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 80.65nmol/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 γ-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.

The bikunin peptide chain of the protease inhibitor inter-α-inhibitor (∣α∣) has been reported to be an inhibitor of calcium oxalate (CaOx) crystallization, and hence has been proposed as having a role in CaOx kidney stone formation. However, further experimental evidence is required to assess if fragments of ∣α∣ other than bikunin may play a role in the regulation of crystallization events in stone formation. The aim of the present study was to assess the effects of ∣α∣ and several of its derivatives on CaOx crystallization in a seeded inorganic system and to compare these effects with those of a known inhibitor of crystallization, prothrombin. ∣α∣ was purified from a preparation of human plasma and fragmented by alkaline hydrolysis, and two of its peptide chains, bikunin and heavy chain 1 (H1), were purified further by HPLC. Their purity was confirmed by SDS/PAGE. Using Coulter counter and [ 14 C]oxalate analysis and scanning electron microscopy, ∣α∣, its H1 chain and bikunin from urine and from plasma were shown to be relatively weak inhibitors of CaOx crystallization in vitro at expected physiological concentrations. It was concluded that members of the ∣α∣ family may not be as important in kidney stone formation as has been generally proposed, although further studies are required before a possible role for ∣α∣ and its fragments in stone formation can be unambiguously discounted.

We have studied the kinetics of oxalate-induced turbidity in fresh human urine and artificial urine. Assays are performed in 96-well plates, which allows many oxalate concentrations to be studied, repeatedly, in a short time. The metastable limit is defined in terms of the lowest oxalate concentration that gives a rate of change of attenuance significantly greater than the control. Interpretation of rates above this limit is based on ln/ln plots of initial rates against added oxalate concentration. This approach has a good theoretical basis, is well supported by our results and gives a turbidity rate index that is related to the product of the growth rate constant and a factor relating to the number and characteristics of the heteronuclei responsible for initiation of crystallization. This interpretation is posited upon the assumptions that second-order crystallization kinetics occur in unseeded urine when supersaturation exceeds the metastable limit and that aggregation during the initial phase of crystallization does not significantly contribute to changes in turbidity. Metastable limits of urine from healthy volunteers corresponded to a calcium oxalate supersaturation ratio of approx. 10. The turbidity rate index was higher in human urine than in artificial urine. The metastable limit, based on either oxalate concentration or supersaturation, for induction of calcium oxalate crystallization in normal human urine is higher than is likely to be found in normal subjects in vivo . The shape of the relationship between the metastable limit (based on oxalate concentration) and calcium concentration emphasizes the benefit of achieving a low urine calcium concentration. Comparison of the turbidity rate indices for human and artificial urine suggests that the role of nucleation promoters is more dominant than that of growth inhibitors.

1. Dietary calcium restriction, an efficient practice in reducing urinary calcium excretion, has been reported to induce either an increase or no change in oxalate excretion, questioning its use in hypercalciuric stone-forming patients. In addition, calcium restriction has been previously demonstrated to induce other urinary changes which might influence the relative supersaturation of calcium oxalate. So the overall effect of calcium deprivation on the relative supersaturation of calcium oxalate is unpredictable. 2. The aim of the study was to evaluate the effect of dietary calcium restriction on the relative supersaturation of calcium oxalate in the urine of stone-forming patients utilizing a computer methodology which takes into account the main soluble complex species of oxalate. 3. We studied 34 stone-forming patients on both a free-choice diet, whose Ca and oxalate content (24 and 1.2 mmol respectively) was assessed by dietary inquiry, and after 30 days on a prescribed low-calcium and normal oxalate diet (11 and 1.1 mmol respectively). Under both conditions, the excretion of the main urinary parameters related to dietary composition, electrolytes, oxalate and daily citrate urinary excretion, were measured. The relative supersaturation of calcium oxalate was calculated by means of an iterative computer method which takes into account the main soluble complex species on which the solubility of calcium oxalate is dependent. In addition, intact parathyroid hormone and 1,25-dihydroxyvitamin D blood levels were also evaluated. In 13 of the patients intestinal calcium absorption was evaluated during both a free- and a low-calcium diet, utilizing kinetics methodology. 4. The low-calcium diet induced, together with an expected reduction of calcium excretion, a marked increase in oxalate urinary output. This finding was independent of the presence or otherwise of hypercalciuria and of the serum levels of parathyroid hormone and vitamin D. Intestinal calcium absorption was also stimulated by calcium deprivation and its levels were well correlated with oxalate excretion. Minor changes in magnesium and citrate excretion were also observed. The overall effect on the relative supersaturation of calcium oxalate consisted in a substantial increase in this parameter during the low-calcium diet. 5. In conclusion, our data reinforce the concept that dietary calcium restriction has potentially deleterious effects on lithogenesis, by increasing the relative supersaturation of calcium oxalate.

