It has been reported that low doses of

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1 Aqueous humor turnover rates in the cat I. Effect of acetazolamide Frank J. Macri,* Robert L. Dixon,** and David P. Rail*'*' Aqueous humor turnover in the cat eye determined by intracameral injection of inulin- 1 *C was found to be 2.1 per cent per minute, a value which is in close agreement with that reported for other species. The inhibition of aqueous humor formation induced by acetazolamide was dose dependent bid the quantities required were much higher than those reported necessary to lower the eye pressure. The data acquired in these experiments also alloived comparisons to be made between intraocular pressure and (1) anterior chamber volume; and (2) rate of aqueous humor formation. No statistically significant regression was found between the IOP and these two factors. In addition, an experimentally induced rise of IOP did not reflexly decrease the rate of aqueous humor turnover as has sometimes been suggested. It has been reported that low doses of acetazolamide lower the eye pressure of the cat by a mechanism involving local vasoconstriction and that an inhibition of aqueous humor formation could not be discerned. 22 " 2 ' 1 Davson 0 concludes on the basis of 2l Na turnover studies that the action of acetazolamide is not primarily due to an inhibition of aqueous humor secretion. However, Becker 5 ' s and Langham 20 report that aqueous humor inflow is decreased after acetazolamide. Because From the Ophthalmology Branch, National Institute of Neurological Diseases and Blindness, and Laboratory of Chemical Pharmacology, National Cancer Institute, National Institutes of Health, Public Health Service, United States Department of Health, Education and Welfare, Bethesda, Md. "Ophthalmology Branch, National Institute of Neurological Diseases and Blindness. *'Laboratory of chemical Pharmacology, National Cancer Institute. 927 of this uncertainty we have reinvestigated the effects of acetazolamide on aqueous humor formation. As a second part to this study, aqueous humor turnover rates were correlated with eye pressure and anterior chamber volume. Methods Male and female cats weighing from 1.5 to 3.0 kilograms were used. The animals were anesthetized with pentobarbital sodium (35 mg. per kilogram, intraperitoneally). Eye and blood pressures were measured manometrically with Sanborn Model 267B pressure transducers and continuously recorded on a 4 channel polygraph. 21 One hundred microliter quantities of buffer containing tracer amounts of 1J C-labeled inulin* were injected into the anterior chamber by means of a leakproof syringe microburet.f 21 A second similar syringe arrangement was connected to the same eye so that aqueous humor could be with- Tracer amounts of inulin- 1 'C was dissolved in a buffer solution of the following composition (all values are milliequivalents per liter): NaCl = 123.5, KC1 = 3, CaCh = 1.3, Mg Cls = 0.8, NaHCOa = 25, NaHjPO, = 0.5, Na^HPOi = 0.5, (iiuilin-carboxyl-c-14 [New England Nuclear Corp.]). t Model SB;, Micro-metric Instrument Co.

2 928 Maori, Dixon, and Rail Investigative O phthalmology October 1965 drawn. In this manner injection and withdrawal could be made within 10 to 20 seconds, and by observing the eye pressure recording no change in the intraocular pressure (IOP) was introduced. The needles for the injection and withdrawal syringes were inserted at opposite sides of the cornea at the limbus and pierced the cornea obliquely for a distance of 1 to 2 mm. The plungers and barrels of the syringes were securely fixed so that no loss of aqueous humor could be effected by passive movement of either unit. The third needle, used for eye pressure measurement, was inserted through the cornea at the outer canthus. Exactly one hour after the inulin- 14 C was injected, the cat was killed by an overdose of pentobarbital sodium and the aqueous humor was completely withdrawn. The animal was killed in an attempt to stop aqueous humor formation and to prevent contamination of the sample. Not more than two minutes elapsed between the time the animal was killed and the aqueous humor withdrawn. The needle shaft which served for the measurement of IOP was used to aspirate the aqueous humor. The attached No. 10 PE tubing was cut and a No. 27 needle on a calibrated 2.0 c.c. syringe immediately inserted into its open end. The dead space in the tubing was measured and varied from 5 to 10 til. At most this introduced a dilution error of 1 per cent which was not considered in the calculations. The aqueous humor was withdrawn as completely as possible and its volume recorded with an accuracy of ±5 per cent. Inulin-i'C was determined for duplicate 100 fil samples of aqueous humor with a Packard liquid