Calcium Oxalate Monohydrate Growth Research Articles

Calcineurin B inhibits calcium oxalate crystallization, growth and aggregation via its high calcium-affinity property

Calcineurin inhibitors (CNIs) are widely used in organ transplantation to suppress immunity and prevent allograft rejection. However, some transplant patients receiving CNIs have hypocitraturia, hyperoxaluria and kidney stone with unclear mechanism. We hypothesized that CNIs suppress activities of urinary calcineurin, which may serve as the stone inhibitor. This study aimed to investigate effects of calcineurin B (CNB) on calcium oxalate monohydrate (COM) stone formation. Sequence and structural analyses revealed that CNB contained four EF-hand (Ca2+-binding) domains, which are known to regulate Ca2+ homeostasis and likely to affect COM crystals. Various crystal assays revealed that CNB dramatically inhibited COM crystallization, crystal growth and crystal aggregation. At an equal amount, degrees of its inhibition against crystallization and crystal growth were slightly inferior to total urinary proteins (TUPs) from healthy subjects that are known to strongly inhibit COM stone formation. Surprisingly, its inhibitory effect against crystal aggregation was slightly superior to TUPs. While TUPs dramatically inhibited crystal-cell adhesion, CNB had no effect on this process. Ca2+-affinity assay revealed that CNB strongly bound Ca2+ at a comparable degree as of TUPs. These findings indicate that CNB serves as a novel inhibitor of COM crystallization, growth and aggregation via its high Ca2+-affinity property.

Factors Differentiating the Effectiveness of Polyprotic Acids as Inhibitors of Calcium Oxalate Crystallization in Kidney Stone Disease

Pathological crystallization of calcium oxalate monohydrate (COM) is a critical process in human kidney stone disease. Methods of curtailing COM growth involve the action of natural and synthetic polyprotic acids, which selectively bind to crystal surfaces and inhibit the anisotropic rates of growth. Here we use a combination of bulk crystallization, in situ scanning probe microscopy, density functional theory, and physiologically relevant in vitro assays to elucidate the molecular origins of COM growth inhibition, focusing on citrate (CA) and its molecular analogues hydroxycitrate (HCA) and isocitrate (ICA). These three molecules differ only in the number and/or placement of hydroxyl groups, yet each exhibit unique binding modes to COM crystal surfaces. A major breakthrough in the development of potential therapies came from the recent discovery of HCA being a potent inhibitor possessing an ability to dissolve crystal surfaces in supersaturated media. Using a combination of experiments and computational calculations, we examine the differentiating factors for isostructural analogues of citrate to prompt dissolution and introduce a vectorized displacement parameter that accounts for strain induced on a crystal lattice as a result of inhibitor adsorption. We also compare the inhibitory effects of all three polyprotic acids in a human urine assay that surprisingly revealed HCA to be a far superior inhibitor than laboratory estimates owing to an apparent synergistic cooperative effect with species in the urine milieu. Collectively, these findings identify new inhibitors of calcium oxalate crystallization and demonstrate that HCA and ICA are promising alternatives to citrate, the current therapy.

Elucidating the Effects of Polyprotic Acid Speciation in Calcium Oxalate Crystallization

Polyprotic acids tend to be very effective modifiers of crystals in synthetic, natural, and biological systems. Examples include calcium biomineralization where proteins and organic acids decorated with carboxylic acids act as inhibitors of crystal growth. For crystals implicated in pathological diseases, large variations in the pH of the growth medium can alter the speciation of polyprotic acids. This is particularly true for calcium minerals comprised of polyprotic counterions wherein changes in solute speciation affect supersaturation, and thus the kinetics of crystal growth. Here, we explore the combined effects of solute and modifier speciation, selecting calcium oxalate monohydrate (COM) as a representative system for calcification. COM is a major constituent of human kidney stones where crystallization in vivo occurs over a broad range of pH spanning 5–8. Common modifiers of COM and its solute (oxalate) are polyprotic acids. Few studies report the effects of oxalate speciation on COM growth. Moreover, it remains to be determined how pH influences the efficacy of polyprotic molecules used to inhibit COM growth, such as citrate (CA) and its molecular analogue hydroxycitrate (HCA). Here, we show that there is a dramatic reduction in the rate of COM growth in the lower limit of physiological pH, commensurate with the loss of oxalate net negative charge. Our findings reveal that CA and HCA exhibit dual modes of action as promoters and inhibitors of COM crystallization at low and high pH, respectively. We also observe distinct differences in the efficacy of each modifier and discuss how local changes in pH near charged crystal interfaces can have a marked impact on local supersaturation and the speciation of adsorbed modifiers. On the basis of our observations of COM crystallization, it is reasonable to expect that changes in pH, or more specifically the speciation of solute and modifier(s), could similarly impact the growth (and growth inhibition) of other crystals employing polyprotic acids.

