TY - JOUR
T1 - Advances in the stable isotope-mass spectrometric measurement of DNA synthesis and cell proliferation
AU - Neese, Richard A.
AU - Siler, Scott Q.
AU - Cesar, Denise
AU - Antelo, Fernando
AU - Lee, Dan
AU - Misell, Lisa
AU - Patel, Ketan
AU - Tehrani, Shandiz
AU - Shah, Payal
AU - Hellerstein, Marc K.
N1 - Funding Information:
These studies were funded in part by grants to M.K.H. from the National Institutes of Health (AI41401, AI43866, and AI44767) and a gift from KineMed, Inc. Human studies were supported by Grant 5-M01-RR00082-36 from the National Center for Research Resources, National Institutes of Health, to the General Clinical Research Center at San Francisco General Hospital.
PY - 2001/12/15
Y1 - 2001/12/15
N2 - 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 isotope-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 limitations, however. Here, we describe technical improvements that increase yield, stability, sensitivity, and reproducibility of the method. The purine deoxyribonucleoside, deoxyadenosine (dA), is directly isolated from hydrolysates of DNA by using an LC18 SPE column. Two derivatives were developed for analyzing the dR moiety of dA alone (without the base), an aldonitrile-triacetate derivative, and a reduced pentosetetraacetate (PTA) derivative. The PTA derivative in particular exhibited greater stability (no degradation after 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), compared to previous derivatives of dA. The need for complex, multidimensional abundance corrected standard curves was thereby avoided. Using the PTA derivative, dR enrichments from DNA of fully turned over cells of rodents with 2H2O enrichments in body water of 2.2-2.8% were 9.0-9.5%, and less than 1.0 μg DNA (ca. 2 × 105 cells) was required for reproducible analyses. In summary, these methodologic advances allow measurement of stable isotope incorporation into DNA and calculation of cell proliferation and death rates in vivo in humans and experimental animals, with fewer cells, greater reproducibility, and less labor. Many applications of this approach can be envisioned.
AB - 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 isotope-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 limitations, however. Here, we describe technical improvements that increase yield, stability, sensitivity, and reproducibility of the method. The purine deoxyribonucleoside, deoxyadenosine (dA), is directly isolated from hydrolysates of DNA by using an LC18 SPE column. Two derivatives were developed for analyzing the dR moiety of dA alone (without the base), an aldonitrile-triacetate derivative, and a reduced pentosetetraacetate (PTA) derivative. The PTA derivative in particular exhibited greater stability (no degradation after 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), compared to previous derivatives of dA. The need for complex, multidimensional abundance corrected standard curves was thereby avoided. Using the PTA derivative, dR enrichments from DNA of fully turned over cells of rodents with 2H2O enrichments in body water of 2.2-2.8% were 9.0-9.5%, and less than 1.0 μg DNA (ca. 2 × 105 cells) was required for reproducible analyses. In summary, these methodologic advances allow measurement of stable isotope incorporation into DNA and calculation of cell proliferation and death rates in vivo in humans and experimental animals, with fewer cells, greater reproducibility, and less labor. Many applications of this approach can be envisioned.
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U2 - 10.1006/abio.2001.5375
DO - 10.1006/abio.2001.5375
M3 - Article
C2 - 11700973
AN - SCOPUS:0035892726
SN - 0003-2697
VL - 298
SP - 189
EP - 195
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -