Calcium Oxalate Monohydrate Crystal Growth Research Articles

The inhibitory effects of epigallocatechin-3-gallate on calcium oxalate monohydrate crystal growth, aggregation and crystal-cell adhesion

Epigallocatechin-3-gallate (EGCG), a predominant phytochemical in tea plant, has been reported to prevent kidney stone formation but with vague mechanism. We investigated modulatory effects of EGCG (at 0.1–100 µM) on calcium oxalate monohydrate (COM) crystals at various stages of kidney stone development. EGCG significantly increased crystal size (at 1–100 µM), but decreased crystal number (at 10–100 µM), resulting in unchanged crystal mass and volume. Interestingly, EGCG at 10–100 µM caused morphological change of the crystals from typical monoclinic prismatic to coffee-bean-like shape, which represented atypical/aberrant form of COM as confirmed by attenuated total reflection - Fourier transform infrared (ATR-FTIR) spectroscopy. EGCG at all concentrations significantly inhibited crystal growth in a concentration-dependent manner. However, only 100 µM and 10–100 µM of EGCG significantly inhibited crystal aggregation and crystal-cell adhesion, respectively. Immunofluorescence staining (without permeabilization) revealed that surface expression of heat shock protein 90 (HSP90) (a COM crystal receptor) on MDCK renal cells was significantly decreased by 10 µM EGCG, whereas other surface COM receptors (annexin A1, annexin A2, enolase 1 and ezrin) remained unchanged. Immunoblotting showed that 10 µM EGCG did not alter total level of HSP90 in MDCK cells, implicating that its decreased surface expression was due to translocation. Our data provide a piece of evidence explaining mechanism underlying the anti-lithiatic property of EGCG by inhibition of COM crystal growth, aggregation and crystal-cell adhesion via reduced surface expression of HSP90, which is an important COM crystal receptor.

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.

Effects of Escherichia coli and Proteus mirabilis on the Growth and Aggregation of Calcium Oxalate Crystal under Microaerobic Conditions.

Escherichia coli and Proteus mirabilis are common single- and polymicrobial urinary tract infections which can survive under various oxygen levels, including inside of stone matrices. Therefore, we aimed to investigate and compare the calcium oxalate monohydrate (COM) lithogenic activities including COM crystal growth and aggregation under microaerobic conditions of E. coli and P. mirabilis isolated from the same stone matrix. The crystal growth was analyzed at the delta crystal area while the crystal aggregation was analyzed as the number of crystal aggregates. The results showed that compared to blank control, E. coli, P. mirabilis and the co-culture of E. coli and P. mirabilis were able to significantly promote COM crystal growth under microaerobic conditions. Interestingly, the delta crystal area in the co-culture under microaerobic conditions was larger than that of E. coli alone and P. mirabilis alone. In addition, only P. mirabilis alone and the co-culture were able to significantly increase COM aggregates. This study demonstrated that single- and co-culture of E. coli and P. mirabilis could promote COM crystal growth and aggregation under microaerobic conditions. The co-culture of E. coli and P. mirabilis may provide the combination effect on COM crystal interactions. The bacterial surfaces and the important effects on bacteria-crystal interactions should be further evaluated.

Trigonelline prevents kidney stone formation processes by inhibiting calcium oxalate crystallization, growth and crystal-cell adhesion, and downregulating crystal receptors

