Journal Article

<sup>32</sup>P-postlabeling high-performance liquid chromatography (<sup>32</sup>P-HPLC) adapted for analysis of 8-hydroxy-2′-deoxyguanosine

Magnus Zeisig, Tim Hofer, Jean Cadet and Lennart Möller

in Carcinogenesis

Volume 20, issue 7, pages 1241-1245
Published in print July 1999 | ISSN: 0143-3334
Published online July 1999 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/20.7.1241
32P-postlabeling high-performance liquid chromatography (32P-HPLC) adapted for analysis of 8-hydroxy-2′-deoxyguanosine

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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.

Keywords: 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.

Journal Article.  3967 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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