Effect of Omega-3 Fatty Acid Ethyl Esters on the Oxylipin Composition of Lipoproteins in Hypertriglyceridemic,Statin-Treated Subjects

Background

Oxylipins mediate inflammation, vascular tension, and more. Their presence in lipoproteins could explain why lipoproteins mediate nearly identical activities.

Methods

To determine how oxylipins are distributed in the lipoproteins of hypertriglyceridemic subjects, and whether omega-3 fatty acids alter them in a manner consistent with improved cardiovascular health, we recruited 15 dyslipidemic subjects whose levels of low density lipoprotein cholesterol (LDL-C) were at goal but who remained hypertriglyceridemic (200–499 mg/dL). They were treated them with the indicated dose of 4 g/d omega-3 acid ethyl esters (P-OM3) for 8 weeks. Measured oxylipins included mid-chain alcohols (HETEs, HEPEs and HDoHEs), ketones (KETEs), epoxides (as EpETrEs, EpETEs, and EpDPEs).

Results

At baseline, arachidonate-oxylipins (HETEs, KETEs, and EpETrEs) were most abundant in plasma with the greatest fraction of total abundance (mean |95% CI|) being carried in high density lipoproteins (HDL); 42% |31, 57| followed by very low density lipoproteins (VLDL); 27% |20, 36|; and LDL 21% |16, 28|. EPA- and DHA-derived oxylipins constituted less than 11% of total. HDL carried alcohols and epoxides but VLDL was also rich in ketones. Treatment decreased AA-derived oxylipins across lipoprotein classes (−23% |−33, −12|, p = 0.0003), and expanded EPA−(322% |241, 422|, p<0.0001) and DHA-derived oxylipins (123% |80, 176|, p<0.0001).

Conclusions

Each lipoprotein class carries a unique oxylipin complement. P-OM3 treatment alters the oxylipin content of all classes, reducing pro-inflammatory and increasing anti-inflammatory species, consistent with the improved inflammatory and vascular status associated with the treatment.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00959842","term_id":"NCT00959842"}}NCT00959842     

Streptozotocin-induced diabetes in human apolipoprotein B transgenic mice. Effects on lipoproteins and atherosclerosis

The effects of diabetes and lipoprotein lipase (LpL) on plasma lipids were studied in mice expressing human apolipoprotein B (HuBTg). Our overall objective was to produce a diabetic mouse model in which the sole effects of blood glucose elevation on atherosclerosis could be assessed. Mice were made diabetic by intraperitoneal injection of streptozotocin, which led to a 2- to 2. 5-fold increase in plasma glucose. Lipids were assessed in mice on chow and on an atherogenic Western type diet (WTD), consisting of 21% (wt/wt) fat and 0.15% (wt/wt) cholesterol. Plasma triglyceride and cholesterol were the same in diabetic and non-diabetic mice on the chow diet. On the WTD, male diabetic HuBTg mice had a >50% increase in plasma cholesterol and more very low density lipoprotein (VLDL) cholesterol and triglyceride as assessed by FPLC analysis. A Triton study showed no increase in triglyceride or apolipoprotein B production, suggesting that the accumulation of VLDL was due to a decrease in lipoprotein clearance. Surprisingly, the VLDL increase in these mice was not due to a decrease in LpL activity in postheparin plasma. To test whether LpL overexpression would alter these diabetes-induced lipoprotein changes, HuBTg mice were crossed with mice expressing human LpL in muscle. LpL overexpression reduced plasma triglyceride, but not cholesterol, in male mice on WTD. Aortic root atherosclerosis assessed in 32-week-old mice on the WTD was not greater in diabetic mice. In summary, diabetes primarily increased plasma VLDL in HuBTg mice. LpL activity was not decreased in these animals. However, additional LpL expression eliminated the diabetic lipoprotein changes. These mice did not have more atherosclerosis with diabetes.     

