Nucleation of calcium oxalate crystals by albumin: involvement in the prevention of stone formation.

BACKGROUND: Urine is supersaturated in calcium oxalate, which means that it will contain calcium oxalate crystals that form spontaneously. Their size must be controlled to prevent retention in ducts and the eventual development of a lithiasis. This is achieved, in part, by specific inhibitors of crystal growth. We investigated whether promoters of crystal nucleation could also participate in that control, because for the same amount of salt that will precipitate from a supersaturated solution, increasing the number of crystals will decrease their average size and facilitate their elimination. METHODS: Albumin was purified from commercial sources and from the urine of healthy subjects or idiopathic calcium stone formers. Its aggregation properties were characterized by biophysical and biochemical techniques. Albumin was then either attached to several supports or left free in solution and incubated in a metastable solution of calcium oxalate. Kinetics of calcium oxalate crystallization were determined by turbidimetry. The nature and efficiency of nucleation were measured by examining the type and number of neoformed crystals. RESULTS: Albumin, one of the most abundant proteins in urine, was a powerful nucleator of calcium oxalate crystals in vitro, with the polymers being more active than monomers. In addition, nucleation by albumin apparently led exclusively to the formation of calcium oxalate dihydrate crystals, whereas calcium oxalate monohydrate crystals were formed in the absence of albumin. An analysis of calcium oxalate crystals in urine showed that the dihydrate form was present in healthy subjects and stone formers, whereas the monohydrate, which is thermodynamically more stable and constitutes the core of most calcium oxalate stones, was present in stone formers only. Finally, urinary albumin purified from healthy subjects contained significantly more polymers and was a stronger promoter of calcium oxalate nucleation than albumin from idiopathic calcium stone formers. CONCLUSIONS: Promotion by albumin of calcium oxalate crystallization with specific formation of the dihydrate form might be protective, because with rapid nucleation of small crystals, the saturation levels fall; thus, larger crystal formation and aggregation with subsequent stone formation may be prevented. We believe that albumin may be an important factor of urine stability.

Protein crystals orientation in a magnetic field.

Nucleation and crystal growth of hen egg-white lysozyme, bovine pancreatic trypsin inhibitor and porcine pancreatic alpha-amylase were carried out in the presence of a magnetic field of 1.25 T produced by small permanent magnets. Crystals were oriented in the magnetic field, except when heterogeneous nucleation occurred. The orientation of protein crystals in the presence of a magnetic field can be attributed to the anisotropic diamagnetic susceptibility of proteins resulting from the large anisotropy of the alpha-helices due to the axial alignment of the peptide bonds.

Pancreatic lithostathine as a calcite habit modifier.

Most biological fluids are supersaturated with calcium salts. A mechanism controlling crystal growth is therefore necessary to prevent excessive precipitation and development of a lithiasis. In pancreatic juice, calcite precipitation is prevented by lithostathine, a glycoprotein that inhibits calcite crystal growth. We describe here the interaction of lithostathine with calcite crystals. Without lithostathine, calcite crystals grew as rhombohedra showing six (104) faces. At low concentration (1 microM), lithostathine already altered crystal growth by generating new (110) faces. At physiological concentrations (3-10 microM), adsorption resulted in a transition from rhombohedral to sub-cubic habits. Immunochemical localization demonstrated that, although all (104) faces are equivalent, lithostathine binding was restricted to the face edges distal to the c axis. Scanning electron microscopy showed that, at the site of lithostathine binding, spreading of new CaCO3 layers during crystal growth was arrested before reaching the crystal diad axis-bearing edges. The successive kinks generated during crystal growth formed the new, striated (110)faces. Similar modifications were observed with the N-terminal undecapeptide of lithostathine that bears the inhibitory activity. With 100 microM lithostathine, (110) faces could reach the c axis outcrop of the former rhombohedron, resulting in an olive-shaped crystal. Finally, the number of crystals increased and their average size decreased when lithostathine concentration increased from 0.1 to 100 microM. Decreased Ca2+ concentration during crystal growth was delayed in the presence of lithostathine. It was concluded that lithostathine controls lithogenesis 1) by triggering germination of numerous calcite crystals and 2) by inhibiting the rate of Ca2+ ion apposition on the nuclei and therefore interfering with the apposition of new layers on calcite. Formation of smaller crystals, whose elimination is easier, is thereby favored.

Calcium carbonate crystals promote calcium oxalate crystallization by heterogeneous or epitaxial nucleation: possible involvement in the control of urinary lithogenesis.

