Removal Of Macromolecules Research Articles

Function of the Nuclear Transport Machinery in Maintaining the Distinctive Compositions of the Nucleus and Cytoplasm.

Although the separation of transcription and translation, mediated by the nuclear envelope, is the defining characteristic of Eukaryotes, the barrier between the nuclear and cytoplasmic compartments needs to be semipermeable to enable material to be moved between them. Moreover, each compartment needs to have a distinctive complement of macromolecules to mediate specific functions and so movement between them needs to be controlled. This is achieved through the selective active transport of macromolecules through the nuclear pores that stud the nuclear envelope, and which serve as a conduit between these compartments. Nuclear pores are huge cylindrical macromolecular assemblies and are constructed from the order of 30 different proteins called nucleoporins. Nuclear pores have a central transport channel that is filled with a dense network of natively unfolded portions of many different nuclear pore proteins (nucleoporins or nups). This network generates a barrier that impedes, but does not entirely prevent, the diffusion of many macromolecules through the pores. The rapid movement of a range of proteins and RNAs through the pores is mediated by a range of transport factors that bind their cargo in one compartment and release it in the other. However, although as their size increases the diffusion of macromolecules through nuclear pores is progressively impaired, additional mechanisms, including the binding of some macromolecules to immobile components of each compartment and also the active removal of macromolecules from the inappropriate compartment, are needed to fully maintain the distinctive compositions of each compartment.

Performance of restricted access copper-imprinted poly(allylthiourea) in an on-line preconcentration and sample clean-up FIA-FAAS system for copper determination in milk samples

A new on-line solid phase preconcentration method using an ion-imprinted polymer with restrict access material based on copper-imprinted poly(allylthiourea) modified with 2-hydroxyethyl methacrylate and bovine serum (IIP-HEMA-BSA) to preconcentrate Cu2+ ions and exclude protein from milk samples via FIA-FAAS was developed. The samples were on-line preconcentrated at pH 4.5 through a mini-column packed with 50.0 mg of IIP-HEMA-BSA at a flow rate of 7.6 mL min−1 followed by a counter-current elution with 1.00 mol L−1 HCl towards the FAAS detector. The interference of ions commonly found in milk samples and the presence of BSA were investigated in the Cu2+ ions preconcentration. The method provided an analytical curve ranging from 3.6 to 100.0 μg L−1, preconcentration factor of 24-fold, limit of detection of 1.1 μg L−1 and sample throughput of 20 h−1. The accuracy of the method was attested by Cu2+ ions addition/recovery tests as well as by using GFAAS of samples microwave-assisted digested. The proposed method was successfully applied to the copper determination in milk samples, requiring only pH adjustment followed by preconcentration step as sample pretreatment. Copper content in bovine milk sample was 0.635 ± 0.042 mg kg−1, while to soybean milk samples were between 0.048 ± 0.008 and 0.094 ± 0.005 mg kg−1. To the best of our knowledge, the potential of IIP-HEMA-BSA for Cu2+ ions extraction from biological samples and simultaneous removal of macromolecules employing minimal sample pretreatment was demonstrated for the first time. The proposed extraction method stands out by being simple, fast and a low-cost analytical strategy when compared to the conventional microwave-assisted acid digestion.

Rapid, Reproducible, Quantifiable NMR Metabolomics: Methanol and Methanol: Chloroform Precipitation for Removal of Macromolecules in Serum and Whole Blood.

