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Background: Neutrophils release leukotriene (LT)B 4 and myeloperoxidase (MPO) that may be important mediators of chronic inflammation in chronic kidney disease (CKD). The n-3 fatty acids (n-3 FA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have the potential to attenuate inflammation through production of LTB 5 and the Specialized Proresolving Lipid Mediators (SPM) that promote the resolution of inflammation. In animal models, coenzyme Q10 (CoQ) also attenuates inflammation by reducing MPO and LTB 4 ., Objective: This study evaluated the independent and combined effects of n-3 FA and CoQ supplementation on neutrophil leukotrienes, the pro-inflammatory eicosanoid 5-hydroxyeicosatetraenoic acid (5-HETE), SPM, and plasma MPO, in patients with CKD., Design: In a double-blind, placebo-controlled intervention of factorial design, 85 patients with CKD were randomized to either n-3 FA (4 g), CoQ (200 mg), both supplements, or control (4 g olive oil), daily for 8 weeks. Plasma MPO and calcium ionophore-stimulated neutrophil release of LTs, 5-HETE and SPM were measured at baseline and after 8 weeks., Results: Seventy four patients completed the intervention. n-3 FA, but not CoQ, significantly increased neutrophil LTB 5 (P < 0.0001) and the SPM 18-hydroxyeicosapentaenoic acid (18-HEPE), resolvin E1 (RvE1), resolvin E2 (RvE2) and resolvin E3 (RvE3) that derive from EPA, as well as 17-hydroxydocosahexaenoic acid (17-HDHA) and resolvin D5 (RvD5) that derive from DHA (all P < 0.01). Neutrophil LTB4 and its metabolites, and 5-HETE were not significantly altered by n-3 FA or CoQ. Plasma MPO was significantly reduced with n-3 FA alone (P = 0.013) but not when given in combination with CoQ., Conclusion: n-3 FA supplementation in patients with CKD leads to increased neutrophil release of LTB 5 and several SPM, as well as a reduction in plasma MPO that may have important implications for limiting chronic inflammation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Future feed for farmed fish are based on untraditional feed ingredients, which will change nutrient profiles compared to traditional feed based on marine ingredients. To understand the impact of oils from different sources on fish health, n-6 and n-3 polyunsaturated fatty acids (PUFAs) were added to salmon head kidney cells, in a fully crossed design, to monitor their individual and combined effects on gene expression. Exposing salmon head kidney cells to single fatty acids, arachidonic acid (AA) or decosahexaenoic acid (DHA), resulted in down-regulation of cell signaling pathway genes and specific fatty acid metabolism genes as well as reduced prostaglandin E2 (PGE2) secretion. Eicosapentaenoic acid (EPA) had no impact on gene transcription in this study, but reduced the cell secretion of PGE2. The combined effect of AA + EPA resulted in up-regulation of eicosanoid pathway genes and the pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α), Bclx (an inducer of apoptosis) and fatty acid translocase (CD36) as well as increased cell secretion of PGE2 into the media. Adding single fatty acids to salmon head kidney cells decreased inflammation markers in this model. The combination AA + EPA acted differently than the rest of the fatty acid combinations by increasing the inflammation markers in these cells. The concentration of fatty acid used in this experiment did not induce any lipid peroxidation responses., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Omega-3 fatty acids (n-3 FA) may have beneficial clinical and immune-modulating effects in surgical patients. In a randomized, double-blind, prospective, placebo-controlled trial, 148 patients referred for elective colorectal cancer surgery received an n-3 FA-enriched oral nutritional supplement (ONS) providing 2.0 g of eicosapentaenoic acid (EPA) and 1.0 g of docosahexaenoic acid (DHA) per day or a standard ONS for seven days before surgery. On the day of operation, there was a significant increase in the production of leukotriene B5 (LTB5) (p < 0.01) and 5-hydroxyeicosapentaenoic acid (5-HEPE) (p < 0.01), a significant decrease in the production of leukotriene B4 (LTB4) (p < 0.01) and a trend for a decrease in the production of 5-hydroxyeicosatetraenoic acid (5-HETE) (p < 0.1) from stimulated neutrophils in the active group compared with controls. There was no association between LTB4 values and postoperative complications. In conclusion, oral n-3 FA exerts anti-inflammatory effects in surgical patients, without reducing the risk of postoperative complications.

Introduction: Marine n-3 polyunsaturated fatty acids (PUFA) have a variety of anti-inflammatory properties. This study evaluated the effect of n-3 PUFA in a low, but recommended cardioprotective dosage on the formation of 5-lipoxygenase pathway metabolites in overweight subjects., Materials and Methods: Fifty subjects were randomized to 1.1g of n-3 PUFA or olive oil for 6 weeks., Results: Leukotriene B(4) formation decreased by 14% in the n-3 PUFA group which proved to be significant within the group (p=0.005) but not between groups (p=0.25). The formation of 5-hydroxyeicosatetraenoic acid (5-HETE) did not differ significantly between the groups. In the n-3 PUFA group, both 5-hydroxyeicosapentaenoic (5-HEPE) acid and leukotriene B(5) increased significantly compared to the control group (p<0.001)., Conclusion: In conclusion, we did not observe any significant net anti-inflammatory effect on the 5-lipoxygenase pathway from a daily supplement of 1.1g marine n-3 PUFA for 6 weeks., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objective: Compared with soybean oil, a fish oil-enriched emulsion can improve the clinical outcomes of patients requiring parenteral nutrition. However, the superiority of fish oil emulsion to medium-chain triacylglycerols/long-chain triacylglycerols for short-term administration has seldom been discussed., Methods: Sixty-four adult patients with gastrointestinal diseases were randomly assigned to receive isocaloric and isonitrogenous total parenteral nutrition with an ω-3 fatty acid-enriched emulsion (Lipoplus; study group, n = 32) or medium-chain triacylglycerols/long-chain triacylglycerols (Lipofundin; control group, n = 32) for 5 d after surgery. Safety and efficacy parameters were assessed on postoperative days 1, 3, and 6., Results: Clinical outcomes including infectious complications and systemic inflammatory response syndrome were comparable between the two groups. Total bilirubin decreased over time in the study group versus an increase in the control group (P = 0.017). Activated partial thromboplastin time in the study group was prolonged significantly compared with the control group from days 1 to 3 (P = 0.002), although the prolongation stopped at the study termination. There were no differences in changes of C-reactive protein, interleukin (IL)-1, IL-8, IL-10, vascular endothelial growth factor (VEGF), and the distribution of the T-cell subpopulation between the two groups. However, fish oil consumption led to an increase in leukotriene B5/ leukotriene B4 and significant decreases in IL-6, tumor necrosis factor-α, and nuclear factor-κB. Furthermore, the overall changes in tumor necrosis factor-α and nuclear factor-κB were positively associated (R(2) = 0.295, P < 0.001)., Conclusions: Gastrointestinal surgery patients benefited from a fish oil-enriched emulsion rather than medium-chain triacylglycerols/long-chain triacylglycerols in the amelioration of liver function and immune status. The positive association of tumor necrosis factor-α and nuclear factor-κB might be involved in the potential anti-inflammation mechanism of fish oil., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The effect of diets containing either 18-carbon n-3 fatty acids (FA) or 20/22-carbon n-3 FA on canine plasma and neutrophil membrane fatty acid composition, superoxide and leukotriene B₄ and B₅ production when fed at the same n-6:n-3 fatty acid ratio was investigated. Four groups of ten dogs each were fed a low fat basal diet supplemented with safflower oil (SFO), beef tallow (BTO), linseed oil (LSO), or Menhaden fish oil (MHO) for 28 days. Dietary fat provided 40.8% of energy and the n-6:n-3 of the diets were ~100:1, 9.7:1, 0.38:1, and 0.34:1 for the SFO, BTO, LSO and MHO groups, respectively. The MHO and LSO groups had increased incorporation of EPA and DPA in both the plasma and neutrophil membranes compared to the BTO and SFO groups. DHA was observed in the MHO but not in the LSO group. Neutrophils from the MHO diet fed dogs had less LTB₄ and greater LTB₅ than the other three groups. The LSO group also showed a reduction in LTB₄ and greater LTB₅ production compared to the SFO and BTO groups. Both LSO and MHO groups had lower superoxide production compared to the SFO and BTO groups. Diets containing 18 or 20/22 carbon n-3 FA fed at the same n-6:n-3 resulted in differential incorporation of long chain n-3 FA into neutrophil membranes. Thus, fatty acid type and chain length individually affect neutrophil membrane structure and function and these effects exist independent of dietary total n-6:total n-3 FA ratios.

