British Journal of Urology (1992), 70,240-246 01992 British Journal of Urology

Agglomeration of Calcium Oxalate Monohydrate in Synthetic Urine F. GRASES, L. MASAROVA, 0. SOHNEL and A. COSTA-BAUZA Department of Chemistry, University of the Balearic Islands, Palma de Mallorca, Spain

Summary-The development of agglomerated particles of calcium oxalate monohydrate (COM) on the semi-batch precipitation from a synthetic urine carried out at physiological conditions (37"C, pH = 5.5) was studied by optical and electron scanning microscopy. COM agglomerates develop by primary and secondary agglomeration proceeding simultaneously; the latter mechanism is, however, less important than the former. Citrate ions modify slightly the COM crystal shape and inhibit primary agglomeration. Mucin particles serve a s a substrate for preferential formation (nucleation) of new COM crystals. The structure of formed agglomerates closely resembles that of a certain type of COM renal calculi. A combination of primary agglomeration of crystals forming stones and nucleation of new crystals on a mucoprotein layer partially covering their surface constitutes the possible mechanism of s u c h stone development. Experimental data support this mechanism.

The agglomeration of calcium oxalate monohydrate (COM) crystals is recognised as an important step in renal stone development (Robertson et al., 1976, 1981; Schneider, 1985). The mechanism governing this process, however, still remains to be clarified. The only mechanism of agglomeration that has been described quantitatively proceeds through crystal-crystal collisions induced by hydrodynamic forces in a liquid medium containing crystals (von Smoluchowski, 1918). This process, usually termed secondary agglomeration, cannot represent a significant mechanism of renal calculi development due to conditions prevailing in the kidney (Finlayson, 1978). Recent experimental results of the semi-batch precipitation of COM indicated primary agglomeration as a possible mechanism for the development of calculi (Grases et al., 1992; Millan et al., 1992). Primary agglomeration represents a sort of crystal rnal-growth that takes place on the surface and/or tips of already developed crystals, the socalled parent crystals (Buckley, 1951;Jones, 1989). This process results in concretions consisting of Accepted for publication 29 August 1991

intergrown crystals with a complex crystal arrangement that closely resembles the structure of certain types of COM renal calculi. Primary agglomeration of COM crystals forming a stone, in combination with the effect of mucoproteins adsorbed on the exposed calculus surface, appear to constitute a development mechanism for certain types of COM renal calculi (Grases et al., 1992). This conclusion was based mainly on results achieved following the semi-batch precipitation of pure calcium chloride and sodium oxalate solutions, i.e. on experiments carried out under conditions not fully consistent with physiological ones. Therefore, further experiments in a urine-like liquor were needed in order to corroborate the feasibility of this mechanism of COM calculi development. We present the results of the semi-batch precipitation of COM carried out in uitro under conditions as close as possible to those expected in uiuo.

Materials and Methods

A semi-batch precipitation was carried out in a magnetically stirred unbaffled beaker of 1 L capacity immersed in a constant temperature water bath with the temperature adjusted to 37°C; 50 ml of a

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solution saturated with respect to calcium oxalate (see below) were placed in the beaker and agitated until 37°C was reached. Two solutions, A and B, were then introduced simultaneously at the combined rate of 200ml/h (i.e. 100 ml/h for each solution) on to a liquid surface through separate inlets. The inlets were situated on opposite sides of the reaction beaker. The samples of reaction mixture withdrawn throughout each run were immediately observed under an optical microscope and solid phase was viewed by an electron scanning microscope. One litre of solution A contained 11.02 g Na2S04.10 H 2 0 , 1.46g MgS0,.7 H20, 4.64g NH4C1, 12.13 g KC1 and 1.413 g CaC12.3.5 H 2 0 . Solution B contained 6.80 g NaH2P0,.2 H 2 0 , 2.18 g Na2HP04.12 H 2 0 , 13.05 g NaC1, 0.287 g Na2C204 and, unless stated otherwise, 1.16 g Na3C6H5O7.2H 2 0 per litre. These substances of Analytical grade purity were dissolved in distilled water. Some experiments were performed with solutions containing double the quantity of calcium chloride and sodium oxalate indicated above, i.e. 2.82 g CaC12.3.5H 2 0and 0.537 g Na2C204respectively. A series of experiments in the presence of mucin (from porcine stomach (Sigma Chem. Co., USA) was also performed. In this case 0.2g/l of mucin were dissolved in solution A, whereas the composition of solution B was not changed. The pH of both solutions was adjusted prior to each

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experiment to the value of 5.5 by adding HC1 and NaOH, as appropriate. The saturated solution charged into the reaction beaker at the beginning of each experiment was prepared by filtering the suspension resulting from the semi-batch precipitation carried out under conditions of a respective experiment for approximately 5 h.

