Ca-selective Electrode Research Articles

Application of Feedback-Based Flow Ratiometry to Simultaneous Determination of Calcium and Magnesium Ions by Chelatometry Using Photometric and Potentiometric Detectors

We advanced the principle of feedback-based flow ratiometry to stepwise titration using two detectors set in tandem in a flow system. The method was applied to the simultaneous determination of calcium and magnesium ions (Ca and Mg, respectively) by a chelatometry. The flow rate (FM) of titrand containing Ca and/or Mg was varied in response to controller output signals (Vc) having triangular wave profile, while the total (titrand + titrant + indicator solution) flow rate (FT) was held constant. Titrant solution (EDTA or EGTA, pH 10) was aspirated to the confluence point of the flow system at the flow rate of FT FM FI, where FI was a constant flow rate of indicator solution (EBT), and then mixed with the titrand. Downstream, the analytical signals of the mixed solution were monitored. First, a high-throughput photometric titration of a single analyte (Ca or Mg) was examined by a feedback-based flow ratiometry coupled with a subsequent fixed triangular wave controlled flow ratiometry. Maximally 17 titrations could be done per minute with reasonable precision (RSD = ca. 1 %). Next, the algorithm of the former ratiometry was modified so that a photometric detector and a potentiometric detector (Ca-selective electrode) could detect the total concentration of both ions and the concentration of Ca, respectively, using EGTA as a titrant. The equivalence points of the photometry and the potentiometry were each estimated from the Vc values at the instant of the sensing of the corresponding equivalence signal in the course of upward and downward scans of Vc. Good throughput (ca. 31 s per determination) was attainable with allowable precision (RSD < 1.6 %).

Stability of the Ion Pair Between Ca2+ and 2-(Hydroxymethyl)-3-Deoxy-D-erythro-Pentonate (α-Isosaccharinate)

Ion-pair formation between Ca2+ and α-isosaccharinate, Ca2+ + ISA-CaISA⇄+, was studied by two independent methods: an ion-exchange and a potentiometric method (Ca-selective electrode). The two methods gave similar values for the complexation constant, log K oCaISA+ at I = 0, (22 ± 1)°C. The ion-exchange method gave a value of log K CaISA+o = (1.8 ± 0.1) and the potentiometric method resulted in logK oCaISA+ = (1.78 ± 0.04). These values are in good agreement with the estimated value, log K oCaISA+ = 1.7, based on the formation of a Ca-gluconate ion pair.

Biomimetic Crystallization of Calcium Phosphates under Constant Conditions from the Revised SBF

Biomimetic Crystallization of Calcium Phosphates under Constant Conditions from the Revised SBF

Preparation of Phosphopeptides Derived from αs-Casein and β-Casein Using Immobilized Glutamic Acid-Specific Endopeptidase and Characterization of Their Calcium Binding

Phosphopeptides that were derived from αs-CN or β-CN were prepared with immobilized glutamic acid-specific endopeptidase, and their Ca2+ binding was characterized. αs-Casein or β-CN was hydrolyzed in a fluidized bed bioreactor containing 2ml of immobilized glutamic acid-specific endopeptidase by recirculating 20ml of αs-CN or β-CN solution (10 mg/ml in 50mM Tris·HCl and 0.02% NaN3, pH 8.0) for 3h at 20°C. The molecular masses of casein peptides were monitored by SDS-PAGE. Each hydrolysate was applied to an anion-exchange column using stepwise elution with various concentrations of KCl to separate peptides. The casein phosphopeptide content of the elution profile was monitored by analysis of protein and P concentrations. Calcium binding in phosphopeptide-enriched fractions was determined by CaCl2 titration and measurement of free Ca2+ with a Ca-selective electrode. The electrophoresis patterns showed four major peptides having molecular masses of 10.8, 9.0, 6.6, and 3.6 kDa in the αs-CN hydrolysate and 9.3, 8.2, and 6.2 kDa in the β-CN hydrolysate. The highest concentrations of P were detected in the fractions that eluted with 0.4 and 0.5M KCl for the αs-CN hydrolysate and with 0.4 M KCl for the β-CN hydrolysate. The calcium-binding ability was found only in the fraction that was eluted with 0.4 M KCl; the maximum Ca2+ binding and the apparent binding constant were 0.24 mmol/mg of protein and 75 M–1, and 0.14 mmol/mg of protein and 148 M–1, respectively. αs-Casein phosphopeptides had different patterns for Ca2+ binding than did β-CN phosphopeptides as the total Ca concentration was increased. Calcium binding to these casein phosphopeptides differed from that previously characterized for the tryptic peptides.

