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Ion-selective electrodes and enzyme electrodes in environmental and clinical studies.

Electrodes have been developed for the assay of glucose, urea, amino acids, uric acid, phosphate, nitrate and perchlorate. The electrodes for the organic compounds are enzyme electrodes which are prepared by chemically immobilizing an enzyme over the outside of a conventional ion-selective electrode. These electrodes will be discussed in depth. The progress and the development of the electrodes that show sensitivity and selectivity for phosphate, nitrate and perchlorate will be outlined. The basis of these sensors is a complex of a transition metal of either an analog of thiourea or an organic chelator, such as 1,10-phenanthraline. Such electrodes respond linearly to phosphate, nitrate or perchlorate, and show selectivity over sulphate, halides and acetate. The linear range of all these electrodes is approx. 10(-1)-10(-5) M with a near Nernstian slope and a reproducibility of 1%. The electrodes are stable and can be used continuously.

Amidohydrolases

Activity and interference effects in measurement of ionized calcium with ion-selective electrodes.

We assessed the magnitude of the errors in ionized-calcium measurements resulting from changes in electrolyte composition for three different ion-selective electrodes: the Orion Model 93-20, the Radiometer F2112, and the Simon neutral carrier membrane electrode. We attempted to distinguish between errors arising from changes in calcium ion activity and those due to interferences in the electrode response. Variation in sodium ion concentration over the range 100--180 mmol/liter produces changes in apparent ionized-calcium concentration that are largely attributable to activity effects for the Radiometer and Simon electrodes. The Orion electrode is subject to an additional sodium-interference effect. Apparent ionized calcium concentration measurements are independent of pH for the Radiometer electrode but not for the Orion electrode; the Simon electrode exhibits intermediate pH response, which is probably clinically negligible. Magnesium and potassium ions have little effect on ionized calcium concentration measurements, particularly when these ions are incorporated into calibration standards.

Calcium

Determination of total carbon dioxide in serum and plasma using a carbonate ion-selective membrane electrode.

A new manual method for the determination of carbon dioxide content in serum and plasma, based on the potentiometric measurement of carbonate, is proposed and evaluated. The new method offers good precision and accuracy in the physiological concentration range as shown by detailed studies on synthetic samples, reference standards, and actual patient samples. Moreover, the method yields results which correlate very well with analyses carried out using various conventional techniques. It is expected that the proposed method could be used for manual determinations of CO2 content, especially where rapid start-up time, minimal sample treatment, and low cost are of importance.

Carbon Dioxide

Continuous-flow analysis of ammonia in perchloric acid supernate of blood or plasma using an ammonia-selective electrode.

A simple automated method for the estimation of ammonia in perchloric acid supernate of blood or plasma using an ion-selective electrode (Orion Ammonia-selective electrode, Model 95-10) is described. The reliability of the proposed method has been checked against an ion-exchange resin procedure, which has been chosen as a standard procedure. Regression equation and correlation coefficient for the proposed method are y = 0.7 X + 10 and 0.945, respectively, as compared with the chosen standard method. Within-run and between-run precision are 2.1% and 3.5% respectively. The average percent recovery is 97.5% and a tentative range is 13-73 microgram/dl (9-52 micronmol/l) ammonia nitrogen.

Adult

Interaction of amino acids with silver(I) ions.

The reactivity of silver(I) ions towards twenty amino acids has been studied in aqueous unbuffered solutions using an ion-selective electrode as a highly sensitive monitor. Contrary to general belief, silver ions are not completely specific for cysteine, but also react with lysine, arginine, methionine and, to a minor extent, cystine. Nevertheless the interaction with all of these species except cysteine is too weak to affect significantly the determination of thiol in proteins by the customary argentometric titration method. The possible origin of errors arising in such titrations is discussed.

Amino Acids

Measurement of ionised calcium in body fluids-a review.

This paper reviews the techniques available to the clinical biochemist for measuring ionised calcium concentrations in biological fluids with particular reference to serum. At present ionised calcium may be measured colorimetrically, using tetramethyl murexide, or potentiometrically, using a calcium ion-selective electrode. These techniques compare favourably in terms of precision with existing methods for measuring total calcium. Advantages of measuring ionised calcium in preference to total calcium are (a) that there is no effect of venous occlusion or change of posture on the ionised fraction of the effect on total calcium, (b) that ionised calcium is the physiologically active form of the element, and (c) that the ionised calcium concentration is a more reliable indicator of the calcium status of patients in certain clinical conditions. The main problem in the measurement of ionised calcium is the marked dependence of the ionised fraction of the pH of the sample. Extreme care must be taken to avoid loss of CO2 or build-up of acid during the handling of the blood sample.

Blood Specimen Collection

Calcium measurements in serum and plasma--total and ionized.

