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Glycosaminoglycans as inhibitors of calcium oxalate crystal growth and aggregation
23
Clinica Chi~ica Acta, 95 (1979) 23-28 0 E~sevier~North-Holland Biomedical Press
CCA 1016
GLYCOSAMINOGLYCANS AS INHIBITORS CRYSTAL GROWTH AND AGGREGATION
OF CALCIUM OXALATE
R.C. BOWYER a**, J.G. BROCKIS b and R.K. McCULLOCH a a Royal Perth Hospital and b University of Western Australia, Perth (Australia) University of Western Australia, Perth (Australia) (Received December 14th, 19’78)
Summary Evidence is presented that the glycosaminoglycans, chondroitin 6-sulphate and chondroitin 4-sulphate, are the major inhibitors of calcium oxalate crystal growth and aggregation in dilute normal urine.
Introduction In recent years a number of theories have been advanced to explain the aetiology of calcium stone formation in the urinary tract. Boyce and co-workers [l] have concentrated their research on the role of the organic matrix in initiating stone formation while others [ 2,3] have stressed the importance of supersaturation of the stone-forming salts. The role of inhibitors of crystal nucleation, growth and aggregation has also attracted much research [4-6]. Generally, these inhibitors appear to act by either reducing the level of supersaturation of the stone-forming ions or by adsorbing on to the crystal faces where they act as crystal poisons. Recent emphasis has been placed on the role of inhibitors of aggregation following the observations of Robertson et al. [6] and Baumann et al. [ 71 that normal people are capable of excreting smaller aggregates of calcium oxalate than stone-formers during periods of excessive supersaturation of the stone-forming ions. Despite widespread interest in this topic the nature of these crystal inhibitor has not been fully elucidated. It is generally believed that there are at least two major inhibitors of crystal growth and aggregation. One, a low molecular weight material shown by Fleisch and Bisaz [4] to be pyrophosphate, accounts for a small percentage of the total inhibitory activity. The other, in the macromolecular fraction of urine and believed by some to be an acidic glycosaminoglycan *To
whom
correspondence shouidbe
addressed.
24
(GAG) {8], appears to be the major inhibitor. Characterisation of this molecule should therefore be an import~t step towards elucidating one of the factors influencing calcium oxalate stone formation. The presence of all the types of GAGs that occur in human tissues with the possible exception of heparin, has been demonstrated in normal urine by many workers [9,10]. There is general a~eement that chondroitin sulphate (both the 4-sulphate and 6-sulphate) is the major component with hepaxan sulphate also present in significant amounts. In vitro studies by others [ll,lZ] have shown palyelec~olytes of this class to be powerful inhibitors of calcium oxalate and calcium phospha~ crystal growth and aggregation. In this work we present evidence strongly supporting a major role for GAGs as inhibitors of calcium oxalate crystal growth and aggregation in noxmal urine and we identify the individual GAGS responsible for this effect. Materials and methods Al&an Blue 8GX, ~hondroitin 4-sulphate (C-4-S) No. C-4134 and chondroitin 6-sulphate (C-6-S) No. C-4384 were obtained from Sigma Chemical Co., St, Louis, U.S.A. (The purity of the chondxoitin sulphates was not specified and has been assumed to be 100% for calculation purposes.) All other reagents were A.R. grade. Cellulose ~hromato~aphy plates (10 X 20 cm, 0.10 mm thickness) were supplied by Merck. Pooled mid-morning urine samples were obtained from 6 healthy adult male laboratory workers and were preserved over chloroform. Pools from the same donors but collected on different days have been labelled Pool 1, Pool 2, etc. elsewhere in this paper.
