Deleterious effects of calcium indicators within cells; an inconvenient truth.

The study of cellular Ca2+ signalling is indebted to Roger Tsien for the invention of fluorescent indicators that can be readily loaded into living cells and provide the means to measure cellular Ca2+ changes over long periods of time with sub-second resolution and microscopic precision. However, a recent study [1] reminds us that as useful as these tools are they need to be employed with caution as there can be off-target effects. This article summarises these recent findings within the wider context of confounding issues that can be encountered when using chemical and genetically-encoded Ca2+ indicators, and briefly discusses some approaches that may mitigate against misleading outcomes.

[Determination of ethylene glycol bis (2-aminoethyl) ether-N,N,N,N-tetraacetic acid in medicine using high performance liquid chromatography].

A method was developed for the determination of ethylene glycol bis(2-aminoethyl) ether-N,N,N,N-tetraacetic acid (EGTA) by high performance liquid chromatography (HPLC). The content of EGTA can be determined by that of EGTA-Cu through the complexation between EGTA and Cu2+. The chromatographic separation was performed on an Ultimate-AQ C18 analytical column (250 mm x 4.6 mm, 5 µm) using the mobile phase of acetonitrile-ion-pairing reagents (with 0.3% tetrabutyl ammonium hydroxide in mass fraction adjusted to pH 6.50 using acetate)-buffered saline (35 mmol/L sodium acetate of pH 6.50) (20:20:60, v/v/v). The chromatographic conditions were as follows: flow rate, 1.50 mL/min; detection wavelength, 245 nm; injection volume, 100 µL; column temperature, 40 °C. Under the conditions, good linear relationships between the mass concentration and the peak area of EGTA were observed in the range of 0.10-15.00 mg/L (R = 0.9998). The limit of detection (LOD, S/N = 3) and limit of quantitation (LOQ, S/N = 10) were determined as 0.05 mg/L and 0.17 mg/L, respectively. The average recoveries were 98.34%-99.03% with the RSDs of 1.08%-3.33% (n = 9). The results showed that the developed method is sensitive, accurate, reproducible and suitable for the analysis of EGTA in medicine.

[Investigation vesicle cycle in nerve formations in somatic muscle of the earthworm Lumbricus terrestris].

Luminous spots with a diameter of 1-2 microm, which are clusters of "synaptic buds", were revealed in the muscular wall of the earthworm using endocytotic fluorescent dyes FM1-43, FM2-10 and FM4-64. Application of the membrane probe Dil that is capable of being subjected to anterograde axonal transport to abdominal ganglia of the nervous chain, and subsequent (in a day) staining of nerve formations by endocytotic dye FM4-64 showed complete imposition of the emission data of the dyes that fluoresce in different parts of the spectrum. Using fluorescent marker DiBAC4(3) showed an increased emission of neural elements with increasing concentration of K+ in the extracellular environment. Application of FM2-10 showed that the higher concentration of K+ in solution, and hence the depolarization of the nerve cells, the faster the upload of the dye, and vice versa, the process slowed down in the absence of K+ in the medium. The seizure and removal of FM2-10 were blocked in calcium-free solutions in the presence of Ca2+ buffers, BABTA or BABTA-AM, but only after a preliminary 40 min incubation. The processes of exo- and endocytosis occurred in the clusters of synaptic "buds" and were preserved in conditions of "rest". This vesicle cycle depends on membrane potential and concentration of K+ and Ca2+, and, it is very likely that the calcium sensor operates on the principle "all or nothing".

Determination of 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA free acid) in rat plasma, urine and feces by liquid chromatography with UV and tandem mass spectrometric detection.

