Chen H J, Zhang L, Cox J, Cunningham J A, Chung F L
American Health Foundation, Valhalla, New York 10595, USA.
Chem Res Toxicol. 1998 Dec;11(12):1474-80. doi: 10.1021/tx980107o.
2,3-Epoxy-4-hydroxynonanal (EH) is a bifunctional aldehyde formed by epoxidation of trans-4-hydroxy-2-nonenal, a peroxidation product of omega-6 polyunsaturated fatty acids. EH is mutagenic and tumorigenic and capable of modifying DNA bases forming etheno adducts in vitro. Recent studies showed that etheno adducts are present in tissue DNA of humans and untreated rodents, suggesting a potential endogenous role of EH in their formation. A sensitive assay is needed so we can determine whether EH is involved in etheno adduct formation in vivo and study the biological significance of the etheno adducts in DNA. In this study, we developed a gas chromatography/negative ion chemical ionization/mass spectrometry assay for the analysis of 1, N6-ethenoadenine (epsilonAde) and 7-(1', 2'-dihydroxyheptyl)-3H-imidazo[2,1-i]purine (DHH-epsilonAde) in DNA; both are products from the reaction of adenine with EH. The assay entails the following sequence of steps: (1) addition of [15N5]epsilonAde and [15N5]DHH-epsilonAde to DNA as internal standards, (2) acid hydrolysis of DNA, (3) adduct enrichment by C18 solid phase extraction (SPE), (4) derivatization by pentafluorobenzylation (PFB), (5) separation of PFB-epsilonAde and PFB-DHH-epsilonAde on a Si SPE column, (6) acetonide (ACT) formation of PFB-DHH-epsilonAde, and (7) GC/MS analysis with selective ion monitoring (SIM). The limit of detection by on-column injection for PFB-epsilonAde monitoring of the (M - PFB)- ion at m/z 158 was 30 amol and for ACT-PFB-DHH-epsilonAde monitoring of the (M - PFB)- ion at m/z 328 was 0.4 fmol; the detection limits for the entire assay were 6.3 fmol for epsilonAde and 36 fmol for DHH-epsilonAde. In calf thymus DNA modified with EH at 37 degreesC for 50 h, both epsilonAde and DHH-epsilonAde were detected at high levels by this method, 4.5 +/- 0.7 and 90.8 +/- 8.7 adducts/10(3) adenine, respectively. These levels were also verified by HPLC fluorescence analysis, indicating that EH extensively reacts with adenine in DNA, forming etheno adducts. The high sensitivity of the assay suggests that it may be used in the analysis of ethenoadenine adducts in vivo.
2,3-环氧-4-羟基壬醛（EH）是一种双功能醛，由反式-4-羟基-2-壬烯醛环氧化形成，反式-4-羟基-2-壬烯醛是ω-6多不饱和脂肪酸的过氧化产物。EH具有致突变性和致癌性，能够在体外修饰DNA碱基形成乙烯基加合物。最近的研究表明，乙烯基加合物存在于人类和未处理啮齿动物的组织DNA中，这表明EH在其形成过程中可能具有潜在的内源性作用。因此需要一种灵敏的检测方法，以便我们能够确定EH是否参与体内乙烯基加合物的形成，并研究DNA中乙烯基加合物的生物学意义。在本研究中，我们开发了一种气相色谱/负离子化学电离/质谱分析法，用于分析DNA中的1,N6-乙烯基腺嘌呤（εAde）和7-(1',2'-二羟基庚基)-3H-咪唑并[2,1-i]嘌呤（DHH-εAde）；这两种物质都是腺嘌呤与EH反应的产物。该分析方法包括以下步骤：（1）向DNA中添加[15N5]εAde和[15N5]DHH-εAde作为内标，（2）DNA的酸水解，（3）通过C18固相萃取（SPE）富集加合物，（4）通过五氟苄基化（PFB）进行衍生化，（5）在硅胶SPE柱上分离PFB-εAde和PFB-DHH-εAde，（6）PFB-DHH-εAde形成丙酮化物（ACT），（7）采用选择性离子监测（SIM）进行气相色谱/质谱分析。通过柱上进样监测m/z 158处的（M - PFB）-离子对PFB-εAde的检测限为30 amol，监测m/z 328处的（M - PFB）-离子对ACT-PFB-DHH-εAde的检测限为0.4 fmol；整个分析方法对εAde的检测限为6.3 fmol，对DHH-εAde的检测限为36 fmol。在37℃下用EH修饰小牛胸腺DNA 50小时后，用该方法检测到εAde和DHH-εAde的含量都很高，分别为4.5±0.7和90.8±8.7个加合物/10(3)个腺嘌呤。这些含量也通过高效液相色谱荧光分析得到了验证，表明EH与DNA中的腺嘌呤广泛反应，形成乙烯基加合物。该分析方法的高灵敏度表明它可用于体内乙烯基腺嘌呤加合物的分析。