Urine and other extracts were filtered using an Amicon apparatus with UM10 membr~es which had a nominal cut-off of 10 000 daltons. Acidification of urine
The pH of a portion of pooled urine was adjusted to 1,5 with concentra~d hydrochlo~c acid and maint~ned at this pH for 24 h. It was then readjusted to pH 5.8 with 10 mol/l sodium hydroxide. The volume change was minimal. ~solatiun
of GAGS from
urine
GAGS were isolated from urine by the method of ~itern~ [ 131 and reconstituted in 0.15 mol/l saline. The concentration of GAGS in urine and other aqueous solutions was estimated by the method of Whiteman [ 131 using C-4-S as a standard. ~ix~res of GAGS were resolved using the ~quenti~ thin-layer ~hxo~to~phy method of Humbel and Chamoles [14]. I&a-red spectra of GAGS were recorded on a Perkin-Elmer 283 instrument using potassium bromide discs. Adsorption of inhibitor by calcium oxalate crystals
To 250 ml of pooled urine stirred at 37”C, 15 ml of 0.2 mol/l calcium chloride and 15 ml of 0.2 mol/l sodium oxalate were added dropwise during 1 h. Stirring was continued for another 3 h and then the resulting suspension was
25
filtered and washed sparingly with water. The calcium oxalate crystals were dissolved in 0.1 mol/l hydrochloric acid and ultrafiltered. The retentate was washed with water to remove the acid and dissolved ions and concentrated to a final volume of 2 ml. To 1 ml of this extract 4 ml of ethanol was added and this mixture was centrifuged at 2000 X g for 1 h. The supernatant was discarded and the precipitate was dissolved in 20 (~1 of water and subjected to sequential thin-layer chromatography. The remainder was kept for testing in the crystal growth system described below.
Measurement of crystal growth and aggregation Inhibition of crystal growth and aggregation was measured using the method of Robertson et al. [ 151. Crystal sizing was performed using a Coulter counter (Model B 100 I.trn orifice) over the diameter range 4.3 to 30 pm. All estimations were determined in quadruplicate. Glassware used for these procedures was washed in Decon detergent and then soaked in 1 mol/l hydrochloric acid overnight followed by rinsing with distilled water. It was kept out of general laboratory use. The incubation procedure was performed using 200 ml ‘E-MIL’ boro A flasks to which the crystals would not adhere during the 4-h period. (With some brands of glassware some workers have found it necessary to treat the surface with surfactants to prevent sticking. However, this procedure should be strongly discouraged as we have found surfactants such as Calgon (sodium hexametaphosphate) to be potent inhibitors of crystal growth and aggregation at concentrations as low as 0.04 ppm (unpublished results).) Results
Inhibitory activity of urinary fractions In order to quantitate the inhibitory activity of both high and low molecular weight components of urine, a series of pooled urines was fractionated using an Amicon UM-10 membrane with a cut-off of 10 000 daltons. The results in Table I show that the retentate, when reconstituted with water to its original volume, accounts for a high percentage of the total inhibitory activity present in the original urine.
Isolation of GAGS from normal urine GAGS were precipitated from urine as a complex with Alcian Blue and then most of the complex was disaggregated and the soluble GAGS isolated and reconstituted in 0.15 mol/l sodium chloride. However, approximately lo-20%
TABLE I FRACTIONATION
OF INHIBITORY
ACTIVITY
Results expressed as % inhibition f S.D. (n = 4).
Pool 1 Pool 2 Pool 3
1% original urine
1% Ultrafiltrate
1% Reconstituted retentate
82.7 12.4 84.0 i 2.5 79.5 zt 4.9
9.2 * 3.2 14.0 _c4.6 11.8 f 2.1
79.4 f 4.0 81.3 f 3.8 74.6 f 4.8
26 TABLE II ACTIVITY
OF GAGS ISOLATED
FROM NORMAL
URINE
Results expressed as % inhibition f S.D. (n = 4).
