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 effect of glycosaminoglycans on the crystallisation of calcium oxalate

The effect of glycosaminoglycans on urinary stone formation was evaluated using a mixed suspension, mixed product removal (MSMPR) crystallisation system together with scanning electron microscopy (SEM) to examine the resulting crystals. Chondroitin sulphate was found to decrease the nucleation rate and to promote both the growth rate and suspension density. Results obtained with hyaluronic acid, although inconclusive, are similar to those given by chondroitin sulphate. Heparin sodium salt had a powerful inhibitory effect on both the nucleation rate and the suspension density, the effect increasing in proportion to the heparin concentration. SEM examination showed that the octahedral habit of calcium oxalate dihydrate was modified by the addition of heparin sodium salt and confirmed that the average crystal size in the presence of chondroitin sulphate and hyaluronic acid was significantly greater than the control or that found in the presence of heparin sodium salt.     

The effect of pH on the risk of calcium oxalate crystallization in urine

The risk of calcium oxalate (CaOx) crystallization at different pH levels was determined in urine from recurrent CaOx-stone formers and normal subjects. The highest crystallization risk was observed between pH 4.5 and 5.5. In the pH range 6.5-7.5, there was a marked increase in crystallization of calcium phosphate (CaP). The results suggest the beneficial effect of moderate alkalinization in terms of a reduced CaOx crystallization. Reduced CaOx crystallization occurs at the expense of an increased formation of CaP crystals. Whether this increases the risk of CaP-stone formation is not known, but the CaP crystals were usually small, at least below pH 7.5.     

尿液成分对草酸钙结石的影响

目的 探讨尿液成分对草酸钙尿结石形成的影响。方珐 应用红外光谱仪对50份尿结石标本进行成分检测；对16例一水草酸钙（COM）与10例二水草酸钙（COD）尿结石患者的24h尿液进行生化检测，并比较两组生化指标。结果 87．5％的c0M结石患者和90％的c0D结石患者24h尿量减少；COM结石患者尿钙（4．94±2．11）mmol／24h，COD结石患者尿钙（9．43±3．78）mmol／24h；差异有统计学意义（P〈0．01）；COM结石患者尿磷（20．50±8．76）mmol／24h，COD结石患者尿磷（28．38±10．21）mmol／24h，差异有统计学意义（P〈0．05）；87．5％的COM结石患者尿枸橼酸低于正常水平。结论 COD结石患者尿钙、尿磷高于COM结石患者，表明COD结石的形成与高钙尿和高磷尿有关；COM结石的形成可能与低尿枸橼酸有关。     

Inhibition of calcium oxalate crystallisation by pentosan polysulphate in control subjects and stone formers

In vitro studies showed that sodium pentosan polysulphate (SPP) is an active inhibitor of calcium oxalate (CaOx) crystal growth and agglomeration and that it acts by increasing the negative zeta potential on the surface of CaOx crystals. Oral administration of SPP to control subjects, recurrent stone formers and patients with primary hyperoxaluria resulted in an overall increase of 8% (P less than 0.01) in the polyanionic inhibition of CaOx crystallisation in urine as measured by the zeta potential. SPP could provide a novel approach to the medical prevention of recurrent CaOx stone disease.     

Effects of different doses of alkaline citrate on urine composition and crystallization of calcium oxalate

Summary Prophylactic treatment with alkaline citrate in patients with recurrent calcium oxalate (CaOx) stone disease results in reduced CaOx supersaturation and increased urinary citrate. The effects of a single evening dose were compared with those of two and three daily doses in six recurrent CaOx stone formers with hypercalciuria, hypocitraturia or raised calcium/citrate quotients. While on a standardized hospital diet the patients were given 7.5 g (28 mmol) of sodium potassium citrate (URALYT-U) in one, two, and three doses. Fractional urine collections during 24 hours were analyzed for pH, composition, and crystallization risk (CR). All dosage regimens had favourable effects on urinary calcium, citrate, calcium/citrate quotients, and CaOx-CR. The most sustained effect was recorded with three divided doses. Single evening doses resulted in the most pronounced effects between 22.00–06.00 h, thereby counteracting the increased risk of CaOx crystallization during that period. In terms of 24 h urine composition the best effect was recorded with alkaline citrate administered three times daily, but because of the favourable response by a single evening dose between 22.00–06.00 h the assumption was made that this dosage regimen might be sufficient to reduce the risk of CaOx crystallization and stone formation. However, the validity of such an assumption can only be established by long-term clinical studies.     

