Long-Term Enteral Arginine Supplementation in Rats with Intestinal Ischemia and Reperfusion

Journal of Surgical Research 175, 67–75 (2012) doi:10.1016/j.jss.2011.02.003

Long-Term Enteral Arginine Supplementation in Rats with Intestinal Ischemia and Reperfusion Chien-Hsing Lee, M.D., Ph.D.,*,† Chien-Chou Hsiao, M.D., M.S.,‡ Ching-Yi Hung, M.S.,* Yu-Jun Chang, M.S.,§ and Hui-Chen Lo, Ph.D.k,1 *Department of Surgery, Division of Pediatric Surgery, Changhua Christian Hospital, Changhua, Taiwan; †School of Medicine, Chung Shan Medical University, Taichung, Taiwan; ‡Department of Pediatrics, Changhua Christian Hospital, Changhua, Taiwan; §Epidemiology and Biostatistics Center, Changhua Christian Hospital, Changhua, Taiwan; and kDepartment of Nutritional Science, Fu Jen Catholic University, Taipei County, Taiwan Originally submitted October 1, 2010; accepted for publication February 3, 2011

intestinal ischemia and reperfusion injury remains questionable and requires further investigation. Ó 2012

Background. The effects of short-term enteral arginine supplementation on intestinal ischemiareperfusion (IR) injury have been widely studied, especially the ischemic preconditioning supplementation. The aim of this study was to investigate the effects of long-term intra-duodenal supplementation of arginine on intestinal morphology, arginine-associated amino acid metabolism, and inflammatory responses in rats with intestinal IR. Materials and Methods. Male Wistar rats with or without three hours of ileal ischemia underwent duodenal cannulation for continuous infusion of formula with 2% arginine or commercial protein powder for 7 d. The serological examinations, plasma amino acid and cytokine profiles, and intestinal morphology were assessed. Results. Intestinal IR injury had significant impacts on the decreases in circulating red blood cells, hemoglobin, ileum mass, and villus height and crypt depth of the distal jejunum. In addition, arginine supplementation decreased serum cholesterol and increased plasma arginine concentrations. In rats with intestinal IR injury, arginine supplementation significantly decreased serum nitric oxide, plasma citrulline and ornithine, and the mucosal protein content of the ileum. Conclusions. These results suggest that long-term intra-duodenal arginine administration may not have observable benefits on intestinal morphology or inflammatory response in rats with intestinal ischemia and reperfusion injury. Therefore, the necessity of long-term arginine supplementation for patients with

Elsevier Inc. All rights reserved.

Key Words: intestinal ischemia and reperfusion; arginine; cytokine; intestinal histology; nitric oxide.

INTRODUCTION

Arginine has been demonstrated to have a variety of physiological functions. For example, it plays critical roles in the modulation of cardiovascular and immune systems [1]. In addition, arginine is the sole source of nitric oxide (NO), a labile nitroso compound in the body [2]. Several human and animal studies have shown that NO has protective effects on injuries resulting from ischemia and reperfusion in the kidneys, heart, liver, and colon [3–7]. This protective ability of NO is closely related to its regulation of vascular tonus. Recent work has shown that NO exhibits intense vasodilatory activity and prevents the aggregation and adhesion of platelets and neutrophils [8, 9], two cell types which interact with both physical and metabolic responses during inflammation and thrombosis. However, the mechanism of NO’s protective role in the status of ischemic precondition is still under investigation [10, 11]. Intestinal ischemia and reperfusion (IR) is encountered in a number of clinical disease entities such as hemorrhagic shock, necrotizing enterocolitis (NEC), sepsis, vascular surgery, small bowel transplantation, cardiopulmonary bypass, and abdominal aortic surgery. Early enteral supplementation of arginine, for example, 2 h after a burn injury, may increase the

1 To whom correspondence and reprint requests should be addressed at Department of Nutritional Science, Fu Jen Catholic University, #510 Jhongjheng Rd., Sinjhuang City, Taipei 24205, Taiwan. E-mail: [email protected].

67

0022-4804/$36.00 Ó 2012 Elsevier Inc. All rights reserved.

