Determination of pyrimidine ribotide and deoxyribotide pools in cultured cells and mouse liver by high-performance liquid chromatography.
High-performance liquid chromatographic (HPLC) assay for determining tissue pools of uridine, deoxyuridine, cytidine, deoxycytidine, and thymidine mono-, di-, and triphospates is presented. The method utilizes anion-exchange and, after conversion of nucleotides to nucleosides by acid phosphatase, reversed-phase chromatography on a preparative column with UV detection at 254 and 280 nm. The yield of this procedure is 80 +/- 2% with a sensitivity limit of 100 pmole nucleotide per sample. A sensitivity of 10 pmole can be achieved for each compound by rechromatographing appropriate nucleoside fractions on analytical columns. The recovery, including this step, is 66 +/- 7%. The assay is reproducible and highly selective, with a lower sensitivity limit of approximately 0.1 muM using 150--250 mg (wet weight) tissue samples. Nucleotide pools have been determined in Balb/c mouse liver and in mouse lymphoma (S-49) cell culture, the latter with and without addition of 5-fluorouracil (5-FUra) to the medium. Data obtained with this assay are similar to those using alternative methodologies. Observed depletion of dTXP pools and expansion of dUMP and dCXP pools after 5-FUra treatment are in agreement with published observations. Pools of dUDP and dUTP were not detectable (less than 10 pmole/10(8) cells) in any tissue sample. These data illustrate the utility of the present method in studying actions of pyrimidine antimetabolites.
D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008099:Liver / Q000032:analysis*; D051379:Mice; D008807:Mice, Inbred BALB C; D011741:Pyrimidine Nucleosides / Q000032:analysis*; D013942:Thymine Nucleotides / Q000032:analysis
D011741:Pyrimidine Nucleosides; D013942:Thymine Nucleotides