Lipoxygenase-derived oxylipins are enriched in anti-citrullinated protein antibody (ACPA)-positive individuals at risk for developing rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is typically preceded by an extended preclinical period where circulating autoantibodies, particularly anti-citrullinated protein antibodies (ACPA), are detectable in the absence of clinical arthritis. Increased dietary intake of anti-inflammatory omega-3 (ω3) polyunsaturated fatty acids (PUFA) has been shown to be associated with a lower the risk of developing incident RA in large epidemiological studies. It is currently not known how changes in fatty acid (FA) metabolism may impact on the progression towards RA in at-risk individuals. To begin to address this question, we profiled serum FAs and oxylipins in an established cohort of at-risk ACPA-positive first-degree relatives (FDR) of RA patients (N = 31), some of whom developed RA (N = 4), and compared their profile to ACPA-negative FDR from the same population (N = 10). METHODS: Gas chromatography (GC) was used for FA quantitation. Oxylipins were extracted and quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS). RESULTS: Although we did not detect any meaningful differences in overall FA content between ACPA + and ACPA - FDR, the levels of oxylipins derived from FA metabolism demonstrated significant differences between the two groups, with the ACPA + group demonstrating enrichment in circulating arachidonic acid- and eicosapentaenoic acid-derived molecules. Compared with the ACPA - FDR group, the ACPA + FDR, including those who progressed into inflammatory arthritis, displayed higher levels of LOX-derived oxylipins. CONCLUSION: ACPA seropositivity in otherwise unaffected individuals at-risk for developing future RA based on family history (FDR) is associated with alterations in the serum oxylipin profile that suggests dysregulated LOX activity.

Phospholipase A2 enzymes differently impact PUFA release and oxylipin formation ex vivo in rat hearts.

Phospholipase A2 (PLA2) enzymes cleave cell membrane phospholipids and release polyunsaturated fatty acids (PUFA), which can be converted into oxylipins. However, little is known about PLA2 preference for PUFA, and even less is known about how this further impacts oxylipin formation. Therefore, we investigated the role of different PLA2 groups in PUFA release and oxylipin formation in rat hearts. Sprague-Dawley rat heart homogenates were incubated without or with varespladib (VAR), methyl arachidonyl fluorophosphonate (MAFP) or EDTA. Free PUFA and oxylipins were determined by HPLC-MS/MS, and isoform expressions by RT-qPCR. Inhibition of sPLA2 IIA and/or V by VAR reduced the release of ARA and DHA, but only DHA oxylipins were inhibited. MAFP reduced the release of ARA, DHA, ALA, and EPA, and the formation of ARA, LA, DGLA, DHA, ALA, and EPA oxylipins. Interestingly, cyclooxygenase and 12-lipoxygenase oxylipins were not inhibited. mRNA expression levels of sPLA2 and iPLA2 isoforms were highest whereas levels of cPLA2 were low, consistent with activity. In conclusion, sPLA2 enzymes lead to the formation of DHA oxylipins, while iPLA2 is likely responsible for the formation of most other oxylipins in healthy rat hearts. Oxylipin formation cannot be implied from PUFA release, thus, both should be evaluated in PLA2 activity studies.

Oils Rich in α-Linolenic Acid or Docosahexaenoic Acid Have Distinct Effects on Plasma Oxylipin and Adiponectin Concentrations and on Monocyte Bioenergetics in Women with Obesity.

