Crystal growth of calcium oxalate in urine of stone-formers and normal controls

Summary In this study, the relative crystal growth rate (Vcr) of calcium oxalate (Caox) and a number of other parameters were determined in 17-h daily (d) and 7-h nocturnal fractions (n) of whole urine from 20 recurrent Caox stone formers (SF) and 29 age-matched male normal controls (NC). Vcr, which was determined by the gel crystallization method (GCM), showed the largest difference between SF and NC among all parameters under investigation. Mean values (±SD) obtained for Vcr were: 0.73±0.58 (SF-d)/0.21±0.22 (NC-d; P<0.001) and 0.63±0.58 (SF-n)/0.24±0.25 (NC-n; P<0.01). Significantly higher concentrations of Ca and lower concentrations of thermodynamic and kinetic effectors of Caox crystal growth were responsible for the higher crystal growth rates observed in SF as compared with NC, i.e., they should be partially causative in Caox urolithiasis. However, other properties of urine or the urinary tract (potentially, crystal agglomeration and adhesion) must be accounted for in the genesis of Caox stones.     

Interaction between nephrocalcin and calcium oxalate monohydrate: A structural study

Summary Study of crystals of calcium oxalate monohydrate grown from gels exposed to 0, 5.6×, 12.5×, 26.2×, 52.5×, 100×, 200×10⁻⁷ M nephrocalcin indicate that this protein profoundly affects their habit, size, and crystal structure. By the time nephrocalcin concentration is 26.2×10⁻⁷ M calcium oxalate monohydrate undergoes a phase change in its basic structure and both crystal size as well the resolution of its diffraction pattern are severely curtailed. These effects are magnified when the protein is 52.5×10⁻⁷ M, since long-range disorder becomes extreme and, out of the entire diffraction pattern, only the 0k0's, h00's and a few other nonaxial reflections remain from the ordered part of the crystal structure. Finally, once the concentration of nephrocalcin is raised to 100 and 200 ×10⁻⁷ M, growth is so inhibited that calcium oxalate monohydrate no longer grows as distinct individuals but rather as aggregates of very small crystallites. All of this is caused by the ability on the part of nephrocalcin to disturb the juxtaposition of the layers alongc by disrupting the organization of both the C(3)−C(4) oxalate groups and the water molecules. Such interaction is modulated by the efficiency with which nephrocalcin adsorbs upon the planes; this process is stereospecific.     

The inhibition of calcium oxalate crystal growth by multidentate organic phosphonates

Summary The effect of a number of structurally related multidentate organic phosphonates on the rate of crystal growth of calcium oxalate was studied as a function of pH. Rate constants were obtained at various concentrations for the phosphonates ethane-1-hydroxy-1,1-diphosphonate (EHDP), nitrilotri(methylenephosphonic acid) (NTMP), N,N,N,N-ethylenediaminetetra(methylenephosphonic acid) (ENTNP), and N,N,N,N-hexamethylenediaminetetra(methylenephosphonic acid (HMTMP), at pH 5.00, 6.00, and 7.00. The effect of pH on the inhibitory activity of each of the phosphonates was considerable with effective concentrations of inhibitor decreasing two orders of magnitude, in some cases, as the pH was increased. At a given pH the potentially hexadentate ligands, ENTMP and HMTMP, were generally the most effective inhibitors. The results suggested that EHDP, at currently administered doses, provides only a moderate increase in the capacity of human urine to inhibit calcium oxalate crystal growth.This investigation was supported in part by Research Grant AM-17717 from the National Institutes of Health, Public Health Service.     

The effect of crystalline monosodium urate on the crystallisation of calcium oxalate in whole human urine

Summary (1) Samples of undiluted urine from normal men were preincubated with crystalline monosodium urate and their metastable limits and responses to a standard oxalate challenge were compared with results obtained from control samples preincubated without urate. (2) Preincubation with urate had no significant effect on the metastable limits of the urines, the morphology, size, or growth rates of calcium oxalate crystals precipitated from the urines, or on the total amount of calcium oxalate deposited in a given time. (3) It was concluded that particulate monosodium urate is unlikely to influence calcium oxalate stone formation by binding to and attenuating the potency of urinary inhibitors.     