Results Individual crystals formed in the reaction mixture always behaved in a manner typical of calcium oxalate monohydrate. The development of agglomerates during the semi-batch precipitation of calcium oxalate was, in principle, similar for all reaction conditions examined. Tiny and often individual crystals were detected in the system by an optical microscope within 10 to 20 min after the onset of introducing reactant solutions into the reactor (Fig. 1A). On continuing such introduction, individual crystals gradually developed into agglomerates, “loose” at the beginning and compact later (Fig. 1B). The size of the agglomerates increased with progressing precipitation. The presence of citrate suppressed agglomeration to some extent. The rate of agglomerate development was slower and the average size reached

Fig. 1 Development of COM agglomerates in a synthetic urine containing no citrate. ( x 400). (A) Initial COM crystals. (B) Compact agglomerates.

242 within a certain time was smaller than at precipitation of solutions containing no citrate. The presence of mucin significantly altered the course of agglomerate formation when compared with precipitation carried out without mucin. COM crystals were preferentially formed on mucin particles and the nucleation in the solution bulk was substantially suppressed (Fig. 2A). The first COM crystals could be observed on the surface of amorphous mucin particles after a certain time had elapsed. With progressing precipitation, the mucin particles became completely “surrounded” by COM crystals and thereafter could not be distinguished (Fig. 2B). The size of agglomerates increased during further precipitation, but the increase was less than in the system containing no mucin particles. Citrate present in the mucincontaining system, even when 3 times as high as in the standard solution B, did not exert any significant influence on the agglomeration process. Agglomerates formed on mucin particles were of the same character as COM agglomerates formed without mucin. Details of agglomerate structure during development were revealed by electron scanning microsCOPY. At the start of precipitation, COM formed individual elongated crystals and small intergrown composites of different shapes (Fig. 3A). “Loose” agglomerates consisting mainly of plate-like inter-

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grown crystals developed from these crystals at early stages of precipitation (Fig. 3B). Compact agglomerates, around 50 pm in diameter, which appeared at later stages of precipitation, were composed of large interconnected crystals and a number of smaller plate-like crystals. Small crystals were mostly intergrown and originated both on the surface and tips of the parent crystals; in the latter case typical “rosette” structures were formed (Fig. 3C). When citrate ions are present in a precipitating system the shape of both initial and agglomerateforming crystals is changed when compared with the situation without citrate (Fig. 3D). Intergrown crystals forming an agglomerate are apparently less clearly defined and the extensive occurrence of new small crystals on the parent crystals is almost absent. If mucin is present in a synthetic urine, small and unconnected COM crystals form first on their surface (Fig. 4A). These particles later become completely covered by a “crust” of intergrown plate-like COM crystals, many of them clearly originating on the parent crystals. Small spherical “sub-formations” consisting of plates arranged in star-like structures can be observed on the surface of large agglomerates (Fig. 4B). Such formations were not observed on agglomerates formed without mucin. Citrate ions do not markedly influence the shape, size and arrangement of COM crystals in

Fig. 2 Development of COM aggregates in a synthetic urine containing citrate and 0.2 g/1 mucin. ( x 400). (A) COM crystals on mucin particle. (B) Agglomerates developed on much particles.

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Fig. 3 Development of COM aggregates in a synthetic urine without (A-C) and with citrate (D) present. (A) Individual crystals formed at the commencement of the process. (B) Loose agglomerates. (C) Detail of a compact agglomerate. (D) Compact agglomeratesformed from a citrate-containingsystem.

agglomerates formed from a mucin-containing synthetic urine. Precipitation of solutions containing increased concentrations of CaCl, and Na2C20, proceeded in the same way as precipitation of the standard A and B solutions, but the development of agglomerates was faster. Their crystalline structure was, however, identical in both conditions.

Discussion Crystals formed on the semi-batch precipitation from a synthetic urine examined under these conditions were typical of calcium oxalate monohydrate (COM) crystals. Formation of calcium oxalate dihydrate (COD) was not observed. In contrast, the formation of a mixture consisting of

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Fig. 4 Development of COM agglomerates in a synthetic urine containing citrate and 0.2 g/1 m u c h (A) First crystals formed on mucin particle. (B) Spherical formations on agglomerate surface.

