Comparison of bovine serum albumin and chitosan effects on calcium phosphate formation in the presence of silver nanoparticles.

The precipitation of calcium phosphates (CaPs) in the presence of more than one type of additive is of interest both from a fundamental point of view and as a possible biomimetic route for the preparation of multicomponent composites in which the activity of the components is preserved. In this study, the effect of bovine serum albumin (BSA) and chitosan (Chi) on the precipitation of CaPs in the presence of silver nanoparticles (AgNPs) stabilized with sodium bis(2-ethylhexyl)sulfosuccinate (AOT-AgNPs), poly(vinylpyrrolidone) (PVP-AgNPs), and citrate (cit-AgNPs) was investigated. In the control system, the precipitation of CaPs occurred in two steps. Amorphous calcium phosphate (ACP) was the first precipitated solid, which transformed into a mixture of calcium-deficient hydroxyapatite (CaDHA) and a smaller amount of octacalcium phosphate (OCP) after 60 min of ageing. Both biomacromolecules inhibited ACP transformation, with Chi being a stronger inhibitor due to its flexible molecular structure. As the concentration of the biomacromolecules increased, the amount of OCP decreased both in the absence and presence of AgNPs. In the presence of cit-AgNPs and two highest BSA concentrations, a change in the composition of the crystalline phase was observed. Calcium hydrogen phosphate dihydrate was formed in the mixture with CaDHA. An effect on the morphology of both the amorphous and crystalline phases was observed. The effect depended on the specific combination of biomacromolecules and differently stabilized AgNP. The results obtained suggest a simple method for fine-tuning the properties of precipitates using different classes of additives. This could be of interest for the biomimetic preparation of multifunctional composites for bone tissue engineering.

Can Differently Stabilized Silver Nanoparticles Modify Calcium Phosphate Precipitation?

Calcium phosphates (CaPs) composites with silver nanoparticles (AgNPs) attract attention as a possible alternative to conventional approaches to combating orthopedic implant-associated infections. Although precipitation of calcium phosphates at room temperatures was pointed out as an advantageous method for the preparation of various CaP-based biomaterials, to the best of our knowledge, no such study exists for the preparation of CaPs/AgNP composites. Motivated by this lack of data in this study we investigated the influence of AgNPs stabilized with citrate (cit-AgNPs), poly(vinylpyrrolidone) (PVP-AgNPs), and sodium bis(2-ethylhexyl) sulfosuccinate (AOT-AgNPs) in the concentration range 5-25 mg dm-3 on the precipitation of CaPs. The first solid phase to precipitate in the investigated precipitation system was amorphous calcium phosphate (ACP). The effect of AgNPs on ACP stability was significant only in the presence of the highest concentration of AOT-AgNPs. However, in all precipitation systems containing AgNPs, the morphology of ACP was affected, as gel-like precipitates formed in addition to the typical chain-like aggregates of spherical particles. The exact effect depended on the type of AgNPs. After 60 min of reaction time, a mixture of calcium-deficient hydroxyapatite (CaDHA) and a smaller amount of octacalcium phosphate (OCP) formed. PXRD and EPR data point out that the amount of formed OCP decreases with increasing AgNPs concentration. The obtained results showed that AgNPs can modify the precipitation of CaPs and that CaPs properties can be fine-tuned by the choice of stabilizing agent. Furthermore, it was shown that precipitation can be used as a simple and fast method for CaP/AgNPs composites preparation which is of special interest for biomaterials preparation.

Particle Size Modulates Silver Nanoparticle Toxicity during Embryogenesis of Urchins Arbacia lixula and Paracentrotus lividus.