Background: Though blood is an excellent biofluid for metabolomics, proteins and lipids present in blood can interfere with 1d-1H NMR spectra and disrupt quantification of metabolites. Here, we present effective macromolecule removal strategies for serum and whole blood (WB) samples. Methods: A variety of macromolecule removal strategies were compared in both WB and serum, along with tests of ultrafiltration alone and in combination with precipitation methods. Results: In healthy human serum, methanol:chloroform:water extraction with ultrafiltration was compared to methanol precipitation with and without ultrafiltration. Methods were tested in healthy pooled human serum, and in serum from patients with sepsis. Effects of long-term storage at −80 °C were tested to explore the impact of macromolecule removal strategy on serum from different conditions. In WB a variety of extraction strategies were tested in two types of WB (from pigs and baboons) to examine the impact of macromolecule removal strategies on different samples. Conclusions: In healthy human serum methanol precipitation of serum with ultrafiltration was superior, but was similar in recovery and variance to methanol:chloroform:water extraction with ultrafiltration in pooled serum from patients with sepsis. In WB, high quality, quantifiable spectra were obtained with the use of a methanol: chloroform precipitation.

Novel Approaches to Block Complement.

The complement system may contribute in many ways to transplant injury, being a promising target for specific therapeutic interventions. There is evidence that the monoclonal anti-C5 antibody eculizumab is effective in the prevention and treatment of early antibody-mediated rejection, but terminal complement blockade might be of limited efficiency in chronic rejection. Given the diversity of immunological events triggered by activation steps upstream to C5, in particular, opsonin and anaphylatoxin formation through C3 cleavage, one may argue that, in the specific context of antibody-mediated rejection, inhibition of antibody-triggered classical pathway (CP) activation might be beneficial. Strategies to interfere with key CP component C1 are currently under clinical evaluation and include the therapeutic use of purified C1-inhibitor, which, besides targeting the integrity and function of the C1 complex, also affects components of the LP, the contact system, the coagulation cascade or surface molecules mediating leukocyte-endothelial interactions. In addition, a monoclonal anti-C1s antibody (BIVV009) has now entered clinical evaluation and was shown to effectively block antibody-triggered CP activation in rejecting kidney allografts. Moreover, modified apheresis techniques for preferential removal of macromolecules, including C1q, may allow for efficient complement depletion, in addition to antibody removal. The availability of effective strategies to interfere with the CP, as well as innovative approaches targeting other pathways, some of them already being tested in clinical trials, will help us figure out how complement contributes to acute and chronic graft injury, and hopefully provide us with new ways to more efficiently counteract rejection.

The Pathophysiological Role of Microglia in Dynamic Surveillance, Phagocytosis and Structural Remodeling of the Developing CNS.

In vertebrates, during an early wave of hematopoiesis in the yolk sac between embryonic day E7.0 and E9.0, cells of mesodermal leaflet addressed to macrophage lineage enter in developing central nervous system (CNS) and originate the developing native microglial cells. Depending on the species, microglial cells represent 5–20% of glial cells resident in adult brain. Here, we briefly discuss some canonical functions of the microglia, i.e., cytokine secretion and functional transition from M1 to M2 phenotype. In addition, we review studies on the non-canonical functions of microglia such as regulation of phagocytosis, synaptic pruning, and sculpting postnatal neural circuits. In this latter context the contribution of microglia to some neurodevelopmental disorders is now well established. Nasu-Hakola (NHD) disease is considered a primary microgliopathy with alterations of the DNAX activation protein 12 (DAP12)-Triggering receptor expressed on myeloid cells 2 (TREM-2) signaling and removal of macromolecules and apoptotic cells followed by secondary microglia activation. In Rett syndrome Mecp2-/- microglia shows a substantial impairment of phagocytic ability, although the role of microglia is not yet clear. In a mouse model of Tourette syndrome (TS), microglia abnormalities have also been described, and deficient microglia-mediated neuroprotection is obvious. Here we review the role of microglial cells in neurodevelopmental disorders without inflammation and on the complex role of microglia in developing CNS.