Modulation of blood oxylipin levels by long-chain omega-3 fatty acid supplementation in hyper- and normolipidemic men

IntroductionLong chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) such as EPA and DHA have been shown to possess beneficial health effects, and it is believed that many of their effects are mediated by their oxygenated products (oxylipins). Recently, we have shown that serum levels of several hydroxy, epoxy, and dihydroxy FAs are dependent on the individual status of the parent FAs in a cohort of normo- and hyperlipidemic subjects. So far, the effect of an increased dietary LC n-3 PUFA intake on hydroxy, epoxy, and dihydroxy FA levels has not been investigated in subjects with mild combined hyperlipidemia.Subjects and methodsIn the present study, we compared oxylipin patterns of 10 hyperlipidemic (cholesterol >200 mg/dl; triglyceride >150 mg/ml) and 10 normolipidemic men in response to twelve weeks of LC n-3 PUFA intake (1.14 g DHA and 1.56 g EPA). Levels of 44 free hydroxy, epoxy and dihydroxy FAs were analyzed in serum by LC-MS. Additionally, oxylipin levels were compared with their parent PUFA levels in erythrocyte membranes; a biomarker for the individual PUFA status.ResultsDifferences in the oxylipin pattern between normo- and hyperlipidemic subjects were minor before and after treatment. In all subjects, levels of EPA-derived oxylipins (170–4800 pM) were considerably elevated after LC n-3 PUFA intake (150–1400%), the increase of DHA-derived oxylipins (360–3900 pM) was less pronounced (30–130%). The relative change of EPA in erythrocyte membranes is strongly correlated (r≥0.5; p<0.05) with the relative change of corresponding epoxy and dihydroxy FA serum levels. The effect on arachidonic acid (AA)-derived oxylipin levels (140–27,100 pM) was inconsistent.Discussion and conclusionsThe dietary LC PUFA composition has a direct influence on the endogenous oxylipin profile, including several highly biological active EPA- and DHA-derived lipid mediators. The shift in oxylipin pattern appears to be dependent on the initial LC PUFA status particularly for EPA. The finding that also levels of other oxylipins derived from ALA, LA or AA are modified by LC n-3 PUFA intake might suggest that at least some of the effects of EPA and DHA could be mediated by a shift in the entire oxylipin profile.     

Very low density lipoprotein (VLDL) receptor-deficient mice have reduced lipoprotein lipase activity. Possible causes of hypertriglyceridemia and reduced body mass with VLDL receptor deficiency.

Although very low density lipoprotein (VLDL) receptor (VLDLr) knockout mice have been reported to have no lipoprotein abnormalities, they develop less adipose tissue than control mice when fed a high calorie diet. Mice that are deficient in adipose tissue expression of lipoprotein lipase (LpL) also have less fat, but only when crossed with ob/ob mice. We hypothesized that the VLDLr, a protein that will bind and transport LpL, is required for optimal LpL actions in vivo and that hypertriglyceridemia due to VLDLr deficiency is exacerbated by either LpL deficiency or VLDL overproduction. Fasted VLDLr knockout (VLDLr0) mice were more hypertriglyceridemic than controls (2-fold greater triglyceride levels). The hypertriglyceridemia due to VLDLr0 was even more evident when VLDLr0 mice were crossed with heterozygous LpL-deficient (LpL1) and human apolipoprotein B (apoB) transgenic mice. This was due to an increase in apoB48-containing VLDL. [(3)H]VLDL turnover studies showed that VLDL-triglyceride clearance in VLDLr0/LpL1 mice was impaired by 50% compared with LpL1 mice. VLDLr0/LpL1 mice had less LpL activity in postheparin plasma, heart, and skeletal muscle. Infection of mice with an adenovirus-expressing receptor-associated protein, an inhibitor of the VLDLr, reduced LpL activity in wild type but not VLDLr0 mice. Therefore, the VLDLr is required for normal LpL regulation in vivo, and the disruption of VLDLr results in hypertriglyceridemia associated with decreased LpL activity.     