A large proportion of urinary stones have calcium oxalate (CaOx) as the major mineral phase. In these stones, CaOx is generally associated with minor amounts of other calcium salts. Several reports showing the presence of calcium carbonate (CaCO3) and calcium phosphate in renal stones suggested that crystals of those salts might be present in the early steps of stone formation. Such crystals might therefore promote CaOx crystallization from supersaturated urine by providing an appropriate substrate for heterogeneous nucleation. That possibility was investigated by seeding a metastable solution of 45Ca oxalate with vaterite or calcite crystallites. Accretion of CaOx was monitored by 45Ca incorporation. We showed that (1) seeds of vaterite (the hexagonal polymorph of CaCO3) and calcite (the rhomboedric form) could initiate calcium oxalate crystal growth; (2) in the presence of lithostathine, an inhibitor of CaCO3 crystal growth, such accretion was not observed. In addition, scanning electron microscopy demonstrated that growth occurred by epitaxy onto calcite seeds whereas no special orientation was observed onto vaterite. It was concluded that calcium carbonate crystals promote crystallization of calcium oxalate and that inhibitors controlling calcium carbonate crystal formation in Henle's loop might play an important role in the prevention of calcium oxalate stone formation.

[Preparation and analysis by x-ray diffraction of three crystalline forms of calcium oxalate in a mixture]. / Préparation et dosage par diffraction de rayons X des trois formes cristallines de l'oxalate de calcium en mélange.

The present work describes a method for analysing, quantitatively, mixtures of the three calcium oxalate hydrates which are the most important components of human kidney stone. The method consists in measuring and comparing the relative intensities of X-ray diffraction peaks obtained by means of the so-called powder method. We first give the composition of the solutions from which the three hydrates were grown separately. Second, we briefly describe the principle of the X-ray quantitative phase analysis and give the results we obtained by analysing binary and ternary mixtures of the three solid phases of calcium oxalate. We show that the proportion of either phase can be estimated with an accuracy of about 2 or 3 wt%.

[*calcium-oxalate lithogenesis: crystallization in the presence of urine from healthy subjects]. / Lithogenèse oxalo-calcique: cristallisation en présence d'urine de sujets sains.

In the present study we aim at describing the influence of urine of non-lithiasic subjects on the different crystallization stages of calcium oxalates. The experimental method consists in comparing the turbidimetric curves obtained by crystallization in pure synthetic urine to the curves obtained after addition of natural urine to the solution. Natural urine plays an important role on nucleation, crystal growth and agglomeration even if only small amounts (4% v/v) are added to the mother solution. Nucleation is favoured (decrease of the induction period and increase of the crystal number) by the presence of solid particles, such as cellular fragments, which play the role of substrates for heterogeneous nucleation. On the other hand, both the growth rate of the crystals and their degree of agglomeration are reduced. Moreover, the physical nature of the crystals which precipitate is different from that of the crystals which nucleate in synthetic urine. Under our conditions of high super-saturation, natural urine favours the nucleation of calcium oxalate dihydrate at the expenses of calcium oxalate trihydrate which forms in pure synthetic urine. A hypothesis on the origin of the lithogenesis process is made and a correlation between the localization of the calcium oxalate stones in the urinary tracts and their main constituents is proposed.

Influence of polydispersity on protein crystallization: a quasi-elastic light-scattering study applied to alpha-amylase.

The early stages of the crystallization process of porcine pancreatic alpha-amylase were investigated by quasi-elastic light scattering. It is shown that at 288 and 293 K the diffusion coefficient does not monotonically change with increasing protein concentration but passes through a maximum at 10 mg ml(-1). In supersaturated solutions, prior to nucleation, the protein is strictly monodisperse. Nucleation induces the formation of aggregates and a polydispersity of, for example, 18% for an initial supersaturation C/C(e) = 5.8. Monodispersity is restored after the nuclei have grown and partially consumed the solute. On the other hand, polydispersity increases up to 20% at 298 K if the protein concentration decreases to 3-4 mg ml(-1), values at which the solutions are under-saturated. When the protein concentration exceeds 5-6 mg ml(-1) the protein becomes monodisperse again. These results, confirmed by those of another system we are studying (bovine pancreatic trypsin inhibitor), are at variance with the statements that supersaturation is always at the origin of aggregation and polydispersity, and that in undersaturated solutions the diffusion coefficient should remain constant for obtaining crystals once the solutions are supersaturated.

[Renal lithostathine: a new protein inhibitor of lithogenesis]. / La lithostathine rénale: un nouvel inhibiteur protéique de la lithogénèse.

