title: Mass spectrometric quantitation of deoxyguanosine and leukotriene A4-deoxyguanosine adducts of DNA.
abstract: An assay was developed using electrospray ionization negative ion tandem mass spectrometry (MS) to identify and quantitate the major product in the reaction of leukotriene A(4) (LTA(4)) with deoxyguanosine (dGuo). A second quantitative assay was established using the same separation and detection techniques to determine the amount of dGuo isolated from enzymatically processed DNA. The amount of LTA(4)-dGuo adduct could then be analytically determined in DNA samples and normalized to the amount of dGuo that had been simultaneously derived from the DNA sample. Stable isotope-labeled internal standards used for these quantitative assays were readily synthesized from isotopically labeled [(15)N(5)(13)C(10)]deoxyguanosine triphosphate and analyzed for isotopic purity using MS. A comparison of fragment ions formed from stable isotope analogs of dGuo revealed the loss of deoxyribose and secondarily the loss of a series of stable neutral small molecules in a fashion similar to patterns described previously for the collisional fragmentation of protonated guanine determined by positive ion fast atom bombardment/MS/MS. The combined quantitative assays were used for the determination of the amount of endogenously formed LTA(4)-dGuo adducts observed in DNA when isolated human neutrophils that had been incubated with arachidonic acid were stimulated with calcium ionophore to initiate leukotriene biosynthesis.
authors.affiliation: Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206, USA.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Hankin
authors.forename: Robert C
authors.initials: RC
authors.lastname: Murphy
mesh_terms: D016718:Arachidonic Acid / Q000494:pharmacology; D002478:Cells, Cultured; D018736:DNA Adducts / Q000737:chemistry*; D003849:Deoxyguanosine / Q000737:chemistry*; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D017572:Leukotriene A4 / Q000096:biosynthesis / Q000737:chemistry*; D009504:Neutrophils / Q000737:chemistry / Q000378:metabolism; D012015:Reference Standards; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D018736:DNA Adducts; D007476:Ionophores; D017572:Leukotriene A4; D016718:Arachidonic Acid; D003849:Deoxyguanosine