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From the Department of Ophthalmology, Herlev Hospital, University of Copenhagen, Denmark.
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Abstract |
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METHODS. The effect of AZM on the blood-retina barrier function was assessed by differential vitreous spectrofluorometry using fluorescein as a tracer. The study included fourteen healthy subjects in a randomized double-masked crossover trial with 3 days’ treatment with AZM (500 mg/d) and placebo, respectively. The two examinations were separated by at least 1 week. Fluorescein concentration was determined separately from its metabolite fluorescein glucuronide. The passive permeability of fluorescein was determined by computerized modeling and curve-fitting to the preretinal curve and the plasma concentration curve obtained at 30 to 60 minutes after the injection of fluorescein. The unidirectional permeability due to outward active transport from vitreous to blood was estimated from the preretinal gradient and the plasma concentration at 7 to 10 hours after injection.
RESULTS. Treatment with AZM was associated with significant increases in passive permeability and unidirectional permeability of fluorescein. For the passive permeability the increase was on average 0.3 ± 0.4 nm/s (mean ± SD; range, -0.8–1.0 nm/s), and for the unidirectional permeability the increase was on average 7.4 nm/s ± 7.0 (mean ± SD; range, -3.3–19.0 nm/s).
CONCLUSIONS. Acetazolamide caused an increase in passive permeability. Unidirectional permeability was increased by AZM, indicating a stimulation of the outward active transport of fluorescein. It has been proposed that the edema-reducing effect of AZM is due to stimulated ion and fluid removal from the retina to the choroid. The results of this study are consistent with AZM affecting the blood-retina barrier with stimulation of at least one ion transport mechanism.
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Introduction |
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Vitreous fluorometry has documented that AZM reduces blood-retina barrier leakage in uveitis and in retinitis pigmentosa complicated by macular edema.4 5 Vitreous fluorometry has also been used to estimate the elimination kinetic of fluorescein from vitreous to blood.6 7 8 9 Differential spectrofluorometry enables determination of fluorescein and its metabolite fluorescein glucuronide in the vitreous and in the blood. Engler et al.9 used this modified vitreous fluorometry technique to obtain a more precise assessment of the carrier-mediated outward transport of fluorescein. We have recently examined the effects of AZM in nine patients with retinitis pigmentosa and macular edema using this method.5 Results from that study indicated that AZM stimulates active transport of fluorescein from retina to blood. To improve our understanding of the mechanism whereby AZM reduces retinal edema, the effects of AZM on passive permeability and active transport of fluorescein in healthy subjects were investigated in this study.
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Methods |
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The first permeability investigation occurred on day 4, after the intake of one capsule containing AZM (Diamox Retard, 250 mg) or placebo in the morning, in the evening the first 3 days, and in the morning the fourth day. After an interval of 3 to 14 days with no treatment, the second period of treatment commenced, with the second and final examination on day 4. Thus, the washout period between each permeability investigation was at least 1 week.
When the passive permeability and unidirectional permeability of fluorescein had been calculated from the data, the code was broken, and the AZM concentrations in frozen plasma samples were measured (analysis conducted at the National Poisons unit, Guy’s & Street Thomas’ Hospital, London, UK) to ensure that the drug had been taken as prescribed.
The research followed the tenets of the Declaration of Helsinki, and the protocol was approved by the Local Ethics Committee and the National Board of Health.
Determination of Passive and Unidirectional Permeability
Fluorescein is assumed to be transported from the blood to the
retina by pure passive diffusion as previously
discussed.10
Based on animal studies, the transport of
fluorescein in the direction from retina to blood is assumed to include
active transport.11
12
To our knowledge there is no
evidence of other contributors to the transport of fluorescein across
the blood-retina barrier. A significant posteriorly directed bulk flow
has been refuted in several studies.13
14
Any contribution
to the transport of fluorescein due to an electrical potential is
supposed to be small.6
The method and assumptions for determination of the passive permeability due to passive diffusion have previously been described in detail.15 16 17 The method used to quantify the unidirectional permeability due to active transport is with some modification identical to the method developed by Engler et al.9 A summary follows below.
