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Fan T.W.-M.,University of Louisville | Fan T.W.-M.,Center for Regulatory Environmental Analytical Metabolomics | Fan T.W.-M.,Brown Cancer Center | Tan J.,Brown Cancer Center | And 4 more authors.
Metabolomics | Year: 2012

We have developed a simple NMR-based method to determine the turnover of nucleotides and incorporation into RNA by stable isotope resolved metabolomics (SIRM) in A549 lung cancer cells. This method requires no chemical degradation of the nucleotides or chromatography. During cell growth, the free ribonucleotide pool is rapidly replaced by de novo synthesized nucleotides. Using [U- 13C]-glucose and [U- 13C, 15N]-glutamine as tracers, we showed that virtually all of the carbons in the nucleotide riboses were derived from glucose, whereas glutamine was preferentially utilized over glucose for pyrimidine ring biosynthesis, via the synthesis of Asp through the Krebs cycle. Incorporation of the glutamine amido nitrogen into the N3 and N9 positions of the purine rings was also demonstrated by proton-detected 15N NMR. The incorporation of 13C from glucose into total RNA was measured and shown to be a major sink for the nucleotides during cell proliferation. This method was applied to determine the metabolic action of an anti-cancer selenium agent (methylseleninic acid or MSA) on A549 cells. We found that MSA inhibited nucleotide turnover and incorporation into RNA, implicating an important role of nucleotide metabolism in the toxic action of MSA on cancer cells. © 2011 Springer Science+Business Media, LLC.


Lorkiewicz P.,University of Louisville | Higashi R.M.,University of Louisville | Higashi R.M.,Center for Regulatory Environmental Analytical Metabolomics | Higashi R.M.,Brown Cancer Center | And 5 more authors.
Metabolomics | Year: 2012

Fourier transform-ion cyclotron resonance-mass spectrometry (FTICR-MS) is capable of acquiring unmatched quality of isotopologue data for stable isotope resolved metabolomics (SIRM). This capability drives the need for a continuous ion introduction for obtaining optimal isotope ratios. Here we report the simultaneous analysis of mono and dinucleotides from crude polar extracts by FTICR-MS by adapting an ion-pairing sample preparation method for LC-MS analysis. This involves a rapid cleanup of extracted nucleotides on pipet tips containing a C 18 stationary phase, which enabled global analysis of nucleotides and their 13C isotopologues at nanomolar concentrations by direct infusion nanoelectrospray FTICR-MS with 5 min of data acquisition. The resolution and mass accuracy enabled computer-assisted unambiguous assignment of most nucleotide species, including all phosphorylated forms of the adenine, guanine, uracil and cytosine nucleotides, NAD +, NADH, NADP +, NADPH, cyclic nucleotides, several UDP-hexoses, and all their 13C isotopologues. The method was applied to a SIRM study on human lung adenocarcinoma A549 cells grown in [U- 13C] glucose with or without the anti-cancer agent methylseleninic acid. At m/z resolving power of 400,000, 13C-isotopologues of nucleotides were fully resolved from all other elemental isotopologues, thus allowing their 13C fractional enrichment to be accurately determined. The method achieves both high sample and high information throughput analysis of nucleotides for metabolic pathway reconstruction in SIRM investigations. © 2011 Springer Science+Business Media, LLC.

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