Comparative Effects of Dietary Fat Manipulation on Fatty Acid Composition of Rat Stomach, Jejunum, and Colon Phospholipids

Transcription

1 J. Clin. Biochem. Nutr., 22, , 1997 Comparative Effects of Dietary Fat Manipulation on Fatty Acid Composition of Rat Stomach, Jejunum, and Colon Phospholipids Manohar GARG,* Robert BLAKE, and Brenda REINHARD Discipline of Nutrition and Dietetics, Faculty of Medical and Health Sciences, University of Newcastle, Callaghan, NSW 2308, Australia (Received November 11, 1996) Summary The phospholipid fatty acid composition of three parts of gastrointestinal tract (GIT) possessing different functions (stomach, jejunum, and colon) was examined to determine if these gut segments consistently respond to dietary fat. Three groups of eight weanling male Sprague-Dawley rats were fed one of three isocaloric, semi-synthetic, nutritionally adequate diets for six weeks. The fat type in the control diet was primarily beef tallow supplemented with sufficient linoleic acid (18: 2n-6) to prevent essential fatty acid deficiency, the n-6 diet contained high levels of 18:2n-6, and the n-3 diet provided high levels of eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids with similar levels of 18:2n-6 as in the control diet. Feeding the n-3 diet resulted in the incorporation of 20:5n-3 and 22:6n-3 into all the GIT segments examined; however, the incorporation was significantly higher in the jejunum and the colon, compared with that in the stomach, phospholipids. The arachidonic acid (20:4n-6) content was lower in the stomach, jejunum, and colon phospholipids following the feeding of the n-3 diet. The 18: 2n-6 content was found to be higher following consumption of the n-3 diet compared with that found for the control diet. The 20:4n-6 content was significantly higher in the jejunum than in the colon or stomach phospholipids, whereas the 18:2n-6 content was consistently higher in all the GIT segments in animals fed the n-6 diet. This diet group also exhibited lower levels of monounsaturated fatty acids in the stomach and lower levels of saturated fatty acids in the jejunum and the colon phospholipids. Thus, dietary fat manipulations inconsistently influence the phospholipid fatty acid composition of various parts of the GIT, suggesting that the functioning of the stomach, jejunum, and colon may be affected differently by alterations in the type of dietary fat ingested. *To whom correspondence should be addressed. 101

2 102 M. GARG, R. BLAKE, and B. REINHARD Key Words: dietary fats, n-6 fatty acids, n-3 fatty acids, fatty acid composition, gastrointestinal tract Changes in dietary fatty acid composition alter phospholipid fatty acid composition in a variety of tissue membranes including liver, kidney, heart, brain, and skeletal muscle [l-3]. The gastrointestinal tract (GIT) is a highly specialized organ, providing the first barrier between the external and internal environment of the body. The epithelial cells of GIT are exposed to continuously changing compositions of the nutrients in the lumen. The lipid components of these biological membranes, particularly the fatty acid constituents of the phospholipids, are one of the main determinants of the integrity and functionality of these tissues specialized in the digestion, absorption, and excretion of nutrients. Most fatty acids associated in phospholipids can be derived from stored fatty acids (adipose tissue) or synthesized de novo or may originate from the dietary sources or from the enterohepatic circulation of the bile phospholipids. In contrast, the "essential" 18:2n-6 and 18:3n-3 polyunsaturated fatty acids (PUFA) are not synthesized de novo and must be ingested as part of the diet. The longer chain desaturated and chain elongated products, such as 20:4n-6, 20:5n-3, and 22:6n-3, can either originate from the diet or, bile phospholipids, or be synthesized from their respective substrates (18:2n-6 and 18:3n-3) [4-6]. PUFAs play several roles in the function of epithelial cells of the GIT. Not only are PUFAs structural components of enterocyte membrane phospholipids and thus regulate membrane physicochemical properties, but they also serve as components of phospholipids required to form the chylomicron coat and as substrates for prostanoid (or eicosanoid) synthesis [7]. Eicosanoid biosynthesis has been demonstrated in every segment of the GIT and has been attributed numerous regulatory functions [8]. The majority of endogenous eicosanoids are derived from 20:4n-6, which is converted to a number of potent biological derivatives by the cyclooxygenase or lipoxygenase pathways. Dietary n-3 fatty acids, such as 20:5n-3 and 22:6n-3 are readily incorporated into the membrane phospholipids of various tissues at the expense of 20:4n-6, and have been shown to act as competitive inhibitors of the cyclooxygenase enzymes leading to decreased conversion of 20:4n-6 to prostaglandins in the gastric mucosa of rats [9]. These alterations may have important implications for eicosanoids as regulators of gastrointestinal function. Since the acyl content of phospholipids is highly specific, and the profile distribution of the molecular species is characteristic of tissue type, organelle, and the metabolic state [10], the purpose of this study was to explore the effects of the n-3 fatty acid from dietary fish oil and n-6 fatty acids from sunflower oil upon the fatty acid composition of membrane phospholipids contained in the stomach, jejunum, and colon of rats. The present study characterizes the fatty acid composition of phospholipids from segments of the GIT after a 6-week dietary interven- J. Clin. Biochem. Nutr.

