Advances in the stable isotope-mass spectrometric measurement of DNA synthesis and cell proliferation

Methods for measuring rates of DNA synthesis, and thus cell proliferation, in humans had not been available until recently. We (D. C. Macallan, C. A. Fullerton, R. A. Neese, K. Haddock, S. S. Park, and M. K. Hellerstein, 1998 , Proc. Natl. Acad. Sci. USA 95, 708-713) recently developed a stable isoto pe-mass spectrometric technique for measuring DNA synthesis by labeling the deoxyribose (dR) moiety of purine deoxyribonucleotides through the de novo nucleotide synthesis pathway. The original analytic approach had limitatio ns, however. Here, we describe technical improvements that increase yield, stability, sensitivity, and reproducibility of the method. The purine deoxy ribonucleoside, deoxyadenosine (dA), is directly isolated from hydrolysates of DNA by using an LC18 SPE column. Two derivatives were developed for ana lyzing the dR moiety of dA alone (without the base), an aldonitrile-triacet ate derivative, and a reduced pentose-tetraacetate (PTA) derivative. The PT A derivative in particular exhibited greater stability (no degradation afte r several weeks), greater GC/MS signal, and much less abundance sensitivity of isotope ratios (i.e., less dependence of mass isotopomer abundances on the amount of material injected into the mass spectrometer source), compare d to previous derivatives of dA. The need for complex, multidimensional abu ndance corrected standard curves was thereby avoided. Using the PTA derivat ive, dR enrichments from DNA of fully turned over cells of rodents with (H2 O)-H-2 enrichments in body water of 2.2-2.8% were 9.0-9.5%, and less than 1 .0 mug DNA (ca. 2 x 10(5) cells) was required for reproducible analyses. In summary, these methodologic advances allow measurement of stable isotope i ncorporation into DNA and calculation of cell proliferation and death rates in vivo in humans and experimental animals, with fewer cells, greater repr oducibility, and less labor. Many applications of this approach can be envi sioned. (C) 2001 Academic Press.