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Ocular and systemic effects of
acetazolamide in nephrectomized rabbits
Zvi Friedman,* Theodore Krupin, and Bernard Becker
The effects of acetazolamide on intraocular pressure (IOP) were studied on rabbits previously
nephrectomized to eliminate the renal effects of the drug. Administration of acetazolamide (5
mg/kg i.v.) reduced IOP from a baseline of 15.2 to 12.2 mm Hg 2 hr later. This dose was found
not to alter arterial blood pH, pCO2, bicarbonate, or base excess. However, 4 hr after drug
administration anterior chamber aqueous humor showed significant reductions in bicarbonate,
pH, and base excess, whereas aqueous humor ascorbate was significantly elevated. Administration of acetazolamide (15 to 50 mg/kg i.v.) to nephrectomized rabbits caused significant
acidosis and pCO2 retention, presumably related to red blood cell carbonic anhydrase inhibition. IOP reduction at these higher doses was greater than that which followed the 5 mg/kg
administration. (INVEST OPHTHALMOL VIS Sci 23:209-213, 1982.)
Key words: intravenous acetazolamide, nephrectomy, rabbits,
intraocular pressure, aqueous humor dynamics
A,
present in other tissues, including the red
blood cell and the kidney. Renal carbonic anhydrase inhibition results in systemic metabolic acidosis, which may play a role in lowering IOP, since acidosis by itself decreases
aqueous humor production and IOP. 3 ' 6> 9 We
have studied the dose response of acetazolamide on IOP and on ocular and blood chemistries in rabbits nephrectomized to eliminate
the renal effects of this agent.
kCetazolamide lowers intraocular pressure
(IOP) in normal and glaucomatous subjects1
as well as in experimental animals.2* 3 Although the decrease in IOP is associated with
a reduction in the rate of aqueous humor
formation,2 the mechanism of action of acetazolamide is still debatable. Acetazolamide is
a potent carbonic anhydrase inhibitor. 4 Since
carbonic anhydrase is present in the ciliary
epithelium, 5 the direct inhibition of this enzyme is assumed to be the site of the ocular
effect. However, carbonic anhydrase is also
Materials and methods
From *the Eye Department, Rothschild Hospital, Haifa,
Israel, and the Department of Ophthalmology and
the Oscar Johnson Institute, Washington University
School of Medicine, St. Louis, Mo.
This study was supported in part by research grants EY
00004 and EY 01942, and a Research Career Development Award EY 00082 (Dr. Krupin) from the National Eye Institute, Bethesda, Md.
Submitted for publication April 9, 1981.
Reprint requests: Theodore Krupin, M.D., Department
of Ophthalmology, Washington University School of
Medicine, 660 South Euclid Ave., St. Louis, Mo.
63110.
Albino rabbits weighing 2 to 3 kg were used. IOP
was measured using a manometrically calibrated
pneumotonograph and topical proparacaine hydrochloride (0.5%) anesthesia. Arterial blood samples
were collected in a heparinized syringe. Blood
chemistries, including pH, pCO 2 , bicarbonate,
total CO 2 , and base excess were determined immediately after collection using a Corning Analyzer
(Model No. 165/2). Plasma osmolality was measured with a vapor pressure osmometer (Model
5100; Wescor Osmometer). Anterior chamber
paracentesis was performed with similar topical
anesthesia. Aqueous humor chemistries were determined on the Corning Analyzer. In addition,
0146-0404/82/080209+ 05$00.50/0 © 1982 Assoc. for Res. in Vis. and Ophthal., Inc.
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209
Invest. Ophthalmol. Vis. Sci.
August 1982
210 Friedman et al.
Table I. Effect of intravenous acetazolamide on IOP and blood chemistry in
nephrectomized rabbitsA
Acetazolamide
Parameter
IOP (mm Hg)
Blood pH
Blood pCo 2 (mm Hg)
Blood bicarbonate (mEq/L)
Base excess (mEq/L)
Dose (mg/kg)
No.
2.5
5.0
15
50
2.5
5.0
15
50
2.5
5.0
15
50
2.5
5.0
15
50
2.5
5.0
15
50
6
6
4
5
6
6
4
5
6
6
4
5
6
6
4
5
6
6
4
5
Baseline
13.7 dt
15.2 dt
17.0 db
15.1 dt
7.44 dt
7.43 dt
7.41 dt
7.34 dt
26.6 dt
28.5 dt
24.6 dt
22.7 dt
19.7 dt
23.9 dt
18.8 dt
15.3 dt
- 2 . 1 dt
- 0 . 7 dt
0.4
0.6
0.6
0.4
0.03
0.02
0.03
0.03
0.8
0.6
1.8
1.1
1.8
2.7
1.9
1.1
1.9
2.0
-4.1 ± 1.6
-9.1 ± 1.5
A
Bilateral nephrectomy was performed 18 hr before baseline measurements.
