title: A LC-MS/MS method for the analysis of intracellular nucleoside triphosphate levels.
abstract: OBJECTIVE
  To simultaneously quantify intracellular nucleoside triphosphate (NTP) and deoxynucleoside triphosphate (dNTP) pools and to assess their changes produced by interfering with ribonucleotide reductase (RNR) expression in leukemia cells.
  
  
  METHODS
  A HPLC-MS/MS system was used to quantify intracellular NTP and dNTP pools.
  
  
  RESULTS
  The assay was linear between 50 nM, the lower limit of quantification (LLOQ), and 10 muM in cell lysate. The within-day coefficients of variation (CVs, n = 5) were found to be 12.0-18.0% at the LLOQ and 3.0-9.0% between 500 and 5,000 nM for dNTPs and 8.0-15.0% and 2.0-6.0% for NTPs. The between-day CVs (n = 5) were 9.0-13.0% and 3.0-11.0% for dNTPs and 9.0-13.0% and 3.0-6.0% for NTPs. The within-day accuracy values were 93.0-119.0% for both NTPs and dNTPs. ATP overlapped with dGTP and they were analyzed as a composite. This method was applied to measure basal intracellular dNTPs/NTPs in five leukemia cell lines exposed to the RNR antisense GTI-2040. Following drug treatment, dCTP and dATP levels were found to decrease significantly in MV4-11 and K562 cells. Additionally, perturbation of dNTP/NTP levels in bone marrow sample of a patient treated with GTI-2040 was detected.
  
  
  CONCLUSIONS
  This method provides a practical tool to measure intracellular dNTP/NTP levels in cells and clinical samples.
authors.affiliation: College of Pharmacy, The Ohio State University, 500 W. 12th Avenue, Columbus, OH 43210, USA.
authors.forename: Ping
authors.initials: P
authors.lastname: Chen
authors.forename: Zhongfa
authors.initials: Z
authors.lastname: Liu
authors.forename: Shujun
authors.initials: S
authors.lastname: Liu
authors.forename: Zhiliang
authors.initials: Z
authors.lastname: Xie
authors.forename: Josephine
authors.initials: J
authors.lastname: Aimiuwu
authors.forename: Jiuxia
authors.initials: J
authors.lastname: Pang
authors.forename: Rebecca
authors.initials: R
authors.lastname: Klisovic
authors.forename: William
authors.initials: W
authors.lastname: Blum
authors.forename: Michael R
authors.initials: MR
authors.lastname: Grever
authors.forename: Guido
authors.initials: G
authors.lastname: Marcucci
authors.forename: Kenneth K
authors.initials: KK
authors.lastname: Chan
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000627:therapeutic use; D001854:Bone Marrow Cells / Q000187:drug effects / Q000378:metabolism; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D003561:Cytarabine / Q000627:therapeutic use; D006801:Humans; D007938:Leukemia / Q000378:metabolism* / Q000628:therapy*; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism* / Q000628:therapy*; D016014:Linear Models; D009711:Nucleotides / Q000032:analysis* / Q000302:isolation & purification; D009838:Oligodeoxyribonucleotides / Q000627:therapeutic use; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000964:Antimetabolites, Antineoplastic; D009711:Nucleotides; D009838:Oligodeoxyribonucleotides; D003561:Cytarabine; C464617:GTI2040