Macrophage lipoprotein lipase modulates the development of atherosclerosis but not adiposity

The role of macrophage lipoprotein lipase (LpL) in the development of atherosclerosis and adiposity was examined in macrophage LpL knockout (MLpLKO) mice. MLpLKO mice were generated using cre-loxP gene targeting. Loss of LpL in macrophages did not alter plasma LpL activity or lipoprotein levels. Incubation of apolipoprotein E (ApoE)-deficient β-VLDL with peritoneal macrophages from ApoE knockout mice lacking macrophage LpL (MLpLKO/ApoEKO) led to less cholesteryl ester formation than that found with ApoEKO macrophages. MLpLKO/ApoEKO macrophages had reduced intracellular triglyceride levels, with decreased CD36 and carnitine palmitoyltransferase-1 mRNA levels compared with ApoEKO macrophages, when incubated with VLDL. Although both MLpLKO/ApoEKO and ApoEKO mice developed comparable hypercholesterolemia in response to feeding with a Western-type diet for 12 weeks, atherosclerosis was less in MLpLKO/ApoEKO mice. Epididymal fat mass and gene expression levels associated with inflammation did not differ between the two groups. In conclusion, macrophage LpL plays an important role in the development of atherosclerosis but not adiposity.     

Serum oxylipin profiles in IgA nephropathy patients reflect kidney functional alterations

Immunoglobulin A nephropathy (IgAN) is a leading cause of chronic kidney disease, frequently associated with hypertension and renal inflammation. ??-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in fish oil (FO) improve kidney function in animal models, but have inconsistent metabolic effects in humans. Oxylipin profiles in serum from IgAN patients supplemented with either FO or corn oil (CO) placebo were analyzed by liquid chromatography coupled to tandem mass spectrometry. EPA cyclooxygenase and lipoxygenase metabolites, and EPA and DHA epoxides and diols were increased in response to FO supplementation, as were total epoxides and epoxide/diol ratios. Several of these metabolites were drivers of separation as assessed by multivariate analysis of FO patients pre- versus post-supplementation, including 17,18-dihydroxyeicosatrienoic acid, prostaglandin D₃, prostagalandin E₃, Resolvin E1, 12-hydroxyeicosapentaenoic acid, and 10(11)-epoxydocosapentaenoic acid. In patients whose proteinuria improved, plasma total oxylipins as well as several hydroxyoctadecadienoic acids, hydroxyeicosatetraenoic acids, and leukotriene B₄ metabolites were among the metabolites that were significantly lower than in patients whose proteinuria either did not improve or worsened. These data support the involvement of oxylipins in the inflammatory component of IgAN as well as the potential use of oxylipin profiles as biomarkers and for assessing responsiveness to ??-3 fatty acid supplementation in IgAN patients.     

Heparin binding triggers human VLDL remodeling by circulating lipoprotein lipase: Relevance to VLDL functionality in health and disease

Hydrolysis of VLDL triacylglycerol (TG) by lipoprotein lipase (LpL) is a major step in energy metabolism and VLDL-to-LDL maturation. Most functional LpL is anchored to the vascular endothelium, yet a small amount circulates on TG-rich lipoproteins. As circulating LpL has low catalytic activity, its role in VLDL remodeling is unclear. We use pre-heparin plasma and heparin-sepharose affinity chromatography to isolate VLDL fractions from normolipidemic, hypertriglyceridemic, or type-2 diabetic subjects. LpL is detected only in the heparin-bound fraction. Transient binding to heparin activates this VLDL-associated LpL, which hydrolyses TG, leading to gradual VLDL remodeling into IDL/LDL and HDL-size particles. The products and the timeframe of this remodeling closely resemble VLDL-to-LDL maturation in vivo. Importantly, the VLDL fraction that does not bind heparin is not remodeled. This relatively inert LpL-free VLDL is rich in TG and apoC-III, poor in apoE and apoC-II, shows impaired functionality as a substrate for the exogenous LpL or CETP, and likely has prolonged residence time in blood, which is expected to promote atherogenesis. This non-bound VLDL fraction increases in hypertriglyceridemia and in type-2 diabetes but decreases upon diabetes treatment that restores the glycemic control. In stark contrast, heparin binding by LDL increases in type-2 diabetes triggering pro-atherogenic LDL modifications. Therefore, the effects of heparin binding are associated negatively with atherogenesis for VLDL but positively for LDL. Collectively, the results reveal that binding to glycosaminoglycans initiates VLDL remodeling by circulating LpL, and suggest heparin binding as a marker of VLDL functionality and a readout for treatment of metabolic disorders.     

