- Email: [email protected]
Dietary fish oils decrease bradykinin-induced secretion in rat distal colon
Nuhition Research. W. 17, No. 4. pp. 693-702.1997 Copyright(B 1997 Elsevier Science Inc. F’rintedin the USA. AU rights resawd 0271-5317/97 $17.00+ .oo
ELSEVIER
PI1 SO271-5317(97)0003f&9
DIETARY FISH OILS DECREASE BRADYKININ-INDUCED RAT DISTAL COLON
SECRETION
IN
Kumiko Yamazaki, B.Sc., Takaji Yajima,* Ph.D. and Tamotsu Kuwata, Ph.D. Department of Nutritional Research, Nutrition Science Institute, Meiji Milk Products Co., Ltd., Tokyo, Japan
ABSTRACT This study aimed to determine whether or not dietary fish oils can modulate colonic chloride (Cl-) secretion induced by bradykinin, an inflammatory mediator, via the synthesis of prostaglandin Ez (PGE2) in a mucosal preparation of rat distal colon. Three diets containing 10 wt% fat composed of different oils, 100% safflower oil, and 40% safflower oil plus 60% sardine oil (rich in eicosapentaenoic acid) and 60% tuna oil (rich in docosahexaenoic acid), respectively, were administered for 21 d to rats. The fish oil supplements did not affect the rate of growth. Bradykinininduced Cl- secretion was monitored as the change in the short-circuit current (I,,) across a mucosal preparation of the distal colon. The increase in I, during 10 min in response to bradykinin 10m7 M was significantly lower for the fish oil-fed rats than for the safflower oil-fed ones. The production of PGEz in response to bradykinin was drastically decreased in the colonic mucosa of fish oil-fed rats compared to that in safflower oil-fed ones. The results suggest that fish oil supplements are valuable for preventing prostaglandin-induced intestinal secretion. Coeydehte1997ElWiaSdacokr. Key words: Sardine Oil, Tuna oil, Colon, Chloride Secretion, Prostaglandin E2
INTRODUCTION Dietary n-3 polyunsaturated fatty acids (PUFA), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), cause a change in the ratio of n-3 to n-6 in the membrane fatty acid composition, via not only direct incorporation into membrane lipid but also reduction of de nouo synthesis of n-6 PUFA, such as arachidonic acid (AA) (1). The change in the ratio could have broad biological effects on whole organs due to alteration of dc nouo synthesis of prostanoids from each PUFA.
??Address correspondence and reprint requests to: Takaji Yajima, Ph.D. Department of Nutritional Research, Nutrition Science Institute, Meiji Milk Products Co., Ltd., 1-21-3 Sakae, Higashimurayama, Tokyo 189, Japan. TEL: 0423-97-5679. FAX: 0423-95-182s. E-mail: [email protected]
693
694
K.YAMAZAKI et al.
It is well known that prostaglandinsplay important roles in the intestinal ion and water handling in physiological and pathophysiologicalconditions (2). Prostaglandins are synthesized de nouo on chemical and mechanical perturbation of the cell membrane, AA being released tbrough the action of phospholipase 4 on membrane phospholipids. Cyclooxygenase converts AA into metabolites that are precursors for prostaglandins and thromboxanes. Kinins, mediators of inflammation, are potent secretagogues in the small intestine and colon. Bradykinin has been shown to cause increases in the short-circuit current (I,) and tissue conductance of the colon reflecting net chloride (Cl-) secretion at a concentration as low as 0.1 nM (3-4). The secretory effect of kinins is mediated by prostaglandins because indomethacin, a potent inhibitor of cyclooxygenase, abolishes the kinin-induced Cl- secretion in the colon (5). The aim of this study is to determine whether or not the intake of fish oil can reduce bradykinin-induced Cl secretion via the de nouo synthesis of PGE, in the rat colon. Sardine oil and tuna oil were used as sources of EPA end DHA, respectively, in this study. MATEXIALS&METHODS Materials
Edible sardine oil and tuna oil were supplied by NOF Co. (Tokyo). Safflower oil was supplied by Binoru Gil Mills Co., Ltd. (Nagoya). Bradykinin, indomethacin and tetrodotoxin were obtained from Sigma (St. Louis, MO). Animals and diets
Twelve male Sprague-Dawley rats (6 weeks old) were obtained from Japan SLC (Shizuoka). The rata were housed in our animal facility with controlled temperature (25 f 2 “C), humidity (55 f 2% relative humidity), and light (lights on 0700 to 1900). They were fed CLEA Rodent Laboratory Chow CE-2 (CLEA-Japan) ud Zibitum for 10 d, and then were randomly assigned into three dietary groups: safflower oil, sardine oil (rich in EPA) and tuna oil (rich in DHA). The sardine oil contained 17.1% of EPA and 10.9% of DHA, and the tuna oil 6.2% of EPA and 22.1% of DHA. The nutrient contents of the semipurified diets were the same except for the types of fat (Table 1). The fatty acid compositions of diets were shown in Table 2, which were calculated by using the analytical data of fatty acid contents in each oil. The fish oil diets contained 4g/lOOg stiower oil, which ensured the essential fatty acid requirements of the rats. Three groups of rats were fed each diet ad libitum for 21 days, and then used for experiments. Three weeks feeding is suf&ient to alter the fatty acid compositions of colonic mucosa (6), and peripheral lymphocytes, mast cells and macrophages (7). At the termination of the experiment, rats were killedby dmining the blood with a syringe from the aorta abdominalis under anesthesia with ether. The blood was collected in tube containing heparin. Plasma was stored at -80 “C until analyzed.
