Assays for Monitoring the Effects of Nicotinamide Supplementation on Mitochondrial Activity in Saccharomyces cerevisiae

The single-celled yeast Saccharomyces cerevisiae is one of the most valuable laboratory models that has been used successfully to identify factors and pathways involved in several cellular processes, the counterparts of which are evolutionarily conserved. Furthermore, it is also a powerful tool for analyzing the effects of molecules of nutraceutical interest with the view of leading to human health benefits and improving the quality of aging. In this context, we present some of the methods that have allowed us to assess the beneficial influence of a form of vitamin B3, namely nicotinamide, on mitochondrial functionality during yeast chronological aging. Mitochondrial dysfunctions are considered to be hallmarks of aging, and of several metabolic and neurodegenerative diseases. More specifically, these methods concern the determination of the respiratory parameters in intact cells in order to evaluate the efficiency of mitochondrial respiration in concert with the risk of superoxide anion (O₂⁻) production, which results from inefficient respiration. In addition, we describe fluorescent staining specific for O₂⁻ detection and mitochondrial membrane potential, as well as a simple clonogenic assay based on the ability of cells to grow on a carbon source that requires a functional mitochondrial metabolism.