Compensatory mechanism for homeostatic blood pressure regulation in Ephx2 gene-disrupted mice

Arachidonic acid-derived epoxides, epoxyeicosatrienoic acids, are important regulators of vascular homeostasis and inflammation, and therefore manipulation of their levels is a potentially useful pharmacological strategy. Soluble epoxide hydrolase converts epoxyeicosatrienoic acids to their corresponding diols, dihydroxyeicosatrienoic acids, modifying or eliminating the function of these oxylipins. To better understand the phenotypic impact of Ephx2 disruption, two independently derived colonies of soluble epoxide hydrolase-null mice were compared. We examined this genotype evaluating protein expression, biofluid oxylipin profile, tissue oxylipin production capacity, and blood pressure. Ephx2 gene disruption eliminated soluble epoxide hydrolase protein expression and activity in liver, kidney, and heart from each colony. Plasma levels of epoxy fatty acids were increased, and fatty acid diols levels were decreased, while measured levels of lipoxygenase- and cyclooxygenase-dependent oxylipins were unchanged. Liver and kidney homogenates also show elevated epoxide fatty acids. However, in whole kidney homogenate a 4-fold increase in the formation of 20-hydroxyeicosatetraenoic acid was measured along with a 3-fold increase in lipoxygenase-derived hydroxylation and prostanoid production. Unlike previous reports, however, neither Ephx2-null colony showed alterations in basal blood pressure. Finally, the soluble epoxide hydrolase-null mice show a survival advantage following acute systemic inflammation. The data suggest that blood pressure homeostasis may be achieved by increasing production of the vasoconstrictor, 20-hydroxyeicosatetraenoic acid in the kidney of the Ephx2-null mice. This shift in renal metabolism is likely a metabolic compensation for the loss of the soluble epoxide hydrolase gene.     

Reciprocal oxylipin-mediated cross-talk in the Aspergillus–seed pathosystem

In Aspergilli, mycotoxin production and sporulation are governed, in part, by endogenous oxylipins (oxygenated, polyunsaturated fatty acids and metabolites derived therefrom). In Aspergillus nidulans , oxylipins are synthesized by the dioxygenase enzymes PpoA, PpoB and PpoC. Structurally similar oxylipins are synthesized in seeds via the action of lipoxygenase (LOX) enzymes. Previous reports have shown that exogenous application of seed oxylipins to Aspergillus cultures alters sporulation and mycotoxin production. Herein, we explored whether a plant oxylipin biosynthetic gene ( ZmLOX3 ) could substitute functionally for A. nidulans ppo genes. We engineered ZmLOX3 into wild-type A. nidulans , and into a Δ ppoAC strain that was reduced in production of oxylipins, conidia and the mycotoxin sterigmatocystin. ZmLOX3 expression increased production of conidia and sterigmatocystin in both backgrounds. We additionally explored whether A. nidulans oxylipins affect seed LOX gene expression during Aspergillus colonization. We observed that peanut seed pnlox2–3 expression was decreased when infected by A. nidulans Δ ppo mutants compared with infection by wild type. This result provides genetic evidence that fungal oxylipins are involved in plant LOX gene expression changes, leading to possible alterations in the fungal/host interaction. This report provides the first genetic evidence for reciprocal oxylipin cross-talk in the Aspergillus –seed pathosystem.     

