Oxidized DNA Bases in Breast Cancer
Abstract
5-Hydroxymethyl-2'-deoxyuridine (HMdU), an oxidized DNA base, has been proposed as a biomarker of breast cancer risk, but there are no facile assays to measure HMdU. We have developed a simple and sensitive fluorometric method for HMdU quantitation, which should be useful in the epidemiological studies. This method utilizes 7-dimethylamino-coumarin-3-acetic-acid (DMACA) as the fluorescent probe for the post-column HMdU derivatization. DMACA postlabeling of HMdU results in the formation of two baseline-separated products, while reaction with dU or dT results in a single fluorescent product. This indicates that post-labeling occurs on primary alcohols. Mass spectral analysis suggests that dT*, dU* and HMdU*I products contain the fluorofore on the 5'-CH2OH group (sugar moiety), while HMdU*II on the 5-CH2Oll of the base. The product quantitation is linear within the range of 2 pmole to 5 fmole, while limit of detection is about 0.01 fmole. Further physiochemical characterization of the products is underway. To quantitate HMdU, cellular DNA will be enzymatically digested to nucleosides, which are well separated by reverse-phase HPLC-1. HMdU-containing fractions are concentrated, subjected to coumarin post-labeling and quantitated using normal phase HPLC-2 with fluorescence detection at (lambda)(ex) = 380 nm and (lambda)(em) = 460 nm. We are in the process of method validation.
Document Details
- Document Type
- Technical Report
- Publication Date
- Aug 01, 2000
- Accession Number
- ADA385945
Entities
People
- Mehul Desai
Organizations
- New York University