Crystallization Kinetics Of Calcium Oxalate Research Articles

In vitro effects on calcium oxalate crystallization kinetics and crystal morphology of an aqueous extract from Ceterach officinarum: Analysis of a potential antilithiatic mechanism.

Ceterach officinarum Willd is a plant widespread throughout Europe and used in southern Italy as a diuretic. Beliefs in the benefits of C. officinarum aqueous extract in the treatment of calcium oxalate kidney stones are widely held. Little is known, however, about the actual mechanism of its antilithiatic action. Our results in this in vitro study corroborate C. officinarum aqueous extract as a good source of antioxidants with a high antioxidant effects. Our results also demonstrate a major impact of C. officinarum aqueous extract on in vitro induced calcium oxalate crystallization kinetics and crystal morphology, showing its critical role in kidney stone formation and/or elimination. We show that progressively increasing doses of C. officinarum aqueous extract cause a sequence of effects. A powerful inhibitory action on calcium oxalate monohydrate (COM) growth and aggregation is first observed. C. officinarum aqueous extract also appears highly effective in stimulating nucleation increasing the number and reducing the size of COM crystals, which become progressively thinner, rounded and concave in a dose-dependent manner. These shape-modified COM crystals are known to be less adherent to renal tubular cells and more easily excreted through the urinary tract preventing kidney stone formation. Further, C. officinarum aqueous extract promotes the formation of calcium oxalate dihydrate (COD) rather than the monohydrate so that, at the highest concentrations used, only COD crystals are observed, in significant greater numbers with a clear reduction in their size, in a dose-dependent manner. Furthermore, AFM analyses allowed us to reveal the presence of C. officinarum component(s) on the surfaces of COD and modified COM crystals. The crystal surface adsorbed component(s) are shown to be similarly active as the total aqueous extract, suggesting a trigger factor which may direct crystal modification towards COD forms. In urolithiasis pathogenesis COD crystals are less dangerous than the COM forms due to their lower affinity for renal tubular cells. Our results are important in understanding the mechanisms which guide the modification induced by C. officinarum on the crystallization process. Based on these data, together with no adverse toxic effect being observed on the in vitro model of human intestinal enterocytes, C. officinarum aqueous extract could represent an attractive natural therapy for the treatment of urolithiasis.

Thermodynamics and kinetics of calcium oxalate crystallization in the presence of amino acids

Specific features of the crystallization of calcium oxalates (CaC2O4) in the presence of amino acids have been established based on thermodynamic and experimental modeling. Phase formation in the Ca2+–C2O4 2-–H2O–amino acid system in a wide range of variation in the component concentrations and solution pH have been theoretically investigated. The influence of pH on the thermodynamic stability of crystallizing compounds is considered. The effect of amino acids of different structures on the formation of the CaC2O4 solid phase in a prototype of physiological solution is analyzed. The kinetic crystallization parameters (induction period and rate constant) and crystallite growth are determined.

Crystallization Kinetics of CaOx in Artificial Urine and in Saline System

The crystallization kinetics of calcium oxalate (CaOx) was comparatively studied by detecting the change of free Ca2+ ions concentration with the reaction time in artificial urine and in saline system. The dynamics equations of CaOx crystallization was r=kcα, and the average reaction order (α) was 3.3 regardless of the relative suprasaturation degree (RS) of CaOx in the range of RS=10.58~17.53. The average reaction rate constant (κ) was (0.97±0.1)×109 in artificial urine and κ=(3.1±1.8)×109 in saline system, due to the presence of inhibitors to CaOx crystallization in artificial urine.

Effect of Unitary, Binary and Ternary Carboxylates on Crystallization Kinetics of Calcium Oxalate in Artificial Urine

Effect of Unitary, Binary and Ternary Carboxylates on Crystallization Kinetics of Calcium Oxalate in Artificial Urine

Effects of an aqueous extract fromAmmodaucus leucotrichuson calcium oxalate crystallizationin vitro

