Award
Dr Larissa Balakireva, CEO & Founder of NovoCIB, was awarded with the Trophy of
"Femmes en Or 2011, Femme de l'Innovation"
in September 2011
.

NMP, NDP, NTP Quantification by HPLC

Ref. # WCS-Nov 7
Quantity Price*
1 sample
€ 620.00

* Pricing updated May 29th, 2015

For different number of samples contact us

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Download "HPLC Analysis Antifolates" 

Cellular Assay for Antifolates

NOVOCIB's Cellular Assay for Antifolates allows to validate inhibition of de novo biosynthesis of purine nucleotides in cultured cells. After incubation of cultured cells with the inhibitor, this high performance liquid chromatography analysis of nucleotides consists in:

  • Extraction of NMP, NDP, and NTP by SPE (Solid Phase Extraction) procedure
  • Separation by ion-paired HPLC of 13 nucleotides (Agilent 1100)
  • Quantification by ion-paired UV-HPLC (peak area at 254nm)

This Cell-Based Assay was validated with methotrexate.



Methotrexate (MTX) is an immunosuppressive agent that has been in clinical use for over 50 years. Although originally introduced for chemotherapy in cancer and leukaemia, methotrexate was coincidentally found to have immunosuppressive properties and is currently used in treating rheumatoid arthritis(1). Methotrexate was first believed to be an inhibitor of the enzyme dihydrofolate reductase (DHFR), the enzyme required for reduction of dihydrofolate (FH2) to tetrahydrolate (FH4). However, as shown in the 80's, methotrexate is actually a prodrug which is polyglutamated and accumulated in cells(2). In contrast to unmodified methotrexate, its polyglutamated derivatives were found to be efficient inhibitors of the ninth folate-dependent step of purine synthesis catalysed by 5-amino-4-imidazolecarboxamide riboside 5’-monophosphate transformylase (AICAR-T)(3) and the thymidylate synthase (TS)(4).
Figure 1. Nucleotide profiles of methotrexate-treated HeLa cells (10µM, 20h)
Ratio between nucleotide content in drug-treated and untreated cells are shown

Figure 2. Effect of methotrexate on cellular pool of purines and pyrimidines

Results
As illustrated by Figure 1 and HPLC spectra, intracellular level of ATP, ADP, GTP and GDP is much lower in methotrexate-treated cells than in untreated control, while cellular contents of UTP and UDP are not affected. Another remarkable change concerns the accumulation of dUMP in methotrexate-treated HeLa cell and depletion of dTTP pool. All these results are in perfect agreement with previously published data(5) showing that MTX inhibits de novo synthesis of purine nucleotides through AICART enzyme and synthesis of thymidylate through thymidilate synthase.


Download this document "HPLC Analysis Antifolates

References
1. L. D. Fairbanks et al. (1999): Methotrexate inhibits the ?rst committed step of purine biosynthesis in mitogen-stimulated human T-lymphocytes: a metabolic basis for ef?cacy in rheumatoid arthritis? Biochem. J. 342(Pt 1), 143-152
2. B. A. Chabner et al. (1985): Polyglutamation of Methotrexate- Is Methotrexate a Prodrug? J. Clin. Invest. 76(3), 907-912
3. C. J. Allegra et al. (1987): Evidence for direct inhibition of de novo purine synthesis in human MCF-7 breast cells as a principal mode of metabolic inhibition by methotrexate J. Biol. Chem. 262(28), 13520-6
4. C. J. Allegra et al. (1985): Enhanced inhibition of thymidylate synthase by methotrexate polyglutamates J. Biol. Chem. 260(17), 9720-6
5. G. P. Budzik et al. (2000): Effects of methotrexate on nucleotide pools in normal human cells and the CEM T cell line Life Sci. 66(23), 2297-307
et al.(1995): 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(2), 407-412


Related Links
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Cellular Assay for RNR inhibitors
Customized Nucleotide Profiling Analysis
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