Characterization of Lipid Profiles after Dietary Intake of Polyunsaturated Fatty Acids Using Integrated Untargeted and Targeted Lipidomics

Abstract

Illuminating the comprehensive lipid profiles after dietary supplementation of polyunsaturated fatty acids (PUFAs) is crucial to revealing the tissue distribution of PUFAs in living organisms, as well as to providing novel insights into lipid metabolism. Here, we performed lipidomic analyses on mouse plasma and nine tissues, including the liver, kidney, brain, white adipose, heart, lung, small intestine, skeletal muscle, and spleen, with the dietary intake conditions of arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) as the ethyl ester form. We incorporated targeted and untargeted approaches for profiling oxylipins and complex lipids such as glycerol (phospho) lipids, sphingolipids, and sterols, respectively, which led to the characterization of 1026 lipid molecules from the mouse tissues. The lipidomic analysis indicated that the intake of PUFAs strongly impacted the lipid profiles of metabolic organs such as the liver and kidney, while causing less impact on the brain. Moreover, we revealed a unique lipid modulation in most tissues, where phospholipids containing linoleic acid were significantly decreased in mice on the ARA-supplemented diet, and bis(monoacylglycero)phosphate (BMP) selectively incorporated DHA over ARA and EPA. We comprehensively studied the lipid profiles after dietary intake of PUFAs, which gives insight into lipid metabolism and nutrition research on PUFA supplementation.

Keywords: arachidonic acid; dietary fat; fatty acid metabolism; lipidomics; mass spectrometry; omega-3 fatty acids.

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).

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.

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/µL plasma instead of ng/mg tissue.

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.

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 rearrangement in the fragmentation process.