Advances in the Stable Isotope–Mass Spectrometric Measurement of DNA Synthesis and Cell Proliferation

Neese, Richard A.; Siler, Scott Q.; Cesar, Denise; Antelo, F.; Liu, Dan; Misell, Lisa; Patel, Ketan; Tehrani, Shandiz; Shah, Payal D.; Hellerstein, Marc K.

doi:10.1006/abio.2001.5375

Cited by 100 publications

(155 citation statements)

References 21 publications

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“…Enrichment of 2 H2O in body water (blood) was measured by a new GC-MS technique, after chemical conversion to tetrabromoethane (25). Briefly, the hydrogen atoms in H 2O were first transferred to acetylene by addition of 1-10 l water via syringe to a chip of calcium carbide in a sealed vial, equipped with a 3-ml syringe inserted into the septum.…”

Section: Animal Studiesmentioning

confidence: 99%

“…GC-MS anaylsis was with a DB-225, 30-m column at 220°C, with the use of methane chemical ionization with selected ion monitoring. The C 2H2Br3ϩ fragment [mass-to-charge ratio (m/z) 265 and 266, representing the M 0 and the Mϩ1 ion, respectively, of the 79 Br 79 Br 81 Br isotopologue] was used for calculating 2 H enrichment, by comparison to standard curves generated by mixing 100% 2 H2O with natural abundance H2O in known proportions (25).…”

Section: Animal Studiesmentioning

confidence: 99%

“…Bone marrow nDNA was isolated by use of a Qiamp column (Qiagen), as described previously (15,25,22). mtDNA was also isolated from cardiac muscle, hindlimb muscle, and platelets by using the Qiagen kit (Qiagen) after isolation of the mitochondrial fraction from the tissue (3,4).…”

Section: Dna Measurements and Calculationsmentioning

confidence: 99%

“…In contrast, previously described techniques for labeling DNA utilized the nucleoside salvage pathway from labeled pyrimidine nucleosides ( 3 H-dT or bromodeoxyuridine). The stable isotope, de novo nucleotide synthesis pathway labeling technique has technical advantages, as discussed previously (22,24,25), in addition to being without toxicity and, therefore, applicable to humans (22,24). Here we apply the 2 H 2 O incorporation method to the measurement of mtDNA synthesis in rodents and humans.…”

mentioning

confidence: 97%

“…Our laboratory recently described a nonradioactive, stable isotope-mass spectrometric method for measuring DNA replication (14,22,24,25). Purine deoxyribonucleotides of replicating DNA are labeled through the de novo nucleotide synthesis pathway from deuterated glucose or 2 H 2 O.…”

mentioning

confidence: 99%

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Measurement of mitochondrial DNA synthesis in vivo using a stable isotope-mass spectrometric technique

Collins

Eng

Hoh

et al. 2003

Journal of Applied Physiology

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. Measurement of mitochondrial DNA synthesis in vivo using a stable isotope-mass spectrometric technique. J Appl Physiol 94: 2203-2211, 2003. First published January 31, 2003 10.1152/japplphysiol.00691. 2002-We describe here a new stable isotope-mass spectrometric technique for measuring mitochondrial DNA (mtDNA) synthesis. Growing (2-4 mo old) and weight-stable (8-10 mo old) Sprague-Dawley rats were primed with 2 H2O (deuterated water) to 2.0-2.5% body water enrichment, via intraperitoneal injection, and then given 4% 2 H2O in drinking water for 3-11 wk. Mitochondria were isolated from cardiac and hindlimb muscle, and mtDNA was isolated and enzymatically hydrolyzed to deoxyribonucleosides. PCR confirmed the absence of nuclear DNA contamination. The isotopic enrichment of the deoxyribose moiety of deoxyadenosine was determined by GC-MS analysis, and percent new mtDNA was calculated by comparison to genomic DNA enrichments in a tissue with nearly complete turnover (bone marrow). Initial label incorporation into deoxyadenosine of mtDNA was linear, and turnover of mtDNA was observed in nongrowing adult female rats (1.1-1.3% new mtDNA per day in cardiac and skeletal muscle). Die-away curves of mtDNA after discontinuing 2 H2O administration gave a similar turnover rate constant. Human subjects were also given 2 H2O for up to 6 wk, and mitochondria from platelets were isolated.

