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Effects of Dietary Lipids on Recovery From Mucosal Injury
GASTROENTEROLOGY 1990;98:1226-1231
Effects of Dietary Lipids on Recovery From Mucosal Injury JON A. VANDERHOOF, JUNG H. Y. PARK, HAMID MOHAMMADPOUR, and DARCY BLACKWOOD Department of Pediatrics, Creighton University School of Medicine, Omaha, Nebraska
The present studies were conducted to determine if diets containing a large amount of fat stimulate the regeneration of damaged intestinal mucosa in the presence or absence of essential fatty acid deficiency. To simulate injury, male Sprague-Dawley rats were given methotrexate, 2.5 mg/kg body wt, subcutaneously for 3 consecutive days. Twenty-four hours after the last methotrexate injection, rats were placed on diets containing either 00/0,1 %, or 10% samower oil. Mucosal weight, protein, deoxyribonucleic acid, maltase, sucrase, lactase, alkaline phosphatase, leucine aminopeptidase, and fatty acids were all determined 3 and 12 days after methotrexate. Crypt-cell production rates were also determined. Essential fatty acid deficiency was confirmed in the 0% safflower oil group, in which triene-tetraene ratios were >0.4. Mucosal weight, deoxyribonucleic acid, protein content, and villus height were all greater in the 1 % samower oil group than in the 0% group at 12 days. In the ileum, l-h thymidine incorporation was greater in the 0% sam ower oil group than in the other two groups. No differences in any of the parameters studied were observed between the 1 % and 10% groups. These results suggest that diets deficient in essential fatty acids may impair the recovery of intestinal mucosa from injury.
W
orldwide, infectious diarrhea causes the deaths of more children than any other disease process (1). This is especially true in underdeveloped countries, where the preexistence of malnutrition markedly worsens the prognosis (2). Most intestinal viruses, as well as many bacteria and parasites, cause severe injury to the small intestine. Enteral feedings administered early in the course of therapy may minimize malnutrition and improve prognosis (3). However, little is known about how specific nutrients affect the regeneration process. Previous studies in our laboratory have demonstrated that long-chain triglycerides
are effective in stimulating mucosal hyperplasia following massive resection of the small intestine (4). Further studies have suggested that these changes may be partly related to linoleic acid content in the diet, and essential fatty acid deficiency might impair mucosal adaptation (5). In other studies, animals fed diets containing 5% linoleic acid had enhanced mucosal adaptation in the remaining intestine compared with animals fed diets containing only 1 % linoleic acid (6). Therefore, the present studies were conducted to determine (a) if diet containing 10% safflower oil is advantageous to the regeneration of intestinal mucosa from methotrexate-induced injury compared with diet containing 1 % safflower oil and (b) if dietary essential fatty acid deficiency impairs this process. Materials and Methods
Materials Sodium methotrexate was obtained from Lederle Laboratories Division (Pearle River, N.Y.). [Methyl3H]thymidine was obtained from ICN Radiochemicals (Irvine, Calif.). Arachidonic acid methyl ester was purchased from Alltech Associates, Inc. (Deerfield, 111.). Eicosatrienoic acid was purchased from Cayman Chemical Co. (Ann Arbor, Mich.). The fat-free, 1 % safflower oil, and 100/0 safflower oil diets were obtained from Teklad (Madison, Wis.).
Animal Models To simulate the injury produced by an enteric virus, male Sprague-Dawley rats were given methotrexate, 2.5 mg/kg body wt, subcutaneously for 3 consecutive days. Preliminary studies in our laboratory have demonstrated that this dosage produces a consistent mucosal injury within 24 h after the last injection which subsequently resolves over a 2-wk recovery period (Figures 1 and 2). Severe inflammatory changes are apparent after 3 days with both polymorpho© 1990 by the American Gastroenterological Association
0016-5085/90/$3.00
May 1990
DIETARY LIPIDS AND MUCOSAL INJURY 1227
containing 00/0, 1%, or 10% safflower oil (Table 1). The fat-free diet was designed to maintain the essential fatty acid-deficient state. The 1 % safflower oil diet was chosen because safflower oil contains >75% linoleic acid and adequately meets the essential fatty acid requirements for the rat (7). The 10% safflower oil diet was designed to provide a higher concentration of fatty acids to determine whether this diet would stimulate mucosal healing and regeneration faster than 1 % fat diet. Animals were pair-fed to insure identical protein, calorie, vitamin, fiber, and mineral intake.
Biochemical, Physiological, and Histological Analyses Three days after the last injection of methotrexate, the rats were killed by cervical dislocation under CO 2 anesthesia. The small intestine, from the ligament of Trietz to the ileocecal valve, was removed and separated into proximal (jejunum) and distal (ileum) halves. The bowel was rinsed with ice-cold saline. The proximal 2 cm of each segment was fixed in buffered formalin for histological analysis. Tissue was fixed, sectioned (4-~m thick), mounted on glass slides, and stained with H&E. A total of 10 villi and crypts were measured in
Figure 1. Jejunal biopsy 3 days after methotrexate in the rat (original magnification x 100). Marked mononuclear cell infiltrate is seen in the lamina propria with scattered polymorphonuclear cells and crypt abscess formation. Villi are severely blunted with a 1:1 crypt-villus ratio. Surface epithelium is severely damaged.
nuclear and lymphocytic infiltrates in the lamina propria, marked villus blunting, loss of surface epithelium, and crypt abscess formation. Nearly all changes resolved by day 12. This effect occurs primarily in the proximal small intestine, but it is also produced to a lesser degree in the distal small intestine.
Experimental Design The animals were cared for according to the guidelines of the Animal Review Committee at the University of Nebraska Medical Center. Forty-two male, weanling, Sprague-Dawley rats (49 ± 4 g body wt) were obtained from Sasco, Inc. (Omaha, Neb.). The rats were housed individually and fed a non purified diet (Rodent Laboratory Chow; Ralston Purina Co., St. Louis, Mo.) and tap water ad libitum for 3 days to acclimate them to laboratory conditions. After this acclimation period, the animals were fed a modified AIN-76 diet (Table 1) containing no fat for 34 days to induce a biochemical essential fatty acid-deficient state. Animals were then given methotrexate as described above. Twentyfour hours after the last methotrexate injection, the animals were divided into three groups of 14 and placed on diets
Figure 2. Jejunal biopsy 12 days after methotrexate in the rat (original magnification x 100). Inflammation is resolved, surface epithelium is normal, and the crypt-villus ratio has normalized at 1:5.
