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Dietary fish oil and fish and borage oil suppress intrapulmonary proinflammatory eicosanoid biosynthesis and attenuate pulmonary neutrophil accumulation in endotoxic rats

Mancuso, Peter PhD; Whelan, Jay PhD; DeMichele, Stephen J. PhD; Snider, Carolyn C. BS; Guszcza, Julie A. BS; Karlstad, Michael D. PhD

Laboratory Investigation

Objective  Proinflammatory eicosanoids and cytokines are important mediators of local inflammation in acute lung injury. We determined if enteral nutrition with anti-inflammatory fatty acids, eicosapentaenoic acid, and gamma-linolenic acid would reduce the intrapulmonary synthesis of proinflammatory eicosanoids and cytokines and pulmonary neutrophil accumulation in a rat model of acute lung injury.

Design  Prospective, randomized, controlled, double-blind study.

Setting  Research laboratory at a university medical center.

Subjects  Male Long-Evans rats (250 g).

Interventions  Rats were randomly assigned to three dietary treatment groups and fed nutritionally complete diets (300 kcal/kg/day) containing 55.2% of the total calories from fat with either 97% corn oil, 20% fish oil, or 20% fish and 20% borage oil for 21 days. On day 22, bronchoalveolar lavage was performed 2 hrs after an intravenous injection of Salmonella enteritidis endotoxin (10 mg/kg) or saline. Bronchoalveolar lavage fluid was analyzed for leukotriene B4, leukotriene C4/D4, thromboxane B2, prostaglandin E2, 6 keto-prostaglandin F1 alpha, tumor necrosis factor (TNF)-alpha, and macrophage inflammatory protein-2 (MIP-2). Lung myeloperoxidase activity (a marker for neutrophil accumulation) and phospholipid fatty acid composition were also determined.

Measurements and Main Results  Lung phospholipid concentrations of arachidonic acid were lower and the concentrations of eicosapentaenoic acid and docosahexaenoic acid were higher with fish oil and fish and borage oil as compared with corn oil. Dihomo-gamma-linolenic acid, the desaturated and elongated intermediate of gamma-linolenic acid, increased with fish and borage oil as compared with fish oil and corn oil. The levels of leukotriene B4, leukotriene C4/D4, 6-keto-prostaglandin F1 alpha and thromboxane B2 with corn oil were significantly increased with endotoxin as compared with saline. In contrast to the corn oil group, endotoxin did not significantly increase bronchoalveolar lavage levels of leukotriene B4, leukotriene C4/D4, and thromboxane B2 above those of saline-treated rats with fish oil and fish and borage oil. Lung myeloperoxidase activity was significantly increased in endotoxin-treated rats compared with those rats given saline in all dietary treatment groups. However, lung myeloperoxidase activity was significantly lower with either fish oil or fish and borage oil as compared with corn oil after endotoxin. Although endotoxin increased the levels of TNF-alpha and MIP-2 with all dietary treatment groups as compared with saline-treated rats, there were no significant differences in the levels of either cytokine between the dietary treatment groups.

Conclusions  These results indicate that dietary fish oil and fish and borage oil as compared with corn oil may ameliorate endotoxin-induced acute lung injury by suppressing the levels of proinflammatory eicosanoids (but not TNF-alpha or MIP-2) in bronchoalveolar lavage fluid and reducing pulmonary neutrophil accumulation. (Crit Care Med 1997; 25:1198-1206)

From the Life Sciences Program in Physiology, University of Tennessee (Dr. Mancuso), the Department of Nutrition (Dr. Whelan), University of Tennessee, Knoxville, TN; Ross Products Division, Abbott Laboratories (Dr. DeMichele), Columbus, OH; and the Department of Anesthesiology, Graduate School of Medicine, University of Tennessee Medical Center (Dr. Karlstad, Ms. Snider, and Ms. Guszcza), Knoxville, TN.

Supported, in part, by the T.K. Beene Memorial Gift Fund, Department of Anesthesiology, University of Tennessee Medical Center and Ross Products Division, Abbott Laboratories.

