Effects of Human Urine on Aggregation of Calcium Oxalate Crystals

0022-5347 /86/1351-0069$02.00/0 THE JOURNAL OF UROLOGY

Vol. 135, January

Copyright © 1986 by The Williams & Wilkins Co.

Printed in U.S.A.

EFFECTS OF HUMAN URINE ON AGGREGATION OF CALCIUM OXALATE CRYSTALS KURT E. SPRINGMANN, GEORGE W. DRACH,* BETH GOTTUNG

AND

ALAN D. RANDOLPH

From the Departments of Surgery and Chemical Engineering, University of Arizona, Tucson, Arizona

ABSTRACT

The importance of aggregation in calcium oxalate urolithiasis, although not fully understood, has long been postulated. Previous investigators of calcium oxalate crystal aggregation have applied static crystallization rather than continuous flow techniques to their studies. We describe the use of a Couette agglomerator in series with our previously reported continuous flow mixed suspensionmixed product removal crystallization system. We compared synthetic urine controls with 5 per cent volume-in-volume human urine additions from normal persons or patients with calcium oxalate stones. There was no significant difference in nucleation, linear crystal growth rate or total crystal mass between normal persons and those with stones. Control nucleation rate was significantly higher than in either human urine addition group. Comparison of aggregator particle size distributions revealed significant differences in aggregation among the control, normal and stone groups. We concluded that urine inhibitors to aggregation are somewhat deficient in patients with stones, resulting in the generation of larger particle masses or eventually stones. erator placed in series. 14 The technique of continuously seeding the Couette flow agglomerator with fresh seed generated continuously in the crystallizer was more convenient, accurate and more nearly representative of physiological conditions than batch preparation of some artificial seed with a narrow distribution of aged calcium oxalate dihydrate particles. Crystallizer input oxalate supersaturation was 0.6 mM. and crystallizer output supersaturation (and, therefore, agglomerator input) was 0.080 to 0.208 mM. Agglomerator output supersaturation was not measured. Figure 1 illustrates the schematic construction of this apparatus. The inner spindle was 51 mm. in diameter, with an annular space between the inner and outer cylinders of 9.2 mm. Thus, at the typical spindle rotation of 80 revolutions per minute an average shear rate of 23 seconds-1 would be expected. These levels are above the physiological rates of 3 to 5 seconds- 1 in small urinary tubules. 15 Experiments were done after the crystallizer reached steady state conditions, with temperature, pH, flow rates, composition and crystallizer volume held constant. Artificial urine solutions used in all experiments were prepared by diluting concentrated stock solutions followed by filtration through 3 µ. filters. The diluted synthetic urine solutions had calcium and oxalate concentrations of 6.0 and 0.6 mM., respectively, which lie within the range of calcium to oxalate ratios and concentrations reported in human urine. 16- 18 Table 1 lists feed solution compositions. In human urine runs a first morning voided urine specimen was filtered through 3 µ. filters and added to the calcium solution to result in a final 5 per cent volume in the crystallizer. All human urine samples had been plated previously on blood agar plates and none yielded any bacterial growth by 48 hours. For these experiments we have compared specimens from 10 normal subjects and 8 patients with stones, none of whom was on specific therapy. At least 2 experiments were performed with each urine specimen. Tau (average duration in crystallizer) was set at 10 minutes and the agglomerator was set at 80 revolutions per minute, both values derived from previous observations by Gottung. 14 Alternately, crystallizer and agglomerator outputs were analyzed instantly by a zone-sensing particle counting analyzer,t with data reduction on a PDP-8/A 16K digital microprocessor. This resulted in values for nucleation rate, linear crystal growth rate, total crystal mass produced3 and several measurements of aggregation.

The simplest theory of calcium oxalate urinary stone formation incorporates spontaneous crystal nucleation, followed by crystal growth and aggregation, resulting in a particle of adequate size to cause obstruction in the urinary tract. Lack of inhibitors, or the presence of promoters of 1 or more of these processes has been advanced as a theoretical difference between normal persons and those with stones. 1 During the last 10 years we have been conducting experiments of continuous crystallization in the calcium oxalate dihydrate system. 2- 5 The chief disadvantage of our use of the continuous crystallizer to date has been the requirement that aggregation be kept to a minimum. Therefore, while our previous studies intimated the importance of aggregation, we had been unable to measure its significance directly. Several investigators have studied previously the importance of aggregation in static systems of calcium oxalate crystal formation but they have not used continuous flow techniques. 6- 9 We have used our continuous mixed suspension-mixed product removal crystallizer system in series with a Couette agglomerator to analyze further the effects on aggregation of addition of human urine to a synthetic urine (fig. 1). Other investigators have shown that patients with calcium oxalate stones tend to excrete larger numbers of calcium oxalate dihydrate particles of larger size than normal persons, often up to 200 µm. in diameter. 10• 11 In vitro studies of the effects of certain highly charged organic anions on calcium oxalate aggregation by Robertson6 and Ryall12 and their associates have showed striking inhibitory effects. These observations support the theory that normal human urine contains crystal aggregation inhibitors that are deficient in urine from patients with stones. Based upon these observations we predicted that significant differences in aggregation behavior would exist between urine specimens from normal subjects and patients with stones. METHODS