The proinflammatory leukotriene B₄ (LTB₄) may be of importance in the progression of chronic kidney disease (CKD). We investigated whether n-3 polyunsaturated fatty acids (PUFA) decrease LTB₄ and increase the formation of the less inflammatory leukotriene B₅ (LTB₅) in patients with CKD. Fifty-six patients with CKD stage 2-5 were randomised to 2.4 g n-3 PUFA or olive oil for 8 weeks. Compared to controls, n-3 PUFA significantly decreased release of LTB₄ (p<0.001) and 5-hydroxyeicosatetraenoic acid (5-HETE) (p<0.01) and significantly increased release of LTB₅ (p<0.001) and 5-hydroxyeicosapentaenoic acid (5-HEPE) (p<0.001) from stimulated neutrophil granulocytes. Kidney function evaluated by creatinine clearance and proteinuria did not improve. In conclusion, n-3 PUFA supplementation for 8 weeks in patients with CKD stage 2-5 significantly decreased LTB₄ and 5-HETE and significantly increased LTB₅ and 5-HEPE. No effect was seen on kidney function., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The modulatory activity of dietary n-3 fatty acids on inflammation and immune response in domestic cats is unknown. Mature female cats (n=14/treatment) were fed control, fish oil or flaxseed oil diets with n-6:n-3 fatty acid ratios of 20:1, 5:1 and 5:1, respectively, for 12 wk. Immune response was assessed on wk 0, 6 and 12, and skin hypersensitivity response on wk 6 and 12. Fish oil increased (P<0.01) eicosapentaenoic and docosahexaenoic acids in plasma and skin, whereas flaxseed oil increased α-linolenic acid. Fish and flaxseed oils decreased (P<0.01) skin inflammatory response to histamine. Cats fed fish but not flaxseed oil had higher (P<0.05) skin leukotriene LTB(5), but not LTB(4). Fish and flaxseed oils lowered B, total T and T(h) subset populations, and leukocyte proliferative response to PWM (P<0.05). In contrast, there was no change in ConA- or PHA-induced lymphocyte proliferation, Tc and MHC II cell populations, DTH response, NK cytotoxicity, IL-2 production, or plasma IgG concentrations. Therefore, fish and flaxseed oil can reduce skin inflammatory responses in cats, however, flaxseed oil appears less immunosuppressive than fish oil., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: This study investigated the effects of parenterally administered fish oil (FO) on the fatty acid composition in rats to determine the optimal omega-6:omega-3 polyunsaturated fatty acid (PUFA) ratio of fat emulsions to achieve an anti-inflammatory effect., Methods: Male Sprague-Dawley rats were infused a parenteral nutrition (PN) solution containing fat emulsions with different omega-6:omega-3 PUFA ratios. The fatty acid content of phospholipids in the membranes of splenocytes was analyzed by gas chromatography (experiment 1). In addition, the amounts of leukotriene (LT) B(4) and LTB(5) released from peritoneal polymorphonuclear leukocytes (PMNs) were measured by high-performance liquid chromatography (experiment 2)., Results: In experiment 1, after infusion of the fat emulsion containing FO, the omega-3 PUFA content in cell membranes rose to 70% of the peak value on day 1 and nearly reached a plateau on day 3. The highest ratio of eicosapentaenoic acid (EPA) to arachidonic acid (AA) was achieved by administering a PN solution with the smallest omega-6:omega-3 PUFA ratio. In experiment 2, a larger amount of LTB(5) was released from Ca-ionophore-stimulated PMNs taken from rats given a larger quantity of FO. The ratio of LTB(5):LTB(4) released from PMNs correlated positively with the EPA:AA ratio in the membranous phospholipid and in serum., Conclusions: The omega-3 PUFAs were readily incorporated into the cell membrane within 3 days of infusion with the fat emulsion. The EPA:AA ratio in membranous phospholipid in PMNs was positively correlated with the LTB(5):LTB(4) production ratio and was a good indicator of anti-inflammatory effects.

The beneficial effects of fish oil on inflammation have been attributed to the content of eicosapentaenoic (EPA)/docosahexaenoic acid. EPA is also a substrate for arachidonic acid (AA) cascade enzymes, but it induces the production of alternative eicosanoids such as 3-series prostanoids and 5-series leukotrienes, which are considered to be less proinflammatory than AA metabolites. However, the molecular basis of this action is poorly understood. In this study, we compared the effects of prostaglandin (PG) E(2) and PGE(3) on endothelium permeability, and the effects of leukotriene (LT) B(4) and LTB(5) on endothelium permeability and mononuclear adhesion and migration. In our study, both prostaglandins increased trans-endothelial Evans blue-albumin (EBA) permeability in a concentration-dependent manner. It is interesting that the effect of PGE(3) was significantly more pronounced than the effect of PGE(2), and both were antagonized by EP(1) and EP(2) antagonists. LTB(4) and LTB(5) had a slight effect on EBA extravasation. However, we observed the enhancement of endothelial permeability in the presence of polymorphonuclear (PMN) cells, probably a consequence of an interplay between leukotriene and prostanoid effects. LTB(4) caused significant increases in the number of PMN cells adhering to endothelial cells, whereas LTB(5) did not induce a significant effect. This effect of LTB(4) appears BLT1 receptor-dependent and was mediated through the enhancement of lymphocyte function-associated antigen-1, membrane attack complex-1, E-selectin, and intercellular adhesion molecule-1 expression. Finally, we observed that, unlike LTB(5), which had a weak effect, LTB(4) was a highly potent chemoattractant. An understanding of the differences in the effects of LTB(4)/LTB(5) on PMN cell adhesion and migration may help to explain the beneficial impact of omega-3 fatty acids in inflammatory processes.

Chronic inflammation has long been associated with neoplastic progression. Our group had recently shown that the addition of a large number of apoptotic tumor cells to the tumor microenvironment induces a potent acute inflammatory reaction capable of promoting melanoma growth; however, primarily necrotizing cells do not cause such a reaction. Here, we show that potent inflammatory agents, such as lipopolysaccharide (LPS) and carrageenan, also promote growth of subtumorigenic doses of melanoma cells, having no effect on melanoma proliferation in vitro. Inhibition of 5-lipoxygenase (5-LOX) seems to have a pivotal role in this model because caffeic acid and MK886, a FLAP (5-LOX-activating protein) inhibitor, partially hindered tumor growth induced by apoptotic cells or LPS. Other enzymes of the arachidonic acid pathway, cyclooxygenase-1 and cyclooxygenase-2, seem to have no participation in this tumor promoter effect, as the inhibitor of both enzymes (indomethacin) did not alter melanoma growth. Leukotriene B4 (LTB4), the main product of the 5-LOX pathway, was able to induce growth of subtumorigenic inocula of melanoma cells, and a LTB4 receptor antagonist inhibited acute inflammation-associated tumor growth. Addition to the tumor inflammatory microenvironment of eicosapentaenoic acid, an omega3-polyunsaturated fatty acid with anti-inflammatory properties, or leukotriene B5, an eicosapentaenoic acid-derived leukotriene, significantly inhibited tumor development. These results give new insights to the mechanisms through which inflammation may contribute to tumor progression and suggest that LOX has an important role in tumor progression associated with an inflammatory state in the presence of apoptosis, which may be a consideration for apoptosis-inducing treatments, such as chemotherapy and radiotherapy.

n-3 PUFA supplementation helps in the prevention or treatment of inflammatory diseases and CVD. However, many supplementations reported sofar are either a combination of n-3 PUFA or used large daily amounts of n-3 PUFA dosages. The present study investigated the influence of increasing dose intake of DHA on the fatty acid composition of phospholipids in neutrophils and on their capability to produce leukotrienes(LT) B4 and B5 in vitro. Twelve healthy volunteers were supplemented with increasing daily doses of DHA (200, 400, 800 and 1600 mg, each dose in TAG containing DHA as the only PUFA and for a 2-week period). At the end of each supplementation period, neutrophil fatty acid composition,and LTB4 and LTB5 production were determined by GC and liquid chromatography-tandem MS, respectively. The DHA/arachidonic acid ratio increased in a dose-dependent manner with respect to the increasing doses of DHA supplementation and was significantly different from baseline after supplementation with either 400, 800 or 1600 mg DHA. The LTB5/LTB4 ratio was significantly increased compared to baseline after supplementation with 800 and 1600 mg DHA. LTB5/LTB4 and DHA/arachidonic acid ratios were correlated (r 0.531, P<0.0001). The present data suggest that both changes in neutrophil lipid composition and LT production occurred with daily supplementation with 800 and 1600 mg DHA. The clinical benefits associated with these doses of DHA in inflammatory diseases remain to be investigated.