Address requests for reprints to: Dr. Michael D. Karlstad, Department of Anesthesiology, University of Tennessee Medical Center, 1924 Alcoa Highway, Knoxville, TN 37920.

KEY WORDS: acute lung injury; adult respiratory distress syndrome; arachidonic acid; enteral nutrition; eicosapentaenoic acid; docosahexaenoic acid; dihomo-gamma-linolenic acid; borage oil; fish oil

The acute respiratory distress syndrome (ARDS) is characterized by pulmonary edema, increased microvascular permeability and pulmonary vascular resistance, decreased lung compliance, and progressive hypoxemia [1,2]. These functional alterations have been associated with the excessive release of arachidonic acid-derived mediators from inflammatory cells. The systemic injection of endotoxin in the rat has been used as a model for sepsis-induced ARDS since it increases the concentrations of intrapulmonary proinflammatory mediators and produces the functional and morphologic pulmonary pathophysiology associated with ARDS in humans. Alveolar macrophages have the potential to release a wide variety of proinflammatory mediators (cytokines and eicosanoids) and have long been considered to be central cells in orchestrating local inflammatory events in ARDS and experimental models of pulmonary inflammation [3]. The release of chemotactic agents (leukotriene B4 and macrophage-inflammatory protein-2) from alveolar macrophages initiates neutrophil recruitment and activation in the pulmonary vasculature [4,5]. Neutrophils damage vascular endothelium by the release of proteolytic enzymes and reactive oxygen species during adherence and diapedesis through vascular tissue [6].

Recent treatment modalities for ARDS have been focused on down-regulation of proinflammatory mediators by pharmacologic intervention [7,8]. However, the efficacy of nutritional support to modulate the local inflammatory response to pulmonary injury requires further investigation. Optimal nutritional support of the patient at risk of developing ARDS remains a complex issue. A primary goal of nutritional support has been to lessen the demands on the respiratory system in these patients. For example, high fat, low carbohydrate enteral diets have been shown to reduce minute ventilation and ventilatory demand by lowering the respiratory quotient and CO2 production in ventilator-dependent patients [9]. Moreover, the use of the anti-inflammatory fatty acids, eicosapentaenoic acid and gamma-linolenic acid, would provide added benefits. Animal and clinical studies [10-12] have shown that nutritional intervention with diets containing fish oils containing eicosapentaenoic acid can favorably modulate proinflammatory eicosanoid production. Similarly, there has been recent interest in the potential benefits of dietary supplementation with gamma-linolenic acid found in borage oil [13]. The gamma-linolenic acid is rapidly elongated to dihomo-gamma-linolenic acid and subsequently incorporated in tissue phospholipids. Dihomo-gamma-linolenic acid is an inhibitor of leukotriene biosynthesis [14] and is further metabolized to prostaglandin E1. Prostaglandin E1 has been shown to increase cardiac output and systemic oxygen delivery while reducing pulmonary arterial pressure and vascular resistance in patients with ARDS [15].

Previously, we [16] have shown that alveolar macrophages from rats fed enteral formulas containing eicosapentaenoic acid and gamma-linolenic acid vs. a control diet produced less proinflammatory mediators (leukotriene B4, thromboxane B2 and prostaglandin E2) when stimulated in vitro. Along with these effects on eicosanoid metabolism, eicosapentaenoic acid and gamma-linolenic acid provided protection against increases in lung microvascular permeability and hemodynamic insufficiency in endotoxic rats [16]. However, we have yet to demonstrate that enteral formulas containing eicosapentaenoic acid and gamma-linolenic acid affect local intrapulmonary production of proinflammatory eicosanoids in vivo. Therefore, to further characterize the anti-inflammatory properties of diets containing eicosapentaenoic acid and gamma-linolenic acid, intrapulmonary proinflammatory mediators were assessed in the bronchoalveolar lavage fluid of endotoxin-challenged rats.

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MATERIALS AND METHODS

Approval of this study was granted by the Animal Care Committee of the University of Tennessee Graduate School of Medicine in accordance with guidelines set forth in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

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Experimental Design.

