Prevalence, pathophysiological mechanisms and factors affecting urolithiasis.

The formation of urinary stone, urolithiasis, is one the oldest known disease affecting human throughout different civilizations and times. The exact pathophysiological mechanism of urolithiasis is not yet clear, as these calculi are of various types and too complex for simple understanding. A single theory cannot explain its formation; therefore, different theories are presented in various times for its explanation like free particle, fixed particle, Randall's plaque theory. In addition, various factors and components are identified that play an important role in the formation of these urinary calculi. In this review, composition of kidney stones, its prevalence/incidence, explanation of pathophysiological mechanisms and role of various factors; urinary pH, uric acid, parathyroid hormone, citrate, oxalate, calcium and macromolecules; osteopontin, matrix Gla protein, kidney injury molecules, urinary prothrombin fragment-1, Tamm-Horsfall protein, inter-α-inhibitors, have been discussed in detail.

Thrombin Generation in Patients With Suspected Venous Thromboembolism.

Increasing number of patients with clinically suspected venous thromboembolism is referred to radiological departments for definitive diagnosis. A simple assay to exclude the diagnosis and avoid radiological examinations is needed. We have reported correlations between D-dimer and prothrombin fragment 1 + 2 measured in plasma and urine. To further develop an analysis based on urine, more understanding of thrombin generation in these patients is needed. The aim of this study was to compare ex vivo thrombin generation with in vivo markers in plasma and urine in patients with and without venous thromboembolism. Urine and blood samples were collected from patients with suspected venous thromboembolism. Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to analyze the samples for in vivo thrombin generation. The ex vivo thrombogram parameters were measured by the calibrated automated thrombogram assay. Venous thromboembolism was verified with compression ultrasound of the lower extremity deep veins or with computer tomography of the pulmonary arteries. Venous thromboembolism was diagnosed in 117 of 591 patients, and they had significantly higher levels of urine and plasma prothromin fragment 1 + 2, D-dimer, lag time, time to peak, and endogenous thrombin potential when adjusted for covariates. The pattern of ex vivo and in vivo thrombin generation in patients with suspected venous thromboembolism was comparable when adjusted for covariates. Prothrombin fragment 1 + 2 in plasma and urine reflects thrombin generation ex vivo in the same manner. This indicates that urine may be an alternative substrate to quantify a procoagulant state.

D-Dimer and prothrombin fragment 1 + 2 in urine and plasma in patients with clinically suspected venous thromboembolism.

Increased levels of urine prothrombin fragment 1 + 2 was recently reported to be associated with imaging-verified venous thromboembolism. In this study we evaluated the relationship between plasma D-dimer and plasma and urine prothrombin fragment 1 + 2 in patients with suspected venous thromboembolism. Urine and blood samples were collected from patients with suspected pulmonary embolism or deep vein thrombosis. The samples were analysed with commercially available ELISA kits. The diagnosis of venous thromboembolism was verified with contrast-enhanced computer tomography of the pulmonary arteries or lower extremity deep vein compression ultrasound and venography as appropriate. Venous thromboembolism was diagnosed in 150 of 720 patients. Significantly higher levels of plasma D-dimer and prothrombin fragment 1 + 2 in plasma and urine were found in those with imaging-confirmed venous thromboembolism versus those without (P < 0.001). The correlation between the three biomarkers was statistically significant (range of rs values 0.45-0.65, P < 0.001). Plasma D-dimer had the highest diagnostic accuracy followed by prothrombin fragment 1 + 2 in plasma. Further development of ELISA analyses for urine testing of prothrombin fragment 1 + 2 may improve its diagnostic accuracy.

Quantitative urinary proteomics using stable isotope labelling by peptide dimethylation in patients with prostate cancer.

