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Ochronosis Oculi in Alkaptonuria*
OCHRONOSIS OCULI IN ALKAPTONURIA* -ONES, M . D . Washtni on, D.C.
BENJAMIN
The purpose of this paper is to record apparently for the first time the histologie findings on an eyeball from a patient whose ochronosis resulted from the congenital type of alkaptonuria. A search of the literature reveals only two histologie studies on ocular tissue, both 1 ' 2 on excised material. The case reported by Skinsnes3 is worthy of note, since it represented two tragic errors, one of diagnosis and one of commission. The pa tient's only eye was enucleated because of a mistaken diagnosis of malignant mela noma. The eye, fixed in formalin, was sent to a pathologic laboratory where merely a gross examination was made. The sciera was found to be darkly pigmented, but no neoplastic growth or other abnormality was noted. The discoloration was thought to be due to improper fixation or to fixation arte fact, and the specimen discarded. One year later he died, and autopsy showed general ized ochronosis, with striking pigmentation of all cartilages examined. Hereditary ochronosis may be defined as an inbred disorder of phenylalanine and tyrosine intermediary metabolism, in which the initial expression is usually the spon taneous urinary excretion of homogentisic acid. Ochronosis and alkaptonuria were at one time considered to be synonymous terms, but now it is generally agreed that the alkap tonuria or urinary excretion of homogentisic acid is only one symptom of ochronosis. The disease is relatively infrequent, mani festing itself in early life and shows a famalial pattern often associated with consan guinity. Genetically, ochronosis and albinism are similar, since both are Mendelian reces sive traits. This similarity also extends to the conversion of tyrosine into pigment, for in * From the Laboratory of Ophthalmic Pathology, Washington Hospital Center. Read at the meeting of the Wilmer Residents Association, Baltimore, March, 19S9.
albinism this transition mechanism is entirely lacking, while in ochronsis the mechanism for breaking down tyrosine further than homogentisic acid is at fault. Homogentisic acid is a normal intermediary compound in tyrosine metabolism, and it is accepted that the excretion of homogentisic acid in alkap tonuria is due to a defect in the enzyme cata lyzing the oxidation of homogentisic acid, homogentisic acid oxidase. LaDu and co-workers4 utilizing liver bi opsy tissue obtained from the presently re corded case were not able to detect any homogentisic acid oxidase activity, appar ently due to a failure to synthesize the active enzyme, rather than to the presence of an inhibitor or to the absence of any known co-factor. Thus, this disorder is another addition to those metabolic derangements known to be due to a defect in a single enzyme system, such as glycogen storage disease, galactosemia, and phenylketonuria. There is also experimental evidence5 indi cating that enzymes are under gene control and that single genes may control biochemi cal reactions through the mediation of spe cific enzymes. Thus inherited gene alterations or defects in genes may lead to loss of spe cific enzymes. Boedeker6 (1859) found in the urine of a diabetic a second reducing substance, which turned brown on the addition of an alkali and which he termed alkapton. This reducing substance was later indentified as a homo logue of gentisic acid, and thus was called homogentisic acid. It was noted that urine containing this matter was of normal color when first voided, but after contact with air turned brown or black, and that linen or woolen articles moistened with the urine took these stains. Virchow7 observed on post-mortem ex amination a discoloration of cartilage due to pale yellow or "ochre" pigment granules,
440
OCHRONOSIS OCULI IN ALKAPTONURIA
and thus the name "ochronosis" was applied. In 1902, Albrecht8 suggested that ochrono sis was caused by alkaptonuria, but Osier 9 (1904) was the first to describe the clinical features of the disease and make the diag nosis during life, pointing out the pigmenta tion of the eyes, nose, ears, skin and nails. The earliest clinical recognition of this disorder may be due to the darkened carti lages of the ear or of the nose, or to the brown pigment spots in the sciera near the insertion of the rectus tendons. Since this is an inherited condition, the defective oxida tion of homogentisic acid and its urinary excretion is present from birth, but during the early years there are no harmful effects. Later, however, a percentage of patients develop secondary changes due to the gradual deposition of the homogentisic acid in certain tissues of the body as a peculiar pigment. The disorder is not a harmless curiosity, for a deforming arthritis and spondylitis, severe arteriosclerotic changes in the heart with pigmentation of the valves, the aorta and coronary arteries, and ochronotic nephrosis may appear and progress. The first comprehensive ophthalmologic publication, including the first slitlamp de scription, was by von Sallmann.10 He con sidered the symmetric arrangement of pig ment granules in the corneal periphery and the scierai pigmentation to be of diagnostic significance. Smith11 (1942) published the best review of this subject in the ophthalmologic litera ture, and contributed four case reports. At this time he was able to state that the total number of cases recorded, including his own, was 82. As stated in the opening of this presentation, there have been only two histo logie reports on excised ocular tissue. Seitz1 (1954) biopsied the corneoconjunctival margin of an eye and stained the sec tions with hematoxylin-eosin and with other special stains. He found that in the subconjunctival tissue there were areas in which the elastic fibers were swollen and frag mented, and adjoining were plump yellowish-
