*2.2. Animals*

Male C57BL/6J mice (Japan SLC, Inc., Shizuoka, Japan) were purchased at 10 weeks of age, and housed under controlled temperature and lighting (12 h light/dark cycle) with free access to water and a controlled diet (fish-meal-free F1, Funabashi Farm, Chiba, Japan). The fatty acid composition and other nutritional factors for fish-meal-free F1 are described in Supplementary Table S1. In this study, the fish-meal-free F1 was used as an ARA-, EPA-, and DHA-free diet, and each PUFA was supplemented into the feed in its ethyl ester form. After 1 week of acclimation, mice were assigned to four groups (n = 5) and fed control diet (control group), control diet supplemented with 1% EPA-E (*w*/*w*) (EPA-fed group), 1% DHA-E ( *w*/*w*) (DHA-fed group), or 1% ARA-E ( *w*/*w*) (ARA-fed group) for 2 weeks. Animals were dissected under isoflurane anesthesia, after which organs, such as the liver, kidney, white adipose, skeletal muscle, heart, small intestine, lung, brain, and spleen, were collected and plasma was isolated. For the lipidome analyses, four biological replicates (n = 4) were used for liver tissue, and five biological replicates (n = 5) were used for the other tissues and plasma. Two of the white adipose samples from the EPA-fed group and one of the skeletal muscle samples from the ARA-fed group in were excluded from the targeted lipidomics results owing to LC-MS analysis failure. All animal experiments were carried out in accordance with the guidelines for the use and care of laboratory animals of Mochida Pharmaceutical, and were approved by the Institutional Animal Care and Use Committee (identification code: PMS14-041).

#### *2.3. Determination of Plasma Triglyceride, Cholesterol, and Fatty Acid Composition*

Standard enzymatic methods were used to determine plasma total cholesterol (TC) and triglyceride (TAG) with commercially available kits purchased from Wako Pure Chemical Industries Ltd. Total fatty acids in plasma were analyzed by gas chromatography (Japan SLC, Inc., Shizuoka, Japan). C16:0, C16:1, C18:0, C18:1, C18:2, C20:3, C20:4, C20:5, C22:5, and C22:6 levels were determined using the standard substances: palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, dihomo-γ-linoleic acid, arachidonic acid, eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid, respectively.

#### *2.4. Untargeted LC-MS*/*MS-Based Lipidomics*

Total lipids from organs were extracted as previously described [16] using internal standards: 1 μM TAG (8:0/8:0/8:0)-13C3 (Larodan, Inc., Monroe, MI, USA), 0.5 μM phosphatidyl glycerol (17:0/14:1) (Avanti Polar Lipids, Inc., Alabaster, AL, USA), 0.5 μM lyso phosphatidylcholine (17:1) (Avanti Polar Lipids, Inc.), 5 μM acylcarnitine (18:0)-d3 (Larodan, Inc., Monroe, MI, USA), 25 μM palmitic acid-d3 (Olbracht Serdary Research Laboratories, Toronto, ON, Canada), 25 μM stearic acid-d3 (Olbracht Serdary Research Laboratories, Toronto, ON, Canada), 5 μM cholic acid-d4 (Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA), and 0.5 μM Cer/Sph mixture I (Avanti Polar Lipids, Inc., Alabaster, AL, USA) (Supplementary Table S2). Untargeted analysis was performed using an ACQUITY UPLC system (Waters, Milford, MA, USA) coupled with a quadruple time-of-flight/MS (TripleTOF 5600+, SCIEX, Framingham, MA, USA). LC separation was performed using a reverse-phase column (Acquity UPLC BEH peptide C18; 2.1 × 50 mm, 1.7 μm particle size; Waters, Milford, MA, USA) with a gradient elution of mobile phase A (methanol: acetonitrile: water = 1:1:3, *v*/*v*/*v* for volume ratio containing 5 mM ammonium acetate and 10 nM EDTA) and mobile phase B (100% isopropanol containing 5 mM ammonium acetate and 10 nM EDTA), and the composition was produced by mixing those solvents. LC gradient consisted of holding solvent (A/B: 100/0) for 1 min, then linearly converting to solvent (A/B: 60/40) for 4 min, linearly converting to solvent (A/B:36/64) for 2.5 min and holding for 4.5 min, then linearly converting to solvent (A/B: 17.5/82.5) for 0.5 min, linearly converting to solvent (A/B: 15/85) for 8.5 min, and linearly converting to solvent (A/B: 5/95) for 1 min followed by returning to solvent (A/B: 100/0) and holding for 5 min for re-equilibration. The flow rate and column temperature were set to 0.3 mL/min and 45 ◦C, respectively. Data dependent MS/MS acquisition mode was applied as previously described [16]. Briefly, the temperature and ion spray voltage floating were set to 300 ◦C

and −5.5 kV, respectively. The accumulation times for MS1 and MS/MS were set to 100 ms and 50 ms, respectively, for scanning a mass range from *m*/*z* 75 to *m*/*z* 1250. The collision energy (CE) was set to 35 eV with a CE spread of 15 eV in high-resolution mode, and other settings of DDA mode were as follows: 10 most intense ions, 100 cps intensity threshold, and 100 sec exclusion time.

#### *2.5. Targeted LC-MS*/*MS-Based Lipidomics*

The LC-MS/MS analysis was performed as described previously [12]. Samples were extracted by solid-phase extraction using Sep-Pak Vac 3cc C18 cartridges (Waters) with deuterium-labeled internal standards at a final concentration of 10 pg/μ<sup>L</sup> ARA-d8, 10 pg/μ<sup>L</sup> 15-HETE-d8, 10 pg/μ<sup>L</sup> LTB4-d4, 10 pg/μ<sup>L</sup> PGE2-d4 per samples. The targeted analysis was performed using a UPLC system (Waters UPLC, Waters, Milford, MA, USA) with a triple quadruple linear ion trap mass spectrometer (QTRAP 5500; SCIEX, Framingham, MA, USA), equipped with Acquity UPLC BEH C18 column (1.0 × 150 mm, 1.7 μm particle size; Waters, Milford, MA, USA). Samples were eluted with a mobile phase composed of water/acetate (100:0.1, *v*/*v*) and acetonitrile/methanol (4:1, *v*/*v*) (73:27) for 5 min, and ramped to 30:70 over 15 min, to 20:80 over 25 min and held for 8 min, ramped to 0:100 over 35 min, and held for 10 min with flow rates of 70 μL/min (0–30 min), 80 μL/min (30–33 min), and 100 μL/min (33–45 min). The MS/MS analyses were performed in negative ion mode, and oxylipins were identified and quantified by multiple reaction monitoring (Supplementary Table S3). Compounds were quantified by using stable internal standards. The calibration curves of all compounds were acquired in triplicate (Supplementary Figure S1). The extraction and matrix recovery were calculated with internal standards (IS) and the compound quantification was conducted with the calibration curves. The lipid compounds of (group 1) free fatty acids, (group 2) monohydrides and epoxides, (group 3) diols, leukotrienes, thromboxanes, lipoxines, and resolvins, and (group 4) prostaglandins and others were corrected with ARA-d8, 15-HETE-d8, LTB4-d4, and PGE2-d4, respectively. The compound concentrations were calculated as:

$$\text{Compound concentration} = \frac{(\text{target compound peak area}) / (\text{slope of calibration curve})}{(\text{IS peak area}) / (\text{IS slope of calibration curve})}$$
