Measuring Equilibrium Bicarbonate Concentrations Directly in Cellular Mitochondria and in Human Serum Using Europium/Terbium Emission Intensity Ratios

A series of Eu and Tb complexes of four different chiral ligands incorporating an azaxanthone sensitiser has been evaluated as probes for the bicarbonate anion. Their binding affinities were assessed at ambient pH with bicarbonate, lactate, citrate, phosphate and serum albumin. Binding was signalled by modulation of circularly polarised luminescence and apparent affinity constants were measured by examining changes in emission intensity ratios. Competition experiments show that with these species and ATP present at normal physiological values, bicarbonate can be determined selectively over the concentration range 10 to 35 mM. Bicarbonate levels are also reported by using a mixture of Eu and Tb complexes of a common ligand, examining the ratio of red/green emitted light. These methods have been adapted for the determination of bicarbonate in human serum and used for the assessment of mitochondrial levels of bicarbonate in several different cell types with confocal microscopy.