441
brown bodies of variable density. These masses varied considerably in shape and contour and frequently multinucleated for eign-body giant cells were found adjacent to them. In the tiny corneal fragment he found round, yellowish depositions, sharply defined and without a surrounding cellular reaction. Rodenhäuser 2 reported the other biopsy material in 1957. He details the clinical and laboratory findings in a 61-year-old man, in whom he excised a segment of bulbar con junctiva from the temporal side. The sec tions were stained with hematoxylin-eosin, brilliant cresyl-violet and orcein-hematoxylin-picrofuchsin. H e found that beneath the epithelium, the connective tissue fibers were partly fragmented and homogeneous, ad jacent to numerous lumps of varying size and without definite structure. He felt that in their appearance and staining reactions these lumps resembled the hyaline masses of a pinguecula, and regarded the disturbance caused by homogentisic acid as a premature senile degeneration of connective tissue. The case presented here has been studied clinically at the National Institute of Health and published by Bunim12 and co-workers in 1957. C A S E REPORT
The patient was a 56-year-white man who had been diagnosed as having alkaptonuria in 1949. In infancy his parents noticed that his diapers took on a brownish-black color soon after they were soiled with urine. His urine continued to turn dark upon exposure to air. In 1946, an erroneous diag nosis of diabetes mellitus was made because of the presence of a urinary reducing substance. Since this could not be eliminated by rigid dietary restriction, further investigations established the correct diag nosis. He had progressive symptoms of joint dis ease dating back to 1934, with a gradual loss of spinal mobility and stiffness of his knees. For about 10 years there has been a gradual deposition of brownish-black pigment in the sciera of each eye, both nasal and lateral to the limbus. There has also been a progressive grayish discoloration of the tip of the nose and both ears, chiefly the conchae. In 1956, he was thoroughly studied at the National In stitute of Arthritis and Metabolic Diseases, and the diagnosis of alkaptonuria, orchronosis and arthritis verified.12 In February, 1957, an esophageal hiatus hernia was repaired, at which time a liver biopsy had
BENJAMIN RONES
442
been performed for enzyme studies.4 He was ad mitted into the Washington Hospital Center on June 18, 1958, because of mental depression and died two days later. An autopsy was performed and showed deposition of finely granular brown pigment in the trachea! cartilages in a narrow band, diffusely in the costochondral junction, and throughout the substance of the intervertébral disc. The left eye was removed for pathologic ex amination. PATHOLOGIC EXAMINATION OF EYE GROSS
The specimen consists of a firm formalinfixed eye measuring 25 by 25 by 24.5 mm. There are two brown spots on the sciera, 2.0 to 3.0 mm. behind the limbus on the nasal and temporal sides, corresponding to the 9- and 3-o'clock positions. The largest, which measures 3.0 mm. in diameter, is situated on the lateral side of the globe between the limbus and lateral rectus. Part of the lesion has a granular appearance. There are num erous brown punctate spots at the corneoscleral margin between the 3- and 4-o'clock positions. The posterior portion of the sciera on the temporal side has a brownish tint. Transillumination is very good except in the area of scierai discoloration where a faint shadow is seen. The eye is opened in the
horizontal plane. An opaque stringy ma terial is present in the vitreous. There is a retinal fold in the macular region. There are no intraocular lesions. MICROSCOPIC
Examination of the celloidin-embedded tissue shows the corneal architecture to be normal, except that near the temporal limbus there are several subepithelial structureless golden-colored bodies (fig. 1) in the stroma separating the lamellae. There are no cellular reactions in their environs. The anterior chamber is deep and the angles are widely open. The iris is normal. The lens shows no abnormalities. The pos terior portion of the globe is normal. There is pigmentation of the anterior sciera, both nasally and temporally, in the outer layers overlying the anterior portion of the pars plana. This is most marked on the temporal side. The pigmentation is diffuse, and of a golden color in contrast to the black of the pigment epithelium of the retina and iris. The connective tissue bundles of the sciera are swollen in this area, and are distorted by pigmented clumps (fig. 2) of translucent, structureless substance ranging in size from granules to coalescent masses.
