Knock-In Mice Expressing a 15-Lipoxygenating Alox5 Mutant Respond Differently to Experimental Inflammation Than Reported Alox5-/- Mice.

Arachidonic acid 5-lipoxygenase (ALOX5) is the key enzyme in the biosynthesis of pro-inflammatory leukotrienes. We recently created knock-in mice (Alox5-KI) which express an arachidonic acid 15-lipoxygenating Alox5 mutant instead of the 5-lipoxygenating wildtype enzyme. These mice were leukotriene deficient but exhibited an elevated linoleic acid oxygenase activity. Here we characterized the polyenoic fatty acid metabolism of these mice in more detail and tested the animals in three different experimental inflammation models. In experimental autoimmune encephalomyelitis (EAE), Alox5-KI mice displayed an earlier disease onset and a significantly higher cumulative incidence rate than wildtype controls but the clinical score kinetics were not significantly different. In dextran sodium sulfate-induced colitis (DSS) and in the chronic constriction nerve injury model (CCI), Alox5-KI mice performed like wildtype controls with similar genetic background. These results were somewhat surprising since in previous loss-of-function studies targeting leukotriene biosynthesis (Alox5-/- mice, inhibitor studies), more severe inflammatory symptoms were observed in the EAE model but the degree of inflammation in DSS colitis was attenuated. Taken together, our data indicate that these mutant Alox5-KI mice respond differently in two models of experimental inflammation than Alox5-/- animals tested previously in similar experimental setups.

Oxylipin patterns in human colon adenomas.

OBJECTIVE: Cyclooxygenase (COX)-derived prostaglandin E2 (PGE2) is an important lipid mediator in colorectal carcinoma (CRC) pathogenesis. Other lipid mediators derived from lipoxygenases (LOX) have also been implicated in neoplastic processes in the colon. In this study we aimed to characterize lipid mediators, so called oxylipins, in human colon adenomatous polyps. DESIGN: We quantified oxylipins in healthy colon tissue and colorectal adenoma tissue procured during routine colonoscopy examinations. Lipid metabolite profiles were analyzed by liquid chromatography-tandem mass spectrometry. RESULTS: Adenoma tissue showed a distinct prostaglandin profile as compared to normal colon mucosa. Interestingly, PGE2 was not higher in adenoma tissue as compared to normal mucosa. In contrast, we found significantly lower levels of prostaglandin D2, prostaglandin J2, and prostaglandin D1 in adenoma tissue. Furthermore, levels of 5-LOX and 12-LOX pathway products were clearly increased in adenoma biopsy samples. We also investigated the effect of aspirin treatment on prostaglandin profiles in adenoma tissue in a subset of patients and found a trend towards decreased prostaglandin levels in response to aspirin. CONCLUSION: The human data presented here show specific changes of oxylipin profiles in colon adenoma tissue with decreased prostaglandin D2 levels as well as increased 5- and 12-LOX metabolites.

Effect of dietary EPA and DHA on murine blood and liver fatty acid profile and liver oxylipin pattern depending on high and low dietary n6-PUFA.

The intake of long-chain n3-polyunsaturated fatty acids (PUFA), which are associated with beneficial health effects, is low in the Western diet, while the portion of dietary n6-PUFA and hence the n6/n3-PUFA ratio is high. Strategies to improve the n3-PUFA status are n3-PUFA supplementation and/or lowering n6-PUFA intake. In the present study, mice were fed with two different sunflower oil-based control diets rich in linoleic (n6-high) or oleic acid (n6-low), either with low n3-PUFA content (∼0.02%) as control or with ∼0.6% eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). The n6-low diet had only little or no effect on levels of arachidonic acid (ARA) and its free oxylipins in liver tissue. Supplementation with EPA or DHA lowered ARA levels with an effect size of n6-high < n6-low. Blood cell %EPA + DHA reached >8% and >11% in n6-high and n6-low groups, respectively. Elevation of EPA levels and EPA derived oxylipins was most pronounced in n6-low groups in liver tissue, while levels of DHA and DHA derived oxylipins were generally unaffected by the background diet. While the n6-low diet alone had no effect on blood and liver tissue ARA levels or n3-PUFA status, a supplementation of EPA or DHA was more effective in combination with an n6-low diet. Thus, supplementation of long-chain n3-PUFA combined with a reduction of dietary n6-PUFA is the most effective way to improve the endogenous n3-PUFA status.

