Formation of F-ring isoprostane-like compounds (F3-isoprostanes) in vivo from eicosapentaenoic acid

Eicosapentaenoic acid (EPA, C20:5, omega-3) is the most abundant polyunsaturated fatty acid (PUFA) in fish oil. Recent studies suggest that the beneficial effects of fish oil are due, in part, to the generation of various free radical-generated non-enzymatic bioactive oxidation products from omega-3 PUFAs, although the specific molecular species responsible for these effects have not been identified. Our research group has previously reported that pro-inflammatory prostaglandin F2-like compounds, termed F2-isoprostanes (IsoPs), are produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid and represent one of the major products resulting from the oxidation of this PUFA. Based on these observations, we questioned whether F2-IsoP-like compounds (F3-IsoPs) are formed from the oxidation of EPA in vivo. Oxidation of EPA in vitro yielded a series of compounds that were structurally established to be F3-IsoPs using a number of chemical and mass spectrometric approaches. The amounts formed were extremely large (up to 8.7 + 1.0 microg/mg EPA) and greater than levels of F2-IsoPs generated from arachidonic acid. We then examined the formation of F3-IsoPs in vivo in mice. Levels of F3-IsoPs in tissues such as heart are virtually undetectable at baseline, but supplementation of animals with EPA markedly increases quantities up to 27.4 + 5.6 ng/g of heart. Interestingly, EPA supplementation also markedly reduced levels of pro-inflammatory arachidonate-derived F2-IsoPs by up to 64% (p < 0.05). Our studies provide the first evidence that identify F3-IsoPs as novel oxidation products of EPA that are generated in vivo. Further understanding of the biological consequences of F3-IsoP formation may provide valuable insights into the cardioprotective mechanism of EPA.     

Quantification of F-ring isoprostane-like compounds (F4-neuroprostanes) derived from docosahexaenoic acid in vivo in humans by a stable isotope dilution mass spectrometric assay

Lipid peroxidation has been implicated in the pathophysiological sequelae of human neurodegenerative disorders. It is recognized that quantification of lipid peroxidation is best assessed in vivo by measuring a series of prostaglandin (PG) F2-like compounds termed F2-isoprostanes (IsoPs) in tissues in which arachidonic acid is abundant. Unlike other organs, the major polyunsaturated fatty acid (PUFA) in the brain is docosahexaenoic acid (DHA, C22:6 omega-6), and this fatty acid is particularly enriched in neurons. We have previously reported that DHA undergoes oxidation in vitro and in vivo resulting in the formation of a series of F2-IsoP-like compounds termed F4-neuroprostanes (F4-NPs). We recently chemically synthesized one F4-NP, 17-F4c-NP, converted it to an 18O-labeled derivative, and utilized it as an internal standard to develop an assay to quantify endogenous production of F4-NPs by gas chromatography (GC)/negative ion chemical ionization (NICI) mass spectrometry (MS). The assay is highly precise and accurate. The lower limit of sensitivity is approximately 10 pg. Levels of F4-NPs in brain tissue from rodents were 8.7 +/- 2.0 ng/g wet weight (mean +/- S.D.). Levels of the F4-NPs in brains from normal humans were found to be 4.9 +/- 0.6 ng/g (mean +/- S.D.) and were 2.1-fold higher in affected regions of brains from humans with Alzheimer's disease (P = 0.02). Thus, this assay provides a sensitive and accurate method to assess oxidation of DHA in animal and human tissues and will allow for the further elucidation of the role of oxidative injury to the central nervous system in association with human neurodegenerative disorders.     

