FACTORS INFLUENCING THE MEASUREMENT OF CALCIUM CONCENTRATIONS IN BIOLOGICAL-FLUIDS BY F-19 NMR-SPECTROSCOPY

Fluorinated derivatives of fluorescent calcium probes have been recent ly used to measure free calcium concentrations in biological fluids by F-19 n.m.r. spectroscopy. To measure calcium levels a method utilizes the change in chemical shift experienced by the fluorine resonance of the calcium chelator amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacet ic acid upon complexation of cations. The procedure assumes that (A) t he fluorinated probe Is in equilibrium between the calcium complex and the free acid form; (B) the chemical exchange between different sites has similar effects on the intensities of the observable resonances; and (c) the presence of macromolecules affects equally the intensities of the resonances arising from different complexes. Experiments desig ned to test these assumptions were carried out in plasma and in model systems containing mixtures of cations and macrormolecules. The result s indicated that in biological fluids the calcium probe is involved in a multisite equilibrium in which 'invisible' (very broad) peaks affec t the equilibrium intensities of the 'observable' peaks. Binding to ma cromolecules affected differently the intensities of the resonances ar ising from different complexes in the spectrum of the chelator, and in fluenced significantly the calculated free calcium concentrations.