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Acid Phosphatase in Eyes with Pseudoexfoliation
ACID PHOSPHATASE IN EYES WITH PSEUDOEXFOLIATION
K. MIZUNO, M.D., S. HARA, M.D., S. ISHIGURO, PH.D., AND Y. TAKEI, M.D. Serulai, Japan
Early description by Vogt! indicated that pseudoexfoliation was caused by the separation and exfoliation of the pericapsular membrane. A similar mechanism involving the zonular lamella has been proposed by Gifford'' and Sugar." In contrast, Bussaca's" histologic study did not reveal any evidence of capsular dehiscence, which led to the interpretation that it is a deposition of an unidentified material of unknown origin. To reconcile these somewhat disparate opinions and to distinguish the disorder from thermal exfoliation of the lens capsule, the term pseudoexfoliation was introduced by Dvorak-Theobald. 5 Recent transmission and scanning electron microscopic studies have showed that a true shedding of the superficial capsule may indeed occur.s-? Other electron microscopic studies of the pseudoexfoliative materials reveal important features such as the presence of cell organelles," lysosomal vacuoles," and Iysosomes? scattered within pseudoexfoliative fibrils. Takei and Mizuno" noticed abnormal increases in the number of lysosomes in the iris and ciliary body, and Dark, Streeten, and Cornwall? proposed the lysosomal role on the formation of the pseudoexfoliative material. In this study, we examined the correlation between pseudoexfoliation and acid phosphatase, a marker enzyme of the lysosomal enzymes, by using histochemical and biochemical methods. From the Department of Ophthalmology, Tohoku University School of Medicine, Sendai, Japan. This study was supported by grant 5041 from the Ministry of Education, Tokyo, Japan. Reprint requests to K. Mizuno, M.D., Department of Ophthalmology, Tohoku University School of Medicine, Sendai 980, Japan. 482
MATERIAL AND METHODS
Histochemistry of acid phosphataseSeven cataractous lenses and seven irises were used in the histochemical study of acid phosphatase. All were initially examined by slit-lamp biomicroscopy after mydriasis. Five of the lenses and irises were obtained from five patients with pseudoexfoliation who had normal intraocular pressure. Two control lenses and an iris were obtained from two patients with senile cataract. Lenses were extracted intracapsularly by cryosurgery without enzymatic zonulolysis. Irises were obtained by peripheral iridectomy. They were fixed for 30 minutes in cold 2.5% glutaraldehyde in O.lM cacodylate buffer (p H 6.8) immediately after extraction, and rinsed with several changes of the same buffer containing 0.33M suerose. For en bloc staining, the lenses with pseudoexfoliation and senile cataract were cut sagittally in half with a razor blade; one half was used for en bloc histochemical examination for acid phosphatase activity, after being incubated in ~-glycerophosphate as substrate at 37°C and pH 5.0 for 30 minutes using Comori's method.t? A control specimen was incubated in the reaction medium without the substrate. After the incubation, a semicircular incision was made along the postequatorial region under the dissecting microscope, and the posterior capsule, nucleus, and most of the cortex were discarded. The remaining half cup, which consisted of the equator, anterior capsule, epithelium, and superficial cortex, was mounted with glycerine jelly on a glass slide after several cuts at its equator. For histochemistry of acid phosphatase by light microscopy, the lens and iris
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were fixed and washed as previously described. The lens was cut in half, and the nucleus was discarded. The materials were mounted in Tissue-Tek II a.C.T. compound, and sagittal sections of 8 urn thickness were made on a cryostat at -25°C. Sections from each specimen were incubated for acid phosphatase activity by using l3-glycerophosphate as substrate at 37°C and pH 5.0 for 30 minutes using Cornori's method.l'' Control sections were incubated in the reaction medium without the substrate. For electron microscopic histochemistry of acid phosphatase, the method of Miller and Palade"! was used. After 30 minutes of prefixation in buffered glutaraldehyde solution, the lens was cut into several pieces through the polar axis, and the nucleus and most of the cortex were discarded. The material was then washed several times with cold O.lM cacodylate buffer (pH 6.8) containing 0.33M sucrose. The pieces were further cut sagittally with a razor blade into slices approximately 10 urn thick. The specimens were incubated for 30 minutes at 20°C in Cornori's medium.!" Before each experiment, the medium was freshly prepared by dissolving 0.12 g of lead nitrate in 100 ml of 0.05M sodium acetate buffer (pH 5.0) containing 7.5% of sucrose (0.22M) and then slowly adding 10 ml of 3% of l3-g1ycerophosphate. Before use, the mixture was warmed at 60°C for one hour, cooled to room temperature, and filtered to eliminate the slight precipitate. After incubation, the materials were washed twice for one minute in a cold 0.05M sodium acetate buffer (pH 5.0) containing 7.5% sucrose, and postfixed in 1% buffered osmium tetroxide at O°C for one hour. The materials were dehydrated in graded ethanol, cleared in propylene oxide, and embedded in Epon or Spurr. Most of the materials were then oriented and sagittal sections were obtained. Ultrathin sections were collected on grids,
483
stained with uranyl acetate and lead nitrate, and examined on an electron microscope. Controls were run by omitting the substrate from, or by adding O.OlM sodium fluoride to the incubation medium. Some of the ultrathin sections were observed on the electron microscope without any electron staining.