DIETARYFISH OILAND FATTYACIDS
695
TABLE 1 Composition of the experimental diets Diets Safflower Oil
Sardine Oil gl1OOg
Casein dl-Methionine Starch Sucrose Cellulose Safflower oil Sardine oil Tuna oil Mineral mix (AIN) Vitamin mix (AIN) Choline bitartrate
Tuna Oil
diet
20.0 0.3 15.0 45.0 5.0 10.0
20.0 0.3 15.0 45.0 5.0 4.0 6.0
20.0 0.3 15.0 45.0 5.0 4.0
3.5 1.0 0.2
3.5 1.0 0.2
6.0 3.5 1.0 0.2
TABLE 2 Fatty acid composition of the experimental diets Diets
Fatty acid Safflower Oil
c14:o C16:O C16:l C18:O C18:l C18:2n-6 C18:3n-3 c2o:o C20:5n-3 c22:o C22:6n-3
2.40 15.20 74.70 0.50 0.20
n-6/n-3
149.4
6.40
0.60
Sardine Oil g/lOOg total fatty acid
3.54 10.60 4.74 2.28 13.40 30.66 0.74 0.08 10.26 0.24 6.54 1.7
Tuna Oil
1.80 13.36 2.94 3.90 17.72 30.72 0.50 0.08 3.72 0.24 13.26 1.8
K.YAMAZAKI et al.
696 Fatty acid analysis of plasma
The lipid of the plasma was extracted by the methods Folch et al (8). The fatty acid composition of the plasma lipid was determined by capillary gas-liquid chromatography (9).
Tissueprep&ration The preparation of an epithelial sheet without the submucosa from the distal colon was previously described (10-11). The epithelialsheet was mounted on ring filter paper (i.d. 8 mm), which was coated with vinyl chloride on one side, and was clamped between the each two halves of an Ussing chamber with a window size of 0.5 cm2. The chamber had a water-jacket to maintain the temperature at 37 “C. Krebs bicarbonate saline solution was used as the bathing solution. The volume of the bathing solution on each side was 10 ml.
The bathing solution comprised (in n&I): NaCl, 123.5; KCl, 4.7; CaCl,, 2.5; MgS04, 1.2; KH2P0,, 1.2; NaHCOa, 20; glucose, 11.1. The solution was bubbled with a mixture of 95% O2 and 5% CO2 (pH 7.4 at 37 “c). Measurement
of colonic electrical activity
The short-circuit current (IBe) was measured using an automatic voltage clamping device (CEZ-9100; Nihon Kohden, Tokyo) that compensates for solution resistance between the potential measuring electrodes. The transepithelial potential was recorded through 1 M KCl-agar bridges connected to a pair of calomel half cells, and the transepithelial current was applied across the tissue via a pair of Ag/AgCl electrodes, that were kept in contact with the mucosal and serosal bathing solutions with a pair of 1 M NaCl-agar bridges. All the experiments were performed under shortcircuit conditions. I, is taken as positive when the current flows from the mucosa to the serosa. The increase in I, was monitored for a lo-min and given as the equivalent flux of monovalent ions. In all experiments, tetrodotoxin (5 x lo-’ M) was added to the serosal solution to prevent the possible influence of tonic nerve activity. Measurement
of prostaglandin production
The production of PGE2 was determinedby measuring the total amount of that secreted into the serosal bathing solution before and after bradykinin stimulation. The tissue was incubated for two successive 15 min periods (control and experimental periods) after a stable period of 30 min in an Ussing chamber. Immediately after the control period bradykinin was added to the serosal bath to a concentration of 19’ M, and then the tissue was incubated for the experimental period. One ml samples were taken from the serosal bath, which were replaced with fresh buffer solution. The samples were kept at -25 “C until prostaglandin measurements. The PGEa contents of the incubation solutions were determined by direct immunoassaying using an EIA system (Amersham, Tokyo) with lo-50 pl samples.
