RESUMO
This study was designed to get information on aggregation (AGN) of urinary calcium oxalate crystals (CaOx) which seems to occur in stone formation despite a protecting coat of urinary macromolecules (UMs). CaOx crystallization was directly produced in urine, control and albumin solution by Ox titration and was spectrophotometrically followed. A rapid decrease of optical density indicating AGN was absent in 14 of 15 freshly voided urines of 5 healthy controls. However, in the presence of UM-coated hydroxyapatite all urines with relative high sodium concentration, being an indicator of concentrated urine, showed a pronounced AGN which was abolished when these urines were diluted. Albumin relatively found to be an inhibitor of AGN showed after temporary adsorption on Ca Phosphate (CaP) massive self-AGN and changed to a promoter of CaOx AGN. Self-AGN after adsorption on surfaces especially of CaP, being an important compound of Randall's plaques, can thus explain this paradoxical behavior of UMs. Aggregated UMs probably bridge zones of electrostatic repulsion between UM-coated crystals with identical electrical surface charge. These zones extend by urine dilution which decreases ionic strength. Diminution of urinary concentration by increasing diuresis seems, therefore, to be important in stone metaphylaxis.
Assuntos
Oxalato de Cálcio/metabolismo , Diurese , Substâncias Macromoleculares/urina , Nefrolitíase/etiologia , Nefrolitíase/prevenção & controle , Cristalização , Humanos , Nefrolitíase/urinaRESUMO
To get information on the consequences of adsorption of urinary macromolecules (UMs) on crystals, Ca phosphate (CaP) precipitation was performed in urine of 15 stone patients and 15 controls. In solutions of dissolved precipitates (DPU), Ca oxalate (CaOx) crystallization and aggregation (AGN) of latex beads were spectrophotometrically studied and compared to results obtained in urine and in UMs isolated by hemofilter dialysis (HD). Tests were repeated with a 20 µg/mL albumin solution (AS). Inhibition of CaOx growth and AGN was significantly reduced in DPU as well as in HD. Furthermore, DPU and HD showed an AGN of latex beads which in patients was more pronounced than in controls. Identical effects observed in DPU and HD can be explained by temporary high concentrations of UMs by adsorption on surfaces favoring self AGN. Temporary adsorption of AS on CaP produced polymers of a limited size, significantly reduced the inhibition of CaOx AGN and accelerated latex AGN. In DPU, AGN of latex beads occurred despite a surface potential of -37 mV, which within short distances produces areas of massive electrostatic repulsion and normally inhibits AGN. Such areas might be bridged by self-aggregated UMs. The retardation of CaOx AGN measured in DPU could be decisive whether during crystalluria crystals are washed out from the kidney by diuresis or are attached to kidney calcifications or pre-existing stones.
Assuntos
Fosfatos de Cálcio/química , Substâncias Macromoleculares/química , Urina/química , Urolitíase/etiologia , Albuminas/química , Estudos de Casos e Controles , Hemofiltração , HumanosRESUMO
Kidney stones probably grow during crystalluria by crystal sedimentation and aggregation (AGN) on stone surfaces. This process has to occur within urinary transit time (UT) through the kidney before crystals are washed out by diuresis. To get more information, we studied by spectrophotometry the formation and AGN of Ca oxalate (Ca Ox) crystals which were directly produced in urine of 30 stone patients and 30 controls by an oxalate (Ox) titration. Some tests were also performed after removing urinary macromolecules (UMs) by ultrafiltration. To induce rapid crystallization, high Ox additions (0.5-0.8 mM) were necessary. The most important finding was retardation of crystal AGN by UM. In urine of 63% of controls but only 33% of patients, no AGN was observed during an observation of 60 minutes (P < 0.05). Also growth and sedimentation rate of crystals were significantly reduced by UM. For stone metaphylaxis, especially for posttreatment residuals, avoiding dietary Ox excesses to prevent crystal formation in the kidney and increasing diuresis to wash out crystals before they aggregate are recommended.