Significant difference from baseline paired t test, p < 0.05.
Significant difference from baseline paired t test, p < 0.005.
aqueous humor ascorbate was measured by microtitration.7
Bilateral nephrectomy was performed with pentobarbital sodium (30 mg/kg i.v.). Eighteen hours
later, one femoral artery was cannulated under
local xylocaine hydrochloride (1%) anesthesia. The
unanesthetized rabbit was placed in a box holder.
Baseline IOP was obtained 1 hr later and an arterial blood sample was obtained. Acetazolamide
was administered via the marginal ear vein in
doses from 1 to 50 mg/kg. IOP and arterial blood
samples were remeasured 15, 30, 60, and 120 min
later. In six rabbits baseline measurements were
followed by anterior chamber paracentesis on one
eye for aqueous humor chemistries. Acetazolamide
(5 mg/kg i.v.) was then administered, and IOP,
arterial blood chemistries, and paracentesis of
the remaining eye for chemistries were performed
4 hr later.
The paired t test was used for statistical analysis.
Results
Baseline IOPs and blood chemistries were
within normal limits for the rabbit 18 hr after
nephrectomy. 6 The administration of acetazolamide (1 mg/kg) did not alter either IOP
or arterial blood chemistries in four rabbits.
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The responses to higher doses of acetazolamide are shown in Table I. IOP was reduced
after 2.5 mg/kg acetazolamide and was markedly lowered after higher doses. Percent decrease in IOP from baseline value 2 hr after
acetazolamide administration demonstrated a
dose-dependent reduction, which was maximal after the 15 mg/kg dose (Fig. 1).
Blood pH was not altered (p > 0.2) at any
time after the 2.5 or 5 mg/kg doses. Systemic
acidosis occurred after larger doses of acetazolamide (Table I). Minor decreases in
blood pCO 2 occurred after 2.5 mg/kg, but no
significant changes in blood pCO 2 followed
doses of 5 or 15 mg/kg. A dose of 50 mg/kg
caused a significant and persistent elevation
of arterial pCO 2 . There was a tendency toward reduction of blood bicarbonate levels
and base excess; however, changes were
small and variable. Plasma osmolality was
unchanged at all times after any dose of
acetazolamide (p > 0.2).
Anterior chamber aqueous humor chemistries were studied in six nephrectomized
rabbits before and 4 hr after administration of
Volume 23
Number 2
Ocular and systemic effects of acetazolamide
211
Time after acetazolamide (min)
30
15
12.2 :t
12.2 :t
12.0 :t
10.9 dt
7.45 dt
7.41 dt
7.34 dt
7.27 dt
23.9 :b
28.6 ±
25.2 ±
30.1 ±
18.0 dt
23.3 dt
16.4 dt
17.0 d:
- 3 . 3 d:
- 0 . 1 d:
- 7 . 2 d:
- 9 . 6 d:
0.6B
0.5 c
0.3 c
0.3 c
0.03
0.02
0.04B
0.03c
1.0"
0.9
1.2
1.1
2.0
2.6
1.6
0.7
1.8
2.0
1.4
1.2
12.7 ± 0.4
10.2 ± 0.2c
10.8 ± 0.7°
9.9 :t 0.4c
7.46 dt 0.03
7.40 db 0.03
7.32 :b 0.04B
7.25 :b 0.02c
24.2 :b 0.96
28.0 dt 0.6
25.9 dt 1.1
30.0 :b 1.4C
18.7 db 1.6
22.4 db 2.4
15.9 ± 1.9
16.4 ± 0 . 5
- 2 . 5 ± 1.6
- 0 . 6 ± 1.9
- 8 . 4 ± 1.7
-10.6 ± 0.9
acetazolamide (5 mg/kg i.v.) (Table II). The
time of 4 hr was chosen to ensure anterior
chamber steady-state conditions. This dose
was selected because it lowered IOP significantly without altering blood chemistries
(Table I). Four hours after drug administration IOP was significantly reduced, whereas
blood pH, pCO 2 , base excess, and osmolality
were unaltered (Table II). Blood bicarbonate
was decreased slightly. Of great interest was
the change in aqueous humor chemistries;
anterior chamber aqueous humor pH, bicarbonate, and base excess were lowered significantly, whereas pCO 2 was unchanged.