Prostate cancer (PCa) is the most commonly diagnosed malignancy in men. The current prevalent diagnosis method, prostate-specific antigen (PSA) screening test, has low sensitivity, specificity and is poor at predicting the grade of disease. Thus, new biomarkers are urgently needed to improve the PCa diagnosis and staging for the management of patients. The aim of this study is to investigate the first voided urinary sample after massage for biomarker discovery for PCa. In this work, untargeted metabolomic profiling of the first voided urinary sample after massage from 28 confirmed prostate cancer patients, 20 benign enlarged prostate patients and 6 healthy volunteers was performed using liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS). Single and multiple peptide protein and cross-linking molecules were identified using PEAKS software. Analytical and diagnostic performance was tested using the Student's t test, Benjamini Hochberg correction and the receiver operating characteristic (ROC) curves. Using differential display analysis to compare peptides and cross-linking molecules of urinary samples between patients with benign, enlarged prostate and malignant cancer, we identified multiple peptides derived from osteopontin (SPP1) and prothrombin (F2) that are lower in PCa patients than in benign and enlarged prostate. The diagnosis accuracies of SPP1 and F2 peptides are 0.65-0.77 and 0.68-0.72, respectively. In addition to this, there are significant differences between PCa and benign/enlarged prostate patients in pyridinoline (PYD) and deoxypyridinoline (DPD) (p value = 0.001). Differences also, as shown in the excretion of these molecules for different stages of PCa (p value = 0.04) as the level of DPD and DPD/PYD ratio, were high in patients with locally advanced tumours. The study underscores the importance of proteomics analysis, and our results demonstrate that a urinary-based in depth proteomic approach allows the potential identification of dysregulated pathways and diagnostic biomarkers.

Prothrombin fragment 1+2 in urine as a marker on coagulation activity in patients with suspected pulmonary embolism.

INTRODUCTION: We have recently reported that increased levels of urine prothrombin fragment 1+2 reflected radiologically verified deep vein thrombosis. In this study we evaluated whether urine prothrombin fragment 1+2 was associated with pulmonary embolism in non-selected patients. MATERIALS AND METHODS: Patients with clinical suspected pulmonary embolism were interviewed on comorbidities and medications. Urine was collected from each patient before radiological examination and snap frozen until analysed on urine prothrombin fragment 1+2 with an ELISA kit. Imaging of the pulmonary arteries were conducted with contrast enhanced computer tomography. RESULTS: Pulmonary embolism was diagnosed in 44/197 patients. Non-significantly higher urine prothrombin fragment 1+2 levels were found in non-selected patients with pulmonary embolism vs. those without (p=0.324). Significantly higher urine prothrombin fragment 1+2 levels were found in the pulmonary embolism positive patients without comorbidities (n=13) compared to the control group (n=28) (p=0.009). The calculated sensitivity, specificity and negative predictive value using the lowest detectable urine prothrombin fragment 1+2 level was 82%, 34% and 87%, respectively. CONCLUSIONS: There was no significant urine prothrombin fragment 1+2 level difference in patients with and without pulmonary embolism. In non-comorbide pulmonary embolism positive patients the urine prothrombin fragment 1+2 levels were significantly higher compared to the control group. The negative predictive value found in this study indicates that uF1+2 has the potential to identify patients with a low risk of PE.

Thrombin split products (prothrombin fragment 1 + 2) in urine in patients with suspected deep vein thrombosis admitted for radiological verification.

INTRODUCTION: The appearance of prothrombin fragment 1 + 2 (F1 + 2) in urine has been associated with postoperative hypercoagulability and thromboembolism. We wanted to assess if F1 + 2 was released in urine (uF1 + 2) in patients with procoagulant disorders, and if higher levels were found in patients with radiological verified deep vein thrombosis (DVT). MATERIALS AND METHODS: Consecutive patients were interviewed on comorbidities and medications. An unselected total cohort (n = 534) and a control cohort (n = 177) were analysed. A urine sample (10 ml) was collected and snap frozen before levels of uF1 + 2 were measured with an ELISA kit. Visualisation of DVT was done with compression ultrasound, supplied with venography when feasible. All patients were followed up for 3-6 months. RESULTS: DVT was diagnosed in 108/534 patients. Statistical significant higher uF1 + 2 levels were found in patients with DVT (p < 0.001), in DVT positive patients with ongoing malignancy (p = 0.034) and in pregnant women compared to the control cohort (p < 0.001). Non-significant increased urine concentrations were found in DVT positive vs. DVT negative patients with infections and traumas. CONCLUSIONS: Levels of uF1 + 2 was associated with DVT both in the total cohort and in the control cohort as well as in most patients with coexisting conditions.

