Analysis of novel hydroperoxides and other metabolites of oleic, linoleic, and linolenic acids by liquid chromatography-mass spectrometry with ion trap MSn.
Linoleate is oxygenated by manganese-lipoxygenase (Mn-LO) to 11S-hydroperoxylinoleic acid and 13R-hydroperoxyoctadeca-9Z,11E-dienoic acid, whereas linoleate diol synthase (LDS) converts linoleate sequentially to 8R-hydroperoxylinoleate, through an 8-dioxygenase by insertion of molecular oxygen, and to 7S,8S-dihydroxylinoleate, through a hydroperoxide isomerase by intramolecular oxygen transfer. We have used liquid chromatography-mass spectrometry (LC-MS) with an ion trap mass spectrometer to study the MSn mass spectra of the main metabolites of oleic, linoleic, alpha-linolenic and gamma-linolenic acids, which are formed by Mn-LO and by LDS. The enzymes were purified from the culture broth (Mn-LO) and mycelium (LDS) of the fungus Gaeumannomyces graminis. MS3 analysis of hydroperoxides and MS2 analysis of dihydroxy- and monohydroxy metabolites yielded many fragments with information on the position of oxygenated carbons. Mn-LO oxygenated C-11 and C-13 of 18:2n-6, 18:3n-3, and 18:3n-6 in a ratio of approximately 1:1-3 at high substrate concentrations. 8-Hydroxy-9(10)epoxystearate was identified as a novel metabolite of LDS and oleic acid by LC-MS and by gas chromatography-MS. We conclude that LC-MS with MSn is a convenient tool for detection and identification of hydroperoxy fatty acids and other metabolites of these enzymes.
Department of Pharmaceutical Biosciences, Uppsala Biomedical Center, Uppsala University, Sweden. Ernst.Oliw@bmc.uu.se
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D002853:Chromatography, Liquid / Q000379:methods*; D005231:Fatty Acids, Unsaturated / Q000032:analysis* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000032:analysis* / Q000378:metabolism; D019746:Intramolecular Oxidoreductases / Q000378:metabolism; D008041:Linoleic Acids / Q000032:analysis / Q000378:metabolism; D008042:Linolenic Acids / Q000032:analysis / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D009829:Oleic Acids / Q000032:analysis* / Q000378:metabolism
D005231:Fatty Acids, Unsaturated; D008041:Linoleic Acids; D008042:Linolenic Acids; D009829:Oleic Acids; D006861:Hydrogen Peroxide; D019746:Intramolecular Oxidoreductases; C021377:hydroperoxide isomerase