3 DIETARY FAT AND PHOSPHOLIPIDS OF GASTROINTESTINAL TRACT 103 tion. This information is important when considering the role of dietary fats in the prevention of gastric ulcers, gastritis (stomach) and ulcerative colitis (large bowel); nutrient uptake by the enterocytes (small bowel) and colon cancer cells; etc. MATERIALS AND METHODS Animals and diets. All experiments were approved by the University of Newcastle Ethics Committee. Male Sprague Dawley rats (6 weeks of age) were housed individually in a well-ventilated room maintained at 22±2 C with light/ dark cycle of 12 h/ 12 h, and assigned randomly to one of 3 diet groups. Groups of 8 rats were fed a control diet, or diets enriched with either sunflower oil (n-6 diet) or fish oil (n-3 diet). Table L Composition of experimental diets.a a All diets were identical except for the type of fat. b Basal diet consisted of starch (20%, w/w), casein (27%), non-nutritive cellulose (5%), vitamin mix (1%), mineral mix (5%), L-methionine (0.25%), inositol (0.625%), and choline (0.275%). Table 2. Fatty acid composition of experimental diets. Vol. 22, No. 2, 1997

4 104 M. GARG, R. BLAKE, and B. REINHARD All diets were identical except for their lipid component. Each diet contained 20% fat, but the type of fat varied (Table 1). The control contained primarily hydrogenated beef tallow (90%) with sunflower seed oil (10%), containing 18:2n-6 to prevent essential fatty acid deficiency. The n-6 diet contained sunflower seed oil (100%) as a source of n-6 fatty acids. The n-3 diet contained beef tallow (65%), sunflower seed oil (10%) and MaxEPA oil (25%) (kindly donated by R.P. Scherer Pty. Ltd., Huntingdale, Melbourne, Australia) and therefore was enriched in n-3 Fig. 1. n-3 fatty acid content of rat stomach, jejunum, and colon phospholipids. Values without a common superscript are significantly different, p<0.05. Fig. 2. Linoleic acid content of rat stomach, jejunum, and colon phospholipids. Values without a common superscript are significantly different, p <0.05. J. Clin. Biochem. Nutr.

5 DIETARY FAT AND PHOSPHOLIPIDS OF GASTROINTESTINAL TRACT 105 Fig. 3. Arachidonic acid content of rat stomach, jejunum, and colon phospholipids. Values without a common superscript are significantly different, p<0.05. fatty acids. The fatty acid compositions of the experimental diets are listed in Table 2. Fresh diets were supplied daily, and the rats had free access to water. Food intake and body mass were recorded throughout the study. Following a 6-week feeding period, rats were sacrificed by COZ asphyxiation. The stomach, jejunum, and colon were excised and flushed with ice-cold PBS to remove contents inside the gastrointestinal tract. After removal of contaminating materials, tissues were stored at -20 C until analyzed. Analyses of phospholipids. The excised tissue segments were cleaned thoroughly to remove any particulate matter and homogenized in 8-10 ml PBS. Total lipids were extracted from individual samples as well as from the experimental diets with chloroform : methanol (2: 1, v/v) containing 0.005% (w/v) butylated hydroxytoluene by a modification of the method of Folch et al. [11]. Phospholipids were isolated on thin-layer chromatography plates (silica gel G 30 X 20, Alltech, Sydney, Australia) by use of a solvent system comprising hexane : diethyl ether : acetic acid (85: 15: 1, v/v/v) [12]. Phospholipid spots were scraped off the plates and methylated with 14% (w/ w) BF3-methanol reagent at 100 C for 60 min [13]. Fatty acid methyl esters were extracted in 200 pl toluene and analyzed by duplicate injection into a flame ionization capillary gas chromatograph (5890A, Hewlett Packard, Little Falls, DE, USA) connected to an integrator (3392A, Hewlett Packard) [14]. The chromatograph was equipped with a 30-m fused carbon-silica capillary column (0.25 mm internal diameter) coated with cyanopropylphenyl (type DB-225, 25pm thickness, 25% w/w coating) (J & W Scientific, CA). For an overall run time of 25 min, the initial temperature was 170 C (for 2 min) which was then increased to 210 C at a rate of 5 C/min, followed by an increase to 212 C at a rate of 0.2 C/min, Vol. 22, No. 2, 1997