Silver nanoparticles represent a threat to biota and have been shown to cause harm through a number of mechanisms, using a wide range of bioassay endpoints. While nanoparticle concentration has been primarily considered, comparison of studies that have used differently sized nanoparticles indicate that nanoparticle diameter may be an important factor that impacts negative outcomes. In considering this, the aim of the present study was to determine if different sizes of silver nanoparticles (AgNPs; 10, 20, 40, 60 and 100 nm) give rise to similar effects during embryogenesis of Mediterranean sea urchins Arbacia lixula and Paracentrotus lividus, or if nanoparticle size is a parameter that can modulate embryotoxicity and spermiotoxicity in these species. Fertilised embryos were exposed to a range of AgNP concentrations (1−1000 µg L−1) and after 48 h larvae were scored. Embryos exposed to 1 and 10 µg L−1 AgNPs (for all tested sizes) showed no negative effect in both sea urchins. The smaller AgNPs (size 10 and 20 nm) caused a decrease in the percentage of normally developed A. lixula larvae at concentrations ≥50 µg L−1 (EC50: 49 and 75 µg L−1, respectively) and at ≥100 µg L−1 (EC50: 67 and 91 µg L−1, respectively) for P. lividus. AgNPs of 40 nm diameter was less harmful in both species ((EC50: 322 and 486 µg L−1, for P. lividus and A. lixula, respectively)). The largest AgNPs (60 and 100 nm) showed a dose-dependent response, with little effect at lower concentrations, while more than 50% of larvae were developmentally delayed at the highest tested concentrations of 500 and 1000 µg L−1 (EC50(100 nm); 662 and 529 µg L−1, for P. lividus and A. lixula, respectively. While AgNPs showed no effect on the fertilisation success of treated sperm, an increase in offspring developmental defects and arrested development was observed in A. lixula larvae for 10 nm AgNPs at concentrations ≥50 µg L−1, and for 20 and 40 nm AgNPs at concentrations >100 µg L−1. Overall, toxicity was mostly ascribed to more rapid oxidative dissolution of smaller nanoparticles, although, in cases, Ag+ ion concentrations alone could not explain high toxicity, indicating a nanoparticle-size effect.

Special Issue: Biomimetic Organic-Inorganic Composites.

Throughout history, the welfare and prosperity of civilizations have depended on the development of novel, more advanced materials [...].

Preparation of Multifunctional N-Doped Carbon Quantum Dots from Citrus clementina Peel: Investigating Targeted Pharmacological Activities and the Potential Application for Fe3+ Sensing.

Carbon quantum dots (CQDs) have recently emerged as innovative theranostic nanomaterials, enabling fast and effective diagnosis and treatment. In this study, a facile hydrothermal approach for N-doped biomass-derived CQDs preparation from Citrus clementina peel and amino acids glycine (Gly) and arginine (Arg) has been presented. The gradual increase in the N-dopant (amino acids) nitrogen content increased the quantum yield of synthesized CQDs. The prepared CQDs exhibited good biocompatibility, stability in aqueous, and high ionic strength media, similar optical properties, while differences were observed regarding the structural and chemical diversity, and biological and antioxidant activity. The antiproliferative effect of CQD@Gly against pancreatic cancer cell lines (CFPAC-1) was observed. At the same time, CQD@Arg has demonstrated the highest quantum yield and antioxidant activity by DPPH scavenging radical method of 81.39 ± 0.39% and has been further used for the ion sensing and cellular imaging of cancer cells. The obtained results have demonstrated selective response toward Fe3+ detection, with linear response ranging from 7.0 µmol dm-3 to 50.0 µmol dm-3 with R2 = 0.9931 and limit of detection (LOD) of 4.57 ± 0.27 µmol dm-3. This research could be a good example of sustainable biomass waste utilization with potential for biomedical analysis and ion sensing applications.

Precipitation at Room Temperature as a Fast and Versatile Method for Calcium Phosphate/TiO2 Nanocomposites Synthesis.

The constantly growing need for advanced bone regeneration materials has motivated the development of calcium phosphates (CaPs) composites with a different metal or metal-oxide nanomaterials and their economical and environmentally friendly production. Here, two procedures for the synthesis of CaPs composites with TiO2 nanoplates (TiNPl) and nanowires (TiNWs) were tested, with the immersion of TiO2 nanomaterials (TiNMs) in corrected simulated body fluid (c-SBF) and precipitation of CaP in the presence of TiNMs. The materials obtained were analyzed by powder X-ray diffraction, spectroscopic and microscopic techniques, Brunauer-Emmett-Teller surface area analysis, thermogravimetric analysis, dynamic and electrophoretic light scattering, and their hemocompatibility and ability to induce reactive oxygen species were evaluated. After 28 days of immersion in c-SBF, no significant CaP coating was formed on TiNMs. However, the composites with calcium-deficient apatite (CaDHA) were obtained after one hour in the spontaneous precipitation system. In the absence of TiNMs, CaDHA was also formed, indicating that control of the CaP phase formed can be accomplished by fine-tuning conditions in the precipitation system. Although the morphology and size of crystalline domains of CaDHA obtained on the different nanomaterials differed, no significant difference was detected in their local structure. Composites showed low reactive oxygen species (ROS) production and did not induce hemolysis. The results obtained indicate that precipitation is a suitable and fast method for the preparation of CaPs/TiNMs nanocomposites which shows great potential for biomedical applications.

