The Effect of Saline Bladder Washings on Calcium Oxalate Crystal Growth and Aggregation

0022-534 7/85/1341-0158$02.00/0 Vol. 134, July Printed in U.S.A.

THE JOURNAL OF UROLOGY

Copyright © 1985 by The Williams & Wilkins Co.

THE EFFECT OF SALINE BLADDER WASHINGS ON CALCIUM OXALATE CRYSTAL GROWTH AND AGGREGATION KATHERINE A. EDYV ANE* ROSEMARY L. RY ALL AND VILLIS R. MARSHALL From the Urology Unit, Department of Surgery, Flinders Medical Centre, Bedford Park, South Australia

ABSTRACT

Saline bladder washouts were obtained from 31 normal patients undergoing routine cystoscopy. A urine control was prepared by diluting a urine sample to the same creatinine concentration as the bladder washout. The inhibitory activities of the samples were then measured in a calcium oxalate seeded crystallization system. Washouts from the first 9 patients inhibited crystal aggregation more strongly than did the controls. This was attributable to microscopic blood contamination, since in the following 22 subjects, in whom contamination was excluded by the use of sensitive haemoglobin test strips, no increase in inhibition of aggregation was seen. The inhibition of calcium oxalate crystal growth by the washouts was consistently greater than that by the urine controls (p <0.05). This suggests that the bladder mucosa is a source of inhibitor(s) of crystal growth but not crystal aggregation. The role of inhibitory substances in urine that may actively prevent calcium renal stones has been the focus of intensive investigation for many years. Yet whilst it has been clearly shown in many different systems that urinary macromolecules, in particular the soluble glycosaminoglycans (GAGs), have powerful anti-crystallization properties in vitro, 1- 3 the significance of these properties in vivo has never been established. The postulated role of these substances in the prevention of renal stones is supported by the observation of Robertson and co-workers 4 of reduced levels of GAGs in stone-formers' urines, but this observation has not been substantiated by others. 5- 7 Although some reports have found a difference between stone-formers and normals with respect to urinary inhibitory activity, 8 • 9 others have not been able to confirm the observation. 6 • 10, 11 The failure to demonstrate consistent differences between the urines from these 2 groups might occur if part of the inhibitory activity of urine were derived from the bladder itself. Under these circumstances the contribution from the bladder might mask any other difference present when urine leaves the kidney. Historically, studies on inhibitors have assumed that the bladder does not contribute to the urinary inhibitory activity. However, recent studies have shown that GAGs and mucosubstances are present in the mucosal layer of the bladder. 12- 14 Also, the ability of the mucosal layer to regenerate itself within 24 hours after removal by acid washing suggests that the production of mucosubstances into bladder urine could be quite significant. 15 In view of the strong evidence for the existence of these macromolecules in bladder mucosa and the possibility of their being released into urine, the present study was undertaken to examine whether the bladder contributes to urinary inhibitory activity. MATERIALS AND METHODS

Saline bladder washings were obtained from 31 patients undergoing routine cystoscopy. Patients were excluded if there was a history of bladder infection or neoplasm. An initial urine sample was collected via the cystoscope and the bladder rinsed of residual urine by filling and emptying twice with sterile normal saline. The bladder was then filled and lavaged via the Accepted for publicat.ion February. 26 , 1985 . * Requests for reprints: Urology Unit, Dept. of Surgery, Flinders Medical Centre, Bedford Park, South Australia 5042.

cystoscope for 30 seconds using a 50 ml. syringe, and the washout collected. In the first 9 patients, washout and urine samples were checked visually for blood contamination. In the remaining 22, the presence of blood was excluded using Multistix test strips (Miles Laboratories) which were capable of detecting blood contamination levels as low as 0.00015 per cent. The concentration of creatinine in the washout and urine samples was determined (Autoanalyser). To account for urinary contamination in the washout, a urine control was prepared by diluting the urine sample, collected when the cystoscope was introduced, with saline to the same creatinine concentration as the washout. After centrifugation and filtration both the urine control and the washout were brought to a final concentration of 1.00 mM Ca and 0.20 mM (C00Na)2 by the addition of calcium and oxalate. The inhibitory effect of the samples on calcium oxalate crystal growth and aggregation were then measured in a standard crystalseeded system3 using a model T All Coulter Counter fitted with a Population Accessory. The growth of calcium oxalate was expressed as the increase in crystal diameter. 16 Initial rates of growth and aggregation were calculated from plots of 1/LlD and 1/LlN versus 1/T, where LlD = cumulative increase in crystal diameter, LlN =percent change in crystal number and T = time. The inhibition in each sample was then expressed as a percentage of the rate occurring in a saline control. Thus, samples with inhibition values of O per cent indicate no inhibition, whilst values of 100 per cent indicate complete inhibition. The Wilcoxon Rank Sum Test was used for all statistical comparisons. RESULTS

Figures 1A and lB show the results obtained from the first 9 patients where blood contamination was excluded by visual examination only. In 8 of the 9 subjects the percentage inhibition of crystal growth by the washout was significantly greater (p <0.05) than the diluted urine (fig. IA). The median value for inhibition of growth in the urine controls was 14. 76 per cent (range 2.19 to 56.85 per cent) and in the washouts was 38.92 per cent (range 9.93 to 62. 73 per cent). The bladder washouts (fig. lB) also consistently produced greater inhibition of aggregation co~pa!e?. to the urine c~ntr~ls (p <0.~1). The median value for mh1b1t10n of aggregat10n m the urme controls was

