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Turnover of hyaluronate in the aqueous humour and vitreous body of the rabbit
Eccp. Eye Res. (19833 36, 493-504
Turnover
of Hyaluronate Vitreous ULLA
* Departments
f University
Body
B. G. LAURENT*
in the Aqueous of the Rabbit AND
Humour
and
J. R. E. FRASER7
and Medical and Physiological Chemistry, of Uppsala, Uppsala, Sweden, and Department of Medicine, Royal Melbourne Hospital, Victoria 3050, Australia
of Ophthalmology
University of Melbourne
(Received 1 May 1982 and accepted 1 December
1982, New York)
Sodium hyaluronates with molecular weights of 18000, 500600 and 4 x 106, labelled with either 3H or i4C, were injected into the anterior chamber 01‘ into the centre of the vitreous body of rabbits and their rates of disappearance were followed. The out-flow from the anterior chamber in anaesthetized animals was virtually independent of the molecular weight indicating that the disappearance of the polysaccharide is controlled by bulk flow. The rate constant for the disappearance of sodium hyaluronate was 0,0094/min and with after treatment with indomethacin @OOSl/ min. These figures are in general agreement published flow-rates of aqueous humour in the rabbit. The disappearance from the vitreous body was strongly molecular weight dependent indicating a diffusion controlled transport. The rate constant for hyaluronate with mean molecular weight of 18000 was O.iCi/day and of 500006, O,024/day. The rate constant for endogenous hyaluronate was estimated to be about O.Ol/day. A calculation using these rate constants shows t,hat the turnover of sodium hyaluronate in the rabbit anterior chamber is about 3 pg per 24 hr while the turnover in the vitreous body is only 15% of that. This confirms an earlier conclusion (Laurent and Granath, 1983) that the preponderant part of the hyaluronate in aqueous humour is not a general degradation product from the vitreous body. Key words : hyaluronic acid ; turnover : aqueous humour ; vitreous body ; bulk flow ; diffusion.
1. Introduction Sodium hyaluronate is one of the major components of the vitreous body. Numerous reports have been published on its concentration, distribution in the tissue and its macromolecular parameters (for reviews see Balazs, 1965; Berman and Voaden, 1970). Relatively little is, however, known about its metabolism. It is believed that the polysaccharide is synthesized by the hyalocytes in the cortica,l layer of the vitreous -.. (Gsterlin and Jacobson, 1968a; Jacobson, 1978a, b) or extracellularly from intermediates produced by the cells (osterlin and Jacobson, 1968b; Jacobson, 197gb). When radioactive glucosamine is administered to owl monkeys, it becomes ineorporated into hyaluronate in the vitreous and remains there for at least 72 days without any significant turnover (osterlin, 1968). Another indication of a very slow turnover comes from the regeneration time of hyaluronate after removal of vitreous in the owl monkey. Sweeney and Balazs, (see Balazs, 1965) found that six months were required for the concentration to rise to that of the control eye. The concentration of hyaluronate in the aqueous humour is low in most species when compared to the concentration in the vitreous (Balazs, Laurent, Laurent, DeRoche and Bunney, 1959; Laurent, 1981). The owl is an exception; in this animal. Please Hospital,
address S-75185
0614~4835/83/040493
correspondence to Dr Uppsala, Sweden. + 11 $03.00/O
Ulla
Laurent,
Department
0 1983 Academic
of Ophthalmology,
Press Inc. (London)
University
Limited
494
U.B.G.LBUREKTAPL‘DJ.R.E.FRABER
hyaluronate is found covering the cornea1 endothelium (Barany, Berggren and Vrabec, 1957; Balazs et al., 1959). The origin of the hyaluronate in the aqueous humour is unknown but it was recently shown in rabbit and cattle that the polysaccharide has a high-molecular weight component, which is not present in the vitreous body. Therefore the hyaluronate in the aqueous humour does not seem to be a general degradation product from the vitreous (Laurent and Granath, 1983). The interest in turnover studies on hyaluronate in the eye has increased owing to the introduction of highly concentrated hyaluronate as an aid in ophthalmic surgery. Viscous solutions of the polysaccharide are used both as a replacement for vitreous tissue (Balazs et al., 1972) and in anterior segment surgery (Pape and Balazs, 1980). Widder (1962) injected hyaluronate (4 mg/ml; mol. wt 1.4-28 x 106) into rabbit vitreous and found that its half-life was approximately two weeks. Hultsch reported seemingly similar results in owl monkeys (Hultsch, 1979). Denlinger and Balazs give values of half-life for implanted hyaluronate in the vitreous of owl monkeys and rhesus monkeys of 30-40 days (Denlinger and Balazs, 1980) and 60-65 days (pers. comm.). However, when the anterior cortical gel in owl monkeys is damaged half-lives as short as 2-3 days are observed (For earlier references see Balazs and Hultsch, 1976; Hultsch, 1979; Balazs and Denlinger, 1982). According to Denlinger and Balazs (1980) the out-flow route of hyaluronate implanted in the vitreous body of owl monkeys is via the aqueous humour. The results of &terlin (1971, 1978) which showed a decreased concentration of hyaluronate in the vitreous of the aphakic eye are in agreement with the concept that there is a natural barrier against transport of hyaluronate from the vitreous to the aqueous humour. High-concentration (10 mg/ml), high-molecular weight hyaluronate injected into the anterior chamber of owl monkeys passes through the trabecular meshwork within 1-2 days (Schubert, Denlinger and Balazs cited by Balazs and Denlinger, 1982). The availability in our laboratory of [3H]- and [r4C]-labelled hyaluronate preparations of molecular weights varying between 18000 and several millions (Fraser, Laurent, Pertoft and Baxter, 1981), has enabled us to study the disappearance of trace amounts of hyaluronate from both vitreous body and anterior chamber in the rabbit. The results are reported in this communication. 2. Materials
and
Methods
Materials Sodium hyaluronate labelled either with 3H in the acetyl group of N-acetylglucosamine or with 14C in the sugar rings was produced by tissue culture of synovial cells as described by Fraser et al. (1981). The purified hyaluronate was dissolved in phosphate-buffered saline and maintained sterile (Fraser et al., 1981). The molecular weight distributions of the samples were determined by gel chromatography as described by Wik, Andersson, Jacobsson and Granath (1979) (see also Laurent and Granath, 1983). The molecular weight parameters and the specific radioactivities of the preparations are given in Table I. Radioactive hyaluronate samples are slowly degraded by radiolysis and therefore data on the total radioactivity in the solutions and the interval between the molecular weight determination and the animal experiments are included. The degree of radiolysis can be inferred ff_om sample 2 for which the molecular weight was determined at 4 months’ interval and M, dropped from 83 x lo5 to 47 x 105. For simp1icit.y each preparation has been assigned an approximate molecular weight. With few exceptions, the turnover studies were made with [aH]and [‘*Cl-labelled hyaluronate in the same experiments. The polysaccharides were mixed in such proportions that the solutions contained &20-times more 3H than r4C expressed in d/min. The injected volume (30 ~1) contained approximately @5 ,ug of sodium hyaluronate.
TURNOVER All weight
OF
experiments were carried out of these animals was 33 kg.
HYALURONATE on female
rabbits
IN (New
THE Zealand
495
EYE White).