Quantification of F2-isoprostanes as a biomarker of oxidative stress

Oxidant stress has been implicated in a wide variety of disease processes. One method to quantify oxidative injury is to measure lipid peroxidation. Quantification of a group of prostaglandin F(2alpha)-like compounds derived from the nonezymatic oxidation of arachidonic acid, termed the F2-isoprostanes (F2-IsoPs), provides an accurate assessment of oxidative stress both in vitro and in vivo. In fact, in a recent independent study sponsored by the National Institutes of Health (NIH), F2-IsoPs were shown to be the most reliable index of in vivo oxidant stress when compared against other well known biomarkers. This protocol details our laboratory's method to quantify F2-IsoPs in biological fluids and tissues using gas chromatography-mass spectrometry (GC-MS). This procedure can be completed for 12-15 samples in 6-8 h.     

Measurement of F(2)-isoprostanes as an index of oxidative stress in vivo

In 1990 we discovered the formation of prostaglandin F(2)-like compounds, F(2)-isoprostanes (F(2)-IsoPs), in vivo by nonenzymatic free radical-induced peroxidation of arachidonic acid. F(2)-IsoPs are initially formed esterified to phospholipids and then released in free form. There are several favorable attributes that make measurement of F(2)-IsoPs attractive as a reliable indicator of oxidative stress in vivo: (i) F(2)-IsoPs are specific products of lipid peroxidation; (ii) they are stable compounds; (iii) levels are present in detectable quantities in all normal biological fluids and tissues, allowing the definition of a normal range; (iv) their formation increases dramatically in vivo in a number of animal models of oxidant injury; (v) their formation is modulated by antioxidant status; and (vi) their levels are not effected by lipid content of the diet. Measurement of F(2)-IsoPs in plasma can be utilized to assess total endogenous production of F(2)-IsoPs whereas measurement of levels esterified in phospholipids can be used to determine the extent of lipid peroxidation in target sites of interest. Recently, we developed an assay for a urinary metabolite of F(2)-IsoPs, which should provide a valuable noninvasive integrated approach to assess total endogenous production of F(2)-IsoPs in large clinical studies.     

Formation of highly reactive A-ring and J-ring isoprostane-like compounds (A4/J4-neuroprostanes) in vivo from docosahexaenoic acid

Free radical-initiated oxidant injury and lipid peroxidation have been implicated in a number of neural disorders. Docosahexaenoic acid is the most abundant unsaturated fatty acid in the central nervous system. We have shown previously that this 22-carbon fatty acid can yield, upon oxidation, isoprostane-like compounds termed neuroprostanes, with E/D-type prostane rings (E(4)/D(4)-neuroprostanes). Eicosanoids with E/D-type prostane rings are unstable and dehydrate to cyclopentenone-containing compounds possessing A-type and J-type prostane rings, respectively. We thus explored whether cyclopentenone neuroprostanes (A(4)/J(4)-neuroprostanes) are formed from the dehydration of E(4)/D(4)-neuroprostanes. Indeed, oxidation of docosahexaenoic acid in vitro increased levels of putative A(4)/J(4)-neuroprostanes 64-fold from 88 +/- 43 to 5463 +/- 2579 ng/mg docosahexaenoic acid. Chemical approaches and liquid chromatography/electrospray ionization tandem mass spectrometry definitively identified them as A(4)/J(4)-neuroprostanes. We subsequently showed these compounds are formed in significant amounts from a biological source, rat brain synaptosomes. A(4)/J(4)-neuroprostanes increased 13-fold, from a basal level of 89 +/- 72 ng/mg protein to 1187 +/- 217 ng/mg (n = 4), upon oxidation. We also detected these compounds in very large amounts in fresh brain tissue from rats at levels of 97 +/- 25 ng/g brain tissue (n = 3) and from humans at levels of 98 +/- 26 ng/g brain tissue (n = 5), quantities that are nearly an order of magnitude higher than other classes of neuroprostanes. Because of the fact that A(4)/J(4)-neuroprostanes contain highly reactive cyclopentenone ring structures, it would be predicted that they readily undergo Michael addition with glutathione and adduct covalently to proteins. Indeed, incubation of A(4)/J(4)-neuroprostanes in vitro with excess glutathione resulted in the formation of large amounts of adducts. Thus, these studies have identified novel, highly reactive A/J-ring isoprostane-like compounds that are derived from docosahexaenoic acid in vivo.     

