Plasma oxylipin profiling identifies polyunsaturated vicinal diols as responsive to arachidonic acid and docosahexaenoic acid intake in growing piglets.

The dose-responsiveness of plasma oxylipins to incremental dietary intake of arachidonic acid (20:4n-6; ARA) and docosahexaenoic acid (22:6n-3; DHA) was determined in piglets. Piglets randomly received one of six formulas (n = 8 per group) from days 3 to 27 postnatally. Diets contained incremental ARA or incremental DHA levels as follows (% fatty acid, ARA/DHA): (A1) 0.1/1.0; (A2) 0.53/1.0; (A3–D3) 0.69/1.0; (A4) 1.1/1.0; (D1) 0.66/0.33; and (D2) 0.67/0.62, resulting in incremental intake (g/kg BW/day) of ARA: 0.07 ± 0.01, 0.43 ± 0.03, 0.55 ± 0.03, and 0.82 ± 0.05 at constant DHA intake (0.82 ± 0.05), or incremental intake of DHA: 0.27 ± 0.02, 0.49 ± 0.03, and 0.81 ± 0.05 at constant ARA intake (0.54 ± 0.04). Plasma oxylipin concentrations and free plasma PUFA levels were determined at day 28 using LC-MS/MS. Incremental dietary ARA intake dose-dependently increased plasma ARA levels. In parallel, ARA intake dose-dependently increased ARA-derived diols 5,6- and 14,15-dihydroxyeicosatrienoic acid (DiHETrE) and linoleic acid-derived 12,13-dihydroxyoctadecenoic acid (DiHOME), downstream metabolites of cytochrome P450 expoxygenase (CYP). The ARA epoxide products from CYP are important in vascular homeostatic maintenance. Incremental DHA intake increased plasma DHA and most markedly raised the eicosapentaenoic acid (EPA) metabolite 17,18-dihydroxyeicosatetraenoic acid (DiHETE) and the DHA metabolite 19,20-dihydroxydocosapentaenoic acid (DiHDPE). In conclusion, increasing ARA and DHA intake dose-dependently influenced endogenous n-6 and n-3 oxylipin plasma concentrations in growing piglets, although the biological relevance of these findings remains to be determined.

Venous blood was collected at day 28, the morning after last formula feeding at day 27. Details have been described elsewhere ( 18 ). Plasma levels of free fatty acids were analyzed by LC-MS/MS. In short, protein in the samples was precipitated by adding 50 µl of internal standard solution and 1050 µl of methanol to 50 µl of plasma. After centrifugation, the supernatant was transferred into an injection vial. For (relative) quantifi cation, a mixture of internal standards [32 exogenous calibrants and 12 internal standards] was used. The free fatty acids were analyzed by LC-MS/MS using a Waters AQCUITY UltraPerformance LC (UPLC) System with an ACQUITY UPLC HSS T3 1.8 µm, 2.1 × 100 mm column (Waters, Milford, MA) and an Agilent Q-TOF 6530 High Resolution Mass Spectrometer (Agilent, Santa Clara, CA) using reference mass correction. Data preprocessing was performed using Agilent Mass-Hunter Quantitative Analysis software; all peaks were checked manually. Samples were analyzed in batches with QC samples added after every 10 samples. The QC samples were used to assess data quality and to correct for instrument response variations. Free fatty acid levels were reported as relative quantitative values of target compound to internal standard.