The
average
Methods Determination of turnover in the anterior chamber. The rabbits were anaesthetized by intravenous injection of pentobarbital (initial dose 30 mg/kg body weight) and smaller doses were given to maintain anaesthesia. Tetracaine drops were used topically to supplement the anaesthesia. The animal was kept warm during the experiment with the aid of a heating pad. At. the end of the experiment, the animal was sacrificed by an overdose of pentobarbital. Two series of experiments were performed. In one series, the animals received no anti-inflammatory drugs. In the other series, they were pretreated with an intravenous dose of indomethacin (Sigma, St. Louis, MO, USA; 20 mg/kg body weight dissolved in 10 ml of 91 M-phosphate buffer pH 7.2 and 0.05 iw-NaCl). Indomethacin inhibits the synthesis of prostaglandins which are involved in the disruption of the blood-aqueous barrier (Beitch and Eakins, 1969; Podos, Becker and Kass, 1973). Fifteen minutes were allowedto pass after the injection before start of the experiment. The animals were laid on their sides and the experiments were performed on the exposed eyes. Needles 45 mm long with outer and inner diameters of 645 and 0.2 mm, respectively, and with divided inlets (Y-shaped) were used. The needles were fired through the periphery of the cornea into the anterior chamber using a needle gun (Sears, 1960). One inlet was joined by a plastic tubing to an Agla Precision Syringe by which 36 ,ul of the aqueous humour was withdrawn. The other inlet was joined to another Agla Syringe by which 30,~d of the hyaluronic acid solution was subsequently injected into the chamber. The tip of the needle was then fired through the contralateral side of the cornea so that it remained outside the anterior chamber during the experiment. This was preferred to withdrawing the needle from the eye which could cause leakage. Each experiment was terminated by withdrawing the aqueous humour through a 0.4 mm needle into a 1 ml syringe. The chamber was washed once with 0.2 ml of physiological saline and the washing was either analysed separately or combined with the main portion. It was planned to use both eyes at the same time. After injection in the right eye, the rabbit was turned so that the eye with inserted needle faced the table and another experiment was carried out on the left eye. When analysing the experiments, it became apparent that the right eye exhibited a faster (approximately 50 %) turnover rate presumably due to pressure exerted on the eye. The data on the right eyes have, therefore, been excluded from calculations of turnover rates. However, they could still be used to calculate the relative difference in disappearance rates of two hyaluronates injected at the same time. The radioactivity in the aqueous humour samples was determined in a Searle Nuclear Chicago Isocap/300 liquid scintillation counter. The samples were diluted to 1 ml with water and mixed with 10 ml of scintillant 299 (Packard-Becker B.V., Breda, The Netherlands). l4C was read with an efficiency of 64% (as determined by a commercial toluene standard) and < 0.1 y0 of the 3H counts appeared in the r4C channel. 3H was read with an efficiency of 40 0h (also determined with a commercial standard). From the counts in the aH channel the fraction of 14C counts appearing in this channel was subtracted. The fractional value was determined with a pure [%-hyaluronate sample. As contamination of the aqueous humour by blood serum could disturb the analysis, rabbit serum (which has a protein content one hundred times higher than aqueous humour) was added in 25-200 ~1 to the scintillation vials. The effect on the counts was a reduction of < 2 o/0 in the r4C channel and < 5 o/0 in the 3H channel. The results have been expressed in terms of the fraction of injected material remaining in the aqueous humour after a given time. The injected dose was determined by transferring 30 pl from the Agla Syringe directly into scintillation vials at the same time as the animal experiment was carried out and treating the samples the same way as the aqueous humour samples. The reproducibility of dispersing high-molecular weight hyaluronate (mol. wt 4 x 106) by the Agla Syringe was determined in a series of ten samples and was found to have a standard deviation of 5.4%. The same experiment with the low-molecular weight hyaluronate(mo1. wt 18000) gave a standard deviation of 3.4 y’, Determination of turnover in the vitreous body. The rabbits were anaesthetized as above. The eyes were proptosed in order to facilitate injection behind the equator of the eyeball. A needle
18000 500000*
3H w
3H 3H
weight
1 2
3 4
* The molecular
x x x x x
Indomethacin Indomethacin Indomethacin Indomethacin
18000 500000 4x106 All
Number of eyes
4 7 2 13
4 10 6 20
four
(lo’
x x x x x
TABLE
months
52 1.6 1.3 33 8.1
S.D.K
the anterior
S.D.,
0.97
0.4 6.1
0.05
0.03 0.08
0.08 0.05 0.05 0.04
(44-109) (63-83) (69-103) (6683)
114 (99-133)
111 (91-138) 118 (94-158)
62 71 83 74
--.-__-_data in Fig. 1 according to equation ln (C/C’“) = - K’ t + In n where a is the extrapolated value of C/C, from the experimental values.
0.92 0.98
o-87 0.95 0.79 0.87
a
2-4 months 3 -5 months il mouth 34 months 1 month
-_ __ C and ti is the
Interval between mol. wt determination and animal experiments
t: (min) (with 95 “/, confidence limits)
7.5 x 104 3.7 x 105
4x105 12 x 106
chamber
1.3 x 105 2.5 x 10’
Specific radioactivity WmWm
preparations
2x 106 2x 10’
0.3 0.6
1.1 0.6 0.6 @5
( lo3 x min-I)
fwm,
11
interval.
103 lo5 10” 105 105
Radioactivity of solution d/min/ml
of hyaluronate
63 5.9
11.1 97 8.4 9.4
x min-‘)
K
of hyaluronate
with
labelling
Number-average mol. wt
and radioactive
I
The rate constant (K) for the disappearance has been calculated from the experimental C, are the concentrations in the anterior chamber at time t and time zero, respectively. time required for the concentration to reach half its original value (half-life).
None None None None
Treatment of animal
twice
103 lo5 105 106 106
L9isappeurance
on this preparation
185 6.3 47 3.9 4.7
Weight average mol. wt
weight parameters
18000 500000 4x106 All
Hyaluronate preparation
was determined
40 x 106 40 x IO”
Designation
Label
Preparation IlO.
Molecular
TABLE
TURNOVER
OF
IN THE
HYALURONATE
EYE
497
with the same dimensions as described earlier but with a single inlet was fired into the middle of the vitreous body. The position of the needle could be followed visually through the transparent media. Thirty ,LL~of hyaluronate solution was injected with the aid of an Agla Syringe during a 3 min period and 2 min later, with released external pressure on the eye, the needle was withdrawn. The animal was allowed to regain consciousness and was returned to the animal quarters. The eyes were examined repeatedly by ophthalmoscopy. In one case the lens was damaged and the eye was excluded from the material. PITo bleedings in the vitreous or retina were noted. At predetermined times, the animals were sacrificed and the eyes enucleated and freed from surrounding tissues and blood. They were dried in an oven at 5660% and stored dried until analysis. The eyes were combusted in a Packard Model 306 Tri-Carb Sample Oxidizer in accordance with t,he instructions from the manufacturer. WO, and 3H,0 were collected separately and the counts recorded in a liquid scintillation spectrometer (Packard Tri-Carb 2425). The same amount of hyaluronate, which had been injected in the eyes, was also added to 500 mg of filter paper which was combusted and analysed as the samples. The reproducibility of pipetting high-molecular weight hyaluronate (mol. wt 4 x 106) on filter paper, and of combusting and analysing it was determined in a series of ten controls. The standard deviation was found to be 5 y0 compared with 3 y0 for low-molecular weight hyaluronat,e (mol. wt 18000). 3. Results Disappearance
of hyaluronate
from
the anterior
chamber
The experimental results are plotted in Fig. 1. The regression lines calculated from the plots in the figure are tabulated in Table II. The disappearance rates for the
a
b
0.1 ' 0
100
200
300
Min
FIG. I. The disappearance of hyaluronates from the anterior chamber of untreated (a) or indomethncintreated (b) rabbits. C/C, is the fraction of that originally injected remaining in the chamber. The following hyaiuronates were used : preparation 1, mol. wt 18000 (A) ; preparation 3, mol. wt 500000 (0); preparation 3, mol. wt 4 x lo6 ( x ); and preparation 4, mol. wt 4 x lo6 (m).
