Measurement of ionized calcium in blood platelets with a new generation calcium indicator

Fura 2, a new generation calcium indicator, has a 30 fold brighter fluorescence than Quin 2, shows wavelength shifts upon calcium binding and has a relatively low buffering capacity for free calcium. Quin 2, the most widely used fluorophore, on the other hand, shows no wavelength shifts and has a very high affinity for free calcium. Therefore, we have compared the relative merits of these two fluorophores for monitoring agonist induced alterations in platelet cytosolic calcium. Platelets loaded with Fura 2 showed a significant rise in cytosolic calcium when stirred with agonists such as epinephrine, arachidonate and thrombin, whereas Quin 2 loaded platelets demonstrated a rise in cytosolic calcium only with thrombin stimulation. A rise in agonist induced calcium in Fura 2 loaded platelets was prevented when the cells were exposed first to antagonists such as aspirin or prostaglandin E1. Arachidonate refractory platelets, upon stirring with a single agonist, did not show a significant elevation in cytosolic calcium. However, when refractory platelets were first exposed to epinephrine and then challenged with arachidonate, they revealed a significant elevation in cytosolic calcium. Unlike Quin 2, Fura 2 at the highest concentration tested did not inhibit platelet function. Improved properties of Fura 2 suggest that it may be a useful agent to study agonist induced alterations in cytosolic calcium levels in blood platelets.     

Measurement of ionized calcium in normal human blood platelets

To measure the concentrations of cytosolic ionized calcium in platelets we used a calcium-sensitive fluorophor (Fura-2) with two different spectrofluorometers (Perkin-Elmer LS-5 and Fluorolog 222). Values obtained by these two instruments for the basal cytosolic ionized calcium concentration of resting platelets and those of agonist-activated platelets did not differ significantly. Both instruments were capable of monitoring the shifts in wavelengths induced by the dye-calcium complex, the ratio between absorbances at the two wavelengths (340/380 nm), and calcium concentrations continuously during agonist-induced platelet activation. We conclude that a relatively inexpensive instrument may be adequate for measuring ionized calcium in cells by this method, although sophisticated kinetic studies may require analytical or research-grade instruments.     

Microelectrode of the Thomas type using a liquid membrane electrode

By replacing the glass-based pH electrode (L. R. Pucacco, S. K. Corona, H. R. Jacobson, and N. W. Carter (1986) Anal. Biochem. 153, 251-261) with a liquid membrane-based pH electrode, a relatively easy-to-manufacture modified Thomas electrode has been developed. The liquid membrane-based modified Thomas electrode can be manufactured without the special equipment (forge) and materials (glass) required to make the glass membrane pH microelectrode (L. R. Pucacco and N. W. Carter (1976) Anal. Biochem. 73, 501-512). The sensitivity (57.4 +/- 0.22 mV/pH unit), response time (20.0 +/- 2.67 s), and electrical resistance (3.48 +/- 0.67 X 10(11) ohm) of this electrode are similar to those of the glass-based version.     

Measurement of ionized calcium in blood platelets with the photoprotein aequorin. Comparison with Quin 2

The Ca2+-sensitive photoprotein aequorin (Mr = 20,000) was introduced into human blood platelets by incubation with 10 mM EGTA and 5 mM ATP. Platelet cytoplasmic and granule contents were retained during the loading procedure, and platelet morphology, aggregation, and secretion in response to agonists were normal after aequorin loading. Luminescence indicated an apparent resting cytoplasmic ionized calcium concentration [( Cai2+]) of 2-4 microM in media containing 1 mM Ca2+ and of 0.8-2 microM in 2-4 mM EGTA. The Ca2+ ionophore A23187 and the enzyme thrombin produced dose-related luminescent signals in both Ca2+-containing and EGTA-containing media. Peak [Cai2+] after A23187 or thrombin stimulation of aequorin-loaded platelets was 2-10 microM, while peak [Cai2+] determined using Quin 2 as the [Cai2+] indicator was at least 1 log unit lower. In platelets loaded with both aequorin and Quin 2, the aequorin signal was delayed but not reduced in amplitude. Aequorin loading of Quin 2-loaded cells had no effect on the Quin 2 signal. Ca2+ buffering by Quin 2 (intracellular concentration greater than 1 mM) is also supported by a reciprocal relationship between [Quin 2] and peak [Cai2+] stimulated by A23187 in the presence of EGTA. Parallel experiments with Quin 2 and aequorin may identify inhomogeneous [Cai2+] in platelets and give a more complete picture of platelet Ca2+ homeostasis than either indicator alone.     