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     

Measurement of the risk of calcium oxalate crystallization in urine

Summary The risk of calcium crystallization (CaOx-CR) in urine was analyzed by means of crystal counting following standardized addition of oxalate. CaOx-CR was determined in 24h urine samples from 21 stone formers and 26 normal subjects following dilution of urine to a creatinine concentration of 5 mol per ml. The mean (±SD) CaOx-CR was in stone formers 1.42±0.57 and in normal subjects 1.29±0.40. CaOx-CR was also analyzed in 16 fresh urine samples diluted to 80 per cent of the original concentration whereby values between 0.36 and 3.6 were recorded. There was a good correlation between CaOx-CR and estimates of the ion-activity product of CaOx, both in urine diluted to 5 mol of creatinine per ml and in 80 per cent diluted urine. It ist suggested that the method described is of value for evalution and follow up of patients with CaOx urolithiasis.     

Evaluation of urine composition and calcium salt crystallization properties in standardized volume-adjusted 12-h night urine from normal subjects and calcium oxalate stone formers

The volume of 12-h night urine from ten normal men (NM), ten normal women (NW) and 31 male calcium stone formers (SFM) was adjusted to 750 ml and analysed with respect to supersaturation with calcium oxalate (CaOx) and calcium phosphate (CaP), inhibition of CaOx crystal growth and aggregation, as well as the CaOx and CaP crystallization propensity. Concentrations of oxalate and glycosaminoglycans and AP(CaOx) index, an estimate of the CaOx ion-activity product, were higher and the concentration of citrate lower in NM than in NW. In SFM the directly assessed risk of CaOx crystallization was higher and the inhibition of CaOx crystal growth lower than in NM. There were no differences between the groups regarding inhibition of CaOx crystal growth by 74% dialysed urine or inhibition of CaOx crystal aggregation. SFM with mixed CaOxCaP stones had a higher concentration of phosphate and a higher AP(CaP) index at pH 7.0 than SFM with CaOx stones.     

The effect of mineral water containing calcium on supersaturation of urine with calcium oxalate

The influence of mineral waters with different calcium content on the urine composition was investigated. Twelve healthy male test persons were housed for 3 x 24 h (once a week) in a climatic chamber under constant resting conditions. The standardized food intake was distributed equally over 24 h. Six 4-h urine samples were collected and chemically analyzed to study the risk of stone formation. The total amount of mineral water (Staatl. Fachingen or Bad Wildunger Helenenquelle) or of tapwater was administered in three portions (700 ml at 9.00, 350 ml at 13.00, 350 ml at 17.00). After mineral water intake, the calcium concentration in the urine was slightly higher. However, the more highly mineralized water caused a significant increase in the urinary magnesium concentration, which persisted into the night. The citrate concentration was increased as a result of alkalization of the urine. After mineral water intake the oxalate excretion was slightly (but not significantly) higher. However, no increased risk of calcium oxalate stone formation was seen when different supersaturation ratios were used as the basis of calculation. In a second study, further 12 volunteers of both sexes were given an oxalate-rich standard meal and 500 ml of the test waters in addition. The urine oxalate concentration was measured hourly for 6 h after the meal, and the total oxalate excretion was calculated. The renal oxalate excretion after mineral water intake was significantly reduced compared with tapwater controls. This effect may be caused by intestinal binding of oxalate by calcium and indicates a further lowering of the risk of calcium oxalate crystallization.     

Different estimates of the risk of calcium oxalate crystallization in urine

Different mathematical expressions of urinary calcium, oxalate, magnesium, citrate and urine volume (V), formulated in order to reflect supersaturation with respect to calcium oxalate, were compared with the computer-calculated ion-activity product of calcium oxalate (APCaOx). A good correlation (r = 0.99) was demonstrated for the following two simplified relationships and APCaOx:Ca0.71 X Ox and (Ca0.71 X Ox)/V1.2. The correspondence between Ca/Mg quotients and APCaOx was generally less satisfactory.     

