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Hyaluronate in aqueous humour
Exp. Eyes Res. (1981) 33, 147 155
Hyaluronate in Aqueous Humour ULLA B. G. LAURENT*
Department of Biochemistry, Monash University, Clayton, Victoria, 3168, Australia (Received 25 July 1980 and accepted 18 November 1980, New York) The sodium hyaluronate concentration of aqueous humour has been determined in 10-200~tl samples by a specific radioassay. The accuracy of the technique was studied on pooled bovine aqueous humour. Analyses of untreated samples, dialysed samples and samples digested with papain and then dialysed all gave the same results within the error of the method. The mean concentration determined in 22 assays was 4"4+0"2#g/ml while independent chemical analysis of the eetylpyridinium precipitable material gave a hexuronate content correspondingto 3"9#g/ml of sodium hyaluronate. Hyaluronidase treatment removed the material reacting in the assay. Addition of exogenous hyaluronate to the aqueous humour could be determined quantitatively. It was concluded that determinations performed on untreated aqueous humour gave valid values of the hyaluronate content in the fluid. The concentration of hyaluronate in bovine aqueous humour increased after death presumably due to a release of hyaluronate from neighbouring tissues. The concentration of hyalmonate in fresh aqueous humour from single eyes (in a few experiments pooled material) was studied in man, cattle, sheep, pig, rabbit, opossum, rat, chicken and mutton bird. Considerablespecies variation was found (between 0"2 and 4'0#g/ml). The level in man was found to be 1"1_+0-1/tg/ml. Analysis of material from bovine foetuses showed lower levels than in adult animals. Key words:hyaluronate ; radioassay; aqueous humour; determination ; human; animals.
1. I n t r o d u c t i o n The presence of h y a l u r o n a t e in aqueous h u m o u r and neighbouring tissues aroused early interest in the physiological role of h y a l u r o n a t e in aqueous flow (see e.g. Bdrdny, 1956; Berggren and Vrabee, 1957). This interest has been renewed recently with the i n n o v a t i o n of intraoeular injection of sodium h y a t u r o n a t e which is now applied in experimental and clinical ophthalmology. I t has been used for the replacement of vitreous h u m o u r (Balazs et al., 1972; P r u e t t , Sehepens and Swann, 1979; Denlinger and Balazs, 1980; Denlinger, E1-Mofty a n d Balazs, 1980) and it has also been deposited in the anterior chamber to protect the corneal endothelium during and after anterior segment surgery (Miller, O'Connor a n d Williams, 1977; Miller and Stegmann, 1980; Graue, Polaek and Balazs, 1980). This development has created a need for a technique which can be used to analyse the h y a l u r o n a t e concentration in the aqueous h u m o u r from single eyes. We have recently described a specific radioassay for h y a l u r o n a t e in n a n o g r a m quantities (Tengblad, !980; L a u r e n t and Tengblad, 1980) a n d the method has now been modified for work on aqueous humour. The occurrence of h y a l u r o n a t e in aqueous h u m o u r was first reported by Meyer and Palmer (1936). A n a t t e m p t to measure its concentration in single eyes (Meyer, S m y t h and Gallardo, 1938) was based on hexosamine d e t e r m i n a t i o n s b u t it is now k n o w n t h a t the hexosamine : h e x u r o n a t e ratio in dialysed aqueous h u m o u r exceeds 1 : 1, the expected ratio for h y a l u r o n a t e (Balazs, L a u r e n t , Laurent, De l~oehe and B u n n e y , 1959b). The h e x u r o n a t e c o n t e n t of pooled aqueous h u m o u r samples from different species was determined by the earbazole method and the results indicate t h a t the h y a l u r o n a t e c o n t e n t varies considerably (Balazs et al., 1959b). Hexuronie acid is, * Present address: Department of Ophthalmology, University Hospital, S-750 14 Uppsala, Sweden. 0014-4835/81/080147+09 801.00/0
9 1981 Academic Press Inc. (London) Limited 147
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however, a c o n s t i t u e n t of o t h e r p o l y s a c c h a r i d e s a n d t h e colorimetric t e c h n i q u e which was used could be affected b y nonspecific colour f o r m a t i o n . F u r t h e r m o r e , t h e a m o u n t of h e x u r o n a t e in t h e a q u e o u s h u m o u r from single eyes is close to t h e lower l i m i t of d e t e c t a b i l i t y . A specific isotope d i l u t i o n m e t h o d in t h e m i c r o g r a m range was d e v e l o p e d b y L a u r e n t , B~rs Carlsson a n d T i d a r e (1969) a n d this was used for t h e analysis of aqueous h u m o u r from calf, cow a n d m o n k e y . The m e t h o d is, however, n o t a p p l i c a b l e for r o u t i n e work. T h e r a d i o a s s a y d e s c r i b e d in this p a p e r has been t e s t e d for a c c u r a c y a n d r e l i a b i l i t y on pooled b o v i n e aqueous h u m o u r . I t can be used for analysis of 10-200/~1 s a m p l e s which enables a s s a y of s a m p l e s from single eyes. The t e c h n i q u e has been used to d e t e r m i n e t h e s o d i u m h y a l u r o n a t e c o n t e n t in t h e aqueous h u m o u r of different species a n d age groups to c o m p l e m e n t earlier w o r k on t h e c o m p a r a t i v e b i o c h e m i s t r y a n d d e v e l o p m e n t of t h e v i t r e o u s b o d y (Balazs et al., 1959b ; Balazs, L a u r e n t a n d L a u r e n t , 1959a).
2. M a t e r i a l s a n d M e t h o d s
Aqueous humour Human fluid was aspirated into a syringe from the anterior chamber before the eyes were opened for cataract surgery. Aqueous humour from single cattle eyes in situ was collected at a local abattoir immediately after the animals were killed. The fluid was withdrawn in two portions with a syringe through a tangential puncture in the cornea ; the first was 0"5 ml and the second was all the remaining liquid which could be aspirated. Fluid was collected similarly from eyes which had been stored at 4~ for varying time periods. Pooled aqueous humour was collected from a large number of bovine eyes approximately 2 hr after death. Calf foetuses were collected 5-10 min after death; all liquid was removed from the eyes in situ. The age of the foetuses was determined by the parameters given by Winters, Green and Comstock (1942). Pig aqueous humour was collected at t h e abattoir from eyes in situ approximately I hr after death, while sheep eyes were brought back to the laboratory and processed within 1-2 hr. Material from rabbit, opossum (Trichosurus vulpecula), rat, domestic fowl and mutton bird (Pujfinus tenuirostris) was usually obtained at the time of deathl All samples were weighed and kept frozen until used.