BACKGROUND: Omega-3 fatty acids, including DHA and α-linolenic acid (ALA), are proposed to improve metabolic health by reducing obesity-associated inflammation. Their effects are mediated in part by conversion to oxylipins. ALA is relatively understudied, and direct comparisons to other omega-3 fatty acids are limited. OBJECTIVES: We compared the effects of equal doses of ALA and DHA on plasma oxylipins and markers of metabolic health in women with obesity. METHODS: We carried out a randomized, double-blind, crossover clinical trial where women aged 20-51 with a BMI of 30-51 kg/m2 were supplemented with 4 g/day of ALA or DHA for 4 weeks in the form of ALA-rich flaxseed oil or DHA-rich fish oil. The primary outcome, the plasma oxylipin profile, was assessed at Days 0 and 28 of each phase by HPLC-MS/MS. Plasma fatty acids, inflammatory markers, and the monocyte glucose metabolism were key secondary outcomes. Data were analyzed using a mixed model. RESULTS: Compared to the baseline visit, there were higher plasma levels of nearly all oxylipins derived from DHA (3.8-fold overall; P < 0.001) and EPA (2.7-fold overall; P < 0.05) after 28 days of fish-oil supplementation, while there were no changes to oxylipins after flaxseed-oil supplementation. Neither supplement altered plasma cytokines; however, adiponectin was increased (1.1-fold; P < 0.05) at the end of the fish-oil phase. Compared to the baseline visit, 28 days of flaxseed-oil supplementation reduced ATP-linked oxygen consumption (0.75-fold; P < 0.05) and increased spare respiratory capacity (1.4-fold; P < 0.05) in monocytes, and countered the shift in oxygen consumption induced by LPS. CONCLUSIONS: Flaxseed oil and fish oil each had unique effects on metabolic parameters in women with obesity. The supplementation regimens were insufficient to reduce inflammatory markers but adequate to elicit increases in omega-3 oxylipins and adiponectin in response to fish oil and to alter monocyte bioenergetics in response to flaxseed oil. This trial was registered at clinicaltrials.gov as NCT03583281.

Oxylipin profiles and levels vary by skeletal muscle type, dietary fat and sex in young rats.

Polyunsaturated fatty acids (PUFA)-derived bioactive lipid mediators called oxylipins have been shown to influence muscle growth, inflammation and repair in select muscles. Since individual oxylipins have varying effects and potencies, broad profiling in differing muscle types is required to further understand their overall effects. In addition, diet and sex are key determinants of oxylipin levels. Therefore, to provide comprehensive data on oxylipin profiles in rat soleus (SO), red gastrocnemius (RG), and white gastrocnemius (WG) muscles, female and male weanling Sprague-Dawley rats were provided control or experimental diets enriched in n-3 (ω-3) or n-6 (ω-6) PUFA for 6 weeks. Free oxylipin analysis by HPLC/MS/MS revealed that SO muscle had 25% more oxylipins and 4-13 times greater oxylipin mass than WG muscle. Dietary n-3 PUFA (α-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid) each increased n-3 oxylipins derived directly from their precursors and several that were not direct precursors, while reducing arachidonic acid derived oxylipins. Dietary linoleic acid had few effects on oxylipins. Oxylipins with a sex effect were higher in females in SO and RG. Oxylipins generally reflected the effects of diet and sex on PUFA, but there were exceptions. These fundamental oxylipin profile data provide groundwork knowledge and context for future research on muscle oxylipin functions. Novelty: Rat SO compared with RG and WG muscles have a higher number and greater mass of oxylipins. Oxylipins generally reflect diet effects on PUFA in all muscles, but there are notable exceptions. Oxylipins in SO and RG are higher in females.

Impact of Age, Menopause, and Obesity on Oxylipins Linked to Vascular Health.

OBJECTIVE: Cardiovascular disease, a major cause of mortality and morbidity, exhibits sexual dimorphism since the onset of cardiovascular disease occurs later in women than in men. The loss of cardioprotection in older women may be due to an increase in arterial stiffness after menopause. Free fatty acid metabolites of polyunsaturated fatty acids, called oxylipins, are known to impact vessel function and may be responsible for the vascular benefits of polyunsaturated fatty acids. The objectives of this study were to compare the plasma oxylipin profiles of young females (20-55 years), older females (55+), and older males (55+) and to identify associations between oxylipins and cardiovascular disease risk factors, such as obesity and arterial stiffness. Approach and Results: We quantified plasma oxylipins by high-performance liquid chromatography-tandem mass spectrometry in archived samples taken from completed clinical trials. We identified 3 major 12-lipoxygenase products, 12-hydroxy-eicosatetraenoic acid, 12-hydroxy-eicosapentaenoic acid, and 14-hydroxy-docosahexaenoic acid, that are present at high levels in young females compared with older females and males. These oxylipins also decreased with obesity and displayed robust negative associations with arterial stiffness as assessed by brachial-ankle pulse wave velocity. According to multiple linear regression modeling, these associations were maintained even after correcting for body mass index category combined with either age, menopausal status, or estradiol levels. Using linear discriminant analysis, the combination of these 3 oxylipins effectively distinguished participants according to both brachial-ankle pulse wave velocity risk group and age. CONCLUSIONS: Higher 12-lipoxygenase oxylipin plasma concentrations associated with lower arterial stiffness in premenopausal females may be an important contributing factor to sex differences in cardiovascular disease. Registration: URL: https://www.clinicaltrials.gov; Unique identifiers: NCT01661543, NCT01562171, NCT01890330, NCT02571114 and NCT02317588.