Male Long-Evans rats, weighing 150 +/- 10 g, were purchased from Charles River Laboratories (Wilmington, MA) and allowed to acclimate to the animal facility before the start of experiments 1 and 2. Three high fat, low carbohydrate enteral diets were fed to rats for 21 days in both experiments. The fatty acid composition of lung phospholipids was assessed in control rats in experiment 1. Eicosanoid and cytokine concentrations recovered in bronchoalveolar lavage fluid and lung neutrophil accumulation (myeloperoxidase activity) in endotoxic and control rats were assessed in experiment 2.

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Enteral Nutrition.

Rats were randomly assigned to one of three dietary groups. Rats within the same dietary group were housed two per cage in a temperature (21[degree sign]C to 23[degree sign]C)- and light-controlled facility (12 hrs light/12 hrs dark). Animals were fed isovolemic, isocaloric (300 kcal/kg/day), and isonitrogenous enteral diets containing corn oil, fish oil, or fish and borage oil for 21 days. Enteral diets were provided in sterilized feeding bottles and a separate bottle provided water ad libitum. Diets were changed daily to minimize the potential for bacterial growth. The diets provided 16.7% of calories from protein, 28.1% of calories from carbohydrate, and 55.2% of calories from lipid. The corn oil diet represents a standard high fat, low carbohydrate enteral formula enriched with linoleic acid devoid of medium chain triglycerides. The fish oil and fish and borage oil diets had similar concentrations of fish oil and medium-chain triglycerides. To maintain a similar n-6 to n-3 fatty acid ratio between these three diets, linoleic acid was replaced with gamma-linolenic acid in the fish and borage oil diet. The fatty acid composition of the enteral diets was determined by gas chromatography (Table 1). The diets were enriched with vitamins and minerals at or above the recommended daily requirement for humans. Diets were formulated and supplied by Ross Products Division, Abbott Laboratories, Columbus, OH.

Table 1

Table 1

In experiment 1 (n = 19), rats were randomly assigned to three dietary groups: corn oil (n = 6), fish oil (n = 6), or fish and borage oil (n = 7). After 21 days of feeding, rats were anesthetized with isoflurane (Abbott Laboratories, North Chicago, IL) and their lungs were removed for phospholipid fatty acid analysis.

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Lung Phospholipid Fatty Acid Analysis.

Lungs were perfused with cold phosphate buffered saline and the lipids were extracted [17]. Lungs (100 mg) were homogenized in cold saline (0.9%) and the homogenates were resuspended in 3 mL methanol-chloroform (2:1, volume/volume), extracted once with chloroform-saline (1:1, volume/volume), and twice with chloroform. The pooled chloroform fractions were evaporated to dryness under nitrogen, redissolved in chloroform, and the phospholipids were separated by thin layer chromatography, using a chloroform/methanol (8:1, volume/volume) solvent system. The phospholipids were recovered by scraping the appropriate band from the thin layer chromatography plate and suspending the scrapings in toluene. The phospholipids were then saponified with 0.5 normal potassium hydroxide in methanol for 8 mins at 86[degree sign]C. Free fatty acids were acidified in 0.7 normal hydrochloric acid in methanol, extracted twice with hexane, and evaporated to dryness under nitrogen. After methylating with ethereal diazomethane, the fatty acid methyl esters were resuspended in hexane and analyzed by gas chromatography (5890 Series II, Hewlett-Packard, Palo Alto, CA), using a DB 23 capillary column (0.25 mm x 30 m) (J & W Chromatography, Folsum, CA) with hydrogen as the carrier gas. Free fatty acids were quantified, using pentadecanoic acid as an internal standard and expressed as mol percent.

In experiment 2 (n = 48), rats were assigned randomly to one of three dietary groups: corn oil (n = 16), fish oil (n = 16), or fish and borage oil (n = 16), and fed for 21 days. On the morning of day 22, rats were anesthetized with isoflurane and given an intravenous (penile vein) injection (1.0 mL/kg body weight) of either Salmonella enteritidis endotoxin (10 mg/kg) (n = 8) or saline (control) (n = 8). The rats were then returned to their cages and given free access to water until the bronchoalveolar lavage procedure was performed.