Coagulation activity in elderly patients with hip fracture measured by prothrombin fragment 1 + 2 in urine.

OBJECTIVE: The purpose of this study was to document the uF1 + 2 excretion in elderly patients during and after a hip fracture (HF). METHODS: The study was a prospective pilot study. Spot urine samples were collected immediately after admission and every morning until surgery. After surgery, urine samples were collected on days 1, 5, 7, 14, and at follow-up on day 90 (±10). RESULTS: A total of 24 women and 7 men with HF completed the study. The median uF1 + 2 level was significantly increased on the day of admission relative to the median level at follow-up. Maximum levels were seen on day 1 with a decreasing tendency until follow-up. Patients treated with a hemiarthroplasty had higher median uF1 + 2 levels on all days compared with patients treated with osteosynthesis. CONCLUSION: A substantial coagulation activity, indicated by high median levels of uF1 + 2, was seen at admission and during the first week after HF.

Differences in urinary prothrombin fragment 1 + 2 levels after total hip replacement in relation to venous thromboembolism and bleeding events.

BACKGROUND: Prothrombin fragment 1 + 2 is excreted in urine (uF1 + 2) as a result of thrombin generation and, therefore, may be a useful marker of coagulation status. OBJECTIVES: To assess uF1 + 2 levels after total hip replacement (THR) in patients with venous thromboembolism (VTE) and bleeding events. PATIENTS/METHODS: This study was conducted in parallel with a prospective, dose-finding study evaluating the efficacy and safety of different doses of rivaroxaban (Xarelto, Bayer HealthCare AG, Wuppertal, Germany) for thromboprophylaxis, relative to enoxaparin. Deep vein thrombosis was diagnosed by mandatory venography performed 5-9 days after THR, or earlier if symptomatic. Symptomatic pulmonary embolism was diagnosed by objective testing. Bleeding complications were registered and stratified into major bleeding, clinically relevant, non-major bleeding, and minor bleeding, using predefined criteria. RESULTS: Eighty-four patients had a VTE and 57 patients had a bleeding event (n = 722). Significantly higher median uF1 + 2 levels were observed in the VTE group on day 3 after THR (P = 0.03), compared with control. Median uF1 + 2 levels were lower in the bleeding group on day 3 after THR (P = 0.005) and on the day of venography (P = 0.36), compared with control. Comparisons between the VTE and bleeding groups showed significantly lower median uF1 + 2 levels in the bleeding group on day 3 after THR and on the day of venography (P < 0.0001 and P = 0.006, respectively). CONCLUSIONS: Measurement of uF1 + 2 could provide a simple clinical test to evaluate non-invasively the intensity of coagulation activation after THR. However, further studies are required to confirm these encouraging preliminary results.

Prothrombin fragment 1+2 in urine as an indicator of sustained coagulation activation after total hip arthroplasty.