Anterior chamber aqueous humor ascorbate
was elevated significantly.
Discussion
Carbonic anhydrase catalyzes the reversible hydration and dehydration of CO 2 by the
following reaction: CO 2 + H2O ^ H 2 CO 3 ^
H + + HCO 3 ". Carbonic anhydrase is present
in many tissues, including the renal cortex,
gastric mucosa, red blood cell, lung, pancreas,
central nervous system, and eye. 8 Inhibition
of the enzyme lowers IOP and also produces
systemic acidosis. The mechanism by which
acetazolamide lowers IOP is confused by the
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60
12.8
12.5
10.4
9.5
7.44
7.40
7.32
7.24
22.5
27.5
26.2
29.3
16.8
22.8
16.4
15.9
-4.4
-1.2
-7.8
-11.0
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
120
0.5
0.8 c
0.2c
0.3 c
0.05
0.02
0.03c
0.01 c
1.1B
0.6
1.8
0.8°
1.8C
2.9 s
2.3
0.6
2.0 8
2.4
1.2
0.9
13.0 d:
12.2 d:
10.8 d:
9.5 i:
7.42 :b
7.40 :b
7.32 :b
7.25: b
24.6 :b
26.2 :b
25.6 :b
31.1 :b
17.6 :b
22.4 :b
16.3 :b
17.3 :b
- 4 . 6 :b
- 1 . 8 :b
- 7 . 9 :b
- 9 . 8 :b
0.7
0.5 c
0.3 c
0.3 c
0.04
0.02
0.04c
0.02c
1.2
0.9
1.9
1.2B
1.8B
2.4
2.3
0.8
2.2B
1.9
1.6B
1.2
associated systemic acidosis, which is capable
by itself of lowering IOP.3> 6> 9
Nephrectomy avoids much of the systemic
acidosis and fall in serum bicarbonate. However, nephrectomy does not eliminate the
inhibition of red blood cell carbonic anhydrase, with resultant impairment of CO 2 elimination from the pulmonary capillary blood.
Our results in nephrectomized rabbits
show that acetazolamide in a dose of 5 mg/kg
can lower IOP without an accompanying alteration in arterial blood pH, bicarbonate, or
pCO 2 . These findings are in contrast to those
of Bietti et al., 10 who studied acetazolamide
in normal rabbits. They report that a dose of
2.5 mg/kg decreases IOP as well as blood
pH, bicarbonate, and base excess, and their
conclusion is that the effect of acetazolamide
on IOP is only due to the metabolic acidosis it
produces. Our findings are consistent with
those of Benedikt et al., u who show that IOP
is lowered even when acetazolamide is given
after the administration of sodium bicarbonate to avoid systemic acidosis.
Higher doses of acetazolamide in our nephrectomized animals produced a systemic
acidosis (15 or 50 mg/kg) and an increase in
pCO 2 (50 mg/kg). These effects are presum-
Invest. Ophthalmol. Vis. Sci.
August 1982
212 Friedman et at.
IJ
a:
q
LxJ
CO
<
-20
a:
o
LU
Q
-40
O
cr
UJ
-60
<
10
100
ACETAZOLAMIDE (mg/kg I.V.)
Fig. 1. IOP response to intravenous acetazolamide in nephrectomized rabbits. Each point
represents the mean percent decrease in IOP (±S.E.M.) for six to eight animals.
Table II. Effect of acetazolamide (5 mg/kg i.v.) on aqueous humor and blood chemistries
(mean ± S.E.M.) in six nephrectomized rabbitsA
Parameter
IOP (mm Hg)
Arterial blood
pH
pCO 2 (mm Hg)
Bicarbonate (mEq/L)
Base excess (mEq/L)
Anterior chamber aqueous humor
pH
pCO 2
Bicarbonate
Base excess
Ascorbate (mg/dl)
Baseline
16.2 ± 0.5
7.40
28.1
17.4
-4.8
7.84
28.2
50.1
+33.4
20.0
±
±
±
±
4 hr after acetazolamide
10.0 ± 0.3 c
0.02
1.4
1.4
1.5
7.38
25.3
14.9
-6.5
± 0.02
± 1.2
±3.6
± 3.5
± 1.9
7.62
27.3
29.1
+ 10.4
25.2
±
±
±
±
0.02
1.3
1.08
1.3
± 0.05 c
±0.6
±3.1C
± 3.8C
± 3.4B
A
Bilateral nephrectomy was performed 18 hr before baseline measurements.
