Effect of intravenous infusion of omega-3 and omega-6 lipid emulsions on equine monocyte fatty acid composition and inflammatory mediator production in vitro.

The effect of intravenous administration of lipid emulsions enriched with omega-3 (n3) and omega-6 (n6) fatty acids on equine monocyte phospholipid fatty acid composition and the synthesis of inflammatory mediators in vitro was evaluated. In a randomized crossover design, horses were infused intravenously with 20% lipid emulsions containing n3 or n6 fatty acids. Monocytes were isolated from the horses before and 0 h, 8 h, 24 h, and 7 days after lipid infusion. Monocyte fatty acid analysis demonstrated incorporation of the parenteral n3 and n6 fatty acids in monocyte phospholipids immediately after infusion, with changes in the fatty acid composition persisting for up to 7 days after infusion. In vitro production of the inflammatory mediators thromboxane B2/thromboxane B3 (TXB(2/3)) and tumor necrosis factor-alpha (TNFalpha) by peripheral blood monocytes was diminished by n3 lipid infusion and was unchanged or increased by n6 lipid infusion. The results of this study demonstrate that short-term infusions of n3 and n6 fatty acid-enriched lipid emulsions alter the fatty acid composition of equine monocyte phospholipids and modify the inflammatory response of these cells in vitro. These results also support further investigation into the use of parenteral n3 fatty acids as part of the supportive therapy of patients with multiple organ dysfunction (MODS) or systemic inflammatory response syndrome (SIRS).

Omega-3 fatty acid-based lipid infusion in patients with chronic plaque psoriasis: results of a double-blind, randomized, placebo-controlled, multicenter trial.

BACKGROUND: Profound changes in the metabolism of eicosanoids with increased concentrations of free arachidonic acid (AA) and its proinflammatory metabolites have been observed in psoriatic lesions. Free eicosapentaenoic acid (EPA) may compete with liberated AA and result in an antiinflammatory effect. OBJECTIVE: Our purpose was to assess the efficacy and safety of intravenously administered fish-oil-derived lipid emulsion on chronic plaque-type psoriasis. METHODS: A double-blind, randomized, parallel group study was performed in eight European centers. Eighty-three patients hospitalized for chronic plaque-type psoriasis with a severity score of at least 15 according to the Psoriasis Area and Severity Index (PASI) participated in a 14-day trial. They were randomly allocated to receive daily infusions with either a omega-3 fatty acid-based lipid emulsion (Omegavenous; 200 ml/day with 4.2 gm of both EPA and docosahexaenoic acid (DHA); 43 patients) or a conventional omega-6-lipid emulsion (Lipovenous; EPA+DHA < 0.1 gm/100 ml; 40 patients). The groups were well matched with respect to demographic data and psoriasis-specific medical history. Efficacy of therapy was evaluated by changes in PASI, in an overall assessment of psoriasis by the investigator, and a self-assessment by the patient. In one center neutrophil 4- versus 5-series leukotriene (LT) generation and platelet 2- versus 3- thromboxane generation were investigated and plasma-free fatty acids were determined. RESULTS: The total PASI score decreased by 11.2 +/- 9.8 in the omega-3 group and by 7.5 +/- 8.8 in the omega-6 group (p = 0.048). In addition, the omega-3 group was superior to the omega-6 group with respect to change in severity of psoriasis per body area, change in overall erythema, overall scaling and overall infiltration, as well as change in overall assessment by the investigator and self-assessment by the patient. Response (defined as decrease in total PASI of at least 50% between admission and last value) was seen in 16 of 43 patients (37%) receiving the omega-3 emulsion and 9 of 40 patients (23%) receiving omega-6 fatty acid-based lipid emulsion. No serious side effects were observed. Within the first few days of omega-3 lipid administration, but not in the omega-6 supplemented patients, a manifold increase in plasma-free EPA concentration, neutrophil leukotriene B5 and platelet thromboxane B3 generation occurred. CONCLUSION: Intravenous omega-3-fatty acid administration is effective in the treatment of chronic plaque-type psoriasis. This effect may be related to changes in inflammatory eicosanoid generation.

Differential inhibition of human platelet aggregation and thromboxane A2 formation by L-arginine in vivo and in vitro.

