Determination of Calcium in Meat Products by Automatic Titration with 1,2-Diaminocyclohexane-N,N,N',N'-tetraacetic Acid.

Mechanically separated meat (MSM) is a by-product of the poultry industry that requires routine quality assessment. Calcium content is an indirect indicator of bone debris in MSM but is difficult to determine by EDTA titration due to the poor solubility of calcium phosphate. Therefore, 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid was used instead, which has two orders of magnitude higher affinity for calcium ions. In addition, the auxiliary complexing agents triethanolamine and Arsenazo III, an indicator that is sensitive to low calcium concentrations, were used. Automatic titration endpoint detection was performed using an immersion probe at 660 nm. It has been shown that the color change in Arsenazo III can also be read with an RGB camera. The CDTA titration procedure has been tested on commercial Bologna-type sausages and the results were in line with AAS and ICP reference data. The content of calcium in sausages turned out to be very diverse and weakly correlated with the content of MSM. The tested MSM samples had a wide range of calcium content: from 62 to 2833 ppm. Calcium-rich poultry by-products include fat and skin (115 to 412 ppm), articular cartilage (1069 to 1704 ppm), and tendons (532 to 34,539 ppm). The CDTA titration procedure is fully suitable for small meat processing plants due to its simplicity of use and low cost.

Vapor Generation of Rare-Earth Elements via Reaction with Tetrahydroborate: Chemical Identities and Mechanisms of Formation.

This technical note reports for the first time the simultaneous vapor generation of 16 rare-earth elements (REEs) via reaction with sodium tetrahydroborate (THB). Significant improvement in REE vapor generation efficiency was discovered in the presence of arsenazo III (ARS). Subsequent to a critical evaluation on the impacts of various experimental variables, including the concentrations of acids, THB, and ARS, blank-limited (reagent contamination and impurity from ARS) detection limits (LODs) in the range of 0.004-0.15 µg L-1 were achieved based on three times the standard deviation of the method blank by the proposed vapor generation inductively coupled plasma mass spectrometry (ICP-MS). The nature of released REE species was studied by various approaches and identified to be REE nanoparticles and REE-ARS chelates; a dual-route mechanism for the vapor generation of REEs was thereby proposed.

Effect of phytate on crystallization on ureteral stents and bacterial attachment: an in vitro study.

The use of double J ureteral stents can lead to several adverse effects, as urinary infection. Bacteria tend to colonize the stent surface, leading to the formation of bacterial biofilms. The presence of urease-producing bacteria increase the urine pH leading to the incrustation and blockage of the stent. On the other hand, these large crystalline masses function as niduses, allowing the attachment of even more bacteria and decreasing its exposure to antibiotics. The aim of this in vitro study was to assess the effect of phytate on the attachment of bacteria to the catheter surface under conditions that favor crystallization. Catheter sections were incubated in a synthetic urine medium (pH 6.5) in the presence or absence of Pseudomonas aeruginosa and phytate. Amount of calcium deposits was measured using an Arsenazo III colorimetric method and the number of attached bacteria to the stent was determined. Differences were assessed using an ANOVA with a Bonferroni post hoc test. The formation of calcium phosphate deposits (brushite and hydroxyapatite) and oxalate crystals (COM), as were as the amount of bacteria decreased when phytate was present. Thus, phytate successfully decreased bacterial adhesion by inhibiting the formation of crystalline deposits.

Estimation and in-situ detection of thorium in human liver cell culture by arsenazo-III based colorimetric assay.

To understand the biological effects of Thorium-232 (Th) in human cells and animal models as well as to assess mitigation strategies for its detoxification, there is a need to develop a sensitive, specific, high-throughput and easily-implementable assay for detection and estimation of Th in biological samples. Here, we have optimized arsenazo-III dye based colorimetric assay to detect Th in biological samples. The concentration of arsenazo-III (i.e. 50 µM) was optimized, which can reliably estimate Th in the concentration range of 2.5 to 40 µM. The optimized assay can specifically detect Th without interference from other metal ions (La, Ce, U, Fe, Ca, Cu, Zn and Mn). A significant correlation (R2 = 0.999) was found between arsenazo-III-based detection of Th and total reflection X-ray fluorescence. The conditions of present assay successfully estimated Th in cell culture medium, cell harvesting (trypsin-EDTA) solution and cell lysate obtained from human liver cell culture. Moreover, for the first time, we detected Th in-situ in adherent liver cells in culture after staining with arsenazo-III. This study confirms that Th can be specifically determined in biological samples using arsenazo-III with the sensitivity, which is relevant to thorium toxicity research.