Na-Hyaluronate assay The method described previously for the analysis of sodium hyaluronate in tissue samples (Laurent and Tengblad, 1980) was used. For the assay proteins with affinity for hyaluronate were prepared from bovine nasal cartilage and labelled with 125I. The proteins were equilibrated between hyaluronate in free solution and an agarose gel substituted with hyMuronate. The radioactivity bound to the gel was a function of the amount of free hyMuronate in the system. Tissues and body fluids were pretreated with papain digestion and dialysis before the analysis in order to remove interfering substances and raise the salt concentration to 1"5 M-NaC1, 0'025 M-phosphate buffer, pH 7 which was used in the incubation mixture. In the analysis of aqueous humour an important modification was introduced as the samples in most cases were not pretreated. The sample volume was between 10#1 and 200#1 which corresponds to 1-15 % of the incubation volume in the assay. The omission of the dialysis step introduced small variations in ionic strength in the incubations. If not otherwise stated, all analyses are means of triplicate determinations.
Pre-treatment of aqueous humour In control experiments aqueous humour was dialysed against 1"5 M-NaC10-025 M-phosphate buffer pH 7-0. This was also done after papain digestion as described previously (Laurent and Tengblad, 1980). Digestion with hyaluronidase from Streptomyces hyalurolyticus (Seikagaku Fine Biochemicals, Tokyo, J a p a n ; 2000 T R U / m g ) was performed on aqueous humour
HYALURONATE IN AQUEOUS HUMOUR
149
dialysed against 0-05 M-sodium acetate pH 5-0. Two ml of fluid was incubated with 2 TRU of enzyme for 16hr at 37~ heated to 100~ for 15 min and dialysed against I'5M-NaC1 0"025 M-phosphate buffer pH 7"0.
Determination of hyaluronidase activity Viscosity measurements were performed at 25~ in an Ostwald viscometer with an outflow time for water of 29 sec. Sodium Hyaluronate (Healon | AB Pharmacia, Uppsala, Sweden) was dissolved in a concentration of 0-25 mg/ml in 0"145 M-NaC1, 2"5 raM-phosphate buffer, pH 7"2 ; in 1'5 M-NaC1, 0"025 M-phosphate buffer pH 7"0; or in the assay medium including protease inhibitors and l m g / m l of bovine serum albumin (Laurent and Tengblad, 1980). Ovine testicular hyaluronidase (Type III, 500 NF/mg, Sigma, St Louis, Missouri, U.S.A.) was added in a concentration of 2/~g/ml and the change in viscosity was followed as a function of time.
Chemical analysis Pooled bovine aqueous humour (54 ml) was dialysed against distilled water, lyophilized and then dissolved in 2ml of 0"lM-phosphate buffer pH 5'7 containing 5mM-EDTA and 5 mM-cysteine hydrochloride. Crude papain (BDH Chemicals, Poole, England) was activated in the same buffer by heating to 60 ~ for 30 rain and 6 mg in 0"2 ml added to the sample. The solution was incubated at 60~ for 18hr. The digest was dialysed against water, and sodium chloride was added to a concentration of 0"03 M. A small precipitate was removed by centrifugation and 100/A containing 1 mg of cctylpyridinium chloride (Scott, 1960) added to the supernatant. The precipitate was allowed to develop at room temperature over night and collected by centrifugation. It was dissolved in 100#l of 0"5 M-NaC1 and re-precipitated by 400#1 of 95 % ethanol (Fraction I). The supernatant from the ethanol precipitation was also saved (Fraction II). The supernatant from the precipitation with cetylpyridinium chloride was mixed with four volumes of 95 ~o ethanol and the precipitate was allowed to form over night at 4~ It was recovered by centrifugation (Fraction III). Hexuronic acid was determined in Fractions, I, II and I I I by the Dische carbazole method modified by Bitter and Muir (1962) using glucuronolactone as standard. Fractions I and II gave the expected colour development for hexuronic acid while Fraction I I I showed a high non-specific absorption. A spectrum was read in the latter case and the background absorbancy at 535 nm (about 60%) estimated from the shape of the curve. The remaining absorbancy at this wavelength could be due to hexuronic acid. Na-hyaluronate was calculated as 2"28 x the glucuronolactone content.
3. R e s u l t s a n d D i s c u s s i o n
The methods The radioassay described recently by L a u r e n t and Tengblad (1980) was used with the modification t h a t the samples were not pretreated to remove interfering factors such as enzymes. The assay medium does contain protease inhibitors but another possible source of error in the radioassay could arise from hyaluronidase activity. However, it is known t h a t most hyaluronidases have low p H optima (Meyer, 1971), testicular hyaluronidase being the only mammalian enzyme with a n y activity at neutral pH. Lysosomal hyaluronidase detected in rabbit iris has no activity above p H 5 (Hayasaka and Sears, 1978). An experiment was performed in which testicular hyaluronidase activity was measured in physiological salt solution; in I'5M-NaC1 buffered at p H 7 with phosphate; or in the standard assay medium, which also contains 1"5 M-NaC1. The specific viscosity of hyaluronate dropped to 50 % in 1.5 rain in the physiological solution. A similar effect was obtained after 160 rain in 1'5 ~-NaC1 and after 400 min in the assay medium. The assay medium has thus a strong inhibitory activity on testicular hyaluronidase, mainly due to the high ionic strength. I t is, therefore, unlikely t h a t hyaluronidase activity will interfere with the assay. As
150
U.B.G. L A U R E N T
mentioned above, small variations in the ionic strength of the assay incubations occurred due to the omission of the dialysis step. As shown by Tengblad (1980) the protein binding to h y a l u r o n a t e is not sensitive to moderate variations in ionic strength and the d a t a presented below confirm t h a t this cannot he a source of error. Experiments with pooled bovine aqueous humour Pooled aqueous h u m o u r was collected and analysed to test the accuracy and reproducibility of the method. P a r t of the material was concentrated and analysed for sodium h y a l u r o n a t e b y cetylpyridinium chloride precipitation and hexuronic acid analysis. The remaining material was analysed to determine (a) the reproducibility within a single assay; (b) the reproducibility between several assays; (e) the importance of sample volume (d) the need for proteolytic digestion and dialysis of the samples ; (e) the hyaluronidase sensitivity of the material reacting in the assay, and (f) the yield in the assay of exogenously added hyaluronate. A direct analysis of hexuronic acid by the carbazole m e t h o d on aqueous h u m o u r dialysed and concentrated 27 x was difficult to perform due to a high b a c k g r o u n d colour. After subtraction of the b a c k g r o u n d the sodium hyaluronate content was roughly estimated to be in the order of 4 # g / m l in the original aqueous humour. The concentrated material was, therefore, digested with papain and precipitated with cetylpyridinium chloride. The precipitable material (Fraction I) a m o u n t e d to 3 . 6 8 # g / m l original fluid. To this should be added a small a m o u n t lost in the final ethanol precipitation step (Fraction II) corresponding to 0' 18 # g / m l . The analysis thus gave a value of 3"9#g sodium h y a l u r o n a t e / m l aqueous humour. However, when the s u p e r n a t a n t fraction which should have contained no h y a l u r o n a t e (Fraction I I I ) was analysed by the carbazole method, there was a colour development which could p a r t l y TABLE I
Analysis of the sodium hyaluronate content in pooled bovine aqueous humour
Material analysed Cetylpyridinium precipitable hexuronic acid-containing polysaecharide (hexuronic acid analysis) (a) 50/~1samples of untreated aqueous humour analysed 13 times in one radioassay (b) 50#1 samples of untreated aqueous humour analysed in 22 independent radioassays (c) 10, 20, 30, 40, 50, 60, 70, 100 and 200#1 samples of untreated aqeuous humour analysed in one radioassay (d) Aqueous humour digested with papain and dialysed against 1'5 ~-NaC1, 0'25 M-phosphate buffer pH 7 Aqueous humour dialysed against 1"5.~-NaC1, 0-025 M-phosphate buffer pH7 (e) Aqueous humour digested with hyaluronidase from Streptomyces hyalurolyticus (f) Untreated aqueous humour with addition of 2'5 #g Na-hyaluronate 5#g Na-hyaluronatc 10#g Na-hyaluronate
Concentration of iXahyaluronate ~g/ml)* Mean _+~ 3'9 (n = 1) 4"5+0"2 (n = 13) 4"4+0'2 (n = 22) 4"5___0'1 (n = 9) 4'7_+0-4 (n = 2) 4"4_+0"1 (n = 2) < 0.03 (n = 1) 7.1 (n = 1) 9'9 (n = 1) 14.8 (n = 1)
* As described in the Methods section every single analytical value is the mean of triplicate or in a few cases duplicate determinations, n = number of triplicate or duplicate determinations.
