Comparison of the Levels of 8-Hydroxyguanine in DNA as Measured by Gas Chromatography-Mass Spectrometry Following Hydrolysis of DNA by Escherichia Coli Fpg Protein or Formic Acid

Published

July 1, 2000

Author(s)

H Rodriguez, J Jurado, J Laval, M. Dizdaroglu

Abstract

8-hydroxyguanine (8-OH-Gua) is one of many lesions generated in DNA by oxidative processes including free radicals. It is the most extensively investigated lesion, due to its miscoding properties and its potential role in mutagenesis, carcinogenesis and aging, and also to the existence of analytical methods using HPLC and gas chromatography mass spectrometry (GC/MS). Some studies raised the possiblity of artifacts generated during sample preparation. We investigated several experimental conditions in order to eliminate possible artifacts during the measurement of 8-OH-Gua in acid -hydrolysates of DNA, although the extent of artifacts seems to depend on experimental conditions. For removal of 8-OH-Gua from DNA, we used either formic acid hydrolysis or specific enzymatic hydrolysis with Escherichia coli Fpg protein. Derivation of enzyme-hydrolysates should not generate additional 8-OH-Gua because of the absence of guanine, which is not released by the enzyme, whereas guanine released by acid may be oxidized to yield 8-OH-Gua. The measurement of 8-OH-Gua in calf thymus DNA by GC/isotope-dilution MS (GC/IDMS) using these two different hydrolyses yielded similar levels of 8-OH-Gua. This indicated that no artifacts occurred during derivatization fo acid-hydrolysates of DNA. Pyridine instead of acetonitrile and room temperature were used during derivatization. Pyridine reduced the level of 8-OH-Gua, when compared with acetonitrile, indicating its potential to prevent oxidation. Two different stable-isotope labeled analogs of 8-OH-Gua used as internal standards for GC/IDMS analysis yielded similar results. A comparison of the present results of recent trials by the European Standards Committee for Oxidative DNA Damage (ESCODD) is also presented.

Citation

Nucleic Acids Research

Volume

28

Issue

No. 15

Pub Type

Journals

Keywords

8-hydroxyguanine, biomarkers, gas chromatography/mass spectrometry, measurment standards, oxidative DNA damage, oxidative stress

Citation

Rodriguez, H. , Jurado, J. , Laval, J. and Dizdaroglu, M. (2000), Comparison of the Levels of 8-Hydroxyguanine in DNA as Measured by Gas Chromatography-Mass Spectrometry Following Hydrolysis of DNA by Escherichia Coli Fpg Protein or Formic Acid, Nucleic Acids Research (Accessed July 26, 2024)