Detection of 8-hydroxydeoxyguanosine in K562 human hematopoietic cells by high-performance capillary electrophoresis.
A method for the detection of 8-hydroxydeoxyguanosine by high-performance capillary electrophoresis (HPCE) was developed. Separations were performed in an uncoated silica capillary (44 cm x 75 microns i.d.) with a P/ACE system with diode-array detector. The separation of purine deoxynucleosides and 8-hydroxydeoxyguanosine was optimized with regard to pH, temperature, applied potential and hydrodynamic injection time. Optimum conditions were 20 mM borate buffer (pH 9.5), 25 degrees C, 25 kV, 20 s load and detection at 254 nm. This method allowed the detection of 8-hydroxydeoxyguanosine in the presence of a 10(5)-fold higher amount of deoxyguanosine. Isolated nuclei from K562 human hematopoietic cells were treated with 15 mM hydrogen peroxide for 2 h. The nuclei were extensively dialyzed and DNA was isolated, enzymatically hydrolyzed to the deoxynucleosides and analyzed by HPCE. DNA from hydrogen peroxide treated nuclei had a 4-fold higher content of 8-hydroxydeoxyguanosine than untreated controls. HPCE analysis of 8-hydroxydeoxyguanosine is fast and simple. Furthermore, it requires a very small sample volume, which makes it useful for biomedical and clinical applications.
Ullrich
O
O
Clinic of Physical Medicine, Medical Faculty (Charité), Humboldt University Berlin, Germany.
Grune
T
T
D000080242:8-Hydroxy-2'-Deoxyguanosine; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis; D004560:Electricity; D019075:Electrophoresis, Capillary; D006413:Hematopoietic System / Q000737:chemistry / Q000166:cytology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D012680:Sensitivity and Specificity; D013696:Temperature
D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D003849:Deoxyguanosine