ASSESSMENT OF DNA OXIDATIVE DAMAGE BY QUANTIFICATION OF THYMIDINE GLYCOL RESIDUES USING GAS-CHROMATOGRAPHY ELECTRON-CAPTURE NEGATIVE IONIZATION MASS-SPECTROMETRY

A technique to assess DNA oxidative damage by quantification of thymid ine 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-tertbutyldimethy lsilyl (PFB-TBDMS) derivative and analyzed by gas chromatography/elect ron capture negative ionization mass spectrometry. [H-2(4)]Thymine gly col was used as an internal standard. The derivatization chemistry was assessed by using [C-14-methyl]glycerate. Successful esterification w as achieved with 75-80% yield using tetrabutylammonium sulfate-assiste d anhydrous pentafluorobenzylation. The PFB-TBDMS derivative exhibits excellent chromatographic and detection properties with a detection li mit of 41 amol injected on column. Freshly dissolved calf thymus DNA w as used to test the method performance. The background level of thymid ine glycol detected in this DNA was 11.7 +/- 0.3 x 10(-6) mol thymidin e glycol per 1 mol thymidine. The thymidine glycol background in undam aged DNA establishes a lower limit of oxidative damage below which bio logical oxidation events would not be measured by this method. The met hod was linear for 4-20 mu g DNA added per tube. The minimum measurabl e amount of thymidine glycol in DNA sample was 36 fmol. An increased l evel of thymidine glycol was measured in salmon sperm DNA which had au toxidized during storage in a refrigerated aqueous solution, 71.2 +/- 14.3 x 10(-6) mol thymidine glycol per 1 mol thymidine. (C) 1996 Acade mic Press, Inc.