68

JOURNAL OF SURGICAL RESEARCH: VOL. 175, NO. 1, JUNE 1, 2012

proliferation of intestinal mucosa in burned rats [12]. Harisb et al. have also indicated that arginine supplementation reduces the incidence of NEC in premature infants [13]. This beneficial effect might be closely associated with locally elevated NO in the small intestine, as suggested by the ability of intestinal NO to maintain mucosal integrity, intestinal barrier function, and intestinal mucosal blood flow during inflammation and injury [14–18]. In addition, arginine supplementation can suppress the release of pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-a, interleukin (IL)-1, and IL-6, after intestinal mesenteric ischemia [19]. The metabolites of arginine, including citrulline, ornithine, and other derivatives, such as polyamines, NO, and agmatine, might also play a role in regulating the production of these cytokines. However, it has also been shown that administering arginine immediately after the intestinal IR injury appears to have either no beneficial effects on survival or even worsen the outcome. This negative effect could be a result of excessive priming and activation of circulating myeloid cells and expression of inducible nitric oxide synthase [20]. These inconsistent effects of arginine supplementation on intestinal IR injury might be due to the severity of the conditions examined and therapeutic dosing, timing, and route. Little is known about the effects of long-term oral administration of arginine on systemic inflammatory and amino acid metabolism in intestinal IR. Therefore, we investigated the effects of long-term intra-duodenal arginine administration on intestinal morphology, circulating arginine-associated amino acid profiles, and inflammatory response in rats with intestinal IR injury. We aimed to find a new therapeutic strategy to improve the morbidity and mortality of patients with clinical conditions associated with intestinal ischemia and reperfusion injury. MATERIALS AND METHODS

Sham-P

350

Sham-A

IR-P

IR-A

320 290 g 260 230 200 -3

0

2

3

4

5

6

7 day

FIG. 1. Body weight changes from 3 d prior to the day of IR injury (d 0) to d 7. Values are means 6 SEM; n ¼ 6 per group. There was no significant difference in body weight among groups during the experimental period.

operation as described in an earlier publication [21]. In brief, 10 cm of the ileum, from 5 to 15 cm proximal to the cecum, received 3 h of total ischemia by ligations of the corresponding mesenteric arteries and vein, both ends of the terminal ileum, and the marginal vessels, using silk sutures. The loss of normal mesenteric pulsation and a color change in the bowel confirmed the vascular occlusion. The forwardly displaced ileum was then returned to the abdominal cavity, and the abdominal wall was temporarily closed. Three hours after the vascular occlusion, the abdomen of the rats was reopened and the silk sutures were removed to release the occlusion. Reperfusion was confirmed by the return of mesenteric pulsation, after which the abdomen was closed. During the experimental period, animals received isonitrogenous and isocaloric enteral nutrition solution via intra-duodenal infusion for 7 d. The formulas (Ensure, Abbott Laboratories, Abbott Park, IL) were supplemented with 2% (wt/wt) of a commercially available protein powder (20% whey and 80% casein) or arginine, which provided 1.169 kcal per mL. Each animal was provided with approximately 65 kcal per day. Seven days after the intra-duodenal feeding, rats were euthanized under anesthesia, with intramuscular injections of 100 mg ketamine and 10 mg xylazine per kg of body weight. The order of euthanasia was randomized among the groups. Blood was collected by cardiac puncture, after which the serum and whole blood were isolated for further assays. The heart, lung, liver, thymus, spleen, kidneys, and gastrocnemius muscles were dissected and weighed and the results were recorded. After removing the above-mentioned organs and tissues, the carcass weights were recorded, and the carcasses were stored at –20 C for composition analysis of water, fat, and protein.

Animals and Study Design Analytic Measurements The animal facilities and protocols were approved by the Laboratory Animal Care and Use Committee of Changhua Christian Hospital in Changhua, Taiwan. Male Wistar rats (5 to 6 weeks old) initially weighing 200–250 g were acclimated to the animal facility and were given free access to water and a chow diet in a room maintained at 22 C on a 12:12-h light-dark cycle for 2 wk prior to surgery. These animals were randomly assigned to four groups: intra-duodenal administration of arginine with [IR-A group] or without [Sham-A group] 3 h of intestinal ischemia and intra-duodenal administration of commercial protein powder with [IR-P group] or without [Sham-P group] 3 h of intestinal ischemia (n ¼ 6 per group). After fasting overnight, the animals were anesthetized by an intramuscular injection with 80 mg ketamine and 8 mg xylazine per kg of body weight; next, intra-duodenal cannulation was performed (d 0). The IR-A and IR-P groups received an additional ileal ischemia

Body weights and the amounts of formula infused were recorded daily. The procedures for determining the amounts of water, protein, and fat in the carcass were previously described [22]. The number of circulating red blood cells, white blood cells, and platelets and the concentration of hemoglobin and percentages of hematocrit were determined using a hematology analyzer (GEN; Coulter Inc., Miami, FL). Serum concentrations of glucose, albumin, triglyceride, cholesterol, blood urea nitrogen (BUN), creatinine, glutamic oxaloacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT) were measured using an automatic analyzer (Hitachi 747; Tokyo, Japan). Intestinal mucosa and muscularis were homogenized in 10 volumes of ice-cold PBS by polytron and the protein concentrations were determined by a commercially available bioinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL). The serum levels of TNF-a,