Oxylipin analysis
Oxylipin profi ling was done according to the method as described by Strassburg et al. ( 27 ). Thus, 250 µl aliquots of piglet plasma were taken and subjected to solid phase extraction with hydrophilic-lipophilic-balanced material (Oasis HLB, Waters, Etten-Leur, The Netherlands) using ethyl acetate to elute analytes. To concentrate, the eluate was gently dried under a nitrogen stream and reconstituted in 50 µl of injection solvent (acetonitrile and methanol, 1:1 v/v). A 5 µl aliquot was injected into the LC-MS for analysis. Separation was done by HPLC (Agilent 1260, San Jose, CA) using an Ascentis Express column (2.1 × 150 mm, 2.7 µm particles; Supelco, Bellefonte, PA) with 0.35 ml/min fl ow rate during a 28 min gradient. The HPLC was coupled to electrospray ionization on a triple-quadrupole mass spectrometer (Agilent 6460, San Jose, CA). Oxylipins were detected in negative ion mode using dynamic multiple reaction monitoring (MRM). The raw data were preprocessed using Agilent MassHunter Quantitative Analysis software (Version B.04.00), and quantitation of oxylipin response was calculated as the peak area ratios of the target analyte to the respective internal standard. To obtain actual concentrations, calibration samples with spiked oxylipin levels were included in the measurement series. Oxylipins were classifi ed according to the LIPID MAPS Structure Database ( 33 ). acids to ARA and DHA is estimated to be low in infants and not as effi cient as preformed ARA and DHA in sustaining ARA and DHA plasma levels (3)(4)(5)(6). Many studies indicate that DHA and ARA-enriched formula from birth to one year of age may confer benefi ts on visual acuity maturation and aspects of cognitive development compared with unenriched formula, particularly in premature infants (7)(8)(9)(10)(11)(12).
Oxylipins are currently the focus of considerable interest as they can act as intercellular signaling molecules and are involved in the regulation of many cell and tissue responses ( 20 ). Oxylipins can exert a wide range of effects in biological tissues, including the regulation of cellular proliferation ( 21,22 ), infl ammation or infl ammation resolution (23)(24)(25), and vascular function ( 23,26 ). Novel analytical methodologies to quantify the large spectrum of plasma lipids have emerged and reveal a remarkable diversity of oxylipins in human plasma ( 19,27 ). The biological roles of many of these oxylipins have not yet been fully elucidated. Over the past decade there has been growing interest in pharmacological modulation of oxylipin enzymatic pathways to mediate benefi cial vascular and infl ammatory effects (28)(29)(30)(31)(32). In previous studies with mice ( 30 ) and healthy volunteers ( 19,31 ), it was shown that dietary intake of n-3 LCPUFA was able to alter plasma oxylipin levels. Despite intense interest in the biological effects of dietary LCPUFA in infant nutrition, the infl uence of dietary DHA and ARA on plasma oxylipin levels has not previously been explored in growing mammals, including humans. We report here a lipidomicsbased study of plasma oxylipin profi le using a dose-response design in growing piglets.

Effect of DHA intake on plasma oxylipins
DHA intake dose-dependently increased the plasma CYP/sEH-catalyzed DHA metabolite 19,20-DiHDPE after the multiple comparison procedure ( Table 3 and Fig. 2A ), although no effect of DHA was apparent on the precursor metabolite 19(20)-EpDPE. Dietary DHA also increased the CYP/sEH-catalyzed EPA metabolite 17,18-DiHETE ( Fig. 2B ), but the precursor 17(18)-EpETE was not detected in Statistics All statistical analyses were performed using R version 2.13.0. Relative response ratios of oxylipins were converted into actual concentrations (nM) using the R chemCal package (free software, R-project). Second-order (quadratic) regression models were applied to examine dose-response relationships between the 24 day mean dietary DHA or ARA intake and log plasma oxylipin or log PUFA levels. As predictor, variable dietary levels were corrected for effective intake per body weight (BW) throughout growth by expressing as g/kg BW/day. Linear mixed modeling was performed for varying dietary DHA intake (at fi xed ARA intake), for gender, and their interactions as independent (fi xed) variable, and litter as a random variable. To remove nonsignificant terms, stepwise regression was used. Some dose-response curves followed a curvilinear (quadratic) relationship. Therefore, we also included a quadratic term in our stepwise regression approach. Modeling for varying ARA intake was performed in the same way but with DHA intake as the fi xed factor. The maternally reared group was excluded from the analysis as the sow's diet differed from the formula diets in more aspects than only DHA and ARA concentrations. The LC-MS/MS data were log-transformed before modeling. False discovery rate (FDR) control was used to correct for multiple comparisons. Data are presented as the mean ± SD.

Effect of ARA on plasma PUFAs
Dietary intervention groups receiving incremental dietary ARA levels, consumed on average 0.07 ± 0.01, 0.43 ± 0.03, 0.55 ± 0.03, and 0.82 ± 0.05 g of ARA/kg BW/day, respectively, at constant DHA (0.82 ± 0.05g/kg BW/day) over the course of 24 days of formula feeding ( Table 1 ). Mixed model regression analysis showed that incremental ARA intake by the piglets signifi cantly increased the plasma levels of ARA ( Table 1 ). The signifi cant increase in plasma DHA, linoleic acid, and ␣ -linolenic acid levels that occurred with increasing ARA intake disappeared after correcting for multiple testing ( Table 1 ). Supplementary Table I shows the coeffi cients, P values, and FDRs obtained from linear mixed modeling for the relationships between ARA intake and plasma fatty acid levels.