Human lipoxygenase isoforms form complex patterns of double and triple oxygenated compounds from eicosapentaenoic acid.

Lipoxygenases (ALOX) are lipid peroxidizing enzymes that catalyze the biosynthesis of pro- and anti-inflammatory lipid mediators and have been implicated in (patho-)physiological processes. In humans, six functional ALOX isoforms exist and their arachidonic acid oxygenation products have been characterized. Products include leukotrienes and lipoxins which are involved in the regulation of inflammation and resolution. Oxygenation of n3-polyunsaturated fatty acids gives rise to specialized pro-resolving mediators, e.g. resolvins. However, the catalytic activity of different ALOX isoforms can lead to a multitude of potentially bioactive products. Here, we characterized the patterns of oxygenation products formed by human recombinant ALOX5, ALOX15, ALOX15B and ALOX12 from eicosapentaenoic acid (EPA) and its 18-hydroxy derivative 18-HEPE with particular emphasis on double and triple oxygenation products. ALOX15 and ALOX5 formed a complex mixture of various double oxygenation products from EPA, which include 5,15-diHEPE and various 8,15-diHEPE isomers. Their biosynthetic mechanisms were explored using heavy oxygen isotopes (H218O, 18O2 gas) and three catalytic activities contributed to product formation: i) fatty acid oxygenase activity, ii) leukotriene synthase activity, iii) lipohydroperoxidase activity. For ALOX15B and ALOX12 more specific product patterns were identified, which was also the case when these enzymes reacted in concert with ALOX5. Several double oxygenated compounds were formed from 18-HEPE by ALOX5, ALOX15B and ALOX12 including previously identified resolvins (RvE2, RvE3), while formation of triple oxygenation products, e.g. 5,17,18-triHEPE, required ALOX5. Taken together our data show that EPA can be converted by human ALOX isoforms to a large number of secondary oxygenation products, which might exhibit bioactivity.

Stability of oxylipins during plasma generation and long-term storage.

Oxidized unsaturated fatty acids - i.e. eicosanoids and other oxylipins - are lipid mediators involved in the regulation of numerous physiological functions such as inflammation, blood coagulation, vascular tone and endothelial permeability. They have raised strong interest in clinical lipidomics in order to understand their role in health and diseases and their use as biomarkers. However, before the clinical translation, it is crucial to validate the analytical reliability of oxylipins. This notably requires to assess the putative artificial formation or degradation of oxylipins by (unsuitable) blood handling during plasma generation, storage and sample preparation. Using a liquid chromatography-mass spectrometry method covering 133 oxylipins we comprehensively analyzed the total (free + esterified) oxylipin profile in plasma and investigated the influence of i) addition of additives during sample preparation, ii) different storage times and temperatures during the transitory stage of plasma generation and iii) long-term storage of plasma samples at -80 °C. Addition of radical scavenger butylated hydroxytoluene reduced the apparent concentrations of hydroxy-PUFA and thus should be added to the samples at the beginning of sample preparation. The concentrations of all oxylipin classes remained stable (within analytical variance of 20%) during the transitory stage of plasma generation up to 24 h at 4 °C or 4 h at 20 °C before centrifugation of EDTA-whole blood and up to 5 days at -20 °C after plasma separation. The variations in oxylipin concentrations did not correlate with storage time, storage temperature or stage of plasma generation. A significant increase of potentially lipoxygenase derived hydroxy-PUFA compared to immediate processing was only detected when samples were stored for longer times before centrifugation, plasma separation as well as freezing of plasma revealing residual enzymatic activity. Autoxidative rather than enzymatic processes led to a slightly increased concentration of 9-HETE when plasma samples were stored at -80 °C for 15 months. Overall, we demonstrate that total plasma oxylipins are robust regarding delays during plasma generation and long-term storage at -80 °C supporting the application of oxylipin profiling in clinical research.

Long-term stimulation of toll-like receptor-2 and -4 upregulates 5-LO and 15-LO-2 expression thereby inducing a lipid mediator shift in human monocyte-derived macrophages.

