CaOx Crystal Aggregation Research Articles

The role of osteopontin on calcium oxalate crystal formation.

We evaluated whether osteopontin (OPN) and other proteins with the RGD sequence as in OPN (RGD family proteins) that are present in renal tubular cells (fibronectin [FN], Tamm-Horsfall glycoprotein [THP], vitronectin [VN], and laminin [LN]) inhibit the aggregation and growth of calcium oxalate (CaOx) crystals by a novel seed crystal method using collagen granules (CG) with and without OPN adhered on the surface. We also evaluated the effect of solid phase OPN, FN and THP in which the relationship between their proteins and CaOx crystallization was reported. Moreover, the state and time-course changes in CaOx crystals adhered to CG were observed under scanning electron microscopy (SEM). The inhibitory activity (IA) on the aggregation and growth of CaOx crystals was measured in vitro by the conventional seed crystal method using isotopes. In this study, the following nine samples were used: OPN alone; FN alone; THP alone; VN alone; LN alone; CG alone; and CG with OPN, FN, or THP adhered on the surface (OPN/FN/THP-immobilized CG). In addition, the state and time-course changes in CaOx crystals adhered to CG were evaluated by SEM. Using the conventional seed crystal method, the following values of IA were obtained: 91.7% (37.5 micro g/ml) for OPN, 5.0% (100 micro g/ml) for FN, 2.0% (100 micro g/ml) for THP, 3.0% (100 micro g/ml) for VN, and 1.0% (100 micro g/ml) for LN. However, the value of IA obtained by our seed crystal method using CG was 92.1% (180cm(2)/5ml PBS) when CG alone was used. Although the value of IA was decreased by 33.6% when OPN-immobilized CG was used, it did not significantly change when FN/THP-immobilized CG was used. When CG alone was used, the evaluation of CaOx crystallization by SEM demonstrated mild adherence and aggregation of CaOx crystal suspension (seed crystals) on the CG surface, although newly formed crystals only slightly adhered to the CG surface. When OPN-immobilized CG was used, marked adherence and aggregation of seed crystals were observed, in addition to the relatively increased adherence of newly formed crystals. When FN/THP-immobilized CG was used, newly formed crystals only slightly adhered to the CG surface, although the degree of seed crystal adherence and aggregation did not significantly change. These findings suggest that the immobilization of OPN to the CG surface enhances the adherence and aggregation of seed crystals, as well as enhancing the adherence of newly formed crystals, resulting in decreased IA of CG (overall promotion of crystal deposition). Therefore, the results of this study clarified that OPN enhances the formation and aggregation of CaOx crystals in this experimental system.

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

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). 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. 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. 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.

Effects of an aqueous extract from Phyllantus niruri on calcium oxalate crystallization in vitro.

Phyllanthus niruri is a plant used in Brazilian folk medicine for the treatment of urolithiasis. It was previously observed that P. niruri shows no toxicity, potentially increases calculus voiding by stone forming patients and inhibits the endocytosis of calcium oxalate (CaOx) crystals by MDCK cells. In addition, in a rat model of urolithiasis it reduced calculus growth. In the present study, we evaluated the effect of an aqueous extract of P. niruri on CaOx crystallization in vitro. CaOx precipitation was induced by the addition of 0.1 M sodium oxalate to unfiltered urine samples from Wistar rats (n=14) and normal humans (n=18) in the presence or absence of P. niruri extract (0.25 mg/ml of urine). The presence of CaOx crystals was evaluated immediately and 24 h later. In vitro crystallization of human urine produced typical mono- and dihydrated CaOx crystals, but only a few typical CaOx crystals were found in rat urine. The presence of P. niruri extract did not inhibit CaOx precipitation and even more crystals were obtained, although they were significantly smaller than those in the control urine. Crystal aggregation observed 24 h after crystallization was also inhibited by P. niruri extract. The results showed an inhibitory effect of P. niruri extract on CaOx crystal growth and aggregation in human urine, suggesting that it may interfere with the early stages of stone formation and may represent an alternative form of treatment and/or prevention of urolithiasis

An oxalate-binding protein with crystal growth promoter activity from human kidney stone matrix.