6 106 M. GARG, R. BLAKE, and B. REINHARD and then finally to 220 C at l0 C/min. Fatty acid methyl ester peaks were identified by reference to injected authentic standard mixtures of fatty acid methyl esters (Nu Chek Prep Inc., Elysian, MN), and to an internal standard, nonedecanoic acid (C 19;0, Nu Chek Prep Inc.). Statistical analysis. All results were expressed as means +SEM. A one-way ANOVA was used to detect significant differences between diet type with a chosen level of significance of p<0.05. When ANOVA was significant, the Scheffe F-test was employed to detect individual differences within groups at 95%. Statistical analysis was carried out with a Stat-view software program. RESULTS Food consumption and body weights Rats fed the three diets did not differ significantly in their patterns of food consumption throughout the 6-week feeding period, during which approximately 25 g/day was consumed. All groups gained weight; however, the fish oil treatment group did not gain as much as the other diet groups. The mean body mass increased by g in the control, 236±10 g in the n-6 treatment group, and 191 ± 12 g in the n-3 diet group. A similar observation of comparatively lower body mass in a dietary fish oil group was reported previously [15]. Fatty acid composition in the stomach phospholipids The phospholipid fatty acid composition of the stomach (Table 3) was slightly influenced by the fatty acid composition of the diet. There were no Table 3. Fatty acid composition of stomach phospholipids of rats fed fat-manipulated diets. Values without a common superscript are statistically different at 95% (Scheffe F-test). J. Clin. Biochem. Nutr.

7 DIETARY FAT AND PHOSPHOLIPIDS OF GASTROINTESTINAL TRACT 107 significant differences in phospholipid saturated fatty acids (SFAs) among the three dietary groups, however, differences were observed in the relative abundance of mono- and polyunsaturated fatty acids (MUFAs and PUFAs, respectively) of the phospholipids. In the stomach of the rats fed the sunflower (n-6) diet, there was a marked increase in 18:2n-6 (Fig. 2) with a corresponding decrease in 18:In-9, compared with these levels in the control; but the fish oil (n-3) group had similar levels of these fatty acids. The level of 20:4n-6 was relatively lower with the n-3 treatment group compared with the control one, with the n-6 treatment group having intermediate values (Fig. 3). As expected, rats fed the fish oil diet contained more 20:5n-3 (EPA) and 22:6n-3 (DHA) in their phospholipids compared with the other diet groups (Fig. 1). Fatty acid composition in the jejunum phospholipids The phospholipid fatty acid composition of the jejunum (Table 4) was strongly influenced by, but did not directly reflect, the fatty acid composition of the diet. Differences were observed in all fatty acid types including SFA, MUFA, and PUFA. In the jejunum of rats fed the fish oil diet, the phospholipid SFA content was significantly lower than that in the control group (with the exception of 18:0), with the n-6 diet group sharing similarities with the fish oil treatment (16:0 and 20:0) and the control (22:0 and 24:0). A trend of increased phospholipid 18:2n-6 corresponding to decreased phospholipid 18:1n-9 was observed across the Table 4. Fatty acid composition of jejunum phospholipids of rats fed fat-manipulated diets. Values without a common superscript are statistically different at 95% (Scheffe F-test). Vol. 22, No. 2, 1997