Significant difference from baseline paired t test, p < 0.05.
Significant difference from baseline paired t test, p < 0.005.
B
c
ably related to the acetazolamide effect on
red blood cell and pulmonary carbonic anhydrase. The IOP reduction is greater with this
associated addition of systemic acidosis (Fig.
1). Recent studies demonstrate that increasing pCO2 by primary respiratory methods elevate IOP.12 This effect of acetazolamide
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after the 50 mg/kg dose did not increase IOP
in our animals.
Rabbit aqueous humor has an excess bicarbonate when compared with blood levels.
The decrease in anterior chamber aqueous
humor bicarbonate after 5 mg/kg acetazolamide is similar to that reported by Becker7
Volume 23
Number 2
Ocular and systemic effects of acetazolamide 213
and Zimmerman et al.13 Since this occurs in
the absence of alteration of blood bicarbonate, it suggests a direct inhibition of ocular
enzyme, with resulting decrease of aqueous
humor bicarbonate, pH, and base excess. The
increase in anterior chamber ascorbate is
consistent with a decreased entry of water into
the eye.7 Our results clearly demonstrate
that in nephrectomized rabbits, acetazolamide in a dose of 5 mg/kg can lower IOP and
decrease aqueous humor bicarbonate and pH
in the face of unaltered blood acid-base balance. However, reduction in IOP after higher
doses of acetazolamide in this animal model
and after administration of the larger doses in
animals and humans with normal renal function may well be a combined effect of carbonic anhydrase inhibition in the eye and
systemic acidosis. Both of these actions can
reduce aqueous humor production and together may result in a greater decrease of
IOP (Fig. 1). In human subjects acetazolamide in a dose of 500 mg twice a day results
in a greater decrease of IOP than that produced by methazolamide in doses of 25 or 50
mg twice a day.14 The increased effectiveness
of acetazolamide is associated with a metabolic acidosis and significant decreases in
plasma pH and bicarbonate.
REFERENCES
1. Becker B: Decrease in intraocular pressure in man
by a carbonic anhydrase inhibitor, Diamox. Am J
Ophthalmol 37:13, 1954.
2. Becker B: The mechanisms of the fall in intraocular
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3.
4.
5.
6.
pressure induced by the carbonic anhydrase inhibitor, Diamox. Am J Ophthalmol 39:178, 1955.
Langham ME and Lee PM: Action of diamox and
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Maren TH: Carbonic anhydrase: chemistry, physiology and inhibition. Physiol Rev 47:595, 1967.
Wistrand PJ: Carbonic anhydrase in the anterior
uvea of the rabbit. Acta Physiol Scand 24:144, 1951.
Krupin T, Oestrich CJ, Bass J, Podos SM, and
Becker B: Acidosis, alkalosis, and aqueous humor
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7. Becker B: The effect of the carbonic anhydrase inhibitor, acetazolamide, on the composition of the
aqueous humor. Am J Ophthalmol 40(part 2): 129,
1955.
8. Ashby W: Carbonic anhydrase in mammalian tissue.
J Biol Chem 151:521, 1943.
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anhydrase inhibition on intraocular pressure of rabbits with different blood CO 2 equilibria. Am J Ophthalmol 50:291, 1960.
10. Bietti G, Virno M, Pecori-Giraldi J, and Pellegrino
N: Acetazolamide, metabolic acidosis and intraocular pressure. Am J Ophthalmol 80:360, 1975.
11. Benedikt O, Zirm M, and Harnnoncourt K: Relation
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after inhibition of carbonic anhydrase with acetazolamide. Albrecht von Graefes Arch Klin Exp Ophthalmol 190:247, 1974.
12. Kielar RA, Teraslinna P, Kearney JT, and Barker D:
Effect of changes in pCO 2 and intraocular pressure.
INVEST OPHTHALMOL VIS SCI 16:534, 1977.
13. Zimmerman TJ, Garg LC, Vogh BP, and Maren TH:
The effect of acetazolamide on the movements of
anions into the posterior chamber of the dog eye. J
Pharmacol Exp Ther 196:510, 1976.
14. Stone RA, Zimmerman TJ, Shin DH, Becker B, and
Kass MA: Low-dose methazolamide and intraocular
pressure. Am J Ophthalmol 83:674, 1977.