Effect of DHA on plasma PUFAs
The three dietary DHA levels resulted in a mean DHA intake of 0.27 ± 0.02, 0.49 ± 0.03, and 0.81 ± 0.05 g/kg BW/day, respectively, at constant ARA intake (0.54 ± 0.04 g/kg BW/day) over the 24 days of feeding. The plasma levels of DHA dose-dependently increased with higher amounts of DHA ingested ( Table 1 ). Other plasma fatty acids were not signifi cantly affected. Dietary DHA in our study did not signifi cantly affect plasma EPA levels, but heart and liver tissue phospholipid EPA levels increased with DHA (data not shown). The coeffi cients, P values, and FDRs for the effects of dietary DHA intake on plasma fatty acids are shown in supplementary Table I.

Detected plasma oxylipins
The metabolites, identifi ed in pig plasma after 24 days of dietary intake, are shown in Tables 2 and 3 supplementary Table III.

DISCUSSION
Pigs are an accepted model of human nutrition since they are very similar to humans with respect to anatomy, physiology ( 34,35 ), and fatty acid metabolism ( 36 ). Based on the high homology between the pig and human genome, high sequence homology between human and porcine COX and LOX enzymes can be expected. Some porcine CYP enzymes showed about 70-83% homology to human forms and metabolized the same test substrates ( 37 ). This is the fi rst study to report on the dose responsiveness of fatty acid-derived oxylipins to dietary intake of ARA and DHA during early postnatal development in piglets. The use of metabolomics technology in this study allowed us to characterize the detectable piglet oxylipin metabolome. The data show that some endogenous n-6 and n-3 oxylipin levels can be dose-dependently modulated by dietary DHA and ARA levels. Most oxylipin curves did not follow Michaelis-Menten enzyme kinetics, which could be explained by the involvement of multiple downstream enzymes in their production and the nonclassical enzyme kinetics as described for some CYP enzymes ( 38 ). The extent to which these oxylipins may mediate benefi cial health effects remains to be explored. These data help to focus such studies on particular oxylipin species in various dietary circumstances of ARA/DHA intake during early development.

Arachidonic acid metabolites
Eicosanoids generated via COX and LOX exert many important functions, as exemplifi ed by various pathologies manifested in mice lacking LOX and COX expression ( 39,40 ). Despite the traditional belief that ARA-derived eicosanoids generated via the COX or LOX pathway contribute to the initiation of infl ammation, emerging data suggest that these eicosanoids also contribute to the resolution of infl ammation ( 23,(41)(42)(43)(44). In perinatal development, COX-catalyzed PGE 2 is an important lipid metabolite not only in immune cell homeostasis ( 45 ) but also in intestinal crypt proliferation ( 45 ) and preservation of neural function ( 46 ). Results of this study in growing piglets demonstrate that increasing dietary ARA levels do not affect plasma concentrations of the LOX-or COXsynthesized ARA metabolites, including PGE 2 , in spite of the increased levels of ARA in plasma and tissue ( 18 ). Also, plasma infl ammation markers remained unchanged by increasing dietary ARA, as was previously reported ( 47 ). Although DHA may compete with ARA for membrane phospholipid incorporation and subsequent eicosanoid formation, dietary DHA also did not infl uence any of LOX-or COX-synthesized ARA metabolites, In several studies, dietary n-3 LCPUFA was shown to reduce tissues may cause only a transient or nondetectable change in circulating concentrations due to dilution in the greater volume of blood. Increasing ARA intake dose-dependently raised plasma ARA levels in parallel with plasma 5,6-DiHETrE and 14, 15-DiHETrE concentrations. The dose-dependent increases in these vicinal diols suggest increased conversion of ARA via the CYP/sEH enzymatic pathway. The precursor epoxides 5(6)-EpETrE and 14(15)-EpETrE were however not detected. EpETrEs may be diffi cult to detect; most EpE-TrE are esterifi ed into phospholipids, whereas nonesterifi ed EpETrEs are rapidly metabolized by sEH to their vicinal diols ( 26 ). In humans, 14(15)-EpETrE concentrations were on par with their corresponding vicinal diols ( 52 ). However, the analysis we employed had a one-third lower sensitivity for epoxides than for diols ( 27 ). Therefore, ARA-derived LOX and COX products from ex vivo immune cells in the presence of an infl ammatory stimulus (48)(49)(50)(51). Our data provide no evidence that ARA-rich or DHA-rich formula infl uence circulating COX or LOX eicosanoids in healthy growing piglets. As physiologically comparable levels of ARA are found in human breast milk and infant formula, comparable effects in humans may be inferred. The LC-MS/MS method employed is suffi ciently sensitive to detect multiple LOX-or COXsynthesized eicosanoids from ARA. The COX-and LOXderived metabolites are rapidly inactivated and excreted, and some of these inactivation products are released to and/or created in the circulatory system. At continuous elevated production of eicosanoids, stable metabolites may accumulate to detectable levels in peripheral blood. However, a low-level production of mediators in peripheral sion of linoleic acid to 12,13-DiHOME via the CYP/sEH pathway may be regulated by physiological demand due to ARA and not the level of substrate availability.

