Analysis of 8-oxodG by HPLC/EC

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Procedure

8-oxodG was measured by high-performance liquid chromatography with electrochemical detection (HPLC/EC) following DNA extraction, RNase treatment, and enzymatic hydrolysis as previously described (7). Briefly, DNA was extracted by a high-salt protein precipitation method that avoids possible artifacts.  DNA, resuspended in 10 mM Tris-EDTA, was digested with nuclease P1 (Boehringer Mannheim, Monza, Italy) for 2 hr and with alkaline phosphatase (Boehringer Mannheim) for 1 hr. Enzymes were precipitated by addition of CHCl3, and the upper layer was stored for analysis of 8-oxodGua at 80 °C under N2. The DNA hydrolysate was analyzed by HPLC/EC (Coulochem; ESA Inc., Thermo Scientific) with a C18 5 μm Uptishere column (250 by 46 mm; Interchim) equipped with a C18 guard column. The eluent was 50 mM ammonium acetate, pH 5.5, containing 9% methanol, at a flow rate of 0.7 ml/min. The potentials applied were 150 and 400 mV for E1 and E2, respectively. The retention time of 8-oxodG was 23 min. Deoxyguanosine was measured in the same run of corresponding 8-oxodGwith a UV detector (model SPD-2A; Shimadzu) at 256 nm; the retention time was 17 min.

References

ref 7: De Luca,G., Russo,M.T., Degan,P., Tiveron,C., Zijno,A., Meccia,E., Ventura,I., Mattei,E., Nakabeppu,Y., Crescenzi,M. et al. (2008) A role for oxidized DNA precursors in Huntington's disease-like striatal neurodegeneration. PLoS Genet., 4(11), e1000266

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