Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in biological samples by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (original) (raw)
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Analytical Biochemistry, 1999
tude lower than the corresponding NTP. Hence, the quantitation of dNTP in cells is generally performed after selective oxidation or removal of the major NTP. The procedures reported so far are lengthy and cumbersome and do not enable the simultaneous determination of NTP. We report the development of a simple, direct HPLC method for the simultaneous determination of dNTP and NTP in colon carcinoma WiDr cell extracts using a stepwise gradient elution ion-pairing HPLC with uv detection at 260 nm and with a minimal chemical manipulation of cells. Exponentially growing WiDr cells were harvested by centrifugation, rinsed with phosphate-buffered saline, and carefully counted. The pellets were suspended in a known volume of ice-cold water and deproteinized with an equal volume of 6% trichloroacetic acid. The acid cell extracts (corresponding to 2.5 ؋ 10 6 cells/100 l) were centrifuged at 13,000g for 10 min at 4°C. The resulting supernatants were stored at ؊80°C prior to analysis. Aliquots (100 l) were neutralized with 4.3 l saturated Na 2 CO 3 solution prior the injection of 40 l onto the HPLC column (injection speed 250 l/min). Chromatographic separations were performed using two Symmetry C18 3.5-m (2 ؋ 3.9 ؋ 150 mm) columns (Waters), connected in series equipped with a Sentry guard column (3.9 ؋ 20 mm i.d.) filled with the same packing material. The HPLC columns were kept at 30°C. The mobile phase was delivered at a flow rate of 0.5 ml/min, with the following stepwise gradient elution program: % solvent A/solvent B, 100/0 at 0 min 3 100/0 at 1 min 3 36/64 at 5 min 3 31/69 at 90 min 3 31/69 at 105 min 3 0/100 at 106 min 3 0/100 at 120 min; 50/50 MeOH/solvent B from 121 to 130 min; 100% solvent A from 131 to 160 min. Solvent A contained 0.01 M KH 2 PO 4 , 0.01 M tetrabutylammonium chloride, and 0.25% MeOH and was adjusted to pH 7.0 (550 l 10 N NaOH for 1 liter solvent A). Solvent B consisted of 0.1 M KH 2 PO 4 , 0.028 M tetrabutylammonium chloride, and 30% MeOH and was neutralized to pH 7.0 (1.4 ml 10 N NaOH for 1 liter solvent B). Even though dNTPs are minor components of cell extracts, satisfactory regression coefficients were obtained for their calibration curves (r 2 > 0.99) established with the addition-calibration methods up to 120 pmol/40-l injection. The applicability of the method was demonstrated by in vitro studies of the modulation of NTP and dNTP pools in WiDr colon carcinoma cell lines exposed to various pharmacological concentrations of cytostatic drugs (i.e., FMdC, IUdR, gemcitabine). In conclusion, this optimized, simplified, analytical method enables the simultaneous quantitation of NTP and dNTP and may represent a valuable tool for the detection of minute alterations of cellular dNTP/NTP pools induced by anticancer/antiviral drugs and diseases.
Quantitation of cellular deoxynucleoside triphosphates
Nucleic Acids Research, 2010
Eukaryotic cells contain a delicate balance of minute amounts of the four deoxyribonucleoside triphosphates (dNTPs), sufficient only for a few minutes of DNA replication. Both a deficiency and a surplus of a single dNTP may result in increased mutation rates, faulty DNA repair or mitochondrial DNA depletion. dNTPs are usually quantified by an enzymatic assay in which incorporation of radioactive dATP (or radioactive dTTP in the assay for dATP) into specific synthetic oligonucleotides by a DNA polymerase is proportional to the concentration of the unknown dNTP. We find that the commonly used Klenow DNA polymerase may substitute the corresponding ribonucleotide for the unknown dNTP leading in some instances to a large overestimation of dNTPs. We now describe assay conditions for each dNTP that avoid ribonucleotide incorporation. For the dTTP and dATP assays it suffices to minimize the concentrations of the Klenow enzyme and of labeled dATP (or dTTP); for dCTP and dGTP we had to replace the Klenow enzyme with either the Taq DNA polymerase or Thermo Sequenase. We suggest that in some earlier reports ribonucleotide incorporation may have caused too high values for dGTP and dCTP.
