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Secretion of Inorganic Phosphorus in Tears*
ULTRASTRUCTURE OF PIGMENT GRANULES
Upon exposure to light, somehow, per haps through photochemical changes, the fila ment on the inner side may be made to be come more twisted so that the granule moves inward toward the more twisted filament. When dark adapted, the filament on the in ner side may become less twisted, so that the migrated granules can move back to their original positions. CONCLUSION
An electron-microscope study of the ret inal pigment granules of several vertebrate eyes (dog, fish, frog, monkey, mouse, and pig) produced the following observations: 1. The pigment granules revealed a num ber of forms, usually cigar-shaped, but also egg-shaped and spherical.
941
2. These granules were composed of num erous microgranules with the same shape as the parent granules. 3. The cigar-shaped pigment granules had filamentous processes at both extremities, and these filaments averaged four to five times the length of the granule itself. An hypothesis concerned with a partial explanation of the migration of pigment granules in the retinal epithelial cells is sug gested. ACKNOWLEDGMENT
I wish to acknowledge the kind assistance of L. W. Freeman, M.D., Luis Lorenz Estella, and R. E. Smith in the collection of animal eyes and the help of Fred M. Wilson, M.D., and T. F. Schlaegel, Jr., M.D., in the preparation of the paper.
REFERENCES
1. Taniguchi, Y.: Ultrastructure of pigment granules of retinal epithelium: I. Cow's eye. Am. J. Ophth., 48:221, 19S9. 2. Sebruyns, M.: Study of the ultrastructure of the retinal epithelium by means of the electron micro scope. Am. J. Ophth., 34:989, 1951. 3. Binder, R., and Orth, E.: Elektronenophtische Studie an Pigmentkornern and Zonulafasern des menschilichen Auges. Arch, f. Ophth., 154:266, 1953. 4. Fra^ois, J., Rabaey, M., and Vandermeerssche, G.: L'ultrastructure des Grains Pigmentaires de L'epithelium Retinien. Ann. ocul., 186 :896, 1953. 5. Kinugasa, H.: Electron microscopic study of eye tissues. Acta Ophth. Japon., 57 :436, 1953. 6. Kion, G., Maertens, C, and Vandermeerssche, G.: Submicroscopic morphology of the retinal pig ment epithelium. Electron Microscopy: Proc. Stockholm Conference. New York, 1957. 7. Takeuchi, H.: Morphological studies of the pigment granules of retina and choroides by electron microscope. Acta Ophth. Japon., 60 :813,1956. 8. Heydenreich, Andreas: Das morphologische and mikrochemische Verhalten der Pigmentgranula des Auges. Arch. f. Ophth., 159:162, 1957. 9. Iwaki, S.: The fine structure of the retinal pigment epithelium and capillary of choriocapillary layer, studied with the electron microscope. J. Kurme M. A., 21:1, 1958. 10. Birbeck, M. S. C, Mercer, E. H., and Barnicot, N. A.: The structure and formation of pigment granule in human hair. Exper. Cell. Research, 10:505, 1956. 11. Kolmer, W.: Handbuch der Mikroscopischen Anatomic des Menschen. Berlin, 3:2, 1936. 12. Uyama, Y.: Die Retina des Saugetieres. Med. f. Osaka LTniv., 2:3, 1951.
SECRETION O F INORGANIC P H O S P H O R U S IN TEARS* JOSEPH BALIK,
M.D.
Prague, Cscchoslovakia
The level of inorganic phosphates has not yet been determined. Magaard was not able to prove them in a qualitative examination of the tear fluid, although Frerichs and Arlt * From the First Ophthalmic Clinic of the Charles University. Director: Prof. Dr. E. Dienst bier.
and Lerch summarize the value of the salt components, as well as the phosphates, so dium, potassium, and ammonia; Frerichs in cluded the chlorides, Arlt and Lerch deter mined them separately. deRoetth mentions phosphates but Ridley in his discussion of the analytical survey of Brown says nothing
942
JOSEPH BALiK
to prove examination of phosphates in the tear fluid. Phosphates exist in body fluids as soluble primary phosphates and as alkali phosphates. In addition they are components of such solid structures as bones, teeth, muscles, and so forth. Finally they are components of organic substances in which they have their particular importance. Phosphates dissolved in the body fluids may form a buffer system, which is of less importance than the system of proteins or bicarbonates (Hamsik, San ta vy) . It seemed probable that they could be found in tears but to prove this posed tech nical difficulties. Proof of their presence in tears would have been only relative, for only minute quantities were available for investi gation, since only minute quantities are pres ent in the conjunctival sac during normal tearing.
