Biomedical subjects

R L Ryall

Publications and source records attributed to R L Ryall.

At least 19 recordsLinked to original sources

PubMed1996

Further evidence linking urolithiasis and blood coagulation: urinary prothrombin fragment 1 is present in stone matrix.

The fact that organic material is always present and distributed throughout each renal calculus suggests that it may play a role in stone formation. The organic matrix of calcium oxalate (CaOx) crystals freshly generated in urine in vitro contains urinary prothrombin fragment 1 (UPTF1) as the principal protein. In this initial study, matrix was extracted from 12 renal calculi and evaluated for the presence of UPTF1 using Western blotting. UPTF1 was present in all eight stones whose principal component was CaOx, and in one of two stones which consisted mainly of calcium phosphate (CaP). UPTF1 was absent from the two struvite calculi examined. The relationship between CaP and UPTF1 was explored further. Matrix harvested from CaP crystals freshly generated in urine in vitro was also shown to contain UPTF1 as its principal component. Our inability to detect UPTF1 in one mixed CaOx/CaP stone may be related to our methods of matrix retrieval, while its absence from two struvite stones argues against it being present in the other stones merely as a consequence of passive inclusion. This absence may be related to the alkaline environment typical of struvite stone growth. The finding that UPTF1 is present in some renal stones provides the first direct evidence that links blood coagulation proteins with urolithiasis.

Adult↗

PubMed1995

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.

Calcium Oxalate↗

PubMed1995

Re-evaluation of the "week-end effect" data: possible role of urinary copper and phosphorus in the pathogenesis of renal calculi.

Early morning urinary concentrations of 10 elements which had demonstrated a "week-end effect" in a previous study, were subjected to a normalization procedure thereby allowing a re-assessment of their potential role in urolithiasis. After transformation of each concentration to a weighted proportion of the total concentration on each day, only Cu and P values were significantly different for kidney stone formers and healthy controls on all three days indicating that these elements may play a role in the pathogenesis of renal calculi. The results obtained in this study demonstrate that a more meaningful picture of the possible differences in the urinary concentrations of stone formers and normal controls might emerge if "proportional" rather than "raw" concentrations are compared.

Bromine↗

PubMed1995

The urinary F1 activation peptide of human prothrombin is a potent inhibitor of calcium oxalate crystallization in undiluted human urine in vitro.

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 14C-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.

Calcium Oxalate↗

PubMed1995

Blood coagulation proteins and urolithiasis are linked: crystal matrix protein is the F1 activation peptide of human prothrombin.

OBJECTIVES: To determine the relationship between prothrombin and crystal matrix protein (CMP). CMP is the predominant protein found in the organic matrix of calcium oxalate (CaOx) crystals generated from human urine and is a 31 kDa glycoprotein, whose N-terminal amino acid sequence shares homology with human prothrombin. MATERIALS AND METHODS: CaOx crystallization was induced in ultrafiltered (UF) human urine containing either plasma or serum derived from the same healthy donor, by the addition of sodium oxalate. The crystals were demineralized and the resulting protein extracts analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, using antibodies raised against human prothrombin and the C-terminus of prothrombin fragment 1 + 2 (F1 + 2). RESULTS: Prothrombin was detected in extracts of crystals precipitated from the UF urine in the presence of plasma, while CMP was completely absent. Crystals precipitated from UF urine supplemented with serum contained relatively large amounts of F1 + 2 and a protein with the same electrophoretic mobility as CMP. Analysis of a standard preparation of F1 + 2 which also contained prothrombin fragment 1 (F1) as a minor contaminant, showed a protein with electrophoretic and staining properties comparable to CMP. CONCLUSION: CMP is a urinary form of F1, a degradation product of prothrombin possessing the domain rich in gamma-carboxyglutamic acid, which may have undergone some molecular modification either before or after its release into the urine.

Adult↗

PubMed1995

Is nephrocalcin related to the urinary derivative (bikunin) of inter-alpha-trypsin inhibitor?