Experiments were done in the mixed suspension -mixed product removal continuous crystallization system as described previously, 2• 13 with the added modification of a Couette agglomAccepted for publication June 5, 1985. Supported in part by National Science Foundation Grant CPE8117753. *~equests for reprints: Department of Surgery, Section of Urology, University of Arizona, Arizona Health Sciences Center, Tucson, Arizona 85724.

t Particle Data, Inc. 69

70

SPRINGMANN AND ASSOCIATES

Couette Agglomerator

size distribution is dependent upon input particle size distribution from the crystallizer. 14 Therefore, we normalized the results from the agglomerator output by dividing by the total number of particles in the crystallizer input to the agglomerator. The total number was calculated as all particles above the lower size threshold of 5 µm. Following this normalization, patients with stones had significantly greater numbers of large particles (p <0.015 and <0.05 for particles > 15 and >24 µm., respectively) than normals, and both were much less (p <0.001) than control experiments (fig. 4). DISCUSSION

Discharge

After addition of 5 per cent volume-in-volume human urine to synthetic urine supersaturated with calcium oxalate, values for crystal growth and total mass did not differ significantly

MSMPR Crystallizer FIG. 1. Schematic of mixed suspension-mixed product removal crystallizer with agglomerator. PDP8/A 16K microprocessor not shown.

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Composition of artificial urine solution

Ammonium chloride Sodium sulfate Potassium chloride Magnesium sulfate Calcium chloride Sodium citrate Sodium chloride Sodium oxalate Sodium phosphate Sodium biphosphate

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FIG. 2. Crystallization observations. S.F., patients with stones. N.L., normal subjects. C, control artificial urine. G, linear crystal growth rate (µ. per minute). B 0 , nucleation rate (particles per cc per minute). Mr, slurry density (mg./1.). RESULTS

Basic crystallization measurements for the 3 groups are presented in figure 2. Addition of human urine resulted in increased growth and decreased nucleation rates compared to control experiments. However, only the control nucleation rate differed significantly from human urine addition experiments. Figure 3 illustrates the effects of diverting crystallizer output through the agglomerator. The presence of human urine, either from normal subjects or stone patients, markedly inhibited the process of aggregation. However, mean particle size of the specimens from patients with stones was significantly larger than that in normal urine, suggesting immediately that the former specimens allow larger crystal aggregates. However, it appeared that numbers of aggregated particles more than 15 and 24 µ. from human urine experiments were not significantly different (fig. 3). It is known that the aggregator output particle

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FIG. 4. Aggregation observations normalized by crystallizer input to aggregator. For particles larger than 15 and 24 µ. (N> 15 and N>24) control values are significantly different from normals (N.L.) and patients with stones (S.F.) (p <0.001). Patients with stones show significantly greater value than normals for difference in mass-weighted average particle size between crystallizer and aggregator (M4 ,3 } and N > 15 (p <0.015), and for N >24 (p = 0.05). Deviation shown is standard deviation. C, control artificial urine.

OXALATE CH.YSTP,_LS

EFFECTS OF HUMAN URINE ON AGGREGA'HON OF

between control artificial urine, and that from normal and patients with stones. Values for nucleation rates were significantly higher in control experiments than for either urine group. This finding contrasts with our earlier observations. 4 •5 Kraljevich produced these earlier crystallization observations with only high molecular weight fractions derived from urine. 19 Significant technical modifications have occurred since these earlier studies. 14 It seems likely, then, that these different observations in our present experiments are results of method changes and the resolution of data is improved simply by technical improvements in our methodology. Kraljevich also produced decreases in aggregation of calcium oxalate crystals with additions of high molecular weight urinary extracts. However, she used scanning electron microscopy as the measurement tool and reported only qualitative results. 19 Gottung established that one can measure calcium oxalate crystallization and aggregation parameters with the mixed suspension-mixed product removal crystallizer and Couette agglomerator serially attached to a particle analyzer. 14 Her human urine addition experiments, which were done in the manner of this study, failed to show significant differences in crystallization or aggregation between normal subjects and patients with stones. Subsequent theoretical analysis of the aggregation system revealed that this was owing to use of too great a rotation in the aggregator (150 revolutions per minute), with resulting disaggregation of the particles. Adjustment of our present aggregator to 80 revolutions per minute has overcome this problem. We can now say that urine contains an inhibitor to aggregation of calcium oxalate crystals, in agreement with previous investigators. 12• 20 Our serial analysis system allows us to hypothesize that l major difference in calcium oxalate crystallization between normal subjects and patients with stones rests in a deficiency in aggregation inhibition in the latter, so that they are capable of generating larger particle masses. The presence of a bimodal particle size distribution for crystals from urine of patients with stones with a second mode of large particles has been noted by ourselves and others, 21 • 22 and we believe it is the result of their aggregation inhibition deficiency. It seems likely that the high molecular weight fractions of urine account for this deficiency. 5 • 12 • 19 • 23 Future knowledge in this regard will depend on further identification of the compound(s) responsible for strong or weak inhibition. Qualitative and quantitative differences in both factors seem likely. 5• 19• 24 However, it may not be possible to reproduce aggregation observations with extracts of urineuse of single glycoprotein isolates or subfractions may not result in whole urine effects. Use of dilute urine may be questioned in these ex1oe1rm1e11ts but requirement of throughput volumes of 4 L use of whole urine impossible at this time. Certainly, this experiment confirms that continuous flow methods of nearly simultaneous study of crystallization and aggregation offer powerful tools for further analysis of calcium oxalate precipitation. REFERENCES 1. Robertson, W. G., Scurr, D. S. and Bridge, C. M.: Factors influ-

encing the crystallization of calcium oxalate in urine-critique. J. Crystal Growth, 53: 182, 1981.