We have developed a method for measuring leukotriene B4 glucuronide, a marker of systemic leukotriene B4 biosynthesis, in human urine. This method involves the separation of two positional isomers of leukotriene B4 glucuronide by high-performance liquid chromatography, followed by hydrolysis with beta-glucuronidase and then leukotriene B4 quantification by enzyme immunoassay after purification by high-performance liquid chromatography. One of two positional isomers of leukotriene B4 glucuronide was predominantly present in urine. The concentration of the isomer increased in urine from aspirin-intolerant asthma patients after aspirin challenge. Urinary leukotriene E4 and leukotriene B4 glucuronide concentrations in 13 normal healthy adults were 94.6 pg/mg-creatinine (median) and 22.3 pg/mg-creatinine, respectively. Urinary LTE4 concentration increased during the first 3h after allergen inhalation in atopic patients. However, allergen-induced bronchoconstriction was not associated with an increased concentration of LTB4 glucuronide in urine. The method enabled us to precisely determine urinary leukotriene B4 glucuronide concentration.

The effect of feeding different amounts of n-6 and n-3 fatty acids (FA) to hens on immune tissue FA composition and leukotriene production of hatched chicks was investigated. Hens were fed diets supplemented with either 3.0% sunflower oil (Diet I), 1.5% sunflower+1.5% fish oil (Diet II), or 3.0% fish oil (Diet III) for 46 days. The hatched chicks were fed a diet containing C18:3n-3, but devoid of longer chain n-6 and n-3 FA, for 21 days. Spleen docosahexaenoic acid (DHA) content was higher in chicks from hens fed Diet III (P<0.05). The bursa content of arachidonic acid was lower in chicks hatched from hens fed Diet III (P<0.05), and the ratio of n-6 to n-3 FA was significantly higher in bursa of chicks hatched to hens fed Diet I (P<0.05). Eicosapentaenoic acid (EPA) and DHA contents were higher in bursa of chicks hatched from hens fed Diet III (P<0.05). Thrombocytes from chicks hatched to hens fed Diet III produced the most leukotriene B(5) (LTB(5)). The ratio of LTB(5) to LTB(4) concentrations was also highest (P<0.05) in chicks hatched to hens fed Diet III. These results indicate that modulating maternal dietary n-6 and n-3 FA may alter leukotriene production in chicks, which could lead to less inflammatory-related disorders in poultry.

Background and Aims: Various anti-inflammatory therapies, including dietary omega-3 polyunsaturated fatty acids (PUFA) supplementation, have been investigated in cystic fibrosis (CF) patients. To further explore this nutritional approach, biological effects of an omega-3 PUFA oral liquid supplementation were measured in 17 CF patients in a double-blind, randomized, crossover without a washout period and placebo-controlled study., Methods: CF patients (age: 18+/-9 year; weight: 43+/-13 kg) received a liquid dietary supplementation either enriched or not in omega-3 PUFA (390-1170 mg/day according to patient weight) during two 6-month periods., Results: Increase in eicosapentaenoic acid was observed in neutrophil membrane following omega-3 PUFA dietary supplementation (from 0.7+/-0.6 to 1.6+/-0.6 micromol%, P<0.01). The leukotriene B(4) (LTB(4))/leukotriene B(5) (LTB(5)) ratio was decreased (from 72+/-27 to 24+/-7, P<0.001) in CF patients taking omega-3 PUFA supplements. In contrast, omega-3 PUFA supplementation affected neither internalization of IL-8 receptors following IL-8 exposure, nor IL-8-induced neutrophil chemotaxis., Conclusion: Our results show that omega-3 PUFA are incorporated in neutrophil membranes. The subsequent decrease in LTB(4)/LTB(5) ratio suggests that, in such conditions, neutrophils may produce less pro-inflammatory mediators from the acid arachidonic pathway. These data indicate that omega-3 PUFA intake may have anti-inflammatory effect that still need to be assessed by long-term studies following large groups of patients.

The aim of this study was to investigate the interaction of a series of novel compounds with leukotriene B(4) receptors (BLT) and vanilloid receptor (TRPV1). First, we characterized leukotriene B(4) (LTB(4)) ethanolamide. In guinea pig isolated lung parenchyma, LTB(4) ethanolamide antagonized the contractile action of LTB(4) with an apparent K(B) value of 7.28 nM. Using a Boyden chamber assay, we demonstrated that this compound stimulated human neutrophil migration in a similar manner to LTB(4) but with lower efficacy. In rat TRPV1 (rTRPV1)-expressing Chinese hamster ovary (CHO) cells and dorsal root ganglion (DRG) neurons, LTB(4) and LTB(4) ethanolamide acted as low-efficacy agonists, increasing intracellular calcium concentration ([Ca(2+)](i)) in a capsazepine-sensitive manner. These results prompted us to hypothesize that a molecule may possess pharmacophores such that it is capable of dual antagonism of BLT and TRPV1 receptors. Two novel compounds, N-[2-fluoro-4-[3-(11 hydroxyheptadec-8-enyl)-thioureiomethyl]-phenyl]-methanesulfonamide (O-3367) and N-[4-[3-(11 hydroxyheptadec-8-enyl)-thioureio-methyl]-phenyl]-methanesulfonamide (O-3383), were synthesized. In human neutrophils, both compounds acted as antagonists, significantly attenuating the BLT receptor-mediated ability of LTB(4) to induce migration, with pIC(50) values of 7.22 +/- 0.17 and 5.95 +/- 0.16, respectively. In rTRPV1-expressing CHO cells, they caused a significant rightward shift in the log concentration-response curve for the TRPV1 receptor agonist capsaicin (3-methoxy-4-hydroxy)benzyl-8-methyl-6-nonenamide). In DRG neurons O-3367 significantly attenuated the capsaicin-induced increases in [Ca(2+)](i) with a pIC(50) value of 5.94 +/- 0.004. O-3367 and O-3383 represent novel structural templates for generating compounds possessing dual antagonism at BLT and TRPV1 receptors. In view of the crucial role of both TRPV1 and BLT receptors in the pathophysiology of inflammatory conditions, such compounds may betoken a novel class of highly effective therapeutics.

Background: Previous research has demonstrated that fish oil supplementation has a protective effect on exercise-induced bronchoconstriction (EIB) in elite athletes, which may be attributed to its antiinflammatory properties. Since EIB in asthma involves proinflammatory mediator release, it is feasible that fish oil supplementation may reduce the severity of EIB in asthmatic subjects., Study Objectives: To determine the efficacy of fish oil supplementation on severity of EIB in subjects with asthma., Design: Randomized, double-blind, crossover study., Setting: Lung function and exercise testing in a university research laboratory., Patients and Measurements: Sixteen asthmatic patients with documented EIB entered the study on their normal diet and then received either fish oil capsules containing 3.2 g of eicosapentaenoic acid and 2.0 g of docohexaenoic acid (fish oil diet, n = 8) or placebo capsules (placebo diet, n = 8) daily for 3 weeks. At the beginning of the study (normal diet) and at the end of each treatment phase, the following pre-exercise and postexercise measures were assessed: (1) pulmonary function; (2) induced sputum differential cell count percentage and proinflammatory eicosanoid metabolite (leukotriene C4 [LTC4]-leukotriene E4 [LTE4] and prostaglandin D2 [PGD2]) and cytokine (interleukin [IL]-1beta and tumor necrosis factor [TNF]-alpha) concentrations; and (3) eicosanoid metabolites leukotriene B4 (LTB4) and leukotriene B5 (LTB(5)) generation from activated polymorphonuclear leukocytes (PMNLs)., Results: On the normal and placebo diet, subjects exhibited EIB. However, the fish oil diet improved pulmonary function to below the diagnostic EIB threshold, with a concurrent reduction in bronchodilator use. Induced sputum differential cell count percentage and concentrations of LTC4-LTE4, PGD2, IL-1beta, and TNF-alpha were significantly reduced before and following exercise on the fish oil diet compared to the normal and placebo diets. There was a significant reduction in LTB4 and a significant increase in LTB5 generation from activated PMNLs on the fish oil diet compared to the normal and placebo diets., Conclusion: Our data suggest that fish oil supplementation may represent a potentially beneficial nonpharmacologic intervention for asthmatic subjects with EIB.

