The effect of experimental conditions on the levels of oxidatively modified bases in DNA as measured by gas chromatography-mass spectrometry: How many modified bases are involved? Prepurification or not?

Recently, an artifactual formation of a number of modified DNA bases has be en alleged during derivatization of DNA hydrolysates to be analyzed by gas chromatography-mass spectrometry (GC-MS). These modified bases were 8-hydro xyguanine (8-OH-Gua), 5-hydroxycytosine (5-OH-Cyt), 8-hydroxyadenine (8-OH- Ade), 5-hydroxymethyl-uracil (5-OHMeUra), and 5-formyluracil, which represe nt only a small percentage of more than 20 modified DNA bases that can be a nalyzed by GC-MS. However, relevant papers reporting the levels of these mo dified bases in DNA of various sources have not been cited, and differences in experimental procedures have not been discussed. We investigated the le vels of modified bases in calf thymus DNA by GC-MS using derivatization at three different temperatures. The results obtained with GC/isotope-dilution MS showed that the levels of 5-OH-Cyt, 8-OH-Ade, 5-OH-Ura, and 5-OHMeUra w ere not affected by increasing the derivatization temperature From 23 degre es C to 120 degrees C. The level of 8-OH-Gua was found to be higher at 120 degrees C. However, this level was much lower than those reported previousl y. Formamidopyrimidines were readily analyzed in contrast to some recent cl aims. The addition of trifluoroacetic acid (TFA) adversely affected the lev els of pyrimidine-derived lesions, suggesting that TFA is not suitable for simultaneous measurement of both pyrimidine- and purine-derived lesions. Th e data obtained were also compared with those previously published. Our dat a and this comparison indicate that no artifactual formation of 5-OH-Cyt, 8 -OH-Ade, and 5-OHMeUra occurred under our experimental conditions in contra st to recent claims, and no prepurification of DNA hydrolysates by a tediou s procedure is necessary for accurate quantification of these compounds. Th e artifactual formation of 8-OH-Gua can be eliminated by derivatization at room temperature for at least 2 h, without the use of TFA. The results in t his article and their comparison with published data indicate that differen t results may be obtained in different laboratories using different experim ental conditions. The data obtained in various laboratories should be compa red by discussing all relevant published data and scientific facts, includi ng differences between experimental conditions used in different laboratori es. (C) 1999 Elsevier Science Inc.