Exploring calcium oxalate crystallization: a constant composition approach.

Crystal growth rates have been extensively studied in calcium oxalate monohydrate (COM) crystallization, because COM crystals are the principal component in most kidney stones. Constant composition methods are useful for studying growth rates, but fail to differentiate concurrent nucleation and aggregation events. A constant composition method coupled with particle size determinations that addresses this deficiency was previously published for a calcium phosphate system, and this method was extended to COM crystallization in this report. A seeded constant composition experiment was combined with particle size determination and a separate near-equilibrium aggregation experiment to separate effects of growth rate, nucleation, and aggregation in COM crystal formation and to test the effects of various inhibitors relevant to stone formation. With no inhibitors present, apparent COM growth rates were heavily influenced by secondary nucleation at low seed crystal additions, but growth-related aggregation increased at higher seed crystal densities. Among small molecule inhibitors, citrate demonstrated growth rate inhibition but enhanced growth-related aggregation, while magnesium did not affect COM crystallization. Polyanions (polyaspartate, polyglutamate, or osteopontin) showed strong growth rate inhibition, but large differences in nucleation and aggregation were observed. Polycations (polyarginine) did not affect COM crystal growth or aggregation. Mixtures of polyanions and polycations produced a complicated set of growth rate, nucleation, and aggregation behaviors. These experiments demonstrated the power of combining particle size determinations with constant composition experiments to fully characterize COM crystallization and to obtain detailed knowledge of inhibitor properties that will be critical to understanding kidney stone formation.

Natural Promoters of Calcium Oxalate Monohydrate Crystallization

Crystallization is often facilitated by modifiers that interact with specific crystal surfaces and mediate the anisotropic rate of growth. Natural and synthetic modifiers tend to function as growth inhibitors that hinder solute attachment and impede the advancement of layers on crystal surfaces. There are fewer examples of modifiers that operate as growth promoters, whereby modifier-crystal interactions accelerate the kinetic rate of crystallization. Here, we examine two proteins, lysozyme and lactoferrin, which are observed in the organic matrix of three types of pathological stones: renal, prostatic, and pancreatic stones. This work focuses on the role of these proteins in the crystallization of calcium oxalate monohydrate (COM), the most prominent constituent of human kidney stones. Using a combination of experimental techniques, we show that these proteins, which are rich in l-arginine and l-lysine amino acids, promote COM growth. The synthesis and testing of peptides derived from contiguous segments of lysozyme's primary amino acid sequence revealed subdomains within the protein that operate either as an inhibitor or promoter of COM growth, with the latter exhibiting efficacies that nearly match that of the protein. We observed that cationic proteins promote COM growth over a wide range of modifier concentration, which differs from calcification promoters in the literature that exhibit dual roles as promoters and inhibitors at low and high concentration, respectively. This seems to suggest a unique mechanism of action for lysozyme and lactoferrin. Possible explanations for their effects on COM growth and crystal habit are proposed on the basis of classical colloidal theories and the physicochemical properties of peptide subdomains, including the number and spatial location of charged or hydrogen-bonding moieties.

Specificity of Growth Inhibitors and their Cooperative Effects in Calcium Oxalate Monohydrate Crystallization

The molecular recognition and interactions governing site-specific adsorption of growth inhibitors on crystal surfaces can be tailored in order to control the anisotropic growth rates and physical properties of crystalline materials. Here we examine this phenomenon in calcium oxalate monohydrate (COM) crystallization, a model system of calcification with specific relevance for pathological mineralization. We analyzed the effect of three putative growth inhibitors--chondroitin sulfate, serum albumin, and transferrin--using analytical techniques capable of resolving inhibitor-crystal interactions from interfacial to bulk scales. We observed that each inhibitor alters surface growth by adsorbing on to distinct steps emanating from screw dislocations on COM surfaces. Binding of inhibitors to different crystallographic faces produced morphological modifications that are consistent with classical mechanisms of layer-by-layer crystal growth inhibition. The site-specific adsorption of inhibitors on COM surfaces was confirmed by bulk crystallization, fluorescent confocal microscopy, and atomic force microscopy. Kinetic studies of COM growth at varying inhibitor concentrations revealed marked differences in their efficacy and potency. Systematic analysis of inhibitor combinations, quantified via the combination index, identified various binary pairings capable of producing synergistic, additive, and antagonistic effects. Collectively, our investigation of physiologically relevant biomolecules suggests potential roles of COM inhibitors in pathological crystallization and provides guiding principles for biomimetic design of molecular modifiers for applications in crystal engineering.

