Nucleation Of Calcium Oxalate Monohydrate Research Articles

Nucleation kinetics of calcium oxalate monohydrate as a function of pH, magnesium, and osteopontin concentration quantified with droplet microfluidics.

A droplet-based microfluidic platform is presented to study the nucleation kinetics of calcium oxalate monohydrate (COM), the most common constituent of kidney stones, while carefully monitoring the pseudo-polymorphic transitions. The precipitation kinetics of COM is studied as a function of supersaturation and pH as well as in the presence of inhibitors of stone formation, magnesium ions (Mg2+), and osteopontin (OPN). We rationalize the trends observed in the measured nucleation rates leveraging a solution chemistry model validated using isothermal solubility measurements. In equimolar calcium and oxalate ion concentrations with different buffer solutions, dramatically slower kinetics is observed at pH 6.0 compared to pHs 3.6 and 8.6. The addition of both Mg2+ and OPN to the solution slows down kinetics appreciably. Interestingly, complete nucleation inhibition is observed at significantly lower OPN, namely, 3.2 × 10-8 M, than Mg2+ concentrations, 0.875 × 10-4 M. The observed inhibition effect of OPN emphasizes the often-overlooked role of macromolecules on COM nucleation due to their low concentration presence in urine. Moreover, analysis of growth rates calculated from observed lag times suggests that inhibition in the presence of Mg2+ cannot be explained solely on altered supersaturation. The presented study highlights the potential of microfluidics in overcoming a major challenge in nephrolithiasis research, the overwhelming physiochemical complexity of urine.

Effect of amino acids and B-group vitamins on nucleation of calcium oxalate monohydrate crystals

Abstract Studied is the influence of amino acids (L-Asp, L-Arg, L-Thr) and B-group vitamins (B1, B6, B12) on the processes of nucleation and the value of surface energy of calcium oxalate monohydrate (COM) crystals. It is established that L-Asp and L-Arg molecules inhibit nucleation of COM, that increases their induction time tenfold in comparison with the one of the pure crystals. L-Thr amino acid is shown to promote the processes of COM nucleation and to diminish the induction time by two times. At the introduction of L-Asp and L-Arg molecules into the solution the degree of COM crystal growth inhibition is 32.0–90.2% and 10.2–86.0% respectively. It is found that B1 and B6 vitamins inhibit the growth of COM crystals practically completely (the inhibition degree exceeds 95%) and lead to 70-fold increase in the induction time of pure COM. The value of surface energy of COM crystals diminishes to 19.8–20.8 mJ/m2 at the introduction of the crystallization inhibitor molecules (L-Asp, L-Arg, B1, B6) into COM solution.

Direct Nanoscale Imaging of Calcium Oxalate Crystallization on Brushite Reveals the Mechanisms Underlying Stone Formation

A mixture of calcium oxalate and calcium phosphate is a source of chronic human disease, forming kidney stones. However, the mechanisms of pathological biomineralization and its modulation by natural inhibitors such as osteopontin (OPN) proteins are poorly defined at the nanoscale. Here, the in vitro formation of calcium oxalate monohydrate (COM) concretions having brushite nidi is observed using in situ atomic force microscopy (AFM) in a simulated acidic urinary milieu. We quantify the dissolution kinetics of the [101]Cc, [1̅00]Cc, and [101̅]Cc steps on the brushite (CaHPO4·2H2O, DCPD) (010) surfaces by oxalate, two urinary constituents. In support of clinical observations, we further demonstrate the inhibitory effect of phosphorylated OPN peptides on the step retreat rates through step-specific interactions, this in turn regulating the kinetics of COM nucleation and aggregation at the expense of brushite crystals by means of the interfacial mineral replacement reactions. The definition of respective roles for DCPD and OPN peptides thereby offers general insights concerning the control of kidney stone formation and the mechanisms through which aberrant crystallization kinetics is inhibited.

Aggregation of Calcium Phosphate and Oxalate Phases in the Formation of Renal Stones.

The majority of human kidney stones are comprised of multiple calcium oxalate monohydrate (COM) crystals encasing a calcium phosphate nucleus. The physiochemical mechanism of nephrolithiasis has not been well determined on the molecular level; this is crucial to the control and prevention of renal stone formation. This work investigates the role of phosphate ions on the formation of calcium oxalate stones; recent work has identified amorphous calcium phosphate (ACP) as a rapidly forming initial precursor to the formation of calcium phosphate minerals in vivo. The effect of phosphate on the nucleation of COM has been investigated using the constant composition (CC) method in combination with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Our findings indicate COM nucleation is strongly promoted by the presence of phosphate; this occurs at relatively low phosphate concentrations, undersaturated with respect to brushite (dicalcium phosphate dehydrate, DCPD) formation. The results show that ACP plays a crucial role in the nucleation of calcium oxalate stones by promoting the aggregation of amorphous calcium oxalate (ACO) precursors at early induction times. The coaggregations of ACP and ACO precursors induce the multiple-point nucleation of COM. These novel findings expand our knowledge of urinary stone development, providing potential targets for treating the condition at the molecular level.

