[Quantitative mineralogical analyzes of kidney stones and diagnosing metabolic disorders in female patients with calcium oxalate urolithiasis].

OBJECTIVE: To conduct a complex examination of female patients with calcium oxalate urolithiasis to detect metabolic disorders, leading to stone formation. MATERIALS AND METHODS: The study was carried out using complex physical and chemical methods, including quantitative X-ray phase analysis of urinary stones, pH measurement, volumetry, urine and blood spectrophotometry. RESULTS: Quantitative mineralogical composition of stones, daily urine pH profile, daily urinary excretion of ions of calcium, magnesium, oxalate, phosphate, citrate and uric acid were determined in 20 female patients with calcium oxalate stones. DISCUSSION: We have shown that most of the stones comprised calcium oxalate monohydrate or mixtures of calcium oxalate dihydrate and hydroxyapatite. Among the identified abnormalities, the most frequent were hypocitraturia and hypercalciuria - 90 and 45%, respectively. Our findings revealed that the daily secretion of citrate and oxalate in patients older than 50 years was significantly lower than in younger patients. SUMMARY AND CONCLUSION: In conclusion, daily urinary citrate excretion should be measured in female patients with calcium oxalate stones. This is necessary both to determine the causes of stone formation, and to monitor the effectiveness of citrate therapy.

Differentiating calcium oxalate and hydroxyapatite stones in vivo using dual-energy CT and urine supersaturation and pH values.

RATIONALE AND OBJECTIVES: Knowledge of urinary stone composition can guide therapeutic intervention for patients with calcium oxalate (CaOx) or hydroxyapatite (HA) stones. In this study, we determined the accuracy of noninvasive differentiation of these two stone types using dual-energy CT (DECT) and urine supersaturation (SS) and pH values. MATERIALS AND METHODS: Patients who underwent clinically indicated DECT scanning for stone disease and subsequent surgical intervention were enrolled. Stone composition was determined using infrared spectroscopy. DECT images were processed using custom-developed software that evaluated the ratio of CT numbers between low- and high-energy images. Clinical information, including patient age, gender, and urine pH and supersaturation profile, was obtained from electronic medical records. Simple and multiple logistic regressions were used to determine if the ratio of CT numbers could discriminate CaOx from HA stones alone or in conjunction with urine supersaturation and pH. RESULTS: Urinary stones (CaOx n = 43, HA n = 18) from 61 patients were included in this study. In a univariate model, DECT data, urine SS-HA, and urine pH had an area under the receiver operating characteristic curve of 0.78 (95% confidence interval [CI] 0.66-0.91, P = .016), 0.76 (95% CI 0.61-0.91, P = .003), and 0.60 (95% CI 0.44-0.75, P = .20), respectively, for predicting stone composition. The combination of CT data and the urinary SS-HA had an area under the receiver operating characteristic curve of 0.79 (95% CI 0.66-0.92, P = .007) for correctly differentiating these two stone types. CONCLUSIONS: DECT differentiated between CaOx and HA stones similarly to SS-HA, whereas pH was a poor discriminator. The combination of DECT and urine SS or pH data did not improve this performance.

Supersaturation of body fluids, plasma and urine, with respect to biological hydroxyapatite.

Supersaturation of body fluids, specifically of plasma and urine, with respect to biological hydroxyapatite was evaluated taking into account calcium complexation, fraction of total phosphorus present as hydrogen phosphate ions, solubility of carbonated hydroxyapatite and the size dependency of equilibrium solubility. Plasma is always supersaturated with respect to apatitic solid phase and thus calcific deposits are formed unless a sufficient quantity of potent inhibitors is present. When urinary pH is lower than 6.3 for normal urine hydroxyapatite cannot appear in renal stones, at higher pH apatitic renal stones can be formed. Predictions based on supersaturation calculated for different conditions correspond well with clinical observations.

Aggregation of freshly precipitated calcium oxalate crystals in urine of calcium stone patients and controls.