Calcium Binding of Phosphopeptides Derived from Hydrolysis of a αs-Casein or β-Casein Using Immobilized Trypsin

Calcium binding to casein phosphopeptides that were derived from αs-CN or β-CN was studied. Purified αs-CN or β-CN was prepared from fresh skim milk using an anion-exchange column. Peptides were prepared by casein hydrolysis using a fluidized bed bioreactor containing 2ml of immobilized trypsin (activity: 49.4 U/g of beads). The disappearance of intact protein and the appearance of products of low molecular mass were monitored by SDS-PAGE. αs-Casein and β-CN hydrolysates were loaded on an anion-exchange column, followed by stepwise elution with 0, 0.1, 0.2, 0.4, and 0.5 M KCl in equilibration buffer to separate the phosphopeptides from the other casein peptides. Protein and P were measured in the elution peaks. Calcium binding to each fraction was determined with a Ca-selective electrode. Electrophoresis showed that intact proteins were hydrolyzed rapidly, and peptides appeared on the gel in greater concentrations as the incubation time increased. The major products were a main band with a molecular mass of 6.2 kDa from β-CN hydrolysates and a series of bands from 4.0 to 12.8 kDa from a αs-CN hydrolysate. The greatest yield and concentration of phosphate from β-CN hydrolysate were found in the peak that eluted with 0.4 M KCl in equilibration buffer and for αs-CN in the peak that eluted with 0.1 M KCl. The αs-CN phosphopeptides showed greater Ca2+ binding than the phosphopeptides from β-CN. Separation of casein phosphopeptides using anion exchange was not specific. However, results showed that each peak containing high concentrations of phosphate had Ca2+-binding ability. Further characterization of these casein phosphopeptides might result in a Ca-complexing food ingredient.

Use of a Ca-selective electrode in the determination of total Ca in the production of crude sugar

The determination of the total calcium in juice, syrups, and other products of the sugar industry is investigated. Total calcium and free calcium is determinated by AAS and employing Ca-selective electrode respectively. A coefficient is obtained for the relation of total calcium with respect to free calcium. The coefficient is employed to determine the content of total calcium in accordance with the following equation. Relative error is less than 5 %.

Use of a Ca-selective electrode in the determination of total Ca in the production of crude sugar. Preliminary studies

The determination of the total calcium in juice, syrups, and other products of the sugar industry is investigated. Total calcium and free calcium is determinated by AAS and employing Ca-selective electrode respectively. A coefficient is obtained for the relation of total calcium with respect to free calcium. The coefficient is employed to determine the content of total calcium in accordance with the following equation.&lt;img src="http:/img/fbpe/eq/v22/15e.gif" alt="15e.gif (673 bytes)" align="middle"&gt;

A new, long-lived Ca-selective electrode

The preparation and performance of a new Ca-selective electrode based on a Nafon ® membrane modified by means of the new ionic carrier N-ethyl- N-(2-trimethylammoniumethyl)- N′-heptyl- N′-methyl-succinamide are described. This electrode is characterized by a wide linear range extending down to pCa⩾5, acceptable selectivity towards alkaline-metal cations, a nearly equal response to Mg 2+ ion (which makes it suitable for water hardness determinations) and an extremely long in-service lifetime.

Recording maximal Ca-ATPase activity in intact human erythrocytes by means of a Ca-selective electrode

Recording maximal Ca-ATPase activity in intact human erythrocytes by means of a Ca-selective electrode

Indo-1 binding to protein in permeabilized ventricular myocytes alters its spectral and Ca binding properties

We have examined the binding of the fluorescent Ca indicator indo-1 to cellular protein in permeabilized ventricular myocytes and also to soluble and particulate myocyte protein. Using either a filtration technique or equilibrium dialysis, and conditions similar to those in a cardiac myocyte patch clamped with 100 microM indo-1 in the patch pipette, we found that 72% of the total indo-1 was bound to myocyte protein at a protein concentration of 100 mg/ml. This corresponds to a binding of 3.8 +/- 0.5 nmol indo-1/mg protein. Separation of the myocyte protein into a soluble and a particulate fraction showed that 63% of the bound indo-1 was bound to soluble protein, corresponding to a binding of 3.22 +/- 0.99 nmol/mg, whereas 37% of the bound indo-1 was bound to particulate protein (0.85 +/- 0.14 nmol/mg) at a low [Ca] (pCa approximately 9). Binding of indo-1 in permeabilized myocytes was approximately 60% higher at a saturating Ca concentration (pCa = 3), than under Ca free conditions (1 mM EGTA). Simultaneous measurements of free [Ca] with a Ca selective electrode and indo-1 fluorescence showed that, the dissociation constant (Kd) for Ca was increased 4-5 fold in the presence of permeabilized myocytes as compared to the value obtained in vitro. In agreement with the binding experiments we estimate that the true Kd and the apparent Kd (using ratiometric measurements) for Ca binding to indo-1 are increased approximately four fold, at a myocyte protein concentration of 100 mg/ml.