TEAR EXAMINATION
Whatman No. 1 Chromatographie paper is soaked with tears. This paper which does not contain any phosphorus, is inserted into 1.0 ml. of trichloracetic acid. Special deproteination is not necessary because the solu tion will not become turbid, probably because of the small quantity of mucin which adheres to the paper. After 10 minutes, 4.0 ml. of sodium acetate are added. After another 10 minutes, the paper is extracted and pressed out on the wall. Any error developing during this treatment would be slight with regard to the total volume because a paper absolutely saturated during taking of the tear fluid con tains not even 100 μΐ. of fluid. Next 0.5 ml. of ascorbic acid and 0.5 ml. of ammonium molybdate are added and at the 10th minute are studied colorimetrically. BLOOD EXAMINATION
EXAMINATION AND OBSERVATIONS
The usual technique of taking of tears was used. To avoid errors due to very small amounts of tears, tear fluid was taken from both eyes and the heavier sample was always examined. The quantities of tear fluid were sufficient for the examination and repeated collections were avoided so as not to change the correlations and results. A group of 30 normal persons (14 men between 18 and 75 years of age and 16 women between 15 and 82 years of age) were examined. Blood sam ples from the elbow vein were collected at the same time as the tears. The technique of Lowry and Lopez, modi fied for present needs, was used for the ex amination ; the principle of this method is the development of a blue color of phosphomolybdate acid, as determined by the spectrophotometer at 700 πιμ. These authors say that it is possible by their method to determine in organic phosphorus in the presence of labile phosphate esters. Reagents were: 1. 2. 3. 4.
5.0-percent solution of trichloracetic acid 0.1 N sodium acetate 1.0-percent solution of ascorbic acid 1.0-percent solution of ammonium molybdate in 0.05 N sulfuric acid.
One ml. of serum is mixed with 9.0 ml. of trichloracetic acid. After standing for 10 minutes, it is centrifugated for 10 minutes. Eight ml. of sodium acetate, 1.0 ml. of as corbic acid, and 1.0 ml. of ammonium molyb date are added to the 2.0 ml. of clear super natant and colorimetrically determined under the same conditions as for tears. In both examinations (tears and blood) control samples containing the same reagents without phosphorus were colorimetrically determined in the same manner at the 10th minute and compared with the distilled water, their color being subtracted from the color of the unknown samples. Calibration. 0.439 gr. K H 2 P 0 4 is dis solved in 100 ml. of distilled water (1.0 ml. contains 1.0 mg. of P ) , 0.1 ml. of this solu tion is adjusted to 100 ml. with trichlorace tic acid (1.0 ml. contains 5.0 μg. of P ) . Then 0.5, 1.0, 1.5, and 2.0 ml. of the last solution are adjusted to 10 ml. with acetate and 1.0 ml. of ascorbic acid and 1.0 ml. of molybdate solution are added. The control sample con tains, instead of the phosphorus solution, 2.0 ml. of trichloracetic acid. The color is inves tigated in the same manner as the samples of
PHOSPHORUS IN TEARS
943
TABLE 1 SURVEY OF RESULTS
Tears
Name
Age (yr.)
Collected (mg.)
P-level /'g./mg.
B.J. K. A. K. B.
42 46 42 33 48 78 71 24 58 21 15 18 30 30 74 65 64 42 37 23 28 18 53 26 21 61 46 65 70 30
18 29 12 88 8 24 11 47 13 13 42 47 45 11 31 11 31 50 85 83 67 76 92 5 7 23 21 22 7 74
0.132 0.082 0.185 0.035 0.284 0.114 0.213 0.067 0.111 0.141 0.072 0.055 0.034 0.125 0.101 0.302 0.100 0.068 0.040 0.034 0.034 0.030 0.036 0.320 0.274 0.102 0.088 0.094 0.313 0.032
C J.