The influence of hydroxyapatite and pyrophosphate on the formation product of calcium oxalate at different pHs

Summary The nucleating effect of hydroxyapatite (HAP) and the inhibitory effect of pyrophosphate (PPi) on calcium oxalate crystallization have been studied at different pH's in solution metastabely supersaturated with respect to calcium oxalate but saturated with respect to HAP. Crystallization was monitored by a decrease of calcium in the supernatant and formation products were calculated. At a pH above 6.0 already minimal HAP concentrations proved to be a suitable substrate for heterogeneous nucleation and growth of calcium oxalate. PPi showed a pronounced inhibitory effect on spontaneous as well as on HAP induced crystallization of calcium oxalate, this effect being highly pH dependent. HAP was found to neutralize the inhibitory effect of PPi in a molar ratio of 10:1.     

Simultaneous measurements of calcium oxalate crystal nucleation and aggregation: impact of various modifiers

Rates of nucleation and aggregation of calcium oxalate crystals were derived from 20-min time course measurements of OD₆₂₀ after mixing solutions containing CaCl₂ and K₂C₂O₄ at 37°C, pH 5.7, ionic strength (IS) 0.21, with constant stirring (500 rpm); final assay concentrations were 4.25 mM calcium and 0.5 mM oxalate, respectively. The maximum increase of OD₆₂₀ with time, termed S _N, mainly reflects maximum rate of formation of new particles and thus crystal nucleation. After equilibrium has been reached, OD₆₂₀ progressively decreases despite ionized calcium staying constant and no new particles being formed, due to crystal aggregation. Rate of aggregation, S _A, is derived from the maximum decrease in OD₆₂₀ with time. S _N and S _A are not independent, as indicated by a positive correlation (r=0.844, P=0.0001). Among the modifiers studied, citrate at 0.5–2.5 mM lowered both S _N and S _A in a concentration-dependent manner (P<0.01 for all comparisons vs control). Chondroitin-6-sulfate at 6.25–25 mg/l moderately lowered S _N, whereas it strongly inhibited aggregation (P<0.01 vs control). At 6.8–20.4 mg/l, albumin did not affect nucleation, whereas it inhibited aggregation in a concentration-dependent manner (P<0.005 vs control for all comparisons).     

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

Summary 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 solutionin 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.     

Crystallisation of calcium oxalate dihydrate in normal urine in presence of sodium copper chlorophyllin

Summary A method is described for the growth of calcium oxalate dihydrate in normal urine. Soluble chlorophyllin, at a concentration of 20 g/ml inhibited the crystallisation and the growth kinetics of the dihydrate crystals. The inhibitory capacity of chlorophyllin was compared with previous results. Data obtained suggest that the food and drug colourant chlorophyllin might be useful in the treatment of calcium oxalate stone disease.     

The effect of serum on the crystallization of calcium oxalate in whole human urine: Inhibition disguised as apparent promotion

Summary The effect of serum on calcium oxalate crystallization was studied in whole human urine. At concentrations between 0.005% to 0.10% (v/v), serum had no effect on the metastable limit of urine as detected by the Coulter Counter. However, serum caused a marked increase in the number and volume of calcium oxalate crystals deposited in response to an oxalate load. That this increase in volume reflected true deposition of calcium oxalate was confirmed by determining calcium concentration and by including control urines containing added serum but no exogenous oxalate. Analysis of particle size distributions showed that the crystals deposited in the presence of serum were smaller than those occurring in the control. Since serum did not affect the limit of metastability, the enhancement of crystal volume is unlikely to be a result of heterogeneous nucleation. We hypothesize that the apparent promotion effect of serum is due to its inhibition of crystal aggregation which increases the crystal surface area available for calcium oxalate deposition.     