Mimicking the Biomolecular Control of Calcium Oxalate Monohydrate Crystal Growth: Effect of Contiguous Glutamic Acids

Scanning confocal interference microscopy (SCIM) and molecular dynamics (MD) simulations were used to investigate the adsorption of the synthetic polypeptide poly(l-glutamic acid) (poly-glu) to calcium oxalate monohydrate (COM) crystals and its effect on COM formation. At low concentrations (1 μg/mL), poly-glu inhibits growth most effectively in ⟨001⟩ directions, indicating strong interactions of the polypeptide with {121} crystal faces. Growth in <010> directions was inhibited only marginally by 1 μg/mL poly-glu, while growth in <100> directions did not appear to be affected. This suggests that, at low concentrations, poly-glu inhibits lattice-ion addition to the faces of COM in the order {121} > {010} ≥ {100}. At high concentrations (6 μg/mL), poly-glu resulted in the formation of dumbbell-shaped crystals featuring concave troughs on the {100} faces. The effects on crystal growth indicate that, at high concentrations, poly-glu interacts with the faces of COM in the order {100} > {121} > {010}. This mirrors MD simulations, which predicted that poly-glu will adsorb to a {100} terrace plane (most calcium-rich) in preference to a {121} (oblique) riser plane but will adsorb to {121} riser plane in preference to an {010} terrace plane (least calcium-rich). The effects of different poly-glu concentration on COM growth (1-6 μg/mL) may be due to variations between the faces in terms of growth mechanism and/or (nano)roughness, which can affect surface energy. In addition, 1 μg/mL might not be adequate to reach the critical concentration for poly-glu to significantly pin step movement on {100} and {010} faces. Understanding the mechanisms involved in these processes is essential for the development of agents to reduce recurrence of kidney stone disease.

Citrate Modulates Calcium Oxalate Crystal Growth by Face-Specific Interactions

Because of its ability to inhibit the growth of calcium oxalate monohydrate (COM) crystals, citrate plays an important role in preventing the formation of kidney stones. To determine the mechanism of inhibition, we studied the citrate-COM interaction using a combination of microscopic and simulation techniques. Using scanning confocal interference microscopy, we found that addition of citrate preferentially inhibits crystal growth in <100> and, to a lesser extent, <001> directions, suggesting that citrate adsorbs to the faces of COM in the order {100} > {121} > {010}. Scanning electron microscopy showed that the resulting crystals are plate shaped, with large {100} faces and rounded ends. Molecular-dynamics simulations predicted, however, that citrate interacts with the faces of COM in a different order, i.e. {100} > {010} > {121}. Our simulations showed that citrate molecules align with the rows of Ca²⁺ ions on the {010} face but do not form close contacts, presumably because of electrostatic repulsion by the carboxylate groups that project from the Ca²⁺-rich plane. We propose that this weak interaction is responsible for citrate’s limited inhibition of COM growth in <010> directions. Overall, these findings indicate that electrostatic interactions with the Ca²⁺-rich faces of COM crystals are responsible for the growth-modulating properties of citrate.

Kinetics of Calcium Oxalate Crystal Growth in the Presence of Osteopontin Isoforms: An Analysis by Scanning Confocal Interference Microcopy