Biochemical analysis of acid phosphatase in aqueous humor-To assay the activity of acid phosphatase in the aqueous humor, seven eyes with pseudoexfoliation, from patients ranging in age from 65 to 72 years, were selected. As a control group, five eyes of patients with senile cataract but without pseudoexfoliation, ranging in age from 61 to 75 years, were used. They were examined routinely including slitlamp biomicroscopy to exclude other complications. Approximately 0.2 ml of the aqueous humor was aspirated with a 26-gauge needle and a microsyringe. The aqueous was stored in a freezer until used; 0.1 ml of the aqueous humor with 0.01 ml of 1% of Triton X-100 was incubated in a total volume of 1.0 ml for 20 minutes at 37°C adding 0.1 ml of 1M citrate-sodium citrate buffer at pH 4.5 with 0.2 ml of 50 mM of p-nitrophenyl phosphate as substrate and 0.6 ml of distilled water. The reaction was stopped by adding 10% trichloroacetic acid. After centrifugation, 3.0 ml of 0.5N sodium hydrochloride was added to 1.0 ml of fhe supernatant. The resulting concentration of p-nitrophenol separated was estimated spectrophotometrically in an alkali-solution at 420 nm by using a spectrophotometer.P RESULTS
Histochemical examination for acid phosphatase-Examination of the flat preparation of the anterior superficial lens of pseudoexfoliation revealed that amorphous flecks of lead phosphate, showing either strong or moderate activity for acid phosphatase, were deposited in the pe-
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Fig. 1 (Mizuno and associates). Light microscopic histochemistry for acid phosphatase of flat preparation of the lens capsule with pseudoexfoliation. Note reaction product in peripheral zone (x 1,000).
Fig. 2 (Mizuno and associates). Light microscopic histochemistry for acid phosphatase of the lens with pseudoexfoliation. Reaction product is intermingled with pseudoexfoliative materials in peripheral zone (x2,000).
ripheral zone on the lens surface (Fig. 1). However, no appreciable reaction products were encountered in the central disk. Histochemistry for acid phosphatase in cryostat sections of the peripheral zone also revealed conspicuous activity of the enzyme in the mass of pseudoexfoliative material lying on the lens capsule (Fig. 2), whereas only a few reaction products appeared in the central disk. Lead precipitate was also found dispersed within the pseudoexfoliative material attached to the posterior surface of the iris (Fig. 3). In control preparations, no deposits of lead precipitate were observed on the surface of the lens capsule. Electron microscopic observation of the iris and lens capsule of eyes with pseudoexfoliation revealed an extraordinary variety in localization and intensity of acid phosphatase reaction (lead phosphate). There were two types of iris pigment epithelial cells on the iris. The first type consisted of cells of which the cell membrane facing the posterior chamber was well preserved and continuous, though disorganization of endoplasmic reticulum and vacuolization had already taken place in the cytoplasm. In these cells, reaction products of acid phosphatase appeared sharply localized in a num-
ber of phagolysosorne concentrated in the posterior part of the cell. Consequently, pseudoexfoliative fibrils adjacent to the cell rarely contained acid phosphatase positive products (Fig. 4). The second type consisted of the pigment epithelial cells of which the cell membrane facing the posterior chamber had partially disappeared and was discontinuous, and a number of vacuoles increased in the cytoplasm. The reaction products were scattered in and out of the cells. Additionally,
Fig. 3 (Mizuno and associates). Light microscopic histochemistry for acid phosphatase of the iris of patient with pseudoexfoliation. Reaction product is dispersed within pseudoexfoliative materials that are attached to the posterior surface of the iris (x 1,000).
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Fig. 4 (Mizuno and associates). Electron microscopic histochemistry for acid phosphatase of the iris with pseudoexfoliation. Lead precipitate is seen in a phagolysosome (P) concentrated in the posterior half of cytoplasm. Cell membrane is well preserved to prevent dispersion of acid phosphatase positive particles into pseudoexfoliative fibrils (PE) (x6,600).