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Bronchoalveolar Lavage and Tissue Collection.

Two hours following the intravenous injection of either endotoxin or saline, rats were anesthetized with isoflurane before performing the bronchoalveolar lavage procedure. The trachea was cannulated with a Jelco[trademark symbol] 14-gauge Teflon intravenous catheter placement unit (Critikon, Tampa, FL) and secured in placed with ligatures. The lungs were lavaged with six separate 6-mL aliquots of ice cold phosphate buffered saline using a 6-mL syringe. The lavage fluid was pooled for each animal. The pulmonary artery was isolated and perfused with 12 mL of cold phosphate buffered saline to flush the blood from the lung. The lungs were then excised and the left lung was quick frozen in liquid nitrogen for assay of myeloperoxidase activity.

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Eicosanoid Extraction of Bronchoalveolar Lavage Fluid.

The total recovered bronchoalveolar lavage fluid from each rat was centrifuged at 400 x g for 10 mins at 4[degree sign]C to pellet the cells. The supernatant was diluted 1:1 (volume/volume) with 20% methanol containing 3 mM formic acid. Eicosanoids were isolated by solid phase extraction using a C-18 cartridge (Burdick and Jackson, Muskegon, MI), washed with distilled water, and eluted off the cartridge with methanol. The remaining bronchoalveolar lavage fluid was aliquoted into 1.5-mL polypropylene tubes and stored at -80[degree sign]C until use for cytokine analysis.

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Prostaglandin and Leukotriene Analysis.

Eicosanoids were analyzed as described previously [18] with the following modifications: the eluents were evaporated to dryness under nitrogen and resuspended in phosphate buffered saline containing 0.1% (weight/volume) gelatin. Each sample was analyzed for leukotriene B4, leukotriene C4/D4, thromboxane B sub 2, prostaglandin E2, and 6-keto-prostaglandin F1 alpha by radioimmunoassay according to the manufacturer's instructions (Advanced Magnetics, Cambridge, MA) and tritiated eicosanoids were obtained from New England Nuclear (Du Pont, Boston, MA). Cross reactivities at half maximum binding of various leukotrienes with leukotriene C4/D4 antiserum are as follows: leukotriene C4 (100%), leukotriene D4 (100%), leukotriene E4 (20%), and leukotriene B4 (<1%). Cross reactivities at half maximum binding of various leukotrienes with leukotriene B4 antiserum are as follows: leukotriene B4 (100%), leukotriene C4 (<1%), and leukotriene D4 (<1%). Cross reactivities at half maximum binding of various prostanoids with 6-keto-prostaglandin F1 alpha antiserum are as follows: 6-keto-prostaglandin F1 alpha (100%), prostaglandin F1 alpha (7.8%), 6-keto-prostaglandin E sub 1 (6.8%), prostaglandin F2 alpha (2.2%), prostaglandin E2 (<1%), and thromboxane B2 (<1%). Cross reactivities at half maximum binding of various prostanoids with prostaglandin E2 are as follows: prostaglandin E2 (100%), prostaglandin E1 (50%), 6-keto-prostaglandin F1 alpha (<1%), and thromboxane B2 (<1%). Cross reactivities at half maximum binding of various prostanoids with thromboxane B2 antiserum are as follows: thromboxane B2 (100%), prostaglandin E2 (<1%), and 6-keto-prostaglandin F1 alpha (<1%).

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Tumor Necrosis Factor (TNF)-alpha and Macrophage Inflammatory Protein (MIP-2) Assays.

TNF-alpha and MIP-2 levels were determined on bronchoalveolar lavage fluid using enzyme-linked immunosorbent assay kits specific for rat cytokines (Biosource Cytoscreen Immunoassay[trademark symbol], Camarillo, CA) according to the manufacturer's instructions. Standards and samples were done in triplicate and the absorbance was read at 450 nm on a Minireader II (Dynatech Laboratories, Chantilly, VA) 96-well plate reader. The minimum detectable amount of TNF-alpha and MIP-2 was 30 and 10 pg/mL, respectively.