This work focuses on the behavior of in vitro calcium oxalate crystallization. The effects of plant ammodocus on the kinetics of calcium oxalate crystallization were examined. The synthetic urine was prepared immediately before use by mixing with a T-type mixing chamber equal volumes of solutions A and B. Both solutions were prepared by dissolving chemicals of reagent-grade purity in deionised and redistilled water. A: Na2C2O4 (2 m mol/l) B: CaCl22H2O (10 m mol/l). Mixture agitation was maintained to prevent sedimentation. The crystal size development was monitored by polarized microscopy at different time intervals by proceeding as follows: Sample drops were examined every five minutes by polarising optical microscopy. We followed the same experimental procedure for the study of crystallization in the presence of inhibitors. We focussed on crystallisation because it may be the nucleating agent for many stones. As shown with ammodocus, the inhibitory effect was not dose dependent; the best inhibitory concentrations (97.25% and 97.85%) was observed at concentration of [75%] and [100%] mM/L, respectively. The inhibitory effect of ammodocus plant involved growth crystal phases. The growth and agglomeration of calcium oxalate crystals are differently modulated by Ammodocus leucotrichus.

Impact of various modifiers on calcium oxalate crystallization

This work focuses on the behavior of in vitro calcium oxalate crystallization. The effects of several compounds on the kinetics of calcium oxalate crystallization were examined. Rates of nucleation and aggregation of calcium oxalate crystals were derived from 30-min time-course measurements of optic density at 620 nm after mixing solutions containing calcium chloride and sodium oxalate at 37 degrees C, pH 5.7. The maximum increase of optic density with time, termed S(N), mainly reflects maximum rate of formation of new particles and thus crystal nucleation. After equilibrium has been reached, optic density decreases. No new particles were formed due to crystal aggregation. S(A) (the maximum slope of decrease of optic density at 620 nm with time, representing crystal aggregation) is derived from the maximum decrease in optic density. Among the modifiers studied, citrate decreased both S(N) and S(A) (P < 0.001). Magnesium was also found to inhibit the rate of nucleation and crystal aggregation, but it appeared in a non-concentrated manner. Nucleation and aggregation inhibition ratios were related inversely to concentration of albumin (P < 0.001). The growth and agglomeration of calcium oxalate crystals are differently modulated by various compounds. The treatments aiming at inhibiting crystallization of calcium oxalate can be better defined by these findings. And new treatment modalities can be developed.

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.

Decreased Renal Expression of the Putative Calcium Oxalate Inhibitor Tamm-Horsfall Protein in the Ethylene Glycol Rat Model of Calcium Oxalate Urolithiasis.

Tamm-Horsfall protein is believed to inhibit calcium oxalate crystallization, aggregation or adhesion to the renal epithelium. We determined whether ethylene glycol induced urolithiasis changes the expression of renal and urinary Tamm-Horsfall protein. For comparison the expression of another calcium oxalate inhibitor, osteopontin, was also analyzed. Male rats were treated with 0.75% ethylene glycol plus an AIN-76 diet (Dyets, Bethlehem Pennsylvania) (ethylene glycol group) or standard rat chow and water (control group) for up to 8 weeks (6 per group for 8 weeks and 3 per group for 3 days to 6 weeks). Kidneys and urine (8 weeks only) were harvested and analyzed by Northern and Western blot analysis, and immunohistochemistry. Tamm-Horsfall protein message and protein (membrane bound form) were decreased, while those of osteopontin were increased in the kidneys of rats treated with ethylene glycol for 8 weeks. As judged by immunochemistry Tamm-Horsfall protein and osteopontin were consistently present in a few tubules in rats in the ethylene glycol and control groups, respectively. In urine expression of the free form of Tamm-Horsfall protein (approximately 75 kDa.) was decreased but detectable in ethylene glycol treated rats. Although readily detected in tissue, osteopontin was not detected in the urine of control or ethylene glycol treated rats. In the time course experiment Tamm-Horsfall protein did not decrease until 4 weeks, when calcium oxalate crystals were detectable in the kidneys of treated rats. In contrast, osteopontin was increased, although inconsistently, beginning at 3 days. Unlike other calcium oxalate inhibitors, such as osteopontin, renal message and protein for Tamm-Horsfall protein was decreased in ethylene glycol treated rats. Tamm-Horsfall protein expression did not decrease until aggregates of crystals had been deposited in the kidneys, while osteopontin expression began to increase almost immediately. Comparisons of the data on kidneys and urine obtained by RNA or protein blot analysis and immunochemistry underscore the need to examine tissue and urine by multiple techniques to obtain the most accurate assessment of how protein expression is changed by a given treatment.

