title: Novel liquid chromatography-mass spectrometry method shows that vitamin E deficiency depletes arachidonic and docosahexaenoic acids in zebrafish (Danio rerio) embryos.
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 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.
authors.affiliation: Linus Pauling Institute, Oregon State University, Corvallis, OR 97331, USA ; School of Biological and Population Health Sciences, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Katie M
authors.initials: KM
authors.lastname: Lebold
authors.affiliation: Linus Pauling Institute, Oregon State University, Corvallis, OR 97331, USA ; College of Pharmacy, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Jay S
authors.initials: JS
authors.lastname: Kirkwood
authors.affiliation: Linus Pauling Institute, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Alan W
authors.initials: AW
authors.lastname: Taylor
authors.affiliation: Linus Pauling Institute, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Jaewoo
authors.initials: J
authors.lastname: Choi
authors.affiliation: Sinnhuber Aquatic Research Laboratory, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Carrie L
authors.initials: CL
authors.lastname: Barton
authors.affiliation: Linus Pauling Institute, Oregon State University, Corvallis, OR 97331, USA ; Environmental & Molecular Toxicology, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Galen W
authors.initials: GW
authors.lastname: Miller
authors.affiliation: Sinnhuber Aquatic Research Laboratory, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Jane
authors.initials: J
authors.lastname: La Du
authors.affiliation: Linus Pauling Institute, Oregon State University, Corvallis, OR 97331, USA ; School of Biological and Population Health Sciences, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Donald B
authors.initials: DB
authors.lastname: Jump
authors.affiliation: Linus Pauling Institute, Oregon State University, Corvallis, OR 97331, USA ; College of Pharmacy, Oregon State University, Corvallis, OR 97331, USA ; Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Jan Frederik
authors.initials: JF
authors.lastname: Stevens
authors.affiliation: Sinnhuber Aquatic Research Laboratory, Oregon State University, Corvallis, OR 97331, USA ; Environmental & Molecular Toxicology, Oregon State University, Corvallis, OR 97331, USA ; Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Tanguay
authors.affiliation: Linus Pauling Institute, Oregon State University, Corvallis, OR 97331, USA ; School of Biological and Population Health Sciences, Oregon State University, Corvallis, OR 97331, USA ; Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Maret G
authors.initials: MG
authors.lastname: Traber
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D002784:Cholesterol / Q000032:analysis / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D004281:Docosahexaenoic Acids / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000032:analysis / Q000378:metabolism; D015227:Lipid Peroxidation / Q000187:drug effects; D013058:Mass Spectrometry; D052616:Solid Phase Extraction; D014810:Vitamin E / Q000494:pharmacology; D014811:Vitamin E Deficiency / Q000378:metabolism / Q000473:pathology; D015027:Zebrafish / Q000254:growth & development / Q000378:metabolism*; D024502:alpha-Tocopherol / Q000032:analysis
chemical_list: D005231:Fatty Acids, Unsaturated; D014810:Vitamin E; D004281:Docosahexaenoic Acids; D016718:Arachidonic Acid; D002784:Cholesterol; D024502:alpha-Tocopherol
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