COM, COD and COT from batch and continuously precipitated solutions of similar composition to that employed in the present work, but with a slightly higher pH, was reported by Gardner (1978), Drach et al. (1978), Rodgers and Garside (1981), BreEeviC etal. (1986) andSkrtiCetu1. (1987). Thisdiscrepancy may be due to slight differences in reaction conditions, but the different mode of precipitation may also play a part. The formation and development of COM agglomerates on the semi-batch precipitation of a synthetic urine and of pure solutions as described by Millan et al. (1992) and Grases et al. (1992) were identical. Briefly, individual elongated COM crystals appearing first in the precipitating system had served as a substrate upon which new crystals were formed. This process resulted in small aggregates composed of elongated intergrown crystals that were often arranged in peculiar patterns within a particle. During further precipitation these crystals developed by lateral growth into plate-like interconnected crystals. New crystals that originated on the surface and/or tips of the parent crystals gradually became integrated into a complex agglomerate structure. After some time, particles consisting of innumerable intergrown and interconnected crystals were formed. The complex crystalline structure of these agglomerates cannot be wholly explained by a mechanism of secondary agglomeration, i.e.

by an incidental sticking together of crystals and their later cementation in place, although individual crystals formed separately in the solution attached to an agglomerate can also be observed. Such crystals, however, represent a small fraction of the total number of crystals forming an agglomerate. Hence, primary agglomeration represents an important, if not decisive, mechanism of COM agglomerate development and secondary agglomeration is of lesser consequence. Citrate ions prevent the formation of large COM agglomerates by inhibiting either crystal growth or agglomeration or, more probably, both processes simultaneously (Curreri et al., 1981 ; Nakayawa et al., 1981; Ryall et al., 1981; Grases et ul., 1988; Grases and Costa-BauzSr, 1990). Due to a qualitative evaluation of the agglomeration process employed in the present work, no firm conclusion can be drawn in that respect, but the observed suppression of new crystals forming on the parent crystals in the presence of citrate ions is consistent with the reported inhibition of agglomeration. Mucin forms in water a colloidal solution containing small amorphous particles of this compound dispersed in the volume of liquid. Mucin particles, when present in a precipitating system, cause preferential formation of COM crystals on their surface and suppress crystal formation in the solution bulk. Hence, the surface of mucin particles

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effectively promotes nucleation of COM crystals agglomeration in conjunction with secondary agfrom a system supersaturated with respect to glomeration that, however, plays a comparatively calcium oxalate. minor role. The surface of mucoprotein particles These observations enable us to draw the follow- strongly promotes nucleation of COM crystals. ing conclusions on the mechanism of COM renal Experimental results support the mechanism of stone development: (i) COM is formed on the semi- renal stone development based on a combination batch precipitation carried out at physiological of primary agglomeration and nucleation promoted conditions ; (ii) primary agglomeration plays an by a mucoprotein layer partially covering the important role in the development of COM agglom- exposed surface of a calculus. erates from a synthetic urine; (iii) citrate ions inhibit, but do not stop, the agglomeration process ; (iv) mucoprotein particles serving as a substrate Acknowledgement promote the formation of new COM crystals; (v) Financial support from the University of the Balearic Islands agglomeration proceeds faster in a system contain- (L. Masirova) and the Direccion General de Investigacion ing a higher concentration of calcium oxalate. Cientifica y Tttcnica, grants No. SAB 91-0040 (0.Sohnel), PBConclusions (ii), (iii) and (iv) are consistent with 89-0423 is gratefully acknowledged. results reported by Lonsdale (1968), Curreri et al. (1981), Nakayawa et al. (1981), Ryall et al. (1981), Grases et ul. (1988) and Grases and Costa-Bauzii References (1990). BreEeviE, L., Skrtk., D. and Garside, J. (1986). Transformation Although information on the crystalline structure of calcium oxalate hydrates. J . Crystal Growth, 74,399-408. of COM renal calculi is limited, at least 2 different Buckley, H. E. (1951). CrystalGrowth. New York: Wiley. types of stone can be distinguished: (i) calculi Cifuentes, L. (1984). Composicion y Structura de 10s Calculos Renales. Barcelona: Salvat Editors. exhibiting a compact fracture surface where crystals Curreri, P. A.,Onoda, G. and Finlayson, B. (1981).A comparative are arranged in an apparently unorganised manner appraisal of adsorption of citrate on whewellite seed crystals. with no evidence of fine crystalline structure (Oren J . Crystal Growth, 53,209-214. et al., 1984); (ii) calculi displaying a clearly visible Drach,G. W.,Randolph, A. D. andMiller, J. D. (1978). Inhibition of calcium oxalate dihydrate crystallization by chemical fine crystalline structure on the fracture surface modifier. J . Urol., 119,99-103. (Kim and Johnson, 1981; Cifuentes, 1984). The Finlayson, B. (1978). Physicochemical aspects of urolithiasis. crystalline structure of renal stones of the latter type Kidney Int., 13,344-360. closely resembles the structure of agglomerates that Gardner, G. L. (1978). Effect of pyrophosphate and phosphonate anions on the crystal growth kinetics of calcium oxalate were formed in this study. Therefore, the second hydrates. J . Phys. Chem., 82,864-870. type of COM calculi, at least, could be formed by F. and Costa-Bauza, A. (1990). Study of factors affecting the mechanism suggested by Grases et al. (1992): a Grases, calcium oxalate crystalline aggregation. Br. J . Urol., 66, 240polycrystalline nucleus of a renal calculus develops 244. through increasing the number of composing Grases, F., Millan, A. and Garcia-Raso, A. (1988). Polyhydroxycarboxylic acids as inhibitors of calcium oxalate crystal crystals by primary agglomeration, whereas the size growth: relation between inhibitory capacity and chemical of crystals increases due to ordinary crystal growth. structure. J . Crystal Growth, 89,496-500. The mucoproteins adsorbed on a part of the exposed Grases, F., Millan, A. and Sohnel, 0. (1992). Role of agglomerasurface of the calculus stop further growth of tion in calcium oxalate monohydrate uroliths development. Nephron. (In press). covered crystals as they are no longer in contact with a supersaturated fluid. However, the mucopro- Jones, A. G. (1989). Agglomeration during crysallization and precipitation from solution. In Fifth International Symposium tein layer promotes the formation of new crystals on Agglomeration. Institute of Chemical Engineers Symposium which then start to grow and multiply by the same Series. Pp. 131-143. Rugby: Institute of Chemical Engineers. mechanism as described above. The conclusions Kim, K. M. and Johnson, F. B. (1981). Calcium oxalate crystal growth in human urinary stones. ScanningElectron Microscopy, inferred from these experiments support, or at least 111, 147-154. are consistent with, the suggested mechanism of Lonsdale, K. (1968). Epitaxy as a growth factor in urinary calculi the development of COM renal calculi. and gallstones. Nature, 217,5658. The COM agglomerates formed on the semi- Millan, A., Grases, F. and Sohnel, 0. (1992). Semi-batch precipitation of calcium oxalate monohydrate. Cryst. Res. batch precipitation from a synthetic urine exhibit Technol. (In press). a crystalline structure similar to the type of renal Y., Abram, V., K&zdy,F. J. etal. (1981). Purification stones displaying a fine crystalline structure on the Nakayawa, and characterization of the principal inhibitor of calcium fracture surface. The mechanism responsible for oxalate monohydrate crystal growth in human urine. J . Biol. the development of these agglomerates is primary Chem., 258,12594-12600.