Macrophage polarization switches during the course of inflammation along with the lipid mediators released. We investigated the lipid mediator formation in human monocyte-derived macrophages during in vitro differentiation and pathogen stimulation. For this, peripheral blood monocytes were differentiated into M1 (CSF-2/IFNγ) or M2 (CSF-1/IL-4) macrophages followed by stimulation with the toll-like receptor (TLR) ligands zymosan (TLR-2), Poly(I:C) (TLR-3) or bacterial lipopolysaccharides (TLR-4) mimicking fungal, viral and bacterial infection, respectively. Expression of enzymes involved in lipid mediator formation such as 5- and 15-lipoxygenases (LO), the 5-LO activating protein and cyclooxygenase-2 (COX-2) was monitored on mRNA and protein level and lipid mediator formation was assessed. In addition, cytokine release was measured. In vitro differentiation of human peripheral blood monocytes to M1 and M2 macrophages considerably attenuated 5-LO activity. Furthermore, while TLR-2 and -4 stimulation of M1 macrophages primarily triggered pro-inflammatory cytokines and lipid mediators, persistent stimulation (16 h) of human M2 macrophages induced a coordinated upregulation of 5- and 15-LO-2 expression. This was accompanied by a marked increase in IL-10 and monohydroxylated 15-LO products in the conditioned media of the cells. After additional stimulation with Ca2+ ionophore combined with supplementation of arachidonic, eicosapentaenoic and docosahexaenoic acid these cells also released small amounts of SPM such as lipoxins and resolvins. From this we conclude that activation of TLR-2 or -4 triggers the biosynthesis of pro-inflammatory 5-LO and COX-2 derived lipid mediators in human monocyte-derived M1 macrophages while persistent stimulation of M2 macrophages induces a shift towards pro-resolving 15-LO derived oxylipins.

Functional Characterization of Knock-In Mice Expressing a 12/15-Lipoxygenating Alox5 Mutant Instead of the 5-Lipoxygenating Wild-Type Enzyme.

Aims: Most mammalian genomes involve several genes encoding for functionally distinct arachidonate lipoxygenase (ALOX isoforms). Proinflammatory leukotrienes are formed via the ALOX5 pathway, but 12/15-lipoxygenating ALOX isoforms have been implicated in the biosynthesis of pro-resolving mediators. In vitro mutagenesis of the triad determinants abolished the leukotriene synthesizing activity of ALOX5, but the biological consequences of these alterations have not been studied. To fill this gap, we created Alox5 knock-in mice, which express the 12/15-lipoxygenating Phe359Trp + Ala424Ile + Asn425Met Alox5 triple mutant and characterized its phenotypic alterations. Results: The mouse Alox5 triple mutant functions as arachidonic acid 15-lipoxygenating enzyme, which also forms 12S-hydroxy and 8S-hydroxy arachidonic acid. In contrast to the wild-type enzyme, the triple mutant effectively oxygenates linoleic acid to 13S-hydroxy linoleic acid (13S-HODE), which functions as activating ligand of the type-2 nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ). Knock-in mice expressing the mutant enzyme are viable, fertile, and develop normally. The mice cannot synthesize proinflammatory leukotrienes but show significantly attenuated plasma levels of lipolytic endocannabinoids. When aging, the animals gained significantly more body weight, which may be related to the fivefold higher levels of 13-HODE in the adipose tissue. Innovation: These data indicate for the first time that in vivo mutagenesis of the triad determinants of mouse Alox5 abolished the biosynthetic capacity of the enzyme for proinflammatory leukotrienes and altered the catalytic properties of the protein favoring the formation of 13-HODE. Conclusion:In vivo triple mutation of the mouse Alox5 gene impacts the body weight homeostasis of aging mice via augmented formation of the activating PPARγ ligand 13-HODE.

Targeting esterified oxylipins by LC-MS - Effect of sample preparation on oxylipin pattern.