Z. M. E. M. S. M. T. F. K. B. V. A. S.V.
S. J.
M.S. S.A. N. V. H. F. V. M. H. E.
J. E.
R. V. M. B. W. H. S.A.
V.J.
B. B. S.A. N. M. S.A. H. M. P. B.
-
Blood P-level (mg.%)
Fs
4.38 2.97 4.37 4.65 4.33 3.40 3.95 3.95 2.97 4.02 5.00 4.30 3.52 3.54 5.98 5.62 5.31 6.10 4.38 4.38 3.95 4.68 3.40 3.40 4.18 4.65 3.87 3.16 3.67 3.87
3.02 2.77 4.25 0.77 6.54 3.36 5.93 1.69 3.75 3.52 1.43 1.27 0.97 3.53 1.69 5.37 1.88 2.06 0.90 0.77 0.87 0.65 1.05 9.40 6.55 2.20 2.26 2.98 8.53 0.82
takes half the quantity). The result indicates the quantity of inorganic phosphorus in each sample of tear fluid. Dividing by the weight of tears in mg. the concentration of phos phorus in 1.0 mg. of tear fluid is obtained. Blood. The quantity calculated in mg. is multiplied by 500 and the concentration of phosphorus in mg. percent is obtained. The P secretory factor (Fs) is calculated from these two values. A survey of results is given ξ = 576,64 e exp (-0,0249084 x) in Table 1. Table 2 surveys the calculated relationships. The relation of the secretory factor (Fs) to the mg. of tears is shown graphically in Figure 1.
tears and blood. The mean error of the de termination averages around three percent. Calculation of tears. The quantity of phos phorus per μg., calculated from the calibra tion equation, is divided by two (the reaction
50Q 400 3JQ 200. 10Q
DISCUSSION
f 20
40
60
80 100
mg
Fig. 1 (Balik). Relationship of secretory factor (Fs) to the mg. of tears.
The first mean value confirms the princi ple which had to be used in the investigation in order to avoid as far as possible mistakes in examining the very minute samples. The
944
JOSEPH BALiK TABLE 2 CALCULATED RELATIONSHIPS
N Average of tearing (mg.) Average quantity of P in the tears (/ag.) Average level of P in serum (mg.%) Average concentration of P of tears (mg.) Average of secretory factor (Fs) Correlation of total quantity of P in tears and blood level of P Correlation of the concentration of P in tears and blood level of P Concentration dependence of P in tears on tearing/?;* = log conc.10 3
30.00 37.43 s= 2.49 s= 4.213 s= 0.1206 s = 3.02 0.59 t= 0.034
29.05 0.593 0.817 0.0928 3.867 for 28 degrees of freedom
η*
= 2.3612-0.0105755 X b=-0.0105755
t = 20.62 for 28 degrees of freedom Dependence of the secretory factor on tearing/fl- ■log Fs. 102 9* = 2.7609-0.01081757 X b=0.0108175F t = 12.08 for 28 degrees of freedom
higher average, however, differs little from the mean value of the secretory factor, which fluctuates around 30 mg., showing that the level of inorganic phosphorus in tears is probably three times higher than in blood. This is an interesting observation. The cor relation of the total quantity of phosphorus in the conjunctival sac to the phosphorus level in blood is likewise interesting. It shows that about 60 percent of it probably depends on the blood level. The second source of phosphorus—the conjunctival goblet cells— could account for the remainder. As far as the concentration of phosphorus in tear fluid is concerned, this rather remarkable correlation could not be proved but differ ences were so slight that further tests seemed unnecessary. The concentration of phos phorus in tear fluid seems not, therefore, to depend on its blood level. Logarithmic and exponential analyses show, moreover, that the concentration falls with increased tear ing. The secretory factor behaves similarly. Its value is not constant either, but changes with a change in the amount of tear fluid. The decrease of both values can be statistic ally verified. Examination of phosphates in the tear fluid showed the mechanism of secretion compared to chlorides. Both are anions and
both seem to be secreted into tears in the same manner. However, a comparison seems to provide convincing evidence that, with chlorides, the secretory factor increases from the first tearing, perhaps because of in creased function of the conjunctival goblet cells. In phosphates quite the reverse seems true. Their concentration in tears falls as the secretion increases. The importance of phos phates as a buffer system in tears may be slight, as in serum, because their quantity is meager as compared to proteins. Phosphates could be used to adjust the pH of eyedrops. A slight decrease in concentration of phos phorus could be related to the consumption of these compounds in the production of the materials necessary as a source of energy for cell/secretory/activity. SUMMARY
Colorimetric examination of tear samples from 30 normal persons (method of Lowry and Lopez) showed that tears contain inor ganic phosphates in the amount of about 2.49 \>.g. in the average tearing of 37 mg. The con centration of phosphorus in 1.0 mg. of tears does not depend on the blood level but on the change of tearing. The secretory factor (Fs), showing that the concentration of in organic phosphorus in tears is about three times higher in average tearing, gives simi-
PHOSPHORUS IN TEARS lar results. The two values (concentration and secretory factor) have similar exponential correlation in increased tearing. This differs fundamentally from the secretion of
945
chlorides into tears; in increased tearing their secretory factors increase slowly and linearly, Jerevanskä 8/702 (13) Vrsovice.