Aspects on estimation of the risk of calcium oxalate crystallization in urine.

A previously formulated simplified estimate of the ion activity product of calcium oxalate, AP(CaOx) index, was modified in order to better correspond numerically to the ion activity product obtained by computed iterative approximation with the EQUIL 2 program. The new index, AP(CaOx) index EQ, was given the following form for a 4-hour urine: 1.9 x Ca(0.84) x Ox x Mg-(0.12) x Cit-(0.22) x V-(1.03), where the excretion of calcium, oxalate (Ox), magnesium and citrate (Cit) was expressed in millimoles excreted during the period, and urine volume (V) in liters. There was a good correlation between AP(CaOx) index and AP(CaOx) index EQ (r = 0.98). A standardized index calculated for a 24-hour urine volume of 1.5 liters, AP(CaOx) index EQ(s), was significantly higher in stoneforming men (p less than 0.001) and women (p less than 0.001) than in normal subjects. The mean ion activity products of CaOx at the start of crystallization in salt solutions and dialyzed urine were 2.74 +/- 0.25 x 10(-8) and 3.50 +/- 0.33 x 10(-8) (mol/l)2, respectively. In the urine from normal subjects, AP(CaOx) index EQ at the point of crystallization was 4.3 +/- 1.1 and in the urine from stone formers 2.8 +/- 0.5 (p less than 0.001). From calculation of AP(CaOx) index EQ in hourly collected urine, it was assumed that the risk of exceeding the level where crystallization starts during periods with the highest supersaturation can be anticipated when the 24-hour AP(CaOx) index EQ value exceeds 2.0.     

Possible role of Tamm-Horsfall glycoprotein in calcium oxalate crystallisation

The role of Tamm-Horsfall glycoprotein (THGP) in the crystallisation of calcium oxalate was investigated. The results showed THGP to have a weak inhibitory effect on crystal growth. In contrast, urinary macromolecules showed a strong inhibitory effect. THGP should, therefore, not be considered the main component of urinary macromolecules which have a strong inhibitory effect on crystal growth. However, THGP did enhance crystal production in the early phase of crystallisation. It appears that THGP should be viewed as a promoter rather than an inhibitor of calcium oxalate crystallisation at physiological concentrations.     

正常人和草酸钙结石病人尿草酸钙晶体基质

以改良Ｍｏｒｓｅ和Ｒｅｓｎｉｃｋ法提取１０例上尿路草酸钙结石病人和１１例正常人的尿草酸钙晶体基璺，用双向聚丙烯酰胺凝胶电泳对晶体基质及结晶前后大分子物的蛋白质组成进行了比较分析。     

The dual effect of urinary macromolecules on the crystallization of calcium oxalate endogenous in urine

Summary The nucleation-promoting and growth-inhibiting activities of urinary macromolecules on the crystallization of calcium oxalate endogenous in urine of stoneformers and normal controls were studied by freezing the ultrafiltrate and retentate fractions of concentrated whole urine (pH 5.3, 1,250 mosmol/kg). Among the normal controls, macromolecules nominally of 10–20 kDa showed nucleation-promoting and growth-inhibiting activities; the 5–10 kDa population was incapable of such effects but did cooperate with molecules >10 kDa in enhancing the effect. In the case of stone-formers, molecules in the nominal ranges of 5–10 kDa and 10–20 kDa when considered separately were not active in the aspects studied but collectively could cooperate to produce pronounced effects. Application of the test to urine ultrafiltrate reconstituted with polyanionic macromolecules recovered from urine indicated that molecules from stoneformers were more powerful than those from normal controls in bringing about promotion of nucleation and inhibition of growth of crystals from urinary calcium oxalate.     

