Download Calcium Oxalate Retinopathy Associated with

Calcium oxalate retinopathy associated with
generalized oxalosis: x-ray diffraction and
electron microscopic studies of
crystal deposits
John D. Bullock, Daniel M. Albert, H. Catherine W. Skinner,
William H. Miller, and John H. Galla
This reports crystallographic and light and electron microscopic findings in a previously
undescribed entity: calcium oxalate retinopathy associated with generalized oxalosis. The
affected patient was a 66-year-old vohite male with a previous history of mild hypertensive
renal disease who had undergone prolonged abdominal surgery under methoxyfiurane (MOF;
Penthrane) anesthesia. He subsequently developed acute irreversible renal failure caused
by renal oxalosis as shown by renal biopsy. He was maintained on renal dialysis and shortly before his death three years later he was noted to have changes in the fundus oculi
suggestive of the flecked retina syndrome. Widespread oxalosis was found at autopsy.
Light and electron microscopy revealed particularly heavy depositions within the retinal
pigment epithelium (RPE). Positive identification of the deposits as the monohydrate form
of calcium oxalate was obtained by x-ray diffraction investigations.
A
pected to be due to methoxyfiurane (MOF;
Penthrane) anesthesia which he had received three and one-half years prior to
his death and which was followed by the
immediate development of irreversible renal failure. At subsequent postmortem
examination, diffuse deposition of calcium
oxalate crystals was found in the retinal
pigment epithelium (RPE) as well as in
the kidney, thyroid, epididymis, myocardium, and bronchus. This report describes
the x-ray diffraction and histochemical
characteristics of the systemic and ocular
crystals and electron microscopic study
of the involved retinal pigment epithelium.
fundus appearance resembling that
of the "flecked retina syndrome" and particularly suggestive of fundus albipunctatus
was observed in a patient with irreversible
renal failure. His renal disease was sus-
From the Department of Ophthalmology, Sections
of Orthopedics and Dentistry, and Department
of Medicine, Yale University School of Medicine, New Haven, Conn.
This study was supported by United States Public Health Service Grant EY000108-04, the
United States Public Health Service Vision
Center Grant EY00785-03, and the Connecticut Lions Eye Research Foundation, Incorporated.
Submitted for publication Oct. 19, 1973.
Reprint requests: Dr. John D. Bullock, Department of Ophthalmology and Visual Science,
Yale University School of Medicine, 333 Cedar
St., New Haven, Conn. 06150.
Results
Case report. The patient was first seen
at Yale-New Haven Hospital in 1967, when
256
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he was 61-years-of-age with complaints of
arthritis and ocular discomfort. A careful
review of his history revealed that he had
been seen by ophthalmologists in 1955,
1962, and 1966. Except for the presence of
a presumed bacterial conjunctivitis in 1966,
his eye examinations were reported as being
entirely within normal limits at each of
these times. His visual acuity on each occasion was better than 20/20 OU and intraocular pressures were within normal limits.
Findings on slit lamp examination were
unremarkable and the fundi were entirely
normal in appearance. At the time of his
1967 visit he had an extensive evaluation,
the results of which included a normal
urinalysis and a serum creatinine of 1.5
mg. per 100 ml. A diagnosis of seronegative
rheumatoid arthritis was made. He had a
mild episcleritis, but his eye examination
was otherwise within normal limits. The
episcleritis cleared rapidly with the use
of topical steroids.
In January, 1969, the patient was admitted to Yale-New Haven Hospital with
a clinical presentation of obstructive jaundice. The patient was otherwise in good
general health except for mild hypertensive
nephropathy with a serum creatinine of
3.5 mg. per 100 ml. He underwent a four
and one-half hour long exploratory laparotomy under MOF anesthesia during
which time correction of an intermittently
obstructing duodenal diverticulum was performed. Although at the time of surgery
the patient appeared to tolerate the procedure well, he developed acute irreversible
nonoliguric renal failure immediately postoperatively, and a percutaneous renal biopsy performed on the thirty-eighth postoperative day showed extensive renal oxalate deposition. A diagnosis of acute renal
failure associated with massive oxalate
crystal deposition was made. The patient
was subsequently maintained on hemodialysis for three and one-half years. The
patient's general medical condition deteriorated and he died in September, 1972.