69

LEE ET AL.: ARGININE AND INTESTINAL ISCHEMIA AND REPERFUSION

TABLE 1 The Relative Weights of Tissues and Organs in Rats with Intestinal Ischemia and Reperfusion Group

Heart g/kg

Liver g/kg

Lung g/kg

Kidney g/kg

Thymus g/kg

Spleen g/kg

Muscle g/kg

Sham-P Sham-A IR-P IR-A

2.76 6 0.18 2.84 6 0.12 2.94 6 0.05 2.86 6 0.11

36.51 6 1.94 32.86 6 2.27 39.26 6 1.36 35.36 6 1.06y

4.70 6 0.25 5.36 6 0.27 5.36 6 0.34 5.35 6 0.36

7.11 6 0.39 7.06 6 0.39 7.28 6 0.34 7.15 6 0.25

1.39 6 0.10 1.44 6 0.11 1.24 6 0.05 1.33 6 0.11

3.10 6 0.20 3.08 6 0.30 4.08 6 0.29* 3.52 6 0.38

10.73 6 0.67 10.66 6 0.30 9.42 6 0.35 10.62 6 0.52

Values are means 6 SEM. Significant differences between Sham-P and IR-P groups and between Sham-A and IR-A groups (one-way ANOVA with least significant difference, P < 0.05). y Significant differences between Sham-P and Sham-A groups and between IR-P and IR-A groups. Gastrocnemius muscle was used to represent the muscle. *

interferon (IFN)-g, IL-6, and IL-10 were measured by a commercially available enzyme-linked immunosorbent assays (ELISA; Pharmingen and R and D Systems, Inc., Minneapolis, MN). Serum concentrations of NO were measured as nitrite/nitrate by using the Griess reaction [23]. Plasma amino acid concentrations, including arginine, citrulline, ornithine, proline, and glutamate were determined using high performance liquid chromatography (HPLC, PICO-TAG method, Milford, MA). The harvested small intestine without gut contents was divided into three parts: proximal jejunum, distal jejunal, and ileum. Approximately 1 cm of small intestine from the middle area of each segment was fixed in 10% buffered formalin for routine paraffin embedding, hematoxylin and eosin (H and E) staining, and morphometric measurement under a light microscope. The villus height, crypt depth, muscularis thickness, and villus density were determined. At least 10 villus-crypt axes were measured per animal. One investigator performed all the measurements to prevent inter-observer differences in measuring technique. To determine the mucosal and muscularis wet and dry weights, 3 cm of each of the proximal jejunal, distal jejunal, and ileal segments were utilized. The mucosa was obtained by scraping the small intestine with a glass slide. This procedure was performed by the same individual to reduce variation. The remaining 3 cm of the proximal jejunal, distal jejunal, and ileal segments were used to analyze the protein (Bioinchoninic Acid Protein Assay; Pierce Chemical Co.) and DNA contents.

Statistical Analysis All groups were compared by one-way analysis of variance (ANOVA) using the SAS general linear models program (SAS Institute Inc. Cary, NC). Values were reported as means 6 SEM. Group means

were considered to be significantly different at P < 0.05, as determined by the technique of protective least-significant difference (LSD) when the ANOVA indicated an overall significant treatment effect, P < 0.05. A post hoc power calculation was determined by the statistical power analyses G*Power 3.1 [24].

RESULTS Body Weight, Relative Weights of Organs and Tissues, and Carcass Composition

Body weight changes are shown in Figure 1. There were no significant differences in body weight among groups before surgery, on the day of surgery (d 0) and after postoperative d 7. The final sample size of each group was six with a 100% survival rate. The relative weights of organs and tissues are shown in Table 1. The relative weights of the liver were significantly lower in the IR-A group compared with the IR-P group, and those of the spleen were significantly higher in the IR-P group compared with the Sham-P group. There were no significant differences in the relative weights of heart, lung, kidneys, thymus, and gastrocnemius muscle among groups. In addition, carcass composition, including the amounts of water, protein, and fat, were not significantly different among groups (data not shown).

TABLE 2 The Complete Blood Counts in Rats with Intestinal Ischemia and Reperfusion Group

WBC 103/mL

RBC 106/mL

Hemoglobin g/dL

Hematocrit %

Platelet 103/dL

Sham-P Sham-A IR-P IR-A

3.95 6 0.42 5.23 6 1.27 6.18 6 0.43* 6.07 6 0.77

6.89 6 0.09 6.91 6 0.14 6.15 6 0.14* 6.31 6 0.10*

13.39 6 0.22 13.66 6 0.34 12.28 6 0.28* 12.38 6 0.21*

38.92 6 0.71 39.32 6 1.10 36.68 6 0.70 36.63 6 0.51

1498 6 34 1368 6 128 1677 6 111 1457 6 73

Values are means 6 SEM. WBC ¼ white blood cells; RBC ¼ red blood cells. * Significant differences between Sham-P and IR-P groups and between Sham-A and IR-A groups (one-way ANOVA with least significant difference, P < 0.05).