Synthesis and evaluation of N-allylthiourea-modified chitosan for adsorptive removal of arsenazo III dye from aqueous solutions.

N-allylthiourea chitosan (ATUCS), a chelating material, was prepared, characterized, and studied for the removal of arsenazo III (As (III)) dye from aqueous solution. Scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), and 1H- and 13C-nuclear magnetic resonance (NMR) were used to characterize the prepared adsorbent and to investigate the adsorption mechanism. Furthermore, the adsorption behavior of chitosan (CS) and ATUCS were studied under various conditions. The equilibrium adsorbed amount of As (III) onto ATUCS was found to be 116.3 mg/g, compared to 87.3 mg/g with respect to CS. The regeneration of the loaded CS and ATUCS were studied using 1:1 solution of H2O2-H2SO4 and reused with certain change in efficiency after the third cycle. The adsorption process was found to fit well with pseudo-second-order kinetic model. The equilibrium data were better described with the Freundlich isotherm. The monolayer adsorption capacity was found to be 204.08 and 90.90 mg/g for the As (III)/ATUCS and As (III)/CS systems, respectively, at 25 °C. The pH of the higher uptake of As (III) onto ATUCS and CS was 4-5 and 8.0, respectively. The results demonstrated improved adsorption of As (III) using ATUCS as compared to the CS.

Facile Arsenazo III-Based Assay for Monitoring Rare Earth Element Depletion from Cultivation Media for Methanotrophic and Methylotrophic Bacteria.

Recently, methanotrophic and methylotrophic bacteria were found to utilize rare earth elements (REEs). To monitor the REE content in culture media of these bacteria, we have developed a rapid screening method using the Arsenazo III (AS III) dye for spectrophotometric REE detection in the low µM (0.1 to 10 µM) range. We designed this assay to follow LaIII and EuIII depletion from the culture medium by the acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum strain SolV. The assay can also be modified to screen the uptake of other REEs, such as PrIII, or to monitor the depletion of LaIII from growth media in neutrophilic methylotrophs such as Methylobacterium extorquens strain AM1. The AS III assay presents a convenient and fast detection method for REE levels in culture media and is a sensitive alternative to inductively coupled plasma mass spectrometry (ICP-MS) or atomic absorption spectroscopy (AAS).IMPORTANCE REE-dependent bacterial metabolism is a quickly emerging field, and while the importance of REEs for both methanotrophic and methylotrophic bacteria is now firmly established, many important questions, such as how these insoluble elements are taken up into cells, are still unanswered. Here, an Arsenazo III dye-based assay has been developed for fast, specific, and sensitive determination of REE content in different culture media. This assay presents a useful tool for optimizing cultivation protocols, as well as for routine REE monitoring during bacterial growth without the need for specialized analytical instrumentation. Furthermore, this assay has the potential to promote the discovery of other REE-dependent microorganisms and can help to elucidate the mechanisms for acquisition of REEs by methanotrophic and methylotrophic bacteria.

Selectivity enhancement of Arsenazo(III) reagent towards heavier lanthanides using polyaminocarboxylic acids: a spectrophotometric study.

A new study has been conducted to quantify lanthanide(III) ions using Arsenazo III-polyaminocarboxylic acid (PACA) system. The study disclosed two different analytically important information: (i) λmax of lanthanide-Arsenazo III complexes for lighter lanthanides like Ce(III) and Nd(III) did not shift from its original position on addition of PACA and (ii) for heavier lanthanides like Dy(III), Tm(III) and Lu(III) a new λmax at 538 nm was observed, while wavelengths at 610 nm and 654 nm were disappeared in presence of ethylenediaminetertracetic acid (EDTA) and trans-1,2-Diaminocyclohexane-N,N,N',N'-tetraacetic acid (DCTA), further the intensity of peak decreased with increase in lanthanide(III) ion concentration. Effect of ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and N-(2-hydroxyethyl) ethylenediamine-N,N',N'-triacetic acid (EDTA-OH) on Arsenzo(III)-Ln(III) complex is very weak and there is no analytically importance of such interaction. Moreover, this work confirms that Nd(III) and heavy lanthanides can be successfully determined with high accuracy in the working range of concentration of these metal ions.

