Sevsen Kulaksizoglu md, Baskent University, Department of Biochemistry, Merkez, 42080, Konya, Turkey. Email: email@example.com
Objective: 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.
Methods: 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°C, pH 5.7. The maximum increase of optic density with time, termed SN, 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. SA (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.
Results: Among the modifiers studied, citrate decreased both SN and SA (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).
Conclusion: 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.
Renal stone disease has afflicted people throughout history. Although the chemical nature of stones has been known for a century, little has been known until recently as to why they occur at all. The explosion of molecular biology and knowledge of kidney development have now elucidated a number of mechanisms for kidney stone development.
Stone formation results from a combination of three main events: (i) crystal nucleation; (ii) crystal growth; and (iii) aggregation.1 Supersaturation is a necessary condition for the occurrence of crystallization. However, it does not itself explain crystal growth and aggregation because it has been shown that the urine of normal people and stone-formers had a similar level of supersaturation.2 Moreover, many recurrent stone-formers with no detectable metabolic abnormality show deficiencies in their urine of the naturally occurring inhibitors of crystallization.3,4 The effect of inhibitors of crystallization has received particular attention because in vitro experiments can be done to investigate the contribution of various inhibitors. But many are time consuming or require expensive and specialized equipment.5,6
The present study reports a quick, easy and reproducible method for the quantitating the inhibition of calcium oxalate monohydrate crystal growth by various compounds. Some of these compounds occur naturally in the urine or are already used in the treatment of renal stone disease.7,8
Materials and methods
Calcium oxalate monohydrate crystallization kinetics were determined as previously described by monitoring the increase in turbidity after mixing solutions of calcium chloride and sodium oxalate to generate a supersaturated calcium oxalate solution. Stock solutions of calcium chloride (8 mmol/L) and sodium oxalate (1 mmol/L), containing 200 mmol/L sodium chloride and 10 mmol/L sodium acetate, were adjusted to pH 5.7. These concentrations were chosen because they are close to physiological urinary concentrations. A pH of 5.7 was selected because it is a pH value frequently observed in the first morning urine of calcium stone-formers.9 For crystallization experiments, 1 mL of the calcium chloride solution was transferred into a 10-mm light-path cuvette in a cell holder maintained at 37°C by a circulating bath of constant temperature, with constant stirring at 28 g (using a Teflon-covered stirring bar, 7 mm × 2 mm). An additional 1 mL of sodium oxalate solution was then added to reach final assay concentrations of 4 mmol/L for calcium and 0.5 mmol/L for oxalate, respectively. Separate solutions of 4 mmol/L calcium chloride and 1.5 mmol/L sodium oxalate were also prepared. Control experiments were performed with three different calcium/oxalate concentration ratios (2 mmol/L : 0.5 mmol/L, 4 mmol/L : 0.5 mmol/L, 4 mmol/L : 0.75 mmol/L).
All experiments with modifiers of calcium oxalate crystallization were performed at assay concentrations of 4 mmol/L calcium, 0.5 mmol/L oxalate, 200 mmol/L sodium chloride and 10 mmol/L sodium acetate, pH 5.70. Concentrated solutions of modifiers were pipetted into the calcium containing solution before oxalate was added.
Trisodium citrate (Merck, Darmstadt, Germany) was studied at final concentrations of 0.05 mmol/L, 0.15 mmol/L and 0.25 mmol/L. Magnesium chloride (Merck) was pipetted into the standard solution in order to get final concentrations of 4 mmol/L, 8 mmol/L and 12 mmol/L. A concentration of 4 mmol/L was chosen because it is very close to physiological urinary concentrations.10 Starting with an inhibitor concentration of 4 mmol/L, the concentration of magnesium was increased up to 12 mmol/L as depicted in the studies of Bongartz et al. and Laube et al.6,8 Bovine serum albumin (96–99% Sigma Chemical, St Louis, MO, USA) with a molecular weight of 68 kDa was also added in small volumes in order to reach concentrations of 6.8, 13.6 and 20.4 mg/L.
