Novel liquid chromatography-mass spectrometry method shows that vitamin E deficiency depletes arachidonic and docosahexaenoic acids in zebrafish (Danio rerio) embryos

Redox Biol. 2013 Dec 17:2:105-13. doi: 10.1016/j.redox.2013.12.007. eCollection 2013.


To test the hypothesis that embryogenesis depends upon α-tocopherol (E) to protect embryo polyunsaturated fatty acids (PUFAs) from lipid peroxidation, new methodologies were applied to measure α-tocopherol and fatty acids in extracts from saponified zebrafish embryos. A solid phase extraction method was developed to separate the analyte classes, using a mixed mode cartridge (reverse phase, π-π bonding, strong anion exchange), then α-tocopherol and cholesterol were measured using standard techniques, while the fatty acids were quantitated using a novel, reverse phase liquid chromatography-mass spectrometry (LC-MS) approach. We also determined if α-tocopherol status alters embryonic lipid peroxidation products by analyzing 24 different oxidized products of arachidonic or docosahexaenoic (DHA) acids in embryos using LC with hybrid quadrupole-time of flight MS. Adult zebrafish were fed E- or E+ diets for 4 months, and then were spawned to obtain E- and E+ embryos. Between 24 and 72 hours post-fertilization (hpf), arachidonic acid decreased 3-times faster in E- (21 pg/h) compared with E+ embryos (7 pg/h, P<0.0001), while both α-tocopherol and DHA concentrations decreased only in E- embryos. At 36 hpf, E- embryos contained double the 5-hydroxy-eicosatetraenoic acids and 7-hydroxy-DHA concentrations, while other hydroxy-lipids remained unchanged. Vitamin E deficiency during embryogenesis depleted DHA and arachidonic acid, and increased hydroxy-fatty acids derived from these PUFA, suggesting that α-tocopherol is necessary to protect these critical fatty acids.

Keywords: ARA, 20:4 ω-6, arachidonic acid; Arachidonic acid; CNS, central nervous system; DHA, 22:6 ω-3, docosahexaenoic acid; Embryogenesis; HDHA, hydroxy-DHA; HETEs, hydroxy-eicosatetraenoic acids; Hybrid quadrupole-time of flight MS; LTA4, leukotriene A4; Neurogenesis; Vitamin E; dn, deuterium-labeled; hpf, hours post-fertilization; δT3, delta-tocotrienol.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Arachidonic Acid / analysis*
  • Arachidonic Acid / isolation & purification
  • Arachidonic Acid / metabolism
  • Cholesterol / analysis
  • Cholesterol / isolation & purification
  • Chromatography, High Pressure Liquid*
  • Docosahexaenoic Acids / analysis*
  • Docosahexaenoic Acids / isolation & purification
  • Docosahexaenoic Acids / metabolism
  • Embryo, Nonmammalian / metabolism
  • Fatty Acids, Unsaturated / analysis
  • Fatty Acids, Unsaturated / metabolism
  • Lipid Peroxidation / drug effects
  • Mass Spectrometry*
  • Solid Phase Extraction
  • Vitamin E / pharmacology
  • Vitamin E Deficiency / metabolism
  • Vitamin E Deficiency / pathology
  • Zebrafish / growth & development
  • Zebrafish / metabolism*
  • alpha-Tocopherol / analysis


  • Fatty Acids, Unsaturated
  • Vitamin E
  • Docosahexaenoic Acids
  • Arachidonic Acid
  • Cholesterol
  • alpha-Tocopherol