Stability of two resin combinations used as sealants against toothbrush abrasion and acid challenge in vitro.

OBJECTIVE: To test the stability of two conventional adhesives when combined with a low-viscosity caries infiltrant used for sealing sound enamel against toothbrush abrasion and acid challenge in vitro. MATERIALS AND METHODS: Bovine enamel discs (Ø = 3 mm) randomly assigned to three groups (n = 10/group) were etched with 37% phosphoric acid for 30 s and treated with resins of different monomer contents forming three test groups: (1) Untreated specimens (Control); (2) Infiltrant (Icon, DMG) + conventional enamel bonding adhesive (Heliobond, Ivoclar Vivadent); and (3) Infiltrant + conventional orthodontic adhesive (Transbond XT Primer, 3M Unitek). All specimens were immersed in hydrochloric acid (pH 2.6) for up to 9 days, during which they were exposed to 1825 toothbrush-strokes per day. Calcium dissolution was assessed using Arsenazo III method at 24-h intervals. Data were analyzed by Kruskal-Wallis and Wilcoxon signed ranks tests. RESULTS: Cumulative calcium dissolution for the untreated specimens (39.75 ± 7.32 µmol/ml) exceeded the sealed groups (Icon + Heliobond: 23.44 ± 7.03 µmol/ml; Icon + Transbond XT Primer: 22.17 ± 5.34 µmol/ml). Untreated specimens presented a relatively constant calcium dissolution rate throughout the experimental period, whereas the sealed groups presented a gradual increase indicating weakening of the seal by toothbrush abrasion. Both sealed groups presented significantly lower daily calcium dissolution at all time points compared to the control, except for Group 2 on the last measurement day. CONCLUSIONS: Low-viscosity caries infiltrant application on sound enamel prior to conventional resin application provided a protective effect against enamel demineralization, but this effect was not stable when challenged mechanically by toothbrush abrasion.

Influence of gamma irradiation on uranium determination by Arsenazo III in the presence of Fe(II)/Fe(III).

Arsenazo III is a widely used reagent for the concentration measurement of uranium and other actinides in aqueous samples. This study indicates that, for routine aqueous samples, due to the strong complexing ability with Arsenazo III, Fe(III) can significantly decrease the UV-Vis absorbance of the U(VI)-Arsenazo III complex, whereas the influence of Fe(II) on the absorbance is negligible. However, when Fe(II) is present in a gamma-irradiated U(VI) aqueous sample, it can give rise to the Fenton reaction, which produces oxidizing radicals that decompose the subsequently added Arsenazo III, leading to a sharp decrease in the absorbance of the U(VI)-Arsenazo III complex. The decrease in absorbance depends on the iron content and irradiation dose. Furthermore, the oxidizing radicals from the Fenton reaction induced by gamma irradiation can be continually produced. Even if the irradiated solution has been aged for more than one month in the absence of light at room temperature and without the exclusion of oxygen, the reactivity of the radicals did not decrease toward the subsequently added Arsenazo III. This finding demonstrates that the presence of Fe(II) in gamma-irradiated U(VI) aqueous samples can lead to incorrect U(VI) measurement using the Arsenazo III method, and a new method needs to be developed for the quantitative determination of U(VI) in the presence of gamma radiation and ferrous iron.

Optimization of the arsenazo-III method for the determination of uranium in water and plant samples.

This work reports a reliable, fast and optimized photometric technique based on the specific chemical complexation of uranyl ion with arsenazo-III. In the case of solid samples (plant samples), for which mineralization under acidic and oxidative conditions was used, addition of ascorbic acid led to stabilization of the arsenazo-uranyl complex over time. The results, in total agreement with data obtained from α and γ spectrometries, demonstrate that the present technique is able to precisely quantify uranium in water as well as in plant samples, within the µg/L and mg/g ranges respectively.