Influence of Fluid Dynamic Conditions on the Crystallization Kinetics of Calcium Oxalate

Influence of Fluid Dynamic Conditions on the Crystallization Kinetics of Calcium Oxalate

Citrate determines calcium oxalate crystallization kinetics and crystal morphology-studies in the presence of Tamm-Horsfall protein of a healthy subject and a severely recurrent calcium stone former.

The aim of this study was to measure the effects of normal (nTHP) and abnormal stone former Tamm-Horsfall protein (SF-THP) on calcium oxalate (CaOx) nucleation and aggregation as well as on crystal morphology, in presence or absence of citrate. Nucleation and aggregation of CaOx crystals from a supersaturated, stirred solution (200 mM NaCl, 10 mM Na-acetate, pH 5.70, 5 mM Ca and 0.5 mM Ox) were studied by spectrophotometric time-course measurements of OD at 620 nm (OD(620)). Measured parameters were induction time t(I) (time to induce formation of detectable particles), S(N), (slope of increase of OD(620), mainly due to crystal nucleation), and S(A), (slope of decrease of OD(620) after equilibrium has been reached, due to crystal aggregation). Effects of citrate, nTHP and SF-THP on these parameters were measured, and scanning electron microscopy (SEM) was performed. At 1.5, 2.5 and 3.5 mM, citrate increased t(I) and inhibited crystal nucleation (by 78-87%) as well as aggregation (by 63-70%), and smaller CaOx crystals (length/width ratio 1.7+/-0.1) than under standard conditions (length/width 3.9+/-0.5) were visible (P<0.001). Normal THP at 30 and 40 mg/l inhibited crystal nucleation and, more strongly, aggregation (inhibition 76-81%). SEM revealed a decrease in length/width ratio to 2.6+/-0.4 (P=0.051 vs standard conditions) and less aggregation than without nTHP. At all concentrations tested, SF-THP reduced t(I) (P=0.0001 vs standard conditions) and promoted aggregation (inhibition -48 to -33%); crystals were elongated with a length/width ratio of 4.3+/-0.6 (P<0. 05 vs nTHP). When simultaneously present with nTHP, citrate enhanced the inhibitory effects of nTHP, producing the smallest (length/width 1.5+/-0.1) and least aggregated crystals. Finally, 3.5 mM citrate turned promotory SF-THP into a crystallization inhibitor with abundant small and clustered, but not aggregated crystals. Citrate appears to be the main determinant of CaOx crystallization rates and crystal morphology in the presence of nTHP as well as SF-THP. Its effects appear to predominate over those of THP, since even promotory SF-THP is turned into a crystallization inhibitor in the presence of citrate. This re-emphasizes at a morphological level what has been concluded from functional as well from clinical studies, namely that citrate is needed in urine at equimolar concentrations to calcium in order to prevent the formation of large crystal aggregates in presence of abnormal THP.

Calcium oxalate crystallization kinetics studied by oxalate-induced turbidity in fresh human urine and artificial urine

Calcium oxalate crystallization kinetics studied by oxalate-induced turbidity in fresh human urine and artificial urine

Calcium oxalate crystallization kinetics studied by oxalate-induced turbidity in fresh human urine and artificial urine

We have studied the kinetics of oxalate-induced turbidity in fresh human urine and artificial urine. Assays are performed in 96-well plates, which allows many oxalate concentrations to be studied, repeatedly, in a short time. The metastable limit is defined in terms of the lowest oxalate concentration that gives a rate of change of attenuance significantly greater than the control. Interpretation of rates above this limit is based on ln/ln plots of initial rates against added oxalate concentration. This approach has a good theoretical basis, is well supported by our results and gives a turbidity rate index that is related to the product of the growth rate constant and a factor relating to the number and characteristics of the heteronuclei responsible for initiation of crystallization. This interpretation is posited upon the assumptions that second-order crystallization kinetics occur in unseeded urine when supersaturation exceeds the metastable limit and that aggregation during the initial phase of crystallization does not significantly contribute to changes in turbidity. Metastable limits of urine from healthy volunteers corresponded to a calcium oxalate supersaturation ratio of approx. 10. The turbidity rate index was higher in human urine than in artificial urine. The metastable limit, based on either oxalate concentration or supersaturation, for induction of calcium oxalate crystallization in normal human urine is higher than is likely to be found in normal subjects in vivo. The shape of the relationship between the metastable limit (based on oxalate concentration) and calcium concentration emphasizes the benefit of achieving a low urine calcium concentration. Comparison of the turbidity rate indices for human and artificial urine suggests that the role of nucleation promoters is more dominant than that of growth inhibitors.