70

JOURNAL OF SURGICAL RESEARCH: VOL. 175, NO. 1, JUNE 1, 2012

TABLE 3 Serum Substrate and Cytokine Concentrations in Rats with Intestinal Ischemia and Reperfusion Group Sham-P Sham-A IR-P IR-A

Glucose mg/dL Albumin mg/dL Cholesterol mg/g TNF-a pg/mL IFN-g pg/mL IL-6 pg/mL IL-10 pg/mL Nitric oxide mmol/L 353.5 6 11.1 279.4 6 23.7y 311.4 6 20.2 281.0 6 15.9

3.59 6 0.07 3.66 6 0.11 3.36 6 0.05* 3.57 6 0.09

86.3 6 3.2 74.6 6 3.8y 83.8 6 1.4 75.5 6 1.7y

4.11 6 0.23 4.456 0.16 4.28 6 2.70 4.92 6 1.29

2.96 6 0.14 3.22 6 0.22 2.98 6 0.21 3.17 6 0.24

62.9 6 2.2 68.3 6 5.3 70.2 6 5.4 67.6 6 4.3

53.8 6 7.5 44.5 6 7.3 51.3 6 13.5 42.4 6 6.8

162.0 6 12.9 220.0 6 49.0 230.3 6 20.0* 156.2 6 14.4y

Values are means 6 SEM. TNF ¼ tumor-necrosis factor; IFN ¼ interferon; IL ¼ interleukin. * Significant differences between Sham-P and IR-P groups and between Sham-A and IR-A groups (one-way ANOVA with least significant difference, P < 0.05). y Indicates significant differences between Sham-P and Sham-A groups and between IR-P and IR-A groups.

Blood and Serum Substrate Concentrations

Plasma Amino Acid Concentrations

Circulating numbers of white blood cells were significantly higher in the IR-P group than the Sham-P group (Table 2), whereas the hematocrit was significantly lower in the IR-A group than the Sham-A group. The circulating numbers red blood cells and the levels of hemoglobin were significantly lower in the IR-P and IR-A groups than in the Sham-P and Sham-A groups, respectively. The number of platelets was not significantly different among groups. Serum glucose was significantly lower in the Sham-A group and serum albumin was significantly lower in the IR-P group compared with the Sham-P group (Table 3). In addition, serum cholesterol was significantly lower in rats with arginine administered, that is, the ShamA and IR-A groups, compared with those with commercial protein powder, that is, the Sham-P and IR-P groups. Serum concentrations of triglyceride, BUN, creatinine, GOT, and GPT were not significantly different among groups. However, serum NO was significantly higher in the IR-P group than the Sham-P group and was significantly lower in the IR-A group than the IRP group. There were no significant differences in serum concentrations of TNF-a, interferon (IFN)-g, IL-6, and IL-10 among groups.

Plasma concentrations of arginine, citrulline, ornithine, proline, and glutamate are shown in Table 4. Plasma arginine concentrations were significantly increased in rats with arginine administration (i.e., the Sham-A and IR-A groups) than those without arginine administration (i.e., the Sham-P and IR-P groups). However, plasma citrulline and ornithine were significantly lower in the IR-A group than in the IR-P group, and plasma glutamine was significantly lower in the IR-A group than in the IR-P and Sham-A groups. There were no significant differences in plasma proline concentrations among groups. Composition and Architecture of the Small Intestine

The results of histological assessments of small intestine are shown in Figure 2. In the proximal jejunal segment (Fig. 2A), there were no significant differences in the villus height, crypt depth, or muscularis thickness among groups. In the distal jejunal segment (Fig. 2B) the intestinal IR injury had significant impacts on villus height and crypt depth. For example, the IR-P group had significantly increased villus height and decreased crypt depth compared with the Sham-P group, and the

TABLE 4 Plasma Amino Acid Concentrations in Rats with Intestinal Ischemia and Reperfusion Group

Arginine mmol/L

Citrulline mmol/L

Ornithine mmol/L

Proline mmol/L

Glutamate mmol/L

Sham-P Sham-A IR-P IR-A

281.6 6 19.2 480.3 6 57.5y 265.9 6 10.7 451.1 6 35.0y

94.1 6 5.0 85.7 6 3.0 101.8 6 2.2 91.2 6 3.3y

382.2 6 7.1 354.4 6 12.9 369.3 6 12.5 329.7 6 5.8y

454.1 6 22.1 393.0 6 63.0 486.2 6 27.8 392.8 6 62.7

454.0 6 13.6 434.4 6 14.0 451.8 6 34.4 331.6 6 43.4*y

Values are means 6 SEM. * Indicates significant differences between Sham-P and IR-P groups and between Sham-A and IR-A groups (one-way ANOVA with least significant difference, P < 0.05). y Indicates significant differences between Sham-P and Sham-A groups and between IR-P and IR-A groups.