1. The aim of this study was to determine whether seed crystals of uric acid or monosodium urate promote the epitaxial deposition of calcium oxalate in undiluted human urine. The effects of seed crystals of uric acid, monosodium urate or calcium oxalate on calcium oxalate crystallization induced in pooled 24-h urine samples collected from six healthy men were determined by [ 14 C]oxalate deposition and Coulter counter particle analysis. The precipitated crystals were examined by scanning electron microscopy. 2. Seed crystals of uric acid, monosodium urate and calcium oxalate increased the precipitated particle volume in comparison with the control containing no seeds by 13.6%, 56.8% and 206.5% respectively, whereas the deposition of [ 14 C]oxalate in these samples relative to the control was 1.4% ( P < 0.05), 5.2% ( P < 0.01) and 54% ( P < 0.001) respectively. The crystalline particles deposited in the presence of monosodium urate seeds were smaller than those in the control samples. Scanning electron microscopy showed that large aggregates of calcium oxalate were formed in the presence of calcium oxalate seeds, which themselves were not visible. In contrast, monosodium urate and, to a lesser extent, uric acid seeds were scattered free on the membrane surfaces and attached like barnacles upon the surface of the calcium oxalate crystals. 3. It was concluded that seed crystals of monosodium urate and uric acid do not promote calcium oxalate deposition to a physiologically significant degree in urine. Howsever, binding of monosodium urate and uric acid crystals and their subsequent enclosure within actively growing calcium oxalate crystals might occur in vivo , thereby explaining the occurrence of mixed urate/oxalate stones.

1. The urinary F1 activation peptide of prothrombin is the predominant protein incorporated into calcium oxalate crystals precipitated from human urine. The aim of this study was to examine the effect of pure urinary prothrombin F1 on calcium oxalate crystallization in human urine. 2. Urinary prothrombin F1 was purified from demineralized calcium oxalate crystals precipitated from human urine, and its effects on calcium oxalate crystallization induced by addition of an oxalate load were tested in undiluted, ultrafiltered urine from healthy men, at final concentrations of 0 to 10 mg/l. 3. Urinary prothrombin F1 did not affect the amount of oxalate required to induce crystallization, but the volume of material deposited increased in proportion to increasing concentrations of urinary prothrombin F1. However, the mean particle size decreased in reverse order: this was confirmed by scanning electron microscopy, which showed it to be the result of a reduction in crystal aggregation rather than in the size of individual crystals. Analysis of 14 C-oxalate data revealed a dose-dependent decrease in calcium oxalate deposition with an increase in urinary prothrombin F1 concentration, indicating that the increase in particle volume recorded by the Coulter Counter resulted from inclusion of urinary prothrombin F1 into the crystalline architecture, rather than increased deposition of calcium oxalate. 4. It was concluded that urinary prothrombin F1 may be an important macromolecular determinant of stone formation.

1. Increasing the concentration of dissolved urate promotes calcium oxalate crystallization in urine from which Tamm-Horsfall mucoprotein, an inhibitor of calcium oxalate crystal aggregation, has almost completely been removed. This study aimed to determine whether the effect of urate could be reduced or abolished by a physiological concentration of Tamm-Horsfall mucoprotein. This was approached in two ways. 2. The effect of Tamm-Horsfall mucoprotein on calcium oxalate crystallization induced by urate was tested in ultrafiltered (10 kDa) urine samples from 10 healthy men. Tamm-Horsfall mucoprotein (35 mg/l) was added to half of each specimen, the urate concentration was increased by the addition of sodium urate solution and crystallization was induced by a standard load of oxalate. The remainder of each urine specimen was used as a control; these specimens were treated with an identical amount of urate solution, but contained no Tamm-Horsfall mucoprotein. Tamm-Horsfall mucoprotein had no effect on the urinary metastable limit or on the deposition of calcium oxalate, but significantly reduced the size of the particles precipitated. 3. The effect of increasing the urate concentration in the presence of Tamm-Horsfall mucoprotein was tested. Tamm-Horsfall mucoprotein (35 mg/l) was added to 10 ultrafiltered urine samples as before, the samples were divided, and the concentration of urate was increased in half of each specimen. Compared with the control to which no urate was added, urate significantly reduced the amount of oxalate required to induce spontaneous calcium oxalate nucleation and increased the median volume and the particle size of the material deposited. 4. It was concluded that, in vivo , (a) hyperuricosuria would encourage the formation of calcium oxalate stones by promoting calcium oxalate crystallization, (b) Tamm-Horsfall mucoprotein would not lessen the effect of urate on calcium oxalate nucleation or bulk deposition but would reduce its effect on crystal aggregation; it could therefore reduce the likelihood of stone formation in patients with hyperuricosuria.