Electrolytic regeneration of NADH from NAD with a liquid crystal membrane electrode

Enzymatically active NADH was electrolytically regenerated from its oxidized from (NAD⁺) with a liquid crystal membrane electrode made by coating a hanging drop or a pool type of mercury electrode with a cholesteryl oleate membrane which could be in the liquid crystal state. A cholesteryl oleate membrane was employed to prevent dimerization of the NAD radical supposed to be an intermediate of electrolytic reduction of NAD⁺.     

Low plasma concentrations of ionized calcium in patients with asthma

It has been suggested that calcium homeostasis is abnormal in the vascular smooth muscle of hypertensive patients and in the bronchial smooth muscle in asthmatics. We have found the mean baseline concentration of plasma ionized calcium to be significantly lower both in 12 asthmatics with exercise-induced asthma (EIA) [1.16 +/- 0.01 (SE) mmol/l, P less than 0.001] and in 20 asthmatics without EIA (1.16 +/- 0.01; P less than 0.001) compared with 42 healthy subjects (1.24 +/- 0.01). The mean concentrations of plasma ionized calcium were not significantly different in asthmatics with and without EIA when measured either before treadmill exercise, during the last seconds of this exercise, or 10 or 20 min after exercise but were significantly lower than in another seven healthy subjects who undertook the same exercise protocol. Total plasma calcium concentrations in the three exercising groups were not significantly different at any point in time. The results suggest that in bronchial asthma an alteration of calcium metabolism may be important, but they also suggest that there is no simple relationship between the plasma ionized calcium concentration and acute exercise-induced bronchoconstriction.     

Determination of diprivan in urine by a supported liquid membrane technique and liquid chromatography-electrochemical detection

A supported liquid membrane technique was used for the extraction and enrichment of propofol in a spiked sample of urine. An acidic solution of propofol and thymol as an internal standard was passed over the membrane and after enrichment the acceptor solution was analyzed by LC with an electrochemical detector. The acceptor and donor pH, flow-rate, and volume of donor and different membrane solvents were varied to optimize the extraction efficiency. The detection limit for 100 ml of a spiked urine sample was 10 ppt of propofol.     

Determination of lignans in human plasma by liquid chromatography with coulometric electrode array detection

The presence of mammalian lignans, mainly enterolactone, in human plasma has been related to lower incidence of certain cancers and cardiovascular disease. The plant lignans secoisolariciresinol and matairesinol have been reported to be precursors of mammalian lignans, but recently other plant lignans relatively abundant in the diet have also been identified as precursors. To evaluate the importance and contribution of these new dietary precursors to the mammalian lignan formation in vivo, metabolic studies in human subjects must be carried out. For this purpose a method based on high-performance liquid chromatography using coulometric electrode array detection for the simultaneous determination of nine plant lignans and two mammalian lignans in human plasma was developed, validated, and shown to fulfill the reliability criteria.     

Determination of urate in undiluted whole blood by enzyme electrode

An amperometric enzyme electrode is described for the assay of urate in undiluted, unstirred whole blood. The electrode used Aspergillus flavus uricase (EC.1.7.3.3) cross-linked to bovine serum albumin by means of glutaraldehyde, sandwiched between a dimethyldichlorosilane-treated microporous polycarbonate membrane and an inner cellulosic H2O2-selective membrane. The resulting device had a low pH dependence, was capable of repeated use in blood, and gave an acceptable correlation with a standard spectrophotometric method. Electrode steady state and dynamic response were found to be dependent upon the amount of enzyme loading, and could be further optimised by the incorporation of catalase in the enzyme layer.     

Determination of the intravesicular ionized sodium concentration in a cell-free brain membrane vesicle preparation using the fluorescent indicator, SBFI.