GASTROENTEROLOGY Yol. 98, No.5
1228 YANDERHOOF ET AL.
Table 1. Composition of Diets (g/kg)
Fat-free
Safflower oil
10% Safflower oil
200.0 3.0 546.98 150.0 50.0 35.0 3.0 10.0 2.0 0.02 0.0
203.0 3.05 530.07 152.3 50.8 35.53 3.05 10.15 2.03 0.02 10.0
229.2 3.44 380.69 172.0 57.3 40.11 3.44 11.5 2.3 0.02 100.0
10/0
Casein, "vitamin-free" test DL-methionine Dextrose, monohydrate Maltodextrin Cellulose (fiber) Mineral mix, AIN-76 (170915) Calcium carbonate, CaC0 3 Yitamin mix, AIN-76A (40077) Choline bitartrate Ethoxyquin (antioxidant) Safflower oil
All diet ingredients except safflower oil (ICN Biomedicals, Costa Mesa, Calif.) were purchased from Teklad, Madison, Wis.
each segment with a micrometer eyepiece to determine villus height and crypt depth. The mucosa was scraped from a 25-cm portion of each segment using a glass slide. This tissue was weighed and used to perform several analyses. These included mucosal protein according to Lowry et al. (8J; maltase, sucrase, and lactase activities according to Dahlqvist (9J; alkaline phosphatase activity by the method of Sommer (10J; and leucine aminopeptidase activity by the method of Goldbarg and Rutenburg (11J. Deoxyribonucleic acid was extracted and measured according to the method of Burton (12J, as modified by Giles and Myers (13J. Total lipids in the mucosa were extracted with chloroformmethanol (2:1J in the presence of 1 % NaCI according to the method of Folch et al. (14J. The organic layer was dried, and methyl esters of fatty acids were formed in the presence of 6 N HCI-methanol (1:5J and dried under nitrogen. Methyl esters of fatty acids were analyzed by gas liquid chromatography (Perkin Elmer Sigma 2, Norwalk, Conn.J. Integrated peaks for eicosatrienoic acid (20:3, n-9J and eicosatetraenoic (20:4, n-6J acid were compared to determine triene-tetraene ratios according to the method of Holman (15J.
dietary linoleic acid early in the course of therapy for mucosal injury. The cumulative effects of therapy over a subsequent convalescent period were then studied by repeating the entire set of studies 12 days after methotrexate administration. All animal groups, feeding protocols, and analyses were conducted exactly as described above except that animals were killed 12 days, rather than 3 days, after completion of methotrexate administration.
Analysis of Deoxyribonucleic Acid Synthesis A second experiment was performed to determine how dietary safflower oil influences epithelial cell turnover following mucosal injury. These animals were injected intraperitoneally with [methyVH]thymidine (2 /LCi/g body wtJ 7 days after the last injection of methotrexate. One hour after this injection, the rats were killed and the small intestines were removed and rinsed with ice-cold saline. A small sample (1.5 cmJ of intestine was obtained from the jejunum and ileum to determine the amount of radioactivity incorporated into DNA per crypt. The tissue was fixed in cold Carnoy's solution for 2 h, stored in 70% ethanol, and then transferred to 500/0 and 30% ethanol for 5 min each. The samples were hydrolyzed in 1 N HCI at 60°C for exactly 7 min. This reaction was stopped by transferring the tissue to cold Schiff's reagent. The staining process continued for 1 h, and the microdissection of 50 crypts was performed by the method similar to that of Wimber et al. (16J. The crypts were dissolved in base and neutralized with acetic acid, and the radioactivity was determined by liquid scintillation counting.
Statistical Analysis All data were calculated as mean ± SEM and were analyzed by analysis of variance. The significant differences among means were further analyzed using Duncan's multiple range test (17J.
Results
Twelve-Day Study Evaluation of the animals 3 days after methotrexate administration allowed for a comparison of the effect of
Mucosal weight and DNA and protein content in the jejunum and ileum are shown in Table 2. These parameters did not show any significant differences
Table 2. Effects of Dietary Lipids on Mucosal Weight and Deoxyribonucleic Acid 3 Day 0%0 Jejunum Mucosal wt (mg/cm) DNA (lLg/cm) Protein (mg/cm) Ileum Mucosal wt (mg/cm) DNA (lLg/cm) Protein (mg/cm)
42.4 132 4.28 43.9 168 5.44
±
1%
3.9 b
± 21
c
± 0.74c ± 2.7c ± 15
12 Day
c
± 0.46
c
°Percentage of safflower oil in diets. bMean (p < 0.05).