To fractionate renal-stone matrix proteins, identify the presence of oxalate-binding protein and assess its effect in a calcium oxalate (CaOx) crystal growth system. Proteins were isolated from the matrix of kidney stones containing CaOx as the major constituent, using EDTA as a demineralizing agent. The solubilized proteins were subjected to cellulose-column chromatography by eluting with increasing sodium chloride concentrations in Tris-HCl buffer. Three protein fraction peaks were eluted, i.e. fraction I in buffer, fraction II in 0.05 mol/L NaCl in buffer and fraction III in 0.3 mol/L NaCl in buffer. The protein fractions were tested for their effects on CaOx crystal growth. All three fractions had maximum CaOx binding activity at pH 7.4 but fraction II also had activity at pH 4.5. Fraction I promoted in vitro CaOx crystal growth, while fractions II and III were inhibitory. When fraction I was further separated on a Sephadex G-200 column, two protein fractions (Ia and Ib) were obtained. Fraction Ia protein had high and fraction Ib low CaOx-binding activity. Fraction Ia had a molecular weight of 48 kDa on gel electrophoresis and Western blotting. The 48 kDa protein did not cross-react with crystal matrix protein antibody, band-3 protein antibody, or albumin. The protein promoted CaOx crystal growth, with an optimum temperature of 37 degrees C and pH 6.5. The inhibitory effect of citrate on crystal growth was significantly lower in the presence of the 48 kDa protein. The protein promoted nucleation and aggregation of CaOx crystals in the in vitro crystallization system at pH 6.5, whereas fraction Ib (29 kDa) inhibited both nucleation and aggregation. Using the 48 kDa antibody, the yield of the protein from the stone matrix was 32% by EDTA extraction and only 3% with other methods. The protein was also detected in the nucleus and mitochondria, and in other matrix fractions of calcium phosphate and uric acid stones. The 48 kDa protein isolated from stone matrix is a potent promoter of CaOx crystal growth with high oxalate-binding activity; it is enriched in the nucleus and mitochondria.

Influence of Urinary Sialic Acid on Calcium Oxalate Crystal Formation

Using seed crystal method, whole-urine method, and scanning electron microscopy, the inhibitory effects of sialic acid and osteopontin (OPN) on aggregation/growth of CaOx crystals were investigated. Using the seed crystal method, sialic acid showed an inhibitory effect on CaOx crystal aggregation/growth in a concentration-dependent manner, but almost no effect was observed using the whole-urine method. OPN showed an inhibitory effect on aggregation/growth in both experimental systems. The inhibitory effect of asialo-OPN on aggregation/growth was approximately 20% lower than that of OPN in the experiment using the seed crystal method and approximately 15% lower in the experiment using the whole-urine method. Scanning electron microscopy showed that OPN and sialic acid inhibit the aggregation of CaOx crystals. The above findings show that sialic acid accounts for about 15–20% of the involvement of OPN in CaOx crystallization.

Effect of prothrombin and its activation fragments on calcium oxalate crystal growth and aggregation in undiluted human urine in vitro: relationship between protein structure and inhibitory activity