697
DIETARY FISH OIL AND FAlTY ACIDS
Stat&i& analysis The results were expressed as means with standard errors. The computer program, Stat view, for a Macintosh computer was used for statistical analysis. One way analysis of variance was performed to compare the three diet groups. Specific differences were detected with Fisher’s PSLD, with a significance level of pcO.05. RESULTS & DISCUSSION E@ts
on growth
The body weight gains, on three weeks feeding, of the three groups, safflower oil, sardine oil and tuna oil, were 125.5 f 4.6 g, 133.3 f 16.6 g and 127.8 + 8.8 g, respectively, which were not significantly different. None of the rats showed symptoms of intestinal disorders such as diarrhea. These kbngs indicate that the fish oil supplements did not affect the growth or gut physiology of the rats. Eficts of dietary fit on fatty acid composition of rat plasma Feeding of sardine oil and tuna oil diets significantly increased EPA (C20:5 n-3) and DHA (C22:6 n-3) levels in the plasma lipid,respectively, compared with safflower diet, and contrary decreased n-6 fatty acids (linoleic acid and AA) levels (Table 3). TABLE 3 Effects of dietary fat on fatty acid composition of rat plasma Fatty acid Safllower Oil
Sardine Oil
Tuna Oil
g/lOOg total fatty acid c14:o C16:O C16:l C18:O C18:l Cl8:2 n-6
0.87 f 0.09 21.13 It 0.95a 2.12 f 0.37 13.26 f 1.40 12.43 f 0.71 27.03 f 4.33’
C20:4 n-6 C20:5 n-3
17.00 f 3.5ga
C22:5 n-3
o.oa o.oa
0.96 f 0.06 25.68 zk3.54b 2.71+ 0.95 13.34 k 1.49 11.23 f 0.84 16.81 f 1.7ab 11.07 _+0.87b
0.90 f 0.40 25.36 5 0.9gb 2.30 k 0.67 12.49 k 0.59 12.46 f 1.58 19.70 k 2.24b
6.39 f 1.91b 1.71 k 0.2ab
3.46 k 0.92’
12.01* 2.20b 1.00 f 0.2ab
4.66 k 0.47b 6.29 + 1.12’ C22:6 n-3 1.11 f 0.3ga n-6/n-3 2.4 f 0.7b 42.8 f 12.6’ 3.1+ 0.7b Values are mean f SD (n=4). Mean values within a row with different superscripts are significantly different at ~~0.05.
K.YAMAZAKI et al.
696
Feeding of both f&h oil diets significantly decreased the n-6/n-3 fatty acid ratio in the plasma compared with safnower diet (Table 3). Eficts on colonic Cl- secretion The bradykinin-induced I*,-responses of mucosal preparations were compared among the three diet groups after three weeks feeding. All the colonic tissues from the three groups responded to the serosal additionof bradykinin, which increased of I, in a dose-dependent manner (data not shown). The concentration of bradykinin of 10m7M was employed in this study since 10” M indomethacin abolished the I,,-response to bradykinin until the concentration of the latter reached 10m7 M. Colonic tissues of sardine oil-fed rats and tuna oil-fed rats showed significantly lower increases in the &+sponse to bradykinin than that in safflower oil-fed rats (Fig. 1). It is well established that the bradykinin-inducedincrease in Gc is primarily due to electrogenic Cl- secretion (12). This indicates therefore that the intake of sardine oil or tuna oil in this study was associated with reduction of bradykinin-inducedCl secretion inthedistal colon of the rat,
5
T
;
b
b
T
OISafflower
Sardine
Tuna
Oil
Oil
Oil
Fig. 1. Effects of dietary fish oils on the short-circuit current (I,) in response to bradykinin in rat distal colon. The increase in I,, in response to bradykinin (10-T M>was integrated for 10 min and given as the equivalent flux of monovalent ions. The values are means and standard error (n=4). The bars with different superscripts show significant differences (p
DIETARY FISH OIL AND FATTY ACIDS
699
Bradykinin-induced Cl- secretion occurs through the following three pathways; through prostaglandin synthesis (13), through the activation of enteric nerves (4), and through bradykinin receptors on enterocytes (14). The mediation through enteric nerves is neglected in the present study because the I,,-responses to bradykinin were measured using mucosal preparations (11) in the presence of a neuroblocker, tetrodotoxin. The abolition of the bradykinin-induced increase in I,, in the presence of indomethacin (10m6 M) strongly suggests mediation through prostaglandins in this study. In fact, prostaglandin E, (PGE& was detected in the serosal bathing solution after bradykinin-stimulation of the colonic mucosa (Fig. 2). This is compatible to the kallidin-induced release of prostaglandins into the serosal but not the mucosal solution bathing rat colonic mucosa (15), PGEz being the predominant product among the synthesized prostanoids (13). The production of PGE, in response to bradykinin stimulation was drastically decreased in the colonic mucosa of rats fed the fish oil-diets compared to in those fed the safflower oil-diet (Fig. 2). Those results indicate that dietary fish oils decrease the net accumulation of PGE, from AA in the colonic mucosa. The lamina propria, but not enterocytes, is the major source of kinin-generated prostaglandins (13), therefore, fibroblasts, lymphocytes, mast cells and vascular endothelial cells are candidates producing prostaglandins in response to bradykinin ( 12 ), This may be supported by much evidence that dietary fish oil supplements suppress the release of PGE, in peripheral lymphocytes, mast cells and peripheral macrophages (7).