The study objective was to determine the effect of feeding food enriched in (n-3) fatty acids (FA) on plasma FA profiles and leukotriene B (LTB) synthesis by stimulated peripheral blood neutrophils from dogs. For 36 weeks, two groups of dogs (n = 5) were fed food that contained either a low ratio of (n-6)-(n-3) FA (1.31:1; fish oil-enriched food) or a high ratio of (n - 6)-(n-3) FA (40.6:1; corn oil-enriched food). Consumption of food enriched in fish oil resulted in higher plasma concentrations of eicosapentaenoic acid and docosahexaenoic acid and lower concentrations of arachidonic acid. Neutrophils from dogs fed fish oil-enriched food produced 7.6-fold more LTB(5)(P = 0.002), and the ratio of LTB(5)-LTB(4) concentrations was 8.3-fold higher (P < 0.001) compared with dogs fed corn oil-enriched food. Dietary FA can modulate leukotriene production by neutrophils in dogs, and suggests that foods enriched in (n-3) FA from fish oil may have value in the treatment of canine inflammatory diseases.

A validated high-performance liquid chromatography (HPLC)-mass spectrometry method has been developed for the simultaneous assay of leukotrienes (LTs) B4 and B5, derived from omega-6 arachidonic acid and omega-3 polyunsaturated fatty acids (PUFA), respectively, produced by human polymorphonuclear leukocytes (PMNLs) stimulated with calcium ionophore A23187. The HPLC separation of PMNL ether extracts was performed on a reversed-phase column using a gradient elution program of 15 mM ammonium acetate and MeOH. Detection was performed by electrospray ionization-single quadripole mass spectrometry using single ion reaction monitoring in the negative mode at m/z 333.3 [M-H](-) and m/z 335.2 for prostaglandin B2/LTB5 and LTB4, respectively. The calibration curves for LTB4 and LTB5 were linear over the ranges 165-990 and 0.825-13.2 ng/ml, respectively. The lower limit of quantification for LTB5 was 0.66 ng/ml. The mean absolute recoveries for LTB4 and LTB5 were 81+/-4.8% and 82+/-5.9%, respectively. The method is precise with mean interday CVs for LTB4 and LTB5 within 7.1-10.7, and 3.8-9.4%, respectively, and accurate (range of interday deviations for LTB4 and LTB5 were -7.8 to 1, and -5 to 9% , respectively). The method has been validated and is being applied to the simultaneous quantification of the leukotrienes B4 and B5 in stimulated PMNLs in a clinical protocol studying the influence of a diet enriched in omega-3 PUFA on various surrogate markers of inflammation in young cystic fibrosis patients.

Background: Aspirin challenge of aspirin-intolerant asthma (AIA) patients causes a significant increase in leukotriene E4 (LTE4) concentration in urine. However, knowledge on leukotriene B4 (LTB4) generation in patients with AIA is insufficient. Recent research has demonstrated that exogenously administered LTB4 is excreted as glucuronide into the urine in human healthy subjects., Objective: The purpose of this study is to estimate urinary LTB4 glucuronide (LTBG) concentration in the clinically stable condition in healthy subjects and asthmatic patients and to investigate changes in urinary LTBG concentration in patients with AIA after aspirin challenge., Methods: A provocation test was performed by intravenous aspirin challenge. After urine was hydrolysed by beta-glucuronidase, the fraction containing LTB4 was purified by high-performance liquid chromatography and LTB4 concentration was quantified by enzyme immunoassay. Urinary LTBG concentration was calculated as the difference between the concentration obtained with hydrolysis and that without hydrolysis., Results: (1) After hydrolysis, the presence of urinary LTB4 was verified by gas chromatography-mass spectrometry-selected ion monitoring. (2) The urinary LTBG concentration was significantly higher in the asthmatic patients than in the healthy subjects (median, 5.37 pg/mg creatinine [range 1.2-13] vs. 3.32 pg/mg creatinine [range, 0.14-10.5], P = 0.0159). (3) The patients with AIA (n = 7), but not those with aspirin-tolerant asthma (n = 6), showed significant increases in LTBG and LTE4 excretions after aspirin challenge. (4) When the concentrations after aspirin challenge were analysed simultaneously, a significant linear correlation was observed between urinary LTBG concentration and urinary LTE4 concentration in patients with AIA (Spearman's rank correlation test, r = 0.817, P = 0.0003)., Conclusion: LTBG is present in human urine, albeit at a concentration lower than urinary LTE4. In addition to a marked increase in cysteinyl-leukotriene production, aspirin challenge induced LTB4 production in AIA patients.

Systemic hypoxia produces a rapid microvascular inflammatory response characterized by increased reactive oxygen species (ROS) levels, leukocyte-endothelial adherence and emigration, and increased vascular permeability. The lipid inflammatory mediator leukotriene B(4) (LTB(4)) is involved in the early hypoxia-induced responses (ROS generation and leukocyte adherence). Whether other lipid inflammatory mediators participate in this phenomenon is not known. The objective of these experiments was to study the role of platelet-activating factor (PAF) in the microvascular inflammatory response to hypoxia and its potential interactions with LTB(4) in this response. Intravital microscopy was used to examine mesenteric venules of anesthetized rats. We found that WEB-2086, a PAF receptor antagonist, completely prevented the increase in ROS levels and leukocyte adherence during a brief reduction in inspired Po(2) to anesthetized rats; administration of either WEB-2086 or the LTB(4) antagonist LTB(4)-DMA attenuated leukocyte emigration and the increase in vascular permeability to the same extent during prolonged systemic hypoxia in conscious rats. Furthermore, no additive effect was observed in either response when both antagonists were administered simultaneously. This study demonstrates a role for PAF in the rapid microvascular inflammatory response to hypoxia, as well as contributions of PAF and LTB(4) to the slowly developing responses observed during sustained hypoxia. The incomplete blockade of the hypoxia-induced increases in vascular permeability and leukocyte emigration by combined administration of both antagonists indicates that factors in addition to LTB(4) and PAF participate in these phenomena.

Acute systemic hypoxia produces rapid leukocyte adherence in the rat mesenteric microcirculation, although the underlying mechanisms are not fully known. Hypoxia is known to increase reactive oxygen species (ROS) generation, which could result in formation of the lipid inflammatory mediator leukotriene B(4) (LTB(4)). The goal of this study was to examine the role of LTB(4) in hypoxia-induced microvascular alterations. Using intravital microscopy, we determined the effect of the LTB(4) antagonist, LTB(4)-dimethyl amide (LTB(4)-DMA), on ROS generation and leukocyte adherence in mesenteric venules during hypoxia. Exogenous LTB(4) increased ROS generation to 144 +/- 8% compared with control values and also promoted leukocyte adherence. These responses to LTB(4) were blocked by pretreating the mesentery with LTB(4)-DMA. Leukopenia did not significantly attenuate the LTB(4)-induced increase in ROS generation (142 +/- 12.1%). LTB(4)-DMA substantially, though not completely, reduced hypoxia-induced ROS generation from 66 +/- 18% to 11 +/- 4% above control values. Hypoxia-induced leukocyte adherence was significantly attenuated by LTB(4)-DMA. Our results support a role for LTB(4) in the mechanism of hypoxia-induced ROS generation and leukocyte adherence in the rat mesenteric microcirculation.

Sjögren-Larsson syndrome (SLS) is an autosomal recessively inherited neurocutaneous disorder caused by a deficiency of the microsomal enzyme fatty aldehyde dehydrogenase (FALDH). We report the clinical characteristics and the results of molecular studies in 19 SLS patients. Patients 1-17 show the classical triad of severe clinical abnormalities including ichthyosis, mental retardation and spasticity. Most patients were born preterm, and all patients exhibit ocular abnormalities and pruritus. Electro-encephalography shows a slow background activity, without other abnormalities. MRI of the brain shows an arrest of myelination, periventricular signal abnormalities of white matter and mild ventricular enlargement. Cerebral (1)H-MR spectroscopy reveals a characteristic, abnormal lipid peak. The degree of white matter abnormality in the MRIs and the height of the lipid peak in (1)H-MR spectra do not correlate with the severity of the neurological signs. The clinical presentation and the clinical course is strikingly similar in these patients. Patient 18 shows a mild phenotype that essentially contains the same, but less severe, clinical features. Patient 19 exhibits the typical, but very mild, dermatological and ocular abnormalities, without any clinical neurological involvement. The diagnosis of SLS was confirmed by demonstration of the enzyme defect in cultured skin fibroblasts. Furthermore, as might be predicted from the essential role of FALDH in leucotriene B(4) (LTB(4)) metabolism, elevated urinary concentrations of LTB(4) and 20-OH-LTB(4) were found in all patients studied. Molecular studies of the FALDH gene revealed eight different mutations, including three new ones: a large 26-base pair deletion (21-46del), a missense mutation (80C-->T) and an insertion mutation (487-488insA). The vast majority of SLS patients seem to be severely affected independent of their genotype.

