Dietary omega-3 fatty acids modulate the eicosanoid profile in man primarily via the CYP-epoxygenase pathway

Abstract

Cytochrome P450 (CYP)-dependent metabolites of arachidonic acid (AA) contribute to the regulation of cardiovascular function. CYP enzymes also accept EPA and DHA to yield more potent vasodilatory and potentially anti-arrhythmic metabolites, suggesting that the endogenous CYP-eicosanoid profile can be favorably shifted by dietary omega-3 fatty acids. To test this hypothesis, 20 healthy volunteers were treated with an EPA/DHA supplement and analyzed for concomitant changes in the circulatory and urinary levels of AA-, EPA-, and DHA-derived metabolites produced by the cyclooxygenase-, lipoxygenase (LOX)-, and CYP-dependent pathways. Raising the Omega-3 Index from about four to eight primarily resulted in a large increase of EPA-derived CYP-dependent epoxy-metabolites followed by increases of EPA- and DHA-derived LOX-dependent monohydroxy-metabolites including the precursors of the resolvin E and D families; resolvins themselves were not detected. The metabolite/precursor fatty acid ratios indicated that CYP epoxygenases metabolized EPA with an 8.6-fold higher efficiency and DHA with a 2.2-fold higher efficiency than AA. Effects on leukotriene, prostaglandin E, prostacyclin, and thromboxane formation remained rather weak. We propose that CYP-dependent epoxy-metabolites of EPA and DHA may function as mediators of the vasodilatory and cardioprotective effects of omega-3 fatty acids and could serve as biomarkers in clinical studies investigating the cardiovascular effects of EPA/DHA supplementation.

Keywords: cytochrome P450; lipidomics; nutrition.

Fig. 1.

Effects of EPA/DHA supplementation on the Omega-3 Index and fatty acid composition in RBCs. A: Frequency distribution of the basal Omega-3 Index in the prescreened group of 38 healthy volunteers. Twenty subjects with an Omega-3 Index ≤6 (dashed line) were included into the study. B: Time- and dose-dependent changes of the Omega-3 Index during the whole course of the study. C: Correlation of the Omega-3 Index achieved after 8 weeks of EPA/DHA supplementation with the product of the baseline Omega-3 Index and the dose per kilogram of body weight. D: Changes in the abundance of individual fatty acids comparing their percentages of total fatty acid in RBCs at week 8 and week 0 of EPA/DHA supplementation. Data are given as mean ± SEM, n = 19. A general linear model for repeated measurements was used for analysis (B, D) and significant changes are indicated as: *P < 0.05 versus basal level [week 0 (W0)] and #P < 0.05 versus maximum treatment [week 8 (W8)]. For dosage dependency (C), a Pearson correlation was performed: r = 0.599, P < 0.01.

Fig. 2.

Effect of EPA/DHA supplementation on the profile of circulating CYP-epoxygenase metabolites. A: Time- and dose-dependent changes of the CYP-epoxyeicosanoid index during the whole course of the study. B: Regioisomeric composition of epoxygenase products derived from AA, EPA, and DHA at baseline [week 0 (W0)], after maximal EPA/DHA supplementation [week 8 (W8)], and after discontinuation of supplementation [week 16 (W16)]. C: Correlation of the relative plasma levels of EPA- and AA-derived metabolites with the EPA/AA precursor fatty acid ratio. D: Correlation of the relative plasma levels of DHA- and AA-derived metabolites with the DHA/AA precursor fatty acid ratio. Metabolite levels are given as the sum of the primary CYP-epoxygenase products and the corresponding vicinal diols produced by sEH-mediated hydrolysis of the primary epoxides. Total metabolite levels (free + esterified) were determined after alkaline hydrolysis. Data are given as mean ± SEM, n = 19. A general linear model for repeated measurements was used for analysis (A, B) and significant changes are indicated as: *P < 0.05 versus basal level (W0) and #P < 0.05 versus maximum treatment (W8). For relative efficiencies, a Pearson correlation was performed: y = 8.60x − 0.06; r = 0.826 with P < 0.001 (C) and y = 2.21x − 0.21; r = 0.587 with P < 0.001 (D).