Trigonelline is the second most abundant bioactive alkaloid found in coffee. It is classified as a phytoestrogen with similar structure as of estradiol and exhibits an estrogenic effect. A previous study has reported that fenugreek seed extract rich with trigonelline can reduce renal crystal deposition in ethylene glycol-induced nephrolithiatic rats. However, direct evidence of such anti-lithogenic effects of trigonelline and underlying mechanisms have not previously been reported. Our study therefore addressed the protective effects and mechanisms of trigonelline against kidney stone-forming processes using crystallization, crystal growth, aggregation and crystal-cell adhesion assays. Also, proteomics was applied to identify changes in receptors for calcium oxalate monohydrate (COM), the most common stone-forming crystal, on apical membranes of trigonelline-treated renal tubular cells. The analyses revealed that trigonelline significantly reduced COM crystal size, number and mass during crystallization. Additionally, trigonelline dose-dependently inhibited crystal growth and crystal-cell adhesion, but did not affect crystal aggregation. Mass spectrometric protein identification showed the smaller number of COM crystal receptors on apical membranes of the trigonelline-treated cells. Western blotting confirmed the decreased levels of some of these crystal receptors by trigonelline. These data highlight the protective mechanisms of trigonelline against kidney stone development by inhibiting COM crystallization, crystal growth and crystal-cell adhesion via downregulation of the crystal receptors on apical membranes of renal tubular cells.

Systematic analysis of modulating activities of native human urinary Tamm-Horsfall protein on calcium oxalate crystallization, growth, aggregation, crystal-cell adhesion and invasion through extracellular matrix

Functions of Tamm-Horsfall protein (THP), the most abundant human urinary protein, have been studied for decades. However, its precise roles in kidney stone formation remain controversial. In this study, we aimed to clarify the roles of native human urinary THP in calcium oxalate monohydrate (COM) kidney stone formation. THP was purified from the human urine by adsorption method using diatomaceous earth (DE). Its effects on stone formation processes, including COM crystallization, crystal growth, aggregation, crystal-cell adhesion and invasion through extracellular matrix (ECM), were examined. SDS-PAGE and Western blotting confirmed that DE adsorption yielded 84.9% purity of the native THP isolated from the human urine. Systematic analyses revealed that THP (at 0.4–40 μg/ml) concentration-dependently reduced COM crystal size but did not affect the crystal mass during initial crystallization. At later steps, THP concentration-dependently inhibited COM crystal growth and aggregation, and prevented crystal-cell adhesion only at 40 μg/ml. However, THP did not affect crystal invasion through the ECM. Sequence analysis revealed two large calcium-binding domains (residues 65–107 and 108–149) and three small oxalate-binding domains (residues 199–207, 361–368 and 601–609) in human THP. Immunofluorescence study confirmed the binding of THP to COM crystals. Analyses for calcium-affinity and/or oxalate-affinity demonstrated that THP exerted a high affinity with only calcium, not oxalate. Functional validation revealed that saturation of THP with calcium, not with oxalate, could abolish the inhibitory effects of THP on COM crystal growth, aggregation and crystal-cell adhesion. These data highlight the inhibitory roles of the native human urinary THP in COM crystal growth, aggregation and crystal-cell adhesion, which are the important processes for kidney stone formation. Such inhibitory effects of THP are most likely mediated via its high affinity with calcium ions.

Mechanistic Pathways for the Molecular Step Growth of Calcium Oxalate Monohydrate Crystal Revealed by In Situ Liquid-Phase Atomic Force Microscopy.

Calcium oxalate monohydrate (COM) crystal is the most common crystalline component of human kidney stones. The molecular-scale inhibitory mechanisms of COM crystal growth by urinary biomolecules such as citrate and osteopontin adsorbed onto the crystal surface are now well understood. However, the pathways by which dissolved calcium and oxalate ions are incorporated into the molecular step of the COM crystal surface, leading to COM crystal growth-a prerequisite to be elucidated for developing effective therapeutics to inhibit COM stones-remain unknown. Here, using in situ liquid-phase atomic microscopy along with a step kinetic model, we reveal the pathways of the calcium and oxalate ions into the COM molecular step via the growth speed analysis of the molecular steps with respect to their step width at the nanoscale. Our results show that, primarily, the ions are adsorbed onto the terrace of the crystal surface from the solution-the rate-controlling stage for the molecular step growth, i.e., COM crystal growth-and then diffuse over it and are eventually incorporated into the steps. This primary pathway of the ions is unaffected by the model peptide D-Asp6 adsorbed on the COM crystal surface, suggesting that urinary biomolecules will not alter the pathway. These new findings rendering an essential understanding of the fundamental growth mechanism of COM crystal at the nanoscale provide crucial insights beneficial to the development of effective therapeutics for COM kidney stones.