1. Increasing the concentration of dissolved urate promotes the crystallization of calcium oxalate from urine. The possibility was investigated that this effect may be caused by heterogeneous nucleation of calcium oxalate by particles of crystalline urate. 2. Urine samples were collected from 10 healthy men, centrifuged and filtered, and a solution of sodium urate was added to increase the medium urate concentration from 2.2 to 5.6 mmol/l. Calcium oxalate crystallization was induced by the addition of oxalate, followed by incubation for 90 min in a shaking waterbath at 37°C. The crystalline material was filtered out and the urate concentration was determined in the filtrate. 3. No difference in the urate concentration before and after induction of calcium oxalate crystallization was observed. These findings were confirmed by infra-red spectroscopy, X-ray powder diffraction and ultraviolet wet chemical analysis with detection limits of 5-10, 1.0 and 0.055%, respectively; urate was not detected in the calcium oxalate crystals. 4. In addition, three urine samples were collected and passed through 10 kDa ultrafiltration membranes to remove any colloidal particles which might have been present. The urate concentration was increased and an oxalate load was added as before, prior to incubation at 37°C in a shaking water bath for 5 min, followed by passage through 10 kDa ultrafiltration membranes. Scanning electron microscopy revealed no particles on the membranes thereby indicating that colloidal or crystalline urate was not formed early in the crystallization experiments. 5. It was concluded that the promotion of calcium oxalate crystallization by urate, and thereby the formation of calcium oxalate renal stones, is not caused by heterogeneous nucleation and subsequent epitaxial growth of calcium oxalate upon particles of urate.

1. Urinary glycosaminoglycans were recovered from the papain digest of polyanions precipitated sequentially by cetylpyridinium chloride and sodium acetate-saturated ethanol. Those from the early morning urine of 48 stone formers and 43 normal control subjects measured 11 and 16 μg of uronic acid/ml of urine, respectively. 2. Preparative agarose gel electrophoresis of the recovered glycosaminoglycans in barium acetate buffer (pH 5.8) yielded fractions containing purely chondroitin sulphate, co-polymeric chondroitin/dermatan sulphates and heparan sulphate. Identification was based on the susceptibility of the fractions to chondroitinase or nitrous acid treatment. Similar compositions of glycosaminoglycan classes were observed in samples from stone formers and normal control subjects. 3. The fractionated glycosaminoglycans were dissolved in urine ultrafiltrate to assay for nucleation-promoting and growth-inhibiting activities towards crystallization of urinary calcium oxalate. When compared at the same uronic acid concentration, both the urinary chondroitin sulphate isomers and heparan sulphates of stone formers demonstrated the capacity to enhance crystal nucleation from calcium oxalate endogenous in urine ultrafiltrates, whereas only urinary heparan sulphates of normal control subjects demonstrated this capacity. 4. Tissue-derived reference chondroitin sulphate, dermatan sulphate and heparin, when similarly tested, showed negligible crystal nucleation-promoting activity. The tissue-derived heparan sulphate was similar to the urinary heparan sulphates in showing marked crystal nucleation-promoting activity. 5. Crystal-growth inhibitory activity was evident in all urinary glycosaminoglycan fractions studied. In particular, urinary heparan sulphate of normal control subjects showed higher activity than that of stone formers or the chondroitin sulphate isomers of both stone formers and normal control subjects ( P <0.005).