Two weeks prior to his death the patient
was examined as part of a program in
which the eyes of terminal patients are
Calcium oxalate retinopathy 257
Fig. 1. Fundus photograph, RE, showing numerous
yellowish white punctate lesions in the posterior
pole.
examined and clinical and pathologic correlations are made when possible.1 The
corrected visual acuity was 20/30 OU and
mild bilateral nuclear sclerosis was noted.
Dilated funduscopic examination revealed
numerous yellow-white punctate lesions
diffusely scattered throughout the posterior
poles and midperiphery of both eyes. Fundus photography was performed (Fig. 1),
but the patient died before fluorescein
angiography and electroretinography could
be performed.
Eye pathology. The globes were removed
six hours after death. The right globe
was fixed in 5 per cent glutaraldehyde
phosphate buffered to pH 7.4 and a collote
was removed and postfixed in 1 per cent
osmium for electron microscopic study.
The left globe was fixed in 10 per cent
buffered formalin. The eyes were of normal size and external appearance. Both
were opened in the horizontal plane of
section. Positive findings on gross examination of the right eye were nuclear sclerosis
and extensive crystalline deposits in the
pigment epithelium. These were particularly apparent when the overlying neurosensory retina was removed (Fig. 2).
Using a No. 25-gauge needle one of these
deposits was removed and examined microscopically between crossed polarized filters.
The crystal exhibited marked birefringence.
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Investigative Ophthalmology
April 1974
258 Bullock et dl.
Fig. 2. Magnified gross photograph of retinal pigment epithelium showing numerous crystalline deposits. (Reflected polarized light, x40.)
Fig. 3. Microscopic appearance showing birefringent crystalline deposits in the retinal pigment epithelial cell layer (H & E). A, low power, partially polarized light (x25). B, partially
polarized light (xlOO).
Gross examination of the left eye was
entirely similar to the right.
Microscopic examination of hematoxylin
and eosin-stained sections of the right
eye showed the following positive findings:
iris stromal atrophy, hyalinization of the
ciliary processes, occasional birefringent
crystals in the area of the pigmented epithelium of the ciliary body, lenticular nu-
clear sclerosis, and minimal cortical degeneration. The most striking finding was
the presence of multiple birefringent crystalline deposits in the retinal pigment epithelium (Fig. 3). A few additional crystals
were noted in the inner layers of the retina
and occasional drusen were seen. Examination of alizarin red- and Von Kossa-stained
sections not only failed to show positive
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Calcium oxalate retinopathy 259
Fig, 4. A and B, consecutive one micron-thick section viewed with light microscopy and crossed
Polaroid filters (A) and thin section viewed with electron microscopy (B). C, electron microscopic appearance of a ciystal that remained in a thin section. D, Tangential section of pigment epithelium showing hexagonal outlines of cells. Note that the large empty space occupied by the crystal appears to be completely within one pigment epithelial cell in this plane
of section. The pigment granules in A through D are 1 micron in diameter.
calcium crystals but also frequent empty
spaces corresponding to crystals in the
hematoxylin and eosin-stained sections
were noted.
The findings on examination of the left
eye were entirely similar.
Electron microscopy and light microscopy of epon sections. Alternate thick
(Fig. 4, A) and thin (Fig. 4, B) oblique
sections of the pigment epithelium were
examined by light and electron microscopy.
The thick microscopic sections were
scanned for birefringent crystals under the
polarizing microscope (Fig. 4, A) and
when these were positively identified the
corresponding thin sections (Fig. 4, B)
were examined using an RCA electron
microscope. The pigment epithelium was
sectioned obliquely and appears in the
electron micrographs as a sheet of cells.
The crystals nearly always appeared elongated and arranged in a butterfly configuration as in Fig. 4, B. Most of the
crystals were located in the apical region
of the RPE close to Bruch's membrane.
