Lee Seon Hwa, Oe Tomoyuki, Arora Jasbir S, Blair Ian A
Center for Cancer Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, 19104-6160, USA.
J Mass Spectrom. 2005 May;40(5):661-8. doi: 10.1002/jms.838.
Intracellular Fe(II), which is up-regulated during oxidative stress and during iron overload, induces the formation of a hydroxyl radical by Fenton chemistry. The hydroxyl radical can convert the prototypic omega-6 polyunsaturated fatty acid, linoleic acid, to 13-hydroperoxy-9,11-(Z,E)-octadecadienoic acid (13-HPODE). Cyclooxygenases can also convert linoleic acid to 13(S)-HPODE during oxidative stress. Subsequent Fe(II)-mediated decomposition to protein- and DNA-reactive bifunctional electrophiles was examined by normal-phase liquid chromatography (LC)/atmospheric pressure chemical ionization (APCI)/mass spectrometry. The potential individual bifunctional electrophiles trans-4,5-epoxy-2(E)-decenal (EDE), cis-EDE, 4-oxo-2(E)-nonenal (ONE) and 4-hydroxy-2(E)-nonenal (HNE) exhibited protonated molecular ions at m/z 169, 169, 155 and 157, respectively. The MH(+) ion at m/z 173 for 4-hydroperoxy-2(E)-nonenal (HPNE) was very weak with an ion corresponding to the loss of OH at m/z 156 as the major ion in the APCI mass spectrum. The bifunctional electrophiles were all separated under normal-phase LC conditions. Interestingly, ions corresponding to ONE and HNE were detected at the same retention time as HPNE, suggesting that it decomposed in the source of the mass spectrometer to ONE and HNE. All five bifunctional electrophiles were formed when 13-HPODE was treated with 50 microM Fe(II). At this concentration of Fe(II), the addition of vitamin C resulted in increased bifunctional electrophile formation. At higher concentrations of Fe(II) (500 microM to 2 mM), no HPNE was detected and there was no additive effect of vitamin C. Additional experiments with synthetic HPNE revealed that it was quantitatively converted to a mixture of ONE and HNE by Fe(II). The HNE is thought to arise from a one-electron reduction of an alkoxy radical derived from HPNE. In contrast, ONE can arise through an alpha-cleavage of the HPNE-derived alkoxy radical or by direct dehydration of HPNE.
细胞内的亚铁离子（Fe(II)）在氧化应激和铁过载期间会上调，通过芬顿化学反应诱导羟基自由基的形成。羟基自由基可将典型的ω-6多不饱和脂肪酸亚油酸转化为13-氢过氧-9,11-(Z,E)-十八碳二烯酸（13-HPODE）。在氧化应激期间，环氧化酶也可将亚油酸转化为13(S)-HPODE。随后，通过正相液相色谱（LC）/大气压化学电离（APCI）/质谱法研究了Fe(II)介导的13-HPODE分解为对蛋白质和DNA具有反应性的双功能亲电试剂的过程。潜在的单个双功能亲电试剂反式-4,5-环氧-2(E)-癸烯醛（EDE）、顺式-EDE、4-氧代-2(E)-壬烯醛（ONE）和4-羟基-2(E)-壬烯醛（HNE）的质子化分子离子分别出现在m/z 169、169、155和157处。4-氢过氧-2(E)-壬烯醛（HPNE）的m/z 173处的MH(+)离子非常弱，在APCI质谱图中，对应于失去OH的m/z 156处的离子为主要离子。在正相LC条件下，所有双功能亲电试剂均被分离。有趣的是，在与HPNE相同的保留时间检测到了对应于ONE和HNE的离子，这表明它在质谱仪的离子源中分解为ONE和HNE。当用50 microM的Fe(II)处理13-HPODE时，所有五种双功能亲电试剂均形成。在此Fe(II)浓度下，添加维生素C会导致双功能亲电试剂的形成增加。在较高浓度的Fe(II)（500 microM至2 mM）下，未检测到HPNE，且维生素C没有相加作用。用合成的HPNE进行的额外实验表明，它被Fe(II)定量转化为ONE和HNE的混合物。据认为，HNE是由HPNE衍生的烷氧基自由基的单电子还原产生的。相比之下，ONE可通过HPNE衍生的烷氧基自由基的α-裂解或HPNE的直接脱水产生。