Nonenzymatic free radical-catalyzed generation of 15-deoxy-Δ(12,14)-prostaglandin J₂-like compounds (deoxy-J₂-isoprostanes) in vivo

15-Deoxy-Δ^12,14-prostaglandin J₂ (15-d-PGJ₂) is a reactive cyclopentenone eicosanoid generated from the dehydration of cyclooxygenase-derived prostaglandin D₂ (PGD₂). This compound possesses an α,β-unsaturated carbonyl moiety that can readily adduct thiol-containing biomolecules such as glutathione and cysteine residues of proteins via the Michael addition. Due to its reactivity, 15-d-PGJ₂ is thought to modulate inflammatory and apoptotic processes and is believed to be an endogenous ligand for peroxisome proliferator-activated receptor-γ. However, the extent to which 15-d-PGJ₂ is formed in vivo and the mechanisms that regulate its formation are unknown. Previously, we have reported the formation of PGD₂ and PGJ₂-like compounds, termed D₂/J₂-isoprostanes (D₂/J₂-IsoPs), produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid (AA). Based on these findings, we investigated whether 15-d-PGJ₂-like compounds are also formed via this nonenzymatic pathway. Here we report the generation of novel 15-d-PGJ₂-like compounds, termed deoxy-J₂-isoprostanes (deoxy-J₂-IsoPs), in vivo, via the nonenzymatic peroxidation of AA. Levels of deoxy-J₂-IsoPs increased 12-fold (6.4 ± 1.1 ng/g liver) in rats after oxidant insult by CCl₄ treatment, compared with basal levels (0.55 ± 0.21 ng/g liver). These compounds may have important bioactivities in vivo under conditions associated with oxidant stress.     

Formation of novel D-ring and E-ring isoprostane-like compounds (D4/E4-neuroprostanes) in vivo from docosahexaenoic acid

Free radical-mediated oxidant injury and lipid peroxidation have been implicated in a number of neural disorders. We have reported that bioactive prostaglandin D2/E2-like compounds, termed D2/E2-isoprostanes, are produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid. Docosahexaenoic acid, in contrast to arachidonic acid, is the most abundant unsaturated fatty acid in brain. We therefore questioned whether D/E-isoprostane-like compounds (D4/E4-neuroprostanes) are formed from the oxidation of docosahexaenoic acid. Levels of putative D4/E4-neuroprostanes increased 380-fold after oxidation of docosahexaenoic acid in vitro from 15.2 +/- 6.3 to 5773 +/- 1024 ng/mg of docosahexaenoic acid. Subsequently, chemical approaches and liquid chromatography electrospray ionization tandem mass spectrometry definitively identified these compounds as D4/E4-neuroprostanes. We then explored the formation of D4/E4-neuroprostanes from a biological source, rat brain synaptosomes. Basal levels of D4/E4-neuroprostanes were 3.8 +/- 0.6 ng/mg of protein and increased 54-fold after oxidation (n = 4). We also detected these compounds in fresh brain tissue from rats at levels of 12.1 +/- 2.4 ng/g of brain tissue (n = 3) and in human brain tissue at levels of 9.2 +/- 4.1 ng/g of brain tissue (n = 4). Thus, these studies have identified novel D/E-ring isoprostane-like compounds that are derived from docosahexaenoic acid and that are formed in brain in vivo. The fact that they are readily detectable suggests that ongoing oxidative stress is present in the central nervous system of humans and animals. Further, identification of these compounds provides a rationale for examining their role in neurological disorders associated with oxidant stress.     