REFERENCES
Balik, J.: Vylucoväni chloridovych iontü do slz. Cs. Of thai., 11:256, 1955. Duke-Elder, S.: Textbook of Ophthalmology, London, Kimpton, 1946, v. 1, p. 648. Lowry, O. H., and Lopez, J. A.: Investigation of inorganic phosphorus in presence of labil phosphate esters. J. Biol. Chem., 162:421, 1946. Magaard, H.: Ueber das Secret und die Secretion der menschlichen Thränendrüse. Arch. f. pathol. Anat. & Physiol., 89:258, 1882. Ridley, F.: The intraocular pressure and drainage of the aqueous humour. J. Exper. Path., 11:217, 1930. Hamsik, A., and Santavy, F.: Lekafska chemie. IV. dil, 1956, Praha.
CONGENITAL
APHAKIA*
J. R E I M E R W O L T E R , M.D., AND M A N O S A . PETROHELOS, M . D .
Ann Arbor, Michigan Congenital aphakia is an extremely rare anomaly which is usually found in eyes with other serious congenital defects. Most of the cases of this rare anomaly in the literature are clinical observations without histologic confirmation. The reported case represents a complete clinical and histopathologic study of an eye with congenital aphakia. C A S E HISTORY
This 37-year-old white man was first seen on April 25, 1957. He gave a history of having been born with his right eye blind and much smaller than normal. There was no history of any eye trauma. There was no other anomaly known in his family and there was no parental consanguinity. The pa tient had been a full-term baby. The eye examination revealed his vision to be: O.D., blindness; O.S., 20/20, with —2.0D. sph. Z +0.25D. cyl. ax. 90°. No nystagmus was seen. The right eye appeared microphthalmic and the hori zontal corneal diameter was 8.5 mm. The globe was white, conjunctiva and sclera were normal. The cornea of the right eye exhibited a white central scar in the subepithelial layers of the stroma. This scar extended horizontally toward the limbus and thus simulated a band keratopathy. The anterior * From the Department of Ophthalmic Surgery and from the Laboratory of Neuropathology of the University of Michigan Medical Center. Supported by Grants No. 2B-5163 (C) and B-475-C5 of the United States Department of Public Health Edu cation and Welfare.
chamber was deep. The pupil was central. No flare or cells were seen. The iris was atrophic. A whitish membrane was seen in the place of the lens. It was not possible to see into the posterior chamber or to visualize the fundus. However, the globe could be well transilluminated. The left eye was essentially normal. The intraocular pressure was normal in the left eye. Tonometer measurements in the right eye indicated an increased intraocular pressure. How ever it was felt that the tonometer was of no value in this microphthalmic eye with an old degenerative central scar in a small and thickened cornea. The intraocular pressure appeared normal at finger pal pation. The patient expressed the desire to have the right eye removed for cosmetic reasons. An enucleation was performed December 18, 1958. HISTOLOGIC EXAMINATION METHOD
The eye was cut in two halves both of which were imbedded in paraffin and cut in serial sections. These sections were stained with hematoxylin eosin. All pictures of this paper are unretouched photomicrographs. RESULTS
The macroscopic examination revealed the eye to be of normal shape and to measure 16 by 15 by 15 mm. The horizontal corneal diameter was 8.5 mm. After opening the globe the anterior chamber was found to be
Upon exposure to light, somehow, per haps through photochemical changes, the fila ment on the inner side may be made to be come more twisted so that the granule moves inward toward the more twisted filament. When dark adapted, the filament on the in ner side may become less twisted, so that the migrated granules can move back to their original positions. CONCLUSION
An electron-microscope study of the ret inal pigment granules of several vertebrate eyes (dog, fish, frog, monkey, mouse, and pig) produced the following observations: 1. The pigment granules revealed a num ber of forms, usually cigar-shaped, but also egg-shaped and spherical.
