Department of Pharmacology, Weill Cornell Medical College, New York, NY, USA.
Methods Mol Biol. 2021;2310:271-285. doi: 10.1007/978-1-0716-1433-4_15.
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.
NAD 是一种氧化还原辅助因子，对多种重要代谢途径的正常功能至关重要，包括线粒体能量代谢中的关键步骤。此外，它还是 sirtuins 等酶和多聚 ADP 核糖聚合酶家族酶的信号底物。Sirtuins 是 NAD 依赖性蛋白去乙酰化酶，利用细胞内 NAD 浓度的变化来产生蛋白酰化状态的变化，从而影响下游功能，包括能量代谢、应激抵抗和细胞存活。因此，细胞中 NAD 的可用性，或在线粒体等特定细胞器中的可用性，调节下游信号和关键生物过程。这一概念促使研究人员需要能够轻松、准确地测量生物样本中的 NAD 浓度。我们在此描述了几种测量组织、细胞或亚细胞区室（如线粒体）中 NAD 和 NADH 浓度的方案。这些方案包括一种循环测定法，该方法可使用配备荧光测量功能的平板读数器快速测量 NAD 或 NADH 水平。该平板测定法除了所需的感兴趣的生物样本外，仅依赖于市售的材料。此外，我们还描述了一种使用稳定同位素标记的 NAD 作为内标通过同位素稀释法来确定生物 NAD 含量的方案。该方法需要质谱来比较内源性 NAD 与外源性同位素标记的 NAD，然后使用 HPLC 和质谱进行定量。
