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PMID: 19291372 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Validation Study

A LC-MS/MS method for the analysis of intracellular nucleoside triphosphate levels.

Pharmaceutical research ·Vol. 26 ·No. 6 ·2009-06-00 ·Pages 1504-15

Chen P, Liu Z, Liu S, Xie Z, Aimiuwu J, Pang J, Klisovic R, Blum W, Grever MR, Marcucci G, Chan KK

Abstract

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. A HPLC-MS/MS system was used to quantify intracellular NTP and dNTP pools. 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. This method provides a practical tool to measure intracellular dNTP/NTP levels in cells and clinical samples.

MeSH Terms
Antimetabolites, Antineoplastic/therapeutic use Bone Marrow Cells/drug effects,metabolism Cell Line, Tumor Chromatography, Liquid/methods Cytarabine/therapeutic use Humans Leukemia/metabolism,therapy Leukemia, Myeloid, Acute/metabolism,therapy Linear Models Nucleotides/analysis,isolation & purification Oligodeoxyribonucleotides/therapeutic use Sensitivity and Specificity Tandem Mass Spectrometry/methods
Chemicals
Antimetabolites, Antineoplastic Nucleotides Oligodeoxyribonucleotides Cytarabine GTI2040
Authors & Affiliations
11 authors, click to expand affiliations / ORCID
Chen Ping
College of Pharmacy, The Ohio State University, 500 W. 12th Avenue, Columbus, OH 43210, USA.
Liu Zhongfa
Liu Shujun
Xie Zhiliang
Aimiuwu Josephine
Pang Jiuxia
Klisovic Rebecca
Blum William
Grever Michael R
Marcucci Guido
Chan Kenneth K
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Article Info
Journal
Pharmaceutical research
Abbr.
Pharm Res
ISSN
1573-904X
DOI
10.1007/s11095-009-9863-9
Published
2009-06-00
Epub
2009-00-17
Pages
1504-15
Language
English
Region
United States
NLM ID
8406521
PMCID
PMC3071250
Subset
IM
Grants
NCI NIH HHS · R01 CA102031 · United States
NCI NIH HHS · R01 CA 102031 · United States
NCI NIH HHS · R01 CA102031-03 · United States
NCI NIH HHS · R01 CA102031-01A1 · United States
NCI NIH HHS · R01 CA102031-07 · United States
NCRR NIH HHS · UL1 RR025755 · United States
NCI NIH HHS · R01 CA102031-06 · United States
NCI NIH HHS · R01 CA102031-04A1 · United States
NCI NIH HHS · R01 CA102031-05 · United States
NCI NIH HHS · R01 CA102031-02 · United States
External Links
PubMed source DOI: 10.1007/s11095-009-9863-9 PMC Full Text View journal details
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