The biochemistry of the isoprostane, neuroprostane, and isofuran pathways of lipid peroxidation

F2-isoprostanes are prostaglandin F2-like compounds that are formed nonenzymatically by free radical mediated peroxidation of arachidonic acid. Intermediate in the pathway of the formation of isoprostanes are labile prostaglandin H2-like bicyclic endoperoxides (H2-isoprostanes), which are reduced to F2-isoprostanes and also undergo rearrangement in vivo to form E-ring and D-ring isoprostanes, isothromboxanes, and highly reactive acyclic gamma-ketoaldehdyes (isoketals). Docosahexaenoic acid (C22:6omega3) is highly enriched in neurons in the brain and is highly susceptible to oxidation. Free radical mediated oxidation of docosahexaenoic acid results in the formation of isoprostane-like compounds (neuroprostanes). F4- and E4/D4-neuroprostanes as well as neuroketals have been shown to be produced in vivo. Finally, we recently discovered a new pathway of lipid peroxidation that forms compounds with a substituted tetrahydrofuran ring (isofurans). Oxygen concentrations differentially modulate the formation of isoprostanes and isofurans; at elevated oxygen concentrations, the formation of isofurans is favored whereas the formation of isoprostanes is disfavored.     

Electrophilic cyclopentenone neuroprostanes are anti-inflammatory mediators formed from the peroxidation of the omega-3 polyunsaturated fatty acid docosahexaenoic acid

The omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) possesses potent anti-inflammatory properties and has shown therapeutic benefit in numerous inflammatory diseases. However, the molecular mechanisms of these anti-inflammatory properties are poorly understood. DHA is highly susceptible to peroxidation, which yields an array of potentially bioactive lipid species. One class of compounds are cyclopentenone neuroprostanes (A(4)/J(4)-NPs), which are highly reactive and similar in structure to anti-inflammatory cyclopentenone prostaglandins. Here we show that a synthetic A(4)/J(4)-NP, 14-A(4)-NP (A(4)-NP), potently suppresses lipopolysaccharideinduced expression of inducible nitric-oxide synthase and cyclooxygenase-2 in macrophages. Furthermore, A(4)-NP blocks lipopolysaccharide-induced NF-kappaB activation via inhibition of Ikappa kinase-mediated phosphorylation of IkappaBalpha. Mutation on Ikappa kinase beta cysteine 179 markedly diminishes the effect of A(4)-NP, suggesting that A(4)-NP acts via thiol modification at this residue. Accordingly, the effects of A(4)-NP are independent of peroxisome proliferator-activated receptor-gamma and are dependent on an intact reactive cyclopentenone ring. Interestingly, free radical-mediated oxidation of DHA greatly enhances its anti-inflammatory potency, an effect that closely parallels the formation of A(4)/J(4)-NPs. Furthermore, chemical reduction or conjugation to glutathione, both of which eliminate the bioactivity of A(4)-NP, also abrogate the anti-inflammatory effects of oxidized DHA. Thus, we have demonstrated that A(4)/J(4)-NPs, formed via the oxidation of DHA, are potent inhibitors of NF-kappaB signaling and may contribute to the anti-inflammatory actions of DHA. These findings have implications for understanding the anti-inflammatory properties of omega-3 fatty acids, and elucidate novel interactions between lipid peroxidation products and inflammation.     

Measurement of F2-isoprostanes, hydroxyeicosatetraenoic products, and oxysterols from a single plasma sample

