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NAD+ Content and Its Role in Mitochondria

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Mitochondrial Regulation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1241))

Abstract

Nicotinamide adenine dinucleotide (NAD+) is a central metabolic coenzyme/cosubstrate involved in cellular energy metabolism and energy production. It can readily be reduced by two electron equivalents and forms the NADH form, which is the minority species to NAD+ under most physiologic conditions. NAD+ plays an important role in not only oxidation–reduction reactions in cells but also as a signaling molecule. For example, NAD+ plays a key role in mitochondrial function via participation in pyruvate dehydrogenase, tricarboxylic acid cycle, and oxidative phosphorylation chemistries. It also serves as a substrate for deacylases SIRT3, SIRT4, and SIRT5, which modify protein posttranslational modifications on lysine within the mitochondrial compartment. Recent work has highlighted the biological significance of dynamic changes to mitochondrial NAD+. This has increased the need for standardized and effective methods to measure NAD+ contents in this organelle. To determine NAD+ concentrations in cells, and specifically in mitochondria, we describe two assays for NAD+ determinations: An Enzymatic Cycling Assay and Isotope Dilution. The cycling assay contains sample NAD+, lactate, lactate dehydrogenase, diaphorase, and resazurin. The isotope dilution assay uses synthetic 18O-NAD+ as an internal standard, and treated samples are fractionated by HPLC and then NAD+ concentration determined by the 16O- and 18O-NAD+ peak (664/666) ratio in positive mode MS.

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Acknowledgements

This work was supported by R01 DK74366, R21 DK094001-01A1, and 1R01 GM106072-01.

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Correspondence to Anthony A. Sauve Ph.D. .

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Li, W., Sauve, A.A. (2015). NAD+ Content and Its Role in Mitochondria. In: Palmeira, C., Rolo, A. (eds) Mitochondrial Regulation. Methods in Molecular Biology, vol 1241. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1875-1_4

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  • DOI: https://doi.org/10.1007/978-1-4939-1875-1_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1874-4

  • Online ISBN: 978-1-4939-1875-1

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