The DNA synthesis rate is such a fundamental parameter of biological function that expanding ways to measure it would have substantial health consequences. A nontoxic method that measured DNA synthesis rate in vivo would apply to such fields as: hematology and oncology to monitor damage to various tissues, especially the blood-forming cells; immunology, where lymphocyte proliferation rates could be quantified; and aging, where effects of various therapeutic strategies to slow or stimulate certian processes could be determined at the cellular level. However, no suitable method for making this measurement in humans exists. The purpose of this this applicaiton is to develop a safe method for measuring DNA synthesis rates in vivo that can be applied to humans. For this purpose, a newly-developed instrumental configuration consisting of a High Performance Liquid Chromatograph, a Chemical Reaction Interface, and an Isotope Ratio Mass Spectrometer (HPLC/CRI/IRMS) will be used. The strategy is to administer a stable-isotopically labeled precursor to DNA, then remove the DNA from certain cell populations, hydrolyze it, and measure the 13C isotopic enrichment in one or more of the nucleosides contained in the DNA. Based on data presented as Preliminary Studies, this measurement is feasible. This proposal will optimize the method by evaluating three different 13C-labeled precursors to DNA , first in cell culture systems, and then in animal models. The goals of the research plan answer questions of: the appropriate level of enrichment needed; the dose and schedule for the labeled material; and timing for cell population sampling. The ability of the stable-isotope method to monitor changes in DNA synthesis rates in comparison to 3H-thymidine will be proved. The bulk of this research is not hypothesis-driven, but technology-driven. Beyond the technical components, experiments are proposed to answer an important biological uncertainty-whether cells taken from the body and examined in vitro are comparable to their in viv behavior. When the research is completed, a well-grounded method to determine DNA synthesis rates in vivo will be available for the next step-testing in humans.
| Black, Gavin E; Abramson, Fred P (2003) Measuring DNA synthesis rates with stable isotopes. Anal Chem 75:56A-63A |
| Black, G E; Boller, Y C; Kennedy, K A et al. (2001) Labeling DNA with stable isotopes: economical and practical considerations. Biotechniques 30:134-8, 140 |
| Abramson, F P (2001) The use of stable isotopes in drug metabolism studies. Semin Perinatol 25:133-8 |
| Abramson, F P; Black, G E; Lecchi, P (2001) Application of high-performance liquid chromatography with isotope-ratio mass spectrometry for measuring low levels of enrichment of underivatized materials. J Chromatogr A 913:269-73 |
