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Enantiomeric separation and analysis of unsaturated hydroperoxy fatty acids by chiral column chromatography-mass spectrometry
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Biochemical pharmacology)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Biochemical Pharmacology)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Biochemical pharmacology)
2008 (English)In: Journal of chromatography. B, ISSN 1570-0232, Vol. 872, 90-98 p.Article in journal (Refereed) Published
Abstract [en]

Hydroperoxides of 18:2n-6 and 20:4n-6 were obtained by autoxidation and photooxidation. The enantiomers Were separated as free acids (Reprosil Chiral-NR column, eluted with hexane containing 1-1.2% alcoholic modifier) and analyzed by on line UV detection (234 nm) and liquid chromatography-MS/MS/MS of carboxylate anions (A(-) -> (A(-)-18) -> full scan) in an ion trap. The combination of UV and MS/MS/MS analysis facilitated identification of hydroperoxides even in complex mixtures of autoxidized or photooxidized fatty acids. The signal intensities increased about two orders of magnitude by raising the isolation width of A(-) from 1.5 amu to 5 or 10 amu for cis-trans conjugated hydroperoxy fatty acids, and one order of magnitude of more for non-conjugated hydroperoxy fatty acids. The S enantiomer of 8-, 9-, 10-, and 13-hydroperoxyoctadecadienoic acids and the S enantiomer of cis-trans conjugated hydroperoxyeicosatetraenoic acids eluted before the corresponding R enantiomer with two exceptions (11-hydroperoxylinoleic acid and 8-hydroperoxyeicosa-5Z,9E,11Z,14Z-tetraenoic acid). The separation of enantiomers or regioisomers could be improved by the choice of either isopropanol or methanol as alcoholic modifier.

Place, publisher, year, edition, pages
2008. Vol. 872, 90-98 p.
Keyword [en]
autoxidation, hydroperoxy eicosanoids, hydroperoxy linoleic acids, MS/MS fragmentation, normal phase HPLC, photooxidation, Reprosil Chiral-NR
National Category
Pharmaceutical Sciences
Research subject
Biochemical Pharmacology
Identifiers
URN: urn:nbn:se:uu:diva-86646DOI: 10.1016/j.jchromb.2008.07.013ISI: 000259764800011OAI: oai:DiVA.org:uu-86646DiVA: diva2:126745
Available from: 2008-11-20 Created: 2008-11-20 Last updated: 2009-08-28Bibliographically approved
In thesis
1. Mass Spectrometric Analysis of Oxylipins: Application to Cytochrome P450-Dependent Metabolism
Open this publication in new window or tab >>Mass Spectrometric Analysis of Oxylipins: Application to Cytochrome P450-Dependent Metabolism
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cytochrome P450 (CYP) family 4 constitutes monoxygenases responsible for hydroxylation of fatty acids and other lipids. For example, CYP4F3 metabolizes leukotrienes and CYP4F8 prostaglandin H. Importantly, six of the twelve CYP4 enzymes are orphans, i.e., with an unknown biological function. The catalytic activity of the enzyme CYP4F8 is known in seminal vesicles, but not in skin or psoriatic lesions, where CYP4F8 is highly expressed. The orphan CYP4F22 is also expressed in skin, and mutations in its gene has been linked to the rare skin disease lamellar ichthyosis, together with, inter alia, mutations in the genes of 12R-LOX and eLOX3. These enzymes appear to constitute a pathway producing hydroperoxides and epoxyalcohols from arachidonic acid. CYP4F22 is hypothesized to act in a consecutive step within this pathway.

The aim of this thesis was to develop analytical methods to prepare and analyze hydroperoxides and epoxyalcohols derived from fatty acids by LC-MS/MS, and to investigate the catalytic performance of CYP4F8 and CYP4F22 for these substrates. The 12R-hydroperoxide of arachidonic acid (12R-HPETE) was prepared by autoxidation and separated from other hydroperoxides by chiral HPLC. MS/MS analysis showed that the hydroperoxides were unstable within the ion trap, but were stabilized by an increase in the isolation width. From the hydroperoxides, epoxyalcohols were generated by hematin treatment, and separated by normal phase HPLC. MS/MS spectra of several epoxyalcohols, derived both from arachidonic acid and linoleic acid, were characterized with aid of [2H]isotopomers and MS3 analysis. Apart from metabolic studies the thesis also include detailed information on MS/MS analysis of several oxygenated fatty acids, with proposed fragmentation mechanisms.

The open reading frame of CYP4F22 was expressed in a recombinant yeast system, and LC-MS/MS analysis revealed that CYP4F22 catalyzed ω3 hydroxylation of arachidonic acid, but not any of the tested epoxyalcohols. In contrast, CYP4F8 metabolizes an epoxyalcohol derived from 12R-HPETE, 11R,12R-epoxy-10-hydroxyeicosatrienoic acid, to the ω3 hydroxy metabolite.

Conclusively, it was demonstrated that LC-MS/MS could be used for the analysis and separation of hydroperoxides and epoxyalcohols for metabolic studies.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 114
Keyword
epoxyalcohols, fatty acids, hydroperoxides, electrospray ionization, ichthyosis, CYP4, linear ion trap, fragmentation mechanism
National Category
Pharmacology and Toxicology
Research subject
Biochemical Pharmacology
Identifiers
urn:nbn:se:uu:diva-109715 (URN)978-91-554-7645-8 (ISBN)
Public defence
2009-12-04, B41, Biomedicinskt centrum, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2009-11-13 Created: 2009-10-22 Last updated: 2011-05-11Bibliographically approved

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