Search

Free radical-induced oxidation products of polyunsaturated fatty acids esterified to phospholipids have been implicated in a number of human diseases including atherosclerosis and neurodegenerative diseases. Some of these phospholipid oxidation products have potent biological activities and likely contribute to human pathophysiological conditions. Oxidation products have also been used as markers of oxidative stress in vivo. Identification and quantification of phospholipid oxidation products are often performed by analyzing the oxidized free fatty acid moieties after hydrolysis from the phospholipids head groups by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS). We now describe the definitive identification of intact oxidized products of glycerophospholipids including glycerophosphatidylcholine (GPC), glycerophosphatidylethanolamine (GPE), and glycerophosphatidylserine (GPS) in vitro and in vivo using iontrap MS. For these analyses, the negative ions of the oxidation products of phospholipids are fragmented to MS(n) and unequivocal structural characterization is obtained based on collision-induced dissociation (CID) of the sn-2 carboxylate ion. This technique overcomes the need to hydrolyze fatty acids from phospholipids in the analysis. The method has been used to identify a number of oxidation products of glycerophospholipids including hydroxyeicosatetraenoates (HETEs) and isoprostanes (IsoPs) esterified to different classes of glycerophospholipids in vitro and in vivo. These studies thus provide a new approach to identify the intact oxidation products of glycerolphospholipids., (2009 John Wiley & Sons, Ltd.)

Electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) were used to analyze epoxyeicosatrienoic acids (EETs) and monohydroxyeicosatetraenoic acids (HETEs) isolated from human red blood cell membranes following base hydrolysis. ESI results in the formation of an abundant isobaric carboxylate anion at m/z 319 for both of these oxidized metabolites of arachidonic acid. The product ion spectra from the collision-induced dissociation of this carboxylate anion could be used to identify each of the isomeric eicosanoids from the unique fragment ions of each eicosanoid. The observed product ion spectra were identical with those previously obtained by fast atom bombardment ionization; however, ESI required less EET and HETE for analysis. Both EET and HETE phospholipids were present in human red blood cells (RBCs) and their abundance could be substantially increased by treatment under conditions that would induce free radical oxidation of membrane phospholipids. Following incubation of human RBCs with tert-butyl hydroperoxide (tBuOOH), phospholipids were extracted and purified by normal-phase high-performance liquid chromatography (HPLC) as to glycerophospholipid class containing ethanolamine (GPE), serine (GPS) and choline (GPC) as the polar head group. Each class of phospholipid was hydrolyzed to yield the free carboxylic acid prior to on-line HPLC/ESI-MS/MS analysis. The formation of oxidized arachidonic acid esterified to phospholipids in treated RBCs was found to increase significantly for both esterified EETs in GPE, GPS and GPC which increased 49-, 34- and 59-fold, respectively, and also for esterified HETEs in GPE, GPS and GPC which increased 3-, 4- and 11-fold, respectively, compared with untreated RBCs. These results provide the first characterization of EETs formed non-enzymatically as intact phospholipids in a lipid peroxidation model system.

Arachidonic acid is oxidized to regioisomeric 5(S)-, 12(S)- and 15(S)-hydroxyeicosatetraenoic acids by the corresponding 5-, 12- and 15-lipoxygenases. These hydroxylated fatty acids can then be incorporated into cellular phospholipids. Negative liquid secondary ion tandem mass spectrometry using a high-energy collision regime in a tandem four-sector mass spectrometer was used to characterize regioisomeric hydroxyeicosatetraenoic acids and the corresponding hydroxyeicosatetraenoic phosphatidylcholine species. Collision-induced dissociation (CID) of the [M-H]- negative ion at m/z 319 from the hydroxyeicosatetraenoic acids regioisomers produced some similar product ions, such as m/z 301 [M-H-H2O]- and m/z 257 [M-H-(H2O + CO2)]-. In addition, product ions characteristic of the particular hydroxyeicosatetraenoic acid were formed from alpha-cleavages adjacent to the hydroxyl moieties. Negative liquid secondary ion mass spectrometry of purified hydroxyeicosatetraenoate phosphatidylcholine species gave an ion at m/z 810 [M-CH3]-. CID of the m/z 810 ion gave product ions at m/z 283 and m/z 319, corresponding to stearate at the sn-1 position and hydroxyeicosatetraenoate at the sn-2 position, respectively. From CID of the negative ion at m/z 319 and examination of the product ion spectra, the hydroxyeicosatetraenoate regioisomer present in the phosphatidylcholine could be identified.