Muscle α-tocopherol concentrations. Zebrafish from the same cohort were fed a vitamin E-sufficient (E+, blue bars, n = 5 per group) or -deficient (E−, red striped bars, n = 5 at 12 months and n = 4 at 18 months) diet from age 45 days until 12 or 18 months. At the indicated times, fish were sacrificed, the muscles harvested, frozen, and kept frozen until analysis. α-Tocopherol skeletal muscle concentrations (mean ± SD) show a diet main effect (p ≤ 0.0001, no significant interaction or main effect of time).

Analysis of E+ fish muscles at 12 vs. 18 months. The heatmap (A) shows the top 25 metabolites separating groups and their relative abundances per sample (red represents a higher concentration per sample while blue indicates a lower one). The partial least squares-discriminant analysis (PLS-DA) scores plot (B) separates 12- and 18-month E+ muscle samples by feature differences and displays a 95% confidence region around samples. Important features plot displays variable importance in projection (VIP) (C) of the PLS-DA and provides an estimation of the discriminatory power of each metabolite with a score. Larger VIP scores indicate greater discriminatory power in the PLS-DA models. The table (D) lists the top values shown in the graph of pathway analysis, while figure (E) displays pathway analysis by ranking metabolites by p-value (shown as −log10(p)) and pathway impact referenced against the KEGG (Kiyoto Encyclopedia of Genes and Genomes) Pathway Database. Red indicates highest impacts, clear the lowest, yellow and orange are intermediate.

Metabolomic differences between E+ and E− fish muscles at 12 months. Shown are the heatmap (A) and PLS-DA scores plot (B), which separates E+ and E− muscle samples by feature differences and displays a 95% confidence region around samples and VIPs (C). There were insufficient statistically significant differences in the data from muscles of E+ and E− fish at 12 months for statistically meaningful pathway analysis.

Metabolomic differences between E+ and E− fish muscles at 18 months. Shown are the heatmap (A) PLS-DA scores plot (B) and VIP scores (C). The table (D) lists the top values shown in the graph of pathway analysis. (E) displays pathway analysis by ranking metabolites by p-value (shown as −log10(p)) and pathway impact referenced against the KEGG Pathway Database. Red indicates highest impacts, clear the lowest, yellow and orange are intermediate.

Purine metabolism is altered in E− zebrafish muscle at 18 months. Bar graphs showing Student’s t-test of 14 metabolites involved in KEGG pathway of zebrafish purine metabolism. Relative response (mean ± SD, n = 5 per group, E+ (blue bar), E− (red stripped bar), group = 18 months). The right graphic is a visualization of the changes in various purines within the purine metabolism pathway. Right graphic created with BioRender.com.

Metabolomic differences between E− fish at 12 and 18 months. Shown are the heatmap (A) PLS-DA scores plot (B,C) VIP scores. There was insufficient statistically significant differences in the data from muscles of E− fish at 12 and 18 months for statistically meaningful pathway analysis.