BAPTA free acid was identified as the main metabolic product of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(actoxymethyl ester) (BAPTA-AM), a neuroprotective agent in cerebral ischemia, in rats. In this paper, liquid chromatography-ultraviolet (LC-UV) and mass spectrometry/mass spectrometry (LC-MS/MS) methods were employed for the determination of BAPTA free acid in rat urine and feces and rat plasma, respectively. By liquid-liquid extraction and LC-UV analysis, a limit of quantitation of 1000 ng/ml using 0.2 ml rat urine for extraction and 250 ng/ml using 1 ml rat fecal homogenate supernatant for extraction could be reached. The assay was linear in the range of 1000-50,000 ng/ml for rat urine and 250-10,000 ng/ml for rat fecal homogenate supernatant. Because the sensitivity of the LC-UV method was apparently insufficient for evaluating the pharmacokinetic profile of BAPTA in rat plasma, a LC-MS/MS method was subsequently developed for the analysis of BAPTA free acid. By protein precipitation and LC-MS/MS analysis, the limit of quantitation was 5 ng/ml using 0.1 ml rat plasma and the linear range was 5.0-500 ng/ml. Both methods were validated and can be used to support a thorough preclinical pharmacokinetic evaluation of BAPTA-AM liposome injection.

Study of solid-phase extraction for the determination of sequestering agents in river water by high-performance liquid chromatography.

Anion-exchange solid-phase extraction accompanied with high-performance liquid chromatography has been developed for the determination of six kinds of aminopolycarboxylic acids (APCAs) in river water [N-(2-hydroxyethyl)ethylenediaminetriacetate (HEDTA), ethylenediaminetetraacetate (EDTA), 1,3-propanediaminetetraacetate (PDTA), diethylenetriaminepentaacetate (DTPA), 1,2-propanediaminetetraacetate (MeEDTA), and O,O'-bis(2-aminoethyl)ethyleneglycoltetraacetate (GEDTA)]. The enrichment of APCAs using an anion-exchange cartridge was successfully done by the removal of anions, which competed with APCAs in anion-exchange processes. Barium chloride solution was added to river water and the mixture was passed through On Guard II Ag and H cartridges and then a Bond Elut Jr.SAX cartridge to enrich APCAs. After elution, APCAs were analyzed on two reversed phase C30 columns connected in series and detected with ultraviolet detection. The enrichment using solid-phase extraction permitted the determination of APCAs in river water at concentrations as low as 1nM. Good recoveries (83-111%) were obtained for each APCA by the standard addition method on three river water samples with high accuracy (RSD 1.8-9.5%). Applying this method, two kinds of APCAs, EDTA and DTPA, were determined in samples from the Oyabe and Senbo Rivers in Japan.

Determination of sequestering agents in cosmetics and synthetic detergents by high-performance liquid chromatography with ultraviolet detection.

Using a fast reversible reaction of aminopolycarboxylic acids (APCAs) into Fe(III)-APCA complexes in the presence of Fe(III) ions, seven kinds of APCAs [nitrilotriacetate (NTA), N-(2-hydroxyethyl)ethylenediamine-triacetate (HEDTA), ethylenediamine-tetraacetate (EDTA), 1,3-propanediamine-tetraacetate (PDTA), diethylenetriamine-pentaacetate (DTPA), 1,2-diaminopropane-tetraacetate (MeEDTA), and O,O'-bis(2-aminoethyl)ethyleneglycol-tetraacetate (GEDTA)] in cosmetics and synthetic detergents were separated on two reversed-phase C30 columns connected in series and detected with ultraviolet detection. Simple pretreatment, consisted of thousand times dilution of samples and addition of 100 microl of the Fe(III) solution containing 10 mM Fe(III) chloride and 0.5 M sulfuric acid to 10 ml of diluted samples, permitted the determination of APCAs in cosmetics and synthetic detergents at concentration level of 0.1 mM, except 0.3 mM for GEDTA. APCAs except GEDTA could be detected at concentration level of 0.03 mM and GEDTA could be detected at concentration level of 0.09 mM. Good recoveries (95-110%) were obtained for each APCA by the standard addition method on two diluted samples with high accuracy (RSD 0.2-9.1%). Three APCAs (EDTA, HEDTA and NTA) were detected in various concentrations in cosmetics and synthetic detergents and the other APCAs were not detected in any of the samples. This method requires no tedious pretreatment and takes only 15 min for one analysis, so it is useful for determination of APCAs.

Tuning caged calcium: photolabile analogues of EGTA with improved optical and chelation properties.