10
I
a
Safflower oil
Sardine oil
Tuna oil
Fig. 2. Effects of dietary fish oils on prostaglandin E2 (PGE2) release into the serosal solution in response to bradykinin (10-T M) in rat distal colon. The values are means and standard error (n=4). The bars with different superscripts show significant differences (~~0.05) .
700
K.YAhJAZAKl et al.
Evidence is accumulating that diets supplemented with n-3 PUFA decrease the n-6/n-3 fatty acids ratio in plasma and tissues (7,16-19). This study also demonstrated that the diets supplemented with sardine oil (rich in EPA) and tuna oil (rich in DHA) drastically decreased the n-6/n-3 fatty acids ratio in rat plasma compared with the diet supplemented with &Bower oil (Table 3). The extra n-3 PUFA compete for the rate limiting enzyme, hepatic Abdesaturase and A6-desaturase, which normally converts dietary linoleic acid to AA (1). This results in reduced production of prostanoids derived &om AA. In the present study, the drastic decrease in the release of PGEZ from the colonic mucosa after bradykinin-stimulation should be reflected by the decrease in the n-6/n-3 fatty acids ratio induced by a diet with more n-3 PUFA (fish oils) than n-6 PUFA k&lower oil). Tuna oil is richer in DHA than sardine oil, both of which were used in this study. Dietary supplementation of both fish oils, however, had similar inhibitory effects on Clsecretion and PGEz production (Figs. 1 and 2), although DHA has a particularly inhibitory effect on the activity of A6-desaturase that forms AA from linoleic acid (20). It is able to speculate that the effects seen in here were due simply to the incorporation of EPA and DHA, which induce the decreased in AA level in the tissue lipid (17), rather than due to competition for transacylase enzymes. In diarrhea associated with mucosal inflammation, such as ileitis, colitis and other forms of inflammatory bowel disease, increased levels of prostanoids in the intestinal mucosa and blood have been observed (5). Therapeutic intervention has been attempted at several different sites along the pathway for prostanoid synthesis. This study demonstrated that nutritional intervention may successfully prevent prostanoids-induceddiarrhea through a decrease in the AA level due to dietary fish oils. REFERENCES 1.
Kinsella JE. a-Linolenic acid: Functions and effects on linolenic acid metabolism and eicosanoid-mediated reactions. San Diego: Academic Press, 1991,1-184.
2.
Gaginella TS. Eicosanoid-mediatedintestinal secretion. New York: Raven Press, 1990, 15-30.
3.
Cuthbert AW, Margolius HS. Kinins stimulate net chloride secretion by the rat colon. Br J Pharmacol 1982; 75:587-598.
4.
Diener M, Bridges RI, Knobloch SF, Rummel W. Indirect effects of bradykinin on ion transport in rat colon descendens: mediated by prostaglandins and enteric neurons. Naunyn-Schmiedeberg’s Arch Pharmacoll988; 337:69-73.
5.
Gaginella TS. Inhibition of eicosanoid-inducedsecretion. New York: Raven Press, 1990, 395-408.
6.
Lee D-YK, Lupton JR, Aukema HM, Chapkin RS. Dietary fat and fiber alter rat colonic mucosal lipid mediators and cell proliferation.J Nutr 1993; 123:18081817.
DIETARY FISH OIL AND FATTY ACIDS
701
7.
Black JM, Kinsella JE. Dietary n-3 fatty acids alter murine peritoneal macrophage cytotoxicity. Ann Nutr Metab 1993; 37:110-120.
8.
Folch J, Lees M, Slone-Stanley GH. A simple method for the isolation and purification of total lipid from anima tissue. J Biol Chem 1957; 226:497-509.
9.
Yonekubo A, Honda S, Hagiwara M, Okano M, Yamamoto Y. The effects of dietary fish oil on the serum lipids and tissue fatty acids composition of rats. Agri Biol Chem 1990; 54:1829-1833.
10.
Yajima T. Contractile effect of short-chain fatty acids on the isolated rat colon. J Physiol 1985; 369:667-678.
11.
Yajima T. Luminal propionate-induced secretory response in the rat distal colon in vitro. J Physiol 1988; 403:559-575.
12.
Gaginella TS, Kachur JF. Kinins as mediators of intestinal secretion, Am J Physiol 1989; 256:Gl-G15.