941
2. These granules were composed of num erous microgranules with the same shape as the parent granules. 3. The cigar-shaped pigment granules had filamentous processes at both extremities, and these filaments averaged four to five times the length of the granule itself. An hypothesis concerned with a partial explanation of the migration of pigment granules in the retinal epithelial cells is sug gested. ACKNOWLEDGMENT
I wish to acknowledge the kind assistance of L. W. Freeman, M.D., Luis Lorenz Estella, and R. E. Smith in the collection of animal eyes and the help of Fred M. Wilson, M.D., and T. F. Schlaegel, Jr., M.D., in the preparation of the paper.
REFERENCES
1. Taniguchi, Y.: Ultrastructure of pigment granules of retinal epithelium: I. Cow's eye. Am. J. Ophth., 48:221, 19S9. 2. Sebruyns, M.: Study of the ultrastructure of the retinal epithelium by means of the electron micro scope. Am. J. Ophth., 34:989, 1951. 3. Binder, R., and Orth, E.: Elektronenophtische Studie an Pigmentkornern and Zonulafasern des menschilichen Auges. Arch, f. Ophth., 154:266, 1953. 4. Fra^ois, J., Rabaey, M., and Vandermeerssche, G.: L'ultrastructure des Grains Pigmentaires de L'epithelium Retinien. Ann. ocul., 186 :896, 1953. 5. Kinugasa, H.: Electron microscopic study of eye tissues. Acta Ophth. Japon., 57 :436, 1953. 6. Kion, G., Maertens, C, and Vandermeerssche, G.: Submicroscopic morphology of the retinal pig ment epithelium. Electron Microscopy: Proc. Stockholm Conference. New York, 1957. 7. Takeuchi, H.: Morphological studies of the pigment granules of retina and choroides by electron microscope. Acta Ophth. Japon., 60 :813,1956. 8. Heydenreich, Andreas: Das morphologische and mikrochemische Verhalten der Pigmentgranula des Auges. Arch. f. Ophth., 159:162, 1957. 9. Iwaki, S.: The fine structure of the retinal pigment epithelium and capillary of choriocapillary layer, studied with the electron microscope. J. Kurme M. A., 21:1, 1958. 10. Birbeck, M. S. C, Mercer, E. H., and Barnicot, N. A.: The structure and formation of pigment granule in human hair. Exper. Cell. Research, 10:505, 1956. 11. Kolmer, W.: Handbuch der Mikroscopischen Anatomic des Menschen. Berlin, 3:2, 1936. 12. Uyama, Y.: Die Retina des Saugetieres. Med. f. Osaka LTniv., 2:3, 1951.
SECRETION O F INORGANIC P H O S P H O R U S IN TEARS* JOSEPH BALIK,
M.D.
Prague, Cscchoslovakia
The level of inorganic phosphates has not yet been determined. Magaard was not able to prove them in a qualitative examination of the tear fluid, although Frerichs and Arlt * From the First Ophthalmic Clinic of the Charles University. Director: Prof. Dr. E. Dienst bier.
and Lerch summarize the value of the salt components, as well as the phosphates, so dium, potassium, and ammonia; Frerichs in cluded the chlorides, Arlt and Lerch deter mined them separately. deRoetth mentions phosphates but Ridley in his discussion of the analytical survey of Brown says nothing
942
JOSEPH BALiK
to prove examination of phosphates in the tear fluid. Phosphates exist in body fluids as soluble primary phosphates and as alkali phosphates. In addition they are components of such solid structures as bones, teeth, muscles, and so forth. Finally they are components of organic substances in which they have their particular importance. Phosphates dissolved in the body fluids may form a buffer system, which is of less importance than the system of proteins or bicarbonates (Hamsik, San ta vy) . It seemed probable that they could be found in tears but to prove this posed tech nical difficulties. Proof of their presence in tears would have been only relative, for only minute quantities were available for investi gation, since only minute quantities are pres ent in the conjunctival sac during normal tearing.
