Evaluation of mag-fura-5, the new fluorescent indicator for free magnesium measurements

Abstract

The new fluorescent indicator, mag-fura-5, was evaluated for its ability to measure accurately physiological changes in cytosolic free magnesium. The apparent dissociation constants (Kd) of the fluorochrome for Mg2+, Mg2+/EGTA and Ca2+/EGTA solutions were 14.7 mM, 15.4 mM, and 1.8 mM respectively. The calculated difference in the fluorescence ratios and in the resulting pMg between the standards with low-Ca2+ or low H+ backgrounds and the corresponding samples with approximately physiological levels were not significant. In contrast, the changes due to an increased Ca2+ or H+ content were statistically significant, with mean pMg differences of 0.10 +/- 0.09 (P < 0.02) and 0.33 +/- 0.26 (P < 0.01) respectively. Repetitive measurements on 3 consecutive days yielded comparable data with differences not exceeding 4%. Because of the good reproducibility, it is suggested that the new fluorescent probe may be suitable for free cytosolic magnesium determinations in isolated cells.

Magnesium: The Fifth but Forgotten Electrolyte

“…66 Other fluorescent probes for ionized magnesium have been described. 67 ' 68 London has reviewed the use of fluorescent probes, mainly mag fura-2, for the determination of intracellular ionized magnesium. 69 At present, this technique is limited to the research laboratory, but may have a future place in the clinical laboratory.…”

Magnesium Measurement in Routine Clinical Practice

“…The methodology of measurement of intracellular free magnesium has been reviewed by Alvarez-Leefmans et al, and interested readers are referred to their review.V A wide variety of fluorescent probes which are sensitive to chelation state are suitable for the measurement of free magnesium inside living cells in both the stimulated and unstimulated state, but none has been used in clinical applications to date." 60,62,63 Because of its propensity to form ATP complexes, magnesium measurement has proved a suitable application of 31p magnetic resonance spectroscopy, which has the additional potential benefit of being capable of measuring equilibrium state information i/1 vivD. 6I.…”

Polycarboxylate Fluorescent Indicators as Ion Concentration Probes in Biological Systems

“…(4). Two more stilbene-type indicators, Stil-1 and Stil-2 mentioned in the same article, that mentioned Fura-2 and Indo-1, were considered to be of limited utility due to [23] and Mag-Indo-1 [37]. The prefix "Mag" was used since these indicators were designed as magnesium probes.…”

“…Magnesium indicators were designed with the same logic used for the calcium probes with an optimum response at the intracellular Mg 2+ levels which range from 0.1 mM to 6.0 mM [20,21] vs. normal serum levels ranging from 0.44 mM to 1.5 mM [22]. The use of magnesium probes is more complicated than that of the calcium probes since changes of Mg 2+ levels are relatively small [23], and magnesium probes also bind to calcium ions, due to the greater "flexibility" of Ca 2+ compared to Mg 2+ [24]. Cytosolic Ca 2+ levels, however, are four orders of magnitude lower than those of Mg 2+ and do not usually interfere, although some complications have been reported in the literature [25,26].…”

“…The probe, when excited by the argon laser at 488 nm has a decreasing fluorescence response to increasing levels of Mg 2+ [12]. Mag-Fura-2 [50] and Mag-Fura-5 [23] Fig. (10) are excitable by UV light ratioable indicators used preferably in fluorescence microscopy whereas the emission ratioable MagIndo-1 is preferred for flow cytometry.…”