1. The effect of hyperuricosuria, simulated by increasing the concentration of dissolved urate, on the crystallization of calcium oxalate in human urine was examined. 2. Twenty urine samples were studied. Ten of these, designated type A, spontaneously precipitated calcium oxalate dihydrate crystals upon the addition of a solution of sodium urate solution which raised the median urate concentration from 3.1 to 7.0 mmol/l. 3. Adding dissolved urate to the remaining type B samples raised the median urate concentration from 2.2 to 6.2 mmol/l, but did not cause the precipitation of calcium oxalate. This was induced in these samples by the addition of a standard load of oxalate above an empirically determined metastable limit. 4. In the type B urine samples, the addition of urate decreased the median metastable limit from 125 to 66 μmol of oxalate, trebled the median volume of crystalline calcium oxalate deposited from 35 000 to 105 000 μm 3 /μl and significantly increased the overall size of the particles precipitated. Calcium oxalate monohydrate was exclusively precipitated, and the individual crystals deposited in the presence of urate were markedly smaller, more numerous, and more highly aggregated than those produced in its absence. 5. These results constitute the most convincing evidence yet obtained that hyperuricosuria may be a powerful promoter of calcium oxalate stone formation.

1. Because urinary prostaglandin excretion could play a role in idiopathic hypercalciuria (IH), we studied the excretion of prostaglandin E (PGE), calcium and sodium at various urine flows in 21 patients (14 males) with urolithiasis and IH, seven stone formers (five males) with normal calciuria and 20 controls (11 males). Dietary composition was comparable and sodium intake was restricted to 100–120 mmol/day. 2. Analyses were performed on 30 min urine collections obtained after overnight water deprivation and during water diuresis. Male IH patients had increased levels of urinary PGE at all ranges of urine flow. PGE excretion correlated directly with urine flow in patients and controls, but the slope of this relationship in individual IH male patients was steeper than in controls ( P < 0.01). Calciuria correlated directly with urine output in patients with IH but not in controls. Calcium and sodium excretion were directly correlated ( P < 0.0001) in patients and controls. There were no significant differences between absorptive IH (seven patients) and renal IH (eight patients). There were no significant differences between stone formers with normocalciuria and control subjects. 3. The findings suggest that increased urinary PGE could play a role in the hypercalciuria syndrome, possibly by promoting natriuresis.

1. The binding of heparin, chondroitin sulphate and the low molecular weight heparin analogue pentosan polysulphate to sodium urate and uric acid crystals was studied by the use of competitive binding technique. Trace amounts of radioactively labelled glycosaminoglycans were used in the binding step, and varying amounts of unlabelled glycosaminoglycans for the competition experiments. 2. The experiments were carried out in 140 mmol/l NaCl at pH 6, with or without 5 mmol/l CaCl 2 in the solution. 3. A reversible and almost complete binding of heparin and chondroitin sulphate to sodium urate crystals did occur in the presence of calcium, whereas pentosan polysulphate bound incompletely. The binding was much less pronounced in the calcium-free conditions. 4. Uric acid crystals did not bind any of the three inhibitors, not even with calcium present. 5. The clinical relevance depends on whether sodium urate microcrystals are present in the urine of calcium stone patients, to cause a binding and thereby a masking or inactivation of these inhibitors in the urine, which seems to be possible in the presence of calcium. However, the potential of pentosan polysulphate for the treatment of calcium stone patients does not seem to be at risk from this effect.

1. Eight recurrent renal stone-forming patients were housed in a metabolic ward and fed on a low (LPD) and a high (HPD) animal protein diet, which were isoenergetic. Metabolic studies were made after 2 weeks on each diet. 2. There was a 90% increase in urinary urate on HPD compared with LPD, whereas serum urate did not change consistently. 3. The urinary acid excretion increased by 200%, including a 100% increase in ammonium ion excretion. A fall in urine pH by 0.9 unit was also seen. 4. The calculated ion activities of the urines revealed a profound increase in the uric acid supersaturation, from undersaturation to supersaturation, and in some cases even surpassing the formation product ratio. The ammonium urate supersaturation also increased. The sodium urate supersaturation was unchanged, despite an induced natriuresis. 5. The risk of forming uric acid or ammonium urate crystals or stones in the urine was increased on a high protein diet, whereas the risk of forming sodium urate crystals was no greater than on a low protein diet. 6. As uric acid and ammonium urate crystals under certain conditions may adsorb a macromolecular fraction of the human urine, which inhibits calcium oxalate crystal growth, it is proposed that this mechanism, along with a decrease in urine pH, may also interfere with the inhibitory activity of calcium oxalate crystal growth and aggregation.