The effect of a combination of aspirin and fish oil on eicosanoids was studied. Four subjects were given 37.5 mg aspirin orally, and 6 weeks later they received a natural, stable fish oil daily for 1 week before taking the same single dose of aspirin. Blood samples for determination of whole blood production of eicosanoids were taken before and after each experimental period. Serum thromboxane A(2)was decreased by 40% (P<0.05) after aspirin alone, but by 62% (P<0.01) after fish oil + aspirin. Serum prostacyclin (measured as 6-keto PGF(1a)) was decreased by aspirin in both cases. The sum of 6-keto PGF(1a)and its equipotent fish oil-derived analogue Delta(17)-6-keto PGF(1a)was reduced after aspirin intake (55%, NS), but after fish oil + aspirin the reduction was smaller (33%, NS). Leukotriene B(4)was increased by 19% (P<0.05) after aspirin, and decreased by 69% (P<0.05) after fish oil + aspirin. A combination of stable fish oil and aspirin thus improves the eicosanoid pattern more than aspirin alone., (Copyright 2001 Harcourt Publishers Ltd.)

The Sjögren-Larsson Syndrome (SLS) is a neurocutaneous disorder, caused by deficient activity of the microsomal enzyme fatty aldehyde dehydrogenase (FALDH). FALDH catalyzes the oxidation of medium- and long-chain fatty aldehydes to their corresponding carboxylic acids. SLS is diagnosed by demonstrating the enzyme deficiency or by mutation analysis of the FALDH gene, while laboratory investigations of plasma, urine, and cerebrospinal fluid do not reveal any diagnostic abnormality. Leukotriene (LT) B4 is a pro-inflammatory mediator synthesized from arachidonic acid. LTB4 is inactivated by microsomal omega-oxidation, successively yielding 20-OH-LTB4, 20-CHO-LTB4 and 20-COOH-LTB4. Since FALDH is involved in LTB4 degradation, we have analyzed LTB4 and its metabolites in urine and cerebrospinal fluid as well as the degradation capacity for LTB4 in fresh polymorphonuclear leukocytes (PMN) of SLS patients. The urinary concentrations of LTB4, 20-OH-LTB4 and 20-COOH-LTB4 are below the detection limit in healthy controls. The urine of all SLS patients (n=13) exhibited highly elevated concentrations of LTB4 and 20-OH-LTB4, while 20-COOH-LTB4 was absent. Cerebrospinal fluid levels of LTB4, 20-OH-LTB4 and 20-COOH-LTB4 were found to be normal (n=7). PMN isolated from four patients were shown to be unable to convert 20-OH-LTB4 to 20-COOH-LTB4. Our findings provide unambiguous evidence for defective LTB4 degradation in SLS patients, and offer new and non-invasive diagnostic tools. Moreover, they open new pathophysiological considerations, with the prospect of rational treatment strategies.

Sprague-Dawley rats were fed eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) ethyl esters at the 2% level for 3 weeks to clarify their effects on immune functions. In the rats fed EPA or DHA, serum cholesterol, triglyceride, and phospholipid (PL) levels were significantly lower than those in the rats fed safflower oil. In PL fractions of serum, liver, lung, splenocytes, and peritoneal exudate cells (PEC), increases in linoleic and dihomo-gamma-linolenic acid contents and a decrease in arachidonic acid (AA) content were observed in the rats fed EPA or DHA. In addition, the EPA content increased in the rats fed EPA and DHA. In the rats fed EPA or DHA, a decrease of LTB4 productivity and an increase of LTBs productivity were observed in the PEC, in response to the treatment with 5 microM calcium ionophore A23187 for 20 min. The changes in leukotriene production were more marked in EPA-fed rats than in DHA-fed rats. These results suggest that dietary EPA affects lipid metabolism and leukotriene synthesis more strongly than DHA.

Male Wistar rats were given purified diets containing safflower (SAF), perilla (PER), or palm (PAL) oils with or without 1% tea polyphenols (TP) for 3 weeks, and chemical mediator releasing activity from rat peritoneal exudate cells (PEC) was measured. Histamine releasing activity was not influenced by TP, while histamine release and intracellular histamine content were significantly increased in the PAL-fed group. On the contrary, leukotriene B4 (LTB4) release was significantly lower in rats fed PER than in those fed SAF and PAL, and TP significantly decreased the release in all fat groups. TP also significantly inhibited the release of LTB5, which was generated only in rats fed PER. TP significantly decreased the proportion of arachidonic acid (AA) in PEC in the SAF-fed group and that of eicosapentaenoic acid (EPA), the precursor of LTB5 in the PER-fed group, but did not influence that of AA in the PAL- and PER-fed group. These results suggest that ingestion of TP improves type I allergic symptom through the inhibition of LT release though the inhibition by TP could not be totally explained by the reduction of substrate fatty acid.

Synthesis of 10,11-dihydro-, 10,11,14,15-tetrahydro-, and 10, 11-dihydro-12-oxoleukotriene B(4) compounds (2, 4, 5) was accomplished stereoselectively by using the nickel-catalyzed coupling reaction illustrated in Scheme 1. The C(1)-C(7) fragments, TBS ether 10a for 2 and 4 and ethyoxyethyl (EE) ether 10b for 5, were prepared in enantiomerically pure forms (>99% ee) by a modified literature procedure (ref 11a). On the other hand, boronate esters 11a and 11b, which correspond to the C(8)-C(20) parts of 2 and 4, respectively, were synthesized from (R)-epichlorohydrin (18) of 99% ee. Briefly, 18 was converted into acetylenes 24 and 32 through epoxide ring-opening with LiC triple bond CC(5)H(11)/BF(3).OEt(2) or C(7)H(15)MgBr/CuCN. Hydroboration of these acetylenes with (+)-(Ipc)(2)BH followed by reaction with MeCHO afforded the corresponding diethyl boronates, which upon ligand exchange with Me(2)C(CH(2)OH)(2) furnished boronate esters 11a and 11b in 75% and 77% yields, respectively. In a similar manner, racemic boronate ester rac-11a, an intermediate for synthesis of 5, was prepared from racemic epichlorohydrin. For synthesis of 2, borate 25 was generated from 11a (1.5 equiv) and MeLi (1.6 equiv). Without isolation, 25 was submitted to reaction with 10a (1 equiv) in the presence of a Ni(0) species at room temperature overnight to afford 26, which upon treatment with TBAF furnished 2 in 64% yield from 10a. Similarly, 11b and 10a furnished 4 in good yield. To synthesize 5, rac-11a and EE ether 10b were joined by the coupling reaction to produce 39, which was transformed into 40 by desilylation with TBAF. After hydrolysis of 40, oxidation with PDC followed by deprotection of the EE group furnished 5 in 36% yield from 40. In addition, 2 was converted into amide 3 in 92% yield.

The peroxisome proliferator activated receptors (PPARs) represent a group of ligand-activated transcription factors that mediate the biological effects of various drugs, such as fibrates and thiazolidinediones. Three PPAR subtypes can be distinguished, alpha, beta and gamma, each of which has an unique pattern of expression among vertebrate tissues. The PPAR alpha receptor is activated by hypolipidemic drugs of the fibrate class, and regulates the expression of numerous genes involved in fatty acid catabolism. The PPAR gamma receptor is activated by hypoglycaemic drugs of the thiazolidinedione class, and is an important determinant of adipocyte differentiation. Little is currently known about PPAR beta. A heavy research effort is currently directed towards the identification of novel high-affinity, high-specificity agonists and antagonists that may be used in the treatment of hyperglycaemia, hyperlipidemia, and other diseases of metabolic origin.

The human 15-lipoxygenase (15-LO) gene was transfected into rat kidneys in vivo via intra-renal arterial injection. Three days later, acute (passive) or accelerated forms of antiglomerular basement membrane antibody-mediated glomerulonephritis were induced in transfected and nontransfected or sham-transfected controls. Studies of glomerular functions (filtration and protein excretion) and ex vivo glomerular leukotriene B(4) biosynthesis at 3 hr, and up to 4 days, after induction of nephritis revealed preservation or normalization of these parameters in transfected kidneys that expressed human 15-LO mRNA and mature protein, but not in contralateral control kidneys or sham-transfected animals. The results provide in vivo-derived data supporting a direct anti-inflammatory role for 15-LO during immune-mediated tissue injury.

