Carcinogenesis, Vol. 20, No. 7, 1241-1245,
July 1999
© 1999 Oxford University Press
Carcinogenesis |
32P-postlabeling high-performance liquid chromatography (32P-HPLC) adapted for analysis of 8-hydroxy-2'-deoxyguanosine
Karolinska Institutet, Department of Biosciences at Novum, Unit for Analytical Toxicology, SE-141 57 Huddinge, Stockholm, Sweden and
1 Département de Recherche, Fordamensole sur la Matière Condensée,SCIB/LAN, CEA/Grenoble, FR-38054 Grenoble Cedex 9, France
8-Hydroxy-2'-deoxyguanosine (8-OH-dG) is a promutagenic lesion in DNA caused by reactive oxygen species. It normally exists at a level of 0.1–1 per 105 2'-deoxyguanosines (dG). To analyze the lesion in easily obtainable biological samples, a very sensitive analytical method is required. The method should also handle the problem with potential oxidation of dG to 8-OH-dG during workup and analysis. 32P-postlabeling high-performance liquid chromatography (32P-HPLC) is an analytical method previously used to analyze lipophilic DNA adducts at levels as low as 1 per 109 normal nucleotides when analyzing microgram amounts of DNA. This method was adapted for analysis of 8-OH-dG. The aim was to develop an analytical method that provided a high sensitivity and good reproducibility, prevented oxidation of dG present in samples to 8-OH-dG, was capable of analyzing DNA from very small samples and still offered high sample throughput and ease of use. In analysis of calf thymus DNA, the method had a detection limit of 0.1 8-OH-dG per 105 dG when 1 µg of DNA was used. The standard deviation of repeated analyses of the same sample was ±10% and the result corresponded well with the established analytical method using HPLC with electrochemical detection. 32P-HPLC is sensitive enough to enable analysis of low levels of 8-OH-dG in biological samples such as small volumes of blood, needle biopsies and tissue swabs. It also substantially reduces oxidation of dG to 8-OH-dG during sample workup and analysis.
Abbreviations: 32P-TLC, 32P-postlabeling thin-layer chromatography and autoradiography; 8-OH-dG, 8-hydroxy-2'-deoxyguanosine; dG, 2'-deoxyguanosine; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; GC–MS, gas chromatography with on-line mass spectrometry; HPLC, high-performance liquid chromatography; HPLC–EC, high-performance liquid chromatography with on-line electrochemical detection; ISA, immunoslot blot assay; MN, micrococcal nuclease; PNK, T4 polynucleotide kinase; SDS, sodium dodecyl sulphate; SPD, spleen phosphodiesterase; TLC, thin-layer chromatography; Tris, tris[hydroxymethyl] aminoethane.
2 To whom correspondence should be addressed Email: lennart.moller{at}cnt.ki.se
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  E. Nagy, M. G. Cornelius, and L. Moller Accelerated 32P-HPLC for bulky DNA adducts Mutagenesis, March 1, 2009; 24(2): 183 - 189. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-L. Ravanat, T. Douki, P. Duez, E. Gremaud, K. Herbert, T. Hofer, L. Lasserre, C. Saint-Pierre, A. Favier, and J. Cadet Cellular background level of 8-oxo-7,8-dihydro-2'-deoxyguanosine: an isotope based method to evaluate artefactual oxidation of DNA during its extraction and subsequent work-up Carcinogenesis, November 1, 2002; 23(11): 1911 - 1918. [Abstract] [Full Text] [PDF]
|
|