8 108 M. GARG, R. BLAKE, and B. REINHARD Table 5. Fatty acid composition of colon phospholipids of rats fed fat-manipulated diets. Values without a common superscript are statistically different at 95% (Scheffe F-test). diet groups, where the n-6 fed rats had the highest 18:2n-6 content (Fig. 2) and lowest 18:ln-9, the control had the highest 18:ln-9 and lowest 18:2n-6, and the n-3 fed rats showed values in between these ranges. Phospholipid 20:4n-6 was highest in the jejunum of n-6 diet-fed rats (Fig. 3). Only the n-3 diet-fed rats contained 20:5n-3, 22:5n-3, and 22:6n-3 was observed with all diets, with the highest level occurring in the fish oil group (Fig. 1). Effect of dietary fat on phospholipid fatty acid composition in the colon In one aspect similar to the jejunum, the phospholipid fatty acid composition of the colon (Table 5) was strongly influenced by the fatty acid composition of the diet. Rats fed the sunflower and fish oil diets had significantly lower short chain (16:0, 18:0, and 20:0) SFA compared with the control treatment. Phospholipid 18:2n-6 was highest and 18:1 n-9 lowest in the n-6 fed rats. The colon of rats fed the fish oil diet contained lower levels of 20:4n-6, whereas the control and n-6 diet treatments equally contained higher levels (Fig. 3). Only the n-3 diet-fed rats contained EPA and 22:5n-3. DHA was significantly higher in the phospholipids of the fish oil diet group (Fig. 1). DISCUSSION This study demonstrated that the phospholipid fatty acid composition of the stomach, jejunum, and colon can be influenced by dietary fat manipulation. After J. Clin. Biochem. Nutr.

9 DIETARY FAT AND PHOSPHOLIPIDS OF GASTROINTESTINAL TRACT weeks, the n-3 and n-6 diet groups produced striking changes in phospholipid fatty acids, but the magnitude of changes was not uniform in the three segments of the GIT examined. As predicted, the feeding of the n-3 diet resulted in the enrichment of all three gastrointestinal segments examined, in 20:5n-3 and 22:6n-3. Incorporation of n-3 fatty acids was considerably higher in the jejunum and the colon, compared with that in the stomach, phospholipids following the feeding of the n-3 diet despite the fact that n-3 fatty acid values were similar in all three parts of the GIT in the n-6 diet-fed animals (Fig. 1). This is suggestive evidence that different parts of the GIT differ in their ability to incorporate dietary n-3 fatty acids and that it is not a simple case of replacement of n-6 PUFA by n-3 PUFA due to competition between the two families of fatty acids for incorporation into the membrane phospholipids. Feeding the n-6 diet resulted in higher incorporation of linoleic acid into stomach, jejunum, and colon phospholipids (Fig. 2). The 20:4n-6 content in the jejunum was the highest with the n-6 diet but was similar for all three diets in the stomach and the colon phospholipids (Fig. 3). The content of 20:2n-6, a chain elongation product of 18:2n-6, was higher in the jejunum and the colon; whereas 20:2n-6 was not detectable in the stomach phospholipids in the n-6 diet-fed animals. Thus, feeding a diet rich in 18:2n-6 does not induce similar increases in 20:2n-6 or 20:4n-6 throughout the GIT but appears to be specific to the proximal small intestine (jejunum) and/or colon. Consumption of the n-3 diet resulted in a lower 20:4n-6 content in the stomach and colon phospholipids. In the jejunum phospholipids, the fish oil-fed group exhibited a trend towards a reduced arachidonic acid content. In the GIT, arachidonic acid may originate from the diet (meat membrane phospholipids), bile (phospholipid) reabsorption and synthesis through desaturation and chain elongation of linoleic acid. As the rat diets were devoid of meat, the 20:4n-6 thus originated from either the bile or endogenous synthesis. A diet rich in n-3 PUFA may inhibit synthesis of 20:4n-6 in the intestinal mucosa or may reduce the 20:4n-6 concentration in the bile phospholipids, as previously demonstrated [16]. The GIT actively synthesizes eicosanoids (prostaglandins and leukotrienes) using 20:4n-6 as a substrate, and eicosanoid synthesis is increased in certain GIT diseases including ulcerative colitis, colon cancer, and other inflammatory conditions. Since n-3 PUFA consumption reduces 20:4n-6 levels in the GIT segments, diets rich in these fatty acids may play an important role in the prevention of these diseases. It is noteworthy that the 18:2n-6 content of phospholipids in all segments of the GIT was higher in rats fed the n-3 diet than in those given the control diet, despite the fact that the dietary content of 18:2n-6 was similar (10%) in the two diets. This is consistent with the previous observations that dietary n-3 fatty acids inhibit the conversion of 18:2n-6 into 20:4n-6 [16]. The functions of the three parts of the GIT examined are vastly different: the stomach acts as a food reservoir and secretes acid and enzymes (pepsinogen) for Vol. 22, No. 2, 1997