Biochemical composition of carious dentin and different layers of sound dentin.

The aim of this study was to evaluate the concentrations of calcium (Ca), inorganic phosphate (Pi) and fluoride (F) in carious dentin and in different layers of sound dentin. The samples examined were 52 permanent teeth (26 sound and 26 carious), which were subjected to two experiments to assess the mineral content of: 1) two layers (internal and external) of sound dentin and 2) sound and carious dentin. Ca and Pi were analyzed using a colorimetric method with arsenazo III (C22H18As2N4O14S2) and molybdate reagents, and F was analyzed using a specific electrode. A non-parametric test, the Mann-Whitney test, was used to verify differences between groups. Sound dentin showed a higher concentration of fluoride in the internal layer than in the external layer (P = 0.03), but no inter-layer differences in Ca or Pi concentration were evident. Lower concentrations of Ca, Pi and F were observed in carious dentin than in sound dentin (P < 0.05). The results of this study suggest that the internal layer of sound dentin has a higher fluoride content than the external layer, and that carious dentin has lower concentrations of Ca, Pi and F than sound dentin.

Flow injection online spectrophotometric determination of uranium after preconcentration on XAD-4 resin impregnated with nalidixic acid.

In this work, spectrophotometer was used as a detector for the determination of uranium from water, biological, and ore samples with a flow injection system coupled with solid phase extraction. In order to promote the online preconcentration of uranium, a minicolumn packed with XAD-4 resin impregnated with nalidixic acid was utilized. The system operation was based on U(VI) ion retention at pH 6 in the minicolumn at flow rate of 15.2 mL min(-1). The uranium complex was removed from the resin by 0.1 mol dm(-3) HCl at flow rate of 3.2 mL min(-1) and was mixed with arsenazo III solution (0.05 % solution in 0.1 mol dm(-3) HCl, 3.2 mL min(-1)) and driven to flow through cell of spectrophotometer where its absorbance was measured at 651 nm. The influence of chemical (pH and HCl (as eluent and reagent medium) concentration) and flow (sample and eluent flow rate and preconcentration time) parameters that could affect the performance of the system as well as the possible interferents was investigated. At the optimum conditions for 60 s preconcentration time (15.2 mL of sample volume), the method presented a detection limit of 1.1 µg L(-1), a relative standard deviation (RSD) of 0.8 % at 100 µg L(-1), enrichment factor of 30, and a sample throughput of 42 h(-1), whereas for 300 s of the preconcentration time (76 mL of sample volume), a detection limit of 0.22 µg L(-1), a RSD of 1.32 % at 10 µg L(-1), enrichment factor of 150, and a sampling frequency of 11 h(-1) were reported.

Impact of laminar flow velocity of different acids on enamel calcium loss.

OBJECTIVE: The aim of the study was to evaluate the impact of flow velocity under laminar flow conditions of different acidic solutions on enamel erosion. MATERIAL AND METHODS: A total of 240 bovine enamel specimens were prepared and allocated to 30 groups (n = 8 each). Samples of 18 groups were superfused in a flow chamber system with laminar flow behavior using 1 ml of citric acid or hydrochloric acid (HCl) of pH 2.0, 2.6 or 3.0. Flow rates in the sample chamber were adjusted to 10, 60 or 100 µl/min. To simulate turbulent flow behavior, samples of six groups were immersed in 1 ml of the respective solution, which was vortexed (15 min, 600 rpm). For simulating non-agitated conditions, specimens of the remaining six groups were immersed in 1 ml of the respective solution without stirring. Calcium in the solutions, released from the enamel samples, was determined using Arsenazo III method. RESULTS: For acidic solutions of pH 2.6 and 3.0, erosive potential of citric acid was equivalent to that of HCl at a flow of 100 µl/min. The same observation was made for the samples subjected to turbulent conditions at pH 3. At all other conditions, citric acid induced a significantly higher calcium loss than HCl. CONCLUSION: It is concluded that under slow laminar flow conditions, flow rate variations lead to higher erosive impact of citric acid compared to hydrochloric acid at pH 2.0, but not at pH ≥ 2.6 and increasing laminar flow or turbulent conditions. CLINICAL RELEVANCE: Erosive enamel dissolution under laminar flow conditions is a complex issue influenced by flow rate and acidic substrate.