Lipolysis-induced degradation of apolipoproteins B and E of human very low density lipoproteins

We have found that in vitro lipolysis of human very low density lipoproteins (VLDL) by purified bovine milk lipoprotein lipase (LpL) promotes degradation of the apolipoprotein (apo) B moiety of VLDL. Analysis by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis showed that lipolysis of VLDL by purified LpL for 1 h at 37 degrees C induced the selective degradation of the high Mr apo-B (apo-B-100) from most hypertriglyceridemic VLDL and from a few normolipidemic VLDL into several small fragments with molecular weights ranging from 90,000-490,000. No detectable degradation of apo-B occurred in control VLDL when incubated without LpL. The apo-E moiety of VLDL from certain individuals was also degraded following lipolysis of VLDL, and the extent of degradation of apo-B and -E in VLDL was varied among the individual VLDL. The major degradation products of apo-E, identified from the gel, were 31,000- and/or 28,000-Da species. In contrast to the apo-E moiety of VLDL, purified apo-E was not degraded when incubated with LpL. Incubation of low density lipoproteins (LDL) with LpL showed only a minimal effect on the apoproteins of LDL. When high density lipoprotein (HDL) was included in the lipolysis mixture as an acceptor of lipolytic surface remnants, the apoproteins of HDL remained unaltered, while the apo-B moiety of VLDL remnants in the mixture was degraded. Inclusion of protease inhibitors in the lipolysis mixture prevented the degradation of apo-B, but the hydrolysis of VLDL-triglyceride was minimally affected. A selective degradation of apo-B in VLDL also occurred during lipolysis of VLDL when VLDL was perfused through rat hearts. These results suggest that conformational changes in apo-B and apo-E caused by VLDL lipolysis may increase the susceptibility of apo-B and apo-E to degradation by the proteases co-isolated with VLDL. The consequences of the lipolysis-induced degradation of apo-B and apo-E on changes in metabolic properties of VLDL remnants remain to be determined.     

Adipocyte lipid storage and adipokine production are modulated by lipoxygenase-derived oxylipins generated from 18-carbon fatty acids

Generation of oxylipins (oxygenated metabolites of fatty acids) by lipoxygenases may be responsible for the beneficial effects of 20- and 22-carbon n-3 fatty acids on adipose tissue dysfunction in obesity, but the potential actions of oxylipins derived from 18-carbon fatty acids, which are generally at higher levels in the diet, are unknown. We therefore compared the effects of select lipoxygenase-derived oxylipins produced from α-linolenic acid (ALA, C18:3 n-3), linoleic acid (LA, C18:2 n-6), and arachidonic acid (AA, C20:4 n-6) on key adipocyte functions that are altered in obesity. Individual oxylipins were added to the culture medium of differentiating 3T3-L1 preadipocytes for 6 days. Lipid accumulation was subsequently determined by Oil Red O staining, while Western blotting was used to measure levels of proteins associated with lipid metabolism and characteristics of adipocyte functionality. Addition of all oxylipins at 30 nM was sufficient to significantly decrease triglyceride accumulation in lipid droplets, and higher levels completely blocked lipid production. Our results establish that lipoxygenase-derived oxylipins produced from 18-carbon PUFA differentially affect multiple adipocyte processes associated with lipid storage and adipokine production. However, these effects are not due to the oxylipins blocking adipocyte maturation and thus globally suppressing all adipocyte characteristics. Furthermore, these oxylipin species decrease the lipid content of adipocytes regardless from which precursor fatty acid or lipoxygenase they were derived. Consequently, adipocyte characteristics can be altered through the ability of oxylipins to selectively modulate levels of proteins involved in both lipid metabolism and adipokine production.     