scintillation counter, Model 3,000. Turner's solution 12 was used as the scintillation medium, and quenching monitored by channel ratios. The aqueous humor withdrawn synchronously with the intracameral inulin- 14 C injection was counted in one vial. Calculations During the intracameral injection of inulin-^'c (counts in), aqueous humor was withdrawn so that the steady-state eye pressure would not be disturbed; as a consequence of this procedure a small amount of inulin- 14 C was lost to the aspirating syringe (counts out). The concentration in the anterior chamber at time zero (C o ) therefore was: Counts in - Counts out Co = Volume A.C. Kout (minute- 1 ) was calculated by the formula used by Barany and Kinsey 1 : Kout = ; t V 2 = half-life (in minutes) of inulin in the anterior chamber. Results Normal rates of aqueous humor turnover. A total of sixteen eyes from sixteen cats was studied (Table I). The normal rate of inuli.n- 14 C turnover (Kout) was found to be 2.1 per cent per minute. Since the average aqueous humor volume in this series was 853 {.d, aqueous humor was being formed at the rate of 17 p\ per minute. Action of acetazolamide on aqueous humor turnover rate. Three groups of experiments were performed in this series (Tables IIA, IIB, IIC): Group I consisted of one eye from each of 10 cats administered acetazolamide in an intravenous dose of 100 mg. per kilogram; Group II, the same as Group I, except that the dose was 250 mg. per kilogram; Group III consisted of 5 cats which were administered acetazolamide in a dose of 600 mg. per kilogram. Since the maximum reduction of IOP with i able I. Aqueous humor volume and turnover rate in unmedicated, anesthetized cats No \ 699 \ 702 \ 703 \ 704 \ 705 \ 706 \ 707 \ \ \ 719 \ 720Meant S.D. Volume ±80 Kout (a) (min.- 1 ) ±0.008 (b) Aqueous (/il/min.) humor (a x b) flow

3 Volume 4 Number 5 Aqueous humor turnover rates 929 Table IIA. Aqueous humor volume and turnover rate in anesthetized cats administered acetazolamide. Group 1 (100 mg. per kilogram, intravenously) No \ 735 \ 736 \ \ 739 \ 740 \ 765 Meant S.D. Volume () Kout (a) (min.-i) (b) Aqueous (/ul/min.) humor flow (a x b) ±4 Table IIB. Aqueous humor volume and turnover rate in anesthetized cats administered acetazolamide. Group II (250 per kilogram, intravenously) No \ 758 \ 759 \ 760 \ 761 \ 762 \ 763 Meant \ 764 S.D. Volume (a: Kout (min.-i) ±.004 (b) Aqueous (M/min.) humor flow (a x b) ± 4 Table IIC. Aqueous humor volume and turnover rate in anesthetized cats administered acetazolamide. Group III (600 mg. per kilogram intravenously) Volume Kout Aqueous humor flow (Ml) (a) (min.- 1 ) (b) (/ul/min.) (axb) Nc i Meant S.D. 920 ± ±.002 5±2 acetazolamide is not obtained until approximately 30 minutes after drug administration, 23 the inulin- 1J C determinations were begun at that time. The eye pressures remained generally constant during the time interval of the turnover study, indicating that acetazolamide was continuously effective. Group I (acetazolamide, 100 mg. per kilogram ): The mean inulin- 14 C turnover rate was 1.8 per cent per minute. This was not a statistically significant difference from the control values of 2.1 per cent per minute and represented a 14.3 per cent inhibition of aqueous formation. Group II (acetazolamide, 250 mg. per kilogram): The mean inulin-^'c turnover rate of this group was 1.1 per cent per minute which was statistically significantly different (P <.001) from control values. The degree of inhibition of aqueous formation amounted to 47.6 per cent. Group III (acetazolamide, 600 mg. per kilogram): Inulin- T1 C turnover rate at this high dose level was 0.6 per cent and represented a 71.4 per cent inhibition in the formation of aqueous humor. Plotting a dose-response curve of the logarithm of the dose versus per cent inhibition of aqueous humor formation yielded a straight line function (Fig. 1). It can be seen from the curve that a dose

4 930 Maori, Dixon, and Rail Inoestigatioe Ophthalmology October 1965 cr «= 100 ACETAZOLAMIDE (mg/kg) I I 500 Fig. 1. Log dose-response relationship of acetazolamicle on turnover rate (Kout) of intracamerally administered inulin-^'c. Table III. Intraocular pressures during inulin- 1J C turnover studies estimated at 300 mg. per kilogram would be required to decrease aqueous humor formation by 50 per cent, and that no inhibition is attainable at doses below 60 mg. per kilogram. Correlation of aqueous humor turnover rate with eye pressure, eye venous pressure, iris artery pressure, iris artery pressure less eye venous pressure, and with femoral artery pressure. Venous pressure of the eye (VP) and ills artery pressure (IAP) were calculated for all eye pressures reported here from previously published 20 steadystate relationships: IOP = 0.87 VP and IOP = IAP Bivariate correlation analysis between aqueous humor turnover rate and the functions: (1) eye pressure, (2) eye venous pressure, (3) iris artery pressure, and (4) iris artery pressure less eye venous pressure were performed on data from the four individual groups, as well as on the com- Acetazolamicle (mg./ Control None If Ave Group I 100 I Average "Administered 30 minutes before beginning of Turnover study. I [ = IOP at beginning of turnover study. F LOP at completion of turnover study. Ave. =: IOP averaged at 5 minute intervals during the turnover study. 6.0 F Ave Group II 250 I F Ave Group III 600 I F Ave. Kg)

