Lipid quantitation and metabolomics data from vitamin E-deficient and -sufficient zebrafish embryos from 0 to 120 hours-post-fertilization

The data herein is in support of our research article by McDougall et al. (2017) [1], in which we used our zebrafish model of embryonic vitamin E (VitE) deficiency to study the consequences of VitE deficiency during development. Adult 5D wild-type zebrafish (Danio rerio), fed defined diets without (E–) or with VitE (E+, 500 mg RRR-α-tocopheryl acetate/kg diet), were spawned to obtain E– and E+ embryos that we evaluated using metabolomics and specific lipid analyses (each measure at 24, 48, 72, 120 hours-post-fertilization, hpf), neurobehavioral development (locomotor responses at 96 hpf), and rescue strategies. Rescues were attempted using micro-injection into the yolksac using VitE (as a phospholipid emulsion containing d6-α-tocopherol at 0 hpf) or D-glucose (in saline at 24 hpf).


Subject area
Biology More specific subject area

Nutrition; Antioxidants
Type of data Graphs; Figures How data was acquired LC-MS (liquid chromatography-mass spectrometry) using a Shimadzu Nexera system (Shimadzu; Columbia, MD, USA) coupled to a high-resolution hybrid quadrupole-time-of-flight mass spectrometer (TripleTOF s 5600; SCIEX; Framingham, MA, USA); Embryos were assessed for viability, developmental progression and spontaneous movements (earliest behavior in zebrafish), using the zebrafish acquisition and analysis program (ZAAP Rescue studies using microinjection into the yolksac may be compared to other methods of compound/nutrient delivery to developing zebrafish.

Data
Fig. 1. shows data from quantitative analyses of LA (linoleic acid, 18:2, omega-6); ARA (arachidonic acid, 20:4, omega-6); EPA (eicosapentaenoic acid, 20:5, omega-3); DHA (docosahexaenoic acid, 22:6, omega-3) in fatty acid extracts from samples with and without alcoholic saponification of E-and E þ embryos collected at 24, 48, 72, and 120 hpf. Tables 1and 2 provide detailed targeted metabolomics datasets for E-and Eþ embryos collected at 24, 48, 72, and 120 hpf. Relative response intensity metabolomics data for choline and methylation pathway intermediates in E-and Eþ embryos are shown in Fig. 2. Relative response intensities of antioxidant network components from metabolomic analyses, as well as quantification of α-tocopherol and ascorbic acid, in E-and E þ embryos (pmol/ embryo) are shown in Fig. 3. Relative response intensities of glycolytic and tricarboxylic acid cycle intermediates in E-and E þ embryos are shown in Fig. 4. Relative response intensities of free saturated fatty acids and coenzyme A from metabolomics data in E-and Eþ embryo are shown in Fig. 5. Fig. 6 shows locomotor activity data from E-and Eþ embryos micro-injected into the yolksac at 0 hpf with either saline or a VitE-emulsion. Fig. 7 shows locomotor activity data from E-and Eþ embryos micro-injected into the yolksac at 24 hpf with either saline or D-glucose.

Study design
All experiments (i.e. lipid quantifications, targeted metabolomics analyses, and micro-injection rescue studies) were performed in duplicate and have been reported in detail [1].

Zebrafish husbandry and diets
The Institutional Animal Care and Use Committee of Oregon State University approved this protocol (ACUP Number: 4344). Tropical 5D strain zebrafish were housed in the Sinnhuber Aquatic Research Laboratory and complete details of the housing and husbandry have been reported [1].

Analyses
Diet and embryo α-tocopherol [2] and ascorbic acid [3] were determined using high-pressure liquid chromatography with electrochemical detection as reported [1].
Extraction and sample preparation for metabolomic analysis were performed following 24, 48, 72, and 120 hpf, embryos (n ¼15 per replicate, n ¼4 replicates per group), as described [1]. Chromatography was performed with a Shimadzu Nexera system (Shimadzu; Columbia, MD, USA) coupled to a high-resolution hybrid quadrupole-time-of-flight mass spectrometer (TripleTOF s 5600; SCIEX; Framingham, MA, USA). Two different LC analyses using reverse phase and HILIC columns were used, as described [1].
Analysis of total DHA, EPA, ARA, and LA were performed as described [2] with modifications, as described [1]. Chromatographic separations were carried out on 4.6 Â 250 mm J'sphere ODS-H80 (4 mm, YMC Co, Kyoto, Japan) for negative ion analysis. TOF-MS and TOF-MS/MS were operated with same parameters as for metabolomics, as described [1].

Microinjection rescue studies
Embryos were microinjected as described and criteria used to assess supplementation tolerance of zebrafish embryos using ZAAP at 24, 48, and 120 hpf, as described [1].      Locomotor response assay activity data showing neurobehavioral impairment. E-and E þ embryos (96 hpf) were analyzed in 96-well plates (128 embryos per group). Locomotor activities following a series of light stimuli (every 6 for 24 min) were measured as distance moved (mm) over time (seconds). E-(red) embryos treated with saline (upper panel) were 84% less responsive to light than were E þ (blue) embryos (E-area-under-curve, AUC: 5727 72 E þ AUC: 3580 7387; p o 0.0001). Embryos with morphological defects were not included in data analysis. E-behavior was partially restored by approximately 50% following glucose injection into the yolk at 24 hpf (lower panel; E-AUC: 2502 7150; E þ AUC: 3734 7359; p o 0.0001). Statistical significance was determined using a Kolmogorov-Smirnov test (po 0.01).

Behavioral assessments
Locomotor activity was measured in a total of n ¼128 embryos per group using Viewpoint Zebrabox [4,5], as described [1].

Data processing and statistical analyses
All data processing and statistical analyses were performed as described in [4][5][6], with modification made as reported [1].