Ocular zinc concentration

Miles A. Galin, Hugo D. Nano, and Theodore Hall

Human and rabbit ocular tissues contain high concentrations of zinc. In descending order in
human globes, the metal concentration appears to be: retina-choroid, ciliary body, iris, optic
nerve, sclera, cornea, and lens. Diamox does not alter the concentration of ocular zinc. In
preliminary studies dithizone, a known zinc chelator, does not appear to alter ocular zinc
concentration as well. The role of this metal in ocular tissue requires further clarification.

I t has been presumed by a variety of

authors that carbonic anhydrase is involved
in aqueous secretion.1 Although little direct
evidence is available as to the mechanisms
involved in aqueous secretion, it is well
known that inhibition of carbonic anhydrase
by a variety of related substances will re-
duce aqueous production.2'3 A consider-
able number of studies on this subject have
been reported; however, there have been
few ophthalmologic studies limited to the
enzyme structure itself.4-5
The carbonic anhydrase molecule is a
polypeptide with a prosthetic group con-
taining zinc. The number of moles of zinc
per enzyme moiety is not absolutely cer-
tain, but it is known that zinc constitutes
approximately 0.33 per cent of carbonic
anhydrase by weight.0
Although it would be of interest to evalu-
ate drugs that might conceivably alter the
status of zinc in carbonic anhydrase or
quantitatively reduce it, exact measure-
From the Department of Surgery (Ophthalmology)
of The New York Hospital-Cornell Medical
Center, and the Biophysics Division of the
Sloane-Kettering Institute for Cancer Research,
New York, N. Y.
Aided in part by grants from The National Society
for the Prevention of Blindness, and The Na-
tional Council to Combat Blindness.
ments of the concentration of zinc in ocular
tissue must first be known. It is the purpose
of this paper to report on the concentra-
tions of zinc in the various components of
ocular tissue, the effect of Diamox on zinc
concentrations, and some preliminary data
concerning dithizone, a known zinc
chelator.
Materials and methods
Studies were conducted on human and albino
rabbit ocular tissues. Fresh, postmortem human
globes and enucleated rabbit eyes were analyzed.
Tissue dissections were carried out under a Zeiss
operating microscope. No homogenates were used,
and an effort was made. to obtain tissue samples
from similar areas. The choroid contained the
pigment layer of the retina in all cases. An area of
ciliary body containing ciliary processes was sub-
mitted for that category of tissue.
Tissues were kept frozen at -10° C. At the time
of analysis the specimen was sliced into segments
0.3 mm. thick. At least three 0.3 mm. segments
for each specimen were analyzed.
The specimens were mounted on specially pre-
pared thin nylon films and then vacuum pumped
at least 12 hours to dryness. They were then
analyzed in vacuo by x-ray fluorescence.7 Exciting
radiation was obtained from a molybdenum anode
at 30 kv., which utilized currents in the range of
20 Ma. Fluorescent radiation from the specimen
was detected in custom-built proportional counters.
The resulting electrical pulses were amplified in
nonoverloading amplifiers (Beva models 152 and
154), sorted for pulse height in single-channel
analyzers (AIC model 510), and counted by
Baird-Atomic sealers (models 1050A, 131A, and

142

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 Volume 1
Number 1 Ocular zinc concentration 143