Nucleic Acids Research, 2021
Information about the cellular concentrations of deoxyribonucleoside triphosphates (dNTPs) is instrumental for mechanistic studies of DNA replication and for understanding diseases caused by defects in dNTP metabolism. The dNTPs are measured by methods based on either HPLC or DNA polymerization. An advantage with the HPLC-based techniques is that the parallel analysis of ribonucleoside triphosphates (rNTPs) can serve as an internal quality control of nucleotide integrity and extraction efficiency. We have developed a Freon-free trichloroacetic acid-based method to extract cellular nucleotides and an isocratic reverse phase HPLC-based technique that is able to separate dNTPs, rNTPs and ADP in a single run. The ability to measure the ADP levels improves the control of nucleotide integrity, and the use of an isocratic elution overcomes the shifting baseline problems in previously developed gradient-based reversed phase protocols for simultaneously measuring dNTPs and rNTPs. An optional...
ABSTRACTDeoxyribonucleotide triphosphates (dNTPs) are vital for the biosynthesis and repair of DNA. Their cellular concentration peaks during the S phase of the cell cycle. In non-proliferating cells dNTP concentrations are low, making their reliable quantification from tissue samples of heterogeneous cellular composition challenging. Partly because of this, the current knowledge related to regulation of and disturbances in cellular dNTP concentrations derive from cell culture experiments with little corroboration at the tissue or organismal level. Here, we fill the methodological gap by presenting a simple non-radioactive microplate assay for the quantification of dNTPs with a minimum requirement of 10 to 30 mg of biopsy material. In contrast to published assays, this assay is based on long (~200 nucleotides) synthetic single-stranded DNA templates, an inhibitor-resistant high-fidelity DNA polymerase, and the double-stranded-DNA-binding EvaGreen dye. The assay quantifies reliably a...
Analytical Biochemistry, 2011
Quantification of nucleotides is an important part of metabolomics but has been hampered by the lack of fast, sensitive, and reliable methods. We present a less time-consuming, more sensitive, and more precise method for the quantitative determination of nucleoside triphosphates (NTPs), 5-ribosyl-1-pyrophosphate (PRPP), and inorganic pyrophosphate (PP i ) in cell extracts. The method uses one-dimensional thin-layer chromatography (TLC) and radiolabeled biological samples. Nucleotides are resolved at the level of ionic charge in an optimized acidic ammonium formate and chloride solvent, permitting quantification of NTPs. The method is significantly simpler and faster than both current two-dimensional methods and high-performance liquid chromatography (HPLC)-based procedures, allowing a higher throughput while common sources of inaccuracies and technical problems are avoided. For determination of PP i , treatment with inorganic pyrophosphatase (PPase) of the radiolabeled phosphate is employed for removal of contaminating pyrophosphate. Biological examples performed in triplicates showed standard deviations of approximately 10% of the mean for the determined concentrations of NTPs.
Cell-cycle-dependent variations of deoxyribonucleoside triphosphate pools in chinese hamster cells
Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1973
Variation of levels of the four deoxyribonucleoside triphosphate pools has been examined as synchronized Chinese hamster cells complete mitosis and traverse the cell cycle in preparation for division. The results indicate that; 1. Mitotic cells have the largest pools of dATP, dGTP, and dTTP and that all four deoxyribonucleoside triphosphates are degraded as cells exit from mitosis. 2. The pool of dCTP is maximal in late S-early G2, and degradation begins in G2. 3. G1 cells have little or no deoxyribonucleoside triphosphates. 4. The pool size of all four deoxyribonucleoside triphosphates increases just prior to initiation of DNA synthesis and increases throughout S. 5. Cells treated with hydroxyurea in G1 accumulate dTTP, dCTP, and dGTP at the time that cells would normally have initiated DNA synthesis, but accumulation of dATP is completely inhibited as is initiation of DNA synthesis. 6. The four deoxyribonucleoside triphosphates are not present in equimolar concentrations, pools in mid-S being 10 pmoles/106 cells, 27 pmoles/106 cells, 104 pmoles/106 cells, and 76 pmoles/106 cells for dGTP, dATP, dTTP, and dCTP, respectively. The pools of dGTP, dATP, dTTP, and dCTP are sufficient to support DNA synthesis for 1.0, 1.3, 5.2, and 3.8 min, respectively.