Morphology of crystals in calcium oxalate monohydrate kidney stones

Both scanning electron microscopy and atomic force microscopy (AFM) have shown that calcium oxalate monohydrate kidney stones are made up from arrangements of sub micron crystals. The purpose of this investigation was to determine the morphology of these crystals which was obscured by the presence of organic matrix in our earlier study. Sections of stones were treated to remove the protein component of the matrix and then imaged using AFM. Images obtained after proteolysis show that the crystals are in the form of plates stacked on (100) surfaces. These results were confirmed by scanning electron microscopy observations from selected regions of calcium oxalate kidney stone surfaces. The observed crystal sizes are consistent with both the known matrix mass fraction and crystallite growth in the passage through the collecting duct.     

Is citrate an inhibitor of calcium oxalate crystal growth in high concentrations of urine?

The effect of citrate on calcium oxalate (CaOx) crystal growth was studied in a system in which series of samples containing [⁴⁵Ca]calcium chloride were brought to different levels of supersaturation with various concentrations of oxalate. The crystallization was assessed by measuring the amount of isotope remaining in solution 30 min after the addition of CaOx seed crystals to samples containing citrate in concentrations corresponding to those in final urine. The experiments were carried out both in pure salt solutions and in solutions with dialysed urine. Increased concentrations of citrate resulted in a reduced crystallization of CaOx in both the presence and absence of dialysed urine, but with the lowest rate of crystallization in the samples containing urine. The increased concentration of ⁴⁵Ca remaining in solution reflected a reduced crystallization, which could possibly be explained both by a reduced supersaturation and by an increased inhibition of CaOx crystal growth. The direct effects of citrate on CaOx crystal growth were assessed by calculating the ion-activity product of CaOx (AP_CaOx) at corresponding degrees of crystallization. The AP_CaOx recorded at a 30% reduction of the amount of isotope in solution increased with increasing concentrations of citrate between 1.0 and 1.5 mmol/l in samples both with and without dialysed urine. These findings indicate that citrate has a weak direct inhibitory effect on CaOx crystal growth, which adds to the reduced growth rate brought about by urinary macromolecules and a decreased supersaturation.     

The effect of traditional risk factors for stone disease on calcium oxalate crystal adherence in the rat bladder

Crystal adherence in the urinary tract has been studied using the chemically injured rat bladder and cell cultures. These studies have provided evidence that mucin prevents adherence and have studied various compounds for their ability to promote or inhibit crystal adherence. Little work has been done examining the effect on crystal adherence of traditional risk factors for stone disease. The study reported here examined the effect hypercalciuria, hyperoxaluria and pH on calcium oxalate crystal adherence using the intact rat bladder model. Calcium at levels seen in hypercalciuric stone formers was associated with increased adherence. Oxalate at levels seen in stone formers had no effect on adherence. There was a tendency to increased crystal adherence at higher pH values only when phosphorus was present as the buffer. Hypercalciuria is a risk factor for stone disease by increasing the level of saturation of calcium oxalate and calcium phosphate in the urine and by decreasing inhibitor function. This study suggests that it may also play a role by increasing crystal adherence within the urinary tract.     

Identification of proteins extracted from calcium oxalate and calcium phosphate crystals induced in the urine of healthy and stone forming subjects