the limiting membrane of phagolysosorne in the cells also became obscure in part, and particles of lead phosphate were likely to be released from it, intermingling with pseudoexfoliative fibrils adjacent to the cells (Fig. 5). The distribution of acid phosphatase positive precipitates in the lens capsule of pseudoexfoliation differed significantly between the central disk and peripheral zone. In the central disk, only occasionally a slight precipitate formed in pseudoexfoliative fibrils surrounding melanin containing phagolysosome, whereas other pseudoexfoliative fibrils rarely contained the precipitates (Fig. 6). The peripheral zone frequently adhered to the posterior surface of the pigment epithelial cell, resulting in pigmented pseudoexfoliative
material on the extracted lens. In such cases, fine dense precipitates appeared within pseudoexfoliative fibrils that firmly adhered to the group of pigment granules separated from the iris pigment epithelium. The precipitates also appeared in and out of phagolysosorne that was possibly separated from the pigment epithelial cells. However, acid phosphatase positive precipitates were rarely seen in pseudoexfoliative fibrils distant from the pigment granules (Fig. 7). In the peripheral zone of another case of pseudoexfoliation, the product of acid phosphatase reaction mainly appeared in a large aggregate within phagolysosome on the lens capsule. The dispersed product also appeared within pseudoexfoliative fibrils and pigment granules that were closely
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Fig. 5 (Mizuno and associates). Electron microscopic histochemistry for acid phosphatase of the iris with pseudoexfoliation. Lead phosphate is seen in and out of phagolysosome (P) of which limiting membrane is ruptured, so that it intermingled with pseudoexfoliative fibrils (PE) (x6,600).
attached to the lens capsule. These situations were clearly proven in electron microscopy processed without any counterstaining (Fig. 8). In specimens of normal subjects, the acid phosphatase positive product was limited to a small number of lysosomes in the iris pigment epithelium and lens epithelium. In controls that omitted the sub-
strate from the medium or which added sodium fluoride to the medium, either none or a significantly reduced number of lead phosphate precipitates were found. Biochemistry of acid phosphataseThe enzyme activity in the aqueous humor was expressed as the amount of p-nitrophenol released in 0.1 ml of the aqueous humor with incubation for 60
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Fig. 6 (Mizuno and associates). Electron microscopic histochemistry for acid phosphatase of the central disk of the lens with pseudoexfoliation. Lead phosphate slightly precipitates within pseudoexfoliative fibrils (PE) around melanin containing phagolysosome (P) (x30,OOO).
minutes at 37°C. The average value of seven eyes with pseudoexfoliation was 127 ± 49.6 f..lg/ml (mean ± S.D.), whereas that of five eyes with senile cataract was 38.4 ± 4.8 ug/rnl (Table). The latter is considered to be within the error of measurement. Thus, the acid phosphatase activity was significantly higher (P < .005) in the aqueous humor of eyes with pseudoexfoliation. DISCUSSION
Our histochemical and biochemical observations have shown the presence of acid phosphatase, a marker enzyme of lysosomal enzymes, in the pseudoexfoliative material of the lens capsule and iris, and in the aqueous humor of patients with pseudoexfoliation. These observations, in part, support the previous study" in which we predicted from electron microscopic observations that the original
487
site of pseudoexfoliation might be in the iris, ciliary process, and zonule, and that lysosomes might account for the pathogenesis of pseudoexfoliative disease. Lysosomal enzymes have an important extracellular as well as intracellular role in the digestion of macromolecules. The high activity of lysosomal enzymes such as acid phosphatase, l3-glucuronidase,12 cathepsin B,13 cathepsin D,14 and acid lipase 15 have been detected in the iris and ciliary body of the bovine eye, but not in the aqueous humor. It is assumed that lysosomal enzymes are barely detectable in the aqueous humor under normal conditions even in cataractous eyes. Under some pathologic conditions, such as pseudoexfoliation, it is not unreasonable to assume that lysosomal enzymes may appear in the aqueous humor. A significant increase in acid phosphatase in the aqueous humor of eyes with pseudoexfoliation is of interest when considering that the pseudoexfoliative material attaches to the surfaces that are facing the aqueous humor; Davenger-f found it attached to the iris, to the ridge of the ciliary processes, to the zonules, and along the radial strips on the anterior hyaloid surface. With reference to the release of acid phosphatase from the iris and ciliary body into the aqueous humor, separation of lysosomes from the epithelial cells is suggested. The present electron microscopic histochemical study has shown acid phosphatase activity not only in phagolysosome but also within pseudoexfoliative fibrils. Additionally, the frequency of reactive sites and intensity of reaction of acid phosphatase is likely more significant in the following: (1) pseudoexfoliative fibrils nearer to the disorganized pigment epithelial cell of the iris, and (2) pseudoexfoliative fibrils nearer to the ruptured phagolysosorne whether it is in or out of the pigment epithelium. Thus, there is a definite association between lead phosphate over
Fig. 7 (Mizuno and associates). Electron microscopic histochemistry for acid phosphatase of the lens with pseudoexfoliation. Lead phosphate is seen in pseudoexfoliative fibrils (PE) near pigment granules and broken phagolysosome (P) separated (x6,600).
Fig. 8 (Mizuno and associates). Electron microscopic histochemistry for acid phosphatase of the lens with pseudoexfoliation without counterstaining. Lead phosphate is aggregated in phagolysosorne (P) and dispersed within pseudoexfoliative fibrils (PE) (x20,OOO).
