Assays for Determination of Cellular and Mitochondrial NAD and NADH Content.

NAD is a redox cofactor essential to the proper functioning of a variety of important metabolic pathways, including key steps in mitochondrial energy metabolism. In addition, it serves as a signaling substrate for enzymes such as sirtuins and the poly-ADP ribosyl-polymerase family of enzymes. Sirtuins, which are NAD-dependent protein deacylases, harness changes in cellular NAD concentrations to produce changes in protein acylation status, thereby affecting downstream functions including energy metabolism, stress resistance, and cell survival. Thus, the availability of NAD in cells, or in specific organelles such as the mitochondrion, regulates downstream signaling and key biological processes. This concept has driven a need for researchers to easily and precisely measure NAD concentrations in biological samples. We herein describe several protocols for the measurement of NAD and NADH concentrations in tissues, cells, or subcellular compartments such as mitochondria. These protocols include a cycling assay that can quickly measure NAD or NADH levels using a plate reader equipped with fluorescence measurement capabilities. This plate assay relies only upon commercially available materials in addition to the biological samples of interest. In addition, we describe a protocol employing stable isotope-labeled NAD as an internal standard to determine biological NAD content by isotope-dilution methods. This method requires mass spectrometry to ratio endogenous NAD with exogenous isotope-labeled NAD to obtain quantification using HPLC and mass spectrometry.