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.

Caffeine prevents kidney stone formation by translocation of apical surface annexin A1 crystal-binding protein into cytoplasm: In vitro evidence.

Recent large 3 cohorts have shown that caffeinated beverage consumption was associated with lower risk of kidney stone disease. However, its protective mechanisms remained unknown and had not been previously investigated. We thus evaluated protective effects of caffeine (1 μM–10 mM) on calcium oxalate monohydrate (COM) kidney stone formation, using crystallization, crystal growth, cell-crystal adhesion, Western blotting, and immunofluorescence assays. The results showed that caffeine reduced crystal number but, on the other hand, increased crystal size, resulting in unchanged crystal mass, consistent with crystal growth that was not affected by caffeine. However, caffeine significantly decreased crystal-binding capacity of MDCK renal tubular cells in a dose-dependent manner. Western blotting and immunofluorescence study of COM crystal-binding proteins revealed significantly decreased level of annexin A1 on apical surface and its translocation into cytoplasm of the caffeine-treated cells, but no significant changes in other COM crystal-binding proteins (annexin A2, α-enolase, HSP70, and HSP90) were observed. Moreover, caffeine decreased intracellular [Ca2+] but increased [Ca2+] secretory index. Taken together, our findings showed an in vitro evidence of the protective mechanism of caffeine against kidney stone formation via translocation of annexin A1 from apical surface into cytoplasm to reduce the crystal-binding capacity of renal tubular epithelial cells.

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.

In vitro evaluation of calcium oxalate monohydrate crystals influenced by Costus igneus aqueous extract

Calcium oxalate monohydrate (COM) crystals are frequently found in urinary calculi (stones). COM crystals were grown by the single diffusion gel growth technique and the inhibitory effects of aqueous extracts of leaves, stems and rhizome of Costus igneus on the growth of COM crystals were studied. With an increase in the concentration of aqueous extract of C. igneus, the weight of the formed crystals was gradually reduced from 2.15 to 0.07 g(leaves), 0.06 g (rhizome) and 0.03 g (stem). The crystals harvested from the COM were characterized by Fourier transform infrared spectroscopy to confirm the functional groups and powder X-ray diffraction analyses to confirm the crystalline phases of COM crystals. A scanning electron microscopy study confirmed that the morphology of the crystals changed from hexagonal (COM) to bipyramids of calcium oxalate dihydrate (COD). This study confirms that using aqueous extracts of stem and rhizome of C. igneus can promote the formation of COD crystals and reduce the nucleation rate of COM crystals, a major component of calcium urinary stones.

Gallotannin Suppresses Calcium Oxalate Crystal Binding and Oxalate-Induced Oxidative Stress in Renal Epithelial Cells

Calcium oxalate monohydrate (COM) crystals bind avidly to the surface of proliferating and migrating renal endothelial cells, perhaps a key event in kidney stone formation. Oxalate-induced pre-oxidative stress can further promote crystal attachment cells. Natural products including gallotannins found in green teas have been studied as potentially novel treatments to prevent crystal retention and kidney stone formation. Gallotannin significantly inhibited COM crystal growth and binding to Madin-Darby Canine Kidney Cells type I (MDCK I) renal epithelial cells at non-toxic concentrations. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that gallotannin significantly attenuated oxalate-induced mRNA and protein expressions of monocyte chemoattractant protein 1 (MCP-1), osteopontin (OPN), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit p22(phox) and p47(phox) in human primary renal epithelial cells (HRCs). Gallotannin also reduced the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) as well as enhanced antioxidant enzyme superoxide dismutase (SOD) activity in oxalate treated HRCs. Taken together, our findings suggest that gallotannin can contribute to nephrolithiasis prevention via direct effects on renal epithelial cells including suppression of COM binding and MCP-1 and OPN expression, along with augmenting antioxidant activity.