Phosphorylation of Osteopontin Is Required for Inhibition of Calcium Oxalate Crystallization

Under near-physiological pH, temperature, and ionic strength, a kinetics constant composition (CC) method was used to examine the roles of phosphorylation of a 14 amino acid segment (DDVDDTDDSHQSDE) corresponding to potential crystal binding domains within the osteopontin (OPN) sequence. The phosphorylated 14-mer OPN peptide segment significantly inhibits both the nucleation and growth of calcium oxalate monohydrate (COM), inhibiting nucleation by markedly increasing induction times and delaying subsequent growth by at least 50% at concentrations less than 44 nM. Molecular modeling predicts that the doubly phosphorylated peptide binds much more strongly to both (-101) and (010) faces of COM. The estimated binding energies are, in part, consistent with the CC experimental observations. Circular dichroism spectroscopy indicates that phosphorylation does not result in conformational changes in the secondary peptide structure, suggesting that the local binding of negatively charged phosphate side chains to crystal faces controls growth inhibition. These in vitro results reveal that the interactions between phosphorylated peptide and COM crystal faces are predominantly electrostatic, further supporting the importance of macromolecules rich in anionic side chains in the inhibition of kidney stone formation. In addition, the phosphorylation-deficient form of this segment fails to inhibit COM crystal growth up to concentrations of 1450 nM. However, at sufficiently high concentrations, this nonphosphorylated segment promotes COM nucleation. Dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) results confirm that aggregation of the nonphosphorylated peptide segment takes place in solution above 900 nM when the aggregated peptide particles may exceed a well-defined minimum size to be effective crystallization promoters.

Templating the Nucleation of Calcium Oxalate Monohydrate via Micropatterned Self-Assembled Monolayers on Gold

Controlling both the nucleation and the growth of crystalline materials requires the confinement of a space wherein nucleation is induced and necessitates transport of ions to the newly formed nucleus. Micropatterned self-assembled monolayers (µP SAMs) of alkanethiolates on gold have been used at length to mimic natural organic templates as they are a facile way to create spatially constrained microenvironments for nucleation and consequently control ion flow to active nucleation sites. Here, we present µP SAMs of ω-functionalized alkanethiolates on gold as effective templating agents for the nucleation of calcium oxalate monohydrate (COM). COM crystals nucleate on carboxyl- and hydroxyl-terminated µP SAMs at a critical ion concentration regardless of growth solution pH. Mixed µP SAMs of mercaptohexadecane (MHD) and mercaptohexadecanoic acid (MHA) nucleated lower densities of COM while decreasing average crystal size. Shortening SAM chain length from MHA to mercaptoundecanoic acid (MUA) nucleated fewer COM crystals with poorly defined faces, while µP hydroxyl-terminated SAMs both lowered the nucleation density and increased the size of the COM crystals.

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.

Modifications on the morphology of synthetically-grown calcium oxalate crystals by crystal-associated proteins isolated from bean seed coats ( Phaseolus vulgaris)

Higher plants form crystals of calcium oxalate in well-controlled conditions. This control is performed in specialized cells by an organic matrix composed by soluble and insoluble water macromolecules. The water-soluble protein matrix associated with the calcium oxalate crystals from bean seed coat ( Phaseolus vulgaris) contains many polypeptides of different molecular masses. In this study, we observed that the water-soluble matrix macromolecules strongly inhibited the nucleation of calcium oxalate crystals (this inhibition was concentration dependent). Additionally, two proteins were isolated and purified from the water-soluble matrix with masses of 250 kDa called calcium oxalate macromolecule associated 1 (COMA1) and 27 kDa called calcium oxalate macromolecule associated 2 (COMA2). These proteins were also strong inhibitors of the nucleation of calcium oxalate monohydrate. On the other hand, we found that the modification in the morphology of calcium oxalate crystals due to the presence of water-soluble macromolecules (altogether) affected mainly the {1 2 0} face. Particularly, COMA1 and COMA2 separately had the same influence on the morphology of these crystals.