Comparison of the effects of different blood purification methods on removal of macromolecules in uremia

Objective To compare the effects of different blood purification methods on the removal of large and medium molecular toxins in patients with maintenance hemodialysis(MHD), and to provide the clinical basis for selecting suitable dialysis modalities to prevent or delay the long term complications of uremic patients. Methods 60 MHD patients with long term and stable hemodialysis were enrolled in this study. All of them were selected from the patients with insufficient hemodialysis.According to the digital table, 60 patients were randomly divided into high flux hemodialysis (HFHD)group and hemodiafiltration (HDF) group, 30 cases in each group. And before, 3 months and 6 months after dialysis, the serum β2-MG, PTH and Cys C after dialysis were tested. Results The clearance of PTH in the HFHD group[(286.34±127.33)pg/mL] was significantly higher than that in the HDF group[(376.04±141.74)pg/mL], the difference was statistically significant(t=2.45, P<0.05). The clearance of β2 -MG in the HDF group[(11.34±1.96)mg/L]was significantly higher than that in the HFHD group[(15.41±3.02)mg/L], the difference was significant(t=5.88, P<0.05). The clearance of Cys C in the HFHD group[(263.67±98.72)μg/dL]was significantly higher than that in the HDF group[(345.63±105.00)μg/dL], the difference was statistically significant(t=2.96, P<0.05). Conclusion Both two dialysis methods are very effective in the removal of large and medium molecular uremic toxins in patients. In the removal of patients with increased PTH, HFHD is more effective. In the removal of patients with increased β2-MG, HDF is more effective. In the removal of Cys C, HFHD is better. Key words: Hemodiafiltration; Uremia

ABO Antibody and Complement Depletion By Immunoadsorption Combined With Membrane Filtration - A Randomized Controlled Crossover Trial.

Potent antibody depletion techniques have paved the way to successful ABO-incompatible transplantation. Considering its efficiency regarding IgG removal, the use of non-antigen-specific semiselective immunoadsorption (IA) has been advocated. One attractive strategy to overcome the caveat of incomplete IgM depletion and to interfere with complement activation could be the adjunctive use of membrane filtration (MF) to enhance the removal of macromolecules. To investigate the depletion efficiency of semiselective IA plus MF, we conducted a randomized crossover trial including 14 patients treated for autoimmune disease. Two treatment sequences, a single session of IA plus MF followed by IA alone after ≥7 days (and vice versa), were analyzed. IA plus MF markedly enhanced the median percent reduction of ABO-specific IgM as determined by flow cytometry (59% versus 23%, P<0.001) and hemagglutination (2 versus 1 titer steps, P<0.001). Combined treatment also substantially lowered C1q concentrations (86% versus 58% reduction, P<0.001) and the functionality of classical complement as reflected by impaired in vitro C3 activation capability. IgG was strongly reduced without any additional effect of MF. In conclusion, we demonstrate enhanced antibody elimination by adding MF to IA. This and the observed impact on classical complement could markedly enhance the efficiency of apheresis in ABO- and/or HLA-incompatible transplantation. DISCLOSURES:Boehmig, G.: Grant/Research Support, Unrestricted grant.

Removal of phosphorus from wastewaters using ferrous salts – A pilot scale membrane bioreactor study

A pilot scale membrane bioreactor (3.7 m3/day capacity), configured for alternate point ferrous sulphate addition, was evaluated in a fourteen month trial to comply with an effluent discharge requirement of less than 0.15 mg-P/L at the 50th percentile and less than 0.30 mg-P/L at the 90th percentile. Ferrous sulphate was added at a molar ratio (Fe(II):PO4) of 2.99 in the filtration chamber for 85 days and 2.60 in the primary anoxic zone for 111 days. Addition of ferrous salts to the anoxic zone achieved a final effluent phosphorous concentration (mg-P/L) of <0.05 (29%), <0.15 (77%) and <0.30 (95%), while addition of ferrous salts in the filtration zone achieved <0.05 (18%), <0.15 (63%) and <0.30 (95%). Analysis of the concentration of iron(II) in the supernatant indicated that phosphorus was mainly removed via adsorption to amorphous iron oxyhydroxides particles in both dosing scenarios. However, analysis of residence time distribution (RTD) data of the reactor indicated that severe short-circuiting from the dosing point to the membrane outlet could occur when the ferrous salts were added to the membrane zone while the reactor behaved close to a completely mixed reactor when dosing to the primary anoxic zone, resulting in improved phosphorus removal. The addition of ferrous salt was also found to delay the onset of severe increase in trans-membrane pressure as a result of the removal of macro-molecules. However, detailed analysis of the form and concentration of iron species in the supernatant and permeate indicated that the presence of fine iron particles resulted in a higher fouling rate when Fe(II) was added to the membrane zone rather than the primary anoxic zone and could cause more severe irreversible fouling in long-term operation.