TEAR EXAMINATION
Whatman No. 1 Chromatographie paper is soaked with tears. This paper which does not contain any phosphorus, is inserted into 1.0 ml. of trichloracetic acid. Special deproteination is not necessary because the solu tion will not become turbid, probably because of the small quantity of mucin which adheres to the paper. After 10 minutes, 4.0 ml. of sodium acetate are added. After another 10 minutes, the paper is extracted and pressed out on the wall. Any error developing during this treatment would be slight with regard to the total volume because a paper absolutely saturated during taking of the tear fluid con tains not even 100 μΐ. of fluid. Next 0.5 ml. of ascorbic acid and 0.5 ml. of ammonium molybdate are added and at the 10th minute are studied colorimetrically. BLOOD EXAMINATION
EXAMINATION AND OBSERVATIONS
The usual technique of taking of tears was used. To avoid errors due to very small amounts of tears, tear fluid was taken from both eyes and the heavier sample was always examined. The quantities of tear fluid were sufficient for the examination and repeated collections were avoided so as not to change the correlations and results. A group of 30 normal persons (14 men between 18 and 75 years of age and 16 women between 15 and 82 years of age) were examined. Blood sam ples from the elbow vein were collected at the same time as the tears. The technique of Lowry and Lopez, modi fied for present needs, was used for the ex amination ; the principle of this method is the development of a blue color of phosphomolybdate acid, as determined by the spectrophotometer at 700 πιμ. These authors say that it is possible by their method to determine in organic phosphorus in the presence of labile phosphate esters. Reagents were: 1. 2. 3. 4.
5.0-percent solution of trichloracetic acid 0.1 N sodium acetate 1.0-percent solution of ascorbic acid 1.0-percent solution of ammonium molybdate in 0.05 N sulfuric acid.
One ml. of serum is mixed with 9.0 ml. of trichloracetic acid. After standing for 10 minutes, it is centrifugated for 10 minutes. Eight ml. of sodium acetate, 1.0 ml. of as corbic acid, and 1.0 ml. of ammonium molyb date are added to the 2.0 ml. of clear super natant and colorimetrically determined under the same conditions as for tears. In both examinations (tears and blood) control samples containing the same reagents without phosphorus were colorimetrically determined in the same manner at the 10th minute and compared with the distilled water, their color being subtracted from the color of the unknown samples. Calibration. 0.439 gr. K H 2 P 0 4 is dis solved in 100 ml. of distilled water (1.0 ml. contains 1.0 mg. of P ) , 0.1 ml. of this solu tion is adjusted to 100 ml. with trichlorace tic acid (1.0 ml. contains 5.0 μg. of P ) . Then 0.5, 1.0, 1.5, and 2.0 ml. of the last solution are adjusted to 10 ml. with acetate and 1.0 ml. of ascorbic acid and 1.0 ml. of molybdate solution are added. The control sample con tains, instead of the phosphorus solution, 2.0 ml. of trichloracetic acid. The color is inves tigated in the same manner as the samples of
PHOSPHORUS IN TEARS
943
TABLE 1 SURVEY OF RESULTS
Tears
Name
Age (yr.)
Collected (mg.)
P-level /'g./mg.
B.J. K. A. K. B.
42 46 42 33 48 78 71 24 58 21 15 18 30 30 74 65 64 42 37 23 28 18 53 26 21 61 46 65 70 30
18 29 12 88 8 24 11 47 13 13 42 47 45 11 31 11 31 50 85 83 67 76 92 5 7 23 21 22 7 74
0.132 0.082 0.185 0.035 0.284 0.114 0.213 0.067 0.111 0.141 0.072 0.055 0.034 0.125 0.101 0.302 0.100 0.068 0.040 0.034 0.034 0.030 0.036 0.320 0.274 0.102 0.088 0.094 0.313 0.032
C J.
Z. M. E. M. S. M. T. F. K. B. V. A. S.V.
S. J.
M.S. S.A. N. V. H. F. V. M. H. E.