A study of primary nucleation of calcium oxalate monohydrate: II. Effect of urinary species

AbstractKidney stones consist of various organic and inorganic compounds. Calcium oxalate monohydrate (COM) is the main inorganic constituent of kidney stones. However, the mechanisms for the formation of calcium oxalate kidney stones are not well understood. In this regard, there are several hypotheses including nucleation, crystal growth and/or aggregation of formed COM crystals. The effect of some urinary species such as oxalate, calcium, citrate, and protein on nucleation and crystallization characteristics of COM is determined by measuring the weight of formed crystals and their size distributions under different chemical conditions, which simulate the urinary environment. Statistical experimental designs are used to determine the interaction effects among various factors. The data clearly show that oxalate and calcium promote nucleation and crystallization of COM. This is attributed to formation of a thermodynamically stable calcium oxalate monohydrate resulting from supersaturation. Citrate, however, inhibits nucleation and further crystal growth. These results are explained on the basis of the high affinity of citrate to combine with calcium to form soluble calcium citrate complexes. Thus, citrate competes with oxalate ion for binding to calcium cations. These conditions decrease the amount of free calcium ions available to form calcium oxalate crystals. In case of protein (mucin), however, the results suggest that no significant effect could be measured of mucin on nucleation and crystal growth. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

A study of primary nucleation of calcium oxalate monohydrate: II. Effect of urinary species

AbstractKidney stones consist of various organic and inorganic compounds. Calcium oxalate monohydrate (COM) is the main inorganic constituent of kidney stones. However, the mechanisms for the formation of calcium oxalate kidney stones are not well understood. In this regard, there are several hypotheses including nucleation, crystal growth and/or aggregation of formed COM crystals. The effect of some urinary species such as oxalate, calcium, citrate, and protein on nucleation and crystallization characteristics of COM is determined by measuring the weight of formed crystals and their size distributions under different chemical conditions, which simulate the urinary environment. Statistical experimental designs are used to determine the interaction effects among various factors. The data clearly show that oxalate and calcium promote nucleation and crystallization of COM. This is attributed to formation of a thermodynamically stable calcium oxalate monohydrate resulting from supersaturation. Citrate, however, inhibits nucleation and further crystal growth. These results are explained on the basis of the high affinity of citrate to combine with calcium to form soluble calcium citrate complexes. Thus, citrate competes with oxalate ion for binding to calcium cations. These conditions decrease the amount of free calcium ions available to form calcium oxalate crystals. In case of protein (mucin), however, the results suggest that no significant effect could be measured of mucin on nucleation and crystal growth. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

A study of primary nucleation of calcium oxalate monohydrate: I‐Effect of supersaturation

AbstractThere are various organic and inorganic constituents in kidney stones. Among them, calcium oxalate monohydrate (COM) is the primary inorganic constituent of kidney stones. However, the mechanisms of formation of kidney stones are not well understood. In this regard, a basic study is carried out for better understanding of nucleation, crystal growth and/or aggregation of formed COM crystals. The primary nucleation of calcium oxalate monohydrate is studied at the laboratory scale using turbidity measurements. Calcium chloride and potassium oxalate solutions are mixed and then added to a Turbidimeter tube for continuous recording of turbidity. Induction time (time to induce formation of detectable crystals) is estimated from time‐turbidity graphs. The effect of some urinary species, such as oxalate and calcium, on nucleation and crystallization characteristics of COM is determined by particle size distribution analysis, measuring weight of crystals and calculation of relative supersaturation. The classical nucleation theory is applied at high supersaturation ratios (SR) ranging from 1.6 to 2.2. The results indicate that nucleation rate increases with increasing supersaturation ratio from 0.81 × 1028 nuclei/cm3.sec at 1.6 SR, to 18.02 × 1028 nuclei/cm3.sec at 2.2 SR. On the other hand, free energy change and radius of critical nucleus are decreased as supersaturation ratio is increased. The nucleation rates are higher than those reported in literature. Such discrepancy is discussed on the bases of differences in experimental techniques. (© 2004 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Nucleation of calcium oxalate crystals on an imprinted polymer surface from pure aqueous solution and urine.