Fig. 1 (Rones). X350. Subepithelial deposit of golden-colored material in corneal stroma. No surrounding tissue reaction.
OCHRONOSIS OCULI IN ALKAPTONURIA
443
Fig. 2 (Rones). χΙΟΟ. Deposits in sciera and episclera. (Hematoxylin-eosin.)
There is no cellular reaction in this area. In the neighboring episclera (fig. 2) there are convoluted masses of this golden material which in some sections appear to be in flat platelike shapes. There is a sparse infiltration of large mononuclear cells in this area, and the overlying epithelium is intact, The hematoxylin-eosin stain was used as the routine technique on these sections. In
an attempt to distinguish the ochronotic pigment from melanin, a battery of staining techniques was employed. There were no differences noted in the preparations which were bleached, stained for melanin, iron, fats, acid and neutral mucopolysaccharides, calcium, phosphate and connective tissues. The use of special stains did, however, offer some suggestive evidence of the nature
Fig. 3 (Rones). Degenerated elastica in episclera. (Weil myelin stain, [top] XSO; [bottom] X300.)
444
BENJAMIN RONES
Fig. 6 (Rones). X300. Laminations in episcleral masses of degenerated elastica. (Weil myelin stain.)
Fig. 4 (Rones). X30Ô. Degenerated elastica and collagen in sciera. (Weil myelin stain.) of the tissue reaction, if not of the pigment itself. It is apparent from the study of the cornea, sciera and episclera, that it is the deposition of the melaninlike substance in the collagen bundles of the sciera and cornea, and in the elastic tissue of the conjunctiva and episclera that produces the patterns de scribed. The most rewarding of these stains
are the Weil myelin sheath stain (figs. 3 and 4) and the Verhoeff elastic tissue stain (fig. 5). With both of these stains, the pig ment takes a blackish color. In the subconjunctival tissue the elastic tissue fibrils cart be traced from their normal wavy fibrils through stages of swelling, fragmentation and mass formation. In the sciera these masses show the same relationship to the bundles of collagen fibers. It can be seen that these masses are not solid but show laminations (fig. 6) as of age and accretion, and that their surface is not smooth
* ♦
Fig. 5 (Rones). X70. Degenerated elastica and collagen. (Verhoeff elastic tissue stain.)
OCHRONOSIS OCULI IN ALKAPTONURIA
445
Fig. 7 (Rones'). χ485. Irregular "furry" surface of degenerated elastica. (Verhoeff elastic tissue stain.)
Fig. 8 (Rones). XS7S. Degenerated elastica and surrounding mast cells. (PAS stain.)