Aggregation (AGN) of freshly precipitated calcium oxalate crystals was photometrically studied in urine of 30 calcium stone patients and 30 controls, in solutions containing urinary macromolecules (UMS) and in an inhibitor free control solution (CS). Crystals were produced by oxalate titration and crystallization was monitored measuring optical density (OD). Tests were repeated adding hydroxyapatite (HAP) to urine and UMS and adding citrate and pyrophosphate (PPi) to UMS of the controls. AGN was recognized as a rapid OD decrease being at least three times faster than sedimentation of single crystals (p < 0.001) and used to calculate an extent of AGN (EA%). The time between the end of titration and the beginning of AGN was determined as suspension stability (SS). The main effect of urinary inhibitors was retardation of AGN without changing EA, SS being higher in urine than UMS (p < 0.001) and in UMS than CS (p < 0.001). In urine of 63% of controls but only in 33% of patients, no AGN was recorded (p < 0.05). The high inhibitory activity of urine could not be reproduced in UMS even in combination with 3.5 mM citrate or 0.05 mM PPi. 0.05 mg/mL HAP reduced SS in all urine samples to low values and increased the rate of rapid OD decrease, being a measure for the size of aggregates. Retarding AGN of crystals during their passage through the kidney seems to be an important mechanism to prevent stone formation during crystalluria. The promotion of AGN by HAP reveals a new role of Randall's plaques in nephrolithiasis.

Proteomic analysis of proteins selectively associated with hydroxyapatite, brushite, and uric acid crystals precipitated from human urine.

The aim of this study was to compare the intracrystalline protein profiles of hydroxyapatite (HA), brushite (BR), and uric acid (UA) crystals precipitated from the same urine samples. HA, BR, and UA crystals were precipitated on two different occasions from the same pooled healthy urine. Crystals were washed to remove surface-bound proteins, and their composition was confirmed using Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM) coupled with energy dispersive X-ray analysis (EDAX). SDS-PAGE was used for visual comparison of the protein content of the demineralised crystal extracts, which were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). HA comprised nanosized particles interspersed with organic material, which was absent from the BR and UA crystals. The number and type of individual proteins differed between the 3 minerals: 45 proteins were detected in the HA crystal extracts and 77 in the BR crystals, including a number of keratins, which were regarded as methodological contaminants. After excluding the keratins, 21 proteins were common to both HA and BR crystals. Seven nonkeratin proteins were identified in the UA extracts. Several proteins consistently detected in the HA and BR crystal extracts have been previously implicated in kidney stone disease, including osteopontin, prothrombin, protein S100A9 (calgranulin B), inter-α-inhibitor, α1-microglobulin bikunin (AMBP), heparan sulfate proteoglycan, and Tamm-Horsfall glycoprotein, all of which are strong calcium binders. We concluded that the association of proteins with HA, BR, and UA crystals formed in healthy urine is selective and that only a few of the numerous proteins present in healthy urine are likely to play any significant role in preventing stone pathogenesis.

Physiopathology and etiology of stone formation in the kidney and the urinary tract.

All stones share similar presenting symptoms, and urine supersaturation with respect to the mineral phase of the stone is essential for stone formation. However, recent studies using papillary biopsies of stone formers have provided a view of the histology of renal crystal deposition which suggests that the early sequence of events leading to stone formation differs greatly, depending on the type of stone and on the urine chemistry leading to supersaturation. Three general pathways for kidney stone formation are seen: (1) stones fixed to the surface of a renal papilla at sites of interstitial apatite plaque (termed Randall's plaque), as seen in idiopathic calcium oxalate stone formers; (2) stones attached to plugs protruding from the openings of ducts of Bellini, as seen in hyperoxaluria and distal tubular acidosis; and (3) stones forming in free solution in the renal collection system, as in cystinuria. The presence of hydroxyapatite crystals in either the interstitial or tubule compartment (and sometimes both) of the renal medulla in stone formers is the rule and has implications for the initial steps of stone formation and the potential for renal injury.

Calcium oxalate aggregation in whole urine, new aspects of calcium stone formation and metaphylaxis.