Current topics in therapeutic plasmapheresis

Therapeutic plasmapheresis has been used for intractable diseases that cannot be cured by conventional drug therapy. Currently, the use of therapeutic plasmapheresis has been approved for 27 diseases by Japan's National Health Insurance system and is mainly categorized into three modalities: plasma exchange (PE), double-filtration plasmapheresis (DFPP), and plasma adsorption (PA). Plasma separators and/or fractionators are essential for the therapy. PE is performed for two purposes: removal of pathogenic antigens or substances in the plasma fraction and supplementation of essential factors, such as albumin and coagulation factors. PE can be used for thrombotic microangiopathy and acute hepatic failure. DFPP can be performed for selective removal of macromolecules while avoiding the use of substitution fluid (i.e., albumin or fresh frozen plasma). DFPP has now been used for conditions involving relatively larger plasma molecules, including hyperviscosity syndrome and ABO-incompatible kidney transplantation. PA can specifically remove pathogenic agents, such as low-density lipoprotein or autoantibodies, in the IgG fractions by the adsorption column and does not require substitution fluids. PA has now been used for a wide variety of neurological diseases, including chronic inflammatory demyelinating polyneuropathy. This review describes the characteristics of each modality, seeking to improve the efficacy and specificity of removal of the target substance.

Macroautophagy: a mechanism for mediating cell death or for promoting cell survival?

Macroautophagy is a ubiquitous mechanism for the bulk removal of macromolecules and cell organelles from the cell. Periyasamy-Thandavan and colleagues report that cisplatin activates autophagy in renal tubular cells and that autophagy plays a role in decreasing apoptosis of tubular cells induced by cisplatin. This finding provides novel evidence that autophagy may play a role in ameliorating the effects of acute injury on the kidney.

Lymphangiogenesis in the bone–implant interface of orthopedic implants: Importance and consequence

The lymphatic system plays an essential physiological role in homeostasis, interstitial fluid composition, and immunity while impaired lymphatic function has been implicated in a number of pathological conditions, including arthritis and delayed wound healing. This study investigated lymphatic capillary growth and lymphangiogenesis pathways in the bone-implant interface from patients with aseptically loosened prosthetic joints. The newly developed lymphatic specific marker, podoplanin, has enabled the first demonstration of lymphatic capillaries in peri-prosthetic tissues (60% of cases contained podoplanin positive vessels). The pro-lymphangiogenic factor (VEGF-C) and its receptor VEGFR-3 showed high level of expression in these tissues, (often in areas of high levels of wear debris). However despite the upregulation of the lymphangiogenesis pathway by a VEGF-C/VEGFR-3-mediated mechanism, there were relatively few podoplanin positive lymphatic vessels in the bone-implant interface (3.4% of total vessels). This may have important pathological consequences in terms of perpetuating inflammation and edema by inhibiting the removal of macromolecules, cells, and interstitial fluid. The identification of lymphatic vessels with internalized polyethylene wear particles provides evidence of this route of wear debris transportation to distal sites. This paper highlights the importance of lymphatic vessels in the maintenance of local and distal inflammatory responses to prosthetic wear particles.