but presents a "furry" appearance (fig. 7). With the PAS stain (fig. 8) it is evident that the cellular reaction around the de generated elastic tissue fibers is of the mast cell variety, and the cells are PAS positive. Cresyl violet also stained the pigment almost black, but was not as clearcut as the above two stains. DISCUSSION
These results were consistent with the observations reported by Cooper and Moran 13 who utilized electron microscopy and special stains to study ochronotic pigment in vari ous locations in comparison with melanin of normal white skin and melanin of a heavily pigmented nevus. Their chemical studies in cluded extraction and isolation of the ochro notic pigment from cartilage, liver and kid ney. They could detect no differences be
tween ochronotic pigment and melanin, and even though they feel that the exact nature and chemical composition of the ochronotic pigment has not been established it is to be accepted as a melanin or melaninlike sub stance. The observations in the present case are in agreement with the biopsied material of Seitz 1 as concerns the episcleral tissue. How ever, no explanation can be offered as to the localization of the pigment deposits in the eye, particularly the predilection for the palpebral aperture. SUMMARY
The histopathologic study of an eye con taining the ochronotic pigment in a patient with alkaptonuria is offered as apparently the first presentation in the literature. Special stains did not reveal any differences of be-
BENJAMIN RONES
446
havior of this pigment from melanin. These stains, however, did suggest that the pigmented material present was the result of degeneration of the elastic tissue fibers and the collagen bundles, apparently the reaction of these tissues to the deposited pigment. T h e reason for the deposition at the particu lar sites in the eyes cannot be determined,
nor does speculation offer any fruitful an swers. 1302 18th Street, N.W. (6).
I wish to express my deep appreciation to Dr. Henry W. Edmonds for providing the globe from the autopsy, and for his suggestions on the key arti cles in the realm of general pathology.
REFERENCES
1. Seitz, R.: Ueber die ochronosischen Pigmentierungen am Auge Klin. Monatsbl. f. Augenh. 124:432, 1954. 2. Rodenhäuser, J. H.: Pigmentation of the eyes in alkaptonuria. Klin. Monatsbl. f Augenh., 131:202, 1957. 3. Skinsnes, O. K.: Generalized ochronosis. Arch. Path., 45:552, 1948. 4. La Du, B. N., Zannoni, V. G., Laster, L., and Seegmiller, J. E. : The nature of the defect in tyrosine metabolism in alcaptonuria. J. Biol. Chem., 230:251, 1958. 5. Beadle, G. W., and Tatum, E. L. : Genetic control of biochemical reactions in neurospora. Proc. Nat. Acad. Sc, 27:499, 1941. 6. Boedeker, A.: Ueber des Alkapton. Ztschr. f. rat. Med., 7:130, 1859. 7. Virchow, R.: Ein Fall von allgemeiner Ochronose der Knorpel und Knorperähnlichen Teile. Virchows Arch. f. path. Anat, 37:212, 1866. 8. Albrecht, H.: Ueber Ochronose, Ztschr. f. Heilk., 23:366, 1902. 9. Osier, W.: Ochronosis: The pigmentation of cartilages, sclerotics and skin in alkaptonuria. Lancet, 1:10,1904. 10. v. Sallmann, L.: Ueber die Augenpigmentierung bei endogener Ochronose, Ztschr. f. Augenh., 60: 164, 1926. 11. Smith, J. W.: Ochronosis of the sciera and cornea complicating alkaptonuria. J.A.M.A., 120:1282, 1942. 12. Bunim, J. J., McGuire, J. S. Jr., Hilbish, T. F., Laster, L., La Du, B. N. Jr., and Seegmiller, J. E.: Alcaptonuria. (Clin. Staff Conf. National Inst. Health.) Ann. Int. Med., 47:1210, 1957. 13. Cooper, J. A., and Moran, T. J.: Studies on ochronosis. Arch. Path., 64:46, 1957.
EFFECTS OF ELECTROLYTE MIXTURES ON FROM HUMAN IRIS* JOHN
Y.
HARPER, JR., CAPT.
(MC)
CELLS
U.S.A.F.
San Antonio, Texas AND C.
M.
POMERAT,
PH.D.
Galveston, Texas INTRODUCTION
" N o r m a l " saline is commonly employed in the armamentarium of the ophthalmolo gist as an irrigating solution during the per formance of intraocular surgery. This solu tion has the advantages of being readily available, low in cost and easily sterilized. Several investigators have noted that this socalled "physiologic" saline is actually dam aging when placed in contact with living ani mal cells. T h e argument that there is little
gross evidence of direct injury should not prevent efforts to investigate the more subtle * From the United States Air Force Base, San Antonio, and the Department of Ophthalmology and the Tissue Culture Laboratory, Department of Anatomy, University of Texas Medical Branch, Galveston. Aided by a grant from the National Society for the Prevention of Blindness, July 26, 1957. The contents of this article are the personal views of the authors and are not to be construed as statement of official Air Force policy nor as Air Force endorsement of any commercial product described.