Delivery and turnover of plasma-derived essential PUFAs in mammalian brain

Polyunsaturated fatty acids (PUFAs) are critical to nervous system function and structure, but their rates of incorporation from plasma into brain have not been evaluated. In the adult rat, calculations based on our model show that at least 3;-5% of esterified brain arachidonic acid (AA) and 2;-8% of esterified brain docosahexaenoic acid (DHA) are replaced daily by unesterified PUFAs in plasma. These rates, when related to unlabeled brain PUFA composition, give half-lives of 1-2 weeks for plasma-brain exchange of AA and DHA. In the human brain, the arachidonate replacement rate is 0.3% per day. Although unesterified plasma PUFA concentrations are low, their rates of incorporation into brain are sufficient to compensate for metabolic and efflux losses, so that PUFA transport from plasma into brain as a component of a lipoprotein is unnecessary. Dietary supplementation, by altering plasma unesterified PUFA concentrations, can regulate brain PUFA content and may help to treat brain diseases involving PUFA imbalance.     

Oxylipin profiling of the hypersensitive response in Arabidopsis thaliana. Formation of a novel oxo-phytodienoic acid-containing galactolipid, arabidopside E

Oxidation products of unsaturated fatty acids, collectively known as oxylipins, function as signaling molecules in plants during development, wounding, and insect and pathogen attack. Certain oxylipins are also known to have direct cytotoxic effects on pathogens. We used inducible expression of bacterial avirulence proteins in planta to study the involvement of oxylipins in race-specific defense against bacterial pathogens. We demonstrate that recognition of the Pseudomonas syringae avirulence protein AvrRpm1 induces 9- and 13-lipoxygenase-dependent oxylipin synthesis in Arabidopsis thaliana. The major oxylipins accumulated were jasmonic acid, 12-oxo-phytodienoic acid, and dinor-oxo-phytodienoic acid. The majority of the newly formed oxylipins (>90%) was found to be esterified to glycerolipids, whereby 12-oxo-phytodienoic acid and dinor-oxo-phytodienoic acid were found to be esterified to a novel galactolipid. The structure of the substance was determined as a monogalactosyldiacylglycerol containing two 12-oxo-phytodienoic acids and one dinor-oxo-phytodienoic acid acyl chain and was given the trivial name arabidopside E. This substance accumulated to surprisingly high levels, 7-8% of total lipid content, and was shown to inhibit growth of a bacterial pathogen in vitro. Arabidopside E was formed also after recognition of the avirulence protein AvrRpt2, suggesting that this could be a conserved feature of defense reactions against bacterial pathogens. In conclusion, the data presented suggest a role of enzymatically formed oxylipins, especially the octadecanoids and arabidopside E in race-specific resistance against bacterial pathogens.     

Anti-inflammatory effects of α-linolenic acid in M1-like macrophages are associated with enhanced production of oxylipins from α-linolenic and linoleic acid

Chronic inflammation, mediated in large part by proinflammatory macrophage populations, contributes directly to the induction and perpetuation of metabolic diseases, including obesity, insulin resistance and type 2 diabetes. Polyunsaturated fatty acids (PUFAs) can have profound effects on inflammation through the formation of bioactive oxygenated metabolites called oxylipins. The objective of this study was to determine if exposure to the dietary omega-3 PUFA α-linolenic acid (ALA) can dampen the inflammatory properties of classically activated (M1-like) macrophages derived from the human THP-1 cell line and to examine the accompanying alterations in oxylipin secretion. We find that ALA treatment leads to a reduction in lipopolysaccharide (LPS)-induced interleukin (IL)-1β, IL-6 and tumor necrosis factor-α production. Although ALA is known to be converted to longer-chain PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), DHA oxylipins were reduced overall by ALA treatment, as was LPS-induced secretion of EPA oxylipins. In contrast, we observed profound increases in oxylipins directly derived from ALA. Lipoxygenase products of linoleic acid were also dramatically increased, and LPS-induced production of AA oxylipins, particularly prostaglandin D2, was reduced. These results suggest that ALA may act to dampen the inflammatory phenotype of M1-like macrophages by a unique set of mechanisms distinct from those used by the long-chain omega-3 fatty acids EPA and DHA. Thus, there is strong rationale for investigating the functions of ALA oxylipins and lesser-known LA oxylipins since they hold promise as anti-inflammatory agents.     

