Assessment of DNA oxidative damage by quantification of thymidine glycol residues using gas chromatography/electron...

A technique to assess DNA oxidative damage by quantification of thymidine glycol residues is described. 2-Methylglycerate was released from thymidine glycol in DNA by alkaline cleavage/borodeuteride reduction, then derivatized to form a combined pentafluorobenzyl-tertbutyldimethylsilyl (PFB-TBDMS) derivative and analyzed by gas chromatography/electron capture negative ionization mass spectrometry. [2H4]Thymine glycol was used as an internal standard. The derivatization chemistry was assessed by using [14C-methyl]glycerate. Successful esterification was achieved with 75-80% yield using tetrabutylammonium sulfate-assisted anhydrous pentafluorobenzylation. The PFB-TBDMS derivative exhibits excellent chromatographic and detection properties with a detection limit of 41 amol injected on column. Freshly dissolved calf thymus DNA was used to test the method performance. The background level of thymidine glycol detected in this DNA was 11.7 +/- 0.3 x 10(-6) mol thymidine glycol per 1 mol thymidine. The thymidine glycol background in undamaged DNA establishes a lower limit of oxidative damage below which biological oxidation events would not be measured by this method. The method was linear for 4-20 micrograms DNA added per tube. The minimum measurable amount of thymidine glycol in DNA sample was 36 fmol. An increased level of thymidine glycol was measured in salmon sperm DNA which had autoxidized during storage in a refrigerated aqueous solution, 71.2 +/- 14.3 x 10(-6) mol thymidine glycol per 1 mol thymidine.