OBJECTIVE: To isolate, purify, sequence and characterize nephrocalcin (NC), a urinary protein that may be an important determinant of calcium oxalate (CaOx) kidney-stone disease. MATERIALS AND METHODS: Proteins were isolated from human urine using cellulose and resin columns and were sequenced using Edman degradation and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Inhibition of CaOx crystal growth by the isolated proteins was assessed by measuring the deposition of 14C-labelled CaOx. RESULTS: A protein assumed to be NC on the basis of SDS-PAGE, inhibitory and gel filtration properties was isolated from healthy human urine. Its molecular weight and the amino acid sequences of two of its peptides suggested it was identical to fragment HI-14 of the light chain (bikunin) of inter-alpha-trypsin inhibitor (ITI). CONCLUSIONS: NC represents a portion of the light chain of ITI, although this conclusion must remain tentative until confirmed using authentic NC.

Alpha-Globulins↗

PubMed1994

Tamm-Horsfall mucoprotein reduces promotion of calcium oxalate crystal aggregation induced by urate in human urine in vitro.

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium Oxalate↗

PubMed1994

Urinary glycosaminoglycans are selectively included into calcium oxalate crystals precipitated from whole human urine.

Urinary glycosaminoglycans are selectively included into calcium oxalate (CaOx) crystals precipitated from whole human urine: The presence of glycosaminoglycans (GAGs) in the organic matrix of urinary stones, and their known effects on CaOx crystallization have prompted speculation regarding their role in CaOx urolithiasis. The aim of this study was to examine the involvement of GAGs in the early stages of CaOx crystallization in human urine. Urine samples were collected from healthy men and CaOx crystallization was induced by the addition of a sodium oxalate load. The crystals were harvested and demineralized, and the GAG content of the resulting extract analysed by cellulose acetate electrophoresis. Only one GAG, heparan sulphate (HS) was detected in the organic matrix of the crystals; chondroitin sulphate (ChS), the most abundant urinary GAG, was conspicuously absent. Further experiments, in which varying amounts of HS and ChS were added to ultrafiltered (10,000 Da) urine prior to induction of calcium oxalate crystallization, showed that ChS was included into the crystals only when HS was absent from the urine. It was concluded that the selective inclusion of GAGs into crystals and stones is a function related more to relative binding affinity than to ambient GAG concentration and that HS and ChS compete for specific binding sites on the crystal surface.

Adult↗

PubMed1994

Urate and calcium oxalate stones: from repute to rhetoric to reality.

A critical appraisal of the evidence commonly cited to support a link between high urate excretion and calcium oxalate (CaOx) urinary calculi is presented. Two theories have been invoked to provide a scientific explanation for urate's apparent promotory effect. The first proposes that urinary urate crystals promote CaOx precipitation by the phenomenon of epitaxy; the second hypothesis is that colloidal particles of urate reduce the inhibitory activity of urinary glycosaminoglycans (GAGs) which normally prevent the crystallization of CaOx. However, to the present, neither has been verified experimentally. More recent research from our group has revealed that at normal physiological pH values dissolved urate directly promotes CaOx precipitation by the classic 'salting-out' effect by enhancing nucleation, growth and aggregation of CaOx crystals. It is therefore suggested that the beneficial effect of allopurinol in reducing CaOx stone recurrences may be attributed to its lowering the urinary output of urate and thereby reducing the probability that CaOx will be salted out of urine, rather than to epitaxy or inactivation of urinary GAGs.

Allopurinol↗

PubMed1994

Crystal matrix protein--getting blood out of a stone.

The short history of crystal matrix protein began in 1991, when it was shown to be the predominant protein present in the organic extract of calcium oxalate crystals precipitated from fresh human urine. Here, we review what has subsequently come to be known about the protein, from its highly specific immunohistochemical distribution in the human nephron, to its finding in kidney stones, to the discovery of its relationship with the human blood coagulation zymogen prothrombin, and, finally, its identification as a urinary form of prothrombin activation fragment 1. A vitamin K-dependent glycopeptide, fragment 1 possesses the so-called GLA domain of its parent molecule; its known properties suggest that it may fulfil a determinant role in calcium oxalate urolithiasis as a potent urinary inhibitor of crystal growth and aggregation.

Animals↗

PubMed1993

Crystal matrix protein is related to human prothrombin.