A major part of oxygenated metabolites of polyunsaturated fatty acids - i.e. eicosanoids and other oxylipins - in biological samples is found in the esterified form. Yet, their biological role is only poorly understood. For quantification of esterified oxylipins in biological samples current protocols mostly apply alkaline hydrolysis with or without prior lipid extraction to release oxylipins into their free form which can be subsequently quantified via liquid chromatography-mass spectrometry. Herein, a detailed protocol for precise and reproducible quantification of esterified oxylipins in plasma is presented comprising i) extraction of lipids and removal of proteins with iso-propanol, ii) base hydrolysis with potassium hydroxide to saponify lipids and iii) solid phase extraction of the liberated oxylipins on C8/anion exchange mixed mode material. Unequal extraction of internal standards and lipid classes during lipid extraction before hydrolysis led to distorted concentrations, emphasizing that the choice of solvent used in this step is important to minimize discrimination. Regarding the hydrolysis conditions, at least 30 min incubation at 60 °C is required with 0.1 M KOH in sample. Drying of the SPE cartridges is a critical parameter since autoxidation processes of PUFA, which are present in high concentrations after cleavage, lead to artificial formation of epoxy fatty acids. With the developed protocol, inter-day, intra-day and inter-operator variance was <21% for most oxylipins including hydroxy-, dihydroxy-, and epoxy-PUFA. The applicability of the developed methodology is demonstrated by investigating the changes in the oxylipin pattern following omega-3 fatty acid feeding to rats.

Single-Dose SDA-Rich Echium Oil Increases Plasma EPA, DPAn3, and DHA Concentrations.

The omega-3 (n3) polyunsaturated fatty acids (PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are associated with health benefits. The primary dietary source of EPA and DHA is seafood. Alpha-linoleic acid (ALA) has not been shown to be a good source for EPA and DHA; however, stearidonic acid (SDA)-which is naturally contained in echium oil (EO)-may be a more promising alternative. This study was aimed at investigating the short-term n3 PUFA metabolism after the ingestion of a single dose of EO. Healthy young male subjects (n = 12) ingested a single dose of 26 g of EO after overnight fasting. Plasma fatty acid concentrations and relative amounts were determined at baseline and 2, 4, 6, 8, 24, 48, and 72 h after the ingestion of EO. During the whole examination period, the participants received standardized nutrition. Plasma ALA and SDA concentrations increased rapidly after the single dose of EO. Additionally, EPA and DPAn3 concentrations both increased significantly by 47% after 72 h compared to baseline; DHA concentrations also significantly increased by 21% after 72 h. To conclude, EO increases plasma ALA, SDA, EPA, DPAn3, and DHA concentrations and may be an alternative source for these n3 PUFAs.

Development of an Optimized LC-MS Method for the Detection of Specialized Pro-Resolving Mediators in Biological Samples.

The cardioprotective and anti-inflammatory effects of long chain omega-3 polyunsaturated fatty acids (n3 PUFA) are believed to be partly mediated by their oxygenated metabolites (oxylipins). In the last two decades interest in a novel group of autacoids termed specialized pro-resolving mediators (SPMs) increased. These are actively involved in the resolution of inflammation. SPMs are multiple hydroxylated fatty acids including resolvins, maresins, and protectins derived from the n3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as well as lipoxins derived from arachidonic acid (ARA). In the present paper, we developed an LC-MS/MS method for a comprehensive set of 18 SPMs derived from ARA, EPA, and DHA and integrated it into our targeted metabolomics platform. Quantification was based on external calibration utilizing five deuterated internal standards in combination with a second internal standard for quality assessment of sample preparation in each sample. The tandem mass spectrometric parameters were carefully optimized for sensitive and specific detection. The influence of source parameters of the used AB Sciex 6500 QTRAP instrument as well as electronic parameters and the selection of transitions are discussed. The method was validated/characterized based on the criteria listed in the European Medicines Agency (EMA) guideline on bioanalytical method validation and method performance is demonstrated regarding recovery of internal standards (between 78 ± 4% and 87 ± 3% from 500 µL of human serum) as well as extraction efficacy of SPMs in spiked plasma (intra-day accuracy within ±20 and ±15% at 0.1 and 0.3 nM in plasma, respectively). Based on the lower limit of quantification of 0.02-0.2 nM, corresponding to 0.18-2.7 pg on column, SPMs were generally not detectable/quantifiable in plasma and serum supporting that circulating levels of SPMs are very low, i.e., <0.1 nM in healthy subjects. Following septic shock or peritonitis, SPMs could be quantified in the samples of several patients. However, in these studies with a small number of patients no clear correlation with severity of inflammation could be observed.

Activation of Lipid Mediator Formation Due to Lipoprotein Apheresis.