Eicosapentaenoic acid metabolites
In the current study, dietary DHA signifi cantly increased EPA levels in tissue phospholipids, although this was not refl ected in the plasma free fatty acid pool. Dietary DHA also increased the EPA-derived vicinal diol 17,18-DiHETE metabolized in the CYP/sEH pathway, suggesting that DHA retroconversion to EPA may occur to some extent. Due to competitive metabolic effects between n-3 and n-6 fatty acids, the effect of DHA on retroconversion to EPA and subsequent metabolites may be less pronounced when ARA is concomitantly present in the diet, as has been reported for pigs fed both DHA and ARA ( 65 ). Although little is known about the role of 17,18-DiHETE, it has been described to have anti-infl ammatory properties ( 66 ). The EPA-derived COX-2-mediated PGF 3 ␣ and LOX-5-mediated 5(s)-HEPE appeared unresponsive to dietary DHA changes and may require a stimulus for production.

Docosahexaenoic acid CYP metabolites
The most obvious fi nding was the dose-responsiveness of plasma 19,20-DiHDPE to dietary DHA intake of the piglets. This is similar to increased plasma 19,20-DiHDPE observed in healthy humans who consumed EPA and DHA from concentrated fi sh oil ( 31 ). However, in the latter study, a much broader range of oxylipins was signifi cantly affected by EPA and DHA than in our study, possibly due to the much larger group sizes used. Little is known about this DHA-derived 19,20-DiHDPE diol, but recent research in sEH knockout mice showed that sEH metabolites, particularly 19,20-DiHDPE, play an important role in normal retinal vascularization ( 67 ). To what extent this metabolite is involved in the maturation of the retina remains to be established. changes in the circulating concentrations of ARA-derived epoxides cannot be ruled out.
The epoxygenases of the CYP2 gene family have prominent roles in vascular regulation and generate the epoxides 5,6-, 8,9-, 11,12-, and 14,15-EpETrE from ARA ( 53 ). EpETrEs are produced by several tissues, including neural ( 54 ) and vascular tissues ( 55 ). Data from in vitro and in vivo studies demonstrate manifold biological activities of EpETrEs, including anti-infl ammatory, neuroprotective, and vascular-protective effects like vasodilation (reviewed in Refs. 56,57 ). Little is known about CYP metabolism in pigs, but porcine ovary was found to highly express CYP2J and sEH and produce EpETrEs and EpOMEs, as well as DiHETrEs and DiHOMEs during ovulation ( 58 ). In cultured endothelial cells of porcine origin, generation of 8,9-, 11,12-, and 14,15-EpETrE has also been observed ( 59,60 ). In growing pigs, EpETrEs may play an important role in vascular formation, as they were shown to be involved in proliferation of porcine endothelial cells ( 61 ). These EpETrE metabolites are further metabolized by sEH to their corresponding vicinal diols (DiHETrEs), which have less biological activity than their precursor metabolites when sensitive endpoints are considered ( 62 ).