J. E.
R. V. M. B. W. H. S.A.
V.J.
B. B. S.A. N. M. S.A. H. M. P. B.
-
Blood P-level (mg.%)
Fs
4.38 2.97 4.37 4.65 4.33 3.40 3.95 3.95 2.97 4.02 5.00 4.30 3.52 3.54 5.98 5.62 5.31 6.10 4.38 4.38 3.95 4.68 3.40 3.40 4.18 4.65 3.87 3.16 3.67 3.87
3.02 2.77 4.25 0.77 6.54 3.36 5.93 1.69 3.75 3.52 1.43 1.27 0.97 3.53 1.69 5.37 1.88 2.06 0.90 0.77 0.87 0.65 1.05 9.40 6.55 2.20 2.26 2.98 8.53 0.82
takes half the quantity). The result indicates the quantity of inorganic phosphorus in each sample of tear fluid. Dividing by the weight of tears in mg. the concentration of phos phorus in 1.0 mg. of tear fluid is obtained. Blood. The quantity calculated in mg. is multiplied by 500 and the concentration of phosphorus in mg. percent is obtained. The P secretory factor (Fs) is calculated from these two values. A survey of results is given ξ = 576,64 e exp (-0,0249084 x) in Table 1. Table 2 surveys the calculated relationships. The relation of the secretory factor (Fs) to the mg. of tears is shown graphically in Figure 1.
tears and blood. The mean error of the de termination averages around three percent. Calculation of tears. The quantity of phos phorus per μg., calculated from the calibra tion equation, is divided by two (the reaction
50Q 400 3JQ 200. 10Q
DISCUSSION
f 20
40
60
80 100
mg
Fig. 1 (Balik). Relationship of secretory factor (Fs) to the mg. of tears.
The first mean value confirms the princi ple which had to be used in the investigation in order to avoid as far as possible mistakes in examining the very minute samples. The
944
JOSEPH BALiK TABLE 2 CALCULATED RELATIONSHIPS
N Average of tearing (mg.) Average quantity of P in the tears (/ag.) Average level of P in serum (mg.%) Average concentration of P of tears (mg.) Average of secretory factor (Fs) Correlation of total quantity of P in tears and blood level of P Correlation of the concentration of P in tears and blood level of P Concentration dependence of P in tears on tearing/?;* = log conc.10 3
30.00 37.43 s= 2.49 s= 4.213 s= 0.1206 s = 3.02 0.59 t= 0.034
29.05 0.593 0.817 0.0928 3.867 for 28 degrees of freedom
η*
= 2.3612-0.0105755 X b=-0.0105755
t = 20.62 for 28 degrees of freedom Dependence of the secretory factor on tearing/fl- ■log Fs. 102 9* = 2.7609-0.01081757 X b=0.0108175F t = 12.08 for 28 degrees of freedom
higher average, however, differs little from the mean value of the secretory factor, which fluctuates around 30 mg., showing that the level of inorganic phosphorus in tears is probably three times higher than in blood. This is an interesting observation. The cor relation of the total quantity of phosphorus in the conjunctival sac to the phosphorus level in blood is likewise interesting. It shows that about 60 percent of it probably depends on the blood level. The second source of phosphorus—the conjunctival goblet cells— could account for the remainder. As far as the concentration of phosphorus in tear fluid is concerned, this rather remarkable correlation could not be proved but differ ences were so slight that further tests seemed unnecessary. The concentration of phos phorus in tear fluid seems not, therefore, to depend on its blood level. Logarithmic and exponential analyses show, moreover, that the concentration falls with increased tear ing. The secretory factor behaves similarly. Its value is not constant either, but changes with a change in the amount of tear fluid. The decrease of both values can be statistic ally verified. Examination of phosphates in the tear fluid showed the mechanism of secretion compared to chlorides. Both are anions and
both seem to be secreted into tears in the same manner. However, a comparison seems to provide convincing evidence that, with chlorides, the secretory factor increases from the first tearing, perhaps because of in creased function of the conjunctival goblet cells. In phosphates quite the reverse seems true. Their concentration in tears falls as the secretion increases. The importance of phos phates as a buffer system in tears may be slight, as in serum, because their quantity is meager as compared to proteins. Phosphates could be used to adjust the pH of eyedrops. A slight decrease in concentration of phos phorus could be related to the consumption of these compounds in the production of the materials necessary as a source of energy for cell/secretory/activity. SUMMARY
Colorimetric examination of tear samples from 30 normal persons (method of Lowry and Lopez) showed that tears contain inor ganic phosphates in the amount of about 2.49 \>.g. in the average tearing of 37 mg. The con centration of phosphorus in 1.0 mg. of tears does not depend on the blood level but on the change of tearing. The secretory factor (Fs), showing that the concentration of in organic phosphorus in tears is about three times higher in average tearing, gives simi-
PHOSPHORUS IN TEARS lar results. The two values (concentration and secretory factor) have similar exponential correlation in increased tearing. This differs fundamentally from the secretion of
945
chlorides into tears; in increased tearing their secretory factors increase slowly and linearly, Jerevanskä 8/702 (13) Vrsovice.
