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.     

Growth retardation of calcium oxalate by sodium copper chlorophyllin

Earlier reports from our laboratories described the inhibitory effect of Mg++ and pyrophosphate on the dissolution and growth of calcium oxalate. In this report, growth of Ca oxalate in presence of very low concentration of chlorophyllin was followed by the Coulter counter and a multichannel analyser. The effect of chlorophyllin concentration on crystal formation was studied at different levels of supersaturation. A concentration of 0.1 microgram/ml produced a remarkable retardation of the induction period and the growth rate. The findings obtained in this study are discussed in the light of the crystal poisoning theory. Comparison was made between chlorophyllin and other growth inhibitors.     

Kinetics of dissolution of calcium oxalate calculi with calcium-chelating irrigating solutions

Dissolution of calcium oxalate urinary calculi was studied in vitro. Rate constants were calculated by measuring the concentration of calcium in solution during dissolution. Various irrigating agents, including ethylenediaminetetraacetic acid (EDTA) and other calcium-chelating substances, were tested at different concentrations and pH values. EDTA was the most effective agent in dissolving calcium oxalate calculi. When EDTA was used, the rate of dissolution depended on both concentration and pH. At a pH of 7.5 or 10.0, the rate of dissolution increased progressively with the EDTA concentration. At each concentration tested, dissolution was faster at a pH of 10.0 than at a pH of 7.5. Calculated rate constants were extrapolated to determine whether in vivo irrigation with EDTA solutions is clinically practical. Even at an EDTA concentration of 0.03 M at a pH of 7.5, a two mm. calculus could be dissolved within 48 hr. Although dissolution of calcium oxalate calculi is not a practical first-line treatment, it might be a useful adjunct to percutaneous stone removal or extracorporeal shock wave lithotripsy.     

Submicroscopic investigations on calcium oxalate stone genesis.

Scanning electron microscopy of calcium oxalate stones indicates a zonary structure of the Weddellite crystals of urine concrements which is due to concentration-dependent growth oscillations representing the precipitation of various calcium oxalate hydrates. In addition to the phenomenological demonstration of Weddellite crystal shapes, incubation experiments with different solutions give rise to the postulation of a dehydration process of primary Weddellite crystals via dissolution and recrystallization to Whewellite. The solvent attack starts at energetically preferred corners, edges and lattice disorders of the Weddellite crystals. The Weddellite-stabilizing role of urinary Mg ions is emphasized.     

Dissolution of calcium oxalate renal stones in patient with jejunoileal bypass and after reanastomosis

A patient formed multiple calcium oxalate renal stones after jejunoileal bypass. Following reanastomosis of the small bowel, she was placed on a high fluid intake and a thiazide diuretic which resulted in a urine undersaturated for calcium oxalate. Stone dissolution occurred in situ.     

Studies on crystalluria in calcium oxalate stone formers

Summary The excretion of calcium oxalate and calcium phosphate crystals was studied in fractionated 24 h urine from 7 men with recurrent calcium oxalate stone disease, both before and during daily administration of 5 mg bendroflumethiazide. Urinary calcium, oxalate, magnesium, citrate, phosphate, pH, and inhibition of calcium oxalate crystal growth rate were analyzed in all samples. Exclusively calcium oxalate crystals were found in 30 per cent of the samples, all with a pH below 6.25, whereas calcium phosphate was the crystal type encountered in urine with a pH above 6.50. Bendroflumethiazide decreased the volume of calcium phosphate but not of calcium oxalate crystals. During the period of observation there was no correlation between calcium oxalate supersaturation and calcium oxalate crystal volume, but a relationship was demonstrated between calcium phosphate supersaturation and calcium phosphate crystal volume.     

The effect of pH on the urinary inhibition of calcium oxalate crystal growth

Urinary inhibition of calcium oxalate crystal growth was measured in metastable solutions of sodium oxalate and calcium chloride at different pH. Inhibition of calcium oxalate crystal growth in urine from patients with calcium oxalate stone disease increased with increasing pH. The increase was most pronounced between pH 5.5 and 7.0. Sodium pyrophosphate in a similar way inhibited calcium oxalate crystal growth in this pH interval, and between pH 6.0 and 7.5 there was also a slight increase in the inhibiting activity by chondroitin sulphate and citrate. Approximative correction factors were calculated in order to obtain a more appropriate value of the inhibition index when urine pH was different from 6.0, and a reasonably good correlation was obtained between inhibition indices calculated by means of the correction factor and inhibition indices derived from direct measurements of the crystal growth rate in solutions with different pH.     

Effect of diphosphonate on crystallization of calcium oxalate in vitro.