The physico-chemical properties of several Ca(2+)-selective, photolabile chelators are described. These molecules have been developed as part of an effort to produce a caged Ca(2+) that improved upon the Ca(2+) chelation properties and light absorption capability of nitrophenyl-EGTA (NP-EGTA). Four dimethoxy-ortho-nitrophenyl derivatives of EGTA (called DMNPE-1 through -4), and one analogue of EGTA (DMNPE-5) have been characterized, each of which is bisected upon irradiation. One of these cages has a higher affinity than NP-EGTA: DMNPE-4 has a K(d) for Ca(2+) of 48 nm at pH 7.2 (19 nM at pH 7.4). Furthermore, this cage has a large extinction coefficient of 5120 M(-1)cm(-1) at 350 nm (cf. 975 M(-1)cm(-1) for NP-EGTA). The other physico-chemical properties of DMNPE-4 are: quantum yield of photolysis of 0.09; bipasic Ca(2+) release kinetics (70% released with a rate of about 48,000 s(-1) and 30% at 1.5s(-1)) and photoproducts that bind Ca(2+) with very low affinity (K(d) in the range of 2mM, pH 7.2), hence most of the bound Ca(2+) is released rapidly and efficiently upon photolysis. Thus, DMNPE-4 has a unique combination of properties that make it an extremely effective Ca(2+) cage.

Smear layer removal and chelated calcium ion quantification of three irrigating solutions.

The purpose of this study was to evaluate, by scanning electron microscopy (SEM), smear layer removal and quantify, by atomic absorption spectrophotometry, the amount of calcium ion present in the chelating solutions after their use. Sixteen extracted canines were instrumented using the step-back technique and were assigned to 3 groups according to the irrigating solution used: G1: 1 mL 17% EDTAC between each file; G2: 1 mL 17% CDTA; G3: 1 mL 17% EGTA. The solutions were collected after use. The teeth were cleaved longitudinally, evaluated under SEM and assessed for smear layer by blinded examiners and scored from 1 to 4. In order to quantify calcium ion release, the collected solutions were examined by atomic absorption spectrophotometry. Freidman's test was used for statistical analysis of SEM values and showed that canals irrigated with 17% EDTAC and 17% CDTA had significantly less smear layer throughout the canals than 17% EGTA (p<0.01). For analysis of the collected solutions, Tukey's test was used and showed that EDTAC and CDTA had a greater amount of calcium ions (22.8+/-7.54 and 60.6+/-20.67 microg/mL, respectively) compared to EGTA (70.5+/-14.2 microg/mL) (p<0.01). The association both methodologies may contribute to the understanding of how these solutions act in the root canal.

Smear layer removal and chelated calcium ion quantification of three irrigating solutions

The purpose of this study was to evaluate, by scanning electron microscopy (SEM), smear layer removal and quantify, by atomic absorption spectrophotometry, the amount of calcium ion present in the chelating solutions after their use. Sixteen extracted canines were instrumented using the step-back technique and were assigned to 3 groups according to the irrigating solution used: G1: 1 mL 17 percent EDTAC between each file; G2: 1 mL 17 percent CDTA; G3: 1 mL 17 percent EGTA. The solutions were collected after use. The teeth were cleaved longitudinally, evaluated under SEM and assessed for smear layer by blinded examiners and scored from 1 to 4. In order to quantify calcium ion release, the collected solutions were examined by atomic absorption spectrophotometry. Freidman's test was used for statistical analysis of SEM values and showed that canals irrigated with 17 percent EDTAC and 17 percent CDTA had significantly less smear layer throughout the canals than 17 percent EGTA (p<0.01). For analysis of the collected solutions, Tukey's test was used and showed that EDTAC and CDTA had a greater amount of calcium ions (22.8±7.54 and 60.6±20.67 æg/mL, respectively) compared to EGTA (70.5±14.2 æg/mL) (p<0.01). The association both methodologies may contribute to the understanding of how these solutions act in the root canal.

