John P. Kavanagh

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

Abstract 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.

Does Fish Oil Benefit Stone Formers

The possibility that dietary fish oil supplementation may benefit patients with hypercalciuric urolithiasis by decreasing calcium excretion and enhancing protective mechanisms has been studied in rats and humans. In experiments on rats in metabolic cages, fish oil inhibited experimental nephrocalcinosis induced by intraperitoneal calcium gluconate. There were no significant changes in urinary biochemistry. In a clinical study on 18 hypercalciuric recurrent stone patients fish oil significantly decreased urinary calcium excretion. This effect was accompanied by decreases in the excretion of magnesium and citrate. Oxalate excretion and urinary fibrinolytic activity were unchanged. Overall, fish oil had a limited impact on the risk profile for recurrent urolithiasis.

In vitro calcium oxalate crystallisation methods

In vitro calcium oxalate crystallisation has been, and will continue to be, of fundamental importance to urolithiasis research. Many different methods have been employed which differ qualitatively and quantitatively in the extent that they reproduce aspects of the renal system or in their ability to distinguish different aspects of crystallisation activity. Whatever system is used there are three key aspects that are worth bearing in mind. Firstly, a major controlling factor will be the prevailing supersaturation and other physicochemical considerations, secondly, during the course of the reaction different processes may come into play and thirdly, the processes we are trying to model take place in a dynamic biological environment. Different approaches to the study of crystallisation can be classified in many ways, such as the process or analytical technique but at a more fundamental level it is helpful to focus on the changes in supersaturation during the course of the reaction. A steady state supersaturation is more likely to be representative of the intra-renal situation than a system which decays to the equilibrium position. The constant composition method and the mixed suspension mixed product removal method both achieve a steady supersaturation.

Supersaturation and renal precipitation: the key to stone formation?

A number of different crystallisation processes can be distinguished. Much emphasis is attached to nucleation, growth and aggregation. Generally, nucleation involves a foreign surface (heterogeneous nucleation). Homogeneous nucleation will only occur under carefully controlled conditions and will not apply to crystallisation within the renal system. The supersaturation required for homogeneous nucleation is much higher than the equilibrium saturation and foreign particles can act as nucleation catalysts, thus reducing the supersaturation required to initiate crystallisation. Nevertheless, there is still a supersaturation barrier that must be overcome before nucleation can occur. This enables metastable supersaturated solutions to exist in which nuclei do not readily form, but if crystals are present then they will grow (Fig. 1). Growth in the context of crystallisation is usually taken to mean enlargement of crystals by direct incorporation of solution species into the solid crystal lattice. The rate at which this takes place is also dependent on supersaturation and in the case of calcium oxalate the relationship is described by second order kinetics:

Why does the Bonn Risk Index discriminate between calcium oxalate stone formers and healthy controls

PURPOSE The BRI has been shown to discriminate between calcium oxalate stone formers and controls. BRI is the ratio of the concentration of ionized calcium and the amount of oxalate that must be added to 200 ml urine to initiate crystallization. Higher BRI values are predictive of being a stone former and a value of 1.0 has been found to be the cutoff value to distinguish stone formers and controls. It is not easy to present a consistent argument based on the thermodynamics of calcium oxalate crystallization to account for the success of this index. For instance, why should 2 samples sharing the same BRI but with different ionized calcium and oxalate values have the same likelihood of being obtained from a stone former? MATERIALS AND METHODS Using data on 195 samples the distribution and interrelationships of measured variables were examined. They were used to calculate illustrative data with which it was possible to examine the effects of varying the parameters and their relationships. RESULTS Data simulations identified 3 necessary and sufficient conditions that must be met for BRI to be an effective discriminator between stone former and nonstone former urine samples. CONCLUSIONS The success of BRI can be explained as the natural outcome of there being significantly different distributions (stone formers vs nonstone formers) of the concentration of ionized calcium and the formation product minus activity product difference as well as the correlation between these 2 variables.

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 persons urine to maintain a lower supersaturation is a crucial protective factor distinguishing non-stone formers from stone formers.

Lessons from a Stone Farm

The stone farm is a system for measuring macroscopic stone growth of 12 calcium stones simultaneously. It is based on mixed suspension, mixed product removal continuous crystallization principles and the stones are grown continuously for about 500 hours or more. The growth of the stones follows a surface area dependent pattern and the growth rate constants are very similar irrespective of whether the stating materials are fragments of human stone or pieces of marble chip. Increasing citrate from 2mM to 6mM caused a significant growth inhibition which persisted in the presence of urinary macromolecules. Phytate was a very effective inhibitor (about 50% at sub‐μM concentrations) but the effective concentration was increased by an order of magnitude in the presence of urinary macromolecules. The effective concentration for inhibition in a crystallization assay was a further two orders of magnitude higher. Urinary macromolecules or almost whole urine were also strongly inhibitory although neither human serum ...

Calcium Stone Growth in Urine from Cystic Fibrosis Patients and Healthy Controls

Cystic fibrosis patients have an increased risk of renal stone disease. There is some evidence that this may be related to a different excretory pattern of stone risk factors, but an alternative hypothesis, that the urine of cystic fibrosis patients is deficient in urinary inhibitors of crystallization and stone formation has not been tested. Here we have grown calcium stones, in vitro, in the presence of urine from healthy controls and compared this with growth in the presence of urine from cystic fibrosis patients. A stone farm was used to grow twelve calcium stones simultaneously, firstly in artificial urine for about 200 hours and then in 90% whole human urine for another 500 hours. Six of the stones received urine from healthy controls and six received urine from adult cystic fibrosis patients. There were no significant differences in stone mass at any of the key time points or in the overall growth pattern (p>0.05) between stones destined for, or treated with, urine from CF patients and the controls...