Poraplot q 25 0.32 column

Genomic DNA was isolated from sorted cell samples (typically consisting of >250,000 cells) using the ReliaPrep Blood gDNA Miniprep system (Promega) according to manufacturer’s instructions. ²H incorporation in DNA was measured by gas chromatography and mass spectrometry (GC/MS) as previously described (16 (link)) with minor modifications. Briefly, after enzymatic hydrolysis of the DNA, purine deoxyribonucleotides were derivatized to perfluorotriacetyl (PFTA) and injected into the gas chromatograph (DB-17MS column; 7890A GC System, Agilent Technologies). The mass of the derivate was measured by negative chemical ionization mass spectrometry (5975C inert XL EI/CI MSD; Agilent Technologies) at m/z 435 (M0) and 436 (M1). Standards of known isotopic enrichments were used to control for varying sample concentrations, as reported previously (17 (link)). Plasma was obtained by centrifugation of whole blood samples and deuterium enrichment was measured on the same GC/MS system (using a PoraPLOT Q 25 × 0.32 column, Varian) as described by Westera et al. (18 (link)).

Deuterium enrichment in plasma and DNA was measured by GC/MS using an Agilent 5973/6890 GC/MS system (Agilent Technologies). Plasma was derivatized to acetylene (C₂H₂, M = 26) as previously described (29 (link)). The derivative was injected into the GC/MS equipped with a PoraPLOT Q 25 × 0.32 column (Varian), and measured in SIM mode monitoring ions m/z 26 (M+0) and m/z 27 (M+1). From the ratio of ions, plasma deuterium enrichment was calculated by calibration against ²H₂0 standards of known enrichment. DNA obtained from sorted lymphocytes and granulocytes was hydrolyzed to deoxy-ribonucleotides and derivatized to penta-fluoro-triacetate (PFTA, M = 435) (29 (link)). The derivative was injected into the GC/MS equipped with a DB-17 column (Agilent Technologies) and measured in SIM mode monitoring ions m/z 435 (M+0), and m/z 436 (M+1). From the ratio of ions, we calculated the deuterium enrichment in the DNA by calibration against deoxyadenosine standards of known enrichment as previously described (6 (link)).