O objetivo do presente estudo foi avaliar a remoção de smear layer por meio da microscopia eletrônica de varredura (MEV) e quantificar a liberação de íons cálcio resultante da irrigação com as soluções quelantes estudadas, por meio da espectrofotometria de absorção atômica. Dezesseis caninos extraídos foram instrumentados com a técnica step-back e divididos em 3 grupos de acordo com a solução irrigadora utilizada: G1: 1 mL de EDTAC a 17 por cento entre cada lima; G2: CDTA a 17 por cento; e G3: EGTA a 17 por cento. As soluções foram coletadas após o uso. Os dentes foram secionados longitudinalmente e as raízes examinadas por MEV para verificação de smear layer nos terços por meio de escores (variando de 1 a 4), e avaliadas por três examinadores calibrados "cegos". Para quantificar a liberação de íons cálcio, as soluções coletadas foram avaliadas por espectrofotometria de absorção atômica. Com relação ao smear layer, o teste de Friedman evidenciou diferença estatisticamente significante (p<0,01) comparando-se o EGTA 17 por cento ao EDTAC 17 por cento e ao CDTA 17 por cento, sendo que os canais irrigados com estas duas soluções apresentaram menor quantidade de smear layer que aqueles irrigados com EGTA. As soluções de EDTAC 17 por cento (70,5±14,2 æg/mL Ca) e CDTA 17 por cento (60,6±20,67 æg/mL Ca) apresentaram maiores quantidades de íons cálcio (p<0,01) quando comparadas ao EGTA 17 por cento (22,8±7,54 æg/mL Ca). Desta forma, pode-se concluir que a associação destas metodologias pode contribuir para o entendimento da ação das soluções quelantes no interior dos canais radiculares.

Avaliação da biocompatibilidade do EDTA, EGTA e ácido cítrico em tecido subcutâneo de ratos / Evaluation of biocompatibility of EDTA, EGTA and citric acid in vivo

O objetivo deste trabalho foi avaliar as reações teciduais induzidas por alguns ácidos, durante a fase exsudativa do processo inflamatório. Utilizando-se do teste edemogênico, o EDTA, EGTA, ácido cítrico e soro fisiológico foram avaliados nos períodos de tempo de 12, 24 e 48 horas. A análise dos dados demostrou que não houve diferença estatisticamente significante entre o EGTA eo ácido cítrico - 12 h, entre o EGTA e soro - 24 h entre as 3 substâncias - 48 h. Notou-se diferença significante entre o EDTA e todos os demais grupos, sendo o EDTA a substância mais irritante independente do tempo analisado.

Fluorometric determination of EDTA and EGTA using terbium-salicylate complex.

A method for determining EDTA (ethylenediaminetetraacetic acid) based on a highly fluorescent terbium-EDTA-salicylic acid complex formation was developed. EDTA from as low as a few picomoles to as high as several nanomoles can be determined in a microtiter plate in 10-20 min. Ethyleneglycol-bis(2-aminoethoxy)-tetraacetic acid (EGTA) also can be determined by the same method, but its sensitivity is ca. 14-fold lower. Interestingly, diethylenetriamine tetraacetic acid did not form fluorescent complex with terbium under the same conditions.

The unusual gradient elution for reversed phase HPLC of a strong chelator as an active drug substance.

The new approach to drug development, especially for cardiovascular and brain diseases, brought to synthesis of new lipophillic derivatives of strong calcium chelator BAPTA - DP-b99 and DP-109. Due to their chelating ability, these compounds require metal-free stationary phases, and their high hydrophobicity resulted in unusually steep gradient elution. Novel HPLC methods for analysis of these two compounds were developed. Purospher RP-C18, 5 microm, 125 x 3.0 mm and XTerra RP18, 3.5 microm, 100 x 4.6 mm columns with a steep gradient from: 1% acetic acid to acetonitrile were used for DP-b99, and Hypersil HyPurity C4, 5 microm, 100 x 4.6 mm column with a steep gradient from 1% Acetic acid to 5% THF in methanol -- for DP-109. Versatile detection techniques could be used with these LC procedures. The methods appeared to be sensitive, selective, reproducible and stability indicating. They could be easily upgraded to bioanalytical methods with LC-MS technique.