PURPOSE: Prothrombin fragment 1+2 measured in spot urine (uF1+2) is an indicator of thrombin generation. We examined whether measured levels of uF1+2 can be used to differentiate between patients who do and do not acquire sustained coagulation activation after total hip arthroplasty (THA). METHODS: We performed two separate studies in patients undergoing THA. Study 1 was a prospective pilot study aiming to roughly estimate the extent of pre- and postoperative fluctuations in the uF1+2 concentration. Study 2 was a larger prospective cohort study aiming to verify the findings of Study 1 and to examine the association between the uF1+2 concentrations and risk of vascular thrombotic complications (VTC) or death. Finally, we sought to define a cut-off concentration value that could be used to identify patients with a sustained uF1+2 elevation after the first postoperative week. The urine samples were analysed by ELISA. In both studies thromboprophylaxis was used for at least 7 days after the operation. RESULTS: The operative trauma resulted in elevation of the uF1+2 level in all patients compared with the preoperative level and levels in the healthy volunteers. Ten out of 113 patients (8.8%) in the second study suffered VTC or death, assumed to be caused by a coagulation problem. Analysis of variance revealed the following statistically significant associations: pre- vs. postoperative log uF1+2 levels (P<0.0001), log uF1+2 levels comparing patients with and without events (P=0.004); and the individual log uF1+2 levels (P<0.0001). A cut-off value of uF1+2 concentration between 0.3 and 0.5 nmol/l had a sensitivity and a negative predictive value between100% and 90%, and specificity between 45% and 63% and overall accuracy between 50% and 65%. This value was obtained by the analysis of a receiver operating characteristic curve with the purpose of identifying patients with sustained coagulation activation on day 5 after operation. CONCLUSION: Our studies suggest that measured levels of uF1+2 can be potentially used to assess the individual risk of VTC after THA and to test for non-invasive detection of sustained coagulation activation.

Effect of urine fractionation on attachment of calcium oxalate crystals to renal epithelial cells: implications for studying renal calculogenesis.

Our aim was to determine whether fractionation of human urine affects the attachment of calcium oxalate monohydrate (COM) crystals to renal cells. Urine collected from six healthy subjects was fractionated into sieved (S), centrifuged (C), centrifuged and filtered (CF), or ultrafiltered (UF). Attachment of [(14)C]COM crystals to Madin-Darby canine kidney (MDCK) cells was studied after precoating the crystals or the cells with the urine fractions and by using the same fractions as the binding medium. Protein content of the fractions and precoated crystals was analyzed with SDS-PAGE and Western blotting. All urine fractions inhibited crystal attachment. When fractions from the six urine samples were used to precoat the cells, the median inhibitions of crystal adhesion ( approximately 40%) were not significantly different. Median inhibition after preincubation of crystals was the same for the S, C, and CF fractions ( approximately 40%) but significantly greater than for the UF fraction ( approximately 28%). When fractions were used as the binding medium, median inhibitions decreased from 64% in the S fraction to 47 (C), 42 (CF), and to 29% (UF). SDS-PAGE analysis showed that centrifugation and filtration reduced the amount of Tamm-Horsfall glycoprotein (THG), which was confirmed by Western blotting. Human serum albumin, urinary prothrombin fragment 1, and osteopontin, but not THG, were present in demineralized extracts of the precoated crystals. Fractionation of human urine affects the attachment of COM crystals to MDCK cells. Hence future studies investigating regulation of crystal-cell interactions should be carried out in untreated urine as the binding medium.

Sialylation of urinary prothrombin fragment 1 is implicated as a contributory factor in the risk of calcium oxalate kidney stone formation.

Urinary glycoproteins are important inhibitors of calcium oxalate crystallization and adhesion of crystals to renal cells, both of which are key mechanisms in kidney stone formation. This has been attributed to glycosylation of the proteins. In South Africa, the black population rarely form stones (incidence < 1%) compared with the white population (incidence 12-15%). A previous study involving urinary prothrombin fragment 1 from both populations demonstrated superior inhibitory activity associated with the protein from the black group. In the present study, we compared N-linked and O-linked oligosaccharides released from urinary prothrombin fragment 1 isolated from the urine of healthy and stone-forming subjects in both populations to elucidate the relationship between glycosylation and calcium oxalate stone pathogenesis. The O-glycans of both control groups and the N-glycans of the black control samples were significantly more sialylated than those of the white stone-formers. This demonstrates a possible association between low-percentage sialylation and kidney stone disease and provides a potential diagnostic method for a predisposition to kidney stones that could lead to the implementation of a preventative regimen. These results indicate that sialylated glycoforms of urinary prothrombin fragment 1 afford protection against calcium oxalate stone formation, possibly by coating the surface of calcium oxalate crystals. This provides a rationale for the established roles of urinary prothrombin fragment 1, namely reducing the potential for crystal aggregation and inhibiting crystal-cell adhesion by masking the interaction of the calcium ions on the crystal surface with the renal cell surface along the nephron.

