abstract
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 Eþ diets for 4 months, and then were spawned to obtain E and Eþ embryos.
Between 24 and 72 hours post-fertilization (hpf), arachidonic acid decreased 3-times faster in E (21 pg/h)
compared with Eþ embryos (7 pg/h, Po0.0001), 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.
& 2013 The Authors. Published by Elsevier B.V. All rights reserved.