The composition of four-hour urine samples from patients with calcium oxalate stone disease

Urine collected during a 24-h period between 06.00 and 10.00 h from 25 patients with recurrent CaOx stone disease was analysed with respect to calcium, oxalate, magnesium, citrate and creatinine. Urinary excretion of oxalate in relation to creatinine was slightly higher in 24-h urine but the correlation between 24-h and 4-h values was good. Good correlations were also recorded for calcium and citrate, whereas a more variable result was obtained for magnesium. In terms of the risk of forming a supersaturated urine (CaOx risk index), a good correlation was observed between 24-h and 4-h urine samples, although the highest values were found in 24-h urine. As a result of a low mean urine flow between 06.00 and 10.00 h, the highest supersaturation in terms of the AP (CaOx) index was observed in these samples. When the risk of calcium oxalate crystallisation (CaOx-CR) was determined by means of the increment in oxalate concentration required for precipitation of CaOx, 7 of 11 samples had the highest values in the 4-h urine. Samples collected during a 4-h period might thus be useful in the evaluation and follow-up of CaOx stone formers and further studies will show to what extent they can replace 24-h urine collections.     

Intestinal oxalate and calcium absorption in recurrent renal stone formers and healthy subjects

The fractional intestinal absorption of oxalate and calcium was investigated by isotope techniques in 20 normal subjects and in 12 idiopathic calcium oxalate stone formers. The greatest amount of 14C-oxalate was excreted during the first six hour period in controls as well as in stone formers. The stone formers had a greater intestinal uptake of oxalate (11 +/- 5.1%) than the controls (6.2 +/- 3.7%; p less than 0.01). There was no significant relationship between the fractional absorption of oxalate and the total urinary oxalate excretion. The stone formers also had a higher fractional uptake of calcium compared to the controls (55 +/- 11% vs. 47 +/- 9.1%; p less than 0.05). There was a positive relationship (r = 0.47) between the urinary excretions of calcium and oxalate in the stone formers. During these conditions no correlation could be demonstrated between the fractional absorptions of oxalate and calcium, neither in the stone formers nor in the controls. In conclusion, patients with recurrent formation of calcium oxalate containing stones appear to have an enhanced intestinal uptake of both oxalate and calcium. This disturbance could be of primary pathogenic importance for their stone forming propensity.     

The effect of warfarin on urine calcium oxalate crystal growth inhibition and urinary excretion of calcium and nephrocalcin

Summary Urine contains inhibitors of calcium oxalate (CaOx) crystal growth. One such inhibitor is nephrocalcin (NC), a glycoprotein which is made in the kidney and contains several residues of gamma-carboxyglutamic acid (Gla) per molecule. The presence of Gla may be important to its ability to inhibit crystal growth. Several studies suggest that vitamin K-dependent proteins may also play a role in renal calcium (Ca) handling, and that vitamin D deficiency may lead to excess urinary Ca loss, but the effect of the vitamin K antagonist warfarin on urinary Ca excretion and CaOx growth inhibition in humans is not known. We studied 11 men while they were taking warfarin for a mean of 252 days, and again a mean of 64 days after its discontinuation. Urinary Ca excretion did not differ between those on or off warfarin, or between those on warfarin and normal controls. The ability of the subjects' urine to inhibit CaOx crystal growth did not differ on or off warfarin, or from that of control urine, and the excretion of immunoreactive NC also did not differ between these groups. NC was found to be responsible for approximately 16% of the CaOx growth inhibition seen. These results do not suggest that vitamin K-dependent proteins play a major role in renal Ca excretion in men, or that interference with vitamin K alters NC excretion or inhibitory activity of the urine.     

Calcium oxalate crystallisation kinetics and the effects of calcium and gamma-carboxyglutamic acid

gamma-carboxyglutamic acid (GLA) is an amino acid with a high affinity for calcium. It is found in urine both as the free amino acid and incorporated into proteins such as osteocalcin. Free and bound GLA have been reported to be found at higher concentrations in the urine of stone formers than controls. We have investigated the effect of GLA and calcium, at physiological levels, on the crystallisation of calcium oxalate using a mixed suspension mixed product removal continuous crystalliser. GLA caused very significant changes in the crystallisation kinetics, but the effect was dependent on the calcium concentration. At 4 mM calcium, GLA decreased the growth rate and increased the nucleation rate; at 12 mM the reverse occurred. At all concentrations of calcium tested, GLA caused a significantly increased crystal mass to be produced. Our evidence supports the hypothesis that GLA modifies calcium oxalate crystallisation and could be a promoter of stone formation in vivo, particularly at moderately elevated levels of calcium excretion.     

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.