The intravesicular ionized Na concentration (Nai) was measured using the fluorescent Na indicator, SBFI, in microsacs, a cell-free brain vesicle preparation. SBFI fluorescence was monitored by a dual excitation-wavelength method at the same wavelengths commonly employed for Fura-2 determination of intracellular ionized calcium concentrations (Cai). Calibration of SBFI fluorescence was reliably performed in brain microsacs in situ. Resting Nai was dependent on the extravesicular Na concentration (Nao) and was about 36 mM in the presence of 120 mM extracellular Nao. In the presence of ouabain, an inhibitor of the plasma membrane Na/K-ATPase, Nai increased by 27 mM over 60 s. Nai was also increased by resuspension of microsacs in buffers of low free Ca concentrations (0 to 0.8 mM), indicating that the extravesicular Ca concentration (Cao) is an important regulator of Nai. Alkaloids active at voltage-sensitive Na channels, veratridine and aconitine, also increased Nai. These results demonstrate the presence of homeostatic mechanisms for neuronal Nai regulation and show that Nai can be measured in a cell-free brain vesicle preparation using SBFI.     

The control of ionized calcium in squid axons

Measurements of the Ca content, [Ca](T), of freshly isolated squid axons show a value of 60 μmol/kg axoplasm. Axons in 3 mM Ca(Na) seawater show little change in Ca content over 4 h, while axons in 3 mM Ca(Na) seawater show little change in Ca content over 4 h, while axons in 10 mM Ca(Na) seawater show gains of 18 μmol/Ca/kgxh. In 10 Ca (Choline) seawater the gain is 2,400 μmol/kgxh. Using aequorin confined to a dialysis capillary in the center of an axon, one finds that [Ca](i) is in a steady state with 3 Ca (Na) seawater, and that both 10 Ca (Na) and 3 Ca (choline) seawater cause increases in [Ca](i). In 3 Ca (Na) seawater-3 Ca (choline) seawater mixtures, 180 mM [Na](0) (40 perecent Na) is as effective as 450 mM [Na](0) (100 percent Na) in maintaining a normal [Ca](1); lower [Na] causes an increase in [Ca](i). If axons are injected with the ATP-splitting enzyme apyrase, the resulting [Ca](1) is not loading with high [Ca](0) or low [Na](0) solutions. Depolarization of an axon with 100 mM K (Na) seawater leads to an increase in the steady-state level of [Ca](1) that is reversed upon returning the axon to normal seawater. Freshly isolated axons treated with either CN or FCCP to inhibit mitochondrial Ca buffering can still maintain a normal [Ca](i) in 1 Ca (Na) seawater.     

Detection of extracellular calcium gradients with a calcium-specific vibrating electrode

We have developed a vibrating calcium-specific electrode to measure minute extracellular calcium gradients and thus infer the patterns of calcium currents that cross the surface of various cells and tissues. Low-resistance calcium electrodes (routinely approximately 500 M omega) are vibrated by means of orthogonally stacked piezoelectrical pushers, driven by a damped square wave at an optimal frequency of 0.5 Hz. Phase- sensitive detection of the electrode signal is performed with either analogue or digital electronics. The resulting data are superimposed on a video image of the preparation that is being measured. Depending on the background calcium concentration, this new device can readily and reliably measure steady extracellular differences of calcium concentration which are as small as 0.01% with spatial and temporal resolutions of a few microns and a few seconds, respectively. The digital version can attain a noise level of less than 1 microV. In exploratory studies, we have used this device to map and measure the patterns of calcium currents that cross the surface of growing fucoid eggs and tobacco pollen, moving amebae and Dictyostelium slugs, recently fertilized ascidian eggs, as well as nurse cells of Sarcophaga follicles. This approach should be easily extendable to other specific ion currents.     

Development of a multiple-bile-ion-sensing membrane electrode

A multiple-bile-ion-sensing polyvinyl chloride-based membrane electrode capable of monitoring any of the three common bile ions in humans, namely, cholate, deoxycholate, and chenodeoxycholate, was developed and characterized. Compared to single-bile-ion-sensing electrodes, it showed a sub-Nernstian response. All other electrode properties were, however, similar, making this a successful replacement for three individual electrodes. With appropriate conditioning, this electrode could repeatedly change selectivity without losing membrane activity. It was reproducible, was stable for 5 months, had low response time, and could be used to measure critical micelle concentrations. The lower limit of detection was 10 nM. Selectivity coefficients for various anions with respect to bile ions more or less followed the Hoffmeister series. Plots of R ((Nernst equivalent of slope in the presence of primary ion and a fixed amount of interfering ion)/(slope in the presence of only the primary ion)) vs square root of ionic strength for an interfering ion were linear. One major application of this electrode is its use in kinetics. We have tested its ability to monitor continuously changing bile ion concentrations during their interactions with a biocompatible polymer, polyethylene glycol (6000), and determined rate constants.