13.
Phillips JA, Hoult JRS. Secretory effects of kinins on colonic epithelium in relation to prostaglandins released from cells of the lamina propria. Br J Pharmacol 1988; 95:701-712.
14.
Cuthbert AW, Kirkland SC, MacVinish LJ. Kinin effects on ion transport in monolayers of HCA-7 cells, a line from human colonic adenocarcinoma. Br J Pharmacol 1985; 86:3-5.
15.
Hoult JRS, Phillips JA. Kinin-induced prostaglandin release in rat colon does not display serosal/mucosal “sidedness” after epithelial removal. Br J Pharmacol 1986; 88:3-5.
16.
Kinsella JE, Lokesh B, Broughton S, Whelan J. Dietary polyunsaturated fatty acids and eicosanoids: Potential effects on the modulation of inflammatory and immune cells: An Overview. Nutrition 1990; 6:24-44.
17.
Yonekubo A, Honda S, Okano M, Takahashi K, Yamamoto Y. Dietary fish oil alters rat milk composition and liver and brain fatty acid composition of fetal and neonatal rats. J Nutr 1993; 123:1703-1708.
18.
Cao J-M, Blond JP, Juaneda P, Durand G, Bezard J. Effect of low levels of dietary fish oil on fatty acid desaturation and tissue fatty acids in obese and lean rats. Lipids 1995; 30:825-832.
19.
Freyland L, Vaagenes H, Asiedu DK, Garras A, Lie 0, Berge RK. Chronic administration of eicosapentaenoic acid and docosahexaenoic acid as ethyl esters reduced plasma cholesterol and changed the fatty acid composition in rat blood and organs. Lipids 1996; 31:169-178.
702 20.
K.YAMAZAKl
etal.
Cook HW, Spence MW. Interaction of (n-3) and (n-6) fatty acids in desaturation and chain elongation of essential fatty acids in cultured glioma cells. Lipids 1987; 22:613-619.
Accepted for publication December 16, 1996.
ELSEVIER
PI1 SO271-5317(97)0003f&9
DIETARY FISH OILS DECREASE BRADYKININ-INDUCED RAT DISTAL COLON
SECRETION
IN
Kumiko Yamazaki, B.Sc., Takaji Yajima,* Ph.D. and Tamotsu Kuwata, Ph.D. Department of Nutritional Research, Nutrition Science Institute, Meiji Milk Products Co., Ltd., Tokyo, Japan
ABSTRACT This study aimed to determine whether or not dietary fish oils can modulate colonic chloride (Cl-) secretion induced by bradykinin, an inflammatory mediator, via the synthesis of prostaglandin Ez (PGE2) in a mucosal preparation of rat distal colon. Three diets containing 10 wt% fat composed of different oils, 100% safflower oil, and 40% safflower oil plus 60% sardine oil (rich in eicosapentaenoic acid) and 60% tuna oil (rich in docosahexaenoic acid), respectively, were administered for 21 d to rats. The fish oil supplements did not affect the rate of growth. Bradykinininduced Cl- secretion was monitored as the change in the short-circuit current (I,,) across a mucosal preparation of the distal colon. The increase in I, during 10 min in response to bradykinin 10m7 M was significantly lower for the fish oil-fed rats than for the safflower oil-fed ones. The production of PGEz in response to bradykinin was drastically decreased in the colonic mucosa of fish oil-fed rats compared to that in safflower oil-fed ones. The results suggest that fish oil supplements are valuable for preventing prostaglandin-induced intestinal secretion. Coeydehte1997ElWiaSdacokr. Key words: Sardine Oil, Tuna oil, Colon, Chloride Secretion, Prostaglandin E2
INTRODUCTION Dietary n-3 polyunsaturated fatty acids (PUFA), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), cause a change in the ratio of n-3 to n-6 in the membrane fatty acid composition, via not only direct incorporation into membrane lipid but also reduction of de nouo synthesis of n-6 PUFA, such as arachidonic acid (AA) (1). The change in the ratio could have broad biological effects on whole organs due to alteration of dc nouo synthesis of prostanoids from each PUFA.
??Address correspondence and reprint requests to: Takaji Yajima, Ph.D. Department of Nutritional Research, Nutrition Science Institute, Meiji Milk Products Co., Ltd., 1-21-3 Sakae, Higashimurayama, Tokyo 189, Japan. TEL: 0423-97-5679. FAX: 0423-95-182s. E-mail: [email protected]
693
694
K.YAMAZAKI et al.