In recent years there has been great interest in the putative role of prothrombin and its activation peptides, especially the urinary form of prothrombin fragment 1, in the pathogenesis of calcium oxalate (CaOx) urolithiasis. Previously, we showed that prothrombin and its activation peptides inhibit CaOx crystallization in inorganic conditions in vitro. The aim of the present study was to determine if this inhibitory activity is retained in undiluted human urine and, therefore, whether it is likely to have any influence under physiological conditions. A secondary objective was to assess the relationship between the structures of the proteins and their inhibitory activities. Prothrombin was purified from Prothrombinex-HT, cleaved with thrombin and the resulting fragment 1 (F1) and fragment 2 (F2) were purified. The purity of each protein was confirmed by SDS/PAGE, and their effects on CaOx crystallization in undiluted ultrafiltered human urine were determined at a final concentration 80.65 nmol/l using Coulter Counter and [(14)C]oxalate analysis. The precipitated crystals were visualized using scanning electron microscopy. The Coulter Counter data revealed that, whereas prothrombin and its activation peptides did not affect the urinary metastable limit and the size of the precipitated particles, F1 did significantly reduce the latter. These findings were corroborated with scanning electron microscopy which also revealed that the reduction in particle size caused by F1 resulted from a decrease in the degree of crystal aggregation, rather than in the size of the individual crystals. The [(14)C]oxalate data showed that none of the proteins added significantly inhibited the mineral deposition. It was concluded that with the exception of F1, which does inhibit CaOx crystal aggregation, prothrombin and its activation peptides do not alter the deposition and aggregation of CaOx crystals in ultrafiltered human urine in vitro. Also, the gamma-carboxyglutamic acid domain of prothrombin and F1, which is absent from thrombin and F2, is the region of the molecules that determines their potent inhibitory effects. The superior potency of F1, compared with prothrombin, probably results from the molecule's greater charge-to-mass ratio.

Comparison of Fibronectin Content in Urinary Macromolecules between Normal Subjects and Recurrent Stone Formers

Objectives: Fibronectin (FN: 230 kD) is a multifunctional α₂–glycoprotein distributed throughout the extracellular matrix and body fluids. Recent studies have shown that a variety of molecules, including FN, inhibit the endocytosis of calcium oxalate (CaOx) crystals in vitro. We recently reported that FN was oversecreted from the renal tubular cells as a result of the stimulation of CaOx crystals, and inhibited the aggregation of CaOx crystals and the adhesion of CaOx crystals to the renal tubular cells. In the present study, we investigated the difference of FN content in urinary macromolecules (UMMs) between normal subjects and recurrent stone formers. Materials and Methods: Urinary parameters in relation to urolithiasis of normal subjects and recurrent stone formers were measured. Proteins in extracted UMMs from urine of normal subjects and recurrent stone formers were measured with a BioRad protein assay, GAGs in each UMMs with a modified DMB assay and the FN content with the ELISA method. Results: In urinary parameters, citrate was significantly higher in urine from normal subjects (female) than normal subjects (male) or recurrent stone formers, and the other parameters showed no differences between each group. The protein concentrations in UMMs showed no differences between each group. Normal subjects (male and female) showed a significantly higher concentration of GAGs than recurrent stone formers (with and without silent stone). Compared with normal subjects and recurrent stone formers without silent stones, higher FN levels were found in recurrent stone formers with silent stones. Normal subjects showed a significantly higher concentration of FN than recurrent stone formers without silent stones. No difference in FN level was shown between normal subjects (male) and normal subjects (female). Conclusion: Recurrent stone formers with silent stones showed a significantly higher concentration of FN in UMMs than normal subjects. This finding suggests that FN might be oversecreted from the renal tubular cells as a result of the stimulation of CaOx stones in vivo. Recurrent stone formers without silent stones showed a significantly lower concentration of FN in UMMs than normal subjects. From this finding it is suggested that FN might play a role as a potent inhibitor of CaOx urolithiasis in a clinical setting.

The effect of osteopontin immobilized collagen granules in the seed crystal method.

Osteopontin (OPN) is a urinary protein which inhibits calcium oxalate (CaOx) crystal growth in the seed crystal system. The aim of this study was to evaluate the effects of OPN immobilized on collagen granules (CG) for CaOx crystal growth and aggregation in this system. OPN-immobilized CG showed a 30% decrease in inhibitory activity compared with non-OPN-immobilized CG. Scanning electron microscopy revealed that 1) OPN-immobilized CG showed more marked aggregation and adhesion of seed crystals (smaller than 0.5 microm in diameter) than non-OPN-immobilized CG, 2) the amount of adherent large type crystals (2-3 microm in diameter) did not differ between OPN immobilized and non-OPN-immobilized CG. In conclusion, immobilized OPN promotes aggregation and adhesion of CaOx crystals smaller than 0.5 microm on CG. These observations suggest that crystal adhesion to immobilized OPN plays a role in the retention of crystals in the kidney.

