A new highly selective physicochemical assay to measure NAD+ in intact cells.
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.
D000818:Animals; D002478:Cells, Cultured; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis; D008099:Liver / Q000166:cytology; D008297:Male; D009243:NAD / Q000032:analysis*; D011064:Poly Adenosine Diphosphate Ribose / Q000032:analysis; D011506:Proteins / Q000032:analysis; D051381:Rats
D011506:Proteins; D009243:NAD; D011064:Poly Adenosine Diphosphate Ribose; D004247:DNA