HYALURONATE IN AQUEOUS HUMOUR
151
have been due to hexuronic acid. This colour development corresponded to that of 2/~g/ml of hyaluronate in the original fluid. The nature of the material is not known as the amount was too small to allow further identification. The analytical results from the hexuronic acid analysis are compared with the results obtained with the radioassay in Table I. The radioassay gave a mean value of 4"5 #g/ml whether the assay was run on a large number of samples of equal volume at the same time or when identical samples were determined in a large number of independent assays. The agreement between the chemical analysis (assuming that all cetylpyridinium precipitable hexuronic acid material is hyaluronate) and the radioassay is acceptable in view of the inherent errors especially in the chemical analysis. When samples of pooled aqueous humour with different volumes between 10 and 200/~1 were analysed, similar values were obtained within the expected error limits and there was no apparent variation with sample volume. This confirms the assumption that the small variations in ionic strength introduced by varying the sample volume does not influence the assay. The aqueous humour was also dialysed extensively against I'5M-NaC1 0"025Mphosphate buffer pH 7'0 directly or after papain digestion. The amount of hyaluronate remaining in the sample was not significantly different from that obtained by direct analysis (Table I) ruling out the possibility that proteins interferring with the assay were present in the aqueous humour or that the hyaluronate was of such low molecular weight that it was readily dialysable. Further confirmation that it is hyaluronate which was being assayed was obtained by digestion with hyaluronidase from Streptomyces hyalurolyticus which removes the reacting material quantitatively. This enzyme is specific for hyaluronate (Ohya and Kaneko, 1970). Addition of exogenous sodium hyaluronate to the aqueous humour could also be assayed quantitatively (Table I).
Hyaluronate content of aqueous humour collected post mortem I t was reported (BMazs, 1965) that the hyaluronate content of the aqueous humour increases post mortem. Experiments were, therefore, carried out using bovine eyes which had been stored for different time periods after death of the animal. The aqueous fluid was withdrawn from each eye in two portions; first approximately 0"5 ml and then all the remaining fluid that could be aspirated. The latter portion presumably contained more of the fluid from less accessible compartments, such as the posterior TABL~II
Analysis of the sodium hyaluronate content of aqueous humour from single bovine eyes stored for different time periods after death at 4~
Time a f t e r death Immediately 4hr 12hr 24hr
Volume of aqueous Numberof humour(ml) eyes Mean-+o'Tx 18 20 11 11
1"50_+0'08(n = 12)* 1"51_+0"04 1'30_+0"04 1.21_+0"07
Concentration of Na-hyaluronate ~ag/ml) Mean-+~ru In the first 0"5ml In the remaining portion fluid 4.0_+0-2 5'9_+0-3 12'6_+1"3 11"2___0-6
4.0_+0"2 7.9_+0'5 14-1_+1"6 19.8-+4.4
* Because of difficultes in removingthe fluid quantitatively in the abattoir six eyeswere not included in the volume determination.
U. B. G. L A U P ~ E N T
152
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HYALURONATE IN AQUEOUS HUMOUR
153
chamber. The total volume aspirated from each eye was recorded and the hyaluronate content determined in the two portions. The results are displayed in Table H. Aqueous humour collected immediately after death contained 4 # g / m l and there was no difference between the first and second portions withdrawn. With increasing time of storage there was a marked increase in aqueous hyaluronate concentration and the largest effect was seen in the last portion aspirated. Hyaluronate was apparently released from neighhouring structures, e.g. the vitreous body, into the aqueous fluid. The volume of the aqueous humour decreases post mortem, which apparently reflects the swelling of neighbouring tissues such as the cornea and lens. The volume changes are, however, too small to explain the changes in hyaluronate concentration (see Table II). In view of the results from studies on post mortem eyes it is necessary to sample the aqueous humour from living animals or immediately after death if the normal hyaluronate level should be determined.
Aqueous humour from different species The analytical data on aqueous humour from different species are shown in Table I I I . I t has been possible in most species to analyse samples of aqueous humour from single eyes hut in two cases (rat, mutton bird), it was necessary to pool material from several animals. Whenever possible the analyses were made in triplicate on the individual eyes and the mean values were used (cattle, sheep, pig, rabbit). In other species the material from each eye was often sufficient only for a single analysis (man, opossum). Large species differences were noted varying from 0"2 # g / m l in the rat and opossum up to 4 # g / m l in cattle. Only a few values are available from the literature for comparison. Balazs et al. (1959b) report the hyaluronate level of cattle aqueous humour from uronic acid analysis to be 2"3 # g / m l and Balazs and Falbe-Hansen (1965) found a concentration of 4'7/~g/ml. Laurent et al. (1969) using a specific isotope dilution technique found 4'2 and 7"1/~g/ml in calf and cow samples, respectively. These values are compatible with our results in view of the errors in the techniques and the post mortem changes discussed above. Hexuronate estimates on aqueous humours from sheep, rabbit and fowl (Balazs et al., 1959b) corresponded to appreciably higher values of hyaluronate than those reported here but these earlier analyses were uncertain due to the limits of the carbazole technique. The species variation presumably mirrors the hyaluronate release from neighbouring tissues, e.g. the vitreous body. The concentration of hyaluronate in the vitreous body, known from previous studies or estimates by the present radioassay (Table I I I ) seems to correlate to the content in the aqueous humour. Of special interest are the low levels found in the rat and the opossum. These animals are known to have very large lenses allowing only a small vitreous cavity (see Duke-Elder, 1958).