5 Volume 4 Number 5 Aqueous humor turnover rates 931 bined data from all groups. In no instance was a correlation coefficient found which indicated a significant (P <.05) correlation between the variables tested. Correlation of intraocular pressure and anterior chamber volume. The intraocular pressure measured immediately before each cat was killed (Table III) was tested against the anterior chamber volume determined immediately after death (Tables I and II). A bivariate correlation analysis was performed for each individual group of experiments. In no case was a significant (P <.05) correlation coefficient found which could imply the dependence of one variable upon the other. Effect of anterior ciliary vein occlusion on inulin- Ul C turnover rate. The anterior ciliary vein (ACV) was occluded by a No. 6-0 suture. Thirty minutes were allowed for equilibration of the homeostatic mechanisms before the beginning of the turnover rate determination. The average IOP at this time was mm. Hg. The mean turnover rate for a one-hour period was 2.3 per cent per minute (Table IV), which is not statistically different from that of normal controls. Analysis of the regression, turnover rate Table IV. Aqueous humor turnover rate in eyes with the anterior ciliary vein occluded IOP Kout (minr 1 ) Mean Table V. Specific activity of plasma after intracameral injection of inulin- 14 C 1 2 Counts Arterial 10.5* 50.9f per minute \ Eye venous 225.9* 73.6f "Average for a one-hour interval after injection. f Average for the 30 to 60 minute interval after injection. versus IOP, was not statistically significant (P >.05). Outflow pathway of inulin- n C. In two heparinized cats, inulin- 14 C was injected into the anterior chamber of the eye. In one of these eyes the anterior ciliary vein (ACV) was cannulated with No. 10 polyethylene tubing and the venous blood collected over the one-hour interval of the determination. In the second eye, a similar collection of venous blood was made by a microcannula placed in the intrascleral veins of the circle of Hovius. Arterial blood was collected from the femoral artery at ten-minute intervals in volumes of 2 c.c. The activity of the plasma of the arterial samples and the venous blood are seen in Table V. The venous effluent from the eye was in both cases significantly greater than that measurable in the arterial system. Discussion Inulin is a heavy molecular weight, starchlike polymer ideal for studies of extracellular space and fluid turnover. Inulin is not metabolized or bound, and is physiologically inert. The molecular weight and spatial configuration of inulin produce a diffusion coefficient equivalent to a molecular weight of 15,000 and insignificant intracellular penetration. 27 Inulin is very rapidly cleared by the kidney so that only insignificant amounts are found in the blood as the result of flow from the anterior chamber. Movement of inulin back to the aqueous humor from the blood, therefore, must also be veiy low and should not represent any appreciable source of error. Movement of inulin from the anterior chamber injection site to the posterior chamber is very unlikely not only because of the valvelike arrangement of the iris upon the lens but also because of the continuous streaming of aqueous humor from the posterior chamber to the anterior. Unless there is significant diffusion through the blood vessels of the anterior surface of the iris, or through the cornea or the lens, which is also unlikely, because of the large