The purpose of our study was to identify the proteins and investigate the differences, if any, between protein components of the matrices of calcium oxalate (CaOx) and calcium phosphate (CaP) crystals induced in␣vitro in whole human urine of healthy individuals and kidney stone patients. In addition, preliminary studies were performed to understand the effect of centrifugation and filtration of urine on its protein contents. Crystallization in urine was induced by addition of an oxalate or phosphate load. Crystals were collected, washed, and analyzed by scanning electron microscopy, X-ray diffraction, and energy dispersive X-ray microanalysis. Matrix proteins were obtained by demineralization with ethylene diamine tetraacetic acid (EDTA), analyzed by polyacrylamide gel electrophoresis, and identified by western blotting technique. No significant differences were detected between protein components of the matrices of CaOx and CaP crystals and between the crystal matrices obtained from the urine of normal and stone forming subjects. Albumin (AB), inter-α-inhibitor (IαI) related proteins, α-1 microglobulin (α-1 m), osteopontin (OPN), prothrombin (PT)-related proteins and Tamm-Horsfall protein (THP) were identified in matrices of both CaOx and CaP crystals induced in urine from both the normal subjects and stone formers. AB, PT-related proteins and OPN were the main constituents. The other proteins were present in smaller but detectable amounts. However, CaP crystal matrix, contained a large amount of THP. In addition CaP crystals contained significantly more proteins than CaOx crystals. Centrifugation and/or filtration of the urine resulted in reduction of many high molecular weight proteins including THP, AB and OPN in the urine. Received: 24 July 1997 / Accepted: 2 January 1998     

Effects of urinary macromolecules on the crystallization of calcium oxalate

Summary The macromolecular fraction of urine with a molecular weight above 3,000 was isolated by dialysis. In the dialysed urine the rate of calcium oxalate (CaOx) crystallization was reduced both in the presence and absence of CaOx seed crystals. There was a clear relationship between crystallization and the relative concentration of the dialysed urine, with the highest crystallization propensity at the lowest concentration of macromolecules. Dilution of dialysed urine also affected crystal size distribution, with a predominance of small (2.8–4.5 m) crystals in 100% dialysed urine and of large (5.6–14.0 m) crystals in 5% dialysed urine. This is consistent with a macromolecular inhibition of both crystal growth and aggregation. Analysis of the crystal size distribution 120 min after supersaturation of whole urine to a level at which approximately 100 crystals in the size interval 3.5–5 m were detected in a Coulter counter surprisingly disclosed a higher mean crystal volume in urine samples from normal subjects than from stone formers. This gives support to the assumptions that macromolecules might be of importance during the initial phase of CaOx crystallization and that urine from stone formers and normal subjects might be different in this respect.     

The in-vivo effect of sodium-potassium citrate on the crystal growth rate of calcium oxalate and other parameters in human urine

Summary In this study, an efficient microtechnique (gel crystallization method) was used to investigate the in-vivo effect of sodium-potassium citrate on the crystal growth rate of calcium oxalate (Vcr) in human urine samples of 6 healthy volunteers. With a daily dose of 3x11 mmol of alkali citrate, Vcr decreased by 70%. This could have been due to the decrease of calcium excretion, which caused 50–60% of the total change, and to the increase of citrate and pH, each contributing about 20–25% to the decline of Vcr. The findings explain the clinical advantages of alkali citrates in the prevention of recurrent calcium oxalate stone formation.     

Kinetic factors influencing the dissolution behavior of calcium oxalate renal stones: A constant composition study

Summary A constant composition method has been used to examine the dissolution kinetics of calcium oxalate renal stones over a wide range of undersaturationin vitro. Demineralization experiments have been carried out with the concentrations of calcium and oxalate ions and ionic strength (hence the solution undersaturation) held constant by the potentiometrically controlled addition of medium electrolyte solution as diluent, triggered by a calcium ion electrode. Kinetic data for renal stones have been compared with results obtained for synthetic calcium oxalate. In addition, constant composition results have been directly compared with results obtained using conventional dissolution methods for both calculi and synthetic calcium oxalate. Overall, calcium oxalate renal stones exhibited markedly different kinetic dissolution behavior as compared with synthetic controls. The renal stone samples dissolved more slowly at all undersaturations, exhibited increased kinetic orders of reaction, and showed reduced sensitivity to solution hydrodynamics. Stones composed of mixed hydrates of calcium oxalate (mono- and di-) came to dihydrate equilibrium in conventional experiments and underwent net dissolution in solutions supersaturated to monohydrate under constant composition conditions. No conversion of di- to monohydrate was observed under these experimental conditions. These results indicate that stone dissolution is strongly influenced by adsorbed inhibitors, presumaly including matrix components, which may complicate efforts to develop systemic and/or irrigation measures effective forin situ solubilization.     

