Abstract
Deuterated water (2H2O) is a label commonly used for safe quantitative measurement of deuterium enrichment into DNA of proliferating cells. More recently, it has been used for labeling proteins and other biomolecules. Our in vitro - in vivo research reports important stable isotopic labeling enrichment differences into the DNA nucleosides and their isotopologues (e.g. deoxyadenosine (dA) M + 1, dA M + 2, dA M + 3), as well as tumor cell proliferation effects for various forms of commercially available stable heavy water (2H2O, H218O, and 2H218O). Using an in vitro mouse thymus tumor cell line, we determined that H218O provides superior DNA labeling enrichment quantitation, as measured by GC-positive chemical ionization (PCI)-MS/MS. In addition, at higher but physiologically relevant doses, both 2H218O and 2H2O down modulated mouse thymus tumor cell proliferation, whereas H218O water had no observable effects on cell proliferation. The in vivo labeling studies, where normal mouse bone marrow cells (i.e. high turnover) were evaluated post labeling, demonstrated DNA enrichments concordant with measurements from the in vitro studies. Our research also reports a headspace-GC-NCI-MS method, which rapidly and quantitatively measures stable heavy water levels in total body water.
Highlights
Deuterium oxide (2H2O or D2O) has been shown to be a safe and stable form of heavy water used for cell kinetics studies, as it constitutively incorporates into the DNA nucleosides of proliferating cells[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15]
Previous T cell kinetics research from our group focused on using D2O in a pre-clinical mouse model of graft-versus-host disease (GVHD), with GC-PCI-mass spectrometry (MS)/MS quantitation of the deuterium enrichments into the DNA base deoxyadenosine and its dA M + 1 isotopologue[10]
We hypothesized that using a form of stable heavy water that would lead to DNA isotopic enrichments in the dA M + 2 or dA M + 3 isotopologue would be advantageous for MS/MS quantitation of dA and its isotopologues
Summary
Deuterium oxide (2H2O or D2O) has been shown to be a safe and stable form of heavy water used for cell kinetics studies, as it constitutively incorporates into the DNA nucleosides of proliferating cells[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15]. Other investigators using stable heavy water for their research have determined the level in total body water (TBW)[27, 28, 30,31,32,33,34,35,36,37,38,39] using infrared absorption or by utilizing test methods for plasma and urine, which involve using a metal catalyst (e.g. uranium), high temperatures (e.g. 600 °C), lengthy overnight incubations, costly solvents (e.g. 13C3-Acetone) and MS measurements of the deuterium moiety, which exchanges from the stable heavy water to a flammable gas (e.g. acetylene, hydrogen)[27, 33]. The test method is based on a rapid gas phase isotopic exchange of the hydrogen:deuterium (H:D) and 16O:18O moieties between the stable heavy water sample at a basic pH (~13–14) and acetone solvent, with quantitative measurements using headspace-GC-NCI-MS in full scan mode
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