Proteins that inhibit the growth and aggregation of calcium oxalate crystals play important roles in the prevention of kidney stone disease. One such protein is osteopontin (OPN), which inhibits the formation of calcium oxalate monohydrate (COM) in a phosphorylation-dependent manner. To determine the role of phosphate groups in the inhibition of COM growth by OPN, we used scanning confocal interference microscopy to compare the effects of highly phosphorylated OPN from cow milk, less phosphorylated OPN from rat bone, and nonphosphorylated recombinant OPN. COM growth was measured in the principal crystallographic directions <001>, <010>, and <100>, representing lattice-ion addition to {121}, {010}, and {100} faces, respectively. While the shapes of growth curves were very consistent from crystal to crystal, absolute growth rates varied widely. To control for this, results were expressed as changes in the aspect ratios <010>/<001> and <100>/<001>. Compared to control, bone OPN increased <010>/<001> and had no effect on <100>/<001>; milk OPN had no effect on <010>/<001>and decreased <100>/<001>; recombinant OPN had no significant effect on either aspect ratio. These findings indicate that milk OPN interacts with COM crystal faces in order of preference {100} > {121} approximately {010}, whereas bone OPN interacts in order of preference {100} approximately {121} > {010}. As {100} is the most Ca(2+)-rich face of COM, while {010} is the least Ca(2+)-rich, it appears that the OPN-mediated inhibition of COM growth occurs through a nonspecific electrostatic interaction between Ca(2+) ions of the crystal and phosphate groups of the protein.

Molecular modulation of calcium oxalate crystallization

Calcium oxalate monohydrate (COM) is the primary constituent of the majority of renal stones. Osteopontin (OPN), an aspartic acid-rich urinary protein, and citrate, a much smaller molecule, are potent inhibitors of COM crystallization at levels present in normal urine. Current concepts of the role of site-specific interactions in crystallization derived from studies of biomineralization are reviewed to provide a context for understanding modulation of COM growth at a molecular level. Results from in situ atomic force microscopy (AFM) analyses of the effects of citrate and OPN on growth verified the critical role of site-specific interactions between these growth modulators and individual steps on COM crystal surfaces. Molecular modeling investigations of interactions of citrate with steps and faces on COM crystal surfaces provided links between the stereochemistry of interaction and the binding energy levels that underlie mechanisms of growth modification and changes in overall crystal morphology. The combination of in situ AFM and molecular modeling provides new knowledge that will aid rationale design of therapeutic agents for inhibition of stone formation.

Crystallization kinetics of calcium oxalate hydrates studied by scanning confocal interference microscopy

Scanning confocal interference microscopy (SCIM) is an optical technique that allows the visualization of structures below the limits of classical optical microscopy (⪡250 nm). This study represents the first use of SCIM to analyze the formation of calcium oxalate crystals, the major constituent of kidney stones. Crystals were nucleated and grown on the glass bottom of Petri dishes in the presence and absence of the polyelectrolyte inhibitor poly- l-aspartic acid (poly-asp). In the absence of poly-asp, monoclinic calcium oxalate monohydrate (COM) nucleated from {1 0 0} or {0 1 0} faces. The first observed particles were 70–120 nm in diameter and grew by a step-like progression in the [0 0 1] and [0 1 0] directions. Addition of poly-asp had several effects on calcium oxalate formation. First, the number of particles was increased, but their sizes were decreased. Second, the rate of COM growth in the [0 0 1] direction was decreased to a greater extent than the rate along [0 1 0]. Third, the formation of tetragonal calcium oxalate dihydrate (COD) crystals was favored. Fourth, the rates of COD growth along 〈1 1 0〉 and allied directions were decreased, whereas that parallel to 〈0 0 1〉 is increased. Sequences of highly resolved growth fronts show step displacement for COM and moving crystal edges for COD. Analysis of image sequences suggested that growth is strongly affected by competing and alternating processes, in which diffusion processes are rate-limiting and induce nonlinear growth. This study shows that SCIM is a powerful technique for the quantitative analysis of crystallization processes and for determining the mode of action of inhibitors.

Modulation of Calcium Oxalate Crystallization by Linear Aspartic Acid-Rich Peptides

Calcium oxalate monohydrate (COM) kidney stone formation is prevented in most humans by urinary crystallization inhibitors. Urinary osteopontin (OPN) is a prototype of the aspartic acid-rich proteins (AARP) that modulate biomineralization. Synthetic poly(aspartic acids) that resemble functional domains of AARPs provide surrogate molecules for exploring the role of AARPs in biomineralization. Effects of linear aspartic acid-rich peptides on COM growth kinetics and morphology were evaluated by the combination of constant composition (CC) analysis and atomic force microscopy (AFM). A spacer amino acid (either glycine or serine) was incorporated during synthesis after each group of 3 aspartic acids (DDD) in the 27-mer peptide sequences. Kinetic CC studies revealed that the DDD peptide with serine spacers (DDDS) was more than 30 times more effective in inhibiting COM crystal growth than the DDD peptide with glycine spacers (DDDG). AFM revealed changes in morphology on (010) and (-101) COM faces that were generally similar to those previously described for OPN and citrate, respectively. At comparable peptide levels, the effects of step pinning and reduced growth rate caused by DDDS were remarkably greater. In CC nucleation studies, DDDS caused a greater prolongation of induction periods than DDDG. Thus, nucleation studies link changes in interfacial energy caused by peptide adsorption to COM to the CC growth and AFM results. These studies indicate that, in addition to the number of acidic residues, the contributions of other amino acids to the conformation of DDD peptides are also important determinants of the inhibition of COM nucleation and growth.