134). The counting rate for scattered incident
quanta was established simultaneously, and, after
suitable calibration, the ratio of fluorescent to
scattered quanta provided a direct measure of the
concentration of an element in each specimen. The
technique is in routine use for the measurement
of amounts of element down to 10"10 Gm.s
The presence of blood may affect the observed
zinc concentrations in certain blood-rich tissues,
such as the choroid and the ciliary body. Since
the technique measures directly the concentration
of zinc in the entire dried specimen rather than
the total amount of zinc, and since the concen-
tration of zinc in blood (approximately 50 ppm
in dried blood) is much lower than the concentra-
tions observed in these tissues, the presence of
blood could only decrease the observed zinc level.
If one third of the mass of the tissues in question
consisted of blood (undoubtedly a gross over-
estimate in view of their pallid appearance), cal-
culations reveal that a tissue with a zinc level
of about 600 ppm exclusive of blood would yield
an observed level of about 500 pprn in its
entirety. For the lower levels generally reported
in this article, the maximum eifect would be less
than 10 per cent.
Diamox studies of an acute and chronic nature
were carried out. Studies in human beings after
administration of Diamox were limited to analysis
of the aqueous. In acute studies, Diamox, 10 mg.
per kilogram, was administered intravenously to
patients scheduled for cataract extraction, and
aqueous was obtained 2 hours later. In rabbits,
similar doses were used, and, after paracentesis,
the globes were enucleated and submitted to dis-
section for subsequent zinc analysis.
Similar fluid and tissue analysis was carried out
in human and rabbit specimens after 1 week of
therapy with oral Diamox in doses of 5 mg. per
kilogram 4 times daily. Rabbits were fed granu-
lated Diamox without difficulty.
Dithizone, a dibasic acid, was prepared for
solution in a manner described elsewhere,9 and
was administered to rabbits in a dose of 50 mg.
per kilogram intravenously at weekly intervals for
three doses. Two animals that exhibited chorio-
retinal lesions and the development of diabetes
mellitus are included in this study. The systemic
and ocular effects of dithizone will form the basis
of a subsequent report.
Results
Table I lists the zinc and copper con-
centration in parts per million of diy mass
in three human globes at postmortem. The
descending order of concentration in the
ocular tissue appears to be: retina-choroid,
ciliary body, iris, optic nerve, sclera,
cornea, and lens.
Table II lists similar data for the albino
rabbit. The descending order of concentra-
tion of zinc in the segments studied from
this species appears to be: choroid, ciliary
body, retina, iris, and lens.
Table III lists the zinc concentration in
parts per million of dry mass in the aqueous
of patients undergoing cataract extraction.
The remainder of the ocular status of these
patients was normal.
Table IV shows the effect of intravenous
Diamox on concentration of zinc in a
similar group of patients undergoing cata-

Table I. Concentration of zinc and copper in parts per million of dry mass in
human ocular tissue
Ciliary Optic
Cornea Iris Lens body Retina Choroid nerve Sclera
No. Zn Cu Zn Cu Zn Cu Zn Cu Zn Cu Zn | Cu Zn j Cu Zn Cu
1 25 0 127 2 19 0 288 57 571 72 562 21 112 23 47 38
2 62 0 140 0 26 1 205 39 435 61 435 36 67 23 52 11
3 35 0 130 0 17 0 189 28 385 32 419 31 161 11 49 20

Table II. Concentration of zinc and copper in parts per million of dry mass in
rabbit ocular tissue
Iris Lens Ciliary body Retina Choroid
Zn Cu Zn Cu Zn Cu Zn Cu Zn Cu
111 17 30 220 40 199 24 440 20
168 6 50 269 37 202 6 500 32

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 Investigative Ophthalmology
144 Galin, Nano, and Hall February 1962

Table III. Aqueous zinc concentration in ract extraction. There does not appear to
parts per million of dry mass in human be any significant change following admin-
cataractous eyes istration of the drug. Similarly, as is shown
in Table V, no change occurred after 1
Patient Zn
week of Diamox orally in a dose of 5 mg.
T. B. 100
W. F. 60 per kilogram 4 times daily.
F.L. 100 Tables VI and VII record the zinc and
A. B. 180 copper concentration in rabbit aqueous 2
S. B. 120
M. T. 110 hours after intravenous administration of
R. S. 130 Diamox in doses of 10 mg. per kilogram
J.D. 70 and after oral Diamox in doses of 5 mg.
J.S. 70 per kilogram 4 times daily for 7 days, re-
spectively. Here too, no significant change
Table IV. Aqueous zinc concentration in occurs after such therapy.
parts per million of dry mass in human Table VIII lists zinc and copper concen-
cataractous eyes 2 hours after intravenous tration in some components of rabbit ocular
administration of Diamox in a dose of 10 tissue after 7 days of Diamox orally in
mg. per kilogram doses of 5 mg. per kilogram 4 times daily.
No significant change in concentration of
Patient Zn these elements occurs.
R. V. 122 Table IX lists the concentration of zinc
L. B. 155
A. M. 118 and copper in various segments of rabbit
CD. 88 ocular tissue after intravenous dithizone,
G. R. 100 given on 3 occasions in doses of 50 mg. per
kilogram. All rabbits selected for analysis
Table V. Aqueous zinc concentration in exhibited a systemic response to dithizone,
parts per million of dry mass in human manifested by chorioretinal lesions and
cataractous eyes after 7 days of Diamox diabetes mellitus. However, no change in
administered orally in a dose of 5 mg. per zinc concentration in ocular tissues ap-
kilogram 4 times daily peared to result.

