Oxidized DNA Bases in Breast Cancer
Abstract
Recent studies have correlated higher levels of 5-hydroxy-methyl-2'-deoxyuridine (HMdU), an oxidized DNA base, with an increased risk of cancer when compared to controls. HMdU has been proposed to be a biomarker of breast cancer risk. We have developed a simple and sensitive fluorometric HPLC method for HMdU quantitation, which will be useful in the epidemiological studies. This method utilizes 7-dimethylamine-coumarin-3-acetic-acid (DMACA) as the fluorescent probe for the pre-column derivatization in anhydrous acetonitrile in the presence of catalyst at 65 deg C for 1 h. The method was optimized for various parameters involved in reaction. The linearity of the fluorescent labeling method was established by labeling low levels of HMdU (in the range of 5 pmol to 0.2 pmol). Commercially available salmon testes DNA was utilize to develop the method for HMdU quantitation from DNA, which is in final stage of development. To quantitate HMdU, DNA was enzymatically digested to nucleosides, which are well separated by HPLC-1. After passing through cation exchanger, HMdU fractions are concentrated, subjected to coumarin post-labeling and quantitated using normal phase HPLC-2 with fluorescence detection. This method will utilize to qunatitate HMdU from blood clots. Recent studies have correlated higher levels of 5-hydroxy- methyl-2'-deoxyuridine (HMdU), an oxidized DNA base, with an increased risk of cancer when compared to controls. HMdU has been proposed to be a biomarker of breast cancer risk. We have developed a simple and sensitive fluorometric HPLC method for HMdU quantitation, which will be useflil in the epidemiological studies. This method utilizes 7-dimethylamino- coumarin-3-acetic-acid (DMACA) as the fluorescent probe for the pre-column derivatization in anhydrous acetonitrile in the presence of catalyst at 650C for 1 h. The method was optimized for various parameters involved in reaction.
Document Details
- Document Type
- Technical Report
- Publication Date
- Aug 01, 2001
- Accession Number
- ADA405218
Entities
People
- Krystyna Frenkel
- Mehul K. Desai
Organizations
- New York University