BENJAMIN
The purpose of this paper is to record apparently for the first time the histologie findings on an eyeball from a patient whose ochronosis resulted from the congenital type of alkaptonuria. A search of the literature reveals only two histologie studies on ocular tissue, both 1 ' 2 on excised material. The case reported by Skinsnes3 is worthy of note, since it represented two tragic errors, one of diagnosis and one of commission. The pa tient's only eye was enucleated because of a mistaken diagnosis of malignant mela noma. The eye, fixed in formalin, was sent to a pathologic laboratory where merely a gross examination was made. The sciera was found to be darkly pigmented, but no neoplastic growth or other abnormality was noted. The discoloration was thought to be due to improper fixation or to fixation arte fact, and the specimen discarded. One year later he died, and autopsy showed general ized ochronosis, with striking pigmentation of all cartilages examined. Hereditary ochronosis may be defined as an inbred disorder of phenylalanine and tyrosine intermediary metabolism, in which the initial expression is usually the spon taneous urinary excretion of homogentisic acid. Ochronosis and alkaptonuria were at one time considered to be synonymous terms, but now it is generally agreed that the alkap tonuria or urinary excretion of homogentisic acid is only one symptom of ochronosis. The disease is relatively infrequent, mani festing itself in early life and shows a famalial pattern often associated with consan guinity. Genetically, ochronosis and albinism are similar, since both are Mendelian reces sive traits. This similarity also extends to the conversion of tyrosine into pigment, for in * From the Laboratory of Ophthalmic Pathology, Washington Hospital Center. Read at the meeting of the Wilmer Residents Association, Baltimore, March, 19S9.
albinism this transition mechanism is entirely lacking, while in ochronsis the mechanism for breaking down tyrosine further than homogentisic acid is at fault. Homogentisic acid is a normal intermediary compound in tyrosine metabolism, and it is accepted that the excretion of homogentisic acid in alkap tonuria is due to a defect in the enzyme cata lyzing the oxidation of homogentisic acid, homogentisic acid oxidase. LaDu and co-workers4 utilizing liver bi opsy tissue obtained from the presently re corded case were not able to detect any homogentisic acid oxidase activity, appar ently due to a failure to synthesize the active enzyme, rather than to the presence of an inhibitor or to the absence of any known co-factor. Thus, this disorder is another addition to those metabolic derangements known to be due to a defect in a single enzyme system, such as glycogen storage disease, galactosemia, and phenylketonuria. There is also experimental evidence5 indi cating that enzymes are under gene control and that single genes may control biochemi cal reactions through the mediation of spe cific enzymes. Thus inherited gene alterations or defects in genes may lead to loss of spe cific enzymes. Boedeker6 (1859) found in the urine of a diabetic a second reducing substance, which turned brown on the addition of an alkali and which he termed alkapton. This reducing substance was later indentified as a homo logue of gentisic acid, and thus was called homogentisic acid. It was noted that urine containing this matter was of normal color when first voided, but after contact with air turned brown or black, and that linen or woolen articles moistened with the urine took these stains. Virchow7 observed on post-mortem ex amination a discoloration of cartilage due to pale yellow or "ochre" pigment granules,
440
OCHRONOSIS OCULI IN ALKAPTONURIA
and thus the name "ochronosis" was applied. In 1902, Albrecht8 suggested that ochrono sis was caused by alkaptonuria, but Osier 9 (1904) was the first to describe the clinical features of the disease and make the diag nosis during life, pointing out the pigmenta tion of the eyes, nose, ears, skin and nails. The earliest clinical recognition of this disorder may be due to the darkened carti lages of the ear or of the nose, or to the brown pigment spots in the sciera near the insertion of the rectus tendons. Since this is an inherited condition, the defective oxida tion of homogentisic acid and its urinary excretion is present from birth, but during the early years there are no harmful effects. Later, however, a percentage of patients develop secondary changes due to the gradual deposition of the homogentisic acid in certain tissues of the body as a peculiar pigment. The disorder is not a harmless curiosity, for a deforming arthritis and spondylitis, severe arteriosclerotic changes in the heart with pigmentation of the valves, the aorta and coronary arteries, and ochronotic nephrosis may appear and progress. The first comprehensive ophthalmologic publication, including the first slitlamp de scription, was by von Sallmann.10 He con sidered the symmetric arrangement of pig ment granules in the corneal periphery and the scierai pigmentation to be of diagnostic significance. Smith11 (1942) published the best review of this subject in the ophthalmologic litera ture, and contributed four case reports. At this time he was able to state that the total number of cases recorded, including his own, was 82. As stated in the opening of this presentation, there have been only two histo logie reports on excised ocular tissue. Seitz1 (1954) biopsied the corneoconjunctival margin of an eye and stained the sec tions with hematoxylin-eosin and with other special stains. He found that in the subconjunctival tissue there were areas in which the elastic fibers were swollen and frag mented, and adjoining were plump yellowish-