The hyaluronate content of aqueous humour in foetal development Cattle foetuses at different times of gestation were collected and aqueous humour could be analysed over the period 5-9 months. The results are shown in Table IV. The hyaluronate levels are lower than in the adult animals. A lower level of hyaluronate was also found in the vitreous body during foetal life compared with adult levels (Balazs et al., 1959a). Furthermore, the concentrations of hyaluronate both in the aqueous humour and the vitreous body are constant over the time period studied. 6
E E R 33
154
U.B.G. L A U R E N T TABLE IV
Sodium hyaluronale content in aqueous humour of bovine foetuses Age of Aspirated volume from foetus Number of single eyes (ml) (months) eyes Range 5 6 7 8 9
7 13 4 4 8
Na-hyaluronate concentration (#g/ml) Mean • ~7 Range*
0"06-0"13 0"13~)'30 0-21~)'53 0'26~'51 0"184)'71
0"9 + 0"2 0-8 • 0"1 1-2• 1"4• 0"4 0"9•
0-4/0-6-1"0/1'4 0'5/0'6-0"8/1"6 0'7/0-8-1-4/2-1 0'7/1'3-1"6/1"9 0'6/0"8-0'9/1"2
* The concentrations in the two eyes from the same animal are given as x/y. ACKNOWLEDGMENTS The author wishes to thank Professors D. A. Lowther and B. N. Preston and their co-workers for help and facilities during her stay at Monash University. Human material was kindly supplied by Professor Gerard Crock, Melbourne and by Professor Lennart Berggren and co-workers, Uppsala; and material from opossum and mutton bird by Drs Magda Weiss and John Baldwin, Monash University. Miss Agneta Laurent gave skilful technical assistance. The project was supported by grants from the Swedish Medical l~esearch Council and AB Pharmacia. REFERENCES Balazs, E. A. (1965). Amino sugar-containing macromolecules in the tissues of the eye and the ear. In The Amino Sugars. Vol. IIA. (Eds Balazs, E. A. and Jeanloz, R. W.). Pp. 401-60. Academic Press, New York and London. Balazs, E. A. and Falbe-Hansen, J. (1965). Unpublished data. Cited in The Amino Sugars. Vol. IIA. (Eds Balazs, E. A. and Jeanloz, R. W.). P. 450. Academic Press, New York and London. Balazs, E . A . , Freeman, M.I., K15ti, R., Meyer-Schwickerath, G., Regnault, F. and Sweeney, D . B . (1972). Hyaluronic acid and replacement of vitreous and aqueous humour. In Modern Problems in Ophthalmology. Vol. 10. (Ed. Streiff, E. B.). Pp. 3-21. S. Karger, Basel. Balazs, E. A., Laurent, T. C. and Laurent, U. B. G. (1959a). Studies on the structure of the vitreous body. VI. Biochemical changes during development. J. Biol. Chem. 234,422-30. Balazs, E. A., Laurent, T. C., Laurent, U. B. G., DeRoche, M. H. and Bunney, D. M. (1959b). Studies on the structure of the vitreous body. VIII. Comparative biochemistry. Arch. Biochem. Biophys. 81, 464-79. Bs E. H. (1956). The action of different kinds of hyaluronidase on the resistance of flow through the angle of the anterior chamber. Acta Ophthalmol. 34, 397-403. Berggren, L. and Vrabec, F. (1957). Demonstration of a coating substance in the trabecular meshwork of the eye : and its decrease after perfusion experiments with different kinds of hyaluronidase. Am. J. Ophthalmol. 44, 200-8. Bitter, T. and Muir, H. M. (1962). A modified uronic acid carbazole reaction. Anal. Bioehem. 4, 330-4. Denlinger, J. L. and Balazs, E. A. (1980). Replacement of the liquid vitreous 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 vitreous with sodium hyaluronate in monkeys. II. Long term evaluation. Exp. Eye Res. 31, 101-17. Duke-Elder, S. (1958). The eye in evolution. In System of Ophthalmology, Vol. I. P. 605. Henry Kimpton, London.
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Graue, E. L., Polack, F. M. and Balazs, E. A. (1980). The protective effect of Na-hyaluronate to corneal endothelium. Exp. Eye Res, 31, t t9-27. Hayasaka, S. and Sears, L. (1978). The presence oflysosomal hyaluronidase in the rabbit iris. Invest. Ophthalmol. 17, 639-44. Laurent, T. C., Bs E., Carlsson, B. and Tidare, E. (1969). Determination of hyaluronic acid in the microgram range. Anal. Biochem. 31~ 133--45. Laurent, U. B. G. and Tengblad, A. (1980). Determination of hyaluronate in biological samples by a specific radioassay technique. Anal. Biochem. 108, 386-94. Meyer, K. (1971). Hyaluronidases. In The Enzymes, Vol. V 3rd edn. (ed. Boyer, P. D.). Pp. 307-20. Academic Press, New York. Meyer, K. and Palmer, J. W. (1936). On the nature of the ocular fluids. Am. J. Ophthalmol. 19, 859-65. Meyer, K., Smyth, E. M. and Gallardo, E. (1938). On the nature of the ocular fluids. II. The hexosamine content. Am. J. Ophthalmol. 21, 1083-90. Miller, D., O'Connor, P. and Williams, J. (1977). Use of Na-hyaluronate during intraocular lens implantation in rabbits. Ophthalmic Surg. 8, 58-61. Miller, D. and Stegmann R. (1980). Use of Na-hyaluronate in auto-corneal transplantation in rabbits. Ophthalmic Surg. 11, 19-21. Ohya, T. and Kaneko, Y. (1970). Novel hyaluronidase from Streptomyces. Biochim. Biophys. Acta 198, 607-9. Pruett, R. C., Schepens, C. L. and Swarm, D. A. (1979). Hyaluronic acid vitreous substitute. Arch. Ophthalmol. 97, 2325-30. Scott, J. E. (1960). Aliphatic ammonium salts in the assay of acidic polysaccharides from tissues. Methods Biochem. Analysis 8, 145-97. Tengblad, A. (1980). Quantitative analysis of hyaluronate in nanogram amounts. Biochem. J. 185, 101-5.
