title: Liquid chromatography/mass spectrometry analysis of bifunctional electrophiles and DNA adducts from vitamin C mediated decomposition of 15-hydroperoxyeicosatetraenoic acid.
abstract: Reactive oxygen species convert the omega-6 polyunsaturated fatty acid arachidonic acid into 15-hydroperoxy-5,8,11,13-(Z,Z,ZE)-eicosatetraenoic acid (15-HPETE). Cyclooxygenases and lipoxygenases can also convert arachidonic acid into 15-HPETE. Vitamin C mediated decomposition of 15(S)-HPETE to protein- and DNA-reactive bifunctional electrophiles was examined by normal-phase liquid chromatography/atmospheric pressure chemical ionization/mass spectrometry (LC/APCI-MS). The individual bifunctional electrophiles, trans-4,5-epoxy-2(E)-decenal (t-EDE), cis-4,5-epoxy-2(E)-decenal (c-EDE), 4-oxo-2(E)-nonenal (ONE), and 4-hydroxy-2(E)-nonenal (HNE), exhibited protonated molecules 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. All five bifunctional electrophiles were formed when 15-HPETE was treated with vitamin C. The LC/MS-based methodology showed that t-EDE was the major bifunctional electrophile formed during vitamin C mediated 15(S)-HPETE decomposition. Stable isotope dilution LC/MS studies revealed that this did not result in the formation of increased levels of unsubstituted etheno-dGuo adducts in calf thymus DNA when compared with 13(S)-hydroperoxy-9,10-(Z,E)-octadecadienoic acid [13(S)-HPODE], a lipid hydroperoxide derived from linoleic acid. However, the formation of heptanone-etheno-dGuo adducts in calf thymus DNA was reduced when compared with the 13(S)-HPODE. This was attributed to the reduced formation of ONE from 15-HPETE when compared with its formation from 13-HPODE. In contrast to reactions with dGuo or DNA conducted using 13(S)-HPODE, no carboxy-containing adducts were observed with 15(S)-HPETE.
authors.affiliation: Center for Cancer Pharmacology, University of Pennsylvania School of Medicine, 854 BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104-6160, USA.
authors.forename: Michelle V
authors.initials: MV
authors.lastname: Williams
authors.forename: Seon Hwa
authors.initials: SH
authors.lastname: Lee
authors.forename: Ian A
authors.initials: IA
authors.lastname: Blair
mesh_terms: D001205:Ascorbic Acid / Q000032:analysis / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D018736:DNA Adducts / Q000032:analysis* / Q000737:chemistry*; D004583:Electrons; D015289:Leukotrienes / Q000032:analysis / Q000737:chemistry*; D008054:Lipid Peroxides / Q000032:analysis / Q000737:chemistry*; D010084:Oxidation-Reduction; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D018736:DNA Adducts; D015289:Leukotrienes; D008054:Lipid Peroxides; C025086:15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; D001205:Ascorbic Acid