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A LC-MS/MS Method for the Analysis of Intracellular Nucleoside Triphosphate Levels

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Abstract

Purpose

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 μM 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.

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References

  1. W. Y. Gao, D. G. Johns, and H. Mitsuya. Enzymatic assay for quantification of deoxynucleoside triphosphates in human cells exposed to antiretroviral 2′,3′-dideoxynucleosides. Anal. Biochem. 222:116–122 (1994).

    Article  PubMed  CAS  Google Scholar 

  2. B. Roy, C. Beuneu, P. Roux, H. Buc, G. Lemaire, and M. Lepoivre. Simultaneous determination of pyrimidine or purine deoxyribonucleoside triphosphates using a polymerase assay. Anal. Biochem. 269:403–409 (1999).

    Article  PubMed  CAS  Google Scholar 

  3. L. Skoog. An enzymatic method for the determination of dCTP and dGTP in picomole amounts. Eur. J. Biochem. 17:202–208 (1970).

    Article  PubMed  CAS  Google Scholar 

  4. P. Ferraro, V. Bianchi, M. R. Biasin, and L. Celotti. Deoxynucleotide pools and DNA synthesis in resting and PHA-stimulated human lymphocytes treated with mutagens. Exp. Cell Res. 199:349–354 (1992).

    Article  PubMed  CAS  Google Scholar 

  5. G. W. Aherne, A. Hardcastle, F. Raynaud, and A. L. Jackman. Immunoreactive dUMP and TTP pools as an index of thymidylate synthase inhibition: effect of tomudex (ZD1694) and a nonpolyglutamated quinazoline antifolate (CB30900) in L1210 mouse leukaemia cells. Biochem. Pharmacol. 51:1293–1301 (1996).

    Article  PubMed  CAS  Google Scholar 

  6. E. M. Piall, G. W. Aherne, and V. Marks. The quantitative determination of 2′-deoxycytidine-5′-triphosphate in cell extracts by radioimmunoassay. Anal. Biochem. 154:276–281 (1986).

    Article  PubMed  CAS  Google Scholar 

  7. L. A. Decosterd, E. Cottin, X. Chen, F. Lejeune, R. O. Mirimanoff, J. Biollaz, and P. A. Coucke. Simultaneous determination of deoxyribonucleoside in the presence of ribonucleoside triphosphates in human carcinoma cells by high-performance liquid chromatography. Anal. Biochem. 270:59–68 (1999).

    Article  PubMed  CAS  Google Scholar 

  8. D. R. Cross, B. J. Miller, and S. J. James. A simplified HPLC method for simultaneously quantifying ribonucleotides and deoxyribonucleotides in cell extracts or frozen tissues. Cell Prolif. 26:327–336 (1993).

    Article  PubMed  CAS  Google Scholar 

  9. F. Arezzo. Determination of ribonucleoside triphosphates and deoxyribonucleoside triphosphates in Novikoff hepatoma cells by high-performance liquid chromatography. Anal. Biochem. 160:57–64 (1987).

    Article  PubMed  CAS  Google Scholar 

  10. D. Di Pierro, B. Tavazzi, C. F. Perno, M. Bartolini, E. Balestra, R. Calio, B. Giardina, and G. Lazzarino. An ion-pairing high-performance liquid chromatographic method for the direct simultaneous determination of nucleotides, deoxynucleotides, nicotinic coenzymes, oxypurines, nucleosides, and bases in perchloric acid cell extracts. Anal. Biochem. 231:407–412 (1995).

    Article  PubMed  Google Scholar 

  11. J. Harmenberg, A. H. Karlsson, and G. Gilljam. Comparison of sample preparation methods for the high-performance liquid chromatographic analysis of cell culture extracts for triphosphate ribonucleosides and deoxyribonucleosides. Anal. Biochem. 161:26–31 (1987).

    Article  PubMed  CAS  Google Scholar 

  12. S. Palmer, and S. Cox. Comparison of extraction procedures for high-performance liquid chromatographic determination of cellular deoxynucleotides. J. Chromatogr A. 667:316–321 (1994).

    Article  PubMed  CAS  Google Scholar 

  13. R. A. Rimerman, G. D. Prorok, K. L. Cordel, A. M. Shahwan, and W. P. Vaughan. Improved high-performance liquid chromatographic analysis of intracellular deoxyribonucleoside triphosphate levels. J. Chromatogr. 619:29–35 (1993).

    Article  PubMed  CAS  Google Scholar 

  14. D. S. Shewach. Quantitation of deoxyribonucleoside 5′-triphosphates by a sequential boronate and anion-exchange high-pressure liquid chromatographic procedure. Anal. Biochem. 206:178–182 (1992).

