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Digestive Diseases and Sciences, Vol. 45, No. 10 (October 2000), pp. 2044 –2050

Chronic Diarrhea Impairs Intestinal Antioxidant Defense System in Rats at Weaning ´ PEZ-PEDROSA, PhD, MARIÁ DOLORES MESA, BS, NATALIA NIETO, PhD, JOSE MARIÁ LO ´ NDEZ, PhD, ANTONIO RIÓS, PhD, MARIÁ ISABEL TORRES, PhD, MARIÁ ISABEL FERNA ´ ´ ´ NGEL GIL, PhD MARIA DOLORES SUAREZ, PhD, and A

The aim of the present study was to evaluate the influence of severe protein– energy malnutrition on the antioxidant defense system in the small and large intestine in rats at weaning. Chronic diarrhea and the subsequent malnutrition were induced by oral intake of a lactose-enriched diet. Twenty rats were weaned at 21 days of age, and the control group was fed a semipurified synthetic diet for two weeks. The malnourished group was fed the same diet but carbohydrates were replaced by lactose, and they developed diarrhea one day after. Rats were killed, and macroscopic and histological features were analyzed, DNA content was measured, and alkaline phosphatase, myeloperoxidase, and ␥-glutamyltranspeptidase activities were determined to assess the degree of intestinal injury. Glutathione levels as well as the activities of intestinal glutathione transferase, glutathione reductase, total glutathione peroxidase, selenium-dependent glutathione peroxidase, superoxide dismutase, and catalase were measured to study the antioxidant defense system. Malnourished rats showed loss of body weight and an increase in length and weight in jejunum and ileum, while no significant changes were observed in colon. Epithelial cells showed fewer and shorter microvilli, larger mitochondria with low inner density and loss of cristae, dilated endoplasmic reticulum, and Golgi apparatus. The protein-to-DNA ratio was higher in the jejunum, ileum, and colon of malnourished rats. Glutathione levels decreased 40% in jejunum and 50% in colon of malnourished rats. A 40 –50% decrease in the activity of all the enzymes of the antioxidant defense system was observed in the jejunum and ileum of malnourished rats, while only catalase and glutathione transferase activities decreased 50% in colon. These results suggest that early chronic diarrhea and severe protein– energy malnutrition impair the antioxidant defense system in both the small and large intestine, which may have a role in the pathogenesis and maintenance of the vicious circle of malabsorption– diarrhea–malnutrition in infancy. KEY WORDS: antioxidant defense system; reactive oxygen species; protein– energy malnutrition; intestine.

Manuscript received August 9, 1999; accepted December 28, 1999. From the Department of Biochemistry and Molecular Biology, and Department of Cell Biology, University of Granada, Granada, Spain. These studies were supported by the CI1-CT91-0078 grant from the European Union. Natalia Nieto is currently working in the Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, New York.

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Jose Marıá Lo ´pez-Pedrosa is currently working in the R&D Department, Abbott Laboratories, Granada, Spain. ´ ngel Gil, Department of Address for reprint requests: Prof. A Biochemistry and Molecular Biology, Facultad de Farmacia, Universidad de Granada, Campus Universitario de Cartuja s/n, 18071 Granada, Spain.

Digestive Diseases and Sciences, Vol. 45, No. 10 (October 2000) 0163-2116/00/1000-2044$18.00/0 © 2000 Plenum Publishing Corporation

ANTIOXIDANT DEFENSE IN MALNUTRITION

Chronic diarrhea and malnutrition are frequently associated in childhood. The exposure to different environmental agents that can cause gastrointestinal infections as well as a number of genetic diseases, namely disaccharide intolerance and food allergies, usually leads to diarrhea and malnutrition (1, 2). Protein– energy malnutrition dramatically affects the development of the intestinal mucosa during the postnatal period. Malnutrition and intestinal dysfunction are related to multiple factors such as impaired immunity and alteration in the metabolic activity of the gastrointestinal tract (3, 4). Thus, low protein diets reduce the rate of protein synthesis in most of the body tissues, especially in the intestine (5). Malnutrition also reduces the intestinal surface and the uptake of amino acids and alters the activity of membranebound enzymes (6); a reduction in the segmental activities of the small intestine disaccharidases and severe histological alterations of intestinal mucosa have been reported in rats at weaning affected by chronic diarrhea induced by dietary intake of lactose (7, 8). There is evidence that oxidative stress may be a major cause of intestinal dysfunction during malnutrition (9, 10), and reactive oxygen species (ROS) are thought to play a key role in electrolyte loss and enhanced mucosal permeability occurring in chronic diarrhea (11, 12). Malnutrition increases the infiltration of lymphocytes within the lamina propria, generating oxidative stress damage (13). Oxidative stress in patients with altered antioxidant defense increases intestinal water secretion which contributes to the persistence of diarrhea (14). Glutathione (GSH) levels as well as the activity of glutathione peroxidase in erythrocytes are markedly decreased in malnourished children with kwashiorkor when compared to healthy or marasmic children, suggesting a compromised capacity to scavenge free radicals (15). Experimental protein deprivation reduces the activity of the antioxidant defense enzymes in rat liver and enhances lipid peroxidation in plasma and other tissues (16). However, there are no reports on the effects of chronic diarrhea in early infancy on the activities of the antioxidant enzymes of the intestine. Those type of studies would contribute to correlating both the systemic and the local response to protein– energy malnutrition. Therefore, an experimental model of chronic diarrhea and protein– energy malnutrition induced by dietary intake of lactose in rats at weaning was used to evaluate the changes in the intestinal antioxidant defense system. Total glutathione levels and the acDigestive Diseases and Sciences, Vol. 45, No. 10 (October 2000)