An approximate estimate of the ion-activity product of calcium oxalate in rat urine

The objective of this report was to derive a simplified approximate estimate of the ion-activity product of calcium oxalate (AP_CaOx) in rat urine. The relative effect of each urine variable was assessed by means of iterative computerised approximation with the EQUIL2 program. A basic urine composition was chosen from literature and experimental data. The most pronounced influence on AP_CaOx was recorded for urinary calcium, oxalate, citrate, magnesium and volume. Based on these calculations, an AP(CaOx) index_RAT was formulated: . For a 24-h urine sample, factor A takes the value 4067 and factor F should be set to 0.015. Conclusion. A simplified approximate estimate of AP_CaOx was derived for rat urine. There was a reasonably good correspondence between AP(CaOx) index_RAT and AP_CaOx, as derived from EQUIL2 (r=0.890), provided the other urine variables do not deviate very much from that in the basic composition.     

凝血酶原分子中γ-羧基谷氨酸对草酸钙结晶的抑制作用研究

目的：探讨凝血酶原(PT)分子中γ-羧基谷氨酸(Gla)在抑制草酸钙结晶形成中的作用。方法：采用加入不同浓度的吗啉和甲醛(1：10000)或(1：100)，体外化学修饰PT，将其分子内8个或2个γ-羧基谷氨酸残基修饰转变成γ-亚甲基谷氨酸残基(γ-MGlu)，草酸钙种晶抑制试验测定化学修饰后PT对草酸钙结晶抑制作用的变化。结果：PT蛋白转变为2γ-MGlu的PT后对草酸钙结晶的抑制活性从正常对照的14．2降低至128(10％)；PT蛋白转变为8γ-MGlu后其抑制活性降低至5．0(65％)。结论：凝血酶原分子内γ-羧基谷氨酸(Gla)在抑制草酸钙结晶形成中具有重要作用。     

The dual role of polyelectrolytes and proteins as mineralization promoters and inhibitors of calcium oxalate monohydrate

Summary Polyelectrolytes and protein molecules appear to be able to act not only as crystallization inhibitors when present in solution, but also as promoters of crystal growth when immobilized onto surfaces. Because this is especially relevant for systems in which heterogeneous nucleation can occur, the influence of poly-L-glutamic (PGlu) acid, poly-L-aspartic (PAsp) acid, and human serum albumin (HSA) on the nucleation and growth inhibition of calcium oxalate monohydrate (COM) was studied using the Constant Composition (CC) kinetics technique. The overgrowth of COM on hydroxyapatite (HAP) seed crystals pretreated with HSA was also investigated. Pronounced differences in inhibiting and nucleating potential were found for the various additives. HSA, a relatively poor growth inhibitor when present in solution, was found to nucleate very regular, hexagonal COM crystals when immobilized on a surface and to enhance the overgrowth of COM when adsorbed on HAP surfaces.     

Determination of GOT activity on nucleation and crystal growth of calcium oxalate

Summary Crystal Size Distribution (CSD) and the yield of Calcium Oxalate Crystals in solutions with an admixture of 5 normal and 3 stone forming urines, were determined. A positive correlation was found between the median size, the number of particles and the overall inhibitory potentials of the urines toward calcium oxalate precipitation in vitro as reflected by Discriminating Index (DI) measurements. Incubation of two samples of stone formers' (SF) urines with glutamic-oxalacetic-transaminase (GOT) caused a reduction of aspartic acid concentration, an increase in glutamic acid concentration and a parallel decrease in the DI values. After 90 min of SF urine incubation with GOT the DI in three samples was improved and both the median size and number of particles reduced, by 28% and 45% respectively. These results could indicate that GOT activity changes the inhibitory power of the SF urine by transforming aspartic acid into glutamic acid, having thus most probably a part in the inhibition of CaOx stone fromation.