A new highly selective physicochemical assay to measure NAD+ in intact cells |
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| Authors: | R Alvarez-Gonzalez R Eichenberger P Loetscher F R Althaus |
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| Institution: | 1. State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China;2. Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China;3. Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China |
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| Abstract: | A simple, fast, and highly specific chromatographic method for measuring the content of NAD+ in intact cells has been developed. This procedure involves the separation of NAD+ from the bulk of acid-soluble nucleosides, nucleotides, and other pyridine containing molecules by affinity chromatography on dihydroxyboronyl-Bio-Rex. The boronate purified preparations were utilized for the quantification of NAD+ by strong anion exchange high-pressure liquid chromatography under isocratic conditions using a low salt buffer system. The overall recovery of the method exceeded 80%. This new method was applied to determine the extent of NAD+ consumption in intact hepatocytes following treatment with two different DNA damaging agents. A major advantage of this method is that it allows for the simultaneous determination of poly(ADP-ribose) in the acid-insoluble fraction of the same sample. |
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