Calcium oxalate crystallization kinetics at different concentrations of human and artificial urine, with a constant calcium to oxalate ratio.

The effect of in vitro dilution of artificial urine or human urine on the crystallization of calcium oxalate was examined in a mixed suspension, mixed product removal crystallization system. Direct growth inhibition by components of artificial urine was not significant and supersaturation was the dominant factor in determining crystal nucleation and growth rates. Dilution of human urine caused a decrease in crystal growth rate that was independent of the input calcium and oxalate concentrations, suggesting that dilution of growth inhibitors could be physiologically more important than any reduction in supersaturation. This loss of growth inhibition was counteracted by a reduction in nucleation promotion, with the net effect that the mass of crystals declined. Correlation of crystallization measurements with urinary concentration (osmotic pressure) confirmed these observations, with a negative relationship for growth rate and a positive relationship for nucleation rate and suspension density. Increasing the concentration of urine shifts the crystallization balance from low nucleation/high growth to high nucleation/low growth. Calcium oxalate crystalluria in healthy urine is therefore less likely at early stages of urine development in the nephron and the likelihood can be further reduced by increased fluid output. Our results suggest that lowering the heterogeneous nucleation activity by dilution is more than sufficient to override the loss of growth inhibition.

Nucleation of calcium oxalate crystals by albumin: Involvement in the prevention of stone formation

Urine is supersaturated in calcium oxalate, which means that it will contain calcium oxalate crystals that form spontaneously. Their size must be controlled to prevent retention in ducts and the eventual development of a lithiasis. This is achieved, in part, by specific inhibitors of crystal growth. We investigated whether promoters of crystal nucleation could also participate in that control, because for the same amount of salt that will precipitate from a supersaturated solution, increasing the number of crystals will decrease their average size and facilitate their elimination. Albumin was purified from commercial sources and from the urine of healthy subjects or idiopathic calcium stone formers. Its aggregation properties were characterized by biophysical and biochemical techniques. Albumin was then either attached to several supports or left free in solution and incubated in a metastable solution of calcium oxalate. Kinetics of calcium oxalate crystallization were determined by turbidimetry. The nature and efficiency of nucleation were measured by examining the type and number of neoformed crystals. Albumin, one of the most abundant proteins in urine, was a powerful nucleator of calcium oxalate crystals in vitro, with the polymers being more active than monomers. In addition, nucleation by albumin apparently led exclusively to the formation of calcium oxalate dihydrate crystals, whereas calcium oxalate monohydrate crystals were formed in the absence of albumin. An analysis of calcium oxalate crystals in urine showed that the dihydrate form was present in healthy subjects and stone formers, whereas the monohydrate, which is thermodynamically more stable and constitutes the core of most calcium oxalate stones, was present in stone formers only. Finally, urinary albumin purified from healthy subjects contained significantly more polymers and was a stronger promoter of calcium oxalate nucleation than albumin from idiopathic calcium stone formers. Promotion by albumin of calcium oxalate crystallization with specific formation of the dihydrate form might be protective, because with rapid nucleation of small crystals, the saturation levels fall; thus, larger crystal formation and aggregation with subsequent stone formation may be prevented. We believe that albumin may be an important factor of urine stability.

Determination of urinary calcium oxalate crystallization mechanisms and kinetics using flow cytometry.

This paper describes the application of flow cytometry to the determination of calcium oxalate crystallization kinetics and mechanisms in real urine. The technique has the unique advantage of simultaneously providing quantitative particle number-size distributions and qualitative data concerning particle structure and morphology. Twenty-four-hour urines from 10 healthy male subjects were treated with sodium oxalate and the ensuing crystallization of calcium oxalate was monitored by flow cytometry. In 7 specimens crystallization was accompanied by increasing size and by a fundamental change in particle morphology. This suggests that nucleation and aggregation occurred in these specimens. In the remaining 3 samples, increasing particle numbers occurred without any changes in size or morphology, indicating that nucleation was the sole mechanism in these specimens. These results demonstrate that flow cytometry is able to differentiate between nucleation, growth and aggregation mechanisms thereby making it an extremely useful analytical tool for stone researchers.

