Improved high-performance liquid chromatographic analysis of intracellular deoxyribonucleoside triphosphate levels.
The ability to measure intracellular deoxyribonucleoside triphosphate (dNTP) pool sizes is important for understanding the intracellular metabolism of DNA synthesis and repair. We have developed an improved method for measuring intracellular dNTP pool size by high-performance liquid chromatography (HPLC). Previous methods have enabled accurate measurement of dNTPs only in concentrations greater than approximately 10 pmol per 10(6) cells due to the inability to partially purify cell extracts, to the inability to apply extracts from extremely large numbers of cells, to the lack of efficient columns, to the presence of incompatible solvents, and to the inability to inject large volumes. We have modified a low-pressure strong anion-exchange column pre-step developed by others to concentrate and partially purify oxidized cell extracts while at the same time eluting them in a more compatible solvent for HPLC injection. The HPLC column is a YMC ODS-AQ column operating in a combined hydrophobic-interaction chromatography-reversed-phase chromatography mode. The injection and elution solvents are both phosphate-based. Using this method it is possible to measure intracellular dNTP levels well below 0.5 pmol per 10(6) cells or at the sensitivity of the DNA polymerase assay.
Rimerman
R A
RA
Department of Internal Medicine, University of Nebraska Medical Center, Omaha 68198.
Prorok
G D
GD
Cordel
K L
KL
Shahwan
A M
AM
Vaughan
W P
WP
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003845:Deoxycytosine Nucleotides / Q000032:analysis; D003853:Deoxyribonucleosides / Q000032:analysis*; D006801:Humans; D007939:Leukemia L1210 / Q000378:metabolism; D008214:Lymphocytes / Q000737:chemistry / Q000378:metabolism; D009000:Monocytes / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010504:Periodic Acid; D014407:Tumor Cells, Cultured / Q000378:metabolism
D003845:Deoxycytosine Nucleotides; D003853:Deoxyribonucleosides; D010504:Periodic Acid; C024107:2'-deoxycytidine 5'-triphosphate