Short bowel syndrome is a malabsorptive state that occurs because of resection of the small intestine and the reduced surface area that results (1-2). Long-term survival after bowel resection is highly dependent on the process of mucosal adaptation, a process of hyperplasia in the remaining intestine that gradually improves nutrient absorption (3-6). This process is dependent on enteral nutrition, which contains luminal nutrients that provide stimulation for intestinal adaptation (7). Enteral feedings are therefore the key feature of the therapy for short bowel syndrome. In cases in which this treatment fails, intestinal transplantation or long-term home parental nutrition remain the only options.
Recently, specific nutrients and other trophic factors have been shown to be highly sensitive stimulants of intestinal adaptation (8-11). Several trophic factors may be important regulators, including insulin-like growth factor (IGF)-I, enteroglucagon precursors, and prostaglandins (13,14). Certain lipids, primarily long chain fatty acids and especially polyunsaturated long chain fatty acids from marine oil, are also highly trophic to the small intestine. For example, past studies have shown that menhaden oil stimulates adaptation (12). The main purpose of this study was to determine whether dietary long chain polyunsaturated fatty acids (LCPUFAs) improve adaptation after resection and to define the quantity needed to promote optimal response. The study was conducted in two parts. First we evaluated the effects of various degrees of resection, 60%, 70%, and 80% on mucosal adaptation in rats using two diets with different lipid composition. In a second experiment, a dose response to lipids was assessed after an 80% bowel resection.
MATERIALS AND METHODS
In experiment 1, 40 male Sprague-Dawley rats (mean weight, 150 ± 10 g; Sasco, St. Louis, MO, U.S.A.) were acclimated for 3 days to laboratory conditions and cared for according to the guidelines of the Animal Review Committee at the University of Nebraska Medical Center, Omaha, Nebraska, U.S.A. Rats were divided into three groups receiving 60%, 70%, and 80% small bowel resection to determine the appropriate degree of resection that results in the most stimulation of adaptation. Rats were then further divided into two dietary groups and fed diets in pairs: one containing safflower oil as the primary fat source, the other containing relatively high levels of docosahexaenoic acid (DHA) and arachidonic acid (AA) as the primary fat source (Bio-Serv, Frenchtown, NJ, U.S.A.). All diets contained 30% of energy as fat.
In the second experiment, 41 rats received an 80% small bowel resection and were fed one of our diets in pairs, each differing in the fat blend providing 30% of energy as fat. Six control rats received sham operations and were fed the control diet A, which contained a basic rat diet with a fat source of 10% soy oil and 90% hydrogenated coconut oil. The fat in diet B consisted of 5% AA, 3.3% DHA, 10% soy oil, and 81.7% hydrogenated coconut oil. Diet C consisted of 15% AA, 10% DHA, 10% soy oil, and 65% hydrogenated coconut oil. Diet D consisted of 45% AA, 30% DHA, 10% soy oil, and 15% hydrogenated coconut oil (Bio-Serv).
All rats were anesthetized with an intermuscular injection of 9.75 mg/kg xylazine (Burns Veterinary Supply, Rockville Centre, NY, U.S.A.) and 60 mg/kg ketamine (Fort Dodge Laboratories, Fort Dodge, IA, U.S.A.). The abdomen was shaved and scrubbed with povidone-iodine (Betadine; Purdue Frederick, Norwalk, CT, U.S.A.). A mid-line incision was made, and the small intestine isolated. In the 80% resection, the bowel was resected between a point 3 cm distal to the ligament of Treitz and 7 cm proximal to the ileocecal valve. In the 70% resection, the bowel was resected from 4 cm distal to the ligament of Treitz to 10 cm proximal to the ileocecal valve. In the 60% resection, the bowel was resected from 5 cm distal to the ligament of Treitz to 15 cm proximal to the ileocecal valve. Intervening mesenteric vessels were ligated with 5-0 silk. The remaining jejunum was reanastomosed to the distal ileum using interrupted sutures of 6-0 silk. Control animals were subjected to a sham operation consisting of a division of the bowel and subsequent reanastomosis. All animals were fed no food for 24 hours after surgery but received 5% glucose water containing 0.0225% tetracycline ad libitum.
All animals in experiments 1 and 2 were killed 14 days after surgery by cervical dislocation. The small intestine from the pylorus to the ileocecal valve was removed, stripped from its mesentery, and divided at the anastomosis. The bowel was drenched with cold isotonic saline, and the ends 1 cm from the anastomosis were removed, because this tissue is subject to hyperplastic artifact due to vascular neoplasia. Both the remaining duodenojejunum and ileum were measured for length and subsequently scraped with a glass slide on a cold glass surface to remove mucosa from underlying tissue. Tissue was then homogenized in 50 mg/ml deionized water for each sample. Mucosal protein was determined by the method of Lowry et al. (15) Deoxyribonucleic acid (DNA) was extracted and measured according to Burton (16) as modified by Giles and Myers (17). Sucrase activity was determined as described by Dahlqvist (18). All results were expressed as mean ± standard error of the mean for each group of animals. Significant differences among the means were analyzed using analysis of variance and Tukey's posttest. Differences were considered statistically significant at p < 0.05.
