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Nutrition, Department of Family and Consumer Sciences, University of Wyoming, Laramie, WY 82071-3354
2To whom correspondence should be addressed. E-mail: broughto{at}uwyo.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • total fat • (n-3) polyunsaturated fatty acid • eicosanoids • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Thus, manipulation of dietary PUFA intake serves as a means of altering tissue lipid composition. Tissue arachidonic acid (AA) [20:4(n-6)], a desaturation-elongation product of dietary linoleic acid (LA) [18:2(n-6)], serves as a precursor for eicosanoids, i.e., the prostaglandins (PG) and leukotrienes (LT) collectively. Alterations in dietary lipids provide a nonpharmacologic means of altering eicosanoid synthesis. For example, ingestion of fish oil can modify tissue lipid PUFA composition by increasing tissue eicosapentaenoic acid (EPA) [20:5(n-3)] and docosahexaenoic acid (DHA) [22:6(n-3)] and reducing AA content (2
–4
). This tissue alteration exerts a significant effect on eicosanoid synthesis due to EPA competition with AA as a substrate for eicosanoid formation.
Currently, Americans derive much of their PUFA from vegetable oils, rich in the essential fatty acid (EFA) LA. A minimum intake of 50 mg/(kg · d), i.e., 1.5 energy percent, has been shown to relieve overt symptoms of EFA deficiency (EFAD) (5
). In the 1950s to 1960s, research demonstrated the benefit of LA consumption and the hypocholesterolemic effect of PUFA. In light of the relationship between plasma cholesterol and coronary heart disease, (n-6) PUFA consumption has been promoted. Current estimates report LA consumption levels of 250–300 mg/(kg · d), five to six times more than required to prevent EFAD.
Current PUFA consumption levels in the United States are vastly different from those in previous times. As hunters and gatherers, we consumed lower total fat with less (n-6) PUFA. Possibly more important was the fact that (n-3) and (n-6) intakes were approximately equal (6
). Increased technology and the industrial era have led to elevated consumption of total fat and (n-6) PUFA, with reductions in (n-3) PUFA intake. This has altered the (n-3):(n-6) ratio dramatically, which may alter key physiological processes influenced or regulated by eicosanoids (6
).
Upon cell activation, AA is released and eicosanoids of both the prostanoid (7
) and leukotriene (8
) families are synthesized. Related to high intakes of LA, the potentially detrimental effect of excess eicosanoid synthesis from AA has generally been overlooked. An unbalanced state of eicosanoid synthesis may promote such diseases as atherosclerosis, heart disease, thrombosis, tumor growth and immune-related pathophysiologies (6
).
Previous studies suggest that while (n-3) PUFA ingestion decreases AA-based eicosanoid synthesis and increases EPA-based LT synthesis, differences in total dietary fat levels may also have the potential to alter total eicosanoid production (2
,9
). As the total fat content in two mouse studies conducted in the same laboratory (9
,2
) was changed from 10 to 15 g fat/100 g diet in the experimental diets, eicosanoid synthesis seemed to be inversely related to total fat energy. These findings suggest a need to carefully evaluate the effect of varying levels of total dietary fat intake on eicosanoid synthesis. Furthermore, there is a specific need to examine the effect of simultaneous alterations in levels of total fat and (n-3) PUFA, to ascertain whether there is an interactive effect on eicosanoid production. Although a high fat diet is not generally recommended, elevated dietary fat may serve as a means for regulating eicosanoid overproduction. This study examined the effect of differing total fat intakes of 5, 10, 15, 20 g fat/100 g diet, along with alterations in the (n-3) to (n-6) PUFA ratio (0, 0.1:1, 0.2:1, 0.4:1, 1:1) on the fatty acid composition of hepatic and peritoneal cell phospholipids and in vivo eicosanoid synthesis by mouse peritoneal cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Mice.
Two hundred forty male C57BL/6 mice weighing 18–20 g (Charles River, Wilmington, MA) were randomly housed at four mice per cage in a temperature- (25oC) and humidity-controlled room on a 12-h light/dark cycle upon arrival. After 1 wk maintenance on nonpurified laboratory diet (Rodent Laboratory Chow 5001; Purina Mills, St. Louis, MO), 12 mice were randomly assigned to each experimental diet for 2 wk. Initial body weights did not differ among dietary groups.
Diets.