498
U. 3. G. LAURENT
AND
J. R. E. FRASER
hyaluronates of different molecular weights were similar although the rates for the high-molecular weight material tend to be somewhat lower. To further explore the dependence of the rate of disappearance on molecular weight we have plotted the ratio 3H/14C as a function of time in those experiments where r3H]- and [14C]-labelled hyaluronates were iiljected simultaneously. The data are shown in Fig. 2. In these plots, we have also included the series of experiments on the right eyes (see Methods) which were excluded from calculations of rate constants.
‘,O t-----s0
2.0
05--
2.0~-
b
0 l
0
0 .
c
A A A
A
o.sJ 0
100
200
300
FIG. 2. The variation in 3H/14C ratio with time when both [3H]- and [‘%I-labelled hyaluronates were injected simultaneously. The concentrations of 3H and 14C have been expressed as fractions of the injected material: (a) and (b), untreated; (c) indomethacin-treated animals. Mixture of preparations 1 (3H; mol. wt 18000) and 2 (W; mol. wt 500900) (0, 0, A, A). Mixture of preparations 2 (Y!; mol. wt 500000) and 4 (3H; mol. wt 4 x 106) (0,m). Open symbols, right eyes. Filled symbols, left eyes. The slope of the plot for the left eyes in (a) is 126 x 10-4. It is positive within 95 y0 confidence limits. The slopes of the plots in (b) and (c) do not significantly differ from zero.
It is apparent that when [‘*Cl-labelled hyaluronate with molecular weight of 500000 is injected together with [3HJ-labelIed hyaluronate of molecular weight 18000, the ratio (3H/14C) is essentially constant during the experiment indicating that the two fractions were removed at the same rate. When [3H]-labelled hyaluronate with molecular weight 4 x lo6 was injected with the [14C]-labelled material, the ratio increased with time in both series of experiments [Fig. 2(a)] indicating that the most high-molecular weight material was removed at a slower rate. The rates of disappearance in untreated and indomet,hacin-treated rabbits differed significantly (P < 0.01) (Fig. 1, Table II). The constant a tabulated in Table II represents the extrapolated value of C/C, at the beginning of the experiment. It is less than unity (0.7990.98) and is a measure of
TURNOVER
OF
HYALUROSATE
IN
the eEiciency by which the radioactive hyaluronate chamber by the experimental procedure. A recovery regarded as excellent in this type of experiment. Disappearance
of hyaluronate
from
the vitreous
THE
499
EYE
is removed from the anterior in the order of 90% must be
body
The removal of two different hyaluronates from the eyeball after injection into the vitreous body is described in Fig. 3. The parameters for the linear regressions are given in Table III. The half-life of the hyaluronate in the eye increases with increasing molecular weight, from 4 days for the sample with 18000 mol. wt to 30 days for the 500000 mol. wt sample. The constant a in Table III is 0.83 and 0.84 which indicates a lower recovery in the experiments on the vitreous body than those on the aqueous humour. This may be accounted for by a rapid initial loss of some radioactive hyaluronate through the channel in the gel formed by the injection needle.
0.01-j 0
10
FIG.~. The disappearance of hyaluronates body. C/C!, is the fraction of that originally were used: preparation 1, mol. wt 18000
Hyaluronate preparation
of hyaluronate Number of eyes
18000 500 000
9 9
For explanations
see Table
III
from the eyeball after injection K
(lo3 x day-‘) 159 23.5 II.
30
from the eyeball after injection into the centre of the vitreous injected remaining in the eyeball. The following hyaluronates (A); preparation 2, mol. wt 500000 (0). TABLE
Disappearance
20
Days
into the vitreous
S.D.K
(lo3 x day-‘)
a
S.D.,
12.5 91
0.83 0.84
0.12 0.10
body
ti (days) (with 95 y0 confidence limits) 44 (3.7-5.5) 295 (154-347)
500
U. B. G. LAURENT
Ah-D
J. R. E. FRASER
A hyaluronate of high-molecular weight but low specific radioactivity (preparation 3, Table I) was also used. There was a considerable spread of the experimental points for this sample owing to the low counts, and possibly experimental difficulties with the preparation, and no reliable K-value could be calculated but the disappearance rate was slow.
4. Discussion If a tracer is introduced into a compartment of constant volume and its disappearance from the compartment is proportional to its actual concentration then, the concentration, C: will decrease according to
C -=e co
-K.t ’
(1)
where C,, is the original concentration at time zero, t is the time and K is the rate constant. This equation can be applied to tracers introduced into the aqueous humour (see e.g. Bill a,nd Hellsing, 1965). If it is assumed that the disappearance of the tracer is governed by bulk flow then, 1 dv K=ppp (2) where V, is the volume of the compartment and dv/dt is the flow-rate. From Eqns. (1) and (2) it follows that the half-life (4, i.e. when the tracer has reached half its original concentration), is obtained by:
(3) The rate constant for the disappearance of hyaluronate from aqueous humour is given in Table II. The disappearance is approximately the same for materials of different molecular weights which indicates that it is governed mainly by bulk flow. The high-molecular weight hyaluronate disappears at a slightly slower rate (about 15 %) which may be due to a small sieving effect in the out-flow channels for particles of this size (Fig. 2). The volume of the anterior chamber in the rabbit is approximately 250 ,LL~and the flow-rate is of the order of 3 pl/min (Kinsey and Reddy, 1964). Using these figures the half-life of a compound removed by bulk flow calculated according to Eqn. (3) should be 60 min. This is in agreement with the value in Table II for untreated rabbits. However, it has been demonstrated (Becker, Krupin and Podos, 1970) that when barbiturate anaesthesia is used in rabbits, the flow-rate of aqueous humour decreases. Conversely the inflammatory response in the rabbit eye during the experiment would be expected to increase the flow-rate by leakage of blood plasma from the uvea and also the facility of out-flow (Podos et al., 1973). Evidence to support this may be adduced from the slower turnover of hyaluronate jn the indomethacin-treated animals. It is possible that the barbiturate effect and the inflammatory response counteract each other and that this is the reason for a turnover rate of hyaluronate very close to the known bulk flow rate of aqueous humour. We conclude that our results are in general agreement with the disappearance of hyaluronate by bulk flow, If the disappearance had been governed by diffusion, one should expect a preferential transport of the low-molecular weight hyaluronate and ifit had been receptor-mediated one could expect an increasing mass uptake per receptor with increasing molecular weight.