TABLE 1. COMPOSITION

OF

DIETS

Component

Amount (g/kg)

Casein Starch* Saccharose* Fat† Cellulose L-methionine Choline chloride Mineral supplement‡ Vitamin supplement§

220.51 485.24 150.0 37.51 80.0 4.0 2.0 20.72 0.20

*The lactose-enriched diet was made substituting the starch and saccharose for lactose. †The fat consisted of 66.5% of olive oil, 10.5% of sunflower oil, and 23% of soybean oil. ‡American Institute of Nutrition, 1977. §Composition of the vitamin supplement (g/100 g): thiamine chlorhydrate 0.6, riboflavin 0.6, pyridoxine chlorhydrate 0.7, nicotinamide 3; calcium pantothenate 1.6, folic acid 0.2, retinol acetate 0.12, cholecalciferol 0.0025, biotin 0.02, cyanocobalamine 1.0, vitamin E 10, vitamin K1 0.1.

tivity of glutathione transferase (GT), glutathione reductase (GR), selenium-dependent glutathione peroxidase (Se-GPX), total glutathione peroxidase (GPX), catalase (CAT), and superoxide dismutase (SOD) were measured in jejunum, ileum, and colon. Tissue injury in the small and large intestine was assessed by electron microscopy, the protein-to-DNA ratio, and the activity of enzymes such as myeloperoxidase (an indicator of the presence of neutrophils within the mucosa), alkaline phosphatase, and ␥-glutamyltransferase. These data suggest that chronic diarrhea markedly decreases the intestinal enzymatic antioxidant defense system, and this may contribute to the maintenance of the vicious circle of malabsorption– diarrhea–malnutrition in early infancy. MATERIALS AND METHODS Experimental Design and Sample Collection. The protocol for this study was approved by the University of Granada (Spain), and animals received humane treatment according to the European Union regulations for animal research. Twenty male Wistar rats (Animals’ Institute, University of Granada) were weaned at 21 days of age and fed either a semipurified synthetic diet (Table 1) for two weeks (control group), or the same diet but replacing the carbohydrates by lactose (malnourished group). The latter group developed diarrhea after one day. Two weeks later, rats were anesthetized with 20% urethane, at a dose of 1 ml/100 g of body weight, and killed. A midline abdominal incision was made, and the small and large intestine were removed and trimmed of fat and mesentery. The small intestine was divided into two equal segments from the Treitz angle to the ileocecal valve; the proximal portion was considered the jejunum, and the distal portion, the ileum. After cleaning the segments with ice-

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NIETO ET AL TABLE 2. INTESTINAL WEIGHT, LENGTH, DNA, AND PROTEIN/DNA IN EXPERIMENTAL MODEL BY DIETARY INTAKE OF LACTOSE IN RATS AT WEANING* Jejunum

Weight (g) Length (cm) DNA (mg/cm) Protein/DNA (mg/mg)

MALNUTRITION INDUCED

Ileum

Colon

Control

Malnourished

Control

Malnourished

Control

Malnourished

1.56 ⫾ 0.10 45.50 ⫾ 1.31 0.20 ⫾ 0.01

3.27 ⫾ 0.20a 59.83 ⫾ 1.09a 0.17 ⫾ 0.01

1.49 ⫾ 0.07 46.30 ⫾ 0.80 0.20 ⫾ 0.01

2.51 ⫾ 0.12a 55.33 ⫾ 1.44a 0.17 ⫾ 0.01

0.59 ⫾ 0.04 12.25 ⫾ 0.54 0.23 ⫾ 0.01

0.58 ⫾ 0.05 12.78 ⫾ 0.47 0.15 ⫾ 0.02b

5.37 ⫾ 0.12

7.41 ⫾ 0.32b

5.67 ⫾ 0.33

7.52 ⫾ 0.46b

5.15 ⫾ 0.17

6.03 ⫾ 0.27b

*Results are expressed as mean ⫾

SEM;