The Influence of Different Classes of Amino Acids on Calcium Phosphates Seeded Growth.

Amino acids (AAs) attract attention for elucidating the role of proteins in biomineralization and the preparation of functionalized biomaterials. The influence that AAs exert on calcium phosphate (CaP) mineralization is still not completely understood, as contradictory results have been reported. In this paper, the influence of the addition of different classes of AAs, charged (L-aspartic acid, Asp; L-lysine, Lys), polar (L-asparagine, Asn; L-serine, Ser; L-tyrosine, Tyr), and non-polar (L-phenylalanine, Phe), on CaP growth in the presence of octacalcium phosphate (OCP) and calcium hydrogenphosphate dihydrate (DCPD) seeds was investigated. In control systems (without AAs), a calcium-deficient apatite (CaDHA) layer was formed on the surface of OCP, while a mixture of CaDHA and OCP in the form of spherical aggregates was formed on the surface of DCPD crystals. Charged and non-polar promoted, while polar AAs inhibited CaDHA formation on the OCP seeds. In the case of DCPD, Lys, Asp, and Phe promoted CaP formation, while the influence of other AAs was negligible. The most efficient promotor of precipitation in both cases was non-polar Phe. No significant influence of AAs on the composition and morphology of precipitates was observed. The obtained results are of interest for understanding biomineralization processes and additive controlled material synthesis.

Impact of surface functionalization on the toxicity and antimicrobial effects of selenium nanoparticles considering different routes of entry.

Selenium nanoparticles (SeNPs) were first designed as nutritional supplements, but they are attractive also for use in diagnostic and therapeutic systems owing to their biocompatibility and protective effects. This study aimed to examine if different SeNPs stabilization strategies affect their (i) antimicrobial activity against bacteria Escherichia coli and Staphylococcus aureus and yeast Saccharomyces cerevisiae and (ii) toxicity to human cells of different biological barriers i.e., skin, oral and intestinal mucosa. For surface stabilization, polyvinylpyrrolidone (PVP), poly-L-lysine (PLL) and polyacrylic acid (PAA) were used rendering neutral, positively and negatively charged SeNPs, respectively. The SeNPs (primary size ~80 nm) showed toxic effects in human cells in vitro and in bacteria S. aureus, but not in E. coli and yeast S. cerevisiae. Toxicity of SeNPs (24 h IC50) ranged from 1.4 to >100 mg Se/L, depending on surface functionalization (PLL > PAA > PVP) and was not caused by ionic Se. At subtoxic concentrations, all SeNPs were taken up by all human cell types, induced oxidative stress response and demonstrated genotoxicity. As the safety profile of SeNPs was dependent not only on target cells (mammalian cells, bacteria, yeast), but also on surface functionalization, these aspects should be considered during development of novel SeNPs-based biomedical products.

Tailoring the stability/aggregation of one-dimensional TiO2(B)/titanate nanowires using surfactants.

The increased utilization of one-dimensional (1D) TiO2 and titanate nanowires (TNWs) in various applications was the motivation behind studying their stability in this work, given that stability greatly influences both the success of the application and the environmental impact. Due to their high abundance in aqueous environments and their rich technological applicability, surfactants are among the most interesting compounds used for tailoring the stability. The aim of this paper is to determine the influence of surfactant molecular structure on TNW stability/aggregation behavior in water and aqueous NaBr solution by dynamic and electrophoretic light scattering. To accomplish this, two structurally different quaternary ammonium surfactants (monomeric DTAB and the corresponding dimeric 12-2-12) at monomer and micellar concentrations were used to investigate TNW stability in water and NaBr. It was shown that TNWs are relatively stable in Milli-Q water. However, the addition of NaBr induces aggregation, especially as the TNW mass concentration increases. DTAB and 12-2-12 adsorb on TNW surfaces as a result of the superposition of favorable electrostatic and hydrophobic interactions. As expected, the interaction of TNWs with 12-2-12 was stronger than with DTAB, due to the presence of two positively charged head groups and two hydrophobic tails. As a consequence of the higher adsorption of 12-2-12, TNWs remained stable in both media, while DTAB showed an opposite behavior. In order to gain more insight into changes in the surface properties after surfactant adsorption on the TNW surface, a surface complexation model was employed. With this first attempt to quantify the contribution of the surfactant structure on the adsorption equilibrium according to the observed differences in the intrinsic log K values, it was shown that 12-2-12 interacts more strongly with TNWs than DTAB. The modelling results enable a better understanding of the interaction between TNWs and surfactants as well as the prediction of the conditions that can promote stabilization or aggregation.