OBJECTIVES: To assess the influence of pH, Ca(2+)-concentration, hydroxyapatite (HAP) and preformed calcium oxalate (CaOx) aggregates on the aggregation (AGN) of CaOx crystals directly produced in unpretreated whole urine (U) by oxalate loads (OL). METHODS: After OL at pH 5.0 and pH 6.5 minimal sedimentation time of precipitates (ST = minutes for 0.05 optical density [OD] decrease) was measured in 40 U of 5 healthy men by spectrophotometry. An ST(P) (< or =2.8) was taken as indicator for primary AGN and an ST(S) (< or =1.4) as one for secondary AGN. In 20 U Ca(2+) was determined initially, Ca(2+) at pH 6.5 was readjusted by adding CaCl(2) to the value measured at pH 5.0 and an OL of 1.5mM performed. OL of 0.25-0.75 mM were given to 20 U either with 0.05 mg/ml HAP or after a primary OL of 2mM. RESULTS: Alkalinization of U from pH 5.0 to 6.5 decreased Ca(2+) by 44+/-15% (mean+/-S.D.) and, in U with total Ca <3mM, below a crucial value of 1mM where no ST(P) was observed. At identical Ca(2+), pH had no influence on ST. With HAP, an ST(P) was found after an OL of 0.5mM in 10% and of 0.75 mM in 35%, predominantly at pH 5.0. An ST(S) was observed after a second OL of 0.5mM in 55% and of 0.7 5mM in 75% of experiments. CONCLUSIONS: Provided that AGN is important for stone formation, calcium nephrolithiasis might be initiated at high urinary Ox and low pH by HAP of kidney calcifications, prevented at moderate calciuria by alkali treatment and augmented during relative hyperoxaluria by secondary AGN.

Synergism between the brushite and hydroxyapatite urinary crystallization inhibitors.

The aim of this paper is to study possible synergic effects between crystallization-inhibitor molecules of low molecular weight on the hydroxyapatite and brushite crystal nucleation. Kinetic-turbidimetric measurements were performed to follow the nucleation process in synthetic urine at 37 degrees C. Only pyrophosphate + phytate mixture manifested synergic effects on the brushite nucleation, whereas the mixture pyrophosphate + citrate exhibited synergic effects only on the hydroxyapatite nucleation. It seems clear that synergic effects between the crystallization inhibitory capacity of some substances in urine can take place and as a consequence, the high crystallization inhibitory capacity of healthy urine could be assigned not only to the individual inhibitory capacity of each product but also to the synergic effects between different products.

A novel type of milk calcium has higher calcium bioavailability compared with the hydroxyapatite type of milk calcium.

We prepared a novel type of milk calcium from milk whey, and evaluated its calcium bioavailability as compared with conventional industrial milk calcium obtained from milk whey, which has been reported to be an excellent calcium source. An X-ray diffraction analysis showed that the chemical form of conventional industrial milk calcium from milk whey through the neutralization and precipitation method (NP-MCa) is hydroxyapatite type, and that of a novel type of milk calcium obtained using the ultrafiltration method (UF-MCa) is different from hydroxyapatite. Growing rats were fed either NP-MCa or UF-MCa as the source of calcium for 33 d. The calcium absorption rate in the UF-MCa diet group was significantly higher than that in the NP-MCa diet group. The calcium content of the femur in the UF-MCa diet group was significantly higher than that in the NP-MCa diet group. The breaking force of the excised femur in the UF-MCa diet group was also significantly higher than that in the NP-MCa diet group. The serum calcium level, alkaline phosphatase activity and calcitonin level were not significantly different between the experimental groups, but the PTH level of the serum in the UF-MCa diet group was significantly lower than that in the NP-MCa diet group. These results indicate that the calcium bioavailability of the novel type of milk calcium (UF-MCa) is higher than that of the hydroxyapatite type of milk calcium (NP-MCa).

Hydroxyapatite induction and secondary aggregation of calcium oxalate, two important processes in calcium stone formation.

Stone formation has often been ascribed to crystal aggregation and fixed particle growth on kidney calcifications. In this paper, the influence of hydroxyapatite (HAP) and of preformed calcium oxalate (CaOx) aggregates on CaOx crystallization was studied in freshly voided urine. Crystallization was induced by different oxalate loads and precipitates were analyzed by the spectrophotometric measurement of sedimentation time (ST), which decreases with increasing particle size. The fact that the ST of aggregates (STA) is significantly lower than the ST of other particles demonstrates that STA is a useful indicator for aggregation. At relatively low oxalate loads the addition of HAP to urine increased STA by a factor of 4.3 (P < 0.001). After a second oxalate load, STA decreased by 56% (P < 0.001), indicating secondary growth of the preexisting aggregates. HAP induced and primary CaOx aggregation occurred at low pH at which a high ionic calcium concentration (Ca2+) was measured. In urine, crystals are coated by macromolecules creating a negative surface potential with a consecutive accumulation of cations such as Ca2+. This Ca2+ accumulation could be responsible for the enhancement of aggregation by preexisting particles, which seems to be important for stone formation and which can otherwise hardly be explained in the presence of coated crystals.

