**3. Results**

#### *3.1. Lipid Profiles after Dietary Intake of ARA, EPA, or DHA*

We first examined the profiles of total cholesterol, triacylglycerol (TAG), and total fatty acids in mouse plasma. Dietary intake of 1% (*w*/*w*) ARA-EE, EPA-EE, or DHA-EE for 2 weeks significantly decreased plasma cholesterol and decreased triacylglycerol levels as compared to the levels of the control group (Figure 1a,b). There were no body weight loss, suggesting that PUFA supplementation did not affect the amount of dietary consumption (Figure 1c). Total fatty acid levels, including C16:0 (palmitic acid), C18:1, and C18:2, were decreased after dietary intake of PUFAs, while C20:4 (ARA), C20:5 (EPA), and C22:6 (DHA) were increased in the respective dietary conditions (Figure 1d). Here, abbreviations of C18:1 and C18:2 were used because of the unclear chromatogram separation from several isomers including vaccenic acid (18:1 n-7), oleic acid (18:1 n-6), linoleic acid (n-6), and conjugated linoleic acids. In this study, we fed PUFAs in synthetic ethyl ester form (PUFA-EE) because this method of producing ethyl esters can achieve a high degree of fatty acid purity. However, the bioavailability of the ethyl ester forms has been reported to be lower than the TAG form, which is the major form of PUFAs in natural fish oil [19,20]. Dietary PUFA-EE and TAG are both hydrolyzed to free fatty acid by pancreatic lipase and passively transported into enterocytes. In the enterocytes, free fatty acids are re-esterified to TAG and then transferred into lymph and blood circulation. Among those processes, the digestion of the ester bond and re-esterification may cause the difference of bioavailability [21]. However, the plasma PUFA levels were increased as expected after dietary intake of PUFA-EEs (Figure 1d), supporting the validity of this study design.

**Figure 1.** Effects on mouse plasma and body tissues following dietary supplementation with polyunsaturated fatty acids (PUFAs). (**a**) The total triglyceride and (**b**) total cholesterol in plasma were analyzed after two weeks of each PUFA diet. (**c**) The body weight in each PUFA diet condition was traced for two weeks, where the intake was started at 11 weeks of age. (**d**) The concentrations of total fatty acids in plasma were analyzed. The abbreviations ARA, EPA, and DHA, refer to arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, respectively. The statistical significance was evaluated by Tukey's testing (\* *P* < 0.05 and \*\* *P* < 0.01) where one-way ANOVA (analysis of variance) showed the statistical significance among groups (*P* < 0.05).

Next, we performed untargeted and targeted lipidomic analysis on the plasma and the nine tissues. A total of 915 molecules from 33 lipid classes were characterized in the untargeted analysis, while 111 oxylipins derived from LA, ALA, ARA, EPA, and DHA were identified in the targeted analysis (Table 1) The score plots of the principal component analysis (PCA) from untargeted lipidomics were clearly clustered by dietary-fed group in the metabolic organs, such as the liver and kidney, and the factor loadings showed that the profiles of glycerolipids and glycerophospholipids contributed to the principal components (Figure 2a and Supplementary Figure S2). On the other hand, the lipidome in the brain and muscle tissues, including the heart and skeletal muscle, was not significantly changed between different dietary conditions. Interestingly, the score plots of DHA dietary intake were classified independently from other conditions in the skeletal muscle, adipose, and heart. According to the factor loadings, the profile of DHA-containing lipids substantially contributed to these score plot dimensions. In fact, the muscle tissues were enriched in DHA present in membrane phospholipids [22], and our results suggested that the efficiency of DHA incorporation into membrane phospholipids was higher in muscle tissues compared to other tissues, and that the PC, PE, and PI lipid classes highly incorporated DHA as an acyl chain component in both the skeletal muscle and heart. In contrast, the membrane phospholipids incorporating DHA and the score plots of dietary DHA intake were not significantly changed in the brain.

