Modifiers of calcium oxalate crystallization found in urine. III. Studies on the role of Tamm-Horsfall mucoprotein and of ionic strength.

The effect was measured of incubating crystals of calcium oxalate monohydrate (COM) with 10 per cent normal urine and 10 per cent artificial urine on the surface zeta potential (ZP) produced on the crystals by various inhibitors of the agglomeration of COM. These studies showed that natural urine contains some constituent, not present in the artificial urine, which interferes with the ability of the inhibitors tested to produce a highly negative ZP on the crystal surface. A second series of studies showed that there are two factors in urine which modify the effect of the polyanionic inhibitors, Tamm-Horsfall mucoprotein (THM) and high ionic strength. At concentrations greater than 10(-8) mol./l., THM interfered with the binding of other polyanions to the surface of COM and reduced the maximum negativity of the ZP achievable on the crystals. This effect was very marked over the urinary concentration range (5 X 10(-8) to 6 X 10(-7) mol./l.; four to 48 mg./l.). By increasing the ionic strength of the incubation medium over the urinary range, the electrical double layer around the COM crystals was compressed and the interference caused by THM accentuated. Within most of the urinary range of ionic strength, THM interfered with the binding of other polyanions at concentrations greater than 10(-9) mol./l. In highly concentrated urine, at the top end of the urinary range of ionic strength, THM polymerized readily and to such an extent that it overwhelmed the inhibitors in urine and strongly promoted the agglomeration of COM crystals. The polymerization point of THM was found to be inversely related to the ionic strength of the solution in which it was dissolved.

Modifiers of calcium oxalate crystallization found in urine. II. Studies on their mode of action in an artificial urine.

The relative potencies of various modifiers of the crystallization of calcium oxalate (CaOx) were determined under "whole urine equivalent" conditions using a batch crystallizer. The system was used to measure changes in the degree of agglomeration of CaOx crystals produced spontaneously at a level of supersaturation within the range found in the urines of recurrent CaOx stone-formers. The modifiers tested included RNA, heparin, chondroitin-4-sulphate, pyrophosphate (at pH 5, 6 and 7), Tamm-Horsfall mucoprotein and a greater than 10,000 dalton fraction of macromolecules obtained from pooled normal urine by dialysis. The effects of these urinary constituents were also measured on the zeta potential produced at the surface of CaOx crystals. In the case of pyrophosphate, there was a clear correlation between the degree of inhibition of agglomeration and the zeta potential observed on the crystal surface indicating that, for this ion, repulsive electrostatic forces dominate the tendency for CaOx crystals to agglomerate. For the other modifiers tested, the tendency to agglomerate appeared to be dependent on the balance between the positive viscous binding ("sticky") forces and the negative electrostatic forces produced by these macromolecules on the CaOx crystal surface.

Modifiers of calcium oxalate crystallization found in urine. I. Studies with a continuous crystallizer using an artificial urine.

Various modifiers of the rate of crystallization of calcium oxalate (CaOx) known to be present in urine were studied in a continuous crystallizer system under conditions approximating those in whole urine. Of the small ions examined, magnesium and citrate were only weak inhibitors of the crystal growth rate and degree of agglomeration of CaOx within the urinary range of concentration of these ions. At and beyond the upper limit of the normal, however, both ions became much more active mainly through their ability to complex oxalate and calcium ions respectively. Pyrophosphate showed similar characteristics although its ability to act as an inhibitor at high concentrations was not due to complexation of calcium ions but to adsorption onto the crystal surface of the freshly generated CaOx crystals. The polyanionic inhibitors, chondroitin-4-sulphate, heparin and ribonucleic acid (RNA), also acted as adsorption inhibitors of growth and agglomeration. Of all the inhibitors tested RNA was the most active within the concentration range found in normal urine. Under the conditions of ionic strength employed, it was not possible to measure the effect of Tamm-Horsfall mucoprotein satisfactorily since it polymerized to form particles sufficiently large to interfere with the true crystal counts of CaOx in the test system.

Sodium pentosan polysulphate as a polyanionic inhibitor of calcium oxalate crystallization in vitro and in vivo.

Studies in vitro showed that sodium pentosan polysulphate (SPP) is an active inhibitor of calcium oxalate crystal growth and agglomeration and that it acts by increasing the negative zeta potential on the surface of calcium oxalate crystals. Oral administration to human subjects resulted in an increase in the negative zeta potential, which is consistent with an increase in the polyanionic inhibitory activity of urine. SPP may offer a novel approach to the medical management of recurrent calcium oxalate stone disease.

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.

Polyanionic inhibitors of calcium oxalate crystal agglomeration in urine.

The excretions and relative potencies of various macromolecular inhibitors of the crystallisation of calcium oxalate (CaOx) were measured in the urines of idiopathic calcium stone-formers and normal subjects. The stone-formers excreted significantly less polyanionic macromolecules in their urine than did the normals, the difference being attributable to the lower excretions of glycosaminoglycans (GAGS), ribonucleic acid (RNA) and Tamm-Horsfall mucoprotein (THM), all of which are precipitable with alcian blue. The relative potencies of the various inhibitors measured under 'whole urine equivalent' conditions using a batch crystallisation system, showed that the order of inhibitory activity towards CaOx crystal agglomeration was RNA greater than GAGS greater than THM greater than pyrophosphate (PPi). This paralleled the order of ability of these inhibitors to produce a high negative zeta potential on the surface of CaOx crystals.

The determination of oxalate in urine and urinary calculi by a new ion-chromatographic technique.

A new method is described for the measurement of oxalate in urine and urinary calculi using medium pressure ion-chromatography. The technique is simple, requires minimal sample preparation and only 20 min analysis time. The minimum detection limit for oxalate is 0.5 mumol/l in the diluted sample as applied to the analyser. The intra-run coefficient of variation for the chromatography stage alone is 3.8%. The overall intra-run and inter-run coefficients of variation, including the sampling and dilution of urine, are 6.5 and 8.3% respectively. The method compares well with an established colorimetric technique for the analysis of oxalate both in urine and in urinary calculi.