Crystallisation properties in stone forming and normal subjects' urine diluted using a standardised procedure to match the composition of urine in the distal part of the distal tubule and the middle part of the collecting duct.

Using a standardised procedure, we assessed the crystallisation properties of calcium phosphate in urine with a composition matching that in the distal part of the distal tubules (DTd) and of calcium oxalate in urine with a composition matching that in the mid-collecting duct (CDm). We used 8-h urine samples collected between 2200 h and 0600 h with sodium azide as preservative. Urine from ten patients with recurrent CaOx stone formation and from ten normal subjects was used for the measurements. The DTd and CDm samples were obtained by diluting the voided 8-h urine to 3000 ml and 1750 ml per 1.73 m2 body surface area, respectively. The nucleation was studied in DTd urine following supersaturation with CaP. The crystal size distribution was assessed with a Coulter counter both following supersaturation of DTd urine with CaP and of CDm urine with CaOx. The crystallisation of CaP in DTd urine as well as that of CaOx in CDm urine, in the presence of CaP crystals that had been precipitated in DTd urine, was measured with the isotope technique. The inhibition of CaOx and brushite crystal aggregation in standardised diluted aliquots of DTd and CDm urine was assessed spectrophotometrically as the rate of sedimentation. There was a slightly increased sedimentation rate and a lower initial absorbance in DTd urine from stone formers supersaturated with CaP. Although these findings might reflect a state of increased crystal aggregation in stone formers' urine, this could not be confirmed by crystal size measurements in the Coulter counter. The inhibition of brushite crystal aggregation in DTd urine was significantly in stone formers' urine than in normal subjects' urine (P < 0.001). Moreover, all inhibition values in DTd samples from stone formers were negative, suggesting a promoter effect on crystal aggregation. The inhibition of CaOx crystal aggregation in CDm urine also was significantly higher in CDm urine from normal subjects than in CDm urine from stone formers (P < 0.05). For all other variables the level was similar when urine samples from the two groups were compared. Although this series of crystallisation assessments was carried out on a small number of standardised diluted urine samples only, the results nevertheless emphasise a defect in aggregation inhibition as one important determinant for an abnormal calcium salt crystallisation in patients with recurrent stone formations. This difference obviously includes aggregation of both CaP crystals in DTd urine and CaOx crystals in CDm urine. The results also show that assessment of crystallisation properties of this kind can be carried out in standardised, diluted 8-h night urine samples, which accordingly can be used in the routine work-up of patients with calcium stone disease. Such an approach might prove useful in order to get information on the combined effects of the driving force of supersaturation and crystallisation modifying properties accomplished by urinary macromolecules and other modifying agents.

Citrate determines calcium oxalate crystallization kinetics and crystal morphology-studies in the presence of Tamm-Horsfall protein of a healthy subject and a severely recurrent calcium stone former.

