver, Biochem. Med. 5: Eye Institute. Submitted for publication April 67, 1971. 29, 1974. Reprint requests: Dr. S. Futterman, De- 17. Leitner, Z. A., Moore, T., and Sharman, I. M.: partment of Ophthalmology, University of Wash- Vitamin A and vitamin E in human blood, ington School of Medicine, Seattle, Wash. 98195. Br. J. Nutr. 14: 157, 1960.

Key words: retinol, retinol-binding protein, retinitis pigmentosa, vitamin A, retina, carotene. REFERENCES A possible cause of decreased vision in CARL KUPFER 1. Gouras, P., Carr, R. E., and Gunrel, R. D.: cryptococcal meningitis. Retinitis pigmentosa in abetalipoproteinemia: AND EDNA MCCRANE. effects of vitamin A, INVEST. OPHTHALMOL. 10: 784, 1971. The optic nerves, chiasm, optic tracts, and lateral 2. Smith, F. R., Underwood, B. A., Denning, C. geniculate nuclei of six patients having fatal cryp- R., et al.: Depressed plasma retinol-binding tococcal meningitis were examined histopathologi- protein levels in cystic fibrosis, J. Lab. Clin. cally using the Smith-Quigley stain for myelin and Med. 80: 423, 1972. periodic acid-Shiff stain for crytococcal organisms. 3. Petersen, R. A., Petersen, V. S., and Robb, R. A correlation was made between the invasion of M.: Vitamin A deficiency with xerophthalmia visual pathways by the cryptococcal organism, the and night blindness in cystic fibrosis, Am. J. Dis. Child. 116: 662, 1968. presence or absence of , and the pres- 4. McLaren, D. S., and Zerian, B.: Failure of ervation or loss of visual acuity. In three patients cleavage of /3-carotene, Am. J. Dis. Child. with papilledema and loss of visual acuity, multiple 121: 278, 1971. cryptococcal abscesses were present in the optic

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