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TABLE
REFERENCES
ACID PHOSPHATASE ACTIVITY IN AQUEOUS HUMOR WITH AND WITHOUT PSEUDOEXFOLIATION*
1. Vogt, A.: Ein neues Spaltlampenbild. Abschilferung der Linsenvorderkapsel als wahrscbeinliche Ursache von senilem chronischem Glaukom. Schweiz. Med. Wochenschr. 56:413, 1926. 2. Gifford, H.: A clinical and pathologic study of exfoliation of the lens capsule. Trans. Am. Ophthalmol. Soc. 55:189,1957. 3. Sugar, H. 5.: The exfoliation syndrome. Source of the fibrillar material on the capsule. SUTV. Ophthalmol. 21:59, 1976. 4. Busacca, A.: Struktur und Bedeutung der Hautchenniederschlage in der vordern und hinteren augenkammer. Albrecht von Graefes Arch. Klin. Exp. Ophthalmol. 119:135, 1928. 5. Dvorak-Theobald, G.: Pseudo-exfoliation of the lens capsule. Relation to "true" exfoliation of the lens capsule as reported in the literature and role in the production of glaucoma capsulocuticulare. Am. J. Ophthalmol. 37:1,1954. 6. Ghosh, M., and Speakman, J. 5.: Inclusions in human lens capsule and their relationship to senile exfoliation. Can. J. Ophthalmol, 7:413, 1972. 7. Dark, A. J., Streeten, B. W., and Cornwall, C. C.: Pseudoexfoliative disease of the lens. A study in electron microscopy and histochemistry. Br. J. Ophthalmol, 61:462, 1977. 8. Davenger, M., and Pedersen, 0.0.: Pseudoexfoliation material on the anterior lens surface. Demonstration and examination of an interfibrillar ground substance. Acta Ophthalmol, 53:3, 1975. 9. Takei, Y., and Mizuno, K.: Electron microscopic study of pseudo-exfoliation of the lens capsule. Albrecht von Graefes Klin. Exp. Ophthalmol, 205: 21.'3, 1978. 10. Weissenfels, N.: Modified lead nitrate method for phosphatase. In Pearse, A. G. F. (ed.): Histochemistry, Technical and Applied, 3rd ed. London, J. and A. Churchill, 1968, p. 729. 11. Miller, F., and Palade, G.: Lytic activities in renal protein absorption droplets. J. Cell BioI. 23: 519,1964. 12. Hayasaka, 5.: Distribution of lysosomal enzymes in the bovine eye. Jpn. J. Ophthalrnol. 18:233, 1974. 13. Hayasaka, 5., Hara, 5., Takaku, Y., and Mizuno, K.: Distribution and some properties of cathepsin B in the bovine eyes. Exp. Eye Res. 26:57, 1978. 14. Hayasaka, 5., Hara, 5., and Mizuno, K.: Distribution and some properties of cathepsin D in the retinal pigment epithelium. Exp. Eye Res. 21:.307, 1975. 15. Hayasaka, 5., Hara, 5., Takaku, Y., and Mizuno, K.: Distribution of acid lipase in the bovine retinal pigment epithelium. Exp. Eye Res. 24:1, 1977. 16. Davenger, M.: The pseudo-exfoliation syndrome. A scanning electron microscopic study. 1. Anterior lens capsule. Acta Ophthalmol. 53:809, 1975. 17. Litty-Benderitter, E.: Corps autophagiques intra-cellulaires au niveau de l'epithelium pigmentaire de !'iris au cours de la pseudoexfoliation capsulaire. J. Fr. Ophtalmol. 1:466, 1978.
ILg p-Nitrophenol Released/ 60 min 37°C/ml Case No. 1 2 3 4 5 6 7 Mean ± S.D.
PE Disease
Control
240 108 96 120 96 144 84 127 ± 49.6
36 36 36 36 48 38.4
t
4.8
*PE designates pseudoexfoliation.
pseudoexfoliative fibrils and disorganization of the pigment epithelium and phagolysosome. The same lysosome and lysosome-like vacuoles were frequently found in the other parts of the anterior segment in eyes with pseudoexfoliation. 8 •9 , 16 , 1 7 Therefore, the lysosomal limiting membrane in the pigment epithelium of the iris or ciliary body, or both, may be broken down in pseudoexfoliation, consequently releasing acid phosphatase into the aqueous humor. However, we do not know whether or not the atrophic process of the pigment epithelium in the iris and ciliary body is the primary or secondary event of pseudoexfoliation. However, a lysosomal enzyme may be directly or indirectly involved with some particular pathologic processes of this disorder. SUMMARY
We studied the activity of acid phosphatase in the anterior segment of the eye with pseudoexfoliation histochemically and biochemically. Light and electron microscopic histochemistry revealed lead precipitates indicating acid phosphatase activity in pseudoexfoliative materials. Biochemical assay for acid phosphatase was significantly higher in the aqueous humor of eyes with pseudoexfoliation than in that of cataractous eyes without pseudoexfoliation.