Pool 4 Pool 5 Pool 6
1% Original urine
Urinary GAGS of same concentration
84.6 * 2.2 82.4 + 6.6 76.9 + 2.7
85.0 + 3.2 80.9 f 3.9 79.0 * 3.1
of the Alcian Blue complex remained insoluble and the composition of this fraction was not further investigated. The GAG concentrations of the saline solution and the original urine were determined by the method of Whiteman [13]. Aggregation studies were performed using the original urine at 1% dilution and the saline solution containing an equivalent amount of GAG. The results of three such experiments, summarised in Table II, show that the difference in inhibitory activity between the 1% urine and the GAG solution is negligible. Characterisation of GAGS in urine The GAG mixture isolated above was separated into its components by a sequential thin-layer chromatography technique [ 141 which afforded excellent resolution. This method showed C-6-S as the major component with C-4-S present in significant amounts and a slower moving fraction, believed to be heparan sulphate, present in trace amounts. The presence of C-6-S and C-4-S in the mixture was confirmed by infra-red studies. Inhibitory activity of commercial C-4-S and C-6-S Commercial preparations of C-4-S and C-6-S were each tested at concentrations equivalent to the total GAG level found in 1% urine. The results of these aggregation studies, shown in Table III, confirm that these two compounds could account for the majority of the natural protective effect found in urine at this dilution. Identification of GAGS adsorbed by calcium oxalate crystals in urine Crystals of calcium oxalate were generated in urine by the simultaneous addition of solutions of sodium oxalate and calcium chloride. The filtered crystals were dissolved in 0.1 mol/l hydrochloric acid and this solution was ultrafiltered through a UM-10 membrane. The retentate then contained macromolecules from urine which had been adsorbed on to calcium oxalate during the TABLE III A COMPARISON OF THE INHIBITORY ACTIVITY + S.D. (n = 4) OF C-4-S SOLUTION CONTAINING THE SAME CONCENTRATION OF GAGS
Pool 7 Pool 8 Pool 9
1% Urine
c-4-s
C-6-S
76.1 f 3.3 81.8 f 2.8 80.8 * 3.8
75.6 f 2.00 78.3 c 6.3 81.9 + 4.9
77.2 k 6.6 19.3 * 6.3 84.0 it 2.7
C-6-S AND 1% URINE
27
crystallisation process. A portion of this extract, when tested in the crystal growth and ag~egation system, showed marked ~hibito~ activity. A further part of the extract was treated with ethanol and the precipitate obtained was identified by thin-layer chromatography as a mixture of C-4-S and C-6-S in almost equal amounts. The amount of GAGS removed from urine during the crystallisation of calcium oxalate was estimated both from solution depletion and crystal adsorption to be approximately 5%. Discussion The observation that normal people as well as stone-former-s are capable of excreting crystals in their urine suggests that inhibitors of aggregation rather than nucleation play a more significant role in the aetiology of urolithiasis. and “growth and aggregation” are used synony(The terms “aggregation” mously in this discussion as it is experimentally difficult to distinguish between the two.) In this work we have used the method of Robertson et al. [ 151 to assess the factors in urine which affect the growth and aggregation of calcium oxalate crystals. As part of this study we have confirmed previous findings [8,16] that the majority of the inhibitory activity resides with the macromolecular components of urine. While it has been demonstrated by others that GAGS are potent inhibitors of crystal growth and aggregation [11,X2] there have been no previous attempts to isolate and quantitate the effect of urinary GAGS. Results in Table II show that the GAGS isolated from urine by Alcian Blue account for most of the protective effect found in diluted urine. At present there are no satisfactory tests for measuring inhibition in undiluted urine and so there must remain some uncertainty as to the significance of the role played by GAGS in the prevention of crystal aggregation in the urinary tract. (If urine is tested for inhibitory activity at concentrations much greater than 5%, the endogenous levels of calcium and oxalate ions may significantly affect the relative levels of calcium and oxalate ions which have been s~d~dised in the experimental procedure.) We have also investigated the identity of the individual urinary GAGS responsible for the observed protective effect. Inhibitors of aggregation generally