Crystal matrix protein (CMP) is the principal protein found in calcium oxalate (CaOx) crystals precipitated from whole human urine. It is a potent inhibitor of crystal aggregation and may therefore be important in the aetiology of kidney stone disease. CMP was isolated from CaOx crystals by EDTA dissolution and purified by Sephacryl S-200 column chromatography and reversed-phase high pressure liquid chromatography. Edman degradation revealed 81.8% sequence identity of the 11 N-terminal amino acids of CMP with the N-terminus of human prothrombin, which contains 10 gamma-carboxyglutamic acid residues in the first 32 amino acids. The apparent relationship between CMP and prothrombin was confirmed when an antibody to human prothrombin reacted with CMP upon Western blotting of sodium dodecyl sulphate polyacrylamide gels of the protein.

Amino Acid Sequence↗

PubMed1993

The scientific basis of calcium oxalate urolithiasis. Predilection and precipitation, promotion and proscription.

The documentation of no other human disease threads as far into antiquity as that of urinary stones. However, despite this arcane history and the development of novel means of treating the condition, the basic mechanisms of stone formation and the identity of indicators of recurrence remain largely shrouded in uncertainty. This review is concerned with what scientific information is known about the cause and formation of calcium oxalate stones--the most common component of human uroliths. Stone pathogenesis can be broadly divided into two main processes: (1) nucleation of insoluble crystals in urine and (2) retention of those crystals within the urinary tract. The first section of the article presents the various factors that are known or surmised to influence the likelihood that crystals will nucleate within the renal collecting system, and these are considered from the perspective of both their relation to metabolic disorders and their usefulness as diagnostic and therapeutic indicators. A discussion of factors that may influence the probability that newly formed crystals will be retained within the nephron forms the second part of the review. In developing this more mechanistic aspect of the disease the epitaxy, matrix and inhibitor theories of stone formation are presented, with particular emphasis being placed on their relation to crystal nucleation, growth or aggregation, and experimental evidence both for and against the hypotheses are discussed.

Calcium Oxalate↗

PubMed1993

Immunohistochemical distribution and quantification of crystal matrix protein.

The aim of this study was to determine the immunohistochemical distribution and quantification of crystal matrix protein (CMP). CMP, a 31 kDa glycoprotein, is the principal macromolecule found in calcium oxalate crystals generated in human urine, and is a potent inhibitor of crystal aggregation. A polyclonal rabbit anti-human CMP antibody was used to examine renal tissue by immunohistochemical techniques and light microscopy (N = 45). Twenty-five other human organs were similarly assessed. Quantification was performed using a visual analogue scale. CMP was visible as cytoplasmic staining in the epithelial cells of the TALH and the distal convoluted tubule including the macula densa in a subgroup of nephrons. CMP was not identified elsewhere in the urinary tract or in the extrarenal organs examined. Despite a trend indicating that the kidneys of normal men had more CMP than those of normal women, the difference failed to reach significance (P = 0.11). There was, however, more CMP in the stone formers group compared with either normal men (P < 0.01) or normal women (P < 0.01). This protein may be an important determinant of calcium oxalate kidney stone disease.

Aged↗

PubMed1993

Dissolved urate promotes calcium oxalate crystallization: epitaxy is not the cause.

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 degrees 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 degrees 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1992

Calcium oxalate crystallization in urine: role of urate and glycosaminoglycans.

Increasing the concentration of urate promotes the crystallization of calcium oxalate in human urine. In this study the possibility that this effect might be attributable to the attenuation of the inhibitory activity of urinary glycosaminoglycans (GAGs) was explored. Urine sample were collected from 20 men with no history of urolithiasis and the intact GAGs removed by 10 kDa ultrafiltration. Ten of these specimens, designated type A, spontaneously precipitated calcium oxalate crystals when the median urate concentration was increased from 3.13 to 7.33 mmol/liter by the addition of a saturated solution of sodium urate. In the remaining more dilute urines, which were designated type B, spontaneous calcium oxalate crystallization did not occur when the median urate concentration was raised from 2.20 to 6.40 mmol/liter. In these samples crystallization was induced by a standard load of oxalate above the empirically determined metastable limit. Addition of urate significantly reduced the median metastable limit from the control value of 125 to 46 mumol oxalate, and the volume of calcium oxalate deposited was increased fourfold from 25,000 to 104,000 microns 3/microliters. The median size of the precipitated particles was also increased in the presence of urate from 12.06 microns to 14.3 microns; this was confirmed by scanning electron microscopy, which demonstrated that the crystals precipitated in the presence of added urate, though individually smaller, were markedly more numerous and more highly aggregated than those deposited in the control. Re-ultrafiltration of the urines to which urate had been added did not alter the urate concentration, and SEM examination of the ultrafiltration membranes did not reveal the presence of any particulate material.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1991

Effects of chondroitin sulphate, human serum albumin and Tamm-Horsfall mucoprotein on calcium oxalate crystallization in undiluted human urine.