Lithostathine is a protein of pancreatic secretion inhibiting calcium carbonate crystal growth. Antibodies to lithostathine were used to identify a related protein in urine and kidney stones. Western blot analysis of proteins extracted from concentrated normal urine or kidney stones demonstrated the presence of a protein with an apparent molecular weight of 23 kDa. The same antibodies were used in immunolocalization experiments on fresh human nephrectomy specimens cryosections. A positive signal was observed in the cells of proximal tubules and thick ascending limbs of Henle's loop. Protein extracts of renal stones inhibited calcium carbonate crystal growth. Because of its structural and functional similarities with pancreatic lithostathine, it was called renal lithostathine.

[Crystallization of calcium phosphate in the presence of magnesium]. / Cristallisation des phosphates de calcium en présence de magnésium.

Formation and evolution of calcium phosphates are investigated, at 37 degrees C, in urine and aqueous solutions whose concentrations in calcium and magnesium are close to those of urine. Although the solutions are supersaturated with respect to all calcium phosphates, only an amorphous phase (ACP) and brushite (B) precipitate at time zero. Later on, ACP transforms either into whitlockite (W) or into apatite (HAP) depending on the solution composition. Phase transformations are discussed in terms of supersaturation, parameter which includes concentrations and pH. It is also shown that magnesium is a powerful inhibitor of the evolution towards HAP.

[Model of calcium oxalate lithogenesis by kinetic turbidimetry]. / Modèle d'étude de la lithogénèse oxalo-calcique par cinétique turbidimétrique.

The crystallization process of calcium oxalates in synthetic urine at 37 degrees C is followed by turbidimetry. As the solution absorbance is very sensitive to the number of particles in suspension, it is possible to distinguish nucleation and growth from agglomeration. In the former case, the slopes of the turbidimetric curves are positive whereas in the latter case, they are negative. Poisoning the solutions by some active additives induces changes of the slopes so that it becomes possible to know whether the additive is a promotor or an inhibitor of nucleation, growth or agglomeration. As an example, the model is tested for checking the influence of human urine on the crystallization process. It is shown that urine fractions of non stone-formers inhibit agglomeration when they contain macromolecules of high molecular weight (> 30,000 Da).

Evidence that human kidney produces a protein similar to lithostathine, the pancreatic inhibitor of CaCO3 crystal growth.

Pancreatic juice is supersaturated in calcium carbonate. CaCO3 crystal growth is controlled by lithostathine, a secretory protein synthesized by pancreatic acinar cells, first described as a constituent of pancreatic stones. It was recently reported that, in the thin descending limb of the Henle's loop, urine was supersaturated in CaCO3 (Coe FL, Parks JH: Defenses of an unstable compromise: crystallization inhibitors and the kidney's role in mineral regulation. Kidney Int. 1990: 38, 625-631. This observation suggested the presence in kidney of a similar inhibitor. In this study, we show that a protein immunologically related to lithostathine is actually present in urine of healthy subjects and in renal stones. Immunocytochemistry of kidney sections localized the protein to cells of the proximal tubules and thick ascending limbs of the Henle's loops. Protein extracts of renal stones inhibited CaCO3 crystal growth in vitro and this inhibition was significantly lifted by incubating the extracts with antibodies to lithostathine. The protein is not immunologically related to nephrocalcin. Because of its structural and functional similarities with pancreatic lithostathine, it was called renal lithostathine.

Urinary supersaturation with respect to brushite in patients suffering calcium oxalate lithiasis.

The urines of 23 stone-formers presenting repeated calcium oxalate lithiasis and 12 control subjects were collected at six different time periods daily. Supersaturations for calcium oxalate and brushite (DCPD) were calculated using ionic and solubility products. Urines of both groups were supersaturated for calcium oxalate but only urines of the stone-formers were supersaturated for brushite, the most simple calcium phosphate which nucleates very easily at the urinary pH. This fact suggests that the core of the calcium oxalate stone could be made of either a calcium oxalate crystallite or a brushite seed onto which hetergeneous nucleation of calcium oxalate can take place.

[Respective effect of calcium and oxalate on calcium oxalate crystallization in the urine]. / Influence respective du calcium et de l'oxalate sur la cristallisation des oxalates de calcium dans l'urine.

Our aim is to determine the respective role of calcium (Ca) and of oxalate (ox) in the formation of calcium oxalate (Caox) crystals. Two urine samples are mixed after increasing either the calcium concentration (Ca) in one sample or the oxalate concentration (ox) in the other. The crystals formed are identified by their habits or by x-ray. Similarly, urine from 37 hypercalciuric stone-formers is analysed. Caox crystallizes at an oversaturation beta greater than or equal to 13,5, critical beta. An almost linear relationship exists between beta and (ox), the critical beta being obtained at an (ox) approximately 0,26 mmol/l. No simple relationship links beta and (Ca). These results point out the predominant role of oxalate in the formation of Caox crystals.