NAD/NADH Assay Kit-WST enables quantification of the amount of total NAD+/NADH, NADH and NAD+ in cells and measurement of their ratio.
Measurement of NAD+ and NADH
Cell lysate from a cell culture can be easily prepared via Deproteinzation using the extraction buffer and filtration tubes found within this kit. Intracellular NADH levels can be quantified by heat treatment of cell lysate. Additionally, intracellular NAD+levels can be determined by subtracting NADH levels from independently measured total NAD+/NADH levels.
Study of NAD+/NADH as Markers
Recently, it has become clear that Sirtuin is linked to longevity and plays a role in NAD+ level regulation. Also Sirtuin has been recognized as a marker necessary to understanding biological states, such as obesity & diabetes, as well as cellular differentiation.
In the experiment shown below, the NAD+ /NADH levels & ratio were determined using HeLa cells.
Standard curves were constructed using different concentrations of HeLa cells (2.5×105 and 5.0×105 cells) cultured in growth media. The standard curves were then used to determine the intracellular NAD+ and NADH levels. As a result, NAD+ and NADH levels varied depending on cell number while the change in cell number had no effect on the NAD+/NADH ratio.
Measurement of NAD+/NADH in Combination with Lactate Assay Kit
Change in metabolic activity was observed when the glycolytic inhibitor 2-Deoxy-D-glucose was added to HeLa cells.
2-Deoxy-D-glucose was added to HeLa cells (1×106 cells) to obtain a final concentration of 6 mmol/l 2-Deoxy-D-glucose. After 24 hours of incubation, lactate levels in the supernatant were quantified using the Lactate Assay Kit-WST (Item#: L256), and the NAD+/NADH ratio was determined with the cell pellet after removing the supernatant using the NAD/NADH Assay Kit-WST.
As a result, intracellular glycolysis was inhibited by 2-Deoxy-D-glucose, which led to decreased lactate levels and an increase in the NAD+/NADH ratio.
Experimental Example: Change in Metabolism in Liver Tissue of NASH-Induced Mouse
It is known that non-alcoholic steatohepatitis (NASH) results in decreased ATP, α-ketoglutarate (α-KG), and NAD levels in affected tissues. ATP, α-KG, and NAD levels were measured in the liver tissue of type 1 diabetic model mice (STAM model) that were treated with a high-fat diet (NASH induction) since 4 weeks old. Measurement at 10 weeks old confirmed that ATP, α-KG, and NAD levels indeed decreased in the tissue samples after NASH induction.
Note: for more details about the experimental procedure, please refer to Q&A “Are there any examples of experiments using tissue samples?”
- Cellular ATP ：ATP Assay Kit-Luminescence (Code：A550).
- Cellular α-KG：α-Ketoglutarate Assay Kit-Fluorometric (Code：K261).
- Cellular NAD ：NAD/NADH Assay Kit-WST (Code：N509).
|ATP||Francesco Bellanti, et al., “Synergistic interaction of fatty acids and oxysterols impairs mitochondrial function and limits liver adaptation during nafld progression“, Redox Biology, 2018, 15, 86-96.|
|α-KG||Jianjian Zhao, et al., “The mechanism and role of intracellular α-ketoglutarate reduction in hepatic stellate cell activation“, Bioscience Reports, 2020, 40, (3).|
|Ali Canbay, et al., “L‑Ornithine L‑Aspartate (LOLA) as a Novel Approach for Therapy of Non‑alcoholic Fatty Liver Disease“, Drugs, 2019, 79, 39-44.|
|NAD||Jinhan He, et al., “Activation of the Aryl Hydrocarbon Receptor Sensitizes Mice to Nonalcoholic Steatohepatitis by Deactivating Mitochondrial Sirtuin Deacetylase Sirt3“, Mol. and Cell. Biol., 2013, 33, (10), 2047-55.|
(Intestinal epithelial organoids in old mice)
|R. Uchida, Y. Saito, K. Nogami, Y. Kajiyama, Y. Suzuki, Y. Kawase, T. Nakaoka, T. Muramatsu, M. Kimura and H. Saito , “Epigenetic silencing of Lgr5 induces senescence of intestinal epithelial organoids during the process of aging”, NPJ Aging Mech Dis., 2018,doi：10.1038/s41514-018-0031-5.|
(Adult T-cell leukemia cell line)
|T. Kozako, A. Aikawa, T. Ohsugi, Y. Uchida, N. Kato, K. Sato, K. Ishitsuka, M. Yoshimitsu and S. Honda, “High expression of NAMPT in adult T-cell leukemia/lymphoma and anti-tumor activity of a NAMPT inhibitor”‘, Eur. J. Pharmacol.., 2019, 865, 172738.|
(Sirt 7 knockout fibroblasts)
|S. U. Sobuz, Y. Sato, T. Yoshizawa, F. Karim, K. Ono, T. Sawa, Y. Miyamoto, M. Oka and K. Yamagata, “SIRT7 regulates the nuclear export of NF-κB p65 by deacetylating Ran.”, Biochim Biophys Acta Mol Cell Res.., 2019, 1866, (9), 1355.|
(Human iPS-derived neural cells)
|K. Hayakawa, K. Nishitani and S. Tanaka, “Kynurenine, 3-OH-kynurenine, and anthranilate are nutrient metabolites that alter H3K4 trimethylation and H2AS40 O-GlcNAcylation at hypothalamus-related loci.”, Sci Rep, 2019, 9(1), 19768.|
|5)||Cell (HuH-7)||M. Mikeli, M. Fujikawa, K. Nagahisa, S. Yasuda, N. Yamada and T. Tanabe,“Contribution of GPD2/mGPDH to an alternative respiratory chain of the mitochondrial energy metabolism and the stemness in CD133-positive HuH-7 cells.”, Genes Cells, 2020, 25(2), 139.|
|6)||Cell (HeLa)||J.Thapaa, K. Hashimoto, S. Sugawara, R. Tsujikawa, T. Okubo, S. Nakamura and H.Yamaguchi, “Hypoxia promotes Chlamydia trachomatis L2/434/Bu growth in immortal human epithelial cells via activation of the PI3K-AKT pathway and maintenance of a balanced NAD+/NADH ratio”, Microbes Infect., 2020, DOI:10.1016/j.micinf.2020.04.010.|