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Myeloperoxidase Tissue Extraction.

A 100-mg sample of the lower lobe of each left lung was homogenized for 30 secs with 5 mL of 80 mM potassium phosphate buffer (KH2 PO4), pH 7.4, and centrifuged at 40,000 x g (18,000 rpm) for 20 mins at 4[degree sign]C. The supernatants were discarded and the pellets were resuspended in 5 mL of 50 mM potassium phosphate buffer with 0.5% hexadecyltrimethylammonium, pH 6.0 and sonicated for 1 min. The sample tubes were frozen in liquid nitrogen for 20 mins and then incubated in a 60[degree sign]C water bath for 2 hrs and centrifuged. The tubes were centrifuged and the supernatants were aliquoted and frozen at -80[degree sign]C.

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Myeloperoxidase Activity Assay.

Myeloperoxidase activity was measured by measuring the hydrogen peroxide-dependent oxidation of 3,3 prime,5,5 prime-tetramethylbenzidine. Samples were thawed at room temperature and centrifuged at 250 g for 10 mins to remove any particulate matter. An aliquot (50 micro L) of each sample and standard were added to a flat bottom 96-well plate in triplicate. Following the addition of 3,3 prime,5,5 prime-tetramethylbenzidine (100 micro L) to each well, the plates were agitated for 30 mins on a plate shaker. The reaction was terminated by the addition of 20 micro L of 10 M sulfuric acid and shaken for an additional 2 mins. Plates were read at 450 nm using a Minireader II 96-well plate reader (Dynatech Laboratories).

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Statistical Analysis.

Values were analyzed using a one-way analysis of variance and the Student-Newman-Keuls' test for differences between groups. Data are expressed as mean +/- SEM. A p <or=to .05 was considered significant. Nonparametric data were analyzed using Kruskal-Wallis analysis of variance on ranks.

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RESULTS

Caloric Intake and Body Weight Gain.

There were no differences in total caloric intake or the percentage of initial body weight gained between the dietary groups over the 21-day feeding period in experiments 1 and 2. There were no visible signs of intolerance to the diets in either experiment.

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Lung Phospholipid Fatty Acid Composition.

Lung phospholipid fatty acid composition in control rats is presented in Table 2. The gamma-linolenic acid was present at a low concentration with fish and borage oil but was not detected with corn oil or fish oil. Dihomo-gamma-linolenic acid was significantly higher with fish and borage oil as compared with the other groups. Arachidonic acid was substantially lower with fish oil and fish and borage oil as compared with corn oil. In addition, there was a significantly higher concentration of arachidonic acid with fish and borage oil as compared with fish oil. Eicosapentaenoic, docosapentaenoic, and docosahexaenoic acids were significantly higher with fish oil and fish and borage oil as compared with corn oil. Eicosapentaenoic acid was not detected in the corn oil group. The n-6/n-3 fatty acid ratio was significantly higher with corn oil as compared with fish oil and fish and borage oil. The ratio of dihomo-gamma-linolenic acid to arachidonic acid was significantly higher with fish and borage oil as compared with the other groups.

Table 2

Table 2

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Bronchoalveolar Lavage Fluid Leukotriene Levels.

Leukotriene B4 and leukotrienes C4/D4 in recovered bronchoalveolar lavage fluid of control and endotoxic rats are presented in Figure 1. Leukotriene B4 was not significantly affected by diet in the saline control groups (Figure 1). Following endotoxin administration, leukotriene B4 was significantly increased with corn oil; however, leukotriene B4 synthesis with fish and fish and borage oil failed to respond to endotoxin challenge since leukotriene B4 did not significantly increase in either of these groups. Moreover, leukotriene B4 was significantly lower with fish oil and fish and borage oil as compared with corn oil following endotoxin administration. The basal level (saline control) of leukotriene C4/D4 was significantly higher with corn oil as compared with fish oil but was not different between corn oil and fish and borage oil. Following endotoxin administration, leukotriene C4/D4 increased significantly (approximately three-fold) over the basal level (saline control) with corn oil, but leukotriene C4/D4 synthesis with fish oil or fish and borage oil failed to respond to endotoxin challenge as leukotriene C4/D4 did not significantly increase in either of these groups. Moreover, leukotriene C4/D4 was significantly lower with fish oil and fish and borage oil as compared with corn oil following endotoxin administration.