The aim of this study was to measure the effects of normal (nTHP) and abnormal stone former Tamm-Horsfall protein (SF-THP) on calcium oxalate (CaOx) nucleation and aggregation as well as on crystal morphology, in presence or absence of citrate. Nucleation and aggregation of CaOx crystals from a supersaturated, stirred solution (200 mM NaCl, 10 mM Na-acetate, pH 5.70, 5 mM Ca and 0.5 mM Ox) were studied by spectrophotometric time-course measurements of OD at 620 nm (OD(620)). Measured parameters were induction time t(I) (time to induce formation of detectable particles), S(N), (slope of increase of OD(620), mainly due to crystal nucleation), and S(A), (slope of decrease of OD(620) after equilibrium has been reached, due to crystal aggregation). Effects of citrate, nTHP and SF-THP on these parameters were measured, and scanning electron microscopy (SEM) was performed. At 1.5, 2.5 and 3.5 mM, citrate increased t(I) and inhibited crystal nucleation (by 78-87%) as well as aggregation (by 63-70%), and smaller CaOx crystals (length/width ratio 1.7+/-0.1) than under standard conditions (length/width 3.9+/-0.5) were visible (P<0.001). Normal THP at 30 and 40 mg/l inhibited crystal nucleation and, more strongly, aggregation (inhibition 76-81%). SEM revealed a decrease in length/width ratio to 2.6+/-0.4 (P=0.051 vs standard conditions) and less aggregation than without nTHP. At all concentrations tested, SF-THP reduced t(I) (P=0.0001 vs standard conditions) and promoted aggregation (inhibition -48 to -33%); crystals were elongated with a length/width ratio of 4.3+/-0.6 (P<0. 05 vs nTHP). When simultaneously present with nTHP, citrate enhanced the inhibitory effects of nTHP, producing the smallest (length/width 1.5+/-0.1) and least aggregated crystals. Finally, 3.5 mM citrate turned promotory SF-THP into a crystallization inhibitor with abundant small and clustered, but not aggregated crystals. Citrate appears to be the main determinant of CaOx crystallization rates and crystal morphology in the presence of nTHP as well as SF-THP. Its effects appear to predominate over those of THP, since even promotory SF-THP is turned into a crystallization inhibitor in the presence of citrate. This re-emphasizes at a morphological level what has been concluded from functional as well from clinical studies, namely that citrate is needed in urine at equimolar concentrations to calcium in order to prevent the formation of large crystal aggregates in presence of abnormal THP.

Structural characteristics of osteopontin for calcium oxalate crystal

I investigated which structural segment of osteopontin (OPN), a matrix component of urinary stones, is significantly related to the formation of urinary stones. I prepared several kinds of OPNs under various conditions and compared the effects of these OPNs on calcium oxalate (CaOx) crystal using RI counts obtained by the seed crystal method and diluted urine method. Furthermore, I performed scanning electron microscopic (SEM) observation of CaOx crystals used in these experiments and evaluated the effects of OPN based on morphological changes in CaOx crystals. The following OPNs were used in this study: human recombinant OPN (rOPN), human native OPN (nOPN) purified from human milk, denatured OPN (dOPN) obtained by adding organic solvent during the course of nOPN purification, and asiaro OPN (aOPN) obtained by removing sialic acid after enzymatic digestion of nOPN. When the effects of OPNs (15 micrograms/ml) were evaluated by the seed crystal method, the following inhibitory activities were observed: nOPN (82%), aOPN (56%), dOPN (49%) and rOPN (15%). When the effects of OPNs (150 micrograms/ml) were evaluated by the undiluted urine method, the following inhibitory activities were observed: nOPN (38%), aOPN (27%), dOPN (21%) and rOPN (0%). Furthermore, using nOPN, I performed SEM observation of CaOx crystals and found that nOPN mainly inhibited CaOx crystal aggregation. Since the inhibitory activity of nOPN was observed not only in the seed crystal method, but also in the undiluted urine method, it was suggested that nOPN may play an important role in the living body during the course of urinary stone formation. Moreover, the inhibitory activity of OPN was not due to its primary structure, but it was closely related to its higher-order structure and side chains including sialic acid. Furthermore, it was clarified that the inhibitory activity of OPN mainly resulted from inhibition of CaOx crystal aggregation rather than growth inhibition in these crystals.

Evidence that calgranulin is produced by kidney cells and is an inhibitor of calcium oxalate crystallization.

