Analysis of oxidative damage to DNA bases by GC-MS enables identification of a range of base oxidation products, but requires a derivatization procedure. However, derivatization at high temperature in the presence of air can cause 'artifactual' oxidation of some undamaged bases, leading to an overestimation of their oxidation products, including @entity25469 . Therefore derivatization conditions that could minimize this problem were investigated. Decreasing derivatization temperature to 23 degrees C lowered levels of @entity25469 , @entity38495 , @entity70375 and @entity28576 measured by GC-MS in hydrolysed @entity291 thymus DNA. Addition of the reducing agent @entity48311 (5%, v/v) to DNA samples during trimethylsilylation at 90 degrees C also decreased levels of these four oxidized DNA bases as well as @entity70376 . Removal of @entity2774 from hydrolysed DNA samples by treatment with guanase, prior to derivatization, resulted in @entity25469 levels (54-59 pmol/mg of DNA) that were significantly lower than samples not pretreated with guanase, independent of the derivatization conditions used. Only hydrolysed DNA samples that were derivatized at 23 degrees C in the presence of @entity48311 produced @entity25469 levels (56+/-8 pmol/mg of DNA) that were as low as those of guanase-pretreated samples. Levels of other oxidized bases were similar to samples derivatized at 23 degrees C without @entity48311 , except for @entity70375 and @entity70376 , which were further decreased by @entity48311 . Levels of @entity25469 , @entity38495 and @entity70375 measured in hydrolysed @entity291 thymus DNA by the improved procedures described here were comparable with those reported previously by HPLC with electrochemical detection and by GC-MS with prepurification to remove undamaged base. We conclude that artifactual oxidation of DNA bases during derivatization can be prevented by decreasing the temperature to 23 degrees C, removing air from the derivatization reaction and adding @entity48311 .

Measurement of oxidative DNA damage by gas chromatography-mass spectrometry: XXXX prevents artifactual generation of oxidized DNA bases.

@entity25469 :: ('MESH:C024829', 'Chemical') :: ['8-hydroxyguanine'] @entity70376 :: ('MESH:C045975', 'Chemical') :: ['5-hydroxyuracil'] @entity28576 :: ('MESH:C008055', 'Chemical') :: ['5-(hydroxymethyl)uracil'] @entity70375 :: ('MESH:C017400', 'Chemical') :: ['5-hydroxycytosine'] @entity38495 :: ('MESH:C002992', 'Chemical') :: ['8-hydroxyadenine'] @entity2774 :: ('MESH:D006147', 'Chemical') :: ['guanine'] @entity48311 :: ('MESH:C007638', 'Chemical') :: ['ethanethiol'] @entity291 :: ('9913', 'Species') :: ['calf']
@entity48311 :: (MESH:C007638,Chemical) :: ['ethanethiol']