Urine protein markers distinguish stone-forming from non-stone-forming relatives of calcium stone formers.

We have investigated urine protein inhibitors of calcium oxalate crystallization to determine whether variations in these proteins are associated with kidney stone disease and whether protein measurements improve the identification of stone formers compared with conventional risk factors (RF). Using Western blotting, we studied variations in the electrophoretic mobility patterns and relative abundances of crystallization-inhibitory proteins in urine from 50 stone-forming (SF) and 50 non-stone-forming (NS) first-degree relatives of calcium SF patients, matched by gender and age. Standard urine chemistry stone risk measurements were also made. Multivariate discriminant analysis was used to test the association of these proteins with nephrolithiasis. Differences in form and abundance of several urine proteins including inter-alpha-trypsin inhibitor (ITI), prothrombin fragment 1 (PF1), CD59, and calgranulin B (calB) were found to be associated with stone formation. By multivariate discriminant analysis, measurements of forms of PF1, ITI, and calB in men and ITI and CD59 in women, classified 84% of men and 76% of women correctly by stone status. In contrast, standard urine chemistry RF identified only 70% of men correctly and failed to distinguish female SF from NS. Thus a small subset of protein measurements distinguished SF from NS far better than conventional RF in a population of relatives of calcium SF, illustrating the significant association of these proteins with stone disease. Variations in these proteins may serve as markers of stone disease activity or vulnerability to recurrence and may provide new insights into mechanisms of stone formation.

Effects of urinary prothrombin fragment 1 in the formation of calcium oxalate calculus.

PURPOSE: We investigated the effects of urinary prothrombin fragment 1 in the formation of calcium oxalate urolithiasis. MATERIALS AND METHODS: Fresh urine and renal parenchyma from patients with calcium oxalate calculus and normal controls were collected. Urinary prothrombin fragment 1 was isolated and purified from urine. It was identified by sodium dodecyl sulfide-polyacrylamide gel electrophoresis and analysis of its first 13 N-amino acids. The inhibitory activity of urinary prothrombin fragment 1 on calcium oxalate crystal growth was tested by the seeded crystallization technique. Meanwhile, the gamma-carboxyglutamic acid composition of urinary prothrombin fragment 1 was analyzed by a previously described method and genetic mutation of the gamma-carboxyglutamic acid domain of urinary prothrombin fragment 1 from renal parenchyma was detected by polymerase chain reaction-single strand conformational polymorphism sequencing. RESULTS: The gamma-carboxyglutamic acid composition of urinary prothrombin fragment 1 was significantly decreased from normal (24.4 to 1.7 mol/1,000 amino acids) in patients with calcium oxalate calculus. The mean growth index +/- SD of urinary prothrombin fragment 1 to calcium oxalate crystals was 42.3 +/- 4.2 compared with the normal index of 19.2 +/- 2.8 (p <0.01). The polymerase chain reaction-single strand conformational polymorphism sequencing technique revealed no genetic mutation of the gamma-carboxyglutamic acid domain of urinary prothrombin fragment 1 in patients with calcium oxalate calculus. CONCLUSIONS: The gamma-carboxyglutamic acid composition of urinary prothrombin fragment 1 as well as its ability to inhibit calcium oxalate crystal growth was significantly decreased in patients with calcium oxalate calculus. This was not caused by genetic mutation of the gamma-carboxyglutamic acid domain of urinary prothrombin fragment 1. It is important to elucidate the mechanisms of calcium oxalate stones in view of urinary prothrombin fragment 1.

Clinical significance of urinary N1,N12-diacetylspermine levels in patients with hepatocellular carcinoma.