RESUMO
Aggregation (AGN) of freshly precipitated calcium oxalate crystals was photometrically studied in urine of 30 calcium stone patients and 30 controls, in solutions containing urinary macromolecules (UMS) and in an inhibitor free control solution (CS). Crystals were produced by oxalate titration and crystallization was monitored measuring optical density (OD). Tests were repeated adding hydroxyapatite (HAP) to urine and UMS and adding citrate and pyrophosphate (PPi) to UMS of the controls. AGN was recognized as a rapid OD decrease being at least three times faster than sedimentation of single crystals (p < 0.001) and used to calculate an extent of AGN (EA%). The time between the end of titration and the beginning of AGN was determined as suspension stability (SS). The main effect of urinary inhibitors was retardation of AGN without changing EA, SS being higher in urine than UMS (p < 0.001) and in UMS than CS (p < 0.001). In urine of 63% of controls but only in 33% of patients, no AGN was recorded (p < 0.05). The high inhibitory activity of urine could not be reproduced in UMS even in combination with 3.5 mM citrate or 0.05 mM PPi. 0.05 mg/mL HAP reduced SS in all urine samples to low values and increased the rate of rapid OD decrease, being a measure for the size of aggregates. Retarding AGN of crystals during their passage through the kidney seems to be an important mechanism to prevent stone formation during crystalluria. The promotion of AGN by HAP reveals a new role of Randall's plaques in nephrolithiasis.
Assuntos
Oxalato de Cálcio/urina , Substâncias Macromoleculares/urina , Nefrolitíase/urina , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Ácido Cítrico/urina , Cristalização , Difosfatos/urina , Durapatita/urina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
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.
Assuntos
Cálcio/química , Substâncias Macromoleculares/química , Substâncias Macromoleculares/urina , Fenômenos Bioquímicos , Cristalização , Humanos , MasculinoRESUMO
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.
Assuntos
Oxalato de Cálcio/urina , Substâncias Macromoleculares/urina , Urolitíase/prevenção & controle , Oxalato de Cálcio/química , Citratos/química , Cristalização , Hemofiltração , Humanos , Masculino , Microscopia Eletrônica de Varredura , Propriedades de Superfície , Fatores de Tempo , Urolitíase/urinaRESUMO
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.
Assuntos
Oxalato de Cálcio/metabolismo , Cristalização , Durapatita/metabolismo , Cálculos Urinários/química , Cálculos Urinários/urina , Adulto , Oxalato de Cálcio/urina , Durapatita/urina , Humanos , Concentração de Íons de Hidrogênio , Masculino , Valores de Referência , Sensibilidade e Especificidade , Espectrofotometria , Cálculos Urinários/fisiopatologiaRESUMO
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.
Assuntos
Oxalato de Cálcio/química , Oxalato de Cálcio/urina , Durapatita/química , Durapatita/urina , Cálculos Urinários/química , Cálcio/urina , Cristalização , Humanos , Concentração de Íons de Hidrogênio , Masculino , Cálculos Urinários/etiologiaRESUMO
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.
Assuntos
Oxalato de Cálcio , Cálculos Urinários/química , Cálculos Urinários/urina , Oxalato de Cálcio/análise , Oxalato de Cálcio/química , Oxalato de Cálcio/urina , Cristalização , Humanos , Microscopia Eletrônica de Varredura , EspectrofotometriaRESUMO
Nucleation, growth and aggregation are thought to be the most important crystallization processes in stone formation. Since crystallization properties change with urinary dilution, centrifugation and filtration, crystallization should always be studied in freshly voided and not pretreated urine. Recently we developed an automated method where calcium oxalate crystallization is induced in native urine by an exogenous oxalate load and nucleation and growth are monitored by an ion-selective calcium electrode. The method has now been supplemented with the spectrophotometric measurement of crystal aggregation. Repeated experiments in the same urine with different oxalate loads enable the determination of the critical oxalate additionable to induce crystallization (metastable limit) and the calculation of an oxalate load-independent growth rate constant. Preliminary results obtained in the native urine of healthy controls showed an extremely high limit of metastability and a complete absence of crystal aggregation. These findings may explain why, despite frequent urinary calcium oxalate supersaturation, healthy people do not form stones.
Assuntos
Oxalato de Cálcio/urina , Cristalização , Humanos , Técnicas In Vitro , Eletrodos Seletivos de Íons , Masculino , EspectrofotometriaRESUMO
A new variant of von Willebrand's disease has been discovered in 2 members of a Macedonian family of 6. The proband, an 8-year-old boy, showed a prolonged bleeding episode on 1 occasion. Ristocetin-induced platelet aggregation and bleeding time were normal. In plasma, ristocetin cofactor activity (RCo) and von Willebrand factor (vWf) antigen were reduced to the same clearly low level. The determination of vWf antigen of platelets resulted in borderline values, while RCo was clearly reduced. Low- and intermediate-resolution agarose gel electrophoresis showed absence of the largest multimers in plasma vWf, and slight reduction in platelet vWf. High-resolution gels revealed abnormal multimeric structure only in plasma vWf. The smaller multimers could be resolved in a broad central band flanked by 4 fainter satellite bands; however, satellite bands close to the central band were more intense, and more distant ones were fainter, compared to normal plasma. The central band of the fastest-moving multimer was markedly intensified, and the mobility of the whole quintuplet was slightly reduced. Heredity seems to be autosomal-dominant. No mutation was found in exon 28 of the vWf gene. Because there was only 1 mild bleeding episode in the family, this structural variation seems to have only little clinical consequence. We conclude that this vWf abnormality is different from those observed in other type II variants previously described. Based on the revised classification by the International Society on Thrombosis and Haemostasis, we proposed designation type 2A-Bern for this new subtype.