Renal tubular cell membranes inhibit growth but promote aggregation of calcium oxalate monohydrate crystals

Cell membranes have been proposed to serve as promoters for calcium oxalate monohydrate (COM) kidney stone formation. However, direct evidence to demonstrate the modulatory effects of renal tubular cell membranes on COM crystals does not currently exist. We thus examined the effects of intact MDCK cells and their fragmented membranes on COM crystal growth, aggregation and transformation. COM crystals were generated in the absence (control) or presence of intact MDCK cells or their membrane fragments. Intact MDCK cells and their membrane fragments significantly inhibited COM crystal growth (22.6% and 25.2% decreases in size, respectively) and significantly reduced COM total crystal mass (23.1% and 25.6% decreases, respectively). In contrast, both of them markedly promoted crystal aggregation (1.9-fold and 3.2-fold increases, respectively). Moreover, both intact cells and membrane fragments could transform COM to calcium oxalate dihydrate (COD) crystals. Finally, COM crystal growth inhibitory activities of both membrane forms were successfully confirmed by a spectrophotometric oxalate-depletion assay. Our data provide the first direct evidence to demonstrate the dual modulatory effects of MDCK membranes on COM crystals. Although growth of individual COM crystals was inhibited, their aggregation was promoted. These findings provide additional insights into the mechanisms of COM kidney stone formation.

Red Blood Cell Membrane Fragments but Not Intact Red Blood Cells Promote Calcium Oxalate Monohydrate Crystal Growth and Aggregation

Cell membranes are thought to promote calcium oxalate kidney stone formation but to our knowledge the modulating effect of red blood cell membranes on calcium oxalate crystals has not been previously investigated. Thus, we examined the effects of red blood cell membrane fragments on calcium oxalate monohydrate and calcium oxalate dihydrate crystal growth and aggregation. Calcium oxalate monohydrate and calcium oxalate dihydrate crystals were treated with red blood cell membrane fragments or intact red blood cells from a healthy donor. Phase contrast microscopy was performed to evaluate crystal morphology and aggregation. We used ImageMaster 2D Platinum software to evaluate crystal size and spectrophotometric oxalate depletion assay to monitor crystal growth. Red blood cell membrane fragments had significant promoting activity on calcium oxalate monohydrate crystal growth with an approximately 75% increase in size and aggregation with an approximately 2.5-fold increase in aggregate number compared to the control without membrane fragments or cells. Approximately 50% of calcium oxalate monohydrate crystals were adhered by red blood cell membrane fragments. Intact red blood cells had no significant effect on calcium oxalate monohydrate crystal growth or aggregation but they could transform calcium oxalate monohydrate to calcium oxalate dihydrate crystals. Red blood cell membrane fragments and intact red blood cells had no effect on calcium oxalate dihydrate crystals. The promoting activity of red blood cell membrane fragments on calcium oxalate monohydrate crystal growth was successfully confirmed by spectrophotometric oxalate depletion assay. To our knowledge our data provide the first direct evidence that red blood cell membrane fragments are a promoting factor for calcium oxalate monohydrate crystal growth and aggregation. Thus, they may aggravate calcium oxalate stone formation.