Lipoprotein apheresis reliably reduces low-density lipoprotein (LDL) cholesterol in patients with atherosclerotic disease and therapy-refractory hypercholesterolemia or elevated lipoprotein (a) (Lp(a)). Besides lowering lipoproteins and triglycerides, apheresis also decreases levels of essential omega-6 and omega-3 polyunsaturated fatty acids (n-6 and n-3 PUFAs) in blood plasma. In contrast, heparin-induced extracorporeal low-density lipoprotein precipitation (HELP) lipid apheresis might increase the formation of potentially pro-inflammatory and pro-thrombotic lipid mediators derived from n-6 and n-3 PUFAs. The study presented here analyzed lipid mediator profiles in the plasma of patients with hyperlipidemia treated by one of three different apheresis methods, either HELP, direct absorption (DA), or membrane filtration (MDF), in a direct pre- and post-apheresis comparison. Using gas chromatography and liquid chromatography tandem mass spectrometry (LC-MS/MS) we were able to analyze fatty acid composition and the formation of lipid mediators called oxylipins. Our data illustrate-particularly in HELP-treated patients-significant decreases of essential omega-6 and omega-3 polyunsaturated fatty acids in blood plasma but significant increases of PUFA-derived lipoxygenase-, as well as cyclooxygenase- and cytochrome P450-derived lipid mediators. Given that n-3 PUFAs in particular are presumed to be cardioprotective and n-3 PUFA-derived lipid mediators might limit inflammatory reactions, these data indicate that n-3 PUFA supplementation in the context of lipid apheresis treatment might have additional benefits through apheresis-triggered protective n-3 PUFA-derived lipid mediators.

Development of an LC-ESI(-)-MS/MS method for the simultaneous quantification of 35 isoprostanes and isofurans derived from the major n3- and n6-PUFAs.

Misregulation of oxidative and antioxidative processes in the organism - oxidative stress - contributes to the pathogenesis of different diseases, e.g. inflammatory or neurodegenerative diseases. Oxidative stress leads to autoxidation of polyunsaturated fatty acids giving rise to prostaglandin-like isoprostanes (IsoP) and isofurans (IsoF). On the one hand they could serve as biomarker of oxidative stress and on the other hand may act as lipid mediators, similarly as the enzymatically formed oxylipins. In the present paper we describe the development of an LC-ESI(-)-MS/MS method allowing the parallel quantification of 27 IsoP and 8 IsoF derived from 6 different PUFA (ALA, ARA, EPA, AdA, n6-DPA, DHA) within 12 min. The chromatographic separation was carried out on an RP-C18 column (2.1 × 150 mm, 1.8 µm) yielding narrow peaks with an average width at half maximum of 3.3-4.2 s. Detection was carried out on a triple quadrupole mass spectrometer operating in selected reaction monitoring mode allowing the selective detection of regioisomers. The limit of detection ranged between 0.1 and 1 nM allowing in combination with solid phase extraction the detection of IsoP and IsoF at subnanomolar concentrations in biological samples. The method was validated for human plasma showing high accuracy and precision. Application of the approach on the investigation of oxidative stress in cultured cells indicated a distinct pattern of IsoP and IsoF in response to reactive oxygen species which warrants further investigation. The described method is not only the most comprehensive approach for the simultaneous quantification of IsoP and IsoF, but it was also integrated in a targeted metabolomics method (Ostermann et al. (2015) Anal Bioanal Chem) allowing the quantification of in total 164 oxylipins formed enzymatically and non-enzymatically within 30.5 min.

Effects of a low and a high dietary LA/ALA ratio on long-chain PUFA concentrations in red blood cells.