±
47.5 142 4.47
± 14
44.0 162 6.14
± l1
SEM, n
=
10% c c
± 3.7
± 0.61c ± 3.1c
c
± 0.35
c
0% c c
44.2 137 4.51
± 5.9
45.0 163 5.70
± 4.3
± 15 ±
0.70 c c
± 15 c ± O.73
c
1%
54.3 179 5.35
± 1.1c
40.4 174 4.61
± 1.3
±
8e
± 0.42c
±
c
9c
± 0.17c
62.0 192 6.73
±
2.2 d
±
0.46 d
49.0 205 5.29
± ±
1.2 d 4d
±
0.24d
c ± 15
10% 57.5 181 6.00 46.5 194 5.51
± ± ± ± ± ±
1.8c.d 7C 0.36 c.d 2.5 d gc.d 0.34d
7. c.dYalues with different superscripts are statistically different among dietary groups
May 1990
DIETARY LIPIDS AND MUCOSAL INJURY 1229
among the three dietary groups in the 3-day study. However, in the 12-day study, mucosal weight and DNA and protein content were higher in 1 % safflower oil group than in the fat-free group except that the difference in DNA in the jejunum did not reach statistical significance. There were no differences in mucosal weight or DNA and protein content between the 1 % and 10% safflower oil groups. The differences in the jejunum were not statistically significant between the 0% and 10% safflower oil groups. There were no differences in crypt depth among the three dietary groups at both 3 and 12 days (data not shown). However, measurements of villus height in the ileum for both the 1 % (506 ± 19 ~m) and 10% safflower oil groups (520 ± 17 ~m) were higher than for the fat-free group (445 ± 14 ~m) at 12 days (p < 0.05). Table 3 shows the results of triene-tetraene ratios in intestinal mucosa . At 3 days, the ratios were > 0.4 in the groups that received diets containing 0% and 1 % safflower oil. At 12 days, the ratios were returned to normal in the 1 % group but increased in the fat-free group. Results of enzyme analyses are shown in Table 4. At 3 days after methotrexate treatments, there were no differences in the activity of enzymes in the jejunum and ileum among the three dietary groups (data not shown). At 12 days, the activities of these mucosal enzymes in the jejunum were lower in the 10% safflower oil group than in the other two groups. However, the differences in leucine aminopeptidase activities were not significantly different between the 0% and 10% groups. These differences were not observed in the ileum. The effects of dietary safflower oil on radiolabeled thymidine incorporation into isolated crypts from intestinal mucosa are shown in Table 5. As described in Materials and Methods, rats were injected with radiolabeled thymidine 1 h before they were killed. Thymidine incorporation into jejunal mucosa was higher in the 0% group than in the 10% group. In the ileum, 1-h thymidine incorporation was higher in 0% group than in the other two groups.
Table 4. Effects of Dietary Lipids on Mucosal Enzyme Activity 12 Days 1%
0% °
Jejunum Sucrase Lactase Maltase ALP LAP Ileum Sucrase Lactase Maltase ALP LAP
0.05 b.c O.Ol c
1.71 0.24 7.04 6.29 0.25
±
0.46 0.18 3.70 0.74 0.27
± 0.05 c
±
± 0.16 ± ±
c
0.27c 0.01 c.d
± O.Ol
c
± 0.27c ± 0.12c
± O.Ol c
1.64 0.21 7.03 6.15 0.29 0.56 0.18 4.35 0.57 0.29
100/0
± 0.04 ± ±
± 0.23 ±
c
0.02 c
± 0.03 ±
c
O.Old 0.16c
c
O.Ol c
c c ± 0.16 c ± O.Ol ± 0.15
1.29 0.17 5.49 5.25 0.24
± 0 .06 d
0.47 0.16 3.64 0.63 0.27
± 0.06 c
± ± ± ±
0.01' 0.19d 0.22d O.Old
± O.Ol c ± 0.34c ± 0.13
c
± 0 .02 c
Enzyme activity is expressed in micromoles/ per centimeter per minute. ALP, alkaline phosphatase; LAP, leucine aminopeptidase. "Percentage of safflower oil in diets. bMean ± SEM, n = 7. c.dValues with different superscripts are statistically different among dietary groups (p < 0.05J.
ery from mucosal injury. Rats fed the 0% diet did not have clinical evidence of essential fatty acid deficiency. However, triene-tetraene ratios in intestinal mucosa were >0.4 (Table 3), indicating that these animals were essential fatty acid deficient (18). Mucosal weight, DNA and protein content (Table 2), and villus height were lower in the ileum at 12 days in this group than in the 1 % group. These results are consistent with the of our previous study (5) in which resected animals receiving an essential fatty aciddeficient diet were shown to have impaired intestinal mucosal hyperplasia response in the remaining small bowel compared with resected controls. Previous studies from our laboratory and others have demonstrated that dietary fats have a significant influence on intestinal hyperplasia following massive resection of the small intestine (4,5). We have also observed that animals fed diets containing 5% linoleic acid have adaptive changes in the remaining intestine that exceed those in animals fed diets containing only 1 % linoleic acid, the amount thought to satisfy growth requirements in the rat (6). However, in the present studies, a diet containing 10% safflower oil did not stimulate mucosal regeneration after methotrexateinduced injury compared with a diet containing 1 % safflower oil. The high-fat diet had rather exacerbat-
Discussion
The present studies have demonstrated that dietary essential fatty acid deficiency impairs recovTable 3. Effects of Dietary Lipids on Triene-Tetraene Ratios
12 Day
3 Day 0%° Jejunum Ileum
1.23 1.32
± ±
0.18b.c 0.15 c
10%
1% 0.82 1.17
"Percentage of safflower oil in diets. bMean groups (p < 0.05J.
±
0.07d
±
0.12c
±
SEM, n
0.26 0.28 =
± ±
0% e
0.05 0.08 d
2.69 2.69
± ±
10%
1% c
0.13 0.19 c
0.16 0.16
± 0.02d
0.10
:t
O.Old
± 0.03
0.09
±
O.Old
d
7. c.d·' Values with different superscripts are statistically different among dietary
1230 VANDERHOOF ET AL.
Table 5. 1-Hour Incorporation in Intestinal Crypts
GASTROENTEROLOGY Vol. 98, No.5
of Radiolabeled Thymidine
00/0"
Jejunum Ileum
58.7 71.7
± ±
5.7b.c 3.9c
10/0
48.2 46.8
± ±
7.3 c.d 9.1 d
100/0
43.0 44.2
±
±
1.7d 3.9d
Incorporation is expressed in disintegrations per minute per crypt. "Percentage of safflower oil in diets. bMean ± SEM. c.dValues with different superscripts are statistically different among dietary groups (p < 0.05).
