Abstract
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.
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Acknowledgments
The work described in this manuscript are in part supported by NIH grant GM R01-106072 (A.A.S.) and from NY Spinal Cord Research Board Contract C32098GG (A.A.S.).
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Yang, Y., Sauve, A.A. (2021). Assays for Determination of Cellular and Mitochondrial NAD+ and NADH Content. In: Palmeira, C.M., Rolo, A.P. (eds) Mitochondrial Regulation. Methods in Molecular Biology, vol 2310. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1433-4_15
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DOI: https://doi.org/10.1007/978-1-0716-1433-4_15
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