441
brown bodies of variable density. These masses varied considerably in shape and contour and frequently multinucleated for eign-body giant cells were found adjacent to them. In the tiny corneal fragment he found round, yellowish depositions, sharply defined and without a surrounding cellular reaction. Rodenhäuser 2 reported the other biopsy material in 1957. He details the clinical and laboratory findings in a 61-year-old man, in whom he excised a segment of bulbar con junctiva from the temporal side. The sec tions were stained with hematoxylin-eosin, brilliant cresyl-violet and orcein-hematoxylin-picrofuchsin. H e found that beneath the epithelium, the connective tissue fibers were partly fragmented and homogeneous, ad jacent to numerous lumps of varying size and without definite structure. He felt that in their appearance and staining reactions these lumps resembled the hyaline masses of a pinguecula, and regarded the disturbance caused by homogentisic acid as a premature senile degeneration of connective tissue. The case presented here has been studied clinically at the National Institute of Health and published by Bunim12 and co-workers in 1957. C A S E REPORT
The patient was a 56-year-white man who had been diagnosed as having alkaptonuria in 1949. In infancy his parents noticed that his diapers took on a brownish-black color soon after they were soiled with urine. His urine continued to turn dark upon exposure to air. In 1946, an erroneous diag nosis of diabetes mellitus was made because of the presence of a urinary reducing substance. Since this could not be eliminated by rigid dietary restriction, further investigations established the correct diag nosis. He had progressive symptoms of joint dis ease dating back to 1934, with a gradual loss of spinal mobility and stiffness of his knees. For about 10 years there has been a gradual deposition of brownish-black pigment in the sciera of each eye, both nasal and lateral to the limbus. There has also been a progressive grayish discoloration of the tip of the nose and both ears, chiefly the conchae. In 1956, he was thoroughly studied at the National In stitute of Arthritis and Metabolic Diseases, and the diagnosis of alkaptonuria, orchronosis and arthritis verified.12 In February, 1957, an esophageal hiatus hernia was repaired, at which time a liver biopsy had
BENJAMIN RONES
442
been performed for enzyme studies.4 He was ad mitted into the Washington Hospital Center on June 18, 1958, because of mental depression and died two days later. An autopsy was performed and showed deposition of finely granular brown pigment in the trachea! cartilages in a narrow band, diffusely in the costochondral junction, and throughout the substance of the intervertébral disc. The left eye was removed for pathologic ex amination. PATHOLOGIC EXAMINATION OF EYE GROSS
The specimen consists of a firm formalinfixed eye measuring 25 by 25 by 24.5 mm. There are two brown spots on the sciera, 2.0 to 3.0 mm. behind the limbus on the nasal and temporal sides, corresponding to the 9- and 3-o'clock positions. The largest, which measures 3.0 mm. in diameter, is situated on the lateral side of the globe between the limbus and lateral rectus. Part of the lesion has a granular appearance. There are num erous brown punctate spots at the corneoscleral margin between the 3- and 4-o'clock positions. The posterior portion of the sciera on the temporal side has a brownish tint. Transillumination is very good except in the area of scierai discoloration where a faint shadow is seen. The eye is opened in the
horizontal plane. An opaque stringy ma terial is present in the vitreous. There is a retinal fold in the macular region. There are no intraocular lesions. MICROSCOPIC
Examination of the celloidin-embedded tissue shows the corneal architecture to be normal, except that near the temporal limbus there are several subepithelial structureless golden-colored bodies (fig. 1) in the stroma separating the lamellae. There are no cellular reactions in their environs. The anterior chamber is deep and the angles are widely open. The iris is normal. The lens shows no abnormalities. The pos terior portion of the globe is normal. There is pigmentation of the anterior sciera, both nasally and temporally, in the outer layers overlying the anterior portion of the pars plana. This is most marked on the temporal side. The pigmentation is diffuse, and of a golden color in contrast to the black of the pigment epithelium of the retina and iris. The connective tissue bundles of the sciera are swollen in this area, and are distorted by pigmented clumps (fig. 2) of translucent, structureless substance ranging in size from granules to coalescent masses.