Precipitation and Solubility of Calcium Hydrogenurate Hexahydrate.

Solid phases formed in the quaternary system: uric acid—calcium hydroxide —hydrochloric acid—water aged for 2 months at 310 K were studied to determine conditions for calcium hydrogenurate hexahydrate, Ca(C5H3N4O)2 · 6H2O precipitation. The precipitates were identified by chemical and thermogravimetric analyses, x-ray powder diffraction, infrared spectroscopy, light microscopy, and scanning electron microscopy. In the precipitation diagram the concentration region in which calcium hydrogenurate hexahydrate precipitated as a single solid phase was established.The solubility of calcium hydrogenurate hexahydrate was investigated in the pH range from 6.2 to 10.1 at different temperatures. The total soluble and ionic concentration of calcium (atomic absorption spectroscopy and Ca-selective electrode), total urate concentration (spectrophotometry), and pH were determined in equilibrated solutions. The data are presented in the form of tables and chemical potential diagrams. By using these data the thermodynamic solubility products of calcium hydrogenurate hexahydrate, Ks = a(Ca2+) · a2(C5H3N4O3−), were determined: pKs=10.12±0.07at288K,pKs=9.81±0.09at298K,pKs=9.28±0.04at310K,andpKs=9.01±0.03at318K.The formation of calcium hydrogenurate hexahydrate crystals in urinary tract of patients with pathologically high concentrations of calcium and urates (hypercalciuria and hyperuricosiuria) is possible.

Tryptic phosphopeptides from whole casein. II. Physicochemical properties related to the solubilization of calcium.

Casein phosphopeptides (CPP) were produced by tryptic hydrolysis of sodium caseinate and further purified by precipitation and chromatography on QAE-Sephadex A-25. Their physico-chemical properties were compared with the properties of an enzymically dephosphorylated equivalent preparation (DPP). Binding of Ca2+ to the peptides was measured using a Ca selective electrode and was found to increase with pH and to show 1/1 stoicheiometry Ca/Porg in CPP at pH 6.5 and 7.6. Klotz plots indicated equivalent binding sites at these two pH values, but some heterogeneity was seen at pH 3.5. In contrast, DPP did not bind significant amounts of Ca2+. CPP effectively inhibited the formation of insoluble calcium phosphates at different Ca/P ratios. The effective CPP concentration was 10 mg/l and complete stability of calcium phosphate solutions was obtained at about 100 mg/l. This stabilizing effect was dependent on the presence of organic P.

Charakterisierung und Quantifizierung von Mineralstoffbindungen in Lebensmitteln

Ion-exchange on Chelex-100, donnan dialysis and the method of ion-retardation are applied to characterize the bonding strengths of the minerals Ca, Mg and Zn in milk and extracts of meat and flour. Another ion-exchange procedure and direkt potentiometry with an Ca-selective electrode are applicate for the quantification of weak bindings. By use of the above mentioned methods it is possible to calculate element species stabilities, which represent characteristic values. Both methods give comparable results for milk, extracts of meat and spinach, grape-juice, red and white wine. Detailed examinations of binding conditions in milk proved citrate to the strength binding compound for Ca and Mg. Model-examinations with citrate, caseine and phosphate agree well with calculations and measurements in milk. Furthermore differences in the binding of Ca and Mg in raw and cream spinach are examined. The above mentioned methods are suitable to characterize and quantify the binding of elements in foodstuffs. The results give informations about chemical changes due to technological processes e.g., in regard to storage life.

Ability of the Ca2+ -selective microelectrodes to measure fast and local Ca2+ transients in nerve cells

The ability of the Ca2+-selective microelectrode to measure fast Ca2+ transients intracellularly is reviewed. In vitro, Ca microelectrodes can respond to Ca2+ injections with time to peaks as small as 40 ms. We present methods to improve the dynamic response of Ca microelectrodes and to make Ca-buffered solutions in high ionic strength. Examples of measurements of intracellular free Ca2+ [( Ca2+]i) transients in Aplysia neurons and in Limulus photoreceptors are shown. To show the validity of those measurements, simultaneous recordings of the Arsenazo III (AIII) absorbance and of the Ca-selective electrode potential were made in voltage-clamped neurons of the abdominal ganglion of Aplysia californica. Pressure injection of AIII to a concentration of 300-500 microM induced a rise in resting [Ca2+]i; injection of higher [AIII] led to buffering of [Ca2+]i transients. Both techniques responded to changes in resting [Ca2+]i in the same direction except that AIII showed an increase in absorbance in 0 [Ca2+]o. Voltage-clamp pulses transiently increased both the AIII absorbance and the Ca2+ electrode potential. Reducing or increasing the driving force for Ca2+ entry changed the magnitude of both signals in the right direction. Examples of spatial localization of [Ca2+]i increases and Ca2+ gradients within the cytoplasm were demonstrated using the Ca electrode. The use of optical techniques to measure local [Ca2+]i changes is briefly reviewed.