Physiological and biochemical effect of silver on the aquatic plant Lemna gibba L.: Evaluation of commercially available product containing colloidal silver.

This paper aims to evaluate the effects of a product containing colloidal silver in the aquatic environment, using duckweed Lemna gibba as a model plant. Therefore, growth parameters, photosynthetic pigments content and protein content as physiological indices were evaluated. Changes in the content of non-enzymatic antioxidants and activity of several antioxidant enzymes, alongside with the accumulation of hydrogen peroxide and lipid peroxidation end-products were assessed to explore the potential of colloidal silver to induce oxidative stress. The commercially available colloidal silver product contained a primary soluble form of silver. The treatment with colloidal silver resulted in significant physiological and biochemical changes in L. gibba plants and a consequent reduction of growth. Accumulation of silver caused altered nutrient balance in the plants as well as a significant decrease in photosynthetic pigments content and protein concentration. The antioxidative response of L. gibba plants to treatment with colloidal silver was inadequate to protect the plants from oxidative stress caused by metal accumulation. Silver caused concentration-dependent and time-dependent hydrogen peroxide accumulation as well as the elevation of lipid peroxidation levels in L. gibba plants. The use of commercially available products containing colloidal silver, and consequent accumulation of silver, both ionic and nanoparticle form in the environment, represents a potential source of toxicity to primary producers in the aquatic ecosystem.

Effect of Protein Corona on Silver Nanoparticle Stabilization and Ion Release Kinetics in Artificial Seawater.

In parallel with the growing use of nanoparticle-containing products, their release into the environment over the coming years is expected to increase significantly. With many large population centers located in near-coastal areas, and increasing evidence that various nanoparticles may be toxic to a range of organisms, biota in estuarine and coastal waters may be particularly vulnerable. While size effects may be important in cases, silver nanoparticles have been found to be toxic in large part due to their release of silver ions. However, there is relatively little data available on how nanoparticle coatings can affect silver ion release in estuarine or marine waters. We have found that albumin, as a model for biocorona-forming macromolecules which nanoparticles may encounter in wastewater streams, stabilizes silver colloids from agglomeration in high salinity marine waters by electrosteric repulsion for long time periods. A minimum mass ratio of about 130 for albumin:silver nanoparticles (40 nm) was required for stable dispersion in seawater. Increasing albumin concentration was also found to reduce dissolution of nanoparticles in seawater with up to 3.3 times lower concentrations of silver ions noted. Persistent colloids and slow sustained ion release may have important consequences for biota in these environmental compartments.

Comparison of in vitro toxicity of silver ions and silver nanoparticles on human hepatoma cells.

Scientific information on the potential harmful effects of silver nanoparticles (AgNPs) on human health severely lags behind their exponentially growing applications in consumer products. In assessing the toxic risk of AgNP usage, liver, as a detoxifying organ, is particularly important. The aim of this study was to explore the toxicity mechanisms of nano and ionic forms of silver on human hepatoblastoma (HepG2) cells. The results showed that silver ions and citrate-coated AgNPs reduced cell viability in a dose-dependent manner. The IC50 values of silver ions and citrate-coated AgNPs were 0.5 and 50 mg L(-1) , respectively. The LDH leakage and inhibition of albumin synthesis, along with decreased ALT activity, indicated that treatment with either AgNP or Ag ions resulted in membrane damage and reduced the cell function of human liver cells. Evaluation of oxidative stress markers demonstrating depletion of GSH, increased ROS production, and increased SOD activity, indicated that oxidative stress might contribute to the toxicity effects of nano and ionic forms of silver. The observed toxic effect of AgNP on HepG2 cells was substantially weaker than that caused by ionic silver, while the uptake of nano and ionic forms of silver by HepG2 cells was nearly the same. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 679-692, 2016.