Mice consumed 1 of 20 diets ad libitum containing 5, 10, 15, or 20 g fat/100 g diet, with (n-3):(n-6) PUFA ratios approximating 0, 0.1:1, 0.2:1, 0.4:1, 1:1 with (n-6) PUFA maintained at 
1.5 g/100 g of diet (actual diet composition shown in Table 1
). The balance of the diet consisted of 3 g/kg dl-methionine, 2 g/kg choline bitartrate, 35 g/kg diet AIN-76 Salt Mix, 10 g/kg vitamin mix, and fat free mix (Dyets, Bethlehem, PA) added to specific diets at levels of 900, 850, 800, and 750 g/kg of diet in the 5, 10, 15, and 20 g/100 g diets, respectively. The fat in the diets was composed of safflower or sardine oil with a mixture of olive oil and tripalmitin (Sigma, St. Louis, MO) serving as the filler oils. Safflower oil contained 75.25% LA as a primary source of (n-6) fatty acids, while the sardine oil (Shaklee Corporation, Pleasanton, CA) was 27.1% EPA, 2.5% 22:5(n-3), 12.0% 22:6 DHA, and 5.4% 18:4(n-3) as a source of long-chain (n-3) PUFA with a small amount of 18:2(n-6) at 1.28%. Diets were prepared in bulk and partitioned into daily rations packaged in Whirl-Pak bags (Nasco, Fort Atkinson, WI), flushed with nitrogen to minimize oxidation and stored at -4oC. Fresh diets were fed daily just before the dark cycle with uneaten food discarded and food cups cleaned before refeeding.
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After two weeks of consuming the experimental diets, using a 21-gauge needle, 1 mg opsonized zymosan in 0.5 mL saline (9 g/L NaCl) was injected into the peritoneum of the mice to stimulate macrophages and induce eicosanoid biosynthesis (10
,11
). Opsonized zymosan was prepared by the method of Maroussem et al. (12
) as modified by Broughton et al. (2
). Briefly, zymosan was boiled at 100oC in distilled water at 10 g/L for 1 h. The zymosan was then cooled and pelleted, and the water decanted. The zymosan was then opsonized by suspending it at 10 g/L and incubating at 37o in rabbit serum for 30 min. After incubation, the zymosan was recovered by centrifugation, washed twice with saline and suspended at 2 g/L in saline (9 g/L NaCl).
After stimulation of eicosanoid synthesis for 30 min, mice were anesthetized with Metofane (Methoxyflurane, Pitman-Moore Washington Crossing, NJ) and the thoracic cavity was opened. After whole blood isolation over sodium citrate (38 g/L sodium citrate) for subsequent platelet and plasma use, the mice were killed by ether inhalation. The peritoneum was opened and washed with 3 mL EDTA-saline (5 mmol/L EDTA in saline) containing 100 ng prostaglandin B1 as an internal standard. A second 2-mL wash of EDTA-saline was used and the two peritoneal washes were pooled and stored on ice. After isolation of a portion of the peritoneal wash for cell quantitation with a hemocytometer, the saline extract was centrifuged (700 x g, 4 min, 25oC) to pellet the cells. The supernatant was decanted and acidified with formic acid to a final concentration of 3 mmol/L formic acid and diluted with methanol to a final concentration of 10% methanol (9
). Ten percent of the final volume was used for quantification of PGE2 and 6-keto-prostaglandin F1
(PGF1) by enzyme-linked immunoassay (EIA) with the remaining fraction used for leukotriene analysis by reverse phase HPLC (RP-HPLC). The pelleted peritoneal cells were resuspended in saline for subsequent phospholipid fatty acid analysis. Livers were perfused with saline-EDTA buffer (9 g/L NaCl, 1 mmol/L EDTA) and removed. A portion of the hepatic tissue was homogenized in saline at a concentration of 100 g/L and extracted for eventual phospholipid fatty acids analysis with the remaining portion wrapped and quick frozen in liquid nitrogen.
Leukotriene isolation and analysis.