Intracellular localization of the caffeinesensitive form of Ca++-dependent ATPase of the sarcoplasmic reticulum

A Ca-selective electrode was used to study the effect of caffeine on different fractions of sarcoplasmic reticulum membranes of rabbit skeletal muscles. Caffeine was found to uncouple Ca2+ transport and ATP hydrolysis in a fraction, which is enriched with fragments of terminal cisterns according to the electron microscopy data. Caffeine does not produce any effect on the light fraction containing no fragments of terminal cisterns. It is concluded that caffeine-sensitive Ca2+-dependent ATPase is localized in terminal cisterns of the sarcoplasmic reticulum.

Calcium binding by lithocholic acid derivatives.

Lithocholic acid (LCA) and its sulfate (LCS) and glucuronide (LCG) derivatives are potent cholestatic agents. During the course of LCG-induced cholestasis in rats, calcium (Ca) salts of LCG precipitate in bile. To characterize the affinity of bile salts for Ca, solutions of selected bile salts were titrated with Ca. Apparent equilibrium constants (KcaBS) were determined from the unbound Ca ion concentrations that were measured spectrophotometrically with metallochromic indicators antipyrylazo III or murexide or with a Ca-selective electrode. KCaBS values were 1.12 +/- 0.04 X 10(-4) M for LCS, 2.88 +/- 0.26 X 10(-4) M for LCG, 3.09 +/- 0.21 X 10(-4) M for LCA, 1.93 +/- 0.07 X 10(-3) M for taurocholic acid (TC), 2.69 +/- 0.08 X 10(-3) M for glycocholic acid (GC), and 6.07 +/- 0.27 X 10(-3) M for taurolithocholic acid sulfate (TLCS). The KCaBS for LCG measured by a Ca-selective electrode under identical conditions was 5.53 +/- 2.75 X 10(-4) M. Comparing relative cholestatic potential with affinity for Ca, cholestatic bile salts LCS, LCG, and LCA bind Ca 10-60 times more avidly than TC, GC, and TLCS. At the unbound Ca ion concentrations of serum or bile (approx 1 mM), only LCS, LCG, and LCA would be expected to bind significant amounts of Ca.

Ion-selective Electrode Determination of Calcium in Apple Fruits1

Abstract A rapid method for estimating total Ca of apple (Malus domestica Borkh.) peel and flesh was developed using a Ca-selective electrode to measure ionic juice Ca++. Ionic juice Ca++ ranged from 3.2 to 7.2 ppm. Significant correlations were found between total Ca and ionic juice Ca++ for flesh (r = 0.91) and peel (r = 0.87).

The calcium selective electrode--a possible tool in dental research.

Ion selective potentiometry is widely used for analytical purposes in biologic research. The Ca selective electrode offers an inexpensive and easily handled means for studying ionized calcium. It does not affect the process studied, and may, for example, combined with an automatic recorder, give running information on dynamic systems. The calcium electrode responds in accordance with the Nernst equation and has a theoretical range of 1--10(-6) M Ca2+. The electrode is influenced by changes in temperature, in pH and also in concentration of buffer solution. The Ca sensitive membrane is adversely affected by acetic acid but tolerates lactate/lactic acid buffers. Best linearity in calibration curves for Ca2+ in lactate buffers is found in concentrations from 10(-4) M to 10(-2) M Ca. In a 5% Na lactate/lactic acid buffer, pH 5, with 0.5 M KNO3 as an ionic strength adjuster, a straight calibration curve, Nernstian slope factor 27 mV/decade, is found from 10(-4) to 1 M Ca.

Determination of free Ca ion concentrations with an ion-selective electrode in the presence of chelating agents in comparison with calculated values

Experimentally determined free Ca ion concentrations, measured with a Ca-selective electrode, were compared with values calculated with a computer program utilizing stability constants of the chelating agents: NTA, EDTA, and EGTA used to set the free ion concentration in the range of 10 −3 to 10 −6 m. In the presence of 0.1 m KCl, 2 m m MgCl 2, 20 m m Hepes (pH 7.4), 2 m m ATP, 0.1 m m CaCl 2 (total concentration), and various ligand concentrations the measured free Ca 2+ levels were found to be approximately six to seven times greater than the computer-derived values. Apparent stability constants for Ca-ATP, Ca-EDTA, and Ca-EGTA were determined under these experimental conditions.