Instrumentation
Ocular fluorescence measurements were performed by use of a
differential spectrofluorometry technique using a commercially
available fluorometer (Fluorotron, Ocumetics, CA) equipped with a
modified light source that excites light at rapidly interchanging
wavelengths of 458 and 488 nm.16
For the plasma
fluorescence analysis a cuvette spectrofluorometer (Perkin–Elmer LS
50) was used.
Procedure
All investigations commenced in the morning. An intravenous (IV)
catheter was positioned in a superficial antecubital arm vein, and the
pupils were dilated using topical 10% phenylephrine plus 1%
topicamide. Ocular fluorescence measurements were conducted before the
injection of an IV bolus injection of sodium fluorescein (14 mg/kg body
weight) and 30 minutes and 1, 7, 8, 9, and 10 hours after injection
(three scans per measurement until 2 hours after injection, thereafter
4 scans per measurement). Sufficient dilation of the pupil (more than 7
mm) was ensured before every fluorescence measurement by supplementary
eye drops. The fluorescein concentration in plasma was measured from
blood samples drawn from the catheter before injection, after
approximately 5, 7, 10, 15, and 30 minutes, and after 1, 2, 7, and 9
hours after injection. From each blood sample the first 10 ml was
thrown out. For the blood samples, heparin-coated vials were used. The
blood samples were centrifuged for 15 minutes at 3000 rpm
(1006g) followed by ultrafiltration of the plasma for 20
minutes by centrifugation at 3000 rpm (1006g) with
ultrafiltration filters, nominal cutoff at 30,000 Da (Millipore). The
ultrafiltrate was frozen immediately for later analysis. A plasma
sample taken before the injection of fluorescein was frozen to
determine the AZM concentration later.
Plasma Concentration Determinations
Diluted samples of ultrafiltrate were examined by use of a cuvette
spectrophotometer with excitation at 458 and 488 nm and emission at 515
nm. The concentration of fluorescein in plasma ultrafiltrate was
determined separately from that of fluorescein glucuronide by use of
differential spectrofluorometry.
Vitreous Concentration Determination
Data analysis included alignment of the scans after fluorescence
landmarks (cornea and the lens), correction for autofluorescence of the
eye, correction for absorption in the lens based on calculations of the
lens transmittance, and correction for absorption in extrinsic
fluorophores.16
The lens transmittance was calculated from
an average of lens autofluorescence data from the first and second
investigations and assuming an intrinsic ratio between the front and
back lens peaks of 1.2. Ocular fluorescence data were used to calculate
fluorescein concentration separately from fluorescein glucuronide as
described by Larsen et al.16
Fluorescence coefficients for
fluorescein and fluorescein glucuronide were determined on the basis of
measurements of fluorescence in cuvettes with known concentrations and
physiological pH (pH, 7.34).