N ⫽ 10; a, P ⬍ 0.001; b, P ⬍ 0.05; malnourished vs controls.

cold 0.9% NaCl, they were measured and weighed. Mucosa was scraped off, weighed, homogenized in 10 mM pH 7.4 sodium phosphate buffer (1/10, w/v), centrifuged at 3000 g for 10 min at 4°C, and supernatants were used for enzymatic assays. For the determination of total GSH, mucosa was homogenized in 5% trichloroacetic acid and centrifuged at 8000 g for 5 min at 4°C. Total proteins of intestinal mucosa were measured by the method of Bradford (17), and the DNA content was quantified by the method of Labarca and Paigen (18). Ultrastructural Analysis. Samples for electron transmission microscopy were fixed in 3% glutaraldehyde/0.1 M pH 7.3 cacodylate buffer, postfixed in 1.5% osmium tetroxide, dehydrated in acetone, and embedded in Epon 812 resin. Ultrathin sections were double stained with uranyl acetate and lead citrate, and examined under a Zeiss 902 transmission electron microscope. Antioxidant Defense System Analysis. GT, GR, GPX (selenium-dependent and total), SOD, and CAT were determined according to Habig et al (19), Calberg and Mannervick (20), Flohe´ and Gu ¨nzler (21), Paoletti and Mocali (22), and Cohen et al (23), respectively. The method of Anderson (24) was used to determine total glutathione. Statistical Analysis. Results are given as mean ⫾ standard error of the mean (SEM). Comparisons among groups were done with the Student’s t test for unpaired data using the 3D program of the BMDP PC-90 version (BMDP Statistical Software, Los Angeles, California).

RESULTS Rats fed the lactose-enriched diet showed a significant loss of body weight (96 ⫾ 6 g in the malnourished group vs 116 ⫾ 7 g in the control group; P ⬍ 0.05). Table 2 shows the intestinal weight, length, DNA, and protein-to-DNA ratio in the experimental model of chronic diarrhea and malnutrition. An increase in the small intestine weight and length was observed in the malnourished group, while no significant changes were observed in the colon. The DNA content was lower only in colon mucosa, while the protein-to-DNA ratio was higher in jejunum, ileum, and colon. Chronic diarrhea caused by malnutrition altered the aspect of the mucosa of ileum and colon at the

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ultrastructural level. Epithelial cells showed less and shorter microvilli, enlarged mitochondria with low inner density and loss of cristae, and dilated endoplasmic reticulum and Golgi apparatus (Figure 1). GSH levels are shown in Figure 2, and the activities of GR, GT, GPX, Se-GPX, CAT, and SOD are shown in Figure 3. Results are expressed as units of segmental activity to indicate better the degree of enzymatic changes in the whole organ. Malnutrition and the consequent chronic diarrhea caused a decrease in GSH levels in jejunum and colon when compared to control groups. GPX activity (total and selenium-dependent) was significantly lower in both the small and large intestine during malnutrition. GR and SOD activities decreased in jejunum and ileum in malnourished rats when compared to control groups. GT decreased 40% in both jejunum and ileum and 50% in the colon of malnourished rats. CAT activity was 50% lower in both the small and large intestine of rats fed the lactose-enriched diet. DISCUSSION Since along with the onset of severe protein– energy malnutrition, biochemical markers indicate an increased lipid peroxidation with a decrease of plasma antioxidants and decreased proportions of plasma polyunsaturated fatty acids and red cell phospholipids (25), the aim of the present study was to evaluate whether chronic diarrhea due to a lactoseenriched diet intake could affect the antioxidant defense system in the intestine of rats at weaning. The ultrastructural analysis and the enzymatic activities of alkaline phosphatase, ␥-glutamyltranspeptidase, and myeloperoxidase (data not shown) confirmed that rats fed the lactose-enriched diet showed a protein– energy malnutrition syndrome (7, 8, 26). The increase in weight and length in both jejunum and ileum together with a decrease in body weight, may be a result of a compensatory mechanism to Digestive Diseases and Sciences, Vol. 45, No. 10 (October 2000)