Patient Zn Discussion
120 At the outset, it must be noted that the
N.J. 160 results reported here are for total amounts
E.F. 120
R. P. 200 of zinc. No effort has been made to sepa-
CD. 74 rate the zinc contained in enzyme systems
from that in other cellular structures. Con-
Table VI. Aqueous zinc and copper con- sequently, zinc concentration for the vari-
centration in parts per million of dry mass ous tissue substrates may in no way reflect
in one eye of rabbits before and in the enzyme concentration. For example, it is
other 2 hours after administration of 10 mg. known that lens tissue has considerably
per kilogram of Diamox intravenously more carbonic anhydrase than choroid
tissue, but far less zinc. However, the
Zn Cu literature varies considerably with respect
No. Before After Before After to reported carbonic anhydrase content of
1 115 120 93 97 ocular tissue as well as to zinc concentra-
2 170 110 126 104 tion.
3 164 174 128 82
4 102 128 58 94 Green and co-workers10 showed that of
5 115 119 110 75 the total carbonic anhydrase present in the
6 125 135 100 111 anterior uvea of the rabbit, the ciliary

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 Volume 1
Number 1 Ocular zinc concentration 145

processes contained about 60 per cent, Table VII. Aqueous zinc and copper con-
while the remaining 40 per cent was found centration in parts per million of dry mass
in the iris. However, Ballintine and Maren11 in one eye of rabbits before and in the
estimated that the carbonic anhydrase in other after administration of 5 mg. per
rabbit ciliary processes was approximately kilogram of Diamox parenterally 4 times
three times that found in the iris. daily for 7 days
Carmichael and associates12 utilized histo-
chemical methods to demonstrate the Zn Cu
presence and location of carbonic anhy- No. Before After Before After
drase. However, their results were incon- 1 111 120 83 111
sistent, and inhibition of the enzyme could 2 178 150 146 130
3 175 140 129 120
not be demonstrated histologically follow- 4 91 123 46 80
ing acetazolamide. Tauber and Krause13 5 109 120 109 114
listed ocular zinc concentrations which 6 123 111 111 108
were reviewed by Bowness and associ-
ates.14' 15 The latter authors found that in
order from highest concentration, ocular Table VIII. Concentration of zinc and cop-
zinc concentrations in cattle eyes were: per in parts per million of dry mass in
iris and ciliary body, choroid and pigment rabbit eyes after administration of 5 mg.
epithelium, retina minus pigment epi- per kilogram of Diamox parenterally 4
thelium, lens, aqueous humor, vitreous times daily for 7 days
humor, sclera, cornea, and optic nerve. It
Ciliary body Retina Choroid
should be noted that the highest concentra-
Zn Cu Zn Cu Zn Cu
tions of zinc were found in pigmented
tissues. This was further substantiated by 212 55 180 40 510 30
264 31 175 28 440 34
noting less zinc concentration per unit of 160 23 195 35 470 36
tissue in albino rabbits as compared with 185 26 200 27 368 20
the pigmented type.
The findings of this study differ slightly
from those of previous authors. In order Table IX. Concentration of zinc and copper
from highest concentration human ocular in parts per million of dry mass in rabbit
tissues contain zinc as follows: retina- eyes after intravenous administration of 50
choroid, ciliary body, iris, optic nerve, mg. per kilogram of dithizone on 3 oc-
sclera, cornea, and lens. The technique of casions, 1 week apart, with the develop-
x-ray fluorescence is quite accurate, and is ment of chorioretinal lesions and diabetes
not open to several of the errors inherent in- mellitus
techniques utilized by previous investi- Ciliary body Choroid Retina
gators. However, species difference may
Zn Cu Zn Cu Zn Cu
exist since previously published studies
have primarily referred to cattle and sheep 210 20 602 24 152 44
155 34 619 38 110 15
eyes. 171 32 658 36 190 17
Although it was necessary to accumulate 166 28 550 76 107 20
base-line data of ocular concentration of
zinc, it was not anticipated that Diamox
would alter these levels. There is a con- NH2 are carbonic anhydrase inhibitors, it
siderable body of evidence indicating that would appear logical that acetazolamide, a
sulfanilamide inhibits carbonic anhydrase compound of this group, would act simi-
activity by inhibition of the prosthetic larly. It is well known, furthermore, that
group of this molecule.16 Since aromatic Diamox inhibition is reversible. Therefore,
sulfonamides unsubstituted on the —SOL. one should not expect to find permanent