1. The short- and longer-term effects of ethane-1-hydroxy-1,1-diphosphonate (EHDP), an inhibitor of crystal growth and potential preventive agent against urinary tract stones in man, have been studied. 2. Measurement of urinary excretion of EHDP was used to define the best dosage regimen. When 4·4 mmol of EHDP was given daily in four divided doses the urinary concentration of EHDP achieved was high enough (10 −5 mol/l) to inhibit the crystallization of calcium crystals throughout the day. 3. Nine patients with recurrent calcium stones were given this dose of EHDP daily for 12 months and seven were then studied for a further 12 months under placebo. During treatment with EHDP, inhibitory activity in urine towards precipitation of calcium phosphate was restored from low values to greatly above normal. This could be accounted for by the inhibitory effect of EHDP itself, coupled with an increase in urinary inorganic pyrophosphate. After stopping EHDP the excretion of EHDP rapidly fell to undetectable levels but the excretion of pyrophosphate remained elevated throughout the 12 months of placebo treatment. EHDP also induced a rise in plasma phosphate and an increase in the urinary excretion of oxalic acid and uric acid, but these changes were all fully reversible when EHDP was stopped. 4. The average rate of stone formation per patient per year decreased from 2·4 to 0·2 during treatment with EHDP and remained low during the following 24 months. However, the dose needed for this effect is known to affect bone turnover and mineralization.

1. In order to assess the relative importance of possible pathogenetic factors in the formation of calcium-containing renal stones, a group of 18 patients (12 men, six women) with active, recurrent stone disease were compared with 16 age-matched control subjects (10 men, six women) given an identical diet. 2. Fifteen (83%) of the patients showed at least one, eight (44%) showed two, and one (6%) patient showed three abnormalities that might predispose to stone formation. 3. Increased urinary calcium excretion was the most common abnormality (11 patients, 61%), particularly in the women (83%). 4. A diminished excretion of inhibitors of crystal formation of calcium phosphate was the next most common abnormality, which occurred in eight patients (44%), all of whom were men. It was largely attributable to a diminished excretion of inorganic pyrophosphate (PP i ). The PP i /orthophosphate ratios were also lower in the stone-formers. Significant differences in residual inhibitory activity after enzymatic removal of PP i between control subjects and stone-formers could not be found in 24 h urine samples but were present during certain times of the day. Pyrophosphate showed a higher inhibitory activity in urine than in control solutions, this enhancement being absent in stone-formers. 5. Nine (50%) of the patients, but only one of the control subjects, produced crystal aggregates greater than 50 μm in diameter after an oral load of oxalate.

1. Whewellite (calcium oxalate monohydrate) crystals were found to induce epitaxially the heterogeneous nucleation of brushite (calcium monohydrogen phosphate dihydrate) from its metastable supersaturated solution in approximately one-quarter of the time required for spontaneous precipitation in the absence of added nucleating agents. Scanning electron-microscope observation of the crystalline phase showed brushite crystals originating from the whewellite seed crystals. 2. Crystal growth, upon nucleation, proceeded rapidly, and the metastable solutions quickly approached saturation. 3. Brushite crystals also induced the precipitation of calcium oxalate crystals in about one-quarter of the time required for spontaneous precipitation; however, the rate of crystal growth was considerably slower. In support of the chemical data, scanning electron micrographs showed few crystals of calcium oxalate nucleated on the surface of the brushite seed. 4. The results provide some insight into the cause of stones containing calcium oxalate or calcium phosphate (or both), which form in the normally acid environment of human urine.

Chemical kinetic data, complemented with scanning electron-microscope observations of the crystalline phase, show that seed crystals of hydroxyapatite have the ability to induce the growth of calcium oxalate monohydrate crystals epitaxially from a metastable supersaturated solution of calcium oxalate. The rate of growth of calcium oxalate crystals is dependent on the surface area of the seed material and follows a second-order rate law. It is suggested that there may be a causal relationship between the occurrence of apatite crystals in the urinary tract and the formation of both ‘pure’ and mixed urinary stones containing calcium oxalate. Under similar experimental conditions, however, seed crystals of calcium oxalate monohydrate appeared unable to induce epitaxially the growth of calcium phosphate crystals from a supersaturated calcium phosphate solution, indicating the absence of an epitaxial relationship between calcium oxalate monohydrate and the initially precipitating calcium phosphate phase(s).