Crystallization during volume reduction of solutions with a composition corresponding to that in the collecting duct: the influence of hydroxyapatite seed crystals and urinary macromolecules.

To examine the effect of hydroxyapatite (HAP) seed crystals and urinary macromolecules on the crystallization under conditions similar to those in the collecting duct, we evaporated 100 ml samples of salt solutions with an ion composition assumed to correspond to that in the collecting duct without and with HAP seed crystals. The crystallization in seeded solutions was assessed both with and without dialysed urine (dU). After evaporation the number and volume of crystals were recorded in a Coulter Multisizer and the crystal morphology examined with scanning electron microscopy (SEM) and X-ray crystallography. Addition of HAP crystals was apparently followed by an approximately 15-20% increase in heterogeneous nucleation of calcium oxalate (CaOx). In these experiments SEM and X-ray crystallography showed a high percentage of CaOx in the precipitate. In samples reduced to 40-69 ml, addition of dU to the collecting duct solution containing HAP seed resulted in a greater mean (SD) number of crystals; 3895 (1841) in samples with dU and 1785 (583) in samples without. This was mainly explained by an increased mean (SD) number of small crystals. The mean crystal volume was 17.8 (1.1) and 34.3 (9.1) in samples reduced to 40 69 ml with and without dU, respectively. This might reflect the inhibitory effect of dU on the growth and/or aggregation of the CaOx-CaP precipitate or a promoted nucleation resulting in a large number of small crystals. It is concluded that calcium phosphate formed above the collecting duct might induce heterogeneous nucleation of CaOx at lower levels of the renal collecting system, and that urinary macromolecules are powerful modifiers of these processes.

The effects of citrate and urinary macromolecules on the aggregation of hydroxyapatite crystals in solutions with a composition similar to that in the distal tubule.

The effects of citrate and dialysed urine (dU) on the aggregation of hydroxyapatite (HAP) crystals in solutions with different pH and otherwise with an ion composition assumed to correspond to that in the distal part of the distal tubule were studied by spectrophotometric assessment of the rate of crystal sedimentation. When the concentration of dU was increased from 1% to 20% we recorded an increased inhibition of HAP crystal aggregation at pH 6.5. There were no differences in the inhibition accomplished by 10% dU when the pH was varied between 5.5 and 7.0, but a lower inhibition was recorded at pH 7.5. Citrate in the range 0.05-4 mmol/l had a concentration-dependent inhibitory effect on HAP crystal aggregation. In the presence of 10% dU the net inhibitory effect of citrate was reduced at all pH levels. In the pH interval 5.5-7.0 a higher inhibition was recorded with 0.5 mmol/l citrate than with 10% dU, but in the presence of dU there was only a minor additional effect of citrate at concentrations below 0.5 mmol/l. These findings indicate that urinary macromolecules present in dU strongly inhibit HAP crystal aggregation under solution conditions corresponding to those in distal tubular urine. At the same nephron level citrate might have a direct inhibitory influence on the aggregation of HAP crystals, but in the presence of normal urinary macromolecules the additive inhibitory effect of citrate is probably only marginal.

Circadian rhythms of urinary saturation levels of calcium oxalate and calcium phosphate in normal male individuals.

The circadian rhythms of the calcium oxalate, calcium phosphate, and brushite saturation levels as estimated by the AP(CaOx), AP(CaP), and AP(Bru) indices (Tiselius), respectively, were studied in 5 healthy males on three different occasions. These indices were calculated from the data of urinary specimens collected in 2.5-hour fractions except during sleep. The calcium oxalate and brushite saturation levels peaked between 5:30 and 8:00 am, and these were significantly higher than in the other periods. The calcium phosphate saturation level had two peaks that occurred between 8:00 and 10:30 am and between 1:00 and 6:00 pm. The ion-activity products of octacalcium phosphate and hydroxyapatite, calculated from the AP(CaP) index, exhibited a pattern similar to the AP(CaP) values, indicating a high risk of crystallization for both substances at around the same peak periods. In conclusion, early morning was found to be the high risk period for calcium oxalate and brushite crystallization, while two high risk periods for octacalcium phosphate and hydroxyapatite were detected between 8:00 and 10:30 am and between 1:00 and 6:00 pm.