It is well known that prostaglandinsplay important roles in the intestinal ion and water handling in physiological and pathophysiologicalconditions (2). Prostaglandins are synthesized de nouo on chemical and mechanical perturbation of the cell membrane, AA being released tbrough the action of phospholipase 4 on membrane phospholipids. Cyclooxygenase converts AA into metabolites that are precursors for prostaglandins and thromboxanes. Kinins, mediators of inflammation, are potent secretagogues in the small intestine and colon. Bradykinin has been shown to cause increases in the short-circuit current (I,) and tissue conductance of the colon reflecting net chloride (Cl-) secretion at a concentration as low as 0.1 nM (3-4). The secretory effect of kinins is mediated by prostaglandins because indomethacin, a potent inhibitor of cyclooxygenase, abolishes the kinin-induced Cl- secretion in the colon (5). The aim of this study is to determine whether or not the intake of fish oil can reduce bradykinin-induced Cl secretion via the de nouo synthesis of PGE, in the rat colon. Sardine oil and tuna oil were used as sources of EPA end DHA, respectively, in this study. MATEXIALS&METHODS Materials
Edible sardine oil and tuna oil were supplied by NOF Co. (Tokyo). Safflower oil was supplied by Binoru Gil Mills Co., Ltd. (Nagoya). Bradykinin, indomethacin and tetrodotoxin were obtained from Sigma (St. Louis, MO). Animals and diets
Twelve male Sprague-Dawley rats (6 weeks old) were obtained from Japan SLC (Shizuoka). The rata were housed in our animal facility with controlled temperature (25 f 2 “C), humidity (55 f 2% relative humidity), and light (lights on 0700 to 1900). They were fed CLEA Rodent Laboratory Chow CE-2 (CLEA-Japan) ud Zibitum for 10 d, and then were randomly assigned into three dietary groups: safflower oil, sardine oil (rich in EPA) and tuna oil (rich in DHA). The sardine oil contained 17.1% of EPA and 10.9% of DHA, and the tuna oil 6.2% of EPA and 22.1% of DHA. The nutrient contents of the semipurified diets were the same except for the types of fat (Table 1). The fatty acid compositions of diets were shown in Table 2, which were calculated by using the analytical data of fatty acid contents in each oil. The fish oil diets contained 4g/lOOg stiower oil, which ensured the essential fatty acid requirements of the rats. Three groups of rats were fed each diet ad libitum for 21 days, and then used for experiments. Three weeks feeding is suf&ient to alter the fatty acid compositions of colonic mucosa (6), and peripheral lymphocytes, mast cells and macrophages (7). At the termination of the experiment, rats were killedby dmining the blood with a syringe from the aorta abdominalis under anesthesia with ether. The blood was collected in tube containing heparin. Plasma was stored at -80 “C until analyzed.
DIETARYFISH OILAND FATTYACIDS
695
TABLE 1 Composition of the experimental diets Diets Safflower Oil
Sardine Oil gl1OOg
Casein dl-Methionine Starch Sucrose Cellulose Safflower oil Sardine oil Tuna oil Mineral mix (AIN) Vitamin mix (AIN) Choline bitartrate
Tuna Oil
diet
20.0 0.3 15.0 45.0 5.0 10.0
20.0 0.3 15.0 45.0 5.0 4.0 6.0
20.0 0.3 15.0 45.0 5.0 4.0
3.5 1.0 0.2
3.5 1.0 0.2
6.0 3.5 1.0 0.2
TABLE 2 Fatty acid composition of the experimental diets Diets
Fatty acid Safflower Oil
c14:o C16:O C16:l C18:O C18:l C18:2n-6 C18:3n-3 c2o:o C20:5n-3 c22:o C22:6n-3
2.40 15.20 74.70 0.50 0.20
n-6/n-3
149.4
6.40
0.60
Sardine Oil g/lOOg total fatty acid
3.54 10.60 4.74 2.28 13.40 30.66 0.74 0.08 10.26 0.24 6.54 1.7
Tuna Oil
1.80 13.36 2.94 3.90 17.72 30.72 0.50 0.08 3.72 0.24 13.26 1.8
K.YAMAZAKI et al.
696 Fatty acid analysis of plasma
The lipid of the plasma was extracted by the methods Folch et al (8). The fatty acid composition of the plasma lipid was determined by capillary gas-liquid chromatography (9).
Tissueprep&ration The preparation of an epithelial sheet without the submucosa from the distal colon was previously described (10-11). The epithelialsheet was mounted on ring filter paper (i.d. 8 mm), which was coated with vinyl chloride on one side, and was clamped between the each two halves of an Ussing chamber with a window size of 0.5 cm2. The chamber had a water-jacket to maintain the temperature at 37 “C. Krebs bicarbonate saline solution was used as the bathing solution. The volume of the bathing solution on each side was 10 ml.