Fig. 3.

Effect of EPA/DHA supplementation on the plasma levels of CYP-ω-hydroxylase metabolites derived from AA (20-HETE), EPA (20-HEPE), and DHA (22-HDHA). Metabolite levels were determined after alkaline hydrolysis and thus represent the sum of free and esterified metabolites. Bars show mean ± SEM, n = 19. A general linear model for repeated measurements was used for analysis and significant changes are indicated as: *P < 0.05 versus basal level [week 0 (W0)] and #P < 0.05 versus maximum treatment [week 8 (W8)].

Fig. 4.

Effect of EPA/DHA supplementation on the plasma levels of LOX-dependent monohydroxy-metabolites. A, B: Plasma levels of 5-LOX-dependent metabolites derived from AA (5-HETE), EPA (5-HEPE), and DHA (4-HDHA and 7-HDHA) and their relative abundance in correlation with the corresponding precursor fatty acid ratios (filled circles for EPA/AA; open circles for DHA/AA). C, D: Plasma levels of 12-LOX-dependent metabolites derived from AA (12-HETE), EPA (12-HEPE), and DHA (14-HDHA) and their relative abundance in correlation with the corresponding precursor fatty acid ratios (filled circles for EPA/AA; open circles for DHA/AA). E, F: Plasma levels of 15-LOX-dependent metabolites derived from AA (15-HETE), EPA (15-HEPE), and DHA (17-HDHA) and their relative abundance in correlation with the corresponding precursor fatty acid ratios (filled circles for EPA/AA; open circles for DHA/AA). Metabolite levels were determined after alkaline hydrolysis and thus represent the sum of free and esterified metabolites. Bars show mean ± SEM, n = 19. A general linear model for repeated measurements was used for analysis (A, C, E) and significant changes are indicated as: *P < 0.05 versus basal level [week 0 (W0)] and #P < 0.05 versus maximum treatment [week 8 (W8)]. For relative efficiencies a Pearson/Spearman Rho correlation was performed: 5-HEPE/5-HETE, y = 4.02x − 0.04, r = 0.790 with P < 0.001 and 4-HDHA/5-HETE, y = 1.89x − 0.20, r = 0.696 with P < 0.001 (B); 12-HEPE/12-HETE, y = 3.37x − 0.05, r = 0.821 with P < 0.001 and 14-HDHA/12-HETE, y = 3.30x − 0.30; r = 0.493 with P < 0.001 (D); and 15-HEPE/15-HETE, y = 1.87x − 0.02, r = 0.795 with P < 0.001 and 17-HDHA/15-HETE, y = 3.15x − 0.32, r = 0.688 with P < 0.001 (F).

Fig. 5.

Effect of EPA/DHA supplementation on 18-HEPE formation. Total plasma levels of 18-HEPE were determined after alkaline hydrolysis. Data are given as mean ± SEM, n = 19. A general linear model for repeated measurements was used for analysis and significant changes are indicated as: *P < 0.05 versus basal level [week 0 (W0)] and #P < 0.05 versus maximum treatment [week 8 (W8)].

Fig. 6.

Effect of EPA/DHA supplementation on leukotriene and prostanoid formation after calcium ionophore stimulation. The levels of free metabolites were determined after A23187-mediated stimulation of whole blood samples and thus reflect the metabolic capacity and substrate specificity of the corresponding enzymes expressed in blood cells. A, B: Generated levels of AA-derived LTB4 and EPA-derived LTB5 and the relative abundance of LTB5 and LTB4 in correlation with the EPA/AA precursor fatty acid ratio. C, D: Levels of AA-derived PGE2 and EPA-derived PGE3 and the relative abundance of PGE3 and PGE2 in correlation with the EPA/AA precursor fatty acid ratio. E, F: Levels of AA-derived TXB2 and EPA-derived TXB3 and the relative abundance of TXB3 and TXB2 in correlation with the EPA/AA precursor fatty acid ratio. Data are given as mean ± SEM, n = 19. A general linear model for repeated measurements was used for analysis (A, C, E) and significant changes are indicated as: *P < 0.05 versus basal level [week 0 (W0)] and #P < 0.05 versus maximum treatment [week 8 (W8)]. For relative efficiencies, a Pearson/Spearman Rho correlation was performed: LTB5/LTB4, y = 0.58x − 0.01, r = 0.864 with P < 0.001 (B); PGE3/PGE2, y = 0.16x − 0.002, r = 0.712 with P < 0.001 (D); TXB3/TXB2 no correlation (F).