act by poisoning the crystal surface [ 17 J. We have used this property to aid in the isolation of the inhibitor by adsorbing it on to crystals of calcium oxalate artificially generated in urine. As this technique involved recovering the inhibitor from calcium oxalate crystals under acidic conditions it was first necessary to establish that the inhibitor was stable to acid. Results in Table IV show that urine loses little of its activity following acid treatment. Thin-layer chromatography of the material isolated from the crystals, and of the urinary GAGS, show C-6-S and C-4-S to be the major GAGS present in each case. Inhibition studies using commercial preparations of C-4-S and C-6-S show these GAGS to have almost the same activity as those extracted from urine. Combined, this evidence strongly suggests that C-6-S and C-4-S are the major inhibitors of calcium oxalate crystal growth and aggregation in dilute urine. It is our observation that only 5% of the total urinary GAGS was adsorbed
28 TABLE IV ACID STABILITY
OF URINARY
INHIBITOR
Results expressed as % inhibition + S.D. (n = 4). -_-I Pool 1 Pool 2
1% IJrine 82.7 r 2.4 84.0 + 2.5
wl_--
1% Urine following acidification
77.4 + 5.6 79.1 t 6.3
by the calcium oxalate crystals generated in urine. Althou~ the experiment conditions may not have favoured maximum adsorption of GAGS during the crystallisation process, normal urine would nevertheless appear to have a large reserve of inhibitor for such episodes of crystal growth and ag~e~tion, These results were obtained using urine from normal adult males and further investigation is required to explore differences in the GAG excretion patterns between normal subjects and stone-former%
The authors would like to acknowledge financial support for this work from the National Health and Medical Research Council, the Australian Kidney Founda~on and Wellcome Australasia and to the Royal Perth Hospital Research Foundation for use of its laboratory facilities. References 1. Boyce. W.H. (1969) in Renal Stone Research Symposium (Hodgkfnson, A. and Nordm. B.E.C.. eds.), pp. 93-102, Churchill, London 2 Pak, C.Y.C. (1969) J, Clin. Invest. 48,1914-1922 3 Robertson, W.G., Peacock, M. and Nordin, B.E.C. (1971) Clin. Sci. 40,365-374 4 Fleiach, H. and Bimz, S. (X962) Am. J. Physiol. 203.671675 5 Smith, L.H.. Meyer, J.L. and McCall, J.T. (1973) in Wrinsry Calculi
Clinica Chi~ica Acta, 95 (1979) 23-28 0 E~sevier~North-Holland Biomedical Press
CCA 1016
GLYCOSAMINOGLYCANS AS INHIBITORS CRYSTAL GROWTH AND AGGREGATION
OF CALCIUM OXALATE
R.C. BOWYER a**, J.G. BROCKIS b and R.K. McCULLOCH a a Royal Perth Hospital and b University of Western Australia, Perth (Australia) University of Western Australia, Perth (Australia) (Received December 14th, 19’78)
Summary Evidence is presented that the glycosaminoglycans, chondroitin 6-sulphate and chondroitin 4-sulphate, are the major inhibitors of calcium oxalate crystal growth and aggregation in dilute normal urine.
Introduction In recent years a number of theories have been advanced to explain the aetiology of calcium stone formation in the urinary tract. Boyce and co-workers [l] have concentrated their research on the role of the organic matrix in initiating stone formation while others [ 2,3] have stressed the importance of supersaturation of the stone-forming salts. The role of inhibitors of crystal nucleation, growth and aggregation has also attracted much research [4-6]. Generally, these inhibitors appear to act by either reducing the level of supersaturation of the stone-forming ions or by adsorbing on to the crystal faces where they act as crystal poisons. Recent emphasis has been placed on the role of inhibitors of aggregation following the observations of Robertson et al. [6] and Baumann et al. [ 71 that normal people are capable of excreting smaller aggregates of calcium oxalate than stone-formers during periods of excessive supersaturation of the stone-forming ions. Despite widespread interest in this topic the nature of these crystal inhibitor has not been fully elucidated. It is generally believed that there are at least two major inhibitors of crystal growth and aggregation. One, a low molecular weight material shown by Fleisch and Bisaz [4] to be pyrophosphate, accounts for a small percentage of the total inhibitory activity. The other, in the macromolecular fraction of urine and believed by some to be an acidic glycosaminoglycan *To
whom
correspondence shouidbe
addressed.