Oxidized lipids such as F2-isoprostanes (F2-IsoPs), hydroxyeicosatetraenoic acid products (HETEs), and cholesterol oxidation products (COPs) are widely believed to be involved in multiple diseases. Usually, each product is measured individually in separate blood samples. In this study we describe a method allowing us to measure F2-IsoPs, HETEs, COPs, and arachidonate using a single sample. Plasma (1 ml) samples from healthy volunteers were diluted with heavy isotopic standards, hydrolyzed in alkali with organic solvent, and then subjected to anionic-exchange solid-phase extraction (SPE). After the SPE column was washed, hexane and hexane/ethyl acetate portions were collected and combined for COPs measurement. Thereafter the column was loaded with hexane/ethanol/acetic acid and fractions were collected for total F2-IsoPs, total HETEs, and arachidonate measurement. All compounds in the eluates were measured by gas chromatography-mass spectrometry. The efficiency of SPE and reproducibility for all compounds measured were high. Levels of total F2-IsoPs (0.45+/-0.26 ng/ml (n=157)), total HETEs (34.06+/-16.35 ng/ml (n=21)), total arachidonate (68.36+/-24.45 microg/ml (n=33)), and COPs (7-ketocholesterol, 12.25+/-6.56 ng/ml; 7beta-hydroxycholesterol, 6.32+/-3.46 ng/ml; 7alpha-hydroxycholesterol, 15.06+/-7.06 ng/ml; 24-hydroxycholesterol, 41.39+/-18.22 ng/ml; and 27-hydroxycholesterol, 29.08+/-16.79 ng/ml (n=26)) were recorded in healthy subjects (age range 20 to 66 years; average male to female ratio 1:1).     

Identification of novel autoxidation products of the omega-3 fatty acid eicosapentaenoic acid in vitro and in vivo

Increased intake of fish oil rich in the omega-3 fatty acids eicosapentaenoic acid (EPA, C20:5 omega-3) and docosahexaenoic acid (DHA, C22:6 omega-3) reduces the incidence of human disorders such as atherosclerotic cardiovascular disease. However, mechanisms that contribute to the beneficial effects of fish oil consumption are poorly understood. Mounting evidence suggests that oxidation products of EPA and DHA may be responsible, at least in part, for these benefits. Previously, we have defined the free radical-induced oxidation of arachidonic acid in vitro and in vivo and have proposed a unified mechanism for its peroxidation. We hypothesize that the oxidation of EPA can be rationally defined but would be predicted to be significantly more complex than arachidonate because of the fact that EPA contains an addition carbon-carbon double bond. Herein, we present, for the first time, a unified mechanism for the peroxidation of EPA. Novel oxidation products were identified employing state-of-the-art mass spectrometric techniques including Ag(+) coordination ionspray and atmospheric pressure chemical ionization mass spectrometry. Predicted compounds detected both in vitro and in vivo included monocylic peroxides, serial cyclic peroxides, bicyclic endoperoxides, and dioxolane-endoperoxides. Systematic study of the peroxidation of EPA provides the basis to examine the role of specific oxidation products as mediators of the biological effects of fish oil.     

Identification of the major urinary metabolite of the highly reactive cyclopentenone isoprostane 15-A(2t)-isoprostane in vivo

The cyclopentenone isoprostanes (A(2)/J(2)-IsoPs) are formed in significant amounts in humans and rodents esterified in tissue phospholipids. Nonetheless, they have not been detected unesterified in the free form, presumably because of their marked reactivity. A(2)/J(2)-IsoPs, similar to other electrophilic lipids such as 15-deoxy-Delta(12,14)-prostaglandin J(2) and 4-hydroxynonenal, contain a highly reactive alpha,beta-unsaturated carbonyl, which allows these compounds to react with thiol-containing biomolecules to produce a range of biological effects. We sought to identify and characterize in rats the major urinary metabolite of 15-A(2t)-IsoP, one of the most abundant A(2)-IsoPs produced in vivo, in order to develop a specific biomarker that can be used to quantify the in vivo production of these molecules. Following intravenous administration of 15-A(2t)-IsoP containing small amounts of [(3)H(4)]15-A(2t)-IsoP, 80% of the radioactivity excreted in the urine remained in aqueous solution after extraction with organic solvents, indicating the formation of a polar conjugate(s). Using high pressure liquid chromatography/mass spectrometry, the major urinary metabolite of 15-A(2t)-IsoP was determined to be the mercapturic acid sulfoxide conjugate in which the carbonyl at C9 was reduced to an alcohol. The structure was confirmed by direct comparison to a synthesized standard and via various chemical derivatizations. In addition, this metabolite was found to be formed in significant quantities in urine from rats exposed to an oxidant stress. The identification of this metabolite combined with the finding that these metabolites are produced in in vivo settings of oxidant stress makes it possible to use this method to quantify, for the first time, the in vivo production of cyclopentenone prostanoids.     