Assuntos
Variação Genética , Doenças de von Willebrand/sangue , Doenças de von Willebrand/genética , Fator de von Willebrand/genética , Sequência de Bases , Tempo de Sangramento , Plaquetas/metabolismo , Criança , Primers do DNA , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Agregação Plaquetária/efeitos dos fármacos , Reação em Cadeia da Polimerase , Ristocetina/farmacologia , Doenças de von Willebrand/fisiopatologia , Fator de von Willebrand/biossíntese , Fator de von Willebrand/isolamento & purificaçãoRESUMO
Monitoring of crystallization of calcium salts with ion-selective electrodes has turned out to be a very sensitive method. The difficulties of handling these electrodes in native whole urine and other biological fluids have been eliminated by new calcium analyzers, which clean and calibrate the electrodes after each measurement. To study crystallization kinetics, repeated calcium ion measurements have to be performed at regular intervals. For this purpose we have developed a special sampler and software. The sampler brings a thermostat-controlled crystallization chamber to the analyzer at preselected intervals. The computer directs and coordinates the sampler and the analyzer, stores the received results and prints out growth curves. Furthermore it calculates the half-time (h) and the maximum decrease of ionic calcium at infinite incubation time (delta Ca2+ infinity). Both values are shown to characterize the growth of calcium oxalate monohydrate in urine. Results are obtained within 40 min.
Assuntos
Oxalato de Cálcio/urina , Cálcio/urina , Cristalização , Eletrodos , Processamento Eletrônico de Dados/métodos , Humanos , Microcomputadores , SoftwareRESUMO
To detect von Willebrand factor multimers in plasma samples and factor VIII concentrates, a vertical discontinuous SDS electrophoresis was developed. A vacuum blotting system allowed to improve the transfer to the nitrocellulose membrane. The visualization of the separated multimers was sensitized by applying an alkaline phosphatase anti-alkaline phosphatase staining technique. The reported method clearly shows structural abnormalities of von Willebrand factor and deficiency of high multimers, the vacuum transfer is efficient and the sensitivity of the staining system is very high.
Assuntos
Fator de von Willebrand/química , Fosfatase Alcalina , Anticorpos Monoclonais , Eletroforese em Gel de Ágar/métodos , Eletroforese Descontínua/métodos , Humanos , Coloração e Rotulagem/métodos , Fator de von Willebrand/análiseRESUMO
In order to have a rapid method of measuring the inhibition of calcium-oxalate monohydrate growth in freshly voided whole urine, a test system by Meyer and Smith that has originally been developed for diluted urine was modified. The crystallization processes were monitored by an ion-selective calcium electrode, which allowed determination of the half-life value of the decrease in calcium within 25 min. Even given the high inhibitory activity of whole urine, the test gave reliable results when a high seed concentration was used. Inhibition was expressed as the ratio between the half-lives of the calcium decrease obtained in the presence and in the absence of inhibitors. This approach allowed kinetic studies of individual inhibitors in model solutions. Furthermore, the measurements of inhibitors in urine could be performed before the chemical composition was determined.
Assuntos
Oxalato de Cálcio/urina , Oxalato de Cálcio/antagonistas & inibidores , Cristalização , Eletrodos , Humanos , Técnicas In Vitro , Cálculos Urinários/urinaRESUMO
The nucleating effect of hydroxyapatite (HAP) and the inhibitory effect of pyrophosphate (PPi) on calcium oxalate crystallization have been studied at different pH's in solution metastabely supersaturated with respect to calcium oxalate but saturated with respect to HAP. Crystallization was monitored by a decrease of calcium in the supernatant and formation products were calculated. At a pH above 6.0 already minimal HAP concentrations proved to be a suitable substrate for heterogeneous nucleation and growth of calcium oxalate. PPi showed a pronounced inhibitory effect on spontaneous as well as on HAP induced crystallization of calcium oxalate, this effect being highly pH dependent. HAP was found to neutralize the inhibitory effect of PPi in a molar ratio of 10:1.