The cDNA for leukotriene B(4) (LTB(4)) receptor (BLT) was cloned from a guinea-pig leucocyte cDNA library. The cloned receptor cDNA encodes 348 amino acid residues and shares 73% identity with the amino acid sequence of human BLT. Northern blot analysis showed the highest expression of the receptor mRNA in leucocytes, followed by lung and spleen. The membrane fractions of HEK-293 and Cos-7 cells transfected with the cDNA showed specific LTB(4)-binding activities, with K(d) values of 0.27 and 0.17 nM respectively. Xenopus laevis oocytes injected with the cRNA of guinea-pig BLT showed LTB(4)-induced Cl(-) currents, indicating that the cloned receptor is functional. LTB(4) is metabolized to 20-hydroxy-LTB(4) and then to 20-carboxy-LTB(4), a transformation considered as a major inactivation pathway of the compound. Using the cloned receptor, we analysed the agonistic effects of LTB(4) and these two metabolites. 20-Carboxy-LTB(4) is a much weaker agonist, with a K(d) value higher than that of LTB(4) by three orders of magnitude, corresponding to a much weaker chemotactic activity. Although 20-hydroxy-LTB(4) is as potent as LTB(4) in inhibiting [(3)H]LTB(4) binding and cAMP formation, it is less potent than LTB(4) in the mobilization of intracellular Ca(2+) and the chemotaxis of Chinese hamster ovary cells expressing the guinea-pig BLT. The present study demonstrated that although LTB(4) and 20-hydroxy-LTB(4) bind to the receptor with similar affinities, they do differ in activating intracellular signalling.

Eicosapentaenoic acid (EPA) was used to modulate the activation of alveolar macrophages, to examine its potential anti-inflammatory effect in addition to its anti-arteriosclerotic or anti-thrombotic effects. Wistar strain rat alveolar macrophages (2 x 10(6) cell) obtained by bronchoalveolar lavage were preincubated with EPA (0-20 microM), and further incubated with 1 mg of silica for 90 min. Leukotriene (LT) B4 and LTB5 of the supernatant were analyzed by reverse phase HPLC. EPA inhibited the production of LTB4 dose-dependently. The production of LTB5, a metabolite from EPA, was increased at low concentrations of EPA (0-10 microM) and decreased at high concentrations (>10 microM). These results suggest that EPA is competitive with arachidonic acid (AA) at low concentrations, and that EPA may inhibit AA metabolism via inhibition of 5-lipoxygenase or phospholipase A2 at high concentrations.

The mouse leukotriene B4 receptor (m-BLTR) gene was cloned. Membrane fractions of human embryonic kidney 293 cells stably expressing m-BLTR demonstrated a high affinity and specific binding for leukotriene B4 (LTB4, Kd = 0.24 +/- 0.03 nM). In competition binding experiments, LTB4 was the most potent competitor (Ki = 0.23 +/- 0.05 nM) followed by 20-hydroxy-LTB4 (Ki = 1.1 +/- 0.2 nM) and by 6-trans-12-epi-LTB4 and LTD4 (Ki > 1 microM). In stably transfected Chinese hamster ovary cells, LTB4 inhibited forskolin-activated cAMP production and induced an increase of intracellular calcium, suggesting that this receptor is coupled to Gi- and Go-like proteins. In Xenopus laevis melanophores transiently expressing m-BLTR, LTB4 induced the aggregation of pigment granules, confirming the inhibition of cAMP production induced by LTB4. BLT receptors share significant sequence homology with chemokine receptors (CCR5 and CXCR4) that act as human immunodeficiency virus (HIV) coreceptors. However, among the 16 HIV/SIV strains tested, the human BLT receptor did not act as a coreceptor for virus entry into CD4-expressing cells based on infection and cell-cell fusion assays. In 5-lipoxygenase-deficient mice, the absence of leukotriene B4 biosynthesis did not detectably alter m-BLT receptor binding in membranes obtained from glycogen-elicited neutrophils. Isolation of the m-BLTR gene will form the basis of future experiments to elucidate the selective role of LTB4, as opposed to cysteinyl-leukotrienes, in murine models of inflammation.

The eicosanoid generating potential of tunic, branchial basket, intestine, ovary and tadpole larvae from the sea squirt, Ciona intestinalis, was examined using a combination of reverse phase high performance liquid chromatography, gas chromatography-mass spectrometry and enzyme immunoassay. All organs examined synthesized the lipoxygenase products 12-hydroxyeicosapentaenoic acid (12-HEPE) and 8-HEPE implying that both 8- and 12-lipoxygenase activity are widely distributed in this species. In addition, tunic and branchial basket generated significant amounts of 8,15-diHEPE and smaller amounts of 8,15-dihydroxyeicosatetraenoic acid (8,15-diHETE), while tunic alone generated small amounts of conjugated tetraene-containing material with a UV chromophore and mass ion characteristic of a lipoxin-like compound. The broad range lipoxygenase inhibitors, esculetin and nordihydroguaiaretic acid, both caused a significant dose dependent inhibition of 12-HEPE and 8,15-diHEPE biosynthesis in tunic, while the specific 5-lipoxygenase inhibitor, REV-5901, and the specific 5-lipoxygenase activating protein inhibitor, MK-866, had no observable effect on the lipoxygenase profile of this tissue. Tunic, branchial basket, intestine and ovary all generated significant amounts of prostaglandin (PG) E and PGF immunoreactive material and smaller amounts of thromboxane B immunoreactive material as measured by enzyme immunoassay. The non-specific cyclooxygenase (COX) inhibitor, indomethacin, the selective COX-1 inhibitors, resveratrol and valerylsalicylate, and the specific COX-2 inhibitors, NS-398, etolodac and DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl) phenyl-2(5H)-furanone) all caused a significant dose dependent inhibition of the biosynthesis of PGE immunoreactive material. However, the specific COX-2 inhibitors were most effective, perhaps implying that a COX-2-like enzyme may be present in this species.

The dietary effect of fish oils (FOs) rich in eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) on the immune function of Sprague-Dawley rats was compared with that of safflower oil. After 3 weeks of feeding at the 10% level of a dietary fat, the IgG and IgM production by splenocytes and IgG production by mesenteric lymph node (MLN) lymphocytes were significantly higher in the FO-fed rats, while no significant difference was found in IgA or IgE productivity by both the spleen and MLN lymphocytes. In the FO-fed rats, peritoneal exudate cells released a lower amount of LTB4, reflecting their lower arachidonic acid level, and a higher amount of LTB5, reflecting their higher EPA level in phospholipids. On these EPA-rich FO exerted a stronger effect than DHA-rich FO immune functions.

Leukotriene B4 (LTB4), an arachidonic acid derivative, is a potent proinflammatory agent whose actions are terminated by catabolism via a microsomal omega-hydroxylation pathway. Although the liver serves as the principal site for LTB4 clearance from the systemic circulation, the attributes of hepatic LTB4 metabolism are ill defined in humans. Thus, we examined metabolism of LTB4 to its omega-hydroxylated metabolite 20-hydroxyleukotriene B4 (20-OH LTB4) by human liver microsomes and also purified the hepatic P450 enzyme underlying this reaction. Liver microsomes from 10 different subjects converted LTB4 to 20-OH LTB4 at similar rates (1.06 +/- 0.3 nmol/min/nmol P450; 0.25 +/- 0.1 nmol/min/mg protein). Analysis of the microsomal LTB4 20-hydroxylation reaction revealed kinetic parameters (apparent Km of 74.8 microM with a VMAX of 2.42 nmol/min/nmol P450) consistent with catalysis by a single P450 enzyme. Conventional chromatography combined with immunochemical screening with rat CYP4A1 antibodies was then used to isolate a P450 enzyme from human liver microsomes with a molecular weight of 57,000 and an NH2-terminal amino acid sequence 94% homologous (12Trp --> 12Gly) over the first 17 residues with the human CYP4F2 cDNA-derived sequence. Upon reconstitution with P450 reductase and phospholipid, CYP4F2 converted LTB4 to 20-OH LTB4 at a turnover rate of 392 pmol/min/nmol P450, whereas the other human liver P450s tested, including CYP4A11, exhibited neglible LTB4 omega-hydroxylase activity. Polyclonal antibodies to CYP4F2 were found to markedly inhibit (91.9 +/- 5%; n = 5) LTB4 20-hydroxylation by human liver microsomes. Microsomal 20-OH LTB4 formation was also inhibited 30% by arachidonic acid, a known CYP4F2 substrate, and 50% by prostaglandin A1 but was unaffected by lauric acid, palmitic acid, and PGF2alpha. Finally, a strong correlation (r = 0.86; P < 0.002; n = 10) was observed between CYP4F2 content and LTB4 20-hydroxylase activity in the human liver samples. Our results indicate that CYP4F2 is the principle LTB4 omega-hydroxylating enzyme expressed in human liver and, as such, may play an important role in regulating circulating as well as hepatic levels of this powerful proinflammatory eicosanoid., (Copyright 1998 Academic Press.)