Winters, L. M., Green, W. W. and Comstock, R. E. (1942). Prenatal development of the bovine. Minn. Tech. Bull. 151, 41.
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Hyaluronate in Aqueous Humour ULLA B. G. LAURENT*
Department of Biochemistry, Monash University, Clayton, Victoria, 3168, Australia (Received 25 July 1980 and accepted 18 November 1980, New York) The sodium hyaluronate concentration of aqueous humour has been determined in 10-200~tl samples by a specific radioassay. The accuracy of the technique was studied on pooled bovine aqueous humour. Analyses of untreated samples, dialysed samples and samples digested with papain and then dialysed all gave the same results within the error of the method. The mean concentration determined in 22 assays was 4"4+0"2#g/ml while independent chemical analysis of the eetylpyridinium precipitable material gave a hexuronate content correspondingto 3"9#g/ml of sodium hyaluronate. Hyaluronidase treatment removed the material reacting in the assay. Addition of exogenous hyaluronate to the aqueous humour could be determined quantitatively. It was concluded that determinations performed on untreated aqueous humour gave valid values of the hyaluronate content in the fluid. The concentration of hyaluronate in bovine aqueous humour increased after death presumably due to a release of hyaluronate from neighbouring tissues. The concentration of hyalmonate in fresh aqueous humour from single eyes (in a few experiments pooled material) was studied in man, cattle, sheep, pig, rabbit, opossum, rat, chicken and mutton bird. Considerablespecies variation was found (between 0"2 and 4'0#g/ml). The level in man was found to be 1"1_+0-1/tg/ml. Analysis of material from bovine foetuses showed lower levels than in adult animals. Key words:hyaluronate ; radioassay; aqueous humour; determination ; human; animals.
1. I n t r o d u c t i o n The presence of h y a l u r o n a t e in aqueous h u m o u r and neighbouring tissues aroused early interest in the physiological role of h y a l u r o n a t e in aqueous flow (see e.g. Bdrdny, 1956; Berggren and Vrabee, 1957). This interest has been renewed recently with the i n n o v a t i o n of intraoeular injection of sodium h y a t u r o n a t e which is now applied in experimental and clinical ophthalmology. I t has been used for the replacement of vitreous h u m o u r (Balazs et al., 1972; P r u e t t , Sehepens and Swann, 1979; Denlinger and Balazs, 1980; Denlinger, E1-Mofty a n d Balazs, 1980) and it has also been deposited in the anterior chamber to protect the corneal endothelium during and after anterior segment surgery (Miller, O'Connor a n d Williams, 1977; Miller and Stegmann, 1980; Graue, Polaek and Balazs, 1980). This development has created a need for a technique which can be used to analyse the h y a l u r o n a t e concentration in the aqueous h u m o u r from single eyes. We have recently described a specific radioassay for h y a l u r o n a t e in n a n o g r a m quantities (Tengblad, !980; L a u r e n t and Tengblad, 1980) a n d the method has now been modified for work on aqueous humour. The occurrence of h y a l u r o n a t e in aqueous h u m o u r was first reported by Meyer and Palmer (1936). A n a t t e m p t to measure its concentration in single eyes (Meyer, S m y t h and Gallardo, 1938) was based on hexosamine d e t e r m i n a t i o n s b u t it is now k n o w n t h a t the hexosamine : h e x u r o n a t e ratio in dialysed aqueous h u m o u r exceeds 1 : 1, the expected ratio for h y a l u r o n a t e (Balazs, L a u r e n t , Laurent, De l~oehe and B u n n e y , 1959b). The h e x u r o n a t e c o n t e n t of pooled aqueous h u m o u r samples from different species was determined by the earbazole method and the results indicate t h a t the h y a l u r o n a t e c o n t e n t varies considerably (Balazs et al., 1959b). Hexuronie acid is, * Present address: Department of Ophthalmology, University Hospital, S-750 14 Uppsala, Sweden. 0014-4835/81/080147+09 801.00/0
9 1981 Academic Press Inc. (London) Limited 147
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however, a c o n s t i t u e n t of o t h e r p o l y s a c c h a r i d e s a n d t h e colorimetric t e c h n i q u e which was used could be affected b y nonspecific colour f o r m a t i o n . F u r t h e r m o r e , t h e a m o u n t of h e x u r o n a t e in t h e a q u e o u s h u m o u r from single eyes is close to t h e lower l i m i t of d e t e c t a b i l i t y . A specific isotope d i l u t i o n m e t h o d in t h e m i c r o g r a m range was d e v e l o p e d b y L a u r e n t , B~rs Carlsson a n d T i d a r e (1969) a n d this was used for t h e analysis of aqueous h u m o u r from calf, cow a n d m o n k e y . The m e t h o d is, however, n o t a p p l i c a b l e for r o u t i n e work. T h e r a d i o a s s a y d e s c r i b e d in this p a p e r has been t e s t e d for a c c u r a c y a n d r e l i a b i l i t y on pooled b o v i n e aqueous h u m o u r . I t can be used for analysis of 10-200/~1 s a m p l e s which enables a s s a y of s a m p l e s from single eyes. The t e c h n i q u e has been used to d e t e r m i n e t h e s o d i u m h y a l u r o n a t e c o n t e n t in t h e aqueous h u m o u r of different species a n d age groups to c o m p l e m e n t earlier w o r k on t h e c o m p a r a t i v e b i o c h e m i s t r y a n d d e v e l o p m e n t of t h e v i t r e o u s b o d y (Balazs et al., 1959b ; Balazs, L a u r e n t a n d L a u r e n t , 1959a).
2. M a t e r i a l s a n d M e t h o d s
Aqueous humour Human fluid was aspirated into a syringe from the anterior chamber before the eyes were opened for cataract surgery. Aqueous humour from single cattle eyes in situ was collected at a local abattoir immediately after the animals were killed. The fluid was withdrawn in two portions with a syringe through a tangential puncture in the cornea ; the first was 0"5 ml and the second was all the remaining liquid which could be aspirated. Fluid was collected similarly from eyes which had been stored at 4~ for varying time periods. Pooled aqueous humour was collected from a large number of bovine eyes approximately 2 hr after death. Calf foetuses were collected 5-10 min after death; all liquid was removed from the eyes in situ. The age of the foetuses was determined by the parameters given by Winters, Green and Comstock (1942). Pig aqueous humour was collected at t h e abattoir from eyes in situ approximately I hr after death, while sheep eyes were brought back to the laboratory and processed within 1-2 hr. Material from rabbit, opossum (Trichosurus vulpecula), rat, domestic fowl and mutton bird (Pujfinus tenuirostris) was usually obtained at the time of deathl All samples were weighed and kept frozen until used.