LEE ET AL.: ARGININE AND INTESTINAL ISCHEMIA AND REPERFUSION

A 2.0

Sham-P

Sham-A

IR-P

IR-A

1.6

mm

1.2

0.8

0.4

0.0 Villus height

B 2.0

Crypt depth

Sham-P

Muscularis thickness

Sham-A

IR-P

IR-A

1.6

mm

1.2

0.8

0.4

0.0 Villus height

C 2.0

Crypt depth

Sham-P

Muscularis thickness

Sham-A

IR-P

IR-A

1.6

mm

1.2

71

significantly higher muscularis thicknesses than the Sham-P group. The results of jejunal and ileal wet and dry weights are shown in Figure 3. The mucosal wet weights of the IR-P group were significantly higher in the proximal and distal jejunum segments than the Sham-P group (Fig. 3A). In the ileum, the mucosal wet weights of the IR-P and IR-A groups were significantly higher than the Sham-P and Sham-A groups, respectively. There were no significant differences in the mucosal dry weights (Fig. 3C) and muscularis wet (Fig. 3B) or dry (Fig. 3D) weights among groups in the proximal and distal jejunum or ileum. The protein and DNA content of the small intestine is shown in Figure 4. In the mucosa, the protein content of the proximal jejunum was significantly higher in the IR-A group than the Sham-A group, (Fig. 4A). In the ileum, the mucosal protein content was significantly higher in the IR-P group than the Sham-P group; it was also higher in the IR-A group than the Sham-A and IR-P groups. The muscularis protein content of the proximal and distal jejunum and ileum were not significantly different among groups (Fig. 4B). The mucosal DNA content of the proximal jejunum was significantly lower in the IR-P group than the Sham-P group and was significantly higher in the IR-A group than the IR-P group (Fig. 4C). In the ileum, the intestinal IR injury had significant impacts on the level in mucosal DNA content as the IR-P and IR-A groups had significantly greater levels than the Sham-P and Sham-A groups, respectively. The mucosal DNA contents of the distal jejunum and muscularis DNA contents of the proximal jejunum and ileum were not significantly different among groups. However, the muscularis DNA content of the distal jejunum was significantly lower in the Sham-A group than the Sham-P group (Fig. 4D).

0.8

Power Calculation 0.4

0.0 Villus height

Crypt depth

Muscularis thickness

FIG. 2. Villus height, crypt depth, and muscularis thickness of the proximal (A) distal (B) segments of jejunum and ileum (C). Values are means 6 SEM; n ¼ 6 per group. Values with a * symbol indicate that there were significant differences between Sham-P and IR-P groups and between Sham-A and IR-A groups (one-way ANOVA with least significant difference, P < 0.05).

IR-A group had significantly lower villus height and higher crypt depth than the Sham-A groups. In the ileal segment (Fig. 2C), villus height and crypt depth were not significantly affected by intestinal IR injury and arginine supplementation; however, the IR-P group had

A power calculation using a sample size of six in each group showed the powers were 0.997, 0.777, 0.893, 0.772, and 0.785 for circulating levels of arginine, citrulline, ornithine, glutamate, and NO, respectively, to detect a group effect. For the other parameters with statistical significances in a one-way ANOVA, most of the powers were very close to or even larger than 0.8. For serum cytokines, such as, TNF-a, (IFN)-g, IL-6, and IL-10, the powers were less than 0.1. DISCUSSION

Arginine and NO play critical roles in modulating the physiological function of the gastrointestinal tract and in maintaining the mucosal integrity of the intestine