Antibody-based donor-acceptor spatial reconfiguration in decorated lanthanide-doped nanoparticle colloids for the quantification of okadaic acid biotoxin.

With the increasing movement away from the mouse bioassay for the detection of toxins in commercially harvested shellfish, there is a growing demand for the development of new and potentially field-deployable tests in its place. In this direction we report the development of a simple and sensitive nanoparticle-based luminescence technique for the detection of the marine biotoxin okadaic acid. Photoluminescent lanthanide nanoparticles were conjugated with fluorophore-labelled anti-okadaic acid antibodies which, upon binding to okadaic acid, gave rise to luminescence resonance energy transfer from the nanoparticle to the organic fluorophore dye deriving from a reduction in distance between the two. The intensity ratio of the fluorophore: nanoparticle emission peaks was found to correlate with okadaic acid concentration, and the sensor showed a linear response in the 0.37-3.97 µM okadaic acid range with a limit of detection of 0.25 µM. This work may have important implications for the development of new, cheap, and versatile biosensors for a range of biomolecules and that are sufficiently simple to be applied in the field or at point-of-care.

Effect of silver nanoparticles on Mediterranean sea urchin embryonal development is species specific and depends on moment of first exposure.

With the ever growing use of nanoparticles in a broad range of industrial and consumer applications there is increasing likelihood that such nanoparticles will enter the aquatic environment and be transported through freshwater systems, eventually reaching estuarine or marine waters. Due to silver's known antimicrobial properties and widespread use of silver nanoparticles (AgNP), their environmental fate and impact is therefore of particular concern. In this context we have investigated the species-specific effects of low concentrations of 60 nm AgNP on embryonal development in Mediterranean sea urchins Arbacia lixula, Paracentrotus lividus and Sphaerechinus granularis. The sensitivity of urchin embryos was tested by exposing embryos to nanoparticle concentrations in the 1-100 µg L(-1) range, with times of exposure varying from 30 min to 24 h (1 h-48 h for S. granularis) post-fertilisation which corresponded with fertilized egg, 4 cell, blastula and gastrula development phases. The most sensitive species to AgNP was A. lixula with significant modulation of embryonal development at the lowest AgNP concentrations of 1-10 µg L(-1) with high numbers of malformed embryos or arrested development. The greatest impact on development was noted for those embryos first exposed to nanoparticles at 6 and 24 h post fertilisation. For P. lividus, similar effects were noted at higher concentrations of 50 µg L(-1) and 100 µg L(-1) for all times of first exposure. The S. granularis embryos indicated a moderate AgNP impact, and significant developmental abnormalities were recorded in the concentration range of 10-50 µg L(-1). As later post-fertilisation exposure times to AgNP caused greater developmental changes in spite of a shorter total exposure time led us to postulate on additional mechanisms of AgNP toxicity. The results herein indicate that toxic effects of AgNP are species-specific. The moment at which embryos first encounter AgNP is also shown to be an important factor in the development of abnormalities, and future applications of the sea urchin embryo development test for nanoparticle toxicity testing should carefully address the specific phase of development of embryos when nanoparticles are first introduced.

Role of subcolloidal (nanosized) precursor species in the early stage of the crystallization of zeolites in heterogeneous systems.

A critical analysis was carried out for the purpose of understanding the role of subcolloidal (nanosized) (alumino)silicate precursor species in the early stage of crystallization of zeolites in heterogeneous systems (hydrogels). The formation and evolution of these subcolloidal species in both the solid and the liquid phases were investigated by various experimental methods such a scanning electron microscopy (SEM, FE-SEM), transmission electron microscopy, atomic force microscopy, particle size analysis, pH measurement, atomic absorption spectroscopy, and dynamic light scattering, after careful separation of intermediates from reaction mixture by two-step centrifugation treatment. The results revealed that a chain of processes (i) the formation of low-molecular-weight (LMW) silicate species, by dissolution of Al-enriched amorphous silica, and their aggregation into about 3 nm sized primary precursor species (PPSs), (ii) the formation of larger (∼3 to ∼15 nm sized) silicate precursor species (LSPSs) by a rapid aggregation/coalescence of PPSs, (iii) the formation of "gel" (primary amorphous precursor) by a random aggregation of LSPSs at room temperature, and (iv) the formation of the worm-like particles (secondary amorphous precursor) occurred in the solid phase during heating of the reaction mixture (hydrogel) from room temperature to 170 °C. It is interesting that almost the same processes occur in the liquid phase but with decreased rate according to the relative low concentration of LMW silicate species. With the above described findings, it is highly expected that the manipulation of crystallization pathway through controlling the formation/evolution of precursor species in the initial stage of the process can be achieved.