10 110 M. GARG, R. BLAKE, and B. REINHARD digestion; the jejunum is involved in digestion, absorption, and transport of nutrients; and the colon absorbs water and electrolytes. Thus, the requirements of the three gut segments for n-6 and n-3 PUFA are different. The n-6 and n-3 PUFA requirements of the jejunal enterocyte phospholipids are unique, i.e., they are continuously required for the chylomicron coat formation for fat transport from the small intestine. Rapid rate of cell turnover in the jejunum (48-72 h) requires an equally early rapid supply of n-6 and/or n-3 PUFA for membrane phospholipid biogenesis. The rate of cell turnover in the stomach is not as rapid as in the jejunum (48-72 h) and the colon (3-8 days). It is therefore likely that 6 weeks of feeding fat-manipulated diets has resulted in maximum alterations in the jejunum and the colon phospholipids but that in the stomach, it is only partly achieved due to slower rate of cell turnover. In conclusion, these results suggest that the magnitude of dietary fatty acid incorporation into phospholipids and the impact of dietary fat manipulations on fatty acid composition vary in different parts of the GIT. These differences should be taken into consideration when assessing the biochemical and clinical effects of dietary fat manipulations in the prevention of gastrointestinal disorders. This project was funded by the Australian Research Council Small Grant Scheme. REFERENCES 1. Ayre, K.J., and Hulbert, A.J. (1996): Dietary fatty acid profile influences the composition of skeletal muscle in rats. J. Nutr., 126, Charnock, J.S., Abeywardena, M.Y., Poletti, V.M., and McLennan, P.L. (1992): Differences in fatty acid composition of various tissues of the marmoset monkey (Callithrix jacchus) after different lipid supplemented diets. Comp. Biochem. PhysioL, 92A, Clandinin, M.T., Cheema, S., Field, C.J., Garg, M.L., Venketraman, J., and Clandinin, T.R. (1991): Dietary fat: Exogenous determination of membrane structure and cell function. FASEB J, 5, Garg, M.L., Keelan, M., Thomson, A.B.R., and Clandinin, M.T. (1988): Fatty acid desaturation in the intestinal mucosa. Biochim. Biophys. Acta, 958, Xu, N., and Nilsson, A. (1996): Uptake and interconversion of plasma unesterified 14C linoleic acid by gastrointestinal tract and blood forming tissues: An experimental study in rat. J. Nutr. Biochem., 7, Nilsson, A., and Becker, W. (1995): Uptake and interconversion of plasma unesterified n-3 linoleic acid by gastrointestinal tract of the rat. Am. J. Physiol., 268, G732-G Garg, M.L., Keelan, M., Thomson, A.B.R., and Clandinin, M.T. (1990): Intestinal microsome: Polyunsaturated fatty acid metabolism and regulation of enterocyte transport properties. Can. J. Physiol. Pharmacol., 68, Whittle, B.J. (1989): Role of eicosanoids and related mediators in gastric mucosal ulceration. Scand J. Gastroenterol (Suppl.), 162, Croft, K.D., Beilin, L.J., Vandongen, R., and Mathews, E. (1984): Dietary modification of fatty acid and prostaglandin synthesis in the rat. Effect of variations in the level of dietary fat. Biochim. Biophys. Acta, 795, Yorek, M.A. (1993): Biological distribution, in Phospholipid Handbook, ed. by Ceve, G., Marcel Dekker, New York, pp Folch, J., Lees, M., and Slone-Stanley, G.H. (1957): A simple method for the isolation of J Clin. Biochem. Nuts:

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