Mathematical Analysis of Passive Permeability
Passive permeability, P(pas), across the
blood-retina barrier and the vitreous diffusion coefficient, D, was
calculated as average values based on the plasma concentration decay
curves and the ocular axial fluorescence scans recorded at 30 minutes
and 1 hour after fluorescein injection (totally 6 scans) by applying a
numerical solution to a mathematical diffusion model.15
The calculations are based on the assumption that the barrier between
blood and vitreous consists of a homogeneous spherical shell of
negligible thickness and that the concentration profiles for
fluorescein within the first hour are determined by the passive
permeability, the concentration curve for non–protein bound plasma
fluorescein, and the diffusion characteristics of fluorescein in the
vitreous.17
The unidirectional flux due to outward active
transport of fluorescein is assumed to be negligible within the first
hours.8
Mathematical Analysis of Active Transport
The unidirectional permeability of fluorescein in the direction
from the retina to the blood, P(uni), was
determined from scans recorded at 7, 8, 9, and 10 hours after
fluorescein injection (4 scans per hour per eye), where the fluorescein
concentration decreases toward the retina and the net transport of
fluorescein is from the vitreous to the blood. The calculations assume
that P(uni) is independent of the vitreous
concentrations at the late measurements. Nonsaturated active transport
has been assumed in other studies within this area.8
9
The flux of fluorescein across the blood-retina barrier in the
direction from the vitreous to the blood at the time, t, is the sum of
passive flux, J(pas), and the
unidirectional flux,*J(uni). The outward flux across the
barrier was determined based on Fick’s law of diffusion from the
vitreous concentration gradient at the retina surface and the diffusion
coefficient of fluorescein in the vitreous.9
In the
spherical eye with radius 0 
r a,
where (
C/r)a denotes
the preretinal gradient in the distance, a, the outward flux
at the time t can be expressed as follows:
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As illustrated in Figure 1 the preretinal gradient (in the direction from the vitreous to the retina) was estimated by linear regression to vitreous concentration data at the distance of 1 to 5 mm from the surface of the retina, and the concentration at the surface of the retina was calculated by linear extrapolation of the regression line. The plasma concentrations were measured at 7 and 9 hours. The concentration at 8 and 10 hours was determined by linear inter- and extrapolation, respectively. The average value of the diffusion coefficients for the two periods was used in the calculations. P(uni) was calculated as an average of the values determined at 7, 8, 9, and 10 hours.
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Additionally, in the evaluation of the method to determine unidirectional permeability, Friedman’s rank sum test18 was used to assess any dependence on time of the 7, 8, 9, and 10 hours’ P(uni) measurements during the two treatment periods.
Subjects
Fifteen subjects gave their informed consent to participate in the
study. Enrollment required the subjects to be in good health without
known systemic illness including diabetes, hypertension (blood pressure
more than 160/90 mm Hg), pregnancy, multiple allergies or adverse
reactions to fluorescein or sulfa drugs, prior episode of kidney
stones, or medication with diuretics. Furthermore, participants were
excluded if they had had eye surgery or had any current eye disease,
including significant cataract. Visual acuity had to be normal and any
refractive anomaly had to be less than ±5 D.
The subjects underwent an ordinary eye examination with slit-lamp biomicroscopy, ophthalmoscopy, tonometry, and fundus color photography (60°). One participant was excluded after the first treatment period due to a moderate adverse reaction to fluorescein injection. The remaining 14 subjects included 8 women and 6 men with an average age of 22.9 years (range, 20–26 years).
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Effects of AZM on P(pas) and P(uni)
For each patient, the values of P(pas) and
P(uni) after treatment with AZM and placebo,
respectively, are shown in Table 1
. For P(pas) there was no statistically
significant period effect or treatment-period interaction
(P = 0.61 and P = 0.097, respectively).
Acetazolamide led to a significant increase in
P(pas), of 0.3 ± 0.4 nm/s (mean ±SD;
range, -0.8–1.0 nm/s; P = 0.030).
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The control of AZM in plasma showed measurable values after AZM treatment of 9.8 ± 1.2 mg/l (mean ± SD; range, 7.1–12.0 mg/l) and no measurable values after placebo.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Effects of AZM on Active and Passive Transport
Effect of pH on the Dissociation of Fluorescein.
The calculations of the unidirectional permeability assume a uniform
passive permeability in the directions vitreous to blood and blood to
vitreous. A selective increase in the outward passive permeability from
vitreous to blood could cause an overestimation of the unidirectional
permeability. We therefore considered whether the increase in
unidirectional permeability could be due to a pH-induced increase of
the passive permeability in the direction vitreous to blood because a
drop in the pH of the retina and vitreous has been seen in cats within
the first hours after a bolus injection of AZM.19
Fluorescein can exist as a cation, neutral molecule, mono- and dianion
with pKa values of 6.7, 4.4, and
2.2.20
At physiological pH, fluorescein is found as mono-
and dianion, and a drop in pH, which increases the proportion of the
monoanion form, will lead to increased lipid solubility.20
However, studies indicate that fluorescein passes the blood-retina
barrier through water-filled pores,21
22
and a change in
fluorescein’s lipid solubility caused by a decline in pH is therefore
unlikely to affect the results significantly.