2. l\1Iiller J. D. Randolph) A. D. and. Drach, 10-J Observations calciu:n1 oxalate crystallization kinetics in sinrulated urine. 117: 1977. 3. Drach, G. W., no.uu.u,,,u, J. D.: Inhibition of 1

4.

5. 6. 7.

8. 9.

10. 11.

1

calcium oxalate I. Pyrophosphate methylene blue. J. Drach, G. W., Thorson, S. and Randolph, A.: of urinary organic macromolecules on crystallization of calcium oxalate: enhancement of nucleation. J. Urol., 123: 519, 1980. Drach, G. W., Kraljevich, Z. and Randolph, A. D.: Effects of high molecular weight urinary macromolecules on crystallization of calcium oxalate dihydrate. J. Urol., 127: 805, 1982. Robertson, W. G., Peacock, M. and Nordin, B. E. C.: Inhibitors of the growth and aggregation of calcium oxalate crystals in vitro. Clin. Chem. Acta, 43: 31, 1973. Adamthwaite, D. N.: Urinary pa1ticulate activity in urinary calculus disease. Brit. J. Urol., 55: 95, 1983. Koide, T., Takemoto, M., Itatani, H., Takaha, M. and Sonoda, T.: Urinary macromolecular substances as natural inhibitors of calcium oxalate crystal aggregation. Invest. Urol., HI: 382, 1981. Ryal!, R. L., Bagley, C. J. and Marshall, V. R.: Independent assessment of the growth and aggregation of calcium oxalate crystals using the Coulter counter. Invest. Urol., 18: 401, 1981. Robertson, W. G., Peacock, M. and Nordin, B. E. C.: Calcium crystalluria in recurrent renal stone-formers. Lancet, 2: 21, 1969. Suzuki, K.: Studies on urolithiasis: crystal aggregation in calcium oxalate stone formers. Nippon Hinyokika Gakkai Zasshi, 72:

842, 1981. 12. Ryall, R. L., Hamet, R. M. and Marshall, V. R.: The effect of urine,

on pyrophosphate, citrate, magnesium and -"y'rJ",2,a"-,I'u'uv 5 ,y the grovvth and aggregation of calcium oxalate Clin. Chim. Acta, 112: 349, 1981. 13. Randolph, A. D. and Larson, M.A.: Themy of Particulate Processes: Analysis and Techniques of Continuous Crystallization. New York: Academic Press, 1971. 14. Gottung, B.: Calcium Oxalate Agglomeration in Urine-Like Mother Liquors. Thesis, University of Arizona, 1983. 15. Adair, J. H.: Coagulation of Calcium Oxalate Monohydrate Suspension. Ph.D. Dissertation, University of Florida, Gainesville, 1981. 16. Robertson, W. G., Peacock, M., Heyburn, P. J., Marshall, D. H. and Clark, P. B.: Risk facto:rs in calcium stone disease of the urinary tract. Brit. J. Urol., 50: 449, 1978. 17. Hargreave, T. B., Sali, A., rvi:ackay, C. and Sullivan, M.: Diurnal variation in urinary oxalate. Brit. J. Urol., 49: 597, 1977. 18. Robertson, W. G., Peacock, M. and Nordin, B. E. C.: Calcium

19.

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oxalate crystalluria and urine saturation in recurrent renal stoneformers. Clin. Sci., 40: 365, 1971. Kraljevich, Z.: Effects of Urinary Macromolecules on Crystallization of Calcium Oxalate in Synthetic Urine Solutions, Thesis, of Arizona, 1981. G. and Peacock, M.: Calcium oxalate auuu.,w.,~ of crystallization in recurrent renal Clin. Sci., 43: 499, 1972. Miller, J. D.. Kinetics of Calcium Oxalate in Simulated Urine. University of Arizona, 1976_ Robertson, Vi!. G.: A method for measuring cakium crystalluria. Clin. Chim. Acta, 26: 105, 1969. White, D. J., Jr., Christoffersen, J., Herman, T. S., Lanzalaco, A. C. and Nm:icollas, H.: Effects of urine nri2trea1;m,2nt on calcium oxalate crystallization inhibition J. UroL, 129: 175,

1983. 24. Randolph, A. D. and Drach, G.

·w.: Some rneasurements of calcium oxalate nucleation and growth rates in urine-like liquors. J. Crystal Growth, 53: 195, 1981.