There is a debate about the optimal dietary ratio of the parent n6 fatty acid linoleic acid (LA) and n3 fatty acid alpha-linolenic acid (ALA) to promote an efficient conversion of ALA to EPA and DHA, which have implications for human health. The aim of the present study was to compare the effects of a low-LA/high-ALA (loLA/hiALA) diet with a high-LA/low-ALA (hiLA/loALA) diet on fatty acid concentrations in red blood cells (RBCs). Fifteen omnivore healthy men (mean age 26.1 ± 4.5 years) with a low initial EPA/DHA status (sum (∑) EPA + DHA% of total fatty acids in RBC at baseline: 4.03 ± 0.17) received both diets for two weeks with a nine-week wash-out phase in between. Fatty acid intake of the subjects was tightly controlled. Concentrations [µg mL-1] and relative amounts [% of total fatty acids] of fatty acids in RBCs were analyzed at baseline (day 0), day 7 and 14 by means of GC-FID. The dietary LA/ALA ratios were 0.56 ± 0.27 : 1 and 25.6 ± 2.41 : 1 and led to significantly different changes of ALA, LA, EPA and ∑EPA + DHA concentrations in RBCs. In the course of the loLA/hiALA diet ALA and EPA concentrations and relative amounts of ∑EPA + DHA increased, whereas LA concentrations decreased. The DHA concentration was unaffected. The hiLA/loALA diet led to slightly decreased EPA concentrations, while all other fatty acid concentrations remained constant. Compared to our previous study, where we simply increased the ALA intake, our results show that ALA supplementation combined with a reduced LA intake (loLA/hiALA diet) more efficiently enhanced EPA blood concentrations. The absence of changes in the PUFA pattern in consequence of a LA/ALA ratio of 25.6 ± 2.41 : 1 suggests that the high LA/ALA ratio of the Western diet already leads to a saturation and a further increase of the ratio does not affect the PUFA pattern.

Aspirin alone and combined with a statin suppresses eicosanoid formation in human colon tissue.

Eicosanoids, including prostaglandins (PGs) and thromboxanes, are broadly bioactive lipid mediators and increase colon tumorigenesis possibly through chronic inflammatory mechanisms. Epidemiological and experimental data suggest that acetylsalicylic acid (ASA) helps prevent colorectal cancer (CRC), possibly through cyclooxygenase (COX)-mediated suppression of eicosanoid, particularly PGE2, formation. Recent studies suggest that statins prevent CRC and improve survival after diagnosis. We identified patients on ASA and/or statin treatment undergoing routine colonoscopy and measured eicosanoid levels in colonic mucosa with targeted metabolomics technology (LC-MS/MS). ASA-treated individuals (n = 27) had significantly lower tissue eicosanoid levels of most COX-derived metabolites than untreated individuals (n = 31). In contrast, COX-derived lipid metabolites tended to be higher in patients with statin treatment (n = 7) as compared with those not receiving statins (n = 24). This effect was not discernible in subjects treated with ASA and statins (n = 11): Individuals treated with both drugs showed a pronounced suppression of COX-derived eicosanoids in colon tissue, even compared with subjects treated with ASA alone. Our data from a routine clinical setting support the hypothesis that ASA and statins could inhibit CRC development via lipid mediator modification. Further studies should directly investigate the effect of dual ASA and statin treatment on colon tumorigenesis in humans.

Effects of a 12-week high-α-linolenic acid intervention on EPA and DHA concentrations in red blood cells and plasma oxylipin pattern in subjects with a low EPA and DHA status.