    Article  PubMed  CAS  Google Scholar 

  15. J. Chi, A. Jayewardene, J. Stone, J. G. Gambertoglio, and F. T. Aweeka. A direct determination of thymidine triphosphate concentrations without dephosphorylation in peripheral blood mononuclear cells by LC/MS/MS. J. Pharm. Biomed. Anal. 26:829–836 (2001).

    Article  PubMed  CAS  Google Scholar 

  16. G. Hennere, F. Becher, A. Pruvost, C. Goujard, J. Grassi, and H. Benech. Liquid chromatography-tandem mass spectrometry assays for intracellular deoxyribonucleotide triphosphate competitors of nucleoside antiretrovirals. J. Chromatogr. 789:273–281 (2003).

    Article  CAS  Google Scholar 

  17. E. N. Fung, Z. Cai, T. C. Burnette, and A. K. Sinhababu. Simultaneous determination of Ziagen and its phosphorylated metabolites by ion-pairing high-performance liquid chromatography-tandem mass spectrometry. J. Chromatogr. 754:285–295 (2001).

    Article  CAS  Google Scholar 

  18. T. W. North, R. K. Bestwick, and C. K. Mathews. Detection of activities that interfere with the enzymatic assay of deoxyribonucleoside 5′-triphosphates. J. Biol. Chem. 255:6640–6645 (1980).

    PubMed  CAS  Google Scholar 

  19. F. Becher, D. Schlemmer, A. Pruvost, M. C. Nevers, C. Goujard, S. Jorajuria, C. Guerreiro, T. Brossette, L. Lebeau, C. Creminon, J. Grassi, and H. Benech. Development of a direct assay for measuring intracellular AZT triphosphate in humans peripheral blood mononuclear cells. Anal. Chem. 74:4220–4227 (2002).

    Article  PubMed  CAS  Google Scholar 

  20. C. Chin, Z. P. Zhang, and H. T. Karnes. A study of matrix effects on an LC/MS/MS assay for olanzapine and desmethyl olanzapine. J. Pharm. Biomed. Anal. 35:1149–1167 (2004).

    Article  PubMed  CAS  Google Scholar 

  21. K. Smid, C. J. Van Moorsel, P. Noordhuis, D. A. Voorn, and G. J. Peters. Interference of gemcitabine triphosphate with the measurements of deoxynucleotides using an optimized DNA polymerase elongation assay. Int. J. Oncol. 19:157–162 (2001).

    PubMed  CAS  Google Scholar 

  22. W. M. Stadler, A. A. Desai, D. I. Quinn, R. Bukowski, B. Poiesz, C. G. Kardinal, N. Lewis, A. Makalinao, P. Murray, and F. M. Torti. A phase I/II study of GTI-2040 and capecitabine in patients with renal cell carcinoma. Cancer Chemother. Pharmacol. 61:689–694 (2008).

    Article  PubMed  CAS  Google Scholar 

  23. A. A. Desai, R. L. Schilsky, A. Young, L. Janisch, W. M. Stadler, N. J. Vogelzang, S. Cadden, J. A. Wright, and M. J. Ratain. A phase I study of antisense oligonucleotide GTI-2040 given by continuous intravenous infusion in patients with advanced solid tumors. Ann. Oncol. 16:958–965 (2005).

    Article  PubMed  CAS  Google Scholar 

  24. Y. Lee, A. Vassilakos, N. Feng, V. Lam, H. Xie, M. Wang, H. Jin, K. Xiong, C. Liu, J. Wright, and A. Young. GTI-2040, an antisense agent targeting the small subunit component (R2) of human ribonucleotide reductase, shows potent antitumor activity against a variety of tumors. Cancer Res. 63:2802–2811 (2003).

    PubMed  CAS  Google Scholar 

  25. J. Shao, B. Zhou, B. Chu, and Y. Yen. Ribonucleotide reductase inhibitors and future drug design. Current Cancer Drug Targets. 6:409–431 (2006).

    Article  PubMed  CAS  Google Scholar 

  26. S. Auriola, J. Frith, M. J. Rogers, A. Koivuniemi, and J. Monkkonen. Identification of adenine nucleotide-containing metabolites of bisphosphonate drugs using ion-pair liquid chromatography-electrospray mass spectrometry. J. Chromatogr. 704:187–195 (1997).

    Article  CAS  Google Scholar 

  27. H. Monkkonen, P. Moilanen, J. Monkkonen, J. C. Frith, M. J. Rogers, and S. Auriola. Analysis of an adenine nucleotide-containing metabolite of clodronate using ion pair high-performance liquid chromatography–electrospray ionisation mass spectrometry. J. Chromatogr. 738:395–403 (2000).

