Analysis of Epoxyeicosatrienoic and Monohydroxyeicosatetraenoic Acids Esterified to Phospholipids in Human Red Blood Cells by Electrospray Tandem Mass Spectrometry
✍ Scribed by Nakamura, Tatsuji; Bratton, Donna L.; Murphy, Robert C.
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 396 KB
- Volume
- 32
- Category
- Article
- ISSN
- 1076-5174
No coin nor oath required. For personal study only.
✦ Synopsis
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 puriÐed 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 esteriÐed to phospholipids in treated RBCs was found to increase signiÐcantly for both esteriÐed EETs in GPE, GPS and GPC which increased 49-, 34-and 59-fold, respectively, and also for esteriÐed HETEs in GPE, GPS and GPC which increased 3-, 4-and 11-fold, respectively, compared with untreated RBCs. These results provide the Ðrst characterization of EETs formed non-enzymatically as intact phospholipids in a lipid peroxidation model system.