Figure 1

Figure 1

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Bronchoalveolar Lavage Fluid Prostaglandin Levels.

Thromboxane B2 (stable metabolite of thromboxane A2), prostaglandin E2, and 6-keto-prostaglandin F1 alpha (stable metabolite of prostacyclin) in recovered bronchoalveolar lavage fluid of control and endotoxic rats are presented in Figure 2. Thromboxane B2 and 6-keto-prostaglandin F1 alpha were not significantly affected by diet in the saline control groups (Figure 2). However, prostaglandin E2 was significantly affected by diet in the saline control groups. It was significantly lower with fish oil as compared with corn oil and fish and borage oil (Figure 2). Thromboxane B2 and 6-keto-prostaglandin F1 alpha increased significantly, whereas the increase in prostaglandin E2 was not significantly different with corn oil following endotoxin administration as compared with their respective saline controls (Figure 2). Thromboxane B2 and prostaglandin E sub 2 failed to respond to endotoxin challenge with fish oil or fish and borage oil as compared with their respective saline controls. In addition, thromboxane B2 was significantly lower with fish oil and fish and borage oil and prostaglandin E2 was significantly lower with fish oil as compared with corn oil following endotoxin administration. 6-ketoprostaglandin F1 alpha was significantly lower with fish oil as compared with corn oil, and fish and borage oil was not significantly different from either corn oil or fish oil following endotoxin administration.

Figure 2

Figure 2

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Bronchoalveolar Lavage Fluid Cytokine Levels.

Macrophage inflammatory protein-2 and TNF-alpha in recovered bronchoalveolar lavage fluid of control and endotoxic rats are shown in Figure 3. Macrophage inflammatory protein-2 and TNF-alpha were not significantly affected by diet in the saline control groups. Macrophage inflammatory protein-2 and TNF-alpha significantly increased to approximately the same level in all three dietary groups following endotoxin administration as compared with their saline control groups.

Figure 3

Figure 3

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Lung Myeloperoxidase Activity.

Lung myeloperoxidase activity of control and endotoxic rats is presented in Figure 4. Lung myeloperoxidase activity was not significantly affected by diet in the saline control groups. Lung myeloperoxidase activity was significantly increased in all three diet groups following endotoxin administration as compared with their respective saline control groups. Lung myeloperoxidase activity was significantly lower in the fish oil and fish and borage oil groups as compared with corn oil following endotoxin administration. Lung myeloperoxidase activity was not statistically different between the fish oil and fish and borage oil groups following endotoxin administration.

Figure 4

Figure 4

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DISCUSSION

The rationale for nutritional support with a high fat, low carbohydrate enteral formula containing fish oil alone or in combination with borage oil instead of corn oil is based on previous studies [16,19] in endotoxin-induced acute lung injury. Fish oil and fish and borage oil attenuated proinflammatory eicosanoid production by alveolar macrophages in vitro and protected against increases in lung microvascular permeability in rats [16] and improved gas exchange and oxygen delivery in pigs [19]. In the current study, we examined the effects of feeding enteral diets containing corn oil, fish oil, or a mixture of fish and borage oil for 21 days on the incorporation of fatty acids into lung phospholipids and on lung myeloperoxidase content and levels of proinflammatory eicosanoids and cytokines in bronchoalveolar lavage fluid 2 hrs after the intravenous administration of endotoxin or saline in rats. We demonstrated that fish oil alone or in combination with borage oil, as compared with corn oil, reduced lung myeloperoxidase content and suppressed the levels of thromboxane B2, leukotriene B4, and leukotriene C4/D4 but not TNF-alpha and MIP-2 in bronchoalveolar lavage fluid of endotoxic rats. These results, in conjunction with our previous study using enteral nutrition with fish oil alone or in combination with borage oil [16], indicate that the decrease in lung neutrophil accumulation and protection against increased lung microvascular permeability may be due to the suppression of proinflammatory eicosanoid synthesis but not the proinflammatory cytokines, TNF-alpha and MIP-2, in endotoxin-induced acute lung injury.