Na-Hyaluronate assay The method described previously for the analysis of sodium hyaluronate in tissue samples (Laurent and Tengblad, 1980) was used. For the assay proteins with affinity for hyaluronate were prepared from bovine nasal cartilage and labelled with 125I. The proteins were equilibrated between hyaluronate in free solution and an agarose gel substituted with hyMuronate. The radioactivity bound to the gel was a function of the amount of free hyMuronate in the system. Tissues and body fluids were pretreated with papain digestion and dialysis before the analysis in order to remove interfering substances and raise the salt concentration to 1"5 M-NaC1, 0'025 M-phosphate buffer, pH 7 which was used in the incubation mixture. In the analysis of aqueous humour an important modification was introduced as the samples in most cases were not pretreated. The sample volume was between 10#1 and 200#1 which corresponds to 1-15 % of the incubation volume in the assay. The omission of the dialysis step introduced small variations in ionic strength in the incubations. If not otherwise stated, all analyses are means of triplicate determinations.
Pre-treatment of aqueous humour In control experiments aqueous humour was dialysed against 1"5 M-NaC10-025 M-phosphate buffer pH 7-0. This was also done after papain digestion as described previously (Laurent and Tengblad, 1980). Digestion with hyaluronidase from Streptomyces hyalurolyticus (Seikagaku Fine Biochemicals, Tokyo, J a p a n ; 2000 T R U / m g ) was performed on aqueous humour
HYALURONATE IN AQUEOUS HUMOUR
149
dialysed against 0-05 M-sodium acetate pH 5-0. Two ml of fluid was incubated with 2 TRU of enzyme for 16hr at 37~ heated to 100~ for 15 min and dialysed against I'5M-NaC1 0"025 M-phosphate buffer pH 7"0.
Determination of hyaluronidase activity Viscosity measurements were performed at 25~ in an Ostwald viscometer with an outflow time for water of 29 sec. Sodium Hyaluronate (Healon | AB Pharmacia, Uppsala, Sweden) was dissolved in a concentration of 0-25 mg/ml in 0"145 M-NaC1, 2"5 raM-phosphate buffer, pH 7"2 ; in 1'5 M-NaC1, 0"025 M-phosphate buffer pH 7"0; or in the assay medium including protease inhibitors and l m g / m l of bovine serum albumin (Laurent and Tengblad, 1980). Ovine testicular hyaluronidase (Type III, 500 NF/mg, Sigma, St Louis, Missouri, U.S.A.) was added in a concentration of 2/~g/ml and the change in viscosity was followed as a function of time.
Chemical analysis Pooled bovine aqueous humour (54 ml) was dialysed against distilled water, lyophilized and then dissolved in 2ml of 0"lM-phosphate buffer pH 5'7 containing 5mM-EDTA and 5 mM-cysteine hydrochloride. Crude papain (BDH Chemicals, Poole, England) was activated in the same buffer by heating to 60 ~ for 30 rain and 6 mg in 0"2 ml added to the sample. The solution was incubated at 60~ for 18hr. The digest was dialysed against water, and sodium chloride was added to a concentration of 0"03 M. A small precipitate was removed by centrifugation and 100/A containing 1 mg of cctylpyridinium chloride (Scott, 1960) added to the supernatant. The precipitate was allowed to develop at room temperature over night and collected by centrifugation. It was dissolved in 100#l of 0"5 M-NaC1 and re-precipitated by 400#1 of 95 % ethanol (Fraction I). The supernatant from the ethanol precipitation was also saved (Fraction II). The supernatant from the precipitation with cetylpyridinium chloride was mixed with four volumes of 95 ~o ethanol and the precipitate was allowed to form over night at 4~ It was recovered by centrifugation (Fraction III). Hexuronic acid was determined in Fractions, I, II and I I I by the Dische carbazole method modified by Bitter and Muir (1962) using glucuronolactone as standard. Fractions I and II gave the expected colour development for hexuronic acid while Fraction I I I showed a high non-specific absorption. A spectrum was read in the latter case and the background absorbancy at 535 nm (about 60%) estimated from the shape of the curve. The remaining absorbancy at this wavelength could be due to hexuronic acid. Na-hyaluronate was calculated as 2"28 x the glucuronolactone content.
3. R e s u l t s a n d D i s c u s s i o n
The methods The radioassay described recently by L a u r e n t and Tengblad (1980) was used with the modification t h a t the samples were not pretreated to remove interfering factors such as enzymes. The assay medium does contain protease inhibitors but another possible source of error in the radioassay could arise from hyaluronidase activity. However, it is known t h a t most hyaluronidases have low p H optima (Meyer, 1971), testicular hyaluronidase being the only mammalian enzyme with a n y activity at neutral pH. Lysosomal hyaluronidase detected in rabbit iris has no activity above p H 5 (Hayasaka and Sears, 1978). An experiment was performed in which testicular hyaluronidase activity was measured in physiological salt solution; in I'5M-NaC1 buffered at p H 7 with phosphate; or in the standard assay medium, which also contains 1"5 M-NaC1. The specific viscosity of hyaluronate dropped to 50 % in 1.5 rain in the physiological solution. A similar effect was obtained after 160 rain in 1'5 ~-NaC1 and after 400 min in the assay medium. The assay medium has thus a strong inhibitory activity on testicular hyaluronidase, mainly due to the high ionic strength. I t is, therefore, unlikely t h a t hyaluronidase activity will interfere with the assay. As
150
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mentioned above, small variations in the ionic strength of the assay incubations occurred due to the omission of the dialysis step. As shown by Tengblad (1980) the protein binding to h y a l u r o n a t e is not sensitive to moderate variations in ionic strength and the d a t a presented below confirm t h a t this cannot he a source of error. Experiments with pooled bovine aqueous humour Pooled aqueous h u m o u r was collected and analysed to test the accuracy and reproducibility of the method. P a r t of the material was concentrated and analysed for sodium h y a l u r o n a t e b y cetylpyridinium chloride precipitation and hexuronic acid analysis. The remaining material was analysed to determine (a) the reproducibility within a single assay; (b) the reproducibility between several assays; (e) the importance of sample volume (d) the need for proteolytic digestion and dialysis of the samples ; (e) the hyaluronidase sensitivity of the material reacting in the assay, and (f) the yield in the assay of exogenously added hyaluronate. A direct analysis of hexuronic acid by the carbazole m e t h o d on aqueous h u m o u r dialysed and concentrated 27 x was difficult to perform due to a high b a c k g r o u n d colour. After subtraction of the b a c k g r o u n d the sodium hyaluronate content was roughly estimated to be in the order of 4 # g / m l in the original aqueous humour. The concentrated material was, therefore, digested with papain and precipitated with cetylpyridinium chloride. The precipitable material (Fraction I) a m o u n t e d to 3 . 6 8 # g / m l original fluid. To this should be added a small a m o u n t lost in the final ethanol precipitation step (Fraction II) corresponding to 0' 18 # g / m l . The analysis thus gave a value of 3"9#g sodium h y a l u r o n a t e / m l aqueous humour. However, when the s u p e r n a t a n t fraction which should have contained no h y a l u r o n a t e (Fraction I I I ) was analysed by the carbazole method, there was a colour development which could p a r t l y TABLE I
Analysis of the sodium hyaluronate content in pooled bovine aqueous humour
Material analysed Cetylpyridinium precipitable hexuronic acid-containing polysaecharide (hexuronic acid analysis) (a) 50/~1samples of untreated aqueous humour analysed 13 times in one radioassay (b) 50#1 samples of untreated aqueous humour analysed in 22 independent radioassays (c) 10, 20, 30, 40, 50, 60, 70, 100 and 200#1 samples of untreated aqeuous humour analysed in one radioassay (d) Aqueous humour digested with papain and dialysed against 1'5 ~-NaC1, 0'25 M-phosphate buffer pH 7 Aqueous humour dialysed against 1"5.~-NaC1, 0-025 M-phosphate buffer pH7 (e) Aqueous humour digested with hyaluronidase from Streptomyces hyalurolyticus (f) Untreated aqueous humour with addition of 2'5 #g Na-hyaluronate 5#g Na-hyaluronatc 10#g Na-hyaluronate
Concentration of iXahyaluronate ~g/ml)* Mean _+~ 3'9 (n = 1) 4"5+0"2 (n = 13) 4"4+0'2 (n = 22) 4"5___0'1 (n = 9) 4'7_+0-4 (n = 2) 4"4_+0"1 (n = 2) < 0.03 (n = 1) 7.1 (n = 1) 9'9 (n = 1) 14.8 (n = 1)
* As described in the Methods section every single analytical value is the mean of triplicate or in a few cases duplicate determinations, n = number of triplicate or duplicate determinations.