Crystallization Kinetics of Calcium Oxalate in Fresh, Minimally Diluted Urine: Comparison of Recurrent Stone Formers and Healthy Controls in A Continuous Mixed Suspension Mixed Product Removal Crystallizer

A reproducible method has been developed for studying calcium oxalate crystallization from fresh, minimally diluted (92%) urine with the mixed suspension mixed product removal continuous crystallization technique. All samples were adjusted to give the same starting calcium and oxalate concentrations. Twenty-one recurrent male stone formers were compared with twenty-two healthy controls. There was no difference in crystal growth rates but crystal nucleation rates were much higher in the control group (p = 0.003). Using growth rate and nucleation rate results, the amount of crystalline material in suspension was shown to be lower in the urine from stone formers, and therefore the equilibrium supersaturation in the crystallizer was lower in the control group (p = 0.001). We propose that the ability of a healthy person’s urine to maintain a lower supersaturation is a crucial protective factor distinguishing non-stone formers from stone formers.

Modulation of calcium oxalate monohydrate crystallization kinetics in vitro

The effects of several low and high molecular weight (mol wt) compounds on the kinetics of calcium oxalate crystallization were examined using a seeded crystal growth method in which the solubility, the growth and the agglomeration of calcium oxalate crystals were measured as three separate and system-independent parameters. Citrate, magnesium, phosphate, pyrophosphate, chondroitinsulphate, pentosanpolysulphate and heparin were tested in a wide range of concentrations. The solubility of calcium oxalate crystals was increased only by citrate and magnesium. The crystal growth was inhibited by all compounds tested, but those with the high mol wt had the greatest effect at low concentrations. In contrast, inhibition of crystal agglomeration was achieved only by the low mol wt compounds; citrate was found to be the most potent inhibitor at concentrations likely to be present in normal urine. The high mol wt substances, despite their potent crystal growth inhibitory activity, had no effect on agglomeration. The results show that growth and agglomeration of calcium oxalate crystals are separate processes which are differently modulated by various compounds. They further provide a possible explanation for the pathogenetic role of citrate in hypocitraturic renal stone disease.

Triamterene and renal stone formation: the influence of triamterene and triamterene stones on calcium oxalate crystallization.

A constant composition method has been used to compare the effects of triamterene renal stone material, synthetic triamterene precipitates, and soluble triamterene on the nucleation and crystallization kinetics of calcium oxalate in aqueous solution in vitro. Crystallization studies have been carried out with the concentrations of calcium and oxalate ions maintained constant by the potentiometrically controlled addition of concentrated reagent solutions containing these ions. Triamterene renal stones were found to be much less effective than synthetic triamterene towards promoting the nucleation and crystallization of calcium oxalate from supersaturated solution. Renal stones composed of triamterene and matrix did not significantly enhance the deposition of calcium oxalate compared to nonseeded controls. The triamterene stones were also found to be ineffective in promoting calcium oxalate crystallization compared to other precipitates thought to be involved in the etiology of stone disease such as calcium hydroxyapatite. For stones of mixed triamterene/calcium oxalate composition, the enhancement of the nucleation and crystallization of calcium oxalate was directly related to the calcium oxalate content of the stone seed material. The presence of soluble triamterene or its metabolites in solution did not influence the crystallization kinetics of pure calcium oxalate seed materials. The results of this study indicate that triamterene in stones does not significantly contribute to further stone development through the enhancement of calcium oxalate crystallization processes.

Effect of trace elements on the kinetics of calcium oxalate crystallization--research on urolithiatic inhibitors.

In order to find effective urolithiasis inhibiting factors, calcium oxalate stones were investigated by spectrum analysis. Of the composite trace elements nine were selected to test their effect on the kinetics of calcium oxalate crystallization in artificial and in urinary stone patient’s urine, and in experimental animals respectively. It was found that Mo and Sr could effectively inhibit calcium oxalate crystallization and decrease the renal total and crystalline calcium. The inhibitive effect of the trace elements, the mechanism of action, the required conditions and the way of their affecting the kinetics of calcium oxalate crystallization are discussed. Their clinical significance and the possibility of practical application are referred to.

Calcium oxalate crystal growth in normal urine: role of contraceptive hormones.

Oral contraceptives inhibited the growth of calcium oxalate crystals in female urine, and the growth rate inhibition depended on the dose of ethinyl-oestradiol in the oral contraceptive agent. The results suggest that the crystallization kinetics of calcium oxalate in urine could be under the control of oestrogenic hormones by an unidentified mechanism.