This study demonstrates that both dietary fish oil and borage oil can significantly alter the fatty acid composition of lung phospholipids by decreasing arachidonic acid and increasing eicosapentaenoic acid with fish oil and increasing dihomo-gamma-linolenic acid when combined with borage oil. These changes in fatty acid composition are consistent with the overall suppression of eicosanoids associated with systemic inflammatory response syndrome, viz. 2-series prostaglandins and 4-series leukotrienes. Furthermore, it has been shown that the incorporation of eicosapentaenoic acid into phospholipids shifts eicosanoid metabolism, to the less biologically active 3-series prostaglandins and 5-series leukotrienes [20-22]. The fact that fish and borage oil increased the ratio of dihomo-gamma-linolenic acid to arachidonic acid in lung phospholipids suggests that gamma-linolenic acid is desaturated and elongated to dihomo-gamma-linolenic acid and is not entirely converted to arachidonic acid. These changes in fatty acid composition are directly associated with the overall suppression of important eicosanoid mediators of the systemic inflammatory response syndrome, the 2-series prostaglandins and 4-series leukotrienes. Palombo et al. [23] showed significant incorporation of eicosapentaenoic acid and gamma-linolenic acid and displacement of arachidonic acid in lung and alveolar macrophage phospholipids after 3 days of enteral feeding with fish oil and borage oil. Increasing phospholipid content of dihomo-gamma-linolenic acid has been associated with an expected increase in the biosynthesis of prostaglandin E1 and an increase in the 15-lipoxygenase metabolite 15-hydroxyeicosatrienoate. The 15-hydroxyeicosatrienoate has been shown to inhibit 5-lipoxygenase and attenuate leukotriene biosynthesis [14].

Leukotrienes have been found in high concentrations in the bronchoalveolar lavage fluid of patients with ARDS [24] and are considered to be key mediators of pulmonary inflammation in ARDS. Leukotrienes are released from a number of inflammatory cells including alveolar macrophages, neutrophils, and mast cells in the lung [25]. It is likely that these sources contributed to the increased concentrations of leukotrienes in bronchoalveolar lavage fluid of endotoxic rats. The cysteinyl-leukotrienes, C4 and D4, have been shown to increase both lung microvascular permeability [26,27] and pulmonary arterial pressure [26] when administered into the pulmonary circulation of animals. Cohn et al. [26] showed that a leukotriene C4/D4 antagonist significantly decreased lung extravascular water accumulation in endotoxic pigs. In addition, pulmonary edema formation can be attenuated by a leukotriene D sub 4 receptor antagonist in endotoxic rats [28]. Thus, suppression of leukotriene C4/D4 biosynthesis may limit the severity of pulmonary microvascular protein leakage in endotoxin-induced acute lung injury [16].

Similarly, inhibition of leukotriene B4 formation has been shown to significantly reduce lung microvascular permeability and edema formation in the rat [29] and dog [30]. Leukotriene B4 is a potent chemoattractant of neutrophils and activated neutrophils are thought to mediate vascular endothelial cell damage by the release of proteases, reactive oxygen species, hypochlorous acid, leukotrienes, cytokines, and platelet activating factor [31]. We [16] have shown previously, using the same enteral diets as in this study, that the synthesis of leukotriene B4 from A23187-stimulated alveolar macrophages was significantly less with diets containing fish oil and fish and borage oil as compared with corn oil. This study suggests that suppressed synthesis of leukotriene B4 in bronchoalveolar lavage fluid may be due to a reduced capacity of alveolar macrophages to synthesize leukotriene B4 in vivo. Lower levels of leukotriene B4 would reduce the chemotactic signal for neutrophil recruitment and are consistent with our current findings of reduced myeloperoxidase activity and neutrophil accumulation in the lung and our previous findings of protection against increased lung microvascular protein permeability in endotoxic rats [16].

