Mass spectrometric quantitation of deoxyguanosine and leukotriene A4-deoxyguanosine adducts of DNA.
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.
Hankin
Joseph A
JA
Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206, USA.
Murphy
Robert C
RC
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*
D018736:DNA Adducts; D007476:Ionophores; D017572:Leukotriene A4; D016718:Arachidonic Acid; D003849:Deoxyguanosine