Reciprocal oxylipin-mediated cross-talk in the Aspergillus-seed pathosystem.

In Aspergilli, mycotoxin production and sporulation are governed, in part, by endogenous oxylipins (oxygenated, polyunsaturated fatty acids and metabolites derived therefrom). In Aspergillus nidulans, oxylipins are synthesized by the dioxygenase enzymes PpoA, PpoB and PpoC. Structurally similar oxylipins are synthesized in seeds via the action of lipoxygenase (LOX) enzymes. Previous reports have shown that exogenous application of seed oxylipins to Aspergillus cultures alters sporulation and mycotoxin production. Herein, we explored whether a plant oxylipin biosynthetic gene (ZmLOX3) could substitute functionally for A. nidulans ppo genes. We engineered ZmLOX3 into wild-type A. nidulans, and into a DeltappoAC strain that was reduced in production of oxylipins, conidia and the mycotoxin sterigmatocystin. ZmLOX3 expression increased production of conidia and sterigmatocystin in both backgrounds. We additionally explored whether A. nidulans oxylipins affect seed LOX gene expression during Aspergillus colonization. We observed that peanut seed pnlox2-3 expression was decreased when infected by A. nidulansDeltappo mutants compared with infection by wild type. This result provides genetic evidence that fungal oxylipins are involved in plant LOX gene expression changes, leading to possible alterations in the fungal/host interaction. This report provides the first genetic evidence for reciprocal oxylipin cross-talk in the Aspergillus-seed pathosystem.     

Dietary ALA,EPA and DHA have distinct effects on oxylipin profiles in female and male rat kidney,liver and serum

There is much data on the effects of dietary n-3 fatty acids on tissue fatty acid compositions, but comparable comprehensive data on their oxygenated metabolites (oxylipins) is limited. The effects of providing female and male rats with diets high in α-linolenic acid (ALA), EPA or DHA for 6 weeks on oxylipins and fatty acids in kidney, liver and serum were therefore examined. The oxylipin profile generally reflected fatty acids, but it also revealed unique effects of individual n-3 fatty acids that were not apparent from fatty acid data alone. Dietary ALA increased renal and serum DHA oxylipins even though DHA itself did not increase, while dietary EPA did not increase DHA oxylipins in kidney or liver, suggesting that high EPA may inhibit this conversion. Oxylipin data generally corroborated fatty acid data that indicated that DHA can be retroconverted to EPA and that further retroconversion to ALA is limited. Dietary n-3 fatty acids decreased n-6 fatty acids and their oxylipins (except linoleic acid and its oxylipins), in order of effectiveness of DHA > EPA > ALA, with some exceptions: several arachidonic acid oxylipins modified at carbon 15 were not lower in all three sites, and EPA had a greater effect on 12-hydroxy-eicosatetraenoic acid and its metabolites in the liver. Oxylipins were predominantly higher in males, which was not reflective of fatty acids. Tissue-specific oxylipin profiles, therefore, provide further information on individual dietary n-3 fatty acid and sex effects that may help explain their unique physiological effects and have implications for dietary recommendations.     

Oxylipin analysis methods

Practical guidelines for monitoring and measuring compounds such as jasmonates, ketols, ketodi(tri)enes and hydroxy-fatty acids as well as detecting the presence of novel oxylipins are presented. Additionally, a protocol for the penetrant analysis of non-enzymatic lipid oxidation is described. Each of the methods, which employ gas chromatography/mass spectrometry, can be applied without specialist knowledge or recourse to the latest analytical instrumentation. Additional information on oxylipin quantification and novel protocols for preparing oxygen isotope-labelled internal standards are provided. Four developing areas of research are identified: (i) profiling of the unbound cellular pools of oxylipins; (ii) profiling of esterified oxylipins and/or monitoring of their release from parent lipids; (iii) monitoring of non-enzymatic lipid oxidation; (iv) analysis of unstable and reactive oxylipins. The methods and protocols presented herein are designed to give technical insights into the first three areas and to provide a platform from which to enter the fourth area.