ing effects on methotrexate-induced mucosal damage if the diet was introduced at the beginning of methotrexate treatments. In similar studies, we introduced the 10% safflower oil diet at the beginning of methotrexate treatments and observed that sucrase activity was 0.54 ± 0.29 JImol/g mucosa per min in the 100/0 safflower oil group compared with 10.22 ± 3.74 JImol/g mucosa per min in the 0% safflower oil group. Therefore, in the present studies, we introduced dietary safflower oil 24 h after the last injection of methotrexate to minimize the effects of safflower oil on mucosal damage. There was a small trend where 10% safflower oil was detrimental to mucosal injury from methotrexate treatment at 3 days, although the differences were not statistically significant. Deoxyribonucleic acid synthesis estimated by radiolabeled thymidine incorporation in the ileal mucosa was highest in the 0% group (Table 5). However, villus height and mucosal DNA and protein content (Table 2) were lower in these essential fatty acid-deficient animals at 12 days. These results are consistent with the results of other studies in which dermal crypt cell mitotic labeling indices and exfoliation are both increased in essential fatty acid-deficient skin lesions (19). We have not determined exfoliation rates, but the present data suggest that both DNA synthesis and degradation increased in the essential fatty aciddeficient mucosa and that the degradation rates exceeded the synthetic rates. The reason ileal DNA synthesis increased in essential fatty acid deficient mucosa is not clear. One possible explanation could be changes in prostaglandin synthesis, because prostaglandins are involved in regulation of the proliferative activities of intestinal epithelium (20). Linoleic acid and arachidonic acid are the precursors of prostaglandin synthesis. In essential fatty acid deficiency, prostaglandin synthesis from endogenous substrates has been shown to decrease (21,22). DeRubertis et al. (23) have observed that colonic mucosal DNA synthesis was increased by aspirin treatment, in correlation with inhibition of colonic prostaglandin synthesis by aspirin. There may have been decreased prostaglandin synthesis in our essential fatty acid-deficient rats. In addition to decreased prostaglandin synthesis, there may have been
changes in the membrane compositions of enterocytes in essential fatty acid-deficient rats. Because we observed differences in the fatty acid composition of intestinal mucosa among dietary groups, and the life span of enterocytes is very short, it is reasonable to assume that such differences in the fatty acid profile also existed in enterocyte membranes. These changes in the membrane may have resulted in the alteration of tissue metabolism. In summary, a diet containing 10% safflower oil did not stimulate the regeneration of intestinal mucosa after methotrexate-induced injury in rats compared with a diet containing 1 % safflower oil. However, a diet deficient in essential fatty acids was detrimental to the recovery of intestinal mucosa from injury. References 1. Fagundes-Neto U, Pacheco IP, Patricio FRdS, Wehba J. Ultrastructural study of alterations in the small intestinal epithelium of children with acute diarrhea. J Pediatr Gastroentrol Nutr 1984;3:510-515. 2. Martorell F, Habicht J-p, Yarbrough C, Lechtig A, Klein RE, Western KA. Acute morbidity and physical growth in rural Guatemalan children. Am J Dis Child 1975;129:1296-1299. 3. Brown KH, MacLean WC. Nutritional management of acute diarrhea: an appraisal of the alternatives. Pediatrics 1973;73:119125. 4. Vanderhoof JA. Grandjean q, Kaufman SS, Burkley KT, Antonson DL. Effect of high percentage medium chain triglyceride diet on mucosal adaptation following massive bowel resection in rats. JPEN 1984;8:685-689. 5. Hart MH, Grandjean q, Park JHY, Erdman SH, Vanderhoof JA. Essential fatty acid deficiency and postresection mucosal adaptation in the rat. Gastroenterology 1988;94:682-687. 6. Park JHY, Grandjean q, Hart MH, Baylor J, Vanderhoof JA. Effect of dietary linoleic acid on mucosal adaptation and prostaglandin (PG) synthesis in rats after 700/0 jejunoileal resection. Gastroenterology 1987;92:1566. 7. Nutrient requirements of the laboratory rat. In: Nutrient requirements of laboratory animals. 3rd ed. Washington, D.C.: National Academy of Sciences, 1978;7-37. 8. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J BioI Chern 1951;193:265-275. 9. Dahlqvist A. Method for assay of intestinal disaccharidases. Anal Biochem 1964;7:18-25. 10. Sommer AJ. The determination of acid and alkaline phosphatase using nitrophenyl phosphate as substrate. Am J Med Technol 1954;20:244-253. 11. Goldbarg JA, Rutenburg AM. The colorimetric determination of leucine aminopeptidase in urine and serum of normal subjects and patients with cancer and other diseases. Cancer 1958;11:283291. 12. Burton K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 1956;62:315-323. 13. Giles KW, Myers A. Improved diphenylamine method for estimation of DNA. Nature 1965;206:93. 14. Folch J, Lees M, Sloane-Stanley GH. A single method for isolation and purification of total lipids from animal tissues. J BioI Chern 1957;26:497-509. 15. Holman RT. Biological activities of and requirements for poly-
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16. 17. 18. 19. 20. 21 .
unsaturated acids. In: Progress in the chemistry of fats and other lipids. Volume 9. Elmsford, N.Y.: Pergammon, 1970;611-682. Wimber DE, Quastler H, Stein OL, Wimber DR. Analysis of tritium incorporation into individual cells by autoradiography of squash preparations. J Biophys Biochem CytoI1960;8:327-331. Snedecor GW, Cochran WG. Statistical methods. 7th ed. Ames, Iowa: Iowa State University, 1980. Holman RT. Essential fatty acid deficiency. In: Progress in the chemistry oUats and other lipids. Volume 9. Elmsford, N.Y.: Pergammon, 1968;276. McCullough JL, Schreiber SH, Ziboh VA. Cell proliferation kinetics of epidermis in the essential fatty acid deficient rat. J Invest DermatoI1978;70:318-320. Craven PA, Saito R, DeRubertis FR. Role of local prostaglandin synthesis in the modulation of proliferative activity of rat colonic epithelium. J Clin Invest 1983;72:1365-1375. Boissoneault GA, Johnston PV. Essential fatty acid deficiency,
DIETARY LIPIDS AND MUCOSAL INJURY 1231
prostaglandin synthesis and humoral immunity in Lewis rats. J Nutr 1983;113:1187-1194. 22. Mathias MM, Dupont J. Quantitative relationships between dietary lin oleate and prostaglandin (eicosanoid) biosynthesis. Lipids 1985;20:791-801. 23. DeRubertis FR. Craven PA, Saito R. 16,16-dimethyl prostaglandin E, suppresses the increases in the proliferative activity of rat colonic epithelium induced by indomethacin and aspirin.Gastroenterology 1985;89:1054-1063.
Received March 27, 1989. Accepted November 3, 1989. Address requests for reprints to: Jon A. Vanderhoof, M.D., 601 North 30th Street, Suite 6850, Omaha, Nebraska 68131. This study was supported by a grant from the Thrasher Foundation.