This article will review the methods currently employed for measuring the concentrations of total and ionized calcium in serum or plasma. As far as total calcium is concerned, various techniques such as atomic absorption spectrometry, spectrophotometry, fluorometry, complexometric titration, and flame photometry will be described and compared. Particular emphasis will be given to the accuracy and precision of each technique. Possible sources of error and interfering agents will be identified and the various procedures for the taking and handling of blood samples evaluated. Inter-laboratory variation in the measurement of calcium will be studied. An assessment will be made of a new reference method for measuring total calcium in serum using isotope-dilution mass spectrometry. The usefulness of the total calcium measurement in clinical medicine will be briefly discussed. Within the last decade the refinement of spectrophotometric techniques and the improvements in ion-selective electrode technology have revolutionized the measurement of ionized calcium in serum, such that it may now be possible to replace total calcium measurements with ionized calcium measurements on a routine basis. The various techniques currently in use for measuring ionized calcium will be described and evaluated. Particular attention will be paid to the preparation of standards, the procedures for taking blood samples, and the handling of the samples prior to and during measurement. An assessment of the relative value of measuring total and ionized calcium will be presented.

Calcium

[Coupled transport of K+, Cl-, H+ and OH- ions in a liposome suspension].

By means of the system of ion-selective electrodes the coupling transport of K+, H+ and Cl- ions in liposome suspension was studied. Nigericin and tributyl tin (TBT) were used as inductors of cation and anion exchange. Effect of pH gradient created on the membrane on ionic transport was studied. The latter was shown to control the antiport exchange of both, cations and anions. At the joint effect of nigericin and TBT the yield of K+ and Cl- ions from liposomes independent of the pH gradient was observed.

Biological Transport

Measurement of free calcium ion in capillary blood and serum.

We describe a new calcium ion-selective electrode for measurement of the substance concentration of free calcium ion [Ca2+] in the plasma phase of whole blood and in serum at 37 degrees C. A sample volume of 50 microliter suffices to obtain simultaneous values of pH and [Ca2+]. We found the within-series analytical standard deviation for serum to be 0.013 mmol/litre (CV, 1.1%) and day-to-day precision to be 0.022 mmol/litre (CV, 1.7%). The reference interval for [Ca2+] (at pH 7.40) in serum was found to be 1.184 +/- 0.054 mmol/litre (2 SD) from measurements on sera from 121 healthy blood donors. Measurements on capillary blood from 29 healthy volunteers gave a mean (+/- 2 SD) value for [Ca2+] (at pH 7.40) of 1.22 +/- 0.072 mmol/litre.

Bicarbonates

Differential effects of cortical speading depression on epileptic foci induced by various convulsants.

Interaction between epileptic foci and spreading depression (SD) was studied in the cerebral cortex of rats anesthetized with Nembutal. At comparable discharge rates, picrotoxin and penicillin caused complete and partial SD blockade respectively, strychnine and Metrazol were ineffective and Aldactone facilitated SD. Conversely, the duration of SD-induced blockade of epileptic activity was maximal for Aldactone and minimal for picrotoxin. Treatment of the picrotoxin focus with tetrodotoxin (10(-4)M) reduced the discharge rate and reinstated SD propagation into the focus. [K+]e measured with ion-selective K+ electrodes 1 mm below the cortical surface increased to 8 mM in penicillin foci blocking SD and remained below 5 mM in Aldactone foci. It is concluded that the differential effect of various convulsants on SD propagation depends on the potassium concentration in the depth of the focus rather than on the discharge rate or on the mechanism of the the epileptogenic effect.

Animals

Physicochemical properties of a liquid ion exchanger microelectrode and its application to biological fluids.

Double-barreled potassium or chloride ion-selective microelectrodes were constructed using a liquid ion exchanger. Technical details of the fabrication of double-barreled microelectrodes, having a PD sensor as one barrel and an ionic sensor as the other, are described. The sensitivity of K+ or CI- ion-selective microelectrodes exhibited an approximately Nernstian response over a temperature range of 7 degrees-37 degrees C, and the electromotive force (EMF) was stable within +/- mV for a few hours. The rise time was less than 1 sec. The effect of pH on the electrode response was negligible over a physiological range of pH 5.6 to 7.8. The selectivity constants of the K+ microelectrode to other cations were 0.011 for Na+, 0.200 for NH+4, and less than 0.002 and 0.001 for Ca++ and Mg++, respectively, while that of the CI- microelectrode was 0.067 for HCO-3. Glucose or urea has no effect on the EMF. Protein has a significant effect on ion exchanger membrane only when the concentration of the tested ion is low and protein is high. On the basis of this background the determination of K+ and CI- activity was carried out both in vivo and in vitro on several biological samples, such as serum, tissue and cellular fluids, and other protein-containing fluids. The values obtained with the microelectrode were consistent with those obtained with the other conventional methods or with the current theory on electrolyte solutions. These results were taken to assure the practical application of these electrodes to biological studies in many fields.

Animals