A LC-MS/MS method for the analysis of intracellular nucleoside triphosphate levels.
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.
Chen
Ping
P
College of Pharmacy, The Ohio State University, 500 W. 12th Avenue, Columbus, OH 43210, USA.
Liu
Zhongfa
Z
Liu
Shujun
S
Xie
Zhiliang
Z
Aimiuwu
Josephine
J
Pang
Jiuxia
J
Klisovic
Rebecca
R
Blum
William
W
Grever
Michael R
MR
Marcucci
Guido
G
Chan
Kenneth K
KK
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*
D000964:Antimetabolites, Antineoplastic; D009711:Nucleotides; D009838:Oligodeoxyribonucleotides; D003561:Cytarabine; C464617:GTI2040