Gas chromatography-mass spectrometry method for determination of phospholipid peroxides; I. Transesterification to form methyl esters

Frederik J.G.M. van Kuijk, David W. Thomas, Robert J. Stephens, Edward A. Dratz

Research output: Contribution to journalArticlepeer-review

61 Scopus citations

Abstract

The purpose of this study is to develop methods for determining the chemical species of lipid peroxides that occur in various types of tissue pathology. Experiments are aimed at determining the phospholipid peroxides associated with retinal degeneration as the initial test case. Phospholipid hydroperoxides are synthesized by photosensitized oxidation, chemically characterized and used to develop and effective and simplified method to identify and measure phospholipid hydroperoxides by gas chromatography-mass spectrometry (GC-MS). An sensitive reverse phase high performance liquid chromatography (HPLC) method is also presented to separate peroxidized phospholipids from phospholipids. For GC-MS, phospholipid peroxides are reduced with sodium borohydride and transesterified to form fatty acid methyl esters using a mild quaternary ammonium hydroxide catalyst. The hydroxyl groups produced by reducing the hydroperoxides are formed into trimethylsilyl ethers and GC-MS is employed (with electron ionization and negative ion chemical ionization) to identify oxidized fatty acids at the 10 ng level. Photooxidation of (palmitoyl)(linoleoyl) phosphatidylcholine yielded equal amounts of the conjugated (9 and 13 isomers) and the nonconjugated (10 and 12 isomers) linoleoyl hydroperoxides. Photooxidation of rat retina total lipids yielded oxidation products of oleolyl (18:1) esters as well as the conjugated and nonconjugated oxidation products of arachidonoyl (20:4) and docosahexaenoyl (22:6) esters virtually all of which arise from phospholipids. The nonconjugated products are of interest as indicators of photosensitized light damage in retina and other tissues. It is notable that all the possible singly oxidized products are found with the exception of the 4, 5 and 7 hydroperoxides of 22:6 and the 5 hydroperoxide of 20:4. It appears that the approach of singlet oxygen is strongly inhibited in the sterically hindered region near the phospholipid head groups.

Original languageEnglish (US)
Pages (from-to)215-225
Number of pages11
JournalJournal of Free Radicals in Biology and Medicine
Volume1
Issue number3
DOIs
StatePublished - 1985

Keywords

  • Fatty acid methyl esters
  • Gas chromatography-mass spectrometry
  • High pressure liquid chromatography
  • Lipid peroxidation
  • Phospholipid HPLC
  • Phospholipid hydroperoxides
  • Singlet oxygen
  • Transesterification

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