Round window accessibility during cochlear implantation.

OBJECTIVE: To assess data regarding round window (RW) visibility and surgical approaches in cochlear implant cases, and to describe and analyze surgical steps relevant for the RW approach in cochlear implantation. STUDY DESIGN: Prospective clinical study. METHODS: A questionnaire was completed by surgeons after each of altogether 110 cochlear implantations. Round window membrane (RWM) visibility was graded according to the St Thomas Hospital (STH) classification. RESULTS: Performing different surgical steps during the preparation of the RW niche, the RWM could be fully exposed (STH Type I) in 87%. A RW approach could be used for electrode insertion in 89% of the adult and 78% of the pediatric cases. The distribution of RW types differed significantly between adults and children. Drilling of the superior bony lip was the surgical step most frequently needed in adult as well as pediatric cases to obtain optimal RW exposure. CONCLUSION: In children, optimized surgical exposure of the RW niche resulted in only 52% full RWM visibility; whereas in adults, this could be achieved in 87%. The facial nerve (FN) had to be exposed at the level of the posterior tympanotomy in more than 70% of pediatric cases with full RWM visibility; while in adult cases with 100% visibility, such specific exposure was necessary in only 33%. Thus, surgical preparation of the RW niche seems to be more demanding in children than in adults.

Spleen Oxylipin and Polyunsaturated Fatty Acid Profiles are Altered by Dietary Source of Polyunsaturated Fatty Acid and by Sex.

As the largest secondary lymphoid organ, the spleen plays an important role in immune responses. The role of arachidonic acid (ARA) and its 20-carbon eicosanoids in modulating immune function has long been of interest. However, recent advances have enabled the identification of numerous other n-6 and n-3 polyunsaturated fatty acid (PUFA)-derived oxylipins. Here, we investigate the effects of diet and sex on the spleen nonesterified oxylipin profiles and phospholipid and neutral lipid PUFA composition in Sprague-Dawley rats supplemented with oils rich in α-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or linoleic acid. Dietary ALA, EPA, and DHA resulted in lower levels of ARA and ARA oxylipins. Oxylipins derived from other n-6 PUFA were also reduced despite no or opposite effect on their PUFA levels. Each diet also resulted in higher levels of oxylipins almost exclusively derived from the supplemented PUFA, despite PUFA in the same biosynthetic pathway also often being increased. Further, while oxylipin differences often reflected changes to phospholipid PUFA, there were instances where they corresponded more closely to changes in neutral lipid PUFA. With respect to sex effects, >50% of lipoxygenase ARA-derived oxylipins were higher in males in at least one diet group, while multiple DHA oxylipins were lower in males only in rats provided the DHA diet. This fundamental description of oxylipin composition in the spleen, including the influence of diet and sex and the relationship to PUFA composition, will help inform future studies examining the functions of these oxylipins under physiological and pathological conditions.

Alpha-linolenic acid enhances the phagocytic and secretory functions of alternatively activated macrophages in part via changes to the oxylipin profile.

Alternatively activated macrophages are innate immune cells that contribute to resolution of inflammation and maintenance of homeostasis. Modulation of available fatty acid sources is thought to affect cellular physiology through a variety of mechanisms, including through alterations to the profile of oxygenated free fatty acid metabolites, called oxylipins, produced in a cell type specific manner. Here, we investigated how treatment with the plant-sourced omega-3 fatty acid α-linolenic acid (ALA) affects the oxylipin profile and functional capacity of a cell culture model of human alternatively activated (M2a-like) macrophages. In a targeted but unbiased screen, ALA enhanced the production of oxylipins from all polyunsaturated fatty acid (PUFA) precursors, with oxylipins derived from ALA being enhanced the most. Consistently, ALA treatment enhanced the expression of both cytoplasmic and calcium-independent phospholipase A2. At a functional level, ALA treatment increased phagocytic activity and altered production of the chemokine MCP-1 by M2a-like cells in a manner dependent on the time of treatment. ALA treatment during polarization increased MCP-1 secretion, which was sensitive to pharmacological inhibition of 15-LOX-1 by ML351. Thus, ALA modulates the phenotype of alternatively activated macrophages, likely through its own LOX-derived oxylipins and/or through general modulation of oxylipin biosynthesis. These effects likely contribute to the overall anti-inflammatory benefit observed with ALA supplementation.