246 Oren, A., Husdan, H., Cheng, P.-T. ef d (1984). Calcium oxalate kidney stones in patients on continuous ambulatory peritoneal dialysis. Kidney Znt., 25, 534-538. Robertson, W. G., Peacock, M., Marshall, R. W. ef d (1976). Saturation-inhibition index as a measure of the risk of calcium oxalate stone formation in the urinary tract. N . Engl. J . Med., 294,249-252. Robertson, W. G., Scurr, D. S. and Bridge, M. (1981). Factors influencing the crystallization of calcium oxalate in urineCritique. J . Crystal Growth, 53, 182-194. Rodgers, A. L. and Garside, J. (1981). The nucleation and growth kinetics of calcium oxalate in the presence of some synthetic urine constituents. Invest. Urol., 18,484488. Ryall, R. L., Harnett, R. M. and Marshall, V. R. (1981). The effect of urine, pyrophosphate, citrate, magnesium and glucosaminoglycanson the growth and aggregation of calcium oxalate crystals in vitro. Clin. Chim. Acta, 112, 349-356. Schneider,H. J. (1985). Pathogenesisofurolithiasis.In Urolithiasis, Etiology, Diagnosis, ed. Schneider, H. J., Pp. 206-207. Berlin: Springer-Verlag.

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Skrtif, D., Fiiredi-Milhofer, H. and MarkoviC, M. (1987) Precipitation of calcium oxalate from high ionic strcngth solutions. VII. The influence of glutamic acid. J . C'rrml Growth, 80,113-120. von Smoluchowski, M. (1918). Mathematical theory of the kinetics of coagulation of colloidal systems. Z . Physik. C h m , 92,129-168.

The Authors F. Grases, PhD, Professor, Department of Chemistry. L. Mashova, PhD, Department of Inorganic and Organic Chemistry, Comenius University, Bratislava, Ciechoslovakia. 0. Sohnel, DSc, Department of Inorganic Processes, vs('hT Pardubice, Czechoslovakia. A. Costa-Bauzi, PhD, Department of Chemistry. Requests for reprints to: F. Grases, Department of Chemistry. Faculty of Sciences, University of Balearic Islands, 0707 1 Palma de Mallorca, Spain.

Agglomeration of calcium oxalate monohydrate in synthetic urine.

The development of agglomerated particles of calcium oxalate monohydrate (COM) on the semi-batch precipitation from a synthetic urine carried out at p...
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