Novel n-3 fatty acid oxidation products activate Nrf2 by destabilizing the association between Keap1 and Cullin3

Consumption of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) can mitigate the progression of diseases in which oxidative stress represents a common underlying biochemical process. Nrf2-regulated gene expression regulates detoxification of reactive oxygen species. EPA and DHA were subjected to an in vitro free radical oxidation process that models in vivo conditions. Oxidized n-3 fatty acids reacted directly with the negative regulator of Nrf2, Keap1, initiating Keap1 dissociation with Cullin3, thereby inducing Nrf2-directed gene expression. Liquid chromatography-tandem mass spectrometry analyses of oxidized EPA demonstrated the presence of novel cyclopentenone-containing molecules termed J3-isoprostanes in vitro and in vivo and were shown to induce Nrf2-directed gene expression. These experiments provide a biochemical basis for the hypothesis that formation of J-ring compounds generated from oxidation of EPA and DHA in vivo can reach concentrations high enough to induce Nrf2-based cellular defense systems.     

Role of omega-3 polyunsaturated fatty acids on cyclooxygenase-2 metabolism in brain-metastatic melanoma

Cyclooxygenase-2 (COX-2) is important in the progression of epithelial tumors. Evidence indicates that omega-6 PUFAs such as arachidonic acid (AA) promote the growth of tumor cells; however, omega-3 fatty acids [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] inhibit tumor cell proliferation. We investigated the effects of omega-3 PUFA on the expression and function of COX-2 in 70W, a human melanoma cell line that metastasizes to the brain in nude mice. We show that 1) tumor necrosis factor-alpha upregulates the expression of both COX-2 mRNA and prostaglandin E2 (PGE2) production, and 2) omega-3 and omega-6 PUFA regulate COX-2 mRNA expression and PGE2 production. AA increased COX-2 mRNA expression and prostaglandin production in omega-6-stimulated 70W cells. Conversely, COX-2 mRNA expression decreased in cells incubated with EPA or DHA. AA increased Matrigel invasion 2.4-fold, whereas EPA or DHA did not. Additionally, PGE2 increased in vitro invasion 2.5-fold, whereas exposure to PGE3 significantly decreased invasion. Our results demonstrate that incubation of 70W cells with either AA or PGE2 increased invasiveness, whereas incubation with EPA or DHA downregulated both COX-2 mRNA and protein expression, with a subsequent decrease in Matrigel invasion. Taken together, these results indicate that omega-3 PUFA regulate COX-2-mediated invasion in brain-metastatic melanoma.     

Isoprostanes and the liver

The liver has been central to our understanding of the physiology and biology of the F2-isoprostanes. The discovery of F2-IsoPs and the initial demonstration that they could be used to localize oxidative stress was first demonstrated in a rat model of oxidative liver injury (carbon tetrachloride), and the first demonstration that plasma concentrations are increased in a human disease was in patients with liver failure and the hepatorenal syndrome [J. Clin. Invest. 90 (6) (1992b) 2502; J. Lipid Mediat. 6 (1/3) (1993) 417]. This article will cover the measurement of F2-IsoPs as markers of lipid peroxidation in vivo in liver disease, and review their biological activity as mediators of disease.     

CYP-eicosanoids--a new link between omega-3 fatty acids and cardiac disease?