In general, eicosanoid formation in bronchoalveolar lavage fluid was significantly lower with fish oil and fish and borage oil as compared with corn oil following endotoxin challenge. Moreover, a striking feature of this dietary study is the lack of response in eicosanoid production by endotoxin-challenged rats fed fish oil and fish and borage oil. As observed with leukotrienes, thromboxane B2 (stable metabolite of thromboxane A2) and prostaglandin E2 failed to increase above their saline controls. Thromboxane A2 has been shown to increase lung microvascular permeability and pulmonary arterial pressure in acute lung injury [32]. Murray et al. [11,19] demonstrated that short-term feeding (8 days) with fish oil or fish and borage oil significantly attenuated plasma thromboxane B2 concentrations and alleviated pulmonary arterial hypertension in endotoxic pigs. Prostaglandin E2 is immunosuppressive at concentrations >10 sup -8 and at low concentrations <10 sup -9 is required for normal T-cell function [33]. Thus, modulation of prostaglandin E2 synthesis may normalize immune function. The results of this study paralleled our previous study where we showed that alveolar macrophages from rats fed fish oil alone or in combination with borage oil synthesized less prostaglandin E2 as compared with corn oil [16]. In contrast to thromboxane B2 and prostaglandin E2, 6-keto-prostaglandin F1 alpha (stable metabolite of prostacyclin) was not completely suppressed following endotoxin challenge with fish oil and fish and borage oil as compared with their saline controls. Prostacyclin opposes the vasoactive and platelet-aggregating activities of thromboxane A2 and therefore may represent a beneficial response in the endotoxic lung.

Tumor necrosis factor-alpha is another important mediator of endotoxin-induced inflammation [34] and acute lung injury [35]. Infusion of recombinant human TNF-alpha induces acute lung injury [35] and symptoms that mimic ARDS in rats. TNF-alpha is increased in the bronchoalveolar lavage fluid and serum in patients with ARDS [33]. Diet had no effect on attenuating TNF-alpha levels in bronchoalveolar lavage fluid following endotoxin challenge in this study. These results are in contrast to studies reporting that dietary fish oil enhances the in vitro production of TNF-alpha from peritoneal macrophages in mice [36] and rats [37].

The MIP-2 is a member of the alpha branch of the supergene family of chemokines [38]. It is rapidly produced by alveolar macrophages and pulmonary epithelial cells in response to endotoxin and is a potent chemoattractant of neutrophils [39]. We show that endotoxin-induced increase in MIP-2 levels in bronchoalveolar lavage fluid was not affected by diet. However, it is likely that MIP-2 played a major role in attracting neutrophils to the pulmonary microvasculature following endotoxin in this study. It appears that the reduction in pulmonary neutrophil accumulation, as shown by a decrease in myeloperoxidase activity, with fish oil alone or in combination with borage oil as compared with corn oil, was due to the suppression of leukotriene B4 synthesis but not MIP-2 in the bronchoalveolar lavage fluid of endotoxic rats. In summary, changes in lung fatty acid phospholipid composition with dietary fish oil and fish and borage oil were associated with suppressed synthesis of proinflammatory 2-series prostaglandins and 4-series leukotrienes in bronchoalveolar lavage fluid of endotoxic rats. These results suggest that the reduction in pulmonary neutrophil accumulation and the previously reported attenuation of lung microvascular protein permeability [16] may be related to the reduced synthesis of proinflammatory eicosanoids but not TNF-alpha or MIP-2 in bronchoalveolar lavage fluid of endotoxic rats. Enteral nutrition with high fat, low carbohydrate formulas containing anti-inflammatory fatty acid lipid blends may provide protection against increases in lung microvascular permeability and pulmonary neutrophil accumulation in critically ill patients at risk of developing ARDS.

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ACKNOWLEDGMENTS

The authors wish to acknowledge Normanella DeWille, PhD, Gregory Snowden, BS, and Jeffrey Morris, BS, for their expertise in the manufacture of the experimental diets.

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