Fig 1. Electron transmission micrographs of ileal mucosa (A, control; B, malnourished) and colonic mucosa (C, control; D, malnourished). 3: microvilli; ✶: mitochondria (⫻8000).

increase the absorption of nutrients since histological features showed fewer and shorter microvilli in the small intestine epithelial cells. The DNA content itself, but not the protein content was lower in the malnourished group, suggesting Digestive Diseases and Sciences, Vol. 45, No. 10 (October 2000)

that the overall process of DNA synthesis is impaired, causing mucosal atrophy and a lower cell turnover, typical features of chronic diarrhea in infancy. The significant increase in the protein-to-DNA ratio in both the small and the large intestine might be related

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Fig 2. GSH levels in jejunal, ileal, and colonic mucosa in control (C) and malnourished (M) rats. Results are expressed in micromoles per centimeter of intestine and are mean ⫾ SEM. N ⫽ 10; *P ⬍ 0.05, M vs C.

to the lower levels of GSH and some of the antioxidant enzymes. GSH, the main component of the antioxidant defense system, is an effective free radical scavenger that participates in important metabolic functions such as maintenance of protein sulfhydryl groups in the reduced state, enzymatic reactions catalyzed by GT, GR, and GPX, transport of amino acids, and protein and DNA synthesis. Malnourished rats with chronic diarrhea showed a marked decrease in GSH levels in the jejunum and colon. Similar results were obtained by Davis et al (27) and Pelissier et al (28) in rats fed low-protein diets. The decrease observed in GSH levels may be the result of an unbalanced turnover involving increased consumption of GSH in de-

Fig 3. Total GPX, Se-GPX, GR, GT, SOD, and CAT activities in jejunum, ileum and colon mucosa in control (C) and malnourished (M) rats. Results are expressed in units per centimeter of intestine and are mean ⫾ SEM. N ⫽ 10; *P ⬍ 0.05, **P ⬍ 0.005, ***P ⬍ 0.001; M vs C.

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Digestive Diseases and Sciences, Vol. 45, No. 10 (October 2000)


toxifying peroxides, a reduction in ␥-glutamylcisteinyl synthetase activity, and the lack of availability of cysteine, glycine, and methionine, the limiting amino acids for GSH synthesis. Darmon et al (29) have shown that weanling rats fed a low-protein diet had the same intestinal GSH content when compared to controls. This discrepancy may indicate that each experimental model of malnutrition could produce different degrees of oxidative injury. GPX reduces lipid and nonlipid hydroperoxides as well as hydrogen peroxide via a GSH-dependent mechanism. The inefficient removal of organic hydroperoxides increases lipid peroxidation and leads to cell death (30). Since reduced glutathione is required for detoxifying peroxides via GPX activity, the low levels of GSH found in the intestinal mucosa of rats fed the lactose-enriched diet may be an explanation for the lower activity of GPX in chronic diarrhea. GT is principally involved in detoxification. These enzymes can be induced by an increase flux of substrate, such as occurs during prooxidative stress or antioxidant deficiency. It has been described that weanling rats fed a protein-deficient diet exhibited low hepatic GT activity and increased lipid peroxidation (31). These results are in agreement with the decrease in GT activity observed in the small and the large intestine of rats fed the lactose-enriched diet; the consequence could be a magnification of the intestinal dysfunction because of the accumulation of aldehydes, epoxides, and peroxides within the mucosa. The data showed a decrease in GR activity with malnutrition in the small intestine. An extreme and prolonged diarrhea may cause thiamine and nicotinamide deficiencies that could limit the supply of NADPH required by GR to regenerate GSH from its oxidized form in vivo. A mechanism such as this would explain why micronutrient deficiencies induce a constant low level of GSH in the jejunum and colon. It has been described that erythrocytes from fooddeprived rats had low GR activity without changes in GSH, while hepatic GSH was lower than in the control groups (32). SOD activity decreases in malnutrition in weaning rats (33). In this model, total SOD activity decreased in the small intestine during chronic diarrhea. The lower activity of CAT in both the small and the large intestine could be due to the lower amount of peroxisomes, or to changes in the biogenesis of peroxisomes during intestinal cell differentiation along the cript– villus axis in response to malnutrition (6, 34). Taken together, these results suggest that chronic diarrhea due to a lactose-enriched diet, and therefore Digestive Diseases and Sciences, Vol. 45, No. 10 (October 2000)

malnutrition, cause an imbalance of the physiological steady state between ROS production and the antioxidant defense system in both the small and large intestine. ACKNOWLEDGMENTS We thank the R&D Department of Abbott Laboratories S.A, Granada, Spain, for supplying the diets.

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