24
(GAG) {8], appears to be the major inhibitor. Characterisation of this molecule should therefore be an import~t step towards elucidating one of the factors influencing calcium oxalate stone formation. The presence of all the types of GAGs that occur in human tissues with the possible exception of heparin, has been demonstrated in normal urine by many workers [9,10]. There is general a~eement that chondroitin sulphate (both the 4-sulphate and 6-sulphate) is the major component with hepaxan sulphate also present in significant amounts. In vitro studies by others [ll,lZ] have shown palyelec~olytes of this class to be powerful inhibitors of calcium oxalate and calcium phospha~ crystal growth and aggregation. In this work we present evidence strongly supporting a major role for GAGs as inhibitors of calcium oxalate crystal growth and aggregation in noxmal urine and we identify the individual GAGS responsible for this effect. Materials and methods Al&an Blue 8GX, ~hondroitin 4-sulphate (C-4-S) No. C-4134 and chondroitin 6-sulphate (C-6-S) No. C-4384 were obtained from Sigma Chemical Co., St, Louis, U.S.A. (The purity of the chondxoitin sulphates was not specified and has been assumed to be 100% for calculation purposes.) All other reagents were A.R. grade. Cellulose ~hromato~aphy plates (10 X 20 cm, 0.10 mm thickness) were supplied by Merck. Pooled mid-morning urine samples were obtained from 6 healthy adult male laboratory workers and were preserved over chloroform. Pools from the same donors but collected on different days have been labelled Pool 1, Pool 2, etc. elsewhere in this paper.
Urine and other extracts were filtered using an Amicon apparatus with UM10 membr~es which had a nominal cut-off of 10 000 daltons. Acidification of urine
The pH of a portion of pooled urine was adjusted to 1,5 with concentra~d hydrochlo~c acid and maint~ned at this pH for 24 h. It was then readjusted to pH 5.8 with 10 mol/l sodium hydroxide. The volume change was minimal. ~solatiun
of GAGS from
urine
GAGS were isolated from urine by the method of ~itern~ [ 131 and reconstituted in 0.15 mol/l saline. The concentration of GAGS in urine and other aqueous solutions was estimated by the method of Whiteman [ 131 using C-4-S as a standard. ~ix~res of GAGS were resolved using the ~quenti~ thin-layer ~hxo~to~phy method of Humbel and Chamoles [14]. I&a-red spectra of GAGS were recorded on a Perkin-Elmer 283 instrument using potassium bromide discs. Adsorption of inhibitor by calcium oxalate crystals
To 250 ml of pooled urine stirred at 37”C, 15 ml of 0.2 mol/l calcium chloride and 15 ml of 0.2 mol/l sodium oxalate were added dropwise during 1 h. Stirring was continued for another 3 h and then the resulting suspension was
25
filtered and washed sparingly with water. The calcium oxalate crystals were dissolved in 0.1 mol/l hydrochloric acid and ultrafiltered. The retentate was washed with water to remove the acid and dissolved ions and concentrated to a final volume of 2 ml. To 1 ml of this extract 4 ml of ethanol was added and this mixture was centrifuged at 2000 X g for 1 h. The supernatant was discarded and the precipitate was dissolved in 20 (~1 of water and subjected to sequential thin-layer chromatography. The remainder was kept for testing in the crystal growth system described below.
Measurement of crystal growth and aggregation Inhibition of crystal growth and aggregation was measured using the method of Robertson et al. [ 151. Crystal sizing was performed using a Coulter counter (Model B 100 I.trn orifice) over the diameter range 4.3 to 30 pm. All estimations were determined in quadruplicate. Glassware used for these procedures was washed in Decon detergent and then soaked in 1 mol/l hydrochloric acid overnight followed by rinsing with distilled water. It was kept out of general laboratory use. The incubation procedure was performed using 200 ml ‘E-MIL’ boro A flasks to which the crystals would not adhere during the 4-h period. (With some brands of glassware some workers have found it necessary to treat the surface with surfactants to prevent sticking. However, this procedure should be strongly discouraged as we have found surfactants such as Calgon (sodium hexametaphosphate) to be potent inhibitors of crystal growth and aggregation at concentrations as low as 0.04 ppm (unpublished results).) Results
Inhibitory activity of urinary fractions In order to quantitate the inhibitory activity of both high and low molecular weight components of urine, a series of pooled urines was fractionated using an Amicon UM-10 membrane with a cut-off of 10 000 daltons. The results in Table I show that the retentate, when reconstituted with water to its original volume, accounts for a high percentage of the total inhibitory activity present in the original urine.
