Determination of intracellular concentrations of the TRPM2 agonist ADP-ribose by reversed-phase HPLC.

Since the NAD metabolite ADP-ribose (ADPR) has recently gained attention as a putative messenger, a method was established for the quantification of intracellular ADPR by reversed-phase HPLC. Cellular nucleotides were extracted with trichloroacetic acid, and crude cell extracts purified by solid phase extraction using a strong anion exchange matrix. After optimization of the extraction procedure, cellular ADPR levels were determined using two different reversed-phase columns (C18 versus C12), operated in ion pair mode. Intracellular ADPR concentrations in human Jurkat T-lymphocytes and murine BW5147 thymocytes were determined to be 44+/-11 microM and 73+/-11 microM, respectively.