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151
have been due to hexuronic acid. This colour development corresponded to that of 2/~g/ml of hyaluronate in the original fluid. The nature of the material is not known as the amount was too small to allow further identification. The analytical results from the hexuronic acid analysis are compared with the results obtained with the radioassay in Table I. The radioassay gave a mean value of 4"5 #g/ml whether the assay was run on a large number of samples of equal volume at the same time or when identical samples were determined in a large number of independent assays. The agreement between the chemical analysis (assuming that all cetylpyridinium precipitable hexuronic acid material is hyaluronate) and the radioassay is acceptable in view of the inherent errors especially in the chemical analysis. When samples of pooled aqueous humour with different volumes between 10 and 200/~1 were analysed, similar values were obtained within the expected error limits and there was no apparent variation with sample volume. This confirms the assumption that the small variations in ionic strength introduced by varying the sample volume does not influence the assay. The aqueous humour was also dialysed extensively against I'5M-NaC1 0"025Mphosphate buffer pH 7'0 directly or after papain digestion. The amount of hyaluronate remaining in the sample was not significantly different from that obtained by direct analysis (Table I) ruling out the possibility that proteins interferring with the assay were present in the aqueous humour or that the hyaluronate was of such low molecular weight that it was readily dialysable. Further confirmation that it is hyaluronate which was being assayed was obtained by digestion with hyaluronidase from Streptomyces hyalurolyticus which removes the reacting material quantitatively. This enzyme is specific for hyaluronate (Ohya and Kaneko, 1970). Addition of exogenous sodium hyaluronate to the aqueous humour could also be assayed quantitatively (Table I).
Hyaluronate content of aqueous humour collected post mortem I t was reported (BMazs, 1965) that the hyaluronate content of the aqueous humour increases post mortem. Experiments were, therefore, carried out using bovine eyes which had been stored for different time periods after death of the animal. The aqueous fluid was withdrawn from each eye in two portions; first approximately 0"5 ml and then all the remaining fluid that could be aspirated. The latter portion presumably contained more of the fluid from less accessible compartments, such as the posterior TABL~II
Analysis of the sodium hyaluronate content of aqueous humour from single bovine eyes stored for different time periods after death at 4~
Time a f t e r death Immediately 4hr 12hr 24hr
Volume of aqueous Numberof humour(ml) eyes Mean-+o'Tx 18 20 11 11
1"50_+0'08(n = 12)* 1"51_+0"04 1'30_+0"04 1.21_+0"07
Concentration of Na-hyaluronate ~ag/ml) Mean-+~ru In the first 0"5ml In the remaining portion fluid 4.0_+0-2 5'9_+0-3 12'6_+1"3 11"2___0-6
4.0_+0"2 7.9_+0'5 14-1_+1"6 19.8-+4.4
* Because of difficultes in removingthe fluid quantitatively in the abattoir six eyeswere not included in the volume determination.
U. B. G. L A U P ~ E N T
152
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HYALURONATE IN AQUEOUS HUMOUR
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chamber. The total volume aspirated from each eye was recorded and the hyaluronate content determined in the two portions. The results are displayed in Table H. Aqueous humour collected immediately after death contained 4 # g / m l and there was no difference between the first and second portions withdrawn. With increasing time of storage there was a marked increase in aqueous hyaluronate concentration and the largest effect was seen in the last portion aspirated. Hyaluronate was apparently released from neighhouring structures, e.g. the vitreous body, into the aqueous fluid. The volume of the aqueous humour decreases post mortem, which apparently reflects the swelling of neighbouring tissues such as the cornea and lens. The volume changes are, however, too small to explain the changes in hyaluronate concentration (see Table II). In view of the results from studies on post mortem eyes it is necessary to sample the aqueous humour from living animals or immediately after death if the normal hyaluronate level should be determined.
Aqueous humour from different species The analytical data on aqueous humour from different species are shown in Table I I I . I t has been possible in most species to analyse samples of aqueous humour from single eyes hut in two cases (rat, mutton bird), it was necessary to pool material from several animals. Whenever possible the analyses were made in triplicate on the individual eyes and the mean values were used (cattle, sheep, pig, rabbit). In other species the material from each eye was often sufficient only for a single analysis (man, opossum). Large species differences were noted varying from 0"2 # g / m l in the rat and opossum up to 4 # g / m l in cattle. Only a few values are available from the literature for comparison. Balazs et al. (1959b) report the hyaluronate level of cattle aqueous humour from uronic acid analysis to be 2"3 # g / m l and Balazs and Falbe-Hansen (1965) found a concentration of 4'7/~g/ml. Laurent et al. (1969) using a specific isotope dilution technique found 4'2 and 7"1/~g/ml in calf and cow samples, respectively. These values are compatible with our results in view of the errors in the techniques and the post mortem changes discussed above. Hexuronate estimates on aqueous humours from sheep, rabbit and fowl (Balazs et al., 1959b) corresponded to appreciably higher values of hyaluronate than those reported here but these earlier analyses were uncertain due to the limits of the carbazole technique. The species variation presumably mirrors the hyaluronate release from neighbouring tissues, e.g. the vitreous body. The concentration of hyaluronate in the vitreous body, known from previous studies or estimates by the present radioassay (Table I I I ) seems to correlate to the content in the aqueous humour. Of special interest are the low levels found in the rat and the opossum. These animals are known to have very large lenses allowing only a small vitreous cavity (see Duke-Elder, 1958).