Leukotrienes were purified by solid phase extraction on C-18 cartridges (Supelclean LC-18; Supelco, Bellfonte, PA). After sequential washing with water and hexane, LT were isolated from the C-18 cartridges by elution with methanol (9
). The methanol eluate was evaporated to dryness under a nitrogen stream, and LT were resuspended in the HPLC solvent of methanol:water (65:35 v/v) pH 4.7, containing 5 mmol/L ammonium acetate and 1 mmol/L EDTA. LT were separated by RP-HPLC using a Partisphere C-18 column (6 mm x 12.5 cm; Whatman, Hillsbro, OR) with quantification spectrophotometrically using a Hewlett-Packard 1040A Diode Array spectrophotometer (Hewlett Packard, Liverpool, NY) monitoring at 280 nm. All LT were identified by their distinctive ultraviolet absorption spectra and retention times compared with known standards. Leukotrienes were quantified using PGB1 as an internal standard and extinction coefficients of authentic standards (13
). Leukotrienes C4, D4, E4, C5, and E5 were purchased from Cayman Chemical (Ann Arbor, MI).
Prostaglandin separation and analysis.
Prostaglandins were triple extracted with equal volumes of ethyl acetate (14
), pooled and evaporated to dryness under a nitrogen stream and resuspended in EIA buffer. The prostaglandins were then analyzed using EIA kits (Caymen Chemical, Ann Arbor, MI). Prostacyclin (PGI2) was assayed as its stable metabolite PGF1.
Fatty acid separation and analysis.
Macrophage and liver analyses were carried out as described previously (15
). Lipids were extracted from isolated peritoneal cells and liver homogenate by the methods of Bligh and Dyer (16
) sequentially with chloroform-methanol (1:2, v/v), chloroform-saline (1:1, v/v), and one part chloroform (2X). Chloroform fractions were isolated and pooled, evaporated to dryness and resuspended in minimal quantities of chloroform. Phospholipids were separated by TLC using a chloroform-methanol (8:1, v/v) solvent system and visualized with 8-hydroxy-1,3,6-pyrene-trisulfonic acid trisodium salt (Eastman Kodak, Rochester, NY) in methanol at 100 mg/L. After separation, the phospholipid band was scraped from the plate and saponified in toluene and 0.5 mol/L potassium hydroxide in methanol for 8 min at 86oC to liberate free fatty acids, cooled on ice, and acidified with 0.7 mol/L hydrochloric acid in methanol. The free fatty acids were twice extracted with hexane, evaporated to dryness and methylated with ethereal diazomethane. The methyl esters were then redissolved in hexane and analyzed by gas chromatography using a Hewlett-Packard 5890 gas chromatograph with a DB23 capillary column (0.25 mm x 30 m; J & W Chromatography, Folsom, CA) with hydrogen as the carrier gas. Fatty acids were quantified using pentadecanoic acid (NuCheck Prep, Elysian, MN) as an internal standard.
Statistical analysis.
The experimental design was a randomized complete block. Differences in means for weight gain, tissue (n-3) and (n-6) PUFA, and specific leukotrienes and prostaglandins were assessed by two-way ANOVA for the ratio of dietary (n-3):(n-6) PUFA and total fat intake with the SAS statistical package (Statistical Analysis Systems Institute, Cary, NC). When overall differences were detected, specific treatment differences were assessed using Tukey’s studentized test (17
). Significance of difference was determined at P < 0.01. Values are expressed in the text as means ± SEM, n = 12.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Tissue fatty acid composition.
As ingestion of (n-3) PUFA increased, both hepatic and peritoneal cell tissues were enriched with (n-3) PUFA (Tables 2
and 3). Generally, as (n-3) PUFA intake approached a 1:1 (n-3):(n-6) ratio, tissue (n-3) PUFA levels increased 2- to 3-fold at each level of total fat intake. With high (n-3) PUFA ingestion, hepatic (data not shown) and peritoneal cell (Fig. 1
) EPA levels rose from virtually undetectable to 5% and 3.5% of the overall phospholipid fatty acid composition, respectively. Increases in (n-3) PUFA were attributable to tissue enrichment with EPA, docosapentaenoic acid [22:5(n-3)] and DHA with reductions in AA and its elongation products 22:4 and 22:5(n-6) (Tables 2
and 3)
.
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, peritoneal cell (n-6) PUFA were progressively reduced by increasing total fat intake (Table 3)
. As (n-3) PUFA intake increased, (n-6) levels were reduced in both tissues. As dietary (n-3) PUFA increased from no (n-3) PUFA to a 1:1 (n-3):(n-6) PUFA ratio, hepatic AA levels were reduced by one-half at each fat intake level, with a progressive reduction being evident at the intermediate (n-3):(n-6) PUFA ratios (data not shown). At each fat intake, peritoneal cell AA levels were reduced by 18–31%. Furthermore, as dietary fat energy increased, peritoneal cell AA decreased (Fig. 2
). The AA decrease with increased (n-3) PUFA intake was 30% in the mice consuming the 5 and 10 g/100 g diets and 20% in the 15 and 20 g/100 g diets. Lowest peritoneal cell (n-6) PUFA levels were found in mice consuming diets high in fat and (n-3) PUFA.
