Title | Assays for Determination of Cellular and Mitochondrial NAD+ and NADH Content. |
Publication Type | Journal Article |
Year of Publication | 2021 |
Authors | Yang Y, Sauve AA |
Journal | Methods Mol Biol |
Volume | 2310 |
Pagination | 271-285 |
Date Published | 2021 |
ISSN | 1940-6029 |
Keywords | Cell Fractionation, Cells, Cultured, Chromatography, High Pressure Liquid, Energy Metabolism, Indicator Dilution Techniques, Mass Spectrometry, Mitochondria, NAD, Spectrometry, Fluorescence |
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. |
DOI | 10.1007/978-1-0716-1433-4_15 |
Alternate Journal | Methods Mol Biol |
PubMed ID | 34096008 |
Grant List | R01 GM106072 / GM / NIGMS NIH HHS / United States |