**Figure 2.** Principal component analyses (PCA) in untargeted and targeted lipidomics data. (**a**) The first and second principal components of PCA were described for plasma and all tissue metabolites obtained in the untargeted analysis. (**b**) The first and second components were also shown by the targeted lipidomics data. F1: fish-meal-free diet not containing ARA, EPA, or DHA.


**Table 1.** Summary of characterized lipids in untargeted and targeted lipidomics.

The nomenclature of lipid classes in untargeted analysis is described in RIKEN PRIMe website (http://prime.psc. riken.jp/). FFA: free fatty acid; LA-O, ALA-O, GLA-O, DGLA-O, MA-O, ARA-O, EPA-O, and DHA-O: oxylipins derived from linoleic acid, alpha-linolenic acid, gamma-linolenic acid, dihomo-gamma-linolenic acid, mead acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, respectively.

#### *3.2. Oxylipin Profiles in Targeted Lipidomics*

According to the PCA score plots of the targeted lipidomic analysis, the oxylipins and their related molecules of plasma and tissues, except for the brain, were significantly a ffected by the dietary intake of PUFAs (Figure 2b and Supplementary Figure S2). Further detail of the oxylipin profiles is given in Figure 3. In most organs, except for the small intestine, lung, and spleen, the concentrations of 11,12-epoxyeicosatrienoic acid (EET) and 14,15-EET were higher than those of other oxylipins in ARA-derived metabolites, including epoxides, hydroxides, prostaglandins (PGs), and others. In contrast, COX-derived PGs such as PGE2, PGD2, and 6-keto-PGF1α were enriched in the small intestine, lung, and spleen, and these tissues contained higher amounts of hydroxyeicosatetraenoic acids (HETEs).

**Figure 3.** Oxylipin profiles in mouse tissues under each dietary condition. The total amounts of monoepoxides, hydroxides, and cyclized oxidized fatty acids including prostaglandins derived from ARA, EPA, and DHA are described. The results are given for each tissue in the four groups, i.e., the control, ARA-fed, EPE-fed, and DHA-fed groups. The ratio of oxidized fatty acids in each metabolite group is described by the stacked bar chart. The error bar indicates the standard deviation from the total amount values, and the statistical significance was evaluated by Tukey's testing (\* *P* < 0.05 and \*\* *P* < 0.01) where one-way ANOVA showed the statistical significance among groups (*P* < 0.05). For plasma, the y-axis unit becomes ng/μ<sup>L</sup> plasma instead of ng/mg tissue.

The levels of EPA and EPA-derived oxylipins were in fact lower than those of ARA and DHA in plasma and all tissues (Figure 1d and Supplementary Figure S3), while their amounts were substantially increased by dietary EPA intake as indicated by EPA-derived metabolites (Figure 3). Among the EPA-derived oxylipins, epoxyeicosatetraenoic acids (EpETEs) were the primary oxylipins present in the liver, kidney, white adipose tissue, skeletal muscle, heart, lung, and brain, while PGs and hydroxyeicosapentaenoic acids (HEPEs) were enriched in the small intestine and spleen. A similar pattern was observed in dietary DHA intake; epoxydocosapentaenoic acid (EpDPE) and hydroxydocosahexaenoic acid (HDoHE) were characteristically present in tissues.

The ARA-, EPA-, and DHA-derived epoxides, which are known as CYP metabolites, were enriched in metabolic tissues such as the liver, kidney, and white adipose tissue. ARA-derived epoxides activate various signaling pathways, and elicit functional responses such as vasorelaxation and anti-inflammation [23]. EPA-derived epoxides have anti-allergic and anti-inflammatory properties: 17,18-EpETE inhibits mast cell degranulation [24,25] and 12-hydroxy-17,18-EpETE suppresses neutrophil infiltration and eosinophilic inflammation [26,27]. These epoxides are converted into diols by soluble epoxide hydrolase, and the levels of diols and the diols/epoxide ratio were high in the small intestine (Supplementary Figure S4).