The essential omega-3 fatty acid alpha-linolenic acid (ALA, 18:3n3) can be converted into EPA and DHA. The aim of the present study was to determine the effect of a high-ALA diet on EPA and DHA levels in red blood cells (RBCs) and their oxylipins in the plasma of subjects with a low EPA and DHA status. Fatty acid concentrations [µg mL-1] and relative amounts [% of total fatty acids] in the RBCs of 19 healthy men (mean age 26.4 ± 4.6 years) were analyzed by means of GC-FID. Free plasma oxylipin concentrations were determined by LC-MS based targeted metabolomics. Samples were collected and analyzed at baseline (week 0) and after 1 (week 1), 3 (week 3), 6 (week 6), and 12 (week 12) weeks of high dietary ALA intake (14.0 ± 0.45 g day-1). ALA concentrations significantly (p < 0.001) increased from 1.44 ± 0.10 (week 0) to 4.65 ± 0.22 (week 1), 5.47 ± 0.23 (week 3), 6.25 ± 0.24 (week 6), and 5.80 ± 0.28 (week 12) µg mL-1. EPA concentrations increased from 6.13 ± 0.51 (week 0) to 7.33 ± 0.33 (week 1), 8.38 ± 0.42 (p = 0.021, week 3), 10.9 ± 0.67 (p < 0.001, week 6), and 11.0 ± 0.64 (p < 0.001, week 12) µg mL-1. DHA concentrations unexpectedly decreased from 41.0 ± 1.93 (week 0) to 37.0 ± 1.32 (week 1), 36.1 ± 1.37 (week 3), 35.1 ± 1.06 (p = 0.010, week 6), and 30.4 ± 1.09 (p < 0.001, week 12) µg mL-1. Relative ΣEPA + DHA amounts were unchanged during the intervention (week 0: 4.63 ± 0.19, week 1: 4.67 ± 0.16, week 3: 4.61 ± 0.13, week 6: 4.73 ± 0.15, week 12: 4.52 ± 0.11). ALA- and EPA-derived hydroxy- and dihydroxy-PUFA increased similarly to their PUFA precursors, although in the case of ALA-derived oxylipins, the concentrations increased less rapidly and to a lesser extent compared to the concentrations of their precursor FA. LA-derived oxylipins remained unchanged and arachidonic acid and DHA oxylipin concentrations were not significantly changed. Our results confirm that the intake of ALA is not a sufficient source for the increase of EPA + DHA in subjects on a Western diet. Specifically, a high-ALA diet results in increased EPA and declined DHA concentrations. However, the changes effectively balance each other out so that ΣEPA + DHA in RBCs - which is an established marker for health protective effects of omega-3-PUFA - remains constant. The PUFA levels in RBCs reflect the concentration and its changes in plasma hydroxy- and dihydroxy-PUFA concentrations for ALA and EPA.

A diet rich in omega-3 fatty acids enhances expression of soluble epoxide hydrolase in murine brain.

Several studies suggest that intake of omega-3 polyunsaturated fatty acids (n3-PUFA) beneficially influences cognitive function. However, effects on the adult brain are not clear. Little is known about the impact of dietary intervention on the fatty acid profile in adult brain, the modulation in the expression of enzymes involved in fatty acid biosynthesis and metabolism as well as changes in resulting oxylipins. These questions were addressed in the present study in two independent n3-PUFA feeding experiments in mice. Supplementation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA, 1% each in the diet) for 30days to adult NMRI and C57BL/6 mice led to a distinct shift in the brain PUFA pattern. While n3-PUFAs EPA, n3 docosapentaenoic acid and DHA were elevated, many n6-PUFAs were significantly decreased (except, e.g. C20:3 n6 which was increased). This shift in PUFAs was accompanied by immense differences in concentrations of oxidative metabolites derived from enzymatic conversion of PUFAs, esp. arachidonic acid whose products were uniformly decreased, and a modulation in the activity and expression pattern of delta-5 and delta-6 desaturases. In both mouse strains a remarkable increase in the soluble epoxide hydrolase (sEH) activity (decreased epoxy-FA concentrations and epoxy-FA to dihydroxy-FA-ratios) as well as sEH expression was observed. Taking the high biological activity of epoxy-FA, e.g. on blood flow and nociceptive signaling into account, this finding might be of relevance for the effects of n3-PUFAs in neurodegenerative diseases. On any account, our study suggests a new distinct regulation of brain PUFA and oxylipin pattern by supplementation of n3-PUFAs to adult rodents.

Mammalian ALOX15 orthologs exhibit pronounced dual positional specificity with docosahexaenoic acid.

Mammalian lipoxygenases (LOX) have been implicated in cell differentiation and in the pathogenesis of inflammatory, hyperproliferative and neurological diseases. Although the reaction specificity of mammalian LOX with n-6 fatty acids (linoleic acid, arachidonic acid) has been explored in detail little information is currently available on the product patterns formed from n-3 polyenoic fatty acids, which are of particular nutritional importance and serve as substrate for the biosynthesis of pro-resolving inflammatory mediators such as resolvins and maresins. Here we expressed the ALOX15 orthologs of eight different mammalian species as well as human ALOX12 and ALOX15B as recombinant his-tag fusion proteins and characterized their reaction specificity with the most abundantly occurring polyunsaturated fatty acids (PUFAs) including 5,8,11,14,17-eicosapentaenoic acid (EPA) and 4,7,10,13,16,19-docosahexaenoic acid (DHA). We found that the LOX isoforms tested accept these fatty acids as suitable substrates and oxygenate them with variable positional specificity to the corresponding n-6 and n-9 hydroperoxy derivatives. Surprisingly, human ALOX15 as well as the corresponding orthologs of chimpanzee and orangutan, which oxygenates arachidonic acid mainly to 15S-H(p)ETE, exhibit a pronounced dual reaction specificity with DHA forming similar amounts of 14- and 17-H(p)DHA. Moreover, ALOX15 orthologs prefer DHA and EPA over AA when equimolar concentrations of n-3 and n-6 PUFA were supplied simultaneously. Taken together, these data indicate that the reaction specificity of mammalian LOX isoforms is variable and strongly depends on the chemistry of fatty acid substrates. Most mammalian ALOX15 orthologs exhibit dual positional specificity with highly unsaturated n-3 polyunsaturated fatty acids.