Effect of pH on Fluorescence
Because the fluorescence of fluorescein decreases significantly
even with minor reductions in pH, a decline in the pH of the vitreous
would cause an underestimation of the vitreous concentrations of
fluorescein. An underestimation of the vitreous concentration of
fluorescein would cause an overestimation of the unidirectional
permeability (and underestimation of passive permeability). A pH
decline of the magnitude observed in cats (0.14 pH U), would seem
unable to explain the increase in unidirectional permeability of 29%
found in this study as appears from the following. Fluorescence
coefficients established from measurements of fluorescein standards at
physiological pH (pH 7.34) are used for the determination of
fluorescein concentrations in the blood and vitreous. A previous study
examined fluorescence intensities at varying pHs (interval 6.5–7.5)
from a 1 µM fluorescein solution and calculation of the apparent
fluorescein concentration based on fluorescence intensity measurements
and fluorescence coefficients determined at physiological
pH.9
The apparent concentration as a function of pH is
approximately linear, and based on the linear regression output (slope,
0.046 µM/pH U; constant, -0.024 µM) it can be calculated that a
drop of 0.14 pH U compared to physiological pH leads to an
underestimation of the fluorescein concentration of approximately 6%,
which will lead to an overestimation of P(uni) of
roughly the same magnitude. Previous studies have been limited to the
study of the acute effects of AZM on retinal and vitreous pH, and it is
possible that pH is normalized after some time due to mechanisms that
counter this immediate effect of AZM.
Electrochemical Forces.
We also considered whether AZM-induced changes of the electrochemical
forces could play a role. The fluorometric calculations assume the same
potential on both sides of the blood-retina barrier. Acetazolamide
given IV is followed immediately by a decline in the electrochemical
potential of the eye. However, the effect seems to be relatively brief
because the electrochemical forces in the eye assessed from an
electrooculogram in healthy subjects is approximately
normalized 1 hour after a bolus injection of AZM despite AZM having a
half-life of 15 hours.23
Changes in electrochemical forces
are therefore assumed not to influence the transport of fluorescein
across the blood-retina barrier.
As can be seen from the considerations noted above, AZM-induced changes in vitreous and retinal pH and electrochemical gradient are inadequate explanations for the observed effect of AZM on P(uni) and P(pas).
Considerations of the AZM Effect on Passive Transport
We found an increase in passive permeability during AZM treatment.
A previous study of leakage assessed from the fluorometry penetration
ratio in five healthy subjects did not show any effect after an IV
bolus injection of AZM.24
The increase in passive
permeability in healthy subjects in the present study contrasts with
the statistically significant decrease in passive permeability found in
a study of 7 patients with retinitis pigmentosa and a decrease in the
posterior penetration ratio in a study of 30 patients with chronic
iridocyclitis complicated by macular edema after 2-week and 1-month
tablet treatments with AZM, respectively.4
5
We have no solid explanation for the increase in P(pas). Based on the previous studies, a limited decline or no change in P(pas would have been expected. It was considered whether the increase in P(pas) could have been related to capillary dilation caused by an AZM-induced increase in the blood’s content of CO2, PaCO2. Inhibition of carbonic anhydrase leads to increased loss of HCO3- in the kidneys and to metabolic acidosis in consequence. The metabolic acidosis is compensated for by increased ventilation, and a study of healthy subjects using the same daily doses of AZM as in the present study showed a reduced PaCO2 after 4 days of medication.25 Changes in the blood’s acid-base status can therefore hardly explain the increase in passive permeability.
An AZM-induced dilation of arterioles related to a reduced pH in the retinal extracellular fluid has been proposed.26 It could be speculated that an increased capillary area (recruitment of capillaries) could be an explanation for the observed increase in P(pas). Such an effect may be largest in healthy subjects, probably those with the highest metabolic activity and H+ production. Assuming that the retinal pH gradually normalizes over time, the shorter treatment time of this study could have caused a relatively lower retinal pH compared with the cited studies.4 5
Apart from the apical and basolateral membranes of the pigment epithelium, carbonic anhydrase is localized to the endothelium of the retinal capillaries, the Müller cells, and outer segment of the cones.27 28 A final speculation could be that the difference in AZM’s effect on P(pas) is due to a different effect on the barrier cells, causing shrinkage or swelling, respectively, depending on which side of the barrier is affected, implying that AZM’s effect depends on the extent to which it can penetrate the blood-retina barrier. There exists no experimental data to clarify this matter.