The effect of Tamm-Horsfall Mucoprotein on the nucleation, growth and aggregation of calcium oxalate monohydrate crystals in vitro

Objective To determine the effect of Tamm-Horsfall Mucoprotein (THM) on the nucleation, growth and aggregation of calcium oxalate monohydrate crystals (COM) in vitro. Methods THM was isolated from normal individuals according to the method of Tamm and Horsfall with a little modification. The concentration of calcium and the amount of COM crystals were determined in the artificial urine and seed crystal system with 0,10,50,100 mg/L THM. Results Under the concentrations of THM being 0,10,50,100 mg/L,the reductions of the concentration of calcium were 0.81,0.57,0.51,0.96 mmol/L; the amounts of nucleation were 3. 20 × 10~4,2. 32×x 10~4 ,1. 83× 10~4, 2. 85×10~4; the growth rate was 53.9% ,48.7% ,34.6% ,19. 8% ;the aggregation rate was 33. 2% ,11.7% ,9.4% ,and 66. 2% respectively. There was significant differences,P<0. 01. Conclusion THM could prevent the calcium oxalate crystals from nucleating,growing and aggregating with the physiological concentration range,but when the concentration of THM got a top limit of the physiological concentration,THM became a promoter of nucleating and aggregating of calcium oxalate crystals. Key words: Calcium oxalate; Mucoprotein; Growth; Aggregation

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.

Urinary Trefoil Factor 1 is a Novel Potent Inhibitor of Calcium Oxalate Crystal Growth and Aggregation

Crystal growth and aggregation are the important mechanisms of calcium oxalate stone formation in the kidney. Recently we successfully purified trefoil factor 1 from human urine and used an oxalate depletion assay to indirectly infer its inhibitory activity against calcium oxalate crystal growth. We searched for direct evidence to define the inhibitory activity of urinary trefoil factor 1 against calcium oxalate crystal growth. Moreover, we also evaluated whether urinary trefoil factor 1 has any effects on calcium oxalate crystal aggregation and transformation. Isolated and aggregated forms of calcium oxalate monohydrate crystals were produced in the absence or presence of 7, 70 and 700 ng/ml urinary trefoil factor 1, nephrocalcin as a positive control or lysozyme (Sigma-Aldrich) as a negative control. The data clearly indicated that urinary trefoil factor 1 and nephrocalcin at physiological levels could effectively inhibit calcium oxalate monohydrate crystal growth and aggregation, whereas lysozyme did not affect the growth and aggregation of calcium oxalate monohydrate crystals. At a supraphysiological concentration of 4 microg/ml urinary trefoil factor 1 and nephrocalcin could transform calcium oxalate monohydrate crystals to the dihydrate type, which has much less adsorptive capability. To our knowledge these data provide the first direct evidence that urinary trefoil factor 1 is a novel potent inhibitor of calcium oxalate crystal growth and aggregation, and can transform calcium oxalate monohydrate crystals to the dihydrate type.

Circular patterns of calcium oxalate crystals induced by defective Langmuir-Blodgett film

The injury of the renal epithelial cell membrane can promote the nucleation of nascent crystals, as well as adhesion of crystals on it. It thus accelerates the formation of renal calculi. In this paper, the defective Langmuir-Blodgett (LB) films were used as a model system to simulate the injured renal epithelial cell membrane. The microcosmic structure of the defective LB film and the molecular mechanism of the effect of this film on nucleation, growth, deposited patterns and adhesion of calcium oxalate monohydrate (COM) were investigated. The circular defective domains were formed in dipalmitoylphosphatidylcholine (DPPC) LB film after the film was treated by potassium oxalate. These domains could induce ring-shaped patterns of COM crystals. In comparison, the LB film without pretreatment by potassium oxalate only induced random growth of hexagonal COM crystals. As the crystallization time increased, the size of COM crystals in the patterns increased, the crystal patterns changed from empty circles to solid circles, and the number of the circular patterns with small size (5–20 μm) increased. The results would shed light on the molecular mechanism of urolithiasis induced by injury of the renal epithelial membrane at the molecular and supramolecular level.