BACKGROUND/AIM: N1,N12-diacetylspermine (DiAcSpm), a diacetylpolyamine which was recently identified in urine, appeared to be a useful tumor marker for urogenital cancers. Here we examined the clinical significance of urinary DiAcSpm as a tumor marker for hepatocellular carcinoma (HCC). METHODS: Urine samples were collected from patients with HCC and benign liver diseases. Urinary levels of DiAcSpm were measured by ELISA, which was newly developed in order to analyze large numbers of samples. RESULTS: The appropriate threshold value was set at 325 nM/g x creatinine. The sensitivity of the DiAcSpm assay for HCC was 65.5% and the specificity calculated between HCC and liver cirrhosis was 76.0%. The percentage of DiAcSpm-positive HCC patients was similar to that for AFP or PIVKA-II. At more advanced clinical stages, the positive percentage of these three markers increased but the DiAcSpm levels appeared to move independently of AFP and PIVKA-II. In HCC patients, the DiAcSpm levels reflected the progression of disease or the effect of treatment. CONCLUSIONS: DiAcSpm levels were found to reflect the severity, activity or viability of HCC. Urinary DiAcSpm can therefore be considered one of the useful indexes for patients with HCC.

Whole urinary proteins coat calcium oxalate monohydrate crystals to greatly decrease their adhesion to renal cells.

PURPOSE: Adhesion of urinary crystals to renal tubular cells could be a critical event that triggers a cascade of responses ending in kidney stone formation. We clarified the role of urinary macromolecules during calcium oxalate monohydrate (COM) crystal adhesion to cells. MATERIALS AND METHODS: To assess COM crystal binding to cells in the presence of whole urine and fractions thereof we used monolayer cultures of distal nephron derived Madin-Darby canine kidney, type I cells as a model system. RESULTS: COM crystal adhesion to cells was decreased in the presence of whole urine compared with an ultrafiltrate prepared by passing urine through a 10 kDa cutoff membrane. Supplementing the ultrafiltrate with urinary concentrate containing proteins greater than 10 kDa returned crystal adhesion to low levels, similar to whole urine. Macromolecules in whole urine acted to decrease binding to cells by coating crystals and 4 proteins previously implicated in the pathogenesis of nephrolithiasis were detected on coated crystals (bikunin, osteopontin, prothrombin fragment 1 + 2 and Tamm-Horsfall glycoprotein). Crystals precipitated and grown in whole urine also bound less avidly to cells than crystals precipitated in artificial urine. CONCLUSIONS: This study confirms that macromolecules present in whole urine can coat crystals and, thereby, block their adhesion to renal tubular cells. Preventing crystal retention in the kidney could be an important mechanism whereby these macromolecules protect against kidney stones.

Correlates of thrombin generation in patients with advanced prostate cancer.

Thrombin generation is increased in men with advanced prostate cancer. Thrombin has the ability to interact with, and affect the biology of, a variety of cell types including prostate cancer cell lines. We therefore looked for correlations between thrombin generation and other markers of disease activity in spot urine samples obtained from men with advanced prostate cancer. Excretion of part of the prothrombin activation peptide F(1+2) (called here iF2), interleukin-6 (IL-6), the bone turnover marker deoxypyridinoline (DpD), and vascular endothelial growth factor (VEGF) were quantitated in spot urine samples collected from 37 men with hormone-refractory prostate cancer. Following log transformation of the data, significant correlations were found by univariate analysis between the excretion of a marker of thrombin generation (iF2) and IL-6, DpD and VEGF, as well as between IL-6 and DpD or VEGF excretion. No correlation was found between any marker and serum PSA level. After multivariate analysis, a significant correlation remained between thrombin generation and IL-6 excretion. Analysis of a second urine specimen obtained from 19 of the subjects 1 to 7 months after the first also revealed a significant correlation between thrombin generation and IL-6, DpD, and VEGF excretion. These data provide evidence of a correlation between thrombin generation/coagulation system activation and IL-6 generation in patients with cancer. They provide a rationale for studying the effects of inhibitors of thrombin generation upon the biology of prostate cancer.