Dietary n-6 and n-3 PUFA alter the free oxylipin profile differently in male and female rat hearts.

Oxylipins are bioactive lipid mediators synthesised from PUFA. The most well-known oxylipins are the eicosanoids derived from arachidonic acid (ARA), and many of them influence cardiac physiology in health and disease. Oxylipins are also formed from other n-3 and n-6 PUFA such as α-linolenic acid (ALA), EPA, DHA and linoleic acid (LA), but fundamental data on the heart oxylipin profile, and the effect of diet and sex on this profile, are lacking. Therefore, weanling female and male Sprague-Dawley rats were given American Institute of Nutrition (AIN)-93G-based diets modified in oil composition to provide higher levels of ALA, EPA, DHA, LA and LA + ALA, compared with control diets. After 6 weeks, free oxylipins in rat hearts were increased primarily by their precursor PUFA, except for EPA oxylipins, which were increased not only by dietary EPA but also by dietary ALA or DHA. Dietary DHA had a greater effect than ALA or EPA on reducing ARA oxylipins. An exception to the dietary n-3 PUFA-lowering effects on ARA oxylipins was observed for several ARA-derived PG metabolites that were higher in rats given EPA diets. Higher dietary LA increased LA oxylipins, but it had no effect on ARA oxylipins. Overall, heart oxylipins were higher in female rats, but this depended on dietary treatment: the female oxylipin:male oxylipin ratio was higher in rats provided the ALA compared with the DHA diet, with other diet groups having ratios in between. In conclusion, individual PUFA and sex have unique and interactive effects on the rat heart free oxylipin profile.

Oxylipin profile of human low-density lipoprotein is dependent on its extent of oxidation.

BACKGROUND AND AIMS: Atherosclerosis is usually the underlying cause of heart attacks, strokes and peripheral vascular diseases - collectively known as cardiovascular diseases. Oxidation of low density lipoprotein (LDL) and its lipid content has an important role in the formation of lipid-laden atherosclerotic plaques. Not much is known about the impact of oxidative stress on bioactive oxylipin molecules present in LDL. The aim of this study is to understand the changes in oxylipin molecules present in LDL characterized by varying degrees of LDL oxidation. METHODS: LDL was isolated from the pooled plasma of healthy normolipidemic volunteers and was subjected to in vitro copper-catalyzed oxidation for varying time intervals (0 h, 6 h, 12 h, 24 h and 30 h). At each time interval, oxylipins were isolated through solid phase extraction and quantified using a targeted LC/-MS/MS approach employing stable isotope dilution method. RESULTS: Our results demonstrate that different forms of oxidized LDL (OxLDL) are characterized by specific oxylipin distribution and concentration. Compared to non-oxidized LDL, there is a significant increase in oxylipin generation (p ≤ 0.05) in OxLDL subjected to 12 h and 24 h of oxidation. Though linoleate derived oxylipins are the most abundant in OxLDL extracts, the concentration of particular oxylipin species differed with different degrees of oxidation. Specifically, two pro-inflammatory linoleate-derived triols, namely 9,10,13-triHOME and 9,12,13-triHOME, exhibited a concentration increase of ~25 fold in 12h-OxLDL compared to non-oxidized LDL. Moreover, Partial least squares Discriminant Analysis (PLS-DA) identified 10 oxylipins, primarily prostaglandins, which could serve as additional biomarkers for oxidative stress or cardiovascular risk assessment. CONCLUSIONS: Our data suggests that oxidative stress induces profound changes in the oxylipin content of LDL and the pattern of change is based on the extent of oxidation.