72

JOURNAL OF SURGICAL RESEARCH: VOL. 175, NO. 1, JUNE 1, 2012

A 80

Sham-P

Sham-A

IR-P

IR-A

B 60

Sham-P

Sham-A

IR-P

IR-A

50 60

mg/cm

mg/cm

40 40

30 20

20

10 0

0 proximal jejunum

Sham-P

Sham-A

proximal jejunum

ileum

IR-P

IR-A

D 20

16

16

12

12

mg/cm

mg/cm

C 20

distal jejunum

8

8

4

4

distal jejunum

Sham-P

Sham-A

ileum

IR-P

IR-A

0

0 proximal jejunum

distal jejunum

ileum

proximal jejunum

distal jejunum

ileum

FIG. 3. Wet weights of the mucosa (A), muscularis (B), dry weights of the mucosa (C), and muscularis (D) of the proximal and distal jejunum and ileum. Values are means 6 SEM; n ¼ 6 per group. Values with a * symbol indicate that there were significant differences between Sham-P and IR-P groups and between Sham-A and IR-A groups (one-way ANOVA with least significant difference, P < 0.05).

in healthy people and in patients with various intestinal disorders [3–7]. The effects of enteral arginine supplementation on intestinal IR injury have been widely studied in short-term administration [14–18]; however, the results remain inconsistent, partly due to the therapeutic dosing, time, route, and disease severity [20, 22]. In the present study, we investigated the effects of long-term intra-duodenal arginine administration on intestinal morphology, circulating arginine associated amino acid profiles, and inflammatory response in rats with intestinal IR injury. Our results show that long-term intra-duodenal arginine administration may not alter the intestinal morphology and inflammatory response in rats with intestinal ischemia and reperfusion injury. It has been suggested that the mechanism of intestinal damage following an IR injury includes nonspecific damage induced by the ischemia and damage caused by reactive oxygen species (ROS) following reperfusion [5]. In the initial phase of a reperfusion injury, activated macrophages, the primary source of extracellular ROS, may initiate the release of proinflammatory cytokines in tissues causing local damage without affecting the circulating levels of cytokines [19, 25]. In our study,

we did not find significant changes in serum concentrations of either pro-inflammatory cytokines (TNF-a and IL-6) and T-helper 1 (IFN)-g) or T-helper 2 (IL-10) cytokines among groups (Table 3). However, intestinal IR injury significantly increased circulating WBC numbers and decreased circulating RBC numbers and hemoglobin levels (Table 2). The intestinal IR-induced increase in WBC was attenuated, but the decreases in RBC and hemoglobin were not improved in animals that received arginine supplementation. These results imply that rats may have hematological defects and that these defects may not be improved by arginine supplementation for 7 d after the intestinal IR. Yokata et al. have reported that renal IR injury might induce cell damage and splenomegaly [26]. Our previous study revealed that intestinal IR injury increases the possibility of bacterial translocation, which further induces the inflammatory response and hepatomegaly [27]. In the present study, we found that intestinal IR injury may also increase the spleen weights (Table 1), and arginine supplementation may decrease the liver weights in animals with intestinal IR injury. However, we did not find significant impacts of intestinal IR and long-term intra-duodenal arginine supplementation on

73

LEE ET AL.: ARGININE AND INTESTINAL ISCHEMIA AND REPERFUSION

Sham-P

Sham-A

IR-P

IR-A

B 2.5

2.0

2.0

1.5

1.5

mg/cm

mg/cm

A 2.5

1.0

1.0

0.5

0.5

0.0

Sham-A

IR-P

IR-A

0.0 proximal jejunum

C 0.30

distal jejunum

Sham-P

Sham-A

ileum

IR-P

proximal jejunum

IR-A

D 0.30

0.24

0.24

0.18

0.18

mg/cm

mg/cm

Sham-P

0.12

0.12

0.06

0.06

0.00

distal jejunum

Sham-P

Sham-A

ileum

IR-P

IR-A

0.00 proximal jejunum

distal jejunum

ileum

proximal jejunum

distal jejunum

ileum

FIG. 4. Protein content of the mucosa (A), muscularis (B), DNA content of the mucosa (C) and muscularis (D) of the proximal and distal jejunum and ileum. Values are means 6 SEM; n ¼ 6 per group. Values with a *symbol indicate that there were significant differences between Sham-P and IR-P groups and between Sham-A and IR-A groups; those with a y symbol indicate that there were significant differences between Sham-P and Sham-A groups and between IR-P and IR-A groups (one-way ANOVA with least significant difference, P < 0.05).

