Measurement of very low rates of cell proliferation by heavy water labeling of DNA and gas chromatography/pyrolysis/isotope ratio–mass spectrometric analysis

Abstract

DNA replication during S-phase represents a biochemical metric of cell division. We present here a protocol for measuring very low rates of cell proliferation, on the basis of the incorporation of deuterium ((2)H) from heavy water ((2)H(2)O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells, by use of gas chromatography/pyrolysis/isotope ratio-mass spectrometry (GC/P/IRMS). Very low levels of label incorporation (>or=0.002% atom percent excess (2)H) can be quantified by GC/P/IRMS. This protocol thereby permits shorter periods or lower amounts of (2)H(2)O administration than would be required using standard GC/MS techniques for measuring cell proliferation kinetics (see accompanying protocol in this issue). A disadvantage of this approach compared to GC/MS is the requirement for larger numbers of cells (> approximately 10(7)). This protocol enables definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans, even when turnover rates are very low. Indolent hematologic malignancies, such as chronic lymphocytic leukemia, and other relatively quiescent cells represent promising areas of application.

Lymphocyte kinetics in health and disease

“…that there is increased activation of CD8 + T cells in HIV infection [kinetic-heterogeneity model]). Technological developments to improve the sensitivity of DNA analysis will increase our ability to analyse the proliferative dynamics of rare phenotypic lymphocyte subsets [50,54], and this might enable us to evaluate kinetic heterogeneity directly.…”

“…Loss of label during the wash-out phase can be used to provide information about the disappearance of labelled cells. There are currently two approaches for delivering stable isotopes: labelling with deuterated glucose [44,45] and labelling with heavy (deuterated) water [50,54]. There are several methodological differences in how the label is administered and the data generated when using these two labels ( Figure 1 in main text).…”

Measurement of Ribosomal RNA Turnover In Vivo by Use of Deuterium-Labeled Glucose

“…A universal issue in labeling studies is recirculation of label, which is likely to be minimal for deuterated glucose because glucose is rapidly metabolized and deuterium is primarily lost into the very large body water pool after oxidation. Although heavy water ( 2 H 2 O) is useful for labeling DNA in slowly dividing cells, in which incorporation of deuterium takes place over weeks (14,28 ), 2 H 2 O would not be suited to RNA analysis because of the relatively high rates of RNA turnover; RNA pools would become saturated with label and useful information obscured.…”

Modelling deuterium labelling of lymphocytes with temporal and/or kinetic heterogeneity

“…Animals and humans have been labelled with deuterated glucose ( 2 H 2 -glucose) or heavy water ( 2 H 2 O) for 1 or 2 days [2-6], 5 days [7] to one week [8] or several months [9][10][11], and have subsequently been followed to study the loss of label during a washout phase. After deuterium labelling, one sorts the cell population of interest, isolates the DNA from the cells, and uses mass spectrometry to determine the enrichment of deuterium in the DNA [12][13][14][15]. During the labelling period, some fraction of the hydrogen atoms in the body will be replaced by deuterium, and dividing cells will incorporate both deuterium and hydrogen in newly synthesized DNA molecules.…”