Brewster angle microscopy of calcium oxalate monohydrate precipitation at phospholipid monolayer phase boundaries.

The precipitation of calcium oxalate monohydrate (COM) at phospholipid monolayers confined to the air/water interface is observed in situ with the aid of Brewster angle microscopy. COM crystals appear as bright objects that are easily identified and quantified to assess the effects of different conditions on crystallization. Crystal precipitation was monitored at monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in liquid condensed (LC) and liquid expanded (LE) phases. Within the LC phase, higher pressures reduce the incidence of crystallization at the interface, implying that within this phase precipitation is enhanced by higher compressibility or fluidity of the monolayer. Precipitation at biphasic LC/LE and LE/gas (G) monolayers was also studied. COM appears preferentially at phase boundaries of the DPPC LC/LE and LE/G monolayers. However, when an LC/LE phase boundary is created by two different phospholipids that are phase segregated, such as DPPC and 1,2-dimyristoyl-sn-glycero-3-phosphocholine, crystal formation occurs away from the interface within the DPPC LC phase. It is suggested that COM growth at phase boundaries is preferred only when there is molecular exchange between the phases.

Calcium Oxalate Monohydrate Precipitation at Phospholipid Monolayer Phase Boundaries

AbstractThe precipitation of calcium oxalate monohydrate (COM) was observed at biphasic phospholipid Langmuir monolayers with the aid of Brewster angle microscopy. COM appears preferentially at phase boundaries of a monolayer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a state of liquid expanded/liquid condensed coexistance. However, when the phase boundary is created by two different phospholipids that are phase segregated, such as DPPC and 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC), crystal formation occurs away from the interface. It is suggested that COM growth at phase boundaries is preferred only when there is molecular exchange between the phases.

Reduced crystallization inhibition by urine from women with nephrolithiasis

Human urine is known to inhibit growth, aggregation, nucleation, and cell adhesion of calcium oxalate monohydrate (COM) crystals, the main solid phase of human kidney stones. This study tested the hypothesis that low levels of inhibition are present in women with calcium oxalate stones and, therefore, could promote stone production. In 17 stone-forming women and 17 normal women matched in age within five years, inhibition by dialyzed urine proteins of COM growth and aggregation was examined, as well as whole urine upper limits of metastability (ULM) for COM and calcium phosphate (CaP) in relation to the corresponding supersaturation (SS). Compared to normals, patient urine showed a reduced ULM in relation to SS. In contrast to men, there was no difference in growth inhibition. Reduced CaP and CaOx ULM values in relation to SS are a characteristic of female stone formers. This defect could promote stones by facilitating crystal nucleation. Abnormal inhibition may well be a very important cause of human nephrolithiasis.

Role of biomolecules from human renal stone matrix on COM crystal growth.

Human renal calculi surgically removed from kidney stone patients were obtained and chemically analysed. Stones with CaOx (calcium oxalate) as the major component were washed in 0.15 M NaCl with gentle stirring for 48 h and then pulverised to a fine powder. The powder was extracted with 0.05 M EGTA, 1 mM PMSF and 1% beta-mercaptoethanol for 4 days at 4 degrees C, the suspensions and the supernatants obtained were filtered through an Amicon Model 200 apparatus (mol. wt. cut off of 10,000 daltons) under nitrogen at 40 p.s.i. and concentrated to a known volume. The method of Nakagawa et al. [7] was employed to study the ability of > 10 kDa fractions to influence COM growth using metastable solution of CaCl2 and Na2C2O4 containing traces of 14C-oxalic acid. Potent biomolecules having the ability to influence CaOx precipitation were subjected to isolation, purification and characterization. Standard biochemical procedures, e.g. ultracentrifugation, ion-exchange chromatography, molecular sieve chromatography and SDS-PAGE, etc., were employed. Results revealed that human renal calculi extract contains biomolecules that can inhibit as well as stimulate the growth of preformed COM (calcium oxalate monohydrate) crystals. Most potent stimulator of CaOx growth was found to have a molecular weight of 66 kDa.