TURNOVER
OF
HYALURONATE
IN
THE
501
EYE
The disappearance of hyaluronate from the eye ball after injection into the vitreous body is strongly molecular weight dependent (Table III; Fig. 3). This is in agreement with a diffusion controlled transport in the vitreous body. Similar behaviour has also been demonstrated for a number of other compounds of lower molecular weight (see Maurice, 1980). Extrapolation of our data to a hyaluronate mol. wt of 2-3 x lo6 corresponding to hyaluronate in the rabbit vitreous (Laurent and Granath, 1983) (see Fig. 4) gives an approximate rate constant for the disappearance of O.Ol/day (4 - 70 days) in agreement with the very slow turnover noticed in owl monkey by osterlin
-1d
7 -to-*
g Y
10-8 110-3 106
108 106 107 Molecular weight FIG. 4. Procedure to obtain the rate constant, K, for the disappearance of endogenous hyaluronate from the vitreous body. When the diffusion coefficient, D, of hyaluronate (taken from Laurent, Ryan and Pietruszkiewicz, 1960) is plotted vs. mol. wt in a double logarithmic plot a straight line is obtained (a). When the rate constants (0) determined for hyaluronates with molecular weights of 18000 and 500000 are plotted similarly and a line drawn between them it becomes parallel to that of the diffusion coefficient indicating that K is proportional to D and thus diffusion controlled. If the line is extrapolated to the molecular weight of endogenous hyaluronate (2-3 x 10’; Laurent and Granath, 1983) a value of O.Ol/day is obtained. 105
(i968) and Balazs and coworkers (Balazs and Hultsch, 1976; Denlinger and Balazs, pers. comm.). It should be stressed that the tracer hyaluronate was deposited in the centre of the vitreous body and that the rate of disappearance therefore only represents the material in this part of the tissue. The results of asterlin (1968) would, however, indicate that the over-all turnover rate also is of the same magnitude. The rapid disappearance noted by Widder (1962) in rabbits and by Balazs and coworkers (Balazs and Hultsch 1976; Hultsch, 1979) in owl monkeys, when high concentrations of hyaluronate were introduced in the vitreous body, is presumably due to a disintegration of the normal vitreous structure and a break-down of the diffusion controlled transport (Balazs and Hultsch, 1976; Balazs and Denlinger, 1982). The transport parameters measured in these experiments can be used to calculate an approximate turnover per day in the two eye compartments. If we introduce mass Aow instead of volume flow, eqn. (2) can be written in the following form K,‘.d” 011
dm/dt
m,
dt
= K.m,,
(4)
502
U. B. G. LAUREST
AND
J. R. E. FRASER
where m, is the total mass of hyaluronate in the compartment and dm/dt the amount which is flowing through the system per time unit. The total amount of hyaluronate in the anterior chamber is equal to the volume (250 ~1) times the concentration (1.1 pg/ml; Laurent, 1981). Using the rate constants in Table II, one obtains a total turnover of the order of 3 ,ug/24 h in the anterior chamber. A similar calculation for hyaluronate in the vitreous body can be made assuming the volume of the rabbit vitreous is 15 ml (Kinsey and Reddy, 1964); the hyaluronate concentration is about 30 pg/ml (Laurent and Granath, 1983) and K is O*Ol/day. The calculated turnover is 0.45 ,ug/24 h and represents only 15 o/0 of that in the aqueous humour. The present results as well as the observation by Laurent and Granath (1983) that the aqueous humour hyaluronate has a higher molecular weight than that in the vitreous body indicate that the polysaccharide in the aqueous is not a general degradation product from the vitreous body. It could conceivably derive from the most anterior part of the vitreous. It is striking, however, that the owl has such a marked layer of hyaluronate on the endothelial surface of the cornea (Bar&y et al., 1957; Balazs et al., 1959) and the endothelial cells could be a source of the polysaccharide. The reports by Wolf (1968) who observed, by electron microscopy, the formation of a viscous material by the endothelial cells may be adduced in favour of this hypothesis. ACKNOWLEDGMENTS One of us (U. B. G. L) is grateful to Professors Ernst advice and to Professor Barany for placing equipment
Barany and Anders Bill for valuable at her disposal. Dr Kirsti Granath kindly performed the molecular weight analyses of the radioactive hyaluronates. The project has been supported by the Swedish Medical Research Council, The National Health and Medical Research Council of Australia and Carmen and Bertil Regner’s fund. REFERENCES
Balazs, E. A. (1965). Amino sugar-containing macromolecules in the tissues of the eye and theear.InTheAminoSugarsVol.IIA(EdsBalazs,E. A.andJeanloz,R. W.).Pp.401-60. Academic Press, New York and London. Balazs, E. A. and Denlinger, J. L. (1982). The vitreous. In The Eye (Ed. Davson, H.) 3rd edn; Academic Press, London. Balazs, E. A., Freeman, M. I., Klati, R., Meyer-Schwickerath, G., Regnault, F. and Sweeney, D. B. (1972). Hyaluronic acid replacement of vitreous and aqueous humour. Mod. Probl.
Ophthd.
10, 3-21. (Karger,
Basel).
Balazs, E. A. and Hultsch, E. (1976). Replacement of the vitreous with hyaluronic acid, collagen and other polymers. In Advances in F&reous Surgery (Eds Irvine, A. R. and O’Malley, C.). Pp. 601-23. Charles C. Thomas, Springfield. IL. Balazs, E. A., Laurent, T. C., Laurent, U. B. G., DeRoche, M. H. and Bunney, D. M. (1959). Studies on the structure of the vitreous body. VIII. Comparative biochemistry. Arch. Biochem. Biophys. 81, 464-79. Barany, E., Berggren, L. and Vrabec, F. (1957). The mutinous layer covering the cornea1 endothelium in the owl Strix Aluco. Br. J. Ophthalmol. 41, 25-30. Becker, B., Krupin, T. and Podos, S. M. (1970). Phenobarbital and aqueous humour dynamics: Effect in rabbits with intact and transected optic nerves. Am. J. Ophthalmol. 70, 68690. Beitch, B. R. and Eakins, K. E. (1969). The effects of prostaglandins on the intraocular pressure of the rabbit. Br. J. Pharmacol. 37, 158-67. Berman, E. R. and Voaden, M. (1970). The vitreous body. In Biochemistry of the Eye (Ed. Graymore, C. N.). Pp. 373-471. Academic Press, London and New York. Bill, A. and Hellsing, K. (1965). Production and drainage of aqueous humour in the cynomolgus monkey (Macaca irus). Invest. Ophthalmol. 4, 920-6.