K. MIZUNO, M.D., S. HARA, M.D., S. ISHIGURO, PH.D., AND Y. TAKEI, M.D. Serulai, Japan
Early description by Vogt! indicated that pseudoexfoliation was caused by the separation and exfoliation of the pericapsular membrane. A similar mechanism involving the zonular lamella has been proposed by Gifford'' and Sugar." In contrast, Bussaca's" histologic study did not reveal any evidence of capsular dehiscence, which led to the interpretation that it is a deposition of an unidentified material of unknown origin. To reconcile these somewhat disparate opinions and to distinguish the disorder from thermal exfoliation of the lens capsule, the term pseudoexfoliation was introduced by Dvorak-Theobald. 5 Recent transmission and scanning electron microscopic studies have showed that a true shedding of the superficial capsule may indeed occur.s-? Other electron microscopic studies of the pseudoexfoliative materials reveal important features such as the presence of cell organelles," lysosomal vacuoles," and Iysosomes? scattered within pseudoexfoliative fibrils. Takei and Mizuno" noticed abnormal increases in the number of lysosomes in the iris and ciliary body, and Dark, Streeten, and Cornwall? proposed the lysosomal role on the formation of the pseudoexfoliative material. In this study, we examined the correlation between pseudoexfoliation and acid phosphatase, a marker enzyme of the lysosomal enzymes, by using histochemical and biochemical methods. From the Department of Ophthalmology, Tohoku University School of Medicine, Sendai, Japan. This study was supported by grant 5041 from the Ministry of Education, Tokyo, Japan. Reprint requests to K. Mizuno, M.D., Department of Ophthalmology, Tohoku University School of Medicine, Sendai 980, Japan. 482
MATERIAL AND METHODS
Histochemistry of acid phosphataseSeven cataractous lenses and seven irises were used in the histochemical study of acid phosphatase. All were initially examined by slit-lamp biomicroscopy after mydriasis. Five of the lenses and irises were obtained from five patients with pseudoexfoliation who had normal intraocular pressure. Two control lenses and an iris were obtained from two patients with senile cataract. Lenses were extracted intracapsularly by cryosurgery without enzymatic zonulolysis. Irises were obtained by peripheral iridectomy. They were fixed for 30 minutes in cold 2.5% glutaraldehyde in O.lM cacodylate buffer (p H 6.8) immediately after extraction, and rinsed with several changes of the same buffer containing 0.33M suerose. For en bloc staining, the lenses with pseudoexfoliation and senile cataract were cut sagittally in half with a razor blade; one half was used for en bloc histochemical examination for acid phosphatase activity, after being incubated in ~-glycerophosphate as substrate at 37°C and pH 5.0 for 30 minutes using Comori's method.t? A control specimen was incubated in the reaction medium without the substrate. After the incubation, a semicircular incision was made along the postequatorial region under the dissecting microscope, and the posterior capsule, nucleus, and most of the cortex were discarded. The remaining half cup, which consisted of the equator, anterior capsule, epithelium, and superficial cortex, was mounted with glycerine jelly on a glass slide after several cuts at its equator. For histochemistry of acid phosphatase by light microscopy, the lens and iris
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were fixed and washed as previously described. The lens was cut in half, and the nucleus was discarded. The materials were mounted in Tissue-Tek II a.C.T. compound, and sagittal sections of 8 urn thickness were made on a cryostat at -25°C. Sections from each specimen were incubated for acid phosphatase activity by using l3-glycerophosphate as substrate at 37°C and pH 5.0 for 30 minutes using Cornori's method.l'' Control sections were incubated in the reaction medium without the substrate. For electron microscopic histochemistry of acid phosphatase, the method of Miller and Palade"! was used. After 30 minutes of prefixation in buffered glutaraldehyde solution, the lens was cut into several pieces through the polar axis, and the nucleus and most of the cortex were discarded. The material was then washed several times with cold O.lM cacodylate buffer (pH 6.8) containing 0.33M sucrose. The pieces were further cut sagittally with a razor blade into slices approximately 10 urn thick. The specimens were incubated for 30 minutes at 20°C in Cornori's medium.!" Before each experiment, the medium was freshly prepared by dissolving 0.12 g of lead nitrate in 100 ml of 0.05M sodium acetate buffer (pH 5.0) containing 7.5% of sucrose (0.22M) and then slowly adding 10 ml of 3% of l3-g1ycerophosphate. Before use, the mixture was warmed at 60°C for one hour, cooled to room temperature, and filtered to eliminate the slight precipitate. After incubation, the materials were washed twice for one minute in a cold 0.05M sodium acetate buffer (pH 5.0) containing 7.5% sucrose, and postfixed in 1% buffered osmium tetroxide at O°C for one hour. The materials were dehydrated in graded ethanol, cleared in propylene oxide, and embedded in Epon or Spurr. Most of the materials were then oriented and sagittal sections were obtained. Ultrathin sections were collected on grids,
483
stained with uranyl acetate and lead nitrate, and examined on an electron microscope. Controls were run by omitting the substrate from, or by adding O.OlM sodium fluoride to the incubation medium. Some of the ultrathin sections were observed on the electron microscope without any electron staining.