The bathing solution comprised (in n&I): NaCl, 123.5; KCl, 4.7; CaCl,, 2.5; MgS04, 1.2; KH2P0,, 1.2; NaHCOa, 20; glucose, 11.1. The solution was bubbled with a mixture of 95% O2 and 5% CO2 (pH 7.4 at 37 “c). Measurement
of colonic electrical activity
The short-circuit current (IBe) was measured using an automatic voltage clamping device (CEZ-9100; Nihon Kohden, Tokyo) that compensates for solution resistance between the potential measuring electrodes. The transepithelial potential was recorded through 1 M KCl-agar bridges connected to a pair of calomel half cells, and the transepithelial current was applied across the tissue via a pair of Ag/AgCl electrodes, that were kept in contact with the mucosal and serosal bathing solutions with a pair of 1 M NaCl-agar bridges. All the experiments were performed under shortcircuit conditions. I, is taken as positive when the current flows from the mucosa to the serosa. The increase in I, was monitored for a lo-min and given as the equivalent flux of monovalent ions. In all experiments, tetrodotoxin (5 x lo-’ M) was added to the serosal solution to prevent the possible influence of tonic nerve activity. Measurement
of prostaglandin production
The production of PGE2 was determinedby measuring the total amount of that secreted into the serosal bathing solution before and after bradykinin stimulation. The tissue was incubated for two successive 15 min periods (control and experimental periods) after a stable period of 30 min in an Ussing chamber. Immediately after the control period bradykinin was added to the serosal bath to a concentration of 19’ M, and then the tissue was incubated for the experimental period. One ml samples were taken from the serosal bath, which were replaced with fresh buffer solution. The samples were kept at -25 “C until prostaglandin measurements. The PGEa contents of the incubation solutions were determined by direct immunoassaying using an EIA system (Amersham, Tokyo) with lo-50 pl samples.
697
DIETARY FISH OIL AND FAlTY ACIDS
Stat&i& analysis The results were expressed as means with standard errors. The computer program, Stat view, for a Macintosh computer was used for statistical analysis. One way analysis of variance was performed to compare the three diet groups. Specific differences were detected with Fisher’s PSLD, with a significance level of pcO.05. RESULTS & DISCUSSION E@ts
on growth
The body weight gains, on three weeks feeding, of the three groups, safflower oil, sardine oil and tuna oil, were 125.5 f 4.6 g, 133.3 f 16.6 g and 127.8 + 8.8 g, respectively, which were not significantly different. None of the rats showed symptoms of intestinal disorders such as diarrhea. These kbngs indicate that the fish oil supplements did not affect the growth or gut physiology of the rats. Eficts of dietary fit on fatty acid composition of rat plasma Feeding of sardine oil and tuna oil diets significantly increased EPA (C20:5 n-3) and DHA (C22:6 n-3) levels in the plasma lipid,respectively, compared with safflower diet, and contrary decreased n-6 fatty acids (linoleic acid and AA) levels (Table 3). TABLE 3 Effects of dietary fat on fatty acid composition of rat plasma Fatty acid Safllower Oil
Sardine Oil
Tuna Oil
g/lOOg total fatty acid c14:o C16:O C16:l C18:O C18:l Cl8:2 n-6
0.87 f 0.09 21.13 It 0.95a 2.12 f 0.37 13.26 f 1.40 12.43 f 0.71 27.03 f 4.33’
C20:4 n-6 C20:5 n-3
17.00 f 3.5ga
C22:5 n-3
o.oa o.oa
0.96 f 0.06 25.68 zk3.54b 2.71+ 0.95 13.34 k 1.49 11.23 f 0.84 16.81 f 1.7ab 11.07 _+0.87b
0.90 f 0.40 25.36 5 0.9gb 2.30 k 0.67 12.49 k 0.59 12.46 f 1.58 19.70 k 2.24b
6.39 f 1.91b 1.71 k 0.2ab
3.46 k 0.92’
12.01* 2.20b 1.00 f 0.2ab
4.66 k 0.47b 6.29 + 1.12’ C22:6 n-3 1.11 f 0.3ga n-6/n-3 2.4 f 0.7b 42.8 f 12.6’ 3.1+ 0.7b Values are mean f SD (n=4). Mean values within a row with different superscripts are significantly different at ~~0.05.
K.YAMAZAKI et al.