TURNOVER
OF
HYALURONATE
IN
THE
EYE
503
Denlinger, J. L. and Balazs, E. A. (1980). Replacement of the liquid vitreus with sodium hyaluronate in monkeys. I. Short-term evaluation. Exp. Eye Res. 31, 81-99. Denlinger, J. L., El-Mofty, A. A. M. and Balazs, E. A. (1980). Replacement of the liquid vitreus with sodium hyaluronate in monkeys. II. Long-term evaluation. Exp. Eye Res. 31, 101-17. Fraser. J. R. E., Laurent, T. C., Pertoft, H. and Baxter, E. (1981). Plasma clearance, tissue distribution and metabolism of hyaluronic acid injected intravenously in the rabbit. Biochem. J. 200, 415-24. Hultsch, E. (1979). Vitreous structure and ocular inflammation. 2nd 1nl. Symp. on Immunology 1978 (Eds Silverstein, A. M. and and Immunopathology of the Eye, San Fransisco, O’Connor, G. R.). Pp. 97-102. Jacobson, B. (1978a). Biosynthesis of hyaluronic acid in the vitreous. V. Studies on a particulate hyalocyte glycosyl transferase. Exp. Eye Res. 27, 247-58. Jacobson, B. (197813). Biosynthesis of hyaluronic acid in the vitreous. VI. Isolation of a complex containing hyaluronic acid and glycosyl transferase activity and studies on the activity of a soluble glycosyl transferase. Exp. Eye Res. 27, 254-73. Kinsey, V. E. and Reddy, D. V. N. (1964). Chemistry and dynamics of aqueous humour. In The Rabbit in Eye Research (Ed. Prince, J. H.). Pp. 218-319. Charles C. Thomas, Springfield, IL. of hyaluronic acid. Laurent, T. C., Ryan, M. and Pietruszkiewiecz, A. (1960). F ractionation The polydispersity of hyaluronic acid from the vitreous body. Biochim. Biophys. Acta 42, 47685. Laurent, U. B. G. (1981). Hyaluronate in aqueous humour. Exp. Eye Res. 33, 147-55. Laurent, U. B. G. and Granath, K. A. (1983). The molecular weight of hyaluronate in the aqueous humour and vitreous body of rabbit and cattle eyes. Exp. Eye Res. 36, 481-492. Maurice, D. M. (1980). Drug exchanges between the blood and vitreous. In The Blood-Retinal Barriers (Ed. Cunha-Vaz, J. G.). Pp. 165-78. Plenum Press, New York and London. ijsterlin, S. E. (1968). The synthesis of hyaluronic acid in vitreous. III. In vivo metabolism in the owl monkey. Exp. Eye Res. 7, 524-33. &terlin, S. E. (1971). Changes in the macromolecular composition of the vitreous produced by removal of the lens. Exeerpta Medica Intern. Congr. Ser. No. 222; Ophthalmology. Pp. 1626-23. &terlin, S. E. (1978). Macromolecular composition of the vitreous in the aphakic owl monkey eye. Exp. Eye Res. 26, 77-84. &terlin, S. E. and Jacobson, B. (1968a). The synthesis of hyaluronic acid in vitreous. I. Soluble and particulate transferases in hyalocytes. Exp. Eye Res. 7, 497-510. &terlin, S. E. and Jacobson, B. (1968b). The synthesis of hyaluronic acid in vitreous. II. The presence of soluble transferase and nucleotide sugar in the acellular vitreous gel. Exp. Eye Res. 7, 511-23. Pape. L. G. and Balazs, E. A. (1980). The use of sodium hyaluronate (Healon) in human anterior segment surgery. Ophthalmology 87, 6994’05. Podos, S. M., Becker, B. and Kass, M. A. (1973). Prostaglandin synthesis, inhibition, and intraocular pressure. Invest. Ophthalmol. 12, 426-33. Sears, M. L. (1960). Miosis and intraocular pressure changes during manometry. Arch. Ophthalmol. 63, 707-14. Widder, W. (1962). Tierversuche tiber die Verweildauer verschiedener Glaskorperimplantate. Albrecht von. Craefes Arch. Klin. Exp. Ophthalmol. 164, 550-69. Wik, K. O., Andersson, T., Jacobsson, J. 6. and Granath, K. A. (1979). Physicochemical characterization of hyaluronate. In Wik, K. 0. Physicochemical Studies on Hyaluronate. Acta Universitatis Upsaliensis, Abstracts of Uppsala Dissertations from the Faculty of ivedieine, no. 334. Wolf, J. (1968). Inner surface of regions in the anterior chamber taking part in the regulation of the intraocular tension, including the demonstration of the covering viscous substance. Dot. Ophthalmol. 25, 113-49.
Turnover
of Hyaluronate Vitreous ULLA
* Departments
f University
Body
B. G. LAURENT*
in the Aqueous of the Rabbit AND
Humour
and
J. R. E. FRASER7
and Medical and Physiological Chemistry, of Uppsala, Uppsala, Sweden, and Department of Medicine, Royal Melbourne Hospital, Victoria 3050, Australia
of Ophthalmology
University of Melbourne
(Received 1 May 1982 and accepted 1 December
1982, New York)
Sodium hyaluronates with molecular weights of 18000, 500600 and 4 x 106, labelled with either 3H or i4C, were injected into the anterior chamber 01‘ into the centre of the vitreous body of rabbits and their rates of disappearance were followed. The out-flow from the anterior chamber in anaesthetized animals was virtually independent of the molecular weight indicating that the disappearance of the polysaccharide is controlled by bulk flow. The rate constant for the disappearance of sodium hyaluronate was 0,0094/min and with after treatment with indomethacin @OOSl/ min. These figures are in general agreement published flow-rates of aqueous humour in the rabbit. The disappearance from the vitreous body was strongly molecular weight dependent indicating a diffusion controlled transport. The rate constant for hyaluronate with mean molecular weight of 18000 was O.iCi/day and of 500006, O,024/day. The rate constant for endogenous hyaluronate was estimated to be about O.Ol/day. A calculation using these rate constants shows t,hat the turnover of sodium hyaluronate in the rabbit anterior chamber is about 3 pg per 24 hr while the turnover in the vitreous body is only 15% of that. This confirms an earlier conclusion (Laurent and Granath, 1983) that the preponderant part of the hyaluronate in aqueous humour is not a general degradation product from the vitreous body. Key words : hyaluronic acid ; turnover : aqueous humour ; vitreous body ; bulk flow ; diffusion.
