Journal of Colloid and Interface Science 256, 168–174 (2002) doi:10.1006/jcis.2002.8283 Aggregation and Dispersion Characteristics of Calcium Oxalate Monohydrate: Effect of Urinary Species Kimberly G. Christmas, ∗ Laurie B. Gower, ∗ Saeed R. Khan,† and Hassan El-Shall† ,1 ∗ Department of Materials Science and Engineering, and †Department of Pathology, University of Florida, Gainesville, Florida 32611 Received July 2, 2001; accepted February 2, 2002; published online April 10, 2002 In this research, screening and central composite experimental designs are used to determine the effect of various factors on the aggregation and dispersion characteristics of previously grown cal- cium oxalate monohydrate (COM) crystals in artificial urinary en- vironments of controlled variables. The variables examined are pH and calcium, oxalate, pyrophosphate, citrate, and protein concen- trations in ultrapure water and artificial urine. Optical density mea- surements, particle size analysis, optical microscopy, AFM force measurements, and protein adsorption have been used to assess the state of aggregation and dispersion of the COM crystals and to elucidate the mechanisms involved in such a complex system. The data indicate that our model protein, mucin, acts as a dispersant. This is attributed to steric hindrance resulting from the adsorbed mucoprotein. Oxalate, however, promotes aggregation. Interesting interactions between protein and oxalate along with protein and citrate are observed. Such interactions (synergistic or antagonistic) are found to depend on the concentrations of these species. Sur- face responses for these interactions are presented and discussed in this paper. In summary, solution, surface, and interface chemistries interact in a complex manner in the physiological environment to either inhibit or promote aggregation, and an understanding of such interactions may help determine and control the factors affecting kidney stone formation. C  2002 Elsevier Science (USA) Key Words: calcium oxalate; crystal aggregation and disper- sion; artificial urine; AFM force measurements; protein adsorption; citrate; oxalate; kidney stones; experimental design. INTRODUCTION The mechanisms responsible for the formation of kidney stones are not well understood. There are several hypotheses concerning stone formation, but few studies have been designed to critically test them. One possible mechanism is the forma- tion of aggregates in the nephron tubules. The urinary tract is a metastable solution with respect to several minerals, and hetero- geneous nucleation and growth of crystals does occur; however, the flow within the renal tract is sufficiently fast to excrete small crystals before they grow large enough to cause occlusion of the nephron tubules (1, 2). Thus aggregation of crystals, which in essence causes rapid growth of a material, is thought to be 1 To whom correspondence should be addressed. one of the major mechanisms causing stone formation (3). Since recurrent stone formation has a high recurrence rate (60–80%), altering the urinary environment may be a method to help pre- vent the recurrence of the formation of kidney stones. Calcium oxalate monohydrate (COM) is a primary constituent of kidney stone disease. Aggregates of COM and other mineral crystals (such as the dihydrate form of calcium oxalate, and various calcium phosphates), along with lipids and biopolymers, are found within kidney stones; but the aggregation of COM in particular is recognized as an important factor in kidney stone development (4, 5). Previous research on calcium oxalate has conflicting results as to the role of some urinary species. Citrate and urinary pro- teins are of particular interest in this study. For example, Kok et al. (1986) showed that low citrate concentration contributes to crystal aggregation in stone-formers (6). They also showed that at higher citrate concentrations, aggregation was inhibited (7). In contrast, Hess et al. (8) showed that citrate did not affect aggregation at various concentrations. However, it was shown that protein influenced aggregation depending on the pH and protein concentration. For instance, at pH 7.2, Tamm–Horsfall protein (THP) and nephrocalcin (NC) inhibited aggregation. On the other hand, at pH 5.7, THP and NC promoted aggregation. At low THP concentration, crystal growth was not affected by THP (9). Benkovic et al. showed that THP promotes aggrega- tion (10). In more recent work by Hess et al. (11), an increase in citrate (1.5–3.5 mM) caused an increase in inhibition of ag- gregation. In addition, THP with citrate enhanced the inhibitory effect of THP. In summary, there are conflicting conclusions about the role of various species such as citrate and protein in this complex system. Most of these cited experiments were performed by changing one variable at a time instead of changing several variables at the same time to understand the whole system. While this research methodology nicely shows trends with a given variable, a com- bination of variables can sometimes lead to synergistic or antag- onistic effects, which will not be discerned in this methodology. An advantage of experimental design is that multiple variables at different levels can be evaluated with a limited number of experiments (12). As mentioned above, these designs can lead to estimation of main and interaction effects of the variables. This could perhaps be a reason for the discrepancy in results 168 0021-9797/02 $35.00 C  2002 Elsevier Science (USA) All rights reserved.