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With all of the tissue shifts, the hepatic unsaturation index (UI) remained fairly stable. Conversely, the peritoneal cell UI increased with increasing (n-3) PUFA intake (P < 0.01).
Eicosanoid formation.
The quantity and type of LT synthesized were influenced by diet. In the (n-6) PUFA only diets, four-series LT (C4 + E4) synthesis decreased by 37% as fat intake rose from 5 to 20 g/100 g diet (Fig. 3
). This same pattern of fat-induced reductions in four-series LT was observed with consumption of the 0.1 (n-3):(n-6) PUFA ratio diets. Mice consuming the 0.2 (n-3):(n-6) PUFA diets maintained a relatively constant level of four-series LT production regardless of level of fat intake while mice consuming the 0.4 and 1.0 (n-3):(n-6) PUFA ratios generally showed increases in four-series LT synthesis with increasing dietary fat up to 15 g/100 g diet after which 4-series LT synthesis plateaued (P < 0.01). Raising (n-3) PUFA ingestion at each level of fat intake led to a depression in four-series LT production (P < 0.01). These data indicated an interaction between total fat and (n-3) PUFA with respect to four-series LT biosynthesis. However, as total dietary fat increased, overall LT biosynthesis decreased (P < 0.01).
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PGF1 levels were reduced by increasing both dietary fat and by increasing the (n-3):(n-6) PUFA ratio (Fig. 4
). Regardless of total fat intake, increased ingestion of (n-3) PUFA decreased PGF1 levels. Mice consuming the 1:1 (n-3):(n-6) PUFA diets had the lowest PGF1 levels regardless of dietary fat content. The lowest overall synthesis occurred in mice consuming the high total fat, high (n-3) diets.
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). In mice fed the two higher fat diets, PGE2 release was reduced to basal levels with no further reductions due to increased (n-3) PUFA ingestion.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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6 desaturase activity (3
), potentially explaining the elevation of tissue LA levels with elevated (n-3) PUFA ingestion previously reported (4
,9
). In the present study, hepatic LA levels did not vary in response to changes in either (n-3) PUFA intake or total fat intake. Although peritoneal cell LA remained fairly stable with increasing (n-3) PUFA intake, increasing the total fat content of the diet led to an increase in tissue LA levels. Thus, although these data do not support an effect of (n-3) PUFA on 6 desaturase activity, they do indicate that the total fat content of the diet may either influence 6 desaturase activity or tissue LA incorporation, at least in peritoneal cell phospholipids.
Ingestion of (n-3) PUFA decreased eicosanoid synthesis, which was more pronounced at lower fat intakes. However, this study also indicated that if (n-6) PUFA remain low in the diet, elevated total dietary fat is equally as effective in reducing four-series LT biosynthesis and in some regard may influence the effect of (n-3) PUFA. It has been suggested that as fat intake is reduced, the need for linolenic acid and other (n-3) PUFA may be reduced (6
). This is not supported by the presently reported data. There was a greater propensity of (n-3) PUFA to alter eicosanoid production in mice fed low fat compared rather than high fat diets. Although the effect of higher (n-3) PUFA intake in blunting four-series LT production was somewhat reversed by increased fat intake resulting in increased four-series LT synthesis, five-series LT were decreased with increasing fat intake. The LT data for the 1:1 (n-3):(n-6) diets indicate that there was a greater total fat induced decrease in five-series LT compared with the increase in four-series LT.
A reduction in four-series and an increase in five-series LT production associated with (n-3) PUFA ingestion has been shown to ameliorate asthmatic symptoms in over 40% of a tested asthmatic population (18
). Because high levels of dietary fat reduced five-series LT synthesis and increased four-series LT synthesis in mice fed diets with high (n-3):(n-6) ratios, high fat intake may offset some relative benefits of (n-3) PUFA supplementation or ingestion. This study indicates that as the composition of dietary fats are altered, total fat in the diet needs to be monitored as well.
