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

To test he hypothesis that embryogenesis depends upon a-tocopherol (E) to protect embryo polyunsaturated fatty acids (PUEAs) from lipid peroxidation, new methodologies were applied to measure a-tocopherol and fatty acids in extracts from saponified zebratish embryos. A solid phase extraction method was developed to separate the analyte classes, using a mixed mode cartridge (reverse phase, pi-pi bonding, strong anion exchange), then a-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 a-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 a-tocopherol and DHA concentrations decreased only in E- embryos. At 36 hpf, E- embryos contained double the 5-hydroxy-eicosatetraenoic acids and 7-hyclroxy-DHA concentrations, while other hydroxy-lipids remained unchanged. Vitamin E deficiency during ernbryogenesis depleted DNA and arachidonic acid, and increased hydroxy-fatty acids derived from these PUFA, suggesting that alpha-tocopherol is necessary to protect these critical fatty acids. (C) 2013 The Authors. Published by Elsevier B.V. All rights reserved