We compared the effects of L-arginine (L-ARG), the precursor of endogenous NO, on platelet aggregation and thromboxane A2 formation in vivo and in vitro. Human platelet-rich plasma (PRP) was anticoagulated with citrate (which decreases extracellular Ca2+) or with recombinant hirudin (which does not affect extracellular Ca2+). Two groups of 10 healthy male volunteers received intravenous infusions of L-ARG (30 g or 6 g, 30 min) or placebo. Blood was collected immediately before and at the end of the infusions for aggregation by ADP or collagen. Infusion of L-ARG inhibited ADP-induced aggregation in PRP anticoagulated with citrate by 37.5+/-6.3% (P < 0.05). In PRP anticoagulated with hirudin, aggregation was inhibited by 33.6+/-16.0% (P < 0.05). L-ARG infusion also inhibited platelet TXB2 formation and slightly, but not significantly decreased the urinary excretion rate of 2,3-dinor-TXB2; cGMP concentrations in PRP were significantly elevated during L-arginine infusion. In vitro preincubation with L-ARG (10 microM-2.5 mM) inhibited platelet aggregation in PRP anticoagulated with rhirudin, but not citrate. This effect was stereospecific for L-arginine, as D-arginine had no effect. It was dependent upon NO synthase activity, as indicated by increased cGMP levels in PRP. Moreover, both the NOS inhibitor L-NMMA and the inhibitor of soluble guanylyl cyclase ODQ antagonized the effects of L-ARG. Haemoglobin, an extracellular scavenger of NO, partly antagonized the antiplatelet effects of L-ARG. 8-Br-cyclic GMP and the exogenous NO donor linsidomine inhibited aggregation in PRP anticoagulated with citrate or r-hirudin. The inhibitory effects of L-ARG on platelet aggregation in vitro were paralleled by increased cyclic GMP levels; L-ARG also inhibited platelet TXB2 formation in PRP anticoagulated with r-hirudin, but not citrate. We conclude that the L-arginine/NO pathway is present in human platelets as a Ca2+-dependent anti-aggregatory pathway. In vivo the formation of NO from L-ARG by endothelial cells may contribute to the platelet-inhibitory effects of L-ARG. NO-releasing compounds like linsidomine inhibit platelet aggregation in vitro independent of extracellular Ca2+.

Microdetermination of the thromboxane B3 gas chromatography-selected-ion monitoring using [18O]thromboxane B3 as an internal standard.

We devised a simple and effective purification for the microdetermination of thromboxane B3 (TXB3), a hydrolysis product of TXA3- [18O2]TXB3 was synthesized by the repeated base-catalyzed hydrolysis of methyl ester derivatives in [18O]water, to obtain an internal standard (IS) for the gas chromatography/selected ion monitoring (GC/SIM) of TXB3. The methyl ester (ME)-methoxime (MO)-dimethylisopropylsilyl (DMIPS) ether derivative was prepared, then GC/SIM was carried out by monitoring the ion at m/z 668 for TXB3 and that at m/z 672 for IS. A good linear response over the range of 10 pg approximately 10 ng was demonstrated. We were able to detect the levels of TXB3 in the medium of human erythroleukemia (HEL) cell cultured with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). This method can be used to determine 3-series thromboxane in biological samples.

Differential influence of arachidonic vs. eicosapentaenoic acid on experimental pulmonary hypertension.

The impact of the 2- and 3-series prostanoid precursors arachidonic acid (AA) and eicosapentaenoic acid (EPA) on experimental pulmonary hypertension was investigated. The model of buffer-perfused rabbit lungs was stimulated by infusion of Escherichia coli hemolysin (HlyA), which is known to provoke sustained thromboxane (Tx)-mediated pulmonary hypertension. Release of di- and trienoic Tx into the recirculating perfusate was quantified by a post-high-performance liquid chromatography enzyme-linked immunosorbent assay technique. HlyA at 0.08 hemolytic unit/ml caused a sustained rise in pulmonary arterial pressure (PAP; maximum increase 14 +/- 2 mmHg) accompanied by progressive TxB2 liberation (maximum perfusate concn 33 +/- 4 pg/ml, baseline < 2 pg/ml). Between 5 and 30 nM, AA provoked a transient monophasic rise in PAP (maximum pressor response 1.5-15 mmHg) and concomitant TxB2 release (peak concn 2-30 pg/ml). Simultaneous administration of HlyA and AA exhibited additive effects with regard to mediator release and pressor responses. EPA at 200-2,000 nM caused a transient rise in PAP similar to that provoked by 5-30 nM AA (maximum pressor response 3-18 mmHg). This was accompanied by liberation of TxB2 (peak concn 16 +/- 5 and 28 +/- 4 pg/ml after 1,000 and 2,000 nM EPA) and TxB3 (peak concn 9 +/- 4 and 30 +/- 3 pg/ml). Combined application of HlyA and EPA resulted in approximate addition of the TxB2 release reaction to each single compound, and TxB3 liberation more than doubled (maximum concn 59 +/- 12 pg/ml). The pressor responses to HlyA-EPA (200-2,000 nM) did not, however, surpass those to HlyA-AA (5-30 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary excretion of 2,3-dinor-thromboxane B1, a major metabolite of thromboxane B2 in the rat.