Reliable techniques for the calculation of activity product (state of saturation), formation product (limit of metastability) and crystal growth of calcium oxalate were devised. The activity product at saturation was 2.53 times 10 minus 9 M2, and was independent of duration of incubation, solid-to-solution ratio, pH, calcium concentration or ionic strength. These tehcniques were utilized to assess the effect of disodium ethane-1-hydroxy-1, 1-diphosphonate (EHDP) on crystallization of calcium oxalate in an aqueous salt solution in vitro. EHDP increased the formation product of calcium oxalate, indicating an inhibition of nucleation of calcium oxalate. Further, it inhibited the crystal growth of calcium oxalate.     

The role of magnesium in calcium oxalate urolithiasis

The aim of this study was to evaluate the effect of magnesium on calcium oxalate crystal formation, both in physiological conditions and at slightly higher oxalate concentrations, using a mixed suspension mixed product removal crystallizer and scanning electron microscopy. True supersaturation ratios were calculated by allowing for complexation in solution. Magnesium inhibited the nucleation rate at all oxalate concentrations. It also inhibited the growth rate at oxalate concentrations of less than approximately 2.0 mmol/l but promoted the growth rate at higher concentrations. This suggests that, provided the oxalate concentration is sufficiently high, increase of magnesium concentration can increase the crystal growth rate. At physiological concentrations of oxalate, however, magnesium decreases both nucleation and growth rates. The SEM photographs showed that the predominant crystal was calcium oxalate trihydrate at low magnesium concentrations, with calcium oxalate dihydrate being observed in larger quantities at high magnesium concentrations.     

阴极发光技术在尿石分析上的应用

应用地质矿物学的阴极发光技术，研究１７枚含有各种成分的尿石，发现这种方法有独特的优越性。各种结石成分呈现不同颜色的荧光，尿酸和尿酸盐呈亮监紫色，胱氨酸亮蓝绿色，草酸钙灰蓝至深蓝色，磷酸钙淡红紫色，磷酸镁铵亮红紫色，容易区别。尤其对于混在其他成分中的少量晶体或微晶，也可因色调对比而明显看出。用此法还确认了以前用偏光显微镜发现的草酸钙代磷灰石的现象，发现草酸钙交代尿酸盐现象。     

Effects of urinary prothrombin fragment 1 in the formation of calcium oxalate calculus

PURPOSE: We investigated the effects of urinary prothrombin fragment 1 in the formation of calcium oxalate urolithiasis. MATERIALS AND METHODS: Fresh urine and renal parenchyma from patients with calcium oxalate calculus and normal controls were collected. Urinary prothrombin fragment 1 was isolated and purified from urine. It was identified by sodium dodecyl sulfide-polyacrylamide gel electrophoresis and analysis of its first 13 N-amino acids. The inhibitory activity of urinary prothrombin fragment 1 on calcium oxalate crystal growth was tested by the seeded crystallization technique. Meanwhile, the gamma-carboxyglutamic acid composition of urinary prothrombin fragment 1 was analyzed by a previously described method and genetic mutation of the gamma-carboxyglutamic acid domain of urinary prothrombin fragment 1 from renal parenchyma was detected by polymerase chain reaction-single strand conformational polymorphism sequencing. RESULTS: The gamma-carboxyglutamic acid composition of urinary prothrombin fragment 1 was significantly decreased from normal (24.4 to 1.7 mol/1,000 amino acids) in patients with calcium oxalate calculus. The mean growth index +/- SD of urinary prothrombin fragment 1 to calcium oxalate crystals was 42.3 +/- 4.2 compared with the normal index of 19.2 +/- 2.8 (p <0.01). The polymerase chain reaction-single strand conformational polymorphism sequencing technique revealed no genetic mutation of the gamma-carboxyglutamic acid domain of urinary prothrombin fragment 1 in patients with calcium oxalate calculus. CONCLUSIONS: The gamma-carboxyglutamic acid composition of urinary prothrombin fragment 1 as well as its ability to inhibit calcium oxalate crystal growth was significantly decreased in patients with calcium oxalate calculus. This was not caused by genetic mutation of the gamma-carboxyglutamic acid domain of urinary prothrombin fragment 1. It is important to elucidate the mechanisms of calcium oxalate stones in view of urinary prothrombin fragment 1.     

Urine composition following jejunoileal bypass

The urinary excretion of oxalate, calcium, citrate, magnesium, urate and creatinine and the inhibition of calcium oxalate crystal growth were determined in 30 patients operated with three different types of jejunoileal bypass. In addition the ion-activity products of calcium oxalate and calcium oxalate saturation were calculated. 15 of the patients had formed urolithiasis postoperatively. The patients were investigated on an out-patient basis with their ordinary diet. All patients had hyperoxaluria. The oxalate excretion did not seem to decrease with time after operation. The patients operated with a biliointestinal shunt had a significantly higher excretion of oxalate than those with the other two types of operation, indicating that variations in the anatomy of the small intestine after jejunoileal bypass might result in different absorption of oxalate or oxalate precursors. Urinary oxalate, calcium oxalate saturation and ion-activity products were higher whereas the excretion of calcium, magnesium and citrate was lower in patients than in controls. The urine volumes, excretion of creatinine and urate and inhibition of calcium oxalate crystal growth were equal in patients and controls. Analogous urine composition was found in patients both with and without urolithiasis with the exception of a higher magnesium excretion observed in stone formers.     