The optical density of the crystals formed was measured at 2-min intervals over 30 min at 620 nm using a Shimadzu UV 1601 spectrophotometer (Kyoto, Japan).11 The increase in optic density reflects an increase in particle number in the function of time.12 The maximum increase of optic density with time, termed SN, mainly reflects maximum rate of formation of new particles and thus crystal nucleation. Maximum time, namely tmax, corresponds to the time between the addition of oxalate and the moment at which maximum absorbance (equilibrium) is measurable. After equilibrium has been reached, optic density decreases. No new particles were formed due to crystal aggregation. SA (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.
At various times in the experiments, crystals were transferred from the spectrophotometric cuvette and observed by polarized light microscopy. They are photographed at a magnification of ×1000.
All values are means ± SE. The Kruskal–Wallis variance analysis for comparisons between groups and Mann–Whitney U-test for paired comparisons within groups were used. For slope measurements, linear regression analysis was used. All experiments with modifiers of calcium oxalate crystallization were compared with assay concentrations of 4 mmol/L calcium and 0.5 mmol/L oxalate. P < 0.05 was considered significant. Percentage inhibition ratio was calculated as (1-[SNm/SNc]) × 100 for rate of nucleation and (1-[SAm/SAc]) × 100 for the rate of aggregation where m stands for modifier and c for control.
Figure 1 presents time-course measurements of optic density at 620 nm under standard conditions (4 mmol/L calcium, 0.5 mmol/L oxalate). The amount of crystals formed, as measured by the turbidity of the solution, gradually increased with time until a maximum was reached. After equilibrium had been reached at 9.8 min, optic density progressively fell due to crystal aggregation.
Table 1 summarizes values of tmax, SN and SA in control experiments with various calcium and/or oxalate concentration ratios. With increasing calcium and oxalate concentrations, tmax progressively decreased, whereas SN and SA increased (P < 0.001).
4 mmol/L calcium/0.5 mmol/L oxalate compared with 4 mmol/L calcium : 0.75 mmol/L oxalate. tmax, time at maximum absorbance; SN, the maximum slope of increase of optic density at 620 nm with time, representing crystal nucleation; SA, the maximum slope of decrease of optic density at 620 nm with time, representing crystal aggregation.
The inhibitory effect of trisodium citrate on the nucleation of calcium oxalate crystal is shown in Table 2. In the presence of trisodium citrate, tmax increased and the slopes of calcium oxalate crystal growth (SN and SA) decreased (P < 0.001). At the highest concentration of trisodium citrate, nucleation percentage inhibition ratio was 83.5% and crystal aggregation was inhibited by approximately 68.8%.
Table 2. Effects of trisodium citrate on calcium oxalate crystallization
As depicted in Table 3, magnesium altered tmax significantly. It inhibited the rate of nucleation and crystal aggregation in a non-concentrating manner. Crystal aggregation and nucleation were inhibited by approximately 70% at all concentrations. Albumin increased tmax at all concentrations (P < 0.001). Additionally it decreased both SN and SA as demonstrated in Table 4. The effect on the rates of nucleation and aggregation were different from those of trisodium citrate and magnesium. Nucleation and aggregation percentage inhibition ratios fell with increasing concentrations to a level less different that of the control at 20.4 mg/L albumin (23.7% nucleation inhibition and 19.1% aggregation inhibition).
Table 3. Effects of magnesium chloride on calcium oxalate crystallization
It is generally thought that supersaturation of urine is the prime element responsible for calcium oxalate stone formation. As seen in our study, with increasing calcium and oxalate concentrations, supersaturation exceeds the metastability (Table 1). Consequently, management and prevention of renal stone disease has been focused on lowering the urinary calcium and oxalate concentrations. However, it was shown that the urine of normal people and stone-formers had a similar level of supersaturation and the calcium oxalate complex is not different from that in normal urine. It may be that in those patients the effective concentration is increased by diminished excretion of substances which make calcium or oxalate complex.