The actual crystals, with rare exception
(Fig. 4, C), were absent in the thin sections, their presence being indicated by
the empty space they had occupied as
in Figs. 4, B and 4, D. The shape of these
spaces corresponded to that of the crystal
as seen with light microscopy in the corresponding thick section. This is illustrated
by a comparison of the light micrograph
(Fig. 4, A) with the electron micrograph
(Fig. 4, B).
It is usually not possible to determine
whether or not the crystals are intracellular.
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Investigative Ophthalmology
April 1974
260 Bullock et al
Fig. 5. Photomicrographs of autopsy specimens showing extensive crystalline deposition. (H
& E, partially polarized light, x40.) A, kidney. B, pericardium. C, thyroid. D, epididymis. E,
bronchus.
However, in an occasional micrograph, such
as Fig. 4, D, the space occupied by the
crystal appears to be entirely within the
confines of one pigment epithelial cell at
least in the plane of this particular section.
It is our impression that the crystals may
form intracellularly in pigment epithelial
cells and that they tend to be localized
near the part of the cell contiguous to
Bruch's membrane. With increase in size,
the crystals became larger than the cells
themselves and are obviously eventually
extracellular in location.
Crystal examination. Slides of the other
organs from the general autopsy were then
obtained and examined by light microscopy using crossed polarizers. Identical
crystals were noted in the kidney, myocardium, bronchus, thyroid gland, and epididymis (Fig. 5). "Wet" kidney preserved
in formalin was noted to contain multiple
grossly visible whitish crystalline deposits
(Fig. 6). The crystals were insoluble in
water and were teased from the specimen
using small forceps. These crystals were
subjected to x-ray examination utilizing a
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Calcium oxalate retinopathy 261
Fig. 6. Gross photograph of kidney section showing numerous whitish crystalline deposits.
Guinier x-ray powder diffraction camera.
Calculations from the x-ray diffraction pattern gave excellent agreement with the
standard pattern for calcium oxlate-monohydrate (whewellite).2 Comparison of the
renal crystals with the mineral whewellite
(from Bruch-Bohemia, Brush Mineral Collection No. 3788, Yale Geology Department) confirmed the identification (Table
I and Fig. 7). Under the dissecting microscope, crystals were removed from the
retinal pigment epithelium and in addition
to the renal crystals, were optically analyzed in polarized light. The crystals were
biaxial positive, and determination of their
indices of refraction in three geometric
planes positively distinguished the crystals
as the monohydrate form of calcium oxalate
(Table II). 2 ' 4
Having identified the crystals in the
kidney and retinal pigment epithelium as
calcium oxalate, attention was then directed to the other tissues in order to
confirm their crystalline content. X-ray
diffraction and optical studies were not
possible on the crystals obtained from the
other tissues because the concentration of
crystals in these tissues was insufficient.
However, positive identification of calcium
oxalate was made by utilization of the
histochemical "bubble" test of Johnson5
and subsequent alizarin red staining of
the residual crystals. The histochemical
presence of calcium oxalate was confirmed
in the original kidney biopsy, the retinal
pigment epithelium, and in the postmortem
Fig. 7. X-ray diffraction pattern of the renal
crystal (right) and the mineral whewellite (left)
showing the identity as calcium oxalate monohydrate (C&C3O<-H.O).
kidney, heart,
(Table III).
thyroid,
and
bronchus
Discussion
The present study concerns a patient
with crystalline deposits in the retinal
pigment epithelium occurring in association
with diffuse deposition of similar crystals
in the kidney, thyroid, epididymis, myocardium, and bronchus. The clinical appearance of the eye changes was consistent
with the criteria outlined by Krill and
Klein'1 for the "flecked retina syndrome"
in 1965.
In the patient herein described, the crystalline deposition was demonstrated to be
calcium oxalate. The varieties of oxalate
deposition or oxalosis can be subdivided
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262 Bullock et al
Investigative Ophthalmology
April 1974
Table I. X-ray diffraction analysis
CaC,O4 H2O
Whewellite
ASTM
20-231
d (nm.)
hkl.