1. Introduction Sodium hyaluronate is one of the major components of the vitreous body. Numerous reports have been published on its concentration, distribution in the tissue and its macromolecular parameters (for reviews see Balazs, 1965; Berman and Voaden, 1970). Relatively little is, however, known about its metabolism. It is believed that the polysaccharide is synthesized by the hyalocytes in the cortica,l layer of the vitreous -.. (Gsterlin and Jacobson, 1968a; Jacobson, 1978a, b) or extracellularly from intermediates produced by the cells (osterlin and Jacobson, 1968b; Jacobson, 197gb). When radioactive glucosamine is administered to owl monkeys, it becomes ineorporated into hyaluronate in the vitreous and remains there for at least 72 days without any significant turnover (osterlin, 1968). Another indication of a very slow turnover comes from the regeneration time of hyaluronate after removal of vitreous in the owl monkey. Sweeney and Balazs, (see Balazs, 1965) found that six months were required for the concentration to rise to that of the control eye. The concentration of hyaluronate in the aqueous humour is low in most species when compared to the concentration in the vitreous (Balazs, Laurent, Laurent, DeRoche and Bunney, 1959; Laurent, 1981). The owl is an exception; in this animal. Please Hospital,
address S-75185
0614~4835/83/040493
correspondence to Dr Uppsala, Sweden. + 11 $03.00/O
Ulla
Laurent,
Department
0 1983 Academic
of Ophthalmology,
Press Inc. (London)
University
Limited
494
U.B.G.LBUREKTAPL‘DJ.R.E.FRABER
hyaluronate is found covering the cornea1 endothelium (Barany, Berggren and Vrabec, 1957; Balazs et al., 1959). The origin of the hyaluronate in the aqueous humour is unknown but it was recently shown in rabbit and cattle that the polysaccharide has a high-molecular weight component, which is not present in the vitreous body. Therefore the hyaluronate in the aqueous humour does not seem to be a general degradation product from the vitreous (Laurent and Granath, 1983). The interest in turnover studies on hyaluronate in the eye has increased owing to the introduction of highly concentrated hyaluronate as an aid in ophthalmic surgery. Viscous solutions of the polysaccharide are used both as a replacement for vitreous tissue (Balazs et al., 1972) and in anterior segment surgery (Pape and Balazs, 1980). Widder (1962) injected hyaluronate (4 mg/ml; mol. wt 1.4-28 x 106) into rabbit vitreous and found that its half-life was approximately two weeks. Hultsch reported seemingly similar results in owl monkeys (Hultsch, 1979). Denlinger and Balazs give values of half-life for implanted hyaluronate in the vitreous of owl monkeys and rhesus monkeys of 30-40 days (Denlinger and Balazs, 1980) and 60-65 days (pers. comm.). However, when the anterior cortical gel in owl monkeys is damaged half-lives as short as 2-3 days are observed (For earlier references see Balazs and Hultsch, 1976; Hultsch, 1979; Balazs and Denlinger, 1982). According to Denlinger and Balazs (1980) the out-flow route of hyaluronate implanted in the vitreous body of owl monkeys is via the aqueous humour. The results of &terlin (1971, 1978) which showed a decreased concentration of hyaluronate in the vitreous of the aphakic eye are in agreement with the concept that there is a natural barrier against transport of hyaluronate from the vitreous to the aqueous humour. High-concentration (10 mg/ml), high-molecular weight hyaluronate injected into the anterior chamber of owl monkeys passes through the trabecular meshwork within 1-2 days (Schubert, Denlinger and Balazs cited by Balazs and Denlinger, 1982). The availability in our laboratory of [3H]- and [r4C]-labelled hyaluronate preparations of molecular weights varying between 18000 and several millions (Fraser, Laurent, Pertoft and Baxter, 1981), has enabled us to study the disappearance of trace amounts of hyaluronate from both vitreous body and anterior chamber in the rabbit. The results are reported in this communication. 2. Materials
and
Methods
Materials Sodium hyaluronate labelled either with 3H in the acetyl group of N-acetylglucosamine or with 14C in the sugar rings was produced by tissue culture of synovial cells as described by Fraser et al. (1981). The purified hyaluronate was dissolved in phosphate-buffered saline and maintained sterile (Fraser et al., 1981). The molecular weight distributions of the samples were determined by gel chromatography as described by Wik, Andersson, Jacobsson and Granath (1979) (see also Laurent and Granath, 1983). The molecular weight parameters and the specific radioactivities of the preparations are given in Table I. Radioactive hyaluronate samples are slowly degraded by radiolysis and therefore data on the total radioactivity in the solutions and the interval between the molecular weight determination and the animal experiments are included. The degree of radiolysis can be inferred ff_om sample 2 for which the molecular weight was determined at 4 months’ interval and M, dropped from 83 x lo5 to 47 x 105. For simp1icit.y each preparation has been assigned an approximate molecular weight. With few exceptions, the turnover studies were made with [aH]and [‘*Cl-labelled hyaluronate in the same experiments. The polysaccharides were mixed in such proportions that the solutions contained &20-times more 3H than r4C expressed in d/min. The injected volume (30 ~1) contained approximately @5 ,ug of sodium hyaluronate.
TURNOVER All weight
OF
experiments were carried out of these animals was 33 kg.
HYALURONATE on female
rabbits
IN (New
THE Zealand
495
EYE White).
The
average
Methods Determination of turnover in the anterior chamber. The rabbits were anaesthetized by intravenous injection of pentobarbital (initial dose 30 mg/kg body weight) and smaller doses were given to maintain anaesthesia. Tetracaine drops were used topically to supplement the anaesthesia. The animal was kept warm during the experiment with the aid of a heating pad. At. the end of the experiment, the animal was sacrificed by an overdose of pentobarbital. Two series of experiments were performed. In one series, the animals received no anti-inflammatory drugs. In the other series, they were pretreated with an intravenous dose of indomethacin (Sigma, St. Louis, MO, USA; 20 mg/kg body weight dissolved in 10 ml of 91 M-phosphate buffer pH 7.2 and 0.05 iw-NaCl). Indomethacin inhibits the synthesis of prostaglandins which are involved in the disruption of the blood-aqueous barrier (Beitch and Eakins, 1969; Podos, Becker and Kass, 1973). Fifteen minutes were allowedto pass after the injection before start of the experiment. The animals were laid on their sides and the experiments were performed on the exposed eyes. Needles 45 mm long with outer and inner diameters of 645 and 0.2 mm, respectively, and with divided inlets (Y-shaped) were used. The needles were fired through the periphery of the cornea into the anterior chamber using a needle gun (Sears, 1960). One inlet was joined by a plastic tubing to an Agla Precision Syringe by which 36 ,ul of the aqueous humour was withdrawn. The other inlet was joined to another Agla Syringe by which 30,~d of the hyaluronic acid solution was subsequently injected into the chamber. The tip of the needle was then fired through the contralateral side of the cornea so that it remained outside the anterior chamber during the experiment. This was preferred to withdrawing the needle from the eye which could cause leakage. Each experiment was terminated by withdrawing the aqueous humour through a 0.4 mm needle into a 1 ml syringe. The chamber was washed once with 0.2 ml of physiological saline and the washing was either analysed separately or combined with the main portion. It was planned to use both eyes at the same time. After injection in the right eye, the rabbit was turned so that the eye with inserted needle faced the table and another experiment was carried out on the left eye. When analysing the experiments, it became apparent that the right eye exhibited a faster (approximately 50 %) turnover rate presumably due to pressure exerted on the eye. The data on the right eyes have, therefore, been excluded from calculations of turnover rates. However, they could still be used to calculate the relative difference in disappearance rates of two hyaluronates injected at the same time. The radioactivity in the aqueous humour samples was determined in a Searle Nuclear Chicago Isocap/300 liquid scintillation counter. The samples were diluted to 1 ml with water and mixed with 10 ml of scintillant 299 (Packard-Becker B.V., Breda, The Netherlands). l4C was read with an efficiency of 64% (as determined by a commercial toluene standard) and < 0.1 y0 of the 3H counts appeared in the r4C channel. 3H was read with an efficiency of 40 0h (also determined with a commercial standard). From the counts in the aH channel the fraction of 14C counts appearing in this channel was subtracted. The fractional value was determined with a pure [%-hyaluronate sample. As contamination of the aqueous humour by blood serum could disturb the analysis, rabbit serum (which has a protein content one hundred times higher than aqueous humour) was added in 25-200 ~1 to the scintillation vials. The effect on the counts was a reduction of < 2 o/0 in the r4C channel and < 5 o/0 in the 3H channel. The results have been expressed in terms of the fraction of injected material remaining in the aqueous humour after a given time. The injected dose was determined by transferring 30 pl from the Agla Syringe directly into scintillation vials at the same time as the animal experiment was carried out and treating the samples the same way as the aqueous humour samples. The reproducibility of dispersing high-molecular weight hyaluronate (mol. wt 4 x 106) by the Agla Syringe was determined in a series of ten samples and was found to have a standard deviation of 5.4%. The same experiment with the low-molecular weight hyaluronate(mo1. wt 18000) gave a standard deviation of 3.4 y’, Determination of turnover in the vitreous body. The rabbits were anaesthetized as above. The eyes were proptosed in order to facilitate injection behind the equator of the eyeball. A needle
18000 500000*
3H w
3H 3H
weight
1 2
3 4
* The molecular
x x x x x
Indomethacin Indomethacin Indomethacin Indomethacin
18000 500000 4x106 All
Number of eyes
4 7 2 13
4 10 6 20
four
(lo’
x x x x x
TABLE
months
52 1.6 1.3 33 8.1
S.D.K
the anterior
S.D.,
0.97
0.4 6.1
0.05
0.03 0.08
0.08 0.05 0.05 0.04
(44-109) (63-83) (69-103) (6683)
114 (99-133)
111 (91-138) 118 (94-158)
62 71 83 74
--.-__-_data in Fig. 1 according to equation ln (C/C’“) = - K’ t + In n where a is the extrapolated value of C/C, from the experimental values.
