A Self-Contained 48-Well Fatty Acid Oxidation Assay

The modulation of fatty acid metabolism and especially the stimulation of fatty acid oxidation in liver or skeletal muscle are attractive therapeutic approaches for the treatment of obesity and the associated insulin resistance. However, current β-oxidation assays are run in very low throughput, which represents an obstacle for drug discovery in this area. Here we describe results for a 48-well β-oxidation assay using a new instrument design. A connecting chamber links two adjacent wells to form an experimental unit, in which one well contains the β-oxidation reaction and the other captures CO2. The experimental units are sealed from each other and from the outside to prevent release of radioactivity from the labeled substrate. CO2 capture in this instrument is linear with time and over the relevant experimental range of substrate concentration. Cellular viability is maintained in the sealed environment, and cells show the expected responses to modulators of β-oxidation, such as the AMP kinase activator 5...