Lipid Class Specific Quantitative Analysis of n-3 Polyunsaturated Fatty Acids in Food Supplements.

Supplementation products containing n-3 PUFA from marine sources serve a large market. Although the amount of eicosapentaenoic acid and docosahexaenoic acid in the products is provided by the manufacturer, no or little information is available on their lipid pattern. Therefore, we quantitatively analyzed the fatty acid pattern in the lipid fractions triglycerides, phospholipids, ethyl esters, and free fatty acids in supplementation products by means of solid phase extraction and gas chromatography. Twelve products from the European and U.S. markets containing fish, krill, algal, or plant oil were analyzed. Total n-3 PUFA content ranged from 68 g/100 g fat (fish oil) to 42 g/100 g fat (algal oil) to 17 g/100 g fat (krill oil). On the basis of the n-3 PUFA containing lipid class, the supplements can be separated dominantly in ethyl ester, re-esterified triglyceride, triglyceride, and phospholipid containing products. Algae-based products contained natural triglycerides, krill oils a complex mixture of phospholipids, triglycerides, and free fatty acids, and fish oil products either ethyl esters, re-esterified triglycerides, or triglycerides. Even products of the same class and source showed distinct differences in their lipid pattern. A specification of the lipid composition of n-3 PUFA products would allow distinguishing the different (qualities of) supplements.

Effects of docosahexaenoic acid supplementation on PUFA levels in red blood cells and plasma.

INTRODUCTION: Polyunsaturated fatty acids (PUFA) are metabolized in a complex network of elongation, desaturation and beta oxidation. MATERIAL AND METHODS: The short (1 and 3 wk), and long term (6 and 12 wk) effect of 1076mg/d docosahexaenoic acid (DHA, free of eicosapentaenoic acid (EPA)) on (absolute) PUFA concentrations in plasma and red blood cells (RBC) of 12 healthy men (mean age 25.1±1.5 years) was investigated. RESULTS: RBC DHA concentrations significantly (p<0.001) increased from 28±1.6µg/mL to 38±2.0µg/mL (wk 1), 52±3.3µg/mL (wk 3), 68±2.6µg/mL (wk 6), and 79±3.5µg/mL (wk 12). Arachidonic acid (AA) concentrations declined in response to DHA treatment, while the effect was more pronounced in plasma (wk 0: 183±9.9µg/mL, wk 12: 139±8.0µg/mL, -24%, p<0.001) compared to RBC (wk 0: 130±3.7µg/mL, wk 12: 108±4.0µg/mL, -16%, p=0.001). Furthermore, an increase of EPA concentrations in plasma (wk 0: 15±1.5µg/mL, wk 1:19±1.6µg/mL, wk 3: 27±2.3µg/mL, wk 6: 23±1.2µg/mL, wk 12: 25±1.7µg/mL, p<0.001) and RBC (wk 0: 4.7±0.33µg/mL, wk 1: 6.7±1.3µg/mL, wk 3: 8.0±0.66µg/mL, wk 6: 6.9±0.44µg/mL, wk 12: 6.7±0.45µg/mL, n.s.) was observed suggesting a retroconversion of DHA to EPA. CONCLUSION: Based on PUFA concentrations we showed that DHA supplementation results in increased EPA levels, whereas it is not known if this impacts the formation of EPA-derived lipid mediators. Furthermore, shifts in the entire PUFA pattern after supplementation of EPA or DHA should be taken into account when discussing differential physiological effects of EPA and DHA.