    Article  CAS  Google Scholar 

  28. R. L. Claire 3rd. Positive ion electrospray ionization tandem mass spectrometry coupled to ion-pairing high-performance liquid chromatography with a phosphate buffer for the quantitative analysis of intracellular nucleotides. Rapid Commun. Mass Spectrom. 14:1625–1634 (2000).

    Article  PubMed  Google Scholar 

  29. K. Tanaka, A. Yoshioka, S. Tanaka, and Y. Wataya. An improved method for the quantitative determination of deoxyribonucleoside triphosphates in cell extracts. Anal. Biochem. 139:35–41 (1984).

    Article  PubMed  CAS  Google Scholar 

  30. D. H. Rammler. Periodate oxidations of enamines. I. Oxidation of adenosine 5′-monophosphate in the presence of methylamine. Biochemistry. 10:4699–4705 (1971).

    Article  PubMed  CAS  Google Scholar 

  31. A. Steinschneider. Effect of methylamine on periodate-oxidized adenosine 5′-phosphate. Biochemistry. 10:173–178 (1971).

    Article  PubMed  CAS  Google Scholar 

  32. M. Uziel. Periodate oxidation and amino-catalyzed elimination of the terminal nucleoside from adenylate or ribonucleic acid. Products of overoxidation. Biochemistry. 12:938–942 (1973).

    Article  PubMed  CAS  Google Scholar 

  33. D. Huang, Y. Zhang, and X. Chen. Analysis of intracellular nucleoside triphosphate levels in normal and tumor cell lines by high-performance liquid chromatography. J. Chromatogr. 784:101–109 (2003).

    Article  CAS  Google Scholar 

  34. E. A. Vasilyeva, I. B. Minkov, A. F. Fitin, and A. D. Vinogradov. Kinetic mechanism of mitochondrial adenosine triphosphatase. Inhibition by azide and activation by sulphite. Biochem. J. 202:15–23 (1982).

    PubMed  CAS  Google Scholar 

  35. X. Wei, G. Dai, G. Marcucci, Z. Liu, D. Hoyt, W. Blum, and K. K. Chan. A specific picomolar hybridization-based ELISA assay for the determination of phosphorothioate oligonucleotides in plasma and cellular matrices. Pharm. Res. 23:1251–1264 (2006).

    Article  PubMed  CAS  Google Scholar 

  36. D. Chandra, S. B. Bratton, M. D. Person, Y. Tian, A. G. Martin, M. Ayres, H. O. Fearnhead, V. Gandhi, and D. G. Tang. Intracellular nucleotides act as critical prosurvival factors by binding to cytochrome C and inhibiting apoptosome. Cell. 125:1333–1346 (2006).

    Article  PubMed  CAS  Google Scholar 

  37. R. B. Klisovic, W. Blum, X. Wei, S. Liu, Z. Liu, Z. Xie, T. Vukosavljevic, C. Kefauver, L. Huynh, J. Pang, J. A. Zwiebel, S. Devine, J. C. Byrd, M. R. Grever, K. Chan, and G. Marcucci. Phase I study of GTI-2040, an antisense to ribonucleotide reductase, in combination with high-dose cytarabine in patients with acute myeloid leukemia. Clin. Cancer Res. 14:3889–3895 (2008).

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

This work was supported in part by the National Cancer Research Institute, Bethesda, MD, RO1 CA102031 (P.I. GM) and by the Biomedical Mass Spectrometry Laboratory, The Ohio State University.

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Authors and Affiliations

  1. College of Pharmacy, The Ohio State University, 500 W. 12th Avenue, Columbus, Ohio, 43210, USA

    Ping Chen, Zhongfa Liu, Zhiliang Xie, Josephine Aimiuwu & Kenneth K. Chan

  2. Division of Hematology-Oncology, The Ohio State University, Columbus, Ohio, 43210, USA

    Shujun Liu, Rebecca Klisovic, William Blum, Michael R. Grever, Guido Marcucci & Kenneth K. Chan

  3. College of Medicine and Public Health, The Ohio State University, Columbus, Ohio, 43210, USA

    Shujun Liu, Jiuxia Pang, Rebecca Klisovic, William Blum, Michael R. Grever, Guido Marcucci & Kenneth K. Chan

  4. Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, 43210, USA

    Rebecca Klisovic, William Blum, Michael R. Grever, Guido Marcucci & Kenneth K. Chan

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  1. Ping Chen
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Correspondence to Kenneth K. Chan.

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Chen, P., Liu, Z., Liu, S. et al. A LC-MS/MS Method for the Analysis of Intracellular Nucleoside Triphosphate Levels. Pharm Res 26, 1504–1515 (2009). https://doi.org/10.1007/s11095-009-9863-9

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  • DOI: https://doi.org/10.1007/s11095-009-9863-9

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