Effects of various dietary supplements on inflammatory processes in primary canine chondrocytes as a model of osteoarthritis.

The use of dietary supplements as an alternative treatment for joint-related pathologies such as osteoarthritis (OA) is increasing. However, there is little scientific evidence to support the intended use. The aim of this study was to evaluate the anti-inflammatory effects of creatine- and amino acid-based supplements in primary cultured canine chondrocytes (CnCs) as an in-vitro model of OA and compare the effects to more commonly used agents, such as the non-steroidal anti-inflammatory drug (NSAID), carprofen, and the joint supplement, glucosamine (GS). CnCs were stimulated with interleukin-1ß (IL-1ß) and the subsequent release of prostaglandin E2 (PGE2) and tumor necrosis factor alpha (TNFα) was measured using an enzyme-linked immunosorbent assay (ELISA). Changes in oxylipins were also assessed using high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). All compounds examined were able to significantly reduce the release of PGE2 and TNFα and were associated with reductions in cyclooxygenase-2 (COX-2) expression and nuclear factor-kappaB (NF-κB) phosphorylation. The creatine- and amino acids-based supplements also altered the profile of oxylipins produced. All compounds examined were less effective at reducing the release of PGE2 than carprofen. Carprofen significantly increased release of TNFα from CnCs, however, while the other agents reduced TNFα release. This study suggests that creatine- and amino acid-based supplements may have a beneficial role in preventing inflammation within the joint and that further studies are warranted.

L'utilisation de suppléments alimentaires à titre de traitement alternatif pour les pathologies associées aux articulations telle que l'arthrose (OA) est en augmentation. Toutefois, il y a peu d'évidences scientifiques qui supportent l'utilisation proposée. L'objectif de la présente étude était d'évaluer les effets anti-inflammatoires de suppléments à base de créatine et d'acides aminés sur des cultures primaires de chondrocytes canins (CnCs) utilisés comme modèle in vitro d'OA et de comparer les effets à des agents plus communément utilisés, tel que l'agent anti-inflammatoire non-stéroïdien (AINS) carprofen, et le supplément articulaire, glucosamine (GS). Les CnCs furent stimulés avec de l'interleukine-1ß (IL-1ß) et la libération subséquente de prostaglandine E2 (PGE2) et le facteur nécrosant de tumeur alpha (TNFα) fut mesurée par épreuve immuno-enzymatique (ELISA). Les changements dans les oxylipines furent également mesurés par chromatographie en phase liquide à haute performance/spectrométrie de masse tandem (HPLC/MS/MS). Tous les composés examinés étaient en mesure de réduire significativement la libération de PGE2 et de TNFα et étaient associés avec des réductions d'expression de cyclooxygénase-2 (COX-2) et de phosphorylation du facteur nucléaire kappaB (NF-κB). Les suppléments à base de créatine et d'acides aminés ont également altéré le profil des oxylipines produits. Tous les composés examinés étaient moins efficaces que le carprofen pour réduire la libération de PGE2. Le carprofen augmentait significativement la libération de TNFα par les CnCs, alors que les autres agents la réduisaient. La présente étude suggère que les suppléments à base de créatine et d'acides aminés pourraient avoir un rôle bénéfique dans la prévention de l'inflammation dans l'articulation et que des études supplémentaires sont requises.(Traduit par Docteur Serge Messier).

The Brain Oxylipin Profile Is Resistant to Modulation by Dietary n-6 and n-3 Polyunsaturated Fatty Acids in Male and Female Rats.

Oxylipins are bioactive lipids formed by the monooxygenation of polyunsaturated fatty acids (PUFA). Eicosanoids derived from arachidonic acid (ARA) are the most well-studied class of oxylipins that influence brain functions in normal health and in disease. However, comprehensive profiling of brain oxylipins from other PUFA with differing functions, and the examination of the effects of dietary PUFA and sex differences in oxylipins are warranted. Therefore, female and male Sprague-Dawley rats were provided standard rodent diets that provided additional levels of the individual n-3 PUFA α-linolenic acid (ALA), eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), or the n-6 PUFA linoleic acid (LNA) alone or with ALA (LNA + ALA) compared to essential fatty acid-sufficient control diets. Oxylipins and PUFA were quantified in whole brains using HPLC-MS/MS and GC, respectively. Eighty-seven oxylipins were present at quantifiable levels: 51% and 17% of these were derived from ARA and DHA, respectively. At the mass level, ARA and DHA oxylipins comprised 81-90% and 6-12% of total oxylipins, while phospholipid ARA and DHA represented 25-35% and 49-62% of PUFA mass, respectively. Increasing dietary n-3 PUFA resulted in higher levels of oxylipins derived from their precursor PUFA; otherwise, the brain oxylipin profile was largely resistant to modulation by diet. Approximately 25% of oxylipins were higher in males, and this was largely unaffected by diet, further revealing a tight regulation of brain oxylipin levels. These fundamental data on brain oxylipin composition, diet effects, and sex differences will help guide future studies examining the functions of oxylipins in the brain.

