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

Richard A. Neese, Scott Q. Siler, Denise Cesar, Fernando Antelo, Dan Lee, Lisa Misell, Ketan Patel, Shandiz Tehrani, Payal Shah, Marc K. Hellerstein

Research output: Contribution to journalArticle

88 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)189-195
Number of pages7
JournalAnalytical Biochemistry
Volume298
Issue number2
DOIs
StatePublished - Dec 15 2001
Externally publishedYes

Fingerprint

Cell proliferation
Isotopes
Cell Proliferation
Deoxyribose
DNA
Derivatives
Deoxyadenosines
Deoxyribonucleotides
Deoxyribonucleosides
Body Water
Mass spectrometers
Cell death
Labeling
Rodentia
Animals
Cell Death
Nucleotides
Personnel
Degradation
Mortality

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Neese, R. A., Siler, S. Q., Cesar, D., Antelo, F., Lee, D., Misell, L., ... Hellerstein, M. K. (2001). Advances in the stable isotope-mass spectrometric measurement of DNA synthesis and cell proliferation. Analytical Biochemistry, 298(2), 189-195. https://doi.org/10.1006/abio.2001.5375

Advances in the stable isotope-mass spectrometric measurement of DNA synthesis and cell proliferation. / Neese, Richard A.; Siler, Scott Q.; Cesar, Denise; Antelo, Fernando; Lee, Dan; Misell, Lisa; Patel, Ketan; Tehrani, Shandiz; Shah, Payal; Hellerstein, Marc K.

In: Analytical Biochemistry, Vol. 298, No. 2, 15.12.2001, p. 189-195.

Research output: Contribution to journalArticle

Neese, RA, Siler, SQ, Cesar, D, Antelo, F, Lee, D, Misell, L, Patel, K, Tehrani, S, Shah, P & Hellerstein, MK 2001, 'Advances in the stable isotope-mass spectrometric measurement of DNA synthesis and cell proliferation', Analytical Biochemistry, vol. 298, no. 2, pp. 189-195. https://doi.org/10.1006/abio.2001.5375
Neese, Richard A. ; Siler, Scott Q. ; Cesar, Denise ; Antelo, Fernando ; Lee, Dan ; Misell, Lisa ; Patel, Ketan ; Tehrani, Shandiz ; Shah, Payal ; Hellerstein, Marc K. / Advances in the stable isotope-mass spectrometric measurement of DNA synthesis and cell proliferation. In: Analytical Biochemistry. 2001 ; Vol. 298, No. 2. pp. 189-195.
@article{75b3bf7210214f288bbe96cafd247f3e,
title = "Advances in the stable isotope-mass spectrometric measurement of DNA synthesis and cell proliferation",
abstract = "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.",
author = "Neese, {Richard A.} and Siler, {Scott Q.} and Denise Cesar and Fernando Antelo and Dan Lee and Lisa Misell and Ketan Patel and Shandiz Tehrani and Payal Shah and Hellerstein, {Marc K.}",
year = "2001",
month = "12",
day = "15",
doi = "10.1006/abio.2001.5375",
language = "English (US)",
volume = "298",
pages = "189--195",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "2",

}

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.

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.

UR - http://www.scopus.com/inward/record.url?scp=0035892726&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035892726&partnerID=8YFLogxK

U2 - 10.1006/abio.2001.5375

DO - 10.1006/abio.2001.5375

M3 - Article

C2 - 11700973

AN - SCOPUS:0035892726

VL - 298

SP - 189

EP - 195

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 2

ER -