Abstract
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 ω‐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 µM 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 µM 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 α‐cleavage of the HPNE‐derived alkoxy radical or by direct dehydration of HPNE. Copyright © 2005 John Wiley & Sons, Ltd.