Synergism between urinary prothrombin fragment 1 and urine: a comparison of inhibitory activities in stone-prone and stone-free population groups.

South African blacks rarely form kidney stones compared with whites. This study investigated whether purified urinary prothrombin fragment 1 (UPTF1) derived from blacks is a more potent inhibitor of calcium oxalate crystallisation than that from whites. UPTF1 was purified from the urine of both population groups and their inhibitory activities were compared in a cross-over design in which each protein was tested in ultrafiltered urine from both population groups. Coulter Multisizer, [14C]-oxalate deposition and scanning electron microscopy experiments were used to monitor crystallisation. The study has demonstrated for the first time that UPTF1 promotes nucleation and that inhibitory activity is synergistically dependent upon urine composition. The activity of the whites' UPTF1 was greater than that of the blacks in the whites' urine (e.g. particle size decrease: 31.7% vs. 25.2%), while the blacks' UPTF1 was superior to that of the whites in the blacks' urine (e.g. particle size decrease: 46.5% vs. 32.4%). In addition, when tested in their respective endogenous urines, the blacks' UPTF1 demonstrated superior inhibitory activity on an absolute scale (e.g. particle size decrease: 46.5% vs. 31.7%). Thus, the urine composition of black South Africans may influence their UPTF1 conformation, conferring greater efficacy for inhibition of calcium oxalate crystallisation.

Calcium phosphate crystal-associated proteins: alpha-2-HS-glycoprotein, prothrombin fragment 1 and osteopontin.

To study the inhibitory effects of calcium phosphate-associated proteins on calcium oxalate crystallization and urinary concentrations of proteins in people who form stones and healthy controls. From 60 L of urine from healthy men, calcium phosphate-associated proteins (alpha-2-HS-glycoprotein, prothrombin fragment 1 and osteopontin) were obtained. The effects of the proteins on calcium oxalate (CaOx) crystallization were studied with a mixed suspension mixed product removal system. To examine urinary concentrations of the proteins, urine samples were collected from 17 healthy subjects and 15 stone formers and analyzed using anion-exchange chromatography and an enzyme immunoassay. Prothrombin fragment 1 (PTF1) and osteopontin (OPN) had strong inhibitory effects on CaOx crystallization, while alpha-2-HS-glycoprotein had a mild inhibitory effect. Urinary concentrations of PTF1 and OPN were lower in stone formers than in healthy controls. Low urinary concentrations of PTF1 and OPN might be one of the reasons for stone formation.

A study of crystal matrix extract and urinary prothrombin fragment 1 from a stone-prone and stone-free population.

South African blacks are immune to urinary calculi whereas whites have an incidence rate similar to that reported in Western societies. Urinary prothrombin fragment 1 (UPTF1) and the crystal matrix extract (CME) from which it is derived have been shown to be potent inhibitors of crystal growth and aggregation in undiluted human urine. The objective of the present study was to isolate CME and UPTF1 from the urines of black and white subjects in order to assess whether either might contribute to the black population's relative stone immunity. CME was isolated from freshly precipitated calcium oxalate (CaOx) crystals and a crystallization study was conducted in synthetic urine. Coulter Counter, 14C-oxalate deposition, and scanning electron microscopy data demonstrated that the extracts from both race groups strongly inhibited CaOx nucleation. The extract derived from the black subjects inhibited nucleation to a greater extent than that from the whites. A phase conversion from COM to COD in the presence of the extracts, in support of the inhibitory effect of CME, was also observed. Purified UPTF1 isolated from both groups' CME was subjected to rigorous biochemical characterization involving matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, protein sequencing by Edman degradation, and amino acid analyses. No differences in molecular weight or amino acid sequence and composition were identified. It is suggested that the more potent inhibitory activity of the extract derived from the black subjects might be related to this group's relative stone immunity.