Fish oil omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protect against arrhythmia and sudden cardiac death by largely unknown mechanisms. Recent in vitro and in vivo studies demonstrate that arachidonic acid (AA) metabolizing cytochrome P450-(CYP) enzymes accept EPA and DHA as efficient alternative substrates. Dietary EPA/DHA supplementation causes a profound shift of the cardiac CYP-eicosanoid profile from AA- to EPA- and DHA-derived epoxy- and hydroxy-metabolites. CYP2J2 and other CYP epoxygenases preferentially epoxidize the ω-3 double bond of EPA and DHA. The corresponding metabolites, 17,18-epoxy-EPA and 19,20-epoxy-DHA, dominate the CYP-eicosanoid profile of the rat heart after EPA/DHA supplementation. The (ω-3)-epoxyeicosanoids show highly potent antiarrhythmic properties in neonatal cardiomyocytes, suggesting that these metabolites may specifically contribute to the cardioprotective effects of omega-3 fatty acids. This hypothesis is discussed in the context of recent findings that revealed CYP-eicosanoid mediated mechanisms in cardiac ischemia-reperfusion injury and maladaptive cardiac hypertrophy.     

Inhibition of leukocyte-endothelial interactions by oxidized omega-3 fatty acids: a novel mechanism for the anti-inflammatory effects of omega-3 fatty acids in fish oil

Omega-3 fatty acids which are abundant in fish oil improve the prognosis of several chronic inflammatory diseases that are characterized by leukocyte-mediated tissue injury. The omega-3 fatty acids, such as eicosapentaenoic acid (EPA), are highly polyunsaturated and readily undergo oxidation. Our data suggest that the beneficial effects of fish oil may be due to the oxidative modification of omega-3 fatty acids. The oxidized products inhibit leukocyte adhesion receptor expression and leukocyte-endothelial interactions. Oxidized EPA is a potent inhibitor of leukocyte interactions with the endothelium compared to native EPA, both in vitro and in an in vivo context of inflammation. The effects of oxidized EPA are mediated through activation of PPARalpha and subsequent inhibition of NF-kappaB, leading to the down-regulation of leukocyte adhesion receptor expression required for leukocyte-endothelial interactions. We propose that oxidation of EPA and its activation of PPARalpha and subsequent inhibition of NF-kappaB is the underlying mechanism for the beneficial effects of fish oil.     

Modulatory effect of omega-3 polyunsaturated fatty acids on osteoblast function and bone metabolism

Recent investigations indicate that the type and amount of polyunsaturated fatty acids (PUFA) influence bone formation in animal models and osteoblastic cell functions in culture. In growing rats, supplementing the diet with omega-3 PUFA results in greater bone formation rates and moderates ex vivo prostaglandin E(2) production in bone organ cultures. A protective effect of omega-3 PUFA on minimizing bone mineral loss in ovariectomized rats has also been reported. The actions of omega-3 fatty acids on bone formation appear to be linked to altering osteoblast functions. Herein we describe experiments with MC3T3-E1 osteoblast-like cells that support findings in vivo where omega-3 PUFA modulated COX-2 protein expression, reduced prostaglandin E(2) production, and increased alkaline phosphatase activity. Other studies indicate that the dietary source of PUFA may affect protein expression of Cbfa1 and nodule formation in fetal rat calvarial cells.     

Resolvins, docosatrienes, and neuroprotectins, novel omega-3-derived mediators, and their aspirin-triggered endogenous epimers: an overview of their protective roles in catabasis

