DETECTION OF 8-HYDROXYDEOXYGUANOSINE IN K562 HUMAN HEMATOPOIETIC-CELLS BY HIGH-PERFORMANCE CAPILLARY ELECTROPHORESIS

A method for the detection of 8-hydroxydeoxyguanosine by high-performa nce capillary electrophoresis (HPCE) was developed. Separations were p erformed in an uncoated silica capillary (44 cmX75 mu m I.D.) with a P /ACE system with diode-array detector. The separation of purine deoxyn ucleosides and 8-hydroxydeoxyguanosine was optimized with regard to pH , temperature, applied potential and hydrodynamic injection time. Opti mum conditions were 20 mM berate 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 amou nt of deoxyguanosine. Isolated nuclei from K562 human hematopoietic ce lls were treated with 15 mM hydrogen peroxide for 2 h. The nuclei were extensively dialyzed and DNA was isolated. enzymatically hydrolyzed t o the deoxynucleosides and analyzed by HPCE. DNA from hydrogen peroxid e treated nuclei had a 4-fold higher content of 8-hydroxydeoxyguanosin e 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. (C) 1 997 Elsevier Science B.V.