Role of calcium in the aggregation of particles coated by urinary macromolecules.

OBJECTIVES: In urine, aggregation (AGN) of crystal occurs although they are coated by negatively charged urinary macromolecules (UM) and isolated at a distance from each other, where attraction forces become extremely weak. Calcium (Ca) bridges or viscous binding by UM could explain this AGN. METHODS: Suspensions of Ca oxalate monohydrate (COM) and carboxylated latex (CL) were prepared in buffered water and UM solutions which were obtained from the urine of 6 healthy men. Surface potential or zetapotential (ZP) of the particles was measured at various concentrations of Ca and UM. AGN of CL was monitored determining particle size and optical density. RESULTS: The ZP of COM was neutralized by Ca and stabilized around -15 mV by UM. In CL, a stable ZP of about -20 mV was achieved at >3 mM Ca. However, to induce AGN further addition of Ca was necessary. AGN then increased independently of ZP with increasing Ca and decreased with increasing UM concentration. CONCLUSIONS: In CL, two different Ca effects could be distinguished, one on ZP and another on AGN. The latter is probably based on the formation of Ca bridges between particles and is inhibited by a steric barrier of adsorbed UM. A similar mechanism is postulated for crystal aggregation.

Stabilization of calcium oxalate suspension by urinary macromolecules, probably an efficient protection from stone formation.

INTRODUCTION: Crystal aggregation (AGN) destabilizes crystal suspensions and during crystalluria probably favors crystal apposition to kidney calcifications and preexisting stones. We analyzed inhibition of AGN and stabilization of calcium oxalate suspensions by urinary macromolecules (UM), urine and solutions with urinary citrate concentration. MATERIALS AND METHODS: Solutions of UM (UMS) were obtained by a hemofiltration procedure from urine of 6 healthy men. Calcium oxalate suspensions were prepared in all solutions and urine by adjusting Ca2+ to 1.5 mM and by an oxalate titration to 1.0 mM. Crystallization was monitored measuring optical density (OD). Stability of suspensions (SS) was defined as the time without sedimentation and zeta potential (ZP) of crystals was measured. AGN was visualized by scanning electron microscopy and quantified by maximal OD. RESULTS: UMS inhibited AGN and increased ZP and SS. Most inhibitory activity of urine could be attributed to UM. 3.3-fold dilution of UM reduced SS only by 30%. CONCLUSIONS: During crystalluria, UM of healthy men are supposed to protect from stone formation by inhibiting AGN and stabilizing crystal suspensions. As a probably important aspect, this protection was found to be limited in time and may favorably be influenced by an increase of diuresis.

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.

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.

Nucleation and aggregation of calcium oxalate in whole urine; spectrophotometric sedimentation analysis: a new approach to study the aggregation of calcium oxalate dihydrate.

Spectrophotometric and scanning electron microscopic (SEM) studies of oxalate-induced crystallization have been performed in whole urine with and without continuous magnetic stirring and before and after millipore filtration of urine. With continuous stirring, preferential nucleation was observed and this followed second order kinetics. Important crystal aggregation only occurred after an oxalate load above 1 mmol/l and without stirring. Under these conditions and at an ionic calcium concentration of 2 mmol/l, single crystals and aggregates of calcium oxalate dihydrate and monohydrate of well defined sizes were produced. Single dehydrates, their aggregates and the other particles could be distinguished by their significantly different sedimentation rates. From sedimentation curves an aggregation ratio for calcium oxalate dihydrate (aggregated/total dihydrate particles) was extrapolated. Millipore filtration removing important urinary macromolecules increased this aggregation ratio as well as the size of the aggregates on SEM pictures.