Fig. 1 (Rones). X350. Subepithelial deposit of golden-colored material in corneal stroma. No surrounding tissue reaction.
OCHRONOSIS OCULI IN ALKAPTONURIA
443
Fig. 2 (Rones). χΙΟΟ. Deposits in sciera and episclera. (Hematoxylin-eosin.)
There is no cellular reaction in this area. In the neighboring episclera (fig. 2) there are convoluted masses of this golden material which in some sections appear to be in flat platelike shapes. There is a sparse infiltration of large mononuclear cells in this area, and the overlying epithelium is intact, The hematoxylin-eosin stain was used as the routine technique on these sections. In
an attempt to distinguish the ochronotic pigment from melanin, a battery of staining techniques was employed. There were no differences noted in the preparations which were bleached, stained for melanin, iron, fats, acid and neutral mucopolysaccharides, calcium, phosphate and connective tissues. The use of special stains did, however, offer some suggestive evidence of the nature
Fig. 3 (Rones). Degenerated elastica in episclera. (Weil myelin stain, [top] XSO; [bottom] X300.)
444
BENJAMIN RONES
Fig. 6 (Rones). X300. Laminations in episcleral masses of degenerated elastica. (Weil myelin stain.)
Fig. 4 (Rones). X30Ô. Degenerated elastica and collagen in sciera. (Weil myelin stain.) of the tissue reaction, if not of the pigment itself. It is apparent from the study of the cornea, sciera and episclera, that it is the deposition of the melaninlike substance in the collagen bundles of the sciera and cornea, and in the elastic tissue of the conjunctiva and episclera that produces the patterns de scribed. The most rewarding of these stains
are the Weil myelin sheath stain (figs. 3 and 4) and the Verhoeff elastic tissue stain (fig. 5). With both of these stains, the pig ment takes a blackish color. In the subconjunctival tissue the elastic tissue fibrils cart be traced from their normal wavy fibrils through stages of swelling, fragmentation and mass formation. In the sciera these masses show the same relationship to the bundles of collagen fibers. It can be seen that these masses are not solid but show laminations (fig. 6) as of age and accretion, and that their surface is not smooth
* ♦
Fig. 5 (Rones). X70. Degenerated elastica and collagen. (Verhoeff elastic tissue stain.)
OCHRONOSIS OCULI IN ALKAPTONURIA
445
Fig. 7 (Rones'). χ485. Irregular "furry" surface of degenerated elastica. (Verhoeff elastic tissue stain.)
Fig. 8 (Rones). XS7S. Degenerated elastica and surrounding mast cells. (PAS stain.)
but presents a "furry" appearance (fig. 7). With the PAS stain (fig. 8) it is evident that the cellular reaction around the de generated elastic tissue fibers is of the mast cell variety, and the cells are PAS positive. Cresyl violet also stained the pigment almost black, but was not as clearcut as the above two stains. DISCUSSION
These results were consistent with the observations reported by Cooper and Moran 13 who utilized electron microscopy and special stains to study ochronotic pigment in vari ous locations in comparison with melanin of normal white skin and melanin of a heavily pigmented nevus. Their chemical studies in cluded extraction and isolation of the ochro notic pigment from cartilage, liver and kid ney. They could detect no differences be
tween ochronotic pigment and melanin, and even though they feel that the exact nature and chemical composition of the ochronotic pigment has not been established it is to be accepted as a melanin or melaninlike sub stance. The observations in the present case are in agreement with the biopsied material of Seitz 1 as concerns the episcleral tissue. How ever, no explanation can be offered as to the localization of the pigment deposits in the eye, particularly the predilection for the palpebral aperture. SUMMARY
The histopathologic study of an eye con taining the ochronotic pigment in a patient with alkaptonuria is offered as apparently the first presentation in the literature. Special stains did not reveal any differences of be-
BENJAMIN RONES
446
havior of this pigment from melanin. These stains, however, did suggest that the pigmented material present was the result of degeneration of the elastic tissue fibers and the collagen bundles, apparently the reaction of these tissues to the deposited pigment. T h e reason for the deposition at the particu lar sites in the eyes cannot be determined,
nor does speculation offer any fruitful an swers. 1302 18th Street, N.W. (6).