0.593
0.579
0.452
0.378
0.365
0.341
0.312
0.301
0.2966
0.2915
0.2897
0.2840
0.2523
0.2494
0.2447
0.2417
0.2384
0.2355
0.2347
0.2263
0.2254
0.2210
0.2130
0.2089
0.2075
0.1995
0.1978
0.1957
0.1950
0.1933
0.1923
0.1890
0.1859
0.1846
0.1823
0.1813
0.1793
0.1737
Intensity
100
30
4
6
70
2
2
10
45
10
8
10
4
18
4
6
4
30
12
8
6
6
2
2
14
2
10
2
10
8
8
6
4
6
6
4
6
6
Whewellite:
Bruch, Bohemia Yale
Brush ColJB 1 (renal lection No.
3788
crystal)
dhki (nm.) dhti (nm.)
0.591
0.593
0.577
0.579
0.454
0.379
0.378
0.364
0.365
0.3411
0.3325
METHOXYFLURANE
H-C- C-O-CH,
CL F
TPNH
CL
F
H- I -i-OH
CL
Z
TPNH
F
\ 8
H- C -C-F
F
CL
H- C-C-O-CH*
CO,
/
i
OH F
+ HF
HCL
I
+
•>
0F
II I
H- C-C-O-CH,3
I
F
CL
+H20n
CL 0
I ii
H- C -C-OH
0 F
II I
HO- C-C-O-CH3
HF
F
CL
0.300
0.2958
0.2910
0.2892
0.2837
0.2524
0.2492
0.2419
0.2386
0.2352
0.2260
0.2210
0.2072
0.1979
0.1948
0.1933
0.1859
0.1845
0.1822
0.1792
0.1736
0.3020
0.2968
0.2912
0.2899
0.2846
0.2527
0.2497
0.2449
0.2419
0.2383
0.2357
0.2348
0.2264
0.2257
0.2212
0.2133
0.2091
0.2079
0.1995
0.1979
0.1960
0.1951
0.1935
0.1927
0.1893
0.1861
0.1848
0.1826
0.1814
0.1793
0.1737
into two major types: (1) diffuse oxalosis
in which calcium oxalate crystals are noted
in multiple organs; and (2) in localized
form, in which the deposition is confined
to the kidney, eye, or other limited location.
Diffuse oxalosis can occur as an inborn
error of metabolism,7's or as a result of
ethylene glycol poisoning.9 While it is possible that ocular involvement may occur
in such cases, no convincing documentation could be found.1012 Numerous causes
OH
i
0
n
H- C -C-OH
co2
+HCL
0 F
II I
HO- C-C-OH
CL
0 0
n H
H- C -C-OH
HF
+HCL
HO- C-C-F
HoO
HOOC-COOH
OXALIC ACID
Fig. 8. Proposed metabolic pathway for the biotransformation of methoxyflurane to oxalic acid
and ionic fluoride.14
Table II. Optical analysis
n*
ny
n2
Axis
Standard calcium
oxalate-monohydrate crystal*
1.4909
1.5554
1.6502
RPE and
renal crystals
1.490
1.544
1.650
Biaxial +
Biaxial +
of calcium oxalate deposition in the kidney
have been described in the literature including oxalate, intoxication, diethylene
glycol poisoning following glyoxalate or
glycolate administration, and with pyridoxine or thiamine deficiency. Renal oxalosis has also been described in association
with a number of systemic disorders.13
Recently, renal oxalosis with transient
hyperoxaluria has been described following
MOF anesthesia. MOF is a recognized
nephrotoxin, probably as a result of its
metabolic products, ionic fluoride and
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Calcium oxalate retinopathy 263
Table III. Summary of crystal analyses
| Histologic presence \Histochemical test5] Optical analysis \ X-ray diffraction
Tissue
QNS
QNS
Kidney (biopsy specimen)
QNS
QNS
Bronchus
ND
ND
Epididymis
ND
Kidney (autopsy specimen)
QNS
QNS
Pericardium
QNS
Retinal pigment epithelium
QNS
QNS
Thyroid
ND—Not done: tissue block or wet tissue could not be located.
QNS—Quantity not sufficient for analysis.
oxalic acid.14 The presumed intermediary
metabolic products are shown in Fig. 8.
We suspect MOF to be the cause of the
oxalosis in this patient.