The effect of exogenous leukotriene B4 (LTB4) on opsonized zymosan-stimulated human neutrophil formation of 5-lipoxygenase products and arachidonic acid release was directly assessed using reverse-phase HPLC/tandem mass spectrometric methods for quantitation. Stable isotopically labeled LTB4, [1,2-13C2]LTB4, caused a dose-dependent inhibition of LTB4 production in isolated human neutrophils with significant inhibition (60 +/- 7% of control levels) when 0.12 nM [13C2]LTB4 was present. Production of 5-hydroxy-6,8,11,14-eicosatetraenoic acid and release of free arachidonic acid were also dose-dependently inhibited by exogenous LTB4. Metabolites of LTB4, 20-hydroxy-LTB4 and 3(S)-hydroxy-LTB4, also significantly reduced LTB4 production to levels as low as 10 +/- 6% and 10 +/- 7% of control levels, respectively, when present exogenously at 10 nM. Exogenous 5-hydroxy-6,8,11,14-eicosatetraenoic acid at concentrations as high as 10 nM produced no significant reduction in LTB4 biosynthesis during zymosan-stimulated human neutrophil production of LTB4. The inhibitory effect of LTB4 could be partially reversed by the LTB4 receptor antagonist U 75302. Furthermore, an alternative stimulus, N-formyl-methionyl-leucyl-phenylalanine (100 nM), did not inhibit the production of LTB4 in opsonized zymosan-stimulated human neutrophils. These results suggest that activation of the LTB4 receptor on the human neutrophil during phagocytosis limits the ultimate biosynthesis of LTB4. This autocrine effect is opposite to that observed when neutrophils have much of the signal transduction pathways bypassed when stimulated with calcium ionophore A23187 or treated with exogenous free arachidonic acid.

Recombinant human neutrophil leukotriene B4 (LTB4) omega-hydroxylase (cytochrome P450 4F3) has been purified to a specific content of 14. 8 nmol of P450/mg of protein from yeast cells. The purified enzyme was homogenous as judged from the SDS-PAGE, with an apparent molecular weight of 55 kDa. The enzyme catalyzed the omega-hydroxylation of LTB4 with a Km of 0.64 microM and Vmax of 34 nmol/min/nmol of P450 in the presence of rabbit hepatic NADPH-P450 reductase and cytochrome b5. Furthermore, various eicosanoids such as 20-hydroxy-LTB4, 6-trans-LTB4, lipoxin A4, lipoxin B4, 5-HETE and 12-HETE, and 12-hydroxy-stearate and 12-hydroxy-oleate were efficiently omega-hydroxylated, although their Km values were much higher than that of LTB4. In contrast, no activity was detected toward laurate, palmitate, arachidonate, 15-HETE, prostaglandin A1, and prostaglandin E1, all of which are excellent substrates for the CYP4A fatty acid omega-hydroxylases. This is the first time human neutrophil LTB4 omega-hydroxylase has been isolated in a highly purified state and characterized especially with respect to its substrate specificity., (Copyright 1998 Academic Press.)

Structural analogues of leukotriene B4 (LTB4) were designed based on the plausible conformation of LTB4 (1). Joining C-7-C-9 of the conformer A or B into an aromatic ring system led to the discovery of benzene analogues 2, 4 and 6a. Joining C-4-C-9 of the conformer C or D into an aromatic ring system led to the discovery of analogues 3, 5 and 7. The compounds examined in this study were evaluated as to their inhibition of [3H] LTB4 binding to human neutrophils, and by a secondary intact human neutrophil functional assay for agonist/antagonist activity. The first analogues prepared, compounds 2-7, demonstrated moderate potency in the LTB4 receptor binding assay. The modification of these compounds by the introduction of another substituent into the aromatic ring produced a marked increase in receptor binding (28c, IC50 = 0.020 microM; 38c, IC50 = 0.020 microM; 52a, IC50 = 0.020 microM; 52b, IC50 = 0.018 microM). Most of these structural analogues of LTB4 demonstrated agonist activity. Of the analogues prepared in this study, only compound 57 demonstrated weak LTB4 receptor antagonist activity, at 10 microM.

Thirty beagles were placed on diets containing ratios of n-6 to n-3 fatty acids ranging from 5:1 to 100:1 for 12 weeks to determine the effects of these diets on platelet reactivity, coagulation screening assays, and antithrombin III activity. Although small changes were observed in adenosine diphosphate (ADP)-, collagen-, and arachidonic acid-induced platelet aggregation and 14C-serotonin release, fibrinogen concentrations, and antithrombin III activities during the 12-week study, these changes were not of clinical significance and did not correlate with the varying ratios of n-6 to n-3 fatty acids.

A reverse-phase-HPLC/tandem mass spectrometric method (LC/MS/MS) was developed for the quantitation of leukotriene B4 (LTB4), the 5-lipoxygenase product, 5-hydroxyeicosatetraenoic acid (5-HETE), as well as the omega-oxidation metabolites of LTB4, 20-hydroxy-LTB4, and 20-carboxy-LTB4. Electrospray-generated carboxylate anions were collisionally activated and decomposed to specific and abundant product ions and multiple reaction monitoring was used to analyze LTB4 (m/z 335-->195), 20-hydroxy-LTB4 (m/z 351-->195), 20-carboxy-LTB4 (m/z 365-->195), and 5-HETE (m/z 319-->115). A linear correlation was observed in comparison of results obtained from quantitation by LC/MS/MS with quantitation by gas chromatography/MS of the pentafluorobenzyl ester/trimethylsilyl ether derivative of LTB4 produced during opsonized zymosan stimulation of human neutrophils. Detection limits at the low picogram level were obtained for all metabolites. This method was applied to the quantitation of 5-lipoxygenase products produced from zymosan-stimulated human neutrophils in the presence of ethanol. At physiologically relevant concentrations of ethanol, production of all 5-lipoxygenase products generated by stimulated neutrophils was markedly attenuated.

Dual-laser flow cytometry, based on the properties of the DNA-binding dyes Hoechst 33342 and propidium iodide, was used, with light and electron microscopy and DNA fragmentation studies, to define the influence of lipoxygenase-derived eicosanoids on apoptosis of human polymorphonuclear neutrophils (PMN) in vitro. Apoptosis was characterized by progression through an early apoptotic phase characterized by condensation of chromatin and coalescence of nuclear lobes, to a late apoptotic phase characterized by nuclear degradation and evanescence, and secondary necrosis. Prolonged exposure of PMN to leukotriene B4 (LTB4) afforded dose-dependent inhibition of constitutive PMN apoptosis (percentage of normal and apoptotic PMN, respectively, after aging for 18 h: vehicle, 30.5 +/- 2.7% and 61.8 +/- 3.2%; LTB4 10(-7) M, 57.6 +/- 1.2% and 37.6 +/- 1.0%) and apoptosis triggered by the classic peptide chemoattractant FMLP. In contrast, apoptosis was not affected by the LTB4 precursor 5(S)-hydroxyeicosatetraenoic acid (HETE), the omega-oxidation LTB4 metabolites 20-hydroxy-LTB4 and 20-carboxy-LTB4, the cysteinyl leukotriene LTC4, the 15-lipoxygenase product 15(S)-HETE, or the lipoxygenase interaction product lipoxin A4. The anti-apoptotic effect of LTB4 was mimicked by 20,20,20-trifluoro-LTB4, LTB4-dimethylamide, and 14,15-dehydro-LTB4, and was blunted by pertussis toxin and genistein, inhibitors of G alpha i GTP-binding proteins and tyrosine kinases, respectively, but not by staurosporine, 15(S)-HETE, or lipoxin A4. This unique pharmacologic profile suggested that LTB4 attenuated apoptosis through activation of cell surface receptors and signaling events distinct from those involved with PMN trafficking, degranulation, and respiratory bursts.