696
Feeding of both f&h oil diets significantly decreased the n-6/n-3 fatty acid ratio in the plasma compared with safnower diet (Table 3). Eficts on colonic Cl- secretion The bradykinin-induced I*,-responses of mucosal preparations were compared among the three diet groups after three weeks feeding. All the colonic tissues from the three groups responded to the serosal additionof bradykinin, which increased of I, in a dose-dependent manner (data not shown). The concentration of bradykinin of 10m7M was employed in this study since 10” M indomethacin abolished the I,,-response to bradykinin until the concentration of the latter reached 10m7 M. Colonic tissues of sardine oil-fed rats and tuna oil-fed rats showed significantly lower increases in the &+sponse to bradykinin than that in safflower oil-fed rats (Fig. 1). It is well established that the bradykinin-inducedincrease in Gc is primarily due to electrogenic Cl- secretion (12). This indicates therefore that the intake of sardine oil or tuna oil in this study was associated with reduction of bradykinin-inducedCl secretion inthedistal colon of the rat,
5
T
;
b
b
T
OISafflower
Sardine
Tuna
Oil
Oil
Oil
Fig. 1. Effects of dietary fish oils on the short-circuit current (I,) in response to bradykinin in rat distal colon. The increase in I,, in response to bradykinin (10-T M>was integrated for 10 min and given as the equivalent flux of monovalent ions. The values are means and standard error (n=4). The bars with different superscripts show significant differences (p
DIETARY FISH OIL AND FATTY ACIDS
699
Bradykinin-induced Cl- secretion occurs through the following three pathways; through prostaglandin synthesis (13), through the activation of enteric nerves (4), and through bradykinin receptors on enterocytes (14). The mediation through enteric nerves is neglected in the present study because the I,,-responses to bradykinin were measured using mucosal preparations (11) in the presence of a neuroblocker, tetrodotoxin. The abolition of the bradykinin-induced increase in I,, in the presence of indomethacin (10m6 M) strongly suggests mediation through prostaglandins in this study. In fact, prostaglandin E, (PGE& was detected in the serosal bathing solution after bradykinin-stimulation of the colonic mucosa (Fig. 2). This is compatible to the kallidin-induced release of prostaglandins into the serosal but not the mucosal solution bathing rat colonic mucosa (15), PGEz being the predominant product among the synthesized prostanoids (13). The production of PGE, in response to bradykinin stimulation was drastically decreased in the colonic mucosa of rats fed the fish oil-diets compared to in those fed the safflower oil-diet (Fig. 2). Those results indicate that dietary fish oils decrease the net accumulation of PGE, from AA in the colonic mucosa. The lamina propria, but not enterocytes, is the major source of kinin-generated prostaglandins (13), therefore, fibroblasts, lymphocytes, mast cells and vascular endothelial cells are candidates producing prostaglandins in response to bradykinin ( 12 ), This may be supported by much evidence that dietary fish oil supplements suppress the release of PGE, in peripheral lymphocytes, mast cells and peripheral macrophages (7).
10
I
a
Safflower oil
Sardine oil
Tuna oil
Fig. 2. Effects of dietary fish oils on prostaglandin E2 (PGE2) release into the serosal solution in response to bradykinin (10-T M) in rat distal colon. The values are means and standard error (n=4). The bars with different superscripts show significant differences (~~0.05) .
700
K.YAhJAZAKl et al.
Evidence is accumulating that diets supplemented with n-3 PUFA decrease the n-6/n-3 fatty acids ratio in plasma and tissues (7,16-19). This study also demonstrated that the diets supplemented with sardine oil (rich in EPA) and tuna oil (rich in DHA) drastically decreased the n-6/n-3 fatty acids ratio in rat plasma compared with the diet supplemented with &Bower oil (Table 3). The extra n-3 PUFA compete for the rate limiting enzyme, hepatic Abdesaturase and A6-desaturase, which normally converts dietary linoleic acid to AA (1). This results in reduced production of prostanoids derived &om AA. In the present study, the drastic decrease in the release of PGEZ from the colonic mucosa after bradykinin-stimulation should be reflected by the decrease in the n-6/n-3 fatty acids ratio induced by a diet with more n-3 PUFA (fish oils) than n-6 PUFA k&lower oil). Tuna oil is richer in DHA than sardine oil, both of which were used in this study. Dietary supplementation of both fish oils, however, had similar inhibitory effects on Clsecretion and PGEz production (Figs. 1 and 2), although DHA has a particularly inhibitory effect on the activity of A6-desaturase that forms AA from linoleic acid (20). It is able to speculate that the effects seen in here were due simply to the incorporation of EPA and DHA, which induce the decreased in AA level in the tissue lipid (17), rather than due to competition for transacylase enzymes. In diarrhea associated with mucosal inflammation, such as ileitis, colitis and other forms of inflammatory bowel disease, increased levels of prostanoids in the intestinal mucosa and blood have been observed (5). Therapeutic intervention has been attempted at several different sites along the pathway for prostanoid synthesis. This study demonstrated that nutritional intervention may successfully prevent prostanoids-induceddiarrhea through a decrease in the AA level due to dietary fish oils. REFERENCES 1.
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Freyland L, Vaagenes H, Asiedu DK, Garras A, Lie 0, Berge RK. Chronic administration of eicosapentaenoic acid and docosahexaenoic acid as ethyl esters reduced plasma cholesterol and changed the fatty acid composition in rat blood and organs. Lipids 1996; 31:169-178.
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Accepted for publication December 16, 1996.