Targeted and untargeted lipidomic analyses of bioactive and metabolic lipids in plasma in EAE mice
A: Scatter plots of regulated fatty acid associated genes (microarray) in the spinal cord of C57BL6 EAE mice versus control mice (CFA without MOG) 16 days after immunization (GSE GSE60847). Volcano plot overviews in Suppl. Fig. 1.
B: Volcano plot show the log2 difference (= fold difference; X-axis) of metabolic plasma lipids between sapropterin and vehicle treated EAE mice versus the –log10 of the P-value of the t-test (Y-axis). Lipids that were reduced in sapropterin-treated mice appear on the left side of the Y-axis, increased lipids on the right side.
C: The box/scatter plot shows the log2 transformed levels of fatty acids. The data represent the ratios of the mass spectrometry peak areas normalized by the peak areas of the respective standard. The box represents the IQR, whiskers show minimum to maximum, the line is the median. Each scatter is a mouse (n = 10 per group). Data were compared with two-way ANOVA for ”fatty acid X treatment” and subsequent posthoc analysis for treatment using an adjustment of alpha according to Šidák.
D: Canonical discriminant score plots of the first discriminant factors CanDis1 and CanDisc2 for plasma lipids using 28 lipid species of five classes as input. The plasma concentrations were obtained from SJL/J mice immunized with PLP and treated orally with vehicle (2% DMSO), sapropterin (2 mg/d) or DHAP (4 mg/d) via the drinking water starting at the day of immunization (n=7 per group). The final blood sample for lipid analyses was obtained 22 days after immunization. Lipids encompassed ceramides, hexosylceramides, sphingolipids, endocannabinoids and lysophosphatidic acids (individually presented in Suppl. Fig. 3). The dots show individual mice. The ellipses show the 95% confidence.
E: Box/scatter plots of normalized ceramides and endocannabinoids in vehicle, sapropterin (BH4) or DAHP treated SJL/J-EAE mice as in A. Lipids were normalized as percentages of the 90%-quantile (raw concentrations shown in Suppl. Fig. 3). The box represents the interquartile range, whiskers show minimum to maximum, the line is the median. Each scatter is a mouse. Data were compared with two-way ANOVA for ”lipid X treatment” and subsequent posthoc analysis for treatment using an adjustment of alpha according to Šidák (n = 6-8 per group, * P < 0.05, ** P < 0.001).
F: Polar plots show the mean normalized levels (percentages of the 90% quantile) of multiple lipid species analyzed via targeted LC-MS/MS analyses in plasma of SJL/J-EAE mice treated as in A. Most ceramides and some lysophosphatidic acids (LPAs) were increased in sapropterin-treated mice.