The kinetics of crystallization of calcium oxalate trihydrate

The crystal growth of calcium oxalate trihydrate has been studied in supersaturated solutions at 37C under conditions in which the activities of calcium and oxalate ions were maintained constant by the potentiostatically controlled addition of titrant solutions containing these ions. It has been possible to stabilize calcium oxalate trihydrate sufficiently without transformation to the monohydrate, to establish the kinetic behavior. The rate of growth is proportional to the square of the relative supersaturation suggesting a spiral dislocation growth mechanism. Moreover, calcium oxalate trihydrate grows considerably more rapidly than does the monohydrate. Polyphosphate ion inhibits calcium oxalate monohydrate to a much greater extent than trihydrate. In the latter case, the reduction in growth rate in the presence of this ion can be interpreted in terms of a Langmuir isotherm. The stabilization of calcium oxalate trihydrate in the presence of inhibitors by preventing the formation of the transformation product, calcium oxalate monohydrate, may be an important factor in the development of renal stones.     

A study of crystallization on urolithiasis in vitro]

The formation, growth and aggregation of calcium oxalate crystals were evaluated with a Coulter counter. CG-120 and sodium pentosan polysulfate (SPP) has inhibitory activity on calcium oxalate crystal formation, growth and aggregation in the seeded crystal system and whole urine system. In continuous crystallizer system, they inhibit the nucleation rate. A new system of observing the crystal formation and growth of fragmented stones after extracorporeal shock wave lithotripsy has been developed. CG-120 has an inhibitory effect on their growth. The deposition of calcium oxalate crystals in the rat kidney was induced, and the, the volume and number of crystals were estimated using a Coulter counter. SPP has an inhibitory effect on calcium oxalate crystal growth in vivo.     

Effects of oxalate on HK-2 cells,a line of proximal tubular epithelial cells from normal human kidney

PURPOSE: Oxalate, a metabolic end product, is a major constituent of majority of renal stones. Previous studies with LLC-PK1 cells, a line of proximal renal epithelial cells of porcine origin, have shown that oxalate produces time and concentration dependent effects on the growth and viability of these cells. We assessed the possibility that oxalate may be toxic to HK-2 cells, a line of human proximal renal epithelial cells. MATERIALS AND METHODS: HK-2 cells were maintained in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum and antibiotics. Cells were exposed to oxalate for various intervals. Trypan blue exclusion criteria were used to assess membrane integrity, cell morphology was assessed by hematoxylin and eosin staining and crystal violet staining was used to measure cell density. DNA synthesis was measured by [3H]-thymidine incorporation and superoxide production was measured by the nitroblue tetrazolium reduction method. RESULTS: Exposure of HK-2 cells to oxalate produced time and concentration dependent increase in the membrane permeability to trypan blue and changes in the light microscopic appearance of the cells. Long-term exposure to oxalate resulted in an increase in DNA synthesis and alterations in cell viability with net cell loss after exposure to high oxalate concentrations. CONCLUSIONS: To our knowledge the results provide the first direct demonstration of the toxic effects of oxalate in HK-2 cells, a line of human renal epithelial cells, and suggest that hyperoxaluria may contribute to renal tubular damage associated with calcium oxalate stone disease.     

酒石酸钾预防草酸钙肾结石形成的研究

体外实验证明酒石酸钾对一水草酸钙晶体的生长和聚集有抑制作用。动物实验发现酒石酸钾能够降低鼠肾组织中钙及草酸的沉积。２７例患者口服酒石酸钾期间，２４小时尿钙、磷和草酸明显下降，尿枸橼酸和尿ＰＨ显著上升，实验证明，酒石酸钾能够抑制草酸钙肾结石的形成，是一种有希望的防石药物。     

Isolation and partial characterization of crystal matrix protein as a potent inhibitor of calcium oxalate crystal aggregation: evidence of activation peptide of human prothrombin

In order to clarify the characteristics of crystal matrix protein (CMP), which exhibits a remarkable affinity for calcium oxalate crystals and may be important in stone pathogenesis, we have isolated CMP from macromolecular matrix substances of newly-formed calcium oxalate crystals. Purification of CMP consisted of calcium oxalate crystal formation, dissolution of crystals, electrodialysis, anion exchange chromatography and high-performance liquid chromatography. CMP showed the protein band of 31 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CMP was identical to that of human prothrombin. Both anti-CMP polyclonal antibody and antihuman prothrombin antibody cross-reacted well with human prothrombin and CMP in Western blotting. Its amino acid composition and its molecular weight of 31 kDa strongly suggest that CMP is the activation peptide of human prothrombin.     