Biochemical analysis of acid phosphatase in aqueous humor-To assay the activity of acid phosphatase in the aqueous humor, seven eyes with pseudoexfoliation, from patients ranging in age from 65 to 72 years, were selected. As a control group, five eyes of patients with senile cataract but without pseudoexfoliation, ranging in age from 61 to 75 years, were used. They were examined routinely including slitlamp biomicroscopy to exclude other complications. Approximately 0.2 ml of the aqueous humor was aspirated with a 26-gauge needle and a microsyringe. The aqueous was stored in a freezer until used; 0.1 ml of the aqueous humor with 0.01 ml of 1% of Triton X-100 was incubated in a total volume of 1.0 ml for 20 minutes at 37°C adding 0.1 ml of 1M citrate-sodium citrate buffer at pH 4.5 with 0.2 ml of 50 mM of p-nitrophenyl phosphate as substrate and 0.6 ml of distilled water. The reaction was stopped by adding 10% trichloroacetic acid. After centrifugation, 3.0 ml of 0.5N sodium hydrochloride was added to 1.0 ml of fhe supernatant. The resulting concentration of p-nitrophenol separated was estimated spectrophotometrically in an alkali-solution at 420 nm by using a spectrophotometer.P RESULTS
Histochemical examination for acid phosphatase-Examination of the flat preparation of the anterior superficial lens of pseudoexfoliation revealed that amorphous flecks of lead phosphate, showing either strong or moderate activity for acid phosphatase, were deposited in the pe-
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Fig. 1 (Mizuno and associates). Light microscopic histochemistry for acid phosphatase of flat preparation of the lens capsule with pseudoexfoliation. Note reaction product in peripheral zone (x 1,000).
Fig. 2 (Mizuno and associates). Light microscopic histochemistry for acid phosphatase of the lens with pseudoexfoliation. Reaction product is intermingled with pseudoexfoliative materials in peripheral zone (x2,000).
ripheral zone on the lens surface (Fig. 1). However, no appreciable reaction products were encountered in the central disk. Histochemistry for acid phosphatase in cryostat sections of the peripheral zone also revealed conspicuous activity of the enzyme in the mass of pseudoexfoliative material lying on the lens capsule (Fig. 2), whereas only a few reaction products appeared in the central disk. Lead precipitate was also found dispersed within the pseudoexfoliative material attached to the posterior surface of the iris (Fig. 3). In control preparations, no deposits of lead precipitate were observed on the surface of the lens capsule. Electron microscopic observation of the iris and lens capsule of eyes with pseudoexfoliation revealed an extraordinary variety in localization and intensity of acid phosphatase reaction (lead phosphate). There were two types of iris pigment epithelial cells on the iris. The first type consisted of cells of which the cell membrane facing the posterior chamber was well preserved and continuous, though disorganization of endoplasmic reticulum and vacuolization had already taken place in the cytoplasm. In these cells, reaction products of acid phosphatase appeared sharply localized in a num-
ber of phagolysosorne concentrated in the posterior part of the cell. Consequently, pseudoexfoliative fibrils adjacent to the cell rarely contained acid phosphatase positive products (Fig. 4). The second type consisted of the pigment epithelial cells of which the cell membrane facing the posterior chamber had partially disappeared and was discontinuous, and a number of vacuoles increased in the cytoplasm. The reaction products were scattered in and out of the cells. Additionally,
Fig. 3 (Mizuno and associates). Light microscopic histochemistry for acid phosphatase of the iris of patient with pseudoexfoliation. Reaction product is dispersed within pseudoexfoliative materials that are attached to the posterior surface of the iris (x 1,000).
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Fig. 4 (Mizuno and associates). Electron microscopic histochemistry for acid phosphatase of the iris with pseudoexfoliation. Lead precipitate is seen in a phagolysosome (P) concentrated in the posterior half of cytoplasm. Cell membrane is well preserved to prevent dispersion of acid phosphatase positive particles into pseudoexfoliative fibrils (PE) (x6,600).