The ω3 oxylipins of EPA were increased following the dietary intake of DHA, while the amounts of ARA-derived oxylipins were decreased by ω3 PUFA dietary intake. Similar observations have also been reported in several other studies [5,7,28]. Interestingly, the increase in brain EPA levels following DHA intake was relatively high, and this result may represent the retro-conversion from DHA to EPA [29]. In contrast, ARA metabolism was competitively suppressed by a rich intake of EPA and DHA.

#### *3.3. Lipidomic Signatures in Untargeted Analyses*

We used circus plots to display the result of untargeted lipidomics (Figure 4) for the liver and brain, the metabolites of which were the most and least affected by the dietary intakes of PUFAs, respectively (Figure 2a). The heatmap layer and correlation linkages in the circus plot clearly showed that the PUFAs in liver were incorporated into glycerolipids, glycerophospholipids, and cholesterol esters, while the profile of sphingolipids was not affected by any dietary supplement. Moreover, the heatmap layer and the detail of fatty acid compositions indicating the existence of 18:2, 20:4/22:4, 20:5/22:5, and 22:6 as the acyl chain composition in a certain lipid class showed that glycerol (phospho) lipids containing 22:4 and 22:5 were increased in most tissues after the dietary intake of ARA and EPA, respectively. This result indicated that ARA and EPA were elongated to docosatetraenoic acid (DTA, 22:4) and docosapentaenoic acid (DPA, 22:5) by elongases such as elongation of very long chain fatty acids protein (ELOVL) 2 and 5 [30]. In contrast, an increase in lipids containing the elongated product of DHA, i.e., 24:6, was not observed following DHA dietary supplementation in the tissues examined.

A few differences in lipid profiles were observed in the mouse brain: free fatty acids (FA) of ARA, EPA, and DHA were affected by dietary intake (Figure 4). Moreover, several phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and ether linked PE (EtherPE), partially reflected the changes in dietary conditions according to the heatmap layer. Interestingly, the amounts of most lipid classes, including sphingolipids, were increased in the ARA-supplemented mice. Moreover, untargeted lipidomics identified the mono-galactocyl/glycosyl-diacylglycerol (MGDG) lipids, the main component of plant membrane lipids, in the mouse brain, where the MGDG lipids were composed of saturated or monounsaturated fatty acids. A few studies have also shown the existence of MGDG in the brain, and its biosynthesis and physiological role have been examined in several in vitro studies, although their mechanisms are still unclear [31–33]. These results sugges<sup>t</sup> that the annotation of untargeted lipidomics should be performed first in an unbiased manner.

**Figure 4.** Lipid profiles in the liver and brain from the untargeted lipidomics data. In the circus plot, the lipid profiles are described separately by lipid class. The profile has been scaled in each metabolite from −1 to 1 in the heatmap. The ion abundance of each lipid is described by the circle size, where the average value in all conditions was utilized for the ion abundance size. Any lipid with more than two double bonds in an acyl chain was defined as a PUFA-containing metabolite. Lipids containing 18:2, 20:4, 22:4, 20:5, 22:5, and 22:6 are marked by pink, red, orange, blue, sky blue, and green colors, respectively. Metabolites were linked if the ion abundance correlation of two metabolites in biological samples was greater than 0.9.