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Section: Animal Studiesmentioning

confidence: 99%

Section: Animal Studiesmentioning

confidence: 99%

Section: Dna Measurements and Calculationsmentioning

confidence: 99%

mentioning

confidence: 97%

mentioning

confidence: 99%

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Measurement of mitochondrial DNA synthesis in vivo using a stable isotope-mass spectrometric technique

Collins

Eng

Hoh

et al. 2003

Journal of Applied Physiology

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Measurement and modeling of human T cell kinetics

et al. 2003

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The ability to measure, describe and interpret T cell kinetics is pivotal in understanding normal lymphocyte homeostasis and diseases that affect T cell numbers. Following in vivo labeling of dividing cells with 6,6-D 2 -glucose in eight healthy volunteers, peripheral blood T cells were sorted by CD4, CD8 and CD45 phenotype. Enrichment of deuterium in DNA was measured by gas chromatography-mass spectrometry. A novel model of T cell kinetics, allowing for heterogeneity within T cell pools, was used to analyze data on acquisition and loss of label and calculate proliferation and disappearance rates for each subpopulation. CD8+ lymphocytes and CD45RA + CD4 + lymphocytes had slower proliferation rates, 0.5% and 0.6% / day, respectively (doubling time about 4 months). Disappearance rates of labeled cells were similar for all cell types (7%-12% / day) and exceeded corresponding proliferation rates. This disparity may be understood conceptually in terms of either phenotypic heterogeneity (rapid versus slow turnover pools), or history (recently divided cells are more likely to die). The new kinetic model fits the data closely and avoids the need to postulate a large external source of lymphocytes to maintain equilibrium.

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Rapid turnover of T cells in acute infectious mononucleosis

et al. 2003

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During acute infectious mononucleosis (AIM), large clones of Epstein-Barr virus-specific T lymphocytes are produced. To investigate the dynamics of clonal expansion, we measured cell proliferation during AIM using deuterated glucose to label DNA of dividing cells in vivo, analyzing cells according to CD4, CD8 and CD45 phenotype. The proportion of labeled CD8 + CD45R0 + T lymphocytes was dramatically increased in AIM subjects compared to controls (mean 17.5 versus 2.8%/day; p X 0.005), indicating very rapid proliferation. Labeling was also increased in CD4 + CD45R0+ cells (7.1 versus 2.1%/day; p X 0.01), but less so in CD45RA + cells. Mathematical modeling, accounting for death of labeled cells and changing pool sizes, gave estimated proliferation rates in CD8 + CD45R0 + cells of 11-130% of cells proliferating per day (mean 47%/day), equivalent to a doubling time of 1.5 days and an appearance rate in blood of about 5×10 9 cells/day (versus 7×10 7 cells/day in controls). Very rapid death rates were also observed amongst labeled cells (range 28-124, mean 57%/day), indicating very short survival times in the circulation. Thus, we have shown direct evidence for massive proliferation of CD8 + CD45R0 + T lymphocytes in AIM and demonstrated that rapid cell division continues concurrently with greatly accelerated rates of cell disappearance.

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Advances in the Stable Isotope–Mass Spectrometric Measurement of DNA Synthesis and Cell Proliferation

Cited by 100 publications

References 21 publications

Measurement of mitochondrial DNA synthesis in vivo using a stable isotope-mass spectrometric technique

Measurement of mitochondrial DNA synthesis in vivo using a stable isotope-mass spectrometric technique

Measurement and modeling of human T cell kinetics

Rapid turnover of T cells in acute infectious mononucleosis

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