Surface modified liposomes by mannosylated conjugates anchored via the adamantyl moiety in the lipid bilayer.

The aim of the present study was to encapsulate mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides, namely [(2R)-N-(adamant-1-yl)-3-(α,ß-d-mannopyranosyloxy)-2-methylpropanamide and (2R)-N-[3-(α-d-mannopyranosyloxy)-2-methylpropanoyl]-d,l-(adamant-2-yl)glycyl-l-alanyl-d-isoglutamine] in liposomes. The characterization of liposomes, size and surface morphology was performed using dynamic light scattering (DLS) and atomic force microscopy (AFM). The results have revealed that the encapsulation of examined compounds changes the size and surface of liposomes. After the concanavalin A (ConA) was added to the liposome preparation, increase in liposome size and their aggregation has been observed. The enlargement of liposomes was ascribed to the specific binding of the ConA to the mannose present on the surface of the prepared liposomes. Thus, it has been shown that the adamantyl moiety from mannosylated 1-aminoadamantane and mannosylated adamantyltripeptides can be used as an anchor in the lipid bilayer for carbohydrate moiety exposed on the liposome surface.

Influence of pH, temperature and common ion on magnesium hydrogenurate octahydrate solubility.

Physico-chemical investigation of urolithiasis today is mostly focused on applying physico-chemical description of precipitation processes to the stone formation with the aim to distinguish between stone formers and nonstone formers. This is done by calculating supersaturation for different solid phases which can be formed in urine using data obtained by urine analysis and existing solubility product, dissociation and complexation constants. In order for this approach to succeed it is of utmost importance that system is described as detailed as possible, i.e., that all species that can be formed are taken into account. Magnesium hydrogenurate octahydrate, Mg(C5H3N4O3)28H2O (Mg(HU)2.8H2O), is among species which can precipitate in the urine and for which solubility data doesn't exist. In order to fill this void crystals of Mg(C5H3N4O)2.8H2O phase I and phase II have been prepared and characterized. Solubility product constant of Mg(C5H3N4O3)2.8H2O phase I in water at 37 degrees C and phase II at different temperatures, pH and in different solvents have been determined by measuring total concentration of uric acid and magnesium ions in solutions at different time periods. Results show that in water at 37 degrees C thermodynamically less stable phase I is more soluble (Ksp = (5.64 +/- 0.20).10(-9) mol3 dm(-9)) than phase II (Ksp = (1.66 +/- 0.13).10(-9) mol3 dm(-9)). Solubility of Mg(HU)2.8H2O phase II increases with temperature. At equilibrium the solubility of phase II is the lowest in the presence of excess of magnesium ions, while solubility in the presence of uric acid is comparable with the one obtained in water.

Metabolic and physico-chemical urolithiasis parameters in the first morning urine.

The 24-hour urine is golden standard for metabolic assessment of stone formers. However, due to the difficulties in collecting almost 1/3 of the samples can not be used for the analysis. Therefore, we analyzed first morning urine and calculated different risk indexes in order to asses possibility of using it in determining urolithiasis risk. Subjects were divided into 4 groups: male patients (n = 31, age 18-64), female patients (n = 31, age 25-63), male controls (n = 16, age 25-64) and female controls (n = 19, age 21-65). First morning urine pH, concentrations of calcium, magnesium, phosphate, sodium, potassium, chloride, citrate, urate, oxalate, creatinine and glycosaminoglycans were determined. Based on them, ionic concentrations and activity products of calcium oxalate and phosphate were calculated by EQUIL 2. In addition, different risk indices were calculated. The results showed that both patients and control groups had metabolic disorders, but the frequencies of occurrence were statistically independent. Significant difference in concentration of urinary constituents between corresponding patient and control groups was observed only for glycosaminoglycans in female subjects. Ca/Cit ratio and Baggio index could differentiate between both corresponding patients and control groups. The results indicate that interplay between stone formation inhibitors and promotors is responsible for urinary stone formation and that the first morning urine could be used in assessing urolithiasis risk and its prevention.