the limiting membrane of phagolysosorne in the cells also became obscure in part, and particles of lead phosphate were likely to be released from it, intermingling with pseudoexfoliative fibrils adjacent to the cells (Fig. 5). The distribution of acid phosphatase positive precipitates in the lens capsule of pseudoexfoliation differed significantly between the central disk and peripheral zone. In the central disk, only occasionally a slight precipitate formed in pseudoexfoliative fibrils surrounding melanin containing phagolysosome, whereas other pseudoexfoliative fibrils rarely contained the precipitates (Fig. 6). The peripheral zone frequently adhered to the posterior surface of the pigment epithelial cell, resulting in pigmented pseudoexfoliative
material on the extracted lens. In such cases, fine dense precipitates appeared within pseudoexfoliative fibrils that firmly adhered to the group of pigment granules separated from the iris pigment epithelium. The precipitates also appeared in and out of phagolysosorne that was possibly separated from the pigment epithelial cells. However, acid phosphatase positive precipitates were rarely seen in pseudoexfoliative fibrils distant from the pigment granules (Fig. 7). In the peripheral zone of another case of pseudoexfoliation, the product of acid phosphatase reaction mainly appeared in a large aggregate within phagolysosome on the lens capsule. The dispersed product also appeared within pseudoexfoliative fibrils and pigment granules that were closely
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Fig. 5 (Mizuno and associates). Electron microscopic histochemistry for acid phosphatase of the iris with pseudoexfoliation. Lead phosphate is seen in and out of phagolysosome (P) of which limiting membrane is ruptured, so that it intermingled with pseudoexfoliative fibrils (PE) (x6,600).
attached to the lens capsule. These situations were clearly proven in electron microscopy processed without any counterstaining (Fig. 8). In specimens of normal subjects, the acid phosphatase positive product was limited to a small number of lysosomes in the iris pigment epithelium and lens epithelium. In controls that omitted the sub-
strate from the medium or which added sodium fluoride to the medium, either none or a significantly reduced number of lead phosphate precipitates were found. Biochemistry of acid phosphataseThe enzyme activity in the aqueous humor was expressed as the amount of p-nitrophenol released in 0.1 ml of the aqueous humor with incubation for 60
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Fig. 6 (Mizuno and associates). Electron microscopic histochemistry for acid phosphatase of the central disk of the lens with pseudoexfoliation. Lead phosphate slightly precipitates within pseudoexfoliative fibrils (PE) around melanin containing phagolysosome (P) (x30,OOO).
minutes at 37°C. The average value of seven eyes with pseudoexfoliation was 127 ± 49.6 f..lg/ml (mean ± S.D.), whereas that of five eyes with senile cataract was 38.4 ± 4.8 ug/rnl (Table). The latter is considered to be within the error of measurement. Thus, the acid phosphatase activity was significantly higher (P < .005) in the aqueous humor of eyes with pseudoexfoliation. DISCUSSION
Our histochemical and biochemical observations have shown the presence of acid phosphatase, a marker enzyme of lysosomal enzymes, in the pseudoexfoliative material of the lens capsule and iris, and in the aqueous humor of patients with pseudoexfoliation. These observations, in part, support the previous study" in which we predicted from electron microscopic observations that the original
487
site of pseudoexfoliation might be in the iris, ciliary process, and zonule, and that lysosomes might account for the pathogenesis of pseudoexfoliative disease. Lysosomal enzymes have an important extracellular as well as intracellular role in the digestion of macromolecules. The high activity of lysosomal enzymes such as acid phosphatase, l3-glucuronidase,12 cathepsin B,13 cathepsin D,14 and acid lipase 15 have been detected in the iris and ciliary body of the bovine eye, but not in the aqueous humor. It is assumed that lysosomal enzymes are barely detectable in the aqueous humor under normal conditions even in cataractous eyes. Under some pathologic conditions, such as pseudoexfoliation, it is not unreasonable to assume that lysosomal enzymes may appear in the aqueous humor. A significant increase in acid phosphatase in the aqueous humor of eyes with pseudoexfoliation is of interest when considering that the pseudoexfoliative material attaches to the surfaces that are facing the aqueous humor; Davenger-f found it attached to the iris, to the ridge of the ciliary processes, to the zonules, and along the radial strips on the anterior hyaloid surface. With reference to the release of acid phosphatase from the iris and ciliary body into the aqueous humor, separation of lysosomes from the epithelial cells is suggested. The present electron microscopic histochemical study has shown acid phosphatase activity not only in phagolysosome but also within pseudoexfoliative fibrils. Additionally, the frequency of reactive sites and intensity of reaction of acid phosphatase is likely more significant in the following: (1) pseudoexfoliative fibrils nearer to the disorganized pigment epithelial cell of the iris, and (2) pseudoexfoliative fibrils nearer to the ruptured phagolysosorne whether it is in or out of the pigment epithelium. Thus, there is a definite association between lead phosphate over
Fig. 7 (Mizuno and associates). Electron microscopic histochemistry for acid phosphatase of the lens with pseudoexfoliation. Lead phosphate is seen in pseudoexfoliative fibrils (PE) near pigment granules and broken phagolysosome (P) separated (x6,600).
Fig. 8 (Mizuno and associates). Electron microscopic histochemistry for acid phosphatase of the lens with pseudoexfoliation without counterstaining. Lead phosphate is aggregated in phagolysosorne (P) and dispersed within pseudoexfoliative fibrils (PE) (x20,OOO).