Isolation of GAGS from normal urine GAGS were precipitated from urine as a complex with Alcian Blue and then most of the complex was disaggregated and the soluble GAGS isolated and reconstituted in 0.15 mol/l sodium chloride. However, approximately lo-20%
TABLE I FRACTIONATION
OF INHIBITORY
ACTIVITY
Results expressed as % inhibition f S.D. (n = 4).
Pool 1 Pool 2 Pool 3
1% original urine
1% Ultrafiltrate
1% Reconstituted retentate
82.7 12.4 84.0 i 2.5 79.5 zt 4.9
9.2 * 3.2 14.0 _c4.6 11.8 f 2.1
79.4 f 4.0 81.3 f 3.8 74.6 f 4.8
26 TABLE II ACTIVITY
OF GAGS ISOLATED
FROM NORMAL
URINE
Results expressed as % inhibition f S.D. (n = 4).
Pool 4 Pool 5 Pool 6
1% Original urine
Urinary GAGS of same concentration
84.6 * 2.2 82.4 + 6.6 76.9 + 2.7
85.0 + 3.2 80.9 f 3.9 79.0 * 3.1
of the Alcian Blue complex remained insoluble and the composition of this fraction was not further investigated. The GAG concentrations of the saline solution and the original urine were determined by the method of Whiteman [13]. Aggregation studies were performed using the original urine at 1% dilution and the saline solution containing an equivalent amount of GAG. The results of three such experiments, summarised in Table II, show that the difference in inhibitory activity between the 1% urine and the GAG solution is negligible. Characterisation of GAGS in urine The GAG mixture isolated above was separated into its components by a sequential thin-layer chromatography technique [ 141 which afforded excellent resolution. This method showed C-6-S as the major component with C-4-S present in significant amounts and a slower moving fraction, believed to be heparan sulphate, present in trace amounts. The presence of C-6-S and C-4-S in the mixture was confirmed by infra-red studies. Inhibitory activity of commercial C-4-S and C-6-S Commercial preparations of C-4-S and C-6-S were each tested at concentrations equivalent to the total GAG level found in 1% urine. The results of these aggregation studies, shown in Table III, confirm that these two compounds could account for the majority of the natural protective effect found in urine at this dilution. Identification of GAGS adsorbed by calcium oxalate crystals in urine Crystals of calcium oxalate were generated in urine by the simultaneous addition of solutions of sodium oxalate and calcium chloride. The filtered crystals were dissolved in 0.1 mol/l hydrochloric acid and this solution was ultrafiltered through a UM-10 membrane. The retentate then contained macromolecules from urine which had been adsorbed on to calcium oxalate during the TABLE III A COMPARISON OF THE INHIBITORY ACTIVITY + S.D. (n = 4) OF C-4-S SOLUTION CONTAINING THE SAME CONCENTRATION OF GAGS
Pool 7 Pool 8 Pool 9
1% Urine
c-4-s
C-6-S
76.1 f 3.3 81.8 f 2.8 80.8 * 3.8
75.6 f 2.00 78.3 c 6.3 81.9 + 4.9
77.2 k 6.6 19.3 * 6.3 84.0 it 2.7
C-6-S AND 1% URINE
27
crystallisation process. A portion of this extract, when tested in the crystal growth and ag~egation system, showed marked ~hibito~ activity. A further part of the extract was treated with ethanol and the precipitate obtained was identified by thin-layer chromatography as a mixture of C-4-S and C-6-S in almost equal amounts. The amount of GAGS removed from urine during the crystallisation of calcium oxalate was estimated both from solution depletion and crystal adsorption to be approximately 5%. Discussion The observation that normal people as well as stone-former-s are capable of excreting crystals in their urine suggests that inhibitors of aggregation rather than nucleation play a more significant role in the aetiology of urolithiasis. and “growth and aggregation” are used synony(The terms “aggregation” mously in this discussion as it is experimentally