Effects of Dietary Lipids on Recovery From Mucosal Injury JON A. VANDERHOOF, JUNG H. Y. PARK, HAMID MOHAMMADPOUR, and DARCY BLACKWOOD Department of Pediatrics, Creighton University School of Medicine, Omaha, Nebraska
The present studies were conducted to determine if diets containing a large amount of fat stimulate the regeneration of damaged intestinal mucosa in the presence or absence of essential fatty acid deficiency. To simulate injury, male Sprague-Dawley rats were given methotrexate, 2.5 mg/kg body wt, subcutaneously for 3 consecutive days. Twenty-four hours after the last methotrexate injection, rats were placed on diets containing either 00/0,1 %, or 10% samower oil. Mucosal weight, protein, deoxyribonucleic acid, maltase, sucrase, lactase, alkaline phosphatase, leucine aminopeptidase, and fatty acids were all determined 3 and 12 days after methotrexate. Crypt-cell production rates were also determined. Essential fatty acid deficiency was confirmed in the 0% safflower oil group, in which triene-tetraene ratios were >0.4. Mucosal weight, deoxyribonucleic acid, protein content, and villus height were all greater in the 1 % samower oil group than in the 0% group at 12 days. In the ileum, l-h thymidine incorporation was greater in the 0% sam ower oil group than in the other two groups. No differences in any of the parameters studied were observed between the 1 % and 10% groups. These results suggest that diets deficient in essential fatty acids may impair the recovery of intestinal mucosa from injury.
W
orldwide, infectious diarrhea causes the deaths of more children than any other disease process (1). This is especially true in underdeveloped countries, where the preexistence of malnutrition markedly worsens the prognosis (2). Most intestinal viruses, as well as many bacteria and parasites, cause severe injury to the small intestine. Enteral feedings administered early in the course of therapy may minimize malnutrition and improve prognosis (3). However, little is known about how specific nutrients affect the regeneration process. Previous studies in our laboratory have demonstrated that long-chain triglycerides
are effective in stimulating mucosal hyperplasia following massive resection of the small intestine (4). Further studies have suggested that these changes may be partly related to linoleic acid content in the diet, and essential fatty acid deficiency might impair mucosal adaptation (5). In other studies, animals fed diets containing 5% linoleic acid had enhanced mucosal adaptation in the remaining intestine compared with animals fed diets containing only 1 % linoleic acid (6). Therefore, the present studies were conducted to determine (a) if diet containing 10% safflower oil is advantageous to the regeneration of intestinal mucosa from methotrexate-induced injury compared with diet containing 1 % safflower oil and (b) if dietary essential fatty acid deficiency impairs this process. Materials and Methods
Materials Sodium methotrexate was obtained from Lederle Laboratories Division (Pearle River, N.Y.). [Methyl3H]thymidine was obtained from ICN Radiochemicals (Irvine, Calif.). Arachidonic acid methyl ester was purchased from Alltech Associates, Inc. (Deerfield, 111.). Eicosatrienoic acid was purchased from Cayman Chemical Co. (Ann Arbor, Mich.). The fat-free, 1 % safflower oil, and 100/0 safflower oil diets were obtained from Teklad (Madison, Wis.).
Animal Models To simulate the injury produced by an enteric virus, male Sprague-Dawley rats were given methotrexate, 2.5 mg/kg body wt, subcutaneously for 3 consecutive days. Preliminary studies in our laboratory have demonstrated that this dosage produces a consistent mucosal injury within 24 h after the last injection which subsequently resolves over a 2-wk recovery period (Figures 1 and 2). Severe inflammatory changes are apparent after 3 days with both polymorpho© 1990 by the American Gastroenterological Association
0016-5085/90/$3.00
May 1990
DIETARY LIPIDS AND MUCOSAL INJURY 1227
containing 00/0, 1%, or 10% safflower oil (Table 1). The fat-free diet was designed to maintain the essential fatty acid-deficient state. The 1 % safflower oil diet was chosen because safflower oil contains >75% linoleic acid and adequately meets the essential fatty acid requirements for the rat (7). The 10% safflower oil diet was designed to provide a higher concentration of fatty acids to determine whether this diet would stimulate mucosal healing and regeneration faster than 1 % fat diet. Animals were pair-fed to insure identical protein, calorie, vitamin, fiber, and mineral intake.
Biochemical, Physiological, and Histological Analyses Three days after the last injection of methotrexate, the rats were killed by cervical dislocation under CO 2 anesthesia. The small intestine, from the ligament of Trietz to the ileocecal valve, was removed and separated into proximal (jejunum) and distal (ileum) halves. The bowel was rinsed with ice-cold saline. The proximal 2 cm of each segment was fixed in buffered formalin for histological analysis. Tissue was fixed, sectioned (4-~m thick), mounted on glass slides, and stained with H&E. A total of 10 villi and crypts were measured in
Figure 1. Jejunal biopsy 3 days after methotrexate in the rat (original magnification x 100). Marked mononuclear cell infiltrate is seen in the lamina propria with scattered polymorphonuclear cells and crypt abscess formation. Villi are severely blunted with a 1:1 crypt-villus ratio. Surface epithelium is severely damaged.
nuclear and lymphocytic infiltrates in the lamina propria, marked villus blunting, loss of surface epithelium, and crypt abscess formation. Nearly all changes resolved by day 12. This effect occurs primarily in the proximal small intestine, but it is also produced to a lesser degree in the distal small intestine.
Experimental Design The animals were cared for according to the guidelines of the Animal Review Committee at the University of Nebraska Medical Center. Forty-two male, weanling, Sprague-Dawley rats (49 ± 4 g body wt) were obtained from Sasco, Inc. (Omaha, Neb.). The rats were housed individually and fed a non purified diet (Rodent Laboratory Chow; Ralston Purina Co., St. Louis, Mo.) and tap water ad libitum for 3 days to acclimate them to laboratory conditions. After this acclimation period, the animals were fed a modified AIN-76 diet (Table 1) containing no fat for 34 days to induce a biochemical essential fatty acid-deficient state. Animals were then given methotrexate as described above. Twentyfour hours after the last methotrexate injection, the animals were divided into three groups of 14 and placed on diets
Figure 2. Jejunal biopsy 12 days after methotrexate in the rat (original magnification x 100). Inflammation is resolved, surface epithelium is normal, and the crypt-villus ratio has normalized at 1:5.