There were no differences in body weights among the groups of animals at the end of the study. Mucosal mass and DNA, protein, and sucrase activities in the duodenum and ileum are shown in Table 1. Mucosal mass increased significantly in the ileum as the degree of resection increased. The results for 60% resection vary in each dietary group. We conclude that a 60% resection does not provide an appropriate model for short bowel syndrome. Animals undergoing 80% resection and fed the AA-DHA diet provided the highest level of intestinal hyperplasia in the ileum. The diet containing DHA and AA induced an increase in ileal mucosal mass when compared with the results of the control diet containing safflower oil (p = 0.029). Similarly, increases in DNA, protein, and sucrase activities were observed in the ileum, with an increase in activity as the percentage of resection increased and also when DHA and AA were added to the diet. Significant differences were not observed in the duodenum.
There were no differences in body weights among the dietary groups of animals at the end of the study. All resected animals had an increase in mucosal mass and in DNA, protein, and sucrase activities when compared with results in control animals undergoing the sham operation (Table 2). Mucosal mass in the ileum increased significantly with diet D (p = 0.007) when compared with the effect of diet A. Adaptation increased progressively as LCPUFAs were added to the diet. Significant differences were also seen in mucosal protein levels and DNA in the ileum with diet D when compared with levels with the base diet A. However, sucrase activity in the ileum showed no significant differences among the diets. Differences were not observed in the duodenum.
The addition of AA and DHA to the diet of animals with short bowel syndrome stimulates an increase in mucosal adaptation in a dose-dependent manner. In experiment 1, we established that an 80% resection proved to be the best model for studying adaptation, probably because of the greater stimulus for adaptation. Eighty percent resection improved mucosal mass in the ileum significantly when compared with the results of 60% or 70% resection. The addition of AA and DHA to the diet further enhanced adaptation when compared with adaptation with the safflower control diet.
The second experiment confirmed the first observation that the addition of LCPUFAs stimulates intestinal adaptation; additionally, the results defined this as a linear, dose-dependent phenomenon. The diet containing 45% AA and 30% DHA as the main fat source resulted in the highest levels of DNA and protein and the greatest mucosal mass in the ileum after an 80% resection. The results were primarily observed in the ileum, most likely because it has a greater capacity for adaptation than the duodenum and jejunum.
The adaptation process begins within 24 to 48 hours after resection (2) with epithelial hyperplasia in the intestinal crypts. Production of new epithelial cells results in lengthening of intestinal villi, increased absorptive surface area, and improved digestive and absorptive function (2). Mucosal hyperplasia does not occur in the absence of enteral nutrition, and atrophy may occur if enteral nutrition is not provided in an appropriate manner. The means by which nutrients stimulate intestinal adaptation is dependent on several events, including direct contact with the epithelial cells; stimulation of the flow of gastric, pancreatic, and biliary secretions; and stimulation of the production of trophic hormones.
The mechanism by which AA and DHA stimulate adaptation is open to speculation. Long chain polyunsaturated fatty acids are structural components of cell membranes and precursors of eicosanoids (19). Prostaglandins derived from AA are considered local hormones that are short lived and are involved in blood flow regulation, modulation of inflammation, and control of transport of ions across membranes, all of which may be important in the process of adaptation. Rats fed diets deficient in essential fatty acids have an impaired mucosal hyperplasia response (20). Also, intestinal adaptation is reduced when rats are administered aspirin or dexamethasone, which inhibit the synthesis of prostaglandins (21). When 16,16 dimethyl-prostaglandin E2 was administered to rats after resection, it significantly increased the adaptation response (14). Similarly, studies in canine small intestine also show that treatment with prostaglandins increases cell proliferation (22).
These changes are comparable with those observed after the feeding of menhaden oil containing eicosapentaenoic acid and DHA. Our data demonstrate that dietary lipid mixtures of AA and DHA stimulate mucosal hyperplasia after resection. Arachidonic acid is the substrate for synthesis of prostaglandins, and eicosapentaenoic acid may also be a substrate for synthesizing similar compounds with perhaps similar metabolic activities. Previous studies from our laboratory (not reported) showed no evidence that DHA stimulated or inhibited gut hyperplasia. However, AA and DHA were combined in this study to support plasma and erythrocyte lipid levels (23,24). It is interesting that our previous results suggest that diets containing predominantly linoleic acid, a precursor to AA, are not a potent stimulator of hyperplasia. However, that eicosapentaenoic acid and AA, direct prostaglandin precursors, are potent stimulators of adaptation suggests that prostaglandins may play a major role in this process.
Polyunsaturated fatty acids appear to have several important metabolic functions. Most of the interest in this area has been derived from studies in premature animals and infants. Recently, there has been interest in adding LCPUFAs to nutritional products, specifically to premature infant formula (25). Studies have shown that dietary AA and DHA are necessary to support plasma and erythrocyte levels of these fatty acids in formula-fed low birth weight infants. In addition, formulas containing n-3 LCPUFAs have been shown to improve visual acuity and cognitive development in preterm infants when compared with results obtained with control formulas. Several different sources of LCPUFAs have been developed for use in formulas, including fermentation oils, such as the ones employed in the present study. These oils have been shown to be highly bioavailable when incorporated into a low birth weight infant formula (23) and to support normal growth in these infants (24).
The use of LCPUFAs and other trophic factors in patients with short bowel syndrome may play an eventual role in enhancing intestinal adaptation. Additional studies in this area including animal models and well-controlled studies in humans with various nutrients, and trophic factors are important in determining the optimal therapy for patients with short bowel syndrome.
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