REFERENCES
Balik, J.: Vylucoväni chloridovych iontü do slz. Cs. Of thai., 11:256, 1955. Duke-Elder, S.: Textbook of Ophthalmology, London, Kimpton, 1946, v. 1, p. 648. Lowry, O. H., and Lopez, J. A.: Investigation of inorganic phosphorus in presence of labil phosphate esters. J. Biol. Chem., 162:421, 1946. Magaard, H.: Ueber das Secret und die Secretion der menschlichen Thränendrüse. Arch. f. pathol. Anat. & Physiol., 89:258, 1882. Ridley, F.: The intraocular pressure and drainage of the aqueous humour. J. Exper. Path., 11:217, 1930. Hamsik, A., and Santavy, F.: Lekafska chemie. IV. dil, 1956, Praha.
CONGENITAL
APHAKIA*
J. R E I M E R W O L T E R , M.D., AND M A N O S A . PETROHELOS, M . D .
Ann Arbor, Michigan Congenital aphakia is an extremely rare anomaly which is usually found in eyes with other serious congenital defects. Most of the cases of this rare anomaly in the literature are clinical observations without histologic confirmation. The reported case represents a complete clinical and histopathologic study of an eye with congenital aphakia. C A S E HISTORY
This 37-year-old white man was first seen on April 25, 1957. He gave a history of having been born with his right eye blind and much smaller than normal. There was no history of any eye trauma. There was no other anomaly known in his family and there was no parental consanguinity. The pa tient had been a full-term baby. The eye examination revealed his vision to be: O.D., blindness; O.S., 20/20, with —2.0D. sph. Z +0.25D. cyl. ax. 90°. No nystagmus was seen. The right eye appeared microphthalmic and the hori zontal corneal diameter was 8.5 mm. The globe was white, conjunctiva and sclera were normal. The cornea of the right eye exhibited a white central scar in the subepithelial layers of the stroma. This scar extended horizontally toward the limbus and thus simulated a band keratopathy. The anterior * From the Department of Ophthalmic Surgery and from the Laboratory of Neuropathology of the University of Michigan Medical Center. Supported by Grants No. 2B-5163 (C) and B-475-C5 of the United States Department of Public Health Edu cation and Welfare.
chamber was deep. The pupil was central. No flare or cells were seen. The iris was atrophic. A whitish membrane was seen in the place of the lens. It was not possible to see into the posterior chamber or to visualize the fundus. However, the globe could be well transilluminated. The left eye was essentially normal. The intraocular pressure was normal in the left eye. Tonometer measurements in the right eye indicated an increased intraocular pressure. How ever it was felt that the tonometer was of no value in this microphthalmic eye with an old degenerative central scar in a small and thickened cornea. The intraocular pressure appeared normal at finger pal pation. The patient expressed the desire to have the right eye removed for cosmetic reasons. An enucleation was performed December 18, 1958. HISTOLOGIC EXAMINATION METHOD
The eye was cut in two halves both of which were imbedded in paraffin and cut in serial sections. These sections were stained with hematoxylin eosin. All pictures of this paper are unretouched photomicrographs. RESULTS
The macroscopic examination revealed the eye to be of normal shape and to measure 16 by 15 by 15 mm. The horizontal corneal diameter was 8.5 mm. After opening the globe the anterior chamber was found to be