GASTROENTEROLOGY Yol. 98, No.5
1228 YANDERHOOF ET AL.
Table 1. Composition of Diets (g/kg)
Fat-free
Safflower oil
10% Safflower oil
200.0 3.0 546.98 150.0 50.0 35.0 3.0 10.0 2.0 0.02 0.0
203.0 3.05 530.07 152.3 50.8 35.53 3.05 10.15 2.03 0.02 10.0
229.2 3.44 380.69 172.0 57.3 40.11 3.44 11.5 2.3 0.02 100.0
10/0
Casein, "vitamin-free" test DL-methionine Dextrose, monohydrate Maltodextrin Cellulose (fiber) Mineral mix, AIN-76 (170915) Calcium carbonate, CaC0 3 Yitamin mix, AIN-76A (40077) Choline bitartrate Ethoxyquin (antioxidant) Safflower oil
All diet ingredients except safflower oil (ICN Biomedicals, Costa Mesa, Calif.) were purchased from Teklad, Madison, Wis.
each segment with a micrometer eyepiece to determine villus height and crypt depth. The mucosa was scraped from a 25-cm portion of each segment using a glass slide. This tissue was weighed and used to perform several analyses. These included mucosal protein according to Lowry et al. (8J; maltase, sucrase, and lactase activities according to Dahlqvist (9J; alkaline phosphatase activity by the method of Sommer (10J; and leucine aminopeptidase activity by the method of Goldbarg and Rutenburg (11J. Deoxyribonucleic acid was extracted and measured according to the method of Burton (12J, as modified by Giles and Myers (13J. Total lipids in the mucosa were extracted with chloroformmethanol (2:1J in the presence of 1 % NaCI according to the method of Folch et al. (14J. The organic layer was dried, and methyl esters of fatty acids were formed in the presence of 6 N HCI-methanol (1:5J and dried under nitrogen. Methyl esters of fatty acids were analyzed by gas liquid chromatography (Perkin Elmer Sigma 2, Norwalk, Conn.J. Integrated peaks for eicosatrienoic acid (20:3, n-9J and eicosatetraenoic (20:4, n-6J acid were compared to determine triene-tetraene ratios according to the method of Holman (15J.
dietary linoleic acid early in the course of therapy for mucosal injury. The cumulative effects of therapy over a subsequent convalescent period were then studied by repeating the entire set of studies 12 days after methotrexate administration. All animal groups, feeding protocols, and analyses were conducted exactly as described above except that animals were killed 12 days, rather than 3 days, after completion of methotrexate administration.
Analysis of Deoxyribonucleic Acid Synthesis A second experiment was performed to determine how dietary safflower oil influences epithelial cell turnover following mucosal injury. These animals were injected intraperitoneally with [methyVH]thymidine (2 /LCi/g body wtJ 7 days after the last injection of methotrexate. One hour after this injection, the rats were killed and the small intestines were removed and rinsed with ice-cold saline. A small sample (1.5 cmJ of intestine was obtained from the jejunum and ileum to determine the amount of radioactivity incorporated into DNA per crypt. The tissue was fixed in cold Carnoy's solution for 2 h, stored in 70% ethanol, and then transferred to 500/0 and 30% ethanol for 5 min each. The samples were hydrolyzed in 1 N HCI at 60°C for exactly 7 min. This reaction was stopped by transferring the tissue to cold Schiff's reagent. The staining process continued for 1 h, and the microdissection of 50 crypts was performed by the method similar to that of Wimber et al. (16J. The crypts were dissolved in base and neutralized with acetic acid, and the radioactivity was determined by liquid scintillation counting.
Statistical Analysis All data were calculated as mean ± SEM and were analyzed by analysis of variance. The significant differences among means were further analyzed using Duncan's multiple range test (17J.
Results
Twelve-Day Study Evaluation of the animals 3 days after methotrexate administration allowed for a comparison of the effect of
Mucosal weight and DNA and protein content in the jejunum and ileum are shown in Table 2. These parameters did not show any significant differences
Table 2. Effects of Dietary Lipids on Mucosal Weight and Deoxyribonucleic Acid 3 Day 0%0 Jejunum Mucosal wt (mg/cm) DNA (lLg/cm) Protein (mg/cm) Ileum Mucosal wt (mg/cm) DNA (lLg/cm) Protein (mg/cm)
42.4 132 4.28 43.9 168 5.44
±
1%
3.9 b
± 21
c
± 0.74c ± 2.7c ± 15
12 Day
c
± 0.46
c
°Percentage of safflower oil in diets. bMean (p < 0.05).
±
47.5 142 4.47
± 14
44.0 162 6.14
± l1
SEM, n
=
10% c c
± 3.7
± 0.61c ± 3.1c
c
± 0.35
c
0% c c
44.2 137 4.51
± 5.9
45.0 163 5.70
± 4.3
± 15 ±
0.70 c c
± 15 c ± O.73
c
1%
54.3 179 5.35
± 1.1c
40.4 174 4.61
± 1.3
±
8e
± 0.42c
±
c
9c
± 0.17c
62.0 192 6.73
±
2.2 d
±
0.46 d
49.0 205 5.29
± ±
1.2 d 4d
±
0.24d
c ± 15
10% 57.5 181 6.00 46.5 194 5.51
± ± ± ± ± ±
1.8c.d 7C 0.36 c.d 2.5 d gc.d 0.34d
7. c.dYalues with different superscripts are statistically different among dietary groups
May 1990
DIETARY LIPIDS AND MUCOSAL INJURY 1229
among the three dietary groups in the 3-day study. However, in the 12-day study, mucosal weight and DNA and protein content were higher in 1 % safflower oil group than in the fat-free group except that the difference in DNA in the jejunum did not reach statistical significance. There were no differences in mucosal weight or DNA and protein content between the 1 % and 10% safflower oil groups. The differences in the jejunum were not statistically significant between the 0% and 10% safflower oil groups. There were no differences in crypt depth among the three dietary groups at both 3 and 12 days (data not shown). However, measurements of villus height in the ileum for both the 1 % (506 ± 19 ~m) and 10% safflower oil groups (520 ± 17 ~m) were higher than for the fat-free group (445 ± 14 ~m) at 12 days (p < 0.05). Table 3 shows the results of triene-tetraene ratios in intestinal mucosa . At 3 days, the ratios were > 0.4 in the groups that received diets containing 0% and 1 % safflower oil. At 12 days, the ratios were returned to normal in the 1 % group but increased in the fat-free group. Results of enzyme analyses are shown in Table 4. At 3 days after methotrexate treatments, there were no differences in the activity of enzymes in the jejunum and ileum among the three dietary groups (data not shown). At 12 days, the activities of these mucosal enzymes in the jejunum were lower in the 10% safflower oil group than in the other two groups. However, the differences in leucine aminopeptidase activities were not significantly different between the 0% and 10% groups. These differences were not observed in the ileum. The effects of dietary safflower oil on radiolabeled thymidine incorporation into isolated crypts from intestinal mucosa are shown in Table 5. As described in Materials and Methods, rats were injected with radiolabeled thymidine 1 h before they were killed. Thymidine incorporation into jejunal mucosa was higher in the 0% group than in the 10% group. In the ileum, 1-h thymidine incorporation was higher in 0% group than in the other two groups.