Adipose tissue oxylipin profiles vary by anatomical site and are altered by dietary linoleic acid in rats.

Dietary PUFA and their effects on adipose tissue have been well studied, but oxylipins, the oxygenated metabolites of PUFA, have been sparsely studied in adipose tissue. To determine the oxylipin profile and to examine their potential importance in various adipose sites, female and male rats were provided control, high linoleic acid (LA), or high LA and high α-linolenic acid (LA + ALA) diets for six weeks. Analysis of gonadal (GAT), mesenteric (MAT), perirenal (PAT), and subcutaneous adipose tissues (SAT) revealed higher numbers of oxylipins in MAT and SAT, primarily due to 20-22 carbon cytochrome P450 oxylipins, as well as metabolites of cyclooxygenase derived oxylipins. LA oxylipins made up 75-96% of the total oxylipin mass and largely determined the total relative amounts between depots (GAT > MAT > PAT > SAT). However, when the two most abundant LA oxylipins (TriHOMEs) were excluded, MAT had the highest mass of oxylipins and exhibited the most sex differences. These differences existed despite comparable PUFA composition between depots. Dietary LA increased oxylipins derived from n-6 PUFA, and the addition of ALA generally returned n-6 PUFA oxylipins to levels similar to control and elevated some n-3 oxylipins. These data on oxylipin profiles in adipose depots from different anatomical sites and the effects of diet and sex provide fundamental knowledge that will aid future studies investigating the physiological effects of adipose tissue.

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.

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)-1ß, 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.

Distinct effects of dietary ALA, EPA and DHA on rat adipose oxylipins vary by depot location and sex.

Dietary EPA and DHA given together alter oxylipins in adipose tissue. To compare the separate effects of individual dietary n-3 PUFA on oxylipins in different adipose depots (gonadal, mesenteric, perirenal, subcutaneous) in males and females, rats were provided diets containing higher levels of α-linolenic acid (ALA), EPA or DHA. Each n-3 PUFA enhanced its respective oxylipins the most, while effects on other n-3 oxylipins varied. For example: in perirenal and subcutaneous depots, more DHA oxylipins were higher with dietary ALA than with EPA; dietary EPA uniquely decreased 14-hydroxy-docosahexaenoic acid, in contrast to increasing many other DHA oxylipins. The n-3 PUFAs also reduced oxylipins from n-6 PUFAs in order of effectiveness: DHA > EPA > ALA. Diet by sex interactions in all depots except the perirenal depot resulted in higher oxylipins in males given DHA, and higher oxylipins in females given the other diets. Diet and sex effects on oxylipins did not necessarily reflect effects on either their tissue phospholipid or neutral lipid PUFA precursors. These varying diet and sex effects on oxylipins in the different adipose sites indicate that they may have distinct effects on adipose function.

Dietary LA and sex effects on oxylipin profiles in rat kidney, liver, and serum differ from their effects on PUFAs.