Reduced crystallization inhibition by urine from men with nephrolithiasis

Human urine is known to inhibit growth, aggregation, nucleation, and cell adhesion of calcium oxalate monohydrate (COM) crystals, the main solid phase of human kidney stones. This study tests the hypothesis that low levels of inhibition are present in men with calcium oxalate stones and could therefore promote stone production. In 17 stone-forming men and 17 normal men that were matched in age to within five years, we studied the inhibition by dialyzed urine proteins of COM growth, aggregation, and binding to cultured BSC-1 renal cells, as well as whole urine upper limits of metastability (ULM) for COM and calcium phosphate (CaP) in relationship to the corresponding supersaturation (SS). Compared with normals, patient urine showed reduced COM growth inhibition and reduced ULM in relationship to SS. When individual defects were considered, 15 of the 17 patients were abnormal in one or more inhibition measurements. ULM and growth inhibition defects frequently coexisted. Reduced COM growth and CaP and CaOx ULM values in relationship to SS are a characteristic of male stone formers. Both defects could promote stones by facilitating crystal nucleation and growth. Abnormal inhibition may be a very important cause of human nephrolithiasis.

Quantification of Crystal Growth of Calcium Oxalate in Gel and its Modification by Urinary Constituents in a New Flow Model of Crystallization

Purpose To use a new flow model of crystallization to quantify the growth of calcium oxalate monohydrate (COM) in a gel matrix under simulated physiological conditions. Materials and Methods Artificial urine supersaturated in COM was conducted through channels containing a gel with COM seed crystals (flow rate: 0.5 ml. per minute; reaction time: 4 hours; 37 plus/minus 1C). A relative crystal growth parameter, Vcr, was quantified by discontinuous measurement of scattered light intensity by microphotometry. The effects on Vcr of varying a series of urinary parameters were investigated within the system. Results The following order of effectiveness in changing COM growth could be derived: dilution greater than oxalate greater than calcium greater than magnesium approximately citrate. Conclusions Drinking therapy and lowering of urinary oxalate excretion can be concluded to be the most promising means of preventing CaOx urolithiasis. The new crystallization model demonstrated here should be suitable to new applications in the investigation of urinary stone formation.

Crystal adsorption and growth slowing by nephrocalcin, albumin, and Tamm-Horsfall protein.

Urine inhibition of calcium oxalate monohydrate (COM) crystal growth (CG) seems due to a glycoprotein that contains gamma-carboxyglutamic acid and has been named nephrocalcin (NC); however, Tamm-Horsfall protein (THP) and albumin resemble NC and make its measurement and role uncertain. NC in urine is aggregated to molecular mass 64 kDa and higher, similar to albumin (64 kDa) and THP (87 kDa). Albumin and THP are calcium binding, albumin adsorbs to COM crystals, and THP has been described as an inhibitor of COM growth. Antisera to NC have cross-reacted with THP even though the NC was isolated from cultured renal cells. Here we have compared highly purified NC, THP, and albumin adsorption with COM crystals and CG inhibition; also we compared their patterns of cross-reactivities with a new antiserum against NC and a monoclonal antibody to THP. NC adsorbs to COM crystals, THP does not. Albumin and THP do not inhibit CG. Cross-reactivity of albumin and THP to the antiserum is slight by direct enzyme-linked immunosorbent assay and nonexistent by competitive ELISA; reaction of NC to the anti-THP monoclonal antibody is absent.

A technique for the study of growth rates of single crystals of sparingly soluble salts

A photomicroscopic technique has been adapted and implemented for the study of growth rates of single crystals of sparingly soluble salts. Experiments have been conducted to determine the growth of calcium oxalate monohydrate (COM) in an isothermal growth cell used in a continuous-flow system. The experimental apparatus permits the control of temperature and of supersaturation of the solution in which the crystals are grown. Growth rates of COM are found to be constant at a constant supersaturation and show little evidence of growth rate dispersion for the conditions tested. The addition of a growth modifier, L-glumatic acid, at a level of 100 ppm yields a statistically significant increase in COM growth rate. Preliminary results indicate that COM crystal growth obeys the general trend predicted by the theory of Burton, Cabrera, and Frank.