Fig. 7.

Calcium ionophore stimulated monohydroxy-metabolite formation via LOX enzymes. Shown is the formation of free metabolites via 5-LOX (A), 12-LOX (C) and 15-LOX (E) enzymes in whole blood samples at baseline [week 0 (W0)], after maximal EPA/DHA-supplementation [week 8 (W8)], and after discontinuation of supplementation [week 16 (W16)]. Bars represent mean ± SEM, n = 19. A general linear model for repeated measurements was used for analysis (A, C, E) and significant changes are indicated as: *P < 0.05 versus basal level (W0) and #P < 0.05 versus maximum treatment (W8). For relative efficiencies a Pearson/Spearman Rho correlation was performed: 5-HEPE/5-HETE versus EPA/AA: y = 1.2x − 0.02, r = 0.884 with P < 0.001 and 4-HDHA/5-HETE versus DHA/AA, y = 0.01x − 0.00008, r = 0.351 with P < 0.001; 7-HDHA/5-HETE versus DHA/AA: y = 0.02x − 0.0008, r = 0.421 with P < 0.001 (B). 12-HEPE/12-HETE versus EPA/DHA: y = 0.73x − 0.01, r = 0.857 with P < 0.001 and 14-HDHA/12-HETE versus DHA/AA: y = x − 0.35 − 0.01, r = 0.444 with P < 0.001 (D). 15-HEPE/15-HETE versus EPA/DHA: y = 1.26x − 0.01, r = 0.834 with P < 0.001 and 17-HDHA/15-HETE versus DHA/AA: y = 0.93x − 0.03, r = 0.510 with P < 0.001 (F).

Fig. 8.

Effect of EPA/DHA supplementation on the formation of nonenzymatic oxidation products. A: Plasma levels of AA-derived 9-HETE and EPA-derived 9-HEPE at baseline [week 0 (W0)], after maximal EPA/DHA supplementation [week 8 (W8)], and after discontinuation of supplementation [week 16 (W16)]. A general linear model for repeated measurements was used for analysis (A, C) and significant changes are indicated as: *P < 0.05 versus basal level (W0) and #P < 0.05 versus maximum treatment (W8). B: Correlation of the plasma 9-HEPE/9-HETE ratio with the EPA/AA precursor ratio. Metabolite levels in (A) and (B) refer to the total amounts of 9-HETE and 9-HEPE as determined after alkaline hydrolysis. C: Presence of free 9-HETE and 9-HEPE in calcium ionophore treated blood samples.

Fig. 9.

Effect of EPA/DHA supplementation on clinical risk factors. Shown are the changes in triglyceride levels (A) and blood pressure (C) and their dependencies on the respective basal levels [(B) and (D), respectively]. Data are given as mean ± SEM, n = 19. A general linear model for repeated measurements was used for analysis (A, C) and significant changes are indicated as: *P < 0.05 versus basal level [week 0 (W0)] and #P < 0.05 versus maximum treatment [week 8 (W8)].

Fig. 10.

Comparison of the susceptibilities of the three branches of the human AA-cascade to dietary EPA/DHA supplementation. This figure summarizes the linear correlations obtained for the corresponding metabolite precursor fatty acid pairs. The slopes of the correlation lines were taken as a measure for the relative efficiencies by which EPA and AA (A) or DHA and AA (B) were utilized by the different enzymatic pathways of eicosanoid formation.