0.92 0.98
o-87 0.95 0.79 0.87
a
2-4 months 3 -5 months il mouth 34 months 1 month
-_ __ C and ti is the
Interval between mol. wt determination and animal experiments
t: (min) (with 95 “/, confidence limits)
7.5 x 104 3.7 x 105
4x105 12 x 106
chamber
1.3 x 105 2.5 x 10’
Specific radioactivity WmWm
preparations
2x 106 2x 10’
0.3 0.6
1.1 0.6 0.6 @5
( lo3 x min-I)
fwm,
11
interval.
103 lo5 10” 105 105
Radioactivity of solution d/min/ml
of hyaluronate
63 5.9
11.1 97 8.4 9.4
x min-‘)
K
of hyaluronate
with
labelling
Number-average mol. wt
and radioactive
I
The rate constant (K) for the disappearance has been calculated from the experimental C, are the concentrations in the anterior chamber at time t and time zero, respectively. time required for the concentration to reach half its original value (half-life).
None None None None
Treatment of animal
twice
103 lo5 105 106 106
L9isappeurance
on this preparation
185 6.3 47 3.9 4.7
Weight average mol. wt
weight parameters
18000 500000 4x106 All
Hyaluronate preparation
was determined
40 x 106 40 x IO”
Designation
Label
Preparation IlO.
Molecular
TABLE
TURNOVER
OF
IN THE
HYALURONATE
EYE
497
with the same dimensions as described earlier but with a single inlet was fired into the middle of the vitreous body. The position of the needle could be followed visually through the transparent media. Thirty ,LL~of hyaluronate solution was injected with the aid of an Agla Syringe during a 3 min period and 2 min later, with released external pressure on the eye, the needle was withdrawn. The animal was allowed to regain consciousness and was returned to the animal quarters. The eyes were examined repeatedly by ophthalmoscopy. In one case the lens was damaged and the eye was excluded from the material. PITo bleedings in the vitreous or retina were noted. At predetermined times, the animals were sacrificed and the eyes enucleated and freed from surrounding tissues and blood. They were dried in an oven at 5660% and stored dried until analysis. The eyes were combusted in a Packard Model 306 Tri-Carb Sample Oxidizer in accordance with t,he instructions from the manufacturer. WO, and 3H,0 were collected separately and the counts recorded in a liquid scintillation spectrometer (Packard Tri-Carb 2425). The same amount of hyaluronate, which had been injected in the eyes, was also added to 500 mg of filter paper which was combusted and analysed as the samples. The reproducibility of pipetting high-molecular weight hyaluronate (mol. wt 4 x 106) on filter paper, and of combusting and analysing it was determined in a series of ten controls. The standard deviation was found to be 5 y0 compared with 3 y0 for low-molecular weight hyaluronat,e (mol. wt 18000). 3. Results Disappearance
of hyaluronate
from
the anterior
chamber
The experimental results are plotted in Fig. 1. The regression lines calculated from the plots in the figure are tabulated in Table II. The disappearance rates for the
a
b
0.1 ' 0
100
200
300
Min
FIG. I. The disappearance of hyaluronates from the anterior chamber of untreated (a) or indomethncintreated (b) rabbits. C/C, is the fraction of that originally injected remaining in the chamber. The following hyaiuronates were used : preparation 1, mol. wt 18000 (A) ; preparation 3, mol. wt 500000 (0); preparation 3, mol. wt 4 x lo6 ( x ); and preparation 4, mol. wt 4 x lo6 (m).
498
U. 3. G. LAURENT
AND
J. R. E. FRASER
hyaluronates of different molecular weights were similar although the rates for the high-molecular weight material tend to be somewhat lower. To further explore the dependence of the rate of disappearance on molecular weight we have plotted the ratio 3H/14C as a function of time in those experiments where r3H]- and [14C]-labelled hyaluronates were iiljected simultaneously. The data are shown in Fig. 2. In these plots, we have also included the series of experiments on the right eyes (see Methods) which were excluded from calculations of rate constants.
‘,O t-----s0
2.0
05--
2.0~-
b
0 l
0
0 .
c
A A A
A
o.sJ 0
100
200
300
FIG. 2. The variation in 3H/14C ratio with time when both [3H]- and [‘%I-labelled hyaluronates were injected simultaneously. The concentrations of 3H and 14C have been expressed as fractions of the injected material: (a) and (b), untreated; (c) indomethacin-treated animals. Mixture of preparations 1 (3H; mol. wt 18000) and 2 (W; mol. wt 500900) (0, 0, A, A). Mixture of preparations 2 (Y!; mol. wt 500000) and 4 (3H; mol. wt 4 x 106) (0,m). Open symbols, right eyes. Filled symbols, left eyes. The slope of the plot for the left eyes in (a) is 126 x 10-4. It is positive within 95 y0 confidence limits. The slopes of the plots in (b) and (c) do not significantly differ from zero.
It is apparent that when [‘*Cl-labelled hyaluronate with molecular weight of 500000 is injected together with [3HJ-labelIed hyaluronate of molecular weight 18000, the ratio (3H/14C) is essentially constant during the experiment indicating that the two fractions were removed at the same rate. When [3H]-labelled hyaluronate with molecular weight 4 x lo6 was injected with the [14C]-labelled material, the ratio increased with time in both series of experiments [Fig. 2(a)] indicating that the most high-molecular weight material was removed at a slower rate. The rates of disappearance in untreated and indomet,hacin-treated rabbits differed significantly (P < 0.01) (Fig. 1, Table II). The constant a tabulated in Table II represents the extrapolated value of C/C, at the beginning of the experiment. It is less than unity (0.7990.98) and is a measure of
TURNOVER
OF
HYALUROSATE
IN
the eEiciency by which the radioactive hyaluronate chamber by the experimental procedure. A recovery regarded as excellent in this type of experiment. Disappearance
of hyaluronate
from
the vitreous
THE
499
EYE
is removed from the anterior in the order of 90% must be
body
The removal of two different hyaluronates from the eyeball after injection into the vitreous body is described in Fig. 3. The parameters for the linear regressions are given in Table III. The half-life of the hyaluronate in the eye increases with increasing molecular weight, from 4 days for the sample with 18000 mol. wt to 30 days for the 500000 mol. wt sample. The constant a in Table III is 0.83 and 0.84 which indicates a lower recovery in the experiments on the vitreous body than those on the aqueous humour. This may be accounted for by a rapid initial loss of some radioactive hyaluronate through the channel in the gel formed by the injection needle.
0.01-j 0
10
FIG.~. The disappearance of hyaluronates body. C/C!, is the fraction of that originally were used: preparation 1, mol. wt 18000
Hyaluronate preparation
of hyaluronate Number of eyes
18000 500 000
9 9
For explanations
see Table
III
from the eyeball after injection K
(lo3 x day-‘) 159 23.5 II.
