Sensitive GC-MS/MS Method to Measure Deuterium Labeled Deoxyadenosine in DNA from Limited Mouse Cell Populations

Abstract

A rapid and sensitive 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 multi-parameter 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 on-line 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 non-relapse-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, HIV/AIDS, arthritis, inflammatory bowel disease, and multiple sclerosis.

In vivo kinetics and nonradioactive imaging of rapidly proliferating cells in graft-versus-host disease

“…Subsequently, we compared the deuterium enriched fraction of deoxyadenosine (dA [M+1]) to unenriched dA, which allowed calculation of the fraction of new Tregs generated during each labeling period (38). Deuterium enrichment in all 4 DNA nucleosides: dA, deoxythymidine (dT), deoxycytosine (dC), or deoxyguanine (dG), can be measured; however, we selected a single nucleoside to allow high throughput analysis, and dA was chosen because DNA of rodents and humans has a higher dA-dT content than dG-dC (39,40), allowing superior sensitivity of detection when evaluating a small number of cells, as low as 10,000 per analysis (38). As previously shown, 2 H 2 O in TBW incorporates into DNA nucleosides through the de novo nucleotide synthesis pathway (16).…”

“…Deuterated water labeling was performed according to previously published protocol (38). Briefly, deuterated water was provided in drinking water after an initial i.v.…”

“…DNA extraction from nonfixed cells was performed on the Promega Maxwell 16 system as previously described (38). For DNA extraction from sorted fixed cells, EpiSonic 1100 Sonication System was used.…”

“…Quantitative determination of dA, its isotopologue (dA [M+1]) and the internal standard (dA M+5) was measured using validated GC-MS/MS methodology (38). Briefly, DNA extracted from FACS purified T cell subsets was hydrolyzed to its bp using EpiQuick DNA Hydrolysis Kit (EpiGentek).…”

Comparing DNA enrichment of proliferating cells following administration of different stable isotopes of heavy water

“…molecules that differ in isotopic composition, leading to different molecular weights) 10 . Other researchers have used D 2 O (long-term labeling) or D 2 -glucose (short-term labeling), and measured some form of an isotopologue ratio (e.g.…”