Single-chain antibody fragment-based adsorbent for the extracorporeal removal of β2-microglobulin

Dialysis-related amyloidosis (DRA) is a frequent complication of end-stage renal disease (ESRD) that has been associated with the accumulation of beta2-microglobulin (beta2-m). Removal of beta2-m results in the loss of important proteins due to the nonspecific nature of current therapies. Although whole antibodies can potentially be used to confer specificity to beta2-m removal from blood, single-chain variable region (scFv) antibody fragments could potentially offer several advantages as immunoadsorption ligands due to their size, genetic definition, ability to be expressed by microbes, and amenability for in vitro evolution. An antihuman beta2-m scFv was constructed from the BBM.1 hybridoma and expressed by a yeast display vector. The binding affinity of the wild-type scFv fragment was quantified by flow cytometry analysis. Soluble scFv was expressed by a yeast secretion vector, purified, and immobilized onto agarose beads. The binding capacity of the immunoadsorbent was measured by equilibrating samples with saturating quantities of fluorescent beta2-m in serum. The displayed scFv possessed a nanomolar affinity (KD= 0.008 +/- 0.004 mg-beta2-m/L). The immunoadsorbent exhibited an adsorption site density of 0.41 +/- 0.01 mg beta2-m/mL settled gel. Under saturating conditions, the mass ratio of adsorbed beta2-m to immobilized antibody is 70% greater than any previous literature report for whole antibodies. Preliminary specificity experiments suggest that the scFv-based immunoadsorbent is specific toward human beta2-m. Recombinant DNA technology was successfully used to engineer an scFv-based immunoadsorbent. Use of immobilized scFvs during hemodialysis may minimize loss of valuable proteins and facilitate the removal of macromolecules that are significantly larger than the molecular weight cut-off of the membrane.

Mesangial Cell Proliferation Inhibitors for the Treatment of Proliferative Glomerular Disease

AbstractFor Abstract see ChemInform Abstract in Full Text.

Mesangial cell proliferation inhibitors for the treatment of proliferative glomerular disease.

Mesangial cells (MC) serve a number of functions in the renal glomerular capillary including structural support of the capillary tuft, modulation of glomerular hemodynamics, and a phagocytic function allowing removal of macromolecules and immune complexes. The proliferation of MC is a prominent feature of glomerular disease including IgA nephropathy, membranoproliferative glomerulonephritis, lupus nephritis, and diabetic nephropathy. In experimental animal models of nephritis, MC proliferation frequently precedes and is linked to the increase of extracellular matrix in the mesangium and glomerulosclerosis. Reduction of MC proliferation in glomerular disease models by treatment with heparin, low-protein diet, or antibodies to platelet-derived growth factor (PDGF), have been shown to reduce extracellular matrix expansion and glomerulosclerotic changes. Therefore, MC proliferation inhibitors may offer therapeutic opportunities for the treatment of proliferative glomerular disease. It is also known that the MC proliferation is inhibited by many kinds of pharmacological drugs, for example, angiotensin converting enzyme (ACE) inhibitors, leukotriene D(4) (LTD(4)) antagonists, PDGF inhibitors, matrix metalloproteinases (MMP) inhibitors, 3-hydroxy-3 methyl glutaryl-coenzymeA (HMG-CoA) inhibitors, cyclin-dependent kinases (CDK) inhibitors, and others. This review summarizes the recently reported MC proliferation inhibitors with their pharmacological properties on the basis of their chemical structures.

Membrane plasmapheresis in the United States: a review over the last 20 years.