Urinary 2,3-dinor-thromboxane B2 (2,3-dinor-TXB2), an enzymatic degradation product of TXB2, is currently measured for evaluating in vivo thromboxane biosynthesis in rats. We simultaneously measured 2,3-dinor-TXB2 and 2,3-dinor-TXB1, another product of TXB2 metabolism, in the urine of rats by immunoaffinity extraction/gas chromatography negative ion chemical ionization mass spectrometry (GC-NICIMS). In rats under basal conditions, urinary excretion of 2,3-dinor-TXB1 was much higher than that of 2,3-dinor-TXB2 (19.22 +/- 4.86 and 1.64 +/- 0.29 ng/24 h, respectively). The relative abundance of the two metabolites in each animal was fairly constant (91.9 +/- 1.6 and 8.1 +/- 1.6% of their sum, respectively). Urinary excretion of both 2,3-dinor-TXB1 and 2,3-dinor-TXB2 increased in rats undergoing in vivo hepatic ischemia-reperfusion. Other thromboxane metabolites, including 11-dehydro-TXB2 and 11-dehydro-2,3-dinor-TXB2, were measured by GC-NICIMS in selected urines. The resulting profile was: 2,3,4,5-tetranor-TXB1 > 2,3-dinor-TXB1 >> 11-dehydro-TXB2 > 2,3-dinor-TXB2 = TXB2. This study shows that urinary 2,3-dinor-TXB1 is a suitable parameter of TXB2 biosynthesis in vivo in rats. The possible cross-reactivity of 2,3-dinor-TXB1 in immunoassays of urinary 2,3-dinor-TXB2 or even TXB2 in rats should be considered in future studies.

Influence of dietary fish on eicosanoid metabolism in man.

Two groups of 40 volunteers were given a dietary supplement consisting of 135 g of mackerel or meat (control) paste per day for 6 weeks. Compliance was about 80% in both groups and the daily intake of 20:5(n-3) and 22:6(n-3) from the mackerel supplement was about 1.3 and 2.3 g, respectively. In collagen-activated platelet rich plasma, the potency of blood platelet to produce HHT from arachidonic acid (AA) clearly reduced in the mackerel group, whereas the formation of HHTE from timnodonic acid (TA) increased slightly. Changes in the formation of HHT and HHTE, measured by HPLC, correlated significantly with those of TxB2 and TxB3, respectively, measured by GC/MS. Changes in the formation of the lipoxygenase products HETE (ex AA) and HEPE (ex TA) were qualitatively similar to that seen for the cyclo-oxygenase products, but quantitatively the responses were smaller. Formation of ir TxB2 in clotting blood significantly reduced in the mackerel group. In collagen-activated, citrated whole blood, TxB2 formation tended to be reduced in the mackerel-supplemented volunteers. Mackerel consumption was associated with the formation of considerable amounts of PGI3, as judged from the appearance of 2,3-dinor-delta 17-6-keto-PGF1 alpha in urine. The amount of the major metabolite of PGI2, 2,3-dinor-6-keto-PGF1 alpha was not reduced, or even increased. The daily amount of tetranor prostaglandin metabolites in the urine did not change significantly, which indicates that mackerel supplementation did not alter the formation of prostaglandins E and F.

Analysis of thromboxane B3 converted from eicosapentaenoic acid in human platelet rich plasma by gas chromatography/mass spectrometry.

Gas chromatographic mass spectrometric determination of thromboxane B3 (TXB3) synthesized from platelet is described. Eicosapentaenoic acid (EPA) was added to human platelet rich plasma and after the reaction the exstraction was carried out. Plasma thromboxanes were run through an Amberlite XAD-2 and SEP-PAK silica cartridge, and then chromatographed using silicagel thin layer plate to remove interfering materials, such as 6-keto-prostaglandin F1 alpha. Extracted thromboxanes were converted into the methoxime-dimethylisopropylsilylmethyl ester derivatives and they were measured by gas chromatography/ammonia chemical ionization mass spectrometry. Three peaks were obtained on the gas chromatogram which were presumed to be 3-series metabolite product TXB3 and their related substances. Results indicates the human platelet may easily convert EPA to TXB3 by adding EPA to PRP without adding arachidonic acid.

Stimulation of eicosapentaenoic acid metabolism in washed human platelets by 12-hydroperoxyeicosatetraenoic acid.

Washed human platelets were not able to convert eicosapentaenoic acid (EPA) to thromboxane B3 (TXB3) and 12-hydroxyeicosapentaenoic acid (AA) to washed human platelets induced conversion of EPA to TXB3 and 12-HEPE. Esculetin, a specific inhibitor of 12-lipoxygenase, prevented the effect of AA, but cyclooxygenase inhibitor did not. The conversion of AA to TXB2 was not affected by the same dose of esculetin. These data suggest that products of AA formed by 12-lipoxygenase in human platelets have stimulatory effects on EPA metabolism. When AA was preincubated with washed human platelets, its effect on EPA conversion was reduced, suggesting that a labile product of AA formed by 12-lipoxygenase is involved in the facilitation of EPA metabolism. Addition of 12-hydroperoxyeicosatetraenoic acid directly to washed human platelets caused dose-dependent synthesis of TXB3 and 12-HEPE, while addition of 12-hydroxyeicosatetraenoic acid had no effect. Thus, 12-hydroperoxyeicosatetraenoic acid formed from AA promotes the metabolism of EPA in washed human platelets.

Structural verification of 15-oxo-13,14-dihydrothromboxane B2.

The mass spectra of three stable isotope derivatives are presented to confirm ion assignments in the spectrum of the methoxime, trimethylsilyl ether, methyl ester from 15-oxo-13,14-dihydrothromboxane B2.