The effects of physiological concentrations of chondroitin sulphate, human serum albumin and Tamm-Horsfall mucoprotein on the crystallization of calcium oxalate in undiluted, ultrafiltered human urine were investigated using particle size analysis and scanning electron microscopy. Neither the amount of oxalate required to induce detectable calcium oxalate crystal nucleation nor crystal morphology was affected by the presence of any of these macromolecules. Chondroitin sulphate had no effect on the amount of crystalline material deposited or on the size of the particles precipitated in response to a standard oxalate load. Human serum albumin slightly reduced the size of the crystal aggregates and caused a small increase in the amount of crystal matter precipitated. By contrast, Tamm-Horsfall mucoprotein significantly inhibited crystal aggregation and markedly increased the volume of matter deposited, although this could not be attributed to a promotion of solute precipitation. It was concluded that chondroitin sulphate, human serum albumin and Tamm-Horsfall mucoprotein cannot account for the inhibitory effects of macromolecules with a relative mass greater than 10 kDa in spun and filtered urine. Nonetheless, Tamm-Horsfall mucoprotein is likely to inhibit crystal aggregation in whole urine in vivo and may therefore be instrumental in preventing calcium oxalate stone formation.

Adult↗

PubMed1991

Urate and calcium stones--picking up a drop of mercury with one's fingers?

The evidence invariably cited to support the suspicion that urinary urate is a predisposing factor in calcium oxalate (CaOx) stone formation is critically reviewed. Analysis of the relevant literature shows that speculation is based on the clinical impression that CaOx stone-formers appear to excrete more urate than do normal subjects, and that allopurinol reduces the rate of CaOx stone recurrences. On balance, this is sufficient to suggest that a high urinary excretion of urate promotes CaOx stone formation. However, in the past, evidence to disclose the mechanism by which urate could exert this effect has been largely shrouded in confusion and controversy. The evidence for two theories that have dominated thinking in this area are reviewed and new findings are reported that indicate that neither can account for the purported effect of urate. It is concluded that dissolved urate in urine, at normal physiological pH values, directly provokes CaOx crystal nucleation by the phenomenon of salting-out. The possibility that urate promotes CaOx stone formation is further strengthened by its ability to increase significantly the amount of CaOx precipitated from solution and to cause the aggregation of individual crystals into large clusters. Future avenues of investigation that should assist in the formulation of diagnostic and therapeutic guidelines are presented.

Allopurinol↗

PubMed1991

Inhibition by sodium-potassium citrate (CG-120) of calcium oxalate crystal growth on to kidney stone fragments obtained from extracorporeal shock wave lithotripsy.

Retention of fragments within the kidney after extracorporeal shock wave lithotripsy (ESWL) continues to be a major shortcoming of this form of stone treatment. The aim of this study was to evaluate the ability of sodium-potassium citrate to inhibit calcium oxalate crystal nucleation and growth on to stone fragments remaining after ESWL. The continuous flow crystallisation technique was adapted to induce calcium oxalate crystal nucleation and growth on to the surface of fragmented kidney stones and the inhibitory effect of sodium-potassium citrate was assessed by scanning electron microscopy and by determining the relative increase in crystalline mass at final concentrations of 0, 2, 4, 6, 8, 10 mmol/l. Sodium-potassium citrate significantly inhibited the deposition of new crystalline calcium oxalate in a dose-dependent manner above 2 mmol/l; these findings were confirmed by scanning electron microscopy. It was concluded that sodium-potassium citrate may provide an effective means of preventing the formation of new kidney stones by the deposition of calcium oxalate on to residual stone fragments resulting from ESWL and that the technique used is an efficient means of testing the efficacy of therapeutic agents to prevent stone recurrence in patients treated with ESWL.

Calcium Oxalate↗