Table 4. Effects of Dietary Lipids on Mucosal Enzyme Activity 12 Days 1%
0% °
Jejunum Sucrase Lactase Maltase ALP LAP Ileum Sucrase Lactase Maltase ALP LAP
0.05 b.c O.Ol c
1.71 0.24 7.04 6.29 0.25
±
0.46 0.18 3.70 0.74 0.27
± 0.05 c
±
± 0.16 ± ±
c
0.27c 0.01 c.d
± O.Ol
c
± 0.27c ± 0.12c
± O.Ol c
1.64 0.21 7.03 6.15 0.29 0.56 0.18 4.35 0.57 0.29
100/0
± 0.04 ± ±
± 0.23 ±
c
0.02 c
± 0.03 ±
c
O.Old 0.16c
c
O.Ol c
c c ± 0.16 c ± O.Ol ± 0.15
1.29 0.17 5.49 5.25 0.24
± 0 .06 d
0.47 0.16 3.64 0.63 0.27
± 0.06 c
± ± ± ±
0.01' 0.19d 0.22d O.Old
± O.Ol c ± 0.34c ± 0.13
c
± 0 .02 c
Enzyme activity is expressed in micromoles/ per centimeter per minute. ALP, alkaline phosphatase; LAP, leucine aminopeptidase. "Percentage of safflower oil in diets. bMean ± SEM, n = 7. c.dValues with different superscripts are statistically different among dietary groups (p < 0.05J.
ery from mucosal injury. Rats fed the 0% diet did not have clinical evidence of essential fatty acid deficiency. However, triene-tetraene ratios in intestinal mucosa were >0.4 (Table 3), indicating that these animals were essential fatty acid deficient (18). Mucosal weight, DNA and protein content (Table 2), and villus height were lower in the ileum at 12 days in this group than in the 1 % group. These results are consistent with the of our previous study (5) in which resected animals receiving an essential fatty aciddeficient diet were shown to have impaired intestinal mucosal hyperplasia response in the remaining small bowel compared with resected controls. Previous studies from our laboratory and others have demonstrated that dietary fats have a significant influence on intestinal hyperplasia following massive resection of the small intestine (4,5). We have also observed that animals fed diets containing 5% linoleic acid have adaptive changes in the remaining intestine that exceed those in animals fed diets containing only 1 % linoleic acid, the amount thought to satisfy growth requirements in the rat (6). However, in the present studies, a diet containing 10% safflower oil did not stimulate mucosal regeneration after methotrexateinduced injury compared with a diet containing 1 % safflower oil. The high-fat diet had rather exacerbat-
Discussion
The present studies have demonstrated that dietary essential fatty acid deficiency impairs recovTable 3. Effects of Dietary Lipids on Triene-Tetraene Ratios
12 Day
3 Day 0%° Jejunum Ileum
1.23 1.32
± ±
0.18b.c 0.15 c
10%
1% 0.82 1.17
"Percentage of safflower oil in diets. bMean groups (p < 0.05J.
±
0.07d
±
0.12c
±
SEM, n
0.26 0.28 =
± ±
0% e
0.05 0.08 d
2.69 2.69
± ±
10%
1% c
0.13 0.19 c
0.16 0.16
± 0.02d
0.10
:t
O.Old
± 0.03
0.09
±
O.Old
d
7. c.d·' Values with different superscripts are statistically different among dietary
1230 VANDERHOOF ET AL.
Table 5. 1-Hour Incorporation in Intestinal Crypts
GASTROENTEROLOGY Vol. 98, No.5
of Radiolabeled Thymidine
00/0"
Jejunum Ileum
58.7 71.7
± ±
5.7b.c 3.9c
10/0
48.2 46.8
± ±
7.3 c.d 9.1 d
100/0
43.0 44.2
±
±
1.7d 3.9d
Incorporation is expressed in disintegrations per minute per crypt. "Percentage of safflower oil in diets. bMean ± SEM. c.dValues with different superscripts are statistically different among dietary groups (p < 0.05).