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146 Galin, Nano, and Hall
quantitative change in the enzyme mole-
cule, in either the prosthetic group or the
protein moiety. Certainly, the quantitative
data provided by this study indicate that
ocular zinc concentration in those areas
where Diamox action is known to exert
physiologic effects is unaltered. This, of
course, does not rule out nonquantitative
changes, such as those by chelation,
polarity changes, etc.
However, permanent change in the
enzyme system seemed an intriguing pos-
sibility with dithizone for a variety of
reasons. This compound has received con-
siderable attention in recent years in both
laboratory and clinical investigations con-
cerning the elimination of zinc from tis-
sues.17"20 In the prostate gland of man and
certain animals, an organ with high zinc
concentration, it has been shown that
anatomic reduction in the size of the tissue
structure is correlated with loss of zinc
from such tissue following administration
of dithizone. Consequently, this substance
has been used in the clinical treatment of
carcinoma of the prostate with reported
clinical results.
Dithizone is one of a family of drugs that
is known to cause destruction of the beta
cells of the islets of Langerhans with the
concomitant development of diabetes mel-
litus. It has been demonstrated by histo-
logic studies that this necrosis is related to
reduction of zinc concentration in this
tissue.21'22
Reports have appeared that chorioretinal
lesions can be induced in animals follow-
ing administration of dithizone.23 It is
known that the percipient retinal elements
contain lactic acid dehydrogenase, one of
the known enzyme systems containing zinc.
Consequently, it might be adduced that this
compound is acting through the chelation
of zinc in this area as well. Furthermore, it
is known that retinal respiration is reduced
in white rats on a zinc-deficient diet.24
Also, it has been demonstrated that histo-
logic changes in the dog tapetum following
administration of dithizone are correlated
with quantitative reduction in zinc con-
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Investigative Ophthalmology
February 1962
centration in that tissue.-5"-7 In rabbits,
however, little change has been noted in
tapetal tissue, whereas chorioretinal tissue
appears susceptible to the effects of
dithizone.
Theoretically, therefore, there was good
reason to measure the effect of dithizone
on ocular zinc concentration and on intra-
ocular pressure. However, this was at-
tempted with the full realization that it is
extremely difficult to remove zinc from
carbonic anhydrase in vivo. Although the
enzymatic effect of carbonic anhydrase is
negated with removal of zinc from the
molecule,2S turnover of labeled zinc from
this molecule is virtually nil.29
The results of this study indicate that
dithizone in doses sufficient to be diabeto-
genic in rabbits and to produce chorio-
retinal lesions does not significantly reduce
ocular zinc concentration. Furthermore, in
a study to be reported, there is no effect on
intraocular pressure in human beings in the
dosage level used to treat prostatic lesions.
Kurus'30 findings that pathologic tissues
have more zinc than normal tissues might
explain, to a certain extent, why the ocular
zinc concentration did not fall. Further-
more, the fact that homogenates were not
used in this investigation may also help
to explain the lack of response. Very large
variations in metal concentration may occur
from tissues taken 2 to 3 mm. apart. De-
tailed studies with whole-tissue homog-
enates are now under way to help elimi-
nate this discrepancy.
The concentration of zinc in ocular tissue
is rather unusual in comparison with the
usual body soft-tissue concentration of this
element. For example, Mawson and
Fischer31 found an average soft-tissue con-
centration of approximately 100 ppm. Only
in prostatic tissue have concentrations
higher than those found in ocular tissue
been reported. These have reached 2,000
ppm. However, it has been noted that
similar concentrations have occasionally
been found in samples of dog retina. It
appears, therefore, that ocular tissue con-
tains extremely high concentrations of zinc.
 Volume 1
Number 1
The available carbonic anhydrase could
account for only a small amount of this
element. The function of the remaining
zinc is still to be determined.
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