[IP3-sensitive Ca(2+)-channels of endoplasmic reticulum in secretory cells of the rat exorbital lacrimal gland].

The role of inositol-1,4,5-trisphosphate of (IP3)-sensitive Ca2+ channels in Ca2+ homeostasis maintenance under activation of M-cholinergic receptors and P2Y receptors in the secretory cells of the rat lacrimal gland was investigated. The study was carried out on intact and permeabilized secretory cells of exorbital lacrimal glands of rats. The cells were isolated using the modified Herzog, Sides, Miller method (1976) and permeabilized with digitonin (50 mg per 0.5 million cells). The functioning of the Ca(2+)-transport systems was estimated by changes of Ca2+ content in the studied cells, which was determined by the spectrophotometric method using arsenazo III. It was shown that IP3-sensitive Ca2+ channels (IP3Rs) of investigated cells are directly inhibited by 2-APB (10 microM/l). On the other hand, the channels are activated by IP3, cholinomimetic (carbacholine) and purine receptor agonist (ATP). When both M-cholinergic receptors and P2Y receptors were activated Ca2+ was released from the same IP3-sensitive store because the effects of ATP and carbacholine at high concentrations (1mM/l and 10 microM/l, respectively) on the Ca2+ content were non-additive. The presence of the store-operated Ca(2+)-channels in secretory cells of the lacrimal gland is confirmed by the observed increase of cellular Ca2+ content as a result of Ca2+ mobilization from the store by carbacholine or thapsigargin and following restoration of Ca2+ concentration in the extracellular solution.

Plaque fluoride concentrations associated to the use of conventional and low-fluoride dentifrices.

PURPOSE: This double-blind, crossover study evaluated whole plaque fluoride concentration (F), as well as whole plaque calcium concentrations (Ca) after brushing with a placebo (PD - fluoride free), low-fluoride (LFD, 513 microg F/g) and conventional (CD, 1,072 microg F/g) dentifrices. METHODS: Children (n=20) were randomly assigned to brush twice daily with one of the dentifrices, during 7 days. On the 7th day, samples were collected at 1 and 12 hours after brushing. F and Ca were analyzed with an ion-selective electrode and with the Arsenazo III method, respectively. Data were analyzed by ANOVA, Tukey's test and by Pearson correlation coefficient (P< 0.05). RESULTS: The use of the fluoridated dentifrices significantly increased plaque [F]s 1 hour after brushing when compared to PD, returning to baseline levels 12 hours after. Positive and significant correlations were found between plaque [F] and (Ca) under most of the conditions evaluated. The mean increase in plaque [F] observed 1 hour after brushing with the CD were only about 47% higher than those obtained for the LFD. The use of a LFD promotes proportionally higher increases in plaque [F] when compared to a CD. Plaque F concentrations were also shown to be dependent on plaque Ca concentrations.

Effect of safflower oil on the protective properties of the in situ formed salivary pellicle.

AIM: The prevalence of dental erosion is still increasing. A possible preventive approach might be rinsing with edible oils to improve the protective properties of the pellicle layer. This was tested in the present in situ study using safflower oil. METHODS: Pellicle formation was carried out in situ on bovine enamel slabs fixed buccally to individual upper jaw splints (6 subjects). After 1 min of pellicle formation subjects rinsed with safflower oil for 10 min, subsequently the samples were exposed in the oral cavity for another 19 min. Enamel slabs without oral exposure and slabs exposed to the oral cavity for 30 min without any rinse served as controls. After pellicle formation in situ, slabs were incubated in HCl (pH 2; 2.3; 3) for 120 s, and kinetics of calcium and phosphate release were measured photometrically (arsenazo III, malachite green). Furthermore, the ultrastructure of the pellicles was evaluated by transmission electron microscopy (TEM). RESULTS: Pellicle alone reduced erosive calcium and phosphate release significantly at all pH values. Pellicle modification by safflower oil resulted in an enhanced calcium loss at all pH values and caused an enhanced phosphate loss at pH 2.3. TEM indicated scattered accumulation of lipid micelles and irregular vesicle-like structures attached to the oil-treated pellicle layer. Acid etching affected the ultrastructure of the pellicle irrespective of oil rinsing. CONCLUSION: The protective properties of the pellicle layer against extensive erosive attacks are limited and mainly determined by pH. The protective effects are modified and reduced by rinses with safflower oil.