30
from the eyeball after injection into the centre of the vitreous injected remaining in the eyeball. The following hyaluronates (A); preparation 2, mol. wt 500000 (0). TABLE
Disappearance
20
Days
into the vitreous
S.D.K
(lo3 x day-‘)
a
S.D.,
12.5 91
0.83 0.84
0.12 0.10
body
ti (days) (with 95 y0 confidence limits) 44 (3.7-5.5) 295 (154-347)
500
U. B. G. LAURENT
Ah-D
J. R. E. FRASER
A hyaluronate of high-molecular weight but low specific radioactivity (preparation 3, Table I) was also used. There was a considerable spread of the experimental points for this sample owing to the low counts, and possibly experimental difficulties with the preparation, and no reliable K-value could be calculated but the disappearance rate was slow.
4. Discussion If a tracer is introduced into a compartment of constant volume and its disappearance from the compartment is proportional to its actual concentration then, the concentration, C: will decrease according to
C -=e co
-K.t ’
(1)
where C,, is the original concentration at time zero, t is the time and K is the rate constant. This equation can be applied to tracers introduced into the aqueous humour (see e.g. Bill a,nd Hellsing, 1965). If it is assumed that the disappearance of the tracer is governed by bulk flow then, 1 dv K=ppp (2) where V, is the volume of the compartment and dv/dt is the flow-rate. From Eqns. (1) and (2) it follows that the half-life (4, i.e. when the tracer has reached half its original concentration), is obtained by:
(3) The rate constant for the disappearance of hyaluronate from aqueous humour is given in Table II. The disappearance is approximately the same for materials of different molecular weights which indicates that it is governed mainly by bulk flow. The high-molecular weight hyaluronate disappears at a slightly slower rate (about 15 %) which may be due to a small sieving effect in the out-flow channels for particles of this size (Fig. 2). The volume of the anterior chamber in the rabbit is approximately 250 ,LL~and the flow-rate is of the order of 3 pl/min (Kinsey and Reddy, 1964). Using these figures the half-life of a compound removed by bulk flow calculated according to Eqn. (3) should be 60 min. This is in agreement with the value in Table II for untreated rabbits. However, it has been demonstrated (Becker, Krupin and Podos, 1970) that when barbiturate anaesthesia is used in rabbits, the flow-rate of aqueous humour decreases. Conversely the inflammatory response in the rabbit eye during the experiment would be expected to increase the flow-rate by leakage of blood plasma from the uvea and also the facility of out-flow (Podos et al., 1973). Evidence to support this may be adduced from the slower turnover of hyaluronate jn the indomethacin-treated animals. It is possible that the barbiturate effect and the inflammatory response counteract each other and that this is the reason for a turnover rate of hyaluronate very close to the known bulk flow rate of aqueous humour. We conclude that our results are in general agreement with the disappearance of hyaluronate by bulk flow, If the disappearance had been governed by diffusion, one should expect a preferential transport of the low-molecular weight hyaluronate and ifit had been receptor-mediated one could expect an increasing mass uptake per receptor with increasing molecular weight.
TURNOVER
OF
HYALURONATE
IN
THE
501
EYE
The disappearance of hyaluronate from the eye ball after injection into the vitreous body is strongly molecular weight dependent (Table III; Fig. 3). This is in agreement with a diffusion controlled transport in the vitreous body. Similar behaviour has also been demonstrated for a number of other compounds of lower molecular weight (see Maurice, 1980). Extrapolation of our data to a hyaluronate mol. wt of 2-3 x lo6 corresponding to hyaluronate in the rabbit vitreous (Laurent and Granath, 1983) (see Fig. 4) gives an approximate rate constant for the disappearance of O.Ol/day (4 - 70 days) in agreement with the very slow turnover noticed in owl monkey by osterlin
-1d
7 -to-*
g Y
10-8 110-3 106
108 106 107 Molecular weight FIG. 4. Procedure to obtain the rate constant, K, for the disappearance of endogenous hyaluronate from the vitreous body. When the diffusion coefficient, D, of hyaluronate (taken from Laurent, Ryan and Pietruszkiewicz, 1960) is plotted vs. mol. wt in a double logarithmic plot a straight line is obtained (a). When the rate constants (0) determined for hyaluronates with molecular weights of 18000 and 500000 are plotted similarly and a line drawn between them it becomes parallel to that of the diffusion coefficient indicating that K is proportional to D and thus diffusion controlled. If the line is extrapolated to the molecular weight of endogenous hyaluronate (2-3 x 10’; Laurent and Granath, 1983) a value of O.Ol/day is obtained. 105
(i968) and Balazs and coworkers (Balazs and Hultsch, 1976; Denlinger and Balazs, pers. comm.). It should be stressed that the tracer hyaluronate was deposited in the centre of the vitreous body and that the rate of disappearance therefore only represents the material in this part of the tissue. The results of asterlin (1968) would, however, indicate that the over-all turnover rate also is of the same magnitude. The rapid disappearance noted by Widder (1962) in rabbits and by Balazs and coworkers (Balazs and Hultsch 1976; Hultsch, 1979) in owl monkeys, when high concentrations of hyaluronate were introduced in the vitreous body, is presumably due to a disintegration of the normal vitreous structure and a break-down of the diffusion controlled transport (Balazs and Hultsch, 1976; Balazs and Denlinger, 1982). The transport parameters measured in these experiments can be used to calculate an approximate turnover per day in the two eye compartments. If we introduce mass Aow instead of volume flow, eqn. (2) can be written in the following form K,‘.d” 011
dm/dt
m,
dt
= K.m,,
(4)
502
U. B. G. LAUREST
AND
J. R. E. FRASER
where m, is the total mass of hyaluronate in the compartment and dm/dt the amount which is flowing through the system per time unit. The total amount of hyaluronate in the anterior chamber is equal to the volume (250 ~1) times the concentration (1.1 pg/ml; Laurent, 1981). Using the rate constants in Table II, one obtains a total turnover of the order of 3 ,ug/24 h in the anterior chamber. A similar calculation for hyaluronate in the vitreous body can be made assuming the volume of the rabbit vitreous is 15 ml (Kinsey and Reddy, 1964); the hyaluronate concentration is about 30 pg/ml (Laurent and Granath, 1983) and K is O*Ol/day. The calculated turnover is 0.45 ,ug/24 h and represents only 15 o/0 of that in the aqueous humour. The present results as well as the observation by Laurent and Granath (1983) that the aqueous humour hyaluronate has a higher molecular weight than that in the vitreous body indicate that the polysaccharide in the aqueous is not a general degradation product from the vitreous body. It could conceivably derive from the most anterior part of the vitreous. It is striking, however, that the owl has such a marked layer of hyaluronate on the endothelial surface of the cornea (Bar&y et al., 1957; Balazs et al., 1959) and the endothelial cells could be a source of the polysaccharide. The reports by Wolf (1968) who observed, by electron microscopy, the formation of a viscous material by the endothelial cells may be adduced in favour of this hypothesis. ACKNOWLEDGMENTS One of us (U. B. G. L) is grateful to Professors Ernst advice and to Professor Barany for placing equipment
Barany and Anders Bill for valuable at her disposal. Dr Kirsti Granath kindly performed the molecular weight analyses of the radioactive hyaluronates. The project has been supported by the Swedish Medical Research Council, The National Health and Medical Research Council of Australia and Carmen and Bertil Regner’s fund. REFERENCES
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