Effect of Concentration of Structurally‐Different Carboxylic Acids on Growth and Aggregation of Calcium Oxalate in Gel Systems

AbstractThe effect of concentration of structurally‐different carboxylic acids such as ethylene diamine tetraacetic acid (H4edta), citric acid (H3cit), tartaric acid (H2tart), and acetic acid (HOAc) on growth and aggregation of calcium oxalate (CaOxa) in gel systems was comparatively investigated. H2tart and H3cit could change the morphology of calcium oxalate monohydrate (COM) and induce the formation of calcium oxalate dihydrate (COD). H4edta could induce the formation of COD at a lower concentration of 0.33 mmol/L and have the strongest ability to inhibit aggregation of COM. HOAc inhibited COM aggregation only at a higher concentration than 500 mmol/L. With increasing the number of carboxylic groups in an acid or increasing the concentration of carboxylic acid, the capacity of this acid to induce COD formation and to inhibit growth and aggregation of COM crystals increased. That is, this capacity followed the order:H4edta&gt;H3cit&gt;H2tart&gt;&gt;HOAc. The result in this work suggested that the presence of H3cit and H2tart in urine played a role in the natural defense against stone formation.

The Role of Escherichia coliform in the Biomineralization of Calcium Oxalate Crystals

AbstractThe influence of the common bacterium Escherichia coliform (E. coli) on the nucleation and growth of calcium oxalate (CaOxa) in aqueous solution is studied in order to determine its role in the biomineralization of urinary stones. The results show that CaOxa crystals obtained in the presence of E. coli transform more quickly from calcium oxalate dihydrate (COD) into calcium oxalate monohydrate (COM), and they become even larger, than in the absence of the bacterium. On decreasing the concentration of chemical reagents, in other words increasing the ratio bacterium/CaOxa, except for the more rapid phase transition from COD to COM, the CaOxa particles obtained become larger and more regular with a hexagonal morphology. This suggests that E. coli accelerates the crystallization of COM, which is the most stable crystal phase of CaOxa and the major component of urinary stones. Transmission electron microscopy (TEM) images, electron diffraction (ED) patterns, and Fourier‐transform IR (FTIR) spectra show that the biomineralization process of CaOxa crystals takes place both inside and outside the bacterium, which implies that it is induced by both intracellular biomolecules and the bioorganic secretions of the bacteria. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) data and UV/Vis spectra show that the proteins bound to the crystal surface with a molecular weight of around 26.5 kDa have tyrosine, tryptophan, or phenylalanine residues. The zeta potential and FTIR spectra are used to investigate the mineralization mechanism of CaOxa with E. coli and show that, the negatively charged biomolecules inside and outside the bacterium interact with Ca2+ ions to provide nucleation sites and then act as modifiers to induce the nucleation, growth, and aggregation of COM crystals. Therefore, we infer that E. coli mineralizes calcium oxalate and plays a role in the formation of urinary stones.(© Wiley‐VCH Verlag GmbH &amp; Co. KGaA, 69451 Weinheim, Germany, 2007)

Growth of calcium oxalate crystals induced by complex films containing biomolecules

AbstractNucleation and growth of calcium oxalate (CaC2O4) crystals induced by films composed of phosphatidylcholine (PC), cholesterol (CS) and human serum albumin (HSA), and of PC, CS and dextran have been carried out. The products obtained were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy and ultraviolet‐visible spectroscopy. The results indicate that hexagonal calcium oxalate monohydrate (COM) and club‐shaped calcium oxalate trihydrate (COT) crystals are obtained on the PC/CH/HSA film, and the microstructure and properties of the PC/CH/HSA film depend on the weight ratio of PC to CS. With an increase in the PC‐to‐CS ratio, the number of COM crystals decreases gradually, and finally disappear, suggesting that PC inhibits the growth of COM crystals. On the PC/CS/dextran film, irregular COM and COT crystals are formed. The possible formation mechanisms of CaC2O4 on the two complex films are discussed. (© 2007 WILEY‐VCH Verlag GmbH &amp; Co. KGaA, Weinheim)