The effect of inhibitors of calcium oxalate crystallization has received particular attention. Previous investigations used 45Ca tracers or laser probes to study inhibitors of calcium oxalate crystallization kinetics.5,6 These models took into account only the growth of calcium oxalate crystal. The present study of spontaneous precipitation involves both nucleation and crystal growth, which is a better reflection of phenomena in the urine, because generally the two processes occur simultaneously. Additionally, many are also time consuming or require expensive or specialized equipment.5,6 The present method is easy and quick to perform. Furthermore, the reproducibility of the measurements is good (coefficient of variation [CV]%, < 7%), suggesting that under the same experimental conditions, an identical distribution of particle number and size was produced. Hennequin et al. described the maximum increase in optic density at 620 nm with time as being related essentially to crystal growth.11 Indeed, optic density is an exact measure of particle concentration per unit volume.2 We have demonstrated that optic density directly relates to concentrations of calcium oxalate crystals per unit volume in aqueous suspensions as shown in Figure 1. Hennequin et al. also described another kinetic parameter: induction time, ti, indicates only the beginning of nucleation of particles that are detectable by turbidimetry.5 Any prolongation of ti was related to inhibition of nucleation. However, it is not easy to detect ti because calcium oxalate nuclei are of the order of 100 A° and much smaller than what can be detected by turbidimetry. Therefore, we took advantage of the maximum time, namely tmax, corresponding to the time between the addition of oxalate and the moment at which maximum absorbance (equilibrium) is measurable. Any prolongation in the beginning of nucleation (ti) will affect the time that maximum absorbance is reached (tmax) (CV%, < 5%).
Citrate increases tmax and also reduces rates of nucleation and aggregation in a non-concentrating manner. This was expected because citrate chelates calcium ions, which efficiently lowers supersaturation, the driving force for crystallization. These results are in contradiction to those results of Hess in which citrate did not change the rate of crystal aggregation under saturated conditions.13
Magnesium was also found to inhibit the rate of nucleation and crystal aggregation, but it appeared in a non-concentrated manner. In our study, starting with an inhibitor concentration of 4 mmol/L, the concentration of magnesium was increased up to 12 mmol/L as depicted in the studies of Laube et al.8 However, our results are in apparent contradiction to those of the study of Laube in which magnesium changed calcium oxalate crystallization in a dose-dependent manner.14 There is a possibility that under 4 mmol/L, magnesium may inhibit nucleation and aggregation in a concentration-dependent manner. Thus, it would be an alternative study to try the lesser concentration of magnesium than 4 mmol/L. In our study, crystal aggregation and nucleation were inhibited by approximately 70% at all concentrations. Compared with control conditions, tmax increased and the slopes of calcium oxalate crystal growth (SN and SA) decreased (P < 0.001). Its inhibitory effect on calcium oxalate formation is based on a different mechanism. The magnesium ion reduces urinary calcium oxalate saturation by forming magnesium oxalate, which is more soluble than calcium oxalate in the urinary tract.15 After oral administration of magnesium salts, they may form complexes with oxalate in the gastrointestinal tract, thus reducing the absorption of oxalate from the intestine. Additionally, it increases the excretion of citrate. However, magnesium salts alone are not recommended for prophylactic treatment. Kato et al. showed that the combination of citrate and magnesium is more effective than either supplement alone in inhibiting the calcium oxalate crystallization.15 So the combination of citrate and magnesium can be a potential alternative experiment to further study.
Albumin is one of the most abundant proteins in urine and was shown to bind tightly to calcium oxalate crystals in an ionic environment similar to urine.16 Because it is a component of the stone matrix, we thought that it could be involved in calcium oxalate crystal nucleation.17 We found that albumin decreased in both SN and SA. Albumin shares the characteristics of being polyanionic in physiological solutions. As indicated by the studies of Wesson et al., polyanions, either synthetic or naturally occurring, induce disaggregation.18 This is in agreement with Hess et al. who found that albumin did not significantly affect crystal nucleation.19 Also, Ryall et al. showed that albumin, added to ultrafiltered human urine, enabled slight promotion of calcium oxalate crystal nucleation.20 However, nucleation and aggregation percentage inhibition ratios were related inversely to the concentration of albumin in our study. According to the study of Cerini, the absorbance at 620 nm, which reflects the presence of crystallized calcium oxalate, increased spontaneously in the presence of albumin, showing that albumin could promote crystal formation in solution.21 Therefore, these results suggest that the appropriate promotion of calcium oxalate crystals by albumin is observed when the concentration of albumin is increased. However, it would be more appropriate to try at higher concentrations of albumin to strengthen these results.
In conclusion, our findings indicate that growth and agglomeration of calcium oxalate crystals are differently modulated by various compounds and that these must not be considered collectively. Therefore, in future studies, the physicochemical basis of treatments aiming at inhibiting the crystallization of calcium oxalate can be better defined and more rational ways of preventing renal stone formation can be developed.