ing effects on methotrexate-induced mucosal damage if the diet was introduced at the beginning of methotrexate treatments. In similar studies, we introduced the 10% safflower oil diet at the beginning of methotrexate treatments and observed that sucrase activity was 0.54 ± 0.29 JImol/g mucosa per min in the 100/0 safflower oil group compared with 10.22 ± 3.74 JImol/g mucosa per min in the 0% safflower oil group. Therefore, in the present studies, we introduced dietary safflower oil 24 h after the last injection of methotrexate to minimize the effects of safflower oil on mucosal damage. There was a small trend where 10% safflower oil was detrimental to mucosal injury from methotrexate treatment at 3 days, although the differences were not statistically significant. Deoxyribonucleic acid synthesis estimated by radiolabeled thymidine incorporation in the ileal mucosa was highest in the 0% group (Table 5). However, villus height and mucosal DNA and protein content (Table 2) were lower in these essential fatty acid-deficient animals at 12 days. These results are consistent with the results of other studies in which dermal crypt cell mitotic labeling indices and exfoliation are both increased in essential fatty acid-deficient skin lesions (19). We have not determined exfoliation rates, but the present data suggest that both DNA synthesis and degradation increased in the essential fatty aciddeficient mucosa and that the degradation rates exceeded the synthetic rates. The reason ileal DNA synthesis increased in essential fatty acid deficient mucosa is not clear. One possible explanation could be changes in prostaglandin synthesis, because prostaglandins are involved in regulation of the proliferative activities of intestinal epithelium (20). Linoleic acid and arachidonic acid are the precursors of prostaglandin synthesis. In essential fatty acid deficiency, prostaglandin synthesis from endogenous substrates has been shown to decrease (21,22). DeRubertis et al. (23) have observed that colonic mucosal DNA synthesis was increased by aspirin treatment, in correlation with inhibition of colonic prostaglandin synthesis by aspirin. There may have been decreased prostaglandin synthesis in our essential fatty acid-deficient rats. In addition to decreased prostaglandin synthesis, there may have been
changes in the membrane compositions of enterocytes in essential fatty acid-deficient rats. Because we observed differences in the fatty acid composition of intestinal mucosa among dietary groups, and the life span of enterocytes is very short, it is reasonable to assume that such differences in the fatty acid profile also existed in enterocyte membranes. These changes in the membrane may have resulted in the alteration of tissue metabolism. In summary, a diet containing 10% safflower oil did not stimulate the regeneration of intestinal mucosa after methotrexate-induced injury in rats compared with a diet containing 1 % safflower oil. However, a diet deficient in essential fatty acids was detrimental to the recovery of intestinal mucosa from injury. References 1. Fagundes-Neto U, Pacheco IP, Patricio FRdS, Wehba J. Ultrastructural study of alterations in the small intestinal epithelium of children with acute diarrhea. J Pediatr Gastroentrol Nutr 1984;3:510-515. 2. Martorell F, Habicht J-p, Yarbrough C, Lechtig A, Klein RE, Western KA. Acute morbidity and physical growth in rural Guatemalan children. Am J Dis Child 1975;129:1296-1299. 3. Brown KH, MacLean WC. Nutritional management of acute diarrhea: an appraisal of the alternatives. Pediatrics 1973;73:119125. 4. Vanderhoof JA. Grandjean q, Kaufman SS, Burkley KT, Antonson DL. Effect of high percentage medium chain triglyceride diet on mucosal adaptation following massive bowel resection in rats. JPEN 1984;8:685-689. 5. Hart MH, Grandjean q, Park JHY, Erdman SH, Vanderhoof JA. Essential fatty acid deficiency and postresection mucosal adaptation in the rat. Gastroenterology 1988;94:682-687. 6. Park JHY, Grandjean q, Hart MH, Baylor J, Vanderhoof JA. Effect of dietary linoleic acid on mucosal adaptation and prostaglandin (PG) synthesis in rats after 700/0 jejunoileal resection. Gastroenterology 1987;92:1566. 7. Nutrient requirements of the laboratory rat. In: Nutrient requirements of laboratory animals. 3rd ed. Washington, D.C.: National Academy of Sciences, 1978;7-37. 8. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J BioI Chern 1951;193:265-275. 9. Dahlqvist A. Method for assay of intestinal disaccharidases. Anal Biochem 1964;7:18-25. 10. Sommer AJ. The determination of acid and alkaline phosphatase using nitrophenyl phosphate as substrate. Am J Med Technol 1954;20:244-253. 11. Goldbarg JA, Rutenburg AM. The colorimetric determination of leucine aminopeptidase in urine and serum of normal subjects and patients with cancer and other diseases. Cancer 1958;11:283291. 12. Burton K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid. Biochem J 1956;62:315-323. 13. Giles KW, Myers A. Improved diphenylamine method for estimation of DNA. Nature 1965;206:93. 14. Folch J, Lees M, Sloane-Stanley GH. A single method for isolation and purification of total lipids from animal tissues. J BioI Chern 1957;26:497-509. 15. Holman RT. Biological activities of and requirements for poly-
May 1990
16. 17. 18. 19. 20. 21 .
unsaturated acids. In: Progress in the chemistry of fats and other lipids. Volume 9. Elmsford, N.Y.: Pergammon, 1970;611-682. Wimber DE, Quastler H, Stein OL, Wimber DR. Analysis of tritium incorporation into individual cells by autoradiography of squash preparations. J Biophys Biochem CytoI1960;8:327-331. Snedecor GW, Cochran WG. Statistical methods. 7th ed. Ames, Iowa: Iowa State University, 1980. Holman RT. Essential fatty acid deficiency. In: Progress in the chemistry oUats and other lipids. Volume 9. Elmsford, N.Y.: Pergammon, 1968;276. McCullough JL, Schreiber SH, Ziboh VA. Cell proliferation kinetics of epidermis in the essential fatty acid deficient rat. J Invest DermatoI1978;70:318-320. Craven PA, Saito R, DeRubertis FR. Role of local prostaglandin synthesis in the modulation of proliferative activity of rat colonic epithelium. J Clin Invest 1983;72:1365-1375. Boissoneault GA, Johnston PV. Essential fatty acid deficiency,
DIETARY LIPIDS AND MUCOSAL INJURY 1231
prostaglandin synthesis and humoral immunity in Lewis rats. J Nutr 1983;113:1187-1194. 22. Mathias MM, Dupont J. Quantitative relationships between dietary lin oleate and prostaglandin (eicosanoid) biosynthesis. Lipids 1985;20:791-801. 23. DeRubertis FR. Craven PA, Saito R. 16,16-dimethyl prostaglandin E, suppresses the increases in the proliferative activity of rat colonic epithelium induced by indomethacin and aspirin.Gastroenterology 1985;89:1054-1063.
Received March 27, 1989. Accepted November 3, 1989. Address requests for reprints to: Jon A. Vanderhoof, M.D., 601 North 30th Street, Suite 6850, Omaha, Nebraska 68131. This study was supported by a grant from the Thrasher Foundation.