The molecular basis for the beneficial impact of essential omega-3 fatty acids is of considerable interest. Recently, novel mediators generated from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) that displayed potent bioactions were first identified in resolving inflammatory exudates [J. Exp. Med. 192 (2000) 1197; J. Exp. Med. 196 (2002) 1025] and in tissues enriched with DHA [J. Exp. Med. 196 (2002) 1025; J. Biol. Chem. 278 (2003) 14677]. The trivial names Resolvin (resolution phase interaction products) and docosatrienes were introduced for the bioactive compounds belonging to these novel series because they demonstrate potent anti-inflammatory and immunoregulatory actions. The compounds derived from eicosapentaenoic acid carrying potent biological actions (i.e., 1-10 nM range) are designated E series, given their EPA precursor, and denoted as Resolvins of the E series (Resolvin E1 or RvE1), and those biosynthesized from the precursor docosahexaenoic acid are Resolvins of the D series (Resolvin D1 or RvD1). Bioactive members from DHA with conjugated triene structures are docosatrienes (DT) that are immunoregulatory [J. Exp. Med. 196 (2002) 1025; J. Biol. Chem. 278 (2003) 14677], and neuroprotective [J. Biol. Chem., 278 (2003) 43807; Proc. Natl. Acad. Sci. U.S.A. [submitted for publication]] and are termed neuroprotectins. The specific receptors for these novel bioactive products from omega-3 EPA and DHA are abbreviated Resolvin D receptors (i.e., ResoDR1), Resolvin E receptor (ResoER1; RER1), and neuroprotectin D receptors (NPDR), respectively, in recognition of their respective cognate ligands. Aspirin treatment impacts biosynthesis of these compounds and a related series by triggering endogenous formation of the 17R-D series Resolvins and docosatrienes. These novel epimers are denoted as aspirin-triggered (AT)-RvDs and -DTs, and possess potent anti-inflammatory actions in vivo essentially equivalent to their 17S series pathway products. Here, we provide a syntomy overview of the formation and actions of these newly uncovered pathways and products as well as highlight their role(s) as endogenous protective mediators generated in anti-inflammation and catabasis.     

Effect of long-chain fatty acids in the culture medium on fatty acid composition of WEHI-3 and J774A.1 cells

As a first step in determining the mechanism of action of specific fatty acids on immunological function of macrophages, a comparative study of the effect of long-chain polyunsaturated fatty acids (PUFA) in the medium was conducted in two macrophage cell lines, J774A.1 and WEHI-3. The baseline fatty-acid profiles of the two cell lines differed in the % distribution of saturated (SFA) and unsaturated fatty acids (UFA). J774A.1 cells had a higher % of SFA (primarily palmitic acid) than WEHI-3 cells. Conversely, WEHI-3 cells had a higher % of UFA (primarily oleic acid) than J774A.1 cells. Neither cell line had detectable amounts of alpha-linolenic acid (ALA) or eicosapentaenoic acid (EPA). The most abundant polyunsaturated fatty acid in both cells lines was arachidonic acid (AA). The efficiency of transport of fatty acids from the medium to the macrophages by two delivery vehicles (BSA complexes and ethanolic suspensions) was compared. Overall, fatty acids were transported satisfactorily by both delivery systems. Alpha-linolenic acid and doscosahexenoic acid (DHA) were transported more efficiently by the ethanolic suspension system. Linoleic acid (LA) was taken up more completely than ALA, and DHA was taken up more completely than EPA by both cell cultures and delivery systems. A dose-response effect was demonstrated for LA, ALA, EPA and DHA in both J774A.1 and WEHI-3 cells. Addition of polyunsaturated fatty acids (PUFA) to the cell cultures modified the total lipid fatty acid composition of the cells. The presence of ALA in the culture medium resulted in a significant decrease in AA in both cell lines. The omega-3/omega-6 fatty acid ratio (omega-3/omega-6), polyunsaturated/saturated fatty acid ratio (P/S), and unsaturation index (UI) increased directly with the amount of PUFA and omega-3 fatty acid provided in the medium. The results indicate that the macrophage cell lines have similar, but not identical, fatty acid profiles that may be the result of differences in fatty acid metabolism. These distinctions could in turn produce differences in immunological function. The ethanol fatty-acid delivery system, when compared with the fatty acid-BSA complex system, is preferable for measurement of dose-response effects, because the cellular fatty acid content increased in proportion to the amount of fatty acid provided in the medium. Similar dose-response results were observed in a previous in vivo study using flaxseed, rich in ALA, as a source of PUFA.