Plasmapheresis is a general term involving extracorporeal plasma separation by centrifugation or primary membrane plasma separator (MPS). Further plasma processing can be accomplished by the use of secondary membrane plasma fractionation (PF), as in double filtration plasmapheresis, also called cascade filtration, low-density lipoprotein pheresis, thermofiltration, and cryofiltration apheresis. Otherwise, the separated plasma is replaced by colloid solution as in plasma exchange (PE). PE is used, unselectively, to treat patients with immunological, neurological, hematological, renal, and metabolic disorders. Secondary PF may be a more selective alternative. In general, the primary MPS and secondary PF are safe, effective, and biocompatible. The advantages of the primary MPS include its simplicity to use with blood pumps and no observed white blood cell or platelet loss, compared with centrifugation. The disadvantages are lack of versatility, the need to monitor transmembrane pressure to prevent hemolysis, and possible biocompatibility issues such as use of polyvinyl alcohol membranes. The advantages of secondary PF, compared with PE, include selective removal of macromolecules according to molecular weight and filter pore size. No deficiency syndromes or sepsis are observed, nor is replacement solution required. More than 1 plasma volume may be processed, and it is less expensive than PE. Cryofiltration apheresis, using the cryoglobulin filter, selectively removes cryoproteins and is a specific treatment for cryoprecipitate-induced diseases. The disadvantages of PF include biocompatibility, especially with concomitant ACE inhibitor use, and membrane plugging. An important disadvantage is that most PFs are investigational in the United States. This article reviews the availability, safety, efficacy, and biocompatibility of primary MPSs and secondary PF in the United States.

Membrane processes for plasma separation and plasma fractionation: guiding principles for clinical use.

The use of membranes in blood processing range from the use of large-pore structures for filtration of blood for the removal of large particles (>20 microm transfusion filters) to membranes for the dialysis of blood for the removal of low molecular weight solutes in the treatment of renal failure. Within the past 20 years, membranes have been applied in the separation of plasma from whole blood. Compared to centrifugal plasma separation membrane, plasma separation is preferred when used with online plasma fractionation since the plasma is free of cells. In addition, membranes have also been applied in the online treatment of plasma for the selective removal of macromolecules in selected disease states obviating the need for plasma replacement products. The membranes used for plasma separation and fractionation may be distinguished from conventional dialysis membranes and high-flux membranes used in hemofiltration by their very high or select passage of plasma proteins. Membrane techniques are simple and safe to apply and can be competitive to other plasma separation and treatment technologies.

Evaluation of an SOS-Chromotest-based approach for the isolation and detection of sediment-associated genotoxins

A series of experiments was conducted to evaluate an approach advanced by the St. Lawrence Centre (SLC) of Environment Canada for assessing the genotoxic potential of sediments. The SLC method entails the extraction, isolation and solvent exchange of the organic constituents in sediment, and the testing of these solubilized extracts with the SOS Chromotest ( Escherichia coli PQ37). A total of five sediments, three variously contaminated by organic compounds and two reference materials certified for persistent organic chemicals, were Soxhlet-extracted. Each of the five extracts was then split, with a portion remaining in crude form and another portion fractionated into two molecular-weight classes of organic contaminants, thus yielding a total of 15 extract samples. The ability of the SOS Chromotest to detect genotoxins in the various organic extracts was evaluated and compared with that of the Ames Fluctuation Assay ( Salmonella typhimurium, strain TA100). The intra-laboratory variance associated with the SOS Chromotest was also assessed. Procedural details are presented and results are discussed. The SOS Chromotest results were in good agreement with those of the Ames Fluctuation Assay, especially after metabolic activation. However, the E. coli PQ37 system was slightly more sensitive than the Salmonella assay for detecting genotoxins in the sediment extracts. The SOS Chromotest was also the most discriminating of the two assays, generating SOS-induction factors that were consistent with the organic contamination gradient reported in the sediment samples. The removal of macromolecules from the dichloromethane extracts by size-exclusion chromatography prior to testing enhanced the sensitivity of both test systems. The intra-laboratory variance of the SOS Chromotest ranged from 0.24% to 23.82%, depending on the extract sample. As applied in this study, the SOS Chromotest can serve as a sensitive test for screening the genotoxic potential of uncharacterized sediment extracts. A more sensitive assay would be appropriate, however, as a confirmation for definitive investigations, especially for the detection of direct-acting genotoxins.