The hyaluronate content of aqueous humour in foetal development Cattle foetuses at different times of gestation were collected and aqueous humour could be analysed over the period 5-9 months. The results are shown in Table IV. The hyaluronate levels are lower than in the adult animals. A lower level of hyaluronate was also found in the vitreous body during foetal life compared with adult levels (Balazs et al., 1959a). Furthermore, the concentrations of hyaluronate both in the aqueous humour and the vitreous body are constant over the time period studied. 6
E E R 33
154
U.B.G. L A U R E N T TABLE IV
Sodium hyaluronale content in aqueous humour of bovine foetuses Age of Aspirated volume from foetus Number of single eyes (ml) (months) eyes Range 5 6 7 8 9
7 13 4 4 8
Na-hyaluronate concentration (#g/ml) Mean • ~7 Range*
0"06-0"13 0"13~)'30 0-21~)'53 0'26~'51 0"184)'71
0"9 + 0"2 0-8 • 0"1 1-2• 1"4• 0"4 0"9•
0-4/0-6-1"0/1'4 0'5/0'6-0"8/1"6 0'7/0-8-1-4/2-1 0'7/1'3-1"6/1"9 0'6/0"8-0'9/1"2
* The concentrations in the two eyes from the same animal are given as x/y. ACKNOWLEDGMENTS The author wishes to thank Professors D. A. Lowther and B. N. Preston and their co-workers for help and facilities during her stay at Monash University. Human material was kindly supplied by Professor Gerard Crock, Melbourne and by Professor Lennart Berggren and co-workers, Uppsala; and material from opossum and mutton bird by Drs Magda Weiss and John Baldwin, Monash University. Miss Agneta Laurent gave skilful technical assistance. The project was supported by grants from the Swedish Medical l~esearch Council and AB Pharmacia. REFERENCES Balazs, E. A. (1965). Amino sugar-containing macromolecules in the tissues of the eye and the ear. In The Amino Sugars. Vol. IIA. (Eds Balazs, E. A. and Jeanloz, R. W.). Pp. 401-60. Academic Press, New York and London. Balazs, E. A. and Falbe-Hansen, J. (1965). Unpublished data. Cited in The Amino Sugars. Vol. IIA. (Eds Balazs, E. A. and Jeanloz, R. W.). P. 450. Academic Press, New York and London. Balazs, E . A . , Freeman, M.I., K15ti, R., Meyer-Schwickerath, G., Regnault, F. and Sweeney, D . B . (1972). Hyaluronic acid and replacement of vitreous and aqueous humour. In Modern Problems in Ophthalmology. Vol. 10. (Ed. Streiff, E. B.). Pp. 3-21. S. Karger, Basel. Balazs, E. A., Laurent, T. C. and Laurent, U. B. G. (1959a). Studies on the structure of the vitreous body. VI. Biochemical changes during development. J. Biol. Chem. 234,422-30. Balazs, E. A., Laurent, T. C., Laurent, U. B. G., DeRoche, M. H. and Bunney, D. M. (1959b). Studies on the structure of the vitreous body. VIII. Comparative biochemistry. Arch. Biochem. Biophys. 81, 464-79. Bs E. H. (1956). The action of different kinds of hyaluronidase on the resistance of flow through the angle of the anterior chamber. Acta Ophthalmol. 34, 397-403. Berggren, L. and Vrabec, F. (1957). Demonstration of a coating substance in the trabecular meshwork of the eye : and its decrease after perfusion experiments with different kinds of hyaluronidase. Am. J. Ophthalmol. 44, 200-8. Bitter, T. and Muir, H. M. (1962). A modified uronic acid carbazole reaction. Anal. Bioehem. 4, 330-4. Denlinger, J. L. and Balazs, E. A. (1980). Replacement of the liquid vitreous 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 vitreous with sodium hyaluronate in monkeys. II. Long term evaluation. Exp. Eye Res. 31, 101-17. Duke-Elder, S. (1958). The eye in evolution. In System of Ophthalmology, Vol. I. P. 605. Henry Kimpton, London.
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Graue, E. L., Polack, F. M. and Balazs, E. A. (1980). The protective effect of Na-hyaluronate to corneal endothelium. Exp. Eye Res, 31, t t9-27. Hayasaka, S. and Sears, L. (1978). The presence oflysosomal hyaluronidase in the rabbit iris. Invest. Ophthalmol. 17, 639-44. Laurent, T. C., Bs E., Carlsson, B. and Tidare, E. (1969). Determination of hyaluronic acid in the microgram range. Anal. Biochem. 31~ 133--45. Laurent, U. B. G. and Tengblad, A. (1980). Determination of hyaluronate in biological samples by a specific radioassay technique. Anal. Biochem. 108, 386-94. Meyer, K. (1971). Hyaluronidases. In The Enzymes, Vol. V 3rd edn. (ed. Boyer, P. D.). Pp. 307-20. Academic Press, New York. Meyer, K. and Palmer, J. W. (1936). On the nature of the ocular fluids. Am. J. Ophthalmol. 19, 859-65. Meyer, K., Smyth, E. M. and Gallardo, E. (1938). On the nature of the ocular fluids. II. The hexosamine content. Am. J. Ophthalmol. 21, 1083-90. Miller, D., O'Connor, P. and Williams, J. (1977). Use of Na-hyaluronate during intraocular lens implantation in rabbits. Ophthalmic Surg. 8, 58-61. Miller, D. and Stegmann R. (1980). Use of Na-hyaluronate in auto-corneal transplantation in rabbits. Ophthalmic Surg. 11, 19-21. Ohya, T. and Kaneko, Y. (1970). Novel hyaluronidase from Streptomyces. Biochim. Biophys. Acta 198, 607-9. Pruett, R. C., Schepens, C. L. and Swarm, D. A. (1979). Hyaluronic acid vitreous substitute. Arch. Ophthalmol. 97, 2325-30. Scott, J. E. (1960). Aliphatic ammonium salts in the assay of acidic polysaccharides from tissues. Methods Biochem. Analysis 8, 145-97. Tengblad, A. (1980). Quantitative analysis of hyaluronate in nanogram amounts. Biochem. J. 185, 101-5.
Winters, L. M., Green, W. W. and Comstock, R. E. (1942). Prenatal development of the bovine. Minn. Tech. Bull. 151, 41.
~-2