body weight gain (Fig. 1), carcass composition (data not shown), and the relative weights of other organs we collected (Table 1). It has been demonstrated that arginine may induce insulin secretion and decrease blood glucose levels [28]. In the present study, we found that arginine supplementation significantly decreased blood glucose in healthy animals, but not in animals with intestinal IR injury (Table 3). In addition, serum albumin was not significantly altered by arginine in animals with intestinal IR injury. Our findings show that serum concentrations of cholesterol were significantly decreased by arginine administration in healthy and intestinal IRinjured animals. The actions of arginine in decreasing serum cholesterol were also reported in our previous study in animals with sub-acute peritonitis [22]. Several studies have shown that oral arginine supplementation may increase the formation of endogenous NO to maintain the intestinal mucosal integrity, protect the intestine from blood borne toxins and tissue destructive mediators, and improve intestinal recovery in rats with intestinal IR injury [19, 29]. In contrast,

more recent studies have indicated that arginine supplementation is not beneficial and possibly even harmful [20, 30], especially in inflammation, infection, and sepsis. In our study, serum NO was significantly elevated in animals suffering from intestinal IR injury without arginine supplementation (Table 3). However, in animals suffering from intestinal IR, arginine supplementation attenuated the elevation in serum NO. The inefficient utilization of arginine to produce NO in animals with intestinal IR injury is possibly due to the competition for the same substrate, i.e., arginine, between the ureagenesis and NO synthesis, as shown in the study of Bertolo et al. [31]. Even if this is true, the possibilities of excess NO excretion from the urinary and respiratory systems causing the decrease in serum NO cannot be excluded. We found that plasma arginine was significantly affected by arginine supplementation, not by intestinal IR injury (Table 4). Plasma concentrations of citrulline, ornithine, proline, and glutamate were also not altered by intestinal IR injury. Nevertheless, plasma citrulline, ornithine, and glutamate were significantly decreased

74

JOURNAL OF SURGICAL RESEARCH: VOL. 175, NO. 1, JUNE 1, 2012

by arginine supplementation in rats with intestinal IR. These results suggest that long-term arginine supplementation might not be metabolized efficiently in late post-injury stages. The decreases in circulating NO and arginine-associated amino acids in the late stages of intestinal IR injury warrant further investigation. It has been noted that the terminal ileum is the most sensitive segment for IR injury, mostly because the anatomic location. The visible effects of small bowel injuries induced by IR range from the loss of villi to the presence of an extended subepithelial space. Recent evidence indicates that arginine may improve intestinal recovery and accelerate the repair of intestinal mucosa following IR [32], possibly by increasing mucosal mass, villus height, crypt depth, and the thickness of bowel wall. In this study, arginine supplementation increased the mucosal DNA content of the proximal jejunum in animals with intestinal IR (Fig. 4) but decreased both the muscularis DNA content of the distal jejunum in healthy animals and the mucosal protein content of the ileum in intestinal IR-injured animals. Arginine did not have significant impacts on the intestinal histology (Fig. 2) or dry mass (Fig. 3) of healthy and intestinal IR-injured animals. These results suggest that the long-term administration of enteral arginine may not have beneficial effects on intestinal histology and mass in intestinal IR injury. In summary, our results suggest that long-term intraduodenal arginine administration may not have observable benefits on intestinal morphology and inflammatory response in rats with intestinal ischemia and reperfusion injury. Therefore, the necessity of long-term arginine supplementation for patients with intestinal ischemia and reperfusion injuries remains questionable and needs to be further investigated. ACKNOWLEDGMENTS This work was supported by the Changhua Christian Hospital under the grant number CCH-4604.

REFERENCES 1. Hemendra N, George U. Arginine: A clinical perspective. Nutr Clin Pract 2002;17:218–25. 2. Levillain O, Hus-Citharel A, Morel F, et al. Localization of urea and ornithine production along mouse and rabbit nephrons: Functional significance. Am J Physiol 1992;263:878. 3. Ogawa T, Nussler AK, Tuzuner E, et al. Contribution of nitric oxide to the protective effects of ischemic preconditioning in ischemia-reperfused rat kidneys. J Lab Chin Med 2001;138:50. 4. Tokuno S, Thor
en P, L€owbeer C, et al. The role of nitric oxide in ischemia/reperfusion injury of isolated hearts from severely atherosclerotic mice. Life Sci 2001;69:2067. 5. Rhee JE, Jung SE, Shin SD, et al. The effects of antioxidants and nitric oxide modulators on hepatic ischemia-reperfusion injury in rats. J Korean Med Sci 2002;17:502.