The prostanoid data, particularly for PGE2, were unique. Stable PGE2 levels associated with elevated fat intake suggest the possibility of a specific production threshold that may be independent of membrane composition (AA content) and more reflective of the need to maintain minimal PGE2 levels. LA intake necessary to maintain this point in higher fat diets may be above the 1–2% linoleic acid threshold previously described for the prevention of EFAD (5
). When dietary (n-6) PUFA were maintained at minimal levels, high total fat intake seemed to suppress PGE2 biosynthesis with dietary (n-3) PUFA incorporation exerting no additional effect. This effect did not seem to be induced through changes in tissue AA incorporation because peritoneal cell phospholipid AA levels did not reflect PGE2 synthesis levels. PGE2 is important in eliciting a wide range of responses including regulation of immune and inflammatory responses (6
). This study demonstrated that marked alterations in PGE2 are induced not only by (n-3) PUFA ingestion, but also by the amount of fat consumed.
The overall data support the integral involvement of diet and the potential that diet may play in the prevention of infections and long-term diseases. Excessive PGE2 production has been associated with a host of pathophysiologies (6
). Because typical intakes of LA may be excessive, the potential risk of overproduction of PGE2 has been recognized. If LA are maintained at minimal levels to prevent EFAD, increasing dietary fat becomes a viable means of regulating PGE2 overproduction. Although some high fat diets may be atherogenic, if (n-6) PUFA intake is balanced with (n-3) PUFA and total fat intakes, some of the pathophysiologies associated with PGE2 overproduction may be controlled. Furthermore, if filler fats could be derived primarily from monounsaturated fatty acids, high fat intakes would be less atherogenic while the benefits associated with controlled PGE2 production may be realized.
Although PGE2 production was depressed and unaffected by (n-3) PUFA addition at high fat intakes, this same pattern did not hold for PGI2 (assayed as 6-keto-PGF1). PGI2 decreased with fat intake and was further depressed by increasing (n-3) PUFA intake. The PGI2 data for mice fed the highest and lowest fat diets were in agreement with previous studies (2
,9
) in that there were minimal reductions in PGI2 with low (n-3) PUFA intake in comparison to levels observed in mice consuming diets devoid of (n-3) PUFA. However, in mice consuming the 10 and 15 g/100 g fat diets, there were marked reductions in 6-keto-PGF1 even with low (n-3) PUFA intake. As the (n-3) PUFA content of the diet increased to a (n-3):(n-6) ratio of 0.4:1 or greater, there were further reductions in PGI2 biosynthesis at all levels of total fat intake. This pattern of production differed from the pattern exhibited for PGE2 and would seem to indicate a point of control other than AA availability in the production of both PGE2 and PGI2. Furthermore, because the PGE2 and PGI2 patterns of production differed so greatly, these results indicate that there is further need to examine potential regulatory sites.
The relationship of dietary unsaturated fatty acids (amounts, types and ratios) and total fat to eicosanoid synthesis needs further study. Although this study emphasizes that there are changes induced by varying both the total fat and (n-3) PUFA contents of the diet, further studies could provide information on which to base general dietary recommendations. Future studies should further investigate the beneficial effect of various PUFA with regard to both eicosanoid-modulated physiologic functions and for optimizing plasma lipids.
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FOOTNOTES |
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3 Abbreviations used: AA, arachidonic acid [20:4(n-6)]; DHA, docosahexaenoic acid [22:6(n-3)]; EFAD, essential fatty acid deficiency; EIA, enzyme-linked immunoassay; EPA, eicosapentaenoic acid [20:5(n-3)]; LA, linoleic acid [18:2(n-6)]; LT, leukotriene; PG, prostaglandin; PGE2, prostaglandin E2; PGF1, 6-keto-prostaglandin F1; PGI2, prostacyclin; PUFA, polyunsaturated fatty acid; RP-HPLC, reverse phase HPLC; UI, unsaturation index.
Manuscript received 22 April 2001. Initial review completed 4 June 2001. Revision accepted 10 October 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1.
Simopoulos, A. P. (1991) Omega-3 fatty acids in health and disease and in growth and development. Am. J. Clin. Nutr. 54:438-463.
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Garg, M. L., Sebokova, E., Thomson, A.B.R. & Clandinin, M. T. (1988) 6-Desaturase activity in liver microsomes of rats fed diets enriched with cholesterol and/or 
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0.01, Tukey’s studentized test).