Both polymorphonuclear (PMN) leukocytes and metabolites of arachidonic acid, especially lipoxygenase products, have been reported to contribute to myocardial damage after coronary artery occlusion and reperfusion. While canine models of myocardial ischemia were used in many of these studies, very little is known about arachidonic acid metabolism by canine PMNs. Moreover, it is unclear whether arachidonic acid metabolites released by canine PMNs affect vascular tone. Therefore, we characterized arachidonic acid metabolism by canine PMNs and determined the effect of these metabolites on vascular tone of isolated canine coronary arteries. Suspensions of canine PMNs were incubated with [14C]arachidonic acid and the calcium ionophore A23187. The incubation media was extracted, and the metabolites resolved by HPLC. 20-Hydroxy-leukotriene B4 (LTB4), 12,20-dihydroxyeicosatetraenoic acid (diHETE), LTB4, 12-hydroxyheptadeclatrienoic acid (HHT), and 12-(S)-hydroxyeicosatetraenoic acid (HETE) were isolated, and their structures confirmed by gas chromatography/mass spectrometry. There was also evidence for the formation of 20-HETE, thromboxane B2 (TXB2), 5-HETE, and several isomers of LTB4. None of the arachidonic acid metabolites that were isolated from incubates of canine PMNs augmented vascular tone, but material migrating with 12,20-diHETE relaxed canine coronary arteries. Authentic 12(S),20-diHETE also produced a concentration-related relaxation of canine coronary artery. 12(R), 20-diHETE was inactive. 20-HETE inhibited A23187-induced PMN aggregation. Thus, arachidonic acid is metabolized in canine PMNs through the cyclooxygenase, lipoxygenases and cytochrome P-450 pathways. Whether these metabolites contribute to myocardial injury remains to be determined.

Leukotriene B4 (LTB4) is metabolized by beta-oxidation, omega-oxidation and the 12-hydroxyeicosanoid dehydrogenase/delta 10-reductase pathway. We have investigated the effects of metabolites formed by the latter pathway on calcium mobilization and migration in human neutrophils and have compared their potencies with those of other LTB4 derivatives. 12-Oxo-LTB4 and 10,11-dihydro-LTB4 were 60 to 100 times less potent than LTB4 in stimulating neutrophils, whereas 10,11-dihydro-12-oxo-LTB4 and 10,11-dihydro-12-epi-LTB4 exhibited still lower potencies. The 6-trans isomers of 12-oxo-LTB4 and 10,11-dihydro-12-oxo-LTB4 were much less potent than the 6-cis compounds. The EC50 values for biologically and chemically (6-cis) synthesized 12-oxo-LTB4 were similar, indicating that the 6,7-double bond is retained in the cis configuration in the biologically formed compound. Methylation of LTB4 markedly reduced its effect on cytosolic calcium levels, whereas addition of a 3-hydroxyl group had a much more modest effect. Modifications of the omega end of the molecule also resulted in lower potencies for calcium mobilization. Nearly all of the compounds tested desensitized neutrophils to LTB4-induced calcium mobilization, which suggests that their effects were mediated by receptors for the latter compound. However, modifications in the carboxyl end of the molecule had smaller effects on desensitization than on calcium mobilization, whereas the reverse was true for modifications in the omega end of the molecule. This suggests that the structural requirements for agonist-induced desensitization to LTB4 may differ to some extent from the requirements for calcium mobilization.

Incubation of leukotriene B4 (LTB4) with Hep G2 cells (a human-derived hepatoma cell line) resulted in the production of several metabolites indicative of alternative pathways of LTB4 metabolism not previously observed in normal hepatocytes. The major extracellular LTB4-derived metabolites were structurally identified using mass spectrometry and ancillary techniques including electrospray ionization. The major metabolite was 10-hydroxy-4,6,8,12-octadecatetraenoic acid (10-HOTE), an unexpected metabolite which lost the hydroxy group at carbon 5 from the parent LTB4. Two other major metabolites were 3(R)-hydroxy-LTB4 and 3(S)-hydroxy-LTB4. The formation of these three metabolites revealed that beta-oxidation from the carboxyl terminus can be a significant metabolic pathway for degradation of this hydroxy unsaturated fatty acid. The normal hepatocyte LTB4-derived metabolite, 20-carboxy-LTB4, was observed as only a minor product. The metabolic profile for Hep G2 cells suggests that the efficient cytochrome P-450 pathway involved in omega-oxidation in typical hepatocytes is absent in these cells. Several minor metabolites were also identified which included dihydro products resulting from metabolism by a 12-hydroxydehydrogenase/delta 10-reductase pathway. The formation of the major metabolite reveals the operation of steps in beta-oxidation of hydroxy, unsaturated fatty acids not anticipated by previously identified steps of fatty acid beta-oxidation.

The effects of 5- and 15-lipoxygenase products, leukotriene B4 (LTB4) and (8R,15S)-dihydroxyeicosa(5E-9,11,13Z)tetraenoic acid (8R,15S-diHETE), on ischemically sensitive abdominal visceral C fiber afferents were evaluated, because this system is important in sensitizing cutaneous afferents. Single-unit activity of abdominal visceral C fiber afferents was recorded from the right thoracic sympathetic chain of anesthetized cats during 5 min of ischemia. Inhibition of 5-lipoxygenase with WY-50295 tromethamine (5 mg/kg iv) augmented the impulse activity from 0.48 +/- 0.15 to 0.79 +/- 0.24 impulses/s (P < 0.05) in seven ischemically sensitive afferents. Conversely, topical application of LTB4 (125 ng) directly onto the receptive field attenuated impulse activity of 10 ischemically sensitive C fiber afferents from 0.82 +/- 0.23 to 0.42 +/- 0.10 impulses/s (P < 0.05). In additional cats, application of 8R,15S-diHETE (125 ng) onto the receptive field augmented the impulse activity of nine ischemically sensitive C fiber afferents (from 0.48 +/- 0.15 to 0.70 +/- 0.15 impulses/s, P < 0.05) and significantly decreased the mechanical threshold of these nine afferents, whereas application of 8S,15S-diHETE (125 ng), a stereoisomer of 8R,15S-diHETE, attenuated the impulse activity from 0.77 +/- 0.48 to 0.45 +/- 0.13 impulses/s (P < 0.05) in six additional ischemically sensitive C fiber afferents. In animals pretreated with aspirin (50 mg/kg iv, n = 6) or 8S,15S-diHETE (125 ng, n = 6), WY-50295 tromethamine (5 mg/kg iv) still potentiated the impulse activity of ischemically sensitive C fiber afferents. These data indicate that 8R,15S-diHETE interacts with stereospecific receptors to sensitize, whereas LTB4 reduces, the response of abdominal visceral afferents to ischemia. Furthermore the data suggest that the augmented response of afferents to abdominal ischemia after inhibition of 5-lipoxygenase is, at least in part, independent of shunting to the cyclooxygenase or 15-lipoxygenase system.

The signal transduction pathway of leukotriene B4 involves phospholipase D activation in cytochalasin B-primed neutrophils, but leukotriene B4 stimulation of increased phosphatidic acid mass in neutrophils has not been demonstrated. Employing the NIH Image program, we have examined the effect of leukotriene B4 on phosphatidic acid mass in human neutrophils incubated with or without cytochalasin B. Our results show that 0.15 microM leukotriene B4 without cytochalasin B was capable of increasing phosphatidic acid mass in neutrophils by 2-fold after 5 s, 2.5-fold after 1 min, and 2-fold after 5 min incubation. Leukotriene B3, leukotriene B4, and leukotriene B5 were equipotent stimuli for phosphatidic acid mass elevation. Leukotriene B4 induced phosphatidylethanol formation at the expense of phosphatidic acid in cells preincubated with 0.25-1% ethanol, indicating phospholipase D activation. Cytochalasin B enhanced leukotriene B4 stimulation of phosphatidic acid mass elevation and phosphatidylethanol formation. There were no measurable changes in 1,2-diglyceride mass after 5 s, but a 1.7-fold increase occurred after 1 min and declined thereafter. Leukotriene B4 stimulation of [3H]glycerol incorporation into phosphatidic acid, diglyceride and phosphatidylinositol was detectable after a 1-min incubation, suggesting increased de novo synthesis of these lipids. These results suggest that leukotriene B4 stimulation of phospholipase D activity contributes to part of the early increased phosphatidic acid mass and that combined actions of stimulated phospholipases C and D, and de novo phosphatidic acid synthesis contribute to the total increased phosphatidic acid mass.