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6.90 per cent (range Oto 27,47 per cent) and in the washouts was 32.36 per cent (range 16.31 to 56.02 per cent). Findings from this laboratory1 7 show that even microscopic amounts of blood can have a potent effect on crystal aggregation whilst having little effect on growth. Therefore results were obtained from 22 more washouts and urines in which microscopic blood contamination was excluded. There was no increase in inhibition of crystal aggregation in the washouts (fig. 2B, p >0.05), but inhibition of growth in the washouts was greater than in the urine controls (fig. 2A, p <0.05). The initial urine samples had a mean creatinine concentration of 8.83 mmol./1. (S.D. = 5.97 mmol./L), whilst the mean creatinine concentration in the 31 washouts was 0.08 mmol./1. (S.D. = 0.07 mmol./1.). Protein analysis 18 of the washouts and urine controls indicated significantly more protein in the washouts than could be attributed to urinary contamination (fig. 3, p <0.05). The median value for protein concentration in the urine controls was 1.05 µg./ml. (range Oto 3.10 µg./ml.) and for the washouts was 1.27 µg./ml. (range 0.12 to 5.18 µg./ml.).

mscussmN The initial approach in this study was to assume that the kidney urine was the only source of inhibitors. If this were the case, then when bladder urine was diluted to make its creatinine concentration equal to that of the bladder washout it would have been expected that the inhibitory activities would have been the same. However, the washouts had consistently greater inhibitory activity, indicating that the bladder is a source of substances that are inhibitors of calcium oxalate crystal growth. Although the increase in activity was far less than that in whole bladder urine, the washout fluid was only in contact with the bladder mucosa for 30 seconds. Under normal circumstances urine is in contact with the mucosa for many hours and after that time could be expected to contain more of these substancs. It is also possible that urine has a greater solubilizing effect on the mucosa! layer than saline, and could thus wash off more mucosubstances from the bladder wall in a given time. Our findings 17 that microscopic levels of blood could influence crystal aggregation led to the collection of 22 washouts where we excluded contamination by the use of the Multistix.

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The absence of a significant increase in inhibition of aggregation in the washout suggested that our earlier result, where there was an increase in inhibition of aggregation, 19 was due to microscopic amounts of blood. Thus the importance of taking into account microscopic blood contamination when measuring urinary inhibitory activity cannot be overemphasized. Although crystal growth and aggregation proceed simultaneously, they are affected to varying degrees by different inhibitors.3 Urine and all other inhibitors, whose separate effects on crystal growth and aggregation have been studied, have inhibited crystal aggregation more strongly than crystal growth. 3 The observation that the bladder washings retarded crystal growth but not crystal aggregation is therefore unexpected and interesting. There are a number of possible mechanisms by which this could occur. The inhibitor may affect growth simply by ion-binding, and not by actually attaching to the crystal surface. Alternatively, its molecular configuration may be such that it can bind to specific areas on the crystal surface without altering the zeta potential, such that growth is affected but not aggregation. To determine the true mechanism it will be necessary to isolate the inhibitory substance.

The small increase in concentration of protein in the washout compared with the control would suggest that the inhibitor present in the washout may be a protein or glycoprotein. Using available assays, to date it has not been possible to detect GAGs either in the washouts or in samples that have been concentrated 30 times by rotary evaporation. However, Martin and associates 20 in a recent study on ureterostomized dogs found decreased levels of alcian blue-precipitable material in kidney urine compared to bladder urine. This was also associated with reduced calcium oxalate crystal-growth inhibitory activity in the kidney urine. Our data suggest that the bladder is a potential source of inhibitors in urine, although the exact nature of this substance remains to be elucidated. Whilst evidence at present would suggest that its effect may be only minor and the actual contribution by the bladder to urinary inhibitory activity in vivo may therefore be only slight, our preliminary results would suggest that the question is worthy of further investigation. Acknowledgment. The authors would like to express their gratitude to the Department of Clinical Biochemistry, Flinders Medical Centre, for performing the creatinine determinations.

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urine adjusted to same washout creatinine concentration as washout FIG. 3. Protein content in bladder washouts and urines adjusted to same creatinine concentration as washouts. REFERENCES

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the growth and aggregation of calcium oxalate crystals in vitro. Clin. Chim. Acta, 43: 31, 1973. 2. Pak, C. Y. C., Holt, K. and Zerwekh, J. E.: Attenuation by monsodium urate of the inhibitory effect of glycosaminoglycans on calcium oxalate nucleation. Invest. Urol., 17: 138, 1979. 3. Ryall, R. L., Harnett, R. M. and Marshall, V. R.: The effect of urine, pyrophosphate, citrate, magnesium and glycosaminoglycans on the growth and aggregation of calcium oxalate crystals in vitro. Clin. Chim. Acta, 112: 349, 1981. 4. Robertson, W. G., Peacock, M., Heyburn, P. J., Marshall, D. H. and Clark, P. B.: Risk factors in calcium stone disease of the urinary tract. Brit. J. Urol., 50: 449, 1978.

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