Calcium oxalate (CaOx) is the most common component of human kidney stones. Heterogeneous nucleation is regarded as the key mechanism in this process. In this study, we have used an imprinted 6-methacrylamidohexanoic acid/divinylbenzene co-polymer as a biomimetic surface to nucleate CaOx crystal formation. The polymer was imprinted with either calcium oxalate monohydrate (COM) or dihydrate (COD) template crystals. These were washed out of the polymer, which was then immersed in various test solutions. The test solutions were an aqueous solution of calcium chloride and sodium oxalate, artificial urine and a sample of real urine. Crystals that formed on the polymer surface were characterized by X-ray powder diffraction, Fourier transform infrared spectroscopy, atomic absorption spectroscopy and scanning electron microscopy. Results showed that in the aqueous solution the COM-imprinted polymer induced the nucleation of COM. The COD-imprinted polymer induced only trace amounts of COD crystallization, together with larger quantities of COM. In artificial and real urines, COM also specifically precipitated on the COM-imprinted surface. The results show that, at least to some extent, the imprinted polymers direct formation of their morphologically matched crystals. In the case of COD, however, it appears that either rapid hydrate transformation of COD to COM occurs, or the more stable COM polymorph is directly co-precipitated by the polymer. Our results support the hypothesis that heterogeneous nucleation plays a key role in CaOx stone formation and that the imprinted polymer model could provide an additional and superior diagnostic tool for stone researchers to assess stone-risk in urine.

Contribution of human uropontin to inhibition of calcium oxalate crystallization

Uropontin (UP) is known to inhibit the growth and nucleation of calcium oxalate monohydrate (COM) crystals, and it also impedes attachment of calcium oxalate crystals to cultured renal epithelial cells. However, its role in normal defense against renal crystallization, and in pathogenesis of nephrolithiasis is unclear. In this study we determined the effect of UP on aggregation of COM crystals as well as the inhibitory activity of UP on COM crystal growth and nucleation in a series of normal subjects, in order to assess the potential of UP as an important urinary inhibitor. The mean urinary excretion of UP measured by ELISA was 185 +/- 12 nmol/24 hr (mean +/- SEM) with a mean urine UP concentration of 131 +/- 13 nM. Uropontin isolated by immunoaffinity chromatography was a very potent inhibitor of COM crystal aggregation, with a mean UP concentration of 28 +/- 4 nM required for a 50% reduction in aggregation. The kDa for COM crystal growth inhibition determined from Langmuir type isotherms was 21 +/- 3 nM and the concentration required for 50% reduction in COM crystal growth rate was 16 +/- 2 nM. Inhibition of secondary nucleation was measured at a single concentration of 200 nM, which reduced the nucleation rate to 42 +/- 3% of control. Using a theoretical model of growth and aggregation inhibition at varying urine flow rates, we showed that inhibitory activity of UP would be significant for all subjects over a wide range of urine flow rates. Overall, UP is a potent inhibitor of COM aggregation as well as growth and nucleation. The urinary concentration of UP is in the range in which its contribution to inhibition of growth and aggregation are likely to be substantial. Thus, UP appears to be an important natural defense against renal crystallizations and nephrolithiasis.

Nucleation of calcium oxalate monohydrate: use of turbidity measurements and computer-assisted simulations in characterizing early events in crystal formation

To examine the nucleation of calcium oxalate monohydrate (COM) crystallization, we have determined the time interval from initial formation of a supersaturated COM solution to the first appearance of turbidity or crystals. The turbidity measurements were conducted with the unaided eye, whereas crystal formation was analyzed using an inverted and an image analysis system. The lag time kinetics were compared with previous nucleation studies using computer simulations with the Gibbs-Thomson nucleation equation and the parabolic growth rate law. The nucleation behavior can be described as homogeneous or heterogeneous depending on the range of initial relative supersaturation. The calculated interfacial energies for homogeneous and heterogeneous nucleation agreed rather well with earlier investigations. Moreover, this investigation provided the opportunity to verify the utility of our particle size distribution program for simulating precipitating systems, and the experimentally determined surface energy was obtained from simulations of the nucleation kinetics.

Triamterene and Renal Stone Formation

We investigated the influence of triamterene (TA), and its metabolites parahydroxytriamterene (PHTAS), and parahydroxytriamterene sulfate (PHTAS) on the nucleation and crystal growth of calcium oxalate monohydrate (COM), in supersaturated solution at 37C using a new constant composition technique. The spontaneous precipitation of COM is preceded by induction periods which decrease with increasing supersaturation. The addition of the triamterene seed materials substantially reduces these delay periods and induces the crystal growth of COM. Specific surface area and scanning electron microscopic results indicate that the seed materials act as sources for the heterogeneous nucleation of COM. In addition, the surface of the more crystalline PHTAS appears to offer sites from which COM crystals can develop as well formed rosettes. This evidence suggests that in addition to triamterene renal stone formation, TA and its metabolites may catalyze the precipitation of other stone forming minerals with which urines may be supersaturated.