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ACID PHOSPHATASE AND PSEUDOEXFOLIATION
489
TABLE
REFERENCES
ACID PHOSPHATASE ACTIVITY IN AQUEOUS HUMOR WITH AND WITHOUT PSEUDOEXFOLIATION*
1. Vogt, A.: Ein neues Spaltlampenbild. Abschilferung der Linsenvorderkapsel als wahrscbeinliche Ursache von senilem chronischem Glaukom. Schweiz. Med. Wochenschr. 56:413, 1926. 2. Gifford, H.: A clinical and pathologic study of exfoliation of the lens capsule. Trans. Am. Ophthalmol. Soc. 55:189,1957. 3. Sugar, H. 5.: The exfoliation syndrome. Source of the fibrillar material on the capsule. SUTV. Ophthalmol. 21:59, 1976. 4. Busacca, A.: Struktur und Bedeutung der Hautchenniederschlage in der vordern und hinteren augenkammer. Albrecht von Graefes Arch. Klin. Exp. Ophthalmol. 119:135, 1928. 5. Dvorak-Theobald, G.: Pseudo-exfoliation of the lens capsule. Relation to "true" exfoliation of the lens capsule as reported in the literature and role in the production of glaucoma capsulocuticulare. Am. J. Ophthalmol. 37:1,1954. 6. Ghosh, M., and Speakman, J. 5.: Inclusions in human lens capsule and their relationship to senile exfoliation. Can. J. Ophthalmol, 7:413, 1972. 7. Dark, A. J., Streeten, B. W., and Cornwall, C. C.: Pseudoexfoliative disease of the lens. A study in electron microscopy and histochemistry. Br. J. Ophthalmol, 61:462, 1977. 8. Davenger, M., and Pedersen, 0.0.: Pseudoexfoliation material on the anterior lens surface. Demonstration and examination of an interfibrillar ground substance. Acta Ophthalmol, 53:3, 1975. 9. Takei, Y., and Mizuno, K.: Electron microscopic study of pseudo-exfoliation of the lens capsule. Albrecht von Graefes Klin. Exp. Ophthalmol, 205: 21.'3, 1978. 10. Weissenfels, N.: Modified lead nitrate method for phosphatase. In Pearse, A. G. F. (ed.): Histochemistry, Technical and Applied, 3rd ed. London, J. and A. Churchill, 1968, p. 729. 11. Miller, F., and Palade, G.: Lytic activities in renal protein absorption droplets. J. Cell BioI. 23: 519,1964. 12. Hayasaka, 5.: Distribution of lysosomal enzymes in the bovine eye. Jpn. J. Ophthalrnol. 18:233, 1974. 13. Hayasaka, 5., Hara, 5., Takaku, Y., and Mizuno, K.: Distribution and some properties of cathepsin B in the bovine eyes. Exp. Eye Res. 26:57, 1978. 14. Hayasaka, 5., Hara, 5., and Mizuno, K.: Distribution and some properties of cathepsin D in the retinal pigment epithelium. Exp. Eye Res. 21:.307, 1975. 15. Hayasaka, 5., Hara, 5., Takaku, Y., and Mizuno, K.: Distribution of acid lipase in the bovine retinal pigment epithelium. Exp. Eye Res. 24:1, 1977. 16. Davenger, M.: The pseudo-exfoliation syndrome. A scanning electron microscopic study. 1. Anterior lens capsule. Acta Ophthalmol. 53:809, 1975. 17. Litty-Benderitter, E.: Corps autophagiques intra-cellulaires au niveau de l'epithelium pigmentaire de !'iris au cours de la pseudoexfoliation capsulaire. J. Fr. Ophtalmol. 1:466, 1978.
ILg p-Nitrophenol Released/ 60 min 37°C/ml Case No. 1 2 3 4 5 6 7 Mean ± S.D.
PE Disease
Control
240 108 96 120 96 144 84 127 ± 49.6
36 36 36 36 48 38.4
t
4.8
*PE designates pseudoexfoliation.
pseudoexfoliative fibrils and disorganization of the pigment epithelium and phagolysosome. The same lysosome and lysosome-like vacuoles were frequently found in the other parts of the anterior segment in eyes with pseudoexfoliation. 8 •9 , 16 , 1 7 Therefore, the lysosomal limiting membrane in the pigment epithelium of the iris or ciliary body, or both, may be broken down in pseudoexfoliation, consequently releasing acid phosphatase into the aqueous humor. However, we do not know whether or not the atrophic process of the pigment epithelium in the iris and ciliary body is the primary or secondary event of pseudoexfoliation. However, a lysosomal enzyme may be directly or indirectly involved with some particular pathologic processes of this disorder. SUMMARY
We studied the activity of acid phosphatase in the anterior segment of the eye with pseudoexfoliation histochemically and biochemically. Light and electron microscopic histochemistry revealed lead precipitates indicating acid phosphatase activity in pseudoexfoliative materials. Biochemical assay for acid phosphatase was significantly higher in the aqueous humor of eyes with pseudoexfoliation than in that of cataractous eyes without pseudoexfoliation.