Effect on P(uni) and the Implications for the
Edema-Reducing Effect of AZM.
The increase in unidirectional permeability confirms the results from
our study of AZM’s effect on active transport of fluorescein in seven
patients with retinitis pigmentosa, five of whom had varying degrees of
angiographic macular leakage.5
That study gave evidence
that AZM affects the blood-retina barrier cells with stimulation of at
least one pump mechanism. It may be possible that other transport
systems are affected, but this cannot be determined by the present
study, which was limited to quantification of the transport of
fluorescein. It is not known which transport mechanism is responsible
for the active transport of fluorescein.
It has been proposed that the edema-reducing effect of AZM is due to a stimulation of ion and fluid removal.1 A stimulated ion and fluid removal has also been proposed on the basis of experiments in animals.29 30 Animal experiments suggest that the pigment epithelium has a considerable potential for drawing fluids from the retina to the choroid by means of ion transport.31 The transport mechanism for water across the blood-retina barrier remains incompletely understood. A recent study suggests cotransport of lactate and water across the pigment epithelium.32 It is unknown whether other cotransport systems for water are attached to the blood-retina barrier and whether the transport mechanism for fluorescein is involved. Future studies could clarify this.
Methodological Issues
The method used to determine the active component was with some
modification identical to that described by Engler et al.9
As stated in the Methods section, calculation of the active transport
involves the determination of the preretinal gradient from 7 to 10
hours. Compared to Engler et al. we used fluorescence data in a
slightly larger distance from the retina (1–5 mm rather than 0.5–5 mm
from the retina) and used linear rather than quadratic fitting for the
estimation of the preretinal gradient. The purpose in using
fluorescence data at a larger distance was to ensure that all data
points referred to the vitreous despite small differences in alignment
and to minimize the range of the fluorescence from the retina. Linear
fit was used instead of quadratic fit because preretinal curves did not
indicate any significant bend toward the retina. To obtain adequate
precision in the determination of the permeabilities, several scans are
required. For the determination of P(pas), six
scans were recorded, and for the determination of
P(uni) 16 scans were recorded. The high number of
scans used to determine P(uni) was due to the low
concentrations of fluorescein in the blood and in the vitreous, causing
a relatively higher measurement uncertainty. In the original
methodological work, hourly measurements from 7 to 12 hours after
fluorescein injection were used.9
In the present study the
measurement period was limited to 10 hours because in our experience
vitreous measurements in healthy subjects performed after 10 hours are
usually affected by significant noise. There was no statistically
significant time dependence.
Earlier studies use the terminology "outward permeability" and "inward permeability." The term "inward permeability" is susceptible to misinterpretation and corresponds to passive permeability used in this study. "Outward permeability" comprises both the active and passive components. For Table 2 unidirectional permeability for previously published data were calculated by deducting passive permeability from outward permeability. The comparison of previously published data for healthy subjects with the placebo values of this study show a better agreement for passive permeability than for unidirectional permeability.7 8 9 In all studies P(uni) is above zero in accordance with the existence of an active transport.
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Acknowledgements |
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Footnotes |
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Data extracts have been published at the ARVO Congress 1996.
Supported by grants from the Jasha Foundation, the Danish Eye Research Foundation, and the Danish Hospital Foundation for Medical Research, Region of Copenhagen, The Faeroe Islands, and Greenland.
Submitted for publication December 16, 1997; revised December 11, 1998; accepted January 20, 1999.
Proprietary interest category: N.
* Unidirectional flux "frequently denotes the special passive
transport process where the concentration in one phase is kept at
zero." In this article the term is used for the one-way flux that is
caused by the existence of special transport molecules in the
membrane.33 
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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