Evaluación del efecto del hipoclorito de sodio, acido cítrico y acido etileno diaminotetracético en la remoción del barro dentinario de los conductos tratatos endodónticamente / Evolução do efeito do hipoclorito de sódio, acido cítrico e acido etileno diaminotetracético na remoção do barro dentinário nos condutos tratados endodonticamente

La remoción del barro dentinario, que se forma por la instrumentación de los conductos radiculares, es importante para el aumento de la permeabilidad por limpiar los orificios de los túbulos dentinarios. En este estudio "in vitro" se utilizaron 90 dientes unirradiculares de humanos, distribuidos en 3 grupos de 30, instrumentados con limas K (15-40), teniendo el hipoclorito de sodio (NaOCl) al 1 per cent como solución irrigante. En el primer grupo, se utilizó únicamente el NaOCl; en el 2º y 3º grupo, además de la solución irrigante, se anãdieron respectivamente el ácido cítrico, al 50 per cent, y el ácido etileno diaminotetracético (EDTA) como soluciones auxiliares. La penetración del colorante Rodamina B en la masa dentinaria evidenció el aumento de la permeabilidad. Los resultados analizados permitieron concluir que la solución irrigante associada con la solución auxiliar de EDTA fue, entre las probadas, aquella que presentó mejores efectos tras comprobación estadística

Theory, design, and characterization of a microdialysis flow cell for Raman spectroscopy.

The theory, design, and application of a dialysis flow cell for Raman spectroscopy are described. The flow cell permits rapid collection of Raman spectra concurrent with the efflux of small solute molecules or ions into a solution of macromolecules and is well suited to acquisition of data during hydrogen-isotope exchange reactions of biological molecules. Kinetic parameters of the device are described by a diffusion model, which accounts satisfactorily for the observed rates of efflux of deuterium oxide (K2H = 0.30 min-1), calcium ions (KCa = 0.10 min-1) and EGTA (KEGTA = 0.07 min-1). Application to the kinetics of glutamate protonation in a peptide copolymer [poly(Glu, Lys, Tyr)] shows that pH-titration rates as high as 3.3 pH units/min can be monitored. It is also shown that one can extract first-order hydrogen-isotope exchange rate constants from measured second-order exchanges by taking into account the rate of entry of 2H2O effluent into the bulk H2O solution. Deuterium exchanges of the single-stranded polyribonucleotides poly(rA) and poly(rU) and of the double-stranded RNA genome from bacteriophage phi 6 have been investigated. The measured nucleotide base exchange rates are comparable with those determined previously by other methods. The results indicate that base exchanges as fast as approximately 2 min-1 can be determined reliably with the present design. Application of the Raman flow cell to hydrogen-isotope exchange of the basic pancreatic trypsin inhibitor confirms consistency with results obtained previously on this protein by tritiation and NMR techniques.

A simple method for the accurate determination of free [Ca] in Ca-EGTA solutions.

A simple method for the accurate determination of free [Ca] in ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA)-buffered Ca solutions is described. This method is useful for calibration of Ca macro- and microelectrodes to low free [Ca] and should improve the reliability of calculated free [Ca] in more complex solutions. Briefly, free [Ca] in Ca-EGTA solutions is measured with a Ca electrode, bound Ca is calculated, and Scatchard and double-reciprocal plots are resolved for the total [EGTA] and the apparent Ca-EGTA association constant (K'Ca) in the solutions used. The free [Ca] is then recalculated using the determined parameters, giving a more accurate knowledge of the free [Ca] in these solutions and providing an accurate calibration curve for the Ca electrode. These solutions can then be used to calibrate other Ca electrodes (e.g., Ca microelectrodes) or the calibrated Ca electrode can be used to measure free [Ca] in solutions containing multiple metal ligands. This method allows determination of free [Ca], K'Ca, and total [EGTA] in the actual solutions used regardless of pH, temperature, or ionic strength. It does not require accurate knowledge of K'Ca or EGTA purity and circumvents many potential errors due to assumption of binding parameters. K'Ca was found to be 2.45 +/- 0.04 X 10(6) M-1 in 100 mM KCl, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 1 mM EGTA at pH 7.00 and 23 degrees C. Total [EGTA] varied with supplier but was always less than quoted.