Variations in urine composition during the day in patients with calcium oxalate stone disease

The diurnal variations of urine composition with respect to calcium, magnesium, oxalate, citrate and inhibition of calcium oxalate crystal growth were studied in patients with recurrent calcium oxalate stone disease. There was considerable variation in the excretion of the different urine constituents with meal-related peaks, which was most pronounced for calcium. The highest concentration of calcium was observed before noon, and between 7 and 11 p.m. Oxalate concentration was highest between 6 and 10 a.m. Consequently, the highest levels of supersaturation were recorded between 6 and 10 a.m., and 6 and 10 p.m. The inhibition index was at the highest level during the first morning hours and could be important in counteracting crystal growth at that time. The risk of exceeding a theoretical formation product of calcium oxalate appeared to be low, with a 24-hour urine volume more than 2,000 ml.     

Fibronectin as a potent inhibitor of calcium oxalate urolithiasis

PURPOSE: Fibronectin (230 kD.) is a multifunctional alpha2-glycoprotein distributed throughout the extracellular matrix and body fluids. Many investigators have demonstrated that fibronectin, because of its cell adhesive action, is related to biological processes such as morphogenesis, wound healing and metastasis. Recent studies have shown that a variety of molecules, including fibronectin, inhibit endocytosis of calcium oxalate crystals in vitro. We investigated other roles of fibronectin in calcium oxalate stone formation. MATERIALS AND METHODS: Immunoblotting of the crystal surface binding substance obtained from pooled healthy male urine samples was used to analyze whether fibronectin was adsorbed onto the surface of calcium oxalate crystals. To clarify the relationship between fibronectin and calcium oxalate crystals, we performed 6 experiments. Experiment 1 was immunohistochemical examination of fibronectin expression in stone forming rat model kidneys, and experiment 2 examined the fibronectin content of stone forming rat kidney models with the enzyme-linked immunosorbent assay. Experiment 3 was designed to determine fibronectin content of Madin-Darby canine kidney (MDCK) cells stimulated by addition of calcium oxalate crystals and experiment 4 identified the inhibitory effect of fibronectin on calcium oxalate crystal growth by the seed crystal method. For experiment 5 we used an aggregometer system to clarify the inhibitory effect of fibronectin on calcium oxalate crystal aggregation and experiment 6 examined the inhibitory effect of fibronectin on the adhesion of calcium oxalate crystals to MDCK cells. RESULTS: In the crystal surface binding substance immunoreactive bands at 230 kD., which correspond to the molecular weight of fibronectin, were detected by Western blot analysis. In stone forming rat kidneys strong expression of fibronectin was found on the renal tubules to which the crystals were attached. The fibronectin content of these kidneys was significantly greater than that of kidneys without calcium oxalate crystals. The fibronectin content of MDCK cells tended to increase in proportion to the concentration of calcium oxalate crystals added to the culture medium. The growth inhibition assay showed that the inhibitory effect of fibronectin on calcium oxalate crystal growth was small in relation to the quantity of fibronectin excreted. However, fibronectin had inhibitory effects on calcium oxalate crystal aggregation and adhesion of the crystals to MDCK cells. CONCLUSIONS: Fibronectin secretion can be stimulated by calcium oxalate crystals, and this protein, which is excreted from the tubular cells, may inhibit calcium oxalate crystal aggregation and attachment to cells.     

Bladder secretion of inhibitors of calcium oxalate crystal growth

Differences in calcium oxalate crystal growth inhibition were studied in normally voided urine (bladder urine) and in urine collected directly from the kidney (kidney urine) in nine dogs. Urine samples were collected before and 10 days after bilateral ureterostomies. Calcium oxalate crystal growth inhibition was measured in a standard seeded crystal growth system. The alcian blue-precipitable material of the urine samples was determined. Significantly lower values were observed in kidney urine than in bladder urine for calcium oxalate crystal inhibition (mean difference, 0.07 +/- 0.02 inhibitor units/mg creatinine; P less than 0.01) and for the alcian blue-precipitable material (mean difference, 0.07 +/- 0.02 mg/mg creatinine; P less than 0.01). We conclude that the bladder adds calcium oxalate crystal growth inhibition to urine. Glycosaminoglycans from the bladder mucosa may be responsible; however, other acidic polymers such as RNA fragments or glycopeptides have been shown to be a constituent of the alcian blue-precipitable material. These are potent inhibitors of calcium oxalate crystal growth, and their participation in the increase of inhibition observed in bladder urine cannot be excluded. Total calcium oxalate crystal growth inhibition present in normally voided urine may be an overestimation of the actual inhibition present at the level of the kidney, where calculi usually form.