Involvement of Prostaglandin E2 in Clearance of Aggregated Protein via Protein Kinase A in Glomeruli

Recently we immunohistochemically demonstrated that prostaglandin E2 (PGE2) promoted the clearance of aggregated bovine serum albumin (a-BSA) deposited in glomeruli. Herein, we investigated the role of PGE2 and its signal transduction in the disposal of macromolecules in glomeruli. EP2 and EP4 receptor mRNA was detected in glomeruli by RT-PCR analysis. A-BSA was injected twice into mice. Glomeruli were then isolated and incubated. A-BSA gradually disappeared from isolated glomeruli. PGE2 increased the intracellular cyclic AMP and decreased a-BSA level in glomeruli. Additionally, 8-bromo-cyclic AMP evoked a loss of a-BSA in isolated glomeruli. The effect of 8-bromo-cyclic AMP on the clearance of a-BSA was abolished by KT 5720 in glomeruli. PGE2 and 8-bromo-cyclic AMP also prompted disposal of a-BSA in cultured mesangial cells. These findings indicate that PGE2 positively regulates the removal of macromolecules via cyclic AMP and protein kinase A in glomeruli, and they provide insight into how to prevent the development of glomerulonephritis and glomerulosclerosis.

On-line separation of macromolecules by membrane filtration with cryogelation. 1980.

Guest Editor's Introduction: This paper was presented at the International Symposium on Plasma Exchange, Cologne, June 6–7, 1980. It was printed in its proceedings, Plasma Exchange Plasmapheresis and Plasmaseparation, (ed) Sieberth H.G., 1980, pages 133–139. It was published by F.K. Schattauer Verlag, Stuttgart, Germany. This paper describes the first clinical application of cryofiltration. The efficiency of double filtration was enhanced by keeping the plasma fractionator filter (cryofilter) at near zero temperature. Cryogel is formed inside the cryofilter and selective removal of macromolecules was achieved without any supplemental albumin supplement.

Effects of urinary macromolecules on the nucleation of calcium oxalate in idiopathic stone formers and healthy controls

Urinary macromolecules have attracted great interest because of their possible role as both promoters and inhibitors of calcium oxalate (CaOx) crystallization and it remains unclear whether there is any difference, in their nucleating activity, between stone formers and controls. We selected 9 male idiopathic CaOx stone formers whose 24-h urines presented no evidence of common urinary stone risk factors such as hypercalciuria, hyperoxaluria, hyperuricosuria, hypocitraturia, hypomagnesiuria or low glycosaminoglycans excretion and 12 male controls (matched for age and body weight) whose 24-h urines did not differ from those of stone formers. The study of urinary CaOx nucleation was made in freshly voided overnight urines whose biochemical composition was almost identical in the two groups. In filtered (0.22 μm) and ultrafiltered (10 kDa) urine we performed an oxalate tolerance test to determine the permissible increment of oxalate, the oxalate level for nucleation and the permissible increment of CaOx relative supersaturation (CaOx RS). In filtered urine from stone formers the permissible increment of oxalate was lower than controls (30 ± 10.2 vs. 46.7 ± 9.7 mg/l, P = 0.001), the oxalate level for nucleation was lower (64.4 ± 14.2 vs. 79.5 ± 15.6 mg/l, P = 0.035) and the permissible increment of CaOx RS was also lower (9.71 ± 2.59 vs. 13.39 ± 3.62, P = 0.018). In ultrafiltered urine these differences disappeared because the removal of macromolecules in stone formers significantly enhanced the oxalate-tolerance values. The difference between the change of the oxalate permissible increment of filtered and ultrafiltered urine allowed a distinction to be made between stone formers and controls that was not feasible in other ways (7.6 ± 5.3 vs. 3.3 ± 5.9 mg/l, P < 0.0001). The study suggests that, in idiopathic CaOx stone formers free from common urinary risk factors of CaOx crystallization, there is an increased tendency for CaOx nucleation in urine, which is mediated by macromolecular components.