Untargeted lipidomics also revealed unique lipid features in the other tissues. The diacylglycerol (DAG) and TAG lipids were the most abundant species in the white adipose tissue, as has previously been described [34], and the DAG and TAG profiles were significantly a ffected in most tissues. Interestingly, however, we found that the profiles of DAG and TAG were not a ffected in the spleen, although the profiles of free fatty acids, glycerophospholipids, and cholesteryl esters did reflect the intakes of ARA, EPA, and DHA (Supplementary Figure S5). This result suggested that the metabolic pathway has a limited ability to incorporate dietary PUFAs into TAG in the spleen. Indeed, diglyceride acyltransferase (DGAT) activity has been reported to be low in the spleen [35]. Our results also showed that the ether-linked PC and PE (EtherPC and EtherPE) incorporating *O*-alkenyl or *O*-alkyl chains were enriched in the kidney, spleen, small intestine, muscle, heart, and brain (Table 1), while the acyl chain profiles of EtherPC and EtherPE reflected the dietary PUFA in all tissues and plasma. In contrast, the low variety of *O*-alkenyl or *O*-alkyl chains containing PC and PE in the liver can be explained by the low expression of the alkyldihydroxyacetonephosphate synthase (AGPS) [36].

In addition, we found that the levels of phospholipids containing LA (LA-PLs) were substantially decreased after the dietary intake of ARA (Figure 5a), and profound changes were observed in PC and PE. This may have resulted from competition for the PL remodeling enzyme lysophospholipid acyltransferase 3 (LPCAT3), which has a high a ffinity for both ARA and LA [37]. Since free LA was decreased by dietary intake of ARA, in addition to LA-PLs (Supplementary Figure S5), the decrease of LA under ARA supplementation could reflect a potential mechanism whereby total ω6 fatty acid amount, i.e., LA + ARA, is maintained in mammalian cells: in fact, a previous study provided a complementary report in which an ARA decrease is observed in plasma following a higher intake of LA [38]. We also found that the BMP containing DHA (DHA-BMP) was substantially increased under DHA supplementation, while an increase in BMP containing ARA and EPA was not observed in ARA or EPA supplementation (Figure 5b): the annotation was performed by curating both positive and negative ion MS/MS spectra (Figure 5c). While DHA is known to be the predominant fatty acyl chain of BMP in the brain [39], our study suggested that there is a selective mechanism in which DHA is preferably incorporated into BMP in several tissues, including the small intestine, lung, heart, kidney, spleen, and liver. Although the BMP biosynthesis mechanism is still unclear, one study reported that lysocardiolipin acyltransferase 8 (AGPAT8), preferring unsaturated fatty acids as the acyl donor, catalyzed the acylation of BMP [40,41], and the gene expression level of AGPAT8 was high in the small intestine, heart, liver, and kidney in the BioGPS dataset (http://ds.biogps.org/?dataset=GSE10246&gene=225010). Importantly, these results indicate the importance of untargeted lipidomics, where unexpected and novel insights can be discovered in a data-driven manner.

**Figure 5.** Unique lipid modulations in ARA or DHA dietary intakes. (**a**) The profile of phosphatidylcholines containing linoleic acid (18:2) as the longest PUFA chain and the ion abundances of each molecule. Note that a hyphen has been used for the description of acyl chains, e.g., 18:0-18:2, because the *sn1*/*sn2* acyl chain structural isomers were not distinguished in this study. (**b**) The profile of bis(monoacylglycero)phosphate (BMP): the asterisk (\*) means any of the fatty acids and the ion abundances of lipids containing 18:2, 20:4, 20:5, or 22:6 were summed for quantification. Black, red, blue, and green represent the dietary conditions: control, ARA, EPA, and DHA supplementation, respectively. The statistical significance was evaluated by Tukey's testing (\* *P* < 0.05 and \*\* *P* < 0.01) where one-way ANOVA showed the statistical significance among groups (*P* < 0.05). (**c**) The experimental positive and negative ion mode MS/MS spectra (black color) of BMP 22:6-22:6 and PG 16:0-22:6 with the in silico reference spectrum (red color for ESI(+)-MS/MS and blue color for ESI(−)-MS/MS). The cleaved places generating the major fragment ions are described by red color for ESI(+)-MS/MS and blue color for ESI(−)-MS/MS. The product ion of *m*/*z* 695.5 from BMP 22:6-22:6 in positive ion mode was interpreted as the result of structural rearrangemen<sup>t</sup> in the fragmentation process.
