Facts about the artifacts in the measurement of oxidative DNA base damage by gas chromatography mass spectrometry

Recently, several papers reported an artifactual formation of a number of m odified bases from intact DNA bases during derivatization of DNA hydrolysat es to be analyzed by gas chromatography-mass spectrometry (GC/MS). These re ports dealt with 8-hydroxyguanine (8-OH-Gua), 5-hydroxycytosine (5-OH-Cyt), 8-hydroxyadenine (8-OH-Ade), 5-hydroxymethyluracil (5-OHMeUra) and 5-formy luracil that represent only a small percentage of the 20 or so modified DNA bases that can be analyzed by GC/MS. Removal of intact DNA bases by prepur ification of calf thymus DNA hydrolysates using HPLC was shown to prevent a rtifactual formation of these modified bases during derivatization. It need s to be emphasized that the procedures for hydrolysis of DNA and derivatiza tion of DNA hydrolysates used in these papers substantially differed from t he established procedures previously described. Furthermore, a large number of relevant papers reporting the levels of these modified bases in DNA of various sources have been ignored. Interestingly, the levels of modified ba ses reported in the literature were not as high as those reported prior to prepurification. Most values for the level of 5-OH-Cyt were even lower than the level measured after prepurification. Levels of 8-OH-Ade were quite cl ose to, or even the same as, or smaller than the level reported after prepu rification. The same holds true for 5-OHMeUra and 8-OH-Gua. All these facts raise the question of the validity of the claims about the measurement of these modified DNA bases by GC/MS. A recent paper reported a complete destr uction of 2,6-diamino-4-hydroxy-5-formamidopyrimidin (FapyGua) and 4,6-diam ino-5-formamidopyrimidine (FapyAde) by formic acid under the conditions of DNA hydrolysis prior to GC/MS. The complete destruction of FapyGua and Fapy Ade by formic acid is in disagreement with the data on these compounds in t he literature. These two compounds were measured by GC/MS following formic acid hydrolysis for many years in our laboratory and by other researchers w ith no difficulties. These facts clearly raise the question of the validity of the claims made about the previous measurements of these compounds by G C/MS.