posted on 2013-05-07, 00:00authored byDon E. Farthing, Nataliya P. Buxbaum, Catherine
V. Bare, Shirin M. Treadwell, Veena Kapoor, Kirsten M. Williams, Ronald
E. Gress
A rapid and sensitive
gas chromatography-tandem mass spectrometry (GC-MS/MS) method was
developed to quantitatively measure low levels of DNA base deoxyadenosine
(dA) and its isotopologues (e.g., dA M+1) from limited mouse cell
populations. Mice undergoing allogeneic hematopoietic transplantation
(AHSCT) received deuterated water at biologically relevant time intervals
post AHSCT, allowing labeling of DNA upon cell division, which was
detected as the dA M+1 isotopologue. Targeted mouse cell populations
were isolated from lymphoid organs and purified by multiparameter
fluorescence activated cell sorting. Cell lysis, DNA extraction, and
hydrolysis were accomplished using available commercial procedures.
The novel analytical method utilized a hydrophilic–lipophilic
balanced sample preparation, rapid online hot GC inlet gas phase sample
derivatization, fast GC low thermal mass technology, and a recently
marketed GC-MS/MS system. Calibration standards containing dA and
fortified with relevant levels of dA M+1 (0.25–20%) and dA
M+5 (internal standard) were used for sample quantitation. The method
employed a quadratic fit for calibration of dA M+1 (0.25–20%)
and dA, demonstrated excellent accuracy and precision, and had limits
of detection of 100 fg on-column for the dA isotopologues. The method
was validated and required only 20 000 cells to characterize
population dynamics of cells involved in the biology of chronic graft-versus-host
disease, the main cause of late morbidity and nonrelapse-mortality
following AHSCT. The high sensitivity and specificity of the method
makes it useful for investigating in vivo kinetics
on limited and important cell populations (e.g., T regulatory cells)
from disease conditions or in disease models that are immune-mediated,
such as diabetes, human immunodeficiency virus/acquired immunodeficiency
syndrome (HIV/AIDS), arthritis, inflammatory bowel disease, and multiple
sclerosis.