6. Ryouichi T, Katsuhisa T. Role of nitric oxide in the colon of patients with ulcerative colitis. World J Surg 1998;22:88. 7. Zhu L, Huang Y, Pei G. Study of L-arginine-nitric oxide pathway in ischemia-reperfusion injury limbs in rats. Chin J Traumatol 2002;5:16. 8. Kerrigan CL, Daniel RK. Pharmacologic treatment of the failing skin flap. Plast Reconstr Surg 1982;70:541. 9. Pang CY, Forrest CR, Mounsey R. Pharmacologic intervention in ischemia induced reperfusion injury in the skeletal muscle. Microsurgery 1993;14:176. 10. Deo SH, Barlow MA, Gonzalez L, et al. Repeated arterial occlusion, delta-opioid receptor (DOR) plasticity and vagal transmission within the sinoatrial node of the anesthetized dog. Exp Biol Med 2009;234:84. 11. Youssef FF, Addae JI, Stone TW. NMDA-induced preconditioning attenuates synaptic plasticity in the rat hippocampus. Brain Res 2006;16:183. 12. Ren J, Wang S, Li A. An experimental study of the effects of early enteral feeding of L-arginine enriched food on post-burn intestinal mucosal proliferation in rats. Zhonghua Shao Shang Za Zhi 2001;17:219. 13. Harisb J, Samuel A, Duglas D, et al. Arginine supplementation prevents necrotizing enterocolitis in the premature infant. J Pediatr 2002;140:425. 14. Hong JJ, Cohn SM, Peres JM, et al. Prospective study of the incidence and outcome of intra-abdominal hypertension and the abdominal compartment. Brit J Surg 2002;89:591. 15. Alican I, Kubes P. A critical role for nitric oxide in intestinal barrier function and dysfunction. Am J Physiol 1996;270:225. 16. Payne D, Kubes P. Nitric oxide donors reduce the rise in reperfusion-induced intestinal mucosal permeability. Am J Physiol 1993;265:189. 17. Taha MO, Miranda-Ferreira R, Paez RP, et al. Role of L-arginine, a substrate of nitric oxide biosynthesis, on intestinal ischemiareperfusion in rabbits. Transplant Proc 2010;42:448. 18. Taha MO, Miranda-Ferreira R, Fagundes AL, et al. Effects of L-nitro-arginine methyl ester, an inhibitor of nitric oxide biosynthesis, on intestinal ischemia/reperfusion injury in rabbits. Transplant Proc 2010;42:457. 19. Spanos CP, Papaconstantinou P, Spanos P, et al. The effect of L-arginine and aprotinin on intestinal ischemia-reperfusion injury. J Gastrointest Surg 2007;11:247. 20. Yanni AE, Margaritis E, Liarakos N, et al. Time-dependent alterations in serum NO concentration after oral administration of L-arginine, L-NAME, and allopurinol in intestinal ischemia/ reperfusion. Vasc Health Risk Manag 2008;4:437. 21. Lee CH, Hung WT, Lo HC, et al. Could fetal bovine serum protect the small bowel from ischemia and reperfusion injury by using a systemic perfusion. Int Surg 2007;92:257. 22. Lo HC, Wu SC, Li ML, et al. Does pharmacological dose of parenteral arginine have beneficial effect in rats with sub-acute peritonitis? Pediatr Surg Int 2010;26:625. 23. Granger DL, Taintor RR, Boockvar KS, et al. Measurement of nitrate and nitrate in biological samples using nitrate reductase and Griess reaction. Methods Enzymol 1996;268:142. 24. Faul F, Erdfelder E, Lang A-G, et al. G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods 2007;39:175. 25. Cui XL, Iwasa M, Iwasa Y, et al. Arginine-supplemented diet decreases expression of inflammatory cytokines and improves survival in burned rats. JPEN J Parenter Enteral Nutr 2000; 24:89. 26. Yokota N, Daniels F, Crosson J, et al. Protective effect of T cell depletion in murine renal ischemia-reperfusion injury. Transplantation 2002;74:759. 27. Lo HC, Tsai FA, Lin SC, et al. Systemic and local secretions of cytokines and nitric oxide in massive bowel resected rats with or without small bowel segment reversal. Cytokine 2001; 14:112.

LEE ET AL.: ARGININE AND INTESTINAL ISCHEMIA AND REPERFUSION 28. Muniappan L, Ozcan S. Induction of insulin secretion in engineered liver cells by nitric oxide. BMC Physiol 2007;7:11. 29. Igor S, Habib H, Jorge M, et al. Oral arginine improves intestinal recovery following ischemia-reperfusion injury in rat. Pediatr Surg Int 2005;21:191. 30. Wilson AM, Harada R, Nair N, et al. L-arginine supplementation in peripheral arterial disease: No benefit and possible harm. Circulation 2007;116:188.

75

31. Bertolo RF, Brunton JA, Pencharz PB, et al. Arginine, ornithine, and proline interconversion is dependent on small intestinal metabolism in neonatal pigs. Am J Physiol Endocrinol Metab 2003; 284:E915. 32. Fukatsu K, Ueno C, Maeshima Y, et al. Effects of L-arginine infusion during ischemia on gut blood perfusion, oxygen tension, and circulating myeloid cell activation in a murine gut ischemia/reperfusion model. JPEN J Parenter Enteral Nutr 2004;4:224.