Correlation of serum oestrogen with salivary calcium in post-menopausal women with and without oral dryness feeling.

OBJECTIVE: The aim of this study was to verify the association between salivary calcium and serum oestrogen levels with oral dryness in post-menopausal women. Also, the correlation between these variables was evaluated. METHODS: A case-control study was carried out on 60 selected menopausal women with and without oral dryness feeling (30 as case and 30 as control) conducted in the Department of Oral Medicine and Radiology, Maharishi Markandeshar University, Mullana, India. Paraffin-stimulated saliva samples were obtained by expectoration. Salivary calcium concentrations were assessed colorimetrically using Arsenazo III reaction. The serum oestrogen concentration was measured using ELISA. Statistical analysis of Student's t-test and Pearson correlation was used. RESULTS: There was significant difference in mean values of both salivary calcium concentration and serum oestrogen between case and control groups. The result obtained also showed that an inverse correlation was found between salivary calcium concentration and serum oestrogen levels in both the groups and in total sample size. CONCLUSION: Oral dryness in post-menopausal women is associated with high levels of salivary calcium and low levels of serum oestrogen. The concentrations of salivary calcium and serum oestrogen are inversely correlated in post-menopausal women, regardless of the presence or absence of oral dryness.

Assays of osteogenic differentiation by cultured human mesenchymal stem cells.

One of the most noteworthy characteristics of mesenchymal stem cells (MSCs) is their ability to differentiate into osteoblasts in vitro and in vivo. In vitro, this is easily achieved by culturing in the appropriate induction medium. It is because of the reliability and ease of this process that osteogenic differentiation has become a popular assay for the demonstration of MSC plasticity. Although the conditions required for inducing osteogenic differentiation by MSCs typically do not vary particularly between investigators, many methods are employed to measure the extent of differentiation. These methods include, but are not limited to, reverse transcriptase PCR (RT-PCR) for detection of osteogenic transcripts, enzyme linked immunosorbent assay (ELISA) for secreted protein markers, colorimetric assays for osteogenic enzymes, and direct staining of matrix components. This chapter reviews the protocols most commonly utilized for the evaluation of osteogenic differentiation for cultured MSCs.

A simplified suite of methods to evaluate chelator conjugation of antibodies: effects on hydrodynamic radius and biodistribution.

INTRODUCTION: Antibodies covalently conjugated with chelators such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) are required for radioimmunoscintigraphy and radioimmunotherapy, which are of growing importance in cancer medicine. METHOD: Here, we report a suite of simple methods that provide a preclinical assessment package for evaluating the effects of DOTA conjugation on the in vitro and in vivo performance of monoclonal antibodies. We exemplify the use of these methods by investigating the effects of DOTA conjugation on the biochemical properties of the DAB4 clone of the La/SSB-specific murine monoclonal autoantibody, APOMAB, which is a novel malignant cell death ligand. RESULTS: We have developed a 96-well microtiter-plate assay to measure directly the concentration of DOTA and other chelators in antibody-chelator conjugate solutions. Coupled with a commercial assay for measuring protein concentration, the dual microtiter-plate method can rapidly determine chelator/antibody ratios in the same plate. The biochemical properties of DAB4 immunoconjugates were altered as the DOTA/Ab ratio increased so that: (i) mass/charge ratio decreased; (ii) hydrodynamic radius increased; (iii) antibody immunoactivity decreased; (iv) rate of chelation of metal ions and specific radioactivity both increased and in vivo, (v) tumor uptake decreased as nonspecific uptake by liver and spleen increased. CONCLUSION: This simplified suite of methods readily identifies biochemical characteristics of the DOTA-immunoconjugates such as hydrodynamic diameter and decreased mass/charge ratio associated with compromised immunotargeting efficiency and, thus, may prove useful for optimizing conjugation procedures in order to maximize immunoconjugate-mediated radioimmunoscintigraphy and radioimmunotherapy.