difficult to distinguish between the two.) In this work we have used the method of Robertson et al. [ 151 to assess the factors in urine which affect the growth and aggregation of calcium oxalate crystals. As part of this study we have confirmed previous findings [8,16] that the majority of the inhibitory activity resides with the macromolecular components of urine. While it has been demonstrated by others that GAGS are potent inhibitors of crystal growth and aggregation [11,X2] there have been no previous attempts to isolate and quantitate the effect of urinary GAGS. Results in Table II show that the GAGS isolated from urine by Alcian Blue account for most of the protective effect found in diluted urine. At present there are no satisfactory tests for measuring inhibition in undiluted urine and so there must remain some uncertainty as to the significance of the role played by GAGS in the prevention of crystal aggregation in the urinary tract. (If urine is tested for inhibitory activity at concentrations much greater than 5%, the endogenous levels of calcium and oxalate ions may significantly affect the relative levels of calcium and oxalate ions which have been s~d~dised in the experimental procedure.) We have also investigated the identity of the individual urinary GAGS responsible for the observed protective effect. Inhibitors of aggregation generally act by poisoning the crystal surface [ 17 J. We have used this property to aid in the isolation of the inhibitor by adsorbing it on to crystals of calcium oxalate artificially generated in urine. As this technique involved recovering the inhibitor from calcium oxalate crystals under acidic conditions it was first necessary to establish that the inhibitor was stable to acid. Results in Table IV show that urine loses little of its activity following acid treatment. Thin-layer chromatography of the material isolated from the crystals, and of the urinary GAGS, show C-6-S and C-4-S to be the major GAGS present in each case. Inhibition studies using commercial preparations of C-4-S and C-6-S show these GAGS to have almost the same activity as those extracted from urine. Combined, this evidence strongly suggests that C-6-S and C-4-S are the major inhibitors of calcium oxalate crystal growth and aggregation in dilute urine. It is our observation that only 5% of the total urinary GAGS was adsorbed
28 TABLE IV ACID STABILITY
OF URINARY
INHIBITOR
Results expressed as % inhibition + S.D. (n = 4). -_-I Pool 1 Pool 2
1% IJrine 82.7 r 2.4 84.0 + 2.5
wl_--
1% Urine following acidification
77.4 + 5.6 79.1 t 6.3
by the calcium oxalate crystals generated in urine. Althou~ the experiment conditions may not have favoured maximum adsorption of GAGS during the crystallisation process, normal urine would nevertheless appear to have a large reserve of inhibitor for such episodes of crystal growth and ag~e~tion, These results were obtained using urine from normal adult males and further investigation is required to explore differences in the GAG excretion patterns between normal subjects and stone-former%
The authors would like to acknowledge financial support for this work from the National Health and Medical Research Council, the Australian Kidney Founda~on and Wellcome Australasia and to the Royal Perth Hospital Research Foundation for use of its laboratory facilities. References 1. Boyce. W.H. (1969) in Renal Stone Research Symposium (Hodgkfnson, A. and Nordm. B.E.C.. eds.), pp. 93-102, Churchill, London 2 Pak, C.Y.C. (1969) J, Clin. Invest. 48,1914-1922 3 Robertson, W.G., Peacock, M. and Nordin, B.E.C. (1971) Clin. Sci. 40,365-374 4 Fleiach, H. and Bimz, S. (X962) Am. J. Physiol. 203.671675 5 Smith, L.H.. Meyer, J.L. and McCall, J.T. (1973) in Wrinsry Calculi