In a review of the literature, ocular
calcium oxalate deposition has previously
been described only in rare instances; intraretinally in the eyes of patients with longstanding retinal detachment,35 in the lenses
of patients with Morgagnian cataracts,1"
and in phakolytic glaucoma.17 The present
report appears to be the first instance of
ocular deposition of calcium oxalate in
diffuse oxalosis as well as the first instance
related to prolonged anesthesia with MOF.
The retinal pigment epithelium (RPE)
are primitive, multipotential cells which
provide metabolic and functional support
for the visual cells of the retina. Among
the many activities in which these cells
have been implicated are phagocytosis,
proliferation, migration, fibrous metaplasia,
osseous metaplasia, and neoplasia. Our
knowledge of the function and the changes
of the RPE is based primarily on histopathologic observations in enucleated eyes.
The molecular nature and physiologic details of the function of the RPE cells is
not clearly established.
The presence of calcium oxalate crystals
in association with these cells appears to
be a unique finding. The predominance
of crystals in the pigment epithelium and
their almost total absence in other ocular
tissues is not explained by the biochemistry or pathology of the RPE. It should
be noted that in the previous reports of
calcium oxalate crystals within the eye,
involvement of the RPE was not described.
Two pathologic processes involving the
RPE which may bear some relationship
to the present case are: (1) calcification
and bone formation within the eye, and
(2) the occurrence of the flecked retina
syndrome. Intraocular calcium deposition
and ossification are common findings in
pathologic specimens. The RPE together
with the inner surfaces of choroid have
been noted as common sites for the presence of calcium or bone. Calcium salts
are deposited in these tissues in two circumstances: (1) when excessive amounts
of these salts are present in the serum
and metastatic deposition occurs, and (2)
degenerative conditions in which the calcium is laid down in devitalized or "dead"
tissues independently of the blood calcium.18 Such calcification is thought to be
related to a low CO2 tension resulting from
metabolic inactivity or to a local increase
in phosphate ions released from disintegrating nucleoproteins. The RPE cells have
been implicated as the cells of origin for
choroidal bone.19 Fibrous metaplasia along
the inner choroid and proliferation of the
RPE have been described as the most
common changes adjacent to intraocular
bone.20
In numerous histopathologic conditions
the RPE may be degenerated, lose their
pigment, undergo fatty or hyaline degeneration, or may be absent in areas. In
certain pathologic conditions, an abnormal
accumulation of basement membrane material results in the formation of drusen.
The role of lysosomes of the RPE cells
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264
Investigative Ophthalmology
April 1974
Bullock et al.
in the formation of drusen has been mentioned.-1 Calcification of drusen are frequently seen, as is calcification of Bruch's
membrane which commonly occurs as an
aging phenomenon. No information could
be found in the literature regarding the
nature of these calcium salts, and this question is under investigation by the present
authors.
In the eye herein presented it could
not be determined whether the calcium
oxalate crystals were contained within the
cells of the RPE. If they in fact are, their
presence could be due to phagocytosis,
to in situ crystal formation within the
cell, or a combination of the two. The
ability of RPE for phagocytosis has been
well demonstrated in organ and tissue
culture. Young and Bok22 have demonstrated the phagocytosis of rod outer segment discs by frog RPE. Smith23 documented the ability of chick RPE to ingest
melanin pigment granules in tissue culture
and, more recently, in organ cultures of
human choroid and RPE, phagocytosis of
thororast was demonstrated.24 The phagocytic activities of the pigment epithelium
appear to be associated with lysosome-like
organelles termed "lamellar inclusion
bodies" or "phagosomes." An essential
morphologic criterion for the recognition
of phagocytic activity by electron microscopy is the presence of a membrane surrounding the phagocytosed material. Ocular tissue in the present study was fixed
within six hours after the patient's death.
Autolysis and artifactitious changes present
make it somewhat difficult to determine
if the crystals are enveloped by a distinct
membrane. In most areas the size of the
crystals are so large as to suggest that
their location is extracellular, although the
initial formation may have been intracellular.
Further studies are now underway to
further elucidate the pathogenesis and significance of these findings. We believe
this to be important because little is known
regarding oxalic acid metabolism in the
normal human or animal eye.25
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