Urine produced by normal human kidneys is almost always supersaturated with respect to calcium oxalate (CaOx), the most common constituent of human kidney stones. Crystallization, with risk of renal damage and kidney stones, appears to be affected by molecules in urine that retard nucleation, growth, aggregation, and renal cell adherence of CaOx. The repertoire of such molecules is incompletely known. We have purified a 28-kDa protein from urine using salt precipitation, preparative isoelectric focusing, and sizing chromatography. Amino acid composition and NH2-terminal amino sequence analysis showed complete homology to calgranulin. Calgranulin was found to be a potent inhibitor of CaOx crystal growth (44% of control) and aggregation (50% of control) in the nanomolar range. Calgranulin cDNA was cloned from a human kidney expression library. Western analysis of human and rat kidney homogenates and mRNA temporal expression from two independent renal epithelial cell lines showed that calgranulin is produced in the kidney. Given its urinary abundance and potency, calgranulin may contribute importantly to the normal urinary inhibition of crystal growth and aggregation and therefore to the renal defense against clinical stone disease.

Identification of structural domains in inter-α-trypsin inhibitor involved in calcium oxalate crystallization

The urinary glycoprotein that inhibits calcium oxalate (CaOx) crystallization in vitro shows a structural similarity to urinary trypsin inhibitor (UTI; recently termed bikunin), the light chain of inter-alpha-trypsin inhibitor (I alpha I). The functional domains of I alpha I involved in its inhibitory activity of CaOx crystallization have been investigated using isolated intact domains of I alpha I produced from controlled proteolytic digests of the protein. The fragments investigated include the heavy chains of I alpha I, UTI, chondroitinase AC-treated UTI, and the carboxyl-terminal domain of UTI (termed HI-8). The effects of I alpha I and its fragments on the inhibitory activity of CaOx crystallization were evaluated in vitro using CaOx crystal aggregation and growth assays, and seeded crystal generation assay as well as using crystal matrix protein generation assay. UTI, but not the heavy chains of I alpha I, had a discernible effect on CaOx crystallization inhibitory activity. Less requirement of the carbohydrate moiety of UTI is implicated by the observation that chondroitinase AC-treated UTI fragment was also found to inhibit CaOx crystallization with almost the same activity as UTI. HI-8 also efficiently inhibited CaOx crystallization, while I alpha I showed a weak inhibitory activity. The results are almost consistent with a seed crystal generation assay and a crystal adsorption inhibition assay, in which I alpha I or its derivatives inhibits prothrombin fragment 1 (F1) adsorption to CaOx crystals. In conclusion, these results suggest that the part of the I alpha I protein responsible for inhibition of CaOx crystallization is the carboxyl-terminal domain of UTI.

Repeated Urine Analysis in Patients with Calcium Stone Disease

Objective: To study the variability in urine composition with respect to factors of importance for the calcium salt crystallization process and to test the reliability of using one or several urine samples in the clinical evaluation. Patients and Methods: Twelve patients collected 16-hour daytime and 8-hour night urine samples during 4 days of the same week. The urine was analysed for calcium, oxalate, phosphate, magnesium, citrate and pH, and the ion activity products of CaOx [AP(CaOx) index] and CaP were calculated. The risk of CaOx crystallization, as well as the inhibition of CaOx crystal growth and aggregation, were assessed. Results: There was a good correlation between estimates of the AP(CaOx) index in the different samples, as well as between the AP(CaOx) index and the direct assessment of the risk of CaOx crystallization in the night and daytime urine samples. There was, however, a pronounced intra-individual variation of all variables and parameters. With the assumption that an abnormality would appear in at least one of the four samples, we found that in more than 80% of the cases, two 24-hour (16 + 8 h) urine samples were sufficient to establish whether the patient had a normal or an abnormal urine composition. Conclusion: Urine samples collected during two 24-, 16- or 8-hour periods appear to be useful for detecting biochemical abnormalities considered of importance for CaOx stone formation.

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.