I wish to express my deep appreciation to Dr. Henry W. Edmonds for providing the globe from the autopsy, and for his suggestions on the key arti cles in the realm of general pathology.
REFERENCES
1. Seitz, R.: Ueber die ochronosischen Pigmentierungen am Auge Klin. Monatsbl. f. Augenh. 124:432, 1954. 2. Rodenhäuser, J. H.: Pigmentation of the eyes in alkaptonuria. Klin. Monatsbl. f Augenh., 131:202, 1957. 3. Skinsnes, O. K.: Generalized ochronosis. Arch. Path., 45:552, 1948. 4. La Du, B. N., Zannoni, V. G., Laster, L., and Seegmiller, J. E. : The nature of the defect in tyrosine metabolism in alcaptonuria. J. Biol. Chem., 230:251, 1958. 5. Beadle, G. W., and Tatum, E. L. : Genetic control of biochemical reactions in neurospora. Proc. Nat. Acad. Sc, 27:499, 1941. 6. Boedeker, A.: Ueber des Alkapton. Ztschr. f. rat. Med., 7:130, 1859. 7. Virchow, R.: Ein Fall von allgemeiner Ochronose der Knorpel und Knorperähnlichen Teile. Virchows Arch. f. path. Anat, 37:212, 1866. 8. Albrecht, H.: Ueber Ochronose, Ztschr. f. Heilk., 23:366, 1902. 9. Osier, W.: Ochronosis: The pigmentation of cartilages, sclerotics and skin in alkaptonuria. Lancet, 1:10,1904. 10. v. Sallmann, L.: Ueber die Augenpigmentierung bei endogener Ochronose, Ztschr. f. Augenh., 60: 164, 1926. 11. Smith, J. W.: Ochronosis of the sciera and cornea complicating alkaptonuria. J.A.M.A., 120:1282, 1942. 12. Bunim, J. J., McGuire, J. S. Jr., Hilbish, T. F., Laster, L., La Du, B. N. Jr., and Seegmiller, J. E.: Alcaptonuria. (Clin. Staff Conf. National Inst. Health.) Ann. Int. Med., 47:1210, 1957. 13. Cooper, J. A., and Moran, T. J.: Studies on ochronosis. Arch. Path., 64:46, 1957.
EFFECTS OF ELECTROLYTE MIXTURES ON FROM HUMAN IRIS* JOHN
Y.
HARPER, JR., CAPT.
(MC)
CELLS
U.S.A.F.
San Antonio, Texas AND C.
M.
POMERAT,
PH.D.
Galveston, Texas INTRODUCTION
" N o r m a l " saline is commonly employed in the armamentarium of the ophthalmolo gist as an irrigating solution during the per formance of intraocular surgery. This solu tion has the advantages of being readily available, low in cost and easily sterilized. Several investigators have noted that this socalled "physiologic" saline is actually dam aging when placed in contact with living ani mal cells. T h e argument that there is little
gross evidence of direct injury should not prevent efforts to investigate the more subtle * From the United States Air Force Base, San Antonio, and the Department of Ophthalmology and the Tissue Culture Laboratory, Department of Anatomy, University of Texas Medical Branch, Galveston. Aided by a grant from the National Society for the Prevention of Blindness, July 26, 1957. The contents of this article are the personal views of the authors and are not to be construed as statement of official Air Force policy nor as Air Force endorsement of any commercial product described.