A vast literature on fatty acids in mammals exists, but comparable compositional data on oxylipins is lacking. Weanling Sprague-Dawley rats were therefore provided control diets or diets with higher linoleic acid (LA) or with higher LA and α-linolenic acid (LA+ALA) for 6 weeks. Kidneys, livers, and serum were analyzed for oxylipins and fatty acids. The proportion of tissue oxylipins derived from LA was greater than the relative proportion of LA itself, whereas arachidonic acid (AA) oxylipins were overrepresented in serum. Higher dietary LA increased kidney LA and AA oxylipins, despite not altering LA or AA. In liver, both LA and AA and their oxylipins were higher, whereas in serum only LA oxylipins were higher with higher dietary LA. Higher LA resulted in a higher ratio of n-6/n-3 PUFA-derived oxylipins; adding ALA to the LA diet mitigated this and many, but not all, effects of the LA diet. Approximately 40% of oxylipins detected were influenced by sex and, unlike their PUFA precursors, most (>90%) of these were higher in males. These differences in dietary LA and sex on oxylipin and fatty acid profiles further our understanding of the effects of fatty acids and may have implications for dietary LA recommendations.

Generation of Bioactive Oxylipins from Exogenously Added Arachidonic, Eicosapentaenoic and Docosahexaenoic Acid in Primary Human Brain Microvessel Endothelial Cells.

The human blood-brain barrier (BBB) is the restrictive barrier between the brain parenchyma and the circulating blood and is formed in part by microvessel endothelial cells. The brain contains significant amounts of arachidonic acid (ARA), and docosahexaenoic acid (DHA), which potentially give rise to the generation of bioactive oxylipins. Oxylipins are oxygenated fatty acid metabolites that are involved in an assortment of biological functions regulating neurological health and disease. Since it is not known which oxylipins are generated by human brain microvessel endothelial cells (HBMECs), they were incubated for up to 30 min in the absence or presence of 0.1-mM ARA, eicosapentaenoic acid (EPA) or DHA bound to albumin (1:1 molar ratio), and the oxylipins generated were examined using high performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS). Of 135 oxylipins screened in the media, 63 were present at >0.1 ng/mL at baseline, and 95 were present after incubation with fatty acid. Oxylipins were rapidly generated and reached maximum levels by 2-5 min. While ARA, EPA and DHA each stimulated the production of oxylipins derived from these fatty acids themselves, ARA also stimulated the production of oxylipins from endogenous 18- and 20-carbon fatty acids, including α-linolenic acid. Oxylipins generated by the lipoxygenase pathway predominated both in resting and stimulated states. Oxylipins formed via the cytochrome P450 pathway were formed primarily from DHA and EPA, but not ARA. These data indicate that HBMECs are capable of generating a plethora of bioactive lipids that have the potential to modulate BBB endothelial cell function.

Dietary linoleic acid and α-linolenic acid differentially affect renal oxylipins and phospholipid fatty acids in diet-induced obese rats.

Analysis of oxylipins derived from fatty acids may provide insight into the biological effects of dietary lipids beyond their effects on tissue fatty acid profiles. We have previously observed that diets with higher amounts of α-linolenic acid (ALA; 18:3n3) are associated with reduced obesity-related glomerulopathy (ORG). Therefore, to examine the renal oxylipin profile, the effects of dietary linoleic acid (LA; 18:2n6) and ALA on oxylipins and renal phospholipid fatty acid composition, and the relationship between oxylipins and ORG, diet-induced obese rats displaying ORG were fed 8 different diets for 8 wk as follows (oil/oil = combination of two oils) [shown as ALA/LA (in g) per 100 g oil]: canola/flax (20/18), canola (8/18), soy (9/53), high-oleic canola/canola (5/16), high-oleic canola (2/15), lard/soy (1/8), and safflower (0.2/73). Targeted lipidomic analysis by HPLC-tandem mass spectrometry revealed that LA and ALA oxylipins comprised 60% of the total renal oxylipin profile examined. Of the >60 oxylipins screened, only those derived either directly or indirectly from ALA were associated with less glomerulomegaly, indicative of reduced ORG progression. Both the amount and ratio of dietary LA and ALA influenced renal polyunsaturated fatty acids (PUFAs); in contrast, only fatty acid amount altered oxylipins derived from these fatty acids, but there was no apparent competition by LA or ALA on their formation. Dietary LA incorporation into renal phospholipids was higher than for ALA, but ALA oxylipin:ALA ratios were higher than the analogous LA ratios for select lipoxygenase reactions. This indicates that the effect of dietary ALA on renal oxylipins exceeded what was reflected in renal PUFA composition. In conclusion, dietary LA and ALA have differential effects on renal oxylipins and PUFAs, and ALA-derived oxylipins are associated with renoprotection in this model of ORG.