6 932 Maori, Dixon, and Rail Investigative Ophthalmology October 1965 size of the inulin molecule, the values of turnover (Kout) determined in these experiments probably represent, quite closely, the values of the rate of aqueous humor formation. Inulin- 1 ' 1 C, injected into the anterior chamber under conditions which did not alter the intraocular pressure, was found to dischai'ge into the venous channels of the circle of Hovius. Movement of aqueous humor to the suprachoroidal space and its absorption from this area have been suggested 10 ' 2S and remain a possibility. It is more likely, however, that inulin leaves by way of the aqueous veins which have been shown in the cat 23 to be in direct communication with the venous plexus. If this is true, previous conclusions 24 based on other evidence to the effect that bulk outflow of aqueous humor is minimal in the normal eye of the anesthetized cat must be re-evaluated. Inulin- 14 C turnover rate in the eye of the anesthetized cat can give a fairly close estimate of the rate of aqueous humor formation. This is a particularly useful technique in determining effects of drugs on inflow, since the rate of movement of inulin across the aqueous-blood barrier can be presumed to be unchanged; differences in turnover rates can be ascribed to different rates of dilution of the anterior chamber fluid by altered rates of aqueous humor formation. The normal rate of aqueous humor turnover in a series of sixteen eyes was found to be 2.1 per cent per minute. This finding is in close agreement with results in rabbits, 1 ' 4 - Si 1S> 10 guinea pigs, 2 chicks, 3 and humans, 1 '> 15 in which different techniques were employed. Since the volumes of aqueous humor were determined, the absolute formation of aqueous humor per unit time could be calculated and was found to be 17 ^1 per minute. Acetazolamide in intravenous doses of 2 to 50 mg. per kilogram has been shown to be effective in lowering the intraocular 1H> 20> 23 pressure of the cat and with the higher dose the lowering of the IOP was maximal at 30 minutes. In the experiments reported here, eye pressures measured at the 30 minute time interval after drug administration and continuously for the following one hour of the turnover study, showed no tendency to rise. It must be concluded, therefore, that the doses of acetazolamide used (100 to 600 mg. per kilogram) were continuously effective during the experimental time period. The mean IOP of Group I (acetazolamide, 100 mg. per kilogram) is higher than that of the controls which received no medication. On the basis of these data, it should not be inferred that acetazolamide raised the eye pressure of this group rather than having lowered it from its own previous control value. Fairly large variability is normally noted in mean eye pressures of groups with small numbers. This is well illustrated by a review of steady-state eye pressures obtained in 100 eyes of 100 cats in this laboratory in Breaking the data down into consecutive groups of 10 observations each, the following mean IOP's were obtained: 24.50, 29.28, 24.42, 28.43, 18.27, 20.10, 21.15, 20.15, 17.22, and Acetazolamide at a dose of 100 mg. per kilogram did not significantly alter the turnover rates of aqueous humor. Doses of 250 mg. per kilogram and 600 mg. per kilogram reduced the turnover rates by 48 per cent and 71 per cent, respectively. Plotting of a dose-response (per cent inhibition of turnover) curve yielded a straight line with an intercept at 60 mg. per kilogram. The dose of acetazolamide required to produce 50 per cent inhibition of aqueous humor formation as seen from the curve, is 300 mg. per kilogram. Doses of acetazolamide, 2 to 50 mg. per kilogram, previously referred to, which have been demonstrated to lower the IOP are well below even the minimal dose of 60 mg. per kilogram where the inhibition of aqueous humor formation begins. Since acetazolamide has been shown to have no effect on the rate of aqueous humor outflow, one is led to the conclusion that another mechanism must be responsible for the ocular hypotensive effect

7 Volume 4 Number 5 Aqueous humor turnover rates 933 observed with this agent. It has been reported that the IOP of the cat is dependent upon its local venous pressure and that the fall in eye pressure observed with acetazolamide is due to a primary fall of the venous pressure brought about by a constriction of the iris artery. 25 The inability of acetazolamide to decrease the rate of aqueous humor turnover in doses which have been demonstrated to lower the IOP lends further support to the vascular mechanism of action of this agent. A seemingly paradoxical finding reported by Green, 1G> 17 Gloster, 13 and Foss, 11 in which local administration of acetazolamide, either subconjunctivally or intracamerally, had no eye pressure lowering effect but completely inhibited carbonic anhydrase can now be explained on the basis that an effective plasma level of drugs is required to constrict the iris arteries. With local administration, the quantities of acetazolamide which might penetrate the blood vessel walls would be quickly washed away and diluted by the circulating blood and thus would be incapable of exerting a vasoconstrictive, IOP-lowering action. The lack of correlation between intraocular vascular pressures and aqueous humor turnover rates found in these experiments is compatible with current concepts that aqueous humor is a secretion