Sialic acid determinations of serum and urine specimens in calcium-containing urinary stone formers

The aim of the present study was to compare sialic acid concentrations of serum and urine specimens in both calcium (Ca)-containing urinary stone formers and non-stone formers. Moreover, we studied inhibitory activity of sialic acid upon the calcium oxalate (CaOx) crystal aggregation and growth. Sialic acid determinations were done on fresh serum and urine samples of 35 Ca-containing urinary stone formers (stone formers group) and 20 non-stone formers (patient controls group). Inhibitory activity of sialic acid upon the CaOx crystal aggregation and growth was studied by using in vitro assay method of seed crystal system. Serum sialic acid concentrations were found to be similar in the two groups. Urinary sialic acid concentrations were significantly lower in the urine specimens of stone formers than in their patient controls. Sialic acid showed a dose dependent inhibitory activity upon the CaOx crystal aggregation and growth into seed crystal method. It is suggested that urinary sialic acid may play some role during the phase of stone formation from the results of the present study, because sialic acid shows marked inhibitory activity upon the CaOx crystal aggregation and growth at concentrations higher than 100 mg/dl.

Calcium oxalate crystal matrix extract: the most potent macromolecular inhibitor of crystal growth and aggregation yet tested in undiluted human urine in vitro.

Demineralization of calcium oxalate (CaOx) crystals precipitated from human urine in vitro yields an organic crystal matrix extract (CME) consisting predominantly of a single protein which we originally named crystal matrix protein but have subsequently shown to be a urinary form of prothrombin activation peptide fragment 1 (F1). The aim of this study was to determine whether CME is a promoter or inhibitor of CaOx crystallization. The effect of CME on CaOx crystal growth and aggregation was tested using a standard seeded crystallization system, and its effect quantified by use of particle size analysis and a computer model. In addition, the effect of CME on the crystallization of CaOx was tested in undiluted, ultrafiltered human urine using Coulter Counter analysis and scanning electron microscopy. It was shown that CME is a potent inhibitor of CaOx crystal growth and aggregation in a seeded metastable solution. However, of greater significance is that at a concentration of 10 mg/l it completely reversed the formation of large crystalline aggregates that form upon the removal of urinary macromolecules from undiluted urine. It was concluded that CME is the most potent macromolecular urinary inhibitor yet to be tested in urine in vitro. By preventing the aggregation of newly formed crystals, the components of CME may significantly reduce the probability of particle retention in vivo and therefore the occurrence of urolithiasis.

Does Tamm-Horsfall mucoprotein inhibit or promote calcium oxalate crystallization in human urine?

Using two different experimental techniques, Tamm-Horsfall mucoprotein (THM) has been reported both to inhibit and to promote calcium oxalate (CaOx) crystallization in ultrafiltered human urine. In this study, these two techniques were used to compare the effects of THM on CaOx crystallization in the same ultrafiltered urine samples. Urine was collected from 10 healthy men and ultrafiltered (10000 Da). Each sample was divided and to one half was added sufficient human THM to give a final concentration of 35 mg/L. CaOx crystallization was induced in the samples by addition of an oxalate load and by evaporation. Using the evaporation technique THM significantly increased the deposition of CaOx determined as 14C-oxalate, from 9772 cpm to 43652 cpm ( P < 0.01). Using the oxalate load method THM had no effect on the metastable limits of the urine with respect to CaOx, and significantly increased the volume of particulate material deposited from 26000 to 39995 μm 3/μl - an increase of 54%. This increase was reduced to 21% when values were corrected for the volume of THM particles recorded in control samples to which no oxalate load was added. Using 14C-oxalate, it was shown that this increase in volume could not be attributed to an enhanced deposition of crystalline CaOx, but was probably the result of an increased polymerization of THM in the presence of CaOx crystals. Despite this, the average size of the particles precipitated in the presence of THM (6.5 μm) was significantly ( P < 0.01) less than that observed in the absence of THM (12.1 μm). It was concluded that the effect of THM on CaOx crystallization in urine depends upon the methodology used to assess it and that promotion would only be expected in vivo in cases of extreme dehydration. Under usual physiological conditions THM would be expected to inhibit CaOx crystal aggregation and to have little effect, if any, on the amount of crystalline material deposited.