Simultaneous Quantification of 1,N~(2)-Propano-2'-deoxyguanosine Adducts Derived from Acrolein and Crotonaldehyde in Human Placenta and Leukocytes by Isotope Dilution Nanoflow LC Nanospray Ionization Tandem Mass Spectrometry

摘要

Humans are exposed to acrolein and crotonaldehyde due to environmental pollution and endogenous lipid peroxi-dation. These aldehydes react with the 2'-deoxyguanosine moiety of DNA, forming the exocyclic 1,N~(2)-propano-2'-deoxyguanosine adducts AdG and CdG. These adducts are mutagenic lesions, and they play an important role in cancer and neurodegenerative diseases. In this study, a highly sensitive and quantitative assay was developed for simultaneous detection and quantification of AdG and CdG isomers in human placenta and leukocyte DNA by isotope dilution nanoflow LC with nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS). The on-column detection limits (S/N >= 3) of AdG and CdG were 15 and 8.9 amol, respectively. The quantification limits of AdG and CdG for the entire assay were 619 and 297 amol, respectively, corresponding to 9.8 and 4.7 adducts in 10~(9) normal nucleotides, respectively, starting with 20 (mu)g of DNA. Different enzyme hydrolysis methods were compared, and the optimal hydrolysis conditions were employed for the assay. Levels of AdG and CdG in human placental DNA (20 (mu)g) were 108 and 26 in 10~(8) normal nucleotides, respectively, with the respective relative standard deviation (RSD) of 2.6percent and 3.1percent (n velence 3). Levels of AdG and CdG in 9 human leukocyte DNA samples were 78 +- 23 (mean +- SD) and 6.2 +- 3.8 (mean +- SD) in 10~(8) normal nucleotides, respectively, starting from 30 (mu)g of DNA. Using this assay, only 4-6 (mu)g of DNA sample was subjected to this nanoLC-NSI/MS/MS system for analysis. Only 1-1.5 mL of blood is needed for measuring AdG and CdG levels in leukocyte DNA. Thus, it is clinically feasible using this highly sensitive assay to investigate the potential of using these adducts as noninvasive biomarkers for DNA damage resulting from acrolein and crotonaldehyde and to study their roles in cancer development and prevention.