Measuring the mitochondrial status of cells

Mitochondria are cell organelles that have a crucial role in cellular energy metabolism. Mitochondria convert combustion energy in a process called oxidative phosphorylation (OXPHOS) to the universal cellular energy currency, adenosine triphosphate (ATP), by oxidizing fuel molecules with oxygen. This is accomplished by a set of enzyme complexes called respiratory chain. The maintenance of the cellular redox state is another indispensable role for the mitochondria. The functional status of the mitochondria can be assessed by determining 1) the respiration ability, 2) the redox state or 3) the specific enzyme activities. Mitochondrial diseases are a heterogeneous set of disorders resulting from dysfunctional oxidative phosphorylation and the resulting inability of cells to produce energy for its needs. Traditionally, defects in OXPHOS protein complexes have been analyzed with spectrophotometric enzyme activity assays or as mitochondrial oxygen consumption with polarographic oxygen electrodes. Mitochondrial oxygen utilization is also possible to measure with probes whose fluorescence intensity is reversibly quenched by the presence of molecular oxygen. The concept of using oxygen quenching of fluorescence and measurement of fluorescence lifetime has been put forward almost two decades ago but it has not yet gained wide general use. We are developing hardware allowing functional studies on mitochondria. In the presentation we describe a prototype employing a fluorescent probe to measure mitochondrial oxygen consumption in minimal sample volumes compatible with biopsy sample sizes of a few milligrams. At least two approaches are employed, one with a miniaturized cuvette and the other with microfluidics and channels. Experimentation is also going on to utilize photoacoustic spectroscopy for measurement of components of the respiratory chain.