and offers no evidence to indicate that aqueous humor is an ultrafiltrate. No positive correlations were found in these studies between aqueous humor turnover rates and either anterior chamber volume or IOP. These results, therefore, offer no evidence in favor of the view that eye pressure is regulated by aqueous humor secretion. Indeed, aqueous humor production is quite constant whether the individual eye pressure is above or much below average values. The possibility that elevations of IOP would reflexly or otherwise decrease aqueous humor formation has often been proposed. This hypothesis was tested in five experiments in which the IOP was elevated by occlusion of the anterior ciliary vein, a procedure previously demonstrated 23 to produce a 14 mm. Hg rise in IOP. Aqueous humor production under these conditions was found identical to that of eyes at normal pressures and therefore offers no evidence that a reflex mechanism exists for an IOP control of aqueous humor formation. REFERENCES 1. Barany, E., and Kinsey, V. E.: The rate of flow of aqueous humor: I. The rate of disappearance of para-aminohippuric acid, radioactive Rayopake and radioactive Diodrast from the aqueous humor of rabbits, Am. J. Ophth. 32: (Part 2) , Barany, E.: Rate of flow of aqueous humor in normal and scorbutic guinea pigs, Arch. Ophth. 46: , Barany, E.: Rate of flow of the aqueous humor in the chicken (Gallus domesticus), Acta physiol. scandinav. 22: , Barany, E., and Wirth, A.: An improved method for estimating rate of flow of aqueous humor in individual animals, Acta ophth. 32: , Becker, B.: The effects of acetazolamide on ascorbic acid turnover, Am. J. Ophth. 41: , Becker, B.: Carbonic anhydrase and the formation of aqueous humor, Am. J. Ophth. 47: (Part 2) , Becker, B.: The turnover of iodide in the rabbit eye, Arch. Ophth. 65: , Becker, B.: The measurement of rate of aqueous flow with iodide, INVEST. OPHTH. 1: 52-58, Davson, H., and Luck, C. P.: The effect of acetazolamide on the chemical composition of the aqueous humor and cerebral spinal fluid of some mammalian species and on the rate of turnover of 2J Na in these fluids, J. Physiol. 137: , Fowlks, W. L., and Havener, V. F.: Aqueous flow into the perivascular space of the rabbit ciliary body, INVEST. OPHTH. 3: , Foss, R. H.: Local application of Diamox: An experimental study of its effect on the intraocular pressure, Am. J. Ophth. 39: , Gjone, E., Vance, H., and Turner, D. A.: Direct liquid scintillation counting of plasma and tissues, Internat. J. Appl. Radiation & Isotopes 8: 95-97, Gloster, J., and Perkins, E. S.: Effect of a carbonic anhvdrase inhibitor (Dinmnv ^ nn

8 934 Macri, Dixon, and Roll Investigative Ophthalmology October 1965 intra-ocular pressure of rabbits and cats, Brit. J. Ophth. 39: , Goldmann, H.: Das Minutenvolumen der menschlichen Vorderkammer bei Normalen und bei Fallen von primarem Glaukom, Ophthalmologica 120: , Goldmann, H.: Abflussdruck, Minutenvolumen und Widerstand der Kammerwasserstromung des Menschen, Documenta ophth. 5: , Green, H., and Leopold, I. H.: Effects of locally administered Diamox, Am. J. Ophth. 40 (part 2): , Green, H., Bocker, C. A., Calnan, A. F., and Leopold, I. H.: Carbonic anhydrase and the maintenance of intraocular tension, Arch. Ophth. 53: , Kinsey, V. E., and Barany, E.: Rate of flow of aqueous humor. II. Derivation of rate of flow and its physiologic significance, Am. J. Ophth. 32: (Part 2) , Kinsey, V. E.: Ion movements in the eye, Circulation 21: , Langham, M. E.: Specific and comparative activity of the carbonic anhydrase inhibitors Neptazane and Diamox on animal and human eyes, Brit. J. Ophth. 42: , Macri, F. J., Wanko, T., and Grimes, P. A.: The elasticity of the eye, Arch. Ophth. 58: , Macri, F. J.: Further studies on the action of acetazolamide and the venous pressure of the eye, Arch. Ophth. 64: , Macri, F. J.: Acetazolamide and the venous pressure of the eye, Arch. Ophth. 63: , Macri, F. J.: Interdependence of venous and eye pressure, Arch. Ophth. 65: , Macri, F. J., and Brown, J. G.: The constrictive action of acetazolamide on the iris arteries of the cat, Arch. Ophth. 66: , Macri, F. J.: The intraocular and vascular pressures of the cat eye, Exper. Eye Res. 3: , Smith, H. W.: The kidney: Structure and function in health and disease, New York, 1951, Oxford University Press, pp Starling, E. H.: Discussion on physiology of intraocular pressure, Proc. Roy. Soc. Med. 6: 44-82, Erratum In the article, "The ontogeny of lactate dehydrogenase in the chick lens," by Drs. Maisel, Kerrigan, and Syner in the June issue of the JOURNAL, p. 362, the reference Sippel 21 should have been Sippel 23 throughout. Reference 23 should have been as follows: Sippel, T. O.: Energy metabolism in the lens during aging, INVEST. OPHTH. In press.